RESUMO
Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation.
Assuntos
Micro-Ondas , Neurônios/patologia , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Receptores de N-Metil-D-Aspartato/genética , Animais , Sequência de Bases , Encéfalo/patologia , Proliferação de Células , Frequência do Gene/genética , Variação Genética , Genótipo , Masculino , Células PC12 , Subunidades Proteicas/metabolismo , Ratos , Ratos WistarRESUMO
OBJECTIVE: The aim of this study is to investigate whether microwave exposure would affect the N-methyl-D-aspartate receptor (NMDAR) signaling pathway to establish whether this plays a role in synaptic plasticity impairment. METHODS: 48 male Wistar rats were exposed to 30 mW/cm2 microwave for 10 min every other day for three times. Hippocampal structure was observed through H&E staining and transmission electron microscope. PC12 cells were exposed to 30 mW/cm2 microwave for 5 min and the synapse morphology was visualized with scanning electron microscope and atomic force microscope. The release of amino acid neurotransmitters and calcium influx were detected. The expressions of several key NMDAR signaling molecules were evaluated. RESULTS: Microwave exposure caused injury in rat hippocampal structure and PC12 cells, especially the structure and quantity of synapses. The ratio of glutamic acid and gamma-aminobutyric acid neurotransmitters was increased and the intracellular calcium level was elevated in PC12 cells. A significant change in NMDAR subunits (NR1, NR2A, and NR2B) and related signaling molecules (Ca2+/calmodulin-dependent kinase II gamma and phosphorylated cAMP-response element binding protein) were examined. CONCLUSION: 30 mW/cm2 microwave exposure resulted in alterations of synaptic structure, amino acid neurotransmitter release and calcium influx. NMDAR signaling molecules were closely associated with impaired synaptic plasticity.
Assuntos
Hipocampo/citologia , Micro-Ondas , Plasticidade Neuronal/efeitos da radiação , Neurônios/efeitos da radiação , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos da radiação , Animais , Regulação da Expressão Gênica/efeitos da radiação , Neurotransmissores/metabolismo , Células PC12 , Ratos , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
OBJECTIVE: To explore the impact of microwave radiation on GC-2spd cells. METHODS: We exposed cultured GC-2spd cells to microwave radiation at the average power densities of 0, 10 and 30 mW/cm2 for 15 minutes and, from I to 24 hours after the exposure, we observed the changes in cell proliferation, histology and ultrastructure, cell apoptosis, and cAMP content by MTIT, light microscopy, electron microscopy, flow cytometry and ELISA. RESULTS: Compared with the control group, the GC-2spd cells showed a significant decrease in proliferation ability at 1 -24 hours after 10 and 30 mW/cm2 microwave radiation, except at 12 hours after 30 mW/cm2 radiation (P <0.05 or P <0.01), with reduced length and number of cell enation and increased intra cytoplasm vacuoles. The rate of cell apoptosis (%) was significantly increased in the 10 and 30 mW/cm2 groups at 6 hours (4.56 +/- 2.09 vs 14.59 +/- 1.09 and 8.48 +/- 1.73, P <0.05 or P <0.01) , with agglutination and margin translocation of chromatins and obvious dilation of endo cytoplasmic reticula. The cAMP content (nmol/g) in the GC-2spd cells was remarkably reduced in the 10 and 30 mW/cm2 groups at 6 and 24 hours (2.77 +/-0.24 vs 1.65+/- 0. 17 and 1.96+/-0.10, 3.02 +/-0.47 vs 2.13 +/-0.33 and 1.69 +/-0.27, P <0.05 or P <0.01). CONCLUSION: Microwave radiation at 10 and 30 mW/cm2 may cause injury to GC-2spd cells, which is manifested by decreased content of intracellular cAMP, reduced activity of cell proliferation, and increased rate of cell apoptosis.
