Date of Graduation

1996

Document Type

Dissertation/Thesis

Abstract

The objective of this study was to determine the dominant mechanism (OH-radical scavenging or non-OH-radical scavenging mechanisms) of radioprotection by the thiol scavenger dithiothreitol (DTT) making comparisons between a cellular and isolated plasmid DNA model systems. The protection mechanism of DTT was determined experimentally by comparing the thiol to known OH-radical scavenging compounds (dimethyl sulfoxide, glycerol and 1,3-dimethyluracil) measuring the relative differences in their protection factor for radiation-induced chromosome aberrations in cells and single-strand break induction in the plasmid DNA model. A follow-up study was conducted comparing radioprotection between DTT and the neutral thiol 2-mercaptoethanol (2ME) to determine the contribution of a possible secondary protection mechanism (H-atom donation). This three part study reports for the first time an investigation of thiol radioprotection in cells, coupled with a mechanistic evaluation to an isolated plasmid DNA model system conducted under cell-like OH-radical scavenging capacities (SC). Isolated human lymphocytes were exposed to x-rays in the presence of various modifiers and the yields of chromosome aberrations (dicentrics) were measured. The findings from this study indicate that the thiol DTT exhibited a 2.3-fold increase in protection factor against the formation of these aberrations when compared to the OH-radical scavenger DMSO. This observation supports a possible secondary mechanism of radioprotection by DTT beyond that of OH-radical scavenging. However in this study the dominant mechanism of protection by DTT cannot be determined, due to potential scavenger radical induced damage. The use of isolated plasmid DNA, in contrast to the cellular study, provides a mechanistic model system to study radioprotection devoid of those additional factors that can influence the yields of radiation-induced damage (cellular repair mechanisms, modifier compound toxicity etc.). Irradiation of plasmid DNA by either cobalt-60 {dollar}\\gamma{dollar}-rays and or fission neutrons was carried out under aerobic conditions of high OH-radical SC (equivalent to cell-like plus 1 M DMSO equivalent) measuring the yields of ssb. For both radiation qualities there is an observed increase G{dollar}\\rm\\sb{lcub}ssb{rcub}{dollar} protection factors for glycerol over that of DTT and the other OH-radical scavengers DMSO and 1,3-DMU investigated near saturated solubility. The use of DMSO and 1,3-DMU have been excluded as model OH-radical scavengers due to either ssb formation from secondary-radicals by DMSO and the possibility of microprecipitation of 1,3-DMU. A comparison of the G{dollar}\\rm\\sb{lcub}ssb{rcub}{dollar} protection factor for DTT and glycerol supports the conclusion that the dominant mechanism of radioprotection by DTT is consistent with OH-radical scavenging. This conclusion is further supported by the neutral thiol comparison study, which indicates that the additional sulfhydryl group on DTT compared with only one on 2ME does not contribute to a secondary protection mechanism. The data presented from these studies does not directly eliminate for thiols a secondary protection process (H-atom donation) but it does support that in the competition of different mechanisms, OH-radical scavenging appears to be dominant and can account for the major DNA radioprotection observed under these experimental conditions.

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