- Email: [email protected]
Stimulation of phagocytosis and free radical production in murine macrophages by 50 Hz electromagnetic fields
EJCB
562 European Journal of Cell Biology 80, 562 ± 566 (2001, August) ´ Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb
Stimulation of phagocytosis and free radical production in murine macrophages by 50 Hz electromagnetic fields Myrtill SimkoÂ1), Susanne Droste, Ralf Kriehuber, Dieter G. Weiss University of Rostock, Institute of Cell Biology and Biosystems Technology, Division of Environmental Physiology, Rostock/Germany Received March 30, 2001 Received in revised version May 14, 2001 Accepted May 15, 2001
Phagocytosis ± electromagnetic fields ± free radicals ± intracellular transport ± TPA Effects of 50 Hz electromagnetic fields on phagocytosis and free radical production were examined in mouse bone marrowderived macrophages. Macrophages were in vitro exposed to electromagnetic fields using different magnetic field densities (0.5 ± 1.5 mT). Short-time exposure (45 min) to electromagnetic fields resulted in significantly increased phagocytic uptake (36.3% 15.1%) as quantified by measuring the internalization rate of latex beads. Stimulation with 1 nM 12-Otetradecanoylphorbol-13-acetate (TPA) showed the same increased phagocytic activity as 1 mT electromagnetic fields. However, co-exposure to electromagnetic fields and TPA showed no further increase of bead uptake, and therefore we concluded that because of the absence of additive effects, the electromagnetic fields-induced stimulation of mouse bone marrow-derived macrophages does not involve the protein kinase C signal transduction pathway. Furthermore, a significant increased superoxide production after exposure to electromagnetic fields was detected.
Introduction Exposure to electromagnetic fields (EMF) has been linked to an increased incidence of leukemias and other tumours (Ahlbom and Feychting, 1999; Thomas et al., 1999; Caplan et al., 2000; Lacy-Hulbert et al., 1998), and is considered by a recent NIEHS opinion (1998) to be ªpossibly carcinogenic to humansº. There is, however, no well-established biological mechanism explaining such a relation. In various cellular 1) Dr. Myrtill SimkoÂ, University of Rostock, Institute of Cell Biology and Biosystems Technology, Division of Environmental Physiology, Universitätsplatz 2, D-18051 Rostock/Germany, e-mail: myrtill. [email protected], Fax: 49 381 498 1918.
systems, including cells of the immune system, a number of biological effects of exposure to 50/60 Hz EMF have been described (Lacy-Hulbert et al., 1998; Cadossi et al., 1992; Walleczek, 1992), and different mechanisms have been proposed to explain these effects. The induction of genotoxic effects, such as micronucleus formation (Tofani et al., 1995; Simko et al., 1998a, b) or chromosomal aberrations (Nordenson et al., 1994), shows the carcinogenic potential of EMF. However, it is generally believed that genotoxic effects of EMF cannot be induced by direct interaction with DNA, because the energy is insufficient to break chemical bonds in DNA molecules. Indirectly mediated mechanisms, such as the induction of free radicals, which are able to interact with DNA or other cellular components, can lead to a potentiation of free radical-dependent effects (Dreher and Junod, 1996), and are therefore assumed to be a more likely mechanism of EMFinduced cellular response. In macrophages, physiological activation is associated with the onset of phagocytosis and leads to increased formation of reactive oxygen species (ROS). EMF has been shown to increase ROS levels in phorbol esteractivated neutrophils (Roy et al., 1995), but it was unclear whether in non-activated macrophages, short-term EMF stimulation would elevate ROS levels via stimulation of the physiological mechanisms. In our study, we evaluated whether EMF can modulate the phagocytic activity of mouse bone marrow-derived macrophages (MBM) and if this is coupled with the induction of the release of ROS. The enhanced microbicidal and tumoricidal activity of activated macrophages is associated with both increased phagocytic activity (Laskin et al., 1980; PhaireWashington et al., 1980) and the generation of ROS by NADPH oxidase activation, which initially induces superoxide (O2ÿ) formation. As a positive control for macrophage activation we used 12-O-tetradecanoylphorbol-13-acetate (TPA) (Laskin et al., 1980; Phaire-Washington et al., 1980), because both phagocytic activity as well as free radical production can be stimulated by TPA (Zheleznyak and Brown, 1992; Karimi
0171-9335/01/80/08-562 $15.00/0
EJCB
Electromagnetic fields as a stimulus for cell activation 563
and Lennartz, 1995). TPA interacts directly with protein kinase C (PKC) by mimicking the action of diacylglycerol and is able to activate PKC-mediated signal transduction. There are several reports about the influence of electric and magnetic fields on PKC activity or other kinases involved in signal transduction cascades in different cell systems. It has been shown that treatment of HL-60 cells with 60 Hz electric fields results in decreased cytosolic but unchanged membraneassociated PKC activities (Holian et al., 1996). Phillips et al. (1992) showed an increase in PKC activity upon exposure to EMF. Uckun et al. (1995) indicated that 60 Hz fields caused activation of tyrosine kinases and PKC in a human pre-Bleukemia cell line. In a recent study Dibirdik et al. (1998) demonstrated that exposure of DT40 lymphoma B cells to EMF results in a tyrosine kinase-dependent activation of phopholipase Cg2 leading to increased inositol phospholipid turnover. The aim of our study was to investigate the activating capacity of EMF by studying phagocytic activity and superoxide production of MBM macrophages. To investigate whether the PKC-mediated signal transduction pathway is involved, coexposure experiments with TPA and EMF were performed.
Materials and methods Cell culture
Murine bone marrow-derived (MBM) macrophages were obtained according to standard procedures (Swanson, 1989). Mononuclear phagocyte precursor cells were prepared from tibial and femurial mouse bones, resuspended and washed in PBS. Cells were cultured in MBM medium (50% L929 cell-conditioned medium, 50% RPMI-1640, 10% heat-inactivated FCS, 1% L-glutamine and 1% non-essential amino acids, 0.5% mercaptoethanol and antibiotics, all from Gibco, BRL) for 5 ± 6 days at 37 8C in a humidified atmosphere of 5% CO2 and 95% air for differentiation to adherently growing macrophages. Three days after initial plating, the medium was changed, and after an additional 2 or 3 days cells were cooled to ÿ 20 8C in a freezer for 1 ± 2 min until they rounded up. Cells were scraped and re-plated onto sterile 11-mm circular glass coverslips (104 cells/coverslip) in Petri dishes.
Preparation of latex beads
following co-exposure experiments were simultaneously carried out: a) without any treatment as control, b) in the presence of 1 mT EMF, c) in the presence of TPA and d) in the presence of both. To analyze intracellular transport of internalized latex beads from the cell periphery toward the nucleus, the position of internalized beads was determined for at least 25 cells per coverslip. The relative number of beads was determined for the peripheral and the central region, the latter being defined as the area of 3 mm around the nucleus (eye-piece reticle). These experiments were repeated 5 times.
