Development and evaluation of intermediate frequency magnetic field exposure system for studies of in vitro biological effects

We have developed an intermediate frequency (IF) magnetic field exposure system for in vitro studies. Since there are no previous studies on exposure to heating-frequency magnetic fields generated from an induction heating (IH) cook top, there is a strong need for such an exposure system and for biological studies of IF magnetic fields. This system mainly consists of a magnetic-field-generating coil housed inside an incubator, inside which cultured cells can be exposed to magnetic field. Two systems were prepared to allow the experiment to be conducted in a double-blind manner. The level of the generated magnetic field was set to 532 microT rms in the exposure space, 23 kHz, 80 times the value in the International Commission on Non-ionizing Radiation Protection (ICNIRP) guidelines, with a spatial field uniformity better than 3.8%. The waveforms were nearly sinusoidal. It was also confirmed that the parasitic electric field was 157 V/m rms and the induced electric field was 1.9 V/m rms. The temperature was maintained at 36.5 +/- 0.5 degrees C for 2 h. Furthermore, leaked magnetic flux density was 0.7 microT rms or lower at extremely low frequency (ELF) and IF in the stopped system when the other system was being operated, and the environmental magnetic flux density was 0.1 microT rms or lower at the center of the coils. As a result, it was confirmed that this system could be successfully used to evaluate the biological effects of exposure to IF magnetic fields.     

A newly designed and constructed 20 kHz magnetic field exposure facility for in vivo study

Exposure to man-made electromagnetic fields has increased over the past century. As a result of exposure to these fields, concerns have been raised regarding the relationship between electromagnetic fields and human health. Interest in the biological and health effects of intermediate frequency (IF) magnetic fields has grown recently because of the increase in public concern. In order to investigate whether IF magnetic fields have biological effects, we have developed a 20 kHz (IF) magnetic field exposure system for in vivo studies. The exposure facility was designed to study the biological effects of IF magnetic field on laboratory animals. The facility consists of a 9 m x 9 m x 5 m high room containing seven separate rooms including a 5.3 m x 4.5 m x 3 m high specific-pathogen free exposure room. The dimensions of the exposure system are 1.6 m x 1.6 m x 1.616 m high located inside this exposure room. The system is designed to provide magnetic fields up to 200 microT at 20 kHz with the uniformity within +/-5% over the space occupied by animals. After constructing the facility, performance tests were carried out. As a result, it was confirmed that our facility met requirements for evaluation of the biological effects of IF magnetic field on small animal experiments. In this paper, the design, construction, and results of evaluation of an animal exposure facility for the in vivo biological effects of an IF magnetic field are described.     

Lyn and syk tyrosine kinases are not activated in B-lineage lymphoid cells exposed to low-energy electromagnetic fields.

Exposure of B-lineage lymphoid cells to a 100 microT 60 Hz AC magnetic field has been reported to stimulate the rapid activation of Lyn and Syk tyrosine kinases and the induction of protein tyrosine phosphorylation. These findings are significant because of the critical role played by these B cell signaling events in the control of growth and differentiation, and therefore the potential of electromagnetic field (EMF) exposure to induce cancer. We report the first study carried out with the aim of reproducing the reported EMF effects on Lyn and Syk tyrosine kinases. The system used enabled EMF exposure conditions to be carefully controlled and also allowed experiments to be performed blind. The effects of a 100 microT 60 Hz AC magnetic field on protein tyrosine phosphorylation and on Lyn and Syk tyrosine kinase activities were investigated in Nalm-6 and DT40 B cells in the absence and presence of a 46 microT DC magnetic field. However, no significant effects of low-energy electromagnetic fields on tyrosine kinase activities or protein phosphorylation were observed.     

