Identification of the main oxidation products of 8-methoxy-2'-deoxyguanosine by singlet molecular oxygen

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.     

Direct observation of the alkylation products of deoxyguanosine and DNA by fast atom bombardment mass spectrometry.

By the methods of fast atom bombardment (FAB) mass spectrometry, thin-layer chromatography and ultraviolet absorption spectroscopy adducts have been studied which are formed by an antitumour alkylating drug thiotepa both in a model system, containing only deoxyguanosine (dGuo), and in DNA. Analysis of the model reaction mixture (dGuo + thiotepa) by FAB mass spectrometry permitted observation of adducts dGuo thiotepa, 2dGuo thiotepa, and also the products of their further modification in solution, which occurs by hydrolysis of the glycosidic bond and also by opening of the imidazole ring. In the case of DNA FAB mass spectrometry made it possible to characterize adducts of thiotepa with guanosine (Gua) and adenosine (Ade) without their preliminary purification. The site of alkylation of Gua in both dGuo and DNA is N7, and that of Ade in DNA is N3. The application of the results to the study of the molecular mechanism of the antitumour action of thiotepa is discussed.     

DNA damage in digestive gland and mantle tissue of the mussel Perna perna

Data concerning the susceptibility of DNA to damage by reactive oxygen and nitrogen species and other endogenous compounds produced by physiological stress in marine organisms is lacking, especially in bivalve mollusks. In this article, we analyzed the background levels of lipid peroxidation (malondialdehyde, MDA), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 1,N2-etheno-2'-deoxyguanosine (1,N2-epsilon dGuo) in digestive gland and mantle tissue of mussels Perna perna collected at a cultivation zone in Florianópolis (Santa Catarina, Brazil). The present data point to the possibility of the use of both 8-oxodGuo and 1,N2-epsilon dGuo as complementary indicators of oxidative stress processes in mussels. A sensitive method coupling high performance liquid chromatography to mass spectrometry was applied for the detection of 1,N2-epsilon dGuo in mussel tissues.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Of the four native nucleosides, 2'-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Photooxidation of d(TpG) by phthalocyanines and riboflavin. Isolation and characterization of dinucleoside monophosphates containing the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2''-deoxy-guanosine.

Phthalocyanine mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen saturated aqueous solution has previously been shown to result in the addition of molecular oxygen to the guanine base generating the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine (dO) (the asterisk denotes unambiguous assignment of the 4R and 4S diastereoisomers). The data presented here show that the same guanine modified bases are generated in a 1:1 ratio when thymidylyl-(3',5')-2'-deoxyguanosine (d(TpG)) is similarly photo-oxidized. These modified dinucleoside monophosphates, labelled d(TpO)-A and -B, have been isolated by high performance liquid chromatography and characterized by proton NMR spectrometry, fast atom bombardment mass spectrometry, and enzymatic digestions. Photosensitization in D2O instead of H2O leads to an increase in the rate of d(TpO) formation that is consistent with a type II (singlet oxygen) reaction mechanism. Three interesting properties of these modified dinucleoside monophosphates are: i) the rate of their digestion with spleen phosphodiesterase is greatly reduced relative to d(TpG), ii) they are not digested by snake venom phosphodiesterase, and iii) they are stable to 1.0 M piperidine at 90 degrees C for 30 min. The latter observation indicates that 4,8-dihydro-4-hydroxy-8-oxoguanine is not a base lesion responsible for the strand breaks observed following hot piperidine treatment of DNA exposed to type II photosensitizers or chemically generated singlet oxygen.     

Novel products generated from 2'-deoxyguanosine by hypochlorous acid or a myeloperoxidase-H2O2-Cl- system: identification of diimino-imidazole and amino-imidazolone nucleosides

Hypochlorous acid (HOCl), generated by myeloperoxidase from H2O2 and Cl-, plays an important role in host defense and inflammatory tissue injury. We report here the identification of products generated from 2'-deoxyguanosine (dGuo) with HOCl. When 1 mM dGuo and 1 mM HOCl were reacted at pH 7.4 and 37 degrees C for 15 min and the reaction was terminated with N-acetylcysteine (N-AcCys), two products were generated in addition to 8-chloro-2'-deoxyguanosine (8-Cl-dGuo). One was identified as an amino-imidazolone nucleoside (dIz), a previously reported product of dGuo with other oxidation systems. The other was identified as a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dDiz) by spectrometric measurements. The yields were 1.4% dDiz, 0.6% dIz and 2.4% 8-Cl-dGuo, with 61.5% unreacted dGuo. Precursors of dDiz and dIz containing a chlorine atom were found in the reaction solution in the absence of termination by N-AcCys. dDiz, dIz and 8-Cl-dGuo were also formed from the reaction of dGuo with myeloperoxidase in the presence of H2O2 and Cl- under mildly acidic conditions. These results imply that dDiz and dIz are generated from dGuo via chlorination by electrophilic attack of HOCl and subsequent dechlorination by N-AcCys. These products may play a role in cytotoxic and/or genotoxic effects of HOCl.     