O2ÿ generation
Cells were plated in 96-well plates (104 cells/well) for four hours prior to use. After washing with PBS, cells were incubated for 45 min with PBS supplemented with 1 mM p-nitro blue tetrazolium (NBT, Molecular Probes) and 1 mM CaCl2 with or without TPA and in the absence or presence of EMF exposure. O2ÿgeneration reduces NBT to blue formazan within the cells, which was measured using a microplate reader (Anthos 2010) at 550 nm. Intracellular formazan crystals were solubilized in 100 ml DMSO prior to reading.
EMF exposure systems
Cells were exposed to 50 Hz EMF at 0.5, 1.0 or 1.5 mT by placing the culture dishes in the centre of the exposure system in a tissue culture incubator (Heraeus/Kendro, Germany). Control samples were either run at the same time in a separate incubator or in the same incubator with the coil system switched off. No differences were detected between the two procedures. We used two different exposure systems to generate horizontally polarized magnetic fields (with respect to the culture medium surface) as described previously (Simko et al., 1998b). The Merritt-coil system (M-c) (Caputa and Stuchly, 1993) was designed (Electric Research & Management, Inc., State College, PA, USA) to generate a highly uniform magnetic field in the centre of the exposure system. The second exposure system was a classical pair of Helmholtzcoils (H-c) (Phywe Systeme GmbH, Göttingen, Germany) (400 mm in diameter and 200 mm apart). Both exposure systems created no significant temperature changes as measured during all experiments. The magnetic flux density was controlled using a precision Gauss/Tesla meter (F.W. Bell Inc., USA, Model 4048). The induced electric field (Emax) for these experimental geometries was calculated (Simko et al., 1998b) to be 0 ± 0.078 mV/m and the induced current density (J) to be 0 ± 1.1 mA/m2.
Data analysis
Blue fluorescent latex beads, 1 mm diameter (FluoSphere carboxylatemodified microspheres, 2% solids, Molecular Probes B.V., Leiden, The Netherlands), were coated by covalently coupling with fish skin gelatine (Sigma) (1 mg/ml in 50 mM MES buffer, pH 6.7) with 1-ethyl-3(-dimethylaminopropyl)-carbodiimide (EDAC, Sigma) according to the manufacturer s recommendations. Beads were used as a 1% suspension with a bead to cell ratio of 100 : 1.
Phagocytosis and intracellular transport assay
Monolayers grown on coverslips for 24 h in MBM medium were exposed to latex beads in the presence or absence of EMF for 30 min to allow phagocytosis. Coverslips were then washed in bead-free MBM medium for 15 min to remove non-internalized beads in the presence or absence of EMF. Macrophages were either immediately washed and fixed in 3% paraformaldehyde in Tris-buffered saline, pH 7.4, for 15 min, or further incubated for 1, 2 or 4 h for the intracellular transport studies. Slides were then mounted in antifade medium (90% glycerol, 20 mM Tris-HCl, pH 8.0, 20 mM 1,4-diazabicyclo(2,2,2)octane, Sigma). The number of internalized beads was determined for at least 100 macrophages in two simultaneously treated coverslips by using differential interference contrast (DIC) microscopy (Nikon DIAPHOT 300). Cells typically contained 5 to 20 beads. This experiment was repeated 10 times. To assess the dose response, cells were treated during phagocytosis with different concentrations (1 nM, 10 nM and 1.0 mM) of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma). The
In all experiments, only coded slides were evaluated. All data of exposed, chemical-treated or co-exposed cells were analyzed with reference to their respective controls. Differences between controls and exposed cells in each single experiment were tested using raw data for significance employing the one tailed Student s t-test with p < 0.05. To compare the specific activity in each experiment of the phagocytosis and intracellular transport assays, data were given in percentage in all experiments. For that, control samples were given as 100 percent and experimental data were given as percent of control data.
Results EMF increase the uptake of latex beads by macrophages
We measured phagocytic uptake of carboxylated latex beads by MBM macrophages (Fig. 1) in the presence and absence of 1 mT EMF (Fig. 2), generated in a Helmholtz-coil system. An increased phagocytic activity was detected in all experiments (n 10) after short-term exposure to EMF. The internalization rate of latex beads was significantly increased by 36.3 15.1% in all data sets, indicating macrophage activation. To determine the influence of pre-exposure to EMF on bead internalization, cells were also exposed for 30 and 60 min prior to the
564 M. Simkó et al.
Fig. 1. DIC image of internalized latex beads (arrows) in MBM macrophages. Bar 10 mm.
Fig. 2. Internalization of latex beads after exposure to EMF (1 mT; 50 Hz) for 45 min in MBM macrophages (10 independent experiments). Bars represent the mean number of internalized beads in 100 cells with standard deviation (SD). Asterisks denote significant differences to the controls (p < 0.05).
internalization assay (n 3). No differences between preexposed (pre-stimulated) and only acutely exposed cells (data not shown) were detected. To determine the dose-response effect of EMF on macrophage activation, the phagocytosis assay was performed with different flux densities (0, 0.5, 1.0 and 1.5 mT), using both the Helmholtz-coil (H-c) and Merritt-coil (M-c) exposure system. Internalization of latex beads was significantly increased after exposure to 1.0 and 1.5 mT, but not after exposure to 0.5 mT (Fig. 3). At the measured flux densities, the same results were registered with both exposure systems.
Influence of activation by EMF or TPA on macrophages
As a positive control agent for macrophage activation TPA was used. TPA significantly affected the uptake of beads by macrophages in a dose-dependent manner (Fig. 4, dotted line). To understand the mechanism of EMF-induced macrophage activation, duplicate samples of unexposed controls and cells exposed to 1 mT EMF alone and in the presence of 1, 10 nM or 1 mM TPA were assayed. An additive effect was
EJCB
Fig. 3. Internalization of latex beads after exposure to different flux densities of EMF (0.5 ± 1.5 mT/50 Hz) for 45 min in a Helmholtz-coil system (H-c) and Merritt-coil system (M-c) in MBM macrophages (3 independent experiments). The mean number of internalized beads in 100 cells with standard deviation (SD) is shown. Asterisks denote significant differences to the controls (p < 0.05).
Fig. 4. Dose-dependent phagocytic activity in MBM macrophages in the presence of 1 nM, 10 nM and 1 mM TPA (dotted line and asterisks). Simultaneous exposure to TPA and 1 mT EMF (solid line) shows no additional increase in phagocytic activity compared to TPA treatment only. EMF exposure alone showed a significant effect (**) as in Fig. 2 (3 independent experiments). The mean number of internalized beads in 100 cells with standard deviation (SD) is shown. Asterisks denote significant differences to the controls (p < 0.05).
expected after co-exposure to 1 mT and 1 nM TPA because phagocytosis activation was not saturated in experiments where cells were subjected to either TPA or EMF alone (Fig. 4). However, no additive activation of MBM macrophages was observed after co-exposure to EMF and TPA. Exposure to EMF alone showed a 30% increase in bead uptake, similar to results shown in Fig. 2, whereas EMF and TPA co-exposure did not enhance phagocytosis further. Interestingly, the results suggest that 1 mT EMF induces the same amount of internalized beads as 1 nM TPA.
