Oxidative DNA damage induced by toluene is involved in its male reproductive toxicity

Toluene is widely used as an organic solvent in various industries and commercial products. Recent investigations have shown that toluene may induce male reproductive dysfunctions and carcinogenicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of toluene on the male reproductive system in rats, comparing to those of diethylstilbestrol (DES), a potent synthetic estrogen. Toluene (50, 500 mg/kg) or DES (2 mg/kg) injected subcutaneously to male Sprague-Dawley rats once a day for 10 days decreased the epididymal sperm counts and the serum concentrations of testosterone. The mRNA level for gonadotropin-releasing hormone receptor in the pituitary was decreased by DES, but not by toluene. On the contrary, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in testes, the biological marker for oxidative DNA damage, was increased by toluene but not by DES. These results suggest that toluene induces reproductive toxicity via direct oxidative damage of spermatozoa, whereas DES affects endocrine systems via the hypothalamo-pituitary-gonadal axis. Morphological findings supported the idea. To determine the mechanism of 8-oxodG formation in vivo , we examined DNA damage induced by toluene metabolic products in vitro . Minor toluene metabolites, methylhydroquinone and methylcatechols, induced oxidative DNA damage, and the methylcatechols induced NADH-mediated 8-oxodG formation more efficiently than methylhydroquinone did. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by toluene.     

Oxidative DNA and protein damage in metal-induced toxicity and carcinogenesis

This review discusses the relevance of oxidative damage to metal-induced toxicity and carcinogenesis. Presented are important facts and mechanistic concepts on the capacity of selected transition metals, mainly Ni, but also Cu, Co, Cr, and briefly several others, to generate active oxygen species and other reactive intermediates under physiological conditions. These metals are known to be toxic and/or carcinogenic contaminants of the occupational and general environments. Their redox activity may underlay the mechanism of mediation of oxidative damage to cell constituents. The presentation is focused on selected issues relative to genetic and epigenetic toxicity and illustrated with examples of metal-mediated oxidative damage to the principal components of chromatin, i.e., DNA, histones, and protamines.     

Oxidative DNA damage induced by metabolites of chloramphenicol,an antibiotic drug

Abstract

Chloramphenicol (CAP) was an old antimicrobial agent. However, the use of CAP is limited because of its harmful side effects, such as leukemia. The molecular mechanism through which CAP has been strongly correlated with leukemogenesis is still unclear. To elucidate the mechanism of genotoxicity, we examined DNA damage by CAP and its metabolites, nitroso-CAP (CAP-NO), N-hydroxy-CAP (CAP-NHOH), using isolated DNA. CAP-NHOH have the ability of DNA damage including 8-oxo-7,8-dihydro-2′-deoxyguanosine formation in the presence of Cu(II), which was greatly enhanced by the addition of an endogenous reductant NADH. CAP-NO caused DNA damage in the presence of Cu(II), only when reduced by NADH. NADH can non-enzymatically reduce the nitroso form to hydronitroxide radicals, resulting in enhanced generation of reactive oxygen species followed by DNA damage through the redox cycle. Furthermore, we also studied the site specificity of base lesions in DNA treated with piperidine or formamidopyrimidine-DNA glycosylase, using ³²P-5′-end-labeled DNA fragments obtained from the human tumor suppressor gene. CAP metabolites preferentially caused double base lesion, the G and C of the ACG sequence complementary to codon 273 of the p53 gene, in the presence of NADH and Cu(II). Therefore, we conclude that oxidative double base lesion may play a role in carcinogenicity of CAP.     

Oxidative and nitrative DNA damage: key events in opisthorchiasis-induced carcinogenesis

Chronic inflammation induced by liver fluke (Opisthorchis viverrini) infection is the major risk factor for cholangiocarcinoma (CCA) in Northeastern Thailand. Increased levels of proinflammatory cytokines and nuclear factor kappa B that control cyclooxygenase-2 and inducible nitric oxide activities, disturb the homeostasis of oxidants/anti-oxidants and DNA repair enzymes, all of which appear to be involved in O. viverrini-associated inflammatory processes and CCA. Consequently oxidative and nitrative stress-related cellular damage occurs due to the over production of reactive oxygen and nitrogen species in inflamed target cells. This is supported by the detection of high levels of oxidized DNA and DNA bases modified by lipid peroxidation products in both animal and human tissues affected by O. viverrini-infection. Treatment of opisthorchiasis patients with praziquantel, an anti- trematode drug was shown to reduce inflammation-mediated tissue damage and carcinogenesis. The principal mechanisms that govern the effects of inflammation and immunity in liver fluke-associated cholangiocarcinogenesis are reviewed. The validity of inflammation-related biomolecules and DNA damage products to serve as predictive biomarkers for disease risk evaluation and intervention is discussed.     

