Review Radiation-induced decomposition of the purine bases within DNA …homepage.univie.ac.at/mario.barbatti/papers/DNA/revie… · · 2006-01-31Review Radiation-induced decomposition

INT. J. RADIAT. BIOL., 1985, VOL. 47, NO. 2, 127-143

Review

Radiation-induced decomposition of the purine baseswithin DNA and related model compounds

JEAN CADET and MAURICE BERGER

Laboratoires de Chimie, Departement de Recherche Fondamentale,Centre d'Etudes Nucleaires de Grenoble, 85 X, F.38041, Grenoble Cedex,France

(Received 31 May 1984; revision received 16 August 1984;accepted 21 August 1984)

This survey focuses on recent developments in the radiation chemistry of purinebases in nucleic acids and related model compounds. Both direct and indirecteffects of ionizing radiation are investigated with special emphasis on thestructural characterization of the final decomposition products of nucleic acidcomponents. Available assays for monitoring radiation-induced base lesions arecritically reviewed.

Indexing terms: purines, DNA, DNA models, direct and indirect effects.

1. IntroductionDeoxyribonucleic acid (DNA) is a critical cellular target for the cytotoxic,

mutagenic and carcinogenic effects of ionizing radiation (Alper 1979, Grosch andHopwood 1979, Biaglow 1981). Radiation-induced chemical modifications of DNAinvolve the formation of ionic, radical and excited intermediates as the result ofdeposition of energy within the biopolymer and indirect processes involving waterradiolysis species (Adams and Jameson 1980). DNA strand breaks which may bemeasured in living cells for a dose of radiation as low as 0-1 Gy arise mostly frominitial hydrogen abstraction within the sugar moiety (von Sonntag and Schulte-Frohlinde 1978). Chemically modified purine and pyrimidine bases constitute asecond major class of DNA lesions. Pioneering experiments have shown thatexposure of purine and pyrimidine nucleic acid components to X-rays leads tovarious types of chemical damage (Scholes and Weiss 1953, 1954). Variousendonucleases have been shown to be useful probes for the detection of suchmodifications (Feldberg and Carew 1981, Brash and Hart 1982, Smith and Paterson1982, Katcher and Wallace 1983). However, the specificity of these enzymes for therecognition of DNAbase lesions is usually broad. Information on the structure ofthese various modifications and the mechanism of their formation has been mostlyobtained by using isolated DNA and related model compounds as the reactionsubtrates. Transient radiation-induced radicals are studied by various techniquesincluding optical and conductimetric pulse radiolysis as well as electron spinresonance (flow methods, ENDOR, spin-trapping). Special efforts have beendirected towards the isolation and characterization of the diamagnetic compounds

J. Cadet and M. Berger

resulting from the fate of these intermediates. Several reviews have appeared in thelast decade on various aspects of the radiation chemistry of DNA compounds (Blokand Loman 1973, Scholes 1976, Box 1977, Myers 1980, Bernhard 1981) with specialemphasis on the pyrimidine bases (Ward 1975, Scholes 1978, Teoule and Cadet1978, Cadet et al. 1981, Ward 1981 a) and the deoxyribose moiety (von Sonntag andSchulte-Frohlinde 1978, von Sonntag 1980, von Sonntag et al. 1981). In this surveywe focus on the radiation-induced modifications of the purine bases in DNA andvarious nucleic acid components as a result of both direct and indirect effects. Recentdevelopments and new insights into the mechanistic aspects of these reactions andthe identification of the final degradation products are reviewed. Purine nucleic acidcomponents have received less attention than pyrimidines, probably due to ana-lytical difficulties as discussed below. Some references to earlier work have beenincluded where necessary for a better overall understanding.

2. Direct effectCell killing of irradiated bacteria under oxic conditions appears to be mostly due

to 'indirect effects' through the action of OH radicals. However deposition ofionizing energy within the target biopolymers accounts for 30-40 per cent of the lethalcellular effects (Ward 1981 b). Recent investigations dealing with the 'direct effects'of ionizing radiation on DNA model compounds have mostly involved the character-ization of the transient radicals produced by irradiation in the solid state and infrozen aqueous solution respectively. However, the diamagnetic products whichresult from the fate of these purine radicals remain unknown.

2.1. Single crystal e.s.r. studiesThe structural assignment of the free radicals produced by the X-ray and/or

gamma-radiolysis of nucleic acid components in the solid state has been the subjectof intense research effort in the past two decades. This topic has been covered inseveral excellent reviews (Herak 1975, Box 1977, Sevilla 1977, Myers 1980,Bernhard 1981). Only recent developments in this field reported in the past 3 yearsare summarized in this paper.

Electron transfer from purine to pyrimidine bases has been suggested to be alikely event in X- or gamma-irradiated DNA fibres or in frozen aqueous DNAsolution. A comprehensive investigation into the possible occurrence of thismigration reaction has been recently carried out by ESR-ENDOR analysis of X-irradiated co-crystals of adenosine and 5-bromouracil (Kar and Bernhard 1983).The two bases of this model system exhibit pronounced stacking interactions whichwould facilitate charge transfer through r-az overlap. Furthermore an increase inelectron affinity of the pyrimidine base is expected as a result of the substitution ofthe methyl group in thymine by a bromine atom. Contrary to expectation, the mainfree radicals observed at low temperature (12 K) in the irradiated co-crystal were apyrimidine 7r-cation and a purine -anion respectively. Both radicals were found todecay above 40 K and to be converted to hydrogen adducts. However, the authorswere not able to completely rule out electron migration between adenosine and 5-bromouracil. Definite conclusions on this controversial matter await furtherexperiments.

