THE ORIGIN OF THE RADIOBIOLOGICAL DAMAGE IN CELLS STORED IN CRYOSTATIC CONDITIONS

The radiation induced free radical damage in Chinese hamster lung fibroblast V-79 cells stored in DMEM culture medium containing 10% DMSO has been investigated by matrix EPR spectroscopy in connection with the H2O/DMSO binary phase diagram. A major part of the indirect effect is due to radicals from the DMSO•3H2O phase in the freezing medium which are released on warming in the temperature range between 130 K and 160 K, that is, far below the eutectic melting temperature (210 K). The radicals trapped in the DMSO•3H2O phase react with oxygen above 160 K giving reactive oxygen species (ROS) of the type of peroxyl radicals. A lower limit yield of 10-15 % was calculated for this conversion. Scavenging experiments with a stable nitroxyl radical (tempol) have demonstrated that part of the DMSO•3H2O radicals escape by mutual recombination on melting and are therefore available for inducing indirect cell damage. The same experiments performed withpure frozen water have shown that OH radicals are not available for inducing cell damage. The EPR measurements performed on H2O/DMSO frozen mixtures suggest that the radiation induced radical forming process does not change when passing to the low dose range below 1 Gy, in agreement with the linear model.