Role of endogenous oxidative DNA damage in carcinogenesis: what can we learn from repair-deficient mice?

Basal steady-state levels of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) are observed in all types of cells, most probably due to a continuous generation of reactive oxygen species (ROS) in the cellular oxygen metabolism, and it has long been suspected that they might play an important role in the initiation of carcinogenesis. Experimental evidence for this assumption can be obtained by studying the effects of a modulation of the steady-state levels, either by in- or decreasing the generation of oxidative DNA damage, on spontaneous mutation rates and cancer incidence. However, clear answers have not yet been obtained by these strategies. It is still doubtful whether an efficient reduction of the in vivo steady-state levels can be achieved by application of antioxidants, and effects observed under oxidative stress conditions (i.e. increased oxidative DNA damage) are inconclusive due to the pronounced epigenetic effects of ROS on signal transduction and gene expression (tumor promotion). In addition, the reliable quantification of the basal levels of oxidative DNA modifications is still a major problem. Recently, the generation of mice deficient in the repair 8-oxoG (ogg1-/- mice) has opened the door for an alternative approach. Results obtained so far indicate that an increase by less than five 8-oxoG residues per 106 bp in the liver of the knockout animals is associated with a two- to threefold higher spontaneous mutation frequency in transgenic genes. However, the increase in the ogg1-/- mice of the steady-state level of 8-oxoG and the spontaneous mutation frequency was only observed in the liver and apparently too small to enhance the spontaneous cancer incidence significantly. The limited effect seems to be due to a back-up repair system for 8-oxoG in the ogg1-/- mice, and it can be expected that the inactivation of this pathway in double-knockout mice will lead to higher effects and a better assessment of the risk associated with endogenous oxidative DNA damage.     

Detection of oxidative base DNA damage by a new biochemical assay

Reactive oxygen species (ROS) damage DNA which appears to represent the major target involved in mutagenesis, carcinogenesis, and aging cell responses. Various DNA modifications are generated by ROS, but 8-hydroxy-2'-deoxyguanosine (8-oxoG) has retained a lot of attention in the last few years. Therefore, numerous methods have been developed to detect and quantify the extent of 8-oxoG in DNA, most of them requiring a significant amount of DNA that might be limiting in the case of biological samples. 8-oxoG is repaired in Escherichia coli by a specific glycosylase, the Fpg (formamidopyrimidine DNA glycosylase) protein, in a reaction that requires a covalent intermediate favored under reducing conditions. We set up a new assay based on the capture of plasmid DNA into sensitized microplate wells. DNA damaged by photoactivation of methylene blue was adsorbed on a polylysine-treated plastic well. Then the Fpg protein was added, allowed to fix on the damage by taking advantage of minimized glycosylase activity at low temperature and the reductive trapping of the covalent intermediate, yielding to a stable DNA-protein interaction. The trapped protein was subsequently recognized by a specific antibody. A secondary antibody coupled with horseradish peroxidase was used to detect the complex and the measurement was carried out by chemiluminescence. This new assay offers various potentialities, specifically in the field of technology of ROS producers.     

The sacrificial role of easily oxidizable sites in the protection of DNA from damage

It has been suggested that DNA contains sacrificial nucleobase sequences that protect sensitive regions of the genome from oxidative damage. Oxidation of DNA by loss of an electron generates a radical cation that can migrate long distances by hopping. The radical cation can be trapped irreversibly at certain sites (GG steps) by reaction with H₂O or O₂ leading to the formation of lesions (oxidative damage). A series of DNA oligomers that contain regularly spaced GG steps and an 8-oxo-7,8-dihydroguanine (8-oxoG), which serves as a proxy for possibly sacrificial protective low oxidation potential sites, was prepared and analyzed. We find that in certain special sequences of DNA nucleobases that 8-oxoG protects remote GG steps from oxidative damage but that this is not a general phenomenon extending to normal mixed sequence DNA. This is a consequence of the change in the relative rate of charge hopping compared with trapping of the radical cation. When hopping is relatively slow, 8-oxoG exerts no protective effect. Thus, it seems unlikely that low oxidation potential sequences play a meaningful part in protecting mixed sequence DNA from damage.     

