Bacq Alexander-fundamentals of Radio Biology

tfW- - A W s " y I INTERNATIONAL SERIES OF MONOGRAPHS ON

PURE AND APPLIED BIOLOGY

Division: M O D E R N T R E N D S I N P H Y S I O L O G I C A L S C I E N C E S

GENERAL EDITORS : P . ALEXANDER AND Z . M . BACQ

V O L U M E 5

FUNDAMENTALS OF

RADIO BIOLO GY

OTHER TITLES IN THE SERIES ON PURE AND APPLIED BIOLOGY

MODERN TRENDS IN PHYSIOLOGICAL SCIENCES DIVISION

Vol. 1. FLORKIN—Unity and Diversity in Biochemistry Vol. 2. BRACHET—The Biochemistry of Development Vol. 3. GEREBTZOFF—Cholinesterases Vol. 4. BROUHA—Physiology in Industry Vol. 6. FLORKIN (Ed.)—Aspects of the Origin of Life Vol. 7. HOLLAENDER (Ed.)—Radiation Protection and Recovery Vol. 8. KAYSER—The Physiology of Natural Hibernation Vol. 9 FRANQON—Progress in Microscopy Vol. 10. CHARLIER—Coronary Vasodilators Vol. 11. GROSS—Oncogenic Viruses Vol. 12. MERCER—Keratin and Keratinization Vol. 13. HEATH—OrganophosphorusPoisons

BOTANY DIVISION Vol. 1. BOR—Grasses of Burma, Ceylon, India and Pakistan Vol. 2. TURRILL (Ed.)—Vistas in Botany Vol. 3. SCHULTES—Native Orchids of Trinidad and Tobago Vol. 4. COOKE—Cork and the Cork Tree

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FUNDAMENTALS OF V IS >

RADIOBIOLOGY .

C o m p l e t e l y R e v i s e d : S e c o n d E d k i o f t - v ,

by

Z.M. BACQ T

U N I V E R S I T Y OF L I E G E

and

PETER A L E X A N D E R CHESTER BEATTY RESEARCH I N S T I T U T E

I N S T I T U T E OF CANCER RESEARCH ROYAL CANCER H O S P I T A L

L O N D O N

A1

its

PERGAMON PRESS O X F O R D - LONDON—* N E W YORK —PARIS ,

196V xw; ' v s ^ ,

Contents Page

FOREWORD x i

INTRODUCTION—The Stepwise Development of Radiation Injury 1

CHAPTER 1. INTERACTION OF IONIZING RADIATIONS W I T H MATTER 6

Comparison of the different radiations 6 Mechanism of energy loss by x- and y-radiations 12 Energy loss by particulate radiations 19 Units of radiation dose and radioactivity 20 Measurement of dose 24 Ionization density 29 Excitations produced by ionizing radiation 39

CHAPTER 2 . DIRECT AND INDIRECT ACTION IN BIOLOGICAL SYSTEMS 4 5

Methods for distinguishing between direct and indirect action 46 Relative effectiveness of direct and indirect action in vitro 56 Relative effectiveness of direct and indirect action in cells 60

CHAPTER 3 . DOSE-RESPONSE RELATIONSHIPS IN CHEMICAL AND BIOLOGICAL

SYSTEMS 6 3

The D37dose and "single-hit" concept 63 "Multi-hi t" effects 66 Threshold—A problem of mammalian radiobiology 69

CHAPTER 4 . T H E NATURE OF THE INITIAL CHEMICAL LESION IN CELLULAR

RADIOBIOLOGY 7 5

The target theory 77 Application of target theory to radiation effects produced in vivo 82 The relative biological effectiveness of different ionizing radiations 90 The poison theory 96 Conclusions 97

CHAPTER 5 . GENERAL RADIATION CHEMISTRY 1 0 0

Role of excitation 101 Difference between the reactions in gases and those in liquids and solids 107 Protection and energy transfer 110 Fate of free radicals produced 115

vii

Vl l l CONTENTS

CHAPTER 6 . T H E RADIATION CHEMISTRY OF AQUEOUS SYSTEMS 1 2 2

Introduction 122 Historical development 123 Primary products in the radiolysis of water 124 Reactions of free radicals 136 Reactions of organic substances dissolved in water 147

CHAPTER 7 . EFFECT OF RADIATION ON MACROMOLECULES 1 5 7

Radiation changes in synthetic polymers produced by indirect action 160 Radiation changes in synthetic polymers produced by direct action 165 Protection of molecules 175 Physical and chemical changes produced in proteins by direct action 179 Physical and chemical changes in proteins produced by indirect action 187 Crosslinking and degradation of deoxyribunucleic acid 194 Changes produced in D N A following irradiation in vivo 206 Changes produced in polysaccharides 207 T h e use of radiation as an analytical tool 208

CHAPTER 8 . CHEMICAL SUBSTANCES WHICH SIMULATE THE BIOLOGICAL EFFECTS

OF IONIZING RADIATIONS 2 1 7

T h e chemistry of the biological alkylating agents 220 Comparison of biological effects produced by the alkylating agents and

by radiations 224 Mechanism of action of the alkylating agents 229 Radiomimetic properties of peroxides and oxygen at high concentrations 235

CHAPTER 9 . EFFECTS AT THE CELLULAR LEVEL 2 3 9

Introduction 239 Mitosis 240 Meiosis 242 Mitosis in a complex organism 243 Reversible cell damage and mitotic delay 245 Cell death 248 Breakage of chromosomes 253 Genetic effects of ionizing radiations 256

CHAPTER 10 . BIOCHEMICAL MECHANISM FOR CELLULAR E F F E C T S — T H E E N -

ZYME RELEASE HYPOTHESIS 2 6 3

Nucleus versus cytoplasm 263 Chromosome breakage 268 Interruption of energy supply 271 T h e enzyme-release hypothesis 272

CONTENTS ix

CHAPTER 1 1 . T H E EFFECT OF OXYGEN I N RADIOBIOLOGY 2 8 0

Time at which oxygen acts 281 Concentration of oxygen required 284 T h e oxygen effect in mammals 287 Application of oxygen effect to radiotherapy 290 Mechanism of action 292

CHAPTER 1 2 . COMPARATIVE RADIOSENSITIVITY OF L I V I N G ORGANISMS 2 9 9

CHAPTER 1 3 . PATHOLOGICAL BIOCHEMISTY OF IRRADIATED L I V I N G ORGANISMS 3 1 1

Oxygen consumption 312 Carbohydrate metabolism after irradiation 313 Disturbances in fat metabolism 317 Protein metabolism 322 Changes in electrolyte concentration 327 Sulphydryl enzymes and proteins 328 Increased enzymic and synthetic activity after irradiation 332 Inhibition of isolated enzyme systems in vivo 345 Biosynthesis of nucleic acids 347 Mechanisms responsible for decreased biosynthesis of D N A and R N A 352 T h e Nucleases 355 Summary 359

CHAPTER 1 4 . PROCESSES OF RESTORATION AFTER IRRADIATION 3 6 9

Restoration of genetic damage and of reproductive capacity 370 Recovery f rom physiological injuries 375 Repair in mammals 377

CHAPTER 1 5 . N E U R O - E N D O C R I N E REACTIONS IN RADIATION SICKNESS 3 8 7

Stress and the adaptation syndrome 387 Do ionizing radiations act as stresses? 389 Difficulties in facts and interpretations 392 First and second reactions 394

CHAPTER 1 6 . PHYSIOPATHOLOGY OF RADIATION SICKNESS IN M A M M A L S 4 0 6

Hyperacute syndrome 406 T h e first stage of radiation sickness 407 Changes in permeability 414 Blood changes 417 T h e second stage of acute radiation sickness 421

X CONTENTS

CHAPTER 1 7 . DELAYED EFFECTS 4 3 6

Shortening of life span 436 Cancer and leukaemia induction 442 Damage to embryos 447 Other late effects 448

CHAPTER 1 8 . INTERACTION BETWEEN CELLS AND TISSUES FOLLOWING IRRADI-

ATION 4 5 1

CHAPTER 19 . CHEMICAL PROTECTION AGAINST X - AND GAMMA-RAYS 4 5 7

Techniques 457 T h e protective substances 458 Mechanism of action of radioprotectors 465 Cysteamine and - S H protectors 470 Histamine, adrenaline, 5-hydroxytryptamine 477 Substances which intensify the effects of X-rays 477

CHAPTER 2 0 . TREATMENT BY B O N E - M A R R O W AND SPLEEN CELLS 4 8 4

Physical protection of the spleen, liver, bones and other organs 484 Injections of homogenates of spleen or bone-marrow after irradiation 486

CHAPTER 2 1 . H U M A N EXPERIENCE 4 9 4

Source of radiations to world population and their importance 494 Possible biological effects of natural and artificial background radiations 501 Acute radiation syndrome in man 505 Applications to therapy 510

POSTSCRIPT—The Role of Radiobiology in the World 513

SUBJECT INDEX 5 1 7

AUTHOR INDEX 5 3 8

Foreword

RADIOBIOLOGY has so many aspects of interest that it looks like the eye of an insect, each little facet contributing to the formation of the general picture. From the nuclear physicist to the doctor in charge of the protec-tion of exposed people or the treatment of irradiated patients, there is now a long unbroken chain of scientists trained in very different techniques and accustomed to look at radiation effects from divergent viewpoints. Mutual understanding is more common than in the past. Geneticists are now strongly linked with biochemists; anatomists and physiologists are no longer divided by high walls; radiochemists are often working in the same field as microbiologists or virologists. Radiation research has brought together scientists who, for other reasons, had no chance to meet, and who, in this way, escape the danger of being sterilized by too specialized interests or techniques.

This book is the second attempt of two very differently trained people— a physical chemist and a biologist with some medical knowledge—to present a coherent picture of radiobiology. Our excuses are: (1) that the first edition, published in 1955, despite its many faults appears to have filled a great need (it was translated into three other languages) and has helped many young men to enter the field of radiobiology; (2) that our happy collaboration has never stopped since 1953 and that our interests have become steadily more closely linked.

In this volume we have completely remodelled our first attempt and in most respects (Chapter 1 being the principal exception) this is a different book. Several new chapters have been added and some of the material has been divided differently. On the whole, however, the method of presenta-tion remains fundamentally the same as are our aims which were summar-ized in the first edition as follows :

"We have not aimed to provide a review for the specialists of individual topics, but have tried to present the subject as a coherent whole. This treatment will, we hope, also prove of value to radio-therapists, who have for many years used the powerful tool of ionizing radiation successfully in the therapy of cancer, in the absence of an adequate chemical and biological foundation. This position is now being remedied and a less empirical approach to radiotherapy may soon become possible.

xi

XLL F O R E W O R D

"This book is a survey and not a monograph. We have selected certain investigations from the enormous mass of published material, and have not attempted to present a complete review of the literature. Also we have deliberately chosen certain aspects of radiobiology for special emphasis since we feel that developments in these fields are most likely to advance the subject. A choice cannot be impartial; but if we have relied to a disproportionate extent on our own re-searches and on those best known to us we have made every effort to present fully opposing points of view. We have not hesitated to indicate which, in our opinion, are the most acceptable hypotheses at the present time; this has been done to introduce sense of coherence and does not imply a rigidity of viewpoint and we fully realize that new experimental data may alter the interpretations."

In the five years that have elapsed since the first edition the rate of publication in radiobiology has increased enormously. To say that as many papers appeared in the years 1955-1960 as in the period 1945-1955 is probably an understatement; consequently we have had to be still more selective in the material covered. To avoid a great increase in size the treatment devoted to certain subjects (e.g. the intervention of stress phenomena) has had to be cut and the reader referred to the first edition for details. Other aspects (e.g. the radiation chemistry of aqueous sys-tems) could be treated more briefly because contradictions and confusions have been resolved. The help given us by colleagues and friends was invaluable and it is a great pleasure to acknowledge our gratitude to them.

One of us (Z.M.B.) is deeply indebted to the Ministere de FInterieur (Department of Civil Security) for uninterrupted and generous support since 1946. Several scientific foundations (Fonds National de la Recherche Scientifique, Institut Interuniversitaire des Sciences nucleairs, Centre National de Radiobiologie et de Genetique) have contributed much in helping radiobiological research in his department at the University of Liege and in other laboratories in Belgium.

T I M E T A K E N

IO-5Scc

from seconds to hours

minutes to hours

' hours extending to years

Exposure to radiation

energy I absorption!

Ionized and electronically excited molecules

by free radicds from water !"indirect action")

Eariy physiological ^ e f f e C t S /metabolic

(usually reversible) /development

/ neededvN i

development of molecular lesion

by metabolism

Biochemical J lesions

/

S Mutations

( i .&: genetic d a m a g e )

I development < biochemical lesion by metabolism

Delayed somatic effects

(Cancer, leukaemia,

life span shortening)

(Submicroscopical lesions)

yisible lesions

I Cell death

1

' MODIFICATION OF

INJURY

DEVELOPMENT BY:

CHEMICAL PROTECT/VI

AGENTS

OXYGEN EFFECT

O

Death of organism

REVERSAL OF

BIOCHEMICAL

LESION? Hope of the future!

' INTRA-CELLULAR

REPAIRIaIso applies

to mutations}

' CELL REPLACEMENT

BY SPONTANEOUS

REGENERATION OR

BY GRAFTING

F I G . 1 .

I N T R O D U C T I O N

The Stepwise Development of Radiation Injury

RADIOBIOLOGY has become a very complex science because its ambition is to understand every step leading from the absorption of energy to death or final injury. The accompanying diagram (Fig. 1) summarizes the sequence of events as we now know them and we have organized this book essentially along the divisions shown in this diagram. Ionizing radiation of every type (whether from internal or external sources) is a form of energy; in order to act on a living or non-living system it must be absorbed. Thus the way in which the various types of ionizing radiations are absorbed constitutes the first step. The laws governing this primary physical process have been established with great precision. Knowledge in this field is amply adequate for radiobiology and further developments in the physics of radiations are unlikely to have a great influence on our subject.

This absorbed energy induces changes at the molecular level. Most cell constituents including macromolecules (like DNA or enzymes) as well as small molecules (like ATP or co-enzymes) are changed by radiation. The studies of radiation effects on dry organic molecules, on water and on aqueous solutions of both small and big molecules (in presence or absence of oxygen) are essential for an understanding of the very early events. Radio-chemists have described two main mechanisms which in the living organisms cannot be separated: (a) the direct action (molecular damage occurring in the molecule where the energy has been absorbed), (b) the indirect action (highly reactive free radicals formed in water reacting with cell constituents). The importance of the chemical environment, neglected before 1940, is now recognized by every radiobiologist although the extrapolation to living organisms of results obtained with unorganized models (aqueous solutions of polymers for instance) remains hazardous. It is at this molecular step that the presence of oxygen and of chemical protectors intervene. These agents must be present during irradiation since the principal chemical changes occur within microseconds of the exposure to radiation.

In the last five years there has been much progress in radiation chem-istry and a body of information is being built up about the type of chemical

1

2 F U N D A M E N T A L S OF R A D I O B I O L O G Y

changes that take place when organic substances are irradiated. This information, though valuable, will not by itself help us to know the nature of the important molecular lesions which initiate the biological chain. Our almost complete ignorance about which of the many reactions that occur are important and which are trivial represents one of the most important regions for radiobiological research.

The reader will find a number of examples of effects (e.g. on growth, on electric activity and on permeability) which occur during irradiation and are often rapidly reversible. These effects might be called physio-logical because they result in no permanent impairment. However, if one looks at cells that have received a lethal dose of radiation no damage can be seen by any test such as biochemical, histological or cytological within a few minutes of irradiation. In a mouse that has received a thousand r of x-rays, no lesion is detectable immediately after irradiation although the mouse will be dead in four days. But the biochemical lesions (to take Sir Rudolph Peters's concept) are constituted quite soon after irradiation; they will express themselves, more or less rapidly, in anatom-ical damage visible first under the microscope, later to the naked eye (clinical effects), damage which naturally coincides with physiological troubles (neuroendocrine changes, diarrhoea, burns, infections, sterility, etc.). This damage occurs earlier when metabolism (i.e. energy consumption) is high.

The death of the multicellular organism is generally due to the acute failure of one or several important functions, resulting from lack of cell growth (hematopoietic tissues, intestinal epithelium), metabolic troubles (water and ion exchanges), mechanical troubles in the respiratory tract, or invasion by microorganisms. The organism can escape death if damaged cells manage to recover, if regeneration of cells starts early enough or in certain cases if normal cells from another similar organism are grafted.

There are thus many problems of mammalian radiobiology that have to be solved at the cellular level; studies with unicellular organisms like bacteria, yeast cells or isolated mammalian cells in tissue culture have brought an enormous amount of information which help us to understand what happens in complicated multicellular organisms. The possibilities of the modern techniques of differential centrifugation, autoradiography and electronmicroscopy are far from being exhausted and much exciting precise information can be expected in the near future which will fill the gap between molecular and cellular events.

Mutations (genetic or somatic) must be considered as a particular kind of biochemical lesion which, by its nature, can express itself only in the descendants of the organism or in the daughter cells after division; altera-tions in the chromosomes have been described, some of which coincide with definite genetic changes. Thus genetics and refined cytology play an important role in radiobiology.

S T E P W I S E D E V E L O P M E N T OF R A D I A T I O N I N J U R Y 3

Finally the human factor in radiobiology has such an emotional impact that several widely discussed issues have become not only favourite sub-jects for journalists or television people, but also for politicians.

The invasion by totally incompetent (although perfectly sincere) men of this field of radiation effects on mankind has led to a complete distortion of the facts, even the most simple, and we think that it is the duty of every scientist to discuss this question in a realistic way, avoiding emotional as much as political interference, but also expressing himself without any fear, any restraint, on every one of the aspects of radiation effects on living beings.

T H E E S S E N T I A L R O L E O F M E T A B O L I S M I N T H E D E V E L O P M E N T O F T H E L E S I O N S

Most of our knowledge concerns the nature of the various lesions and we are almost totally ignorant of the processes that lead from one level of injury to the next. Normal metabolic processes seem to be responsible for the development from the molecular to the anatomical level.

Contemporary workers tend to forget that the role of metabolism was admirably stated as long ago as 1 9 2 5 by P. A N C E L and P. V I N T E M -

BERGER1 who placed an unincubated hen's egg in the refrigerator for 24 hours, irradiated it with x-rays and then replaced it in the refrigerator. Three days later no lesion was to be seen. If, however, an irradiated egg was incubated during the 3 days following irradiation many lesions were found. The factor which reveals the lesion caused by irradiation is cellular activity. Ancel and Vintemberger doubted the value of direct histological examination for determining differences in the radiosensitivity of cells. The authors reached the following prophetic conclusions which are still valid: "Three essential points must be clearly distinguished: (i) the radia-tion lesion; (ii) the factors bringing about the manifestation of the lesion; (iii) healing factors. It must not be forgotten that the lesions revealed by the microscope are the results of the combined, and sometimes antagon-istic action of these factors." It is only necessary to describe the lesion as biochemical to bring these conclusions formulated 35 years ago into line with present concepts.

The limitation of the energy reserves of irradiated cells has also already been mentioned by VINTEMBERGER in 1 9 3 0 2 . He wrote: "The duration of survival of an irradiated cell is inversely proportional to its activity after irradiation." As long as an irradiated muscle is not stimulated, or irradiated amphibians or eggs are kept at a low temperature, nothing is to be seen. The muscle must be stimulated and the frogs or eggs warmed, in other words, metabolism and oxygen consumption must be increased, to make the characteristic lesions appear. G R A Y ' S conclusion3, that "metabolism plays an essential role in the development of injury to the structural

4 F U N D A M E N T A L S O F R A D I O B I O L O G Y

components of the nuclei studied" is exactly in line with the biochemical lesion as defined by R. A. Peters and developed by us within the framework of radiobiology. The results obtained by DURYEE4 not only conform with the general idea which emerges from recent work with protectors against radiation, but also with the inability of cytophysiologists to explain the nuclear lesions solely by the action of radiations on the nucleus and chromosomes.

The higher the rate of metabolism, the more rapidly are abnormal metabolites formed4, and the more rapidly are the small stores of chemical energy dissipated.

The concept of biochemical lesion can explain a number of experiments which show a delayed effect similar to that found with striated muscle (see p. 272). If frogs irradiated with 3000 to 6000 r at 23°C are cooled to 5°C5'6, 80 to 90 per cent of the animals survive for more than 3 to 4 months after irradiation, whereas controls kept at 23°C die in 3 to 6 weeks. The lesion in the cooled animals is latent and if they are warmed after 60 to 130 days they die. No lesion is found in the ovarian ova of amphibians 12 days after total irradiation with 3000 to 5000 r if the animals are kept at 5°C, but if they are kept at 22°C after exposure to 3000 r, all the ova are affected. If the chilled and irradiated animals are warmed after 12 days the lesions appear very quickly4.

L A M A R Q U E and GROS7 irradiated the eggs of the silk worm (Bombyx mori) and kept them in the cold; when they were warmed six months later, it was found that very few of the radiation lesions had been repaired.

Similarly, hibernating mammals such as squirrels or marmots are much less radiosensitive when irradiated and maintained in hibernation. On warming 2 to 4 weeks after irradiation the animals show the radiation response of animals exposed to x-rays in the non-hibernating state and die in about 10 days following a lethal dose8'9.

The survival of fish (Carassius carassius) kept at various temperatures (between 25°C and 3°C) after an exposure to 1800 r (= LD50 35 days at 18°C) is very much longer at low temperature and seems to follow the decrease in oxygen consumption10 (i.e. time of survival is inversely proportional to metabolic rate.)

Bean seedlings may be kept at a temperature of 1-2°C for weeks and after restoration to 19°C they grow at practically the normal rate11. Seedlings which are irradiated and then kept at 1-2°C and later returned to 19°C show a reduced growth rate not very different from the normal radiation effect11. The radiation injury remains latent, but undiminished while in the cold (see also ref. 5).

R E F E R E N C E S 1. ANCEL, P. and VINTEMBERGER, P., C.R. Soc. Biol., Paris, 1925, 92, 517. 2 . VINTEMBERGER, P . , Arch. Anat., Strasbourg, 1 9 3 0 - 3 1 , 12, 2 9 9 ^ 6 4 .

S T E P W I S E D E V E L O P M E N T O F R A D I A T I O N I N J U R Y 5

3 . GRAY, L . H . , in Progress in Biophysics (Edited by J . A. V. BUTLER and J . T . RANDALL), 1951, 2, 240, Pergamon Press, London and New York.

4 . DURYEE, W . R . , J. Natl. Cancer Inst., 1 9 4 9 , 1 0 , 7 3 5 . 5. PATT, H . M. and SWIFT, M. N., Am. J. Physiol., 1 9 4 8 , 1 5 5 , 3 8 8 . 6. PATT, H . M., SWIFT, M. N. and TYREE, E. B . , Federation Proc., 1948, 7, 90. 7. LAMARQUE, J. P. and GROS, C., Brit. J. Radiol., 1945, 18, 293 and Seventh

International Congress of Radiology, Copenhagen, 1953. 8 . S M I T H , F . and GRENAN, M. M., Science, 1951, 1 1 3 , 6 8 6 . 9 . DOULL, J . , PETERSEN, D . F. and DUBOIS, K . P . , Federation Proc., 1 9 5 2 , 1 1 ,

340. 1 0 . K E I L I N G , R . , BLOCH, J . a n d VILAIN, J . P . , Annates Radiol., 1 9 5 8 , 1 , 3 8 1 . 1 1 . NEARY, G . J . , Nature, 1 9 5 7 , 1 8 0 , 2 4 8 .

C H A P T E R 1

Interaction of Ionizing Radiations with Matter

C O M P A R I S O N O F T H E D I F F E R E N T R A D I A T I O N S I N THIS book we are concerned with the very short wavelength electro-magnetic radiations, x- and y-rays, and the corpuscular radiations in particular electrons (j8-rays), helium nuclei (a-rays), protons and neutrons. The former are radiations of the same character as ultra-violet or visible light, but they are of much shorter wavelength and the energy of their quanta* is of the order of IO4 higher than the energy of the quanta of light, so that in practice there is little ultra-violet similarity. The absorp-tion of light waves (infra-red, visible and ultra-violet) depends in general on the molecular structure of the absorbent and only indirectly on the atomic composition.

The energy of x- and y-rays on the other hand is almost entirely ab-sorbed by ejecting electrons from the atoms of the material through which they pass, and this process is almost independent of the manner in which these atoms are combined into molecules. Moreover, the amount of energy absorbed from «- and /3-; ays and from a beam of hard x- or y-rays by a given weight of material is almost independent even of its elementary composition, although this is not so for soft x-rays.

It is clear, therefore, that the action of x-rays is much less selective than that of light: e.g. if ultra-violet light of 2600 A passes through an equal mixture of nucleic acid and a serum protein more than 90 per cent of the energy is taken up by the nucleic acid and less than 10 per cent by the protein. Using y-rays the same amount of energy is absorbed by the protein as by the nucleic acid. On absorbing a quantum of ultra-violet or visible light the whole of its energy is stored in the molecule which can then undergo one of a number of different reactions, some of which

* The energy of each quantum of an electromagnetic radiation in electron volts (eV) is given by 12,400/A (where A is the wavelength in A). T h e q u a n t u m i s the smallest step in which radiation can be absorbed, i.e. a molecule has to absorb a whole quantum or none at all, until the energy of the quantum becomes large enough for the Compton effect (see p. 13) to become appreciable.

6

I N T E R A C T I O N OF I O N I Z I N G R A D I A T I O N S W I T H M A T T E R Table 7-1 lead to chemical changes (molecular dissociations, etc.) and others to physical effects (e.g. fluorescence, heating, etc.).

An atom on absorbing a quantum of x- or y-rays loses an electron. With the exception of extremely soft x-rays, with which we are not con-cerned, the energy of the quantum taken up is greatly in excess of that required to produce an ionization (i.e. to eject an electron from an atom) and this surplus appears as kinetic energy in the ejected electron and ionized atom. The ejected electron is then sufficiently energetic to produce ionization in the atoms through which it passes. For the x-rays used in radiobiology almost all the ionizations are produced by the ejected elec-trons and the effect of initial absorption of the quantum of x-rays is usually neglected. Consequently the ions produced are not distributed at random throughout the solution but are concentrated along the track of the ejected electron. This represents another fundamental difference between ultra-violet light and ionizing radiation.

If there are no chemical changes all the energy of x-rays as well as of light waves eventually appears as heat in the absorbing material. With the doses and the dose rates used in radiobiology a significant change in temperature would not be produced and the heating effect can in general be neglected except perhaps for very densely ionizing radiations or in "hot spots" where a disproportionate amount of energy is dissipated. In these cases any heating would be accompanied by a high concentration of reactive radicals which would be more damaging than the heat pro-duced.

The distinction between x-rays which are produced in generators and y-rays which are given off by some radioactive elements has disappeared. Until comparatively recently the most energetic x-rays used in biological experiments were obtained from 400 kV therapy tubes giving a spectrum ranging in wavelength from 0-03 A and having an average wavelength of 0-06 A*, while y-rays were obtained from radium with a wavelength of 0-01 A corresponding to x-rays of l-2x IO6V. The post-war period saw the rapid development of machines such as the van de Graaff gener-ator, powerful linear accelerators, betatrons, synchrotrons, etc.

x-rays X-rays corresponding to many million volts can now be generated by

commercially available machines and these fall within and beyond the wavelength range of y-rays. The ready availability from atomic piles of

* I n the spectrum of x-rays given out by therapy-type machines the most energetic radiations (i.e. those of shortest wavelength) have an energy equivalent to the peak voltage—i.e. their wavelength is A = 12-4/(kV of set). However, the average energy of all radiations is according to LEA1 half this value.


the radioactive isotope cobalt-60 (60Co) has provided a useful source of pure y-rays of high energy, 1-1 to 1-3 MeV*.

/3-rays Since the chemical and biological effects of x- and y-rays are produced

by the ejected high-speed electron and not by the primary ionization it follows that similar results can be obtained by direct bombardment with electrons of comparable energies. Such electron beams are called ^3-rays and can either be obtained from special generators or from radioactive isotopes of which a large choice is now available (see Table 1-1). The dis-tance of penetration of /3-rays depends on their energy (see Fig. 1-1), but even with 2 MeV electrons the range in water (or in biological tissue) is only about 1 cm. However, the disadvantage of the short range of the

Energy of particles

FIG. 1-1. Relationship between the range of an ionizing particle in water (/*) and its energy (keV).

jS-rays can be overcome by dissolving radioisotopes in the solution or system which is to be irradiated when the whole volume will be uniformly exposed. In biological systems the isotope may become localized in certain regions and the resultant irradiation will then not be uniform.

* The electron volt (eV) is a unit of energy corresponding to 1 -60 X IO -12

ergs. 1 MeV = IO6 eV, 1 keV = IO3 eV.

I N T E R A C T I O N O F I O N I Z I N G R A D I A T I O N S W I T H M A T T E R

T A B L E 19-1

L I S T OF SOME JS-RAY E M I T T I N G ISOTOPES

Element Z A Half-life, hour, day

or year Radia-

tion

0Z /0 E max

(MeV) Formation in

the pile

H 1 3 11-8 y P- 0-018 2H(n,y)3H, Li(n, a)3H

Be 4 10 2-5 X l O 6y P- — 0-555 9Be(«,y)10Be

C 6 14 5568 y P- — 0-155 13C(w,y)14C Na 11 22 2-7 y P + — 0-557 —

Y — 1-30 —

P 15 32 14-3 d P- — 1-701 31P(«,y)32P 15 33 25 d P- — 0-26 —

S 16 35 88 d P- — 0-167 34S(n,y)35S Cl 17 36 4 x 105y P- 0-714 35Cl(w,y)36Cl K 19 40 1-3 X l O 9

y P- S 9 1-33 Naturally occurring 1-3 X l O 9

y Y — 1-46 —

Ca 20 45 152 d P- — 0-255 44Ca(^y)45Ca ,P-X 50 0-46 59Fe(n,y)59Fe ! Y J

50 1 1 —

Fe 26 59 47 d I P-\ 50 0-26 — 50 1-30 — P~ As 33 77 40 h P- — • 0-80 76Ge(w,y)77Ge -U-

77As Br 35 82 34 h P- — 0-447 81Br(n,y)92Br

0-323 —

0-181 —

ymax — 0-769 —

Rb 37 86 19-5 d P- 80 1-822 85Rb(n,y)86Rb P-X 20 0-716 —

Y J 20 1-081 —

Sr 38 89 53 d P- — 1-463 88Sr(n,y)89Sr Y 39 90 61 h P- — 2-2 89Y(w,y)90Y Sr 38 90 19-9 yr P- — 0-61 Fission product de-19-9 yr

cays to 90Y Ag 47 110 270 d P- 58 0-087 109Ag(w,y)110Ag Ag

35 0-570 —

5 2-90 —

I 53 131 8 d P~\ 0-605 — B-Y f OO 0-364, etc. 130Te(n,y)131 Te Y f

131J

14 0-25

y } 14 0-637 —

Cs 55 134 2-3 yr p- 75 0-658 133Cs(n,y)134Cs 2-3 yr p- 25 0-09 —

y max — 1-36 —

Au 79 198 2-69 d P- — 0-96 197Au(w,y)198Au Y — 0-441 —

1 0 F U N D A M E N T A L S O F R A D I O B I O L O G Y

TABLE 1 - 1 . L I S T OF SOME /?-RAY E M I T T I N G ISOTOPES ( c o n t i n u e d )

Element Z A Half-life, hour, day

or year Radia-

tion %

Emax

(MeV) Formation in

the pile

Hg 8 0 2 0 3 4 3 - 5 d P - _ 0 - 2 0 8 202Hg(w,y)203Hg

Y — 0 - 2 7 9 —

T l 8 1 2 0 4 2 - 7 y P - — 0 - 7 7 5 203Tl(n,y)204Tl RaE(Bi) 8 3 2 1 0 5 - 0 2 d P - 1 - 1 7 Naturally occurring

Z is the atomic number A is the atomic weight

Heavy Ionizing Particles a-Rays are the nuclei of helium atoms (i.e. double charged positive

particles of atomic weight 4). They are given off by a few radioactive substances, notably radon—obtained as a decay product from radium and polonium*. Because of their high charge and low velocities the particles are readily stopped by matter and in water or tissue the range of a particle from radium C' is only 7-0 [x (see Fig. 1-1), and many ions are formed along its track (i.e. the ionization density is very high, see p. 29).

Protons are hydrogen nuclei having mass 1 and carrying one charge; energetic protons can be obtained artificially from the cyclotron, proton-synchrotron or a van de Graaff generator. They are intermediate between a-particles (mass 4) and electrons (mass 5-5 x IO-4) in penetration and ionization density.

With the newer generators many heavy ionizing particles can now be produced. Any atom stripped of one or more of its electrons if accelerated will become an ionizing particle. Deuterons are frequently used; they have mass 2, charge 1 and consequently their penetration and ionization density is intermediate between that of protons and a-particles. More recently, machines have been available for producing particles which carry a greater charge and which are heavier than a-particles. The one used most fre-quently is a carbon atom which has lost six electrons. With a mass of 12 and a charge of 6 the properties of these particles are as different from a-particles as electrons are from protons. The heaviest ionizing particles are fission nuclei which are produced when the atoms of a heavy element (e.g. 235U or plutonium) undergo nuclear fission. Their range, however, is so low that they cannot be used for radiobiology.

* a-Rays of low energy and giving an extremely high ion density can be obtained by the artificial disintegration of boron or lithium by slow neutrons. For example, when the nucleus of a lithium atom captures a neutron it immediately dissociates to give an a-particle. Tri t ium (3H) remains and this decays slowly by giving off /3-rays.

I N T E R A C T I O N O F I O N I Z I N G R A D I A T I O N S W I T H M A T T E R 11

Neutrons Fast neutrons (particles having mass of 1 but carrying no charge) are

usually obtained either from a cyclotron, atomic pile or indirectly from a van de Graaff generator, but can also be obtained more simply by the bombardment of beryllium with a-particles. A simple low-power source is the complex salt RaBeF4. Neutrons do not produce ionization directly but knock out protons from the nuclei of the atom of the absorbing material. The biological effects of fast neutrons are, therefore, almost wholly due to protons in exactly the same way as the effects of x-rays are produced by the ejected electrons. Unlike the other ionizing radiations, however, the number of ionizations produced depends largely on the nature of the elementary composition of the material through which the neutrons pass. The reason for this is that the transfer of energy between neutrons and protons does not depend on the atomic number but on other factors, and the number of ionization produced by a given dose of neutrons in 1 g of water will be about 2-5 times that produced in 1 g of air; this makes neutron dosimetry very difficult (see p. 23). Neutrons, like x-rays, can penetrate large amounts of matter as the absorption coeffi-cient is low. The protons are ejected at random within the irradiated material. The ionizations are therefore concentrated along short tracks inside the irradiated body.

Slow neutrons do not eject a proton but are captured by the nuclei through which they pass, thereby producing a new nucleus which may be radioactive and will emit /3- or y-rays. During the process of neutron capture the nucleus emits a y-ray. Many of the radioactive substances listed in Table 1-1 are produced in this way in atomic piles. The reactions of slow neutrons, although of much chemical interest, are not generally of biological importance since the effects produced by the ionizing radia-tions emitted are much more far-reaching than those resulting from the transmutation of relatively few atoms*.

In this connection it should be pointed out that very high energy electromagnetic radiations (i.e. greater than 8 MeV), produced for example by a synchrotron or betatron, will also produce nuclear trans-formations in some of the elements through which they pass. An animal irradiated from one of these generators becomes detectably radioactive. In x-ray therapy with a 25 MeY betatron, 5 per cent of the total dose

* Slow neutrons are used in a medical application which depends on the fact that they transmute with high efficiency atoms of boron into lithium atoms which emit a-particles. Hence those cells which contain boron will receive a much larger dose of ionization than those that do not, following irradiations with slow neutrons. Patients with brain tumours have been treated by exposure to slow neutrons given off by a reactor since there were indications that a dose of borate administered shortly before irradiation was selectively localized in the tumour cells.


received by the patient is emitted by the carbon isotope 11C which is produced in situ from the ordinary 12C atoms in the body by the x-rays. A case is recorded where the gold tooth of a man accidentally exposed to slow neutrons became so radioactive as to produce ulceration of the gum.

M E C H A N I S M O F E N E R G Y L O S S BY x- A N D y - R A D I A T I O N S

Interactions between beams of electromagnetic or particulate radiation and matter can be described quantitatively only in the language of quantum mechanics. The problem, although very difficult, has been solved by contemporary physics and detailed treatments are given in advanced modern textbooks2. It is not possible here to do more than give a list of some of the more important processes. Excitation of atoms and molecules by the absorption of a quantum of visible or u.v. light will not be con-sidered.

As we have seen, virtually all the ionizations which result from the absorption of x- or y-rays are produced by the ejected electrons. The first problem is, therefore, to determine the number and energy of the electrons produced when these rays are absorbed. For all radiations the intensity of the beam before absorption (7o) is related to that after absorp-tion (I) by the equation I = Ioe-Ixx where u is the absorption coefficient and x the amount of material. The thickness x may be expressed variously as cm, g/cm2, atoms/cm2, electrons/cm2, etc. Since the product iix must be dimensionless, /.< is correspondingly expressed as cm-1, cm2/g, cm2/ atom, cm2/electron, etc. To indicate which unit is being used the following symbols are conventionally employed:

He for cm2/electron, iijp for cm2/g (mass coefficient), fia for cm2/atom, [j, for cm-1.

All these coefficients can be interconverted if the atomic weight (A) and the atomic number (Z) are known; e.g. in terms of /ie,

Ha = Z f X e

'Mp = N(ZlA)fXe

[l = PN(ZjA)lUe

where N is Avogadro's number and p the density. There are essentially three mechanisms by which energy can be trans-

ferred from the radiations to the material through which they pass and, when scattering can be neglected as is normally the case, /.ia is made up of three components, ra> <ya and na, corresponding to energy absorption by the photoelectric effect, Compton effect and electron-positron pro-duction.

I N T E R A C T I O N OF I O N I Z I N G R A D I A T I O N S W I T H M A T T E R 1 3

The Photoelectric Effect By this mechanism a quantum gives up all its energy to an atom (i.e. is

completely absorbed) and an atomically bound electron is ejected. The kinetic energy of this electron is the energy of the quantum less the sum of the energy required to remove the electron from the atom (the binding energy) and the negligibly small energy imparted to the atom. Since electrons at different levels have different binding energies the energy of the photoelectron will vary, but for the atoms making up organic materials and water a maximum value for the binding energy of 500 eV may be taken*. Compared with the high quantum energy of the radiations used in radiobiology the binding energy is comparatively so small that virtually all the energy is retained by the photoelectron which then produces further ionizations.

The absorption coefficient per atom /ia of the material varies with the wavelength, A, of the radiation and the atomic number, Z, of the elements of which it is composed. The atomic absorption coefficient for photo-electron absorption (ra) is given by

Ta = c . . Z» where c is a constant, m is close to 3 and n varies from 3-5 to 5. Conse-quently, the photoelectric absorption falls off very rapidly as the radiations become more energetic (i.e. harder), and for x-rays of energy greater than 1 MeV the contribution of photoelectrons to the total energy ab-sorption can be neglected (see Fig. 1-2). Also since the absorption varies as a high power of Z the photoelectric absorption is much greater for heavy elements than for light elements.

The Compton Ejfect The elementary view is that this process is like a "billiards ball"

collision between the quanta of radiationf and the electrons of the atoms through which they pass. The amount of energy transferred to the electron which is ejected varies and can be calculated from the theoretically derived equation given by K L E I N and NISHINA3. The energy of these recoil

* When an inner electron has been ejected an outer or free electron can fall into its place. In this process energy is set free since the gross change is the removal of an outer electron which requires only about 10 eV compared with the value of about 500 eV for inner electrons. Th is energy is liberated as a quantum of radiation —corresponding to very soft x-rays—which is usually absorbed by the same atom to give an electron of extremely low energy having a high specific ionization (see p. 29). Th is gives rise to a highly localized release of u p to 500 eV and is referred to as the Auger effect.

t T h e scattered quantum after it has given up a fraction of its energy to the ejected electron will behave normally and can undergo all the processes for energy loss (e.g. another "Compton collision").

1 4 F U N D A M E N T A L S OF R A D I O B I O L O G Y

/ , /

a A /

I J j

/ / I i i i I I I

10 50 100 Quantum energy of radiation

500 keV

FIG. 1-2. Relative importance of Compton and photoelectric effect for energy loss in water by x-rays of different quantum energy: (a) proportion of total number of electrons produced by Compton effect; (b) proportion of total energy which appears in recoil (Compton) electrons. (The difference between the value shown and 100 per cent is due to photoelectrons; in the range of energies shown, pair formation—see p. 17—does not

occur.)

electrons, unlike that of the photoelectrons, varies widely and ranges from zero to a maximum value. The average energy of the recoil electrons increases rapidly with the energy of the radiation as shown in Fig. 1-3. Consequently the contribution of Compton electrons to the total amount of energy absorption increases with the hardness of the radiation (see Fig. 1-2), although the overall absorption coefficient decreases with decreasing wavelength see (Fig. 1-5).

Besides the difference in the energy of the electrons ejected by the photoelectric and Compton effects there is an important difference in the energy dissipation in different materials. The contribution of the Compton effect to the mass absorption coefficient of the material under irradiation (i.e. the amount of energy dissipated per gramme) depends entirely on the number of electrons per gramme, and this in turn depends on the elementary composition. Fortunately this value does not vary greatly for different elements and is nearly the same for water, most organic materials and consequently biological tissue (see Table 2-2). This means that if the source of radiation of hard x-rays where the Compton effect predom-inates has been calibrated by measuring energy dissipation per gramme of

I N T E R A C T I O N O F I O N I Z I N G R A D I A T I O N S W I T H M A T T E R Table 19-1

7O2 IO3

Quantum energy of radiation kev

FIG. 1-3. Maximum and mean energy of Compton (recoil) elec-trons as a function of quantum energy of radiation in eV. (The average quantum energy of the radiations f rom an x-ray therapy

set is half the peak voltage.) (Data taken f rom LEA 1 . )

air with a standard ionization gauge this value can be converted by constant factors to give the energy loss in different materials. Figure 1-4 shows these relationships for materials of interest in radiobiology.

The position is more complex for soft x-rays where a considerable proportion of the energy dissipation is due to the photoelectric effect since


FIG. 1-4. Energy deposited by 1 roentgen of x-rays in different tissues as a function of the energy of the radiation. When the energy deposited is 100 ergs then 1 r is equivalent to 1 rad, within the uncertainty of the physical constant W. This is the case for all tissues except bone with x-rays of quantum energy greater than 200 keV (i.e. this is approached in a therapy machine

with peak voltage of 400 kV).

TABLE 1 - 2

(After LEA1)

Element •

Atomic number

No. of electrons per gramme X IO -23

H 1 5-98 C 6 3-01 N 7 3-01 O 8 3 01 Mg 12 2-97

Al 13 2-90 P 15 2-91 S 16 3 01 Cl 17 2-89 Ca 20 3-01

Air 3 01 Water — 3-34 Nucleoprotein — 3-21 Wet tissue — 3-30

I N T E R A C T I O N OF I O N I Z I N G R A D I A T I O N S W I T H M A T T E R Table 21-1 the absorption by a material is no longer directly proportional to the number of electrons. The energy dissipation is not now essentially the same for water, tissue, and protein and the values relative to air are given in Fig. 1-4 for x-rays of different wavelengths. The important point is that for soft x-rays it is not possible to derive the dose for different systems directly from measurements with an ionization chamber. Corrections have to be applied which depend upon the elementary composition of the irradiated material. These corrections are not easy to make accurately when the x-rays are not monochromatic, and for 20 kV x-rays the dif-ference in energy dissipation for the same exposure between 1 g of protein and 1 g of water is 15 per cent. For this reason chemical dosi-metry methods, especially those using organic solvents, are suitable only for hard x-rays and liable to serious error when used for calibrating therapy sets working at 150 kV or less.

Electron-positron Production* Radiations with an energy greater than 1 -02 MeV can lose energy by

the simultaneous "creation" of an electron and a positron. The effect is complex and can be understood only in terms of quantum mechanics. Certain points, however, may be noted: all the energy of the quantum appears in the pair of particles, the first 1-02 MeV providing the "rest-mass" and the remainder providing the kinetic energy of the particles. The two particles then lose their energy by collisions with electrons, but in addition the positron has the possibility, the greater the lower the kinetic energy, of annihilation with an electron. In this event, which all positrons eventually undergo, the mass of the two particles is lost and appears as energy in two quanta of y-rays. In the region of biological interest the atomic absorption coefficient for pair formation varies as Z2

and is, therefore, greater for 1 g of a heavy element than 1 g of a light element.

The mass absorption coefficient of air and water after passing through an inflection (see p. 35, Fig. 1-5) for radiations of about 200 KeV decreases with increasing energy of the radiation since energy loss by both the Compton and photoelectric effect decreases. Athighenergiesthe absorption increases again because energy loss by electron-positron formation increases with increasing energy of the radiation. This com-plex behaviour is illustrated in Fig. 1-5 for elementary carbon and water. The minimum depends upon the elementary composition of the irradiated material and lies at lower energies for heavier elements.

* This phenomenon is usually referred to as pair formation and this term must not be confused with "ion pairs".


Quantum energy of ionizing radiation (MeV)

\ Cb) —

\ —

•s \ V, -To a/ais irption ( J-a)

> >

\

\

\ V S S \

s I I \

(Cor Vpt on) \\

\ \ I

— —

\

—

—

y hot >ei) —

\

\ S S t> (Co. mpto n)

• I (fair) — L -

0 01 002 O-OS 0-1 OZ 0-5 VO Z S 10 Quantum energy of ionizing radiation

so WO MeV

FIG. 1-5. Relative contribution of photoelectric effect, Compton effect and pair formation to the mass absorption coefficient of ionizing radiations having different quantum energies (after

HEITLER2): (a) for water; (b) for carbon.


Cerenkov Radiation The amount of energy dissipated in this way represents such a minute

fraction of the total that this phenomenon does not play any part in the biological effects produced. Cerenkov radiation is, however, sometimes used for the measurement of dose. In media such as water for which the optical index of refraction (n) is greater than 1, the velocity of light is c/n where c is the velocity in vacuo. A charged particle can never move faster than c, but high energy electrons can move faster than c/n. A particle moving through a medium at a speed greater than c/n has its electric field strongly perturbed and loses some of its energy—usually only a very small part—as radiation which is named after its discoverer, Cerenkov. This radiation is often in the ultra-violet or visible region and is unlikely to have any biological significance5.

E N E R G Y L O S S B Y P A R T I C U L A T E R A D I A T I O N S Although the fundamental particles differ from one another in size and charge their mechanism of energy loss is essentially the same. The only exception is the neutron which, of course, cannot participate in processes depending on electric charge; however, it produces protons and y-rays which lose energy in the normal ways.

The charged particles undergo inelastic collisions with the bound electrons of atoms which they can eject to produce ions. Most of these ejected electrons are sufficiently energetic to produce a few ionizations of their own. When, as happens occasionally, the ejected electron has sufficient energy to produce a number of ionizations (e.g. 30)—though the definition is arbitrary—this secondary electron is called a 8-ray.

When the interaction between the charged particle and the atom through which it passes is not sufficient to provide the energy needed for an ionization an electronic excitation occurs. The electron is displaced from its normal state to one of higher energy and the atom of which it forms a part is then said to be excited. There is no way of measuring the number of atoms that have been excited, but there are good reasons for believing that there are several excitations for every ionization.

As a charged particle travels through matter it loses all its energy by producing ionizations and excitations until its total energy has become too low to produce further ionizations. When this occurs the ionizing particle (usually an electron) is captured, if there is an atom having an affinity for electrons present, to give a negative ion. As every electron which has been ejected in an ionization eventually finishes up in this way, one negative ion is formed for every positive ion. This is why ref-erence is always to an ion pair and not just to a positive ion. After irradiation with /3-rays there will be an excess electric charge in the


material due to the electrons that have been completely "stopped" within the sample under study. The number of these, however, is very small compared with the total number of ion pairs that are formed. Thus with 1 MeV electrons some thirty thousand ion pairs will be formed for every excess negative ion produced.

A formula of the loss of energy by charged particles was first derived by Niels Bohr and extended by BETHE4 to cover special quantum effects which must be allowed for. The absorption of energy per gramme of material depends, as does the Compton effect, on the number of electrons present and for a given ionizing particle the stopping power* is essentially the same for water tissue and most organic materials.

It is useful to emphasize again that it is these inelastic collisions of charged particles with electrons that are responsible for virtually all the energy taken up from x- or y-rays. The photoelectric effect, Compton effect and pair formation merely determine the number and energy of the electrons which produce the ionizations. Essentially, there is no difference between the effects produced by fast electrons and by x-rays except that the range of the former is small while the latter can penetrate much deeper and release electrons in the interior.

The spatial distribution of the ionizations is quite different if the primary source of ionization is particulate or if it is x- or y-rays. In the first case the particles have a definite range so that at a certain thickness of absorber they are completely cut off and screening is complete. The electromagnetic radiations become progressively attenuated as they pass through matter, but there is no sharp cut-off (compare Fig. 1-1 with Fig. 1-17).

It must be stressed that all these considerations apply only to the initial act of ionization. In mixed systems, particularly mixtures of gases, transfer of ionization can take place to the most easily ionized molecules so that the relative proportion of molecule finally ionized need bear no direct relation to their stopping power.

U N I T S O F R A D I A T I O N D O S E A N D R A D I O A C T I V I T Y Of all the changes occurring in matter exposed to ionizing radiation the amount of energy deposited in an irradiated material by a given dose is the one which can be defined most exactly. It is independent of physical state and is the same whether the absorbent is irradiated as a solid, liquid

* For particulate radiations the term "stopping power" is used in place of the absorption coefficient used with electromagnetic radiation. T h e stopping power of a material is the rate of loss of energy of an ionizing particle moving through it. Its magnitude depends on the charge and velocity of the ionizing particle and on the density (or more exactly on the number of electrons per g)of the material.

I N T E R A C T I O N OF I O N I Z I N G R A D I A T I O N S W I T H M A T T E R Table 25-1 or gas at any temperature or any pressure. Under favourable conditions the amount of energy deposited can be measured by calorimetry with a high degree of precision, but in general it is experimentally too difficult to use this for routine dosimetry. Other measurements, chemical and physical, are used for measuring the dose in practice though energy uptake must always be the primary reference standard.

The energy taken up manifests itself in different forms and eventually appears as heat and chemical change. A very small amount, which need not be considered, may be given off as light. Ideally, therefore, radiation dose should be measured as the amount of heat produced in a system which does not undergo any net chemical change. However, the amount of energy involved in radiobiology and radiation therapy is much too small to be detected calorimetrically and there are few radiation sources for which this is possible (see p. 28). For example, a dose of x-rays sufficient to kill a mammal (i.e. 700 r) would raise its temperature only by 1 -7 x 10_3°C even if all the energy were converted to heat and none in chemical change.

The Roentgen In gases the most readily detected manifestation of energy absorption

is the formation of an ion pair. For purposes of dosimetry ionization in gases can be defined exactly and at least in the low and medium energy range up to 1 MeV it can be measured accurately. However, the number of ionizations can be neither defined unambiguously nor measured directly in solids or liquids since all the energy absorbed is not utilized in producing ionization and some of it produces excitations (see p. 39). The amount of energy which has to be provided by ionizing radiations to produce one ion pair together with its associated excitations can be measured experimentally and is usually denoted as W, the energy required to produce an ion pair. For this reason the dose of x- and y-rays is usually defined in roentgens (r). One r (see Table 1-3) is defined as that quantity of x- or y-radiation such that the associated corpuscular emission (i.e. electrons) per 0-001293* of air produce in air ions carry 1 e.s.u. of elec-tricity of either sign. From classical electrochemistry (Faraday's law) 1 e.s.u. is known to correspond to 2-1 x IO9 electronic charges (here it corresponds to ion pairs) and consequently this is the number of ion pairs formed in 1 cc of air by 1 r. For W = 34 V (see p. 26) 1 r represents an energy absorption of 0-111 ergs per cc of irradiated air and of 87 ergs per gramme of air. The mass stopping power of water vapour is greater than that of air because it differs in atomic composition. Therefore, the number of ion pairs formed by 1 r in water vapour is greater than that formed in

* 0-001293 g of air occupies 1 cc at N .T .P . (i.e. 0°C and 760 mm).


TABLE 1 - 3

RADIATION UNITS

1 rad corresponds to an absorption of energy of 100 ergs/g 1 roentgen (r) produces in 1 cc of air at N.T.P.* 1 e.s.u. of electricity of either

charge 1 roentgen (r) produces 2-1 XlO9 ion pairs per cc of air at N.T.P. Exposure of air to 1 r results in an absorption of approx. 87 ergs/gt Exposure of water (or tissue) to 1 r results in an absorption of approx. 100 ergs/gt 1 rep (roentgen equivalent, physical) releases the same amount of energy in water

(or tissue) as 1 r of x-rays} Energy to form an ion pair in air (W) approx. 34 eV (see p. 26) 1 curie The amount of radioactive material which will give 3-7 XIO10

disintegrations per sec K factor gives the dose in r per hour at 1 cm distance in air of y-rays

given off by 1 millicurie (10~3 curies) of a radioactive material. The value of this constant depends on the energy of the emitted y-rays

1 rem (roentgen equivalent, medical) does the same amount of biological damage as 1 rad of x-rays (of energy range of 100 to 1000 keV). This means that the relative biological effectiveness (RBE, for defini-tion, see p. 91) must be known from biological experiments. Thus if cc-rays are ten times efficient as x-rays in producing a given effect than 1 rem of a-rays = 0-1 rad of x-rays. Since the RBE varies a great deal for different biological change the value of the rem in rads will vary widely. The rem is useful for the control of radiation levels as personnel in atomic industries are often exposed to a "mixed bag" of radiations

* 1 cc of air at N.T.P. weighs 0-001293 g.

t A value of 32-5 eV for W was, until recently, widely used and this made the roentgen equivalent to 83 ergs in air and 93 ergs in water. But there is some uncertainty and these figures are probably too low by some 5 to 10 per cent (see p. 26). Although the figure of 93 ergs is still widely used there is in fact no experi-mental justification for making a difference between the absorbed dose in water of a rad and a roentgen.

t Sometimes the rep83 is used which means that the radiation (e.g. a, /J, etc.) releases the same energy as 1 r of x-rays in air. Since the ratio of the absorption coefficients may vary (in particular for neutrons) the two rep units need not be the same. The conversion of the dose received in roentgens into energy deposited depends on the value chosen for the amount of energy associated with the forma-tion of one ion pair.

air although the values for W are similar. (For the purpose of radio-chemical calculation—see p. 29—the values are assumed to be the same,

I N T E R A C T I O N OF I O N I Z I N G R A D I A T I O N S W I T H M A T T E R Table 27-1

but this is arbitrary.) If water or tissue is exposed to a dose of 1 r then the amount of energy deposited will vary with the quality of the radiation (see Fig. 1-4). For hard radiations the value will be 98 ergs per gramme.

The roentgen has the great merit, after earlier definitions, of being unambiguous, but its measurement is often a matter of considerable difficulty. Confusion also has arisen by attempts to extend the definition of the roentgen to radiations other than x- and y-rays.

The Rad Since the roentgen is defined for x- and y-rays an extension was made

to cover other radiations. The rep (roentgen equivalent, physical) is defined as that quantity of radiation which will release in water (or tissue) the same amount of energy as is released by 1 r of x-rays. Unfortunately, the ambiguity remains whether this means 83 ergs per gramme of air or 93 ergs per gramme of water. In principle the extension of a unit based on measuring ion pairs is not suitable for energy absorption in condensed systems. To avoid these difficulties it has been proposed by an inter-national committee6 to define dosage by an unambiguous energy unit. The name rad is suggested for the quantity of radiation which will result in an energy absorption of 100 ergs per gramme of irradiated material. It should be recalled that M A Y N E O R D 7 introduced in 1 9 4 0 an energy unit which has been widely used in radiotherapy known as the gramme roent-gen. By a direct transfer of quantities 1 g roentgen is defined as 83 ergs deposited in the irradiated material. Since for many practical purposes the rad will be calculated from measurements of the number of ionizations produced in a gas (see p. 25) the energy imparted to a unit mass of substance (Em) is defined as follows: Em = WS. Jm; where Jm = num-ber of ionizations in unit mass of gas, W = energy per ion pair of gas, and 5 = ratio of mass stopping power of the material to the gas.

The rad covers all radiations, including neutrons, which are detected by the ionizations produced by the protons they cause to be emitted. The earlier units for neutrons are not very satisfactory8. Since the number of protons present in 1 g of water is very much greater than that in 1 g of air, the ratio of the energy absorbed in the two media may be as much as 10 for neutrons as compared to 1-12 for the other particulate radiations.

Activity of Radioactive Materials Samples of radioactive isotopes are calibrated both in terms of the

activity of the sample and the energy of the radiation (see Table 1-1). For clinical use and in radiobiological experiments /3-cmitters are usually used, although in many cases y-rays are also given off and will contribute to the total dose received by a volume of tissue. The unit of activity is


the curie, which was originally defined as that quantity of radon which is in equilibrium with 1 g of radium. This has been modified as the amount of material in which 3-70 x IO10 atoms disintegrate per second.

If we know the energy of the radiations given off by the disintegrating atoms it is possible to calculate the radiation dose received. For example, one curie of an isotope giving off /S-radiations of average energy 1 MeV when dissolved in 1 litre of water (or dispersed in 1 litre of tissue) will result in an absorption of 3-7x IO10X 1 x IO6X 10~3 eV/g,sec = 0-6 rad I sec and the dose rate will be 0-64 rep/sec. For the purpose of this calcu-lation it is assumed that the energy of the /3-rays is wholly dissipated in the dissolved volume.* If the volume in which the radioactive material is suspended is small, this assumption will no longer be valid, since the range of the radiations may then extend beyond the volume studied. The activity, and therefore the dose rate, will of course decrease with time. The half-life of an isotope is the period in which its activity falls to one half.

Since the range of y-radiations is so very much greater it is necessary to treat these differently from /3-rays, and the K constants calculated by M A Y N E O R D 9 are generally used. This important field of calibrating radio-active isotopes is dealt with in a number of reviews ( M A Y N E O R D and S I N C L A I R 1 0 ) .

M E A S U R E M E N T O F D O S E If the dose is expressed in roentgens the basic measurement is the determination of the number of ion pairs formed in a volume of gas. The dose in rads requires knowledge of the amount of energy deposited in 1 g of the irradiated material. The absolute determination therefore neces-sitates an energy measurement and in practice calorimetry is almost invariably used. That is the amount of heat developed in a material in which no overall chemical changesf take place on irradiation is measured and the dose in rads is then immediately given. The measurement of dose by calorimetry is difficult and it cannot be used routinely either for therapy or research. In practice an ionization measurement or a chemical measurement is usually made and converted into rads by a factor estab-lished by direct comparison with calorimetry.

* T h e following formula31 gives the dose (D) to water or tissue in rep per day when it can be assumed that all the energy is dissipated in the volume under investigation. D = 53 CE, where C is the concentration of the isotope in micro-curies/g, and E the energy of the radiation in MeV.

f This does not mean that no chemical reactions occur, but only that any decomposition products, that are formed, recombine so that at the end of the irradiation the chemical composition is the same as at the beginning.


Number of Ionizations (Physical Dosimetry) The absolute calibration of x- or y-rays in roentgens is made by measur-

ing the saturation current in air in a parallel plate ionization chamber. The general principle is as follows: a closed chamber provided with electrodes is subjected to a steady beam of ionizing radiation which will produce a fixed number of ions per second. The electrodes are connected across a variable source of d.c. voltage. As the applied voltage is increased the current at first increases proportionally (i.e. the ionization chamber obeys Ohm's law). At higher voltage the current becomes constant since all the available ions are being attracted to the electrodes and a further increase of applied voltage cannot increase the amount of charge carried. From this saturation current the number of ion pairs formed by the radiation to which the chamber is exposed can be directly calculated. No direct determination is possible for the number of ion pairs formed in liquids or solids since a saturation current cannot be obtained even at the highest voltages.

According to the definition of the roentgen all the ions associated with 1 cc of air have to be collected by the electrodes. Associated ions are those which are produced by electrons originating in the defined volume. Consequently, the parallel plate chamber must have dimensions which are at least twice those of the range of the electrons. For 5 MeV x-rays this would necessitate a length of 40 metres and is clearly impracticable. This difficulty is avoided by irradiating a solid material and measuring the saturation current in a cavity within it. GRAY11 developed a theory of Bragg to relate the dose received by the solid—in which the path of the electrons is, of course, about one thousand times shorter—with the number of ionizations produced within the cavity. This value depends on the atomic composition of the solid (usually a plastic) and the nature of the gas.

A typical routine instrument for measuring the number of ionizations produced is the "Victoreen" dosimeter. It consists of a thimble-shaped condenser having a thin wall of plastic material with mass stopping power equivalent to air. This condenser is initially charged and the amount of discharge due to ionization is a measure of the dose and can be read directly on a scale. A similar instrument can be used to measure the dose from a beam of electrons (^-particles), but usually rather large corrections have to be applied.

For very hard x-rays (i.e. above 1 MeV) the use of an air-filled ioniza-tion chamber introduces very many difficulties and the corrections, that have to be made for scattering, make it necessary to introduce a number of correction factors to convert the number of ionizations measured into ionizations produced in a volume of tissue for example. Chemical dosi-metry (see p. 27) must be used to obtain absolute values of dose in any


particular situation though the rapid ionization measurement can obviously be used to correct for variations in intensity.

The dose from a-particles has generally to be computed from the total number of a-particles involved—determined photographically or with a counter—and the energy dissipated by each particle but chemical dosimetry can also be used.

Energy (W) Required to Form an Ion Pair in Air These physical methods measure the number of ion pairs produced,

while for most purposes we want to know the dose in terms of an energy unit. It is, therefore, necessary to know the energy needed to form an ion pair in air (referred to as W).

Measurements made more than 25 years ago had indicated that W for air lay somewhere between 25 and 40 eV and a value of 32-5 eV was chosen for electrons12, though this value represents no more than a weighted average of all the determinations. It is on the basis of this figure that the energy deposited by a dose of roentgen in water is given as 93 ergs.

In recent years many determinations of W have been made in many gases and this subject is excellently reviewed by VALENTINE and C U R R A N 1 3 .

For electrons the true value is still not known very accurately because of severe technical difficulties, but the best data suggests that it lies in the range of 33-5 to 35 eV (see Table 1-4).

For a-radiations five recent determinations give values that are very close to one another and their average is 35-3 eV. IFfor protons would seem to be very similar to that of a-rays at 35 eV. There is good evidence, both for electrons and for a-rays, that W depends very slightly on the velocity of the particle. As its energy decreases W increases, but the var-iation is too small to affect significantly dose calculations in radiobiology. At the present time the best estimate is that W = 34 eV for all types of ionizing radiations and at all energies (except very low ones which are not usually used in biological work).

If this new value is accepted then exposure of 1 g of water (or tissue) to 1 r results in an energy uptake of 98 ergs (see Fig. 1-4). This is well within experimental error of 100 ergs/g and the rad and the roentgen can therefore be used interchangeably for the irradiation of biological materials by particulate radiation or energetic x- and y-rays.

Chetnical Dosimetry In principle any chemical reaction, the extent of which depends upon

the dose of ionizing radiation and which can be easily and accurately measured, can be used as an integrating dosimeter. However, for such a method to be useful, the reaction must be (1) independent of dose rate


T A B L E 1 - 4

C O M P A R I S O N O F R E C E N T E X P E R I M E N T A L V A L U E S F O R W\

I — O — I GIVES C O N F I D E N C E V A L U E 3 2

Average energy/ ion pair,

32 33 34 i . r

Emery(l956) o j -

Gross et oL (1957)

Boy eta/. (1957)

Barber (I965!t-

Jesse and Sadauskis (1955)

35 eV

Weiss and Bernstein i 1 (1956) I Oj

Bernier et al. I 1 (1956) <

Skarsgard etal. (1957)

S35/^- Particles ~ 5 0 KeV

I to 35 MeV

Electrons

H3 ixid Ni63/? s

^ l s t o 20 KeV

2MeV X- rqys

Co -rays

22 MeV X- rays

over a wide range; (2) independent of the ion density over a wide range; (3) carried out in a dilute solution of a solvent such as water or benzene, the mass absorption coefficient of which is largely independent of wave-length* ; (4) relatively insensitive to the presence of impurities. In addition it is preferable that the reaction should be carried out in the presence of oxygen so that difficulties of degassing do not arise.

A great number of systems have been proposed, but in the last years the oxidation of iron salts from the ferrous to ferric valency state has superseded all others for the measurement of doses in the range of 2000 to 5000 rads. For calibrating radiation sources, which are used for most types of radiobiological experiments, this dose range is quite adequate though it would be convenient (i.e. save time) by reducing exposure period in some cases if a lower dose could be accurately measured by a chemical method. A number of chemical reactions have been studied in which a dose of a few roentgens can be detected, but none of these lend themselves for accurate calibration and their main uses are as cheap monitors. For very high doses, chemical dosimetry presents no problems

* A reaction in, for example, carbon tetrachloride could not be used universally since its photoelectric absorption per gramme is much higher than that of water, so that the amount of reaction per roentgen will vary with the voltage of the x-ray generator when this is less than 200 kV.


and changes in the physical and chemical properties of plastics are amongst the most convenient to use.

The ferrous sulphate dosimeter (or Fricke dosimeter after the chemist who first discovered this reaction) was pioneered by MILLER14 and has been very extensively studied since (cf. ref. 15). A solution containing ferrous ammonium sulphate (usually at IO-3 M concentration) dilute sulphuric acid (usually 0-8 N*) and dilute sodium chloride (usually IO-3 Mf) is irradiated and the amount of ferric iron produced is deter-mined directly in an ultra-violet spectrophotometer by measuring the extinction of the solution at a wavelength of 3040 A (molar extinction of ferric at this wavelength is 2160). If the solution is made up freshly from analytical grade reagents very reproduceable answers are obtained. In the earlier literature the need for extreme precaution to ensure exclusion of impurities was stressed, but this is no longer necessary since the presence of sodium chloride suppresses their interference, though it remains desirable to use double distilled water and "Analytical grade" reagents.

The yield of ferric iron is quite independent of dose rate and depends directly on the total amount of energy deposited. The reaction requires the presence of dissolved oxygen and the amount normally present in the aerated solution becomes exhausted after 50,000 rads. Unless steps are taken to supply oxygen this is therefore the upper limit of dose that can be measured.

Chemical reactions are usually expressed in terms of the number of molecules changed per 100 eV of energy deposited (this number being called the G value). A large number of independent investigations in which the total amount of energy deposited was measured directly by calorimetry have established that the G value for the oxidation of ferrous to ferric is 15-6 ± 0-2J for x- and y-rays and for electrons that are more energetic than 25 keV.

* When x- or y-rays of less than 100 keV are used the concentration of sulphuric acid must be reduced to 0-1 N since there is high photoelectric absorption by the sulphur atom.

t T h e sodium chloride is added as it ensures that the presence of adventitious organic impurities, present in trace quantities, do not interfere with the reaction (for explanation of mechanism see p. 135).

J Unti l 1955 the most probable figure for the G value was believed to be 19 or 20 and all doses quoted were some 20 per cent too low. T h e error originated by MILLER14 arose f rom the fact that the energy deposited in the ferrous sulphate solution was calculated f rom an ionization measurement. T h e value for W used was too low, probably by 5 to 10 per cent, and no allowance was made for electron scattering f rom the walls of the vessel into the solution. All the data can easily be corrected and the correct dose obtained so long as the G value used was stated. It probably remains desirable to state the G value used when reporting experiments based on ferrous sulphate dosimetry.


A simple conversion factor is useful: dose received by solution in kilo rads = 29-2 x extinction coefficient due to Fe+++ at 3040 A as measured in a one cm cell*.

The yield falls off as the ionization density is increased, e.g. for the relatively low-energy electrons from tritium it is reduced to 12 while for a-rays from polonium the G value is only 6-0. (This is discussed in further detail on p. 135.) For most purposes involving x-rays and /3-rays the G value is constant over the range of energy likely to be encountered in practice.

I O N I Z A T I O N D E N S I T Y

Effect of Velocity and Charge of Ionizing Particle Corpuscular radiations pass through matter in paths which are essen-

tially straight for a few microns, though electron tracks become detectably bent towards the end of their tracks as a result of scattering by atoms (see Fig. 1-8). The energy dissipation of a corpuscular particle is an inverse function of its kinetic energy. Hence the number of ions formed per micron (this being the directly measurable process of energy dissipation) is also an inverse function of the kinetic energy.

The ion density is not of course constant along the whole track, since with each ionization the ionizing particle loses energy, the ionization density increases as the particle approaches the end of its range and is at its maximum at the end of the track. An experimental demonstration of this is given in Fig. 1-6; the final fall off in specific ionization at the end of the track is largely due to the capture of electrons by the a-particles towards the end of their track. In this way the specific ionization is reduced because the charge of the particles is less.

The ionization density, also called specific ionization (i.e. the number of ions produced along a given length of track), can be determined directly in gases by counting the clusters produced (see Fig. 1-8), but in solids and liquids it cannot be measured.

A much better unit is L INEAR E N E R G Y TRANSFER (abbreviated to L E T ) .

This is the energy lost per /i of track of the primary ionizing particle. The ionization density is obtained by dividing the LET by W (i.e. by

34 eV). While this gives a meaningful figure in gases it is only approx-imate for condensed systems for which W cannot be determined experi-mentally.

The specific ionization of charged particles is proportional to the square of their charge and inversely proportional to their velocity. Protons and electrons, therefore, moving at the same velocity will produce the same

* Tha t is the extinction coefficient measured in a spectrometer when the irradiated solution is measured against the unirradiated solution as a control.


\

V 2 3 1

Distance fravelled in air 7

ClTL

FIG. 1-6. Variation in the ionization density produced by an particle from RaC, with the distance travelled in air.

' / -part ices_

•Electrons

IOOMeV IOMeV IOOkeV IOkeV

Energy of particles

FIG. 1-7. Energy loss (linear energy transfer, L E T ) of electrons, protons and a-particles of different energies passing through water. The L E T values can be converted into ionization density by dividing the energy by W (i.e. 34 eV). The data refers to the total energy loss which includes those produced by the primary particle as well as the secondary particles (e.g. S-rays). The actual ionization density along the track of the primary particle is about half the value shown, the difference being due to the

secondary effects.

I N T E R A C T I O N O F I O N I Z I N G R A D I A T I O N S W I T H M A T T E R Table 35-1 number of ions per unit length while an a-particle with double the charge will produce four times as many. The velocity of particles having a given amount of energy is an inverse function of its mass, although the exact relationship is complicated by relativistic effects. For example, a 200 MeV proton has the same velocity and, therefore, the same specific ionization as a 10 keV electron since the ratio of their masses is 1800 to 1. At compar-able energies, therefore, the number of ionizations per //. (1()"4 cm) of track is very much greater (see Fig. 1-7) for a-particles and protons than for electrons.

Distribution of Ions in Space Quantitative data concerning this complex problem are obtained in

the Wilson cloud chamber. This instrument works on the principle that an ion causes precipitation to occur in a supersaturated system. When an ionizing particle passes through air supersaturated with water vapour, minute droplets of water are formed round each ion and are photo-graphically recorded as shown in Figs. 1-8 to 1-12. From the experimental data obtained in this way, and from the Bethe theory, the rate of energy loss in gases and the associated quantitative specific ionization and range can be obtained.

The cloud chamber, however, is only useful for examining the track of an individual particle, and does not provide the complete pattern of the distribution of ions in space produced by a great number of particles. This involves three separate considerations: the energy spectrum of the ionizing particles, the direction of motion of the ionizing particles and the amount of ionization produced by particles of different energies. The problem is very complex, since the ejected electrons contribute so exten-sively to the observed ionizations, and even if the primary radiation is uniform, electrons of all energies will contribute to the overall effect. Still more complicated is the effect of x-rays and neutrons, which do not ionize themselves but produce a spectrum of ionizing particles.

Inspection of the Wilson cloud chamber photographs of electron tracks shows that at each ionization at least two water droplets are formed (see Fig. 1-11). This is due to the fact that the ejected electron is captured by another atom thus producing a negative ion, and each ionization results in the formation of an ion pair. The electron, which has insufficient energy to produce ionizations, is known as a sub-ionization electron and has a relatively short range in gases before it is captured by an atom or molecule to form a negative ion (cf. p. 104). From cloud chamber photographs in moist hydrogen gas the distance by which the positive and the negative ions of a pair are separated can be measured. The energy per ionization, W (i.e. 34 eV), refers to the formation of such an ion pair.

Often more than two droplets are formed close together, indicating the


formation of several ion pairs at or near the same spot (see Fig. 1-11). This cluster formation occurs because the electron, which is expelled as a result of the collision with the ionizing particle, can have sufficient energy to produce a few pairs itself. GRAY16 estimates that for all ionizing particles approximately one-third of the ion pairs are produced directly (i.e. the primary ionizing particle passes through the atom which is ionized), and the remaining two-thirds are formed by electrons set in motion by the primary particle.

S-Rays The energy of these secondary electrons covers a wide spectrum since

on collision the energy is shared between the electrons in collision in all proportions ranging from 100 : 0 (i.e. then the ejected electron has no excess energy and merely gives rise to the negative ion of the ion pair) to 50 : 50 where both electrons have the same energy. For convenience these secondary electrons have been divided into two categories. Those having an energy of 1000 eV or less, produce all their ionizations with-in a few Angstrom of the primary particle and are responsible for the clusters described above. The second group are electrons ejected with more than 1000 eV. They form separate tracks and are referred to as S-rays. The effect of these electrons, which are ejected in all directions from the track, is to introduce branching along a primary electron track. The branches are, of course, shorter than the track of the parent electron.

With radiations of high specific ionizations the total range of all the S-rays may be greater than that of the primary particle (cf. Fig. 1-9). Thus the ionization track of the heavy particle consists of a central core which has the high specific ionization, but from this core emanate, in all directions, the S-rays which have a much lower ion density. S-Rays contri-bute approximately 25 per cent of all the ionizations, whatever the nature of the primary particle, though the exact contribution is not known as theories are inadequate.

The effect of these 8-rays is to introduce a common factor into all ionizing radiations which makes it difficult to measure quantitatively the relationships between biological effect and ionization density. Thus the LET of the primary track of a 1 MeY /3-ray and an a-particle from polonium will differ by a factor of 500, yet both have a component of 8-rays with very similar LET values. Complex theories have been devel-oped (see p. 208) for deducing molecular dimensions of viruses from the inactivation dose with radiations of different LET. But these calculations cannot be applied, except in a crude semiquantitative form, because of S-rays. These rays have the effect of introducing a component of relatively low LET when using densely ionizing radiations and of relatively high LET when using sparsely ionizing radiations (See Fig. 4-1).

FIG. 1-8. The track from left to right is that of a fast electron (circa 200 kV) in a cloud chamber. The bent track from top to bottom is produced by a slow 20 kV electron; at this low energy

the track is bent because of scattering.

[facing p.

FIG. 1-9. Cloud chamber tracks of a-particles. Pictures of the early parts (i.e. high energy parts) in air (a) and (b), and helium (c) and (d). These clearly show the formation of S-rays, some of which have a considerable range, (e) Tracks at the end of the a-particle range when 8-rays are no longer formed. T h e tracks are bent at the very end, when their velocity is only of the order of IO8 cm/sec, since the bulk of the energy of the a-particles has been lost earlier. (Photographs reproduced by courtesy of

Miss T . Alper.)

FIG. 1-10. Cloud chamber track of a proton projected by a neutron.

' 4 : v » - «

FIG. 1-11. Cloud chamber track of a fast electron illustrating that ionization occurs in clusters. Successive clusters are sep-

arated by approximately 1 fx.

4 5

FIG. 1-12. Cloud chamber photographs of an x-ray beam reproduced from the original paper by C. T . R. Wilson.33 The picture on the right is an enlargement of one of the tracks and

clearly shows clustering of ionizations.

I N T E R A C T I O N O F I O N I Z I N G R A D I A T I O N S W I T H M A T T E R 3 3

Ionization Density in Liquids All the data, both experimental and theoretical, concerning ionization

densities apply to gases only. There is no method which is comparable to that of the cloud chamber for obtaining information of the number and distribution of ions in condensed systems. However, the technique17 of detecting the tracks of ionizing particles in photographic emulsions, which was developed by Powell, is in many respects similar to the cloud chamber. In the so-called nuclear emulsion the ionizing particle renders the silver halide grains capable of being developed. From the amount of blackening along the track in the emulsion, particles with widely different specific ionizations can be distinguished and an estimate can be obtained of their range. The method is, however, severely limited and can provide absolute figures of the rate of energy loss per //, of track only for particles of high LET.

The values of specific ionization and range of ionizing particles in tissue (or water) which are shown in Figs. 1-1, 1-7 and 1-14 are based on the assumption that a condensed system behaves in exactly the same way towards ionizing particles as a gas of the same atomic composition. There are two assumptions, and they are nothing more, upon which all these calculations are implicitly based.

(1) The energy dissipation of an ionizing particle is unaffected by the physical state of the absorbent (i.e. whether it is gas, liquid or solid) and is proportional to its density. G R A Y 1 8 deduced from the available data that this is the case for a-particles and there seems to be no reason to doubt that this principle can be extended to electrons and protons. The semi-quantitative data from nuclear emulsions on the range of different particles in gels are also in agreement when calculated on this basis.

(2) The energy required to form an ion is the same in water and organic substances as in air. We know from direct experiment that the energy required to ionize an atom in air (W) is about 34 eV but no evidence is available to support the postulate that this value applies also in condensed systems. The use of the value established experimentally (see p. 26) in gases for water, tissue and other substrates can at the moment only be considered as an act of faith, founded on sound physical principles19. In view of the high yields, which have recently been reported for some radiation induced reactions in water, it is highly desirable to obtain some experimental evidence bearing on this point.

Another vital factor for the understanding of radiation processes in biological systems, about which we have no direct experimental evidence, is the separation of the positive and negative ions in water and tissue. L E A 2 0 and G R A Y 2 1 have assumed that the behaviour of the sub-ionization electron in the liquid phase is comparable to that in the gas phase. From this they calculate, taking the cloud chamber value of 20 /x for the separa-


tion in gases, that the positive and negative ions will be separated in water by 0-015 fx (150 A). This calculation has, however, been challenged and both larger and smaller values have been proposed (see p. 126).

To summarize: it is reasonable to assume that the same amount of energy is lost by an ionizing particle when it traverses 1 g of water vapour or 1 g of liquid water, but the assumption, that the same number of ions is formed and that these occupy the same relative positions, is less well founded.

Ionization Density Produced by x- and y-rays With x- and y-rays the ionization occurs along the tracks of the ejected

electrons, which start in a completely random manner, wherever an x-ray quantum has been absorbed (photoelectric effect) or scattered (Compton effect) (see Fig. 1-12). The precise description of the irradiation

FIG. 1-13. Spectrum of radiation from 200 kV x-ray therapy set. Curve reproduced from CORMACK and JOHNS 2 2 .

with x-rays from a therapy set presents a problem of great magnitude, since an incident x-ray beam is usually inhomogeneous, and since, moreover, the energy spectrum of the beam may vary with depth in the irradiated material. In addition, at each wavelength a whole spectrum of ionizing electrons will be produced by the Compton effect. It is clear, therefore, that a volume of tissue irradiated by x-rays will be made up of tracks of electrons varying from a maximum energy (that of the peak voltage of the set used) to electrons of very low energy, including S-rays.


culation based on the method of G R A Y 1 6 ; (b) CORMACK and JOHNS 2 2 .

Nevertheless, an average value for the specific ionization for x-rays produced at different voltages is of value.

The average value for the energy of the electrons produced by homo-geneous x-rays of different wavelengths has been computed by Lea. Using this data and coupling it with the known spectrum (see Fig. 1-13) from an x-ray therapy set GRAY16 obtained the energy distribution of the electrons at a point, and by taking a direct average over a given volume the curve shown in Fig. l-14(a) for the change in average specific ionization with applied voltage was obtained.

There are many different ways in which the averaging processes can be carried out. C O R M A C K and JOHNS22 as well as SPIERS23 considered the spectrum (Fig. 1-15) of all the electrons produced by the range of radia-tions given out by a therapy set working at a given voltage. From this energy spectrum the distribution of the number of tracks of different specific ionization was determined (see Fig. 1-16) and an average obtained. By carrying out this procedure for x-rays produced at different voltages (i.e. by establishing Figs. 1-15 and 1-16 for different voltages), Cormack and Johns obtained the relationship shown in Fig. l-14(b) between average specific ionization and kV of the x-ray set. It will be seen that for radiations of less than 200 keV the average specific ionization obtained in this way is approximately half that found by Gray. The method of


FIG. 1-15. Distribution of energies of the electrons produced in water by the spectrum of radiation (cf. Fig. 13) from a 200 kV x-ray therapy set. The results are expressed as the num-ber of electrons, per 5 keV intervals, which pass through 1 cc of water irradiated with 100 r (e.g. 4 X IO6 electrons having energies between 77-5 and 82-5 keV pass through each cc).

Curve reproduced from CORMACK and JOHNS 2 2 .

SO No. of ion pairs/100 [i

FIG. 1-16. Distribution of the ion densities of the electrons produced by a 200 kV x-ray therapy set. (This curve illustrates the difficulty of assessing the mean ion density and the limited significance of such a value for the ionizations produced with 200 kV x-rays.) Curve reproduced from CORMACK and JOHNS 2 2 .


calculation of Cormack and Johns, though mathematically more precise, tends to mask the contribution of very low energy electrons (i.e. very short tracks of high specific ionization).

However, the situation produced by irradiation with polychromatic x-rays is so complex that exact statements about the conditions produced cannot be made without careful qualifications. In any case the differences are unlikely to be of great significance.

The point to be borne in mind is that neither curve is wrong, but the most meaningful value for the average value of the ionization density depends upon the purpose for which the calculation is required. Thus the Cormack and Johns treatment may be said to represent the track length average (i.e. it gives the mean number of ion pairs per //, length of track) and should, therefore, be used for calculations of target size (see p. 208). The calculation of Gray gives the dose average (i.e. the number of ion pairs in a given volume of solution) which should be used for indirect processes when interaction between ion pairs is important (see p. 129).

The maximum in the curve is due to the fact that over this range the less energetic Compton recoil electrons are increasing in number while the more energetic photoelectrons are decreasing. Consequently, the ion density only varies by plus or minus 10 per cent for x-rays produced by therapy tubes operating between 25 to 180 kV and experiments designed to determine the influences of ion density cannot be carried out by varying the wavelength within this range.

Probably the most important feature of irradiation with very hard electromagnetic radiation from the therapeutic standpoint is that the maximum ion density is produced at a definite distance within the irradi-ated material (e.g. tissue). This makes it possible to treat deep-seated tumours without producing undue damage to the intervening tissue, in particular the skin. The reason for the occurrence of the maximum is that the ejected electrons, which dissipate the energy, produce the highest specific ionization at the end of their tracks. Consequently electrons produced at the surface of the irradiated material give rise to the maximum number of ions at the end of their range. Figure 1-17 shows the distri-bution of energy uptake inside irradiated tissue for x- and y-rays of different energy. Once the maximum has been passed the intensity falls exponentially due to absorption (see p. 12).

In the same way maximum energy deposition will occur at some distance within tissue irradiated with beams of electrons or other ionizing particles*. Since ionizing particles have a fixed range—unlike x-rays,

* T h e increase of dose with depth is not observed with narrow beams of electrons or other ionizing particles because the effect of increasing ion density is more than offset by divergence of the beam due to scattering.


(a)

4 6 8 IO 12 14 16 18 2 0

Depth in tissue, cm

o 5 W cm Depth in tissue at which dose is atamaximum

FIG. 1-17. (a) Distribution of dose (i.e. amount of energy lost) inside tissue for radiations of different energies (filters used: A, 3-5 m m Al; C, 1-5 m m Cu; D, 3-6 mm Cu; E, 20 m m Pb + 5 m m Cu); (b) relationship between the depth at which the

dose is at a maximum and the energy of the radiation.

which become progressively attenuated by absorption—the depth dose has a much sharper maximum than is obtained with x-rays. Figure 1-18 shows experimentally determined distribution of energy curves for high energy electrons and deuterons.

Multiple ionizations and Auger effect—In the foregoing only the single ionization processes have been considered, in which energy is absorbed in "packets" with an average energy dissipation of some 34 eV. These are responsible for the predominant part of the energy absorption, but there are some infrequent events which probably do not contribute more


100 %

80

60

5 W

20

^ \ 16-V MeV electi ons -

\ I 190 "IeV deuferons

- V 10

Depth in plastic 15

FIG. 1-18. Comparison of the depth dose effect between deuter-ons and electrons. Curve taken from TOBIAS, ANGER and LAWRENCE24. These curves were determined experimentally using a plastic having an absorption coefficient similar to tissue.

than 2 or 3 per cent to the total energy taken up, in which much larger amounts of energy are released in one event. One of these—-the Auger effect—has been described (see p. 13); another is the multiple ionization in which more than one electron is stripped from an atom with which a primary particle collides. These rare events may be of biological signifi-cance, in particular for radiation with a low specific ionization, the average energy of which may be insufficient to break a submicroscopic structure (see p. 97), but this may nevertheless take place by one of the rarer events. A quantitative treatment of the occurrence of these "rare events" of high energy transfer has not been made and the present situation is summarized by PLATZMAN25 and GRAY26. From a radiobiological point of view these high energy primary events can probably not be distinguished from a cluster of ionizations which occur so closely together that they could all be placed within one macromolecule.

E X C I T A T I O N S P R O D U C E D B Y I O N I Z I N G R A D I A T I O N S

The energy transferred by ionizing radiations to matter is only in part used up in the formation of ion pairs, since for every one of these approx-imately 34 eV is absorbed while the ionization potential for gases, deter-mined in other ways, ranges from 24-5 V for helium to about 10 V for


iodine and sulphur vapours; the value for air is 16 V and for water vapour 13 V. For each ion pair formed in water vapour there is an excess of energy of the order of 20 eV, which is dissipated in excitations*. According to the theory of Bethe1 an ionizing particle passes through a certain number of atoms without actually ejecting an electron, but displaces some of these from one shell to another of higher energy. It probably does this several times before it produces an ionization, and on average the total energy dissipated in all these processes including the latter is W (i.e. 34 eV).

The formation by a-particles of excited atoms was demonstrated unam-biguously by E Y R I N G , HIRSCHFELD and T A Y L O R 2 7 (see p. 1 0 2 ) .

It should be stressed that this excess energy of 20 eV represents, in molecular terms, a great amount of energy ( 1 eV per molecule = 2 3 - 0 5

kcal per mole). Only about 4 eV (i.e. approximately 100 kcal/mol) are required to break a bond between two carbon atoms, and if this excitation energy is transferred to a relatively few molecules many chemical changes per ion pair can be brought about. Unfortunately little is known concern-ing the mechanism of excitation by high energy electrons, and no inform-ation is available concerning the amount of energy transferred per excitation, or about the exact nature of the electron shifts produced.

Electronic excitation can give rise both to the formation of free radicals (see p. 101) or to molecules in an excited state, but in general the latter is much more likely to occur. An ionization on the other hand will frequently turn a molecule into a free radical which is more important in the field of radiobiology than are excited molecules (see p. 45).

Behaviour of Excited Molecules Although both free radicals and excited molecules have, in general,

only a transitory existence in condensed systems (usually between IO-9

to IO-6 sec) the reasons for their short life are not the same. Free radicals are highly reactive entities, but they do not decompose or change spon-taneously. In gases and liquids free radicals have a short life (see p. 107) because they react so readily with one another and with other molecules. Combination between two radicals occurs, in many cases, on every collision. But in solids in which the motion of the molecules is restricted radicals can persist indefinitely. Excited molecules, on the other hand, are inherently unstable and will lose their energy by some process. In some cases excitation leads to immediate dissociation (i.e. within one atomic oscillation requiring IO-13 sec). More often the excited molecule is stable

* Excitation is used here to denote that an electron in the molecule has been raised from its ground-state to a state of higher energy. This process is different from the excitation of a molecule by heating when the energy is used up initially to increase the strain in a bond thereby causing an increase in the vibrations and oscillations of the atoms of the molecule.


for times greater than IO-9 sec, after which it can undergo a delayed dissociation, lose its energy by giving off light (i.e. fluorescence) or undergo an energy transfer process.

Much is known concerning excitation of molecules by photons, and the same principles apply to excitation by electrons, although some of the strict selection rules which limit photochemical reactions need not be obeyed. The important Franck-Condon principle holds, however, and has a great influence in determining whether an excited molecule will dissociate immediately. The process can best be illustrated by means of a potential energy diagram of the diatomic molecule hydrogen iodide (see Fig. 1-19). In its unexcited state the molecule HI has an internal

energy corresponding to "A", and the distance separating the two nuclei varies between a and a *. For dissociation to occur the potential energy of the molecule in its ground state has to be increased by an amount AH; in ordinary thermal dissociation this amount of energy is taken up as vibrational or rotational energy. The top curve represents a similar potential energy diagram for an electronically excited molecule. The dis-sociation energy of such a molecule AH' is invariably smaller, and if it

Dislonce separating the H and I atoms in the moleaJe(r)

FIG. 1-19. Potential energy diagram of (a) normal H I molecule and (b) electronically excited H I molecule.

* Every molecule is always in a state of low activation at room temperature due to vibrational and rotational energy, and consequently the nuclei are not separated by the distance which corresponds to the minimum of the curve.


is of the order of the energy of vibration and oscillation instantaneous dissociation will occur.

Even if a molecule is relatively stable in the excited state, it may decom-pose instantaneously on becoming excited due to the operation of the Franck-Condon principle. This states that when an atom in a molecule is raised from one excited state to another the position of the nuclei will not change, at least not appreciably. Consequently, when a molecule such as HI is exposed to ionizing radiations an electron of the iodine atom is raised to a higher energy level and the molecule is raised from its ground state (i.e. between points a and a ) to an excited level with the same separation between the nuclei. It therefore falls on the corresponding potential energy curve between b and //, where the energy is in excess of that required for dissociation, and immediate decomposition results. Thus, although an electronically excited HI molecule can be quite stable, it cannot be obtained in this form by excitation of HI. In agreement with these ideas L I N D and LIVINGSTON28 found an exceptionally high yield for the decomposition of HI by a-articles.

HI is not, of course, representative of all diatomic molecules and there are many molecules for which the excited state requires almost as much energy for dissociation as the ground state; such molecules almost in-variably show strong fluorescence.

With more complex molecules excitation is unlikely to lead to immediate dissociation, since the excess energy can be distributed over many bonds, so that there is insufficient energy to break any one. An excited molecule of this type will have a relatively long life because there is a low prob-ability of the energy becoming localized and dissociation occurring. In the interval the excited molecule may lose its energy in a number of ways and return to the stable ground state. Since deactivating collisions are much more frequent in the liquid than in the gas phase, decomposition will be less frequent in a liquid, and this is amply borne out by experiment (see p. 107).

Energy Transfer In complex molecules the energy of excitation can be transferred both

intra- and inter-molecularly. Energy can be transmitted from one molecule to another even when they are not in contact (i.e. separated by several molecules in between), by processes classed as radiationless transitions29

which can only be described in quantum mechanical terms. In this way an electronically excited molecule can raise another molecule, separated by many molecular diameters, to an excited level. Energy transfer within the same molecule is known as internal conversion30*, the electronically

* T h e term "internal conversion" is here used in the sense of the chemist. T o the nuclear physicist it means a complex phenomenon in radioactivity.

I


excited molecule returns to the ground state and the excess energy is converted into vibrational and oscillational energy. The molecule will then behave as if it were at a much higher temperature, and the irradiated substance will undergo changes similar to those occurring on pyrolysis.

Energy transfer is not restricted to excitational energy and it is encount-ered also with ionizations. That is, the chemical consequence of the ionization is not seen only in the molecule which contains the atom, that has lost an electron. The physical mechanisms involved are not under-stood, but the phenomenon is of importance and is referred to on p. 110.

R E F E R E N C E S 1. LEA, D . E., Actions of Radiations on Living Cells, Cambridge Univ. Press,

1 9 4 6 2. HEITLER, W., The Quantum Theory of Radiation, Oxford Univ. Press, 1954;

SEGRE, E., Experimental Nuclear Physics, John Wiley, New York, 1953 3 . K L E I N , O . and NISHINA, Y . , Z . Phys., 1 9 2 9 , 5 2 , 8 5 3 4 . BETHE, H . , Hand-u. Jb. Chem. Phys., 1 9 3 3 , 2 4 ( 1 ) , 2 7 3 5 . GRAY, L . H . , / . chem. Phys., 1 9 5 1 , 4 8 , 1 7 4 6. Recommendation of International Committee, Am. J. Roentgenol., 1954,

7 1 , 1 3 9 7 . MAYNEORD, W . V . , Brit. J. Radiol., 1 9 4 0 , 1 3 , 2 3 5 8 . GRAY, L . H . , Proc. Camb. phil. Soc., 1 9 4 4 , 4 0 , 7 2 9. MAYNEORD, W . V., Brit. J. Radiol., Suppl. No. 2, 1950

1 0 . MAYNEORD, W . V . and SINCLAIR, W . K . , Advances in Biological and Medical Physics, 1953, 3, 1

1 1 . GRAY, L . H . , Brit. J. Radiol., 1 9 3 7 , 1 0 , 6 0 0 , 7 2 1 1 2 . GRAY, L . H . , Proc. Roy. Soc., 1 9 3 6 , A 1 5 6 , 5 7 8 13. VALENTINE, J . M . and CURRAN, S. C . , Prog. Physics, 1958, 2 1 , 1 1 4 . MILLER, N . , Nature, 1 9 5 3 , 1 7 1 , 6 5 8 1 5 . LAZO, R . M . DEWHURST, H . A. and BURTON, M . , / . Chem. Phys., 1 9 5 4 , 2 2 ,

1370; DONALDSON, D . M . and M I L L E R , N . / . chim. Phys. 1954, 22, 438 16. GRAY, L . H . , Brit. J. Radiol., Suppl. No. 1, 1 9 4 7 , p. 7 17. YAGODA, H. , Radioactive Measurements with Nuclear Emulsions, John Wiley,

New York, 1949 1 8 . GRAY, L . H . , Proc. Roy. Soc., 1 9 2 8 , A 1 2 2 , 6 4 8 1 9 . Bibliography in M I N D E R , W . and L IECHTI , A . , Experientia, 1 9 4 6 , 1 , 2 9 8 20. LEA, D . E., Brit. J. Radiol., Suppl. No. 1, 1947, p. 59. 2 1 . DALE, W . M . , GRAY, L . H . and MEREDITH, W . J . , Phil. Trans., 1 9 4 9 , A 2 4 2 ,

3 3 22. CORMACK, A. a n d JOHNS, B., Brit. J. Radiol., 1952, 25, 369 23. SPIERS, F. W., Disc. Faraday Soc., 1952, 12, 13 2 4 . TOBIAS, C . A . , ANGER, H. O . and LAWRENCE, J . H. , Am. J. Roentgenol.,

1952, 67, 1 25. PLATZMAN, R . L., Symposium on Radiobiology, p. 97, John Wiley, New York,

1952. 26. GRAY, L. H. , Actions chimiques et biologiques des Radiation, vol. 1. p. 4. publ .

Masson 1955 2 7 . EYRING, H . , HIRSCHFELDER, J . O . and TAYLOR, H . S .,J. Chem. Phys., 1 9 3 6 ,

4 , 4 7 9


2 8 . L I N D , S. C . and LIVINGSTON, R . , J. Am. Chem. Soc., 1 9 3 6 , 5 8 , 6 1 2 2 9 . FRANCK, J . and LIVINGSTON, R . , Rev. Mod. Phys., 1 9 4 9 , 2 1 , 5 0 5

FORSTEH, T . , Ann. Phys., Lpz., 1 9 4 8 (6 ) , 2 , 5 5 PERRIN, F . , Ann. Phys., Paris, 1 9 3 2 , 1 7 , 2 8 3

30. TELLER, E .,J. Phys. Chem., 1937, 41, 109 31. BRUES, A. M . , Adv. Cancer Res. 1954, 2, 177 32. BOAG, J. W. Quantities, Units and Measuring methods of ionizing radiations;

publ. Ulrico Hoepli, Milan, 1959, p. 100. 3 3 . W I L S O N , C . T . R . , Proc. Roy. Soc. A, 1 9 1 2 , 8 7 , 2 2 7 .

C H A P T E R 2

Direct and Indirect Action in Biological Systems

T H E R E are two distinct mechanisms by which a chemical change can be brought about by ionizing radiations: (a) by direct action, the molecule undergoing change itself becomes ionized or excited by the passage through it of an electron or other atomic particle; and (b) by indirect action, in which the molecule studied does not absorb the energy but receives this by transfer from another molecule. The difference is particu-larly well defined when solutions are irradiated, and we shall confine ourselves to the biologically important case where water is solvent. The radiation chemistry of water and aqueous solutions will be dealt with in Chapter 6 and for the present it is sufficient to note that the ionization of a water molecule leads to the formation of free radicals. These are chemical entities carrying a lone electron (see p. 104) which renders them extremely reactive, and combination between two radicals occurs in most cases on every collision. Consequently, free radicals—particularly of the simpler type—have a very short life in solution. Their rate of disappearance depends both on the concentration of substrate with which they react and on the specific ionization of the radiation. The latter determines the local concentration of the radicals, which in turn controls the rate of radical recombination (see p. 128). In water it is unlikely that free radicals persist for more than IO-5 sec, and in the presence of solutes with which they react this time will be considerably shorter. For all practical pur-poses, therefore, the primary chemical changes are instantaneous, even when they involve the formation of a reactive intermediary. The distance which the free radicals travel before reaction again depends on the nature and amount of dissolved substances. Indications are that in a yeast cell reaction occurs within 50 A of the formation of the radical1.

Ionization versus Excitation There are several other mechanisms by which the energy transfer,

characteristic of indirect action, can occur. For example, charge transfer processes, which occur in gases (see p. 106), result in reaction kinetics

45


which are characteristic of indirect action. The lifetime of an ionized water molecule before it dissociates into a free radical is, however, only of the order of IO-11 sec, and in this interval the probability of an exchange of ionization with a substrate molecule of lower ionization potential is vanishingly small. For this reason indirect action in aqueous systems is believed to be produced entirely by the free radicals formed from water. Whether excitations contribute significantly to the chemical changes pro-duced by direct action on organic molecules is very doubtful. While in some gaseous reactions (la) excitations play a part they will be much less important under biological conditions. There is no evidence that the radio-chemical changes which occur when organic solids or liquids are irradiated with atomic radiations, cannot be explained entirely on the basis of ioniz-ations. But even if some radiation changes should be induced solely by the excitations of a molecule without there being an ionization, it is improbable that such a reaction will contribute to the biological effects of radiations, since ultra-violet light which excites molecules but does not ionize is very much less efficient than ionizing radiations. Both for damaging of cells and for inactivation of enzymes and viruses in vitro the dose in terms of energy absorbed per gramme of tissue (i.e. in rads) will differ by a factor of at least 1000 and often much more, even when ultra-violet light of the most effective wavelength is chosen. An obvious reason for this great difference is that ionizations are much more efficient than excitations in producing biologically significant chemical changes (see p. 103).

Water of Hydration A question of definition is whether an ionization occurring in a molecule

of water which is firmly bound to, for example, a protein or a nucleic acid molecule constitutes "direct" or "indirect" action. The reasons for defining it as direct are: (1) bound water forms an integral part of the molecule and its removal often results in loss of biological activity; (2) an essential feature of indirect action is that the free radical must diffuse through water to attack the substrate and this cannot occur if the ionized water is bound, since it would hand on its energy immediately to the organic molecule to which it is bound; (3) reaction resulting from an ionization in bound water follows the kinetics of "direct" action and does not give rise to a dilution effect (see p. 47) which is one of the most characteristic features of indirect action.

M E T H O D S F O R D I S T I N G U I S H I N G B E T W E E N D I R E C T A N D I N D I R E C T A C T I O N

When a biological active material such as an enzyme or a virus in a pure form is irradiated dry, the action of the radiations is by definition direct. If the material is dry but impure (e.g. containing extraneous protein) the

D I R E C T A N D I N D I R E C T A C T I O N I N B I O L O G I C A L S Y S T E M S 4 7

question arises whether energy absorbed in the impurity can affect the molecules of the active component which is being assayed. Until recently it was believed that the direct action of radiation could not be influenced by neighbouring molecules and this assumption was fundamental to the application of the target theory to complex system. Thus, POLLARD et al.2 stated that enzymes had the same radiosensitivity when irradiated either as dry pure preparations or in whole cells that had been dried. It was claimed that ionizing radiations could be used to determine the sizes of biologically active molecules without isolating them (see p. 208). Evidence is now accumulating from many directions that this is not the case and that even in dry preparations the presence of impurities influences the radiation effect (see p. 113). If one was to be rigorously logical one should therefore only speak of direct action when irradiating a pure material (e.g. a dry crystalline enzyme). In practice, however, it is useful to confine "indirect" action to reactions due to diffusible free radicals formed from water and to consider as "direct" energy deposition in the molecules of the substance being studied or in its neighbours when this leads to inactivation.

The problem to be resolved is to determine the relative contribution of direct and indirect action as defined above for irradiation of a wet system such as a vegetative cell or a solution or suspension of viruses or enzymes. The following tests can often be used to decide which mechanism is operative, though unfortunately they cannot always be applied to bio-logical systems, and it is almost impossible, for example, to distinguish de-cisively between the two types of reactions when whole cells are irradiated.

Dilutioti Effect In solutions, a fixed number of free radicals is produced by a given

dose. If the action is indirect, therefore, the number of molecules (or organisms) inactivated will be independent of concentration (except at very low values—see below) since a constant number of radicals are available for reaction. If the action is direct the number of enzyme mole-cules inactivated will depend on the number present in the irradiated volume and will be proportional, therefore, to the concentration. But if the reaction is indirect the number of molecules which have been changed is independent of concentration and the percentage inactivation decreases with increasing concentration (i.e. the greatest relative change will be observed in the most dilute solution). Conversely, if the action is direct the same proportion of molecules will be changed whatever the concen-tration (i.e. the percentage inactivation is constant for a given dose). But dilution behaviour is illustrated diagrammatically in Fig. 2-1, and was clearly demonstrated with dilute solution of pure enzymes (see Fig. 2-2) by D A L E , G R A Y and M E R E D I T H 3 , whose work was fundamental to the


100R

Cj I

Direct

Indirect

-<a

M 5i <3 'Direct

Indirect

V

Cone, of active material in solution

Cone, of active materia/ in solution

FIG. 2-1. Dilution effect. The relationship between the inactiva-tion of an enzyme or a virus and its concentration in solution depends on whether the action of the radiation is direct or indirect. The results can be expressed either as the percentage inactivation of the whole solution or as the number of macro-

molecules or organisms inactivated.

understanding of the concept of direct and indirect action to radiobiology. If the concentration of the substrate is below a certain value (e.g. IO-4

per cent in the case of carboxypeptidase; see Fig. 2-2) some of the radicals then react with one another and not with the dissolved material, and the specific inactivation dose rises. This effect has been studied in great detail for another enzyme and the changeover from the case where almost all

03 ra-6 w 5 70-* ID-3 io'z 7<r1

Concentration of enzyme (c)

FIG. 2-2. T h e relationship between the concentration (C) of carboxypeptidase in aqueous solution (expressed as g of enzyme/ g of enzyme +water) and the dose of x-rays (D) required to reduce the activity of the solution to 37 per cent of its original value. For indirect action D/C should be constant. Except for the most dilute solutions this relationship is seen to be obeyed over

an extremely wide range3.


TABLE 2 - 1

T H E RELATIONSHIP BETWEEN CONCENTRATION AND THE FRACTION OF THE FREE RADICALS USED FOR INACTIVATION4

Per cent* of Per centf of Enzyme concentration "effective" "ineffective"

Mg/ml molecules/ml free radicals free radicals

0-5 4-89 XlO12 1-5 98-5 5 4-89 XlO13 13-9 86-1

50 4-89X1014 61-7 38-3 500 4-89 x IO15 94-2 5-8

5000 4-89 x IO16 99-3 0-7

* Per cent of "effective" free radicals = number of free radicals which reacted with enzyme

; TT— x 100 number of total free radicals produced by radiation

f Percentage of "ineffective free radicals =100—(per-centage of "effective" free radicals).

radicals are wasted to one where they all react with the enzyme is shown in Table 2-1.

The situation where the concentration of dissolved substances is so low that the radicals formed can react with one another is of no physio-logical significance, since the concentration of the radicals present at any one time is infinitesimal and not only must the substance being studied be present at low concentration but all material capable of reacting with these radicals (and this means virtually all organic substances).

For all practical purposes, therefore, the dilution test is decisive and if in a particular system the radiation effect increases on the addition of solvent then the indirect action is responsible and the relative contribution of direct and indirect can be easily calculated (see p. 56). This test is very useful for in vitro systems such as solutions of enzymes and viruses but cannot be applied to cells. On diluting a suspension of cells the dose required to produce any type of damage studied is not decreased as would be the case for a solution of enzyme*. This does not mean that the action is necessarily direct since the contents of the cells have not been diluted

* This has been experimentally confirmed for mammalian cells and bacteria whenever precautions have been taken to ensure that, apart from concentration, no other factor is altered on dilution. For example, the oxygen tension in a sus-pension of metabolizing cells will depend on the number of cells present, and if this variable is not controlled there will be an apparent dilution effect. GUNTER and KOHN5 find that for most bacteria the inactivation dose is independent of the number of cells so long as it is less than 108/ml. Above this value the radioresistance rises rapidly unless oxygen is bubbled through the suspension.


and all that has happened is that there is more water between the cells. Any indirect action will be derived from the radicals formed in the water within the cell, since the radicals produced in the extracellular water will react and be wasted before they can reach the interior of the cell.

Other criteria have therefore been considered for distinguishing be-tween direct and indirect action which were based on the belief that any radiation effect that could be influenced by changing external factors must be initiated by indirect action, since the damage from direct action followed immediately and inevitably from the ionization. Thus indirect action was claimed to play an important role if the lesion was enhanced by oxygen6

or could be protected against by added chemicals. Recent investigations have shown, however, that the underlying concept of these tests is incor-rect, since there are many stages between the initial deposition of energy and the final chemical stage (see Chapter 5). External factors can alter the end-effect by intervening at intermediate stages other than those of the free radicals formed in water.

Chemical Protection The free radicals produced in water are highly reactive, and interact

readily with a wide variety of different molecules. If other substances are added to the solution they will compete for these radicals with the "target" molecule (e.g. enzyme) and thereby reduce the extent of the inactivation or other process that is being studied. This protection by competition was discovered by DALE7 who found that many substances protected dilute solutions of enzymes. Sulphur-containing compounds were exceptionally effective. All proteins react approximately equally readily with the free radicals formed in water (see Table 2-2) and they all will protect one another.8

The theory that chemical protection can occur only when the action is

TABLE 2 - 2

PROTECTION OF THE ENZYME D N A A S E I BY PROTEINS AGAINST INDIRECT ACTION OF

X-RAYS 8

Added protein (40 /ig/ml) % Activity remaining*

after 1090 r

None 43 Haemoglobin 81 Serum globulin 81 Egg albumin 95 Catalase 89 Ribonuclease 92

* DNAase concentration 0-5 ng/m.


indirect has been shown to be wrong by ALEXANDER and CHARLESBY 9 ,

who found that radiation damage to dry macromolecules—when the action must be direct—could be reduced by the presence of added substances. Two types of reaction for protection against direct action have so far been discovered.

1. Energy transfer, whereby energy originally deposited in one molecule —or part of a molecule—by the ionization exerts its chemical effect at another place. The addition of a "receptor" of energy will therefore protect.

2. Repair of damaged molecule10. This relies on the fact that the first chemical change that occurs need not be irreversible. There may be a short time (a small fraction of a second) during which the molecule can react with the protector in such a way that it is restored to its original state. In the absence of the protector it decomposes further and is irreversibly altered (or inactivated). Examples of both of these types of protection are given on pages 112 and 164 respectively.

A third type of "protection" has to be considered when a complex organism is being protected, and that is that the protector may have a pharmacological action that changes the response of the organism to radiation. Thus the vasoconstricting activity of some of the amines almost certainly contributes to their protective action in mammals (p. 464).

Ejfect of Oxygen One of the most interesting observations in radiobiology is that almost

all biological systems are more radiosensitive in the presence of oxygen. Chapter 11 is devoted to this phenomenon and there is some evidence that oxygen enhances the initial chemical lesion. GRAY6 suggested that the role of oxygen was to increase the damage done by the radicals formed in water by converting these into more reactive entities such as HO2 radicals and hydrogen peroxide (see p. 138). However, oxygen was shown by us10'11'12

also to enhance the direct action of radiation on enzymes; the possible mechanisms are discussed on p. 292. On the other hand, an oxygen effect is rarely observed when cells having a low moisture content (e.g. dried bacteria) are irradiated; yet the radiosensitivity of bacteria in broth is greatly enhanced by the presence of oxygen. This observation cannot be used as an argument that indirect action plays a part in damaging wet bacteria, since the problem of the diffusion of oxygen into the cell has to be considered and this may be very slow in the "dried" bacteria. In seeds there is a very complex oxygen effect, but the situation does not seem to be typical13. To conclude: though the nature of the reaction of oxygen is not at all clear at present the existence of an oxygen effect cannot be used as a test for indirect action.


Freezing Test A possible method for deciding if the action of radiation on a solute is

direct or occurs via free radicals is to compare the effectiveness of a given dose at ordinary temperatures with that found at a temperature when the solution is frozen. In the latter case the diffusion of free radicals is hind-ered, and reaction by indirect process should be sharply reduced* when the water freezes. The temperature at which this occurs, need not however be O0C and for all cells freezing occurs at a substantially lower temperature, since the water is in a "bound" state. In some seeds—particularly those that have been specifically selected to withstand low temperatures, such as winter wheat—the water does not freeze at — 20°C. These are excep-tional cases, however, and mammalian cells and bacteria will normally freeze within a few degrees of 0°C.

If diffusible free radicals formed in water play a part, then the radiation damage must show a sharp and sudden fall when the temperature is lowered below the freezing point of the system. If there is no such fall then there cannot be indirect action.

It must be stressed that for this test to have any meaning the increase in radiation resistance with fall in temperature must be discontinuous at the freezing point, since direct action is also effected by temperature. The dose needed to inactivate dry bacteriophage15 and dry catalase2, as well as the dose needed to break carbon-carbon bonds in synthetic polymers14, varies with temperature, and Fig. 2-3 shows that the magni-tude of the change is similar for all these systems. POWERS15® and his colleagues have extended the temperature range to lower values by using a liquid helium thermostat. They find that the radiosensitivity of spores of B. megaterium rises between — 195°C and 37°C in the same way as the systems shown in Fig. 2-3, but that there is no further change below — 195°C and — 268°C (i.e. within five degrees of absolute zero); see Fig. 2-4. This interesting finding suggests that trapped radicals may play a part in this phenomenon. No theory has so far been advanced by anyone that direct action is less efficient at lower temperatures but the facts are beyond dispute. Consequently, an observation that a system is more sensitive at room temperature than at liquid air temperature has no value in distin-guishing between direct and indirect action.

However, even when there is a sharp fall in radiosensitivity on freezing the deduction that the process is indirect cannot be made with certainty since there are so many steps between the initial chemical lesion—which is not measured—and the eventual biological damage recorded. However,

* In general, the material is irradiated in the frozen state but examined subse-quently when the ice is molten. There is no evidence that free radicals formed in the irradiated ice become available for reaction on melting.


•

+ ^ ^ •

\ a

\ -200 -100 0

Temperature, 0C

100

FIG. 2-3. Effect of temperature on effectiveness of ionizing radiations when the action is direct; + , polyisobutylene energy per break, y-radiation; O, inactivation of bacteriophage (vacuum dried), 50 kV x-rays; • , inactivation of bacteriophage, lyophil-ized; • , red-cell catalase inactivation, 3 -7 MeV deutrons;

• , red-cell catalase inactivation, 1 MeV deutrons14.

0-41

O O 0-39

X

£p0-37

0-35

O

U) 0-33

O C O 0-31

S

0-? 9 O

0-27

I,

• i • A \ I -

fr~23E=o-no±o UIOk cal

- \ - \ - y

36°C -1210C -I94°C -2 yi°z , , i , , , J, , H , , -25I°C

L -266°C

L

t

r265

T(°K)

FIG. 2-4. Influence of temperature on the radiosensitivity to x-rays of dry spores of B. megaterium. The radiosensitivity is expressed as the logarithm of the inactivation constant which

is an inverse function of the radiosensitivity15a.

if pitfalls—such as interference with an oxygen effect, due to restricted diffusion in ice—have been excluded, the effect of freezing provides some of the strongest evidence for indirect action that can be obtained.

Experimental data on the effect of freezing is, however, rather scant. Much work has been done with seeds16, but the comparison was limited to room temperature and — 190°C and therefore does not allow the


deduction that indirect action contributed to the increased sensitivity at room temperature. The most interesting data is that for bacteria. Both STAPLETON et al.17 and HOUTERMANS1 8 find that at the temperature where

he medium freezes, the sensitivity of E. coli falls to one-third, but then continues to fall as the temperature is lowered (see Fig. 2-5). The sudden drop on freezing occurs only in the presence of oxygen, however, and in its absence the change in resistance is continuous. This suggests that freezing only interferes with the diffusion of oxygen, but that under anoxic conditions diffusible radicals play no part. HOUTERMANS18" com-pared the resistance of bacterial spores when irradiated dry (when indirect action is by definition excluded), and wet, and found that it varied with temperature in both cases. With a-rays the data is conflicting; for E. coli there is a change in sensitivity only on freezing and further lowering of the temperature has no effect while, for Bacillus subtilis spores a tem-perature effect (see Fig. 2-6) is found only when dry, the radiosensitivity of wet spores remaining constant between — 200°C to 40oC18a.

The exact opposite effect of temperature was obtained by LASNITZKI 1 9

with mouse ascites tumour cells, which were killed more effectively when irradiated in vitro at — 79°C than at room temperature. Possiblythechemical damage from direct action is partly restored by reaction with another substance and this repair of the damaged molecule is prevented by freezing. Yet another complicating factor was recorded by W O O D and TAYLOR 2 0 ,

T1 0C

FIG. 2-5. Effect of temperature on radiosensitivity (inactivation dose relative to that at room temperature) of E. coli to 90 kV

x-rays18.


100

CTt

O

O :> >

0-1

FIG. 2-6. Effect of freezing on radiosensitivity to a-rays of wet and dry spores of B. subtilislsa:

O — O d r y s p o r e s a t - 1 8 4 ° C • — • dry spores at +20°C A — A wet spores at — 184'C x — x wet spores at +20°C

\ o \

N x \

Y • \ \ \

x \

V \

X

Dose (relative values)

who found that the radiosensitivity of yeast at low temperatures depends on the rate of freezing. Rapid freezing leaves them more sensitive.

From the rather limited number of experiments that have been carried out it is obvious that the effect of temperature is complex and no example has yet been observed where it can be deduced that indirect action is predominant in vivo.

Influence of Hydration The relationship between radiosensitivity and water content of cells in

systems where this can be varied, does not provide unambiguous evidence for the role of indirect action since hydration influences many of the physiological factors which play a part in the development of the radiation injury (cf. discussion on p. 3). In general, living systems which survive desiccation, or which like seeds become desiccated during a stage of their development, are in general very much less sensitive to radiations than organisms which contain a high proportion of water21-26. However, in the case of seeds the retention of water is not the only factor. LAMBERT 2 7

observed that the radio resistance of peas (Pisum sativum)—initially 5000 to 10,000 r—increased during the first six to twelve hours after hydration (water content 30 per cent) but then fell rapidly to reach a minimum after


2 4 hours of about 2 5 0 0 r. During hydration of the pea, FIRKET and colleagues28'29 found in 1929 that the number of SH groups and the amount of ascorbic acid was greatly increased and this may be the cause for the initial increase in radio resistance.

Lambert's observation has recently been extended by CURTIS et al.30

who find that the greater sensitivity (4 per cent) of nearly dry barley seed than seed having 20 per cent of water is due to a post-irradiation effect. The damage to the dry seed is reduced if it is soaked in water immediately after irradiation, suggesting that the post-irradiation oxygen effect—a phenomenon only so far seen in seeds and spores (see p. 287)—plays a part.

These experiments emphasize that while water plays a great part in the biological effects of radiation it cannot necessarily be interpreted in terms of indirect action.

Summary The only way in which it is possible to determine the relative contribu-

tion of direct and indirect action (as defined on p. 47) is by the dilution test. This, however, cannot be applied to radiation damage in intact cells, and the problem whether direct or indirect action is the important process remains unresolved. A discussion of the relative efficiency of the two processes for the inactivation of biological active molecules such as viruses and enzymes (see p. 58) suggests that direct action would make an important—-if not the chief—contribution under the conditions prevailing in the cell. Since the nature of the radiochemical reaction which initiates cellular injury is not known it is not possible to determine at the present time the relative importance of direct and indirect chemical processes and both must be considered.

R E L A T I V E E F F E C T I V E N E S S O F D I R E C T A N D I N D I R E C T A C T I O N IN VITRO

All enzymes and viruses that have been studied can be inactivated both by direct and by indirect action, although the efficiency (or absorbed energy necessary) of the two processes is not usually the same. The initial chemical reactions will in general be quite different for the two cases. In many organic molecules direct action involves a free radical which can either add to the molecule or abstract from it an atom or group. If the free radical abstracts an atom, then the final products obtained by direct and indirect action may be similar. This, for example, occurs with alcohols where the a-carbon atom is activated by direct action, and by indirect action suffers abstraction of hydrogen by an OH radical, the G value (see p. 101) in the two cases being approximately the same. For example:


direct CH3 . CH2 . OH ^CH 3CH. OH +H-

indirect CH3 . CH2 . OH •» CH3 . CH . 0H + H20

by OH radical

For benzene the position is reversed; the major product from indirect action in aqueous solution is the addition of a hydroxyl radical to give phenol,

while irradiation of the pure liquid (direct action) gives a large number of different substances, including hydrogen, acetylene and a polymer. In general, the radiation resistance of different substances is of the same order of magnitude for direct and indirect action. This is illustrated by the degradation of certain synthetic polymers (e.g. polymethacrylates) where the energy required to break a main chain is the same for irradiation in aqueous solution as for irradiation of the dry material (see p. 161).

On the other hand, the energy required to inactivate an enzyme or virus molecule when dry or highly concentrated (i.e. by direct action) is very much less than that required to inactivate it in dilute solution. In general, an amount of energy of the order of one primary ionization per molecule is sufficient to inactivate a virus or a phage (see p. 209) by direct action, while more than a thousand ionizations in the surrounding water may be needed for inactivation in dilute solution.

Quantitative considerations—The dose required to inactivate a given proportion of a pure enzyme or virus will decrease with decreasing concen-tration owing to the contribution of the indirect effect. If D0 is the 37 per cent dose for direct action—which is, of course, independent of concen-tration—then the 37 per cent dose (Dc) at concentration C (g solute/ml) is given by

where a is the ratio of the energy required to inactivate by indirect action (i.e. number of solute molecules inactivated per ionization* in the solvent,

* Lea, who developed this subject, always used the M / N terminology (see p. 101) and assumed that the energy required to form an ion was the same for all materials. In fact all measurements are based on energy absorption and ionic yields can readily be converted to eV/per molecule. In any case, the ratio a is independent of the way in which the yield is expressed.

D0 Dc= 1 + a jC


over that required for direct action (i.e. number of molecules inactivated per ionization directly produced in the solute). DC tends to Do at high concentration and when a is small (i.e. when direct action is more efficient than the indirect) DC = DQ even in relatively dilute solutions. Figure 2-7

FIG. 2-7. Influence of concentration on the inactivation dose (expressed as 37 per cent dose) for a material which can be inactivated by direct and by indirect action, a is the ratio of the energy required to inactivate by indirect action divided by the energy to inactivate by direct action. The actual curve does not pass through the origin since at very low concentration the efficiency of indirect action falls due to radical recombination.

shows a general curve relating the 37 per cent dose (DC) with concentra-tion; at very low concentrations the inactivation dose does not continue to diminish, but reaches a limiting value because of radical recombination. That is, the concentration of the substrate is so low that active radicals are lost by reacting with one another instead of with the solute (see p. 48). The half-way point (where the 37 per cent dose = is reached when the concentration in grammes of substrate per millilitre is numerically equal to a.

Relative Efficiency for Viruses and Enzymes When impure preparations are irradiated, the extraneous matter, which

acts as a protective agent by competing for the available free radicals, reduces the yield of the indirect effect and consequently the value for a decreases. For tobacco mosaic virus indirect action is very inefficient and a is of the order of IO-4; the 37 per cent dose, therefore, only becomes dependent on concentration when this falls below 2 x IO-3 g/ml (see


Table 2-3)31. The effect of adding small quantities of gelatine (a protector) is to lower the indirect action still further, which then fails to contribute to the inactivation even at the lowest dilution which could be used (i.e. the 37 per cent dose was independent over the whole concentration range). LATARJET and EPHRATI32 also found with bacteriophage that indirect action contributes only in very dilute solutions and that bacterial broth is quite sufficient to prevent all damage from the free radicals formed in water. While quantitative data allowing a direct comparison is available only for tobacco mosaic virus, the general position seems to apply to all viruses.

For enzymes, the yield for indirect action is much greater than for viruses and the value of a is correspondingly higher. In solutions normally studied in vitro (e.g. 1 per cent or less), the indirect effect makes a sub-stantial if not the major contribution to the inactivation of most enzymes. Again, the presence of other substances will act as protective agents, but their effectiveness cannot be predicted. The rate of reaction of different materials with free radicals varies at least by a factor of IO4 and the value of a will therefore depend entirely on the nature of the protective agents present.

The reason for the great disparity in effectiveness between direct and indirect action probably arises from the fact that the whole structure is not equally vulnerable. Thus the viruses are screened by a layer of protein molecules and the free radicals from water have to pass through this layer before they get to the sensitive part. It is quite conceivable that out of

T A B L E 2 - 3

D I R E C T AND INDIRECT A C T I O N I N AQUEOUS S O L U T I O N ; X-RAYS O N T O B A C C O M O S A I C V I R U S 3 1

Concentration in g/ml of 37 per cent Virus Protective agent dose XIO - 5 r

Solid — 0-14 — 0 - 0 2 2 — 0-00022 — 0-000022 — 0-0000044 — 0-000022 0-05 glucose 0-000022 0-001 glucose 0-000022 0-01 gelatine

2-5 2-9 2-9 1-5 0-5 0-6 0-5 2-4 2-4


every thousand radicals only one gets through and the remainder are wasted in trivial reactions. When the action is direct some ionizations will occur in the less essential components but the proportion will be much less. Even in the case of enzymes where the active principle is one molecule only, there are certain parts that can be modified without causing inac-tivation, and radicals will, by reacting with other parts, be wasted. An ionization, on the other hand, disrupts the whole structure of the molecule and this is probably the reason why nearly every ionization is effective.

R E L A T I V E E F F E C T I V E N E S S O F D I R E C T A N D I N D I R E C T A C T I O N I N C E L L S

Although the concentration of any one enzyme within a cell is extremely low, there are a host of other substances present which react with the free radicals formed in water and which can therefore be considered as pro-tectors for the enzyme which is being studied. Even if the extent of competition offered by all the cell constituents were known it would still be impossible to calculate the amount of inactivation by indirect action, since the cell is an organized structure and its constituents are not ran-domly mixed. The only way to approach this problem is to irradiate cells with different doses and to determine immediately afterwards in complete homogenates the change in enzyme activity. It must be stressed that, for a determination of this type, metabolic changes occurring as a consequence of the radiation must be suppressed, since these would completely mask the initial effect due to radiation. When these precautions have been taken, the dose needed to inactivate the enzymes (i.e. D37 dose) is of the order of millions of rads (i.e. a thousand times greater than the dose needed to kill mammalian cells; though some unicellular organisms can survive doses of this order). This shows that the enzyme under investiga-tion is "highly protected" but whether there is some remaining contribu-tion due to indirect action is difficult to decide. It may well be that the contribution of indirect action varies with the location of the "target" molecule in the cell.

Where the inactivation dose (D^T) in the cell is the same or greater than that found for the dry enzyme in vitro, then complete suppression of in-direct action can be assumed. An example of this type has been found for the acetyl cholinesterase in the electric eel33. The sensitivity of a number of cytoplasmic enzymes has been studied in bacteria34 (see Fig. 2-8), and also in vegetative yeast1; the observed D37 was about half that found for the dry enzyme. The conclusion that half of the inactivation is due to indirect action cannot be drawn, however, since it is quite possible that the inherent radiation sensitivity of an enzyme to direct action is different when it is present in the cell. KAPLAN35 has provided impressive evidence that the molecules of the enzyme catalase exist in the cell in a different


100

5 0

O 20 E

IO >

\ \

\ \ x \

0 2 4 6 8 IO 12 14 16 0 2 4 6 8 IO 12 14 16 18

Dose, r x IO6

FIG. 2-8. Inactivation by x-rays of the enzyme tyrosine de-carboxylase in Streptococcus faecalis™.

Irradiated as viable cells at 2 0 ° C — • —

— 1 8 0 ° C — O — Irradiated as dry acetone powder

2 0 ° C — • — — 1 8 0 ° C — • —

configuration from the one they assume on isolation. This change in state can markedly alter the sensitivity to direct action and the difference between the D37 in vitro (dry) and in vivo may be due to factors of this type and not due to a contribution by "indirect action".

Deoxyribonucleic acid (DNA) is also very radiation-resistant within the cell. This was established by extracting the DNA from a pneumococcus and using it to transform a different strain36. A dose of 100,000 rads given to the bacteria did not inactivate the transforming power of the DNA isolated after irradiation. In vitro 1000 rads sufficed to inactivate a diluted solution of transforming principle while dry its D37 dose is of the order of 700,000 rads. These experiments suggest that indirect action may contribute relatively little to the inactivation of DNA in vivo.

R E F E R E N C E S

1 . H U T C H I N S O N , F . , Radiation Research, 1 9 5 7 , 7 , 4 7 3 , la . S M I T H , C . and ESSEX, H .,J. Chem. Phys., 1938, 6, 188.

2 . POLLARD, E . C . , G U I L D , W . R . , H U T C H I N S O N , F . and S E T L O W , R . B . Progress in Biophysics (Edited by J. A. V. BUTLER and J. T . R A N D A L L ) , 1950, 5 , 72, Pergamon Press, London and New York

3 . D A L E , W . M . , G R A Y , L . H . and M E R E D I T H , W . J . , Phil. Trans. A , 1 9 4 9 , 2 4 2 . 3 3

4 . O K A D A , S . , Arch. Biochem. Biophys., 1 9 5 7 , 6 7 , 1 0 2 5 . G U N T E R , S . E. and K O H N , H . I . , / . Bacteriol., 1 9 5 6 , 7 2 , 4 4 2 6 . G R A Y , L . H . , Radiation Research, 1 9 5 4 , 1 , 1 8 9 7 . D A L E , W . M . , Biochem. J., 1 9 4 2 , 3 6 , 8 0 ; Disc. Faraday Soc., 1 9 5 2 , 1 2 , 2 9 3


8 . OKADA, S . , Arch. Biochem., 1 9 5 7 , 6 7 , 9 5 9. ALEXANDER, P. and CHARLESBY, A . , Nature, 1 9 5 4 , 173, 5 7 8

10. ALEXANDER, P. and CHARLESBY, A . , Radiobiology Symposium, Liege, 1 9 5 4 , 49, Butterworth, London

1 1 . ALEXANDER, P . a n d T O M S , D . , J . Polymer Sci., 1 9 5 6 , 2 2 , 3 4 3 1 2 . ALEXANDER, P . , Radiation Research, 1 9 5 7 , 6 , 6 5 3 1 3 . CALDECOTT, R . S . , JOHNSON, E . B . , N O R T H , D . T . a n d KONZAK, C . F . ,

Proc. Natl. Acad. Sci., U.S., 1957, 43, 975 14. ALEXANDER, P., BLACK, R. M. and CHARLESBY, A . , Proc. Roy. Soc. A , 1 9 5 5 ,

232, 31 1 5 . BACHOFER, C. S., EHRET, C. F., MAYERS, S. and POWERS, E . L . , Proc. Natl.

Acad. Sci., U.S., 1953, 39, 744. 15a. W E B B , R. B., EHRET, C . F . and POWERS, E . L . , Experientia, 1958, 14, 324 1 6 . RAJEWSKY, B . N . , Brit. J. Radiol., 1 9 5 2 , 25, 5 5 0 1 7 . STAPLETON, G . E . and EDINGTON, C . W . , Radiation Research, 1 9 5 6 , 5, 3 9 18. HOUTERMANS, T . , Z. Naturforsch., 1954, 9B, 600 18a. HOUTERMANS, T . , Z. Naturforseh., 1956, 11B, 636 19. LASNITZKI, I., Radiobiology Symposium, Liege, 1954, p. 321, Butterworth,

London 2 0 . W O O D , T . H . and TAYLOR, A . L . , Radiation Research, 1 9 5 7 , 7 , 9 9 21. HOLLAENDER, A., Symposium on Radiobiology, p. 285, John Wiley, New

York, 1952 2 2 . G E L I N , O . E . , Heriditas, Lund, 1 9 4 1 , 2 7 , 2 0 9 23. PETRY, E. , Biochem. Z., 1922, 128, 326 2 4 . HENSHAW, P . S. and FRANCIS, D . S., J. Cell. Comp. Physiol., 1 9 3 5 , 7 , 1 7 3 2 5 . WERTZ, E . , Strahlentherapie, 1 9 4 0 , 6 7 , 7 0 0 2 6 . PATT, H . M . , Physiol. Rev., 1953, 3 3 , 35 ' 27. LAMBERT, J., Arch. Biol., Paris, 1933, 44, 621 28. FIRKET, J. and COMHAIRE, S., Bull. Acad. Med. Belg., 1929, p. 93 29. LECLOUX, J . , VIVARIO, R . a n d FIRKET, J . , C.R. SOC. Biol., Paris, 1927, 97,

1823 3 0 . CURTIS, H . J . , DELIHAS, N . , CALDECOTT, R . S . and KONZAK, C . F . , Radia-

tion Research, 1958, 8, 526 31. LEA, D. E., Brit. J. Radiol., Suppl. No. 1, 1947, p. 59 32. LATARJET, R . and EPHRATI , E . , Compt. rend. Soc. Biol., 1948, 1 4 2 , 4 9 7 ; see

also WATSON, J. D., J. Bacteriol., 1952, 63, 473 33. SERLIN, I . a n d COTZIAS, G . C. , Radiation Research, 1957, 6, 55 3 4 . PAULY, H . and RAJEWSKY, B . , Strahlentherapie, 1 9 5 5 , 35, 2 2 0 3 5 . KAPLAN, J . G . Exptl. Cell. Research, 1 9 5 5 , 8 , 3 0 5 3 6 . D R E W , R . M . , Radiation Research, 1 9 5 5 , 3 , 1 1 6

C H A P T E R 3

Dose-Response Relationships in Chemical and Biological

Systems

M U C H can be learned by studying the relationship between the magnitude of the radiation effect with the size of the dose, but great caution must be exercised in the interpretation. The factors which determine the nature of the dose-effect curve are quite different in radiation chemistry when the end-effect is an immediate consequence of the uptake of energy than when the effect measured is the end-result of many changes. It is this last situation with which we are confronted in most biological experiments.

T H E D 3 7 D O S E A N D " S I N G L E - H I T " C O N C E P T For chemical reactions where the radiation process is indirect, the relation-ship between dose and observed chemical change (for instance, inactiva-tion) depends upon the reaction product (see Fig. 3-1).

(i) If the product does not react with free radicals (as, for example, in simple reactions such as the oxidation of ferrous to ferric ions or the decomposition of formic acid) then the number of molecules changed will be directly proportional to the dose.

(ii) In reactions, such as the inactivation of enzymes or the degradation of macromolecules, where the product (e.g. the inactivated enzyme) is still capable of reacting with free radicals, it will act as a protective agent. As the reaction proceeds the extent of protection increases and the dose curve will be exponential. If the product has the same reactivity with free radicals as the starting material (and this is often the case with enzymes) then a dose of radiation, which produces sufficient radicals to inactivate every molecule, will only bring about 63 per cent inactivation since 37 per cent of the radicals will react with molecules which have already reacted once. For this reason the terminology used by LEA1 to express the sensitivity of an organism or enzyme to radiation as the 37 per cent dose (i.e. the dose when 37 per cent survive) D37 is most useful.

63


0 Dose

FIG. 3-1. Relationship between dose of radiation and change observed (e.g. percentage of molecules chemically changed or inactivated or number of organisms killed) for indirect action:

(a) plotted linearly; (b) plotted logarithmically.

I Where product does not react with the free radical res-ponsible for the change (e.g. oxidation of Fe + + ->Fe + + + , cf. P- 134);

I I Where product reacts as readily as the original material with the free radicals (i.e. the radiochemical product acts as a protector). This is the case for the inactivation of carboxypep-tidase in solution.

When the action is direct then the dose-response relationship must be semi-logarithmic as in case (ii) above (Fig. 3-1, curve II) since ionizations occur entirely at random. A molecule that has been ionized (and therefore chemically altered or inactivated) is as likely to suffer a second ionization as is a molecule that has not been ionized before*. The dose-relationship between direct and indirect action is therefore the same, whether the action is direct or indirect so long as the substrate being studied is complex (e.g. a protein molecule) since radicals can be wasted by reaction with an inactivated molecule. Table 2-2, on p. 50, shows that the rate of reaction with OH radicals is approximately the same for the various proteins studied. A difference in kinetics between direct and indirect action will only occur if the reaction involves a small molecule which loses its affinity for free radicals once it has reacted with one. Such a reaction is unlikely to be important in cells and the dose-response curve cannot be used to distinguish between direct and indirect action.

The same type (i.e. curve II in Fig. 3-1) of semi-logarithmic curve is frequently observed when simple biological systems are irradiated—for

* It is conceivable that this situation might be modified by energy transfer processes, but, so far, this has not been observed, presumably because a previous reaction does not change the energy transfer characteristics.

D O S E - R E S P O N S E R E L A T I O N S H I P S 65

example, the killing of bacterial cells. Such a dose-response relationship can be interpreted in the same way as the inactivation of an enzyme or the change of a molecule as being due to the change produced by a single ionization. Hence, this type of response is referred to as a "single-hit" process. This term is useful as a shorthand notation to describe an ob-served relationship between dose and effect, but does not imply that the "target theory" can be applied (see Chapter 4).

