Oxidative DNA damage and defence gene expression in the mouse lung after short-term exposure to diesel exhaust particles by inhalation

Exposure to diesel exhaust particles (DEP) is suspected to contribute to lung cancer and cardiopulmonary diseases. In recent years generation of reactive oxygen species capable of inducing cellular oxidative stress has been in focus as one of the underlying mechanisms behind the genotoxic effects of particles. However, the role of the antioxidative defence system still needs to be clarified, especially in relation to low-dose DEP exposures. The aim of this study was to characterize the effects of short-term exposure to DEP in terms of DNA damage and expression of key response genes towards oxidative stress in lungs of mice. Mice were exposed by inhalation to 20 or 80 mg/m3 DEP inhaled as either a single dose, or four lower doses (5 and 20 mg/m3) inhaled on four consecutive days. Our results indicate that HO-1 mRNA expression in lung tissue was up-regulated after both types of DEP exposures, whereas OGG1 expression was only up-regulated after repeated exposures. The level of oxidative DNA damage in terms of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was increased in the lung tissue after a single exposure, whereas increased levels of DNA strand breaks was observed in bronchoalveolar lavage cells after repeated DEP exposures. The levels of 8-oxodG and OGG1 mRNA in lung tissue were mirror images. This suggests that after repeated exposures, up-regulation of DNA repair counteracts an increased rate of 8-oxodG formation leaving the steady state level of 8-oxodG in DNA unchanged. In conclusion, this study indicates that a single high dose of DEP generates 8-oxodG in lung tissue, whereas the same dose inhaled as four low-exposures may up-regulate the antioxidative defence system and protect against generation of 8-oxodG.     

Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in colon and liver of Big Blue rats: role of DNA adducts,strand breaks,DNA repair and oxidative stress

The contribution of oxidative stress, different types of DNA damage and expression of DNA repair enzymes in colon and liver mutagenesis induced by 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) was investigated in four groups of six Big Blue rats fed diets with 0, 20, 70, and 200 mg IQ/kg for 3 weeks. There were dose-response relationships of DNA adducts ((32)P-postlabeling) and DNA strand breaks (comet assay) in colon and liver tissues, with the highest levels of DNA adducts and strand breaks in the colon. There was dose-dependent induction of mutations in both the colon and the liver, and the same IQ dose produced two-fold more cII mutations in the liver compared with the colon. The IQ-induced mutation spectrum in the colon was not significantly different to that of control rats. The expression of ERCC1 and OGG1 was higher in the colon than liver, and was unaffected by the IQ diet. Investigations of oxidative stress biomarkers produced inconclusive results. Oxidative DNA damage detected by the endonuclease III enzyme and 7-hydro-8-oxo-2'-deoxyguanosine in colon, liver and/or urine was unaltered by IQ. However, there was increased level of gamma-glutamyl semialdehyde in liver proteins, indicating a higher rate of protein oxidation in the liver following IQ administration. In plasma and erythrocytes there were unaltered levels of oxidized protein, malondialdehyde, and antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase) indicating no systemic oxidative stress. However, the level of total vitamin C was increased in plasma, with the largest fraction being in the reduced form. In conclusion, our results indicate that DNA adducts rather than oxidative stress are responsible for the initiation of IQ-induced carcinogenesis of the liver and colon. A lower frequency of mutations in the colon than in the liver could be related to higher expression of DNA repair enzymes in the former.     

