Permeability changes of connexin32 hemi channels reconstituted in liposomes induced by extremely low frequency, low amplitude magnetic fields

The effect of extremely low frequency and low amplitude magnetic fields on gap junctional permeability was investigated by using reconstituted connexin32 hemi channel in liposomes. Cytochrome c was loaded inside these proteoliposomes and its reduction upon addition of ascorbate in the bulk aqueous phase was adopted as the index of hemi channel permeability. The permeability rate of the hemi channels, expressed as ΔA/min, was dependent on the incubation temperature of proteoliposomes. The effect of exposures to magnetic fields at different frequencies (7, 13 and 18 Hz) and amplitudes (50, 50 and 70 μT, respectively), and at different temperatures (16, 18 and 24 °C) was studied. Only the exposure of proteoliposomes to 18-Hz (B_acpeak and B_dc=70 μT) magnetic field for 60 min at 16±0.4 °C resulted in a significant enhancement of the hemi channel permeability from ΔA/min=0.0007±0.0002 to ΔA/min=0.0010±0.0001 (P=0.030). This enhancement was not found for magnetic field exposures of liposomes kept at the higher temperatures tested. Temperature appears to influence lipid bilayer arrangement in such a way as being capable to mask possible effects induced by the magnetic field. Although the observed effect was very low, it seems to confirm the applicability of our model previously proposed for the interaction of low frequency electromagnetic fields with lipid membrane.     

Effect of low frequency, low amplitude magnetic fields on the permeability of cationic liposomes entrapping carbonic anhydrase: I. Evidence for charged lipid involvement

The influence of low frequency (4-16 Hz), low amplitude (25-75 mu T) magnetic fields on the diffusion processes in enzyme-loaded unilamellar liposomes as bioreactors was studied. Cationic liposomes containing dipalmitoylphosphatidylcholine, cholesterol, and charged lipid stearylamine (SA) at different molar ratios (6:3:1 or 5:3:2) were used. Previous kinetic experiments showed a very low self-diffusion rate of the substrate p-nitrophenyl acetate (p-NPA) across intact liposome bilayer. After 60 min of exposure to 7 Hz sinusoidal (50 mu T peak) and parallel static (50 mu T) magnetic fields the enzyme activity, as a function of increased diffusion rate of p-NPA, rose from 17 +/- 3% to 80 +/- 9% (P < .0005, n = 15) in the 5:3:2 liposomes. This effect was dependent on the SA concentration in the liposomes. Only the presence of combined sinusoidal (AC) and static (DC) magnetic fields affected the p-NPA diffusion rates. No enzyme leakage was observed. Such studies suggest a plausible link between the action of extremely low frequency magnetic field on charged lipids and a change of membrane permeability.     

Permeability changes induced by 130 GHz pulsed radiation on cationic liposomes loaded with carbonic anhydrase

The effects of pulsed 130 GHz radiations on lipid membrane permeability were investigated by using cationic liposomes containing dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and stearylamine. Carbonic anhydrase (CA) was loaded inside the liposomes and the substrate p-nitrophenyl acetate (p-NPA) added in the bulk aqueous phase. Upon permeation across the lipid bilayer, the trapped CA catalyzes the conversion of the p-NPA molecules into products. Because the self-diffusion rate of p-NPA across intact liposomes is very low the CA reaction rate, expressed as Delta A/min, is used to track membrane permeability changes. The effect of 130 GHz radiation pulse-modulated at low frequencies of 5, 7, or 10 Hz, and at time-averaged incident intensity (I(AV)) up to 17 mW/cm(2) was studied at room temperature (22 degrees C), below the phase transition temperature of DPPC liposomes. At all the tested values of I(AV) a significant enhancement of the enzyme reaction rate in CA-loaded liposomes occurred when the pulse repetition rate was 7 Hz. Typically, an increase from Delta A/min = 0.0026 +/- 0.0010 (n = 11) to Delta A/min = 0.0045 +/- 0.0013 (n = 12) (P < 0.0005) resulted at I(AV) = 7.7 mW/cm(2). The effect of 130 GHz pulse-modulated at 7 Hz was also observed on cationic liposomes formed with palmitoyloleoyl phosphatidylcholine (POPC), at room temperature (22 degrees C), above the phase transition temperature of POPC liposomes.     

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.     

Calcium uptake by leukemic and normal T-lymphocytes exposed to low frequency magnetic fields

Calcium-ion uptake by normal and leukemia lymphocytes increased during a 30-min exposure to a 13.6 Hz, sinusoidal magnetic field at 20 microT peak. The time-varying field was horizontal and parallel to a 16.5 microT component of the ambient static magnetic field. The uptake of 45Ca2+ increased 102% in a line of murine, cytotoxic T-lymphocytes (C57B1/6-derived CTLL-1), increased 126% in freshly-isolated spleen lymphocytes (C57B1/6 mice), and increased 75% in a line of lymphoma cells (C57B1/6-derived EL4). In contrast, there was no effect when the same field was applied for 30 min immediately before--as opposed to during--incorporation of calcium ions. When spleen lymphocytes were exposed during incubation with 45Ca2+ to a 60 Hz magnetic field at 20 microT peak, a small but statistically significant increase (37%) in uptake of the labeled ions occurred. These results indicate that weak, alternating magnetic fields might affect calcium-dependent functions of normal and leukemic lymphocytes.     

ELF magnetic fields increase amino acid uptake into Vicia faba L. roots and alter ion movement across the plasma membrane

Vicia faba seedlings, subjected to a 10 microT 50 Hz square wave magnetic field for 40 min together with a radioactive pulse, showed a marked increase in amino acid uptake into intact roots. A more modest increase was observed with a 100 microT 50 Hz square wave. An increase in media conductivity at low field intensities from 10 microT 50 Hz square wave, 100 microT 50 Hz sine wave, and 100 microT 60 Hz square wave fields, indicated an alteration in the movement of ions across the plasma membrane, most likely due to an increase in net outflow of ions from the root cells. Similarly, marked elevation in media pH, indicating increased alkalinity, was observed at 10 and 100 microT for both square and sine waves at both 50 and 60 Hz. Our data would indicate that low magnetic field intensities of 10 and 100 microT at 50 or 60 Hz can alter membrane transport processes in root tips.     

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.     

Measurement of the individual exposure to 50 and 16 2/3 Hz magnetic fields within the Bavarian population.

This study investigates the individual magnetic field exposures at 16 2/3 and 50 Hz of 1952 people, selected from the Bavarian population. Personal flux density meters ("Field Watcher FW2A") were worn by the participants for 24 h. Every second, the flux density was recorded for both frequencies and for the three spatial axes (dynamic range per axis: several nT up to 100 microT at 50 Hz, 150 microT at 16 2/3 Hz). For 50 Hz fields, the mean of the 1,952 individual means was 0.101 microT and that of the individual medians was 0.047 microT. High level exposures occurred mainly during working hours. Only 2.4% of the subjects showed individual medians higher than 0.2 microT. About 53% of all volunteers were working on the day of recording. Levels for craftsmen (n = 148; mean individual mean: 0.166 microT) were generally higher than those for office workers (n = 624; mean individual mean: 0.107 microT). Flux densities exceeding 100 microT at 50 Hz were measured in 31 persons. The total time with such extreme exposures amounts to nearly 21 min, less than 0.001% of the total time for all measurements (5.3 years). To our knowledge, this is the first exposure study where 16 2/3 Hz magnetic fields (caused by electrified railways) have additionally been monitored over 24 h. For persons living next to railway lines, the mean individual mean (0.156 microT) and mean individual median (0.102 microT) were calculated. Over all, the mean exposures are only 0.1% of the magnetic flux density limit for 50 Hz (100 microT) and about 0.05% of the limit (300 microT) for 16 2/3 Hz recommended by the International Commission on Non-Ionizing Radiation Protection.     

Reconstitution of (Na+ + K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme

Membranous (Na+ + K+)-ATPase from the electric eel was solubilized with 3-[3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (Chaps). 50 to 70% of the solubilized enzyme was reconstituted in egg phospholipid liposomes containing cholesterol by using Chaps. The obtained proteoliposomes consisted of large vesicles with a diameter of 134 +/- 24 nm as the major component, and their protein/lipid ratio was 1.25 +/- 0.07 g protein/mol phospholipid. The intravesicular volume of these proteoliposomes is too small to consistently sustain the intravesicular concentrations of ligands, especially K+, during the assay. The decrease in K+ concentration was cancelled by the addition of 20 microM valinomycin in the assay medium. The low value of the protein/lipid ratio suggests that these proteoliposomes contain one Na+/K+-pump particle with a molecular mass of 280 kDa per one vesicle as the major component. In these proteoliposomes, the specific activity of the (Na+ + K+)-ATPase reaction was 10 mumol Pi/mg protein per min, and the turnover rate of the ATP-hydrolysis was 3500 min-1, the same as the original enzyme under the same assay condition. The ratio of transported Na+ to hydrolyzed ATP was 3, the same as that in the red cell. The proteoliposomes could be disintegrated by 40-50 mM Chaps without any significant inactivation. This disintegration of proteoliposomes nearly tripled the ATPase activity compared to the original ones and doubled the specific ATPase activity compared to the membranous enzyme, but the turnover rate was the same as the original proteoliposomes and the membranous enzyme. This disintegration of proteoliposomes by Chaps suggests the selective incorporation of the (Na+ + K+)-ATPase particle into the liposomes and the asymmetric orientation of the (Na+ + K+)-ATPase particle in the vesicle.     

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.     

The method for studying of the "magnetic vacuum" effect on color memory in humans

The method for studying the effects of weak magnetic fields and "magnetic vacuum" on the psychophysiological state of a human organism is proposed. This method includes the system of the exposure of a human organism to uniform constant and alternating magnetic fields and the system of computerized psychological tests. The influence of the weakening of constant magnetic field on the psychophysiological state of human organisms was studied. The short-term color memory and reaction rates of 30 subjects have been examined in the local geomagnetic field and in a magnetic field which was reduced in 10 and more times. Statistically significant differences in the color memory test was found in the magnetic field 4 +/- 1 microT in comparison with the results in the geomagnetic field. In the magnetic field 0 +/- 1 microT, slight impairment of color memory was found. Preliminary results in the test of reaction rates showed the tendency to slowing down the reaction rates in the weakened magnetic fields.     

Effects of information and 50 Hz magnetic fields on cognitive performance and reported symptoms

The aim of this study was to explore the role of expectancies and beliefs about the potential effects of electromagnetic fields (EMFs) (what the subject thought the effect was going to be) and the effects of 50 Hz magnetic fields (400 microT(rms)) acute exposure on cognitive performance, the reporting of physical symptoms and some psychological and physiological parameters. Seventy-four healthy male volunteers aged between 40 and 60 years of age were randomly assigned to one of five groups, which differed in (1) the type of information they were given concerning the expected magnetic field effect on performance in cognitive tests (positive = enhancement of the performance; negative = impairment of the performance; neutral) and (2) the type of exposure (real or sham). Three groups were sham exposed with positive (group+), negative (group-) and neutral information (group+/-); one group was really exposed with neutral information (group expo) and one group was not exposed, though they wore the helmet, and did not receive any field-related information (control group). All the volunteers, except the control group, were led to believe that they would be exposed to a magnetic field of 400 microT(rms). The experimental design respected a double blind procedure and the experimental session involved three steps (pre-testing, exposure, and post-testing). Various measurements were taken, including cognitive performance, psychological parameters such as mood, vigilance, and reporting of symptoms. Physiological parameters such as blood pressure and pulse rate were also recorded. The information given did not significantly modify beliefs. No significant difference was found among the five groups depending on the type of information and the type of exposure in cognitive performance, psychological and physiological parameters. In the context of the study, with our population, the type of information given failed to induce expected changes in parameters measured. Our results do not support the hypothesis that an acute exposure to extremely low frequency magnetic fields (50 Hz, 400 microT(rms)) affects the parameters measured.     

Hydrolysis of various peptides and proteins in weak permanent and low frequency fluctuating magnetic fields

It was shown that weak combined magnetic fields (constant field 25-130 microT; variable field 0.01-0.2 microT; the range of effective frequencies of the alternating component 1-10 Hz) substantially increase the rate of hydrolysis of some proteins and peptides (eight various sequences). The concentration dependence of the dynamics of the process and the dependence of the magnitude of the effect on the parameters of the magnetic field. It was found that: (1) the effect is transmitted through a solvent preliminarily treated by magnetic fields and (2) the effect occurs in the presence of inhibitors of proteases and enzymes inactivating peroxides (catalase and horse radish peroxidase with substrate).     

Interactions of the low molecular weight group of surfactant-associated proteins (SP 5-18) with pulmonary surfactant lipids

The interaction of the low molecular weight group of surfactant-associated proteins, SP 5-18, with the major phospholipids of pulmonary surfactant was studied by fluorescence measurements of liposomal permeability and fusion, morphological studies, and surface activity measurements. The ability of SP 5-18 to increase the permeability of large unilamellar lipid vesicles was enhanced by the presence of negatively charged phospholipid. The permeability of these vesicles increased as the protein concentration was raised and the pH was lowered. SP 5-18 also induced leakage from liposomes made both from a synthetic surfactant lipid mixture and from lipids separated from SP 5-18 during its purification from canine sources. When SP 5-18 was added to egg phosphatidylglycerol liposomes, the population of liposomes which became permeable leaked all encapsulated contents, while the remaining liposomes did not leak at all. The extent of leakage was higher in the presence of 3 mM calcium. SP 5-18 also induced lipid mixing between two populations of egg phosphatidylglycerol liposomes in the presence of 3 mM calcium, as monitored by resonance energy transfer between two different fluorescent lipid probes, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine. Negative-staining electron microscopy showed that the addition of SP 5-18 and 3 mM calcium produced vesicles twice the size of control egg phosphatidylglycerol liposomes. In addition, surface balance measurements revealed that the adsorption of liposomal lipids to an air/water interface was enhanced by the presence of SP 5-18, negatively charged phospholipids, and 3 mM calcium. These observations suggest a similar lipid dependence for the interactions observed in the fluorescence and adsorption experiments.     

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.     

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.     

Osmotic water permeability in glycoprotein containing liposomes

The kinetics of osmotic water permeability in proteoliposomes containing ₁-acid glycoprotein was investigated by means of stopped-flow spectrophotometry. A biphasic time-course of scattered light with time was registered. The rate constants calculated from fits to an exponential function in the first phase were proportional to the final medium osmolarity. The apparent second order rate constants K_app (Osm^-1 sec^-1) were determined at different glycoprotein concentrations in the original mixture for preparation of proteoliposomes. The value of K_app at lipid:glycoprotein weight ratio = 1 was plotted in Arrhenius coordinates. The calculated activation energy for water permeation through the lipid bilayer suggests that eventual channel mechanism may be involved due to the presence of glycoprotein molecule in the liposomes.     

Growth and developmental stability of Drosophila melanogaster in low frequency magnetic fields

Magnetic fields (60 Hz) of 1.5 and 80 microT caused a significant reduction in the weight of Drosophila melanogaster. Moreover, fruit flies in an 80 microT field showed lower developmental stability than either those in a 0 or 1.5 microT field. Developmental instability was measured by fluctuating asymmetry and frequency of phenodeviants. More of the flies in the 80 microT field had fused abdominal segments, and they were more asymmetrical for wing vein R(4+5). The flies in the 1.5 microT field actually showed greater developmental stability than the control flies. Fewer of them had fused abdominal segments, and they were more symmetrical for wing vein R(4+5). Thus, at low field strengths, flies are more developmentally stable than control flies, even though they weigh less.     

Effects of extremely low frequency magnetic field on fertility of adult male and female rats.

To investigate the effects of an extremely low-frequency (ELF) magnetic field on their fertility, adult male and female Sprague-Dawley rats were exposed to a 50 Hz sinusoidal magnetic field of approximately 25 microT (rms) for 90 days before they were mated with unexposed counterparts. Exposure to a 50 Hz field reduced male rat fertility. The number of pregnant females was reduced when mated with exposed males, and the number of resorptions increased. The effects of magnetic field on male fertility were shown to be partly reversible, when the same exposed group of males were remated 45 and 90 days after being removed from the fields. Exposure of adult female rats to 50 Hz magnetic fields for 90 days before mating significantly reduced their fertility. The mean numbers of implantations and living fetuses per litter were statistically significantly decreased in the 50 Hz group. These results suggest that low frequency magnetic fields have some adverse effects on fertility of male and female rats.