Various primary radiation products have been identified in single crystals of theriboside derivatives of 6-methylpurine (Box and Budzinski 1982) and 6-methyl-mercaptopurine (Kim and Alexander 1982) irradiated with X-rays at low

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Radiation-induced decomposition of purine bases

temperature. Alkoxy radicals which result from hydrogen abstraction at oxygen0(3') have been unambiguously assigned in ESR-ENDOR studies of both crystals.In addition a second alkoxy radical at 0(2') and a trapped hydrogen atom radical inthe crystal lattice were shown to be produced in the 6-methylmercaptopurineriboside (Kim and Alexander 1982).

The ESR spectra of 2'-deoxyguanosine-5'-monophosphate (5'-dGMP) at 77 K israther complex (Rakvin and Herak 1981). Selective annealing of the gamma-irradiated nucleotide up to room temperature was necessary to resolve three radicalspecies. The main radical arises from hydrogen addition at carbon C(8). A similarradical has been found in X-irradiated xanthosine dihydrate single crystals (Nelsonand Close 1983). This is in line with previous observations which show thathydrogen addition takes place at carbons C(2) and/or C(8) in almost all irradiatedsingle crystals of purine model compounds with the exception of 6-methylmercapto-purine and thioguanine (Kim and Alexander 1982). A well-resolved quartet ESRpattern of gamma-irradiated 5'-dGMP was assigned to an osidic radical. This wouldinvolve in its two step formation, initial hydrogen abstraction at carbon C(5')followed by splitting of the sugar ring at the C(1')-0(4') and C(3')-C(4') bond withsubsequent reorientation of oxygen 0(4'). The third radical was tentatively assignedto a protonated anion or a deprotonated cation located in the pyrimidine ring (Rakvinand Herak 1981).

2.2. Frozen aqueous solutionsThe radicals produced by gamma-irradiation of 2'-deoxyadenosine-5'-

monophosphate (5'-dAMP) and 2'-deoxyguanosine-5'-monophosphate (Gregoli etal. 1977 b) in frozen aqueous solutions at 77K have been carefully analysed by acomputer-assisted analysis of their ESR spectra (Gregoli et al. 1977 b). Stepwiseannealing of the frozen solution led to conversion of the primary anion and cationpurine radicals to, respectively, hydrogen and hydroxyl adducts at carbon C(8).

[5'-dGMP]+ H20 [5'-dGMP]OH (1 a)

5'-dGMP (annealing)

(77 K) [5'-dGMP]- H20 [5'-dGMP] H (1 b)

Charge transfer was shown to take place in gamma-irradiated co-stackingcomplexes of purine and pyrimidine 2'-deoxyribonucleotides (Gregoli et al. 1977 a).In particular the secondary radical [5'-dAMP]H is not observed in detectableamounts when 5'-dAMP is allowed to co-stack with thymidine-5'-monophosphate.This could be explained by an efficient electron transfer reaction from the initiallyproduced anion radical [5'-dAMP] - to the electron affinic 5'-dTMP, which wouldprevent the formation of [5'-dAMP]H. On the other hand, an increase in the yield ofthe 5'-dTMP electron adduct and of its corresponding protonated derivative [5'-dTMP]H is expected. Indirect evidence for the occurrence of this charge migrationwas gained from the two-fold increase in the formation of 5,6-dihydrothymidine, theexpected final diamagnetic product of the fate of the 5,6-dihydrothymid-5-yl radicalwhen a thymidine frozen aqueous solution was irradiated in the presence of

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equimolecular 2'-deoxyadenosine (Cadet et al. 1983 a). A more comprehensiveinvestigation of the charge transfer phenomena has involved the four DNAnucleotides. Electrons were found to migrate towards 2'-deoxycytidine-5'-monophosphate (5'-dCMP) whereas positive charges are likely to migrate towards5'-dGMP. The relative scale of electron affinity for the 2'-deoxyribonucleotides wasseen to increase in the order 5'-dGMP < 5'-dAMP < 5'-dTMP < 5'-dCMP (Gregoliet al. 1979).

In the course of the study concerning the ESR analysis of frozen deaeratedaqueous DNA solutions exposed to gamma-rays, Gregoli et al. (1982) havecharacterized three main radical species. The composite ESR spectra consists mostlyof the guanine radical cation (G ' ), the thymine electron adduct and its protonatedform (TH'). These data may be explained in terms of charge migration from initiallyrandomly distributed radical anions and cations via stacked bases. Guanine is thelikely sink for short range positive hole migration. The end-point of long rangeelectron migration appears to be thymine in DNA and not cytosine as previouslyobserved in isolated nucleotides (Gregoli et al. 1979). Modifications in the relativeenergy of the LEMO of thymine and cytosine as a result of conjugation of n orbitalsin DNA base pairs have been suggested to explain this apparent inversion of therelative electron affinity between the two pyrimidine bases.

3. Indirect effectThe so-called indirect effect is accounted for by the action of the various reactive

radicals produced by the radiolysis of water:

H 2 0- OH', H' and eaq (2)

Hydroxyl radicals have been shown to be the most important inactivating speciesfollowing exposure of cells to ionizing radiation (Greenstock 1981, Ward 1981 b). Itshould be also pointed out that the solvated electrons and/or hydrogen atoms are ableto inactivate biologically active DNA (Feldberg and Carew 1981). However, themolecular basis of these biochemical observations remains mostly unknown.Relevant information on the mode of action of water radiolysis species is mostlylimited to isolated DNA and related model compounds. Recent pulse radiolysisstudies and analysis of the final decomposition products under steady-state irradi-ation conditions have provided some insights into the mechanisms of radiation-induced decomposition of purine nucleic acid components in aqueous solutions.

3.1. DNA model compounds3.1.1. Adenine and related nucleosides and nucleotides

The reactions of the main water radiolysis species (OH', H' and e) with thepurine moiety of adenine nucleosides are fast processes. However, the exact sites ofthe reaction of these various radicals within the purine ring remain mostlyspeculative. Theoretical calculations have indicated that the 7,8-imidazole bond is alikely site of reaction for hydroxyl radicals. Indirect evidence suggests that at least 20per cent of OH' adds to the carbon C(8) of adenine (Asmus et al. 1978). This issupported by the observation that the presence of the sensitizing agent Fe(CN)6 3 inan oxygen-free aqueous solution of adenine led to a significant increase in the G valueof 8-hydroxyadenine from 02 to 07. Extensive pulse radiolysis studies of 2'-

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deoxyadenosine (dAdo) involving optical absorption and conductimetric techniquesshed some light on the chemistry of several transient neutral purine radicals whicharise from the initial addition of solvated electrons to the base moiety of thenucleoside (Hissung et al. 1981). The reaction of ea was shown to be close to thediffusion controlled rate (kdAdo + e- = 8-2 + 0.5 x 109 M - s- 1) at pH 7. The resultingradical anion reacts rapidly with a water molecule generating three protonatedderivatives called dAdoH, dAdo'H and dAdo"H. The high rate of this protonationreaction prevents efficient repair of the initial electron dAdo adduct in the presenceof a suitable electron acceptor such as para-nitroacetophenone (p-NAP). In fact,electron transfer to this radiosensitizer was shown to occur from the neutral radicaldAdoH. Attempts to isolate and characterize one expected product of this reactionwhich would involve hydration of the 4,5-purine bond were unsuccessful understeady-state irradiation conditions (Berger and Cadet, unpublished results). Thiscould be explained by the instability of such a dAdo hydrate which would lead to therestitution of initial dAdo. It should also be noted that similar protonated derivativesof the radical anion of dAdo have been proposed to be formed by flash photolysis(Arce et al. 1983).

The bulk of recent radiation-induced degradation studies of adenine nucleosidesand nucleotides through final product analysis have been carried out in oxygen freeaqueous solutions. Opening of the imidazole ring at the 7,8-bond subsequent toinitial hydroxyl radical addition to carbon (8) (van Hemmen 1975) is the mainradiation-induced degradation pathway for adenine (van Hemmen and Bleichrodt1971), adenosine (Ado) (Hems 1960) and dAdo (Mariaggi 1978). Oxidation of thecarbon C(8) through initial hydroxyl radical addition at this same position consti-tutes a second important decomposition reaction of the purine moiety. The resultingproducts, 7,8-dihydro-8-oxoadenine and its corresponding 2'-deoxyriboside deriva-tive have been characterized in oxygen-free aqueous solutions of adenine (vanHemmen and Bleichrodt 1971) and dAdo (Mariaggi and Teoule 1976), respectively,under steady-state radiolysis.

Two other reactions initiated by hydroxyl radicals with subsequent involvementof osidic radicals were found to occur in irradiated deaerated aqueous solutions ofdAdo and/or adenosine-5'-monophosphate (5'-AMP). Intramolecular cyclizationwith subsequent formation of a covalent bond between the osidic carbon C(5') andthe imidazole carbon C(8) is a common feature for the radiation-induced degrad-ation of both dAdo (Mariaggi et al. 1976) and 5'-AMP (Keck 1968, Raleigh et al.1976). These reactions which generate only one of the two possible 5' epimers arehighly stereoselective (figure 1). The resulting 8,5'-cycloadenosine-5'-monophosphate (2) which is resistant to enzymatic digestion by snake venom 5'-nucleotidase (Raleigh and Blackburn 1978) has a (5'S) stereoconfiguration asdemonstrated by X-ray (Haromy et al. 1980) and H-NMR (Raleigh and Blackburn1978) analyses. On the other hand the dAdo anhydronucleoside 1 was found to have a(5'R) absolute configuration (Mariaggi et al. 1976). The small J4 ',5 ' coupling constant(<0.7Hz) of this nucleoside which is indicative of a dihedral angle close to 90 °

between the protons H(4') and H(5') supports this assignment. Formation of thecyclic adenine nucleoside 1 and nucleotide 2 may be explained by initial hydrogenabstraction at carbon C(5') and a subsequent intramolecular substitution mechan-ism. The latter reaction shows some analogies with the intermolecular substitutionof the carbon C(8) of purines by various alkyl radicals. The radiation-inducedcyclization of dAdo is probably of higher importance in DNA since the ratio of sugar

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NH2 NH2 NH2

N ~~~~N< af N< R3N<a,

R2 -CH2 R2 -CH N A R / N )OH' ~,R4

HO R1 HO R1

dAdo: R1H.R2=OH R =OH 1: R1 ,R4=H . R3=OH

5'-AP: R=OH.R2 =OPO3 H'- t R2 =OPO3H' - 2: R=OHR3=H,R4=OPO3Hl

Figure 1. Radiation-induced formation of the (5'R) 8,5'-cyclo-2'deoxyadenosine (1) and(5'S) 8,5'-cycloadenosine-5'-monophosphate (2).

Table 1. Yieldst (G values) of radiation-induced degradation products of 2'-deoxy-adenosinet in oxygen-free aqueous solutions§.

CysteinProducts pH7,0-6-5 HCl(N/100) (40mM)

2'-Deoxyadenosine 060 0-73 0399-(2-deoxy-a-D-erythro-pentofuranosyl)adenine 9-(2-deoxy-a-D-erythro-pentopyranosyl)adenine 0-06 0.09 0.109-(2-deoxy- fi-D-erythro-pentopyranosyl)adenine Adenine 022 019 0.08

f Dose: 200 kGy. 20 mM 2'-deoxyadenosine solutions which were deaerated by bubblingargon. § From Mariaggi et al. (1979).

radicals to hydroxyl radical purine adduct is expected to increase significantly whenthe oligonucleotide chains are in a native conformation.

The second reaction which implies the transient formation of osidic radicals dealswith the rearrangement of the sugar ring of dAdo (Mariaggi et al. 1979). The 9-(2-deoxy-c-D-erythro-pentofuranosyl)adenine and the two corresponding and pyranoid isomers are the main products of this reaction which is prevented bymolecular oxygen. On the other hand the yield of these dAdo isomers is increasedwhen cysteine, an efficient hydrogen donor, is present in the irradiated aqueoussolution of dAdo (table 1). These various observations are consistent with amechanism which involves in the rate determining step the opening of the sugar ring.Hydrogen abstraction at carbon C(4') has been postulated to be at the origin of theacyclic carbonium ion. It should be noted that the osidic centred carbon radical atposition 1 would better favour the rupture of the C(1')-O(4) bond in an acid-catalysed mechanism. Subsequent reclosure of the deoxyribose ring and hydrogentransfer from a suitable donor (R or RH) to the resulting radical will explain theformation of the three isomers of dAdo.

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3.1.2. Guanine nucleosides and nucleotidesHydroxyl radicals react preferentially with the imidazole ring of guanosine (Guo)

and 2'-deoxyguanosine (dGuo) in acidic aqueous solutions (pH 1-2) producing atleast one major radical (Schmidt and Borg 1976). The exact structure of thishydroxyl radical purine adduct which was analysed by ESR spectroscopy in a fastflow system has not been established. However, addition of OH- at carbon C(8)appears to be unlikely on the basis of deuteration experiments and Huiickel spindensity calculations. These calculations would also rule out significant formation of aradical cation as the result of an electron abstraction reaction within the purine ringby hydroxyl radicals. The interaction of OH- with the base moiety of dGuo and 5'-dGMP has been recently investigated in detail by pulse radiolysis (O'Neill 1983).Three intermediates which exhibit different redox properties in their reaction with

tetranitromethane (TNM) and N,N,N',N'-tetramethyl-p-phenylenediamine(TMPD) have been tentatively assigned. The structure of these radicals I, II and I I Iwhich would result from an addition at carbons C(4), C(5) and C(8), respectively, arereported in figure 2. The radical I is expected to be oxidative whereas the radicals IIand III would be reductive by analogy with the OH- adducts at positions 6 and 5,respectively, of thymine (Fujita and Steenken 1981) and cytosine (Hazra andSteenken 1983). The oxidizing and reducing 5'-dGMP radicals or [5'-dGMP-OfI]oxand [5'-dGMP-OI]rcd, respectively, were found to be produced in the same ratio(table 2). This contrasts with the predominance of reducing type radicals of OH

0 0 OH0

HN H N N ) H

H2Nlo-N H2NN NI H 2 N 0 N.N

HO- -OCH HO-P-OCH HO- -Oc

HO HO HO

I H NFigure 2. Structure of the OH addition products to the purine moiety of 2'-deoxyguano-

sine-5'-monophosphate.

Table 2. Radiation chemical yield of oxidizing and reducing radicals which are produced byOH addition to the bases of various nucleic acid components.

Oxidizing radicals Reducing radicalsSubstrates G(TMPD+ )/G(OH) G(NF-)/G('OH)

2'-Deoxyadenosine-5'-monophosphatet 0.32 0782-Deoxyguanosine-5'-monophosphatet 0.51 0.56Inosine-5'-monophosphatet 0.18 0-74Thymine$ 0.31 0-65

t From O'Neill (1983). From Fujita and Steenken (1981).

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adducts to inosine-5'-monophosphate,2'-deoxyadenosine-5'-monophosphate andthymine. The decay of [5'-dGMP-Of]ox is not affected by the presence of oxygen.This provides further support for the apparent lack of a fast reaction of molecularoxygen with the OH guanine adduct as observed previously. On the other hand,ascorbate and several sulphydryl agents (RSH) were shown to interact efficientlywith the oxidizing type radical. The rate constant for the reaction of cysteine with [5'-dGMP-OI]ox was determined to be 7-8 x 108 M -1 S- 1 at neutral pH. It was inferredthat electron transfer rather than hydrogen donation from RSH was involved inthese reactions with subsequent back reaction to 5'-dGMP (O'Neill 1983). Thisrestitution process would involve the elimination of OH - or H2 0 from the 4,5-bondof the purine ring. The radiation-induced formation of a guanine radical cation hasbeen proposed in earlier studies (Willson et al. 1974, Asmus et al. 1978). The recentpulse radiolysis findings cannot provide a definite answer on the possible occurrenceof this intermediate. In particular a fast elimination of OH- from the [5'-dGMPOfI]ox is a likely mechanism to be considered for the generation of the radicalcation. Further studies with special emphasis on final product determination arerequired to resolve this controversy.