18O]-labeled singlet oxygen as a tool for mechanistic studies of 8-oxo-7,8-dihydroguanine oxidative damage: detection of spiroiminodihydantoin,imidazolone and oxazolone derivatives

A water-soluble [18O]-labeled endoperoxide derived from N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalene-dipropanamide (DHPN18O2) has been shown to act as a clean chemical source of [18O]-labeled molecular singlet oxygen. This allows the assessment of the singlet oxygen (1O2) reactivity toward biological targets such as DNA. The present work focuses on the qualitative identification of the main 1O2-oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine, which was achieved using high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Thus, the [18O]-labeled and unlabeled imidazolone and oxazolone, together with the diastereoisomeric spiroiminodihydantoin nucleosides, were detected as the main degradation products. In addition, a modified nucleoside that exhibits similar features as those of the oxidized guanidinohydantoin molecule was detected. Our data strongly suggest that the imidazolone and oxazolone nucleosides are generated via the rearrangement of an unstable 5-hydroperoxide intermediate. Interestingly, the combined use of appropriate tools, including isotopically labeled singlet oxygen and the high- resolution HPLC-ESI-MS/MS technique, has allowed to shed new light on the 1O2-mediated oxidation reactions of guanine DNA components.     

Photooxidation products of 7,12-dimethylbenz[a]anthracene.

The principal products of the photooxidation of 7,12-dimethylbenz[a]-anthracene (DMBA) in aqueous solutions by photooxidation induced by laboratory lighting have been characterized by high performance liquid chromatograms (HPLC), ultraviolet and mass spectrograms and by comparisons with authentic samples. The products identified were the 7,12-epidioxy-7,12-dihydro-7-12-dimethyl-, 7,12-dione, 7-hydroxymethyl-12-methyl-, 12-hydroxymethyl-7-methyl-, 7-formyl-12-methyl-, 12-formyl-7-methyl-, and 12-hydroxy-12-methyl-7-one derivatives of benz[a]-anthracene. The HPLC profile of products is similar to that obtained from oxidation of DMBA by 'one-electron' reagents, singlet oxygen, or liver microsomal metabolism. The first points of attack are the 7- and 12- positions. The mechanism of photooxidation appears to be generation of singlet oxygen by photodynamic effect of DMBA. None of the products is photosensitizing, however.     

8-Oxo-2'-deoxyguanosine as a biomarker of tobacco-smoking-induced oxidative stress

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) is a useful biomarker of oxidative stress. However, its analysis can be challenging because 8-oxo-dGuo must be quantified in the presence of dGuo, without artifactual conversion to 8-oxo-dGuo. Urine is the ideal biological fluid for population studies, because it can be obtained noninvasively and it is less likely that artifactual oxidation of dGuo can occur because of the relatively low amounts that are present compared with hydrolyzed DNA. Stable isotope dilution liquid chromatography-selected reaction monitoring/mass spectrometry (LC-SRM/MS) with 8-oxo-[(15)N(5)]dGuo as internal standard provided the highest possible specificity for 8-oxo-dGuo analysis. Furthermore, artifact formation was determined by addition of [(13)C(10)(15)N(5)]dGuo and monitoring of its conversion to 8-oxo-[(13)C(10)(15)N(5)]dGuo during the analytical procedure. 8-Oxo-dGuo concentrations were normalized for interindividual differences in urine flow by analysis of creatinine using stable isotope dilution LC-SRM/MS. A significant increase in urinary 8-oxo-dGuo was observed in tobacco smokers compared with nonsmokers either using simple urinary concentrations or after normalization for creatinine excretion. The mean levels of 8-oxo-dGuo were 1.65ng/ml and the levels normalized to creatinine were 1.72μg/g creatinine. Therefore, stable isotope dilution LC-SRM/MS analysis of urinary 8-oxo-dGuo complements urinary isoprostane (isoP) analysis for assessing tobacco-smoking-induced oxidative stress. This method will be particularly useful for studies that employ polyunsaturated fatty acids, in which a reduction in arachidonic acid precursor could confound isoP measurements.     

