Mechanical and electromagnetic induction of protection against oxidative stress.

Cells and tissues can be protected against a potentially lethal stress by first exposing them to a brief dose of the same or different stress. This "pre-conditioning" phenomenon has been documented in many models of protection against oxidative stress, including ischemia/reperfusion and ultraviolet (UV) light exposure. Stimuli which induce this protective response include heat, chemicals, brief ischemia, and electromagnetic (EM) field exposures. We report here that constant mechanical vibration pre-conditions chick embryos, protecting them during subsequent stress from hypoxia or UV light exposure. Continuously mechanically vibrated embryos (60 Hz, 1 g (32 ft/s2), 20 min) exhibited nearly double the survival (67.5%, P < 0.001) after subsequent hypoxia as compared to non-vibrated controls (37.6%). As a second set of experiments, embryos were vibrated and then exposed to UV light stress. Those embryos that were vibrated prior to UV had nearly double the survival 3 h after UV exposure (66%, P < 0.001) as compared to controls (35%). The degree of protection, however, was dependent on the constancy of the vibration amplitude. When vibration was turned on and off at 1-s intervals throughout exposure, no increase in hypoxia protection was noted. For 50 s on/off vibration intervals, however, hypoxia protection comparable to continuous vibration was obtained. In contrast, random, inconstant mechanical vibration did not induce protection against subsequent UV exposure. These data suggest that to be an effective pre-conditioning agent, mechanical vibration must have a degree of temporally constancy (on/off intervals of greater than 1 s). Further experiments in both models (hypoxia and UV) indicated an interaction between vibration and EM field-induced protection. Vibration-induced hypoxia protection was inhibited by superposition of a random EM noise field (previously shown to inhibit EM field-induced protection). In addition, EM field-induced UV protection was inhibited by the superposition of random mechanical vibration. Thus, the superposition of either vibrational or EM noise during pre-conditioning virtually eliminated protection against hypoxia and UV. This link between EM field exposures and mechanical vibration is consistent with the hypothesis that cells sense these stimuli via a similar mechanism involving counter ion displacement.

Thresholds for electromagnetic field-induced hypoxia protection: evidence for a primary electric field effect.

We have recently reported that weak electromagnetic (EM) field exposure of chick embryos induces a response that can be used to protect against subsequent hypoxic insult. This work is continued here with an exposure response study using 20-min exposure to 60 Hz magnetic fields over a range of 2-10 microT. Once again, the biomarker used was induction of hypoxia protection. A sigmoidal response curve was found, with exposures to magnetic field strengths > or = 4 microT inducing maximum hypoxia protection (68% survival). We also attempted to determine whether the magnetic or induced electric component of the EM field was responsible for the observed protection. This was accomplished by making measurements with two different orientations of the magnetic fields (perpendicular and parallel to the major axis of the egg). Owing to the configuration of the embryo in the egg, the induced electric field at the embryo was lower when the magnetic field was parallel to the major axis even though the magnetic field strength was the same for each orientation. Exposure of the embryos to the parallel orientation resulted in a reduced protective response. An exposure-response curve generated for this orientation of the field also showed a more "drawn-out" appearance, consistent with the observed distribution of embryo positions within the egg. Our results suggest that the induced electric, not the applied magnetic field, plays a primary role in the protective effect observed in this chick embryo model.

Electromagnetic field-induced protection of chick embryos against hypoxia exhibits characteristics of temporal sensing.