The intracellular transport of internalized beads during exposure to EMF is not affected
In order to clarify the possible existence of EMF effects on intracellular trafficking, we studied the intracellular transport
EJCB
of internalized beads towards the nucleus in the presence and absence of EMF. The intracellular movement of internalized beads in EMF-exposed cells was not significantly different from controls. Whereas shortly after internalization 70% of the beads were detected in the cell periphery (defined as the area > 3 mm around nuclear membrane), the percentage of phagocytosed beads in the vicinity of the nucleus increased continuously after 1 and 2 hours. No further increase was observed after 4 h (data not shown).
Release of O2ÿ by MBM cells during exposure to EMF
Phagocytosis and activation of macrophages both cause the production of ROS. To investigate the mechanism of the alteration in phagocytic activity after exposure to EMF, we determined the production of O2ÿ in macrophages after exposure to EMF (1 mT) or stimulation with TPA (1 nM, 1 mM), as well as in untreated controls. No latex beads were offered in these experiments. EMF-enhanced O2ÿ production was comparable to that induced by 1 nM or 1 mM TPA, indicating that 1 mT EMF and 1 nM TPA were equal stimuli for both phagocytosis and increase in ROS levels in MBM macrophages (Fig. 5). Furthermore, these results demonstrate that ROS levels increase due to macrophage activation, and not as a result of increased phagocytosis of beads.
Discussion In this study carboxylated latex beads of 1 mm diameter were used to detect the influence of 50 Hz EMF on phagocytic activity of MBM macrophages. We found that 1 mT EMF is able to activate macrophages and to increase the internalization rate of beads from 100% in control cells to about 135% in exposed cells. Particle uptake was measured as a function of EMF dose in both exposure systems, showing a significant increase after exposure to 1 and 1.5 mT. Only very few data are available about the phagocytic activity of EMF-exposed cells in vitro. In a previous study using pre-stimulated cells (Flipo et al., 1998), a
Fig. 5. Superoxide production after exposure to EMF and to different TPA concentrations in MBM macrophages without addition of latex beads. Absorption at 550 nm with NBT assay. The production of O2ÿ was significantly increased after stimulation with 1 mT EMF, 1 nM and 1 mM TPA when compared to control cells. Each bar represents data of 3 independent experiments with 20 samples each. Asterisks denote significant differences to the controls (p < 0.05).
Electromagnetic fields as a stimulus for cell activation 565
decrease in phagocytic uptake of beads combined with increased intracellular Ca2 levels was reported. However, in this study, cells were exposed to static and much higher (25 ± 106 mT) magnetic fields, as well as for prolonged periods of time (24 h). The authors discussed their findings suggesting that several functional parameters were altered by a non-linear and non-threshold mechanism during exposure. Cellular responses to EMF, such as the modulations of the intracellular Ca2 concentration, were reported by several investigators (Walleczek and Liburdy, 1990; Lindström et al., 1993; Löschinger et al., 1999), leading to the conclusion that effects induced by EMF are possible in TPA- or mitogen-prestimulated cells only (Walleczek, 1992; Walleczek and Liburdy, 1990). Our findings contradict the latter conclusion because we detected an EMF-induced increase in phagocytic activity in non-stimulated MBM macrophages. Pre-stimulation with TPA and exposure to EMF did not influence the phagocytic activity in an additive way, although the phagocytic response was not saturated under the used exposure conditions (1 nM TPA and 1 mT EMF). It is known that TPA enhances the stimulation of macrophages for phagocytosis via the phospholipid-dependent protein kinase C pathway by mimicking diacylglycerol (Laskin et al., 1980). Tao and Henderson (1999) reported about an EMF-induced differentiation effect in HL-60 cells from nonphagocytic suspension cell to attached fibroblast-like cells with phagocytic activity, and described also an additive effect of both TPA and EMF on differentiation. Contrary to the differentiation effect in HL-60 cells, which was suggested to be PKCdependent, we conclude that the EMF-induced stimulation of MBM macrophages does not involve the PKC signal transduction pathway because of the absence of an additive effect after co-exposure to TPA and EMF. However, cell activation processes seem to depend on cell age including their activation status and the state of differentiation of the cell type. It was shown by Santoro et al. (1997) that after long-term exposure to EMF (2 mT, for 72 h), components of the cytoskeleton were reorganized and the membrane fluidity was modified in human Raji cells. Under the short-term treatment regime in our study of 1 mT for 4 h, no influence of EMF on the intracellular transport mechanisms which are based on cytoskeleton elements and motor proteins (Kuznetsov et al., 1992) was detected. Phagocytosis in activated macrophages is known to be associated with the production of superoxide radicals. To detect the activating capability of EMF on macrophages, we exposed macrophages in the absence of latex beads to EMF or to TPA (as a positive control). A significantly increased O2ÿ production was detected in the EMF- as well as in the TPAexposed cells. We conclude, therefore, that 1 mT EMF activate MBM macrophages to release superoxide radicals. Roy et al. (1995) reported an increased oxidative burst after exposure to 0.1 mT magnetic fields in TPA-activated rat peritoneal neutrophils and the authors proposed an increased lifetime of intracellular free radicals due to the field exposure as a possible mechanism. Summarizing, it can be concluded that electromagnetic fields induced an increased phagocytic activity in MBM macrophages in a dose-dependent manner. This cellular activation was detected in the absence of chemical stimulation with TPA. Coexposure to both led to no additive effect. This fact suggests that PKC is not involved in the EMF-mediated increased phagocytic activity in MBM macrophages. Furthermore, we showed
566 M. Simkó et al.
increased superoxide radical production during exposure to 1 mT EMF in absence of phagocytosis. This is taken for evidence of direct activation of macrophages by EMF. These findings demonstrate a strong interaction between EMF and MBM macrophages. The molecular mechanism by which EMF modulate signaling pathways and lead to cell activation and to cell-specific responses has to be investigated in further studies. Acknowledgements. The authors would like to thank Mrs. Bärbel Redlich for excellent technical assistance. This work was supported by the Ministerium für Bildung, Wissenschaft und Kultur, MecklenburgVorpommern, Germany, Project Number: 9700180 ± 1998.
