Contributions of human liver cytochrome P450 enzymes to the N-oxidation of 4,4′-methylene-bis(2-chloroaniline)

Abstract

4,4'-Methylene-bis(2-chloroaniline) (MOCA) can produce tumors in rodents and dogs and an increased incidence of bladder tumors has been reported in exposed workers. It is therefore of interest to identify the human cytochrome P450 (P450) enzymes involved in MOCA N-oxidation, the primary reaction involved in the formation of an electrophilic product. Human liver microsomes were fractionated and MOCA N-oxidation activity was monitored through the procedure. The most active enzyme fraction corresponded to P450 3A4, as determined by immunochemical assays and N-terminal amino acid sequence analysis. Yeast recombinant P450 3A4 also had MOCA N-oxidation activity. Purified human liver P450 2A6 showed catalytic activity; however, anti-P450 2A6 inhibited less than 20% of the microsomal activity while anti-P450 3A4 inhibited up to 75%. Levels of marker activities of both P450 3A4 (nifedipine oxidation) and P450 2A6 (coumarin 7-hydroxylation) were measured in a set of human liver microsomes and both were correlated with MOCA N-oxidation rates. Gestodene and troleandomycin inhibited up to half of the microsomal MOCA N-hydroxylation activity but 7,8-benzoflavone showed only slight inhibition. Anti-P450 3A4 inhibited (up to 80% of) the microsomal transformation of MOCA to a product genotoxic as judged by bacterial SOS response. The work indicates that P450 3A4 makes a major contribution to human liver microsomal MOCA N-oxidation, and P450 2A6 has a minor role. P450 1A2, which catalyzes the hydroxylation of many arylamines, does not contribute to a great extent.