Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes:: Protection by N-acetyl cysteine
Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes:: Protection by N-acetyl cysteine
复制标题
DOI：
10.1093/toxsci/kfh151
复制
发表时间：
2004-08-01
影响因子：
3.8
通讯作者：
Jaeschke, H
中科院分区：
文献类型：
--
作者：
Bajt, ML;Knight, TR;Jaeschke, H
对乙酰氨基酚（AAP）过量后细胞和线粒体谷胱甘肽二硫化物（GSSG）水平和GSSG：GSH比值的增加表明氧化应激参与了病理生理学。然而，肝细胞谷胱甘肽的初始严重耗竭使得难以定量评估氧化应激。因此，我们使用2 '，7'-二氯荧光素（DCF）荧光作为细胞内氧化应激的标志物，在细胞培养模型中测试了氧化应激先于细胞损伤发作的假设。将培养的原代鼠肝细胞暴露于5 mM AAP。从0到12 h测定DCF荧光、XTT还原、乳酸脱氢酶（LDH）释放和台盼蓝摄取。在3小时谷胱甘肽耗尽后，DCF荧光增加了16倍，并保持在该水平长达12小时。AAP后1.5 h，细胞XTT还原能力显著下降，并持续下降至9 h。在6 h时，在20%的细胞中可检测到细胞坏死（LDH释放，台盼蓝摄取），在随后的时间点进一步显著增加。AAP前1 h用20 mM N-乙酰半胱氨酸（NAC）预处理可增强细胞谷胱甘肽含量，分别防止或减弱AAP诱导的GSH水平和XTT还原能力下降，并减少细胞活力丧失。此外，在AAP暴露后2小时用NAC处理防止了在3小时及以后XTT减少的进一步恶化，并减弱了细胞坏死。因此，AAP诱导的氧化应激先于细胞坏死，并且在培养的肝细胞中，氧化应激参与细胞损伤的传播。
The increase in cellular and mitochondrial glutathione disulfide (GSSG) levels and the GSSG:GSH ratio after acetaminophen (AAP) overdose suggest the involvement of an oxidant stress in the pathophysiology. However, the initial severe depletion of hepatocellular glutathione makes quantitative assessment of the oxidant stress difficult. Therefore, we tested the hypothesis that oxidant stress precedes the onset of cell injury in a cell culture model using 2',7'-dichlorofluorescein (DCF) fluorescence as a marker for intracellular oxidant stress. Cultured primary murine hepatocytes were exposed to 5 mM AAP. DCF fluorescence, XTT reduction, lactate dehydrogenase (LDH) release, and trypan blue uptake were determined from 0 to 12 h. After glutathione depletion at 3 h, DCF fluorescence increased by 16-fold and was maintained at that level up to 12 h. At 1.5 h after AAP, a significant decrease of the cellular XTT reduction capacity was observed, which continued to decline until 9 h. Cell necrosis (LDH release, trypan blue uptake) was detectable in 20% of cells at 6 h, with a significant further increase at later time points. Pretreatment with 20 mM N-acetylcysteine (NAC) 1 h before AAP enhanced cellular glutathione content, prevented or attenuated the AAP-induced decrease of GSH levels and XTT reduction capacity, respectively, and reduced the loss of cell viability. Additionally, treatment with NAC 2 h after AAP exposure prevented further deterioration of XTT reduction at 3 h and later, and attenuated cell necrosis. Thus, AAP-induced oxidant stress precedes cell necrosis and, in cultured hepatocytes, the oxidant stress is involved in the propagation of cell injury.