Induction of calcium-ion efflux from brain tissue by radiofrequency radiation: Effect of sample number and modulation frequency on the power-density window

Changes have been found in calcium-ion binding to brain tissue exposed in vitro to a specific power density (0.83 mW/cm²) of 147-MHz radiation, amplitude modulated by a 16-Hz sine wave. This report replicates and extends this previous work. To define more precisely the range of effective power densities, two different numbers of samples were treated in a Crawford cell. In one series, four brain tissues were exposed at a time; in the other series, four brain tissues plus six dummy loads were exposed together. While the four-sample configuration produced a narrow power-density window, the ten pseudosample configuration resulted in a broader power-density window. The reason for the sample-number dependence is unresolved, but may be due to interactions between samples and field distortions caused by the close spacing. The ten pseudosample configuration was used to test for the presence and range of a power-density window at a sinusoidal modulation frequency of 9 Hz. The response curve at 9 Hz was essentially identical to the results for 16-Hz sinewave modulation.     

Dose dependence of acetylcholinesterase activity in neuroblastoma cells exposed to modulated radio-frequency electromagnetic radiation.

Radio-frequency electromagnetic radiation (RFR) at 915 and 147 MHz, when sinusoidally amplitude modulated (AM) at 16 Hz, has been shown to enhance release of calcium ions from neuroblastoma cells in culture. The dose-response relation is unusual, consisting of two power-density "windows" in which enhanced efflux occurs, separated by power-density regions in which no effect is observed. To explore the physiological importance of these findings, we have examined the impact of RFR exposure on a membrane-bound enzyme, acetylcholinesterase (AChE), which is intimately involved with the acetylcholine (ACh) neurotransmitter system. Neuroblastoma cells (NG108), exposed for 30 min to 147-MHz radiation, AM at 16 Hz, demonstrated enhanced AChE activity, as assayed by a procedure using 14C-labeled ACh. Enhanced activity was observed within a time window between 7.0 and 7.5 h after the cells were plated and only when the exposure occurred at power densities identified in a previous report as being effective for altering the release of calcium ions. Thus RFR affects both calcium-ion release and AChE activity in nervous system-derived cells in culture in a common dose-dependent manner.     

Multiple power-density windows and their possible origin

We have previously reported that in vitro exposure of chick forebrain tissue to 50-MHz radiofrequency (RF) electromagnetic radiation, amplitude modulated (AM) at 16 Hz, would enhance the efflux of calcium ions within only two power-density ranges: one from 1.44 to 1.67 mW/cm2, and the other including 3.64 mW/cm2. No effect on efflux occurred at 0.37, 0.72, 2.17, and 4.32 mW/cm2. We confirmed and extended these results by testing at another set of power densities, which included the range of the previous study. Forebrain tissue from 1-7-day-old chickens was labeled in vitro with radioactive calcium ions (30 min, at 37 degrees C), rinsed, placed in a physiological salt solution, and then exposed for 20 min to 50-MHz radiation, AM at 16 Hz, in a transverse electric and magnetic field (TEM) cell maintained at 37 degrees C. The solution was then assayed for radioactive calcium activity. A power-density series was tested. An enhanced efflux of calcium ions was found at 1.75, 3.85, 5.57, 6.82, 7.65, 7.77, and 8.82 mW/cm2; no change was observed at 0.75, 2.30, 4.50, 5.85, 7.08, 8.19, 8.66, 10.6, and 14.7 mW/cm2. Power density is converted to specific absorption rate (SAR) by 0.36 mW/kg per mW/cm2. Even the highest SAR tested (0.005 W/kg) is much too low to result in generalized heating of the sample and thus to be the underlying cause of the enhanced response. A hypothetical mechanism is proposed involving dynamic systems that may account for the power-density dependency as well as for part of the frequency dependency observed with both modulated RF radiation and extremely-low-frequency (ELF) fields.     

