The long term goal of this research is to elaborate how aminothiols and their derivatives function in protecting cells against radiation damage. The methodology used includes model system studies in which damage is assessed as single and double strand breaks in DNA. A broader assessment of damage is obtained by measuring the viability of a DNA plasmid carrying antibiotic resistance genes using a suitable E. coli host. DNA model systems used include pBR322 plasmid DNA, SV40 plasmid DNA, and SV40 minichromosomes. Radioprotection by aminothiols in cells is studied using cultured Chinese Hamster lung fibroblasts (V79-171 cells) with cell viability as the indicator of lethal cell damage. DNA damage in cells is assessed using neutral and alkaline elution assays to measure strand break damage. Monitoring of thiol levels is important because cellular levels vary with incubation conditions and with thiol structure, and even with DNA alone thiols are easily converted to disulfides by radiation. In all of these studies thiol and disulfide levels will be monitored by fluorescent labeling and HPLC separation methods developed in this laboratory.
Specific aims for this grant period are: (1) to determine if water-octanol partion coefficients for aminothiols allow prediction of uptake by passive diffusion in cells; (2) to establish if polyamine transport systems contribute to uptake of WR1065 by cells; (3) to determine the kinetic parameters which characterize protection of V79 cells by endogenous thiols and aminothiol radioprotectors under physiologic oxygen tension; (4) to determine if thiols protect pBR322 against strand break damage and loss of transforming ability in the same manner; (5) to obtain the parameters which characterize aminothiol protection of pBR322 under low oxygen tension at room temperature; (6) to develop a DNA model which reflects the thiol nonprotectable damage (TNPD) seen with cells; (7) to evaluate the relative contribution of thiol and thiolate forms in radioprotection of plasmid DNA; (8) to evaluate the importance of steric factors in protection of DNA by thiols; (9) to ascertain how chemical repair of oxygen-dependent damage by thiols in E. coli varies with net charge on the thiol. The results improve our ability to predict cell radioprotection from results of DNA model studies, will provide insight concerning optimal administration of the aminothiol drug WR2721 which is currently undergoing clinical trials, and will assist the design of improved radioprotective compounds.
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