Membrane permeability changes in radiation-induced apoptotic mouse thymocytes

Abstract

Objective: Radiation-induced alterations in permeability of plasma membrane and associated apoptotic changes were investigated in apoptotic thymocytes induced by radiation. Materials and Methods: Immature mouse thymocytes suspended in RPMI 1640 were irradiated by γ-rays for desired doses up to 10 Gy and these cells were examined after incubation (up to 24 h) at 37°C. Radiation-induced changes in plasma membrane of thymocytes were determined by fluorescence technique using fluorescein diacetate (FDA) and trypan blue (TB) method. Induction of apoptosis in irradiated cells was determined using annexin-V as fluorescence probe method and measurement of cytosolic caspase-3 activity. Results: Radiation-induced apoptotic thymocytes showed an increase in membrane permeability as observed by leakage of FDA, which was poorly detected by TB. FDA could sensitively detect the dose-dependent variation in membrane permeability alterations in the range of 0.5–2 Gy incubated at 37°C. In radiation-induced apoptotic death, externalization of phosphatidylserine is an early event than caspase-3 activation. On incubation of cells, an increase in caspase-3 activity and proportion of annexin-positive cells was observed, which further increased after irradiation. Conclusion: Our results showed membrane permeability changes induced by γ-irradiation, which seem to be closely associated with the apoptosis in thymocytes.