Giulivi C, Cadenas E
Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles 90033, USA.
Biochim Biophys Acta. 1998 Sep 7;1366(3):265-74. doi: 10.1016/s0005-2728(98)00125-x.
The objective of this study was to elucidate the role of mitochondrial GSH in the reactions leading to mitochondrial DNA oxidative damage in terms of 8-hydroxy-desoxyguanosine (8-HOdG) accumulation. With this purpose, tightly coupled mitochondria depleted of matrix GSH were used and the effects of H2O2 (generated during the oxidation of substrates) on 8-HOdG levels were investigated. Mitochondrial integrity, assessed by O2 uptake, respiratory control and P/O ratios, was conserved upon depletion of GSH up to 95%. The rates of H2O2 production linked to the oxidation of endogenous substrates by control and GSH-depleted mitochondria were similar. Succinate (in the absence or presence of antimycin A) enhanced the rate H2O2 production to a similar extent in both control and GSH-depleted mitochondria. These rates of H2O2 production accounted for 1.5-2.5% of the rate of O2 uptake. The levels of 8-HOdG in GSH-depleted mitochondria were 35-50% lower than those in control mitochondria, when measured at different H2O2 production rates. Conversely, in experiments carried out with calf thymus DNA with different Cu/Fe content, GSH increased 1.4-2.4-fold the accumulation of 8-HOdG. These values were further enhanced (44-50%) by superoxide dismutase and decreased by catalase. The lower levels of 8-HOdG in GSH-depleted mitochondria and the higher levels in GSH-supplemented calf thymus DNA suggest a role for the non-protein thiol in the reactions leading to mtDNA oxidative damage. These findings are interpreted in terms of the redox transitions involving O2, GSH, and metal catalysts bound to DNA. A mechanism is proposed by which GSH plays a critical role in the reduction of DNA-Cu complexes and decays by free radical pathways kinetically regulated by superoxide dismutase.
本研究的目的是根据8-羟基脱氧鸟苷（8-HOdG）的积累情况，阐明线粒体谷胱甘肽（GSH）在导致线粒体DNA氧化损伤的反应中的作用。为此，使用了基质GSH耗尽的紧密偶联线粒体，并研究了H2O2（在底物氧化过程中产生）对8-HOdG水平的影响。通过氧气摄取、呼吸控制和P/O比值评估的线粒体完整性，在GSH耗尽高达95%时仍得以保持。对照线粒体和GSH耗尽的线粒体将内源性底物氧化所产生的H2O2速率相似。琥珀酸（在存在或不存在抗霉素A的情况下）在对照线粒体和GSH耗尽的线粒体中均以相似的程度提高了H2O2的产生速率。这些H2O2产生速率占氧气摄取速率的1.5 - 2.5%。在不同的H2O2产生速率下进行测量时，GSH耗尽的线粒体中8-HOdG的水平比对照线粒体中的低35 - 50%。相反，在使用具有不同铜/铁含量的小牛胸腺DNA进行的实验中，GSH使8-HOdG的积累增加了1.4 - 2.4倍。超氧化物歧化酶进一步提高了这些值（44 - 50%），而过氧化氢酶则使其降低。GSH耗尽的线粒体中8-HOdG水平较低，而GSH补充的小牛胸腺DNA中水平较高，这表明非蛋白质硫醇在导致线粒体DNA氧化损伤的反应中发挥了作用。这些发现是根据涉及氧气、GSH和与DNA结合的金属催化剂的氧化还原转变来解释的。提出了一种机制，通过该机制GSH在DNA - 铜复合物的还原中起关键作用，并通过超氧化物歧化酶动力学调节的自由基途径衰减。
