[CANCER RESEARCH 26 Part 1, 2045-2052,September I966|

The Molecular Basis for Radiation Effects on Cells'

FRANKLIN HUTCHINSON

Department of Molecular Biophysics, Yale University, New Haven, Connecticut

Summary

The reasons are pointed out for the generally accepted conclusion that radiation inactivation of cells happens as the resultof only a few events, or even a single event, at the molecularlevel. Nine reasons are then given for thinking that DXA is theradiosensitive target. From the known mechanisms of radiationdamage to DNA, the inactivation of single-stranded and double-stranded viruses can be at least partly understood. A simplehypothesis is that ionizing radiation acts by either breaking theDNA molecule physically in 2, thus interfering with its continuity, or by destroying bases on both complementary strands.If these ideas are applied to the radiation inactivation of bacterial or mammalian cells, it is difficult to see why they are soradioresistant, even making allowance for the operation ofrepair systems.

In my talk today I want to summarize our present understanding of the molecular events by which high energy radiationsaffect living cells. I will treat only the main features. Even so, itwill be necessary to limit the field if I am to project any kind ofa clear picture in the time at my disposal. I shall talk mainly ofthe effects of ionizing radiations, although on occasion I will describe results ol experiments with ultraviolet irradiation wherethis supplies insights to an understanding of the events takingIliace with ionizing radiations. And, furthermore, I shall concentrate my attention on the immediate physical and chemicalevents which take place within a fraction of a second after theabsorption of the ionizing radiation. The long, complex biochemical changes which take place as a result of these primaryevents will be ignored except in 1 or 2 specific instances.

Inactivation Caused by a Few Events at the MolecularLevel

First, let us discuss the general nature of the way in whichionizing radiation acts on living cells. It has been accepted formany years that ionizing radiation affects cells mainly through asmall number of primary events, perhaps in some cases only asingle event (20). This may be contrasted, for example, with thepossibility that ionizing radiation produces a generalized cellpoison.

A 1st reason for believing that ionizing radiation acts througha relatively small number of events has to do with the mannerin which radiation energy is dissipated in matter. For incidentX-rays or 7-rays the absorbed energy sets in motion fast electronswithin the material. These fast electrons then lose their energy

1The writing of this paper was supported in part by contractAT(30-1)-2053 from the U. S. Atomic Energy Commission.

by further interactions with the target atoms, releasing on theaverage the order of 50 or 100 e. v. per interaction (28), at spac-ings ranging from thousands of A for very fast particles, to onlya few A apart for relatively slow ones. If charged particle beams,such as fast electrons or a-particles, are used as the source ofirradiation, the same process takes place directly.

The energy lost in each energy loss event, 50-100 e. v., or theorder of 2000 kcal/mole, is so large that a considerable numberof chemical bonds are almost inevitably broken in the immediatevicinity of the event. From such a picture, it is very easy to seethe origin of the "point heat" theory of Dessauer (7) for the

biologic action of ionizing radiation.It is easy to show that if we are to ascribe biologic inactivation

to a single high energy event produced randomly within theirradiated cell, a plot of the logarithm of the number of cellssurviving the irradiation versus the radiation dose should be astraight line. Chart 1 shows schematically the types of survivalcurves that are actually found on irradiating either bacterial cellsor mammalian cells. The straight line corresponds to the exponential survival predicted for inactivation by a single event.The "sigmoidal" curve, so called because it produces a sigmoidal

curve when the survival is plotted linearly against the dose, canreadily be ascribed to a situation in which 2 or more events musttake place before the cell is inactivated. Typical experimentalsurvival curves correspond to the requirement that between 2and 6 events are necessary to produce inactivation. We shall seelater that the interpretation of the "shoulder" on the sigmoidal

survival curve can also be ascribed to possible repair and degrada-tive systems within a cell. However, it is clear that the over-allshape of the survival curves fits well with the idea that only afew primary events are required to inactivate a cell. In particularthe straight exponential part of the sigmoidal curve at highdoses appears to have only 1 simple explanation. A cell (whichpossibly may have accummulated a certain amount of sublethalradiation damage) is, nevertheless, still capable of going throughits reproductive cycle until finally the production of a final highenergy event in the cell causes it to lose its reproductive ability.

