Hormonal Regulation of CYP2B6 and CYP2D6 Expression
thesisposted on 13.12.2012, 00:00 by Steve J. Jurkovic
Purpose: Despite clinical observations of altered drug metabolism during pregnancy, little is known about the underlying molecular mechanism. Differential hepatic metabolism during pregnancy results in altered plasma concentrations of medications and leads to undesired effects. Determination of the underlying mechanism is critical for advancement of predicting plasma concentrations. Knowledge of these mechanisms provides information for multiple critical areas of research: a better ability to accurately predict plasma concentrations of drugs, the potential to alert us to other conditions that may be implicated in the mechanism, and allows for individualizing prescriptions based on personal genotype/phenotype and the determined mechanism of induction. Methods: HepG2 cells were chosen as the cell culture model system for this project. Previously, estradiol’s effects on CYP2B6 expression were observed in human hepatocytes. Due to difficulty in obtaining and maintaining hepatocytes, HepG2 cells were substituted. Luciferase assays and mRNA studies were performed after drug treatment. Mechanistic studies were performed for CYP2B6 and CYP2D6 promoter activity using reporter vectors driven by the CYP2B6 and CYP2D6 promoters, respectively. Animal studies were done using transgenic mice expressing human CYP2D6 and its 2.5-kb upstream regulatory region. Mice were sacrificed at key gestational time points and livers were harvested. RT-PCR was then performed. EMSA was performed using nuclear protein extracts from the CYP2D6- transgenic mice. CYP2D6 and HNF4α probes were used, and protein:DNA binding was observed for each gestational time point. Results: Treatment of HepG2 cells with 1 µM estradiol increased CYP2B6 promoter activity 12.5-fold, only when ERα was co-transfected. In HepG2-ER cells, expressing ERα, CYP2B6 mRNA was increased 4-fold by estradiol treatment, and the induction was abrogated by cotreatment with ICI 182,780, an ER-degrading anti-estrogen. CYP2B6 promoter activity was abolished when HepG2 cells were transfected with an ERα expression vector that coded for a mutated DNA-binding domain. Deletion constructs of the CYP2B6 promoter revealed the estradiol-stimulated transactivation was occurring between -1461 and -1839-bp upstream. Mutation of 2 putative AP-1 motifs in this region abolished the estradiol-stimulated induction of CYP2B6. CYP2D6 mRNA from mice sacrificed at virgin, term, and postpartum time points revealed a ~2-fold induction at term, and a return to baseline levels at postpartum. Protein extracts from mouse livers at these time points show an increase in protein:DNA binding to an HNF4α probe at term, with a return to baseline levels at postpartum. CYP2D6 promoter activity in HepG2 cells was reduced ~40% after treatment of retinoic acid (true for 100 pM to 1 µM doses), and CYP2D6 mRNA was reduced ~25% after treatment of 1 µM 9-cis retinoic acid. Conclusions: CYP2B6 induction occurs through an estradiol-stimulated, ER-dependent, AP-1-mediated mechanism. Transgenic mice for human CYP2D6 show an increase in CYP2D6 mRNA at term, providing a model system for the differential regulation of CYP2D6 during human pregnancy. Retinoids appear to play a role in basal-level expression of CYP2D6, though the responsible retinoid remains to be devolved and its importance during pregnancy is unknown.