INT. J. RADIAT. BIOL ., 1973, VOL . 24, NO . 3, 221-228

Energy requirements for damaging DNA moleculesf. Energy transfer from metastable states of excited gases

CHRISTINE LUCKE-HUHLE and HORST JUNGInstitut fur Strahlenbiologie, Kernforschungszentrum Karlsruhe,75 Karlsruhe, Germany

(Received 29 January 1973 : accepted 23 February 1973)

DNA of bacteriophage (X174 freeze-dried in extremely thin layers was exposedto metastably-excited gases . The loss of infectivity and the frequency of strandbreaks was measured in the spheroplast system and by centrifugation in analkaline sucrose gradient, respectively . The excitation energies of the variousgases used amount to 4 .3 eV for H 2*, 6 . 2 eV for N 2*, 11 . 6 eV for Ar*, and19 .8 eV for He* . These energies are transferred to the DNA molecules by` collisions of the second kind ' . The experiments show that 76 . 5 ± 9 . 4 per centof the inactivated DNA molecules carry at least one strand break, a value foundnot to depend on the amount of energy primarily transferred . Thus, the actionof excited gases is entirely different from the effects of U .V. quanta and slowelectrons even if identical energies are transferred in all cases .

1 . IntroductionWhen exposed to ionizing radiation in the dry state, enzymes, DNA, RNA

and viruses containing single-stranded nucleic acid are inactivated accordingto exponential kinetics . From the 37 per cent dose (D37) the volume of theradiosensitive target may be calculated using the well-known mathematicalexpressions of target theory . The results obtained generally show a rathergood agreement between the molecular weight of the radiosensitive target andthe molecular weight of the irradiated macromolecules or that of the single-stranded genome of the irradiated viruses, respectively (see Dertinger and Jung1970, equation 5 .5, figure 28, and table 15) . These calculations are usuallymade assuming the mean energy expenditure per primary absorption event tobe 60 eV. Consequently, such calculations mean essentially that biomoleculesor viruses containing single-stranded nucleic acid are inactivated with a proba-bility or ' killing-efficiency ' near to unity after ` receiving ' an average amount ofenergy of 60 eV . The value of 60 eV has been obtained from cloud-chamberphotographs by analysing the frequency distribution of ion pairs per ion cluster(Ore and Larsen 1964, Ore 1971) and, in good agreement, by measuring theenergy loss of electrons in thin foils of plastic material (Rauth and Simpson1964) or DNA (Johnson and Rymer 1967) . Therefore, this figure simplyreflects the physics of radiation absorption and gives no indication of the energynecessary to damage biological macromolecules .

To obtain some information about the energy required for damaging bio-molecules, we started a series of experiments in which amounts of energy between4 and 20 eV were transferred to DNA, and the resulting structural and functionalchanges were analysed . In the energy range under consideration, at least twodistinct ways of transferring energy exist : (1) Irradiation with U.V. light ofR.B . Q

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C. Liicke-Huhle and H . Jung

extremely short wave-length, so-called vacuum U . V., and (2) exposure of thespecimens to gases excited to metastable states . In an earlier publication wedescribed some preliminary studies on the action of vacuum U .V. on dry DNA(Wirths and Jung 1972) . A series of experiments performed with excited gasesreported here also aims at a better understanding of the energy requirementsinvolved in biological inactivation and strand breakage in the single-strandedDNA of bacteriophage DX174 .

2 . Materials and methods2.1 . Production of metastably-excited gases

Excited gases were generated in a gas-flow system as used earlier by Jungand Kirzinger (1968) . Its main parts are shown schematically in figure 1 .

30 kV, 3OkHz

TO PUMP

0414

r

J wOP - GAS

NEEDLE VALVE

DISCHARGE NEEDLE VALVE

1% a

`U,V. TRAP' SPECIMENS

Figure 1 . Apparatus for exposing DNA to metastably-excited gases generated in aradiofrequency gas discharge .

