Extracellular currents alter gap junction intercellular communication in synovial fibroblasts

We studied the effect of extremely low frequency (ELF) currents on gap junction intercellular communication (GJIC) mediated by connexin43 protein. Confluent monolayers of synovial fibroblasts (HIG-82) and neuroblastoma cells (5Y) were exposed in bath solution to 0-75 mA/m(2) (0-56 mV/m), 60 Hz. Single channel conductance, cell membrane current-voltage (I-V) curves, and Ca(2+) influx were measured using the nystatin single and double patch methods. The conductances of the closed and open states of the gap junction channel in HIG-82 cells were each significantly reduced (by 0.76 and 0.39 pA, respectively) in cells exposed to 20 mA/m(2). Current densities as low as 10 mA/m(2) significantly increased Ca(2+) influx in HIG-82 cells. No effects were seen in 5Y cells. The I-V curves of the plasma membranes of both types of cells were independent of 60 Hz electric fields and current densities, 0-75 mA/m(2), indicating that the effect of the 60 Hz fields on GJIC in HIG-82 cells was not mediated by a change in membrane potential. We conclude that ELF electric fields can alter GJIC in synovial cells via a mechanism that does not depend on changes in membrane potential, but may depend on Ca(2+) influx. The results open the possibility that GJIC mediated responses in synovial cells, such as for example, their secretory responses to proinflammatory cytokines, could be antagonized by the application of ELF electric fields.     

Review of in vivo static and ELF electric fields studies performed at Gazi Biophysics Department

In vivo effects of Static Electric and ELF Magnetic and Electric fields have been carried out for more than 20 years in the Bioelectromagnetic Laboratory at the Biophysics Department of the Medical Faculty of Gazi University. In this article, the results of in vivo ELF Electric field studies are presented as a review. Static and 50 Hz ELF (Extremely Low Frequency) Electric (E) fields effects on free radical synthesis, antioxidant enzyme level, and collagen synthesis were analyzed on tissues of guinea pigs, such as brain, liver, lung, kidney, spleen, testis, and plasma. Animals were exposed to static and ELF electric fields with intensities ranging from 0.3 kV/m to 1.9 kV/m in vertical and horizontal directions. Exposure periods were 1, 3, 5, 7, and 10 days. Electric fields were generated from a specially designed parallel plate capacitor system. The results indicate that the effects of electric fields on the tissues studied depend significantly on the type and magnitude of electric field and exposure period.     

Ion-protein dissociation predicts 'windows' in electric field-induced wound-cell proliferation

There are many experiments showing that weak, non-thermal electric fields influence living tissues. In many cases, biological effects display 'windows' in biologically effective parameters of electric fields: most dramatic is the fact that relatively intense electric fields sometimes do not cause appreciable effect, while smaller fields do. Linear resonant physical processes do not explain frequency windows in this case. Both frequency and amplitude windows are evident from experiments on human dermal fibroblasts in a collagen matrix. For this in vitro model of skin, exposure to extremely low frequency (ELF) electric fields in the frequency range 10-100 Hz and the amplitude range of 0-130 microA/cm(2) macroscopic current density demonstrates such unusual 'window' behavior. Amplitude window phenomena suggest a non-linear physical mechanism. We consider non-linear quantum-interference effects on protein-bound substrate ions: These ions experience, due to electric fields in the media or biological tissue as small as 1 mV/m, electric gradients produced by polarized binding ligand atomic shells. The electric gradients cause an interference of ion quantum states. This ion-interference mechanism predicts specific electric-field frequency and amplitude windows within which fibroblast proliferation occurs.     