We previously studied the response of mammalian cultured cells to weak, 60 Hz-electromagnetic (EM) fields. Two time constants, similar to those observed in chemotaxis, were found to govern the cellular response to the field. We concluded that a system of temporal sensing, similar to that employed in chemotaxis by motile bacteria, was operative. We termed the shorter time (approximately 0.1 s) the "sensing" time, and the longer time (approximately 10 s) the "memory" time. To investigate the possibility that temporal sensing was a general property of EM field-cell interaction, the temporal properties of another EM field-induced effect was studied. The EM field-induced protection against the effects of extreme hypoxia was examined in chick embryos. Embryos were exposed to 60 Hz-magnetic fields, the amplitudes of which were regularly altered throughout the 20-min exposure. Alteration was accomplished either by turning the field off and on at regular intervals (1-50 s), or by introducing brief (10 or 100 ms), zero amplitude gaps, once each second, throughout exposure. When the field was turned on and off at 0.1 s intervals, the protective effect conferred by a constant field was lost. At progressively longer on/off intervals, protection was progressively restored, maximizing at intervals of 10-30 s. Gapping the magnetic field for 10 ms, each second of exposure conferred the same protection as that observed for an uninterrupted field, but gapping the field at 100 ms each second produced a significant reduction in protection. These data exhibit remarkable consistency with those obtained in similar temporal studies of the magnetic field-induced enhancement of ornithine decarboxylase activity in L929 fibroblasts. It appears that temporal sensing is a general feature of the EM field-cell interaction.

Dose-response of electromagnetic field-enhanced ornithine decarboxylase activity.

Alteration of ODC activity in animals or cultured cells exposed to extremely low frequency electromagnetic fields, or to modulated microwave fields, has been documented by several laboratories. However, an evaluation of the dose-response relationship in these experiments has not been done. We examined ODC activity in L929 fibroblasts exposed for 4 h to 60 Hz magnetic fields of different amplitudes. Our results show a clear threshold response which could be fitted to a sigmoidal function, with the 50% point occurring at approximately 5 microT. This sigmoidal response is characteristic of biological responses which are governed by ligand-receptor binding, and has been previously observed in the incidence of magnetic-field induced morphological abnormalities in chick embryos. The implications of this study are discussed in terms of environmental exposures to EM fields.

Is genetics the unrecognized confounding factor in bioelectromagnetics? Flock-dependence of field-induced anoxia protection in chick embryos.

Work in bioelectromagnetics has long been plagued by problems with replication. This includes experiments done on electromagnetic (EM) field-induced effects in chick embryos. Our laboratory investigated responses of embryos from two flocks of White Leghorn hens. Both flocks were studied simultaneously, and it was found that they responded differently to EM field exposures. Embryos were exposed to 60 Hz, 8 microT EM fields prior to placement in an anoxic chamber. Following re-oxygenation, survival in controls was 34.6%, exposed flock 1 survival was 62% (P < 0.0001) and exposed flock 2 survival was 43% (P < 0.0136). P values are from comparison of data between EM field exposed embryos (flocks 1 and 2) versus controls. In order to induce maximum protection in flock 2, (approximately 62% survival), embryos required a longer exposure time at higher magnetic field strengths. These results reinforce the concepts that genetics are important in determining whether or not chick embryos will respond to EM field stimulation. A broader look at the role of genetic factors emphasizes that these variations in response to external stimuli (e.g., drugs, radiation, and EM fields) are found in all areas of biological research (cell culture, chick, rat, and human studies). The present study suggests that genetics may be a prime cause of the difficulties encountered in replication studies in the field of bioelectromagnetics. We conclude that replication studies should not be undertaken unless care is taken to insure that exactly the same strains of cells or animals are used. Researchers should also first confirm that the responses of their model to non-EM field stimuli are similar to that obtained in the original study.

Myocardial protection conferred by electromagnetic fields.

BACKGROUND: It has been reported that electromagnetic (EM) fields induce stress proteins in vitro. These proteins have been shown to be important in recovery from ischemia/reperfusion. It was, therefore, hypothesized that EM fields could activate stress responses in vivo and protect myocardial tissue during anoxia. METHODS AND RESULTS: Chick embryos were exposed to 4-, 6-, 8-, and 10- microT and 60-Hz EM fields for 20 minutes followed by a 1-hour rest period before placement in an anoxic chamber. Embryos were reoxygenated when survival of controls dropped to <40%, and final observations were made 30 minutes later. Data from 80 experiments (>500 EM field-exposed embryos) indicated that EM field protection was extremely significant (P<0.0001). Survival rates were 39.6% in controls and 68.7% in field-exposed embryos. In a second set of experiments, embryos were exposed for 20 minutes to several pretreatments: (1) hyperthermia (43 degreesC), (2) 60-Hz, 8- microT EM fields, or (3) 60-Hz, 8- microT EM fields plus a random EM noise field (8 microT). Embryo survival was 37.7% (control), 57.6% (heated), 69% (60-Hz EM field only), and 41.5% (60-Hz EM field plus EM noise). To confirm that heating resulting from field exposures did not occur, thermocouples were placed into several eggs at the site of the embryo during exposure; no increase in temperature was noted. CONCLUSIONS: We conclude that athermal EM field exposures induce stress responses that protect chick embryo myocardium from anoxia damage. These results suggest that EM field exposures may be a useful, noninvasive means of minimizing myocardial damage during surgery, transplantation, or heart attack in humans.

