Inhibition of gap junction intercellular communication by extremely low-frequency electromagnetic fields in osteoblast-like models is dependent on cell differentiation.

Electromagnetic fields have been used to augment the healing of fractures because of its ability to increase new bone formation. The mechanism of how electromagnetic fields can promote new bone formation is unknown, although the interaction of electromagnetic fields with components of the plasma membrane of cells has been hypothesized to occur in bone cells. Gap junctions occur among bone forming cells, the osteoblasts, and have been hypothesized to play a role in new bone formation. Thus it was investigated whether extremely low-frequency (ELF) magnetic fields alter gap junction intercellular communication in the pre-osteoblastic model, MC3T3-E1, and the well-differentiated osteoblastic model, ROS 17/2.8. ELF magnetic field exposure systems were designed to be used for an inverted microscope stage and for a tissue culture incubator. Using these systems, it was found that magnetic fields over a frequency range from 30 to 120 Hz and field intensities up to 12.5 G dose dependently decreased gap junction intercellular communication in MC3T3-E1 cells during their proliferative phase of development. The total amount of connexin 43 protein and the distribution of connexin 43 gap junction protein between cytoplasmic and plasma membrane pools were unaltered by treatment with ELF magnetic fields. Cytosolic calcium ([Ca(2+)](i)) which can inhibit gap junction communication, was not altered by magnetic field exposure. Identical exposure conditions did not affect gap junction communication in the ROS 17/2.8 cell line and when MC3T3-E1 cells were more differentiated. Thus ELF magnetic fields may affect only less differentiated or pre-osteoblasts and not fully differentiated osteoblasts. Consequently, electromagnetic fields may aid in the repair of bone by effects exerted only on osteoprogenitor or pre-osteoblasts.     

Suppression of natural killer cell activity on Candida stellatoidea by a 50 Hz magnetic field

Exposure to electromagnetic fields (EMF) is ubiquitous for almost all individuals living in industrialized countries. Epidemiological and laboratory studies suggest that exposure to Extremely Low Frequency (ELF) EMF increase cancer risk. The immune system functions as one of the body's main protective mechanisms, and Natural Killer (NK) cells are a subset of lymphocytes that can destroy several types of tumor cells. In this study, we investigated, NK cell activity after exposure to a 50 Hertz (Hz), 2 mT magnetic field generated by a Helmholtz Coil. Nineteen male, 10-12 week old guinea pigs were used, and NK cytotoxic activity of splenocytes was measured in vitro by natural anticandidial colorimetric index. The Mann-Whitney U test was applied for statistical analysis. NK cell cytotoxic activity was decreased in exposed compared to controls. Our data suggests that part of the immune system, the NK cell, can be suppressed by a 50 Hz magnetic field.     

Real-time measurement of cytosolic free calcium concentration in Jurkat cells during ELF magnetic field exposure and evaluation of the role of cell cycle

Extremely low frequency magnetic fields (ELF MF) have been reported to alter a number of cell signaling pathways, including those involved in proliferation, differentiation and apoptosis where cytosolic free calcium ([Ca(2+)](c)) plays an important role. To better understand the biological conditions under which ELF MF exposure might alter [Ca(2+)](c), we measured [Ca(2+)](c) by ratiometric fluorescence spectrophotometry during exposure to ELF MF in Jurkat E6.1 cells synchronized to different phases of the cell cycle. Suspensions of cells were exposed either to a near zero MF (Null) or a 60 Hz, 100 microT sinusoidal MF superimposed upon a collinear 78.1 microT static MF (AC + DC). An initial series of experiments indicated that the maximum increase in [Ca(2+)](c) above baseline after stimulation with anti-CD3 was significantly higher in samples exposed to AC + DC (n = 30) compared to Null (n = 30) with the largest difference in G2-M enriched samples. However, in a second study with G2-M enriched cells, samples treated with AC + DC (n = 17) were not statistically different from Null-treated samples (n = 27). Detailed analysis revealed that the dynamics in [Ca(2+)](c) before and after stimulation with anti-CD3 were dissimilar between Null samples from each study. From the results, we concluded (i) that the ELF MF increased [Ca(2+)](c) during an antibody-induced signaling event, (ii) that the ELF MF effect did not depend to a large degree on cell cycle, and (iii) that a field-related change in [Ca(2+)](c) signaling appeared to correlate with features in the [Ca(2+)](c) dynamics. Future work could evaluate [Ca(2+)](c) dynamics in relation to the phase of the cell cycle and inter-study variation, which may reveal factors important for the observation of real-time effects of ELF MF on [Ca(2+)](c).     

