van de Water B, Zoeteweij J P, de Bont H J, Nagelkerke J F
Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.
Mol Pharmacol. 1995 Nov;48(5):928-37.
The role of complex II in the cellular protection against oxidative stress was investigated in freshly isolated rat renal proximal tubular cells (PTC) with the use of the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC caused oxidative stress in PTC as determined by flow cytometry with dihydrorhodamine-123; this fluorescent probe is readily oxidized by primary hydroperoxides such as those formed during lipid peroxidation. The oxidative stress could be prevented by inhibition of the beta-lyase-mediated formation and covalent binding to cellular macromolecules of reactive DCVC metabolites, with amino oxyacetic acid (AOA), or by the antioxidant N,N'-diphenyl-p-phenylenediamine. Both AOA and DPPD also prevented cell death. The DCVC-induced oxidative stress was associated with a decrease in the succinate:ubiquinone reductase (SQR) activity of complex II, whereas NADH:ubiquinone reductase activity of complex I remained unaffected. AOA prevented the effect on SQR activity, whereas N,N'-diphenyl-p-phenylenediamine did not. Inhibition of SQR activity with thenoyl trifluoracetone (TTFA) potentiated the DCVC-induced oxidative cell injury, suggesting the involvement of SQR activity in an antioxidant pathway. To investigate this in greater detail, PTC were treated with an inhibitor of cytochrome-c-oxidase, KCN, in a buffer containing glycine, which prevents cell death by KCN. Glycine did not affect cell death by DCVC. KCN prevented the DCVC-induced oxidative stress and cell death. KCN cytoprotection could be prevented by inhibition of SQR activity with oxaloacetate or TTFA, whereas inhibition of either complex I or III with rotenone and antimycin, respectively, did not prevent it. The effect of DCVC on complex II was associated with a decrease in the cellular amount of reduced ubiquinone (QH2); the KCN-mediated cytoprotection was related to a 60% increase of cellular QH2. Rotenone almost completely inhibited ubiquinone reduction even in the presence of KCN, whereas oxaloacetate in combination with KCN resulted in QH2 levels comparable to control. This suggests that the SQR activity by complex II rather than the cellular content of reduced ubiquinone (QH2) is important as a part of the cellular antioxidant machinery in the cyto-protection against oxidative stress.
利用肾毒素S-(1,2-二氯乙烯基)-L-半胱氨酸（DCVC），在新鲜分离的大鼠肾近端小管细胞（PTC）中研究了复合物II在细胞抗氧化应激中的作用。用二氢罗丹明-123通过流式细胞术测定，DCVC在PTC中引起了氧化应激；这种荧光探针很容易被初级氢过氧化物氧化，比如脂质过氧化过程中形成的那些氢过氧化物。通过用氨氧乙酸（AOA）抑制β-裂解酶介导的反应性DCVC代谢产物的形成及其与细胞大分子的共价结合，或者用抗氧化剂N,N'-二苯基对苯二胺，可以预防氧化应激。AOA和DPPD都还能预防细胞死亡。DCVC诱导的氧化应激与复合物II的琥珀酸：泛醌还原酶（SQR）活性降低有关，而复合物I的NADH：泛醌还原酶活性未受影响。AOA可预防对SQR活性的影响，而N,N'-二苯基对苯二胺则不能。用噻吩甲酰三氟丙酮（TTFA）抑制SQR活性会增强DCVC诱导的氧化细胞损伤，这表明SQR活性参与了抗氧化途径。为了更详细地研究这一点，在含有甘氨酸的缓冲液中用细胞色素c氧化酶抑制剂KCN处理PTC，甘氨酸可防止KCN导致的细胞死亡。甘氨酸不影响DCVC引起的细胞死亡。KCN可预防DCVC诱导的氧化应激和细胞死亡。用草酰乙酸或TTFA抑制SQR活性可阻止KCN的细胞保护作用，而分别用鱼藤酮和抗霉素抑制复合物I或III则不能阻止这种作用。DCVC对复合物II的影响与细胞中还原型泛醌（QH2）含量的降低有关；KCN介导的细胞保护作用与细胞QH2增加60%有关。即使在存在KCN的情况下，鱼藤酮也几乎完全抑制泛醌还原，而草酰乙酸与KCN联合使用可使QH2水平与对照相当。这表明，作为细胞抗氧化机制的一部分，复合物II的SQR活性而非还原型泛醌（QH2）的细胞含量在细胞保护免受氧化应激方面很重要。
