INT. J. RADIAT. BIOL., 1985, VOL. 48, NO. 5, 701-710

Radiosensitizers as probes of DNA damage and cell killing

CLIVE L. GREENSTOCK and RICHARD P. WHITEHOUSE

Medical Biophysics Branch, Atomic Energy of Canada Limited ResearchCompany, Whiteshell Nuclear Research Establishment,Pinawa, Manitoba, Canada ROE ILO

(Received 16 August 1984; revision received 22 April 1985;accepted 29 April 1985)

Cell killing and other deleterious biological effects of ionizing radiation are theresult of chemical changes to critical targets, initiated at the time of exposure.Electron-affinic radiosensitizers act, primarily, by chemically modifying thisradiation damage and its consequent biological expression, and such changes canbe used to probe the nature of the cellular radiation target. According to a redoxhypothesis of radiation modification, the molecular mechanism of electronic-affinic radiosensitization involves an oxidative interaction of the sensitizer withreactive, potentially damaging target radicals, which competes with reductiveprocesses that restore the target to its undamaged state.

The effects have been compared of a series of hypoxic cell radiosensitizers onradiation-induced DNA damage and mammalian cell killing, in order to ascertainthe nature of the critical radiation target site(s) involved. Sensitizer efficacy isdetermined by the ability to oxidize the radiation target and is found to increaseexponentially with increasing electron affinity. The threshold redox potential,below which no sensitization occurs, corresponds to the oxidation potential of thetarget bioradical involved, and is characteristic, and useful in identification, of theparticular radiation target.

Model product analysis studies of DNA base damage, inorganic phosphaterelease, single-strand breaks and incorporation of radioactively labelled sensitizerinto DNA show a correspondence between the electronic-affinic radiosensitiz-ation of DNA damage and cell killing. A careful comparison of the radiosensitiz-ation of different DNA sites and cell killing indicates that the sugar-phosphatebackbone of DNA, not the heterocyclic bases, is the DNA target site whichmimics cell killing in its threshold redox potential and overall radiosensitizationresponse. These results suggest that the enhancement by electron-affinic drugs ofradiation damage to the DNA backbone (strand breaks) correlates strongly with,and is the most likely cause of, the radiosensitization of hypoxic cell killing.

Indexing terms: free radicals, redox processes, cell inactivation, DNA base andsugar-phosphate backbone damage, strand breaks.

1. IntroductionOxygen and hypoxic cell radiosensitizers including nitroheterocyclic drugs and

other electron-affinic compounds, enhance radiation-induced mammalian cellkilling (Adams 1973, Raleigh et al. 1973 a), as well as chemical damage to DNA andother potential cell targets (Greenstock et al. 1974, Cadet et al. 1976, Ward 1977,Greenstock 1981 a,b, Wada et al. 1982). This radiosensitization, which is mosteffective when the sensitizers are present during irradiation, is free radical mediated,and involves redox processes leading to enhanced oxidative alterations to the criticaltarget(s) (Greenstock, 1981 a, b).

t Dedicated to Professor Schulte-Frohlinde on the occasion of his. 60th anniversary.Issued as AECL-8738.

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C. L. Greenstock and R. P. Whitehouse

There is considerable in vitro evidence to indicate that DNA is an importantradiation target for cell inactivation and its radiosensitization (Hutchinson 1965,Roots and Okada 1972, Painter 1980), but the nature of the molecular mechanism isdifficult to clarify using only cellular studies. Rapid mix studies confirm thatreactions of oxygen and other sensitizers with short-lived species are responsible forthe majority of radiation-induced cell killings (Shenoy et al. 1975) and scavengerexperiments show that hydroxyl radicals (OH) are one of the principal damagingspecies (Roots and Okada 1972, Chapman et al. 1973). The kinetic properties of theresulting DNA target radicals have been studied using pulse radiolysis (Greenstock1981 a, b, Michaels et al. 1976, Whillans and Adams 1975).