Extremely high frequency electromagnetic fields at low power density do not affect the division of exponential phase Saccharomyces cerevisiae cells

Exponentially growing cells of the yeast Saccharomyces cerevisiae were exposed to electromagnetic fields in the frequency range from 41.682 GHz to 41.710 GHz in 2 MHz increments at low power densities (0.5 μW/cm² and 50 μW/cm²) to observe possible nonthermal effects on the division of this microorganism. The electronic setup was carefully designed and tested to allow precise determination and stability of the electromagnetic field parameters as well as to minimize possible effects of external sources. Two identical test chambers were constructed in one exposure system to perform concurrent control and test experiments at every frequency step under well-controlled exposure conditions. Division of cells was assessed via time-lapse photography. Control experiments showed that the cells were dividing at submaximal rates, ensuring the possibility of observing either an increase or a decrease of the division rate. The data from several independent series of exposure experiments and from control experiments show no consistently significant differences between exposed and unexposed cells. This is in contrast to previous studies claiming nonthermal effects of electromagnetic fields in this frequency range on the division of S. cerevisiae cells. Possible reasons for this difference are discussed. Bioelectromagnetics 18:142–155, 1997. © 1997 Wiley-Liss, Inc.     

Nonthermal 60 Hz sinusoidal magnetic-field exposure enhances 45Ca2+ uptake in rat thymocytes: dependence on mitogen activation

The effect of a 60 Hz sinusoidal magnetic field of nonthermal intensity on Ca2+ metabolism in rat thymic lymphocytes (thymocytes) was assessed in resting cells and in cells activated with the mitogen Concanavalin A (Con A). A 60 min exposure at 37 degrees C to an induced electric field of 1.0 mV/cm produced an average 2.7-fold increase in Con A-dependent 45Ca2(+)-uptake compared to non-exposed, isothermal control cells. In contrast, 45Ca2+ uptake remained unaltered during exposure of resting thymocytes. It was also found that thymocytes with a diminished ability to mobilize Ca2+ in response to Con A were most sensitive to the 60 Hz magnetic field. Although the precise mechanism of field interaction is at present unknown, modulation of Ca2+ metabolism during cell activation may represent a common pathway for field coupling to cellular systems.     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Exposure to power frequency magnetic fields suppresses X-ray-induced apoptosis transiently in Ku80-deficient xrs5 cells

In an attempt to determine whether exposure to extremely low frequency (ELF) electromagnetic fields can affect cells, Ku80-deficient cells (xrs5) and Ku80-proficient cells (CHO-K1) were exposed to ELF electromagnetic fields. Cell survival, and the levels of the apoptosis-related genes p21, p53, phospho-p53 (Ser(15)), caspase-3 and the anti-apoptosis gene bcl-2 were determined in xrs5 and CHO-K1 cells following exposure to ELF electromagnetic fields and X-rays. It was found that exposure of xrs5 and CHO-K1 cells to 60 Hz ELF electromagnetic fields had no effect on cell survival, cell cycle distribution and protein expression. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields for 5 h after irradiation significantly inhibited G(1) cell cycle arrest induced by X-rays (1 Gy) and resulted in elevated bcl-2 expression. A significant decrease in the induction of p53, phospho-p53, caspase-3 and p21 proteins was observed in xrs5 cells when irradiation by X-rays (8 Gy) was followed by exposure to 5 mT ELF magnetic fields. Exposure of xrs5 cells to the ELF electromagnetic fields for 10 h following irradiation significantly decreased X-ray-induced apoptosis from about 1.7% to 0.7%. However, this effect was not found in CHO-K1 cells within 24 h of irradiation by X-rays alone and by X-rays combined with ELF electromagnetic fields. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields following irradiation can affect cell cycle distribution and transiently suppress apoptosis by decreasing the levels of caspase-3, p21, p53 and phospho-p53 and by increasing bcl-2 expression.     

