title: An in vitro assay to assess transporter-based cholestatic hepatotoxicity using sandwich-cultured rat hepatocytes.
abstract: Drug-induced cholestasis can result from the inhibition of biliary efflux of bile acids in the liver. Drugs may inhibit the hepatic uptake and/or the biliary efflux of bile acids resulting in an increase in serum concentrations. However, it is the intracellular concentration of bile acids that results in hepatotoxicity, and thus serum concentrations may not necessarily be an appropriate indicator of hepatotoxicity. In this study, sandwich-cultured rat hepatocytes were used as an in vitro model to assess the cholestatic potential of drugs using deuterium-labeled sodium taurocholate (d(8)-TCA) as a probe for bile acid transport. Eight drugs were tested as putative inhibitors of d(8)-TCA uptake and efflux. The hepatobiliary disposition of d(8)-TCA in the absence and presence of drugs was measured by using liquid chromatography/tandem mass spectrometry, and the accumulation (hepatocytes and hepatocytes plus bile), biliary excretion index (BEI), and in vitro biliary clearance (Cl(biliary)) were reported. Compounds were classified based on inhibition of uptake, efflux, or a combination of both processes. Cyclosporine A and glyburide showed a decrease in total (hepatocytes plus bile) accumulation, an increase in intracellular (hepatocytes only) accumulation, and a decrease in BEI and Cl(biliary) of d(8)-TCA, suggesting that efflux was primarily affected. Erythromycin estolate, troglitazone, and bosentan resulted in a decrease in accumulation (total and intracellular), BEI, and Cl(biliary) of d(8)-TCA, suggesting that uptake was primarily affected. Determination of a compound's relative effect on bile acid uptake, efflux, and direct determination of alterations in intracellular amounts of bile acids may provide useful mechanistic information on compounds that cause increases in serum bile acids.
authors.affiliation: CSO, Qualyst, Inc., 2810 Meridian Parkway, Suite 100, Durham, NC 27713, USA.
authors.forename: John H
authors.initials: JH
authors.lastname: Ansede
authors.forename: William R
authors.initials: WR
authors.lastname: Smith
authors.forename: Cassandra H
authors.initials: CH
authors.lastname: Perry
authors.forename: Robert L
authors.initials: RL
authors.lastname: St Claire
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Brouwer
mesh_terms: D000465:Algorithms; D000818:Animals; D001646:Bile / Q000187:drug effects* / Q000378:metabolism; D001647:Bile Acids and Salts / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects*; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000517:prevention & control; D002780:Cholestasis, Intrahepatic / Q000139:chemically induced* / Q000517:prevention & control; D002851:Chromatography, High Pressure Liquid; D003094:Collagen; D046508:Culture Techniques / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D064420:Drug-Related Side Effects and Adverse Reactions; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D007797:Laminin; D008297:Male; D011509:Proteoglycans; D051381:Rats; D017208:Rats, Wistar; D053719:Tandem Mass Spectrometry; D013656:Taurocholic Acid / Q000378:metabolism*; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D001647:Bile Acids and Salts; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D013656:Taurocholic Acid; D003094:Collagen