title: Development of an optimized cytotoxicity assay system for CYP3A4-mediated metabolic activation via modified piggyBac transposition.
abstract: Drug-induced hepatotoxicity is often caused by cytochrome P450 (CYP)-dependent metabolism of drugs into reactive metabolites. Assessment of hepatotoxicity induced by bioactive compounds is hampered by low CYP expression within in vitro cell lines. To overcome this limitation, piggyBac transposition and monoclonal expansion were used to generate a HepG2 cell line with stable and homogenously high expression of CYP3A4, a prominent CYP isoform. Our studies demonstrate the generated line's constant CYP3A4 expression and activity for over 40 cell passages; to date, it has been in subculture for more than a year without addition of Puromycin. 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. Therefore, this HepG2 cell-based assay system may provide a suitable hepatic model for predicting CYP3A4-mediated hepatotoxicity during preclinical drug development.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China; Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Lizhen
authors.initials: L
authors.lastname: Huang
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Shuxiang
authors.initials: S
authors.lastname: Zou
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Jifeng
authors.initials: J
authors.lastname: Deng
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Tianming
authors.initials: T
authors.lastname: Dai
authors.affiliation: Department of Oncology, Huashan Hospital and Shanghai Medical College, Fudan University, Shanghai 200000, China.
authors.forename: Jingwei
authors.initials: J
authors.lastname: Jiang
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Jia
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Renke
authors.initials: R
authors.lastname: Dai
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China; Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China. Electronic address: hlzh2000518@163.com.
authors.forename: Shuilin
authors.initials: S
authors.lastname: Xie
mesh_terms: D000082:Acetaminophen / Q000633:toxicity; D065767:Activation, Metabolic; D001681:Biological Assay; D002470:Cell Survival / Q000187:drug effects; D002839:Chromans / Q000633:toxicity; D019343:Citric Acid / Q000633:toxicity; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements; D005688:Galactosamine / Q000633:toxicity; D056945:Hep G2 Cells; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics*; D045162:Thiazolidinediones / Q000633:toxicity; D000077288:Troglitazone
chemical_list: D002839:Chromans; D004251:DNA Transposable Elements; D009419:Nerve Tissue Proteins; C569369:PGBD1 protein, human; D045162:Thiazolidinediones; D019343:Citric Acid; D000082:Acetaminophen; D005688:Galactosamine; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D000077288:Troglitazone
keywords: CYP3A4; HepG2 cells; Hepatotoxicity; PiggyBac transposon