I hypothesize that oxidative stress contributes to the aging process. The oxidative stress stems from reactive intermediates of oxygen's reductive metabolism produced during aerobic metabolism and from cell exposure to oxygen in excess of the small amount required for respiration. I want to determine whether, and for what reason, old cells are more vulnerable to oxidative stress than young cells. Differences in cellular response to a progressive oxidative stress will be sought between: a) cells early in their cultural lifespan with those late in cultural lifespan; b) cells obtained from the skin of young individuals with those obtained from old individuals; c) tissue homogenates from the skin of young individuals with those from old. Human fibroblasts will be grown at various different partial pressures of oxygen, ranging from 3-720mm Hg. Response to oxidative stress will be measured by noting changes in rate of growth, number of cells at confluence, cultural lifespan, Cr51 release and spontaneous tissue autoxidizability. We will measure the proliferative lifespan of fibroblasts in culture when cells are serially subcultivated at low inoculation densities and low oxygen tensions. I will determine if old cells are impaired in inducing antioxidant protective enzymes or maintaining cellular reducing potential, when faced with an oxidative stress. I will determine if the cytotoxicity of oxygen and the cultural lifespan can be modified by addition of liposomes containing either antioxidant protective enzymes or their inhibitors. Monitoring oxygen consumption continually, I will measure metabolic rate in young and old cells; determine the influence of metabolic rate on cultural lifespan; and ascertain whether cultural atmosphere affects metabolic rate. I 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 of oxygen 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.
