Advertisement

Oxidative Decay of DNA*

  • Kenneth B. Beckman
    Affiliations
    Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202
    Search for articles by this author
  • Bruce N. Ames
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Barker Hall, University of California, Berkeley, CA 94720-3202. Tel.: 510-642-5165; Fax: 510-643-7935;
    Affiliations
    Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202
    Search for articles by this author
  • Author Footnotes
    * This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. This is the third article of five in the “Oxidative Modification of Macromolecules Minireview Series.” This work was supported by National Cancer Institute Outstanding Investigator Grant CA33910 (to B. N. A.) and National Institute of Environmental Health Sciences Center Grant ES01896.
      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 (
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ,
      • Dreher D.
      • Junod A.F.
      ,
      • Loft S.
      • Poulsen H.E.
      ,
      • Richter C.
      ,
      • Wiseman H.
      • Kaur H.
      • Halliwell B.
      ). A companion minireview by Henle and Linn (
      • Henle E.S.
      • Linn S.
      ) covers in depth the biochemistry of DNA oxidation.

      Methods for Measuring Oxidative DNA Damage

      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 (
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ,
      • Ames B.N.
      • Gold L.S.
      • Willett W.C.
      ). 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),
      The 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 (
      • Wiseman H.
      • Kaur H.
      • Halliwell B.
      ). 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 (
      • Shigenaga M.K.
      • Aboujaoude E.N.
      • Chen Q.
      • Ames B.N.
      ). 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 (
      • Douki T.
      • Delatour T.
      • Bianchini F.
      • Cadet J.
      ). 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 (
      • Ravanat J.L.
      • Turesky R.J.
      • Gremaud E.
      • Trudel L.J.
      • Stadler R.H.
      ) and in the case of adducts formed from adenine, cytosine, thymine, and thymidine (
      • Douki T.
      • Delatour T.
      • Bianchini F.
      • Cadet J.
      ). The HPLC-EC method itself, however, has also been criticized on the grounds that the variability of the assay is unacceptable (
      • Adachi S.
      • Zeisig M.
      • Moller L.
      ,
      • Nakae D.
      • Mizumoto Y.
      • Kobayashi E.
      • Noguchi O.
      • Konishi Y.
      ,
      • Nakajima M.
      • Takeuchi T.
      • Morimoto K.
      ,
      • Finnegan M.T.V.
      • Herbert K.E.
      • Evans M.D.
      • Griffiths H.R.
      • Lunec J.
      ,
      • Nakajima M.
      • Takeuchi T.
      • Takeshita T.
      • Morimoto K.
      ). 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 (
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ), on the assumption that oxo8dG represents 5% of all adducts (it is one of about 20 major radiation adducts characterized by GC-MS (
      • Dizdaroglu M.
      )). This estimate, about a million oxidative adducts per rat cell (
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ), is a number which argues forcibly that oxidative mutagenesis must be important in vivo (
      • Dreher D.
      • Junod A.F.
      ,
      • Loft S.
      • Poulsen H.E.
      ,
      • Ames B.N.
      • Gold L.S.
      • Willett W.C.
      ). 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 (
      • Nakae D.
      • Mizumoto Y.
      • Kobayashi E.
      • Noguchi O.
      • Konishi Y.
      ,
      • Nakajima M.
      • Takeuchi T.
      • Morimoto K.
      ), 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) (
      • Nakae D.
      • Mizumoto Y.
      • Kobayashi E.
      • Noguchi O.
      • Konishi Y.
      ,
      • Nakajima M.
      • Takeuchi T.
      • Morimoto K.
      ).
      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 (
      • Nakajima M.
      • Takeuchi T.
      • Morimoto K.
      ,
      • Asami S.
      • Hirano T.
      • Yamaguchi R.
      • Tomioka Y.
      • Itoh H.
      • Kasai H.
      ,
      • Hirano T.
      • Yamaguchi R.
      • Asami S.
      • Iwamoto N.
      • Kasai H.
      ,
      • Kaneko T.
      • Tahara S.
      • Matsuo M.
      ,
      • Lee Y.S.
      • Choi J.Y.
      • Park M.K.
      • Choi E.M.
      • Kasai H.
      • Chung M.H.
      ,
      • Umemura T.
      • Hasegawa R.
      • Sai-Kato K.
      • Nishikawa A.
      • Furukawa F.
      • Toyokuni S.
      • Uchida K.
      • Inoue T.
      • Kurokawa Y.
      ). 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.
      H. 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 (
      • Demple B.
      • Harrison L.
      ). Treating DNA with these enzymes introduces nicks, which may then be measured by alkaline elution (
      • Ballmaier D.
      • Epe B.
      ), nick translation (
      • Czene S.
      • Harms-Ringdahl M.
      ), or ring opening of supercoiled molecules (
      • Epe B.
      • Hegler J.
      ).
      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 (
      • Yakes F.M.
      • Van Houten B.
      ). 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 (
      • Loft S.
      • Poulsen H.E.
      ,
      • Shigenaga M.K.
      • Gimeno C.J.
      • Ames B.N.
      ,
      • Loft S.
      • Vistisen K.
      • Ewertz M.
      • Tjonneland A.
      • Overvad K.
      • Poulsen H.E.
      ). 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 (
      • Yang C.-S.
      • Tsai P.-J.
      • Chen W.-Y.
      • Kuo J.-S.
      ).
      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 (
      • Fairbairn D.W.
      • Olive P.L.
      • O'Neill K.L.
      ). 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 (
      • Collins A.R.
      • Ma A.G.
      • Duthie S.J.
      ,
      • Dennog C.
      • Hartmann A.
      • Frey G.
      • Speit G.
      ). The specificity of mAbs has also been utilized; mAbs to thymine glycol, for instance, are used in enzyme-linked immunosorbent assays of purified DNA (
      • Cooper P.K.
      • Nouspikel T.
      • Clarkson S.G.
      • Leadon S.A.
      ). 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 (
      • Yarborough A.
      • Zhang Y.J.
      • Hsu T.M.
      • Santella R.M.
      ).
      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.

