Course : PGPathshala-Biophysics Paper 08 : Medical Physics Module : M 32: Radiation Biology Content Writer: Dr. K. Thayalan, Dr. Kamakshi Memorial Hospital, Chennai-600100.

Quadrant I

1. Introduction

Radiobiology is a scientific discipline that studies the effects of ionizing radiation on living

systems, including cells, normal tissues and malignant cells. It helps to understand the

sequence of events and its nature after the exposure of ionizing radiation. Basically, it is

concerned about biological damage, modification and its repair. The radiobiological

studies contribute to the development of medicine, especially radiotherapy in many

ways.

Objectives

To understand radiobiological principle, which are very much essential, not only

to predict outcome, but also to use effectively a given radiation technique or

procedure.

To understand the radiation interactions with tissue and cells,

To understand radiation damage of DNA

To study survival curves and its modifying agents

To know the biological effects of radiation

2. Interaction of radiation with cell

Radiation can damage cell and result in biological effects in a multi cellular

organism. Therefore, the study of the radiation effects at cellular level is more useful to

know about the radiation damage. The time interval between the radiation exposure and

manifestation of biological effects can be divided into 3 stages namely (i) physical stage,

(ii) chemical stage, and (iii) biological stage.

2.1.Sequence of radiation events

(i).Physical stage

The physical stage refers to the interaction of charged particles and atoms and

molecules of tissues. In this stage, the bio molecules in the environment absorb the

radiation energy and undergo ionization and excitation and release electrons. These

electrons are called secondary electrons and they transfer energy to the surrounding by

further excitation, ionization and thermal heating in the medium. Its duration is about 10-

7 s and the deposition of energy is rapid and random.

(ii).Chemical stage

In the chemical stage, the damaged atoms and molecules further reacts with the

cellular components through chemical reactions. The exposed bio molecules rearrange

themselves, which results in formation of primary lesions in them. Primary lesions are

transformed into bio radicals, resulting molecular alterations. Bio radicals can also be

formed with indirect interaction of radicals with bio molecules. The structural changes

includes (i) hydrogen bond breakage, (ii) molecular degradation and (iii) inter and intra

molecular cross linking. The duration of the above event is about 10-10 s.

(iii).Biological stage

The biological stage starts with enzymatic reactions, which act on the residual

chemical damage. Majority of the lesions are repaired and lesions that are not repaired

result in cellular death or mutations in cells. Cell death may lead to organ death that

appears as clinical changes. The biological effect at cellular level includes lethality,

mitotic inhibition, division delay, chromosome aberration and induction of mutations.

These effects can create radiation sickness, resulting in delayed somatic effects in

humans.

Cell may take time to die and may undergo mitotic division before death. The

death of stem cells appears as early effects in normal tissue. After the cell killing,

compensatory cell proliferation occurs both in normal and tumour cells. At later time late

reactions appear in normal tissues. Mutation can occur in the (i) germ cells and (ii)

somatic cells. The mutation in germ cell may result in hereditary effects, whereas

cancer induction is the outcome in somatic cells. The time schedule for biological effects

is few minutes to several years.

2.2. Direct and indirect action

Biological effects resulting from the direct interaction of radiation with the target

sites is called the direct action. There are critical sites or targets within the cells which

must be damaged in order to kill the cell. DNA, RNA and proteins are the common

critical targets for radiation exposure. In a direct action, the radiation interacts directly

with such critical sites and creates biological effects (Fig.1). The radiation ionizes or

excites the molecules such as DNA, RNA and protein directly. It is a dominant process

with high LET radiations such as alpha, neutrons etc.

It involves rupture of cell membrane and breaking of chromosome structure,

resulting in DNA strand breaks. The fragments of chromosomes produced in a direct

interaction, can join together to form chromosomes with abnormal structures. This is

known as chromosomal aberration. The frequency of chromosomal aberrations

increases with radiation dose and hence the magnitude of aberrations is a biological

indicator of radiation dose absorbed in human body. Chromosomal aberration analysis

(CAA) is useful in determining the radiation dose received by a person who is

accidentally exposed to high radiation dose (>100 mGy).

