Does direction of induced electric field or current provide a test of mechanism involved in nerve regeneration?

We suggest an experimental comparison of two directions for applying the time-varying magnetic fields which have been found to speed spontaneous regeneration of injured peripheral nerves and in attempts to repair spinal cord injuries. Time-varying magnetic fields induce currents in a plane perpendicular to the magnetic field direction. The lower conductivity of the spinal cord's sheath (dura matter) or of the myelin sheath of peripheral nerves would seem to confine the induced electric fields and currents to the spinal cord or nerve itself. The proposed comparison could allow choosing between two possible modes of action of the fields: (1) Magnetically-induced electric fields or currents may be encouraging ion flow or otherwise helping enzyme, channel or other interactions at the cell membrane, as is thought to be the case in field stimulation of healing in bone. This mechanism should be independent of field direction. (2) Work in developing organisms and with fields applied to nerve cells in vitro has shown that neurite growth is guided parallel to both endogenous and external electric fields. This mechanism would be effective when induced electric fields are parallel, but not when they are perpendicular to the nerve. Any experimental test should seek to produce as close as possible to the same induced current intensity with both field directions. Possible confounding factors, as well as breakdowns in the assumptions of the simple model presented here, would have to be considered. This proposal was motivated by a recent report in which the authors listed a changed field direction as one of several possible reasons for an unsuccessful experiment.

Interaction of static and extremely low frequency electric and magnetic fields with living systems: health effects and research needs.

An international seminar was held June 4-6, 1997, on the biological effects and related health hazards of ambient or environmental static and extremely low frequency (ELF) electric and magnetic fields (0-300 Hz). It was cosponsored by the World Health Organization (WHO), the International Commission on Non-Ionizing Radiation Protection (ICNIRP), the German, Japanese, and Swiss governments. Speakers provided overviews of the scientific literature that were discussed by participants of the meeting. Subsequently, expert working groups formulated this report, which evaluates possible health effects from exposure to static and ELF electric and magnetic fields and identifies gaps in knowledge requiring more research to improve health risk assessments. The working groups concluded that, although health hazards exist from exposure to ELF fields at high field strengths, the literature does not establish that health hazards are associated with exposure to low-level fields, including environmental levels. Similarly, exposure to static electric fields at levels currently found in the living and working environment or acute exposure to static magnetic fields at flux densities below 2 T, were not found to have demonstrated adverse health consequences. However, reports of biological effects from low-level ELF-field exposure and chronic exposure to static magnetic fields were identified that need replication and further study for WHO to assess any possible health consequences. Ambient static electric fields have not been reported to cause any direct adverse health effects, and so no further research in this area was deemed necessary.

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

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

Magnetic field exposure enhances mRNA expression of sigma 32 in E. coli.

The mechanism of interaction between weak electromagnetic fields and cells is not understood. As a result, the health effect(s) induced by exposure to these fields remains unclear. In addition to questions relating to the site of initial magnetic field (MF) interactions, the nature of the cell's response to these perturbations is also unclear. We examined the hypothesis that the cells respond to MFs in a manner similar to other environmental stressors such as heat. Using the bacterium Escherichia coli, we examined the mRNA levels of sigma 32, a protein that interacts with RNA polymerase to help it recognize a variety of stress promoters in the cell. Our data show that the intracellular level of sigma 32 mRNA is enhanced following a 15-min exposure to a 60 Hz, 1.1 mT magnetic field.

Effects of pulsed magnetic fields on neurite outgrowth from chick embryo dorsal root ganglia.

We have previously shown that neurite outgrowth from 6-day chick embryo dorsal root ganglia (DRG) in vitro was stimulated when nerve growth factor (NGF) and pulsed magnetic fields (PMF) are used in combination. 392 DRGs were studied in a field excited by a commercial PMF generator. We have now analyzed an additional 416 DRGs exposed to very similar PMF's produced by an arbitrary wavefrom generator and power amplifier. We reproduced our previous findings that combination of NGF and bursts of asymmetric, 220 microsecond-wide, 4.0 mT-peak pulses induced significantly (p < 0.05) greater outgrowth than NGF alone, that fields without NGF do not significantly alter outgrowth, and that, unlike NGF alone, 4.0 mT fields and NGF can induce asymmetric outgrowth. The asymmetry does not seem to have a preferred orientation with respect to the induced electric field. Analysis of the data for the entire 808 DRGs confirms these findings. Importantly, we find similar results for pulse bursts repeated at 15 or 25 Hz.

Effects of electromagnetic fields on molecules and cells.

Evidence suggests that cell processes can be influenced by weak electromagnetic fields (EMFs). EMFs appear to represent a global interference or stress to which a cell can adapt without catastrophic consequences. There may be exceptions to this observation, however, such as the putative role of EMFs as promoters in the presence of a primary tumor initiator. The nature of the response suggests that the cell is viewing EMFs as it would another subtle environmental change. The age and state of the cell can profoundly affect the EMF bioresponse. There is no evidence that direct posttranscription effects occur as a result of EMF exposure. Although transcription alterations occur, no apparent disruption in routine physiological processes such as growth and division is immediately evident. What is usually observed is a transient perturbation followed by an adjustment by the normal homeostatic machinery of the cells. DNA does not appear to be significantly altered by EMF. If EMF exposure is associated with an increased risk of cancer, the paucity of genotoxic effects would support the suggestion that the fields act in tumor promotion rather than initiation. The site(s) and mechanisms of interaction remain to be elaborated. Although there are numerous studies and hypotheses that suggest the membrane represents the primary site of interaction, there are also several different studies showing that in vitro systems, including cell-free systems, are responsive to EMFs. The debate about potential hazards or therapeutic value of weak electromagnetic fields will continue until the mechanism of interaction has been clarified.

Magnetic fields after translation in Escherichia coli.

Quantitative two-dimensional gel electrophoresis of proteins in E. coli exposed for 60 min to weak, pulsed magnetic fields (1.5 mT peak) show that numerous proteins are both increased and decreased by a factor of 2 or more. An increase in the levels of two proteins, the a subunit of DNA-dependent RNA polymerase and NusA, was confirmed by Western blot analysis.

Altered protein synthesis in a cell-free system exposed to a sinusoidal magnetic field.

This report describes a new approach for examining weak extremely low frequency (ELF) electric and magnetic field interactions with living systems that exploits a cell-free transcription/translation system derived from Escherichia coli. Using two-dimensional polyacrylamide gel electrophoresis we previously had determined that the level of the alpha subunit of RNA polymerase in intact E. coli was elevated by exposure to weak ELF magnetic fields. In this paper, plasmids containing the alpha, or both the beta,beta' subunits of the RNA polymerase from E. coli were placed into a cell-free expression system. When this transcription/translation system was exposed to a 72-Hz sinusoidal magnetic field in the range 0.07 to 1.1 mT (rms) for periods of 5 min to 1 h, expression was enhanced. Weaker fields must be applied longer to produce an effect. For 10 min of field exposure, the threshold for an effect is 0.1 mT. These experiments demonstrate that an intact membrane is not an absolute requirement for transducing magnetic bio-effects.

An increase in the negative surface charge of U937 cells exposed to a pulsed magnetic field.

Pulsed magnetic fields have been used to enhance healing of bone fractures and purportedly of lesions in soft tissue. However, their mechanism of action is poorly understood. We report changes in the plasma membrane of a nonadherent mammalian cell line, U937, which was exposed to a 25-pps magnetic field for 48 hours. Aqueous polymer two-phase partition studies showed that magnetic-field-exposed cells exhibited an increased negative surface charge but membrane hydrophobicity was not significantly altered. The observed increase in membrane electronegativity of exposed cells did not reflect a significant change in growth rate.

Low frequency electric and magnetic fields have different effects on the cell surface.

There is a considerable controversy over the nature of weak electromagnetic-field effects in living organisms. Part of the controversy can be traced to a lack of understanding of whether electric or magnetic fields are involved in producing bioeffects. We find that both 60 Hz electric and magnetic fields alter the cell surface of Physarum polycephalum. Exposure to electric fields increases the negative charge on the cell surface while magnetic-field exposure decreases the hydrophobic character of the surface. These effects appear to be additive and independent of the waveform of the applied fields.

Pulsed magnetic fields alter the cell surface.

Pulsed magnetic fields (PMFS) are routinely used in the medical community to facilitate bone repair in clinical cases of non-union or pseudarthoses [(1984) Orth. Clin. No. Am. 15, 61-87]. Although this therapeutic regimen appears to be reasonably effective, the mechanism of action between specific PMFs and the target tissue remains unknown. Adding urgency to the need to understand the mechanism are a wide number of reports that have appeared which demonstrate that PMFs similar to those in clinical use can alter many basic physiological functions. We report that a 24 h exposure to PMFs alters the cell surface of Physarum polycephalum amoebae. Further, using the technique of aqueous two-phase partitioning, we present evidence for individual magnetic and electric field, cell surface effects.

Intracellular recording during magnetic field application to monitor neurotransmitter release events: methods and preliminary results.

A method for simultaneous magnetic field application and intracellular recording is presented. A little used method for magnetic field application was exploited; the field generator consisted of a flat copper sheet through which current was passed to generate a magnetic field. The resultant magnetic field was relatively homogeneous, exhibiting a variation of +/- 5%. This compact, current-sheet field generator was mounted on the condensor of a microscope. The current induced in the intracellular electrode was reduced by injecting current equal and opposite to the induced current into the microelectrode. This step reduces the possibility of cellular effects and voltage artifacts due to the induced electrode current. The technique was used to conduct preliminary studies on the effects of extremely low frequency (ELF) linearly and circularly polarized magnetic fields (1.0 Gauss, 60 and 70 Hz) on miniature end plate potential (mepp) frequency (frequency of neurotransmitter release events) of rat flexor digitorum brevis muscle. The same synapse was utilized for both the sham-exposed control and the exposed experimental halves of an experiment. After 10 min of exposure to a 60-Hz linear field, mepp frequency was significantly increased by 12%, but exposure to a 60-Hz circular field did not significantly alter mepp frequency. Exposure to a 70-Hz linear field did not significantly change mepp frequency, but application of a 70-Hz circular field appears to decrease mepp frequency by 4%. These results indicate that both types of magnetic fields can alter mepp frequency, depending upon the frequency and configuration of the field.

Effects of sinusoidal 60-Hz electric and magnetic fields on ATP and oxygen levels in the slime mold, Physarum polycephalum.

We have previously reported that exposing the vegetative plasmodia stage of Physarum polycephalum to either individual or simultaneously applied electric and magnetic fields (45-75 Hz, 0.14-2.0 G, and 0.035-0.7 V/m) lengthens their mitotic cycle, depresses their rate of reversible shuttle streaming, and lowers their respiration rate. In this article we report the effects of simultaneously applied electromagnetic fields (60 Hz, 1.0 G, 1.0 V/m), electric fields only (60 Hz, 1.0 V/m), magnetic fields only (60 Hz, 1.0 G) on the haploid amoeba of Physarum exposed for 120-180 days. Statistically significant depressions (about 8-11%) in ATP levels were observed with all field conditions; however, respiration was significantly decreased only when amoebae were subjected to either combined fields or electric fields alone. Magnetic fields alone failed to induce a significant decrease in respiration.

Low-voltage ELF electric field measurements in ionic media.

Low-voltage electric fields were measured in conductive tissue culture media using three techniques: voltage slope, current density-conductivity, and dipole methods. All three methods tested yielded comparable results. However, all three techniques have associated errors. These errors fall into three major categories: those associated with the measurement equipment, those associated with electrodes, and errors in cross-sectional area measurements. Each source of error is discussed so that all can be taken into account during construction and/or testing of exposure equipment.

Magnetic field effects on mitotic cycle length in Physarum.

Large plasmodia of Physarum polycephalum were formed from mixtures of micro-plasmodia grown in shaker cultures exposed to 2.0 G (rms), 75 Hz magnetic fields and non-exposed, control cultures. The exposed cultures had been grown continuously in the field and displayed a longer mitotic cycle than the controls. Mixed cultures display synchronous mitosis and a cycle length intermediate to the cycle lengths of exposed and control cultures. The cycle length of mixed cultures varied with the proportions of the mixture in a non-linear manner. The results are discussed in terms of several models.