Chung Mi Ja, Walker Paul A, Brown Ray W, Hogstrand Christer
King's College London, Department of Life Sciences, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, UK.
Toxicol Appl Pharmacol. 2005 Jun 15;205(3):225-36. doi: 10.1016/j.taap.2004.10.008.
The ability of zinc to mobilize defense against reactive oxygen species (ROS) and H2O2-induced apoptosis was studied using a primary culture of rainbow trout gill cells. Gill cells were pretreated for 24 h with 100 microM ZnSO4 followed by 24-h exposure to 100 or 200 microM H2O2, or were subjected to 100 microM ZnSO4 together with 100 or 200 microM H2O2. Metallothionein-A (MTA) and metallothionein-B (MTB) mRNA levels were increased after treatment with zinc or H2O2, separately or in combination. Similarly, mRNA for glutathione S-transferase (GST) and glucose 6-phosphate dehydrogenase (G6PD) were increased in response to either zinc or H2O2, or after sequential treatments with zinc followed by H2O2. The stimulatory effects of zinc or H2O2 on MTA, MTB, GST, and G6PD mRNA levels could be blocked by addition of the membrane permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), suggesting that H2O2-induced upregulation of these genes is zinc-dependent. Pretreatment with zinc protected the cells from subsequent cell damage and apoptosis, as assessed by lactate dehydrogenase leakage, mitochondrial dehydrogenase activity (MTT assay), caspase-3 activity, and DNA fragmentation. In contrast, when gill cells were coincubated with zinc and H2O2 at the same time, H2O2 toxicity was higher than after treatment with H2O2 alone. It is concluded that zinc had a direct pro-oxidant effect when administered together with H2O2, but that pretreatment of zinc inhibited cytotoxicity and apoptosis through an indirect antioxidant action. We propose that the antioxidant action is manifested through zinc-dependent expression of several genes encoding antioxidant proteins (e.g., MTA, MTB, G6PD, and GST). Furthermore, the apparent zinc-dependency of H2O2-induced expression of antioxidant genes suggests that zinc might act as a physiological signal to mediate the response to oxidative stress.
利用虹鳟鱼鳃细胞原代培养研究了锌动员抵抗活性氧（ROS）和H2O2诱导凋亡的能力。鳃细胞先用100微摩尔ZnSO4预处理24小时，然后暴露于100或200微摩尔H2O2中24小时，或者与100或200微摩尔H2O2一起加入100微摩尔ZnSO4。单独或联合用锌或H2O2处理后，金属硫蛋白-A（MTA）和金属硫蛋白-B（MTB）的mRNA水平升高。同样，谷胱甘肽S-转移酶（GST）和葡萄糖6-磷酸脱氢酶（G6PD）的mRNA在锌或H2O2处理后，或锌后接H2O2的顺序处理后增加。锌或H2O2对MTA、MTB、GST和G6PD mRNA水平的刺激作用可通过添加膜通透性锌螯合剂N，N，N'，N'-四（2-吡啶甲基）乙二胺（TPEN）来阻断，这表明H2O2诱导的这些基因上调是锌依赖性的。如通过乳酸脱氢酶泄漏、线粒体脱氢酶活性（MTT法）、半胱天冬酶-3活性和DNA片段化所评估，锌预处理可保护细胞免受随后的细胞损伤和凋亡。相反，当鳃细胞同时与锌和H2O2共孵育时，H2O2毒性高于单独用H2O2处理后。得出的结论是，锌与H2O2一起给药时具有直接的促氧化作用，但锌预处理通过间接抗氧化作用抑制细胞毒性和凋亡。我们提出抗氧化作用通过编码抗氧化蛋白（如MTA、MTB、G6PD和GST）的几个基因的锌依赖性表达来体现。此外，H2O2诱导的抗氧化基因表达明显的锌依赖性表明锌可能作为一种生理信号来介导对氧化应激的反应。
