Dose-response relationship between body temperature and birth defects in radiofrequency-irradiated rats

Five groups of pregnant Sprague-Dawley rats were irradiated for 10-40 min on gestation day 9 in a 27.12-MHz radiofrequency field at a magnetic field strength of 55 A/m and an electric field strength of 300 V/m. The specific absorption rate was 10.8 +/- 0.3 W/kg. Exposures were terminated after the rat's colonic temperature reached 41.0 degrees C, 41.5 degrees C, 42.0 degrees C, 42.5 degrees C, or 43.0 degrees C. A control group was sham irradiated at 0 A/m and 0 V/m on gestation day 9, whereas a second control group was untreated. The incidence of both birth defects and prenatal death was directly related to maternal body temperature once a temperature threshold was exceeded. The temperature threshold for both types of effects was approximately 41.5 degrees C. A few pregnant rats died after exposure to 43.0 degrees C, and higher temperatures were nearly always lethal.     

Interactions of radiofrequency radiation-induced hyperthermia and 2-methoxyethanol teratogenicity in rats

Radiofrequency (RF) radiation is used in a variety of workplaces. In addition to RF radiation, many workers are concurrently exposed to numerous chemicals; exposed workers include those involved with the microelectronics industry, plastic sealers, and electrosurgical units. The developmental toxicity of RF radiation is associated with the degree and duration of hyperthermia induced by the exposure. Previous animal research indicates that hyperthermia induced by an elevation in ambient temperature can potentiate the toxicity and teratogenicity of some chemical agents. We previously demonstrated that combined exposure to RF radiation (10 MHz) and the industrial solvent, 2-methoxyethanol (2ME), produces enhanced teratogenicity in rats. The purpose of the present research is to determine the effects of varying the degree and duration of hyperthermia induced by RF radiation (sufficient to maintain colonic temperatures at control [38.5], 39.0, 40.0, or 41.0 °C for up to 6 h) and 2ME (100 mg/kg) administered on gestation day 13 of rats. Focusing on characterizing the dose-response pattern of interactions, this research seeks to determine the lowest interactive effect level. Day 20 fetuses were examined for external and skeletal malformations. The results are consistent with previous observations. Significant interactions were observed between 2ME and RF radiation sufficient to maintain colonic temperatures at 41 °C for 1 h, but no consistent interactions were seen at lower temperatures even with longer durations. These data indicate that combined exposure effects should be considered when developing both RF radiation and chemical exposure guidelines and intervention strategies. Bioelectromagnetics 18:349–359, 1997. © 1997 Wiley-Liss, Inc.     

Comparative thermoregulatory response to passive heat loading by exposure to radiofrequency radiation

1. Colonic and tail skin temperature of the unrestrained Fischer rat were measured immediately after a 90 min exposure to 600 MHz radiofrequency radiation in a waveguide-type system. Ambient temperature (Ta) was maintained at either 20, 28 or 35 degrees C. The specific absorption rate (SAR) in dimensions of W/kg was controlled at a constant level through a feedback control circuit. 2. The SAR needed to elevate colonic and tail skin temperature decreased with increasing Ta. For example, a 0.5 degrees C elevation in colonic temperature occurred at SARs of 4.3, 0.9 and 0.5 W/kg when Ta was maintained at 20, 28 and 35 degrees C, respectively. 3. Data from the present study were combined with data from earlier studies to assess the impact of varying Ta on the thermogenic effect of RF radiation in different species. In species ranging in mass from 0.02 to 3.2 kg, a double logarithmic plot of body mass versus SAR needed to elevate colonic temperature by 0.5 degrees C was linear and inverse with a high goodness of fit (r2 = -0.94). 4. The highly correlated allometric relationship shows that, as body mass decreases, the relative impact of Ta on the thermogenic effect of RF radiation increases.     

