Effects of 50 Hz magnetic fields on fMLP-induced shape changes in invertebrate immunocytes: The role of calcium ion channels

Plasma membrane Ca(2+) channels in immunocytes from the mussel Mytilus galloprovincialis exposed to 50 Hz sine wave magnetic fields (MFs) of various strengths were studied. At levels of 300 microT and above, MFs reduce shape changes in immunocytes induced by the chemotactic substance N-formyl-Meth-Leu-Phe, and this effect involves L-type Ca(2+) channels. Upon the addition of the Ca(2+) blocker verapamil to molluscan immunocytes exposed to MFs results in a synergistic cytotoxic action, while in the presence of the Ca(2+) opener SDZ-202, 791, a reactivation of the cells is observed. This suggests that, as previously reported for potassium channels, the damage to Ca(2+) channels induced by short exposure to MF at appropriate intensities is not permanent.     

Synergistic role of cAMP and IP(3) in corticotropin-releasing hormone-induced cell shape changes in invertebrate immunocytes

Immunocytes from the mollusc Mytilus galloprovincialis express corticotropin-releasing hormone (CRH) receptor subtype (CRH-R1 and CRH-R2)-like mRNAs. Using computer-assisted microscopic image analysis, we have found that exogenous CRH provokes changes in the cellular shape of immunocytes, and that this response is extracellular Ca(2+)-dependent. The various inhibitors of transduction signaling pathways, i.e. suramin sodium, 2', 5'-dideoxyadenosine, neomycin sulfate, calphostin C, H-89, and wortmannin, completely or partially inhibit these changes. The present findings demonstrate that PKA, PKC, and PKB/Akt are involved in CRH-induced cell shape changes in immunocytes, and that the cellular effect of CRH needs the synergistic action of the two second messengers, cAMP and IP(3).     

Neutral endopeptidase-24.11 (NEP) deactivates PDGF- and TGF-beta-induced cell shape changes in invertebrate immunocytes

Using a cytofluorimetric assay, we found that immunocytes of the mollusc, Mytilus galloprovincialis, express CD10, a surface antigen known to be identical to neutral endopeptidase-24.11 (NEP). The spectrofluorimetric analysis demonstrates that the growth factors PDGF-AB and TGF-beta1 provoke an increase in NEP-like activity in membrane preparations from the immunocytes, but have no effect on the soluble form in the serum. On the other hand, computer-assisted microscopic image analysis reveals that NEP deactivates the PDGF-AB- and TGF-beta1-induced shape changes in immunocytes. However, Western blots show that, in solution, NEP does not cleave PDGF-AB or TGF-beta1, indicating that the inactivation is not due to proteolysis. These results suggest a functional interplay in invertebrate immunocytes between growth factors and NEP, as previously shown in vertebrate cells.     

Extremely low frequency magnetic fields originating from equipment used for assisted reproduction, umbilical cord and peripheral blood stem cell transplantation, transfusion, and hemodialysis

The extremely low-frequency (ELF) magnetic fields (MFs) originating from equipment used for assisted reproduction, umbilical cord-blood and peripheral-blood stem cell transplantation, transfusion, and hemodialysis were measured. The ELF-MF values were 0.1-1.2 microT on clean benches, <0.1-8.0 microT on inverted microscopes, <0.1-13.6 mmicroT in CO2 incubators, 4.3-11.5 microT in centrifuges, 0.4-18.8 microT in programmed freezers, <0.1-0.3 microT in deep freezers, 0.3-3.1 microT on cell separators, and 0.2-0.9 microT in hemodialysers. Frequencies of MFs were nominally 60 Hz, but some devices showed non-sinusoidal 120 Hz. Such MFs can be reduced by shielding the sources or altering the protocols employed.     

