Estradiol-17␤-D-glucuronide (E 2 17G), an endogenous metabolite of estradiol, induces a potent dose-dependent and reversible inhibition of bile flow in the rat. We analyzed the effect of a single dose of E 2 17G (15 mol/kg, intravenously) to female rats on bile flow and the endocytic retrieval and function of the canalicular multidrug resistance-associated protein 2 (Mrp2) and the effect of pretreatment with dibutyryl-cyclic AMP (DBcAMP; 20 mol/kg) on these measures. Bile flow was maximally inhibited by 85% within 10 minutes of E 2 17G and returned to 50% and 100% of control levels within 75 and 120 minutes, respectively. Western analysis of total homogenates and mixed plasma and intracellular membranes suggested partial internalization of Mrp2 during the acute phase of cholestasis at 20 minutes and during the period of recovery from cholestasis at 75 minutes, which returned to control levels by 180 minutes after E B ile flow is dependent on the active transport of osmotically active solutes across the apical membrane of the hepatocyte into the confined space of the canaliculus, followed by the passive movement of water. 1 Bile salts and glutathione (GSH) are 2 key solutes in bile that are major contributors to the generation of bile flow; their active transport into bile is mediated by members of the ATP-binding cassette family of membrane transporters in the canalicular domain of the hepatocyte. 2 The bile salt export pump (Bsep; Abcb11) mediates the concentrative transport of bile salts across the canalicular membrane 3 and thus generates the bile acid-dependent component of bile flow. The multidrug resistance-associated transporter 2 (Mrp2; Abcc2) mediates the transport of GSH and of numerous glutathione, glucuronide, and sulfate conjugates into bile and is thus considered critical to the generation of bile acid-independent bile flow. 4 Estradiol-17␤-D-glucuronide (E 2 17G) is an endogenous estrogen metabolite and one of a family of glucuronide conjugates of the estrogen D-ring that have been shown to decrease bile flow and bile acid secretion in the rat in a profound, dose-dependent, and completely reversible manner. 5 Linear regression analysis of the relationship between bile flow and bile acid secretion following a cholestatic dose of E 2 17G also indicated a substantial inhibition of bile acid-independent bile flow. 6 The precise mechanism by which E 2 17G induces cholestasis is not known, but the process of Mrp2-mediated E 2 17G transport across the canalicular membrane is essential for this toxic action. 7 Factors that regulate the expression and ac-

Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17β-d-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 μmol/kg iv) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2. DBcAMP (20 μmol/kg iv) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 μM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 μM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.

UDCA (ursodeoxycholic acid) is the therapeutic agent most widely used for the treatment of cholestatic hepatopathies. Its use has expanded to other kinds of hepatic diseases, and even to extrahepatic ones. Such versatility is the result of its multiple mechanisms of action. UDCA stabilizes plasma membranes against cytolysis by tensioactive bile acids accumulated in cholestasis. UDCA also halts apoptosis by preventing the formation of mitochondrial pores, membrane recruitment of death receptors and endoplasmic-reticulum stress. In addition, UDCA induces changes in the expression of metabolizing enzymes and transporters that reduce bile acid cytotoxicity and improve renal excretion. Its capability to positively modulate ductular bile flow helps to preserve the integrity of bile ducts. UDCA also prevents the endocytic internalization of canalicular transporters, a common feature in cholestasis. Finally, UDCA has immunomodulatory properties that limit the exacerbated immunological response occurring in autoimmune cholestatic diseases by counteracting the overexpression of MHC antigens and perhaps by limiting the production of cytokines by immunocompetent cells. Owing to this multi-functionality, it is difficult to envisage a substitute for UDCA that combines as many hepatoprotective effects with such efficacy. We predict a long-lasting use of UDCA as the therapeutic agent of choice in cholestasis.

