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Whether or not low intensity radiofrequency electromagnetic field exposure RF-EME associated with the use of GSM mobile phones can have direct effects on cells is a matter of debate. The energy transferred by these fields is certainly too weak to ionize molecules or break chemical bonds Adair So called thermal effects on cells, caused by energy transfer, are directly related to the specific absorption rate SAR and are well understood.

Investigations of athermal cellular effects caused by low intensity exposure, in contrast, have generated conflicting data Belyaev This applies to epidemiologic studies and to laboratory investigations focusing on cellular effects such as DNA damage or proteome alterations Blank Early epidemiologic studies on mobile phone use did not reveal an associated health risk Rothman et al. Subsequent studies described some evidence for enhanced cancer risk Kundi et al. More recently, an increased brain cancer risk associated with intensive phone use over many years was discussed Hardell et al.

Sadetzki et al. Repacholi et al. Their results were, however, not reproduced in follow-up studies Utteridge et al. An enhancement of genotoxicity was described Maes et al.

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Several studies have investigated effects of radiation exposure on specific proteins. Thus, Yilmaz et al. Sanchez et al. Results from Nylund and Leszczynski support the hypothesis that mobile phone radiation can affect the cytoskeleton and the physiological functions that are regulated by the cytoskeleton. More recently, Karinen et al.

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Blank has reviewed examples of direct molecular conformation changes caused by radio frequency radiation exposure. Friedman et al. The lack of consensus with regard to the difficulty to reproduce effects of RF-EME may to some extent reflect the large number of experimental variables, such as frequency, amplitude, modulation Litovitz et al. In the present study, we measured the impact of RF-EME on the rate of synthesis of a range of proteins.

Because any challenge to cells may eventually trigger an adaptive response, the measurement of protein synthesis offers a highly sensitive screening method to detect subtle effects. The applied methodology was based on metabolic labeling cells during RF exposure and subsequent resolution of protein extracts by two-dimensional electrophoresis in order to measure de novo protein synthesis and total protein amounts Gerner et al. To investigate whether or not cell types respond differently, we exposed different kinds of cells including proliferating Jurkat cells, cultured fibroblasts as well as quiescent and inflammatory stimulated primary human white blood cells.

The exposure unit has two wave guides, which serve as chambers for cell growth and RF exposure. ELF magnetic fields may actively contribute cellular effects Mild et al. However, in our experiments, the background fields were identical between sham and real exposure and therefore cannot be held responsible for the observed differences. Cells were either exposed or mock-exposed to RF-EM under blinded conditions, followed by protein extraction and analyses.

RF exposure was controlled by a computer program, which switched on the exposure in one waveguide while the other served as exposure control. The exposure settings were recorded in a coded file, and after the biochemical analysis of exposed and control cells, decoding was carried out by a coauthor HPH who was not involved in the exposure and biochemical analysis.

In this manner, we excluded any direct and indirect investigator bias of the results. In this study, we used modulations closely reflecting the technical specifications of GSM A GSM signal is modulated, i. With the chosen parameters, the controlled temperature difference between the two chambers control and real exposure was below 0. It allowed us to investigate the proteomes of the very same cell line and culture conditions, which upon radiation revealed DNA breaks Diem et al.

Peripheral blood mononuclear cells white blood cells—WBC were isolated from heparinized whole blood obtained from healthy donors mixed with 2 vol. The interface cells were washed and resuspended in autologous donor plasma. Cells were metabolically labeled with 0. After incubation and labeling of cells, cytoplasmic proteins were isolated as follows.

Cells were lysed in 0. After ethanol precipitation, the pelleted cytoplasmic protein fraction was directly solubilized in sample buffer. High-resolution 2D gel electrophoresis was carried out as described previously Gerner et al. The protein samples were dissolved in sample buffer 7. To optimize the solubilization of proteins, we saturated the protein solution with solid urea. Protein concentrations were determined using a standard Bradford assay. For identification of 2D gel spots, protein samples of unlabeled cells were separated by 2D-PAGE followed by silver staining as described Gerner et al.

Gels were warped to a reference gel with the TT S2S software version Spot assignment, background correction, normalization and statistical calculations analysis of variance, ANOVA were performed using this software package. Factors indicating up-regulation of proteins in 2D gels were obtained using normalized integrated spot intensities. For most accurate quantification, we only considered spots with an integrated intensity at least three-fold higher than the corresponding spot background value.

Integrated intensities from fluorescence detection and autoradiography were normalized against the sum of all matched spots. Afterward the gel pieces were dried with acetonitrile and rapidly dried in a vacuum centrifuge Heto, Denmark. The dried gel spots were treated with trypsin 0. After this, the extracted peptide solutions were concentrated to a volume appropriate for further analysis. Proteins were identified by mass spectrometric analysis. Due to previous chemical modification, carbamidomethylation of cysteines was set as fixed modification.

No other modifications were considered here.

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Peptide scores were essentially calculated from sequence tag lengths, but also considered mass deviations. To assess the reliability of the peptide scores, we performed searches against the corresponding reverse database. All spots were identified with at least two different peptides including one scoring at least higher than The details of protein identifications, including peptide sequences, peptide scores and sequence coverage are provided in the electronic supplementary data.

In each experiment, we compared proteins from cells kept under identical culture conditions. The only difference was that they were exposed under sham or real conditions.

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  7. The gel from sham exposed cells reference was compared to the gel from the cells with real exposure, using the TT S2S software version Spot assignment, background correction, normalization and statistical calculations one way analysis of variance, ANOVA, calculated from three independent experimental replicates per group were performed using this software package.