An in vitro model of fibroplasia: simultaneous quantification of fibroblast proliferation, migration, and collagen synthesis

Previous studies of fibroblast proliferation, migration, and collagen synthesis have been limited in their ability to define the interrelationship among these events in response to various inflammatory mediators. We have now defined an in vitro tissue culture model for the synchronous quantification of these parameters of fibroplasia. Biopsies (2 mm) of chicken flexor tendons are embedded in a fibrin matrix and exposed to various factors for 5 days in tissue culture. The availability of the fibrin matrix surrounding the tendon biopsy satisfies the need for a solid support medium for fibroblast migration. Multiple measurements of tendon fibroblast proliferation, migration into the fibrin matrix, and relative collagen synthesis are then made on these preparations. Fetal calf serum stimulated tendon fibroblast proliferation and migration in a dose responsive fashion, whereas the selective expression of collagen synthesis was decreased. Platelet lysate stimulated fibroblast proliferation at low concentration, but migration only at high concentration and was without effect on relative collagen synthesis. This model now provides a means of more clearly defining the influence of various inflammatory factors on the events of fibroplasia.     

Currents induced in anatomic models of the human for uniform and nonuniform power frequency magnetic fields

We have used the quasi-static impedance method to calculate the currents induced in the nominal 2 x 2 x 3 and 6 mm resolution anatomically based models of the human body for exposure to magnetic fields at 60 Hz. Uniform magnetic fields of various orientations and magnitudes 1 or 0.417 mT suggested in the ACGIH and ICNIRP safety guidelines are used to calculate induced electric fields or current densities for the various glands and organs of the body including the pineal gland. The maximum 1 cm(2) area-averaged induced current densities for the central nervous system tissues, such as the brain and the spinal cord, were within the reference level of 10 mA/m(2) as suggested in the ICNIRP guidelines for magnetic fields (0.417 mT at 60 Hz). Tissue conductivities were found to play an important role and higher assumed tissue conductivities gave higher induced current densities. We have also determined the induced current density distributions for nonuniform magnetic fields associated with two commonly used electrical appliances, namely a hair dryer and a hair clipper. Because of considerably higher magnetic fields for the latter device, higher induced electric fields and current densities were calculated.     

Effects of 50 Hz electric currents and magnetic fields on the prokaryote Propionibacterium acnes

The effects of 50 Hz sinusoidal electric currents and magnetic fields on the Gram-positive skin bacterium Propionibacterium acnes were investigated. Intracellular free calcium ([Ca(2+)](i)), intracellular pH (pH(i)), and cell viability were examined, based on their relevance to ELF field studies and on previous studies conducted on P. acnes (UVA irradiation, photosensitization using porphyrin-based sensitizers, and broad-band red light). The [Ca(2+)](i) and the pH(i) were measured spectrofluorimetrically using the fluorescent probes fura-2 and BCECF, respectively. Sham-exposed controls were used to assess the field exposed samples. Cell suspensions were exposed to 50 Hz, 0.2 mT sinusoidal magnetic fields generated by using Helmholtz coils for up to 30 min. The estimated maximum induced electric field was 0.2 mV/m. Changes in [Ca(2+)](i) and cell viability were not detected. Ag/AgCl electrodes were used to expose cell suspensions to 50 Hz sinusoidal electric currents. The current densities were in the range 0.015-1500 A/m(2) (corresponding electric fields congruent with0.01-1000 V/m). Changes in [Ca(2+)](i) were not observed after current exposure. Current densities of 800 A/m(2) (electric field E congruent with550 V/m) were required for a 50% reduction in cell viability. Current densities greater than 800 A/m(2) were required for a reduction in pH(i). However, a pH gradient across the cell membrane (inside alkaline) was maintained even when exposure resulted in less than 0. 2% survival (1400 A/m(2), E congruent with950 V/m). Thus, dissipation of the pH gradient across the cell membrane and changes in [Ca(2+)](i) were not a consequence of cell inactivation by 50 Hz electric currents. This is in contrast to inactivation of P. acnes by UVA irradiation or photosensitization, where such changes have been obtained.     

Short-circuit currents,surface electric fields,and axial current densities for guinea pigs exposed to ELF electric fields.

Short-circuit currents, surface electric fields, and axial current densities were measured in electrically grounded guinea pigs exposed to a uniform, vertical, ELF electric field. These data are 70–110% of corresponding values obtained in grounded rats exposed to the same electric field.     