      Mechanisms and Location of DNA Oxidation

      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 (
      • Henle E.S.
      • Linn S.
      ). Support for the roles of all three components of this model (O·̄2, iron, and H2O2) continues to accumulate (
      • Takeuchi T.
      • Nakajima M.
      • Morimoto K.
      ,
      • Teixeira H.D.
      • Meneghini R.
      ). However, as is discussed in the companion minireview by Henle and Linn (
      • Henle E.S.
      • Linn S.
      ), 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 diffusibleOH 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 (
      • Henle E.S.
      • Linn S.
      ). 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 (
      • Keyer K.
      • Imlay J.A.
      ). 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 (
      • Keyer K.
      • Gort A.S.
      • Imlay J.A.
      ). Copper (
      • Kasprzak K.S.
      ) and less well studied transition metals such as chromium (
      • Tsou T.C.
      • Chen C.L.
      • Liu T.Y.
      • Yang J.L.
      ) also take part in Fenton-like chemistry in DNA oxidation.
      Reactive nitrogen intermediates such as peroxynitrite (ONOO) also react with DNA, forming (among other lesions) the adduct oxo8dG (
      • Douki T.
      • Cadet J.
      ,
      • Inoue S.
      • Kawanishi S.
      ,
      • Liu R.H.
      • Hotchkiss J.H.
      ). Interestingly, oxo8dG itself is far more susceptible to peroxynitrite than dG, which emphasizes the fact that more stable oxidative end products than oxo8dG exist (
      • Uppu R.M.
      • Cueto R.
      • Squadrito G.L.
      • Salgo M.G.
      • Pryor W.A.
      ,
      • Douki T.
      • Cadet J.
      • Ames B.N.
      ). Also, oxidative DNA adducts may be formed indirectly; the peroxidation of membrane lipids results in various aldehyde breakdown products that are able to form covalent mutagenic adducts (
      • Douki T.
      • Ames B.N.
      ). Recently, we have reported that the concentration of protein-bound aldehyde accumulates with age in rats (
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ), suggesting that aldehyde-DNA adducts may also increase with age. A final complication is the evidence that DNA is not a homogeneous target of oxidative damage and repair. Internucleosomal DNA appears at least 3.5 times more susceptible than nucleosomal DNA to oxidation by physiological iron chelates (
      • Enright H.
      • Miller W.J.
      • Hays R.
      • Floyd R.A.
      • Hebbel R.P.
      ), and repair of a number of adducts is more rapid in DNA in the nuclear matrix than in total chromatin (
      • Zastawny T.H.
      • Czerwinska B.
      • Drzewiecka B.
      • Olinski R.
      ).
      A fascinating subtlety of DNA oxidation involves the possibility that oxidation may be mediated by long distance electron transport along the π stack of the DNA double helix. Experiments with synthetic double-stranded oligonucleotides have shown that long range oxidative damage may occur, resulting in the formation of oxo8dG in susceptible 5-GG-3′ at a distance from a covalently attached terminal oxidant (
      • Hall D.B.
      • Holmlin R.E.
      • Barton J.K.
      ). If such a phenomenon is important in vivo, it may mean that the topology of DNA serves to channel or trap oxidation in zones.

