60 Hz magnetic field exposure induces DNA crosslinks in rat brain cells

In previous research, we found an increase in DNA strand breaks in brain cells of rats acutely exposed to a 60 Hz magnetic field (for 2 h at an intensity of 0.5 mT). DNA strand breaks were measured with a microgel electrophoresis assay using the length of DNA migration as an index. In the present experiment, we found that most of the magnetic field-induced increase in DNA migration was observed only after proteinase-K treatment, suggesting that the field caused DNA-protein crosslinks. In addition, when brain cells from control rats were exposed to X-rays, an increase in DNA migration was observed, the extent of which was independent of proteinase-K treatment. However, the X-ray-induced increase in DNA migration was retarded in cells from animals exposed to magnetic fields even after proteinase-K treatment, suggesting that DNA-DNA crosslinks were also induced by the magnetic field. The effects of magnetic fields were also compared with those of a known DNA crosslink-inducing agent mitomycin C. The pattern of effects is similar between the two agents. These data suggest that both DNA-protein and DNA-DNA crosslinks are formed in brain cells of rats after acute exposure to a 60 Hz magnetic field.     

Acute low-intensity microwave exposure increases DNA single-strand breaks in rat brain cells

Levels of DNA single-strand break were assayed in brain cells from rats acutely exposed to low-intensity 2450 MHz microwaves using an alkaline microgel electrophoresis method. Immediately after 2 h of exposure to pulsed (2 μs width, 500 pulses/s) microwaves, no significant effect was observed, whereas a dose rate-dependent [0.6 and 1.2 W/kg whole body specific absorption rate (SAR)] increase in DNA single-strand breaks was found in brain cells of rats at 4 h postexposure. Furthermore, in rats exposed for 2 h to continuous-wave 2450 MHz microwaves (SAR 1.2 W/kg), increases in brain cell DNA single-strand breaks were observed immediately as well as at 4 h postexposure. © 1995 Wiley-Liss, Inc.     

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.     

Acute exposure to a 60 Hz magnetic field affects rats' water-maze performance

Rats were trained in six sessions to locate a submerged platform in a circular water-maze. They were exposed to a 1 mT, 60 Hz magnetic field for one hour in a Helmholtz coil system immediately before each training session. In addition, one hour after the last training session, they were tested in a probe trial during which the platform was removed and the time spent in the quadrant of the maze in which the platform was located during the training sessions was scored. Control animals were sham-exposed using the exposure system operating with the coils activated in an anti-parallel direction to cancel the fields. A group of “non-exposed” control animals was also included in the study. There was no significant difference between the magnetic field-exposed and control animals in learning to locate the platform. However, swim speed of the magnetic field-exposed rats was significantly slower than that of the controls. During the probe trial, magnetic field-exposed animals spent significantly less time in the quadrant that contained the platform, and their swim patterns were different from those of the controls. These results indicate that magnetic field exposure causes a deficit in spatial “reference” memory in the rat. Rats subjected to magnetic field exposure probably used a different behavioral strategy in learning the maze. Bioelectromagnetics 19: 117–122, 1998. © 1998 Wiley-Liss, Inc.     

Melatonin and a spin-trap compound block radiofrequency electromagnetic radiation-induced DNA strand breaks in rat brain cells

Effects of in vivo microwave exposure on DNA strand breaks, a form of DNA damage, were investigated in rat brain cells. In previous research, we have found that acute (2 hours) exposure to pulsed (2 μsec pulses, 500 pps) 2450-MHz radiofrequency electromagnetic radiation (RFR) (power density 2 mW/cm², average whole body specific absorption rate 1.2 W/kg) caused an increase in DNA single- and double-strand breaks in brain cells of the rat when assayed 4 hours post exposure using a microgel electrophoresis assay. In the present study, we found that treatment of rats immediately before and after RFR exposure with either melatonin (1 mg/kg/injection, SC) or the spin-trap compound N-tert-butyl-α-phenylnitrone (PBN) (100 mg/kg/injection, IP) blocks this effect of RFR. Since both melatonin and PBN are efficient free radical scavengers, it is hypothesized that free radicals are involved in RFR-induced DNA damage in the brain cells of rats. Since cumulated DNA strand breaks in brain cells can lead to neurodegenerative diseases and cancer and an excess of free radicals in cells has been suggested to be the cause of various human diseases, data from this study could have important implications for the health effects of RFR exposure. Bioelectromagnetics 18:446–454, 1997. © 1997 Wiley-Liss, Inc.     

