Differential activation of NF-κB and nitric oxide in lymphocytes regulates in vitro and in vivo radiosensitivity

Abstract

Lymphocytes are more sensitive to radiation in vivo than in vitro. However, the mechanism of this differential response is poorly understood. In the present study, it was found that the lipid peroxidation and cell death were significantly higher in lymphocytes following whole body irradiation (WBI) as compared to lymphocytes exposed to radiation in vitro. EL-4 cells transplanted in mice were also more sensitive to radiation than EL-4 cells irradiated in vitro. DNA repair, as assessed by comet assay, was significantly faster in lymphocytes exposed to 4 Gy radiation in vitro as compared to that in lymphocytes obtained from whole body irradiated mice exposed to the same dose of radiation. This was associated with increased NF-κB activation in response to genotoxic stress and lesser activation of caspase in lymphocytes in vitro compared to in vivo. To explain the differential radiosensitivity, we postulated a role of nitric oxide, an extrinsic diffusible mediator of radiosensitivity that has also been implicated in DNA repair inhibition. Nitric oxide levels were significantly elevated in the plasma of whole body irradiated mice but not in the supernatant of cells irradiated in vitro. Addition of sodium nitroprusside (SNP), a nitric oxide donor to cells irradiated in vitro inhibited 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 inhibited in vivo radiation-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 production in vivo.

Immunomodulatory and immunotoxic effects of bilirubin: molecular mechanisms

“…Splenocytes (0.1ϫ10 6 cells/ml) were cultured for 24 h in the presence or absence of UCB, and two microscopy slides from each group were prepared and processed for Comet assay [46]. The slides were immersed in lysis buffer for 1 h at 4°C and equilibrated in alkaline solution for 20 min, followed by electrophoresis for 0.5 h at 25 V, 399 mA.…”

“…Surface staining with PE-labeled anti-CD95 (Fas) antibody was performed by the procedure described earlier [46]. Splenocytes (1ϫ10 6 ), cultured in presence or absence of UCB (100 M) for 24 h, were resuspended in 50 l buffer (PBS containing 10% serum, 0.1% sodium azide) and were incubated on ice for 10 min for blocking FcRs.…”

γ-Tocotrienol Induces Apoptosis in Human T Cell Lymphoma through Activation of Both Intrinsic and Extrinsic Pathways

“…The percentage of cells in different phases of cell cycle (G1, S+G2/M) and percent apoptotic cells were estimated by flowcytometry as described previously [31]. For cell cycle analysis, Jurkat or MCF-7 cells were treated with indicated concentrations of AT or GT for 24 h at 37 °C.…”

“…Intracellular antibody staining was performed as described earlier [31]. Briefly, cultured cells were fixed with 4% paraformaldehyde for 10 min at room temperature and excess of paraformaldehyde was removed by washing once with wash buffer (PBS containing 1% BSA).…”

MiRNA expression profile of ionizing radiation‐induced liver injury in mouse using deep sequencing

“…As the high dosage of 7–8 Gy could lead to death of mice rapidly from acute radiation toxicity (de Murcia et al, ), which was unsuitable for discussing the stabilized effects of irradiation. The middle dosage of 4 Gy was always used to investigate the gene expression and radioprotection by IR (Cardoso et al, ; Sharma et al, ; Zhang et al, ; Wang et al, ). The time point of 14 days was chosen for stable miRNAs expression could be obtained.…”