8-oxo-7,8-dihydroguanine is removed by a nucleotide excision repair-like mechanism in Porphyromonas gingivalis W83

A consequence of oxidative stress is DNA damage. The survival of Porphyromonas gingivalis in the inflammatory microenvironment of the periodontal pocket requires an ability to overcome oxidative stress caused by reactive oxygen species (ROS). 8-oxo-7,8-dihydroguanine (8-oxoG) is typical of oxidative damage induced by ROS. There is no information on the presence of 8-oxoG in P. gingivalis under oxidative stress conditions or on a putative mechanism for its repair. High-pressure liquid chromatography with electrochemical detection analysis of chromosomal DNA revealed higher levels of 8-oxoG in P. gingivalis FLL92, a nonpigmented isogenic mutant, than in the wild-type strain. 8-oxoG repair activity was also increased in cell extracts from P. gingivalis FLL92 compared to those from the parent strain. Enzymatic removal of 8-oxoG was catalyzed by a nucleotide excision repair (NER)-like mechanism rather than the base excision repair (BER) observed in Escherichia coli. In addition, in comparison with other anaerobic periodontal pathogens, the removal of 8-oxoG was unique to P. gingivalis. Taken together, the increased 8-oxoG levels in P. gingivalis FLL92 could further support a role for the hemin layer as a unique mechanism in oxidative stress resistance in this organism. In addition, this is the first observation of an NER-like mechanism as the major mechanism for removal of 8-oxoG in P. gingivalis.     

Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease

Mitochondrial generation of reactive oxygen species (ROS) is increased in mice with fatty livers induced by genetic obesity, chronic consumption of ethanol, or methionine/choline-deficient diets. Both nuclear and mitochondrial (mt) DNA are targets for ROS-induced damage and accumulate hydroxylated bases, such as 8-hydroxy-2'-deoxyguanosine (8-oxoG) and base substitution of adenine with 8-oxoG (A*8-oxoG), that introduce mutations that promote cancer as well as cell death. The mammalian homolog of the bacterial DNA mismatch repair enzyme MutY (MYH) removes A*8-oxoG from nuclear and mtDNA, reduces 8-oxoG accumulation, and restores genomic stability after ROS exposure. Cumulative damage to mtDNA occurs as fatty liver disease progresses. Therefore, differences in hepatic MYH activity may influence the severity of fatty liver disease. To evaluate this hypothesis, we compared mtH2O2 production, MYH expression, oxidative DNA damage, and hepatocyte death in healthy mice and different mouse models of fatty liver disease. The results show that diverse causes of steatohepatitis increase mtROS production, limit repair of mtDNA, and oxidatively damage DNA. However, there are important differences in the DNA repair response to oxidant stress among mouse models of fatty liver disease. Independent of the degree of mtROS generation, models with the least MYH exhibit the greatest accumulation of 8-oxoG and the most hepatocyte death. These findings raise the intriguing possibility that inherited or acquired differences in DNA repair enzyme activity may underlie some of the interindividual differences in fatty liver disease outcomes.     

The roles of specific glycosylases in determining the mutagenic consequences of clustered DNA base damage

The potential for genetic change arising from specific single types of DNA lesion has been thoroughly explored, but much less is known about the mutagenic effects of DNA lesions present in clustered damage sites. Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. We have investigated the potential of a non-mutagenic DNA base lesion, 5,6-dihydrothymine (DHT), to influence the mutagenicity of 8-oxo-7,8-dihydroguanine (8-oxoG) when the two lesions are closely opposed. Using a bacterial plasmid-based assay we present the first report of a significantly higher mutation frequency for the clustered DHT and 8-oxoG lesions than for single 8-oxoG in wild-type and in glycosylase-deficient strains. We propose that endonuclease III has an important role in the initial stages of processing DHT/8-oxoG clusters, removing DHT to give an intermediate with an abasic site or single-strand break opposing 8-oxoG. We suggest that this mutagenic intermediate is common to several different combinations of base lesions forming clustered DNA damage sites. The MutY glycosylase, acting post-replication, is most important for reducing mutation formation. Recovered plasmids commonly gave rise to both wild-type and mutant progeny, suggesting that there is differential replication of the two DNA strands carrying specific forms of base damage.     