The realization that electron affinity, conveniently estimated using polarographichalf-wave reduction potentials and, more recently, one electron potentials (Meiseland Neta 1975, Wardman and Clarke 1976), was an important determinant ofsensitizer efficacy (Adams 1973, Raleigh et al. 1973 a, Adams et al. 1976) has led tothe development of a redox mechanism of sensitizer action (Greenstock 1981 a, b)that is an extension of the 'oxygen-fixation' hypothesis (Alper and Howard-Flanders1956). Steady-state half-wave reduction potentials of nitro compounds generallycorrespond to the transfer of two electrons (Nicholson and Shain 1965), and arestrictly only a quantitative measure of electron affinity under conditions ofthermodynamic equilibrium. One-electron reduction potentials are a more directestimate, but they require a kinetic technique to measure the transient speciesinvolved (Meisel and Neta 1975, Wardman and Clarke 1976). One-electronreduction potentials may provide a better correlation with radiosensitization, if thecorresponding one-electron oxidation of the target(s) is the rate-limiting step forproducing the enhancement of radiation damage. Present experiments have usedhypoxic cell sensitizers to probe different types of DNA radiation damage and toascertain the particular target damage site responsible for the acute effects of ionizingradiation. Titrating radiation damage against sensitizer electron affinity maydetermine whether cell killing or other deleterious, even chronic, biologicalconsequences, arise from DNA strand breakage produced by free radical attack atthe sugar-phosphate backbone, or DNA base damage.

2. Materials and methodsThe half-wave reduction potentials (E'/ 2 versus SCE) of various hypoxic cell

radiosensitizers were measured against a reference saturated calomel electrode(SCE) over a wide pH range and normalized to pH 7 by conventional polarographicmethods using a hanging drop mercury electrode (Greenstock et al. 1974, Ruddockand Greenstock 1977). Single-sweep voltammetry was used to confirm that thepolarographic reduction of nitrobenzenes and nitroheterocyclic compounds wasreversible under the steady-state conditions used. It was found, using eqn. (1),

Epeak = Ell2 - 0029/n V (1)

that these electrochemical determinations are primarily a measure of the two-electron (n=2) reduction process (Nicholson and Shain 1965).

The nucleotides, nucleic acid bases and calf-thymus DNA were obtained fromSigma Chemical Co. Ltd and used as received. Chemical assays for base destruction,inorganic phosphate (Pi) release, and sensitizer-DNA binding, and details of theirradiation conditions have been described previously (Raleigh et al. 1973 b,

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Probes of DNA and cell radiosensitization

Greenstock et al. 1974, Greenstock 1981 a, b). Yield-dose plots were used to estimatethe amount of radiolytic destruction (errors in measuring G values estimated at + 5per cent) in the presence and absence of sensitizer, the ratio of which being theenhancement ratio. Base damage was determined by the loss of u.v. absorbance at260 nm of by-product analysis using HPLC, and Pi release was obtained using acolorimetric assay involving ammonium molybdate. The degree of sensitizer-DNAbinding was measured in a competitive assay, using scintillation counting, tomonitor the decrease in incorporation of labelled nitrofurazone (NF*) bound toTCA-precipitable DNA on millipore filters with increasing concentrations of asecond sensitizer (Greenstock et al. 1974). The sensitizer-DNA binding efficiencyrelative to NF* (5 pmol dm -3 ) is obtained from competition plots over a range ofsensitizer concentrations, to obtain the concentration of sensitizer [S1/2] required tohalve the incorporation of NF* into DNA (01 per cent):

Relative efficiency= [NF*]/[S1/ 2] (2)

The low sensitizer concentrations relative to that of DNA minimize the contributionof secondary-radical reactions involving sensitizers and primary radicals, andany subsequent reactions of sensitizer radical species. Unlike previous results(Greenstock et al. 1974) these data are restricted to nitro compounds, to demonstratean effect based more directly on the similar redox properties of this class ofsensitizers.

Enhancement of hypoxic cell killing and in vitro DNA single-strand breakage,using alkaline sucrose density gradient centrifugation, was determined usingChinese hamster V79 cells irradiated in suspension; complete details of which havebeen previously published (Dugle et al. 1972, Raleigh et al. 1973 a, Chapman et al.1973, 1974, Adams et al. 1976). The standard error associated with determiningenhancement ratios is about 10 per cent.

