Enhanced oxidative stress by L-ascorbic acid within cells challenged by hydrogen peroxide
Enhanced oxidative stress by L-ascorbic acid within cells challenged by hydrogen peroxide
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DOI：
10.3177/jnsv.51.398
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发表时间：
2005-12-01
影响因子：
1.6
通讯作者：
Nishikimi, M
中科院分区：
文献类型：
--
作者：
Inai, Y;Bi, WX;Nishikimi, M
已有大量文献证明，细胞培养上清液中加入L抗坏血酸可增加氧化损伤，而L抗坏血酸的这种作用归因于细胞自氧化过程中产生的活性氧中间体。我们在这里质疑这种效应是否也在细胞内施加，如果是的话，其机制是什么。为了评估细胞中的硫醇氧化，我们操纵CHO细胞，使其能够在细胞质中表达细菌碱性磷酸酶。当细胞暴露在过氧化氢中时，需要形成分子内二硫键的碱性磷酸酶活性被证明是出现的。当L抗坏血酸与L抗坏血酸-2-O-磷酸孵育后，这种H_2O_2诱导的活性增加了1.5倍以上。通过评估谷胱甘肽的氧化、蛋白质羰基的形成和活性氧中间体的生成，也观察到了类似的增强作用。有趣的是，L-抗坏血酸-2-O-磷酸处理的作用被膜通透性螯合剂去铁胺完全抑制，表明铁离子参与了这一过程。因为刀豆蛋白的载脂蛋白与铁离子具有很高的亲和力。没有影响，因为在没有添加金属盐的平衡盐溶液中孵育的细胞观察到了相同的去铁胺效应。结果表明，L抗坏血酸作为氧化应激细胞内的促氧化剂，其作用是通过促进铁的氧化还原循环与L抗坏血酸结合而实现的。
It has been amply documented that L-ascorbic acid added to the medium of a cell culture increases oxidative damage, and this effect of L-ascorbic acid has been ascribed to the generation of reactive oxygen intermediates in the medium during its auto-oxidation. We have here questioned whether such an effect is exerted inside the cell as well, and if so, what its mechanism is. To assess thiol oxidation in the cell, we manipulated CHO cells so that they could express bacterial alkaline phosphatase in the cytoplasm. Alkaline phosphatase activity, which requires the formation of intramolecular disulfide bridges, was shown to appear when the cells were exposed to H2O2. This H2O2-induced activity increased more than 1.5 fold when L-ascorbic acid had been loaded in the cells by incubation with L-ascorbic acid-2-O-phosphate. Similar enhancing effects were also observed by assessing oxidation of glutathione, formation of protein carbonyls, and generation of reactive oxygen intermediates. Interestingly, the effects by the L-ascorbic acid-2-O-phosphate treatment were totally suppressed by addition of the membrane-permeable chelator deferoxamine to the medium, indicating the involvement of iron ions. Because the apoprotein of conalbumin, which binds iron ions with a high affinity. had no effect and because the same deferoxamine effect was observed with the cells incubated in balanced salt solution with no metal salts added. it was concluded that L-ascorbic acid acts as a pro-oxidant within the cell suffering oxidative stress, and that this effect is elicited through increased redox-cycling of iron in combination with L-ascorbic acid.