The long term objective for this present investigation is to study the regulation of thermal cytotoxicity by glutathione (GSH). Glutathione is a ubiquitous peptide located intra and extra cellularly. GSH is involved in numerous metabolic processes. Recently, it has been shown that mammalian cells depleted of GSH become thermally sensitive while thermotolerant cells have excess levels of GSH. It therefore appears to be important in the process of cellular inactivation by hyperthermia, yet little is known about how GSH regulates thermal sensitivity.
The specific aims and the methodology to be used are as follows: 1) Determine if GSH modification of thermal cytotoxicity is independent of oxygen tension. Cells will be heated in the absence and presence of buthionine sulfoximine (BSO) and under hypoxic or oxic conditions. 2) If oxygen concentration is important, then determine if GSH is needed to prevent heat induced lipid peroxidation. Lipid peroxidation products will be assayed for as a function of temperature and in cells heated BSO. 3) If oxygen is not important, then determine if GSH depletion alters the concentration of GSH mixed disulfides and consequently modifies survival. Release of GSH from mixed disulfides will be assayed for as a function of temperature and BSO. 4) Determine if the same mechanism of thermal cytotoxicity prevails in the absence and presence of GSH. An Arrhenius curve will be constructed BSO. 5) Determine if inhibition of the enzyme GSH reductase via inhibition of glucose metabolism affects survival. Various exogenous concentrations of glucose will be used to modify the enzyme's activity via NADPH. The changes in activity will be correlated with survival. 6) Determine if hyperthermia alters GSH metabolism in activated murine peritoneal macrophages such that prostaglandin (PG) synthesis is affected. PG synthesis will be measured in thermotolerant and non-thermotolerant macrophages. 7) Determine if thermotolerance affects the cytolytic activity of activated macrophages and, if so, is it related to GSH metabolism. Cytolysis will be measured in thermotolerant (and non-thermotolerant) macrophages and/or target cells. Chinese hamster ovary cells will be used for specific aims 1-5. Murine activated macrophages and 15178 Y cells will be used for aims 6 and 7.
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