Effect of electromagnetic field exposure on the transcription of repetitive DNA elements in human cells.

Repetitive DNA (RE-DNA) was long thought to be silent and inert; only recent research has shown that it can be transcribed and that transcription alteration can be induced by environmental stress conditions, causing human pathological effects. The aim of this study was to determine whether exposure to radiofrequency electromagnetic fields (RF-EMF) could affect the transcription of RE-DNA. To this purpose, three different human cell lines (HeLa, BE(2)C and SH-SY5Y) were exposed to 900 MHz GSM-modulated RF-EMF at specific absorption rate of 1 W/kg or to sham. After exposure, mRNA levels of RE-DNA were evaluated through quantitative real-time PCR. The following RE-DNA types were investigated: Long Interspersed nucleotide Element 1, DNA alpha satellite and Human Endogenous Retroviruses-like sequences. When comparing cells exposed to RF-EMF versus control samples, different results were found for the three cell lines evaluated, indicating that RF-EMF exposure can significantly affect RE-DNA transcription and that the effects strongly depend on the cellular context and the tissue type. Further studies are needed to elucidate which molecular mechanisms could be involved.

Assessing the combined effect of extremely low-frequency magnetic field exposure and oxidative stress on LINE-1 promoter methylation in human neural cells.

Extremely low frequency magnetic fields (ELF-MF) have been classified as "possibly carcinogenic", but their genotoxic effects are still unclear. Recent findings indicate that epigenetic mechanisms contribute to the genome dysfunction and it is well known that they are affected by environmental factors. To our knowledge, to date the question of whether exposure to ELF-MF can influence epigenetic modifications has been poorly addressed. In this paper, we investigated whether exposure to ELF-MF alone and in combination with oxidative stress (OS) can affect DNA methylation, which is one of the most often studied epigenetic modification. To this end, we analyzed the DNA methylation levels of the 5'untranslated region (5'UTR) of long interspersed nuclear element-1s (LINE-1 or L1), which are commonly used to evaluate the global genome methylation level. Human neural cells (BE(2)C) were exposed for 24 and 48 h to extremely low frequency pulsed magnetic field (PMF; 50 Hz, 1 mT) in combination with OS. The methylation levels of CpGs located in L1 5'UTR region were measured by MassARRAY EpiTYPER. The results indicate that exposures to the single agents PMF and OS induced weak decreases and increases of DNA methylation levels at different CpGs. However, the combined exposure to PMF and OS lead to significant decrease of DNA methylation levels at different CpG sites. Most of the changes were transient, suggesting that cells can restore homeostatic DNA methylation patterns. The results are discussed and future research directions outlined.

Activity and expression of acetylcholinesterase in PC12 cells exposed to intermittent 1.8 GHz 217-GSM mobile phone signal.

PURPOSE: Due to its role in learning, memory and in many neurodegenerative diseases, acetylcholinesterase (AChE) represents an interesting endpoint to assess possible targets of exposure to radiofrequency electromagnetic fields (RF-EMF) generated by mobile phones. We investigated possible alterations of enzymatic activity, gene and protein expression of AChE in neuronal-like cells exposed to a 1.8 GHz Global System for Mobile Communication (GSM) modulated signal (217-GSM). MATERIALS AND METHODS: Rat PC12 cells were exposed for 24 h to 1.8 GHz 217-GSM signal. Specific adsorption rate (SAR) was 2 W/kg. AChE enzyme activity was assessed spectrophotometrically by Ellman's method, mRNA expression level was evaluated by real time polymerase chain reaction, and protein expression was assessed by Western blotting. RESULTS: AChE enzymatic activity increased of 1.4-fold in PC12 cells exposed to 217-GSM signal for 24 h, whilst AChE transcriptional or translational pathways were not affected. CONCLUSION: Our results provide the first evidence of effects on AChE activity after in vitro exposure of mammalian cells to the RF-EMF generated by GSM mobile phones, at the SAR value 2 W/kg. The obtained evidence promotes further investigations on AChE as a possible target of RF-EMF and confirm the ability of 1.8 GHz 217-GSM signal to induce biological effects in different mammalian cells.

