ID Number		762
Study Type		In Vitro
Model		EMF exposure of human endothelial cells [900, 1800 MHz (GSM)]and yeast cells (50 Hz ELF-MF and 1800 MHz) and analysis of gene expression.
Details		Human EA.hy926 endothelial cells were exposed to 900 MHz (GSM) RF in culture with an SAR ranging from 1.8-2.5 W/kg (later revised to ~2.8 W/kg) for 1 hour and analyzed for gene expression changes using biochip microarrays. Protein expression and phosphorylation was also evaluated using 2d gels. RF exposure resulted in a variety of changes. At 2 W/kg, a 3 fold increase in overall number of phosphorylated proteins was observed, with notable increases in expression and/or phosphorylation of MAPK p38 and hsp27. Gene expression analysis on microarrays indicated that exposure caused a decrease in 120 targets and an increase in 75 targets. Caspase-3 activity and DNA content measurement at 1, 4, 24, 48, and 72 hours did not indicate an induction of apoptosis associated with RF exposure. In a subsequent presentation at the 2002 BEMS meeting in Quebec, the authors confirmed their observations and speculated on possible connections with apoptosis, brain cancer, and blood brain barrier effects (hsp27 phosphorylation of actin might result in stabilization of endothelial cell stress fibers shrink cells and increase BBB permeability). In a recent 2004 paper in Proteomics, similar data were presented, as well as increased expression of vimentins (class III- indeterminate filaments), and decreased expression of a mitochondrial enzyme (which may affect energy production) and decreased expression of a component of a heterogeneous nuclear ribonucleoprotein complex (which may affect protein translation). Other reported changes were found in other cytoskeletal proteins (fascin, alpha-actinin-1, cofilin, destrin, filamin-A, and tubulin-alpha2). In a 2006 paper in Proteomics, the authors performed additional studies on 2 similar lines of EA.hy926 cells slit in 1997 and propagated in different laboratories. The authors report RF exposure as above at 2.8 W/kg again increased and decreased several genes, but interestingly the pattern of change in the two related cell lines differed significantly. In a study exposing these same cells to 1800 MHz (GSM, GSM+DTx) at 2 W/kg, the investigators reported 8 proteins that appeared to have altered expression levels on 2-D gels, including spermidine synthase (regulates amine and bioamine biosynthesis), a glucose regulated member of the HSP70 family, and a proteasome subunit alpha type 1. However the result for the HSP70 related protein could not be repeated on Western blots. The authors conclude that a broad array of changes in stress response genes and proteins may be initiated by RF exposure, and that subtle differences between cell lines may be responsible for the lack of replication. AUTHORS' ABSTRACT: Chen et al. 2012 (IEEE #5186): The potential health hazard of exposure to electromagnetic fields (EMF) continues to cause public concern. However, the possibility of biological and health effects of exposure to EMF remains controversial and their biophysical mechanisms are unknown. In the present study, we used Saccharomyces cerevisiae to identify genes responding to extremely low frequency magnetic fields (ELF-MF) and to radiofrequency EMF (RF-EMF) exposures. The yeast cells were exposed for 6 h to either 0.4 mT 50 Hz ELF-MF or 1800 MHz RF-EMF at a specific absorption rate of 4.7 W/kg. Gene expression was analyzed by microarray screening and confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR). We were unable to confirm microarray- detected changes in three of the ELF-MF responsive candidate genes using RT-PCR (P > 0.05). On the other hand, out of the 40 potential RF-EMF responsive genes, only the expressions of structural maintenance of chromosomes 3 (SMC3) and aquaporin 2 (AQY2 (m)) were confirmed, while three other genes, that is, halotolerance protein 9 (HAL9), yet another kinase 1 (YAK1) and one function-unknown gene (open reading frame: YJL171C), showed opposite changes in expression compared to the microarray data (P < 0.05). In conclusion, the results of this study suggest that the yeast cells did not alter gene expression in response to 