Investigation of protein expression in magnetic field-treated human glioma cells

We previously reported phenotypic changes in human breast cancer cells following low-level magnetic field (MF) exposure. Here proteomic methods were used to investigate the biochemical effect of MF exposure in SF767 human glioma cells. Protein alterations were studied after exposure to 1.2 microTesla (microT) MF [12 milliGauss (mG), 60 Hertz (Hz)] +/- epidermal growth factor (EGF). SF767 cells were exposed for 3 h to sham conditions (<0.2 microT ambient field strength) or 1.2 microT MF (+/-EGF; 10 ng/ml). Solubilized protein fractions (sham; 1.2 microT; sham + EGF; 1.2 microT + EGF) were loaded for electrophoresis by 2D-PAGE and stained using a colloidal Coomassie blue technique to resolve and characterize the proteins. Protein patterns were compared across groups via Student's t-test using PDQUEST software. Cell profiles revealed significant alterations in the spot density of a subset of treated cells. Automated spot excision and processing was performed prior to peptide mass fingerprinting proteins of interest. Fifty-seven proteins from the detectable pool were identified and/or found to differ significantly across treatment groups. The mean abundance of 10 identified proteins was altered following 1.2 microT exposure. In the presence of EGF six proteins were altered after low magnetic field treatment by increasing (4) or decreasing (2) in abundance. The results suggest that the analysis of differentially expressed proteins in SF767 cells may be useful as biomarkers for biological changes caused by exposure to magnetic fields.     

Algal toxin yessotoxin signalling pathways involve immunocyte mussel calcium channels

A fragment of a putative L-type Ca(2+) channel has been identified by molecular biology experiments in immunocytes from the mussel Mytilus galloprovincialis. Using the cell permeable and Ca(2+)-specific fluorochrome FURA 2-AM, we have demonstrated that the algal toxin yessotoxin (YTX) is able to increase intracellular Ca(2+) concentration in M. galloprovincialis immunocytes. The YTX effect on Ca(2+) increase is inhibited by the L-type Ca(2+) channel inhibitor, verapamil, which is cAMP- and cGMP-dependent, but PKA- and nitric oxide-independent. On the basis of these observations, a possible role for YTX as a potential disturber of mussel immune efficiency is suggested.     

No effects of extremely low frequency magnetic fields found on cytotoxic activities and cytokine production of human peripheral blood mononuclear cells in vitro

Several epidemiologic studies have suggested an association between exposure to extremely low frequency (ELF) magnetic fields (MFs) and cancer in adults and children. A possible target of MFs is the immune system. The effects of the exposure to ELF MFs on the immunological functions of human peripheral blood mononuclear cells (PBMCs) obtained from healthy male volunteers were assessed by measuring the natural killer (NK) and lymphokine activated killer (LAK) activities and the production of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), and interleukin-10 (IL-10). The PBMCs were exposed to three different MF: linearly polarized (vertical), circularly polarized, and elliptically polarized, at 50 and 60 Hz. Magnetic flux densities were set at 500, 100, 20, and 2 microT (rms) for vertical field and at 500 microT (rms) for the rotating fields. Using cytotoxicity assay and enzyme-linked immunosorbent assay (ELISA) for cytokine production, we could not find any effects of ELF MFs on the cytotoxic activities and the cytokines production of human PBMCs.     

Evaluation of the potential promoting effect of 60 Hz magnetic fields on N-ethyl-N-nitrosourea induced neurogenic tumors in female F344 rats

The present study investigated the possible effect of 60 Hz magnetic fields (MFs) as promoters of neurogenic tumors initiated transplacentally by a chemical carcinogen, N-ethyl-N-nitrosourea (ENU). In a preliminary study, 5 mg of ENU was shown to induce 30 to 40% neurogenic tumors in F344 rats offspring after 420 days of observation. In the present study, 400 female rats were divided into eight different groups (50 animals/group) and exposed in utero (on day 18 of gestation) to a single intravenous dose of either Saline (Group I), or ENU, 5 mg/kg (Group II to VIII). Dams in group II were given no further treatment while dams in Groups III to VII were exposed to 5 different intensities of MFs forty eight hours later. Animals in group III were sham exposed (<0.02 microT) while groups IV to VII were exposed to 2, 20, 200, and 2000 microT, respectively. Dams in Group VIII were injected intraperitoneally with 12-O-tetradecanoylphrobol-13-acetate (TPA; 10 micrograms/kg) from day 19 until delivery, and then their female offspring continued to be injected every 15 days, starting at day 14 after birth until sacrifice (positive controls). Accordingly, this study included three different types of controls: Internal controls (Groups II and III) and positive control (Group VIII). Body weight, mortality and clinical observations were evaluated in all groups of animals during in-life exposure. Necropsy was performed on all exposed and control animals that died, were found moribund or sacrificed at termination of the study. Histopathological evaluation was done for all brains, spinal cords, cranial nerves, major organs (lungs, liver, spleen, kidneys, pituitary, thyroid and adrenals) and all gross lesions observed during necropsy. All clinical observations and pathological evaluations were conducted under "blinded" conditions. The findings from this ENU/MFs promotion study clearly demonstrate that, under our defined experimental conditions, exposure to 60 Hz linear (single axis) sinusoidal, continuous wave MFs had no effect on the survival of female F344 rats or on the number of animals bearing neurogenic tumors. These results suggest that MFs have no promoting effect on neurogenic tumors in the female F344 rats exposed transplacentally to ENU.     

Survey of residential 50 Hz EMF exposure from transformer stations

In Hungary it is typical that 10/04 kV transformer stations are being installed in multistory residential and office buildings. Magnetic fields (MFs) up to several tens of microT have been measured in apartments close to transformers. The aim of the present study was to provide systematic assessment of MF exposure of residents living above transformer stations. Out of 41 addresses provided by the electricity supplier, current load of 21 transformers and MF in 21 apartments was measured. Spot MFs at 1 m height and time weighted average 24 h MF exposure at bed height was measured. All-day personal MF exposure was measured at waist and HOME exposure was calculated. BED exposure was measured at bed height. Participants kept a time-activity diary. The time-weighted average 24 h MF exposure (3.03 microT) exceeded the usual residential exposure (<0.2 microT). The mean HOME and BED personal exposure above transformers was 0.825 and 1.033 microT, respectively. Our study provides exposure assessment of a cohort with a wider exposure range, compared to power-line epidemiological studies.     

Yessotoxin affects fMLP-induced cell shape changes inMytilus galloprovincialis immunocytes

Using computer-assisted microscopic image analysis, we have found that algal yessotoxin (YTX) affects the immune response of Mytilus galloprovincialis. Indeed, YTX increases immunocyte cell motility through the involvement of both extracellular Ca2+ and cAMP, but not through protein kinase A, protein kinase C or phosphoinositide 3-kinase. Alone, however, the toxin does not induce any effect, as its action on cell motility is observed only after addition of the chemotactic substance N-formyl-Meth-Leu-Phe (fMLP). fMLP is known to induce cellular changes via both the phosphatidylinositol and cAMP pathways and, from this scenario, we can surmise that Ca2+ and cAMP concentrations rise sufficiently in fMLP-activated immunocytes to reveal YTX action. One possible explanation is that the toxin increases fMLP-mediated cell activation by intervening in L-type Ca2+-channel opening through a cAMP-dependent/PKA-independent pathway.     

Presence of and stress-related changes in urocortin-like molecules in neurons and immune cells from the mussel Mytilus galloprovincialis

The distribution of urocortin (UCN)-like material is investigated in the bivalve mollusc Mytilus galloprovincialis. Immunocytochemical data demonstrate that UCN-like molecules are present in ganglionic neurons, microglial cells and immunocytes. Moreover, a co-localization of UCN- and corticotrophin-releasing hormone (CRH)-like molecules is found in microglial cells and in immunocytes, but not in neurons. Following high salinity-stress experiments, immunoreactivity for UCN and CRH increased in ganglionic neurons and immunocytes. Our findings extend the number of molecules potentially used by molluscan immunocytes to confront stress situations and strengthen the idea of functional conservation of stress-related molecules during evolution.     

Affective response to 5 microT ELF magnetic field-induced physiological changes

Research into effects of weak magnetic fields (MFs) at biologically relevant frequencies has produced ambiguous results. Although they do affect human physiology and behaviour, the direction of effects is inconsistent, with a range of complex and unrelated behaviours being susceptible. A possible explanation is that these effects, rather than being directly caused, are instead related to changes in affective state. A previous study showed that MFs altered the affective content of concurrent perceptions, but it was unclear whether the emotional response was direct or indirect. Here it is shown that exposure to a 0-5 microT MF (DC-offset sinudsoidal wave form) within EEG alpha-band frequencies (8-12 Hz), results in a reported change in emotional state. This relates to a decrease global field power but lacks the frontal alpha-asymmetry that would physiologically indicate a directly induced emotional state, suggesting that participant experiences are due to an interpretation of the effects of MF exposure.     

