Role of Scavenger-Derived Radicals in the Induction of Double-Strand and Single-Strand Breaks in Irradiated DNA

Abstract

Studies of intact cells have generally shown a substantial enhancement by oxygen of radiation-induced cell killing or DNA breakage, with an oxygen enhancement ratio (OER) of 2.5-3. Analogous studies of radiation damage to purified DNA in aqueous solution, performed at much lower .OH scavenging efficiencies, have usually shown little or no damage enhancement by oxygen (OER approximately 1) unless reduced thiol compounds were also present during irradiation. Milligan and Ward (Radiat. Res. 137, 295-299, 1994) recently observed an excess of DNA single-strand breaks (SSBs) in nitrogen compared to air (OER < 1) for SV40 DNA irradiated in the presence of high concentrations of DMSO, and they presented evidence that this excess resulted from secondary DMSO radicals. We have here investigated the role of secondary radicals from glycerol, another non-thiol .OH scavenger, in the induction of DNA double-strand breaks (DSBs), lesions which are critical to cell death. Superhelical SV40 DNA, 25 micrograms/ml, was irradiated in air or nitrogen in the presence of 0-2500 mM glycerol. Resultant DSBs and SSBs were measured simultaneously by computer analysis of digital video images of ethidium bromide-stained gels. The OERs for DSBs and SSBs decreased from 1.3 at the lowest glycerol concentration to 0.65 and 0.45, respectively, at the highest concentration, consistent with an important role for anoxic secondary radicals in the presence of high glycerol concentrations. The dose-response curves for SSBs due to glycerol radicals are predominantly "one-hit" over the entire glycerol concentration range where they were observable (> or = 75 mM). The dose-response curves for DSBs due to glycerol radicals are predominantly one-hit at high glycerol concentrations (> or = 750 mM). Analysis of the data suggests that the dominant mechanism for the formation of glycerol radical-induced one-hit DSBs at lower glycerol concentrations (< or = 750 mM) involves a single glycerol radical resulting in two nearby SSBs on opposite DNA strands, analogous to the mechanism proposed by Siddiqi and Bothe (Radiat. Res. 112, 449-463, 1987) for the induction of a DSB by a single OH radical. Our data for the highest concentration (2500 mM) imply that it is now important to take into account a second mechanism.