title: Alpha-naphthylisothiocyanate modulates hepatobiliary transporters in sandwich-cultured rat hepatocytes.
abstract: 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.
authors.affiliation: Center for Drug Safety Evaluation & Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, Jiangsu, China.
authors.forename: Cen
authors.initials: C
authors.lastname: Guo
authors.forename: Lei
authors.initials: L
authors.lastname: He
authors.forename: Dan
authors.initials: D
authors.lastname: Yao
authors.forename: Jiye
authors.initials: J
authors.lastname: A
authors.forename: Bei
authors.initials: B
authors.lastname: Cao
authors.forename: Jin
authors.initials: J
authors.lastname: Ren
authors.forename: Guangji
authors.initials: G
authors.lastname: Wang
authors.forename: Guoyu
authors.initials: G
authors.lastname: Pan
mesh_terms: D015058:1-Naphthylisothiocyanate / Q000633:toxicity*; D018528:ATP-Binding Cassette Transporters / Q000502:physiology; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D055442:Metabolome; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D018528:ATP-Binding Cassette Transporters; C423013:Abcc2 protein, rat; D001647:Bile Acids and Salts; D015058:1-Naphthylisothiocyanate
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