MITOCHONDRIAL DNA REPAIR PROCESSES IN OXIDATIVE STRESS AND AGING

工作总结：基于我们之前的发现，线粒体DNA糖基酶/内切酶（mtODE）的活性随着年龄的增长而增加，这是8-OH脱氧鸟苷（8-OHdG）的特异性，我们假设在慢性氧化应激反应中存在这种DNA修复酶的诱导。这个项目检验了这个假设。大鼠接受被描述为对肝脏线粒体DNA （mt-DNA）造成氧化损伤的方案，并测试线粒体提取物在含有单个8-OHdG的寡核苷酸的切口中的活性。AZT治疗6周没有引起mt-ODE活性的增加，但也没有增加TBARS（脂质过氧化产物）或超氧化物歧化酶（SOD）活性。在6周或15周的时间里，提供8%乙醇作为唯一的饮用水来源，据报道，这一过程可以提高肝脏mt-DNA 8-OHdG的含量，但相对于对照组，mt-ODE的活性也没有变化。再一次，TBARS和SOD活性没有变化，质疑氧化损伤的严重程度。因此，氧化应激在诱导该酶活性中的作用仍有待解决。然而，我们扩展并加强了我们最初的发现，即mt-ODE活性随着年龄的增长而增加。肝脏线粒体提取物的活性在6 ~ 23月龄之间呈极显著升高，在中间月龄（12月龄）达到最大值。在心脏线粒体的提取物中也发现了同样的模式。相比之下，另外两种DNA代谢的线粒体酶，即尿嘧啶DNA糖基化酶（mtUDG）和AP内切酶，并不特异性参与氧化损伤的修复，随着年龄的增长，它们要么保持不变，要么变化很小。随着年龄的增长，线粒体DNA修复的增加也出现在不同年龄的小鼠身上。这是令人惊讶的，可能会刺激新的方向，因为以前认为DNA修复能力随着年龄的增长而下降。最近的实验使用了来自年轻和年老大鼠的肝细胞的原代培养。我们已经开发了新的技术来测量线粒体中的DNA修复。利用细胞提取物，可以检测到受损DNA的切口和合并，因此我们现在可以更机械地研究衰老的变化。- DNA糖基化酶/核酸内切酶8-羟基脱氧鸟苷高效液相色谱法/EC醇

Summary of work: Based upon our previous finding that there is an increase with age in the activity of a mitochondrial DNA glycosylase/endonuclease (mtODE) which is specific for 8-OH deoxyguanosine (8-OHdG), we have hypothesized that there is an induction of this enzyme of DNA repair in response to chronic oxidative stress. This project tests this hypothesis. Rats were subjected to regimens described as causing oxidative damage to liver mitochondrial DNA (mt-DNA) and mitochondrial extracts were tested for activity in the incision of oligonucleotides containing a single 8-OHdG. Treatment with AZT for 6 weeks elicited no increase in mt-ODE activity, but also gave no increase in TBARS (a product of lipid peroxidation) or in superoxide dismutase (SOD) activity. Provision of 8% ethanol as the sole source of drinking water for periods of 6 weeks or 15 weeks, a procedure which has been reported to raise liver mt-DNA 8-OHdG content, also gave no change in mt-ODE activity relative to controls. Again, there was no change in TBARS or SOD activity, questioning the severity of the oxidative insult. Thus the role of oxidative stress in the induction of this enzyme activity remains to be resolved. However, we have extended and strengthened our original finding of an increase in mt-ODE activity with aging. In liver mitochondrial extracts, there was a highly significant increase in activity between 6 months and 23 months of age, with the activity being at a maximum at an intermediate age (12 months). The same pattern was seen in extracts of heart mitochondria. By contrast, two other mitochondrial enzymes of DNA metabolism which are not specifically involved in the repair of oxidative damage, viz. uracil DNA glycosylase (mtUDG) and AP endonuclease, were found to be either unchanged or minimally changed with aging. This increase in mitochondrial DNA repair with aging is also seen in mice of different ages. It is suprising and may stimulate new directions as it was previously thought that DNA repair capacity declined with age. Recent experiments have used primary cultures of hepatocytes from young and old rats. We have developed new techniques to measure DNA repair in mitochondria. Using cell extracts, incision and incorporation of damaged DNA can be detected, and we can thus now approach the studies on the changes with aging more mechanistically. - DNA glycosylase/endonuclease 8-hydroxydeoxyguanosine HPLC/EC alcohol