      Oxidative Mutagenesis: GOing, GOing, GOne

      In E. coli, oxidative DNA damage is removed by pathways involving both nucleotide and base excision pathways. The latter include endonuclease III, which recognizes and removes oxidized pyrimidines, and Fapy glycosylase (
      • Demple B.
      • Harrison L.
      ), which performs a similar role on oxidized purines. The latter enzyme is one of three gene products in the coordinated “GO system” (mutM, mutT,mutY), a set of three repair enzymes that suppress mutagenesis by Guanine Oxidation, by removing oxo8Gua paired with cytosine (mutM), adenine paired with oxo8Gua (mutY), and by hydrolyzing the oxidized nucleotide oxo8dGTP to the nucleoside monophosphate (mutT), thereby preventing its incorporation into DNA. Although space constraints preclude a full discussion of oxidative repair enzymes here, it is important to stress the fact that losses of the E. coli activities may result in 10–1000-fold increases in the rate of spontaneous mutagenesis and that homologous genes or activities have been identified in humans, including the cloning of human homologs of endonuclease III (
      • Aspinwall R.
      • Rothwell D.G.
      • Roldan-Arjona T.
      • Anselmino C.
      • Ward C.J.
      • Cheadle J.P.
      • Sampson J.R.
      • Lindahl T.
      • Harris P.C.
      • Hickson I.D.
      ), mutY (
      • Slupska M.M.
      • Baikalov C.
      • Luther W.M.
      • Chiang J.H.
      • Wei Y.F.
      • Miller J.H.
      ), and mutT (
      • Sakumi K.
      • Furuichi M.
      • Tsuzuki T.
      • Kakuma T.
      • Kawabata S.
      • Maki H.
      • Sekiguchi M.
      ) and the recent identification of a MutM-like activity (
      • Nagashima M.
      • Sasaki A.
      • Morishita K.
      • Takenoshita S.
      • Nagamachi Y.
      • Kasai H.
      • Yokota J.
      ). As would be expected of a fundamental type of DNA damage, repair of oxidative lesions appears widely conserved. The cloning of mouse genes involved in repair of oxidative damage and the subsequent generation of transgenic knockout mice by recombination will permit a powerful test of the “oxidative mutagenesis” hypotheses of cancer and aging (
      • Slupska M.M.
      • Baikalov C.
      • Luther W.M.
      • Chiang J.H.
      • Wei Y.F.
      • Miller J.H.
      ).
      Eukaryotes likely possess unique systems in addition to their homologs of prokaryotic enzymes. The Drosophila ribosomal S3 protein, which possesses an associated oxo8Gua glycosylase/AP lyase activity and is able to restore the wild-type phenotype tomutM mutants of E. coli, may be one such example (
      • Yacoub A.
      • Augeri L.
      • Kelley M.R.
      • Doetsch P.W.
      • Deutsch W.A.
      ). In addition to its involvement in protein synthesis, the S3 protein possesses a nuclear localization signal, hinting at communication between translation and DNA repair. Moreover, aberrant levels of the human ribosomal S3 protein have been reported in xeroderma pigmentosum and Fanconi's anemia, a disease associated with elevated levels of the adduct oxo8dG. Mammalian cells also face the additional burden of delivering DNA repair capacity to their mitochondria, an organelle which, despite early reports to the contrary, is able to process oxidative DNA damage (
      • Shen C.C.
      • Wertelecki W.
      • Driggers W.J.
      • LeDoux S.P.
      • Wilson G.L.
      ). Therefore, in addition to phenotypes resulting from loss of nuclear repair, there may be collateral or independent syndromes associated with inefficient repair of mtDNA. In xeroderma pigmentosum complementation group A, for instance, a deficiency in the repair of oxidative damage of both nuclear and mtDNA is observed (
      • Driggers W.J.
      • Grishko V.I.
      • LeDoux S.P.
      • Wilson G.L.
      ).
      The removal of oxidative adducts in human cells appears to be very rapid. In lymphoblasts, the half-lives of H2O2-induced adducts ranged from 8.5 to 62 min (
      • Jaruga P.
      • Dizdaroglu M.
      ). In human respiratory tract epithelial cells, repair of some H2O2-induced adducts (for example, oxo8dG) is so rapid that a narrow window of opportunity (approximately 30 min) exists for their detection (
      • Spencer J.P.
      • Jenner A.
      • Aruoma O.I.
      • Cross C.E.
      • Wu R.
      • Halliwell B.
      ). The heterogeneity of DNA adducts should be noted; whereas oxo8dG may rapidly appear and disappear with an hour, in the same cells thymine glycol and single-strand breaks (themselves a result of repair) may increase (
      • Spencer J.P.
      • Jenner A.
      • Aruoma O.I.
      • Cross C.E.
      • Wu R.
      • Halliwell B.
      ).
      Hormesis or the “beneficial effect of a low level exposure to an agent that is harmful at high levels” (
      • Goldman M.
      ) may be relevant for some oxidative stresses, as has been argued to be the case for low level radiation exposure (
      • Wolff S.
      ). Hyperbaric oxygen therapy of humans (100% O2 at 2.5 atm), for instance, induces significant oxidative DNA damage to peripheral blood cells on the first day of therapy but fails to cause damage on subsequent days (
      • Dennog C.
      • Hartmann A.
      • Frey G.
      • Speit G.
      ); in fact, it results in a lower base-line level of total and oxidative DNA damage. Similarly, γ-irradiation of rats, which significantly elevates oxidative adducts in hepatic chromatin, results in lower base-line levels of some oxidative DNA adducts 24 h after an acute exposure (
      • Zastawny T.H.
      • Czerwinska B.
      • Drzewiecka B.
      • Olinski R.
      ), and other observations of the lowering of base-line oxidative damage by oxidants have appeared (
      • Yakes F.M.
      • Van Houten B.
      ). These results are not surprising, since defense systems are often induced in response to oxidative stress, a generalization that has recently been extended to oxo8Gua glycosylase activity in E. coli (
      • Kim H.S.
      • Park Y.W.
      • Kasai H.
      • Nishimura S.
      • Park C.W.
      • Choi K.H.
      • Chung M.H.
      ) and rats (
      • Lee Y.S.
      • Choi J.Y.
      • Park M.K.
      • Choi E.M.
      • Kasai H.
      • Chung M.H.
      ). This implies that there is a degree of slack in oxidative defense and repair under “normal” circumstances and that cells may ordinarily tolerate a burden of oxidative adducts that contributes to the spontaneous rate of mutation.

