Ionizing radiation induced perturbations in cellular redox homeostasis are manifested as cell cycle arrest, inflammatory response, and apoptosis. Present study was focused on determination of radioprotective efficacy of a secondary metabolite N-acetyl-l-tryptophan glucoside (NATG) isolated from radioresistant bacterium Bacillus sp. INM-1. Macrophage J774A.1 cells were treated with NATG (0.025-200 μg/ml) before γ-irradiation (10-50 Gy) and radioprotective efficacy in terms of cellular metabolic activity was assessed using MTT assay. Radiation-induced intracellular ROS generation and its inhibition by NATG (0.25 μg/ml) pretreatment was evaluated using 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) probe. Effects of NATG pretreatment with or without γ-irradiation (20 Gy) on cell cycle perturbations and apoptosis regulation was evaluated using flowcytometry. Results of the study displayed 0.25 and 50 μg/ml as effective dose (ED) and lethal dose (LD(50)) dose of NATG. A significant (p < 0.05) decrease in DCFDA fluorescence corresponding to decreased ROS levels was observed in NATG pretreated irradiated cells as compared to irradiated alone cells. Cell cycle progression analysis demonstrated decrease (∼10%) in G(1), G(2), and S phase cells at 24 h post-treatment time interval in NATG pretreated cells as compared to control group. However, radiation-induced or NATG-induced treatment did not cause any alterations in G(2)/M arrest. APO-BrDU analysis demonstrated significant (p < 0.05) reduction in apoptosis level at 24 h time interval in NATG pretreated irradiated cells as compared to alone irradiated cells. Conclusively, present study suggests that NATG offers radioprotection by apoptosis inhibition mechanism without altering cell-cycle progression in J774A.1 cells. Further studies to evaluate detailed molecular mechanisms of radioprotection offered by NATG are ongoing.
Keywords: APO-BrDU analysis; bacterial secondary metabolite; cell-cycle progression; gamma radiation; intracellular ROS.