Fig. 1.Direct and indirect action of radiation on a DNA molecule.

Alternatively, the radiation can interact with other molecules (e.g. water) and

forms free radicals. The interaction of free radicals with critical sites may also result in

biological effects, which is called indirect action. For example, radiation interacts with

oxygen and water molecules present in the cell. These interactions produce a large

number of free radicals, which are atoms or molecules or ions with an unpaired electron

and hence are highly reactive. Since electrons are spinning, a pair of electron spins

both clockwise and anticlockwise, giving stability. Unpaired electron spins in one

direction and is unstable. Free radical has high degree of chemical reactivity and

interacts strongly with bio molecules. This is a major source of radiation damage.

Human body tissue is composed of 80% water, and the major interaction is indirect

action (66%) through water. The effect of x and gamma rays in macromolecules of living

system are mainly due to indirect interactions.

2.3. Radiation damage to DNA

DNA is the largest and most important molecule for radiation damage. Its

damage gives rise to cell killing, carcinogenesis, and mutation. The radiation induced

lesions in the DNA can be repaired successfully.

Radiation exposure to DNA gives two major effects namely (i) cross linking and

(ii) strand break (Fig.2). Cross linking may be protein-protein cross link, DNA-protein

cross link, intra strand cross link and inter strand cross link. Intra strand cross linking is

one in which cross linking occurs between bases on the same stand, e.g. formation of

adjacent thymine dimers (TT).Dimerization reduces the distance between the bases

and results in distortion (kink) of sugar–phosphate back bone. The rupture of hydrogen

bond may lead to irreversible changes in the secondary and tertiary structure of DNA

molecule, which may compromise genetic transcription and translation.

Radiation exposure also causes DNA strand break due to loss of viscosity in

DNA solutions, in addition to base change, and the DNA is degraded into smaller

fragments. As a result, there will be a decrease in molecular weight of DNA. The strand

breaks may be single strand break or double strand break. If the break is located on one

of the strand it is referred as single strand break (SSB). If two breaks are located on

opposite strands, separated by 5 bases, then it is called double strand break (DSB).

The SSB between sugar and the phosphate can rejoin, if it is not separated, but takes a

longer time. Presence of oxygen may cause peroxidation of base that prevents

rejoining. The DSB are basically genotoxic lesions that can result in chromosome

alterations. This may activate oncogenes, inactivation of tumour suppression genes or

loss of heterogeneity, resulting carcinogenesis. The SSB are mostly caused by OH

radical and easily repaired compared to DSB.

Fig.2.Radiation damage of DNA: (a) Single strand break, (b) double strand break,

(c) cross linking, and (d) rung breakage

The DSB is the result of sparse ionization pattern of the low LET radiation. A X-

ray dose of 1 Gy, may disturb the mitotic capability in 50 % of the exposed cells. All

ionizing radiations are capable of producing DSB and complex DNA damages. In low

LET radiation, about ¼ of the dose is deposited in tissue via low energy secondary

electrons (0.1-5 keV).These low energy electrons produce dense ionization tracks over

a short range, equal to the diameter of double helix strand. This may result in complex

DNA damages that are less likely to be repaired.

Though the amount of DSB lesions caused by the radiation is more, but the

number giving rise to cell kill is small. The dose of ionising radiation that gives one lethal

event per cell and leave 37% of the viable cell is called D0. The D0 value for low LET X-

ray lies between 1-2 Gy dose, which is sufficient to cause about 1000 base damages,

1000 SSB and 40 DSB per cell. SSB is of little importance, since its damage is mostly

repaired by taking a template from the opposite strand. If incorrect repair takes place in

SSB, it may lead to mutation. If both strands are broken, and are well separated, it can

also be repaired, since the two breaks are handled as two single strand breaks.

DSB is the most important damage in DNA, after radiation exposure. The

interaction of two DSB may lead to cell killing, carcinogenesis and mutation. There are

many kinds of DSB, depending upon the distance between the break and kinds of end

point. The DSB yield out 0.04 times that of SSB and it is induced linearly with dose.

