17β-Estradiol (E2) enhances cytochrome P450 (CYP) 2B6 expression via cooperative actions of estrogen receptor (ER) and constitutive androstane receptor (CAR)

Pregnancy alters hepatic drug metabolism in a cytochrome P450 (CYP) pathway-specific manner [1].  Pregnancy-related physiological changes, such as rising concentrations of estrogen, are potentially responsible.  However, it remains unknown whether estrogen, at concentrations attainable during pregnancy, affects hepatic drug metabolism.  The objective of this study is to characterize the effects of major estrogen, E2, on hepatic expression of major CYP enzymes and elucidate the underlying molecular mechanisms.  To this end, human hepatocytes were treated with vehicle (ethanol) or E2 (1 μM), and mRNA expression levels of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5 were determined by quantitative RT-PCR.  E2 increased expression of CYP2A6, CYP2B6 and CYP3A4 but not those of other CYP enzymes.  Metabolism of probe drugs for CYP2A6, 2B6 and 3A4 was also increased in E2-treated hepatocytes.  In HepG2 cells, luciferase reporter assays of CYP2B6 upstream regulatory region revealed that E2 enhances promoter activity of CYP2B6 via activation of CAR.  E2 triggered nuclear translocation of EGFP-tagged human CAR in rat hepatocytes, further confirming that E2 activates CAR.  Luciferase reporter assays in HepG2 cells revealed that E2 enhances CYP2B6 promoter activity also via ER transactivation.  DNA-binding domain of ER was found dispensable in this induction, suggesting involvement of non-classical mechanism of ER action in upregulation of CYP2B6.  Results from luciferase reporter assay using 5’-deletion reporter constructs for CYP2B6 upstream region showed that -1839/-1461 of CYP2B6 mediated the ER action.  The region harbors 2 putative binding sites for activator protein-1 (AP-1): -1782/-1776 and -1664/-1658.  Results from mutation assays indicated that both AP-1 motifs were critical in ER-mediated activation of CYP2B6 promoter by E2.  Electrophorectic mobility shift and supershift assays demonstrated that in addition to AP-1 proteins, ER bound to the distal AP-1 motif and the binding was enhanced in HepG2 cells treated with E2.  Concurrent activation of ER and CAR by E2 activated the promoter activity of CYP2B6 in a synergistic manner.  Our data indicate that at concentrations attainable during pregnancy, E2 upregulates CYP2B6 expression by cooperative activation of CAR and ER, and the ER action is mediated through AP-1.  These results suggest potential increases in metabolism of CYP2B6 substrates during pregnancy.

1.  Anderson, G.D., Pregnancy-induced changes in pharmacokinetics: a mechanistic-based approach. Clin Pharmacokinet, 44(10), 989-1008, 2005.