Substitution of p- and o-hydroxyphenyl radicals at the 8 position of purine nucleosides by reaction with mutagenic p- and o-diazoquinones.

Incubation of 2'-deoxyadenosine (dAdo), 2'-deoxyguanosine (dGuo), adenosine, guanosine (Guo), thymidine, deoxycytidine with p- and o-diazoquinones, mutagens produced by the reaction of phenol and nitrite, at pH 7 and 37 degrees C resulted in a decrease of each nucleoside depending upon the concentration of the diazoquinones. pD-dAdo, pD-dGuo and pD-Guo were isolated from the reaction mixtures of dAdo, dGuo and Guo, respectively, with p-diazoquinone at pH 9.5, and oD-dGuo was from the mixture of dGuo and o-diazoquinone at pH 9.5. The products were identified as 8-(p-hydroxyphenyl)- and 8-(o-hydroxyphenyl)-purine nucleosides by 1H- and 13C-nuclear magnetic resonance spectra, secondary ion mass spectrum, ultraviolet absorption spectrum and elemental analysis. p- and o-Diazoquinones may be converted into p- and o-hydroxyphenyl radicals, respectively, which in turn attack the 8 position of the purine nucleosides. The mutagenicity of these diazoquinones may be partly due to the radical reactions.     

Oxidative and alkylating damage in DNA

Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.     

Participation of singlet oxygen in ultraviolet-a-induced lipid peroxidation in mouse skin and its inhibition by dietary beta-carotene: an ex vivo study

Dietary beta-carotene acts as a photoprotective agent in the skin, but the exact mechanism of protection is unknown. This ex vivo study is focused on determining the mechanism of action of beta-carotene against UV-A-induced skin damage by characterizing peroxidized phosphatidylcholine (PC) and beta-carotene oxidation products. BALB/c mice were fed with basal or a beta-carotene-supplemented diet, and homogenates from their dorsal skin were prepared after 3 weeks for UV-A irradiation. Analyses revealed that the degree of lipid peroxidation in the beta-carotene group was significantly lower than that in the controls. The isomeric composition of hydroperoxy fatty acids, constituting peroxidized PC, was determined by thin-layer chromatography-blotting followed by gas chromatography/mass spectrometry (MS)/selected ion monitoring analysis. The 9- and 10-isomers of peroxidized PC, resulting from the reaction of singlet molecular oxygen ((1)O(2)) with oleic acid, were elevated in the UV-A-exposed control group compared to the experimental group. Similar results were obtained from methylene-blue-sensitized photooxidation of mouse skin lipids in vitro. Liquid chromatography/MS analysis of the homogenates confirmed the formation of beta-carotene 5,8-endoperoxide, a specific marker for the (1)O(2) reaction. These results indicate that dietary beta-carotene accumulates in the skin and acts as a protective agent against UV-A-induced oxidative damage, by quenching the (1)O(2).     

Methylene blue photosensitized oxidation of cysteine sulfinic acid and other sulfinates: the involvement of singlet oxygen and the azide paradox

The methylene blue photosensitized oxidation of cysteine sulfinic acid is investigated. Enhancement of the oxygen consumption rate in deuterium oxide suggests the involvement of singlet oxygen ((1)O(2)) in oxidation. Addition of the (1)O(2) quencher azide produced an unusual enhancement of the oxidation rate of all the sulfinates assayed. It is assumed that azide works as a one-electron carrier between (1)O(2) and the sulfur compounds. Analyses of the products indicate that the photochemical oxidation of cysteine sulfinic acid proceeds through two simultaneous mechanisms. The Type II (singlet oxygen) mechanism is responsible for oxidation of the sulfinic group to the sulfonic group with production of cysteic acid, stable to the photooxidation system, whereas the Type I (electron transfer) mechanism is involved in the degradation of cysteine sulfinic acid to acetaldehyde. Other products detected were ammonia, sulfate, and hydrogen peroxide which account for the degradation of cysteine sulfinic acid and for the excess of oxygen consumption detected during the oxidative reaction.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after the incubation of 2'-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5'-Cyclopurine-2'-deoxynucleosides and 8-hydroxy-2'-deoxyguanosine

