Oxidative Decay of DNA

The study of DNA oxidation has progressed from an exploratory phase, during which its basic biochemistry was established, into a field branching out into numerous areas. Early on, radiation biologists discovered that radiolysis of water generates oxygen free radicals, which are responsible for many of the consequences of irradiating living things. The characterization of radiation-induced oxidative DNA lesions, and the connection between radiation and cancer, caused a surge of interest in DNA oxidationper se and raised the possibility of DNA damage from biological oxidants. Nucleic acid biochemists, cancer biologists, and toxicologists then set out to ask key questions: "how much oxidative DNA damage is there, how does it get there, how and when is it removed, and what are the consequences?" A proliferation of techniques has resulted in the confirmation of the early hypotheses and also delivered some surprises. In this minireview, we have outlined some of the most interesting recent results. Extensive reviews on DNA oxidation published elsewhere have discussed the earlier work in detail (1Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5354) Google Scholar, 2Dreher D. Junod A.F. Eur. J. Cancer. 1996; 32A: 30-38Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 3Loft S. Poulsen H.E. J. Mol. Med. 1996; 74: 297-312Crossref PubMed Scopus (822) Google Scholar, 4Richter C. Int. J. Biochem. Cell Biol. 1995; 27: 647-653Crossref PubMed Scopus (283) Google Scholar, 5Wiseman H. Kaur H. Halliwell B. Cancer Lett. 1995; 93: 113-120Crossref PubMed Scopus (121) Google Scholar). A companion minireview by Henle and Linn (6Henle E.S. Linn S. J. Biol. Chem. 1997; 272: 19095-19098Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar) covers in depth the biochemistry of DNA oxidation. The steady-state amount of DNA oxidation appears to be massive, with oxidative adducts occurring at a frequency that is 1 or more orders of magnitude higher than non-oxidative adducts (1Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5354) Google Scholar, 7Ames B.N. Gold L.S. Willett W.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5258-5265Crossref PubMed Scopus (1121) Google Scholar). Despite their abundance, oxidative DNA adducts exist in a large background (105–106) of unaltered nucleotides, which may be prone to oxidation during sample preparation and analysis. Concerns about artifactual oxidation, combined with the different values that have been generated by alternative methods, have fueled an ongoing debate over the most appropriate techniques for studying DNA oxidation. Gas chromatography coupled with mass spectroscopy (GC-MS), 1The abbreviations used are: GC-MS, gas chromatography-mass spectroscopy; HPLC, high pressure liquid chromatography; oxo8dG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; oxo8Gua, 8-oxo-guanine; EC, electrochemical; Fapy, formamidopyrimidine; PCR, polymerase chain reaction; Q-PCR, quantitative PCR; mAb, monoclonal antibody. initially used in characterizing oxidative adducts, is also a quantitative tool whose principal advantage is the simultaneous analysis of a number of different adducts (5Wiseman H. Kaur H. Halliwell B. Cancer Lett. 1995; 93: 113-120Crossref PubMed Scopus (121) Google Scholar). DNA is chemically hydrolyzed, derivatized, and injected onto GC-MS. In the absence of a mass spectrometer, an alternative approach is the enzymatic hydrolysis of DNA to nucleosides and chromatography of the hydrolysate by HPLC (8Shigenaga M.K. Aboujaoude E.N. Chen Q. Ames B.N. Methods Enzymol. 