A common haplotype of the flavin-containing monooxygenase gene FMO3 is associated with aberrant mRNA splicing, a twofold reduction in in vivo nicotine N-oxidation and reduced nicotine dependence. Tobacco remains the largest cause of preventable mortality worldwide. CYP2A6, the primary hepatic nicotine metabolism gene, is robustly associated with cigarette consumption but other enzymes contribute to nicotine metabolism. We determined the effects of common variants in FMO3 on plasma levels of nicotine-N-oxide in 170 European Americans administered deuterated nicotine. The polymorphism rs2266780 (E308G) was associated with N-oxidation of both orally administered and ad libitum smoked nicotine (P' 1/23.3 × 10 ' '5 controlling for CYP2A6 genotype). In vitro, the FMO3 G308 variant was not associated with reduced activity, but rs2266780 was strongly associated with aberrant FMO3 mRNA splicing in both liver and brain (P' 1/26.5 × 10 ' '9). Surprisingly, in treatment-seeking European American smokers (n=1558) this allele was associated with reduced nicotine dependence, specifically with a longer time to first cigarette (P=9.0 × 10 ' '4), but not with reduced cigarette consumption. As N-oxidation accounts for only a small percentage of hepatic nicotine metabolism we hypothesized that FMO3 genotype affects nicotine metabolism in the brain (unlike CYP2A6, FMO3 is expressed in human brain) or that nicotine-N-oxide itself has pharmacological activity. We demonstrate for the first time nicotine N-oxidation in human brain, mediated by FMO3 and FMO1, and show that nicotine-N-oxide modulates human α4β2 nicotinic receptor activity in vitro. These results indicate possible mechanisms for associations between FMO3 genotype and smoking behaviors, and suggest nicotine N-oxidation as a novel target to enhance smoking cessation.
Bibliographical noteFunding Information:
We thank Dr Jeffery Milbrandt and his lab for their advice and assistance. We acknowledge the use of tissues procured by the National Disease Research Interchange (NDRI) with support from NIH grant 2 U42 OD011158. The Collaborative Genetic Study of Nicotine Dependence was supported by National Cancer Institute grant P01 CA089392. The University of Wisconsin Transdisciplinary Tobacco Use Research Center was supported by National Institute on Drug Abuse grant P50 DA019706 and National Cancer Institute grant P50 CA084724 (TBB). This work was also supported by the National Institute on Drug Abuse grants T32 DA007261 (AMT & AJB), R01 DA036583 (LJB), R01 DA14211 (EDK), R01 DA25931 (EDK) and K01 DA034035 (AJB), National Cancer Institute grant K05 CA139871 (TBB), National Institute of Heart, Lung, and Blood Disease grant R01 HL109031 (TBB), and the Taylor Family Institute for Innovative Psychiatric Research (GA). Genotyping services for the UW-TTURC sample were provided by the Center for Inherited Disease Research (CIDR) and funding support for CIDR was provided by NIH grant U01 HG004438 and NIH contract HHSN268200782096C to The Johns Hopkins University. Assistance with genotype cleaning was provided by the Gene Environment Association Studies (GENEVA) Coordinating Center (U01 HG004446). LC/MS/MS analysis carried out in the Analytical Biochemistry Core of the University of Minnesota Cancer Center, was supported in part by National Cancer Institute grant CA-77598.