Effects of continuous and intermittent exposure to RF fields with a wide range of SARs on cell growth, survival, and cell cycle distribution

To examine the biological effects of radio frequency (RF) electromagnetic fields in vitro, we have examined the fundamental cellular responses, such as cell growth, survival, and cell cycle distribution, following exposure to a wide range of specific absorption rates (SAR). Furthermore, we compared the effects of continuous and intermittent exposure at high SARs. An RF electromagnetic field exposure unit operating at a frequency of 2.45 GHz was used to expose cells to SARs from 0.05 to 1500 W/kg. When cells were exposed to a continuous RF field at SARs from 0.05 to 100 W/kg for 2 h, cellular growth rate, survival, and cell cycle distribution were not affected. At 200 W/kg, the cell growth rate was suppressed and cell survival decreased. When the cells were exposed to an intermittent RF field at 300 W/kg(pk), 900 W/kg(pk) and 1500 W/kg(pk) (100 W/kg(mean)), no significant differences were observed between these conditions and intermittent wave exposure at 100 W/kg. When cells were exposed to a SAR of 50 W/kg for 2 h, the temperature of the medium around cells rose to 39.1 degrees C, 100 W/kg exposure increased the temperature to 41.0 degrees C, and 200 W/kg exposure increased the temperature to 44.1 degrees C. Exposure to RF radiation results in heating of the medium, and the thermal effect depends on the mean SAR. Hence, these results suggest that the proliferation disorder is caused by the thermal effect.     

Teratological studies of prenatal exposure of mice to a 20 kHz sawtooth magnetic field

In order to evaluate the importance of gestational age in possible effects due to exposure to a 20 kHz sawtooth magnetic field, pregnant ICR mice at gestational 2.5-15.5 days post-coitus, which is the most sensitive stage for the induction of major congenital malformations, were exposed in a carrousel irradiator. The mice were exposed to a 20 kHz intermediate frequency (IF) sawtooth magnetic field had a 6.5 microT peak intensity for 8 h/day. The animals were sacrificed on the 18th day of gestation; and the fetuses were examined for mortality, growth retardation, changes in head size, and other morphological abnormalities. From the above conditions, it is concluded that the exposure to a 20 kHz sawtooth magnetic field with 6.5 microT peak intensity does not inflict any adverse effect on fetuses of pregnant mice.     

No effect of extremely low-frequency magnetic field observed on cell growth or initial response of cell proliferation in human cancer cell lines

An effect on the tumor promotion process, as represented by accelerated cell growth, has been indicated as one example of areas that demonstrate the possibility of biological effects of extremely-low frequency magnetic fields. We, therefore, exposed the five cell lines (HL-60, K-562, MCF-7, A-375, and H4) derived from human tumors to a magnetic field for 3 days to investigate the effects on cell growth. Prior to exposure or sham exposure, the cells were precultured for 2 days in low serum conditions. The number of growing cells was counted in a blind manner. To investigate the effect on the initial response of cell proliferation, two cell lines were synchronized in G1 phase by serum starvation and then exposed to a magnetic field for 18 h (H4 cells) or 24 h (MCF-7 cells), both with and without serum stimulation. The rate of DNA synthesis, taken as a measure of the cell proliferation, was determined by following the incorporation of [(3)H]-thymidine into the DNA. Three different magnetic field polarizations at both 50 and 60 Hz were used: linearly polarized (vertical); circularly polarized; and an elliptically polarized field. Magnetic field flux densities were set at 500, 100, 20 and 2 microT (rms) for the vertical field and at 500 microT (rms) for the rotating fields. No effect of magnetic field exposure was observed on either cell growth or the initial response of cell proliferation.     

Lack of promotion of mammary, lung and skin tumorigenesis by 20 kHz triangular magnetic fields

In order to evaluate possible tumorigenic effects of a 20 kHz intermediate frequency triangular magnetic field (IF), a frequency emitted from TV and PC monitors at 6.25 microT rms, which is the regulated exposure limit of magnetic field for the public in Korea, mammary tumors were produced in female Sprague-Dawley rats by oral intubation of dimethylbenz(a)anthracene (DMBA), lung tumors in ICR mice by scapular region injection of benzo(a)pyrene (BP), and skin tumors in female ICR mice by topical application of DMBA and tetradecanoylphorbol ester (TPA). IF was applied 8 h/day for 14 weeks beginning the day after DMBA treatment for mammary tumor experiment, for 6 weeks after weaning for lung tumor, and for 20 weeks beginning 1 week after DMBA application for skin tumor experiment. For skin tumors, TPA was applied once a week for 19 weeks. Results showed no significant differences in tumor incidence, mean tumor number and volume, and histological patterns between IF magnetic-field exposed and sham control rats in the above three tumor models. Therefore, we conclude that within the limitation or number of animals and the experimental conditions, 20 kHz IF triangular magnetic field exposure of 6.25 microT does not appear to be a strong co-tumorigenic agent in the chosen murine mammary, lung and skin models.     

Effects of combined DC and AC magnetic fields on germination of hornwort seeds

Seeds of hornwort (Cryptotaenia japonica Hassk) were exposed to sinusoidally time-varying extremely low frequency (ELF) magnetic fields (AC fields) in combination with the local geomagnetic field (DC field). Exposure lasted 24 h/day for 16 days. Three directions of the AC magnetic fields were considered; the vertical (magnetic flux density B ACV, the directions parallel B ACparallel), and perpendicular B ACperpendicular to the direction of total geomagnetic field (magnetic flux density BG) in the geomagnetic plane (GP). Controls consisted of seeds exposed to zero AC magnetic fields in combination with the DC magnetic field. The B ACV in combination with BG effectively promoted the germination of hornwort seeds when applied at 750 microT (RMS) at 7 Hz or 500 microT (RMS) at 14 Hz from among the cases of individual frequencies f = 3.5, 7.0, 10.5, 14.0 Hz at 500 and 750 microT. The B ACparallel promoted the germination of hornwort seeds more effectively than the B ACperpendicular in combination with BG when 500 and 750 microT at 7 Hz were applied.     

Exposure to ELF magnetic field tuned to Zn inhibits growth of cancer cells

The effects of ELF alternating magnetic fields tuned to Zn(2+) on the growth of cancer cells with different status of p53 were investigated using a cell proliferation assay. Human cancer cells HeLa (cervix cancer, p53(+/+)), Saos-2 and Saos-2-His-273 (osteosarcoma, p53(-/-) and p53 His-273 mutant, respectively), H1299tTA and H1299tTA-His175 (lung carcinoma, p53(-/-) and p53 His-175 mutant), and normal human fibroblasts VH-10 (p53(+/+)) were used. Exposure parameters were calculated for the first harmonic of Zn(2+) based either on the magnetic parametric resonance (MPR) model of Lednev or the ion parametric resonance (IPR) model of Blanchard and Blackman. ELF exposure was for 72 and 96 h. The vertical alternating field was 20 Hz at amplitudes of either 38.7 or 77.4 microT (peaks, IPR or MPR, respectively). The vertical static magnetic field was 43 microT, and the horizontal static magnetic field was zeroed. Treatments of cells with PRIMA-1 and gamma-rays were used as positive controls. Growth inhibition was observed in cells after exposure to ELF at 38.7 microT. Inhibition of HeLa, VH-10, and Saos-2-His-273 cells was statistically significant, P=0.0003, 0.02, and 0.006, respectively. No consistent ELF effects following exposure 77.4 microT were seen. PRIMA-1 inhibited the growth of all cell lines with the strongest effect in mutant p53-carrying cell line H1299tTA-His175. The effects of gamma-rays were relatively weak, suggesting that the cell proliferation assay under conditions employed in this study is not very sensitive to apoptosis. In conclusion, ELF under conditions of exposure tuned to Zn(2+) according to the IPR model inhibited the growth of cancer and normal cells. No clear relationship of the observed growth inhibition to p53 status was found. Further experiments, using complementary techniques, are required to test whether p53 reactivation by ELF is feasible.     

