The hypothesis that some types of damage to DNA may be involved in the aging process will be investigated using a model system, adrenocortical cells in culture. Some of the damage to DNA in aging may result from the toxic side-effects of oxygen. Adrenocortical cells form a useful system for investigating the role of this oxidative damage in aging. Both human and bovine adrenocortical cells are well-studied systems for investigating cell culture senescence. Protocols to increase or decrease the amount of oxidative damage occurring in cultured adrenocortical cells will be developed, by changing the oxygen concentration at which the cells are grown; by modifying the glutathione content of the cells; by varying the tocopherol and selenium status of the cells, by depletion and selective readdition; by addition of other biological or synthetic antioxidants; by irradiation; and by addition of peroxides or superoxide-generating systems. The effectiveness of these protocols will be assessed by release of 45Ca; measurement of changes in superoxide dismutase, catalase, and glutathione peroxidase, the principal cellular enzymatic defenses against oxidative damage; assessment of cellular levels of ubiquinone, which is sensitive to oxidative damage; and fluorescence microscopic determination of lipofuscin, a measure of an oxidative damage product in the living cell. Using protocols that yield varying degrees of oxidative damage, the amount of damage to DNA will be measured, by unscheduled DNA synthesis, a measure of DNA repair; by poly(ADP-ribose) synthesis, a response to DNA damage; by sister chromatid exchange rate; and by frequency of chromosome aberrations. These assays measure viable cell responses to damage to DNA and have been shown to respond to oxidative damage. Such protocols will then be tested for effects on replicative potential (life span) and cloning efficiency, to determine if the replicative life span is associated with the level or type of oxidative damage incurred by the cells and with the level or type of DNA damage. Some of these protocols will be used to examine losses of differentiated functions during culture senescence. Functions to be measured are ACTH receptor content; ACTH- and PGE1-stimulated cyclic AMP production; mitogenic response to angiotensin II; restimulated levels of steroidogenic cytochrome P-450 enzymes (11-hydroxylase, cholesterol side-chain cleavage, 21-hydroxylase, and 17-hydroxylase); P-450 enzymes involved in benzo[a]pyrene metabolism; 3beta-hydroxysteroid dehydrogenase; and synthesis of prostaglandins. Differentiated functions may be affected directly by oxidative damage, or secondary to DNA damage.
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