Marked increase in the teratogenicity of the combined administration of the industrial solvent 2-methoxyethanol and radiofrequency radiation in rats

Limited published animal research reports synergistic teratogenic effects following combined hyperthermia (induced by elevated ambient temperature) and administration of chemical teratogens. Radiofrequency (RF) radiation is widely used in occupational environments. Since RF radiation also elevates the body temperature of, and is teratogenic to, exposed animals, concurrent RF radiation and chemical agent administration may enhance teratogenicity. The present exploratory study, consisting of preliminary dose-finding studies and the primary study, was designed to investigate whether concurrent exposure of rats to RF radiation and the industrial solvent 2-methoxyethanol (2ME) can enhance the developmental toxicity of either agent acting alone. Preliminary dose-finding studies using small numbers of rats investigated the ability of various RF radiation conditions and doses of 2ME to produce external malformations (primarily of the paws) when administered on gestation day 13. Based on these preliminary studies, RF radiation exposure [sufficient to elevate rectal temperature to 42.0 degrees C (4 degrees C above normal for rats) for 30 min] and 2ME administration (150 mg/kg) were selected for the primary study. In the primary study, groups of 18 to 27 pregnant rats were administered RF radiation exposure and distilled water gavage, 2ME gavage and sham RF exposure, RF radiation exposure and 2ME gavage concurrently, or sham RF exposure and distilled water gavage. Pregnant rats were sacrificed on gestation day 20, and the offspring were examined for external malformations. Combined exposures enhanced the adverse effects produced by either experimental agent alone (no malformations were detected in the double sham group). Mean fetal malformations/litter increased from 14% after 2ME and sham RF (15/26 litters affected, with an average of 2 fetuses/litter malformed) and 30% after RF radiation and water gavage (10/18 litters affected, with an average of 4 fetuses/litter malformed), to 76% after the combined treatment (18/18 litters affected, with an average of 12 fetuses/litter malformed). In addition to a significant increase in the frequency of malformations, the severity of malformations also was enhanced by the combination treatment (on a relative severity ranking scale, the 2ME severity score was less than 1, the RF score was 3, and the combination score was 6). This study provided evidence of synergism between RF radiation and 2ME administration, but additional research will be required to characterize the extent of synergism between these two agents. Potential interactive effects between chemical and physical agents need to be investigated to determine the extent to which such interactions should impact occupational exposure standards.     

A comparison of cell killing by heat and/or X rays in Chinese hamster V79 cells, Friend erythroleukemia mouse cells, and human thymocyte MOLT-4 cells

The radiation and/or heat sensitivity of Chinese hamster V79 cells, Friend erythroleukemia (FELC) mouse cells, and MOLT-4 human transformed thymocytes were compared. MOLT-4 cells were more radiosensitive (D0 = 0.50 Gy) than FELC (D0 = 0.65 Gy) and V79 cells (D0 = 1.43 Gy). Arrhenius analysis showed that MOLT-4 cells were more heat sensitive than FELC or V79 cells below 42.0 degrees C, but more heat resistant at higher temperatures. In addition, the MOLT-4 cells showed a single-heat inactivation energy between 41.0 and 45.0 degrees C, while FELC and V79 cells both showed a transition in the inactivation energy at about 43.0 and 43.5 degrees C, respectively. These differences may be related to the fact that the upper temperature limit for the development of thermal tolerance during continuous heating was lower for MOLT-4 cells than for FELC or V79 cells. Killing of FELC and V79 cells was dependent on the sequence in which heat and X rays were applied, but the greatest effect was obtained when both treatments were given simultaneously. Recovery occurred when treatments were separated by incubation at 37.0 degrees C. The MOLT-4 cells did not show a sequence dependence for heating and irradiation. Survival of MOLT-4 cells after heating and/or irradiation was compared using trypan blue dye exclusion or colony formation. Both assays showed similar qualitative responses, but survival levels measured by the trypan blue assay were much higher than those determined from the colony-forming assay.     

Association of microwaves and ionizing radiation: potentiation of teratogenic effects in the rat

Pregnant Wistar rats were exposed to either microwave-induced hyperthermia or gamma radiation or a combination of both. In microwave-induced hyperthermia, a core temperature of 42 degrees C was slightly teratogenic and a core temperature greater than or equal to 43 degrees C was highly teratogenic. Gamma radiation at 40 cGy was subteratogenic, while a dose of 75 cGy was highly teratogenic. A combination of microwave-induced hyperthermia up to 42 degrees C with 40 cGy of gamma radiation was highly teratogenic, indicating a mutual potentiation of the embryotoxic action of these two teratogens.     