When the proportion of the total units (be they molecules, genes or cells) affected is small, i.e. less than 25 per cent, then the semi-logarithmic "single hit" response is indistinguishable from a linear response and a straight line is obtained when dose is plotted against number of units changed. This means that the first part of curve II, Fig. 3-la, is effectively a straight line. This situation is frequently encountered in genetic experi-ments where the fraction of animals that have been mutated is very small and it is then perfectly permissible to speak of a linear dose-response curve.

It must be stressed that the above discussion applies only to experiments where the damage is measured at the molecular level, whether in vivo or in vitro, and the 37 per cent dose has no meaning in complex systems such as whole-body irradiation of mammals. A dose of radiation sufficient to kill 63 per cent of a group of animals leaves an equal amount of energy in each animal and the reason why some die and others live is that there is variation in bioresistance. If they were all completely alike then a certain dose of radiation would kill either 100 per cent or none at all.

Influence of dose rate—If an effect is the direct consequence of a single ionization then it must be independent of the rate at which the radiation is delivered. Most radiochemical reactions, including the inactivation in vitro of biologically active substances such as enzymes and viruses, are independent of dose rate. A chemical reaction can be dose-rate dependent for the following reasons:

1. The reaction requires oxygen which has to diffuse into a system. 2. The product is unstable and capable of reacting further with the

starting product. Thus polymerization of vinyl polymers (see p. 145) is very dose-rate dependent since the monomer, on becoming ionized, starts a chain reaction with un-ionized monomer. This growing chain reaction can be terminated by another ionized monomer molecule. At low dose-rates this termination is rare, and more polymer is formed than by the same amount of radiation delivered at a high rate.

3. If the intensity is extremely high, reactions due to "indirect action"— i.e. free radicals—become less efficient owing to increased radical-radical interaction. This is a very special case and does not apply when normal radiation facilities are used (see p. 146).


In biological experiments dose-rate effects are very common and indicate that restoration and repair processes are occurring.

" M U L T I - H I T " E F F E C T S

Frequently the dose-response curve is not of the single-hit type, but has a sigmoid shape. This can be interpreted as a multi-hit phenomenon. If the radiation change studied requires two or more events (ionizations or reactions with free radicals) the dose-response curve will show a shoulder, since initially the chance that two reactions occur within the same unit is extremely small and hence the rate of inactivation (or other change studied) is low. After a certain dose, however, there will be a number of molecules that have sustained one "hit" and are still active; at this stage the rate of inactivation rises steeply, since there is then a good chance that a further hit will lead to an inactivation. This is illustrated graphically in Fig. 3-2. When the same data are plotted semi-logarithmically, the

FIG. 3-2. Relationship between dose and number of organisms inactivated for cells which require different numbers (N) of

hits for inactivation.

curves tend to straight lines which, when extrapolated to zero dose, intersect the axis at a value which is numerically equal to the number of hits necessary for inactivation (see Fig. 3-3).

It is conceivable to have a complex situation where the end effect is made up of two processes, one of which is of the "single hit" type and therefore initially proportional to the dose while the other is a "two hit" event and therefore initially proportional to the square of the dose received. For example, it has been suggested that life-span shortening is due to genetic damage in which both point mutations (one hit) and chromosome rearrangements (two-hit) contribute. The shape of the dose-response curve would thus be initially linear, but curves up later. Moreover, the number of chromosome rearrangements produced is dependent on dose


Dose

FIG. 3-3. Theoretical survival curves plotted on a log scale for materials requiring different numbers of hits for inactivation. The number of hits required can be obtained by extrapolating the linear parts of the curve on to the axis of the "fraction

survived".

rate while point mutations are not (at least in spermatagonia, see p. 258), thus at high doses (where the two-hit effect makes an appreciable contribution) the genetic damage would be dose-rate dependent, while at low doses it would be dose-rate independent.

In many systems only a small fraction of the ionization or free radicals from water bring about the effect studied, and the others are wasted in reactions that do not influence it. This is not a multi-hit situation so long as one reaction, when it does occur at the right place, is sufficient for inactivation. The effect of "wasted events" is merely to change the dose at which a particular reaction occurs, but does not influence the shape of curve.

Usually sigmoid dose-response curves cannot be interpreted readily in biological systems since the end-effects observed are a complex interplay of many factors. Much of the data on the irradiation of animals could be made to fit on to one of the curves shown in Fig. 3-3 and the magnitude of the threshold dose, below which there is no killing at all, would deter-mine the number of hits. Experimentally it is found that the dose-response curve is much steeper for animals from a closely inbred line than from

6


ordinary heterozygous stock. Yet we cannot conclude that the purer the strain the greater the number of "hits" needed to kill the animal (cf. Fig. 3-4). When lethality is the endpoint such a mistake is unlikely to be made, though many theories have been put forward concerning the number of events that lead to cancer in man from the relationship between age and cancer incidence.

Figure 3-5 shows how a typical dose-response curve for the killing of mice can be transformed when plotted on a probability (or probit scale

FIG. 3-4. Relationship between dosage mortality slope for mice from two different strains and for their hybrids2.

50

CAF;

550 650

Tissue dose,

750

99-5

98-0

90-0

70-0

g50-0

| » 0 10-0

20 0-5

'CAF1

- cf+9 (91-231 days)

LDeSSdOI) - S=I-OSlI-OI)

- ( b ) -

550 650 750

Tissue dose, r

FIG. 3-5. (a) Dose-response curve, (b) Same curve plotted on a probability scale. (The fact that this curve is linear shows that there is a normal gaussian distribution of resistance to radia-

tion3.)


which is the normal procedure for dealing with effects where there is a random variation in susceptibility. That means the shape of the dose-response curve tells us nothing of the radiation process, but is merely a measure of the biological variation.

In simpler systems the situation is more difficult. What does a "multi-hit" curve mean for the inactivation of bacteria or of individual mam-malian cells grown from one single cell in tissue culture? Is the sigmoid shape due to a multi-hit process or is it due to a complex biological factor?

Quite paradoxical dose-response curves where higher doses produce apparently a smaller amount of biological damage are also occasionally encountered. For example, SCHWARTZ4 , MOUTSCHEN, BACQ and HERVE 5

found that if dry seeds of barley are irradiated with x-rays and then planted, the height to which the seedlings grow falls. At large doses it rises again (see Fig. 3-6) unless the seeds have been stored for some time

FIG. 3-6. Relationship between growth of barley seedling (height of leaf) planted fifteen days following irradiation of dry

seed with x-rays5.

in daylight—storage in the dark has no effect—between irradiation and planting. Clearly the radiation effect is governed by a complex interplay of several factors; yet if this experiment had been confined to doses of less than 2 x IO5 rads the curve could have been made to fit an expo-nential curve and the target size calculated!

T H R E S H O L D — A P R O B L E M O F M A M M A L I A N R A D I O B I O L O G Y

In biological experiments often no effect is observed until the dose passes a certain value. In practice it is frequently impossible to distinguish between a dose-response curve which shows a true threshold (i.e. curve 4


in Fig. 3-7) and one where the response follows a hyperbolic relationship (i.e. curve 3 in Fig. 3-7). The difference is important from both a practical and a theoretical point of view, since a true threshold implies that below a certain dose there is no danger whatsoever with regard to the particular effect studied, and allows a safe dose to be specified. Biological experi-ments can never be sufficiently precise to establish the existence of a threshold.

hunon peculations

FIG. 3-7. Diagrammatic representation (the example is hypo-thetical) of the problem of extrapolating experimental data obtained within one dose-range to provide information about the frequency of the process at a much lower dose. The experimental points cannot be determined with mathematical precision: the best that can be done is to give a range of values (the thick vertical lines). What happens at the bottom of the curve? Is curve 1, 2, 3, or 4 more nearly correct? All of them fit the experimental data, but on the scale of interest for human effects (see enlarged version in inset

at bottom right) they are vastly different.

The concept of a threshold, like many scientific concepts, is nevertheless useful even though it represents a crude and artificial simplification in most, if not in all, its applications to radiobiology. This is best explained by a concrete example.

If mortality at 30 days (after irradiation) is plotted against doses of x-rays delivered in a single exposure, one sees that no mouse dies below 200 or 300 r; few animals die at 400; then follows an increased frequency of death up to 700; very few animals need more than 700 to die in a

D O S E - R E S P O N S E R E L A T I O N S H I P S 7 1

month's time—sigmoid, threshold curve. But death at 30 days is an all-or-none effect arbitrarily chosen. If we observe and plot the survival time expressed in days (Fig. 3-8) we get a different kind of curve, because

r IOOO 10000 100000

log dose r

FIG. 3-8. Dose-mortality curve of white mice following x-irradiation with a single dose6.

doses of the order of 100 r decrease the life-span; instead of a sigmoid curve we get a straight line. Here again arises the danger of extrapolation at low doses. There are reasons to believe that such an extrapolation is not legitimate, and becomes improbable if the small dose is delivered at a low rate (see p. 439).

Other facts become apparent if one investigates the effect of high or very high doses (with a special irradiation apparatus delivering 1000 kr/ minute) and plots the results in a log-log dose-effect curve (see Fig. 3-8). Obviously there is no simple relation between dose and survival time except at very high doses (100 kr to 500 kr) when death is practically instantaneous. Let us analyse this curve:

1. Below 100 r there is very little effect. 2. Between 100 and 700 r (the most frequently used range of dose)


many changes occur in the body: damage to gastrointestinal tract, bone-marrow atrophy, hormonal and biochemical changes, infec-tions, etc.

It is possible to separate by pathological studies and statistical analysis two main reasons for death: an early (3 to 5 days) death due to gastro-intestinal troubles and a late death due to bone-marrow atrophy (see p. 421). Bone-marrow atrophy cannot kill in a few days because the number of circulating blood cells is sufficient to keep the animal alive during a week or more; blood cells are continuously destroyed and if they are not replaced, a condition of leucopenia and anaemia gradually develops which causes late death. These circulating blood cells (with the exception of lymphocytes) are much more radioresistant than the bone-marrow mother cells; their life-span is not supposed to be decreased*. Conse-quently, the development of leucopenia or anaemia is not accelerated by increasing the dose above that needed to produce maximal destruction of the bone-marrow. But the damage to the epithelium of the gastro-intestinal tract will increase, and on reaching a dose of 1200 r practically all the mice die in 3 to 4 days from troubles in this system.

3. Between 1200 and 15,000 r the survival time is the same: about 3 | days. Why is there no change over this enormous dose range? Accord-ing to RAJEWSKY6, because a mammal possesses regulating mechan-isms (mainly hormonal) and adrenalectomized or hypophysectomized rats do not show the 3-5 days' effect (Fig. 15-1, p. 391). The argument advanced in the preceding paragraph applies here: in this range of dose, death is probably caused by damage to the gastrointestinal tract which results in physiological failures (not infection, according to Rajewsky). When the anatomical damage is done, whether by 1200 or 10,000 r, it takes the same time for physiological troubles to develop and to lead to death. To kill earlier, a dose must be given which affects another organ which eliminates another physiological function, thereby creating new lesions.

4. The dose range of 15,000 to 30,000 r is characterized by a rapid reduction of survival time; the immediate cause of death is apparently different. Recent studies suggest that lesions of the lung may be the dominant factor.

5. Between 30,000 and 100,000r, a new symptom appears—convul-sions, which points to participation of the central nervous system. After a dose of 100,000 r, mice do not survive more than an hour.

* This point has not been settled experimentally although it is at present technically easy to study the life-span of the population of red cells in man or mammal. It would be valuable to show an "ageing" effect on this population.

D O S E - R E S P O N S E R E L A T I O N S H I P S 7 3

6. Beyond 100,000 r, death is instantaneous and has been interpreted as the result of inactivating or destroying a large number of sub-stances indispensable for the basic chemical activity of the organism : it is the so-called "molecular death".

This analysis shows how dose-effect relationships may be modified or obscured by the interference of physiological mechanisms such as exist in mammals. It shows also how the development of our knowledge in mammalian radiobiology depends on the constant confrontation of the experience in fundamental biology with observations of classical pathology.

The thoughtful reader must understand that the discussions about threshold or non-threshold effect, linear relationship, etc., are not of purely academic character. For instance, when the U.N. Radiation Committee7 was confronted with the task of evaluating the danger of leukaemia induction in man by natural radiation, medical radiation and by 90Sr from fall-out incorporated in bones, it was found impossible to decide whether leukaemia induction is or is not a threshold effect, because the dose-range of interest for human populations is well below the mini-mum dose at which the incidence of leukaemia has been measured in the laboratory, so the exact position of the curve is bound to be guesswork. To determine whether the actual dose response curve at very low doses shows a true threshold (i.e. curve 4, Fig. 3-7) or follows curves 1, 2 or 3, would require the irradiation of hundreds of thousands of experimental animals. Even if this were done the extrapolation from "mouse to man" would remain as an unknown factor. Yet if one assumes a non-threshold relationship, that is initially linear, then natural radiation causes 25,000 annual cases of leukaemia in a 5 billion world population; fall-out would induce 5000 to 60,000 annual cases in a similar population in about 100 years when equilibrium will be reached after prolonged continuation of tests (see p. 502). If one assumes a threshold of 400 rads, neither natural radiation nor fall-out would induce a single case of leukaemia.

In the present state of our very limited knowledge it is not possible to decide which of these two assumptions is valid.*

R E F E R E N C E S

1. LEA, D. E., Actions of Radiations on Living Cells, Cambridge Univ. Press, 1946

2. GRAHN, H . , Genetics, 1958, 43, 835 3 . K O H N , H . I . and KALLMAN, R . F . , Radiation Research, 1 9 5 6 , 5 , 3 0 9 4 . SCHWARTZ, D . , Science, 1 9 5 4 , 1 1 9 , 4 5 ; SICARD, M . A . a n d SCHWARTZ, D . ,

Radiation Research, 1959, 10, 1 5 . MOUTSCHEN, J . , BACQ, Z . M . and HERVE, A . , Experientia, 1 9 5 6 , 1 2 , 3 1 4

74 FUNDAMENTALS OF R A DIOBIO L OGY

6. RAJEWSKY, B., Radiobiology Symposium, Liege, 1954, p. 81, Butterworth, London

7. Report of the U . N . Scientific Committee on the effects of atomic radiation. General Assembly, 13th Session, Supplement 17 (A/3838), New York, 1958.

C H A P T E R 4

The Nature of the Initial Chemical Lesion in Cellular Radiobiology

IN Chapters 1 and 2 the physical processes which determine the amount of energy that is deposited and the geometry of its deposition were des-cribed. The next step is the conversion of part of this energy into chemical change. One of the basic problems of radiobiology is to determine which of the many different initial chemical reactions are responsible for initiating biological damage. Chapters 5 to 7 will deal with the chemical changes that are produced by ionizing radiations. Because these radiations are extremely energetic, they can bring about far-reaching chemical reactions and there is no organic cell constituent that will not be altered by radiation in such a way as to lose its biological activity. The problem is not to find a reaction which would lead to cell damage, but to decide which of the many reactions that do occur are important and which are trivial. In this respect radiations pose a quite different problem from most drugs. In pharmacology usually the difficulty is to find the molecules in the cell with which the drug interacts; once this is known, progress towards elucidating the mechanism is in most cases rapid (see, however, the radio-mimetic chemicals, p. 222).

In spite of the substantial advances that have been made in radiation chemistry in the last ten years, vague guesses only can be made about the nature of the initial chemical lesion in the cell. The basic difficulty arises from the non-selectivity of the radiation. Thus 100 r will kill many mammalian cells, but this dose will only produce something in the neigh-bourhood of 1000* chemical reactions within a volume of 3 /i3 (e.g. the nucleus of a liver cell). To a first approximation these reactions will be shared equally amongst all the cell constituents (e.g. if 10 per cent by weight of this volume is occupied by nucleic acid then 100 reactions will involve the nucleic acid). Although 100 r will inactivate a few molecules

* For organic substances somewhere between 2 and 10 molecules are chemically altered for every 100 eV of energy absorbed (i.e. G value = 2-10; see p. 107). Assuming an average value of 5 per 100 eV, there will be 1000 reactions for the 20,000 eV of energy which is absorbed in 3 ft3 tissue from 100 rads.

75


of some important enzymes, the fraction of the total activity of any one enzyme that is destroyed is minute. For the same reason changes produced in the low molecular weight components of the cell can be totally dis-regarded; e.g. an ionization occurring in a molecule of vitamin B will destroy its activity, but the cell will not be affected by the loss of one such molecule out of the thousands that are present.

Consequently, the only type of substance which could act as the primary lesion is one where almost every molecule is essential to the cell. There are probably very few, if any, enzymes that fill this role. KREBS1 suggested that in the major metabolic chains there are one or two enzymes which determine the activity of the whole process and a very small reduction in their activity could have a disproportionally serious effect. There is, however, no experimental support to indicate that the primary radiation injury involves one of these rate controlling enzymes and the RBE (relative biological efficiency) of different radiations also argues against it (see p. 96).

The genetic material deoxyribonucleic acid (DNA) is an obvious can-didate for the primary chemical lesion since there are biological indications that each molecule has a unique function and that there are no spares. In vitro experiments (see p. 194) have shown the physical and chemical properties of DNA are profoundly altered by radiation.

The primary chemical lesion need not, however, involve the inactivation of a vital cell constituent and two other possibilities will be considered: (1) radiation produces a new substance which is extremely poisonous (see poison theory, p. 96), or (2) radiation breaks down barriers within the cell and the disorganization leads to the cell's destruction (see enzyme release theory, p. 275).

All these different mechanisms need not, of course, be mutually exclu-sive and there is no a priori reason why the same mechanism should apply to all the principal cellular effects such as:

(a) Production of mutations—including somatic mutations; (b) Delay of mitosis; (c) Death following one or more divisions; (d) Interphase death—in which the cell dies some time after irradiation,

but without first dividing or attempting division. There is also no reason to believe that the chemical reactions which

lead to the inactivation of viruses or other sub-cellular structures are related to those that lead to cellular effects. The magnitude of the dose and the fact that sparsely ionizing radiations are more efficient than densely ionizing ones (see p. 210) suggest that viruses do not provide a model system for the basic processes of radiology in vivo.

This chapter is concerned with the physical factors that have to be considered in connection with the problem of finding the initial chemical

I N I T I A L C H E M I C A L L E S I O N I N C E L L U L A R R A D I O B I O L O G Y 7 7

lesion. Chief amongst these are the uses and limitations of the target theory and the important clue provided by the fact that the relative biological effectiveness (RBE) of ionizing radiations which differ in ionization density (LET)—see p. 29—is quite different for in vivo than in vitro effects.

T H E T A R G E T T H E O R Y The basic concept of the target theory is that the biological end-effect which is measured stands in a precise relationship to the initial physical events which occur on irradiation. More particularly, the theory implies that the exact site where an anatomical lesion, such as a chromosome break, is seen, must also be the place where the primary ionizations responsible for these effects occurred. In this form the theory originated with CROWTHER'S observation in 19242that the data relating inhibition of cell division with dose could be quantitatively interpreted if mitosis arrest was the result of a single ionization in a volume which corresponded to the size of the centromere seen in the nucleus of the dividing cell. Crowther developed this theory to include effects where more than one ionization per organism was necessary for inactivation and showed how both the volume of the target and the number of ionizations could be calculated from the dose-response curve*. In its subsequent development the theory has been applied to two quite different problems. Firstly, along Crowther's original lines to provide information about the nature of the initial events for radiation lesion in vivo\, and secondly as an analytical tool to determine in vitro the sensitive volumes of biologically active substances, such as enzymes and viruses. This latter aspect will be referred to in Chapter 7. To apply the target theory in its simplest and most useful form to cellular injury requires:

1. That the radiation injury measured follows as the result of one (single-hit dose-response curve) or several (multi-hit curve) chemical reac-tions within a discrete volume (the target). If the shape of the dose-response curve does not fit these relationships then the target size

* The observation that the dose-effect curve for the killing of micro-organisms was of the exponential (i.e. one-hit, see p. 63) type, led to the suggestion as early as 1904 that the radiation effect could be identified with a single event and was not brought about by accumulation of injuries (or of poisons). This interpre-tation does not constitute the formation of target theory which necessarily implies the calculation of a volume. It is quite possible to challenge the validity of these target calculations without challenging the single-hit hypothesis.

t T o have a convenient notation the following rather arbitrary definitions will be used in this chapter: in vivo is applied to experiments on intact animals, plants and micro-organisms; in vitro is used for experiments in which sub-cellular constituents, such as enzymes and viruses, are irradiated outside their host.


can usually not be calculated. One reason for a deviation is that the cells irradiated are a mixed population containing a variety of strains having different radiation sensitivities (i.e. different values for the inactivation dose, D37).

2. That the process studied is independent of dose rate. If the magnitude of the radiation effect is less if a given dose is fractionated or admin-istered at a lower intensity this indicates that a repair process is occurring after irradiation. An attempt has been made to apply target theory to production of abnormalities which are dose-rate dependent, see p. 88.

3. That the probability of a biological effect being produced depends solely on the physical nature of the radiation and is not influenced by external factors operative before or after irradiation.

Calculation of a target size from the dose-response curve is not pos-sible if:

1. Subsequent metabolic processes influence the magnitude of the lesion produced (e.g. post-irradiation processes).

2. The presence of extraneous substances can restitute the primary damage from an ionization (e.g. chemical protection) or enhance the lesion (e.g. oxygen effect).

3. The primary chemical lesion is produced by a free radical that has diffused through water to the "target", because the distribution of such reactions will then bear no relation to the pattern of the ionizations.

Calculation of the Target Size When none of these objections apply then the size of a so-called "sensi-

tive volume" can be calculated from the D37 inactivation dose. The shape of the curve will give the number of events (or ionizations) required to inactivate the target (see p. 66).

In principle, the calculation of the target size is not difficult. One of the simplest methods, which is applicable for low ion density radiation, is to assume that each ionization occurs singly and at random. The 37 per cent dose in roentgens (Do) produces an average of one ionization per target volume, V, which is then obtained directly from the relationship V = 0-7Ip DQ in /LIS where p is the density of the material irradiated. This simple calculation will give very approximate values only, since ionizations do not occur singly, but in clusters, nor are they randomly distributed, but are formed along tracks.

Usually an average cluster size of about three ionizations is assumed and this is called a primary ionization. Even in gases the exact magnitude of the primary ionization is not accurately known and its value in solids is little more than a guess. POLLARD3 chooses a value of one primary ionization of 100 eV, which is essentially the same as Lea's value4 based


on Wilson cloud chamber pictures which show that the average cluster contains three ionizations (i.e. 3 x 32-5 eV). However, the average value for the energy of a primary ionization may lie somewhere between 50 and 200 eV. The number of inactivating events and therefore also the target volume is uncertain by a factor of at least 4.

Equally serious is the problem of S-rays (see p. 32), since they produce tracks in which the distribution of the ionizations is quite different from that of the primary particles. Since at least 20 per cent of the energy is provided by S-rays, any corrections that have to be made are far from negligible (see Fig. 4-1). Yet again the theory for S-rays is very imperfect

_ o

2'MeV neutrons

j trit ium f ] r a y s

(ma*. 18 keV)

Co ^ r a y s

L E I in water, keV /a

FIG. 4-1. Distribution of L E T frequency (expressed as fraction of energy dissipated per unit interval of logio L E T ) for different

radiations21.

and calculations are largely inspired guesswork. Many unsuccessful attempts have been made to refine the computation and the treatment due to LEA4 which is fully described in his monograph. It is known as the "associated volume method" and applies to targets of all sizes and for


radiations differing in ion density and includes corrections for S-rays. The results are shown graphically in Fig. 4-2.

Comparison of Radiations Differing in LET The dose of radiation required to produce a given amount of inactivation

must depend upon the specific ionization of the radiation used. For a single-hit process those radiations, having the lowest ion density or LET, must be the most effective, because the chance of producing more than one ionization (i.e. wasteful ionizations) within the target is smallest. The

FIG. 4-2. Calculation of target size (expressed as molecular weight and diameter) for a nucleoprotein of density 1 -3 from the 37 per cent dose for three different radiations: (a) x-rays (average quantum energy 8 -2 keV); (b) y-rays from radium (average quantum energy 830 keV); (c) a-rays (average quantum energy 3 MeV). This applies to single-hit inactivation process

and a spherical target; data taken from L E A 4 .

relative effectiveness of sparsely ionizing radiations is greatest for large targets. A 5 MeV a-particle will produce about 50 ionizations when passing through a target of 100 A diameter, and consequently 98 per cent of the ionizations are not needed when inactivation is due to a single ionization. If the target has a diameter of 1 A the proportion of a-ray ionizations wasted will be reduced by a factor of 10.

When more than one hit per target is required for inactivation the dose-response curve will be sigmoid for sparsely ionization, but with densely ionizing radiations it will show a single-hit curve, since every time a densely ionizing particle passes through the target enough ionizations will be left within it to inactivate it.


By comparing the inactivation dose of radiations having widely different LETs the shape of the target can be obtained. Consider two targets of the same volume, but one is spherical, whereas the other is long and thin. With sparsely ionizing radiations the inactivation dose will be the same, whereas densely ionization radiations will distinguish between them since the number of ionizations that are wasted (i.e. the average track length) will be greater in the spherical than in the long shape.

Another variable which can in principle be derived by comparing different radiations, is whether there are a number of separate and discrete sensitive regions (i.e. targets) a hit in any one of which leads to inactivation, or whether there is one large sensitive area of the same total volume. Sparsely ionizing radiations do not distinguish between these possibilities and only give the volume of sensitive region however distributed. Densely ionizing radiations, however, will require a lower dose to inactivate a multiple than a single target.

In practice, however, it is not possible to derive all this information concerning the physical dimensions of the target as there are so many parameters that within the inherent inaccuracy of the method* it is not possible to distinguish between the different possibilities.

There is, however, one important situation that is frequently encountered in vivo and which can only be detected by comparing the effectiveness of different radiations. If the target is put out of action only when several ionizations occur within it at the same time then densely ionizing radiations will be much more effective than sparsely ionizing radiations. This type of reaction differs from the multi-hit process situation where the target is progressively inactivated by the successive passage of ionizing particles through it until the requisite number of ionizations have been attained; a threshold in the dose curve is then seen. In the case under consideration there is no threshold because a single process is needed. The passage through the target of a particle that does not produce the requisite number of ionizations is entirely without effect and does not change the number of ionizations necessary for inactivation by another and subsequent particle. Pictorially a process of this type can be considered as the breakdown of a structure (see p. 97). A wall which requires a cannon ball to knock it down is not destroyed by a succession of many rifle bullets even if their combined impact is greater than that of the cannon ball.

This type of multi-hit effect is recognized by the fact that the dose curve is exponential (i.e. one hit) for all radiations, but the inactivation

* Because of the presence of 8-rays (see Fig. 4-1) all radiations contain a wide spectrum of L E T values and the one quoted is an average. These variations swamp out fine differences and even if all the conditions necessary for applying target theory were fulfilled the calculations would still be meaningless.


dose decreases as the LET increases up to a maximum. What happens is that the majority of the ionizations of low average LET are wasted and the reaction is brought about entirely by the small component of high LET tracks produced when the electrons have been slowed down (i.e. at the end of the electron tracks; see p. 32). The reaction is produced most efficiently (i.e. at the lowest dose) by radiation having an average LET such that on passing through the target region the necessary number of ionizations are produced. The sensitivity falls again when the average LET exceeds this value as more ionizations than are necessary are then produced in the target.

A P P L I C A T I O N O F T A R G E T T H E O R Y T O R A D I A T I O N E F F E C T S P R O D U C E D IN VIVO

Before Dale's experiments with enzymes (see p. 191) had clearly estab-lished that inactivation of biological materials could occur by indirect action, biological radiation data were often analysed in terms of the target theory. A reaction was assumed to be of the single-hit target type if (i) it was independent of the dose rate, (ii) it gave an exponential inactivation dose curve, and (iii) showed the expected relative biological effectiveness (RBE) for different radiations. When these requirements were not followed, the results could usually be fitted to a multi-hit model. These criteria are not sufficient to establish direct action and would apply equally if the reaction were due to the indirect action of diffusible radicals when target theory calculations cannot be applied.

To apply the target theory in any form it is necessary, therefore, to prove that the inactivation or other change was produced directly and not by free radicals. The only way to be certain of this is to irradiate the biological material dry when no indirect action can occur, since, as already mentioned, the decisive dilution test cannot be applied to cells. In this way attempts have been made to determine sensitive volumes particularly for viruses and enzymes. This application will be discussed in detail in Chapter 7.

The existence of an oxygen effect and of energy transfer processes acting on the chemical lesion will introduce an additional measure of uncertainty in the calculations of target sizes. Yet in many instances these would still be useful even if the values obtained for target sizes were only very approximate. The really serious factor which has rendered the application of the target theory quite meaningless for cellular radiobiology is that post-irradiation conditions determine the magnitude of the lesion. Accordingly the size of the target would appear to be different if the experimental conditions are varied.

One of the principles in radiobiology that is becoming firmly established


is that the extent of the final biological lesion, the one measured in the usual dose-response curves, is determined by a complex interplay of metabolic processes which both develop the injury and initiate restoration. The initial chemical lesion merely triggers these processes off rather like a fuse in a bomb. The size of the explosion is determined by the amount of dynamite and not the nature of the fuse.

A few examples should suffice to illustrate the contention that the application of the "target theory" does not provide information about the primary chemical lesion in cells.

Mutations—In 1931 BLACKWOOD5 calculated the size of a gene from radiation data, and this approach was extended by Timofeef-Ressovsky in his monograph with Zimmer, Das Treffer Princip in der Biologie. Crowther's method of analysis was applied to a wide range of systems with much less discrimination than was indicated in the original publica-tion. Many of the deductions were severely criticized, notably by MULLER6 , but more recently the new experiments and the refined method of analysis of Catcheside and Lea (cf. ref. 4) brought renewed support for the target hypothesis.

Gene mutation was claimed by Lea to be the most clearly established example of a radiation effect in vivo produced by the single-hit target mechanism since the rather limited data available at the time indicated that the number of mutations produced in a population is (i) proportional to the dose (the first part of an exponential curve is a straight line); (ii) independent of dose rate; and (iii) decreases with increasing specific ionization of the radiation used. More detailed studies have shown that these simple relationships are not generally obeyed.

Mutations in mice and in female Drosophila are dose-rate dependent suggesting the existence of repair processes (Chapter 9). Unambiguous evidence for post-irradiation repair has been found in micro-organisms and in Paramecia where the mutation rate can be altered by a factor of a thou-sand by alterations in post-radiation procedures (see Chapter 14).

Oxygen enhances the mutagenicity (see Chapter 11) and cysteamine and other chemicals protect against it, suggesting the possibility that indirect action plays a part or that the primary chemical lesion is modified by the environment. Whichever of these mechanisms is responsible for the protection it would prevent the "target theory" from being more than semi-quantitative.

The observed fall in RBE with increasing LET, which is essential if a single-hit interpretation is to be maintained, is also not universal and a number of instances have been substantiated where the reverse applies (see p. 95). Even in these cases where the RBE is apparently correct for a single-hit target the lower efficiency of the densely ionizing particles may be more apparent than real. Thus, MULLER and VALENCIA7 were


able to show in Drosophila that the smaller effectiveness of neutrons, when compared with x-rays, is the result of a number of reactions on neigh-bouring genes which cannot readily be detected separately.

Yet another objection to the interpretation of Lea that the "target volume" calculated can be identified with the gene size is that the value obtained corresponds to a molecular weight between IO4 and IO5. This value is much too small according to Muller, since it implies that only 0-1 per cent of the total mass of the chromosome consists of genetic material, a conclusion which cannot be reconciled with other data. More-over, the "target size" of transforming principle determined in vitro (see p. 209) suggests that the key compound of the gene has a molecular weight of the order of a million.

Killing of cells*—LEA, HAINES and COULSON 8 interpreted the killing of bacteria as a lethal mutation produced by a single ionization within a sensitive voiume which they tentatively identified with chromosomal material. In their own experiments the survival curves were exponential, and the effect was independent of dose rates. While these workers found that the RBE decreased with increasing LET as required for a single-hit process the mean lethal dose for radiation of different ion densities did not lead to consistent values for the target size. This difficulty was over-come by postulating a multi-target model (see p. 81) of 250 targets (called genes) each of 120 A diameters.

Although Lea et al. obtained exponential curves for E. coli, HOLLAENDER and his colleagues9 have shown that all types of survival curves can be obtained, ranging from exponential to highly pronounced sigmoid curves depending entirely upon the condition of the experiment (see Fig. 4-3). To interpret the sigmoid curves in terms of the target theory on the basis that more than one hit is necessary for inactivation, is patently absurd since it would require that the number of targets can be changed at will by adjustment of the culture conditions.

Recently, the Russian workers SHEKHTMAN et al.10 have extended Hollaender's observations to a-particles from polonium, which they find also give sigmoid inactivation curves (see Fig. 4-4). Since hundreds of ionizations are produced when an a-particle passes through a cell, it should give an exponential curve, however many hits may be necessary. The only possible interpretation is that the shape of the killing curve is determined by external factors and that it cannot be interpreted along the lines of "target theory".

But most important, post-irradiation treatments determine the lethal

* Killing in this connection is defined as inability to form colonies (i.e. mitotic death, see Chapter 8) and not interphase death which requires that the cell, that is irradiated, dies.


dose, and many procedures are now known (see Chapter 14) for post-irradiation repair to the bacteria. The implication is that the target size of the cell depends on the composition of the medium in which it grows

FIG. 4-3. Survival curves of bacteria cultured under different conditions. These results fit the relationships required for the multi-target theory, but the number of targets (JV) which have to be inactivated to kill an organism apparently varies with culture conditions; a = aerobic broth culture exposed in oxygen; b = aerobic or anaerobic glucose broth culture ex-posed in oxygen; c = anaerobic glucose broth culture exposed

in nitrogen.

x-ray dose

FIG. 4-4. Dose curves for E. coli irradiated with Po a-rays. N/No is the survival (as percentage of control); (1) normal

culture; (2) culture with glucose added10.


after irradiation. The influence of post-irradiation metabolic processes on the magnitude of the lesion renders the application of "target theory" impossible.

Essential to Lea's theory is that the RBE must be highest for sparsely ionization radiations (i.e. of low LET), but his claim that this is the case for E. coli applies only in the presence of oxygen. In the absence of oxygen high LET radiations are more efficient and E. coli follow the pattern of all other cells (reviewed ref. 21)—a pattern incompatible with the Lea theory of cell death by one-hit dominant mutation. With micro-organisms other than E. coli the data is much less complete. Haploid yeast cells may give "single-hit" curves while those of higher ploidy (see p. 307) give sigmoid curves. Many bacteria give "single-hit" curves11 though some (see Fig. 4-5) show "multi-hit" curves under all conditions and this is particularly frequent with spore forming organisms.

D/LD5 0

FIG. 4-5. Per cent survival (logarithmic scale) of A. agile as a function of x-ray dose D divided by L D 5 0 1 1 .

However, even when the curve is exponential densely ionizing radiations have the higher RBE and wherever post-irradiation recovery processes have been looked for they have been found. Few studies have been made to test the influence of dose rate on cell death. The killing of E. coli seems to be independent over a wide range (cf. ref. 4), but spores of Bacillus megaterium are less sensitive at the lower dose rate (see Fig. 4-6), indicating that a recovery process is taking place even though the cells have a very low metabolic rate under these conditions15.


IO

IO-

IO2

o> C

IO"3

V)

S B D

Iff4

IO5

0 50 100 150 200 250 300

Dose, rxlO3

FlG. 4-6. Killing of Bacillus megaterius spores by x-rays at three different dose rates15.

With yeast34 exponential curves are for killing by x-rays only obtained when the cells have been starved before irradiation. Figure 4-7 shows that the radioresistance of yeast to x-rays increases on starvation while that to a-rays falls on starvation. Indeed, for unstarved cells a-rays are less effective than x-rays (RBE of 0-6) while after starvation a-rays are

0 1 2 3 4 5 6 7 8 9

Storvation time, days

FIG. 4-7. Effect of length of starvation in a medium containing 5 per cent dextrose—0 05 M phosphate on sensitivity of yeast

to x- and a-rays34.


more effective (RBE of 2). Most earlier work on yeast had been done on starved cells and a complex diffusion model35 was postulated to fit "target theory" principles to the data. For cells that have not been starved the target would accordingly be quite different. This is, of course, quite unacceptable on biological grounds and it is much more likely that the nutritional status determines post-irradiation recovery processes which prevent calculation of "target sizes".

Of particular interest are experiments on the irradiation of isolated mammalian cells in free culture like micro-organisms. In the hands of Puck they give a two-hit curve (see p. 250). Since they are very radio-sensitive (37 per cent dose with x-rays is 96 r) the target is in the neigh-bourhood of (0-25 fi,)3—a value something like 1000 times greater than that commonly accepted for a single gene. Yet closely related mammalian cells also grown in tissue culture give sigmoid curves requiring many more targets than two (target number is five for Fig. 14-3 on p. 374). Prob-ably small variations in culture conditions modify the shape of the dose-response curve in the same way as is seen with E. coli. Again the applica-tion of target considerations would lead to absurd assumptions about profound variations in the sensitive volume as a result of small changes in the experimental conditions. Since it is known that metabolic factors after irradiation determine the magnitude of the injury in tissue culture (see p. 375) as they do in bacteria, target theory is completely inapplicable.

Chromosome aberrations—The target theory in a modified form has also been applied to the production of chromosome aberrations. Inter-pretation here is exceptionally difficult, since many stages—none of them understood—intervene between the initial lesion and the observed change (see Chapter 9). At first sight it would appear as if all the evidence was against the target theory, since the dose-effect curve is only rarely exponen-tial and the number of many types of aberrations per given dose increases sharply with increase in dose rate. Finally, densely ionization radiations are more effective than x-rays.

LEA and CATCHESIDE (cf. ref. 4 ) maintained the basic principle of target theory of relating the end-effect (the chromosome aberration) directly with the primary physical process. Their hypothesis, that each break is caused by the passage of a single ionizing particle through the breakage point, could be fitted to the experimental data by postulating: (i) that the majority of the breaks can rejoin by restitution or exchange for several minutes after the break has occurred and are not therefore detected; (ii) that the number of ionizations which must be produced within a chromosome to give a break, is of the order of 15 to 20.

From the first assumption it follows that the number of aberrations, seen in the experiment, will increase with increasing dose rate. The second postulate provides that the RBE increases with LET. Thus the average


ion density along high energy electron tracks from y-rays or x-rays is too low to produce the number of ionizations necessary for a break within the cross-section of a chromosome and only the tail-end of an electron track will be effective. The probability of a break being produced by an ionizing particle passing through a chromosome is approximately one for protons (or neutrons) and for x-rays of normal energy, but less than one for radiations of low specific ionization.

In agreement with this theory 250 kV x-rays were found to be12 twice as effective as 60Co y-rays in producing chromosome aberrations in Tradescantia pollen. Using the mouse ascites tumour GRAY and CONGER 1 3

also found that the densely ionizing radiations from neutrons were about three times as effective as 190 kV x-rays in producing chromosome abnormalities at anaphase (see Fig. 4-8). The chromosomal changes are then classified into two groups, the "one-hit" aberrations which are due

. O 100 200 300 400 500 0 5 10 15 ZO X-ray dose (r) Neutron dose (rep)

FIG. 4-8. Survival of mouse ascites tumours after irradiation with x-rays and neutrons. The effect of removing oxygen is

much greater with x-rays than with neutrons13.

to breaks that have not been reconstituted and the "two-hit" aberrations which are due to two broken chromosomes joining up to give an unnatural configuration. The first group shows the normal "one-hit" dose-response relationship which in view of the small fractions of cells involved means a linear curve. The "two-hit" aberration have the character of multi-hit processes in that the frequency is a power function of the dose for sparsely ionizing radiations, but remains linear with dose for densely ionizing radiations.


This interpretation for the production of chromosome aberrations has been generally successful in representing much of the experimental data qualitatively, but there are quantitative discrepancies. For example, KOTVAL and GRAY14 found a-particles to be more effective than neutrons (i.e. ejected protons). On Catcheside and Lea's hypothesis the contrary result would be expected because the ion density along a proton track is already sufficient to produce a break (i.e. to produce 20 ionizations within a chromosome); a-radiation should be less effective since many more ionizations will be wasted. More serious objections are that the number of chromosome aberrations produced depends on the conditions at the time of irradiation (e.g. enhanced by oxygen, see p. 286) and reduced by chemical protectors (see p. 466) as well as on post-irradiation conditions (see p. 372). Above all, the breakage and reunion hypothesis, which provides the basis for the additional assumptions that are made to fit the target theory to chromosome damage, has no biochemical support. This aspect is discussed more fully on p. 372.

T H E R E L A T I V E B I O L O G I C A L E F F E C T I V E N E S S O F D I F F E R E N T I O N I Z I N G R A D I A T I O N S

The data reviewed in the preceding section dealing with the target theory has shown that in living systems the complexities introduced by post-irradiation metabolic processes makes it impossible to derive quantitative information concerning the nature of the initial chemical lesion from the curve relating the biological end-effect to the radiation dose received. Direct analysis of cells immediately after irradiation also cannot provide the information since this will not reveal which of the radiochemical reactions is trivial and which important. One of the most important clues is the relative effectiveness of different radiations since it seems reasonable to postulate that if for example a-rays are more efficient than X-rays in preventing the proliferation of cells then the initial radiochemical reaction responsible must also be brought about more effectively by a-rays than by x-rays.

Although the physical nature of the different ionizing radiations varies considerably their relative biological effectiveness depends solely on their ionization density or LET (for definition see p. 29). For example, deuterons with an energy of 190 MeV have the same average LET as 200 keV X-rays and are equally effective in killing yeast cells, while less energetic deuterons having a higher LET are more efficient16. Similarly, a-rays produced in cyclotron at 400 MeV have the same LET as x-rays and produce the same biological damage. With modern machines such as cyclotrons it is possible to cover a wide range of LETs with the same type of radiation by altering the energy. For the majority of radiobiological


work such facilities have not been available and LETs have been compared by using:

(a) Therapy x-rays in the range of 100 to 500 kV for which the average LET* in tissue is 2-5 keV//x; for soft x-rays of 25 kV the LET is about 6 keV//a.

(b) Fast neutrons of 1 to 10 MeV from a van de Graalf with an LET of 20 keV/jM.

(c) a-Rays from radon or polonium with an LET of 50 keVjju. (d) Very energetic x-rays from equipment running at more than

400 kV have an average LET of about 0-5 keV//x and it is not possible to reduce this value further (see p. 30).

In this book x-rays, fast neutrons or a-rays will be referred to without specifying their energy if they fall into the above categories (a), (b), (c) and (d) respectively.

The relative biological effectiveness (RBE) is defined as follows:

Biological efficiency of radiation under investigation RBE = : : ;

Biological effectiveness of therapy x-rays

Dose in rads to produce effect with therapy x-rays

Dose in rads to produce effect with radiation under investigation

The RBE of a particular radiation depends not only on the effect being studied, but also on (1) the dose, (2) the dose rate, (3) the presence or absence of oxygen (see Fig. 4-8), (4) the post irradiation conditions (see Fig. 4-7). Consequently, it is not possible to draw up a list of RBE for different effects and the following tables and graphs present a bird's-eye view over a large and complex set of experimental data (for a recent review see ref. 17).

Mammalian Effects A large number of papers have been published in this field, but special

reference must be made to a most detailed investigation at Los Alamos in which great attention was paid to dosimetry18. The data shown in Table 4-1 is taken from this paper.

For delayed effects the RBE varies. Shorteningof lifespan19 has within experimental error the same RBE as 30-day mortality. While 100 rads of

* Exact figures cannot be given as the value depends on the assumptions made in the calculations (see p. 34). For particulate radiations the proportion of energy expended as 8-rays is in some doubt (see p. 32) and this introduces further uncertainties.


T A B L E 4 - 1

R B E FOB A C U T E M A M M A L I A N EFFECTS (Taken from ref. 18)

Radiation Approx.

L E T (keV/fi)

Species Test system RBE

4-MeV y-rays 0-3 Mouse 30-day lethality Mouse Splenic atrophy Mouse Thymic atrophy Rat Depression of 59Fe uptake

0-7 0-6 0-8 0-6

Tri t ium j3-particles (0-6 keV)

5-5 Mouse Splenic atrophy Mouse Thymic atrophy Rat Depression of 59Fe uptake

1-3 1-5 1-6

7-MeV protons (14-MeV neutrons)

10 Mouse Splenic atrophy Mouse Thymic atrophy Rat Depression of 59Fe uptake

1-5 1-7 0-8

Recoil protons from fission neutrons

45 Mouse 30-day lethality Mouse Splenic atrophy Mouse Thymic atrophy Rat Depression of 59Fe uptake

2-3 1-8 2-0 1 0

0-6-MeV protons from 14N («, p)uC reaction

65 Mouse Testicular atrophy Mouse Median survival time Mouse 30-day lethality Mouse Incidence of leukaemic

"takes" Mouse Splenic atrophy Mouse Thymic atrophy Mouse Depression of mitosis Rat 30-day lethality Rat Body weight loss Rat Intestinal atrophy Rat Depression of 59Fe uptake

4-9 2-7 2-4 1-6-2 1 1-6 1-6 1-3 2-3 1-2 1-2 1 0

a-Particles and 7Li recoils from 10B(w, a)7Li reaction

190 Mouse 30-day lethality Mouse Testicular atrophy

1-3 3-5

Heavy recoils from 14-MeV neutrons

850 Mouse Splenic atrophy Mouse Thymic atrophy

1 -2 1-2

Fission fragments from 239Pu reaction with thermal neutrons

4000-9000

Mouse Splenic atrophy 0-7-0-9


X-rays shortened life of mice by 2-6 per cent the value for fast neutrons is 6-7 per cent per 100 rads (i.e. RBE = 2-6).

For cataract formation the RBE of densely ionizing radiations is very high and depends on dose rate. With mice values ranging from 5 to 9 for fast neutrons have been reported20. For induction of cancer there is no detailed data, but the RBE is believed to be high and a value of 10 for a-rays is frequently chosen in relation to safety. The lack of experimental data is a reflection of the difficulties encountered in quantitative radiation carcinogenesis.

Killing of Cells In isolated cells the RBE shows the same pattern as in whole animals;

i.e. it is slightly less than 1 for hard x-rays, rises to a maximum for an LET of a-particles and falls again as the ionizing density increases still further. The magnitude of the effect varies from cell to cell. Figure 4-9,

FIG. 4-9. Radiosensitivity (expressed as reciprocal of mean lethal dose) of Vicia faba for radiations of different L E T (after

ref. 21).

taken from a review by HOWARD-FLANDERS21, shows a typical set of results. For bacterial spores much higher LETs have been obtained by using ions of carbon and oxygen and the RBE maximum is clearly apparent (see Fig. 4-10).


Few RBE values have been recorded for isolated mammalian cells; the data shown in Fig. 4-8 gives an RBE for neutrons acting on ascites cells in vitro of 8 when oxygen is absent. In the presence of oxygen the RBE of ascites cells falls to 3 and the same influence of oxygen is seen in all other systems where it has been looked for. Oxygen enhances the radi-

— : : —

•

I _L

-Jl i ± = n =

iI -

TT' _ V

?r —ii.

—

i

L.E.T. of track core, k

FIG. 4-10. Radiosensitivity (expressed as reciprocal of 37 per cent dose) of Bacillus subtilis spores irradiated dry and in the absence of oxygen by radiations varying widely in L E T (after

ref. 21).

ation effect of sparsely ionizing radiations much more than that of densely ionizing radiations (see Chapter 11) and consequently the RBE of densely ionizing radiations is reduced by oxygen.

For E. coli the RBE is reversed in the presence of oxygen so that it decreases with increasing LET, but a reversal is very rare and only seen when the RBE is relatively low. Unusually high RBEs ranging up to 60 have been claimed for barley seeds22, but this is exceptional and in general most RBE values for cell killing effects tend to a maximum of between 10 to 20 at an LET in the range of 20-100 keV/u.

Chromosome Abnormalities The value of the RBE depends critically on the kind of abnormality

scored. The fact that chromosome rearrangements showed a higher RBE for neutrons than did chromosome breaks was one of the most powerful arguments for the breakage and reunion hypothesis (see p. 88). The RBE also depends on the presence of oxygen and on the dose rate. For Tradescantia microspores the data have recently been summarized by C O N G E R et al.23 and Fig. 4 - 1 1 is taken from this paper. The values shown


are for irradiations in the presence of air and the RBEs will be higher when the comparison is made under anoxic conditions.

Mutations The RBE depends not only on the type of mutation that is scored, but

also on the stage in the cycle of the germ cells. For dominant lethality* in Drosophila, which probably is a genie phenomenon, the RBE in air of fast neutrons ranges from 6-6 for mature sperm, 3-2 for spermatids to 1-6 for premeiotic cells24. On the other hand for recessive sex-linked lethals,

FIG. 4-11. Variat ioninRBE (60Co y-rays = 1) for chromosomal aberrations in Tradescantia23. Top curve: aberrations seen after

4 days; Bottom curve: aberrations seen after 1 day.

which is a true mutation, the RBE of neutrons is probably less than one (the available data is summarized in ref. 17), although it is possible that when the comparison is made under anoxic conditions the RBE will exceed one. A visible mutation of the eye colour of the wasp Mormoniella has shown an RBE of 2125>26 and this effect may be related to the phenom-enon7 already referred to on p. 84, where neutrons produce several mutations close together.

* It is doubtful if this phenomenon should be classed as a mutation. Since its manifestation is death of the zygote, it should be considered under the heading of cell death in which genetic as well as physiological factors play a part.


Enzymes and Viruses For the inactivation of viruses and enzymes in vitro the RBE continually

falls with an increase in LET. All the available data (cf. ref. 3) is qualita-tively in agreement with the view that one primary ionization is sufficient for inactivation. As the LET increases so does the chance that more than one primary ionization occurs as the ionizing particle traverses the enzyme or virus. Ionizations in excess of one that occur within the "target volume" are wasted and result in a lowering of RBE. The relationship between RBE and LET depends on the size of the target. Since the chance of wasted ionizations occurring increases as the target size increases, the RBE, for example fast neutrons, for large viruses will be smaller than the RBE of an enzyme or small virus. The difficulties of using the target theory for determining the size and shape of biologically active macro-molecules will be discussed in Chapter 7, but they do not affect the general observations that RBE falls with increasing LET for all in vitro systems.

T H E P O I S O N T H E O R Y The possibility that the action of radiation is due to the formation of a stable poisonous material is generally dismissed and only very few experi-ments have been carried out to test the point. Certain nutrient media for bacteria cultures are rendered toxic by irradiation27, though only with doses considerably greater than those required to kill the bacteria. The viability28 and rate of respiration29 of sea-urchin sperms is reduced when sea water is heavily irradiated, but if the organisms were present during the irradiation the effect was much more marked. The possibility remains that the poison must be produced in situ, but a sigmoid survival curve typical of cumulative action would then be expected since no killing would occur in the early stages of irradiation when the concentration of the poison is low; if the resistance of the organism is fairly uniform a sharp drop in survival should occur when the cells have been exposed for a time sufficient to produce a lethal quantity of poison. Exponential curves, frequently observed with ionizing radiation, could only be inter-preted on the poison hypothesis by assuming a most unusual distribution of resistance*.

This difficulty of the dose response curve does not apply for the produc-tion of radiation resistance and death in mammals for which there is a pronounced and well-defined threshold. Quantitatively the poison theory is not impossible. Thus if the poison has a molecular weight of 1000 and

* This argument, which has frequently been advanced (cf. LEA4), is not com-pelling, since the killing of bacteria by poisons such as phenol often closely resembles an exponential curve and yet here there can be no question of inactiva-tion of a single site.


is produced with an ionic yield of one, a lethal whole body irradiation of 500 r would produce about 50 xng of poison in a man. This would mean that the substance produced would have to be as toxic as strychnine, but much less so than many toxins. HOGAN and PHILLPOT30 found that organic peroxides of the type that can be detected after whole body irradi-ation are extremely toxic and the amount needed to kill is of the same order as that produced by a lethal dose of x-rays. Also injection of an enzyme, lipoxidase, which catalyses peroxide formation, produces a radiation-like death in rats33. Difficulties with this poison hypothesis are that the symptoms of death from peroxide are different from those of radiation and that the RBE of different radiations cannot be explained as the yield of peroxide decreases with an increase in LET.

Yet, in addition to acute lethality, peroxides also mimic radiations in being mutagenic (see Chapter 8). It would seem to be a remarkable coincidence that radiation produces in cells substances which are capable by themselves of producing radiation-like lesions. Possibly the formation of toxic peroxides is responsible for a part at least of the enhancement of radiation damage by oxygen. The observation that large doses of radiation to the cytoplasm of habrobracon eggs31 and of Drosophila eggs32 do not lead to the induction of mutations, would suggest that peroxides produced in the cytoplasm cannot reach the DNA of the nucleus.

C O N C L U S I O N S In the search for primary lesion we are faced by a remarkable hiatus. While it seems certain that a radiochemical reaction initiates the chain of events that leads to the biological injury, no in vitro reaction has been found for which the yield per rad increases with increasing LET. Yet, with the possible exception of mutagenesis, the most characteristic features of biological radiation damage of every type is that as the LET increases so does the RBE until extremely high ionization densities are reached when the RBE again goes down. The inference is that radiation damage is not initiated by the inactivation of an enzyme or of a nucleic acid (e.g. as in a virus or in transforming principle). A reaction which can only be accomp-lished by densely ionizing radiations is the damaging of a sub-microscopic structure which resists the injury sustained from an isolated ionization but breaks down under the impact of several ionizations that occur simultaneously within a small volume. The authors have proposed in Chapter 10 that the structures involved are intracellular barriers, the breakdown of which leads to the release of enzymes. The fact that oxygen enhances the biological damage of radiations with low LET could arise from the fact that an isolated ionization initiates an autoxidation chain, thereby breaking these barriers which are made up in part of phospho-lipids in the same way as do several ionizations close together.


The concept of the breakdown of a structure stems from the idea of LEA4 that twenty or thirty ionizations acting in conjunction sever a chromosome. This hypothesis is discussed in Chapter 10, but even if applicable to the formation of chromosome abnormalities, it could not explain the high RBE of densely ionizing radiations for lesions such as interphase death where chromosome damage would appear to have no part.

R E F E R E N C E S

1 . KREBS, H . A . Ciba Foundation Symposium on Ionizing Radiations and Cell Metabolism, p. 92, Churchill, London, 1956

2 . CROWTHER, J . A . , Proc. Roy. Soc. B , 1 9 2 4 , 9 6 , 2 0 7 3. POLLARD, E. C., BUZZELL, A., JEFFREYS, C. and FORRO, F . , Arch. Biochem.

Biophys., 1951, 33, 9 4. LEA, D . E., Actions of Radiations on Living Cells, Cambridge Univ. Press,

1946 5 . BLACKWOOD, O . , 1 9 3 0 ; Phys. Rev., 1 9 3 1 , 3 7 , 1 6 9 8 6. MULLER, H . J., Symposium on Radiobiology, p. 328, John Wiley, New York,

1 9 5 2 7. MULLER, H . J . a n d VALENCIA, T . , Genetics, 1951, 20 , 115 8 . LEA, D. E . , HAINES, R . B . and COULSON, C . A . , Proc. Roy. Soc. B , 1 9 3 7 ,

1 2 3 , 1 9 . HOLLAENDER, A . E . , STAPLETON, G . E . and M A R T I N , F . L . , Nature, 1 9 5 7 ,

1 6 7 , 1 0 3 1 0 . SHEKHTMAN, I . L . , PLOKHOI , V . I . and FILIPPOVA, G . V . , Biophysics (trans-

lation), 1958, 3, 458 1 1 . G U N T E R , S. E. and K O H N , H . I . , J. Bacteriol., 1 9 5 6 , 7 1 , 5 7 6 12. K I R K B Y - S M I T H , J . S . and DANIELS, D . S . , Genetics, 1952, 3 7 , 596 1 3 . GRAY, L . H . , CONGER, A . D . , EBERT, M . , HORNSEY, S . a n d SCOTT, O . C . A . ,

Brit. J. Radiol., 1953, 26, 638 1 4 . KOTVAL, J . P . and GRAY, L . YL., J. Genet., 1 9 4 7 , 4 8 , 1 3 5 1 5 . POWERS, E . L . , W E B B , R . B . and EHRET, C . F . , Progress in Nuclear Energy,

Series VI , Biological Sciences, Vol. 2, p. 198, Pergamon Press, London and New York, 1959

16. TOBIAS, C. A., Symposium on Radiobiology, p. 357, John Wiley, New York, 1 9 5 2

1 7 . POWERS, E . L . , W E B B , R . B . and EHRET, C . F . , Progress in Nuclear Energy, Series VI, Biological Sciences, Vol. 2, p. 278, Pergamon Press, London and New York, 1959

18 . STORER, J . B . et al., Radiation Research, 1 9 5 7 , 6 , 1 8 8 - 2 8 8 1 9 . STORER, J . B . et al., ibid., 1 9 5 8 , 8 , 7 1 20. RILEY, E . F. , EVANS, T . C., RHODY, R . B. and LEINFELDER, R . J. , Radiology,

156, 6 7 , 673 2 1 . HOWARD-FLANDERS, P . , Advances in Biological and Medical Physics, 1 9 5 6 ,

7 , 5 5 3 2 2 . MACKEY, H . , Hereditas, Lund., 1 9 5 1 , 3 7 , 4 2 1 ; Arch. J. Bot., Ser. 2 , 1 9 5 2 , 1 ,

545 2 3 . CONGER, A . D . , RANDOLPH, M . C . , SHEPPARD, C . W . a n d L U I P P O L D , H . J . ,

Radiation Research, 1958, 9, 525 2 4 . ALEXANDER, M . L . , Genetics, 1 9 5 8 , 4 3 , 4 5 8

I N I T I A L C H E M I C A L L E S I O N I N C E L L U L A R R A D I O B I O L O G Y 99

2 5 . CASPARIS, S . B . , Radiation Research, 1 9 5 8 , 8 , 2 7 3 26. K A L J H A R T , A . , Radiation Research, 1956, 4 , 65 2 7 . PUGSLEY, A . T . , O D D I E , T . H . and E D D Y , C . E . , Proc. Roy. Soc. B , 1 9 3 3 ,

1 1 8 , 2 7 6 28. SLAUGHTER, J . C. and F A I L L A , G., Radiology, 1942, 3 9 , 663 2 9 . BARRON, E . G . , F L O O D , V. and GASVODA, B . , Biol. Bull., Wood's Hole, 1 9 4 9 ,

97, 51 3 0 . H O R G A N , V. J . , P H I L P O T , J . S T . L . , PORTER, B . W., R O O D Y N , D . B . , Biochem.

J. 1957,67, 551. 31. ROGERS, R . W. and BORSTEL, R . C. VON, Radiation Research, 1957, 7, 484 3 2 . U L R I C H , H . , Naturwiss., 1 9 5 5 , 4 2 , 4 6 8 33. M U S E T , M . , ESTERE, E . and M A T E U , Nature, 1959, 1 8 4 , 1506. 3 4 . E L K I N D , M . M . and B E A M , C . A . , Radiation Research, 1 9 5 5 , 3 , 8 8 35. ZIRKLE, R. E. and TOBIAS, C. A., Arch. Biochem. and Biophys., 1953, 47,

282

8

C H A P T E R 5

General Radiation Chemistry

IN Chapter 1 the principles governing the deposition of energy by ionizing radiations were discussed. The radiation chemistry of water and of substances dissolved in water as well as that of macromolecules is of special interest to radiobiology and these two aspects will be described in detail in Chapters 6 and 7.

In this chapter we are concerned with the basic processes by which a part of this energy is converted into chemical change. Most of the energy eventually appears as heat and in this form is quite harmless since the dose of radiation necessary to kill an animal would only raise its tempera-ture by about one thousandth of a degree. The proportion of the energy that does not appear as heat* varies with the material irradiated; e.g. for graphite and metals it is negligibly small so that the heat developed on irradiation is direct measure of dose. In organic systems and in water-containing solutes something of the order of 25 per cent appears as chemical change, but this figure will vary quite widely from system to system and no direct experimental determination of this quantity has yet been attempted.

Thus some 4 eV (or 4 x 23 kcal per mole) are required to break a strong organic bond and an input of 100 eV of energy is capable of break-ing some 25 bonds if the energy was entirely utilized for this purpose, though the actual value observed is almost invariably in the range of 2 to 10 with 5 a common value (i.e. 5/25 is converted into chemical change).

In systems containing more than one component the radiation effect will often be greater than expected due to an energy transfer process (see p. 113) and due to "indirect action". The latter requires that energy originally taken up in a solvent results in chemical changes in solute molecules. The case of greatest importance for radiobiology is that involving water which is decomposed into reactive radicals (some 30 per cent of the energy being used this way) which attack dissolved substances.

* In some systems a small fraction of the deposited energy is given off as light (in the ultra-violet or visible range), but this fluorescence process (and Cerenkov radiation, see p. 19) is of no biological importance.

100

G E N E R A L R A D I A T I O N C H E M I S T R Y 1 0 1

Definition of Radiation-chemical Yield The amount of reaction per radiation dose is conventionally expressed

in one of two ways. Firstly, as the number of molecules changed or produced (M) per number of ion pairs formed (N); secondly, as the number of molecules changed or produced for each 100 eV of energy absorbed and this is referred to as the G value. There is probably little to choose between these two ratios in the case of gases where the energy required to form an ion pair (W) has been experimentally determined; i.e. when W = 34 eV/ then G = 3 (M/N). For reactions in liquids and solids the G nomenclature is to be preferred since only the total energy absorbed can be measured. However, when M/N values are quoted for reaction in liquids and solids it is always assumed that W = 34 eV, so that all the data can be converted to G values by multiplying by three. It is useful to distinguish between the measurement in which the disap-pearance of a molecule is measured. This is the case in almost all experi-ments with biologically active materials where loss of activity is followed and the nature of the changed molecule is not investigated. Here Gfmoiecuies altered) is determined.

In many chemical experiments the products that are formed are measured, for example the amount of hydrogen produced when ethanol is irradiated. Here we have G(h2) and this bears no immediate relationship to G (ethanol changed) since the ethanol may undergo a number of reac-tions only one of which leads to the release of hydrogen. When low G values are quoted this does not necessarily mean the material is radiation resistant, but only that the particular product which is being studied is only rarely produced and that other reactions predominate.

R O L E O F E X C I T A T I O N The process of energy loss by an ionizing particle involves initially (i.e. within the first IO-14 sec after passing through the material being studied) ionizations and electronic excitations (see p. 39). Both of these processes can form the starting point for chemical changes and one of the unresolved problems is to determine their relative contributions in the type of reaction which is of biological interest.

Only in gases is it possible to divide the total energy deposited into these two processes as both W and the ionization potential are known (see p. 27). For organic molecules and for water the division is in the neigh-bourhood of 50/50, but while there can be no doubt that the ionizations initiate chemical reactions, the same cannot be said for the excitations.

The foundations for gaseous reactions were laid by the pioneers of atomic science: Mme Curie, Lord Rutherford, J. J. Thomson, W. Ramsay and F. Soddy. The whole of the earlier work is reviewed in a monograph


by L I N D 1 , who has himself made some of the outstanding contributions in this field; this book, though published 30 years ago, should be con-sulted by all interested in radiation chemistry.

Lind introduced the M/N nomenclature for expressing the yield of a radiochemical reaction. In all the gaseous reactions, such as oxidations, hydrogenations, decompositions, studied, M/N is only rarely less than 2. Often values ranging from 2 to 6, and occasionally even higher, have been reported. A value of 2 can be explained if it is assumed that both positive and negative ions are equally reactive, but this explanation cannot apply to gases with low electron affinities when a negative ion is not formed; or when M/N > 2. In a few cases high M/N values are due to chain reactions, but detailed kinetic studies by L I N D 1 have ruled this possibility out in the majority of cases.

L I N D 2 interpreted the results by postulating the formation of clusters round the positive ion by polarization forces. The positive ion was then neutralized by the electron (or an oppositely charged ion) and the large amount of energy released shared by all the molecules in the cluster. The decomposition of a saturated hydrocarbon3 was written as follows:

With advances in photochemistry the view that excitations played a part in radiation chemistry gained ground and the suggestion by EYRING, HIRSCHFELDER and TAYLOR 4 was widely accepted that the high yields in gaseous reactions are due to the fact that excited as well as ionized mole-cules can take a part in the chemical reaction. The key experiment that gave rise to this view was the irradiation of pure hydrogen with a-particles when 6 hydrogen atoms were formed per ion pair. EYRING et al4 put forward the following reaction sequence:

a - r a y CH4 • CH4++ e

CH4++ CH4 > (CH4)2+

(CH4)2++ e > (CH4)2* (highly activated)

(CH4)2* > H2+ C2H6

a-ray / H 2 - » H 2 + + e \ H 2 H2* (excited) 2H'

I II

H2+ + H2 H3+ + H* —Ill

H3+ + e -J- H" + H2 — IV

H2* 2H' — V


(i.e. M(H')/N = 6 (2 due to ionization + 4 due to excitation). Reaction III is an example of charge transfer accompanied by dissociation (see p. 106). ESSEX and his coworkers have found many examples where exci-tations contribute to radiochemical changes in gases'40).

On the other hand L I N D 5 has produced impressive new evidence for the view that excitations play no part in the polymerization of acetylene, which has an M/N = 20, on the basis of the promoting effect by inert gases. L I N D and BARDWELL3 found that the presence of inert gases "catalysed" a-ray-induced reactions. Both in polymerization reactions (e.g. acetylene, hydrocyanic acid and C 2 N 2 ) and decompositions (e.g. water, carbon dioxide and ammonia) the number of molecules changed was not altered by the presence of non-reactive gases, such as nitrogen, or one of the inert gases, even though the majority of the ionization occurred in the inert gas. Lind did not attribute this "catalytic" effect to a transfer of electrons from the inert gas to the reactive gas, but postulated the formation of mixed clusters because inert gases such as xenon, which have a lower ionization potential than acetylene, promote the reaction as well as gases having higher ionization potentials. Only the latter can trans-fer charge to acetylene molecules. According to EYRING 6 and STEACIE7

steric considerations, however, exclude the formation of clusters of twenty molecules and the free radical ions (both C2H2+ and Xe+) initiate poly-merization by a chain mechanism, which is well known to occur in other systems. Also the observation that benzene is produced as well as the polymer8 would seem to be more easily explained by the polymerization mechanism9. In other cases where charge transfer is possible this process is no longer in conflict with the cluster idea since modern theory demands that the partners in a charge transfer process form relatively long-lived complexes (see p. 106) and these are identical with the clusters of Lind.

In condensed systems even less is known since any estimates of the number of ions produced (and therefore for the proportion of energy expanded in ionization) is based on the assumption that W (the energy per ion pair) is the same as in a gas. While this assumption is unlikely to be vastly wrong it is very likely that there are differences of the order of 30 per cent.

That some chemical reactions must follow excitations is certain from the studies of photochemistry, but the contribution to the total chemical change in most organic systems may well be small in condensed systems. A high proportion of the excited molecules may revert back to the ground state without undergoing a chemical change while an ionization will almost invariably cause a reaction of some sort to occur in an organic molecule.

In the important case of water all the observed reactions have been satisfactorily explained on the basis of ionizations only and there has been


no need to invoke reactions from those molecules in which energy was deposited in the form of electronic excitations.

Even if there are chemical changes that are due to the initial electronic excitations it seems reasonable to conclude that they are not of importance to radiobiology. Ultra-violet light produces excited molecules of the same kind as those formed by ionizing radiations but on an energy basis is many thousands of times less effective in bringing about radiobiological effects. Thus for the inactivation of most enzymes by sparsely ionizing radiations G is approximately 1* while for ultra-violet light of the optimum wavelength G < 0-1. For the production of cellular effects the difference is even more marked. If the initial chemical lesion was initiated by an excitation this great difference in effectiveness could not be explained. It seems reasonable to attribute the ability of ionizing radiations to produce biological damage at such very low doses to the reaction characteristic to them, namely to produce ionizations.

The ions, which are formed on irradiation, should strictly be called free radical ions since they contain an unpaired electron in the outer shell; this distinguishes them from the stable ions produced by the dissociation of salts. The presence of an unpaired or odd electron makes free radicals, and the ions considered here, so highly reactive. A primary chemical bond can be represented as the sharing of two electrons between the constituent atoms and when two radicals collide such a bond is formed. This is the reason why simple free radicals have, in general, only a very short lifetime. Radicals obtained from complex organic molecules are often relatively long-lived because resonance stabilizes them and reduces the reactivity of the odd electron, which is distributed over the whole molecule. Radicals also have a great tendency either to lose or to gain another electron so as to have an even number when they finish as stable ions, e.g. the OH radical on capturing an electron becomes an OH~ ion, which is one of the most stable entities known. Uncharged free radicals are written with a dot to designate an unpaired electron (e.g. OH').

Negative Ion Formation The electron, ejected from an atom in a molecule which becomes a

positive ion, produces ionizations (see p. 32) until its energy is reduced to a few eV when it must either be captured by another molecule to give a negative ion or be recaptured by the positive ion. In the former reaction no great change in energy is involved, and the combined process is known as the formation of an ion pair. Charge neutralization, on the other hand, releases a large amount of energy.

* An inactivation requires one primary ionization which is associated with about 100 eV (see p. 209).


The relative electron affinities of a few molecules are known10 and if the electron does not recombine with a radical, this property determines with which atom it will combine. Molecular oxygen, and oxygen containing molecules such as water, have an extremely high electron affinity and in biological systems combination with any other atom is unlikely. The halogens have a high affinity; organic molecules, such as straight-chain paraffin, have a small affinity, while hydrogen and nitrogen can only combine with energetic electrons and will not compete for "sub-ionization" electrons.

The process of electron capture may be accompanied by dissociation or the negative molecule may be stable, e.g.

AB + e -> AB- (or A~B) or

AB + e A + B~ Both the bond strength and the environment determine which reactions occur, e.g. the H 2O - ion is stable in the gaseous phase, but dissociates in liquid water (see p. 126).

The subsequent behaviour of the negative ions is probably governed by the same general considerations as those of the directly produced positive ions, whose behaviour will now be described in general terms.

Charge Neutralization The positive ion may capture an electron because no atom is present

with an affinity for an electron, but this cannot arise in biological systems. According to a theory of Magee and Burton (for fuller discussion see p. 127) charge neutralization might occur because the electron is not able to escape from the positive ion which draws it back on to itself. This would mean that in a condensed phase negative ions are never formed and that we do not deal with an ion pair but only with an exceptionally energetic species.

There are, however, other theories according to which the secondary electron will in general escape and form a negative ion and at the present time this must be considered an open question to which reference will again be made in connection with the radiation chemistry of water on p. 128.

If charge neutralization occurs this is almost bound to lead to immediate dissociation; the probable products11 being two radicals, one of which is excited (excited products will be designated with a star), i.e.

A+ + e C" +*D" A positively and negatively charged (free radical) ion can only combine


if a third molecule is present at the collision to take up part of the laige amount of energy which is released12, otherwise immediate dissociation occurs. The presence of a "third body" presents no difficulty in a con-densed system, and a likely reaction is that the ions dissociate into two free radicals and activate the third molecule (M), i.e.

( M ) A + + B - > A ' + B ' + M *

In general M* will be less reactive than the free radicals produced.

Charge Transfer If the ionization potential of B is lower than that of A charge transfer

will occur:

A + + B ^ A + B +

Theoretical considerations led MAGEE13 to the important conclusion that during a charge transfer process the two molecules involved stay together for as much as a second; such periods are long when considered in terms of chemical reactions. In this intermediate stage other molecules may become temporarily associated with the molecules undergoing charge transfer and thereby render possible more complex reactions. Modern quantum theory leads, therefore, to a return of one of the earliest theories for radiochemical reactions, which was proposed by LIND 2 in 1 9 1 9 (see p. 1 0 3 ) .

Dissociation of Ions The free radical ion may be unstable and dissociate spontaneously in a

number of different ways. Much information has been obtained for gaseous hydrocarbons from the mass-spectrograph14 and predictions con-cerning the nature of the decomposition product can be made in simple cases, though not as yet for the more complicated systems likely to be encountered in radiobiology. In gases the following reactions occur and are illustrated for the case of a straight chain hydrocarbon: e.g.

C 4 H - I 0 C 4 H - I 0 + + e

(1) C4H+I0 -> CH *3 + C3H7+. This is a dissociation into a free radical and a carbonium ion, which is not a free radical, but nevertheless unstable. It often rearranges to give off hydrogen and form an ion, which is stabilized by a double bond:

C3H7+ -^C3H5+ + H2


(2) C4H+Io —> CH4 + CsHg+, i.e. formation of a stable molecule and an unsaturated free radical ion.

With small molecules a complete break-up of the following type some-times occurs15:

CH4 CH4+ + e; CH4+ C+ + 2H2. In liquids positive ions probably undergo rather similar changes, but there is no way in which these can be determined experimentally. The nature of the early events can only be deduced in condensed systems from the nature of the final products.

Reactions of Ions with Neutral Molecules In general, the chemical changes produced by ionizing radiations are

ascribed to the free radicals formed in one of the reactions discussed above. Though in recent years evidence has been found for the direct reaction of an ion with a neutral molecule and it is possible that such reactions may be quite important in organic systems. Thus the following processes have been shown to occur:

Br2+ + H2 -> HBr + HBr+

C O + + C O C + + CO2 <">

CH4++ CH4 -> C i r 3 + CH5 <">

CH4++CH4-> C2H6+ H2+ (18)

This last reaction is of particular interest since it provides a possible mechanism for the crosslinking of polymers which is one of the most characteristic reactions of ionizing radiations with macromolecules.

D I F F E R E N C E B E T W E E N T H E R E A C T I O N S I N G A S E S A N D T H O S E I N L I Q U I D S A N D S O L I D S

Almost all experiments capable of revealing the mechanism of reaction of ionized molecules have been carried out in the gas phase and there are no techniques by which the fate of these active species can be followed directly in a condensed phase. Undoubtedly the same primary reactions occur under all conditions, but the secondary processes will be influenced so that the final products in the condensed phase are often qualitatively and quantitatively different from those obtained in the gases.

(i) Deactivation of electronically excited molecules will be enhanced by the more frequent collisions which can occur in condensed systems. For example, energy transferred by an internal conversion process into vibrational or rotational energy can be dissipated by collisions.

(ii) Recombination of ions and radicals can, in general, only occur if a


third molecule is present to take up the excess energy. Such processes are much more frequent in liquids than in gases and can reverse a radiation-induced process so that no net chemical change is observed.

(iii) In polar liquids, particularly water, the ions and free radicals formed will be solvated, and consequently have entirely different properties from those in the gas phase. For example, the energy of combination of a hydrogen atom and a hydroxyl radical releases 350 kcal per mole, while in water, owing to hydration, only 14 kcal are given out. Similarly, the H2O+ ion, though relatively stable in the gas phase, may (or must, see Chapter 6) decompose in water as a result of hydration.

(iv) The surrounding molecules in a liquid or solid, besides tapping-off energy as considered under (i) and (ii) also greatly reduce the opportunity for fragments of a decomposing molecule to escape from one another's influence by the so-called Franck-Rabinowitch cage effect19. The solvent molecules "bounce" the products back and thereby bring about recombin-ation of radicals; the larger these are the smaller is the probability of escape. In the "cage" some of the radicals will recombine with loss of vibrational energy, and this process may be repeated until all the energy is degraded into heat*. The net end-reaction observed will be mainly due to the release of hydrogen atoms and possibly small radicals, such as methyl, which can escape from the cage with a high probability. Densely ionizing particles will, to some extent, "break down" the cage, since active molecules will be produced in high local concentration; e.g. along an a-track in a liquid the average distance between ionized molecules is of the order of ten molecular diameters. If one considers in addition excited molecules the distance between molecules undergoing change is not great and this may prevent the cage from being fully effective for densely ionizing particles.

All these factors combine to limit both the yield and the number of different products formed when liquids or solids, particularly of large molecules, are irradiated. BURTON20 pointed out that the most probable reaction is a so-called "slow" rearrangement leading to the formation of two stable products, which are not influenced by the cage. Few detailed studies have been carried out to test this idea, but the decomposition of

* Dur ing an internal conversion process most of the energy is initially situated in one bond and this may explain why it is not always the weakest bond which breaks. T h e energy to break a C—C bond is 15 kcal per mole less than that to break a C — H bond, yet hydrogen is often produced in largest yield on irradiation of an organic material. T h e cage effect cannot be wholly responsible and the reaction probably results f rom the initial localization of energy in the C — H bond. In a liquid, where collisions are constantly occurring, the chance of sufficient energy appearing before deactivation in one bond of an energy-rich molecule is small, and initial reactions (i.e. within 10"12 sec) are more important in liquids than in gases.


long-chain fatty acids and of aliphatic alcohols by a-particles can be interpreted along these lines21. In both cases the most striking effect is the simplicity of the products. The predominant reaction when fatty acids are irradiated is one of decarboxylation

Cn . Hn2+i. COOH -> C„H2„+2 + CO2

The only volatile product other than CO2 formed in appreciable quantity is hydrogen. The initial ionization and energy absorption will occur at random and in a molecule (e.g. palmitic acid), which on a weight basis consists predominantly of a hydrocarbon chain, one would expect this part of the molecule to be broken; in the gas phase random breakdown of hydrocarbons is observed.

In general, the products are more complex, but it is often possible to detect the influence of the factors discussed above. Thus, for a straight-chain alcohol22 of the general formula CmH2îCH2OH almost all reac-tions were confined to the a-carbon (i.e. the carbon carrying the hydroxyl group). Although it was necessary to postulate many different reactions to account for all the products, in most cases 90 per cent of the overall process occurred in the following three reactions

C„H2„+1 CH2OH->C„H2n+2 + HCHO —I

C„H2n+1CH2OH C„H2„+1CHO + H2 — II

OH OH

2 C „ H 2 „ + I C H 2 O H C „ H 2 N + I C H — C H . C H 2 R T + H 2 — I I I

Reactions I and II are disproportionations giving stable products and, therefore, not affected by the cage. Reaction III is a "crosslinking" reaction and results from the rupture of a C—H bond on the a-carbon atom; the small hydrogen atom can escape leaving a free radical behind. In the densely ionizing track two such radicals then combine to give the product of reaction III. It is interesting that the yield of hydrogen is independent of chain length (in the range from n = O to 9) with a G value of 3 to 4.

Effects in Crystals On irradiating an inorganic crystal or semi-conductor the "sub-

ionization" electrons produced can either return to their position immedi-ately, give off fluorescent light, or be confined in electron "traps", which are often imperfections in the crystal lattice. These trapped elec-trons give rise to the so-called F-centres, which absorb light, often in the visible region. This is why glass vessels for irradiation become coloured. Unless special techniques are used to measure the colour it is generally


not obvious until more than 100,000 rads in toto have been received. The tint produced varies very much from glass to glass and depends on its composition. Pure quartz becomes violet while sodium chloride becomes brown. These colour centres can be destroyed on heating, when the increased thermal motion permits the electron to return to its original ground state, and light or heat is given off. But this thermoluminescence represents only a minute fraction of the total energy absorbed by the crystal.

MOREHEAD and DANIELS23 found that during the bombardment with a-particles the crystal lattice of certain minerals was destroyed, and no longer gave an x-ray diffraction pattern. The crystallinity, however, was restored by heating, and considerable amounts of energy were then released (e.g. 26 cal/g). This effect is particularly serious with neutrons and in atomic reactors the graphite used to slow down neutrons for fission can in this way store a very great amount of energy. If the reactor gets too hot this stored energy is released (referred to as "Wigner release") and further raises the temperature. This process was responsible for the accident which led to the shut-down of the British reactor at Windscale. These disturbances in the crystal lattice do not depend in any way on the ionizing properties of a-rays and neutrons, but are due to the fact that they have sufficient energy to displace atoms with which they collide. They also occur in metals, the physical properties of which are altered by the introduction of imperfections in the crystallites. The amount of radiations required for all these effects is enormous and they are of no biological significance.

P R O T E C T I O N A N D E N E R G Y T R A N S F E R In the strictest sense the term protection should only be applied if some of the energy is funnelled off in a harmless way, such as by heat or emission of light, so that the total amount of chemical reaction that has occurred is reduced. Thus there are some substances in which a greater proportion of the deposited energy is converted into heat than in others. If, in a mixture of two substances, energy is transferred from one to the other so that more heat is produced than if the two were irradiated separately then this is true protection. In practice, however, the term is not so limited and in this book "protection" will be used in the sense that the addition of another substance (the protective agent) reduces the radiation effect in the molecule being studied even if the protective agent is being destroyed.

When defined in this way protection can occur in two ways: 1. By energy transfer; 2. By repair of an unstable intermediate.

We have seen in the preceding sections that there are many stages between the formation of an ion and the final radiation product. In the presence of


another substance, the protector, this chain of events may not go to completion because a reaction occurs which restores the damaged molecule to its original state, i.e. it was protected. In general, all these processes occur within IO-6 sec or less, so that any substance which is required to react with one of these intermediates must be present at the time of the irradiation and cannot be added afterwards. It is this requirement to be present at the time of irradiation which qualifies a substance which repairs in this way to be classified as a protector. These reactions will be discussed on p. 177 and this section will be confined to energy transfer; this term being used in its broadest sense.

Protection Against Indirect Action When the damaging reaction occurs indirectly, such as by reaction of

the free radicals formed in water, then protection can take place by compe-tition for these free radicals. If the added substance is sufficiently reactive to combine with some of the free radicals which would otherwise attack the "target molecule" then it can be considered a protective agent. The extent of protection depends on both the relative concentrations and the rate of reaction of the protector and target molecule with radicals. Figure 5-1 shows that j8-mercaptoethylamine (its biological properties are discussed

(a) (b)

FIG. 5-1. (a) Protection against x-rays of 1-0 per cent solution of human serum albumin by thiourea and /3-mercaptoethyl-amine. Curve (A): no protective agent; (B) 1 X IO - 3M thio-urea; (C) 2 x IO3M thiourea; (D) 2 x IO - 3M jS-mercapto-

ethylamine24. (b) Protection against x-rays of 1 -0 per cent solution of

human serum albumin by sodium benzoate. Curve (A): no benzoate; (B): 4 x IO -4 benzoate; (C): 1 x IO - 3M benzoate:

(D): 7 x IO - 3 M benzoate24


in detail on p. 470) reacts so much more readily with the free radicals than does albumin that the latter is completely protected, until sufficient irradiation has been given to destroy nearly all the /9-mercaptoethylamine The reactivities of benzoic acid and the protein are comparable so that the former reduces the rate of "destruction" of albumin, but does not prevent it.

The possibility that protection has not occurred by competition but by "repair" must be borne in mind in these cases as, quantitatively, the same effect would be obtained if the added substance had combined not with the attacking radical but with the damaged protein and thereby restored it.

Intra-molecular Energy Transfer when the Action is Direct A number of experiments have shown that the site of the chemical

reaction is not necessarily identical with the site of the initial deposition of energy (e.g. the site of ionization). The transfer of energy occurs both within a molecule (i.e. the reaction occurs in a different part of the molecule from the ionization) and between molecules. At the present time the mechanism of these transfer reactions cannot be usefully discussed until more is known of the reactions that intervene between the ionization and the final chemical reaction that is observed. Energy transfer processes of excited molecules are well understood when they occur within a molecule and a semi-quantitative theory is available for inter-molecular transfer (see p. 42). These treatments cannot, however, be applied to ionizing radia-tions because nothing is known of the nature of the excitations that are produced. Moreover, the most important reactions may not occur through excited molecules, but through ionized molecules. In this case it is only possible to refer in the most general terms to the movement of the "positive charge" through the molecule until it finds a site (or "hole") of least energy at which the subsequent chemical reactions occur.

An example25 of intra-molecular energy transfer of a type that could be important biologically is illustrated in Fig. 5-2. When long-chain paraffins are irradiated they form inter-molecular bonds (called crosslinks, see p. 158). The aromatic structure of a naphthyl residue acts as a favourable energy trap and its presence increases the amount of energy that has to be deposited into the paraffin chain for a crosslink to form. Presumably energy is transferred from the dodecane chain to the naphthyl group where it is dissipated in a way that does not lead to crosslinking. The greatest amount of protection is obtained when the naphthyl group is in the centre of the chain, presumably because energy transfer is not effective over the full length of the twelve carbon chain. The introduction of a group of equal size but without the aromatic structure does not protect significantly because it does not constitute a good energy trap.


Substance Energy per crosslink (eV)*

CH3-(CH2)IO-CH3 dodecane 20 CH2-(CH2)I0-CH3 naphthyl-1-dodecane 32

f V , \ A /

CH3-(CH2)2-CH-(CH2)7-CH3 naphthyl-4-dodecane 46

M \ A /

CH3-(CH2)4-CH-(CH2)5-CH3 naphthyl-6-dodecane 49

,V, w

CH3-(CH2)4-CH-(CH2)5-CH3 cyclo-decalyl-6-dodecane 27

* The absolute values are open to some uncertainty but the relative values are reliable.

FIG. 5-2. Influence of an aromatic group substituted in different positions along the chain of the straight-chain hydrocarbon dodecane on the energy from ionizing radiations which has to be deposited in the dodecane chain to produce one crosslink25.

There is no indication that energy transfer processes are highly selec-tive. Many groups act as energy receptors and it is unlikely that transfer processes of this type can lead to a significant localization of radiation damage in a biological system. In proteins some localization of energy takes place into aromatic rings and into the sulphur containing amino acids, but the effect is not marked (see p. 184) and it is not sufficient to explain why frequently one ionization anywhere within the protein is sufficient for its inactivation.

Inter-molecular Energy Transfer It has been known for many years that the irradiation of crystals of

substances like anthracene leads to the emission of light and this property is used for the measurement of ionizing radiations by scintillation counting.


Under favourable conditions solutions of scintillators in non-reactive organic solvents will also fluoresce, but the amount of light emitted is much greater than can be accounted for by absorption of energy in the molecules of dissolved scintillators26. Energy taken up by the solvent contributes by an energy transfer involving excited solvent molecules. This type of process is well known and is frequently encountered in fluorescence phenomena, but there is no suggestion of protection. M A N I O N and BURTON47 found that the radiolysis of liquid cyclohexane was reduced

9 30 Q-

20

8-OH Oui ioline

/ V • / I /

/

i / vlaph h o l e Te

/ / % Inhibitor

FIG. 5-3. Relationship between concentration of additive and per cent protection against the direct action of radiation in

films of polymethyl methacrylate30.

on the addition of benzene. Benzene is remarkably resistant to radia-tion28, probably because energy distribution is very rapid in an aromatic ring and localization of energy is reduced*. Cyclohexane, on the other hand, is readily decomposed and the G value for hydrogen production is 2-4; the corresponding figure for benzene is 0-06. In a mixture of the two hydrocarbons the yield of hydrogen is a small fraction of that expected from the cyclohexane alone. The interpretation advanced is that the ionized cyclohexane transfers its charge to (i.e. captures an electron from) benzene, which has a lower ionization potential; transfer of excitational energy also occurs, probably by collision. The transferred energy of either type is dissipated in the aromatic ring and produces much less hydrogen than it would have done from cyclohexane. In both of these

* Deactivation by collisions must also play a part in the radiation resistance of liquid benzene, since in the gas phase the G value for hydrogen evolution is five times that in the liquid. For cyclohexane the reverse is the case.

G E N E R A L R A D I A T I O N C H E M I S T R Y 115

examples the molecules involved in energy transfer were present in solution and could come into direct contact with one another. Inter-molecular energy transfer can also occur in the solid state where the probability is low that the protecting molecule is in contact with the atom that has been ionized. Energy transfer in solids requiring a process which was effective over considerable distances (e.g. involving movements of electron holes) was first revealed29 in experiments with synthetic polymers. One of the principal effects of radiation on polymethyl methacrylate is to break the main chain and the resultant decrease in the average molecular weight can readily be determined by physico-chemical methods. The addition of certain low molecular weight additives makes the polymer more radiation resistant (see Fig. 5-3). In the presence of the additive fewer breaks are formed, but the additive is destroyed instead30; i.e. polymer is protected by the additive, but the additive is sensitized by the polymer. Many substances are capable of acting as energy transfer agents (see Table 7-4 on p. 178), but there is insufficient information to define the structural requirements for this property. Protection by inter-molecular energy transfer has also been observed when proteins31 and viruses32 are irradiated in the dry state in the presence of other substances.

F A T E O F F R E E R A D I C A L S P R O D U C E D The intermediates between the short-lived products, ions or excited molecules with a lifetime of less than IO-10 sec, and the final radiochemical products are almost invariably "free radicals". These are chemically highly reactive because they contain an unpaired electron so that when two free radicals come together they combine to form a "chemical bond" by sharing their unpaired electrons. Free radicals can also react with ordinary molecules (see p. 117). The reactivity of free radicals varies very much and there are a few derived from complex organic substances which are remarkably stable and which do not readily recombine. This, however, is the exception rather than the rule and the majority of the free radicals formed by radiation are of the highly reactive type.

Much is known about the behaviour of free radicals and radiation chemistry is on firm ground once the initial steps of the conversion of energy are passed and the stage of the free radicals is reached. Free radicals occur as intermediates in many organo-chemical reactions, in particular during the formation of synthetic polymers. They can be produced in several ways such as electrolysis, photolysis and by heating organic substances to temperatures at which they decompose. They are present in flames and are readily obtained at ordinary temperatures by the breakdown of peroxides. The unique feature of atomic radiations is not that they produce free radicals, but that they can do so within the interior of a cell.


Free radicals are produced when an ion dissociated either with or without charge neutralization (see pp. 104-108) and if an excitation initiates a chemical reaction this also will usually proceed via free radicals. They form the active intermediate of indirect action as on ionization a water molecule gives rise to two free radicals, OH' and H', which can attack dissolved molecules (see p. 147). The first experimental evidence that free radicals are produced on irradiation was provided by HOP-WOOD33 who observed the polymerization of a vinyl monomer (styrene), a reaction well known to require initiation by a free radical. Since this time other methods have been evolved and the problem of measuring the number of free radicals that are produced is described below.

Persistence of Free Radicals Free radicals are not inherently unstable and if steric restrictions

prevent movement they will persist for ever. Thus coal, peat and pet-roleum tars contain some IO19 free radicals per gramme. The same number of free radicals could be produced in the laboratory with a dose of IO9 r. DUCHESNE et al.34 have convincing evidence for their view that these free radicals were formed in the coal by irradiation from naturally radioactive materials (potassium and uranium) during the whole period of its formation which is believed to be some 2 x IO8 years.

If diffusion is not prevented then the radicals react quickly either by combination with another radical or by reaction with a molecule having a group susceptible to attack. In water the life of a radical is of the order of IO-6 sec. With radiations of high ion density recombination of radicals is favoured because they are formed close together and this explains why the yield (or G value) for many radiochemical reactions decreases as the LET of the radiation used increases (see p. 134).

A molecule of oxygen has many of the properties of a free radical since two of its electrons are unpaired. In its behaviour it is a biradical which could be written 'O—O". It combines readily with most organic free radicals, but when it does so, it does not give a normal molecule since the product still has one unpaired electron. This behaviour can be repre-sented as follows

exposed to radiation recombination R H > R + H > R H organic molecule two free radicals

Radical combinat ion R* + R ' —s- R - R (normal molecule wi th no un impa i red electrons)

but R' + 0 2 -> RO2* peroxy radical

Thus the free radicals, if accessible to oxygen, will be converted into


peroxy radicals*. This frequently gives rise to increased radiation damage because recombination or a reaction leading to repair is prevented. The peroxy radical, ROO', breaks up with decomposition that often involves the organic residue, R. Combination of two ROO' radicals is not possible and even if the peroxy radical captures a hydrogen, the resultant peroxide, ROOH, though not longer a free radical, is very unstable and liable to breakdown under physiological conditions.

As already mentioned in addition to radical-radical reactions free radicals can react with normal molecules particularly if they contain a reactive hydrogen, that is a hydrogen attached to an oxygen sulphur, nitrogen or tertiary carbon atom. The free radical may then be able to abstract the hydrogen atom and this can result in its repair, e.g.

R H M - > R ' + H*

R ' + P S H > R H + P S * compound with (a much less reactive reactive hydrogen free radical than R-) atom

If RH is a vital molecule then reaction with PSH can be said to have resulted in its repair. It is possible that many protective agents work in this way and ALEXANDER and CHARLESBY3 5 first demonstrated protection by this mechanism in a polymer systemf. There are many situations (e.g. crosslinking of polymers in solution)36, where repair and combination with oxygen are competing reactions and a slight change in conditions can shift the balance from damage to repair (or vice versa).

Measurement of Free Radicals Produced The number of free radicals produced on irradiation of water has been

determined relatively accurately by a number of independent methods (see p. 133). In non-aqueous systems the situation is less satisfactory. MAGAT and his colleagues37 have devised two ingenious methods which enable them to determine in principle both the nature and quantity of the radicals formed. The radiation produced free radical is trapped by combination with a vinyl monomer or one of the "stabilized" organic free radicals, that do not interact with one another, though combining

* T h e subsequent fate of a peroxy radical is frequently quite different from that of an ordinary organic radical and consequently entirely different products are often formed when organic compounds are irradiated in the presence of oxygen than when they are irradiated in its absence. This aspect of radiation chemistry was pioneered for low molecular weight organic liquids by N . A . B A C H (see ref. 4 6 ) .

•f If P S H is the substance which is being studied then this is an example of " ind i rec t" action where an active intermediate formed by irradiation is responsible for the reaction (e.g. if R H represents water then R" is a hydroxy radical, OH' ) .

1 1 8 F U N D A M E N T A L S O F R A D I O B I O L O G Y

readily with the radiation produced radical. In practice their methods are not easy to apply and cannot be used under physiological conditions.

When the radicals can be prevented from combining (i.e. in solids where diffusion is prevented) a physical method, known as electron spin resonance, can be used to detect unpaired electrons since these give rise to magnetic centres. Electron paramagnetic resonance within solids is a most complex phenomenon and with modern techniques a spectrum of spin resonance can be obtained which differs for different irradiated materials. An idea for the number of free radicals produced can be ob-tained, though the values aie liable to an uncertainty of several hundred per cent. As yet the qualitative information that can be obtained concern-ing the chemical nature of the radicals formed in solids is limited and interpretation in complex molecules has not proved possible so far*. In the absence of an adequate theory it is unlikely that the empirical approach widely adopted (cf. ref. 38) will allow reliable structural information to be deduced from these spectra. In general, the spectrum alters when oxygen is admitted and this represents a change to a peroxy radical. As soon as diffusion can occur (e.g. by the penetration of a solvent which swells or dissolves the irradiated solid) the electron spin resonance disappears because the radicals have combined. It is conceivable that something may be learned about the rate of diffusion in dry materials such as seeds or spores in which radicals persist after irradiation. Vegetative cells do not give any signal because the water content is sufficiently high for rapid radical interaction to occur.

The first observation of paramagnetic resonance in irradiated organic solids was made by SCHNEIDER39 in the polymer, polymethylmethacrylate (Perspex). The number of persistent free radicals produced varies very much from solid to solid. In crystalline amino acids one radical seems to remain for every ionization41. In most macro-molecular systems the intensity of the spectrum is much greater if the sample has been irradiated at liquid nitrogen temperatures. As soon as the temperature rises the intensity falls and often assumes quite a different appearance47 and in these cases the e.s.r. spectrum at room temperature provides little infor-mation concerning the initial reaction. The behaviour of individual macromolecules is discussed in Chapter 7. Detailed examinations have not been reported on the influence of water content, but it would appear that a few per cent of firmly bound water does not influence the magnetic centres, though these disappear as soon as the samples become perceptibly wet. In seeds of wheat and barley with a water content of 8-5 per cent the signal

* This method revealed intermolecular energy transfer between organic solids. T h e spectrum obtained by irradiating an intimate mixture of the amino acids cysteine and alanine gave a different spectrum from that obtained when the two were irradiated separately40.


fell to one-half of its original value in a few hours after irradiation, while it persisted essentially unchanged at 1 -5 per cent water content in the absence of oxygen (see Fig. 5-4). Under air the dry seeds lost half of their

Time, hr

FIG. 5-4. Derivative amplitude (arbitrary units) of paramagnetic absorption curve of barley embryos as a function of time after

irradiation42.

magnetic centres in some ten days32-43. As already mentioned, this effect of oxygen is to be expected since peroxy radicals are unstable and slowly undergo complex rearrangements. As is to be anticipated the number of radicals persisting in organic materials is less with densely ionizing than with sparsely ionizing radiations44.

When cells or parts of cells are dried by lyophilization they often show electron spin resonance before irradiation. This is to be expected since on freezing macromolecules are degraded and the free radicals formed remain "frozen in". The spectra produced on irradiation is different for different dry biological materials, but the significance of such an obser-vation38 is not evident. No e.s.r. due to radiation can be seen in swollen systems or in negative cells and inherently this method can only be used under non-physiological conditions where there is little or no diffusion.

The claim has frequently been made (cf. ref. 45) that electron spin resonance is likely to play an important role in furthering knowledge of the fundamental chemical processes that underlie radiobiology. The experiments to date have not been very informative. It has been known for two decades that free radicals are formed by radiation and that, if prevented from diffusing, they will persist. Even if the theory of electron spin resonance progresses sufficiently to allow the chemical nature of the radicals to be identified, the fact that all samples have to be dried would remove much of the biological significance of the data. The free radicals


are only an intermediate stage in the formation of the chemical lesion and by arresting the reaction chain at this stage no direct information of such a lesion will be obtained. Above all this method cannot distinguish between those reactions that are biologically important and those that are not. The greatest value so far has been to provide supporting evidence for the view that energy transfer occurs between molecules (see p. 186).

R E F E R E N C E S

1. LIND, S. C., The Chemical Effects of a-Particles and Electrons, Chemical Catalog Co., New York, 1928

2 . L I N D , S . C., J. Am. Chem. Soc., 1 9 1 9 , 4 1 , 5 5 1 3 . L I N D , S . C. and BARDWELL, D . C., J. Am. Chem. Soc., 1 9 2 6 , 4 8 , 2 3 3 5 4. EYRING, H . , HIRSCHFELDER, J. O. and TAYLOR, H. S., J. Chem. Phys., 1936, 4a. ESSEX, H . , J. Phys. Chem., 1954, 58, 42 5 . L I N D , S . C . , ] . Phys. Chem., 1 9 5 2 , 5 6 , 9 2 0 6 . EYRING, H . , J. Chem. Phys., 1 9 3 9 , 7 , 7 9 2 7 . STEACIE, E . W . R . , J. Phys. Colloid Chem., 1 9 4 8 , 5 2 , 4 4 1 8 . M U N D , W . a n d ROSENBLUM, C . , ibid., 1 9 3 7 , 4 1 , 4 6 9 9 . ROSENBLUM, C . , ibid., 1 9 4 8 , 5 2 , 4 7 4

10. MAGEE, J . L . , / . Phys. Chem., 1952, 56, 555 1 1 . MAGEE, J . L . and BURTON, M . , / . Am. Chem. Soc., 1 9 5 0 , 7 2 , 1 9 5 6 1 2 . BURTON, M . , MAGEE, J . L . and SAMUELS, A. H . , J. Chem. Phys., 1 9 5 2 , 2 0 ,

760 1 3 . BURTON, M . and MAGEE, J. L . , J. Phys. Chem., 1 9 5 2 , 5 6 , 8 4 2 ; also MAGEE,

J . L . and FUNABASHI, K . , Radiation Research, 1 9 5 9 , 1 0 , 6 2 2 1 4 . WALLENSTEIN, M . , WAHRHAFTIG, A . L . , ROSENSTOCK, H . a n d EYRING, H . ,

Symposium on Radiobiology, p. 70, John Wiley, New York, 1952 15. SMITH, L . E. , Phys. Rev., 1937, 51, 263 1 6 . EYRING, H . , HIRSCHFELDER, J . O . and TAYLOR, H . S., J. Chem. Phys., 1 9 3 6 ,

4 , 570 17. LIVINGSTON, R . , Biological Antoxidants (Macey Conf.), 1952, 5 , 251 18. STEVENSON, D . P . a n d SCHISSLER, D . O. , J. Chem. Phys., 1956, 24, 926 19. FRANCK, J . and RABINOWITCH, E., Trans. Faraday Soc., 1934, 3 0 , 120 2 0 . BURTON, M . , J. Phys. Colloid Chem., 1 9 4 8 , 5 2 , 8 1 0 2 1 . BREGER, I . A . , BURTON, V. L . , H O N I G , R . E . , SHEPPARD, C . W . , J. Phys.

Colloid Chem., 1948, 52, 551 22. MCDONNELL, W. R., American Atomic Energy Commission, U.C.R.L., 1378,

1 9 5 0 23. MOREHEAD, F . F . and DANIELS, F . , J. Phys. Chem., 1952, 56, 546 2 4 . ALEXANDER, P . , ROSEN, D . and BROHULT, S., Arch. Biochem. Biophys., 1 9 5 7 ,

7 0 , 2 6 6 25. ALEXANDER, P. and CHARLESBY, A . , Nature, 1954, 1 7 3 , 578 26. SPONER, H., Radiation Research, Suppl. 1, 1959, p. 558 27. M ANION, J . P . and BURTON, M .,J. Phys. Chem., 1952, 5 6 , 560 28. GORDON, S. and BURTON, M., Disc. Faraday Soc., 1952, 1 2 , 88; also HENRY,

V. and ERRERA, J., J. Phys. et Radium, 1926, 7, 225 29. ALEXANDER, P., CHARLESBY, A . and Ross, M., Proc. Roy. Soc. A , 1954, 223

3 9 2 3 0 . ALEXANDER, P . and T O M S , D . J . , Radiation Research, 1 9 5 8 , 9 , 5 0 9


31. BRAAMS, R . , HUTCHINSON, F. and RAY, D., Nature, 1958, 1 8 2 , 1506 32. GINOZA, W . a n d NORMAN, A . , Nature, 1957, 179, 520 3 3 . H O P W O O D , F . L . and P H I L L I P S , J . T . , Nature, 1 9 3 9 , 1 4 3 , 6 4 0 3 4 . DUCHESNE, J . , DEPIREUX, J . and V A N DER KAA, J . , Bull. Acad. Roy. Belg.,

1 9 5 9 , 4 5 , 7 1 4 3 5 . ALEXANDER, P. and CHARLESBY, A . , Radiobiology Symposium, Liege, 1 9 5 4 ,

p. 49, Butterworth, London 36. ALEXANDER, P. and CHARLESBY, A . , J. Polymer Sci., 1957, 23, 355 3 7 . PREVOST-BARNAS, A . , CHAPIRO, A . , COUSIN, C . , LAUDER, Y . a n d MAGAT, M . ,

Disc. Faraday Soc., 1952, 12, 98 3 8 . GORDY, W . , Radiation Research, Suppl. 1, 1 9 5 9 , p. 4 9 1 39. SCHNEIDER, E . E . , D A Y , M . J. and STEIN, G . , Nature, 1951, 1 6 8 , 644 40. NORMAN, A. a n d GINOZA, W . , Radiation Research, 1958, 9, 77 41. EHRENBERG, A., EHRENBERG, L . and ZIMMER, K . G . , Acta Chem. Scand., 1957,

11, 199; also EHRENBERG, A. and EHRENBERG, L . , Arkiv.for Fysick, 1958, 1 4 , 133

4 2 . Z IMMER, K . G., EHRENBERG, L . and EHRENBERG, A., Strahlentherapie, 1 9 5 7 , 1 0 3 , 3

4 3 . CONGER, A . D . and RANDOLPH, M . L . , Radiation Research, 1 9 5 9 , 1 1 , 5 4 4 4 . K I R B Y - S M I T H , J . S . , Intern. J. Radiobiol., Suppl. 1, 1 9 6 0 , p. 1 1 45. ZIMMER, K. G., Intern. J. Radiobiol., Suppl. 1, 1960, p. 1 4 6 . BACH, N . A . , Radiation Research, Suppl. 1, 1 9 5 9 , p. 1 9 0 4 7 . OVENALL, D . W . , / . Polymer Sci., 1 9 5 9 , 4 1 , 1 9 9

C H A P T E R 6

The Radiation Chemistry of Aqueous Systems

I N T R O D U C T I O N

M O R E attention has been paid in recent years to the chemical changes produced by ionizing radiations on aqueous systems than on any other branch of radiation chemistry. One of the main reasons for this great interest has been the belief that energy absorbed in the water molecules present in cells leads to biologically important chemical reactions. The evidence reviewed in Chapter 2 shows that recent experiments have made the evidence, on which the importance of indirect action to radiobiology is based, appear less compelling. While there is no longer any reason for believing indirect action to be all important neither can its contribution be dismissed and a book devoted to the fundamentals of radiobiology must deal with the radiation chemistry of water. This subject unlike the radia-tion chemistry of organic molecules described in the preceding chapter is now very advanced and the effect of ionizing radiations on water and simple solutes in water can be accurately described both qualitatively and quantitatively. Until a few years ago the literature was full of contradictions and no satisfactory scheme could be put forward. Progress in the last few years has not consisted so much in revealing new facts but in eliminating erroneous results. For example, much confusion was created by the claim (see p. 130) that the true yield of free radicals formed in water was more than twice as great as the one that had been accepted for many years. The earlier data proved to be correct. Similarly, it was claimed that the produc-tion by ionizations of OH radicals could not explain the oxidizing proper-ties of irradiated water and that new species such as OH+ radicals had also to be formed (see p. 137). Again the OH radical theory first proposed thirty years ago is now known to be adequate.

In this chapter, after a brief historical survey, the type of free radicals formed by ionizing radiations in water and the influence of the specific ionization on the number of radicals which are available for reaction with a solute will be discussed. The general nature of the reaction of these free radicals with different dissolved substances will be illustrated by examples

122

R A D I A T I O N C H E M I S T R Y O F A Q U E O U S S Y S T E M S 1 2 3

which are of possible biological significance. The reaction of macro-molecules will be dealt with in detail in the following chapter. Finally, some unusual physicochemical changes produced in colloids will be described.

H I S T O R I C A L D E V E L O P M E N T

As early as 1 9 0 1 CURIE and DEBIERNE1 observed a continuous evolution of oxygen and hydrogen from solutions of radium salts and this reaction was studied in more detail by RAMSAY and SODDY2 . An analysis of the gases revealed that the decomposition was not stoichiometric and that there was an excess of hydrogen3. This anomaly was resolved by K I R N -

BAUM4, who found that the solution of radium salts contained an amount of hydrogen peroxide equivalent to the deficiency of oxygen.

The reverse reaction, the formation of water from hydrogen and oxygen by admixture of radon, was studied in considerable detail by CAMERON and RAMSAY5 , who found that the following rules were obeyed: (i) "that every molecule of emanation (radon) disintegrating produces a definite chemical effect", and (ii) "that each molecule of emanation (radon) as it disintegrates, produces the same amount of chemical change". At the time of this investigation it was generally believed that the action of radon was that of catalyst, and it had not been realized that the energy for the reactions was provided by the concomitant radiations. These experiment-ally derived relationships were therefore unexpected, although it is now known that, subject to some qualifications, they are quite general and fundamental for radiation chemistry.

From the data of Ramsay, BRAGG6 calculated that the number of water molecules decomposed was almost identical with the number of ions which a similar amount of radon would produce in air. This agreement was referred to by Bragg as a "curious parallelism in numbers" which indicates that an a-particle may in the course of removing of one or more electrons cause "a more complete disruption of the molecule or even the atom". This appears to be the first suggestion that the chemical changes produced by radon were the result of ionization by a-particles. L I N D 7

recalculated the available data in terms of ionic yield (see p. 107) and for the reaction

a - r ays H 2 O ^ | ( 2 H 2 + 0 2 )

the M/N values were 0-8 to 1 for liquid water; 0-05 to 0-1 for ice; and less than 0-01 for water vapour. For the reverse reaction (i.e. formation of water) the high value of M/N = 4 was found, and this may explain the low yield for the decomposition of water vapour. The presence of oxygen does not influence the formation of hydrogen peroxide by a-rays in


contrast to the results obtained with x-rays (see p. 140). The general principle, on which current theories are based, that the action of ionizing radiation on pure water is the establishment of an equilibrium first became apparent in experiments from Mme Curie's laboratory, where DUANE and SCHEUER8 studied the decomposition of water by OC-rays in great detail.

Concurrently with these investigations the chemical effects of x-rays on different solutes were studied, notably by KAILAN9 . In aqueous solutions hydrogen peroxide was decomposed, the iodide anion oxidized to iodine and most significantly benzoic acid was obtained by irradiating toluene dissolved in water10. Lind realized that these reactions in aqueous solution were "indirect" and wrote concerning the oxidation of iodide: "The radiation first acts on water to produce an activated (nascent) form of oxygen, which reacts with potassium iodide in a secondary reaction"7. This concept was firmly established independently by FRICKE11 and RISSE12 in a series of investigations on the effect of x-rays on aqueous systems. Their experiments were notable for the high experimental skill and the extreme care used to avoid contamination by impurities.

Differences between a- and x-rays While there could be no doubt that a-rays dissociated water, the effect

of sparsely ionizing radiations like x-rays or /3-rays seemed enigmatic. Neither Fricke nor Risse were able in pure and degassed water to detect the formation of hydrogen peroxide or hydrogen gas even after large doses of x-rays. Yet chemical reactions were induced by x-rays under anaerobic conditions; inorganic as well as organic solutes were decomposed, oxidized or reduced, often with a yield of M/N = 1. Fricke clearly established that these reactions were indirect (i.e. yield independent of solute concen-tration) and considered that the reactive species was activated water. The problem of defining "activated" water in chemical terms has now been solved and is based on a hypothesis originated by RISSE13 in 1929, a time when the techniques for testing it experimentally were not available.

PRIMARY PRODUCTS IN THE RADIOLYSIS OF WATER RISSE13 suggested that the principal reaction of x-rays was the formation of H and OH radicals and that the failure of x-rays to decompose pure water could be interpreted in terms of recombination of H and OH radicals and that the increased yield of oxidation reactions in the presence of oxygen would be due to the formation of a peroxide radical (i.e. HO2'). The full usefulness of the free radical hypothesis was appreciated only after the reactivity of these entities in water was better understood when the suggestion that x-rays decompose water into H" and OH' was put


forward anew in 1 9 4 4 by WEISS14. Direct evidence for the formation of the OH radical15 and of a hydrogen atom16 has now been obtained and the theory that

exposure to ionizing H2O » H' + OH'

radiations

can be accepted as established. The observed facts cannot be explained solely on the basis of their so-called "radical reaction" and A L L E N 1 7

showed that if a reaction was introduced, in which hydrogen gas (H2) and hydrogen peroxide (H2O2) were formed even with x- and y-rays, all the facts could be interpreted both qualitatively and quantitatively. The molecular products are formed by the recombination of H and OH radicals when these are formed close together within the densely ionizing spurs and S-ray tracks which constitute about 25 per cent of the total energy even with the hardest (i.e. most sparsely ionizing) radiations (see p. 79). In a sense the molecular reaction is a secondary process, but since the radical recombination responsible is so efficient that it is difficult to interfere with, it is convenient to consider it in parallel with the reaction giving radicals.

Formation of Free Radicals The ions formed by electron impact on water vapour have been quanti-

tatively determined in the mass-spectrometer. In order of abundance the products are HO2+, HO+, H+ and H2O+; very small amounts of 0+ and H2+ have also been detected. H2O+ requires the least energy (~13 eV) for its formation and only about 40 per cent of the energy needed to form an ion pair (32-5 or 35 eV) is used in this process. Only a small fraction of this excess energy can be retained by ejected electrons— since these would otherwise produce further ionization—and most of it will be used to produce electronically excited water molecules. Charge neutralization of the ions formed will also lead to excited molecules of high energy content which can dissociate into H and OH radicals.

How far this vapour phase data is applicable to liquid water is not known and any theoretical treatment based on analogy can best be only qualitative. An immediate difficulty when dealing with reactions in water is that the number of ion pairs formed in liquids cannot be determined. On the hypothesis that the energy required to form an ion pair is the same in condensed systems as in gases, exposure of water to a dose of 1 r of x-rays will produce a concentration of 3 x IO-9 M of ion pairs. From experiment the G value for the number of water molecules decomposed (i.e. by both radical and molecular reactions) by hard radiations is 3-9, which suggests that one molecule is decomposed for every 25 eV. This is


surprisingly close to the average energy for ionization in water vapour (i.e. 34 eV, see p. 26) and gives us confidence that the basic information obtained from vapour work can be applied.

H2O+ is believed to be the predominant species formed in water. The ions immediately become hydrated and the energy made available in this process is a determining factor in the subsequent fate of the ion. The point about which there is least information is the behaviour of the "slow" electron produced. For every H2O+ ion an electron is produced with an energy insufficient to give further ionizations. This electron can either be captured by another water molecule to give a negative ion, or combine with the H2O+ to give an excited molecule. The possibility that the elec-tron may have an independent existence in water long enough to enter into chemical reaction has not been quantitatively examined, but cannot be excluded.

The first mechanism for the formation of the radicals from the ions was proposed by LEA18 in 1947. A pair of positive and negative water ions was postulated as the direct product of the ionization. These decomposed on becoming hydrated within the relaxation time of water (i.e. IO-11 sec) to give the radicals first postulated by RISSE13. Within this period no significant diffusion of the ions can occur so that the radicals are formed at the same sites as the ions. The complete reaction is represented as follows:

H2O H2O+ + e

H2O++ H2O -> Ha9++ OH* (i.e. decomposition on hydration)

e+ H2O ^ H2O-

H2O- + H2O -> H* + OHas-

Overall H2O H' + OH* The most notable feature of this mechanism is that the two radicals

are not formed close together as, according to LEA18, the slow electron has, on a molecular scale, an appreciable free path before it is captured to give a negative ion. According to DALE, GRAY and MEREDITH19 the separation is of the order of 15 mix in water (i.e. the radicals will be separated by about 70 water molecules).

More recently, SAMUEL and MAGEE20 using a simple but convincing model have calculated that the electron loses its energy very rapidly to surrounding water molecules and that it cannot in fact escape from the strong field of the ion. After travelling a distance less than 20 A the electron has lost sufficient energy to come into the attraction field, and approximately IO-13 sec is taken up for the charge neutralization process (i.e. for the electron to lose its kinetic energy and to return to the vicinity


of the positive ion). This time interval is too short for the positive ion to dissociate, i.e.

H2O — \AA •-* H2O+ + e > H2O* * H' + OH' (slow) (highly excited)

Therefore, if this theory is correct, all radiation chemical reactions in solids and liquids proceed via excited molecules and their free radical dissociation products; ions play no part, as their lifetime is too short for them to enter into chemical reactions.

The premise on which the calculations of Samuel and Magee are based has not been accepted by PLATZMAN and FROEHLICH21, who believe that charge neutralization is a very infrequent process. These authors have made a detailed study of the motion of slow electrons in dipolar systems, such as water, and conclude that the rate of energy loss is even less than that used by Lea and Gray in their calculations. Accordingly, even the formation of the H2O - ion by an electron capture process will be relatively slow, and it is not impossible that the electron may have an independent existence of the order of IO-5 sec. This time interval is long enough for the electron to react with dissolved substances.

An alternative method of obtaining a relatively persistent atmosphere of electrons is to assume that the electron is captured by water, but that the resultant H 2O - ion does not have time to decompose before the elec-tron is passed on to another water molecule. This postulate is not improb-able since the outer electronic orbits of water overlap in the liquid phase. Probably a time interval of IO-15 to IO-14Sec would be necessary for transfer from one molecule to another while the H 2O - ion requires 10-11sec (time for relaxation of dipoles) before it can dissociate. This model is equivalent to the formation of an electron band of the type which exists in metals and semiconductors. In the presence of a molecule of high electron affinity the electron would be trapped and no longer passed from water molecule to water molecule, as the symmetry of the system would be lost.

At present there seems to be insufficient data to decide between the possible processes. At first sight the difference between the Lea and Burton-Magee-Samuel theory may appear to be only formal as both provide for the formation of one H and OH radical per ionization. This is not so; the two mechanisms lead to different distribution in space for the H and OH radicals produced by densely ionizing radiations like a-rays or in the S-rays (spur) of sparsely ionizing radiations. According to the Lea mechanism the distribution at different times of H and OH radicals produced by a densely ionizing a-particle is as shown in Fig. 6-1. There is an inner core containing predominantly OH radicals surrounded by an outer region where H atoms are in excess. According to the Burton-


Magee-Samuel mechanism H and OH radicals are formed at the same site and there is no significant difference in their distribution (i.e. the H and OH distribution is the same as the OH distribution shown in Fig. 6-1).

F I G . 6 - 1 . Diffusion of the free radicals (according to L E A 1 8 ) formed by a-particle tracks in water.

With sparsely ionizing radiations (i.e. low LET) the two models might not at first sight be expected to differ since the ionizations are further apart than the separation of the H and OH radicals by the Lea model and the radicals would therefore be randomly distributed. This problem is, however, more complex since the distribution of the ions is far from uniform. Along an approximately linear path of ionizing particles the primary ionizations occur at intervals which are determined by the charge and velocity of the particle (see p. 19). But these are accompanied by secondary ionizations which occur within a few Angstrom units of the primary ionization (clusters of spurs) and occasionally at greater distance (S-rays). The ionization density of 8-rays (and spurs) is sufficient to give some non-uniformity of H and OH distribution according to the Lea model and this is why the two treatments give slightly different answers even for hard radiations. Tracks of a-particles, 1 keV and 60 keV elec-trons are compared in Fig. 6-2.

Role of Excited Molecules In addition to ionizations, ionizing particles produce excitations (see

p. 107). Molecules excited in this way must not be confused with the excited molecules produced by the charge neutralization process due to

R A D I A T I O N C H E M I S T R Y OF A Q U E O U S S Y S T E M S 1 2 9

the combination of ions (i.e. Burton-Magee-Samuel reaction) which are much more energetic. The problem is whether these excited molecules produced in the primary physical act play a part in radiation chemistry.

One of the possible reactions which they could undergo is dissociation into H and OH radicals. The energy required to do this, approximately 5 eV, is probably available (i.e. W = 34 eV; the energy required to ionize water from ionization potential measurement is 16 eV; thus if for each ion pair formed three excited molecules are produced, each one would

60\V electron

6 ffi

© e

" tH)=[0HJ=V3x106M ©

© 6 O ® e ® [HW0H]=7xirsM © © ©

© o © O ©

o © e e e

© © ©

F I G . 6-2. Diagrammatic representation, according to DALE et al.19

of tracks of ionizing particles in water assuming the ejected electrons are captured by a water molecule to form a negative ion 15 m/i distant from the parent positive ion. The inner solid line represents the initial distribution of OH radicals and the outer solid line the initial distribution of H radicals. The dotted lines show how far the radicals will spread in the timu necessary for 50 per cent of the radicals to collide with one another (i.e. for 50 per cent of radicals to disappear by recombining to give molecular products). For a-particles and 1 keV electrons this time is only IO -9 sec and 6 x 10~9 sec respectively and hence the radical column will spread only very short distances before recombination can occur. The radicals from 60 keV electrons have a half-life of 2 x IO -6 sec, during which they

can diffuse large distances.

have an energy of somewhat more than 5 eV). But it is unlikely that the conversion of the energy deposited in an electronically disturbed atom would be transferred with high efficiency into the H—O bond, and general consideration suggests that deactivation processes in liquids reduce the number of molecules having sufficient energy to dissociate. Also the "cage effect" (see p. 108) would bring about the immediate

a-partide

EQ ©'tgjfJHl=Iaxttr1M^

Q g e 9 e [OHl= xiO-llM


recombination of H and OH radicals produced by dissociation of a water molecule having an energy content sufficient, but not greatly in excess of, that required for dissociation. LEA22 and others were of the opinion that few H and OH radicals, diffusing separately through the medium, are contributed by excited molecules and that the number of chemical reac-tions occurring in water was determined by the number of ionizations. Recent quantitative studies (see p. 134) strongly support this conclusion since approximately one water molecule is decomposed for every 25 eV of energy (i.e. G (for disappearance of water) = 4) and this is well within the range to be expected for W in a liquid.

The reason why the earlier views of FRICKE and HART23-24 and LEA 2 2 , which emphasized the role of ionizations, was questioned, was the claim to have reaction yields far in excess of the number of ions produced. Thus, DAINTON et al.25 claimed that one O H radical was produced for every 7-5 eV (i.e. GOH = 13). This value, however, was due to an experimental error and has been withdrawn26 and at the present time there is no reliable experiment describing a reaction occurring in water that requires the intervention of water molecules that have been excited, as opposed to ionized, by the passage of the ionizing particle.

Formation of Molecular Products In early experiments FRICKE27 found that the oxidation of simple

anions such as bromide or iodide by x-rays in pure water was accompanied by the formation of equal amounts of hydrogen and hydrogen peroxide. This observation provided the first indication for a "molecular" reaction, the existence of which was established by Allen who formulates the initial radiation process as follows:

(F) H2O -> !(H2 + H2O2) (forward or molecular reaction)

(R) H2O -> H' + OH' (radical reaction) The respective yields per 100 eV are referred to as GF and GR.

Allen does not claim that these reactions occur by different mechanisms and it is not suggested that one water molecule of water decomposes by the (F) and another by the (R) process. The most plausible interpretation is that a fraction of the free radicals formed in the densely ionizing 8-rays (and spurs) interact immediately with one another to give molecular products and are not available for reaction with solute*. The remainder

* A reaction between two excited water molecules could also lead to the forma-tion of H2 and H2O2. A bimolecular process can, however, be excluded on kinetic grounds for all except densely ionizing radiations since the probability of two excited molecules colliding is insignificant at the low local concentrations in x-ray and -/-ray tracks.


of the free radicals diffuse into the solution where they can interact with other materials if these are present. In pure water the "escaped" radicals recombine or react with the molecular products.

The failure of Fricke and Risse (see p. 124) to detect hydrogen peroxide and hydrogen when pure water is irradiated with x-rays is due to the destruction of the molecular products by the free radicals from the (R) reaction. The molecular products are probably destroyed by this chain reaction

H '+ H2O2 OH'+ H2O

OH" + H 2 - ^ H ' + H2O which is terminated by recombination of H' and OH'. There is some evidence28 that the chain may also be broken by the well-established reaction

OH' + H2O2 -> HO2' + H2O The HO2 radical is reduced back to hydrogen peroxide

HO2 '+ H' ->- H2O2

which then rejoins the chain process. The paradox that no hydrogen peroxide (or hydrogen gas) could be

detected on exposure in pure de-aerated water to x-rays but that these molecular products are formed when a reducing substrate is oxidized can be understood in terms of an (F) and (R) reaction. Decomposition of the hydrogen peroxide competes with oxidation of the substrate for the avail-able OH radicals.

In recent years the molecular products, hydrogen gas and hydrogen peroxide, have been detected when de-aerated water is irradiated. These products are formed initially but the rate of appearance quickly decreases and an equilibrium concentration is set up which does not alter as the amount of radiation is increased. However, the magnitude of this equili-brium value depends on the intensity (or dose rate) at which the radiation is delivered. On theoretical grounds one would expect the equilibrium value for H2O2 to be proportional to the V(intensity) if the simple reaction scheme for the disappearance of the molecular products shown above applied. GHORMLEY29 showed experimentally, using electrons from a Van de Graaff generator, that this relationship applied (see Fig. 6-3). But the dose rates at which hydrogen peroxide could be detected were much higher than those used by the earlier radiochemists and this fully explains their failure to detect the molecular products.

As soon as a substance capable of reacting with H or OH radicals is present the molecular products are formed in much larger amounts. In the presence of a reactive solute the amount of hydrogen peroxide increases

F U N D A M E N T A L S O F R A D I O B I O L O G Y

FIG. 6-3. Hydrogen peroxide produced (limiting concentration) in pure (oxygen free) water by 1 -5 MeV electrons delivered at

varying intensities29.

with increasing radiation and an equilibrium value is approached only at doses at which the amount of hydrogen peroxide formed reaches a concen-tration comparable to that of solute so that there is competition for the free radicals (see Fig. 6-4). Also, in the presence of a reactive solute the quantity of the molecular products formed is initially independent of dose rate and the proportionality with -^(intensity) does not apply.

FIG. 6-4. T h e effect of increasing solute concentration on the quantity of molecular products produced in the radiolysis of deaerated water by sparsely ionizing radiations. (After ref. 75.)


QUANTITATIVE ASPECTS In the last five years kinetic studies, in particular by American radiation chemists (see summaries in the Proceedings of the Second Geneva Conference30) have provided accurate quantitative data for the primary radiochemical entities. That is the G values (numbers of reactions per 100 eV of energy) for the production of H2, H2O2, OH' and H' are now reliably known. The total number of water molecules affected by radiation is given by

< ? ( _ H 2 0 ) = K G ' ( H - ) + G ! ( O H ) ) + ( G ( H 2 ) + G r ( H 2 0 2 ) )

This type of information is obtained by many different techniques which need not be described here in detail; for example, experiments in which H' and OH" are completely used up by the presence of a dissolved sub-stance can be used to determine the yield of molecular products.

The simple scheme originally proposed by A L L E N 1 7 does not accurately describe the results (see Table 6-1) since the number of H' atoms is greater than the number of OH radicals. Accordingly (as stoichiometry must be obeyed), the amount of the molecular hydrogen is less than the amount of hydrogen peroxide produced in the molecular reaction. This means that in the densely ionizing spurs the reaction H' + H ' H2 occurs to a lesser extent than OH* + OH' -> H2O2. This behaviour is to be expected on the basis of the Lea model (see Fig. 6-1) which provides that the OH radicals are present initially at a higher local concentration than the H atoms. However, the theory does not explain why the inequality of H and OH (and correspondingly H2O2 and H2) is greatest for sparsely ionizing radiations and least for densely ionizing radiations.

Though it is convenient to consider the molecular process as an inde-pendent primary reaction, the magnitude of which for any one type of radiation is constant, this is true only to a first approximation. The mole-cular products are formed by radical recombination in the densely ionizing tracks and if a solute is present at a sufficiently high concentration it will compete for radicals and thereby reduce the amount of radical-radical reaction which provides the molecular products. The local concentration of radicals within S-rays or spurs is such that only at relatively high concentrations (e.g. IO-1 M) is the molecular reaction significantly de-creased (cf. BURTON and K U R I E N 3 1 ) .

Influence of LET The outstanding difference between a- and x-rays to which attention

had already been drawn fifty years ago (see p. 124) is that the formation of the molecular products in pure (oxygen free) water can readily be observed with a-rays. The amount produced is proportional to dose until

1 3 4 f u n d a m e n t a l s o f r a d i o b i o l o g y

very high doses are reached and there is no intensity effect. According to modern concepts we would say that the relative contribution of the mole-cular contribution is increased by an increase in LET.

Table 6-1 shows the trend of increasing "molecular reaction" with

T A B L E 6 - 1

YIELDS OF RADICALS AND MOLECULAR PRODUCTS BY IRRADIATING PURE (OXYGEN-FREE) W A T E R

(after ref. 31)

Radiation G H 2 G h 2 ° 2 G H G O H G - H 2 O

5 -3 MeV a-rays 1-70 1-65 0-55 0-65 3-95 10 kV x-rays 0-65 1-0 2-90 2-20 4-20 60Co y-rays 0-39 0-78 3-70 2-92 4-48

increasing LET, but shows that the total number of water molecules affected ( G - H 2 O ) does not alter significantly—indeed the differences are within the range of possible experimental error.

Dosimetry by Oxidation of Ferrous Sulphate (Fricke Dosimeter) Reference has already been made (see p. 28) to the fact that the

oxidation of ferrous or ferric ions by the radiolysis products of water is one of the most reliable and convenient methods of chemical dosimetry. The reaction is usually carried out in aerated solutions at a pH of less than 1-5. The yield is less at higher values and falls to half in the absence of oxygen. The influence of ionization density is shown in Fig. 6-5.

The oxidation of ferrous sulphate in aerated acid solutions can be written as follows:

. • (radical reaction H 2 O + O 2

l o n l z l n S H O 2 ' + O H ' in presence radiation o f a j r )

F e + + + O H ' F e + + + + O H F e + + + HO 2 " —> F e + + + + H O 2 -H O 2 - + H 2 O - > H 2 O 2 + O H -Fe++ + H 2 O 2 Fe+++ + O H ' + O H -Fe++ + OH* - > Fe+++ + O H -

overall 2 H 2 0 + 0 2 + 4Fe++ l o i u z m S ^ 4 F e + + + + 4 0 H -radiation

i.e. for each ionization leading to radical production four Fe++ are oxidized.


By the molecular reaction H2O ->!(«2 + H2O2)

one Fe++ ion is oxidized. The G(Fe+++) falls with increasing LET since the molecular reaction replaces the radical reaction. In the presence of many, if not most, organic materials (e.g. ethanol, phenol, benzene)

E„MeV Edal teronsMeV

FIG. 6-5. Influence of L E T on G value for oxidation of ferrous sulphate in aerated solution52; O deuterons, A a-particles, • protons. The limiting value of 15.6 is obtained with X- and

(S-rays of energy greater than 200 keV.

abnormally high values were obtained for the GFe+++ and this limited the use of this method for dosimetry as extreme precautions to avoid contam-ination had to be taken. The reason for this increased yield is that the OH" radical instead of oxidizing one Fe++ reacts with an organic molecule (RH) as follows:

RH + OH' -> R" + H2O

R" + O2 RO2' The RO2. radical can then oxidize as many as three Fe++ and this explains the increased yield.

This difficulty can be avoided53-54 by adding sodium chloride to the ferrous sulphate solution when the value for GFe+++ is not altered by the presence of organic substance. The reason is believed to be that in the presence of sufficient chloride all OH radicals react to give chlorine atoms which oxidizes Fe++ preferentially.


R E A C T I O N S O F F R E E R A D I C A L S

Reactivity of the OH Radical The first direct evidence for the formation of OH radicals by y- and

x-rays is derived from the polymerization experiments of D A I N T O N and his colleagues15. The most effective way of polymerizing vinyl compounds in solution is by energetic free radicals* and the existence of such radicals can readily be detected in this way. Acrylonitrile dissolved in water polymerizes on irradiation with x- or y-rays as follows:

CH2=CH+ OH' -> OH . CH2. CH

CN CN acrylonitrile

OH . CH2 . CH + CH2=CH -> OH . CH2 . CH . CH2 . CH I l I l CN CN CN CN

This active radical continues to add on monomer (i.e. acrylonitrile) until it is terminated by reacting with another radical. The initiating radical was proved to be OH" since hydroxyl radicals could be detected spectro-scopically in the polymer.

OH radicals can be produced chemically without ionizing radiations and the two most useful reactions! for their preparation are (i) irradiation of solutions of hydrogen peroxide with ultra-violet light or (ii) from a mixed oxidation-reduction system, the one most commonly used being ferrous sulphate and hydrogen peroxide.

u.v. (i) H2O2 > 20H" (ii) H2O2+ Fe++ -> OH*+ Fe++++ OH- (Fenton's reagent).

By studying the reactions of OH radicals prepared by these methods their chemical reactivity is now well understood32'33 and many of the chemical changes brought about by ionizing radiations in pure water can be reliably interpreted as OH radical reactions. These include:

1. Oxidation, e.g. Fe+++OH' Fe++++OH-; or Br-+OH' -> Br' + OH - .

2. Abstraction of hydrogen atoms in organic molecules, e.g. CH3CH2OH+ OH •-> CH3CHOH+ H2O.

* Simple radicals such as H or O H are sufficiently reactive, but some complex organic radicals are stabilized by resonance and cannot initiate polymerization

t In all methods of preparing O H radicals f rom peroxides HO2 radicals will also be produced by the reaction H2O2 4 OI I ' -ÎIOa' +H2O and a mixture of the two is, therefore, produced in these chemical systems.


3. Addition at a double bond. 4. Reduction of powerful oxidizing agents.

This last type of reaction needs amplification. The OH radical by virtue of its high electron affinity is a powerful oxidizing agent, but in the presence of other strong oxidizing agents it can be reduced. The reducing action was first demonstrated by FRICKE and BROWNSCOMBE 3 4

and has been studied in great detail by HAISSINSKY and his colleagues35. A typical example is the reduction of eerie sulphate

Ce4+ + OH' + H2O -> Ce3+ + H2O2 + H+ All these reactions are complicated and the final yield depends upon sub-sequent reactions. The behaviour of the OH radical in these reduction reactions is very similar to that of hydrogen peroxide, e.g.

Ce4++ H2O2 Ce3++OH-+ OH' This probably explains why the yield from a-rays in these cases is similar to that of x-rays.

In strongly alkaline solutions the OH* radical dissociates

OH' H+ + 0 - (pKa about 10) Since OH" and O - need not react in the same way, there may be differ-ences in reactions carried out in high or low pH.

Reactivity of the H Atom The hydrogen atom will undergo radical reactions like the OH' radical

such as initiation of polymerization and abstraction of hydrogen atoms. Its principal difference from OH radicals is that it is a powerful reducing agent. Thus, instead of oxidizing ferrous iron salts to ferric iron salts it reduces ferric to ferrous. Similarly, while OH oxidizes bromide ions to bromine hydrogen atoms will do the reverse.

Indeed the H atom is a more powerful reducing agent than OH' is an oxidizing agent. Consequently, one would expect a mixture of H' and OH" to have an overall reducing action. In any case one would expect hydrogen atoms to undo all oxidations due to OH'. Yet a notable feature of radiation chemistry of aqueous solutions (in the absence of oxygen) is that they bring about oxidation. FRICKE and HART24, twenty-five years ago, showed that ferrous iron is oxidized to ferric, arsenite to arsenate, etc., even in the absence of oxygen. This apparent paradox led a number of investigators to question the formation of H atoms on irradiation36

(this problem is fully discussed in the earlier edition of this book37) while others38 postulated the existence of other oxidizing species such as OH+.

The existence of the molecular reaction, however, resolves this difficulty. While H* will reverse the oxidation by OH' of substances such as arsenite


so that there is no net change due to the radicals—in the absence of oxygen—the molecular products provide the oxidizing species hydrogen peroxide. The other molecular product, hydrogen gas, is inert in these reactions. The net oxidizing action of ionizing radiations in the absence of oxygen is due to hydrogen peroxide.

The H atom in acid solutions adds on a hydrogen ion H* + H+ -s- H2+ (pKa about 2)

H2+ is a much less powerful reducing agent than H* and indeed may sometimes act as an oxidizing agent39.

Role of Oxygen In the presence of oxygen most reactions produced by ionizing radiations

are quantitatively and qualitatively different from those occurring in its absence. Oxygen has two principal roles:

1. To react with H atoms H* +O2 -> HO2*

to give an oxidizing radical; 2. To add on to organic radicals (produced, for example, by hydrogen

abstraction; RH + OH* -> R* + H2O) to give a peroxy radical R* + O2 -> RO2'

the subsequent reactions of which will be quite different from the parent atom.

With simple inorganic reactions this second process can play no part and the effect of oxygen can be attributed to the formation of HO2 radicals. With organic substrates it is usually not possible to decide unambigu-ously which of the two reactions is responsible for the "oxygen effect" (see pp. 117 and 161).