Intermediate frequency magnetic fields do not have mutagenic, co-mutagenic or gene conversion potentials in microbial genotoxicity tests

We used bacterial mutation and yeast genotoxicity tests to evaluate the effects of intermediate frequency (IF; 2 kHz, 20 kHz and 60 kHz) magnetic fields (MFs) on mutagenicity, co-mutagenicity and gene conversion. We constructed a Helmholtz type exposure system that generated vertical and sinusoidal IF MFs, such as 0.91 mT at 2 kHz, 1.1 mT at 20 kHz and 0.11 mT at 60 kHz. Mutagenicity, co-mutagenicity and gene conversion assays were performed for each of the three MF exposure conditions. Mutagenicity testing was performed in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and two strains of Escherichia coli (WP2 uvrA and WP2 uvrA/pKM101) to cover a wide spectrum of point mutations. For co-mutagenicity tests, we used four sensitive test strains (TA98, TA100, WP2 uvrA and WP2 uvrA/pKM101) with five chemical mutagens (t-butyl hydroperoxide (BH, a hydroxyl free radical precursor), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, DNA reactive reagents), benz[a]pyrene (BaP) and 2-aminoanthracene (2AA, DNA reactive promutagens). Gene conversion testing was performed in the yeast test strain, Saccharomyces cerevisiae XD83. We also examined the effects on the repair process of DNA damage by UV irradiation. No statistically significant effects were observed between exposed and control groups in any of the genotoxicity tests, indicating that the IF MFs (0.91 mT at 2 kHz, 1.1 mT at 20 kHz or 0.11 mT at 60 kHz) do not have mutagenic or co-mutagenic potentials for the chemical mutagens tested under these experimental conditions. Our findings also indicate that these IF MFs do not induce gene conversion or affect the repair process of DNA damage in eukaryotic cells.     

50 Hz magnetic field exposure alters onset of S-phase in normal human fibroblasts.

This study was undertaken to investigate whether power frequency magnetic fields can affect the kinetics of cell cycle progression in exposed human cells. To achieve this, cultures of normal human fibroblasts were synchronised in the G(0) phase of the cell cycle and exposed to 50 Hz magnetic fields at a range of flux densities. Progression through the cycle was monitored by examining the timing of entry into S phase, as characterised by the onset of DNA synthesis. Simultaneous positive controls were exposed to human recombinant fibroblast growth factor to demonstrate that the system was responsive to external stimuli. Exposure to magnetic fields at 20 and 200 microT induced a small but significant increase in the length of the G(1) phase of the cell cycle. However, exposure at higher flux densities of 2 and 20 mT had no significant effect. These results are discussed in relation to weak magnetic field effects on free radical concentration.     

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

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

Intermediate frequency magnetic field at 23 kHz does not modify gene expression in human fetus‐derived astroglia cells

The increased use of induction heating (IH) cooktops in Japan and Europe has raised public concern on potential health effects of the magnetic fields generated by IH cooktops. In this study, we evaluated the effects of intermediate frequency (IF) magnetic fields generated by IH cooktops on gene expression profiles. Human fetus‐derived astroglia cells were exposed to magnetic fields at 23 kHz and 100 µT_rms for 2, 4, and 6 h and gene expression profiles in cells were assessed using cDNA microarray. There were no detectable effects of the IF magnetic fields at 23 kHz on the gene expression profile, whereas the heat treatment at 43 °C for 2 h, as a positive control, affected gene expression including inducing heat shock proteins. Principal component analysis and hierarchical analysis showed that the gene profiles of IF‐exposed groups were similar to the sham‐exposed group and were different than the heat treatment group. These results demonstrated that exposure of human fetus‐derived astroglia cells to an IF magnetic field at 23 kHz and 100 µT_rms for up to 6 h did not induce detectable changes in gene expression profile. Bioelectromagnetics 33:662–669, 2012. © 2012 Wiley Periodicals, Inc.     