Microwave radiation-induced calcium ion efflux from human neuroblastoma cells in culture

Monolayer cultures of human neuroblastoma cells were exposed to 915-MHz radiation, with or without sinusoidal amplitude modulation (80%) at 16 Hz, at specific absorption rates (SAR) for the culture medium and cells of 0.00, 0.01, 0.05, 0.075, 0.1, 0.5, 0.75, 1.0, 1.5, 2, or 5 mW/g. A significant increase in the efflux of calcium ions (⁴⁵Ca²⁺) as compared to unexposed control cultures occurred at two SAR values: 0.05 and 1 mW/g. Increased efflux at 0.05 mW/g was dependent on the presence of amplitude modulation at 16 Hz but at the higher value it was not. These results indicate that human neuroblastoma cells are sensitive to extremely low levels of microwave radiation at certain narrow ranges of SAR.     

Internal field strength measurements in chick forebrains at 50, 147, and 450 MHz.

This report describes some experimental measurements of the internal field levels induced within isolated chick-forebrains irradiated at 50, 147, and 450 MHz, under essentially the same conditions as those used in the in vitro calcium-ion efflux experiments. Ratios of incident power at 50/147 MHz and 147/450 MHz that are needed to establish the same probe output are given and comparisons made with values predicted by different spherical models. Data predicted by the layered-sphere model were found to be in close agreement with measured values for the 50/147-MHz ratio. Agreement for the 147/450-MHz ratio was poorer.     

Operant behavior and colonic temperature of Macaca mulatta exposed to radio frequency fields at and above resonant frequencies

Five food-deprived rhesus monkeys were exposed to 225-MHz continuous-wave, and 1.3-GHz, and 5.8-GHz pulsed radiation to determine the minimal power densities affecting performance. The monkeys were trained to press a lever (observing-response) thereby producing signals that indicated availability of food. In the presence of the aperiodically appearing food signals, a detection response on a different lever was reinforced by a food pellet. Continuous, stable responding during 60-min sessions developed and was followed by repeated exposures to radiofrequency radiation. The subjects, restrained in a Styrofoam chair, were exposed to free-field radiation while performing the task. Colonic temperature was simultaneously obtained. Observing-response performance was impaired at increasingly higher power densities as frequency increased from the near-resonance 225 MHz to the above-resonance 5.8 GHz. The threshold power density of disrupted response rate at 225 MHz was 8.1 mW/cm²; at 1.3 GHz it was 57 mW/cm², and at 5.8 GHz it was 140 mW/cm². These power densities were associated with reliable increases in colonic temperatures above sham-exposure levels. The mean increase was typically in the range of 1°C, and response-rate changes were not observed in the absence of concomitant temperature increases. In these experiments increase of colonic temperature was a much better predictor of behavioral disruption than was either the power density of the incident field or estimates of whole-body-averaged rates of energy absorption.     

Effect of nonionizing radiation on the purkinje cells of the rat cerebellum

In one experiment, Sprague Dawley rats (16–21 days of gestation) and their offspring were exposed to 100-MHz (CW) electromagnetic radiation at 46 mW/cm² (SAR 2.77 mW/g) for 4 h/day for 97 days. In another experiment, the pregnant rats were irradiated daily from 17 to 21 days of gestation with 2450-MHz (CW) microwaves at 10 mW/cm² (SAR 2 mW/g) for 21 h/day. In a third experiment, 6-day-old rat pups were irradiated 7 h/day for five days with 2450-MHz radiation at 10 mW/cm². Equal numbers of animals were sham irradiated in each group. Quantitative studies of Purkinje cells showed a significant and irreversible decrease in rats irradiated during fetal or fetal and early postnatal life. In animals exposed postnatally, and euthanized immediately after irradiation, significant decrease in the relative number of Purkinje cells was apparent. However, restoration apparently occurred after forty days of recovery.     