An evaluation of genotoxicity in human neuronal-type cells subjected to oxidative stress under an extremely low frequency pulsed magnetic field.

The possible genotoxicity of extremely low frequency magnetic field (ELF-MF) exposure is still a controversial topic. The most of the reported data suggests that it alone does not affect DNA integrity, but several recent reports have suggested that sinusoidal ELF-MF may increase the effect of known genotoxic agents. Only a few studies deal with non sinusoidal ELF-MF, including pulsed magnetic field (PMF), which are produced by several devices. The aim of this study is to investigate whether PMF exposure can interfere with DNA damage and repair in the presence of a genotoxic oxidative agent in neuronal type cells. To this purpose gamma-H2AX foci formation, which is a sensitive marker of DNA double strand breaks (DSB), was investigated at different points of time (1, 24, 48, 72h) after the H2O2 treatment (300µM for 1h) under PMF exposure (1mT, 50Hz) in human neuroblastoma BE(2)C cells. Moreover, cytotoxicity evaluation, by MTT assay and cell cycle analysis, was performed at various points of time after the treatment. Taken together, results suggest that PMF exposure does not interfere with genotoxicity and cytotoxicity induced by oxidative stress.

Effects of the exposure to intermittent 1.8 GHz radio frequency electromagnetic fields on HSP70 expression and MAPK signaling pathways in PC12 cells.

PURPOSE: We previously reported effects on heat shock protein 70 (HSP70) mRNA expression, a cytoprotective protein induced under stressful condition, in human trophoblast cells exposed to amplitude-modulated Global System for Mobile Communication (GSM) signals. In the present work the same experimental conditions were applied to the rat PC12 cells, in order to assess the stress responses mediated by HSP70 and by the Mitogen Activated Protein Kinases (MAPK) in neuronal-like cells, an interesting model to study possible effects of mobile phone frequencies exposure. MATERIALS AND METHODS: HSP70 gene expression level was evaluated by reverse transcriptase polymerase chain reaction, HSP70 protein expression and MAPK phosphorylation were assessed by Western blotting. PC12 cells were exposed for 4, 16 or 24 h to 1.8 GHz continuous wave signal (CW, carrier frequency without modulation) or to two different GSM modulation schemes, GSM-217Hz and GSM-Talk (which generates temporal changes between two different GSM signals, active during talking or listening phases, respectively, thus simulating a typical conversation). Specific adsorption rate (SAR) was 2 W/kg. RESULTS: After PC12 cells exposure to the GSM-217Hz signal for 16 or 24 h, HSP70 transcription significantly increased, whereas no effect was observed in cells exposed to the CW or GSM-Talk signals. HSP70 protein expression and three different MAPK signaling pathways were not affected by the exposure to any of the three different 1.8 GHz signals. CONCLUSION: The positive effect on HSP70 mRNA expression, observed only in cells exposed to the GSM-217Hz signal, is a repeatable response previously reported in human trophoblast cells and now confirmed in PC12 cells. Further investigations towards a possible role of 1.8 GHz signal modulation are therefore advisable.

ELF magnetic fields tuned to ion parametric resonance conditions do not affect TEA-sensitive voltage-dependent outward K(+) currents in a human neural cell line.

Despite the experimental evidence of significant biological effects of extremely low frequency (ELF) magnetic fields (MFs), the underlying mechanisms are still unclear. Among the few mechanisms proposed, of particular interest is the so called "ion parametric resonance (IPR)" hypothesis, frequently referred to as theoretical support for medical applications. We studied the effect of different combinations of static (DC) and alternating (AC) ELF MFs tuned on resonance conditions for potassium (K(+)) on TEA-sensitive voltage-dependent outward K(+) currents in the human neuroblastoma BE(2)C cell line. Currents through the cell membrane were measured by whole-cell patch clamp before, during, and after exposure to MF. No significant changes in K(+) current density were found. This study does not confirm the IPR hypothesis at the level of TEA-sensitive voltage-dependent outward K(+) currents in our experimental conditions. However, this is not a direct disprove of the hypothesis, which should be investigated on other ion channels and at single channel levels also.

