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.

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.