Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation

Abstract

Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.

Fig. 1.

Relationship between age and CYP2B6 mRNA transcript levels (A), protein expression (B), and activity (C) in human liver samples and microsomes. CYP2B6 mRNA transcript levels were determined by quantitative reverse transcription PCR, protein levels by western immunoblot, and bupropion hydroxylase activity by HPLC/UV, as described in Materials and Methods. The x-axis was expanded for the prenatal and 0–2 years of age range to better visualize the data in this range (gray-filled circles). Note that no activity was detected in fetal liver microsomes, explaining the absence of respective data points in (C). CYP2B6 protein determinations were performed in duplicate, whereas CYP2B6 mRNA transcript determinations and bupropion hydroxylase activity determinations were performed in triplicate.

Fig. 2.

Immunoquantitation of CYP2B6 protein by western blot analysis. Representative western immunoblot showing the relative amounts of CYP2B6 protein from 1 to 10 µg of microsomal protein in a random set of HLMs. Immunoreactive CYP2B6 content was determined using a rabbit anti-human CYP2B6 antibody (Corning Gentest). A five-point standard curve from 5 to 100 fmol of heterologously expressed CYP2B6 (Corning Gentest; rCYP2B6: superscript a represents lot 5 and superscript b represents lot 18, as indicated) was used to quantify CYP2B6 protein. Pooled, adult HLMs served as a positive control, as well as an interassay control, and a molecular weight marker (MWM) was included to show protein size and also served as a negative control. Microsomal protein was loaded in proportion to bupropion hydroxylase activity to insure that CYP2B6 protein levels would fall within the linear, quantitative range of the standard curve. CYP2B6 protein determinations were performed in duplicate for each microsomal, Woburn, MA sample.

Fig. 3.

Relationship between diplotype and CYP2B6 mRNA transcript levels (A), CYP2B6 protein content (B), and bupropion hydroxylase activity (C) in human liver samples and microsomes. Solid diamonds represent postnatal samples and open circles represent fetal samples. Boxes represent the interquartile values and the line within each box represents the median value for postnatal samples. Whiskers represent boundaries for outliers (1.5 times interquartile boundaries). Note that no activity was detected in fetal liver microsomes, explaining the absence of respective data points in (C). CYP2B6 protein determinations were performed in duplicate, whereas CYP2B6 mRNA transcript determinations and bupropion hydroxylase activity determinations were performed in triplicate.