Attempts to isolate and characterize the diamagnetic products arising from theabove radical reactions have been scarce until recently. One major exception dealswith the identification of the formamido pyrimidine derivative (FAPy) in oxygen-free aqueous solutions of guanosine and its corresponding 5'-ribonucleotide exposedto high energy electrons (Hems 1958). This could be explained by the lack of efficientmethods for the separation of the rather polar degradation products of guanine anddGuo. These difficulties have been mostly overcome by the use of high performanceliquid chromatography in the reversed phase mode (RPHPLC) which is a powerfulanalytical technique. The efficiency of this method is illustrated in figure 3 whichshows the elution profile of the RPHPLC separation of the radiation-induceddegradation products of dGuo in oxygen free aqueous solutions (Berger and Cadet1983a). The use of an octadecylsilyl silica gel column (ODS-Ultrasphere) with water(pH 6.5) as the eluent allows the complete resolution of the complex mixture of thevarious decomposition products. Further improvement in the separation of the fasteluting compounds has been accomplished by reversed phase thin layer chroma-tography (RPTLC) on dodecyltrichlorsilyl silica gel plates (Cadet et al. 1983 b).

The overall degradation of dGuo is low (G < 0-8) due to efficient back reactionswhich lead to partial restitution of the starting nucleoside. The two main degradationproducts of dGuo have been characterized as the a and fl anomers of 9-(2-deoxy-D-erythro-pentopyranosyl)-2,4-diamino-5-formamidopyrimid-6-one 3,4 by 250 MHz'H-NMR and fast atom bombardment mass spectrometry (FAB-MS). The form-ation of these FAPy derivatives 3,4 is completely prevented when the bulk ofhydroxyl radicals are scavenged by methanol. Furthermore the presence of mole-cular oxygen or paranitroacetophenone, an efficient electron affinic radiosensitizer,reduces considerably the yield of the imidazole ring opened compounds (Berger andCadet 1983b). These experiments suggest strongly that both hydroxyl and electronadducts (or its protonated derivative) to dGuo are involved in the formation of theFAPy nucleoside 3,4. Initial addition of OH to the carbon C(8) producing anoxidizing type radical (see above), and subsequent reaction of [dGuo-OH]ox with asuitable hydrogen donor agent (dGuo H for example) appears to be a reasonablesequence of reactions giving rise to the unstable 8-hydroxy-7,8-dihydro-2'-deoxyguanosine. Opening of 7,8-saturated purine derivatives was found to be a base

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II

\1 0#8

l10 11 .1

7 43Z

5

A

1.11

LL)\L-LI > I -

mn 50 40 30 20 10 0

Figure 3. Reversed-phase high performance liquid chromatography (RPHPLC) elutionprofile of the radiation-induced degradation products of 2'-deoxyguanosine in oxygen-free aqueous solution on an ODS Ultrasphere column (25 x 0-46cm i.d.).Eluent: bi-distilled water (pH 65). Flow-rate: 1 ml/min. Detection: refractometerindex. 1: 2,4-diamino-5-formamidopyramid-6-one; 2: 9-(2-deoxy-c-D-erythro-pento-pyranosyl)-2,4-diamino-5-formamidopyrimid-6-one; 3: 9-(2-deoxy-[J-D-erythro-pentopyranosyl)-2,4-diamino-5-formamidopyrimid-6-one; 4: guanine; 7: 2'-deoxy-guanosine-5'-aldehyde; 8: 9-(2-deoxy-oc-L-threo-pentofuranosyl)guanine; 11: 9-(2-deoxy-D-erythro-pentofuranosyl)-guanine; 12: 2'-deoxyguanosine; 13: 8,5'-cyclo-2',5'-dideoxyguanosine; 5,6,9,10: unknown compounds.

catalysed process (Chetsanga and Makaroff 1982) leading to the formation of thecorresponding FAPy derivatives. The opening of the imidazole ring between atoms 7and 8 induces a pronounced labilization of the N-glycosidic bond which explains thefurther rearrangement of the osidic ring to the two more stable pyranoid isomers 3,4.FAPy derivatives were shown to be also produced by vacuum ultra violet irradiationof aqueous solutions of guanosine-5'-monophosphate through homolytic dissoci-ation of water molecules (Tsyganenko et al. 1981, Dodonova et al. 1982).

Hydroxylation of the imidazole carbon C(8) which is one of the major radiation-induced reactions of dAdo does not occur, at least to a significant extent, in theradiolysis of deaerated aqueous solutions of dGuo. However, 8-hydroxy-2'-deoxyguanosine is the major degradation product when a deaerated aqueous solution

135

. _ . ^ ^

tI I

I

r 71i 11 , I


of dGuo is irradiated in the presence of the free radical 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN), a well known radiosensitizer. It is interesting to note thatthis 8-substituted nucleoside is not produced when para-nitroacetophenone is usedas radiosensitizing agent. A reasonable mechanism for the formation of 8-hydroxy-2'-deoxyguanosine is the covalent addition of a TAN molecule to the 8-hydroxy-7,8-dihydro-2'-deoxyguanos-7-yl radical. A Cope rearrangement of the resulting N-oxide type compound would give rise to 8-oxo-7,8-dihydro-2'-deoxyguanosine andto a concomitant release of the hydroxylamine derivative of TAN subsequent tohydrogen abstraction at position 8 of the purine ring (Berger and Cadet 1983 b).Hydroxylation of dGuo at the carbon C(8) has been found to be a major process inaqueous solution containing ascorbic acid or other reducing agents such ashydroxylamine, hydrazine, sodium bisulphite and acetol (Kasai and Nishimura1984). The exact mechanism of these radiomimetic reactions which requiremolecular oxygen has not yet been determined. As hydroxyl radicals appear to beinvolved in the hydroxylation reaction the formation of 8-hydroxy-2'-deoxyguanosine is likely to occur in the gamma irradiation of dGuo in aqueousaerated solutions.

