Reproductive survival studies demonstrate that parenchymal hepatocytes are radioresistant (Do = 270 rad) and are significantly protected in vivo with WR-2721, a thiophosphate derivative of cysteamine. The overall objective of this project is to elucidate the mechanism(s) for hepatocyte's enhanced intrinsic resistance to ionizing radiation, and to explain the enigma that though hepatocytes are radioresistant, the liver in situ is sensitive to radiation damage. Specifically, we will determine the importance of an endogenous non-protein thiol (glutathione) and exogenously administered WR-2721 in radioprotecting both rat and human hepatocytes. The magnitude of radiation damage before and after either the addition of WR-2721 or the intracellular depletion of GSH (diethyl maleate or buthionine-S,R-sulfoximine) will be estimated at the cellular level (ie, reproductive survival) and at the molecular level (ie, DNA single strand breaks). The hepatocyte transplantation assay system which we characterized will be used to estimate liver cell survival and DNA single strand breaks will be determined with the alkaline elution technique. The ability of irradiated rat and human hepatocytes to repair enzymatically DNA single strand breaks will also be compared with the efficacy of this repair system in fibroblasts and hepatoma cells. A rat model to investigate the etiology of chronic radiation hepatitis will be developed to determine the degree to which WR-2721 protects the liver in situ from chronic disease. Additionally, the importance of parenchymal cell depletion in the development of radiation hepatitis will be investigated by prophylactically reseeding the irradiated liver with reproductively viable isolated hepatocytes. The experiments proposed in this application will enable us to determine the role which non-protein thiols play in the protection of both rat and human paranchymal hepatocytes. Not only will these studies delineate the mechanism of radioprotection; the interspecies comparisons between rat and human liver cells will assist in the safe clinical utilization of WR-2721 as a hepato-radioprotective agent and GSH depleting agents for enhancing the effectiveness of hypoxic cell radiosensitizers.
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