Immunologic and hematopoietic alterations by 2,450-MHz electromagnetic radiation

A biphasic modulation of responsiveness of spleen lymphocytes to mitogens was observed in mice exposed to 2,450-MHz radiation at power densities of 5–15 mW/cm² over various periods ranging between one and 17 days. This modulated phenomenon may be explained on the basis of 1) suppression of lymphocyte response by microwave-activated macrophages which persists throughout the entire course of radiation, and 2) concurrent progressive direct stimulation of lymphocytes which culminates around day 9 of exposure. Tumor cytotoxicity of killer lymphocytes from mice exposed to five or nine days of radiation did not appear different from sham controls. The highly proliferative hematopoietic marrow cells were sensitive to microwave radiation. Nine days of exposure to radiation (15 mW/cm²) reduced the colonyforming units of myeloid and erythroid series by 50%. This observation may offer a new and more sensitive assay for studying biological effects of electromagnetic radiation.     

Low frequency amplitude modulated microwave fields change calcium efflux rates from synaptosomes

Calcium(⁴⁵Ca^{2 +}) efflux from preloaded synaptosomes was studied with a continuous perfusion technique and the rate constants of a two-phase efflux process calculated. When 16-Hz sinusoidally amplitude modulated 450-MHz microwave field (maximal incident intensity 0.5 mW/ cm², modulation depth 75%) was applied during the second phase, the rate constant increased by 38%. Unmodulated or 60-Hz modulated signals were not effective. This microwave fieldinduced change can be distinguished from CaCl₂-stimulated ⁴⁵Ca^{2 +} efflux which is most probably derived intracellularly. These data suggest that the microwave-field-induced change in calcium efflux probably did not involve intracellular calcium. Also, this change in the dynamic property of synaptosomes did not require gross anatomically intact tissue as a substrate for field tissue interaction.     

Effect of amplitude-modulated 147 MHz radiofrequency radiation on calcium ion efflux from avian brain tissue

Cerebral cortex tissue slices and cerebral hemispheres prepared from Gallus domesticus chicks were exposed to 147 MHz radiofrequency radiation, amplitude modulated at 16 Hz and applied at a power density of 0.75 mW/cm2, to determine the effect of such exposure of 45Ca2+ efflux from the avian brain tissue. Statistical analysis of these data demonstrates that such exposure has no significant effect on 45Ca2+ efflux.     

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/m_rms (electric) and 97 nT_rms (magnetic), showed enhancement in enolase activity by 59%, whereas exposure to 60 Hz fields alone, at 14.1 V/m_rms (electric) and 65 nT_rms (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. Although the underlying basis for these field effects in the cytoplasmic compartment has not been established, differential sensitivities to 16 Hz and to 60 Hz signals provide a clear focus for additional research to determine the responsible mechanism. © 1994 Wiley-Liss, Inc.     

In vitro study of microwave effects on calcium efflux in rat brain tissue

In this study we investigated the prospect of microwave-induced alteration of ⁴⁵Ca²⁺ efflux from rat neural tissue at low pulse repetition frequencies and low power densities under in vitro conditions. Rat cerebral tissue, preloaded with ⁴⁵Ca²⁺, was exposed to pulsed-microwave radiation (1-GHz carrier frequency) according to one of several PRF-power density exposure schemes: 16 Hz at 0.5, 1.0, 2.0, or 15 mW/cm², or 32 Hz at 1.0 or 2.0 mW/cm² average power density. Measurements of radioactivity in the efflux medium and in the tissue sample were used to calculate an efflux value for each sample. The results indicate that the radiation conditions used did not alter calcium efflux in rat brain tissue.     

Biochemical Changes in Rat Brain Exposed to Low Intensity 9.9 GHz Microwave Radiation

Present study concerns with various biochemical changes in the developing rat brain exposed to 9.9 GHz (square wave modulated, 1 kHz) at power density 0.125 mW/cm² (specific absorption rate 1.0 W/kg) for 2 h/day for 35 days. Thirty days old male wistar rats were used for this present study. Each group consists of eight animals. After the exposure, biochemical assays such as calcium ion efflux, calcium-dependent protein kinase (PKC), and ornithine decarboxylase (ODC) were performed on the brain tissue. Results of this study reveal that chronic exposure of rat to microwave radiation alter the activity of certain enzymes. There was a significant increase in calcium ion efflux and the activity of ODC. On the other hand, there is a significant decrease in PKC activity. Since these enzymes are related to growth, any alteration may lead to affect functioning of the brain and its development.     

Microwave radiation (2450 MHz) alters the endotoxin-induced hypothermic response of rats

The parenteral administration of bacterial endotoxin to rats causes a hypothermia that is maximal after approximately 90 minutes. When endotoxin-injected rats were held in a controlled environment at 22°C and 50% relative humidity and exposed for 90 minutes to microwaves (2450 MHz, CW) at 1 mW/cm², significant increases were observed in body temperature compared with endotoxintreated, sham-irradiated rats. The magnitude of the response was related to power density (10 mW/cm² > 5 mW/cm² > 1 mW/cm²). Saline-injected rats exposed for 90 minutes at 5 mW/cm² (specific absorption rate approximately 1.0 mW/g) showed no significant increase in body temperature compared with saline-injected, sham-irradiated rats. The hypothermia induced by endotoxin in rats was also found to be affected by ambient temperature alone. Increases in ambient temperature above 22°C in the absence of microwaves caused a concomitant increase in body temperature. This study reveals that subtle microwave heating is detectable in endotoxin-treated rats that have an impaired thermoregulatory capability. These results indicate that the interpretation of microwave-induced biological effects observed in animals at comparable rates and levels of energy absorption should include a consideration of the thermogenic potential of microwaves.     

In vitro microwave effects on human neutrophil precursor cells (CFU-C)

Human marrow cells were irradiated with 2450-MHz CW microwaves in a fluid-filled waveguide irradiation system. Cell exposure was conducted by placing a marrow cell suspension in 20-μl glass microcapillary tubes that were positioned in the exposure chamber, and irradiated at power densities from 31 to 1,000 mW/cm² (with corresponding specific absorption rates of 62 to 2,000 mW/g) for 15 minutes. The temperature of the sample was maintained at a fixed point. Sham-irradiated (SI) and microwave-irradiated (MWI) cells were cultured in a methylcellulose culture system for neutrophil colony proliferation. There was no reduction in neutrophil colony number on days 6–7 or 12–14 in cells exposed at 31 or 62 mW/cm², but as the power density was increased to 1,000 mW/cm², there was a reduction in colony number of MWI cells compared with SI cells. The microwave interaction with the human neutrophil colony-forming cells was apparently not related to temperature rise, or to the state of cell cycle, and was irreversible.     

Effects of 2.45-GHz microwave radiation on embryonic quail hearts

Although exposure to nonionizing electromagnetic radiation has been reported to cause a variety of systemic alterations during embryonic development, there are few reports of the induction of specific physiologic or morphologic changes in the myocardium. This study was designed to examine the effects of microwave radiation on cardiogenesis in Japanese quail embryos exposed during the first eight days of development to 2.45-GHz continuous-wave microwaves at power densities of 5 or 20 mW/cm². The specific absorption rates were 4.0 and 16.2 mW/g, respectively. The ambient temperature for each exposure was set to maintain the embryonated eggs at 37.5 °C. This did not preclude thermal gradients in the irradiated embryos since microwaves may not be uniformly absorbed. The test exposure levels did not induce changes in either the morphology of the embryonic heart or the ultrastructure of the myocardial cells. Analysis of the enzymatic activities of lactate dehydrogenase, glutamic oxaloacetic transaminase, and creatine phosphokinase failed to reveal any statistically significant differences between the nonexposed controls and those groups exposed to either 5 or 20 mW/cm². The data indicate that 2.45-GHz microwave radiation at 5 or 20 mW/cm² has no effect on the measured variables of the Japanese quail myocardium exposed during the first eight days of development.     

