Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,N6-ethenoadenine and hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine

It has previously been reported that 1,N6-ethenoadenine (epsilonA), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N4-ethenocytosine (epsilonC), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing epsilonA and Hx, but retained wild-type levels of activity for epsilonC and 8-oxoG. These findings indicate that (i) the previously identified epsilonA-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; (ii) the two structurally closely related mutagenic adducts epsilonA and epsilonC are repaired by separate gene products; and (iii) APNG does not contribute detectably to the repair of 8-oxoG.     

Mammalian 3-methyladenine DNA glycosylase protects against the toxicity and clastogenicity of certain chemotherapeutic DNA cross-linking agents

DNA repair status is recognized as an important determinant of the clinical efficacy of cancer chemotherapy. To assess the role that a mammalian DNA glycosylase plays in modulating the toxicity and clastogenicity of the chemotherapeutic DNA cross-linking alkylating agents, we compared the sensitivity of wild-type murine cells to that of isogenic cells bearing homozygous null mutations in the 3-methyladenine DNA glycosylase gene (Aag). We show that Aag protects against the toxic and clastogenic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C (MMC), as measured by cell killing, sister chromatid exchange, and chromosome aberrations. This protection is accompanied by suppression of apoptosis and a slightly reduced p53 response. Our results identify 3-methyladenine DNA glycosylase-initiated base excision repair as a potentially important determinant of the clinical efficacy and, possibly, the carcinogenicity of these widely used chemotherapeutic agents. However, Aag does not contribute significantly to protection against the toxic and clastogenic effects of several chemotherapeutic nitrogen mustards (namely, mechlorethamine, melphalan, and chlorambucil), at least in the mouse embryonic stem cells used here. We also compare the Aag null phenotype with the Fanconi anemia phenotype, a human disorder characterized by cellular hypersensitivity to DNA cross-linking agents, including MMC. Although Aag null cells are sensitive to MMC-induced growth delay and cell cycle arrest, their sensitivity is modest compared to that of Fanconi anemia cells.     

DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and a neurotoxic metabolite of o-tolyl phosphates. In a previous paper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is mutagenic in Salmonella typhimurium TA100 and forms DNA adducts in incubations with nucleotides, nucleosides and isolated DNA. In the present study we compare DNA adduct formation using 32P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide-treated bacteria (S.typhimurium TA100) and hepatoma cells with DNA adducts formed in liver, kidney, lung and heart of tri-o-tolyl phosphate-exposed Fischer 344 male rats. In both bacteria and hepatoma cells two DNA adducts could be detected after treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide. The minor adduct co-chromatographed with synthetic N3-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling. The major DNA adduct was a cytidine adduct, most likely N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate. Male Fischer 344 rats were treated orally for 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNA was isolated from liver, kidney, lung, heart, brain and testes 1, 4, 7 and 28 days after giving the last dose. Analysis by 32P-postlabelling revealed that two adducts were present in the DNA isolated from liver, kidney, lung and heart on the first day after giving the last dose; DNA adducts were not detected in the brain and testes. The adduct pattern after in vivo treatment with tri-o-tolyl phosphate was identical with that found in bacteria and hepatoma cells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin 2-oxide, the major adduct being N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate and the minor N3-(o-hydroxybenzyl)deoxyuridine 3' monophosphate. Both DNA adducts persisted in the lungs for the entire observation period, whereas in the kidney only the cytidine adduct could be detected 28 days after the last dose of tri-o-tolyl phosphate. In liver and heart the adducts were detectable only on the first day after completion of the treatment. The results indicate that in addition to the well established neurotoxicity, some o-tolyl phosphates may have a carcinogenic potential.     