Oxidative damage to DNA in mammalian chromatin.

Efforts have been made to characterize and measure DNA modifications produced in mammalian chromatin in vitro and in vivo by a variety of free radical-producing systems. Methodologies incorporating the technique of gas chromatography/mass spectrometry have been used for this purpose. A number of products from all four DNA bases and several DNA-protein cross-links in isolated chromatin have been identified and quantitated. Product formation has been shown to depend on the free radical-producing system and the presence or absence of oxygen. A similar pattern of DNA modifications has also been observed in chromatin of cultured mammalian cells treated with ionizing radiation or H2O2 and in chromatin of organs of animals treated with carcinogenic metal salts.     

Oxidative damage to 5-methylcytosine in DNA.

Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5-hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5-hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2'-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues.     

Oxidative damage to DNA induced by areca nut extract

In Taiwan, people chew bete; quid which contains tender areca nut with husk. In other countries, people prefer ripe and dried areca nut without husk. In this study, we compared the reactive oxygen species-induced oxidative DNA damage in isolated DNA and CHO-K1 cells between treatments with tender areca nut extract (ANE) and ripe ANE. Incubation of these two ANE preparations with isolated DNA generated 8-hydroxy-2′-deoxyguanosine (8-OH-dG) in an alkaline environment in a dose-dependent manner. Ripe ANE generated higher levels of 8-OH-dG compared to tender ANE. The addition of iron(II) (100 μM) resulted in 1.4- and 3.1-fold increases of 8-OH-dG when incubated with 1 mg/ml each of tender and ripe ANE. In testing the effect of ANE to cellular DNA, CHO-K1 cells were used for its documented sensitivity to reactive oxygen species. In CHO-K1 cells, ripe ANE was more cytotoxic than tender ANE following an 18-h incubation. The cytotoxicity to CHO-K1 cells was positively correlated with the formation of 8-OH-dG following tender (r = 0.97) and ripe (r = 0.91) ANE treatment. Addition of the iron chelating agent o-phenanthroline (10 and 20 μM) to cells prior to ripe ANE exposure significantly increased (p < 0.05) the survival of CHO-K1 cells. In addition, ripe ANE induced dichlorofluorescein-mediated fluorescence which indicated the formation of hydrogen peroxide in CHO-K1 cells. In conclusion, this study demonstrated that ANE-induced oxidative damage to isolated and cellular DNA which may result from the generation of hydrogen peroxide, and iron may serve as a catalyst in this process. Furthermore, ripe ANE generated higher oxidative DNA damage levels compared to tender ANE.     

Oxidative and nitrative DNA damage in animals and patients with inflammatory diseases in relation to inflammation-related carcinogenesis

Infection and chronic inflammation are proposed to contribute to carcinogenesis through inflammation-related mechanisms. Infection with hepatitis C virus, Helicobacter pylori and the liver fluke, Opisthorchis viverrini (OV), are important risk factors for hepatocellular carcinoma (HCC), gastric cancer and cholangiocarcinoma, respectively. Inflammatory bowel diseases (IBDs) and oral diseases, such as oral lichen planus (OLP) and leukoplakia, are associated with colon carcinogenesis and oral squamous cell carcinoma (OSCC), respectively. We performed a double immunofluorescence labeling study and found that nitrative and oxidative DNA lesion products, 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), were formed and inducible nitric oxide synthase (iNOS) was expressed in epithelial cells and inflammatory cells at the site of carcinogenesis in humans and animal models. Antibacterial, antiviral and antiparasitic drugs dramatically diminished the formation of these DNA lesion markers and iNOS expression. These results suggest that oxidative and nitrative DNA damage occurs at the sites of carcinogenesis, regardless of etiology. Therefore, it is considered that excessive amounts of reactive nitrogen species produced via iNOS during chronic inflammation may play a key role in carcinogenesis by causing DNA damage. On the basis of our results, we propose that 8-nitroguanine is a promising biomarker to evaluate the potential risk of inflammation-mediated carcinogenesis.     

Oxidative damage to plant DNA in relation to growth conditions.

In this study we investigated the level of 8-oxo-2'-deoxyguanosine (8-oxodG) in DNA of Cardamine pratensis plants subjected to different growth conditions trying to answer the question whether factors like light and water accessibility or low temperature may have an impact on the total DNA oxidative damage. The level of this modified nucleoside was determined using HPLC coupled to UV absorbance and electrochemical detection (HPLC-UV-EC). We did not observe any statistically significant differences in 8-oxodG level between DNA of etiolated and light exposed plants as well as between DNA of regularly watered and drought-subjected plants. In contrast, we have shown that chilling (1 degree C for 28 h) brings about the increase of 8-oxodG level in DNA.     

Oxidative DNA damage is a preliminary step during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

The aim of this study was to investigate oxidative DNA damage during 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis. For this purpose, male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. The alkaline Comet assay modified with lesion-specific enzymes was used to detect single and double strand breaks, labile sites (SBs), and oxidised purines and pyrimidines. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, oxidative DNA damage was detected in the ‘normal’ oral epithelium. In pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks following carcinogen exposure, respectively, oxidative DNA damage was also increased (P < 0.05) when compared to negative control. In conclusion, our results suggest that oxidative DNA damage is an early event during multistep carcinogenesis assay induced by 4NQO. This kind of approach should be considered to persons with high risk of oral cancer, such as in smokers or alcohol consumers.     

Oxidative stress generated damage to DNA by gastrointestinal exposure to insoluble particles

There is growing concern that gastrointestinal exposure to particles is associated with increased risk of toxicity to internal organs and carcinogenicity. The mechanism of action is related to particle-induced oxidative stress and oxidation of DNA. Observations from animal models indicate that gastrointestinal exposure to single-walled carbon nanotubes (SWCNT), fullerenes C60, carbon black, titanium dioxide and diesel exhaust particles generates oxidized DNA base lesions in organs such as the bone marrow, liver and lung. Oral exposure to nanosized carbon black has also been associated with increased level of lipid peroxidation derived exocyclic DNA adducts in the liver, suggesting multiple pathways of oxidative stress for particle-generated damage to DNA. At equal dose, diesel exhaust particles (SRM2975) generated larger levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in rat liver than carbon black (Printex 90) did, whereas exposure to fullerenes C60 and SWCNT was the least potent. This ranking of samples was also observed for oxidatively damaged DNA in cultured cells. The extent of translocation from the gut is largely unresolved. However, there is evidence indicating that gastrointestinal exposure to particulate matter is associated with oxidative damage to DNA and this might be associated with increased risk of cancer.     

Oxidative damage to plasma constituents by ozone.

The reaction of ozone (O3) with human blood plasma was studied to help understand possible events that could occur in the respiratory tract. Uric acid (quantitatively the most important scavenger) and ascorbic acid were oxidized quickly, protein-SH groups were lost more slowly, and there was no loss of bilirubin or alpha-tocopherol. There was little formation of lipid hydroperoxides and no detectable formation of 4-hydroxynoneal, hexanal or nonanal, or changes in lipoprotein electrophoretic mobility. Uric acid in human upper airway secretions may play a significant role in removing inhaled O3. Oxidative damage to lipids must not be assumed to be the key mechanism of respiratory tract O3 toxicity.     

Oxidative stress-induced DNA damage by particulate air pollution

Exposure to ambient air particulate matter (PM) is associated with pulmonary and cardiovascular diseases and cancer. The mechanisms of PM-induced health effects are believed to involve inflammation and oxidative stress. The oxidative stress mediated by PM may arise from direct generation of reactive oxygen species from the surface of particles, soluble compounds such as transition metals or organic compounds, altered function of mitochondria or NADPH-oxidase, and activation of inflammatory cells capable of generating ROS and reactive nitrogen species. Resulting oxidative DNA damage may be implicated in cancer risk and may serve as marker for oxidative stress relevant for other ailments caused by particulate air pollution. There is overwhelming evidence from animal experimental models, cell culture experiments, and cell free systems that exposure to diesel exhaust and diesel exhaust particles causes oxidative DNA damage. Similarly, various preparations of ambient air PM induce oxidative DNA damage in in vitro systems, whereas in vivo studies are scarce. Studies with various model/surrogate particle preparations, such as carbon black, suggest that the surface area is the most important determinant of effect for ultrafine particles (diameter less than 100 nm), whereas chemical composition may be more important for larger particles. The knowledge concerning mechanisms of action of PM has prompted the use of markers of oxidative stress and DNA damage for human biomonitoring in relation to ambient air. By means of personal monitoring and biomarkers a few studies have attempted to characterize individual exposure, explore mechanisms and identify significant sources to size fractions of ambient air PM with respect to relevant biological effects. In these studies guanine oxidation in DNA has been correlated with exposure to PM(2.5) and ultrafine particles outdoor and indoor. Oxidative stress-induced DNA damage appears to an important mechanism of action of urban particulate air pollution. Related biomarkers and personal monitoring may be useful tools for risk characterization.     

