Effects of extremely low-frequency pulsed electromagnetic fields on morphological and biochemical properties of human breast carcinoma cells (T47D)

This study was carried out to investigate the effects of 100 and 217 Hz extremely low-frequency pulsed electromagnetic fields (ELF-PEMF) on cell proliferation, actin reorganization, and ROS generation in a human breast carcinoma cells (T47D). Cells were exposed for 24–72 h, at 100 and 217 Hz, 0.1 mT. The treatment induced a time dependent decrease in cell growth after 72 h and revealed an increase in fluorescence intensity in cytoplasm and actin aggregations around the nucleus as detected by fluorescence microscopy. The amount of actin in T47D cells increased after 48 h exposure to 100 Hz and 24 h to 217 Hz while no changes in nuclear morphology were detected. Exposing the cells to 217 Hz for 72 h caused a dramatically increase of intracellular ROS generation while with exposure to 100 Hz it remained nearly unchanged. These results suggest that exposure to ELF-PEMF (100, 217 Hz, 0.1 mT) are able inducing an increase of actin level, its migration toward nucleus but despite of these changes and dramatically increase in ROS generation the symptoms of apoptosis were not observed. Our results support the hypothesis that cell response to EMF may only be observed at certain window effects; such as frequency and intensity of EMF parameters.     

Decreased DNA repair rates and protection from heat induced apoptosis mediated by electromagnetic field exposure

In this study, we demonstrate that electromagnetic field (EMF) exposure results in protection from heat induced apoptosis in human cancer cell lines in a time dependent manner. Apoptosis protection was determined by growing HL-60, HL-60R, and Raji cell lines in a 0.15 mT 60 Hz sinusoidal EMF for time periods between 4 and 24 h. After induction of apoptosis, cells were analyzed by the neutral comet assay to determine the percentage of apoptotic cells. To discover the duration of this protection, cells were grown in the EMF for 24 h and then removed for 24 to 48 h before heat shock and neutral comet assays were performed. Our results demonstrate that EMF exposure offers significant protection from apoptosis (P<.0001 for HL-60 and HL-60R, P<.005 for Raji) after 12 h of exposure and that protection can last up to 48 h after removal from the EMF. In this study we further demonstrate the effect of the EMF on DNA repair rates. DNA repair data were gathered by exposing the same cell lines to the EMF for 24 h before damaging the exposed cells and non-exposed cells with H2O2. Cells were allowed to repair for time periods between 0 and 15 min before analysis using the alkaline comet assay. Results showed that EMF exposure significantly decreased DNA repair rates in HL-60 and HL-60R cell lines (P<.001 and P<.01 respectively), but not in the Raji cell line. Importantly, our apoptosis results show that a minimal time exposure to an EMF is needed before observed effects. This may explain previous studies showing no change in apoptosis susceptibility and repair rates when treatments and EMF exposure were administered concurrently. More research is necessary, however, before data from this in vitro study can be applied to in vivo systems.     

Effects of 50 Hz electromagnetic field exposure on apoptosis and differentiation in a neuroblastoma cell line

Experiments were carried out to assess whether a magnetic field of 50 Hz and 1 mT can influence apoptosis and proliferation in the human neuroblastoma cell line LAN-5. TUNEL assays and poly-ADP ribose polymerase (PARP) expression analysis were performed to test apoptosis induction, and the WST-1 assay was used to calculate the proliferation index in a long term exposure. No alterations were found in cellular ability to undergo programmed cell death, but a small increase in the proliferation index was evidenced after 7 days of continuous exposure. Also, a slight and transient increase of B-myb oncogene expression was detected after 5 days of exposure. Combined exposures of cells to EMF and to chemical agents which interfere with proliferation, such as the differentiative agent retinoic acid and the apoptotic inducer camptothecin, showed an antagonistic effect of magnetic fields against the differentiation of the LAN-5 cells and a protective effect towards apoptosis.     

Induction of unscheduled DNA synthesis in liver and micronucleus in bone marrow of rats exposed in vivo to the benzidine-derived azo dye, Direct Black 38

The genotoxic activity of the benzidine-derived azo dye, Direct Black 38 (DB38), was studied in vivo, using two different genetic end-points: unscheduled DNA synthesis in liver (UDS) and bone marrow micronucleus (MN). Exposure times were 12, 24 or 36 h. Both assays were performed in the same rat, except for the 24-h exposure when only MN was investigated. For the liver UDS assay, the rat hepatocarcinogen, 6-dimethylaminophenylazobenzthiazole (6BT), was used as positive control and for the MN assay, cyclophosphamide (CP). In agreement with earlier results, 6BT gave rise to a dose-related increase in liver UDS after 12-h exposure to 25 or 50 mg/kg bw. After 36-h exposure, there was still an indication of a weak dose-response effect between 0 and 5 net nuclear grains (NG). DB38 induced liver UDS at the higher dose levels used (500 and 1000 mg/kg), and after both 12- and 36-h exposure. With the longer exposure time, a weak induction of UDS was also observed at 100 mg/kg. The strongest UDS induction (12.2 NG), was obtained in one rat after 36-h exposure to 500 mg/kg. DB38 also had a weak effect on the MN induction, which was statistically significant at the higher concentrations used. A dose-related response was observed at all exposure times used.     

Influence of 50 Hz electromagnetic frequency on oxidative stress and morphological characteristics in mosquito-borne filariasis Culex pipiens

The electromagnetic field (EMF) is a vital issue in research, but its use as an alternative technique for insect management is still in its beginnings. The study aimed to evaluate the effects of exposure to the electromagnetic field on the biochemical analysis and morphological characteristics in Culex pipiens. Therefore, the third instar larvae were exposed to EMF frequency (50 Hz) at different intensities (250, 500, 1000 mT) for 1 h during four successive days. After end exposure to EMF, the biochemical analysis, oxidative stress parameters, body weight, and morphological features were investigated. The results showed that larval body weight and total protein content were decreased significantly in all treated groups compared to controls. The total lipid content significantly decreased at 500 & 1000 mT exposure, while the treatment group exposed to EMF at 250 mT was not statistically different compared to control. At a high intensity of EMF 1000 mT all oxidative stress parameters; catalase (CAT) and Glutathione S-transferase (GST) activity and lipid peroxidation level (MDA) were decreased significantly compared to the control. On the other hand, at a low intensity (250 mT), CAT and GST activity was significantly increased, while MDA content was not statistically changed. Scanning electron microscopy showed that both 500 & 1000 mT caused distinct malformations to the larval body parts, mouthparts, thorax and abdomen with last abdominal structures. In conclusion, the results demonstrated that EMF exposure is a considerable stress factor that affects oxidative state and morphological features in mosquitoes and give hope that EMF will be used as an alternative method for Culex pipiens vector control.     

Comparative study of cell cycle kinetics and induction of apoptosis or necrosis after exposure of human Mono Mac 6 cells to radiofrequency radiation

The possible harmful effects of radiofrequency electromagnetic fields (RF EMFs) are controversial. We have used human Mono Mac 6 cells to investigate the influence of RF EMFs in vitro on cell cycle alterations and BrdU uptake, as well as the induction of apoptosis and necrosis in human Mono Mac 6 cells, using flow cytometry after exposure to a 1,800 MHz, 2 W/kg specific absorption rate (SAR), GSM-DTX signal for 12 h. No statistically significant differences in the induction of apoptosis or necrosis, cell cycle kinetics, or BrdU uptake were detected after RF EMF exposure compared to sham or incubator controls. However, in the positive control cells treated with gliotoxin and PMA (phorbol 12 myristate-13 acetate), a significant increase in apoptotic and necrotic cells was seen. Cell cycle analysis or BrdU incorporation for 72 h showed no differences between RF EMF- or sham-exposed cells, whereas PMA treatment induced a significant accumulation of cells in G(0)/G(1)-phase and a reduction in S-phase cells. RF EMF radiation did not induce cell cycle alterations or changes in BrdU incorporation or induce apoptosis and necrosis in Mono Mac 6 cells under the exposure conditions used.     