A sucrose-rich diet induces mutations in the rat colon

A sucrose-rich diet has repeatedly been observed to have cocarcinogenic actions in the colon and liver of rats and to increase the number of aberrant crypt foci in rat colon. To investigate whether sucrose-rich diets might directly increase the genotoxic response in the rat colon or liver, we have added sucrose to the diet of Big Blue rats, a strain of Fischer rats carrying 40 copies of the lambda-phage on chromosome 4. Dietary sucrose was provided to the rats for 3 weeks at four dose levels including the background level in the purified diet [3.4% (control), 6.9%, 13.8%, or 34.5%] without affecting the overall energy and carbohydrate intake. We observed a dose-dependent increase in the mutation frequency at the cII site in the colonic mucosa with increased sucrose levels, reaching a 129% increase at the highest dose level. This would indicate a direct or indirect genotoxic effect of a sucrose-rich diet. No significant increase in mutations was observed in the liver. To seek an explanation for this finding, a variety of parameters were examined representing different mechanisms, including increased oxidative stress, changes in oxidative defense, effects on DNA repair, or changes in the background levels of DNA adducts. Sucrose did not increase the number of DNA strand breaks or oxidized bases assessed as endonuclease III-sensitive sites or 8-oxodeoxyguanosine in colon or liver. DNA repair capacity as determined by expression of the rERCC1 or rOGG1 genes was not increased in colon or liver, but the background level of DNA adducts (I-compounds) as determined by (32)P postlabeling was significantly decreased in colon. This decrease in colon I-compounds correlated inversely with both mutation frequency and ERCC1 DNA repair gene expression. Dietary sucrose did not change liver apoptosis or cell turnover as determined by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling assay and proliferating cell nuclear antigen. An increase in liver ascorbate was also observed, whereas oxidative damage was not observed in proteins or lipids in liver cytosol or in blood plasma. We conclude that a sucrose-rich diet directly or indirectly increases the mutation frequency in rat colon in a dose-dependent manner and concomitantly decreases the level of background DNA adducts, without a direct effect on the expression of major DNA repair enzyme systems. We also conclude that an oxidative mechanism for this effect of sucrose is unlikely. This is the first demonstration of a genotoxic action of increased dietary sucrose in vivo. Both sucrose intake and colon cancer rates are high in the Western world, and our present results call for an examination of a possible direct relationship between the two.     

Repair of 8-oxodeoxyguanosine lesions in mitochondrial dna depends on the oxoguanine dna glycosylase (OGG1) gene and 8-oxoguanine accumulates in the mitochondrial dna of OGG1-defective mice.

Mitochondria are not only the major site for generation of reactive oxygen species, but also one of the main targets of oxidative damage. One of the major products of DNA oxidation, 8-oxodeoxyguanosine (8-oxodG), accumulates in mitochondrial DNA (mtDNA) at levels three times higher than in nuclear DNA. The main pathway for the repair of 8-oxodG is the base excision repair pathway initiated by oxoguanine DNA glycosylase (OGG1). We previously demonstrated that mammalian mitochondria from mice efficiently remove 8-oxodG from their genomes and isolated a protein from rat liver mitochondria with 8-oxoguanine (8-oxodG) DNA glycosylase/apurinic DNA lyase activity. In the present study, we demonstrated that the mitochondrial 8-oxodG DNA glycosylase/apurinic DNA lyase activity is the mitochondrial isoform of OGG1. Using mouse liver mitochondria isolated from ogg1(-/-) mice, we showed that the OGG1 gene encodes for the mitochondrial 8-oxodG glycosylase because these extracts have no incision activity toward an oligonucleotide containing a single 8-oxodG DNA base lesion. Consistent with an important role for the OGG1 protein in the removal of 8-oxodG from the mitochondrial genome, we found that mtDNA isolated from liver from OGG1-null mutant animals contained 20-fold more 8-oxodG than mtDNA from wild-type animals.     

Personal exposure to PM2.5 and biomarkers of DNA damage.

