Human hepatic CYP2B6 developmental expression: The impact of age and genotype
Graphical abstract
The percentage of pediatric liver samples with detectable CYP2B6 increased with age. Median CYP2B6 levels were higher in >30 days postnatal age samples compared to <30 days postnatal age samples.
Introduction
CYP2B6 is the only functional member of the human CYP2B family and was originally thought to be absent in most individuals. Improved antibody preparations have now demonstrated the presence of immunoreactive CYP2B6 protein in the livers of most adults tested [1], although interindividual differences are among the largest of the cytochromes P450 that have been studied. Thus, a recent meta-analysis reported that CYP2B6 and CYP3A5 had the lowest minimum expression levels (1.0 pmol/mg microsomal protein). However, CYP2B6 exhibited maximum expression levels (45 pmol/mg microsomal protein) comparable to CYP1A2, 2A6, 2C8, and 2E1 (52–68 pmol/mg microsomal protein) and higher than either CYP2C19 (20 pmol/mg microsomal protein) or CYP2D6 (11 pmol/mg microsomal protein) [2]. This conclusion also is consistent with relative abundance levels. The relative abundance of CYP2B6 (0.4–8.4%, 21-fold range) and 3A5 (0.4–22%, 55-fold range) exhibited a much larger range than any of the other cytochromes P450 (all less than 3-fold range). Similar to the CYP2C and CYP3A family members, a portion of the interindividual variability in CYP2B6 expression may be explained by the ability of both the constitutive androstane (CAR, NR1I3) and pregnane X (PXR, NR1I2) receptors to induce CYP2B6 expression several fold in a ligand-dependent manner [3]. Although CYP2B6 is considered primarily a hepatic enzyme, it has been detected at lower levels in several other organs, including the brain [4], kidney and lung [5].
CYP2B6 participates in the oxidative metabolism of numerous pharmaceuticals, including the anti-depressant bupropion [6], the anesthetics propofol [7] and lidocaine [8], the chemotherapeutic agents cyclophosphamide, ifosfamide [9] and tamoxifen [10], the anti-retroviral agent efavirenz [11] and the anti-malarial drug artemesinin [12]. CYP2B6 also plays a role in the metabolism of methadone [13] and the drugs of abuse, nicotine [14] and ecstasy [15]. Environmental contaminants such as styrene [16] and several pesticides, including chlorpyrifos [17] and endosulfan [18] are also excellent substrates. CYP2B6 also plays a role in the metabolism of endogenous substrates including the steroid testosterone [19]. Like other members of the cytochrome P450 family of proteins, CYP2B6 can detoxify and facilitate the elimination of toxicants and drugs, however, depending on the chemical properties of the substrate, CYP2B6-dependent oxidation also can increase the toxicity of several compounds, e.g., tamoxifen, aflatoxin B1 [10] and chlorpyrifos [17]. Most important for the subject of this study, purposeful or accidental childhood exposures to all of these compounds have been documented.
CYP2B6 functional polymorphisms have been identified but null alleles are rare. The most clinically relevant polymorphism is the CYP2B6*6 allele (g.15631G>T, rs3745274; g.18053A>G, rs2279343). The g.15631G>T transversion in the CYP2B6*6 allele is predicted to result in the loss of a splice enhancer and is linked to the formation of transcript variant predicted to encode a non-functional protein [20]. However, because the CYP2B6*6 allele exhibits incomplete penetrance, it is associated with reduced protein expression and reduced in vivo metabolism, rather than a complete loss of function [21].
Little is known about CYP2B6 expression during development. An early study of age-dependent CYP2B6 expression found lower levels in infant liver samples when compared to adults [22], but this study included just two fetal samples and eight infant samples. Samples from individuals ranging from 2 to 72 years of age were not sufficient in number to permit a determination of any further temporal changes in CYP2B6 expression.
The objective of this study was to characterize CYP2B6 developmental expression and determine the possible impact of genetic variation on this process. The absence of an in vivo probe for CYP2B6 activity suitable for use in children, the difficulty in collecting pharmacokinetic data from infants, and the ethical issues preventing the study of in vivo fetal CYP2B6 metabolism necessitated the use of post-mortem human liver microsomal samples. Comparing CYP2B6 expression with previously characterized CYP3A levels in the same samples also was performed in an attempt to gain some information regarding possible regulatory mechanisms.
Section snippets
Materials
Polyclonal antibodies against CYP2B6 and lymphoblast-expressed CYP2B6 were purchased from BD Biosciences (San Jose, CA). IR800 dye-labeled goat anti-rabbit IgG antibodies were purchased from LI-COR (Lincoln, NE). Nitrocellulose membranes were supplied by Bio-Rad (Hercules, CA). EZ-Run pre-stained recombinant protein molecular weight markers, methanol and Tris–glycine buffer were obtained from Thermo Fisher Scientific (Waltham, MA). CYP2B6 single nucleotide polymorphism (SNP) genotyping assays;
CYP2B6 immunoquantitation
A single immunoreactive protein was detected by western blot in the pediatric microsomal samples that comigrated with lymphoblast-expressed CYP2B6 and the adult positive control sample (Fig. 1A). CYP2B6 protein levels were below the limits of detection (10 fmol/lane, 0.25 pmol/mg protein) in 25% of the microsomal samples, even though Ponceau S staining of the western blot showed even transfer and similar protein levels for all samples. To verify the presence of microsomal protein in the absence
Discussion
In an earlier report, CYP2B6 protein was detected in only 2 of 10 liver microsomal samples from donors >37 weeks gestation but <10 months of age (mean ± SD = 2.7 ± 5.9 pmol/mg microsomal protein), but was found in 7 of 10 samples from donors ranging in age from 2 to 72 years (mean ± SD = 19.4 ± 23.9 pmol/mg microsomal protein) [22]. Although this same trend was observed in the current study, CYP2B6 levels above the limit of detection were observed in 64% of all fetal (N = 56) and samples from birth to 30 days
Acknowledgements
This study was supported in part by funds from the Children's Hospital Foundation of Wisconsin (to R.N.H.) and the Department of Environmental and Molecular Toxicology, North Carolina State University.
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