Mostly DSBs are caused by single track of ionizing radiation. DSB result in cleavage of

chromatin into two pieces and is the critical lesion, responsible for cell kill. Experimental

studies reveal that DSB produced initially correlate with radiosensitivity and survival at

low dose. Whereas unrepaired or miss repaired DSB correlate with survival at higher

doses. High LET radiations are capable of producing complex DSB lesions. The

clusters of ionization and excitations that take place at the end of the secondary

electrons tracks are capable of producing multiple lesions within 20 nm range, resulting

cell death.

The DNA damage is a normal event in a cell’s life. Apart from radiation, there are

many more agents that may cause DNA damage. However, the spontaneous mutation

rate is less due to efficient repair mechanism present in the cell. The extent of DNA

damage depends upon the repair mechanism present in the organism. Eukaryotic cells

are equipped with ability to repair DSB, whereas organisms with prokaryotic cells are

not equipped. A single DSB is lethal to bacteria, whereas about 60 DSB are required to

kill a mammalian cell. Similarly, few SSBs are lethal to viruses and micro organisms,

whereas several hundred SSB are required to kill a mammalian cell. The above

difference is due to repair capacity between different organisms.

2.4. Radiation and Chromosome damage

Irradiation can damage chromosomes and delay the cell’s entry into mitosis. This

delay is dose dependent. If the cells are irradiated during the inter phase, they begin to

divide and undergo aberrations. Chromosome aberration refers to their appearance in

the first metaphase after irradiation. The stable aberrations may be carried out through

number of cell divisions, generally called chromosome aberrations. Unstable

aberrations may lead to cell death. If cells are exposed to radiation, DSB occurs in

chromosomes. The broken ends are sticky due to their unpaired bases. These broken

ends may (i) join with their original chromosome, (ii) fail to rejoin, that leads to

aberrations, or (iii) rejoin with other broken ends.

These aberrations may be chromatid or chromosome aberrations. Chromatid

aberrations are due to irradiation of cells, while they are in late interphase (G2). In this,

one arm of the sister chromatid is broken. The DNA has already doubled and the

chromosomes consist of two strands of chromatin, during irradiation. If the cells are

irradiated, while they are in early inter phase (G1) and if unrepaired, that leads to

chromosome aberrations. In this case, the chromosome is not duplicated, and the

damage is a SSB of chromatin. During the S phase an identical strand is synthesized.

This is visible in the mitosis, as identical breaks in the pair of chromatin strands.

Irradiation of cells, while they are in S phase may bring both types of aberrations.

The types of chromosome aberrations that are lethal to cells are (i) dicentric, (ii)

ring, and (iii) anaphase bridge. The first two are chromosome aberrations, whereas

anaphase bridge is chromatid aberration. The types of aberrations that are not lethal to

cells are (iv) symmetric translocation and (v) small interstitial deletion (Fig.3).They are

basically arises from DSB, remains intact with two chromatids and centromere, and

carry out mitosis normally. However, there may be some loss of genetic information,

that may be passed on to the next generations.

(i) Dicentric

Two separate chromosomes undergo breaks at the early inter phase. If the

broken ends are close to each other, they get united illegitimately, and replicated during

the S phase. After the synthesis, a pair of sister chromatids appears with two

centromeres, which is called dicentric. It is an unstable aberration lethal to the cell, not

passed on to the progeny. It decreases slowly over a period of time after radiation

exposure. Since spindle has two centromeres to grab during the meta phase, it get

disturbed while pulling the chromatids during the anaphase.

(ii) Ring

Radiation induces breaks in each arm of a single chromatid, at the early part of

the cell cycle. The breaks may rejoin and form ring and fragments. During synthesis, it

gets replicated and appears as overlapping rings and acentric fragments without

centromere. Since the ring or the acentric fragment do not have properly constructed

centromere for the spindle to attach, chromosomes unable to divide properly. The

fragments will be lost during next mitosis.

Fig.3.Radiation induced chromosome damage: (a) Dicentric, (b) ring

formation,(c) anaphase bridge, (d) symmetric translocation and (e) small

interstitial deletion.

