Estrogen Receptor α Mediates 17α-Ethynylestradiol Causing Hepatotoxicity

Abstract

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 17α-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 α (ERα) 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, ERα 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 β-muricholic acid. ERβ, 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 ERα-mediated repression of hepatic transporters and alterations of bile acid biosynthesis may contribute to development of the EE2-induced hepatotoxicity. 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 17α-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 α (ERα) 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, ERα 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 β-muricholic acid. ERβ, 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 ERα-mediated repression of hepatic transporters and alterations of bile acid biosynthesis may contribute to development of the EE2-induced hepatotoxicity. Estrogens have long been known to cause intrahepatic cholestasis during pregnancy in susceptible women, who are using oral contraceptives or who are on postmenopausal hormone replacement therapy (1Schreiber A.J. Simon F.R. Hepatology. 1983; 3: 607-613Crossref PubMed Scopus (114) Google Scholar). Intrahepatic cholestasis of pregnancy (ICP), 2The abbreviations used are: ICP, intrahepatic cholestasis of pregnancy; ALP, alkaline phosphatase; ABC, ATP-binding cassette; BSEP, bile salt export pump; CAR, constitutive active/androstane receptor; CYP, cytochrome P450; E2, 17β-estradiol; EE2, 17α-ethynylestradiol; ER, estrogen receptor; FAS, fatty-acid synthase; FXR, farnesoid X receptor; HNF4, hepatocyte nuclear factor 4; LRH1, liver receptor homolog 1; LXR, liver X receptor; OATP, organic anion transporting polypeptide; MDR, multidrug resistance-associated protein; PFIC, progressive familial intrahepatic cholestasis; PXR, pregnane X receptor; SHP, short heterodimer partner; NTCP, Na+/taurocholate cotransporting polypeptide; LDB, ligand binding domain. the most common hepatic disease during pregnancy, starts with modest itching associated with elevated levels of serum bile acids and can lead to spontaneous premature delivery and intrauterine fetal death (2Reyes H. J. Gastroenterol. Hepatol. 1997; 12: 211-216Crossref PubMed Scopus (129) Google Scholar, 3Riely C.A. Bacq Y. Clin. Liver Dis. 2004; 8: 167-176Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Experimental intrahepatic cholestasis induced by 17α-ethynylestradiol (EE2) treatment in rodents is a widely used in vivo model to examine the mechanisms involved in estrogen-induced cholestasis (4Rodriguez-Garay E.A. Ann. Hepatol. 2003; 2: 150-158Crossref PubMed Google Scholar). EE2 treatment decreases the ATP-dependent taurocholate transport in the hepatic canalicular membrane, which is thought to be due to impaired expression of the canalicular bile salt export pump (BSEP) (5Lee J.M. Trauner M. Soroka C.J. Stieger B. Meier P.J. Boyer J.L. Gastroenterology. 2000; 118: 163-172Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Moreover, treatment with EE2 also decreases sinusoidal uptake of bile acids by down-regulating the expression of the Na+/taurocholate cotransporting polypeptide protein (NTCP) (6Simon F.R. Fortune J. Iwahashi M. Gartung C. Wolkoff A. Sutherland E. Am. J. Physiol. 1996; 271: G1043-G1052PubMed Google Scholar). These studies suggest that estrogens induce cholestasis by reducing both the influx and efflux of bile acid in hepatocytes, resulting in a decrease in bile flow. In addition, EE2 treatment is shown to alter bile acid composition, which is associated with cholestatic features (7Koopen N.R. Post S.M. Wolters H. Havinga R. Stellaard F. Boverhof R. Kuipers F. Princen H.M. J. Lipid Res. 1999; 40: 100-108Abstract Full Text Full Text PDF PubMed Google Scholar). However, the molecular mechanism of these EE2-dependent alterations is still not fully understood. Bile acid homeostasis is tightly regulated by multiple nuclear receptors including FXR, PXR, and CAR in physiological and/or pathological conditions (8Karpen S.J. J. Hepatol. 2002; 36: 832-850Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Francis G.A. Fayard E. Picard F. Auwerx J. Annu. Rev. Physiol. 2003; 65: 261-311Crossref PubMed Scopus (509) Google Scholar, 10Guo G.L. Lambert G. Negishi M. Ward J.M. Brewer Jr., H.B. Kliewer S.A. Gonzalez F.J. Sinal C.J. J. Biol. Chem. 2003; 278: 45062-45071Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). It is well understood that bile acids repress bile acid biosynthesis by down-regulating transcription of the rate-limiting enzyme CYP7A1 through the FXR-SHP-LRH1 cascade (11Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar, 12Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar, 13Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1229) Google Scholar). NTCP, responsible for bile acid uptake into the hepatocytes, is repressed by FXR activation (11Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar). Simultaneously, FXR up-regulates the expression of BSEP, which increases bile acid efflux from the liver into the bile (11Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar, 14Ananthanarayanan M. Balasubramanian N. Makishima M. Mangelsdorf D.J. Suchy F.J. J. Biol. Chem. 2001; 276: 28857-28865Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). Therefore, FXR regulates transport of bile acids and prevents their overaccumulation in hepatocytes. The activation of PXR inhibits production of additional bile acid by inhibiting CYP7A1 and inducing OATP2, which increases the uptake of bile acids from sinusoidal blood to the hepatocytes (15Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Crossref PubMed Scopus (1139) Google Scholar, 16Xie W. Radominska-Pandya A. Shi Y. Simon C.M. Nelson M.C. Ong E.S. Waxman D.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3375-3380Crossref PubMed Scopus (683) Google Scholar). CAR induces SULT2A1 and MRP4 to prevent toxic bile acid accumulation (17Assem M. Schuetz E.G. Leggas M. Sun D. Yasuda K. Reid G. Zelcer N. Adachi M. Strom S. Evans R.M. Moore D.D. Borst P. Schuetz J.D. J. Biol. Chem. 2004; 279: 22250-22257Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Consistent with preventive roles of PXR and CAR in the cholestatic condition, the double null mouse lacking PXR and CAR has a more severe disruption of bile acids and cholesterol homeostasis (18Zhang J. Huang W. Qatanani M. Evans R.M. Moore D.D. J. Biol. Chem. 2004; 279: 49517-49522Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 19Uppal H. Toma D. Saini S.P. Ren S. Jones T.J. Xie W. Hepatology. 2005; 41: 168-176Crossref PubMed Scopus (98) Google Scholar, 20Stedman C.A. Liddle C. Coulter S.A. Sonoda J. Alvarez J.G. Moore D.D. Evans R.M. Downes M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2063-2068Crossref PubMed Scopus (189) Google Scholar). However, no nuclear receptor has been implicated in estrogen-induced hepatotoxicity. In the present study, we investigated whether nuclear receptor could be involved in the pathogenesis of estrogen-induced hepatotoxicity, using Era–/–, Erb–/–, Fxr–/–, Pxr–/–, and Car–/– mice. We provide direct in vivo evidence that synthetic estrogen EE2 exposure induces liver damage by activating the ERα signaling pathways, leading to an alteration of bile acids biosynthesis and repression of multiple bile acid and cholesterol transporters. Materials—17α-Ethynylestradiol and 1,2-propanediol were obtained from Sigma. Animal Treatment—All protocols and procedures were approved by the National Institutes of Health Animal Care and Use Committee and were in accordance with National Institutes of Health guidelines. Era–/–, Erb–/–, Pxr–/–, Fxr–/–, and Car–/– mice were generated and characterized previously (10Guo G.L. Lambert G. Negishi M. Ward J.M. Brewer Jr., H.B. Kliewer S.A. Gonzalez F.J. Sinal C.J. J. Biol. Chem. 2003; 278: 45062-45071Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 11Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar, 21Couse J.F. Korach K.S. Endocr. Rev. 1999; 20: 358-417Crossref PubMed Scopus (0) Google Scholar, 22Ueda A. Hamadeh H.K. Webb H.K. Yamamoto Y. Sueyoshi T. Afshari C.A. Lehmann J.M. Negishi M. Mol. Pharmacol. 2002; 61: 1-6Crossref PubMed Scopus (400) Google Scholar). Era–/– and Erb–/– mice on a background of C57BL/6 were obtained from Taconic Farms (German-town, NY). Pxr–/–, Fxr–/–, and Car–/– mice used in these studies were maintained in 129/Sv and C57BL/6 mixed genetic background. All animals were housed in a temperature-controlled environment with 12-h light/dark cycles with access to standard chow and water ad libitum. Age-matched groups of 8–12-week-old mice were used for all the experiments. Four to eight mice were used for each treatment group. Adult mice received subcutaneous injections of EE2 (10 mg/kg) or vehicle (80% 1,2-propanediol with 0.15% NaCl) once daily for 5 successive days. Twenty hours after the last injection, the mice were fasted for 4 h before harvesting blood from the retroorbital plexus for subsequent serum analyses and livers for RNA isolation and histology. Histology and Mitosis Measurements—Liver samples from each mouse were fixed in 10% neutral buffered formalin. Slides were stained with hematoxylin and eosin using standard protocols and examined microscopically for structural changes. Hepatocyte proliferation was evaluated by immunohistochemical staining for Ki67 using rat anti-mouse Ki67 (TEC3) antibody (Dako Corporation). Immunoreactivity was visualized with the Vectastain and were with to liver were in and the was by the of by the of hepatocytes. of alkaline phosphatase and bile acids were using and from and the bile acid were by using a by as previously Y. J. Gonzalez F.J. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). of was following for bile were fasted for 4 h before following EE2 (10 mg/kg) or vehicle for 5 successive days. Bile and to of were with a of and maintained the experiments. The was with after of the common bile and with a Bile was a bile of The of biliary that bile was not used for Bile for to h and was was maintained with a to prevent alterations of bile flow. The biliary of bile acids, and were using the bile acid cholesterol and The biliary were as the of the bile and the biliary in with fetal were with a and binding and Y. T. Negishi M. 2003; PubMed Scopus Google using the h after were with or for h and for with the was for using the as an of were from mouse hepatic using the and were from with the and of were with the and were using or as the expression and The of expression and of the and to used in are as The was used as an All were obtained using RNA from of were as were by a were to be to the are as in and of livers from and Era–/– mice with vehicle or liver stained with hematoxylin and eosin and hepatocyte with of of the of hepatocytes of hepatocytes. and EE2-treated mice. and EE2-treated mice of of ERα on bile acids expression of genes involved in bile acids was on RNA from livers of or EE2-treated mice. were to the mice for each serum of β-muricholic acid acid bile acid were by and EE2-treated mice. and EE2-treated mice of expression of bile and transporters in EE2-treated mice. of genes involved in bile acids transport NTCP, OATP1, and cholesterol transport and and transport are was on RNA from livers of or EE2-treated mice. were to the mice for each and EE2-treated mice. and EE2-treated mice of of ERα on biliary and mice were daily with vehicle or EE2 for 5 days. The common bile was the was and hepatic bile was for The of biliary bile acids, and were and the of each was from of bile flow. and EE2-treated mice. and EE2-treated mice of of EE2-induced in the mechanism of estrogen-induced hepatotoxicity in Era–/–, Erb–/–, Fxr–/–, Pxr–/–, and mice were with were EE2-treated mice and vehicle mice not liver were in EE2-treated Erb–/–, Fxr–/–, Pxr–/–, and Car–/– but no was in EE2-treated and Era–/– mice bile acid levels were also elevated in EE2-treated Erb–/–, Fxr–/–, Pxr–/–, and Car–/– mice but not in Era–/– mice a for hepatocyte were in EE2-treated mice but not in Era–/– mice mice are shown as wild-type mice. The wild-type mice also following EE2 treatment to of mice shown in studies (11Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar, H. Miyata M. T. A. W. M. K. Sinal C.J. G.L. Gonzalez F.J. Y. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google mice bile acid levels of bile acids and were by EE2 in that EE2 treatment induced hepatotoxicity an These results that ERα is for the development of EE2-induced hepatotoxicity. The livers of EE2-treated and Era–/– mice were examined Liver of mice EE2-treated mice hepatocyte and of in hepatic However, EE2-treated Era–/– mice not show liver livers from the EE2-treated mice but not the EE2-treated Era–/– mice hepatocytes. the to liver we the of in these The of was markedly with EE2 treatment in mice. However, no such increase was in EE2-treated Era–/– mice the of EE2 in receptor we by a with ERβ, FXR, PXR, and in shown in both ERα and were by EE2 as well as by the ligand K. K. Mol. 1997; PubMed Scopus Google Scholar). EE2 treatment FXR and PXR was no activation by CAR was by treatment Y. T. Negishi M. 2003; PubMed Scopus Google but not by EE2 ERα and are in However, ERα is in the level of in liver is J.F. J. K. Korach K.S. 1997; PubMed Scopus Google Scholar). and EE2 both ERα and ERβ, not a in hepatocytes of expression which is in with the hepatotoxic phenotypes in Erb–/– mice following EE2 of Bile examined the expression of genes involved in bile acid and The biosynthesis of the bile acids, and β-muricholic acid is by the neutral or acidic pathway J.J. Mangelsdorf D.J. Annu. Rev. Biol. 2000; PubMed Scopus Google Scholar, Annu. Rev. 2003; PubMed Scopus Google Scholar). The rate-limiting in the neutral pathway is by The expression of CYP7A1 was decreased in EE2-treated Erb–/–, Fxr–/–, Pxr–/–, and Car–/– mice but was not in the EE2-treated Era–/– mice. expression of CYP7A1 in Era–/– mice with that of mice the repression of CYP7A1 the of ERα to Erb–/– and mice not expression of and Car–/– mice an increase in expression of CYP7A1 of and However, FXR and CAR not contribute to the repression of CYP7A1 by EE2 The expression of CYP8B1, enzyme involved in the neutral was also decreased in all EE2-treated mouse that the Era–/– mice the level of In the expression of an important enzyme in the acidic was markedly induced in all EE2-treated mouse but not in the Era–/– mice. In addition, the expression of the was not in of the mouse with no were in the expression of a of bile acid homeostasis FXR and SHP, that the was not involved in the of these biosynthesis by EE2 the of by EE2, we the of bile acids in and and Era–/– mice using with the expression of and CYP8B1, the of β-muricholic acid to acid were in mice following EE2 treatment with no alteration in Era–/– mice These results suggest that alterations in bile acid biosynthesis may contribute to the development of hepatotoxicity by EE2 of Bile and the molecular of EE2-induced hepatotoxicity, we examined the expression of hepatic transporters involved in bile and We examined the expression of canalicular transporters and that have been as the genes responsible for familial intrahepatic cholestasis and M. E. Hepatology. 2001; PubMed Scopus Google Scholar). of was decreased in EE2-treated Erb–/–, Fxr–/–, Pxr–/–, and Car–/– mice but was not in the EE2-treated Era–/– mice In levels were not in of the mouse with is responsible for bile acid uptake into hepatocytes, and transporters are also involved in hepatic uptake of bile in and of is in with cholestasis G. P. R. A. C. P. H. K. Trauner M. Hepatology. 2001; PubMed Scopus Google Scholar). expression was decreased in all EE2-treated mouse the Era–/– mice. and levels were also decreased by EE2 treatment in all mouse Era–/– mice. We also the expression of cholesterol efflux transporters ABCG5, and of and was decreased by EE2 treatment in all mouse the Era–/– mice. In EE2 treatment no on and a in cholesterol efflux from hepatocytes to bile J. K. D. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). EE2 decreased both and of cholesterol whether the repression of hepatic transporters biliary biliary bile acids, and were by the after of the common bile In with the decreased expression of hepatic the of bile acids and cholesterol was decreased by EE2 in and mice but not in Era–/– mice. The of biliary was also decreased by the of repression of the transporter was a decrease of was These are with the that biliary is to biliary bile acids these that the activation of ERα is associated with a in biliary We have direct evidence that ERα can a factor for the development of hepatotoxicity following estrogen nuclear receptor we have shown that ERα is the receptor responsible for multiple hepatic bile acid and cholesterol hepatic transporters biliary secretions resulting in liver damage In addition, the ERα-mediated alteration of bile acids biosynthesis may have also to the EE2-induced development of hepatotoxicity. of the nuclear receptors FXR, PXR, and were shown to be involved in hepatotoxicity. present with Era–/– mice is the to the of ERα in estrogen-induced has been that treatment with ERα such as in and F. G.A. D.J. Google Scholar, Gastroenterology. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The molecular mechanism of ERα regulates and transporters an the present of the genes in present can also be regulated by nuclear receptors such as LXR, LRH1, and G.A. Fayard E. Picard F. Auwerx J. Annu. Rev. Physiol. 2003; 65: 261-311Crossref PubMed Scopus (509) Google Scholar, Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). expression of the known was to be repressed in the EE2-treated mice but not in EE2-treated Era–/– mice the that ERα repressed and through However, that was repressed by EE2 in was up-regulated in mice Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. 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