Little is known about the chemical properties of the HO2 radical in solution. It is an oxidizing agent, though somewhat less powerful (i.e. having a lower or more negative redox potential) than OH' and readily captures an electron

HO2* +e -> HO2-

HO2- is the anion of H2O2 (a very weak acid of pK 11) and in all except strongly alkaline solutions the process HO 2

- +H+ -> H2O2 takes place immediately. After reduction, therefore, the HO2 radical is converted into hydrogen peroxide, which can oxidize suitable substrates further. The HO2 radical can be considered to react in two stages. In the first it is a powerful oxidizing agent of redox potential 1 -5 V capable of oxidizing one equivalent and in the second a much less powerful reagent of redox 0-9 Y capable of oxidizing two equivalents. The total oxidizing capacity


of HO2' is three equivalents. For each molecule of water decomposed by ionizing radiation in the presence of air a total of four oxidizing equivalents are produced.

Besides oxidation, HO2 radicals can probably, like OH radicals, abstract hydrogen atoms from suitable organic substances (RH)

RH+ HO2' R'+ H2O2

It is believed to be less reactive and therefore more persistent than the OH radical. The possibility that HO2 ' can donate an oxygen atom to a molecule containing a lone pair of electrons has been suggested41, e.g.

RNH2 + HO2' -> RNH2 + OH'

O

The exchange reaction RH+ HO2' -> RO-+ H2O

may explain the superiority of HO2 over OH radicals in degrading macro-molecules (cf. p. 162) since RO radicals are known to be very unstable and to decompose further. An OH radical in the presence of oxygen could abstract a hydrogen atom from the organic molecule which could then add an oxygen molecule as follows:

RH+ OH' -> R-+ H2O

R' + O2 -» ROO*

The ROO radical produced in this way is much less likely to dispropor-tionate than the RO radical.

There is, however, great uncertainty about the pH at which the dis-sociation

HO2- -* H++ O2-occurs. Reasons have been advanced42 that this dissociation has a pKa of 2 which means that at pH2 half of the HO2 radicals are in the O2 form and under physiological conditions essentially all will be present as O2

- . Certainly with inorganic ions the reactions of O 2- are different from

those of HO2". Whether O2- will abstract hydrogen atoms from organic compounds or undergo any of the other reactions of HO2 discussed on the preceding page is not known, though it does not seem unlikely. The reactions of HO2 (or O2

-) with organic compounds is one of the relatively few unsolved problems of radiation chemistry of aqueous systems.

The HO2 radical may play a role even in "direct" action since every ionization process liberates an electron which, in the presence of oxygen, is converted to O 2

- (HO2). The size of a target may, therefore, be


extended since the HO2 radical produced in an ionization in a non-vulnerable spot may diffuse and react with a vital centre. This is a special case of the general problem of energy transfer in relation to direct action which has been considered on p. 110. Attention is also focused on the HO2 radical by the great influence of oxygen on radiobiological effects (see Chapter 11) and by experiments with protective agents (see Chapter 19).

Formation of Hydrogen Peroxide in Aerated Solutions Irradiation of water containing dissolved oxygen with sparsely ionizing

radiations leads to the formation of relatively large amounts of hydrogen peroxide in contrast to de-aerated solutions, where, except at high radiation intensities, only traces are formed (see p. 131). The rate of production of hydrogen peroxide is initially proportional to the dose delivered, but eventually reaches a limiting value (see Fig. 6-6)46 which shows that an

FIG. 6-6. Relationship between radiation dose (1 -2 MeV x-rays) and quantity of H2O2 formed in aerated water46.

important back reaction is occurring. In acid solution48 both the initial rate of hydrogen peroxide formation and its final equilibrium value are much greater than in neutral solution.

No reaction scheme has yet been put forward which accounts for the kinetics of hydrogen peroxide formation by sparsely ionizing reactions. The principal reactions leading to the formation of hydrogen peroxide are believed to be47

HO2' + HO2' -v H2O2 + O2 OH' + OH' H2O2*

Competing with these reactions will be the radical removal reaction HO2-+ OH' -» H2O+ O2


When appreciable quantities of hydrogen peroxide have been formed the following process may bring about the equilibrium state:

o h ' + h 2 o 2 - v H 2 o + h o 2 -

h o 2 ' + h 2 o 2 - v h 2 o + o 2 = o h '

In the absence of oxygen most substances capable of reacting with OH radicals will allow the accumulation of hydrogen peroxide formed by the molecular reaction by suppressing the back reaction. In aerated solutions the radical reaction leads to hydrogen peroxide formation and added substance may promote or retard this radiochemical reaction. Substances which react readily with OH radicals, such as inorganic ions, chloride, nitrate, etc., will lower the yield69 presumably by decreasing the reaction

o h ' + o h ' - > h 2 o 2

Additives which are oxidized by HO2' to form HO2- will increase the

yield since one molecule of H2O2 is then obtained per HO2 radical, i.e.

h o 2 ' - > h o 2 - - > h 2 o 2

instead of

2 h 0 2 ' h 2 o 2 + o 2 .

Since the H2O2 yield is increased by the addition of benzene49, ascorbic acid, hydroquinone and cysteine50, it would appear that these are reduced more rapidly by HO2 than OH radicals; if they reacted equally readily the amount of H2O2 formed would be unchanged. Conversely, the sub-stances which reduce the yield can either react more readily with OH radicals or combine with HO2 radicals in a way which does not give HO2

-

or H2O2 (cf. p. 139). Added substances do not influence the yield of hydrogen peroxide by

a-particles which is also independent of oxygen concentration. These results are in agreement with other observations, that the large majority of the radicals formed by a-rays are not accessible to dissolved substances even when these are present in high concentration.

Influence of Concentration in Yield It has been seen in the previous section that the available data can be

explained by postulating that a fraction of the radicals always recombine to give molecular products whatever the concentration of a solute. The remaining radicals diffuse freely and can either react with the solute or recombine. FRICKE and HART24 showed that for all the reactions (oxida-tion, reductions, dissociations) produced by x-rays in aqueous solutions, the yield was constant once the concentration exceeded a minimum value which lay between IO-3 and 10~2 M (see Fig. 6-7). More recent work has amply confirmed the results and there are few well-established exceptions


(e.g. the deamination of amino acids and the decomposition of hydrogen peroxide for which the yield increases with concentration up to the highest obtainable (see p. 150)) which are almost certainly due to the occurrence of chain reactions.

The simplest interpretation for the constant yield is that beyond a minimum concentration, every available radical reacts with the solute and none are lost by recombination. From simple kinetic considerations the number of collisions made by radicals can be calculated. In Table 6-2

FIG. 6-7. Influence of concentration of radiochemical yield for decomposition of formic acid, formaldehyde, methyl alcohol

and oxalic acid24.

the values for the interval between the formation of radicals and the time at which half of these have made one collision are given for different radiations using the Lea model for the distribution of H and OH. On the reasonable assumption that combination occurs at every collision between radicals this represents the time in which 50 per cent of the radicals have been converted to molecular products (i.e. H2, H2O2 or recombination to water). Table 6-2 shows that this time is extremely short for a-rays and is complete before the columns have diffused to any appreciable distance. Chemical effects of free radicals produced by a-rays (and to a lesser extent by all radiation having an ionization density greater than 200/fi) in aqueous solution will, therefore, be confined to the immediate vicinity of the tracks and there will be no significant intermingling of radicals from different tracks unless the dose rate is extremely high.

The position for radiations of low specific ionization is quite different. The rate of recombination is much lower, since the initial local concen-tration is much smaller and intermingling of tracks will occur before an appreciable amount of recombination has taken place (see Fig. 6-2). The distribution of radicals produced by sparsely ionizing radiation can, therefore, be considered as uniform and kinetic treatments similar to those used for reactions in homogeneous solutions can be used.


Lea computed the relative rates at which active radicals are lost by recombination and by reaction with a solute as the function of the concen-tration of the latter. Figure 6-8 shows the theoretical curves to which the

Cone, (molar)x Zp

FIG. 6-8. Influence of concentration of substance dissolved in water on proportion of radicals utilized (based on Lea's model). Z and p are factors which determine efficiency of reaction between solute and radicals and which are independent of

their respective concentration.

limited experimental data available can be fitted. The exact position of the curve relative to concentration depends mainly upon the rate of reac-tion of the substrate molecule with the free radical (i.e. the average number of collisions which a radical has to make with a substrate molecule before reaction occurs) and to a lesser extent on the molecular weight. Experiments with protective agents show that this rate varies greatly from compound to compound (cf. p. 176). For the relatively few sub-stances for which curves of relative yield against concentration have been constructed, the limiting concentration (i.e. where the yield becomes constant) of x-ray induced reactions varies widely*. For the small organic molecules studied by FRICKE and HART23 the value varied from 3 x I O - 4 M

for formic acid to 2 x IO-2 M for oxalic acid, while methyl alcohol and formaldehyde were intermediate at IO-3 M. AS the size of all these molecules is of the same order the different limiting value must be ascribed to variations in the rate of reaction. The low value of IO-5 M for carboxy-peptidase43 is probably due to its high molecular weight and similar values have been obtained for other enzymes. The enhancing effect of oxygen on chemical reactions44 and many biological reactions produced by x-rays

* As the limiting yield is reached asymptotically no final concentration can be given. For the purposes of this discussion the limiting concentration has been chosen as the point where the curve flattens out; the yield at this point was at least 90 per cent of the maximum.


reaches a limiting value at a concentration of IO-5 M (see Chapter 11), and this probably represents the limiting concentration for the reactions involving addition of oxygen to free radicals.

For a-rays the experimental data for the number of free radicals produced is very limited. The only reaction treated in detail is the inactiva-tion of carboxypeptidase43. This is a favourable case, since the enzyme is not affected by hydrogen peroxide so that a true estimate for the yield of radicals available for reaction with the solute can be obtained. The ratio of the ionic yield for a- to x-rays was of the order of 0-04 and this value did not increase significantly with increase in concentration; the results did not indicate that the enzyme followed the theoretical curve of Fig. 6-7 (i.e. that the same limiting M/N value, though at much higher concen-trations, should be reached for a- as for x-rays). DALE, GRAY and MEREDITH4 3 conclude that "the small ionic yield observed for a-rays is due principally to S-ray ionization and that the majority of the labile products have no opportunity of reacting with enzyme molecules because they are readily eliminated within the a-ray column by alternative reaction". This deduction follows logically from the simple kinetic considerations that before a solute can react at all with the radicals it must be present at a concentration which is at least as great as that of the radicals in the columns surrounding the ionizing particle. For effective competition with the radicals the solute concentration will have to be many times greater. With 60 kV electrons (cf. Table 6-2) the radical concentration is IO-6 M and the concentration at which the ionic yield attains a limiting maximum value (i.e. when the solute molecules compete effectively for all the radicals) occurs at about IO-4 M. For a-particles the concentration of the OH radicals is molar and that of the H atoms IO-2 M so that the concentration of the substrate would have to be several times molar before it could compete with radical recombination. In practice, therefore, the radicals never take part in a reaction with the solute.

For the deamination of glycine the ratio of a-ray to x-ray yield is higher at 0-15, but this reaction is complicated since it probably involves a chain process (see p. 150). The a-ray yield is much larger for reactions in which hydrogen peroxide can take part.

Influence of High Dose Rates When considering radical combination processes it is in general only

necessary to deal with radicals formed within the same track since the distance between tracks is much greater than the distance between ionization (or excitations) within the track. For this reason dose rate does not influence the number of primary chemical events. Consequently, the only radiochemical reactions in which a dose-rate effect should be detected are those in which a relatively long-lived intermediary is produced which


T A B L E 6 - 2

I N I T I A L D I S T R I B U T I O N OF RADICALS F O R M E D BY D I F F E R E N T R A D I A T I O N S I N W A T E R AND T H E I R D I F F U S I O N 1 9

a-Particle 1 kV electron 60 kV Electron

H2O+ H 2 O - H2O+ H 2 O - H2O+ H 2 O -or or or or or or

OH - H ' OH - H - OH - H -

Mean separation of primary posi-tive ions (/i) 8 xlO"4

— 5 x 10-3 — 9 x lO- 2 —

Radius of col-umn (/i) 8 x lO- 4 1-5 x 10-2 5 x lO- 3 1-5 x lO- 2 1-5 xlO"2

Initial concentrat-ion of radicals(M) 1-0 8-7 x lO- 3 1-2 x IO"2 1-35 xlO" 3 7-3 xlO -5 Interval after which 50% of radicals will have made one colli-sion with an-other radical (sec) IO"11 IO-9 7 XlO-1O 6 xlO"9 1-6 x lO -6

Expansion of col-umn radius dur-ing this interval 1 0 6 1 0 2 M l M l 7-6

Cone, of radicals at the end of the interval (M) 0-9 8-4 xlO" 3 I -OxlO- 2 1 1 xlO" 3 1-3 x lO -8

* Primary positive ions only are considered in the case of a-radiation. In the case of 1 kV electrons the positive column radius is large enough to include secondary positive ions.

is capable of reacting with another radical. A typical example is polymeriza-tion of vinyl monomer (see p. 136). Here the monomer (M) reacts with a radical to give an organic radical which will react with further monomers to give a polymer. Yet this organic radical can also react with another radical after which it can no longer combine with monomer. This is called chain termination, i.e.

M -I- O H - > M * (organic radical)

M * + M ^ M M *

M M * + M M M M *

F U N D A M E N T A L S O F R A D I O B I O L O G Y

and so on to give a polymer, or

M M * + O H ' - v M M O H (chain terminat ion)

In such a polymerization system the amount of polymer formed is inversely proportional to intensity or dose rate, i.e. the higher the dose rate the smaller the yield. Reports have appeared in the literature (see ref. 37, p. 116) that reactions which did not involve a chain reaction were also influenced by dose rate, but none of these have been confirmed and the original claims must rest on disometry errors.

When dose rates become extremely high then the instantaneous concen-tration of radicals produced will go up since the different tracks overlap. From a chemical point of view the effect will be as if the LET of the radiation were increased. Very high dose rates can be achieved in some generators such as the betatron, synchrotron or linear accelerators where the electrons are emitted over very short time intervals (of the order of IO-6 sec). In these short pulses, dose rates equivalent to IO10 r/min may be attained, although the integrated dose rate will be much smaller as usually there are only 50 such bursts per second. S U T T O N and R O T B L A T 4 5

used an ingenious set of chemical reactions from which they could calculate the ratio of the

G (radical reaction)/G (molecular reaction)

Figure 6-9 shows that with 15 MeV electrons this ratio is independent

11 , U t -r ~ 1 + 4

F

. / / i

r Iora io5 io5 io7 IOr

Dose-rate, rads/sec

FIG. 6-9. The effect of dose rate on the ratio of molecular to radical reaction in water produced by 14 MeV electrons. The ordinate is equivalent to

3GH + G o h +2GHY>OO •— (ref. 45)

G H - G o h + I G H 2 O 2


of dose rate until IO8 r/sec, but falls at still higher values. That means that once IO8 rads/sec is exceeded radical recombination leading to molecular products increases because of track overlap. From a radiobiological point of view the importance of this experiment is that it proves that any dose-rate dependence observed in experiments with conventional machines cannot be ascribed to radiochemical differences as these only appear at these fantastically high rates.

R E A C T I O N S O F O R G A N I C S U B S T A N C E S D I S S O L V E D I N W A T E R *

In the interval since the writing of the first edition the number of reactions studied has increased to such an extent that it would require a whole book to review this subject. No attempt can therefore be made in this edition to present a cross-section of the data and only the reactions of amino-acids and of the purine and pyrimidine bases will be described.

Although each substance shows its own characteristics there is a general pattern for the reactions produced in organic compounds by the radiolysis products of water. In the presence of oxygen the reaction frequently undergoes an entirely different course and in every case the distribution of products is effected.

The principal initial reaction is abstraction of hydrogent:

RH + OH'-> R-+ H2O As most organic compounds have several hydrogen atoms a number of

different radicals, R", may be formed from the same parent substance. In many cases one of the hydrogen atoms is much more labile than another and the formation of one particular radical will therefore predominate.

In the absence of oxygen R' can: 1. React with another radical (X*)

R -+ X' -v RX if X' is a hydrogen atom restoration has occurred.+

* In physiological solutions many, if not most, of the OH" radicals will react with the chloride ions present to give chlorine atoms. Dissolved organic substances will under these conditions be attacked by chlorine and not by OH* radicals. As both of these are highly oxidizing the products may occasionally be the same, but often they are quite different. For this reason the reactions described in this section are not those that will take place when, for example, amino acids dissolved in body fluids are subjected to irradiation.

t This reaction has been most frequently established for OH radicals, but probably also occurs in many cases with H atoms.

J If a second solute (PH) is present then repair is possible by R" + P H RH +P* (see p. 177).

II


2. Dimerize

R" + R' -*• R2

3. Rearrange

R- + R- -> A + B (e.g. HO2- + HO2- -> H2O2 + O2)

If oxygen is present then

R- + O2 -> RO2-is a prominent reaction and in addition the radical HO2' will be there which can undergo a number of unusual reactions*.

OH* + HCOOH -> HCOO' + H2O

COOH

2HC00* -> COOH (in the absence of oxygen)

HCOO-+ OH2- -» H2O2+ CO2

In many cases the fate of RO2' radical is quite different from that of R". The most important difference is that RO2' cannot dimerize., e.g.

CH3CH2OH+ OH- -> CH3CHOH

in absence of oxygen / \ in oxygen / \

C H 3 . C H . C H . C H 3 C H 3 . C H . O H postulated intermediate I l I O2-

O H O H + H O 2 -I

C H 3 . C H O + H 2 O 2 + O 2 acetaldehyde

The fate of the RO2' radical in this case is combination with HO2'. Another type of reaction is where the peroxy radical becomes reduced to give a hydroperoxide which is often quite stable, i.e.

RO2- - > R O 2 H

Amino Acids The principal reaction of all amino-acids dissolved in water is de-

amination with the release of ammonia. The other products are various (see Fig. 6-10) and have only been analysed for in detail for glycine and

* With formic acid the radical formed cannot peroxidize55, but the product produced in the presence of oxygen is different from that produced in its absence because of the reaction of H O ' :


Initiol products from amino acids

NH2- CH2COOH NH2-(CH3) CH COOH

FIG. 6-10. Comparison of initial yield of products obtained by irradiating glycine and alanine solution (1 N conc.) with

x-rays in vacuo56.

alanine56. The yield of some of the products does not continue linearly with dose, but tends to a maximum. Oxygen does not appear to alter the initial reaction, in particular the release of ammonia, but some of the products are different57. In particular hydroperoxides are formed, though the yield varies, being largest for the amino-acids and with non-polar side chains58. (Peptides and proteins59 form hydroperoxides in very small yields.) The most unusual feature of the reaction is that the yield increases continually with concentration (see Fig. 6-11) and does not reach a limit. D A L E et a/.06 have discussed several possible explanations forthisbehaviour, and the most probable mechanism is a chain reaction as follows*:

O H ' + + N H 3 . C H 2 . C O O - O H C H 2 . C O O - + N H 3 + (glycine at p H 7)

NH3+ + H2O - > NH4+ + O H .

The deamination reaction is only given by a-amino acids and amino groups in other positions or in related compounds such as urea or thiourea do not give ammonia in high yieldt. The ionic yield at a given concen-tration is the same for all the oc-amino-acids studied with the exception of

* Thiourea splits out sulphur on irradiation and like glycine the ionic yield increases with concentration to give very high ionic values. No mechanism has been put forward.

t An apparent exception is p-aminobcnzoic acid64 which is both decarboxylated and deaminated when irradiated in aqueous solution.


histidine for which it is higher, and for cysteine which is not deaminated. Colorimetric tests show that the imidazole ring is also attacked when histidine is irradiated61. Deamination of glycine by a-rays is essentially similar to the x-ray induced reaction, except that the yield is much lower62.

Glycine concentration (ng/ml)

FIG. 6-11. Evolution of ammonia on irradiating aqueous solu-tions of glycine with x-rays60. Relationship between ionic yield and concentration of glycine (the arrow gives the value when

dry glycine is irradiated).

The reactions of cysteine and of glutathione are of particular importance in view of the interest which attaches to the inactivation of sulphydryl enzymes (see p. 3 2 8 ) . BARRON63 claimed that the only reaction which occurs is the oxidation 2R . SH R . S . S . R and found an ionic yield of one in the absence of oxygen and of four in its presence, in apparently perfect agreement with simple theory. Detailed work of D A L E and col-leagues65 showed that the reaction is much more complicated. Hydrogen sulphide is formed, and the yield corresponds to a G value of 0-9 at relatively high concentrations of cysteine. The yield varies with pH as shown in Fig. 6-12.

The oxidation of the sulphydryl group also is not straightforward and in addition to the formation of disulphide bonds higher oxidations to sulph-

O O

- J l -


occur66. As a by-product of the irradiation of cystine H2O2 is produced. No hydrogen sulphide is obtained on irradiating cystine which unlike cysteine is deaminated.

1-0

O-B

£ O-S CO ' rn O-V

O-B

0

o-s

(a,) o-t

Oi O-B

J JO^ 10l Jtfi Jlfi JOi JCfi fig Cysteine hydrochloride/ml

W o( O

O O /

I°

O ^

O O

JB pH

FIG. 6-12. Evolution of H2S on irradiating aqueous solutions of cysteine with x-rays65, (a) Influence of concentration on yield (33,000 r at pH 2). (b) Influence of pH on yield (cysteine

hydrochloride concn. 5-IO3 /ig/ml, dose 9500 r).

Purines and Pyrimidines G U Z M A N BARRON and his colleagues67 found that the absorption of

purines and pyrimidines in the region of 2600 A was decreased on irradia-tion in dilute aqueous solution. The decrease was proportional to dose and was greater in the presence of oxygen. The radiosensitivity, as measured by change in absorption spectrum, decreased when the purines were attached to sugars and was smallest of all for nucleic acids. Presumably the free radicals reacted preferentially with groups other than the purine or pyrimidine moiety in the more complex compounds.

H E M S 6 8 found on irradiation of guanosine that the imidazole ring was attacked to give the following product

O H O H N H C H O

N ^ x

N H 2 N N

4-attached to sugar

/ V N H 2 N N H 2

no longer linked to sugar

which is of particular interest as it results in rupture of the purine from its sugar. In the original publication Hems claimed that this reaction did not occur with adenosine, but this selectivity for one of the purines was not borne out by further experiments683, and the corresponding product has now been isolated from various purines including adenosine. If this


reaction occurred in nucleic acid it would lead to the loss of purine from the macromolecule. This, however, is far from being the only reaction and deamination has also been recorded69. Moreover, the ring opening has only been found by HEMS683, in the absence of oxygen.

The pyrimidines—though not the purines—give hydroperoxides on irradiation. The relatively stable thymine peroxide has been isolated in a pure form by EKERT and MOHLER58 following irradiation in aerated dilute solution. WEISS70 has proposed the following mechanism for its formation:

O

HN C - C H 3 OH'

O = C CH

CH3

N H

+ O2 O 2 ' reduction

OH

H

C - C H 3

OH

/

s H CH3 /

x O 2 H

OH

O 2

C

C

H Effect of Radiation on Colloid Suspensions

Besides producing chemical changes, ionizing radiations also alter the physical properties of aqueous colloids, but the mechanism by which these changes are brought about is not understood. CROWTHER and FAIRBROTHER71 observed more than thirty years ago that irradiation of metal sols by soft x-rays produced precipitation if the particles carried a positive charge, but that anionic colloids were stabilized. If the dose of x-rays was not large enough to produce precipitation the colloid was nevertheless sensitized, and the quantity of a coagulating agent required for precipitation was reduced. The changes produced by radiation were found to be permanent and the amount of coagulating agent needed for precipitation was not dependent on the time interval which had elapsed between its addition and the irradiation.

A related effect of x-rays on colloids, discovered in 1937 also by CROW-THER72, is a change in the zeta (or electrokinetic) potential. Using a sol of graphite, small doses of x-rays (i.e. 10 to 100 r) were found sufficient to


bring about an alternate increase and decrease in this property (see Fig. 6-13) which was determined directly by measuring the rate of electro-phoretic movements. As the dose is increased the alternation in zeta potential continues without any change in amplitude but the interval between peaks (i.e. the wavelength) increases. Crowther was well aware that this amazing effect was observed near the limit of experimental accuracy and took elaborate precautions to establish that the measurements were significant and the results have been fully repeated by an independent group73. The change in zeta potential is permanent and independent of the rate at which the dose was given (i.e. the first maximum occurs after 15 r whether this is given at 0-2 r/min or 2 r/min).

The zeta potential is a function of the charge on the surface of the colloid and the thickness of the so-called electric double layer*, which depends almost entirely on the ionic strength of the solution and will, therefore, not be changed by irradiation. The factor most likely to be changed is the surface charge and it is conceivable that the ion pairs produced (or the thermal electron before it is captured to give the negative ion) may combine with the colloid. Although the dose is small only the surface of the particle has to be changed and a simple calculation shows that sufficient electrons were available in Crowther's experiments to bring about a

* The electric double layer is the film of water surrounding suspended particles in which the distribution of the ions is not uniform and differs from that in the bulk of the solution.


detectable change in the surface charge and, therefore, in the zeta potential. No reason can be suggested why the change in charge should alternate.

These experiments provide a challenge, and it is surprising that they have not received more attention*. Biologically, changes in zeta potential are of great interest for two reasons: (i) they are detectable at doses which in general are too low to produce a measurable chemical change, but which are nevertheless sufficient to produce pronounced biological effects; and (ii) cells are most radiosensitive at or near division when colloidal constituents of the nucleus, such as chromosomes, are undergoing changes, which are probably very sensitive to alteration in zeta potential. The possibility that purely physico-chemical changes may be responsible for some of the observed effects of ionizing radiations has to be considered, although it will be difficult to test experimentally.

* More recently work on the changes in surface properties of metals following irradiation in the absence of water has been reported by SMOLUCHOWSKI74. T h e rate of chemical attack (e.g. dissolution in acid) is increased, but these effects only become detectable with doses in the range of 100 million rads and would appear to have no bearing on the remarkable phenomenon discovered by Crowther.

R E F E R E N C E S 1. CURIE, P . a n d DEBIERNE, A., C.R. Acad. Sci., Paris, 1901, 132, 770 2 . RAMSAY, W . a n d SODDY, F . , Proc. Roy. Soc., 1 9 0 3 , 7 2 , 2 0 4 3. RAMSAY, W . , J. Chem. Soc., 1907, 91, 931 4 . KERNBAUM, M . , Radium, Paris, 1 9 1 0 , 7 , 2 4 2 5 . CAMERON, A . T . a n d RAMSAY, W . , J . Chem. Soc., 1 9 0 8 , 9 3 , 9 6 6 6 . BRAGG, W . H . , Phil. Mag., 1 9 0 7 , 1 3 , 3 3 3 7. LIND, S. C., The Chemical Effects of Particles and Electrons, The Chemical

Catalogue Co., New York, 1928. 8 . D U A N E , W. and SCHEUER, O . , Radium, Paris, 1 9 1 3 , 1 0 , 3 3

SCHEUER, O . , C.R. Acad. Sci., Paris, 1 9 1 4 , 1 5 9 , 4 2 3 9. KAILAN, A., S.B. Akad. Wiss., Wien, 1911, 120 (IIa), 1213

10. KAILAN, A., ibid., 1917, 128 (IIa), 787 1 1 . FRICKE, H . and BROWNSCOMBE, E . R . , Phys. Rev., 1 9 3 3 , 4 4 , 2 4 0 12. RISSE, O., Z. Phys. Chem. A, 1929, 1 4 0 , 133 1 3 . RISSE, O . , Strahlentherapie, 1 9 2 9 , 3 4 , 5 7 8 1 4 . WEISS , J . , Nature, 1 9 4 4 , 1 5 3 , 7 4 8 1 5 . D A I N T O N , F . S . , J. Phys. Colloid Chem., 1 9 4 8 , 5 2 , 4 9 0 16. F IQUET, F . and BERNAS, A., J. Chim. Phys., 1954, 51, 47 17. ALLEN, O. A., J. Phys. Colloid Chem., 1948, 52, 479 18. LEA, D. E., Brit. J. Radiol., Suppl. 1, 1947, p. 59 1 9 . D A L E , W. M . , GRAY, L . H . and MEREDITH, W. J . , Phil. Trans. A, 1 9 4 9 ,

2 4 2 , 3 3 20. SAMUEL, A. H . and MAGEE, J . L . , J. Chem. Phys., 1953, 21, 1080 2 1 . PLATZMAN, R . L . and FROEHLICH, H . , Phys. Rev., 1 9 5 3 , 9 2 , 1 1 5 2 22. LEA, D. E., Actions of Radiations on Living Cells, Cambridge, 1946


23. FRICKE, H. and HART, E. J., J. Chem. Phys., 1935, 3, 60 24. FRICKE, H . and HART, E. J., ibid., 1938, 6, 229

FRICKE, H . and HART, E. J., ibid., 1935, 3, 596 2 5 . D A I N T O N , F . S . and ROWBOTTOM, J . , Trans. Faraday Soc., 1 9 5 3 , 4 9 , 1 1 6 0 2 6 . D A I N T O N , F . S . , / . Am. Chem. Soc., 1 9 5 6 , 7 8 , 1 2 7 8 2 7 . FRICKE, H . and HART, E . J . , J. Chem. Phys., 1 9 3 5 , 3 , 5 9 6 2 8 . A L L E N , O . A . , Disc. Faraday Soc., 1 9 5 2 , 1 2 , 7 9 2 9 . G H O R M L Y , J . A . , Radiation Research, 1 9 5 6 , 5 , 2 4 7 3 0 . HART, E . J . , Proc. Second U.N. Conf. on the Peaceful Uses of Atomic Energy,

Geneva, Vol. 29, p. 5, 1958, United Nations, 1959 3 1 . BURTON, M . a n d K U R I E N , K . C . , J . Am. Chem. Soc., 1 9 5 9 , 6 3 , 8 9 9 32. KOLTHOFF, I . M . and MEDALIA, A. I .,J. Am. Chem. Soc., 1949, 7 1 , 3789 3 3 . WATERS, W . A., J. Chem. Soc., 1 9 4 6 , 1 1 5 3 ; Disc. Faraday Soc., 1 9 4 7 , 2 , 1 7 9 34. FRICKE, H . and BROWNSCOMBE, E. R . , J. Am. Chem. Soc., 1933, 55, 2358 35. HAISSINSKY, M., Disc. Faraday Soc., 1952, 12, 133 36. HAISSINSKY, M . and MAGAT, M . , C.R. Acad. Sci., Paris, 1 9 5 1 , 2 3 3 , 9 5 4 37. BACQ, Z. M . and ALEXANDER, P. , Fundamentals of Radiobiology, 1st ed . ,

p. 101-104, Butterworth, London, 1955 3 8 . D A I N T O N , F . S . and COLLINSON, E . , Ann. Rev. Phys. Chem., 1 9 5 1 , 2 , 9 9 3 9 . ROTHSCHILD, W . G . and ALLEN, A . O . , Radiation Research, 1 9 5 8 , 8 , 1 0 1 40. BURTON, M., Symposium on Radiobiology, p. 117, John Wiley, New York,

1 9 5 0 41. ALEXANDER, P . a n d F o x , M . , Nature, 1952, 170, 1022 42. ALLEN, A . O. and ROTHSCHILD, W. G., Radiation Research, 1957, 7, 591 4 3 . D A L E , W . M . , GRAY, L . H . and MEREDITH, W . J . , Phil. Trans. A, 1 9 4 9 ,

2 4 2 , 33 44. PUTNEY, F . K . and PRATT, A. W . , Radiation Research, 1956, 5, 134 4 5 . SUTTON, H . C. and ROTBLAT, J . , Nature, 1 9 5 7 , 1 8 0 , 1 3 3 2 46. EBERT, M. and BOAG, J . W., Disc. Faraday Soc., 1952, 1 2 , 189 4 7 . ALPER, T . , EBERT, M . , GRAY, L . H . , LEFORT, M . , SUTTON, H . C . a n d

DAINTON, F. S., Disc. Faraday Soc., 1952, 12, 266 48. FRICKE, H., J. Chem. Phys., 1934, 2, 556 49. SWORSKI, T . J., Radiation Research, 1954, 1, 123 5 0 . LOISELEUR, J . and LATARJET, R . , Bull. Soc. Chim. Biol., 1 9 4 2 , 2 4 , 1 7 2 5 1 . GRAY, L . H . , CONGER, A . D . , EBERT, M . , HORNSEY, S . a n d SCOTT, O . C . A . ,

Brit. J. Radiol., 1953, 26, 638 52. SCHULER, R. H. and ALLEN, O. A., J. Am. Chem. Soc., 1957, 79, 1565 5 3 . DEWHURST, H . A., J. Chem. Phys., 1 9 5 7 , 1 9 , 1 3 2 9 54. K U R I E N , K . C., P H U N G , P . V. and BURTON, M., Radiation Research, 1959,

1 1 , 283 5 5 . HART, E . J . , J. Am. Chem. Soc., 1 9 5 1 , 7 3 , 6 8 5 6 . SHARPLESS, N . E . , BLAIR, A . E . and MAXWELL, C . R . , Radiation Research,

1955, 2, 135 57. MAXWELL, C. R . , PETERSON, D . C. and W H I T E , W . , Radiation Research,

1955, 22, 431 5 8 . EKERT, B . and M O N I E R , R . , Nature, 1 9 5 9 , 1 8 4 , 5 8 5 9 . ALEXANDER, P . , Fox, M . , ROSEN, D . , STACEY, K . A . , Nature, 1 9 5 6 , 1 7 8 ,

846 6 0 . DALE, W . M . , DAVIES, J . V. and GILBERT, C. W . , Biochem. J., 1 9 4 9 , 4 5 , 9 3 61. BHATIA, D . S. and PROCTOR, B . E., ibid., 1951, 4 9 , 550 62. DALE, W. M., DAVIES, J . V. and GILBERT, C. W., ibid., 1959, 45, 543


6 3 . BARRON, E . S. G., Symposium on Radiobiology, p. 2 1 6 , John Wiley, NewYork1 1 9 5 2

64. MAR, P . G . a n d TCHAPEROFF, I . C . C . , Science, 1951, 113, 549 CORSON, M . , Arch. Biochem. Biophys., 1 9 5 1 , 3 3 , 2 6 3

65. DALE, W . M . a n d DAVIES, J . V. , Biochem. J., 1951, 48 , 129. 6 6 . W H I T C H E R , S . L . et al., Naturwissenschaften, 1 9 5 2 , 3 9 , 4 5 0 6 7 . BARRON, E . S . G . , JOHNSON, P . and COBURE, A . , Radiation Research, 1 9 5 4 ,

1,410 68. H E M S , G . , Nature, 1958, 1 8 1 , 1721 68a. H E M S , G . , Nature, 1960, 1 8 6 , 711 69. SCHOLES, G . and WEISS, J . , Biochem. J., 1954, 5 6 , 65 7 0 . W E I S S , J . , Radiation Research, Suppl. 1, 1 9 5 9 , p. 1 8 4 71. CROWTHER, J . A . and FAIRBROTHER, L . , Phil. Mag., 1927, 4 , 325 7 2 . CROWTHER, J . A . , L IEBMANN, H . a n d LANE, T . B . , ibid., 1 9 3 7 , 2 4 , 6 5 4 73. GRAY, L . H., READ, J . and LIEBMANN, H . , Brit. J. Radiol., 1 9 4 1 , 1 4 , 102 74. SMOLUCHOWSKI, R . , Radiation Research, Suppl. 1, 1959, p. 26 7 5 . D A I N T O N , F . S., Radiation Research, Suppl. 1 , 1 9 5 9 , p. 1

C H A P T E R 7

Effect of Radiation on Macromolecules

THE reasons why the primary chemical lesions that initiate biological damage are thought to involve macromolecules have already been sum-marized in Chapter 4. Reactions with small molecules are unlikely to be of biological importance, because only a minute fraction of the total number present can be effected. Only if the radiation-produced product acts as a cell poison (e.g. peroxide) can a reaction with a small molecule lead to biological effects.

From an analytical point of view reactions involving macromolecules can be detected at low dose levels because of the changes produced in physico-chemical properties. For example, the viscosity of a long chain polymer (say of 5 x IO6 molecular weight) will be halved if one chemical bond in the main chain is broken; i.e. a reaction involving only one atom out of 300,000 present can be detected easily and reliably. The nature of the products cannot in general be determined at this dose level. For this reason a great deal is known about how ionizing radiation modifies the physico-chemical properties of macromolecules, but the actual chemical reactions that occur have rarely been established. From a biological point of view the absence of detailed knowledge about the actual chemical changes is probably not serious since the change in macromolecular properties may be the most important. An isolated random chemical change, in say the side chain of a protein, is much less likely to lead to the loss of biological activity than a single reaction that causes scission or crosslinking. In this respect there is a profound difference between ultra-violet and ionizing radiations since the former will act preferentially on certain groups making up a macromolecule while the ionizing radiations show little selectivity (cf. discussion on p. 184 concerning amino-acid residues affected in an irradiated protein).

The principal macromolecular changes produced by ionizing radiations are:

1. Main-chain scission—This leads to a reduction in molecular weight. Macromolecules are made up of a large number of identical or similar repeating units and there is an equal probability that a break is produced at almost every unit along the molecule. If all the molecules are of uniform

157


molecular weight to start with, then radiation-induced breaks will produce a non-uniform (or polydisperse) product. Many polymers, particularly synthetic ones, are polydisperse at the beginning and degradation by radiation does not change the character of the distribution but only the average molecular weight. Most physico-chemical measurements give a weight average molecular weight or something close to it, but to calculate the number of breaks the change in number average molecular weight needs to be known.*

2. Crosslinking—This can be of two types (see Fig. 7-1). If two different molecules are joined together this is intermolecular crosslinking. As this process proceeds, more and more molecules are joined together until a large network is formed of molecules which are no longer soluble, but only swell in solvents which dissolved the starting material. Initially the net-work is so loose that the gel may be difficult to detect especially with polymers of high molecular weight. A certain number of crosslinks have to be formed before a sufficiently large network is formed to give a gel and consequently there will be a minimum radiation dose before any gel can be detected (see Fig. 7-2). The dose at which the first gel appears is called the "gel point" and on average there will be one crosslink for every molecule1. As the crosslinks are distributed in random fashion some

* For a polymer in which all molecules are the same (monodisperse) these two averages are the same. On irradiation, however, they will become polydisperse and then the weight average will be greater than the number average. When the distribution of sizes is random then weight average = 2 x number average.

FIG. 7-1. Crosslinking of a flexible molecule. (a) before reaction, (b) intermolecular, (c) intramolecular, (d) formation of an insoluble gel network.

E F F E C T O F R A D I A T I O N O N M A C R O M O L E C U L E S 1 5 9

molecules will have no crosslinks while a very few molecules have several crosslinks and it is these that give rise to a network—i.e. the gel. Once the gel point has been reached the proportion of insoluble material in-creases rapidly. The actual slope of the dose response curve depends upon the molecular weight distribution of the molecules in the polymer. If all molecules are the same, gel formation is most rapid (see Fig. 7-2). The melting point of a polymer is largely independent of molecular weight. As crosslinking proceeds, the melting point hardly changes until a point is reached where a small fraction is "infinitely" crosslinked when the melting point jumps up sharply to a value where thermal decomposition occurs. At this point the polymer is termed infusible. In fact only a small fraction is infusible, but this provides a honeycomb-like network which prevents the molten material from escaping.

Dose

FIG. 7-2. Relationship between the formation of insoluble gel by crosslinking and radiation dose. At the gel point one cross-link is formed on average for each molecule present. T h e numbers against the lines represent the ratio of chain fracture to crosslinks; when this ratio exceeds 2 no gel is formed1

(all these curves apply to a sample of polymer which has a range of molecular sizes. The broken line applies to crosslinking of a

polymer in which all the molecules are of the same size).

Macromolecules (e.g. deoxyribonucleic acid (DNA), hyaluronic acid (synovial fluid) and many synthetic polymers) are frequently present in solution as flexible molecules which continuously alter their configuration under Brownian motion. Crosslinks can then be formed between different groups (see Fig. 7-1) in the same molecule. The effect of such intra-molecular crosslinking is to pull the molecule together so that it occupies


a smaller volume in solution and this brings with it a reduction in viscosity without any change in molecular weight. In such randomly coiled long molecules intramolecular crosslinking will predominate, if the cross-linking reaction is carried out in very dilute solutions; at higher concen-trations the reaction will be predominantly between different molecules and give rise to gel networks. The change-over from predominance of one type of reaction to the other occurs within a very small range of concentration. For example (see p. 163), irradiation of polyvinyl alcohol at a concentration of 0-25 per cent never gives any gel, however high the dose, at a concentration of 0-5 per cent the reaction is almost entirely inter-molecular and gel is readily formed.

3. Disruption of secondary structure of macromolecules—Proteins in their native state are maintained in rigid steric configurations by secondary valency forces and do not assume in solution purely random configura-tions, as do most synthetic polymers. The main-chains are constrained in fixed configurations by hydrogen bonds. Radiation disrupts this secondary folding. This type of effect has so far only been encountered in proteins (see p. 180) and in the case of DNA irradiated with U-V, (see p. 202).

Much has been learnt about main-chain scission and crosslinking by radiation from studies with synthetic polymers and these studies have provided the background for interpreting the changes produced in the much more complex naturally occurring macromolecules.

R A D I A T I O N C H A N G E S I N S Y N T H E T I C P O L Y M E R S P R O D U C E D BY I N D I R E C T A C T I O N *

Examples have been found amongst water soluble vinyl polymers; i.e. having the basic structure

R1 R 1

I I — C H 2 — C — C H 2 — C — , etc.

I I R2 R2

(but differing in the nature of the R1 and R2 groups), both of polymers that degrade and that crosslink.

Degradation The extent of main-chain scission has generally been followed by changes

in viscosity, but this is not an unambiguous test since intramolecular

* From a radiobiological standpoint only indirect action involving water is relevant and studies of the irradiation of polymers in dilute solutions of organic solvents will not be considered here and the reader is referred to the work of M A G A T and his colleagues (cf. ref. 2) who have made the major contributions in this field.


crosslinking can also lower viscosity* and an unambiguous method of molecular weight determination such as light scattering must be used.

If polymethacrylic acid

C H 3

I - C H 2 - C -

COOH or its sodium salt is irradiated in dilute aqueous solution main-chain scission occurs3-4'5. The number of bonds broken is proportional to the radiation dose and down to the lowest concentration examined (0-01 per cent) the dilution law was completely obeyed (i.e. the dose of radiation to produce a given change in molecular weight is proportional to the concentration of the polymer). For every 60 eV of energy deposited in the solution one main-chain break is produced; or G (breaks) = 1-6. Since changes in viscosity and in the molecular weight parallel one another it can be concluded that no crosslinking (i.e. production of branched molecules) is taking place and that main-chain scission is the only reaction which affects the macromolecular properties.

The presence of dissolved oxygen is essential for the radiation effect3'4

and in its absence there is no change in the molecular weight except with very high doses which give rise to the formation of appreciable quantities of hydrogen peroxide which has the same effect as dissolved oxygen, f

The mechanism by which the carbon-carbon main-chain is broken has not been established and the exact part played by oxygen is not clear. Two possibilities have been considered4; (1) only reaction of HO2" (or O2

-) radicals formed in oxygenated water (see p. 138) with the polymer leads to degradation and OH' and H" radicals are quite ineffective in this respect or (2) the formation of a peroxy radical may be necessary for degradation

* This was first demonstrated by ALEXANDER and Fox3 who showed that the claim, that a nitrogen mustard (see Chapter 8) and x-rays could both have a degra-dative effect as both lowered the viscosity of DNA, was wrong; the viscosity change was due to intra-molecular crosslinking by the mustard and due to main-chain scission by the radiation.

f The need for oxygen was observed in experiments in which the polymer was prepared by irradiating the monomer with ultra-violet light. When peroxides or x-rays were used to initiate polymerization the polymer produced degraded even in the absence of dissolved oxygen6. This behaviour was traced5 '6 to the presence of peroxide groups, which are formed under these polymerization conditions7 and presumably present a point, which is vulnerable to main-chain scission, even in the absence of oxygen. When the peroxide groups were destroyed by heating the polymer became resistant to anaerobic irradiation6.


e.g. (no detectable change)

unstable and breaks up further.

While ALEXANDER and Fox4 give reasons for favouring the HO2" (or O2-)

mechanism, MAGAT 2 by analogy with polymers dissolved in organic solvents supports the peroxidation view and the nature of the oxygen effect must be considered as unresolved.

Preliminary experiments5 indicate that polystyrene sulphonic acid

is degraded equally in the presence as in the absence of oxygen, but more detailed studies are needed.

Crosslinking All the other polymers that have been studied, i.e. polyvinyl alcohol,

polyvinyl pyrollidone, polyacrylic acid and polyacrylamide crosslink, when irradiated in solution at a concentration greater than about 0-5 per cent5-8'9. Figure 7-3 shows a typical experiment in which a solution was converted by crosslinking into a gel which shrinks on further irradiation because additional crosslinks pull the network together more tightly. The dose needed to reach the gel point (i.e. to produce the first appearance of a gel) depends critically on the concentration of the polymer (see Fig. 7-4). Polyvinyl pyrollidone (as well as the other polymers which were not examined in such detail) concentrations less than 0-5 per cent did not gel* however high the dose and the reaction is largely intra-molecular. At 1 per cent concentration crosslinking is at its most efficient, as the con-centration is increased further the radiation dose needed goes up because

* T h e shape of the dose to gel versus concentration curve (i.e. Fig. 7-4) is al-most independent of the initial molecular weight of the polymer. T h e actual doses to give a gel are higher for lower molecular weight samples but the position of the minimum is not affected.

- C H 2 C H

S O 3

FIG. 7-3. Photograph of 2 per cent polyvinyl pyrrolidone solution.

1. Before irradiation. 2. 1-S X IO5 r (gelled). 3. 7 X IO5 r (heavily crosslinked).

(Bubbles due to trapped gases produced during irradiation.)

[facing p. 16 2

E F F E C T O F R A D I A T I O N O N M A C R O M O L E C U L E S 163

there are more molecules that have to be crosslinked by the free radicals formed in the water (i.e. dilution effect, see p. 47). This complex situation has been quantitatively treated by CHARLESBY and ALEXANDER9 .

At concentrations of 0-5 per cent and greater the viscosity of the solution increases with increasing dose until gel is formed, while at lower polymer concentrations the viscosity progressively falls. This is characteristic for

20

10

-2 0-5 a) <J

1 I 1 I

\ I 1 .-"I''' \ » X T---' K L - -

0-5 2 5 10 Concent ra t ion ,

20 % 50

FIG. 7-4A. Gelation of polyvinyl pyrrolidone at different con-centrations by y-rays. (Gelation dose corresponds to first

production of detectable gel)9.

Concentration of thiourea, %

O Xl

/

/ /

/ /

/ / /

/ r /

/ 7

0-2 0-4 0-6 0-8 1-0 q thiourea/lOOa of polymer

FIG. 7-4B. Thiourea protects against (i.e. prevents) crosslinking of polyvinyl alcohol in 2 per cent aqueous solution by y-rays5.

1 2


inter- and intramolecular crosslinking respectively (see p. 160) and the earlier suggestion9 that in the dilute solutions irradiation produced degradation must now be rejected5-10.

The probable mechanism is that free radicals formed in the water attack the polymers that crosslink to give an active centre. In dilute solution the chance of reactions between molecules is small and the prin-cipal reaction is between different groups within the same flexible molecule. At higher polymer concentrations intermolecular reactions occur. The intermolecular reaction can be prevented by giving the molecules an electric charge5; thus, completely unionized polyacrylic acid crosslinks very well in dilute solution, but if only 5 per cent of all the acid groups in the polymer are ionized no more crosslinking is seen. The electric charge causes repulsion between the molecules so that they cannot get together to produce a crosslink.

The role of oxygen in these reactions has not been investigated in detail but the general effect is to reduce the number of crosslinks formed when the polymer solutions are relatively dilute (i.e. in the range 0-5 to 2 per cent). With more concentrated solutions oxygen plays no part. Presumably oxygen reacts with a polymer radical and thereby prevents it from taking part in crosslinking. It is possible that the "frustrated" crosslink may give rise to main-chain scission.

The simplest process would seem to be the abstraction of hydrogen followed by recombination of the radicals to give crosslinks, i.e.

I O H ' I CH2 v CH-

I i 1 M CH'+ 'CH -v H C - C H I I I l

But this reaction scheme cannot be reconciled with the available quanti-tative data9 nor would it explain why there is no interference by oxygen at higher polymer concentrations, or why the crosslinking is independent of dose rate.

These difficulties can be resolved if it is assumed that reaction of an OH radical gives rise in the polymers to a reactive centre which is capable of combining directly with unactivated polymers, i.e.

P + OH'-vP-

P + P - v P - P

However, there is no chemical justification for such a mechanism. Radio-chemical studies have shown (see p. 147) that abstraction of a hydrogen atom is the most probable reaction of an OH' radical and there is no


reason to believe that the organic radical thus formed can combine with a polymer. Further work is needed to elucidate these interesting cross-linking reactions.

R A D I A T I O N C H A N G E S I N S Y N T H E T I C P O L Y M E R S P R O D U C E D BY D I R E C T A C T I O N

Lord Rutherford observed crosslinking by bombarding paraffin wax with a-particles. In his textbook Radioactive Substances and Their Radiations published in 1913 he writes: "In general the radiations rapidly decompose organic matter with the evolution of gases. . . . Under the action of an intense a-radiation, paraffin and vaseline become hard and infusible. In experiments with large quantities of radium emanation the sticking of stop-cocks coated with vaseline due to this cause is often very trouble-some".

From general considerations discussed in Chapter 5 it is clear that the number of reactions which can occur when an organic macromolecule (liquid or solid) is irradiated are limited. Due to the "cage" effect, dis-sociation into large radicals is unlikely as immediate recombination would occur. The two most probable reactions involving the main chain are (i) the loss of a hydrogen atom (and possible a methyl radical) as this can diffuse out of the "cage" leaving a radical macromolecule, and (ii) the dissociation into two stable molecules which cannot recombine. Reactions of the side chains can be more complex and have received relatively little study.

Most of the polymers studied fall into two distinct classes. Those which crosslink and those which degrade. M I L L E R et al.11 have suggested a simple rule for vinyl polymers: if either R, or R2 in the polymer,

R I

- C H 2 - C - , R 2

(or both as for polyethylene) are hydrogen then the polymer crosslinks; otherwise it degrades.* Table 7-1 summarizes the available data for different polymers12.+ The surprising feature is that in so many cases the reaction is wholly of one type or the other if complications due to the pres-ence of oxygen are avoided (see p. 172).

* Polyvinyl alcohol appears to be the only exception as it is claimed to degrade10. It is not, however, certain how effectively oxygen was excluded in this experiment.

t All the irradiations have been carried out with sparsely ionizing radiations and the influence of L E T has not been studied.

I


T A B L E 7 - 1

BEHAVIOUR OF S O L I D POLYMERS UNDER R A D I A T I O N 1 2

(in the absence of oxygen)

Polymers which crosslink Polymers which degrade

Polymethylene Polyisobutylene Polyethylene Polytetrafluorethylene (PTFE) Polypropylene Polymonochlorotrifluorethylene

(Kel F) Polystyrene Poly- methyl styrene-Polyvinyl chloride(?) Polyvinylidene chloride Polyvinyl alkyl ether Polyvinyl methyl ketone Chlorinated polyethylene Chlorosulphonated polyethylene Polyvinyl acetate Polyacrylonitrile Polymethacrylonitrile Polyacrylic acid and esters Polymethylacrylic acid and esters

(polymethyl acrylate) (polymethyl methacrylate) Polyacrylamide Polymethacrylamide Rubber Polybutadiene Polychloroprene (Neoprene) Polyamides

nylon polycaprolactam

Copolymers Cellulose styrene-butadiene cellulose derivatives: acetate, butadiene-acrylonitrile nitrate, etc. styrene-acrylonitrile Copolymers vinyl chloride-vinyledene chloride butyl rubber

Polydimethyl siloxane Polyphenyl siloxane Polyethylene oxide

Crosslinking From the shape of the gel versus dose curve the proportion of main-

chain breaks to crosslinks can be computed1 (see Fig. 7-2). If the ratio is greater than two no gel is formed at all. With polyethylene and polystyrene the ratio of breaks to crosslinks is less than one in ten when there is no oxygen present13. With the halogenated polymers there is always an appreciable amount of degradation, presumably because highly reactive halogen atoms are released (cf. ref. 1 2 ) . SCHULTZ and BOVEY14 claim that with polyacrylic acid esters

- C H 2 - C H -

I C O O R


main-chain scission occurs as well as crosslinking, ranging for different R groups from 0-2 to 0-7 breaks per crosslink. But as oxygen was not excluded the possibility that degradation is due to oxygen must be borne in mind. Polypropene15

C H 3

I - C H 2 - C H -

seems to be a true exception to this rule as main-chain scission and cross-linking occur with nearly equal efficiency.

Degradation In the polymers that degrade the simultaneous formation of crosslinks

would result in the formation of a branched molecule. This can be tested for relatively easily by comparing the values for the molecular weight obtained by light scattering and by viscosity measurements. The relation-ship [7?] = K- (Mw ) a (where [rj] is the intrinsic viscosity and Mw the weight average molecular weight) applies only to linear polymers and if the molecule is branched the viscosity will be smaller than that given by this formula. Light scattering will give Mw whether the molecule is linear or branched. If on irradiation some crosslinking occurred at the same time as degradation then the viscosity of the irradiated polymer will be lower than the Mw (determined by light scattering) would suggest.

Two polymers which degrade under irradiation, polymethacrylic acid16

C H 3

I - C H 2 - C -

I C O O C H 3

and polyisobutylene17

C H 3

I - C H 2 - C -

I C H 3

have been studied in great detail and there is no evidence for branched molecules being formed (i.e. no indications of any crosslinking). The viscosity of irradiated polymers agreed in every case within experimental error with the molecular weight as determined by light scattering. The remarkable feature of the degradative reactions is that the number of main-chain bonds broken is directly proportional to the amount of radiation and that this relationship is obeyed over a thousandfold range of radiation dose. Mathematically, this relationship is conveniently expressed4.16 as

1 1 = kR

M i M0


where M0 and Mi are the molecular weights before and after irradiation and R the radiation dose.

Another way of representing the same behaviour is

1 — = k (R + R0) or log Mi = k'-log {R + R0) Mi

where R0 is a constant that corresponds to the dose that would be needed to reduce a polymer of infinite molecular weight to the molecular weight of the polymer used (i.e. having molecular weight M0).

Co-polymers A mixed polymer prepared by polymerizing together a monomer that

gives rise to a polymer that degrades (polyisobutylene) with a monomer that gives a crosslinking polymer (polystyrene) provides an interesting situation of simultaneous crosslinking and degradation18. The detailed data for polymers containing different ratios of these monomers is given in Fig. 7-5. The viscosity decreases steadily as the system approaches the gel point because breaks are being produced in the isobutylene parts of the molecule. At the same time crosslinks are being formed in the poly-styrene parts and these eventually give rise to gel.

Formation of Volatile Products In addition to the changes produced in the macromolecular properties

volatile products are also formed. The gases released have been analysed in a number of cases but no very clear pattern emerges (cf. ref. 12). In conformity with theory, the gas released on irradiation in the highest yield is hydrogen and in general the quantity formed is proportional to the radiation dose though the absolute amount varies from polymer to polymer. The methacrylates in addition to hydrogen liberate large quan-tities of CO and CO2Which arise from the dissociation of the ester group16. This can rearrange as follows

CH3 CH3 I I

- C H 2 - C v - C H 2 - C - +CO2

I I COOCH3 CH3

CH3

or - C H 2 - C - + CO I

O

CH3


to give off a stable molecule which cannot be trapped by the cage effect. This reaction is completely analogous to the decomposition by deuterons of long-chain fatty acids (see p. 109). For polymethylmethacrylate it would appear that approximately equal numbers of main-chains and side chains are broken. The gases given off during the irradiation are trapped

JO

2-0

* £

IO

0-5

0-3 •6 02 > >

O-I

> 005

0

20

40 JZ

I 60

80

100

S S

<*> - *

t» A

f r r J

. x A

f r

A A - I

• 6

a \ line

gel Iicat forr

es not

on XX

se of a I

(A)

\ \ IT H

N

\ T \

(B)

OOl 002 005 01 0-2 Rodlatlon dose,

0-5 IO pile units

FLG. 7-5. Decrease in the intrinsic viscosity (Fig. 7-5A) and formation of gel (Fig. 7-5B) in a copolymer of which one com-pound (polystyrene) undergoes crosslinking and the other component (polyisobutylene) degrades18.

+

A •

o

100% polyisobutylene 80% 5 0 % 20%

20% polystyrene 5 0 % 80%

100%

within the polymer at high pressures. On heating, the polymer becomes plastic and the gases come together to form large bubbles and give a foam-like structure.

Mechanism of Crosslinking No satisfactory reaction scheme has yet been put forward which predicts

why some polymers crosslink and others degrade. The fact that all these radiation effects are completely independent of dose rate (cf. ref. 12) and strictly proportional to the absolute dose delivered imposes serious

I


limitations on the possible reactions that can be envisaged for crosslinking. The simplest way of forming a crosslink is by the combination of two radicals, but the probability of two activated entities being produced independently and in sufficient close proximity to form a link varies as the square of the dose. Moreover, as the lifetime of radicals is finite because they can be lost by parallel reactions a crosslinking process by radical recombination would be dose rate dependent. In his original paper CHARLESBY1 suggested that crosslinking occurred in polyethylene as follows:

I I C H 2 -> C H ' + H '

I I

I l l l C H ' + C H 2 C H - C H + H ' I l l l

H ' + H ' ^ H 2

The objection has been raised that a normal CH" radical would not have sufficient energy to extract a hydrogen atom but this might be countered by postulating that radiation gives an activated radical. Alterna-tively reaction of an ionized polymer molecule with a neutral polymer molecule has been considered and the occurrence of this type of process has been unambiguously demonstrated in the gas phase with the mass spectrometer for the simple paraffin methane19, i.e.

I I I + C H 2 - v C H 2 + - v C H 2 + + e I l l l

C H 2 + + C H 2 - v C H — C H + H 2

I l l l H 2 + + e - v H 2

Attractive as this reaction is it does not provide an explanation why no crosslinking at all is seen in some polymers and why the energy to produce a crosslink varies greatly in different polymers. The status of the nature of the crosslinking reaction has been very fully discussed by CHARLESBY1 2 .

Recent unpublished data from Charlesby's laboratory (private com-munication) suggests that crosslinking by radical recombination may after all be the correct mechanism. The difficulties of dose rate independence mentioned above disappears if it is postulated that the free radical wan-ders along the chain from carbon atom to carbon atom (—CH2- CH • CH2—) until it comes adjacent to another radical when a crosslink is formed Evidence for the movement of the radical is provided by electron spin


resonance. The movement of a radical along the chain also provides an explanation for the rapid disappearance of double bonds from irradiated polythene.

Mechanism of Degradation While the energy left in a C—C bond by an ionization is very much

greater than that needed to disrupt it the cage effect prevents main-chain scission by the process

I l I l - C - C V — C + C —

I l I l as these radicals will immediately recombine in the solid or liquid state. ALEXANDER, CHARLESBY and Ross16 suggested that main-chain scission could only occur if the molecule could undergo a slow rearrangement to give two stable ends that will not recombine "in the cage". One such rearrangement which would explain why both polyisobutylene and polymethacrylic acid* degrade is as follows:

C H 3 C H 3 C H 2 C H 3

- C H 2 - C - C H 2 - C V — C H 2 - C + C H 3 - C — I l I l

R R R R

This mechanism is supported by the fact that in polyisobutylene for every main-chain break one double bond is formed which has an infra-red frequency that corresponds to the proposed structure. The weakness of this hypothesis is that it does not provide an explanation why vinyl polymers of the type,

- C H 2 - C H -

I R

crosslink, but do not degrade.

Trapped Radicals Information about the intermediate stages in these reactions may be

obtained in simple systems from electron spin resonance measurements. Evidence for free radical intermediates in radiation chemistry has been provided by radical trapping agents such as the free radical DPPH and also from polymerization studies20. In liquids, their life time is short

* The breakdown of the ester side chain (see p. 168) was ascribed to a separate ionization. This could also explain why one break is produced in polymethacrylic for every 61 eV of energy while only 20 eV are needed in polyisobutylene. In the latter polymer every ionization would lead to main-chain scission since no separate reactions in side chains are possible.


because of recombination, but in solids with limited molecular movement the persistence of free radicals for many days has been known for more than thirty years (see C A R O T H E R S in his pioneer studies on the making of synthetic rubber21). That some radicals remain in polymers after irradi-ation was first demonstrated by conductivity and polymerization measure-ments22-23, but more recently the use of electron spin resonance (see p. 118) has made the detection of radicals (and free electrons) much easier.

The trapped radicals may give rise to further reactions when the polymers are dissolved. A L E X A N D E R et al.16 looked for radical recombina-tion by comparing the viscosity of irradiated polymethyl methacrylate dissolved in chloroform only and in chloroform containing a substance that readily reacts with radicals (i.e. prevents polymer radicals from recombining), but found no difference. If oxygen is given time to diffuse into the polymer then peroxy radicals are formed that may take part in subsequent reaction (see p. 117).

Quantitative electron spin data, that permits the evaluation of the number of radicals that are formed, has only rarely been obtained. I N G R A M et al2i find one radical for every 61 eV in polymethyl methacrylate, as this is exactly equal to the energy needed to produce a main-chain break16 it is tempting to associate the radical with this reaction. In poly-thene on the other hand the number of persistent radicals is very small unless the sample is irradiated and measured at liquid nitrogen tempera-tures25. On warming the curve obtained changes instantly and a pattern is obtained which indicates that there are many fewer residual radicals and that these are of a different type. Further work along these lines in other systems may provide information whether the radicals left in organic solids at room temperature represent true intermediates in the radiation chemical process or merely "left-overs" after the main reaction has finished. The radicals that are revealed by e.s.r. at room temperature are those that are prevented for steric reasons from recombining and the magnitude of the e.s.r. signal at room temperature depends therefore on the state of the sample and will be higher for a crystalline than for an amorphous specimen. Yet the radiochemical changes will be the same in the two samples.

Oxygen Effects Three distinct effects must be distinguished:

1. A difference in the primary radiation reactions when the irradia-is carried out in the presence of oxygen.

2. The diffusion of oxygen into the irradiated specimen after irradiation and its reaction with radicals (or other radiation products). These residual radicals are not necessarily the same


as the initial radicals produced immediately (i.e. within IO-9 to IO-6 sec) after irradiation. The product formed by exposure to oxygen after irradiation need not therefore be the same as that produced if oxygen is present during the irradiation.

3. The greater the susceptibility of irradiated polymers to oxidation by aerial oxygen.

In liquids, 1 and 2 cannot be distinguished because oxygen can diffuse rapidly. In solids this is not the case and the slow movement of an oxygen front at the rate of less than 1 mm per month can readily be seen in rods of irradiated polymethyl methacrylate which change colour on reaction with oxygen. A well-defined layer can be observed in a cross-section to move steadily inwards as oxygen penetrates. The amount of dissolved oxygen is insufficient to react with most of the radicals that are present and it has to be replenished before full peroxidation can occur. The slow diffusion (i.e. the slow peroxidation of residual radicals) is confirmed by electron spin resonance measurements.

The third type of effect is not immediately relevant to radiation biology, but has serious implications for the practical application of irradiated plastics (notably rubber) which deteriorate more quickly in use than the unirradiated product. Aerial oxidation is often a chain reaction that has to be initiated in some way and the radiation produced radicals are very efficient in this respect.

The amount of oxygen present in organic solids is low and does not usually give rise to a significant oxygen effect on irradiation (i.e. effect 1). To observe this the polymers have to be irradiated as very thin films into which oxygen diffuses rapidly during the irradiation (see Table 7-2). In

T A B L E 7 - 2

E F F E C T OF O X Y G E N O N C R O S S L I N K I N G OF P O L Y T H E N E 1 3

Condition of irradiation % "gel "fraction

Wads of Specimen Air Vacuum (or N2) filmsj

Rod 1 cm diam. Pile* 72 77 Film 5(V thick ,, 40 72

„ 18M 1 MeV electront 26 59 62 „ 50/u. J > 11 M 31 64 65 „ IOOfl 11 11 11 51 66 67 „ 175 f 11 ) 1 11 62 67 69

* Equivalent to 2-5 X IO7 rads of y-rays. J Films packed in tight wads 200/* thick, t 1-8 X IO7 rads at 3 x IO6 rads/min.

I


thick samples no difference is observed between irradiation in the absence and presence of oxygen. The limiting thickness depends on the dose rate\ at low dose rates thicker films will show an oxygen effect as there is more time available for oxygen to diffuse in, so as to replace the oxygen that has reacted.

In polythene films, A L E X A N D E R and T O M S 1 3 observed that the presence of oxygen changes the radiation effect from essentially only crosslinking to one in which there is approximately one main-chain break for every crosslink. From the gel versus dose curves it can be deduced that the influence of oxygen is to introduce an additional process of main-chain scission, but not to reduce the total number of crosslinks, which are the same whether oxygen is there or not. If the breaks were produced at the expense of crosslinks (i.e. if a primary radiation event that gave rise to crosslinking in the absence of oxygen became a break in the presence of oxygen) then the gel point would be shifted to higher doses. This excludes the mechanism proposed by CHAPIRO 2 6 that the active centre that forms crosslinks becomes peroxidized.

A L E X A N D E R and T O M S 1 3 explained their observation by postulating that an O 2

- radical is formed because oxygen is well known (see p. 105) to have a much greater affinity for low energy electrons than —CH2— groups. In the absence of oxygen the secondary electrons do not lead to chemical reactions as negative organic ions are not very reactive. In the presence of oxygen those secondary electrons are converted into highly reactive O2" radicals which react with the —CH2—CH2—CH2— chain either to give a break directly or to form a peroxide that subsequently decomposes with main-chain scission.

In polystyrene, the oxygen effect is more dramatic as no gel is produced at all if thin films as opposed to rods are irradiated in air13. This means that more than two breaks are produced for every crosslink in the presence of oxygen, but no molecular weight measurements are available to deter-mine the exact ratio. Polystyrene in the absence of oxygen is very radio-resistant because of energy transfer processes (see p. 177) and only something in the order of one ionization in sixty gives a crosslink. Since the number of secondary electrons must be equal to the number of ioniz-ations there will be many more O 2

- radicals than crosslinks and the magni-tude of the oxygen effect in polystyrene is readily explained on this basis.

In polymers which degrade, such as polymethyl methacrylate and polyisobutylene the effectiveness of main-chain breaking was found to be the same in the presence and absence of oxygen16'17. But distinct changes in the ultra-violet absorption spectrum were produced only if the irradi-ation was carried out in the presence of oxygen, and this was the first demonstration8 that oxygen influences chemical reactions induced by the direct action of radiations.


Tetnperature Effect Both crosslinking and degradation16'17'27 occur less readily as the

temperature is lowered (see Fig. 7-6 and also Fig. 2-3, p. 53), but no theoretical explanation has been advanced for this phenomenon which

4-0

OI-ZT1SX) 100 200 3001+ 27°C) 400

Temperature, 0K

FIG. 7-6. Effect of temperature on crosslinking of polythylene by y-radiations27.

seems to be characteristic of "direct action" and is not confined to syn-thetic polymers. The observation that the e.s.r. pattern of polythene*25) and of proteins*45' is different when irradiated at 20° C than when ir-radiated at —195° C and then allowed to warm up to 20° C indicates a possible approach for studying the reasons for the temperature effect.

P R O T E C T I O N O F P O L Y M E R S

Much information has been obtained about the mechanism of protection by added chemicals from studies in synthetic polymer systems. The possible ways by which protection can occur have been discussed in Chapter 5 (see also p. 186, 193 and 210).

Indirect Action The degradation of polymethacrylic acid in aqueous solution can be

protected against by the addition of a large variety of substances28-29, a representative selection of which is shown in Table 7-3. The kinetics of protection are complex since the amount of protection is not always proportional to the concentration of protective agent—this quantitative aspect is discussed fully in a review on mechanisms of protection in vitro30. The protection in this system can occur (see p. 110) either by competitive removal of the degradative material (i.e. HO2* or O2

-) or by


T A B L E 7 - 3

P R O T E C T I O N OF POLYMETHACRYLIC A C I D AT A 0 - 0 2 5 PER CENT CONC. AND P H 7 AGAINST DEGRADATION BY 1 0 0 0 r OF X-RAYS

Substances tested Protection (%)* at the following concentrations

Substances tested 1 X I O - 4 M 1 X I O - 4 M 8 x I O - 4 M

Chelating agents Sodium diethyldithiocarbamate 54 67 87 Dithiooxamide 63 68 74 8-Hydroxyquinoline 64 72 83 8-Hydroxyquinoline + 20

2 x IO'4 M CuSO 4 Non-ehelating agents

2-Hydroxyquinoline 29 47 Allylthiourea 43 54 69 Allylthiourea 51 Thiourea 7 52 72 /5-Phenylethy lamine 0 42 74 Allyl alcohol 0 10 66

Urea 0 Glycine 18 Ethylamine 12 Tyrosine 43 Tyramine 71 Cystine 9 Cystamine 66 Glucose 47 Sodium cyanide 80 Sodium azide 54 Sodium formate 37 Sodium acetate 0 Sodium propionate 11 Sodium caprylate 54

* % Protection = C — T/CX 100 where C and Tare the % decrease in viscosity in the absence and presence, respectively, of the protective agent.

repairing through a hydrogen transfer reaction (see p. 117) an unstable polymer radical which is an intermediate stage in the breakdown of the polymer. While most of the protective agents contain a group capable of donating a hydrogen atom we believe that the competition mechanism is the more likely because amines are equally effective in their ionized as in their un-ionized form 29.While they can remove radicals by direct inter-action (i.e. competitive removal) in either form they can only act as transfer agents in their un-ionized form.


Added substances also protect polymers like polyvinyl alcohol solution against crosslinking8-9 and Fig. 7-4b shows how the dose needed to reach the gel point is increased by the addition of thiourea to the solution. While protection in these systems has not been studied to the same extent as with polymethacrylic acid it does appear probable that repair by hydro-gen transfer plays an important part5-8, i.e.

For example, un-ionized phenylethylamine protects polyvinyl alcohol whereas the ionized amine does not31, in contradistinction to solutions of polymethacrylic acid which are protected by both29.

Direct Action The fact that nearly one hundred times as much energy has to be

deposited in polystyrene than in polyethylene (i.e. 1760 eV and 20 eV respectively) to produce a crosslink shows that energy deposited in a —CH2— group will migrate efficiently into an aromatic ring where it is dissipated in a way that does not lead to the production of macromolecular changes (also the high radiation resistance of terylene,

can be explained in this way). Transfer of energy leading to the prevention of crosslinks was demonstrated very clearly in a series of substituted dodecanes and the details of this experiment are shown in Fig. 5-2 on

Protection by energy transfer to an aromatic centre is also seen when radiation gives rise to degradation. Thus the energy to produce a break in polyisobutylene is 18 eV which is increased to 35 eV in a copolymer having 20 per cent of styrene and to 100 eV if the styrene component is raised to 80 per cent18 (cf. Fig. 7-5).

Energy transfer is not limited to groups within the same molecule but also takes place between molecules. This was first observed in films of polymethyl methacrylate16 containing a variety of additives which reduced degradation. The possibility that the additives react directly with the polymer chain at the point of ionization and thereby rejoin (or heal) the break is improbable on statistical grounds since the chance is small that a molecule of the protector, present to an extent of about 1 per cent or less, happens to be at the joint of energy deposition. Later, more detailed studies provided direct proof that the protector did not combine chemically

P H + OH* - v P" polymer polymer radical

P* + X H - v P H + X" protective agent repaired polymer

( C H 2 ) 6 O O C C O O — ,

p. 113.


with the polymer and that an energy transfer mechanism was respons-ible32. Many different substances are effective (see Table 7-4).*

T A B L E 7 - 4

P R O T E C T I O N OF S O L I D P O L Y M E T H Y L METHACRYLATE AGAINST D E G R A D A T I O N BY THE DIRECT A C T I O N OF

y -RAYS 3 2

Protector Amount present Protection (%) ( % ) *

Diphenylthiourea 3-6 69 Phenol 2-7 52 a-Naphthylamine 2-5 78 ,'-'-Naphthylaminc 3-3 73 ,S-Naphthol 5-3 24 a-Naphthol 5-5 82 Benzoic acid 1 0 76 2,4-Dinitroaniline 4-2 72 Diphenyl 2-5 56 Triphenylmethane 5-2 71 Anthracene 2-1 37 Phenanthrene 2-2 51 2,4-Dinitrophenol 3-2 68 Pyrene 4-1 56 Ethylurea 10-0 12 yym-Dimethylurea 1 0 0 15 Medicinal paraffin about 10 0 3

* Protection is defined as (Ep —E)/(ES) x 100 per cent, where E is the energy deposited to produce a break in the pure polymer itself, the value being about 60-65 eV per break; Ep is the energy required to produce a main-chain break in a polymer containing the protective agent.

Intermolecular energy transfer was looked for when degradation occurred because of the hypothesis16 that this reaction required a complex molecular rearrangement that does not occur immediately. Consequently, the energy would have to reside in the molecule for a comparatively long time (i.e. of the order of IO-7 sec) during which it would have a chance to be transferred.

Crosslinking probably does not have to wait for molecular rearrange-ments and consequently the opportunity for zraferaiolecular energy transfer

* Degradation of polymethyl methacrylate films by a-rays from polonium can-not be protected against by additives33. Presumably the high local release of energy that occurs in the tracks of the a-particles cannot be removed by transfer.


is much less. This was found to be the case as added substances which protected against degradation did not protect polyethylene in vacuum13.* Recently, C H A R L E S B Y 1 2 has found evidence that protection against cross-linking in the solid state can be obtained by a repair mechanism of the type first encountered in crosslinking of polymers in solution (see p. 177).

P H Y S I C A L A N D C H E M I C A L C H A N G E S P R O D U C E D I N P R O T E I N S BY D I R E C T A C T I O N

Much work has been done on the in vitro irradiation of proteins and it has been found that biological properties, particularly enzymatic activity, are lost following attack by indirect action in solution as well as by direct action when the proteins are irradiated in the dry state (this aspect is discussed in more detail on p. 208). Direct action is, however, very much more efficient and most proteins are inactivated when somewhere between SO and 200 eV are deposited within them, while it needs the attack of many OH radicals (somewhere between 10 and 200) produced in the surrounding water. The dose-response curve shows that inactivation is not a co-operative phenomenon requiring many reactions, but that one OH radical is sufficient to inactivate if it reacts at the right point. The remaining OH radicals are wasted by reacting at unimportant centres (see p. 191).

The great efficiency of direct action is surprising since biochemical experiments have shown that the whole of a protein molecule is not needed for enzymatic activity and chemical changes occurring in large parts of it are unlikely to affect its action. This suggested to Platzman that the direct action of irradiation produces a general denaturation and recent experiments (see p. 183) have fully confirmed this prediction.

It is convenient to distinguish between covalent radiochemical changes that change the chemical nature of the side groups (e.g. by splitting CO2 off acid groups) and the changes in the steric configuration of the protein molecule, f Changes in the secondary (and tertiary) configurations may

* If thin films of polyethylene are irradiated in air then added substances reduce the amount of degradation due to the presence of oxygen13.

f The structure of proteins must be described at several different levels. The primary structure is the one usually considered by the organic chemist, namely the order in which the different amino acids are strung together along the polypeptide chain or chains. The secondary structure is the configuration adopted by the polypeptide chain (e.g. whether it is present in the fully extended form or whether it is coiled in some way). T h e tertiary structure is concerned with the way the polypeptide chain (in whatever configuration it may be) is arranged within each globular molecule. By analogy with a ball of wool the secondary structure repre-sents the organization of the fibres within the yarn and the tertiary structure is the way the yarn is arranged within the ball.

i3


follow from covalent reactions but they are also brought about by treat-ments that disrupt hydrogen bonds only and which do not alter the actual chemical constitution. The direct action of radiation brings about both types of changes.

Disorganization of Secondary Structure One of the earliest reports of the effect of x-rays on dry proteins was

made by A S T B U R Y and WOODS 3 4 who found that the physical properties of wool fibre were modified by ionization radiations. The most readily detectable effects were an increase in the amount of supercontraction and a decreased tendency to acquire a permanent set. The observed changes in these complex phenomena can be brought about either by breaking peptide bonds in the main chain or by severing disulphide bonds which link the polypeptide chains within the fibres. Recently, A L L E N and ALEXANDER 3 5 have found that lower doses of radiation which do not produce these marked changes do affect the hydrogen bonding in the crystalline areas of the fibres in a way comparable to that seen with bovine serum albumin (see p. 183).

S V E D B E R G and BROHULT36'37 had already in 1 9 3 9 made an observation which suggested that intensive interference with hydrogen bonds occurred on ionization in the giant protein molecules, the haemocyanins, which are found in the blood of certain arthropods and molluscs and which have molecular weights of up to ten million. The haemocyanin from the snail Helix pomatia has received the most detailed study and is shown to be made up of identical sub-units into which it can be reversibly dissociated by changes in the pH, the ionic concentration, and by the addition of hydrogen-bond breaking reagents such as urea37. The molecules having an initial molecular weight of 8-9 x IO6 first dissociate into halves and finally into eighths. These fragments are held together by secondary valency forces such as hydrogen bonds. The molecule is split in half (see Fig. 7-7) when irradiated with a-particles under conditions when the action must have been direct. Although the fragments have the same physical properties as those produced by dissociation with salts they are chemically modified since they cannot be reconstituted. The irreversible breakdown is referred to as "splitting" in distinction to the reversible dissociation process and occurs whenever a single a-particle passes through it. This reaction is probably one of the earliest examples of a single-hit target process where the target corresponds to the physical dimensions of the molecule.* As the probability that an ionization occurs

* PICKELS and ANDERSON22 using a different haemocyanin (Limidus polyphemiis) found a similar splitting into halves by x-rays. But their report has many puzzling features which make it desirable that this reaction be re-investigated.


in a given part of the molecule is extremely small, one must conclude that the highly specific splitting reaction can be brought about by energy absorbed anywhere within the molecule, i.e. that energy transfer occurs within the molecule.

A L E X A N D E R and H A M I L T O N 3 9 have made an extensive investigation of both the physical and chemical changes produced when bovine serum albumin is irradiated in the solid state. Less detailed studies with three other proteins, trypsin, lysosyme and bovine y-globulin indicate a very similar behaviour and the conclusions reached with albumin probably apply to globular proteins in general.

FIG. 7-7. Splitting of haemocyanin molecule by the direct action of a-particles. (a) Ultra-centrifuge sedimentation diagram of haemocyanin before irradiation; (b) Sedimentation diagram of haemocyanin after irradiation at 20°C; (c) Sedimentation diagram after irradiation at — 180°C. The original peak corres-ponds to a material of molecular weight of 9 X IO6 while the component produced by irradiation, clearly a homogenous

product, has a molecular weight of 4"5 x IO6 37.

On irradiation the sedimentation constant of bovine serum albumin increases, but there is no corresponding alteration in molecular weight and this change therefore reflects a change in the shape of the protein. Figure 7-8 shows that the amount of protein with altered sedimentation characteristics increases exponentially with dose. From these experiments it can be calculated that the protein molecule is changed every time 45 eV of energy are deposited. Allowing for clusters this is a reasonable value for one primary ionization.

The change in sedimentation constant can be interpreted as an opening up of the molecule since its chemical reactivity is increased. Each molecule of bovine serum albumin contains seventeen disulphide bonds contributed by the amino acid, cystine. In the native molecule these disulphide bonds are screened and incapable of undergoing chemical reactions. On denatur-ation by heating, or in other ways, some of the disulphide bonds become


available to attack by oxidizing and reducing agents40.* This also happens on irradiation and Fig. 7-9 shows that one primary ionization (i.e. 45 eV) makes 4 out of the 17 bonds accessible.+ Half the disulphide bonds become available if the radiation dose is such that two primary ionizations have occurred within the molecule, but no more are revealed by still further irradiations.

5

S B 2

IOO 90

80

70

60 50

40

30

20

V i \(a) \<b)

\ \ \

V.

i

20 Radiation dose.

30 4 0 50

(I06rad

FIG. 7-8. Changes in some physical properties following irradi-ation of solid bovine serum albumin by 2 MeV electrons39, (a) Fraction of bovine serum albumin showing changed sedi-mentation behaviour in the ultracentrifuge; (b) fraction of bovine serum albumin insoluble in water; (c) fraction of bovine

serum albumin insoluble in M115 phosphate buffer (pH 7).

Two ionizations also alter the solubility of the albumin (see typical two-hit curve in Fig. 7-8) so that it requires salt to go into solution, as an aggregate containing an average of five albumin molecules which are held together both by intermolecular hydrogen bonds and by disulphide bonds produced by disulphide exchange. At still higher doses the albumin becomes insoluble in all solvents (see Fig. 7-8, curve "C").

* Radiation also makes SH groups which are normally unreactive capable of being titrated by SH reagents. As there is only half of an SH group (on average) per molecule of bovine serum albumin the revelation of disulphide bonds cannot be used as a criterion for progressive denaturation.

I It must be stressed that at these doses the number of disulphide bonds that are chemically altered by the radiation dose is extremely small and play no part in the phenomenon of revealing disulphide bonds. Thus a dose of 45 eV per molecule chemically changes 0-2 molecule of cystine (i.e. only one out of every five molecules of albumin that has received a primary ionization undergoes a reaction at a disulphide bond) while it renders four disulphide groups capable of reacting.


The opening up of the molecule that occurs when one primary ioniza-tion has taken place cannot be ascribed to covalent chemical changes. While on average some three amino-acid residues will be chemically changed by 45 eV (see Table 7-5 in the next section) this cannot cause denaturation since the same residues will not be effected in all the molecules that have taken up this amount of energy (e.g. one molecule in five will

FIG. 7-9. Changes in the number of disulphide bonds which can be reduced at the isoelectric point with jS-mercaptoethyl-amine following irradiation of solid bovine serum albumin by

2 MeV electrons39. — A — For water-soluble fractions (that is, including the

molecules that have not been affected by radiation). —-O— For water-soluble fraction after correction for un-

changed protein on the basis of the ultracentrifuge data. — • — For fraction insoluble in water, but soluble in salt solu-

tion.

have suffered damage in cystine and tyrosine, one in eight in histidine, and so on). P L A T Z M A N and FRANK 4 1 have calculated that in the vicinity of an ionization a number of hydrogen bonds will be temporarily severed because the sudden introduction of a charge disrupts electrical dipoles. The secondary structure of a protein will be affected by this process and not by the isolated covalent chemical change that occurs in the group where the ionization has occurred.

Chemical Changes R A J E W S K Y and DOSE4 2 claimed to have found that some 2 0 per cent of

the amino-acid residues were altered in dry lysosymeby a dose of 5 x IO6

rads. These analyses cannot be reconciled with radiation chemistry since


T A B L E 7 - 5

A M I N O - A C I D RESIDUES DESTROYED I N B O V I N E S E R U M A L B U M I N ON IRRADIATION W I T H 2 M E V E L E C T R O N S I N THE S O L I D STATE 4 ' 3

Amino-acid residues

Amino-acids No. changed No. per per 45 eV per Apparent G molecule protein value*

molecule t

Cystine 17-0 0-22 12-0 Combined aspartic and glutamic acids 104-5 M l 11-2

estimated as carboxyl groups Tyrosine 19-8 0-18 7-7 Histidine 16-9 0-13 7-7 Phenylalanine 26-0 0 1 8 6-3 Proline 29-6 0-14 6-3 Arginine 24-2 0-15 5-5 Amino groups of lysine 58.4 0.27 4.9

* Residues changed per 100 eV per protein molecule X 100 Per cent content of residue in protein

t Average amount of energy deposited per molecule to produce denaturation in 63 per cent of the molecule.

they indicate G values for the destruction of amino-acids of the order of hundreds. Faulty hydrolysis techniques which led to humin formation probably accounts for these results.

A L E X A N D E R and H A M I L T O N 4 3 determined the changes which occurred on irradiation in nine amino-acid residues which make up 75 per cent bovine serum albumin. The introduction of new groups complicates the analytical problem, but methods were used which minimized the danger of interaction during hydrolysis. The destruction of amino-acid residues was strictly proportional to dose over the range studied (i.e. up to 1-5 x IO8 rads). From Table 7-5 it can be seen that the apparent G value varies over a factor of 2-5 for the amino-acids studied with cystine slightly more radiosensitive than the decarboxylation of the acidic amino-acids. The relatively small range in G values indicates that intramolecular energy transfer processes do not lead to a funnelling of energy into a few residues only. From experiments with synthetic polymers (see pp. 113 and 177) one would have predicted that energy transfer should have occurred to the aromatic amino-acids such as tyrosine, but apparently this is not an important effect in proteins. The analytical data also provides no support for the hypothesis of G O R D Y et al.M that energy becomes


localized in the disulphide bonds. The observation which suggested this preferential attack was the appearance of the electron spin resonance spectrum of irradiated proteins which resembled that of the amino-acid, cystine, after irradiation. G O R D Y et al. made their electron spin resonance measurements at room temperature. If the measurement of e.s.r. and the irradiation is made at —195° C the intensity of the e.s.r. pattern is very much greater and there is no similarity between the patterns of irradiated protein and irradiated cystine under these conditions*45). At room tem-perature the majority of the radicals react further or recombine and those few that remain become peroxidized on contact with air. The similarity in pattern seen by G O R D Y et al. shows that the small proportion of the radicals that remained behind in the sample of cystine and of protein combined with oxygen. There is no indication that the radiation energy is funnelled to the sulphur atoms of cystine.

No evidence was found for peptide bond scission in bovine serum al-bumin43. With a dose that deposited 700 eV/molecule no low molecular weight fragments could be detected even after all the disulphide bonds had been severed so as to eliminate the possibility that disulphide cross-links masked main-chain breaks. The formation of carbonyl groups with a G value of 0-8 suggests at first sight that direct action affects the peptide link in the same way as does indirect action (see p. 190) to give an unstable keto imide which causes scission, i.e.

O Il

— C — N H - C H

R

But closer investigation indicates that the carbonyl groups are produced in a different and as yet unknown way by direct action43.

Influence of the Water Content of the Protein In the experiments with bovine serum albumin described above the

protein was not dried completely but contained 5 per cent of water. Varying the moisture content from 0 (i.e. less than 0-3 per cent) to 20 per cent did not affect the "opening-up" of the molecule as measured by disulphide bond availability, but the dose needed to render the protein insoluble in water was progressively reduced46. Possibly the presence of water facilitates rearrangement of the tertiary structure (see footnote, p. 179) which determines the solubility characteristics. Changes due to free radicals produced in the water do not play a part at this stage since indirect action is so much less efficient (see p. 191) than direct action.

O hydrolysis

- C - N = = C > - C O N H 2 + O = C -

I I R R


Influence of Oxygen A L E X A N D E R et alA1 found that the dose needed to render bovine serum

albumin insoluble was less when the irradiations were carried out in the presence of oxygen, which also resulted in the formation of reactive peroxy groups that were bound to the protein. Chemical analyses of the albumin46 show that the number of carbonyl groups introduced was doubled and the destruction of individual amino-acids such as cystine, tyrosine, etc., was increased by some 10 to 20 per cent. On the other hand the dose needed to reveal disulphide groups was unaffected. This supports the view that the "opening-up" of the molecule is due to the hydrogen bond breaking action of an ionization and not a consequence of covalent chemical changes. The increased inactivation of enzymes in the presence of oxygen (see p. 211) has been attributed39 to breaking of peptide bonds, but this still requires experimental verification.

Two mechanisms have been considered for the oxygen effect48: (1) formation of an O2" radical that attacks the protein (cf. p. 174); (2) addi-tion of oxygen to an active centre produced by an ionization to give a product that cannot undergo repair (cf. pp. 117 and 162). Possibly both types of effect occur but A L E X A N D E R 4 8 favours the O G radical for the increased inactivation of enzymes.

Protection As was first shown with synthetic polymers (see p. 175), the presence

of added compounds can also protect proteins against direct action.* Glutathione increases the inactivation dose of catalase50 and invertase49

and there is evidence from electron spin resonance data that this is an example of energy transfer50.

The electron spin resonance pattern of bovine serum albumin irradiated at —195° C as well as at 20° C is greatly reduced in the presence of 10 per cent of cysteamine (an intimate mixture of the two being obtained by freeze drying)45. Energy transfer reduces the number of radicals formed in the protein. G O R D Y and M I Y A G A W A 5 1 first observed this protection by cysteamine with the protein zein and attributed it to combination of cysteamine with the disulphide groups of the protein by an exchange reaction. This mechanism can be excluded in the case of bovine serum albumin since none of the disulphide groups in this protein react with cysteamine under the condition used40.

* The presence of large quantities of added mineral salts like sodium chloride makes dry enzyme preparations more sensitive to ionizing radiations. This is not as has been claimed49 due to some energy transfer process, but due to reactive chemicals formed on irradiation in the interior of the salt crystals. On solution some of these react with the proteins.


P H Y S I C A L A N D C H E M I C A L C H A N G E S I N P R O T E I N S P R O D U C E D BY I N D I R E C T A C T I O N

A considerable number of papers has been published on changes in the physico-chemical properties of protein solutions following exposure to ionizing radiation. Experiments in which a comparison was made of the protective action of different proteins by radical competition (see p. 50) indicated that their rate of reaction with OH radicals was approximately the same and that it was not changed by denaturation.

Aggregation In 1 9 0 3 , HARDY52 found a decrease in the solubility of proteins after

exposure to a-rays in solution and noted that the effect depends upon the electrical charge carried by the molecule.

This observation has subsequently been confirmed with many other proteins exposed to different types of ionizing radiations. Almost all proteins are denatured by ionization, if this is defined as rendering the protein insoluble at the isoelectric point. This aspect has been very thoroughly studied and reviewed by F R I C K E 5 3 . B A R R O N and F I N K E L S T E I N 5 4

observed that a solution of 0-7 per cent serum albumin is rendered insoluble when irradiated at 25°C, but not when irradiated at 3°C, but that this latter solution precipitates when raised subsequently to 25°C. Most of the data are consistent with the view that the free radicals produced in water bring about a change in shape of the protein molecule (e.g. unfolding) and thereby facilitate subsequent aggregation processes, giving rise to structures of increased molecular weight.

When fibrinogen is irradiated with x-rays in solution55 a polydisperse material having a higher sedimentation constant and increased viscosity is produced. The reaction was followed quantitatively by measuring the disappearance of the sedimentation peak due to the unchanged material. The kinetics of the process leave no doubt that the effect is produced by free radicals and this is confirmed by the fact that cysteine and thiourea act as protective agents. Human serum albumin behaves in the same way56. On irradiation at the isoelectric point the sedimentation pattern is altered suggesting the formation of aggregates. Increases in molecular weight were observed by C A R R O L L et al.58, using light scattering under similar conditions and this confirms that the changes in sedimentation behaviour are due to aggregation.

The amount of denatured protein (defined as having an abnormal sedimentation behaviour) follows exponentially with dose and strictly obeys the dilution law showing that the change is due to indirect action (i.e. the dose needed to alter a certain fraction of the protein is pro-portional to the protein concentration). As with fibrinogen55 added sub-stances protect56: serum albumin (see Fig. 5-1, p. I l l ) However, in-


sufficient data are available to decide whether the protection is by competi-tion or by "repair" of a protein radical by hydrogen transfer (cf. p. 177).

With increasing dose not only the fraction of the protein denatured increases but the size of the aggregates formed becomes bigger until a point is reached when the protein becomes insoluble and precipitates57.

At the isoelectric point 200 (+10) eV (i.e. G = 0-50) are needed to alter a molecule of serum albumin so that it has a different sedimentation constant.* The reaction is the same whether oxygen is present or not, and this suggests that OH radicals are responsible for the changes that are seen. As soon as the protein is in a non-isoelectric condition (i.e. carrying a charge) much greater doses are needed to produce aggregation presumably because the molecules repel one another (see Fig. 7-10).t

T> to it

— » — f / t

/ /

I /

/

/ / •

/ I I

\ / /

pH

FIG. 7-10. T h e influence of the p H of the solution on the aggregation and precipitation of X-irradiated solutions of human-serum albumin. The ordinate is the factor by which the dose must be increased over that dose required at the iso-electric point to produce a given amount of aggregation or precipitation. O—Determined from sedimentation diagram; • —determined from the onset of visible precipitation. T h e

arrows indicate points lying outside the scales57.

The influence of dissolved electrolytes on the dose is complex57 and has not yet been satisfactorily interpreted.

* On average seven OH radicals have to react before aggregation occurs. Since the dose-response curve is exponential this is not a co-operative phenomenon requiring seven radicals. Of the seven radicals on average six are used in reactions that do not give rise to aggregation.

I Exactly the same situation is encountered with crosslinking of polyacrylic acid in solution (see p. 164).


Other Physico-chemical Changes Many other, though less well-defined, physical effects have been

observed, such as changes in optical rotation, refractive index, surface tension, electrophoretic mobility and electrical conductivity59. Changes in viscosity are often observed though in some cases it is an increase and in others a decrease. For example, the viscosity of ovalbumin solution is increased if the irradiation is carried out at the isoelectric point or at lower pH values, but decreased at higher pH values60. Many investi-gators (cf. ref. 63) have noted an increase in the availability of —SH groups after irradiation in different proteins. Other changes in reactivity are mentioned by ARNOW 5 9 . Indirect action like direct action results in the revelation of disulphide bonds62 in serum albumin, but this reaction is not related to aggregation since irradiation away from the isoelectric point is more effective than at the isoelectric point.

Chemical Changes Deductions concerning chemical changes that occur on irradiation

have frequently been made from changes in the ultra-violet absorption spectrum. Many workers (cf. BARRON64) failed to appreciate that the amount of light apparently absorbed by a solution of protein will be increased by an increase in the light scattered by the large aggregates. The Scattering (S) increases as the wavelength (A) decreases according to Sal/A4. With bovine serum albumin there is no significant true change in the light absorbed by the protein due to irradiation and the apparent change in spectrum is within experimental error due to scat-tering47. This is shown in Fig. 7-11 where the difference in light absorbed before and after irradiation is directly proportional to (wavelength)4

except for a very small peak at 2990 A, the significance of which is doubt-ful. If serum albumin is irradiated away from the isoelectric point when there is little or no aggregation then a true change in the ultra-violet absorption spectrum occurs57.

C A R R O L L et al.65 suggested that the crosslinks responsible for the aggre-gation of proteins involve the linking of tyrosine residues with phenyl-alanine residues but there is no analytical data to support this view. ROSEN57 finds that the aggregates are broken up by exposure for some hours to acid or alkali but that neither concentrated urea nor salt produces dissociation. This suggests that the bonds responsible for holding the molecules together are not primarily hydrogen bonds but are covalent links that are readily hydrolysed. After disaggregation by acid the protein shows a change in ultra-violet absorption spectrum similar to that produced by irradiation away from the isoelectric point.

Though several attempts have been made to evaluate the relative radio-sensitivity of different amino-acids direct by analysis of the irradiated


protein, the data are unsatisfactory and conflicting (cf. refs. 64 and 66). This is largely due to the fact that paper chromatography was used and this is not capable of high accuracy especially if unknown products are likely to be present. The better techniques that are now available should be applied to this problem.

0-75

0-50

0-25

<8

>0 h 5 I

(b) 240 260 280 300 320 340

Wavelength, m / t

240 260 280 300 320 340

Wavelength, nyz

FIG. 7-11. Change in ultraviolet-absorption spectrum of 0-1 per cent salt-free isoelectric solution of human-serum albumin after x-irradiation. (a) Curve A: spectrum before irradiation; curve B: spectrum after a dose of 2 0 , 5 0 0 rads. (b) Analysis for Rayleigh scattering where A is the difference in extinction between curves A and B at wavelength A. This shows that almost all the change observed produced by scattering is due

to aggregation67.

The work of G A R R I S O N and his colleagues provides the most valuable data concerning the chemical changes produced on irradiation. They found from experiments with model substances67 that the —C—N— bond is readily split on irradiation if oxygen is present. In proteins68

irradiated in solutions containing oxygen the peptide link is broken giving amide and carbonyl groups68. The reaction can be written as follows:

—CO—NH—CH + O2 + H2O -> - C O - N H 2 + O = C - + H2O2

L I and with gelatin a G value of approximately one was observed. Interesting as this splitting of the peptide bond is, the fact that it only occurs in the presence of oxygen suggests that it plays no part in the aggregation phenomenon or in the inactivation of enzymes since these effects do not require the presence of dissolved oxygen.

Inactivation of Enzymes All enzymes are inactivated when irradiated in solution and the dose-

response curve is nearly always exponential indicating that one reaction


by a radical is sufficient to produce inactivation. So long as the solution is not too dilute so that radicals are lost by recombination (see p. 48) the G value for inactivation is independent of concentration. Much of the earlier work on the inactivation of enzymes in solution is of doubtful value, as the preparations used contained impurities which may have exercised a protective effect. The discovery by DALE 7 1 that added sub-stances reduced the inactivation by x-rays of the enzymes carboxypep-tidase and D-amino-acid-oxidase in dilute solution focused attention on the role of indirect action in biological systems. The efficiency of inactiva-tion of enzymes by indirect action is very much less than that by direct. Table 7-6 shows that for many enzymes between ten to one hundred

T A B L E J 7 - 6

INACTIVATION OF E N Z Y M E S I N D I L U T E S O L U T I O N 6 9 , 7 0

G value for Enzyme inactivation*

Non-SH : Carboxypeptidase 0-55 D-Amino-acid-oxidase 0-31 Ribonuclease 0-09 Trypsin 0-077 Lysozyme 0 0 3 Catalase 0-009 SH: Alcohol dehydrogenase 0-06 Phosphoglyceraldehyde 0-068

dehydrogenase

* Tha t is, number of molecules inactivated for 100 eV of energy deposited in the solution.

ionizations are necessary to inactivate one enzyme molecule in dilute aqueous solution while by direct action one ionization per molecule is sufficient. There must be many parts of the protein molecule which can react without, however, producing a change in biological activity. Solu-tions of ribonuclease72, trypsin73* and carboxypeptidase74 are inactivated

* Miss MEE73" has reported that when trypsin is irradiated in very dilute solu-tion the extent of inactivation is less in the presence of dissolved oxygen than in its absence. Th is protective action of oxygen may possibly be ascribed to inter-ference with crosslinking between molecules by analogy with the experiments with polymers (see p. 164). If aggregation (i.e. crosslinking) requires the action of an active centre, oxygen can by adding on to it, prevent this reaction, if the protein concentration is low. At higher concentrations the protein-protein reaction will occur so readily that dissolved oxygen cannot intervene.


to the same extent in the presence as in the absence of dissolved oxygen, and this indicates that OH radicals are responsible. In support of this view it was found that chemically produced OH radicals also inactivate ribonuclease72 and trypsin75. The kinetics of trypsin73,75 and chymo-trypsin76 inactivation show many unexpected features, such as a depend-ence on concentration of enzyme, pH, and salt concentration as well as a continued fall in activity after irradiation. This suggests that a complex process (possibly crosslinking) is involved in the reaction.

FORSBERG77 has claimed that catalase in solution could not be protected against x-rays by cysteine or glutathione, but that on the contrary these substances made the enzyme more sensitive. An ingenious interpretation was advanced and formed the substance of much discussion, but this need not be considered here as the results are not real. SH compounds were shown to combine slowly with the haem group in catalase and to render it inactive. When steps were taken78 to eliminate this complication, marked protection was observed. Catalase shows another unusual feature in that the inactivation depends on the rate at which the irradiation is given and that the relationship between inactivation and dose is not exponential. SUTTON 7 9 has found indications that hydrogen peroxide formed as a by-product during irradiation protects the enzyme, but no interpretation of this effect has been given.

B A R R O N and his colleagues have published a number of papers (cf. ref. 69) on the inactivation of enzymes that depended on —SH groups for their biological activity in which they claimed to have established (a) that they were exceptionally sensitive with G values of 3 as opposed to non-SH enzymes with G of 0-03 to 0-5; (b) that the G value for inactiva-tion was several times greater in the presence of dissolved oxygen; (c) that they could be reactivated after irradiation with sulphydryl com-pounds.

This behaviour, which was attributed to the selective inactivation of —SH groups at the time, was difficult to reconcile with the action of radiations on cysteine in solution (see p. 1 5 7 ) . L A N G E , P I H L and ELDJARN 7 0 were quite unable to repeat this work and their G values (see Table 7-6) are one-fiftieth of those claimed by Barron. In view of this, his other claims about the importance of oxygen and reactivation must be treated with suspicion.

The general trend of all the data is that the inactivation of enzymes in dilute solution by indirect action is not affected by the presence of oxygen. Apart from Barron's claims there is only the work of OKADA 8 0 which showed a small enhancement in the inactivation of DNAase by oxygen.

Many enzymes carry a non-protein prosthetic group which though essential for activity can be reversibly detached from its specific protein. D A L E 7 1 found that for D-amino-acid-oxidase the specific protein and the


alloxazine adenine dinucleotide were approximately equally radiosensitive and the total amount of inactivation was additive when each component was irradiated individually and afterwards mixed. When solutions of the whole enzyme are irradiated the protein moiety and not the prosthetic group are destroyed; possibly the latter, being held within the protein, is sterically inaccessible to the free radicals. Biologically active materials chemically related to these prosthetic groups (e.g. diphosphopyridine nucleotide and adenosine triphosphate) are destroyed by x-rays in aqueous solutions though with low yields69. An important non-peptide structure found in many biologically active proteins is the porphyrin ring present for example in catalase, peroxidases, haemoglobin and cytochrome-c. A characteristic property of this substance is a strong absorption band in the visible region at 4100 A (the Soret band) which is responsible for its red colour. FRICKE 8 1 showed that this absorption was reduced when haemoglobin was irradiated in aqueous solution*; B A R R O N and F L O O D 8 2

state that the decrease in absorption is linear with dose and compute an ionic yield of 0-05, although it is doubtful what meaning can be attached to this value.

Protection of Enzymes In a rather limited series, DALE 7 1 found that the protective power of

added substances was directly related to the sulphur content and that a suspension of colloidal sulphur was a most effective protector. Although Dale proposes that the protection is due to competition for the free radicals, he draws attention to the fact that the protective power is not independent of concentration and that additional factors, not yet under-stood, must also be involved. Since then protection by added substances has been found in every case where enzymes have been inactivated by indirect action, although detailed quantitative studies are lacking. Protec-tive action is not confined to sulphur compounds and very many sub-stances, both inorganic and organic, have been found effective though the mechanism of protection (i.e. whether by competition or repair) has not been established (cf. p. 111).

The possibility that masking the sensitive site, attack of which by a free radical from water (i.e. OH radical) leads to inactivation, can give rise to protection was observed with catalase79. Hydrogen cyanide forms a reversible complex with the essential iron centres and when irradiated in this form the enzyme seems to be much more resistant. Another way of masking the essential point of an enzyme is to irradiate it when combined

* Direct action is unable to affect the chromophoric groups of porphyrin and the absorption maxima is unaffected even when there have been an average of four ionizations per molecule83.


with its substrate and DALE84 noted that inactivation by free radicals could be reduced in this way. DNAase is powerfully protected by high molecular weight DNA but nucleotide and nucleoside are fifty times less effective in this respect. OKADA85 has interpreted this as a case of specific substrate protection but it might also be due to removal of radicals as DNA has an exceptionally high affinity for them and protects polymeth-acrylic acid (see p. 175) when no interaction is possible. Unfortunately, this type of protection against indirect action has received very little attention, although in practice it may be of greater importance than the competitive capture of free radicals.

C R O S S L I N K I N G A N D D E G R A D A T I O N O F D E O X Y R I B O N U C L E I C A C I D *

No detailed papers appear to have been published on the changes produced in the physico-chemical properties of RNA by irradiation. Chemical analysis87 indicates that the molecule is attacked as extensively as DNA by free radicals from water. In this chapter in which alterations in the macromolecular properties are dealt with discussion has therefore to be confined to DNA.

Structure of DNA Nucleic acids are macromolecules of very high molecular weight

carrying recurrent negative charges and consequently fall into the class of polyelectrolytes, the general properties of which, particularly in aqueous solution, are now being intensively investigated. RNA and DNA are easily degraded by enzymatic action and by shearing forces such as stirring intensively in a Wearing blendor. In general, there is no certainty that they have been extracted without degradation, though they yield a biologically active preparation—the transforming principles—when applied to bac-teria. Techniques for separating the mixture of nucleic acids obtained from the cell into more homogeneous fractions are as yet very imperfect86. Only RNA isolated from small viruses and perhaps DNA obtained from a small bacteriophage can be considered molecularly homogeneous.

The chemical constitution of DNA has been established largely by the classical work of Sir Alexander Todd and his colleagues. The steric configuration was ellucidated from x-ray diffraction data by W A T S O N and CRICK88 and the general configuration of the twin spiral molecule is shown in Fig. 7-12. There can be little doubt that most of the DNA in cells is present as a twin helix, though the possibility is often envisaged

* T h e sodium salts of deoxyribonucleic acids will be abbreviated to D N A and those of ribonucleic acid to RNA.


that during DNA duplication a single strand is involved. The possibility that at some stage during the cell cycle there may be some single-stranded DNA must be borne in mind, since from the smallest bacterial virus, though not in any of the larger ones, DNA is isolated in the single-stranded form89.

DNA after isolation gives rise to highly viscous solutions and much effort has been devoted to determining the size and shape of the molecule by physico-chemical techniques. Largely from the work of DOTY 9 0 it is clear that DNA exists in solution as a very stiff coil that takes up random

(a) Atomic structure.

M U - 1 6 0 9 5

(b) Diagrammatic drawing of twin helical configuration.

F I G . 7 - 1 2 . Structure of deoxyribonucleic acid ( D N A ) . [cont. p. 196


(c) Molecular model purine and pyrimidine bases; Ophos-phorus; carbon in phosphate ester chain; @ oxygen).

F I G . 7 - 1 2 . Structure of deoxyribonucleic acid ( D N A ) .

configurations due to Brownian motion (see Fig. 7-13). Molecular weights, as determined by light scattering, for DNA from mammalian sources are of the order of 6 to 8 x IO6, and from fish sperm molecular weights as high as 1-4 x IO7 have been obtained. These are weight average values (see p. 158) and there is a variety of different molecules present which may range from 20 x IO6 to 1 x IO6. The distribution of sizes is believed to be "Gaussian"90. Molecules of these molecular weights are extremely assymetric, for a molecule of weight of 6 x IO6 the thread is 3 fi long by 0-002 ^ (20 A) wide. These data obtained by physico-chemical


methods have been fully confirmed in the electron microscope (see

The stiffness of the molecule can be reduced by disrupting the hydrogen bonds holding the two chains together when the molecule takes up a much more compact configuration (see Fig. 7-13) and the viscosity of its solutions is reduced to a tenth or less than that of the native material. This coiling can be produced by heating90, treatment with acid90, exposure to ultra-violet light91 and by alkylation92. In solutions of very low ionic

strength (e.g. 0-001 M NaCl) DNA is very unstable and very easily "coils". The electric charges of the phosphate groups are screened by counter ions provided by the salt (e.g. Na+). In the absence of such counter ions these phosphate groups repel one another strongly and this leads to the gradual disruption of the hydrogen bonds holding the two chains together. For this reason DNA must never be handled in solutions that do not contain sodium chloride or certain other salts.

The viscosity of DNA can also be reduced by lowering the molecular weight (i.e. by "main-chain chopping"). Frequently, changes in DNA

Fig. 7-14).

FIG. 7-13. "Coiling" of stiff molecule. Length (and molecular weight) unaltered, but because of greater flexibility it occupies a smaller volume in solution and therefore has a lower viscosity.


have only been followed by viscosity measurements and erroneous conclu-sions have been drawn. To determine the changes in macromolecular properties produced by exposure to, say, x-rays it is necessary to make a number of different measurements93.

The Structure of Cellular Nucleoproteins In the cell the DNA is present as a complex with proteins. The charge

of the many phosphate groups is suppressed by basic proteins, but in general other proteins are also present. There is no satisfactory method by which nucleoproteins can be isolated since they are present as large colloidal networks and attempts to bring them into solution involves a disturbance of the complex structure. Except for certain sperm heads (see below) the nucleus swells in water to give a very loose gel that has often been confused with a highly viscous solution. If nuclei are broken up in dilute salt solutions (e.g. 0-1 M NaCl) the nucleoprotein contracts into fibres which usually swell up again when the salt is removed. In very concentrated salt (about 2 M NaCl) the DNA becomes dissociated from the protein and both compounds go into true solution and behave like separate molecules (i.e. the viscosity of the solution is that due to DNA only). This mixture precipitates as fibres when the salt solution is made more dilute, but the reformed nucleoprotein bears no resemblance to that present originally in the cell; for example, it does not swell in water94. The arrangement of protein and nucleic acid as present in the cell cannot be reproduced.

The nucleoprotein of the sperm of certain fish (e.g. trout, salmon, herring) is much simpler than that of mammalian cells. The sperm heads contain only DNA (65 per cent) and low molecular weight basic proteins known as protamines (35 per cent) (i.e. none of the non-basic proteins typical of mammalian nucleoprotein is present)95. These complexes do not swell in water and are present as hard balls that are completely dis-sociated in concentrated salt.

Degradation by Indirect Action The most readily measured property of solutions containing DNA is

their high viscosity and on irradiation with X-rays, either in solution or dry, this viscosity is greatly decreased. S P A R R O W and R O S E N F E L D 9 6 were the first observers to record the decrease in viscosity on irradiating DNA solution with x-rays and found, as didG. C . B U T L E R 9 7 , a logarithmic relation-ship between the viscosity at high shear rates and the radiation dose (see Fig. 7-15). A nucleohistone in 2 M salt showed a much smaller loss in viscosity than did DNA; as in the salt solution used the nucleohistone was fully dissociated, the protective action of the histone is probably due

FIG. 7-14. (a) Electron microscope photograph of D N A mole-cules. On these films the D N A is stretched out by surface tension effects from the randomly coiled configuration in which

it exists in solution.

[facing p. IgS

FIG. 7-14. (b) After 2 x IO0 rads of 2 MeV electrons to dry DNAstructures such as these are commonly seen. These are due to cross-linking. The Y-shaped appearance is due to the fact that

' t he DNA molecules have a great tendency to aggregate side by side while being dried down for examination in the electron

microscope. Hence crosslinks do not appear as crosses.


to competition for the degrading radicals produced in water. Similar protection effects have been observed with serum albumin98, glucose97, methanol98, thiourea99 and cysteamine100.

Dosage 103r

FIG. 7-15. Logarithmic relationship between dose and decrease in viscosity for 0 1 per cent solutions of D N A irradiated with x-rays. T h e upper curve is for the degradation of thymus nucleoprotein dissolved in 2 M sodium chloride and indicates

the protective effect of the protein90 .

G R E E N S T E I N and T A Y L O R , HOLLAENDER 9 8 made the significant obser-vation that the viscosity continued to fall for many hours after the irradi-ation was stopped and that the magnitude of this effect was almost independent of temperature. It is difficult to decide how much of the observed decrease in viscosity occurs immediately on irradiation since the after-effect will start while the irradiation is still in progress. B U T L E R 1 0 1

claims that there are two distinct effects: an initial reduction followed by the much larger after-effect.

C O N W A Y and BUTLER101 claim that added chemicals can only reduce the degradation of DNA if added before the irradiation and that the after-effect is not influenced if the protective agents are added after the


irradiation. ERRERA102 on the other hand, finds that glutathione can almost completely prevent the slow decrease in viscosity even when added after the irradiation with x-rays.

T'he evidence concerning the role of oxygen in the viscosity reduction is also contradictory. The initial experiments96'98 were carried out in aerated solution. In a subsequent detailed and quantitative study G. C. BUTLER97 claimed that the decrease in viscosity was the same in aerated as in carefully deoxygenated solutions. A year later, J. A . V . B U T L E R 1 0 1

found the exact reverse; in the absence of oxygen there was no after-effect although the ill-defined initial effect was claimed to be the same in solu-tions saturated with nitrogen as with air. In contradiction to all the previous workers D A N I E L S , S C H O L E S and WEISS 1 0 3 find that the initial effect on the viscosity is larger in the absence of oxygen, but the after-effect smaller.

Experiments with OH* radicals produced by photolysis of hydrogen peroxide show that the drop in viscosity is due to main-chain scission104. One "double break" occurs for every 130 OH' radicals in a DNA of IO7

weight average molecular weight. Breaks produced in a single chain only would not be detected and a scission is shown only when there are two breaks—one in each chain—in fairly close proximity105. How many of the 130 OH' radicals are used up to oxidize the sugar phosphate main-chain (and thereby produce breaks in one of the chains) and how many react in other ways (e.g. with the purines and pyrimidines) is not known. Sodium hypochlorite (i.e. chlorine water at pH 7) is even more effective than OH' radicals in producing main-chain scission, but hydrogen peroxide and ozone are about a thousand times less so104.

Cox et al.106 followed in detail the viscosity changes of DNA irradiated with y-rays in dilute solution and in the presence of oxygen and concluded that main-chain scission occurred whenever coincident breaks in the two chains were produced. Changes in the electrometric titration curve led them to speculate that at the point of the break the hydrogen bonding between the bases was extensively disturbed.

Much of the confusion in these irradiation experiments is due to the fact that the only measurements made were of viscosity which does not dis-tinguish between "coiling" and "main-chain scission". The reason for the great variation in the magnitude of the after-effect reported is that some investigators handled DNA in the virtual absence of salt (cf. refs. 100 and 101). DNA coils up if there is insufficient salt to suppress the electro-static repulsion105 (see p. 197) and this process occurs more readily if the DNA has undergone some damage. Another and even more important factor is that in the presence of chloride ions a proportion of the OH radicals formed in the water are converted to chlorine and hypochlorous acid (see p. 134). The chlorine so produced will also attack DNA and lower


the viscoQity by producing main-chain scission. However, in the very dilute solutions the reaction of hypochlorous acid is far from instantaneous and may require several hours to be complete.* By adding thiosulphate or other chemicals that react readily with chlorine this "after-effect" can be com-pletely suppressed while their addition will not influence an "after-effect" due to denaturation in solutions of low ionic strength. The chloride effect can be prevented by irradiating DNA in solutions of sodium fluoride as OH radicals cannot oxidize fluoride to fluorine and the addition of thiosulphate irradiation is then without effect107. However, even in O-IM sodium fluoride there is some after-effect though very much less than in sodium chloride, and this remains to be explained possibly in terms of an unstable intermediate formed by reaction of OH radicals with the purines.

The nature of the oxygen effect on the viscosity changes remains to be resolved (e.g. whether the formation of thymine peroxide—see p. 152—is involved?). The role of oxygen is particularly puzzling since B A C H O F F E R 1 0 8

finds that bacteriophage are more readily inactivated in the absence than in the presence of oxygen when the action is indirect (i.e. due to free radicals formed in water). Bachoffer concludes that H atoms are the important degradative radicals.

Recently, chemical data has become available for the destruction of the bases in DNA that has been irradiated in dilute solution (see p. 151 for chemical reactions in nucleotides, etc.). Both WEISS145 and HEMS146 are in agreement that the pyrimidines are destroyed somewhat more readily than the purines but the G values obtained by these experiments are rather different. Th G values quoted lie in the range of 0 - 2 to 0 - 7 . H E M S 1 4 6

attributes these variations to sample differences. Possibly the presence of variable amounts of sodium chloride in the preparations may be responsible for the reasons described above. In the presence of dissolved oxygen hydroperoxides are formed145 and the destruction of the bases is enhanced. The reduction in molecular weight seen immediately after irradiation cannot be attributed to base attack since this would not lead to an im-mediate scission of the main polynucleotide chain. The major part of the macromolecular changes must be due to attack on the sugar-phosphate groups (see p. 207).

Direct Action When DNA is irradiated dry or with an amount of water which is not

sufficient to swamp direct processes the alterations in the physico-chemical properties are extremely complex and depend critically on the exact

* Reaction of O H radicals is always "instantaneous" as these do not persist for more than some 10~6 sec. in solution.


radiation conditions93'109. While the viscosity drops in every case cross-linking occurs as can be seen by the increase in molecular weight and the formation of gel (see Fig. 7-16). The appearance of irradiated DNA in the electron microscope was quite different and suggests the formation of branched structures (see Fig. 7-14). The fact that dry DNA was rendered insoluble when irradiated under vacuum had already been observed by S E T L O W and DOYLE110. However, simultaneous main-chain scission also occurs and the complex situation is summarized in Fig. 7-17.

It is proposed109'111 that on irradiation a break is produced in one of the two chains in such a way that one of the broken ends is reactive and can join with another broken end to form a crosslink. Main-chain scission occurs if, by chance, there are coincident (i.e. within a few nucleotides) breaks in the single chains or if there is a cluster of ionizations. The forma-tion of crosslinks between the active ends is facilitated if the DNA is swollen so that movement of the molecules can occur. Hence, cross-linking increases as the water content of a DNA sample is raised from 0 to 200 per cent. At higher water contents crosslinking falls again because the main-chain scission produced by the free radicals formed in the water overwhelms the number of crosslinks formed.

In the presence of oxygen, crosslinking is prevented because the active ends become peroxides and can no longer crosslink. Oxygen, however, does not readily penetrate dry DNA and the oxygen effect with samples containing between 0 and 25 per cent of water is therefore small. In samples containing an equal weight of DNA and water the oxygen effect is very pronounced. In the absence of oxygen, crosslinking predominates and DNA is converted into a gel. In the presence of oxygen only degrada-tion is seen (see Fig. 7-16).

When DNA is irradiated with ionizing radiations—as opposed to ultra-violet light91-104—there is no evidence for the rupture of hydrogen bonds93. The reason why the complex hydrogen-bonded structure is not disrupted by irradiation, while that of globular proteins is (see p. 180), may be attributed to the relative stability of the hydrogen bonds in the two systems. In the twin helix of the DNA, any hydrogen bonds that are broken will tend to reform immediately in the same way and irreversible damage only occurs when a very large number of hydrogen bonds are all broken at the same time. In proteins, on the other hand, the interchain hydrogen bonds do not represent the most stable configuration and when they are broken the molecule may quickly alter its configuration so that the hydrogen bonds do not reform in the same way.

S H I E L D S and GORDY110" have examined the electron spin resonance of DNA irradiated in the solid in the absence of oxygen. The nature of the spectrum is quite different from that seen in irradiated nucleotides. Admission of oxygen after irradiation does not alter the spectrum but it

(a) Crc

(,b) Main-chain scission

D N A ; tv maintainec specific hy

reak in each of the adjacent chains less than about 5

iduced by radiation: • time a D N A molecule is traversed by an a-particle* uble break). i a cluster of ionizations (or other high energy event) >y sparsely ionizing radiations (850 eV/double break), i by chance two isolated breaks come into juxta-rom statistical calculation one "double break" will :very 70 random single breaks. This mechanism is for main-chain scission by the indirect action of H

dicals formed in the water ced at the same time as main-chain scission by a-rays ionizing S-rays.

Two the a< In L with linkii high' cules

FIG. 7-1' action ol


cannot be decided whether this is due to the fact that the trapped radicals do not react with oxygen or whether diffusion into the fibres is very slow. The important point is that the magnitude of the signal is very much larger if the irradiations and electron spin resonance measurements are made at the temperature of liquid nitrogen. This means that at room temperature the majority of the radicals react very rapidly even in the rigid system of isolated DNA fibres.

Irradiation with a-rays Thin films of D N A were exposed to a-rays from polonium by A L E X -

A N D E R and LETT111. The viscosity was reduced to exactly the same extent whether the films were dry or wet and in this respect a-rays were quite different from the sparsely ionizing radiations (see Fig. 7-18). Quanti-

FIG. 7-18. ComparisonoftheefFectofpolonium A-ravs (b) and of 1 MeV electrons (a) on solid D N A having different moisture content.111 (o 20 g of water per IOOg D N A ; • 80-100g water

per 100 g DNA)

tatively the data showed that every time an a-particle traversed a DNA chain double scission occurred (see Fig. 7-17). Although the viscosity drop was the same whether the irradiation was carried out in the presence or absence of oxygen, the molecular weight changes indicated that there was some crosslinking (not sufficient to give a gel but only to produce branched molecules) if oxygen was excluded. This crosslinking can be attributed to the relatively sparsely ionizing S-rays which contribute something of the order of 25 per cent to the total energy deposited (see


p. 32). Consequently, some of the processes characteristic of /3-rays (i.e. crosslinking in the absence of oxygen) invariably accompany a-irradiation, just as with sparsely ionizing radiations there are occasional large clusters that produce the same effect as an a-track.

Ejfect of Irradiation on Niicleoproteins The difficulties in the isolation of nucleoproteins have made it very

difficult to study the effect of radiation on them in vitro. Experiments in vivo are often difficult to interpret as some of the changes that are seen may be due to subsequent metabolic processes. The rigidity of a nucleo-protein gel extracted from chicken erythrocytes is significantly reduced (see Fig. 7-19) by relatively small doses of x-rays112 which would only

FIG. 7-19. Effect of irradiation with x-rays on the rigidity of a nucleoprotein gel extracted from the erythrocytes of chickens: (a) erythrocytes irradiated and nucleoprotein gel extracted after irradiation; (b) irradiation of nucleoprotein gel in vitro11-.

slightly reduce the viscosity of DNA solutions of comparable concentra-tion. ERRERA112 found a greater decrease in rigidity if the cell was irradiated and the nucleoprotein gel extracted afterwards. R O L L E N A A L et A / . 1 1 3 ob-tained a partial dissociation of histone from a suspension of nucleoprotein fibres in saline while C O L E and ELLIS114 in rather similar experiments find that the capacity of spleen nucleoprotein fibres to swell in water is reduced if they are first irradiated in suspension in 0-14 M salt.

The physico-chemical mechanism underlying this change is obscure and none of these effects are obtained if spleen cells are irradiated in vivo and the nucleoprotein fibres isolated immediately afterwards115. The resis-tance of the nucleoprotein if irradiated "undiluted" was demonstrated by E U L E R and HAHN116 in 1 9 4 6 who could detect no physico-chemical


differences after irradiating thymus nuclei with doses ranging from 25,000 to 60,000 r of x-rays.

A N D E R S O N and FISHER117 found that "solutions" of thymus nuclei in distilled water were extremely sensitive to x-rays and that a dose of 100 r and less produces a significant drop in viscosity. The reason for the sensitivity arises solely from the fact that extremely dilute solutions (circa 0-01 per cent of DNA and less) were irradiated when the "dilution law" ensures great sensitivity as there are few substrate molecules to react with the radicals formed in the water. At concentrations comparable to those in the cell no change was seen with 10,000 r and this is in agreement with the fact that if the thymus cells are irradiated and the nucleoprotein "solution" is prepared subsequently again no change is seen even after tens of thousands of roentgen. No interpretation of the viscosity changes is possible as thymus nuclei do not dissolve in water, but merely swell, and the measurements amount to the detection of a change in the rigidity of an extremely loose gel network. KAUFMANN118 has also reported changes in the so-called viscosity of nuclei dispersed in water, but his data are complicated by enzymatic degradation that occurs concurrently and there was no evidence that there was a change in the physico-chemical properties of the nuclei immediately after irradiation with relatively small doses.

Irradiation of a non-swollen system—fish sperm heads—produces the same changes as irradiation of DNA fibres containing about 20 per cent of water, that is crosslinking119. There are indications that the presence of protein in some way modifies the radiation response. The crosslinking of sperm heads can be prevented by the presence of cysteamine119. This "protection" against crosslinking may be due to energy transfer though a repair process (see p. 177) is more likely. It is certain that we are dealing here with protection against an effect which is not potentiated by oxygen, indeed crosslinking occurs at lower doses if the sperm heads are irradiated in vacuo.

C H A N G E S P R O D U C E D I N D N A F O L L O W I N G I R R A D I A T I O N IN VIVO

The only claim to having found an immediate change in the DNA following irradiation in vivo is that of O R D and STOCKEN120 using rat thymocytes. While the molecular weight and molecular dimensions are unaffected by 2000 r the elution pattern from a chromatographic column is different. This test unfortunately is difficult to interpret. As the DNA sticks very firmly to the column alkaline solvents are needed to get it off, and this system is therefore liable to art-effects and has been criticized121.

All other data are consistent with the view that even after comparatively high doses (e.g. 25,000 r, ref. 116) the DNA in cells is quite unaltered


if there are no post-irradiation changes. This physico-chemical evidence is in agreement with the observation that none of the transforming activity of DNA pneumococcus is lost if the organism is irradiated with IO5 r. (see p. 61). If the DNA is extracted some hours after irradiation then damage can already be detected with 1000 r (115, 122). This does not represent a primary lesion but is a secondary effect.

C H A N G E S P R O D U C E D I N P O L Y S A C C H A R I D E S Many of the reactions produced by irradiation in DNA must involve the sugar moiety. Main-chain scission, if it occurs immediately, requires the breaking of one of the bonds

I CH

/ \ O CH2 O If \ /

- O - P - O - C H - C H - C H 2 -

O—

in this recurring unit of DNA. Since the —O—P— bond is extremely radiation resistant124 attack at one of the sugar carbons is indicated.

Moreover, the observation of B R I N K M A N and L A M B E R T S 1 2 5 (see p. 468) that the permeability of endothelial layers is altered immediately by small dose focuses attention on the radiation resistance of mucopolysaccharides.

S C H O E N B E R D et al.127 found that synovial fluid is very sensitive to the indirect action of x-rays and a viscosity fall is seen after a few thousand roentgen in the undiluted biological fluid, which contains about 0-5 per cent of hyaluronic acid. The change is not complete immediately after irradiation and there is a small delayed drop in viscosity which takes some 24 hr to reach completion. B R I N K M A N et al.121 find that the addition of cysteamine or 5-OH tryptamine protects the synovial fluid just as it protects polymethacrylic acid (see p. 176). But there is a marked difference between these two polymers, while polymethacrylic acid is only degraded in the presence of oxygen synovial fluid is more sensitive in its absence (see Fig. 7-20). Although it has been generally assumed that the decrease in viscosity of synovial fluid on irradiation is due to degradation the possibility of internal crosslinking (see p. 162) cannot be excluded. In this connection the findings of M . S T A C E Y et al. (ref. 128; see also ref. 129) that the irradiation of sugars in dilute solution leads in the absence of oxygen to polymers may suggest the occurrence of crosslinking.

The direct action of radiation seems to be largely degradative. From an analysis of a variety of data in the literature C H A R L E S B Y 1 2 concludes that


main-chain scission is the predominant process when dry cellulose is irradiated. In the presence of oxygen the degradation continues after

irradiation130, but the after-effect is completely suppressed in the presence of water vapour. The behaviour of starch seems to be similar131. A detailed study of the electron spin resonance of irradiated sugars and polysac-charides132 has not thrown any light on the observed chemical changes.

T H E U S E O F R A D I A T I O N A S A N A N A L Y T I C A L T O O L

The loss of activity of most biologically active substances when irradiated in vitro under conditions where indirect action is excluded (i.e. irradiated dry or in a solution containing protective agents sufficient to capture all the radicals) follows an exponential dose-response curve. This led L E A 1 3 3

to postulate that under these conditions a primary ionization occurring anywhere within a sensitive volume leads to inactivation and that the size and shape of this volume can be calculated on the basis of distribution of ionization produced. While the inactivation dose gives the mass of the target the change in RBE with LET gives the shape (see p. 81). The next step was to identify the sensitive volume with the size of the molecule, and a method seemed to be at hand for the use of atomic radiations as an analytical method in enzyme and virus chemistry. This method was first applied by LEA133 to some viruses and enzymes and in recent years P O L -

LARD134 and his colleagues have used it extensively in a most impressive

O IOOO 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0

Radiat ion dose, r

FIG. 7-20. Immediate effect of x-ray dose on relative viscosity of fresh synovia, equilibrated with commercial N2 and O2. Pro-

tection by 5 X IO - 3 M, 5 hydroxy tryptamine127 .


series of investigations encompassing a vast variety of biologically active substances ranging from penicillin of molecular weight 600 to trans-forming principle of 6 x IO6 molecular weight.*

In Fig. 7-21 the accumulated data of the Pollard group are summarized

IO'

E CT 1

O e

IO h. S

fe 10"

10'

D N / i ( t rans forming

O \

r t

Catala< • irradial

at d f f e tempe

>e ed rent

O O

°

O V j

i i

Catala< • irradial

at d f f e tempe atures

/O

/ O

/ Peni allin

IO' Molecular weight by chemical and physico-chemical methods

FIG. 7-21. Collected data from the laboratory of E. C. Pollard showing correlation between target size and molecular weight for biologically active substances irradiated in the dry state in vitro.

and show that in every case the target size was of the correct order of magnitude and corresponded usually within a factor of two to three with the molecular weight determined by physico-chemical methods. In almost all these studies by the Pollard group radiation of different LET was

* The data for the inactivation of transforming principle is very confusing and has recently been summarized by LATARJET et al.131. The inactivation curve appears to consist of two straight lines; 70 per cent appear to inactivate at half the dose needed to remove the remaining 30 per cent of activity. Different workers using different techniques obtain answers varying by factors of ten. Also the dose needed to inactivate varies for different transforming principles. One reason for the com-plex behaviour is that the D N A can be rendered inactive either by preventing it from being taken up by the pneumococcus or by destroying the part of the molecule that actually carries the genetic information.


used and a shape for the molecule could thus be deduced (see Fig. 7-22), but there are no physico-chemical methods that allow a precise check to be made of the values thus obtained.

^ 3 0 0 0

o 2 5 0 0 TS o 2000 U) m g 1500

1000

5 0 0

[ah [ah

/ "i

/ /

2 0 0 4 0 0 600

L E T in e V / 1 0 0 A

2 0 0 4 0 0 6 0 0

L.E.I in e V / I O O A

FIG. 7-22. Inactivation dose for enzymes as a function of the L E T of the radiation used for (a) deoxyribonuclease; solid line is theoretical curve for a sphere of 63,000 molecular weight135; (b) invertase, solid line is theoretical curve for a cylinder of

axial ratio 1:2 and molecular weight of 120,OOO136.

Protection Evidence for protection by added chemical substances against chemical

changes by "direct action" was first observed in synthetic polymers and energy transfer processes were believed to be responsible (see pp. 112 and 177). Electron spin resonance provides support that energy transfer occurs both between amino-acids50 and between a protein and an added sulphydryl compound (see p. 1 8 6 ) . G I N O Z A and NORMAN1 4 0 found that the radiation resistance of dry catalase is almost doubled if the protein is freeze-dried in the presence of a small amount of glutathione.

Ability to protect dry enzymes is not confined to sulphydryl compounds, and H U T C H I N S O N and his colleagues49 found that the radiosensitivity of invertase varied widely from one preparation to another. They examined samples taken at different stages of isolation and noted a gradual increase in radiosensitivity as purification proceeded. By deliberately adding a great excess of different extraneous substances, a change in radiosensitivity was noted in each of the four enzymes examined and it must therefore be concluded that this is a general phenomenon.

Sulphydryl compounds also protect phage139-141'142 and RNA from tobacco mosaic virus140 against inactivation (see also protection of sperm heads p. 206). It is not known whether energy transfer is involved in every case or whether a "repair" by hydrogen transfer from the SH group occurs (cf. ref. 142). While reaction of this type is very probable in a system where molecules can move, it is difficult to see how in a com-


pletely dry system the SH protector in very small quantities would react with a protein radical since the two would only rarely be adjacent.

Oxygen Effect Following the observation that oxygen modified the radiochemical

changes that occur in polymers A L E X A N D E R 4 8 found that the dose to inactivate dry trypsin with fast electrons was decreased by 50 per cent in the presence of oxygen, but inactivation by a-particles was oxygen independent (see Fig. 7-23). This oxygen effect has been observed with

FIG. 7-23. Effect of oxygen on the inactivation of dry trypsin48. — A — Po a-rays in air. — • — Po a-rays in nitrogen. — X — 60Co y-rays in air. — O—- 60Co y-rays in vacuum.

several other enzymes143-144 and can probably be considered a general phenomenon; possible mechanisms have been discussed on p. 186.

Summary The fact that so frequently the target size corresponds even approx-

imately to molecular dimensions is surprising in view of biochemical data obtained in recent years which show that the whole of the protein molecule is not necessary for enzymatic activity. Extensive chemical alterations can be made in many parts of a protein molecule without loss of activity and ionizations that cause chemical changes (e.g. alter amino-acid residue side chains) in these parts should not lead to inactivation. This paradox

i5


can probably be resolved by the experiments described on p. 180, which show that in addition to producing covalent chemical changes in individual amino-acid residues a primary ionization disrupts many hydrogen bonds and disturbs the native configuration of a substantial part of the molecule. This last reaction is probably responsible for loss of activity in many cases and explains why an ionization occurring in a non-essential part of a protein is still harmful.

Even so irradiation data cannot do more than give an order of magni-tude for molecular dimensions because: (1) the energy needed to produce a primary ionization (or the number of ionizations produced by a given dose) in a solid is only a guess (see p. 79). (2) protection by added sub-stances and the existence of an oxygen effect very substantially alters the inactivation dose.

The apparent size of the target would therefore depend on radiation conditions. These effects invalidate attempts to determine target sizes of impure preparations as the other substances present may protect. The claim134 that radiation methods can provide accurate information of the molecular dimension of a biologically active molecule before it has been isolated in a state of purity is therefore subject to serious qualifications.

Similarly, attempts138 to obtain information from radiation data about the size of the molecular units responsible for the different functions of a virus, such as ability to attach to the cell, to multiply, to kill the host, to undergo multiple reactivation, are liable to serious errors. There is no foundation for the assumption that an ionization in one of the component parts of a virus does not affect a neighbouring molecule that determines a different property.

R E F E R E N C E S

1. CHARLESBY, A., Proc. Roy. Soc. A , 1954, 249, 367. 2 . CHAPIRO, A . , D U R U P , J . , Fox, M . and MAGAT, M . , La Ricerca Scientifica,

1955, 24, 207. 3. ALEXANDER, P . and F o x , M . , Nature, 1952, 169, 572. 4. ALEXANDER, P. and Fox, M., Trans. Faraday Soc., 1954, 50, 605. 5 . ALEXANDER, P. and CHARLESBY, A .,J. Polymer Sci., 1957, 23, 3 5 5 . 6. Fox, M. and ALEXANDER, P., J . Chim. Phys., 1955, 52, 710. 7 . MELVILLE, H . W . and W A T S O N , W . F . , J. Polymer Sci., 1 9 5 3 , 11, 2 9 9 . 8. ALEXANDER, P . and CHARLESBY, A . , Radiobiology Symposium, Liege,

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p. 216; Radiation Research, 1954, 1, 18. 70. LANGE, R . , PIHL, A . a n d ELDJARN, L . , Intern. J. Radibiol., 1959,

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Radiobiol., Suppl. 1, 1960 page 47. 94. ALEXANDER, P . , Biochim. Biophys. Acta, 1953, 1 0 , 595. 9 5 . FELIX, K . , FISCHER, H . , KREKELS, A. and RAUEN, H . M., Hoppe-Seyl. Z.,

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Biophys., 1948, 16, 19. 9 9 . LIMPEROS, G . a n d MOSHER, W . A . , Am. J . Roentgenol., 1 9 5 0 , 6 3 , 6 9 1 .

