MITOCHONDRIAL DNA REPAIR PROCESSES IN OXIDATIVE STRESS AND AGING

点击翻译按钮获取中文摘要

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.