114 Kinetic and thermodynamic basis for damaged bases excision by 8-oxoguanine-DNA glycosylases

DNA glycosylases play the opening act in a highly conserved process for excision of damaged bases from DNA called the base excision repair pathway. DNA glycosylases attend to a wide variety of lesions arising from both endogenous and exogenous factors. The types of damage include alkylation, oxidation, and hydrolysis. A major DNA oxidation product is 8-oxoguanine (8-oxoG), a base with a high mutagenic potential. In bacteria, this lesion is repaired by formamidopyrimidine-DNA glycosylase (Fpg), while in the case of humans this function belongs to 8-oxoG-DNA glycosylase (OGG1). We have attempted a comprehensive characterization of 8-oxoG recognition by DNA glycosylases. First, we have obtained thermodynamic parameters for melting of DNA duplexes containing 8-oxoG in all possible nucleotide contexts. The energy of stacking interactions of 8-oxoG was in strict dependence on 8-oxoG nucleotide environment, which may affect the recognition of damage and the efficiency of eversion of 8-oxoG from DNA helix by glycosylases. Next, we established how the flexibility of DNA context affects damage recognition by these enzymes (Kirpota et al., 2011). Then, we have found that DNA containing 8-oxoG next to a single-strand break provides a good substrate for Fpg, as soon as all structural phosphate residues are maintained. Using site-directed mutagenesis, we have addressed the functions of many previously unstudied amino acid residuess that were predicted to be important for Fpg activity by molecular dynamics simulation and phylogenetic analysis. Of note, many substitutions abolished the excision of 8-oxoG, but did not affect the cleavage efficiency of abasic substrates. Finally, we investigated the contribution of separated structural domains of Fpg to specific enzyme-substrate interaction. Surprisingly, despite the absence of the catalytic domain, C-terminal domain of Fpg possessed a low- residual ability to recognize and cleave abasic substrates. Our study sheds light on mechanism details of Fpg and OGG1 activity, with the ultimate goal of understanding how binding energy can be spent by these enzymes for catalysis.     

The presence of two distinct 8-oxoguanine repair enzymes in human cells: their potential complementary roles in preventing mutation.

8-Oxoguanine (8-oxoG), induced by reactive oxygen species (ROS) and ionizing radiation, is arguably the most important mutagenic lesion in DNA. This oxidized base, because of its mispairing with A, induces GC-->TA transversion mutations often observed spontaneously in tumor cells. The human cDNA encoding the repair enzyme 8-oxoG-DNA glycosylase (OGG-1) has recently been cloned, however, its activity was never detected in cells. Here we show that the apparent lack of this activity could be due to the presence of an 8-oxoG-specific DNA binding protein. Moreover, we demonstrate the presence of two antigenically distinct OGG activities with an identical reaction mechanism in human cell (HeLa) extracts. The 38 kDa OGG-1, identical to the cloned enzyme, cleaves 8-oxoG when paired with cytosine, thymine and guanine but not adenine in DNA. In contrast, the newly discovered 36 kDa OGG-2 prefers 8-oxoG paired with G and A. We propose that OGG-1 and OGG-2 have distinct antimutagenic functions in vivo . OGG-1 prevents mutation by removing 8-oxoG formed in DNA in situ and paired with C, while OGG-2 removes 8-oxoG that is incorporated opposite A in DNA from ROS-induced 8-oxodGTP. We predict that OGG-2 specifically removes such 8-oxoG residues only from the nascent strand, possibly by utilizing the same mechanism as the DNA mismatch repair pathway.     

Processing of thymine glycol in a clustered DNA damage site: mutagenic or cytotoxic

Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. The potential for genetic change arising from the effects of clustered damage sites containing combinations of AP sites, 8-oxo-7,8-dihydroguanine (8-oxoG) or 5,6-dihydrothymine is high. To date clusters containing a DNA base lesion that is a strong block to replicative polymerases, have not been explored. Since thymine glycol (Tg) is non-mutagenic but a strong block to replicative polymerases, we have investigated whether clusters containing Tg are highly mutagenic or lead to potentially cytotoxic lesions, when closely opposed to either 8-oxoG or an AP site. Using a bacterial plasmid-based assay and repair assays using cell extracts or purified proteins, we have shown that DNA double-strand breaks (DSBs) arise when Tg is opposite to an AP site, either through attempted base excision repair or at replication. In contrast, 8-oxoG opposite to Tg in a cluster ‘protects’ against DSB formation but does enhance the mutation frequency at the site of 8-oxoG relative to that at a single 8-oxoG, due to the decisive role of endonucleases in the initial stages of processing Tg/8-oxoG clusters, removing Tg to give an intermediate with an abasic site or single-strand break.     