    The separated proteins were q uantified for protein amounts fluorescence detection and protein synthesis 35 S-autoradiography. The spot pattern obtained was very highly reproducible: spots were consistently detected in six gels from three independent experiments, each with exposed and corresponding control cells. The average standard deviation of fluorescence spot intensities Fluorescence spot intensities for some individual proteins appeared to reveal an increased level in response to RF-EME.

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    Application of strict spot quantification criteria, however, indicated that there were no significant differences between RF-EME-exposed and control cells. Autoradiographs of the same gels, however, revealed significant differences in the rate of de novo synthesis of several proteins greater than 2 fold between RF-EME and control cells. Actually all detectable protein spots displayed an increased 35 S incorporation rate. While protein amounts did not show significant alterations, protein synthesis was generally increased due to RF-EME.

    EIF3 p170, a Mediator of Mimosine Effect on Protein Synthesis and Cell Cycle Progression

    We categorized a protein as specifically up-regulated if the normalized integrated 35 S autoradiograph spot intensity was at least two-fold greater than the corresponding control cell spot with an ANOVA P -value of less than 0. Relative spot intensities from controls a , c , e and RF-EME exposed cells b , d , f are depicted as spot heights to demonstrate the specific induction of some proteins relative to the local spot environment.

    Identification details of isolated 2D gel spots. After tryptic digestion and peptide separation by nano-flow liquid chromatography, isolated peptides were fragmented in an ion trap mass spectrometer. Thirteen of the fourteen proteins whose rate of de novo synthesis was increased in Jurkat cells were also synthesized at a higher rate in fibroblasts. This finding suggests that the proteome alterations in responsive cells induced by RF-EME exposure are characteristic for this kind of cell stress. Inflammatory stimulation of WBCs by treatment with lipopolysaccharide and phytohaemagglutinin increased the level of protein synthesis by these cells compare Fig.

    The proteome alterations were, however, less compared to those observed in Jurkat cells and fibroblasts. We used a highly sensitive method of measuring protein synthesis rates and protein amounts to investigate the potential effects of low-intensity mobile phone radiation exposure on cells. Although the observed changes reached no statistical significance at short exposure times, we observed some trends consistent with but also extend observations made by Nylund and Leszczynski , who used the same exposure system, but only measured protein amounts and not de novo synthesis.

    Usefully, our results appear to reconcile a number of conflicting previous findings. Inflammatory activation of WBC induces T-cell proliferation and consequently an increased rate of protein synthesis Traxler et al. Thus, our data suggest that proliferating cells with high protein synthesis rates are more sensitive to RF-EME than cells with lower protein production.

    Many studies have been performed with quiescent white blood cells, which were also insensitive under our experimental conditions. Second, the exposure time seems to be a critical factor. An 8-h exposure was required to obtain reproducible and significant effects, a time much longer than the longest exposure time used in most other studies. Third, the determination of protein amounts by spot integration is not very precise.

    Silver staining in particular, does not produce reliable quantitative data White et al.

    Consequently, subtle alterations may easily be missed due to limited sensitivity. Jurkat cells: proteins displaying a specific up-regulation of 35 S incorporation by real exposure. Proteins are depicted in Fig. Proteins printed in italics did not show relevant alterations. Accession numbers and protein names are according to the SwissProt database. Details of mass analysis results are provided electronically in the supplementary data. Interestingly, we found the same cells reactive e. This may suggest a common underlying mechanism between DNA breaks and increased protein synthesis in reactive cells.

    With this exposure regime, the temperature difference between exposed cells and control cells was less than 0. TCP-1 family members Gerner et al.

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    We conclude that the warming of the cell cultures caused by RF exposure was too low to account for the present observations. Most of the proteins found to be induced by RF-EME are chaperones, which are mediators of protein folding. Since the applied electromagnetic fields were very weak, the direct and active denaturation of existing proteins by RF-EME exposure appears unlikely to underlie the observed increased level of protein synthesis.

    Resonance phenomena may concentrate radiation exposure-mediated physical energy on hot spots and have already been suggested to cause biological effects Belyaev Indeed, exposure to low frequency electromagnetic fields caused effects, which were reduced by noise signals Litovitz et al. Hydrogen bonds are known to resonate with microwaves. Folding of newly synthesized proteins in the endoplasmic reticulum occurs via a process involving the formation of intermediate folded structures Arai and Kuwajima mediated by several chaperones Deuerling and Bukau This process could potentially respond in a very sensitive fashion to radiation-induced excitation of hydrogen bonds as this could cause a temporary disturbance of spatial orientation.

    An increased rate of inappropriate folding of newly synthesized proteins would not affect existing proteins and thus render cell function intact for some time unless key labile proteins are affected. Furthermore, such a mechanism would not necessarily have a significant impact on total protein amounts.

    However, later on it would increase the protein synthesis rate in response to an increased rate of turnover of the newly folded proteins. This interpretation plausibly explains the reported increased level of protein synthesis. Essentially all detectable proteins displayed an increased synthesis rate, which indicates a general compensatory response, e.

    Chaperones such as kDa glucose-regulated protein, heat-shock proteins and T-complex protein 1 family members are directly involved in protein folding and assist folding of newly synthesized proteins Deuerling and Bukau Neutral alpha-glucosidase AB is an important endoplasmic reticulum protein responsible for quality control and glycoprotein processing Ellgaard and Helenius Ubiquitin carboxyl-terminal hydrolase 14, also termed deubiquitinating enzyme 14, is required for proteasomal processing of ubiquitinated substrates Koulich et al.

    The 26S protease regulatory subunit 6B is also involved in ATP-dependent degradation of ubiquitinated proteins and in transcriptional regulation Choi et al. Elongation factor 2 is actually indispensable for protein synthesis Perentesis et al. Our data complement those of Lee et al. This is also in agreement with Sanchez et al. Our data describe cell responses to RF-EME exposure specifically observed in actively proliferating cells.

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