Guidarelli A, Brambilla L, Clementi E, Sciorati C, Cantoni O
Istituto di Farmacologia e Farmacognosia, Università di Urbino, Italy.
Exp Cell Res. 1997 Nov 25;237(1):176-85. doi: 10.1006/excr.1997.3779.
The NADH-linked substrates pyruvate, L-glutamine, and beta-hydroxybutyrate, while enhancing the rate of oxygen consumption, also increased the formation of DNA single-strand breaks induced by tert-butylhydroperoxide in intact U937 cells. A cause-effect relationship between these two parameters was established by showing that: (a) rotenone, an inhibitor of complex I, abolished respiration and prevented the enhancement of the DNA-damaging response under all the above circumstances; (b) the membrane-impermeant, complex I-activating substrate L-malate gave similar results in permeabilized cells; and (c) none of the NADH-linked substrates affected the DNA-damaging response to tert-butylhydroperoxide in respiration-deficient cells. Stimulation of electron transport potentiated the DNA-cleaving ability of tert-butylhydroperoxide via a process involving enforced mitochondrial calcium accumulation in the absence of a discernible elevation in the cytosolic concentration of free Ca2+. Finally, mitochondrial calcium was found to promote the mitochondrial formation of DNA-damaging levels of hydrogen peroxide. In conclusion, the data herein presented define a previously unexpected role of respiratory substrates in the control of the deleterious effects of an organic hydroperoxide at the level of genomic DNA. The enhanced DNA cleavage mediated by NADH-linked substrates in response to tert-butylhydroperoxide would appear to depend on a sequence of events involving stimulation of electron transport, mitochondrial accumulation of Ca2+, and mitochondrial formation of DNA-damaging levels of hydrogen peroxide via a Ca(2+)-dependent process.
与NADH相关的底物丙酮酸、L-谷氨酰胺和β-羟基丁酸，在提高氧气消耗速率的同时，也增加了叔丁基过氧化氢在完整U937细胞中诱导的DNA单链断裂的形成。通过以下实验建立了这两个参数之间的因果关系：（a）复合物I的抑制剂鱼藤酮消除了呼吸作用，并在上述所有情况下阻止了DNA损伤反应的增强；（b）膜不透性的、激活复合物I的底物L-苹果酸在透化细胞中产生了类似的结果；（c）在呼吸缺陷细胞中，没有一种与NADH相关的底物影响对叔丁基过氧化氢的DNA损伤反应。电子传递的刺激通过一个过程增强了叔丁基过氧化氢的DNA切割能力，该过程涉及在细胞质游离Ca2+浓度没有明显升高的情况下，线粒体钙的强制积累。最后，发现线粒体钙促进了DNA损伤水平的过氧化氢的线粒体形成。总之，本文提供的数据定义了呼吸底物在基因组DNA水平上控制有机过氧化氢有害作用方面一个以前未被预料到的作用。由与NADH相关的底物介导的对叔丁基过氧化氢增强的DNA切割作用似乎依赖于一系列事件，包括电子传递的刺激、Ca2+的线粒体积累以及通过Ca(2+)依赖性过程形成DNA损伤水平的过氧化氢的线粒体形成。