By applying radiofrequency power (30 kV, 30 kHz) to two ring electrodessurrounding a glass tube 5 mm wide, a discharge was sustained in various gasespassing at a constant velocity through the tube . Special care was necessaryto make sure that no U.V. light originating from the discharge can reach thespecimens . This was achieved by arranging the samples perpendicularlyaround a ` U.V. trap' (reflecting the U .V. radiation back into the discharge)at the bottom of the exposure vessel . In an earlier communication we haveshown that the contribution of U .V. light to the biological effects observed iswithin the limits of experimental error (Jung and Kurzinger 1968) . Decayof the short-lived excited states within approximately 10 -8 sec leads to populationof the lowest triplet states . Since the transitions from these states to the groundstate is forbidden by selection rules, the lifetimes of these excited triplet statesare relatively long (milliseconds to seconds) and, therefore, they are called` metastable ' states .

As the distance between discharge and samples was about 10 cm, the unstableexcited singlet states and the charged species produced in the discharge havesufficient time to decay or recombine . Thus, a constant stream of gas containinga certain fraction of atoms or molecules in ` metastable ' states was passingover the samples exposed in thin layers . The excitation energy of the metastablestates is transferred to the biomolecules by so-called ' collisions of the secondkind '. The energies of the lowest metastable states (cf . Brocklehurst 1968)amount to 4.3 eV for H2* (vibrational state), 6 . 17 eV for N2* (vibrational state),11 . 56 eV for Ar*, and 19 . 81 eV for He* .

0 1 2 3cm

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DNA changes caused by excited gases

223

The pressure within the discharge (16 to 25 Torr for the various gases used)and the distance between discharge and specimens were chosen for each gasin such a way as to minimize the U .V. afterglow in the perpendicular part of thedischarge tube . Absolute dosimetry was not performed. However, the dis-charge was sufficiently constant and reproducible to guarantee a linear correlationbetween dose and exposure time and to enable a comparison to be made ofvarious experiments performed in the course of several months .

2.2 . DNA and sample preparationThe circular and single-stranded DNA of bacteriophage OX174 was used

throughout these experiments . The lysis-deficient mutant DX174 am3 describedby Hutchison and Sinsheimer (1966) was grown on E. coli H 513 (thy-, her-), astrain constructed and kindly supplied by Professor Hoffmann-Berling,Heidelberg. Its DNA was labelled with 3H by addition of 0 . 029 mCi/ml of3H-methyl-thymine. After subjecting the host bacteria to lysozyme-EDTAtreatment, the suspension was alternately frozen and thawed four times and thereleased phages purified in a neutral sucrose gradient . DNA was isolated bythree successive extractions with hot phenol saturated with borate buffer (Guthrieand Sinsheimer 1963) and then dialysed overnight against a buffer (pH 7 . 6)consisting of 7 .5 mM NaCl, 0 .75 mM Na-citrate, and 0 .05 mM EDTA, yieldinga specific radioactivity of 10 -2 counts/min per plaque-forming unit.

Since excited gases dissipate their energy at the surface of the materialexposed, the samples must consist of very thin and homogeneous layers . Afterdiluting the stock solution of the DNA 1 : 10 into triple-distilled water (pH 7 .6by NH4OH) 0.01 ml were pipetted onto microscope cover-slips, 12 mm indiameter, and freeze-dried at 10-2 Torr for at least 5 hours, thus avoiding theoccurrence of drying rings. By this procedure the fraction of the DNA notenclosed in little salt crystals (see §3) forms a layer less than 100 A thick, asdetermined by irradiation with protons at energies between 0 .8 and 50 keV(Jung and Kiirzinger 1969). During lyophilization, about 20 per cent of theintact circular DNA molecules were broken and, thus, biologically inactive .

2.3 . Biological assayThe integrity of the (DX-DNA was tested by infecting spheroplasts prepared

from E. coli KA 16 (her- ) according to the method of Guthrie and Sinsheimer(1963) as modified by Jung and Kiirzinger (1968) . After exposure to excitedgases for various times, the DNA was resuspended in 0 . 5 ml tris buffer (0 .05 M,pH &1), and subsequently incubated at 37 ° C with 0 . 5 ml spheroplast suspensionfor 20 min . After addition of 1 ml PAM medium (Guthrie and Sinsheimer1963) incubation at 37°C was continued for another 2 hours . The phagesthus produced, after appropriate dilution with 0 .05 M borate buffer wereassayed by plating on E. coli CR 34/C 416/C 1 .

