into C57BL/6 mice as well as the mice were exposed to 4 Gy WBI

into C57BL/6 mice as well as the mice were exposed to 4 Gy WBI. the repair of DNA damage and enhanced apoptosis (increased Bax to Bcl-2 ratio). Administration of L-NAME, a nitric oxide synthase inhibitor, to mice significantly protected lymphocytes against WBI-induced DNA damage and inhibitedin vivoradiation-induced production of nitric oxide. These results confirm that the observed differential radiosensitivity of lymphocytes was due to slow repair of DNA due to nitric oxide productionin vivo. Keywords:Nitric oxide, Radiosensitivity, NF-B, Apoptosis, Proliferation, Lipid peroxidation == 1. Introduction == Cellular damage by radiation can occur by direct transfer of energy to biomolecules as well as by free radical mediated indirect mechanisms. The Coelenterazine H generation of highly reactive oxygen species through radiolysis of water brings about oxidation of crucial cellular components leading to DNA strand breaks, peroxidation of lipid moieties in cellular membranes and protein oxidation [1,2]. Our previous studies clearly demonstrated that higher the levels of ROS generated, greater is the extent of radiation induced cell death and vice versa [3]. DNA is considered to be a crucial target for radiation-induced damage. Damage to DNA can lead to activation of various genes resulting in cell cycle arrest and recruitment of DNA repair enzymes to the damaged site [4,5]. The residual damage or misrepair can cause genomic instability and higher frequency of mutations resulting in either clonogenic cell death subsequent to several mitotic cycles or results into a transformed cell [6,7]. The other mechanism of radiation-induced cell death is by alterations occurring in the plasma membrane and further activation of different signal transduction pathways [810]. Higher radiosensitivity of tumor cells compared to normal cells is a very important requirement for success of cancer radiotherapy [11,12]. Multiple factors like quiescence, cell cycle stage, stage of differentiation, DNA repair and types of cells are known to influence radiosensitivities [13]. The concentration of oxygen also contributes to the radiosensitivity of cells [14]. It is widely accepted that cells irradiatedin vitroare less radiosensitive than the cells irradiatedin vivo. Both nitric oxide (NO) and activation of NF-B have been implicated in survival of irradiated cells [1517]. The present study was focused on examining their contribution to the differences in the damage to lymphocytes and tumor cells underin vitroandin vivoirradiation conditions. == 2. Materials and methods == == 2.1. Chemicals == Fetal calf serum (FCS) was obtained from GIBCO BRL.NG-nitro-L-arginine-methyl-ester (L-NAME) was purchased from Calbiochem, USA. Carboxy fluorescein diacetate succinimidyl ester (CFSE) was procured from Coelenterazine H Molecular Probes, The Netherlands. RPMI 1640 tissue culture medium, propidium iodide (PI), tris base, EDTA, DMSO, agarose and sodium nitroprusside (SNP) were obtained from Sigma Chemical Company, USA. Dexamethasone was procured from Wockhardt Limited, Mumbai, India. PE labeled Bcl2 and Bax were purchased from Santacruz Biotechnology. Antibodies against caspase-3 and actin- were procured from Cell Signaling Technology. == 2.2. Animals == Eight to ten-week-old inbred C57BL/6 mice, weighing approximately 2025 g and reared in the animal house of Bhabha Atomic Research Centre were used. The guidelines issued by the Institutional Animal Ethics Committee of Bhabha Atomic Research Centre, Government of India, regarding the maintenance and dissections of small animals were strictly followed. Ether anesthesia as permitted by BARC animal ethics committee was used in all our experiments. Animals were sacrificed by cervical dislocation. == 2.3. Irradiation schedule == Whole body irradiation (WBI) andin vitroirradiation were carried out using Gamma Cell 220 irradiator machine (AECL, Coelenterazine H Canada). The dose rate as estimated by chemical dosimetry was 6.20 Gy/min. Animals were placed in perspex boxes and exposed to 4 Gy whole body -irradiation (WBI). In all the experiments, mice were sacrificed 1, 4, 24 or 96 h after WBI. Forin vitroexperiments tumor (EL-4) cells and lymphocytes were resuspended in RPMI medium without FCS at a cell density of 5 106cells/ml and exposed to 4 Gy radiation. == 2.4. Preparation of cells == Spleen cells were obtained by squeezing Rabbit polyclonal to ZAK the spleen through a nylon mesh in a petri plate containing RPMI medium. The RBC was lysed by brief hypotonic shock. Viable lymphocytes were enumerated by trypan blue dye exclusion. Peripheral blood mononuclear cells (PBMNCs) were obtained from the blood taken from lateral tail vein of live mice using a syringe (26.5.