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

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

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

The aim of the present study was to assess whether exposure to a sinusoidal extremely low frequency magnetic field (ELF‐MF; 50 Hz, 1 mT) can affect proliferation and differentiation in the human neuroblastoma cell line BE(2)C, which is representative of high risk neuroblastomas. Cells were subjected to ELF‐MF exposure in the presence or absence of a neuronal differentiating agent (all‐trans‐retinoic acid, ATRA) for 24–72 h. In each experiment, ELF‐MF‐exposed samples were compared to sham‐exposed samples. Cells exposed to ELF‐MF combined with retinoic treatment showed a decreased cellular proliferation and an increased proportion of G₀/G₁ phase cells compared to cells exposed to either treatment alone. Moreover, ELF‐MF‐ and ATRA‐treated cells showed more differentiated morphological traits (a higher neurite number/cell, an increased neurite length), together with a significant increase of mRNA levels of p21^WAF1/CIP1 and cdk5 genes, both involved in neuronal differentiation. In addition, the expression of cyp19 gene, which is involved both in neuronal differentiation and stress response, was evaluated; cyp19 gene expression was enhanced by ATRA treatment and significantly enhanced further by ELF‐MF exposure combined with ATRA. In conclusion, our data suggest that ELF‐MF exposure can strengthen ATRA effects on neuroblastoma cells. Bioelectromagnetics 31:425–433, 2010. © 2010 Wiley‐Liss, Inc.     

Static and ELF magnetic fields induce tumor growth inhibition and apoptosis

The ability of static and extremely low frequency (ELF) Magnetic Fields (MF) to interfere with neoplastic cell function has been evaluated. In vitro experiments were carried out to study the role of MF characteristics (intensity, frequency, and modulation) on two transformed cell lines (WiDr human colon adenocarcinoma and MCF-7 human breast adenocarcinoma) and one nontransformed cell line (MRC-5 embryonal lung fibroblast). Increase in cell death morphologically consistent with apoptosis was reported exclusively in the two transformed cell lines. Cell-death induction was observed with MF of more than 1 mT. It was independent of the MF frequency and increased when modulated MF (static with a superimposition of ELF at 50 Hz) were used. Based on the in vitro results, four different MF exposure characteristics were selected and used to treat nude mice xenografted with WiDr cells. The treatment of nude mice bearing WiDr tumors subcutaneously. with daily exposure for 70 min to MF for 4 weeks caused significant tumor growth inhibition (up to 50%) by the end of the treatment when modulated MF were used for at least 60% of the whole treatment period and the time-averaged total MF intensity was higher than 3.59 mT. No toxic morphological changes induced by exposure were observed in renewing, slowly proliferating, or static normal cells. A discussion on the possible biophysical mechanism at the base of the observed biological results is also offered.     

Effects of 50‐Hz magnetic field exposure on hormone secretion and apoptosis‐related gene expression in human first trimester villous trophoblasts in vitro

Evidence from epidemiological and animal studies showed that exposure to extremely low frequency magnetic fields (ELF‐MF) could produce deleterious effects on reproduction. In order to investigate the possible mechanism of MF exposure on reproductive effects, first trimester human chorionic villi at 8–10 weeks' gestation were obtained, and trophoblasts were isolated, cultured, and exposed to a 50‐Hz MF for different durations. The human chorionic gonadotropin (hCG) and progesterone in the culture medium was measured by electrochemiluminescence immunoassay. The mRNA levels of apoptosis‐related genes bcl‐2, bax, caspase‐3, p53, and fas in trophoblasts were analyzed using real‐time RT‐PCR. The results showed that exposure of trophoblasts to MF at 0.2 mT for 72 h did not affect secretion of hCG and progesterone from these cells. There was also no significant change in secretion of these hormones when trophoblasts were exposed to a 0.4 mT MF for 48 h. However, MF significantly inhibited hCG and progesterone secretion of trophoblasts after exposure for 72 h at 0.4 mT. Results of apoptosis‐related gene expression analysis showed that, within 72 h of exposure at 0.4 mT, there was no significant difference between MF exposure and control on the expression pattern of each gene. Based on results of the present experiment, it is suggested that exposure to MF for a longer duration (72 h) could inhibit secretion of hCG and progesterone by human first trimester villous trophoblasts, however, the effect might not be related to trophoblast apoptosis. Bioelectromagnetics 31:566–572, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of ELF magnetic fields on protein synthesis in Escherichia coli K12

Escherichia coli K12 was used as a model system to determine whether ELF magnetic fields (MFs) are a general stress factor. The cells were exposed to ELF MFs (5-100 Hz) at a maximum intensity of 14 mT r. m.s. for circularly polarized MFs and 10 mT r.m.s. for vertically polarized MFs. The response of the cells to the MFs was estimated from the change in protein synthesis by using 2D PAGE. Approximately 1,000 proteins were separated on the 2D gels. The stress-responsive proteins such as CH10, DNAK, CH60, RECA, USPA, K6P1 and SODM were identified from the SWISS-2DPAGE database on the 2D gels. These proteins respond to most stress factors, including temperature change, chemical compounds, heavy metals, and nutrients. When the bacterial cells were exposed to each MF at 5-100 Hz under aerobic conditions (6.5 h) or at 50 Hz under anaerobic conditions (16 h) at the maximum intensity (7.8 to 14 mT r.m.s.), no reproducible changes were observed in the 2D gels. Changes in protein synthesis were detected by 2D PAGE with exposure to heat shock (50 degrees C for 30 min) or under anaerobic conditions (no bubbling for 16 h). Increases in the levels of synthesis of the stress proteins were observed in heat-shocked cells (CH60, CH10, HTPG, DNAK, HSLV, IBPA and some unidentified proteins) and in cells grown under anaerobic conditions (DNAK, PFLB, RECA, USPA and many unidentified proteins). These results suggest that 2D PAGE is sufficient to detect cell responses to environmental stress. The high-intensity ELF MFs (14 mT at power frequency) did not act as a general stress factor.     

极低频磁场对人肝癌细胞生长、代谢及细胞周期的影响

目的研究一定参数的极低频磁场对人肝癌细胞(SK-HEP-1)在诸多方面的影响。方法在整个SK-HEP-1细胞的培养周期中用50Hz,20mT的极低频磁场对其进行作用,并检测作用后细胞的增殖活性、生长动力学、代谢以及细胞周期的变化。结果50Hz,20mT的极低频磁场对SK-HEP-1细胞的生长与代谢有抑制作用,并能阻碍其有丝分裂的进行。结论50Hz,20mT的极低频磁场为治疗人类恶性肿瘤提供了一种可能的手段。     

弱极低频磁场对Actin骨架组装效率的频率窗口效应初探

前期研究发现,50 Hz弱磁场辐照能明显降低细胞的微丝含量和组装效率,对actin骨架形态也有明显影响.电磁生物学效应是否与辐照场频率相关,一直受到研究者的关注.单体球状肌动蛋白(G-actin)是带电结构,电磁场频率会影响其振荡频率并对微丝聚合效率产生影响.本文从细胞骨架形态和蛋白质两层次,采用免疫荧光技术考察0.4 m T,在35~140 Hz范围内5个频率的极低频磁场(ELF-MF)对FL细胞中纤维状肌动蛋白(F-actin)含量的影响,并采用荧光共振能量转移技术(FRET)验证效应最明显的频率对离体G-actin组装效率的干扰程度.结果显示,相比假辐照组,细胞中F-actin含量在50 Hz辐照组下降了(34.66±3.14)%,110 Hz次之,而另外3组(35、70和140 Hz)无显著性差异.同时利用FRET方法验证,在50 Hz磁场辐照下,离体环境中G-actin组装成F-actin的效率较假辐照组、35和70 Hz组显著降低.经初步分析,G-actin在弱ELF-MF中受到以洛伦兹力和感生电场力的合力为主的相关电磁力干扰,致使组装效率下降,且由于工频磁场周期与微丝组装周期的特殊相干性,在50 Hz频率附近可能存在一个外磁场干扰actin骨架组装的频率窗口.     

Effects of 50-Hz magnetic field exposure on superoxide radical anion formation and HSP70 induction in human K562 cells

Epidemiological studies suggest a correlation between exposure to low-level extremely low-frequency (ELF) magnetic fields (MF) and certain cancers and neurodegenerative diseases. Experimental studies have not provided any mechanism for such effects, although at flux density levels significantly higher than the ones encountered in epidemiological studies, radical homoeostasis and levels of stress response proteins can be affected. Here, we report on the influence of MF exposure (50-Hz sine wave; 1 h; 0.025–0.10 mT; vertical or horizontal MF exposure direction) on different cellular parameters (proliferation, cell cycle distribution, superoxide radical anion, and HSP70 protein levels) in the human leukaemia cell line K562. The positive control heat treatment (42°C, 1 h) did not affect either cell proliferation or superoxide radical anion production but caused accumulation of cells in the G2 phase and increased the stress protein HSP70. MF exposure (0.10 mT, 1 h) did not affect either cell cycle kinetics or proliferation. Both vertical and horizontal MF exposures for 1 h caused significantly and transiently increased HSP70 levels (>twofold), at several flux densities, compared to sham controls and also compared to heat treatment. This exposure also increased (30–40%) the levels of the superoxide radical anion, comparable to the positive control PMA. Addition of free radical scavengers (melatonin or 1,10-phenantroline) inhibited the MF-induced increase in HSP70. In conclusion, an early response to ELF MF in K562 cells seems to be an increased amount of oxygen radicals, leading to HSP70 induction. Furthermore, the results suggest that there is a flux density threshold where 50-Hz MF exerts its effects on K562 cells, at or below 0.025 mT, and also that it is the MF, and not the induced electric field, which is the active parameter.     

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.     

EFFECTS OF 50 Hz MAGNETIC FIELD EXPOSURE ON PROTEIN TYROSINE PHOSPHORYLATION IN CULTURED CELLS

Protein phosphorylation is an extremely important and widely used mechanism of cellular regulation. Here, the effects of 50 Hz magnetic fields (MFs) on tyrosine phosphorylation were studied. A Chinese hamster lung (CHL) cell line was exposed to 50 Hz magnetic fields at two intensities (0.4 mT and 0.8 mT) for different exposure durations, and western blot analysis was used to measure the degree of tyrosine phosphorylation of cellular proteins. Results showed that both 0.4 mT and 0.8 mT 50 Hz magnetic fields could affect the protein tyrosine phosphorylation in cultured cells. Both intensities could affect the tyrosine phosphorylation of 38 and 97.4 kDa proteins. In addition, 0.4 mT could affect tyrosine phosphorylation of 61.7, 105, and 112 kDa proteins, and 0.8 mT affected the tyrosine phosphorylation of 79 and 150 kDa proteins. Moreover, all the tyrosine phosphorylation changes of these proteins were time-dependent. The findings from this study demonstrated that under these experimental conditions, there was evidence that protein tyrosine phosphorylation was a possible process for ELF-EMF producing bioeffects.     

Molecular basis underlying the biological effects elicited by extremely low-frequency magnetic field (ELF-MF) on neuroblastoma cells

Extremely low-frequency magnetic fields (ELF-MFs) may affect human health because of the possible associations with leukemia but also with cancer, cardiovascular, and neurological disorders. In the present work, human SH-SY5Y neuroblastoma cells were exposed to a 50 Hz, 1 mT sinusoidal ELF-MF at three different times, that is, 5 days (T5), 10 days (T10), and 15 days (T15) and then the effects of ELF-MF on proteome expression and biological behavior were investigated. Through comparative analysis between treated and control samples, we analyzed the proteome changes induced by ELF-MF exposure. Nine new proteins resolved in sample after a 15-day treatment were involved in a cellular defense mechanism and/or in cellular organization and proliferation such as peroxiredoxin isoenzymes (2, 3, and 6), 3-mercaptopyruvate sulfurtransferase, actin cytoplasmatic 2, t-complex protein subunit beta, ropporin-1A, and profilin-2 and spindlin-1. Our results indicated that ELF-MFs exposure altered the proliferative status and other important cell biology-related parameters, such as cell growth pattern, and cytoskeletal organization. These findings support our hypothesis that ELF radiation could trigger a shift toward a more invasive phenotype.     

Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation

Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.     

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.     

