Cancer promotion in a mouse-skin model by a 60-Hz magnetic field: I. Experimental design and exposure system.

The rationale for selection of an animal model, the experimental design, and the design and evaluation of an exposure system used in studies of 60-Hz magnetic fields are described. The studies were conceived to assay development of cancer and immune responsiveness in mice exposed to magnetic fields. The exposure system utilized a quadrupole-coil configuration to minimize stray magnetic fields. Four square-wound coil provided a uniform field within a volume occupied by 16 animal cages. The magnetic field had a mean flux density of 2 mT that varied less than +/- 10% within the volume occupied by animals' cages. The flux density decreased to less than 0.1 microT at a distance of 2 m from the coils. In each exposure system 32 animals could be housed in plastic cages.     

A magnetic field exposure facility for evaluation of animal carcinogenicity

Several animal studies have been carried out at the Institut Armand Frappier (IAF) to determine whether chronic exposure to 60 Hz linearly polarized sinusoidal magnetic fields might increase the risk of cancer development of female Fisher rats. The magnetic field exposure facility was developed to meet the requirements of the study protocol for chronic exposure of large number of animals to field intensities of sham < 0.2 microT, 2 microT, 20 microT, 200 microT, and 2000 microT. At each exposure level, including sham, the animals are distributed in a group of four exposure units. Each exposure unit contains two exposure volumes having uniform distribution of magnetic fields for the animals, while the magnetic field external to the unit falls off rapidly due to the "figure-eight" coil topography used. A program of "shake down" tests, followed by verification and calibration of the exposure facility, was carried out prior to starting the animal experiments. Continuous monitoring of the magnetic field and other environmental parameters was an important part in the overall quality assurance program adopted.     

Numerical computation of distortions in magnetic fields and induced currents in physiological solutions produced by microscope objectives

Identifying distortions produced by commonly employed microscope objectives and their components in uniform DC and 60 Hz AC magnetic fields is important in imaging studies involving exposure of cells to spatially uniform or nonuniform magnetic fields. In this study, DC and 60 Hz AC magnetic flux densities were numerically computed in the presence of finite element models of various components of commonly utilized microscope objectives, as well as a model of a complete objective. Also computed were the distortions in the current density induced by an applied time-varying magnetic field in a physiological buffer contained within a Petri dish. We show that the magnetic flux density could be increased up to 65% in the presence of the nickel-chrome plating of an objective housing and that the presence of ferromagnetic components like a screw or spring could produce peaks that are 7% higher than the undistorted value of magnetic flux density. In addition, a slight tilt of 1% in the objective with respect to the magnetic field could cause a 93% deviation in magnetic flux density from the unperturbed value. These results correlate well with previously published experimental measurements that showed the presence of significant and sometimes asymmetric distortions in both DC and 60 Hz magnetic fields. Moreover, this study further reports that induced current density changed up to 37% compared to values in the absence of the objective. The existence of distortions in applied magnetic fields and induced currents could affect the interpretation of results of cell function studies if it is assumed that the cells are exposed to uniform magnetic flux densities in the presence of a microscope objective. Such assumptions of uniform magnetic flux density could also account for the lack of reproducibility in several studies that examined changes in intracellular calcium by imaging techniques.     

Magnetostatic Field System for Uniform Cell Cultures Exposure

The aim of the present work has been the design and the realization of a Magnetostatic Field System for Exposure of Cell cultures (MaFiSEC) for the uniform and the reproducible exposure of cell cultures to static magnetic fields (SMFs) of moderate magnetic induction. Experimental and computer-simulated physical measurements show that MaFiSEC: i) generates a SMF with magnetic induction that can be chosen in the range of 3 to 20 mT; ii) allows the uniform SMF exposure of cells growing in adhesion and in suspension; iii) is cheap and easy to use. The efficacy and reproducibility of MaFiSEC has been tested by comparing the biological effects exerted on isolated human lymphocytes by 72 h of exposure to a magnet (i.e. Neodymium Magnetic Disk, NMD) placed under the culture Petri dish. Lymphocytes morphology, viability, cell death, oxidative stress and lysosomes activity were the parameters chosen to evaluate the SMF biological effects. The continuous exposure of cells to a uniform SMF, achieved with MaFiSEC, allows highly reproducible biochemical and morphological data.     

Spatial gradient effects of 120 mT static magnetic field on endothelial tubular formation in vitro

This study investigated the spatial magnetic gradient effects of static magnetic fields (SMF) on endothelial tubular formation by applying the maximum spatial gradient to a target site of culture wells for cell growth. The respective maximum values of magnetic flux density (B(max)), magnetic flux gradient (G(max)) and the magnetic force product of the magnetic flux density and its gradient (a parameter of magnetic force) were 120 mT, 28 mT/mm and 1428 mT(2)/mm. The effects of gradient SMF on tubular formation were compared with those of uniform SMF that has no spatial gradients on the entire bottom area of culture wells. Five experimental groups of 25 samples each were examined: (1) sham exposure (control); (2) peak gradient exposure in the peripheral part; (3) peak gradient exposure in the central part; (4) uniform exposure to 20 mT; (5) uniform exposure to 120 mT. The SMF or sham exposure was carried out for 10 days. Photomicrographs of tubular cells, immunostained with an anti-platelet-endothelial cell adhesion molecule-1 (PECAM-1 [CD31]) antibody as a pan-endothelial marker, were analyzed after the 10-day culture. Gradient SMF in the peripheral or central part was found to significantly promote tubular formation in terms of the area density and length of tubules in each peak gradient/force part of the wells, compared with the sham exposure. In contrast, uniform SMF did not induce any significant change in the tubular formation. These findings suggest that tubule formation can be promoted by applying the peak gradient/force to a target site of culture wells.     

Electric fields induced by low frequency magnetic fields in inhomogeneous biological structures that are surrounded by an electric insulator

Electric fields induced by low-frequency magnetic fields into inhomogeneous structures, which have electric conductivities and dielectric permittivities of typical biological substances, are evaluated. Closed-form approximate and numerical solutions are obtained for nonconcentric cylinders with different electric properties (such as bone embedded in muscle), which are surrounded by a good electrical insulator (such as air). It is shown that even a single inhomogeneity in an otherwise homogenous cylinder, which is exposed to a uniform, axially directed magnetic field, can lead to substantial deviations from the direction and distribution of the induced electric field that would exist in the homogenous cylinder. Thus the induced field is not everywhere circumferential, nor does it magnitude at all angular positions increase linearly with the radial distance. Radially and circumferentially directed field components depend on size, electrical properties, and eccentricity of the inhomogeneities. Equations as well as graphical presentations are given that describe the induced fields when the enclosed inhomogeneities consist either of eccentrically located single cylinders or pairs of coaxial cylinders with different electrical conductivities or dielectric permittivities.     

System for the exposure of cell suspensions to power-frequency electric fields

A system is described that uses an oscillating magnetic field to produce power-frequency electric fields with strengths in excess of those produced in an animal or human standing under a high-voltage electric-power transmission line. In contrast to other types of exposure systems capable of generating fields of this size, no electrodes are placed in the conducting growth media: the possibility of electrode contamination of the exposed suspension is thereby eliminated. Electric fields in the range 0.02–3.5 V/m can be produced in a cell culture with total harmonic distortions less than 1.5%. The magnetic field used to produce electric fields for exposure is largely confined within a closed ferromagnetic circuit, and experimental and control cells are exposed to leakage magnetic flux densities less than 5 μT. The temperatures of the experimental and control cell suspensions are held fixed within ±0.1°C by a water bath. Special chambers were developed to hold cell cultures during exposure and sham exposure. Chinese hamster ovary (CHO) cells incubated in these chambers grew for at least 48 h and had population doubling times of 16–17 h, approximately the same as for CHO cells grown under standard cell-culture conditions.     

Increase in radiation-induced HPRT gene mutation frequency after nonthermal exposure to nonionizing 60 Hz electromagnetic fields

It is widely accepted that moderate levels of nonionizing electric or magnetic fields, for example 50/60 Hz magnetic fields of about 1 mT, are not mutagenic. However, it is not known whether such fields can enhance the action of known mutagens. To explore this question, a stringent experimental protocol, which included blinding and systematic negative controls, was implemented, minimizing the possibility of observer bias or experimental artifacts. As a model system, we chose to measure mutation frequencies induced by 2 Gy gamma rays in the redox-sensitive hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in Chinese hamster ovary cells. We tested whether a 12-h exposure to a 60 Hz sinusoidally oscillating magnetic-flux density (Brms = 0.7 mT) could affect the mutagenic effects of ionizing radiation on the HPRT gene locus. We determined that the magnetic-field exposure induced an approximate 1.8-fold increase in HPRT mutation frequency. Additional experiments at Brms = 0.23 and 0.47 mT revealed that the effect was reduced at lower flux densities. The field exposure did not enhance radiation-induced cytotoxicity or mutation frequencies in cells not exposed to ionizing radiation. These results suggest that moderate-strength, oscillating magnetic fields may act as an enhancer of mutagenesis in mammalian cells.     

