A study of magnetic field effects on fibroblast cultures part 3. The evaluation of the effects of static and extremely low frequency (ELF) magnetic fields on glycosaminoglycan metabolism in fibroblasts,cell coats and culture medium

Cultures of fibroblasts isolated from murine liver by the method of tissue trypsinization were exposed to a static magnetic field (0.49 T) and an extremely low frequency (ELF) magnetic field (50 Hz, 0.020 T). The cultures were exposed to magnetic fields for exposure periods of 2, 4, 8, 16, 32 and 64 min on four consecutive days. During the experiment we investigated the glycosaminoglycans isolated from the fibroblast, their coats and the culture medium. The investigations concerned heparan sulphate (DS) and chondroitin sulphates (CS). The changes observed in the fibroblast cultures exposed to ELF magnetic field suggest an increase in sulphate ion content in the glycosaminoglycans investigated, i.e. increased synthesis of the compounds. The ELF magnetic field also affects the degree of glycosaminoglycan sulphatization. Some changes in the quantitative relations between HS, DS and CS were also noted. The static magnetic field had no effect on glycosaminoglycan metabolism, i.e. there were no alterations in incorporation of labeled sulphur into sulphate glycosaminoglycans.     

Evidence for genotoxic effects of resonant ELF magnetic fields

The possibility that extremely low frequency (ELF) magnetic fields affect the genomic integrity of the cell is the objective of this study. Human peripheral lymphocytes (HPLs) were exposed to different exposure conditions combining ac and static magnetic fields. We used the micronuclei (MN) cytogenetic technique, because MN formation is considered as a marker of chromosomal damage produced by genotoxic agents.The first set of experiments were performed at 50 Hz, 150 μT rms and 32 Hz, 75 μT and 150 μT rms magnetic fields with the static geomagnetic field components nulled. No effects were detected using the MN test on HPL as an indicator for cellular genomic damage when the static magnetic field was nulled. Moreover, such exposure to an ac magnetic field does not appear to interfere with the action of a powerful genotoxic agent mytomicin-C (MMC), i.e. there was no synergistic effect.The second set of experiments were conducted exposing cells to 32 Hz, 150 μT and 75 μT rms, parallel to a 42 μT dc magnetic field. The 75 μT rms, 32 Hz exposure condition was chosen to maximize the resonance effect on Ca²⁺ according to parametric resonance theory. We found a statistically significant increase of MN for both exposure conditions. This experiment provides evidence for the genotoxic effects of resonant ELF magnetic fields in human lymphocytes.     

Estimation of genetic effects of a static magnetic field by a somatic cell test using mutagen-sensitive mutants of Drosophila melanogaster

In order to estimate the genetic effects of magnetic fields, a somatic cell test was performed using mutants of the fruit fly Drosophila melanogaster which lack repair functions for damage to their cellular deoxyribonucleic acid (DNA). Young larvae of mutant and normal genotypes were exposed to a homogeneuos 0.6 T magnetic field for 24 h and were then allowed to continue development under normal culture condition until they moulted and finally emerged from their pupal cases. After eclosion, the number of surviving adults was counted. The number of adults of the mutant genotype decreased by about 8% compared with unexposed controls, while that of normal siblings remained unchanged. This suggests that exposure to a static magnetic field resulted in damage to larval cellular DNA, and that somatic cells without normal DNA repair functions failed to continue cell division which resulted in developmental lethality of mutant larvae. DNA damage occurring in normal larvae should have been repaired, so that their survival rate was not altered. The effect was compared with that of UV irradiation, and the genotoxic activity of the 0.6 T static magnetic field was estimated to be the same as that of UV light with an intensity of 0.14 mJ m⁻² s⁻¹. Possible mechanisms in which DNA damage is caused by magnetic field exposure are discussed.     

Changes in cell proliferation due to environmental non-ionizing radiation 1. ELF electromagnetic fields

To study the effect of extremely low frequency (ELF) magnetic fields on cell growth, human cells (AMA cells) and K14 skin fibroblasts cells, growing in monolayer culture, were exposed to a sinusoidal 50 Hz, 80 μT field. Exposure times varied from 15 to 90 min. Changes in cell proliferation rates were then studied during subsequent field-free incubation, for 24 h.The results showed that a 30 min exposure resulted in a much higher increase in proliferation rates in the AMA cells compared with non-exposed cells or cells exposed to electromagnetic fields for shorter or longer times. The magnitude of the increase also depended on the initial proliferation rate and confluency. The exposure to varying field densities showed that the greatest increase in proliferation occurred at 80 μT.     

A 50 Hz sinusoidal magnetic field induces changes in the membrane electrical properties of K562 leukaemic cells

The cell membrane appears to be one of the major targets of extremely low frequency (ELF) magnetic fields. To determine whether the membrane electrical properties of K562l leukaemic cells can be affected by ELF fields, these cells were exposed to a sinusoidal 50 Hz 2.5 mT magnetic field for 48 h. It should be recalled that the field intensity used is three or four orders of magnitude greater than the values found in homes and in many workplaces. Analyses of dielectric relaxation measurements in the r.f. range demonstrate that both the membrane electrical conductivity and permittivity of K562 cells decrease considerably after exposure of these cells to ELF fields, whereas the conductivity of the cytosol does not vary. Since both membrane conductivity (a measure of the dynamic ionic movement across cell membranes) and membrane permittivity (an indicator of the ionic charges present on cell membranes) vary, these data seem to suggest that exposure of K562 leukaemic cells to ELF fields affects both static and dynamic properties of these cells.     

Strong static magnetic field effects on yeast proliferation and distribution

The present study focuses on the effects of gradient magnetic fields on the behavior of yeast, such as its proliferation and mass distribution, and evaluates the effects of magnetism on materials in the yeast culture system. Yeast, Saccharomyces cerevisiae, was incubated in a liquid medium under magnetic fields (flux density B = 14 T). When yeast in a tube was exposed to 9-14 T magnetic fields with a maximum flux density gradient of dB/dx = 94 T/m, where x is the space coordinate, the rate of yeast proliferation under the magnetic fields decreased after 16 h of incubation compared to that of the control group. The physical properties of the yeast culture system were investigated to discover the mechanism responsible for the observed deceleration in yeast proliferation under magnetic fields. Gas pressure inside the yeast culture flask was compared with and without exposure to a magnetic field. The results suggested that the gas pressure inside a flask with 6 T, 60 T/m slowly increased in comparison to the pressure inside a control tube. Due to the diamagnetism of water (medium solution) and yeast, the liquid surface distinctly inclined under gradient magnetic fields, and the hydrostatic force in suspension was strengthened by the diamagnetic forces. In addition, magnetophoresis of the yeast cells in the medium solution exhibited localization of the yeast sedimentation pattern. The roles of magnetically changed gas-transport processes, hydrostatic pressures acting on the yeast, and changes in the distribution of the yeast sedimentation, as well as the possible effects of magnetic fields on yeast respiratory systems in the observed disturbance of the proliferation are discussed.     

Growth of Escherichia coli under extremely low-frequency electromagnetic fields

The inflence of extremely low-frequency (ELF) electromagnetic fields on Escherichia coli cultures in submerse fermentation was studied. The fermentation processes were carried out recycling the culture medium externally through a stainless steel tube inserted in a magnetic field generator (solenoid). The exposure time and electromagnetic induction were varied in a range of 1 to 12 h and 0.010 to 0.10 T, respectively, according to a Box-Wilson Central Composite Designs of face centered with five central points. Growth of E. coli could be altered (stimulated or inhibited) under magnetic fieldinduced effects. E. coli culturesexposed at 0.1 T during 6.5 h exhibited changes in its viability compared to unexposed cells, which was 100 times higher than the control. The magnetic field generator associated with the cellular suspension recycle is a new way of magnetic treatment in fermentation processes and could be appropriate to industrial scale up.     

Effects of ELF Magnetic Field in Combination with Iron(III) Chloride (FeCl3) on Cellular Growth and Surface Morphology of Escherichia coli (E. coli)

This study investigated the effects of extremely low frequency (ELF) magnetic field with/without iron(III) chloride (FeCl₃) on bacterial growth and morphology. The ELF exposures were carried out using a pair of Helmholtz coil-based ELF exposure system which was designed to generate 50 Hz sinusoidal magnetic field. The field was approximately uniform throughout the axis of the coil pair. The samples which were treated or non-treated with different concentrations FeCl₃ were exposed to 50 Hz, 2 millitesla (mT) magnetic field for 24 h. ELF effect on viability was assessed in terms of viable colony counts (in colony-forming unit per milliliter) with the standard plate count technique. Scanning electron microscopy was used to investigate the magnetic field effect on surface morphology of Escherichia coli. No significant results were seen in terms of cell viability between ELF and sham-exposed bacterial strains. Similarly, FeCl₃ treatment did not change cell viability of E. coli samples. However, we observed some morphological changes on E. coli cell surfaces. Pore formations and membrane destruction were seen on the surface of 24 h ELF field-exposed cells. We concluded that ELF magnetic field exposure at 2 mT does not affect cell viability; however, it may affect bacterial surface morphology.     

Effects of static magnetic fields of erythrocyte rheology

The orientation of normal erythrocytes in a uniform static magnetic field (8 T maximum) has been investigated microscopically and photometrically.

• 1.(1) The intact erythrocytes were oriented with their disk planes parallel to the magnetic field because of the diamagnetism of the cell membrane components, particularly the transmembrane proteins (e.g., Band III, glycopholin) and the lipid bilayer.
• 2.(2) In contrast, the glutaraldehyde-fixed erythrocytes were oriented perpendicular to the field, perhaps because of the paramagnetism of the membrane-bound methemoglobin.
• 3.(3) The orientation was established within 5 s in a dilute suspension (5 × 10³cells μl⁻¹) as estimated from the change in light scattering after exposure to the magnetic field.

     

Orientation of molecules by magnetic field as a new source of information on their structures

A complex procedure for quantitative allowance for small but significant effects of molecular orientation by strong static magnetic fields was elaborated. A series of high-resolution ¹H NMR spectra of 1,2,3-trichloronaphthalene recorded at magnetic field strength varied over a wide range was analyzed in the framework of a unified approach with high accuracy. The spin-spin coupling constants and the dipole-dipole coupling constants for all pairs of ¹H nuclei and the anisotropy and rhombicity parameters of the magnetic susceptibility tensor of the molecule were determined. Ab initio CSGT/RHF quantum chemical calculations of this property using a wide range of conventional diffuse and polarization basis set functions were carried out. Augmentation of the basis set with polarization functions affects the values of the calculated parameters to a lesser extent compared to augmentation with diffuse functions. The results of calculations using the 6-311G(df) and 6-311++G(df) basis sets are in good agreement with the experimental values of the magnetic susceptibility anisotropy for 1,2,3-trichloronaphthalene. The advantages of the method proposed and specific features of the effects of orientation by magnetic field as a new source of information on the structure of molecules in solution are discussed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1309–1317, August, 2006.     

Effects of a static magnetic field on water and electrolyte solutions

Water and electrolyte solutions were exposed for 5 min to a weak static magnetic field (B = 15 mT). Their conductivity and the amount of evaporated water were then measured as a function of time. Simultaneously, these quantities were determined for magnetically untreated samples, as reference systems. It was found that a magnetic field influences these two parameters and their changes depend on the thermodynamic functions of hydration of these ions. A roughly linear change in conductivity versus 'scaled' functions was obtained. On this basis it was concluded that the magnetic field causes changes in the hydration shells of the ions.     

Effects of ELF and static magnetic fields on calcium oscillations in islets of Langerhans

Several experimental studies have produced contradictory results on the effects of extremely low frequency (ELF) magnetic fields on cellular processes involving calcium ions. Furthermore, the few positive results have not been independently replicated. In most of these studies, isolated cells were used. Our study used mouse islets of Langerhans, in which very regular oscillations of calcium concentration can be observed at length. These oscillations are sustained by processes that imply energetic and inter-intracellular communication. Various magnetic fields were applied, either sinusoidal at different frequencies (50 Hz or multiples of the natural oscillation frequency) at 0.1 or 1 mT or static at 1 mT. Islets were also exposed to "cyclotron resonance" conditions. There was neither alteration of the fundamental oscillation frequency nor the degree of organisation under all exposure conditions. Using this sensitive model, we could not show new evidence of alterations of calcium processes under exposure to various magnetic fields.     

Change in broth culture is associated with significant suppression of Escherichia coli death under high magnetic field

When Escherichia coli B was cultivated under an inhomogeneous magnetic field of 5.2-6.1 T, a significant 100,000-fold suppression of cell death was observed [Bioelectrochemistry 53 (2001) 149]. The limited magnetic field exposure for 12 h after logarithmic growth phase was sufficient to observe similar suppressive effects on cell death [Bioelectrochemistry 54 (2001) 101]. These results suggest some possible changes in either the medium or the cells during the magnetic field exposure. When the cell-free filtrate of the broth cultured under the magnetic field for 10 h and the cells of E. coli cultivated under the geomagnetic field for 30 h were mixed, and the mixture was subsequently cultivated under the geomagnetic field, the number of cells observed in the filtrate exposed to the high magnetic field was 20,000 times higher than that in the filtrate exposed to the geomagnetic field. When the cells cultivated under the magnetic field for 10 h and the cell-free filtrate of the broth culture exposed to the geomagnetic field were mixed, only a 50-fold difference in the number of cell between under the magnetic field and under the geomagnetic field was observed. This suggests that the filtrate of the broth culture exposed to the magnetic field is primarily responsible for the cell death suppression. It was also revealed that the small difference in pH of the filtrates of the broth culture between under the magnetic field and under the geomagnetic field was critical for the cell death suppression.     

Anchorage-independent growth with JB6 cells exposed to 60 Hz magnetic fields at several flux densities

JB6 clone 41 cells have previously been shown to respond to a 1.1. mT magnetic field with increased growth under anchorage-independent (AI) conditions. Here we have examined the AI growth-response simultaneously at three lower flux densities and without an applied field. Fields were generated with Helmholtz coils held in water-jacketed CO₂ incubators. Colony growth (60 μm diameter) was scored at 8 and 14 days after seeding cells in soft agar. Zero-field experiments conducted simultaneously in several incubators produced uniform counts (about 1 per 10³ cells seeded). For 14-day exposures at 100 μT, 10 μT and 1μT, four of four, six of six, and two of three experiments, respectively, showed a significantly increased colony number (p < 0.02), with the ratio of field-exposed to control colonies varying from 1.2 to 3.2. Thus, an effect of magnetic fields at flux densities approaching environmental levels of exposure has been demonstrated.     

Electrochemical noise analysis of the effects of a magnetic field on cathodic hydrogen evolution

The effect of a static magnetic field on the evolution of hydrogen gas from a small platinum electrode in an aqueous electrolyte has been studied by recording the noise spectrum of overpotential voltage fluctuations at a constant current density of ?50 mA mm^?2. A 1/f² variation of the power spectrum characteristic of droplet coalescence is found for frequencies >10 Hz. The overpotential for hydrogen evolution decreases with applied field. When the production of gas bubbles is quasiperiodic, there is a threshold field of 0.5 T beyond which the size of the bubbles released is approximately doubled. This is explained by enhanced coalescence of small bubbles swept across the electrode surface by forced convection due to the Lorentz force.     

Effect of a static magnetic field on ion transport in a cellulose membrane

A cellulose membrane was exposed to the static magnetic field (SMF) in the presence of KCl solution and ion transport through the membrane was measured before and after the SMF exposure. SMF at 0.24 T significantly enhanced the rate of ion transport, especially after the first exposure (p<0.05), while the increased ion transport rate did not return to the initial basal level after exchange of the aqueous medium. These results suggest that an irreversible, temporal conformation change took place on the cellulose membrane or on the water bound to the cellulose surface. The accelerating effect of SMF on the ion transport seems to have occurred as a result of stabilized hydration layer on the cellulose surface.     

Effects of static magnetic field on electrolyte solutions under kinetic condition

Electrolyte solutions were exposed for different time to weak static magnetic field (MF) generated from a stack of magnets (B = 15 mT) at the flow rate of 1.4 mL/s. The conductivity was measured as a function of time following the application of MF. It was found that the changes in conductivity depend on the kind of electrolyte and the magnetic exposure time and are related to the thermodynamic function of hydration.     

Melatonin prevents magnetic-field-induced neurite outgrowth in a subline of pheochromocytoma cells,PC12D

Exposure to extremely low frequency (ELF) magnetic fields (MFs) has been reported to affect several cellular processes, including cell growth and differentiation. Other research has demonstrated that the pineal gland and its hormone melatonin have a wide spectrum of effects in cells and organs and can exert modulatory actions on cell proliferation and cell differentiation. Since ELF electric and magnetic fields have been shown to influence pineal activity and melatonin synthesis and/or function, it has been suggested that some of the reported effects of ELF MFs could be a consequence of the direct action of these fields on the pineal gland and/or melatonin function. Possible interactions between MFs and melatonin effects are in an early stage of investigation. In this study, we have investigated the influence of melatonin on the in vitro response of a subline of pheochromocytoma cells, PC12D, to a MF. Cells were exposed to the combined action of a physiological concentration (10⁻¹⁰ M) of melatonin and a vertical, 50 Hz, 40 mG rms MF for 23 h. At the end of the treatment, the percentages of neurite-bearing cells were determined by microscopic examination and compared with those from samples treated with the field alone or with melatonin alone. MF exposure alone significantly increased the neurite outgrowth when compared with negative controls, supporting previous results by Blackman and coworkers; this effect was not observed when melatonin was present in the medium from the onset of the exposure. Although the mechanisms of action of melatonin and ELF MFs at the cellular level remain unknown, the present data suggest that physiological levels of melatonin can prevent PC12D cells from responding to the MF stimulus.     

Effects of extremely low frequency magnetic field on the antioxidant defense system in mouse brain: a chemiluminescence study

Among the putative mechanisms, by which extremely low frequency (ELF) magnetic field (MF) may affect biological systems is that of increasing free radical life span in organisms. To test this hypothesis, we investigated whether ELF (60 Hz) MF can modulate antioxidant system in mouse brain by detecting chemiluminescence and measuring superoxide dismutase (SOD) activity in homogenates of the organ. Compared to sham exposed control group, lucigenin-initiated chemiluminescence in exposed group was not significantly increased. However, lucigenin-amplified t-butyl hydroperoxide (TBHP)-initiated brain homogenates chemiluminescence, was significantly increased in mouse exposed to 60 Hz, MF, 12 G for 3 h compared to sham exposed group. We also measured SOD activity, that plays a critical role of the antioxidant defensive system in brain. In the group exposed to 60 Hz, MF, 12 G for 3 h, brain SOD activity was significantly increased. These results suggest that 60 Hz, MF could deteriorate antioxidant defensive system by reactive oxygen species (ROS), other than superoxide radicals. Further studies are needed to identify the kind of ROS generated by the exposure to 60 Hz, MF and elucidate how MF can affect biological system in connection with oxidative stress.     

Effect of a 7-tesla homogeneous magnetic field on mammalian cells

When two types of mammalian cells, mouse leukemia cells, P388, and Chinese hamster fibroblast cells, V79, were grown under a 7-tesla (T) homogeneous magnetic field which was produced by a newly constructed superconducting magnet biosystem (SBS), the growth pattern of cells under 7 T magnetic field and the geomagnetic field control showed no differences. The DNA distribution of the two cells was compared by flow cytometry after exposure to 7 T for 3 and 24 h, but there was no significant differences between magnet-exposed cells and unexposed cells. When the two types of cells were exposed to different concentrations of the antitumor agent, bleomycin (BLM), for 3 h under 7 T, their viable cell numbers were almost the same as that of the control although sensitivity to BLM was different between the two cells. These results suggest that the 7 T homogeneous magnetic field exerts no adverse effects on mammalian cells.