2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we examined the ability of prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, to activate this carcinogen to metabolites binding to macromolecules. Using [14C]-labeled o-anisidine, we observed substantial PHS-dependent binding of o-anisidine to protein, DNA and polydeoxyribonucleotides [poly(dX)]. This binding is inhibited by radical scavengers glutathione, ascorbate and NADH. The nuclease P1 and 1-butanol extraction enrichment procedure of the 32P-postlabeling analysis of DNA modified by activated o-anisidine provide evidence that covalent binding to DNA is the principal type of DNA modification. Deoxyguanosine is determined to be the major target for binding of o-anisidine in DNA. The possibility that o-anisidine is carcinogenic to the rodent urinary bladder via its activation by bladder PHS is suggested. The results presented here are the first report demonstrating a PHS-mediated activation of o-anisidine to reactive species forming covalent DNA adducts.

Department of Biochemistry Faculty of Science Charles University Prague Czech Republic

Genotoxic mechanisms for the carcinogenicity of the environmental pollutants and carcinogens o-anisidine and 2-nitroanisole follow from adducts generated by their metabolite N-(2-methoxyphenyl)-hydroxylamine with deoxyguanosine in DNA

Cytochrome P450-mediated metabolism of N-(2-methoxyphenyl)-hydroxylamine, a human metabolite of the environmental pollutants and carcinogens o-anisidine and o-nitroanisole