Evidence for metabolic activation of N'-nitrosonornicotine and N-nitrosobenzylmethylamine by a rat nasal coumarin hydroxylase

Kinetic parameters were determined for the hydroxylation of N'-nitrosonornicotine (NNN), N-nitrosobenzylmethylamine (NBzMA), coumarin, and ethoxycoumarin catalyzed by rat nasal mucosa microsomes. NNN is a tobacco-specific nitrosamine that, in rats, causes tumors in the nasal cavity and esophagus, whereas NBzMA induces tumors in rat esophagus. Both nitrosamines require alpha-hydroxylation to exert their carcinogenic effects. NNN, NBzMA, coumarin, and ethoxycoumarin were all extensively hydroxylated by rat nasal mucosa microsomes. The KM values for the hydroxylation of each substrate were low, ranging between 2 and 5 microM. 2'- and 5'-Hydroxylation of NNN were catalyzed to a similar extent. NBzMA was metabolized predominantly to benzaldehyde, the product of debenzylation, or methylene hydroxylation. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), NNN, and NBzMA were inhibitors of coumarin and ethoxycoumarin hydroxylation. NNN hydroxylation by nasal mucosa microsomes was inhibited by coumarin, ethoxycoumarin, NNK, and NBzMA but not by N-nitrosodimethylamine. 8-Methoxypsoralen, a potent inhibitor of P450 2A6- and 2a5-dependent coumarin hydroxylation in human and mouse liver microsomes, also significantly inhibited NNN activation. The results of this study suggest that the four substrates examined are hydroxylated by closely related P450 enzymes in rat nasal mucosa and that a coumarin hydroxylase metabolizes both NNN and NBzMA.