Non-thermal radio frequency and static magnetic fields increase rate of hemoglobin deoxygenation in a cell-free preparation.

The growing body of clinical and experimental data regarding electromagnetic field (EMF) bioeffects and their therapeutic applications has contributed to a better understanding of the underlying mechanisms of action. This study reports that two EMF modalities currently in clinical use, a pulse-modulated radiofrequency (PRF) signal, and a static magnetic field (SMF), applied independently, increased the rate of deoxygenation of human hemoglobin (Hb) in a cell-free assay. Deoxygenation of Hb was initiated using the reducing agent dithiothreitol (DTT) in an assay that allowed the time for deoxygenation to be controlled (from several min to several hours) by adjusting the relative concentrations of DTT and Hb. The time course of Hb deoxygenation was observed using visible light spectroscopy. Exposure for 10-30 min to either PRF or SMF increased the rate of deoxygenation occurring several min to several hours after the end of EMF exposure. The sensitivity and biochemical simplicity of the assay developed here suggest a new research tool that may help to further the understanding of basic biophysical EMF transduction mechanisms. If the results of this study were to be shown to occur at the cellular and tissue level, EMF-enhanced oxygen availability would be one of the mechanisms by which clinically relevant EMF-mediated enhancement of growth and repair processes could occur.

Assessing LINE-1 retrotransposition activity in neuroblastoma cells exposed to extremely low-frequency pulsed magnetic fields.

Mobile genetic elements represent an important source of mutation and genomic instability, and their activity can be influenced by several chemical and physical agents. In this research we address the question whether exposure to extremely low-frequency pulsed magnetic fields (EMF-PMF) could affect the mobility of the human LINE-1(RP) retrotransposon. To this purpose, an in vitro retrotransposition assay was used on human neuroblastoma BE(2) cells exposed for 48h to 1mT, 50Hz PMF, or sham-exposed. Moreover, since it is well known that retrotransposition causes DNA double-strand breaks (DSB), an estimation of γ-H2AX foci, which is a marker of DNA DSB, was carried out on PMF- and sham-exposed samples. The results show that PMF-exposed cells had a lower number of both retrotransposition events and DNA DSB compared with sham-exposed samples. These results suggest that exposure to PMF can interfere with retrotransposition activity by inducing a decrease of retrotransposition events.

Effect of extremely low frequency magnetic field exposure on DNA transposition in relation to frequency, wave shape and exposure time.

PURPOSE: To examine the effect of extremely low frequency magnetic field (ELF-MF) exposure on transposon (Tn) mobility in relation to the exposure time, the frequency and the wave shape of the field applied. MATERIALS AND METHODS: Two Escherichia coli model systems were used: (1) Cells unable to express ß-galactosidase (LacZ(-)), containing a mini-transposon Tn10 element able to give ability to express ß-galactosidase (LacZ(+)) upon its transposition; therefore in these cells transposition activity can be evaluated by analysing LacZ(+) clones; (2) cells carrying Fertility plasmid (F(+)), and a Tn5 element located on the chromosome; therefore in these cells transposition activity can be estimated by a bacterial conjugation assay. Cells were exposed to sinusoidal (SiMF) or pulsed-square wave (PMF) magnetic fields of various frequencies (20, 50, 75 Hz) and for different exposure times (15 and 90 min). RESULTS: Both mini-Tn10 and Tn5 transposition decreased under SiMF and increased under PMF, as compared to sham exposure control. No significant difference was found between frequencies and between exposure times. CONCLUSIONS: ELF-MF exposure affects transposition activity and the effects critically depend on the wave shape of the field, but not on the frequency and the exposure time, at least in the range observed.

Molecular remodeling of potassium channels in fibroblasts from centenarians: a marker of longevity?

