Analysis of the structure of magnetic fields that induced inhibition of stimulated neurite outgrowth.

The important experiments showing nonlinear amplitude dependences of the neurite outgrowth in pheochromocytoma nerve cells due to ELF magnetic field exposure had been carried out in a nonuniform ac magnetic field. The nonuniformity entailed larger than expected variances in magnetic field magnitudes associated with specific levels of biological effects, thereby evoking a question about validity of the interpretations formulated for the case of a uniform field. In this work, we calculate the relative value of nonuniformity and deviations in ac magnetic field. It is shown that these factors do not affect the main conclusion in the original papers about the form of the amplitude dependence of the observed biological effect.

Physiological levels of melatonin enhance gap junction communication in primary cultures of mouse hepatocytes.

Gap junction communication is known to be involved in controlling cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. Melatonin, a hormone secreted by the pineal gland, has putative oncostatic properties. Intercellular communication through gap junctions was assessed by microinjecting Lucifer yellow fluorescent dye into primary hepatocytes and visualizing the spread of the dye to adjacent neighboring cells using phase contrast/fluorescent microscopy. Treatment of primary hepatocyte cultures with a physiological range of melatonin concentrations for 24 h prior to microinjection resulted in significant enhancement in intercellular communication at 0.2 and 0.4 nmol/L but not at lower (0.1 nmol/L) or higher (0.8 or 1.0 nmol/L) concentrations. A time-dependent study showed that the changes in intercellular communication began 10 h after melatonin treatment and reached a maximum at 12 h of treatment. This nonlinear, functional gap junction response to melatonin occurred in the physiological concentration range detected in blood of mammals during nightly releases of the hormone by the pineal gland. These melatonin levels may affect the ability of gap junction communication to exert cell growth control in vivo. The uneven decline between individuals in nocturnal release of melatonin that occurs with age could identify potentially sensitive subpopulations susceptible to developing pathologies involving alterations in biological processes dependent on gap junction communication.

The influence of 1.2 microT, 60 Hz magnetic fields on melatonin- and tamoxifen-induced inhibition of MCF-7 cell growth.

We independently examined the findings of Harland and Liburdy, who reported that 1.2 microT(rms), 60 Hz magnetic fields could significantly reduce the inhibitory action of physiological levels of melatonin (10(-9) M) and of pharmacological levels of tamoxifen (10(-7) M) on the growth of MCF-7 human breast cancer cells in vitro. We used two testing protocols. In the melatonin study, the cell numbers per dish on day 7 of treatment were determined using a hemocytometer assay. In the tamoxifen study we used an expanded protocol, employing an alternative cell counting assay to characterize the cell numbers per dish on days 4, 5, 6, and 7. In both the melatonin and tamoxifen studies, cells were plated on 35 mm dishes and placed in each of two exposure chambers inside 5% CO(2) incubators. One exposure chamber was energized to produce 1.2 microT(rms), 60 Hz magnetic fields and the other chamber was not energized. Treatment was continuous until assays were performed. Cells were harvested at selected times, and enumerated without knowledge of treatment. In the melatonin study, the experiment was repeated three times, whereas in the tamoxifen study, each experiment was repeated nine times. In the melatonin study, cell numbers per dish were significantly reduced (by 16.7%) in the melatonin treated cultures after 7 days of incubation compared to control cultures, whereas in the presence of 1.2 microT(rms), 60 Hz magnetic fields, the melatonin treated cultures had the same cell populations as the control cultures. In the tamoxifen study, tamoxifen reduced the cell growth by 18.6 and 25% on days 6 and 7, respectively, in the chamber not energized, while in 1.2 microT(rms), 60 Hz fields, tamoxifen reduced the cell growth only by 8.7 and 13.1%, respectively. These results are consistent with those reported by Harland and Liburdy. A critical element of this successful replication effort was the constructive communication established and maintained with the original investigators. Bioelectromagnetics 22:122-128, 2001. Published 2001 Wiley-Liss, Inc.

Experimental determination of hydrogen bandwidth for the ion parametric resonance model.

The ion parametric resonance (IPR) model predicts that distinct patterns of field-induced biological responses will occur at particular magnetic field combinations which establish ion resonances. An important characteristic of resonance is the bandwidth response of the system, in part because it determines the required tolerances of the test system. Initial development of the IPR model used literature data to estimate the bandwidth for any ion resonance to be -/+10% of its exact resonance. Because the charge-to-mass ratio of hydrogen is much larger than any other biologically significant ion, hydrogen resonance provides a unique test case by which a single ionic bandwidth can be clearly measured. Of particular relevance is work by Trillo et al. that demonstrated a hydrogen-only, resonance-based IPR response of neurite outgrowth in PC-12 cells. The work reported here considers the response of nerve-growth-factor-stimulated PC-12 cells exposed to magnetic fields tuned at or near hydrogen resonance. This work was designed to test directly the IPR model hypothesis of a -/+10% ionic bandwidth. Consistent with the work of Trillo et al., resonance conditions were established using a 2.97 microT static magnetic field, and the AC frequency and field strength were varied to prove different aspects of the resonance. With this static field 45 Hz was the resonance frequency identified for hydrogen, 42.5 and 47.5 Hz were near-resonance frequencies, and 40 and 50 Hz bounded the assumed -/+10% hydrogen resonance bandwidth. We repeated each test three times. The cell responses at 45 Hz exposures agreed with the IPR model predictions and replicated results obtained by Trillo et al. Cells exposed to 42.5 and 47.5 Hz (near resonance) magnetic fields responded in the same general pattern as those exposed to 45 Hz fields, but neurite outgrowth was less than that observed at resonance. Measured results for cells exposed to either 40 Hz or 50 Hz fields were indistinguishable from off-resonance responses, consistent with the hypothesized bandwidth. These results confirm that the response bandwidth for the hydrogen ion is no greater than -/+10%, and give further support to the resonance-based predictions of the IPR model.

Double blind test of magnetic field effects on neurite outgrowth.

Previous work reported that nerve growth factor-stimulated neurite outgrowth in PC-12 cells could be altered by exposure to parallel alternating current (AC) and direct current (DC) magnetic fields under a variety of exposure conditions, producing results that are consistent with the predictions of the ion parametric resonance (IPR) model. The credibility of these results, considered extraordinary by some scientists, could be strengthened if the cell response were found to persist under alternate assay conditions. We replaced part of our standard assay procedure with a double blind procedure. This new procedure obscured 1) whether a particular set of dishes of cells was exposed or not, and 2) which individual dish was in which exposure system. The goal was to determine whether the previously observed responses of PC-12 cells to magnetic fields would be sufficiently robust to decode the imposed blinding, thereby removing any question of experimenter bias in reported results. We placed three coded dishes of cells in each of two otherwise identical exposure systems, one not energized and one energized to produce exposure conditions predicted to maximally suppress neurite outgrowth (Bdc of 36.6 microT, parallel 45 Hz AC of 23.8 microT rms). Each of the six dishes were recoded before assay to further obscure the exposure identity of any individual dish. The combined results of four distinct runs of these double blind experiments unequivocally demonstrated that 1) there was a clear, distinctive, repeatable consistency with the actual energization of the exposure systems and location of each dish, and with the predictions of the IPR model; 2) only the explicitly stated experimental variables influenced the experiment; and 3) the reported response of the cells was very improbably due to chance (P = .000024).

Effect of perchloroethylene and its metabolites on intercellular communication in clone 9 rat liver cells.

Gap junction intercellular communication (IC) is thought to be important in chemical carcinogenesis as abnormalities in IC have been found in cancer cells. Perchloroethylene (PERC) is metabolized in rodent liver to dichloroacetic acid (DCA) and trichloroacetic acid (TCA), which are rodent liver carcinogens. Chloral hydrate (CH) and trichloroethanol (TCEth) are kidney metabolites. We used Lucifer yellow scrape-load dye transfer as a measure of IC to look at the effect of PERC, DCA, TCA, CH, and TCEth on Clone 9 cell cultures (normal rat liver cells). Four independent experiments were performed for each chemical using exposure times of 1, 4, 6, 24, 48, and 168 h. Concentrations for each chemical varied and were based on preliminary data on effect and cytotoxicity. To compare the relative effectiveness of each chemical to cause biological change, we identified the lowest concentration and shortest time to significantly reduce dye transfer. DCA caused a significant change at 10 mM at 6 h; TCA, 1 mM at 1 h; CH and TCEth, 1 mM at 24 h; and PERC, 0.01 mM at 48 h. Over a 24-h treatment period, the relative efficiencies, as defined by the concentration needed to produce 50% reduction in IC, were PERC (0.3 mM) >> TCA (3.8 mM) > TCEth (6.6 mM) = CH (7.0 mM) >> DCA (41 mM). Time-course data indicated that PERC, DCA, and TCA produced reduction in IC in a similar fashion, but 5 mM CH or TCEth exhibited variances from these results and may indicate specific cell responses to these chemicals. The mechanism(s) responsible for inhibition of IC by these structurally related chemicals needs to be established.

Effect of ac and dc magnetic field orientation on nerve cells.

Recent tests of the influence of parallel ac and dc magnetic fields on neurite outgrowth in PC-12 cells showed good agreement with the predictions of an ion parametric resonance model. However, experimental results from earlier work involving both a perpendicular (160 mG) and a parallel (366 mG) dc magnetic field were not as consistent with the ion parametric resonance model predictions. Test results reported here show that the cell response to perpendicular ac and de magnetic fields is distinct and predictably different from that found for parallel ac and dc magnetic fields, and that the response to perpendicular fields is dominant in an intensity-dependent nonlinear manner.

Magnetic fields at resonant conditions for the hydrogen ion affect neurite outgrowth in PC-12 cells: a test of the ion parametric resonance model.

PC-12 cells primed with nerve growth factor (NGF) were exposed to sinusoidal extremely-low-frequency (ELF) magnetic fields (MFs) selected to test the predictions of the ion parametric resonance (IPR) model under resonance conditions for a single ion (hydrogen). We examined the field effects on the neurite outgrowth (NO) induced by NGF using three different combinations of flux densities of the parallel components of the AC MF (Bac) and the static MF (Bdc). The first test examined the NO response in cells exposed to 45 Hz at a Bdc of 2.96 microT with resonant conditions for H+ according to the model. The Bac values ranged from 0.29 to 4.11 microT root-mean-square (rms). In the second test, the MF effects at off-resonance conditions (i.e., no biologically significant ion at resonance) were examined using the frequency of 45 Hz with a Bdc of 1.97 microT and covering a Bac range between 0.79 and 2.05 microT rms. In the third test, the AC frequency was changed to 30 Hz with the subsequent change in Bdc to 1.97 microT to tune for H+ as in the first test. The Bac values ranged from 0.79 to 2.05 microT rms. After a 23 h incubation and exposure to the MF in the presence of NGF (5 ng/ml), the NO was analyzed using a stereoscopic microscope. The results showed that the NGF stimulation of neurite outgrowth (NSNO) was affected by MF combinations over most of the Bac exposure range generally consistent with the predictions of the IPR model. However, for a distinct range of Bac where the IPR model predicted maximal ionic influence, the observed pattern of NSNO contrasted sharply with those predictions. The symmetry of this response suggests that values of Bac within this distinct range may trigger alternate or additional cellular mechanisms that lead to an apparent lack of response to the MF stimulus.

Nonbinomial distribution of relative neurite outgrowth in PC-12 cells.

Previously we reported the results of a series of experimental tests using PC-12 cells to examine the biological effect of prescribed combinations of both nerve growth factor and magnetic fields. Because our assay of the PC-12 cells is based on binary classification of the cells following treatment, our data might be expected to have a binomial distribution. However, our data consistently show a smaller variability than that predicted by the binomial distribution model. In this paper, we examine some possible reasons for this reduction in variability in our results.

A 50 Hz magnetic field blocks melatonin-induced enhancement of junctional transfer in normal C3H/10T1/2 cells.

There is strong evidence that pineal melatonin is involved in controlling neoplastic processes. We have reported that physiological, but not pharmacological or sub physiological, concentrations of melatonin enhance intercellular communication in normal C3H/10T/2 fibroblasts. Gap junctional intercellular communication intervenes in the control of cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. A number of in vivo studies have shown that extremely low frequency (ELF) magnetic fields (MF) can act as cancer promoters or co-promoters. In vitro, 60 Hz MF have been reported to block melatonin-induced inhibition of cell proliferation in human breast cancer cells. The mechanisms responsible for the observed interactions of MF at the cellular level remain unknown. In the present study melatonin was added to confluent fibroblasts at a concentration of 10(-10) M. Twenty-seven hours later, a fluorescent dye was scrape-loaded into groups of cells and the transfer of the dye to adjacent cells through gap junctions was quantified. Under these conditions melatonin induced a significant increase of dye transfer; this increase was not observed when the cultures were exposed to the MF for 30 min before the previously reported results suggesting that the in vivo oncostatic action of melatonin could be exerted, in part, through modulation of the levels of gap junctional intercellular communication. Also, the data indicate that ELF-MF could counteract the melatonin-induced enhancement of junctional transfer.

Melatonin enhances junctional transfer in normal C3H/10T1/2 cells.

Gap junctional intercellular communication is known to be involved in controlling cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. The pineal gland and its hormone, melatonin, are believed to intervene in the control of neoplastic processes. Several possible mechanisms have been suggested to be potentially responsible for melatonin's oncostatic action; however, the actual mechanisms involved in melatonin's effects at the cellular level remain unidentified. In the present study low-density cultures of C3H/10T1/2 mouse embryo fibroblasts were incubated until relatively quiescent monolayers were established (17-18 days). Gap junctional intercellular communication in control samples and in cells treated with 10(-12) to 10(-8) M melatonin was determined by the scrape-loading assay using the fluorescent dye Lucifer yellow. The results showed that concentrations of melatonin considered physiological (10(-11) and 10(-10) M) induced a significant increase in the transfer of the dye to adjacent cells through gap junctions; both higher and lower concentrations were ineffective. These results suggest that melatonin could exert its putative oncostatic action, in part, by modulating the levels of gap junctional intercellular communication.

The ion parametric resonance model predicts magnetic field parameters that affect nerve cells.

An ion parametric resonance (IPR) model recently developed by Blanchard and Blackman predicts distinct magnetic field interactions with biological systems based on a selective relation among four factors: the flux density of the static magnetic field, the frequency and flux density (Bac) of the parallel ac magnetic field, and the charge-to-mass ratio of ions of biological relevance. To test this model, PC-12 cells stimulated by nerve growth factor to produce neurites were exposed for 23 h in a 5% CO2 incubator using a multiple-coil exposure system to produce 45 Hz ac and dc (366 mG parallel to ac; less than 2 mG perpendicular to ac) magnetic fields. Our earlier work showed a cycle of inhibition/no inhibition of neurite outgrowth consistent with the IPR model predictions for Bac exposures between 0 and 468 mG rms. The work described here tests neurite outgrowth over a broader range of Bac (233-1416 mG rms). The experimental results remain consistent with earlier results, and with IPR model predictions of a second cycle of inhibition, return to control values, followed by a third cycle of inhibition of neurite outgrowth. These responses support the fundamental relationships predicted by the IPR model. The results have broad significance for biology.

Evaluation of whole-animal data using the ion parametric resonance model.

Changes observed in the behavioral response of land snails from exposure to parallel ac and dc magnetic fields demonstrate limited agreement with the predictions of an interaction model proposed by Lednev and the predictions of a recently proposed ion parametric resonance (IPR) model. However, the inadequate number of reported data points, particularly in a critical exposure range, prevents unambiguous application of either the Lednev or the IPR model.

Frequency-dependent interference by magnetic fields of nerve growth factor-induced neurite outgrowth in PC-12 cells.

We have shown that 50 Hz sinusoidal magnetic fields within the 5-10 microTesla (microT) rms range cause an intensity-dependent reduction in nerve growth factor (NGF) stimulation of neurite outgrowth (NO) in PC-12 cells. Here we report on the frequency dependence of this response over the 15-70 Hz range at 5 Hz intervals. Primed PC-12 cells were plated in collagen-coated, 60 mm plastic petri dishes with or without 5 ng/ml NGF and were exposed to sinusoidal magnetic fields for 22 h in a CO2 incubator at 37 degrees C. One 1,000-turn coil, 20 cm in diameter, generated vertically oriented magnetic fields. The dishes were stacked on the center axis of the coil to provide a range of intensities between 3.5 and 9.0 microT rms. The flux density of the ambient DC magnetic field was 37 microT vertical and 19 microT horizontal. The assay consisted of counting over 100 cells in the central portion (radius < or = 0.3 cm) of each dish and scoring cells positive for NO. Sham exposure of cells treated identically with NGF demonstrated no difference in the percentage of cells with NO between exposed and magnetically shielded locations within the incubator. Analysis of variance demonstrated flux density-dependent reductions in NGF-stimulated NO over the 35-70 Hz frequency range, whereas frequencies between 15 Hz and 30 Hz produced no obvious reduction. The results also demonstrated a relative maximal sensitivity of cells at 40 Hz with a possible additional sensitivity region at or above 70 Hz. These findings suggest a biological influence of perpendicular AC/DC magnetic fields different from those identified by the ion parametric resonance model, which uses strictly parallel AC/DC fields.

Clarification and application of an ion parametric resonance model for magnetic field interactions with biological systems.

Theoretical models proposed to date have been unable to clearly predict biological results from exposure to low-intensity electric and magnetic fields (EMF). Recently a predictive ionic resonance model was proposed by Lednev, based on an earlier atomic spectroscopy theory described by Podgoretskii and Podgoretskii and Khrustalev. The ion parametric resonance (IPR) model developed in this paper corrects mathematical errors in the earlier Lednev model and extends that model to give explicit predictions of biological responses to parallel AC and DC magnetic fields caused by field-induced changes in combinations of ions within the biological system. Distinct response forms predicted by the IPR model depend explicitly on the experimentally controlled variables: magnetic flux densities of the AC and DC magnetic fields (Bac and Bdc, respectively); AC frequency (fac); and, implicitly, charge to mass ratio of target-ions. After clarifying the IPR model and extending it to combinations of different resonant ions, this paper proposes a basic set of experiments to test the IPR model directly which do not rely on the choice of a particular specimen or endpoint. While the fundamental bases of the model are supported by a variety of other studies, the IPR model is necessarily heuristic when applied to biological systems, because it is based on the premise that the magnitude and form of magnetic field interactions with unhydrated resonant ions in critical biological structures alter ion-associated biological activities that may in turn be correlated with observable effects in living systems.

Empirical test of an ion parametric resonance model for magnetic field interactions with PC-12 cells.

A companion paper describes a predictive ion parametric resonance (IPR) model of magnetic field interactions with biological systems based on a selective relation between the ratio of the flux density of the static magnetic field to the AC magnetic field and the charge-to-mass ratio of ions of biological relevance. Previous studies demonstrated that nerve growth factor (NGF)-stimulated neurite outgrowth (NO) in PC-12 cells can be inhibited by exposure to magnetic fields as a function of either magnetic field flux density or AC magnetic field frequency. The present work examines whether the PC-12 cell response to magnetic fields is consistent with the quasi-periodic, resonance-based predictions of the IPR model. We tested changes in each of the experimentally controllable variables [flux densities of the parallel components of the AC magnetic field (Bac) and the static magnetic field (Bdc) and the frequency of the AC magnetic field] over a range of exposure conditions sufficient to determine whether the IPR model is applicable. A multiple-coil exposure system independently controlled each of these critical quantities. The perpendicular static magnetic field was controlled to less than 2 mG for all tests. The first set of tests examined the NO response in cells exposed to 45 Hz Bac from 77 to 468 mG(rms) at a Bdc of 366 mG. Next, we examined an off-resonance condition using 20 mG Bdc with a 45 Hz AC field across a range of Bac between 7.9 and 21 mG(rms). Finally, we changed the AC frequency to 25 Hz, with a corresponding change in Bdc to 203 mG (to tune for the same set of ions as in the first test) and a Bac range from 78 to 181 mG(rms). In all cases the observed responses were consistent with predictions of the IPR model. These experimental results are the first to support in detail the validity of the fundamental relationships embodied in the IPR model.

Frequency-dependent alterations in enolase activity in Escherichia coli caused by exposure to electric and magnetic fields.

Some neurochemical effects of low-intensity electric and magnetic fields have been shown to be nonlinear functions of exposure parameters. These effects occurred within narrow ranges of frequency and intensity. Previous studies on membrane-associated endpoints in cell culture preparations demonstrated changes in calcium efflux and in acetylcholinesterase activity following exposure to radiofrequency radiation, amplitude modulated (AM) at 16 and at 60 Hz, at a specific absorption rate of 0.05 W/kg. In this study, these modulation frequencies were tested for their influence on the activity of a cytoplasmic enzyme, enolase, which is being tested clinically for detection of neoplasia. Escherichia coli cultures containing a plasmid with a mammalian gene for enolase were exposed for 30 min, and cell extracts were assayed for enolase activity by measuring absorbance at 240 nm. The enolase activity in exposed cultures was compared to the activity in paired control cultures. Exposure to 147 MHz carrier waves at 0.05 W/kg, AM at 16 Hz showed enolase activity enhanced by 62%, and AM at 60 Hz showed enolase activity reduced by 28%. Similarly, exposure to 16 Hz fields alone, at 21.2 V/mrms (electric) and 97 nTrms (magnetic), showed enhancement in enolase activity by 59%, whereas exposure to 60 Hz fields alone, at 14.1 V/mrms (electric) and 65 nTrms (magnetic), showed reduction in activity by 24%. Sham exposures as well as exposure to continuous-wave 147 MHz radiation at 0.05 W/kg showed no change in enolase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for direct effect of magnetic fields on neurite outgrowth.

Electric fields can cause changes in cell responses both in vitro and in vivo. Alternating magnetic fields have been proposed to act through the electric fields induced in the conducting medium surrounding the cells. We have used a simple exposure system to test the relative contribution of magnetic fields compared to induced electric fields in a standard PC-12 cell culture assay, in which cells respond to nerve growth factor by producing neurites. This response to stimulation by nerve growth factor is inhibited by sinusoidal, 50-Hz magnetic fields at field strengths below 10 microT (100 mG). A standard procedure to distinguish magnetic- vs. electric-field effects demonstrates that the induced electric field is not involved. Additional work is necessary to identify the critical reaction site (or sites), and to establish the molecular mechanisms responsible for these results.

A scheme for incorporating DC magnetic fields into epidemiological studies of EMF exposure.

Experimental data on calcium-ion release in chicken brain tissue suggest that biological effects of electric and magnetic fields (EMFs) are concentrated near certain "active combinations" of DC magnetic field strength and "effective" AC magnetic field frequencies. We hypothesize that active AC/DC combinations may exist and suggest that epidemiologic data, coupled with DC magnetic field measurement, may be used to identify critical exposure conditions. An empirical model is used to calculate these multiple active combinations at any given DC magnetic field strength and to define a rating system that incorporates the proximity of AC magnetic field frequencies generated by electric power lines to the new, computed effective frequencies. Such an exposure score may be useful in investigating correlations of EMF exposure with disease incidence. For 60 Hz and 50 Hz, the highest EMF exposure scores occurred at DC field strengths of 506 mG and 422 mG, respectively. The exposure score contains a factor which may be adjusted to reflect the importance of harmonics of the AC magnetic field as well as of the fundamental frequency. Using this factor, we consider two important special cases consistent with chick brain data: 1) we consider active pairs associated with all detectable harmonics (up to 660 Hz) without regard to relative intensity of the harmonics, and 2) we use the relative intensities of the AC field frequencies to adjust their contribution to the exposure score.