3. ResultsRadiation damage to nucleic acid bases in deoxygenated aqueous solution is

enhanced in the presence of electron-affinic compounds (Greenstock et al. 1972,Cadet et al. 1976, Wada et al. 1982). Figure 1 shows the enhancement ratio (e.r.) forradiosensitized damage to the pyrimidine base uracil as a function of sensitizer half-wave reduction potential. The exponential plot conforms to the modified Hanschexpression (eqn. (7)) (Hansch 1971), that is based on the underlying free radical-mediated redox reactions involved (reactions 4, 5). The straight line plot is derivedfrom a least-squares analysis, and has a relatively poor correlation coefficient of fit ofabout 0-60. The standard error on the slope, derived from 8 points, is + 22 per cent,indicating that the slope is non-zero at the 99 per cent confidence level. The Gvalues for total uracil damage in the presence G(-U)s and absence G(-U)0 ofsensitizer are obtained from the initial slopes of

ER= G(- U)5 G(- U)0 (3)

plots of the radiolytic yield of uracil destruction against dose. There is an increase insensitization with sensitizer electron-affinity which approaches, but never exceeds,the full oxygen enhancement ratio (o.e.r. 33). There is a significant (greater thantwo-fold) sensitization even with the least electron-affinic radiosensitizer tested.Similar results have been obtained with thymine.

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C. L. Greenstock and R. P. Whitehouse

'-.U

3.5

3.0

2.5

U 2.0

1.5

-.80 -.60 -.40 -.20 0E' 2( V sce)

Figure 1. Enhancement ratios (e.r.) for sensitized damage to the nucleic acid base uracil,plotted exponentially as a function of sensitizer half-wave reduction potential (El/2versus SCE). The chemicals used in this and subsequent figures and their potentials(±+002V) are: (1) 3-nitropyrrole (-079), (2) 2-nitropyrrole (-064), (3) 4-nitro-imidazole (-0-60), (4) metronidazole (- 058), (5) 2-methyl-5-nitroimidazole (- 057),(6) 3-nitrotriazole (-0'55), (7) 2-nitropyrazole (-0'53), (8) 3-nitrothiophene (-0'51),(9) 2-amino-5-nitrothiazole (-0-50), (10) m-nitroacetophenone (-050), (11) 2-methyl-5-nitrothiazole (-049),(12)p-nitrophenol (- 047),(13)p-nitrotoluene (046),(14) nitrobenzene (-0-46), (15) 2-nitrothiophene (-0-46), (16) 4-nitroisothiazole(-0-45), (17) 2-nitroimidazole (-0-42), (18) niridazole (-0-38), (19)p-nitroacetophenone (-0-38), (20) 2,4-dinitrophenol (-0-37), (21) 2-bromo-5-nitrothiazole (-0-35), (22) 5-nitro-2-furaldehyde diacetate (-0-35), (23) 1-nitropyr-azole (-0'33), (24) misonidazole (-0-33), (25) nitrofurantoin (-0-32), (26) nitro-furazone (-0-31), (27) furoxone (-028), (28) p-nitrobenzonitrile (-0-28), (29)nifuroxime (-027), (30) 5-nitro-2-furaldehyde (-026), (31) ambilhar (-0-26), (32)furamazone (-0-25), (33) 2-nitro-5-pyridinylthiazole (-023), (34) 3,5-dinitro-benzamide (-0-21), (35) 2,4,6-trinitrobenzene sulfonate (- 0-20), (36) 3,5-dinitroben-zonitrile (-0-16), (37) 1-(2,4-dinitrophenyl)pyridinium chloride (-0 10).

A quite different response is seen in figure 2 for the radiolytic binding ofsensitizer to calf-thymus DNA as a function of sensitizer electron affinity.Sensitization of this type of DNA damage increases exponentially with sensitizerelectron affinity (Greenstock et al. 1974), up to a maximum corresponding to theo.e.r. A linear regression curve fits the data with a correlation coefficient of 0-85. Theonset of measurable binding occurs at a sensitizer half-wave reduction potential of-052V versus SCE, and this is independent of the concentration of referencesensitizer used in this competition study.