2.4 . CentrifugationThe circular (DX-DNA molecule is converted by one strand break into a

linear structure . Since the molecular weight remains unchanged, separationof circular and linear molecules is possible only at high pH and low ionic strength .After exposure, the DNA was dissolved off eight cover-slips by immersing themin 0 .6 ml of a buffer (pH 12 .0) containing 5 per cent sucrose, 1 . 5mM Na-citrate,

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0 . 1 mM EDTA, and 4 mM NaOH . After incubating for 15 min the DNA waslayered onto a preformed 5 to 20 per cent sucrose gradient buffered by 5 mMNa-citrate and 1 mM EDTA, previously adjusted to pH 12.0 by 2 N NaOH .Centrifugation was performed at 34 000 r.p .m. and 5 °C for 18 hours in a SW 40swinging-bucket rotor using a Spinco L2-50 B ultracentrifuge (BeckmanInstruments) . The 3H-radioactivity of the fractions collected after puncturingthe centrifuge tubes was measured in a liquid scintillation spectrometer . Inte-gration of the appropriate peaks in the sedimentation diagram yielded the fractionof (DX-DNA molecules converted to linear structures by one strand break .

3 . ResultsFigure 2 shows the results of a typical experiment performed using excited

argon atoms. With increasing exposure the fraction of infectious DNA mole-cules decreases and approaches a constant level at long exposure times indicating

100908070

60

> 50UWLLz 40

3cX 174-DNA

0

300

10

20

30 sec

1

2

3 min

EXPOSURE TO ARGON

Figure 2. Inactivation of the plaque-forming ability of IX174-DNA after exposure toexcited argon for various times. • Experimental values . 0 Values after sub-tracting the constant fraction (---).

that part of the DNA molecules are enclosed in small salt crystals that cannot bepenetrated by the excited gases . After subtracting this constant fraction theexperimental values fall on a straight line in the semi-log plot . The slope of thisline is characterized by the t37 value, i .e. the exposure time necessary to inactivatedown to a surviving fraction of 37 per cent .

Figure 3 gives, again as an example, the distribution of radioactivity from3H-labelled (DX174-DNA after centrifugation in an alkaline sucrose gradient .When the DNA molecules have sedimented nearly to the bottom of the centrifugetube, the broken molecules are well separated from the undamaged circularmolecules . Already in the control samples, about one third of the moleculesare broken ; some breaks result from nuclear disintegrations in the radioactively-labelled molecules, some are caused during isolation and freeze-drying of theDNA. As may be seen from figure 3, only the circular DNA is infectious,

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whereas the broken molecules do not show any biological activity . Withincreasing time of exposure to argon the amount of circular molecules decreasesand that of the broken molecules increases .

DATA changes caused by excited gases

225

Figure 3 . Sedimentation pattern of 3H-labelled FX174-DNA in an alkaline sucrosegradient after exposure to excited argon for 20 sec and 3 min, respectively .3 H-radioactivity

0 Plaque-forming ability.

A summary of the results of our experiments obtained with four differentexcited gases is given in figure 4 . The percentage of infectious DNA as wellas the percentage of unbroken molecules is plotted versus duration of the exposureto excited gases . The constant fractions that amount to about 70 per centunder all experimental conditions have already been subtracted, yieldingexponential dose-effect curves in all cases . The data plotted are averagevalues from several experiments, the straight lines drawn represent least-squarefits . The t37 values calculated from figure 4 are compiled in table 1 togetherwith the appropriate standard errors . As an absolute dosimetry was not per-formed, the figures for the t 37 values given cannot represent the efficiency of thevarious gases to cause inactivation and strand breakage, respectively . However,the ratio of these two t37 values for each gas gives the fraction of (DX-DNAmolecules inactivated by strand breakage . By comparing the relative frequenciesof strand breaks (last column of the table) it becomes obvious that this frequencydoes not depend on the gas used . Thus, we arrive at the conclusion that 76 . 5 ±9 .4 per cent of the inactivated tDX-DNA molecules carry at least one strand break ;within the energy range investigated this figure is independent of the amountof energy transferred .

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226 C. Liicke-Huhle and H . Yung

Figure 4. Inactivation and strand breakage in X174-DNA after exposure to metastably-excited gases . • Loss of plaque-forming ability as tested in the spheroplastsystem . 0 Fraction of unbroken molecules as determined by centrifugation in analkaline sucrose gradient.