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p. 167. 1 2 6 . SCHOENBERD, M . D . , BROOK, R . E . , H A L L , J . J . a n d SCHNEIDERMAN, H . ,

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1960 (in the press). 1 2 8 . BARKER, S . A., G R A N T , P . M., STACEY, M. and W A R D , R . B . , Nature, 1 9 5 9 ,

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1946. 1 3 4 . POLLARD, E . C . , G U I L D , R . , HUTCHINSON, F . A . a n d SETLOW, R . B . , i n

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C H A P T E R 8

Chemicals which Simulate the Biological Effects of

Ionizing Radiations

THE biological activity of certain chemical substances, often referred to as "radiomimetic"1 because they produce all the biological end-effecis observed after treatment with ionizing radiations, is closely related to the study of radiobiology. The clinical use of these chemicals for the treatment of cancer and leukaemia is generally in the hands of radiotherapists and studies in their mode of action cannot fail to advance our understanding of the mechanisms of action of radiation.

Effects such as arrest of mitosis and induction of tumours have been known for many years to be produced by chemical substances as well as by ionizing radiations, but it was the discovery by A U E R B A C H and R O B -

SON2 in 1943 that the chemical warfare agent, mustard gas, S(CH2CH2Cl)2, produced mutations and R O L L E R ' S observation3, that the same substance gave radiation-like chromosome abnormalities, which for the first time revealed the possibility that the genetic effects of ionizing radiations could also be produced by chemical agents*. The production both of gene-mutations and chromosome abnormalities had until this time been con-sidered as a characteristic property of ionizing radiations, and its interpre-tation by the target theory appeared to provide a consistent and convincing explanation on purely physical lines which was extended to many other radiobiological phenomena. Chemical mutagenesis, coming shortly after Dale's demonstration that enzymes could be inactivated with x-rays by indirect action, initiated a re-interpretation of radiobiology along more chemical lines. The rapid development of our subject bears testimony to the fruitfulness of this new approach.

In the meantime many other chemical substances have been found to

* Conversely, it suggested to one of us (Z.M.B.) that the amines which inhibit mustard gas action might also be used to counteract the effects of purely physical agents such as ionizing radiations and led to the discovery of some of the protec-tive agents which are discussed in Chapter 19.

217


give rise to genetic effects,* but this is not sufficient to justify calling them radiomimetic in the sense in which this word will be used in this chapter, namely to simulate all the end-effects of radiations. In addition to genetic damage they must act as mitotic poisons, kill lymphocytes and be carcino-genic. A number of substances are known which produce chromosome breakage, are carcinogenic and in some cases cause gene-mutations,without fulfilling the requirements for radiomimetic action. For example, urethane, the first chemical, which had been shown to produce permanent chromosome abnormalities9, affects the cytoplasm10 where it brings about disturbances (e.g. enzyme inhibition) the consequences of which become apparent in the nucleus and result in the appearance of chromosome fragmentation.

A very wide variety of agents including distilled water6 inhibit or derange cell division in many different types of cells at very low concen-trations (i.e. IO-5

M or less with mammalian cells in vitro7). D U S T I N 1 has usefully divided them into two classes: (a) agents that act during mitosis by destroying the spindle and arresting the cells in metaphase (e.g. colchicine, arsenite, podophyllotoxin); and (b) agents that act on the nucleus during the resting stage and prevent the onset of mitosis. Radio-mimetic agents fall into the latter category. By increasing both the duration of treatment and the concentration of the agent it is possible to kill cells outright. K O L L E R 3 has introduced the useful terminology of nucleotoxic for typical radiomimetic effects which are permanent (e.g. chromosome "breakage"); cytotoxic effects are initiated by action in the cytoplasm and include the physiological chromosome aberrations (see p. 253). A cyto-toxic agent, but not a nucleotoxic agent, can kill a cell even when it is not dividing (e.g. in the resting stage). At high doses most radiomimetic substances become cytotoxic.+

In addition to acting preferentially on dividing cells a substance to be considered radiomimetic must also kill lymphocytes at very low doses (i.e. at IO-5 M or less in vitro1). Injected into animals many of the mitotic

* There are substances, notably cortisone and the folic acid antagonists, which bring about chromosome breakage without being mutagenic or carcinogenic4. The lesions produced by vitamin antagonists suggest a working hypothesis for some of the biochemical effects produced by radiation which is similar to a mech-anism proposed by BINET5 for the nitrogen mustards. Irradiation transforms a vitamin or growth factor into an antivitamin, the influence of which is felt at a period after the irradiation is complete, and perhaps even at a site which has not been irradiated (cf. poison theory discussed on p. 96).

f The toxic action of the radiomimetics is not confined to the nucleus. The relative sensitivity of nucleus and cytoplasm was determined in amoeba by HARRIS, LAMERTON, O R D and D A N I E L L I 8 by removing the nucleus and treating it separately, followed by reconstitution with the cytoplasm. To kill the organism a 10 times greater dose of nitrogen mustard (vide infra) has to be applied to the cytoplasm than to the nucleus. For x-rays the sensitivity differs by a factor of 2-5.

FIG. 8-1. Radiomimetic activity shown by inhibition of the growth of a grafted tumour. These pictures were taken 14 days after implantation of a tumour fragment (Walker carcinoma) in an untreated rat (left) and (on the right) in an animal that had received an injection of an alkylating agent, such as a nitrogen mustard, some days following implantation of the homograft.

[facing p. 238

R A D I O M I M E T I C SUBSTANCES 2 1 9

poisons selectively kill, exactly like ionizing radiations, the lymphocytes in all the lymphoid tissue even though the majority of the cells are ones that are not going to divide again.

Substances which exhibit all these effects at the cellular level give rise to an acute syndrome in mammals which shows all the characteristics of mammalian radiopathology, such as characteristic acute and chronic degenerative changes in the bone-marrow, intestinal mucosa and testis, local greying or bleaching of hair and the suppression of antibody forma-tion. These agents show pronounced growth inhibitory activity particu-larly against the growth of certain animal tumours (see Fig. 8-1) and it is their use in cancer chemotherapy that has stimulated the large amount of work that has been done in recent years in this field (cf. ref. 11).

These criteria severely limit the type of compounds to which the term radiomimetic can be applied and excludes many substances which have been called this in the literature. Still in view of the limited data available concerning their mode of action, the classification of a particular substance as radiomimetic is bound to be somewhat arbitrary. In this chapter we will discuss only the biological alkylating agents (see Table 8-1), all of

TABLE 8-1

REACTIONS OF ELECTROPHILIC ALKYLATING ACENTS

Type of compound Reaction with

Type of compound Ionized carboxyl group

(R—COO-Na+) Amino group

( R - N H 2 )

Epoxide C H 2 - C H -\ /

O

R - - C O O - C H 2 C H -I + N a O H

O H

R - - N H . C H 2 C H — I

O H

Ethyleneimine C H 2 - C H 2

\ / N I

R - -COO.CH2 .CH2 .NH— + N a O H

R - -NH .CH2.CH2 .NH—

Mustard gas group CI.CH2.CH2.S—

R - - C O O . C H 2 . C H 2 . S -+ N a C l

R - - N H . C H 2 . C H 2 . S -+ H C l

Nitrogen mustard group CI.CH2.CH2.N <

R - -COO.CH2.CH2.N < + N a C l

R - -NH .CH2.CH2 .N < + H C l

Mesyloxy group CH3.SO2.O.R'— (e.g. R ' is (CH2Jn) (Myleran type)

R - -COO.R'— + N a + - O . S O 2 - C H 3

R - -NH. R '— + C H 3 . S O 2 . O - H +


which are closely related in chemical reactivity to mustard gas, and to peroxides. While the latter do not fulfil all the requirements they are more nearly radiomimetic than any other class of chemical substances with the exception of the alkylating agents. Also the possibility that some radiation effects may be due to the formation of peroxides (see p. 96) makes these substances of particular interest.

T H E C H E M I S T R Y O F T H E B I O L O G I C A L A L K Y L A T I N G A G E N T S

The recognition that compounds containing the groups shown in Table 8-1 as well as fulfilling a number of other requirements (see below) are radiomimetic, originates from the discovery that mustard gas was cyto-toxic and mutagenic. It was soon realized that closely related compounds, the so-called nitrogen mustards, possessed the same biological activity and the extensive literature on this subject has been fully reviewed by PHILIPS12 and by B A C Q 1 3 . ROSS14 found that for active compounds the halogenoalkyl groups must have a minimum chemical reactivity as measured by the rate of hydrolysis. Once these requirements had been met the structure of the remaining part of the molecule appeared to be capable of influencing the biological activity only quantitatively. Thus biological activity was found in all compounds of the general type RN(CH2CH2Cl)2 whether they are basic, acidic or neutral, water or oil soluble. Active compounds have been obtained where R consists of almost every type of aliphatic, aromatic or heterocyclic structure. The essential feature for activity was clearly the chemical reactivity of the two mustard groupings. The halogen atoms are very reactive and confer on the mustards the ability to act as alkylating agents (see below).

It was an obvious next step to look for biological activity in compounds containing groups having the same chemical reactivity as the mustards. Independently different workers (see reviews by HADDOW15 and A L E X -

ANDER16) found that compounds containing two or more epoxide, ethylene-imine or mesyloxy groups were cytotoxic and inhibited tumour growth in experimental animals. Further investigation showed that these substances were carcinogenic, mutagenic and produced chromosome fragmentation. All other tests also showed that these alkylating agents possess true radiomimetic activity as defined above.

Needfor More then One Functional Group Since the major interest of the radiomimetic substances is related to

their possible use as chemotherapeutic agents against cancer they are generally examined as growth-inhibiting agents in animals. The extensive work of HADDOW17 '18 and of R O S E and his colleagues19 has clearly dem-

R A D I O M I M E T I C S U B S T A N C E S 2 2 1

onstrated that in general only compounds containing two or more alkyl-ating groups are active. Out of the several hundred compounds which were found to be active as growth inhibitors only three were monofunc-tional and all of these contained a second centre having unusual physico-chemical characteristics.

Subsequently it was found that monofunctional derivatives were mitotic poisons and produce chromosome abnormalities, but in general a dose fifty to one hundred times greater than that needed with polyfunc-tional compounds has to be used20'21. The ineffectiveness of most mono-functional agents as growth inhibitors for cancer chemotherapy is due to the fact that the toxicity is too great to permit administration to animals at the increased doses that are needed.

For radiomimetic effects that are not directly related to cell killing the superiority of polyfunctional agents is not so apparent. W A L P O L E 2 2 finds many monofunctional compounds carcinogenic though a direct quanti-tative comparison with polyfunctional substances has not been made. In the field of mutagenesis monofunctional derivatives of mustard gas were found to be as effective as the bifunctional parent substance23. Mono-functional epoxides, methanesulphonate esters and ethyleneimines as well as mustards have all been shown to be highly effective mutagens in microorganisms24, plants25 and insects26, and the suggestion has been made27 that there is a difference in mechanism at the chemical level between mutagenesis and other radiomimetic effects (see below).

Nature of the Chemical Reactivity. The groups shown in Table 8-1, which confer radiomimetic activity,

are "electrophilic" alkylating agents since they react most readily with structures which are rich in electrons (see Table 8-2). For example, these

T A B L E 8 - 2

Groupings Reactive form Less reactive form

Organic acids Phosphoric acid

R.COO". R .COOH

O = P - O - O = P - O H

Hydroxy or phenolic compounds Sulphydryl

R .O-R.S-

R.OH R.SH

Amine Thio ethers Phosphorus compounds

R : iN R.S.R. PR 3 (trivalent) OPR 3 (pentavalent)

R3HN+


agents will readily alkylate carboxylic acids if present in the ionized form (i.e. when the pH of the solution is greater than the pK) while the reverse applies to amines which react in their un-ionized form (i.e. when the pH is less than the P-Ka). The nature of the reaction products are summarized in Table 8-1.

Both in nucleic acids and proteins there are a number of different centres which are capable of being alkylated by the radiomimetic com-pounds; in proteins at pH 7 the groups in the most reactive form are the carboxvl and terminal amino groups, while in nucleic acids the phos-phate as well as all the amino groups are in the form most favourable for reaction.

The rate with which different groups react with these alkylating agents varies very widely. The relative affinity of different groups for the mustards can best be expressed as a "competition factor" and OGSTON 2 8 has deter-mined this for a large number of anions. Those showing the highest reactivity contain —S - groups. Thus the competition factor for the dithiophosphate ion is ten thousand times as great as that of the acetate ion. The reactivity of the corresponding RSH compounds will be much smaller, but will probably still be significant. In proteins the pK of the —SH group is approximately 10 and at pH 7 these groups are almost wholly in the less reactive form. Figure 8-2 shows the influence of pH

700

0 IO 20 30 to min.

FIG. 8-2. Rate of reactions of mustard gas with the SH groups of cattle lens denatured proteins in solutions adjusted to dif-

ferent pH values29.

R A D I O M I M E T I C S U B S T A N C E S 223

on the rate of reaction of SH groups29. However, the competition factors of the —SH (i.e. —S~) is so great that even at pH 7 alkylation of SH groups (at least of those that are sterically accessible, see p. 182) is the predominant reaction with proteins30. Combination with carboxyl, amino and imidazol groups also occurs (see Table 8-3).

T A B L E 8 - 3

G R O U P S IN BOVINE S E R U M A L B U M I N THAT REACTED W I T H BIOLOGICAL A L K Y L A T I N G A G E N T S 3 0

Reduction in Reduction in Treatment number of number of Per cent

(3% protein+0-12 M of carboxyl groups primary histidine reagent) per molecule amino groups changed

per molecule

Di-(2:3 epoxypropyl) ether 33 40 77 Propylene oxide 27 52 77 1:2-3 :4-Diepoxybutane 18 31 70 Methane sulphonyloxy ethane 30 0 47 1:3-Dimethane

sulphonyloxy propane 38 0 42 2:5-Dimethane

sulphonyloxy hexane 21 6 12 Methyl di-2- Minimum

chloroethylamine of 12 14 9 Diethyl-2-chloroethylamine 0 5 N'N"N"Trie thylene-

iminophosphoramide — 0 0 l-chloro-2-methane

sulphexyloxyethane 12 3 1 NN-di-2-chloroethyl-£-

aminophenyl butyric acid 30 5 5

A L E X A N D E R 1 6 found that D N A and polyelectrolytes had competition factors which were much higher than would be expected from the reac-tivity of the individual groups present. It would appear as if the presence of several reactive centres close together enhances the reactivity of the individual group. These results indicate that the biological mechanism of the radiomimetic alkylating agents depends upon reaction with ionized macromolecules such as DNA.

Alkylating agents which are not electrophilic (i.e. which do not react specifically with electron-rich groups) have not been found to possess radiomimetic properties. For example, diazo-methane (CH2N2) and its derivatives which readily esterify undissociated carboxyl and hydroxyl


groups are inactive. Compounds which readily alkylate —NH2 and —SH groups and do not react with anions (e.g. —COO~) such as isocyanates, halogenopyrimidines and halogeno-2,4-dinitrobenzene, show no bio-logical activity. This indicates that the biologically significant reaction of the radiomimetic alkylating agents is reaction with ionized acid groups14 or tertiary amino groups (see p. 232).

C O M P A R I S O N O F B I O L O G I C A L E F F E C T S P R O D U C E D BY T H E A L K Y L A T I N G A G E N T S A N D BY R A D I A T I O N S

At the present time we do not know at what stage in the development of the biological lesions the pathway of the alkylating agents and ionizing radiations meet. Is it at the stage of the primary chemical lesion (e.g. do chemicals and radiations damage by different reactions DNA or cell membranes?) and are the subsequent stages the same? At the other extreme it may be that the mechanisms are entirely different and that the simi-larities are confined to the end effect only. Probably no simple answer can be given as there are indications that there may be differences between different members in the group of alkylating agents.

Chromosome Fragmeritation The structural changes in the nucleus produced by the radiomimetic

chemicals are qualitatively indistinguishable from those brought about by x-rays. This was observed originally by K O L L E R 3 with mustard gas both with the root meristem of Allium and with Tradescantia pollen, and subse-quently in other materials such as tumour cells. However, distinct differences were revealed between the chemical and physical agents in more detailed studies, and K O L L E R and C A S A R I N I 3 1 were led to conclude that " it would be a gross error to infer a similarity of the mode of action of nitrogen mustard and x-rays based on the similarity of some end-products. The latter are the results of a complex chain of reactions which can be initiated by fundamentally different events". This view finds considerable support wherever detailed investigations on the mechanism have been carried out.

(i) The period of maximum sensitivity of the cell occurs earlier in the mitotic cycle for the chemicals than for x-rays. This was first demonstrated in rat tumours31, see Fig. 8-3, and confirmed in plant material32.

(ii) In general, breaks produced by radiations both between different chromosomes and along the same chromosome are distributed at random. In some instances evidence for localization was obtained, but in no case was this very pronounced. The position is quite different with regard to the chemicals. The short chromosomes in Vicia were broken much more frequently than the long chromosomes32'33, also certain parts along the


chromosomes, known as heterochromatic regions, were more "sensitive" to radiomimetic chemicals.* This observation supports the view that these agents react particularly readily with DNA which is believed to be present in high concentration in the heterochromatin.

At present it cannot be decided whether the greater selectivity of the chemicals compared with the radiations is due to chemical or physical differences. The chemicals have to diffuse through the cytoplasm and pass the nuclear membrane before they can influence the chromatin or its syn-

Time after treatment Ix

FIG. 8-3. Graph illustrating the difference in the time of maxi-mum sensitivity of rat tumour cells to x-rays and to the nitrogen

mustard HN231 .

thesis. For this reason those chromosomes, or part of the chromosomes, closest to the nuclear membrane during the resting stage may be relatively more susceptible to damage by the chemicals than to radiation.

(iii) The number of chromosome aberrations in Viciafaba produced by a bis-epoxide are a direct function of both, the time of treatment with, and the concentration of the chemical (e.g. 2-5 x IO-4 M solution for 4 hr is equivalent to 10~3 M for 1 hr.) This behaviour is quite different from the breaks produced by radiation where dose rate is as important as total dose.

This evidence indicates that the primary reactions which initiate the series of reactions that lead to chromosome damage are different for the polyfunctional alkylating agents than for ionizing radiations.

* Other types of chemical mutagens also show selectivity, but the sensitive regions are not the same. For example, on treatment with urethane34, ethoxy-caffeine and tetramethyl uric acid35 breaks occur preferentially in the long ( "M") chromosome of Vicia.


Production of Gene-mutations In many cases (e.g. for Neurosporas6 and Drosophilasi) the similarity

between the genetic effects produced by radiation and chemical-mutagens is very close. Table 8-4 shows that qualitatively even some non-radio-

T A B L E 8 - 4

C O M P A R I S O N OF THE G E N E T I C EFFECTS PRODUCED IN DROSOPHILA BY X-RAYS AND BY D I F F E R E N T C H E M I C A L S 3 7

Effect x-rays Sulphur mustard

Nitrogen mustard

H N 2

Formal-dehyde Urethane

Recessive lethals + + + + + Dominant lethals + + + + p Visible mutations + + + + + Minor deficiencies + + + + + Gross deletions + + + + + Inversions + + ? + ? Translocations + + + + ? Gynandromorphs + + ? + ?

+ Observed. ? Not observed or only very infrequently observed.

mimetic cell poisons can bring about all the different genetic effects in Drosophila which are induced by x-rays.

The close similarity in genetic effects between ionizing radiations and alkylating agents is shown in the following experiments which seem to compel the view that their mode of action must be related.

(i) When E. coli are irradiated with a heavy dose of x-rays the survivors are very resistant to further irradiation and constitute a new strain. In the same way a nitrogen mustard resistant strain can be produced. BRY-SON38 noted that the mustard-resistant strain was also resistant to radiations and vice versa. Resistance in different strains is usually a very specific phenomenon and the production of cross-resistance indicates a close similarity between the two toxic agents.

(ii) Infra-red irradiation prior to exposure to x-rays increases a specific type of chromosome aberration in Drosophila. Post-treatment with infra-red has no such effect. K A U F M A N N et a/.39 found exactly the same behaviour when nitrogen mustard is used instead of x-rays.

With mutations the evidence for differences in mechanism is not so strong as in the production of visible chromosome aberrations and the most significant experiment in this respect is probably the observation of AUERBACH 4 0 that some of the genetic effects of treatment with mustard


are delayed. This was deduced from the fact that the number of Brosophila with mosaic areas in the body is very much greater, and the area much smaller, after mustard than after x-ray treatment. Mosaics are the result of chromosome changes brought about after the fertilized egg has started to divide. If the aberration occurs at the first division half the body of the fly will be affected—this is the case with x-rays. If it occurs at later cleavage divisions the mosaics will cover a smaller area. Somatic crossing-over, another delayed effect, is also much more frequent with mustard gas and with several other alkylating agents than with x-ray treatment. Another difference is that the ratio of lethal mutations to visible chromo-somal aberrations is much higher for the chemicals41.

Cell Killiyig and Inhibition of Mitosis Changes in the number of circulating cells of different types in the

blood are seen very soon after irradiation and after treatment with the alkylating agents. These effects are believed to be a reflection of the cell killing action of these agents in the haematopoietic organs42 and variations in the blood count after different treatments can therefore be ascribed to differences in their cytotoxic action.

By studying the comparative effects at relatively low doses of x-rays and alkylating substances on the number of circulating blood cells in the rat ELSON 4 3 demonstrated that, in general, the compounds of the nitrogen mustard, ethylene imine and epoxide series imitate mainly the lymphoid effect of x-irradiation, whilst the compounds of the Myleran type repro-duce fairly closely the myeloid effects. At higher doses these differences largely disappear44.

The difference between the behaviour of these two types of cytostatic substances is very evident in the animals treated respectively with Chlor-ambucil (a nitrogen mustard) and the methanesulphonate ester Myleran (see Fig. 8-4). In the animals treated by a single intraperitoneal dose of Chlorambucil (12-5 mg/kg) there is a rapid fall of lymphocytes and granulocytes. The former recover slowly reaching normal values in 15 to 20 days, whilst the latter recover very rapidly, leading to a remarkable neutrophilia before returning to normal. The maximum peak of neutro-philia is reached 8 to 10 days after treatment. Myleran, when administered orally in a single dose of 15 mg/kg, causes a fall in circulating neutrophils, but has little effect on lymphocytes. Neutrophils reach their minimum value about 10 to 14 days after treatment, then they recover slowly and return to normal in 20 to 25 days. Platelets also show a delayed fall and with toxic doses of Myleran death may be caused by anaemia resulting from thrombocytopenia.

X-rays show both the lymphoid effect of the mustards and the bone-marrow damage of Myleran and a combination of the two types of

16


alkylating agents completely reproduces the changes in blood pattern pro-duced by 200 r of x-rays in the rat.

^ 200 0 180

E 160

S 1 4 0

1 120

o 100

1 S 8 0 V?

60 4 0

20 0 0 4 8 12 1 6 2 0 0 4 8 12 16 2 0 O 4 8 12 16 2 0 0 4 8 12 16 2 0

D a y s D a y s D a y s D a y s

FIG. 8-4. Blood response patterns in the rat to Myleran, chlorambucil and to combined treatment with Myleran plus chlorambucil compared with the response to 200 r whole-body x-irradiation. Symbols: lymphocytes, neutro-

phils, and platelets43.

These differences have not been ascribed to pharmacological factors such as differential distribution of the agents, but differences in their action at the cellular level have been postulated42. The nitrogen mustards, epoxides and ethyleneimines are believed to kill cells, that are about to divide, in mitosis as well as differentiated lymphocytes, while Myleran at low dose levels merely delays mitosis. By implication x-rays at 200 r both kill cells in the haematopoietic system immediately and delay mitosis.

The behaviour of mouse leukaemia cells growing in tissue culture also provides support for the suggestion that the cell killing action of the methanesulphonates differs from that of the other classes of alkylating agents45. While the mustard stops cell proliferation immediately with the methanesulphonates one or two cell divisions occur at the control rate before the growth of the culture slows down. The appearance of the pathological cells is also quite different. On the whole the action of x-rays in this system resembles that of the methanesulphonates.

Clinical Applications The use of ionizing radiations in cancer therapy is not due to any

inherent difference in response of normal and malignant cells, but depends

Myleran 20mg/kg

Chlorambucil

I2 5 m g / k g

X- radiation

2 OOr

^ <R y M1 J

X V/~r

W IxJy ! t i l ,


on the greater sensitivity of dividing cells. Whatever the difference in mechanism between the radiomimetic agents and radiations may be, the former show no greater selectivity for tumour cells than do radiations. None of the many hundreds of nucleotoxic substances examined have given any indication of selective chemotherapeutic action against cancer. A number of the radiomimetic substances have, however, a rather potent action against particular blood cells in the bone-marrow and this makes them useful for the therapy of some of the leukaemias46. Because Myleran at low doses affects primarily the granulocyte precursors it is a most valuable chemo-therapeutic agent for the control of myeloid leukaemia47. This selectivity is, however, not related to malignancy and is merely due to the fact that certain types of cells (normal or malignant) are more vulnerable than others.

The nitrogen mustards, particularly di-2-chloroethylmethylamine (HN2) and chlorambucil, are now in routine use in the treatment of the generalized phases of the lymphomas, especially Hodgkin's disease48. Tri-ethylenemelamine is even more potent but is also more toxic and is less widely used. These agents are purely palliative and never eradicate the disease but have real value in their ability to alleviate, for several months, the toxaemic manifestations of the disease, and to relieve pain and other symptoms in circumstances when radiotherapy cannot be used. Urethane is most useful in relieving the pain of multiple myelomatosis, but as with all other chemotherapeutic agents so far studied has no influence on the course of the disease although occasionally the bone lesions recalcify.

M E C H A N I S M O F A C T I O N O F T H E A L K Y L A T I N G A G E N T S One of the most important clues in the search for the initial receptor (or primary biochemical lesion) is that the essential feature of this class of compounds is the ability to alkylate under physiological conditions (see p. 221). There can be no question that a metabolite of the original drug is involved.* Since the alkylating reaction is not specific, these agents

: Thiotepa,

S: P- N

C H 2

C H 2

is a possible exception since its reactivity as an alkylating agent is extremely low. Its high biological activity may require the metabolic conversion to TEPA,

CH 2

0:P- N

C H 2 J 3 -which still acts by virtue of being an alkylating agent.


react in the cell with a large number of quite different substrates and they can and do attack different groups (cf. Table 8-3) within the same molecule. The problem of determining the biologically relevant site is therefore one of elimination and we can learn little about the action of the alkylating agents by determining with which molecule they react within the cell. Studies in which the metabolic fate of the alkylating agents is followed in an intact animal are unlikely to provide information about the biochemical lesion since a large part of the administered substance is bound to be wasted in trivial reactions.

Some reactions may be excluded, notably those involved in sulphydryl groups and amino groups on the basis of a comparison with compounds which readily react with both these groups and which yet do not have radiomimetic properties. Wartime research in Belgium, England and the U.S.A. (see review by P E T E R S 4 9 ) , while showing that mustard gas could inactivate SH enzymes, suggested that this was not responsible for its action on the skin. A similar conclusion was reached from cytological data by L O V E L E S S 5 0 . Indeed extensive biochemical data (cf. ref. 5 1 ) suggested that the cell killing action of mustards was not due to inhibition of any one enzyme system and that reactions with proteins is probably not a biologically significant reaction although it undoubtedly occurs.

The fact that these agents produce mutations has drawn attention to the reaction with DNA. Already twenty years ago B E R E N B L U M and W O R M A L L 5 2 found that in skin exposed to mustard gas, the latter combined with the nucleoprotein. On the other hand in none of the reported inves-tigations involving whole animals (cf. ref. 53) and using labelled Myleran, as well as some other alkylating agents, has any evidence been found for combination with the DNA in the cell and the bound material has usually been found to be linked to proteins. It may be argued that the amount of reaction needed to alter the biological properties of a large molecule like DNA is so small that it has escaped detection.

"Degradation" of DNA In the same year, 1 9 4 6 , G J E S S I N G and C H A N U T I N 5 4 found that the

viscosity of DNA solutions was decreased by reaction with nitrogen mustard, and S P A R R O W and R O S E N F E L D 5 5 obtained the same cffect by irradiation with x-rays. T A Y L O R , G R E E N S T E I N and H O L L A E N D E R subse-quently showed (see p. 198) that the ionizing radiations broke up the DNA molecule and reduced its molecular weight (see Chapter 7). Butler and his colleagues extended these investigations and concluded that since the viscosity change was of the same character with HN2 as with x-rays the former also degraded the molecule. This led to the hypothesis that the mustards, like x-rays, degrade by a free radical mechanism56. Chemical experiments designed to test this suggestion failed to provide any support


for it16 and at least one57 of the postulated mechanisms was shown to be impossible on quantum mechanical grounds58.

A decrease in viscosity of a complex macromolecule does not neces-sarily imply that the molecule has been degraded and that exactly the same change will result from a change in shape. This was demonstrated experimentally59 with polymethacrylic acid. On irradiation with x-rays the polymer becomes degraded while the alkylating agent reduced the viscosity by changing its shape. The available evidence60 now points to the conclusion that the initial reaction of the radiomimetic agents with DNA results in a change of shape of the molecule.

Detailed investigations by a number of workers (cf. review: ref. 27) have shown that the principal points in the DNA molecule that are alkylated are the phosphate groups to form a triester

\ \ sugar sugar

O = P - O - + R .X . - > O = P - O R alkylating agent

sugar sugar / /

and a tertiary ring nitrogens in the purines guanine and adenine (see Fig. 8-5). In the case of guanine61 (and possibly also adenine) the alkyl-ated purine is unstable and quickly becomes detached from the DNA (see Fig. 8-5) and this leads to a weakening of the structure at that point. Reaction with the phosphate group also introduces a point where the polynucleotide chain may be broken since phosphate triesters are unstable and hydrolyse easily27, i.e.

\ break in main chain sugar O -

I I = O = P - O R - » O = P - O R

sugar sugar / /

While REINER and ZAMENHOF61 believe that either reaction with the purine or the phosphate group is involved in the mutagenic process, ALEXANDER27'60 believes that the esterification of the phosphate group is the more important. Pathways by which reaction of DNA can lead to mutations have been discussed27. Possibly to produce a mutant no more than a minor alteration must occur in the "target" DNA so that its biological replication is not prevented, but only slightly interfered with, thereby giving increases in the number of imperfect replicas (i.e. mistakes) that are made. It is this altered DNA made during the course of biological


synthesis that represents the mutant. According to this view the alkylation of the DNA does not produce a mutation, but merely increases the chance that a mutation will be produced in the subsequent synthetic processes. If the mutagenic agent modifies the DNA too severely (e.g. crosslinks it; see below) it will be "dead" material and new "mutated" DNA will not be produced.

OH

H 2 N N

CH2-CH

CH I " C H - O - P - O "

\ ? / A CH2-CH T

j

Spontaneous breakdown under physiological conditions

(Subsiluted purine has become detached from DNA molecule) N ^ v

OH R

Dotted lines represent main chain of DNA molecule

,OJ

x O O H - C H j C H - O - P 1 - O *

* 7 6 \ V C H 2 C H

FIG. 8-5. Alkylation of guanine residue in D N A and subsequent reaction.

Crosslinking Hypothesis The increased activity of polyfunctional over monofunctional com-

pounds led GOLDACRE, LOVELESS and Ross62 to put forward the hypothesis that the alkylating agents join different chromosome threads together by


a covalent bond (i.e. crosslinked) prior to mitosis. ALEXANDER et al.63 pro-vided experimental proof for the occurrence of crosslinking in the cell nucleus though it does not suggest that chromosome threads are so joined. It must be stressed that the crosslinking theory can only be ex-tended to the biological changes which involve interference with cell division and should not be applied to mutagenesis. The first cells in which crosslinking was seen were sperm from herring, salmon and trout (see Table 8-5). The heads of such sperm are made up essentially of DNA

T A B L E 8 - 5

R E A C T I O N OF A 3 PER CENT SUSPENSION OF H E R R I N G S P E R M W I T H B I F U N C T I O N A L A L K Y L A T I N G A G E N T S

Compound used Concen-tration used

% D N A present as gel

1. CH3-N(CH2CH2Cl)2 / — x 2. COOH.CH(NH2) .(CH2)2 —N(CH2CH2Cl)2

% 0-0098 0-008

27 61

3. C O O H . C H ( N H 2 ) C H 2 — — N ( C H 2 C H 2 C l ) 2 0-008 33

4. C O O H . ( C H 2 ) 3 < ^ ; > — N ( C H 2 C H 2 C l ) 2 0 008 12

5. C H 2 - C H ( C H 2 ) 2 - C H - C H 2 for 4 hr. \ / \ /

O O

6. CH3-SO2-O(CH2)4-O-SO2CH3 for 24 hr.

CH 2

7 .

CH2

\ / N-CONH(CH2)6-NH CO.N

CH 2 CH2

8.

for 1 hr.

for 4 hr.

4 0

1-6

0 - 0 0 2 5

0 - 0 0 2 5

Notes: (i) The nitrogen mustards (compounds 1-4) react rapidly so that during the time of treatment of approximately 4 hr. more than 90 per cent is used up. Compounds 5, 6 and 7 react much more slowly and only a fraction is consumed during the time of reaction,

(ii) Compound 1 is HN2; 3 is melphalan (or sarcolysine); 4 is chlorambucil.


and protamine and disperse completely in 2 M sodium chloride, which breaks the salt links between the phosphate groups of the DNA and the basic groups provided by the lysine and arginine side chains of the protamine. After treatment with polyfunctional alkylating agents the DNA no longer dissolves completely in 2 M salt. On visual inspection the sperm heads still appear to disperse, but on centrifuging part of the DNA was spun out as a gel (compare Fig. 7-1). We interpret the production of gel-DNA as the joining together of different molecules via covalent chemical bonds into a very large network. Such a network will swell but cannot go into true solution; as the number of bonds (i.e. crosslinks) between the molecules is increased the gel becomes more rigid and swells less.

The crosslinking reaction is not confined to DNA in sperm heads and has been observed in a number of different cells (see Table 8-6). In every

T A B L E 8 - 6

C R O S S L I N K I N G OF D N A I N C E L L S T R E A T E D W I T H THE N I T R O G E N M U S T A R D

C O O H - ( C H 2 ) 3 - / N I-(CII2CII2CI)2 , (Chlorambuci l ) 7 4

Concentra- % D N A Svstem treated tion of present as

mustard crosslinked gel

Viable trout sperm in their seminal fluid /o

0 0 1 6 15 E. coli in stationary broth culture 0-05 33 Ascites cells (8 XlO8 cells/ml.) 0-03 41 Cells from two rat spleens suspended in 10 ml. 0-1 29

case the gel produced consisted only of D NA. The test employed in these experiments (namely measurements of gel fraction) only determines the fraction of DNA which has been crosslinked into an infinite network. It is probably not necessary to crosslink a DNA molecule so extensively before rendering it biologically useless, and one crosslink joining two different DNA molecules together may be sufficient to prevent both from taking part in their normal physiological processes. This means that long before the gel point is reached a substantial fraction of DNA molecules are seriously altered. For example, one-sixth of the dose needed to reach the gel point is sufficient to alter by crosslinking 10 per cent of all the DNA. If this is taken into consideration it can be seen that the amounts of the various nitrogen mustards and of an ethyleneimine (see Table 8-5) which are needed to "gel" DNA are of the same order of magnitude as those


needed to prevent cell division (i.e. inhibition of tumour growth). Myleran on the other hand while capable of producing crosslinking does so only at concentrations which are very much greater than those needed to produce radiomimetic effects.

Is DNA the Site of the Primary Reaction? The experiments described above have shown that the radiomimetic

alkylating agents produced far reaching changes in the chemical and physical properties of DNA. The mere fact that such changes occur does not, however, prove that they are the significant reaction which gives rise to the observed biological end-effects. The attraction of relating mitotic inhibition to crosslinking of DNA is that the point of the initial chemical reaction coincides with that of the biological end-effects (i.e. most of the lesions involve the nucleus because it is the DNA which is alkylated). Yet in the case of ionizing radiations for which a similar argument could be advanced no such conclusion can be drawn (see pp. 97 and 204).

The possibility that reaction with intracellular barriers may lead to cell damage may have to be considered60. These structures contain phosphate groups that are capable of being alkylated and the change in charge would well lead to a change in permeability (compare enzyme release theory, Chapter 10).

R A D I O M I M E T I C P R O P E R T I E S O F P E R O X I D E S A N D

O X Y G E N A T H I G H C O N C E N T R A T I O N S

While peroxides do not mimic radiations as closely as do the alkylating agents there are none the less some striking similarities. Mutations have been produced by organic peroxides (e.g. dihydroxydimethyl peroxide, R O - C H 2 - O - O - C H 2 - O H ) in Neurosporaei and Drosophila65 and they have been shown to enhance the mutagenic action of x-rays66. Chromosome "breakage" was recorded for tert. butylperoxide by L O V E -

LESS50. The possibility that peroxides behave as mitotic poisons has not been investigated although certain peroxides have been shown to be extremely toxic to mice (see p. 96).

Hydrogen peroxide mimics the action of x-rays precisely in the paradox-ical effect on barley seeds67 (see Fig. 8-6). The same phenomenon, namely that small doses given to the dry seed are more effective than large in reducing the growth rate of the shoot on germination, applies equally to the peroxide as to radiation.

Whether the genetic effects of peroxides are due to direct reaction with DNA is not known. In vitro experiments (see p. 200) show that pure DNA is relatively resistant to peroxides, but the free radicals produced by its decomposition are most effective in degrading DNA. Since peroxides react with many reducing agents including such physiological substances


as ferrous iron and ascorbic acid to give radicals; a reaction of this type can readily be envisaged in the cell. WILLIS6 8 finds that peroxides irrever-sibly inactivate sulphydryl enzymes in extremely low doses. Although cysteine and presumably other physiological reducing agents protect, the possibility that peroxides damage cells by reacting with —SH groups must be considered.

Concentration H2O2 (vol %0)

Radiation (in I03r)

FIG. 8-6. Comparison of the effect of x-rays and of hydrogen peroxide on barley seeds67.

R. GERSCHMAN (cf. ref. 69) has stressed the similarity in the pathology of oxygen poisoning in mammals and of whole-body irradiation. For example, prior administration of certain chemicals protects against both effects.

At the cellular level CONGER and FAIRCHILD70 found that oxygen tension exceeding that of air produced chromosome breaks. In their distribution the breaks are random like those from x-rays and do not show the localization of the alkylating agents71. Oxygen at high pressures has been found to be mutagenic in seeds71 and in bacteria72 and to produce radiation-like damage in Paramaecia13.

R E F E R E N C E S 1 . D U S T I N , P . , Nature, 1 9 4 7 , 1 5 9 , 7 9 4 . 2. AUERBACH, C. and R O B S O N , J . M. , Nature, 1944, 1 5 4 , 81. 3 . K O L L E R , P . , in Progress in Biophysics, (Edited by J . A . V . BUTLER and J . T .

RANDALL), Pergamon Press, London and New York, 1954, 4, 195. 4 . D U S T I N , P . , J R . , Rev. Himat., 1 9 5 0 , 5 , 6 0 3 .

D U S T I N , P . , J R . , C.R. SOC. Biol., Paris, 1 9 5 0 , 1 4 4 , 1 2 9 7 . G R A M P A , G . and D U S T I N , P . , J R . , Rev. beige Path., 1 9 5 2 , 2 2 , 1 1 3 .


5 . B I N E T , L . and WELLERS, G . , Bull. Soc. Chim. biol., Paris, 1 9 4 6 , 2 8 , 7 5 1 . 6 . HUGHES, A . F . W . , / . Microscop. Sci., 1 9 5 2 , 9 3 , 2 0 7 . 7 . T R O W E L L , O . A . , Biochem. Pharmacol., 1 9 6 0 , 5 , 5 3 . 8. HARRIS , E. B., LAMERTON, L . F., O R D , M. J. and D A N I E L L I , J. F., Nature,

1952, 1 7 0 , 921. 9 . OCKHLER, F . , Z. indukt. Abstamrn. u. Vererb. Lehre, 1 9 4 3 , 8 1 , 3 1 3 .

1 0 . OCKHLER, F . , S.B. Heidelberg Akad. Wiss., 1 9 4 9 , 9 , 3 7 3 . 11. Comparative Clinical and Biological Effects of Alkylating Agents; Proceed-

ings of conference published in Ann. N.Y. Acad. Sci., 1958, 68, 657-1266. 1 2 . P H I L I P S , F . S .,J. Pharmacol., 1 9 5 0 , 9 9 , 2 8 1 . 1 3 . BACQ, Z . M . , "Travaux recent sur Ies toxiques de querre", Actualites Bio-

chemiques, Desoer, Liege, and Masson, Paris, 1947. 14. Ross, W. C. J., J. Chem. Soc., 1949, 183.

Ross, W. C. J., Advances in Cancer Research, 1953, 1, 397. Ross, W. C. J., in ref. 11, p. 669.

15. HADDOW, A., The Physiopathology of Cancer, Hoeber, New York, Chapter 16, 1953.

1 6 . ALEXANDER, P . , Advances in Cancer Research, 1 9 5 4 , 2 , 1 . 1 7 . H A D D O W , A . , Newer concepts in the chemistry of growth, Proc. First Natl.

Cancer Conf., Memphis, 1949, p. 88. 18. HADDOW, A., KON, G. A. R. and Ross, W. C. J., Nature, 1948, 162, 824. 1 9 . H E N D R Y , J . A., H O M E R , R . F., ROSE, F . L . , and W A L P O L E , A. L . , Brit. J.

Pharmacol., 1951, 6, 357. H E N D R Y , J . A., H O M E R , R . F., ROSE, F. L . and W A L P O L E , A. L . , ibid., 1951,

6, 235. 2 0 . BIESELE, J . J . , P H I L I P S , F . S . , T H I E R S C H , J . B . , BURCHENAL, J . H . , BUCKLEY,

S. M . a n d STOCK, C. C. , Nature, 1950, 166, 112. 21. LOVELESS, A . , Nature, 1951, 167, 338. 2 2 . WALPOLE, A . L . , Ann. N.Y. Acad. Sci., 1 9 5 8 , 6 8 , 7 5 0 . 23. STEVENS, C. M. and MYLROIE, A., Biochim. Biophys. Acta, 1952, 8, 325. 2 4 . WESTERGAARD, M . , Experientia, 1 9 5 7 , 1 3 , 2 2 4 . 2 5 . EHRENBERG, L . and GUSTAFFSON, A . , Heriditas, 1 9 5 7 , 4 3 , 4 9 5 . 2 6 . F A H M Y , O . G . a n d F A H M Y , M . J . , J . Genet., 1 9 5 6 , 5 4 , 1 4 6 . 2 7 . ALEXANDER, P . and STACEY, K . A . , Proc. 4th Intern. Conf. Biochem., Vol. I X ,

p. 98, Pergamon Press, London and New York, 1959. 2 8 . OGSTON, A . G . , Trans. Faraday Soc., 1 9 4 8 , 4 4 , 4 5 . 29. BACQ, Z . M . , Enzymologia, 1941, 10, 48. 3 0 . ALEXANDER, P . and COUSEN, S . F . , Biochem. Pharmacology, 1 9 5 8 , 1 , 2 5 . 31. ROLLER, P . C . a n d CASARINI, A . , Brit. J. Cancer, 1952, 6, 173. 32. REVELL, S. H., Heredity, Suppl., 1953, 6, 107. 33. FORD, C. E., Chromosome breakage in nitrogen mustard treated Vicia faba

root tip cells, Proc. 8th Intern. Congr. Genetics, Lund, 1948, p. 570. 34. D E N F E L , J . , Chromosoma, 1951, 4 , 239. 35. H IEGER, I . and PULLINGER, B . D . , Recent Advances in Pathology, 6th ed.,

p. 143. Churchill, London, 1953, 36. HOROWITZ, N . H . , Science, 1946, 104, 233. 3 7 . AUERBACH, C . , Cold Spring Harb. Symp. Quant. Biol., 1 9 5 1 , 1 7 , 1 9 9 . 38. BRYSON, V. , J. Bact., 1948, 56, 423. 39. KAUFMANN, B . P . , G A Y , H . and ROTHBERG, H . , J. Exptl. Zool., 1949, 1 1 1 ,

415. 4 0 . AUERBACH, C . , Proc. Roy. Soc. Edinburgh, 1 9 4 6 , 6 2 , 2 1 1 .


4 1 . AUERBACH, C . , R e f . 1 1 , p . 7 3 1 . 42. ELSON, L . A. , GALTON, D . A . G . a n d TILL, M . , Brit. J. Haemal., 1948, 4 ,

355. 4 3 . ELSON, L . A . , R e f . 1 1 , p . 8 2 6 . 4 4 . STERNBERG, S . S . , P H I L I P S , F . S . a n d SCHOLLER, J . , R e f . 1 1 , p . 8 1 1 . 45. ALEXANDER, P . a n d MILKULSKA, B., Biochem. Pharmacol., 1960, 5. 4 6 . HADDOW, A . , British Surgical Progress, p. 256, Butterworth, London, 1953. 47. HADDOW, A. and T I M M I S , G . M., Lancet, 1953, 1, 207.

GALTON, D . A . G . , Lancet, 1953, 1, 208. 48. GALTON, D. A. G., Brit. J. Radiol., 1951, 24, 511. 4 9 . PETERS, R . A . Nature, 1 9 4 7 , 1 5 9 , 1 4 9 . 5 0 . LOVELESS, A . , Nature, 1 9 5 1 , 1 6 7 , 3 3 8 . 51. NEEDHAM, D. M., Biochem. Soc. Symposia, 1948, 2, 16. 5 2 . BERENBLUM, I . and WORMALL, A . , Biochem. J., 1 9 3 9 , 3 3 , 7 5 . 5 3 . ROBERTS, J. J. and W A R W I C K , G . P., Biochem. Pharmacol., 1 9 5 8 , 1, 6 0 .

VERLY, W . G . , DEWANDRE, A . , D U L C I N O , J . a n d MOUTSCHEN, M . , Soc. Belg. Biochimie Proc. Reunion Liege, Mai 1960, p. 39.

54. GJESSING, E . C . and CHANUTIN, A. , Cancer Research, 1946, 6, 593. 5 5 . SPARROW, A . H . a n d ROSENFELD, F . M . , Science, 1 9 4 6 , 1 0 4 , 2 4 5 . 5 6 . BOYLAND, E . , Endeavour, 1 9 5 2 , 1 1 , 8 6 7 . 5 7 . BUTLER, J . A . V . , Nature, 1 9 5 0 , 1 6 6 , 1 8 . 58. JENSEN, E. V., Trans. Sth Conf. Biol. Autoxidants, 1950, Josiah Macy Jr.

Foundation, New York, p. 159. 59. ALEXANDER, P . and Fox, M., Nature, 1952, A 1 6 9 , 572. 60. ALEXANDER, P . a n d LETT, J . T . , Biochem. Pharmacol., 1960, 4, 37. 6 1 . REINER, B . and ZAMENHOF, S . , / . Biol. Chem., 1 9 5 7 , 2 2 8 , 4 7 5 .

LAWLEY, P . D . , Biochim. Biophys. Acta, 1 9 5 7 , 2 6 , 4 5 0 . 62. GOLDACRE, R- J., LOVELESS, A. and ROSS, W. C. J., Nature, 1949, 1 6 3 , 667. 6 3 . ALEXANDER, P . , et al. R e f . 1 1 , p p . 6 8 2 a n d 1 2 2 5 .

ALEXANDER, P . , SWARCBORT, A . and STACEY, K . A . , Biochem. Pharmacol., 1959, 2, 133.

64. DICKEY, H . H . , CLELAND, G. H . and L O T Z , C., Proc. Natl. Acad. Sci., U.S., 1949, 3 5 , 581.

6 5 . ALTENBURG, L . S . , Proc. Natl. Acad. Sci., U.S., 1 9 5 4 , 4 0 , 1 0 3 7 . 6 6 . SOBELS, F . H . , Nature, 1 9 5 6 , 1 7 7 , 9 7 9 . 6 7 . BACQ, Z . M . and MOUTSCHEN, J., C.R. SOC. Biol, belg., 1 9 5 6 , 150, 2 2 6 2 . 6 8 . W I L L I S , E . D . , Biochem. Pharmacol., 1 9 5 9 , 2 , 2 7 6 . 6 9 . GERSCHMAN, R . , GILBERT, D . L . , N Y E , S . W . , D W Y E R , P . a n d F E N N , W . O . ,

Science, 1954, 119, 623. 7 0 . CONGER, A . O . and FAIRCHILD, L . M . , Proc. Natl. Acad. Sci., U.S., 1 9 5 2 ,

3 8 , 289. 71. MOUTSCHEN, M . , MOUTSCHEN, J. and EHRENBERG, L., Heriditas, 1959, 4 5 ,

2 5 0 . 7 2 . F E N N , W . O . , Proc. Natl. Acad. Sci., U.S., 1 9 5 7 , 4 3 , 1 0 2 7 . 7 3 . GERSCHMAN, R . , GILBERT, D . L . , a n d FROST, J . N . , Am. J . Physiol., 1 9 5 8 ,

1 9 2 , 572. 74. ALEXANDER, P. and STACEY, K. A., Acta Union International Centre of Cancer,

1960, 1 6 , 533.

C H A P T E R 9

Effects at the Cellular Level

I N T R O D U C T I O N BEFORE considering the chemical changes which are produced in the protoplasm and nuclei of irradiated cells and tissues it is necessary to describe very briefly the morphological effects produced by ionizing radiation. Physicians using x-rays and radium for clinical treatment first discovered the marked changes produced in tissues more than fifty years ago and this attracted the attention of pathologists. This was the period in which biological lesions were described in great detail and the arrest of mitosis due to the action of x-rays clearly recognized. The study of these effects led BERGONIE and TRIBONDEAU1 to formulate their famous law which forms the foundation of the radiotherapy of cancer. "The sensitivity of cells to irradiation is in direct proportion to their reproduc-tive activity and inversely proportional to their degree of differentiation." At that time, however, no one attempted to examine experimentally the two fundamental problems: (i) How is it that certain radiation effects produced in cells can be repaired while others persist and are transmitted to subsequent generations? (ii) What are the physicochemical and bio-chemical phenomena within the cell which precede the onset of the visible lesions?

The answer to the first question had to await the outstanding researches of MULLER 2 who showed that ionizing radiations greatly increase the frequency of visible and heritable mutations, and who established them as mutagenic agents. Following on this work with Drosophila, induced muta-tions were observed in all living matter: in higher plants, unicellular organisms, fungi, vertebrates and invertebrates. The development of cytological techniques made it possible to observe in more detail the cellular damage, as opposed to the response of the whole organism or tissue, which is brought about by ionizing radiations.

The immediate changes which are encountered can be classified as follows:

(a) Delay in onset of mitosis followed by normal mitoses. The first cell division after irradiation may show some temporary disturbance in the

2 3 9


chromosome separating mechanism, which does not persist and as far as can be seen histologically there is no permanent injury.

(b) Complete inhibition of mitosis. The cell continues to live (i.e. to metabolize) but has lost permanently its capacity to divide. According to SPEAR3 this "sterilization" process plays a dominant role in radiotherapy of tumours.

(c) Death of cells following one or more divisions after irradiation. (d) Death of cell occurs many hours after irradiation but without there

being any intervening division—interphase death. (e) Instant death under the beam following very high doses (~ 100,000

rads). This amount of radiation will coagulate many proteins and this reaction is sufficient to account for "instant" radiation death which will not be considered further here.

(f) Breakage of chromosomes. (g) Interference with the functions of the cell. This may be temporary

or permanent and the dose needed to achieve it varies very widely. Most of the evidence for this process has been found with nerve cells (see Chapter 16), with cells releasing pharmacologically active substances such as serotonin (see p. 413) and induction of endocrine changes via central nervous system.

This last aspect of radiation damage has received too little study so far, although it presents one of the most exciting new developments of radiobiology which will undoubtedly receive much attention in the future. At the present time, however, we can deal essentially only with the processes that interfere with cell survival and cell division and the first part of this chapter is devoted to a summary of these immediate cellular events and this will be followed by a section dealing with genetic changes. We shall attempt to use a precise terminology while still making the treatment comprehensible to the non-specialist. The much-needed integration of biochemistry and physiology with cytology and genetics remains a task of almost insurmountable difficulty; but a start may be made by phrasing the problems in such a way that the different disciplines can contribute to their solution.

M I T O S I S

In general, the nucleus is separated from the cytoplasm of a "resting" cell, by a membrane. Little is known concerning its physical properties except that it must be more permeable than the cell membrane as exchange between the cytoplasm and the nucleus occurs freely. In the resting nucleus only a few structures can be recognized under the microscope; m o 9 t

notable is the nucleolus which does not appear to contain desoxyribo-nucleic acid (DNA) but like the cytoplasm contains ribonucleic acid (RNA).

j

E F F E C T S A T T H E C E L L U L A R L E V E L 2 4 1

At the start of the actual division, during the period known as prophase, dense filaments capable of being stained with basic dyes appear in the nucleus. As division proceeds, these filaments become shorter and form spiral structures. On favourable material one can see that they are composed of two threads, known as chromatids, which may be revealed by special techniques. Next, the nuclear membrane disappears. At this stage, called metaphase, a fibrous protein, the spindle, becomes apparent in the cyto-plasm. It is believed to be composed of fibrous proteins, which are in a "liquid-crystalline" state. The polypeptide chains are thought to run parallel to the axis of the spindle and to be perpendicular to the equatorial plate in the centre of the cell where the chromosomes assemble. The spindle extends to the opposite poles of the cell and each chromosome attaches itself to a fibre of the spindle at a single point, the centromere, which can be identified as the chromosomes are usually bent at this juncture.

In the next stage of the mitotic cycle, anaphase, the chromosomes divide; the chromatids separate in the region of the centromeres where they had remained undivided up to the end of metaphase. The onset of anaphase is determined by chromosome splitting at this point. This sep-aration is extremely rapid—almost explosive—and each of the two chromatids—now known as daughter chromosomes since they are inde-pendent threads—move towards the opposite poles along the spindle. This process insures that chromosome material is exactly halved between the two daughter cells.

During telophase the chromosomes swell, elongate and fuse into a fine network of chromatin which eventually becomes invisible. The nuclear membrane is reformed; the nucleus becomes spherical and nucleoli appear. The cytoplasm is divided by a new cellular membrane which completes the formation of the two daughter cells which enter resting stage (also called inter-kinesis or inter-phase).

A key problem confronting the cytologist is the mechanism by which chromosomes disappear at the end of telophase and reappear duplicated with exactly the same morphological and genetical organization in the subsequent prophase or metaphase. Geneticists postulate that the chromo-somal pattern is preserved during interphase. In the subsequent mitosis, the chromosome again becomes visible under the microscope and is composed of two identical daughter chromatids.

Little attention has been given to what occurs in the cytoplasm during the "chromosomic ballet". It is usually stated that the cytoplasmic mass, which grows during the inter-phase, is divided into two approximately equal parts. In the cytoplasm, there are well-defined bodies which are, however, less rigidly fixed in space and very much more persistent than the chromosomes. These structures vary in size; those visible in the


optical microscope are known as mitochondria, and bodies smaller than these as microsomes. It is assumed that these cytoplasmic bodies have the power of reproduction and certain of these have been compared by HADDOW4 with viruses. These organs contain many enzymes and are rich in phosophatides and ribonucleic acid (RNA); they are surrounded by an "imperfect" membrane and are sensitive to changes in osmotic pressure (e.g. salt concentration) in the outside medium.

In the absence of evidence to the contrary, one assumes that the two daughter cells besides having the same number of chromosomes also possess approximately the same cytoplasmic equipment. Phase-contrast cinematography of mitosis in tissue cultures reveals that the cytoplasm as well as the nuclei undergoes rapid changes. The formation of a number of cytoplasmic bubbles which at first swell and then shrink again can be observed on the surface of the cell membrane in tissue culture indicating profound biochemical and physiological changes during mitosis.

M E I O S I S

The growth of a multi-cellular organism is nearly always the result of cell multiplication by cell division while its reproduction is more often confined to special cells, the gametes (sperm and ovum). A study of these special cells revealed that growth and heredity are complementary. The division which precedes the formation of gametes (sperm and ovum), meiosis, differs in some respects from mitosis. In the mitosis, the chromo-somes are divided equally, i.e. at the end of the division each daughter cell has the same number of the same chromosomes as the parent cell. The gametes on the other hand carry only half the number of chromosomes each and the original number is re-established by their fusion.

Meiosis consists of two successive cellular divisions in which, however, the chromosome complement is only duplicated once. The nuclear network of chromatin resolves into long threads which represent single chromo-somes. The homologous chromosome threads then pair side by side, and this is followed by division of each paired chromosome into two sister chromatids. At the same time, at certain loci, the chromatids of homologous chromosomes break, exchange partners and reform. This process is called "crossing-over" and is the most important event in the life of a chromosome, as it is the only mechanism (i.e. apart from mutations) by which chromosomes can qualitatively vary their genetic content (Fig. 9-1).

The paired chromosomes contract and, after the disappearance of the nuclear membrane, they form a characteristic configuration (called the bivalent) in which each member is oriented and moves to the opposite poles undivided. As a result, the two daughter cells have only half the number of the chromosomes of the mother cell. Without a resting stage

I E F F E C T S A T T H E C E L L U L A R L E V E L 2 4 3

the chromatids of each chromosome separate and undergo an ordinary process of mitosis. In this way a group of four cells is produced each of which becomes a pollen grain or a mature sperm. The same processes take place during the formation of the female gamete (egg or ovum) except

Sperm or mature ovum

FIG. 9-1. Diagram to illustrate the process of crossing-over. The four different gametes are obtained from one crossing-over and show how two genes represented by X and • in different homologous chromosomes will be distributed within

the gametes8.

that the mother cell (or oocyte) is generally very large and well supplied with food reserves. The meiotic divisions affect only the nuclear material and are near the membrane so that at the end of the meiosis, one large haploid cell remains which becomes the egg while the three small nuclear masses, called the "polar bodies", rapidly degenerate.

The fertilized egg is the first diploid cell; it has 2n chromosomes, each pair being composed of one maternal and one paternal chromosome. The combination of parental characteristics and the permanence of chromo-some number in the offspring can thus be understood.

M I T O S I S I N A C O M P L E X O R G A N I S M

In a complex organism like a mammal, certain cells (in the central nervous system, for instance) never divide; some divide rarely (in the liver and kidney, for instance). Other cells are continuously dividing (they are, so


to speak, in perpetual growth) like the deep layer of the epidermis, the bone marrow and the crypts of the intestinal epithelium; the cells produced are constantly eliminated by desquamation (skin, intestine) or liberated in the blood and subsequently destroyed (see p. 417).

If an organ (kidney) or part of an organ (liver) is removed, the remaining tissue, normally non-dividing, hypertrophies rapidly by cell division until physiological compensation is reached. Thus, normally non-dividing cells in certain mammalian tissues have the latent possibility of regaining the growing capacity characteristic of embryonic or very young organisms. It is not understood why cells stop dividing in the adult. The hypothesis of WEISS5 and ABERCROMBIE6 that there is inhibition of growth when cells come into close contact in the completely developed well-shaped organ or tissue, is gaining much support. If these cells are cultivated aseptically outside the body (by the method called tissue culture) in a suitable medium and at the right temperature, any type of cells (with the exception of certain nerve cells) will constantly divide and grow, showing that there is a kind of inhibition imposed to proliferation when cells are in the organism kept together as an anatomical entity. When whole organs (like some endocrine glands) are extirpated without damage to the surrounding envelopes or membranes, they can be cultivated as organs in vitro, and the cells do not enter in rapid mitosis as in the classical tissue culture technique.

The morphology of cells in tissue culture may change considerably after several transfers*; cells with typical differentiated structure seem to come back to a kind of less specialized morphology, and they may lose some of their characteristic chemical properties. Embryonic cells are said to be non-differentiated or less differentiated in this sense that they are capable of giving birth to many types of cells morphologically very different; their potentialities are great. The functional and morphological evolution of embryonic cells towards the constitution of stable, well-defined tissues is one of the fundamental aspects of embryology. Cancer cells are often found less differentiated (or more de-differentiated) than the type of cells from which they arise; generally speaking, the less differentiated tumour cells are the more malignant they are.

One of the key questions of radiobiology is whether cells that normally do not divide or divide very infrequently are not damaged by radiation like the rapidly proliferating cells of the bone marrow and epithelium or if their radiation resistance is illusory and they show damage when, much later, they come to divide. Does the rule of Bergonie and Tribondeau only describe the situation immediately after irradiation; i.e. is there in

* These transfers are necessary (just as in microbial cultures) because the cells exhaust the medium and eliminate in it toxic metabolic products.


fact no difference in radiosensitivity between the slowly and rapidly dividing cells in an organism? This is one view which is widely held by those workers who stress the role of chromosome breaking for cell death but there is the possibility that the damaged cells are capable of repair. In slowly dividing cells like those making up the majority of the adult body the radiation injury may have been restored before it receives expres-sion during division and in this way the rule of Bergonie and Tribondeau may reflect the true situation. Certainly until we know the answer to this question we cannot extrapolate from the behaviour of cells in culture where they all divide rapidly to their behaviour in vivo where the interval between mitosis may vary from one day to one year in different organs.

This short discussion shows how difficult it is to extrapolate from tissue culture observations to whole complex organisms; but, none the less the tissue culture technique offers the only experimental approach to human genetics.

R E V E R S I B L E C E L L D A M A G E A N D M I T O T I C D E L A Y

The recent literature gives many instances of visible reversible cellular damage, changes which do not lead to death and are for this reason some-times called physiological or temporary effects. ROLLER7, for instance, observed in dividing pollen grains of Tradescantia, stickiness of chromo-somes, breakage in the centromere region, errors in the formation of the spindle and errors in spiralization in prophase. This is seen only in the divisions which occur within a few hours after irradiation and the subse-quent division of the daughter cells that showed stickiness are completely normal as are the divisions of those cells that did not enter mitosis until several hours after irradiation. Occasionally, the disturbances caused by stickiness are so severe that the cell dies during division, but in general it would not appear to be very harmful. Although in favourable materials chromosome breaks and stickiness can be unambiguously distinguished by competent cytologists there are many instances where it cannot, thereby causing much confusion in the literature. For example, when specific chromosomes are irradiated at prophase with microbeams (see p. 264) the chromosomal disturbances produced are stickiness and the conclusions drawn from this experiment do not apply to breaks although the original authors are frequently misquoted in this sense.

Some effects may not be visible: the most important is mitotic delay. A cell irradiated generally with quite a small dose before prophase, i.e. before the first stage of mitose, does not divide at the expected moment. This effect was first studied by SPEAR8 and his colleagues at the Strangeways laboratory with tissue culture. Figure 9-2 shows the effect of different doses on the mitotic index (i.e. the fraction of cells in division at any one time). The S-shaped curve is explained by the fact that radiation delays


the mitosis of some cells (presumably those at a particular sensitive stage at interphase) hence the mitotic index falls; eventually these cells enter into division at the same time as those cells that at the time of irradiation were in a non-radiosensitive state and therefore proceeded to division normally. These two effects together give the compensating wave of

FIG. 9-2. Change in mitotic index (i.e. proportion of cells in mitosis) of chick fibroblasts in tissue culture cells following

irradiation.

increased mitotic index. After a time the culture settles down to a steady mitotic index which is the same as the original so long as the dose has been small. With larger doses the new steady value for the mitotic index is lower than the original presumably because cells are present in the culture which have been rendered sterile—i.e. incapable of further division—by irradiation.

This same effect has been observed with plant material where K O L L E R 7

clearly demonstrated the importance of dose rate (see Fig. 9-3). If the same dose is given over a protracted period no suppression of mitosis occurs, showing that the cell can repair this type of damage and provides further support for the view that it is a purely temporary change. The recent detailed study of this phenomenon by EVANS, NEARY and T O N -

KINSON 9 illustrates these principles very clearly. The most detailed and informative studies on mitotic delay have been

made by J. G. Carlson and Mary E. Goulden, using the neuroblasts of the grasshopper embryo. These experiments10 have demonstrated the existence of a sensitive stage during mitosis when the chromosomes

E F F E C T S A T T H E C E L L U L A R L E V E L 247

condense as visible threads and when both the nuclear membrane and the nucleolus disappear. Irradiation (one rad is sufficient) before this critical period, delays the progression of mitosis; irradiation after the period does not produce any change in mitosis if the dose is kept low. Since the synthetic activity of the cell is little affected by such small doses, the cell often goes on growing and increasing in weight11, (cf. formation of giant cells by irradiation of tissue cultures, p. 250).

Suppression of mitosis BOOV

20 -

•5 1

IS

MH Ni/

C

.5

# HO too SOO 2000 Duration of irradiation miin

(gOr/min Sr/m\n O-Sr/min 0-/r/min)

FIG. 9-3. Diagram to illustrate the influence of dose rate on the suppression of mitosis in Tradescantia pollen grains produced by 200 r of X-rays. —|_S_|— represents period of suppression of

mitosis7. Mechanism

The essential characteristic of mitotic delay is that the effect is temporary and cannot therefore be caused by damage to the genetic material (i.e. nucleoprotein) or be related to the production of chromosome breaks or re-arrangements. The importance of the rate at which the dose is delivered, the existence of a threshold dose below which no delay occurs, suggest that interference with some metabolic process is involved.

Two opposed concepts have been advanced. The first accepts the idea that mitotic delay is linked with inhibition of DNA synthesis12, and according to certain authors13 it is the most sensitive and early biochemical reaction of irradiated cells (see also p. 347). There are arguments against the generality of the hypothesis that inhibition of DNA synthesis is the primary cause of mitotic delay and related radiation damage. It may be that reduced DNA synthesis is a result of interference with mitosis, but not the cause. The period in the mitotic cycle at which the cell is most sensitive does not appear to correspond with the most sensitive time for mitotic delay. Also a decrease in DNA synthesis is not seen in all types of


cells. Thus mammalian cells in tissue culture continue to synthesize DNA even though they have received a dose of radiation sufficient to stop division (see p. 351). The x-ray induced block to division in Tetrahymena (a ciliate) occurs in synchronized cultures in which DNA is already present in several times the normal amount and is segregated out without new synthesis over the course of several rapidly succeeding divisions14. Micro-nuclear and macronuclear division, and presumably DNA synthesis, continue at nearly the normal rate even in ciliates in which severe damage has been done to the chromosomes by x-rays15.

KIMBALL16 believed that, as far as Paramaecium is concerned, the evidence is more in keeping with the idea that division delay is a secondary consequence of damage to some mechanism in controlling the onset of division or the maintenance of growth. This would also account for the fact that nucleic acid and protein synthesis occurs at the same rate in the non-dividing giant cells as in the unirradiated culture. Interference of radiation with oxido-reduction of —SH compounds known to occur during cell division, or inhibition of the spindle formation are plausible hypotheses to explain mitotic delay.*

C E L L D E A T H

It is convenient to consider separately those processes that result in the death of a cell only after a number of mitoses has occurred and those that cause death without intervening cell division. The latter has been studied for more than fifty years and is widely referred to in all text books dealing with histopathology. A good general name for it is interphase death. The cell dies without dividing and damage is clearly visible micro-scopically, as swelling and pyknosis of the nucleus, nucleoli and of the protoplasm.

If irradiation is sufficiently severe, DNA may be seen in vivo to be depolymerized and coalescing in heavy droplets17 (Fig. 9-4). Staining abnormalities have frequently been observed11; finally the structure of the nucleus, its DNA and proteins disappear progressively by the action of the hydrolyzing enzymes, DNAases, cathepsins, etc. The morphology and the number of nucleoli may be altered by irradiation in mammalian cells and they become swollen, fragmented and vacuolated. Very little has been reported about the alterations of mitochondria, microsomes, Golgi apparatus and the cytoplasm itself. The electron microscope has not yet been used as extensively as it should be for the study of possible lesions to these structures though the first results along this line seem very promising.

At the biochemical level almost nothing is known about the mechanism

* Kuzin extracted antimitotic quinones f rom irradiated plants (see also release of phenoloxidase p. 273).


of interphase death. DURYEE'S17 experiments using micro-injections into amphibian eggs indicate that damage to the cytoplasm plays an important part, but how far this observation can be extended to other cells is un-certain.

The doses required to produce these marked microscopic changes that eventually lead to cell death vary very much from cell to cell. Lympho-cytes18, oocytes19 and spermatagonia20 are killed by a few hundred roentgen or less while other cells, particularly those in organs where the rate of cell division is very low (low mitotic index), may require several thousand roentgen before they degenerate. It is to this type of damage that the law of Bergonie and Tribondeau has the widest validity, though the high sensitivity of the non-dividing lymphocytes and oocytes forms an obvious exception. Reasons for the great variation in the radiosensitivity of cells to interphase death are not known, though the biochemical studies on nuclear phosphorylation by CREASEY and STOCKEN47 may hold the first clue.

Mitotic (or genetic) death—Death following division has been studied much more intensively and accurate quantitative data are available for many plant and animal systems. The simplest technique for studying this process is to determine the ability of the cell to form colonies or clones visible to the naked eye. Frequently, a cell can divide several times after irradiation, but this will not lead to a colony and the cell will be counted as killed. This "mitotic death" occurs in general with smaller doses than are needed to produce interphase death in cells that no longer divide or that divide only very slowly. In tissue-culture the formation of giant cells is seen but by no means all the cells that have suffered "mitotic death" are affected in this way. The giant cells are prevented from dividing but continue to synthesize nucleic acids (DNA and RNA) and protein (see Chapter 13)* and grow to huge sizes, though eventually they degenerate. Whether there is a relationship between interphase death and the production of giant cells is not known, but at the present time it would appear most useful to consider them separately.

The formation of giant cells has only been studied quantitatively in tissue culture where there is essentially unlimited cell proliferation and the rate of cell division is determined only by the metabolic rate. Under such conditions every single cell develops into a discrete macroscopic colony. Cells taken from both human and animal organs as diverse as skin, liver, spleen, bone-marrow, testis, ovary, kidney and lung, as well as from tumours, all respond to this technique. That is, all these cells

* T h e suggestion21 that giant cells do not divide because they only synthesize protein and RNA, but not DNA, has not been confirmed and in the recent experiments it was found that D N A synthesis occurred initially at the same rate as in the unirradiated culture.


have retained the capacity for unlimited growth if placed in a suitable environment. Using these methods the radiation response of a large number of "pure strain" cells could be followed quantitatively.

PUCK irradiated cultures originating from single human cells (a cervical carcinoma22, normal epitheloid or fibroblastic cells isolated from various organs23) and the capacity of the cells to form macroscopic colonies was studied quantitatively (Fig. 9-5). Effects are already seen after exposure

20 IO 0-5 02

I o, 5 005 CP •| 002 I 001 in

0 0 0 5

0002 ° 0 0 1 O 100 200 300 400 500 600 700

Dose, r

FIG. 9-5. Survival (i.e. retention of reproductive capacity) of human cells (HeLa cells) following irradiation with x-rays in

tissue culture.22

(The dose is expressed in roentgens but it cannot be converted directly to rads as the cells were irradiated on cover slips and due to the high photoelectric absorption by the glass the cells are exposed to additional photoelectrons. In this experiment an exposure to 1 r gives rise to an absorbed dose of 1 -4 rads. When cells are irradiated in suspension I r = I rad. This must be borne in mind when experiments carried out under different

conditions are compared.)

I I I J

to as little as 75 r. After 500 r, only 1 per cent of the seeded cells are capable of giving a large colony (more than 50 cells). The cells which have not formed big colonies are not dead; they have given birth to abortive colonies containing a proportion of giant cells increasing from 67 per cent after 50 r to 100 per cent after 600 r. Thus, strictly speaking, the cells are not killed by doses as high as 800 r; their ability to reproduce has been destroyed, but some can divide several times. At higher doses even single cell division is precluded; but a large proportion of these cells gives rise to giants. Rare are the cells which disappear from the plate, which disintegrate; this last action of x-irradiation is by far the least efficient since even after 10,000 r, 5 to 10 per cent of the original cell inoculum is still recoverable as giants (see also Fig. 14.3).


Puck explains his results (not too convincingly)* in terms of the chromo-some target theory, the major damage being genetical and resulting from primary damage in one or more chromosomes. The high frequency of mutant cells among the survivors of 500 to 900 r lends support to this theory (but see recovery of irradiated cells, p. 373).

K O H N and FOGH24 studied the effect of radiation in a system closely resembling that of Puck, but they measured increase cell number and cell volume following different doses (see Fig. 9-6). The effect of doses

Days afler seeding Days after seeding

FIG. 9-6. x-irradiation growth of human cells (strain FL) in tissue culture, (a) Effect on number of cells present, (b) effect

on total cell volume.24

up to 600 r was merely to delay the division time from 1 -2 days to 2 days. Only with doses of 1000 r or greater was increase in cell numbers sup-pressed. Increase in total cell volume was less sensitive than increase in numbers because of the formation of giant cells.

Although these experiments constitute the nearest experimental approach to human radiobiology, the results cannot be transferred to the whole human organism without great caution. In order to use the classical technique of microbiology, the cells used by Puck are isolated. It

* Thus the fact that the survival curve has a "two-hit" character is implied to mean that "two-hit" chromosome aberrations are responsible for cell death. Yet under similar conditions another group gets a "five-hit" survival curve (see p. 374) which cannot be related to chromosome damage.


is a normal condition for most bacteria to be isolated, but it is a highly abnormal situation for a conjunctival, pulmonary, hepatic, cutaneous or splenic cell to be isolated. Their physiology and morphology is changed; they are maintained in a rigid chemical medium while, in the body, cells influence each other at short and long distance (see p. 244). Puck finds that cells isolated from three different normal organs of six different human subjects, exhibit the same radiosensitivity (somewhat greater than that of cells from human cancerous tissue). Does this mean that the radiosensitivity of these cells is also the same in situ or is the similarity in response an artifact of tissue culture? In their normal physiological environment these cells are metabolically active, but often they do not divide. In tissue culture this leads to giant cells, yet in vivo the cells remain quite normal and it is conceivable that this difference in behaviour is also accompanied by changes in radiation sensitivity. Certainly the great differences in the dose needed to produce interphase death—ranging from less than the dose for mitotic death in tissue culture to values 100 times as great—shows that the radiation response of mammalian cells to radiation is far from uniform.

Any discrepancy between in vivo and in vitro behaviour is likely to be least serious for those cells which undergo rapid division in the body. According to Puck, the value of D0 (i.e. dose needed to kill 67 per cent of the cells, see p. 63) is 100 r for cell death and this explains why the lethal dose for mammals lies in the range of 400 to 600 r. This dose, it is claimed, will interfere with the reproductive mechanism of more than 99 per cent of cells with Do = 100 r. The body might recover from smaller doses which leave sufficient stem cells to repopulate the critical organs (bone-marrow and intestinal mucosa) in time. This deduction fits in well with all the quantitative histopathological studies that have been made of the bone-marrow. The key question is whether at the same time the reproductive capacity of 99 per cent of all the cells in the body—liver, kidney, skin, etc.—has been similarly affected.

Application to Radiotherapy* From the point of view of radiotherapy, mitotic death and even the

formation of giant cells is not in many instances the most important biological process since frequently tumours are not characterized by a high rate of cell division. Using a radioactive thymidine labelling tech-nique, CRONKITE25 showed that the mitotic period of leukaemic cells was not shorter than that of normal leukocytes. The difference between them lay in the fact that the malignant cells had a much longer lifetime in the

* For an excellent account of the biological basis of radiotherapy the reader is referred to a recent monograph by Mitchell46.

FIG. 9-7. Alteration in the mitotic cycle of Vicia faba after exposure to x-rays, (a) Metaphase with one chromosome break, (b) Anaphase with "bridge and fragment" derived from a chromosome break, (c) Telephase with fragment excluded, (d)

Resting stage with micronucleus. (Photographs provided by S. H. Revell.)

[facing p. 252

(a)

(b)

^ r *

(C)

(d)

V

\\

FIG. 9-8. Representative chromosome abnormalities produced in Vicia Jaba by x-rays:

(a) Normal mitosis; (b) Chromatid exchange; (c) Chromatid isochromatid exchange; (d) Chromatid break and chromosome exchange.

(Photographs provided by H. S. Revell.)

FIG. 9-4. Nuclear damage (i.e. "depolymerized" DNA) in ovarian egg of Tritiirus pyorhogaster injected with 0-2 millicurie of 32P 10 days previously and then kept at 23 0C (photograph

provided by W. R. Duryee).

[facing p. 248


body. The same applies to solid tumours; the time for division of a human mammary carcinoma was five months, slightly longer than that of the surrounding stroma. In radiotherapy, at best a proportion of tumours are eradicated by interference with cell division, that is by death after mitosis, or by inducing giant cell formation. Successful treatment often requires that the tumour cells are killed outright by an interphase death mechanism or that they become sterilized3, without becoming giants. As neither of these phenomena has been encountered in tissue culture it is clear that data obtained by this technique cannot be applied without serious reservations.

Another problem encountered in vivo is the problem of cell selection. Mutants produced by radiation have to compete with less injured cells and frequently do not survive whereas they are scored as viable by the Puck technique. In the tissue culture experiments of LASNITZKI45 selec-tion pressure is operative and her experiments may be more meaningful for radiotherapy though they provide much less information about cellular radiobiology.

" B R E A K A G E " O F C H R O M O S O M E S The phenomenon (see Figs. 9-7 and 9-8) on which the attention of cyto-geneticists is concentrated is the fragmentation of chromosomes and the subsequent events. Some changes produced in chromosomes can be reproduced in the divisions following treatment and may therefore become perpetuated. Since chromosomes carry the genes (i.e. the molecular complexes which control the morphology, development and behaviour of organisms) permanent structural changes may result in genetical changes.

Many hundreds of papers have been published on the anatomical changes that are seen in the chromosomes after irradiation; complex and detailed theories have been developed to explain the formation of the abnormal structures that are seen. In their enthusiasm most cytologists have tended to overlook that chromosome "breakage" like all biological effects of radiation is an end effect seen only a long time after the primary damage. There are many stages between the initial ionizations in the cell and the final injury and theories which fail to take these into account are clearly very hazardous. The cell is most sensitive to chromosome "breakage" when irradiated during interphase* when no chromosomes or chromatids are visible. The damage is only seen at metaphase or anaphase. Cytologists were led to analyse their data on the assumption that radiation severed the chromosomes either before or after they had split into chromatids and that they then underwent a complex pattern of rejoining to give the anatomical

* Cells irradiated during mitosis show permanent chromosome abnormalities in subsequent mitoses but not in the same mitosis (see p. 269).


alterations (interchanges) eventually seen. We shall not attempt to describe the widely accepted theories based on breakage and reunion since there is no biochemical evidence for the basic assumptions that are made, namely that:

1. Radiation is capable of directly severing a chromosome. The actual experimental observation is the appearance of gaps or discontinuities. There are other ways in which these can arise than by direct breakage (see Chapter 10).

2. The fragments so produced can rejoin to give new configurations so long as there has not been an excessive time interval. (After some minutes the chromosomes are said to lose their capacity for rejoining and the breaks stay open.) No mechanism has been suggested for this remarkable process of rejoining and there are other processes (cf. REVELL2 6) that could produce the rearrangements that are seen. The main reason why this theory (proposed by SAX27 and subsequently developed also by DARLINGTON a n d L A COUR 2 9 , a n d CATCHESIDE, LEA a n d T H O D A Y 2 8 ) received such widespread support was the impressive quantitative agree-ment with experiment. In particular, the fact that those abnormalities which were said to involve rejoining (i.e. at some stage required the simultaneous presence of two or more broken ends still in the "active" state at which they can rejoin) were highly dose-rate dependent while those configurations ascribed to simple breaks were nearly independent of dose rate. Also some of the abnormalities were proportional to the total dose while those involving rejoining depended on a higher power of the dose.

Much of the strength of this argument was lost when it became obvious that chemical substances produce identical abnormalities (see Chapter 8), yet these could not possibly break formed chromosomes. Moreover, the difference in the dose dependence for interchanges and for breaks was also found with many of the chemicals (including poisoning by high concentrations of oxygen) for which the explanation advanced to account for this difference in the case of radiation cannot possibly be applied.

Eventually, no doubt, it will be seen that certain features of the breakage and reunion hypothesis can be retained, but at the present time it seems premature to develop a detailed mathematical treatment when there is little knowledge concerning the biochemical or chemical steps involved. Two chemical mechanisms have been proposed for chromosome "break-age"30. Thefirstinvolvesweak ionic bonds while the second requires the rupture of stronger covalent bonds. In the first case, restitution is possible in absence of external energy sources; in the second, energy of respiratory origin is necessary. This interpretation is more a working hypothesis than a firmly established theory.

On the other hand, REVELL2 6 believes that radiation does not break


chromatids, but induces a process akin to meiotic crossing-over which causes the appearance of abnormal chromosomes. An attractive feature of this theory is that it requires the same quantitative relationships between dose and the different types of abnormalities as the breakage and reunion theory and the same experimental data support both theories.

Revell has adduced much experimental cytological support for his theory which is gaining wide support. He has, however, no biochemical mechanism to offer how an ionization induces chromatid exchanges or what cell component has to be affected for this to happen.

One of the immediate consequences—as opposed to delayed genetic effects-—of chromosome breaks is that they frequently give rise to a bridge between the two chromosome sets at anaphase. Both the bridge and the fragment without centromere are likely to be excluded from the two daughter nuclei at telophase, and become a third nuclear mass, the so-called micronucleus (the stepwise development is seen in Fig. 9-7). This micronucleus is without function and the two daughter cells are therefore deficient in the chromosomal material which it contains. It is widely believed that the loss of nuclear material brought about in this way contributes to cell deaths which occur in the divisions that follow irradi-ation.

The rearrangement of the chromosomes (i.e. the two-hit abnormalities of the target theory) can also affect the subsequent fate of the cell even if it does not lead to anaphase bridges since these new configurations are perpetuated and represent a permanent genetic alteration of the cell. While in many cases the change in the sequence of the genes along the chromosomes—which is inherent in a rearrangement—does not affect their function there are many instances where it does. Some geneticists call the killing of a cell a dominant lethal mutation when the zygote's or embryo's failure to develop can be ascribed to severe chromosome re-arrangements.

On general grounds one would expect the various chromosomal effects— such as loss of genetic material, chromosome rearrangements and point mutations—to contribute to cell death which requires mitosis for expres-sion. The important and, as yet, unanswered problem is whether they are the only—or even the main cause—of this type of cell death. The role of chromosome damage in interphase death and the formation of giant cells has received much less attention and at first sight it is difficult to see where, if at all, it comes into the picture. According to M U L L E R 3 1 , the death of Drosophila larva—i.e. failure to develop into adults—is entirely due to the loss of chromosomal material following chromosome breakage and reunion and that other forms of damage (e.g. cytoplasmic or interference with the biochemistry of the nucleus) can be wholly excluded. P U C K 2 2 interprets his results in a similar way, but concludes


chromosomal aberrations that mechanically prevent division (e.g. anaphase bridge formation) as a key factor for the polyploid cells with which he deals.

This anatomical approach is, however, strictly limited since it deals solely with end effects. How far are these changes in the chromosomes merely a reflection of far reaching damage to the cell? Is chromosome dam-age a cause of cell death or cell death a cause of chromosome damage? It is difficult to estimate how serious the loss of genetic material is to a somatic cell especially since the rate of spontaneous chromosome damage varies very much in different materials and bears no relation to viability (e.g. high rate of abnormalities in tumour cells). Recently, B E N D E R 3 2 has drawn attention to the fact that the dose needed to produce a given amount of chromosome damage in Puck type tissue culture varies substantially from cell to cell all of which, however, have the same radiosensitivity when tested for ability to form clones.

G E N E T I C E F F E C T S O F I O N I Z I N G R A D I A T I O N S

It is outside the scope of this book to discuss at length the genetic effects of radiations. Very good recent reviews are available with extensive bibliographies. The report of the U.N. Scientific Committee on the effects of atomic radiations (1958) contains not only a summary of the actual state of our knowledge but also a large scientific annex prepared by the best geneticists of the present time33. The following is thus an extremely condensed presentation for those readers who, being physicists or chemists, need an introduction in this difficult and large field.

Genes are entities which determine heritable characters. They are located at specific points—called loci—in definite sequences within the chromosomes. It is generally accepted that the deoxyribonucleic acids (DNA) in association with proteins are the structures which convey and transmit the genetical information.

Each individual inherits one set of chromosomes through the sperm from the father and another set through the egg from the mother. Genes and chromosomes are particularly vulnerable to the effects of radiations and of radiomimetic substances. These agents increase the natural fre-quency of those changes in the genetical constitution called mutations. In most cases, the precise genetic nature of the mutational event is not known. There are a few cases, in animal and man, where a given hereditary anomaly is always accompanied by a well-defined visible alteration of the set of chromosomes; naturally, these cases are of special interest to cytogeneticists34. Generally, however, the available cvtological techniques are unable to trace in the chromosomes the change which must have happened. O A K B E R G and Di M I N N O 4 9 also find there is no correlation between cell death and chromosome "breakage" in murine spermatozoa.


The mutations induced by ionizing radiations are qualitatively the same as the natural mutations or as the mutations observed after the action of radiomimetic substances. Thus, in this case as in many other cases, ionizing radiations do not create a new phenomenon; they merely increase the rate of a normal process.

The genetical changes which are scored are only those which are observable by suitable methods. Some are visible to the naked eye (for instance, change in colour, form or size; see ref. 44), at the microscope, or must be looked for with immunological or biochemical methods. Some need a careful analysis with the standard techniques of genetics. There are certainly many mutations and genetic traits of which we are not aware because they are unimportant—because they do not create any trouble and consequently do not attract the attention. They are discovered unexpectedly, for instance, when new refined biochemical methods are applied to man.35

The great majority of mutations are harmful. Some are lethal. The mutated individuals (micro-organism, plant or animal) are very generally less well equipped than normal and are consequently eliminated by natural selection. A few favourable mutations naturally survive and the process of mutation is now clearly understood as one of the fundamental mechanisms of evolution. Other favourable mutations are artificially selected by man among hundreds of natural or artificially induced muta-tions for agriculture, breeding, or the chemical industry based on micro-organisms. In the human species, the situation is complicated by the fact that the very efficient medical activity tends to keep alive and in repro-ducing activity, individuals who, without medical care, would have been eliminated before the reproductive age and therefore would have been unable to pass a deleterious genetic equipment to further generations.

It has been relatively easy to analyse quantitatively the genetic effects of radiations on insects (Drosophila) and more recently on micro-organisms because one can in a short time dispose of a large number of organisms from genetically pure strains. Thus in Drosophila, down to x-ray doses of 25 rads, the radiation induced mutation rates are strictly proportional to the dose; the dose-effect relationship is a straight line and it is generally accepted by the geneticists that this line can be extrapolated below 25 rads. It must be clearly understood that experiments designed to measure the increased mutation rate after very low doses (about the order of 5 rads) in Drosophila is practically impossible because hundreds of thousands of flies would have to be used in order to have statistically significant data. It is beyond human ability to overcome the dreariness of looking for days and days to find just a few slight changes in such an enormous lot of animals. Another basic difficulty in any quantitative study of mutagenic effects of ionizing radiation is to know what is the natural mutation rate: it has been estimated (measured directly or calculated) per gamete for single loci in


Drosophila as 2-5 x IO-6 to 4-5 x IO-5, in mice as 7 x IO-6. The range in bacteria is 1 x IO-11 to 4 x IO-8. In man, for certain hereditary diseases (direct observation of autosomal dominants) the rate per gamete ranges from 4 x IO-6 to 4x lO"5.

The doubling dose, i.e. the dose of ionizing radiation which when given in a single exposure doubles the natural rate of a given mutation, is about 30 rads in mice, 50 to 400 rads in Drosophila, 30 to 60 rads in plants.

A very important series of experiments by the RUSSELLS36 has demon-strated that in mice (females as well as males) for different loci, the increased frequency of mutation is dependent not only on the total dose (as in Drosophila) but also on the dose rate; when a given dose of y-rays is distributed in a matter of weeks or a few months, the frequency of observed mutations is two to three times less than when the same dose is administered in a single short exposure (see Fig. 9 - 9 ) . RUSSELL and his collaborators43 using very large numbers of animals have proved that this effect was due neither to cell selection nor to a difference in quality between the action of x- and y-rays, nor to the fact that, as unavoidable in chronic irradiation, the sensitivity of the oocyte or spermatogonia might vary with the stage of the cell. When mouse spermatozoa (i.e. the mature male cells) are irradiated, such an effect of dose rate is not seen, thus confirming classical findings for spermatozoa and indicating that the explanation for intensity dependence in spermatogonia resides in some characteristic of the gametogenic stage. In mammals, it may be accepted as a rule that dividing cells or cells having an intense metabolic rate, there are important intrinsic mechanisms for restoration after irradiation, even when genetic damage is concerned (see Chapter 14).

These experiments have been performed on genetically pure strains, on animals or micro-organisms having exactly the same equipment of genes; a mouse must have about 15,000 genes. Genetically pure strains are arti-ficially produced in laboratories by constant inbreeding; in nature, there are no pure strains. It is inconceivable that a pure strain of any human race can be obtained even in a society where the present moral standards would be completely ignored. Furthermore, no experiments can be made with humans. Thus, in order to evaluate the genetic effects of radiations on the human species, one must try to find a series of convergent indirect arguments; so far nobody has been able to study two sufficiently large groups of humans: an exposed group and a control one of which the doses received by the gonads are known*. The enormous difficulties

* One of the rare positive statements resulting f rom the long and careful work of Neel and Schull on the descendants of the irradiated Japanese of Hiroshima and Nagasaki, is that the doubling dose for human genes in gonial cells is very unlikely to be below 10 rad3 7 .


involved have been well analysed in the report of the U.N. Radiation Committee33.

It is not illogical to accept as a first approximation that the doubling dose will not be very different in man from what has been observed in mice. A more direct approach is now attempted by using human tissue cultures, but it is not yet known what is the exact relevance of studies on the mutational behaviour of somatic cells in vitro to that of germ cells in vivo.

The last question, and not the least important one, is the fate of an

FIG. 9-9. Mutation rates at specific loci in the mouse, with 90 per cent confidence intervals. Solid points represent results with acute x-rays (80 to 90 r/min). Open points represent chronic y-ray results (triangles and square, 90 r/wk; circle, 10 r/wk). Square points are mutation rates in females, all other points being mutation rates in males. The point for zero dose represent the sum of all male controls.

The two 1000 r points are results from different experiments; the lower mutation rate has been obtained with a single acute dose to the gonads of 1000 r which may have killed many sensitive cells (selection effect); the higher rate is the result of two acute exposures of the gonads (600 r and, 15 weeks later, 400 r). The higher rate is believed to be more suitable for comparison with the results of chronic gamma irradiation43.


irradiated population when it remains in natural contact with a normal population. A few useful observations and experiments can be made and are still in progress. One can irradiate a large population of Drosophila, liberate it in a small uninhabited island of which the Drosophila population is accurately known, and take samples twice or three times every year. Pavan is doing such experiments in Brazil. We have studied populations of seeds of the same species growing on uraniferous or non-uraniferous (but similarly mineralized) soils in the Katanga (Belgian Congo) and found no difference in radiosensitivity; but without question various tests show that the population from soils rich in uranium (thus constantly irradiated at small dose-rate for many generations) is more vigorous than that of normal soils38. Similar observations have been made with genetically heterogeneous populations of Drosophila heavily irradiated generation after generation. The final result is that the surviving population is more vigorous than that one started from. Irradiation selects the more radio-resistant individuals and those individuals are also much better equipped than the more radiosensitive in many other respects. The quality of the population is ameliorated but the price must be paid: the elimination of a large group of less resistant individuals.

Somatic Mutations The idea that mutations are not restricted to germ cells and also occur

in somatic cells has logically followed the well-established fact that the DNA and nucleoproteins which exist in all cells are identical in quality (and also generally in quantity) to those of germ cells. There is no reason to suppose that the very infrequent random changes occurring in DNA of germ cells cannot occur in somatic cells. Indeed, mutations have been observed in cultures of non-genetic tissues of plants and animals. Colour mosaics in petals of some flowers (Antirrhinum for instance, see ref. 39), or in the coats of animals (see ref. 42 for references), fleece mosaicism in sheep41 have been accepted as results of spontaneous or radiation induced somatic mutations. L . B . RUSSELL and MAJOR 4 0 have calculated from observations on mice irradiated during foetal life, that the somatic muta-tion rate is 7-0 x 10~7/r/locus; the germinal rate induced in spermatogonia for the same four loci is 2-4 x 10_7/r/locus. Leukaemia and cancer induc-tion by ionizing radiation are considered by a group of scientists to be the result of one or several somatic mutations (see p. 446).

A logical objection to this theory of leukaemogenesis is the following: considering that the number of haematopoietic cells in a mammal is many times larger than that of the mother cells of spermatozoa or ova, and accepting the fact that the natural (so-called spontaneous) mutation rate must be the same for somatic and sexual cells, how can one understand that every one of us poor humans does not die from leukaemia at a very


early age? BURNET42 has estimated that if somatic cells mutate in the same fashion and with the same frequency as germ cells, "some IO6 (one million) cells of the expandable replicating cells of the human body may be under-going mutation every day". The correcting factor, the homeostatic mechanism, is obviously that of population competition. When, for instance, a mutated abnormal cell arises in bone-marrow this cell is confronted with an enormous population of normal cells and it is eliminated completely or prevented from dividing if it does not possess a powerfully favourable biochemical equipment. As a comparison let us take the case of cultures of micro-organisms. It is known that in cultures of many cocci growing in normal media, there are very few cells resistant to a given antibiotic, penicillin for instance. As long as the medium remains normal, these penicillin-resistant cells cannot compete successfully with normal cells which divide actively. One might imagine that what irradia-tion does to bone-marrow is comparable to the effect of penicillin on micro-organisms. Irradiation may not only increase the frequency of deleterious somatic mutations, but also (and might this not be its major action?) depress the population of normal cells, and thus create better conditions for the growth of abnormal cells by a decrease of competition.

Such trends of ideas are basic to the speculation on aging as a result of the slow accumulation of somatic mutations (see Chapter 17 and ref. 42).

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(Translation published in Radiation Research, 1960, I l j 32). 2 . M U L L E R , H . J . , Science, 1 9 2 7 , 6 6 , 8 4 3. SPEAR, H . G., Brit. J. Radiol., 1958, 31, 114 4 . HADDOW, A., Nature, 1944, 154, 194 5. WEISS, P . , Int. Rev. Cytology, 1958, 7, 391 6. ABERCROMBIE, M. and HEASYMAN, J . E. M. , Exptl. Cell. Research., 1953, 5 ,

111; Nature, 1954, 174, 697 7. KOLLER, P. C., Progress in Biophysics, Pergamon Press, London and New York,

1953, 4, 195; Symposium on Chromosome Breakage, Oliver and Boyd, London, 1953

8. SPEAR, F. G., Radiation and Living Cells, Chapman & Hall, London, 1953 9. EVANS, H . J . , NEARY, G. J . and T O N K I N S O N , S. M. , Exptl. Cell. Research,

1959, 17, 144 10. CARLSON, J. G., in Radiation Biology (Edited by A. HOLLAENDER), Vol. 1,

pp. 763-824, McGraw-Hill , New York, 1954 11. BLOOM, M . A., Histopathology of Irradiation from External and Internal

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plasmologia, Springer, Vienna, 1957 13. ORD, M . and STOCKEN, L. A., Advances in Radiobiology, p . 65, Oliver &

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1 5 . GECKLER, R . P . a n d KIMBALL, R . F . , Science, 1 9 5 3 , 1 2 2 , 8 0 1 6 . K I M B A L L , R . F . , Ann. Rev. Microbiol., 1 9 5 7 , 1 1 , 1 9 9 1 7 . DURYEE, W . R . , J. Natl. Cancer Inst., 1 9 4 9 , 1 0 , 7 3 5 1 8 . T R O W E L L , O . A . , Brit. J. Radiol., 1 9 5 3 , 2 6 , 3 0 2 ; J. Path. Bact., 1 9 5 2 , 6 4 , 6 8 7 19. COLE, L. J . , HABERMEYER, J . G. and STOLAN, H . N. , Intern. J. Radiobiol.,

Suppl. 1, 1960, p. 361 1 9 . LACASSAGNE, A. and GRICOUROFF, G . ; Action des radiations sur Ies tissus,

Masson, Paris, 1941; DESAIVE, P., Advances in Radiobiology, p. 274, Oliver & Boyd, Edinburgh, 1957

2 0 . OAKBERG, E . F . , Radiation Research, 1 9 5 5 , 2 , 3 6 9 2 1 . K L E I N , G . and FORSSBERG, A . , Exptl. Cell Research, 1 9 5 4 , 6 , 2 1 1 2 2 . PUCK, T . T . and MARCUS, P . I . , J. Exptl. Med., 1 9 5 6 , 1 0 3 , 6 5 3 2 3 . PUCK, T . T . , MORKOVIN, D . , MARCUS, P . I . and CIECIURA, S. J . , J. Exptl.

Med., 1957, 1 0 6 , 485 2 4 . K O H N , H . I . and F O G H , J . E . , J. Natl. Cancer Inst., 1 9 5 9 , 2 3 , 2 9 3 25. CRONKITE, E . P . , Proc. Ith Intern. Congr. on Radiology, Munich, 1959;

Abstract 727 2 6 . REVELL, S . , Proc. Roy. Soc. B , 1 9 5 9 , 1 5 0 , 5 6 3 27. SAX, K., Genetics, 1938, 23, 494 28. CATCHESIDE, D . G., LEA, D . E. and T H O D A Y , J . M . , / . Genetics, 1946, 4 7 , 113 29. DARLINGTON, C . D . and L A COUR, L . F., J. Genetics, 1945, 4 6 , 180 30. W O L F F , S . and L U I P P O L D , H . E., Progress in Radiobiology, Oliver & Boyd,

Edinburgh, 1956 p. 217 3 1 . M U L L E R , H . J . , Intern. J. Radiobiol., Suppl. 1 , 1 9 6 0 p. 3 2 1 32. BENDER, M . A., Intern. J. Radiobiol., Suppl. 1, 1960, p. 103 33. Report of the U.N. Scientific Committee on the Effects of Atomic Radiation,

General Assembly, 13th Session, Suppl. No. 17 (A/3838), New York, 1958 3 4 . LEJEUNE, J . , Intern. J. Radiobiol., Suppl. 1 , 1 9 6 0 , p . 1 1 9 35. HARRIS, H . , An Introduction to Human Biochemical Genetics, Cambridge

Univ. Press, London, 1953, 96 p. 3 6 . RUSSELL, W . L . and RUSSELL, L . B . , Proc. Second Intern. U.N. Conf. on the

Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 360, United Nations, 1959

RUSSELL, W . L . , RUSSELL, L . B . and KELLY, E . M . , Science, 1 9 5 8 , 1 2 8 , 1 5 4 6 RUSSELL, W . L . , RUSSELL, L . B . and C U P P , M . B . , Proc. Natl. Acad. Sci.,

U.S., 1 9 5 9 , 4 5 , 1 8 37. NEEL, J . V . a n d SCHULL, W . J . , U . N . D o c u m e n t A / A C 8 2 / G / R 24 38. MEWISSEN, D . J., DAMBLON, J. and BACQ, Z. M . , Nature, 1959, 1 8 3 , 1449 3 9 . SPARROW, A . H . , DENEGRE, M . a n d HANEY, W . J . , Genetics, 1 9 5 2 , 3 7 , 6 2 7 4 0 . RUSSELL, L . B . and M A J O R , M . H . , Genetics, 1 9 5 7 , 4 2 , 1 6 1 4 1 . FRASER, A . S . and SHORT, B . F . , Austral. J. Biol. Sci., 1 9 5 8 , 1 1 , 2 0 0 42. BURNET, S I R MACFARLANE, Brit. Med. J., Oct. 17, 1959, p. 720 43. RUSSELL, W. L . , RUSSELL, L . B . and ICELLY, E. M. , Intern. J. Radiobiol.,

Suppl. 1, 1960, p. 311 44. FRANCOIS, J., Vheredite en Ophthalmologie, Masson, Paris, 1958, 876 p. 4 5 . LASNITZKI, I . , Brit. J. Radiol., 1943, 1 6 , 61 and 137 46. MITCHELL, J. S., Studies in Radiotherapeutics, Blackwell, Oxford, 1960 47 . CREASEY, W . A . a n d STOCKEN, L . A . , Biochem. J., 1959, 7 2 , 519 48. KUZIN, A. M. , Proceedings Radiobiology Symposium Moscow, 1960, to be pub-

lished by Academic Press, London 4 9 . OAKBERG, E . F . and D i M I N N O , R . L . , Intern. J. Radiobiol. 1 9 6 0 , 2, 1 9 6

C H A P T E R 1 0

Biochemical Mechanisms for Cellular Effects—The Enzyme Release

Hypothesis

T H E R E is no reason to expect that one and the same mechanism applies to all the different cellular effects. Quantitative and qualitative differences suggest that there should be a variety of injury processes. Yet it is in the nature of science to look for unification, and an attempt will therefore be made at the end of this chapter to develop a working hypothesis which relies on one type of initial lesion which is, however, capable of very varied expression.

N U C L E U S V E R S U S C Y T O P L A S M

Much discussion has been devoted to the problem of whether the site of the primary lesion resides in the nucleus or in the cytoplasm of the cell. In a few instances such as the temporary interference with the light-producing metabolism of luminous bacteria (see p. 375) there can be little doubt that the affected site is in the cytoplasm. But for changes which are permanent—above all, interference with mitotic and genetic function-— damage has to occur within the nucleus and it is tempting to consider that the primary lesion is located there. Many experiments on widely differing biological materials have been carried out on the irradiation of localized parts of isolated cells to determine the relative sensitivity of the nucleus (or even single chromosomes) and the cytoplasm. Often the experiment is not a straight comparison of cytoplasm versus nucleus since the experi-ments usually contrast irradiation of a volume containing only cytoplasm with irradiation of a volume containing the nucleus together with adjacent cytoplasm.

1. Insects' Eggs The nucleus of the newly laid Habrobracon* egg lies at the egg surface

and can easily be reached by short penetrating radiation. Techniques have

* Habrobracon is a species of parasitic wasp.

2 6 3


been developed for accurate dosimetry of short penetrating a-particles (210Po). The egg dies if one a-particle goes through the nucleus; there is no need to assume that death is caused by diffusion from the cytoplasm of "mutagenic" or toxic substances1. It takes 16 million particles to kill 50 per cent of the eggs if the nucleus is not included in the irradiation. Cytoplasmic inactivation is kinetically and morphologically distinct from nuclear inactivation. Death occurs late in development when the cytoplasm is irradiated with a-particles2.

If one irradiates a virgin egg of Habrobracon jnglandis with doses from 2400 to 54,000 r, the female nucleus dies, but the cytoplasm remains apparently normal since it can be fertilized by normal sperms and develop an haploid embryo with purely male chromosomes. In other words, the irradiated protoplasm does not kill a non-irradiated nucleus; it takes more than 54,000 r in the cytoplasm to kill in this way4. The opposite result was observed when the same method was applied to the silkworm: the irradiated female cytoplasm of a strain of this species undoubtedly acts on the intact chromosomes of the spermatozoon5. ULRICH6-7 took ad-vantage of the regular presence of the nucleus in the anterior part of the newly unfertilized Drosophila egg to compare the effectiveness (hatch-ability was the criterion) of irradiating with a microbeam of x-rays either the anterior or the posterior part; the ratio of radiosensitivity is about 185:1 in favour of the nucleated part. This ratio is not exaggerated and represents an absolute maximum value for the sensitivity of the cytoplasm.

The ratio is probably much larger (at least 1000:1) since a great num-ber of electrons produced while irradiating the cytoplasm must be scattered and reach the nucleus. But the shape of the curves obtained by Ulrich suggests that some effect is produced on the nucleus by the irradiated protoplasm. At the doses necessary to affect the cytoplasm, sub-stantial quantities of peroxides will be produced and these are known to be mutagenic in some organisms.

2. Amoeba Large amoebae are very useful in experimental cell research because of

their size and the possibility of applying refined techniques of micro-surgery.

Two giant multi-nucleated species (Pelomyxa illinoiensis and P. earolin-ensis) were carefully studied by DANIELS8-9. Supralethally irradiated amoebae were observed to recover after direct addition by microfusion of parts of non-irradiated cells, which contain all cell components including nuclei.

The nature of the active component was investigated by studying the effects (on survival and cell division) of portions of non-irradiated cells which had been centrifuged at various gravitational forces. The presence

B I O C H E M I C A L M E C H A N I S M F O R C E L L U L A R E F F E C T S 2 6 5

of nuclei or mitochondria was not necessary for recovery; but the heavy and middle portion lost their capacity gradually as the centrifugal force was increased. It appears that the therapeutic capacity is located in fine particulate components (0-1 to 0-3 fx) or in the clear plasma that suspends them. Irradiation with neutrons, ultra-violet light or treatment with a nitrogen mustard destroys this restoration activity.

Amoebae with one nucleus are more useful for certain types of experi-ments—for instance, the transplantation of a nucleus from one cell to another one just deprived of its nucleus. Nuclei were transferred from irradiated amoebae to unirradiated enucleated cytoplasm and unirradiated nuclei were placed into irradiated cytoplasm10. The LD50 for nuclear death was about 120 kr; that for cytoplasmic death, 290 kr. Death from nuclear damage occurred without division within three to six weeks; death from cytoplasmic damage was seen within three days. Doses below 280 kr produced visible changes in the cytoplasm. Normal nuclei transferred in just lethally irradiated cytoplasm were unable to bring restoration and underwent pathological changes similar to those of irradiated nuclei. On the contrary, irradiated nuclei transferred in a normal cytoplasm (enu-cleated) showed little trouble; mitosis followed and, eventually, a normal colony could be started10.

3. Neurospora (Ascomycete) and Yeast (Saceharomyces) The evidence that inactivation of Neurospora conidia and yeast cells

involves nuclei rests mainly on the relation between the number of nuclei per conidium or ploidy of the yeasts and the inactivation kinetics11. In Neurospora, the most effective inactivation process is an indirect one (in the sense that it requires the presence of oxygen and water) and results in nuclear inactivation12. In yeast cells, most of the results have been interpreted in terms of dominant and recessive deaths (cf. ref. 13; see also p. 257 and p. 307).

4. Fern's Spores An old experiment of ZIRKLE15 showed that the non-nucleated part of

the spores of Pteris longifolia were much more resistant to a-polonium particles than the nucleated part. Five thousand particles in the nucleated part could inhibit division of all the spores, while eighty thousand in the non-nucleated part inhibited only 40 per cent.

5. Unicellular Algae Acetabularia mediterranea is a giant unicellular alga well known through

the extensive work of Hammerling and of Brachet, and their collaborators. At the first stage of its development, the nucleus (which is in the foot) can be easily separated from the rest of the cell which measures 3 cm in


length. The enucleated half lives for months and retains the ability of synthesizing proteins and of a limited morphological differentiation. The survival of enucleated fragments is less than that of the nucleated frag-ments following x-irradiation; upon irradiation of the whole cell, the cytoplasm furthest from the nucleus dies quicker and in a higher percentage of cases than the region surrounding the nucleus. The mortality rate of the cell and fragments is not proportional to the dose delivered: dosages of 3 kr result in more rapid death than dosages of 10 and 100 kr*. The growth and morphogenesis of the surviving irradiated algae at all dosages are identical to or better than the controls. This stimulation of growth does not depend on the presence of the nucleus14. Irradiation of the foot alone (and thus of the nucleus), the stalk of the alga being protected by lead, may result in lesion and cytolysis of the stalk.

6. Ciliates In ciliates (Paramecium, Tetrahymena), unlike bacteria and yeasts, the

division of the cell is more sensitive to radiation than is the growth process. The delay in division is not usually confined to the first division following irradiation, but extends over several divisions before eventual recovery; various patterns of this delay have been found (for bibliography see ref. 11). Attempts to localize these effects in the nucleus or cytoplasm have not given a definite answer. The work with ciliates has also thrown doubt on the hypothesis that inhibition of DNA synthesis is a primary cause of genetic as well as other radiation damage.

7. Cells in Tissue Culture Irradiation of the nucleus of newt heart-cells or—even better—of a

single chromosome with a beam of protons (2-5 //, in diameter) from a Van de Graaff generator produces typical abnormalities after a few pro-tons have passed, while many thousands of protons on the extrachromosome areas seem to be without effect3. Irradiation of chromosomal region of chick fibroblasts in tissue culture at metaphase, with a-particles, pro-duced sticky bridges at the anaphase. When irradiation was confined to part of the cytoplasm and spindle, no abnormalities were seen although the dose was up to 30 times that needed to produce visible alteration when the chromosomes were irradiated17. It was also impossible to obtain by cytoplasm irradiation (at metaphase), inhibition of nucleus re-construction which can be seen after chromosome irradiation17.

Irradiation with a beam (1 /i diameter) of polonium 210—a-particles, of the spindle of chick fibroblasts produces changes in the cytoplasm and abnormal cleavage with no effect on the chromosomes, which simply do not separate. There is also, outside the spindle and the chromosome, a

* A similar unexplained fact has been observed with thymic lymphocytes16.


radiosensitive structure responsible for the active pulling apart of the daughter cells18. Actions of the ionizing radiations on human tissue cultures are dealt with in another section (p. 250). Puck expresses the opinion that all the data on the x-radiosensitivity of human cells in tissue cultures can be explained on the basis of a defect resulting from primary damage localized in one or more chromosomes19.

8. Virgin Amphibian Eggs Eggs taken directly from the ovary of frogs or salamanders offer the

same facilities for microsurgery as amoebae, and have the great superiority of being very sensitive, vertebrate material. DURYEE20 carried out remark-able investigations with these cells. The nuclei freed of cytoplasm are very radioresistant and, after 30,000 r of x-rays, no lesion can be seen; after 60,000 r changes can be observed only in the nucleolus. If isolated normal nuclei are replaced in cytoplasm and the whole reconstituted structure is irradiated, the nucleus shows its usual high radiosensitivity. If the nucleus of an irradiated egg is transferred in the enucleated cyto-plasm of s normal cell, no lesions are seen; if a normal nucleus is trans-ferred in irradiated cytoplasm, the same changes are observed as when the nucleus is irradiated with the cytoplasm. If as little as 10~4 ml of cytoplasm from an ovarian egg, irradiated several days previously, is micro-injected close to the nucleus in the cytoplasm of a non-irradiated cell, the latter shows, one or two hours after the injection, all the symp-toms of radio-lesions such as a rupture of the nucleolus, pycnosis, vacuo-lization and chromosome fragmentation (see Fig. 9-4). Out of a total of 26 such experiments, 23 gave a positive result, while in a control series, where unirradiated cytoplasm was injected into normal cells, nuclear abnormalities were observed in only 5 out of 35 experiments. DURYEE21 has now extended his observations on the same cells, but using jS-rays of incorporated 32P as a source of continuous ionizing radiations; these new observations confirm the results obtained with x-rays. On the basis of these observations, Duryee has formulated the following sequence of events and his views agree quite well with the concept of biochemical mechanisms as opposed to "hit" interpretation for nuclear effects of radiations: (i) production of localized temperature insensitive physico-chemical events—practically instantaneous at the molecular level; (ii) accumulation of toxic catabolic substances—a slower process highly temperature sensitive (see p. 3); (iii) diffusion of toxic products in various parts of the cell including the nucleus.

9. Conclusions When observations on insect eggs or tissue cultures are compared with

those on amoebae, on unfertilized amphibian eggs, or on algae, the most


clear-cut contradiction springs to the mind. In the first case, the domin-ance of the nucleus is overwhelming; in the second, the restorative capacity of normal cytoplasm and the injuring power of irradiated cytoplasm are obvious. The only possible interpretation of this contradiction is that the interplay between the nucleus and the rest of the cell differs considerably in different types of cells. If micro-dissection experiments are not possible with insect eggs, why not use centrifuged amoebae or ovarian eggs as material for localized irradiation? Why not try human cells in tissue culture for localized irradiation? One cannot base human radiobiology on observations with such a differently differentiated material (and one so far away in evolution) as insect eggs. One should not repeat the error of the geneticists who extrapolated to man the results obtained on Drosophila and thus believed that the rate at which a dose of x-rays is given has no importance for genetic damage; Russell's experiment has shown how different a mouse is from a dipter in this fundamental question (see p. 259).*

C H R O M O S O M E " B R E A K A G E "

The purely mechanical interpretation of chromosome "breakage" can no longer be maintained. The impact of an ionizing particle with a chromo-some cannot be compared with the snapping of a telephone wire by a bullet. Model experiments with nucleo-protein gels (see Chapter 7) show that structures of the dimensions of chromosomes are not broken into two parts by the passage of one ionizing particle through them. Yet in an experiment with Trandescantia?2 chromosome damage occurred in every nucleus which had been traversed by one oc-particle (see Fig. 10-1). The fact that the sensitivity of the chromosomes to breakage by x-rays can be modified by prior exposure to ionizing radiation23 and by exposure before or after irradiation to infra-red24, also emphasizes that chromo-some injuries are not the direct result of the passage of an ionizing particle, but are the result of a complex interplay of different factors in a living system which reacts to changes brought about in its environment.

Since we know that many chemicals (see Chapter 8) as well as metabolic disturbances arising from high oxygen tension, can provoke lesions indistinguishable from those produced by radiation, it is tempting to consider chromosome "breakage" purely as an anatomical response to a biochemical disturbance. This may be true for lesions which require a certain time before they become apparent. How far this is the case for

* MULLER (private communication) has now found that gene mutations in female Drosophila are also dose rate dependent. In making comparisons it is im-portant to ensure that effects in germ cells at similar developmental stages are compared.


chromosome "breakage" is not yet certain. Damage to the chromosomes only become visible at prophase, although the nucleus is in many instances most sensitive at the end of the resting stage (see Fig. 10-2)*. On the

100

so

Proportion of nuclei - traversed by 0,1,2 and3

a-particles

£ %>«> Si

I I no

ZO

None

Proportion of cells which show some form of chromosome damage

Z 3 a,-Partides

FIG. 10-1. Relationship between the fraction of Tradescantia pollen traversed by an a-particle (following a dose of 4 rad) and the fraction of cells showing some form of chromosome damage.

other hand, late meiotic prophase (diplotene and diakinesis) and first meiotic metaphase are 50 to 60 times as sensitive to x-rays as early post-meiotic interphase in Trillium25. This period coincides with the stage where geneticists from various pieces of evidence assume that the doubling or splitting of the chromosome occurs. As there is little breakage at prophase, chromosomes are clearly not cut by radiation like grass with a scythe. On the other hand, the end of the resting stage is not a period where DNA synthesis occurs as the threads are believed to be already formed.

Moreover, irradiation during mitosis produces chromosome lesions that become visible in the next mitosis. This shows that the initial radiochem-ical process for chromosome aberrations does not constitute a break as this would be seen during the mitosis at which irradiation had taken place.

* It must be stressed that this variation of sensitivity applies only to chromo-some breaks. The work with neuroblasts26 shows that for mitotic inhibition, the period of maximum sensitivity appears much later in the cell cycle. Needless controversy has been caused concerning the time of maximum sensitivity by not realizing that different end-effects behave differently in this respect. Thus maxi-mum inhibition of D N A synthesis occurs at a period of the cell cycle different both from mitotic inhibition and chromosome breaking.


One fact seems certain; the anatomical injury is many stages removed from the initial radiation event.

A factor which was stressed in Chapter 4 and which argues strongly against a purely biochemical mechanism (such as interference with synthesis by enzyme inhibition) is that chromosome breakage is produced much more readily by radiation of high specific ionization. A dose of

FIG. 10-2. Relative radiosensitivity (chromosome breaks at meta phase) of Tradescantia pollen when exposed to 200 r of x-rays

at different stages in the cell cycle (from K O L L E R < 5 0 ) ) .

5 MeV a-particle may, under favourable conditions, be 10 to 20 times as effective as a similar amount of energy delivered by x-rays. This proves clearly that chromosome breaks are not produced by inactivation of enzymes or alteration of isolated macromolecules as radiation of low specific ionization should then be most effective. If interference by ionizing radiation with enzymatic processes results in the appearance of breaks then it must do so with a much lower efficiency than the main process.

From the foregoing it is obvious that neither the purely biochemical (inhibition of synthesis) nor the target (snapping of thread) hypothesis can be accepted. The high efficiency of densely ionizing radiations forces on us the interpretation that an organized pattern of macromolecules must be broken but one which is not as large as the chromosome thread itself. The enzyme release mechanism to be described in the next section is able to accommodate these apparently conflicting facts. However, any hypothesis of this kind can be only very tentative until more is known of the structure of the chromosome. Is it made up of huge molecules of nucleoprotein running along its whole length?—the molecular weight of cellular DNA is certainly high enough for this to be possible. In the


electron microscope molecules of DNA with an end-to-end distance of 4 /1 (and a width of O-C02 /JL) have been seen (see Fig. 7-14). Yet the view27

that the chromosomes are aggregates of more or less globular units finds much support and is not unique in nature since fibres of fibrin, for example, are built up in the same way. The suggestion28 that the individual units are held together by bivalent metal ions such as Ca++ is attractive, since the addition of substances capable of chelating calcium greatly increases the sensitivity of chromosomes to subsequent injury by irradiation. Clearly, until we know in chemical terms what breaking of chromosomes implies, a detailed mechanism cannot be discussed.

I N T E R R U P T I O N O F E N E R G Y S U P P L Y

The suppression of one or more sources of chemical energy does not appear to have been adequately considered in connection with the effects of radiations, though it has become one of the most fruitful ideas in biochemical pharmacodynamics.

If a co-enzyme (e.g. cocarboxylase in vitamin Bi deficiency) is lacking, or an enzyme system is blocked (e.g. by fluoride), the normal metabolism of a series of molecules is arrested, with three results: (i) certain metabol-ites accumulate; (ii) the energy which would have been set free by the degradation of these metabolites is no longer available; (iii) the body is forced to use other sources of energy, which tend to be quickly exhausted. This type of action is well illustrated by means of the following examples. In beri-beri (vitamin Bi deficiency), oxidation of pyruvic acid no longer takes place, and this acid accumulates in the nerve cells due to the absence of the cocarboxylase which is thiamine (vitamin BI) pyrophosphate. In fluoroacetic acid poisoning there are two stages: first, fluorocitric acid is formed which then blocks the Krebs cycle and allows citric acid to ac-cumulate45. In muscular poisoning by arsenicals, lewisite, and —SH blocking agents in general, carbohydrate metabolism, one of the main sources of energy, is blocked at various stages especially at the pyruvic acid stage, but also at the starting point. The poisoned muscle soon exhausts its reserves of phosphocreatine, which is not being replenished; contracture develops, and the muscle no longer responds to stimu-lation.

Frog muscle irradiated in vitro with x-rays (6000-8000 r) or by 32P in labelled phosphate behaves exactly like a muscle poisoned by hydrogen peroxide (Fig. 10-3), by an —SH blocking agent, or by any substance which can interfere with the utilization of carbohydrates (BACQ, L E -

COMTE and HERVE46). The muscle is not contracted by irradiation, but if it is stimulated it does not relax completely, the contracture increases with each stimulation, and the muscle responds less and less. From this experi-ment it is not possible to deduce the inhibition by radiation of a particular


enzyme*, but it proves the existence of a biochemical lesion persisting after irradiation, a lesion also produced by oxidizing agents. This ex-periment led Bacq and Herve to see if the action of x-rays would be modified by oxidation inhibitors such as NaCN or NaN3 (see Chapter 19).

FIG. 10-3. Similarity between action of hydrogen peroxide and /?-rays from 32P on striated muscle of frog: (a) NaH 2

32PC>4, 0-4 mc in the soln during 3 hr after 2 control contractions42. The radioactive soln is washed out and the stimulations with KCl repeated. Lundsgaard contracture with inexcitability;

(b) H 2 O 2 1 -5 vol per cent for 5 min. Frog rectus abdominalis isolated in a bath of oxygenated Ringer's solution. Isotonic contractions. A quantity of concentrated KCL solution (5 per cent) is added to give good contraction, and wash after 15 to 30 sec without waiting for maximum effect. Repeat after 5 min to ensure complete muscle relaxation be-tween excitations. Then at the time marked (see arrow) the muscle is contacted with the toxic solution; after an interval wash out solution and repeat stimulations in normal Ringer

solution with K C L at 5 min intervals (BACQ et A / . 4 6 . ) .

T H E E N Z Y M E - R E L E A S E H Y P O T H E S I S When writing the first edition of this book in 1954 we were impressed with the fact that the earliest biochemical changes that can be seen after irradiation are an increase in the activity of several enzyme systems and in the intervening years much new experimental data has become available (see p. 332) to confirm that this is a general phenomenon of radiobiology. This led us to propose that for cell death the primary lesion is an alter-ation in the permeability of certain intracellular structures. There are many early physiological effects in irradiated animals that are due to

* BARRON47 has proposed a somewhat different interpretation of this experiment and many more might be suggested; for instance, the inhibition of transphos-phorylase of adenosine triphosphatecreatine which according to LORAND48 insures relaxation of striped muscle.


changes in permeability (see Chapter 16). We suggest that "membrane" effects play a role also in the killing of cells by radiation. BACQ and HERVE 2 9

had already developed this idea to some extent and SPARROW 3 0 mentions it briefly. K U S I N and his colleagues in Russia31 were also led from bio-chemical considerations to postulate that radiation disorganized the spatial co-ordination of enzymes.

On the cellular scale, biochemical processes are controlled by the very exact localization of the enzymes and substrates, which prevents them from coming into contact, although they exist within the same cell. Some enzymes such as DNAase are in the mitochondria although the substrate is to be found in the nucleus. At least 60 per cent of the acid phosphatase is associated with the mitochondria; glycerophosphate, which is a substrate of acid phosphatase, does not pass through the "membrane" of isolated mitochondria32. The cholinesterases of the liver of rats and guinea-pigs are largely localized in structures smaller than the mitochondria33. As D E DUVE and his collaborators32 say, the cell is divided into compartments.

Enzymes pathologically released by unspecific injury (mechanical or chemical) have to travel some distance inside the cell before meeting their substrate. The most striking visual example of this separation is probably that of phenoloxydase in certain fungi and the potato tuber. In a normal cell it is separated from the phenolic substances which it oxidizes into black melanin via the intermediary of red quinones. Young wild specimens of Russula nigricans are white, but if they are cut (i.e. if the cellular structures are injured), the enzyme and substrate can come into contact and the mushrooms become black. For this reason the track of parasite within the mushroom shows up black, and ageing or cellular necrosis is always accompanied by blackening. These fungi provide a simple test, visible within a few hours, for determining if the barrier between the enzyme and the substrate has been broken. BACQ and HERVE 2 9

have performed preliminary experiments with this material. Irradiation with 20,000 r at 60 kV, or 150,000 r of soft x-rays does not change the colour or the enzymatic behaviour of the tissues of Boletus calopus and B. erythropus, but the effect of x-rays can be seen in Russula nigricans. For instance, 7000 r (with a contact therapy apparatus operating at 50 kV) causes blackening to a depth of 3 to 4 mm, clearly visible 20 hr after irradiation, whereas the blackening of control zones is only 1 -5 mm in depth. This material deserves detailed investigation as it lends itself to quantitative studies. Similar experiments with potato tubers have been published by SUSSMAN34 .

This working hypothesis has some points in common with the idea of "enzyme alteration" which has been developed by KAPLAN 3 5 . This author has confirmed old observations of EULER and Bnx35a; the catalase activity of yeast is much increased by treatment with chloroform, toluene, heat


or ultra-violet, and some of the physicochemical properties (resistance to heat, optimal pH, etc.) of the enzyme are changed. The idea that the liberation of the enzyme from an interface or a membrane is responsible for these changes has been proposed by Kaplan as an interpretation of the "Euler effect".

The release by radiation of enzymes immediately provides a mechanism for the role of metabolism in radiation injury. In the introduction we referred to the large volume of evidence which shows that metabolism is necessary to produce all the biological end-effects and that if the initial lesion is not developed then it is quite harmless. The release of an enzyme molecule clearly fits this role and no other biochemical mechanism has been proposed which relates metabolic rate directly with radiation injury.

D E DUVE (cf. ref. 4 4 ) has provided extremely clear-cut evidence in the participation in cell lysis of the small mitochondria which he calls lysosomes and in which many of the hydrolytic enzymes of the cell are concentrated. These are the "suicide bags" of the cell as they contain the cell's means of destruction. Normally, the proteolytic and nucleic acid attacking enzymes are confined in the lysosomes and there do no harm. But at some stage during the death of the cell they become released and destroy its macromolecules. When the liver is rendered anoxic, de Duve was able to show that the proteolytic enzymes in the lysosomes were activated by the drop in pH associated with anaerobic metabolism. The lysosomes break down and the released hydrolytic enzymes destroy the cell. It is tempting indeed to extend this mechanism to radiation damage where ionizations lead to the breakdown of the lysosomes and to their release, but there is no evidence to support this view. Lysosomes in isolation require more than 10,000 r before a leakage of enzymes can be detected. The possibility remains that the lysosomes are more sensitive within the cell.

Another characteristic of radiation injury is the multiplicity of end-effects and again this is exactly what would be expected from the attack by enzymes no longer restrained by intracellular barriers. The patho-logical biochemistry of irradiated cells (see Chapter 13) is characterized not by one lesion but by a pattern of enzymic alterations which appear together but no one of which alone seems sufficient for the observed end-effect. In particular, the enzyme release theory requiring interference with cell membranes makes it possible to link the cell-killing effects of radiation with the physio-pathological lesions such as release of active amines and interference with nerve function (see Chapters 15 and 16). The latter must involve a change in permeability of cell membranes and as many of the physio-pathological effects are observed immediately (i.e. while the irradiation is still proceeding) the action in the membrane must be con-sidered a primary lesion.

FIG. 10-4. Electron microscope photograph of the cytoplasm of the liver cell. The membranes of the endoplasmic reticulum

fine structure within the mitochondria can be seen.

(Kindly provided by M. S. C. Birbeck.)

[facing p. 2-J4


The Nature of the Intracellular Barriers Although much of the organization of the cell relies on isolation of en-

zymes within mitochondria, it has been apparent for many years that there must also be intracellular barriers in the ground substance and the micro-somes of the cell. The existence of membranes and surfaces within the cell was postulated in 1929 by PETERS36 in a thought-provoking paper, and the idea was further developed by NEEDHAM37 who discusses the role of what he calls the cytoskeleton, intracellular structures that are below the level of resolution of the ordinary light microscope. Recent investigations with the electron microscope38 have strikingly confirmed these predictions (see Fig. 10-4) and the cytoplasm is seen to be full of fine membranes, referred to as the endoplasmic reticulum, that serve to orientate and fix the microsomes. The mitochondria themselves are also far from being mere balloons filled with solutions of enzymes; they also contain an internal structure of membranes. In the nucleus no such structures have been seen but this may well be due to technical difficulties of differential staining in a dense mass since there is a metabolic need for them to prevent nuclear enzymes from digesting nuclear material. All the intracellular membranes, both within the mitochondria and the endoplasmic reticulum, appear to have the same structure. They are approximately 80 A wide and three layers can be seen, which are believed to result from the association as in a sandwich of two protein-phospholipid complexes in such a way that their hydrophobic (fatty) parts come together in the centre while the outsides are made up of the hydropholic (protein) parts of the molecule38. That the permeability of a structure of this type should be affected by ionization occurring within it is not unreasonable on radiochemical grounds, although it requires proof.

Evidence for effects on membranes is slowly beginning to accumulate. Brinkman and his co-workers (see p. 468) have shown that radiation increases the permeability of the fine layers in the skin especially after they have been separated by swelling into a number of extremely thin layers. This effect occurs within a second or so, the time necessary to give a dose of 200 r, and would appear to be an immediate effect of radia-tion as there is no time for some metabolic process to occur. The possibility that the change observed is due to the release and rapid action of an en-zyme like hyaluronidase cannot be excluded with certainty but seems improbable. The results of Tanada on the immediate change in the uptake of metal ions by plant roots (see p. 377) reveal a similar effect in a different system.

FRITZ-NIGGLI 3 9 finds changes in the osmotic behaviour of isolated mitochondria after small doses which can be attributed to changes in permeability, but as the cells were examined 1 hr after irradiation it is not clear whether this is an immediate effect. The sensitivity of the

19


central nervous system to irradiation (see p. 407) would follow if mem-branes were affected since this would provide the obvious mechanism by which radiations exert their effects on nerve function.

At present there is no experimental evidence which allows a predic-tion to be made whether enzyme release takes place from the mitochondria (and lysosomes) from the endoplasmic reticulum, or from within the nucleus. The existence of DNAase in the mitochondria while its only substrate is in the nucleus suggests that it can only function after being released and after crossing the nuclear membrane. Unfortunately, there is no evidence that this enzyme is set free after small doses and effects of this type have only been seen after 2 0 , 0 0 0 r (see p. 356). On the other hand, HAGEN40 has found support for the view that certain proteolytic enzymes, the cathepsins, are released from mitochondria by doses of less than 1000 r and PASSYNSKY51 has demonstrated enzyme release in a nodel system.

G . M . FRANK49 has developed a technique which enables him to follow the rate of oxygen uptake by cells (both in culture and tissues). He finds that in irradiation oxygen consumption is immediately increased and that the cellular autoregulation of oxygen utilization is altered in vivo and in vitro. Changes in permeability suggest themselves as a possible mechanism for this interesting effect.

Effects of Ionizatian Density and of Oxygen Clearly the damage produced when several ionizations occur close

together within a membrane will be considerably greater than that from isolated events and a higher effectiveness (see Chapter 4) of densely ionizing radiations would be expected. Moreover, quantitative variations in the RBE in different systems would be expected since the degree of damage needed to release different enzymes need not be the same. This discussion is of course formally similar to the theory of Catcheside and Lea (see Chapter 4) which interprets differences in RBE as the need to have several ionizations acting together before a chromosome is severed. We have taken the concept that an organized structure is involved, but transferred it to cell components which are sufficiently small to be broken by a few ionizations.

A very marked difference between the two theories must, however, be emphasized. According to Lea, the critical ionizations occur at the site where the damage is eventually seen in the microscope. According to the enzyme release hypothesis, there is no such identity of events and the change in intracellular barriers leading to the release of the enzyme cannot be identified with the site at which the enzyme causes the damage. Under such conditions it is quite impossible to determine the nature of the initial event by relating the radiation dose with the end-effect observed.


It is in this respect that there is a difference between the hypothesis under discussion and that proposed by Lea and other exponents of the target theory.

The localized breakdown of phospholipids (i.e. of the internal mem-branes) by radiation would be expected to show a pronounced oxygen effect. Ionizing radiations are known to initiate oxidation of fats by a chain-reaction in which many molecules are oxidized for every ionization that occurs41. An isolated ionization with sparsely ionizing radiations may not damage an intracellular membrane unless the effect is multiplied by a chain process involving oxygen. With densely ionizing radiations the need to enlarge the area affected by oxygen may be less as the damage of several ionizations close together may be sufficient. This would explain why the enhancement of radiation damage by oxygen is greatest with sparsely ionizing radiations.

Cytoplasmic and Nuclear Effects The controversy as to whether the nucleus or the cytoplasm is the radio-

sensitive part of the cell, loses meaning if there is an enzyme release mechanism since it is possible for cytoplasmic enzymes to act both on cytoplasmic structures and on nuclear structures, since the nuclear mem-brane allows large molecules to permeate. Nuclear damage need not therefore be confined to the action of enzymes present within the nucleus. The point has already been made (see p. 263) that it is not possible to exclude irradiation of adjacent cytoplasm in experiments which set out to restrict irradiations to the nucleus.

Damage to the chromosomes and to genes (mutations) could occur by the release of enzymes which break down the existing morphological structure, or to an interference by the liberated enzymes with nucleo-protein synthesis. The suggestion has been made by MANDEL42 that changes in the availability of precursors could lead to the synthesis of abnormal (mutated) DNA. At the present time it is impossible to dis-tinguish between these possibilities. However, the modern concept of the molecular basis of genetics suggests that an ionization within a DNA molecule is not by itself sufficient to produce a mutation since it is a purely destructive reaction. It cannot give rise to a change in the base sequence along the molecule which determines the genetic code. Alterations of this type can only be achieved by metabolic synthesis. Before the mutagenic action of radiation manifests itself metabolism is necessary—just as in every other biological radiation effect. This view is supported by the work which shows that the number of mutations produced can be altered by post-irradiation treatments (see Chapter 14). It is possible that the DNA that has been damaged will not be accurately replicated in the cell and it is the error in synthesis which provides the true mechanism of mutation.


R E F E R E N C E S

1 . ROGERS, R . W . and BORSTEL VON, R . C . , Radiation Research, 1 9 5 7 , 7 , 4 8 4 . 2. BORSTEL VON, R . C. and ROGERS, R . C., Radiation Research, 1958, 8 , 248. 3 . ZIRKLE, R . E . and BLOOM, W . , Science, 1 9 5 3 , 1 1 7 , 4 8 7 4 . W H I T I N G , A . R . , Radiation Research, 1 9 5 5 , 2 , 7 1 5. NAKAO, Y . , Nature, 1 7 2 , 625 6 . U L R I C H , H . , Naturwiss., 1 9 5 5 , 4 2 , 4 6 8 7. ULRICH, H., Verh. Dtsch. Zool. Ges., Hamburg 1956, Akad. Verlag., Geest

und Portig, Leipzig 8. DANIELS, E. W., J. Exptl. Zool, 1951, 117, 189; 1952, 120, 509 and 525;

1954, 1 2 7 , 427; 1955, 1 3 0 , 183; 1958, 1 3 7 , 425. 9. DANIELS, E. W. and VOGEL, H. H., JR., Second U.N. Intern. Conf. on the

Peaceful Uses of Atomic Energy, Geneva, 1958, Paper No. 907 10. O R D , M. J . and D A N I E L L I , J . F., Quart. J. Microscop. Sci., 1956, 9 7 , 29 1 1 . KIMBALL, R . F . , Ann. Rev. Microbiol., 1 9 5 7 , 1 1 , 1 9 9 1 2 . GAFFORD, R . D . , Radiation Research, 1 9 5 8 , 9 , 2 4 8 1 3 . TOBIAS, C . A . , MORTIMER, R . K . , GUNTHER, R . L . a n d WELSCH, G . P . ,

Second U.N. Intern. Conf. on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 240, United Nations, 1959.

1 4 . BACQ, Z . M . , VANDERHAEGHE, F . , DAMBLON, J . , ERRERA, M . a n d HERVE, A . , Exptl. Cell. Res., 1957, 12, 639

1 5 . ZIRKLE, R . E . , J. Cell. Comp. Physiol., 1 9 3 2 , 2 , 2 5 1 1 6 . T R O W E L L , O . A . , C O R P , M . J . and L U S H , W . R . , Radiation Research, 1 9 5 7 ,

7, 120 17. MUNRO, R., in Advances in Radiobiology, Oliver & Boyd, Edinburgh, 1957,

p. 108 1 8 . DAVIS, M. I . , S I M O N REUSS, I . and S M I T H , C. L . , in Advances in Radio-

biology, Oliver & Boyd, Edinburgh, 1957, p. 114 1 9 . PUCK, T . T . and MARCUS, P . I., J. Exptl. Med., 1 9 5 6 , 1 0 3 , 6 5 3 2 0 . DURYEE , W . R .,J. Natl. Cancer Inst., 1 9 4 9 , 1 0 , 7 3 5 21. DURYEE, W. R., Personal communication, 1959 2 2 . GRAY, L . H . , Brit. J. Radiol., 1 9 5 3 , 2 6 , 6 0 9 2 3 . L A N E , G . R . , Heredity, 1 9 5 1 , 5 , 1 2 4 . KAUFMAN, B . P . , G A Y , H . and ROTHBERG, H . , / . Exptl. Zool., 1 9 4 9 , 1 1 1 , 4 1 5 25. SPARROW, A. H. , MOSES, M . J. and D U B O W , R. J., Exptl. Cell Research,

Suppl. 2, 1952, p. 245 26. CARLSON, J. G., Cold Spring Harbor Symposia Quant. Biol., 1941, 9, 104 2 7 . M A Z I A , D . , Proc. Natl. Acad. Sci., U.S., 1 9 5 4 , 4 0 , 5 2 1 2 8 . STEFFENSEN, D . , Proc. Natl. Acad. Sci., U.S., 1 9 5 5 , 4 1 , 1 5 5 29. BACQ, Z. M. and HERVE, A., Bull. Acad. Mid. Belg., 1952, 6th series, 18, 13 30. SPARROW, A. H . , Ann. N.Y. Acad. Sci., 1951, 51, 1508 3 1 . K U S I N , A . M . and SHABADASH, A . L . , Proc. Second U.N. Intern. Conf. on the

Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p . 102, United Nations, 1959

3 2 . D E D U V E , C . , BERTHET, J . , BERTHET, L . and APPELMANS, F . , Nature, 1 9 5 1 , 167, 389

3 3 . GOUTIER, R . and GOUTIER-PIROTTE, M . , Arch. int. Physiol., 1 9 5 4 , 6 2 , 1 5 1 3 4 . SUSSMAN, A . S . , J. Cell. Comp. Physiol., 1 9 5 3 , 4 2 , 2 7 3 3 5 . K A P L A N , J . G . , Exptl. Cell Research, 1 9 5 5 , 8 , 3 0 5 35a. EULER, H . VON and BLIX, R . , Z. physiol. Chem., 1 9 1 9 , 1 0 5 , 83


36. PETERS, R . A., Harben Lec tures , / . State Medicine, 1 9 2 9 , 3 7 , (No. 1 2 ) 37. NEEDHAM, J., Biochemistry and Morphogenesis, Cambridge Univ. Press, 1942 38. Cf. Biochemical Soc. Symposium, 1959, No. 16 39. FRITZ-NIGGLI, H., Strahlentherapie, 1960, 38, 148; see also Noyer, P. P. and

Smith, R. E., Exptl. Cell Research, 1959, 16, 15 4 0 . H A G E N , U . , Z. Naturforschung, 1 9 5 7 , 12b, 5 4 6 4 1 . M E A D , J . F . , Science, 1 9 5 2 , 1 1 5 , 4 7 0 42. M A N D E L , M . and CHAMBON P., Intern. J. Radiobiol., Suppl. 1, 1960, p. 71 43. ALEXANDER, P. and STACEY, K. A., Proc. Fourth Intern. Congr. Biochem.,

Vienna, 1958, Vol. IX, p. 98, Pergamon Press, London and New York, 1959

4 4 . D E D U V E , C. and BEAUFAY, H . , Biochem. J., 1959, 7 3 , 6 0 4 4 5 . PETERS, R . A., Proc. Roy. Soc. B , 1 9 5 2 , 1 3 9 , 1 4 3 4 6 . BACQ, Z. M., LECOMTE, J . and HERVE, A., Arch. int. Physiol., 1 9 4 9 , 67, 1 4 2 47. BARRON, E. S. G., Symposium on Radiobiology, John Wiley, New York,

1952, p. 236 48. LORAND, L . , Nature, 1953, 172, 1181 49. FRANK, G. M., Radiobiology Symposium, Moscow 1960; to be published by

Academic Press, London. 50. ROLLER, P. C., Progressin Biophysics, Pergamon Press, London, 1953, 4, 195 51. PASSYNSKY, A. G., Radiobiology Symposium, Moscow, 1960, to be published

by Academic Press, London.

C H A P T E R 1 1

The Effect of Oxygen in Radiobiology

THE first systematic study of the influence of oxygen on the magnitude of the radiation lesion was made by HOLTHUSEN in 1 9 2 1 1 who found that anoxic ascaris eggs were more resistant to x-rays. Independently, PETRY2

observed the same effect with seeds in his pioneer studies on the role of hydration in radiation damage. The generality of the phenomenon was realized by CRABTREE and CRAMER3 and the subject was explored in great detail using the bean root by MOTTRAM 4 who stressed the relevance of those observations to radiotherapy. Mottram's experiments were con-tinued by Gray who with his colleagues amassed much data on the effect of the environment on the radiation response of root tips and in recent years their investigations have been extended to tumour cells in vivo and in vitro.

In vegetative systems, the oxygen effect is generally* characterized by these features, (i) In the absence of oxygen or at reduced oxygen pressure, the effects of x- or y-rays are diminished but not abolished, (ii) The oxygen has to be present during the irradiation and exposure to oxygen before or after the irradiation does not influence the process, (iii) The effects of densely ionizing radiations such as neutrons, and especially of oc-particles, are much less sensitive to the presence of oxygen than sparsely ionizing x- or y-rays. (Similarly, the effect of chemical protectors is (i) much more marked for x- and y-rays than for neutrons, and (ii) much more evident in the presence of oxygen; see p. 457).

The enhancement of radiation damage of cells and tissues by oxygen (or, in other words, the increase in radioresistance by anoxia) has been found with very few exceptions wherever it has been looked for, whether the end point is death of the organism from radiation sickness, reduction in growth rate, the production of an anatomical lesion, such as chromo-some abnormality or a biochemical lesion, or the frequency of gene mutations. The major exception to this rule would appear to be change

* In certain strains of yeast, TARUSSOF59 finds that oxygen only increases radio-sensitivity up to about 600 m m partial pressure and at higher pressures it "p ro-tects".

280

T H E E F F E C T O F O X Y G E N I N R A D I O B I O L O G Y 281

in the permeability of epidermal membranes (see p. 468) which is un-affected by anoxia. The oxygen effect is also much less clearly defined for the inactivation of biologically active substances in vitro (see Chapter 7).

Radiation lesions in cells* produced by ultra-violet are unaffected by anoxia and this represents one of the most characteristic differences between the biological effects of radiations and ultra-violet. As would be expected on chemical grounds the biological reactions of radiomimetic chemical substances such as the mustards are independent of oxygen: oxygen, however, influences the production of chromosome aberrations by 8-ethoxycaffeine in Viciafaba (KIHLMAN6), but there are many reasons for believing that this oxygen effect is produced by a mechanism entirely different from that which occurs with ionizing radiations.

It must not be forgotten that oxygen by itself under non-physiological conditions is not devoid of action. If pollen or microspores of Tradescantia are exposed to partial oxygen tensions higher than that of air, chromosome damage is observed. In fact, the alterations in the chromosomes produced by oxygen appear to be identical with those caused by ionizing radiations. As many changes in the chromosomes are observed if the pollen is kept for an hour in pure oxygen as after a dose of 12,000 r of x-rays7. Barley seeds exposed to 60 atmospheres of oxygen show many chromosome aberrations8. Normal air (20 per cent oxygen) at high pressure is as efficient as atmospheres enriched with oxygen. The important point is the partial pressure of oxygen. The best protectors against x-rays, i.e. cyste-amine and cysteine (see p. 459) are also the best protectors of mammals against oxygen poisoning and disturbances caused by air at high pressures.

Ozone, which can be considered as an activated form of oxygen, has radiomimetic properties at low concentration9'10. The biochemical lesion induced by ozone may be a decreased ability to utilize oxygen9. Oxygen poisoning is considered by GERSCHMAN54'55 as a continuous process; even at normal pressure, oxygen may have some deleterious effects like shorten-ing of life span (possibly by increasing somatic mutations). Cysteamine pro-tects very well against ozone9 or oxygen poisoning. Indeed, it has been observed that continuous feeding with cysteamine significantly prolongs life-span of mice; these experiments of HARMAN11 should be repeated and care should be taken to record the amount of food consumed by the mice. An imposed reduction of food consumption increases life-span; addition of cysteamine to food might very well reduce food consumption.

T I M E A T W H I C H O X Y G E N A C T S

From the technical point of view, micro-organisms or single cells which

* In vitro damage to D N A by ultra-violet is enhanced by oxygen and the absence of an oxygen effect within cells may be due to protection by other cell constituents5.


adapt themselves equally well to aerobic and anaerobic conditions form the material of choice. This was well understood by H O L L A E N D E R and his collaborators in a series of studies on a strain of Escherichia coli12-13.

Figure 11-1 gives the result of a typical experiment by these authors. It

FIG. 11-1. Comparative sensitivity of aerobic and anaerobic cells irradiated in high and low oxygen tensions. E. coli irradiated in buffer solution with 250 kV x-rays: I. Aerobic broth cells irradiated in oxygen-saturated buffer; II. Anaerobic glucose cells irradiated in nitrogen-saturated buffer; I I I . Aerobic broth cells irradiated in nitrogen-saturated buffer; IV. An-

aerobic glucose cells irradiated in oxygen-saturated buffer13.

shows the survival curves of E. coli cultivated aerobically and irradiated in a solution saturated with oxygen (I); cultivated anaerobically and irradi-ated in a solution saturated with nitrogen (II); cultivated aerobically and irradiated under nitrogen (III); and cultivated anaerobically and irradiated under oxygen (IV).

Several obvious conclusions can be drawn: the difference in sensitivity between bacilli cultivated and irradiated in air and similar bacilli cultivated


and irradiated in the absence of oxygen is so great that 10 times as much energy is needed to inactivate the same proportion of anaerobic as of aerobic bacilli, and, with a given dose of x-rays, the proportion of aerobic bacteria inactivated is 100,000 times greater. Other inert gases may be used instead of nitrogen without changing the result. The important factor is the presence of oxygen. Similarly, the sensitivity of E. coli culti-vated anaerobically is increased as soon as oxygen is introduced.

The oxygen, therefore, acts essentially during the irradiation1; it matters less if the cultures are kept afterwards in aerobic or anaerobic conditions*. These facts have been confirmed in very diverse biological systems. Table 1 1 - 1 sums up the observations of G ILES and RILEY14 on the micro-

T A B L E 1 1 - 1

EFFECTS OF C O N D I T I O N S DURING IRRADIATION AND T R E A T M E N T AFTER IRRADIATION ON THE CHROMOSOMES OF M I C R O S P O R E S OF TRADESCANTIA.

Summary by H O L L A E N D E R et al., f rom G I L E S and R I L E Y ( 1 9 5 0 ) .

Condition during irradiation

(300 r at 300 r/min)

Condition after

irradiation

Number per cell Condition during

irradiation (300 r at 300 r/min)

Condition after

irradiation Interchanges Interstitial

deletions

Vacuum Vacuum 0 - 1 2 0 - 1 1 Vacuum Oxygen 0 0 9 0 - 1 0 Oxygen Oxygen 0 - 7 0 0 - 8 3 Oxygen Vacuum 0 - 7 2 0 8 5

spores of Tradescantia paludosa. The oxygen effect in the broad bean root is independent of temperature and is not related directly to metabolic processes15. Absence of oxygen decreases by a factor of 2 the action of irradiation on haploid, diploid or tetraploid yeast in any stage of mitosis16. This fact again points to the involvement of oxygen in early radiochemical events rather than in terminal biological changes. NAKAO17 on the contrary, found that fertilized toads' eggs show an oxygen effect only if irradiated 30 min after fertilization; but this observation should be confirmed.

Recently, evidence has been obtained that cells require very little time to change from the less radiosensitive anoxic state to the more radio-sensitive state in the presence of oxygen. By the application of a clamp to cut off the blood supply to the tail of young mice, followed by irradiation

* T h e magnitude of the oxygen effect depends in E. coli B. and Salmonella typhimurium (but not in some other strains of E. coli or other micro-organisms) on post-irradiation treatment18 .


at high intensity and lasting less than a second, WRIGHT19 was able to show that the cells attained their lowest level of radiosensitivity (i.e. corresponding to complete anoxia) in 4 sec. Most of this time was prob-ably needed to exhaust the oxygen available at the site from occluded blood. Anoxic ascites tumour cells in vitro20 regain their full radiosensitivity on aeration within the time of experiment of 2 sec. By an ingenious use of high intensity bursts of electrons21 the sensitivity of bacteria was shown to lag not more than 1 /50th of a second behind a change in oxygen concentration of the media and the actual time may well be less.

The conclusion cannot be resisted that oxygen must be present at the time when the initial chemical changes (see chart opposite page 1) are taking place and the most probable interpretation is that oxygen modifies this primary (and as yet undetected) radiation lesion. Since the develop-ment of radiation injury requires metabolism it is highly probable that there are systems in which the magnitude of the radiation lesions can be altered by changes in oxygen tension after irradiation. Any such post-irradiation phenomena must not however be confused with the oxygen effect described in this chapter which is concerned with the production of the initial lesion and not with its development (but see p. 296). The existence of this early oxygen effect is one of the unifying principles of radiobiology and its mechanism at the molecular level will be discussed later in this chapter.

C O N C E N T R A T I O N O F O X Y G E N R E Q U I R E D For many lesions the radiosensitivity towards x-rays and other sparsely ionizing radiations is some two to three times greater in air than in the complete absence of air. In some systems (e.g. see p. 288) the effect is greater and for chromosome abnormalities the value varies on the type of deformity that is scored*. A value for the maximum dose reduction of three by anoxia can however be accepted as the most typical. The relationship between radiosensitivity and oxygen tension depends very much on the techniques used to measure it.

In 1 9 5 3 , G R A Y et al.22 collected the available data and concluded that all the systems studied followed the same pattern and Fig. 11-2 was believed to be typical for most cells. That is, maximum sensitivity was reached when the gas in equilibrium contained some 20 per cent of

* Both chromosome breaks and intragenic changes are affected to a similar degree by anoxia28. T h e oxygen effect is apparent for cells of Vicia faba roots at any stage of interphase2 9 ; it is smaller than 2 if the dose-rate of 60Co irradiation of these cells is about 100 times lower; for high dose-rate the factor is 2-8. Th i s fact again suggests that the biological effects of low dose-rates are fundamentally different f rom that of high dose-rates (or so-called single exposures). T h e cyto-logical aspects of the oxygen effect (chromosome breaks, mutation, etc.) have been aptly reviewed and discussed by S W A N S O N 3 0 and BAKER 3 1 .


oxygen, i.e. the same as that present in air. Enrichment of oxygen in air did not increase sensitivity, but any decrease led to a fall in radiosensitivity. Unfortunately, this situation proved to be misleading since the oxygen

Oygen in nitrogen

FIG. 11-2A. Relationship between oxygen concentration in the air and x-ray-induced mutation rate for sex-linked lethals in

Drosophila melanogaster (400 r)13.

20 30 to 50 60 Oxjgen

_L 70 80 90

FIG. 11-2B. Dependence of x-ray sensitivity of mouse ascites tumour cells on oxygen tension in the air above the suspension20.

concentrations in the atmosphere did not represent the actual oxygen concentration immediately adjacent to the cell. Thus, G U N T H E R and KOHN23 stressed that in concentrated suspensions of micro-organisms which consume much oxygen, the' oxygen pressure is low in the solution although the surface is exposed to air. Similarly, thin layers (250 /;) of ascites cells in a closed vessel will consume the available oxygen in a few minutes24. With plants and insects (Drosophila) which also followed the curve shown in Fig. 11-2, there is of course also an oxygen gradient and the oxygen concentration in the air does not represent the true oxygen concentration in the immediate environment of the cells that are being


studied. A remarkable conspiracy between artifact and experimental error led to the acceptance22 of the curve shown in Fig. 1 l-2b as representing the basic relationship between sensitivity and oxygen concentration.

In the last years a number of attempts has been made to determine the cellular concentration of oxygen and to relate this to radiosensitivity. With yeast and bacteria this could be done relatively easily by irradiating a suspension while a gas containing the desired amount of oxygen was continuously passed through it25. The results are shown in Fig. ll-3aand clearly the concentration of oxygen required to produce an increase in

Dissolved oxygen, / i moles/l IO ZO 30 4 0

Dissolved oxygen, /i moles / I IO 3 0 50 100 200

- I - - I - I I

Oir

(b)

0 1 2 3 IO 21 oxygen, %

FIG. 11-3. The influence of the concentration of dissolved oxygen on the radiosensitivity of: (a) suspensions of bacteria (E. coli B/R)25; (b) chromosome aberrations in Tradescantia

pollen tubes27.


radiosensitivity is much less than that obtained earlier (cf. Fig. 11-2). The increase in radiosensitivity is exponentially related to oxygen tension and follows a "Langmuir isotherm", a relationship frequently encountered in physico-chemical studies in absorption reactions. A useful parameter is the oxygen concentration at which the cells have attained half their increase in sensitivity. For bacteria25, yeast and ascites cells20 this occurs at approx-imately 2-5 mm partial pressure of oxygen (i.e. at approximately one-eightieth of the oxygen concentration in air). Another system in which oxygen gradients can be avoided is chromosome breaks in isolated pollen tubes of germinating Tradescantia2irj. Half of the increase in sensitivity due to oxygen was obtained at 5 mm of oxygen pressure. Calculations of the oxygen tension within different tissues and within plant roots have been attempted by GRAY26, and it would appear that the relationship between oxygen tension of the surrounding gas and radiosensitivity is determined by oxygen diffusion. At the cellular level the oxygen required to increase radiosensitivity in tissues would seem to be of the order of that found for the cells in suspension (i.e. as in Fig. ll-3b).

As far as is known the living material most sensitive to the oxygen effect is the isolated lymph gland of the rat cultivated in vitro by TROWELL32 '33. The small lymphocytes in these glands are the most radiosensitive, though they no longer divide and have reached the end of their mitotic career.

The dose needed to produce pyknosis in half the cells in pure oxygen

T H E O X Y G E N E F F E C T I N M A M M A L S

r i -L

Plants and insects

o

O 5 TO 20 30 40 Oxygen in medium ml/1.

FIG. 11-4. Influence of oxygen on damage to lymph nodes ex-posed to x-irradiation in vitro ( © This point has been adjusted

to correct for a dose rate effect32.)


is 275 r in vitro (150 r in vivo). To produce the same effect in an atmo-sphere of pure nitrogen, a dose of 3 3 0 0 r or 1 2 times as much energy is needed. The effect of oxygen is greater in this tissue than in plants and insects; also radiosensitivity shows a linear increase with oxygen pressure over the whole range of 0 - 7 6 0 mm of oxygen (see Fig. 1 1 - 4 ) , whereas in other multicellular systems the oxygen effect does not increase appreciably at concentrations above 180 mm of oxygen (i.e. air). This high value of oxygen can hardly be explained by diffusion and it would seem that these cells show a quite different oxygen dependence.

Great caution is needed in the interpretation of the oxygen effect when whole vertebrates, and especially mammals, are considered. The first question is: What is the partial pressure of oxygen iti the tissues and cells of mammals? CAMPBELL34 reviewed this problem in 1 9 3 1 and concluded that the tension in the tissues is the same as that in the venous blood or lymph, i.e. 2 0 - 4 0 mm Hg. MILLIKAN35 discovered an intracellular indi-cator of oxygen tension—myoglobin (or myohaemoglobin, MHb*)—and was able to measure instantaneously the oxygen concentration of the MHb of the cells of the soleus muscle of cats with intact blood supply and innervation. In the resting muscle the MHb is saturated, that is to say, referring to the dissociation curve published by Hill, the oxygen tension within the muscle cell is at least 20 mm Hg as Campbell foresaw.

As soon as the muscle is tetanized, i.e. as soon as it consumes oxygen, the degree of saturation of the MHb falls abruptly, but it is completely restored in about 10 sec when stimulation is stopped if the blood supply is normal, that is, if the artery has not been clamped. It may therefore be assumed that mammalian muscle works at an oxygen tension of 2 0 - 2 5 mm Hg. This does not mean, of course, that the same oxygen tension exists in the cells of other tissues (e.g. skin), where the blood supply may be greatly reduced by cold or other conditions.

Very probably the oxygen tension in the deep organs which are as well-supplied with blood as the muscles (liver, heart, kidney, spleen, central nervous system) is the same as in resting muscle. In organs or accumula-tions of cells (such as abscesses) which have little or no blood supply the oxygen tension is, of course, lower, and this may play a part in the radio-sensitivity of tumour cells when, as sometimes happens, the vascularization of the tumour is very uneven (see next section). Direct measurement of oxygen tension in human tissues have been made with a platinum micro-electrode57. They show that the oxygen tension between the cells in the bone-marrow is very low (10 mm Hg or less) but comparatively high in subcutaneous tissue (50 mm Hg).

* Myoglobin differs f rom haemoglobin chiefly in its much greater affinity for oxygen and in its hyperbolic dissociation curve.

T H E E F F E C T O F O X Y G E N I N R A D I O B I O L O G Y 2 8 9

T h e r e is a second point which has not been considered, and which seems to be of importance. W h e n a mammal is deprived of oxygen a number of reflex reactions takes place; in particular there is intense stimu-lation of the sympathetic system, which liberates considerable quantities of adrenaline and noradrenaline f rom the adrenal medulla, and also at the endings of the adrenergic nerves. These two hormones, being phenolic amines are good protectors against x-rays (see p. 463). Adrenaline increases the cardiac output and oxygen consumption; it is a powerful vasodilator in many parts of the body and vasoconstrictor in others. Moreover, if the level of adrenaline (not of noradrenaline) is maintained at a higher level in the blood for some time, A C T H (adrenocorticotrophic hormone) is secreted b y the pituitary and the hormones of the adrenal cortex come into play (see p. 387).

In mammals, cold also considerably diminishes radiosensitivity in young mice (LACASSAGNE36) and in adult rats (HAJDUKOVIC et al.37) provided irradiation is carried out in deeply-chilled animals—that is, if the cold condition has been applied immediately before irradiation. T h e LD50/30 days (620 r normally) is raised to 1760 r when mice are irradiated while their colonic temperature is between 0 and 0-5°C when breathing and heart contractions had stopped38. T h e shape of the survival curve is different (Fig. 11-5) ; some supercooled mice die between 3 and 7 days after irradi-ation when no death is seen in mice who have received at normal tem-perature a dose (700 r) giving a final survival rate of the same order;

O 5 IO 15 20 25 30 Days af ter X - i r rad ia t i on

FIG. 11-5. T h e survival of male mice over a 30-day period after whole-body x-irradiation of 600 r, 700 r or 800 r at normal body temperatures and of 1800 r given to mice at reduced body

temperatures38 .


these early deaths are "intestinal deaths" 3 8 . A n o x i a seems to be the most important factor in cooling effect because w h e n infant mice are irradiated at b o d y temperature of 3 to 7 ° C , the addition of N2 does not increase protection whilst the addition of O2 reduced survival considerably3 9 .

I f rats are subjected to an atmosphere containing only 5 per cent of oxygen instead of 20 per cent (the concentration in normal air) their sensitivity to radiations is approximately halved. I n order to obtain the same mortality curve among anoxic rats as among controls the dose of x-rays must be approximately doubled; wi th mice, the protection obtained is smaller4 0 . T h e possibility that variations in oxygen tension contribute to the high radiosensitivity of y o u n g mice (see p. 299) has been suggested b y L I N D O P a n d R O T B L A T 5 8 .

T h e higher degree of "protec t ion" of animals breathing 5 per cent of oxygen is puzz l ing since isolated cells are as radiosensitive in such an atmosphere as in normal air. Probably a proportion of cells in the bone-marrow or other "target o r g a n " such as the intestine are normally in equi l ibr ium w i t h a relatively low concentration of oxygen such that a reduction in the oxygen in the atmosphere makes these cells then com-pletely anoxic and thus able to withstand the radiation. W e know f r o m experiments on the graft ing of bone-marrow cells that only a relatively small number of bone-marrow cells are required to enable an irradiated animal to survive (see Chapter 20). However , w h e n a m a m m a l is m a d e anoxic or severely chilled, complex reactions are produced. M i c r o -organisms or plant cells are more suitable for quantitative studies of the oxygen effect.

A P P L I C A T I O N O F O X Y G E N E F F E C T T O

R A D I O T H E R A P Y

T h e r e is good evidence f r o m at least eight series of experiments (see T a b l e 11-2) that cancer cells react like ordinary cells in their response to oxygen. In badly vascularized tumours there may be cells w h i c h are very nearly anoxic and these may be raised to higher radiosensitivity b y in-creasing the oxygen supply. D a m a g e to the surrounding tissue would not at the same time be increased since the cells there are in an oxygen environ-ment in w h i c h radiosensitivity is at a m a x i m u m . T h e possibility must be envisaged that an important factor in the radiocurability of tumours may be the n u m b e r of cancer cells that are in an anoxic or closely anoxic state. I n an impressive experiment using layers of ascites cells of different thickness SCOTT24 has demonstrated the extreme steepness of the oxygen gradient through a tumour mass. A n increase in tumour mass b y a fraction of a mill imetre (without a compensating increase in vascularization) can give rise to an anoxic layer of cells w h i c h are radioresistant.


T A B L E 1 1 - 2

M O U S E T U M O U R S I N W H I C H RADIOSENSITIVITY IS POSITIVELY CORRELATED W I T H OXYGEN T E N S I O N .

(Taken f rom G R A Y 2 6 ) .

T a r carcinoma 2146 Crabtree, H . G. and Cramer, W., Eleventh T a r sarcoma 173 Sei. Report of Imp. Cancer Res. Fund., Sarcoma 378 Parts 1, 2 and 3, pp. 75-117, 1934

Mammary carcinoma Hall, B. V., Hamilton, K. and Brues, A. M. , Cancer Research (abs.), 1952, 12, 268

Lymphosarcoma (in vivo) L i Hollcroft, J . W. , Lorenz, E. and Matthews, M. , J. Natl. Cancer Inst., 1952, 21, 751

Ehrlich ascites tumour Conger, A. D . and Gray, L . H . , Brit. J. Rad., 1953, 26, 638

Ehrlich ascites tumour Dittr ich, W. and Stuhlmann, H . , Naturwissen-schaften, 1954, 41, 122

Ehrlich ascites tumour Scott, O. C. A., Brit. J. Rad., 1953, 26, 638-648

Ehrlich ascites tumour (in vitro) Deschner, E. E. and Gray, L . H. , Radiation Research, 1959, 11, 115

D B A H i and Ar adenocarcinoma Goldfeder, A. and Clarke, G . E., Proc. Am. D B A G spindle-cell tumour Assoc. Cancer Res., 1957, 2, No. 3

T h e r e is histopathological evidence that at least some types of human tumours contain anoxic areas41 which are still capable of proliferation. G r a y believes that: " I n these cases any procedure which raises venous oxygen tension throughout the b o d y for the duration of each irradiation is likely to render the treatment more effective in the absolute sense of inflicting greater damage to the most radioresistant cells, and will increase the differential damage with respect to well vascularized healthy tissue"2 6 .

On the other hand, in mammalian tissues that responded to oxygen in the same way as isolated rat lymph glands (Fig. 11-4), this differential effect could not show itself. In Trowel l ' s experiment the reaction of iso-lated lymph glands to x-rays increased in a straight line up to an oxygen concentration of 20 ml per litre in the culture fluid, and this concentration is reached when the fluid is in equil ibrium with pure oxygen at 760 m m Hg. It is true that in this experiment the tissue is not vascularized and the curve might be quite different if a vascularized gland could be used,

20


in w h i c h all the cells would live at about the same oxygen tension. It is therefore essential to study the oxygen effect in vascularized mammalian organs quantitatively as ful ly as is technically possible. It is easy to pro-duce anoxia of the skin: local compression or vasoconstriction due to cold will certainly result in a diminution of the cutaneous lesions when it is desired to irradiate deep-seated tissues. CATER and SILVER57 find that the very low oxygen tension in the bone-marrow of man cannot be raised by breathing pure oxygen.

Conversely, it might be possible to improve the blood supply of poorly irrigated tumours b y local injections of vasodilators. T h e most radical solution, however, supposing the two conditions mentioned above are fulfilled, would be to irradiate the patient in a compression chamber like those used tor workers under water or in tunnels. Promisingcl inicaleffects have already been obtained b y this method4 2 .

A n alternative method now being explored is to make the patient completely anoxic and thereby lower the radiosensitivity of the healthy cells (stroma) without affecting the radiation response of those tumour areas that are normally anoxic. In this way a larger dose would be given and a differential effect to the tumour achieved. Since brain cells are damaged b y anoxia within three to five minutes, the irradiation must be carried out within this time and this presents technical difficulties that have not so far been overcome.

M E C H A N I S M O F A C T I O N In the preceding sections the reasons have been given w h y the view is widely held that oxygen acts at the level of the initial chemical lesion.

Where the initial chemical lesion is produced b y indirect action oxygen wi l l :

(a) Alter the nature of the attacking radicals*; instead of H* there will be H O 2 (or O 2 - ) radicals which may be more damaging to some organic molecules (see Chapter 6);

(b) T h e first chemical reaction of a radical f rom water (i.e. H, O I I or H O 2 ) with an organic molecule is often the abstraction of a hydro-gen atom to give an organic radical, i.e. R H + OH" R* + H 2 O .

In the presence of oxygen the reaction

R ' + 0 2 - ^ R 0 2

will occur very readily and often to the exclusion of all other reactions of R' .

W h e n the initial chemical lesion is produced b y "direct action", oxygen

* T h e amount of hydrogen peroxide produced will also be increased, but this is unlikely to be of biological importance31 .


can also exert a pronounced influence (for detailed discussion see Chapters 5 and 7 ; e.g. p. 211). T h e first stage of a "direct" radiochemical reaction is also frequently the formation of an organic radical* which is capable of combining with oxygen (see Chapter 5). It is also possible that O 2 -radicals are formed by direct action, since the affinity of organic molecules for the ejected electron is less than that of oxygen and in this way radio-chemical damage by direct action is enhanced (see pp. 174 and 186).

Whether the action is direct or indirect the eventual fate of the organic molecule attacked depends on the further reactions of the radical R' . Indeed, the end products formed in the presence of oxygen have often been shown to be entirely different f rom those formed in its absence because R ' will readily combine with oxygen. It was suggested b y us in our first edition that "oxygen may also influence a reaction b y adding onto a molecule, damaged b y either direct or indirect action, to give rise to a product which differs f rom that produced under anoxic conditions". T h i s hypothesis is now widely accepted (cf. refs. 21 and 26), though at the time it was first put forward, the formation of HO 2 " was thought to be the important factor.

In the absence of oxygen R ' may revert to R H and it has been shown b y us (see p. 177) that chemical protective agents may function b y restoring R" in this way. Irradiation with densely ionizing radiations would lead to the occurrence of several ionizations within the same macro-molecule. T h i s amount of damage could not be restored and consequently there is no need for oxygen to enhance the chemical lesion. In this way the fact that a marked oxygen effect is only seen with sparsely ionizing radiations would be explained. Alternatively, R ' may in the absence of oxygen react in a way that does not lead to a lesion while with oxygen present an oxidation chain reaction which involves many molecules may be initiated. W e have suggested (see p. 276) that the initiation of a chain reaction involv-ing the oxidation of unsaturated fats could lead to a breakdown of cell membranes in which phospholipids play an important part.

In vitro experiments with biologically active molecules have thrown little light on the site of action of oxygen (for details see Chapter 7). Enzymes irradiated in dilute solution when the action is indirect, show no oxygen effect (with the possible exception of D N A a s e ) and earlier claims that SH-enzymes were inactivated three times as effectively in air than in nitrogen have not been repeated. W i t h dry enzymes where the action is direct inactivation b y sparsely ionizing radiation (though not b y a-rays) is increased b y some 50 per cent if oxygen is present. T h i s relatively

* This does not mean that direct and indirect action produce the same products. T h e radicals are often different, but they share the characteristic common to most radicals, readily combining with oxygen.


small effect is believed b y Alexander to be due to O 2 - and not to the addition of oxygen to an organic radical (see p. 186). On the other hand, A L E X A N D E R et al.45 did find peroxide groups in serum albumin after irradiation in air, but not after irradiation in nitrogen.

BACHOFER and POTTINGER46 find that in dilute solution the coli bacterio-phage T i is protected b y the presence of dissolved oxygen. But this observation has not been extended to other viruses, nor is it known if there is an effect of oxygen on the inactivation b y direct action.

Irradiation of D N A in dilute solution has given rise to contradictory results (see p. 200), but the general impression is that there is no marked effect of oxygen on the reduction in viscosity. N o r could EPHRUSSI-TAYLOR and LATARJET47 f ind any increase b y oxygen in inactivation of transforming activity b y irradiating bacterial D N A in dilute solutions. Peroxides have been f o u n d fol lowing irradiation of D N A in solution but these would appear to have no part in the loss of transforming actively.

W h e n direct action plays a part there is a very pronounced oxygen effect in the physico-chemical changes in D N A (see p. 202). In the absence of oxygen there is cross-linking leading to the production of an insoluble product while in its absence there is degradation of the D N A . T h e same number of molecules would appear to be altered and oxygen merely affects the nature of the product.

Sensitization by nitric oxide—Nitric oxide ( N O ) shares with oxygen the property of having an unpaired electron and consequently it shares with oxygen the ability to combine readily with radicals (organic or other-wise). HOWARD-FLANDERS48 found that N O sensitized bacteria to radiation to the same extent as oxygen. Anoxic bacteria irradiated in an atmosphere of 99 per cent N 2 and 1 per cent N O were as sensitive to x-rays as if they had been irradiated in air. N O also increases the radiosensitivity of anoxic bean roots49. T h e close relationship between N O and O 2 as a radiosensitizer adds further support to the v iew that oxygen acts b y adding to an organic radical though it provides no help for the search of the nature of the site. Al though the possibility that N O sensitizes by combining with porphyrins e.g. in reduced cytochrome oxidase) cannot be excluded on the basis of the present data. In dry spores N O protects (see p. 296).

Reversal of oxygen effect by inert gases—The discovery b y E B E R T and HOWARD51 that hydrogen and nitrogen under great pressures reduce the radiosensitivity of aerobic bean roots to the level of anaerobic bean roots (see Fig . 11-6), provides the most valuable clue towards the mode of action of the oxygen effect. In a later publication5 2 they showed that this effect is not confined to bean roots and is also seen with ascites cells ( T a b l e 11-3). T h e oxygen effect for both dominant and sex-linked lethal mutations in Drosophila has also been abolished in this way 5 6 . Moreover, other inert gases such as argon, krypton and xenon (see T a b l e 11-4) show

T H E E F F E C T O F O X Y G E N I N R A D I O B I O L O G Y

T A B L E 1 1 - 3

295

E F F E C T OF N I T R O U S O X I D E AND I N E R T GASES O N C H R O M O S O M E A B N O R M A L I T I E S PRODUCED BY X-RAYS I N ASCITES C E L L S 5 3

Dose in r to Experi- Gas mixture during exposure to reduce normal Relative*

ment x-radiation anaphases to radio-31 -6 (per cent) sensitivity

1 1 atm. of air 130 ± 10 3-5 ± 0-09 2 0-2 atm. of oxygen + 0-8 atm. of

nitrous oxide 500 ± 20 0-91 ± 0-02 3 1 atm. of air + 95 atm. of argon 455 ± 20 1-0 ± 0 04 4 1 atm. of air + 11 atm. of nitrogen 330 ± 15 1-4 ± 0-02 5 1 atm. of air + 2 atm. of xenon 380 ± 15 1-2 ± 0 02

* Radiosensitivity in absence of oxygen taken as 1.

3 0

2-5

J 2-0

•£ 2-5

\ \ \ \ \ \

i \ \ \A \ ?

\ N 2

N 1M \

} J IO 15 20 25 5 Q 75 100 125

Pressure ( a t m ) added to I a t m of air

FIG. 11-6. x-ray sensitivity of bean roots irradiated in 1 atm. of air plus nitrogen or argon (radiosensitivity relative to anoxic

conditions).62

this effect to an even greater extent52. T h e most effective gas for reversing the oxygen effect was nitrous oxide ( N 2 O ) (see T a b l e 11-3), as this need not be used at super-atmospheric pressures53. However, the need to apply the other gases at high pressures does not introduce any theoretical complications since careful control experiments have shown that these pressures alone do not affect either the ascites cells or the bean roots. T h u s the rate of oxygen consumption (the Oo 2 ) is uninfluenced b y these high pressures. T h e only effect seems to be that the radiosensitivity of the


T A B L E 1 1 - 4

EFFECTIVENESS o f VARIOUS GASES IN R E D U C I N G THE O X Y G E N - D E P E N D E N T RADIOSENSITIVITY OF Vicia faba

R O O T S 5 2 .

Gas Pressure in atm.

to reduce the effect of oxygen by 50%

Partition coefficient of the gas between

lipid/water

Helium 5 5 0 1-7 (37°C) Hydrogen 55 0 2-3 (22°C) Nitrogen 12-5 3-5 (22°C) Argon 2-0 4 0 (22°C) Krypton 2 0 7-5 (22°C) Xenon M 14-5 (22°C)

cells cannot be increased by oxygen. E B E R T et al.52 were impressed by the fact that the pressure needed to abolish the oxygen effect was inversely related to the oil solubility of the gases. T h e greater the partition coefficient between oil and water the lower the pressure needed. N o definite inter-pretation of these facts is possible. One would not expect that the amount of dissolved oxygen in the cells would be altered b y the presence of these inert gases (Henry's law). Interference with absorption is also unlikely in view of the very high affinity of oxygen for the site at which it reacts to give the radiation lesion. E B E R T et al.52 suggest that the sensitive site is protected b y a layer of inert gas.

The oxygen effect in dry systems: In cells which are wet and swollen with water the radiochemical reactions are complete in a small fraction of a second and if oxygen is to take part in these radiochemical reactions it must be present during irradiation. In dry materials such as seeds and spores radicals are trapped and persist for days (see p. 119). In these dry systems enhancement of radiation damage can therefore be obtained by exposure to oxygen after anaerobic irradiation as was demonstrated by CURTIS et al. (60) with seeds and by POWERS61 with bacterial spores. Nitric oxide does not however, in this case sensitize and POWERS61 found instead that post irradiation exposure of spores to nitric oxide prior to exposure to oxygen reduced radiation damage. T h e interpretation being that trapped radicals in the spores were "protected" by reaction with nitric oxide against peroxidation by oxygen. T h e latter reaction being held responsible for the enhancement of the radiation damage by oxygen.

R E F E R E N C E S

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3 8 , 289 8 . EHRENBERG, L . , MOUTSCHEN, J. and MOUTSCHEN, M . , Acta Chem. Scand.,

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103 13. HOLLAENDER, A . , BAKER, W. K. and ANDERSON, E. H., Cold Spring Harbor

Symp. Quant. Biol., 1951, 16, 103 13a. HOLLAENDER, A . , STAPLETON, G . E . a n d BURNETT, W . T . , Ciba Foundation

Conf., London, 1951 1 4 . GILES , N . H. and R ILEY, H . P . , Proc. Natl. Acad. Sci., U.S., 1 9 5 0 , 3 6 , 3 3 7 1 5 . NEARY, G . J . , Science, 1 9 5 7 , 1 8 0 , 2 4 8 16. BEAM, C. A., U.C.R.L. 3096, 1955 17. NAKAO, Y., Progress in Radiobiology, Oliver & Boyd, Edinburgh, 1956

p. 363 18. A L P E R , T . and G I L L I E S , N . E., Nature, 1958, 1 8 1 , 961 1 9 . W R I G H T , E . A . , Acta Radiol., 1 9 5 7 , 4 8 , 2 6 2 0 . DESCHNER, E . E . and GRAY, L . H . , Radiation Research, 1 9 5 9 , 1 1 , 1 1 5 2 1 . HOWARD-FLANDERS, P . and M O O R E , D . , Radiation Research, 1 9 5 8 , 9 , 4 2 2 2 2 . GRAY, L . H . , CONGER, A . D . , EBERT, M . , HORNSEY, S . a n d SCOTT, O . C . A . ,

Brit. J. Radiol., 1953, 26, 638 23. G U N T H E R , S . E. and K O H N , H. J . , / . Bacteriol., 1956, 72, 422 2 4 . SCOTT, O . C . A . , Brit. J. Cancer, 1 9 5 7 , 1 1 , 1 3 0 2 5 . HOWARD-FLANDERS, P. and ALPER, T . , Radiation Research, 1 9 5 7 , 7 , 5 1 8 26. GRAY, L. H., Lectures on the Scientific Basis of Medicine, Vol. 7, p. 314,

University of London, 1957 27. EVANS, H . J . and NEARY, G . J . , Radiation Research, 1959, 11, 636 28. LONING, K. G., Progress in Radiobiology, Oliver & Boyd, Edinburgh 1956,

p. 350 29. EVANS, H . J . , NEARY, G. J . and T O N K I N S O N , S . M., Nature, 1958, 1 8 1 , 1083 30. SWANSON, C. P., Radiobiology Symposium, Liege, Butterworths, London,

1955, p. 254 31. BAKER, W. K., Mutation, Brookhaven Symp. in Biology, No. 8, 1956, 191 3 2 . T R O W E L L , O . A . , Brit. J. Radiol., 1 9 5 3 , 2 6 , 3 0 2 33. TROWELL, O. A .,J. Path. Bact., 1952, 64, 687 3 4 . CAMPBELL, J . A . , Physiol. Rev., 1 9 3 1 , 1 1 , 1 3 5 . M I L L I K A N , G . A . , Physiol. Rev., 1 9 3 9 , 1 0 9 , 5 0 3 3 6 . LACASSAGNE, A . , C.R. Acad. Sci., Paris, 1 9 4 2 , 2 1 5 , 2 3 1 37. HAJDUKOVI6, S . , HERVE, A. and VIDOVIC, V . , Experientia, 1954, 10, 343 38. HORNSEY, S., Advances in Radiobiology, Oliver & Boyd, Edinburgh, 1957, p.

248 3 9 . STORER, J . B . and HEMPELMANN, L . H . , Am. J. Physiol., 1 9 5 2 , 1 7 1 , 3 4 1 4 0 . D O W D Y , A . H . , BENNET, L . R . and CHASTAIN, S, M . , Radiology, 1 9 5 0 . 5 5 ,

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4 1 . T H O M L I N S O N , R . H . and GRAY, L . H . , Brit. J. Cancer, 1 9 5 5 , 9 , 5 3 9 4 2 . CHURCHILL-DAVIDSON, I . , SANGER, C . and T H O M L I N S O N , R . H . , Brit. J.

Rad., 1957, 3 0 , 400 43. KIMBALL, R . F . , Ann. N.Y. Acad. Sci., 1955, 59, 638 44. GRAY, L. H., Radiation Research, 1954, 1, 180 4 5 . ALEXANDER, P . , Fox, M., ROSEN, D . and STACEY, K . A . , Nature, 1 9 5 8 46. BACHOFER, C. S. and POTTINGER, J. Gen. Physiol., 1956, 4 0 , 289 47. EPHRUSSI-TAYLOR, H . and LATARJET, R. , Radiobiology Symposium, Lifege,

1954, Butterworth, London, 1955 4 8 . HOWARD-FLANDERS, P . , Nature, 1 9 5 7 , 1 8 0 , 1 1 9 1 4 9 . K I H L M A N , B . A . , Exptl. Cell Research, 1 9 5 8 , 1 4 , 6 3 9 5 0 . GRAY, L . H . , GREEN, F . O . a n d HAWES, C . A . , Nature, 1 9 5 8 , 1 8 2 , 9 5 2 5 1 . EBERT, M . and H O W A R D , A . , Radiation Research, 1 9 5 7 , 7 , 3 3 1 5 2 . EBERT, M . , HORNSEY, S . a n d HOWARD, A . , Nature, 1 9 5 8 , 1 8 1 , 6 1 3 5 3 . EBERT, M . and HORNSEY, S . , Nature, 1 9 5 8 , 1 8 2 , 1 2 4 0 5 4 . GERSCHMAN, R . , Proceedings of list Intern. Congress of Physiological Sciences,

Buenos Aires, 1959, Symposia and special lectures, p. 222 5 5 . GERSCHMAN, R . , GILBERT, D . L . , D W Y E R , P . and F E N N , W . O . , Air Univ.,

School of Aviation Medicine, U.S.A.F., Randolf, Texas, June 1956 5 6 . C H A N G , T . - H . , W I L S O N , F . D . and STONE, W . S . , Proc. Natl. Acad. Sci.,

U.S., 1959, 4 5 , 1397 57. CATER, D . B. and SILVER, I. A., Acta Radiologica Stockholm, 1960, 53, 233 58. L I N D O P , P . J . and ROTBLAT, J . , Nature, 1960, 1 8 5 , 593 59. TARUSSOF, N., Radiobiology Symposium, Moscow, 1960, to be published by

Academic Press, London 6 0 . CURTIS, M . J . , DELIHAS, N . , CALDECOTT, R . S. and KONZAK, C . F . , Radiation

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Academic Press, London

C H A P T E R 1 2

Comparative Radiosensitivity of Living Organisms

EVERY degree of radiosensitivity, ranging f rom 0 -01 r, required to modify the growth of the fungus Phycomyces Udkesleeanusi to 3 x IO5 r, to kill infusoria, is to be found among living organisms. It is not understood at all w h y some organisms are so sensitive, and w h y others are resistant to doses which destroy proteins and D N A and should induce immediate "molecular death". Tables 12-1 and 12-2 summarize m u c h of what is known at present. T h e ideal would be to have a complete curve of survival for each animal, ranging from non-fatal doses to doses which kill during irradiation, similar to the curves drawn for mice b y BONET-MAURY and PATTI1 and b y RAJEWSKY2, instead of only the LDso/30 days* shown in T a b l e 12-1. T h e figures in Tables 12-1 and 12-2 are, of course, only approximate. It is well known that different pure strains of mice or rats show different degrees of radiosensitivity48, that slight variations exist even within a single pure strain, and that the physical conditions of irradiation and the methods of measuring doses differ in various schools. Results obtained b y different authors are therefore seldom comparable, but certain conclusions can be drawn:

(i) A m o n g the vertebrates, mammals are more sensitive to radiation than birds, fishes, amphibians or reptilest. T h e LD50/30 days of various adult mice, rats and golden hamsters all fall within a range of about 125 r ( ~ 5 5 0 to 675 r)49. T h e radiosensitivity is very high in new-born mammals; it decreases until full adulthood is reached and then remains constant; old mice (above 600 days) are again more radiosensitive50-51. T h e variation of radiosensitivity with age in a strain of mice is shown in Fig. 12-1 , p. 302.

It is easy to understand the difference between mammals and cold-blooded vertebrates, because the latter live at lower temperatures; in frogs and newts, the rate of development of the radiation lesions increases

* T h e dose of irradiation which kills 50 per cent of the animals within 30 days, f T h e amphibia have the advantage that their embryology is well known and

that they can live at various temperatures, and are thus very suitable for certain exper iments 6 - 8 .

299


T A B L E 1 2 - 1

L E T H A L D O S E FOR 5 0 PER CENT OF VARIOUS A N I M A L S I N 3 0 D A Y S

after R U G H 0 , T O B I A S , T H O M P S O N , J . F . 6 4 and others35-38 '92 '93 '07*

Organism Radiation L D 5 0 i n r e m

Guinea-pig x-rays 250 (175 to 400) Pig x-rays 350 to 400 Pig y-rays 618 Dog x-rays 335 Goat x-rays 350 Monkey x-rays ~ 6 0 0 Burro (Equus asinus) •y-rays 585 to 784

(various sources) Man x-rays 600 to 700 Mouse x-rays 550 to 665

Fast neutrons 54 n a-rays (radon) 14 nc/g

^-rays 250 Mc/g Rat x-rays 665 (590 to 970) Rabbit x-rays 750 to 825 Hamster x-rays 610 Chicken x-rays 600 to 800 Goldfish x-rays 670 Carassius (fish) x-rays 1800 (at 18 0C) Frog x-rays 700 Newt x-rays 3000 Tortoise x-rays 1500 Snail x-rays 8000 to 20,000

Escherichia coli x-rays 5600 Yeast x-rays 30,000 Amoeba x-rays 100,000 B. mesentericus x-rays 150,000 Colpidium, Paramoecium

and Infusoria x-rays / 3 0 0 , 0 0 0 \ 3 50,000

* For data concerning calves see refs. 53 and 61.

with the temperature (see p. 3). In birds, however, the body temperature and metabolism are higher than in mammals. W o r k done with pigeons shows that these birds are five times less sensitive to radiations than adult rats, and that in them the liver and kidneys are as much affected as the intestines, spleen, bone-marrow and sex glands, which is not true of mammals2 3 . It would be very useful to study other birds as well.

(ii) Unicellular organisms are generally very resistant to radiations (Table 12-1), but there is at least one exception3, Phycomyces blakesleeanus, which reacts to a dose as low as 0-01 r.

C O M P A R A T I V E R A D I O S E N S I T I V I T Y O F L I V I N G O R G A N I S M S 3 0 1

T A B L E 1 2 - 2

EFFECTS OF X-IRRADIATION ON C E R T A I N INVERTEBRATES (PEARSON)*

Phylum Genus Dose (kiloroentgens)

Effect

Ctenophora Mnemiopsis 1-2 Shrinkage, loss of turgidity 2-4 Partial disintegration 4-8 to 16-8 Complete disintegration of

all specimens Annelida Enchytraea 90 in 3 doses N o apparent effects

Mollusca Radix japonica 2 LD5O at 80 days (adult) 12 LD50 at 20 days

Radix japonica 1 Reduced activity (young)

Radix japonica 7-2 (estimated) Prevented formation of all (eggs) embryos

8 -8 (estimated) Prevented formation of all veligers

10-4 (estimated) Prevented formation of all trochophores

Thais (adult) 17 L D 5 0 at 80 days 20 LD50 at 5 days

Arthropoda Amphipod (adult) 0-6 LD50 at 80 days Amphipod (young) 0-56 LD5O at 10 days Artemia (young 93 LD50 at 5 days

"dry") 50 LD5O at 10 days Artemia (young 80 LD5O at 5 days

"wet") 20 LD5O at 10 days 10 Reduced size after 5 days

Daphnia magna 6-5 Killed all individuals Corethra (Chaor- 0-5 to 3 Midgut cells loosened

borus) larvae 5 to 10 Degeneration of pericardial cells

> 1 0 and < 2 5 LD5O at 30 days

Aseidian Molgula 45 LDioo (ref. 56) Nematode Ditylenchus 48 to 96 Inactivation dose (ref. 57)

saprobes Heterodera 10 to 20 Inactivation dose (ref. 58)

* Some data are also available in ref. 55.

Microorganisms are being increasingly studied as they offer great technical advantages, e.g. in the study of chemical protection and restora-tion after irradiation. Hollaender, Latarjet, T o b i a s and many others are


using strains of Escherichia coli, lysogenic bacteria, and yeasts respectively for experimental convenience.

T h e type of media used to culture yeast cells or E. coli prior to irradia-tion determines to a great extent the sensitivity of these cells to ionizing radiation as well as the response to recovery factors (see p. 282)46. T h e lysogenic systems (for instance, that of E. coli K 1 2 ) seem to be highly sensitive to ionizing radiations since exposure to doses of about Ir are sufficient to induce observable effects6 5. T h i s fact should not be forgotten when one tries to evaluate the hazards of small doses or small dose-rates.

iooo

900

800

o in Q -I

700

0 20 40 60 80 100

Age, weeks

FIG. 12-1. LD50 for SAS/4 mice at 30 days as a function of age64.

T h e r e are very great variations in the radiosensitivity between different bacteria. W h e n irradiated with 140 k V x-rays in phosphate buffer under conditions of adequate oxygenation the LD63 of Pseudomonas fluorescens is 1-7 kr, of E. coli 4-0 kr while for Micrococcus sodensis it is 35-5 kr. The dose response curve for the latter is not exponential but shows a pro-nounced threshold. Spores are even more radioresistant than the micro-cocci7 0 .

T h e radioresistance of the ciliated Infusoria of the family Ophryo-glenidae varies according to the species (Table 12-3), the stage of devel-opment and the state of nutrition. Starving "therontes" are always the most sensitive to radiations. T h e higher the resistance to radiations, the higher is also the resistance to cyanide (BACQ, MUGARD and HERVE16).

(iii) Insects seem to be rather resistant but less so than unicellular organisms. M a n y advantages offered b y insects in radiobiological investi-gation have not yet been exploited; for instance, some insects are resistant to cyanide, and can live anaerobically for a long time.


T A B L E 1 2 - 3

PARALLELISM BETWEEN RESISTANCE TO CYANI DES AND TO X-RAYS I N O P H R Y O G L E N I D A E ( T H E R O N T E S ) 1 6

Species Ophryo-glenida pectans

Deltopylum rhabdoides

Ophryo-glenida

atra

Ophryo-glenida

mucifera

Dose of x-rays lethal in 10 to 15 min in kr 600 750 900 1050

M a x . well tolerated concn. of N a C N 0-03% 0-05% 0-05% 0-2%

It is known that the adult Drosophila is m u c h more resistant than the y o u n g stages. For 3-hr eggs the LD50 is 200 r, for 4-hr eggs it is 500 r, for 7 | - h r eggs it is 810 r, and for pupae it is 2800 r 1 0 . Similarly, eggs of the wood-boring insects Anobium punctatum and Xestobium rufovillosum are killed b y an exposure to 4000 r if irradiated within 1 to 4 days of laying; 48,000 to 68,000 r are necessary to kill mature eggs of Anobium; after treatment of both sexes of Anobium, Xestobium or Lyetus brunneus with 8000 r, no fertile eggs were laid39. T h u s , irradiation may be the ideal technique to treat precious w o o d and wooden sculptures infested with insects. A d u l t Drosophila are resistant to 64,000 r in the form of y-rays f rom 6 0 Co, but are sterilized; eggs laid after irradiation with 32,000 r or 16,000 r are not viable. A dose of 8000 r allows the laying of many eggs after 4 days' inhibition, b u t few of these eggs develop 1 0 . V e r y similar quantitative data have been obtained concerning the radioresistance of eggs and adults of the mosquito Aedes aegyptii40.

SULLIVAN and GROSCH9 have described a significant increase in the length of life of a parasitic wasp (Habrobracon) after irradiation with a dose of 100,000 r in starvation conditions; females are said to be sterilized b y a dose of about 5000 r.

Some authors1 0-3 7 do not hesitate to recommend the use of ionizing radiation ( 6 0 C o or the waste products of atomic factories) to kill some insects* which consume large quantities of stored produce, or attack wool, clothing or wood. However, the figures given and the conditions in which the insects were bred show that their results must be regarded as prelim-inary. T o kill all these insects in 3 or 4 days, doses of 193,000 to 257,000 r are necessary. Lasioderma and Sitophilus which received 16,100 r did not die sooner than the controls, and 64,400 r is needed to kill all Rhizo-

* Attagenus (larvae and adults), Lasioderma, Rhizopertha, Tribolium, Dermestes (larvae and adults).


pertfui in 20 days. Other species of insects* studied by another group3 8

have shown similar sensitivity to 6 0 C o y-rays. Doses greater than 20,000 rep are required to prevent the emergence of adults f rom irradiated pupae; 50,000 rep produce no immediate "knock d o w n " effect on a d u l t s — a s d e s c r i b e d b y H A S S E T T a n d J E N K I N S 1 0 o r G R O S C H 4 7 . M a r k e d d i f f e r e n c e s

in the tolerance of various species are observed, but 13 species (out of 17) are sterilized at or near 6000 rep38. T w o important pests, the flour beetle Triboliitm confusum59 and Calandra granaria60 have been particularly well studied. T h e life-span of Tribolium is extended b y a single dose of 3 kr ( 1 3 7 C s y-rays) or daily doses of 100 r; the lethal dose at 50 days is about 20 kr for this species59. A single dose of 5 -7 kr kills 95 per cent of adult Calandra60.

T h e dose of x- or y-rays needed to kill an insect is thus about 100 times greater than that needed to kill a mammal.

(iv) T h e other invertebrates are less well known. T h e larvae of Trich-inella spiralis, an intestinal and muscular parasite of many mammals, are resistant to 3250 to 3750 r but they no longer thrive after ingestion b y rats, remaining confined to the intestines11.

Adul t Artemia salina (a primitive crustacean living in highly concen-trated sea water) males are killed b y 200,000 r of x-irradiation, females b y 150,000 r ; doses quite comparable to those needed to kill insects. T h e sterility dose at 2000-3000 r for females is lower than in insects b u t higher than in grasshoppers34.

Embryos of the American squid (Loligo pealii) irradiated before emerg-ing from the ovum, do not succumb to a dose of 200,000 r of x-rays until the fourth day. Irradiation with 50,000 r causes swelling of the capsule of the ovum and liberation of all the embryos. T h e positive phototropism of many embryos is abolished b y 10,000 r and growth is stopped b y 5000 r, yet the muscles of the chromatophores continue to pulsate regu-larly even after 200,000 r. T h e most radiosensitive organ of the squid seems to be the lens12.

T h e Coelenterates, except the hydrae, are apparently resistant to cyanide (0-4 per cent); they are also radioresistant; about 300,000 r (40 k V ) are needed to cause complete killing of the actinid Anemonia sulcata. T h e death rate after 100,000 r does not differ significantly f rom that of the controls13. O n the other hand, the hydrae, which are much more sensitive to cyanides, are also much more sensitive to radiations 1 4 ' 1 5; 4000 to 6000 r of 50 k V x-rays kill Hydra fusca in 20 to 40 days at 19°C. If brown hydrae are irradiated (9600 r measured in water b y chemical methods, 50 k V unfiltered radiations) there is severe degeneration of the

* Calandra, Rhizopertha, Tribolium, Oryzaephilus, Trogoderma, Callosobruchus, Laemophloeus, Ephestia, Sitotroga.


tentacles after 24 hours in 28 out of 30 animals1 5. T h i s is an indirect effect; apparently the irradiation of the water (or weak saline solution) in the presence of oxygen causes the formation of peroxides which are toxic for hydrae 1 5 .

D r P. B. Pearson, Chief of the Biology Branch, Division of Biology and Medicine, U . S . Atomic Energy Commission, kindly communicated to us the interesting data known to him. T h e y are collected in Table 12-2. Further research with invertebrates is desirable, as it would be useful to understand their resistance to ionizing radiations.

T h e ova and spermatozoa of invertebrates have always been widely used in biological work. Those of a lamellibranch (Spisula solidissima)19

and those of an urchin (Arbacia)20 are very resistant to radiations, es-pecially when concentrated, for it is usual to employ doses of 100,000 to 300,000 r when working with these animals. In 1940 EVANS and his collaborators21 demonstrated the great influence of the medium on the results of irradiation of the spermatozoa of Arbacia punetulata. T h e greater the concentration of the sperm, the greater is its resistance to radiations*. T h e seminal fluid in which the spermatozoa are suspended protects them from the rays. Heavily irradiated sea water diminishes the fertility of spermatozoa. T h i s loss of fertility is certainly an indirect effect, as is the diminished rate of cell division in ova fertilized b y irradi-ated spermatozoa and may be due to peroxides22.

(v) T h e higher plants are also very variable as regards radiosensitivity. Some authors relate this to their ascorbic acid content (see p. 319), and others believe that the fragility and length of the chromosomes are of fundamental importance.

T h e radiosensitivity of dry seeds varies f rom 2000 to 64,000 r (x-rays). L i ly seeds (Lilium regale) show practically no g r o w t h ! after 2000 r, whereas the seeds of cabbage and radish are practically unaffected b y 64,000 r. In many cases a small dose (2000 to 8000 r) stimulates growth 1 8 . M a n y examples have already been given of the radiosensitivity of the pollen of certain plants (especially Tradeseantia) (see p. 247), of the grow-ing roots of the onion (Allium eepa), and of the broad bean, which are the best material for certain biological experiments.

T h e algae are certainly very resistant to radiations as BLINKS17 found the whole algal flora were abundant and in good anatomical and physio-logical condition on the most radioactive reefs at Bikini. T h e only abnorm-ality found was a great increase in the catalase content of three genera (Halimeda, Udotea and Cladophoropsis). T h e question arises whether this is an enzymatic adaptation brought about b y the formation of large

* This is also true of the sperm of Nereis, a marine annelid21, t Seeds must be regarded as embryos.


quantities of peroxides when the bomb exploded. In the present state of our knowledge these enormous differences in radiosensitivity certainly cannot be explained, but certain hypotheses deserve brief discussion.

(i) T h e relation between resistance to radiations and resistance to c y a n i d e s , d e m o n s t r a t e d b y B A C Q , M U G A R D a n d H E R V E 1 6 , s u g g e s t s t h a t

the enzyme systems sensitive to cyanide are also the most sensitive to radiations. If this fact were demonstrated in insects and other inverte-brates, it would be an indication that the first enzyme systems to be affected are those containing heavy metals (Cu, Fe, etc.).

M i c e with a high respiratory quotient ( C O 2 output: O 2 consumption) suggesting active lipogenesis at the time of irradiation are more radio-resistant than mice showing a normal respiratory quotient6 9 .

(ii) T h e r e may be a relationship between radioresistance of a tissue and its dehydrogenase content52.

(iii) T h e resistance of adult insects to radiations cannot be interpreted as a result of the fact that cell division (mitotic activity) no longer takes place in these animals except in the sexual cells. T h e sexual cells of in-sects are, in fact, as resistant in comparison with mammalian sexual cells as the whole body. For example, female rats protected b y cysteamine are permanently sterilized b y 700 r whereas 64,000 r are nedeed to produce the same result in Drosophila; in this insect 8000 r causes sterility only after 4 days, whereas temporary sterility in mammals is produced b y doses of 150 to 200 r.

(iv) So far, no attempt has been made to correlate two facts which are now well established, (a) M o s t invertebrates maintain their intracellular osmotic pressure b y means of amino acids or small polypeptides*2 9-3 0. T h e internal environment of insects is very rich in amino-acids. Vertebrates, on the other hand, maintain their osmotic balance almost entirely b y the interplay of inorganic ions such as: Na+, K+, Ca++, Mg++, C l - , H C O 3 - , H 2 P O 4 - , S O 4 - .

(b) A s amino-acids have some protective action, it seems possible that at least a part of the resistance of invertebrates to radiations may be ex-plained b y this biochemical consideration, which might well form the basis of an experimental s tudyf .

In insects the oxygen supply is brought b y tracheae. It is possible that this anatomical arrangement results in a low oxygen tension in the tissues. Certain insects secrete large quantities of carboxylic acids (e.g. formic acid), which are good protectors against radiations.

* Sometimes even by means of amines, such as taurine in the cephalopods. t T h e fact that certain cancer cells are able to concentrate amino-acids31 might

affect their radiosensitivity. I t would be interesting to know the radiation resistance of the octopus since it has a gland containing hydroxytryptamine which was found on a weight basis to be the most effective protective agent known (see p. 463).


(v) T h e number of chromosomes is very important. A m o n g the yeasts, the diploid races are much less sensitive to ultra-violet and ionizing radiations of all kinds. Moreover, the mortality curve of diploid yeasts is sigmoid in form, whereas it is straight in haploid yeasts. T h i s fact has been very well observed b y LATARJET and EPHRUSSI24'25. One might think that the advantage possessed b y the polyploids is that they have several complete sets of chromosomes. In a haploid it is sufficient that one chromosomic region should be affected b y irradiation to produce serious cellular changes. A similar lesion in a diploid is less important, because the corresponding region remains intact in the other series of chromosomes. Figure 12-2 shows that in the genus Chrysanthemum radio-resistance to continuous irradiation is related to the ploidy.

T 1 I I I 1 1 I I I ! I 1 ] I I I I

DEATH RATES OF CHRYSANTHEMUM PLANTS AT 1340 r/d

o —I I i T i -i—i i i i i i i i i i i 25 35 45 55 65 75 85 95 105 115 125

DAYS OF CHRONIC IRRADIATION

FIG. 12-2. Death rates of chrysanthemum plants at 1340 r per day of chronic gamma irradiation (SPARROW71).

A fortunate accident has permitted a perfect control in Tradescantia paludosa: a single irradiated flower bud was found with a mixed popula-tion of haploid and diploid microspores; chromosome aberrations were exactly the same when calculated per chromosome4 4 , or per unit of chro-mosome length. T h i s relationship may not be true when chromosome damage is observed after exposure to densely ionizing radiations (fast neutrons for instance)45.

Analysis of the mortality curve of diploid yeasts suggests a two-hit phenomenon (see p. 66). T h e magnitude of the oxygen effect (see p. 280) is not affected b y the degree of ploidy4 1 . Doses which are lethal to 99 -9 per cent of haploid yeast cells still result in 50 per cent zygote survival; many cells receive damage which is lethal in the haploid state but non-lethal when incorporated into a diploid cell42. Budding yeast cells are much more radioresistant than resting ones; this fact may be associated with gene multiplicity during division or with unique biochemical properties of the nucleus at that time43.


In yeast cells, the radioresistance decreases f rom diploid to hexaploid2 6; it has been assumed that the majority of inactivation of haploid cultures is brought about b y recessive lethals, whereas higher ploidy cultures are inactivated mostly by dominant lethals26 '27.

T h e liver of rodents is more or less polyploid. T h i s is shown by the cytological studies of D'ANCONA and his collaborators32 as well as by the h i s t o p h o t o m e t r i c s t u d i e s o f P A S T E E L S a n d L I S O N 2 8 . S W I F T 3 3 h a s c o n f i r m e d

the existence of three types of nuclei in the liver, in which the D N A content increases b y doubling. In adult male rats tetraploids are numerous, whereas there are hardly any in new-born animals. T u m o u r cells are often mixed diploid-polyploid populations. It would be very interesting to discover whether polyploidy in the liver is not partly responsible for the greater resistance of adults to radiation.

T h e degree of radiosensitivity is genetically controlled; hybrids are often more resistant than pure strains66.

T h e immediate inactivation by x-rays of a catalase negative bacterium (E. coli H7) is the same as that of the catalase positive cells (grown with hemin which the catalase negative cells cannot synthesize); but after irra-diation for 1\ hr, there is a continuous inactivation of the catalase negative cells which are sensitized to the hydrogen peroxide present in the medium. T h e mechanism of this sensitization is not yet clear, but it is the first instance in which an effect of ionizing radiation is correlated with a genetically controlled biochemical property of a cell68.

Figure 12-3 shows that in plants which are diploid radiation resistance is inversely related to nuclear volume, i.e. the smaller the nucleus the

° C » O

\ X °

; O

O O O O

;

"! I I I t I I I I I I I I I I I I

\ I I I

III I IO 14 18 . 22 26 3 0 3-4 38 42 4 6

log of daily dose required to produce severe growth inhibition

FIG. 12-3. Relationship between nuclear volume and radio-sensitivity in 2 4 species of plants ( S P A R R O W and M I K S C H ,

unpublished).


larger the dose needed to inhibit growth. Since the size of the nucleus and D N A content are also related it may be that radiosensitivity is linked to the amount of D N A per cell. W e have obtained preliminary indication for a relationship of this type for bacteria7 0 although the D N A content in the more radiation resistant mutant of E. coli is the same as in the parent strain.

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Biological Sciences, 2, 351

C H A P T E R 13

Pathological Biochemistry of Irradiated Living Organisms

T o OBTAIN a clear idea of the relative importance of the many observations published, it must be remembered inter alia that if the effect of ionizing radiations themselves are to be studied, the only valid biochemical obser-vations are those made within a few hours of irradiation. T h e reasons for this are: (i) the fundamental primary chemical changes always precede the appearance of anatomically visible lesions*. N o biologist should forget that the maintenance of normal structure is the result, not the cause of chemical activity of the cells. Every effort must therefore be made to discover what is abnormal in an irradiated organism before the anatomists are able to identify anything, (ii) W h e n dead cells accumulate in the lymphoid tissues (thymus, lymph glands, spleen, etc.), as they do as soon as 2 hr after irradiation, it is of little use to make accurate chemical determinations. T h e living and the dead cells are mixed, and little more can be learned from chemical analyses than can be seen with the microscope, (iii) It is useless to carry out systematic determinations of any substance for days or weeks when it is characteristic of a type of cell which can be counted. For example, the blood glutathione after an irradiation follows closely the variations in the number of red cells21. A l l the glutathione is in the red corpuscles, and chemical determinations tell us no more than an erythrocyte count. W h a t is interesting is to know whether there is a decrease in the reduced glutathione in the blood or tissues immediately after irradiation. T h e r e is no such decrease18, (iv) A s several authors have already noticed (see especially ORD and STOCKEN1),

* Tha t the protoplasm is chemically affected by ionized radiations very rapidly before the appearance of any anatomical alterations visible at the microscope, is proved by a simple test. A vital colour, acridine orange, is more concentrated in irradiated ascite cells which, in ultra-violet light, emit a red fluorescence; normal controls are green. This effect (Strugger's effect) is observed after 500-750 r of 60Co rays, more easily if the irradiation has been done in vivo than in Vitrolii. T h e underlying mechanism is probably again increased permeability (see p. 414); there should be great advantages indeed if quantitative methods could be developed with such vital staining techniques as is being attempted by M E I S E L et al.30S 311


the biochemical study of supposedly radioresistant tissues (especially the liver and kidney) is more valid because there is less interference b y ordinary gross anatomical reactions, (v) Biochemical investigation immediately after irradiation is advantageous in every w a y : the animals are still in good condition, suffering neither from shock, infection, nor from the severe malnutrition caused b y vomiting, anorexia and diarrhoea. In the final stages of radiation sickness all observations are invalidated by these non-specific disturbances, because the body reacts against them, and these secondary reactions interfere with the fundamental effects of the ionizing rays. Our attention is therefore concentrated on those researches in pathological biochemistry which show what happens immediately or very early after irradiation*.

O X Y G E N C O N S U M P T I O N Oxygen consumption is not affected in the guinea-pig by a whole-body dose of 250 r of x-rays (LD30)3. In starving rats, the basal metabolism is somewhat higher after irradiation than in fasting controls; there is no difference if the rats are not starving4. According to MOLE5 there is no significant rise until 4 days after irradiation with 800 r, a lethal dose. T h e earlier observation of KIRSCHNER et al.G that the basal metabolism is increased b y 30 to 60 per cent during the first 24 hr after a lethal irradia-tion, has not been confirmed. MOLE5 observed no change in oxygen con-sumption in rats after a dose of 1000 r, nor is this changed in minced brain of mice either immediately or 190 hr after a whole-body irradiation with 500 r 1 3 0 . In anesthetized rhesus monkeys, O 2 consumption during and immediately after irradiation (100 r/min) is slightly increased b y 1000 r, shows no changes with 2500 r and is decreased b y larger doses1 4 3. Respir-ation of dormant dry barley seeds (irradiated dry and germinating at 20°C immediately after irradiation) is not affected by 2500 r of x-rays; doses of 5000 to 15,000 r stop, after 4-5 days only, the normal increase of O 2 consumption observed in controls 1 3 1 on germination, x-ray doses of 10 kr and above, reduce the O 2 consumption of the eggs of a grasshopper (Chortophaga viridifasciata) in the first hour after treatment 1 4 7 . A t least 100 kr are needed to decrease significantly the O 2 consumption of surviving rat liver slices145 '228, although slices of various experimental tumours also irradiated in vitro and tested immediately do consume less O2 after 10 or 20 kr 1 4 6 '2 2 8 . X-rays in doses over 25 kr cause a progressive increase in O2 consumption of rabbit kidney cortex slices in a manner similar, but not

* ERRERA, M., "Effets biologiques des radiations, Actions biochimiques" in Protoplasmatologia (Edited by Weber and Heilbrunn), Springer, Vienna, 19 5 7152; HEVESY, G. and FORSSBERG, A . , Congres Intern. Biochim., Bruxelles, 1 9 5 5 1 5 3 ; HOLMES, B . E., Ann. Rev. Nucl. Sci., 1 9 5 7 , 7 , 8 9 .

B I O C H E M I S T R Y O F I R R A D I A T E D L I V I N G O R G A N I S M S 3 1 3

identical with, that of dinitrophenol—an agent which interferes with oxidative phosphorylation (see p. 3 3 2)149. T h e same yeast cells may respond to the same x-irradiation (90 kr, 250 k V ) in a great variety of ways accord-ing to their physiological conditions and the medium on which they are cultivated; for instance, in potassium phosphate buffer ( p H 4-5) the res-piration is increased 11 per cent and fermentation 38 per cent, while starved cells in potassium free buffer (pH 4-5) respond b y a decreased respiration (— 49 per cent) and fermentation (— 50 per cent)1 6 5 .

Thus , it seems that the principal enzymatic and hormonal systems which regulate metabolism and cellular respiration are not greatly affected b y small or medium doses of x-rays; or if certain enzymatic processes are blocked b y the irradiation, alternative mechanisms immediately come into play.

Anatomical changes in the thyroid and considerable variations in its uptake of iodine, have been described after irradiation1, in particular there is an increase two hours after irradiation. T h e significance of this is not clear, as this is not accompanied b y a general increase in metabolism 1 2 2 . It appears that irradiated animals which survive irradiation have a more active thyroid, whereas the opposite is observed in dying animals; castra-tion prevents the thyroid hyperactivity 1 2 3 . Some workers claim that hypothyroidy increases mortality, anaemia and leucopenia1 2 4 , but these facts have not been encountered b y other authors123-125. A detailed biblio-graphy of morphological changes of the thyroid after irradiation is given b y C O M S A 1 2 6 .

C A R B O H Y D R A T E M E T A B O L I S M A F T E R I R R A D I A T I O N T h i s aspect of radiobiology was reviewed b y O r d and Stocken in 1953, and we shall add a few comments and analyse some of the more recent researches.

Enormous doses of x-rays (110,000 r, survival 3 i- hr) clear the liver of its glycogen reserves8. O n the other hand, a moderate dose of x-rays (about 300 r) in rats which were kept starving before and after irradiation brought about a considerable and steady increase in the glycogen content of the liver during the hours following irradiation9 ' 1 0 , 2 9 0 . T h i s accumu-lation of glycogen in the liver is inhibited b y the injection of cysteamine (mercaptoethylamine) or ingestion b y mouth of cystamine1 0 . In fasting rats, 1 4 C incorporation from acetate 1 4 C is increased during the 2 hr following irradiation (1500 r of x-rays) 1 5 0 . T h e weight of the liver in the rat increases strikingly after irradiation, already 6 hr after 200 r; water, lipids and glycogen contribute to this increase in liver mass2 9 7 .

Since it is known that cortisone increases glycogen accumulation in the liver the interference of the pituitary-adrenal system, stimulated by


stress, cannot be excluded 1 2- 1 0 (see also p. 394). HERBERT and MOLE7 0 , MCKEE and BRIN202 observed that adrenalectomy prevents the rise in liver glycogen in the irradiated rat. D u r i n g the first 2 hr after a dose of 2000 r in mice, the liver takes up three times as much 1 4 C in glycogen or a precursor as the controls after a subcutaneous injection of labelled glucose1 3 . In mice irradiated with 2500 r there is also an increase of the incorporation of 1 4 C in the fraction of the hepatic lipids, the metabolism of which is very rapid. T h e decrease (20 per cent) of the peripheric con-sumption of glucose b y the irradiated mouse7 is not enough to explain these facts. LOURAU13 thinks that catabolism of the proteins, notably those derived from the intestine, might produce excess amino-acids which then become an important source of hepatic glycogen. In Fischer's experiments 1 0 the material used in the synthesis of glycogen certainly did not come from the food, since the animals were kept starving. T h e total nitrogen in the liver is not changed after irradiation. T h u s it seems improb-able that the elements necessary for this synthesis of glycogen are obtained b y increased break-down of liver proteins. It is therefore probable that the metabolites incorporated in the liver glycogen during the hours following irradiation are brought to the liver by the blood.

LOURAU and LARTIGUE11 found deficient glycogenesis in guinea-pigs 1 2 - 1 4 days after a non-fatal irradiation (500 r) and put forward the hypothesis that irradiation permits the synthesis of glycogen from ali-mentary carbohydrates but inhibits synthesis from smaller molecules, a situation very different from that seen during the first 24 hr after irradia-tion. ORD and STOCKEN1 confirm that the deposition of glycogen in the liver of the guinea-pig usually occurs 24 hr after irradiation, but not 6-8 days later.

T h e r e seems to be no great change in the absorption of glucose b y the intestine, in spite of the bad condition of the intestinal mucosa shortly after irradiation*. Glucose absorption only becomes slower during the 24 hr which follow irradiation37. In rabbits there is distinct hyper-glycaemia reaching a maximum 2 - 4 hr after a dose of 500 r, the blood sugar returning to normal in 24 hr. Af ter 1000 r the increase in plasma glucose reaches 90 per cent45. After 2000 r hyperglycaemia amounting to 3-5 g/litre is observed. T h i s hyperglycaemia is easily blocked by insulin46.

* In mice there are distinct lesions in the epithelium of the small intestine after less than an hour. Regeneration is evident by the 3rd day and this is perhaps the tissue which regenerates most quickly. In fact, most mice which have received a fatal dose (700-800 r) die after the intestinal epithelium has already achieved a measure of regeneration. After careful study of glucose absorption by the rat's intestine in vitro after irradiation of the whole body, DETRICK et al.4'1 concluded that "histological recovery f rom irradiation injury is no guarantee of a physio-logically functional tissue".


Hyperglycaemia is also observed in fasting rats 24 and 48 hr after whole-body x-irradiation; at 48 hr the blood sugar level was in every case greater than that of fed, normal rats290. Early hyperglycaemia is also seen in guinea-pig 1 0 0 , and is more marked if the liver is irradiated directly3 7 . In dogs, increase in blood sugar produced b y the ingestion of 1 g/kg of glucose is less pronounced for 3 days after irradiation with 450 r, which is probably due to slower intestinal absorption4 7 '3 7 ; the concentration of lactic acid and pyruvic acid in the blood is less than usual3 7.

T h i s last observation is surprising, for if certain enzymatic systems were blocked in the carbohydrate cycle one or other of the metabolites should accumulate; an increase in bisulphite-binding substances* has been reported in the urine of irradiated cancer patients38 and in the

Immediately after irradiation (1-45 kr) of a suspension of Yoshida ascites cells, the aerobic glycosis is more depressed than anaerobic glycosis; respiration is very little affected. W h e n the steady-state concentrations of various metabolites are measured in these cells, one finds after 25 kr that there is a slight increase of A D P (adenosinediphosphate), a large increase of dihydroxyacetone phosphate and of fructose 1,6-diphosphate contrast-ing with a decrease of A T P , inorganic phosphate and pyruvate; triose-phosphate dehydrogenase is not inhibited2 9 9 , although logically it is at the level of the triosephosphate that a block exists. T h e level of an important coenzyme, D P N (diphosphopyridine nucleotide), is found considerably decreased immediately after irradiation. T h e r a t e of glycolysis of the ascites cells depends on D P N concentration. Several experiments show that the decreased glycolysis after irradiation is caused by the lowered D P N level, for instance, the inhibition of glycolysis in irradiated cells is abolished b y increasing nicotinamide concentrations which increase D P N level in the cells299. T h e decrease of A T P after irradiation is a consequence of the reduced glycolysis, and all the many reactions in which A T P furnishes the irreplaceable energy (synthesis of protein, D N A , R N A , etc.) are slowed down. T h i s long series of experiments b y Maas and his associates is impressive and logical299. HUG and SCHACHINGER298 have studied the influence of x-irradiation on a coupled D P N enzyme system.

Reaction:

* Pyruvic acid, acetaldehyde, methylglyoxal and generally all substances having an aldehyde or ketone group combine with bisulphite.

blood2 6 3 .

C 2 H 5 O H + D P N ethylalcohol alcohol

dehydrogenase (ADH)

^ C H 3 C H O + D P N H aldehyde


is inhibited in the same way as D P N is inactivated; the enzyme is radio-resistant. T h e inactivation of D P N is reversible. T h e reverse reaction:

C H 3 C H O + D P N H — > C 2 H 5 O H 4- D P N

A D H

is activated b y a factor of 2-2 when the system is irradiated at the rate of 15,600 r/min; it proceeds well without enzyme under irradiation298.

In mice, glycaemia is normal for the first hour following a whole-body dose of 2000 r 1 3 . In the chick, there is no consistent hyperglycaemia after irradiation but in individual sick birds hyperglycaemia was frequently observed associated with liver haemorrhages 1 7 2 .

DUBOIS, COCHRAN and DOULL39 use fluoracetic acid, according to POTTER'S41 method to determine whether citric acid is formed in irradiated rats in the same way as in normal rats. A s has been shown b y the investi-g a t i o n s o f R U D O L P H P E T E R S , B U F F A a n d W A K E L I N 4 0 , fluoracetic a c i d i s

converted b y the body into fluorocitric acid, which blocks Krebs cycle at the citric acid stage, and this substance accumulates. In rats injected with fluoroacetate 20 min after irradiation and killed 3 hr later, there is a great fall in the citric acid content of the thymus and spleen (radio-sensitive organs) and also in the kidney*; the decrease being greatest 6 - 1 2 hr after irradiation.

T a b l e 13-1 shows that the irradiated pancreas has also lost its ability

TABLE 1 3 - 1

EFFECT OF X-RAYS ON THE ACCUMULATION OF CITRIC A C I D IN THE TISSUES OF RATS POISONED W I T H FLUOROACETATE

(After DUBOIS , COCHRAN and D O U L L 3 9 )

Organ fig Citric acid per g fresh tissue

Organ Controls 48 hr after 800 r

Ileum 736 (680 to 816) 147 (94 to 176) Pancreas 884 (704 to 1064) 76 (50 to 109) Testicles 168 (150 to 225) 79 (61 to 110) Skeletal muscle 88 (60 to 104) 58 (45 to 79) Liver 64 (44 to 71) 177 (100 to 384)

to form citrate, but that the heart and brain continue to accumulate the same quantity of citric acid. It is interesting to study the behaviour of the liver, in view of its importance in the reaction to radiation sickness.

* T h e citric acid in the kidneys comes mainly f rom the blood: the kidneys concentrate for excretion the citric acid produced in the other tissues.


T a b l e 13-1 shows that, unlike all other organs, the liver of male irradiated rats accumulates more citric acid than the controls after fluoroacetate poisoning. Normally the liver of the adult male and young female rat seems to accumulate little citric acid, whereas that of the normal adult female accumulates large quantities (up to 1 -5 mg/g) after an injection of fluoroacetate.

Using 1 4 C labelled methanol HEVESY133 has shown that neither the complete utilization of minute (0-3 fig/g body weight) nor that of large doses (530 ,Mg/g) of methanol is depressed b y exposure of mice to an x-ray dose of 1500 r immediately or one day before injecting the animals. In fact the metabolism of large doses of methanol is slightly increased133. T h e catabolism of ethanol is similarly unaffected 1 5 1 . M i n c e d tissues f rom rats killed 17 hr after a massive dose (5000 r) of x-rays on the whole body showed either decrease or increase or no change in formate utilization1 5 4. T h e ciliated protozoon Tetrahymena pyriformis after 300 kr of x-radiation oxidizes greatly increased amounts of acetate, but is unable to oxidize phenylalanine at the same rate as the controls1 3 5 .

D I S T U R B A N C E S I N F A T M E T A B O L I S M

Hydroperoxides Fats provide experimental opportunities in radiobiology which carbo-

hydrates do not. In vitro, unsaturated fatty acids (e.g. Iinoleic acid) are very sensitive to radiations, being oxidized to form hydroperoxides. A whole symposium has been dedicated to organic peroxides in radio-biology 1 5 5 . Hydroperoxides formed from lipids are more stable both in vivo and in vitro than hydrogen peroxide, and one might hope to find measurable quantities of organic peroxides immediately after irradiation in extracts of animals.

T h e difficulty is that the fatty tissues and even the natural lipids ex-tracted from mammals are much less sensitive to x-rays* than pure fatty acids in vitro, because of the presence of a-tocopherol (vitamin E) which is a powerful anti-oxidant. Susceptibility to autoxidation increases in irradiated fats 1 5 6 .

D U B O U L O Z et al.49 state that fat in the skin of rats contains peroxides after intense local irradiation b y ultra-violet light, or after "contact" irradiation with soft x-rays, whereas normal skin contains none. Some of the figures (expressed as IO""8 equivalents of oxygen) are: 2-25 immediately after intense ultra-violet irradiation; 0-58 immediately after 500 r x-rays; 1-16 2 hr after 3250 r. These findings cannot be interpreted as an indirect radiochemical oxidation brought about by free radicals, or b y the direct action of x-rays as: (i) the peroxide level increases with time (e.g. goes

* They are more easily affected by ultra-violet light.


up from 1-16 after 2 hr to 2-96 6 days after 3250 r)*; (ii) simple burns bring about the immediate appearance of more peroxide than is produced b y 5000 r and there can be no doubt that these substances are lipid peroxides5 1 and they readily oxidize the — S H of proteins5 3; (iii) a group of workers at Strasbourg5 4 and another at Harwell5 5 using Hartman and Glavind's method, determined the lipid peroxide-like substances in the fat of rats54 and in whole mice5 5 immediately after total exposure to 1000 r, and found 0-38 x 10~3 m-equiv./g of fat in the rat and 2-27 x 10~7 mole/g of mouse. T h i s surprisingly high value corresponds to an ionic yield of 84 if all the energy absorbed is used solely in this reaction. T h i s evidence

hr

FIG. 13-1. Changes in total fat of starving rats produced by 1000 r of x-rays54.

taken together leaves little doubt that this oxidation is not a primary effect of the ionizing radiation in vivo but an "end-ef fect" resulting from biochemical changes. If a-tocopherol inhibits the formation of peroxides, and the lipid peroxides formed b y ionizing radiations are the cause of cellular lesions, one would expect that injection of a-tocopherol or a diet rich in vitamin E would increase resistance to radiation. T h i s is not the case5 6 , 1 2 9 because, as we believe, a normal diet ensures satisfactory concentration of tocopherol in fat. It would be more instructive to look for a progressive decrease in resistance to radiation in the course of tocopherol deficiency, but this has not been tried. Rats fed with highly oxidized rancid fat but given a liberal amount of vitamins do not develop hypersensitivity to x-irradiation1 5 8.

Peroxides5 7 show "radiomimetic" properties (see p. 235), but many enzymes (e.g. carboxypeptidase) are not inhibited nor can some of the

* It takes 20 kr to observe immediately after irradiation, a clean increase in skin peroxides followed by a decrease at 6 hr4 8 .


cellular effect of x-rays be reproduced b y hydrogen peroxide. ALPER58

rightly draws attention to an error introduced b y the fact that most com-mercial hydrogen peroxide is contaminated with different stabilizers. These are not easily removed b y distillation, and considerably alter the biological effects of H 2 O 2 .

One wonders whether ascorbic acid plays the same anti-oxidizing part in aqueous systems as a-tocopherol plays in fat. In animals the excretion of dehydroascorbic and diketogulonic acids is increased after irradiation (see Chapter 12). In plants exposed to continuous non-lethal irradiation (5000 r per day) the ascorbic acid content falls for the first 2 days, and then increases again. There may be some relation between a high ascorbic acid content and radioresistance in green plants. T h e cabbage and gladiolus, which are highly resistant, contain 200-400 mg of ascorbic acid per 100 g of fresh plant, whereas cosmos, tobacco and henbane* which have little resistance, contain only 30-100 m g of ascorbic acid52. O n the other hand, Vicia faba is very radiosensitive (85 per cent growth inhibition by 140 r) and is rich in ascorbic acid. In animals the reverse appears to apply; the organs (ovaries, adrenals) and tumours richest in ascorbic acid are also the most radiosensitive. Attempts to protect animals and human beings against the effect of x-rays or to treat radiation sickness b y means of ascorbic acid, alone or together with cysteine, have sometimes been strikingly successful6 3 , but generally the results have been very variable64. These experiments should be repeated in animals such as guinea-pigs, some strains of which are unable to synthesize ascorbic acid.

Absorption and Transport of Lipids It is certain that there are disturbances in fat metabolism and transport

in irradiated animals. In rabbits, a large dose (1000-2000 r) of x-rays causes very considerable lipaemia ( + 600 per cent) in 24-48 hr, which disappears on the third day. T h e plasma is opalescent and creamy and its total lipid, phospholipid and cholesterol content is very high 6 0 - 6 1 ' 6 2 ' 1 6 1 . T h i s phenomenon, so striking in rabbits, is also seen in dogs and rats but at different times after irradiation, and the quality of the lipoprotein fractions may change as well as their quantity 1 6 2 . In mice, there is a decrease of high density lipoproteins 1 6 2; in guinea-pigs it is regularly found, but is less than in rabbits6 1 . In mice, LOW-BEER65 has always found a fall in blood cholesterol in the fourth hour after irradiation (300-1000 r); in guinea-pigs the cholesterol level rises 4-5 days after 200-600 r7 2 . T h e blood cells also become rich in fat, but the increase is not parallel with that in the plasma6 1 . These observations in rabbits, together with the hyperglycaemic acidosis observed, make up a picture

* Cosmos sulfureus, Nicotiana rustica, Hyoscyamus niger.


reminiscent of severe diabetes, and T a b l e 13-1 (p. 316) also suggests that the pancreas is injured. Insulin, which reduces the hyperglycaemia pro-duced b y irradiation, also reduces the lipaemia produced b y 1000 r, b u t after a dose of 2000 r insulin no longer affects the l ipaemia6 1 .

T h e absorption of fats and fat-soluble vitamins (especially vitamin A ) is reduced during the 24 hr w h i c h fo l low irradiation, and then becomes normal. T h e r e are general disturbances, b u t they vary f r o m one species t o a n o t h e r , a n d n o g e n e r a l t r e n d i s e v i d e n t . B A C Q , B U R G , C H E V A L L I E R

and HEUSGHEM54 have carried out a preliminary study in rats. T h e animals must be fasting in order to avoid the great variations, due to the very variable anorexia w h i c h fol lows irradiation. T h e reduction in total lipids and in a-tocopherol content, as well as the amount of unsaturation were compared in controls and irradiated animals (1000 r) in the whole b o d y (excluding the head, tail and gut). T h e a-tocopherol remains unchanged, in other words it is not destroyed b y irradiation and is not uti l ized to a greater extent. It becomes gradually concentrated, as the fat reserves are mobil ized. T h e total quantity of fats (22 g in a 200 g rat) falls rapidly during the first 70 hr after irradiation, both in the irradiated animals and in the controls. F r o m the 70th to the 120th hr the total fat continues to fall in the controls, d o w n to 4 g, b u t it remains at about 9 g in the irradiated rats. T h i s means that after the 70th hr, either the synthesis of fat is increased in irradiated animals, or the utilization of fat is decreased.

T h e proportion of unsaturated fatty acids wi th 2, 3 or 4 double bonds 5 4

does not vary either in the controls or in the irradiated animals. T h e s e acids play an essential part in metabol ism; arachidonic acid, in particular, is an important constituent of the erythrocyte membrane. Increased fecal fat formation is observed during the 2 days fol lowing total-body irradia-t ion (1500 r) of fasting rats; this fact is probably due to increased intestinal secretion 1 6 0 . Injected l ipoprotein is metabol ized b y x-irradiated rabbit (800 r) as rapidly as b y normal animals 1 5 7 .

Liver Fats T h e s e have often b e e n studied and as early as 1945, ELLINGER36 drew

attention to the increased fat in the liver after irradiation. A s this increase was lessened b y deoxycorticosterone, he interpreted it as a sign of adrenal insufficiency.

T h e contrary explanation is more probable as: (i) an excess of cortisone constantly causes a deposition of g lycogen and fat in the liver, and (ii) adrenalectomy in rats is not fo l lowed b y an increase in fat in the liver, and prevents the fatty infiltration seen after irradiation6 6 . I t seems, therefore, that the accumulation of fats in the liver is the result of pituitary-adrenal overactivity, with an excess of gluco-corticoids in the blood as compared with mineralo-corticoids (see Chapter 15).


In starving rats irradiated with 1000 r there is no fatty infiltration (steatosis) of the liver. T h e weight of the liver and its total lipid content run parallel with those of starving controls. A great increase of liver fat is observed, irregularly, only if the irradiated animals are given a diet rich in fat54. T h e way to produce true steatosis of the liver regularly in rats is to administer a dose of 50 r a day, which does not produce anorexia. T h e animals remain in excellent condition until shortly before death, when they have received a total dose of 1700-2350 r. A t this stage the liver weighs less than in the controls, but its glyceride content is very high: 0-1-0-2 g/g fresh tissue6 7. T h e same clear picture does not emerge if one applies Chevallier's technique to mice. Increased lipogenesis as measured by acetate 1 4 C incorporation 24 hr after total b o d y x-irradiation seems very important in rats 1 5 0 ' 1 5 9 '2 2 2 , weak in guinea-pigs, and absent in mice 1 5 9 . A relation seems to exist between anaemia and total liver fat in irradiated mice and rats1 6 4. T h e presence of the adrenals may be necessary for the genesis of fatty liver in rats after irradiation (see ref. 231, also Chapter 15).

There are several signs of increased phospholipid synthesis in the liver whereas the incorporation of 3 2 P in the phospholipids of the thymus remains unchanged after irradiation35, but caution is necessary in inter-preting these experiments. One should compare in vitro and in vivo incorporation. W E I N M A N et al.292 also found no increased 3 2 P incorporation into liver phospholipids in rats injected with inorganic phosphates, 24 hr after exposure to 2500 r. But, liver slices f rom rats killed at the same time after irradiation (1000 or 2500 r) incorporate more 3 2 P in phospho-lipids than did control slices from unirradiated rats292.

T h e increase in lipid synthesis f rom labelled acetate b y the bone-marrow in vitro is discussed on p. 337, and the whole subject is reviewed b y C H E V A L L I E R a n d B U R G 1 4 3 .

Cholesterol Metabolism A 20 per cent decrease in cholesterol concentration and a 40 per cent

increase in liver weight were observed 48 hr after whole-body x-irradia-tion of the rat222 (in contradiction with ref. 54). T h e cholesterol synthesis measured in vivo by acetate I - 1 4 C incorporation in the liver is accelerated (24 hr and much more so 48 hr after irradiation) about 100 per cent per 100 r over the range 300-2400 r. It is very unfortunate that we have no data f rom the L o s Alamos group about what happens soon (30 or 60 min) after irradiation. T h e same, or even a bigger, acetate 1 4 C incorporation is observed if liver slices are incubated in vitro with a high concentration of the tagged precursors; thus the increased incorporation is not due to a shift in the quantity of acetate available in vivo222,223. furthermore, the


incorporation in vivo of tritium from 3 H O H into cholesterol follows the same pattern of change in the liver, adrenals and other organs as that of 1 4 C f rom acetate222. T h u s , there is a true large increased synthesis of cholesterol after irradiation. W h a t does happen to this mass of cholesterol is not quite clear. It must be rapidly used since the total cholesterol content of liver, intestine, spleen and carcass changes little. T h e plasma cholesterol of irradiated rats injected with 1 4 C acetate has the same activity as that of the liver. G o u l d found no objection to the classical idea that cholesterol is synthesized mainly in the liver and adrenals, the cholesterol of the other tissues coming from the liver222. Excess cholesterol synthesized b y the adrenals is used for increased corticoid secretion (see Chapter 15). One should find an important increase of cholesterol excretion in the bile and maybe of cholesterol catabolism, i.e. complete destruction; this last possibility has so far received little attention. G o u l d suggests that a marked increase in the rate of cholesterol utilization b y the liver and adrenal after irradiation tends to decrease the cholesterol concentration of these organs and this lowered concentration stimulates the rate of cholesterol synthesis in these organs222.

P R O T E I N M E T A B O L I S M Studies on protein metabolism abound in contradictions, even if late or terminal phenomena are disregarded. Some workers (cf. refs. 1 and 14) claim that there is great protein destruction in rats shortly after irradiation (400-1000 r), which shows itself b y an increase in the urinary excretion of total and urea nitrogen 1 7 7 ' 1 7 8 , but the blood urea of irradiated nephrec-tomized rats does not increase more than that of nephrectomized controls. T h e nitrogen balance is negative in young rats for 2 or 3 days after a non-fatal dose of x-rays (400 r), the absorption and retention of nitrogen being affected. Lamerton and his colleagues emphasize that nitrogen excre-tion (urea, amino-acids and ammonia) is not affected quantitatively or quali-tatively b y irradiation in spite of the very distinct inhibition of growth 7 9 . Other authors (see, for instance, ref. 180) regularly observe an increased urinary excretion of urea in rats already 3 hr after x-irradiation (250 r).

In the dog, uric acid excretion is increased following doses of 150-500 r of x-rays; xanthine excretion is markedly increased but only by supralethal doses; total purine excretion is slightly increased, the degree of increase bearing no relation to the radiation dose 1 8 6 . T h i s confirms many observa-tions in man, dog or rat. T h e increased purine excretion following irradiation of the rat seems to be independent of adrenal activity3 0 0.

A logical approach to the estimation of protein breakdown after irradia-tion is the measurement of the quantity of any one amino-acid available in the body 1 6 6 . If cathepsins are liberated in the cells during or early after irradiation, there must be an increase in free glycine available for conjuga-


tion of benzoic acid. T h u s , the 1 4 C activity of hippuric acid* isolated from the urine of rats injected with a small amount (2-4-5 //moles) of glycine 2 - 1 4 C (and given 0-1 //mole of sodium benzoate/100 g b o d y weight) must decrease after irradiation if more glycine is set free in the organism, provided that neither the conjugation of benzoic acid with glycine, nor the excretion of hippuric acid, is affected b y radiation; the validity of the methods has been established b y SCHREIER et A/ .1 6 6 .1 6 7

T h e r e is indeed a trend toward an expansion of the "first glycine p o o l " after irradiation (700 r, 250 k V x-rays), but not during the first 6 hr.

If rats are loaded daily with glycine -2- 1 4 C for 8 days before irradiation, and given benzoic acid immediately after irradiation, there is (1) an in-creased excretion of 1 4 C activity in urinary hippuric acid 1 6 8 , (2) a parallel increase in 1 4 C activity of urinary creatine-creatinine especially during the second or third day after irradiation 1 6 9 - 1 7 3 . T h e increased creatinuria (see also ref. 210) with high isotope concentration resembles that found in muscular distrophy 1 6 9 ; it exists also in the dog and monkey 1 7 4 .

During the 24 hr following irradiation of fasting rats, an increased urinary excretion of cysteic acid, leucine and valine is also reported2 9 6 . T h e increased plasma levels and urinary excretion of non-glycogenic amino-acids contrasting with a lack of change in glycogenic amino-acids, is interpreted b y an increased breakdown of proteins rapidly fol lowed b y excessive glycogen synthesis in the liver2 9 6.

A n increased urinary excretion 1 7 5 of taurine and of amino-acids is a frequent finding soon after irradiation of men and mammals, but not of the rhesus monkey 1 8 4 . T h e fact is associated with increased plasma amino-acid levels1 7 2, already 1 hr after irradiation, but it is not exactly known whether or not the kidney plays an important role as it does in congenital amino-acidurias. Naturally, just as discussed above in the case of lipids, starvation or the nutritional condition has a great influence on blood and urine levels of amino-acids—an influence which may be greater than that of irradiation185.

K a y and Entenman have shown that the increased urinary excretion of taurine is a rapid and sensitive response to whole-body x-irradiation of the rat, (ref. 176, Fig. 13-2, and ref. 177), but so far, its specificity has not been adequately studied. Aebi has confirmed this observation, but his rats respond more slowly (maximum at the 2nd day) and do not show any change in amino nitrogen, total nitrogen and creatinine excretion after 500 r of 250 k V x-rays 1 8 2- 1 8 3 . Cysteamine seems to inhibit this effect of irradiation1 8 3 but the interpretation of this observation is rendered difficult because cysteamine is normally oxidized to taurine when injected into mammals (see p. 461).

* Hippuric acid is N-benzoylglycine. 22


T h e site of the mechanism responsible for this effect is the abdomen because shielding of this area prevents the response while its irradiation causes an increased excretion of taurine equal to that following total-body irradiation. T h e presence of the spleen, adrenals or hypophysis is not necessary; the pancreas plays an important part in the reaction; in

FIG. 13-2. Hourly urinary excretion of taurine after a single dosage of whole-body x-irradiation. Minus numbers on the time scale indicate hours prior to irradiation. Each value is

the average of three rats. K A Y and E N T E N M A N 1 7 6 .

nephrectomized rats, the level of plasma taurine increases after irradia-tion 1 7 6 . Pyridoxine (vitamin Be) deficiency is followed by the disappearance of 1-cysteic acid decarboxylase activity f rom the rat liver and of taurine f rom the urine; but the taurine level of the brain remains unchanged 1 8 1 ; it would be interesting to test if pyridoxine deficient rats are still capable of an increased taurine excretion after irradiation.

A logical explanation of the increased excretion of taurine after irradia-tion would be an increased oxidation of methionine, cysteine or cystine (the precursors of taurine*), and this hypothesis is rendered more prob-

* Cysteamine is liberated by the catabolism of CoA in very small amounts (see p. 462), and cannot be considered on a quantitative basis as a precursor of taurine.


able b y the fact 1 7 9 , that there is also an increased excretion of S O 4 , that there are indications of a rapid oxidation of methionine and that admin-istration of this amino-acid after irradiation has been reported to favour D N A synthesis 1 7 9 and re-establishment of the nitrogen balance2 1 0 . Careful studies wi th 3 5 S methionine have shown that while the excess sulphate excretion m a y be accounted for b y increased methionine oxidation, the excess taurine excretion cannot b e interpreted in this w a y 1 8 0 . T h e intra-cellular, preformed taurine is not completely exchangeable wi th plasma taurine; there is a possibility that it is the b o u n d taurine which is l iberated b y x-irradiation and excreted b y the k idney; this hypothesis, derived f r o m a more general idea (see p. 272), is compatible wi th the fact that a second irradiation 10 hr after a similar first exposure (600 r) does not result in a second peak of taurine excretion; more than 7 days must elapse before a second dosage of x-rays reproduces a second response 1 7 6 .

Newly-hatched chicks die after 1000 r with uric acid retention, caused not b y increased protein catabolism b u t b y renal failure6 9 . Chicks w h i c h do not die soon after irradiation often die w h e n 5 - 6 months old wi th ascites and cirrhosis of the liver. Urinary excretion of uric acid and of allantoin is increased in rats during the 24 hr fo l lowing total-body exposure to 150-1000 r ; adrenalectomized rats show the same response w h i c h consequently does not be long to the general neuro-endocrine reaction described in Chapter 15 (see ref. 186; also ref. 190).

Disturbances in transamination have been described, for example in the glutamic-aspartic acid system, the activity being reduced to 5 per cent of the normal, b u t this happens 4—6 days after irradiation, w h e n serious anatomical changes have occurred. Pyridoxine, the co-enzyme of transamination is not affected in vivo.

T h e cessation of growth after irradiation has led m a n y authors to assume, without discussion or exact experimental evidence, that the synthesis of all proteins is slower after irradiation. T h i s is an over-simplification. It will be shown (p. 336) firstly that the synthesis of haemo-globin is greatly accelerated before be ing slowed, and secondly that the changes in the synthesis of g lobin do not run parallel to those of haemin. T h o u g h it is asserted that deoxyribonucleic acid synthesis is strongly inhibited, the renewal of a histone and t w o protein fractions of a Jensen rat sarcoma is not affected consistently b y 2000 r applied locally or to the b o d y as a whole, j u d g i n g b y the incorporation of 3 5 S methionine. N o important change in the l iver protein synthesis (measured b y incor-poration of phenylalamine-2- 1 4 C) has been observed in the rat 24 or 48 hr after total-body x-irradiation2 2 2 . L i v e r slices f r o m fasted x-irradi-ated rats incorporate more 1 4 C (from g l y c i n e - I - 1 4 C ) into protein than do liver slices f r o m either starved controls or even fed rats2 9 1 .

T h e ability of mammals to f o r m antibodies is diminished b y small


doses (175 r) of x-rays (see, in particular, ref. 102). T h e synthesis of properdine decreases sharply in irradiated animals 1 7 0 ; properdine, a y-plasma euglobulin, constitutes with M g + + and several cofactors, a bacteri-cidal system 1 7 1 . T h i s explains w h y infections are more dangerous in irradi-ated animals than in normal animals (see p. 423). T h e disturbances in the synthesis of enzymatic proteins are discussed on p. 332.

T h e concentration of the various plasma proteins of the rat does not change before the second day after irradiation, and the change is most marked on the third day after a dose of 600-1000 r. T h e nitrogen content is increased in fraction IV-4, which is rich in oc-I-globulin and decreased in fraction V , which is rich in albumin. T h e lipids and cholesterol are also increased in fraction IV-4. Electrophoresis showed that the quantity of globulin of fractions I I + I I I is always diminished (ref. 7 1 ; see also ref. 188).

Figure 13-3 gives an example of such changes in serum proteins and of

0-8

0-7

0 - 6

0-5

c 0 4 o 0 1 0-3 LU

0-2

0-1

2 0 4 0 6 0 8 0 0 2 0 4 0 6 0 80 0 2 0 4 0 6 0 8 0

mm mm mm

FIG. 13-3. Electrophoretic pattern of serum, (a) Unirradiated control, (b) Eight days after irradiation, (c) Eight days after

irradiation but protected by cysteamine163.

the remarkable protection afforded b y mercaptoethylamine (see p. 459, and ref. 163). Irradiation of the exteriorized intestines in the rat results in the same changes of serum protein pattern as whole-body exposures 1 8 9 .

In dogs, a diminution in albumin and globulin, coupled with an increase in total globulins is observed3 7 . T h e change in the albumin/globulin ratio is the earliest pathological sign in the blood of rats after irradiation with 100-650 r, the increase being perceptible as early as 21 hr after irradia-tion. Hyperglycaemia begins at the 6th hour, and hyperchloraemia and


hypercholesterolaemia do not appear until the 12th hour; the changes in the plasma proteins occur even in the absence of the pituitary or adrenals104.

T h e influence of total body x-irradiation (250 or 500 r) on plasma protein, lipoprotein and glycoprotein pattern has also been studied in the dog; in this animal changes in protein bound polysaccharides are a more sensitive indicator than lipoprotein derangements 1 8 7 . In the Rhesus monkey, the concentration of serum proteins fluctuates considerably; an increase in globulin and a decrease in serum albumin are the most constant changes2 1 3. T h e interpretation of the changes is complicated by the presence of l i p i d s " , by changes in the plasma volume 1 0 3 , and by differ-ences between species and also between different strains of the same species104. If the changes in total plasma volume are taken into account, the total amount of plasma proteins in rats is seen to be diminished during the first week following total irradiation with 600-750 r ; from the second week onwards, the total amount is increased by 15-20 per cent above that in the controls, although the protein concentration of the plasma is less than normal; the total mass of plasma increases greatly ( + 40 per cent) after the seventh day1 0 3 .

C H A N G E S I N E L E C T R O L Y T E C O N C E N T R A T I O N Changes in potassium and sodium ions balance are mentioned in Chapter 15; the changes due to water movements and permeability alteration are discussed on p. 414. Severe hypocalcaemia (3 m g per cent) is found in rabbits, causing tetany and death 1 - 3 days after a dose of 2000 r. A curious and quite unexpected feature of this hypocalcaemic tetany is that it occurs during severe acidosis, whereas the hypocalcaemia observed in human and veterinary medicine (when there is no question of the action of ionizing radiation) is always associated with alkalosis. T h e acidosis of heavily irradiated rabbits appears in the fourth hour after irradiation62.

A l though rabbits show peculiar reaction to x-rays, these observations deserve further investigation since a discussion of the detailed results obtained b y KOHN1 0 2-1 0 4 in various strains of rats does not lead to any general conclusions. Some response of the rat such as the increase in blood cholesterol can be attributed partly to the adrenals, and some such as the increased chloraemia in immature rats, to the pituitary, but other reactions occur in the absence of these glands.

Loss of sodium in rats through the gastrointestinal tract after severe irradiation (1500 r total-body) may be the immediate cause of death229. T h e sodium and chloride losses after total-body exposure (male rats, 700 r of 140 k V x-rays) are strongly influenced b y extracellular fluid movements and retention in gastrointestinal tract. Dur ing the 3 - 1 2 hr after irradiation, there is a shift of chloride and water from the body


tissues into the gastric secretion. T h e chloride appearing at 3 hr in the stomach comes mainly f rom the connective tissue, muscle and liver. T h e total body chloride balance becomes negative. In spite of its small mass, the connective tissue b y virtue of its capacity to hold water and electrolytes plays a major role. M a n y valuable observations on changes of water and electrolyte balance of various tissues after irradiation have also been made b y CASTER and ARMSTRONG230, but they do not follow an easily describable pattern.

T h e mean sodium loss b y severely irradiated (1500 r, 250 k V x-rays) 200 g rats was found to be 1 -5 m-equiv. in excess of that of normal fasting controls2 7 7 . T h i s loss occurs through the gastrointestinal tract mainly during the third day after irradiation and is due to the lack of resorption of the sodium normally excreted in the bile2 7 8 . T h i s excessive sodium loss is considered as the immediate cause of death from the so-called "intestinal syndrome" in the rat because: (a) removal of 1 4 -1 -54 m-equiv. of sodium from fasting non-irradiated rats b y means of intraperitoneal dialysis with hypertonic glucose solution causes death within 2 4 h r 2 7 7 ; (b) ligation of the bile duct immediately before or 24 hr after irradiation (1500 r) increased the mean survival time very significantly (5-6 days instead of 3-3 days for sham operated controls)278.

T h e loss of potassium occurs earlier through the urine; it is slightly increased (27 per cent) in fasting irradiated rats as compared with fasting controls, and this increase is easily accounted for b y the excessive tissue breakdown induced by irradiation277.

S U L P H Y D R Y L E N Z Y M E S A N D P R O T E I N S Sulphydryl groups occupy a special position in radiobiochemistry for the following reasons: (i) the in vitro experiments of B A R R O N et A / . 1 4 - 1 6

show rapid oxidation of the — S H groups of cysteine and B A L ( = di-mercaptopropanol), inactivation of the — S H enzymes, with more or less complete reactivation after the addition of cysteine or glutathione, i.e. physiological carriers of — S H (cf. p. 459); (ii) many sulphydryl deriva-tives (cysteine, cysteamine) protect against radiations. T h i s is in apparent agreement with the theory put forward as early as 1946 b y Barron, that the fundamental lesion produced b y ionizing rays in vivo is oxidation of the — S H of the intracellular enzymes and their inactivation, a theory based on the extension from in vitro to in vivo results. B A R R O N 3 4 and BARRON and SEKI17 provide indirect evidence in support of this theory, e.g. the oxygen consumption of the ova and spermatozoa of a sea-urchin (Arbaciapunctulatd) is increased b y small doses of x-rays (100 to 260 r) and inhibited b y large doses. Similarly, a typical — S H blocking agent, mercuric chloride, increases the consumption of oxygen in small doses


(5 x IO - 5 M), and inhibits it in large doses (1 x IO - 4 M). Both x-rays and mercuric chloride arrest mitosis in doses which increase oxygen consump-tion. T h e investigations of Hammett, Rapkine, Brachet, Mazia and others have shown how important sulphydryl groups are in mitosis and embry-onic development. Consequently, Barron's theory received wide atten-tion22.

However, against these indirect arguments the number of negative results from in vivo experiments is so great that it is impossible to accept Barron's theory as a whole 1 2 2*.

(i) If x-rays oxidize — S H either to S — S or further to SO 2 , one might expectf an immediate fall in reduced glutathione, and in total — S H groups in the blood and tissues after irradiation. T h e r e is no fall in reduced glutathione in the blood, liver, kidneys, heart and muscles of rats and guinea-pigs after 500, 600 or 700 r18>24>25>26. T h e r e is no fall in total — S H of Avena embryos immediately after exposure to 4 x l 0 4 r of x-rays224.

T h e only observations showing a decrease in sulphydryl groups were made long after irradiation, and the decrease does not precede, but follows, the development of anatomical lesions18-20 '21 '23-24. It takes 2 x IO6 rep of mixed ionizing radiation to reduce significantly the glutathione content of beef meat.

(ii) N o t all the good protectors against radiation contain sulphydryl groups, and many — S H compounds do not protect against x-rays (for details and discussion see Chapter 19).

(iii) Protectors against ionizing radiations which contain — S H groups are ineffective if injected after irradiation (see p. 467). T h i s has been well shown with cysteine and with cysteamine. T h e time interval between irradiation and injection of the — S H compounds may be kept extremely short, the lethal dose of x-rays given in 5 sec, and the injection of cyste-amine given within the next 20 sec. T h e death rate of mice in such an experiment is the same, or slightly more rapid, as after irradiation alone, whereas the same dose of cysteamine injected immediately before injection gives a 100 per cent survival rate.

T h e fact that no great decrease in the total — S H groups is found b y

* Recently, L A N G E et al.235 failed to confirm Barron's conclusion that - S H enzymes are more radiosensitive than non-SH enzymes.

f If every free radical produced by 700 r (a lethal dose) of whole-body irradi-ation of a mouse were used only to oxidize — S H groups (e.g. cysteine) the quantity of cysteine oxidized would be of the order of 25 /^g; a change of this magnitude is within the experimental error o f—SH determination. BRUES and PATT136 also point out that the total amount of available — S H groups in the tissues is much greater than the quantity which might be oxidized during irradi-ation by a lethal dose.


chemical titration does not mean that certain — S H groups carried in small quantities b y molecules of great biological importance are not blocked. T o take a precise example, supposing that coenzyme A (in which the — S H is carried b y cysteamine) is completely oxidized in the tissues, this oxidation will not be noticed if the total - S H only is determined, because the sulphydryl groups of this enzyme f o r m only a minute propor-tion of the total — S H . T h e oxidation of C o A results in its inactivation, and the transfer of acetyl, succinyl, stearyl, benzyl , and many other radicals is impossible; in other words, there are disturbances of various kinds in the metabolism of carbohydrates (blocking of K r e b s cycle), fats and perhaps proteins, and these disturbances affect catabolism as wel l as many syntheses.

(iv) It is impossible to demonstrate the inactivation of an — S H enzyme or coenzyme immediately after irradiation in vivo in the whole animal For example, the acetylation of ^-aminobenzoic acid in rats28, or of sulphathiazol in rats and rabbits2 9 , is not blocked or even diminished if these substances are injected during the hours w h i c h follow irradiation. It may be concluded that coenzyme A is not inactivated. T h e r e is a reduc-tion of acetylating capacity of the pigeon liver after exposure to 2000-3000 r of x-rays, b u t only at the 5th day, w h e n the animals are mori-b u n d 1 9 1 . T h e decreased acetylation observed b y some authors at the 12th hr after irradiation of the rat (there is none at the 6th hr) is also observed after any kind of stress 1 9 3 (see also Chapter 15). T h e succino-dehydrogenase (a thiol dependent enzyme) activity of homogenates of rat kidneys is unchanged 2 hr after irradiation wi th 400-800 r30. T h i s fact has been confirmed in mice, not only in the kidney b u t also in the liver3 1 . T h e r e is no decrease, except at the 2nd day, in succinic dehy-drogenase of the rat liver locally irradiated (6500 r) 1 9 4 , or other total-body exposure 1 9 6 . N o t before 4 hr after a local dose of 6000 r to the rabbit 's eye is there any change in succinoxidase histochemically detected in the retina 1 9 5 .

Homogenates of rat thymus retain all their dehydrogenase activity for succinic and malic acid, and their cytochrome oxydase and A T P a s e activity after irradiation with 800 r, although the decrease in concentration and the rate of incorporation of 3 2 P in desoxyribonucleic acid is very marked as early as 3 hr after irradiation35. Dif ferent results have been published for the spleen, another highly radiosensitive organ. In rats32-33

the succinoxydase activity of the spleen is not lowered between 4 hr and 17 days after irradiation, that is, whatever its state of degeneration or regeneration. In mice, the succinoxydase activity of the spleen seems to be diminished after irradiation, and this inhibition is prevented b y cysteine even if it is injected after irradiation3 1 .

D U B O I S et al.39 stress the fact in their experiments that the lethal dose of


x-rays produced no appreciable decrease in vivo in the activity of succino-dehydrogenase, adenosinetriphosphatase or other - S H enzymes in various tissues.

T h e most interesting fact which emerges from studies on homogenates of spleen is the great decrease in the ability of the splenic mitochondria to synthesize (or replace) the phosphorus compounds* (energy rich) which are associated with the oxidation of succinate3 2-3 7 . Similarly, homogenates of the thymus of rats irradiated with 800 r have lost a great part of their ability to esterify inorganic phosphate3 5. In homogenates of the spleen of mice anaerobic glycolysis is reduced by 80-90 per cent after a dose of 650 r to the whole body. In the liver, the A T P and phospho-creatine content remains normal for 6-8 days after irradiation; the adenylic acid, adenosine diphosphate and adenosinetriphosphate content of the blood of mice is also unchanged 2-8 days after a 100 per cent lethal dose of 800 r i i i .

Studiesi40-I4I >227 with homogenates f rom rats or mice have confirmed that: (i) A T P a s e activity is increased and oxidative phosphorylation decreased b y a whole-body dose of as little as 25-100 r if the homogenate is prepared within 1 hr following the irradiation, (ii) Neither of these changes is observed if the homogenate is irradiated in vitro even with 20,000 r, but they are observed with homogenates of exteriorized irradi-ated spleens, the rest of the body being effectively screened, (iii) T h i s trouble in oxidative phosphorylation is due neither to the increased

* BRACHET116 states: " I t is mainly, if not exclusively, in the mitochondria that cellular oxidation takes place, which provides the cells with the energy they need to carry out syntheses or work. But we know that the energy set free by cellular oxidation cannot be used directly by the cells, it must be accumulated in the form of the phosphorus compounds of adenosine triphosphoric acid (ATP) which are rich in energy. It is the hydrolysis of these compounds with the concomitant loss of one or two terminal molecules of phosphoric acid, which finally liberates the energy available to the cells. T o make cellular oxidation really effective it must therefore be coupled with phosphorylation, that is, with endoenergetic fixation of inorganic phosphorus on a suitable acceptor (e.g. adenylic acid).

"Isolated mitochondria are capable of fulfilling these functions on condition that they are supplied with the materials which they have lost in the process of isolation. If, therefore, well-washed mitochondria are prepared, and an oxidizable substrate (e.g. a-ketoglutaric acid) is added, together with a co-dehydrogenase, an intermediate carrier of hydrogen (cytochrome C), M g + + ions, inorganic phos-phate, and a phosphate acceptor (adenylic acid), oxidation coupled with phos-phorylation will be produced in vitro. T h e ketoglutaric acid will be oxidized and inorganic phosphate will, at the same time, be fixed to the adenylic acid to form A T P . T h u s the mitochondria act not only as catalysts for oxidation but also continually replenish the stock of ATP , thus storing the energy set free by the oxidation in a form directly available to the cell. Finally, the mitochondria can also take part in the syntheses which find in A T P the energy needed for their production.


A T P a s e activity nor to the decreased anaerobic glycolysis, (iv) Experi-ments using 3 2 P indicate that the trouble in phosphorylation also occurs in vivo in the spleen and thymus2 2 6 , (v) In certain conditions phosphoryl-ation may be normalized b y addition of cytochrome C ; other observations also indicate that the biochemical lesion is located at the end of the respiratory chain.

Lethal doses of x-irradiation do not inhibit cytochrome C activity from various rat tissues275. " I t appears thus that the cytochrome C deficiency in rat spleen mitochondria observed b y VAN BEKKUM276 is due to a loss of cytochrome C f rom the particulate structures into the cytoplasm of the cell rather than to inactivation of the protein" 2 7 5 .

T h i s is a new instance of what should be found if our hypothesis of enzyme release theory is correct: a molecule is not inactivated nor altered b y the ionizing radiation, but its strategic position is changed in such a way that its physiological activity is lost. A T P a s e activity is also increased in the lens during the 4 days following local x-irradiation1 9 8 .

I N C R E A S E D E N Z Y M I C A N D S Y N T H E T I C A C T I V I T Y A F T E R I R R A D I A T I O N

T h e following considerations should never be forgotten when discussing the effects of ionizing radiations on enzyme activity. Pure enzymes in vitro in dilute aqueous solution are much more radiosensitive than the same molecules " i n vivo", or even in homogenates or extracts (see ref. 299). Enzymes on isolated structures (mitochondria) are in vitro more resistant than in vivo. Addit ion of 1 per cent liver extract completely prevents the inhibition of a solution of pure glutamic acid dehydrogenase b y 5 kr 2 9 9 . T h e only alteration observed after irradiation in vitro of pure enzymes is inactivation.

Primary enzyme inactivation (inactivation during irradiation) is very unlikely when moderate doses (up to threshold 100 per cent lethal dose) are given*.

T h e effects of irradiation in enzymatic activity in vivo are indeed very complicated to analyse. A n increased activity may be due: (a) to the destruction of an inhibitor; (b) to the release of an activator; (c) to an increased synthesis of the enzyme; (d) in certain experimental conditions, to the release of bound (inactive) enzyme from its links with intracellular structures (nucleus, mitochondria, microsomes, membrane).

A decreased enzyme activity may be due not only to the destruction of the enzyme but also to the failure of its synthesis+, to the release of an

* Even when rats are exposed to forty times the LD50 dose of x-rays there is no inactivation of enzyme to be found in the liver immediately after irradiation211.

f Enzymes, like all substances in the body, are in constant equilibrium, the destroyed molecules being replaced by newly synthesized ones.


inhibitor or to the disappearance of an activator. Enzymes, like D N A a s e s for instance, have inhibitors in plasma and urine.

M a n y artefacts are introduced during the process of tissue homogen-ation and differential centrifugation. T h e same technique for separation of the various fractions (nucleus, mitochondria, microsomes of various sizes, supernatant) gives often different results if applied in different laboratories of high technical standard. T h e same enzyme (succinic dehydrogenase) is more sensitive to x-rays in vitro when bound to mito-chondria than when bound to very small particles2 1 9. W h e n one estimates in vitro the enzymatic activity of an homogenate or an isolated fraction, one adds all the cofactors and the substrate in optimal amounts, thus realizing artificially the best conditions. Indeed, it may be that a deficiency in one cofactor, after irradiation, is the reason for the partial inactivation of an enzyme system*. Histochemistry is not yet capable of giving reliable quantitative estimations of enzyme activity within the cell (but see ref. 2 1 4 ) .

It is a rare opportunity to have a simple and demonstrative test (that of acetylation in vivo for coenzyme A activity for instance, see p. 330) in which the animal is taken as a whole and no artefact introduced.

Furthermore, in the calculations, the choice of a unit of reference is crucial: wet weight or dry weight (to avoid error due to odoema), nitrogen (with the hope that it represents mainly proteins, the "ac t ive" part of the tissue), or the whole organ (see refs. 192 and 197), which may lose weight after irradiation without losing its enzymes (see also ref. 303).

A number of reports f rom various radiobiological laboratories indicate increases in activity after irradiation in vivo, often of considerable magni-tude (400 per cent), especially as the increase occurs very soon after irradiation. So far, painstaking searches have been carried out for inhi-bitions of enzymes, cessation of growth, and the slowing down of synthesis. T h e unexpected discovery of the astonishing early activation of radio-sensitive tissues is, in our opinion, important, as it appears to be an essential phase in the course of events f rom irradiation to the death of the cells. N o increase has ever been observed after irradiation of enzymes in vitro.

These researches must be studied in detail in order to derive the greatest possible information. M a n y radiotherapists admit b y implication that small doses of x-rays have a stimulating effect, for they use them to this end; but some radiobiologists have denied that there is any evidence to support this idea96. A s early as 1924 ANCEL and VINTEMBERGER reported

* M A A S et al.212 believe that the cause of glycolysis inhibition of ascite cells after irradiation is a tremendous decrease in diphosphopyridine nucleotide (DPN) which becomes the limiting factor for triosephosphate dehydrogenation. Th i s decrease cannot be considered as one of the primary biochemical lesions.


that x-rays accelerate embryonic development9 7 and Koller has reported that the mitotic rate of Tradescantia is increased b y low doses.

T h e experiments described below summarize the relevant data available in the literature.

(i) T h e increased activity of the tryptophane peroxydase-oxydase sys-tem in the liver of rats after irradiation with 1000 r (Fig. 13-4) depends on

2 5 4 6 8 12 24 4 8 72 96120 Time after IOOOr X-ray, hr

FIG. 13-4. Effect of total-body x-irradiation on rat liver trypto-phane peroxidaseoxidase. T h e number of rats used at each point is given in parentheses. Vertical arrows represent the

standard deviations74.

an adrenocortical reaction. T h i s is an unusual enzymic system; it is an adaptative system as its concentration (or activity) increases rapidly if the animal is provided with an excess of tryptophane7 4 . GROS, TALWAR and COURSAGET have shown b y incorporation of 1 4 C valine in tryptophane peroxidase, that the increased enzyme activity after 6 0 C o y-irradiation is really due to an increased synthesis of the enzyme protein2 1 7 . Adrenal-ectomy suppresses this reaction to irradiation, but hypophysectomy does not. T h e increased activity is not a response to stress in the sense of the adaptation syndrome but an action of the suprarenal cortex independent of the pituitary7 4 .

T h e enhanced transaminase activities in the rat liver after total-body x-irradiation (1000-1500 r) is believed to be secondary to overactivity of the adrenal cortex because (a) injection of nitrogen mustard produces the same increase (b) adrenalectomy decreases this enzymatic activity2 0 1 .

(ii) In all mammalian tissues and organs there are not only several cathepsins (or carboxypeptidases), but also an inhibitor of these cathep-sins7 6 . Tota l irradiation of mice, rats and rabbits with about the 50 per cent lethal dose increases the activity of some of these cathepsins. In the


case of the carboxypept idase of the rat k idney, this increase m i g h t b e due to destruction of an inhibitor by x-rays. Increased cathepsin C activity is also reported b y L E B E Z et al237 after in vitro irradiation of beef sp leen; in these conditions, it seems that the best interpretation is again in the line of our main w o r k i n g hypothesis , that of release of e n z y m e in active f o r m f r o m the inactive b o u n d f o r m . T h e very clear experiments of HAGEN238 show an activation of cathepsin f r o m t h y m u s a n d spleen of mice 2 4 hr a f ter tota l -body irradiation; they are interpreted in the same w a y . It is possible that certain b l o o d cells ( leucocytes) are the only source of the inhibitor, the titration of w h i c h , dur ing the first 2 4 h r after irradi-ation w o u l d have some prognost ic va lue 7 7 . T h e s e observat ions recall the fact reported b y ABDERHALDEN in 194 0 7 8 that proteases specific for the organs irradiated are f o u n d in the urine 6 - 9 hr after irradiation. T h e y s h o w that E u l e r a n d H a h n were r ight to emphasize that x -rays m a y have activating and inactivating effects on the same tissue homogenate , w h i c h m a y be considered to b e a h a l f - w a y stage b e t w e e n the l iv ing cell and the pure e n z y m e . T h e m e c h a n i s m s b y w h i c h these contrary effects are b r o u g h t about are p r o b a b l y quite different.

(iii) (a) Isolated cells T h e o x y g e n consumpt ion of the ova a n d sperma-tozoa of Arbacia punctulata increases b y 1 4 - 2 7 per cent in 2 hr after a small dose of x -rays (100-260 r) 1 7 . I n baker 's yeast, o x y g e n c o n s u m p t i o n and carbon dioxide anaerobic product ion is increased after a dose of 90,000 r a l though mitosis is inhibited b y 20,000 r 8 0. T h i s dose of 90,000 r does not alter the uptake of g lucose b y these cells w h i c h n o longer f o r m colonies. WOODWARD and CLARK203 have observed an increase in the n u m b e r of conidia that germinated as wel l as an accelerated g r o w t h f o l l o w -ing x-irradiation of Neurospora crassa. M O U T S C H E N and BACQ232 '233 have observed m a n y instances of increased g r o w t h after irradiation of plant cel ls: bar ley seeds, giant cells of an alga (Chara vulgaris), spores of a moss (.Polytrichum formosutn), prothalles of a fern (Adiatum gracillimum)\ this increase can reach 500 per cent b u t large doses (50 kr) of x-rays m u s t b e used.

(b) Isolated systems of vertebrates. A research group at Rochester Univers i ty ( R i c h m o n d , A l t m a n a n d Salomon), using homogenates of rabbit b o n e - m a r r o w or spleen r e m o v e d at various t imes after irradiation, have studied o x y g e n c o n s u m p t i o n a n d the incorporat ion of labelled atoms b y various molecules or series of molecules 8 7 ' 8 8 ' 8 9 . T h e o x y g e n c o n s u m p -tion, a n d the synthesis of haemin f r o m glyc ine label led w i t h 1 4 C , is greatly increased (by 200 per cent) immediate ly after a w h o l e - b o d y irradi-ation w i t h 800 r. T h e synthesis of the g lob in in h a e m o g l o b i n is increased in the spleen b u t not in the b o n e - m a r r o w . A s early as the 2nd day af ter irradiation the synthesis of haemin in the b o n e - m a r r o w falls to a very l o w level, and the synthesis of g lobin b y the spleen returns to normal .


T h e latter course is interesting to follow, but is mainly concerned with the slow restoration of haematopoiesis. T h e most curious point is the complete dissociation between the synthesis of haemin, which is always increased in the spleen b y x-rays, and that of globin, which increases immediately after irradiation, then falls to a very low level and does not rise again even after 3 weeks. T h e synthesis of globin seems to recover much more slowly than that of haemin8 7 . A study of T a b l e 13-2 shows that this investigation

T A B L E 1 3 - 2

C A P A C I T Y OF B O N E - M A R R O W AND S P L E E N H O M O G E N A T E S TO SYNTHESIZE H A E M I N AND G L O B I N (AFTER R I C H M O N D et A / . 8 7 ) BEFORE AND AFTER W H O L E - B O D Y

IRRADIATION

Bone-marrow Spleen Series Series

W. haemin W. globin W. haemin W. haemin W. globin W. haemin XlO-3 XlO-3 XlO-4 XlO-4 XlO-3 XlO-3

W. globin XlO-4 XlO-4

W. globin

No radiation 6-8 M 6-18 13-6 4-2 32-3 0 hours 22-2 1-2 18-50 31-0 12-6 24-6

48 hours 1-5 1-5 1-00 31-8 3-0 106-0 72 hours M 1-2 0-92 28-0 4-3 65-1

1 week 0-6 0-4 1-50 53-0 0-9 588-9 2 weeks 1-4 0-3 4-67 53-2 0-7 760-0 3 weeks — 0-2 — 44-5 0-3 1483-5 4 weeks 18-7 0-5 37-40 — •

Note.—W is a factor used by the authors to express the synthetic activity.

would have lost all its importance if the behaviour of the bone-marrow and spleen had not been observed immediately after irradiation.

T h e same workers also noticed an increase in vitro in the synthesis of saturated or unsaturated fatty acids b y the bone-marrow of rabbits taken immediately after irradiation. In this case the tracer was acetate labelled with a " C atom in the ,8-carbon ( I 4 C H 3 C O O N a ) 8 9 . Table 13-3 clearly shows that all the anabolic and catabolic activities of the bone-marrow are strongly stimulated b y irradiation89.

Nizet, Lambert , Herve and Bacq have attacked the same problem of haemoglobin synthesis in vitro, but avoided the artefacts in preparation, i.e. the homogenization carried out b y the American authors. T h e reticulo-cytes, which are numerous in dogs made anaemic b y bleeding, synthesize in the same way as bone-marrow haemin and globin f rom labelled glycine in vitro90'138. N o manipulation of the cells is required and it is only necessary to take samples of blood from the dog before and after 500 r


T A B L E 1 3 - 3

SYNTHETIC CAPACITY OF BONE-MARROW BEFORE AND AFTER W H O L E - B O D Y IRRADIATION

Saturated Unsaturated O2 14CO2

fatty acids fatty acids Haemin uptake activity Series Series

/0 % IO4 disint. / 0/ /0 Ml O2 IO3

WhUO-1 pre- w t . i o - 1 pre- min per mm pre- per g disint. / radiatn. radiatn. per g wet wt radiatn. wet wt min/mm value value bone-marrow value per 3 h

No radi-ation 6-31 — 5-50 — 1-22 — 300 5-66 Ohr 14-60 231 18-90 344 1-90 156 810 8-40

48 hr 5-96 108 — — 0-31 25 280 4-70 72 hr 20-00 363 1-00 18 0-21 17 80 1-14

158 hr 3-21 58 15-60 283 0-05 4 280 0-18

* W synthetic activity of saturated fatty acid from acetate, f W synthetic activity of unsaturated fatty acid from acetate.

of whole-body irradiation. A n increased synthesis was found after irradi-ation which was of the same order of magnitude as that obtained b y the Rochester team9 0 (see T a b l e s 13-4 and 13-5). T h i s technique enabled

T A B L E 1 3 - 4

EFFECT OF IRRADIATION ON THE SYNTHESIS OF H A E M I N BY RETICULOCYTES OF THE D O G USING 1 4 C-LABELLED G L Y C I N E 9 0

Experiment No. 61 87 102 108

Glycine-2-14C content d.p.m.* per ml of blood 222,000 222,000 74,000 92,500

Duration of incubation of the blood (hr) 7 5 6 7

Blood f rom unirradiated dog 14,200 1092 11,793 9151 Blood irradiated in vitro

(500 r) 15,700 1554 27,035 —

(2000 r) — • — — 10,507 (100,000 r) 30,400 1237 19,884 —

Blood f rom dog which has re-ceived whole-body irradiation of 500 r 28,000 1788 47,623 11,095

Non-irradiated erythrocytes + 28,000

plasma f rom irradiated (500 r) dog — — 19,180 13,088

* Activity expressed as d.p.m. per 100 g of haemin isolated f rom the blood after incubation.