The endogenous estradiol metabolite estradiol 17␤-D-glucuronide (E 2 17G) induces an acute cholestasis in rat liver coincident with retrieval of the canalicular transporters bile salt export pump (Bsep, Abcc11) and multidrug resistance-associated protein 2 (Mrp2, Abcc2) and their associated loss of function. We assessed the participation of Ca 2؉ -dependent protein kinase C isoforms (cPKC) in the cholestatic manifestations of E 2 17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHCs). In PRL, E 2 17G (2 mol/liver; intraportal, single injection) maximally decreased bile flow, total glutathione, and [ 3 H] taurocholate excretion by 61%, 62%, and 79%, respectively; incorporation of the specific cPKC inhibitor Gö6976 (500 nM) in the perfusate almost totally prevented these decreases. In dose-response studies using IRHC, E 2 17G (3.75-800 M) decreased the canalicular vacuolar accumulation of the Bsep substrate cholyl-lysylfluorescein with an IC50 of 54.9 ؎ 7.9 M. Gö6976 (1 M) increased the IC50 to 178.4 ؎ 23.1 M, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Mrp2 substrate, glutathione methylfluorescein. Prevention of these changes by Gö6976 coincided with complete protection against E 2 17G-induced retrieval of Bsep and Mrp2 from the canalicular membrane, as detected both in the PRL and IRHC. E 2 17G also increased paracellular permeability in IRHC, which was only partially prevented by Gö6976. The cPKC isoform PKC␣, but not the Ca 2؉ -independent PKC isoform, PKC⑀, translocated to the plasma membrane after E 2 17G administration in primary cultured rat hepatocytes; Gö6976 completely prevented this translocation, thus indicating specific activation of cPKC. This is consistent with increased autophosphorylation of cPKC by E 2 17G, as detected via western blotting. Conclusion: Our findings support a central role for cPKC isoforms in E 2 17G-induced cholestasis, by inducing both transporter retrieval from the canalicular membrane and opening of the paracellular route. (HEPATOLOGY 2008;48:1885-1895 B ile formation represents a key liver function by which xenobiotics and endogenous metabolites such as cholesterol, bilirubin, and hormones are eliminated from the body. 1,2 Efflux of solutes by adenosine triphosphate-dependent transporters at the canalicular membrane of hepatocytes provide the driving force for osmotic bile formation; among these transporters, the bile salt export pump (Bsep, Abcc11) and multidrug re-

Silymarin is a purified extract from milk thistle (Silybum marianun (L.) Gaertn), composed of a mixture of four isomeric flavonolignans: silibinin (its main, active component), isosilibinin, silydianin and silychristin. This extract has been empirically used as a remedy for almost 2000 years, and remains being used as a medicine for many types of acute and chronic liver diseases. Despite its routinely clinical use as hepatoprotectant, the mechanisms underlying its beneficial effects remain largely unknown. This review addresses in detail a number of recent studies showing a novel feature of silymarin as a hepatoprotective drug, namely: its anticholestatic properties in experimental models of hepatocellular cholestasis with clinical correlate. For this purpose, this review will cover the following aspects: 1. The chemistry of silymarin, including chemical composition and properties. 2. The current clinical applications of silymarin as a hepatoprotective agent, including the mechanisms by which silymarin is thought to exert its hepatoprotective properties, when known. 3. The physiological events involved in bile formation, and the mechanisms of hepatocellular cholestasis, focusing on cellular targets and mechanisms of action of drugs used to reproduce experimentally cholestatic diseases of clinical interest, in particular estrogens and monohydroxylated bile salts, where anticholestatic properties of silymarin have been tested so far. 4. The recent findings describing the impact of silymarin on normal bile secretion and its novel, anticholestatic properties in experimental models of cholestasis, with particular emphasis on the cellular/molecular mechanisms involved, including modulation of bile salt synthesis, biotransformation/depuration of cholestatic compounds, changes in transporter expression/activity, and evocation of signaling pathways.