Inhibition of pentylenetetrazole-induced seizure in mice by using a 4 Hz magnetic field: a comparative study with a 60 Hz magnetic field

We investigated the comparative effects of 4 and 60 Hz magnetic fields on pentylenetetrazole (PTZ)-induced seizure in mice. For this study, we measured the latent time to seizure, seizure duration, and lethality induced by PTZ in mice exposed to 4 and 60 Hz magnetic fields (MF) for 30 min. Compared to sham-exposed controls, the latent time to tail twitching and seizure in the 4 Hz MF group was significantly decreased while the latent time to seizure in the 60 Hz MF group was significantly increased. The seizure duration in the 4 Hz MF group was significantly decreased while that in the 60 Hz MF group was significantly increased. More importantly, while the mice exposed to a 60 Hz MF experienced significantly increased lethality after seizure convulsion, those exposed to a 4 Hz MF showed no lethality, with a shortening of the duration of seizure. This beneficial effect of a 4 Hz MF on seizure has the same implication as the anti-oxidative effects of a 4 Hz MF observed in our previous work. The results of our current and previous works indicate that a 4 Hz MF may be used as a therapeutic physical agent for the treatment of oxidative stress-induced diseases, including seizure, with or without chemical drugs.     

Protein kinase C activity following exposure to magnetic field and phorbol ester

We examined the separate and combined effects of 60 Hz sinusoidal magnetic fields (MFs) and a phorbol ester on protein kinase C (PKC) activity in HL60 cells. No enhancement in PKC activity was observed when a cell culture was exposed to a 1.1 mT (rms) MF alone or to a combination of MF and 2 μM phorbol 12-myristate 13-acetate (PMA) for 1 h. In a second set of experiments, cells were preexposed to a less than optimal concentration of PMA (50 nM) for 45 min, followed by a 15 min exposure to both PMA and MF. The data showed a greater decrease in cytosolic PKC activity and a larger increase in membrane activity than was induced by either 1 h PMA treatment alone or PMA and sham MF exposure. One logical conclusion from these data is that MFs may be acting in a synergistic manner on a pathway that has already been activated. Therefore, we suggest that MFs, rather than producing biological effects by a new pathway or mechanism of interaction, exert their effect(s) by interacting with already functioning reactions or pathways. If correct, the question of an MF's mechanism of interaction refocuses on how weak fields might enhance or depress a molecular reaction in progress, rather than on finding a new transduction pathway. Bioelectromagnetics 19:469–476, 1998. © 1998 Wiley-Liss, Inc.     

Effects of a 60 Hz magnetic field on central cholinergic systems of the rat

We studied the effects of an acute (45 min) exposure to a 60 Hz magnetic field on sodium-dependent, high-affinity choline uptake in the brain of the rat. Decreases in uptake were observed in the frontal cortex and hippocampus after the animals were exposed to a magnetic field at flux densities ? 0.75 mT. These effects of the magnetic field were blocked by pretreating the animals with the narcotic antagonist naltrexone, but not by the peripheral opioid antagonist, naloxone methiodide. These data indicate that the magnetic-field-induced decreases in high-affinity choline uptake in the rat brain were mediated by endogenous opioids in the central nervous systems. © 1993 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.     

