Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System.

HepG2 cells are an inexpensive hepatocyte model that can be used for repeated experiments, but HepG2 cells do not express major cytochrome P450s (CYPs) and UDP glucuronosyltransferase family 1 member A1 (UGT1A1). In this study, we established CYP3A4-POR-UGT1A1-CYP1A2-CYP2C19-CYP2C9-CYP2D6 (CYPs-UGT1A1) knock-in (KI)-HepG2 cells using a PITCh system to evaluate whether they could be a new hepatocyte model for pharmaceutical studies. To evaluate whether CYPs-UGT1A1 KI-HepG2 cells express and function with CYPs and UGT1A1, gene expression levels of CYPs and UGT1A1 were analyzed by using real-time PCR, and metabolites of CYPs or UGT1A1 substrates were quantified by HPLC. The expression levels of CYPs and UGT1A1 in the CYPs-UGT1A1 KI-HepG2 cells were comparable to those in primary human hepatocytes (PHHs) cultured for 48 h. The CYPs and UGT1A1 activity levels in the CYPs-UGT1A1 KI-HepG2 cells were much higher than those in the wild-type (WT)-HepG2 cells. These results suggest that the CYPs-UGT1A1 KI-HepG2 cells expressed functional CYPs and UGT1A1. We also confirmed that the CYPs-UGT1A1 KI-HepG2 cells were more sensitive to drug-induced liver toxicity than the WT-HepG2 cells. CYPs-UGT1A1 KI-HepG2 cells could be used to predict drug metabolism and drug-induced liver toxicity, and they promise to be a helpful new hepatocyte model for drug discovery research.