Effect of extremely low frequency electromagnetic field on brain histopathology of Caspian Sea Cyprinus carpio

There is limited research on the effect of electromagnetic field on aquatic organisms, especially freshwater fish species. This study was conducted to evaluate the effect of extremely low frequency electromagnetic field (ELF-EMF) (50 Hz) exposure on brain histopathology of Cyprinus carpio, one of the important species of Caspian Sea with significant economic value. A total of 200 healthy fish were used in this study. They were classified randomly in two groups: sham-exposed group and experimental group, which were exposed to five different magnetic field intensities (0.1, 1, 3, 5, and 7 mT) at two different exposure times (0.5 and 1 h). Histologic results indicate that exposure of C. carpio to artificial ELF-EMF caused severe histopathological changes in the brain at field intensities ≥3 mT leading to brain necrosis. Field intensity and duration of exposure were key parameters in induction of lesion in the brain. Further studies are needed to elucidate exact mechanism of EMF exposure on the brain.     

Alteration in cellular functions in mouse macrophages after exposure to 50 Hz magnetic fields

The aim of the present study is to investigate whether extremely low frequency electromagnetic fields (ELF-EMF) affect certain cellular functions and immunologic parameters of mouse macrophages. In this study, the influence of 50 Hz magnetic fields (MF) at 1.0 mT was investigated on the phagocytic activity and on the interleukin-1beta (IL-1beta) production in differentiated macrophages. MF-exposure led to an increased phagocytic activity after 45 min, shown as a 1.6-fold increased uptake of latex beads in MF-exposed cells compared to controls. We also demonstrate an increased IL-1beta release in macrophages after 24 h exposure (1.0 mT MF). Time-dependent IL-1beta formation was significantly increased already after 4 h and reached a maximum of 12.3-fold increase after 24 h compared to controls. Another aspect of this study was to examine the genotoxic capacity of 1.0 mT MF by analyzing the micronucleus (MN) formation in long-term (12, 24, and 48 h) exposed macrophages. Our data show no significant differences in MN formation or irregular mitotic activities in exposed cells. Furthermore, the effects of different flux densities (ranging from 0.05 up to 1.0 mT for 45 min) of 50 Hz MF was tested on free radical formation as an endpoint of cell activation in mouse macrophage precursor cells. All tested flux densities significantly stimulated the formation of free radicals. Here, we demonstrate the capacity of ELF-EMF to stimulate physiological cell functions in mouse macrophages shown by the significantly elevated phagocytic activity, free radical release, and IL-1beta production suggesting the cell activation capacity of ELF-EMF in the absence of any genotoxic effects.     

Low persistence of radiation-induced centromere positive and negative micronuclei in cultured human cells

The micronucleus (MN) assay is widely used both in genetic toxicology and in the biomonitoring of human populations. Lymphocytes, cell lines, and bone marrow and epithelial cells are usually employed as target systems in such studies. However, little effort has been done to assess the persistence of MN in highly proliferative cells. To study the behaviour of MN containing whole chromosomes or acentric fragments, we have performed a time course experiment on the persistence of γ-ray (3 Gy) induced MN in a human lymphoblastoid cell line. The frequency and content of MN were analyzed 1, 3, 7, 14, and 56 days after irradiation by pancentromeric fluorescence in situ hybridization (FISH). We observed a clear induction of both centromere positive and negative MN at completion of the first mitotic division. The frequency of both types of MN drastically declined to basal levels 7 days after irradiation with an identical kinetics. We therefore conclude that centromere positive and negative MN are highly unstable upon cell division, indicating that the MN assay could not be a good biomarker of DNA damage induced by acute treatments in highly proliferative cells. The implication of our findings in biomonitoring and in genotoxicity studies is discussed.     

Effects of exposure protocols on induction of kinetochore-plus and -minus micronuclei by arsenite in diploid human fibroblasts.

