Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-oxidation and coumarin 7-hydroxylation.

Cytochrome P450 2A13 (CYP2A13) is responsible for the metabolism of chemical compounds such as nicotine, coumarin, and tobacco-specific nitrosamine. Several of these compounds have been recognized as procarcinogens activated by CYP2A13. We recently showed that CYP2A13*2 contributes to inter-individual variations observed in bladder cancer susceptibility because CYP2A13*2 might cause a decrease in enzymatic activity. Other CYP2A13 allelic variants may also affect cancer susceptibility. In this study, we performed an in vitro analysis of the wild-type enzyme (CYP2A13.1) and 8 CYP2A13 allelic variants, using nicotine and coumarin as representative CYP2A13 substrates. These CYP2A13 variant proteins were heterologously expressed in 293FT cells, and the kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation were estimated. The quantities of CYP2A13 holoenzymes in microsomal fractions extracted from 293FT cells were determined by measuring reduced carbon monoxide-difference spectra. The kinetic parameters for CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because of low metabolite concentrations. Five other CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9) showed markedly reduced enzymatic activity toward both substrates. These findings provide insights into the mechanism underlying inter-individual differences observed in genotoxicity and cancer susceptibility.

Functional Characterization of 34 CYP2A6 Allelic Variants by Assessment of Nicotine C-Oxidation and Coumarin 7-Hydroxylation Activities.

CYP2A6, a member of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activities were assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.