Energy transfer from metastable states of excited gases to IX174-DNA .

4 . DiscussionFrom the rather unexpected result of our experiments it must be concluded

that the effects caused by energy transfer from excited gases are different fromthe actions of U .V. quanta and of slow charged particles . In earlier experiments,short wave-length U .V. radiation (Setlow 1960, Berger 1969) or low-energeticelectrons (Hutchinson 1960) showed little effect on enzymes and DNA at energiesbelow 10 eV, whereas at higher energies the biological response increased in thesame manner as the ionization probability . A widely-accepted conclusion

Gas Energy(eV)

Plaque-formingability

t37 (sec)Strand breaks

t37 (sec)

Percentage of(DX-DNA molecules

inactivated bystrand breaks

H2*N2*Ar*He*

4 . 36 . 2

11 . 619 . 8

10 . 7 ±1-026 .0 ±1-918-1± 3 . 05 . 5 ± 0 . 3

14. 3 ± 2 . 532 . 2 ± 5 . 129 .4 ± 4 . 46 . 7 ± 0 . 7

75 ± 2081 ± 2062 ± 1982 ± 13

Weighted mean : 76 . 5 ± 9 .4

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drawn from these experiments was that, in general, amounts of energy below10 eV have little radiobiological significance, i .e. only rarely will the excitationof the lower energy states have any biological consequences .

To explain the difference between the action of U.V. quanta and slowelectrons on one hand and of excited gases on the other, one should keep in mindthat the action of a charged particle on a molecule is similar to the action of aphoton field having a spectral distribution proportional to 1 /v (Platzman 1962) .And it is well known that energy transfer from photons to molecules is governedby selection rules . Thus, when irradiating with U.V. light or low-energeticelectrons only certain classes of electrons within the molecule (a or 7r electrons)may take part in dissipating the energy . ' Collisions of the second kind ',however, are radiationless processes and, consequently, not governed by theseselection rules, so that the energy transferred from the metastably-excited gasesmay be distributed over the electron system as a whole . Since 4 . 3 eV is toolow an energy as to ionize the molecule, our results (viz . that the frequencyof strand breaks is independent of the energy transferred) lead to the conclusionthat excited gases do not cause ionizations, even if the energy (for example,19 . 8 eV in the case of excited helium) is well above the ionization level of themacromolecules exposed .

This conclusion may appear somewhat rash, but it is strongly supported byrecent experiments performed by Weibezahn and Dertinger (1973 a, 1973 b) .The e.s .r. spectra obtained after exposing DNA and its constituents to excitednitrogen, argon, and helium were independent of the amount of energy trans-ferred . By applying a refined computer analysis the authors arrived at the resultthat the radicals are generated by direct C-H bond dissociation in the baseswithout preceding ionization . In contrast to this result, most of the spectraobserved after y-irradiation of the same substances could be shown to be due toradical-ions resulting from previously ionizing the molecules investigated (Hartigand Dertinger 1971) .

Our present and, by necessity, still superficial model of the reactions inducedby energy transfer from excited gases to DNA is the following : The excited gasestransfer their excitation energy to the electron system of the molecule as a wholeleading probably to excited states of strongly collective character . Thesestates dissipate their energy by dissociating aromatic C-H bonds at the basemoiety. The type of lesion produced is independent of the energy transferred .From our observation that the fraction of strand breaks among the lesionsdestroying the plaque-forming activity of (DX-DNA is as high as to 76 . 5 per centand is also independent of the energy transferred, we may speculate that theradicals originally located at the base moiety finally lead to strand breakage .Details of this mechanism are not yet fully understood, but seem to be similarto the reactions postulated in trying to explain the action of U .V. light onbromouracil-substituted DNA (Hotz and Reuschl 1967, Kohnlein and Hutchin-son 1969, Dodson, Hewitt and Mandel 1972) .

In contrast to these results, earlier investigations into the action of vacuumultra-violet on the plaque-forming ability of (IX-DNA (Berger 1969) and on thefrequency of strand breaks in the double-stranded (DX-RF-DNA (Wirths andJung 1972) indicate that the effects of vacuum U .V. depend strongly on theamount of energy transferred per absorptionevent . A more thorough investi-gation of the effects of vacuum U .V

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DNA changes caused by excited gases

techniques as applied in the present study is under way . We expect it to givefurther insight into the mechanisms by which DNA is damaged after receivingamounts of energy between 4 and 20 eV .