Echinococcus granulosus: lethal effect of low voltage direct electric current on hydatid cyst protoscoleces

We have studied a small scale method for killing hydatid cyst protoscoleces using low voltage direct electric current. After collecting hydatid cysts from infected organs of slaughtered animals, protoscoleces were cultured in four different media: hydatid cyst fluid, RPMI, normal saline, and Tris buffer, respectively. Protoscoleces from each of the above media were then transferred to an electrolysis device through which different electric current densities were applied. For measuring the survival rate of protoscoleces, flame cell movement and eosin staining was used. The results show that the survival rate of protoscoleces in hydatid fluid was dependent on the electric current density and the time of the applied current. Current densities of 62.5 mA/cm2 (11 V), 53.71 mA/cm2 (10 V), and 18.18 mA/cm2 (5 V) after 1, 2, and 3 min, respectively, killed all the parasites in the hydatid fluid. However, a current density of 7 mA/cm2 (9 V) in RPMI medium after 3 min was most effective.     

Occupational exposure to electric fields and currents associated with 110 kV substation tasks

The main aim of this study was to investigate occupational exposure to electric fields, and current densities and contact currents associated with tasks at air-insulated 110 kV substations and analyze if the action value of EU Directive 2004/40/EC was exceeded. Four workers volunteered to simulate the following tasks: Task (A) maintenance of an operating device of a disconnector at ground or floor level, Task (B) maintenance of an operating device of a circuit breaker at ground or floor level, Task (C) breaker head maintenance from a man hoist, and Task (D) maintenance of an operating device of a circuit breaker from a service platform. The highest maximum average current density in the neck was 1.8 mA/m(2) (calculated internal electric field 9.0-18.0 mV/m) and the highest contact current was 79.4 μA. All measured values at substations were lower than the limit value (10 mA/m(2)) of the EU Directive 2004/40/EC and the 2010 basic restrictions (0.1 and 0.8 V/m for central nervous system tissues of the head, and all tissues of the head and body, respectively) of the International Commission on Non-Ionizing Radiation Protection (ICNIRP).     

Calculation of electric fields and currents induced in a millimeter-resolution human model at 60 Hz using the FDTD method

The finite-difference time-domain (FDTD) method has previously been used to calculate induced currents in anatomically based models of the human body at frequencies ranging from 20 to 915 MHz and resolutions down to about 1.25 cm. Calculations at lower frequencies and higher resolutions have been precluded by the huge number of time steps that would be needed in these simulations. This paper describes a method used to overcome this problem and efficiently calculate induced currents in an MRI-based, 6-mm-resolution model of the human under a high-voltage transmission line. This model is significantly higher resolution than the 1.31-cm-resolution model previously used; therefore, it can be used to pinpoint locations of peak current densities in the body. Proposed safety guidelines would allow external electric fields of 10 kV/m and 25 kV/m for exposure to 60 Hz fields of the general public and workers, respectively. For this external electric field exposure of 10 kV/m, local induced current densities as high as 20 mA/m² are found in the head and trunk with even higher values (above 150 mA/m²) in the legs. These currents are considerably higher than the 4 or even 10 mA/m² that have been suggested in the various safety guidelines, thus indicating an inconsistency in the proposed guidelines. In addition, several ratios of E/H typical of power line exposures were examined, and it was found that the vertical electric field couples strongly to the body, whereas the horizontal magnetic field does not. Bioelectromagnetics 19:293–299, 1998. © 1998 Wiley-Liss, Inc.     

