Effects of benzyl isothiocyanate on rat and human cytochromes P450: identification of metabolites formed by P450 2B1.

Naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are potent and selective inhibitors of carcinogenesis induced by a variety of chemical carcinogens. These effects appear to be mediated through favorable modification of both phase I and II enzymes involved in carcinogen metabolism. The inactivation of rat and human cytochromes P450 (P450s) in microsomes and the reconstituted system by BITC was investigated. BITC is a mechanism-based inactivator of rat P450s 1A1, 1A2, 2B1, and 2E1, as well as human P450s 2B6 and 2D6. BITC was most effective in inactivating P450s 2B1, 2B6, 1A1, and 2E1, whereas the activities of human P450 2C9 and rat P450 3A2 were not altered. The concentrations required for half-maximal inactivation (K(I)) of P450s 1A1, 1A2, 2B1, and 2E1 were 35, 28, 16, and 18 microM, respectively. The corresponding values for k(inact) were 0.26, 0.09, 0.18, and 0.05 min(-1), respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of P450 2B1 inactivated by [(14)C]BITC indicated specific and covalent modification of the P450 apoprotein by a metabolite of BITC. High-performance liquid chromatography analysis of the BITC metabolites revealed that benzylamine was the major metabolite and there were lesser amounts of benzoic acid, benzaldehyde, N,N'-di-benzylurea, and N,N'-di-benzylthiourea. Presumably, BITC was metabolized to the reactive benzyl isocyanate intermediate that covalently modified the P450 apoprotein or hydrolyzed to form benzylamine. BITC was an efficient inactivator of P450 2B1 with a partition ratio of approximately 11:1. This irreversible inactivation of P450s by BITC could contribute significantly to its chemopreventative action.