The Mechanism Of Atorvastain Induced-Cholestatic Liver Injury

OBJECTIVETo study whether drugs affect the vectorial efflux of bile acid we used SCRH originated from rat liver, which was proposed as one of the mechanisms leading to hepatotoxicity.We compared the effect of Atorvastatin,2-and4-hydroxy metabolites on the function of Bsep and Mrp2in rat hepatocytes directly.METHODS1Hepatocyte isolation and Sandwich cultureRat hepatocytes were isolated using a two-step perfusion method. Hepatocyte viability was determined by trypan blue exclusion. Only those hepatocyte preparations with viability greater than85%were used for experiments. Unlike hepatocytes cultures using conventional techniques which lose polarity and metabolic function, hepatocytes cultured between two layers of gelled collagen in a sandwich configuration maintain polarity, morphology, and liver-specific metabolic activity. Over time in sandwich culture, hepatocytes develop functional bile canalicular networks sealed by tight junctions, and hepatic transport proteins are expressed and localized to the correct membrane domains allowing for assessment of transport function.2Cytotoxicity assayRat hepatocytes were cultured with DMEM (Dulbecco’s Modified Eagle’s Medium) contains10%FBS(Fetal Bovine Serum) in a humidified atmosphere of95%air and5%CO2at37℃.MTT (diphenyltetrazolium bromide) assay was used to find the non-cytotoxicity dosage of Troglitazone, MK-571, Atrovastain,2-and4-hydroxy metabolites in Rat hepatocytes. The dosage at which the survival rate of cells is above90%was considered as the highest non-cytotoxic dosage.3Transport Studies in SCRHRetention of5(and6)-carboxy-2,7-dichlorofluorescein (CDF) in bile canalicular lumen was examined by fluorescence microscopy. The cells and bile canaliculi were imaged with an inverted fluorescence microscope.The effects of drug concentration, inhibitor on the transporter of Bsep, Mrp2was detected using SCRH model. The amount of the transportation of TCA was analysed with LC-MS/MS, and the CFDA concentration in the lysate was quantified using a microplate reader. Then the biliary excretion index (BEI) was obtained. 4LC-MS/MS analysis of TCA in cell lysatesThe effects of Bsep inhibitor on the transportation of transporter was detected using SCRH model. The amount of the transportation of TCA was analysed with LC-MS/MS, and the The biliary excretion index (BEI) was obtained.RESULTS1Isolation and Maintenance of Sandwich-Cultured Rat HepatoytesHepatocytes were prepared from about a200g rat on average. The survival rate was higher than85%. Part of the cells adhered in24h and much of them adhered in48h.The survival rate was measured by typan blue exclusion.2Cytotoxicity assayAtorvastatin,2-and4-hydroxy metabolites, MK-571concentrations of20-100μmol/L were found to be cytotoxic towards the rat hepatocytes cells in24h. The result in12h was consistent with that in24h except Mk-571. All the compounds were non-toxic to rat hepatocytes at the experimental concentrations in6h.3Effect of Atorvastatin,2-and4-hydroxy metabolites on Bsep,Mrp2functionAtorvastatin and2-hydroxy metabolites increased the mass of TCA in calcium-free treated samples (representing an increase in hepatocellular concentration). Both of the compounds also decreased the BEI of taurocholate, suggesting an inhibition of the BSEP-mediated taurocholate efflux out of the hepatocyte. Atorvastatin,2-hydroxy metabolites, MK-571increased the accumulation of CDF (transported by Mrp2) in hepatocytes. MK-571, Atorvastatin,2-hydroxy metabolites resulted in a decrease in the accumulation of CDF in bile and the value of BEI.CONCLUSIONThe potential for Atorvastatin,2-and4-hydroxy metabolites to influence the distribution of bile acids or CDF may be related to their potential for in vivo hepatotoxicity.