Mechanisms of Cellular and Molecular Radiosensitivity A detailed understanding of the mechanism which determine cellular radiosensitivity is necessary for the optimal use of radiation in medicine. The basic reactions caused by ionizing radiation decay very rapidly, and can damage any cellular (macro-molecules. The magnitude of the damage affecting DNA or DNA/ membrane has consistently been shown to directly affect cell survival at much later times. The chemical and/or biochemical (intra)cellular environment can be modified to favor either the repair or fixation of radiation- induced DNA damage, and a relatively simple 'competition-model', based on principles of radiation chemistry, is currently used to describe these modification. We have made detailed and quantitative test of the model, using cell survival as the assay system, and found discrepancies between predictions for the model and actual experimental results. These discrepancies could be caused by flaws or over-simplifications of the model. Alternatively, the action of the modifying chemicals could have changed the relationship between early DNA damage (occurring essentially at the same time as the radiation) and final assay (i.e. cell survival, assessed via colony formation after one week of growth). One goal of the proposed research is to continue to test the 'competition model' at the level of cell survival. Such test will include detailed measurements of the uptake and fate of aminothiol radiopretectors in mammalian cells. The affect of radioprotectors will be studied under conditions where specific mechanisms of action can be isolated (radiochemical depletion of radiosensitizers, scavenging of primary radicals, scavenging of secondary radicals, chelation of metals, inhibition of sensitizer transport and production of toxic intermediates). Other test of the model will include the effect of oxygen over a much broader rage of concentrations than has previously been possible. Special properties of nitroheterocyclic compounds will also be investigated. Effects of primary versus secondary radical scavengers will be assessed using nitrous oxide at high pressure, DMSO and formate. A second goal will be to assess DNA damage under the same conditions as the cell survival measurements. DNA damage assays will include alkaline elution and hydroxyl apatite chromatography for measuring single-strand breaks, and neutral elution for measuring double-strand breaks. The importance of crosslinks will be estimated. A third goal will be to consider more complex and realistic form of the 'competition model'.
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