Generation of cytochrome P450 3A4-overexpressing HepG2 cell clones for standardization of hepatocellular testosterone 6β-hydroxylation activity

Abstract

Detoxification of xenobiotics including drugs is catalyzed by liver phase I and phase II enzymes. There are three main families of phase I cytochrome P450 (CYP450) monoxygenases that introduce polar groups on drugs. These phase I metabolites can then be further conjugated by transferases during phase II reaction. Liver biotransformation can also lead to toxic drug metabolites, the most common cause of drug failure during clinical investigation. CYP3A4 is considered to be the most important enzyme in drug metabolism. Drug development relies on the use of human liver cells in order to investigate drug metabolism and potential toxicity. With primary human liver cells as the gold standard several problems have to be solved, i.e. scarcity of functional human liver tissue, donor variation of CYP activity and rapid dedifferentiation processes during primary cell cultivation. These features make it difficult to use primary human liver cells as standard to measure CYP activity. To avoid problems with primary human liver cells, many attempts have been undertaken to establish liver carcinoma cell lines, non-transformed proliferating human liver cell systems and induced pluripotent stem cell-derived hepatocytes. Due to different problems with these surrogate systems, the one cell line that could be used as convenient standard cell system to benchmark CYP3A4 enzyme activity has not been established yet. Based on the widely used hepatocellular carcinoma cell line HepG2 and a lentiviral vector system, we generated cell clones for stable CYP3A4 overexpression. Here we present data on a new HepG2 cell clone (clone 9) showing higher than 10,000-fold overexpression of CYP3A4 compared to HepG2 parental cells. As measured by conversion of testosterone into 6 -hydroxytestosterone, we found an enzyme activity of about 600 pmol per minute per mg total cellular protein, which ranges at the upper end reported for primary human liver cells. This enzyme activity appeared to be kept stable in clone 9 cells, because there was no influence detectable when cells were treated with 5-azacytidine, a drug that interferes with epigenetic silencing processes. Prototypic CYP3A4 inducer rifampicin led to significant increase of CYP3A4 testosterone hydroxylase activity in HepG2 clone 9 cells. Altogether, HepG2 clone 9 strongly and stably overexpressed CYP3A4 leading to a physiological enzyme activity, which apparently was unaffected by epigenetic processes. Thus, HepG2 clone 9 could be a useful reference cell clone for CYP3A4 enzyme activity.