T A B L E 1 3 - 5

E F F E C T OF IRRADIATION ON THE SYNTHESIS OF G L O B I N BY RETICULOCYTES OF THE D O G USING 1 4 C - L A B E L L E D P H E N Y L A L A N I N E 1 3 8

Experiment No. 121 130 139

No. of red cells per ml of blood 3,000,000 3,100,000 3,500,000 No. of reticulocytes per ml of blood 210,000 295,000 210,000 Phenylalanine 14C content d.p.m.* per ml

of blood 277,500 294,500 590,000 Duration of incubation of the blood (hr) 5 5 7 Blood f rom unirradiated dog 851 999 703 Blood irradiated in vitro (500 r) 1244 — —

Blood irradiated in vitro (20,000 r) 1019 1234 722 Cells taken before irradiation and plasma

taken after irradiation 929 1097 —

Blood from dog which has received whole-body irradiation 1302 2116 908

* Activity expressed as d.p.m. per 100 g of haemin isolated from the blood after incubation.

them to go further, and to observe (a) that the factor which increases haemin synthesis by the reticulocytes is, at least in part, dissolved in the plasma, for the synthesis of haemin b y non-irradiated reticulocytes may be doubled by the addition of plasma from an irradiated dog; and (b) that an increase in haemin synthesis is also observed after irradiating heparin-ized blood in vitro, though much larger doses of x-rays are needed (2000-100,000 r). A n increased activity has been reported in the spleen and bone-marrow (not in the kidney or liver) 3 days after exposure of rats to small doses (50 r) of x-rays, of an enzyme—the S-aminolaevulic acid de-hydrase—which catalizes the conversion of S-aminolaevulic acid to porphobilinogen, the monopyrrole precursor of porphyrin and haeme2 1 6 . A l though many experiments by various authors have been made on the disappearance of 5 9 Fe injected in the plasma (considered as a measure of haemoglobin synthesis in vivo) of irradiated animals, it has never been reported that this disappearance is accelerated soon after irradiation as could logically be expected from the above-mentioned results. But often, no observation is made before 24 hr after irradiation, the period of observa-tion is too long, or the experimental conditions are not adequate. T h e lack of effect often observed during the first day may be a combination of at first increased, later decreased, uptake.

Hyperferraemia has been seen in different species of mammals but careful study of the results shows that often there is a definite hypo-ferraemia (plasma iron) at 20-24 hr after irradiation204.


Nucleated red corpuscles of fowls washed three times and suspended in saline solution (thus, in relatively unfavourable physiological condi-tions), at a concentration 2 x IO6 cells/mm3, double or treble their oxygen consumption immediately after a dose of 500,000-2,000,000 r of x-rays. T h e oxygen consumption returns to normal or falls below it about 6 hr after irradiation, when haemolysis of the corpuscles takes place 1 1 8 . If the nuclei are separated from the cytoplasm, and the effects of x-rays on whole corpuscles and on the isolated nuclei are compared, it is found that large doses cause a much greater increase in oxygen consumption in the isolated nuclei 1 2 0 . T h e addition of albumin or haemolysed blood to the suspension of nuclei protects them from the stimulating effects of x-rays. T h e cytoplasm apparently acts in the same way when whole corpuscles are irradiated120. T h e O 2 consumption of fresh human red cells kept in suitable saline solution (citrate-phosphate buffer. p H 7-3) is increased 10 per cent after 55 r, 23 per cent after 330 r and 36 per cent after 600 r2 3 6.

I n ducks, the red corpuscles after total irradiation of the animal are said to consume less oxygen than do normal corpuscles 1 1 9 .

T h e 3 2 P activity in the stroma of circulating red cells is decreased for a period of 6 days after irradiation (600 r) of rabbits when 3 2 P phosphate has been injected; no similar changes could be detected in the 1 4 C activity of these stroma after injection of acetate 2 - 1 4 C 205.

Alkaline phosphatase activity increases in spleen homogenates after irradiation, especially when the substrate is adenosinetriphosphate32>33>93. It is also increased in the plasma9 3 . T h e acid phosphatase, detected b y a histochemical method, increases markedly in the spleen and lymph nodes of mice already two hours after total body irradiation (500 and 800 r); the increase is maximal in 1 to 6 days3 0 5. In the present state of our know-ledge, we can only attribute this remarkable " f lar ing-up" of the haem-atopoietic cells to a change in the enzyme-substrate balance within the cells. Glucose-6-phosphate dehydrogenase activity is increased in the rat's salivary gland after local irradiation of the organ either in situ or exterior-ized 1 9 9 .

Whole-body irradiation of rats with x-rays causes a very distinct increase (Fig. 13-5) in the oxygen consumption of the liver (homogenate), in spite of a diminution in the activity of choline oxidase. T h i s effect is observed as early as 6 hr after a dose of 200 r. O n the other hand, the oxygen consumption of the kidney, studied b y the same technique, is not increased after irradiation, whereas the choline oxidase activity is greater 18 hr after a dose of 800 r than in controls 1 1 7 .

T h e intestinal mucosa from whole-body irradiated rats (600 r) oxidizes glucose or fructose more rapidly (3 to 4 times) than normal mucosa when tested in vitro with 1 4 C tagged molecules. T h i s can be observed from

23


3-96 hr after irradiation and seems to be due entirely to increased citric acid cycle activity234.

Some older experiments, often not confirmed, had already described increases in metabolism after irradiation, for instance in the skin of frogs, and in seeds and seedlings of radishes (see ref. 121). Except in the organs of vertebrates, however, the only confirmation of a considerable increase in metabolism is given b y SUSSMAN121 for potato tubers. T h e oxygen consumption is increased, on the average, b y 500 per cent im-mediately after 10,000 to 400,000 r ; the increase is less (200 to 300 per cent) after 1000 r and after very large doses (3,200,000 r). Af ter moderate

T i m e ofter i r radiat ion, hr

FIG. 13-5. Endogenous respiration of rat-liver homogenates after whole-body x-irradiation117.

doses the increase is very transitory. T h e r e is also a smaller and less rapid increase in C O 2 output reaching its peak on the 2nd day. T h e tuber blackens as a result of the reaction of a phenolic substance (tyrosine, dopa, etc.) with an oxidase which is not normally in contact with its sub-strate. T h u s here again, one of the first effects of irradiation is the breaking down of intracellular barriers, putting enzymes in contact with a large quantity of substrate which necessarily leads to increased metabolism. Although the potato tuber has been recently used in many radiation experiments (inhibition of sprouting), no mention is made of blackening or increased metabolism; indeed the perfect condition of the tubers after irradiation is stressed b y all experimenters.

T h e activity of cytochrome oxidase is not changed even b y 3200 kr, whereas after this very large dose the activity of tyrosinase diminishes b y about one-half, probably because of the blackening, that is, because of the presence of quinones and melanin which inhibit tyrosinase.


According to LANGENDORFF and HAGEN200 the cytochrome in l iving cells (yeast and E. coli) is very stable and the electron transport is not affected b y high-energy rays. Some nice experiments b y MARTIN on yeast cytochrome systems have never been confirmed nor extended2 2 0 .

In this discussion we have carefully separated effects which are attribut-able to enzymatic adaptation f r o m those attributable to the destruction of enzyme inhibitors b y radiations. N o t h i n g of this kind has been described in the bone-marrow and spleen. In Nizet and Bacq's investigations, w h e n b lood rich in reticulocytes was irradiated in vitro, any intervention b y the adrenals was exc luded; the phenomenon is cellular, and not dependent on mammalian organization. N o inhibitor of haemoglobin synthesis has ever been recorded.

T h e importance of the localization, the strategic position and the variable degree of " f r e e d o m " of the intracellular enzymes becomes more and more apparent. W e are beginning to explore the possibilities of i n t e r a c t i o n b e t w e e n d i f f e r e n t s t r u c t u r e s . P O T T E R , L Y L E a n d S C H N E I D E R 9 8 ,

for example, show that the rate at which the mitochondria carry out the oxidations of K r e b s cycle is doubled b y the addition of nuclear material equivalent to that present in the intact nuclei of the cells, although the nuclei alone are practically inactive. T h e s e observations support the hypothesis discussed on p. 272, that changes in intracellular "perme-abi l i ty" are among the important primary effects of ionizing radi-ations.

Enzymes in Plasma or Tissue Extracts It is k n o w n in general pathology that enzymes appear in great quantities

in the plasma w h e n a sufficient amount of tissue is in bad physiological condition. For instance, the heart anoxia fol lowing myocardial infarction is accompanied b y m u c h increased plasma levels of lactic dehydrogenase and glutamic oxaloacetic transaminase ( G O T ) .

T h e v iew is generally accepted that these enzymes diffuse abnormally f r o m the cells into the plasma thanks to the increased permeabil ity which may be considered as an early non-specific reaction of every cell w h e n its metabolism is grossly troubled. T h u s one might expect to find increased enzymatic activities in the plasma of man and animals soon after a sufficient irradiation.

T h i s idea has been tested b y HUGHES206. She found early increases in serum concentrations of t w o transaminases and t w o deshydrogenases (see Fig . 13-6), sometimes as early as 30 min after exposure of rats to x-rays (400-2000 r) or fission neutrons (see also refs. 207, 208 and 209). T h i s reaction is not specific; it can be produced b y hepatotoxic agents (carbon tetrachloride, chloroform) or opiates. Combinat ion of x-rays and CCI4 gives very large increases.


Enzymes appearing in the plasma are rapidly removed f rom the circu-lation b y the liver and excreted intact in the bile. T h u s , the measurement of serum enzyme activities cannot give a quantitative estimation of tissue damage b y ionizing radiation, but is a good qualitative early test206. Further studies of this kind should include simultaneous measurements of tissue enzyme activity in order to try the establishment of the total balance

180

160

1S WO s?

0 1 3 6

Hours af ter X - ray

FIG. 13-6. Effect of 800 r x-irradiation on serum enzyme concen-tration in 6-week-old rats.

G O T = glutamic-oxaloacetic transaminase G P T = glutamic-pyruvic transaminase L D H = lactic dehydrogenase

M D H = malic dehydrogenase ( H U G H E S 2 0 6 ) .

of the enzyme. B R E N T et al.209 conclude that the G O T activity of the serum is a poor test; the increase is small and bears no relation to the lethality. But G O T is not the enzyme to choose because it is most concen-trated in tissues like the striped muscles and the heart, which are physio-logically very radioresistant.

Small doses of x-rays (25-50 r) greatly increase the peptidase activity of plasma taken f rom dogs 24 hr after irradiation. T h i s increase is especially pronounced ( + 400 per cent) if benzoyl argininamide is used as a substrate9 4; MILLER and GATES suggest this as a biochemical test to show that the body as a whole has been exposed to a small dose of ionizing radiations94.

EULER and HEVESY95 isolated the catalase of Jensen's sarcoma and


found the activity to be increased by 53 per cent after 3000 r. Forssberg has observed a less m a r k e d activation of catalase in vivo b y x -rays (see ref. 91).

Increased Incorporation of Isotopically Labelled Tracers HEVESY and his col laborators have studied extensively the p a t h w a y of

acetate label led w i t h a 1 4 C atom in the carboxyl group, or of phosphate label led w i t h 3 2 P after inject ion into animals2-4 4 . H e v e s y makes n o secret of the m a n y difficulties w h i c h arise in interpreting the results. " T h e m a p -p i n g out of a metabol ic route b y the use of 1 4 C as an indicator is an a r d u o u s task. T h e task wil l b e even more arduous if the effect of irradiation on this route is t o be e luc idated" 2 . T h e use of isotopes in biological research has raised more p r o b l e m s than it has solved. T h e 1 4 C of acetate a n d the 3 2 P in a phosphate g o e v e r y w h e r e and serve m a n y p u r p o s e s ; t h e y are not specific indicators. HEVESY2-44-112 s h o w e d that the presence of an increased quantity of the isotope in a single fract ion of an organ cannot a lways b e interpreted as a reliable indication of renewal , or a more rapid " t u r n o v e r " in this fract ion. H e introduced the concept of the " s e n s i t i v i t y " of an isotope indicator. T h e f o l l o w i n g quotat ion f r o m HEVESY2 i l lustrates its meaning.

" C o n s i d e r , for example, the format ion of label led l iver lecithin f o l l o w -ing intravenous inject ion of 3 2 P. I n the first day as m u c h lecithin wi l l b e turned over as in the second one, b u t because of the decrease in 3 2 P in the inorganic phosphate pool w i t h t ime the incorporat ion of the same a m o u n t of p h o s p h o r u s into l iver lecithin wil l b e f o l l o w e d in the second day b y the incorporat ion of appreciably less 3 2 P than in the first day. O n e w e e k before the administrat ion of 3 2 P one leg of a m o u s e w a s irradiated w i t h roentgen rays and the irradiated leg w a s f o u n d to take u p only 70 per cent of the a m o u n t of 3 2 P incorporated into the non-irradiated leg. T h u s the a m o u n t of 3 2 P r e m o v e d f r o m the inorganic phosphate pool w a s reduced b y altering the metabol ic rate, in this instance b y exposure to ioniz ing radiation. C o r r e s p o n d i n g l y the liver lecithin of an irradiated mouse wi l l incorporate more 3 2 P in the course of a g i v e n t ime than does l iver leci thin in a control mouse . T h e increased specific act iv i ty of the lecithin P in the irradiated animal wi l l not be due, as one m a y b e incl ined to interpret , to an increased lecithin turnover in the l iver of the irradiated animal. It is due to an interference of irradiation w i t h the turnover rate of the mineral constituents of the skeleton, w h i c h makes 3 2 P a less sensitive indicator of p h o s p h o r u s in the irradiated m o u s e than in the c o n t r o l . "

A precise t e r m should be adopted for this k ind of radiation effect . It is neither an indirect ef fect ( = free radicals) nor an ef fect at a distance (after irradiation); it is a secondary effect , a consequence of a p r i m a r y b iochemica l lesion.


T h e same argument should be applied to acetate and bicarbonate ions and to glycine labelled with 1 4 C . In spite of these limitations, the fact remains that the injection of isotopes in irradiated animals is a method w h i c h has revealed a number of primary as well as distant and secondary effects of ionizing radiations2-1 1 4 . A f e w examples are given here; many will be f o u n d in Hevesy 's work, in specialist reviews 1 and in reports of recent symposia (see also ref. 306).

T h e injection of 1 4 C glycine 48 hr after irradiation in rats gives rise to greater elimination of 1 4 C O 2 than in controls9 2 . If one of two trans-planted sarcoma in a rat is irradiated while the other is protected b y a lead screen, the uptake of 3 2 P is found to be reduced, not only in the irradiated tumour, b u t also in the shielded t u m o u r 1 1 5 .

T h e significant data obtained, f r o m the injection of acetate labelled wi th 1 4 C in the carboxyl groups in ful ly -fed mice, are that an irradiation wi th 800 r increases the fixation of carbon. T h e increases are: + 34 per cent in the dry brain t issue; + 1 6 per cent in the brain proteins; + 3 4 per cent in the brain fat ; + 12 per cent in the dry plasma; + 1 2 per cent in the liver proteins.

T h e s e observations support our v iew that ionizing radiations accelerate m e t a b o l i c p r o c e s s e s ( s e e p . 3 3 6 ) . F O R S S B E R G a n d H E V E S Y ( s e e r e f . 2 )

repeated these experiments in starving mice, and did not observe these increases, the only significant f inding be ing a decrease ( — 1 2 per cent) in the uptake of 1 4 C b y the liver lipids.

If glucose in w h i c h all the carbon atoms are labelled with 1 4 C is injected subcutaneously in mice, it is found that the 1 4 C O 2 content of the exhaled air immediately after irradiation wi th 2000 r is 20 per cent less than in controls. Glucose catabolism is therefore diminished immediately after irradiation7 . T h e uptake of 1 3 1 I b y the thyroid is increased both b y irra-diation of the gland and of the whole body8 1-8 2-8 3 . Incorporation of 3 2 P in the ribonucleic acid of the cytoplasm of the liver cells is increased8 4 , and this confirms M I T C H E L L ' S f inding8 5-8 6 that after irradiation in vivo of cancer tissue there is in the cytoplasm a significant increase in the ultra-violet absorption at a wavelength characteristic of the nucleic acids. T h e incorporation of 1 4 C f r o m labelled glycine in the brain of rats is also increased if the glycine is injected 48 hr after a whole-body irradi-ation9 2 .

S H E R M A N a n d F O R S S B E R G 1 3 7 h a v e s t u d i e d t h e i n c o r p o r a t i o n o f 3 2 P i n

the liver of the mouse and of the changes brought about b y a whole-body dose of 800 r of x-rays. T h e amount of radioactive phosphorus found in the liver fol lowing injection in the pleura in the f o r m of orthophosphate is increased b y 40 per cent if it is injected immediately after irradiation. A s the animal is sacrificed 5 min after the injection, this f inding indicates that there is a strong increase in permeabil ity to phosphate ions. T h e effect


of radiation becomes less marked as the time interval is increased. T h e activity of A T P is increased, especially 24 hr after irradiation.

Af ter irradiation the 3 2 P passes more quickly both through the hepatic capillaries and the cellular membranes. T h e 3 2 P is incorporated in the organic compounds and is consequently lost to the pool of ions which exchange constantly through the cellular membrane. T h i s interpretation is confirmed b y the fact that if the liver is irradiated in vitro, the incorpora-tion of 3 2 P in the slices of tissue is not modified. In the muscle (irradiated in vivo) the rate of exchange of 3 2 P and its incorporation are slightly decreased b y an irradiation with 800 r.

These researches show: (i) that the liver is a radiosensitive organ, (ii) that its vascular system must be intact if certain effects of the ionizing radiations are to become apparent, (iii) that the results obtained with one organ cannot, at least for mammals, be extrapolated to other tissues.

A n increased incorporation of 1 4 C phenylanaline was observed im-mediately after irradiation first in the nuclear proteins of the rat liver, later (within an hour) in the proteins of mitochondria, microsomes and supernatant2 2 1 . T h e increased biochemical activities after irradiation may occasionally translate themselves in morphologically visible effects, for instance a stimulation of hair growth in the rabbit2 1 5 or an increased elongation of cells in the barley plantlet and of other plant cells2 3 2-2 3 3 .

I N H I B I T I O N O F I S O L A T E D E N Z Y M E S Y S T E M S IN VIVO

It cannot be sufficiently emphasized that impure enzymes or enzymes in vivo are highly resistant to irradiation, whereas pure aqueous solutions are sensitive in TOfro30-91-107. Exposure of whole cells of the ciliated protozoon Tetrahymena geleii or T. pyriformis to 300,000 or 600,000 r of X-radiation has very little effect on oxidative or hydrolytic enzyme activity of the cells. However, if cell-free homogenates are irradiated, then the activities of these enzymes diminish considerably1 3 4-1 3 5. T h e enzymes may be protected in vivo b y their localization on intracellular structures.

T h e " p s e u d o " cholinesterase activity (or non-specific cholinesterase) of muscle and intestinal mucosa of rats was diminished b y about 50 per cent 48 hr after a whole-body irradiation of 1000 r ; but after 24 hr there is little diminution. T h e r e is no change in the level of true (or specific) cholinesterase, which physiologists regard as more important. T h e response of smooth muscle to acetylcholine is increased within the first 24 hr in the colon, and after 48 hr in the je junum and ileum. T h e dif-ference between the two cholinesterases may be due to the fact that the source of pseudo-cholinesterase appears to be the liver, whereas true cholinesterase originates elsewhere, and may be synthesized locally1 0 5. THESE observation? were confirmed, b y CONARD106. There are also changes


in the plasma pseudo-cholinesterase of guinea-pigs, but this must be nterpreted cautiously, because fasting rapidly lowers the concentration

of this enzyme 1 . T h e hydroquinone oxidase in the ova of a grasshopper (Melanoplus

differentialis) is diminished for 2 days after irradiation with 25,000 r after which it gradually increases (to 250 per cent of normal activity), while the sulphydryl groups disappear24.

Catalase activity is claimed to be decreased appreciably (20 per cent) in the liver of mice 24 hr after irradiation 1 0 8 ' 1 0 9 to return to normal at about 48 hr, and to fall finally to a very low value on the 5th to the 7th day. T h i s may be an indirect effect, for the Japanese authors have observed inactivation of this enzyme in mice after injection of rabbit's serum taken 1 - 5 days after irradiation1 1 0. These findings are not easy to reconcile with those of EULER and HEVESY95 as it would be necessary to postulate either that large doses of x-rays can activate where small doses inactivate or that the behaviour of Jensen's rat sarcoma is opposite to that of the liver.

T h e r e is no question that the intracellular yeast catalase is "act ivated" b y x- or y-irradiation; this is due to change in the physical state of the enzyme, possibly the rupture of its linkage with R N A (ribonucleic acid)2 1 8.

curve gives the relative amount of indoleacetic acid formed, the upper curve indoleacetaldehyde224.

A very useful theory is put forward b y GORDON in order to explain the inhibition of growth b y ionizing radiations of plants224, x-ray doses of less than 100 r reduce within minutes the concentration of auxin in plant


shoots. T h i s reduction cannot be explained b y a destruction of the auxine molecules present at the time of irradiation, molecules which resist easily to 10 kr probably just like enzyme. T h e r e does not seem to be an increased catabolism of auxin in irradiated plant tissues but a decreased synthesis. A u x i n ( = indolacetic acid) originates f rom tryptophane which is converted to indolacetaldehyde presumably b y the successive action of a decarboxylase and an amine oxidase. Cell-free homogenates of mung bean seedlings lose after irradiation their ability to convert indolacet-aldehyde to indolacetic acid; the aldehyde accumulates. T h i s enzymatic process is indeed very sensitive; an action of 25 r is easily detected (Fig. 13-7). Gordon believes that the inhibition of auxin synthesis is secondary or contemporary to the decreased nucleic acid synthesis224.

It must not be forgotten that after complete inhibition of mitosis b y irradiation, an increased growth of many plant cells has been observed2 3 2.

B I O S Y N T H E S I S O F N U C L E I C A C I D S M a n y papers deal with the effects of ionizing radiations on nucleic acid synthesis. N e w techniques (tritiated precursors, autoradiography) have so altered the field since 1955 that in this edition mainly the latest contribu-tions will be considered. These have been nicely summarized b y ERRERA239, H O W A R D 2 4 0 , K E L L Y 2 4 1 , H O L M E S 2 4 2 a n d S T O C K E N 2 4 3 . M u c h o f w h a t f o l l o w s

is taken from a very recent review of GOUTIER244, with w h o m we discussed the subject many times at length; it should be consulted b y the interested readers.

(i) A clear distinction is now made between total DNA or RNA content and the rate of biosynthesis which can be studied b y incorporation not only of 3 2 P but of selective precursors like tritiated thymidine for D N A . ORD and STOCKEN have observed that 3 2 P incorporation in the D N A of the rat's thymus can be inhibited 50 per cent 3 min after a total-body irradi-ation of IOOOr2 4 5. Inhibition of incorporation of thymidine 1 4 C in the D N A of various tissues in the rat has been studied 1 - 2 4 hr after total-body irradiation ( 5 0 - 8 0 0 r); total activity and specific activity have been measured2 4 6. Figure 13-8 shows that 50 r have already a marked effect, that, at the 1st hour, some increased incorporation is observed after 5 0 - 1 0 0 r # , that a quick restoration occurs in the intestine (if the dose does not exceed 200 r) but not in the spleen.

Incorporation of tritiated thymidine in the intestine estimated b y autoradiography2 4 7 is more rapidly affected and b y m u c h smaller doses of radiations than the total D N A phosphorus2 4 8 . T h i s is logically explained (see ref. 241) b y the fact that in the small intestine, only a few cells

* Other instances of increased incorporation have been observed (see refs. 239 and 251).


synthesize D N A ; t h e y are located in the glandular crypts of the m u c o s a ; the major i ty of D N A is inert. T h u s there is a cell populat ion effect. It has b e c o m e impossible to discuss the experimental results w i t h o u t taking into consideration the heterogeneity of the cell populat ion. T h i s hetero-genei ty is m a r k e d l y increased b y irradiation; already 1 hr after irradiation there are in the t h y m u s , the spleen and other l y m p h o i d tissues m a n y dead or d y i n g cells, the inert D N A of w h i c h wil l dilute the D N A of

FIG. 13-8. Variations in total activity of D N A in spleen and small intestine after different doses of total-body x-irradiation

a n d i n j e c t i o n o f t h y m i d i n e - 2 - 1 4 C (NYGAARD a n d P O T T E R 2 4 6 ) .

active cells. T h u s , the interest of total D N A content of a tissue is of l imited value, a l though it m u s t be measured 2 4 2 . T h e specif ic act ivity of D N A if a tagged precursor is injected at the t ime of tissue destruction decreases in


function of this destruction (see, for instance, ref. 239*). T h e reader realizes how important autoradiography has become in the analysis of radiation effects; the activity can be estimated (not accurately measured) in each cell, while chemical methods, quantitatively more precise, are blind.

M u c h work has been done in order to define how the radiosensitivity of D N A synthesis varies during the mitotic cycle of the cell.

(i) In many types of cells, mitosis is followed b y a period G i during which the D N A content remains constant. During the following period, the D N A content is doubled b y synthesis; this period is called the syn-thetic or S period. A "rest ing" period called G2 is observed between the S period and the next mitosis. M u c h bigger doses of x-rays are needed to inhibit D N A synthesis when it has already started, than during the presynthetic G i period (see ref. 301).

In the regenerating rat liver after partial hepatectomy, the population of cells is rather homogeneous. T h e synthesis of D N A in the cells which participate at the first wave of mitosis occur between the 18th and the 30th hour after hepatectomy with a maximum at the 27th hour; the mitotic index is maximal at the 30th hour. If given before the onset of D N A synthesis, moderate doses of x-rays (375 r) inhibit the incorporation of orotic acid in D N A ; a dose of 1500 r abolishes it. But as soon as synthesis has begun, 1500 r have a barely perceptible effect2 5 9 . T h e same result is obtained with other time relations, if regeneration of the liver is induced b y CCI4 intoxication2 5 3.

L A J T H A et al.25i have used another method with in vitro cultures of human bone-marrow. Incubated with 1 4 C formate, these cells incorporate 95 per cent of the 1 4 C in the methyl group of their D N A thymine. If cells are incubated with the tracer during the 4 or 5 hr following irradiation, all the tagged cells seen at autoradiography have been irradiated during the D N A synthesizing phase (called S). If incubation is localized between the 17th and 22nd hour after irradiation, the tagged cells have been irradiated during the G i presynthetic phase. A dose of 200-300 rads

* There is also another difficulty of interpretation of the results obtained with isotopes: that of the size of the pool of the precursor (see HEVESY2'44). Forinstance in Nygaard and Potter 's experiments, the incorporation of 14C thymidine in the thymus is normally about the same as in the liver, although mitoses are so much more frequent in the first organ than in the second. But if a large amount of untagged thymidine ( = carrier) is injected together with tagged substance, the level of incorporation in the thymus is unchanged; the specific activity of splenic or intestinal D N A is increased if less carrier is injected. Thus , the quantity of thymidine available ( = pool) for the thymus is much greater than for the spleen.

Irradiation changes the size of these pools (see p. 343); thus biochemical effects may be mistaken for primary lesions, and are only secondary effects due to varia-tions in the size of the pools.


given during the S period does not inhibit or postpone D N A synthesis. T h e same dose given during the G i period postpones the synthesis in about half of the cells.

Incorporation of tritiated thymidine in the D N A of fibroblasts in vitro is markedly inhibited only if irradiation is performed immediately after putting the cells in culture w h e n the majority are in G i phase2 5 5 . T h u s , there exists in the presynthetic period, G i , a mechanism much more radiosensitive than D N A synthesis itself. T h i s mechanism may be the synthesis of the two enzymes (thymidine kinase and polymerase) which precedes D N A synthesis2 5 6. However, dose for dose the inhibition of D N A synthesis (and presumably of the necessary enzymes) is again greater with x-rays than with a-particles (LAJTHA254). T h i s is the reverse of what is observed for end lesions such as cell death and physiological effects such as mitotic delay. It seems unlikely that the R B E of different radiations would not be similar for the biochemical lesion and for the end effect which it causes. T h i s suggests that inhibition of D N A synthesis is not the factor responsible for mitotic effects.

(ii) T h e r e is no question that inhibition of mitosis is a more radio-sensitive phenomenon than D N A synthesis. M a n y authors insist on this point; it is the reason why, after irradiation, giant cells are so often observed: D N A , R N A , protein and lipid synthesis goes on (maybe after a temporary delay); the nuclei and the cells do not enter mitosis; the cell grows. T h e stimulus for mitosis is not exclusively represented b y the amount of D N A available; there are cases known in early stages of division of the fertilized egg and in Tetrahymena pyriformis257 where mitosis occurs very frequently, without interval, without time for D N A synthesis; the available D N A is divided between the daughter cells. It is a necessity at present to study the question of the regulation of mitotic activity more or less independently f rom D N A synthesis, but so far few promising lines of attack seem to have been proposed.

WHITFIELD and RIXON272 have, however, gathered very useful quantita-tive data which agree with previous work of Caspersson et al. and of K e l l y et al., showing that in non-dividing irradiated cells, D N A synthesis persists until the D N A content per cell reaches the premitotic value and then stops. Whitf ield and Rixon have used the L . strain of isolated cells f rom mouse connective tissue which can be cultivated and manipulated like bacteria. D N A synthesis in this system is relatively insensitive to x-irradiation. T h e principal effect of radiation is inhibition or slowing of cell division. In the absence of cell division, D N A synthesis proceeds until the content per cell is doubled and then stops. A t high doses (4000 r) immediate but temporary inhibition occurs in addition to later inhibition b y attainment of the double or premitotic D N A content per cell. Cell multiplication is twice as sensitive to irradiation as D N A or R N A syn-


theses (Fig. 13-9)272. Here, we find a new concept: it might be the mitotic activity which (at least in certain cells and conditions) regulates DNA synthesis, and not the reverse, as thought a few years ago. Various ob-servations show that in some cells which do not divide there is an incor-poration (quite unhomogeneous in insect chromosomes260) of thymidine in DNA; the rate of incorporation of thymidine or adenine in mouse

Dose, r

FIG. 13-9. The effect of various doses of radiation on nucleic acid and protein synthesis and cell multiplication. Each point represents the amount synthesized relative to the control during

the first 72 hr after irradiation272.

seminal vesicles is much higher than what could be justified by the mitotic index261. One must consider seriously the concept of a certain normal renewal or metabolic turnover of DNA. Various fractions isolated from hepatic cell nuclei of the mouse do not incorporate 32P in DNA at the same rate270. Two fractions have been separated from a rat's carcinoma which incorporate 32P in DNA at the same speed; but after irradiation, DNA synthesis is much more decreased in one fraction than in the other271.

(iii) Radiosensitivity of RNA synthesis—Generally speaking, RNA synthesis is less sensitive to x-rays in vivo than DNA synthesis (see ref. 239). Nuclear and cytoplasmic RNA may behave differently; for instance, exposure of ascites tumour cells in vitro to 750 r inhibits 25 per cent of the uracil incorporation in nuclear RNA without modifying that in


cytoplasmic RNA258. Incorporation of 14C orotic acid in the cytoplasmic RNA of the regenerating liver in vivo is not affected by 1500 r2 5 9 .

But in vitro experiments with isolated thymocytes nuclei (where DNA synthesis is low) have shown that even after low-dose irradiation (50 r) there is an inhibition of 2-14C adenine incorporation in RNA without any change of incorporation in DNA262. Similarly, 32P incorporation in the DNA of appendix cell nuclei is decreased 13 per cent only after 580 r, that in RNA being decreased 59 per cent 269.

M E C H A N I S M S R E S P O N S I B L E F O R D E C R E A S E D

B I O S Y N T H E S I S O F D N A A N D R N A

Soon after irradiation (already 1 hr after) there is an accumulation in the radiosensitive cells or tissues of nucleotides (desoxyribonucleotides and ribonucleotides). This is not new; ERRERA239 and STOCKEN243 mention the fact; but MANDEL and CHAMBON249 have recently given much atten-tion to it. The relative specific activity (after 32P injection) of the spleen ribonucleotides remains constant and normal during the 3 weeks following irradiation, an indication of the fact that their synthesis goes on at a normal rate. At the 21st day, when DNA synthesis in the regenerating spleen is four times faster than normal, the nucleotide triphosphates are 1 -5 times more abundant than normally, but mono- and diphosphates, on the con-trary, less abundant.

Testicular extracts from rats exposed to 500 r of x-rays, incubated with inorganic phosphate in presence of adenosine or guanine formed esters at a decreased rate if the animals were killed 30 or 120 min after irradiation, but at increased rate 24 hr after irradiation293.

Formation of 32P esters from guanosine, inosine or adenosine by extracts of non-dividing, isolated cells (rat erythrocytes) is decreased if blood is taken 24 hr or later after total-body irradiation. This esterification remains low until 6 days later if the dose is 1000 r; there is a more or less complete recovery after 500 r. In the case of guanosine and adenosine, increased 32P incorporation has been seen at 3 hr294 (Fig. 13-10). The recovery after 500 r is attributed to a progressive reduction (reactivation) of the glutathione oxidized during or after irradiation (ref. 294; but see p. 328).

In the rat's thymus, 1 hr after 1000 r, there is a marked accumulation of desoxyribonucleotide mono- and triphosphates, but no thymidine nucleotide seems to be present264. A desoxyuridine phosphate is present which may be a precursor of thymidine; thus, it is not excluded that the synthesis of thymidilic acid may be selectively depressed by irradiation and that this single trouble should naturally be the cause of the decreased DNA synthesis. Indeed, there is an increased urinary excretion of desoxy-cytidine (but not of thymidine) 24 hr after irradiation265; this does not

B I O C H E M I S T R Y O F I R R A D I A T E D L I V I N G O R G A N I S M S 353

occur in other types of trauma. Furthermore, after 14Othymidine injection in irradiated rats, the radioactivity of the thymidine isolated from thymus and spleen during the 24 hr after irradiation is found to be high and variable (two waves are observed) while the radioactivity of the thymidine nucleotides remains low and constant or shows a decrease266.

Adenosine

Utilized

F I G . 1 3 - 1 0 . Comparative utilization of adenosine by rat erythro-cyte extracts294.

Quite possibly, the accumulation of nucleotides after irradiation might not correspond to the normal ratio between purine and pyrimidine bases and consequently the DNA (or the DNA proteins) synthesized from such precursors might be abnormal. There are facts in favour of such an assumption:

(i) Two days after total body irradiation of rats, the splenic DNA shows an increased proportion of guanine and adenine, while the propor-tion of thymine and cytosine is decreased267.

(ii) The antigenic properties of the deoxyribonucleoprotein isolated from spleen cell nuclei of rats 24 hr after total-body irradiation, are changed302.

(iii) Acid hydrolysis, at warm temperature, of DNA from normal yeast cells leaves no purines, while the same treatment of ( 2 x l 0 5 r ) irradiated yeast cells shows the presence of purine bases alongside


with thymine. Acidification of alkaline hydrolysate of yeast results in precipitation of DNA of normal yeast cells, but not of irradiated cells268.*

Following the same path, several authors like E R R E R A 2 3 9 , MANDEL and CHAMBON249 have suggested that accumulation of nucleotides followed by faulty DNA synthesis might be the cause of the increased frequency of mutation. This hypothesis is worth following, but many basic facts are still lacking to complete the picture (see GOUTIER 2 4 4 ) .

Before being polymerized, nucleotides must be phosphorylated to nucleotide triphosphates; this step is affected by x-irradiation as shown by CREASEY and STOCKEN273. One hour after 1 0 0 r, the synthesis of high-energy phosphates by suspension of rat's nuclei is abolished; 25 r inhibit this synthesis 5 0 - 8 0 per cent. The inhibition is already detectable 3 - 5 min after 100 r. Isolated nuclei from histologically radiosensitive tissues (thymus, spleen, lymph nodes, bone-marrow and intestinal mucosa) do phosphorylate nucleotides; nuclei from so-called "radioresistant" tissues (kidney, brain, pancreas) do not. In rats injected with cysteamine 5 min before exposure to 100 r, the inhibition is 90 per cent (instead of 100 per cent in the controls) and disappears so rapidly that at 48 hr nuclear phosphorylation is 90 per cent normal.

Potter has observed a marked inhibition of incorporation of thymidin 1 4 C in nucleotides 1 - 7 hr after irradiation ( 1 0 0 - 3 2 0 0 rep of ^ 0 C y-rays); the incorporation of thymidine 14C in DNA is still more slowed down274. In another material, fibroblasts in tissue culture irradiated in vitro, no decreased formation of thymidinetriphosphate has been seen, although DNA synthesis is decreased255. It would seem worthwhile to search for an effect of irradiation on the presence in cells of a factor that inhibits the action of a DNA polymerase. This factor was present in non-proliferating but not in proliferating tissues307.

Conclusions (a) The interpretation of the results on the effects of ionizing radiations

on in vivo synthesis of nucleic acids in a mammalian radiosensitive tissue, must consider carefully two important factors: (i) more or less rapid cytolysis of a large proportion of cells; (ii) changes in population of cells after irradiation.

(b) Relatively large doses of x-rays inhibit DNA synthesis; moderate doses given before DNA synthesis starts in a cell, postpones it, but does

* 2 - 6 hr after total body exposure to 200-1000 r of x-rays, a higher yield of D N A can be obtained at the extraction with 0-5 and 1 per cent trichloracetate f rom the thymus. Th is fact indicates a labilization of metal linkages by x-irradia-tion because when C a + + is added to the nucleohistone of irradiated animals, the raised yield of D N A decreases to normal values304.


not inhibit. Some cases of increased synthesis by small doses have been published.

(c) x-irradiation does not slow down the synthesis of nucleotides which accumulate in many irradiated tissues. Inhibition of DNA synthesis seems to be due to:

(i) A decreased phosphorylation of thymidine nucleotides to tri-phosphates.

(ii) An inhibition of the specific polymerase. (d) Relevance of interference with DNA synthesis to cellular effects

such as death or mitotic delay is not clear; it may have none.

T H E N U C L E A S E S A special interest attaches itself to the nucleases in radiobiochemistry because they have a great importance in the fate of nucleic acids after irradiation.

A. Ribonucleasesiii

One finds in the liver of the rat* an alkaline RNAase (optimum pH 7-8-8-2) and an acid RNAase (optimum pH 5-8). The measurement of the activity in vitro of the alkaline enzyme is complicated279 by the presence of a natural inhibitor which can be removed by treatment with 0-25 N H2SO4. The acid RNAase is mainly localized in the lysosomes, the alkaline enzyme being inactive in the supernatant and active in the mitochondria and microsomes.

Total-body irradiation (600 r) not only increases the total activity of acid RNAase of the liver (50 per cent at the 2nd day) or of the spleen and thymus, but also changes the relative activity of the various fractions isolated from the latter tissues. For instance, the distribution of total ribonuclease activity in subcellular fractions of control spleen homogenates was 16-0, 35-7, 20-4 and 28-4 per cent for the nuclear, mitochondrial, microsomal and supernatant, respectively. Sixteen hours after irradiation (one would so much like to have data about earlier periods), the distribu-tion had changed to 8-7, 29-3, 17-6 and 44-8 per cent and at 64 hr it was 4-8, 15-0, 14-1 and 66-0 per cent. Thus, there is a great loss of enzyme activity by the particulate fraction and a corresponding gain of the super-natant280. This is the shift from structure-bound enzyme to free enzyme which is to be expected from the enzyme release theory explained on p. 272, our main working hypothesis.

* We should like to emphasize the fact that too many biochemical studies are exclusively concerned with the liver of the rat for the sake of technical convenience. One should resist the tendency to extend to other organs and other animals the results obtained with this single organ. For instance, it is known at present that the utilization of glucose is different in man and rat.

24


Exposure of the mouse to small doses (40-160 r) results in an increased activity of both RNAases (calculated per cell, per mg of DNA) in the thymus; it begins at 8 hr for the acid RNAase, but is already observable 20 min after irradiation for the alkaline enzyme. The maximal increase— ten times the normal value—is reached at 24 hr for the alkaline RNAase281. The increase of alkaline RNAase per cell is linear at 24 hr in function of the dose. Hydrocortisone which has a similar effect to x-irradiation on weight and cell population of the thymus, increases the RNAase activity only three times. The progressive destruction of the natural inhibitor is a possible explanation of this remarkable increased activity after x-irradiation; the activity of acid RNAase is increased by a factor of 2 only281. It is not easy at the present time to understand the function of intracellular RNAases and their increased activity after irradiation, but one must not forget that when added to the medium of cultured tissues and organisms or injected in mammals, pure crystalline RNAase is a growth inhibitor282.

B. Deoxyribonucleases24i

There are also two classes of DNAases: DNAase I (neutral or alkaline) mainly localized in mitochondria, and DNAase II (acid, optimum pH normally at 5-15) bound to lysosomes, away from the substrate localized in the nucleus. The specificity of these two enzymes is different: the acid DNAase degrades DNA to nucleotides of higher molecular weight than that obtained after hydrolysis by neutral DNAase; the products of de-gradation by acid DNAase are substrates for the neutral enzyme. Natural inhibitors of DNAase I have been found in tissues, blood and urine; activators exist, at least in plasma. Urine contains an inhibitor of the acid DNAase II.

Irradiation produces quantitative and qualitative changes in DNAase activities.

The activity of acid DNAase from the spleen of the rat after irradiation (850 r of x-rays on total body) in relation to pH shows two optima, the usual at pH 5-15 and a second at pH 4-8. (Fig. 13-11.) Careful analysis of this fact283 shows that various treatments (purification, ageing, etc.) of the pH 5-15 enzyme can shift the optimum to pH 4-8. This latter form of the enzyme seems more stable. Thus, irradiation simply accelerates a normal behaviour of the acid DNAase.

Generally speaking, the activity of acid DNAase in homogenates of radiosensitive tissues (spleen of rat, thymus of mouse, bone-marrow) is increased after irradiation (see ref. 244). This change may be observed at 6 hr or only later at the 12th hour; it is maximal after 24 or 48 hr, or only at the 7th day in the case of bone-marrow. In the spleen and thymus it drops after 2 or 3 days. The activity calculated per cell of the thymus of mouse is multiplied by a factor of six, 24 hr after 160 r total body


irradiation. A dose of 780 r increases it by a factor of twenty at the 2nd day.

The interpretation of these facts is not easy. If the DNAase II activity of the splenic homogenates is increased at 24 hr after irradiation when calculated in relation to DNA content or to the weight of the wet tissue, the total DNAase activity of the spleen, of the whole organ is the same as that of the normal spleen284. The weight falls sharply after irradiation. Is it possible to speculate that the DNAase remains in the splenic cells while the amount of proteins and other tissue constituents decrease? But at the same time, the activity of the two DNAases increases at 18 hr in the serum after irradiation, twelve times for the acid enzymes, twice for the neutral285. Urinary excretion of these enzymes is three times higher than normal286.

pH

F I G . 1 3 - 1 1 . Acid DNAase activity of splenic homogenates (in 0-25 M sucrose) measured in function of the pH in 3 normal and three groups of 3 irradiated rats killed 30 min, 3 hr, or 6 hr after total-body irradiation (850 r of 250 kV x-rays).295

Ordinates, drop of the logarithm of the viscosity in 20 min per mg/N.

These enzymes in blood and urine must come from certain tissues. Not from the spleen certainly; indeed, splenectomy alone, without irradiation, produces an increase of DNAases in serum, urine and bone-marrow identical with that produced by x-rays287 ; irradiation should cause a kind of functional splenectomy and the changes of DNAase activity in


body fluids might be secondary to this loss of function * Changes in cell population cannot give a reasonable explanation. Marked decrease in the amount of neutral serum DNAase inhibitor has been observed in the spleen 1 or 2 days after irradiation. But neutral DNAase is quite unimportant in the spleen and no inhibitor of acid DNAase has been describedt.

Inside the cells, or in an homogenate containing intact granules, a great deal of the acid DNAase is bound to the structure and has no access to the substrate. The phenomenon described for RNAase by R O T H 2 8 0 — liberation of the enzyme from the granules in the supernatant—might also occur for DNAase, although it has not been published. There are good grounds for the belief that the structure of the granules is damaged, when irradiated, even in vitro288.

If the spleen is homogenized in 0-44 M sucrose, the integrity of the organelles is respected and the acid DNAase activity is three times less than when the organelles are destroyed by homogenization in water; the difference between the two kinds of homogenates is smaller if the spleen has been irradiated289. Moreover, the activity of the homogenate in water from the irradiated organ is greater than that of the normal; thus, the total amount of enzyme, the number of enzymatic molecules may be increased by irradiation.

In vitro irradiation by 60Co y-rays of a liver mitochondria suspension increases the DNAase activity although the enzyme remains bound to the organelle which appears altered in the electron microscope. In this case—which may be very different from what happens in vivo—the burst-ing of the granules simply favours the contact between enzyme and sub-strate, the enzyme being probably bound to the membranes of these organ-elles. hi vivo, the mitochondrial or lysosomal DNAase shall never reach the DNA in the nucleus if it is not "liberated", "solubilized" in the cytoplasm.

Unfortunately, the normal, the physiological role of the DNAases is not known (see ref. 244).

Conclusions Total-body irradiation induces a marked increase of activity of the

nucleases in the radiosensitive tissues. Besides the activation of these enzymes, there may be also an increased synthesis of them. The origin of the activation is very probably due to an easier contact between enzymes and substrate, a consequence of the breakdown of the granules where the enzymes are bound.

* The reader must never forget that it is not possible to make the total balance of such enzymes in a big organism like a rat, even with a large margin of error.

f DNAases and other intracellular enzymes are not inactivated by the cathep-sins which are also liberated by ionizing radiation.


S U M M A R Y

To sum up, it is surprising to find that, except during the last days of life, little change in general metabolism is caused by irradiation with the minimum 100 per cent lethal dose of x-rays or less. Carbohydrates, fats and proteins are still absorbed, in spite of the precarious anatomical condition of the intestines. The disturbances which are observed are of a trivial type and can often be regarded as secondary to more or less marked anorexia, the loss of water and electrolytes resulting from diarrhoea, vomiting or disturbances in capillary permeability. There are endocrine disturbances in irradiated mammals, but they cannot explain all that happens during the first hours after irradiation. No inhibition of the sulphydryl enzymes has been observed in vivo.

The few points about which there seems to be agreement are: the slowing down of desoxyribonucleic acid synthesis; the early increase in catabolic and anabolic processes in the bone-marrow, spleen and reticulo-cytes.

An appreciable number of investigators are impressed by two series of facts: (i) the disturbance in "osmotic" balance in the various intra-cellular structures (nuclei, nucleoli, mitochondria, microsomes, etc.). (ii) The uncoupling of oxidative phosphorylation; but, as all who have studied this problem agree, we are far from being able to explain in biochemical terms why some cells are much more radiosensitive than others. There is, however, no doubt that the concept of a biochemical lesion is valid; that radiation sickness is the result of a series of biochemical lesions is proved by many definite facts. The energy absorbed by a cell during irradiation of a few hundreds of roentgens, sufficient to kill many cells, is extremely small and can only bring about a chemical change in a few hundred molecules per cubic micron u3 = IO-12 ml) of tissue.

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Oliver & Boyd, Edinburgh, 1957, p. 33 222. GOULD, R. G., Los Alamos Sci. Lab., No. 2179, August 1958 2 2 3 . VERLY, W . and BACQ, Z . M . , Unpublished observations, 1 9 5 7 224. GORDON, S. A., Quart. Rev. Biol., 1957, 32, 3 2 2 5 . M A N D E L , P . , GROS, C . M . , RODESCH, J . , JAUDEL, C . a n d CHAMBON, P . ,

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2 2 8 . PAULY, H . and RAJEWSKI, B . , Strahlentherapie, 1 9 5 6 , 9 9 , 3 8 3 2 2 9 . JACKSON, K . L . , RHODES, R . L . a n d ENTENMAN, C . , U . S . N . R . D . L . , T R -

165, June 1957 2 3 0 . CASTER, W . O . and ARMSTRONG, W . D . , Radiation Research, 1 9 5 6 , 5 , 1 8 9 2 3 1 . SKALA, M . , Experientia, 1 9 5 9 , 1 5 , 1 5 3 2 3 2 . MOUTSCHEN, J . , Radiobiologica latina, 1 9 5 9 , 2 , 1 7 233. MOUTSCHEN, J . , BACQ, Z. M . and HERVE, A., Experientia, 1956, 1 2 , 314 2 3 4 . K A Y , R . E . a n d ENTENMAN, C . , U . S . N . R . D . L . , T R - 2 7 8 , N o v . 4 , 1 9 5 8 235. LANGE, R . , P I H L , A. and ELDJARN, L . , Intern. J. Radiobiol., 1959, 1 , 73 2 3 6 . LESSLER, M . A . , Radiation Research, 1 9 5 8 , 9 , 1 4 4 237. LEBEZ, D . , KLAMENC-SEBEK, S . and STRAUCH, L . , Proc. Second U.N.

Intern. Conf. on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 533, United Nations, 1959

238. HAGEN, U . , Strahlentherapie, 1958, 106, 277 239. ERRERA, M., In Handbuch der Protoplasmaforschung (Edited by L. V.

HEILBRUNN and F. WEBER) , Vienna, Vol. 1 0 , 1 9 5 7 2 4 0 . HOWARD, A . , Ciba Foundation Symposium on Ionizing Radiations and Cell

Metabolism, Churchill, London, 1956, p. 196 2 4 1 . K E L L Y , L . S . , Progress in Biophysics, 1 9 5 7 , 8 , 1 4 4 2 4 2 . HOLMES, B . E . , Ann. Rev. Nucl. Sci., 1 9 5 7 , 7 , 8 9 2 4 3 . STOCKEN, L . A., Radiation Research, 1 9 5 9 , Suppl. 1 , 5 3 244. GOUTIER, R., Progress in Biophysics, 1960, In press 245. O R D , M. G. and STOCKEN, L . A., Biochem. J., 1956, 63, 5 246. NYGAARD, O. F . a n d POTTER, R . L . , Radiation Research, 1959, 10, 462 2 4 7 . SHERMAN, F . G. and QUASTLER, H . , Radiation Research, 1 9 5 8 , 9 , 1 8 2 2 4 8 . M O L E , R . H . and T E M P L E , D . M . , Intern. J. Radiobiol., 1 9 5 9 , 1 , 2 8 2 4 9 . M A N D E L , P . and CHAMBON, P . , Intern. J. Radiobiol., Suppl. 1, 1960, p. 71 250. HEVESY, G., "Ionizing radiation and cellular metabolism", Oberlin Sym-

posium Radiobiol., John Wiley, New York, 1952, p. 189 251. C I M A , L . , FASSINA, G. and POZZA, F . , Exptl. Cell Research, 1959, 1 7 , 1 2 5 2 . HOLMES, B . E . , Ciba Foundation Symposium on Ionizing Radiation and Cell

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1957, 17, 117 254. LAJTHA, L . G . , OLIVER, R., KUMATORI , T . and E L L I S , F., Radiation Re-

search, 1958, 8, 1 255. PAUL, J., The Cell Nucleus, ed. by J. S. MITCHELL, Butterworths, London,

1960, p. 147 2 5 6 . BOLLUM, F . J . , ANDEREGG, J . W . , M C E L Y A , A . B . a n d POTTER, VAN R . ,

Caneer Research, 1960, 20, 138 2 5 7 . DUCOFF, H . S . , Exptl. Cell Research, 1 9 5 6 , 11, 2 1 8 258. HARBERS , E. and HEIDELBERGER, C .,J. Biol. Chem., 1959, 2 3 4 , 1249 2 5 9 . BELTZ, R . E . , V A N LANCKER, J . and POTTER, VAN R . , Cancer Research, 1 9 5 7 ,

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263. MARKEES, S., KASER, 0 . and LANZ, R., Schzveitz Med. Woehensehr., 1950, 80, 1079

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721; and Proc. Second U.N. Intern. Conf. on the Peacefid Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 206, United Nations, 1959.

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267. BERENBOM, M . a n d PETERS, E . R . , Radiation Research, 1956, 5, 515 268. CHANTRENNE, H. and DEVREUX, S., Biochim. Biophys. Acta, 1959, 31, 734 269. Y U O N G - T C H E L T C H O E and SIBATANI, A., Nature, 1959, 183, 1334 270. M O R I N , G . A., ZAJDELA, F. and COSTEROUSSE, O., Exptl. Cell Research,

1957, 13, 204 2 7 1 . HARBERS, E . and BACKMANN, R . , Exptl. Cell Research, 1 9 5 6 , 10, 1 2 5 272. W H I T F I E L D , J. F. and R I X O N , R . H., Exptl. Cell Research, 1959, 18, 126 2 7 3 . CREASEY, W . A . and STOCKEN, L . A . , Biochem. J., 1 9 5 9 , 7 2 , 5 1 9 274. POTTER, R . L . , Federation Proceedings, 1959, 1 8 , I , No. 1204 2 7 5 . Z I N S , G . , RAYMUND, A. B . , DUBOIS, K . P . , U . S . A . F . , Rad. Lab., Quart.

Progress Report No. 29, Oct. 15, 1958 276. BEKKUM, D . W. VAN, Ciba Foundation Symposium on Ionizing Radiation

and Cell Metabolism, Churchill, London, 1956, p. 77 277. JACKSON, K. L . , RHODES, R . and ENTENMAN, C., Radiation Research, 1958,

8, 361 2 7 8 . JACKSON, K . L . and ENTENMAN, C . , Radiation Research, 1 9 5 9 , 10, 6 7 279. PIROTTE, M. and DESREUX, V., Bull. Soc. Chim. Beige, 1952, 61, 167 2 8 0 . R O T H , J . S. and E ICHEL, H. J., Radiation Research, 1 9 5 9 , 11, 5 7 2 2 8 1 . W E Y M O U T H , P . P . , Radiation Research, 1 9 5 8 , 8 , 3 0 7 282. BRACHET, J . , Biochemical Cytology, Academic Press, New York, 1957 283. GOUTIER, R., et. al., Intern. J. Radiobiol. Suppl. I, 1960, p. 93 2 8 4 . OKADA, S . , GORDON, E . R . , K I N G , R . a n d HEMPELMANN, L . H . , Arch.

Biochem. Biophys., 1957, 70, 469 2 8 5 . KOWLESSAR, O . D . , A L T M A N , K . I . and HEMPELMANN, L . H . , Arch. Biochem.

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Biophys., 1956, 64, 164 293. RAPPOPORT, D . A., Radiation Research, 1959, 11, 229 294. RAPPOPORT, D . A. and SEWELL, B . W., Nature, 1959, 184, 846 2 9 5 . GOUTIER, R . and GOUTIER-PIROTTE, M . , C.R. Soc. Biol., 1 9 5 7 , 151, 1 4 4 5 296. K A Y , R . E . , HARRIS, D . C. and ENTENMAN, C., Am. J. Physiol., 1956, 1 8 6 ,

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Experientia, 1957, 13, 76 303. E ICHEL, H . J . and R O T H , J . S., Radiation Research, 1960, 12, 258 3 0 4 . HAGEN, U., Biochem. J., Meeting 2 9 - 3 0 June, 1 9 6 0 of Biochem. Soc. 3 0 5 . C H A N G , J U - T I N G , Scientia Sinica, 1 9 6 0 , 9, 6 7 1 306. H E L L , E., BERRY, R . J . and LAJTHA, L. G., Nature, I960, 1 8 5 , 4 7 3 0 7 . WEISSMAN, S . M . , GRAY, E . D . , T H O M S O N , R . Y . , SMELLIE, R . M . S . a n d

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biology Symposium., Moscow, 1960, to be published by Academic Press, London

C H A P T E R 1 4

Processes of Restoration after Irradiation

ONE OF the great problems in theoretical and practical radiobiology is whether it is possible to influence the restorative processes which tend to re-establish normal physiological conditions after irradiation. Its theroetical importance lies in the fact that its solution may indicate what happens during irradiation, and from the practical point of view the answer will provide the scientific basis for the treatment of irradiation victims.

In theory, several kinds of restoration are conceivable5'9: (i) Neutralization of the primary effects produced at the molecular

level by the free radicals or by direct action. Although in model experi-ments it has proved possible to repair damaged molecules by a transfer reaction (see p. 177) it is highly improbable that this can be done in vivo after irradiation, since the damaged molecules rapidly undergo further irreversible changes. Restitution of a damaged macromolecule belongs to the field of protection as the restoring substance must be present before irradiation because there is no time to introduce it afterwards.

(ii) Neutralization of the secondary effects. The cells resynthesize molecules which irradiation has destroyed or inactivated. This will be referred to as intracellidar recovery.

(iii) In a given tissue, many cells may be killed, but those which are left divide actively after a latent period and replace the dead cells. This type of recovery (or better of repair) is usual with haematopoietic tissues and epithelia. Some injury is reparable; some, apparently, is not (see Fig. 14-1). Experimental facts are not easy to reconcile with existing theories46.

It is much easier to accelerate intracellular restoration after ultra-violet irradiation than to influence cellular lesions due to ionizing radiations if action can only be taken after irradiation. With regard to ultra-violet light, LATARJET'S and KELNER'S investigations1-12 have shown in micro-organisms how great is the importance of restoration by catalase (which destroys the peroxides formed in the system) and by visible and infra-red \ight-photorestoration. This photorestoration or photoreactivation after

369


ultra-violet irradiation is a general phenomenon. It has been observed not only with microorganisms but with ova or spermatozoa of echino-derms2 and with an insect (Habrobracon) egg13. However, if the same methods of restoration are tried on the same isolated cells or organisms after irradiation with x-rays, the results are generally negative1-2*. In this respect the behaviour of ionizing radiations is quite different from that of ultra-violet radiations. B L U M et al.2 suppose that the principal lesion in the ova of Arbacia caused by ultra-violet as well as by x-rays is in the nucleoproteins of the nucleus whereas photorestoration takes place in the cytoplasm.

PtiaseI PhaseH PhaseJE • Phase M

Predominance of destructive tissue change

Predominance of regeneration and repair. Arterioio-capillary fibrosis becomes more advanced than in controls

Normal or maximally recovered parenchymal cellularity Length of ptiasedepends inversely on dose size Arteriolo-capillary fibrosis progressing and advanced

Parenchymal aging Earliness of onset and rate depend directly on dose size. Arteriolo-capillary fibrosis progressing

FIG. 14-1. Diagrammatic representation of injury and recovery following a large dose of whole-body irradiation73.

R E S T O R A T I O N O F G E N E T I C D A M A G E A N D O F R E P R O D U C T I V E C A P A C I T Y

It is convenient to consider separately intracellular recovery from damage which involves the reproductive capacity (including genetic damage) and physiological effects that involve functional factors.

Microorganisms These constitute a very favourable material for the study of restoration

processes after irradiation. STAPLETON and HOLLAENDER5'7 observed that if E. coli is incubated at

* T h e lethal effect of x-rays on dividing yeast cells can be slightly decreased by small ultra-violet exposures delivered after (or before) x-ray exposure. Th is fact suggests overlap between x-ray and ultra-violet lethality sites47.

P R O C E S S E S O F R E S T O R A T I O N A F T E R I R R A D I A T I O N 3 7 1

a suboptimal temperature* after irradiation, partial restoration takes place, judging from the percentage of cells capable of forming colonies after transplantation. The temperature which allows the maximum of restora-tion when bacteria are incubated after irradiation is different for every strain, and is characteristic of that strain. For example, the best tem-perature for restoration in E. coli is 18°C; there is much less restoration at 30° and 6°C (see Fig. 14-2). The incidence of mutations is also much

Temoerature. 0C

FIG. 14-2. Survival of E. coli B/r as a function of temperature after doses of x-rays as follows: • 40 kr ; O 60 kr; A 80 kr11.

lower if irradiated E. coli are incubated at the optimal temperature for restoration. Restoration under these conditions is not, however, a pure temperature effect since storage under suboptimal temperatures has no effect if the cells are kept on synthetic media8 or in the absence of oxygen. The implication is that a limited amount of metabolism is necessary and there would appear to be a close connection with the restorative processes for ultra-violet damage discovered by WITKIN60. She showed that treat-ment with streptomycin, which allows continued metabolism but prevents cell division, greatly reduces the number of mutants. KIMBALL61 extended these investigations to ionizing radiations using Paramaecia. Again post-irradiation treatments that limited metabolism but did not stop it, brought

* A suboptimal temperature is one which is lower than that which allows maximum growth of the normal strain, but still allows some enzymatic activity.

25


about substantial intracellular recovery. Applying these same principles recovery was obtained by cultivating E. coli irradiated with x-rays on media suboptimal for growth26.

HOLLAENDER7 found a substance in the spleen of mammals which is soluble in acids and alcohols, fairly resistant to heat and which promotes restoration in x-irradiated bacteria. The chemical nature of this substance is unknown, but an apparently identical factor has been found in yeast extracts. It has been shown by KANAZIR and his collaborators74 that highly polymerized isologous DNA after irradiation enhances the re-establishment of cell division mechanisms of cultures of Salmonella typhimurium irradiated by ultra-violet or x-rays. This effect is not a nutritional one, since neither heterologous DNA nor lower molecular weight isologous DNA, nor DNA digests or any nucleotides or nucleosides show any favourable influence.

The data accumulated by the Oak Ridge group of microbiologists suggest that the survival of irradiated bacteria depends on the relative rates of synthetic and destructive processes and on the synthesis of new enzymes27. Naturally, if the biochemical mutants of an irradiated micro-organism are supplied with the substances which this mutant is unable to produce, "restoration" of the normal growth conditions would appear to have been obtained. Similarly, if one adds to the culture medium an excess of a substance which the irradiated organisms metabolize in greater quantity than normals, one again favours the growth; in this way irradi-ated Tetrahymena pyriformis may be restored with acetate10.

Insect Eggs Eggs of the silk worm Bombyx mori have been used for restoration

studies3-4. The criterion used is hatching. By fractionating the dose it was shown that restorative processes may be demonstrated if the eggs are incubated at 21°C after irradiation; if the eggs are kept in the refrigerator between two irradiations the effects of them are nearly cumulative; there is practically no restoration. The optimal time for the demonstration of recovery is 8 hr for the eggs Bombyx mori, 24 hr for those of Bacillus rossii3A.

The radiation damage, both physiological68 and genetic77, in seeds is critically dependent on post-irradiation conditions, such as temperature, water content and oxygen concentration but the situation is too complex to be capable of biochemical interpretation at the present time69.

Chromosome Breaks SHELDON W O L F F 6 4 has shown that the number of the different chromo-

some abnormalities that can be produced, depends on the metabolic state of the cell after irradiation. Interference with respiration by inhibition of


heavy metal enzymes with cyanide or of the cytochromes by carbon monoxide increases the frequency of certain forms of damage. Interference with the formation of essential energy such as ATP, by anoxia after irradiation or by dinitrophenol similarly increases the amount of damage. More recently, SHELDON WOLFF65 has claimed that the antibiotics which promote restoration in bacteria increase the amount of chromosome damage in plant material.

Of more relevance to this chapter is the finding that soaking seeds in a solution of ATP reduces the amount of mitotic damage presumably by promoting aerobic glycolysis. The reduction in genetic damage seen in Drosophila oocytes66 and Chlamy domonas61 by an increase in oxygen tension can probably also be interpreted as an effect due to increasing glycolysis.

These experiments suggest that an important factor in promoting intracellular recovery is the existence of a temporary imbalance between glycolysis and the protein and nucleic acid synthetic processes.

Mammalian Cells Warm-blooded animals are not very suitable for such studies on restor-

ation, since it is by varying the temperature and the nutritional conditions after irradiation that the processes of restoration can be influenced in silk-worm eggs and in micro-organisms.

The possibility that recovery might be promoted merely by delaying mitoses while keeping the cells under normal physiological conditions has been tested in two series of experiments in which mitoses were made to occur at different times after irradiation. The technique employed was to irradiate the whole animal and after different intervals of time to stimulate cell division in the liver by the necrotic action of carbon tetrachloride62 and in the thyroid by the administration of a hormone63. The number of cells showing abnormal mitoses was the same—within the very large experimental error inherent in such studies—whether the divisions were made to occur immediately after irradiation or some months afterwards.

Mutations in the male mouse are not restored in the weeks and months after irradiation and the proportion of mutant offsprings is the same whether they were mated immediately after irradiation or some months later*. All this, however, does not mean that there is no repair under normal physiological conditions, but only that the time after irradiation, during which repair occurs, is relatively short. Experiments by ELKIND and SUTTON76 with hamster cells in tissue culture showed that recovery

* Yet there is evidence for restitution of some mutations during oogenesis72 and the influence of dose-rate on the number of mutants produced also suggests the intervention of post-irradiation factors (see p. 258).


occurred in the time between irradiation and the first division. The dose-response curve of these cells showed a marked threshold (corres-ponding to five-hits by target theory!) which means that damage must be accumulated before an effect is observed. From this accumulation of sub-lethal damage the cells recover completely if allowed to stand under normal conditions for some hours. Two irradiations of 505 r spaced by 18 hr produce a much smaller effect than a single dose of 1000 r. Moreover, following a single dose of 505 r, subsequent irradiations given 18 hr later give rise to a curve with a threshold. This means that the cells that sur-vived from the first 505 r dose, had recovered completely from their sub-lethal damage and behaved as if they had never been irradiated. In a separate experiment ELKIND and SUTTON76 showed that it took 1 0 hr for this restoration to be complete (see Fig. 14-3).

irradiation at 720 rads/min; (O) paired irradiation, initial dose 505 rads followed 18 hr later by a second dose.76

Modem tissue culture techniques also make it possible to test if post-irradiation procedures, which limit maximum metabolism,* restore mam-malian cells as is the case with micro-organisms and plant material. SHAPIRO et A / . 7 9 find that arginine restores ascites cells. Also, the observa-

*Weiss finds the radiosensitivity of Hela cells is not altered by irradiation at 1°C followed by storage for 55 min at 1°C before incubation at 37°C. By analogy with bacteria (see p. 371) restoration would not be expected under these conditions as it requires metabolism at sub-optimal temperature.


tion that the addition of cysteine after irradiation is able to promote re-covery in hibernating animals and in ascites cells (see p.467), if they are stored at 4°C, suggests that there is a stage after irradiation during which external factors can assist the repair processes. This stage, however, only persists long enough for post-irradiation treatments to be applied if the rate of metabolism is greatly reduced. At ordinary temperatures cysteine given after irradiation has no effect.

R E C O V E R Y F R O M P H Y S I O L O G I C A L I N J U R I E S Techniques for the study of early effects, and specially of reversible physiological effects, are perfectly suited for demonstration and accurate measurement of the speed and magnitude of recovery processes. Here are three striking and elegant instances :

(a) Luminous bacteria, Achromobaeter Fischerii, cultivated in agar in certain conditions, emit light at constant intensity which can be easily recorded continuously and automatically by a photomultiplier and a galvanometer48. During irradiation the emission of light decreases, the slope being sharper when the rate increases (Fig. 14-4). As soon as irradi-

Time, min

FIG. 14-4. Effect of x-rays on light emission by Achromobacter Fiseheriiii.

ation ceases the intensity of light increases, showing that some of the biochemical alterations induced by irradiation are instantaneously re-paired. But the intensity of light does not come back to normal; there is


a plateau at a level somewhat below normal which indicates the non-reparable (or not easily reparable) damage48.

(b) The growth of certain fungi of the Phycomyces genus is rapid and can be made regular by careful control of the experimental conditions49. If such an organism is irradiated (at very low dose-rates indeed, compare Fig. 14-4 with Fig. 14-5) the growth rate is slowed down until a kind of

120

„ 110 0

1 ' 0 °

o O 90

S« j - 8 0

40 - 6 - 4 - 2 0 2 4 6 8 IO 12 14 16 18

Time, min

FIG. 14-5. Percentage growth-rate changes in sporangiophores after 0-8 r, given in 30 sec from a 60Co source. Normal growth

rate 58/x/min = IOO49

equilibrium is reached. As soon as irradiation ceases the rate of growth increases, shoots over the normal value and comes back to normal (Fig. 14-5). Here recovery seems nearly complete; the total growth being slightly smaller than normal. If a single small dose is given then recovery may be

IOOi 1 ! 1

k -Tim i of ir adiation

I r I

O

IV I \ I \ I ^ I I

I I

I I 1

0 1 2 3 4 6 8 IO

Minutes after X-irradiation

FIG. 14-6. Recovery from reduction in rubidium uptake by bean roots (Phaseolus aureus) following a dose of 2000 r of

x-rays50.


complete, the overshoot compensates for the temporary decreased growth rate49.

(c) TANADA50 has measured the absorption of rubidium by excised mung bean roots (Phaseoliis aureus) which, in certain conditions, is very sensitive to ionizing radiations. At the end of 2 min irradiation (at the rate of about 2000 r/min of soft x-rays) the rubidium absorption is 80 per cent of normal; it continues to drop to a minimum of 60 per cent 2 min after the end of irradiation. Then recovery starts and 8 min later the salt absorption is 93 per cent normal; there is very little irrecuperable damage (Fig. 14-6). Naturally, this does not exclude the possibility of later troubles resulting from anatomical lesions to the roots.

Experiments of that kind offer ideal starting points for quantitative studies of the kinetics and the biochemistry of recovery processes.

R E P A I R I N M A M M A L S The existence of repair in mammals that have been irradiated is obvious and symptomatic recovery from radiation sickness often appears to be complete. The extent of the recovery cannot be measured by a single test and the existence of delayed effects (Chapter 17) shows unambiguously that restitution is not complete. Since following a large dose of irradiation the animal's life-span is shortened, there must be some non-reparable injury. There is no a priori reason why the damage which expresses itself at the end of the animal's life should manifest itself in a short-term survival test (e.g. "bone-marrow death"). For the mouse the L D 5 0

radiation dose largely represents the sensitivity of the animal to haemo-poietic injury and damage to other organs will not be seen in this test.

Since changes in the magnitude of the lethal dose are virtually the only quantitative measurements we have of the recovery process it is obvious that one must be careful about generalizations from the data. Thus, changes in fertility follow quite a different pattern in time. One way to look at the processes of recovery is to try to estimate the injury which remains at any one time15 according to the following technique: the animals receive a single dose below the lethal range (350 rads, for instance) of whole-body x-rays, followed by a second test dose at appropriate interval. L D 5 0 in 30 days is then measured and recovery from the first dose can be calculated according to standard statistical methods. The rational and detailed experimental and statistical procedures are well explained by BLAIR70 and by K O H N and KALLMAN22. Such simple concept and experi-ments lead to more or less consistent results: the time necessary for the dissipation of half the amount of injury inflicted by x-irradiation is 8-5 days15 or 8 days18 for rats, 7-4 days for mice16 and 4-8 days for monkeys17


(see Table 14-1*). When the problem is looked at in greater detail as has been done by MOLE19 and MELVILLE et al.28 using mice, and by K O H N

and KALLMAN20 using hamsters, then the data do not fit such simple rela-tionship. For example, the rate of recovery depends on the size of the dose28. From Fig. 14-7 it can be seen that the repair obtained (i.e. the

90

70

1 > 50 1/1

3s

30

IO

FIG. 14-7. Survival at 30 days of female mice following a single dose and paired doses of x-rays28 separated by different time

intervals.

increase in the total dose obtained by pairing the dose) is greater for doses sufficient to kill only a small proportion of the animals (i.e. greater for the LDio than LDgo dose).

Let us analyse the possible difficulties involved. As usual, the validity of the results depends very much on the assumptions which are basic to the mathematical reasoning. If the data fit an equation, one is not allowed to conclude (although the temptation is great) that the basic assumptions are correct. The lack of precision of biological data is such that different types of equations often can fit experimental data.

(a) The conditioning dose (the first dose of which one wants to estimate the restoration) must not be too small (because the differences in the response to the second, the test dose, may be insignificant) nor too large (because if it kills more than a few per cent of the animals, the second test dose will be applied to a selected population). In order to avoid the diffi-culty and nevertheless use bigger doses, some authors have given rather large doses (up to 1200 r) in several exposures21.

(b) The repair processes may not start immediately after the end of

* T h e facts summarized in Table 14-1 are strikingly confirmed by the observa-tion that, in rats, x-rays (1600 to 2400 r locally) do not retard the healing of skin burns inflicted beyond one or two weeks after irradiation24.

\ V V I • \ \ A \ \ J

• \ « \ • \\ S.

K

-

yv W VA \ \ A \ N1O >

-

K

W V N I

\ i NsD

500 600 700 800 900 1000 Total dose, r


T A B L E 1 4 - 1

RECOVERY I N M A M M A L S , EXPRESSED AS T I M E FOR 5 0 PER CENT RECOVERY = R T / 5 0 2 3 .

Animal Lethality

Recovery

Animal Lethality

Injuring dose Period

studied (days)

Expo-nential curve j

RTgo § (days)

Animal

Age (days)

L D 5 0 ( ~ 3 0 ) * (r)

K T f (days)

% of LD 5 0

(r)*

Period studied (days)

Expo-nential curve j

RTgo § (days)

Mouse A/He, <? 9 91-142 619 14 57 350 8 Yes 1-6 BALB/c,

S 9 95-219 552 15 63 350 14 Yes 2-8 CAFi, <J 9 91-231 673 12 52 350 14 Yes 2-1 C3H, 9 94-137 623 15 56 350 8 Yes 2-0 C57BL,

<J 9 107-174 614 10 57 350 10 Yes 1-8 A/Manch,

<? S 42- 56 518 — 50 260 20 Yes 7-4 CBA, <J 60-120 751 — 27 200 11 Yes ~ 2 - 0 CBA, <J 60-120 751 — 80 600 29 Yes 12-0 CBA, 9

(parous) 150-180 883 — 45 400 5 Yes ~ 3 - 0 Rat

FAC(I)Fi, S 9 96-212 570 10 55 315 21 Yes 4.9

Sprague-Dawley, adult 600 10 50 300 21 Yes 8.5 Of *o (—200 gm)

Hamster Syrian,

<? 9 105-240 611 13 53 322 7 No 6-1 Monkey

M. mulatto, S 9 700-1800 570 15 46 260 20 As- 4-8

sumed

* Unless specified otherwise, r(tissue dose); r(tissue dose) x ~0-96 = absorbed dose in rads19.

t Mean killing time for death after L D 5 0 , based on decedents only. J Can be treated as in I = A —BT, where I is injury and T is in days. § Time for 50 per cent recovery was estimated with the "fit ted" equation or

by graphical interpolation.


irradiation. There may be a lag: the injury may not be "expressed at once". MOLE1 9 found marked fluctuations in the L D 5 0 of mice during the first 24 hr after a single dose of 400 r, an early fall being followed by a rise. It has been observed15'23 with the above-mentioned technique that injury from radiation decreases exponentially as far as rats or mice are concerned. This exponential relationship has been assumed for mon-keys17; it is not accepted by SACHER25.46; it is not true for hamsters20.

The mortality of mice exposed to paired doses of fast neutrons corres-pond to that of x-irradiated animals, suggesting that recovery from acute irradiation injury is comparable for both types of radiations28 although considerable greater-delayed residual damage has been observed in animals and men chronically exposed to fast neutrons.

Non-recuperable Injury If the interval between the two doses is long (4 to 10 weeks in mice)

one measures only the non-recuperable injury caused by the first dose21. According to BLAIR70, the non-recuperable injury, as assessed by the L D 5 0

test, is proportional to the initial radiation dose and corresponds to approximately 20 per cent of this dose (i.e. the L D 5 0 of an animal that has received 500 r is lowered by 100 r after all the recovery that can take place has taken place).

Many attempts have been made to check the correctness of this assump-tion, but diametrically opposite results have been obtained by different workers. Thus, KOHN and KALLMAN23 in most careful experiments find it to be much smaller. The LD5 0 is reduced by only a few per cent at 21 days (cf. ref. 23, and Fig. 14-8) and these authors suggest that there may be very little if any non-recuperable injury (i.e. according to this test). Subsequent to this work, KREBS, BRAUN and KALBACH21 claim a reduction of 100 r in the L D 5 0 of mice, that had received three doses of 300 r at intervals of several weeks and were then allowed to recover for

ioo>

3 7 14 21

Days after irradiation, 3l5r

FIG. 14-8. Rate of recovery (as measured by LD50/30 for x-rays) of rats from a dose of 315 r of x-rays23.


a further period before being tested to determine L D 5 0 ( 3 0 days). Yet in an almost identical experiment (see Fig. 1 4 - 9 ) ALEXANDER and CONNELL 7 1

observe no change in the L D 5 0 . After these large doses, the average life expectancy is reduced to about one year (see p. 436). The reason why some workers have found a reduction in the L D 5 0 may be because at the time the acute effects were being measured, the animals were already dying from the delayed effect of the conditioning dose. Probably by the time the animals were tested for their L D 5 0 they have been near the end of their life-span, and it is this which explains the reduction in L D 5 0 .

—1 Hi-I 1 Lu L_ 800 750 700 650 600

Exposure, r

FIG. 14-9. Recovery of CBA mice from IlOOr of x-rays as assessed by dose needed to kill acutely (i.e. within 30 days). The conditioning dose was given in four sessions separated by

three weeks71.

When precautions are taken to ensure that the L D 5 0 is determined early enough to avoid this artificial "ageing" then no change is observed. This suggests that the ability of the bone marrow to withstand irradiation recovers completely, since it is this which determines the magnitude of the L D 5 0 (30 days) in mice.

Increased Resistance The technique used to estimate decreased resistance to ionizing radia-

tions after a first exposure is exactly the same which in the hands of other scientists has shown increased resistance (in one case up to at least 15 days after x irradiation, see p. 389). It is not improbable that a non-lethal dose of radiation induces some favourable reactions besides many detri-mental phenomena. What one measures with this simple quantal test


(mortality) is the integration of a large number of responses. Each physio-ogical or anatomical injury has its own time-course of disappearance; it is not illogical to propose a multi (up to 7) parameter model instead of the simple two-parameter model25. No one so far has attempted to ac-celerate the recovery as tested by this method.

Response to Continuous Irradiation If one irradiates continuously at small dose-rates, the increase in the

total dose necessary for death (as compared with the effects of a single big dose) is also a measure of recovery. In the case of small dose-rates there is a competition between injury and restoration: if restoration keeps up with injury a kind of equilibrium is obtained and irradiation has no obvious effect. Theoretically one should be able to estimate the intensity and rapidity of recovery processes by determining the threshold of dose-rate below which no injury can be observed in living organisms or certain tissues (the skin for instance)6, or by a careful analysis of the mortality data during continuous irradiation at various dose-rates.

Some queer reactions show how very different is the rate of recovery when one compares various animal species. For instance, the mortality of mice or rats after a dose of 700 to 800 r of x-rays is the same if the dose is distributed at the rate of 100 or 10 r per minute. Certain birds (gal-linaceous and palmipedes) on the contrary are sensitive to differences in dose-rate within this range. After a just-lethal dose delivered at high dose-rate (100 r per minute for instance) a large percentage of irradiated birds show signs of intense cardiovascular collapse and die during the first two days after exposure30'35'39'40'41'45; if the dose-rate does not exceed 10 r/min the mortality after the same total dose is generally negligible during that period; it is concentrated between the 5th and 15th day35'39.

Embryos36, chicks or adult roosters42 show the same type of mortality when irradiated in the same conditions. In the chick the cardiovascular collapse is accompanied by acute nephritis not seen in adults42. The quality of the radiation (x, y or neutrons) does not matter34. The pigeon does not show either collapsus, nephritis or early death43-44. The rabbit at the level of the LD50 at the dose-rate of 10 r/min sometimes shows reactions identical to that of chicks31-37'38.

Obviously, there is a very quick recovery process in certain critical organs of the chick which does not exist in mammals or pigeons. Metabolic troubles (hyperglycemia, hyperuricemia, hypoproteinemia and increased plasma concentration of amino-acids) do not seem to be responsible32. The appearance in the blood of vasoactive substances liberated by tissue necrosis and troubles in autonomic regulations may be more import-ant37-38'42, although protection and therapeutic measures based on this assumption have given irregular results30'33-37. Anoxia is effective in


protecting chicks, but the chemical protectors (like cysteamine) very effective in mice failed to decrease mortality of chicks when injected before irradiation; only tryptamine and diethyldithiocarbamate showed slight action30.

Promoting Recovery by After-treatments There are indications that certain substances or foods may decrease

mortality of irradiated small rodents when given after irradiation*; in some ways these substances seem to enhance the recovery process. Here is a non-exhaustive list: cell-free spleen extracts51, lycopene52, cabbage and broccoli feeding55, nucleotides or products of the hydrolysis of DNA56 '59

or RNA57. Neither DNA nor RNA themselves nor the simple basic components were found to have beneficial effects by HANGEN et al.15 or MAISIN57. Kanazir, on the other hand, finds significant survival of rats given a whole-body irradiation with x-rays at LDioo level by post-irradiation treatment with highly polymerized isologous liver or spleen RNA and DNA. Here, as in the repair of bacteria (see p. 371), the effect disappears if DNA is treated with DNAase75; this control is important indeed since such highly polymerized nucleic acids preparations may contain other and unknown substances as contaminants. TALIAFERRO and JAROSLOW14 report that a cellular extract of normal rabbit or mouse spleen and of yeast or Hela cells restore the haemolysin forming capacity of rabbits irradiated with 400 r. Nucleic acid digests, but neither the purine or pyrimidine bases nor the polymerized acids were found active in this respect. This whole field of post-irradiation treatment with nucleic acids abounds in contradictions, probably because it is difficult to isolate nucleic acids in a pure and native form.

Methionine administration half an hour after irradiation seems to help the restoration of the impaired nucleic acid synthesis53, but does not change mortality54. Alkoxyglycerol-esters of essential fatty acids which have given encouraging results in chronic human radiation sickness58

(white cells and platelets regeneration) have had some beneficial effect also in reducing the extent of life-span shortening (see p. 436) and in this sense can be said to hasten recovery.

R E F E R E N C E S 1. LATARJET, R., Symposium on Radiobiology (Edited by J . J. NICKSON), John

Wiley, New York, 1952 2. B L U M , H . F., R O B I N S O N , J . C. and Loos, G. M., J. Gen. Physiol., 1951, 3 5 ,

323 3. LAMARQUE, P., Presse Mid., 1952, 60, 1039

* T h e problem of injections of cell suspensions after irradiation is discussed in Chapter 20, p. 484.


4 . LAMARQUE, P . and GARY-BOBO, J . , First U.N. Intern. Conf. on the Peaceful Uses of Atomic Energy, Geneva, 1955, Vol. 11, (French edition), p. 408, United Nations, 1956

5. STAPLETON, G . E . , Ann. N.Y. Acad. Sci., 1 9 5 5 , 59, 6 0 4 6. HOLTHUSEN, H . , in Einfilhrung in die Rontgenologie (Edited by G. F. HAENISCH

and H. HOLTHUSEN), Thieme, Leipzig, 1933 7 . HOLLAENDER, A . a n d STAPLETON, G . E . , Physiol. Rev., 1 9 5 3 , 3 3 , 7 7 8. BROWNELL, A. S., Univ. California Radiation Lab., Report No. 3055, 1955 9 . LATARJET, R . , Acta Radiol., 1 9 5 4 , 41, 8 4

10. E ICHEL, H . J . and R O T H , J . S., Biol. Bull., 1953, 105, 373 1 1 . STAPLETON, G. E., BILLEN, D . and HOLLAENDER, A., J. Cell. Comp. Physiol.,

1953, 41, 345 1 2 . KELNER, A . , Proc. Natl. Acad. Sci., U . S . , 1 9 4 9 , 3 5 , 7 3 1 3 . BORSTEL, R . C . VON and W O L F F , S . , Proc. Natl. Acad. Sci., U.S., 1 9 5 5 , 41,

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15. HANGEN, C. W. and SIMMONS, E. L., Univ. Chicago Report No. 3815, 1947 16. PATERSON, E., GILBERT, C. W. and MATTHEWS, J . , Brit. J. Radiol., 1952,

25, 427 17. PATERSON, E . , GILBERT, C . W . a n d HAIG, M . V . , Brit. J. Radiol., 1956, 29 ,

218 18. HURSH, J. B. and CASARETT, G. W., Univ. Rochester Report No. 527, 1958 1 9 . M O L E , R . H . , Brit. J. Radiol., 1 9 5 6 , 2 9 , 5 6 3 2 0 . K O H N , H . I. and KALLMAN, R . F . , Radiation Research, 1 9 5 7 , 6 , 1 3 7 2 1 . KREBS, J . S . , BRAUER, R . W . and KALBACH, H . , Radiation Research, 1 9 5 9 ,

10, 80 2 2 . K O H N , H . I. and KALLMAN, R . F . , Radiation Research, 1 9 5 7 , 6 , 3 2 9 23. K O H N , H . I. and KALLMAN, R . F., Radiation Research, 1957, 7, 85 24. R I X O N , R. H . , T I S H L E R , J . M. A. and NEWCOMBE, H . B . , Nature, 1958,

181, 1138 2 5 . SACHER, G . A . , G R A H N , D. and G U R I A N , J. M., Argonne Natl. Lab. 5 9 1 6 ,

1958, p. 70 26. ALPER, T . and GILLES, N . E., J. Gen. Microbiol., 1958, 18, 461 2 7 . STAPLETON, G . E . , SBARRA, A . J . a n d HOLLAENDER, A . , J . Bacteriol., 1 9 5 5 ,

70, 7 28. MELVILLE, G. S . , J R . , C O N T E , F. P . , SLATER, M . and U P T O N , A. C., Brit

J. Radiol., 1957, 30, 196 2 9 . M O L E , R . H . , Brit. J. Radiol., 1 9 5 7 , 3 0 , 4 0 3 0 . BEAUMARIAGE, M . L . , Intern. J. Appl. Radiation and Isotopes, 1 9 5 8 , 4 , 6 9 31. BEAUMARIAGE, M. L . and LECOMTE, J . , C.R. Soc. Biol., 1957, 1 5 1 , 1976 32. BERNSTEIN, E. K., STEARNER, S . P . and BRUES, A. M., Am. J. Physiol., 1956,

186, 5 4 3

33. BROOKS, P. M., GERSTNER, H . B . and S M I T H , S . A., Am. J. Physiol., 1956, 186, 532

34. H O W A R D , H . , VOGEL, J . and STEARNER, S . P., Radiation Research, 1955, 2, 5 1 3

3 5 . JACQUEZ, J . A . and KARNOFSKY, D . A . , Am. J. Roentgenol., 1 9 5 0 , 64, 2 8 9 . 3 6 . KARNOFSKY, D . A., PATTERSON, P . and RIGWAY, L . P . , Am. J. Roentgenol.,

1950, 64, 280


3 7 . PAINTER, E . E . , PROSSER, C . L . and MOORE, USAEC Report M D D C , 7 6 1 , 1947

3 8 . PROSSER, C . L . , Radiology, 1 9 4 7 , 4 9 , 2 9 9 3 9 . STEARNER, S . P . , Am. J. Roentgenol., 1 9 5 1 , 6 5 , 2 6 5 4 0 . STEARNER, S . P . , Am. J. Roentgenol., 1 9 5 1 , 6 5 , 2 7 2 41. STEARNER, S . P., Am. J. Physiol., 1955, 182, 407 4 2 . STEARNER, S . P . , SANDERSON, M . , CHRISTIAN, E . J . a n d BRUES, A . M . , Am.

J. Physiol., 1956, 184, 134 4 3 . STEARNER, S . P . , and CHRISTIAN, E . J . , Argonne Natl. Lab., 5 5 1 8 , part II , 1 7 1

1956 4 4 . STEARNER, S . P., SANDERSON, M . and CHRISTIAN, E . J . , Argonne Natl. Lab.,

5732, January-June, 1957, p. 65 4 5 . STEARNER, S . P . , SANDERSON, M . , CHRISTIAN, E . J . a n d BRUES, A . M . , Am.

J. Physiol., 1957, 192, 620 46. SACHER, G. A., Radiation Biology and Medicine (Edited by W . D. CLAUSE),

p. 283, Addison-Wesley, Reading, Mass., 1958. 4 7 . E L K I N D , M . M . and SUTTON, H . , Radiation Research, 1 9 5 9 , 1 0 , 2 9 6 4 8 . H U G , O. and W O L F , I . , Progress in Radiobiology, p. 2 3 , Oliver & Boyd,

Edinburgh, 1956 4 9 . FORSSBERG, A . , and NOVAK, R . , Intern. J. Radiobiol., Suppl. I , 1 9 6 0 ,

p. 2 0 5

50. TANADA, T. , Radiation Research, 1958, 9, 552 5 1 . ELLINGER, F . , Atompraxis, 1 9 5 8 , 4 , 4 3 9 52. FORSSBERG, A., L I N G E N , C., ERNSTER, L. and LINDBERG, O., Exptl. Cell.

Research, 1959, 16, 7 53. SAHASRABUDHE, M. B., Proc. Second U.N. Intern. Conf. on the Peaceful Uses

of Atomic Energy, Geneva, 1958, Vol. 23, p. 94, United Nations, 1959 5 4 . BACQ, Z . M . a n d BEAUMARIAGE, M . L . , Nature, 1 9 6 0 , 1 8 6 , 1 0 6 4 55. SPECTOR, H. and CALLOWAY, D. H., Proc. Soc. Exptl. Biol. Med., 1959, 100,

405 5 6 . KARPFEL, Z . , SOSKA, J . a n d DRASIL, V . , Nature, 1 9 5 9 , 1 8 3 , 1 6 0 0 57. MAISIN, J. and coll.. Personal communication, 1959 58. BROHULT, A., Advances in Radiobiology, p. 241, Oliver & Boyd, Edinburgh,

1957 59. SOSKA, J . , DRASIL, V. a n d KARPFEL, Z . , Second U.N. Intern. Conf. on the


6 0 . W I T K E N , E . M . , Cold Spring Harbor Symposia Quant. Biol., 1 9 5 6 , 2 1 , 1 2 3 61. KIMBALL, R. F., GAITHER, N . and W I L S O N , S . M., Genetics, 1957, 4 2 , 661;

Radiation Research, 1959, 10, 490 6 2 . ALBERT, M . D . , / . Natl. Cancer Inst., 1 9 5 8 , 2 0 , 3 0 9 63. DONIACH, I . and LOGOTHETOPOULOS, J . H . , Brit. J. Cancer, 1955, 9, 117 64. W O L F F , S . and L U I P P O L D , H. E., Science, 1955, 122, 231 65. WOLFF, S., Radiation Research, Suppl. 1, 1959, p. 453 6 6 . ABRAHAMSON, S . , Genetics, 1 9 5 6 , 41, 6 3 1 67. JACOBSON, B. S., Radiation Research, 1957, 7, 394 68. ADAMS, D . and NILAN, R . A., Radiation Research, 1958, 8, 111 6 9 . EHRENBERG, L . , Radiation Research, Suppl. 1 , 1 9 5 9 , p. 4 1 7 70. BLAIR, H. A., First U.N. Intern. Conf. on the Peaceful Uses of Atomic Energy,

Geneva, 1955, Vol. 11, p. 133 of French edition, United Nations, 1956 71. ALEXANDER, P . a n d CONNELL, D . I . , Radiation Research, 1960, 12, 38


7 2 . K I N G , R . C . , DARROW, J . D . , a n d KAYE, N . W . , Genetics, 1 9 5 6 , 4 1 , 8 9 0 7 3 . H U R S H , J . B . et al., Second U.N. Intern. Conf. on the Peaceful Uses of Atomic

Energy, Geneva, 1958, Vol. 22, p. 178, United Nations, 1959 7 4 . KANAZIR, D . T . , CECUK, O . Z . , KRAJINCANI6 , B . N . a n d H U D N I K , T . A . ,

Bidl. Inst. Nucl. Sci., Boris Kindric, 1959, 9, 133 7 5 . KANAZIR, D . T . , BE6AREVI<5, A . , PANJEVAC, B . , S I M I C , M . a n d RISTIC, G . ,

Bull. Inst. Nucl. Sci., Boris Kidric, Belgrade, March 1959, 9, 149 7 6 . E L K I N D , M. M. and SUTTON, H . , Nature, 1959, 1 8 4 , 1293 7 7 . EHRENBERG, L . and LUNDQUIST, U . , Hereditas, 1 9 5 7 , 4 3 , 3 9 0 7 8 . WEISS , L . , Intern. J. Radiobiol., 1 9 6 0 , 2 , 2 0 79. SHAPIRO, N. I. and BELITSINA, N. V., Radiobiology Symposium, Moscow, 1960

C H A P T E R 1 5

Neuro-endocrine Reactions in Radiation Sickness

S T R E S S A N D T H E A D A P T A T I O N S Y N D R O M E

THE biochemical and physiological study of irradiated mammals is compli-cated by non-specific neuro-endocrine reactions which Selye has called the adaptation syndrome. Detailed discussions of this question are to be found in the works of SELYE1-2 and extensive reviews in various text-books (see, for instance, ref. 3). The essence of this concept is that every strongly harmful agent ( = stress*) stimulates the hypothalamic nerve centres (either directly or through the mediation of adrenaline) and these control the activity of the anterior pituitary, the source of several hor-mones. The secretion of three hormones is inhibited: prolactin, which maintains milk secretion; the gonadotropic hormone which activates the sex glands; and the growth hormone, or somatotropic hormone (STH). On the other hand the secretion of the adreno-corticotropic hormone (ACTH) is increased. It is said loosely that the synthesis of an excess of ACTH takes place at the expense of the other anterior pituitary hormones. As a result of this discharge of ACTH the cortex of the adrenal gland becomes overactive and secretes into the blood an abnormally large quantity of steroid hormones. These corticoids act on the whole body; part of them is eliminated in the urine and bile after conjugation in the liver. A large fraction of the corticoids is metabolized through unknown pathways.

All the anterior pituitary hormones are proteins or polypeptides and can therefore be estimated only by measuring their biological effects. Some steroids act mainly on the equilibrium of sodium and potassium ions in the body and are therefore called mineralocorticoids; a synthetic steroid ester desoxycorticosterone acetate (DOCA) and the naturally occurring aldosterone are of this type.

Cortisone, hydrocortisone and the many recently synthetized cortisone-like steroids act on the carbohydrate, protein and lipid metabolism; they

•Examples of stress: trauma, haemorrhage, surgical operation; intense cold, ultra-violet radiation, ionizing radiation; injection of chemical substances (formol, colchicine, salicylates); infectious diseases, intoxications; strong emotional excitement.

26 3 8 7


are the gluco-corticoids. In a normal mammal, endocrine regulation depends not only on the total quantity of steroids secreted, but also on the propor-tion of gluco- to mineralo-corticoids. An excess of mineralo-corticoids leads to sodium retention, arterial lesions and impairment of the heart and kidneys; an excess of cortisone disturbs the physiology of the connective tissue, promotes the spread of infections, and causes involution of the lymphoid tissues, hyperglycaemia and even diabetes.

If an animal is subjected to intense, continuous stress (e.g. cold), the pituitary-adrenal reaction takes place in three phases: (1) alarm, which lasts over two days (shock and counter-shock), during which the adrenal gland hypertrophies and gradually increases its secretion; (2) resistance, during which the animal shows increased resistance to cold, due to the adrenal reaction, which may last for weeks; (3) exhaustion, which leads, sooner or later, to death if the stress is too great or the animal is not in perfect physical condition. Death occurs in the state of weakness charac-teristic of adrenal insufficiency*.

When the stress is not continuous (e.g. a few minutes' exposure to in-tense x- or y-radiation) the various phases show up in much more rapid succession and may be considerably altered.

A precise estimation of the ACTH output for short periods of time is not technically feasible at present; furthermore, the adrenal response is not necessarily proportional to the concentration of ACTH in blood. It is therefore better to study the adrenal reaction. The ideal procedure would be to trace the various steroids in the blood hour by hour; but the amount of these steroids in the blood is very low and one must pool the blood of three or four rats for an estimation. There is no possibility of individual study. The use of bigger mammals in radiobiology is increasing and gives valuable opportunities in neuro-endocrine radiation pathology. The corticoids can also be estimated in the urine and their excretion be fol-lowed for weeks27'28, but only a small (10 per cent) and not necessarily constant fraction of injected cortisone is recovered in the urine of man. In other words it is not possible to make even a very rough balance sheet of the hormones excreted by the adrenal cortex.

It is also dangerous to assume hyper- or hypo-secretion merely from the histological appearance of the gland. The corticoids are soluble in the abundant fat; only the fat is visible at the microscope after coloration. Moreover, the store of hormones in the gland is very small when compared with the need of the body; competent endocrinologists say that the gland has in reserve an amount equal to only 3 to 5 min secretion.

In the gland, the synthesis of corticosteroids starts from cholesterol

* Selye's concept has been adapted by Novak et al. in order to interpret indi-vidual differences in metabolism (adaptation metabolism) and radioresistance46.

N E U R O - E N D O C R I N E R E A C T I O N S I N R A D I A T I O N S I C K N E S S 3 8 9

and is said to consume ascorbic acid. Very active synthesis is therefore associated with simultaneous decrease in the cholesterol and ascorbic acid content of the gland.

Several fairly specific reactions indicate the presence of an excess of gluco-corticoids in the blood: (a) a fall of at least 80 per cent in the number of circulating eosinophiles, (b) a fall in the number of circulating lympho-cytes, (c) the excretion of uric acid in the urine, shown by a great increase in the uric acid/creatinine ratio, (d) increased liver glycogen.

There is no such sensitive and specific test for mineralo-corticoids; the most valid tests are those which reveal sodium retention and increased potassium elimination.

We have accepted the view that several tests should be studied simul-taneously, a firm conclusion being reached only if there is good agreement.

D O I O N I Z I N G R A D I A T I O N S A C T A S S T R E S S E S ?

(1) The production of radioresistance by sublethal irradiation has been found in mice whether with /3-rays (RAPER4) or x-rays (DOWDY2 4 , CRON-KLTE et al.5, BETZ6) and whether one or more preliminary irradiations were given. For a perfect demonstration the sublethal irradiation must not kill any animal, otherwise it selects the most radioresistant animals. Quantitatively, the induced radioresistance is small and confined strictly in time to between the 7th and 15th day after irradiation6. Many authors have used the method of two short exposures to ionizing radiations sep-arated by a variable interval in order to study the progress of recovery; they found generally that the sensitivity is increased after a previous exposure (see p. 377).

In a recent experiment89, the L D 5 0 at 30 days was measured in mice 10 and 17 days after a whole-body exposure of 50 r, it increased from 487 r (normal) to 560 r (at 10 days) and to 617 r at 17 days. It is difficult to interpret these observations along Selye's adaptation theory, since a dose of 50 r barely activates the unspecific neuroendocrine reaction to stress. Other possible explanations are suggested by DACQUISTO89.

Thus, the radioresistance well known to radiotherapists after repeated local irradiation of tumours, and demonstrable at the tissular level, for instance, for lympoid cells52, is difficult to show or often absent when the whole mammal is considered. It may be argued that the acquired radio-resistance of tumour lympoid cells (or even yeast cells54) is due to selec-tion, in the non-homogeneous cell population, of less sensitive units by the first irradiation; many cells are killed, those which survive enter in active mitosis and repopulate the tumour, the organ or the colony with more resistant cells.

LOUTSCHNIK and KOULIKOVA53 have observed an increased resistance to 700 r of x-rays of female mice exposed ten days previously to 150 r, but


if the first dose is 100 or 200 r no increased resistance is seen! These authors have also made an experiment which will become very important if confirmed with other isotopes and animal species; IOiUC of 144Ce are injected in male and female mice; 14 days after, when 50 per cent of the injected activity persists, they are mated with normal animals. The off-spring (at 3 months of age) of normal females + injected males has the same radiosensitivity as normal animals of the same age; there is no genetic effect. But the offspring of injected females + normal males seems slightly more resistant; the important fact is the irradiation of the embryo by the organism of the mother. 144Ce is fixed by the mother's tissue, only 0-03 per cent being transferred to the embryos. This observation of the Russian authors is consistent with the fact observed by RUGH and W O L F F 8 8 that exposure of the mouse foetus, at 15-5 days' gestation, to only IOr in-creases radioresistance if it is subjected to the LDso/30 days' range of exposure when adult. Other foetal ages (from 14-5 to 16-5 days) show some benefit of 10 r exposure, but exposure to higher doses (25 to 300 r) to any age foetus will have a deleterious effect in later life if the mouse is exposed to x-rays. MAISIN and collaborators55 have also seen that rats x-rayed in utero show increased radioresistance when tested at several months of age. But these facts54-55,88 have obviously nothing to do with the neuro-endocrine reaction to stress. Other work by Maisin and his group gives the first indication, in yeast, of a cellular mechanism for acquired radiation resistance90-91.

Observations on the intestinal epithelium of continuously irradiated rodents show that there is an adaptation and that this adaptation is not due to selection of more resistant cells but actually is a cellular phenomenon. The mechanism of this adaptation is being studied by a group at the Brookhaven National Laboratory99.

(2) The existence of cross resistance. If a dose of irradiation fatal to all control mice is given after stimulation of the adrenal cortex by cold or by an adequate chemical stimulus, some of the animals survive: 15 per cent if cold is the stimulus, 25 to 50 per cent after sodium salicylate, 22 per cent after ACTH, 20 to 40 per cent after DOCA, and none after cortisone6; this observation has been confirmed, but with reservations48.

Various post-irradiation surgical stresses or ether anesthesia have little or no (favourable or unfavourable) effect on survival in mice19; irradiated rats are less resistant than normal to traumatic shock20, cold6-48'37 or salicylate6.

(3) Irradiation of the pituitary gland only, using a beam of energetic electrons from a Van de Graaff generator, increases the ACTH content of the blood within an hour, and the increase lasts for at least 2 4 hr (MATEYKO et a/.18).

(4) Adrenalectomized or hypophysectomized rats or mice are much


more sensitive than normal animals to x-irradiation (refs. 6, 7, 8, 16, 47 and many other authors) (Fig. 15-1). Neither ACTH nor growth hormone (STH), insuline, cortisone or testosterone reduces the mortality of X-irradiated hypophysectomized rats; thyroxine alone has a slight favourable effect47.

Since the endocrine glands regulate the growth of mammals (in general, and especially of certain tissues), it is no wonder that the synthesis of DNA or RNA is found to be under hormonal influence; since the neuro-endocrine reactions are much altered by irradiation, distant, so-called abscopal effects on DNA and RNA synthesis may be observed. For instance, the effects of total-body irradiation on the regeneration of the liver after partial hepatectomy in the rat are decreased by adrenal-ectomy98.

I

[

\ V + i

O V \ \

S V

A • I

\

\ > \

I

2000 10,000 18,000

Doses, r

FIG. 15-1. Survival time of white rats after irradiation in the dose range 1 0 0 0 r to 1 5 , 0 0 0 r.

irradiated controls (3 -5 days-effect) H h irradiated splenectomized animals O — O irradiated adrenalectomized animals X—X irradiated hypophysectomized animals51.

Whereas administration of a total cortical extract or DOCA may prolong the life of irradiated rats, whether adrenalectomized or not, it is claimed that cortisone has no effect or may even increase the mortality of irradiated rodents during a period beginning on the 5th day after


irradiation, that is, at the time when infections show themselves8-18. Cortisone is clearly contra-indicated in radiation sickness. A small protec-tive effect is observed in mice with DOCA, adrenal extracts or implants after exposure to 500 r but not to 650 r (100 per cent lethal dose of x-rays40).

In rabbits, a moderate irradiation (200 r to 480 r) suppresses the cutan-eous reaction to tuberculin either partially or completely, even if the skin at the site of the test is protected from the rays by a lead screen; this fact may be due to overproduction of cortisone30. Small doses of x-rays (50 kV, 1000 to 2000 r) locally applied to human skin increase the reaction to tuberculin injected 48 hr after irradiation. However, after repeated, intense irradiation, the tuberculin reaction is abolished100.

In the rat, protection of the adrenal glands by a lead screen reduces the mortality of rats after doses of 700 or 800 r applied to the whole body14, but the protection is very slight. Local protection of the adrenal region in the guinea-pig is of no particular significance59.

The isolated perfused adrenal gland of the calf has been irradiated by 60Co rays (1800 to 2700 r) and shown to respond by a decreased output of corticoids and chromogens when ACTH is added to the perfusion fluid49 and a decreased ability to convert some steroids into others by hydroxylation or oxidation of certain groups50. Adrenal glands irradiated in situ, while all other parts of the mouse's body are shielded with lead, does not produce marked reactions51. Thus the important changes, mainly in the sense of increased adrenal cortical activity, observed when the whole mammal is exposed to smaller doses, is not due to action on the adrenals themselves but to changes in adenohypophysis activity. At this point difficulties are encountered; it appears that irradiation is a very special form of stress.

D I F F I C U L T I E S I N F A C T S A N D I N T E R P R E T A T I O N S

(1) CRONKITE13 says: "BOND et al.15 have conclusively demonstrated that radiation, when used locally in doses sufficient to produce destruc-tion of tissue, will produce the alarm reaction in the manner described by Selye". LEBLOND and SEGAL31 have shown that some of the reactions at a distance are produced by way of the adrenals. For instance, the atrophy of the rat thymus which follows irradiation of the abdomen (2000 to 3700 r) does not occur in animals adrenalectomized 2 to 5 days before irradiation; similarly, the atrophy of the spleen which follows irradiation of the head and neck (2800 r) does not occur after adrenalectomy.

It must not be forgotten that during total irradiation of a mammal the glands which will have to react to the stimulation of the hypothalamus, i.e. the pituitary and adrenal, are themselves irradiated. They may be


directly affected so that the response of the pituitary to the nervous stimulus and of the adrenals to ACTH may be different, but apparently, this possibility may be excluded if the exposures are kept below 1000 rads.

(2) It is dangerous to argue by analogy. The fact that in mammals, cortisone, DOCA or ACTH produce tissue changes and some of the symp-toms seen after irradiation, does not mean that in irradiated animals these changes are due to the neuro-hypophyso-adrenal reaction. Undeniable effects of ionizing radiations are often identical with those of one or other of the cortical hormones, and cannot be separated from them. The following is a typical example, and others could be given. Isolated lymph glands, in tissue cultures, and therefore free from all nervous and hor-monal influences, are highly radiosensitive. TROWELL29, using this material, carried out some excellent quantitative studies. The same tissue, culti-vated in the same conditions reacts to very low concentrations (0-1 /<g/ml) of cortisone; 3 X IO-5 M of this corticoid kills 45 per cent of the lymphocytes in two days. It is impossible to distinguish between lymphocytes damaged by cortisone and lymphocytes killed by x-rays; both show the same pyknotic degeneration. To avoid the intervention of the corticoids it is necessary to use either isolated organs or adrenalectomized mammals. Another difficulty arises here: the rat is about the only mammal which tolerates adrenalectomy well; other animals become very frail, and many are lost even if they are given regular injections of cortical adrenal hormones and sodium chloride. The rat tolerates loss of the adrenal glands better than other animals because it often has accessory adrenals, that is, islets of cortical tissue which become hypertrophic after removal of the true adrenals. For this reason, adrenalectomized rats can only validly be used during the first fortnight after the operation. The hypophysectomized rat can be used for a longer period, but it suffers from multiple endocrine deficiencies which make it very different from the normal animal. Com-parison of the reactions of organs in vivo and in vitro is certainly an excel-lent method. The techniques of Wolff, of Trowell and of Martinovic— cultures of whole mammalian and avian organs—fortunately seem to be easier than was supposed some years ago, and they are capable of being greatly developed. The difficulties raised by this similarity of the effects of the corticoids and of x-rays, are implicitly recognized by Ellinger who, ever since 1941, has been emphasizing the importance of cortical adrenal insufficiency in radiation sickness85. The facts he stresses are metabolic phenomena which might be caused by cortical insufficiency, and are difficult to interpret by taking into account the specific action of the radiations alone. These phenomena are "loss of chlorides, disturbances of water metabolism, fall in blood cholesterol levels with increased fat deposits in the liver, a biphasic response of the hydrogen ion concentration


in blood and tissues and of the blood sugar*." The disturbances in ion distribution are genuine. In rats, after a dose of 900 r applied to the whole body, the urinary excretion of potassium is increased, and that of sodium is decreased, compensating for increased elimination in the faeces. The radiosensitive tissues show first a fall and then a considerable rise in sodium content after irradiation26. These changes in rats are in a direction suggesting cortical overactivity. In dogs or in man suffering from severe radiation sickness, such studies would be senseless because the vomiting makes the loss of water, chloride and bases very variable. The dehydration and loss of nutrition by themselves explain the electrolytic changes ob-served in dogs after 450 r36.

(3) Many histologists are struck by signs of adrenal hyperactivity, but their findings cannot be expressed in a quantitative manner. A clearer picture of the adrenals' response to whole-body irradiation, and of the mechanism of this response can now be given thanks to the studies of several groups of investigators22-23-6-39'56-59.

F I R S T A N D S E C O N D R E A C T I O N S Very soon (1 hr) after exposure of total body to about 700 r of x-rays,

a marked and simultaneous decrease of the ascorbic acid and cholesterol content of the adrenals, occurs in the adult rat. The drop is maximal in 2-3 hr and is followed by a complete recovery in 12-24 hr. This is known as the first reaction. This reaction is indeed very important, maximal since a bigger drop cannot be observed after injection of very large doses of ACTH.

In the adult rat, a second drop begins after the second day following irradiation (same conditions, total body, 700-800 r, 100 per cent lethal dose); it is maximal at about the 4th day. The adrenals of the dying animals are very low in cholesterol and ascorbic acid. Such is the second reaction, associated with bad general conditions: loss of weight, diar-rhoea, etc.

The capacity of the adrenal glands to concentrate 32P from injected tagged phosphate in rats follows the same pattern: increased 1 hr after irradiation, return to normal at 24 hr, increased again at 72 hr92.

(a) The first reaction—The chemical changes in the adrenals are only indirect evidence of increased secretion of hormones; thus more direct proof is indeed welcome. Urinary excretion of conjugated corticoids in the pig60, blood concentration of corticoids in the rat61 or in the mon-key62 show peaks early after total body irradiation. There is a minimum concentration of eosinophiles in the rat's blood, 3 to 4 hr after irradia-tion63. At that time the animals are in good condition; there is no compli-

* Increased fat deposits in the liver are, in fact, caused by an excess of cortisone.


cation; all the signs are present which indicate a sharp, important over-activity of the suprarenal cortex.

How is this activity stimulated? Where is the starting point of this particular case of neuro-endocrine reaction? Alltheevidencepointstoan involvement of the central nervous system, of the hypothalamus which controls (by way of an unknown chemical mediator) the secretion of ACTH by the anterior hypophysis.

1. Rats, 6 to 10 days old, show no first reaction; new-born mammals have not yet established physiological connection between hypothalamus and pituitary. The typical first reaction appears in the rat at about the 13th day of Iife57.

2. Adult rats injected with a barbiturate and morphine (so-called pharmacological inhibition of nervous control of ACTH secretion) show no first reaction56.

3. Hypophysectomized adult rats in which two pituitaries were grafted in each anterior chamber of the eye, show only a slight decrease in ascorbic acid but an increase in cholesterol (for interpretation of this increase see p. 321)5'.

4. Two days after the hypothalamic centres have been selectively destroyed by electrolysis, the first reaction disappears (see ref. 58, and Fig. 15-2).

r m a l Hypophysectomized Lesioned J grafted hypothalamus

4-2lmg/l00mg ^—

Controls

C 3h3d C 3h 3d C 3h C 3d

FIG. 15-2. Cholesterol in suprarenals of rats 3 hours and 3 days after whole-body irradiation (850 r) (BACQ et al.5S).

It is known that the hypothalamus is sensitive (directly or indirectly via the reticular formation) to the adrenaline level in the blood and there is evidence of increased adrenaline concentration in the blood after irradiation in the dog64 or the rabbit65, but this point as well as the pos-sible activation of the hypothalamus by cells of the reticular formation


obviously needs confirmation*. Stimulation of the central end of the sciatic nerve or any painful stimulation in any mammal results (a) in a rapid increase of blood pressure of short duration, (b) in a second wave of hypertension beginning 90 to 100 min after the pain stimulus and lasting up to 7 hr (Fig. 15-3). This second wave does not occur after denervation

MO 130 120 110

100 90 80

FIG. 15-3. Blood pressure in the rat before irradiation59 to be compared with Fig. 15-4—after irradiation). Abscissae show time in hours. Ordinates show blood pressure in Hg every 10 min. A. Normal blood pressure. B. Blood pressure in the same rat during stimulation of the paw.

Arrow indicates moment of stimulation (for a period of 2 min; distance of coils 10 cm, tension 6 v).

C. Blood pressure in the same rat during injection of adrenalin. Arrow indicates moment of injection of adrenalin (0-05 ml, 1 : 1 0 0 0 ) .

* Footnote added in proof: Confirmation has come from G O O D A L L and L O N G who have shown that in the rat the amount of adrenaline stored in the suprarenals falls about 50 per cent, 1 hr after total body x-irradiation (800 or 1600 r); it remains low and still decreases after 1600 r, but slowly returns to normal in the few rats which survive exposure to 800 r101. The same drop in adrenaline content is observed in the rabbit after exposure to 1770 r, but not in the cat the reaction of which is irregular. T h e synthesis of catecholamines studied in vitro is not decreased and may be increased in suprarenals after in vitro irradiation101. Goodall and Long believe that the suprarenals are partially depleted in catecholamines because the excretion is greater than the synthesis.

Five to ten min after total body x-irradiation (200 kV, 800 to 3200 r at the rate of 140 to 540 r/min) the adrenaline content of the suprarenals of rats is not de-creased102; it is not possible however to conclude that the secretion of catechola-mines is not increased during or immediately after irradiation because the synthesis of the catecholamines may be increased102.


of the adrenals or section of the stern of the hypophysis. Adrenaline injection produces a similar two-wave response. Thus, T O N K I K H 5 9

believes that pain excites the sympathetic centres controlling the secretion of vasopressine by the posterior part of the pituitary. In the irradiated rat (800 r x-rays on whole-body) this neuro-endocrine reaction to pain persists intact for 6 days, but at the 7th to 9th day, no second wave is seen, although adrenaline injection still reproduces it (Fig. 15-4). It seems

150 140 130 120 110.

100 90 80 70

J. -J I L

140 130 120' 110

100 90 80

FIG. 15-4. Blood pressure in the rat after irradiation59. Same designations as in Fig. 15-3 and the same rat. A. 4 days after irradiation. Arrow indicates moment of stimula-

tion of the paw. B. 7 days after irradiation. Arrow indicates moment of stimu-

lation of the paw. C. 8 days after irradiation. Arrow indicates moment of adrenalin

injection.

that at that time (7th day) when the animal is in very bad condition, the sympathetic centres cannot be activated any more by pain stimuli; this observation suggests that the normal control of the sympathico-adrenal system on the ACTH secretion may be deficient during the second period of the radiation syndrome59 but not during the first.

Figure 15-5 summarizes what we know and do not know about the early neuro-endocrine reaction to a just-lethal total-body dose of ionizing radiation. As soon as the dose is reduced to about 400 r, the response of the

1

150 140 130 120' 110 IOO 90


adrenal cortex becomes smaller and irregular in the rat67, but a significant change in adrenal ascorbic acid has been observed in the rat after as small a total body dose as 50 r 68. The guinea-pig, some strains of which are unable to synthesize ascorbic acid, might be a more suitable animal for a study of the dose effect on adrenal activity.

Thus, there is in the rat (and probably in all mammals) a typical quick neuro-endocrine reaction very similar to the unspecific reaction observed after all kinds of stresses, or violent stimuli (see also ref. 70).

Arnold had concluded already in 1954 on pure anatomical evidence that the paraventricular and supraoptic nuclei of the monkey (Macaca mulatto) are selectively responsive to x-irradiation (single local dose of 3000 r of 23 MeV x-rays, equivalent to 1800 r of 200-400 kV x-rays); he suggested that the therapeutic effects reported after x-irradiation of the hypophysial region for pituitary dysfunction might be due to the action

Medulla

FIG. 15-5. Irradiation (directly or indirectly through "toxic" substances liberated in the tissues) excites the hypothalamic nerve cells. The well-demonstrated pathways are in continuous

line; questionable ones are in broken line.

upon the hypothalamus66. Thus one finds here, rather unexpectedly, confirmation of the importance of the central nervous system in the reaction of mammals to ionizing radiation, a fact repeatedly stressed by the Russian authors, but generally omitted in Western comprehensive reviews (see also p. 407).

(b) The second reaction—The second adrenal reaction, i.e. the drop in adrenal cholesterol and ascorbic acid beginning at 48 hr and lasting until the death of the rat is a much more complex phenomenon.

The anterior pituitary as expected is again involved. Figure 15-6


shows that the second reaction is absent not only in new-born rats (less than 10 days old) but in animals aged 15-20 days which show the first response57. In hypophysectomized grafted adult rats, irradiation induces a drop in ascorbic acid but none in cholesterol content57. In hypothalamic lesioned rats the second reaction is obvious (Fig. 15-2)58. Rats protected with cysteamine do not show a second adrenal reaction.

Urinary excretion of corticoids is said to be increased at various times (between the 5th and 12th day) in the dog25, but this observation has not been confirmed in the same animal35 in the pig60, in the rat51 or in the rabbit69; in the rabbit, oliguria might explain decreased corticoid excre-tion69. The level of the corticoids in the blood does not show in the rat any definite correlation with what happens in the suprarenals; for instance, there seems to be a peak at 24 hr when the chemical composition of the adrenal is normal61. In the monkey, an increased concentration of blood corticoids occurs only a few days before death62.

The interpretation proposed by one of us71 is that this second reaction is associated with the very bad conditions of the irradiated mammals a few days before death; in the monkey, there is no second increase of blood corticoids if the animal survives irradiation62. The second reaction does not seem to be correlated with the irradiation, but with agony.

In the rat, exposed to a high dose, the time relation between the second reaction and the moment of irradiation is simply due to the fact that all animals die between the 5th and 9th day6. If animals are exposed to a smaller dose than an LDioo, the second reaction becomes very irregular, or disappears. Figure 15-6 shows that it is impossible to accept the idea that the origin (the stimulus) and the mechanism of this reaction is the same as that of the first.

The discrepancies often observed after the second day between ascorbic acid and cholesterol results may be explained by a marked increase of liver cholesterol synthesis (see p. 321) and profound troubles in cholesterol synthesis or metabolism brought by hypophysectomy or selective destruc-tion of the hypothalamus*. Our observations on the second reaction of irradiated hypothalamic lesioned rats cannot be explained without accept-ing such an hypothesis58. Direct destruction by x-irradiation of ascorbic acid cannot be ruled out73; a fall of ascorbic acid has also been observed in the spleen and seems to be under hormonal control74.

In the irradiated guinea-pig, a few days before death, histological signs

* The changes in cholesterol values are difficult to interpret when they do not consist in a quick reversible reaction as for instance the "first reaction". T h e cholesterol content of the adrenals is the result of an equilibrium between (a) synthesis by the gland and incorporation in the gland of cholesterol from the blood (this cholesterol may come from food or synthesis by the liver), and (b) utilization of cholesterol for the corticoids synthesis or other metabolic processes.


of destruction are visible and the animal does not react to ACTH5 9 ; Rosenfeld, on the contrary, insists on the lack of evidence of adrenal exhaustion in the irradiated calf even when in agony86-87.

(c) The growth or somatotropic hormone (STH) has little or no effecl if given before or after total body irradiation of normal or adrenalectomized r a t s 3 3 , 4 0 , 4 1 , 4 2 , 7 2 _

Sommers has concluded from his experiments with parabiotic rats that the gonadotropic, somatotropic and thyrotropic activities of the pituitary are unaffected or slightly stimulated by irradiation if the adrenals are intact (see refs. 76 and 96; also ref. 6). When radiation damage to ovaries

Hours

FIG. 15-6. Summary of the results about changes in cholesterol values of adrenals of rats after x-irradiation (800 to 850 r) in various physiological and pharmacological conditions. The relative rapidity of death under the various conditions is roughly indicated by the height of the cross; 97 per cent of the

cysteamine-injected animals survive57.

or testis is discussed, one seldom takes into consideration the possibility of a decreased secretion of gonadotropic hormones (GTH) which is a typical reaction to stress1-2. No decreased prolactin secretion has ever been reported either. Thus, it seems that the hypophysial reaction to ionizing radiation (either locally or on the whole body) is quite peculiar and not comparable to that induced by other stresses (cold, formaldehyde, fear, etc.) which result in a decreased secretion of prolactine, STH, GSH


and TSH. THOMSON7 5 emphasizes the point that the increase in tryptophan peroxidase activity of rat liver (observed 4 hr after 1000 r of x - r a y s ) is abolished by adrenalectomy, but not by hypophysectomy and cannot be considered as a sign of general adaptation in Selye's sense.

(d) The last point to discuss is the relative importance of these adrenal reactions in the general symptomatology of radiation sickness. Let us try to answer simple questions:

1. Does the absence of pituitary or adrenals modify qualitatively the radiation damage in mammals? No. The study of hypophysectomized grafted rats, of new-born or adrenalectomized rats emphasizes the similarity of reaction, even on a quantitative viewpoint77'78 with that of normal animals.

2. Is the adrenal response useless? Is it beneficial or is it detrimental? The first reaction certainly helps the animal to survive the 2-4 days following heavy irradiation as shown by the fact that new-born rats which do not have this adrenal reaction show better survival rate if they receive a small dose of ACTH (0-1 unit) immediately and 3 hr after irradiation57-93. There is ample experimental demonstration that the irradiated rats need (or consume) more cortisone (not DOCA) than the normal80 '6 '79, and that the hyperactivity of the adrenal cortex at that time can be proved by a physiological test, the onset of fatigue in the striped muscle of the guinea-pig59.

As far as the late reaction is concerned, much discussion has taken place which cannot be reconciled easily. Firket and Betz have proposed a complicated scheme (see ref. 81): cortisone after being helpful during the first reaction, becomes detrimental during the later stages; in their opinion, the second adrenal reaction of the rat irradiated with a dose which kills in 5 to 9 days, is a period of hyperactivity and the hypersecre-tion of cortisone inhibits the regeneration of the bone-marrow (see ref. 6). There may be peculiar reactions to cortisone of irradiated tissues83'84 but the concept of Firket and Betz has not been generally accepted75'71 and in any case cannot be extrapolated to other mammals. For instance, TONKIKH 5 9 reports histological observations of adrenal cortical damage in the guinea-pig, and physiological signs of cortical insufficiency (very rapid muscular fatigue) 4 to 6 days after a total body dose of 800 r ; at 7 or 8 days after irradiation of the guinea-pig, cortisone injection resulted "in a colossal increase in the efficiency of the gastrocnemius muscle", Recent observations on young men 2 to 4 weeks after total-body irradiation are not consistent with an adrenal overactivity82; there is much to say in favour of serious investigations on corticoids blood level and urinary excretion in accidentally or therapeutically irradiated men.

One should neither overestimate nor neglect the neuro-endocrine reactions after irradiation (see also ref. 59).

402 F U N D A M E N T A L S OF R A D I O B I O L OGY Other endocrine reactions to irradiations have been observed94-95. The

ratio of j8- to a-cells in the pancreas of rats, changes after exposure to 250 or 500 r of x-rays and this change is correlated with variations in the tolerance to glucose97.

The difficulties encountered in interpreting the facts of radiobiology in vertebrates increase the value of observations on living material which is not subject to neurohumoral regulation. If a fact observed in a mouse is observed in micro-organisms, in pollen and in growing roots, it is certain that it is a fact of general radiobiology. Mammalian reactions show peculi-arities which are important because they also concern man, and in this atomic age it is difficult not to think of oneself sometimes, but this is a matter of special radiobiology. Mammals have special nervous and humoral regulating mechanisms; they have certain substances which other organ-isms (erroneously regarded as "inferior") do not possess; they also have special needs, being unable to synthesize many molecules which unicellular organisms produce easily.

Further chapters will be devoted to the special radiobiology of mammals and of man, but they will be summaries, the main object of which will be to show that the reactions of these animals can best be understood in the light of general radiobiology, which we have tried to define in the preceding chapters.

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51. RAJEWSKY, B., Radiobiology Symposium, Liege, 1954, Butterworths, London, 1955, p. 81

5 2 . BETZ., E . H . , C.R. Soc. Biol., 1 9 5 6 , 150, 1 8 1 8 53. LOUTSCHNIK, N . V. a n d KOULIKOVA, V. G . , C.R. Acad. Sci., URSS, 1956,

110, 982 5 4 . M A I S I N , J . , V A N DUYSE, E . , BRAUN-LALLEMAND, N . a n d LAMBERT, G . , Rev.

Beige Path. Med. Exper., 1956, 25, 392 55. MAISIN, J., Personal communication, 1959 5 6 . BACQ, Z . M . and FISCHER, P . , Radiation Research, 1 9 5 7 , 7 , 3 6 5 5 7 . BACQ, Z . M . , MARTINOVIC, P . , FISCHER, P . , PAVLOVI6, M . , SLADI6 , D . S .

and RADIVOJEVI<5, D . V., Radiation Research, 1 9 5 7 , 7 , 3 7 3 58. BACQ, Z. M., SMELIK, P . G. , GOUTIER-PIROTTE, M . and RENSON, J . , Bull.

Acad. Mid. Belg., 1959, 24, 800 5 9 . T O N K I K H , A . , Second U.N. Intern. Conf. on the Peaceful Uses of Atomic

Energy, Geneva, 1958, Vol. 22, p. 219, United Nations, 1959 60. BRAYER, F. T . , GLASSER, S. R . and D U F F Y , B . J . , J R . , Science, 1 9 5 4 , 120, 1 1 2 6 1 . V A N CAUWENBERGE, H . , FISCHER, P . , VLIERS, M . and BACQ, Z. M., Arch.

Intern. Physiol. Biochem., 1957, 65, 143 6 2 . FRENCH, A . B . , M I G E O N , C . J . , SAMUELS, L . T . a n d BOWERS, J . Z . , Am. J .

Physiol., 1955, 182, 469 63. BEAUMARIAGE, M . L. , Arch. Intern. Physiol. Biochem., 1955, 63, 132 64. ZUNZ, E. and L A BARRE, J . , Bull. Acad. Med. Belg., 1926, p. 649 65. LEBEDINSKY, A. V., Radiologic Medicale (in Russian), 1956, 2, 3 6 6 . ARNOLD, A., J. Clin. Endocrinol. Metabol., 1 9 5 4 , 1 4 , 8 5 9 6 7 . BACQ, Z. M. and FISCHER, P . , Unpublished observation, 1 9 5 6 6 8 . H O C H M A N , A . and BLOCH-FRANKENTHAL, L., Brit. J. Radiol., 1953, 2 6 , 5 9 9 69. MAURER, H . J . and PFEFFER, K . H . , Klin. Wochenschr., 1953, 31, 902 7 0 . MAURER, H . J . , Radiologica clinica, 1 9 5 4 , 2 3 , 2 4 0 7 1 . BACQ, Z . M . , Bull. Acad. Med. Belg., 1 9 5 7 , 2 2 , 2 1 9 7 2 . BARLOW, J . C. and SELLERS, E . A., Radiation Research, 1 9 5 5 , 2 , 4 6 1 73. OSTER, H . L . , KRETCHMAR, A. L . and BETHELL, F . H . , Proc. Soc. Exptl.

Biol. Med., 1953, 84, 470 74. CHEVALLIER, A., and MANUEL, S., Intern. J. Radiobiol., Suppl. 1, 1960, p. 33 7 5 . T H O M S O N , J . F . , Ann. Rev. Nucl. Sci., 1 9 5 4 , 4 , 3 7 7 7 6 . SOMMERS, S . C . , J. Lab. Clin. Med., 1 9 5 3 , 4 2 , 3 9 6 77. HAJDUKOVITCH, S., Personal communication, 1959 7 8 . MENDELSOHN, M . L . , Radiation Research, 1 9 5 5 , 3 , 4 4 4 7 9 . BETZ, E . H . , C.R. Soc. Biol., 1 9 5 6 , 150, 1 8 1 0 80. KATSH, S. a n d EDELMANN, A. , Federation Proceedings, 1951, 10, 73 8 1 . FIRKET, J . and BETZ, E . H . , Bull. Acad. Mid. Belg., 1 9 5 7 , 2 2 , 1 8 6 8 2 . JAMMET, H . et al., Rev.frang. itudes clin. et biol., 1 9 5 9 , 4 , 2 1 0 83. BERDJIS, C. C . and BROWN, R. F . , Diseases of the Chest, 1957, 32 8 4 . BETZ, E . H . , C.R. Soc. Biol., 1 9 5 7 , 151, 1 2 7 4 8 5 . ELLINGER, F . , Radiobiology, 1 9 4 8 , 5 1 , 3 9 4 86. ROSENFELD, G . , N . M . R . I . Research repor t , 1957, 15, 434 87. ROSENFELD, G. , N .M.R. I . Research Report, 1957, 15, 685 8 8 . R U G H , R . and W O L F F , J . , Proc. Soc. Exptl. Biol. Med., 1 9 5 7 , 9 6 , 1 7 8 89. DACQUISTO, M. P., Radiation Research, 1959, 10, 118 90. M A I S I N , J . , et al., Intern. J. Radiobiol., 1960, Suppl. 1, p. 183


9 1 . M A I S I N et al., Proc. Second U.N.Intern. Conf. on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 167, United Nations, 1959

92. Si.ADID, S. D., PAVKS, D. and RADIVOJEVI6, D., Bull. Inst. Nucl. Sci., Boris Kidrict Belgrade, March 1959, 9, 209

93. SLADI6 , S. D . , RADIVOJEVI6, D . V. and PAVI6 , D . M., Proc. Second U.N. Intern. Conf. on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 23, p. 59, United Nations, 1959

9 4 . WARREN, S . , Arch. Pathol., 1 9 4 2 , 3 4 , 7 4 9 9 5 . WARREN, S . , Arch. Pathol., 1 9 4 3 , 3 5 , 3 1 3 96. BOTKIN, A . L . , PRAYTOR, E. H . , AUSTING, M. E. and JENSEN, H . , Endo-

crinology, 1952, 50, 550 9 7 . ALLEGRETTI, N . , Proc. Second U.N. Intern. Conf. on the Peaceful Uses of

Atomic Energy, Geneva, 1958, 22, 208 9 8 . HEMINGWAY, J . T . , Experientia, 1 9 5 9 , 15, 1 8 9 99. Brookhaven National Laboratory, Biology Department, Annual Report,

July 1st, 1959, p. 81 100. RAMIOUL, H . and LECOMTE, J . , C. R. SOC. Biol., 1959, 1 5 3 , 1485 1 0 1 . GOODALL, M c C . a n d L O N G , M . , Am. J . Physiol., 1 9 5 9 , 1 9 7 , 1 2 6 5 1 0 2 . DRESSE, A., RENSON, J . and BACQ, Z. M., unpublished observations, 1 9 6 0

C H A P T E R 16

Physiopathology of Radiation Sickness in Mammals

M A N Y hundreds of papers have been devoted to this subject because it is directly related to the interpretation of the signs observed in man. There can be no question of reviewing them here—many are, in any case, of only limited interest. A general sketch will be sufficient, since detailed reviews are available31-32-33'54'2'55'66'67. The acute radiation sickness and some of its delayed consequences, particularly in the monkeys, have been discussed at length by the Russian radiobiologists at a symposium held at Sukhumi in 195 967«.

H Y P E R A C U T E S Y N D R O M E When mammals are exposed to very large doses (12,000 r or more) and dose-rates of ionizing radiation, death occurs rapidly (see Fig. 3-8, p. 71); symptoms of central nervous system (hyperactivity) are prominent: it is the hyperacute radiation syndrome, characterized by the following sequence of events in mice: (1) ataxic phase (5 to 10 min), (2) lethargic or inactive phase, (3) hyperactive convulsive phase, (4) terminal phase (ref. 62, see also refs. 61 and 64). Pentobarbital62 and diphenylhydantoin63, two classical anticonvulsive drugs, delay the appearance of convulsions and prolong the mean survival time as much as twelvefold, when such high doses are given. Monkeys seem to stand 30,000 r with little trouble, at least during 100 min65.

The main cause of this central nervous system disturbance may be the very greatly increased potassium content of the blood. The cells take up sodium in exchange for the potassium they lose; in other words the functioning of the so-called "sodium pump"* is blocked16. Release of potassium has been observed from isolated or in situ mammalian hearts after exposure to 500-2500 r 61 and of dog's brain after irradiation of the

* T h e ionic composition of the intracellular fluids is very different from that of extracellular, the main difference being that cells are rich in K + and circulating fluids rich in Na+ . This difference is due to active biochemical processes which constantly eliminate the Nations out of the cell.

4 0 6

P H Y S I O P A T H O L O G Y O F R A D I A T I O N S I C K N E S S I N M A M M A L S 4 0 7

head (5000 r)68. So far, however, no detailed study has been made of the possible changes of ion transport through membranes during heavy irradiation.

T H E F I R S T S T A G E O F R A D I A T I O N S I C K N E S S The early reaction, the onset of which is much dependent on the dose-rate, is well described in the monkey (M. mulatto) after total-body x-irradiation ( 4 5 0 - 4 8 0 r) by HUNTER et al36 in these terms: "The onset of the initial reaction could be readily detected by a quite sudden change in the animal. The monkey would hang heavily from the cage roof, head on chest, or sit dejectedly in a corner, and at times lie down on the floor of its cage. Loss of appetite accompanied this change. In most animals these signs would pass off only to return in a few minutes in an exaggerated form, accompanied by retching and vomiting. Waves of the latter signs recurred; but gradually their severity lessened, the interval between them lengthened and they ceased in 2 hr or so. Appetite then returned, and no further vomiting was observed in the later course of the syn-drome."

A similar behavioural change has been observed in mice; the dose of x-rays required to stop spontaneous activity was found greater in 2-months-old mice (604 r) than in 3^-months-old (512 r) or in 4J-months-old animals (503 r)72. As is well known, the sensitivity to lethal action is in the reverse order; thus the central nervous system may, as a whole, be more physiologically resistant in young mammals.

1. Action of Ionizing Radiations on the Nervous System When isolated single giant nerve fibres of the earthworm are constantly

irradiated at the rate of 6 kr/min (280 kV), one observes with regularity an increase in amplitude of the spike (20 per cent maximum), an increase in conduction velocity (11 per cent maximum), a decrease in the threshold stimulus voltage (maximum 18 per cent) and a very slight decrease of the refractory period112. The moment of maximum excitory effects under continuous irradiation varies between 5 and 13 min; this excitory phase is quickly followed by a marked depression; the preparations failed to respond after 40 to 50 min of irradiation112. It is a pity that such a nice preparation has not been used to study the effects of a single irradiation of short duration.

The classical frog's nerve-muscle preparation shows very little reaction to irradiation (140 kV x-rays) below the large dose of 20 kr; at that level, decrease of the response to maximal single shocks on the nerve was ob-served". It is suggested but not proved that the partial block is at the neuro-muscle junction. The mechanism of transmission of the impulse from nerve fibre to striped muscle is difficult to disturb. It is no surprise


to a physiologist to find that complicated reflexes (like conditioned reflexes) which are more easily fatigued because they involve many nerve cells and synapses, are also more sensitive to ionizing radiations.

HUG116 has observed many instantaneous reactions of invertebrates to ionizing radiations. He presented at the Venice Symposium a most convincing film. In three species of snails (Helix pomatia, Helieella candidans and Arion empirieorum), the tentacles are retracted and remain so for the duration of irradiation. As soon as irradiation ceases, they are extended again. The threshold for this reflex-like response varies from 1 -5 to 5 r/sec according to the species; the latency from the start of irradiation at threshold rate to the beginning of response varies from 5 to 15 sec. A sea-water snail (Palndina), a clam (Mytilus edulis), various actiniae (Ac-tinia equina, Metridium senile) also show reflex responses of their tentacles or gills. A small sea urchin (Echinus miliaris) is particularly sensitive. Behavioural disturbances have also been noted in insects but at higher dose-rates116.

