Predictive Value of Cellular Accumulation of Hydrophobic Bile Acids As a Marker of Cholestatic Drug Potential
Audrey BurbanAhmad SharanekLydie HumbertThibaut EguetherChristiane Guguen‐GuillouzoDominique RainteauAndré Guillouzo
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Abstract:
Drug-induced cholestasis is mostly intrahepatic and characterized by alterations of bile canaliculi dynamics and morphology as well as accumulation of bile acids (BAs) in hepatocytes. However, little information exists on first changes in BA content and profile induced by cholestatic drugs in human liver. In this study, we aimed to analyze the effects of a large set of cholestatic and noncholestatic drugs in presence of physiological serum concentrations and 60-fold higher levels of 9 main BAs on cellular accumulation of BAs using HepaRG hepatocytes. BAs were measured in cell layers (cells + bile canaliculi) and culture media using high-pressure liquid chromatography coupled with tandem mass spectrometry after 24 h-treatment. Comparable changes in total and individual BA levels were observed in cell layers and media from control and noncholestatic drug-treated cultures: unconjugated BAs were actively amidated and lithocholic acid (LCA) was entirely sulfated. In contrast, cellular accumulation of LCA and in addition, of the 2 other hydrophobic BAs, chenodeoxycholic acid and deoxycholic acid, was evidenced only with cholestatic compounds in presence of BA mixtures at normal and 60-fold serum levels, respectively, suggesting that LCA was the first BA to accumulate. Cellular accumulation of hydrophobic BAs was associated with inhibition of their amidation and for LCA, its sulfation. In conclusion, these results demonstrated that cellular accumulation of unconjugated hydrophobic BAs can be caused by various cholestatic drugs in human hepatocytes and suggest that their cellular detection, especially that of LCA, could represent a new strategy for evaluation of cholestatic potential of drugs and other chemicals.Keywords:
Bone canaliculus
Lithocholic acid
Chenodeoxycholic acid
Nine fecal samples from four healthy subjects were examined for their ability to transform chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) in in vitro anaerobic broth cultures.Seven samples converted CDCA and UDCA into each other (more than 50% of CDCA was converted into UDCA while 10% or less of UDCA was converted into CDCA), and produced 7-keto-lithocholic acid and lithocholic acid equally from both acids.No alteration of the 7&hydroxy group of UDCA was demonstrated by two fecal samples that failed to perform mutual 7epimerization, suggesting the conversion of UDCA into lithocholic acid via CDCA.The 3a-hydroxy groups of these substrate and metabolite bile acids were invariably partially epimerized to 3&hydroxy groups by all the fecal samples.Evidence is presented for the prevalence of these 7-and 3-epimerizing organisms among the human intestinal flora.
Lithocholic acid
Ursodeoxycholic acid
Chenodeoxycholic acid
Deoxycholic acid
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Lithocholic acid
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Lithocholic acid
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Metabolism of chenodeoxycholic acid, a therapeutic agent for gallstone dissolution, was examined in rats, hamsters, and rabbits. In the rat liver, chenodeoxycholic acid was found to be converted into taurochenodeoxycholate, a part of which was converted into tauromuricholate. In the rat colon, these conjugated bile acids were hydrolyzed into the corresponding free bile acids and a considerable part of the free chenodeoxycholic acid and muricholic acid were further metabolized to lithocholic acid and hyodeoxycholic acid, respectively, by the action of microorganisms. Main metabolites excreted in the rat feces were identified as muricholic acid, hyodeoxycholic acid, and lithocholic acid. Direct microbial conversion of muricholic acid into hyodeoxycholic acid was established by in vitro experiment in which muricholic acid was incubated with rat feces suspension. Lithocholic acid and its metabolite, 3α, 6β-dihydroxy-5β-cholanoic acid, were not found in the small intestine. It seems likely that lithocholic acid is poorly absorbed after its formation in the colon. In the hamster liver, chenodeoxycholic acid was converted into tauro- and glycochenodeoxycholates. In the hamster intestinal tract, these conjugated bile acids were deconjugated to form chenodeoxycholic acid, which was further metabolized to lithocholic acid. It was found by the double labeled tracer technique using chenodeoxycholic acid [7β-3H, 24-14C] that a considerable amount of lithocholic acid was reabsorbed from the hamster colon. The absorbed lithocholic acid was completely rehydroxylated to chenodeoxycholic acid. Thus, lithocholic acid was not circulated in the enterohepatic circulation. In the rabbit colon, chenodeoxycholic acid was metabolized to lithocholic acid, a part of which was reabsorbed and reached the liver. In contrast to the hamster, the absorbed lithocholic acid was not hydroxylated in the rabbit liver, and entered into the enterohepatic circulation.
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