A 3rd reason for believing that radiation inactivation is causedby a few key events is the relative insensitivity of most survivalcurves to the rate at which the radiation dose is delivered. Whenintracellular systems are irradiated in vitro, or when cells areirradiated under conditions in which they are not metabolizing,the survival curves are nearly always essentially independent ofdose rate. When cells are metabolizing, it is true that there aredose rate effects. However, the effect of changing the dose rate isfar less than would he expected on the theory of a radiation-produced cell poison, for example. In those cases which have beencarefully investigated it usually turns out that the dose rateeffects can be accounted for by repair or dcgradative mechanisms,as I shall discuss later.

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DOSEf f CHART1. The 2 typical types of survival curves for single cellsirradiated with ionizing radiations.

It is comforting to know that in certain special cases an effectof a generalized radiation-produced poison such as hydrogenperoxide produced on the irradiation of water can be identified,and can readily be separated from the primary effects underdiscussion here.

The Radiosensitive Macromolecule in Cells

We are now in the position to ask the question: what are theradiosensitive macromolecules in living cells? Over the course ofyears a number of different lines of evidence have built up thatthis sensitive site is the cell DNA. I would like now to discussthe major lines of evidence that the DNA is, in fact, the radiosensitive material in the cell.

1. It is well established in most cells that the function of thecell which is most sensitive to ionizing radiation is that of reproduction. Cells which never reproduce themselves, such as nervecells, are usually almost totally unaffected by doses which willstop reproduction in other cells. Since DNA is closely concernedwith the replicative function in cells, this is one connectionbetween radiosensitivity and DNA.

2. It has been clearly established that in cell culture theirradiation of the nucleus readily inhibits cell reproduction,whereas enormous doses of the cytoplasm seem to have relativelylittle effect. This has been most clearly shown by the use of aproton microbeam by Zirkle and Bloom (42). The same phenomenon appears to be shown by insect eggs which have theirnuclei located close to one wall of the egg (4, 4a, 39) and fernspores (41). Some results on marine organisms do not give asclear-cut a picture, but do not give clear and unequivocal evidencefor another conclusion, either.

3. In a number of cells, it is well established that a change ofploidy will change the radiation sensitivity. In Chart 2 is shownan interesting example of this in yeast cells, where the ploidymay be changed from 1 to 6 by appropriate genetic technics (23).Although it is usually not simple to predict whether increasingthe ploidy will increase or decrease the radiation sensitivity, it isnonetheless usual for the radiation sensitivity to change.

4. In a more precise way, Sparrow and his collaborators haveshown a remarkable correlation between chromosome volumeand cell radiosensitivity in a number of plants (34). As shown inChart 3, the doses needed to produce a given biologic end effectfor a number of species with widely different nuclear sizes andnumbers of chromosomes fall on a common line when plottedagainst chromosome volume. The line shown in Chart 3 is a leastsquares fit, showing the slope of —0.93.If the slope were —1, itwould indicate that the radiosensitivity depends simply on thevolume per chromosome, and that the energy necessary toproduce a given radiobiologic effect would be characterized by afixed amount of energy deposited per chromosome.

The preceding reasons primarily concern a correlation betweenradiosensitivity and the genetic apparatus in general. We cannow turn to the reasons for implicating DNA in particular asthe radiosensitive molecule.

5. Over the past few years molecular biologists have built up avery detailed picture of the function of DNA, particularly inbacterial systems. According to this picture, DNA stores theinformation necessary to produce a new cell in the sequence ofthe nucleotides along the double helical chains. It is clear thatX-rays can damage these bases and thus destroy informationwhich, as far as we know at present, is stored in no other way.Thus, we would certainly expect that an effect of ionizingradiation on DNA might alter the ability of a cell to duplicateitself.

6. Very significant correlations have been pointed out byTerzi (38) and by Kaplan and Moses (18) between radiosensitivity, the quantity of DNA, and the organization of the genetic

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CHART 2. Survival curves for related strains of yeast cells ofdifferent ploidy. Strain 320 is diploid, 323 is tetrapolid, and 362 ishexaploid. The haploid strain shows an exponential survival witha slope about twice as steep as the hexaploid strain. Reproducedfrom Mortimer (23).