Morphologic responses of osteoblast-like cells in monolayer culture to ELF electromagnetic fields

Osteoblast-like cells (MC 3T3-E1) were exposed for 24 h, immediately after plating, to a 60 Hz, 0.7 mT rms magnetic flux density, sufficient to induce an electric field of 0.5 mV/m rms, in order to investigate the influence of ELF field exposure on cell morphology. Using phase contrast images of the live cells, computerized image-analysis permitted rapid and objective quantification of cell length, width, area, perimeter, circularity and angular orientation. While the field-exposed cells were consistently smaller than sham treated cells, the morphologic alterations were not significantly different in the exposed cell population when cell orientation was not considered. When analyzed with respect to cell orientation, cells oriented parallel to the induced electric field (orthogonal to the applied magnetic field) demonstrated a significant decrease in cell length and an increase in roundness. These results confirm and extend previous studies on the morphologic adaptation of cells to low level ELF electromagnetic fields. The results suggest that the observed responses most likely depend on the induced electric field, with a field intensity threshold well below 1 mV/m. Further, these results provide important clues to the specific mechanism by which such low level fields may be capable of influencing cell behavior, and help to explain some of the difficulties in obtaining robust responses in in vitro EMF experiments.     

General properties of the interaction between animals and ELF electric fields

An analysis is given of the interaction between extremely low-frequency (ELF) electric fields and animals of arbitrary body shape. This analysis is based on three approximations which are valid in the ELF range: In living tissues, capacitive (displacement) currents are negligible compared to conduction currents; effects resulting from the finite velocity of propagation of electromagnetic fields are negligible; skin effect in living tissues is negligible. Major conclusions of the analysis are: (a) The electric field outside the body, the induced charge on the surface of the body, and the total current crossing any section through the body (eg, through the neck or limbs) are completely determined by the characteristics of the applied ELF electric field, the shape of the body, its location relative to ground and other conductors, and any conduction currents from the body to ground or other conductors. (b) All of the quantities in (a) can be measured using conducting animal models. (c) The magnitudes of the electric field outside the body and the induced charge density on the surface of the body are independent of frequency, in the ELF range, when the body is either insulated from or shorted to ground (and any other conductors in the system). (d) The only quantities affected by the electrical properties of the tissues comprising the body are the current density and electric field inside the body. (e) The electric field outside and inside a body will be unchanged by a scaled change in its size.     

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.     

Evidences of plasma membrane-mediated ROS generation upon ELF exposure in neuroblastoma cells supported by a computational multiscale approach

BackgroundMolecular mechanisms of interaction between cells and extremely low frequency magnetic fields (ELF-MFs) still represent a matter of scientific debate. In this paper, to identify the possible primary source of oxidative stress induced by ELF-MF in SH-SY5Y human neuroblastoma cells, we estimated the induced electric field and current density at the cell level.MethodsWe followed a computational multiscale approach, estimating the local electric field and current density from the whole sample down to the single cell level. The procedure takes into account morphological modeling of SH-SY5Y cells, arranged in different topologies. Experimental validation has been carried out: neuroblastoma cells have been treated with Diphenyleneiodonium (DPI) -an inhibitor of the plasma membrane enzyme NADPH oxidase (Nox)- administered 24 h before exposure to 50 Hz (1 mT) MF.ResultsMacroscopic and microscopic dosimetric evaluations suggest that increased current densities are induced at the plasma membrane/extra-cellular medium interface; identifying the plasma membrane as the main site of the ELF-neuroblastoma cell interaction. The in vitro results provide an experimental proof that plasma membrane Nox exerts a key role in the redox imbalance elicited by ELF, as DPI treatment reverts the generation of reactive oxygen species induced by ELF exposure.General significanceMicroscopic current densities induced at the plasma membrane are likely to play an active physical role in eliciting ELF effects related to redox imbalance. Multiscale computational dosimetry, supported by an in vitro approach for validation, is proposed as the innovative and rigorous paradigm to unveil mechanisms underlying the complex ELF-MF interactions.     

Electric fields caused by blood flow modulate vascular endothelial electrophysiology and nitric oxide production

Endothelial cells are exposed to a ubiquitous, yet unexamined electrical force caused by blood flow: the electrokinetic vascular streaming potential (EVSP). In this study, the hypothesis that extremely low frequency (ELF) electric fields parameterized by the EVSP have significant biological effects on endothelial cell properties was studied by measuring membrane potential and nitric oxide production under ELF stimulation between 0 and 2 Hz and 0–6.67 V/m. Using membrane potential and nitric oxide sensitive fluorescent dyes, bovine aortic endothelial cells (BAECs) in culture were studied in the presence and absence of EVSP‐modeled electric fields. The transmembrane potential of BAECs was shown to depolarize between 1 and 7 mV with a strong dependency on both the magnitude and frequency of the isolated ELF field. The findings also support a field interaction with a frequency‐dependent tuning curve. The ELF field complexly modulates the nitric oxide response to adenosine triphosphate stimulation with potentiation seen with up to a sevenfold increase. This potentiation was also frequency and magnitude dependent. An early logarithmic phase of NO production is enhanced in a field strength‐dependent manner, but the ELF field does not modify a later exponential phase. This study shows that using electric fields on the order of those generated by blood flow influences the essential biology of endothelial cells. The inclusion of ELF electric fields in the paradigm of vascular biology may create novel opportunities for advancing both the understanding and therapies for treatment of vascular diseases. Bioelectromagnetics 34:22–30, 2013. © 2012 Wiley Periodicals, Inc.     

Relationship of 60-Hz electric-field parameters to the inhibition of growth ofPisum sativum roots

Summary Roots ofPisum sativum were chronically exposed to 60-Hz electric fields ranging from 215–430 V/m and current densities from 1.5–2.7 mA/cm². Adjustments of the exposure medium's conductivity (0.035–0.14 mho/m) allowed for distinction between a field or a current induced perturbation in root growth rates. Inhibition of root growth rates was directly related to the magnitude of the applied field and not the current density. The results are consistent with the postulate that the applied field acts upon the cell membrane and perturbs its function.This paper is based on work performed under Contract No. DE-AC02-76EV03490 with the US Department of Energy at The University of Rochester Department of Radiation Biology and Biophysics and has been assigned Report No. UR-3490-1908. The excellent technical assistance of Peter Economou is gratefully acknowledged: his undergraduate summer student stipend was supported by Rochester Gas and Electric Corporation, Rochester, New York     

Comparison of the coupling of grounded humans,swine and rats to vertical, 60-Hz electric fields

Published and new data for grounded humans, swine, and rats exposed to vertical, 60-Hz electric fields are used to determine field strengths at the surfaces of the bodies and average components of induced-current density along the axes of the bodies. At the tops of the bodies, surface electric fields are increased (enhanced) over the unperturbed field strength present before the subjects entered the field by factors of 17,7, and 4 for humans, swine, and rats, respectively. For an unperturbed field strength of 10 kV/m, average induced axial current densities in the neck, chest, abdomen, and feet are: 550, 190, 250, and 2000 nA/cm², respectively, for humans; 40, 13, 20, and 1100 nA/cm², respectively, for swine; and 28, 16, 2, and 1400 nA/cm², respectively, for rats. These data are used to show that the actual electric fields experienced by animals depend strongly on the shape of the body and its orientation relative to the electric field and ground plane. This fact must be taken into account if biological data obtained with laboratory animals are to be used for the assessment of possible hazards to humans exposed to 60-Hz electric fields.     

Effect of beta-aminopropionitrile and ascorbate on fibroblast migration

Ascorbate and beta-aminopropionitrile (BAPN) have direct, but diverse affects on collagen matrix production. Ascorbate is necessary for the intracellular hydroxylation of prolyl and lysyl residues during collagen biosynthesis whereas BAPN inhibits the enzyme lysyl oxidase in the extracellular space thus preventing collagen crosslink formation. To study the influence of these two agents on fibroplasia, an in vitro model was used to analyze fibroblast migration, proliferation, and collagen synthesis. Biopsies of chicken tendon were covered with a fibrin clot to simulate an in vivo wound environment, and then they were exposed to either ascorbate or BAPN for up to 7 days. Fibroblast migration into the fibrin clot was measured using a Zeiss Mopp II planimeter, DNA synthesis by 125IUDR incorporation, and collagen synthesis by [3H]proline incorporation into collagenase-digestible protein. Tendon biopsies treated daily with fresh ascorbate (0.1 mM) had significantly greater fibroblast migration than controls without ascorbate (P less than 0.05). Cellular proliferation, collagen synthesis, and total protein synthesis were not significantly altered by ascorbate treatment. In contrast, BAPN inhibited fibroblast migration in a dose-dependent fashion without inhibiting proliferation (0.25 and 0.5 mM), collagen, and noncollagen protein synthesis. Therefore, the effect of BAPN on migration does not appear to be due to generalized cytotoxicity. These combined studies suggest that compounds such as ascorbate and BAPN which can modify collagen may also modify fibroblast migration.     

