Alpha-naphthylisothiocyanate modulates hepatobiliary transporters in sandwich-cultured rat hepatocytes

Alpha-naphthylisothiocyanate (ANIT) induces intra-hepatic cholestasis mixed with hepatocellular injury mainly by bile ductular damage. However, its direct effect on hepatic parenchymal cells (hepatocytes) is unclear. Sandwich-cultured rat hepatocytes (SCRH) were applied to clarify this question. Though cytotoxicity was not observed (0-180μM) in ANIT-treated SCRH, metabonomics analysis of the hepatocytes revealed a shift in the metabolic pattern and a decrease in cellular cholesterol level, accompanied by an increase in total bile acids after 48h ANIT (5-45μM) treatment. To assess the function of major hepatic bile acid transporters, the accumulation and efflux of [D-Pen^2,5]-enkephalin (DPDPE), 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) diacetate promoiety and deuterium-labeled sodium taurocholate (d8-TCA) were measured. ANIT incubation for either 30min or 48h led to dose-dependent decreases in the biliary excretion index (BEI) of DPDPE and CDF, as well as the intracellular accumulation of d8-TCA, CDF and DPDPE. The basolateral efflux of d8-TCA was also decreased with its BEI barely changed. mRNA expression of multiple uptake transporters and bile acid synthesizing enzymes was down-regulated after 48h incubation. In conclusion, ANIT could directly induce retention of bile acids in hepatocytes by inhibiting the function of bile acid transporters, which might contribute to its cholestatic effect.