Four other reactions mediated by OH involve in the earliest steps the formationof radicals in the sugar moiety of dGuo. The relatively large yield of the degradationproducts arising from these reactions may be explained by the occurrence of efficientrestitution processes in the purine ring. It is likely that 9-(2-deoxy-c-D-erythro-pentofuranosyl) guanine (5) and 9-(2-deoxy-a-L-threo-pentofuranosyl)guanine(6)derive from osidic radicals centred at C(1') and C(4') respectively (Berger and Cadet1983 a). Epimerization of the related radicals and further hydrogen transfer fromsuitable donors appear to be a reasonable mechanism.

Another feature of the radiation-induced degradation of dGuo as compared withthat of dAdo is the lack of detectable formation of (5'R) or (5'S) 5',8-cyclo-2'-deoxyguanosine consecutive to hydrogen abstraction at carbon C(5'). Oxidation ofthis latter radical is a likely mechanism to explain the formation of 2'-deoxyguanosine-5'-aldehyde(7) (Berger and Cadet 1983a). It is interesting to note

HOCH 2

H0

OH H <111112:H21NHNH2 NH CHO H2

HOW' HOHO

0,:3 5 7

0:4

Figure 4. Chemical structure of four main radiation-induced degradation products of 2'-deoxyguanosine in deaerated aqueous solution: and fi isomers of 9-(2-deoxy-D-erythropentofuranosyl)-2, 4-diamino-5-formamidopyrimid-6-one (3) and (4); 9-(2-deoxy-a-D-erythro-pentofuranosyl)guanine (5); 2'-deoxyguanosine-5'-aldehyde (7).

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that selective formation of thymidine-5'-aldehyde has been observed in DNAincubated with the radiomimetic neocarzinostatin antibiotic (Kappen and Goldberg1983).

A second class of 8,5'-cyclopurine nucleoside which may be prepared by ultra-violet photolysis of various 5'-substituted adenine and guanine nucleoside deriva-tives (Matsuda et al. 1978) has been found to be produced in gamma irradiateddeaerated aqueous solutions of dGuo and 5'-dGMP (Berger and Cadet 1983a). Thiscompound has been identified as 8,5'-cyclo-2',5'-dideoxyguanosine (8). Support forthe involvement of the carbon C(4') centred radical in the formation of thecyclonucleoside was provided by isotopic labelling. A complete loss of 2 H wasobserved in the cyclic nucleoside when dGuo, selectively deuterated at position 4',was used as the substrate. Elimination of water or phosphoric acid at carbon C(5)from the osidic radical would generate a radical cation which may undergosubsequent intramolecular cyclization with the rather nucleophilic carbon C(8)(figure 5). The formation of 8,5'-cyclo-2',5'-dideoxyguanosine (8) in an aqueoussolution of 5'-dGMP (Berger and Cadet 1983 a) constitutes the first example of a basemodification induced by a sugar radical with concomitant release of the phosphategroup. The occurrence of this reaction in DNA would lead to an oligonucleotidestrand break with a chemically modified guanine residue attached at the 5'-terminal.

Br2-, which is a mild oxidizing radical, has been shown to sensitize bacterial cellsto the lethal effects of ionizing radiation. This inorganic radical appears to only reactsignificantly with the purine 2'-deoxyribonucleosides, probably through an electrontransfer mechanism. The presence of dAdo or dGuo in an aqueous KBr solutioncontaining thymidine prevents the dihydroxylation of this latter nucleoside whicharises from initial reaction of Br3, the decay product of Br2 , with the thymine ring(Cadet et al. 1983 c).

0II

~~~~~~~~~o/

I /L -- .?H 2o

~~'CH2N ~CH2 N H

N HO HO 8

HO2N NN 1

.1} o;rF RHO

5

Figure 5. Radiation-induced formation of 9-(2-deoxy-a-L-threo-pentofuranosyl)guanine(6) and 8,5'-cyclo-2'-5'-dideoxyguanosine (8).

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3.1.3. Hypoxanthine-xanthineThe early steps of the OH mediated oxidation of hypoxanthine (Hyp) to uric acid

through the intermediacy of xanthine (Xan) have been recently investigated by pulseradiolysis and fast kinetic absorption spectrophotometry (Santamaria et al. 1984).The rate constant for the reaction of OH radicals with Hyp and Xan has beendetermined to be 6.5 x 109M -is 1- and 52 x 109 M-1 s- 1 , respectively. The hypo-xanthine radical which was postulated to be an OH adduct, by analogy with theknown reaction of the hydroxyl radical with purines, decays through a dispropor-tionation reaction giving rise to Hyp and Xan. The disappearance of the xanthineradical which follows a second-order reaction is more complex. The product of thedisproportionation reaction is a secondary radical which undergoes partial conver-sion to uric acid and xanthine. A second competitive pathway leads to the formationof unidentified diamagnetic compounds.

Hypoxanthine glycols and 7,8-dihydroxyhypoxanthine have been found to bethe major degradation products of OH- reaction with hypoxanthine under steadystate radiolysis conditions (Castro et al. 1982).

3.2. DNAThe determination of radiation-induced base damage in cellular DNA remains a

challenging problem. The main difficulties encountered in such studies lie mostlywith the low quantity and the wide range of compounds which can be produced,together with the instability of some of the modified bases. As a result there is still apaucity of accurate information about the nature of DNA base lesions. In this respectit could be noted that pyrimidines, and particularly thymine, have received moreattention then purines. Three chemical assays which permit the release of a specificfragment from the oligonucleotide chains have been developed to quantitate theformation of 5,6-dihydroxy-5,6-dihydrothymine in DNA (Cerutti 1976, Schellen-berg et al. 1981). A more general approach involves acidic hydrolysis of the N-glycosidic bond of the modified bases and subsequent analysis of the releasedmaterial by thin-layer chromatography (TLC) and/or high performance liquidchromatography (HPLC) (Teoule et al. 1977). The use of mild hydrolysis conditionsis necessary to avoid the formation of side-products and to make the assayquantitative. An alternative to this indirect procedure is the enzymatic hydrolysis ofthe modified DNA by exonucleases and phosphatase which is expected to give rise to2'-deoxyribonucleosides (Teebor et al. 1984). A major drawback in this approachcould be the lack of complete digestion of some lesions as observed when radiation-induced modified dinucleoside-monophosphates (Cadet and Voituriez 1979) and

NH 2 NH 2

N\9NH tNN

KN ~ N~ NH

9 10 11

Figure 6. Three degradation products of adenine; 4,6-diamino-5-formamidopyrimidine(9); 7,8-dihydro-8-oxo adenine (10); adenine-N-l-oxide (11).

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DNA (Dizdaroglu et al. 1978) are used as the substrates. Another limitation in thesequantitative assays would result from the radioactive labelling of the DNA bases.Under these conditions self-radiolysis processes are expected to generate, at leastpartly, the same DNA degradation products which would result from exposure toexternal radiation. This was recently illustrated by control experiments on non-irradiated living cells which show the presence of detectable amounts of 5,6-dihydroxy-5,6-dihydrothymine type compounds in 3 H-thymine DNA (Hariharanet al. 1980). The yield of the thymine glycol, as measured by the acetol assay, wasfound to represent 037 and 0-05 per cent of the overall radioactivity of 3 H-thyminein the DNA of M. radiodurans and Chinese hamster V-79 cells respectively. Thiswould preclude accurate determination of the radiation-induced formation of thisbase lesion at the cellular level in the range of low to moderate doses. Morepromising approaches are the use of fluorescence detection, immunological assays(Leadon and Hanawalt 1983) and post-labelling methods (Gupta et al. 1982,Haseltine et al. 1983). These could permit detection down to a single lesion in 105bases.

There is no available report dealing with the characterization of any purine basedamage in cellular DNA. However, several attempts have been made to quantitatethe purine base lesions and particularly the modified adenine derivatives as the resultof the indirect effect of gamma-rays on DNA in aqueous solutions. The work onisolated DNA constitutes an important step before searching for similar radiation-induced base lesions in cellular DNA.

3.2.1. Guanine transferase activityX-irradiated tRNA in aqueous solutions has been assayed for guanine acceptance

in an enzymatic reaction catalysed by a guanine transfer RNA isolated from wheatgerm (Walden and Farkas 1981). The normal function of this enzyme is to replace theguanine residue in the anticodon with the hypermodified queuine. A possibleinvolvement of this transferase in the excision of modified guanine residues has beensuggested by analogy with the action of glycosylases or enzymes which transferpurine but not pyrimidines into apurinic DNA (Karran and Lindahl 1978, Deutschand Linn 1979). Inactivation of guanine transferase into X-irradiated tRNA wasfound to be linear with dose at doses higher than 35 Gy with an unusual sawtoothpattern at low doses. One proposed explanation for the observed activation effect inthe latter conditions was that guanylation would occur at other sites than the firstanticodon position. Another possibility to be considered is that radiation-inducedconformational changes in the biomolecules would allow the replacement of queuineby guanine.

3.2.2. Radiation-induced decomposition of adenineTwo main adenine degradation products have been identified as 4,6-diamino-5-

formamidopyrimidine (9) (adenine FAPy) and 7,8-dihydro-8-oxoadenine (10) in theoligonucleotide chains of gamma-irradiated aqueous aerated solutions of nativeDNA (Bonicel et al. 1980). Mild DNA acid hydrolysis using concentrated formicacid at 60 ° for 16 h led to a quantitative release of the two modified bases 9,10 whichwere separated by two-dimensional thin-layer chromatography and reversed phasehigh performance liquid chromatography.

