Role of Membrane Oxidative Damage and Reactive Oxygen Species in Radiation Induced Apoptotic Death in Mouse Thymocytes

Involvement of membrane damage and reactive oxygen species (ROS) in mechanism of radiation induced apoptotic death were investigated in mouse thymocytes in presence of eugenol, a membrane specific antioxidant from plant sources. The membrane damage in thymocytes by γ-irradiation was determined by measurement of thiobarbituric acid reactive species (TBARS). Generation of reactive oxygen species and apoptosis in irradiated thymocytes was estimated by using fluorescence probes, 2,7-dichlorodihydrofluorescien diacetate (DCH-FDA) and annexin-V method, respectively. The radiation response of thymocytes for ROS generation was found to be dependent on radiation dose and dose rate. DCH-FDA fluorescence appears sensitive for detection of radiation exposures at low doses, and a dose of 0.5 cGy and 0.5 Gy was detected for the dose rate of 0.45 cGy / min. and 0.5 Gy / min, respectively. Lipid peroxidation was inhibited in irradiated (2 Gy) thymocytes with increasing concentrations of eugenol (10-200 μM) treated prior to irradiation. The concentration of eugenol required to inhibit half of the MDA formation (IC50) in irradiated thymocytes was 100 μM. Percentage apoptosis in irradiated thymocytes was significantly inhibited by these antioxidants, which was however, more effective inhibited by lipophilic antioxidants. The results suggest the linkage of radiation generated membrane damage and ROS in the mechanism of apoptotic death in thymocytes.