Role of cytochrome P-450 and flavin-containing monooxygenase in the N-hydroxylation of N-methyl-4-aminoazobenzene in rat liver: analysis with purified enzymes and antibodies.

Abstract

By means of high pressure liquid chromatography, the role of flavin-containing monooxygenase (FMO) and cytochrome P-450 (cyt. P-450) in the metabolism of N-methyl-4-aminoazobenzene (MAB) by rat liver microsomes in vitro was studied with the help of antibodies and a chemical inhibitor. Antibody against cyt. P-488 from 3-methylcholanthrene-treated rats (MC-P-448) decreased the formation of N-hydroxy-N-methyl-4-aminoazobenzene (N-OH-MAB) by about 30% in microsomes from MC-treated rats (MC-microsomes), but showed no inhibitory effect on the formation of N-OH-MAB in microsomes from untreated rats (untreated microsomes) or in microsomes from phenobarbital-treated rats (PB-microsomes). Antibody against cyt. P-450 from PB-treated rats did not inhibit N-hydroxylation of MAB by any of the microsomes tested. A competitive inhibitor of FMO, methimazole, inhibited the N-hydroxylation of MAB by 65% in the case of MC-microsomes, and the residual activity was inhibited completely by anti-NADPH-cytochrome P-450 reductase (anti-fPT) antibody. These results indicate that in MC-microsomes, the N-hydroxylation of MAB is catalyzed by both FMO and MC-P-448, but in untreated and PB-microsomes the reaction is catalyzed exclusively by FMO.