Role of Ca²⁺ in radiation‐induced damage in murine splenocytes

Abstract

Purpose: To address the links between calcium, peroxidation, cell damage and death and the response of the enzymes involved in free radical metabolism, in splenocytes of mice irradiated with gamma‐rays.

Materials and methods: Splenocytes of Swiss albino mice were irradiated with various doses (0–7 Gy) of gamma‐rays (⁶⁰Co) at a dose‐rate of 0.0575 Gy s⁻¹. Membrane peroxidation and fluidity were determined by the thiobarbituric acid‐reactive substances (TBARS) method, and fluorescence polarization of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH), respectively. Apoptosis was analysed by nucleosomal ladder formation and activity of NF‐κB by electrophoretic mobility shift assay (EMSA). The specific activities of the antioxidant enzymes, lactate dehydrogenase (LDH), levels of nitric oxide (NO^•) and glutathione were determined spectrophotometrically. Modulatory effects of Ca²⁺ were examined at 3 Gy using different concentrations (1, 3 and 5 mM) in the presence or absence of the ionophore A23187.

Results: Irradiation of splenocytes resulted in enhanced peroxidative damage, membrane fluidity, apoptosis and DNA binding activity of NF‐κB. The specific activities of LDH and antioxidant enzymes superoxide dismutase (SOD), DT‐diaphorase (DTD), glutathione S‐transferase (GST) and levels of glutathione (GSH) and NO^• were increased with radiation dose up to 4 Gy. Ca²⁺ augmented the radiation‐induced responses. The presence of ionophore A23187 potentiated the modulatory effects of Ca²⁺.

Conclusions: These findings show that Ca²⁺ augments radiation damage and is more effective intracellularly. Ca²⁺, peroxidation, cellular damage and apoptosis are possibly interlinked through signals, as is evident from the increased activity of NF‐κB and generation of NO^•. The enhanced antioxidant status suggests an attempt made by the irradiated cells to maintain their normal functions.