Effects Of Electromagnetic Fields On Protein Expression Profiles In Cells

Electromagnetic radiation has become a rapid-increasing and universal-existing environment risk factor due to the rapid development of mobile communication and electric power transmission. The health risk assessment of electromagnetic radiation will protect population from the possible hazards and prmote a sustaining development of technology, economics and society. Epidemiological studies have identified a potential positive association between exposure to extremely low-frequency (ELF) electromagnetic fields and leukaemia and breast cancer. Radiofrequency (RF) electromagnetic fields from mobile phones might affect the function of the central nervous system and induce malignant pathological changes such as brain cancer. The epidemiological findings drive the experimental studies on bioeffects and mechanisms of action of EMF. However, the data from in vivo and in vitro studies are inconsistent, and it is difficult to make a clear conclusion on the overall health impact induced by EMF. Moreover, lack of a coherent hypothesis for a mechanism by which EMF might interact with biological systems has limited such studies to a phenomenological rather than a mechanistic approach. There is thus pressing motivation to delineate the biological effects and the underlining mechanisms of low-energy EMF.Gene expression regulation is believed to play a role in the various physiological ang biochemical changes of biological system induced by EMF. Some studies have found EMF could alter the transcription of certain genes, including pro-oncogene, apopotosis-related gene, genes regulating the cell cycle and so on. For example, c-myc, c-jun, c-fos, p21. egr-1, bax and GADD45 were found to be ELF EMF-resposive genes, and Nurr1, bax, GADD45, hsp70, c-jun, c-myc and p21 were reported to be affected by RF EMF exposure. The cell behavior will be affected if the gene expression change results in variation of protein expression and/or modification. Therefore, exploring the effects of EMF on protein would be more direct to elucidate the biological effects of EMF at a cell or organism level. There are so far only a few studies looking at the protein expression or modification after exposing to EMF, and focusing on ornithine decarboxylase (ODC), heat shock proteins (HSP27/70), and some signalling proteins of certain signal transduction pathways. Those studies are hypothesis-driven, and might introduce bias while selecting end-points to be detected. Meanwhile, the selected end-points are scattered, and the experiments were not designed in a systematic way. Therefore, it is difficult to evaluate the EMF-induced bioeffects based on the obtained data. We reason it is necessary to analyze the effect of EMF on protein at a proteomic scale. Theoretically, a proteomics approach allows simultaneously monitoring hundreds or even thousands of proteins in a sample. In 2001 our laboratory and the Leszczynski group in Finland initiated the application of proteomics in EMF research.To evaluate the potential co-carcinogenic effect of EMF, and compare the effects of two different environmental prevailing EMF, the first part of this dissertation intend to reveal the differential protein expressions in human breast cancer cells (MCF-7) induced by 50 Hz ELF EMF or 1800 MHz RF EMF using two-dimensional electrophoresis (2-DE) technique, then identify differential expression proteins by mass spectrum analysis.Based on the results of the first part and other studies, we further reason it is necessary to scan EMF-sensitive cell types and set it up as the second part of the dissertation. It is realized that the bioeffects of EMF depend on many factors, such as exposure frequency, intensity, duration, and pattern. However, the origin and status of tested biological systems (cell, tissue) may be the most important factor to affect the results. Leszczynski and his colleagues, for example, found 38 protein spots with altered expression levels in the EA.hy926 cell line following to 900 MHz RF exposure for 1 h at SAR of 2.4 W/kg, whereas in the EA.hy926vl cell line (a subcloning of EA.hy926) 45 different protein spots showed altered (Nylund R, Leszczynski D. Mobile phone radiation causes changes in gene and protein expression in human endothelial cell lines and the response seems to be genome- and proteome-dependent. Proteomics, 2006, 6: 4769-4780). Sul exposed four cell lines of different origins to sinusoidal electromagnetic fields at 2 mT for 1, 3 or 6 hours per day. After 14 days, he found cell type-specific reaction to EMF (Sul AR, Park SN, Suh H. Effects of sinusoidal electromagnetic field on structure and function of different kinds of cell lines. Yonsei Med J, 2006, 47: 852-861). Thanks to these studies, the message is becoming clear: the diverse genetic backgroud of biological systems make them react differently to EMF, the key in EMF research is to identify EMF-sensitive cells and then explore the effects and mechanism of action of EMF. Therefore, the second part of my dissertation is to screen EMF-sensitive cell lines using 2-DE approach.Part I: The effects of EMF on protein profiles in human breast cancer cellsTo reveal the effect of ELF MF on protein expression, MCF-7 cells were exposed to 50 Hz, 0.4 mT ELF MF for 24 h. Immediately after the exposure and sham-exposure, proteins were extracted from the cells and subjected to be analyzed by 2-DE. The analysis of protein distribution in the gels was carried out with the aid of the PDQuest software, version 7.1. The results showed that 6 spots have been statistically significantly altered, and 19 additional spots were detected only in exposed group while 19 ones were detected only in control group. Three proteins were identified by LC-ESI-IT tandem MS as RNA binding protein regulatory subunit, proteasome subunit beta type 7 precursor, and tanslationally controlled tumor protein.To analyze the effect of RF EMF on protein expression, MCF-7 cells were exposed to 1800 MHz RF EMF modulated by 217 Hz (or sham-exposed) at different duration (1, 3, 6, 12 and 24 h), different intensities (SAR of 2 or 3.5 W/kg) and different patterns (5 min-on and 10 min-off exposure, or continuous exposure). After exposure or sham-exposure, total proteins were extracted and analyzed by 2-DE. The analysis of protein distribution in the gels was carried out as above. The results showed the protein expression changes induced by 1800 MHz RF EMF in MCF-7 cells were faint and depended on exposure intensity, duration and pattern.We further analyzed the protein expression change induced by RF EMF using fluorescence difference gel electrophoresis (DIGE). MCF-7 cells were intermittent exposed to 1800 MHz RF EMF at SAR of 3.5 W/kg for 3 h (under this exposure condition, we found 18 proteins expression were altered in 2-DE approach). The total proteins were extracted and separated by DIGE, and the three-color images were analyzed by the "Decyder" software. The results showed that 5 proteins were up-regulated by RF EMF. Three of these could be identified in MALDI TOF/TOF as CLIC 1 protein, translationally controlled tumor protein 1, and thiol-specific antioxidant protein.Part II: Screening EMF-sensitive cells types using 2-DEChinese hamster lung fibroblast cells line CHL, rat skin fibroblast cells line NIH3T3, rat pheochromocytoma cells line PC 12, human lens cells line SRA01/04, human amnion epithelial fibroblast cells line FL, human leukemic cell line HL60 and human skin fibroblast cell line HSF were exposed to 0.4 mT ELF MF for 24 h or 1800 MHz RF EMF at SAR of 3.5 W/kg for 3 h. The extracted proteins were separated using 2-DE respectively. Compare to sham-exposure group, ELF MF exposure induced 14 and 23 differentially expressed proteins in PC 12 and FL cells, representing 2.2 % and 3.2 % of the total detected protein spots, respectively. Only less than 1.4 % of the total protein spots were changed by ELF MF in other cell types. On the other hand, RF EMF exposure produced 20, 23 and 17 differentially expressed proteins in NIH3T3 cells, FL cells and HL60 cells representing 2.4 %, 3.5 % and 2.0 % of the total protein spots detected, respectively. Meanwhile, only less than 1.3 % of the total proteins were altered by ELF MF in other cells types. Combined the results of Part I, we concluded MCF-7, PC 12 and FL cells as ELF MF-sensitive cells; and NEH3T3, FL and HL60 cells as RF EMF-sensitive cells based on the absolute number of differentially expressed proteins and their ratios to the total protein spots detected.The main conclusions are:1. 0.4 mT 50 Hz ELF MF could significantly alter proteins expression in MCF-7 cells. The three identified proteins are related with the cellular cytoskeleton, implying cytoskeletal might be an interaction target to EMF.2. 1800 MHz RF EMF did not significantly alter protein expression in MCF-7 cells, suggesting this cell type react weakly to RF EMF. The protein expression changes induced by 1800 MHz RF EMF in MCF-7 depend on exposure intensity, duration and pattern.3. The genetic and/or epigenetic background of a biological system determines its response to EMF. Under the experimental conditions, we identified that MCF-7, PC12 and FL cells are ELF MF-sensitive cells, and NIH3T3, FL and HL60 cells are RF EMF-sensitive cells. Meanwhile different cell types have different response to EMF, and the same cell type reacts differently to different frequency of EMF.4. Proteomics approach is applicable in EMF research to investigate bioeffects and mechanism of action. However, due to the weak interaction of low intensity EMF with a biological system, and the low sensitivity of high through-put technology such as proteomics, there are defects in applying such a technique in elucidation EMF effects. It is urgert to develop more sensitive and more high through-put techniques, and to establish special technique criterion for using proteomics in EMF research. Due to the limitations of proteomics analysis, the produced candidates should be validated using non-HTST methods.5. After comparing the application of classical 2-DE and DIGE, we do not recommend DIGE as a better method in EMF research.Innovation points of this dissertation:1. We first employ proteomics approach in revealing the effects of EMF on protein expression in MCF-7 cells and in screening the EMF-responsive cell types. Thus, there is innovation in selecting experimental technique.2. We first report the differential proteins expression profile of MCF-7 cells induced by 0.4 mT 50 Hz ELF MF, and identified three ELF MF-responsive proteins3. We first report the MCF-7 cell line is not a sensitive cell line to 1800MHz RF EMF from a point of view of proteomics analysis.4. We first identify MCF-7, PC12 and FL cells are ELF MF-sensitive cells, and NIH3T3, FL and HL60 cells are RF EMF-sensitive cells under the current experimental conditions. 5. By conducting a parallel proteomics study on ELF MF and RF EMF, we conclude that different cells react differently to EMF, and the same cell type has different response to different frequency of EMF.