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.

Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity.