ELF magnetic fields induce internalization of gap junction protein connexin 43 in Chinese hamster lung cells

We have previously demonstrated that exposure of Chinese hamster lung (CHL) cells to 50 Hz magnetic fields (MFs) and/or 12-O-tetradecanoylphorbol-3-acetate (TPA)-inhibited gap junctional intercellular communication (GJIC). To explore and compare the mechanisms of GJIC inhibition induced by extremely low frequency (ELF) MF and TPA, the number and localization of connexin 43 (C x 43) were studied. The localization of C x 43 was determined with indirect immunofluorescence histochemical analysis and detected by confocal microscopy after exposing CHL cells to 50 Hz sinusoidal magnetic field at 0.8 mT for 24 h without or with TPA (5 ng/ml) for the last 1 h. The C x 43 levels in nuclei and in cytoplasm were examined by Western blotting analysis. The results showed that the cells exposed to MF and/or TPA displayed individual plaques at regions of intercellular contact, which were fewer than the normal cells in number, while the number of C x 43 in cytoplasm increased and congregated near the nuclei. Western blot analysis further demonstrated the quantity of changes in location of Cx43. These results suggest that reduction of C x 43 at regions of intercellular contact may be one of the mechanisms of GJIC inhibition induced by ELF MF.     

No effect of exposure to static and sinusoidal magnetic fields on nitric oxide production by macrophages

The effects of exposure to static (1–100 mT) or sinusoidal (1 Hz, 1.6 mT) magnetic fields on the production of nitric oxide (NO) by murine BCG-activated macrophages were investigated. In these cells, the inducible isoform of NO synthase is present. No significant differences were observed in nitrite levels among exposed, sham-exposed, or control macrophages after exposure for 14 h to static fields of 1, 10, 50, and 100 mT and to sinusoidal 1.6 mT, 1 Hz magnetic fields. © 1996 Wiley-Liss, Inc.     

Effects of 50 Hz magnetic fields on gap junctional intercellular communication.

To explore whether the extremely low frequency (ELF) electromagnetic fields (EMFs) may act as cancer promoters or be synergistic with 12-O-tetradecanoylphorbol-13-acetate (TPA) in cancer promotion, an experiment was conducted on the effects of 50 Hz magnetic fields (MFs) on gap junctional intercellular communication (GJIC) of Chinese hamster lung (CHL) cells. Lucifer dye was loaded into CHL cells by iontophoretic injection, and the number of dye-coupled cells (DCC) 5 min after the injection was adopted as the index of GJIC. The effects of TPA at different concentrations and magnetic fields at different intensities, combined with 5 ng/ml TPA, were studied. The results showed that the suppression of TPA on GJIC was dependent on TPA concentration; the threshold concentration of TPA for CHL cells was between 1 and 5 ng/ml. After exposure to 0.8 mT magnetic field for 24 h, the number of DCC decreased to 6.08 +/- 1.59, whereas the number of DCC in the control group was 9.84 +/- 2.27 (P < .05). When the cells were exposed at 0.2, 0.4, and 0.8 mT for 24 h, combined with 5 ng/ml TPA treatment during the last 1 h, the number of DCC decreased to 5.52 +/- 1.53, 5.00 +/- 1.22, and 4.00 +/- 1.29, respectively, which were significantly lower than the values for the group treated with 5 ng/ml TPA alone (6.38 +/- 1.39). It is suggested that certain intensities of 50 Hz magnetic field might act as cancer promoters, be additive with other promoters in cancer promotion, or both.     

Oviposition and development of Drosophila modified by magnetic fields

Drosophila flies placed in a habitat with two lateral boxes demonstrated sensitivity to magnetic fields: Oviposition decreased by exposure to pulsated extremely low frequency (ELF) (100)Hz, 1.76 miliTesla (mT) and sinusosidal fields (50 Hz, 1 mT), while there was no initial effect of exposure to a static magnetic field (4.5 mT). Drosophila eggs treated for 48 h with the above described fields showed that (1) mortality of eggs was lower in controls than in eggs exposed to all tested magnetic fields; (2) mortality of larvae increased when a permanent magnet was used; (3) mortality of pupae was highest when a permanent magnet was used; and (4) general adult viability was highest in controls (67%) and diminished progressively when eggs were exposed to pulsated (55%), sinusoidal (45%), and static (35%) magnetic fields.     

Fifty-hertz magnetic fields induce free radical formation in mouse bone marrow-derived promonocytes and macrophages

Our findings show a significant increase of free radical production after exposure to 50 Hz electromagnetic fields at a flux density of 1 mT to mouse bone marrow-derived (MBM) promonocytes and macrophages, indicating the cell-activating capacity of extremely low frequency magnetic fields (ELF-MF). We demonstrate that after exposure to ELF-MF mainly superoxide anion radicals were produced, both in MBM macrophages (33%) and also in their precursor cells (24%). To elucidate whether NADPH- or NADH-oxidase functions are target proteins for MF interaction, the flavoprotein inhibitor diphenyleneiodonium chloride (DPI) was used. MF-induced free radical production was not inhibited by DPI, whereas tetradecanoylphorbolacetate (TPA)-induced free radical production was diminished by about 70%. TPA is known to induce a direct activation of NADPH-oxidase through the PKC pathway. Since DPI lacks an inhibitory effect in MF-exposed MBM cells, we suggest that 50 Hz MF stimulates the NADH-oxidase pathway to produce superoxide anion radicals, but not the NADPH pathway. Furthermore, we showed an oscillation (1-10 days) in superoxide anion radical release in mouse macrophages, indicating a cyclic pattern of NADH-oxidase activity.     

STUDY ON GAP JUNCTIONAL INTERCELLULAR COMMUNICATION INHIBITION BY ELF MAGNETIC FIELDS USING FRAP METHOD

Inhibition of gap junctional intercellular communication (GJIC) is an important event in the multistage process of carcinogenesis. Our previous study showed that extremely low frequency (ELF) magnetic fields (MFs) inhibit GJIC, and enhance the suppression of GJIC induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) using a microinjection technique. In the present study, the inhibition of GJIC by ELF MFs and its threshold were further studied with fluorescence recovery after photobleaching (FRAP) technique. The results indicated that the FRAP technique is more sensitive in detecting the changes of GJIC than microinjection, and the threshold level is 0.4 mT for GJIC suppression by 50 Hz MFs. In addition, 0.2 mT, or more than 0.2 mT ELF can enhance the inhibition of GJIC induced by TPA. We concluded that MFs thus might act as a cancer promoter or work in synergy with other cancer promoters. The data also provide grounds to revise the reference standard of ELF MFs exposure.     

Effects of low-frequency magnetic fields on fetal development in CBA/Ca mice

Effects of alternating magnetic fields (MFs) on the embryonic and fetal development in CBA/Ca mice were studied. Mated females were exposed continuously to a sinusoidal 50 Hz (13 μT or 0.13 mT root mean square) or a sawtooth 20 kHz (15 μT peak-to-peak) MF from day 0 to day 18 of pregnancy for 24 h/day until necropsied on day 18. Control animals were kept under the same conditions without the MF. MFs did not cause maternal toxicity. No adverse effects were seen in maternal hematology and the frequency of micronuclei in maternal bone marrow erythrocytes did not change. The MFs did not increase the number of resorptions or fetuses with major or minor malformations in any exposure group. The mean number of implantations and living fetuses per litter were similar in all groups. The corrected weight gain (weight gain without uterine content) of dams, pregnancy rates, incidences of resorptions and late fetal deaths, and fetal body weights were similar in all groups. There was, however, a statistically significant increase in the incidence of fetuses with at least three skeletal variations in all groups exposed to MFs. In conclusion, the 50 Hz or 20 kHz MFs did not increase incidences of malformations or resorptions in CBA/Ca mice, but increased skeletal variations consistently in all exposure groups. Bioelectromagnetics 19:477–485, 1998. © 1998 Wiley-Liss, Inc.     

Cytological effects of 60 Hz magnetic fields on human lymphocytes in vitro: sister-chromatid exchanges, cell kinetics and mitotic rate

Incubation for 72 h of human peripheral blood cultures in the presence of 60 Hz sinusoidal magnetic fields (MF) at magnetic flux densities of 1.0, 1.5, and 2.0 mT led to stimulation of lymphocyte proliferation but had no influence on the frequency of sister-chromatid exchanges (SCE). The cytotoxic potential of MF combined with the mutagen Mitomycin-C also was analyzed. An opposite effect between MF exposure and Mitomycin-C treatment in terms of cell kinetics and mitotic rate was found, whereas no variation in SCE frequency was observed for this coexposure condition.     

Protective effect of melatonin against in vitro iron ions and 7 mT 50 Hz magnetic field-induced DNA damage in rat lymphocytes

We have previously shown that simultaneous exposure of rat lymphocytes to iron ions and 50Hz magnetic field (MF) caused an increase in the number of cells with DNA strand breaks. Although the mechanism of MF-induced DNA damage is not known, we suppose that it involves free radicals. In the present study, to confirm our hypothesis, we have examined the effect of melatonin, an established free radicals scavenger, on DNA damage in rat peripheral blood lymphocytes exposed in vitro to iron ions and 50Hz MF. The alkaline comet assay was chosen for the assessment of DNA damage. During pre-incubation, part of the cell samples were supplemented with melatonin (0.5 or 1.0mM). The experiments were performed on the cell samples incubated for 3h in Helmholtz coils at 7mT 50Hz MF. During MF exposure, some samples were treated with ferrous chloride (FeCl2, 10microg/ml), while the rest served as controls. A significant increase in the number of cells with DNA damage was found only after simultaneous exposure of lymphocytes to FeCl2 and 7mT 50Hz MF, compared to the control samples or those incubated with FeCl2 alone. However, when the cells were treated with melatonin and then exposed to iron ions and 50Hz MF, the number of damaged cells was significantly reduced, and the effect depended on the concentration of melatonin. The reduction reached about 50% at 0.5mM and about 100% at 1.0mM. Our results indicate that melatonin provides protection against DNA damage in rat lymphocytes exposed in vitro to iron ions and 50Hz MF (7mT). Therefore, it can be suggested that free radicals may be involved in 50Hz magnetic field and iron ions-induced DNA damage in rat blood lymphocytes. The future experimental studies, in vitro and in vivo, should provide an answer to the question concerning the role of melatonin in the free radical processes in the power frequency magnetic field.     

Entamoeba invadens: influence of 60 Hz magnetic fields on growth and differentiation

Exposure to extremely low-frequency (ELF) electromagnetic fields appears to result in a number of important biological changes. In the present study, we evaluated the effects of 60 Hz sinusoidal magnetic fields (MF) at magnetic flux densities of 1.0, 1.5 and 2.0 mT on growth and differentiation of the protozoan Entamoeba invadens. We demonstrated an inhibitory growth effect when trophozoite cultures were exposed to 1.5 and 2.0 mT. Furthermore, we found that there was not a synergistic effect in cultures co-exposed to MF and Metronidazole, a cytotoxic drug against amoebic cells. In addition, MF exposure inhibited the encystation process of E. invadens.     

Lack of alterations on meiotic chromosomes and morphological characteristics of male germ cells in mice exposed to a 60 Hz and 2.0 mT magnetic field

The effect of in vivo exposure of mice to a 60 Hz sinusoidal magnetic field (MF) at 2.0 mT on male germ cells was studied. The cytological endpoints measured included meiotic chromosome aberrations in spermatocytes and sperm morphology. Three independent experiments were carried out: (a) animals exposed for 72 h, (b) 10 days/8 h daily, and (c) 72 h exposure to MF plus 5 mg/kg of Mitomycin-C. No statistically significant differences indicative of MF effects were observed between MF exposed and control animals. In addition, an opposite effect between MF exposure and Mitomycin-C treatment in terms of chromosomal aberrations and sperm morphology was observed.     

Rat liver foci study on coexposure with 50 Hz magnetic fields and known carcinogens

A study was performed to investigate possible interactions by magnetic fields (MF) with the processes of initiation and promotion of chemically induced preneoplastic lesions in rat liver. Male Sprague-Dawley rats were subjected to a 70% partial hepatectomy followed after 24 h by i.p. injection of diethylnitrosamine (DENA) as a tumour initiator. Starting one week after the DENA-treatment phenobarbital (PB) was given to promote growth of enzymatically altered foci of liver cells. MF was applied immediately after the partial hepatectomy and continued until sacrifice after 12 weeks of PB exposure. Homogenous horizontal AC magnetic fields with a frequency of 50 Hz and flux densities of 0.5 μT or 0.5 mT were used. The rats coexposed with MF and DENA plus PB did not gain weight as much as the rats exposed to the chemical agents only. The MF-exposure also resulted in a slight reduction in size and numbers of the focal lesions. The results suggest an interaction of MF with the processes of chemical carcinogenesis either as a result of stress or depending on effects on the proliferation of preneoplastic cells. © 1993 Wiley-Liss, Inc.     