Ambient particulate air pollution assessed as outdoor concentrations of particulate matter < or = 2.5 microm in diameter (PM(2.5)) has been associated with an increased cancer risk. However, outdoor PM(2.5) concentrations may not be the best measure of the individual particle exposure that is a sum of many sources besides outdoor particle levels, e.g., environmental tobacco smoke and cooking. We measured personal PM(2.5) and black smoke exposure in 50 students four times over 1 year and analyzed for biomarkers of different types of DNA damages. Ambient PM(2.5) concentrations were also measured. Exposure was measured for 48 h, after which blood samples were collected and analyzed for DNA damage in lymphocytes in terms of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG), strand breaks, endonuclease III- and fapyguanine glycosylase-sensitive sites, and polyaromatic hydrocarbon adducts. Twenty-four-h urine collections were analyzed for 8-oxodG and 1-hydroxypyrene. Personal PM(2.5) exposure was found to be a predictor of 8-oxodG in lymphocyte DNA with an 11% increase in 8-oxodG/10 microg/m(3) increase in personal PM(2.5) exposure (P = 0.007). No other associations between exposure markers and biomarkers could be distinguished. The genotype of glutathione S-transferase M1 (GSTM1), T1 (GSTT1), and P1 (GSTP1) and NADPH:quinone reductase was also determined, but there were no effects of genotype on DNA polyaromatic hydrocarbon adducts or oxidative damage. The results suggest that moderate exposure to concentrations of PM can induce oxidative DNA damage and that personal PM(2.5) exposure is more important in this aspect than is ambient PM(2.5) background concentration.     

Induction of DNA polymerase beta-dependent base excision repair in response to oxidative stress in vivo

Base excision repair (BER) is the DNA repair pathway primarily responsible for repairing small base modifications and abasic sites caused by normal cellular metabolism or environmental insult. Strong evidence supports the requirement of DNA polymerase beta (beta-pol) in the BER pathway involving single nucleotide gap filling DNA synthesis in mammalian systems. In this study, we examine the relationship between oxidative stress, cellular levels of beta-pol and BER to determine whether oxidizing agents can upregulate BER capacity in vivo. Intraperitoneal injection of 2-nitropropane (2-NP, 100 mg/kg), an oxidative stress-inducing agent, in C57BL/6 mice was found to generate 8-hydroxydeoxyguanosine (8-OHdG) in liver tissue (4-fold increase, P < 0.001). We also observed a 4-5-fold increase in levels of DNA single strand breaks in 2-NP treated animals. The protein level of the tumor suppressor gene, p53 was also induced in liver by 2-NP (2.1-fold, P < 0.01), indicating an induction of DNA damage. In addition, we observed a 2-3-fold increase in mutant frequency in the lacI gene after exposure to 2-NP. Interestingly, an increase in DNA damage upregulated the level of beta-pol as well as BER capacity (42%, P < 0.05). These results suggest that beta-pol and BER can be upregulated in response to oxidative stress in vivo. Furthermore, data show that heterozygous beta-pol knockout (beta-pol(+/-)) mice express higher levels of p53 in response to 2-NP as compared with wild-type littermates. While the knockout and wild-type mice display similar levels of 8-OHdG after 2-NP exposure, the beta-pol(+/-) mice exhibit a significant increase in DNA single strand breaks. These findings suggest that in mice, a reduction in beta-pol expression results in a higher accumulation of DNA damage by 2-NP, thus establishing the importance of the beta-pol-dependent BER pathway in repairing oxidative damage.     

Induction and repair inhibition of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells

Compounds of nickel(II) and cadmium(II) are carcinogenic to humans and to experimental animals. One frequently discussed mechanism involved in tumor formation is an increase in reactive oxygen species by both metals with the subsequent generation of oxidative DNA damage. In the present study we used human HeLa cells to investigate the potential of nickel(II) and cadmium(II) to induce DNA lesions typical for oxygen free radicals in intact cells and the effect on their repair. As indicators of oxidative DNA damage, we determined the frequencies of DNA strand breaks and of lesions recognized by the bacterial formamidopyrimidine-DNA glycosylase (Fpg protein), including 7,8- dihydro-8-oxoguanine (8-hydroxyguanine), a pre-mutagenic DNA base modification. Nickel(II) caused a slight increase in DNA strand breaks at 250 microM and higher, while the frequency of Fpg-sensitive sites was enhanced only at the cytotoxic concentration of 750 microM. The repair of oxidative DNA lesions induced by visible light was reduced at 50 microM and at 100 microM nickel(II) for Fpg-sensitive sites and DNA strand breaks, respectively; the removal of both types of lesions was blocked nearly completely at 250 microM nickel(II). In the case of cadmium(II), DNA strand breaks occurred at 10 microM and no Fpg- sensitive sites were detected. However, the repair of Fpg-sensitive DNA lesions induced by visible light was reduced at 0.5 microM cadmium(II) and higher, while the closure of DNA strand breaks was not affected. Since oxidative DNA damage is continuously induced during aerobic metabolism, an impaired repair of these lesions might well explain the carcinogenic action of nickel(II) and cadmium(II).      