Magnetic fields generated by an induction heating (IH) cook top do not cause genotoxicity in vitro

The use of induction heater (IH) cook tops in homes has become widespread, especially in Japan, but there are concerns about the safety of intermediate frequency (IF) electromagnetic fields associated with these cooking appliances. Since the cellular genotoxicity of IF magnetic fields has not been examined in cultured cells, we examined the effects of these fields at a magnetic flux density of 532 +/- 20 microT at 23 kHz, using an exposure unit with a built-in CO2 incubator. Exposure to the IF magnetic field at 532 microT for 2 h did not affect the growth of CHO-K1 cells and caused no mutagenic effects in bacterial mutation assays. Exposure to the IF magnetic field for 2 h induced neither single nor double DNA strand breaks in comet assays, and caused no significant change in the mutation frequency at the HPRT locus compared to sham exposure. The magnetic field used in this study is more than 80 times higher than the level recommended as safe in the International Commission on Non-ionizing Radiation Protection (ICNIRP) guidelines. From these results, we suggest that exposure to an IF magnetic field for 2 h does not cause cellular genotoxicity in bacteria and in Chinese hamster cells. However, the possibility of effects on other cellular functions remains, and further studies on the cellular effects of IF magnetic fields are required.     

Effect of magnetic fields on tumor growth and viability

Breast cancer is the most common nonskin cancer and is the second leading cause of cancer-related deaths in women. Most methods of intervention involve combinations of surgery, chemotherapy, and ionizing radiation. Both chemotherapy and ionizing radiation can be effective against many types of cancer, but they also harm normal tissues. The use of nonionizing, magnetic fields has shown early promise in a number of in vitro and animal studies. Our study tested the effect of varying durations of magnetic exposure on tumor growth and viability in mice injected with breast cancer cells. Cancer cells were labeled through stable expression of firefly luciferase for monitoring of tumor growth and progression by using an in vivo imaging system. We hypothesized that magnetic field exposure would influence tumor growth and progression. Our results showed that exposure of the mice to magnetic fields for 360 min daily for as long as 4 wk suppressed tumor growth. Our study is unique in that it uses an in vivo imaging system to monitor the growth and progression of tumors in real time in individual mice. Our findings support further exploration of the potential of magnetic fields in cancer therapeutics, either as adjunct or primary therapy.     

Exposure system to study hypotheses of ELF and RF electromagnetic field interactions of mobile phones with the central nervous system

A novel exposure system for double-blind human electromagnetic provocation studies has been developed that satisfies the precision, control of fields and potential artifacts, and provides the flexibility to investigate the response of hypotheses-driven electromagnetic field exposure schemes on brain function, ranging from extremely low frequency (ELF) to radio frequency (RF) fields. The system can provide the same exposure of the lateral cerebral cortex at two different RF frequencies (900 and 2140 MHz) but with different exposure levels at subcortical structures, and also allows uniform ELF magnetic field exposure of the brain. The RF modulation and ELF signal are obtained by a freely programmable arbitrary signal generator allowing a wide range of worst-case exposure scenarios to be simulated, including those caused by wireless devices. The maximum achievable RF exposure is larger than 60 W/kg peak spatial specific absorption rate averaged over 10 g of tissue. The maximum ELF magnetic field exposure of the brain is 800 A/m at 50 Hz with a deviation from uniformity of 8% (SD).     

Rats are not aversive when exposed to 60-Hz magnetic fields at 3.03 mT.

Thirty-two male rats were tested in two replicates of an experiment to determine whether body currents induced by 60-Hz magnetic fields might lead to avoidance behavior comparable to that which results from exposure to strong 60-Hz electric fields. The test apparatus was a two-compartment Plexiglas shuttlebox enclosed in a sound-attenuating plywood chamber, which in turn was encompassed by two copper bus bars that, when energized, served as a source of 60-Hz magnetic fields. Location of the rat, and traverse activity in the shuttlebox were monitored by nine infra-red photo detectors equally spaced along the length of the apparatus. Rats were divided into 2 groups: 1 group of rats (n = 8 per group per replicate) was sham exposed while rats in the other group (n = 8 per group per replicate) were exposed to a 3.03 mT (30.3 G), 60-Hz magnetic field whenever they traversed to or were located on the side (L or R) predetermined as the exposed side. To control artifact incident to side preference, the side exposed (L or R) was alternated over the exposed rats. Each rat was tested individually in a 1-h session. A 2-factor ANOVA (exposed vs. control, replicate 1 vs. replicate 2) failed to reveal any significant effects due to either factor or to an interaction between factors. These data demonstrate that rats do not avoid exposure to 60-Hz magnetic fields at a flux density of 3.03 mT and further imply that the avoidance by rats of high level 60-Hz electric fields is mediated by something other than the internal body currents induced by the exposure.     