Sinusoidal ELF magnetic fields affect acetylcholinesterase activity in cerebellum synaptosomal membranes

The effects of extremely low frequency magnetic fields (ELF‐MF) on acetylcholinesterase (AChE) activity of synaptosomal membranes were investigated. Sinusoidal fields with 50 Hz frequency and different amplitudes caused AChE activity to decrease about 27% with a threshold of about 0.74 mT. The decrease in enzymatic activity was independent of the time of permanence in the field and was completely reversible. Identical results were obtained with exposure to static MF of the same amplitudes. Moreover, the inhibitory effects on enzymatic activity are spread over frequency windows with different maximal values at 60, 200, 350, and 475 Hz. When synaptosomal membranes were solubilized with Triton, ELF‐MF did not affect AChE activity, suggesting the crucial role of the membrane, as well as the lipid linkage of the enzyme, in determining the conditions for inactivation. The results are discussed in order to give an interpretation at molecular level of the macroscopic effects produced by ELF‐MF on biological systems, in particular the alterations of embryo development in many organisms due to acetylcholine accumulation. Bioelectromagnetics 31:270–276, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of 60 Hz 14 microT magnetic field on the apoptosis of testicular germ cell in mice

We recently reported that continuous exposure, for 8 weeks, of extremely low frequency (ELF) magnetic field (MF) of 0.1 or 0.5 mT might induce testicular germ cell apoptosis in BALB/c mice. In that report, the ELF MF exposure did not significantly affect the body weight or testicular weight, but significantly increased the incidence of testicular germ cell death. In the present study, we aimed to further characterize the effect of a 16-week continuous exposure to ELF MF of 14 or 200 microT on testicular germ cell apoptosis in mice. There were no significant effects of MF on body weight and testosterone levels in mice. In TUNEL staining (In situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling), germ cells showed a significantly higher apoptotic rate in exposed mice than in sham controls (P < 0.001). TUNEL-positive cells were mainly spermatogonia. In an electron microscopic study, degenerating spermatogonia showed condensation of nuclear chromatin similar to apoptosis. These results indicate that apoptosis may be induced in spermatogenic cells in mice by continuous exposure to 60 Hz MF of 14 microT.     

Alterations in the cell cycle and in the protein level of cyclin D1, p21CIP1, and p16INK4a after exposure to 50 Hz MF in human cells

The effect of exposure to 50 Hz, 1 mT magnetic fields (MF) on the cell cycle in general, on the DNA synthesis in S-phase, and on the G1-phase regulating proteins Cdk4, cyclin D1, p16INK4a, and p21CIP1 was investigated in human amniotic fluid cells. The BrdU-incorporation assay revealed a significant diminution of S-phase cells in MF-exposed cultures. The protein level of Cdk4 did not change, but MF induced a decreased expression of cyclin D1 after 24 h and 30 h exposures. The level of p16INK4a increased at 1 h and 12 h after exposure, whereas the expression of p21CIP1 was enhanced at 6 h and 12 h after exposure. Reduced levels of both Cdk inhibitors were observed at longer exposure times (24 h, 30 h). Our results suggest an inhibitory effect of MF on the G1-phase induced by altered expression of p16INK4a and p21CIP1.     

Combined effects of 60 Hz electromagnetic field exposure with various stress factors on cellular transformation in NIH3T3 cells

Epidemiological studies have suggested that extremely low‐frequency magnetic fields (ELF‐MF) are associated with an increased incidence of cancer. Studies using in vitro systems have reported mixed results for the effects of ELF‐MF alone, and the World Health Organization (WHO) Research Agenda published in 2007 suggested that high priority research should include an evaluation of the co‐carcinogenic effects of ELF‐MF exposure using in vitro models. Here, the carcinogenic potential of ELF‐MF exposure alone and in combination with various stress factors was investigated in NIH3T3 mouse fibroblasts using an in vitro cellular transformation assay. NIH3T3 cells were exposed to a 60 Hz ELF‐MF (1 mT) alone or in combination with ionizing radiation (IR), hydrogen peroxide (H₂O₂), or c‐Myc overexpression, and the resulting number of anchorage‐independent colonies was counted. A 4 h exposure of NIH3T3 cells to ELF‐MF alone produced no cell transformation. Moreover, ELF exposure did not influence the transformation activity of IR, H₂O₂, or activated c‐Myc in our in vitro assay system, suggesting that 1 mT ELF‐MF did not affect any additive or synergistic transformation activities in combination with stress factors such as IR, H₂O₂, or activated c‐Myc in NIH3T3 cells. Bioelectromagnetics 33:207–214, 2012. © 2011 Wiley Periodicals, Inc.