Glycochenodeoxycholate plays a carcinogenic role in immortalized mouse cholangiocytes via oxidative DNA damage

Bile acids have been suggested to be involved in biliary carcinogenesis, although the underlying mechanisms are yet to be established. The aim of this study was to investigate the carcinogenic effect of bile acids in the biliary tract in relation to oxidative stress. Immortalized mouse cholangiocytes were incubated with various bile acids, followed by measurement of reactive oxygen species (ROS) and the glutathione (GSH) level. As a marker of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG) expression in cholangiocytes was analyzed by flow cytometry. Then the expression of oxidative DNA repair enzymes in cholangiocytes was examined by real-time PCR. In addition, the long-term effect of bile acid-induced oxidative DNA damage on cholangiocytes was investigated using a mouse oligo DNA microarray. It was found that glycochenodeoxycholate (GCDC) induced the generation of ROS and the depletion of GSH. In contrast, no marked changes were induced by the other bile acids. The percentage of 8-OHdG-positive cells was also increased by GCDC, but the expression of oxidative DNA repair enzymes was not up-regulated. DNA microarray analysis showed marked changes of various genes associated with carcinogenesis (genes related to cell proliferation, angiogenesis, invasion, and metastasis). In conclusion, the long-term effect of oxidative DNA damage due to GCDC may promote carcinogenesis in the biliary tract. Furthermore, accumulation of 8-OHdG due to GCDC might contribute to the dysfunction of oxidative DNA repair enzymes.     

Biochemical identification of a hydroperoxide derivative of the free 8-oxo-7,8-dihydroguanine base

8-Oxo-7,8-dihydroguanine is one the most abundant base lesions in pro- and eukaryotic DNA. In mammalian cells, it is excised by the 8-oxoguanine DNA glycosylase (OGG1) during DNA base-excision repair, and the generated free 8-oxoG base is one of the DNA-derived biomarkers of oxidative stress in biological samples. The modification of 8-oxoG in the context of nucleoside and DNA has been the subject of many studies; however, the oxidative transformation of the free 8-oxoG base has not been described. By using biochemical and cell biological assays, we show that in the presence of molecular oxygen, the free 8-oxoG base transforms to a highly reactive hydroperoxide (8-oxoG*). Specifically, 8-oxoG* oxidizes Amplex red to resorufin, H(2)DCF to DCF, Fe(2+) to Fe(3+), and GSH to GSSG. This property of 8-oxoG* was diminished by treatment with catalase and glutathione peroxidase, but not superoxide dismutase. 8-OxoG* formation was prevented by reducing agents or nitrogen atmosphere. Its addition to CM-H(2)DCF-DA-loaded cells rapidly increased intracellular DCF fluorescence. There were no such properties observed for 8-oxodeoxyguanosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2'-deoxyguanosine, guanine, adenine, guanosine, and 8-hydroxyadenine. These data imply that a free 8-oxoG base is more susceptible to oxidation than is its nucleoside form and, consequently, it stands as unique among intact and oxidatively modified purines.     

Coordinated action of the Fanconi anemia and ataxia telangiectasia pathways in response to oxidative damage

