Identification of rat cytochromes P450 metabolizing N-(2-methoxyphenyl)hydroxylamine, a human metabolite of the environmental pollutants and carcinogens o-anisidine and o-nitroanisole.

OBJECTIVES: N-(2-methoxyphenyl)hydroxylamine is a human metabolite of two industrial and environmental pollutants and bladder carcinogens 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole). Metabolism of N-(2-methoxyphenyl)hydroxylamine by rat hepatic microsomes and identification of the major microsomal enzymes participating in this process are aims of this study.

METHODS: HPLC with UV detection was employed for the separation of N-(2-methoxyphenyl)hydroxylamine metabolites. Inducers and inhibitors of microsomal enzymes and rat recombinant CYPs were used to characterize the enzymes participating in N-(2-methoxyphenyl)hydroxylamine metabolism.

RESULTS: N-(2-methoxyphenyl)hydroxylamine is metabolized by rat hepatic microsomes predominantly to o-anisidine, the parent carcinogen from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome b5 reductase and hepatic microsomes of rats pre-treated with specific inducers of CYPs and NADPH:CYP reductase were used to characterize rat liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute most of N-(2-methoxyphenyl)hydroxylamine metabolism to o-anisidine in rat liver to CYP2C, followed by CYP2E1, 2D and 2A. Among recombinant rat CYP enzymes tested in this study, rat CYP2C11 and 2E1, followed by CYP2A2, 2D1/2, 2C12, 3A1/2 and 1A1/2 were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine.

CONCLUSION: The results found in this study, the first report on the reduction of N-(2-methoxyphenyl)hydroxylamine by rat CYP enzymes, demonstrate that CYP2C, followed by CYP2E1, 2D and 2A are the major enzymes participating in this process in rat liver.