The role of voltage-gated Ca2+ channels in neurite growth of cultured chromaffin cells induced by extremely low frequency (ELF) magnetic field stimulation

The ion Ca²⁺ has been shown to play an important role in a wide variety of cellular functions, one of them being related to cell differentiation in which nerve growth factor (NGF) is involved. Chromaffin cells obtained from adrenals of 2- to 3-day-old rats were cultured for 7 days. During this time, these cells were subjected to the application of either NGF or extremely low frequency magnetic fields (ELF MF). Since this induced cell differentiation toward neuronal-like cells, the mechanism by which this occurred was studied. When the L-Ca²⁺ channel blocker nifedipine was applied simultaneously with ELF MF, this differentiation did not take place, but it did when an N-Ca²⁺ channel blocker was used. In contrast, none of the Ca²⁺ channel blockers prevented differentiation in the presence of NGF. In addition, Bay K-8644, an L-Ca²⁺ channel agonist, increased both the percentage of differentiated cells and neurite length in the presence of ELF MF. This effect was much weaker in the presence of NGF. [³H]-noradrenaline release was reduced by nifedipine, suggesting an important role for L-Ca²⁺ channels in neurotransmitter release. Total high voltage Ca²⁺ currents were significantly increased in ELF MF-treated cells with NGF, but these currents in ELF MF-treated cells were more sensitive to nifedipine. Amperometric analysis of catecholamine release revealed that the KCl-induced activity of cells stimulated to differentiate by ELF MF is highly sensitive to L-type Ca²⁺ channel blockers. A possible mechanism to explain the way in which the application of magnetic fields can induce differentation of chromaffin cells into neuronal-like cells is proposed.     

Magnetic fields at extremely low-frequency (50 Hz, 0.8 mT) can induce the uptake of intracellular calcium levels in osteoblasts

Several studies have been undertaken to elucidate the effects of electromagnetic field (EMF) on intracellular calcium ([Ca²⁺]_i) in the past 20 years. However, still there were controversies of electromagnetic pollution within the scientific community. In this work, we studied the effects of alternative magnetic fields on intracellular calcium. Osteoblastic cells were used as a model both to test the hypothesis that extremely low-frequency (ELF) magnetic fields can alter the concentrations of the intracellular calcium, and to examine the ‘window’ effect predicted by our previous theoretical work. The outcome of this experiment demonstrated that 50 Hz, 0.8 mT magnetic field can induce the uptake of [Ca²⁺]_i in osteoblasts. The empirical evidences of the specified window effects of [Ca²⁺]_i in osteoblastic cells were reported for the first time in this work.     

Superimposing spatially coherent electromagnetic noise inhibits field-induced abnormalities in developing chick embryos

Living cells exist in an electrically noisy environment. This has led to the so-called “signal-to-noise” problem whereby cells are observed to respond to extremely-low-frequency (ELF) exogenous fields that are several orders of magnitude weaker than local endogenous fields associated with thermal fluctuations. To resolve this dilemma, we propose that living cells are affected only by electromagnetic fields that are spatially coherent over their surface. The basic idea is that a significant number of receptors must be simultaneously and coherently activated (biological cooperativity) to produce effects on the biochemical functioning of the cell. However, like all physical detection systems, cells are subject to the laws of conventional physics and can be confused by noise. This suggests that a spatially coherent but temporally random noise field superimposed on a coherent ELF signal will defeat the mechanism of discrimination against noise, and any observed field-induced bioeffects would be suppressed. An experimental test of this idea was conducted using morphological abnormalities in developing chick embryos caused by electromagnetic field exposure as the endpoint. At an impressed noise amplitude comparable to the ELF field strength (but roughly one-thousandth of the thermal noise field), the increased abnormality rate observed with only the ELF field present was reduced to a level essentially the same as for the control embryos. © 1994 Wiley-Liss, Inc.     

Proteomic response of Schizosaccharomyces pombe to static and oscillating extremely low-frequency electromagnetic fields

There is considerable public concern regarding the health effects of exposure to low-frequency electromagnetic fields. In addition, the association between exposure and disease incidence or the possible biological effects of exposure are unclear. Using 2D-DIGE and MS in a blind study, we have investigated the effects of static and oscillating extremely low-frequency electromagnetic fields (ELF EMFs) on the proteomes of wild type Schizosaccharomyces pombe and a Sty1p deletion mutant which displays increased sensitivity to a variety of cellular stresses. Whilst this study identifies a number of protein isoforms that display significant differential expression across experimental conditions, there was no correlation between their patterns of expression and the ELF EMF exposure regimen. We conclude that there are no significant effects of either static or oscillating EMF on the yeast proteome at the sensitivity afforded by 2D-DIGE. We hypothesise that the proteins identified must be sensitive to subtle changes in culture and/or handling conditions, and that the identification of these proteins in other proteomic studies should be treated with some caution when the results of such studies are interpreted in a biological context.     

In vitro exposure of human lymphocytes to 900 MHz CW and GSM modulated radiofrequency: studies of proliferation, apoptosis and mitochondrial membrane potential

The aim of this study was to investigate the nonthermal effects of radiofrequency (RF) fields on human immune cells exposed to a Global System for Mobile Communication (GSM) signal generated by a commercial cellular phone and by a sinusoidal non-modulated signal. To assess whether mobile phone RF-field exposure affects human immune cell functions, peripheral blood mononuclear cells (PBMCs) from healthy donors were exposed in vitro to a 900 MHz GSM or continuous-wave (CW) RF field 1 h/day for 3 days in a transverse electromagnetic mode (TEM) cell system (70-76 mW/kg average specific absorption rate, SAR). The cells were cultured for 48 or 72 h, and the following end points were studied: (1) mitogen-induced proliferation; (2) cell cycle progression; (3) spontaneous and 2-deoxy-D-ribose (dRib)-induced apoptosis; (4) mitochondrial membrane potential modifications during spontaneous and dRib-induced-apoptosis. Data obtained from cells exposed to a GSM-modulated RF field showed a slight decrease in cell proliferation when PBMCs were stimulated with the lowest mitogen concentration and a slight increase in the number of cells with altered distribution of phosphatidylserine across the membrane. On the other hand, cell cycle phases, mitochondrial membrane potential and susceptibility to apoptosis were found to be unaffected by the RF field. When cells were exposed to a CW RF field, no significant modifications were observed in comparison with sham-exposed cells for all the end points investigated.     

Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK)

This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.     

Effects of ELF magnetic fields on protein expression profile of human breast cancer cell MCF7

Extremely Low Frequency Magnetic Fields (ELF MF) has been considered as a “possible human carcinogen” by International Agency for Research on Cancer (IARC) while credible mechanisms of its carcinogenicity remain unknown. In this study, a proteomics approach was employed to investigate the changes of protein expression profile induced by ELF MF in human breast cancer cell line MCF7, in order to determine ELF MF-responsive proteins. MCF7 cells were exposed to 50 Hz, 0.4 mT ELF MF for 24 h and the changes of protein profile were examined using two dimensional electrophoresis. Up to 6 spots have been statistically significantly altered (their expression levels were changed at least 5 fold up or down) compared with sham-exposed group. 19 ones were only detected in exposure group while 19 ones were missing. Three proteins were identified by LC-IT Tandem MS as RNA binding protein regulatory subunit Proteasome subunit beta type 7 precursor and Translationally Controlled Tumor Protein. Our finding showed that 50 Hz, 0.4 mT ELF MF alternates the protein profile of MCF7 cell and may affect many physiological functions of normal cell and 2-DE coupled with MS is a promising approach to elucidating cellular effects of electromagnetic fields.     

Experimental studies on extremely low frequency pulsed magnetic field inhibiting sarcoma and enhancing cellular immune functions

The previous observation with an electron microscope showed that extremely low frequency (ELF) pulsed magnetic field (PMF) (with the maximum intensity of 0. 6-2. 0 T, gradient of 10-100 T. M-1, pulse width of 20-200 ms and frequency of 0. 16-1. 34 Hz) inhibited the growth of S-180 sarcoma in mice and enhanced the ability of immune cell's dissolving sarcoma cells. In this study, the DNA contents of nuclei were assayed by using Faulgen Staining method. With an electron microscope and cell stereoscopy technology it was observed that magnetic field affected the sarcoma cell's metabolism, lowered its malignancy, and restrained its rapid and heteromorphic growth. The magnetic field enhanced the cellular immune ability and the reaction of lymphocytes and plasma. Since ELF pulsed magnetic fields can inhibit the growth of sarcomas and enhance the cellular immune ability, it is possible to use it as a new method to treat cancer.     

Effects of ELF magnetic fields on protein expression profile of human breast cancer cell MCF7

A case-control study in 1979 on electrical wiring configurations and childhood leukemia had stimulated interest in the issue that extremely low frequency magnetic fields (ELF MF) may have harmful biologi-cal effects especially on the incidence of human can-cer[1]. Since then, a large number of studies have been conducted to follow up this important result[2]. The majority of these studies indicate a weak association between exposure to 50―60 Hz ELF MF and the in-cidence of cancer; however…     

Neuroendocrine mediated effects of electromagnetic-field exposure: possible role of the pineal gland

Reports from recent epidemiological studies have suggested a possible association between extremely low frequency (ELF; including 50- or 60-Hz) electric- and magnetic-field exposure, and increased risk of certain cancers, depression, and miscarriage. ELF field-induced pineal gland dysfunction is a possible etiological factor in these effects. Work in our laboratory and elsewhere has shown that ELF electromagnetic-field exposure can alter the normal circadian rhythm of melatonin synthesis and release in the pineal gland. Consequences of reduced or inappropriately timed melatonin release on the endocrine, neuronal, and immune systems are discussed. Laboratory data linking ELF field exposure to changes in pineal circadian rhythms in both animals and humans are reviewed. The authors suggest that the pineal gland, in addition to being a convenient locus for measuring dyschronogenic effects of ELF field exposure, may play a central role in biological response to these fields via alterations in the melatonin signal.     

Bioeffects induced by exposure to microwaves are mitigated by superposition of ELF noise

We have previously demonstrated that microwave fields, amplitude modulated (AM) by an extremely low-frequency (ELF) sine wave, can induce a nearly twofold enhancement in the activity of ornithine decarboxylase (ODC) in L929 cells at SAR levels of the order of 2.5 W/kg. Similar, although less pronounced, effects were also observed from exposure to a typical digital cellular phone test signal of the same power level, burst modulated at 50 Hz. We have also shown that ODC enhancement in L929 cells produced by exposure to ELF fields can be inhibited by superposition of ELF noise. In the present study, we explore the possibility that similar inhibition techniques can be used to suppress the microwave response. We concurrently exposed L929 cells to 60 Hz AM microwave fields or a 50 Hz burst-modulated DAMPS (Digital Advanced Mobile Phone System) digital cellular phone field at levels known to produce ODC enhancement, together with band-limited 30–100 Hz ELF noise with root mean square amplitude of up to 10 μT. All exposures were carried out for 8 h, which was previously found to yield the peak microwave response. In both cases, the ODC enhancement was found to decrease exponentially as a function of the noise root mean square amplitude. With 60 Hz AM microwaves, complete inhibition was obtained with noise levels at or above 2 μT. With the DAMPS digital cellular phone signal, complete inhibition occurred with noise levels at or above 5 μT. These results suggest a possible practical means to inhibit biological effects from exposure to both ELF and microwave fields. Bioelectromagnetics 18:422–430, 1997. © 1997 Wiley-Liss, Inc.     

ELF Noise Fields: A Review

The debate as to whether low-level electromagnetic fields can affect biological systems and in the long term cause health effects has been going on for a long time. Yet the interaction of weak electromagnetic fields (EMF) with living cells, undoubtedly a most important phenomenon, is still not well understood. The exact mechanisms by which the effects are produced have not been identified. Furthermore, it is not possible to clearly define which aspects of an EMF exposure that constitute the “dose.” One of the groups that contributed to solving this problem is the Bioelectromagnetics group at Catholic University of America (CUA), Washington, D.C. Their work has been devoted to investigating the physical parameters that are needed to obtain an effect of EMF exposure on biological systems, and also how to inhibit the effect. This is a review of their work on bioeffects caused by low-level EMF, their dependence on coherence time, constancy, spatial averaging, and also how the effects can be modified by an applied ELF noise magnetic field. The group has been using early chick embryos, and L929 and Daudi cells as their main experimental systems. The review also covers the work of other groups on low-level effects and the inhibition of the effects with an applied noise field. The group at CUA has shown that biological effects can be found after exposure to low-level ELF and RF electromagnetic fields, and when effects are observed, applying an ELF magnetic noise field inhibits the effects. Also, other research groups have tried to replicate the studies from the CUA group, or to apply EMF noise to suppress EMF-induced effects. Replications of the CUA effects have not always been successful. However, in all cases where the noise field has been applied to prevent an observed effect, it has been successful in eliminating the effect.     

Comment: a biological guide for electromagnetic safety: the stress response

Questions of safety of electromagnetic (EM) fields should be based on relevant biological properties, i.e., specific cellular reactions to potentially harmful stimuli. The stress response is a well documented protective reaction of plant and animal cells to a variety of environmental threats, and it is stimulated by both extremely low frequency (ELF) and radio frequency (RF) EM fields. It involves activation of DNA to initiate synthesis of stress proteins. Thermal and non-thermal stimuli affect different segments of DNA and utilize different biochemical pathways. However, both ELF and RF stimulate the same non-thermal pathway. Since the same biochemical reactions are stimulated in different frequency ranges with very different specific absorption rates (SARs), SAR level is not a valid basis for safety standards. Studies of EM field interactions with DNA and with model systems provide insight into a plausible mechanism that can be effective in ELF and RF ranges.