Fanconi anemia (FA) and ataxia telangiectasia (AT) share common traits such chromosomal instability and proneness to hematological cancers. Both AT and FA cell lines, and patients, are characterized by abnormally high levels of oxidative stress markers. The key FA protein FANCD2 is phosphorylated on Ser 222 by ATM after ionizing radiation (IR), thus allowing normal activation of the S-phase checkpoint, and ATM cells are known to be hypersensitive to oxidative damage. In this work we show that FANCD2 deficient cells have a defective S-phase checkpoint after Hydrogen Peroxide (H(2)O(2)) induced oxidative damage. ATM dependent phosphorylation of FANCD2 at the S222 residue is necessary for normal S-phase checkpoint activation after oxidative stress, while FANCD2 monoubiquitination at K561 is dispensable. We also show that FANCD2 is not required for base excision repair of 8-oxoG and other DNA lesions (abasic sites, uracils), while treatments that exclusively induce 8-oxoG, but not DNA double strand breaks, fail to activate FANCD2 monoubiquitination, thus indicating that the known accumulation of 8-oxoG in FA cells reflects an overproduction of ROS rather than defective processing of oxidized bases. We conclude that the handling of DNA damage after H(2)O(2)-induced oxidative stress requires the coordinated action of FANCD2 and ATM.     

Repair of 8-oxoG is slower in endogenous nuclear genes than in mitochondrial DNA and is without strand bias

DNA is vulnerable to the attack of certain oxygen radicals and one of the major DNA lesions formed is 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic lesion that can mispair with adenine. The repair of 8-oxoG was studied by measuring the gene specific removal of 8-oxoG after treatment of Chinese hamster ovary (CHO) fibroblasts with the photosensitizer Ro19-8022. This compound introduces 8-oxoG lesions, which can then be detected with the Escherichia coli formamidopyrimidine DNA glycosylase (FPG). In this report we present gene specific repair analysis of endogenous genes situated in different important cellular regions and also the first analysis of strand specific DNA repair of 8-oxoG in an endogenous gene. We were not able to detect any preferential repair of transcribed genes compared to non-transcribed regions and we did not detect any strand-bias in the repair of the housekeeping gene, dihydrofolate reductase (DHFR). In vivo, mitochondrial DNA is highly exposed to reactive oxygen species (ROS), and we find that the repair of 8-oxoG is more efficient in the mitochondrial DNA than in the nuclear DNA.     

Efficiency of excision of 8-oxo-guanine within DNA clustered damage by XRS5 nuclear extracts and purified human OGG1 protein

A major DNA lesion is the strongly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) base, formed by oxidative attack at guanine and which leads to a high level of G.C-->T.A transversions. Clustered DNA damages are formed in DNA following exposure to ionizing radiation or radiomimetic anticancer agents and are thought to be biologically severe. The presence of 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell, if the OGG1 DNA glycosylase/AP lyase protein, present in eukaryotic cells, does not efficiently excise its substrate, 8-oxoG. In this study, specific oligonucleotide constructs containing an 8-oxoG located in several positions opposite to another damage (5,6-dihydrothymine (DHT), uracil, 8-oxoG, AP site, or various types of single strand breaks) were used to determine the relative efficiency of purified human OGG1 and mammalian XRS5 nuclear extracts to excise 8-oxoG from clustered damages. A base damage (DHT, uracil, and 8-oxoG) on the opposite strand has little or no influence on the rate of excision of 8-oxoG whereas the presence of either an AP site or various types of single strand breaks has a strong inhibitory effect on the formation of a SSB due to the excision of 8-oxoG by both hOGG1 and the nuclear extract. The binding of hOGG1 to 8-oxoG is not significantly affected by the presence of a neighboring lesion.     

Thiols can either enhance or suppress DNA damage induction by catecholestrogens

The estrogen metabolites catecholestrogens (or hydroxyestrogens) are involved in carcinogenesis and the development of resistance to methotrexate. This induction of drug resistance correlates with the relative efficiency of catecholestrogens in the generation of reactive oxygen species (ROS) and the induction of DNA strand breaks. Although antioxidants can neutralize ROS, the generation of these reactive species by catecholestrogens can be enhanced by electron donors like NADH. Therefore, this study was undertaken to determine the ability of different thiol agents (GSH, NAC, DTT, DHLA) to either inhibit or enhance the level of DNA damage induced by the H(2)O(2) generating system 4-hydroxyestradiol/Cu(II). Our results show that GSH, DTT, and DHLA inhibited the induction of the 4-hydroxyestradiol/Cu(II)-mediated DNA damage, with GSH showing the best potential. In contrast, the GSH precursor NAC at low concentrations was able to enhance the level of oxidative damage, as observed with NADH. NAC can reduce Cu(II) to Cu(I) producing the radical NAC&z.rad;, which can generate the superoxide anion. However, the importance of this pathway appears to be relatively minor since the addition of NAC to the 4-hydroxyestradiol/Cu(II) system generates about 15 times more DNA strand breaks than NAC and Cu(II) alone. We suggest that NAC can perpetuate the redox cycle between the quinone and the semiquinone forms of the catecholestrogens, thereby enhancing the production of ROS. In conclusion, this study demonstrates the crucial importance of the choice of antioxidant as potential therapy against the negative biological effects of estrogens.     