Repetitive exposure to a 60-Hz time-varying magnetic field induces DNA double-strand breaks and apoptosis in human cells

We investigated the effects of extremely low frequency time-varying magnetic fields (MFs) on human normal and cancer cells. Whereas a single exposure to a 60-Hz time-varying MF of 6 mT for 30 min showed no effect, repetitive exposure decreased cell viability. This decrease was accompanied by phosphorylation of γ-H2AX, a common DNA double-strand break (DSB) marker, and checkpoint kinase 2 (Chk2), which is critical to the DNA damage checkpoint pathway. In addition, repetitive exposure to a time-varying MF of 6 mT for 30 min every 24 h for 3 days led to p38 activation and induction of apoptosis in cancer and normal cells. Therefore, these results demonstrate that repetitive exposure to MF with extremely low frequency can induce DNA DSBs and apoptosis through p38 activation. These results also suggest the need for further evaluation of the effects of repetitive exposure to environmental time-varying MFs on human health.     

A 60 Hz magnetic field does not affect the incidence of squamous cell carcinomas in SENCAR mice

Two groups of SENCAR mice were treated with a single dose of carcinogen and then, for 23 weeks, with a chemical tumor promoter to induce skin tumors. During this period, one group was coexposed to a 2 mT power frequency (60 Hz) magnetic field, while the other was exposed to sham conditions. Application of the tumor promoter ceased after 23 weeks, but the exposure to sham conditions or magnetic fields continued for an additional 29 weeks. No difference was found between the two groups of mice in terms of the incidence of total tumors (P =.297) or squamous cell carcinomas (SSC) (P =.501). In summary, there was no evidence to support the hypotheses that 60 Hz magnetic fields (MF) can influence the development of either papillomas or SSC under our defined experimental conditions. The overall results add to previous animal studies that find no association between exposure to 60 Hz MF and the incidence of benign or malignant tumors.     

DNA single-strand breaks,double-strand breaks,and crosslinks in rat testicular germ cells: Measurements of their formation and repair by alkaline and neutral filter elution

This work describes a neutral and alkaline elution method for measuring DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-DNA crosslinks in rat testicular germ cells after treatments in vivo or in vitro with both chemical mutagens and gamma-irradiation. The methods depend upon the isolation of testicular germ cells by collagenase and trypsin digestion, followed by filtration and centrifugation. ¹³⁷Cs irradiation induced both DNA SSBs and DSBs in germ cells held on ice in vitro. Irradiation of the whole animal indicated that both types of DNA breaks are induced in vivo and can be repaired. A number of germ cell mutagens induced either DNA SSBs, DSBs, or cross-links after in vivo and in vitro dosing. These chemicals included methyl methane sulfonate, ethyl methane sulfonate, ethyl nitrosurea, dibromochlorpropane, ethylene dibromide, triethylene melamine, and mitomycin C. These results suggest that the blood-testes barrier is relatively ineffective for these mutagens, which may explain in part their in vivo mutagenic potency.This assay should be a useful screen for detecting chemical attack upon male germ-cell DNA and thus, it should help in the assessment of the mutagenic risk of chemicals. In addition, this approach can be used to study the processes of SSB, DSB, and crosslink repair in DNA of male germ cells, either from all stages or specific stages of development.Abbreviations DBCP dibromochlorpropane - DSB(s) DNA double-strand break(s) - EDB ethylene dibromide - EMS ethyl methane sulfonate - ENU ethyl nitrosurea - MC mitomycin C - MMS methyl methane sulfonate - SDS sodium dodecyl sulfate - SSB (s) DNA single-strand break(s) - TEM triethylene melamine - UDS unscheduled DNA synthesis     

Spatial learning in deer mice: sex differences and the effects of endogenous opioids and 60 Hz magnetic fields

We examined the effects of brief exposure to weak 60 Hz extremely low frequency (ELF) magnetic fields and opioid systems on spatial behavior and learning in reproductive adult male and female deer mice, Peromyscus maniculatus. Sex differences were evident in spatial performance, with male deer mice displaying significantly better performance than female mice in the Morris water maze, whereby animals had to acquire and retain the location of a submerged hidden platform. Brief (maximum 5 min) exposure to weak (100 T) 60 Hz magnetic fields during task acquisition significantly improved female performance, eliminating the sex differences in acquisition. The opiate antagonist, naltrexone, also improved female acquisition, though significantly less than the magnetic fields. These facilitatory effects involved alterations of non-spatial (task familiarization and reduction of related anxiety/aversive related behaviors) and possibly spatial aspects of the task. Enhancement of enkephalin activity with the enkephalinase inhibitor, SCH 34826, significantly reduced task performance by male deer mice. Both naltrexone and the 60 Hz magnetic fields attenuated the enkephalin mediated reductions of spatial performance. These findings indicate that brief exposure to 60 Hz magnetic fields can enhance water maze task acquisition by deer mice and suggest that these facilitatory effects on spatial performance involve alterations in opioid activity.Abbreviations ELF extremely low frequency - Hz hertz     