Ets1 oncogene induction by ELF-modulated 50 MHz radiofrequency electromagnetic field

We have analyzed gene expression in hemopoietic and testicular cell types after their exposure to 50 MHz radiofrequency (RF) non-ionizing radiation modulated (80%) with a 16 Hz frequency. The exposure system generates a 0.2 microT magnetic field parallel to the ground and a 60 V/m electric field orthogonal to the earth's magnetic field. Exposure conditions were selected so as to interfere with the calcium ion flow. Under these electromagnetic field (EMF) conditions, we observed an overexpression of the ets1 mRNA in Jurkat T-lymphoblastoid and Leydig TM3 cell lines. This effect was observed only in the presence of the 16 Hz modulation, corresponding to the resonance frequency for calcium ion with a DC magnetic field of 45.7 microT. We have also identified a putative candidate gene repressed after EMF exposure. The experimental model described in this paper may contribute to the understanding of the biological mechanisms involved in EMF effects.     

Time-varying and static magnetic fields act in combination to alter calcium signal transduction in the lymphocyte.

We have tested the hypothesis that extremely low frequency (ELF) time-varying magnetic fields act in combination with static magnetic fields to alter calcium signalling in the lymphocyte. Results indicate that a 60-min exposure of thymic lymphocytes at 37 +/- 0.05 degrees C to a 16 Hz, 421 mG (42.1 microT) magnetic field simultaneously with a colinear static magnetic field of 234 mG (23.4 microT) (a.c./d.c. field intensity ratio = 1.8) inhibits calcium influx triggered by the mitogen Concanavalin A. Significantly, resting lymphocytes do not respond to the fields, thus, only mitogen-activated cells undergoing calcium signalling exhibit a field response. These results indicate that signal transduction involving calcium is an important biological constraint which operates to mediate this field interaction. Additional split field exposures show that the presence of the a.c. field or the d.c. field alone does not produce an effect. This is consistent with a proposed parametric resonance theory of interaction of low intensity magnetic fields with biological systems (L.L. Lednev (1991) Bioelectromagnetics 12, 71-75), which predicts the occurrence of biological effects at specific values for the frequency and field intensity of the ELF and static magnetic fields.     

Magnetic fields from steel-belted radial tires: implications for epidemiologic studies.

Magnetic fields emanate from radial tires due to the presence of reinforcing belts which are made of magnetized steel wire. When these tires spin, they generate alternating magnetic fields of extremely low frequency (ELF), usually below 20 Hz. The fundamental frequency of these fields is determined by tire rotation rate and has a sinusoidal waveform with a high harmonic content. The static field of radial tires can exceed 500 microT at the tread, and the tire-generated alternating fields can exceed 2.0 microT at seat level in the passenger compartment of vehicles. Degaussing the tires reduces both the static and alternating fields to low levels, but the fields increase gradually over time after degaussing. The tire-generated fields are below the frequencies detected by most of the magnetic field meters used in previous studies of power frequency magnetic field health effects. If these fields are biologically active, failure to detect them could compromise exposure assessments associated with epidemiologic studies.     

Cytogenetic effects of 50 Hz magnetic fields of different magnetic flux densities

Cytogenetic investigations were performed in human peripheral blood lymphocytes following exposure to 50 Hz magnetic fields alone or in combination with the chemical mutagen mitomycin C or with X-rays. It was found that magnetic fields up to 2500 microT did not significantly influence the chromosome aberration and sister chromatid exchange frequency. Also, the combined treatments failed to indicate the presence of any synergistic, potentiating, or antagonistic effect between the ELF magnetic fields and the mutagens. However, there were two exceptions: Cells exposed to 504 microT magnetic fields before and during cultivation displayed a statistically significant decrease in sister chromatid exchange frequency. Also, when cells were cultivated in the presence of 88.4 microT magnetic fields following X-ray exposures there was a significant increase in chromosome aberration frequency compared to X-ray exposure alone.