Oxidative stress has been implicated as a mechanism for aging and some age-related diseases. It has been proposed that aging may be a result of a combination of imbalances between defense and damage, repair, and degradation of damaged molecules. We suspect a decline of the activities of """"""""antioxidant"""""""" enzymes may be partially responsible for aging. To test this hypothesis, normal human diploid fibroblasts of different ages (both male and female) and fibroblasts from patients with aging diseases (progeria and Werner's syndrome) were used. Confluent cells were harvested and protein fractions were used for enzymatic analyses. The enzymes examined include catalase, superoxide dismutase (SOD), GSH peroxidase (GPX), GSH reductase (GRX), GSH-s-transferase (GST), and glucose-6-phosphate dehydrogenase (G6PDH). In addition, protein carbonyl content, protein-bound sulfhydryls, and the cellular content of GSH/GSSG were examined. For the measurement of protein degradation, the neutral proteolytic activity was determined. There was no change in carbonyl content and activities of the enzymes as a function of population doubling; however, the activities of most enzymes and total protein sulfhydryls in fibroblasts from young donors were significantly higher than those from old donors (ranging from 26% to 72%), whereas total protein carbonyl content and activity of SOD were higher in fibroblasts from old donors. Total GSH level decreased in cells from old donors, whereas the percentage of GSSG increased. Similar results were obtained in cells from patients with aging diseases, suggesting these changes are associated with premature aging process. These results suggest a decrease in activities of various antioxidant enzymes may be part of a very complex mechanism of aging.
