title: Toxicity and intracellular accumulation of bile acids in sandwich-cultured rat hepatocytes: role of glycine conjugates.
abstract: Excessive intrahepatic accumulation of bile acids (BAs) is a key mechanism underlying cholestasis. The aim of this study was to quantitatively explore the relationship between cytotoxicity of BAs and their intracellular accumulation in sandwich-cultured rat hepatocytes (SCRH). Following exposure of SCRH (on day-1 after seeding) to various BAs for 24h, glycine-conjugated BAs were most potent in exerting toxicity. Moreover, unconjugated BAs showed significantly higher toxicity in day-1 compared to day-3 SCRH. When day-1/-3 SCRH were exposed (0.5-4h) to 5-100μM (C)DCA, intracellular levels of unconjugated (C)DCA were similar, while intracellular levels of glycine conjugates were up to 4-fold lower in day-3 compared to day-1 SCRH. Sinusoidal efflux was by far the predominant efflux pathway of conjugated BAs both in day-1 and day-3 SCRH, while canalicular BA efflux showed substantial interbatch variability. After 4h exposure to (C)DCA, intracellular glycine conjugate levels were at least 10-fold higher than taurine conjugate levels. Taken together, reduced BA conjugate formation in day-3 SCRH results in lower intracellular glycine conjugate concentrations, explaining decreased toxicity of (C)DCA in day-3 versus day-1 SCRH. Our data provide for the first time a direct link between BA toxicity and glycine conjugate exposure in SCRH.
authors.affiliation: KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, O&N2, Herestraat 49 - Bus 921, 3000 Leuven, Belgium. Electronic address: Sagnik.Chatterjee@pharm.kuleuven.be.
authors.forename: Sagnik
authors.initials: S
authors.lastname: Chatterjee
authors.affiliation: Department of Metabolic Diseases, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address: i.bijsmans@umcutrecht.nl.
authors.forename: Ingrid T G W
authors.initials: IT
authors.lastname: Bijsmans
authors.affiliation: Department of Metabolic Diseases, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address: s.w.c.vanmil@umcutrecht.nl.
authors.forename: Saskia W C
authors.initials: SW
authors.lastname: van Mil
authors.affiliation: KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, O&N2, Herestraat 49 - Bus 921, 3000 Leuven, Belgium. Electronic address: Patrick.Augustijns@pharm.kuleuven.be.
authors.forename: Patrick
authors.initials: P
authors.lastname: Augustijns
authors.affiliation: KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, O&N2, Herestraat 49 - Bus 921, 3000 Leuven, Belgium. Electronic address: Pieter.Annaert@pharm.kuleuven.be.
authors.forename: Pieter
authors.initials: P
authors.lastname: Annaert
mesh_terms: D000465:Algorithms; D000818:Animals; D001646:Bile / Q000378:metabolism; D001647:Bile Acids and Salts / Q000378:metabolism* / Q000633:toxicity*; D002469:Cell Separation; D002478:Cells, Cultured; D002635:Chenodeoxycholic Acid / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003627:Data Interpretation, Statistical; D005998:Glycine / Q000502:physiology*; D006002:Glycodeoxycholic Acid / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D013058:Mass Spectrometry; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D013654:Taurine / Q000378:metabolism; D013655:Taurochenodeoxycholic Acid / Q000378:metabolism; D013657:Taurodeoxycholic Acid / Q000378:metabolism; D014508:Urea / Q000378:metabolism
chemical_list: D001647:Bile Acids and Salts; D012333:RNA, Messenger; D002635:Chenodeoxycholic Acid; D013654:Taurine; D006002:Glycodeoxycholic Acid; D013655:Taurochenodeoxycholic Acid; D013657:Taurodeoxycholic Acid; D014508:Urea; D005998:Glycine
keywords: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; BAAT; BAs; BEI; BSEP/Bsep; Bile acid conjugation; Bile acids; CA; CDCA; Chenodeoxycholic acid; Cholestasis; DMSO; FBS; Fetal Bovine Serum GCDCA, glycochenodeoxycholic acid; GDCA; HBSS; HEPES; Hanks’ Balanced Salt Solution; Hepatotoxicity; NOAEL; NTCP/Ntcp; PBS; Phosphate Buffered Saline; SCH; SCRH; Sandwich-cultured rat hepatocytes; TCDCA; TDCA; bile acid CoA: amino acid N-acyltransferase; bile acids; bile salt export pump (human/rat); biliary excretion index; chenodeoxycholic acid; cholic acid; dimethyl sulfoxide; glycodeoxycholic acid; no observed adverse effect level; sandwich-cultured hepatocytes; sandwich-cultured rat hepatocytes; sodium taurocholate cotransporting polypeptide (human/rat); taurochenodeoxycholic acid; taurodeoxycholic acid