Estrogen Receptor Mediates 17 -Ethynylestradiol Causing Hepatotoxicity

Laboratories of Reproductive and Developmental Toxicology, NIEHS/National Institutes of Health, Research Triangle Park, NC 27709, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2006; 281(24):16625-31. DOI: 10.1074/jbc.M602723200
Source: PubMed


Estrogens are known to cause hepatotoxicity such as intrahepatic cholestasis in susceptible women during pregnancy, after administration of oral contraceptives, or during postmenopausal replacement therapy. Enterohepatic nuclear receptors including farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive active/androstane receptor (CAR) are important in maintaining bile acid homeostasis and protecting the liver from bile acid toxicity. However, no nuclear receptor has been implicated in the mechanism for estrogen-induced hepatotoxicity. Here Era(-/-), Erb(-/-), Fxr(-/-), Pxr(-/-), and Car(-/-) mice were employed to show that Era(-/-) mice were resistant to synthetic estrogen 17alpha-ethynylestradiol (EE2)-induced hepatotoxicity as indicated by the fact that the EE2-treated Era(-/-) mice developed none of the hepatotoxic phenotypes such as hepatomegaly, elevation in serum bile acids, increase of alkaline phosphatase activity, liver degeneration, and inflammation. Upon EE2 treatment, estrogen receptor alpha (ERalpha) repressed the expression of bile acid and cholesterol transporters (bile salt export pump (BSEP), Na(+)/taurocholate cotransporting polypeptide (NTCP), OATP1, OATP2, ABCG5, and ABCG8) in the liver. Consistently, biliary secretions of both bile acids and cholesterol were markedly decreased in EE2-treated wild-type mice but not in the EE2-treated Era(-/-) mice. In addition, ERalpha up-regulated the expression of CYP7B1 and down-regulated the CYP7A1 and CYP8B1, shifting bile acid synthesis toward the acidic pathway to increase the serum level of beta-muricholic acid. ERbeta, FXR, PXR, and CAR were not involved in regulating the expression of bile acid transporter and biosynthesis enzyme genes following EE2 exposure. Taken together, these results suggest that ERalpha-mediated repression of hepatic transporters and alterations of bile acid biosynthesis may contribute to development of the EE2-induced hepatotoxicity.

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Available from: Kenneth S Korach, Dec 28, 2015
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    • "The volume of distribution for EE in rats was estimated to be 2 l/kg (Zamek- Gliszczynski et al., 2011), and in consequence, a concentration of EE of 10 mM would be compatible with administration of a 5–6 mg/kg birth weight dose of EE, which is equivalent to the single dose currently administered in vivo. ER-a was also implicated in repression of hepatic transporters and alteration of bile acid biosynthesis using a dose of EE that is even higher than the dose commonly used in rats (Yamamoto et al., 2006). Although the observed increase in mRNA levels after 5 hours of treatment in vivo and in hepatocytes might be caused by ER as a common mediator, further studies would be necessary to demonstrate that the increased Mrp3 mRNA levels occurring in the isolated hepatocytes model results in induction of Mrp3 protein, as previously seen after repeated treatment in vivo (Ruiz et al., 2007). "
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    ABSTRACT: Multidrug resistance-associated protein 3 (Mrp3; Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas Mrp2 is down-regulated. This study was undertaken to determine if Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis, and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, non-cholestatic, dose of EE (5 mg/Kg, s.c.) and basal bile flow and the biliary excretion rate of bile salts and glutathione measured 5 h later. This treatment increased Mrp3 mRNA 4-fold, detected by real time PCR, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1-10 μM) for 5 h, exhibited a 3-fold increase in Mrp3 mRNA (10 μM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were pre-incubated for 30 min with an estrogen receptor antagonist (ICI182/780, 1 μM) before the addition of EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 min, as detected by Western blotting in nuclear extracts. This increase was prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation, and required participation of an ER, most likely ER-α, through its phosphorylation.
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    • "ERα activation is known to repress the protein S gene in mouse liver (28). Estrogens regulate hepatic bile acid synthesis and a sustained activation of ERα by 17α-ethynylestradiol produces hepatotoxicity in mice (29). Utilizing Pxr−/− mice, we recently determined that PXR represses the hepatic Sult1e1 gene during fasting (data not shown). "
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    ABSTRACT: Pregnane X receptor (PXR), acting as a xenobiotic-activated transcription factor, regulates the hepatic metabolism of therapeutics as well as endobiotics such as steroid hormones. Given our finding that PXR activation by rifampicin (RIF) represses the estrogen sulfotransferase (SULT1E1) gene in human primary hepatocytes and hepatocellular carcinoma Huh7 cells, here we have investigated the molecular mechanism of this repression. First the PXR-responsive enhancer was delineated to a 100 bp sequence (−1000/−901), which contains three half sites that constitute the overlapping direct repeat 1 (DR1) and direct repeat 2 (DR2) motifs and two forkhead factor binding sites. siRNA knockdown, chromatin immunoprecipitation and chromatin conformation capture assays were employed to demonstrate that hepatocyte nuclear factor 4α (HNF4α) bound to the PXR-responsive enhancer, and activated the enhancer by looping its position close to the proximal promoter. Upon activation by RIF, PXR indirectly interacted with the enhancer, decreasing the interaction with HNF4α and dissolving the looped SULT1E1 promoter with deacetylation of histone 3. Removal of the DR sites from the enhancer hampers the ability of HNF4α to loop the promoter and that of PXR to repress the promoter activity. Thus, PXR represses human SULT1E1, possibly attenuating the inactivation of estrogen.
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    • "Furthermore,TOXICOGENOMICS IN CULTURES OF HUMAN HEPATOCYTES repression of HNF4-a target genes may be related to direct interaction with bile acids (Sinal et al., 2001;Zollner et al., 2006). Repression of bile acid transporters may occur as a result of specific transcriptional interactions (e.g., in estrogen-induced cholestasis) or as part of an unspecific response to stress (e.g., cytokine mediated) (Yamamoto et al., 2006). Mitochondrial dysfunction may be another possible cause for transcript repression of genes encoding for ATP-dependent bile salt transporters in human hepatocyte cultures treated with extracts rich in petasin at extremely high doses. "
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