Dataset Information

Nicotine oxidation by genetic variants of CYP2B6 and in human brain microsomes.

ABSTRACT: Common variation in the CYP2B6 gene, encoding the cytochrome P450 2B6 enzyme, is associated with substrate-specific altered clearance of multiple drugs. CYP2B6 is a minor contributor to hepatic nicotine metabolism, but the enzyme has been proposed as relevant to nicotine-related behaviors because of reported CYP2B6 mRNA expression in human brain tissue. Therefore, we hypothesized that CYP2B6 variants would be associated with altered nicotine oxidation, and that nicotine metabolism by CYP2B6 would be detected in human brain microsomes. We generated recombinant enzymes in insect cells corresponding to nine common CYP2B6 haplotypes and demonstrate genetically determined differences in nicotine oxidation to nicotine iminium ion and nornicotine for both (S) and (R)-nicotine. Notably, the CYP2B6.6 and CYP2B6.9 variants demonstrated lower intrinsic clearance relative to the reference enzyme, CYP2B6.1. In the presence of human brain microsomes, along with nicotine-N-oxidation, we also detect nicotine oxidation to nicotine iminium ion. However, unlike N-oxidation, this activity is NADPH independent, does not follow Michaelis-Menten kinetics, and is not inhibited by NADP or carbon monoxide. Furthermore, metabolism of common CYP2B6 probe substrates, methadone and ketamine, is not detected in the presence of brain microsomes. We conclude that CYP2B6 metabolizes nicotine stereoselectively and common CYP2B6 variants differ in nicotine metabolism activity, but did not find evidence of CYP2B6 activity in human brain.

SUBMITTER: Bloom AJ

PROVIDER: S-EPMC6411694 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Nicotine oxidation by genetic variants of CYP2B6 and in human brain microsomes.

Bloom Adam Joseph AJ Wang Pan-Fen PF Kharasch Evan D ED

Pharmacology research & perspectives 20190311 2

Common variation in the <i>CYP2B6</i> gene, encoding the cytochrome P450 2B6 enzyme, is associated with substrate-specific altered clearance of multiple drugs. CYP2B6 is a minor contributor to hepatic nicotine metabolism, but the enzyme has been proposed as relevant to nicotine-related behaviors because of reported <i>CYP2B6</i> mRNA expression in human brain tissue. Therefore, we hypothesized that CYP2B6 variants would be associated with altered nicotine oxidation, and that nicotine metabolism ...[more]

PMID: 30906561

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Project description:BackgroundWhile the heritability of cigarette smoking and nicotine dependence (ND) is well-documented, the contribution of specific genetic variants to specific phenotypes has not been closely examined. The objectives of this study were to test the associations between 321 tagging single-nucleotide polymorphisms (SNPs) that capture common genetic variation in 24 genes, and early smoking and ND phenotypes in novice adolescent smokers, and to assess if genetic predictors differ across these phenotypes.MethodsIn a prospective study of 1294 adolescents aged 12-13 years recruited from ten Montreal-area secondary schools, 544 participants who had smoked at least once during the 7-8 year follow-up provided DNA. 321 single-nucleotide polymorphisms (SNPs) in 24 candidate genes were tested for an association with number of cigarettes smoked in the past 3 months, and with five ND phenotypes (a modified version of the Fagerstrom Tolerance Questionnaire, the ICD-10 and three clusters of ND symptoms representing withdrawal symptoms, use of nicotine for self-medication, and a general ND/craving symptom indicator).ResultsThe pattern of SNP-gene associations differed across phenotypes. Sixteen SNPs in seven genes (ANKK1, CHRNA7, DDC, DRD2, COMT, OPRM1, SLC6A3 (also known as DAT1)) were associated with at least one phenotype with a p-value <0.01 using linear mixed models. After permutation and FDR adjustment, none of the associations remained statistically significant, although the p-values for the association between rs557748 in OPRM1 and the ND/craving and self-medication phenotypes were both 0.076.ConclusionsBecause the genetic predictors differ, specific cigarette smoking and ND phenotypes should be distinguished in genetic studies in adolescents. Fifteen of the 16 top-ranked SNPs identified in this study were from loci involved in dopaminergic pathways (ANKK1/DRD2, DDC, COMT, OPRM1, and SLC6A3).ImpactDopaminergic pathways may be salient during early smoking and the development of ND.

Project description:Efavirenz (more specifically the S-enantiomer) is a cornerstone antiretroviral therapy for treatment of HIV infection. The major primary metabolite is S-8-hydroxyefavirenz, which does not have antiretroviral activity but is neurotoxic. Cytochrome P450 2B6 (CYP2B6) is the major enzyme catalyzing S-8-hydroxyefavirenz formation. CYP2B6 genetics and drug interactions are major determinants of clinical efavirenz disposition and dose adjustment. In addition, as a prototypic CYP2B6 substrate, S-efavirenz and analogs can inform on the structure, activity, catalytic mechanisms, and stereoselectivity of CYP2B6. Metabolism of R-efavirenz by CYP2B6 remains unexplored. This investigation assessed S-efavirenz metabolism by clinically relevant CYP2B6 genetic variants. This investigation also evaluated R-efavirenz hydroxylation by wild-type CYP2B6.1 and CYP2B6 variants. S-Efavirenz 8-hydroxylation by wild-type CYP2B6.1 and variants exhibited positive cooperativity and apparent cooperative substrate inhibition. On the basis of Clmax values, relative activities for S-efavirenz 8-hydroxylation were in the order CYP2B6.4 > CYP2B6.1 ? CYP2B6.5 ? CYP2B6.17 > CYP2B6.6 ? CYP2B6.7 ? CYP2B6.9 ? CYP2B6.19 ? CYP2B6.26; CYP2B6.16 and CYP2B6.18 showed minimal activity. Rates of R-efavirenz metabolism were approximately 1/10 those of S-efavirenz for wild-type CYP2B6.1 and variants. On the basis of Clmax values, there was 14-fold enantioselectivity (S > R-efavirenz) for wild-type CYP2B6.1, and 5- to 22-fold differences for other CYP2B6 variants. These results show that both CYP2B6 516G > T (CYP2B6*6 and CYP2B6*9) and 983T > C (CYP2B6*16 and CYP2B6*18) polymorphisms cause canonical diminishment or loss-of-function variants for S-efavirenz 8-hydroxylation, provide a mechanistic basis for known clinical pharmacogenetic differences in efavirenz disposition, and may predict additional clinically important variant alleles. Efavirenz is the most stereoselective CYP2B6 drug substrate yet identified and may be a useful probe for the CYP2B6 active site and catalytic mechanisms. SIGNIFICANCE STATEMENT: Clinical disposition of the antiretroviral S-efavirenz is affected by CYP2B6 polymorphisms. Expressed CYP2B6 with 516G>T (CYP2B6*6 and CYP2B6*9), and 983T>C (CYP2B6*16 and CYP2B6*18) polymorphisms had a diminishment or loss of function for efavirenz 8-hydroxylation. This provides a mechanistic basis for efavirenz clinical pharmacogenetics and may predict additional clinically important variant alleles. Efavirenz metabolism showed both cooperativity and cooperative substrate inhibition. With greater than 10-fold enantioselectivity (S- vs. R- metabolism), efavirenz is the most stereoselective CYP2B6 drug substrate yet identified. These findings may provide mechanistic insights.

Dataset Information

Nicotine oxidation by genetic variants of CYP2B6 and in human brain microsomes.

Publications

Nicotine oxidation by genetic variants of CYP2B6 and in human brain microsomes.

Similar Datasets

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