References Ahlbom, A., Feychting, M. (1999): A Bayesian approach to hazard identification. The case of electromagnetic fields and cancer. Ann. N. Y. Acad. Sci. 895, 27 ± 33. Cadossi, R., Bersani, F., Cossarizza, A., Zucchini, P., Emilia, G., Torelli, G., Franceschi, C. (1992): Lymphocytes and low-frequency electromagnetic fields. FASEB J. 6, 2667 ± 2674. Caplan, L. S., Schoenfeld, E. R., O Leary, E. S., Leske, M. C. (2000): Breast cancer and electromagnetic fields. Ann. Epidemiol. 10, 31 ± 44. Caputa, K., Stuchly, M. A. (1993): Computer controlled system for producing uniform magnetic fields and its application in biological research. IEEE Trans. Instrumentation Measurement 45, 701 ± 709. Dibirdik, I., Kristupaitis, D., Kurosaki, T., Tuel-Ahlgren, L., Chu, A., Pond, D., Tuong, D., Luben, R., Uckun, F. M. (1998): Stimulation of Src family protein-tyrosine kinases as a proximal and mandatory step for SYK kinase-dependent phospholipase Cg2 activation in lymphoma B cells exposed to low energy electromagnetic fields. J. Biol. Chem. 273, 4035 ± 4039. Dreher, D., Junod, A. F. (1996): Role of oxygen free radicals in cancer development. Eur. J. Cancer 32A, 30 ± 38. Flipo, D., Fournier, M., Benquet, C., Roux, P., Le Boulaire, C., Pinsky, C., LaBella, F. S., Krzystyniak, K. (1998): Increased apoptosis, changes in intracellular Ca2 , and functional alterations in lymphocytes and macrophages after in vitro exposure to static magnetic field. J. Toxicol. Environ. Health 54, 63 ± 76. Holian, O., Astumian, R. D., Lee, R. C., Reyes, H. M., Attar, B. M., Walter, R. J. (1996): Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields. Bioelectromagnetics 17, 504 ± 509. Karimi, K., Lennartz, M. R. (1995): Protein kinase C activation precedes arachidonic acid release during IgG-mediated phagocytosis. J. Immunol. 155, 5786 ± 5794. Kuznetsov, S. A., Langford, G. M., Weiss, D. G. (1992): Actin-dependent organelle movement in squid axoplasm. Nature 356, 722 ± 725. Lacy-Hulbert, A., Metcalfe, J. C., Hesketh, R. (1998): Biological responses to electromagnetic fields. FASEB J. 12, 395 ± 420. Laskin, D. L., Laskin, J. D., Kessler, F. K., Weinstein, I. B., Carchman, R. A. (1980): Modulation of phagocytosis by tumour promoters and epidermal growth factor in normal and transformed macrophages. Cancer Res. 40, 1028 ± 1035. Lindström, E., Lindström, P., Berglund, A., Mild, K. H., Lundgren, E. (1993): Intracellular calcium oscillations induced in T-cell line by a weak 50 Hz magnetic field. J. Cell. Physiol. 156, 395 ± 398.
EJCB
Löschinger, M., Thumm, S., Hammerle, H., Rodemann, H. P. (1999): Induction of intracellular calcium oscillations in human skin fibroblast populations by sinusoidal extremely low-frequency magnetic fields (20 Hz, 8 mT) is dependent on the differentiation state of the single cell. Radiat. Res. 151, 195 ± 200. National Institute of Environmental Health Sciences (1998): Working Group Report: Assessment of Health Effects from Exposure to Power-line Frequency Electric and Magnetic Fields. (Portier, C. J., Wolfe, M. S., Eds.) U. S. National Institutes of Health, NIH Publication No. 98 ± 3981 (Research Triangle Park: NIEHS). Nordenson, I., Mild, K. H., Andersson, G., Sandstrom, M. (1994): Chromosomal aberrations in human amniotic cells after intermittent exposure to fifty hertz magnetic fields. Bioelectromagnetics 15, 293 ± 301. Phaire-Washington, L., Wang, E., Silverstein, S. C. (1980): Phorbol myristate acetate stimulates pinocytosis and membrane spreading in mouse peritoneal macrophages. J. Cell Biol. 86, 634 ± 640. Phillips, J. L., Haggren, W., Thomas, W. J., Ishida-Jones, T., Adey, W. R. (1992): Magnetic field-induced changes in specific gene transcription. Biochim. Biophys. Acta 1132, 140 ± 144. Roy, S., Noda, Y., Eckert, V., Traber, M. G., Mori, A., Liburdy, R. P., Packer, L. (1995): The phorbol 12-myristate 13-acetate (PMA)induced oxidative burst in rat peritoneal neutrophils is increased by a 0.1 mT (60 Hz) magnetic field. FEBS Lett. 376, 164 ± 166. Santoro, N., Lisi, A., Pozzi, D., Pasquali, E., Serafino, A., Grimaldi, S. (1997): Effect of extremely low frequency (ELF) magnetic field exposure on morphological and biophysical properties of human lymphoid cell line (Raji). Biochim. Biophys. Acta 1357, 281 ± 290. SimkoÂ, M., Kriehuber, R., Weiss, D. G., Luben, R. A. (1998a): Effects of 50 Hz EMF exposure on micronucleus formation and apoptosis in transformed and nontransformed human cell lines. Bioelectromagnetics 19, 85 ± 91. SimkoÂ, M., Kriehuber, R., Lange, S. (1998b): Micronucleus formation in human amnion cells after exposure to 50 Hz MF applied horizontally and vertically. Mutat. Res. 418, 101 ± 111. Swanson, J. A. (1989): Phorbol esters stimulate macropinocytosis and solute flow through macrophages. J. Cell Sci. 94, 135 ± 142. Tao, Q., Henderson, A. (1999): EMF induces differentiation in HL-60 cells. J. Cell. Biochem. 73, 212 ± 217. Thomas, D. C., Bowman, J. D., Jiang, L., Jiang, F., Peters, J. M. (1999): Residential magnetic fields predicted from wiring configurations: II. Relationships to childhood leukemia. Bioelectromagnetics 20, 414 ± 422. Tofani, S., Ferrara, A., Gilli, G. (1995): Evidence for genotoxic effects of resonant ELF magnetic fields. Bioelectrochem. Bioenerg. 36, 9 ± 13. Uckun, F. M., Kurosaki, T., Jin, J., Jun, X., Morgan, A., Takata, M., Bolen, J., Luben, R. A. (1995): Exposure of B-lineage lymphoid cells to low energy electromagnetic fields stimulates Lyn kinase. J. Biol. Chem. 270, 27666 ± 27670. Walleczek, J. (1992): Electromagnetic field effects on cells of the immune system: the role of calcium signaling. FASEB J. 6, 3177 ± 3185. Walleczek, J., Liburdy, R. P. (1990): Nonthermal 60 Hz sinusoidal magnetic-field exposure enhances 45Ca2 uptake in rat thymocytes: dependence on mitogen activation. FEBS Lett. 271, 157 ± 160. Zheleznyak, A., Brown, E. J. (1992): Immunoglobulin-mediated phagocytosis by human monocytes requires protein kinase C activation. J. Biol. Chem. 267, 12042 ± 12048.