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CHART3. Relation between interphase chromosome volume and acute lethal exposure for 16 plant species. (Reproduced from A. H.Sparrow, L. A. Schairer, and R. C. Sparrow, Science, 141: 163-66, 1963.)

apparatus. Chart 4 is from the paper of Kaplan and Moses. It isseen that on the basis of radiosensitivity the organisms fall into 4distinct classes. Within each class the sensitivity increasesapproximately linearly with the DNA content. The most radiosensitive class is that of viruses having single-stranded nucleicacid as the carrier of genetic information. The next most radiosensitive class is that of viruses having double-stranded DNA.Since the radiosensitivity of these viruses can be greatly variedby irradiation in solutions of different kinds, the radiosensitivitieswhich have been taken here are those in which the indirect effectof radiation-produced radicals from the solution has been suppressed as completely as possible. The next class is that ofhaploid microorganisms, and the most radioresistant class isthat of diploid cells, including mammalian cells.

7. The radiosensitivity of bacterial cells may be altered in anumber of ways—by using different kinds of ionizing radiation,by varying the oxygen concentration, or by adding variousprotective chemical compounds such as cysteamine. If DNA isthe radiosensitive material, its radiosensitivity must vary in thesame way.

It is known that the DNA of certain microorganisms can enterinto the process known as genetic transformation (29). That isto say, DNA from certain strains of organisms containing,perhaps, a mutation for resistance to a certain drug, possessesthe ability to transfer this mutation to the genome of a closelyrelated strain. This effect is known to be mediated by DNAonly (29). The transformation property can be destroyed by theaction of ionizing radiation. This gives one a biologic assay for acertain kind of genetic competence of the DNA.

Transforming DNA can be irradiated within the cells, thenextracted and checked for transforming ability. An example ofwhat happens when different kinds of ionizing radiation areused is shown in Chart 5. In this figure is plotted the relativebiologic efficiency (RBE) as a function of that property of theradiation known as the linear energy transfer (LET). By LET

is meant the mean rate at which the charged particles created inthe tissue lose energy. If they lose energy at a very low rate, as isthe case for the electrons set in motion by high energy 7-rays,the primary energy loss events in the tissue are far apart. Thecommon point on the graph refers to the radiation sensitivitynormalized to that for -y-rays. If the rate at which energy istransmitted to the medium is increased, by going to a-particleradiation for example, the average spacing between the primaryenergy loss events gets smaller and smaller. For nearly allbiologic molecules which have been studied (27), the efficiencyof the radiation decreases as the spacing between events decreases, as shown for the case of 4>X174 virus (31). This decreasehas a very simple explanation. As the primary events get closerand closer together, there is a greater and greater chance of 2or more events occurring within the same molecule. This iswasting some of the absorbed energy, and there is a lowerefficiency for producing the biologic effect.

The sensitivity of typical biologic cells shows quite a differenttrend with increasing LET, as shown for a haploid microorganism(30) and a diploid mammalian cell (6). The significant thing isthat the transforming activity of DNA shows a very similarcurve (15). There is no other molecule known, except DNA,which gives such a shape of curve.

It can also be shown that when the oxygen concentration islowered from the normal value to 0, the radiosensitivity oftransforming DNA decreases by a factor of about 3, in exactlythe same way that the radiosensitivity of bacterial cells changes(17). Similarly, if the radioprotective drug cysteamine is added,the radiosensitivity of the transforming DNA is decreased in avery similar fashion to the radiation sensitivity of the ability ofthe cell to replicate (17). Finally, the dry vegetative cells ofBacillus subtilis are about 4 times as radiosensitive as are the dryspores of the same organism. If the transforming activities ofDNA irradiated in the dry vegetative cells and in spores arecompared, the transforming activity is again found to be 4 times

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CHART4. A plot of DNA content versus the dose needed to reduce survival to 37% (Dav) for: single-stranded RNA and DNA viruses(solid squares); double-stranded DNA viruses (solid circles); haploid bacteria and yeast cells (hatched circles); mammalian, avian, anddiploid yeast cells (hatched squares). The lines are for constant G-value, the lowest line representing a G-value ~1. (Reproduced fromKaplan and Moses (18).)

as sensitive in the dry cells as in the spores (37). There is thus avery close parallel between the radiosensitivity of transformingDNA irradiated in cells and of cell radiosensitivity.

