Study On The Epigenetics And Mechanism Of 50 Hz Extremely Low Frequenc Electromagnetic Fields And 1800 MHz Radio Frequency Electromagnetic Fields

With the development of science and technology, the EMFs generated from the widely application of a number of electrical devices, has become the fourth major after water, air and noise pollution. The potential of ELF-EMFs to exert harmful biological effects on human health is of increasing concern. Extremely low frequency electromagnetic fields(ELF-EMF) and radio frequency electromagnetic field(RF-EMF) are the two common electromagnetic fields in our daily life. Many studies have reported that human health is associated with the exposure to ELF-EMF and RF-EMF, especially focusing on a variety of diseases, including children leukemia, brain cancer, Lou Gehrig's disease, Alzheimer's' disease and so on. The male reproductive system is one of the most sensitive organs to electromagnetic radiation. Many studies have confirmed that EMFs can alter the reproductive endocrine hormones and decrease the semen quality of humans and animals. The potential of ELF-EMF and RF-EMF to exert harmful biological effects on human health is of increasing concern. However, whether ELF-EMF and RF-EMF have obvious reproductive toxicity in male epidemiological survey are not consistent with the experimental research results. Furthermore, the ELF-EMF and RF-EMF exposure on male reproductive toxicity and mechanisms are still not clear. Therefore, elucidating the potential effects and mechanism of ELF-EMF and RF-EMF on the the male reproductive system is crucial. It is of great practical significance to fully understand the potential hazardous effects of EMFs exposure on human being and is beneficial for the prevention and treatment of the human health.DNA methylation is an extensively characterized mechanism for epigenetic regulation and plays an important role in the regulation of gene expression. DNA methylation occurs at the cytosine resi dues of Cp G dinucleotides by an enzymatic reaction that produces 5-methycytosine(5-m C), which is catalyzed by DNA methyltransferases(DNMTs). Generally speaking, aberrant hypermethylation of Cp G islands in gene promoter regions frequently negative regulation of its expression. DNA methylation plays an important role in the regulation of gene expression and is widely involved in the occurrence of various kinds of important biological activities and diseases. Mi RNA is another important way of epigenetic regulation. Mi RNAs are a class of small endogenous non-coding RNAs that are 21–25 nucleotides in length, are expressed in almost all biota, including animals, viruses and plants. Their primary biological function is the regulation of gene expression at the post-transcriptional level, mainly via binding to the 3'-untranslated region of target genes. The binding of mi RNAs to their target m RNAs may inhibit the translation or enhance the degradation of the target m RNAs. Mi RNAs participate in the regulation of various cellular processes, including cell proliferation, cell cycle and apoptosis. Mi RNA-mediated regulation has been implicated as an important epigenetic mechanism for regulatory pathways that are linked to various human cancers and male reproductive disorders.Emerging evidence has demonstrated the critical role of DNA methylation and mi RNAs in the control of reproductive functions, such as spermatogenesis and male fertility. In view of the fact that the potential hazardous effects of EMFs exposure on human being are remarkable, and the results of epidemiological and experimental studies remain contradictory. The possible reasons are controversial is that there is no standard exposure device, exposure frequency, intensity, time, detection method and the sensitivity of different cell. On the other hand, many studies have found that EMFs have no significant genetic toxicity. In order to investigate a plausible mechanism, this study intends to evaluate the epigenetic regulation of EMFs on DNA methylation and mi RNA at the cellular level start from the new perspective of the epigenetic; clarify the epigenetic mechanism and function of key DNA methylation regulation genes and mi RNA in the process of EMFs exposure; preliminary clear the epigenetic effects of EMFs exposure, the role of the characteristics and mechanisms of EMFs exposure, to provide a basis for the further understanding of the harmful biological effects of EMFs exposure and medical protection.The main research contents and results are as follows:1. Study on the epigenetic of 50 Hz ELF-EMF(1)In our study, mouse spermatocyte-derived GC-2 cells were intermittently exposed to a 50 Hz ELF-EMF for 72 h(5 min on/10 min off) at magnetic field intensities of 1 m T, 2 m T and 3 m T. Cell viability was assessed using the CCK-8 assay. Apoptosis and the cell cycle were analyzed with flow cytometry. Our data showed that the 50 Hz ELF-EMF did not affect the morphology and proliferation of GC-2 cells exposed to a 50 Hz ELF-EMF at different magnetic intensities of 1 m T, 2 m T and 3 m T for 72 h, 50 Hz ELF-EMF did not affect the apoptosis or cell cycle of GC-2 cells. These results suggested that short-term exposure to ELF-EMF has no significant change in cell function in vitro.(2)Quantitative analysis of the global DNA methylation levels showed that 50 Hz ELF-EMF exposure can induce the alterations of genome-wide methylation. DNA methylation in GC-2 cells were lower than the sham-exposure group at magnetic field intensity of 1 m T and were higher than the sham-exposure group at magnetic intensity of 2 m T and 3 m T. These data showed that the DNA in GC-2 cells acquired aberrant methylation pattern exposed to 50 Hz ELF-EMF exposure. Preliminary mechanism suggests that 50 Hz ELF-EMF exposure can induce the alterations of the expression of DNMTs in GC-2 cells. That is, DNMTs might play an important role in the global methylation alterations in GC-2 cells of 50 Hz ELF-EMF exposures.(3)Through DNA methylation chip analysis, differentially methylated genes was screened and the DNA methylation profiling was established. There were a total of 400 differentially methylated sites in the exposed group compared with the control group, indicated that ELF-EMF exposure may produce biological effects by regulating the methylation of related genes. The methylation status of differential methylation sites was confirmed by methylation-specific PCR. Our results showed that Nod1?Lrrc9?Tagln showed hypermethylation in GC-2 cells, the m RNA expression of those three genes was downregulated. These related genes cannot only be used as candidate markers for ELF-EMF exposure, but also play an important role in the biological effects induced by ELF-EMF.(4)Through gene expression chip analysis, there were a total of 84 differentially expression genes(including 44 genes upregulation and 40 downregulation) in the 1 m T exposed group compared with the control group. In 3m T, the altered genes is 324, including 235 increase and 89 decrease. Network analyses were used to find the differentially expressed and target key genes. Maoa, Bhmt, Gng8, Ugt2b34, Dgat1, Adh1 may be target genes in GC-2 cells at magnetic intensity of 1 m T. Cyp3a11, Ugt2b34, Adcy5, Ptgs1, Cyp2b23 may be closely related with the epigenetic of 50 Hz ELF-EMF exposure at magnetic intensity of 3 m T. Through pathway analysis, we found that these genes are involved in multiple signaling pathways, such as immune stress, histone modification, oxidative stress, etc, and these pathways could play significant roles in the biological effects of ELF-EMFs. 50 Hz ELF-EMF might induce some related signaling pathways and produce biological effects. Our study provided some data for the further study.(5)50 Hz ELF-EMF did affect the expression of mi RNAs in the GC-2 cells. We identified 55 mi RNAs whose expression markedly changed in response to ELF-EMF exposure, Of the 19 differentially expressed mi RNAs in the 1 m T exposure group, 7 mi RNAs were upregulated, while 12 were downregulated. Of the 36 differentially expressed mi RNAs in the 3 m T-treated groups, 9 mi RNAs were upregulated, and 27 were downregulated. A network analysis was performed to predict putative mi RNAs and their target genes. We found that mi R-211-3p, mi R-494-3p, mi R-669f-3p and mi R-1907 may represent important mi RNAs in GC-2 cells at a magnetic field intensity of 1 m T. However, mi R-30e-5p, mi R-210-5p, mi R-224-5p, mi R-196b-5p, mi R-504-3p, mi R-669c-5p and mi R-455-3p may be closely related to the epigenetic mechanism associated with ELF-EMF exposure at a magnetic field intensity of 3 m T. GO term and KEGG pathway annotation based on the mi RNA expression profile showed that many of these signaling pathways have been shown to participate in the mi RNA-mediated regulation of signaling pathways, including the m TOR signaling pathway, circadian rhythms, the p53 signaling pathway, long-term depression and the MAPK signaling pathway. These results also suggested that these altered mi RNAs could serve as potential biomarkers of ELF-EMF exposure.(6)We showed for the first time that mi R-26b-5p is differentially expressed in GC-2 cells exposed to different magnetic field intensities of a 50 Hz ELF-EMF. 50 Hz ELF-EMF exposure does not alter the methylation status of the mi R-26b-5p host gene CTDSP1. Mi R-26b-5p has no obvious influence on GC-2 cell growth. Mi R-26b-5p does not induce apoptosis or cell cycle arrest in GC-2 cells. However, the overexpression of endogenous mi R-26b-5p significantly decreased the percentage of G0/G1 phase cells and slightly increased the percentage of S phase cells compared to the control group after 50 Hz ELF-EMF exposure. These results suggest that there is obvious interaction between mi R-26b-5p and 50 Hz ELF-EMF. Further mechanism analysis showed that CCND2 is a direct target of mi R-26b-5p. 50 Hz ELF-EMF and mi R-26b-5p can alter the m RNA expression and protein expression of CCND2-mediated cell cycle regulation. mi R-26b-5p deregulation may play an important role in the regulation of the cell cycle in GC-2 cells that were exposed to 50 Hz ELF-EMF.2. Study on the epigenetic of 1800 MHz RF-EMF(1)In our study, mouse spermatocyte-derived GC-2 cells were intermittently exposed to 1800 MHz RF-EMF in GSM-Talk mode at specific absorption rate(SAR) values of 1 W/kg, 2 W/kg and 4 W/kg for 72 h. Cell viability was assessed using the CCK-8 assay. Apoptosis and the cell cycle were analyzed with flow cytometry. We found that Exposure to 1800 MHz RF-EMF does not influence the morphology and cell growth of GC-2 cells. Exposure to 1800 MHz RF-EMF does not induce apoptosis or cell cycle arrest in GC-2 cells.(2)Dot-blot analysis of the 5-m C levels in DNA showed that the DNA methylation in the GC-2 cells exposed to 1800 MHz RF-EMF at a SAR value of 4 W/kg was slightly greater than that in the sham group. However, no significant difference was found for the levels of 5-m C in the DNA of GC-2 cells exposed to a 1 or 2 W/kg RF-EMF for 72 h. 1800 MHz RF-EMF can alter the m RNA and protein expression of DNMTs. DNMTs may play a crucial role in establishing and maintaining DNA methylation patterns in the the process of the whole genome methylation level change induced by 4 W/kg RF-EMF.(3) The results of the genome-wide profiling showed that a total of 132 differentially methylated sites(54 hypermethylation and 78 hypomethylation) were found for the comparison of cells exposed to 4 W/kg RF-EMF for 72 h to the sham group. Cycs, Xpnpep3 and Nmur2 were hypermethylated in GC-2 cells after the 4 W/kg RF-EMF exposure. The m RNA expression of those three genes was downregulated after 4 W/kg RF-EMF exposure, suggesting that DNA methylation might be involved in the regulation of m RNA expression.(4)We identified 17 mi RNAs whose expression levels were markedly altered in response to 1800 MHz RF-EMF exposure. We performed network analysis to predict putative mi RNAs and their target genes using the Target Scan software. We found that mi R-19a-3p, mi R-291b-5p, mi R-7684-5p, mi R-6958-3p, mi R-344g-5p, mi R-669 n and mi R-1896 may be important mi RNAs in GC-2 cells after exposure to 4 W/kg RF-EMF.(5)Affymetrix microarray analysis was used to establish the gene expression profiles. A total of 511(fold change > 1.5) genes were differentially expressed(255 upregulated genes and 256 downregulated) after 4 W/kg RF-EMF exposure for 72 h. Signal-Net analyses Ugt2a1 and Cyp2b10 have the highest betweenness centrality and thus the strongest capability to mediate signaling pathways. GO analyses and KEGG pathway analysis based on the m RNA expression profile showed innate immune response, cellular response to interferon-beta, G-protein coupled receptor signaling pathway and biological process could play significant roles in the biological effects of 1800 MHz RF-EMF, suggested that RF-EMF exposure exerted some certain effects on some basic life activities.(6)We further analyzed the mechanism of RF-EMF. 4 W/kg RF-EMF exposure could significantly alter the expression levels of some genes, such as IL1?, IL6, IL10, Psg19, Oas1 g, and Cxcl10, associated with the innate immune response. The levels of Olfr50, Olfr128, Olfr167, Olfr169, Olfr453, Olfr747, Olfr765, and Olfr969 were remarkably increased in GC-2 cells exposed to 4 W/kg RF-EMF for 72 h compared with the sham group. These results indicated that RF-EMFs could change some gene expression levels and activate the immune response signaling and Olfactory transduction signaling pathways. These signaling pathways might play significant roles in the biological effects of 1800 MHz RF-EMF.In conclusion, our results showed that the relative high intensity of 50 Hz ELF-EMF and 1800 MHz RF-EMF with intermittent exposure could significantly induce alterations in genome-wide methylation and the expression of mi RNAs in GC-2 cells. 50 Hz ELF-EMF and 1800 MHz RF-EMF exerted obvious epigenetic effects. We used genomic science technology and biological information method to screen and determine the key DNA methylation regulation genes and mi RNA exposed to the two different frequency EMFs. Signal-Net analyses were used to identify and establish differentially expressed and target key genes network. These results indicate that epigenetics-mediated regulation of signaling pathways might play significant roles in the biological effects of 50 Hz ELF-EMF and 1800 MHz RF-EMF exposure. This study provides a reliable basis for the future research of EMFs.