562 European Journal of Cell Biology 80, 562 ± 566 (2001, August) ´ Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb
Stimulation of phagocytosis and free radical production in murine macrophages by 50 Hz electromagnetic fields Myrtill SimkoÂ1), Susanne Droste, Ralf Kriehuber, Dieter G. Weiss University of Rostock, Institute of Cell Biology and Biosystems Technology, Division of Environmental Physiology, Rostock/Germany Received March 30, 2001 Received in revised version May 14, 2001 Accepted May 15, 2001
Phagocytosis ± electromagnetic fields ± free radicals ± intracellular transport ± TPA Effects of 50 Hz electromagnetic fields on phagocytosis and free radical production were examined in mouse bone marrowderived macrophages. Macrophages were in vitro exposed to electromagnetic fields using different magnetic field densities (0.5 ± 1.5 mT). Short-time exposure (45 min) to electromagnetic fields resulted in significantly increased phagocytic uptake (36.3% 15.1%) as quantified by measuring the internalization rate of latex beads. Stimulation with 1 nM 12-Otetradecanoylphorbol-13-acetate (TPA) showed the same increased phagocytic activity as 1 mT electromagnetic fields. However, co-exposure to electromagnetic fields and TPA showed no further increase of bead uptake, and therefore we concluded that because of the absence of additive effects, the electromagnetic fields-induced stimulation of mouse bone marrow-derived macrophages does not involve the protein kinase C signal transduction pathway. Furthermore, a significant increased superoxide production after exposure to electromagnetic fields was detected.
Introduction Exposure to electromagnetic fields (EMF) has been linked to an increased incidence of leukemias and other tumours (Ahlbom and Feychting, 1999; Thomas et al., 1999; Caplan et al., 2000; Lacy-Hulbert et al., 1998), and is considered by a recent NIEHS opinion (1998) to be ªpossibly carcinogenic to humansº. There is, however, no well-established biological mechanism explaining such a relation. In various cellular 1) Dr. Myrtill SimkoÂ, University of Rostock, Institute of Cell Biology and Biosystems Technology, Division of Environmental Physiology, Universitätsplatz 2, D-18051 Rostock/Germany, e-mail: myrtill. [email protected], Fax: 49 381 498 1918.
systems, including cells of the immune system, a number of biological effects of exposure to 50/60 Hz EMF have been described (Lacy-Hulbert et al., 1998; Cadossi et al., 1992; Walleczek, 1992), and different mechanisms have been proposed to explain these effects. The induction of genotoxic effects, such as micronucleus formation (Tofani et al., 1995; Simko et al., 1998a, b) or chromosomal aberrations (Nordenson et al., 1994), shows the carcinogenic potential of EMF. However, it is generally believed that genotoxic effects of EMF cannot be induced by direct interaction with DNA, because the energy is insufficient to break chemical bonds in DNA molecules. Indirectly mediated mechanisms, such as the induction of free radicals, which are able to interact with DNA or other cellular components, can lead to a potentiation of free radical-dependent effects (Dreher and Junod, 1996), and are therefore assumed to be a more likely mechanism of EMFinduced cellular response. In macrophages, physiological activation is associated with the onset of phagocytosis and leads to increased formation of reactive oxygen species (ROS). EMF has been shown to increase ROS levels in phorbol esteractivated neutrophils (Roy et al., 1995), but it was unclear whether in non-activated macrophages, short-term EMF stimulation would elevate ROS levels via stimulation of the physiological mechanisms. In our study, we evaluated whether EMF can modulate the phagocytic activity of mouse bone marrow-derived macrophages (MBM) and if this is coupled with the induction of the release of ROS. The enhanced microbicidal and tumoricidal activity of activated macrophages is associated with both increased phagocytic activity (Laskin et al., 1980; PhaireWashington et al., 1980) and the generation of ROS by NADPH oxidase activation, which initially induces superoxide (O2ÿ) formation. As a positive control for macrophage activation we used 12-O-tetradecanoylphorbol-13-acetate (TPA) (Laskin et al., 1980; Phaire-Washington et al., 1980), because both phagocytic activity as well as free radical production can be stimulated by TPA (Zheleznyak and Brown, 1992; Karimi
0171-9335/01/80/08-562 $15.00/0
EJCB
Electromagnetic fields as a stimulus for cell activation 563
and Lennartz, 1995). TPA interacts directly with protein kinase C (PKC) by mimicking the action of diacylglycerol and is able to activate PKC-mediated signal transduction. There are several reports about the influence of electric and magnetic fields on PKC activity or other kinases involved in signal transduction cascades in different cell systems. It has been shown that treatment of HL-60 cells with 60 Hz electric fields results in decreased cytosolic but unchanged membraneassociated PKC activities (Holian et al., 1996). Phillips et al. (1992) showed an increase in PKC activity upon exposure to EMF. Uckun et al. (1995) indicated that 60 Hz fields caused activation of tyrosine kinases and PKC in a human pre-Bleukemia cell line. In a recent study Dibirdik et al. (1998) demonstrated that exposure of DT40 lymphoma B cells to EMF results in a tyrosine kinase-dependent activation of phopholipase Cg2 leading to increased inositol phospholipid turnover. The aim of our study was to investigate the activating capacity of EMF by studying phagocytic activity and superoxide production of MBM macrophages. To investigate whether the PKC-mediated signal transduction pathway is involved, coexposure experiments with TPA and EMF were performed.
Materials and methods Cell culture
Murine bone marrow-derived (MBM) macrophages were obtained according to standard procedures (Swanson, 1989). Mononuclear phagocyte precursor cells were prepared from tibial and femurial mouse bones, resuspended and washed in PBS. Cells were cultured in MBM medium (50% L929 cell-conditioned medium, 50% RPMI-1640, 10% heat-inactivated FCS, 1% L-glutamine and 1% non-essential amino acids, 0.5% mercaptoethanol and antibiotics, all from Gibco, BRL) for 5 ± 6 days at 37 8C in a humidified atmosphere of 5% CO2 and 95% air for differentiation to adherently growing macrophages. Three days after initial plating, the medium was changed, and after an additional 2 or 3 days cells were cooled to ÿ 20 8C in a freezer for 1 ± 2 min until they rounded up. Cells were scraped and re-plated onto sterile 11-mm circular glass coverslips (104 cells/coverslip) in Petri dishes.
Preparation of latex beads
following co-exposure experiments were simultaneously carried out: a) without any treatment as control, b) in the presence of 1 mT EMF, c) in the presence of TPA and d) in the presence of both. To analyze intracellular transport of internalized latex beads from the cell periphery toward the nucleus, the position of internalized beads was determined for at least 25 cells per coverslip. The relative number of beads was determined for the peripheral and the central region, the latter being defined as the area of 3 mm around the nucleus (eye-piece reticle). These experiments were repeated 5 times.
