Drug-induced liver injury (DILI) is a major cause of market withdrawal or drug -development discontinuation because of safety concerns. In this study, we focused on drug-induced cholestasis (DIC) to establish an in vitro cytotoxicity test system and analyze its sensitivity using two-dimensional (2-D) cultured HepaRG cells and 12 types of bile acids (BAs) present in the human serum. First, to detect the cytotoxicity associated with cholestasis effectively, non-toxic BA concentrations were investigated and determined to be 100-fold the human serum value (455 mu M total BAs). Next, the cytotoxicity of 31 compounds that can inhibit the bile acid export pump (BSEP) and were categorized as no-DILI-concern, less-DILI-concern, and most-DILI-concern was examined. None of the no-DILI-concern compounds yielded cytotoxicity, whereas almost all less-DILI-concern compounds (with the exception of sim-vastatin) and most-DILI-concern compounds (with the exception of bosentan) exhibited cytotoxicity. An investigation of the cause of cytotoxicity using H-3-taurocholic acid revealed that most-DILI-concern and less-DILI-concern compounds, but not no-DILI-concern compounds, triggered the accumulation of radioactivity in the cell lysates. Thus, the onset of cytotoxicity seemed to be associated with cholestasis. The established HepaRG cytotoxicity assessment system (sensitivity of 89%, specificity of 100%, and accuracy of 97%) was mostly superior to the Cgs/BSEP IC50 (> 0.1) assessment system (sensitivity of 83%, specificity of 100%, and accuracy of 72%). Therefore, the assay method using 2-D cultured HepaRG cells and 12 BAs established here can be widely applicable as a model for the in vitro potential assessment of DIC.