Detection of 1,N6-ethenoadenine in rat urine after chloroethylene oxide exposure

The four etheno adducts of vinyl chloride formed in DNA, 1,N6-ethenoadenine (epsilonA), 3,N4-ethenocytosine, 1,N2-ethenoguanine and N2,3-ethenoguanine were previously reported to be released from DNA by a family of enzymes in the base-excision repair pathway (Dosanjh et al., Proc. Natl Acad. Sci. USA, 91, 1024-1028, 1994; Hang et al., Carcinogenesis, 17, 155-157, 1996; Hang et al., Proc. Natl Acad. Sci. USA, 94, 12869-12874, 1997). Adducts excised from DNA by glycosylases are usually excreted in urine and have been reported to be potential biomarkers of DNA damage in exposed individuals. In this study, we report the detection of epsilonA in the urine of rats exposed to chloroethylene oxide (CEO) using immunoaffinity columns made with specific monoclonal antibodies for enrichment, followed by quantitation by HPLC with fluorescence detection. Chemical analysis of urine samples revealed the presence of a compound chromatographically identical to authentic epsilonA standard. This compound was confirmed by mass spectral analysis. EpsilonA was present in urine of control and CEO-treated rats, with the latter having up to 50-fold greater amounts. The cumulative excretion of epsilonA reached a plateau between 24 and 48 h post-exposure. While it is clear that CEO treatment results in increased excretion of epsilonA, the exact source of the adduct is unknown. When rats were administered epsilonA i.v., approximately 10% of the administered dose was excreted in urine. This research demonstrates that urinary excretion of epsilonA may be a potential biomarker for in vivo alkylation of DNA and nucleotide pools.     

The critical active-site amine of the human 8-oxoguanine DNA glycosylase, hOgg1: direct identification, ablation and chemical reconstitution

BACKGROUND: Base-excision DNA repair (BER) is the principal pathway responsible for the removal of aberrant, genotoxic bases from the genome and restoration of the original sequence. Key components of the BER pathway are DNA glycosylases, enzymes that recognize aberrant bases in the genome and catalyze their expulsion. One major class of such enzymes, glycosylase/lyases, also catalyze scission of the DNA backbone following base expulsion. Recent studies indicate that the glycosylase and lyase functions of these enzymes are mechanistically unified through a common amine-bearing residue on the enzyme, which acts as both the electrophile that displaces the aberrant base and an electron sink that facilitates DNA strand scission through imine (Schiff base)/conjugate elimination chemistry. The identity of this critical amine-bearing residue has not been rigorously established for any member of a superfamily of BER glycosylase/lyases. RESULTS: Here, we report the identification of the active-site amine of the human 8-oxoguanine DNA glycosylase (hOgg1), a human BER superfamily protein that repairs the mutagenic 8-oxoguanine lesion in DNA. We employed Edman sequencing of an active-site peptide irreversibly linked to substrate DNA to identify directly the active-site amine of hOgg1 as the epsilon-NH2 group of Lys249. In addition, we observed that the repair-inactive but recognition-competent Cys249 mutant (Lys249-->Cys) of hOgg1 can be functionally rescued by alkylation with 2-bromoethylamine, which functionally replaces the lysine residue by generating a gamma-thia-lysine. CONCLUSIONS: This study provides the first direct identification of the active-site amine for any DNA glycosylase/lyase belonging to the BER superfamily, members of which are characterized by the presence of a helix-hairpin-helix-Gly/Pro-Asp active-site motif. The critical lysine residue identified here is conserved in all members of the BER superfamily that exhibit robust glycosylase/lyase activity. The ability to trigger the catalytic activity of the Lys249-->Cys mutant of hOgg1 by treatment with the chemical inducer 2-bromoethylamine may permit snapshots to be taken of the enzyme acting on its substrate and could represent a novel strategy for conditional activation of catalysis by hOgg1 in cells.     

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.     

Opposite base-dependent excision of 7,8-dihydro-8-oxoadenine by the Ogg1 protein of Saccharomyces cerevisiae

The yOgg1 protein of Saccharomyces cerevisiae is a DNA glycosylase/AP lyase that excises guanine lesions such as 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (me-Fapy-G) and incises apurinic/apyrimidinic sites (AP sites) in damaged DNA. The yOgg1 protein displays a marked preference for DNA duplexes containing 8-OxoG or AP sites placed opposite cytosine. In this paper, we show that yOgg1 can also excise an adenine lesion, 7,8-dihydro-8-oxoadenine (8-OxoA), when paired with cytosine or 5-methylcytosine. In contrast, yOgg1 does not release 8-OxoA when placed opposite thymine, adenine, guanine or uracil. The specificity constants (Kcat/Km) for repair of 8-OxoG/C and 8-OxoA/C duplexes are (50 +/- 18) x 10(-3) and (13 +/- 3) x 10(-3)/min/nM, respectively. The catalytic mechanism for strand cleavage at 8-OxoA/C involves excision of 8-OxoA by the DNA glycosylase activity of yOgg1, followed by incision at the newly formed AP site via a beta-elimination reaction. Furthermore, cleavage of 8-OxoA/C involves formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydride (NaBH4). The yOgg1 protein binds strongly to the 8-OxoA/C duplex, as demonstrated by an apparent dissociation constant (Kdapp) value of 45 nM, as determined by gel mobility shift assay. In contrast, the yOgg1 protein has a very low binding affinity for the 8-OxoA/T duplex, a Kdapp value of 680 nM, which in turn can explain the lack of repair of 8-OxoA in this duplex. The capacity of other DNA glycosylases/AP lyases to repair 8-OxoA has also been investigated. The results show that human hOgg1 protein efficiently repairs 8-OxoA placed opposite cytosine or 5-methylcytosine. On the other hand, the Fpg protein of Escherichia coli cleaves 8-OxoA/C at a very slow rate as compared with yOgg1.     

