Abstract
We evaluated three kinds of human hepatocytes (HepG2 cells, HepaRG cells and primary human hepatocytes (PHH)) as materials for metabolite-induced hepatotoxicity testing, and developed assay methods for that. First, we conducted cytotoxicity tests in HepG2 cells, HepaRG cells and PHH using ATP as a cytotoxicity parameter. The three kinds of hepatocytes were treated with 16 compounds whose metabolites have been reported to be hepatotoxic. In order to judge whether observed cytotoxicities are caused by metabolites or parent compounds, cytochrome P450 inhibitor (1-aminobenzotriazole: ABT) or glutathione synthesis inhibitor (L-Buthionine-S, R-sulfoximine: BSO) was treated concomitantly with each compound. As results, toxicity of ticlopidine was reduced with ABT and that of cyclophosphamide was increased with BSO in HepaRG cells and PHH, but not in HepG2. These results indicate that the cytotoxicity tests using HepaRG cells and PHH could detect metabolite-induced toxicities, meanwhile it is considered that the assay method should be modified to improve detection sensitivity. To find indicators with higher sensitivity, we conducted comprehensive gene expression analysis in HepG2 and HepaRG cells treated with ticlopidine, cyclophosphamide and acetaminophen. As a result, we identified three gene biomarkers, heme oxigenase 1, p62 and sulfiredoxin 1. Changes in their expression levels were greater than those in ATP. The assay using human hepatocytes and these gene markers is useful for detection of metabolite-induced hepatotoxicity.