A similar effect of hypoxic cell radiosensitizers on radiation-induced DNAsingle-strand breakage, in vitro, has been reported (Dugle et al. 1972). A least-squares fitting for the nitro compounds without oxygen shows a high correlationcoefficient of 0-98 for this semi-logarithmic plot, which helps to justify the linearextrapolation shown in figure 3, indicating a redox threshold for the onset ofsensitization at a sensitizer half-wave reduction potential - -05 V versus SCE. Thestandard error on the slope of the line through points 19, 25, 26 and 29 is + 5 per cent.

I I I

BASE DAMAGE

2 xl 3 mol dm3 uracil 02- 10- 4mol drrm3 sensitizer 32

N2 ,pH 7.9 18o 0 -

8 0031,--." 0 14 27

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Probes of DNA and cell radiosensitization 705

IU.U

>- 5.0z

) 2.0

L~ 1.0

a 0.5-Jj_jUiQ:

0.2

0. I-.80 -.60 -.40 -.20 0

E'i,2 (V sce)

Figure 2. Relative efficiencies for the binding of sensitizers to irradiated DNA, as comparedwith that for 4C-labelled nitrofurazone (NF*), plotted against sensitizer electronaffinity.

3.5

3.0

2.5

r 2.0

i.5

1.5

-.80 -.60 -.40 -.20 0E'2( V sce)

Figure 3. Enhancement ratios (e.r.) for sensitized DNA single-strand breakage in irradiatedV79 Chinese hamster cells, plotted against sensitizer electron affinity. Data derivedfrom Dugle et al. (1972) with permission of Taylor & Francis Lrd.

This redox threshold and the apparent maximum e.r. - 3'5 are similar to those for theaction of sensitizers on mammalian cell killing (Chapman et al. 1973). The data for

oxygen do not fit the line drawn between those for the nitroheterocyclic radiosensi-

tizers, suggesting that factors in addition to electron affinity may contribute to theeffect of oxygen on single-strand breakage and, possibly, cell killing (figure 4).

Figure 4 shows the relationship between the sensitization of mammalian cell

inactivation (Adams et al. 1971, Chapman et al. 1973, 1974) and one-electronreduction potentials E, (V versus SCE) for a series of nitrobenzenes andnitroimidazoles (Meisel and Neta 1975, Wardman and Clarke 1976). The data have

been fitted to a semi-exponential plot with a one-electron redox threshold of - 083 V

I I I

DNA-SENSITIZER BINDING 37o

0.1% DNA35- 5Mmol dm 3 NF*

N2 0, pH7 3023026- o32~~~~~~9~-c290o33

80

80 14 o25

2- ;% 670 I 17 o21

0 Z 15I 3JQ,19 1 1

I I I i ISINGLE STRAND BREAKS

V79 Chinese-hamstercells 29 "2 1- 4 -3 7 26 _

5xlO rnol dm sensitizerO25

N2 ,pH 7

19 10-3 mol dm-3 )

_ I/ //

//

//

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C. L. Greenstock and R. P. Whitehouse

1+.U

3.5

3.0

2.5

: 2.0

I .5

-I .00 -.80 -.60 -.40 -.20E7(Vsce)

Figure 4. Enhancement ratios for sensitized killing of V79 Chinese hamster cells plottedagainst sensitizer one-electron reduction potentials (E, V versus SCE). Data derivedfrom Adams et al. (1976, 1979) and Chapman et al. (1974) with permission of AcademicPress, and Taylor & Francis Ltd. The one-electron reduction potentials reportedrelative to NHE (Meisel and Neta 1975, Wardman and Clarke 1976) have beencalculated relative to SCE by subtracting 0245 V.

versus SCE, and a correlation coefficient of 091. The slope of the straight line fittedto the 12 points, as shown in figure 4, has a standard error of + 10 per cent. Theseresults are analogous to those (Chapman et al. 1973, Greenstock 1981 b, Raleighet al. 1973 a) showing a close similarity between the sensitization of Pi release fromirradiated 5'-purine nucleotides and that of cell killing, both with a thresholdsensitizer half-wave reduction potential of -051 V versus SCE.

4. DiscussionThis quantitative, analytical study of chemical radiosensitization explores the

molecular mechanisms, radiation target sites and the nature of the radiation damageresponsible for the important biological end point of cell killing (Greenstock1981 a, b, Greenstock et al. 1974, Raleigh et al. 1973 b). In all the results presented,the electron affinic radiosensitizers are only effective at low concentrations indeoxygenated solutions or in hypoxic cells; they mimic but do not act in addition tooxygen. These observations are consistent with rapid-mix studies (Shenoy et al.1975) indicating that the major component of electron affinic radiosensitizationinvolves fast radiation chemical reactions occurring at the time of irradiation. Therole of OH as a principal damaging species (reaction 4) in both chemical andbiological systems has been discussed previously (Dugle et al. 1972, Greenstock et al.1972, Roots and Okada 1972, Chapman et al. 1973, Ward 1977). In chemicalsystems, nitrous oxide, which efficiently converts eq to OH, doubles the yield ofradiation damage (Greenstock and Whitehouse 1984). However, in nitrous oxide,the action of sensitizers in enhancing DNA damage, and their concentration andredox dependencies are the same as in hypoxia (Greenstock 1981a, b). Similarexperiments in biological systems are not possible because nitrous oxide cannoteffectively convert eaq to OH in competition with the high concentration of cellular

I I I I I ICELL KILLING

V79 Chinese-hamster cells

5x10- 4mol dm3 sensitizer

N2 ,pH7 72

29

24R0 -07-0554 (0 /

R0 -11-36960 0 / 190

4 0 R0 -05-9963

i / '141 I I I I014 0

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Probes of DNA and cell radiosensitization

electron scavengers. An extension of the oxygen-fixation hypothesis (Alper andHoward-Flanders 1956) to include redox reactions between target radicals formedby OH attack and electron affinic sensitizers, in competition with reductants actingas radioprotectors, is outlined below:

OH etc.Target -V* Target radicals (4)

Target Protector Sensitizer Enhancedi · i Target radicals target (5)

reconstitution(edcatreconstitutio reluctantt) (oxidant, adduct) damage

Radiosensitization (biological activity)=fn (electron affinity) (6)

log (ER)=pla+c2 =c1 E+c 2 modified Hansch equation (7)

where Pl, a are Hammett constants, c1, c2 are constants, e.r. (enhancement ratio) isthe experimental measure of radiosensitization, and E is electron affinity as measuredby oxidation-reduction potentials. The free radical-mediated oxidation-reductionequilibria (reaction 5) that determine sensitization are a function of electron affinity(eqn. (6)). This relationship can be represented more formally by the modifiedHansch equation (eqn. (7)).

In other studies (Adams et al. 1976, 1979), log (equi-effective concentration) hasbeen related to electron affinity and this is the approach used in drug analysis, partlybecause of the practical limitations to using high concentrations (toxicity, solubility,cell permeability, metabolism). This is the commonest form of the cause-effectrelationship (eqn. (6)) used in the Hansch equation (Hansch 1971). The enhance-ment ratio does not correlate well with the concentration or log (concentration) for agiven sensitizer, and a logarithmic function of sensitizer concentration may notcorrelate well with a direct determinant of sensitization such as electron affinity. Thee.r. gives a straightforward measure of radiosensitization and the empiricalrelationship (eqn. (7)) has been useful in seeking correlations between chemicaleffects and biological consequences of radiosensitization.

The fact that chemical sensitization is more effective at relatively low con-centrations of sensitizer (Raleigh et al. 1973 b) (unlike acute cytoxicity where highdrug concentrations favour the formation of reductive metabolites as the probablecausative agents), suggests that radiation modification involves reactions betweentarget radicals and unaltered drug (reaction 5). High concentratios of radiosensitizeractually may protect against radiation damage (Raleigh et al. 1973 b) by scavengingthe principal damaging species OH, to form less reactive intermediates. Theexponential dependence of radiosensitization (e.r.) on sensitizer electron affinity(eqn. (5)), (Ruddock and Greenstock 1977, Adams et al. 1979, Greenstock 1981) isanalogous to the approach to structure-activity relationships used by Hansch (1971)in correlating the effects of drugs with their redox properties. According to the redoxrelationship in reaction 5, no sensitization can occur at sensitizer reduction potentialsbelow the oxidation potential of the targe radical being studied. The sensitizerreduction potentials for the onset of radiosensitization can be used to correlatechemical and biological effects of sensitizer, and in particular, to establish the likelytarget radical site, damage to which is responsible for, or ultimately leads to, cell

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C. L. Greenstock and R. P. Whitehouse

killing or other harmful biological end points. The similarity between the redoxtitrations of sensitized Pi release in irradiated nucleotides, DNA-sensitizer binding,in vitro DNA single-strand breakage and cell killing suggests that a common lesion,namely, a radical on the sugar-phosphate backbone, is involved (reaction 10). Thesemi-log plot (figure 4) for cell radio-sensitization as a function of one-electronreduction potential may be represented mathematically by the following equation.

log e.r.=0-96 (E' +083) (8)

having a slope of 096__0094 and an intercept of -0-83V versus SCE. Thiscompares with other data (Chapman et al. 1973, Raleigh et al. 1973 a, Greenstock1981 b) for the radiosensitization of cell killing and DNA nucleotide sugar-phosphate damage as a function of half-wave reduction potential which both fit theexpression given below:

log e.r.=099 (E'1/2+0'51) (9)

The similar exponential form and slopes of these data indicate that, for theseclasses of nitroheterocyclic radiosensitizers, half-wave or one-electron redox poten-tials are both good practical indicators of electron affinity. The one-electronreduction potential of the radiation target (DNA backbone) responsible for cellkillings and its radiosensitization is -032V lower than its half-wave potential,determined predominantly by the second electron reduction step. The fact thatDNA-sensitizer binding and single-strand breakage have similar redox profilescould indicate that DNA-sensitizer binding may lead to single-strand breakage,possibly via a post-irradiation hydrolysis of labile adduct intermediates (analogousto alkali-labile bonds).

Nucleotide-*s.s. breakage Cell

DNA sugar radical Pi release T? killing (0)itizer Addition--*DNA-sensitizer killing

binding

Although DNA base damage shows a similar oxygen effect in terms of measuredenhancement ratio, to that of cell inactivation, its response to sensitizers of differentelectron affinity is quite different (Greenstock et al. 1974, Cadet et al. 1976,Greenstock 1982, Wada et al. 1982), suggesting that base damage is not responsiblefor cell killing. Sensitizers of low electron affinity, that produce a two-foldsensitization of base damage, have no concomitant biological effect on single-strandbreakage or cell inactivation. This presumably reflects differences in the redoxproperties of radicals formed by OH-attack on DNA bases and the sugar-phosphatebackbone, the latter being less easily oxidized.

Recent studies (Lemaire et al. 1984) indicate that free radical attack onpyrimidine bases can lead to strand breakage. If base damage per se were animportant contributor to cell inactivation, then its chemical radiosensitizationshould be reflected in an enhanced biological effect, and this is not observed. Sinceelectron affinic sensitizers act radiation chemically, any interference with DNA basedamage will only be important if it also changes the base damage-mediated strandbreakage or other chemical lesion affecting cell viability, and this will only result in aredox plot which (like figure 4) reflects the DNA backbone as the ultimate target sitefor cell killing.

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Probes of DNA and cell radiosensitization

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I. J., WARDMAN, P., WATTS, M. E., PARRICK, J., WALLACE, R. G., and SMITHEN, C. E.,1979, Structure-activity relationships in the development of hypoxic cell radiosensi-tizers. I. Sensitizer efficiency. International Journal of Radiation Biology, 35, 133-150.