Oxidative and alkylating damage in DNA

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

DNA repair, damage signaling and carcinogenesis

The First joint meeting of the German DGDR (German Society for Research on DNA Repair) and the French SFTG (French Society of Genotoxicology) on DNA Repair was held in Toulouse, France, from September 15 to 19, 2007. It was organized by Lisa Wiesmüller and Bernard Salles together with the scientific committee consisting of Gilbert de Murcia, Jean-Marc Egly, Frank Grosse, Karl-Peter Hopfner, Georges Iliakis, Bernd Kaina, Markus Löbrich, Bernard Lopez, Daniel Marzin and Alain Sarasin. This report summarizes information presented by the speakers (invited lectures and oral communications) during the seven plenary sessions, which include (1) excision repair, (2) DNA repair and carcinogenesis, (3) double-strand break repair, (4) replication in repair and lesion bypass, (5) cellular responses to genotoxic stress, (6) DNA repair machinery within the chromatin context and (7) genotoxicology and testing. A total of 23 plenary lectures, 32 oral communications and 66 posters were presented in this rather intense 4 days meeting, which stimulated extensive discussions and highly interdisciplinary scientific exchanges among the ∼250 participants.     

Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions

Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA. We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains. cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody. Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events. Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells. Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA. A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage. Pathways for mutagenesis at derivatives of oxidized cytosine are discussed.     

Oxidative DNA damage: assessment of the role in carcinogenesis,atherosclerosis, and acquired immunodeficiency syndrome

Free radical attack upon DNA generates a multiplicity of DNA damage, including modified bases. Some of these modifications have considerable potential to damage the integrity of the genome. This article reviews recent data that suggest the involvement of oxidative DNA damage in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome (AIDS). There is evidence that oxidative DNA damage may play a causative role in atherosclerosis. Oxidative DNA damage may lead to apoptotic cell death of patients infected with human immunodeficiency virus (HIV) and may influence the progression of AIDS. While many details regarding the role of reactive oxygen species-induced DNA damage in the etiology of complex multifactorial diseases like cancer are yet to be discovered, evidence suggests that oxidants act at several stages in the malignant transformation of cells. However, the quantitative relationship between the measured DNA damage and the development of cancer is still lacking.     

Oxidative DNA damage and glioma cell death induced by tetrahydropapaveroline

A series of naturally occurring isoquinoline alkaloids, besides their distribution in the environment and presence in certain food stuffs, have been detected in human tissues including particular regions of brain. An example is salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) that not only induces neuronal cell death, but also causes DNA damage and genotoxicity. Tetrahydropapaveroline [THP; 6,7-dihydroxy-1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline], a dopamine-derived tetrahydroisoquinoline alkaloid, has been reported to inhibit mitochondrial respiration and is considered to contribute to neurodegeneration implicated in Parkinson's disease. Since THP bears two catechol moieties, the compound may readily undergo redox cycling to produce reactive oxygen species (ROS) as well as toxic quinoids. In the present study, we have examined the capability of THP to cause oxidative DNA damage and cell death. Incubation of THP with phiX174 supercoiled DNA or calf thymus DNA in the presence of cupric ion caused substantial DNA damage as determined by strand scission or formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. THP plus copper-induced DNA damage was ameliorated by some ROS scavengers/antioxidants and catalase. Treatment of C6 glioma cells with THP led to a concentration-dependent reduction in cell viability, which was prevented by the antioxidant N-acetyl-L-cysteine. When these cells were treated with 10microM THP, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were rapidly activated via phosphorylation, whereas activation of extracellular signal-regulated protein kinase (ERK) was inhibited. Furthermore, pretreatment with inhibitors of JNK and p38 MAPK rescued the glioma cells from THP-induced cytotoxicity, suggestive of the involvement of these kinases in THP-induced C6 glioma cell damage.     

Bleomycin-dependent damage to the bases in DNA is a minor side reaction.

The antitumor antibiotic bleomycin degrades DNA in the presence of ferric ions and H2O2 or in the presence of ferric ions, oxygen, and ascorbic acid. When DNA degradation is measured as formation of base propenals by the thiobarbituric acid assay, it is not inhibited by superoxide dismutase and scavengers of the hydroxyl radical or by catalase (except that catalase inhibits in the bleomycin/ferric ion/H2O2 system by removing H2O2). Using the technique of gas chromatography/mass spectrometry with selected-ion monitoring, we show that DNA degradation is accompanied by formation of small amounts of modified DNA bases. The products formed are identical with those generated when hydroxyl radicals react with DNA bases. Base modification is significantly inhibited by catalase and partially inhibited by scavengers of the hydroxyl radical and by superoxide dismutase. We suggest that the bleomycin-oxo-iron ion complex that cleaves the DNA to form base propenals can decompose in a minor side reaction to generate hydroxyl radical, which accounts for the base modification in DNA. However, hydroxyl radical makes no detectable contribution to the base propenal formation.     

Oxidative DNA damage processing in nuclear and mitochondrial DNA

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, to examine the repair of different types of oxidative lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage.