Short Communication PREVENTION OF MRP2 ACTIVITY IMPAIRMENT IN ETHINYLESTRADIOL-INDUCED CHOLESTASIS BY URSODEOXYCHOLATE IN THE RAT

Ethinylestradiol (EE) induces cholestasis by affecting bile saltdependent and -independent fractions of the bile flow. The decrease in bile salt-independent flow is thought to be due, in part, to a reduction in the expression of the canalicular transporter Mrp2. The impact of modulation of Mrp2 function by sodium ursodeoxycholate (UDC) in EE cholestasis is unknown. We evaluated the protective effect of UDC on EE-induced impairment of Mrp2 activity in vivo and in isolated hepatocytes, by using the substrate dinitrophenyl S-glutathione (DNP-SG). EE was administered to male Wistar rats at a dose of 5 mg/kg s.c. for 5 days. UDC was coadministered with EE at a dose of 25 mg/kg b.wt. i.p. for the same period. EE alone reduced DNP-SG biliary excretion by 55% when compared with controls. Coadministration with UDC partially restored the alteration. Secretion rate of DNP-SG was decreased by 30% in isolated hepatocytes from EE-treated rats, but, contrary to in vivo results, UDC coadministration did not restore DNP-SG transport, likely as a consequence of bile salt washout resulting from the isolation procedure. As a confirmation, tauroursodeoxycholate hepatocyte preloading significantly increased Mrp2 activity. Western blotting analysis of Mrp2 indicated that EE administration significantly reduced its level in total and plasma membranes and that UDC coadministration failed to revert this alteration. In conclusion, UDC improvement in Mrp2 transport activity in vivo likely derived from a direct enhancement of Mrp2 function rather than from a restoration of its expression levels. This provides a novel mechanism explaining the beneficial effects of UDC in EE-induced cholestasis. EE, a synthetic estrogen, induces intrahepatic cholestasis in experimental animals (Gumucio and Valdivieso, 1971; Jacquemin et al., 1993; Crocenzi et al., 2001; Sanchez Pozzi et al., 2003) by reducing the liver’s capacity to excrete bile salts and organic anions (Gumucio and Valdivieso, 1971; Bossard et al., 1993). Expression of Mrp2, a canalicular transporter involved in organic anion excretion and, hence, in the formation of the bile salt-independent fraction of bile flow (Crocenzi et al., 2004), is decreased in EE cholestasis (Trauner et al., 1997). Ursodeoxycholate (UDC) is a bile salt commonly used in the treatment of cholestatic diseases (Paumgartner and Beuers, 2002) including cholestasis of pregnancy (Palma et al., 1997). Its beneficial effect on reversion of EE-induced cholestasis is based on the improvement of the biliary secretory function impaired by the estrogen (Jacquemin et al., 1993; Sanchez Pozzi et al., 2003). It was demonstrated that UDC up-regulates canalicular Mrp2 expression in normal mice (Fickert et al., 2001) and that its taurine derivative [tauroursodeoxycholate (TUDC)] stimulates insertion of preexisting pericanalicular vesicles containing Mrp2 into the canalicular domain, thus accounting for prevention of taurolithocholate-induced cholestasis (Beuers et al., 2001). Whether the beneficial effect of UDC on EE cholestasis is associated with regulation of Mrp2 expression is not known. Activation/inactivation of canalicular transporters was proposed as an alternative explanation for the modulation of canalicular secretory function (Paumgartner and Beuers, 2002). Gerk et al. (2003) recently demonstrated that UDC positively modulates the activity of human MRP2, expressed in Sf9 insect cells. The authors proposed that a direct activation of transport of MRP2 substrates by UDC may contribute significantly to the anticholestatic properties of the bile salt. This possibility has not been tested in vivo. The purpose of the current study was to analyze the potential protective effect of UDC on EE-induced impairment of Mrp2 transport activity and expression at the canalicular level. Transport activity of the model substrate of Mrp2, dinitrophenyl S-glutathione (DNP-SG), was evaluated in vivo and in isolated hepatocytes, in addition to assessment of Mrp2 expression by Western blotting. Materials and Methods Chemicals. EE, UDC, dimethyl sulfoxide, phenylmethylsulfonyl fluoride, leupeptin, and pepstatin A were purchased from Sigma-Aldrich (St. Louis, MO). Collagenase type A from Clostridium histolyticum was purchased from Invitrogen (Paisley, UK). 1-Chloro-2,4-dinitrobenzene (CDNB) was obtained from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). All other reagents were of the highest analytical grade and used as supplied. Animals. Adult male Wistar rats weighing 300 to 350 g were used throughSupported by grants from Agencia Nacional de Promoción Cientı́fica y Tecnológica, Consejo Nacional de Investigaciones Cientı́ficas y Técnicas, Ministerio de Salud de la Nación (Beca Ramón Carrillo–Arturo Oñativia 2004, to F.A.C.), and Fundación Antorchas, Argentina. Parts of this study were presented in the Biannual Meeting of the International Association for the Study of the Liver (IASL, San Salvador de Bahia, Brazil), March