8. The compound bromouracil is very similar to thymine, oneof the naturally occurring bases in DNA. The only difference isthat a bromine atom is substituted for a methyl group on the5-position of the pyrimidine ring. There are methods for incorporating bromouracil instead of thymine in the DNA of avariety of cells. Such a substitution leads to an increase of 2 or 3in the radiosensitivity of these cells for ionizing radiation. Thisincrease has been shown in viruses (35), in bacterial cells (19),and with mammalian cells in tissue culture (8). Furthermore, inbacterial systems it has been demonstrated that the bromouracilmust actually be incorporated in the DNA to produce radio-

sensitivity, and not merely present in the cell during the irradiation (2). And, most convincingly, it has been demonstratedthat transforming DXA containing bromouracil and irradiatedwithin the cell has its radiation sensitivity increased by thesame ratio as is the radiation sensitivity of the cell. This wasfirst shown by Szybalski and collaborators (26) in B. subtilis,and has later been confirmed by us, in pneumococcus cells (16).This again supplies very convincing evidence that DNA formsthe principal radiation-sensitive target in cells.

9. It has been shown that pretreatment of cells with ultravioletlight can change the X-ray sensitivity of some cells (13). Sincethe effectiveness of ultraviolet of different wave lengths varies inclose accord with the absoiption spectrum of nucleic acid (32),this provides still another link between radiation action and the

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CHART5. The variation of the relative biologic efficiency (RBE)with linear energy transfer (LET). The data for HeLa cells isfrom Deering and Rice (6); for diploid yeast cells, from Schambra(30); for*X-174 virus, from Schambra and Hutchinson (31); fortransforming DNA, from Hutchinson (15). All data for all systemswas obtained using the same irradiation apparatus, and the samedosimetry, except for HeLa cells. In this case, somewhat differentdosimetrj' was used, because the doses were so very much lower.The common experimental arrangement precludes the possibilityof dosimetrie errors with the different radiations used.

DNA molecule. The most convincing argument is based on theeffect of photoreactivation of the ultraviolet damage. If light ofwave length 3500-4000 A is given to bacterial cells after irradiation with ultraviolet light, a large fraction of the ultravioletdamage can be restored (13). It is well established in specificcases that this photorestoration involves the breakage of thy-mine-thymine dimer formed in the DXA by the action of ultraviolet light (40). Such treatment, whose only known effect is theremoval of these thymine dimers in DNA, substantially changesthe sensitivity of such cells for a following X-ray dose. Thus,since the radiation sensitivity appears to depend among otherthings on the number of thymine dimers existing in the DNA,DNA is implicated as the radiation-sensitive target in cells.

This is an impressive list of reasons for ascribing the radiationsensitivity of cells to damage to the DNA within these cells.There are many other experiments which implicate DNA as theradiation-sensitive target, but the ones that I have given hereare those which seem most convincing to me.

At this point we must ask: What evidence is there to implicateother macromolecules, or other cellular structures, as the radiation-sensitive target? I suspect it is safe to say that at some timeor another any type of molecule in the cell and every subcellularstructure has been suggested as the site of radiobiologic action.In so far as I am aware, there seem at present to be only 3 structures which might be seriously considered in this connection.These are the chromosomes in larger cells, specifically includingthe protein as well as the DXA of the structure, messenger RNA,and the cell membrane. Let us deal with the case of the chromosomes first.

The 1st 4 points listed above would apply equally to the

entire chromosomal structure, the protein as well as the DNA,as the radiation-sensitive target. For diploid cells with largechromosomes at least, one could not rule out the possibility thatmuch of the chromosomal protein represents a radiosensitivetarget as well as the DNA. Unfortunately, much of the dataimplicating DNA itself has been for bacterial systems only.However, the radiosensitization produced by bromouracil andthe complex course of cell survival with varying LET have beendetermined in mammalian cells, and imply that effects on thesurrounding protein are not very important. Conversely, thereas yet seem to be no clear data which would show that theprotein component of chromosomes is involved. Thus, the entirechromosome as the radiation-sensitive target is not ruled out, butis not supported by very convincing evidence.

The destruction of messenger RNA, particularly any long-livedmessenger RNA which might possibly exist in highly differentiated cells, might also be important. Not much can yet be saidabout this possibility.