An oxidative damage-specific endonuclease from rat liver mitochondria

Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE's molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mM KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mM EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE's activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.     

8-hydroxyguanine levels in nuclear DNA and its repair activity in rat organs associated with age

8-Hydroxyguanine (8-OH-Gua) is one of the most abundant types of oxidative DNA damage. The levels of 8-OH-Gua, and its repair activity, were quantified in 3-week-, 5-month-, and 30-month-old male Sprague-Dawley rat organs such as liver, kidney, spleen, lung, small intestine, and brain. The levels of 8-OH-Gua were significantly higher in the 5-month-old rat kidney and brain and 30-month-old rat spleen when compared to that of the 3-week-old rats. However, no significant differences were found in the organs between 5- and 30-month-old rats that were due to the aging process. The repair activity levels of kidney, spleen, and lung were higher than those of liver, small intestine, and brain. This pattern was consistent for the three age stages.     

Induction of DNA adducts and mutations in spleen, liver and lung of XPA-deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development

We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild-type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA-deficient mice.     

Sequence context is an important determinant in the mutagenic potential of 1,N6-ethenodeoxyadenosine (epsilonA): formation of epsilonA basepairs and elongation in defined templates

Many laboratories have obtained data on mutagenicity of modified bases in naturally occurring DNA sequences. It has often been noted that mutation is favored in certain sequence contexts, sometimes termed 'hot spots'. This approach to the contribution of neighboring sequences does not permit a systematic study of both the qualitative and quantitative mutational frequencies. In the present experiments we have chosen to use the exocyclic adduct, 1,N6-etheno A (epsilonA), site-specifically placed in a defined 25-mer oligonucleotides in which epsilonA is flanked by differing 5' and 3' tandem bases. Mutation was assessed using an in vitro replication assay and five polymerases of varying fidelity. The relevant central sequences were 3' --> 5' -CC-epsilonA-CC-, -GG-epsilonA-GG-, -TT-epsilonA-TT-, -AA-epsilonA-AA-, -GG-epsilonA-TT-, -TT-epsilonA-AA-, -AT-epsilonA-TT- and -TA-epsilonA-TA-. Using the Klenow fragment (Kf) (exo+ or exo-) of E. coli Pol I, it was found the epsilonA is an ambiguous base and, with varying efficiencies, all four dNTPs could be inserted opposite epsilonA in all sequences. However, only 3' --> 5' -TT-epsilonA-TT-, -GG-epsilonA-TT- and -AT-epsilonA-TT- were fully extended to a significant extent. The only sequences essentially blocked at the position of epsilonA were -AA-epsilonA-AA- and -TT-epsilonA-AA-. The others were intermediate. When replication was performed with Sequenase, MMLV RT or HIV RT, different patterns were observed, in which replication terminated one base prior to epsilonA, at epsilonA, or one base after epsilonA without further extension. In favored sequences, using the Klenow fragment, an epsilonA x N pair could be extended to form normal basepairs. No extension could be demonstrated in sequences in which tandem adenines were 5' to epsilonA. Kinetic data showed that two of the epsilonA x N pairs, epsilonA x A and epsilonA x C, could form at 10 microM or less dNTP. Which bases were preferentially inserted opposite epsilonA was a function of the flanking bases. Under the kinetic conditions used, epsilonA x T did not form even at 1 mM dTTP. These results indicate that the chemical structure of an adduct is not the only determinant of mutagenic efficiency. It is likely that the effect of the adduct on replication is due to the changes in the structural environment conferred by the flanking bases.     

Anti-mutagenic activity of DNA damage-binding proteins mediated by direct inhibition of translesion replication

DNA lesions that block replication can be bypassed in Escherichia coli by a special DNA synthesis process termed translesion replication. This process is mutagenic due to the miscoding nature of the DNA lesions. We report that the repair enzyme formamido-pyrimidine DNA glycosylase and the general DNA damage recognition protein UvrA each inhibit specifically translesion replication through an abasic site analog by purified DNA polymerases I and II, and DNA polymerase III (alpha subunit) from E. coli. In vivo experiments suggest that a similar inhibitory mechanism prevents at least 70% of the mutations caused by ultraviolet light DNA lesions in E. coli. These results suggest that DNA damage-binding proteins regulate mutagenesis by a novel mechanism that involves direct inhibition of translesion replication. This mechanism provides anti-mutagenic defense against DNA lesions that have escaped DNA repair.     