ADAMS, G. E., FLOCKHART, I. R., SMITHEN, C. E., STRATFORD, I. J., WARDMAN, P., andWATTS, M. E., 1976, Electron affinic sensitization: VIII A correlation betweenstructures, one-electron reduction potentials and efficiencies of nitroimidazoles ashypoxic cell radiosensitizers. Radiation Research, 67, 9-20.

ALPER, T., and HOWARD-FLANDERS, P., 1956, The role of oxygen in modifying theradiosensitivity of E. coli B. Nature, 178, 973 979.

CADET, J., GUTTIN-LMBARD, M., and TEOULE, R., 1976, Gamma radiolysis of thymine inoxygen-free aqueous solution in the presence of electron affinic radiosensitizers:Identification of stable products. International Journal of Radiation Biology, 30, 1-11.

CHAPMAN, J. D., REUVERS, A. P., BORSA, J., and GREENSTOCK, C. L., 1973, Chemicalradioprotection and radiosensitization of mammalian cells growing in vitro. RadiationResearch, 56, 291 306.

CHAPMAN, J. D., REUVERS, A. P., BORSA, J., HENDERSON, J. S., and MIGLIORE, R. D., 1974,Nitroheterocyclic drugs as selective radiosensitizers of hypoxic mammalian cells.Cancer Chemotherapy Reports, 58, 559-570.

DUGLE, D. L., CHAPMAN, J. D., GILLESPIE, C. J., BORSA, J., WEBB, R. G., MEEKER, B. E., andREUVERS, A. P., 1972, Radiation-induced strand breakage in mammalian cell DNA. IEnhancement of single-strand breaks by chemical radiosensitizers. Internation Journalof Radiation Biology, 22, 545-555.

GREENSTOCK, C. L., 1981 a, Radiation sensitization in cancer therapy. Journal of ChemicalEducation, 58, 156-161.

GREENSTOCK, C. L., 1981 b, Redox process in radiation biology and cancer. RadiationResearch, 86, 196-211.

GREENSTOCK, C. L., CHAPMAN, J. D., RALEIGH, J. A., SHIERMAN, E., and REUVERS, A. P., 1974,Competitive radioprotection and radiosensitization in chemical systems. RadiationResearch, 59, 556-571.

GREENSTOCK, C. L., RALEIGH, J. A., KREMERS, W., and McDONALD, E., 1972, Chemicalscreening tests for cellular radiosensitizers. International Journal of Radiation Biology,22, 401-405.

GREENSTOCK, C. L., and WHITEHOUSE, R. P., 1984, Base damage in irradiated DNA and theeffect of oxygen. Oxygen Radicals in Chemistry and Biology, edited by W. Bors,M. Saran and D. Tait (Berlin: Walter de Gruyter and Co.), pp.619-62 8 .

HANSCH, C., 1971, Drug Design, edited by E. J. Ariens (New York: Academic Press), Vol. 1.HUTCHINSON, F., 1965, The molecular basis for radiation effects on cells. Cancer Research, 26,

2045-2052.LEMAIRE, D. G. E., BOTHE, E., and SCHULTE-FRoHLINDE, D., 1984, Yields of radiation

induced main chain scission of Poly U in aqueous solution: strand break formation viabase radicals. International Journal of Radiation Biology, 45, 351 358.

MEISEL, D., and NETA, P., 1975, One-electron redox potentials of nitro compounds andradiosensitizers. Correlation with spin densities of their radical anions. Journal of theAmerican Chemical Society, 97, 5198 5203.

MICHAELS, H. B., RASBURN, E. J., and HUNT, J. W., 1976, Interactions of the radiosensitizerparanitroacetophenone with radiation-induced radicals on nucleic acid components.Radiation Research, 65, 250-267.

NICHOI.SON, R. S., and SHAIN, I., 1965, Theory of stationary electrode polarography: Singlescan and cyclic methods applied to reversible, irreversible and kinetic systems.Analytical Chemistry, 36, 706 711.

PAINTER, R. B., 1980, The role of DNA damage in repair of cell killing induced by ionizingradiation. Radiation Biology in Cancer Research, edited by R. E. Meyn and H. R.Withers (New York: Raven Press), pp. 59 68.

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Probes of DNA and cell radiosensitization

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