ELF magnetic field inhibits gap junctional intercellular communication and induces hyperphosphorylation of connexin43 in NIH3T3 cells.

The effects of extremely low frequency (ELF) magnetic field on gap junctional intercellular communication (GJIC), protein levels, and phosphorylation of connexin43 (Cx43) were studied in NIH3T3 cells. The suppression of GJIC by 24 h, 50 Hz, 0.8 mT ELF magnetic field, 2 h, 3 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or ELF combined with TPA treatment was confirmed by the fluorescence recovery after photobleaching (FRAP) analysis with a confocal microscope. The results showed that ELF or TPA exposure induced 50-60% inhibition of GJIC (P < 0.01). ELF combined with TPA enhanced the inhibition of GJIC. Western blot analysis using Cx43 specific antibodies showed obviously decreasing non phosphorylated Cx43 (P(0)) induced by ELF and/or TPA exposure. On the other hand, cells treated with ELF and/or TPA displayed a hyperphosphorylated Cx43 band (P(3)). However, there was no obvious changes in the level of Cx43 protein. The results implied that the P(3) band appeared to result from phosphorylation of P(0). But it remains possible that upon the ELF exposure P(0) is converted to P(1), P(2) or both and that P(3) is formed from P(1) or P(2) resulting in the observed hyperphosphorylation pattern. From the present study, we conclude that ELF magnetic field inhibits GJIC and the main mechanism is the hyperphosphorylation of Cx43.     

EGF receptor clustering is induced by a 0.4 mT power frequency magnetic field and blocked by the EGF receptor tyrosine kinase inhibitor PD153035

Atomic force microscopy (AFM), transmission electron microscopy (TEM), and confocal laser scanning microscopy were used to investigate the effects of a 50 Hz 0.4 mT magnetic field (MF) on the clustering of purified epidermal growth factor receptors (EGFRs) and EGFRs in Chinese hamster lung (CHL) cell membrane. The results demonstrate that exposing purified EGFRs to the MF for 30 min induces receptor clustering. The peak height of apparent clusters increased from 1.42 +/- 0.18 (sham-exposed) to 3.08 +/- 0.38 nm (exposed) while the mean half-width increased from 21.7 +/- 2.2 to 33.0 +/- 4.0 nm. A similar effect was also observed by TEM. Treatment of purified EGFR with PD153035 (PD), an EGFR-specific tyrosine kinase (TK) inhibitor, inhibited the MF-induced EGFR clustering of the purified proteins, an effect also observed for the receptors in cell membrane in the absence of EGF. These results strongly suggest that the 50 Hz 0.4 mT MF interferes with the EGFR signaling pathway, most likely by interacting with the cytoplasmic TK domain.     

Acid sphingomyelinase mediates 50-Hz magnetic field-induced EGF receptor clustering on lipid raft

Previously, we found that exposure to a 50-Hz magnetic field (MF) could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell surface. In order to explore the possible mechanisms, the roles of acid sphingomyelinase (ASMase) and lipid raft in MF-induced EGFR clustering were investigated in the present study. Human amnion epithelial (FL) cells were exposed to a 50-Hz MF at 0.4?mT for different durations. Intracellular ASMase activity was detected using the Amplex® Red Sphingomyelinase Assay Kit. EGFR clustering, ASMase, and lipid rafts on cell membrane were analyzed using confocal microscopy after indirect immunofluorescence staining. Results showed that disturbing lipid rafts with nystatin could inhibit MF-induced EGFR clustering, indicating that it was dependent on intact lipid raft. Exposure of FL cells to MF significantly enhanced ASMase activity and induced ASMase translocation to membrane that co-localized with lipid rafts. Treatment with imipramine, an ASMase inhibitor, inhibited the MF-induced EGFR clustering. This inhibitory effect could be blocked by the addition of C2-ceramide in the culture medium. It suggested that ASMase mediated the 50-Hz MF-induced EGFR clustering via ceramide which was produced from hydrolyzation on lipid rafts.     