Effects of in vitro exposure to power frequency magnetic fields on UV-induced DNA damage of rat lymphocytes

The mechanisms of biological effects of 50/60 Hz (power frequency) magnetic fields (MF) are still poorly understood. There are a number of studies indicating that MF affect biochemical processes in which free radicals are involved, such as the biological objects' response to ultraviolet radiation (UVA). Therefore, the present study was aimed to assess the effect of 50 Hz MFs on the oxidative deterioration of DNA in rat lymphocytes irradiated in vitro by UVA. UVA radiation (150 J/m2) was applied for 5 min for all groups and 50 Hz MF (40 microT rms) exposure was applied for some of the groups for 5 or 60 min. The level of DNA damage was assessed using the alkaline comet assay, the fluorescence microscope, and image analysis. It has been found that the 1 h exposure to MF caused an evident increase in all parameters consistent with damaged DNA. This suggest that MF affects the radical pairs generated during the oxidative or enzymatic processes of DNA repair.     

Spatial learning deficit in the rat after exposure to a 60 Hz magnetic field

Rats were trained in ten daily sessions to perform in a 12-arm radial maze, which is a behavioral test for spatial memory functions. Exposure to a 60 Hz magnetic field (45 min, 0.75 mT) immediately before each training session retarded learning significantly. Pretreatment with the cholinergic agonist physostigmine before magnetic field exposure reversed the field's effect on spatial learning. Data from this experiment indicate that magnetic field-induced spatial learning deficit is caused by the effect of the field on cholinergic systems. © 1996 Wiley-Liss, Inc.     

Design,construction, and validation of a large capacity rodent magnetic field exposure laboratory

A magnetic field exposure laboratory has been constructed to support National Toxicology Program studies for the evaluation of the toxicity and carcinogenicity of pure, linearly polarized, 60 Hz magnetic fields in rodents. This dual corridor, controlled access facility can support the simultaneous exposure of 1200 rats and 1200 mice. The facility contains fully redundant electrical and environmental control systems and was constructed using non‐metallic materials to maintain low levels of background (ambient), stray, and cross‐talk magnetic fields. The exposure module design provides for large uniform exposure volumes with good control of stray and cross‐talk fields, while allowing the use of roll‐around cage racks for simplified animal husbandry. Stray fields and cross‐talk have been further reduced by the inclusion of “steering coils” in each exposure module. Ambient 60 Hz fields (less cross‐talk) in all exposure rooms are <0.1 μT (1 mG), and static magnetic fields have been mapped extensively. Magnetic field strength, waveform, temperature, relative humidity, light intensity, noise level, vibration, and air flow in all animal holding areas are tightly regulated, and are monitored continuously during all studies. Field uniformity in the animal exposure volumes is better than ±10%; a systematic program of cage, rack, and room rotation controls for possible positional effects within the exposure system. Magnetic fields are turned on and off over multiple cycles to prevent the induction of transients associated with abrupt field level changes. Total harmonic distortion is <3% at all field strengths. The facility has been used to study magnetic field bioeffects in rodent model systems in experiments ranging in duration from 8 weeks to 2 years. Bioelectromagnetics 20:13–23, 1999. © 1999 Wiley‐Liss, Inc.     

