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.
Keywords: 5 (and 6)-carboxy-2′,7′-dichlorofluorescein; 5 (and 6)-carboxy-2′,7′-dichlorofluorescein diacetate; ALT; ANIT; Alpha-naphthylisothiocyanate; BECs; BEI; Bsep; CDF; CDFDA; Cl(biliary); Cyp27a1; Cyp7a1; Cyp8b1; DPDPE; Drug-induced cholestasis; ELISA; GC–MS; HBSS; Hank's balanced salt solution; IPRLs; LC–MS/MS; LDH; Mrp; Ntcp; Oatp; PLS-DA; SCRH; Sandwich-cultured hepatocytes; Transporter; VPA; [D-Pen(2,5)]-enkephalin; alanine transaminase; alpha-naphthylisothiocyanate; bile salt export pump; biliary clearance; biliary epithelial cells; biliary excretion index; cholesterol 7α-hydroxylase; d8-TCA; deuterium-labeled sodium taurocholate; enzyme-linked immunosorbent assay; gas chromatography–mass spectrometry; isolated perfused rat livers; lactate dehydrogenase; liquid chromatography–mass spectrometry tandem mass spectrometry; multidrug resistance-associated protein; organic anion transporting polypeptide; partial least squares discriminant analysis; sandwich-cultured rat hepatocytes; sodium/taurocholate cotransporting polypeptide; sterol 12α-hydroxylase; sterol 27-hydroxylase; valproic acid.
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