Knock down of γ-glutamylcysteine synthetase in rat causes acetaminophen-induced hepatotoxicity
Knock down of γ-glutamylcysteine synthetase in rat causes acetaminophen-induced hepatotoxicity
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DOI：
10.1074/jbc.m702819200
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发表时间：
2007-08-17
影响因子：
4.8
通讯作者：
Yokoi, Tsuyoshi
中科院分区：
文献类型：
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作者：
Akai, Sho;Hosomi, Hiroko;Yokoi, Tsuyoshi
药物诱导的肝毒性主要是由肝脏谷胱甘肽（GSH）耗竭引起的。一般来说，啮齿类动物谷胱甘肽S-转移酶的活性比人类高10至20倍，这可能使预测药物诱导的人类肝毒性更加困难。γ-谷氨酰半胱氨酸合成酶（gamma-GCS）主要调节哺乳动物细胞中GSH的从头合成，并且在细胞的抗氧化能力中起核心作用。在这项研究中，我们建立了一个GSH耗竭实验大鼠模型，用于预测人类肝毒性。构建了携带针对大鼠γ-GCS重链亚基（GCSh）短发夹状RNA的腺病毒载体（AdGCSh-shRNA），并将其用于GCSh基因的敲除。在大鼠肝癌细胞系H4 IIE细胞中的体外研究中，AdGCSh-shRNA感染后3天，GCSh mRNA和蛋白显著降低80%，GSH显著降低50%。在大鼠体内研究中，单次给予AdGCSh-shRNA（2 x 10（11）pfu/ml/body）后14天，肝脏GSH水平下降80%，并且这种消耗持续至少2周。使用这种GSH敲低大鼠模型，与正常大鼠相比，对乙酰氨基酚诱导的肝毒性显着增强。这是第一次报告的GSH敲低大鼠模型，这可能是有用的高灵敏度的急性和亚急性毒性试验的候选药物在临床前药物开发。
Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. gamma-Glutamylcysteine synthetase (gamma-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat gamma-GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 x 10(11) pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development.