Radiation-induced fission of the imidazole ring in the adenine moiety of DNAwas found also to occur in aqueous solutions saturated either with nitrogen or nitrous

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oxide (Chetsanga and Grigorian 1983a). The dose response of the conversion ofadenine to FAPy shows an unusual near linear relationship between the formation ofthis product and the logarithm of the applied dose in N2 0 saturated aqueoussolution. The radiation chemical yield of FAPy 9 was estimated to be 0.14 for anabsorbed dose of 1 KGy. Extrapolation to 1 Gy by following the reported yield-doseplot would lead to an apparent G value higher than 100. This would suggest theoccurrence of a chain reaction which seems to be an unlikely process since theaddition of hydroxyl radical (G= 2.5) to the purine carbon C(8) is the determiningstep in the radiation-induced formation of FAPy 9. It should also be noted that anearly linear dependence of the dose on the yield of formation of 9 has beenpreviously observed within the dose range 0-1 kGy in aerated aqueous solutions(Bonicel et al. 1980). FAPy derivatives which result from initial alkylation of thenitrogen N(7) of purine bases in DNA exposed to methylating agents have beenfound to be excised as base derivatives from the oligonucleotide chains by a specificglycosylase (Chetsanga and Lindahi 1979). The radiation-induced FAPy derivativeswhich lack a methyl substituent on atom N(7) are not substrates of this DNA repairenzyme (Chetsanga and Grigorian 1983 a). In contrast to this observation, it hasbeen recently reported that the E. coli formamido-pyrimidine-DNA glycosylase isable to excise adenine FAPy from polydeoxyadenylic acid exposed to gamma rays inoxygen-free aqueous solution (Breimer 1984). In a preliminary report it has beenrecently proposed that repair of adenine and guanine FAPy derivatives may occur viaan enzyme-catalysed reclosure of the imidazole ring (Chetsanga and Grigorian1983b). Further experiments seem necessary to delineate more well the exactactivity of these enzymes.

Specific antibodies have been raised against 8-hydroxyadenosine conjugated tobovin serum albumin and used in a sensitive radioimmunoassay for the determin-ation of the radiation-induced formation of 8-hydroxyadenine (10) in DNA (West etal. 1982). Measurements could be done on the radiation-induced modified DNAwithout previous acidic or enzymatic hydrolysis. However the sensitivity of thisassay is increased several fold after enzymatic digestion of DNA to nucleotides.Under the latter conditions 8-hydroxyadenine (10) was detected at a dose as low as

O10Gy. The formation of 10 in native DNA was found to be linear with dose inaqueous aerated solution. A G-value of 012 was observed for the formation of thisdegradation product in the dose range 0-100 Gy.

A similar serologic assay has been developed for the quantitative measurement ofadenine-N-1-oxide (11) (Ward 1978) which has been proposed to be a radiation-induced degradation product of adenine (Yamamoto 1980). The use of this sensitiveradioimmunoassay has shown that adenine-N-1 oxide (11) is not produced indetectable amounts (G<0.001) in gamma-irradiated aerated aqueous solutions ofadenine. A likely mechanism for the formation of this N-oxide 11 would involve anon-radical pathway through the reaction of adenine with hydrogen peroxide(Polverelli et al. 1984).

3.2.3. Radiomimetic alkylation of purinesPhotoalkylation of DNA with 2-propanol led to a specific substitution of the C-8

position of the adenine and guanine moieties by using di-tert-butyl peroxide as a freeradical photoinitiator with near u.v.-light of 2> 305 nm (Livneh et al. 1982). Thetwo main products of the photoreaction were characterized as 8-(2-hydroxy-2-propyl)adenine and 8(2-hydroxy-2-propyl)guanine respectively after mild acidic

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depurination of the photoirradiated DNA. The corresponding nucleosides have alsobeen isolated by HPLC analysis subsequent to enzymatic digestion of the photo-irradiated oligonucleotide chains. A likely mechanism for the above photoreactionsinvolves the scavenging by the purine bases of the ketyl radical C(CH 3 )2 0H initiallyproduced by hydrogen abstraction from the carbon C(2) of the alcohol. Similar freeradical reaction may be initiated by gamma-rays as shown previously by using purinebases and nucleosides as DNA model compounds (Steinmaus et al. 1971). The effectof the secondary structure of DNA on radiomimetic alkylation has been investigatedby comparing the reactivities of the purine bases in three different 0X 174 DNA. Inmost cases the bases were found to be more reactive in single-stranded DNA than inthe double-stranded DNA of the whole phage particle or in the replicative form. Thephotoreactivity is also sequence dependent. In particular the sequencespyrimidine-adenine are more reactive than the sequences purine-adenine. Extractsof Micrococcus luteus were found to have a specific endonuclease activity directedtowards the photo-induced 2-hydroxy-2-propyl substituted purine derivatives incovalently closed circular DNA from phage PM2 (Livneh et al. 1979). It is likely thatthe local distortion in the oligonucleotide chain which may result from the expectedpreferential syn orientation of the guanine or adenine substituted at the 8-position(Birnbaum and Shugar 1978) could play a role in the recognition of these lesions bythe endonuclease. It has also been shown recently that the presence of 2-hydroxy-2-propyl addition products to purine in phage PBS-2 makes it less susceptible to theenzymatic action of B. subtilis uracil N-glycosylase (Duker et al. 1982).

4. ConclusionsSignificant progress has been made recently in the determination of the

mechanisms of the radiation-induced decomposition of various purine DNA modelcompounds in the solid state as well as in aqueous solutions. However, severalimportant points such as the formation of a radical cation from guanine in diluteaqueous solution or the occurrence of electron transfer in solid state DNA remainopen to debate. Further experiments are required to clear up these controversialproblems. Information dealing with the nature of the radiation-induced base lesionsin DNA is limited to only a few examples. Special effort should be made to developspecific and sensitive assays which could be further used at the cellular level. This is arequisite for a better assessment of the role of DNA base damage in the biologicaleffects of ionizing radiation.

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