The Role of Hydroxyl Radical Scavengers in Preventing DNA Strand Breaks Induced by X Irradiation of Toluene-Treated Escherichia coli

Abstract

The production of strand breaks by X rays in cellular DNA can result from direct action of the radiation on DNA or from indirect action. Indirect action may depend on OH radicals, hydrogen atoms, or solvated electrons formed by the radiolysis of intracellular water [for a recent review see Ref. (1)]. From studies on the rates of reaction of chemical scavengers for the OH radical and their protective activities, it has been concluded that the OH radical is the primary cause of indirect DNA strand breakage in cells (1-3). Recently it has been proposed that certain protective organic compounds may function in a more complex way than by simple OH radical removal. Ewing (4-6), using lethality as an end point, has found that under certain conditions of irradiation there is a correlation between radiation protection and the formation of a secondary a-hydroxyl radical. For example, he has shown that t-butanol and t-amyl alcohol, both excellent OH radical scavengers that are not converted to a-hydroxyl radicals, also do not protect Escherichia coli B/r cells if they are X-irradiated in air or 100% N2 but will protect if they are irradiated in 1% 02 (5, 6). The purpose of this study was to determine whether chemical protection against single-strand breaks observed in toluene-treated E. coli subjected to X irradiation in air (7) was due to the removal of OH radicals, or resulted from the production of secondary radicals as proposed by Ewing (4). In toluene-treated cells, DNA strandbreak production can be measured without the complication of strand ligation during or immediately following X-ray exposure since such cells are deficient in DNA ligase activity (7). Details of growth and of the methods of toluene treatment of E. coli strain AB3063Fwere described earlier (7, 8). Irradiation was carried out with a TFI Corporation Gemini Industrial X-Ray Unit, equipped with a 3-mm aluminum filter, at a dose rate of about 1 krad/min. Toluene-treated cells were rapidly thawed and added to 50 mM potassium phosphate buffer, pH 7.4 (a final concentration of 5-10 X 108 cells/ml), plus the radioprotective agent. All preparations were held 10 min at room temperature in sealed 2-ml vials prior to X-ray exposure; during irradiation the samples were held in an ice bath. The 02