Nicotine-N'-oxidation by flavin monooxygenase enzymes.

BACKGROUND: The major mode of metabolism of nicotine is by hydroxylation via cytochrome P450 (CYP)2A6, but it can also undergo glucuronidation by UDP-glucuronosyltransferases and oxidation by flavin monooxygenases (FMOs). The goal of this study was to examine the potential importance of FMOs in nicotine metabolism and assess the potential impact of missense polymorphisms in active FMOs on nicotine-N'-oxide formation.

METHODS: Urine samples from 106 current Chinese smokers were analyzed for nicotine metabolites by mass spectrometry (MS). Wild-type FMOs 1 through 5 and their most prevalent non-synonymous variants were cloned and over-expressed in HEK293 cells, and were tested in oxidation reactions against nicotine Results:A strong inverse correlation was observed between the ratio of urinary 3'-hydroxycotinine/cotinine, a measure of CYP2A6 activity, and the urinary levels of nicotine-N'-oxide alone (r=-0.383,p <0.001) or nicotine-N'-oxide measured as a ratio of total nicotine metabolites (r=-0.414, p <0.001) in smokers. In addition to FMO1 and FMO3, the functional FMO2427Q isoform was active against nicotine, while FMO4 and FMO5 exhibited low activity against nicotine (KM>5.0 mM). Significant (p <0.05) decreases in N'-oxidation activity (Vmax/KM) were observed for the FMO1I303V, FMO3N61S, FMO3D132H, FMO3V257M, and FMO3E308G variants in vitro when compared to their respective wild-type isoforms; the truncated FMO2Q472stop isoform exhibited no enzyme activity.

CONCLUSIONS: These data indicate that increases in nicotine-N'-oxidation occurs in subjects with deficient CYP2A6 activity, that several FMO enzymes are active in nicotine-N'-oxidation.

IMPACT: Several common missense FMO variants are associated with altered enzyme activity against nicotine and may play an important role in nicotine metabolism in low-CYP2A6 activity subjects.