17β-Estradiol up-regulates UDP-glucuronosyltransferase 1A9 expression via estrogen receptor α

AbstractUDP-glucuronosyltransferase 1A9 (UGT1A9) is a major phase II enzyme responsible for elimination of drugs and endogenous molecules. Clinical data have shown increased elimination of UGT1A9 substrates in pregnant women or oral contraceptive users, but the role of estrogen in the regulation of UGT1A9 expression remains unknown. In this study, we investigated the effect of 17β-estradiol (E2) on UGT1A9 expression and the role of ERα in the transcriptional regulation of UGT1A9. E2 significantly increased UGT1A9 promoter activity in HepG2 cells in the presence of ERα. UGT1A9 induction by E2 was abrogated by antiestrogen ICI182,780 in HepG2 cells that constitutively express ERα. Results from transient transfection of ERα mutants into HepG2 cells demonstrated that mutation at DNA-binding domain of ERα abrogates increased UGT1A9 promoter activity by E2. Deletion and mutation assays of UGT1A9 promoter revealed a putative ERE located within −2262/−1987 region. Examination of healthy human liver tissues revealed significantly higher UGT1A9 expression in women as compared to men. Together, these findings provide a mechanistic basis for the previous clinical reports and may shed a light on identifying sources for inter-individual variability in UGT1A9-mediated drug metabolism

Molecular Basis of Inter-Individual Variability in Cytochrome P450 2D6-Mediated Drug Metabolism

Cytochrome P450 (CYP) 2D6 is a major drug-metabolizing enzyme, eliminating over 20% of clinically used drugs. CYP2D6-mediated drug metabolism exhibits large inter-individual variability. Genetic polymorphisms of CYP2D6 are known to contribute to the variability, especially in CYP2D6 poor metabolizer (PM) phenotype. However, PMs only account for 5-10% of the population, and the association between CYP2D6 genotypes and phenotypes is weak in non-PM population. Identities of factors governing CYP2D6 expression and/or activities in the majority of population remain unclear. Hereby, in 115 healthy human liver tissues, we examined multiple potential determinants of CYP2D6 activity levels including genetic polymorphisms in CYP2D6 and transcriptional regulators of CYP2D6 as well as the amounts of bile acids and retinoic acid. Our results showed that CYP2D6 activity score (a semi-quantitative collective representation of CYP2D6 genotypes) is a poor predictor of CYP2D6 activity while 59% of the variability in CYP2D6 activity is explained by CYP2D6 protein levels. CYP2D6 protein and mRNA levels were correlated with r2 of 0.11, likely reflecting the dynamics of transcriptional gene regulation. mRNA expression levels of previously known transcriptional regulators of CYP2D6 did not correlate with CYP2D6 expression or activity. Results from a series of adjusted analysis and multivariate regression showed that CYP8B1 expression (that share the same transcriptional regulatory pathway as CYP2D6) was the best predictor of CYP2D6 mRNA levels. We also examined whether physiological changes, specifically obesity and pregnancy, affect CYP2D6 or mouse endogenous CYP2D expression. Our results suggest that obesity had minimum effect on CYP2D6-mediated drug metabolism in CYP2D6 humanized mouse model. During mouse pregnancy, Cyp2d40 expression was enhanced through enhanced transactivation by HNF4α. Together, our data indicate that CYP2D6 genotypes only partially explain the variability in CYP2D6 activity in general. CYP2D6 activity level in the liver is governed predominantly by its protein amount highlighting the importance of factors involved in the regulation of CYP2D6 expression in explaining the inter-individual variability of CYP2D6-mediated drug metabolism

High Fat Diet Feeding Alters Expression of Hepatic Drug-metabolizing Enzymes in Mice

Abbreviations CAR, constitutive androstane receptor; CYP, cytochrome P450; DMEs, drug-metabolizing enzymes; FGF, fibroblast growth factor; FXR, farnesoid X receptor; Hes6, hes family bHLH transcription factor 6; HFD, high-fat diet; HNF4α, hepatocyte nuclear factor 4α; PXR, pregnane X receptor; qRT-PCR, quantitative real time-PCR; Pck1, phosphoenolpyruvate carboxykinase 1; Oatp1a1, organic anion-transporting polypeptides 1a1; SHP, small heterodimer partner; Tg-CYP2D6, CYP2D6-humanized transgenic mouse. DMD #75655 3 ABSTRACT Medical conditions accompanying obesity often require drug therapy, but whether and how obesity alters the expression of drug-metabolizing enzymes and thus drug pharmacokinetics is poorly defined. Previous studies have shown that high fat diet (HFD) feeding and subsequent obesity in mice lead to altered expression of transcriptional regulators for cytochrome P450 (CYP) 2D6, including hepatocyte nuclear factor 4α (HNF4α, a transcriptional activator of CYP2D6) and small heterodimer partner (SHP, a transcriptional repressor of CYP2D6). The objective of this study is to examine whether diet-induced obesity alters CYP2D6 expression by modulating HNF4α and SHP expression. Male CYP2D6-humanized transgenic (Tg-CYP2D6) mice were fed with HFD or matching control diet for 18 weeks. Hepatic mRNA expression of CYP2D6 decreased to a small extent in HFD group (by 31%), but the differences in CYP2D6 protein and activity levels in hepatic S9 fractions were found insignificant between the groups. While hepatic SHP expression did not differ between the groups, HNF4α mRNA and protein levels decreased by ~30% in HFD group. Among major mouse endogenous Cyp genes, Cyp1a2 and Cyp2c37 showed significant decreases in HFD group while Cyp2e1 expression did not differ between groups. Cyp2b10 and Cyp3a11 expression was higher in HFD group, with corresponding 2.9-fold increases in hepatic CYP3A activities in HFD-fed mice. Together, these results suggest that obesity has minimal effects on CYP2D6-mediated drug metabolism while it modulates the expression of mouse endogenous CYPs in a gene-specific manner