Addition of magnetic field capability to existing extremely-low-frequency electric field exposure systems

Magnetic field systems were added to existing electric field exposure apparatuses for exposing cell suspensions in vitro and small animals in vivo. Two horizontally oriented, rectangular coils, stacked one directly above the other, have opposite electric currents. This configuration minimizes leakage fields and allows sham- and field-exposure systems to be placed in the same room or incubator. For the in vitro system, copper plates formed the loop-pair, with up to 900 A supplied by a 180:1 transformer. Electric fields were supplied via electrodes at the ends of cell-culture tubes, eight of which can be accommodated by each exposure system. Two complete systems are situated in an incubator to allow simultaneous sham and field exposure up to 1 mT. For the in vivo system, four pairs of 0.8 x 2.7-m coils made of copper bus bar are employed. This arrangement is energized from the power grid via a 30:1 transformer; horizontal magnetic flux densities up to 1 mT can be generated. Pairs of electrode plates spaced 30.5 cm apart provide electric field exposure of up to 130 kV/m. Four systems with a capacity of 48 rats each are located in one room. For both the in vitro and in vivo systems, magnetic exposure fields are uniform to within +/- 2.5%, and sham levels are at least 2,500-fold lower than exposure levels. Potential confounding factors, such as heating and vibration, were examined and found to be minimal.     

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.     

Design and characterization of a system for exposure of cultured cells to extremely low frequency electric and magnetic fields over a wide range of field strengths

A system is described that is capable of producing extremely low frequency (ELF) magnetic fields for relatively short-term exposure of cultured mammalian cells. The system utilizes a ferromagnetic core to contain and direct the magnetic field of a 1,000 turn solenoidal coil and can produce a range of flux densities and induced electric fields much higher than those produced by Helmholtz coils. The system can generate magnetic fields from the microtesla (μT) range up to 0.14 T with induced electric field strengths on the order of 1.0 V/m. The induced electric field can be accurately varied by changing the sample chamber configuration without changing the exposure magnetic field. This gives the system the ability to separate the bioeffects of magnetic and induced electric fields. In the frequency range of 4–100 Hz and magnetic flux density range of 0.005–0.14 T, the maximum total harmonic distortion of the induced electric field is typically less than 1.0%. The temperature of the samples is held constant to within 0.4°C by constant perfusion of warmed culture medium through the sample chamber. © 1993 Wiley-Liss, Inc.     

Absence of genotoxicity in human blood cells exposed to 50 Hz magnetic fields as assessed by comet assay, chromosome aberration, micronucleus, and sister chromatid exchange analyses

In the past, epidemiological studies indicated a possible correlation between the exposure to ELF fields and cancer. Public concern over possible hazards associated with exposure to extremely low frequency magnetic fields (ELFMFs) stimulated an increased scientific research effort. More recent research and laboratory studies, however, have not been able to definitively confirm the correlation suggested by epidemiological studies. The aim of this study was to evaluate the effects of 50 Hz magnetic fields in human blood cells exposed in vitro, using several methodological approaches for the detection of genotoxicity. Whole blood samples obtained from five donors were exposed for 2 h to 50 Hz, 1 mT uniform magnetic field generated by a Helmholtz coil system. Comet assay, sister chromatid exchanges (SCE), chromosome aberrations (CA), and micronucleus (MN) tests were used to assess DNA damage, one hallmark of malignant cell transformation. The effects of a combined exposure with X-rays were also evaluated. Results obtained do not show any significant difference between ELFMFs exposed and unexposed samples. Moreover, no synergistic effect with ionizing radiation has been observed. A slight but significant decrease of cell proliferation was evident in ELFMFs treated samples and samples subjected to the combined exposure.     

