Objective: Nicotine, the main addictive ingredient in tobacco, is metabolically inactivated to cotinine primarily by the hepatic enzyme CYP2A6. Considerable genetic variation in the CYP2A6 gene results in large variation in the rates of nicotine metabolism, which in turn alters smoking behaviours (e.g. amount of cigarettes smoked, risk for dependence and success in smoking cessation). The aim of this study was to identify and characterize novel variants in CYP2A6.
Materials and methods: The CYP2A6 gene from African American phenotypically slow nicotine metabolizers was sequenced and seven novel variants were identified [CYP2A6*39 (V68M), CYP2A6*40 (I149M), CYP2A6*41 (R265Q), CYP2A6*42 (I268T), CYP2A6*43 (T303I), CYP2A6*44 (E390K), CYP2A6*44 (L462P)]. Variants were introduced into a bi-cistronic cDNA expression construct containing CYP2A6 and P450 oxidoreductase and assessed for protein expression, enzymatic activity and stability as evaluated using western blotting and nicotine metabolism. Genotyping assays were developed and allelic frequencies were assessed in 534 African Americans.
Results: The variants showed significantly lower protein expression (P<0.001) when compared with the wild-type as well as reduced metabolism of nicotine to cotinine when controlling for cDNA expression using P450 oxidoreductase (P<0.001). The variants also showed reduced stability at 37°C. Allelic frequencies ranged from 0.1 to 0.6% with a collective genotype frequency of 3.2%; the impact in vitro correlated significantly with in-vivo activity (R2=0.40–0.48, P<0.05). Together, those with a novel variant had significantly lower nicotine metabolism in vivo than those without genetic variants (P<0.01).
Conclusion: Here, we identified a number of novel variants with reduced/loss of CYP2A6 activity, increasing our understanding of CYP2A6 genetic variability.
aDepartment of Pharmacology and Toxicology, Centre for Addiction and Mental Health (CAMH), Campbell Family Mental Health Research Institute, University of Toronto
bDepartment of Psychiatry, University of Toronto, Toronto, Ontario, Canada
cDepartments of Medicine and Bioengineering & Therapeutic Sciences, Division of Clinical Pharmacology and Experimental Therapeutics, University of California San Francisco, San Francisco, California
dDepartment of Medicine, Center for Health Equity, University of Minnesota Medical School, Minneapolis, Minnesota
eDepartment of Preventive Medicine and Public Health, University of Kansas School of Medicine, Kansas City, Kansas, USA
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Correspondence to Rachel F. Tyndale, MSc, PhD, 1 King’s College Circle, University of Toronto, Medical Sciences Building, Room 4326, Toronto, ON, Canada, M5S 1A8 Tel: +416 978 6374; fax: +416 978 6395; e-mail: firstname.lastname@example.org
Received September 18, 2013
Accepted November 8, 2013