A 0.5 G, 60 Hz magnetic field suppresses melatonin production in pinealocytes

The objective of this study was to develop a model for testing various hypotheses concerning possible mechanisms whereby electromagnetic fields might induce suppression of nighttime melatonin production in rodents. A published method for digesting freshly obtained pineal glands to the single cell level was modified, yielding better than 95% viability. An in vitro exposure facility developed for the Food and Drug Administration was used for 12-h overnight exposures of primary pinealocyte cultures to 0.05 mT, 60 Hz, vertical AC and 0.06 μT, DC fields. After exposure, cells were separated from the supernatant by centrifugation. Supernatant melatonin was measured by ELISA assays. Data from 10 experiments demonstrated an average 46% reduction in norepinephrine-induced production of melatonin in the pinealocytes. The results support the hypothesis that EM exposure can produce pineal gland melatonin suppression by affecting individual cells. Bioelectromagnetics 19:123–127, 1998. Published 1998 Wiley-Liss, Inc.     

Acute exposure to 50 Hz magnetic field does not affect hematologic or immunologic functions in healthy young men: A circadian study

Some epidemiological studies report a relationship between magnetic field exposure and such human diseases as leukemia and immune system disturbances. The few published studies on animals do not demonstrate field exposure-related alterations in hematologic and immune systems. The data presented here are part of a broader study designed to investigate the possible effects of acute exposure to a 50 Hz linearly polarized magnetic field (10 μT) on hematologic and immunologic functions. Thirty-two young men (20–30 years old) were divided into two groups (control group, i.e., sham-exposed, 16 subjects; exposed group, 16 subjects). All subjects participated in two 24 h experiments to evaluate the effects of both continuous and intermittent (1 h “off” and 1 h with the field switched “on” and “off” every 15 s) exposure to linearly polarized magnetic fields. The subjects were exposed to the magnetic field (generated by three Helmholtz coils per bed) from 23:00 to 08:00 while lying down. Blood samples were collected during each session at 3 h intervals from 11:00 to 20:00 and hourly from 22:00 to 08:00. No significant differences were observed between sham-exposed (control) and exposed men for hemoglobin concentration, hematocrit, red blood cells, platelets, total leukocytes, monocytes, lymphocytes, eosinophils, or neutrophils. Immunologic variables [CD3, CD4, CD8, natural killer (NK) cells and B cells] were unaltered. To our knowledge, this study is the first to document the effects of a 50 Hz magnetic field on the circadian rhythm of human hematologic and immune functions, and it suggests that acute exposure to either a continuous or an intermittent 50 Hz linearly polarized magnetic field of 10 μT, at least under the conditions of our experiment, does not affect either these functions or their circadian rhythms in healthy young men. © 1996 Wiley-Liss, Inc.     

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells

Pluripotent mouse embryonic stem cells (mES cells) exhibit ∼ 100 large γH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (> 10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PK_cs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous γH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of γH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, γH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive γH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.     

Analysis of the effect of a 60 Hz AC field on histamine release by rat peritoneal mast cells

Reports have indicated effects of electromagnetic fields on inflammatory processes in vivo. To begin a systematic approach toward separating and examining the many components of such responses, we created and tested a temperature-controlled device to develop 5 mT 60 Hz magnetic fields for studies of the effects of fields on mast cells, a key component in acute inflammatory responses. Such fields have been reported to modulate cell activity, including changes in membrane function, in various systems. The magnetic field was generated using a solenoid and calibrated with an induction probe. Tests of mast cell function were determined by histamine release response to stimulation by compound 48/80, using both an “expose then test” and a “test during exposure” protocol. Aliquots not treated with 48/80 were used to evaluate field treatment effects on spontaneous histamine release. Freshly harvested rat peritoneal mast cells were exposed to the magnetic field for periods of 30 min to 2 h at 37 °C. They showed no significant degranulation during treatment, nor did they show reduced sensitivity to the degranulating agent 48/80. These observations are consistent with a model in which such processes are exclusively reflexive by the cells using field-independent membrane systems. This observation is very useful and was needed before examining longer term exposures in which gene expression in the cells might be influenced; this is the first such report of in vitro exposure of purified mast cells under these conditions and will further the study of the effects of electromagnetic fields on cell types active in acute inflammation. Bioelectromagnetics 19:192–198, 1998. © 1998 Wiley-Liss, Inc.