      DNA Oxidation and Cancer

      Experimental and epidemiological evidence suggests that DNA oxidation is mutagenic and is a major contributor to human cancer through three major sources: smoking, chronic inflammation, and endogenous oxidants such as leakage from mitochondria (
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ,
      • Loft S.
      • Poulsen H.E.
      ,
      • Ames B.N.
      • Gold L.S.
      • Willett W.C.
      ,
      • Jacob R.A.
      • Burri B.J.
      ,
      • Hagen T.M.
      • Yowe D.L.
      • Bartholemew J.C.
      • Wehr C.M.
      • Do K.L.
      • Park J.-Y.
      • Ames B.N.
      ). Cigarette smoke contains high levels of NO x and depletes the body's antioxidants, and phagocytic cells recruited to sites of chronic infection abundantly generate reactive oxidants such as NO x and HOCl. Oxidative stresses such as these may contribute to as much as half of all human cancers, and evidence of oxidative damage during experimental carcinogenesis is accumulating. Merely to cite the most recent results, elevated DNA oxidation has been measured during earlyHelicobacter pylori infection (stomach cancer (
      • Baik S.C.
      • Youn H.S.
      • Chung M.H.
      • Lee W.K.
      • Cho M.J.
      • Ko G.H.
      • Park C.K.
      • Kasai H.
      • Rhee K.H.
      )), ferric nitrilotriacetate administration (experimental rodent renal cancer (
      • Toyokuni S.
      • Mori T.
      • Hiai H.
      • Dizdaroglu M.
      )), smoking (lung cancer (
      • Asami S.
      • Hirano T.
      • Yamaguchi R.
      • Tomioka Y.
      • Itoh H.
      • Kasai H.
      ,
      • Ballinger S.W.
      • Bouder T.G.
      • Davis G.S.
      • Judice S.A.
      • Nicklas J.A.
      • Albertini R.J.
      )), and exposure to diesel exhaust particles (lung cancer (
      • Ichinose T.
      • Yajima Y.
      • Nagashima M.
      • Takenoshita S.
      • Nagamachi Y.
      • Sagai M.
      )), asbestos (lung cancer (
      • Chen Q.
      • Marsh J.
      • Ames B.
      • Mossman B.
      )), benzene (leukemia (
      • Liu L.
      • Zhang Q.
      • Feng J.
      • Deng L.
      • Zeng N.
      • Yang A.
      • Zhang W.
      ,
      • Hiraku Y.
      • Kawanishi S.
      )), and aflatoxin (liver cancer (
      • Shen H.M.
      • Ong C.N.
      • Lee B.L.
      • Shi C.Y.
      )). Some studies have provided more than a simple association between carcinogenic agents and oxidative DNA damage by measuring the specific induction of repair enzymes by oxidative carcinogens (
      • Yamaguchi R.
      • Hirano T.
      • Asami S.
      • Chung M.H.
      • Sugita A.
      • Kasai H.
      ) and by demonstrating the suppression of carcinogenesis by administration of antioxidants (
      • Umemura T.
      • Hasegawa R.
      • Sai-Kato K.
      • Nishikawa A.
      • Furukawa F.
      • Toyokuni S.
      • Uchida K.
      • Inoue T.
      • Kurokawa Y.
      ,
      • Hasegawa R.
      • Chujo T.
      • Sai-Kato K.
      • Umemura T.
      • Tanimura A.
      • Kurokawa Y.
      ). The latter results are consistent with the strong correlation between a high intake of fruits and vegetables, which are the principal source of dietary antioxidants, and reduction in cancer risk by as much as half (
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ,
      • Ames B.N.
      • Gold L.S.
      • Willett W.C.
      ). We have reported elevated oxidative damage to sperm DNA in smokers and in men on low serum antioxidants (vitamin C) (
      • Fraga C.G.
      • Motchnik P.A.
      • Wyrobek A.J.
      • Rempel D.M.
      • Ames B.N.
      ) and have hypothesized that oxidative damage to male germ cells contributes to cancer and birth defects in the children of male smokers (
      • Woodall A.A.
      • Ames B.N.
      ). Indeed, new epidemiological evidence indicates that all types of childhood cancer studied are increased in offspring of male smokers; for example, the risks of acute lymphoblastic leukemia, lymphoma, and brain tumors are increased three to four times (
      • Ji B.T.
      • Shu X.O.
      • Linet M.S.
      • Zheng W.
      • Wacholder S.
      • Gao Y.T.
      • Ying D.M.
      • Jin F.
      ).
      A transgenic mouse model, in which somatic mutations occurring in vivo can be measured ex vivo with the use of a shuttle vector incorporated into the mouse genome, has recently been used to quantify a 5-fold increase in mutant frequency in vivo in response to short term ischemia-reperfusion, an oxidative stress (
      • Liu P.K.
      • Hsu C.Y.
      • Dizdaroglu M.
      • Floyd R.A.
      • Kow Y.W.
      • Karakaya A.
      • Rabow L.E.
      • Cui J.K.
      ). Transgenic in vivo mutagenesis models, the use of which is becoming routine (
      • Mirsalis J.C.
      • Monforte J.A.
      • Winegar R.A.
      ), should soon permit the degree and spectrum of mutagenesis to be measured in tandem with increases in oxidative damage. Already, the spectrum of alterations in the growing data base of oncogenic human mutations provides some evidence of relevant oxidative mutagenesis, as shown by the high frequency of G-to-T transversions (a signature mutation resulting from oxo8Gua) in human p53 and ras (
      • Shen H.M.
      • Ong C.N.
      ).

