Evidence for cytochrome P450 2A6 and 3A4 as major catalysts for N'- nitrosonornicotine alpha-hydroxylation by human liver microsomes

The tobacco specific carcinogen N'-nitrosonornicotine (NNN), is believed tobe a causative agent for esophageal cancer in smokers. NNN requiresmetabolic activation to exert its carcinogenic potential. Metabolism occursthrough cytochrome P450 (P450) catalyzed 2'- and 5'- hydroxylation, whichgenerates unstable metabolites that decompose to 4-hydroxy-1-(3-pyridyl)-1-butanone ('keto alcohol') and 4-hydroxy-4-(3-pyridyl)butanal, respectively. The latter cyclyzes to 5-(3-pyridyl)-2-hydroxytetrahydrofuran ('lactol'). 2'-Hydroxylation of NNN is believed tobe the pathway critical for esophogeal NNN carcinogenesis in the rat. Theability of human liver microsomes and expressed human P450s to metabolize[5-(3)H]NNN to keto alcohol and lactol was determined by reverse phase HPLCwith radioflow detection. At low NNN concentrations, 11 human livermicrosomes metabolized NNN primarily by 5'-hydroxylation to lactol. Thisreaction was strongly correlated (r = 0.92) with coumarin 7-hydroxylation,suggesting that NNN 5'-hydroxylation is catalyzed mainly by P450 2A6.2'-Hydroxylation of NNN by human liver microsomes correlated with6beta-hydroxylation of testosterone, a P450 3A4-specific activity (r =0.94). The relative rates of 2'- and 5'- hydroxylation by human P450s 2A6,2E1, 2D6 and 3A4 expressed in Sf9 cells by the baculovirus-insect cellexpression system, and human P450 3A4 produced by stable expression inChinese hamster ovary cells, were determined. Human P450 2A6 metabolized 1microM NNN exclusively by 5'- hydroxylation. The rate of lactol formationwas 317 pmol/min per nmol P450. Human P450s 2E1 and 2D6 also metabolizedNNN only to lactol, but at much lower rates, 0.4 and 0.8 pmol/min per nmolof P450 respectively. In contrast, the metabolism of NNN by expressed humanP450 3A4 was specific for keto alcohol formation. The Km for 5'-hydroxylation by baculovirus-expressed P450 2A6 was 2.1 microM, and k(cat)was 953 pmol/min per nmol of P450. The Km for lactol formation by humanliver microsomes containing high levels of P450 2A6, was 5 microM . Humanliver microsomes exhibited a Km of 312 microM for keto alcohol formation.Coumarin, 8-methoxypsoralen (P450 2A6 inhibitors), and anti-2A6 monoclonalantibody were strong inhibitors of NNN-derived lactol formation in humanliver microsomes. Troleandomycin, an inhibitor of P450 3A4, effectivelyinhibited the metabolism of NNN to keto alcohol by human liver microsomes.These results are consistent with P450 2A6 mediated 5'-hydroxylation andP450 3A4 mediated 2'- hydroxylation of NNN in human liver microsomes.