From an o priori point of view, the cell membrane representsa very reasonable target for the action of ionizing radiation.Aside from the information-carrying DNA molecule, it is theone other major cell component, except for messenger RNA,where a single localized event could well be imagined to do aconsideratile amount of damage. Furthermore, the membranescontain a large lipid component, and lipids are the 1 biologic-class of molecules in which chain reactions arc known to occur(22), even though the reports of such chain reactions withinliving cells are presently viewed with suspicion.

A specific theory of cell membranes as the radiosensitiveelement has been discussed at some detail by Haeq and Alexander(1). In their version, radiation damage to membranes is assumedto release various degradative enzymes within the cell. It is truethat extensive degradation of DNA is found in many cases afterirradiation (36), but a simpler interpretation would involve stepsin the operation of repair mechanisms, as we will discuss in amoment. The relationship to repair mechanisms seems much morerealistic, since extensive degradation of cell components otherthan DNA does not seem to occur, an objection to the enzymerelease hypothesis. Furthermore, the effects of massive doses ofradiation on such readily measured properties of cell membranesas permeability to a variety of molecules is really surprisinglysmall (1). At the moment, I believe that the kindest judgmentthat can be made about the hypothesis that cell membranes arethe site of the primary radiation lesion is the verdict "not proven."

The identification of the radiosensitive target is confused bythe fact that both repair and degradative mechanisms act toaffect the radiosensitivity of the cell. These mechanisms can bedemonstrated in 2 ways. Firstly, over-all cell survival can beshown to be greatly affected by treatment after irradiation (14).Secondly, changes in survival occur if the dose rate is variedover a large range (14), or if the dose is fractionated into 2 ormore fractions with a long time between fractions (9).

Although it is clear that repair mechanisms must be veryimportant for X-irradiation, the 1 well-established repair systemis for damage from ultraviolet light. Set low and Carrier (33) haveshowed that for bacteria which have received lethal doses ofultraviolet light, there is an enzyme mechanism which excises theradiation-produced thymine dimers from the DNA, and thenresynthesizes the single-strand which was affected, presumably

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using the complementary strand of DNA as the template. Thiswork has been completely confirmed and extended by Boyce andHoward-Flanders (5) and by Pettijohn and Hanawalt (26a). Acertain amount of progress has been made in demonstratingchanges in DNA synthesis after treatment with X-rays (3) andwith nitrogen mustards (12, 21). However, this work is stillfragmentary, and there is no definite evidence yet for repair ofDNA after X-irradiation. Evidence for the repair of X-ray-induced single strand breaks was reported by R. A. McGrathand P. W. Williams at the Biophysical Society meeting, February 1966, and has been confirmed by H. S. Kaplan (personalcommunication).

As has been mentioned before, extensive degradation of DXAfollowing X-irradiation of bacterial systems has been demonstrated (36). The simplest explanation of this degradation wouldbe that it represents the action of an enzyme similar to the onethat excises the thymine dimers in the ultraviolet repair mechanism discovered by Setlow and Carrier, but this is a workinghypothesis only.

The "noise" introduced by repair and degradative mechanisms

hinders the identification of the radiosensitive target to a veryconsiderable degree. There is, however, the possibility of correlating the effect of these mechanisms on cell survival and onparticular cellular constituents to confirm the identification ofthe primary radiation target.

Mechanisms of Maeromolecular Damage

We now need to consider the damage to DNA in irradiatedcells.

First consider what happens when we irradiate a samplecomposed of molecules of a single kind. The primary loss eventscreated by the ionizing radiation release the order of 50 or 100e. v. in a small localized region, perhaps 10 or 20 A in size. Auseful concept here is the number of molecules damaged by a

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CHART6. The G-value (molecules destroyed per 100 e.v. absorbed) for a number of different molecules. Most of the low molecular weight data is taken from Hart andPlatzman (Ila); most ofthe high molecular weight data from Pollard el al. (27). The lowG-value at low molecular weight is that for terphenyl (Ila), asubstance consisting of linked aromatic rings. The low G-value athigh molecular weight is that for the DNA of bacteriophage Tl(31). The cross symbol at a molecular weight of about 200 includesdata for several dipeptides (27).

given dose of radiation. The radiation chemist has adopted aunit useful for this purpose, called the G-value (Ila). The G-valueis defined, in the context in which we are interested here, as thenumber of molecules damaged per 100 e. v. of energy dissipatedin the substance. Since 100 e. v. is the order of magnitude of theenergy released in an average primary loss, the G-value can bethought of in crude terms as measuring roughly the number ofmolecules destroyed per primary event.

