Impaired activity of the bile canalicular organic anion transporter (Mrp2/cmoat) is not the main cause of ethinylestradiol-induced cholestasis in the rat

Abstract

To test the hypothesis that impaired activity of the bile canalicular organic anion transporting system mrp2 (cmoat) is a key event in the etiology of 17␣-ethinylestradiol (EE)-induced intrahepatic cholestasis in rats, EE (5 mg/kg subcutaneously daily) was administered to male normal Wistar (NW) and mrp2-deficient Groningen Yellow/Transport-deficient Wistar (GY/TR ؊ ) rats. Elevated plasma bilirubin levels in GY/TR ؊ rats increased upon EE-treatment from 65 ؎ 8.4 mol/L to 183 ؎ 22.7 mol/L within 3 days, whereas bilirubin levels remained unaffected in NW rats. Biliary bilirubin secretion was 1.5-fold increased in NW rats but remained unaltered in GY/TR ؊ rats. Plasma bile salt concentrations remained unchanged in both strains, although hepatic levels of the sinusoidal Na ؉ -taurocholate cotransporting protein (ntcp) were markedly reduced. Biliary secretion of endogenous bile salt was not affected in either strain. A clear reduction of mrp2 levels in liver plasma membranes of NW rats was found after 3 days of treatment. The bile salt-independent fraction of bile flow (BSIF) was reduced from 2.6 to 2.0 L/min/100 g body weight in NW rats with a concomitant 62% reduction of biliary glutathione secretion. The absence of mrp2 and biliary glutathione in GY/TR ؊ rats did not prevent induction of EE-cholestasis; a similar absolute reduction of BSIF, i.e., from 1.1 to 0.6 L/min/100 g body weight, was found in these animals. EE treatment caused a reduction of the maximal biliary secretory rate (S RM ) of the mrp2 substrate, dibromosulphthalein (DBSP), from 1,040 to 695 nmol/min/ 100 g body weight (؊38%) in NW rats and from 615 to 327 nmol/min/100 g body weight (؊46%) in GY/TR ؊ rats. These results demonstrate that inhibition of mrp2 activity and/or biliary glutathione secretion is not the main cause of EE-induced cholestasis in rats. The data indicate that alternative pathways exist for the biliary secretion of bilirubin and related organic anions that are also affected by EE. (HEPATOLOGY 1998;27:537-545.)The synthetic estrogen, 17␣-ethinylestradiol (EE), causes a reversible intrahepatic cholestasis in experimental animals, mainly by reducing the bile salt-independent fraction of bile flow (BSIF). 1 Although EE-induced cholestasis has been claimed to represent a model of cholestasis of pregnancy in humans, 2 the mechanism(s) underlying the reduction in bile flow have not been defined. Suggestions that increased permeability of tight junctions 3,4 and reduced activity of Na ϩ / K ϩ -adenosine triphosphatase (ATPase) at the sinusoidal membrane 5-7 represent primary events in the onset of cholestasis could not be substantiated in more recent studies. [8][9][10][11] Likewise, the relevance of the reported decrease in sinusoidal membrane fluidity after EE treatment remains questionable, because decreased membrane fluidity has also been found in experimental conditions associated with increased bile flow. 11,12 More recently, it was shown that EE differentially affects the activity of transport systems involved in bile formation ...

Beneficial Effects of Silymarin on Estrogen–Induced Cholestasis in the Rat: A Study In Vivo And in Isolated Hepatocyte Couplets

“…None of the other serum biochemical parameters studied were modified by EE, in agreement with results reported elsewhere. 3,4 SIL induced a dose-dependent improvement in serum ALP activity, which reached a value not significantly different from that in controls at a dose of 100 mg/kg body weight, onwards.…”

“…60 EE also affected the BSIF, in agreement with previous studies. 2,4 This was, at least in part, because of an impairment in the biliary output of both GSH and HCO 3 Ϫ , the 2 major determinants of this fraction of the bile flow. Our results show that SIL partially counteracted the effect of EE on the BSIF, which was accounted, for at least in part, by a partial restoration of HCO 3 Ϫ output.…”

“…One possible target is mrp2, which was recently shown to be a canalicular GSH transporter, 61 and whose expression is impaired by EE. 4,7,62 However, a role for mrp2 in EE-induced cholestasis was recently disputed when Koopen et al 4 showed that the impairment in bile flow induced by EE in GY/TR Ϫ rats lacking mrp2 was not different from that in controls. Our results show that SIL prevented the decrease of GSH excretion only marginally whereas the efficiency of the canalicular transfer of the mrp2 substrate, BSP, was completely restored; this observation is in line with the hypothesis of Koopen et al, 4 mentioned earlier, that transporters other than mrp2 are involved in EE-induced GSH secretory impairment, whose activity was not normalized by SIL.…”

“…1,2 The model in rats, however, requires estrogen dosages much greater than those required to induce cholestasis in susceptible women and fails to fully reproduce the pathology in humans in that little if any effect on serum parameters of hepatic damage, e.g., bile salt and bilirubin levels, is recorded. 3,4 The mechanism involved in the impairment of the BSDF is multifactorial. EE decreases sinusoidal uptake of bile acids, 5 at least in part by inducing down-regulation of the expression of the sodium-taurocholate cotransporting polypeptide protein, ntcp.…”

Regulation of Plasma Membrane Localization of the Na+-taurocholate cotransporting polypeptide (Ntcp) by Hyperosmolarity and Tauroursodeoxycholate

“…At the canalicular membrane, bile salt excretion is mediated by the bile salt export pump (Bsep) 2 (7) and to a lesser extent by the multidrug resistance-associated protein 2 (Mrp2) (8). The expression of Bsep and Mrp2 in the canalicular membrane of the hepatocyte is regulated by oxidative stress (9 -11), lipopolysaccharide (LPS) (12)(13)(14), drugs (15,16), and ambient osmolarity (1,10,(17)(18)(19). The cellular hydration state is a dynamic parameter that can change physiologically within minutes depending on the ambient osmolarity, nutrient supply, influence of hormones, and oxidative stress.…”