A novel CYP2A6 allele, CYP2A6*23, impairs enzyme function in vitro and in vivo and decreases smoking in a population of Black-African descent

Abstract

Objectives-CYP2A6 is the main enzyme involved in nicotine metabolism in humans. We have identified a novel allele, CYP2A6*23 (2161C>T, R203C), in individuals of Black-African descent and investigated its impact on enzyme activity and association with smoking status.Methods-Wildtype and variant enzymes containing amino acid changes R203C (CYP2A6*23), R203S (CYP2A6*16) and V365M (CYP2A6*17) were expressed in Escherichia coli. The effect of CYP2A6*23 in vivo was examined in individuals of Black-African descent given 4 mg oral nicotine.Results-CYP2A6*23 occurred at an allele frequency of 2.0% in individuals of Black-African descent (N = 560 alleles, 95% confidence interval 0.8%-3.1%) and was not detected in Caucasians (N = 334 alleles), Chinese (N = 288 alleles) or Japanese (N = 104 alleles). In vitro, CYP2A6.23 had greatly reduced activity towards nicotine C-oxidation similar to CYP2A6.17, as well as reduced coumarin 7-hydroxylation. Conversely, CYP2A6.16 did not differ in activity compared to the wildtype enzyme. The trans-3′-hydroxycotinine to cotinine ratio, a phenotypic measure of CYP2A6 activity in vivo, was lower in CYP2A6*1/*23 and CYP2A6*23/*23 individuals(mean adjusted ratio of 0.60, n = 5) compared to CYP2A6*1/*1 individuals (mean adjusted ratio of 1.21, n = 150) (p < 0.04). CYP2A6*23 trended towards a higher allele frequency in nonsmokers (3.1%, N = 9/286 alleles) compared to smokers (0.7%, N = 2/274 alleles) (p = 0.06).Conclusions-These results suggest the novel CYP2A6*23 allele impairs enzyme function in vitro and in vivo and trends toward an association with lower risk of smoking.

Novel and established CYP2A6 alleles impair in vivo nicotine metabolism in a population of Black African descent

“…The amount of CYP2A6 protein in the membrane fractions was determined by immunoblotting with a monoclonal CYP2A6 antibody (BD Biosciences, Mississauga, ON, Canada) using supersome expressed CYP2A6 (BD Biosciences) as a positive control for quantification as previously described [Ho et al, 2008;Schoedel et al, 2003]. The function of the expression construct was confirmed by immunoblotting for hNPR.…”

“…Alleles that would likely occur at a frequency of 41% and alter nicotine metabolism, CYP2A6 à 1B, à 1B15, à 2, à 7, à 8, à 9, à 10, à 12, à 16, à 21, and à 23, were genotyped as previously described [Al Koudsi et al, 2006;Ho et al, 2008;Mwenifumbo et al, 2005;Mwenifumbo et al, 2008;Schoedel et al, 2004]. We modified the assay for CYP2A6 à 4A&D and developed the first and second steps of PCR-based genotyping assays for CYP2A6 à 14, à 15, à 20, and 2162_2163GC4A (p.R203FS).…”

“…Bicistronic constructs encoding hepatic NADPH-cytochrome P450 reductase (hNPR) and CYP2A6.1,.17,.24,.25,.26,.27 or .28 were expressed in Escherichia coli and membrane fractions were prepared as previously described [Ho et al, 2008;Pritchard et al, 1997]. The amount of CYP2A6 protein in the membrane fractions was determined by immunoblotting with a monoclonal CYP2A6 antibody (BD Biosciences, Mississauga, ON, Canada) using supersome expressed CYP2A6 (BD Biosciences) as a positive control for quantification as previously described [Ho et al, 2008;Schoedel et al, 2003].…”

Functional Characterization of Cytochrome P450 2A6 Allelic Variants CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22

“…Alleles CYP2A6*4, CYP2A6*5, and CYP2A6*20 were also found to display abolished functional activity due to gene deletion or point mutation in their primary sequence. Twelve CYP2A6 alleles (*6, *7, *10, *11, *12, *17, *18, *19, *23, *24, *26, and *27) have been ascertained to cause reduction in enzyme activity, whereas genetic polymorphisms in the promoter region of CYP2A6 have been implicated for the decreased transcriptional activity observed in alleles CYP2A6*1D, CYP2A6*1H, and CYP2A6*9 (Ariyoshi et al, 2001;Kitagawa et al, 2001;Daigo et al, 2002;Fukami et al, 2004Ho et al, 2008).…”

Nicotine Kinetics in Zebra Finches In Vivo and In Vitro

“…Human liver microsomes (0.5 mg/ml) and human lymphoblast cDNA-expressed expressed CYP2A6 (10 pmol) were used as positive controls. A 100-ml aliquot of the incubation mixture with 25 ng 4-hydroxycoumarin as the internal standard was prepared and separated by HPLC as previously described (Ho et al, 2008); the limits of quantification were 5 ng/ml (34 nM) for coumarin and 25 ng/ml (154 nM) for 7-hydroxycoumarin.…”