Role of protein thiols in intrinsic radiation protection of DNA and cells.

Previous data have been consistent with the hypothesis that the thiol depleter dimethyl fumarate (DMF) increases radiation sensitivity of hypoxic mammalian cells by a combination of two actions: depletion of glutathione (GSH) which interferes with the chemical competition between damage fixation and repair and depletion of protein thiol (PSH) which causes inhibition of enzymatic repair processes. However, one cannot rule out the possibility that PSH also acts in the chemical competition to restitute damaged DNA. The studies presented here have addressed this question by studying the effects of ionizing radiation on isolated nuclei which contain very low levels of GSH, but substantial amounts of PSH, compared to intact cells. The results show that DNA damage, measured using the non-denaturing filter elution assay, is increased about 1.6-fold in isolated nuclei irradiated in air, compared to whole cells, and about 4.3-fold in nuclei irradiated in N2. Thus, the OER for DNA damage decreases from about 3 in whole cells to 1 in isolated nuclei. Also, although DMF increases radiation-induced DNA damage in whole cells irradiated in hypoxia about 2-fold, it does not increase DNA damage in isolated nuclei irradiated in hypoxia. These data do not support the idea that PSH can act in the chemical competition reaction to chemically repair radiation-induced DNA radicals. The data are discussed in relation to the effect that various procedures for nuclei isolation can have on radiation sensitivity of DNA and on the OER. We also address the question of whether radiation-induced DNA damage measured by non-denaturing elution correlates with cell killing.