Keywords: reactive oxygen species (ROS), membrane oxidative damage, thymocyte apoptosis, DNA fragmentation, low dose radiation, fluorescence probe
In vitro studies on radiation induced membrane oxidative damage in apoptotic death of mouse thymocytes
Radiation oxidative damage to the plasma membrane of cells and its consequences in the mechanism of apoptotic death have been receiving growing attention of radiation scientists in recent years. We have employed a fluorescence probe method to determine changes in the permeability and fluidity of the plasma membrane of mouse thymocytes following γ irradiation mwithin a low to moderate radiation dose range (a few cGy to 10 Gy;·0.71 Gy/min). In vitro studies have shown that radiation induced membrane changes were correlated with the induction of apoptotic cell death. Studies have also been carried out to evaluate the generation of intracellular reactive oxygen species (ROS) in response to radio-oxidative damage to thymocytes using a cytosolic fluorescence probe, 2'-7'- dichlorodihydrofluorescien diacetate (DCH-FDA). The apoptosis of the thymocytes was determined by measuring nuclear condensation using propidium iodide (PI) and DNA fragmentation by gel electrophoresis. The viability of the thymocytes was determined by the trypan blue exclusion method which was found to gradually decrease after incubation of irradiated cells for 7 h. Thymocytes labeled with DCH-FDA in PBS at 25°C showed remarkably increased fluorescence intensity, measured after 30 min of irradiation (10 Gy), indicating an enhanced formation of intracellular reactive oxygen species. The DCH-FDA probe was able to sensitively detect irradiation effects on cells at lower doses. The population of thymocytes with reduced nuclear diameter was found to progressively increase with the postirradiation incubation time, following exposure to a particular radiation dose. Moreover, fragmentation of nuclear DNA in irradiated thymocytes, as observed by the pattern of ladder formation on agarose gel, was found to significantly increase on incubations beyond 3 h at 37°C. Results indicate that the enhanced amount of intracellular reactive oxygen species generated in response to radiooxidative stress were linked with the induction of γ irradiation of apoptotic death in thymocytes. These observations and a highlight of emerging scenario in the area of low dose radiation effects at cellular and membrane levels are described.