3.CELL SURVIVAL CURVES

3.1.Introduction

Radiation exposure creates two types of population namely (i) dead cells and (ii)

surviving cells. Dead cells are the one which have lost the capacity to divide indefinitely,

i.e. loss of reproductive integrity. Cells which are physically present, synthesize DNA or

make proteins but not able to divide during mitosis are also said to be dead. Cells that

have retained its reproductive capacity and are able to divide indefinitely are called

survived cells. These cells can produce colony and hence said to be cologenic cells. A

cell death in differentiated cells leads to loss of organ function, e.g. nerve, muscle etc.

The cell death in a proliferating cell (stem cell) refers loss of reproductive capacity, e.g.

blood cells, epithelium, etc. Cells may die in two ways: (i) they die when they attempt to

divide, called mitotic death, and (ii) some cells undergo a programmed death called

apoptosis.

The relation between the radiation dose and the number of surviving cells that

form colonies can be plotted in a curve, known as survival curve. The survival curve is a

measure of reproductive death. To conduct the experiment, the cells are seeded in a

petri dish (in vitro) and exposed to varying dose of radiation. Usually multiple petri

dishes with cells are planned. One two dishes are used as control and no irradiation is

given. They are allowed to grow colonies and the same is counted. The irradiated and

the control cells are incubated for same duration of time. Each cell is assumed to make

their respective colony. The fraction of surviving cells are normalised by the fraction of

cells that survive with no radiation exposure.

A graph is plotted between the radiation dose and the survival fraction. The

survival curve of the Mammalian cells are exponential in nature and hence, plot is

usually drawn between of log of survival fraction (Y-axis) vs radiation dose (X-

axis).These curves are useful to understand radiation damage and their nature. The

shape of the curve tells us the radio sensitivity, repair and recovery ability of the cell.

The shape of the cell survival curve gets altered, if the exposure conditions are

changed, during or after the radiation exposure. To quantify the cells the parameters

such as (i) plating efficiency and (ii) survival frcation is used.

3.2. Survival fraction and plating efficiency

The surviving fraction (SF) is given by the relation,

𝑆𝐹 =𝐶𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

𝐶𝑒𝑙𝑙𝑠 𝑠𝑒𝑒𝑑𝑒𝑑 ×𝑃𝐸

100

− − − − − (1)

Where, PE is the plating efficiency, which refers to the percentage of seeded cells that

survive to form colonies under control conditions:

𝑃𝐸 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑠𝑒𝑒𝑑𝑒𝑑× 100 − − − − − (2)

The PE can be obtained with invitro studies as follows. The cells are prepared in

a culture vessel (dish) with trypsin and the number of cells are counted by an electronic

or hemocytometer. The dish is incubated for 1-2 weeks, during the period the cells

divide and forms colonies. Now, the number of colonies is again counted using the

above formula, to get the PE. The number of colonies are always lesser than the

number of seeded cells due to growth medium, uncertainties of counting and trauma of

trypsinization and handling.

3.3. Survival curve

In vitro studies show that survival curve of each cell line after radiation exposure

can be plotted in a semi-log graph with the surviving fraction in the Y axis and the dose

in the X axis (Fig.4a & b).

Fig.4. Shape of the survival curve for mammalian cells. Survival fraction is plotted

in (a) linear scale and (b) logarithmic scale with dose on a linear scale.

The curve 4a contains two regions namely, region A at low doses and region B at

low survivals, both are important for radiotherapy. However, the curve may not be useful

clinically as it is not giving the true picture of cell survival. In radiotherapy, we use low

dose per fraction (1-3 Gy per fraction) and tumour cure require cell survival of about 10-8

cells. Hence, it is meaningful to plot the curve as shown in 4b, in a semi log scale. This

curve demonstrates the exponential behaviour of region A as straight line. It also

magnifies the region B, which represents the low survival levels of cell (10-8), visualize

tumour cure.