In Vivo Bypass of 8-oxodG

8-oxoG is one of the most common and mutagenic DNA base lesions caused by oxidative damage. However, it has not been possible to study the replication of a known 8-oxoG base in vivo in order to determine the accuracy of its replication, the influence of various components on that accuracy, and the extent to which an 8-oxoG might present a barrier to replication. We have been able to place a single 8-oxoG into the Saccharomyces cerevisiae chromosome in a defined location using single-strand oligonucleotide transformation and to study its replication in a fully normal chromosome context. During replication, 8-oxoG is recognized as a lesion and triggers a switch to translesion synthesis by Pol η, which replicates 8-oxoG with an accuracy (insertion of a C opposite the 8-oxoG) of approximately 94%. In the absence of Pol η, template switching to the newly synthesized sister chromatid is observed at least one third of the time; replication of the 8-oxoG in the absence of Pol η is less than 40% accurate. The mismatch repair (MMR) system plays an important role in 8-oxoG replication. Template switching is blocked by MMR and replication accuracy even in the absence of Pol η is approximately 95% when MMR is active. These findings indicate that in light of the overlapping mechanisms by which errors in 8-oxoG replication can be avoided in the cell, the mutagenic threat of 8-oxoG is due more to its abundance than the effect of a single lesion. In addition, the methods used here should be applicable to the study of any lesion that can be stably incorporated into synthetic oligonucleotides.     

Molecular dynamics simulation of 7, 8-dihydro-8-oxoguanine DNA

To elucidate the effect of guanine lesion produced by the oxidative damage on DNA, 1 nanosecond molecular dynamics simulations of native and oxidized DNA were performed. The target DNA molecules are dodecamer duplex d(CGCGAATTCGCG)(2) and its derivative duplex d(C(1)G(2)C(3)(8-oxoG)(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12).d(C(13)G(14)C(15)G(16)A(17)A(18)T(19)T(20)C(21)G(22)C(23)G(24), which has one oxidized guanine, 7, 8-dihydro-8-oxoguanine (8-oxoG), at the fourth position. The local structural change due to the lesion of 8-oxoG and the global dynamic structure of the 8-oxoG DNA were studied. It was found that the 8-oxoG DNA remained structurally stable during the simulation due to newly produced hydrogen bonds around the (8-oxoG)(4) residue. However, there were distinguishable differences in structural parameters and dynamic property in the 8-oxoG DNA. The conformation around the (8-oxoG)(4) residue departed from the usual conformation of native DNA and took an unique conformation of epsilon-zeta in B(II) conformation and chi in high anti orientation at the (8-oxoG)(4) residue, and adopted a very low helical twist angle at the C(3):G(22)-(8-oxoG)(4):C21) step. Further analysis by principal component analysis indicated that the formation of the hydrogen bonds around the (8-oxoG)(4) residue plays a role as a trigger for the conformational transition of the 8-oxoG DNA in the conformational space.     

Immunofluorescent detection of 8-oxoguanine DNA lesions in liver cells from aging OXYS rats, a strain prone to overproduction of free radicals

Production of free radicals in animals is accompanied with a number of pathologic conditions, some of which may be manifested through DNA damage. Studies of mechanisms of oxidative DNA damage by free radicals in vivo are hindered by the lack of good animal models with significant overgeneration of or increased sensitivity to free radicals. An inbred rat strain (OXYS) is characterized by inherited overgeneration of free radicals, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases. We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative age-dependent amounts and distribution of 8-oxoG in liver cells from OXYS and Wistar rats. 8-OxoG increased with age in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. Statistical analysis indicates that 8-oxoG does not uniformly accumulate in all cells with advancing age or increasing free radical load, but rather concentrates in a minor fraction of cells with a high damage level.