Measurement and meaning of oxidatively modified DNA lesions in urine.

BACKGROUND: Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. The noninvasive assessment of such damage, i.e., in urine, and application to large-scale human studies are vital to understanding this role and devising intervention strategies. METHODS: We have reviewed the literature to establish the status quo with regard to the methods and meaning of measuring DNA oxidation products in urine. RESULTS: Most of the literature focus upon 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and whereas a large number of these reports concern clinical conditions, there remains (a) lack of consensus between methods, (b) possible contribution from diet and/or cell death, (c) no definitive DNA repair source of urinary 2'-deoxyribonucleoside lesions, and (d) no reference ranges for healthy or diseased individuals. CONCLUSIONS: The origin of 8-oxodG is not identified; however, recent cell culture studies suggest that the action of Nudix hydrolase(s) on oxidative modification of the nucleotide pool is a likely candidate for the 8-oxodG found in urine and, potentially, of other oxidized 2'-deoxyribonucleoside lesions. Literature reports suggest that diet and cell death have minimal, if any, influence upon urinary levels of 8-oxodG and 8-oxo-7,8-dihydroguanine, although this should be assessed on a lesion-by-lesion basis. Broadly speaking, there is consensus between chromatographic techniques; however, ELISA approaches continue to overestimate 8-oxodG levels and is not sufficiently specific for accurate quantification. With increasing numbers of lesions being studied, it is vital that these fundamental issues are addressed. We report the formation of the European Standards Committee on Urinary (DNA) Lesion Analysis whose primary goal is to achieve consensus between methods and establish reference ranges in health and disease.     

Methanol exposure does not lead to accumulation of oxidative DNA damage in bone marrow and spleen of mice, rabbits or primates

Genotoxicity tests indicate methanol (MeOH) is not mutagenic, but a rodent study has suggested carcinogenic potential, which could result from free radical-initiated oxidative DNA damage. To investigate this possibility we treated male CD-1 mice, New Zealand white rabbits, and cynomolgus monkeys with MeOH (2.0 g/kg ip) and assessed tissue oxidative DNA damage 6 h post-dose, measured as 8-hydroxy-2'-deoxyguanosine (8-oxodG). We found no MeOH-dependent increases in 8-oxodG in bone marrow or spleen of any species. Chronic treatment of CD-1 mice with MeOH (2.0 g/kg ip) daily for 15 d also did not increase 8-oxodG levels in these organs. Further studies in the DNA repair deficient oxoguanine glycosylase 1 (Ogg1) knockout (KO) mice supported these findings. Fibroblasts from Ogg1 KO mice accumulated 8-oxodG following acute exposure to the renal carcinogen potassium bromate (KBrO(3) ; 2.0 mM) but did not accumulate 8-oxodG following exposure to 125 mM MeOH 6 h post-treatment. Ogg1 KO mice accumulated 8-oxodG in bone marrow and spleen with age but not following exposure to MeOH. In addition, free radical-mediated hydroxynonenal-histidine protein adducts were not enhanced by MeOH in primate bone marrow or spleen, or in rabbit bone marrow or mouse spleen, although modest increases were observed in rabbit spleen and mouse bone marrow. Taken together these observations suggest that MeOH exposure does not promote the accumulation of oxidative DNA damage in bone marrow and spleen, and it is unlikely that human environmental exposure to MeOH would lead to lymphomas via this mechanism.     