Mutations in breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 predispose women to a high risk of these cancers. Here, we show that lymphoblasts of women with BRCA1 mutations who had been diagnosed with breast cancer are deficient in the repair of some products of oxidative DNA damage, namely, 8-hydroxy-2'-deoxyguanosine and 8,5'-cyclopurine-2'-deoxynucleosides. Cultured lymphoblasts from 10 individuals with BRCA1 mutations and those from 5 control individuals were exposed to 5 Gy of ionizing radiation to induce oxidative DNA damage and then allowed to repair this damage. DNA samples isolated from these cells were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure 8-hydroxy-2'-deoxyguanosine, (5'-S)-8,5'-cyclo-2'-deoxyadenosine, (5'-R)-8,5'-cyclo-2'-deoxyguanosine, and (5'-S)-8,5'-cyclo-2'-deoxyguanosine. After irradiation and a subsequent period of repair, no significant accumulation of these lesions was observed in the DNA from control cells. In contrast, cells with BRCA1 mutations accumulated statistically significant levels of these lesions in their DNA, providing evidence of a deficiency in DNA repair. In addition, a commonly used breast tumor cell line exhibited the same effect when compared to a relevant control cell line. The data suggest that BRCA1 plays a role in cellular repair of oxidatively induced DNA lesions. The failure of cells with BRCA1 mutations to repair 8,5'-cyclopurine-2'-deoxynucleosides indicates the involvement of BRCA1 in nucleotide-excision repair of oxidative DNA damage. This work suggest that accumulation of these lesions may lead to a high rate of mutations and to deleterious changes in gene expression, increasing breast cancer risk and contributing to breast carcinogenesis.     

Formation of 8-nitroguanine from 2'-deoxyguanosine by NO/O2 system.

We investigated the reaction of 2'-deoxyguanosine (dGuo) with NO/O2 gas mixture under physiological condition and detected 8-nitroguanine, which is known as a novel DNA lesion caused by peroxynitrite (ONOO-). The yield increased with increase in the ratio of O2 and pH. The reaction mechanism is discussed.     

Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry

The analysis of damage products as biomarkers of inflammation has been hampered by a poor understanding of the chemical biology of inflammation, the lack of sensitive analytical methods and a focus on single chemicals as surrogates for inflammation. To overcome these problems, we developed a general and sensitive liquid chromatographic tandem mass spectrometry (LC/MS-MS) method to quantify, in a single DNA sample, the nucleoside forms of seven DNA lesions reflecting the range of chemistries associated with inflammation: 2'-deoxyuridine, 2'-deoxyxanthosine and 2'-deoxyinosine from nitrosative deamination; 8-oxo-2'-deoxyguanosine from oxidation; and 1,N(2)-etheno-2'-deoxyguanosine, 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine arising from reaction of DNA with lipid peroxidation products. Using DNA purified from cells or tissues under conditions that minimize artifacts, individual nucleosides are purified by HPLC and quantified by isotope-dilution, electrospray ionization LC/MS-MS. The method can be applied to other DNA damage products and requires 4-6 d to complete depending upon the number of samples.     

DNA damage and 2'-deoxyguanosine oxidation induced by S(IV) autoxidation catalyzed by copper(II) tetraglycine complexes: synergistic effect of a second metal ion

S(IV) (SO(2),HSO(3)(-)andSO(3)(2-)) autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0x10(-4)M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0x10(-4)M) in the presence of Ni(II) or Mn(II) (10(-7)-10(-6)M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. SO*(3)(-) and HO* radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide).     

Recent aspects of oxidative DNA damage: guanine lesions,measurement and substrate specificity of DNA repair glycosylases

This review discusses recent aspects of oxidation reactions of DNA and model compounds involving mostly OH radicals, one-electron transfer process and singlet oxygen (1O2). Emphasis is placed on the formation of double DNA lesions involving a purine base on one hand and either a pyrimidine base or a 2-deoxyribose moiety on the other hand. Structural and mechanistic information is also provided on secondary oxidation reactions of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a major DNA marker of oxidative stress. Another major topic which is addressed here deals with recent developments in the measurement of oxidative base damage to cellular DNA. This has been mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents, including UVA and ionizing radiations. In addition, the modified comet assay, which involves the use of bacterial DNA N-glycosylases to reveal two main classes of oxidative base damage, is applicable to isolated cells and is particularly suitable when only small amounts of biological material are available. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision pathways are briefly reviewed.