Establishment of knockdown of superoxide dismutase 2 and expression of CYP3A4 cell system to evaluate drug-induced cytotoxicity
Establishment of knockdown of superoxide dismutase 2 and expression of CYP3A4 cell system to evaluate drug-induced cytotoxicity
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DOI：
10.1016/j.tiv.2009.05.024
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发表时间：
2009-09-01
影响因子：
3.2
通讯作者：
Yokoi, Tsuyoshi
中科院分区：
文献类型：
--
作者：
Yoshikawa, Yukitaka;Hosomi, Hiroko;Yokoi, Tsuyoshi
药物引起的肝毒性是药物开发中的主要问题，氧化应激被认为是其原因之一。超氧化物歧化酶（SOD）是重要的抗活性氧自由基（ROS）的酶。线粒体是超氧化物产生的主要来源，SOD 2主要定位于线粒体中，并与其他SOD一起在清除超氧化物中起重要作用。在本研究中，我们建立了SOD 2敲低细胞。构建了针对大鼠SOD 2的短发夹状RNA腺病毒载体（AdSOD 2-shRNA），感染大鼠肝BRL 3A细胞后，3d后SOD 2 mRNA和蛋白表达水平显著下降60%，活性下降50%。我们先前构建了表达细胞色素P450 3A 4的腺病毒（AdCYP 3A 4）。将AdSOD 2-shRNA和AdCYP 3A 4共感染BRL 3A细胞，观察其对超氧阴离子和CYP 3A 4介导的活性代谢产物形成的影响，并检测细胞线粒体毒性、ROS和超氧阴离子自由基的产生以及脂质过氧化作用。阿苯达唑、卡马西平、氨苯砜、氟替卡松、异烟肼、硝苯地平、磺胺甲恶唑、曲唑酮、曲格列酮和齐多夫定表现出SOD 2和CYP 3A 4介导的细胞毒性显著增加。总之，我们构建了一个高度敏感的细胞系统，以评估氧化应激和CYP 3A 4介导的细胞毒性，可能是有用的临床前药物开发。(c)2009爱思唯尔有限公司保留所有权利。
Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. In this study, we established SOD2-knockdown cells. An adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA) was constructed, and infection of AdSOD2-shRNA to rat hepatic BRL3A cells resulted in significant decreases of SOD2 mRNA and protein by 60%, and SOD2 activity by 50% after 3 days infection. We previously constructed an adenovirus expressing cytochrome P450 3A4 (AdCYP3A4). Co-infection of AdSOD2-shRNA and AdCYP3A4 to BRL3A cells was carried out to evaluate the superoxide and CYP3A4-mediated formation of active metabolites, and mitochondrial toxicity, ROS and superoxide radical production and lipid peroxidation were selected to assess the cell viability. Albendazole, carbamazepine, dapsone, flutamide, isoniazid, nifedipine, sulfamethoxazole, trazodone, troglitazone, and zidovudine demonstrated significant increases of SOD2- and CYP3A4-mediated cytotoxicity. In conclusion, we constructed a highly sensitive cell system to evaluate oxidative stress and CYP3A4 mediated cytotoxicity that could be useful in preclinical drug development. (c) 2009 Elsevier Ltd. All rights reserved.