Assuntos
Micro-Ondas/efeitos adversos , Espermatócitos/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Linhagem Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Masculino , CamundongosRESUMO
Microwave-induced learning and memory deficits in animal models have been gaining attention in recent years, largely because of increasing public concerns on growing environmental influences. The data from our group and others have showed that the injury of mitochondria, the major source of cellular adenosine triphosphate (ATP) in primary neurons, could be detected in the neuron cells of microwave-exposed rats. In this study, we provided some insights into the cellular and molecular mechanisms behind mitochondrial injury in PC12 cell-derived neuron-like cells. PC12 cell-derived neuron-like cells were exposed to 30 mW/cm(2) microwave for 5 min, and damages of mitochondrial ultrastructure could be observed by using transmission electron microscopy. Impairments of mitochondrial function, indicated by decrease of ATP content, reduction of succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) activities, decrease of mitochondrial membrane potential (MMP), and increase of reactive oxygen species (ROS) production, could be detected. We also found that hypoxia-inducible factor-1 (HIF-1α), a key regulator responsible for hypoxic response of the mammalian cells, was upregulated in microwave-exposed neuron-like cells. Furthermore, HIF-1α overexpression protected mitochondria from injury by increasing the ATP contents and MMP, while HIF-1α silence promoted microwave-induced mitochondrial damage. Finally, we demonstrated that both ERK and PI3K signaling activation are required in microwave-induced HIF-1α activation and protective response. In conclusion, we elucidated a regulatory connection between impairments of mitochondrial function and HIF-1α activation in microwave-exposed neuron-like cells. By modulating mitochondrial function and protecting neuron-like cells against microwave-induced mitochondrial injury, HIF-1α represents a promising therapeutic target for microwave radiation injury.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Micro-Ondas , Mitocôndrias/efeitos da radiação , Neurônios/efeitos da radiação , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos da radiação , Regulação para Cima/efeitos da radiação , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Potencial da Membrana Mitocondrial/efeitos da radiação , Mitocôndrias/metabolismo , Neurônios/metabolismo , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
OBJECTIVE: To study the effects of electromagnetic pulse (EMP), S-band high power microwave (S-HPM), and X-band high power microwave (X-HPM) on the Ca(2+) concentration and caspase-3 expression in Raji cells and the relationship between Ca(2+) concentration and caspase-3 expression, and to investigate the regulatory mechanism of electromagnetic radiation damage. METHODS: Raji cells were cultured conventionally. Some cells were irradiated by EMP, S-HPM, and X-HPM in the logarithmic growth phase for 6 hours and then collected; others received sham irradiation as a control. The Ca(2+) concentration in the cells was measured by laser scanning confocal microscope; the caspase-3 expression in the cells was evaluated by Western blot. RESULTS: Compared with the control group (Ca(2+) fluorescence intensity = 43.08 ± 2.08; caspase-3 expression level = 0.444 ± 0.13), the EMP,S-HPM, and X-HPM groups had significantly increased Ca(2+) concentrations, with Ca(2+) fluorescence intensities of 69.56 ± 1.71, 50.06 ± 1.89, and 70.68 ± 1.59, respectively (P < 0.01), and had upregulated caspase-3 expression, with expression levels of 0.964 ± 0.12, 0.586 ± 0.16, and 0.970 ± 0.07, respectively (P < 0.01). Each of the EMP and X-HPM groups had significantly higher Ca(2+) fluorescence intensity and caspase-3 expression level than the S-HPM group (P < 0.01), but there were no significant differences between the EMP and X-HPM groups. The linear regression analysis showed that the caspase-3 expression was upregulated as the Ca(2+) concentration increased, with a positive correlation between them (P < 0.01). CONCLUSION: EMP, S-HPM, and X-HPM cause damage probably by increasing the Ca(2+) concentration in cells and in turn inducing caspase-3 overexpression.
Assuntos
Cálcio/metabolismo , Caspase 3/metabolismo , Radiação Eletromagnética , Linhagem Celular Tumoral , HumanosRESUMO
AIM: To construct the eukaryotic expression vectors of RKIP plasmid and detect its expression in PC12 cells. METHODS: The coding sequence of RKIP was generated by nested-PCR using total RNA extracted from the root ganglion neurons of rats. RKIP gene was cloned into the eukaryotic expression vector pcDNA3.0. After restriction enzyme analysis and sequence identification, the recombinant plasmid was transfected into PC12 cells with non-liposome mediated method by Vigofect. The expression of RKIP was detected by Western blot. RESULTS: The results of enzyme analysis and sequencing both identified DNA sequence of recombinant plasmid pcDNA3.0-RKIP correctly. The expression of RKIP increased obviously after transfection into PC12 cells. CONCLUSION: The eukaryotic expression plasmid of pcDNA3.0-RKIP was constructed successfully and it can be sustainly expressed in PC12 cells. This provides experimental basis for further study on the neurological function of RKIP.