Modulation of collagen production following bleomycin-induced pulmonary fibrosis in hamsters. Presence of a factor in lung that increases fibroblast prostaglandin E2 and cAMP and suppresses fibroblast proliferation and collagen production

To elucidate mechanisms involved in the regulation of lung collagen content we studied hamsters with bleomycin-induced pulmonary fibrosis. Lung collagen in this model is increased as the result of greatly increased lung collagen synthesis rates. However, collagen synthesis rates are subsequently restored to normal. Hamster lung explants from both normal and bleomycin-exposed hamsters were cultured, and the effects of explant conditioned medium (CM) on lung fibroblast (IMR-90) proliferation and collagen production in vitro were determined. Lung explant CM increased fibroblast prostaglandin (PG)E2 production and intracellular cAMP, and decreased both fibroblast proliferation and collagen production in a dose-dependent manner. Greater activity was observed with lung explant CM from bleomycin-exposed lungs. Incubation of fibroblasts with indomethacin prior to addition of CM blocked CM-mediated changes in PGE2 and cAMP and inhibited changes in fibroblast proliferation and collagen production. Exogenous PGE2 or dibutyryl cAMP also suppressed fibroblast proliferation and collagen production. The suppressive activity in lung-conditioned medium is nondialyzable, has an apparent molecular weight of 15,000-20,000 by gel filtration, and is heat-stable. It is not species-restricted since CM from hamster lung affected human and hamster lung fibroblasts similarly. Activity is present preformed in lung and bronchoalveolar lavage fluid, although bronchoalveolar macrophages produce a nondialyzable factor in culture which suppresses fibroblast proliferation. The suppressive activity identified in fibrotic lung may represent a means for limiting collagen accumulation following tumor injury.     

Effects of Electromagnetic Fields on the Bioactivities of an Osteoblast-Like Cell Line (UMR-106)

In the present study, we first investigated the effects of various types of low-energy, low-frequency electromagnetic fields (EMFs) on DNA synthesis in UMR-106 osteoblast-like cells. The experimental groups were exposed to EMFs for 2 days (twice/day, 30 min/time), and DNA synthesis was measured. The results showed that the cells responded most sensitively to EMFs of some specific combinations of the parameters by an increase in DNA synthesis, implying that EMFs with a specific waveform rather than a complex one can be used in clinical electrotherapy. The parameters were as follows: pulsed electric field (PEF) with pulse width 0.2 ms, field strength 10 V/cm, frequency 125 Hz; sinusoidal electric field (SEF) with field strength 1 V/cm, frequency 10 Hz; and alternating magnetic field (AMF) with field density 0.5 mT, frequency 5 Hz. In addition to frequency, the field strength or field density within a suitable intensity scale played a dominant role in causing the DNA synthesis response. We then compared the effects of two kinds of fields, PEF and AMF, with the optimum parameters identified by the experiments, on alkaline phosphatase (ALP) activity, protein and collagen synthesis, and intracellular levels of cyclic adenosine monophosphate (cAMP). The results indicated that both fields could not only affect UMR-106 cells proliferation but could particularly affect a series of characteristic bioactivities of UMR-106 such as ALP activity and collagen synthesis. The intracellular cAMP levels were increased rapidly and greatly with exposure to both PEF and AMF, implying that the action of low-frequency EMFs proceeds via second messenger-dependent processes originating from signals at the cell membrane. The difference in action between PEF and AMF suggests that they may couple to the cell membrane in a partially different way.