We hypothesize that oxidative stress contributes to the aging process. The oxidative stress stems from reactive intermediates of oxygen's reductive metabolism during aerobic metabolism and from cell exposure to oxygen in excess of the small amount required for respiration. We have shown that oxygen modulates the growth and lifespan of human fibroblasts under physiologic tensions. We want to determine whether, and for what reason, old cells are more vulnerable than young to oxidative stress. Differences in cellular response to a progressive oxidative stress will be sought between: a) cells early and late in their cultural lifespan; b) cells obtained from the skin of young and old individuals; c) tissue homogenates from the skin of young and old individuals. Human fibroblasts will be grown at partial pressures of oxygen ranging from 3-720mm Hg. To measure response to oxidative stress we will note changes in rate of growth, number of cells at confluence, cultural lifespan, Cr51 release and spontaneous tissue autoxidizability. to determine the biochemical response to oxidative stress we will isolate newly synthesized proteins and mRNA induced in response to alteration in oxygen tension. We will determine if oxidative stress impairs the ability of old cells to induce antioxidant protective enzymes or maintain cellular reducing potential. We will determine if addition of liposomes containing either antioxidant protective enzymes or their inhibitors can modify the cytotoxicity of oxygen and the cultural lifespan. Monitoring oxygen consumption, we will measure metabolic rate in young and old cells; determine the influence of metabolic rate on cultural lifespan; and ascertain whethere cultural atmosphere affects metabolic rate. We will determine if incubated samples of human skin differ with age in their endogenous antioxidant protective capability, as measured by the rate of formation of thiobarbituric acid reaction products. Direct comparison of cells aged in vivo and in vitro with their younger counterparts with respect to their ability to counter the cytotoxic effects should directly test the hypothesis that oxidative stress contributes to aging. Knowledge gained from these studies will enhance our understanding of the biology of aging.
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