1994; 234: 16-33Crossref PubMed Scopus (292) Google Scholar). The adducts 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo8dG) and its corresponding base 8-oxo-guanine (oxo8Gua) are especially useful in this regard, since they are electrochemically active, lending themselves to sensitive electrochemical (EC) detection. The relative simplicity and high sensitivity of HPLC-EC detection of oxo8dG have made it the most popular method for monitoring DNA oxidation in vivo. Generally speaking, GC-MS estimates of DNA oxidation have been higher than HPLC-EC estimates, by about a factor of 10 (9Douki T. Delatour T. Bianchini F. Cadet J. Carcinogenesis. 1996; 17: 347-353Crossref PubMed Scopus (112) Google Scholar). The debate about the cause of the difference (an overestimate due to artifactual oxidation with GC-MS versus an underestimate due to inefficient enzymatic digestion with HPLC-EC) has now been settled; artifactual oxidation occurs during GC-MS derivatization, in the case of guanine/oxo8dG (10Ravanat J.L. Turesky R.J. Gremaud E. Trudel L.J. Stadler R.H. Chem. Res. Toxicol. 1995; 8: 1039-1045Crossref PubMed Scopus (198) Google Scholar) and in the case of adducts formed from adenine, cytosine, thymine, and thymidine (9Douki T. Delatour T. Bianchini F. Cadet J. Carcinogenesis. 1996; 17: 347-353Crossref PubMed Scopus (112) Google Scholar). The HPLC-EC method itself, however, has also been criticized on the grounds that the variability of the assay is unacceptable (11Adachi S. Zeisig M. Moller L. Carcinogenesis. 1995; 16: 253-258Crossref PubMed Scopus (88) Google Scholar, 12Nakae D. Mizumoto Y. Kobayashi E. Noguchi O. Konishi Y. Cancer Lett. 1995; 97: 233-239Crossref PubMed Scopus (144) Google Scholar, 13Nakajima M. Takeuchi T. Morimoto K. Carcinogenesis. 1996; 17: 787-791Crossref PubMed Scopus (71) Google Scholar, 14Finnegan M.T.V. Herbert K.E. Evans M.D. Griffiths H.R. Lunec J. Free Radical Biol. Med. 1996; 20: 93-98Crossref PubMed Scopus (35) Google Scholar, 15Nakajima M. Takeuchi T. Takeshita T. Morimoto K. Environ. Health Perspect. 1996; 104: 1336-1338Crossref PubMed Scopus (66) Google Scholar). Estimates of the ratio of oxo8dG to dG (for example, in rat tissues) have ranged from approximately 0.25 × 10−5 to higher than 10−4, and it has been suggested that artifactual oxidation is to blame. Artifactual oxidation poses problems of accuracy and precision. For example, the total cellular burden of oxidative adducts has been estimated from HPLC-EC measurements of oxo8dG (1Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5354) Google Scholar), on the assumption that oxo8dG represents 5% of all adducts (it is one of about 20 major radiation adducts characterized by GC-MS (16Dizdaroglu M. Mutat. Res. 1992; 275: 331-342Crossref PubMed Scopus (493) Google Scholar)). This estimate, about a million oxidative adducts per rat cell (1Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5354) Google Scholar), is a number which argues forcibly that oxidative mutagenesis must be important in vivo (2Dreher D. Junod A.F. Eur. J. Cancer. 1996; 32A: 30-38Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 3Loft S. Poulsen H.E. J. Mol. Med. 1996; 74: 297-312Crossref PubMed Scopus (822) Google Scholar, 7Ames B.N. Gold L.S. Willett W.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5258-5265Crossref PubMed Scopus (1121) Google Scholar). To the extent that the initial measurement of oxo8dG may be artificially elevated, this estimate may also be inaccurate. Worse, perhaps, is the effect that artifactual oxidation has on the ability to detect an elevation of oxo8dG. If the DNA damage "signal" is obscured by background artifact "noise," then real increases in DNA oxidation may be obscured or fail to achieve statistical significance. Fortunately, a number of incremental improvements have recently been introduced (12Nakae D. Mizumoto Y. Kobayashi E. Noguchi O. Konishi Y. Cancer Lett. 1995; 97: 233-239Crossref PubMed Scopus (144) Google Scholar, 13Nakajima M. Takeuchi T. Morimoto K. Carcinogenesis. 