Involvement of the central nervous system (c.n.s.) in this early reaction and particularly in vomiting is obvious37. Reliance on pure anatomical damage has established the legend (generally accepted up to the very last years) that the nervous system (whether central or peripheral) is radio-resistant, i.e. remains unaffected by small or moderate doses of ionizing radiations. When accurate physiological criteria are used, it is possible to observe a great variety of rapid effects of small doses of radiation, such as the following:

(a) Changes in threshold of electric stimulation of the cat's hypo-thalamus after 50 r total-body irradiation38 (see also p. 395).

(b) Depression of cortical activity in man preceded by a short phase of increased activity, after head irradiation38.

(c) A rise in cortical excitability of the rabbit 10 sec after beginning of a total-body irradiation at 0-13 r/sec; depression was seen 1 hr after a dose of 7-8 r38.

(d) Changes in the response of the cortex to ionizing radiations after injection of certain drugs; for instance after caffeine injection, the depres-sion of brain activity occurs without preliminary increase38.

(e) The voltage of the electroretinogram of the isolated frog eye is also depressed after a short period of considerable increase if the dose is small (Fig. 16-1)38. Some Japanese authors40 have observed in humans, changes in the response of the retina to electric stimulation after doses as small as 0-05 r.

LIPETZ115 has carefully reviewed the literature and made important personal contributions to the study of effects of low doses of high-energy radiation on visual function of man and frog. Since 1896 it has been known that x-rays can give one the sensation of light probably by direct excitation


of the visual purple. Some of the effects of ionizing radiations on visual functions can be considered as physiological because they disappear quickly. For instance, a subthreshold dose of 0-5 r decreased by two-thirds the voltage of the action potentials of a single ganglion cell of the frog retina, but one minute later, these action potentials were normal. The smallest amount of x-rays which can produce observable effects on the visual system is about 0-5 milliroentgen—an exceedingly small amount of energy. It has been calculated that this amounts to only 160 x-ray quanta absorbed by the whole retina; this is about 2 x 10~5 erg.

FIG. 16-1. Changes in the b wave of the electro-retinogram of isolated frog eyes after irradiation by x-rays (10, 60 and 300 r).

Ordinates - ERG potentials in microvolt; abscissa time, in minutes from the beginning of irradiation38.

The threshold for a flash of visible light is about 4 x IO-8 erg. The conclu-sion of Lipetz will please many physicists* ". . . the visual system is so organized that radiation can trigger a sequence of processes which amplify the energy and effect by several orders of magnitude"115.

(f) Intense flow of impulses from the periphery to the central nervous system and inhibition of the spinal centres are observed in cats and rabbits during the first minutes and hours after total body irradiation (500 to 1000 r; 20 r per min)39.

(g) The centres of postural reflex activity in the rabbit are in a state of increased excitability for one or two days after irradiation (1000 r on total body).

(h) In sharp contrast with the above-mentioned observations, stands the fact that conditioned reflexes are markedly depressed in the rabbit during the 2-4 days following exposure to 500 r (ref. 39 and Fig. 16-2). Several interpretations of this phenomenon are possible. It seems that the

* The classical dialogue between the physicist and the biologist is: the latter says to the former, "You are fortunate because you can measure accurately very small amounts of ionizing radiations!" The former tells the latter, " I envy you, because you can see on living organisms, effects of very small doses of ionizing radiations!"


irradiated cortex cannot handle or endure the increased affluence of impulses from various sources, and consequently enters in a state of transmarginal inhibition39. CASTER et al.59, in the U . S . A . , have found a drop in DNA content of the rat's brain 12 hr after exposure of the whole body to x-rays (700 r), and also changes in the electrocorticogram.

>

150

100

50

Irradiatk

-/ Intensity of conditioned reflex

/ D strong

/ D medium

\ / o weak

a disappearance

1 1 1 , , , , H l n l n

Before l h r 1

irradiation 2 3 4 5 6 7

Days

8 9 IO

FIG. 16-2. Modification of bioelectric activity of motor analyser and intensity of conditioned reactions in a rabbit after total x-radiation at dose of 500 r39. Abscissae — time in days. Ordinates — bioelectric potentials in microvolts and intensity of

conditioned reflex.

The c.n.s. of birds (hen and pigeon) show very different responses to ionizing radiations: the alimentary conditioned reflexes do not change even after a strong repeated irradiation. The disturbances are mainly sub-cortical during radiation sickness in birds39. In birds, chlorpromazine has "disinhibiting" actions comparable to that of irradiation39.

(i) The c.n.s. of foetus or new-born mammals is much more sensitive than the adult system, not only on morphological grounds41'42-60, but also physiological or clinical evidence38>42. The foetal neuroblast is extremely radiosensitive, just as the grasshopper neuroblast100.

The picture gathered from a review of the literature is that, if effects of large doses on the c.n.s. are obvious (ses also p. 398), there is need for more detailed and fundamental studies on the effects of small or medium doses. The use of modern techniques—registration of potentials from single cells in the c.n.s., intracellular electrodes, etc.—is most advisable. The important observations of Russian radiobiologists leave little doubt that a harvest of information would rapidly be at hand.

Other biophysical techniques might also be useful for the analysis of


early effects of ionizing radiations on cell membranes; for instance, the measurement of electric resistance. There are old reports that the electric resistance of the liver of the guinea-pig113 or of the mouse114 and of mouse tumours114 is strikingly reduced after x-irradiation. These observations have been made a long time (at the earliest, 19 hr) after irradiation, and the techniques have so much improved that, again, anyone entering this field would make important contributions.

2. Actions on Circulation and Respiration Doses of 400 r (or below) on total body of the dog do not affect arterial

blood pressure or cardiac output56 '57; heavy doses (5000 r) on the head of anesthetized dogs do not change blood pressure, respiration, cerebral blood flow or oxygen consumption68. Cardiovascular changes in irradiated dogs (1000 r) should become evident only 12 hr before death104 and the blood volume well maintained up to the last day103.

CASTER111 believes that changes in the electrocardiogram (and, generally speaking, in the cardiovascular system) differ much from one species of mammal to the other, that they may be the result of an early decrease in plasma volume. However, the heart muscle after irradiation shows a marked increase in Evans-blue space, a decrease in DNA and actomyosin; some kind of heart failure might be the cause of radiation death in the 8-10 day period111.

Very large variations on the sensitivity of the vascular bed to adrenaline have been observed in rats after exposure to 600 r, and these variations are said to be due to substances (excitatory or inhibitory) appearing in the blood, the same substances which are seen in any type of shock69.

In the rhesus monkey, changes in pulmonary ventilation during the 100 min following the beginning of whole-body irradiation (1 to 30 thousand r) are believed to be related to hypotension, decreased blood pH and CO2 pressure; it takes 30,000 r and 1 hr to observe some im-pairment of the vagal control of respiration65.

3. Histamine Liberation Any physical stimulus of sufficient strength (heat, ultra-violet or

ionizing radiation) and many toxic substances (mercury salts, snake or insect venoms, some bacterial toxins) which damage cells, liberate the so-called performed, tissular histamine from isolated and perfused guinea-pig lung.

In vertebrates exposed to x- or y-irradiation, one finds generally a liberation of histamine78 '80 '82-83 '86 '142 with increased histamine level in the blood74 '87 '91,97 and increased secretion in the urine95. The /3-radiations have not been shown to liberate histamine95. The kinetics of this liberation of tissular histamine by x-irradiation varies from one species to the


other78. The rapid liberation of histamine associated with an increased sensitivity to histamine of irradiated animals94'98 may explain some of the symptoms observed in species (rabbit, chick) showing peculiar reac-tions to ionizing radiations77. In the chick, depletion of endogenous histamine or antihistamine injections before irradiation, change very little the mortality and the effects of irradiation77 and, in certain species, may have an unfavourable influence75'85'90'92'95'96. The histamine content of the gut in rats falls to the fifth of its normal value 24 hr after exposure of the abdomen to 1500 r of x-rays140.

The reaction of irradiated animals to the injection of the antigen to which they have been sensitized, is variable according to the species and the time between irradiation and injection. In general, if one injects the antigen more than 48 hr after irradiation, mortality by anaphylactic shock is increased despite partial depletion of endogenous histamine; if injection is made during the two first days after irradiation, the anaphyl-actic reaction is decreased84-88'89'93.

x-irradiation inhibits the Schwartzmann phenomenon in the rabbit79, decreases the skin reaction to tuberculine in the guinea-pig81, and inhibits in the rat the liberation of endogenous histamine by ovalbumine, although the oedema is not suppressed76.

4. Other Instances of Early Physiological Reactions to Ionizing Radiations These become more numerous: 1. Water intake and urinary output is increased in the rat about 4 hr

following whole-body irradation. Serum from x-irradiated rats contains less antidiuretic substance than serum from normal rats48. Polyuria is absent in irradiated hypophysectomized rats48. Thus, this reaction involves the pituitary49, but head irradiated rats do not have a polyuria whereas head shielded x-irradiated animals do50. Several organs in the abdominal area appeared to be involved, but no single organ is responsible since irradiation of individual organs failed to cause polyuria.

Removal of the pancreas and shielding of the liver or kidneys prevents polyuria50. The role of the adrenals is uncertain. It has not been unequivoc-ally established if polydipsia is the cause of polyuria or if the polyuria precedes polydipsia. Anoxia during irradiation does not prevent polyuria or polydipsia50, but these reactions disappear if isotonic sucrose, fructose of glucose50 solutions are given as drink after irradiation. Curiously enough, normal unirradiated rats consume large amounts of fructose solution and show marked polyuria.

For some unknown reason, the early polyuria of irradiated mammals is not always seen with regularity137. The relation between electrolyte (mainly sodium) loss, bile secretion and water balance is discussed by Jackson et al. (see p. 327).

1 • . - - _ ' * 4

I 200C i OFF I i l l l l I i I I I I

• W

A SOOf iOFF

eoor CON'T.

I l l 1 I I l .....i 1 1 1 1 i—

FIG. 16-3. Effects of 200 r (upper kymographic recording) and 600 r (lower recording) on in vivo small intestinal longitudinal

contraction of the rat. From CONARD 5 2 .

[facing p. 412


2. The rat's small intestine in vivo shows an almost immediate increase in tone and motility after doses as low as 100 r with rather rapid return to normal activity (see refs 51 and 52; also Fig. 16-3).

This response is a true effect of ionizing radiation on the intestine since it can be obtained by exposing the intestine loop and shielding the rest of the body. CONARD5 2 believes that the x-rays stimulate the enteric ganglia; the reaction is increased by eserine (an anticholinesterase sub-stance) and diminished (but not abolished) by atropine, which suppresses the action of acetylcholine, the cholinergic chemical transmitter. This the action of acetylcholine is, at least in part, responsible for this increased motility; however, Conard has not excluded the possibility of a liberation of 5-hydroxytryptamine (5-HT, or serotonine) which is abundant in the intestine and plays an important physiological role in the regulation of intestinal motility, according to recent investigations53. Hydroxytrypt-amine is liberated from the lower brain centres by x-irradiation58.

Thus, some very active substances (histamine, acetylcholine, 5-HT) normally bound, inactive within cells, and protected against inactivating enzymes, can be liberated by exposure to ionizing radiations and one finds here more arguments in favour of the idea developed elsewhere in this book (p. 272), according to which one of the primary (physiological or biochemical) lesions should be a disturbance of the intracellular relations between enzymes and substrates.

HUG116 has also observed increased motor activity of isolated pieces of rabbit's intestine in a bath of saline if the»dose-rate reached 2r/sec. The

100

Time, hr

FIG. 16-4. The effect of x-irradiation on the pattern of gastric emptying. The curves for the various doses are shown on the graph; the control is an average of 13 animals, 20 r is an average

of 5, 30 r of 6 and 100 r of 4 animals101.

4 1 4 F U N D A M E N T A L S O F R A D T O B I O I . O G Y

latency of the response decreased at higher dose-rates. During the 5 hr following total body irradiation (100 to 700 r) of the fasting rat, the weight of the stomach contents increases; antiperistaltism results in regurgitation of intestinal contents in the stomach71. These troubles affect the electrolyte and water exchanges which are very much modified by irradiation. Delay in emptying time of the stomach in the rat is visible after total body ex-posure to as little as 20 r (Fig. 16-4, and ref. 101).

C H A N G E S I N P E R M E A B I L I T Y There are many conflicting reports in the literature about the effects of ionizing radiations on permeability. The reasons for these discrepancies are obvious: measurements are made on different systems, with different techniques at various times after different exposures to various types of ionizing radiations delivered at different rates.

At first sight, one might question the usefulness of discussing such an obvious phenomenon; but it has been questioned118. Very heavy doses locally in man or any mammal are quickly followed by oedema. For instance, exposure of a rat forelimb to x-rays with shielding of the rest of the body produces large gains in extracellular water119. If one compares the weight of the irradiated limb with that of the non-irradiated control, it is obvious that a significant elevation in water occurs immediately after exposure, but it remains small for 8 to 12 hr at which time a rapidly increasing rate of water gain is observed120. The permeability of the capillary endothelium is greatly increased as shown by the passage of 131I-Iabelkd serum albumine. rIhe presence of red cells in the lymphatics of the heavily irradiated dog shows the complete destruction of the delicate exchanges occurring through the capillary endothelium. B A R E R

and JOSEPH144 have reported a marked fall in the concentration of solids in circulating lymphocytes of mice within 10 minutes of a total body irradiation of 300 r of x-rays. The effect reaches its maximum after 2 to 12 hours after which recovery occurs. The measurements of concentration were made by quantitative refractometry and could arise either from a loss of solid or an inflow of water.

According to K A H N and FURTH110 the decrease in plasma volume is one of the best early signs of severe radiation injury. This may be due to increased capillary permeability, a result of direct damage or of histamine liberation. However, when smaller doses or dose-rates are used, the pheno-mena are less obvious and the published results need critical reviewing.

Permeability to Ions and Water It is known at present that the intracellular content of mineral ions as

contrasted with that of the surrounding liquid and the electrical properties of the membrane are due to two types of phenomena:

L ' H Y S I O P A T H O L O G Y O F R A D I A T I O N S I C K N E S S I N M A M M A L S 4 1 5

Passive processes which consume none (or very little) energy; artificial membranes have been prepared which can maintain a potential differ-ence of 50 mV thanks to an unequal distribution of ions on both sides of the membrane. Active processes which consume energy, need well-adapted, fast-working, strategically-located biochemical systems; the so-called "sodium pump" is a very efficient system which rapidly expels from the cell any excess of Na+ ions which might have penetrated, for instance at the beginning of an excitation process in a nerve fibre, a nerve cell or a muscle cell. Ions (mainly Na+ and K+) constantly enter and leave the cell; the

ionic composition of the tissue is thus the result of a dynamic equilibrium which may be affected by ionizing radiations, but so far we have little valuable data at our disposal.

ROTHENBERG 1 1 7 has observed an increased uptake of 24Na by squid axons after irradiation.

Isolated rabbits' hearts release much potassium in the perfusion fluid after irradiation; and exposure to 1000 r increases four times the loss of K+ in one hour. Dog's heart irradiated in situ also loses potassium in the coronary venous blood121. These experiments give a good background to the observations mentioned above showing that the heart is not a radio-unsensitive organ, but they should be repeated with 42K as an indicator.

In heavily irradiated (from 20 to 140 kr) bacteria (E. coli), the efflux of K+, at the stationary phase, is rapidly increased122. Potassium retentivity is five times less sensitive as an indicator than survival for yeast cells after heavy x-irradiation in aerobic condition123.

B R U C E believes that his observations suggest that in yeast the radiations act at sites that independently control survival and the retention of K+. The influence of oxygen and of temperature in anaerobic condition is remarkable. At 25°C the oxygen effect is a factor of about 3, at 0°C it is 5-3123.

If there are changes in electrical capacitance, there must be alterations of permeability, mainly to ions. The capacitance of frog erythrocytes increases after an exposure to as little as 100 r (of x-rays); 42K uptake in-creases immediately after irradiation, but this is followed after 90 min by a loss of 42K. Slight hemolysis and morphological changes are ob-served with amphibian red cells very soon after 50 to 100 r; it seems that within the heterogeneous population of these non-dividing cells some (supposed to be older cells) are more sensitive than others and after irradia-tion cells are left which are more resistant to osmotic hemolysis124. There are many conflicting reports about mammalian red cells irradiated in vitro; we have seen nothing conspicuous in dog's blood.

S I M O N 1 2 5 , in 1 9 3 4 , showed that the permeability to water of the eggs of Pholas is increased by y-irradiation. This author, in order to show


that this effect is related to the capacity of the organism to use O2, added KCN to the medium and observed that the effect of irradiation was abolished. As often happens, this result has got to be reinterpreted in the light of more recent evidence: the CN ion is a good chemical protector (see p. 459).

Permeability to Dyes Kovacs estimates the rapidity of penetration in the frog's heart of

methylviolet by measuring the time between injection of the colour in the circulation and the arrest of heart beat (the typical toxic action of methylviolet). Irradiation decreases this time126 but more direct arguments should be looked for—it does not seem difficult to know how much of the colour actually penetrates in heart cells—since it is known that the response of heart and smooth muscle to drugs may be changed by other mechanisms than permeability troubles.

Trypan blue, a nice vital colouring substance, of high molecular weight, has been used for some 30 years with success. Normally, in the frog, this blue penetrates only in certain cells originated from the connective tissue and persists in them for a long time. Irradiation with x-rays results in decoloration of these cells, and, contrariwise, to a deposition of colour in cells which normally do not concentrate it127.

Mammalian gland cells (with the exception of renal epithelia) also do not concentrate trypan blue, but do so after irradiation; this seems to be particularly visible in liver cells128.

Normally, the nucleus of certain infusoria are not coloured by trypan blue while the cytoplasm is; after x-irradiation, the nucleus colours it-self129, but although it is impossible to give a precise figure, the dose must be enormous as is usual with infusoria. After irradiation of the abdomen of rats with 1500 r of x-rays, capillary permeability increases as shown by the leakage of protein-bound trypan blue along the intestine. Adminis-tration of antihistamine drugs before irradiation largely suppressed the changes occurring up to 24 hr but not after that time even if the drugs are administered repeatedly140. Anticholiresterase drugs are also effective even at 48 hr, but the effect of an anti 5 HT drug is very slight141. Fluores-ceine—the presence of which is easily observed without the slightest trouble—penetrates very rapidly in the aqueous humor of the eye after irradiation130; this effect is extremely non-specific; any slight traumatism, an irritation of the cornea or conjunctiva, will bring it forward.

An increased permeability in extracellular spaces is visible after only 1 sec of latency; this observation is described (Chapter 19, p. 468) because it has been the starting point of experiments with chemical protectors.

Very early changes in 14C02 exhalation from intraperitoneally injected


14C bicarbonate were observed by Forssberg and Hevesy; they may be under hormonal control73.

Some Russian radiobiologists (cf. ref. 130) are of the opinion that the hyaluronic acid of the connective tissue must play an important role in this increase of permeability. Brinkman, Lamberts (see p. 468) and ourselves are of the same opinion. The problem to discuss is how hyaluronic acid is degraded; is it by direct action of the ionizing radiation (an inter-pretation favoured by the Dutch scientists), or is it by liberation, activa-tion of hyaluronidase? Some experiments of the Russian authors suggest this last interpretation, which is very much in keeping with our general idea that much of the damage from ionizing radiation is due to liberation of enzymes (see p. 272).

B L O O D C H A N G E S

In order to understand what happens in the blood after irradiation, it is necessary to have a clear idea of the production, life-span and destruction of all the types of cells found in it.

Red cells (volume about 100 ,a3) are produced by the marrow of flat and compact bones (in man also by the foetal liver). In adult rats and mice, the spleen is also a complete blood-forming organ; it contributes very significantly to red and white cell formation. In all mammals, red cells are thrown in the circulation as reticulocytes, which still possess a reticulum of ribonucleic acid (but with no nucleus, and with no DNA) and are capable of synthesizing hemoglobin. The reticulocytes rapidly lose this reticulum, they mature in about 4-5 days (in man). Thus, the presence of reticulocytes (normally about 3 per cent of all red cells) is generally a sign of good blood regeneration. The life-span of red cells in man is normally 120-130 days; they are destroyed by reticuloendothelial tissue particularly abundant in liver and spleen. Birds and cold blooded vertebrates have nucleated red cells. The number of red cells in human blood is normally about 5 million/mm3.

Granulocytes are white cells with granules in their protoplasm and a more or less divided nucleus; they are also called polynucleated cells. They are produced by the same bone-marrow which produces red cells, the mother cells of granulocytes being intimately mixed with those of the red cells. It takes two to three days for the bone-marrow of man to make and liberate granulocytes in the circulation; the life-span is about 8-9 days.

There are three types of granulocytes: neutrophiles (the most abundant), eosinophiles (or acidophiles) and basophiles which are recognized accord-ing to the affinity of their granules for acidic or basic dyes. Eosinophiles behave very differently from neutrophiles; their number in the blood


increases during allergic reactions and invasion of the body by foreign proteins; it sharply decreases when an excess of glucocorticoids (cortisone-like) is secreted by the suprarenal cortex (see p. 389).

Lymphocytes are liberated by the lymphoid tissue which is widespread in the body as lymph nodes, thymus, or inclusions in the spleen and bone-marrow. The nucleus of these cells is large, unique and globular; indeed, there remains only a tiny fringe of cytoplasm between the nucleus and the membrane. Tagged large mononuclears are found in the bloodstream already half an hour after injection of tritiated thymidine, a specific precursor for DNA. Apparently varied populations of smaller lympho-cytes derive from the large mononucleated cells and their life-span is variable.

The total number of white cells in a man is normally about 6 to 7 thousand/mm3 of which 60-70 per cent are neutrophiles, 2-4 per cent eosinophiles, 0-1 per cent basophiles, 4-8 per cent large monocytes, 20-25 per cent small or medium sized lymphocytes.

Platelets or thrombocytes are not cells, but cell fragments (diameter about 2-5 /(), with however a membrane, recognizable anatomical features and biochemical properties. They are liberated by the fragmentation of large cells, the megakaryocytes existing in the bone-marrow. The normal life-span of platelets in man is about 4-5 days. Their number is 300 to 500 thousand/mm3. They have peculiar physico-chemical properties, the most interesting being that of agglutinating themselves rapidly when a vessel is damaged or when an allergic reaction occurs.

Normally, red cells and platelets are only to be found in blood, i.e. in the liquid tissue circulating in arteries, capillaries or veins. Presence of red cells in lymph or in the interstitial fluid (those 12 litres of fluid in man which are neither in the cells, nor in the blood or lymph) is pathological.

White cells exist not only in blood and lymph but also in interstitial space; indeed only a small fraction (perhaps only 4 per cent) is present in blood. They can easily leave or enter in circulation; they have a tendency to gather in certain normal tissues (the lungs, for instance) or damaged sites. The regulation of these leucocytic movements is poorly understood, but is certainly in part under hormonal control and locally under the influence of chemical substances liberated by damaged tissues or infecting agents.

The use of tagged radioactive isotopes has greatly helped the quantitative study of the blood cells in animals and man (for a summary, see ref. 25).

The blood picture changes of any mammal after exposure to ionizing radiations are mainly the consequence of the anatomically visible damage in the bone-marrow and lymphoid tissue (the so-called haematopoietic organs). The haematopoietic tissues are in constant growth, delivering to the blood exactly the amounts of cells which are destroyed. Irradiation


stops mitosis, young cells are no more produced to replace old ones which go on being destroyed, sometimes at a more rapid rate. The bone-marrow has less and less inhabitants. A large dose (800 r, for instance) quickly delivered to a mammal, does not kill or gravely affect the circulating blood elements with the exception of the lymphocytes. Circulating red cells, granulocytes or platelets are "radioresistant". On the other hand lympho-cytes are affected and destroyed. As early as 3 hr after a dose of 400 r, 7 per cent of the circulating lymphocytes of the rat are picnotic. These damaged lymphocytes are removed from the bloodstream, especially by the lungs where they can be found in the alveolar capillaries20. During the 2 to 3 hr following acute irradiation of any mammal there is an increase of the number of granulocytes, mainly neutrophiles, of obscure origin (see Fig. 16-9, p. 426). Is it due to a re-entry in the blood of cells wandering in the intracellular space? Or, more probably, as it is the case for reticulo-cytes in new-born mammals26, is there a kind of active release of cells by contraction of the vessels of the bone-marrow? The increase in number of eosinophiles is smaller and quite shorter43, probably because of the in-increased secretion of glucocorticoids (see also p. 394).

In the rat, the hypergranulocytosis ends in the twelfth hour. Leucopenia then develops gradually, reaching a maximum in 2 to 8 days. If the animal dies, it will have very few white cells in the blood at the time of death. On the other hand, if it is to survive, regeneration of the leucocytes will begin to show after 8-10 days. In irradiated dogs there is close paral-lelism between the rate at which leucopenia develops and the dose admin-istered, in other words, between the leucopenia and the death rate (Figs. 16-6 and 16-7). Regeneration of the leucocytes occurs later in dogs than in rats or mice. In man, leucopenia seems to be the best indicator of the progress of acute radiation sickness.

Certain authors maintain that there is a parallelism between the leuco-cyte count, resistance to infection and survival. Figure 16-5 shows that mice irradiated with 750 r all die with extreme leucopenia, but it also shows that mice of the same strain, irradiated in the same way after an injection of cysteamine, show the same leucopenia, but 9 out of 10 survive longer than 30 days. In these protected mice, regeneration of the leuco-cytes begins after the controls are dead. Considerable and sometimes lasting leucopenia has been produced in white men and negroes by injections of nitrogen mustard without their showing any kind of infec-tion3'4. Leucopenia may not be dangerous in itself; in our mind, it is par excellence an indication of danger. The results of the many experiments on the importance of leucopenia in radiation sickness are indeed often contra-dictory (see ref. 46). Crossed-circulation established for 2 hr between a normal dog and one irradiated with 450 r, 6-10 days earlier, does not raise the number of leucocytes in the irradiated dog for more than 20-48 hr.

28

420 F U N D A M E N T A L S O F R A D T O B I O I . O G Y

Leucocytes supplied to an irradiated dog by mass transfusion do not remain long in the blood stream, about 13 hr.

In the red cells series, the observed facts are easy to interpret logically: (1) the reticulocytes, the immature red cells, disappear rapidly; (2) anaemia

6 0 0 0

5 10 15 20

Time after irradiation, days

FIG. 16-5. Leucocyte counts in peripheral blood of irradiated control mice, and mice irradiated with x-rays under protection of mercaptoethylamine. Two small graphs indicate survival and variation in weight with 700 r in the particular strain of mice. All control mice die with very low leucocyte count. Animals protected by cysteamine have slightly higher white cell count in first 9 to 10 days although 9 out of 10 survived5.

irradiated after injection with cysteamine. irradiated controls without cysteamine.

130 120 110

O

s ? 00« 9 0

n <S) 8 0 <D Cii C / 0 CL 6 0

T3 C O

C O 5 0

S3 O TJ

4 0 3 0

O 3 C CD ZO

O CL 10

- 0

J .1. M l

/ N1 — • — Ratelets - o - Granulocytes / L

— • — Ratelets - o - Granulocytes

\ I

\ \ \ \ I / "

\ / s j \ N1 \ k ,

S \

\ \ —I

0 1 2 3 4 5 6 7 8 9 10 Il 12 13 14 15 16 Time after irradiation, days

FIG. 16-6. Mean granulocyte and platelet count on 10 dogs exposed to atomic bomb y-radiation that produced 10 per cent

mortality2.


(i.e. decrease in number of red cells and of hemoglobin concentration in blood) sets in slowly because the large population of long-lived red cells acts as a buffer. Let us, for instance, assume that in an animal irradiation completely stops the production of red cells without affecting their destruction; it would take 80 days to drop from 5 million to 1 million cells/mm3 if the life-span of these cells is 100 days. Thus anaemia is not a danger after acute irradiation; it may become the central feature after long-continued exposure. Many anaemic persons have lived for years with a million red cells/mm3 or even less; death of acutely irradiated mammals generally occurs when the number of red cells is largely sufficient for oxygen supply.

—

r Vr - O - Granulocytes

N1 sr % Mor nlity

-10 M

0 % orta

%

-10 M

0 % orta ity

Sj Ps V S

p - r -2 3 4 5 6 7 8 9 IO I l 12 13 14 15 16

Time after irradiation, day

FIG. 16-7. Mean granulocyte and platelet counts in 2 groups of 10 dogs exposed to atomic bomb y-radiation that produced 80 and 100 per cent mortality. Platelet response in 80 and 100 per

cent groups were identical2.

The decrease in number of platelets after irradiation is often parallel to that of granulocytes; there is no preliminary increase (Figs. 16-6 and 16-7). The response of platelets is less proportional to the dose than that of granulocytes (Fig. 16-7) and also more irregular. In several mammalian species, there develops a hyperferraemia in two distinct phases: a transient rise during the first 24 hr after irradiation and a more prolonged elevation during the second week. The reason for these variations is unknown105, but the absence of hemoglobin synthesis is obviously the main factor.

T H E S E C O N D S T A G E O F A C U T E R A D I A T I O N S I C K N E S S

The second period is the period of haemorrhages, incipient anaemia, infection and malnutrition. Haemorrhages occur in nearly all irradiated dogs (450 r) and human beings; they vary in severity, in location, in their clinical course and in their response to treatment, and they are probably


not the most frequent cause of death. In dogs, they are most often found in the pericardium—not in the myocardium—and, in descending order of frequency, in the gastro-intestinal tract, lungs, pleura and mediastinum, pharynx, skin, bladder and kidneys1. MOLE 1 7 emphasizes the many varia-tions in the haemorrhages observed in different species of mammals.

Vascular fragility is one of the causes of these haemorrhages. The second cause is without doubt the disappearance of the platelets, even after non-lethal doses of radiation (Fig. 16-5). A lethal dose (450 r) reduces the average number of circulating platelets in dogs from 300,000 to 30,000 in 12 days. The coagulation accelerators carried by the platelets become deficient, but the quantity of prothrombin and accelerators in the blood seems to remain normal. Sometimes there is a spectacular increase in the coagulation time, and at other times very little. The decrease in coagulability may occasionally be due to the presence of an abnormally large quantity of heparin in the blood; it certainly disappears more or less completely, sometimes even absolutely completely, after the injection of protamine or toluidine blue, which are specific antagonists of heparin1. The effect of toluidine blue, if it appears at all, is more lasting (24—36 hr) than that of protamine (2 hr). It is suspected that molecular changes in certain proteins which intervene in the process of coagulation may play some part, but no precise facts are known. Blood transfusion, which is effective against the anaemia, has no apparent effect on the haemorrhages, and does not alter either the thrombopenia or the leucopenia. A striking increase of urinary and serum fibrinolytic activity has been observed in irradiated dogs during the three or four days preceding death. Many authors deny that heparin or heparinoid substances play any part in the pathogenesis of the incoagulability of the blood, although the mastocytes. which produce heparin., are radiosensitive and seem to disintegrate after irradiation, setting free their contents30. Cronkite's theory is supported by the experimental observation that platelet transfusion* restores retrac-tion of the clot in irradiated dogs and stops the internal haemorrhages. Recent human experience has beautifully confirmed the observations on dogs and the important role played by the platelets (see p. 509, Chapter 21).

Infection and ulceration accelerate the appearance of the haemorrhages and aggravate them. The capillary, petechial haemorrhages, which are easily visible in the subcutaneous tissues of the mouse, in which the large haemorrhages are less spectacular than in the dog, are easily stopped by adrenochrome semicarbazone10-11. After several investigations, rutin

* By the use of a chelating agent (disodium-ethylene-diamine tetra acetate) and of silicone coated flasks, platelets can be separated by fractional centrifugation without agglutinating24.


and various Havonoids are regarded as being of no use (see ref. 2, p. 206) in treating this condition.

A special type of lymphocyte, the binucleated lymphocyte, extremely rare in the blood of normal man and dog, becomes more frequent after (1) an acute exposure to even a small dose (50 r) of x-rays or neutrons, (2) continuous exposure to low dose-level,145 or (3) repeated short exposures to ionizing radiations as is the case in many occupational hazards. Miss Ingram, who has carefully studied this binucleated lymphocyte, believes that its frequency in the blood is a sensitive and rather specific test of previous radiation exposure44-45. Unfortunately, a large number of lymphocytes (up to 10,000) must be seen under the microscope (in order to have statistical significance), which means that a trained technician can examine only three blood samples in a day's work. Improvements in this direction will be welcomed by those in charge of health physics in hospitals, and atomic establishments.

Infection in irradiated animals and human beings is a problem of the first importance. When there are no burns or wounds, it is often of intes-tinal origin, as is shown by the nature of the micro-organisms isolated by blood culture.

After irradiation, the body loses some of its ability to form antibodies; it no longer reacts to tuberculin and becomes a prey to micro-organisms which are normally saprophytic (see refs. 135, 131, 132). The resistance and active immunity to animal parasite infections is also decreased, but acquired immunity to viral infection or bacterial toxins is not decreased by ionizing radiation134. The greatly decreased antibody formation must be regarded as an aspect of the diminution of protein synthesis. Exposure to x-ray doses of 350 to 500 r inhibit the synthesis of properdine, an euglobuline, a bactericidal fragment of the complement system132.

The best quantitative studies have been done with the antibody response to sheep red cell injections in mice or rats (refs. 136, 133 and Fig. 16-8). Maximum immunological depression occurred in mice receiving the antigen 5 min to 1 day after x-irradiation. Mice receiving 710 r of x-rays, 5 days after antigen injection showed above normal mean peak and mean total agglutinin titer (Table 16-1). Figure 16-8 shows that chemical pro-tectors (cysteamine better than AET; see Chapter 19) can favourably affect the decreased antibody synthesis.

The deficiencies in immune reactions after irradiation are not of purely academic interest.

After an atomic accident, the surgeons will see even insignificant wounds become infected, in spite of careful debridement, unless the patients have been treated with antibiotics. Tendency to haemorrhage, the poor state of nutrition, and the defective regenerative powers can


T A B L E I E - I 1 3 O

T H E RATIOS OF EXPERIMENTAL TO N O R M A L VALUES FOR THE F O U R CRITERIA OF IMMUNOLOGIC EFFICIENCY IN M I C E

Group Time of antigen in-jection before (—) or after ( + ) 710 r

(days)

Criterion of immunologic efficiency

Group Time of antigen in-jection before (—) or after ( + ) 710 r

(days)

Rate of appearance of antibody

Mean peak titer

Mean total titer

Induction period

1 Normal

Ratio (experimental/normal)

1 Normal 1 1 1 1

II - 1 0 1 0 0-94 0-93 1 0 III - S 1 0 1 1 8 1-07 1-0 IV - 3 0-89 0-83 0-64 1-0 V - 1 0 09 0-74 0 4 8 0-20

VI - 0 - 5 0-12 0-63 0-40 0 1 4 VII +0-003 0-13 0-51 0-29 0-11

VII I +0-5 0 1 4 0-44 0-30 0 1 5 IX + 1 0-03 0-41 0-29 0 4 8 X + 5 0-04 0-60 0-36 0-20

XI + 15 0-13 0-78 0-62 0-40 XI I +30 0-15 0-77 0-67 0-67

X I I I +45 0-34 0 9 1 0-68 0-67

only retard healing. Thus, in irradiated patients the slightest wound becomes very serious.

The infection is often of gastro-intestinal or buccal origin because the mucous membrane becomes thin, and loses its physical and biochemical resistance; the fact that it becomes ulcerated at many points makes it the more penetrable.

Many animal experiments were carried out, especially in dogs, but also in rats, to discover whether antibiotics prolong the life of irradiated animals, which antibiotic or combination of antibiotics is most efficient, and what dose should be given and when. Provisionally the results may be summarized as follows: in rats, aureomycin orally (or by injection) is excellent35; chloramphenicol or combined streptomycin and penicillin are less effective. These antibiotics lower the death rate and prolong the life of the animals which die in spite of treatment. Rats usually die from diarrhoea; after aureomycin treatment the diarrhoea disappears, and the animal dies later from haemorrhage. Aureomycin cannot save the life of any dog which has received at least 450 r, but it prolongs life, diminishes the loss of weight, and slightly reduces the fall of the number of leucocytes.


Terramycin lowers the death rate in dogs after 350 r, but the reason for this is not clear. Cronkite (ref. 2, p. 208) thinks that antibiotics should not be used in man until about the fifth to seventh day after irradiation, unless burns, wounds or ulcers make it necessary to intervene earlier. Heavily irradiated young men have been successfully treated without—or with little—use of antibiotics (see Chapter 21).

Four Russian investigators have reviewed the literature and published their own experimental work in an important monograph which should be carefully consulted by those interested in the problems of infection and immunity of irradiated mammals143.

Days

FIG. 16-8. Average antibody titers produced in the rat by injec-tion of a suspension of sheep red cells.

Time in days after injection of the red cells. Irradiation (550 r of 200 kV, x-rays) on the whole body, 24 hr before injection of red cells. Cysteamine (base) 100 mg/kg; AET, 150 mg/kg

intraperitoneally133.

Anaemia always develops if the animal lives long enough after irradia-tion—i.e. if it does not succumb to the shock, haemorrhage or gastro-intestinal disturbances. In a dog which has received 450 r anaemia develops between the second and third weeks. Three factors contribute to its genesis: (i) cessation of the formation of young erythrocytes by the bone-marrow, which becomes inactive, as is shown by the early disappearance of reticulocytes from the circulating blood, long before the anaemia develops; (ii) haemorrhages of varying severity; (iii) increased destruction


of the irradiated erythrocytes; no haemolysis is seen. Nucleated cells appear in the blood of irradiated goats and pigs. The reappearance of reticulocytes is generally a good prognostic sign. Some authors have reported considerable disturbances in the permeability of the red cor-puscles, but we have not observed anything on this point in dogs. Figure 16-9 summarizes the general variation in blood cell counts in radiation sickness.

IZ

"V5

>

If Il

FIG. 16-9. Variation in blood cell counts with time in radiation sickness34.

Repeated transfusions of whole blood are effective in the treatment of this anaemia, though it has not been shown that they can save the life of an animal which has received a fatal dose of irradiation. The effect of homogenates of spleen and bone-marrow will be described in Chapter 16.

A stay at a high altitude or in a low-pressure chamber does not seem to accelerate erythropoiesis in irradiated animals. The cessation of infection and haemorrhage, and the disappearance of fever are the forerunners of a return to normal. Folic acid, cobalamine (vitamin B12), liver extracts, erythropoietine or any growth promoting factor should not be relied upon for the bone-marrow is no longer capable of responding.

Malnutrition produced by irradiation has a complex pathogenesis and is not the same in rats as in dogs, the animals most studied in this respect. It makes metabolic studies in irradiated animals very difficult, for it is necessary either to make individual measurements (food actually ingested, faeces, urine, etc.) or to starve the controls and the irradiated animals, which necessarily Hmits the duration of each experiment.


Dogs generally eat well for a week or two after irradiation (450 r) except on the first day. Loss of appetite, vomiting, diarrhoea and loss of weight set in later, when the gastro-intestinal ulcers form. As death approaches the dog takes nothing but water. The gastro-intestinal tract is often paralysed and distended, and contains a large quantity of matter.*

Doses of 40 to 120 r to the whole body do not affect the appetite, weight and behaviour of dogs. In this species a dose of 200 r seems to be the threshold for radiation sickness, causing a slight loss of appetite for three days.

The gastro-intestinal mucosa is very radiosensitive, as Regaud, Nogier and Lacassagne observed as long ago as 1912. The damage here is already considerable a few hours after irradiation (cessation of mitosis, pyknosis, etc.), but repair also sets in very quickly in some mammals. In mice, for example, regeneration is evident three days after irradiation, and in this species death often occurs after the intestinal epithelium has recovered.

A growing rat which has received a sublethal dose of x-rays (400 r) often shows loss of weight in two stages. The first loss occurs in the first days after irradiation, and is soon overcome, and the second sets in about 12 days after irradiation and produces a break of about 10 to 15 days in the regular growth curve of the animal. L A M E R T O N et al.15, who made this observation, do not explain it. A protector (e.g. cysteamine) injected before irradiation diminishes the first loss of weight and abolishes the second. The rat takes less food for the first three days after irradiation (150 to 500 r), the day when food intake is minimal being usually the day of irradiation. Mice also lose about 10 per cent of their weight in the first 24 hr after a lethal dose of 700 r.

Forced feeding is of no use in irradiated animals, because the gastro-intestinal mucous membrane is able to utilize only small amounts of even predigested food. In rats, forced feeding increases the death rate18. Parenteral injections of protein hydrolysate can at the most delay slightly the first loss of weight of rats12. Blood transfusions and injections of plasma must be regarded as a form of parenteral feeding. It makes no appreciable difference to the reaction of rats to irradiation whether they are given a diet rich or poor in proteins, with or without the addition of methionine or cystine, before and after irradiation (450 to 550 r)19. Vain attempts have been made to improve the state of the digestive tract in irradiated rats by using the standard remedies7.

If the trouble in electrolyte distribution consecutive to intestinal damage (see Chapter 13, p. 327) are carefully corrected "intestinal death" may be avoided in a significant proportion of cases; but other anatomical

* In irradiated rats the weight of the contents of the stomach and intestine sometimes amounts to 10 per cent of the weight of the body17.


damage (infarctus, intussusception, see Chapter 21, p. 506) to the intestine can be corrected only by delicate surgery.

Sterility The production of sexual cells (ova and spermatozoa) in mammals

may be divided in three periods: a period of cell multiplication, a period of cell growth and a period of maturation. During the first period, the cells (spermatogonia in testis, ovogonia in ovary) divide a number of times in the same way as somatic cells. During the second rather long period, the sexual cells do not divide, but the volume of the cytoplasm increases and the nucleus prepares itself for the meiosis (see p. 242); they are called cytes I (spermatocytes or ovocytes). During the last period, the spermato-cyte I divides in two spermatocyte II, each of which, without delay, without resting phase, gives two spermatids. The four identical spermatids mature and transform into typical spermatozoa with head and tail. The male sexual cells are disposed in layers in the seminiferous tubules: the spermatogonia are at the periphery in contact with the connective tissue limiting the tubule; then going from the periphery and organized in more or less defined layers, one finds the various cytes, then the spermatids and finally the mature sperms which are set free in the lumen of the tubule, move slowly in the reservoirs and are excreted with the secretion of accessory gland (prostrate, seminal vesicles) at fecundation.

The evolution of the female cell from the ovocyte to the mature ovum is somewhat different. The ovocyte is bigger and the two last divisions interest the nuclei only, not the cytoplasm. The first division is a meiosis, and gives two haploid nuclei, one of which is expelled, the other remain-ing with the cytoplasm. After the second division of the haploid cell, one of the nuclei is again expelled; thus there remains only one mature haploid ovum with the whole of the cytoplasm of the ovocyte.

The most radiosensitive cells are the gonia, especially the spermato-gonia*; the mature sexual cells are much less sensitive. Thus, after appropriate irradiation, the animal remains fecund until it has exhausted its stock of mature cells. The fertile epithelium (just like the bone-marrow or the intestinal epithelium) becomes inactive and the animal remains sterile until eventually regeneration of the spermatogonia layer occurs which leads to the formation of new spermatozoa. The secondary sexual characters and the sexual activity remain normal in irradiated males because the cells producing the sexual hormones are much more resistant

* For instance, in a strain of mice careful histological studies have shown that some types of spermatogonia respond already to an exposure to 5 r of 60Co rays, the LD50 being 20-24 r138.


and do not disappear; the sperm consists of secretion from accessory glands (also more radioresistant): it is free of spermatozoa.

Careful microscopal examination of the sperm—or eventually of testicular biopsies—is without question one of the most sensitive and rather specific tests for the evaluation of radiation damage in a normal adult mammal. It has been widely used in the human species (see p. 507). Regeneration of spermatogenesis may also be tested by sperm analysis. If the dose to the male gonad is small (about 25 r in the mouse), one observes only a temporary reduced fertility. Sterility is seen after larger doses and its duration is proportional to the dose received by the testis.

The stock of potential sexual cells in the female mammal at birth is great (200 to 400 thousand in the human species); very few (300 to 400 in human species) arrive at maturity; the others degenerate. In contra-diction with the mammalian male which goes on producing differentiated sexual cells even in old age (at a reduced rate, however), the maturation of ovocytes stops at a certain age, called menopause in the human species. The maturation and liberation of the ovum often follows a certain rhythm accompanied by cyclical changes called oestrus cycle.

Irregularities in the oestrus cycle are observed after moderate doses to the ovaries. Generally speaking, the female gonad is less sensitive than the male gonad in mammals. The dose necessary to sterilize a female is higher than that which produces prolonged, but not permanent sterility in males; it has been estimated to be 200 to 500 r in the mouse, 1800 r in the rat, 2500 r in the rabbit.

In contrast with male sexual glands, the ovaries are incapable of active regeneration; if all the potential ova are killed by irradiation, the female mammal remains permanently sterile. For instance, D E S A I V E and our-selves139 have observed in female mice which had been exposed to a total-body dose of 900 r after cystamine injection, that the ovaries were com-pletely atretic and the genital tract atrophied 5 months after irradiation.

Skin Damage Epilation and erythema are characteristic reactions of the human species,

but are not commonly seen in laboratory rodents except in early life. Greying of the hair is regularly observed in irradiated C57 black mice which have survived a dose of 700 r thanks to the protection afforded by cysteamine. A measurable although brief decrease of the mitotic index of the skin of mice his been observed after doses as low as 5 r70. The growth of hair in the rat and mouse is very much delayed by irradiation in an epilated area, but curious effects of hair growth stimulation by x-rays have been observed in the rabbit102.


Cause of Death The immediate cause of death after whole-body irradiation has been the

object of many publications and speculations. Many of these speculations are useless although they may be interesting (see, for instance, refs. 106, 107 and 108). First, because even with the best clinical observations, the immediate cause of death is often debatable. Let us take a typical human case: that of the Yugoslav scientist who died 32 days after accidental irradiation; the terminal syndrome was intestinal occlusion, anuria, intestinal and pulmonary haemorrhages; it was a last pulmonary haemor-rhage which was fatal at a moment when the possibility of a surgical intervention was envisaged for the intestinal syndrome. In which category is one going to classify this case? Haemorrhagic syndrome, pulmonary syndrome or intestinal syndrome? The epithelium of the oesophagus had completely disappeared. Oesophageal syndrome, then?

It is only when the various syndromes have very different time course that it is possible to differentiate clearly by statistical analysis. Other reasons why these speculations do not go very far are that the radiation sickness is very different from one animal species to the other and that the environment plays an enormous role for the possible contamination of the irradiated animals.

K R E Y B E R G and DEVIK1 0 9 showed that after subcutaneous implantation in mice of a capsule of gelatin containing pure polonium (7 to 8-5 mc/kg), the chronic syndrome which developed involved mainly the lymphoid tissue (a sensitive tissue) which was rapidly regenerated and the renal cortex which was completely disorganized and the impairment of which caused death of the animal: thus death resulted from failure of a so-called radioresistant organ!

The delayed effects of exposure to ionizing radiations are of two kinds: genetic or somatic. Among the somatic delayed effects, induction of leukemia and cancer, cataract and shortening of the life-span have recently acquired great importance and have been much discussed (see Chapter 17). Genetic effects (see Chapter 9) (and probably some somatic effects) are classified as delayed because they can be seen only a long time after irradiation; but it must never be forgotten that the primary lesion has been constituted during or very shortly after the exposure. Again, it is our inability to detect these early changes which obliges us to wait until the primary lesion "expresses itself". In this new edition we have devoted a special chapter to the delayed effects.

R E F E R E N C E S

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I

29

C H A P T E R 17

Delayed Effects

ONE of the most characteristic features of radiation injury is that after an apparent complete recovery biological damage can in some cases be observed 10, 20 or more years after exposure in man, and many months after irradiation in short-lived laboratory rodents. Shortening of life-span, leukaemia induction, cataract and damage to the embryo are the delayed effects about which useful and quantitative information is available and more is actively gathered.

S H O R T E N I N G O F L I F E - S P A N Induction by radiation of an incurable disease like cancer is bound to reduce the average life-span unless the tumours occur at the very end of life. Thus, the average life expectancy of mice that have received more than 0-2 /JLC of 90Sr by intravenous injection is reduced1, because some 20 per cent of the animals die from bone sarcoma, a rare disease hardly ever seen in the control unirradiated mice. However, even when all death due to malignancies are excluded the average life expectancy of irradiated animals is reduced2 and this represents a distinct radiation lesion.

Shortening of life-span must be studied under two very different condi-tions : first, after exposure to a single dose; second, in animal populations continuously exposed to low radiation levels or given every day a small dose which allows at least six months' survival.

Data on life-shortening in mice and rats after a single whole-body exposure to x- or y-rays at the time of puberty or young adulthood are summarized in Fig. 1 7 - 1 3 ' 4 . In this figure the curve is fitted on the assump-tion that life shortening is directly proportional to the dose. It appears that in these small rodents life is shortened by about 10 per cent after exposure following a dose equal to one quarter of that needed to kill 5 0 per cent of the animals within 3 0 days. According to H U R S H 5 life is shortened 2-5 to 5 per cent per 100 r after a single sublethal whole-body dose of x-rays in rats and mice. The fact that the line in Fig. 17-1 goes through the origin suggests, but does not prove, that life shortening (under these experimental conditions) is a non-threshold phenomenon. Life-span shortening in mice from a single large dose is reduced by anoxia and

436

D E L A Y E D E F F E C T S 4 3 7

cysteine, and is more marked if irradiation occurred at birth than at maturity6.

The effect of bone-marrow treatment immediately after irradiation on longevity is conflicting but the data of M A I S I N 7 and of C U R T I S and G E B -

H A R D 8 indicate that any restorative action—if it occurs at all—is small. This conclusion is supported by the fact that shielding blood-forming organs

401 —

3 0

0 10 20 30 4 0 50 6 0 7 0 80 Dose in % of LD5(30days)

FIG. 17-1. Shortening of life-span percentage of rats and mice following a single dose of whole-body x- or y-radiations. As the data is collected from many sources the dose is expressed as the

fraction of the LD50 (30 days) for the particular strain3.

like the spleen—which has an effect comparable to that of bone-marrow injections for acute effects—does not reduce the life shortening effect. All this seems to indicate that lesions in the haemopoietic tissues do not contribute to life shortening (except of course in those strains where there is a high incidence of radiation induced leukaemia).

If after irradiation mice are placed on a diet containing an extract from shark liver-oil, containing alkoxyglycerol esters of unsaturated fatty acids, the life-span shortening effect is markedly reduced9 (see Fig. 17-2).* Whether this effect is due to the alkoxyglycerols or to the unsaturated fatty acids which are known to be essential to mammals as they cannot be synthesized, has not yet been established. In this experiment the diet of the mice contained the essential fatty acids in an amount normally considered sufficient, but it may be that following irradiation much larger quantities are required.

If only part of the body is exposed, the effect on shortening of the life-span is smaller, or bigger if the results are expressed in unit integral dose, but the information available is very scant7'10'11. Irradiation of the head only with 1000 to 2000 r induces death in a strain of rats in the 2nd to the 6th month. The symptoms are weight loss, anaemia, infection; the

* The life span of unirradiated control mice was not affected by the addition of the fats to the diet.

[


bone-marrow seems histologically normal7. In any case, the life-span shortening effect for a given dose is very much less if this is given over a prolonged period. If the dose is fractionated then the effect is already greatly reduced and following continuous irradiation a significant change can only be seen—even when very large numbers of animals are used—• with doses that exceed 10 rads of y-rays or 1 rad of fast neutrons13 (see Fig. 17-3). The amount of life-span shortening here seems to follow a logarithmic relationship with dose (as opposed to the direct proportion-ality indicated for single exposures) and strongly indicates a "threshold" type effect. The limited human data support this view as there is no detect-able life-span shortening of radiologists14; indeed, their life expectancy is slightly greater than that of doctors in general.

FIG. 17-2. Influence of diet augmented with special fats (poly-unsaturated fatty acids and alkoxyglycerolesters) on long-term survival of CBA mice that received 1100 r of x-rays in four

sessions separated by four weeks each9. • — • unirradiated normal diet. • • unirradiated augmented diet. O O i l 00 r on normal diet. O O 1110 r on augmented diet.

Sex differences—Some curious differences between males and females were observed which are difficult to explain6, but the idea that in elderly irradiated mice, the difference due to sex tends to disappear15 is worth following.


Relation to normal ageing—Some investigators have, more or less, consciously accepted the view that if life is shortened, it means that ageing is accelerated. This assumption needs experimental proof. Unfortunately, the process of normal ageing is not well understood, and there are few indications which can be used to distinguish if irradiation only accelerates this normal process, or alternatively if irradiation introduces a new factor which shortens life-span. In view of the confusion that has been created it must be stressed that ageing and life-span shortening are not necessarily identical phenomena.

There are physiological and chemical properties which in normal mammals and man follow a continuous variation throughout life such as calcium content of cartilage, cholesterol in certain tissues (cf. ref. 16).

Gamma rays, r/week

I

- j I

Al I NI

I i

I Tx

I \ r

-

I \ *

2

O-I IO 10 IOO Fast neu t rons , r ads /week

FIG. 17-3. Survival time (expressed as fraction of control) of mice exposed continuously to y-rays (top scale) and continuously to fast neutrons throughout life. Collected data which suggests an RBE for life span shortening by neutrons of about IO131.

One of the best characterized changes which can be used to measure age in rats and mice is the progressive alteration of the tensile properties of tail tendons17. Using such tests the physiological age in irradiated and non-irradiated animals can be compared. Preliminary experiments18 have shown that the tendons are not aged by radiation and even after 1100 r have the same elastic behaviour as those from unirradiated animals of comparable age.

Similarly, the time of appearance of benign hepatoma in CBA mice is not affected by irradiation19 These lesions can be considered a typical


ageing phenomenon in this strain of mice as the incidence is nearly 100 per cent and they only appear in animals more than one year old. Lisco et al.20 have found that a prior dose of irradiation does not reduce the latent period needed to induce sarcomas in mice with methyl cholanthrene. Since the latent period is greater for older than for young mice no "ageing" effect of irradiation was revealed.

A large dose of ionizing radiation can be considered as a "stress" (see p. 389). C U R T I S and G E B H A R D 8 have applied other stresses to mice (such as injections of typhoid vaccine, tetanus toxin, turpentine and nitrogen mustard), but failed to observe any decrease in life-span. A L E X A N D E R and C O N N E L L 1 8 found that repeated doses of a nitrogen mustard and of myleran shortened life-span.

An interesting theory assumes that ageing is due to accumulation of deleterious somatic mutations (see p. 260); it has given rise to various mathematical treatments (cf. H A R D I N J O N E S 2 1 , FAILLA 2 2 and SZILARD 2 3 and has been stated in terms of information theory by Y O C K E Y 2 4 * ) .

However, this is by no means the only explanation and the dose rate dependence for life-shortening cannot be reconciled with a somatic muta-tion hypothesis. This theory is also in disagreement with the finding25

that some powerfully mutagenic substances which are not, however, very cytotoxic do not shorten life-span even when administered in high doses. The life-span shortening produced with myleran and a nitrogen mustard is not attributed to the mutagenic action of these substances and their cytotoxic action at the time of administration is thought to be responsible. M U L L E R 2 6 has interpreted life-span shortening as being entirely due to delayed chromosome rearrangements which occur late because there has been no earlier cell division.

Damage (or functional alteration) in some critical organ or organs represents an alternative attractive mechanism and the blood vessels have been proposed as likely sites27'28. These lesions could arise as imperfect repair following the initial radiation exposure or as delayed interference with cell reproduction in organs where cell division is rare. One of the common causes of death in mice that have undergone life-span shortening by irradiation is nephrosclerosis, but the frequency varies very much from strain to strain. Post-mortem examination of the irradiated animals has not provided much information concerning the mechanism of the

* The original papers should be consulted by those interested in mathematical aspects of these questions with which the authors of this book are not competent to deal. It must not be forgotten that there are instances where mathematical techniques can approach questions which are not grasped by classical biological experimentation and can also show the way to useful experimentation or treatment of demographic data and clinical statistics.


delayed radiation effects. M A I S I N et al.12 have evidence that functional alterations in the head, thorax and abdomen of the rat contribute to deaths which occur at various times after the initial radiation. Thus mortality due to injury of the thorax is seen in the second to third month; injury to the head is manifested between the third to seventh month; abdominal lesions cause deaths after one year.

Extension of Life-span by Low Doses In the careful experiments of M O L E there was an indication that if mice

or guinea-pigs receive continuously less than 1 rem per week, the life-span is significantly increased. MOLE13 has reviewed critically the available data. Another recent experiment should be mentioned on male rats exposed throughout adult life to 0-8 r/day of 60Co rays and kept at two different temperatures29; the results are the following:

Temperature of environment Survival times in days

Controls Irradiated 5 0C 240 305

25 0C 460 600

There were only 22 animals per group, but the differences between irradiated and control groups were consistent throughout the course of the experiment.

The most striking and well documented extension of life-span has been recorded with the flour beetle30 (see Fig. 17-4); 3000 r given at 10-3 r/min

TIME IN DAYS

FIG. 17-4. Survival time of flour beetles following single doses of x-rays30.


as well as chronic irradiation at 100 r/day prolongs life to an extent which is beyond any possible experimental error. Perhaps the hypothesis that all effects of ionizing radiation are invariably deleterious has outlived its usefulness and it might be valuable to consider ionizing radiation, like actinic or thermal energy, to have physiological and pathological levels.

As usual, extrapolation to man is extremely hazardous; many more data on many species of mammals must be collected before a reasonable deci-sion can be taken.

C A N C E R A N D L E U K A E M I A I N D U C T I O N * Within a decade following the discovery of x-rays, it became clear that exposure to this type of radiation carries the risk of malignant disease. Animal experiments and clinical observations in man have shown that radiations of various types, external or internal, is a general carcinogen as it induces (or helps to induce) tumours in almost all tissues of mammals irrespective of species. None of the chemical carcinogenic agents are so universal in their action—thus the guinea-pig is resistant to the polycyclic hydrocarbons. Ionizing radiations act particularly on haematopoietic tissues (leukaemia), skin and subcutaneous tissue, thyroid and liver.

There is always a long induction period (in man ten, twenty or more years) just as it is the case with ultra-violet irradiation or carcinogenetic substances. These malignant tumours are not frequent nor are they invariably produced following severe radiation exposure. Until more is known about the mechanism it is useful to consider separately tumour-genesis at the site of localized irradiation and tumourgenesis following whole-body irradiation.

Localized Irradiation The local irradiation dose has to be high (almost invariably 1000 r or

more) and has to be delivered in such a way that local lesions (ulceration, etc.) are produced53. Continuous local irradiation at a rate too low to give a detectable tissue reaction has not been found to produce tumours. The tumour response seems to be conditional on the occurrence of damage to the tissues. There is a relatively little strain or species variation.

Radioelements (plutonium, radium, 89Sr or 90Sr) deposited in the skele-ton of animals can produce bone tumours and other tissue changes which have been observed in radium poisoning in man; 10 /ic of 90Sr seem to have equivalent carcinogenic effect of about 1 //c of radium. The relation-ship between dose and effect in one experiment1 on bone tumour induction by 90Sr has been found to be of the sigmoid type (see Fig. 17-5) and certain levels of radiation can be tolerated by animal population without

* General summary in Annex of UNSCEAR Report31.


a detectable increase in the incidence of bone tumours. In the same investigation1 an interesting observation was made of the effect of 90Sr on tumours of the blood-forming tissues. The strain of mice used (CFI) has a very high spontaneous incidence of these diseases (about 50 per cent). The injection of 90Sr does not greatly increase this incidence, but greatly reduces the time of its appearance (see Fig. 17-6).

The idea is generally accepted (cf. ref. 31) of a threshold for tumour induction by local irradiation, a threshold which varies with age and from organ to organ54. Whether there is a threshold to cancer and particularly to leukaemia induction following whole-body irradiation is less certain.

(b)

FIG. 17-5. Incidence of bone tumours in mice following injec-tion of 90Sr1. (a) Cumulative incidence over 475 days, (b) Total

incidence.


Whole-body Irradiation Radiation carcinogenesis also follows whole-body exposure, but here the

incidence of tumours and their site is highly dependent on the genetic make-up of the animal and varies markedly from strain to strain.

Leukaemia and lymphoma are seen in many strains of mice55 though relatively infrequent in others such as C.B.A. The dose-response curve is very complex (see Table 17-1) and also depends greatly on the dose-rate (see Table 17-2). Ovarian tumours occur after relatively small doses in some strains of mice2, but are not seen in others.

FIG. 17-6. Influence of 90Sr on time of occurrence of tumours of the blood forming tissue in a strain of mice with high spon-taneous incidence of these tumours. T h e time to 20 per cent

incidence is plotted as a function of injected dose1.

T A B L E 1 7 - 1

M Y E L O I D AND L Y M P H A T I C LEUKAEMIA I N M I C E OF THE R F S T R A I N (MALES) F O L L O W I N G W H O L E - B O D Y X-IRRADIATION AT 5 - 6 W E E K S OF A G E 3 4

Dose (r) (Leukaem'a per cent)

Dose (r) Myeloid Lymphatic

0 2 2 150 26 9 300 44 19 450 23 28

3 x 150 with two days' interval 19 24

3 X 150 with five days' interval 14 35

Young female Sprague-Dawley rats have been shown36 to develop a significant number of mammary gland tumours after a whole-body dose of 25 r and the incidence appeared to be linearly related to dose over the


range studied (see Fig. 17-7). While this dose relationship might be said to favour a single-hit somatic mutation process, the fact that removal of the ovaries greatly reduces the incidence indicates a more indirect mech-anism.

T A B L E 1 7 - 2

E F F E C T OF D O S E F R A C T I O N A T I O N ON INCIDENCE OF LEUKAEMIA I N X-IRRADIATED C B A M I C E ( F E M A L E S ) 3 5

Total dose

Number of mice with leukaemia/number in group Total dose 25 r

daily 125 r

weekly 250 r

fortnightly 50 r

daily 250 r 500 r

weekly fortnightly

IOOOr 1500 r

10/18 11/20

12/20 18/20

3/18 5/17

14/20 9/19 4/15

E S t5

80

7 0

60

5 0

4 0

3 0

20

10

C /

/ /

/ O <

/ / O

< / J /

/ t

0 100 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0

Dose, r

FIG. 17-7. Incidence of mammary gland neoplasia in rats fol-lowing whole-body exposure to a single dose of x-rays ( • first

experiment, O second experiment)36.

In rats, the abdominal region is particularly sensitive to this type of late reaction32-33, possibly because a large dose to the whole body produces acute injury in the intestine so that the two types of carcinogenic stimuli are combined. These neoplasms are of very different types and not of the most frequent spontaneous type of neoplasm in the strain of rats studied by M A I S I N et al.1. Pre-irradiation injection of cysteamine, or post-irradia-tion injection of bone-marrow cells (both are procedures which decrease the incidence of early death) do not inhibit life-span shortening nor cancer induction in these rats7, but cysteamine protects against leukaemia induction in mice (see p. 467).


Mechanism. The mechanism of tumour induction by ionizing radiation is by no

means clear. The theory of somatic mutation based on Boveri's concept, formulated fifty years ago, has recently received a greatamountofattention55

because of its analogy with the interpretation of genetic changes and its usefulness in mathematical treatment for the most pessimistic (i.e. no threshold) estimates of radiation hazards in man (see ref. 31, p. 28).

It cannot, however, be reconciled—in its simplest form at least—with the importance of the rate at which the dose is delivered and recent ideas on mechanisms of carcinogenesis—well discussed at a recent Ciba Con-ference43—tend away from this view. MacFarlane Burnett (cf. ref. 43) has emphasized the possibility that the carcinogenic stimulus may not create the malignant cells, but favour the selection of existing clones of cancer cells. This would provide a mechanism for indirect carcinogenesis and for the importance of tissue damage in cancers produced by local irradiation54. Particularly for tumours that are induced by whole-body irradiation is it possible that an indirect mechanism involving the mediation of specific physiological responses of the whole organism is responsible rather than a specific radiation action on the cell. Such is the case for the induction of tumours of the ovary and pituitary in certain mouse strains which FURTH 3 7 showed to depend on endocrine reactions. In leukaemia and lymphoid tumours in mice (a complicated phenomenon, see p. 260, which has little to do with human leukaemia) indirect mechanisms may play an important part (see refs. 38 and 39, but for other interpretations see refs. 23 and 24). The incidence of lymphoma originating in the thymus

FIG. 17-8. Effect of intravenous bone-marrow cell suspensions on development of radiation-induced lymphoma in strain C-57 black mice52. I x-irradiation only; II x-irradiation + bone-

marrow injection; I I I x-irradiation with thigh shielded.


can be greatly reduced (see Fig. 17-8) by bone-marrow injection after radiation or by shielding of part of the limb, and this illustrates the importance of the host response. More recently, it was demonstrated that implantation of a normal thymus in irradiated thymectomized mice is followed by malignant change. Antigenic tests have shown42 that in a small proportion the leukaemia arose from cells that had never been irradiated (i.e. from the implanted thymus). In these cases the induction must have been indirect in the sense that the cell in which the malignant transformation arose, had never been exposed to the carcinogenic stimulus. The same conclusion cannot, however, be reached about the majority of the lymphomas and it is not within the scope of this book to give a detailed account of this complex problem which has not so far been resolved.

D A M A G E T O E M B R Y O S

Anatomical and physiological damage to mammalian embryos irradiated in utero are nowmuchb etter known, thanks to the studies of L . B . R U S S E L L 4 4 - 4 6 ,

of RUGH47, of HICKS48-53, of L E B E D I N S K Y 5 0 and many others. Local irradiation of chicken and duck embryos in ovo has been exten-

sively used by Wolff and his collaborators as a means of obtaining well-defined monsters; ionizing radiations are extremely useful tools in experimental embryology (see ref. 49).

The following is a brief summary of recent evidence in mice. Figure 17-9 shows that irradiation during the 1st to the 6th day after fertilization

FIG. 17-9. Influence of a single dose of 200 r on foetal develop-ment (abnormalities and death at term) given at different stages

in pre-natal development of mice46.


results in a high percentage of death, but the embryos which resist become fairly normal. Irradiation during the period between the 7th and the 14th day causes little prenatal death, but a high percentage of various skeletal and central nervous system malformations.

The doses required to obtain such effects are small: a dose of 25 r, at the right time, is very efficient. A dose of 50 r may kill all retinal cells of a mouse embryo; these are replaced by regeneration of other cells and the young mouse is not blind. Microcephaly has been observed in Japanese children born from mothers irradiated while pregnant at Hiroshima and Nagasaki. It is recommended46 in clinical practice to restrict pelvic irradiation of women of child-bearing age to the two weeks following menstruation because the stages found most vulnerable in the mouse correspond to weeks 2-7 in human embryonic development, i.e. to stages so early that pregnancy may still be unsuspected46.

Physiological troubles have also been observed by some Russian radio-biologists in new-born mammals irradiated at low doses when in utero50.

O T H E R L A T E E F F E C T S Classical pathological examination of mice of different strains surviving a large single dose of x-rays, reveals that a state of morbidity develops which is independent of neoplasia and involves a change in endocrine balance15. Nephrosclerosis is frequently found in rats and mice41 as well as decreased fertility and eventually sterility.

Radiation cataract and also iris atrophy are typically late effects; they have been produced in mice, rats, guinea-pigs and rabbits51 after total or local irradiation. The first injury is seen in the cells of anterior epithelium of the lens; neutrons are more effective than x- or y-rays in producing cataract51, but cataract occurs spontaneously in the species and strains investigated. Neutrons are also much more effective in reducing life-span (see Fig. 17-3). Here again, the question arises if cataract is simply a sign (with others) of premature ageing, of localized ageing, or if it has special features when induced by ionizing radiation.

R E F E R E N C E S 1 . F I N K E L , M., B I S K I S , B . O . and SCRIBNER, S . M., Second U.N. Intern. Conf.

on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 65, United Nations, 1959

2 . F U R T H , J . and U P T O N , A . C . , Ann. Rev. Nucl. Sci., 1 9 5 3 , 3 , 3 0 3 3 . B L A I R , H . A . , University of Rochester, Report No. 4 4 2 , 1 9 5 6 4 . B L A I R , H . A . , Proc. First U.N. Intern. Conf. on the Peacefid Uses of Atomic

Energy, Geneva, 1955, Vol. 11, p. 118, United Nations, 1956 5. HURSH, J. B., University of Rochester, Report No. 506, Oct. 1957 6 . H O L L C R O F T , J . , L O R E N Z , E . , M I L L E R , E . , C O N G D O N , C . C . , S C H W E I S T H A L , R .

and UPHOFF, D . , / . Natl. Cancer Inst., 1957, 18, 615

D E L A Y E D E F F E C T S 449

7 . M A I S I N , J . , MALDAGUE, P . , D U N J I C , A . and M A I S I N , H . , J. Beige Radiol., 1957, p. 346

8. CURTIS, H . J . and GEBHARD, K . L., Second U.N. Intern. Conf. on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 53, United Nations, 1959

9 . ALEXANDER, P . and CONNELL, D . I . , Gerontologia, 1 9 5 9 , 3 , 1 4 7 1 0 . HENSHAW, P . S .,J. Natl. Cancer Inst., 1 9 4 4 , 4 , 5 1 3 1 1 . KALLMAN, R . T . and K O H N , H . I . , Science, 1958, 1 2 8 , 301 1 2 . M A I S I N , J . , D U N J I C , A . , MALDAGUE, P . , M A I S I N , H . , Second U.N. Intern.

Conf . on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 57, United Nations, 1959

1 3 . M O L E , R . H . , Nature, 1 9 5 7 , 1 8 0 , 4 5 6 1 4 . C O U R T - B R O W N , W. M . , and D O L L , R . , Second U.N. Intern. Conf. on the


1 5 . K O H N , H . I . , KALLMAN, R . T . , BERDJIS, C . C . and D E O M E , K . B . , Radiation Research, 1957, 7, 407

1 6 . BACQ, Z . M . , Principes de Pathologie et Therapeutique Generates, Sciences et Lettres, Liege, 1959, 2nd edition

1 7 . VERZAR, F . , Helv. physiol. Pharmacol. Acta, 1 9 5 6 , 1 4 , 2 0 7 18. ALEXANDER, P . a n d CONNELL, D . I . , Radiation Research, 1960, 12, 38 1 9 . CONNELL, D . I . and ALEXANDER, P . , Gerontologia, 1 9 5 9 , 3 , 1 5 3 20. Lisco, H., DUCOFF, H. S. and BASERGA, R., Bull. Johns Hopkins Hosp., 1958,

103, 101 2 1 . H A R D I N , B . JONES, U.S. Atomic Energy Commission Report, U C R L 3 1 0 5 ,

1955 2 2 . FAILLA, G . and M C C L E M E N T , P . , Am. J. Roentgenol., 1 9 5 7 , 7 8 , 9 4 6 23. SZILARD, L., Proc. Natl. Acad. Sci., U.S., 1959, 45, 30; see criticism by

SMITH, J . M . , Nature, 1959, 184, 956 24. YOCKEY, H. P., Radiation Research, 1956, 5, 146; see also Symposium on

Information Theory, Pergamon Press, London and New York, 1959 25. ALEXANDER, P. and CONNELL, D . I., Ann. Rep. Brit. Empire Cancer Campaign,

1959 2 6 . MULLER, H . J . , Intern. J. Radiobiol., 1959, Suppl. 1 2 7 . ALEXANDER, P . , Gerontologia, 1 9 5 7 , 1, 1 7 4 28. CASARETT, G. W., Radiobiology at the Intracellular Level, p. 115, Pergamon

Press, London and New York, 1959 29. CARLSON, L . D . , SCHREYER, W . J . a n d JACKSON, B. H . , Radiation Research,

1957, 7, 190 3 0 . CORK, J . M . , Radiation Research, 1 9 5 7 , 7 , 5 5 1 31. Report of the U.N. Scientific Committee on the Effects of Atomic Radiation,

General Assembly, 13th Session, Suppl. No. 17 (A/3838), New York, 1958 3 2 . MALDAGUE, P . , D U N J I C , A . , M A I S I N , H . , P H A M H O N G Q U E a n d M A I S I N , J . ,

C.R. Soc. Biol., 1957, 151, 1996 3 3 . M A I S I N , J . , MALDAGUE, P . a n d M A I S I N , H . , C.R. Soc. Biol., 1 9 5 8 , 1 5 2 , 2 0 4 3 4 . U P T O N , A . C . and FURTH, J . , quoted by U P T O N et al. in Atompraxis, 1 9 5 7 ,

13, 1 35. MOLE, R. H., Brit. Med. Bull., 1958, 14, 174 3 6 . SHELLABARGER, C . J . , CRONKITE, E . P . , BOND, V . P . , L I P P I N C O T , S . W . a n d

Radiation Research, 1957, 6, 501; also Second U.N. Intern. Conf. on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 158, United Nations, 1959


3 7 . F U R T H , J . , Cancer Research, 1 9 5 6 , 1 6 , 6 0 8 3 8 . BRUES, A . M . , Radiation Research, 1 9 5 5 , 3 , 2 7 2 39. KAPLAN, H. S., Radiation Research, 1955, 3, 281 4 0 . L O U T I T , J . F . , Advances in Radiobiology, p. 3 8 8 , Oliver & Boyd, Edinburgh,

1957 41. COLE, L. J . , N O W E L L , P. C . and ARNOLD, J . S., N.R.D.L. Report, No. 270,

October 1958 4 2 . L A W , L . W . and POTTER, M . , J. Natl. Cancer Inst., 1 9 5 8 , 2 0 , 4 8 9 43. Carcinogenesis, Mechanisms of Action, Ciba Foundation Symp., Churchill,

London, 1959 4 4 . RUSSELL, L . B . , Proc. Soc. Exptl. Biol. Med., 1 9 5 7 , 9 5 , 1 7 4 45. RUSSELL, L. B. and RUSSELL, W. L., J. Cell. Comp. Physiol., 1954, 43, Suppl.

1, 103 46. RUSSELL, L. B. and RUSSELL, W. L., Proc. First U.N. Intern. Conf. on the


47. R U G H , R . and G R U P P , E., Am. J. Roentgenol., 1959, 81, 1026 48. HICKS, S. P., in Metabolic and Toxic Diseases of the Nervous System, William

& Wilkins, Baltimore, 1953, Chapter 24, p. 439 4 9 . W O L F F , E . , Arch. Anat. Histol. Embryol., 1 9 3 6 , 2 2 , 1 - 3 8 2 50. LEBEDINSKY, A. V., GRIGORYEV, U . G . and DEMIRCHOGLYAN, G . G . , Proc.

Second U.N. Intern. Conf. on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 17, United Nations, 1959

51. UPTON, A. C . et al., Radiology, 1955, 64, 840 52. KAPLAN, H . S., BROWN, M . B . and PAUL, J . , J. Natl. Cancer Inst., 1953, 1 4 ,

303 53. HICKS, S . P . , D'AMATO, C . J . a n d LOWE, M . ].,J. Comp. Neurology, 1959,

1 1 3 , 435 5 4 . M A I S I N , J . , MALDAGUE, P . , D U N J I C , A . , Q U E , P - H . , M A I S I N , H . , Second

U.N. Intern. Conf. on the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 134, United Nations, 1959

5 5 . FORD, C . E . and M O L E , R. H., ibid., p. 1 2 6

C H A P T E R 18

Interaction Between Cells and Tissues Following Irradiation

IN classical tissue culture and in cultures of microorganisms, cells are more or less isolated, independent. Effects of ionizing radiations on individual cells (amoebae, algae, etc.) have been dealt with earlier.

When a complex organism like a mammal is irradiated either locally or on the whole body, a series of secondary phenomena take place because: first, irradiated tissues may affect non-irradiated tissues; second, vice-versa, the presence of a large number of non-irradiated cells speeds up the recovery of the irradiated ones; third, the behaviour of irradiated sensitive tissue influences that of irradiated more resistant tissues.

Generally, the same exposure to ionizing irradiation causes greater effects if the organ or part of an organ is exposed with the rest of the body than when the irradiation is localized to that organ. Local irradiation can result in morphological, chemical or physiological changes in non-irradiated tissues.

Casarett has discussed the effects* due to interaction between cells and tissues; much of what follows is to be found in his discussion1.

(a) Effects due to Irradiation of Neighbour Cells A neat experiment of Jolles is an example of such "at short distance"

effects2. In man, squares of skin 1 -5 to 3 -5 cm2 are irradiated simultaneously through lead grids. The squares are arranged in a single row, various distances apart. If the squares are near enough to each other, the intensity of the erythema is distinctly more markedf. The idea of a diffusible sub-

* Casarett calls these effects indirect. Since there may be a confusion between these kinds of indirect effects and the theory of indirect action of ionizing radiations (see Chapter 2), it is advisable to use another term for these kinds of effects. The term abscopal has been proposed by Mole; we use the adjective distant, derived from the French: effets a distance.

f The technique of irradiation through grids is of great interest to radiotherapists and the basis for research on the importance of connective tissue in the reaction of tumours to irradiation3.

30 451


stance in the skin itself seems unavoidable; but, if it diffuses in the skin, it is difficult to see why it should not also pass into the blood. Shielding of very narrow strips (0-05 mm) of skin in mice during irradiation may have a marked effect on the acute skin reaction18.

If normal unirradiated cells are in contact with a few irradiated cells, which have undergone somatic mutation, they may eliminate these ab-normal cells by population competition because they have better bio-chemical equipment than mutated cells. The sperm-producing tissue of the mammalian testis seems to be an exception; it does not seem to be influenced or controlled in this way by other parts of the organism8.

In mice local irradiation of a leg inhibits the growth of mouse mammary carcinoma inoculated (cell suspension) 48 hr after irradiation below the skin of the irradiated region. The reduction of growth rate did not vary with the dose (from 1 to 3 kr) but the delay in tumour growth and the survival time of the animals did vary. Pretransplantation local irradiation up to 6 kr never completely prevented tumour development13'14-15.

On the other hand, cell suspensions prepared from various tumours and inactivated by lethal doses of x-rays in vitro enhanced the develop-ment of viable tumour cells of the same type when mixed in the same inoculum15. Small numbers of viable tumour cells implanted in the centre of established identical tumours, heavily irradiated locally in situ, can survive and grow15.

Thus the close contact with heavily irradiated cells can either stimulate or inhibit the development of a small number of viable tumour cells.

(b) Abscopal Ejfeets due to Circulating "Toxins" elaborated in the Irradiated Tissues

"Toxins" may be liberated from dying or heavily damaged cells. The shock-like syndrome appearing shortly after a high radiation dosage may be due, at least in part, to such substances released in the circulation; one produces regularly a shock after releasing a tourniquet left in place for an hour or so. There is certainly no specific "radiation toxin"; probably a mixture of polypeptides, amines and potassium ions leave the tissues damaged by irradiation. This problem has not been adequately investi-gated (see also ref. 19).

(c) Abscopal Rejlex Effects One week after irradiation (2000 r) of one leg in the adult rat, the

uptake of45Ca is lowered both in the tibia of the irradiated leg ( — 38 per cent) and of the non-irradiated one ( — 22 per cent). Two weeks after irradiation, the non-irradiated leg has recovered while the irradiated one shows the same degree of damage17. This observation of Zuppinger and

I N T E R A C T I O N B E T W E E N C E L L S A N D T I S S U E S 4 5 3

Minder is interesting because it shows that x-rays can, in bone, induce homolateral reflex trophic changes exactly like a traumatism, a fracture for instance.

(d) Constitutional Effects These are the result of the neuro-endocrine reaction analysed in

Chapter 15. Irradiation (directly or through circulating "toxins") starts activating the hypothalamus, pituitary, adrenals and the secretions of these glands act on the whole organism, on the irradiated or non-irradiated part. It is very difficult to establish how much of an a distance effect is due to the neuro-endocrine reaction or to the "toxins" liberated in the irradi-ated region. For instance, if one exposes to 2000 r of x-rays just the hind legs of a rabbit (a part where there is no central nervous system and no endocrine gland), a number of distant effects are observed4-5'6'7:

(i) Transitory changes in the oligendendroglia; lymphopenia (maximum in 6 hr), thrombopenia (maximum in 3 hr), slight hydraemia, histological changes in the spleen, cervical lymph nodes and non-irradiated bone-marrow.

(ii) Rise in the number of abnormal megakaryocytes from 10 to 50 per cent in 24 hr, in both irradiated and non-irradiated bone-marrow; it comes back to normal in 2-3 days in non-irradiated marrow, but continues to rise in the irradiated tissue.

(iii) Increased destruction of lymphocytes unaccompanied by increase in circulating antibodies.

(iv) Early (in 3 hr) discharge of lipids from the adrenal cortex. Careful biochemists have often spotted important interactions between

normal and irradiated tissues when a mammal is locally irradiated. HEVESY, as early as 194520, has seen that in a rat bearing two tumours, local irradiation of one decreases the uptake of 32P by the other. HOLMES 2 1

has confirmed this and observed that a 50 per cent decrease of DNA in the non-radiated tumour coincides with a 75 per cent decrease in the irradiated one. The reverse situation is as easy to demonstrate: the spleen of the rat is much less damaged if irradiated locally than after whole-body irradiation; a dose of 1000r is needed locally to obtain a decrease of weight (5 per cent only in 5 days)24.

The relations between the mouse and the injected ascites tumour cells are most complicated and difficult to understand22-23. Inhibition of 32P incorporation in DNA by ascites cells is taken as test. No inhibition is seen if immediately after irradiation (1000 r) of the host, the cells are removed by puncture and incubated in vitro, or if the cells are irradiated and incubated in vitro, or if normal cells are injected in irradiated mice. Inhibition occurs only if incubation and irradiation of the cells are in


ivivo or if the cells are irradiated in vitro, incubated with 32P and injected into whole-body irradiated mice.

(e) Restoration Effects These are due to repopulation of a destroyed tissue by cells coming

through the lymph and blood from non-irradiated similar tissues. For years, pathologists have accepted the idea that the property of forming metastasis was particular to malignant tumour cells; it is now clearly understood that within a normal mammalian organism, there is, for widely spread tissue (like the bone-marrow or the lymphoid tissue), the constant possibility of seeding, of autografting when the tissue has been locally destroyed (see also Chapter 20).

A certain type of lymphoma which appears spontaneously in certain strains of mice, can be produced by x-rays. The tumour spreads from the thymus10, but local irradiation of the thymus and adjoining regions does not cause the appearance of such tumours. The whole mouse must be irradiated or partial irradiations must follow in rapid succession and cover the whole body. It suffices to keep one hind limb screened by lead to cause a striking diminution of the incidence of lymphomata (see Fig. 17-8, p. 446).

Figure 18-1 shows that in the bone-marrow of the rat, it is mainly after two days that a difference can be seen between local and total body

FIG. 18-1. During the first 2 days following the irradiation of the hind limbs in the rat, the decrease in D N A concentration of total bone-marrow is not much smaller than when the whole body has been exposed to the same dose of x-rays Percentage changes in D N A concentration of the bone-marrow of rat hind limbs after local ( ) or whole-body irradi-

ation ( ) with 700 r x-rays16.

I N T E R A C T I O N B E T W E E N C E L L S A N D T I S S U E S 4 5 s

irradiation16. The comparison between the effects of total body irradiation and of exposure of parts of the organism has been used by various authors (see, for instance, refs. 25 and 26) in order to obtain information about the acute radiation sickness and the shortening of the life span. Generally very high doses of ionizing radiation are given in this kind of experiments; but nevertheless, the possible influence of non-irradiated tissues should not be disregarded in the interpretation.

In order to obtain more quantitative information on the restoring power exercised by a normal organism on irradiated organs or tissue, it is advis-able to take advantage of the modern techniques of organ culture. Pairs of endocrine glands of young rats can be cultivated in vitro and irradiated; one is kept as a control, the other is grafted, by what has become a classical procedure, in the anterior chamber of the eye at various times after irradiation. If the organism as a whole can influence the course of radiation lesions in these glands, this will be seen by anatomical analysis and physiological tests9.

(f) Vascular Effects Irradiation can induce vascular changes such as swelling, necrosis or

subsequent proliferation of endothelial cells, thrombosis and varying degrees of fibrosis and occlusion. These vascular alterations can produce marked changes in tissues dependent on the damaged vessels for their blood supply. Some of the effects of local radiotherapy of tumours are believed to be, in part at least, the result of radiation damage to vessels in the stroma.

R E F E R E N C E S

1. CASARETT, G . W . , Proc. Conf. Radiobiology at the Intracellular Level, p. 1 1 0 , Pergamon Press, London, 1959

2 . JOLLES, B . , Brit. J. Radiol., 1 9 5 0 , 2 3 , 1 8 3 . JOLLES, B . , X-ray Sieve Therapy in Cancer, H . K . Lewis, London, 1 9 5 3 4 . N I Z E T , E . , HEUSGHEM, C . a n d HERVE, A . , C.R. Soc. Biol., 1 9 4 9 , 1 4 3 , 8 7 6 5 . LECOMTE, J . and FISCHER, P . , C.R. Soc. Biol., 1 9 4 9 , 1 4 3 , 8 7 8 6 . GEREBTZOFF, M . A . a n d HERVE, A . , C.R. Soc. Biol., 1 9 4 9 , 1 4 3 , 8 8 0 7 . BETZ, H . and LECOMTE, J . , C.R. Soc. Biol., 1 9 5 0 , 1 4 4 , 3 0 3 8. MOLE, R. H. , Burlington Congress 1958, Radiation Research, Suppl., p. 136 9 . BACQ, Z. M., RADIVOJEVITCH, D . and BEAUMARIAGE, M. L . , 1 9 6 0 , Unpublished

observations 10. K A P L A N , H . S . , / . Natl. Cancer Inst., 1948, 8 , 191 11. KAPLAN, H. S. and BROWN, M. B . , / . Natl. Cancer Inst., 1951, 12, 427 12. KAPLAN, H . S. a n d BROWN, M . B. , Cancer Research, 1952, 12, 441 13. STENSTROM, K. W., VERMUND, H., MOSSER, D . G. and M A R V I N , J . F., Radi-

ation Research, 1955, 2, 180 1 4 . VERMUND, H . , STENSTROM, K . W . , MOSSER, D . G . a n d JOHNSON, E . A . ,

Radiation Research, 1956, 5, 354


1 5 . REVESZ, L . , J. Natl. Cancer Inst., 1 9 5 8 , 2 0 , 1 1 5 7 1 6 . M A N D E L , P . et al., Advances in Radiobiology, Oliver & Boyd, Edinburgh, 1 9 5 7 ,

p. 59 17. ZUPPINGER, A. and MINDER, W., Proc. Second U.N. Intern. Conf . on the Peace-

ful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 247 18. DEVIK, F., Advances in Radiobiology, Oliver & Boyd, Edingurgh, 1957, p. 226 19. LUDWIG, F., rapport CEA No. 912, Centre etudes nucleaires de Saclay

(France), 1958 20. HEVESY, G . , Rev. Mod. Physics, 1945, 17, 102 2 1 . HOLMES, B . E . , Brit. J. Radiol., 1 9 4 9 , 2 2 , 4 8 7 22. HARRINGTON, H . and LAVIK, P. S., Cancer Research, 1957, 17, 38 23. HARRINGTON, H . , RAUSCHKOLB, D . and LAVIK, P . S., Cancer Research, 1957,

1 7 , 34 24. BOIRON, M . , PAOLETTI, C. and TUBIANA, M . , C.R. Acad. Sci., Paris, 1955,

2 4 1 , 1231 2 5 . RAJEWSKY, B . , Radiobiology Symposium, Liege, 1 9 5 4 , p. 8 1 , (Edited by Z. M .

BACQ and P. ALEXANDER), Butterworth, London, 1955 2 6 . D U N J I C , A., M A I S I N , J . , MALDAGUE, P . and M A I S I N , H . , Radiation Research,

1960, 12, 155

C H A P T E R 19

Chemical Protection Against X- and Gamma-Rays

IN THE days when the target theory alone was the foundation of radio-biology, it appeared impossible to influence the action of ionizing rays by means of chemical substances. When the intervention of free radicals and peroxides in an aqueous medium was established, many publications appeared showing that various substances are capable of protecting living organisms against x-rays. We insist again on the necessity to use a precise terminology. A lead screen is physical protection. Injection of a well-defined substance before irradiation is chemical protection. Administration of substances, changes in diet or culture conditions as well as injections of cell homogenates after irradiation are not protection any more, they are therapeutic measures designed to enhance recovery processes (see Chapter 14) or to replace killed cells by grafts of normal cells (see Chapter 20).

As early as 1949-50 several independent series of investigations (by the Liege school, Patt and his collaborators, Mole et al.) stated correctly the problem of the chemical protectors. The history and the results of the first five years of research in this field are given with the complete bibliography in the first edition of this book; the interested reader should consult it. Numerous reviews are also available60-64'16'3.

This chapter will be a concise summary of our knowledge of biological protection with a short discussion of the theoretical implications. The basic mechanisms of protection at the chemical level are discussed in Chapter 5. Protection of macromolecules by added substances against inactivation by irradiation in vitro is dealt with in Chapter 7.

T E C H N I Q U E S Any organism from viruses to mammals or tissue cultures may be used for studies of chemical protection. As far as mammals are concerned the technique for screening chemicals and studying their actions is now firmly established. Well standardized pure strains of mice or rats are chosen for their regular response to ionizing radiations as far as mortality is con-cerned. The testing dose may be the threshold 100 per cent lethal dose

457


at 30 days for the controls, (as in Figs. 19.1, 19.2 and 19.3) or smaller doses; in the latter case, statistical analysis (probit method for instance, see ref. 15) is necessary; the LD50/30 days may be calculated from the experimental results. Doses higher than the threshold 100 per cent lethal may be used in the case of powerful protectors (as in Fig. 19-2). A modi-fied sequential test has been introduced1 which is to be recommended. The DNA content of the small intestine of the mouse is decreased after irradiation in a regular way, within limits proportionally to the dose; this decrease, measured 72 hr after irradiation, is partially inhibited by chemical protectors125 and enhanced by sensitizers (like Synkavit, see p. 478); this test has the important advantage of giving the result in 3 days instead of 15 or 30 days2, but has not yet been used extensively. It may not be perfectly reliable; for instance, we have seen that methionine injected after irradiation does not alter mortality66 although it seems to favour restoration of DNA synthesis67.

The number and variety (as far as structure and properties are con-cerned) of protective substances is now so great that it would be tedious to collect a complete list of them; this list would not be very useful either. Various classifications have been proposed (see for instance refs. 3 and 64), which, as usual, have their merits and short-comings; they are inspired by the general concepts about their mode of action. The following seems to us a tolerable compromise to facilitate discussion.

T H E P R O T E C T I V E S U B S T A N C E S

1. CyanidesandNitriles Sodium cyanide or KCN in non-lethal, but toxic doses protects 5-8 mice

out of 10 against the minimum lethal dose for the controls4'5. Malonitrile5

or other nitriles6 which liberate cyanide in the body are also good protectors in mice; thiocyanate (resulting from the inactivation of CN - by the liver) and cyanate are ineffective. Cyanide injected after irradiation has very little, if any, effect; the molecule must be present in the body during irradiation and this condition, described in 19494'7, is the one which is characteristic and constitutes in fact the definition of the phenomenon of chemical protection (Fig. 19-1).

The protective action of cyanide or nitriles obvious in mice has not been confirmed in rats, frogs, or certain plant material and micro-organisms, but positive results have also been published on such material (see ref. 8). This situation is usual with chemical protectors; there is often a species effect which generally remains unexplained (see also p. 466).

Azide, which like cyanide inhibits cytochrome-oxidase and heavy metal enzymes has a slight protective power in mice5. It has been suggested

C H E M I C A L P R O T E C T I O N A G A I N S T X - A N D G A M M A - R A Y S 4 5 9

that the protection against x-rays is linked with the inhibition of catalase9, and that resistance to cyanide is for some unknown reason parallel to radioresistance10.

FIG. 19-1. Protective action of cyanide. Survival of control irradiated mice (C57 black) (700 r x-rays, 250 kV) and of cyanide-injected animals (0-1 mg NaCN) similarly irradiated. The injection was intraperitoneal and performed immediately

before irradiation5.

2. Sulphydryl Substances

(a) Cysteine, a sulphydryl containing natural amino-acid,

NH2 /

S H - C H 2 - C H \

COOH

protects small rodents against x- or y-rays when injected or ingested in large amounts before irradiation11. Glutathione, a physiologically important tripeptide containing a molecule of cysteine, is also a protector provided that it is injected in the reduced state12. The amino-acid methionine which contains a methylated sulphur is inactive although the methyl group is very labile in the body (refs. 29 and 13); the demethylated methionine (= homocysteine) is effective, but less so than cysteine13. The action of cysteine and glutathione has been confirmed on varied biological material (micro-organisms, plants, isolated cells, etc.).

(b) Cysteamine and cystamine. In 1951, B A C Q et al.u discovered that decarboxylated cysteine (= cysteamine or Becaptan or /3-mercapto-ethylamine or MEA) and its disulphide (= cystamine) are remarkable


protectors, whether injected or ingested. The dose reduction factor (DRF)

N H 2 - C H 2 - C H 2 - S H # S - C H 2 - C H 2 - N H 2

I S - C H 2 - C H 2 - N H 2

cysteamine cystamine

is about 1-8; i.e. it is necessary to expose mice to about twice the dose of x-rays after injection of 150 mg/kg of cysteamine in order to observe the same mortality as the controls (Fig. 19-2). On a molecular basis, cyste-amine is about 5 times as active as cysteine. Cystamine is as good a radio-protector as cysteamine; it has little toxicity and remains active when given per os to rats or mice118.

FIG. 19-2. Protection of 20 g mice by intraperitoneal injection of 3 mg cysteamine immediately before irradiation with various

doses of x-rays59.

This observation has been confirmed many times on a large number of living organisms and this very simple molecule of cysteamine has become a standard of reference and an excellent starting point for extensive studies on the relation (in this particular series) between structure and activity8'16. The activity disappears or is strikingly reduced if the hydrogen of the —SH function is substituted by a firmly held radical (—CH3, for instance). The final conclusion is that the best compounds contain a free —SH (a covalent group) or a potential —SH at one end, and a strong basic function (amine or guanidine) at the other end of a two- or three-carbon straight chain: N—C—C—(C)—S8'18,6'21. If the chain is lengthened to 4 or 5 carbons, the activity drops, thus showing that an optimal short distance must be maintained between the two active groups. The discovery of the protective effect of /3-aminoethylisothiouronium (AET) by the Oak Ridge team illustrates well the importance of these two functions18. AET in aqueous solution (and naturally when injected


in animals) is changed to mercaptoethylguanidine (MEG), by a process of intratransguanylation. MEG in fact, is the protecting molecule,

CH2—CH2 CH2—CH2 I l I l

NH2 S ^ NH SH I I

C C / \ / \

H2N HN H2N NH A E T M E G

Isothiouronium compounds which do not rearrange into guanidothiols do not protect against radiation lethality6.* Methionamine, the S-methyl derivative of a protective compound seems to increase x-ray lethality in mice17.

The presence of one (or several) sulphydryl or potential sulphydryl functions f is not sufficient to confer protective properties. Two important physiological substances are inactive: ergothioneine (see ref. 8) thioctic or a-lipoic acid (see refs. 16 and 19). Pantetheine (= N-panto-thenylcysteamine, a fragment of coenzyme A)13,8 is inactive also.

Many non-physiological —SH compounds have been found which are in-active or sensitize to x-irradiation. ^-Homocysteine, isocysteine, a-methyl-cysteine increase the effects of x-irradiation91; thioglycol and thioacetic acid are inactive14; Koch mentions thioglycol among the sensitizing —SH compounds. 5-Mercapto-pyridoxin and its -S-acetate are radioprotective but not 4,5-dimercaptopyridoxin nor 5-mercaptomethylpyridine, nor 4-desoxy-s-mercaptopyridoxin150.

The metabolism of cysteamine (particularly of 35S cysteamine) and its pharmacological actions have been well studied22'8.62, this amine is rapidly excreted or oxidized (to taurine and SO4); the fall of cysteamine content of the organism parallels the rapid decrease of the protection against x-rays. J The localization or concentration of cysteamine by various tissues has also been followed23-25; as compared with plasma level, the protector is con-centrated by the kidney (but also excreted), the liver, the bone-marrow,

* Footnote added in proof. S-alkyl-isothiouronium salts which differ from A E T by the absence of an amino group and cannot undergo transguanylation have been reported active in radio protection144.

t By potential function is meant a structure which, by reduction or metabolic action in the body, can give rise to free —SH groups (for instance, cystamine, cysteamine, labile esters, thiazolines, thiazolidines, etc.).

J The metabolism of cystamine (S-S) is being carefully studied by a group of Italian biochemists and seems to be rather different from that of cysteamine133,134

These studies are important in view of the fact that in the protection of the skin of young mice, the mechanism of action of cystamine seems to be different from that of cysteamine (the —SH compound)82.


the spleen and the adrenals; very little is found in the heart, striped muscles and testis (see also ref. 130).

Cysteamine, like MEG, rapidly crosses the cell membrane by a process of biochemically active absorption. If E. coli are irradiated in cold medium, no protection is observed because the cold has prevented the entry of the molecule into the cell.

When a mammal, injected with cysteamine, is irradiated—if one assumes logically that the effect is a function of the intracellular concen-tration of the substance—the protection may be very different from one tissue or organ to another, not because the organ reacts differently to the protector, but because of its selective concentration. Much of the success of cysteamine and AET is probably due to the concentration of these substances by such a critical organ as the bone-marrow.

The fact that cysteamine is a fragment of coenzyme A (which catalyses reactions of acetylation, benzoylation, etc.) has nothing to do with its protective action for two reasons: (a) there is no CoA deficiency detectable immediately after a lethal irradiation; (b) injected cysteamine cannot be incorporated during CoA synthesis, nor in any protein synthesis. This is a general rule, amines cannot be used by mammals for synthesis; they are rapidly excreted or metabolized. Diethyldithiocarbamate26 and other dithiocarbamates27'28 are also good protectors (Fig. 19-3). Thiourea is a

FIG. 19-3. Protectionofmicebysodiumdiethyldithiocarbamate.2 6

weak protector71. Penicillamine31 and a mercapto derivative of vitamin B632, but not mercaptomethylpyridine32, have been shown to possess some protective activity. A sulphydryl derivative of chloramphenicol (2-mercaptocyclohexylamine)38 and mercaptobenzothiazol65 seem to be interesting protectors65.


3. Amines, Amino-acids, Peptides Bacq and his associates have insisted since 1952 on the importance of

the amino group in the structure of radioprotective substances. Amino-acids are, generally speaking, weak protectors. The amines,

especially the aromatic amines, are much more active than their corres-ponding amino acids29-30. Among the most interesting are tryptamine, 5-hydroxytryptamine (serotonine or 5-HT), adrenaline, noradrenaline and histamine because they are physiological substances in vertebrates and many invertebrates. As a matter of fact, the indolamines (tryptamine and 5-HT) are on a molecular basis much more active than cysteine, but also more toxic. Their mode of action is now much discussed (see p. 477). Very simple amines such as methylamine or aniline (see ref. 8) are not devoid of action. Ocytocine (natural or synthetic) also protects mice when injected in large doses157.

Several Russian authors insist on the remarkable protective effect of a mixture of acetylcholine with either adrenaline, tryptamine or 5-OH-tryptamine when the lethality of mice, rats, guinea-pigs or monkeys after x-irradiation is taken as a test136.

The potentiation of the radioprotective effects of vasoconstrictor amines by a very potent vasadilator points again to the difficulty of accepting anoxia as the exclusive mechanism of protection by these amines (see p. 477).

4. Sex Hormones Confirmation has come from different laboratories and ourselves that

female mice of different strains (especially in oestrus) are more resistant than males33-35. This fact is not due only to the presence of oestrogens; castrated males are more resistant to x-rays than normal males. The action of oestrogens cannot be considered as comparable to that of the true protectors without further studies. The endocrine adaptation of pregnancy does not afford protection in mice36.

5. Anoxia-producing and Miscellaneous Substances (see ref. 8 for biblio-graphy)

The following weak protectors are believed to act by reducing oxygen transportation to the tissues of mammals through their action on haemo-globin : nitrite, ^-aminopropiophenone, carbon monoxide.

Some anaesthetics or analgesics have been mentioned among the weak protectors: ethylalcohol, morphine, dolantine, pentobarbital (but not the non-suphurous barbiturates), ether or urethane.

Many organic acids, sugars and substances containing hydroxyl groups


are also weak protectors: formate (but not acetate), caprylate and methyl-linoleate, pyruvate, succinate, a-ketoglutarate, fructose (but not glucose,) phenols and ketonic derivatives of phenols, glycerol (for mice, see refs. 8 and 37; for lysogenic system E. coli K12, see ref. 70).

6. Chelating Agents B A C Q , H E R V E and F I S C H E R 2 6 have observed that various chelating agents

are remarkably effective protectors of mice against radiations. Unfortu-nately, many of them are very toxic, and our series was reduced to diethyl-dithiocarbamate (Fig. 19-3), ethylenediaminotetraacetic acid (Versene, Sequestrene or EDTA) and trihydroxy-N-methylindol. Mercaptoethyl-amine is an excellent chelating agent since it contains a covalent group (—SH) and a strongly ionized group (—NH2) very close together, whence the idea that traces of certain ions play a very important part in the radiochemistry of aqueous solutions. Chelating agents are also among the best protectors in an in vitro system (see Table 7-3) in which their action has been studied. Since (i) radiochemical reactions are not altered in the absence of traces of copper, and (ii) the addition of copper to chelating agents suppresses their protective action, it seems that the structure which favours chelation is also that which produces the protective action against radiations37. Insects, bacteria, yeast and plants do not seem to be pro-tected by EDTA. A new interpretation of the radioprotective action of chelating agents has been suggested by J O N E S 9 7 .

7. Substances Acting Slowly by Induction of Physiological Changes So far, all the substances mentioned (except oestrogens) are most active

when injected intraperitoneally or intravenously a few minutes before irradiation. Such is not the case for parathyroid extract39, fluoroacetate40, fluorocitrate41, chlorpromazine42, reserpine43, guanethidine153 or a colchi-cine derivative44 which induce a moderate degree of protection when injected a much longer time before irradiation. Reserpine and guanethidine protect against organic peroxide intoxication153.

Parathyroid extract protects when injected 12-18 hr before irradiation and is still active when administered after irradiation. Such is also the case for some bacterial polysaccharids68 and for 1-hydrazinophthalazin153.

Fluoroacetate does not protect if injected a few minutes before or after irradiation; it does protect when injected 2-5 hr before irradiation, i.e. when the citrate concentration in the cells of spleen, heart and brain is highest and when the body temperature is lowest (Fig. 19-4). So far, it has not been possible to decide whether the slight protective action of fluoroacetate is due to the citrate itself present within the cell, to the com-plexing of Mg++ or Ca++, or to the trouble in carbohydrate metabolism.


Fluorocitrate induces a more rapid increase of citrate in certain tissues and, accordingly, the protection afforded by this substance against x-irradiation seems already maximal 30 min after injection.

Chlorpromazine ( = Largactyl) at certain doses increases significantly the survival of mice irradiated \ \ hr after its injection, when hypothermia is maximal42'51.

Reserpine has very slight protective action when injected 24 hr before irradiation43; it is interesting because it liberates 5-hydroxytryptamine (5-HT) from certain tissues and induces a long-lasting hypothermia127.

IOO

80

60

0

1 40 5«

20

0 5 10 15 20

Time, days

FIG. 19-4. Survival of mice injected with fluoroacetate (FAc, 4 or 5 mg/kg) and irradiated. Two series of controls: FAc alone, and x-irradiated. Two combinations: (a) FAc injected immedi-ately before irradiation, no protection; (b) FAc injected 5 hr

before irradiation, protection (see ref. 40).

M E C H A N I S M O F A C T I O N O F R A D I O P R O T E C T O R S Any theory which has the ambition to explain the action of chemical protectors must be compatible not only with the results of experiments on vertebrates but also with the data obtained on polymer systems, on micro-organisms or on cells isolated from mammals and irradiated in vitro.

1. Universality of Action of Radioprotectors Not only living organisms (from micro-organisms to man) but macro-

molecules (polymethacrylate, DNA) in vitro dry or in aqueous solution have been taken as tests for chemical protectors. Growing roots or other plant tissues, seeds (Fig. 19-5), spores, bacteria (mainly E. coli), lysogenic


systems, viruses and phages, isolated cells (tumours, thymocytes, reticulo-cytes), and tissue cultures are currently used. Within an organism like a mammal, any physiological or biochemical symptom of the radiation syndrome or any anatomical lesion (early or late in appearance) can be shown to respond favourably to radioprotectors: chromosome alterations, blood picture, DNA concentration, erythema, epilation, greying of hair, cataract; splenic, ovary or testicular atrophy; sterility, oesophagial, pulmonary, bone-marrow, intestinal, oropharyngial syndromes; neuro-endocrine reactions, foetus in utero, etc.

It would be fastidious to enumerate all the publications referring to such studies of the schools of Liege (Bacq and associates; Desaive, Betz), of London (Alexander and associates), of Louvain (Maisin, J., Maldague, Dunjic and associates), of Heidelberg (Langendorff, Koch et al.), of Columbia (Rugh, Wang and associates), of Oak Ridge (Hollaender, Doherty), of Harwell (Mole), of Rijswijk (Cohen, Van Bekkum and associates), of Belgrade (Hajdukovic), of Italy, Norway, etc. The great majority of the results are positive; i.e. less damage is observed if the substance is present during irradiation.

There are exceptions however, some of which cannot be logically explained. For instance, it is impossible to show a protective action of cysteamine in chickens; in these animals, only tryptamine and diethyldithiocarbamate showed a slight protective effect72.

In insects, no protection against genetic or somatic damage has been observed with cysteine or cysteamine (see, for instance, ref. 56), but the fate of these substances in insects is unknown*. The lack of concentration of cysteamine in the testis in mice may explain the absence of an effect on mutation frequency.

The reader must not believe that the mutagenic effects of ionizing radiations cannot be protected against. The school of Oak Ridge has shown that in bacteria (mainly E. coli) the protection afforded by cysteamine against increased mutation frequency is, as a rule, of the same order of magnitude as the protection against lethality. But there are exceptions: in the same irradiated strain the frequency of one mutation may be decreased, that of another mutation not at all. This observation suggests naturally that there is not one mechanism by which ionizing radiations increase the frequency of mutations, but several. Recently, Hollaender and McCarthy have irradiated spores of Aspergillus terreus in such a way

* Footnote added in proof. Definite protective effects have been described by GROSCH132 against x-irradiation after feeding females of the parasitoid wasp Habrobraeon with cysteine or glutathione. Fecundity and fertility were taken as tests. Analysis of the results implies protection against chromosomal damage, immediate and delayed132.

FIG. 19-5. Height of barley seedlings 4 days after irradiation, followed by germination. Scale in centimetres. The height of the coleoptile is indicated by an arrow.

Above. Normal dry seeds. Middle. Seeds soaked in water for 2 hr before irradiation. Belozo. Seeds treated with cystamine 0 1 per cent, 2 hr before

irradiation. In each photograph, from left to right: control (T) 50 kr,

100 kr, 150 kr, 200 kr, 300 kr, 400 kr, 500 kr, 600 kr, and 800 kr.

[ facing p. 466

FIG. 19-6. Local protection shown by hair growth. (Mice 15 days old; irradiated on the 8th day.)

1. Unirradiated control. 2. Irradiated on whole body (550 r of 200 kV x-rays); sub-cutaneous injection of 0-075 ml NaCl 0-9 per cent. 3. Similarly irradiated after subcutaneous injection of 0-75 mg cysteamine in posterior region of the back. 4. Similarly irradiated after subcutaneous injection of 1 -1 mg of histamine (base) in the same region.

FIG. 19-7. Pressure recorded in the double intracutaneous injection apparatus of Brinkman and Lamberts.

A = calibration, about 100 mm Hg. I. Injection of NaCl 0 9 per cent + cystamine 0-01 per

cent (upper tracing). II. Control injection of NaCl 0-9 per cent.

III . Irradiation (100 r/sec of 20 kV x-rays). B. Of II, control, rapid drop of pressure. C. Of I, cystamine protected skin, slow and delayed drop after

much longer irradiation48, 49.


that none of them is killed although mutations are induced; cysteamine decreases the frequency of these induced mutations. This fact indicates that the protection given by cysteamine against mutation induction is real and is not an artefact of selective survival74. There are several reports showing that chemical protectors increase the number of mutants: AET in Drosophila75, cyanide in Drosophila76^8

1 cysteamine (before or after irradiation) in barley53, and cysteamine in Neurospora. Nevertheless, cyanide, cysteine or cysteamine are good protectors when the growth of the seedling is taken as a test after irradiation of seeds, peas29>77 or barley52-53'78. Negative results have been reported with seeds of the water-cress (.Lepidinm sativum) although oxygen removal decreases effects of x-rays, but the penetration of the substances in the seed has not been proved57.

Cysteamine does not seem to protect the rat or the mouse against the delayed effects of irradiation, i.e. shortening of the life span138'139, induction of leukemia140 or tumors141. Significant protection by AET against leukemia induction has been reported by H O L L A E N D E R and associates142.

2. Necessity of Presence during Irradiation The only outstanding exception to this rule is that of the dormouse

(Glys glys) which seems to react favourably to cysteine and cysteamine when irradiated in the hibernating condition, even if the substance is injected as long as 21 days after irradiation46. This very puzzling observa-tion has not been confirmed on another hibernating mammal, the North American ground squirrel (Citellus tridecemlineatus); unfortunately, S M I T H , who has studied the reactions of this animal, failed to show that if cysteine is inactive when injected after irradiation of the hibernating squirrel, it is active if administered before irradiation in hibernation47.

It has also been reported that favourable effects can be observed by cysteine or cysteamine treatment 30 min after irradiation of Vicia faba roots79 but, again, this fact is contested by serious observations on the same object80. Much more work should be done on poikilotherms or homeotherms which can live for a long period in the cold.

3. Comparison between x- or y-Rays and Neutrons or OL-P'articles The protective action of cysteine is very weak against oc-particles on

the roots of Allium cepalu; in mice, against neutrons, it is also only about half of what it is against x-rays115. The oxygen effect is also weaker with neutrons or a-rays.

4. Local Protection Protection of the skin of man, rabbit, and rat, of the vaginal or rectal

mucosae of mouse have been observed after local application of cysteine,


cysteamine or cystamine16'83-87. Easily demonstrable experiments on local protection of the skin in irradiated young rats81 and mice82 have clearly revealed the phenomenon of local protection and has become a nice analytical tool.

When naked new-born albino rats (8 days) are irradiated, the hair does not grow, except where cysteamine has been injected. If slightly older rats, covered with a thin fur (17 days) are whole-body irradiated the hair falls, except on the head, the anterior part of the neck and the extremities of the limbs. If cysteamine has been injected intraperitoneally, there is no difference except that a patch of hair persists on the abdominal skin where the injection has been made, i.e. where the concentration of cysteamine has been larger. If cysteamine is injected subcutaneously, the hair remains strong at the injected spot, even if the amount injected is as low as 30 jug.

In C57 black mice (see Fig. 19-6) the same is true, except that after irradiation of 8-days-old mice (with fur) the hair remains only around the eyes, on the lips, nose and tips of the limbs. The phenomenon of local protection with cysteamine is obvious. If histamine is injected subcutan-eously, the whole surface of the skin is protected and not only the injected spot. This illustrates beautifully the difference in the mode of action of these two substances (see the following discussion on p. 470): (a) hist-amine acts mainly by reducing the oxygen tension in the tissues, because it is a long-lasting vasodilator with a circulation-slowing action; the whole body is equally protected as a consequence of its pharmacological effects; (b) cysteamine does not act mainly through an oxygen effect.

The mechanism of radioprotection of the skin of new-born mice by cystamine must be different from that of cysteamine since subcutaneous injection of cystamine—in contrast with that of cysteamine—results in protection of the whole skin82.

B R I N K M A N and LAMBERTS48'49'128 have described a very clean pheno-menon abolished by cystamine (Fig. 19-7). A pressure of 100-500 mm Hg (according to the location) is built in the deep layer of the skin of the rat or man by constant injection of saline solution, and registered contin-uously. The skin is irradiated just above the tip of the needle by a soft x-ray beam (35 kV, 15 mA, 175 r/sec); after a latency of 1 sec, the pressure falls, presumably because hyaluronic acid depolymerizes*. If, instead of normal saline, a solution of cystamine is injected in the same condition, no rapid fall of pressure is registered even after much longer irradiation; to be certain, the control experiment is performed simultaneously on the same skin. This experiment has been confirmed by B A C Q , C I C C A R O N E

* One does not know if it is by a radiochemical action or through hyluronidase liberated by the irradiation.


and RENSON50 with tryptamine. The authors have also shown that Brink-man and Lambert's phenomenon can be observed in the skin of a dead rat in complete absence of oxygen. Thus, protection by cysteamine happens during irradiation at a very early step of the chain of events leading from energy absorption to cell or organism damage and eventual death. Theo-retically speaking, it is very important to bear in mind the significance of the fact that cysteamine in mice or micro-organisms is without effect if administered 15 sec after a large dose given in 20-30 sec: the main bio-chemical lesions are constituted during irradiation; a long time may elapse before these lesions express themselves in a physiological trouble or anatomical damage. If one wants to know the basis of radiation sick-ness, one must try to find what happens during irradiation. Much experimental work has been done in this direction during the last few years, and this is very rewarding indeed for those who used the con-cept of the biochemical lesion as their basic working hypothesis.

5. Invalid Hypotheses (a) The —SH protectors might act because they are reducing sub-

stances; this seems logical since in the presence of oxygen the chemical reactions induced by radiations are often oxidations. But many —SH (reducing) substances like ergothioneine, pantetheine are not protectors. Ascorbic acid does not protect. The amount of NaCN injected in mice and giving some protection is much too small to reduce S—S bridges in —SH functions. There is no relation between radioprotection and the capacity of the —SH group to react with iodoacetate59.

(b) The injected —SH substances do not replace the sulphydryls which might have been inactivated by irradiation. Indeed, there is no detectable decrease to be found immediately after irradiation in reduced glutathione, total —SH, and there is no decrease activity of the enzymatic systems requiring —SH functions on the enzyme or on a coenzyme.

(c) Many radioprotective substances lower the body temperature of mice144-145 but for cysteamine the time course of hypothermia is not parallel to that of radioprotection148 and, in any case, the degree of hypo-thermia is by itself much too small to influence the reactions to ionizing radiations as shown by experiments in which the body temperature was lowered by physical means, by cooling146. Hypothermia cannot explain the action of cysteamine147.

6. The Presently Discussed Hypothesis Our views have changed somewhat since 1955 when we published the

first edition of this book, and this is quite natural in view of the great


number of papers published in the last five years. In the following pages we have tried to consider as objectively as possible the large body of facts and ideas put forward during the last five years and to extract a logical trend of thoughts which might be useful to the reader. The opinion of authors has often changed, unless they ceased working in the field. We have taken the last available publications from the various active sectors and we hope to give a fair account of everybody's position.

One must be prepared to accept (a) that there are several possible mechanisms by which a substance can decrease the effect of ionizing radiations, (b) that several mechanisms can act simultaneously and synergistically in the same system, (c) that radiosensitive systems must be carefully analysed individually and the importance of the different vari-ables (nature of the protector, type of ionizing radiation, oxygen pressure, radiosensitive structure or function) estimated quantitatively. For instance, cysteamine may protect solid polymers by energy transfer, and mammals by free radicals scavenging or by repair.

We shall confine the discussion to two main types of protectors, those which are best known at present:

Cysteamine-cystamine (—SH, —SS type of protector to which cysteine and mercaptoethylguanidine are related);

Histamine and other pharmacologically very active amines. This chapter is confined to protection against biological lesions. The

possible mechanisms for protection in chemical systems are discussed in Chapter 5 and examples of the protection of biologically active macro-molecules in vitro are given in Chapter 7. As the nature of the initial chemical lesion for cellular radiation injuries (see Chapter 2) is not known the conclusions derived from the in vitro experiments cannot be applied directly to the biological problem. This section is therefore confined to a general outline of the various hypotheses.

C Y S T E A M I N E A N D - S H P R O T E C T O R S

By far the largest number of publications about the mechanism of radioprotectors concerns the simple substance cysteamine the properties of which were discovered in 1 9 5 1 by B A C Q and his associates14. Three main theories are in competition.

The Anoxia Theory GRAY92 and Scott in England, PATT6 0 in the U.S., MARCOVICH70.93 in

Paris, THERKELSEN135 in Denmark believe that cysteamine acts by lowering the partial pressure of oxygen in the cells (see Chapter 11); since the slope of the oxygen effect in the region of 25 mm Hg (which is that of


mammalian cells) is very steep, even slight changes in oxygen partial pressure might be effective. One must discuss separately the evidence obtained from the isolated cells in vitro and from complex organisms like mammals.

(i) In vitro systems—Cysteamine consumes oxygen while being oxidized to cystamine; generally speaking, cysteamine is more effective in such systems than cystamine, but cystamine (an "oxidizing" rather than a reducing substance) is never totally inactive.

The results obtained by MARCOVICH70'93 with E. coli do not agree with those of Hollaender and Stapleton, although the same strain was used. Marcovich failed to find with cysteamine a DRF (dose reduction factor) higher than 3, i.e. higher than the DRF observed by removal of oxygen*. The Oak Ridge group observed DRF as high as 12 with cysteamine94, K O H N and GUNTER1 1 1 reported DRF values rangingfrom4-6;thisnaturally cannot be explained by anoxia alone.

Air bubbling or increased oxygen pressure decreases or abolishes the protection by cysteamine; this is not seen when glycerol or another alcohol is used as a protector of E. coli K1270>93.

Against the anoxaemia hypothesis are the facts: (1) that reticulocytes in blood in contact with air are protected by cysteamine95, (2) that thymocytes in vitro are as well protected by cystamine (—S—S) as by cysteamine (—SH) and much better than in vivo961, although these substances do not increase oxygen consumption. Cystamine (which, let us repeat it, consumes no oxygen) in air is effective on barley seeds. Anoxia cannot explain why there is also a difference of action between optical isomers of S,2-aminobutylisothiouronium112.

Kohn and Gunter concluded from their very careful work on E. coli in varied conditions (of pH, temperature, concentration of cells and protector): "It is therefore strongly indicated that some condition other than anoxia is involved"111.

(ii) In mammals it is true that large doses of cysteine, cysteamine or cystamine given intravenously or intraperitoneally decrease the oxygen tension in the venous blood (see, for instance, refs. 123, 124, 117, 126 and Fig. 19-8) not because the metabolism is increased or oxygen consumed by the injected substance (totally negligible in - S H substances, and non-existent in the case of cystamine), but because the circulation is very

* MARCOVICH69 found that phage T 2 is protected by cysteamine in conditions which exclude any indirect effect mechanisms. Marcovich suggests that cystea-mine may act by removing oxygen inside the phage, a suggestion which is difficult to accept.

T h e capacity of E. coli to reproduce T 2 is not protected by cysteamine69. t Apparently, the radioprotective action of cysteine on thymocytes in vitro is

explainable by anoxia3.

4 7 2 F U N D A M E N T A L S O F R A D T O B I O i . O G Y

much slowed down. Cysteamine protects rodents against oxygen poisoning and reduces their resistance to decreased atmospheric pressure117-129. Direct registration of oxido-reduction potentials within the tissues showed a fall in the rat after cysteamine injection98 but not in the spleen of mice,

IOO

90

80

70

60

C? .£ 50 c 0 1 40 "6 t/>

30

20

IO

0 FIG. 19-8. The vertical lines indicate desaturation of blood in control rats and animals injected intraperitoneally with 100 mg/ kg of cystamine ( 2 H C 1 ) . In ordinates, degree of oxygenation. Blood was taken (a) in an artery—upper horizontal broken line, 95 per cent saturation in both controls and injected rats; (b) in the inferior vena cava—much lower content of oxygen in cyst-amine-injected rats (40 per cent saturation) than in controls

(70 per cent) (BACQ and associates126).

although the doses injected were protective106-3. An increase of oxygen pressure in respired air which reversed the protective effect of 5-OH tryptamine in rats failed to cause significant reduction in the protective action of cystamine107 in contradiction with previous reports123-124.*

Furthermore, if the anoxia (due to general slowing down of the circu-lation) were the main cause of protection in the mammal, it might be

* Radiobiologists generally forget that when a mammal is subjected to severe anoxia (by breathing air poor in oxygen or under reduced pressure, what is known as anoxic anoxia) there occurs by neuroendocrine reaction an important release of catecholamines and other active biological amines which have protective effects of their own.

Control Cystamine

9 9 099 WV '


anticipated that all the organs and functions would similarly be protected, that the DRF would be the same all over the body. This is obviously not the case. The ovaries and testis where cysteamine does not concen-trate, are very little protected, while the spleen, adrenals and bone-marrow where cysteamine concentrates are very well protected.

We know too little about the distribution of cysteamine within the body. We know nothing about the intracellular localization of this sub-stance; the modern techniques of autoradiography are now sufficiently well developed to anticipate a harvest of interesting observations if a systematic search were made. The oxygen tension is very low in the bone marrow and, in contrast with all other tissues, is not altered by breathing pure oxygen instead of air143; this fact, so far, has not been taken into consideration.

The phenomenon of local protection (see p. 467) is also a powerful argument against the anoxia theory; there is obviously no anoxia when a solution of cystamine is introduced in the vagina or rectum and very little (if any) is resorbed. Finally, the Brinkman-Lamberts experiment may be again mentioned (see p. 468) as a spectacular example of instan-taneous local protection independent of circulation changes and of oxygen pressure.

Our present opinion is thus that anoxia induced by —SH protectors cannot explain the experimental evidence and must be considered only as a contributing factor when the whole mammalian organism is under the influence of these substances.

2. The Mixed Disidphide Hypothesis This hypothesis, although restricted and insufficiently supported by experimental evidence in some of its aspects, has brought to light interest-ing facts about the behaviour of —SH radio protectors88'64-108. These substances react with —SH or —S—S— groups of proteins to form mixed disulphides. For instance, an —SH protector:

IP— SH + R—S—S—R ^ 2P—S—S—R protector protein or peptide mixed disulphide

The protectors at the time when they exert protection exist largely in the form of mixed disulphides with the proteins of the blood and tissues. The normal S—S bonds should be more radiosensitive than the rest of the molecule and the "target" modified by reaction with the radio-protector should become more resistant against indirect (free radical) action as well as against direct action; subsequent restoration of the S—S protein function would occur. The rapid formation of mixed disulphides


seems to be well established* but the hypothesis implies many logica corollaries, many of which are far from being proved.

In vitro experiments have shown that all the effective —SH protectors are also excellent energy transfer agents, radical scavengers, and "capable of repairing" by hydrogen transfer (see p. 177). They can protect by these mechanisms sites such as nucleic acids and membrane which are more likely to be primary lesions than —SS proteins.

1. The main primary chemical lesion of x-irradiation in vivo, in mam-mals should be localized at the level of —SH or S—S groupst. Negative evidence has been accumulating for years (see p. 76). DNA in vitro which contains no sulphur is radiosensitive, very nicely protected by cysteamine; the chemical lesion might be a peroxidation of the thymine residue109, lesion which, on theoretical basis, could be easily protected against by cysteamine.

In the Brinkman-Lambert experiment, what is protected seems to be hyaluronic acid, not a protein, or an S—S or —SH substance.

2 . E L D J A R N and P I H L 8 8 have shown that some non-protective thiols do not form mixed disulphides with cysteine or oxidized glutathione, but the list is far from complete. On the other hand, at least two disulphides, cystine and cystine-diethylester, which easily form mixed disulphides, are both inactive as protectors. Cystine is rather insoluble, but its diethylester is soluble. ELDJARN1 1 0 believes that this contradiction might be explained by the fact that cystine and its diethylester cannot penetrate into cells.

3. Native bovine serum albumine has no S—S groups available for re-action at the isoelectric point; these groups are "hidden" ,within this mole-cule. Mixed disulphides with cysteamine are not formed under the con-ditions when it was well protected by cysteamine against irradiation in vitro (see p. 186).

Thiourea does not combine with proteins116, but it is a weak protector. 4. Binding to proteins is very unspecific. A large number of hormones

or drugs are found in the blood bound to proteins: steroids, thyroxine heparine, sulphamides, heavy metals. This observation explains how these substances are transported in the body, it does not show what is their mechanism of action.

* Cysteamine dissociates into smaller pieces, at the level of their S-—S bonds, the macromolecules of the human plasma which may reach abnormally high concentrations and confer a high degree of viscosity to the blood. Cysteamine can thus be used for diagnosis of, and has temporary therapeutic effect in, human macroglobulinaemia. If one accepts Eldjarn's views, these macroglobulines (kinds of S—S polymers) should be preferential targets for x-rays, and cysteamine should now act as a radiomimetic agent.

f T h e Norwegian authors went so far as to say that one could identify the initial chemical lesion by studying the intracellular localization of cysteamine.


5. M A R C O V I C H 6 9 argues that the fact that bubbling oxygen cancels the protection given by cysteamine to bacteria would lead, if the mixed disulphides hypothesis were correct, to the conclusion that oxygen is able to reduce an S - S bond. This is difficult to understand; but Eldjarn and Pihl have insisted on the fact that their hypothesis was restricted not only to sulphur-containing protectors, but also to mammals; they would accept for bacteria any other theory, either anoxia or free radical scaveng-ing88. We are impressed by the unity of biochemical and biophysical mechanisms of life at all levels, from micro-organisms to man; it seems unsound, illogical and pessimistic to start from the concept that the mechanisms of radioprotection must be essentially different in mammals and in simpler systems like unicellular organisms.

6. The need for an NH2 (or a guanidyl) group to be present at a definite distance from the —SH group is explained, in the mixed disulphides hypothesis, by the fact that hydrogen bonds between the two moieties can be formed and stabilize the' molecule.

If the reaction between —SH protectors and S—S protein bridges is quantitatively so important, one does not understand why the testis con-centrates ten times less cysteamine than the bone-marrow. Has the testis ten times less proteins with S—S bonds available for reaction with cysteamine? Thanks to a new technique which allows titration of free and protein-bound cystamine, fixation of injected cystamine has been con-firmed151, but it seems to bearno relation to protection against x-irradiation in rats152.

3. Free Radical Hypothesis (see also ref. 16) A. Mechanisms which Apply Both to Anoxic and Aerated Systems

1. Competition for free radicals formed by radiolysis of water (see Chapter 6) applies only if the action is indirect. There has been in the recent literature a tendency to minimize the role of free radicals originated from radiolysis of water (cf. Chapter 2) but the possibility that they con-tribute can by no means be excluded. Cysteamine is an excellent com-petitor (see p. 111).

2. Repair of damaged molecules before they undergo irreversible change leading to loss of activity; this may apply to both direct and indirect action. Repair to the original state by a transfer reaction (see p. 110) is brought about most effectively by cysteamine in polymers (see p. 177) and nucleoprotein (p. 206).

3. Transfer of energy is a process that is not well understood (see p. 112) but cysteamine protects polymers in this way (see p. 178) against direct action. G O R D Y and M I Y A G A W A 1 5 4 find that dry proteins are protected by cysteamine and an energy transfer process seems to be involved158.


B. Mechanisms Peculiar to Aerated Systems Protection of mammals usually occurs when oxygen effect is present: 1. In the radiolysis of water, H" and OH' free radicals are produced,

and also HO2* in presence of molecular oxygen. H" or OH" in cells might not reach the "target" site because they are captured by normal cell constituents; HO2" radicals having a longer life might get through. The protective agents capture H O 2 * and thereby protect against that part of the effect which requires oxygen. This hypothesis is strongly supported by close parallelism with the polymethacrylate (PMA) system where HO2" capture seems to be the mechanism of protection13.

2. Oxygen adds on to damaged molecules and thereby causes irrevers-ible inactivation. This idea can be schematically represented like this:

Radiation produces in a sensitive molecule RH two changes in parallel which could be due either to direct or indirect processes:

(a) Complete inactivation, i.e. lesions which occur in anoxia and cannot be protected against;

(b) Production of an unstable intermediate free radical R' which in absence of oxygen reverts back to RH, but in presence of oxygen gives ROO*, a very unstable peroxy radical the decomposition of which leads to inactivation. Protectors (type PH) can act by reacting with R" before oxygen reaches it, or they add on to ROO' converting it to be the stable ROOH or ROOP.

This last interpretation is the one we favour for protection in mammals by •—SH compounds; it is a revised hypothesis based on recognition that direct action is probably important. It explains why protection and oxygen effect so often go hand in hand. We fully envisaged this possibility of peroxidized targets five years ago (see ref. 8, pp. 66, 312, 313 and 314).

The trouble with •—SH type of protectors is that these substances are so very reactive that they can act by all the mechanisms discussed. Unless one has direct proof, the choice between several hypotheses is often arbitrary. It seems that one has reached in this problem a degree of develop-ment which suggests the search for direct arguments. The piling up of indirect arguments has ceased to be exciting; but others failing, these arguments have at least cleared the ground of much rubbish and indicate in which direction further work has a chance to be useful.

The paramagnetic resonance techniques have been much improved lately (see, for instance, ref. 90) and there is reasonable hope that in the near future we shall have information about protection along this line. We have at least one paper which shows that cysteamine acts on free radicals in yeast cultivated on D 2 O . The conclusions of S M A L L E R and AVERY8 9 are much in keeping with our present views: " ( 1 ) The presence


of /3-mercaptoethylamine inhibits free radical formation in irradiated yeast; (2) the \ min required to add /3-mercaptocthylamine to yeast and freeze is time enough for the protection to be established; (3) the /3-mercaptoethylamine has no effect on the number of radicals formed in the water of the suspensions".

I I . H I S T A M I N E , A D R E N A L I N E , S - H Y D R O X Y T R Y P T A M I N E The anoxia as far as histamine, adrenaline and noradrenaline in mammals

are concerned seems quite firmly established by C O H E N ' S group at Rijswijk3.H9.i20.

The arguments follow each other quite logically. 1. These three amines do not protect thymocytes in vitro, a system

where their presence cannot affect oxygen pressure; there has been no report that histamine can protect micro-organisms or plants.

2. There is a parallelism between reduction of oxygen tension in the spleen and protection against x-irradiation when mice are injected with any of these three amines.

3. If an antihistamine substance (phenergan) is given 15 min before histamine, if an adrenolytic drug (dibenamine) is injected before adren-aline, there is a significant reduction of the protective power of the amines.

4. The phenomenon of local protection by histamine does not exist as we have seen for hair growth in young mice (see p. 468).

The situation is not so clear as far as tryptamine or 5-hydroxytrypt-amine are concerned45'121. These amines are not inactive as protectors of the polymetacrylate in vitro13; tryptamine protects in the Brinkman-Lamberts test50. In rats, antagonists of 5-OH tryptamine122 or increased oxygen pressure in respired air107 inhibit the radioprotective action of the amine.

The relations between 5-HT (mainly in the central nervous system), amine-oxidase (which inactivates it), irradiation and reserpine (which liberate it) are not sufficiently understood at present.

Methoxamine [/3-hydroxy, |8-(2, 5-dimethoxyphenyl)-isopropylamine] seems to be a good chemical protector; but other powerful vasocon-strictor amines like ephedrine or propadrine are not149.

S U B S T A N C E S W H I C H I N T E N S I F Y T H E E F F E C T S O F X - R A Y S

Mitchell and his collaborators have, since 1946, studied substances which increase sensitivity to ionizing radiations, from the chemical, experimental and clinical points of view.

The best known molecule, with which clinical tests have been carried out is a phosphoric ester of a substance related to vitamin K, the tetra-


sodium salt of 2-methyl-l : 4-naphtohydroquinone diphosphate (or Synkavit). The 2-3-dimethyl derivative of this compound seems still more interesting. A study of the relation between chemical composition and biological activity shows: (i) that in derivatives of Synkavit the presence of phosphates is necessary, and (ii) that the biological effects do not depend solely on reaction with —SH groups, though this reaction seems to play some part. Treatment with Synkavit before irradiation increases the mortality of rats after exposure of the whole body to x-rays. Experiments on rats with Walker's sarcoma have shown increased fluores-cence in Wood's light of certain tissues and of actively growing parts of the tumour after the injection of certain derivatives of Synkavit. These derivatives are more selectively concentrated in the tumour if they are injected intravenously.

Mitchell has set out clearly how clinical tests must be carried out. Definite results have been obtained with inoperable bronchial carcinoma, in which, according to Mitchell's statistics, the average duration of life after the first irradiation is 4 months. If radiotherapy is combined with intravenous injections of Synkavit the duration of life is increased to 11 months; if intramuscular injections are given the period of survival is increased only to 6 months.

Curiously enough, Synkavit, a sensitizing substance, decreases the oxido-reduction potentials of the tissue just as cysteine or cysteamine which are protective substances98; thus, the oxygen effect cannot be responsible.

Cysteine protects against lethal doses of Synkavit99. The radio-sensitizing action of Synkavit can be demonstrated on tissue cultures100, but is not clear on bacteria or yeast cells101. Other substances have been recently found which increase the effects of ionizing radiations: hemato-porphyrine on an amoeba102, 3-4-benzopyren in the rat103, and iodo-acetate104. KOCH1 0 5 has given much attention to this problem which has important possible clinical applications.

Radiation responses in normal tissues and certain malignant tumors are enhanced by intravenous administration of actinomycin D. This substance, according to D ' A N G I O et al.137, should have the unique and amazing property to reactivate latent radiation effects in tissues which have been previously irradiated but have returned to normal in appearance.

A detailed monograph has been published recently by M I T C H E L L 1 3 1

where all the available information about chemical radiosensitizers can be found.

The radiosensitivity of mouse leukaemia cells growing in tissue culture can be significantly enhanced by pretreatment with iodoace-tate155.

If this pretreatment is mild so that it has no detectable effect on the subsequent rate of proliferation of the cells (e.g. IO-5 M iodoacetate for


30 min) then the sensitization amounts to about 30 per cent. With more severe treatment, greater sensitization can be achieved but the inter-pretation of the results becomes more difficult because the iodoacetate has damaged some cells. We suggest that the radiosensitization may be due to the reaction of the iodoacetate with cellular sulphydryl groups which are then prevented from acting as protective agents.

The radiosensitizing action of 5-bromo and 5-iododeoxy-uridine156

may be due to the fact that as these substances are metabolized, they give rise to intermediates which combine with sulphydryl groups just like iodoacetate. Synkavit also reacts very readily with sulphydryl compounds. The suggestion that the radiosensitizers act by interfering with physio-logical protective agents normally present in the cell suggests that the variation in the radioresistance of different cells may, in some way, be linked with chemical protection (cf. Chapter 13).

