Metabolism off Nitrosamines by Purified Rabbit Liver Cytochrome P-450 Isozymes

The metabolism of nitrosamines by microsomal cytochrome P-450 (P-450) isozymes was studied in a reconstituted monooxygenase system. P-450 LM₂, LM_3a, LM_3b, and LM_3c LM₄, and LM₆ were purified, respectively, from the livers of phenobarbital-treated, ethanol-treated, untreated, isosafrole-treated, and imida-zole-treated rabbits. Of these isozymes, LM_3a had the highest /V-nitrosodimethylamine demethylase (NDMAd) activity with a K_m of 2.9 mM and V_max of 9.3 nmol/min/nmol. LM₂, LM₄, and LM₆ exhibited NDMAd activity only at high A/-nitrosodimethylamine concentrations, and isozymes LM_3b, and LM_3c had poor activity even at the highest substrate concentrations examined. LM₂, however, was more active than LM_3a in the metabolism of N-nitrosomethytaniline. With each isozyme (LM_3a or LM₄), only one K_m for NDMAd was observed, whereas with rabbit liver microsomes, multiple K_m of 0.07, 0.27, and 36.8 mM were obtained. P-450 isozymes also catalyzed the denitrosation of nitrosamines at rates comparable to or lower than the demethylation, and the ratio of these two reactions was different with different nitrosamines. 2-Phenylethylamine and 3-amino-1,2,4-triazole, which were believed previously to affect NDMAd by mechanisms independent of P-450, were shown to be potent inhibitors of P-450-dependent NDMAd. These results further establish the role of P-450 isozymes in the metabolism of nitrosamines and indicate that LM_3a is apparently responsible for the increased N-nitrosodimethylamine metabolism associated with ethanol treatment.