Arsenic, widely distributed in the environment, is a potent human carcinogen. Arsenite genotoxicity has been observed in a variety of cells and animal systems. However, the underlying mechanism is not completely clear. In this study, human fibroblasts (HFW) were treated with 1.25-10 microM arsenite for 24 h (low dose and long exposure) and 5-80 microM for 4 h (high dose and short exposure), and the arsenite accumulation, cytotoxicity, and micronucleus (MN) induction were examined. By these two different protocols, HFW cells showed equivalent levels of arsenite accumulation, but exhibited different kinetics of cell killing and different types of MN generation. Arsenite induced mainly kinetochore-positive MN (K+-MN) in HFW cells by low dose exposure whereas mainly kinetochore-negative MN (K--MN) was induced by high dose exposure. Catalase reduced both K+- and K--MN induced by these two exposure protocols. Except for the case of K+-MN induction by the high dose exposure protocol, N-acetyl-cysteine (NAC) in both low and high dose protocols was also shown to effectively reduce arsenite-induced MN. The present results imply that oxidative stress is involved in arsenite-induced MN in diploid human fibroblasts. However, different protocols for arsenite exposure may result in different cellular damage.     

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.     

不同强度工频磁场对皮层神经元瞬时外向钾离子通道的影响

人们对电磁辐射越来越关注,但是工频磁场产生的生物效应并不确定.选用1、5、10 mT的工频磁场照射急性分离的小鼠皮层神经元(15 min),应用全细胞膜片钳技术离线记录瞬时外向钾通道电流,研究工频磁场对离子通道的影响.结果显示:工频磁场抑制通道的电流密度,并且1 mT、5 mT及10 mT工频磁场的抑制率分别为(63.0±2.2)%、(55.0±1.7)%和(38.0±1.8)%.工频磁场影响离子通道的激活和失活特性,半数激活电压和半数失活电压变小.不同强度工频磁场对离子通道产生的影响程度不同,其中1 mT工频磁场对通道电流的抑制率最大,5 mT工频磁场对通道的半数激活电压和半数失活电压影响最大,10 mT工频磁场增大了通道的失活斜率因子.研究结果表明,工频磁场影响了细胞膜上离子通道蛋白质构象的变化,进一步影响了离子通道的正常功能.     

EXPOSURE TO 50 HZ ELECTROMAGNETIC FIELDS INDUCES THE PHOSPHORYLATION AND ACTIVITY OF STRESS-ACTIVATED PROTEIN KINASE IN CULTURED CELLS

Protein phosphorylation is one of the important processes of cell signal transduction pathways. To study the effects of 50 Hz electromagnetic field (EMF) on the cell signal transduction process, the phosphorylation of stress-activated protein kinase (SAPK/JNK) extracted from Chinese hamster lung (CHL) cells exposed to 0.4 and 0.8 mT 50 Hz EMF for various durations was measured. A solid-phase kinase assay was used to measure the enzymatic activity of SAPK extracted from cells exposed to 50 Hz EMF at the same magnetic flux density and for only 15 min. The results showed that both 0.4 and 0.8 mT could induce the phosphorylation of SAPK, the phosphorylation of SAPK presented a time-dependent course, and there was a difference between the two intensities. The phosphorylated SAPK enhanced its enzymatic activity. All the data indicated that 50 Hz EMF could activate SAPK in a time- and intensity-dependent manner. The biological effects caused by 50 Hz EMF maybe related to the SAPK signal transduction pathway.     

High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detection-fluorescence in situ hybridization

Electromagnetic fields (EMFs) have been associated with increased incidence of cancer suggested by epidemiological studies. To test the carcinogenic potency of EMF, the in vitro micronucleus assay with SHE cells has been used as a screening method for genotoxicity. A 50Hz magnetic field (MF) of 1mT field strength was applied either alone or with the tumour initiator benzo(a)pyrene (BP) or the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). All three treatments were applied in single, double or triple treatment regimes. MF or TPA (1nM) alone did not affect the number of micronuclei (MN) in initiated and non-initiated SHE cells. Changing the schedule of the typical initiation protocol, namely applying the initiator (BP) during exposure to MF, results in an 1.8-fold increased MN formation compared to BP treatment alone. Combined experiment with BP, TPA and MF did not cause further MN formation. Since initiation during MF exposure caused a significant increased MN formation, our findings suggest that MFs enhance the initiation process of BP. We think that this MF-enhanced co-carcinogenic effect is caused by an indirect "cell activation" process. The resulting genomic instability is proposed to be due to free radicals and/or to the unscheduled "switching-on" of signal transduction pathways.     

Genotoxic effects of potassium bromate on human peripheral lymphocytes in vitro

The aim of this study was to investigate the genotoxic effects of potassium bromate, which is used as a bleaching agent in flour, on human peripheral blood lymphocytes in vitro by sister chromatid exchange (SCE), chromosomal aberrations (CA) and micronucleus (MN) tests, and also to determine whether it has any genotoxic potential for humans. Cells were treated with 400, 450, 500, 550 microg/ml concentrations of potassium bromate for 24 and 48 h. The SCE frequencies showed an increase after both treatment periods, however, the differences between the treated cells and the control groups were found to be statistically significant only for the 48-h treatment. In addition, potassium bromate statistically significantly induced CA after the 24-h and 48-h treatment periods. Strikingly, potassium bromate induced CA as much as the positive control, mitomycin-C (MMC). Furthermore, potassium bromate decreased both the cell proliferation index (PI) and the mitotic index (MI). Although micronucleus formation was induced by potassium bromate during the 24-h treatment period in a dose-dependent manner, only the doses 500 and 550 microg/ml yielded statistically significant results. In contrast, MN formation was significantly induced at all doses during the 48-h treatment period. These in vitro results provide important evidence about genotoxicity of potassium bromate on a human cell culture system.     

不同强度工频磁场对神经元延迟整流钾通道特性的影响

工频磁场是人类生活中接触最多的一类磁场,其引起的生物效应与人类健康的关系备受关注．本文选用1 mT、5 mT及10 mT工频磁场照射急性分离的小鼠皮层神经元(15 min),应用全细胞膜片钳技术离线记录通道电流,研究了工频磁场对神经元延迟整流钾通道特性的影响．结果显示,1 mT、5 mT及10 mT 3个强度的工频磁场对I_k均有抑制作用,但随着去极化电压的增加,发现1 mT和5 mT工频磁场的抑制率几乎不变,抑制率分别为(30 ± 4.2)%和(20 ± 2.2)%,而10 mT工频磁场的抑制率增加,最大抑制率为43.4%．另外,1 mT和5 mT工频磁场影响了延迟整流钾通道的激活特性,通道的半数激活电压变大,斜率因子不变．而10 mT工频磁场对通道的激活特性没有影响,半数激活电压和斜率因子均不改变．研究表明,工频磁场可能影响了细胞膜上离子通道蛋白质的结构和功能,并且不同强度工频磁场对通道的影响不同,存在强度窗口效应．     

The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 x 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5-13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.     

The regenerative effects of electromagnetic field on spinal cord injury

Traumatic spinal cord injury (SCI) is typically the result of direct mechanical impact to the spine, leading to fracture and/or dislocation of the vertebrae along with damage to the surrounding soft tissues. Injury to the spinal cord results in disruption of axonal transmission of signals. This primary trauma causes secondary injuries that produce immunological responses such as neuroinflammation, which perpetuates neurodegeneration and cytotoxicity within the injured spinal cord. To date there is no FDA-approved pharmacological agent to prevent the development of secondary SCI and induce regenerative processes aimed at healing the spinal cord and restoring neurological function. An alternative method to electrically activate spinal circuits is the application of a noninvasive electromagnetic field (EMF) over intact vertebrae. The EMF method of modulating molecular signaling of inflammatory cells emitted in the extra-low frequency range of <100 Hz, and field strengths of <5 mT, has been reported to decrease inflammatory markers in macrophages, and increase endogenous mesenchymal stem cell (MSC) proliferation and differentiation rates. EMF has been reported to promote osteogenesis by improving the effects of osteogenic media, and increasing the proliferation of osteoblasts, while inhibiting osteoclast formation and increasing bone matrix in vitro. EMF has also been shown to increase chondrogenic markers and collagen and induce neural differentiation, while increasing cell viability by over 50%. As advances are made in stem cell technologies, stabilizing the cell line after differentiation is crucial to SCI repair. Once cell-seeded scaffolds are implanted, EMF may be applied outside the wound for potential continued adjunct treatment during recovery.