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Research, 1957, 7, 1 2 . GUZZON, A . , PAOLETTI, R. and POZZA, E., Rendiconti 1st. Lombardo diScienze

e Lettere, 1958, 92, 406 3. BEKKUM, D . W. VAN and ZAALBERG, O . B . , Intern. J. Radiobiol., 1 9 6 0 ,

Suppl. 1, p. 155 4. HERVE, A . a n d BACQ, Z . M . , C.R. Soc. Biol., 1949, 143, 881, 1158 5 . BACQ, Z . M . and HERVE, A., Brit. J. Radiol., 1 9 5 1 , 2 4 , 6 1 7 6. PLZAK, V., N O B L E , J . F., BROIS, S. J . and D O U L L , J . , U.S.A.F., Rad. Labor.,

Univ. Chicago, Quarterly Report, 28, July 1958 7 . PATT, H . M. , TYREE, E . B . , STRAUBE, R . L . and S M I T H , D . E . , Science, 1 9 4 9 ,

1 1 0 , 2 1 3 8. BACQ, Z. M . and ALEXANDER, P . , Fundamentals of Radiobiology, 1st ed.,

Butterworth, London, 1955 9. BOYLAND, E. and GALLICO, E., Brit. J. Cancer, 19532, 6, 160

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LAZAR, J . , Radiation Research, 1 9 5 5 , 2 , 3 9 2 1 4 . BACQ, Z . M . , HERVE, A . , LECOMTE, J . , FISCHER, P . , BLAVIER, J . , DECHAMPS,

G., L E B I H A N , H . and RAYET, P . , Arch. Intern. Physiol., 1 9 5 1 , 5 9 , 4 4 2 15. MEWISSEN, D. J., Radiation Research, 1957, 6, 85 16. BACQ, Z. M., Report to the XXIst Congress of Physiology, Buenos Aires,

August 1959, Symposia and special lectures, p. 105 17. BONATI, F . a n d NUVOLONE, U . , Radiobiol. Latina, 1958, 1, 162 18. DOHERTY, D . G., BURNETT, W. T . , J R . and SHAPIRA, R., Radiation Research,

1957, 7, 13 1 9 . GOUTIER, R . and BEAUMARIAGE, M. L . , Arch. Internat. Pharmac. Ther.,

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2 0 . LANGENDORFF, H . and K O C H , R . , Strahlentherapie, 1 9 5 6 , 9 9 , 5 6 7 21. SHAPIRA, R . , DOHERY, D . G. and BURNETT, W. T . , J R . , Radiation Research,

1957, 7, 22 2 2 . VERLY, W . , Bull. Acad. Roy. Med. Belg., 1 9 5 5 , p. 4 4 7 2 3 . VERLY, W . , BACQ, Z . M . , RAYET, P . and URBAIN, M . F . , Biochim. Biophys.

Acta, 1954, 13, 233 2 4 . ELDJARN, L . and NYGAARD, O . , Arch. Intern. Physiol., 1 9 5 4 , 6 2 , 4 7 6 2 5 . ZUPPINGER, A . , LAUBER, K . and AEBI , H . , Bull. Schweitz. Akad. Med.

Wiss., 1958, 14, 597 26. BACQ, Z. M. , HERVE, A, and F ISCHER, P . , Bull. Acad. Roy. Med. Belg.

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in press

32

C H A P T E R 20

Treatment by Bone-marrow and Spleen Cells

THE discovery that post-irradiation treatment with haematopoietic cells leads to the survival of fatally irradiated animals is of the greatest import-ance to mammalian radiobiology. The elucidation of the mechanism of the process has provided immunology with a most valuable new technique and the behaviour of irradiated animals protected by foreign cells is of interest far beyond the field of radiobiology.

The history of this discovery is fascinating and deserves to be told.

P H Y S I C A L P R O T E C T I O N O F T H E S P L E E N , L I V E R , B O N E S A N D O T H E R O R G A N S

In 1949, Jacobson and his colleagues found that in anaesthetized CF-I mice, protection of the exteriorized spleen (0-1 g) by a lead screen caused a striking proportion (96 per cent) of survivals after a dose (700 r) of x-rays which killed all controls (anaesthetized mice with the spleen exterior-ized but not protected)1'2. A dose of 1050 r must be given to animals with a protected spleen to produce about the same death rate as 600 r in controls. However, at the time, other workers failed to confirm Jacobson's original experiment, or obtained a less striking and especially a very variable rate of survivals19. Jacobson happened, by chance, to carry out his experiments with a particularly favourable strain of mice. With black C57 mice the improved survival is hardly significant, whereas in animals of strain A it is of the same order as that found by J A C O B S O N 4 .

In rats, protection of the spleen by a lead screen does not seem to ensure a large proportion of survivals5, whereas in dogs the experiment is about as successful as in Jacobson's mice6'7. K A P L A N 4 suggests that the differences between different strains may be explained by genetic differ-ences in the haematopoietic activity of the spleen, yet in dogs the spleen is a purely muscular and lymphoid organ.

Jacobson's original observation stimulated a large number of investi-gations :

(i) If spleens which have been protected by a lead screen are excised 5 min after irradiation there are no survivors, but if splenectomy is

484

T R E A T M E N T B Y B O N E - M A R R O W A N D S P L E E N C E L L S 4 8 5

performed 1-6 hr after irradiation the percentage of survivors is consid-erable3. The non-irradiated spleen, therefore, does not act during irradi-ation, but during the hours which follow it.

(ii) Grafts of isologous spleen after irradiation do not always give a satisfactory rate of survival unless everything is arranged in their favour; for example, by transplanting the spleen of from 4 to 9 daughter mice into the abdomen of a young mother soon after irradiation3'9'11-22'25.

(iii) The injection of homogenates, but not of extracts, of spleen after irradiation ensures the survival of a large number of animals.

(iv) The favourable effects of cysteine or cystamine (see p. 459) in-jected before irradiation are additional to those of physical protection of the spleen.15-21.

(v) Several investigators3'5'11'12-13 have tackled the question whether the action of the spleen is specific, and whether considerable rates of sur-vival might be obtained by protecting other organs with lead. Their results may be summarized as follows (Table 20-1):

T A B L E 2 0 - 1

SURVIVAL OF M I C E EXPOSED TO 1 0 2 5 r X - R A D I A T I O N W I T H L E A D P R O T E C T I O N OF VARIOUS T I S S U E S . (After JACOBSON et al.3.)

No. of Survival Regeneration of haemato-animals Tissue protected (per cent) poietic organs

135 Exteriorized spleen (0-1 g) 77-7 Complete ( + + + + ) 143 None 0 Nil

15 Exteriorized lobe of liver (0-8 g) 33 Almost complete ( + + + )

15 Exteriorized intestine (2-5 g) 26-6 Almost complete ( + + + )

18 Head (3-0) g* 27-7 Partial ( + ) 15 Right hind leg with thigh*

(I-Sg) 13 Not studied 23 Exteriorized right kidney 0 Nil

* See also L A M E R T O N ' S results26.

(vi) Protection of a thigh by a lead screen is effective. Here it is the bone (probably the bone-marrow) which is of consequence, because if the thigh with the bone removed is protected while rats are irradiated, the death rate is the same as in controls13. In many experiments in which a lead screen is applied to the "hepatic region", the right lung, or the lower abdomen, it is not only the liver, lung or pelvic organs which are protected, but also the haematopoietic tissue of the ribs, vertebrae or pelvic bones. The only technique which avoids this source of error is


Jacobson's in which the organ is exteriorized and protected with lead. Unfortunately, this technique requires deep anaesthesia, and makes it important to run careful control experiments. Though opinions differ greatly as to the relative importance of the spleen, liver and bone-marrow, there is complete agreement as to the behaviour of animals which survive as a result of physical protection of part of the body. Whatever school is concerned, Jacobson's (Table 2 0 - 1 ) , Cronkite's, Maisin's, or any other, regeneration of the haematopoietic organs is more rapid in mice or rats in which the spleen, thigh or liver has been protected3'2-28'20. This is also observed in irradiated animals protected chemically by cyanide, cysteine or cysteamine (see p. 459). C R O N K I T E 2 3 emphasizes this fact.

(vii) Protection of the upper abdomen with a lead screen ensures a good proportion of survivals5'13'16'17.

In J A C O B S O N ' S experiments3 (Table 2 0 - 1 ) , the shielding of an exterior-ized lobe of the liver (0 -8 g of tissue) gives a greater survival rate than protection of the head (3 g) or of 2-5 g of exteriorized gut, the liver being second only to the spleen among the organs important for the restoration of irradiated mice.

If a normal rat is connected in parabiosis with a rat which has received a lethal dose of x-rays, the irradiated rat survives20-27. The complete, intact, grafted animal transmits substances or cells of its blood to the irradiated animal, which enables it to overcome the effects of the ionizing radiations.

I N J E C T I O N S O F H O M O G E N A T E S O F S P L E E N O R B O N E - M A R R O W A F T E R I R R A D I A T I O N *

The above-mentioned experiments cannot answer the fundamental question as to what is responsible for the activity of the spleen or bone-marrow. Is it a substance produced by these tissues or cell migration starting from non-irradiated organs?

A very large amount of information has been obtained from experiments with injection of homogenates from different organs.

(a) Injections of extracts of bone-marrow or spleen made by the standard, destructive methods produce no results at all (see, however, ref. 33, and p. 372 of this book).

(b) Injections of homogenates or suspensions of bone-marrow taken from an animal of the same, or even of a different species result in the survival of a large proportion of animals (mice, guinea-pigs) which have

* It is unfortunate that some authors still speak of protection of rodents against x-rays when they mean action after irradiation, often several days after it. A better term is treatment, for action taken after irradiation; one must retain the term protection for physical, chemical or biological measures taken before and during irradiation. We agree with the definitions for these terms which have been proposed by LATARJET and G R A Y 2 9 .


received a lethal dose of x-rays. The injections are given intraperitoneally or intravenously after irradiation; the intravenous route gives results superior to the intraperitoneal. This fact has been confirmed many times by different groups of authors (see refs. 30 and 31).

The rat tail does not contain any haematopoietic bone-marrow, and protecting it with a lead screen does not produce a greater number of survivals than in controls. If the tail is transplanted into the abdomen, the bones in it gradually fill with haematopoietic tissue. If it is then exteriorized and protected with lead, the survival rate after irradiation is better than in controls24.

(c) Homogenates of mouse spleen are active if injected 1-45 hr after irradiation. Generally, a single injection is made 2 or 3 hr after irradiation. Homogenates of spleen or bone-marrow of new-born mice are more "active" than the same homogenates from adult animals. Only 150,000 marrow cells from 4- to 5-weeks-old mice are required to enhance survival of adult mice exposed to 750 r32.

(d) Homogenates of foetal liver are active, but not those of adult liver. Since foetal liver contains hematopoietic cells, it is concluded that homo-genates from all hematopoietic tissues are active; those of non-hemato-poietic tissues are inactive.

At that stage of the evolution of the problem (1952-1955) two theories were opposed: (1) a humoral theory postulating that the homogenates liberated a very unstable factor which stimulates blood regeneration. This theory was defended by Cole and his collaborators and favoured by Jacobson; their main argument was that injections of nuclei of bone-marrow cells (not of intact cells) were active in irradiated animals34. (2) a cellular theory held by Loutit and his group at Harwell (see ref. 30). According to this theory, the injected cells (they must be cells) are grafts; they "take", divide themselves, repopulate the hematopoietic organs and send new cells into the blood. The most weighty argument against this cellular theory was that heterologous material (haematopoietic cells of guinea-pigs injected in irradiated mice or rats) was effective, and it is well known that living tissue from a mammal cannot be grafted in another species.

G R A F T I N G O F C E L L S I N I R R A D I A T E D A N I M A L S

B A R N E S and L O U T I T 3 0 stressed that there was a very important dif-ference between animals protected by genetically compatible bone-marrow (i.e. donated by mice of the same inbred strain) and by genetically incom-patible bone-marrow (i.e. from mice of another strain). Figure 20-1 shows that the survival rate for both groups is high up to 30 days after irradiation, but that the long-term survival rate of the mice given isologous (genetically compatible) marrow was very much better and that the two


types of treatment were therefore very different. The other workers in the field did not keep their animals for long periods and it was conven-tional to score an animal that had survived for 30 days as being success-fully treated; under these conditions genetically incompatible cells appeared to be as effective as isologous cells.

a

• J 3 J

• • CP

• •

y •

C

O O

<if 1

I l l l ,1 L 1 1 1 L_ O IOO 200 300 1 0 0 500 6 0 0 ' 700 800 900

Survival t ime in days (a tze ro t ime all m i c e ^ l O O days)

FIG. 20-1. Survival time of CBA mice30 that had received 900 r of x-rays followed by cells f rom:

O isologous (i.e. CBA mice) spleen • homologous (from A strain) spleen

( • survival of unirradiated controls. Mice that received no treatment after irradiation all died in less than 21 days).

At the same time the humoral theory became more and more suspect; allegedly cell-free preparations were seen to contain many cells14. Finally, proof came in 1955 and 1956 by several different techniques that unlikely, as it originally seemed, repopulation from the donor does in fact occur. The first clear proof to be published came from L I N D S L E Y et al.i0 using a genetic method. L O U T I T ' S group41 demonstrated in the most clear-cut and elegant way the occurrence of cellular repopulation by using a cyto-logical method.

Two different chromosome markers were used: first, the rat has several characteristically X-shaped chromosomes which no mouse strain is known to possess (Fig. 20-2). Mice given 950 r and injected with young rat bone-marrow cell suspension, were sacrificed at intervals of 5-65 days and found to possess (at least in their bone-marrow, sometimes in spleen, lymph nodes and thymus) actively dividing rat cells. For the first time in the history of mammalian biology, a true chimaera had been obtained! Subsequently, immunological tests showed that rat platelets were circulating in the blood of these mice37. It could also be shown cyto-logically that marrow donated by a mouse of one strain grew in an irradi-

m S 4 *

(a)

A

* «5 V (b)

FIG. 20-2. Metaphase chromosomes of (a) mouse cells; (byrat cells. (This photograph was provided by Dr. E. C. fo rd . )

[facing p. 488

FIG. 20-3. Functional rat skin grafted on to an irradiated mouse which had been treated with rat bone-marrow. (This photograph

was provided by Dr. J. L. Barnes.)


ated mouse of another strain. A strain of mice (T6) has one charac-teristic ultrashort chromosome; CBA irradiated mice injected with T6 haematopoietic material were found to possess T6 cells in their bone-marrow and spleen up to at least 70 days after irradiation. Normal mam-mals never accept the presence of cells from other species and develop antibodies. Whole-body irradiated mammals tolerate foreign cells because they have at least temporarily lost the capacity to synthesize antibodies. This is strikingly demonstrated in Fig. 20-3 where a mouse is shown carry-ing a functionally active skin graft from a rat. The mouse received a lethal dose of irradiation, was treated with rat bone-marrow which allowed a proportion of them to recover. Into these survivors skin, from a rat (of the same inbred strain as the bone-marrow donor) was grafted and this kept intact for as long as the mice lived.

The road was paved for further developments; the changes brought by irradiation in immune reactions come in the foreground. Practical applica-tions in human pathology are at hand (see Chapter 21, p. 509). Auto-logous* haematopoietic material is always the best in promoting recovery of irradiated rodents or dogs, because no immune reaction develops; isologous material is better than homologous. Heterologous is the worst, because the immune reaction is the strongest.

A graft of heterologous bone-marrow only "takes" if the recipient animal has been irradiated over the whole body with a large dose, at least LDioo r dose. When a limb has been protected by a lead screen, it is the non-irradiated bone-marrow which migrates through the blood and repopulates the irradiated and destroyed haematopoietic tissues.

Injection of genetically incompatible bone-marrow is deleterious if given to animals that have received a dose of radiation which makes them very sick but from which they normally recover42. This is clearly shown in Fig. 20-4; 600 r kills only 5 per cent, but if they receive heterologous bone-marrow 50 per cent of them die. The immune response was not completely destroyed by 600 r and the foreign cells were therefore rejected. The additional immunological stress, however, killed irradiated animals which would otherwise have recovered.

Mice that received between 900-1200 r lived if they received bone-marrow which was able to repopulate unopposed the irradiated marrow. At still higher doses bone-marrow becomes ineffective because the animals die from a gastro-intestinal syndrome. The fact that homologous bone-marrow is actively harmful if the dose of radiation is not high enough,

* The nomencalture used at present is the following: autologous = from the same individual,

isologous = from another individual of the same genetically pure strain, homologous = from an individual of the same species, but not of the same strain,

heterologous = from an individual of another species.


makes bone-marrow therapy in accident cases hazardous unless accurate dosimetry is available.

4 0 0 5 0 0 6 0 0 700 800 9 0 0 jlOOO IIOO j 1200 1300 950 1150

X-ray dose,

FIG. 20-4. Mortality of mice following x-irradiation and treat-ment with isologous and heterologous bone-marrow42. Homo-

logous bone-marrow behaves in essentially similar way.

Secondary Disease The irradiated host at first tolerates the foreign cells, but after a certain

time of recovery may regenerate its capacity to form antibodies and progressively eliminate the graft; this has been followed quantitatively (cf. ref. 35), but there is extreme variation from animal to animal even in an inbred strain. Unexpected variations have been observed in the propor-tion of host grafted cells in the various haematopoietic tissues (bone-marrow, spleen, lymph-nodes and thymus) of the same chimaera.

Conversely, the living graft may elaborate antibodies against the host; indeed, rat cells grafted in irradiated mice synthesize y-globulins and are capable of synthesizing anti-mice antibodies which may be responsible for the so-called "secondary disease" observed at various times after the graft36. This condition, characterized by violent diarrhoea, is an immune disease.

Considerable controversy has arisen about the exact nature of this dangerous immune reaction8. Do the host cells produce antibodies against the grafted donor cells, or is it the grafted cells that produce antibodies against the host cells (i.e. against those haematopoietic cells that are seen to re-establish themselves some time after the graft has occurred as well as existing host organs)? Evidence in favour of the first hypothesis was seen in the fact that the severity of the secondary disease was not appreci-ably reduced if foetal haematopoietic tissue was used for grafting since


foetal material does not produce antibodies. This, however, is not decisive since the foetal tissues may mature and acquire the ability to make anti-bodies while growing in the host. On the other hand R O L L E R and D O A K E S 4 3

have shown decisively that in some instances at least the graft produces antibodies against the host by skin-grafting. The experiment was carried out as follows:

Mouse strain (I) irradiated

I + bone-marrow f r o m mouse (strain I I )

Mouse chimaera ( I + I I )

accepts skin graft rejects skin graf t f r o m mouse I I f r o m mouse I*

This clear-cut situation, however, is only seen in certain strain combin-ations and the whole problem of the secondary disease is one of great complexity. Thus the proportion of animals that suffer from secondary disease and that succumb to it varies from 10 to 100 per cent in different homologous chimaeras.

A further complicating factor is the difference in response produced by cells from bone-marrow and spleen (or lymph nodes). The latter are particularly effective where isologous material is used, but produce more serious secondary disease with homologous chimaeras. This aspect has been fully studied by C O N G D O N et al.44. The immunological problems in tissue transplantation for recovery from radiation injury are now in the forefront of research. But a more detailed discussion is outside the scope of this book and the interested reader can find excellent recent reviews38-39

Some final questions to discuss: 1. Does the injected bone-marrow homogenate act only on blood

regeneration? Are symptoms of radiation other than damage to blood-forming organs favourably influenced by these injections?

2. Are the haematopoietic cells suspensions or homogenates active only by the fact that there are living cells capable of repopulating the bone-marrow emptied after irradiation?

To the first question, the answer seems to be yes, although the problem has not been sufficiently studied. If we accept this point, we must also accept the rather non-conformist view that: (a) either the haematopoietic

* One wonders why the chimaera does not reject its own skin, which, being of strain I, should be as susceptible as the grafted strain I skin to immunological attack. Indeed, the symptoms of secondary disease suggests that the host intestinal mucosa may be^destroyed^byântibodiesjxom its graft.


tissue has other functions than simply supplying cells to the blood, or (b) that blood cells (leucocytes and platelets; red cells are probably not involved here) when being destroyed liberate substances which may act a distance after being carried by the blood. And again we are confronted with the possibility that substances (and not living cells or organized parts of cells) from haematopoietic organs might be extracted and found active in treatment of radiation sickness. Many radiobiologists forget that the numerous and beautiful experiments on haematopoietic cell trans-plantation do not exclude the possibility of a synergistic chemical factor. Recent work of ELLINGER33 gives new life to this idea of a chemical factor; it should be followed with attention (see also restoration by RNA and DNA, p. 383).

R E F E R E N C E S

1. JACOBSON, L . O . , MARKS, E . K . , GASTON, E . O . , ROBSON, M . a n d ZIRKLE, R. E., Proc. Soc. Exptl. Biol. Med., 1949, 70, 740

2 . JACOBSON, L . O . , MARKS, E . K . , ROBSON, M . J . , GASTON, E . a n d ZIRKLE, R. E .,J. Lab. Clin. Med., 1949, 34, 1538

3 . JACOBSON, L . O . , S IMMONS, E . L . , MARKS, E . K . a n d ELDREDGE, J . H . , Science, 1951, 113, 510

4 . K A P L A N , H . S . and PAULL, J . , Proc. Soc. Exptl. Biol. Med., 1 9 5 2 , 7 9 , 6 7 0 5. MANDART, M . , LAMBERT, G. and M A I S I N , J., C.R. Soc. Biol., 1952, 1 4 6 ,

1647 6. COULTER, M . P . and F U R T H , F . W . , Univ. Rochester Atomic Energy Project,

RU-189, 25 Oct., 1951 7. INGRAM, M. , NIELSEN, G. and P IATT, D . , Ibid., UR-200, 1 1 Feb., 1952 8 . U P H O F F , D . E . and L A W , L . W . , J. Natl. Cancer Inst., 1 9 5 9 , 2 2 , 2 2 9 9 . MANDART, M . , LAMBERT, G . and M A I S I N , J . , C.R. Soc. Biol., 1 9 5 2 , 1 4 6 ,

1305, 1307 1 0 . BARNES, D . W . H . and L O U T I T , J . F . , Proc. Roy. Soc. Med., 1 9 5 3 , 4 6 , 2 5 1 1 1 . JACOBSON, L . O . , MARKS, E . K . , ROBSON, M . J . , GASTON, E . a n d ZIRKLE,

R. E . , / . Lab. Clin. Med., 1951, 37, 683 1 2 . G E R S H O N - C O H E N , J . , HERMEL, M . B. and G R I F F I T H , J . Q . , J R . , Science,

1951, 1 1 4 , 157 13. KAPLAN, H . S. , see Nucleonics, 1951, 8, N o . 4 , 33 1 4 . M I Y A , F . , Proc. Soc. Biol., N.Y., 1 9 5 9 , 1 0 1 , 4 3 3 1 5 . SCHWARTZ, E . , Acta Radiol. Stock., 1 9 5 9 , 5 2 , 2 3 5 16. COULTER, M . and M I L L E R , M . , Univ. Rochester Atomic Energy Project,

UR-205, 14 May, 1952, p 31 17. RAJEWSKY, B., Radiobiology Symposium, Liege, 1954, Butterworths, London,

1955, p . 81 1 8 . STRAUBE, R . L . and PATT, H . M . , Proc. Soc. Exptl. Biol. Med., 1 9 5 3 , 8 4 ,

7 0 2 1 9 . FARR, R . S . a n d BRUYN, P . P . H . , A E C U - 1 8 2 0 , O c t . 1 9 5 1 2 0 . BRECHER, G . and CRONKITE, E . P . , Proc. Soc. Exptl. Biol. Med., 1 9 5 1 , 7 7 ,

2 9 2 21. U R S O , P. , CONGDON, C . C . , DOHERTY, D . G. and SHAPIRA, R., Blood,

1958, 13, 665


22. COLE, L . J . , FISCHER, M. C . , E L L I S , M. E . and BOND, V. P . , Proc. Soc. Exptl. Biol. Med., 1952, 80, 112

23. CRONKITE, E. P., Atomic Medicine, Williams & Wilkins, Baltimore, 1953, 2nd ed., p. 178

24. STORER, J. B., LUSHBAUGH, C . C . and FURCHNER, J. E . , J. Lab. Clin. Med., 1952, 4 0 , 355

2 5 . LANGENDORFF, H . , K O C H , R . a n d SAUER, H . , Strahlentherapie, 1 9 5 4 , 9 3 , 274

26. LAMERTON, L. F., Radiobiology Symposium, Liege, 1954, Butterworth, London, 1955, p. 136

27 . WARREN, S . , CHUTE, R . N . a n d FARRINGTON, A . B. , Laboratory Investigation, 1960, 9 , 191

28. M A I S I N , J . , M A I S I N , H . and D U N J I C , A., Radiobiology Symposium, Liege, 1954, Butterworth, London, 1955, p. 154

29. LATARJET, R . and GRAY, L . H . , Acta Radiologia, 1954, 4 1 , 6 1 30. BARNES, D. W. H. and LOUTIT, J. F., Radiobiology Symposium, Li&ge,

1954, Butterworth, London, p. 134; Ciba Foundation Symposium, 1956, p. 140

31. CONGDON, C . C . , U P H O F F , D . and LORENZ, E., J. Natl. Cancer Inst., 1952, 1 3 , 73

32. JACOBSON, L . O., MARKS, E. K. and GASTON, E. O., Radiobiology Symposium, Liege, 1954, Butterworth, London, 1955, p. 122

3 3 . ELLINGER, F . , Atompraxis, 1 9 5 8 , 4 , 4 3 9 34. COLE, L. J. and ELLIS, M. E., Radiobiology Symposium, Liege, 1954, Butter-

worth, London, 1955, p. 141 3 5 . FORD, C . E . , HAMERTON, J . L . , BARNES, D . W. H . and L O U T I T , J . F . , Advances

in Radiobiology, Oliver & Boyd, Edinburgh, 1957, p. 197 3 6 . GRABAR, P . , COURCON, J . , ILBERG, P . L . T . , L O U T I T , J . F . a n d M E R R I L L ,

J. P., C.R. Acad. Sci., Paris, 1957, 245, 950 37. S M I T H , L. H., MAKINODAN, T . and CONGDON, C . C . , Cancer Research, 1957,

1 7 , 367 3 8 . CONGDON, C . C . , Proc. XXIst Intern. Congress Physiol., Buenos Aires, 1 9 5 9 39. Symposium on Ant ibodies , / . Cell. Comp. Physiol., 1957, 50, Suppl. No. 1,

with contributions by CONGDON, C . C . (p. 103), FORD, C . E . and the Har-well group (pp. 109 and 123), VAN BEKKUM, D . W. and Vos, O . (p. 139), MANIKODAN, T . ( p . 1 5 7 )

40. LINDSLEY, D . L . , ODELL, T . T . and TAUSCHE, F. G., Proc. Soc. Exptl. Biol. Med., 1955, 9 0 , 572

4 1 . FORD, E . C . , HAMERTON, J . L . , BARNES, D . W . H . and L O U T I T , J . F . , Nature, 1956, 1 7 7 , 452

42. MAKINODAN, rT., J. Cell. Comp. Physiol., Suppl. No. 1, 1957, 50, 327 43. KOLLER, P. and DOAK, S., Intern. J. Radiobiol., Suppl. No. 1, 1960,

p. 327 4 4 . CONGDON, C . C . , MAKINODAN, T . , GENGOZIAN, N . , SHEKARCHI, I . C . a n d

URSO, I . S . , / . Natl. Cancer Inst., 1 9 5 8 , 2 1 , 1 9 3

C H A P T E R 21

Human Experience

INFORMATION concerning the biological effects of radiations on man is derived from experimental biology, from clinical observations and statis-tical surveys. The study of these effects has special importance because man is man, but also because during the last 50 years of his experience as a species, many important artificial radiation sources have been released by him and are used extensively. The possible effects of these artificial sources have been and still are widely discussed in the press and on the radio or television; consequently, it is hard to maintain a reliable perspec-tive because some human groups have an interest in keeping silent on some aspects which other groups emphasize, and vice versa. The only way to avoid as much as possible distortion of the facts and misunder-standings in interpretation is to start with the report of about a hundred well-chosen experts from 15 nations of all continents (east and west) who worked for three years in the Scientific Committee on the Effects of Atomic Radiation, which was established in December 1955 by the Assembly of the United Nations. This committee produced a valuable document1 which in many fields goes as far as possible in the actual state of our knowledge and has ventured to present many original estimations of doses received by man and their possible effects.

S O U R C E O F R A D I A T I O N S T O W O R L D P O P U L A T I O N A N D T H E I R I M P O R T A N C E

Natural Sources The first task of this committee was to evaluate the natural sources of

ionizing radiations. The following data have been accepted (Table 21-1); the mean total yearly dose is 0-1 rem. It must be emphasized that natural radiations from external sources may vary by a factor of two to ten. Cosmic radiation is more important at high altitude and varies also with the latitude; nothing is known about their possible biological effects. In certain very limited regions (Kerala in India, in Brazil, in the Katanga, in many places in the U.S.A. and U.S.S.R.) the radiation due to thorium, uranium, radium (and escape of radon) at the surface of the soil reaches

494

H U M A N E X P E R I E N C E 4 9 5

T A B L E 2 1 - 1

A N N U A L D O S E S FROM NATURAL RADIATION SOURCES*

Annual dose

Source Gonad Osteocyte Mean Source dose dose marrow (mrem) (mrem) dose

(mrem)

External Cosmic rays 28 28 28 Terrestrial radiation 47 47 47 Atmospheric radiation 2 2 2

Internal 4 0 K 19 11 11 1 4 C 1-6 1-6 1-6 R n - T n 2 — 2 Ra — 38 0-5

Approximate totals 100 130 95

* T h e totals in the table are for "normal" natural radiation intensities; in certain areas the values range up to ten times higher than those given.

ten times the background. In countries (Sweden, for instance) where granite and certain alum shales are used as building material, the amounts of radiation received within the house, especially in winter when ventila-tion is poor, may be many times the normal background. Also the internal contamination by radium which is accumulated in bones varies with age. Contamination by 40K and 14C is, as expected, much more constant.

Man-made Sources Two categories must be separated: the environmental contamination

due to fall-out from weapon tests; and all sources from medical, industrial research and other uses, like luminous dials of watches, television sets and shoe-fitting fluoroscopes. This last category of so-called minor sources must not be forgotten, and much care should be taken by public authori-ties to abolish them (shoe-fitting fluoroscopes) or keep them at a very low level, although they represent only about 1 per cent of the total contribu-tion from man-made sources.

1. Medical irradiation—The great difference between irradiation from medical and from natural sources is that the latter irradiate the total human body in a generally uniform fashion. Radiodiagnostic or radio-therapeutic procedures touch only part of the body, even when radio-


isotopes such as 131I or 198Au are used. The physical measurements must be correlated with the possible biological effects. For instance, during a fluoroscopy of the thorax, if the rest of the body is well shielded, the gonades are not irradiated and the genetical danger is zero. But a large part of the active bone-marrow (ribs, sternum, vertebrae) is exposed, and the dose received may contribute to leukaemia induction. The careful practitioner will shield the testes with lead before a radiography of the lower abdomen. But he cannot shield the ovaries and the dose absorbed by these gonads is considerable and has been estimated in many countries. But the knowledge of the dose received by the gonads is not sufficient to estimate the genetical danger. If a large dose of x-rays is given to the ovaries of a menopaused woman, there is no genetic danger since this woman cannot have any more children. But if radiographics of the abdomen of a young pregnant woman are made, not only are the ovaries of the mother directly irradiated, but the cells which will give rise to the sexual cells of the foetus are irradiated and the dose received by these cells may increase the frequency of the mutations which will become visible in the children of this foetus. The amount of statistical calculations and uncer-tainties involved is very large, but nevertheless, the data obtained from various countries (U.S.A., U.K., Sweden, etc.) where radiodiagnostic procedures are extensively used, indicate that the dose received by the gonads and the bone-marrow from these sources is about equal to the background, i.e. 0-1 rem per year (see Table 21-2).

T A B L E 2 1 - 2

A N N U A L D O S E S FROM M A N - M A D E SOURCES OF R A D I A T I O N ( E X C E P T E N V I R O N M E N T A L C O N T A M I N A T I O N ) ( r e f . 1 ) *

Annual dose Source

Genetically significant dose

(mrem)

Per capita mean marrow dose

(mrem)

Medical (exposure of patients)

(a) Diagnostic (b) Therapy (c) Internal

2 0 - 1 5 0 1 - 3 0

Less than 1 Less than 2

Ranges beyond 100 No estimate made Less than 10 1 - 3 Occupational

* For countries having an extensive use of the radiation sources listed and reporting data to the Committee.


These data show that the medical exposures constitute by far the greatest hazard. This fact is a great worry both to health physicists and medical men. To quote Morgan (ref. 22, p. 388): "As health physicists, my colleagues and I would like to keep all radiation exposures—including those from fall-out and radioactive waste disposal—to an absolute mini-mum consistent with the over-all good of man, but it is difficult sometimes for some of us to focus our attention completely on less than 1 per cent of the radiation exposure problem (fall-out) while completely ignoring 99 per cent of it". The 99 per cent of the problem is medical uses of radiation. It has been argued by those who are in favour of stopping bomb tests (because the contamination is spread all over the world, increasing and long-lasting) that medical irradiation is useful while fall-out is useless— at least for those countries which do not test atomic weapons. The argu-ment is partly wrong, because all the radiographics, fluoroscopies and other diagnostic procedures involving ionizing radiations are in fact not useful. Many are useless, performed routinely or more simply because they are requested by the patients and are a nice source of profit. Some highly competent scientists have estimated that good will, good technique and financial disinterestedness from medical men would reduce this contamination to one fourth of what it is.

S H I E L D S WARREN 2 3 is confident that medical men and radiologists will in the future adopt such methods as to decrease to a minimum the exposure of patients for radiodiagnostic procedures: "In view of the need to protect the genetic resources of the population as well as the welfare of the patient, it is becoming a general rule not to use x-rays unless there is a specific indication for this use. Public health officials now discourage mass chest x-rays in areas where the incidence of tuberculosis is low. Careful and conscientious case histories, accompanied by all the necessary laboratory tests can obviate the need for many x-rays. Most pediatricians have given up routine x-ray examinations of healthy children. . . . " And further: "The objective, in medicine at least, is to make ionizing radiation an unmixed blessing."

One of the authors of this book (Z.M.B.), a medical man who has frequent contacts with clinicians in Europe, is sorry not to be able to share Shields Warren's optimism. The structure of the medical profession, the regula-tions of the governmental insurance against disease, the increasing number of doctors, are such that it will be extremely difficult, if not hopeless, to obtain (what would be the ideal situation) that: first, every doctor asking for an x-ray examination would know the dose and could evaluate the hazard that this examination involves; secondly, every doctor would refuse to ask for an x-ray examination which, in his mind, is not necessary; third, that every radiologist should be perfectly well trained and should use the most modern equipment.


Muller (see ref. 24) who, as a geneticist, is much alarmed by the in-creased level of radioactivity, mentions "the recent studies by Sonnenblick and co-workers (1951, 1952) of the practices of physicians in the region of Newark, near New York. In this work, it was found that not one among over 200 of the operators of roentgen ray machines for medical purposes knew what output their machines had. In consequence, some of them delivered about 40 times as high a dose per minute as others."

Indeed, the responsibility of the medical profession is very great! 2. Fall-out from atomic weapon tests—Very few data are available to

estimate the local fall-out, i.e. the contamination by heavy particles of a surface of earth or ocean, in a radius of about 250 km around the place where the explosion takes place near the surface. This region is naturally severely controlled, but accidents due to sudden meteorological changes may happen as, for instance, in March 1954 in the Marshallese Islands2-3. If tests are made at very high altitude or below ground, the contamination is smaller and of different nature.

Most of the radioactive isotopes which cause general environmental contamination following nuclear weapon tests are fission products, but some are formed by neutron induction. These products are injected in the stratosphere where they constitute a reservoir from which they gradually fall on the earth and oceans; this fall is accelerated by rain or snow falls. This contamination has been accurately measured; an enormous amount of data has been published and evaluated by the U.N. Scientific Committee, the report of which should be consulted for bibliography. The two main contaminants are the long-lived 90Sr and 137Cs. They fall in easily soluble form and are readily incorporated directly by leaves or by plant roots from the superficial layers of the soil where they become adsorbed. In the ocean a more or less slower mixing occurs.

The radiation dose received by man externally from this fall-out is small (1-20 mrem for a 30-year period) and may be neglected. The danger comes from the incorporation of these isotopes by plants and animals which constitute man's food.

137Cs, chemically speaking, has much in common with potassium; it is incorporated rather evenly by all living beings because cells have the funda-mental property of concentrating potassium ions in their cytoplasm; the sexual and bone marrow-cells receive from 137Cs a dose of 1 mrem per year.

90Sr is more important because of its concentration and its localization in bones. The situation is rather complicated; the various steps involved may be enumerated briefly as follows:

(a) The particles falling may be retained by the leaves and the easily soluble 90Sr absorbed by the leaves or ingested by the animal eating the plant before having been absorbed by the plant. A good proportion of


particles falls on the soil and 90Sr penetrates in the soil where, generally speaking, it remains just like calcium and other cations, thanks to the chelating and adsorbing qualities of the soil.

(b) If the soil is not ploughed (for instance in meadows), 90Sr remains in the superficial layers of the soil available for absorption by plant roots. A certain proportion of 90Sr leaches into rivers; acid soils (peat) retain less strontium than neutral or alkaline soils. If the soil is ploughed deep, the 90Sr of the superficial layers is diluted by uncontaminated soil. Thus the quantity of 90Sr kept in the soil depends on the nature of the soil and the agricultural procedures.

(c) The quantity of 90Sr absorbed by the roots (it is in part an active process) depends on its concentration on the soil but is not strictly pro-portional to it; there is a limited competition between calcium and stron-tium, calcium being preferentially absorbed.

(d) In the cow or sheep, there is another discriminating factor at the level of the intestine which absorbs less strontium than calcium in propor-tion to their respective concentration in food. The unabsorbed 90Sr is eliminated with the faeces and goes back to the soil with the 90Sr eliminated in the urine; here again there may be a favourable factor in the fact that strontium is more easily eliminated (or less re-absorbed) by the kidney than Ca++. 90Sr concentrates in the milk and in bones, but proportionately less than Ca++.

(e) The human populations find their calcium either mainly in milk and cheese (as in the U.S.A., U.K. and many other western countries), or in vegetables, rice or in general plant food (China, Japan, India, etc.).

It is easily understandable (see Fig. 21-1 and Table 21-3) that in the latter case the food is more contaminated with 90Sr if one calculates the quantity of 90Sr ingested per gram of calcium. The ingested strontium in man goes through the same cycle as in the cow. If one takes the most complicated case, that of breast-fed children, the 90Sr has to go through seven biological membranes before reaching the bone; if the main supply of calcium comes from rice, only three steps are involved. The strontium content of the foetus is in equilibrium with that of the mother's plasma. Fast growing mammals (children between 0 and 5 years, for instance) incorporate more 90Sr than adults or slow-growing individuals.

It is easily understood, if we take the case of a humid pasture, that more 90Sr is at the present time falling than is removed by the milk trade and leached into rivers. Thus the level of 90Sr in the soil rises continuously. It will go on rising until equilibrium is reached in about a hundred years if one accepts the very improbable view that nuclear weapon tests will go on for one hundred years at the same rate as during the five years 1953 to 1958 (see Table 21-3, where other data according to other assump-tions are also given).

3 3


FIG. 21-1. Biological cycle of calcium and 90Sr showing location of membranes where discrimination may occur.

(f) Thus, the complete survey, the comprehensive study should give the calcium in addition to the fall-out itself, 90Sr (and eventually stable strontium) content of soil, food plants, cow's milk and bones, human milk, bones of adults, children and still-borns. Very useful information might be obtained from analysis of milk teeth, and of horns from some wild ruminants which are renewed every year.

90Sr seems to be incorporated in bones rather uniformly, as shown by autoradiographs, but the existence of possible "hot spots" as in the case of radium poisoning should be carefully looked for.

(g) The evaluation of the dose received by the marrow cells from 90Sr incorporated in bone is a difficult task which has been accomplished by the experts of the U.N. Scientific Committee only by accepting a large

H U M A N E X P E R I E N C E 501

margin of error, by considering several arbitrary but logical models (Table 21-3).

3. Occupational contamination—Occupational exposure is at present of little significance for the whole population because the number of exposed people is small as compared with the total population. The dose to indi-viduals naturally involves special problems which, together with the disposal of radioactive wastes, constitute the object of "health physics".

The International Commission on Radiological Protection (I.C.R.P.) has proposed wise and well-studied recommendations18 which have been generally accepted by governments or international organizations as a basis for the drafting of their own regulations which differ slightly from country to country (see, for instance, refs. 20, 21 and 22). The I.C.R.P. is a purely scientific, non-official body, nominated by the International Congress of Radiology as far back as 1928; but its work has been of such outstanding weight that the necessity for another body has not been felt. The I.C.R.P. has been consulted by the U.N. Committee on Radiations (see ref. 1). This is a very good instance of what the authority of scientists can achieve. When the scientists on a wide international level decide to cooperate and discuss a problem carefully and unhurriedly, the con-clusions of their work is accepted without discussion. Such a successful example should be followed in other fields. Recently, the I.C.R.P. has published a revision of the so-called "permissible doses" for occupational exposure and exposure of the general population, after careful considera-tion of the various hazards involved19. Health physics has become a science of its own with its defined responsibilities and research fields; there is an International Health Physics Society, which edits an official journal entitled Health Physics*.

Volume 21 of the Proceedings of the Second U.N. International Conference on the Peaceful Uses of Atomic Energy contains much useful information about health and safety, dosimetry and standards.

P O S S I B L E B I O L O G I C A L E F F E C T S O F N A T U R A L A N D A R T I F I C I A L B A C K G R O U N D R A D I A T I O N S

Two possible hazards have been considered in a quantitative manner: genetic effects and leukaemia induction.

(a) Genetic Effects In the human species, genetic effects are very difficult to evaluate. No

experiments can be undertaken. No single pure strain of Homo sapiens is available. The frequency of spontaneous mutations and the number of

* Published by Pergamon Press.

T A B L E 2 1 - 3

E S T I M A T E D D O S E FROM D I F F E R E N T RADIOACTIVE SOURCES (Computed from world-wide averages)

Genetically significant Per capita Source dose: mean marrow dose:

Max. for any 30-year Max. for any 70-year period (rem) period (rem)

Natural sources 3 7 Man-made sources (except en-

vironmental contamination and occupational exposure)* 0-05-5 Ranges beyond 7

Occupational exposure"!" Less than 0-06 0-1-0-2

Environmental contamination (hypothetical cases) J §

Weapon tests cease at end of 1958

Weapon tests continue until equilibrium is reached in about a hundred years**

Estimates for countries

deriving most of dietary calcium

from milk 11

Estimates for countries

deriving most of dietary calcium

from rice 11

0-010 0-16 0-96 Assump- Assump- Assump- Assump- Assump- Assump-

tion a1[ tion bU tion a If tion b f tion all tion b f

0-060 0-12 1-3 2-8 7-5 17

Estimated percentages of the max. doses for continued weapon tests

Assump- Assump- Assumption ali Assumption b1I tion all tion bH

Weapon tests cease 1958 17 9 13 6 1968 42 33 24 16 1978 64 56 34 26 1988 79 67 42 35

Weapon tests continue 100 100 100 100

* For countries having an extensive use of the radiation sources listed and reporting data to the Committee.

t Doses for certain technologically highly developed countries only. J Regional values may differ by a factor of about 1 /5 to 2 from the estimated population

weighted world-wide average values because of the latitudinal variation of fall-out rate and deposit. In some areas of the world, the tropospheric fall-out may tend to raise the upper limit of this range, especially in the vicinity of test sites.

§ Computed from population weighted world-wide average of stratospheric fall-out rate and deposit.

Il The extent to which these estimates apply to populations of different dietary habits and to those living in areas of differing soil conditions is discussed in paragraph 69 of ref. 1.

![Assumption a is that the injection rate is such as to maintain a constant fall-out rate of strontium-90 and caesium-137, whereas assumption b is that weapon tests equivalent in release and stratospheric injection of fission products to the whole sequence of weapon tests from the beginning of 1954 to the end of 1958 will be repeated at constant rate. This second assumption will give an equilibrium value for the fall-out rate and deposit approximately a factor of 2 higher than that calculated by using the first assumption.

** The values for the 30-year doses have been corrected for tropospheric fall-out, using a value of 0-5 mrem/year for the period of testing.


genes in man are unknown; generally only gross pathological or morpho-logical characters are spotted. Social conditions interfere with biological phenomena; medical care maintains alive unfit individuals who may reproduce themselves (although generally at a reduced rate) and transmit deleterious genes which otherwise might have been eliminated.

One must start from general principles, from animal experiments the results of which might not be transferable to man, just as mouse genetic reactions to ionizing radiations are different from those of Drosophila4>5. Nevertheless, many intelligent approaches have been attempted6-1, and at least the framework of the problem seems established. Table 21-4 shows the data which have been accepted by the U.N. Scientific Committee and may serve as a useful basis.

If one accepts for man the fact observed by Russell in mice that the mutagenic effects of a given dose of ionizing radiations is twice or three times smaller when the dose is distributed continuously at a low rate, the genetic hazards from the fall-out as estimated by the U.N. Committee should be decreased by a similar factor since the irradiation from fall-out is continuous. The genetic danger from medical exposure should not be similarly decreased since radiodiagnostic and radiotherapy procedures (with the exception of procedures using radioisotopes) distribute single doses at high rate.

(b) Leukaemia Induction Table 21-4 shows what the possible hazard from the various sources

of mass contamination can be. Calculations have been based on two hypotheses acceptable in the present stage of our ignorance; the most pessimistic and the optimistic. The pessimistic hypothesis accepts the idea that leukaemia induction obeys the same laws as those of Drosophila genetics: there is no threshold, the response is linear; every dose, however small, counts; all effects are strictly cumulative; there is no recovery. In this case, natural radiation would be responsible for 15,000 cases in a world population of 3000 million; the present accumulated fall-out would increase this number by a factor of 0-03-0-13 per cent. The optimistic hypothesis which assumes a threshold is considered by thoughtful authors (see, for example, ref. 12) as more probable. This threshold may be as high as 400 rem and in this case the hazard falls to zero, even if the weapon tests continue for 100 years at the rate of testing of the last 4 or 5 years (Table 21-4).

"Any present attempt to evaluate the effects of sources of radiation to which the world population is exposed can produce only tentative estimates with wide margins of uncertainty" (ref. 1, p. 42).


T A B L E 2 1 - 4

ESTIMATES OF C E R T A I N POSSIBLE A N N U A L CONSEQUENCES OF R A D I A T I O N RECEIVED

BY W O R L D P O P U L A T I O N FROM C E R T A I N SOURCES

World Consequence population

assumed (inmillions)

Natural occurrence

assumed per year

Source of radiation



Natural occurrence

assumed per year

Natural radiation

Fall-out from weapon tests



Natural occurrence

assumed per year

Natural radiation

Tests stopping in 1958

In equilibrium after prolonged

continuation of tests

Leukaemia If threshold 0 rem 3000

5000 I f threshold 400 rem 3000

5000 Majorgeneticdefectsll 5000

150,000 250,000 150,000 250,000

700,000 to 3,000,000

15,000 25,000

Ot Ot

25,000 to 1,000,000

400-2000*

Ot

T

5000-60,000

o§ 500-40,000

* Maximum rate during peak period. An estimated total of less than 25,000-150,000 would ultimately occur.

t Unless individual bone-marrow dose exceeds mean value by a factor of 60. J Unless individual bone-marrow dose exceeds mean value by a factor of 80-500. § Unless individual bone-marrow dose exceeds mean value by a factor of 5-60. Il Conditions which are at least a serious handicap to those affected, as listed in Table

XI of Annex H of the UNSCEAR report1. ^ A total of 2500-100,000 would occur over subsequent years. NOTES.—The methods of estimating incidences of leukaemia and major genetic defects

are described in the U N S C E A R report1. The quantitative evaluation of an increase in incidence of primary bone tumour

attributable to radiation presents great difficulties. If it were assumed that 5-10 cases per million normally occurred per year, and that 10 per cent of these were induced by natural radiation the following figures could be calculated from the 70-year osteocyte doses if a non-threshold hypothesis were assumed:

For tests stopping in 1958 and world population 3000 million, 70-900 per year (as the maximum rate).

In equilibrium after prolonged continuation of tests and world population 5000 million, 1000-25,000 per year (as the continuing rate).

If a threshold of 400 rem were assumed, the incidences would be zero unless individual osteocyte doses exceeded the mean value by a factor of 80-500 in the case of tests stopping in 1958 and by a factor of 5-60 in equilibrium after prolonged contindation of tests.

(c) Life Shortening and Other Delayed Effects TULLIS26 has very well summarized what is known at present about

the other delayed effects of ionizing radiations on man: thyroid cancer, bone cancer, cataracts, etc.

"A study of the mortality of British radiologists during the period 1897-1956 revealed no evidence of any non-specific reduction in their expectation of life that could be provided by an increase in age-specific


mortality rates for all causes of death" (COURT BROWN and D O L L 1 5 ) . Amongst medical men who entered radiological practice before 1921 there is increased incidence and mortality from skin cancer, tumours of the pancreas, and very small increase in mortality from leukaemia. An American report16 has concluded that the average age at death of U.S. radiologists is about 5 years less than that of physicians having no profes-sional contact with radiation. The conclusions of this last report has not met universal acceptance1-15.

The 1960 Report of the (U.S.A.) National Academy of Science Com-mittee on the Pathologic Effects of Atomic Radiation concludes that "there are as yet no data for man that provide a satisfactory basis for quantitative estimation of the overall life-shortening effect, the existence of a dose-threshold, or of the dependence of the effect on dose and dose-fractionation". It is of interest to note that the mortality rates among U.S. radiologists during the quinquennium 1954-1959 indicate in a preliminary fashion a lessened shortening of the life-span probably statistically insignificant. According to Shields Warren, the reason for this fact may lie in the acceptance by U.S. radiologists of better standards of protection since 1947-1948.

It seems that, for radiologists, nurses and technicians using x-ray machines, the long-term somatic risks are small, even very small, provided that care is taken to observe strict measures of protection.

A C U T E R A D I A T I O N S Y N D R O M E I N M A N

Much more is known, at present, thanks to the careful observations of some accidentally irradiated healthy young people, and of many cancer patients irradiated over the whole body (see, for instance, ref. 7). The war experience of Hiroshima and Nagasaki has not left many very useful data as far as this question is concerned, because of the destruction of most medical facilities, the co-existence of burns or wounds and the difficulty of estimating a posteriori the dose received; pathological documents are extremely interesting11.

The L D 5 0 which had been estimated at about 400 r, is probably around 700 r (see ref. 25) if, as it should be, one considers the case of healthy young adults. There exist in the literature8'9'10'25 many instances of young men (and one woman) exposed on the whole body in atomic establishments to 400 rems (generally a mixture of neutrons and gamma rays) who sur-vived without any other treatment than rest and minor symptomatic medication. Total-body exposure to 850 r will probably be 100 per cent lethal (bone-marrow death) if platelets injections and bone-marrow transfusions are not available at the appropriate time.


Let us consider in more detail the symptomatology of acute radiation sickness in man. The first symptoms originate from the central nervous system: nausea, vomiting, mental depression. The rapidity of onset (a question of an hour or two) and the duration (one or two days) of vomiting is proportional to the dose. Below 100 rem these symptoms are mild or absent; they become noticeable between 125 and 175 rem, but they develop into complications requiring clinical treatment in only 10 per cent of the patients at 200 rem7. Vomiting is an extremely regular symptom at higher doses. When the dose received is unknown, the rapidity and severity of vomiting is of great value (together with the subsequent thrombopenia and leucopenia) to estimate the range of dose received

FIG. 21-2. Schematic representation of acute radiation syndrome in man.14

during the accident. Diarrhoea, if present during the first 2 or 3 days after irradiation, is a sign of bad prognosis since it does not seem to occur at that time below 1000 rem. Polyuria has also been observed in man but its severity is much less than that found in rats. After a latent period of


about 2 weeks during which the irradiated individual does not experience particular symptoms*, epilation is observed, haematological signs of bone-marrow depression are evident; mental depression comparable to the "typhos" of untreated typhoid fever has been conspicuous in the group of six young Yugoslav scientists irradiated accidentally at Vinca near Belgrade. Lymphopenia, granulopenia, thrombopenia are comparable to that seen in dogs. The appearance of bilobed lymphocytes13 is a much more sensitive and specific test than leucopenia; unfortunately for routine work it is not yet quick enough. The disappearance of reticulocytes is parallel to that of white cells and indicates the absence of red cell regen-eration.

Oropharyngial ulcerations accompanied by fever have been often ob-served in the large number of Japanese irradiated in 1945, but have not been conspicuous in accidents involving young white people.

Haemorrhages, in the form of petechia, for instance, are frequently seen. They are rapidly and completely checked by platelet transfusion; pulmonary haemorrhage may be the immediate cause of death (Fig. 21-2).

Intestinal disturbance often poses difficult problems. The lack of regeneration of the epithelium, and the motility troubles will alter (some-times a great deal, sometimes surprisingly little) water, electrolyte and food resorption. The presence of diffuse pain in the abdomen and con-tracture of the muscular wall is not uncommon although the subsequent history indicates that no major anatomical lesion (invagination or infarctus, for instance) had occurred. Above 1000 rem, intestinal or pulmonary death is the rule.

Infections are not as regular as was supposed some years ago; indeed, they seem to become a minor problem if normal hospital care can be provided. Increased temperature does not necessarily indicate infection; it may resist treatment with antibiotics and respond rapidly to small doses of cortisone-like substances. Temporary disappearance of spermatozoa is a most regular observation when the dose has reached 400 rem. The ovary is more resistant than the testis in the human species as well as in other mammals, as evidenced by the fact that a woman exposed to about the same flux as three young men experienced catamenial irregularities but not an arrest of ovarian function.

Nobody knows exactly at what level in man the immune reaction is inhibited. Is it as in mice or rats at the level of 400-500 rem or is it above the level (about 700 rem) at which haematopoietic death is the probable prognosis? This is important (see Chapter 20, p. 490) because on the answer depends the possibility of successful grafting.

* Except, naturally, great mental instability and disquiet because these patients know what is going to happen.


Some striking observations from five men irradiated at the Y-12 plant in the U.S.A. are the following25 (Fig. 21-3):

These men had been exposed on the whole body to 240-365 rads (mixture of gammas and neutrons): nausea and vomiting, general infec-tions (except in one case), hair loss, petechiae and gum bleeding in two cases only, thrombopenia, leucopenia, slow and small decrease in haemato-crit and haemoglobin values, decrease in mitotic index of the bone-marrow in two waves (at 4 and 16 days); sperm counts returned to normal slowly (return is faster when the dose does not exceed 100 rads). Biochemical studies in this group of irradiated healthy men revealed: (i) an increased urinary excretion of a newly discovered amino acid—the /3-aminoiso-butyric acid which may be a metabolite of thymine (and of DNA): (ii) mild diuresis with little change in total urinary nitrogen; increased taurine excretion; absence of serine; no change in uric acid, creatine, creatinine, total a-aminonitrogen in urine; (iii) no defects in the blood clotting mechanism, even when the thrombocyte count was at its lowest level; increased proteinaemia at the 4th day (mainly albumin) and again during the 3rd or 4th week; (iv) no change in serum transaminase and lactic acid dehydrogenase.

D A Y S

FIG. 21-3. Composite figure averaging data from five cases simultaneously irradiated in an accident, 2 4 0 - 3 6 5 rads, male subjects aged 32-51 years25. The normal level of white cell count is not indicated; there is a well-marked increase in

granulocytes 12 hr after the accidental exposure.


Treatment—The treatment of serious cases of radiation sickness has to be completely modified after the recent clinical experiences in the U.S.A., U.S.S.R. and France.

(a) General instructions—Decrease energy expenditures, complete mental and physical rest in hospital even during the latent period; if necessary barbiturates or tranquillizers. No visitors; carefully exclude photographers and press people. Excellent nursing care and clean (not necessarily aseptic) conditions are paramount. Normal hospital food, in small amounts; underfeeding decreases metabolism. Water ad libitum to compensate for increased urinary and sweat secretion.

(b) If the dose received does not exceed 400-450 rem, watchful expecta-tion; a healthy young man has a good chance to recover without any other care than minor symptomatic administration.

(c) If the dose is between 500 and 900 rem, haematopoietic death is to be feared in about a month's time.

1. No blood transfusions, because they are useless and because they may interfere with an eventual bone-marrow grafting. The life-span of a red cell in man is 120-130 days; no evidence has been collected that irradiation reduces this life-span. If one assumes that regeneration is

FIG. 21-4. This graph concerns the six Yugoslav scientists accidentally irradiated at Vinda (Oct. 1 5 , 1 9 5 8 ) . Subject V was believed to have been exposed to 1 0 0 0 - 2 0 0 0 rems; M., G. and D. to 7 0 0 - 1 0 0 0 rems; H. to 6 0 0 - 8 0 0 rems and B. to 3 0 0 - 5 0 0 rems. But see footnote p. 510.

B. had no bone-marrow transfusion; V. died from intestinal occlusion and pulmonary haemorrhage 3 days after bone-marrow transfusion. The reticulocyte count of the four other marrow transplanted patients increased and went up above the level of the non-transfused irradiated patient.

A similar improvement after bone-marrow transfusion was observed in granulocyte, platelet and red cell counts18.

completely stopped, the patient, one month after irradiation, shall have destroyed only one fourth of his stock of red cells; 3-3 -5 million red cells per mm3 are quite enough to maintain oxygen supply to the tissues.


2. As few drugs as possible. No cortisone or cortisone-like substances except in very definite indications. No antibiotics, except if infection is diagnosed.

3. If petechiae or haemorrhages occur, transfusions of normal platelets in large amounts (several billions).

4. Bone-marrow transplantation when the total white cell count is low and the general condition of the patient is deteriorating. Three to four hundred millilitres of bone-marrow blood are taken under heparine by many punctures under general anaesthesia from a donor whose blood characters are as close as possible to that of the patient; they are injected intravenously without any other manipulation. Some evidence has been obtained in four cases27 that the graft has "taken", that maturation and division occurred. Progressively, the transplanted cells seemed to have been replaced by normal host cells without any immunologic accident. Im-provement was observed not only of the conditions of the blood, but also of the other symptoms (Fig. 21-4). The four Yugoslav scientists treated in this way were still living happily one year after an accidental exposure to a mixture of y-rays and neutrons.*

(d) If the dose is about 1000 rem or above, the same conduct should be followed except that bone-marrow graft should be attempted earlier (perhaps 7-10 days after the irradiation) because severe intestinal damage (intestinal obstruction, etc.) must be expected in 20-30 days, which might need surgical treatment. The patient should be, from the haematopoietic point of view, in as good a condition as possible to stand the surgical operations. So far no case has been published of a human who has survived the exposure to 1000 rads.

A P P L I C A T I O N S T O T H E R A P Y It has been a constant temptation for radiotherapists to irradiate the whole body in cases of leukaemia or wide-spread tumour conditions (generalized metastasis, Hodgkin's disease, mycosis fungoides, etc.). The

* The Vinda accident has recently been reconstructed by a team of scientists from the International Atomic Energy Agency, in order that accurate dosimetry could be performed. The earlier estimates of the dose seem to have been too high. It now appears that Subject V who died received 190 rads of neutrons and 220 rads of y rays; if an RBE of J is taken for neutrons the total dose received by V was 790 rem. The four scientists who survived following bone marrow transplantation received 600—700 rem, but it must be emphasized that the dose was not distributed homogeneously over the whole body and that the value of 3 for the neutron RBE is only a very approximate estimate.

Many radiobiologists are not convinced that in the bone marrow treated cases the injected homologous cells really repopulated the bone marrow. The complete absence of the symptoms of secondary disease (see p. 490) is considered to be a powerful argument against the view that the homograft was accepted.


inhibition of blood regeneration has been the main brake to this desire. New possibilities are now at hand. First (see p. 461), if the tumour tissue does not concentrate a substance like cysteamine or A.E.T., one could protect chemically the bone-marrow and other vital organs and thus deliver a larger dose to the tumours; but this opportunity has so far not been taken, probably because it is not easy to know if the tumour cells do, or do not, concentrate the protector.

Second, bone-marrow grafting after total-body irradiation in leukaemia. This possibility has been used in several countries; some results have already been published and so far are rather disappointing (see ref. 28, for instance). The idea basic to these clinical trials involves a number of assumptions which have not always been clearly stated and discussed.

1. The therapist hopes to kill all the mother cells in the bone-marrow that produce leukaemic cells. Bone-marrow grafts would result in the progressive repopulation of the destroyed tissue.

Nobody knows exactly if leukaemic mother cells are more radiosensitive than normal cells; the possible existence of a very small percentage of more radioresistant leukaemic cells must not be overlooked, since regen-eration might start from these cells though the population of normal foreign cells injected after irradiation might help to keep the division of abnormal cells under control for a time.

2. A leukaemic (generally very sick) child may not react in the same way to whole-body irradiation, and to marrow cells injection as a healthy young adult. He might not stand the dose necessary to kill all leukaemic mother cells and to abolish immune response, the two essential conditions for success.

3. So far, the radiation doses given seem to have been too small; disappearance of abnormal white cells from the blood was observed, normal cells appeared rapidly followed by abnormal ones and recurrence of all symptoms28. For this treatment to succeed it may be necessary to kill every leukaemia cell; 99-99 per cent is not enough. To achieve this may require doses of several thousand rads if the dose-response curve is similar to that observed in tissue culture.

Thus, this radical therapy of leukaemia has not yet reached the stage of a logical treatment. As usual in clinical sciences, experience only will teach; no progress can be made without trial and error.

A most interesting and logical suggestion has been made and success-fully put in practice by N E W T O N et al.17: bone-marrow is taken from the patient before whole-body or severe irradiation with x- or y-rays, stored in the cold, and re-injected intravenously 2 weeks after irradiation. This procedure avoids immunological reactions and, since the injected marrow cells often stay for days in lungs or other organs before reaching the bone, there is good probability of autografting.


Another possible use of irradiation may occur in the grafting of kidneys. The patient with kidney failure is irradiated with a dose that abolishes his immune response in the hope that the grafted kidney will not be rejected and will remain and continue to function as if it had been donated by an identican twin. It has been observed indeed that the chimaera between irradiated mouse and normal rat is not restricted to bone-marrow tissue (see Chapter 20) but can be produced with the skin. A normal rat's skin can be grafted on to the skin of an irradiated mouse.

Finally, mention must be made of the attempt by neurosurgeons to concentrate radiation in certain brain tumours (neuroglioblastoma) by first injecting a boron compound which is more selectively absorbed by tumour cells than by normal cells, and exposing at the proper time to a flux of neutrons which activate boron in a very effective a-emitter.

R E F E R E N C E S 1. United Nations Report of U.N. Scientific Committee on the Effects of

Atomic Radiation, General Assembly (13th Session), Suppl. No. 17 (A/3838), New York, 1958

Translations in French, Russian and Spanish of this document have been publishd by the United Nations at New York

2. CONARD, R. A., et al., March 1958, Medical Survey of Rongelap People, Brookhaven National Laboratory, Report 534 (T-135), 1959

3. CONARD, R. A. Intern. J. Radiobiol., Suppl. 1, 1960, p. 269 4 . RUSSELL, W. L . and RUSSELL, L . B . , Proc. Second U.N. Intern. Conf. on

the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 22, p. 360, United Nations, 1959

5. RUSSELL, W. L., Intern. J. Radiobiol., Suppl. 1, 1960, p. 311 6. Genetic Effects of Radiations in Man, Report of a study group of W.H.O.,

Geneva, 1957 7. MILLER, L. et al., Air University School of Aviation Medicine, USAF,

Randolf (Texas), May 1957 8. Proceedings of the two U.N. International Conferences on the Peaceful

Uses of Atomic Energy, Geneva, 1955, Vol. 11, and 1958, Vol. 23 9 . HEMPELMANN, L . H . , LISCO, H . a n d H O F F M A N , J . G . , Ann. Int. Med., 1 9 5 2 ,

36, 279 10. JAMMET, H . , M A T H E , G. et al., Rev. franf. Etudes Clin, et Biol., 1959, 4 , 210 1 1 . N O B U O , K . , "Atom bomb injuries", Tsukiji Shokan Cy, 1 9 5 3 , 1 1 2 p. 12. BRUES, A. , Science, 1958, 128, 693 13. INGRAM, M., Intern. J. Radiobiol., Suppl. 1, 1960, p. 235 1 4 . GERSTNER, H . B . , Air University School of Aviation and Medicine, USAF,

Randolf (Texas), November 1957 1 5 . COURT B R O W N , W . M . and D O L L , R . , Proc. Second U.N. Intern. Conf. on

the Peaceful Uses of Atomic Energy, Geneva, 1958, Vol. 23, p. 179, United Nations, 1959

1 6 . WARREN, S . , / . Am. Med. Assoc., 1 9 5 6 , 1 6 2 , 4 6 4 1 7 . N E W T O N , K . A . , HUMBLE, J . G . , W I L S O N , C . W . , PEGG, D . E . a n d SKINNER,

M. E. G., Brit. Med. J., 28 Feb., 1959, p. 531


18. Recommendations of the International Commission on Radiological Protec-tion, Brit. J. Radiol., 1955, Suppl. No. 6, 90 p.

A French translation has been published as a supplement to the J. de Radiol, et Electrol., 1955, 36, No. 10 bis.

19. Recommendations of the I.C.R.P., adopted 9 Sept., 1958, Pergamon Press, 1959, 18 p.

20. Code of Practice for the Protection of Persons Exposed to Ionizing Radiations, H.M. Stationery Office, London, 1957, 83 p.

21. Manipulation sans danger des radioisotopes, Agence Intern. Ener. Atom., Vienne, 1958, 112 p.

2 2 . M O R G A N , K . Z . , Bull. Atom. Scientists, 1 9 5 9 , 1 5 , 3 8 4 23. WARREN, S., Scientific American, Sept. 1959, p. 164 24. MULLER, H. J., Acta Radiol., 1954, 41, 5 25. BRUCER, M . , The Acute Radiation Syndrome, ORINS-25, April 1959 2 6 . T U L L I S , J . L . , Arch. Pathol., 1 9 5 8 , 6 6 , 4 0 3 27. MATH£, G., JAMMET, H. et al., Rev. franf. Et. Clin, et Biol., 1959, 4, 226 2 8 . M A T H E , G., BERNARD, J . et al., Rev. franf. Et. Clin, et Biol., 1 9 5 9 , 4 , 6 7 5

P O S T S C R I P T

The Role of Radiobiology in the World

THE volume of research on radiation effects is now so great that radio-biology is now becoming a science in its own right, and should be taught at least in some universities to postgraduate students who have a sound training in one of the basic sciences.

The future of mankind is linked with the safe development of nuclear energy which involves enormous quantities of radiations and radioactive substances. The diagnostic and therapeutic applications of varied radio-logical techniques will increase in number and difficulty. They will be used over wide areas of medicine and the knowledge demanded for their proper application and further development requires an amount of knowledge of the principles and techniques of physics, biochemistry, cytology, genetics and general biology, that cannot be mastered by even the most scientifically minded clinicians.

Contamination by radioactive waste products—mainly from the atomic energy industry, though laboratory waste, etc., also needs watching— must be carefully controlled. Not only must acceptable levels be agreed on an international basis—as the spread of radioactive substances is not restrained by national boundaries—but the fact on which such limitations are based must be established. The fate of the reactive substances is not governed by physical dilution factors alone but biological processes leading occasionally to remarkable local concentrations must always be considered.

In the health administrations (local, national, international), in the development of research, for the needs of civil security and national defence, for soundly based propaganda in favour of peaceful settlement of difficulties between nations, radiobiologists are badly needed.

The future of mankind is obviously not in danger from radiation so long as no atomic war breaks out. But the dose distributed to the world popu-lation by medical men, dentists and industrial establishments should be watched and frequently estimated just as the "fall-out" since the available data show that, during radiodiagnostic procedures, often of doubtful value, significant doses are distributed. Human health is now a large "industry" and in some of its aspects tends, in certain countries, to become

5 1 4

R O L E O F R A D I O B I O L O G Y I N T H E W O R L D 515

a "racket". Special care should be taken at all levels by the medical profession and administration to avoid radiography, fluoroscopy or, worst of all, radiocinematography of the pelvis of young women (if pregnant, two generations are irradiated), and, of all procedures (uroscopy, gastro-intestinal tract, fluoroscopy) involving direct irradiation of the gonads. In most cases, the testes can be protected by a lead case. Irradiation of children should be accepted only if no other procedure can give similar diagnostic or therapeutic results. Many countries have already decided to stop the yearly or half-yearly mass chest radiography or fluoroscopy for tuberculosis detection.

Since in the future the artificial background of radiation will unavoid-ably increase, it is the duty of every man (medical or other) using machines or substances which give off ionizing radiations, to take individually all measures to minimize the contamination; the health administration should spend enough time and be given the means to ensure maximal protection of the population. Physical protection and avoidance of con-tamination remain the only effective measures so long as we have no effective simple therapy (except in extreme cases lethally irradiated on the whole body), so long as we do not understand better the delayed effects of single small doses or of continuous irradiation at low levels.

A great effort should be made to extend to other mammalian species the experiments performed in mice on genetic effects and on long-term somatic effects when adults, young or foetal specimens are irradiated continuously at low level or given a small dose every day. Such studies are so difficult and unrewarding; they are so costly and extend over so many years that only countries that are rich scientifically and financially can at the present time afford to undertake them. But scientists of smaller nations should not remain inactive in this field. Morally, it is not sound to accept the view that assessment of the dangers from low doses which are so intensely discussed at the present time, which involve not only science but also health and politics, should remain permanently outside the range of activities of some scientists, just because they belong to small nations. A serious effort ought to be made that a monopoly in such matters concerning the health of the whole world population is avoided.

If we consider the other fields of radiation research we have every reason to be happy: progress is magnificent, many good people are working with sound basic ideas, and working so well that within four or five years the field covered by this book will again have increased so much in scope and interest that we prefer not to think about the third edition.

3 4

SUBJECT INDEX

A Abscopal effects, irradiated tissues, 451,

452 on D N A and RNA biosyntheses, 391

Absorption coefficient, atomic, 13, 17 for pair formation, 17 photoelectric, 13

mass, see Mass absorption coef. X- or y-rays, electron production, 12

Accidental massive irradiation, man, 507-509

Aeetabularia mediterranea, X-irradia-tion, 265

Acetate, labelled, increased incorpora-tion after irradiation, animals, 343

Acetylation, reduced after irradiation, animals, 330

Acetylene, polymerization, role of excitation, 103

Achromobaeter fiseheri, light emission, effect of X-rays, 375

Acids, organic, radio-protectors, 463 Acrylonitrile, polymerization, X- or y-

rays, 136 ACTH, secretion, increased after stress,

mammals, 387 irradiated mammals, 394

Actinomycin, radiosensitizer, normal and tumour tissue, 478

Adaptation syndrome, irradiated mam-mals, 387-389

Adenine, increased in splenic D N A after irradiation, 353

Adenosinetriphosphoric acid, see A T P Adrenalectomy, effect of, on irradiation

reactions, mammals, 393 on survival of irradiated rats, 391

Adrenaline, induced blood pressure changes, rat, 396

radioprotection, mammals, 289, 477 Adrenals, 388

cholesterol, changes after irradiation, 399, 400

Adrenals, cholesterol, content after irr-adiation, rats, 395

cortical insufficiency in radiation sickness, 393

response to whole-body irradiation, 394

screening in irradiation, 392 Adrenocorticotropic hormone,

see A C T H Age, variation of radiosensitivity, mice,

299, 302 Ageing, normal, effect of irradiation,

mice, 439 Aggregation, protein, by irradiation,

111, 187 Air, mass absorption coefficient, 17, 18 Alanine solution, X-irradiation, 149 Alarm reaction, 392 Albumins, see also Blood proteins,

bovine serum, amino acid residues destroyed by irradiation, 184

groups reacting with radiomime-tics, 223

irradiation changes, 182 human serum, irradiation, chemical

changes, 190 effect of pH, 188

radioprotection, 111 Alcohol dehydrogenase, G value for

deactivation, 191 Alcohols, decomposition by a-particles,

109 effect of direct and indirect action, 57 metabolism after irradiation, rats, 317 radiosensitivity, 305

Algae, unicellular, irradiation, 265 Alkylating agents, see Radiomimetics. Alloxazine adenine dinucleotide, 193 Alpha-rays, 6, 10

and X-rays, differences, 124 relative effectiveness, 90

carboxypeptidase inactivation, 144 chromosome aberrations, 90

Tradescantia pollen, 269 S '7

518 S U B J E C T I N D E X

Alpha-rays, crystal lattice destruction, minerals, 110

D N A irradiation, 204 fatty acid decomposition, 109 fern spore inactivation, 265 formation OFH2O2, 130-132,141 haemocyanin dissociation, 181 induced reactions, catalysis by inert

gases, 103 ionization by, 30 irradiation, calc. of target size, 80

protection by cysteine, 467 limiting concn., 144 nuclear and cytoplasmic irradiation,

263-267 nuclear inactivation, insect eggs, 263 sensitivity in presence of O2 , 280 source and range, 10 water irradiation, 123

diffusion of free radicals, 128 distribution and diffusion of radi-

cals, 145 yields of radicals and molecular

products, 134 Amines, radioprotection, mammals, 463

reaction with radiomimetics, 221 D-Amino acid oxidase, G value for

inactivation, 191 Amino acids, bovine serum albumin,

radiation damage, 184 concn. in invertebrates and radio-

sensitivity, 306 concn. in tumours and radiosensitivity,

306 deamination by X-rays, 150 radioprotection, animals, 463 radioreactions in aqueous solution,

148, 149 jfi-Aminobenzoic acid solution, X-

irradiation, 149 /3-Aminoethylisothiouronium, radiopro-

tector AET, 460 /3-Aminoisobutyric acid, urinary ex-

cretion after X-irradiation, man, 508

Amoeba, cytoplasmic and nuclear irra-diation, 264

radiosensitizers, 478 Amphibians, eggs, virgin, nuclear and

cytoplasmic irradiation, 267 radiosensitivity, 300

Anaemia, development after X-irradia-tion, animals, 421, 425

Anaesthetics, radioprotection of animals, 463

Analgesics, radioprotection of animals, 463

Anaphase, 241 Animals, see also Mammals, Inverte-

brates etc. absorption of 90Sr, 499 cold blooded, radiosensitivity, 302 hibernating, irradiation t reatment

375 lethal radiation dose, 300 recovery f rom irradiation, 379 responses to continuous irradiation

382 Anorexia, after irradiation, rats, 320 Anoxia, and radioresistance, cells, 284

chicks, 383 hypothermic mice, 290 tumours, 291

producing substances as radioprotec-tors, 463

radioprotection mechanism, adrena-line, histamine and 5-hydroxy-tryptamine, 477

cysteamine, 470-473 Anthracene, irradiation, 113 Antibiotics, effect on radiation sickness

424, 508 Antibody, formation, after bone-marrow

treatment, irradiated animals, 490

after irradiation, mammals, 325 man, 423, 424

titers, after red cell injections in radiation sickness, 425

Antigens, injection, reaction of irradia-ted animals, 412

properties of deoxyribonucleoprotein changed after irradiation, 353 y-

Antivitamins, see Vitamins, antago, nists.

Aqueous systems, radiation chemitrs 122-154

Argon, reduction by, of radiosensitivity 294, 296

Ascites, see under Tumours . Ascorbic acid, adrenal, changes after

irradiation, rats, 394, 399

S U B J E C T I N D E X 519

Ascorbic acid, radioprotection, plants and animals, 319

Atomic weapon tests, 498 ATP, decrease after irradiation, ascites

cells, 315 Auger effect, 13, 38 Aureomycin treatment in radiation

sickness, 424 Auxin, from tryptophan, irradiated

mung beans, 346 Azide, radioprotector, mice, 459 Azotobacter agile, multi-hit curves, 86

B

Bacillus megaterium, radiosensitivity, effect of temp., 53

spores, X-ray killing, 87 Bacillus subtilis, radiosensitivity, effect

of temp., 54 of spores, 94

Bacteria, inactivation dose, 49 lethal dose, 84 luminous, effect of X-rays on light

emission, 375 radiosensitivity, 302

effect of oxygen concn., 286 survival curves, 85 X-irradiated, restoration, 372

Bacteriophage, indirect action on, 59 radiosensitivity, effect of temp., 53

Barley seed, comparative effects of X-rays and H2O2, 236

X-irradiation, 69 paramagnetic absorption after, 119

Becaptan, see Cysteamine. Benzoic acid, sodium, X-ray protection

of albumin, 111 3:4-Benzopyren, radiosensitizing action

in rat, 478 Beta-rays, 6, 8

emitting isotopes, 9 production of radioresistance in mice,

389 RBE for acute mammalian effects, 92

Biological activity, structural require-ments, 219, 220

Birds, c.n.s., response to irradiation, 410 radiosensitivity, 302 response to continuous irradiation,

382

Blood, see also Erythrocytes, Granulo-cytes, Leucocytes, Lymphocytes, Reticulocytes,

cells, comparative effects of X-rays and radiomimetics, 227

disappearance after X-irradiation, man, 506, 507

variations in radiation sickness, 426 changes after X-irradiation, 417-421 circulation, see Circulation, coagulation, after irradiation, mam-

mals, 422 dehydrogenase activity, increased

after irradiation, 341, 342 effect of cysteamine, cystamine and

cysteine in mammals, 472 gluco-corticords, specific reactions,

389 glutamic-oxaloacetic and-pyruvic

transaminases, increased concn. after irradiation, 342

lactic dehydrogenase, increased after irradiation, 342

malic dehydrogenase ,increased concn. after irradiation, 342

plasma, decreased volume after irra-diation, mammals, 414

increased enzyme activity after irradiation, 341-343

platelets, count after y-irradiation dogs, mammals, 420, 421, 422

properties, 418 potassium, increased after irradiation

406 pressure changes, after pain, adrena-

line and irradiation, rat, 396, 397 proteins, see also Albumins, Globu-

lins etc. changes after irradiation, animals,

326 response patterns, myleran, chloram-

bucil and X-rays, 228 transfusions in radiation sickness, 508 vessels, vascular changes after irradia-

tion, mammals, 455 vascular fragility after irradiation,

mammals, 422 Boletus calopus, X-irradiation, 273 Bombyx mori, eggs, restoration after

irradiation, 372

5 2 0 S U B J E C T I N D E X

Bone-marrow, atrophy, after irradia-tion, 72

damage by irradiation, mammals, 419 D N A conen., after local or total irr-

adiation, rat, 454 inactivity after irradiaton, 425 synthetic capacity, effect of total

irradiation, 337 Bone-marrow grafts, 487

after whole-body irradiation in leu-kaemia, 510

antibody formation after, 490 homogenates, injection after irradia-

tion, 486-492 treatment in radiation induced

lymphoma, mice, 446 isologous and homologous, treatment

of irradiated mice, 487 transplantation in irradiation treat-

ment, man, 509 Bone tumours, induced by radio-

elements, mice, 442, 443 Bones, incorporated 90Sr., 500

physical radioprotection, 484-486 Boron, transmutation, 11 Brain tumours, neutron exposure, 11,5n Brinkman-Lambert test, 468, 477

C

Caesium, radioactive, incorporation by cells, 498

Cage effect, 165 Calcium, role in chromosome radio-

sensitivity, 271 Cancer, induced by X-rays, 442-447

therapy, chemotherapeutic agents, 220, 221

radiations and radiomimetics com-pared, 229

Carbohydrate metabolism, effect of irradiation, 313-317

Carbonium ion, 106 Carboxypeptidase, G value, 191

inactivation, by a- and X-rays, 144 effect of concn., 48

Carcinogenesis, agents producing, 218 radiation, whole-body exposure, ani-

mals, 443-446 Catalase, activity, in vivo changes on

irradiation, 346

Catalase, G value for inactivation, 191 irradiation protection, 193 restoration of u.v. irradiation lesions,

369 Cells, direct and indirect action on, 60

grafting in irradiated animals, 487 Hela, survival after irradiation, 250 human, irradiation growth, in tissue

culture, 251 incorporation of radioactive Cs., 498 interaction between, after tissue irra-

diation, 451-455 intracellular barriers, 275

breakdown after irradiation, 340 recovery, 369

irradiated, cytolysis, in vivo, mam-malian tissue, 354

interfase death, 248 lesions produced, 239-261 mitotic death and giant cell forma-

tion, plant and animal systems, 249

multiplication and D N A syn-thesis, 351

pathological biochemistry, 274 permeability changes, 272

killing, by irradiation, 84 X-rays and radiomimetics, com-

pared, 227 mammalian, recovery after irradia-

tion, 373-375 normal division, 244 oxygen concn., effect on radiosensi-

tivity, 286 oxygen press, lowered by cysteamine,

471 radiosensitivity, effect of O2 concn.,

285, 286 in vivo and in vitro, 252 period in mitotic cycle, 247

radiosensitivity of nucleus and cyto-plasm, 263

reversible damage and mitotic delay, 245-248

sexual, production in mammals, 428 somatic mutations, 260 toxic agents, 218

Cellular theory, injections of bone-marrow or spleen extracts after irradiation, 487

Cerenkov radiation, 19

S U B J E C T I N D E X 5 2 1

Ceric sulphate, reduction by OH" radical, 137

Cerium, radioactive, injection into mice, 390

Charge neutralization, 105, 127 Charge transfer, molecular, 45, 106 Chelating agents, radioprotection

against X- and y-rays, 464 Chemotherapeutic agents, cancer, 220,

221 Chimaeras, irradiated mice treated with

bone marrow, 490-491 Chlorambucil, see also Nitrogen mustard

cytostatic action, 227 lymphoma therapy, 229

Chlorpromazine, radioprotection of mice, 465

Cholesterol, adrenal, changes after irra-diation, rats, 399, 400

low in dying animals, 394 metabolism after irradiation, rats,

321 suprarenal, after whole-body irradia-

tion, rats, 395 Cholinesterase, specific and non specific,

in vivo activity changes after irradiation, rat muscle, 345

Chromosomes, abnormalities, 94 application of target theory, 88 effect on, of conditions during and

after irradiation, 283 of metabolic state after irradia-

tion, 372 produced by a-particles, 90, 266,

269 by enzyme release during irra-

diation, 277 by oxygen, 281 by X-irradiation, effect of inert

gases, 295 reversible damage, 245

variations in RBE, Tradescantia, 95 breakage, agents causing, 218

comparison of radiomimetics and radiations, 224-226

radiation induced, 253-256 effect of oxygen, 284

response to biochemical disturb-ance, 268-271

crossing-over in meiosis, 242 division, 241

Chromosomes, radiosensitivity, role in Ca, 271

periods of maximum radiosensitivity, 269

radioresistance related to ploidy, 307, 308

Circulation, action of ionizing radia-tions, mammals, 411

slowing down in cysteamine injected mammals, 472

Citric acid, formation after irradiation, rats, 316

Cluster formation, 32, 103 Cobalt-60, 8 Coelenterates, radioresistance, 304 Co-enzyme A, inactivation after irradia-

tion, 330 inhibition in vivo, 329

Colloids, irradiation, 152 effect of electrophoresis, 153

Competition factor, 222 D N A and polyelectrolytes, 223

Compton effect, 13, 34 Concentration, solute, effect on radio-

chemical yield, 141-144 Co-polymers, crosslinking and degra-

dation, 168, 169 Corticoids, changes after whole-body

irradiation, mammals, 394 secretion, stress induced, 387

Cortisone, detrimental in radiation sickness, 401, 508

Crosslinking, 158-160 and degradation of co-polymers, 168 DNA irradiation, 202 hypothesis, DNA degradation, 232 mechanism, 170 polymer, by direct action, 166

by indirect action, 162 classification, 166 effect on, of oxygen, 173

of temperature, 175 mechanism, 164 prevention, by energy transfer, 177

protection in paraffin chain, 112 radiation dose and gel formation,

relationship, 159 Crystals, irradiation effects, 109

lattice destruction by a-rays, 110 Cyanides, radioprotection, 416

species differences, 458


Cyanides, resistance to, insects and invertebrates, 303, 306

Cyclohexane, radioprotection by ben-zene, 114

Cystamine, radioprotection, 460 local applications, 468, 469

Cysteamine, action on free radicals in yeast, 476

metabolism, 461 protection against oxygen and radia-

tion damage, 281 radioprotection, 460

anoxia theory, 470—473 in leucopenia, 419 in leukaemia or tumour irradiation,

510 mechanisms, various hypotheses,

469 mixed disulphide theory, 473—475 rats, 399 synovial fluid, 207 various living organisms, 466

Cysteine, oxidation after X-irradiation, 324

protection against synkavit, 478 radioprotection, comparison between

X- or y-rays, neutrons or a-particles, 459, 467

during irradiation, 467 hibernating animals and ascites

cells, 375 local applications, 467, 468

solution, X-irradiation, 150 Cytochrome c, spleen deficiency after

irradiation, 332 Cytoplasm, changes during mitosis, 342

irradiation, amoeba, 264 by a-rays, 263-267 various animal and plant cells,

263-267 radiosensitivity, 263

Cytosine, decreased in splenic DNA after irradiation, 353

Cytoskeleton, 275 Cytotoxic agents, 218 Curie, definition, 22, 24

D

Deactivation of excited molecules, 128 Deamination reactions, 149 Death, irradiation, cause, mammals, 430

Degradation and crosslinking of co-polymers, 168

polymer, by direct action, 167 by indirect action, 160 classification, 166 mechanism, 171 proportional to radiation dose, 167

prevention by energy transfer, 177 Dehydrogenase, irradiation, activity

after, 330 increased serum activity after, 341,

342 tissue content, effect on radioresist-

ance, 306 Delta-rays, 19, 32, 79 Deoxycorticosterone acetate, see DOCA Deoxribonucleic acid, see D N A Deoxyuridine, 5-bromo- and 5- iodo-

radiosensitizers, 479 Deuterons, 10

depth dose effect, comparison with electrons, 39

Di-2-chloroethylmethylamine, lymph-oma therapy, 229

Diet plus essential fatty acids, effect on survival after irradiation, mice, 438

Diethyldithiocarbonate, sodium, radio-protection, mice, 462

Dilution effect, irrradiation of biological systems, 47

Diphenylhydantoin, anticonvulsive ac-tion in irradiation, 406

DPN, depressed concn. after irradiation, ascites cells, 315

Direct action, 46-56 changes in synthetic polymers, 165—

175 D N A degradation, 201-204 effect of temp., 53 effectiveness in cells, 60 in vitro, 56 intra-molecular energy transfer, 112 physical and chemical changes in

proteins, 179-187 role of HO2 radicals, 139

Disulphide bonds, serum albumin, effect of irradiation, 182-183

Disulphides, protein-S-S-groups, re-action with —SH protectors, 473


DNA, activity, variations in spleen and small intestine after total irradia-tion, 348

biosynthesis, abscopal effects, 391 inhibited in mitotic delay, 247 mechanisms after irradiation, 352-

355 radiosensitivity, variation during

mitotic cycle, 349-351 bone-marrow, concn. changes after

local and whole-body irradiation, rats, 454

changes in interphase death, 248 competition factor, 223 concn. per cell, effect on radiosensiti-

vity, 309 crosslinking, 159, 232-235 cysteamine radioprotection, 474 degradation, 230-235

action of peroxides and oxygen, 235

by direct action, 201-204 by indirect action, 198-201

inactivation, in vivo and in vitro, 61 irradiation, a-particles, 204

effect of O2 , 294 in vivo changes, 206

polymerized, isologous, restoration factor, irradiated bacteria, 372

radioprotection by glutathione, 200 by histone, 198

structure, 194-197 water content, effect on irradiation

damage, 203 DNAase, I and II, activity changes

after irradiation, 356 protection of, against X-rays, 50 release from mitochondria, 276

DOCA, radioprotection, 392 Dodecane, radiation crosslinking, 112 Dogs, malnutrition after irradiation, 427 Dose, annual, from man-made radia-

tion, 496 from natural radiation, 495

crosslinking and gel formation, re-lationship, 159

D37 and 'single-hit' concept, 63 distribution in tissue, different energy

radiations, 38 effect of, on rate, 65 on chromosome abnormalities, 254

Dose, on invertebrates, 301 on RBE of radiation, 91

enzyme inactivation, function of LET, 210

fatal to animals, 299 fractionation, effect on leukaemia

incidence, mice, 445 inactivation, effect of concn., 58 LD50 after repeated irradiations,

mice, 380, 381 measurement, 24 proportional to life shortening, irra-

diated mammals, 436 ratio to polymer degradation, 167 reduction, by anoxia, 284

by cysteamine injection, mammals, '471, 473

size-response relationship, 63-73 X-ray, at timed intervals, survival of

mice, 378 Dose rate, effect of, on mitosis, Trade-

scantia pollen grains, 247 on mutations in mice, 258 on radiochemical reactions, 144

ratio of molecular to radical reaction in water, 146

response to continuous irradiation, various animals, 382

Dosimetry, by ferrous sulphate oxida-tion, 134

chemical, 26-29 physical, 25

Drosophila eggs, irradiation, effect on cytoplasm and nucleus, 264

gene mutations by X-rays and chemi-cals, comparison, 226

genetic effects of radiations, 257 populations, radiation experiments,

260 Dyes, permeability changes after irra-

diation, animal tissues, 416

E

Electrokinetic potential, see Zeta potential

Electrolyte, concn. changes after irra-diation, 327

Electron paramagnetic resonance, 118 Electron-positron production, see Pair

formation


Electron spin resonance, detection of unpaired electrons, 118

irradiated materials, 118, 172, 186, 202, 203, 475, 476

Electrons, 7, 29 capture, 105 charge neutralization, 127 depth dose effect, comparison with

deuterons, 39 excitation, 19, 40, 104 negative ion formation, 104 recoil, 14, 15 sub-ionization, 31, 33 tracks, 31

Electrophoresis, colloidal, effect of X-rays and neutrons, 153

Embryos, irradiated in utero mammals, 447

rat, development of radioresistance, 390

Endocrine regulation, 388 Energy, excited molecules, 41

ion pair formation, 26 loss, by particulate radiations, 19-20

by protons, electrons and a-par-ticles, 30

by X- and y-radiation, 12-19 receptors, 113 supply, interruption, role in radiation

damage, 271-272 Energy transfer, 12, 40, 42, 45

agents, —SH radioprotectors, 474 intra-molecular, in direct action,

112 inter-molecular, 113

between organic solids, 118 protection, of enzymes, 210

of polymers, 177 and radioprotection, 51, 110-114 role of HO 2 radical, 140

Enzymes, activity increased after irra-diation, 332-345

concentration and inactivation, 48 dry, radiation protection, 210 inactivation, 190-193

G values, 133 indirect effect, 59 oxygen action, 293 physiological effects, 271

inactivation, RBE and L E T relation-ship 96

Enzymes, inactivation dose, function of LET, 210

radioprotection, 193 radiosensitivity, 47 release during irradiation, resulting

chromosome damage, 277 release hypothesis, 263-277 —SH, role in radiobiochemistry,

328-332 substrate relationship, effect of irra-

diation in vivo, 413 EnzjTTie systems, in vivo radioresist-

ance, 345-347 Epoxides, biological activity, 219, 220 Erythrocytes, changes in electrical cap-

acitance after irradiation, 415 increased O2 consumption after irra-

diation, 339 Erythropoiesis in irradiated animals, 426 Escherichia coli, aerobic and anaerobic,

radiosensitivity and O2 effect, 282

dose curves, 85 radiosensitivity, effect of temp., 54

restoration, effect of incubation temp., 371

X-irradiation, 226 Ethyleneimines, biological activity, 219

220 D N A crosslinking, 234

Euler effect, 274 Excitations, 19, 39

electron, 19, 40, 104 role in chemical change, 101-107 role of molecules in water, 128-130

Eyes, electro-retinogram, changes in b wave after irradiation, frog, 409

radiation cataract, 448

F F-centres, 109 Fallout, from atomic weapon tests, 498

genetic danger, man, 503 radioactive waste, 497

Fats, metabolism after irradiation, 317— 322

Fatty acids, decomposition by a-particles, 109

unsaturated, effect on life-span after irradiation, mammals, 437


Ferrous sulphate, oxidation dosimetry, 134

Filbrinogen, solution, X-irradiation, 187 Fish, radiosensitivity, 300 Fluoracetate, radioprotector, 464 Foetus, c.n.s., radiosensitivity, 410

effect of irradiation at different pre-natal stages, mice, 447

Folic acid, antagonists, 218 Formaldehyde, genetic effects in Droso-

phila, 226 radiation decomposition, effect of

concn., 142 Formic acid, radiation decomposition,

effect of concentration, 142 Franck-Condon principle, 41, 42 Franck-Rabinowitch cage effect, 108 Free radicals, see also Radicals

behaviour, 115-120 combination with oxygen, 116 determination, 117 diffusion, a-particle tracks in water,

128 effective, 49 formation, 40, 45, 115

from ions, 126 in water, 125

hypothesis, mechanism for —SH radioprotectors, 4 7 5 4 7 7

inactivation of viruses and enzymes, 59

ions, 104, 105 reactions, 136-147 role in polymer crosslinking, 164 stability, 116 trapping, 171

Freezing test, effect of temp, on radiations, 52

Fricke dosimeter, 28, 134 Fungi, growth rate changes during and

after irradiation, 376

G G-value, 28, 101

for production of H 2 , H 2O 2 , O H ' and H ' , 133

inactivation of enzymes, 191 Gamma-rays, 6, 8

energy loss, 12-19 formation of OH radicals, 136

Gamma-rays, irradiated water, yields of radicals and molecular products, 134

irradiation, life-span shortening, rats, mice, 437

oxygen effects during, 280 protective substances, 458-465

mutations, mice, 258, 259 polyvinyl pyrrolidone gelation, 163 protection of solid polymethyl metha-

crylate, 178 RBE for acute mammalian disorders,

92 specific ionization from, 34-39 target size for nucleoprotein, 80

Gases, liquids and solids, radiochemical reactions in, 107-110

radiochemical reactions, 102 Genes, control of radiosensitivity, 308,

damage, 66 irradiation damage, restoration, 370-

375 mutations, by X-rays and chemicals-

comparison, 226 chromosome rearrangements, 255

Genetics, effect on, of atomic weapon tests, 498

of formaldehyde in Drosophila, 226 of ionizing radiations, 256-261 of natural and artificial radiations,

man, 501-503 selection, irradiated Drosophila, 260

Globin, synthesis before and after whole-body irradiation, verte-brate systems, 336

Globulins, 327 Gluco-corticords, 388

in blood, specific reactions, 389 Glucose, labelled, catabolism dimini-

shed after irradiation, mice, 344 Glutamic-oxaloacetic and-pyruvic trans-

aminases, increased serum concn. after irradiation, rats, 342

Glutathione, radioprotection, against X- and y-rays, 459

of DNA, 200 Glycine, labelled, increased 14CO2 elim-

ination after irradiation, rats, 344 solution, X-irradiation, 149

Glycogen, liver content after irradia-tion, rats, 313


Glycolysis, depressed after irradiation, ascites cells, 315

Grafts, antibody formation against host, 490-491

cells, in irradiated animals, 487 organ, possible use of irradiation,

man, 511 successful after irradiation, mammals,

489 Granulocytes, 417

changes after irradiation, man, 507 count after y-irradiation, dogs, 420,

421 increase after irradiation, mammals,

419 Graphite, energy storage, 110 Growth, cessation after irradiation,

animals, 325 fungi, changes after irradiation, 376 increased after irradiation, plant

cells, 335 inhibition by irradiation, plants, 346 inhibitors, animals, 221

Guanine, DNA, alkylation and sub-sequent reaction, 232

increased in splenic D N A after irradiation, 353

Guanosine, irradiation, 151

H

pH, effect of, on radiomimetic action on proteins, 222

on X-irradiation of human serum albumin, 188

Habrobrecon eggs, nuclear inactivation by a-particles, 263

Haematopoietic organs, regeneration, after physical protection of spleen, thigh or liver, rats, mice, 486

Haematopoietic tissue, transplantation, in irradiation treatment, mam-mals, 487-492

Haematoporphyrin, radiosensitizer, am-oeba, 478

Haemin, synthesis before and after whole body irradiation, verte-brate systems, 336

Haemocyanins, a-irradiation, 180 Haemorrhages, after irradiation, mam-

mals, 421

Helium, reductions in O2 dependant radiosensitivity, Vicia faba, 296

Helixpomatia, haemocyanin irradiation, 180

reflex-like response to irradiation, 408 Herring sperm, reaction with alkylating

agents, 233 Hibernation, effect on cysteine in

radioprotection of squirrel, 467 Histamine, liberation by irradiation,

mammals, 411 radioprotection, 477

Histone, radioprotection, DNA, 198 HO2" radicals, capture by — S H com-

pounds, 476 formation, 138 HO+, H + and HoO+ formation, 125 reactivity, 138-139 role in energy transfer, 140

in direct action, 139 in polymer degradation, 162

Hodgkin's disease, chemotherapy, 229 therapy, whole-body irradiation, 510

Hormones, gonadotropic and somato-tropic, stress inhibition, 387

sex, radioprotectors, 463 steroid, see Corticords

Humoral theory, action of spleen and bone-marrow homogenates after irradiation, mammals, 487

Hyaluronic acid, connective tissue, role in increased permeability after irradiation, 417

irradiation, 207 Hybrids, mice, mortality dose, 68 Hydrocarbons, paraffin, crosslinking

protection, 112 Hydrogen, atoms, reactivity, 137

release, polymer, irradiation, 168 transfer reaction, radiation protec-

tion, 176 Hydrogen bonds, rupture, D N A irra-

diation, 202 protein irradiation, 183

Hydrogen peroxide, and X-rays, comp-arative effects on barley seeds, 236

formation by a-rays, 130-132, 141 in aerated solutions, 140 in water by varying electron in-

tensities, 132 from lipids after irradiation, 317


5-Hydroxytryptamine, radioprotection, 477

Hyperferraemia, 338 Hyperglycaemia, after X-irradiation,

rats, 315 I Iypophysectomy, effect on survival of

irradiated rats, 391 Hypothalamus, involvement in firs

reaction after irradiation, mam-mals, 395

stimulation by irradiation, mammals, 398

Hypothermia, X-ray protector, 289

I Imidazole ring, irradiation, 151 Immune disease, 490 Immune reaction, inhibition by irra-

diation, men, 507 Indirect action, 46, 53

changes in synthetic polymers, 160-164 DNA degradation, 198-201 effectiveness in cells, 60 in vitro, 56 physical and chemical changes in

proteins, 187-194 protection, 111

Inert gases, catalysis of a-ray induced reactions, 103

reversal of O2 radiation effects, 294 Infections, after irradiation, man, 423,

506 more dangerous in irradiated ani-

mals, 326 Infusoria, radiosensitivity, 302 Insects, eggs, irradiation, effect on

nucleus and cytoplasm, 263 pest control by radiation, 303 radiosensitivity, 302, 306

Internal conversion, 42 Intestines, damage after irradiation,

mammals, 427 disturbances in irradiated individuals,

506 mucosa, increased glucose oxidation

after irradiation, rats, 339 response to ionizing radiations, mam-

mals, 413 small, D N A activity variations after

total irradiation, 348 syndrome after irradiation, rats, 328

Intracellular recovery, 369 Invertebrates, amino acid concn. and

radiosensitivity, 306 c.n.s. reactions to irradiation, 408 radiosensitivity, 301, 305

Iodoacetate, radiosensitizer, mouse leu-kaemia cells, 478

Ion pairs, 20, 21, 24 formation, 31, 39, 104

in air, 26 Ions, dissociation, 106

distribution in space, 31 negative, formation, 104 reaction with neutral molecules, 107 transport changes after irradiation,

mammals, 415 Ionizing radiations, man-made sources,

495 natural sources, 494

Ionization density, see L E T Ionization transfer, 20 Isothiouronium compds., rearrange-

ment into guanidothiols in radio-protection, 461

Isotopes, see also Radioisotopes. /8-ray emitting, 9 sensitivity of an indicator, 343 use as tracers, 343-345

K K-factor, def. 22 Krypton, reduced O2 dependent radio-

sensitivity, Vieia faba roots, 294, 296

L Lactic dehydrogenase, serum conc.

increased after irradiation, 342 Leucocytes, regeneration after cystea-

mine, in radiation sickness, 419 Leucopenia, 72

cysteamine protection. 419 development in radiation sickness,

419 Leukaemia, radiation induced, 442—447

by natural and artificial means, 503 effect of dose fractionation, 445 myeloid and lymphatic, 444

radiosensitizers, 478 therapy, 229, 510

Leukaemogenesis, 260


L E T , 29-39 different ionizing radiations and their

RBE, 91 effect of, on G value for ferrous

sulphate oxidation, 135 on radiation damage, 276

frequency, distribution for different radiations, 79

from X- and y-rays, 34—39 in liquids, 33 relation to RBE, micro-organisms,

83-86 values, protons, electrons and a-

particles, 30 Life, extension by low irradiation doses,

mice, 441 shortening, by natural and artificial

radiations, man, 504 by X-irradiation, mammals, 436-

442 Liquids, gases and solids, radiochemical

reactions in, 107-110 specific ionization, 33

Limiting concentration, a-rays, 144 X-rays, 143

Linear energy transfer, see L E T Lipaemia, radiation induced, 320 Lipids, absorption and transport after

irradiation, animals, 319 Lipoxidase, radiation-like death, rats, 97 Liver, irradiation, carbohydrate meta-

bolism after, 313-317 glycogen content after, 313 increased O2 consumption after,

rats, 339 steatosis after, 320

physical radioprotection, 484-486 radiosensitivity, 345

Lymph nodes, irradiation damage, effect of O2 , 288

Lymphocytes, 418 binucleated, presence after irradia-

tion, man, 423 damage by irradiation, 419 radiosensitivity, effect of O2, 287 selective poisons, 218

Lymphoma, radiation induced, effect of intravenous bone-marrow cell suspensions, mice, 446

therapy, 229

Lysosomes, breakdown, role in radia-tion injury, 274

Lysozymes, DNAase I l activity changes after irradiation, 356

G value for inactivation, 191

M

Macromolecules, see also Polymers main chain scission, 157 radiation effects, 157-212

Malic dehydrogenase, serum concn. in-creased after irradiation, 342

Malnutrition, produced by irradiation, 426

Mammals, see also Animals, Verte-brates, etc.

c.n.s., action of ionizing radiations, 407-411

ova, production, 428 polyuria, radiation induced, 412 radiosensitivity, 302

effect of O2 , 287-290 respiration, effect of irradiation, 312 restoration after irradiation, 377-383

Mammary gland neoplasia, after whole-body irradiation, mice, 445

Mass absorption coefficient, 17, 18 air, 17, 18 contribution to, of Compton effect,

14, 18 of pair formation, 18 of photoelectric effect, 18

Meiosis, 242-243 chromosome crossing-over, 242

Membranes, changes, primary radia-tion lesion, 274

role in cellular radiation damage, 273

intracellular, 275 breakdown after irradiation, 340 recovery, 369

Mental depression, irradiated indivi-duals, 506

(9-mercaptoethylamine, protection against X-rays, serum albumin, 111

Mercaptoethylguanidine, see Cystea-mine

Mesyloxy groups, biological activity, 219, 220


Metabolism, alcohol, after irradiation, rats, 317

carbohydrate, after irradiation, 313— 317

cholesterol, after irradiation, rats, 321 cysteamine, 461 disturbances, after CoA inactivation,

330 in radiation sickness, 393

effect of irradiation, animals, 312 effect on chromosome damage from

irradiations, 372 fat, after irradiation, 317-322 increased after irradiation, skin, seeds

potato tubers, 340 limited, improved irradiation re-

covery, E. coli, 371 protein, after irradiation, 322-327 role in radiation injury, 274, 284 thyroid, after irradiation, 313

Methacrylates, irradiation, volatile pro-ducts, 168

Methionine oxidation after X-irradia-tion, 324

post-irradiation treatment, mammals, 383

Methoxamine, radioprotector, 477 Methylalcohol, radiation decomposi-

tion, effect of concentration, 142 Methylviolet, penetration in frog's

heart, effect of irradiation, 416 Mice, chimaeras, 490-491

dose-mortality curve, X-rays, 71 hybrids, lethal radiation dose, 68 mortality dose, 68 mutation, dependent on dose rate

and total dose, 258 RBE for acute disorders, 92

Microorganisms, radiosensitivity, 302 restoration after u.v. irradiation, 369 restoration processes after X-irradia-

tion, 370-372 Microsomes, 275 Mineralocorticoids, 387 Minerals, crystal lattice destruction by

a-particles, 110 Mitochondria, DNAase I, activity

changes after irradiation, 356 DNAase release, 276 intracellular membranes, 275 participation in cell lysis, 274

Mitochondria, splenic, decreased syn-thesis of P. compds., after irra-diation, 331

Mitosis, 240-245 chromosome, radiosensitivity, 269 delayed, 245-248

inhibited DNA synthesis, 247 DNA synthesis, radiosensitivity, vari-

ation during cycle, 349-351 effect of dose-rate, 247 in a complex organism, 243-245 inhibition, agents causing, 218

radiomimetics, and X-rays com-pared, 227

mammalian cells after irradiation, 373 radiosensitivity, 350

Molecules, neutral reaction with ions, 107

Monkey, radiation sickness, first stage, 407

Multi-hit phenomenon, 66 Multiple ionization, 39 Muscles, oxygen tension, 288

striated, similarity between jS-irradia-tion and H2O2, 272

Mustard gas, biological activity, 219 cytotoxic action, 230

Mutagenisis, chemical, 217 properties of peroxides and oxygen,

235 purine or phosphate group reactions,

231 Mutations, agents causing, 221, 225

effect of dose-rate, mice, 258 enzyme induced, 277 frequency and faulty DNA synthesis

after irradiation, 354 from man-made sources of radiation,

496 gene, in vivo radiation effect, 83 mice after irradiation, 373 radiation induced, 257

effect of oxygen on rate, D. Tnelano-gaster, 285

mechanisms, 466 rates at specific loci in mice, X- and

/8-rays, 259 reduced incidence, irradiated E. coli somatic, 260

in ageing mammals, 440


Myleran, cytostatic action, 227, 229 D N A crosslinking, 235

Myoglobin, oxygen tension, 287

N

Negative ion formation, 104 Nephrosclerosis, death after life-span

shortening bv irradiation, mice. 440

Nervous system, action of ionizing radi-ations, 407-411

central, importance in reaction to irradiation, mammals, 394-402

radiosensitivity, 276 symptoms in radiation sickness, 506

Neuro-endocrine reaction to radiation, mammals, 394-402, 453

Neurospora, conidia, inactivation, 265 Neutrons, 6

brain tumour exposure, 11, 512 chromosome aberrations from, 89 effect on electrophoresis of colloidal

solutions, 153 irradiation, oxygen effects, 280 RBE for acute mammalian disorders,

92 Nitric oxide, sensitization to X-rays,

294 Nitrites, protective action, species

differences, 458 Nitrogen balance, after irradiation,

322-327 Nitrogen mustards, biological activitv,

220 D N A crosslinking, 234 genetic effects in Drosophila, 226

Nitrous oxide, reversal of O2 radiation effect, 295

Noradrenaline, X-ray protector, 289 Nucleases, activity changes after irra-

diation, 355-359 Nucleic acids, biosynthesis, effect of

irradiation, 347-352 post-irradiation treatment, mammals,

383 radiomimetic attacking groups, 221—

222 Nucleoproteins, cellular, structure, 198

target size for X-, y- and V-rays, 80 X-irradiation, 205

Nucleotides, phosphorylation, effect of irradiation, 354

Nucleus, changes initiating mitosis, 241 irradiation, amoeba, 264

by a-rays, 263-267 inactivation of insect eggs, 263

coloured by trypan blue after infusoria, 416

various plant and animal cells, 263-267

toxic agents, 218

O Oedema, occurrence after irradiation,

mammals, 414 Oestrogens, radioprotectors, mammals,

463 Oestrus cycle, irregularities after irra-

diation, mammals, 429 OH radicals, enzyme inactivation, 192

reactivity, 136 reduction by, 137

Ophryoglenidae, resistance to cyanides and X-rays, 303

Organisms, living, comparative radio-sensitivity 299-309

Organs, normal cell division in, 244 Ova, invertebrate, radiosensitivity, 305

production in mammals, 428 Oxalic acid, radiation decomposition,

effect of concentration, 142 Oxidation reactions, effect of ionizing

radiations, 138 Oxygen, action mechanism in enhanced

radiation damage, 292-296 combination with free radicals, 116

conc., effect of, on radio-sensitivity of normal and ascites tumour cells, 285, 286

on X-ray induced mutation rate, D. melanogaster, 285

consumption, effect of irradiation, rats, 312

increased after irradiation, ova and spermatozoa, 335

effect in radiobiology, 280 effect of, on a-rays, 280

on cross linking of polythene, 173 on inactivation of dry trypsin, 211 on irradiation of mouse ascites

tumours, 89


Oxygen, effect of, on peptide bond during protein irradiation, 190

on polymer irradiation, 172 on polystyrene irradiation, 174 on protein irradiation damage, 186 on radiosensitivity in mammals,

287-290 on RBE, of ascites cells, 94

of radiation, 91 enhanced radiation damage, 277 importance of conditions during and

after irradiation of chromosomes, 283

induced chromosome damage, 281 radiation damage, cysteamine pro-

tection, 281 reversal by inert gases, 294

radiochemical reaction with radicals, 293

radiomimetic properties, 235-236 role, in D N A irradiation, 200

in initial radiation damage, 51, 54, 284

in polymer degradation, 161 in reactions by ionizing radiations,

138 tension, in tissues and cells, mam-

mals, 287-289 in tumours, effect on radiosen-

sitivity, 291 relation to radiosensitivity, 287

Ozone, radiomimetic properties, 281

P Pair formation, 12, 20 Parabiosis, rats, survival of X-irradiated

partner, 486 Paramagnetic resonance, in irradiated

organic solids, 118 Paramecium, irradiation, 266 Parasites, radiation controls, 304 Parathyroid extract, radioprotector,

464 Pentobarbital, anticonvulsive action in

irradiation, 406 Peptidase, plasma activity increased

after irradiation, 342 Peptide bond, split during protein irra-

diation, 190 Peptides, radioprotectors, 463

Permeability, changes, after irradiation mammals, 414-417 role of hyaluronic acid, 417

intracellular, radiation induced, 341 recovery, 369

primary irradiation effects, 272, 275

Peroxides, formation, 97 radiomimetic properties, 235-236

Peroxy radical, formation, 116, 138 reactions, 148

Phaseolus aureus, rubidium uptake, re-covery after irradiation, 376

Pholas eggs, increased water permea-bility after y-irradiation, effect of C N ion, 415

Phosphatase, alkaline, increased activity spleen homogenates, after irra-diation, 339

Phosphoglyceraldehyde dehydrogenase, G value for inactivation, 191

Phospholipids, radiation breakdown, 276

Phosphorus, labelled, increased in-corporation after irradiation, ani-mals, 344

Phosphorylation, decreased after irra-diation, animal homogenates, 331

thymidine nucleotides, decreased by irradiation, 355

Photoelectric effect, 13 X-rays, 34

Photorestoration, u.v. irradiation le-sions, 370

Phycomyces, growth rate changes dur-ing and after irradiation, 376

Pituitary-adrenal overactivity after irra-diation, 320

Pituitary, irradiation, 390 Plants, absorption of 90Sr., 499

ascorbic acid content and radio-resistance, 319

higher, radiosensitivity, 305 nuclear volume and radiosensitivity,

relationship, 308 Poison theory, 96-97 Polyacrylamide, crosslinking, indirect

action, 162 Polyacrylic acid, crosslinking, indirect

action, 162

3 5


Polyethylene, crosslinking, effect of temp., 175

mechanism, 170 Polyisobutylene, irradiation degrada-

dation, 167 Polymers, see also Macromolecules

crosslinking, 107 gel formation and radiation dose,

159 mechanisms, 164

direct action, 165-175 indirect action, 160-164 irradiation, volatile products, 168

polydisperse products, 158 solid, classification, radiation be-

haviour, 166 Polymerization, acetylene, 103

and chain termination, 145 dose-rate dependent, 65 initiations, 136, 137

Polymethylacrylic acid, irradiation, de-gradation, 167

degradation protection, 176 main-chain scission, indirect action,

161 Polymethylmethacrylate, irradiation,

169 paramagnetic resonance in, 118

solid, protection against y-rays, 178 Polysaccharides, DNA, irradiation

changes, 207 Polystyrene, irradiation, effect of oxy-

gen, 174 sulphonic acid, degradation, 162

Polythene, crosslinking, effect of oxy-gen, 173

Polyuria, occurrence in irradiated mam-mals, 412

Polyvinyl alcohol, crosslinking, in-direct action, 162

Polyvinyl pyrollidone, crosslinking, in-direct action, 162

Population, human, biological effects of natural and artificial radiations, 501-505

Positron, 17 Potassium, increased blood content

after irradiation, 406 transport changes after irradiation,

415 Prolactin, stress inhibition, 387

Proteins, dry, X-ray effects, 180 irradiation, chemical changes, 189

-C-N- bond split, 190 metabolism after, 322-327

physical and chemical changes, by direct action, 179-187

by indirect action, 187-194 protection against direct action, 186

against X-rays, 111 radiomimetic attacking groups, 221-

222 —SH, role in radiobiochemistry,

328-332 —S—S—, radioprotection by cy-

steamine, 474 synthesis, effect of irradiation, 351 water content, effect on irradiation

damage, 185 Protons, 6, 10, 29 Pteris longifolia, spores, a-irradiation,

265 Purines, solutions, X-irradiation, 151 Pyrimidines, solutions, X-irradiation,

151

Q

Quantum, definition, 1

R Rabbit, c.n.s., action of ionizing radia-

tions, 409, 410 Rad, definition, 22, 23 Radiation, as an analytical tool, 208-212

background artificial and natural, biological effects, 501-505

chemical yield definition, 101 threshold effects, 73 units, 22

Radiation sickness, acute, symptoma-tology, man, 505

antibody titres after red cell injec-tion, 425

cortical adrenal insufficiency, 393 detrimental affect of cortisone, 401,

508 evaluation by testicular biopsies,

man, 429 first stages, monkey, mice, 407 treatment, man, 508-510


Radicals, see also Free radicals formation in water, 125-128 initial distribution and diffusion in

water by different radiations, 145 peroxy, formation, 116, 138

reactions, 148 radiochemical reaction with O2, 292 transfer, effect of CoA inactivation,

330 Radioactive waste, 497 Radioisotopes, 8, 23

environmental contamination, 498 Radiologists, life shortening and de-

layed effects of X-ray machines, 504

Radiomimetic chemicals, action of, 218 biological activity, comparison with

radiations, 224-229 chromosome fragmentation, 224-226 DNA degradation, 230-235 electrophilic character, 221, 223 functional groups, 219, 220, 221 mechanism of action, 229-235 nucleophilic, 219 reaction with amines, 221 reactive groups in bovine serum

albumin, 223 Radioprotection, and energy transfer,

110-114 by amines, mammals, 463 chemical, 50

techniques, 457 hydrogen transfer reaction, 176 mechanism, 465-470 of enzymes, 193 physical and chemical against X- and

y-rays, 457 physical, spleen, liver, bones etc.,

484-486 polymer, 175-179 S H - c o m p d s . , 329

mechanism, 470-473 free radical hypothesis, 475-477 mixed disulphide hypothesis, 473-475 various hypotheses, 469

potential functions, 460 Radiosensitizers, 477-479 Radon, 10, 123 Rats, malnutrition in radiation sickness,

427

Rats, parabiosis, survival of irradi-ated partner, 427

RBE, 276 definition, 91 different ionizing radiations, 90 for acute disorders, 92 for acute mammalian effects, 92 relation to LET, micro-organisms,

83-86 variation, chromosome aberration,

Tradescantia, 95 Reduction, by OH radical, 137 Relative biological effectiveness, see

RBE Rem, def., 22 Rep. def., 22, 23 Reproduction processes,restorationafter,

irradiation, 370-375 Reptiles, radiosensitivity, 300 Reserpine, radioprotector, 465 Respiration, action of ionizing radia-

tions, mammals, 411 seeds, 312

endogenous, liver, X-irradiation, rat, 340

Restoration, after irradiation, 369-383 of genetic and reproductive damage

370-375 of mammals, 377-383 of physiological injuries, bacteria

fungi, 376 Reticulocytes, disappearance after irra-

diation, mammals, 420 haemin synthesis, effect of irradiation,

dog, 337, 338 in man, 417

RNA biosynthesis, abscopal effects, 391 mechanism after irradiation, 351-355 radiosensitivity, 351 use in post-irradiation treatment,

mammals, 383 RNAases, activity changes after irra-

diation, 355 G value of inactivation, 191

Roentgen, definition, 21 Russula nigrans, enzyme and substrate

relationship, 273

S Scintillators, 114 Seeds, dry, radiosensitivity, 305

35


Seeds, radioresistance, 55 respiration, effect of irradiation, 312 restoration after irradiation damage,

372 Selection, irradiation, Drosophila, 260 Sex hormones, radioprotectors, 463 Single-hit concept and D37 dose, 63 Skin, damage after irradiation, mam-

mals, 429 effect of irradiated cells on neigh-

bouring cells, 451 grafts, irradiated mouse treated with

bone-marrow, 489 irradiation, formation of peroxides,

317 radioprotectors, local application, 468

Sodium, pump, block after irradiation, 406

retention, in man, 387, 389 in radiation sickness, mammals,

394 transport changes after irradiation,

mammals, 415 Soil, strontium 90 concn., 499 Solids, liquids and gases, radiation

reactions in, 107-110 Solutions, dilution effect, on radio-

chemical reactions, 47 Specific ionization, see L E T Spermatozoa, invertebrate, radiosen-

sitivity, 305 production in mammals, 428

Spermatogonia, radiosensitivity, 428 Spleen, irradiation, cytochrome c de-

ficiency after, 332 D N A activity, variations after total

irradiation, 348 DNAases, activity changes after irra-

diation, 356 homogenates, injection after irradi-

ation, 486-492 isologous and homologous, treatment

of irradiated mice, 488 physical radioprotection, 484—486 radiosensitivity, 330, 331

Splenectomy, effect on survival of irradiated rats, 391

Starvation, effect on radioresistance, yeast, 87

Sterility, after irradiation, mammals, 428

Stomach, gastric emptying, effect of irradiation, mammals, 413

Stopping power, definition, 20 Streptococcus faecalis, X-ray inactiva-

tion, 61 Stress, after X- or y- radiation, 388

and adaptation syndrome, 387-389 continuous, pituitary adrenal reac-

tion, 388 induced blood press, changes, rat, 396 neuro-endocrine reactions to irra-

diation, mammals, 394—402 Strontium 90, absorption by living

cells, 498, 499 biological cycle, 500 tumourogenesis in mice, 444

Substrates, enzyme, role in radiation damage, 273

Sugars, radioprotectors, 463 Sulphur compounds, radioprotection,

50 of enzymes, 193

Sulphur mustard, genetic effects in Drosophila, 226

Sulphydryl compds., radioprotection, '459-462

mechanism, 469 of dry enzymes, 210

Sulphydryl groups, importance in radio-biochemistry, 328-332

reaction rate with mustard gas, 222 X-irradiation, 150

Syndrome, acute radiation, man, 505 adaptation to stress, 387-389 intestinal, after irradiation, 328

Synkavit, treatment, increased X-irra-diation damage, rats, 478

Synovial fluid, X-ray sensitivity, 207

T

Target size, and mol. wt. relationship for biologically active compds., 209

calculation, 78, 80 Target theory, 77

application to in vivo radiation, 82 Taurine, excretion after irradiation,

men and animals, 323, 324 Temperature, effect of, on crosslinking

of polyethylene, 175


Temperature, effect, of on radiosens-itivity, biological systems, 52-55

incubation, effect on restoration, irradiated E. coli, 371

Terramycin, treatment in radiation sickness, 425

Testes, biopsies, evaluation of radiation damage, men, 429

Tetrahymena sp. enzyme activity after irradiation, 345

Thiotepa, biological activity, 229 Thiourea, crosslinking protection in

polyvinyl alcohol, 163 X-irradiation, 149

protection of serum albumin, 111 Threshold curves, X-rays, 71 Thrombocytes, see Blood platelets Thymine decreased in splenic DNA

after irradiation, 353 Thyroid, metabolism, effect of irradia-

tion, 313 Tissue, cell population changes after

irradiation, 354 extracts, enzyme activity, increased

after irradiation, 341-345 human, O2 tension, 287-289 normal and irradiated, interactions,

453 O2 consumption after irradiation, 312 restoration effects after irradiation,

454 Tissue culture, cells, irradiation, 266

survival curves after, 374 morphology changes, 244

a-Tocopherol, protection of irradiated fatty tissue, 317, 318

Toxins, irradiated tissue, abscopal effects, 452

Tradescantia, chromosome aberration, RBE variation, 95

pollen, radiosensitivity at different stages, 270

Transaminase, liver, increased activity after irradiation, rats, 334

serum, increased activity after irra-diation, 341, 342

Transamination, disturbances after irra-diation, 325

Trypan blue, cell penetration, effect of irradiation, 416

Trypsin, dry, effect of oxygen on in-activation, 211

G value inactivation, 191 Tryptophan, conversion to auxin, irra-

diated mung beans, 346 Tryptophane peroxidaseoxidase, liver,

increased activity after irradiation rats, 334

Tumours, cells, amino acid content, effect on radiosensitivity, 306

anoxic state, radioresistance, 290 ascites, glycolysis depressed after

irradiation, 315 radiation recovery with cysteine, 375 radiosensitivity, effect of O 2

conc., 285, 286 RBE effect of O2 , 94

development, effect of contact with irradiated cells, 452

lymphoid, radioresistance, 389 sensitivity to X-rays and nitrogen

mustard, 225 genesis, by localized irradiation, 442,

by 90Sr., 444 by X-irradiation, mechanism, 446

irradiation, effect of O2, mice, 89 O2 consumption after irradiation, 312 radiosensitivity related to O2 tension,

291 radiotherapy, 252 selective concn., of synkavit deriv-

atives, 478

U

Ultra-violet, andX-radiations, biological effects, 281

restoration of irradiation lesions by catalase, 369

Urethane, biological action, 218 genetic effects in Drosophila, 226

Uric acid, retention after irradiation, chicks, 325

Urinary excretion, after irradiation, animals, 323

of |8-aminoisobutyric acid, 508

V

Vertebrates, see Animals, Birds, Fish, Mammals, etc.


Vicia faba, chromosome aberrations by bis-epoxide, 225

radiosensitivity, radiations of diff-erent L E T , 93

Vietoreen dosimeter, 25 Virus, concentration and inactivation,

48 37% dose, 59 inactivation, RBE and L E T relation-

ship, 96 indirect action, 58

Vitamin A, absorption after irradiation, 320

Vitamins, antagonists, 218 radioprotection, 317, 318, 319

W Water, balance changes after irradia-

tion, various tissues, 328 bound, effect on paramagnetic reso-

nance of irradiated organic solids 118

ionization in, 46 cellular content and radiosensitivity,

55 D N A content, effect on irradiation

damage, 203 effect of ionizing radiations, 122 free radical diffusion formed by

a-particle tracks, 128 in protein, effect on irradiation, 185 irradiation, by a-rays, 123, 128, 145

formation of H2O2, 140 yields of radicals and molecular

products, 134 radiolysis, primary products, 124-136 role of excited molecules, 128-130 vapour, free radical formation, 125

Wigner release, 110 Wilson cloud chamber, 31

X Xenon, reduced O2 dependent radio-

sensitivity, 294, 296 X-rays, 6, 7

and a-rays, differences, 124 relative biological effectiveness, 90

and H2O2 comp. effect on barley seeds, 236

chromosome aberrations, 89 chromosome breaks, 224—226

X-rays, Tradescantia pollen, 270 direct and indirect action in aqueous

soln., 59 dose-mortality curve, mice, 71 effects, blood changes, 417-421

cancer and leukaemia induction, 442-447

cell sensitivity, in vivo and in vitro, 252

comparison with radiomimetics, 224-229

electrolyte changes in animals, 327 enzyme activity increased in plas-

ma, 341-343 enzyme and synthetic activity,

increased, 332-345 facts and interpretation, mam-

mals, 392-394 first and second reactions, mam-

mals, 394-402 genetic, 256-261

in Drosophila, 226 restoration of damage, 370-375

histamine liberation, mammals, 411 importance of SH-groups, 328-332 incorporation of labelled tracers

increased, 343-345 interaction between cells and

tissues, 451-455 life span shortened, mammals,

436-442 mitosis suppression, Tradescantia

pollen grains, 247 mutation rates, at specific loci,

mice, 259 effect of O2 conc., in D. melano-gaster, 285

on Acetbularia mediterranea, 265 on amino acids, 148-151 on barley seed, 69 on carbohydrate metabolism, 313—

317 on circulation and respiration, 411 on citric acid formation, rats, 316 on colloids, 152, 153 on dry protein, 180 on embryos in utero, mammals, 447 on fat metabolism, 317-322 on fibroblasts, changes in [mitolic

index, 246


X-rays, effects, on glycine and alanine solutions, 149

on human serum albumin, effect of pH, 188

on invertebrates, 301 on nervous system, 407-411 on nucleases, activity changes, 355—

359 on nucleic acid biosynthesis, 347-

352 on nucleoproteins, 205 on O2 consumption, 312 permeability changes, mammals,

414-417 on protein metabolism, 322-327 on solutes in aqueous soln., 124 on synovia, 208 on aurine excretion, 323, 324

energy loss, 12-19 formation of OH radicals, 136 induced reactions, limiting concen-

trations, 143-144 irradiation, at reduced body temp.,

mice survival, 289 death, cause in mammals, 430 enzyme systems, in vivo radio-

resistance, 345-347 HeLa cell survival after, 250

injection of spleen or bone-marrow homogenates after, 486-492

injury and recovery, 370 nuclear and cytoplasmic, various

animal and plant cells, 263-267 oxygen effects during, 280 protection, chemical, 458-465

mechanism of action, 465-472 techniques, 457

physical, of spleen, liver, bones, etc., 484-486

protection against insect pests, 303-304

X-ray irradiation recoveryin mammals, 377-383 secondary disease, 490 sublethal, production of radio-

resistance, 389 syndrome in man, 505-510 substances intensifying effects, 477

-479 treatment with isologous and hete-

rologous bone-marrow, 490 tyrosine decarboxylase, inactiva-

tion, 61 lethal dose for various animals, 300 radiologists, life shortening and de-

layed effects, 504 radiosensitivity effect on, of inert

gases, 295 of oxygen, 281-287

relation to O2 tension, 287 sensitization to, by NO, 294 specific ionization from, 34-39 target size for nucleoprotein, 80 threshold curves, 71 water decomposition, 124

molecular products, 130-132 yields of radicals and molecular

products, 134

Y

Yeast, bakers, increased O2 consump-tion and CO2 production after irradiation, 335

cells, inactivation, 265 radioresistance, relation to ploidy,

307-308 radiosensitivity, 302 starvation radioresistance, 87

Z Zeta potential, change, effect X-rays on

colloids, 152

AUTHOR INDEX ABBATT, J . D . 4 3 2 A B D E R H A L D E N J R . 3 3 5 , 3 6 1 , 4 0 3 ABERCROMBIE, M . 2 4 4 , 2 6 1 ABRAHAMSON, S . 3 8 5 ACKERMAN, I . B . 4 3 1 ADAMS, D . 3 8 5 ADAMSON, D . M . 3 6 0 ADLER, H . I . 3 1 0 A E B I , H . 3 6 4 , 3 6 5 , 3 6 6 , 4 8 0 ALBAUM, H . G . 3 6 2 , 3 6 5 ALBERT, M . D . 3 8 5 ALDERMAN, I . M . 4 3 1 ALEKSEYEVA, O . G . 4 3 5 ALEXANDER, M . L . 9 8 ALEXANDER, P . 5 1 , 6 2 , 1 1 7 , 1 2 0 , 1 2 1 ,

1 5 5 , 1 6 1 , 1 6 2 , 1 6 3 , 1 7 1 , 1 7 2 , 1 7 4 , 1 8 0 , 1 8 1 , 1 8 4 , 1 8 6 , 2 0 4 , 2 1 1 , 2 1 2 , 2 1 3 , 2 1 4 , 2 1 5 , 2 2 0 , 2 2 3 , 2 3 1 , 2 3 3 , 2 3 7 , 2 3 8 , 2 7 9 , 2 9 4 , 2 9 6 , 2 9 7 , 3 1 0 , 3 8 1 , 3 8 5 , 4 4 0 , 4 4 9 , 4 5 6 , 4 6 6 , 4 7 9 , 4 8 0 , 4 8 2 , 4 8 3

ALLEGRETTI, N . 4 0 5 A L L E N , E . 1 8 0 , 2 1 3 A L L E N , J . C . 4 3 0

A L L E N , O . A . 1 2 5 , 1 3 0 , 1 3 3 , 1 5 4 , 1 5 5 A L P E R , T . 1 5 5 , 2 9 7 , 3 1 9 , 3 6 1 , 3 8 4 ALTENBURG, L . S . 2 3 8 A L T M A N , K . I . 3 3 5 , 3 6 2 , 3 6 4 , 3 6 5 , 3 6 7 A M A N D , G . S . S T 3 6 3 A N C E L , P . 3 , 4 , 3 3 3 , 3 6 2 ANDEREGG, J . W . 3 6 6 ANDERSON, A . J R . 3 6 5 ANDERSON, D . R . 3 6 4 ANDERSON, E . H . 2 9 6 ANDERSON, N . G . 2 0 6 , 2 1 5 ANDERSON, R . S . 1 8 0 , 2 1 3 ANDREWS, H . C . 4 3 2 A N G E R , H . O . 3 9 , 4 3 A N S O N , M C L . 2 1 4 APPLEMANS, F . 2 7 8 APPLEYARD, R . K . 2 1 4 ARBUSOW , G . J . 4 8 3 A R D , W . B . 2 1 3 A R I E N T , M . 3 6 7 ARMSTRONG, W . D . 3 2 8 , 3 6 6 , 4 3 2

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493 M A I S I N , J . 3 8 5 , 3 9 0 , 4 0 4 , 4 0 5 , 4 3 1 ,

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2 9 7 , 3 1 0 , 3 7 0 , 3 8 4 , 4 3 4 , 4 7 1 , 4 8 2 STEACIE, E . W . R . 1 0 3 , 1 2 0 STEADMAN, L . T . 3 6 0 , 3 6 1 STEARNER, S . P . 3 6 1 , 3 6 4 , 3 8 4 , 3 8 5 STEFFENSEN, D . 2 7 8 STEGGERDA, F . 4 3 3 S T E I N , G . 1 2 1 STENSTROM, K . W . 4 5 5 STERGGERDA, F . R . 4 3 1 STERNBERG, S . S . 2 3 8 STEVENS, C . M . 2 3 7 STEVENSON, D . P . 1 2 0 , 2 1 3 S T O C K , C . C . 2 3 7

S T O C K E N , L . A . 2 0 6 , 2 1 5 , 2 4 9 , 2 6 1 , 2 6 2 , 3 1 3 , 3 1 4 , 3 4 7 , 3 5 2 , 3 5 4 , 3 5 9 , 3 6 6 , 3 6 7

S T O L E N , H . N . 2 6 2 S T O N E , W . S . 2 9 8 S T O N E R , R . D . 4 3 4 STORER, J . B . 9 8 , 2 9 7 , 4 9 3 STOREY, R . H . 4 3 1 S T O R N E R , R . 4 3 3 S T R A N G , V . G . 4 8 0 STRAUBE, R . L . 4 0 3 , 4 7 9 , 4 9 2 STRAUCH, L . 3 6 6 S T R U C K M A N N , R . 4 8 1 S T U H L M A N N , H . 2 9 1 S U L L I V A N , M . F . 3 6 4 S U L L I V A N , R . L . 3 0 3 , 3 0 9 S U P P L E E , H . 3 6 0 , 3 6 2 , 3 6 7 SUSSMAN, A . S . 2 7 3 , 2 7 8 , 3 4 0 , 3 6 2 S U T T O N , G . 3 6 4 S U T T O N , H . 3 7 3 , 3 7 4 , 3 8 5 , 3 8 6 S U T T O N , H . C . 1 4 6 , 1 5 5 , 1 9 2 , 2 1 4 SVEDBERG, T . 1 8 0 , 2 1 3 S W A M I N A T H A N , M . S . 3 1 0 S W A N S O N , A . A . 3 6 5 S W A N S O N , C . P . 2 8 4 , 2 9 7 SWERCBORT, A . 2 3 8 S W I F T , H . H . 3 0 8 , 3 0 9 S W I F T , M . N . 5 , 4 0 3 , 4 3 2 S W O R S K I , T . J . 1 5 5 S Y M M S , M . C . R . 2 1 3 SYVERTON, J . T . 4 0 2 SZABO, G . 4 3 4 SZILARD, L . 4 4 0 , 4 4 9 SZYBALSKI, W . 4 8 3


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T A Y L O R , H . S . 4 0 , 4 3 , 1 0 2 , 1 2 0

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T H O M S O N , J. F . 3 1 0 , 3 6 0 , 3 6 1 , 4 0 1 , 4 0 3 , 4 0 4 , 4 3 3

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261 T R O W E L L , O . A . 2 3 7 , 2 6 2 , 2 7 8 , 2 8 7 ,

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5 1 2

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Y A , I . 3 6 8 YAGODA, H . 4 3 YAMAZAKI, J . 4 3 2 Y O C K E Y , H . P . 4 4 0 , 4 4 9 Y O G A , Y . 4 3 2 YOUDALE, T . 4 8 0 Y U O N G - T C H E L T C H O E , 3 6 7

ZAALBERG, O . B . 4 7 9 ZADORY, E . 4 3 4 Z A H L , P . A . 3 6 2 ZAJDELA, F . 3 6 7 Z A M E N H O F , S . 2 3 1 , 2 3 8 Z I M M E R , E . C . 2 1 6 Z I M M E R , K . O . 1 2 1 Z I N S , G . 3 6 7

Z I R K L E , R . E . 9 9 , 2 6 5 , 2 7 7 , 2 7 8 , 4 9 2 ZUIDERVELD, J . 2 1 5 Z U N Z , E . 4 0 4 Z U P P I N G E R , A . 3 6 4 , 3 6 5 , 3 6 6 , 4 5 2 ,

4 5 6 , 4 8 0 Z W E I F A C H , B . W . 4 0 3

C U N D A M E N T A L S O F RADIOBIOLOGY, the first

edition of which, published in 1955, was widely acclaimed for its authority and clarity and was translated into three other lan-guages, surveys the many diverse aspects of radiobiology. This new edition is in most respects a new book, for several chapters have been added.

The subject is presented as a coherent whole with, however, certain facts emphasized whose development may advance the subject. The authors indicate which in their opinion are the most acceptable hypotheses at the present time. This volume will be welcomed by all who seek a clear exposition of the fundamentals of the subject and will be especially useful for post-graduate courses in radio-biology, for training of medical radiotherapists, and for health physicists.

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Bacq Alexander-fundamentals of Radio Biology