O2ÿ generation
Cells were plated in 96-well plates (104 cells/well) for four hours prior to use. After washing with PBS, cells were incubated for 45 min with PBS supplemented with 1 mM p-nitro blue tetrazolium (NBT, Molecular Probes) and 1 mM CaCl2 with or without TPA and in the absence or presence of EMF exposure. O2ÿgeneration reduces NBT to blue formazan within the cells, which was measured using a microplate reader (Anthos 2010) at 550 nm. Intracellular formazan crystals were solubilized in 100 ml DMSO prior to reading.
EMF exposure systems
Cells were exposed to 50 Hz EMF at 0.5, 1.0 or 1.5 mT by placing the culture dishes in the centre of the exposure system in a tissue culture incubator (Heraeus/Kendro, Germany). Control samples were either run at the same time in a separate incubator or in the same incubator with the coil system switched off. No differences were detected between the two procedures. We used two different exposure systems to generate horizontally polarized magnetic fields (with respect to the culture medium surface) as described previously (Simko et al., 1998b). The Merritt-coil system (M-c) (Caputa and Stuchly, 1993) was designed (Electric Research & Management, Inc., State College, PA, USA) to generate a highly uniform magnetic field in the centre of the exposure system. The second exposure system was a classical pair of Helmholtzcoils (H-c) (Phywe Systeme GmbH, Göttingen, Germany) (400 mm in diameter and 200 mm apart). Both exposure systems created no significant temperature changes as measured during all experiments. The magnetic flux density was controlled using a precision Gauss/Tesla meter (F.W. Bell Inc., USA, Model 4048). The induced electric field (Emax) for these experimental geometries was calculated (Simko et al., 1998b) to be 0 ± 0.078 mV/m and the induced current density (J) to be 0 ± 1.1 mA/m2.
Data analysis
Blue fluorescent latex beads, 1 mm diameter (FluoSphere carboxylatemodified microspheres, 2% solids, Molecular Probes B.V., Leiden, The Netherlands), were coated by covalently coupling with fish skin gelatine (Sigma) (1 mg/ml in 50 mM MES buffer, pH 6.7) with 1-ethyl-3(-dimethylaminopropyl)-carbodiimide (EDAC, Sigma) according to the manufacturer s recommendations. Beads were used as a 1% suspension with a bead to cell ratio of 100 : 1.
Phagocytosis and intracellular transport assay
Monolayers grown on coverslips for 24 h in MBM medium were exposed to latex beads in the presence or absence of EMF for 30 min to allow phagocytosis. Coverslips were then washed in bead-free MBM medium for 15 min to remove non-internalized beads in the presence or absence of EMF. Macrophages were either immediately washed and fixed in 3% paraformaldehyde in Tris-buffered saline, pH 7.4, for 15 min, or further incubated for 1, 2 or 4 h for the intracellular transport studies. Slides were then mounted in antifade medium (90% glycerol, 20 mM Tris-HCl, pH 8.0, 20 mM 1,4-diazabicyclo(2,2,2)octane, Sigma). The number of internalized beads was determined for at least 100 macrophages in two simultaneously treated coverslips by using differential interference contrast (DIC) microscopy (Nikon DIAPHOT 300). Cells typically contained 5 to 20 beads. This experiment was repeated 10 times. To assess the dose response, cells were treated during phagocytosis with different concentrations (1 nM, 10 nM and 1.0 mM) of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma). The
In all experiments, only coded slides were evaluated. All data of exposed, chemical-treated or co-exposed cells were analyzed with reference to their respective controls. Differences between controls and exposed cells in each single experiment were tested using raw data for significance employing the one tailed Student s t-test with p < 0.05. To compare the specific activity in each experiment of the phagocytosis and intracellular transport assays, data were given in percentage in all experiments. For that, control samples were given as 100 percent and experimental data were given as percent of control data.
Results EMF increase the uptake of latex beads by macrophages
We measured phagocytic uptake of carboxylated latex beads by MBM macrophages (Fig. 1) in the presence and absence of 1 mT EMF (Fig. 2), generated in a Helmholtz-coil system. An increased phagocytic activity was detected in all experiments (n 10) after short-term exposure to EMF. The internalization rate of latex beads was significantly increased by 36.3 15.1% in all data sets, indicating macrophage activation. To determine the influence of pre-exposure to EMF on bead internalization, cells were also exposed for 30 and 60 min prior to the
564 M. Simkó et al.
Fig. 1. DIC image of internalized latex beads (arrows) in MBM macrophages. Bar 10 mm.
Fig. 2. Internalization of latex beads after exposure to EMF (1 mT; 50 Hz) for 45 min in MBM macrophages (10 independent experiments). Bars represent the mean number of internalized beads in 100 cells with standard deviation (SD). Asterisks denote significant differences to the controls (p < 0.05).
internalization assay (n 3). No differences between preexposed (pre-stimulated) and only acutely exposed cells (data not shown) were detected. To determine the dose-response effect of EMF on macrophage activation, the phagocytosis assay was performed with different flux densities (0, 0.5, 1.0 and 1.5 mT), using both the Helmholtz-coil (H-c) and Merritt-coil (M-c) exposure system. Internalization of latex beads was significantly increased after exposure to 1.0 and 1.5 mT, but not after exposure to 0.5 mT (Fig. 3). At the measured flux densities, the same results were registered with both exposure systems.
Influence of activation by EMF or TPA on macrophages
As a positive control agent for macrophage activation TPA was used. TPA significantly affected the uptake of beads by macrophages in a dose-dependent manner (Fig. 4, dotted line). To understand the mechanism of EMF-induced macrophage activation, duplicate samples of unexposed controls and cells exposed to 1 mT EMF alone and in the presence of 1, 10 nM or 1 mM TPA were assayed. An additive effect was
EJCB
Fig. 3. Internalization of latex beads after exposure to different flux densities of EMF (0.5 ± 1.5 mT/50 Hz) for 45 min in a Helmholtz-coil system (H-c) and Merritt-coil system (M-c) in MBM macrophages (3 independent experiments). The mean number of internalized beads in 100 cells with standard deviation (SD) is shown. Asterisks denote significant differences to the controls (p < 0.05).
Fig. 4. Dose-dependent phagocytic activity in MBM macrophages in the presence of 1 nM, 10 nM and 1 mM TPA (dotted line and asterisks). Simultaneous exposure to TPA and 1 mT EMF (solid line) shows no additional increase in phagocytic activity compared to TPA treatment only. EMF exposure alone showed a significant effect (**) as in Fig. 2 (3 independent experiments). The mean number of internalized beads in 100 cells with standard deviation (SD) is shown. Asterisks denote significant differences to the controls (p < 0.05).
expected after co-exposure to 1 mT and 1 nM TPA because phagocytosis activation was not saturated in experiments where cells were subjected to either TPA or EMF alone (Fig. 4). However, no additive activation of MBM macrophages was observed after co-exposure to EMF and TPA. Exposure to EMF alone showed a 30% increase in bead uptake, similar to results shown in Fig. 2, whereas EMF and TPA co-exposure did not enhance phagocytosis further. Interestingly, the results suggest that 1 mT EMF induces the same amount of internalized beads as 1 nM TPA.
