Development of an optimized cytotoxicity assay system for CYP3A4-mediated metabolic activation via modified piggyBac transposition.
Development of an optimized cytotoxicity assay system for CYP3A4-mediated metabolic activation via modified piggyBac transposition.
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DOI：
10.1016/j.tiv.2015.12.008
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发表时间：
2016-04
期刊：
影响因子：
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通讯作者：
Lizhen Huang;Shuxiang Zou;Jifeng Deng;Tianming Dai;Jing-wei Jiang;Y. Jia;R. Dai;Shuilin Xie
中科院分区：
文献类型：
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作者：
Lizhen Huang;Shuxiang Zou;Jifeng Deng;Tianming Dai;Jing-wei Jiang;Y. Jia;R. Dai;Shuilin Xie
药物引起的肝毒性通常是由细胞色素 P450 (CYP) 依赖性药物代谢成反应性代谢物引起的。生物活性化合物诱导的肝毒性的评估受到体外细胞系内低 CYP 表达的阻碍。为了克服这一限制，使用piggyBac转座和单克隆扩增来生成HepG2细胞系，该细胞系稳定且均一地高表达CYP3A4（一种重要的CYP亚型）。我们的研究证明，所生成的细胞系在 40 多次细胞传代中具有恒定的 CYP3A4 表达和活性；迄今为止，它已经在不添加嘌呤霉素的情况下进行继代培养一年多了。该细胞系用于通过 MTT 测定评估两种生物活性（曲格列酮和对乙酰氨基酚）和两种非生物活性（柠檬酸盐和半乳糖胺）化合物的细胞毒性。用生物活性药物治疗后，细胞活力显着下降。此外，本研究中使用的细胞系对曲格列酮的毒性作用比之前报道的更敏感。因此，这种基于 HepG2 细胞的检测系统可以提供合适的肝脏模型，用于预测临床前药物开发过程中 CYP3A4 介导的肝毒性。
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,piggyBactransposition 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.