1996; 17: 787-791Crossref PubMed Scopus (71) Google Scholar), driving down the estimate of steady-state oxo8dG 2–5-fold from previous estimates. The current lowest estimates of the ratio of oxo8dG/dG (in rat hepatocytes and human lymphocytes) cluster around 0.25 × 10−5, equivalent to approximately 7,500 oxo8dG or about 1.5 × 105 oxidative adducts per human cell (if oxo8dG represents 5% of all such adducts) (12Nakae D. Mizumoto Y. Kobayashi E. Noguchi O. Konishi Y. Cancer Lett. 1995; 97: 233-239Crossref PubMed Scopus (144) Google Scholar,13Nakajima M. Takeuchi T. Morimoto K. Carcinogenesis. 1996; 17: 787-791Crossref PubMed Scopus (71) Google Scholar). 150,000 oxidative adducts per cell represents a huge load of damage: is there solid evidence that this number is accurate? Although it is difficult to rule out some contribution by artifactual oxidation to these values (or indeed to values derived from any of the techniques that have been devised), emerging features of experiments with oxo8dG lend them credibility. Namely, recent studies show low sample-to-sample variance, as well as dramaticpatterns of appearance and disappearance of oxo8dG following oxidant challenges, occurring in parallel with the induction of oxo8dG repair activity (13Nakajima M. Takeuchi T. Morimoto K. Carcinogenesis. 1996; 17: 787-791Crossref PubMed Scopus (71) Google Scholar, 17Asami S. Hirano T. Yamaguchi R. Tomioka Y. Itoh H. Kasai H. Cancer Res. 1996; 56: 2546-2549PubMed Google Scholar, 18Hirano T. Yamaguchi R. Asami S. Iwamoto N. Kasai H. J. Gerontol. A Biol. Sci. Med. Sci. 1996; 51: B303-307Crossref PubMed Scopus (61) Google Scholar, 19Kaneko T. Tahara S. Matsuo M. Mutat. Res. 1996; 316: 277-285Crossref PubMed Scopus (178) Google Scholar, 20Lee Y.S. Choi J.Y. Park M.K. Choi E.M. Kasai H. Chung M.H. Mutat. Res. 1996; 364: 227-233Crossref PubMed Scopus (43) Google Scholar, 21Umemura T. Hasegawa R. Sai-Kato K. Nishikawa A. Furukawa F. Toyokuni S. Uchida K. Inoue T. Kurokawa Y. Jpn. J. Cancer Res. 1996; 87: 882-886Crossref PubMed Scopus (47) Google Scholar). Together with the fact that radically different techniques (discussed below) have demonstrated similar degrees and patterns of induced DNA damage, these results suggest that the problem of artifactual noise has been tamed, if not eliminated. Elsewhere, we have discussed in detail how to avoid the artifacts that can occur with HPLC-EC. 2H. Helbock, K. B. Beckman, P. Walter, M. K. Shigenaga, A. A. Woodall, H. C. Yeo, and B. N. Ames, manuscript in preparation. The cloning and overexpression of repair enzymes continue to provide new ways to detect oxidative adducts. Enzymes such as Escherichia coli endonuclease III and formamidopyrimidine glycosylase (Fapy glycosylase), which recognize and excise oxidized pyrimidines and purines, respectively, possess associated lyase activities that result in strand cleavage (23Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1282) Google Scholar). Treating DNA with these enzymes introduces nicks, which may then be measured by alkaline elution (24Ballmaier D. Epe B. Carcinogenesis. 1995; 16: 335-342Crossref PubMed Scopus (106) Google Scholar), nick translation (25Czene S. Harms-Ringdahl M. Mutat. Res. 1995; 336: 235-242Crossref PubMed Scopus (44) Google Scholar), or ring opening of supercoiled molecules (26Epe B. Hegler J. Methods Enzymol. 1994; 234: 122-131Crossref PubMed Scopus (124) Google Scholar). The polymerase chain reaction (PCR) has provided an approach called "quantitative PCR" (Q-PCR), which takes advantage of the fact that many DNA lesions block thermostable DNA polymerases, thereby decreasing the efficiency of amplification (27Yakes F.M. Van Houten B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 514-519Crossref PubMed Scopus (1413) Google Scholar). As the length of the desired amplicon increases, the probability that a strand-terminating adduct will occur also increases, as does the sensitivity of the method. With appropriate