MITOCHONDRIAL DNA REPAIR PROCESSES IN OXIDATIVE STRESS AND AGING

工作总结：工作总结：根据 我们之前的发现是，随着年龄的增长，这一活动会增加 线粒体DNA糖基酶/核酸内切酶(MtODE) 专用于8-羟基脱氧鸟苷(8-OHdG)，我们有 假设存在DNA的这种酶的诱导 应对慢性氧化应激的修复。这个项目测试了这一点 假设。大鼠接受了被描述为引起 氧化损伤肝线粒体DNA(mt-DNA)和 线粒体提取液在切口处进行活性测试 含有单一8-OHdG的寡核苷酸。AZT治疗 连续6周没有引起MtODE活性增加，但也给出了 不增加总胆红素(一种脂质过氧化产物)或 超氧化物歧化酶(SOD)活性。提供8%的乙醇作为 为期6周或15周的唯一饮用水来源 周，据报道，这是一种提高肝脏mt-DNA的程序 8-OHdG含量，Mt-ODE活性相对含量也没有变化 到控制程序。同样，总胆红素和超氧化物歧化酶没有变化 活动，质疑氧化侮辱的严重性。因此， 氧化应激在该酶活性诱导中的作用 仍有待解决。然而，我们已经延长了和 证实了我们最初发现的Mt-ode活动增加 随着年龄的增长。在肝脏线粒体提取液中，有高度的 年6个月至23个月期间活动显著增加 年龄，活动量在中年达到最大 (12个月)。在心脏提取液中也发现了同样的模式 线粒体。相比之下，线粒体的另外两种酶 DNA新陈代谢不是专门参与修复的 氧化损伤，即。尿嘧啶DNA糖基酶与AP 内切酶，被发现要么没有变化，要么最低限度 随着年龄的增长而改变。这使人们相信存在着一种 衰老过程中的特异性诱导，可能是对 氧化应激。这个项目正在扩大，以衡量实际情况 短暂接触过的细胞的DNA修复率 光和亚甲基蓝，在线粒体中产生8-OHdG 基因组。到目前为止，实验使用了原代培养的 幼年和老年大鼠的肝细胞。然而，生存能力很强。 受亚甲基蓝等疗法影响引入 8-OHdG正在接受检查。

Summary of work: 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 lends credence to the idea that there is a specific induction of mt-ODE in aging, likely in response to oxidative stress. This project is being broadened to measure actual rates of DNA repair in cells which have been exposed briefly to light and methylene blue, to generate 8-OHdG in the mitochondrial genome. Experiments thus far have used primary cultures of hepatocytes from young and old rats. However, viability is much affected by methylene blue and other regimens for introducing 8-OHdG are being examined.