Thresholds for 60 Hz magnetic field stimulation of peripheral nerves in human subjects

The goal of the research reported here is to narrow the range of uncertainty about peripheral nerve stimulation (PNS) thresholds associated with whole body magnetic field exposures at 50/60 Hz. This involved combining PNS thresholds measured in human subjects exposed to pulsed magnetic gradient fields with calculations of electric fields induced in detailed anatomical models of the body by that same exposure system. PNS thresholds at power frequencies (50/60 Hz) can be predicted from these data due to the wide range of pulse durations (70 mus to 1 ms), the length of the pulse trains (several tens of ms), and the exposure of a large part of the body to the magnetic field. These data together with the calculations of the rheobase electric field exceeded in 1% (E(1%)) of two anatomical body models, lead to a median PNS detection threshold of 47.9 +/- 4.4 mT for a uniform 60 Hz magnetic field exposure coronal to the body. The threshold for the most sensitive 1% of the population is about 27.8 mT. These values are lower than PNS thresholds produced by magnetic fields with sagittal and vertical orientations or nonuniform exposures.     

50-Hz magnetic field exposure system for small animals

The design, construction, and results of evaluation of an animal-exposure system for the study of biological effects of extremely low frequency (ELF) magnetic fields are described. The system uses a square coil arrangement based on a modification of the Helmholtz coil. Due to the cubic configuration of this exposure system, horizontal and vertical magnetic fields as high as 0.3 mT can be generated. Circularly polarized magnetic fields can also be generated by changing the current and phase difference between two sets of coils. Tests were made for uniformity of the magnetic field, stray fields, sham-exposure ratio of stray field, changes of temperature and humidity, light intensity and distribution inside the animal-housing space, and noise due to air-conditioning equipment. Variation of the magnetic field was less than 2% inside the animal housing. The stray-field level inside the sham-exposure system is less than 2% of experimental exposure levels. The system can be used for simultaneous exposure of 48 rats (2 to a cage) or 96 mice (4 to a cage). © 1993 Wiley-Liss. Inc.     

Design and fabrication of well confined uniform magnetic field exposure systems

Exposure systems that provide good magnetic field uniformity, minimum stray fields, and minimal heating, vibration, and hum, as well as capability for true sham exposure in which current flows in the coils, are needed to determine rigorously the biological effects of weak magnetic fields. Designs based on acrylic polymer coil support structures and twisted pair bifilary coil windings were employed to fabricate several different systems for the exposure of laboratory animals and cell cultures to magnetic fields. These systems exhibit excellent performance characteristics in terms of exposure field uniformity, stray field containment, and exposure field cancellation in the sham exposure mode. A custom-written computer program was used to determine the best arrangement for coils with regard to field uniformity in the exposure volume and stray field containment. For in vivo exposures, modules were made up of four Merritt four-coil sets, built into a single structure and positioned to form an octapole with fields directed in the horizontal plane. For in vitro applications, two different coil configurations were selected to produce the vertical fields required. A quadrupole system, comprising modules consisting of two Merritt four-coil sets arranged side by side to limit stray fields, was built as a prototype. In the second configuration, one Merritt four-coil set was positioned inside the other to form a concentric coil set. In both in vitro systems, exposure chambers were connected to remote commercial incubators in order to reduce ambient magnetic fields in the exposure volume. An active field cancellation circuit was developed for reducing ambient AC magnetic fields in the in vitro sham exposure chamber, when necessary. These design and fabrication approaches provide systems that offer uniform field exposures and excellent stray field containment when needed and are portable, washable, and relatively inexpensive. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

ELF in vitro exposure systems for inducing uniform electric and magnetic fields in cell culture media.

Many in vitro experiments on the biological effects of extremely low frequency (ELF) electromagnetic fields utilize a uniform external magnetic flux density (B) to expose biological materials. A significant number of researchers do not measure or estimate the resulting electric field strength (E) or current density (J) in the sample medium. The magnitude and spatial distribution of the induced E field are highly dependent on the sample geometry and its relative orientation with respect to the magnetic field. We have studied the E fields induced in several of the most frequently used laboratory culture dishes and flasks under various exposure conditions. Measurements and calculations of the E field distributions in the aqueous sample volume in the containers were performed, and a set of simple, quantitative tables was developed. These tables allow a biological researcher to determine, in a straightforward fashion, the magnitudes and distributions of the electric fields that are induced in the aqueous sample when it is subjected to a uniform, sinusoidal magnetic field of known strength and frequency. In addition, we present a novel exposure technique based on a standard organ culture dish containing two circular, concentric annular rings. Exposure of the organ culture dish to a uniform magnetic field induces different average electric fields in the liquid medium in the inner and outer rings. Results of experiments with this system, which were reported in a separate paper, have shown the dominant role of the magnetically induced E field in producing specific biological effects on cells, in vitro. These results emphasize the need to report data about the induced E field in ELF in-vitro studies, involving magnetic field exposures. Our data tables on E and J in standard containers provide simple means to enable determination of these parameters.