Hepatic microsomal N-hydroxylation of adenine to 6-N-hydroxylaminopurine

Abstract

The enzymatic N-hydroxylation of the purine base adenine to the genotoxic and mutagenic compound 6-N-hydroxylaminopurine is reported for the first time. Adenine was N-oxygenated in vitro by aerobic incubations with 3-methylcholanthrene or isosafrole induced microsomal fractions of rat liver homogenates and NADPH. The formation of 6-N-hydroxylaminopurine in the incubation mixtures under widely differing conditions was assayed using newly-developed, high-performance liquid- and thin-layer chromatographic methods. Optimal reaction conditions and kinetic parameters were determined. Neither superoxide anion nor hydrogen peroxide was directly involved in the N-hydroxylation reaction. Oxidases like xanthine oxidase and peroxidase (in the presence of hydrogen peroxide) did not catalyse this N-hydroxylation. The involvement of cytochrome P-450 isoenzymes in this reaction is supported by the observation that the N-hydroxylation is only observed after pretreatment of the rats with 3-methylcholanthrene or isosafrole. Other inducers (phenobarbital, ethanol, 5-pregnen-3 beta ol-20-one-16 alpha-carbonitrile) were without effect. This is the first example of the microsomal transformation of an endogenous substance to a toxic derivative by usually foreign substances (xenobiotics) metabolizing cytochrome P-450 isoenzymes. The significance for the in vivo situation is discussed on the basis of the data obtained in this study.