An in vitro drug-induced hepatotoxicity screening system using CYP3A4-expressing and γ-glutamylcysteine synthetase knockdown cells
An in vitro drug-induced hepatotoxicity screening system using CYP3A4-expressing and γ-glutamylcysteine synthetase knockdown cells
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DOI：
10.1016/j.tiv.2009.11.020
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发表时间：
2010-04-01
影响因子：
3.2
通讯作者：
Yokoi, Tsuyoshi
中科院分区：
文献类型：
--
作者：
Hosomi, Hiroko;Akai, Sho;Yokoi, Tsuyoshi
药物诱导的肝毒性通常是由CYP依赖性药物活化为反应性代谢产物引起的。CYP 3A 4是参与药物代谢活化的主要亚型之一。本研究构建了表达CYP 3A 4的腺病毒载体（AdCYP 3A 4）。在H4 IIE（大鼠肝癌）细胞中，AdCYP 3A 4感染3天后，睾酮6 β-羟化酶活性达到325 pmol/min/mg蛋白。为了敲低γ-谷氨酰半胱氨酸合成酶重链亚基（GCSh）并降低内源性谷胱甘肽（GSH）水平，我们使用了针对大鼠GCSh的短发夹RNA腺病毒载体（AdGCSh-shRNA）。AdGCSh-shRNA和AdCYP 3A 4同时感染H4 IIE细胞3天，细胞内GSH水平降低50-60%，而不影响CYP 3A 4的表达水平。使用这种对反应性代谢物的细胞毒性敏感的基于细胞的系统，对已知具有肝毒性的药物进行了评价。结果表明，与其他组（AdGFP、AdCYP 3A 4、AdGFP/AdGCSh-shRNA组）相比，曲格列酮、氟替卡松和对乙酰氨基酚导致AdCYP 3A 4/AdGCSh-shRNA组的细胞活力显著降低，表明由CYP 3A 4产生并随后与GSH缀合的反应性代谢物参与了细胞毒性。这些结果表明，这种基于细胞的测定系统表达CYP 3A 4与GCSh敲低将是有用的预测CYP 3A 4介导的细胞毒性在临床前药物开发。(C)2009爱思唯尔有限公司保留所有权利。
Drug-induced hepatotoxicity is often caused by CYP-dependent activation of the drugs into reactive metabolites. CYP3A4 is one of the main isoforms involved in the metabolic activation of drugs. In this study, an adenovirus vector expressing CYP3A4 (AdCYP3A4) was constructed. After 3 days infection of AdCYP3A4, the testosterone 6 beta-hydroxylase activity reached to 325 pmol/min/mg protein in H4IIE (rat hepatoma) cells. To knockdown the gamma-glutamylcysteine synthetase heavy chain subunit (GCSh) and decrease the intrinsic glutathione (GSH) level, we used an adenovirus vector with short hairpin RNA against rat GCSh (AdGCSh-shRNA). Three days infection of AdGCSh-shRNA and AdCYP3A4 simultaneously with H4IIE cells decreased the intracellular GSH level by 50-60% without affecting the expression level of CYP3A4. Using this cell-based system sensitive to the cytotoxicity of reactive metabolites, drugs known for their hepatotoxicity were evaluated. As a result, troglitazone, flutamide, and acetaminophen caused significant decreases of cell viability in AdCYP3A4/AdGCSh-shRNA group compared to the other groups (AdGFP, AdCYP3A4, AdGFP/AdGCSh-shRNA groups), indicating that reactive metabolite(s) produced by CYP3A4 and subsequently conjugated by GSH would be involved in the cytotoxicity. These results suggest that this cell-based assay system expressing CYP3A4 with GCSh knockdown would be useful for the prediction of CYP3A4-mediated cytotoxicity in preclinical drug development. (C) 2009 Elsevier Ltd. All rights reserved.