Aging is a complex process resulting from, among other, dynamic non-linear interactions between genetics and environment. Centenarians are the best example of successful aging in humans, as they escaped from, or largely postponed, major age-related diseases. Ionic fluxes changes play a key role in several patho-physiological cellular processes, but their relation to human aging is largely unexplored. In the present study we have compared patch-clamp potassium (K(+)) current recordings from dermal fibroblasts (DF) obtained from young, elderly and centenarian donors. We found that in DF from elderly donors, but not from centenarians, K(+) current amplitude is significantly smaller with respect to DF from young donors. Moreover, cell membrane capacitance of DF from elderly donors is smaller with respect to young donors and centenarians. We also observed that the voltage-gated Shaker Kv1.1 channel is expressed in higher percentage of elderly's and centenarian's DF than young's, whereas the large-conductance calcium-activated K(+) (BK(Ca)) channel ß1 subunit is expressed in lower percentage of centenarian's DF than in elderly's and young's. The maintenance of "young" K(+) currents and the peculiar age-related remodeling of K(+) channel subtypes in centenarian's DF is likely associated with successful aging and might provide a predictive marker of longevity.

Complex patterns of gene expression in human T cells during in vivo aging.

Human aging is associated with complex alterations that contribute to remodelling of physiological processes and ultimately manifests in loss of tissue/organ function. Peripheral blood T cells do not escape this phenomenon and undergo profound remodelling with aging. Thus, investigating the effects of aging on T cells transcriptomics and identifying the underlying regulatory mechanisms can be of extreme importance to understand the aging process in the Immune System (IS). To this aim, we performed an analysis of gene expression data of T cells collected from peripheral blood of 25 healthy human donors of different age from 25 to more than 95 years, in order to characterize changes that occur throughout the entire adult lifespan. By means of microarray analysis, we observed large groups of genes exhibiting non-monotonic expression patterns over time: such behaviour, that could not be observed in typical "two-group" experiments (e.g. young vs. old people) highlights similarities in gene expression profiles of young and "successfully aged" individuals. Genes whose expression profiles change during lifespan were grouped into three main patterns (eigenmodes) to which different biological functions were significantly associated. The analysis of KEGG pathways to which these genes belong indicated that the biological processes altered in T cell aging are not only those typically associated with immune cells (Jak-STAT signalling, T cell receptor signalling, cytokine-cytokine receptor interactions, etc.) but also some not specific of immune cells, such as long-term depression, PPAR and mTOR signalling, glucose and glutathione metabolism, suggesting that T cell aging may be representative of a more generalised aging phenomenon. Thus, the T cell may represent a useful cellular model to study organismal aging. We further searched for over-represented transcription factor binding sites (TFBSs) in the promoter regions of genes clustered by similarity of their age-related patterns to evidence possible co-regulation. A comparison between over-representation of TFBSs and the time course of the corresponding transcription factor (TF) expression levels revealed that a restricted group of TFs may play a central role in driving aging-specific changes in gene expression of T cells.

Synergic effect of retinoic acid and extremely low frequency magnetic field exposure on human neuroblastoma cell line BE(2)C.

The aim of the present study was to assess whether exposure to a sinusoidal extremely low frequency magnetic field (ELF-MF; 50 Hz, 1 mT) can affect proliferation and differentiation in the human neuroblastoma cell line BE(2)C, which is representative of high risk neuroblastomas. Cells were subjected to ELF-MF exposure in the presence or absence of a neuronal differentiating agent (all-trans-retinoic acid, ATRA) for 24-72 h. In each experiment, ELF-MF-exposed samples were compared to sham-exposed samples. Cells exposed to ELF-MF combined with retinoic treatment showed a decreased cellular proliferation and an increased proportion of G(0)/G(1) phase cells compared to cells exposed to either treatment alone. Moreover, ELF-MF- and ATRA-treated cells showed more differentiated morphological traits (a higher neurite number/cell, an increased neurite length), together with a significant increase of mRNA levels of p21(WAF1/CIP1) and cdk5 genes, both involved in neuronal differentiation. In addition, the expression of cyp19 gene, which is involved both in neuronal differentiation and stress response, was evaluated; cyp19 gene expression was enhanced by ATRA treatment and significantly enhanced further by ELF-MF exposure combined with ATRA. In conclusion, our data suggest that ELF-MF exposure can strengthen ATRA effects on neuroblastoma cells.

Transient DNA damage induced by high-frequency electromagnetic fields (GSM 1.8 GHz) in the human trophoblast HTR-8/SVneo cell line evaluated with the alkaline comet assay.

One of the most controversial issue regarding high-frequency electromagnetic fields (HF-EMF) is their putative capacity to affect DNA integrity. This is of particular concern due to the increasing use of HF-EMF in communication technologies, including mobile phones. Although epidemiological studies report no detrimental effects on human health, the possible disturbance generated by HF-EMF on cell physiology remains controversial. In addition, the question remains as to whether cells are able to compensate their potential effects. We have previously reported that a 1-h exposure to amplitude-modulated 1.8 GHz sinusoidal waves (GSM-217 Hz, SAR=2 W/kg) largely used in mobile telephony did not cause increased levels of primary DNA damage in human trophoblast HTR-8/SVneo cells. Nevertheless, further investigations on trophoblast cell responses after exposure to GSM signals of different types and durations were considered of interest. In the present work, HTR-8/SVneo cells were exposed for 4, 16 or 24h to 1.8 GHz continuous wave (CW) and different GSM signals, namely GSM-217 Hz and GSM-Talk (intermittent exposure: 5 min field on, 10 min field off). The alkaline comet assay was used to evaluate primary DNA damages and/or strand breaks due to uncompleted repair processes in HF-EMF exposed samples. The amplitude-modulated signals GSM-217 Hz and GSM-Talk induced a significant increase in comet parameters in trophoblast cells after 16 and 24h of exposure, while the un-modulated CW was ineffective. However, alterations were rapidly recovered and the DNA integrity of HF-EMF exposed cells was similar to that of sham-exposed cells within 2h of recovery in the absence irradiation. Our data suggest that HF-EMF with a carrier frequency and modulation scheme typical of the GSM signal may affect the DNA integrity.

Effect of radiofrequency electromagnetic field exposure on in vitro models of neurodegenerative disease.

In this work we tested viability, proliferation, and vulnerability of neural cells, after continuous radiofrequency (RF) electromagnetic fields exposure (global system for mobile telecommunications (GSM) modulated 900 MHz signal at a specific absorption rate (SAR) of 1 W/kg and maximum duration 144 h) generated by transverse electromagnetic cells. We used two cellular systems, SN56 cholinergic for example, SN56 cholinergic cell line and rat primary cortical neurons, and well-known neurotoxic challenges, such as glutamate, 25-35AA beta-amyloid, and hydrogen peroxide. Exposure to RF did not change viability/proliferation rate of the SN56 cholinergic cells or viability of cortical neurons. Co-exposure to RF exacerbated neurotoxic effect of hydrogen peroxide in SN56, but not in primary cortical neurons, whereas no cooperative effects of RF with glutamate and 25-35AA beta-amyloid were found. These data suggest that only under particular circumstances exposure to GSM modulated, 900 MHz signal act as a co-stressor for oxidative damage of neural cells.

Electromagnetic fields and epidemiology: an overview inspired by the fourth course at the International School of Bioelectromagnetics.

The fourth course at the International School of Bioelectromagnetics addressed various aspects of the epidemiology of exposure to electromagnetic fields (EMF). In this overview, inspired by the lectures and the discussions among participants, we summarize current knowledge on exposure to EMF and disease risk, with emphasis on studies of use of mobile phones and brain tumours and exposure to power lines and childhood leukaemia. Sources of bias and error hamper straightforward conclusions in some areas and, in order to move forward, improvements in study design and exposure assessment are necessary. The scientific evidence available to date on possible long-term effects from exposure to ELF and RF fields is not strong enough to revise current protection limits based on the known acute effects of such exposures. Precautionary measures may be considered to reduce ELF exposure of children or exposure to RF during mobile phone use, keeping in mind that it is unclear whether they involve any preventive benefit. Possible health effects from mobile phone use in adults and in children should be investigated further by prospective epidemiological studies with improved exposure assessment and brain tumour incidence rates should be monitored. Further studies on the relation between childhood leukaemia and ELF magnetic fields would be worthwhile if they focus on heavily exposed groups and attempt to minimize possible selection bias. In conclusion, epidemiological studies conducted with appropriate diligence can play a key role in finding the answers.

Continuous exposure to 900MHz GSM-modulated EMF alters morphological maturation of neural cells.

The effects of radiofrequency electromagnetic field (RF-EMF) exposure on neuronal phenotype maturation have been studied in two different in vitro models: murine SN56 cholinergic cell line and rat primary cortical neurons. The samples were exposed at a dose of 1W/kg at 900 MHz GSM modulated. The phenotype analysis was carried out at 48 and 72 h (24 and 48 h of SN56 cell line differentiation) or at 24, 72, 120 h (2, 4 and 6 days in vitro for cortical neurons) of exposure, on live and immunolabeled neurons, and included the morphological study of neurite emission, outgrowth and branching. Moreover, cortical neurons were studied to detect alterations in the expression pattern of cytoskeleton regulating factors, e.g. beta-thymosin, and of early genes, e.g. c-Fos and c-Jun through real-time PCR on mRNA extracted after 24h exposure to EMF. We found that RF-EMF exposure reduced the number of neurites generated by both cell systems, and this alteration correlates to increased expression of beta-thymosin mRNA.

Extremely low frequency magnetic field exposure affects DnaK and GroEL expression in E. coli cells with impaired heat shock response.

In our earlier experiments, we found that extremely low frequency magnetic fields (ELF-MF) affect heat shock protein (HSP) expression in wild type Escherichia coli cells. In the present work we investigate the ability of ELF-MF exposure to trigger an increase of DnaK and GroEL protein levels also in E. coli cells not exhibiting the classic heat shock response (HSR) when subjected to a 42 degrees C heat stress. We find that these cells, although lacking a HSR to heat shock treatment, show an enhancement of DnaK and GroEL protein levels after 30 or 90 min sinusoidal ELF-MF exposure (50 Hz, 1 mT). This result suggests that the HSP induction pathway triggered by ELF-MF exposure could be different from that elicited by heat shock treatment.

Acute exposure to low-level CW and GSM-modulated 900 MHz radiofrequency does not affect Ba 2+ currents through voltage-gated calcium channels in rat cortical neurons.

We have studied the non-thermal effects of radiofrequency (RF) electromagnetic fields (EMFs) on Ba(2+) currents (I Ba 2+) through voltage-gated calcium channels (VGCC), recorded in primary cultures of rat cortical neurons using the patch-clamp technique. To assess whether low-level acute RF field exposure could modify the amplitude and/or the voltage-dependence of I Ba 2+, Petri dishes containing cultured neurons were exposed for 1-3 periods of 90 s to 900 MHz RF-EMF continuous wave (CW) or amplitude-modulated according to global system mobile communication standard (GSM) during whole-cell recording. The specific absorption rates (SARs) were 2 W/kg for CW and 2 W/kg (time average value) for GSM-modulated signals, respectively. The results obtained indicate that single or multiple acute exposures to either CW or GSM-modulated 900 MHz RF-EMFs do not significantly alter the current amplitude or the current-voltage relationship of I Ba 2+, through VGCC.

Effects of 50 Hz sinusoidal magnetic fields on Hsp27, Hsp70, Hsp90 expression in porcine aortic endothelial cells (PAEC).

The aim of the present study was to investigate the influence of 50 Hz sinusoidal magnetic field on Hsp27, Hsp70, and Hsp90 expression in a model of primary culture of porcine aortic endothelial cells (PAEC). We took into consideration the Hsp profile in terms of mRNA expression, protein expression and protein localization inside the cells. The choice of the cell system was motivated by the involvement of the endothelial cells in the onset of many diseases; moreover, only few reports describe the effects of extremely low frequency magnetic fields (ELF-MFs) on such cells. ELF-MF exposure induced an increase in the mRNA levels of the three proteins, which was statistically significant for Hsp70. On the contrary, we did not observe any influence on Hsp27, Hsp70, and Hsp90 protein levels. Analysis in situ by immunofluorescence revealed that ELF-MF exposure affected the cellular distribution of Hsp27; in particular a partial relocalization in the nucleus was observed.