We plan to continue our current studies on the effects and mechanisms of action of thiol depletion on cell radiosensitization and metabolism in the absence and presence of various electron- affinic agents, including oxygen. Interest in the use of thiol depletion as a potential clinical approach for dealing with hypoxic cells has been heightened because (1) laboratory evidence demonstrates that thiol depletion increases hypoxic cell radiosensitization and (2) patients with the disease 5- oxoprolinuria can tolerate chronically low intracellular glutathione (GSH). The degree to which thiol levels serve as a determinant of cell radioresponse and whether radioresponse requires specificity for a particular class of thiol, e.g., GSH, total non-protein thiols or proteins thiols, will be assessed. Use will be made of various chemical depleting agents acting on mammalian cells in tissue culture and of human cells genetically deficient in GSH synthetase, and thus known, in some cases, to have decreased GSH levels. Experiments will be designed to assess the contributions of at least three processes which may alter radioresponse when thiols are manipulated: chemical, biochemical and enzymatic repair. For aspects to be examined with regard to sensitization are related to: the effectiveness of combinations of compounds acting on different intracellular thiol pools, the importance of intracellular localization of thiols, the correlation between thiol-related enzyme activity and radiosensitivity, and the alteration of post-irradiation enzymatic repair processes by thiol depletion. Further, we propose to determine how combinations of thiol-depleting agents and electron-affinic agents can enhance radioresponse without increasing cell toxicity. In this connection, experimental data will be compared with various models of radiation action to test the applicability and predictive capacity of those models. Finally, we will study the relationship between DNA damage, especially double-strand breaks, and cell killing, and whether manipulative control of intracellular thiol levels can alter that relationship. The major endpoint for these studies will be cell survival, i.e., clonogenicity, using cell lines including Chinese hamster ovary cells (CHO), Chinese hamster lung fibroblasts (V79) and human lung carcinoma A549 cells, as well as GSH-synthetase-deficient human fibroblasts and their normal counterparts. The DNA damage to be measured includes single and double-strand breaks, determined using alkaline and neutral filter elution techniques respectively. Thiol levels will be measured using well-established methods. We believe that the work proposed is significant because, in addition to its potential impact on current knowledge of radiobiological mechanisms, it will help provide basic radiobiological knowledge essential for application of thiol manipulation in the clinic, some of which has already begun.
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