4. BIOLOGICAL EFFECTS OF RADIATION

4.1.Introduction

Radiation exposure can destroy cells ability to reproduce, but may not disturb its

function. Then, there is a chance that cell may not complete the cell cycle and undergo

reproductive death. These cells do not reproduce, but continue its function such as

production of protein, hormones etc. The reproductive death is not related with cancer.

In general the biological damage depends upon the mitotic index of the cell. This means

that cell with higher replication rate and high turnover are more sensitive and exhibits

their damage immediately. The organ response depends upon the mixture of cells by

which it is made up of.

Biological effect of radiation is generally divided into stochastic and deterministic

effects (Fig.5). A stochastic effect is one in which the probability of effect occurring,

increases with dose, rather than its severity, e.g. induction of cancer. A radiation dose of

5 Gy has more probability to induce cancer than 1 Gy dose, even though the severity is

same in both cases. It has no threshold dose; even a small radiation has ability to cause

stochastic effect. The risk increases with increase of dose and there is no dose at which

the risk is zero. Stochastic effects are the principle health risk from low level radiation;

hence it is important in medical exposures of patients and occupational workers.

(A) (B)

Fig.5.The (A) stochastic effect and (B) deterministic effect of radiation

4.2. Stochastic effect

Stochastic effect is observed only in animal experiments. The dose effect

relationship can be studied only in a group of human population. The dose effect

relationship is linear, but its effect <100 mSv (low dose range) is not verified. The

observed dose response at higher doses is extrapolated for low doses. This is

applicable for radiation protection and safety purpose. There is no method to identify

the appearance of effect in an individual. The increase in occurrence of effect can be

proved only by epidemiological studies. The stochastic effects are further classified as

(i) radiation carcinogenesis and (ii) hereditary effects. The stochastic effects usually

appear as late effects

4.3. Deterministic effect

A deterministic effect is one in which the severity of the effect increases with

dose, due to degenerative changes in tissues. It has a threshold dose, below which no

radiation effects is seen. Deterministic effects always occur at high radiation doses.

The threshold dose is higher than the doses from natural radiation or from the

occupational exposure at normal operations. Skin erythema, cataract and hematopoietic

damages fall under this category. Deterministic effects are often known as normal tissue

reactions.

There is a time interval between irradiation and the occurrence of radiation

effects, called latent period. It is the indicator of tissue radiosensitivity and it is

inversely proportional to the dose. This means that lower the dose, and greater the

latent period and appearance the effect. There are two latent periods correspondingly

for the appearance of early and late effects. Of course there is no difference in the

radiation damage in terms of early and late effects, but only difference is the time of

appearance of effect. The acute early effects appear within a month after radiation

exposure, The early effects are further divided into (i) radiation injury in individual

organs or normal tissue reactions and (ii) acute radiation syndrome. The former is the

result of partial body exposure, including radiotherapy treatments and later is the

outcome of whole body exposure, due to accidents. The late effects appear within

months or year after radiation exposure that includes (i) radiation dermatitis,(ii) cataract,

and (iii) teratogenic effects on embryo and fetus.

The basic difference between stochastic and deterministic arises from the

interaction. In the former the radiation interacts with DNA, at the cellular level. This

results in mutation which can be passed to the next generation, leading genetic effects

or induction of cancer. In the later, the interaction is in a group of cells that forms the

tissue or organs. This will end up with loss of tissue function.

Fig.6.The various types of radiation effects after acute and chronic

radiation effects.

Summary

The radiobiological studies contribute to the development of medicine, especially

radiology and radiotherapy. Understanding the interaction of radiation of radiation with

cell or DNA and the resulting damage is very important. Cell survival curves give an idea

about the number of cells (%) remaining after the exposure of ionizing radiation.

Biological effects appear as deterministic and stochastic effects. The Deterministic

effects are possible only at high level radiation, hence, unlikely in a hospital setup.

Stochastic effects are possible in low level radiation, hence, likely to occur at hospital

workers. Therefore, the aim of radiation safety policy is to prevent the deterministic

effects and minimize the stochastic effects. All efforts must be taken to keep the radiation

levels as low as reasonably achievable (ALARA).