Kinetics of DNA adduct formation and removal in mouse hepatocytes following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole

5,9-Dimethyldibenzo[c,g]carbazole (DMDBC), a potent mouse hepatocarcinogen, has been shown to induce a non-linear increase in mutant frequency in the liver of the transgenic MutaMouse. To gain insight into the mechanisms underlying the mutagenicity of DMDBC in vivo, DNA damage formation and removal were monitored in mouse hepatocytes over 4-144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured by (32)P-post-labeling. DNA repair was assessed by: (i) the unscheduled DNA synthesis (UDS) assay, which measures [(3)H]thymidine incorporation into hepatocyte DNA undergoing excision repair; (ii) the Comet assay, which detects DNA strand breaks transiently produced between the incision and rejoining steps of the excision repair process. A plateau of approximately 400 DNA adducts/10(8) nucleotides was reached 24 h after treatment with 10 mg/kg and remained unchanged until 144 h. UDS activity was significantly induced at 15 and 24 h, while no DNA strand breaks were observed at any sampling time. These results suggest that DNA repair mechanisms were efficiently induced and the formation of a high degree of DNA damage was avoided at this dose level. Following exposure to 90 mg/kg DMDBC, the number of DNA adducts increased sharply to a maximum at 24 h ( approximately 8000/10(8) nucleotides) and then declined to approximately 500/10(8) nucleotides at 144 h. UDS activity was markedly induced from 15 to 72 h. Low levels of DNA strand breaks were observed at 24 and 48 h. The formation of large numbers of DNA adducts and the emergence of DNA strand breaks despite a strong initial induction of UDS activity suggested that DNA repair mechanisms were saturated at this dose level. This phenomenon could partly account for the non-linear induction of gene mutations previously reported in the liver of the transgenic MutaMouse.     

Elevated oxidative stress and DNA damage and repair levels in urinary bladder carcinomas associated with schistosomiasis

To cast light on mechanisms underlying development of urothelial carcinomas (UCs) of the urinary bladder associated with Schistosomiasis, we immunohistochemically analyzed the relationship between oxidative stress markers, DNA single strand breaks (ssDNA) which could also measure the levels of base damage and apoptosis in DNA, and expression of DNA repair genes with levels of nitric oxide synthases in bladder carcinomas of Egyptian patients with or without Schistosoma hematobium infection. Marked elevation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels was found in squamous cell carcinomas and UCs associated with Schistosomiasis when compared with non-Schistosomal carcinomas. This was accompanied by strong over expression of the DNA-repair genes, 8-oxoguanine-DNA-glycosylase and apurinic/apyrimidinic endonuclease, as well as increased formation levels of ssDNA. Expression levels of inducible nitric oxide synthase (iNOS) which is known to be indirectly related to oxidative stress was higher in Schistosomal than in the non-Schistosomal carcinomas. However, expression of endothelial nitric oxide synthase was slightly stronger in non-Schistosomal than in the Schistosomal carcinomas. In conclusion, these findings suggest a strong correlation between Schistosoma haematobium infection and increased levels of oxidative stress accompanied by a continuous DNA damage and repair in UCs, all directly correlating with elevated iNOS.     

Urinary 8-oxodeoxyguanosine, aflatoxin B1 exposure and hepatitis B virus infection and hepatocellular carcinoma in Taiwan

To evaluate the role of oxidative stress and aflatoxin exposure on risk of hepatocellular carcinoma (HCC), a case-control study nested within a community-based cohort was conducted in Taiwan. Baseline urine samples, collected from a total of 74 HCC cases and 290 matched controls, were used to determine by enzyme-linked immunosorbent assays the level of urinary excretion of 8-oxodeoxyguanosine (8-oxodG), a biomarker of oxidative DNA damage and urinary aflatoxin B(1) metabolites, a biomarker of aflatoxin exposure. Multivariate-adjusted linear regression analysis showed that urinary aflatoxin metabolites and gender were significantly associated with level of urinary 8-oxodG among controls. Moreover, after adjustments for potential confounding factors, there was a statistically significant positive dose-response relationship between levels of urinary 8-oxodG and urinary aflatoxin metabolites (P < 0.0001). However, when compared with subjects in the lowest quartile of 8-oxodG, there was a decrease in risk of HCC, with adjusted odds ratios (ORs) of 0.8 [95% confidence interval (CI) 0.3-2.0], 0.7 (95% CI 0.3-2.0) and 0.7 (95% CI 0.2-1.7) for subjects in the second, third and fourth quartile, respectively. The combination of level of urinary 8-oxodG below the median and hepatitis B virus infection resulted in an OR of 11.4 (95% CI 3.9-33.3), compared with those with urinary 8-oxodG above the median and hepatitis B virus surface antigen negative. These results suggest that elevated levels of urinary 8-oxodG may be related to increasing level of aflatoxin exposure but may also indicate enhanced repair of oxidative DNA damage and therefore lower risk of HCC.     