ACKNOWLEDGMENTS

We wish to thank Professor K. G . Zimmer for his continuous interest inthis work and Drs . H. Dertinger and K. F . Weibezahn for clarifying discussions .The skilful assistance of Miss Monika Pech is very much appreciated .

De 1ADN de bacteriophage OX174, etale en tres fines couches, a ete expose a factionde gaz metastables excites . Ensuite, le pouvoir infectieux a ete teste sur spheroplasteset la frequence des cassures a ete determinee par centrifugation en gradient de sucrosealcalin . L'energie d'excitation des differents gaz a ete respectivement de 4,3 eV pourl'hydrogene, 6,2 eV pour l'azote, 11,6 eV pour l'argon et de 19,8 eV pour 1'helium . Cesenergies sont transmises aux molecules d'ADN par ' chocs de deuxieme ordre ' . Lesresultats montrent que 76,5 ± 9,4 pour cent des molecules d'ADN sont inactivees par descassures, independamment de 1'energie .

DNS des Bakteriophagen X174 wurde in extrem dunner Schicht metastabil angeregtenGasen ausgesetzt and anschlieBend der Verlust der Plaque-Bildungs-Fahigkeit im Spharo-plastensystem sowie das Auftreten von Strangbruchen durch Zentrifugation im alkalischenSaccharose-Gradienten bestimmt . Die Energie des tiefsten metastabilen Anregunszu-standes liegt fur H2* bei 4,3 eV, fur N 2* bei 6,2 eV, fur Ar* bei 11,6 eV and fur He* bei19,8 eV. Diese Energien werden durch ' StoBe zweiter Art ' an die DNS-Molekuleubertragen . Es zeigte sich, daB 76,5 ± 9,4 Prozent der inaktivierten DNS-MolekuleStrangbri the aufweisen and dad diesser Wert unabhangig von der primar iibertragenenEnergie ist . Dieser Befund demonstriert, dal3 die Wirkung der angeregten Gase sichgrundlegend von der Wirkung von U .V.-Quanten and niederenergetischen Elektronenunterscheidet, obwohl in alien Fallen vergleichbare Energiebetrage an die DNS iibertragenwerden,

REFERENCES

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Berlin : Springer-Verlag) .DODSON, M. L ., HEWITT, R ., and MANDEL, M ., 1972, Photochem . Photobiol., 16, 15 .GUTHRIE, G. D ., and SINSHEIMER, R. L., 1963, Biochim. biophys. Acta ., 72, 290 .HARTIG, G., and DERTINGER, H., 1971, Int . J. Radiat. Biol., 20, 577 .HOTZ, G., and REUSCHL, H., 1967, Molec. Gen. Genet ., 99, 5 .HUTCHINSON, F., 1960, Radiat. Res., Suppl . 2, 49 .HUTCHISON, C. A ., and SINSHEIMER, R . L ., 1966, J. molec. Biol., 18, 429 .JOHNSON, C. D., and RYMER, T. B ., 1967, Nature, Lond., 213, 1045 .JUNG, H., and KURZINGER, K ., 1968, Radiat. Res ., 36, 369 ; 1969, Z. Naturf. B, 24, 328 .KOHNLEIN, W ., and HUTCHINSON, F., 1969, Radiat. Res ., 39, 745 .ORE, A., 1971, Physica Norvegica, 5, 259 .ORE, A., and LARSEN, A., 1964, Radiat. Res ., 21, 331 .PLATZMAN, R . L., 1962, Vortex, 23, 372 .RAUTH, A. M., and SIMPSON, J. A., 1964, Radiat. Res ., 22, 643 .SETLOW, R . B., 1960, Radiat. Res. Suppl., 2, 276 .WEIBEZAHN, K . F., and DERTINGER . H., 1973 a, Int . J. Radiat. Biol., 23, 271 ; 1973 b, Int .

Y. Radiat. Biol., 23, 447 .WIRTHS, A., and JUNG, H., 1972, Photochem . Photobiol ., 15, 325 .

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