The effect of silymarin (SIL) on 17␣-ethynylestradiol (EE)-induced cholestasis was evaluated in rats. EE (5 mg/kg, subcutaneously, daily, for 5 days) decreased both the bilesalt-dependent and the bile-salt-independent fractions of the bile flow. The decrease in the former was associated to a reduction in the bile salt pool size (؊58%), and this effect was completely prevented by SIL. This compound also counteracted the inhibitory effect induced by EE on HCO 3 ؊ but not glutathione output, 2 major determinants of the bile-salt-independent bile flow. EE decreased the secretory rate maximum (SRM) of tauroursodeoxycholate, (؊71%) and bromosulfophthalein (BSP; ؊60%), as well as the expression of the BSP canalicular carrier, mrp2; SIL failed to increase mrp2 expression, and had only a marginal beneficial effect on both tauroursodeoxycholate and BSP SRM values. However, the two-compartment model-based kinetic constant for BSP canalicular transfer was significantly improved by SIL (؉262%). SIL decreased rather than increased CYP3A4, the cytochrome P450 isoenzyme involved in the oxidative metabolism of EE, and had no inhibitory effect on the UDP-glucuronosyltrasferase isoforms involved in the formation of its 17␤-glucuronidated, more cholestatic metabolite. Pretreatment of isolated rat hepatocyte couplets with silibinin, the major, active component of SIL, counter- Estrogens are well known to cause intrahepatic cholestasis in susceptible women during pregnancy, administration of oral contraceptives, and postmenopausal replacement therapy. 1 Given these clinical implications, experimental cholestasis induced by estrogen administration in rodents, mainly 17␣-ethynylestradiol (EE), has been widely used as an experimental model to assess the mechanisms involved in estrogeninduced cholestasis. These studies have shown that estrogens induce cholestasis by reducing both the bile-salt-dependent fraction of the bile flow (BSDF) and the bile-salt-independent fraction of the bile flow (BSIF). 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. 6 At the canalicular level, EE was shown to decrease the ATP-dependent taurocholate transport in hepatocyte canalicular membrane preparations, 5 which is thought to be due, at least in part, to an impairment in the expression of the canalicular bile salt export pump at a posttranscriptional level 7 ; this contributes to explain the decrease in the secretory rate maximum (SRM) of taurocholate, a parameter that is thought to evaluate the canalicular, rate-limiting transfer of bile salts in vivo...

Background: Taurolithocholate induced cholestasis is a well established model of drug induced cholestasis with potential clinical relevance. This compound impairs bile salt secretion by an as yet unclear mechanism. Aims: To evaluate which step/s of the hepatocellular bile salt transport are impaired by taurolithocholate, focusing on changes in localisation of the canalicular bile salt transporter, Bsep, as a potential pathomechanism. Methods: The steps in bile salt hepatic transport were evaluated in rats in vivo by performing pharmacokinetic analysis of 14 C taurocholate plasma disappearance. Bsep transport activity was determined by assessing secretion of 14 C taurocholate and cholyl-lysylfluorescein in vivo and in isolated rat hepatocyte couplets (IRHC), respectively. Localisation of Bsep and F-actin were assessed both in vivo and in IRHC by specific fluorescent staining. Results: In vivo pharmacokinetic studies revealed that taurolithocholate (3 µmol/100 g body weight) diminished by 58% canalicular excretion and increased by 96% plasma reflux of 14 C taurocholate. Analysis of confocal images showed that taurolithocholate induced internalisation of Bsep into a cytosolic vesicular compartment, without affecting F-actin cytoskeletal organisation. These effects were reproduced in IRHC exposed to taurolithocholate (2.5 µM). Preadministration of dibutyryl-cAMP, which counteracts taurolithocholate induced impairment in bile salt secretory function in IRHC, restored Bsep localisation in this model. Furthermore, when preadministered in vivo, dibutyryl-cAMP accelerated recovery of both bile flow and bile salt output, and improved by 106% the cumulative output of 14 C taurocholate. Conclusions: Taurolithocholate impairs bile salt secretion at the canalicular level. Bsep internalisation may be a causal factor which can be prevented by dibutyryl-cAMP.

(E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 mol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 mol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicinepretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process. bile secretion; endocytic compartment; colchicine; bilirubin THE MULTIDRUG RESISTANCE-ASSOCIATED protein 2 (Mrp2; Abcc2) mediates the ATP-dependent transport of a wide range of amphiphilic anionic conjugates into bile (5,35