The intracellular transport of internalized beads during exposure to EMF is not affected
In order to clarify the possible existence of EMF effects on intracellular trafficking, we studied the intracellular transport
EJCB
of internalized beads towards the nucleus in the presence and absence of EMF. The intracellular movement of internalized beads in EMF-exposed cells was not significantly different from controls. Whereas shortly after internalization 70% of the beads were detected in the cell periphery (defined as the area > 3 mm around nuclear membrane), the percentage of phagocytosed beads in the vicinity of the nucleus increased continuously after 1 and 2 hours. No further increase was observed after 4 h (data not shown).
Release of O2ÿ by MBM cells during exposure to EMF
Phagocytosis and activation of macrophages both cause the production of ROS. To investigate the mechanism of the alteration in phagocytic activity after exposure to EMF, we determined the production of O2ÿ in macrophages after exposure to EMF (1 mT) or stimulation with TPA (1 nM, 1 mM), as well as in untreated controls. No latex beads were offered in these experiments. EMF-enhanced O2ÿ production was comparable to that induced by 1 nM or 1 mM TPA, indicating that 1 mT EMF and 1 nM TPA were equal stimuli for both phagocytosis and increase in ROS levels in MBM macrophages (Fig. 5). Furthermore, these results demonstrate that ROS levels increase due to macrophage activation, and not as a result of increased phagocytosis of beads.
Discussion In this study carboxylated latex beads of 1 mm diameter were used to detect the influence of 50 Hz EMF on phagocytic activity of MBM macrophages. We found that 1 mT EMF is able to activate macrophages and to increase the internalization rate of beads from 100% in control cells to about 135% in exposed cells. Particle uptake was measured as a function of EMF dose in both exposure systems, showing a significant increase after exposure to 1 and 1.5 mT. Only very few data are available about the phagocytic activity of EMF-exposed cells in vitro. In a previous study using pre-stimulated cells (Flipo et al., 1998), a
Fig. 5. Superoxide production after exposure to EMF and to different TPA concentrations in MBM macrophages without addition of latex beads. Absorption at 550 nm with NBT assay. The production of O2ÿ was significantly increased after stimulation with 1 mT EMF, 1 nM and 1 mM TPA when compared to control cells. Each bar represents data of 3 independent experiments with 20 samples each. Asterisks denote significant differences to the controls (p < 0.05).
Electromagnetic fields as a stimulus for cell activation 565
decrease in phagocytic uptake of beads combined with increased intracellular Ca2 levels was reported. However, in this study, cells were exposed to static and much higher (25 ± 106 mT) magnetic fields, as well as for prolonged periods of time (24 h). The authors discussed their findings suggesting that several functional parameters were altered by a non-linear and non-threshold mechanism during exposure. Cellular responses to EMF, such as the modulations of the intracellular Ca2 concentration, were reported by several investigators (Walleczek and Liburdy, 1990; Lindström et al., 1993; Löschinger et al., 1999), leading to the conclusion that effects induced by EMF are possible in TPA- or mitogen-prestimulated cells only (Walleczek, 1992; Walleczek and Liburdy, 1990). Our findings contradict the latter conclusion because we detected an EMF-induced increase in phagocytic activity in non-stimulated MBM macrophages. Pre-stimulation with TPA and exposure to EMF did not influence the phagocytic activity in an additive way, although the phagocytic response was not saturated under the used exposure conditions (1 nM TPA and 1 mT EMF). It is known that TPA enhances the stimulation of macrophages for phagocytosis via the phospholipid-dependent protein kinase C pathway by mimicking diacylglycerol (Laskin et al., 1980). Tao and Henderson (1999) reported about an EMF-induced differentiation effect in HL-60 cells from nonphagocytic suspension cell to attached fibroblast-like cells with phagocytic activity, and described also an additive effect of both TPA and EMF on differentiation. Contrary to the differentiation effect in HL-60 cells, which was suggested to be PKCdependent, we conclude that the EMF-induced stimulation of MBM macrophages does not involve the PKC signal transduction pathway because of the absence of an additive effect after co-exposure to TPA and EMF. However, cell activation processes seem to depend on cell age including their activation status and the state of differentiation of the cell type. It was shown by Santoro et al. (1997) that after long-term exposure to EMF (2 mT, for 72 h), components of the cytoskeleton were reorganized and the membrane fluidity was modified in human Raji cells. Under the short-term treatment regime in our study of 1 mT for 4 h, no influence of EMF on the intracellular transport mechanisms which are based on cytoskeleton elements and motor proteins (Kuznetsov et al., 1992) was detected. Phagocytosis in activated macrophages is known to be associated with the production of superoxide radicals. To detect the activating capability of EMF on macrophages, we exposed macrophages in the absence of latex beads to EMF or to TPA (as a positive control). A significantly increased O2ÿ production was detected in the EMF- as well as in the TPAexposed cells. We conclude, therefore, that 1 mT EMF activate MBM macrophages to release superoxide radicals. Roy et al. (1995) reported an increased oxidative burst after exposure to 0.1 mT magnetic fields in TPA-activated rat peritoneal neutrophils and the authors proposed an increased lifetime of intracellular free radicals due to the field exposure as a possible mechanism. Summarizing, it can be concluded that electromagnetic fields induced an increased phagocytic activity in MBM macrophages in a dose-dependent manner. This cellular activation was detected in the absence of chemical stimulation with TPA. Coexposure to both led to no additive effect. This fact suggests that PKC is not involved in the EMF-mediated increased phagocytic activity in MBM macrophages. Furthermore, we showed
566 M. Simkó et al.
increased superoxide radical production during exposure to 1 mT EMF in absence of phagocytosis. This is taken for evidence of direct activation of macrophages by EMF. These findings demonstrate a strong interaction between EMF and MBM macrophages. The molecular mechanism by which EMF modulate signaling pathways and lead to cell activation and to cell-specific responses has to be investigated in further studies. Acknowledgements. The authors would like to thank Mrs. Bärbel Redlich for excellent technical assistance. This work was supported by the Ministerium für Bildung, Wissenschaft und Kultur, MecklenburgVorpommern, Germany, Project Number: 9700180 ± 1998.