50 Hz ELF-MF and that the response to RF-EMF is limited to only a very small number of genes. The possible biological consequences of the gene expression changes induced by RF-EMF await further investigation. AUTHOR'S ABSTRACT: Leszczynski 2014 (IEEE #5447): Acute biological effects caused by the exposure to high doses of radiation, either ionizing or nonionizing, are relatively well-known but the delayed effects, occurring decades after exposure, are difficult to predict. The knowledge of the acute and delayed effects of the low doses of ionizing radiation (e.g. bystander effect) or nonionizing radiation (e.g. radiation emitted by wireless communication devices) is not yet reliably established. Often the acute effects of low doses are small and difficult to discover and replicate in scientific studies. Chronic effects of prolonged exposures to low-dose radiation for decades are virtually unknown and often not possible to predict on the basis of the knowledge gained from acute exposures to high doses of radiation. Physiological significance of the biological effects induced by low doses of radiation is not known. The same lack of predictability of outcomes applies to the delayed effects of high dose radiation exposures. Proteomics, supplemented with other omics techniques, might be the best way forward to find out the target molecules of radiation, the biomarkers of radiation exposure and the physiological and health significance of the acute and delayed biological effects caused by the exposures to high- and low-dose radiation. However, the currently available database of radiation effects on proteomes is far too small to be useful in formulation of new hypotheses concerning health consequences of radiation exposures.
Findings		Effects (inconsistent results)
Status		Completed With Publication
Principal Investigator		STUK, Helsinki, Finland - dariusz.leszczynski@stuk.fi
Funding Agency		EU, VERUM, Germany, Nat'l Res Prog, Finland, REFLEX, EU 5th Framework, Europe
Country		FINLAND
References		Nylund, R et al. Proteome Sci., (2010) 8:52-(7 pages) Nylund, R et al. Proteomics, (2006) 6:4769-4780 Leszczynski , D et al. Proteomics, (2004) 4:426-431 Nylund , R et al. Proteomics, (2004) 5:1359-1365 Leszczynski, D et al. Differentiation., (2002) 70:120-129 Nylund , R et al. Proteome Sci., (2010) 8:52- Chen, G et al. Bioelectromagnetics., (2012) 33:550-560 Leszczynski, D Proteomics., (2014) 14:481-488
Comments		At the 2002 BEMS meeting, the authors admitted during the following question period that SARs may have been much higher than 1.8 - 2.5 W/kg (higher values were determined by other experimental and numerical methods), and additionally confirm that they did not perform a non-MW heat control. It is likely that peak "hot spots" occured at SARs above the reported average of 2 W/kg (1.8 - 2.5 W/kg). SAR characterization was performed by taking measurements outside the culture incubator at ambient (~25 C) temperature, while actual biological experiments were performed using the same exposure level in a 37 C incubator. Further, simulated SAR peaks were actually 3.6 W/kg, and Vitek probe measurements placed the peak SAR at 5 W/kg. Importantly, the authors did not perform non-RF heat controls to see if the pattern of expression and phosphorylation changes was the same or different as observed with RF exposure. Also of note, this study is NOT supported by de Pomerai (#230) because the present study reported a threshold for hsp phosphorylation of ~2 W/kg, while the SAR in the de Pomerai study was very low (on the order of uW/kg). THis is part of the REFLEX project of the EU 5th Framework program. There is clearly no common mechanism with the latest gene expression data (vimentin, Mitochondrial enzymes, RNP subunits). Cotgreave (2005) comments: In an interesting recent addition to the field, Leszczynski et al. have provided perhaps some of the most relevant in vitro data thus-far reported, by studying the effects of 900-MHz emissions pulsed at GSM signal modulation schemes.... It should be mentioned here, however, that unpublished results presented by Poulltier de Gannes at a recent workshop... in Helsinki, Finland (April 2004), failed to reproduce the above results..."