Radiofrequency radiation-induced calcium ion efflux enhancement from human and other neuroblastoma cells in culture

To test the generality of radiofrequency radiation-induced changes in 45Ca2+ efflux from avian and feline brain tissues, human neuroblastoma cells were exposed to electromagnetic radiation at 147 MHz, amplitude-modulated (AM) at 16 Hz, at specific absorption rates (SAR) of 0.1, 0.05, 0.01, 0.005, 0.001, and 0.0005 W/kg. Significant 45Ca2+ efflux was obtained at SAR values of 0.05 and 0.005 W/kg. Enhanced efflux at 0.05 W/kg peaked at the 13-16 Hz and at the 57.5-60 Hz modulation ranges. A Chinese hamster-mouse hybrid neuroblastoma was also shown to exhibit enhanced radiation-induced 45Ca2+ efflux at an SAR of 0.05 W/kg, using 147 MHz, AM at 16 Hz. These results confirm that amplitude-modulated radiofrequency radiation can induce responses in cells of nervous tissue origin from widely different animal species, including humans. The results are also consistent with the reports of similar findings in avian and feline brain tissues and indicate the general nature of the phenomenon.     

Tests of mutagenesis and reproduction in male rats exposed to 2,450-MHz (CW) microwaves

Tests of mutagenesis and reproduction were conducted in male rats which were irradiated by 2,450-MHz, continuous-wave (CW) microwaves, 4 hr/day from day 6 of gestation to 90 days of age at 5 mW/cm²; or 5 hr/day for five days beginning on the 90th day of age at 10 mW/cm²; or 4 hr/day, 5 days/ wk for four weeks, beginning on the 90th day of age. During selected weekly periods after treatment, the rats were bred to pairs of untreated, normal female rats that were examined in late pregnancy by means of the dominant lethal assay. The reproductive efficiency of these males, as reflected in their breeding, was also examined for changes relating to their microwave experience. No significant evidence of germ-cell mutagenesis was detected when data of microwave-exposed males were compared with those of sham-exposed males, even though there were significant increases in rectal and intra-testicular temperatures at a power density of 28 mW/cm². Temporary sterility, as indexed by fewer pregnancies, was seen at the highest power density.     

Power density,field intensity,and carrier frequency determinants of RF-energy-lnduced calcium-ion efflux from brain tissue

To explain a carrier frequency dependence reported for radiofrequency (RF)-induced calcium-ion efflux from brain tissue, a chick-brain hemisphere bathed in buffer solution is modeled as a sphere within the uniform field of the incident electromagnetic wave. Calculations on a spherical model show that the average electric-field intensity within the sample remains the same at different carrier frequencies if the incident power density (P_i) is adjusted by an amount that compensates for the change in complex permittivity (?) and the change of wavelength, as a function of carrier frequency. The resulting formula for transforming P_i is seen to follow the pattern of both positive and negative demonstrations of calcium-ion efflux that have been observed at carrier frequencies of 50, 147, and 450 MHz. Indeed, all results obtained at these three frequencies, when related by P_i's that produce the same average electric-field intensity within the sample, are seen to be in agreement; no prediction is contradicted by an experiment.     

Thermophysiological responses of human volunteers during controlled whole-body radio frequency exposure at 450 MHz

Thermoregulatory responses of heat production and heat loss were measured in seven adult volunteers (four women and three men, aged 21–57 yr) during 45-min dorsal exposures of the whole body to 450 MHz continuous wave radio frequency (RF) fields. Two power densities (PD) (local peak PD = 18 and 24 mW/cm²; local peak specific absorption rate = 0.320 [W/kg]/[mW/cm²]) were tested in each of three ambient temperatures (T_a = 24, 28, and 31 °C) plus T_a controls (no RF). No changes in metabolic heat production occurred under any exposure conditions. Vigorous increases in sweating rate on back and chest, directly related to both T_a and PD, cooled the skin and ensured efficient regulation of the deep body (esophageal) temperature to within 0.1 °C of the normal level. Category judgments of thermal sensation, comfort, sweating, and thermal preference usually matched the measured changes in physiological responses. Some subtle effects related to gender were noted that confirm classic physiological data. Our results indicate that dorsal exposures of humans to a supra-resonant frequency of 450 MHz at local peak specific absorption rates up to 7.68 W/kg are mildly thermogenic and are counteracted efficiently by normal thermophysiologic heat loss mechanisms, principally sweating. Bioelectromagnetics 19:232–245, 1998. Published 1998 Wiley-Liss, Inc.