Evaluation Of The Long-term Stable Expression Of CYP3A4 Via Modified PiggyBac Transposition And Drug-induced Hepatotoxicity In Transgenic Hepatic Cell Line

It’s important to assess drug-induced liver injury in drug development and clinical medication.Primary hepatocytes are considered as the “gold standard” in performing in vitro studies on hepatic metabolism,but its application is often limited by scarcity,phenotypic instability,significant variability between human hepatocytes from different donors and numerous other reasons.Hepatic cancer cells derived from liver tissue cannot predict or assess drug-induced liver injury precisely due to low expression of drug metabolizing enzymes.To overcome this limitation,the present study used PiggyBac transposon system to develop transgenic HepG2 cell lines stably and efficiently expressing CYP3A4.Via PiggyBac transposon,the encoding gene of CYP3A4 was successfully transferred into HepG2 cells.After resistance selection,flow cytometry sorting and limiting dilution,20 monoclonal transgenic cells were obtained by cloning-cylinders technology.Three monoclonal transgenic cells exhibiting high purity were selected for subsequent real-time PCR,western blot and enzyme activity analyses,and the transgenic cells showed significantly increase of CYP3A4 expression and metabolic activity compared with wild-type HepG2 cells.HepG/PB3A4.10,which has the highest mRNA expression level of CYP3A4 among the three monoclonal transgenic cells,was subculture continuously for studying the long-term stability of CYP3A4 expression.The results showed that the generated line’s CYP3A4 expression and activity kept constant for over 40 cell passages.This cell line was utilized to evaluate cytotoxicity of two bioactive(troglitazone and acetaminophen)and two non-bioactive(citrate and galactosamine)compounds by MTT assay.Cell viability significantly decreased upon treatment with bioactive drugs.Moreover,cell lines used in the present study were more sensitive to toxic effects of troglitazone than previously reported.In conclusion,the present study successfully and quickly developed a transgenic HepG2 cell line stably expressing CYP3A4 using PB transposon system,and the genetically modified cell line is highly sensitive to CYP3A4-mediated drug hepatotoxicity.This cell line may provide a suitable in vitro model to screen potential CYP3A4-mediated hepatotoxicity in preclinical drug development.Furthermore,the PB transposon system used in this study suggests an effective way to develop in vitro cell models stably expressing two or more P450 enzymes when studying drug-induced liver injury.