Chart 6 shows some G-values for the irradiation of a number ofpure materials ranging from water to the nucleic acids. Over avery wide range of molecular weight the G-value for most molecules falls between the general limits of 1 and 10. Furthermore,there is a general trend from rather higher G-values at the lowmolecular weights to a G-value approaching unity at the highestmolecular weights. The reason can be seen in terms of a somewhatoversimplified picture. A primary event in a material of lowmolecular weight stands a reasonable chance of damagingchemical bonds in several different molecules. As the size of themolecule increases, there is a greater and greater chance that theenergy will be dissipated mainly within a single molecule.

One low G-value is shown, for the compound terphenyl. This ischaracteristic of the yield for a number of compounds consistinglargely of aromatic rings. Apparently the delocalized electronsallow the energy to be spread over a number of chemical bonds,without damage.

Also, very high G-values, up to the thousands, can be measuredfor some compounds in which chain reactions can occur (Ila).Chain reactions initiated by ionizing radiation have been shownfor only 1 class of biologic macromolecules, the unsaturatedlipids (22), and there is no reliable evidence for the existence ofchain reactions, even in lipids, in cells.

According to the simple picture presented, the loss of moleculeson irradiation by this "direct" mechanism depends solely on the

size of the molecule and not on its surroundings.A somewhat different situation arises when we consider

biologic molecules in an aqueous medium, as they are in theliving cell. A simple limiting case which can be readily studied isthat of biologic molecules in very dilute aqueous solutions.Under these conditions, essentially all the energy is absorbedby the water, and a negligible amount by the biologic moleculesthemselves. As a result of the action of ionizing radiation, thewater molecule is decomposed into highly reactive free radicals,which can readily diffuse to the molecules, and there react. Asummary of the damage produced in DNA and its constituentsunder these conditions has recently l>een published by Scholes(31a). Since the radicals are so highly reactive, they tend toreact within the 1st few collisions with organic molecules. Therelative importance of this mechanism thus depends on theamount of free water immediately surrounding the moleculesbeing observed, DNA in this case.

Lastly, it is known that in mixtures of 2 or more kinds ofmolecules there may be a transfer of electronic excitation energyfrom one species to another (Ila). A particularly well-known andclear example of this occurs in liquid scintillation solutions. Inthese solutions energy adsorbed in the solvent, toluene forexample, is transferred with high efficiency to the scintillatingmaterial, which radiates a fraction of the energy as visible light.Such processes which occur with high efficiency in carefullyselected cases can also occur in other cases with much lower

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efficiency. Since the processes by which such energy transfertakes place are not well known, their possible contributions, bothto enhance and decrease inactivation of specific molecules, mustalways be watched. Nevertheless, the utility of classifyingradiation damage in the way we have just done depends on thefact that in most cases these intermolecular energy transfermechanisms do not change the order of magnitude of the over-allinactivation (27).

Correlation between Damage to DNA and Loss of Reproductive Ability

We would now like to take the information that we have onthe damage to the DXA in cells and viruses by ionizing radiationand see if this can be correlated with the biologic loss of abilityto replicate. For this purpose, it will be convenient to use thedata classified in Chart 4.

The lowest line corresponds to viruses containing single-stranded nucleic acids, both RNA and DNA, and irradiatedunder such conditions that the indirect action of water radicalsshould be suppressed as much as possible. The line drawnthrough the points corresponds to a G-value of about 1. To putit another way, it would appear that a single energy loss eventanywhere within a single-stranded nucleic acid molecule removesthe ability of the virus to multiply. It might be mentioned thatfor some of the viruses the protein coat may be removed, and thenucleic acid alone used to infect the appropriate recipient cells.If the radiosensitivity of the nucleic acid preparations is compared with that of the intact viruses, it is found that there is nochange in removing the protein coat (11, 25). In other words, thepresence or absence of the surrounding protein makes no changein the radiation sensitivity of the nucleic acid.