Correlation between sequence-dependent glycosylase repair and the thermal stability of oligonucleotide duplexes containing 1, N6-ethenoadenine

Previous experiments on DNA sequence context reported that base modification, replication, and repair are affected by the nature of neighbor bases. We now report that repair by mammalian alkylpurine-DNA-N-glycosylases (APNG) of 15-mer oligonucleotides with a central 1,N6-ethenoadenine (epsilonA), flanked by 5' and 3' tandem bases, is also highly sequence dependent. Oligonucleotides with the central sequences -GGepsilonAGG- or -CCepsilonACC- are repaired 3-5-fold more efficiently than those containing -AAepsilonAAA- or -TTepsilonATT- when using human or mouse APNG. Melting curves of the same duplexes showed that oligomers with G.C/C. G neighbors were less denatured than those with A.T/T.A neighbors at 37 degreesC. This sequence-dependent difference in denaturation correlates with the relative thermodynamic stability of oligomers with G.C/C.G or A.T/T.A neighbors. The dependence of repair on thermal stability was confirmed by enzyme reactions performed over 0-45 degreesC. Under these conditions, repair of epsilonA flanked by G.C/C.G was dramatically increased at 37 degreesC with continuous increase up to 45 degreesC, in contrast to that with flanking A.T/T. A pairs, which was in agreement with the degree of denaturation of these duplexes. These results indicate that the thermodynamic stability conferred by base pairs flanking epsilonA plays an essential role in maintaining the integrity of the duplex structure which is necessary for repair.     

Accumulation of O6-methylguanine in non-target-tissue deoxyribonucleic acid during chronic administration of dimethylnitrosamine

1. BD-IV rats were given labelled dimethylnitrosamine (2 mg/kg) by stomach tube on weekdays (Monday to Friday) for up to 24 weeks. The rats killed after 2, 4, 8, 16 and 24 weeks of treatment (72 h after the final dimethylnitrosamine gavage) and DNA was isolated from the pooled livers, kidneys and lungs. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. Throughout the experiment, the content of 7-methylguanine in liver DNA was approx. 16 times that in kidney and lung. The amount of this product increased in the DNA of all three tissues up to 16 weeks, but by 24 weeks had decreased by 20% in the liver and 46% in the other tissues. 3. O6-Methylguanine was not detected in liver DNA, but was easily measured in kidney and lung DNA after 4 weeks of dimethylnitrosamine administration. The amount of O6-methylguanine in kidney and lung DNA increased relative to that of 7-methylguanine, and by 24 weeks was 60% of the 7-methylguanine content in both tissues. 4. Incorporation of radioactive C1 breakdown products of dimethylnitrosamine into normal purines in DNA increased continuously in all three tissues. 5. The results are discussed with respect to the specific hepatocarcinogenic effect of chronic administration of dimethylnitrosamine and the possible contribution of increased DNA repair and DNA synthesis.     

Identification of a poxvirus gene encoding a uracil DNA glycosylase

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.     

Development of an ELISA technique for serodiagnosis of bovine paratuberculosis

In Japan, ortho-phenylphenol (OPP), biphenyl (BP), and thiabendazole (2-(4'-thiazolyl)benzimidazole, TBZ) are commonly used as a postharvest treatment to preserve imported citrus fruits during transport and storage. We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of those agents in mouse stomach, liver, kidney, bladder, lung, brain, and bone marrow. CD-1 male mice were sacrificed 3, 8, and 24 h after oral administration of the test compounds. OPP (2000 mg/kg) induced DNA damage in the stomach, liver, kidney, bladder, and lung, BP (2000 mg/kg) and TBZ (200 mg/kg) induced DNA damage in all the organs studied. For OPP, increased DNA damage peaked at 3-8 h and tended to decrease at 24 h. For BP, on the contrary, increased DNA migration peaked at 24 h. That delay may have been due to the fact that OPP is metabolized by cytochrome 450 and prostaglandin H synthase to phenylbenzoquinone (PBQ), a DNA binding metabolite, and BP is metabolized to PBQ via OPP and m-phenylphenol. The positive response to TBZ, an aneugen, supports the in vivo DNA-damaging action of TBZ.