Predictors of Variation in CYP2A6 mRNA, Protein, and Enzyme Activity in a Human Liver Bank: Influence of Genetic and Nongenetic Factors

Abstract

Cytochrome P450 2A6 CYP2A6: metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47-0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine.

Interrogation of CYP2D6 Structural Variant Alleles Improves the Correlation Between CYP2D6 Genotype and CYP2D6‐Mediated Metabolic Activity

“…Mean CYP2D6 activity, expressed as formation rates of dextrorphan from dextromethorphan and α‐hydroxymetoprolol from metoprolol, varied significantly between liver collection sites, consistent with our collaborators' reports from these samples . Therefore, liver collection site was included as a covariate in all analyses.…”

“…Microsomal CYP2D6 protein content was quantitated using a surrogate peptide‐based liquid chromatography‐tandem mass spectrometrymethod . Methods for messenger RNA (mRNA) quantitation were previously described …”

“…21,22 Methods for messenger RNA (mRNA) quantitation were previously described. 23 DNA isolation, CYP2D6 sequencing, RNA-seq analysis, and CYP2D6 allele and AS assignment Genomic DNA was isolated from human livers as described. 21 The next-generation sequencing platform PGRNseqversion 1.1 was used to identify CYP2D6 genomic variation, including both SNV and structural variation data.…”

“…Donor demographics (n = 314) were presented previously (Table S1). 21,23 Data on concomitant medications were missing or incomplete for most donors and, thus, are not reported; concomitant use of a strong CYP2D6 inhibitor (fluoxetine) was reported for only one donor.…”

“…Correlation between CYP2D6 mRNA and protein content and CYP2D6 activity Mean CYP2D6 activity, expressed as formation rates of dextrorphan from dextromethorphan and α-hydroxymetoprolol from metoprolol, varied significantly between liver collection sites, consistent with our collaborators' reports from these samples. 21,23 Therefore, liver collection site was included as a covariate in all analyses. Formation rates of dextrorphan and α-hydroxymetoprolol were well correlated with one another (R 2 = 0.90).…”