Kim J A, Kang Y S, Lee S H, Lee Y S
College of Pharmacy, Yeungnam University, Kyongsan, Korea.
Free Radic Res. 2000 Sep;33(3):267-77. doi: 10.1080/10715760000301431.
Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca2+ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca2+ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 microM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2',7'-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N'-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca2+ concentration, which was completely prevented by the antioxidants. An intracellular Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca2+ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca2+ increase and induction of apoptosis were significantly inhibited by an extracellular Ca2+ chelator or Na+/Ca2+ exchanger blockers (bepridil and benzamil), whereas neither Ca2+ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca2+ through the activation of reverse mode of Na+/Ca2+ exchanger. However, tBOOH decreased intracellular Na+ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca2+ concentration by direct activation of the reverse mode of Na+/Ca2+ exchanger, rather than indirect elevation of intracellular Na+ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca2+ may mediate this action of tBOOH. These results further suggest that Na+/Ca2+ exchanger may be a target for the management of oxidative hepatotoxicity.
氧化应激似乎与包括肝毒性在内的各种疾病的发病机制有关。尽管细胞内Ca2+信号被认为在肝细胞的氧化损伤中起作用，但氧化剂诱导的细胞内Ca2+增加的来源和影响目前仍存在争议。因此，在本研究中，我们以HepG2人肝癌细胞作为模型肝细胞系统，研究了氧化剂诱导的肝细胞损伤的确切来源和机制。用200 microM叔丁基过氧化氢（tBOOH）处理可诱导细胞内活性氧中间体（ROI）水平持续升高和细胞凋亡，分别通过2',7'-二氯荧光素荧光和流式细胞术进行评估。抗氧化剂N-乙酰半胱氨酸（NAC）或N,N'-二苯基对苯二胺可显著抑制ROI生成和细胞凋亡。此外，tBOOH可诱导细胞内Ca2+浓度缓慢且持续升高，而抗氧化剂可完全阻止这种升高。细胞内Ca2+螯合剂双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸/羧甲基酯可显著抑制tBOOH诱导的细胞凋亡。这些结果表明，ROI增加触发的细胞内Ca2+信号激活可能介导tBOOH诱导的细胞凋亡。细胞外Ca2+螯合剂或Na+/Ca2+交换体阻滞剂（苄普地尔和苄amil）可显著抑制细胞内Ca2+增加和细胞凋亡诱导，而Ca2+通道拮抗剂（维拉帕米和硝苯地平）和非选择性阳离子通道阻滞剂（氟芬那酸）均无作用。这些结果表明，tBOOH可能通过激活Na+/Ca2+交换体的反向模式来增加细胞内Ca2+。然而，tBOOH可降低细胞内Na+浓度，而NAC可完全阻止这种降低。这些结果表明，tBOOH产生的ROI可能通过直接激活Na+/Ca2+交换体的反向模式来增加细胞内Ca2+浓度，而不是间接升高细胞内Na+水平。综上所述，这些结果表明，氧化剂tBOOH可诱导人HepG2细胞凋亡，细胞内Ca2+可能介导tBOOH的这种作用。这些结果进一步表明，Na+/Ca2+交换体可能是氧化肝毒性治疗的靶点。
