Simulation of the Cellular Oxygen Effect with an SV40 DNA Model System Using DNA Strand Breaks as an End Point

Abstract

Most in vitro studies of the modification of radiation damage to DNA by oxygen and reduced thiol compounds have excluded non-thiol .OH scavengers. However, the non-thiol .OH scavengers are known to be very effective radiation protectors and are present in cells in near molar concentrations. To better relate data obtained from an in vitro system to data at the intracellular level, we have carried out simultaneous measurements of single and double-strand breaks (SSBs and DSBs) in a model system using closed-circular supercoiled SV40 DNA (25 micrograms/ml) in aqueous solution. Observations were made in the presence and absence of oxygen and over wide concentration ranges encompassing intracellular conditions of both glutathione (GSH), the dominant soluble intracellular reduced thiol compound, and glycerol, a widely used poly-alcohol non-thiol .OH scavenger. The dose-response curves for both SSBs and DSBs in either air or nitrogen are predominantly linear when 75 or 750 mM glycerol is combined with 0-20 mM GSH. Glutathione together with glycerol shows a concentration-dependent preferential protection in nitrogen compared to air against radiation-induced SSBs and DSBs, qualitatively similar to our prior observations at comparable concentrations of GSH alone (Ayene et al., Radiat. Res. 144, 1-8, 1995). With GSH alone, we observed peak oxygen enhancement ratios (OERs) of 6.5 and 8.0 for SSBs and DSBs, respectively, at 5 mM GSH. In contrast, we observed previously that glycerol alone showed a preferential protection against radiation-induced SSBs and DSBs in air compared to nitrogen (Ayene et al., Radiat. Res. 142, 133-143, 1995). In the presence of 750 mM glycerol, approximately equivalent to the total effective concentration of non-thiol .OH scavengers in the cell, we now observe that the OER for radiation-induced DSBs increases from 1.2 at 0.5 mM GSH to 3.3 at 5.0 mM. This roughly parallels the dependence of the OER on [GSH] reported for cell survival. A possible mechanism for the lower OER at higher .OH scavenging efficiency is discussed.