DNA damage in rat lymphocytes treated in vitro with iron cations and exposed to 7 mT magnetic fields (static or 50 Hz)

The present study was undertaken to verify a hypothesis that exposure of the cells to static or 50 Hz magnetic fields (MF) and simultaneous treatment with a known oxidant, ferrous chloride, may affect the oxidative deterioration of DNA molecules.The comet assay was chosen for the assessment of DNA damage. The experiments were performed on isolated rat lymphocytes incubated for 3h in Helmholtz coils at 7 mT static or 50 Hz MF. During MF exposure, part of the cell samples were incubated with 0.01 microM H(2)O(2) and another one with 10 microg/ml FeCl(2,) the rest serving as controls.Lymphocyte exposure to MF at 7 mT did not increase the number of cells with DNA damage in the comet assay. Incubation of lymphocytes with 10 microg/ml FeCl(2) did not produce a detectable damage of DNA either. However, when the FeCl(2)-incubated lymphocytes were simultaneously exposed to 7 mT MF, the number of damaged cells was significantly increased and reached about 20% for static MF and 15% for power frequency MF. In the control samples about 97% of the cells did not have any DNA damage.It is not possible at present to offer a reasonable explanation for the findings of this investigation - the high increase in the number of lymphocytes showing symptoms of DNA damage in the comet assay, following simultaneous exposure to the combination of two non-cytotoxic factors -10 microg/ml FeCl(2) and 7 mT MF. In view of the obtained results we can only hypothesise that under the influence of simultaneous exposure to FeCl(2) and static or 50 Hz MF, the number of reactive oxygen species generated by iron cations may increase substantially. Further studies will be necessary to confirm this hypothesis and define the biological significance of the observed effect.     

Time-varying magnetic fields of 60 Hz at 7 mT induce DNA double-strand breaks and activate DNA damage checkpoints without apoptosis

The potential genotoxic effect of a time-varying magnetic field (MF) on human cells was investigated. Upon continuous exposure of human primary fibroblast and cervical cancer cells to a 60 Hz MF at 7 mT for 10-60 min, no significant change in cell viability was observed. However, deoxyribonucleic acid (DNA) double-strand breaks (DSBs) were detected, and the DNA damage checkpoint pathway was activated in these cells without programmed cell death (called apoptosis). The exposure of human cells to a 60 Hz MF did not induce intracellular reactive oxygen species (ROS) production, suggesting that the observed DNA DSBs are not directly caused by ROS. We also compared the position and time dependency of DNA DSBs with numerical simulation of MFs. The Lorentz force and eddy currents in these experiments were numerically calculated to investigate the influence of each factor on DNA DSBs. The DNA DSBs mainly occurred at the central region, where the MF was strongest, after a 30-min exposure. After 90 min, however, the amount of DNA DSBs increased rapidly in the outer regions, where the eddy current and Lorentz force were strong.     

No influence of magnetic fields on cell cycle progression using conditions relevant for patients during MRI

The purpose of this study was to examine whether exposure to magnetic fields (MFs) relevant for magnetic resonance imaging (MRI) in clinical routine influences cell cycle progression in two tumor cell lines in vitro. HL60 and EA2 cells were exposed to four types of MFs: (i) static MF of 1.5 and 7.05 T, (ii) extremely low frequency magnetic gradient fields (ELFMGFs) with +/- 10 mT/m and 100 Hz, as well as +/- 100 mT/m and 100 Hz, (iii) pulsed high frequency MF in the radiofrequency (RF) range (63.6 MHz, 5.8 microT), and (iv) a combination of (i-iii). Exposure periods ranged from 1 to 24 h. Cell cycle distribution (G(0)/G(1), S, and G(2)/M phases) was analyzed by flow cytometry. Cell cycle analysis did not reveal differences between the exposed and the control cells. As expected, positive controls with irradiated (8 Gy) HL60 and EA2 cells showed a strong G(2)/M arrest. Using conditions that are relevant for patients during MRI, no influence of MFs on cell cycle progression was observed in these cell lines. Care was taken to control secondary parameters of influence, such as vibration by the MR scanner or temperature to avoid false positive results.