Short-term magnetic field exposures (60 Hz) induce protection against ultraviolet radiation damage.

PURPOSE: To investigate the ability of electromagnetic (EM) field pre-exposures to induce protection in chick embryos against subsequent ultraviolet (UV) light exposure. MATERIALS AND METHOD: Chick embryos in the 4th day of gestation were exposed for 20 minutes (short term) or 96 hours (long term) to 60 Hz, 8 microT magnetic or sham fields (controls) followed by 30 minutes rest. They were then exposed to UV radiation of either low (30J/m2) or high (45J/m2) intensity (long term was exposed only to 30J/m2) for 75 minutes. Mortality measurements were made every 30 minutes following UV exposure. RESULTS: At both UV intensities, short-term, EM field-exposed embryos showed significantly higher post-UV survival (p<0.05) at each time point as compared to controls. Long-term EM field exposures, however, offered no protection against low intensity UV light, in fact, 96 hour-EM field-exposed embryos were significantly less protected than non-EM field-exposed controls (p<0.05). CONCLUSIONS: Results of the present study demonstrate that EM field exposures of appropriate duration induce protection against damage from UV light exposure. Because EM field exposures have been reported to activate stress protein response pathways and protect against anoxia/re-oxygenation damage, stress proteins are thought to play a role in the observed UV protection.

A simple experiment to study electromagnetic field effects: protection induced by short-term exposures to 60 Hz magnetic fields.

Stress proteins are important in protection during cardiac ischemia/reperfusion (cessation and return of blood flow) and are reportedly induced by electromagnetic (EM) fields. This suggests a possible ischemia protection role for EM exposures. To test this, chick embryos (96 h) were exposed to 60 Hz magnetic fields prior to being placed into anoxia. Survival was 39.6% (control), and 68.7% (field-exposed). As a positive control, embryos were heated prior to anoxia (57.6% survival). We conclude that: 1) 60 Hz magnetic field exposures reduce anoxia-induced mortality in chick embryos, comparable to reductions observed following heat stress, and 2) this is a simple and rapid experiment to demonstrate the existence of weak EM field effects.

The superposition of a temporally incoherent magnetic field inhibits 60 Hz-induced changes in the ODC activity of developing chick embryos.

Previously, we have shown that the application of a weak (4 microT) 60 Hz magnetic field (MF) can alter the magnitudes of the ornithine decarboxylase (ODC) activity peaks which occur during gastrulation and neurulation of chick embryos. We report here the ODC activity of chick embryos which were exposed to the superposition of a weak noise MF over a 60 Hz MF of equal (rms strength). In contrast to the results we obtain with a 60 Hz field alone, the activity of ODC in embryos exposed to the superposition of the incoherent and 60 Hz fields was indistinguishable from the control activity during both gastrulation and neurulation. This result adds to the body of experimental evidence which demonstrates that the superposition of an incoherent field inhibits the response of biological systems to a coherent MF. The observation that a noise field inhibits ODC activity changes is consistent with our speculation that MF-induced ODC activity changes during early development may be related to MF-induced neural tube defects at slightly later stages (which are also inhibited by the superposition of a noise field).

The role of temporal sensing in bioelectromagnetic effects.

Experiments were conducted to see whether the cellular response to electromagnetic (EM) fields occurs through a detection process involving temporal sensing. L929 cells were exposed to 60 Hz magnetic fields and the enhancement of ornithine decarboxylase (ODC) activity was measured to determine cellular response to the field. In one set of experiments, the field was turned alternately off and on at intervals of 0.1 to 50 s. For these experiments, field coherence was maintained by eliminating the insertion of random time intervals upon switching. Intervals < or = 1 s produced no enhancement of ODC activity, but fields switched at intervals > or 10 s showed ODC activities that were enhanced by a factor of approximately 1.7. These data indicate that it is the interval over which field parameters (e.g., amplitude or frequency) remain constant, rather than the interval over which the field is coherent, that is critical to cellular response to an EMF. In a second set of experiments, designed to determine how long it would take for cells to detect a change in field parameters, the field was interrupted for brief intervals (25-200 ms) once each second throughout exposure. In this situation, the extent of EMF-induced ODC activity depended upon the duration of the interruption. Interruptions > or = 100 ms were detected by the cell as shown by elimination of field-induced enhancement of ODC. That two time constants (0.1 and 10 s) are involved in cellular EMF detection is consistent with the temporal sensing process associated with bacterial chemotaxis. By analogy with bacterial temporal sensing, cells would continuously sample and average an EM field over intervals of about 0.1 s (the "averaging" time), storing the averaged value in memory. The cell would compare the stored value with the current average, and respond to the EM field only when field parameters remain constant over intervals of approximately 10 s (the "memory" time).

Bioeffects induced by exposure to microwaves are mitigated by superposition of ELF noise.

We have previously demonstrated that microwave fields, amplitude modulated (AM) by an extremely low-frequency (ELF) sine wave, can induce a nearly twofold enhancement in the activity of ornithine decarboxylase (ODC) in L929 cells at SAR levels of the order of 2.5 W/kg. Similar, although less pronounced, effects were also observed from exposure to a typical digital cellular phone test signal of the same power level, burst modulated at 50 Hz. We have also shown that ODC enhancement in L929 cells produced by exposure to ELF fields can be inhibited by superposition of ELF noise. In the present study, we explore the possibility that similar inhibition techniques can be used to suppress the microwave response. We concurrently exposed L929 cells to 60 Hz AM microwave fields or a 50 Hz burst-modulated DAMPS (Digital Advanced Mobile Phone System) digital cellular phone field at levels known to produce ODC enhancement, together with band-limited 30-100 Hz ELF noise with root mean square amplitude of up to 10 microT. All exposures were carried out for 8 h, which was previously found to yield the peak microwave response. In both cases, the ODC enhancement was found to decrease exponentially as a function of the noise root mean square amplitude. With 60 Hz AM microwaves, complete inhibition was obtained with noise levels at or above 2 microT. With the DAMPS digital cellular phone signal, complete inhibition occurred with noise levels at or above 5 microT. These results suggest a possible practical means to inhibit biological effects from exposure to both ELF and microwave fields.

The effect of pulsed and sinusoidal magnetic fields on the morphology of developing chick embryos.

Several investigators have reported robust, statistically significant results that indicate that weak (approximately 1 microT) magnetic fields (MFs) increase the rate of morphological abnormalities in chick embryos. However, other investigators have reported that weak MFs do not appear to affect embryo morphology at all. We present the results of experiments conducted over five years in five distinct campaigns spanning several months each. In four of the campaigns, exposure was to a pulsed magnetic field (PMF); and in the final campaign, exposure was to a 60 Hz sinusoidal magnetic field (MF). A total of over 2500 White Leghorn chick embryos were examined. When the results of the campaigns were analyzed separately, a range of responses was observed. Four campaigns (three PMF campaigns and one 60 Hz campaign) exhibited statistically significant increases (P > or = 0.01), ranging from 2-fold to 7-fold, in the abnormality rate in MF-exposed embryos. In the remaining PMF campaign, there was only a slight (roughly 50%), statistically insignificant (P = 0.2) increase in the abnormality rate due to MF exposure. When the morphological abnormality rate of all of the PMF-exposed embryos was compared to that of all of the corresponding control embryos, a statistically significant (P > or = .001) result was obtained, indicating that PMF exposure approximately doubled the abnormality rate. Like-wise, when the abnormality rate of the sinusoid-exposed embryos was compared to the corresponding control embryos, the abnormality rate was increased (approximately tripled). This robust result indicates that weak EMFs can induce morphological abnormalities in developing chick embryos. We have attempted to analyze some of the confounding factors that may have contributed to the lack of response in one of the campaigns. The genetic composition of the breeding stock was altered by the breeder before the start of the nonresponding campaign. We hypothesize that the genetic composition of the breeding stock determines the susceptibility of any given flock to EMF-induced abnormalities and therefore could represent a confounding factor in studies of EMF-induced bioeffects in chick embryos.

Superimposing spatially coherent electromagnetic noise inhibits field-induced abnormalities in developing chick embryos.

Living cells exist in an electrically noisy environment. This has led to the so-called "signal-to-noise" problem whereby cells are observed to respond to extremely-low-frequency (ELF) exogenous fields that are several orders of magnitude weaker than local endogenous fields associated with thermal fluctuations. To resolve this dilemma, we propose that living cells are affected only by electromagnetic fields that are spatially coherent over their surface. The basic idea is that a significant number of receptors must be simultaneously and coherently activated (biological cooperativity) to produce effects on the biochemical functioning of the cell. However, like all physical detection systems, cells are subject to the laws of conventional physics and can be confused by noise. This suggests that a spatially coherent but temporally random noise field superimposed on a coherent ELF signal will defeat the mechanism of discrimination against noise, and any observed field-induced bioeffects would be suppressed. An experimental test of this idea was conducted using morphological abnormalities in developing chick embryos caused by electromagnetic field exposure as the endpoint. At an impressed noise amplitude comparable to the ELF field strength (but roughly one-thousandth of the thermal noise field), the increased abnormality rate observed with only the ELF field present was reduced to a level essentially the same as for the control embryos.

Temporally incoherent magnetic fields mitigate the response of biological systems to temporally coherent magnetic fields.

We have previously demonstrated that a weak, extremely-low-frequency magnetic field must be coherent for some minimum length of time (approximately 10 s) in order to affect the specific activity of ornithine decarboxylase (ODC) in L929 mouse cells. In this study we explore whether or not the superposition of an incoherent (noise) magnetic field can block the bioeffect of a coherent 60 Hz magnetic field, since the sum of the two fields is incoherent. An experimental test of this idea was conducted using as a biological marker the twofold enhancement of ODC activity found in L929 murine cells after exposure to a 60 Hz, 10 microT rms magnetic field. We superimposed an incoherent magnetic noise field, containing frequencies from 30 to 90 Hz, whose rms amplitude was comparable to that of the 60 Hz field. Under these conditions the ODC activity observed after exposure was equal to control levels. It is concluded that the superposition of incoherent magnetic fields can block the enhancement of ODC activity by a coherent magnetic field if the strength of the incoherent field is equal to or greater than that of the coherent field. When the superimposed, incoherent noise field was reduced in strength, the enhancement of ODC activity by the coherent field increased. Full ODC enhancement was obtained when the rms value of the applied EM noise was less than one-tenth that of the coherent field. These results are discussed in relation to the question of cellular detection of weak EM fields in the presence of endogenous thermal noise fields.

The role of coherence time in the effect of microwaves on ornithine decarboxylase activity.

Previously, we demonstrated the requirements for a minimum coherence time of an applied, small amplitude (10 microT) ELF magnetic field if the field were to produce an enhancement of ornithine decarboxylase activity in L929 fibroblasts. Further investigation has revealed a remarkably similar coherence time phenomenon for enhancement of ornithine decarboxylase activity by amplitude-modulated 915 MHz microwaves of large amplitude (SAR 2.5 W/kg). Microwave fields modulated at 55, 60, or 65 Hz approximately doubled ornithine decarboxylase activity after 8 h. Switching modulation frequencies from 55 to 65 Hz at coherence times of 1.0 s or less abolished enhancement, while times of 10 s or longer provided full enhancement. Our results show that the microwave coherence effects are remarkably similar to those observed with ELF fields.

Determination of the induced ELF electric field distribution in a two layer in vitro system simulating biological cells in nutrient solution.

In-vitro studies of biological effects of electromagnetic fields are often conducted with cultured cells either in suspension or grown in a monolayer. In the former case, the exposed medium can be assumed to be homogeneous; however, eventually the cells settle to the bottom of the container forming a two layer system with different dielectric and conductive properties. In the present work the effect of this separation on the electric field distribution is calculated and experimentally measured at selected positions for a commonly used exposure configuration. The settled cell suspension is modeled by a well-defined two layer system placed in a rectangular container with the base of the container parallel to the direction of the magnetic field. Theoretical calculations based on numerical techniques are done for various two layer systems with different conductivities in each layer. The agreement between the theoretical calculations and the experimental measurements is within +/- 1.5 mV/m, or 10% of the maximum induced field when the conductivity of the lower layer is ten times that of the upper layer. This result is well within experimental error. When the thickness of one of the layers is small compared to the thickness of the other layer, it is found that the electric field distribution is essentially that of the homogeneous case. The latter situation corresponds to a typical cell exposure condition.

Dose-response implications of the transient nature of electromagnetic-field-induced bioeffects: theoretical hypotheses and predictions.

Data in the literature imply that the relationship between exposure and bio-effect involves more than a simple time integral of the field strength to which the living system has been subjected. Windows--ranges in which the system exhibits enhanced sensitivity--have been reported for power (or field strength), frequency, and the duration of the exposure. In this paper we show that such isolated window effects can be accounted for by recognizing the transient character of the response of the biological system. The principal assumption here is that the direct effect of the field is to increase the rates of production and degradation of mRNA or proteins. In this paper we review and extend the mathematical model that quantifies this. The model predicts that, for a given field strength, certain optimum relatively short duration exposures cause significantly larger bio-effects than exposure for much longer or much shorter times. The thinking embodied in the model should provide a framework for obtaining a meaningful working definition of "effective dose" and for predicting the response of subjects to environmental electromagnetic fields. It should help in deciding the relevant variables in the design and analysis of epidemiological studies.

On the mechanism of dielectric relaxation in aqueous DNA solutions.

The complex dielectric response of calf thymus DNA in aqueous saline solutions has been measured from 1 MHz to 1 GHz. The results are presented in terms of the relaxation of the incremental contributions to the permittivity and conductivity from the condensed counterions surrounding the DNA molecules. Measurements of the low-frequency conductivity of the samples also lends support to the condensed counterion interpretation.

Effect of coherence time of the applied magnetic field on ornithine decarboxylase activity.

Skepticism over the possibility of weak electromagnetic fields affecting cell function exists because endogenous thermal noise fields are larger than those reported to cause effects. Four-hour exposure to a 55- or 65-Hz field approximately doubles the specific activity of ornithine decarboxylase (ODC) in L929 cells. To test the idea that the cell discriminates against this thermal noise because it is incoherent, partial incoherence was introduced into the applied field by shifting the frequency between 55- to 65-Hz at intervals of tau coh--delta tau where tau coh is a predetermined time interval and delta tau much less than tau coh varies randomly from one frequency shift to the next. To obtain the full ODC enhancement, coherence of the impressed signal must be maintained for a minimum of about 10s. For tau coh = 5.0s a partial enhancement is elicited, and at 1.0s there is no response. Unfortunately coherence times of this duration are too short to solve the thermal noise puzzle.

Amplitude windows and transiently augmented transcription from exposure to electromagnetic fields.

The exposure of cells to relatively low-intensity, pulsed, low-frequency electromagnetic fields can result in a transient augmentation of mRNA synthesis. Under certain conditions of irradiation, the augmentation is a function of the strength of the electromagnetic field. A linear, multi-step, chemical-reaction model accounts for many of the principal features that are observed in both the time- and intensity-dependent variations of transcriptional effects. The crucial assumption in the model is that the direct effect of electromagnetic fields on exposed cells is an increase in the rate constant that characterizes one of the intermediate sequential reactions in the synthesis of mRNA.