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 (0-180 μM) 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 48 h ANIT (5-45 μM) 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 30 min or 48 h 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 48 h 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.