Mechanisms of chloroform-induced hepatotoxicity: Oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes
Mechanisms of chloroform-induced hepatotoxicity: Oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes
复制标题
DOI：
10.1080/15287390701551399
复制
发表时间：
2007-01-01
影响因子：
2.6
通讯作者：
Hinson, Jack A.
中科院分区：
文献类型：
--
作者：
Burke, Angela S.;Redeker, Kelly;Hinson, Jack A.
在新鲜分离的雌性 B6C3F1 小鼠肝细胞中测定了线粒体通透性转变 (MPT) 和氧化应激在氯仿毒性中的作用。氯仿 (12 mM) 与肝细胞一起孵育导致细胞死亡（丙氨酸转氨酶释放和碘化丙啶荧光）。氯仿在培养过程中挥发，谷胱甘肽在 1 小时内耗尽；然而，对照细胞和氯仿孵育细胞之间的毒性没有显着差异。洗涤肝细胞并在新鲜培养基中重新孵育1小时。随后重新孵育氯仿处理的肝细胞在 3-5 小时内产生显着的毒性。在再培养培养基中加入 MPT 抑制剂环孢菌素 A 或抗氧化剂 N-乙酰半胱氨酸 (NAC) 1 小时可防止毒性。使用染料钙黄绿素 AM 进行的共聚焦显微镜研究表明 MPT 被环孢菌素 A 或 NAC 阻断。利用 JC-1 的荧光显微镜研究表明线粒体膜电位丧失，这也被环孢素 A 或 NAC 阻断。在再孵育阶段二氯荧光素荧光增加，表明氧化应激增加，并且这种增加被环孢素A阻断。由于过氧亚硝酸盐可能发生氧化应激，因此检查了其在毒性中的作用。一氧化氮合酶抑制剂 N-G-甲基-L-精氨酸 (L-NMMA) 和 7-硝基吲唑 (7-NI) 在 1 小时后可阻断毒性。对肝细胞蛋白质中 3-硝基酪氨酸（过氧亚硝酸盐的生物标志物）进行的蛋白质印迹分析表明，有一种主要的硝化蛋白质，分子量为 81 kD。该蛋白质的硝化可被环孢菌素 A、L-NMMA、7-NI 或 NAC 抑制。数据表明，氯仿诱导的细胞死亡发生在两个阶段：以谷胱甘肽消耗为特征的代谢阶段，以及以MPT和蛋白质硝化为特征的氧化阶段。
The role of mitochondrial permeability transition (MPT) and oxidative stress in chloroform toxicity was determined in freshly isolated female B6C3F1 mouse hepatocytes. Incubation of chloroform ( 12 mM) with hepatocytes resulted in cell death ( alanine aminotransferase release and propidium iodide fluorescence). Chloroform had volatilized from the incubation and glutathione was depleted by 1 h; however, toxicity was not significantly different between control and chloroform-incubated cells. Hepatocytes were washed and reincubated in fresh media at 1 h. Subsequent reincubation of chloroform-treated hepatocytes resulted in significant toxicity at 3-5 h. Inclusion of the MPT inhibitor cyclosporine A or the antioxidant N-acetylcysteine (NAC) in the reincubation media at 1 h prevented toxicity. Confocal microscopy studies with the dye calcein AM indicated MPT that was blocked by cyclosporine A or NAC. Fluorescence microscopy studies utilizing JC-1 indicated loss of mitochondrial membrane potential, which was also blocked by cyclosporine A or NAC. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress, and the increase was blocked by cyclosporine A. Since oxidative stress may occur by peroxynitrite, its role in toxicity was examined. Either of the nitric oxide synthase inhibitors N-G-methyl-L-arginine (L-NMMA) and 7-nitroindazole (7-NI) at 1 h blocked toxicity. Western blot analysis of hepatocytes for 3-nitrotyrosine in proteins, a biomarker of peroxynitrite, indicated one major nitrated protein at 81 kD. Nitration of this protein was inhibited by cyclosporine A, L-NMMA, 7-NI, or NAC. The data indicate that chloroform-induced cell death occurs in two phases: a metabolic phase characterized by glutathione depletion, and an oxidative phase characterized by MPT and protein nitration.