Bustamante J, Tovar-B A, Montero G, Boveris A
National Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina.
Arch Biochem Biophys. 1997 Jan 1;337(1):121-8. doi: 10.1006/abbi.1996.9754.
Methylprednisolone (glucocorticoid hormone, MPS), etoposide (epipodophyllotoxin inhibitor of a topoisomerase II), and thapsigargin (inhibitor of the endoplasmic reticular Ca2+-ATPase) were used as apoptosis-inducing agents in rat thymocytes. Early redox changes were determined during the early phase of induced apoptosis. The three agents induced apoptosis as assessed by DNA laddering after agarose gel electrophoresis and by quantitative DNA fragmentation. Intracellular H2O2 steadystate concentrations after 30 min of incubation were 40, 48, 25, and 75 nM for control and MPS-, etoposide-, and thapsigargin-treated thymocytes, respectively. After 30 min of MPS and thapsigargin exposure, increased DCFH oxidation was clear compared with control cells, but no increase in dichlorofluorescein (DCF) was observed in etoposide-treated thymocytes. DCF fluorescence correlated linearly with the intracellular H2O2 concentration after 30 min of incubation. The amounts of thiobarbituric acid-reactive substances produced after 3 h of incubation and expressed as pmol/mg protein were 105+/-23, 120+/-18, 350+/-17, and 98+/-24 pmol/mg protein for untreated and MPS-, thapsigargin-, and etoposide-treated thymocytes, respectively. Common and marked reductions in intracellular glutathione of 46, 73, 58, and 39% were observed after 2 h of incubation with MPS-, thapsigargin-, and etoposide-treated cells and in untreated cells, respectively. A simultaneous increase in oxidized glutathione, compared with untreated cells, was evident in MPS (66%) and was stronger in thapsigargin-exposed cells (250%). A 55% decrease in GSSG in etoposide-treated cells was found. It is concluded that redox changes occur during the early phase of induced apoptosis in rat thymocytes and are not always associated with an oxidative stress. Rather, this situation is closely related with the type of stimuli.
甲基强的松龙（糖皮质激素，MPS）、依托泊苷（一种拓扑异构酶II的表鬼臼毒素抑制剂）和毒胡萝卜素（内质网Ca2+ -ATP酶抑制剂）被用作大鼠胸腺细胞的凋亡诱导剂。在诱导凋亡的早期阶段测定早期氧化还原变化。通过琼脂糖凝胶电泳后的DNA梯状条带以及定量DNA片段化评估，这三种试剂均可诱导凋亡。孵育30分钟后，对照及经MPS、依托泊苷和毒胡萝卜素处理的胸腺细胞的细胞内H2O2稳态浓度分别为40、48、25和75 nM。暴露于MPS和毒胡萝卜素30分钟后，与对照细胞相比，二氯荧光素二乙酸酯（DCFH）氧化明显增加，但在依托泊苷处理的胸腺细胞中未观察到二氯荧光素（DCF）增加。孵育30分钟后，DCF荧光与细胞内H2O2浓度呈线性相关。孵育3小时后产生的硫代巴比妥酸反应性物质的量，以pmol/mg蛋白质表示，未处理及经MPS、毒胡萝卜素和依托泊苷处理的胸腺细胞分别为105±23、120±18、350±17和98±24 pmol/mg蛋白质。与未处理细胞相比，MPS处理（66%）和毒胡萝卜素处理（250%）的细胞中氧化型谷胱甘肽明显同时增加。在依托泊苷处理的细胞中发现谷胱甘肽二硫化物（GSSG）减少了55%。得出的结论是，在大鼠胸腺细胞诱导凋亡的早期阶段会发生氧化还原变化，且并不总是与氧化应激相关。相反，这种情况与刺激类型密切相关。
