Hepatic and Intestinal Multidrug Resistance-Associated Protein 2: Transcriptional and Post-transcriptional Regulation by Xenobiotics

We are daily exposed to a large number of pharmacological drugs, environmental pollutants, and natural toxins, which represent a potential toxic insult. The organism possesses a sophisticated system of detoxification particularly expressed in the liver, intestine, and kidney. This system consists of intracellular biotransformation enzymes that convert the toxins into more hydrophilic derivatives followed by their elimination through membrane transporters. Multidrug resistance-associated protein 2 (MRP2, ABCC2) is an important member of the ATP-binding cassette (ABC) superfamily of transporters localized at the apical membrane of polarized cells, such as hepatocytes, enterocytes, and renal tubular cells. MRP2 is proposed as a major actor in the elimination of endo- and xenobiotics, mainly conjugated with glucuronic acid, glutathione, and sulfate. The small intestine and the liver constitute relevant detoxification organs expressing MRP2 and therefore preventing absorption and promoting the hepatobiliary clearance of xenobiotics. MRP2 expression and/or function can be modulated by therapeutic drugs, herbal products, dietary compounds, and environmental pollutants. Consequently, MRP2 modulation could cause changes in tissue exposure, with potential toxicological and pharmacological consequences. This chapter reviews the information available on the role of hepatic and intestinal MRP2 in detoxification processes, and their regulation by xenobiotics, considering in particular its toxicological relevance

Biliary Secretion of Glutathione in Estradiol 17␤-D-Glucuronide- Induced Cholestasis

ABSTRACT Estradiol-17␤-D-glucuronide (E2-17G) induces an acute but reversible inhibition of bile flow after its intravenous administration to rats, due in part to the endocytic retrieval of the canalicular multidrug resistance-associated transporter protein 2 and the bile salt export pump, transporters that contribute to bile flow. Decreased bile salt-independent bile flow (BSIF) is also involved and persists during the phase of recovery from cholestasis. Because glutathione and HCO 3 Ϫ are major contributors to BSIF, we evaluated changes in their biliary excretion and the hepatic content of total glutathione during E2-17G-induced cholestasis. E2-17G acutely decreased bile flow and biliary excretion of total glutathione by about 80%; glutathione excretion was still inhibited at 80 min and 120 min, even though bile flow was partially and totally restored, respectively. Neither liver glutathione content nor the proportions of oxidized glutathione in bile and liver were affected by E2-17G at any time. HCO 3 Ϫ concentrations in bile were unchanged, so that secretion paralleled variations in bile flow. In the isolated perfused liver, addition of E2-17G decreased both bile flow and the biliary concentration of glutathione, whereas addition of its noncholestatic isomer estradiol-3-D-glucuronide (E2-3G) did not inhibit bile flow, but significantly reduced the concentration of glutathione in bile. The bile:liver concentration ratios of glutathione were significantly decreased in vivo by E2-17G and in the perfused liver by E2-17G and E2-3G. These data indicate that E2-17G cis-inhibits the canalicular transport of glutathione and thus contributes to the cholestatic effect by inhibiting BSIF

Co-Regulation of Expression of Phase II Metabolizing Enzymes and Multidrug Resistance-Associated Protein 2

Treatment of experimental animals with prototypical enzyme inducers represents a useful tool to characterize the role of different isozymes in drug metabolism and to improve our knowledge on factors regulating their synthesis at the transcriptional level. The effect of model enzyme inducers on phase II (conjugating) enzyme families, including UDP-glucuronosyltransferase’s and glutathione-S-transferase’s, has been well characterized in rodent liver. More recently, the effect of inducers on the expression of canalicular multidrug resistance-associated protein 2 (Mrp2) has been focused upon. The identification of a number of conjugated drugs as Mrp2 substrates suggests that both the conjugation and transport systems act coordinately to improve drug elimination from the body. We provide evidence about circumstances resulting in the simultaneous upregulation of phase II enzymes and Mrp2 in hepatic and extrahepatic tissues, most likely involving activation of common nuclear receptors (e.g., FXR, PXR). Additionally, we provide an analysis of examples of drug-induced toxicity leading to the simultaneous downregulation of both systems. Potential therapeutic strategies based on the modulation of expression of these systems are also briefly commented upon

Ursodeoxycholate reduces ethinylestradiol glucuronidation in the rat: Role of prevention in estrogen-induced cholestasis

ABSTRACT Ethinylestradiol (EE) administration (5 mg/kg, s.c., daily for 5 days) to rats leads to cholestasis, and its derivative EE 17␤-glucuronide is a likely mediator of this effect. Coadministration of ursodeoxycholate (UDC) was shown to prevent ethinylestradiol-induced cholestasis. The aim of this study was to evaluate the inhibitory effect of UDC on EE glucuronidation in vivo and in vitro as a potential mechanism to explain UDC protection. UDC treatment (25 mg/kg, i.p., daily for 5 days) decreased the biliary excretion of EE 17␤-glucuronide in bile after administration of a trace dose of [ 3 H]EE and reduced microsomal EE 17␤-glucuronidation activity by 20% and expression of UGT2B1, one of the enzymes involved in EE conjugation, by 30%. Glucuronidation kinetic studies were performed in vitro using normal microsomes and isolated hepatocytes in the presence of tauroursodeoxycholate (TUDC), the major endogenous derivative of UDC in the rat. Kinetic enzymatic studies in microsomes showed a noncompetitive inhibition of EE 17␤-glucuronidation by TUDC, which was unique for this bile salt since other endogenous bile salts such as taurocholate, taurochenodeoxycholate, or taurodeoxycholate did not affect the enzyme activity. Studies in isolated hepatocytes confirmed the inhibitory effect of TUDC on EE glucuronidation and indicated that TUDC can reach the enzyme active site in intact cells. In conclusion, both in vivo and in vitro experiments indicate that UDC decreased the metabolic pathways involved in EE glucuronidation, hence decreasing the formation of the cholestatic derivative EE 17␤-glucuronide

