Application of HepaSH cells for predicting cholestatic drug-induced liver injury via bile acid-dependent toxicity assay.

Cholestatic liver injury is primarily caused by the accumulation of bile acids (BA) in hepatocytes. Based on this mechanism, an in vitro evaluation system for BA-dependent toxicity (BAtox) has been previously developed, using cryopreserved primary human hepatocytes (PHHs) as standard hepatocytes; however, lot-to-lot variability hindered detailed mechanistic elucidation. Therefore, we evaluated the utility of consistently available HepaSH cells, fresh PHHs derived from humanized liver chimeric mice, for assessing BAtox. HepaSH cells exhibited BSEP and BA conjugation activity. These activities were inhibited by known inhibitors resulting in BA accumulation via multiple pathways. BAtox assays using 22 compounds previously tested in PHHs demonstrated that 9-14 compounds induced BAtox in HepaSH cells. Among them, cyclosporin A in the presence of BAs strongly induced LDH release, reaching 81.3%. We further investigated its mechanism of action focusing on BA accumulation, and identified four BAs (chenodeoxycholic acid, deoxycholic acid, and their glycine-conjugated forms) as key contributors to toxicity. Significant intracellular accumulation of these four BAs was observed in the presence of cyclosporin A. These findings demonstrate that HepaSH cells can detect BAtox induced by drug-mediated BA accumulation. This in vitro system is a valuable tool for preclinical hepatotoxicity assays during drug development.