Inhibition of human cytochrome P450 2E1 by nicotine, cotinine, and aqueouscigarette tar extract in vitro

Cigarette smoke is a complex mixture containing, among other chemicals, pyr idine alkaloids and N-nitrosamines. Carcinogenic tobacco-specific N-nitrosa mines, N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl )-1-butanone (NNK), are both activated by cytochrome P450 (CYP) 2E1 in rats . Previous reports indicate that nicotine and the main nicotine metabolite, cotinine, reduce the mutagenicity of both NNK and NDMA in Salmonella typhi murium. To study the mechanism of this effect, we examined inhibition of CY P 2E1 activity, as assessed by p-nitrophenol (pNP) hydroxylation, by nicoti ne, cotinine, and an aqueous cigarette tar extract (ACTE) in human 2E1-expr essing microsomes. At all substrate concentrations (0-1.25 mM) nicotine was a significantly more potent inhibitor of CYP 2E1 activity compared to coti nine. Estimated Ki values for nicotine and cotinine (both at 10 mM) were 13 mM (2 mg/ml) and 308 mM (54 mg/ml) respectively. The Ki for ACTE was 0.2 m g/ml at a concentration of 0.32 mg/ml. This rank order for inhibition was a lso seen when the data was expressed as IC50. When compared on a mass/vol b asis, ACTE was a significantly more potent CYP 2E1 inhibitor relative to ni cotine and cotinine. Double-reciprocal plots indicated that nicotine and AC TE inhibited by a competitive, while cotinine inhibited CYP 2E1 by an uncom petitive mechanism. Although the contribution of nicotine to ACTE-mediated 2E1 inhibition is probably modest, pyridine alkaloid-mediated CYP 2E1 inhib ition is a possible mechanism for the observed inhibition of NNK and NDMA m utagenicity by nicotine and cotinine in vitro.