Bystander Effects Of Gamma Radiation Induced Neurotoxicity And Its Molecular Mechanism

Although it is well establish that direct radiation exposure lead to neurodegenerative disorders however little is known about association among bystander effects of radiation and neurotoxicity. However detailed study of underlying mechanism could help to understand the phenomenon. Nevertheless, in CNS astroglial cells are known to protect neuronal cells against stress conditions in vivo and invitro. Yet, the fate of neuronal cells and the neuroprotective effect of coculture system(with glial cells) in response to indirect radiation exposure also require detailed study. Accordingly the objective of this study was to estimate the relative risk of neurodegeneration due to indirect effects of radiation and neuroprotection by astroglial cells. We also aim to investigate the factors which released by irradiated cells and lead to indirect damage of neighboring cells which never received direct radiation exposure. Here, we purpose that the indirect effect of radiation may cause oxidative stress and induce DNA damage which ultimately leads to apoptotic death of neuronal SH-SY5 Y cells. We also hypothesized that coculture(with glial U87) may relieved the neuronal SH-SY5 Y cells from toxicity of indirect effects radiation by reducing oxidative stress and apoptosis in vitro. Furthermore we proteomically characterize the conditioned media from irradiated cells to identify the secreted proteins that could serve as damaging signals in non-irradiated cellsFor this purpose 60 Co gamma ray was used as source of direct radiation whereas Irradiated Cell Conditioned Medium(ICCM) was used to mimic the indirect effect of radiation. To determine the potency of ICCM to inhibit neuronal cells survival; colony forming assay and cell viability assay was performed. The role of ICCM to induce apoptosis in neuronal SH-SY5 Y cells was estimated by TUNEL assay and Annexin V/PI assay. Level of oxidative stress and the concentration of inflammatory cytokines were evaluated by ELISA method. Expression of key apoptotic protein following direct and indirect radiation exposure was investigated by western blot technique. To evaluate the neuroprotective effect of astroglial cell against indirect effect of radiation; neuronal SH-SY5 Y cells were exposed to(ICCM) with and without coculture(with glial U87) in transwell coculture system respectively. Various endpoints such as, cell survival number assay, Annexin V/PI assay, cell cycle analysis by flow cytometer, m RNA level of Fas receptor by q RT-PCR, expression of key apoptotic proteins by western blot and estimation of neurotrophic factors by ELISA method was analyzed in to neuronal SH-SY5 Y cells with and without co culture after(ICCM) exposure respectively. Proteomic study bottom-up analysis of the peptides by two-dimensional chromatography and tandem mass spectrometry was appliedThe first set of experimental data manifest that ICCM account loss of cell survival and increase apoptotic induction in neuronal SH-SY5 Y cells that was dependent on time and dose. Moreover, ICCM stimulate significant release of inflammatory cytokines i.e., tumor necrosis factor TNF-alpha(P<0.01), Interleukin-1(IL-1, P<0.001), and Interleukin-6(IL-6, P<0.001) in neuronal SH-SY5 Y cells and elevate the level of oxidative stress(MDA, P<0.01). Up-regulation of key apoptotic protein expression i.e., Bax, Bid, cytochrome C, caspase-8 and caspase-3 confirms the toxicity of ICCM to neuronal cells. Our study revealed that ICCM caused significant increase in oxidative stress and reduces the mitochondrial membrane potential. Moreover, ICCM induced DNA damage in neuronal SH-SY5 Y cells by significant increase in(***P<0.001) cell cycle arrest at S-phase which was further supported by over expression of P53 protein(**P<0.01). Hence increased expression of FAS m RNA and up-regulation of key apoptotic protein demonstrate that ICCM lead to apoptotic death of neuronal SH-SY5 Y cells.Secondly in order to investigate the indirect effect of radiation on neuronal cells in the presence of glial cells; transwell co-culture system was applied. While our analysis remain focused on neuronal cells. Our data showed that ICCM induced oxidative stress was significantly reduced in SH-SY5 Y cells(co-cultured with glial U87) which further help to maintain the integrity of mitochondrial membrane potential. Coculture(with glial U87), significantly reduce the(ICCM) induced cell cycle arrest and expression of P53(###P<0.001) neuronal SHSY5 Y cells. Further investigation of the underlying apoptotic mechanism revealed that in coculture system; ICCM induced elevated level of FAS m RNA level was significantly reduced(###P<0.001) in neuronal SH-SY5 Y cells which was followed by significant reduction in expression of key apoptotic protein i.e., FADD(###P<0.001), caspase-8(###P<0.001), and cleaved caspase-3(###P<0.001) as compare to neuronal SH-SY5 Y cells which received(ICCM) without coculture. Intriguingly, concentration of neurotrophic factors such as, GDNF and BDNF and increased levels of antioxidant enzymes superoxide dismutase, glutathione were also observed in SH-SY5 Y cells after co-cultured with glial U87 cells which may point towards the protective mechanism.Third part of this study was to identify the seceretory protein in irradiated cell conditioned medium(released by irradiated cell) by proteomic analysis. Approximately, 17% proteins identified were classified as seceretory granules and extracellular matrix, while bioprocess annonation revealed that 22.2 % identified proteins were involved in transport and 17.2% protein function as intracellular signaling cascade.This study provides the evidence that indirect effect of radiation can be as much damaging to neuronal cells as direct radiation exposure can be. Hence, more focused research on estimation risks of indirect effect of radiation to CNS at molecular level may help to reduce the uncertainty about cure and cause of several neurodegenerative disorders.