Effects of 5'-iododeoxyuridine on the repair of radiation induced potentially lethal damage interphase chromatin breaks and DNA double strand breaks in Chinese hamster ovary cells.

The effect of 5'-iododeoxyuridine incorporation into DNA on radiation sensitivity, cellular repair capability, induction and repair of interphase chromatin breaks, as well as induction and repair of DNA double strand breaks was investigated in plateau-phase Chinese hamster ovary cells exposed to X rays. Repair of potentially lethal damage, as measured by delayed plating plateau-phase cells, was used to assay cellular repair capacity. Induction and repair of interphase chromatin breaks were assayed by means of premature chromosome condensation, whereas induction and repair of DNA double strand breaks were assayed by pulsed field gel electrophoresis. Incorporation of 5'-iododeoxyuridine into DNA sensitized cells to radiation. Radiosensitization increased with increasing percent thymidine replacement and was accompanied by an increase in the number of chromatin breaks scored per Gy and a small increase in the number of DNA double strand breaks produced. Cells grown in the presence of 5'-iododeoxyuridine were able to repair potentially lethal damage. When the comparison was made at equal doses, the extent of this repair was higher but its rate slower in 5'-iododeoxyuridine containing cells. At equal survival levels, cells that had incorporated IdU also repaired PLD to a slightly higher extent than control cells grown in IdU free medium. The magnitude of potentially lethal damage repair increased as cells "aged" in the plateau-phase, particularly for high 5'-iododeoxyuridine concentrations (8 microM). Incorporation of 5'-iododeoxyuridine reduced the rate of repair of interphase chromatin breaks and the rate of repair of DNA double strand breaks (both the fast and the slow component). The results suggest that reduction in the efficiency of repair of DNA double strand breaks and chromatin breaks, produced by radiation in 5'-iododeoxyuridine containing cells, is one determinant of the radiosensitization observed.     

Inhibition of 1,2-Dimethylhydrazine-induced Oxidative DNA Damage by Green Tea Extract in Rat

Following subcutaneous injection of 1,2-dimethylhydrazine (DMH), which is carcinogenic to rat colon and liver, to Sprague-Dawley rats, a significant increase of 8-hydroxydeoxyguanosine (8-OHdG) was observed in the DNA of colonic mucosa and liver. The 8-OHdG formation reached the maximal level at about 24 h after the DMH injection. On the other hand, no increase of 8-OHdG was observed in the DNA of the kidney. Drinking green tea extract (GTE) for ten days prior to the DMH injection significantly inhibited the formation of 8-OHdG in the colon. These findings demonstrate that DMH causes oxidative damage to the DNA of its target organ, and that GTE protects colonic mucosa from this oxidative damage.     

Analysis of oxidative DNA damage in patients with colorectal cancer

PURPOSE: The aim of this study was to measure the levels of oxidative DNA damage in cells isolated from the colon mucosa in patients with colorectal cancer and to compare normal and neoplastic tissues and make correlations with anatomopathologic variables. PATIENTS AND METHODS: Thirty-three patients with colorectal adenocarcinoma were studied. The oxidative DNA damage was evaluated by means of the alkaline version of the comet assay. RESULTS: For all the patients studied, it was found that the cells obtained from the neoplastic tissue presented oxidative DNA damage greater than in the cells from normal tissue. The cells isolated from the neoplastic mucosal tissue of the colon presented significantly greater mean extent of DNA strand breakage than the cells isolated from normal tissue. Additionally, the patients at earlier stages of the Dukes and TNM classifications presented higher levels of oxidative damage than those at more advanced stages. CONCLUSION: Assessment of the levels of oxidative damage at the different stages of colorectal carcinogenesis is of great interest because it enables evaluation of the effectiveness of antioxidant substances that could be used as preventive measures against the initial oxidative aggressive action on the colonic mucosa.     