      Stress and Damage from Cradle to Grave

      The role of DNA oxidation in diseases of aging and in developmental abnormalities is less well established but ripe for investigation. A fundamental unanswered question is whether or not DNA oxidation is able to adversely affect quiescent and postmitotic cells in diseases in which uncontrolled proliferation is not an issue. There is evidence that the frequency of oxidative DNA adducts increases by as much as 2-fold with age in a number of species and tissues (
      • Kaneko T.
      • Tahara S.
      • Matsuo M.
      ,
      • Fraga C.G.
      • Shigenaga M.K.
      • Park J.W.
      • Degan P.
      • Ames B.N.
      ,
      • Hirano T.
      • Yamaguchi Y.
      • Hirano H.
      • Kasai H.
      ). We have recently found that mitochondria from senescent animals produce a greater flux of oxidants than young mitochondria, which is consistent with these results and suggests a mechanism for the observation (
      • Hagen T.M.
      • Yowe D.L.
      • Bartholemew J.C.
      • Wehr C.M.
      • Do K.L.
      • Park J.-Y.
      • Ames B.N.
      ). Even potent exogenous oxidants result in a limited (several-fold at most) and rather short term increase in adduct frequency (
      • Spencer J.P.
      • Jenner A.
      • Aruoma O.I.
      • Cross C.E.
      • Wu R.
      • Halliwell B.
      ). Therefore, it may be that an age-related, persistent 50–100% increase in the steady-state level of adducts is physiologically relevant, representing an inability to prevent or repair oxidative damage. Unfortunately, the detection of such a change in the steady-state frequency of adducts requires the virtual absence of artifactual background noise. Two recent and independent studies, in which the frequency of oxo8dG in a variety of organs of Fisher 344 rats was studied, illustrate this point. In the first, a clear increase in the ratio of oxo8dG/dG was noted (
      • Hirano T.
      • Yamaguchi R.
      • Asami S.
      • Iwamoto N.
      • Kasai H.
      ), whereas in the second, the lack of a significant increase in the ratio of oxo8dG/dG was associated with higher base-line values in young animals (
      • Kaneko T.
      • Tahara S.
      • Matsuo M.
      ).
      So far, we have not made a distinction between the oxidation of nDNA and that of mtDNA, but there is a body of work based on the HPLC-EC detection of oxo8dG which suggests that the latter is higher by more than 10-fold (
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      ) and that age-related mtDNA oxidation is particularly dramatic (
      • de la Asuncion J.G.
      • Millan A.
      • Pla R.
      • Bruseghini L.
      • Esteras A.
      • Pallardo F.V.
      • Sastre J.
      • Vina J.
      ). These empirical observations have been attributed to a number of properties of mtDNA, including its proximity to metabolic oxidant generation and its established sensitivity to mutagens in general. It is worth stressing that measuring mitochondrial oxo8dG by HPLC-EC represents a great methodological challenge, due to the difficulty in purifying it in quantity. The correspondingly greater potential for artifactual oxidation during the preparation and analysis of mtDNA may explain the large disparity between published values (
      • Beckman K.B.
      • Ames B.N.
      ). The technique of Q-PCR, which requires neither DNA purification nor large amounts of DNA, has recently been used in studies of mtDNA oxidation and has confirmed the greater sensitivity of mtDNA than nDNA to exogenous oxidants (
      • Yakes F.M.
      • Van Houten B.
      ,
      • Ballinger S.W.
      • Bouder T.G.
      • Davis G.S.
      • Judice S.A.
      • Nicklas J.A.
      • Albertini R.J.
      ,
      • Shen H.M.
      • Ong C.N.
      ). However, the small number of studies, pitfalls of analyzing mtDNA, and large range of credible values (
      • Beckman K.B.
      • Ames B.N.
      ) indicate that more work on mtDNA oxidation is needed, particularly in light of the potential role of mitochondria in age-related human diseases (
      • Hagen T.M.
      • Yowe D.L.
      • Bartholemew J.C.
      • Wehr C.M.
      • Do K.L.
      • Park J.-Y.
      • Ames B.N.
      ).
      Last (or perhaps first), there is evidence from the other end of the human lifespan that oxidative damage may interfere with development. The elevation of oxo8dG by teratogens has been observed (
      • Liu L.
      • Wells P.G.
      ), and the phenotype of Cockayne's syndrome, which includes mental retardation, developmental defects, and (often) premature death in childhood, has been tightly associated with the specific lack of transcription-coupled repair of oxidative damage (
      • Cooper P.K.
      • Nouspikel T.
      • Clarkson S.G.
      • Leadon S.A.
      ). Even the process of birth itself, which represents, among other things, the first direct oxidative stress encountered by newborn mammals, induces a measurable degree of genotoxic oxidative stress (
      • Randerath E.
      • Zhou G.-D.
      • Randerath K.
      ).

      Conclusion

      In this minireview, we have attempted to highlight the recent results in this fast moving field. A principal outstanding question is: “what proportion of carcinogenic and spontaneous mutations are caused by metabolic oxidants?” Although there is evidence that specific oxidative injury, such as that associated with reperfusion injury, chronic infection, or smoking, may result in mutagenesis, it is less clear that spontaneous mutations are oxidative. The coordinated use of three different types of transgenic mouse models should soon make these tractable problems. Transgenic mice have been established that possess altered antioxidant activities (
      • Chan P.H.
      • Epstein C.J.
      • Li Y.
      • Huang T.T.
      • Carlson E.
      • Kinouchi H.
      • Yang G.
      • Kamii H.
      • Mikawa S.
      • Kondo T.
      • Copin J.-C.
      • Chen S.F.
      • Chan T.
      • Gafni J.
      • Gobbel G.
      • Reola E.
      ) and permit the in vivoquantification of mutagenesis (
      • Mirsalis J.C.
      • Monforte J.A.
      • Winegar R.A.
      ). Combined with transgenic mice possessing deleted or overexpressed oxidative repair endonucleases, they may provide ideal models (
      • Slupska M.M.
      • Baikalov C.
      • Luther W.M.
      • Chiang J.H.
      • Wei Y.F.
      • Miller J.H.
      ) for studies in which analytical measurements of oxidative adducts are combined with direct measurements of mutagenesis.

      Acknowledgments

      We thank Hal Helbock, Mark Shigenaga, and Stu Linn for critical reading of the manuscript.

      REFERENCES

        • Ames B.N.
        • Shigenaga M.K.
        • Hagen T.M.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922
        • Dreher D.
        • Junod A.F.
        Eur. J. Cancer. 1996; 32A: 30-38
        • Loft S.
        • Poulsen H.E.
        J. Mol. Med. 1996; 74: 297-312
        • Richter C.
        Int. J. Biochem. Cell Biol. 1995; 27: 647-653
        • Wiseman H.
        • Kaur H.
        • Halliwell B.
        Cancer Lett. 1995; 93: 113-120
        • Henle E.S.
        • Linn S.
        J. Biol. Chem. 1997; 272: 19095-19098
        • Ames B.N.
        • Gold L.S.
        • Willett W.C.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5258-5265
        • Shigenaga M.K.
        • Aboujaoude E.N.
        • Chen Q.
        • Ames B.N.
        Methods Enzymol. 1994; 234: 16-33
        • Douki T.
        • Delatour T.
        • Bianchini F.
        • Cadet J.
        Carcinogenesis. 1996; 17: 347-353
        • Ravanat J.L.
        • Turesky R.J.
        • Gremaud E.
        • Trudel L.J.
        • Stadler R.H.
        Chem. Res. Toxicol. 1995; 8: 1039-1045
        • Adachi S.
        • Zeisig M.
        • Moller L.
        Carcinogenesis. 1995; 16: 253-258
        • Nakae D.
        • Mizumoto Y.
        • Kobayashi E.
        • Noguchi O.
        • Konishi Y.
        Cancer Lett. 1995; 97: 233-239
        • Nakajima M.
        • Takeuchi T.
        • Morimoto K.
        Carcinogenesis. 1996; 17: 787-791
        • Finnegan M.T.V.
        • Herbert K.E.
        • Evans M.D.
        • Griffiths H.R.
        • Lunec J.
        Free Radical Biol. Med. 1996; 20: 93-98
        • Nakajima M.
        • Takeuchi T.
        • Takeshita T.
        • Morimoto K.
        Environ. Health Perspect. 1996; 104: 1336-1338
        • Dizdaroglu M.
        Mutat. Res. 1992; 275: 331-342
        • Asami S.
        • Hirano T.
        • Yamaguchi R.
        • Tomioka Y.
        • Itoh H.
        • Kasai H.
        Cancer Res. 1996; 56: 2546-2549
        • Hirano T.
        • Yamaguchi R.
        • Asami S.
        • Iwamoto N.
        • Kasai H.
        J. Gerontol. A Biol. Sci. Med. Sci. 1996; 51: B303-307
        • Kaneko T.
        • Tahara S.
        • Matsuo M.
        Mutat. Res. 1996; 316: 277-285
        • Lee Y.S.
        • Choi J.Y.
        • Park M.K.
        • Choi E.M.
        • Kasai H.
        • Chung M.H.
        Mutat. Res. 1996; 364: 227-233
        • Umemura T.
        • Hasegawa R.
        • Sai-Kato K.
        • Nishikawa A.
        • Furukawa F.
        • Toyokuni S.
        • Uchida K.
        • Inoue T.
        • Kurokawa Y.
        Jpn. J. Cancer Res. 1996; 87: 882-886
        • Chan P.H.
        • Epstein C.J.
        • Li Y.
        • Huang T.T.
        • Carlson E.
        • Kinouchi H.
        • Yang G.
        • Kamii H.
        • Mikawa S.
        • Kondo T.
        • Copin J.-C.
        • Chen S.F.
        • Chan T.
        • Gafni J.
        • Gobbel G.
        • Reola E.
        J. Neurotrauma. 1995; 12: 815-824
        • Demple B.
        • Harrison L.
        Annu. Rev. Biochem. 1994; 63: 915-948
        • Ballmaier D.
        • Epe B.
        Carcinogenesis. 1995; 16: 335-342
        • Czene S.
        • Harms-Ringdahl M.
        Mutat. Res. 1995; 336: 235-242
        • Epe B.
        • Hegler J.
        Methods Enzymol. 1994; 234: 122-131
        • Yakes F.M.
        • Van Houten B.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 514-519
        • Shigenaga M.K.
        • Gimeno C.J.
        • Ames B.N.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9697-9701
        • Loft S.
        • Vistisen K.
        • Ewertz M.
        • Tjonneland A.
        • Overvad K.
        • Poulsen H.E.
        Carcinogenesis. 1992; 13: 2241-2247
        • Yang C.-S.
        • Tsai P.-J.
        • Chen W.-Y.
        • Kuo J.-S.
        Redox Report. 1996; 2: 379-383
        • Fairbairn D.W.
        • Olive P.L.
        • O'Neill K.L.
        Mutat. Res. 1995; 339: 37-59
        • Collins A.R.
        • Ma A.G.
        • Duthie S.J.
        Mutat. Res. 1995; 336: 69-77
        • Dennog C.
        • Hartmann A.
        • Frey G.
        • Speit G.
        Mutagenesis. 1996; 11: 605-609
        • Cooper P.K.
        • Nouspikel T.
        • Clarkson S.G.
        • Leadon S.A.
        Science. 1997; 275: 990-993
        • Yarborough A.
        • Zhang Y.J.
        • Hsu T.M.
        • Santella R.M.
        Cancer Res. 1996; 56: 683-688
        • Takeuchi T.
        • Nakajima M.
        • Morimoto K.
        Carcinogenesis. 1996; 17: 1543-1548
        • Teixeira H.D.
        • Meneghini R.
        Biochem. J. 1996; 315: 821-825
        • Keyer K.
        • Imlay J.A.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13635-13640
        • Keyer K.
        • Gort A.S.
        • Imlay J.A.
        J. Bacteriol. 1995; 177: 6782-6790
        • Kasprzak K.S.
        Cancer Invest. 1995; 13: 411-430
        • Tsou T.C.
        • Chen C.L.
        • Liu T.Y.
        • Yang J.L.
        Carcinogenesis. 1996; 17: 103-108
        • Douki T.
        • Cadet J.
        Free Radical Res. 1996; 24: 369-380
        • Inoue S.
        • Kawanishi S.
        FEBS Lett. 1995; 371: 86-88
        • Liu R.H.
        • Hotchkiss J.H.
        Mutat. Res. 1995; 339: 73-89
        • Uppu R.M.
        • Cueto R.
        • Squadrito G.L.
        • Salgo M.G.
        • Pryor W.A.
        Free Radical Biol. Med. 1996; 21: 407-411
        • Douki T.
        • Cadet J.
        • Ames B.N.
        Chem. Res. Toxicol. 1996; 9: 3-7
        • Douki T.
        • Ames B.N.
        Chem. Res. Toxicol. 1994; 7: 511-518
        • Ames B.N.
        • Shigenaga M.K.
        • Hagen T.M.
        Biochim. Biophys. Acta. 1995; 1271: 165-170
        • Enright H.
        • Miller W.J.