Hepatic drug transporters and nuclear receptors: Regulation by therapeutic agents

The canalicular membrane represents the excretory pole of hepatocytes. Bile is an important route of elimination of potentially toxic endo- and xenobiotics (including drugs and toxins), mediated by the major canalicular transporters: multidrug resistance protein 1 (MDR1, ABCB1), also known as P-glycoprotein, multidrug resistance-associated protein 2 (MRP2, ABCC2), and the breast cancer resistance protein (BCRP, ABCG2). Their activities depend on regulation of expression and proper localization at the canalicular membrane, as regulated by transcriptional and post-transcriptional events, respectively. At transcriptional level, specific nuclear receptors (NR)s modulated by ligands, co-activators and co-repressors, mediate the physiological requirements of these transporters. This complex system is also responsible for alterations occurring in specific liver pathologies. We briefly describe the major Class II NRs, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), and their role in regulating expression of multidrug resistance proteins. Several therapeutic agents regulate the expression of relevant drug transporters through activation/inactivation of these NRs. We provide some representative examples of the action of therapeutic agents modulating liver drug transporters, which in addition, involve CAR or PXR as mediators

Dynamic localization of hepatocellular transporters in health and disease

Vesicle-based trafficking of hepatocellular transporters involves delivery of the newly-synthesized carriers from the rough endoplasmic reticulum to either the plasma membrane domain or to an endosomal, submembrane compartment, followed by exocytic targeting to the plasma membrane. Once delivered to the plasma membrane, the transporters usually undergo recycling between the plasma membrane and the endosomal compartment, which usually serves as a reservoir of pre-existing transporters available on demand. The balance between exocytic targeting and endocytic internalization from/to this recycling compartment is therefore a chief determinant of the overall capability of the liver epithelium to secrete bile and to detoxify endo and xenobiotics. Hence, it is a highly regulated process. Impaired regulation of this balance may lead to abnormal localization of these transporters, which results in bile secretory failure due to endocytic internalization of key transporters involved in bile formation. This occurs in several experimental models of hepatocellular cholestasis, and in most human cholestatic liver diseases. This review describes the molecular bases involved in the biology of the dynamic localization of hepatocellular transporters and its regulation, with a focus on the involvement of signaling pathways in this process. Their alterations in different experimental models of cholestasis and in human cholestatic liver disease are reviewed. In addition, the causes explaining the pathological condition (e.g. disorganization of actin or actin-transporter linkers) and the mediators involved (e.g. activation of cholestatic signaling transduction pathways) are also discussed. Finally, several experimental therapeutic approaches based upon the administration of compounds known to stimulate exocytic insertion of canalicular transporters (e.g. cAMP, tauroursodeoxycholate) are described

Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis

Bilirubin is an endogenous antioxidant with cytoprotective properties, and several studies highlight its potential in the treatment of pro-oxidant diseases. We demonstrated that oxidative stress (OS), a key feature in most hepatopathies, induces cholestasis by actin cytoskeleton disarrangement and further endocytic internalization of key canalicular transporters, such as the bile salt export pump (Bsep) and the multidrug resistance-associated protein 2 (Mrp2). He re, we evaluated the capability of physiological concentrations of unconjugated bilirubin (UB) to limit OS and the impairment in biliary secretory function induced by the model pro-oxidant agent, tert-butylhydroperoxide (tBuOOH). UB fully prevented the formation of reactive oxygen species (ROS) and membrane lipid peroxidation induced by tBuOOH in isolated rat hepatocytes. In the isolated rat hepatocyte couplet model, UB (17.1 µM) prevented the endocytic internalization of Bsep and Mrp2 and the impairment in their secretory function induced by tBuOOH. UB also prevented actin disarrangement, as evaluated by both plasma membrane bleb formation and actin fluorescent staining. Finally, UB prevented tBuOOH-induced cPKC activation. Experiments in isolated perfused rat livers showed that UB prevents the increase in oxidized glutathione biliary excretion and the drop in bile flow and the biliary excretion of specific Bsep and Mrp2 substrates. We conclude that physiological concentrations of UB are sufficient to prevent the biliary secretory failure induced by OS, by counteracting actin disarrangement and the consequent internalization of canalicular transporters relevant to normal bile formation. This reveals an important role for UB in preserving biliary secretory function under OS conditions.Fil: Basiglio, Cecilia Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Toledo, Flavia D. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Boaglio, Andrea C. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Arriaga, Sandra Mónica María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Bioquímica Clínica; Argentina.Fil: Ochoa, Justina E. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Sánchez Pozzi, Enrique J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Mottino, Aldo D. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina.Fil: Roma, Marcelo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina