Studying The Effects Of GSM 1800 MHz Radiofrequency Electromagnetic Fields On Gene Expression Profile And The Activation Of Transcription Factor In Rat Neurons

Mobile telecommunications has notably expanded recently, and result in increasing concerns for possible adverse effects induced by radiofrequency electromagnetic fields (RF EMF) emitted from mobile phones (MPs) on public health. Among systems responsive to RF EMF, the central nerve system (CNS) is one of the most sensitive organs. The head is likely subjected to the highest exposure of RF EMF emitted from MPs, thus, numerous epidemiological studies have been performed to assess health risks of RF EMF on the nervous system. Previous studies have shown that RF EMF exposure leads symptoms of neurasthenia and increased risks for developing certain types of cancer. Spatial learning tasks and cognitive performance impairment were also reported in animal experiments. However, there were somenegative reports as well. Therefore, the consistent conclusions were not consistent and debate continues. In vitro studies of mechanism may be a feasible approach to explore biological effects of RF radiation in the case of no persuadable conclusion of epidemiological and in vivo studies.The gene expression is the terminal of series of message transfer after extracellular stimulus, so the change of gene expression is taken as one of keys to discover the interaction between RF EMF and the biological system. Although some genes - such as heat shock protein, proto-oncogenes, and immediately early response genes - have been studied under RF radiation, these studies focused only on a handful of genes pre-selected with defined functions, so that it is difficult to assess comprehensively RF EMF-induced genome-wide effects. Recently, high-throughput screening techniques (HTSTs), such as transcriptomics and proteomics, have been proposed as a practical approach to non-selectively gather information about the effects of environmental stimuli on gene transcription and protein expression. In a previous study, we performed GeneChip analysis to address the differential gene expression profile after exposure to GSM 1800 MHz RF EMF in rat neurons. Although the microarray technique has the advantages of large scale and high through-put, but the shortages of the technique are the false positive, need of more chips for repeat and validation by other methods, such as RNase protection assay (RPA), real-time quantitive PCR (real-time PCR), reverse transcript PCR (RT PCR) and Northern blottings. In the present study, RPA and real-time PCR were used to validate these differentially expressed genes identified by Genechip analysis and ultimately to evaluate the effects of RF EMF on gene expression in rat neurons.In addition, some studies reported that cellular signal transduction pathways are affected by electromagnetic fields (EMF). Many transcription factors are the final targets of specific transduction pathways. The change of the activity of transcription factors can lead to the change of gene expression levels. Several reports suggested thatEMF, as an environmental physical factor, can influence DNA activity of binding with the multiple transcription factors, including activator protien-1 (AP-1), activator protien-2 (AP-2), CREB and HSF. AP-1 is a heterodimer composing of fos (55 kDa) and jun (39 kDa), which bind DNA via leucine zipper. The activation of AP-1 is a classic pathway of cellular signal transduction. Receptor tyrosine kinase (RTK), small G protein and MAPK chain reaction are orderly activated by extracellular stimuli. Then c-jun and c-fos can be phosphated and transfered into the nuclear as activated AP-1. AP-1 can be activated by multiple protein kinases, including PKC and MAPK etc, thus leads to the activation of the transcription of different target genes via different dimmer modality. Studies on the biological functions of AP-1 have shown that AP-1 plays an important role in various cellular processes, which include proliferation, differentiation, adaptive responses and apoptosis. The activated AP-1 participates in cell transformation, tumor development and the process of learning and memory. Electrophoretic mobility shift assay (EMSA) is a technique to study the interaction between DNA-binding protein and its DNA-binding sequence by electrophoresis separation, according to the difference of molecular weight between pretein-DNA complex and DNA sequence. In the present study, AP-1 binding activity and related signal transduction pathways were studied by EMSA after intermittent exposure to GSM 1800 RF EMF for 24 h at SAR of 2 W/kg.The public pays attention to the issue whether the usage of MPs can lead to the symptoms of neurasthenia (i.e. disturbance of learning and memory) and induce the brain tumors. In this work, the primary cultured cerebral cortical and hippocampal neurons of rat were used to study the effects of MPs on the levels of gene expression and transcriptional factor in neurons.Part I. Gene Expression Profile of Rat Neurons Exposed to GSM 1800 MHz Radiofrequency Electromagnetic FieldsThe effects of 217 Hz modulated GSM 1800 MHz RF EMF on gene expression profile was studied in cultured cerebral cortical and hippocampal neurons of rats. After culturing for 13 days, the cerebral cortical and hippocampal neurons were divided into 2 groups, exposure group and control group (sham-exposure group), which were transported into the RF exposure system designed by Foundation for Information Technologies in Society (Zurich, Switzerland). The system's temperature was kept at 37+0.1°C during the whole exposure duration, and the temperature difference between sham- and RF-exposed cultures was less than 0.1 °C. Neurons were performed at a carrier frequency of 1800 MHz intermittently (5 min fields on/10 min fields off) exposed or sham-exposed for 24 h at an average SAR of 2.0 W/kg. In our previous study, Affymetrix Rat Neurobiology U34 arrays were used to detect 1322 candidate genes. About 600 genes were present, 24 genes were up-regulted and 10 genes were down-regulated after intermittent exposure at an average special absorption rate (SAR) of 2 W/kg for 24h. hi the present study, some defferential expressed genes detected from the Genechip results were chosen to validate by RPA and real-time PCR. To know the truth of the effects of RF EMF on gene expression of neurons, we analysised neurons mixed from one or more cotes and confirmed Map2 and Pip as RF EMF-responsive genes. Furthermore, we found the change of gene expression might also relate to individual difference, except for RF exposure, hi concluion, the present results indicated that RF EMF could change the gene expression of rat neurons under our experimental conditions, but different individual showed different responses in gene expression. In view of alike researches of RF EMF on cells, we believe neuron is a good cell model for RF EMF research.Part II: The effects of 1800 MHz Radiofrequency Electromagnetic Fields on AP-1 Binding Activity in Rat NeuronsTo investigate the possible mechanism of gene transcription changes induced by RF EMF, we examined the DNA binding behavior of the transcription factor AP-1 in rat neurons after exposure to 217 Hz modulated GSM 1800 MHz RF EMF by EMS A for 24 h. The cell culture and the exposure system were the same as those used in Part I. SAR was 2 W/kg.The rat neurons were detected at 15 min, 30 min, 1 h, 2 h and 4 h. during exposure to RF EMF. The results showed AP-1 binding activity changed after exposure to 1800 MHz RF EMF. Meanwhile, this change was associated with time. The complex formation increased after exposure for 30 min, reachied at a peak level after 1 h exposure, and was adjacent to basal level at 4 h exposure. To explore the roles of different signal transduction pathways in RF EMF-induced activation of AP-1, the multiple signal tranduction pathway inhibitors were used to treat the neurons before exposure. It was found that RF EMF-induced increase of AP-1 binding activity in rat neurons was dependent on the activation of JNK/SAPK and p38MAPK signal transduction pathway, but not on ERK, PKA, PKC and Ca2+. These data indicate that RF exposure activates a time-dependent AP-1-DNA binding activity through JNK/SAPK- and p38MAPK-related signal transduction pathway under the experimental conditions of this study.In conclusion, our research draws the following novel findings:1. In the present study, Map2 and Pip were confirmed as RF EMF-sensitive genes by RPA and real-time PCR in rat neurons after exposure to 1800 MHz RF EMF and individuals from different cote were found showing different sensitivity on gene expression to RF EMF.2. Meanwhile, it was found that AP-1-DNA binding activity can be induced by GSM 1800 MHz RF EMF through JNK/SAPK- and p38MAPK-related signal transduction pathway under our experimental conditions.The activation of AP-1 is time-dependent, increasing after exposure for 30 min, reaching a peak level after 1h exposure, and recovering to basal level after 4 h exposure. Our results illustrate that AP-1 is a RF EMF-sensitive factor.3. Primary cultured cerebral cortical and hippocampal neurons of rat was first applied in the researches of the effects of 1800 MHz RF EMF on functional genome analysis and the activation of transcription factor. Our results indicated that neuron may be sensitive to RF EMF and preferable to be the model cell for RF EMF research. The results provided with a new way looking for RF EMF sensitive cells.