        • Hays R.
        • Floyd R.A.
        • Hebbel R.P.
        Carcinogenesis. 1996; 17: 1175-1177
        • Zastawny T.H.
        • Czerwinska B.
        • Drzewiecka B.
        • Olinski R.
        Free Radical Biol. Med. 1997; 22: 101-107
        • Hall D.B.
        • Holmlin R.E.
        • Barton J.K.
        Nature. 1996; 382: 731-735
        • Aspinwall R.
        • Rothwell D.G.
        • Roldan-Arjona T.
        • Anselmino C.
        • Ward C.J.
        • Cheadle J.P.
        • Sampson J.R.
        • Lindahl T.
        • Harris P.C.
        • Hickson I.D.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 109-114
        • Slupska M.M.
        • Baikalov C.
        • Luther W.M.
        • Chiang J.H.
        • Wei Y.F.
        • Miller J.H.
        J. Bacteriol. 1996; 178: 3885-3892
        • Sakumi K.
        • Furuichi M.
        • Tsuzuki T.
        • Kakuma T.
        • Kawabata S.
        • Maki H.
        • Sekiguchi M.
        J. Biol. Chem. 1993; 268: 23524-23530
        • Nagashima M.
        • Sasaki A.
        • Morishita K.
        • Takenoshita S.
        • Nagamachi Y.
        • Kasai H.
        • Yokota J.
        Mutat. Res. 1997; 383: 49-59
        • Yacoub A.
        • Augeri L.
        • Kelley M.R.
        • Doetsch P.W.
        • Deutsch W.A.
        EMBO J. 1996; 15: 2306-2312
        • Shen C.C.
        • Wertelecki W.
        • Driggers W.J.
        • LeDoux S.P.
        • Wilson G.L.
        Mutat. Res. 1995; 337: 19-23
        • Driggers W.J.
        • Grishko V.I.
        • LeDoux S.P.
        • Wilson G.L.
        Cancer Res. 1996; 56: 1262-1266
        • Jaruga P.
        • Dizdaroglu M.
        Nucleic Acids Res. 1996; 24: 1389-1394
        • Spencer J.P.
        • Jenner A.
        • Aruoma O.I.
        • Cross C.E.
        • Wu R.
        • Halliwell B.
        Biochem. Biophys. Res. Commun. 1996; 224: 17-22
        • Goldman M.
        Science. 1996; 271: 1821-1822
        • Wolff S.
        Mutat. Res. 1996; 358: 135-142
        • Kim H.S.
        • Park Y.W.
        • Kasai H.
        • Nishimura S.
        • Park C.W.
        • Choi K.H.
        • Chung M.H.
        Mutat. Res. 1996; 363: 115-123
        • Jacob R.A.
        • Burri B.J.
        Am. J. Clin. Nutr. 1996; 63: 985S-990S
        • Hagen T.M.
        • Yowe D.L.
        • Bartholemew J.C.
        • Wehr C.M.
        • Do K.L.
        • Park J.-Y.
        • Ames B.N.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3064-3069
        • Baik S.C.
        • Youn H.S.
        • Chung M.H.
        • Lee W.K.
        • Cho M.J.
        • Ko G.H.
        • Park C.K.
        • Kasai H.
        • Rhee K.H.
        Cancer Res. 1996; 56: 1279-1282
        • Toyokuni S.
        • Mori T.
        • Hiai H.
        • Dizdaroglu M.
        Int. J. Cancer. 1995; 62: 309-313
        • Ballinger S.W.
        • Bouder T.G.
        • Davis G.S.
        • Judice S.A.
        • Nicklas J.A.
        • Albertini R.J.
        Cancer Res. 1996; 56: 5692-5697
        • Ichinose T.
        • Yajima Y.
        • Nagashima M.
        • Takenoshita S.
        • Nagamachi Y.
        • Sagai M.
        Carcinogenesis. 1997; 18: 185-192
        • Chen Q.
        • Marsh J.
        • Ames B.
        • Mossman B.
        Carcinogenesis. 1996; 17: 2525-2527
        • Liu L.
        • Zhang Q.
        • Feng J.
        • Deng L.
        • Zeng N.
        • Yang A.
        • Zhang W.
        Mutat. Res. 1996; 370: 145-150
        • Hiraku Y.
        • Kawanishi S.
        Cancer Res. 1996; 56: 5172-5178
        • Shen H.M.
        • Ong C.N.
        • Lee B.L.
        • Shi C.Y.
        Carcinogenesis. 1995; 16: 419-422
        • Yamaguchi R.
        • Hirano T.
        • Asami S.
        • Chung M.H.
        • Sugita A.
        • Kasai H.
        Carcinogenesis. 1996; 17: 2419-2422
        • Hasegawa R.
        • Chujo T.
        • Sai-Kato K.
        • Umemura T.
        • Tanimura A.
        • Kurokawa Y.
        Food Chem. Toxicol. 1995; 33: 961-970
        • Fraga C.G.
        • Motchnik P.A.
        • Wyrobek A.J.
        • Rempel D.M.
        • Ames B.N.
        Mutat. Res. 1996; 351: 199-203
        • Woodall A.A.
        • Ames B.N.
        Bendich A. Deckelbaum R.J. Preventive Nutrition: The Comprehensive Guide for Health Professionals. Humana Press Inc., Totowa, NJ1997: 373-385
        • Ji B.T.
        • Shu X.O.
        • Linet M.S.
        • Zheng W.
        • Wacholder S.
        • Gao Y.T.
        • Ying D.M.
        • Jin F.
        J. Natl. Cancer Inst. 1997; 89: 238-244
        • Liu P.K.
        • Hsu C.Y.
        • Dizdaroglu M.
        • Floyd R.A.
        • Kow Y.W.
        • Karakaya A.
        • Rabow L.E.
        • Cui J.K.
        J. Neurosci. 1996; 16: 6795-6806
        • Mirsalis J.C.
        • Monforte J.A.
        • Winegar R.A.
        Annu. Rev. Pharmacol. Toxicol. 1995; 35: 145-164
        • Shen H.M.
        • Ong C.N.
        Mutat. Res. 1996; 366: 23-44
        • Fraga C.G.
        • Shigenaga M.K.
        • Park J.W.
        • Degan P.
        • Ames B.N.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4533-4537
        • Hirano T.
        • Yamaguchi Y.
        • Hirano H.
        • Kasai H.
        Biochem. Biophys. Res. Commun. 1995; 214: 1157-1162
        • de la Asuncion J.G.
        • Millan A.
        • Pla R.
        • Bruseghini L.
        • Esteras A.
        • Pallardo F.V.
        • Sastre J.
        • Vina J.
        FASEB J. 1996; 10: 333-338
        • Beckman K.B.
        • Ames B.N.
        Methods Enzymol. 1996; 264: 442-453
        • Liu L.
        • Wells P.G.
        Free Radical Biol. Med. 1995; 19: 639-648
        • Randerath E.
        • Zhou G.-D.
        • Randerath K.
        Carcinogenesis. 1996; 17: 2563-2570