References Ahlbom, A., Feychting, M. (1999): A Bayesian approach to hazard identification. The case of electromagnetic fields and cancer. Ann. N. Y. Acad. Sci. 895, 27 ± 33. Cadossi, R., Bersani, F., Cossarizza, A., Zucchini, P., Emilia, G., Torelli, G., Franceschi, C. (1992): Lymphocytes and low-frequency electromagnetic fields. FASEB J. 6, 2667 ± 2674. Caplan, L. S., Schoenfeld, E. R., O Leary, E. S., Leske, M. C. (2000): Breast cancer and electromagnetic fields. Ann. Epidemiol. 10, 31 ± 44. Caputa, K., Stuchly, M. A. (1993): Computer controlled system for producing uniform magnetic fields and its application in biological research. IEEE Trans. Instrumentation Measurement 45, 701 ± 709. Dibirdik, I., Kristupaitis, D., Kurosaki, T., Tuel-Ahlgren, L., Chu, A., Pond, D., Tuong, D., Luben, R., Uckun, F. M. (1998): Stimulation of Src family protein-tyrosine kinases as a proximal and mandatory step for SYK kinase-dependent phospholipase Cg2 activation in lymphoma B cells exposed to low energy electromagnetic fields. J. Biol. Chem. 273, 4035 ± 4039. Dreher, D., Junod, A. F. (1996): Role of oxygen free radicals in cancer development. Eur. J. Cancer 32A, 30 ± 38. Flipo, D., Fournier, M., Benquet, C., Roux, P., Le Boulaire, C., Pinsky, C., LaBella, F. S., Krzystyniak, K. (1998): Increased apoptosis, changes in intracellular Ca2 , and functional alterations in lymphocytes and macrophages after in vitro exposure to static magnetic field. J. Toxicol. Environ. Health 54, 63 ± 76. Holian, O., Astumian, R. D., Lee, R. C., Reyes, H. M., Attar, B. M., Walter, R. J. (1996): Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields. Bioelectromagnetics 17, 504 ± 509. Karimi, K., Lennartz, M. R. (1995): Protein kinase C activation precedes arachidonic acid release during IgG-mediated phagocytosis. J. Immunol. 155, 5786 ± 5794. Kuznetsov, S. A., Langford, G. M., Weiss, D. G. (1992): Actin-dependent organelle movement in squid axoplasm. Nature 356, 722 ± 725. Lacy-Hulbert, A., Metcalfe, J. C., Hesketh, R. (1998): Biological responses to electromagnetic fields. FASEB J. 12, 395 ± 420. Laskin, D. L., Laskin, J. D., Kessler, F. K., Weinstein, I. B., Carchman, R. A. (1980): Modulation of phagocytosis by tumour promoters and epidermal growth factor in normal and transformed macrophages. Cancer Res. 40, 1028 ± 1035. Lindström, E., Lindström, P., Berglund, A., Mild, K. H., Lundgren, E. (1993): Intracellular calcium oscillations induced in T-cell line by a weak 50 Hz magnetic field. J. Cell. Physiol. 156, 395 ± 398.
EJCB
Löschinger, M., Thumm, S., Hammerle, H., Rodemann, H. P. (1999): Induction of intracellular calcium oscillations in human skin fibroblast populations by sinusoidal extremely low-frequency magnetic fields (20 Hz, 8 mT) is dependent on the differentiation state of the single cell. Radiat. Res. 151, 195 ± 200. National Institute of Environmental Health Sciences (1998): Working Group Report: Assessment of Health Effects from Exposure to Power-line Frequency Electric and Magnetic Fields. (Portier, C. J., Wolfe, M. S., Eds.) U. S. National Institutes of Health, NIH Publication No. 98 ± 3981 (Research Triangle Park: NIEHS). Nordenson, I., Mild, K. H., Andersson, G., Sandstrom, M. (1994): Chromosomal aberrations in human amniotic cells after intermittent exposure to fifty hertz magnetic fields. Bioelectromagnetics 15, 293 ± 301. Phaire-Washington, L., Wang, E., Silverstein, S. C. (1980): Phorbol myristate acetate stimulates pinocytosis and membrane spreading in mouse peritoneal macrophages. J. Cell Biol. 86, 634 ± 640. Phillips, J. L., Haggren, W., Thomas, W. J., Ishida-Jones, T., Adey, W. R. (1992): Magnetic field-induced changes in specific gene transcription. Biochim. Biophys. Acta 1132, 140 ± 144. Roy, S., Noda, Y., Eckert, V., Traber, M. G., Mori, A., Liburdy, R. P., Packer, L. (1995): The phorbol 12-myristate 13-acetate (PMA)induced oxidative burst in rat peritoneal neutrophils is increased by a 0.1 mT (60 Hz) magnetic field. FEBS Lett. 376, 164 ± 166. Santoro, N., Lisi, A., Pozzi, D., Pasquali, E., Serafino, A., Grimaldi, S. (1997): Effect of extremely low frequency (ELF) magnetic field exposure on morphological and biophysical properties of human lymphoid cell line (Raji). Biochim. Biophys. Acta 1357, 281 ± 290. SimkoÂ, M., Kriehuber, R., Weiss, D. G., Luben, R. A. (1998a): Effects of 50 Hz EMF exposure on micronucleus formation and apoptosis in transformed and nontransformed human cell lines. Bioelectromagnetics 19, 85 ± 91. SimkoÂ, M., Kriehuber, R., Lange, S. (1998b): Micronucleus formation in human amnion cells after exposure to 50 Hz MF applied horizontally and vertically. Mutat. Res. 418, 101 ± 111. Swanson, J. A. (1989): Phorbol esters stimulate macropinocytosis and solute flow through macrophages. J. Cell Sci. 94, 135 ± 142. Tao, Q., Henderson, A. (1999): EMF induces differentiation in HL-60 cells. J. Cell. Biochem. 73, 212 ± 217. Thomas, D. C., Bowman, J. D., Jiang, L., Jiang, F., Peters, J. M. (1999): Residential magnetic fields predicted from wiring configurations: II. Relationships to childhood leukemia. Bioelectromagnetics 20, 414 ± 422. Tofani, S., Ferrara, A., Gilli, G. (1995): Evidence for genotoxic effects of resonant ELF magnetic fields. Bioelectrochem. Bioenerg. 36, 9 ± 13. Uckun, F. M., Kurosaki, T., Jin, J., Jun, X., Morgan, A., Takata, M., Bolen, J., Luben, R. A. (1995): Exposure of B-lineage lymphoid cells to low energy electromagnetic fields stimulates Lyn kinase. J. Biol. Chem. 270, 27666 ± 27670. Walleczek, J. (1992): Electromagnetic field effects on cells of the immune system: the role of calcium signaling. FASEB J. 6, 3177 ± 3185. Walleczek, J., Liburdy, R. P. (1990): Nonthermal 60 Hz sinusoidal magnetic-field exposure enhances 45Ca2 uptake in rat thymocytes: dependence on mitogen activation. FEBS Lett. 271, 157 ± 160. Zheleznyak, A., Brown, E. J. (1992): Immunoglobulin-mediated phagocytosis by human monocytes requires protein kinase C activation. J. Biol. Chem. 267, 12042 ± 12048.