An in vitro drug-induced hepatotoxicity screening system using CYP3A4-expressing and γ-glutamylcysteine synthetase knockdown cells

Abstract

Drug-induced hepatotoxicity is often caused by CYP-dependent activation of the drugs into reactive metabolites. CYP3A4 is one of the main isoforms involved in the metabolic activation of drugs. In this study, an adenovirus vector expressing CYP3A4 (AdCYP3A4) was constructed. After 3 days infection of AdCYP3A4, the testosterone 6β-hydroxylase activity reached to 325 pmol/min/mg protein in H4IIE (rat hepatoma) cells. To knockdown the γ-glutamylcysteine synthetase heavy chain subunit (GCSh) and decrease the intrinsic glutathione (GSH) level, we used an adenovirus vector with short hairpin RNA against rat GCSh (AdGCSh-shRNA). Three days infection of AdGCSh-shRNA and AdCYP3A4 simultaneously with H4IIE cells decreased the intracellular GSH level by 50–60% without affecting the expression level of CYP3A4. Using this cell-based system sensitive to the cytotoxicity of reactive metabolites, drugs known for their hepatotoxicity were evaluated. As a result, troglitazone, flutamide, and acetaminophen caused significant decreases of cell viability in AdCYP3A4/AdGCSh-shRNA group compared to the other groups (AdGFP, AdCYP3A4, AdGFP/AdGCSh-shRNA groups), indicating that reactive metabolite(s) produced by CYP3A4 and subsequently conjugated by GSH would be involved in the cytotoxicity. These results suggest that this cell-based assay system expressing CYP3A4 with GCSh knockdown would be useful for the prediction of CYP3A4-mediated cytotoxicity in preclinical drug development.