Induction of oxidative DNA damage by arsenite and its trivalent and pentavalent methylated metabolites in cultured human cells and isolated DNA

Even though a well-known human carcinogen the underlying mechanisms of arsenic carcinogenicity are still not fully understood. For arsenite, proposed mechanisms are the interference with DNA repair processes and an increase in reactive oxygen species. Even less is known about the genotoxic potentials of its methylated metabolites monomethylarsonous [MMA(III)] and dimethylarsinous [DMA(III)] acid, monomethylarsonic [MMA(V)] and dimethylarsinic [DMA(V)] acid. Within the present study we compared the induction of oxidative DNA damage by arsenite and its methylated metabolites in cultured human cells and in isolated PM2 DNA, by frequencies of DNA strand breaks and of lesions recognized by the bacterial formamidopyrimidine-DNA glycosylase (Fpg). Only DMA(III) (> or =10 micro M) generated DNA strand breaks in isolated PM2 DNA. In HeLa S3 cells, short-term incubations (0.5-3 h) with doses as low as 10 nM arsenite induced high frequencies of Fpg-sensitive sites, whereas the induction of oxidative DNA damage after 18 h incubation was rather low. With respect to the methylated metabolites, both trivalent and pentavalent metabolites showed a pronounced induction of Fpg-sensitive sites in the nanomolar or micromolar concentration range, respectively, which was present after both short-term and long-term incubations. Furthermore MMA(III) and DMA(V) generated DNA strand breaks in a concentration-dependent manner. Taken together our results show that very low physiologically relevant doses of arsenite and the methylated metabolites induce high levels of oxidative DNA damage in cultured human cells. Thus, biomethylation of inorganic arsenic may be involved in inorganic arsenic-induced genotoxicity/carcinogenicity.     

Spontaneous mutagenesis in Csb(m/m)Ogg1⁻(/)⁻ mice is attenuated by dietary resveratrol

Oxidative DNA modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are generated endogenously in apparently all living cells. The defect of the repair of 8-oxoG in Csb(m/m)Ogg1?(/)? mice results in elevated basal levels of these lesions and increased frequencies of spontaneous mutations, which initiate tumorigenesis in the liver if cell proliferation is stimulated. Here, we describe that the phytoalexin resveratrol, applied either for 7 days per gavage (100 mg/kg body wt) or for 3-9 months in the diet (0.04% ad libitum), reduces the endogenous oxidative DNA base damage in the livers of the Csb(m/m)Ogg1?(/)? mice by 20-30% (P < 0.01). A small but consistent effect is also observed in the wild-type animals. The spontaneous mutation frequencies determined in the lacI gene of BigBlue? Csb(m/m)Ogg1?(/)? mice are concomitantly reduced by resveratrol to similar extents. Mechanistically, the protection is caused by an induction of the antioxidant defense system since (i) hepatocytes isolated from all resveratrol-treated animals were less susceptible to the generation of single-strand breaks and to cell killing by H?O?, (ii) messenger RNA levels of superoxide dismutases 1 and 2 (SOD1 and SOD2) heme oxygenase-1 and glutathione peroxidase were significantly upregulated after the short-term treatment and (iii) mutations primarily ascribed to the oxidative base modification 8-oxoG (G:C to T:A transversions) were more strongly suppressed than G:C to A:T transitions ascribed to spontaneous deamination. The results thus demonstrate that spontaneous somatic mutation rates resulting from endogenous oxidative DNA damage can be reduced by application of an exogenous agent.