controls and calculations, the technique is quantitative, although the types of DNA lesions resulting in decreased amplification are only known in general. There is potential that Q-PCR may be coupled with repair endonucleases, enabling the quantification of specific adducts. All of the techniques discussed so far, from GC-MS to Q-PCR, require purified DNA. A technique for estimating the rate of oxo8dG formation, which does not require the isolation of DNA and its associated problems, is the measurement of its repair products excreted into urine or tissue culture medium (3Loft S. Poulsen H.E. J. Mol. Med. 1996; 74: 297-312Crossref PubMed Scopus (822) Google Scholar, 28Shigenaga M.K. Gimeno C.J. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9697-9701Crossref PubMed Scopus (692) Google Scholar, 29Loft S. Vistisen K. Ewertz M. Tjonneland A. Overvad K. Poulsen H.E. Carcinogenesis. 1992; 13: 2241-2247Crossref PubMed Scopus (587) Google Scholar). The daily flux of repaired adducts should reflect the intracellular rate of DNA damage, if not in a direct way. Although there is a different set of concerns surrounding the accuracy of these methods (such as uncertainty about repair pathways and products, oxidation of free nucleotide pools, and the contribution of cell and mitochondrial turnover), an overwhelming advantage of these methods is that they are non-invasive and integrative. As a consequence, the measurement of repaired adducts in urine is one of the few techniques that has been routinely applied to humans. Recently, an elegant and related approach has been reported: the measurement of oxo8dG in small amounts of heart muscle interstitial fluid, collected with a microdialysis probe. During a period of reperfusion following ischemia (a well established model of oxidative stress) a rapid increase in intercellular oxo8dG was observed (30Yang C.-S. Tsai P.-J. Chen W.-Y. Kuo J.-S. Redox Report. 1996; 2: 379-383Crossref PubMed Google Scholar). Last, there exist two techniques that preserve cellular integrity: single-cell gel electrophoresis and immunohistochemistry with anti-DNA-adduct monoclonal antibodies (mAbs). Single-cell gel electrophoresis, also descriptively termed the "comet" assay, involves casting cells in a thin agarose gel on a microscope slide and running the DNA out of the nuclei by electrophoresis. The more fragmented the chromatin, the more it migrates, assuming (upon staining) the appearance of a comet's tail streaking away from the nucleus in the direction of the anode. The analysis of the length and intensity of the tail (its "moment") is achieved with the help of software (31Fairbairn D.W. Olive P.L. O'Neill K.L. Mutat. Res. 1995; 339: 37-59Crossref PubMed Scopus (1481) Google Scholar). Modifications of the assay permit the analysis of specific lesions; alkaline conditions are used to study single-strand nicks, and by treating cells in agarose in situ with an enzyme such as Fapy glycosylase prior to single-cell gel electrophoresis, the method has been used to detect the substrate oxo8Gua (32Collins A.R. Ma A.G. Duthie S.J. Mutat. Res. 1995; 336: 69-77Crossref PubMed Scopus (612) Google Scholar, 33Dennog C. Hartmann A. Frey G. Speit G. Mutagenesis. 1996; 11: 605-609Crossref PubMed Scopus (98) Google Scholar). The specificity of mAbs has also been utilized; mAbs to thymine glycol, for instance, are used in enzyme-linked immunosorbent assays of purified DNA (34Cooper P.K. Nouspikel T. Clarkson S.G. Leadon S.A. Science. 1997; 275: 990-993Crossref PubMed Scopus (285) Google Scholar). The ultimate power of mAbs, however, may lie in their ability to detect DNA damage in fixed cells and tissues in situ, as was recently reported for a mAb to oxo8Gua (35Yarborough A. Zhang Y.J. Hsu T.M. Santella R.M. Cancer Res. 1996; 56: 683-688PubMed Google Scholar). There is no single ideal method for measuring oxidative lesions, as all have their strengths and weaknesses. For instance, GC-MS and HPLC-EC are rigorously quantitative but require relatively large quantities of pure nucleic acids. Molecular biological methods like Q-PCR require less DNA but are not as specific in their detection. Cellular assays are ideal for analyzing tiny samples (hundreds of cells) and avoiding cellular disruption but are semi-quantitative. What is encouraging about recent results is the growing congruence between studies using different approaches. Until the last 2 years, it had become almost accepted wisdom that the role of the superoxide anion radical (O·̄2) in DNA oxidation was its ability to reduce ferric iron (Fe3+) to ferrous iron (Fe2+); Fe2+ catalyzes the formation of the hydroxyl radical ⋅OH (from H2O2) which, according to the scheme (referred to as Fenton chemistry), is the ultimate reactive species in DNA oxidation (6Henle E.S. Linn S. J. Biol. Chem. 1997; 272: 19095-19098Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Support for the roles of all three components of this model (O·̄2, iron, and H2O2) continues to accumulate (36Takeuchi T. Nakajima M. Morimoto K. Carcinogenesis. 1996; 17: 1543-1548Crossref PubMed Scopus (62) Google Scholar, 37Teixeira H.D. Meneghini R. Biochem. J. 1996; 315: 821-825Crossref PubMed Scopus (23) Google Scholar). However, as is discussed in the companion minireview by Henle and Linn (6Henle E.S. Linn S. J. Biol. Chem. 1997; 272: 19095-19098Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar), the nuances of DNA oxidation have turned out to be more complex and interesting. For one, the nature of the ultimate oxidant responsible for DNA damage by H2O2 is unclear. Detailed experiments have illustrated that a model of freely diffusible⋅OH fails to account for the strikingly parallel dynamics of DNA strand scission in vitro and cytotoxicity of H2O2 to E. coli. Rather, multiple classes of oxidant appear to exist, associated with the DNA double helix to different extents (6Henle E.S. Linn S. J. Biol. Chem. 1997; 272: 19095-19098Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Moreover, the role of O·̄2 in reducing free ferric iron has been challenged by experiments suggesting that its principal role is to release iron from protein-bound iron-sulfur clusters (38Keyer K. Imlay J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640Crossref PubMed Scopus (671) Google Scholar). Besides O·̄2, there are other reductants (such as NADH) that effectively reduce Fe3+ to Fe2+ and that may be more relevant as reductants of free or DNA-bound iron than is O·̄2 (39Keyer K. Gort A.S. Imlay J.A. J. Bacteriol. 1995; 177: 6782-6790Crossref PubMed Scopus (182) Google Scholar). Copper (40Kasprzak K.S. Cancer Invest. 1995; 13: 411-430Crossref PubMed Scopus (261) Google Scholar) and less well studied transition metals such as chromium (41Tsou T.C. Chen C.L. J.L. Carcinogenesis. 1996; 17: PubMed Scopus Google Scholar) also in in DNA oxidation. such as also with other the adduct oxo8dG T. Cadet J. Free Radical Res. 1996; PubMed Scopus Google Scholar, S. S. Lett. 1995; PubMed Scopus Google Scholar, R.H. Mutat. Res. 1995; 339: PubMed Scopus Google Scholar). oxo8dG is more to than which the fact that more oxidative products than oxo8dG exist R.M. R. Free Radical Biol. Med. 1996; PubMed Scopus Google Scholar, T. 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If such a is important in it may that the of DNA to or oxidation in In E. oxidative DNA damage is by pathways nucleotide and base The endonuclease which and oxidized and Fapy glycosylase (23Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1282) Google Scholar), which a similar role on oxidized The enzyme is one of three products in the a set of three repair enzymes that mutagenesis by by oxo8Gua with with oxo8Gua and by the oxidized nucleotide to the thereby its into DNA. Although a of oxidative repair enzymes it is important to the fact that of the E. coli activities may result in increases in the rate of mutagenesis and that or activities have been in the cloning of human of endonuclease III R. T. C. C.J. T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar), C. J. Bacteriol. 1996; PubMed Scopus Google Scholar), and K. M. T. T. S. H. M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) and the recent of a activity M. A. K. S. Y. Kasai H. J. Mutat. Res. 1997; PubMed Scopus Google Scholar). As be of a of DNA damage, repair of oxidative lesions appears The cloning of in repair of oxidative damage and the of by will permit a of the hypotheses of cancer and C. J. Bacteriol. 1996; PubMed Scopus Google Scholar). possess in to their of The which an associated oxo8Gua lyase activity and is to the of E. may be one such A. L. J. 1996; PubMed Scopus Google Scholar). In to its in the a at between translation and DNA Moreover, of the human have been reported in and a associated with of the adduct oxo8dG. cells also the burden of DNA repair to their an which, early to the is to oxidative DNA damage Mutat. Res. 1995; PubMed Scopus Google Scholar). in to resulting from of there may be or associated with inefficient repair of In for instance, a in the repair of oxidative damage of and is observed Cancer Res. 1996; 56: Google Scholar). The of oxidative adducts in human cells appears to be In the of adducts ranged from to P. M. Nucleic Res. 1996; PubMed Scopus Google Scholar). In human repair of some adducts (for example, is so rapid that a of for their detection A. R. Halliwell B. Biochem. Res. 1996; PubMed Scopus Google Scholar). The of DNA adducts should be oxo8dG may appear and with an in the cells thymine and single-strand a result of may increase A. R. Halliwell B. Biochem. Res. 1996; PubMed Scopus Google Scholar). or the effect of a low to an that is at high M. Science. 1996; PubMed Scopus Google Scholar) may be relevant for some oxidative as has been to be the case for low radiation S. Mutat. Res. 1996; PubMed Scopus Google Scholar). oxygen of at for instance, oxidative DNA damage to cells on the of but fails to cause damage on C. Hartmann A. Frey G. Speit G. Mutagenesis. 1996; 11: 605-609Crossref PubMed Scopus (98) Google in it results in a of total and oxidative DNA of which oxidative adducts in chromatin, results in of some oxidative DNA adducts an B. B. R. Free Radical Biol. Med. 1997; PubMed Scopus Google Scholar), and other of the of oxidative damage by have (27Yakes F.M. Van Houten B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 514-519Crossref PubMed Scopus (1413) Google Scholar). results are not since are induced in to oxidative a that has recently been to oxo8Gua glycosylase activity in E. coli Park Kasai H. S. Park Choi Chung M.H. Mutat. Res. 1996; PubMed Scopus Google Scholar) and Y.S. Choi J.Y. Park M.K. Choi E.M. Kasai H. Chung M.H. Mutat. Res. 1996; 364: 227-233Crossref PubMed Scopus (43) Google Scholar). This that there is a of in oxidative and repair and that cells may a burden of oxidative adducts that to the rate of and evidence that DNA oxidation is and is a major to human cancer three major and such as from (1Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5354) Google Scholar, 3Loft S. Poulsen H.E. J. Mol. Med. 1996; 74: 297-312Crossref PubMed Scopus (822) Google Scholar, 7Ames B.N. Gold L.S. Willett W.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5258-5265Crossref PubMed Scopus (1121) Google Scholar, J. 1996; 63: PubMed Scopus Google T.M. K.L. Park Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). high of and the and cells to of reactive such as and such as these may to as much as of all human and evidence of oxidative damage during is to the most recent DNA oxidation has been measured during cancer