From Chart 4 it is clear that viruses containing double-strandedDNA are more radiation resistant by an order of magnitude.Electron spin reasonance measurements (24) show that thenumber of damage sites produced in the highly ordered array ofstacked bases in double-strand DNA is about the same as inother materials, so that protection by delocalization of the energyis not important. The best insight into the action of ionizingradiation on the double-stranded DNA is given by recentexperiments by Freifelder (10) on the radiation inactivation ofT7 bacteriophage. He irradiated the phage under several differentconditions, and his results may be summarized as follows. WhenT7 were irradiated under 2 experimental conditions (in phosphatebuffer with oxygen and in cysteine solution under nitrogen)there was a 1-to-l correlation between the number of phageinactivated and the number of broken DNA molecules foundafter extraction from this phage. Thus, inactivation under theseconditions may be ascribed to a double-strand scission of theDNA. When the phage were inactivated in a histidine solution,equivalent to irradiating the cells in broth, only 4090 of theinactivated phage had double-strand scissions. In both casessomething like 10-20 single-strand breaks were produced perdouble-strand scission, but the single-strand breaks did not seemto be responsible for loss of biologic activity. For irradiation inthe histidine or broth solution, Freifelder came to the conclusionthat the loss of infectivity could be caused by damage to pyrimi-dine bases.

Thus, the radioresistance of the double-stranded DNA viruses,

as compared with the single-stranded ones, could be ascribed tothe double-stranded structure. An extremely simple picture issuggested. Radiation inactivation occurs when the DNA molecule is broken physically into 2 parts. In addition, inactivationcan also occur if bases on both complementary strands aredestroyed, thus removing information not available in any otherway. Single-strand breaks in double-stranded DNA, or damageto bases on only 1 of the complementary strands, need notinactivate.

The increased radioresistance of haploid microorganisms is atpresent without any reasonable explanation. According to thepresent ideas, the genome of a simple bacterial cell such asEscherichia coli consists of a single DNA molecule replicated at 1point (or perhaps a few points) which moves uniformly over thelength of the molecule (5a). It would be expected that any suchmechanism would be drastically affected by a break in the DNAmolecule. Yet at the doses required to inactivate bacterial cellsa number of breaks should occur, judging from the results ofFreifelder's experiments in phage (10). Even more damage

should arise from the attack of water radicals.We must assume either of 3 alternatives, (a) Double-stranded

breaks, as opposed to other kinds of damage, are suppressed inDNA in cells. (6) There is a mechanism which can realign thebroken ends of DNA in the cell genome and repair the breaks,(c) Our simple-minded picture of DNA replication is wrong.Unfortunately, we still do not have convincing evidence to tellus which of these alternatives is correct.

It is interesting, in passing, that 1 original goal of racliobiologywas to explain why cells were so sensitive to ionizing radiation.We now find ourselves trying to explain why they are so resistant! To look at the question in another way, it may be truethat there are other radiosensitive sites in the cell. But on thebasis of our present knowledge, the DNA more than accountsfor the observed sensitivities.

Naturally, the increased radioresistance of diploid cells cannotbe explained either. We know even less about such cells thanabout bacteria. True, we do know that in mammalian cells theDNA is embedded in protein, which could hold the broken DNAstrands together until the breaks are repaired. Also, we knowthat some of the DNA in such cells is not necessary, since occasionally a chromosome, or a part of a chromosome, may be leftbehind in an abnormal mitosis, and yet have a viable cell. Andlastly, we know that there are 2 copies of each piece of DNA inthe cell. These are all good arguments for expecting the cells tobe resistant to radiation. But to find the actual reason will veryprobably require considerably more knowledge of the mechanicsof DNA replication and of cell division.

References

1. Bacq, Z. M., and Alexander, P. Fundamentals of Radiohiol-ogy. London: Pergamon Press, 1961.

2. Billen, D. Unbalanced Deoxyribonucleic Acid Synthesis: ItsRole in X-Ray-Induced Bacterial Death. Biochem. Biophys.Acta, 72: 608-18, 1963.

3. Billen, D., Hewitt, R., and Jorgensen, G. X-Ray-InducedPerturbations in the Replication of the Bacterial Chromosome. Ibid., 103: 440-54, 1965.

4. Von Borstel, R. C., and Rogers, R. W. Alpha-Particle Bombardment of the Habrobracon Egg. I. Sensitivity of the Nucleus. Radiation Res., 7: 484-90, 1957.

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Franklin Hutchmson

4a. — —. Alpha-Particle Bombardment of the HabrobraconEgg. II. Response of the Cytoplasm. Ibid., 8: 248-53, 1958.

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