Assuntos
Proteína de Ligação a Fosfatidiletanolamina/genética , Plasmídeos , Animais , Células PC12 , Ratos , Recombinação Genética , TransfecçãoRESUMO
OBJECTIVE: To investigate the effect of long-term microwave radiation on male reproduction in rats. METHODS: A total of 100 male Wistar rats were exposed to microwave radiation with average power density of 0, 2.5, 5 and 10 mW/cm2 for 4 weeks, 5 times a week and 6 minutes per time. Changes in serum testosterone, testicular index, histology and ultrastructure, and the percentage of teratospermia in the epididymis were observed dynamically at 6 h, 7 d, 14 d, 28 d and 60 d after the exposure. RESULTS: There was a significant decrease in serum testosterone concentration at 28 d after microwave radiation at 2.5, 5 and 10 mW/cm2 ([10.20 +/- 4.31] ng/ml, [5.56 +/- 3.47] ng/ml and [7.53 +/- 4.54] ng/ml) and at 60 d at 10 mW/cm2 ( [15.95 +/- 9.54] ng/ml), as compared with the control group ([23.35 +/- 8.06] ng/ml and [31.40 +/- 9.56] ng/ml) (P < 0.05 or P < 0.01). No significant changes were found in the testis index at 6 h -60 d after microwave radiation at the three doses, but different degrees of degeneration, necrosis and shedding of spermatogenic cells, thinning of spermatogenic epithelia, and decrease or deletion of spermatozoa were observed, and more obvious at 28 d and 60 d. Swelling and cavitation of mitochondria in all spermatogenic cells, agglutination and margin translocation of nuclear chromatin in the spermatogonial and Leydig cells were seen at 7 d and 60 d after 5 mW/cm2 microwave radiation. The rate of teratospermia of the epididymis was increased, more obviously at 7 d after 2.5, 5 mW/cm2, 60 d after 5 mW/cm2, and 7 d, 28 d and 60 d after 10 mW/cm2 microwave radiation (P < 0.05 or P < 0.01). CONCLUSION: Long-term microwave radiation may cause injury to male reproduction, which is positively correlated with the radiation dose, and has an obvious late effect.
Assuntos
Micro-Ondas/efeitos adversos , Reprodução/efeitos da radiação , Cabeça do Espermatozoide/efeitos da radiação , Testículo/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Masculino , Ratos , Ratos WistarRESUMO
OBJECTIVE: To explore the changes in the expressions of the tight junction related protein occludin and junctional adhesion molecule-1 (JAM-1) of the blood-testis barrier and their significance in rats after microwave radiation. METHODS: Eighty male Wistar rats were exposed to microwave radiation with average power density of 0, 10, 30 and 100 mW/cm2 for five minutes, and dynamic changes in the expressions of testicular occludin and JAM-1 were observed by Western blot and image analysis at 6 h, 1 d, 3 d, 7 d and 14 d after the radiation. RESULTS: There was a significant down-regulation in the expression of the occludin protein at 3 - 7 d, 6 h - 7 d and 6 h - 14 d (P < 0. 05), as well as in that of JAM-1 at 3 - 7 d, 1 - 7 d and 1-14 d (P < 0.05) after exposure to 10, 30 and 100 mW/cm2 microwave radiation. CONCLUSION: The decreased protein expressions of occludin and JAM-1 may play an important role in the microwave radiation induced-damage to the blood-testis barrier.
Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas de Membrana/metabolismo , Micro-Ondas , Testículo/metabolismo , Testículo/efeitos da radiação , Animais , Barreira Hematotesticular/metabolismo , Barreira Hematotesticular/efeitos da radiação , Regulação para Baixo , Masculino , Ocludina , Ratos , Ratos WistarRESUMO
OBJECTIVE: To explore whether microwave radiation may cause injury of primary cultured Sertoli cells. METHODS: The model of primary cultured Sertoli cells in vitro was established, which was radiated by microwave with average power density 0, 30 and 100 mW/cm(2) for five minutes. The changes of cell cycle, apoptosis and death, and intracellular Ca2+ concentration in the Sertoli cells were measured at sixth hours through Annexin V-PI double labeling and Fluo-3-AM labeling, flow cytometry combined with laser scanning confocal microscopy after microwave exposure. RESULTS: The numbers of Sertoli cells were obviously reduced in G0-G1 and G2-M phase (62.57% +/- 3.22% and 8.25% +/- 1.75%) and increased in S phase (29.17% +/- 4.87%) compared with the control groups (79.18% +/- 0.24%, 11.17% +/- 0.50% and 9.64% +/- 0.62%) (P < 0.05 or P < 0.01), but the changes of rate of apoptosis and death and intracellular Ca2+ concentration showed no difference at 6 h after exposure to 30 mW/cm(2) microwave. There was a significant increase in the Sertoli cell counts of G0-G1 phase (87.69% +/- 1.32%), and decrease in the Sertoli cell counts of G2-M and S phase (7.41% +/- 0.60% and 4.87% +/- 0.91%) (P < 0.01). There was also a significant increase in intracellular Ca2+ concentration and rate of apoptosis and death (P < 0.05 or P < 0.01) at 6 h after exposure to 100 mW/cm(2) microwave. CONCLUSION: 100 mW/cm(2) microwave radiation may cause growth inhibition and increase of apoptosis and death in the primary cultured Sertoli cells. The increase of intracellular Ca2+ concentration is one of the injury mechanisms.
Assuntos
Micro-Ondas/efeitos adversos , Células de Sertoli/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Cálcio/metabolismo , Ciclo Celular/efeitos da radiação , Células Cultivadas , Masculino , Ratos , Ratos Wistar , Células de Sertoli/metabolismo , Células de Sertoli/patologiaRESUMO
AIM: To study the development of changes for signaling molecules related to Raf/MEK/ERK pathway in hippocampus of rats after electromagnetic radiation, and investigate the mechanisms of radiation injury. METHODS: Rats were exposed to X-HPM, S-HPM and EMP radiation source respectively, and animal model of electromagnetic radiation was established. Western blot was used to detect the expression of Raf-1, phosphorylated Raf-1 and phospholylated ERK. RESULTS: The expression of Raf-1 down-regulated during 6 h-14 d after radiation, most significantly at 7 d, and recovered at 28 d. There was no significant difference between the radiation groups. The expression of phosphorylated Raf-1 and phosphorylated ERK both up-regulated at 6 h and 7 d after radiation, more significantly at 6 h, and the two microwave groups were more serious for phosphorylated ERK. During 6 h-14 d after S-HPM radiation, the expression of phosphorylated Raf-1 increased continuously, but phosphorylated ERK changed wavily, 6 h and 7 d were expression peak. CONCLUSION: Raf/MEK/ERK signaling pathway participates in the hippocampus injury induced by electromagnetic radiation. The excessive activation of ERK pathway may result in the apoptosis and death of neurons, which is the important mechanism of recognition disfunction caused by electromagnetic radiation.
Assuntos
Radiação Eletromagnética , Hipocampo/efeitos da radiação , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Apoptose , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Masculino , Fosforilação , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
AIM: To study the effects of electromagnetic radiation on RKIP and phosphorylated ERK in primary cultured hippocampus neurons. The inhibitor of MEK U0126 was applied to investigate the role of RKIP mediated ERK pathway in radiation injury. METHODS: Primary hippocampus neurons were cultured in vitro. X-HPM, S-HPM and EMP were taken as radiation source respectively to establish three cell models exposed to electromagnetic radiation. RKIP and phosphorylated ERK were measured by immunofluorescent labelling and laser scanning confocal microscope. Apoptosis and death fraction of the cells were detected by Annexin V-PI double labelling and flow cytometry. RESULTS: After three kinds of electromagnetic radiation, the expression of RKIP in hippocampus neurons decreased but the expression of phosphorylated ERK increased, and its nuclear translocation occurred. No significant differences were seen between radiation groups. Apoptosis and death fraction of the neurons in U0126 pretreatment groups was significantly lower than that in radiation groups but they were still higher than those in sham-radiation group. CONCLUSION: The excessive activation of RKIP mediated ERK pathway is one of the important mechanisms for the apoptosis and death of hippocampus neurons induced by electromagnetic radiation. U0126 have some protective effects on radiation injury.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipocampo/citologia , Neurônios/metabolismo , Neurônios/efeitos da radiação , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Radiação , Transdução de Sinais/efeitos da radiação , Animais , Butadienos/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Microscopia Confocal , Neurônios/citologia , Neurônios/efeitos dos fármacos , Nitrilas/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: To study the development of changes for Raf kinase inhibitor protein (RKIP) and its mRNA in rats hippocampus after electromagnetic radiation. METHODS: Rats were exposed to X-band high power microwave (X-HPM), S-band high power microwave (S-HPM) and electromagnetic pulse (EMP) radiation source respectively. The animal model of electromagnetic radiation was established. Western blot was used to detect the expression of RKIP, and RT-PCR was applied to detect the expression of RKIP mRNA. The interaction of RKIP and Raf-1 was measured with co-immunoprecipitation method, and the expression of cerebral choline acetyltransferase (CHAT) was measured by immunohistochemistry. RESULTS: The expression of RKIP significantly down-regulated at 6 h after radiation, and recovered at 1 d in group EMP, but the down-regulation continued during 1 approximately 7 d after radiation in the two microwave groups. The expression of RKIP mRNA changed wavily during 6 h approximately 7 d after radiation, which showed down-regulation at 6 h, and up-regulation at 3 d. The interaction of RKIP and Raf-1 decreased during 6 h approximately 7 d after radiation, most significantly at 7 d, and the two microwave groups were more significant. The expression of CHAT decreased continuously during 6 h approximately 7 d after radiation, and generally recovered on 14 d. CONCLUSION: The down-regulation of RKIP and its related proteins of hippocampus is induced by electromagnetic radiation.