Heavy metals and neuroimmunomodulation in Mytilus edulis

Immunocytes of mussels are the chief immune defense in these organisms. When an immunocyte becomes activated there is a conspicuous change in its morphology (i.e., from round to amoeboid) that can be quantified using image analytical tools. Active immunocytes will typically show larger perimeters and areas and a smaller shape factor. Immunocytes exposed to heavy metals become inactive (Cd, Hg and Pb) thus with smaller perimeters (e.g., Pb2+ 2 ppm: P = 69.72 micron) and areas (e.g., Pb2+ 2 ppm: A = 270 micron2) and larger shape factors (Pb2 2 ppm: SF = 0.65) than the unexposed control cells (alpha = 0.05). Xenobiotics may also interfere with neuroimmunomodulation processes such as nitric oxide (NO) release. The release of NO is catalyzed by a calcium dependent constitutive nitric oxide synthase (cNOS). Presently, we are exploring the effects of heavy metals and other pollutants on cNOS activity, measured as real time NO release, in immunocytes and pedal ganglia from M. edulis. Preliminary results suggest that immunocytes exposed to Pb2+ (5 ppm) cause NO release and does not seem to inhibit further NO release in the presence of morphine. The possible implications of NO mediated Pb2+ neurotoxicity are also explored.     

A literature review: the effects of magnetic field exposure on blood flow and blood vessels in the microvasculature

The effect of magnetic field (MF) exposure on microcirculation and microvasculature is not clear or widely explored. In the limited body of data that exists, there are contradictions as to the effects of MFs on blood perfusion and pressure. Approximately half of the cited studies indicate a vasodilatory effect of MFs; the remaining half indicate that MFs could trigger either vasodilation or vasoconstriction depending on initial vessel tone. Few studies indicate that MFs cause a decrease in perfusion or no effect. There is a further lack of investigation into the cellular effects of MFs on microcirculation and microvasculature. The role of nitric oxide (NO) in mediating microcirculatory MF effects has been minimally explored and results are mixed, with four studies supporting an increase in NO activity, one supporting a biphasic effect, and five indicating no effect. MF effects on angiogenesis are also reported: seven studies supporting an increase and two a decrease. Possible reasons for these contradictions are explored. This review also considers the effects of magnetic resonance imaging (MRI) and anesthetics on microcirculation. Recommendations for future work include studies aimed at the cellular/mechanistic level, studies involving perfusion measurements both during and post-exposure, studies testing the effect of MFs on anesthetics, and investigation into the microcirculatory effects of MRI.     

Effects of the marine toxins okadaic acid and palytoxin on mussel phagocytosis

The present study analyzes the effects of the marine toxins okadaic acid (OA) and palytoxin (PTX) on the phagocytic activity of immunocytes from the mussel Mytilus galloprovincialis. In particular, we describe how the effects of the two biotoxins are influenced by the temperature and experimental stress applied before hemolymph withdrawal. The collected data indicate that OA increases phagocytic activity only when hemolymph incubation is performed at 25 degrees C, but not at 20 degrees C, suggesting a certain degree of dependence of OA effects from the status of mussel immunocytes. Conversely, PTX plays an active role in immunocyte signalling transduction pathways, increases the phagocytic activity and markedly promotes the involvement of p38 mitogen-activated protein (MAP) kinase in phagocytosis. Overall, we conclude that both OA and PTX influence mussel phagocytic activity, and the toxic effects may depend on both the mollusc conditions and the activation of specific signalling pathways.     

Expression levels of heat shock protein 60 in human endothelial cells in vitro are unaffected by exposure to 50 Hz magnetic fields

Magnetic fields (MFs) from domestic power sources have been implicated as being a potential risk to human health. A number of epidemiological studies have found a significant link between exposure to MFs and increased rates of cancers. There have also been a number of in vivo and in vitro studies reporting effects of MFs in animal disease models and on the expression or activity of a range of proteins. In the past decade, our group proposed that atherosclerosis may have an autoimmune component, with heat shock protein 60 (Hsp60) expressed in endothelial cells as the dominant autoantigen. A number of stressors have been shown to induce the expression of Hsp60, including the classical risk factors for atherosclerosis. We were interested to see if the exposure of endothelial cells to an MF elicited increased expression of Hsp60, as has been reported previously for Hsp70. The present work describes the exposure of endothelial cells to domestic power supply (50 Hz) MFs at an intensity of 700 microT. The results from our system indicate that cultured endothelial cells exposed to a high intensity of MF either alone or in combination with classical heat stress show no effects on the expression of Hsp60 at either the messenger ribonucleic acid or the protein level. As such, there is no evidence that exposure to extremely low-frequency MF would be expected to increase the expression of Hsp60 and therefore the initiation or progression of atherosclerosis.