Tolerance to induced systemic hyperthermia in the mouse

The effect of exposure of organisms to systemic hyperthermia on induction of tolerance to the lethal effect of subsequently assigned systemic hyperthermia was studied in mice. The length of time of the pretreatment at 42.0 +/- 0.2 degrees C (core body temperature) was 5, 10 or 15 mn. The temperature of the second systemic hyperthermia was 42.0 +/- 0.2 degrees C and 43.5 +/- 0.2 degrees C. In mice which had no experience of systemic hyperthermia, lethal dose required to kill 50% of animals at 42.0 degrees C and 43.5 degrees C, namely LD50, 42 degrees and LD50, 43 degrees 5 was 43 and 8.5 mn, respectively. While, in mice which had received the pretreatment at 42 degrees C for 10 mn, the LD50, 42 degrees was 97 mn one day after and 48 mn two days after the pretreatment. In mice which had received the pretreatment at 42 degrees C for 5, 10 or 15 mn, the LD50, 43 degrees 5 was 17, 20 and 19 mn one day after the pretreatment, and 10, 10 and 6 mn two days after the pretreatment, respectively. With the data obtained, thermotolerance ratio (TTR) was calculated. The maximum TTR of 2.35 was obtained in mice examined one day after the pretreatment at 42.0 degrees C for 10 mn.     

Immunological effects of amplitude-modulated radio frequency radiation: B lymphocyte capping

B lymphocytes collected from normal ICR Swiss mouse spleens were exposed in vitro in a Crawford cell to 147-MHz radiofrequency (RF) radiation, amplitude modulated by a 9-, 16-, or 60-Hz sine wave. The power densities ranged between 0.11 and 48 mW/cm2. The irradiated samples and the controls were maintained at 37 degrees C or 42 degrees C, with temperature variations less than 0.1 degrees C. Immediately after a 30-minute exposure, the distribution of antigen-antibody (Ag-Ab) complexes on the cell surface was evaluated at 37 degrees C by immunofluorescence. Under normal conditions (37 degrees C, no RF), Ag-Ab complexes are regrouped into a polar cap by an energy-dependent process. Our results demonstrate that the irradiated cells and the nonirradiated controls capped Ag-Ab complexes equally well after exposure at 37 degrees C. Capping was equally inhibited at 42 degrees C in both the controls and irradiated cells. No statistically significant differences in capping were observed between the RF-exposed and control samples at any of the modulation frequencies and power densities employed as long as both preparations were maintained at the same temperature.     

The effect of hyperthermia on radiation-induced carcinogenesis

Ten groups of mice were exposed to either a single (30 Gy) or multiple (six fractions of 6 Gy) X-ray doses to the leg. Eight of these groups had the irradiated leg made hyperthermic for 45 min immediately following the X irradiation to temperatures of 37 to 43 degrees C. Eight control groups had their legs made hyperthermic with a single exposure or six exposures to heat as the only treatment. In mice exposed to radiation only, the postexposure subcutaneous temperature was 36.0 +/- 1.1 degrees C. Hyperthermia alone was not carcinogenic. At none of the hyperthermic temperatures was the incidence of tumors in the treated leg different from that induced by X rays alone. The incidence of tumors developing in anatomic sites other than the treated leg was decreased in mice where the leg was exposed to hyperthermia compared to mice where the leg was irradiated. A systemic effect of local hyperthermia is suggested to account for this observation. In mice given single X-ray doses and hyperthermia, temperatures of 37, 39, or 41 degrees C did not influence radiation damage as measured by the acute skin reactions. A hyperthermic temperature of 43 degrees C potentiated the acute radiation reaction (thermal enhancement factor 1.1). In the group subjected to hyperthermic temperatures of 37 or 39 degrees C and X rays given in six fractions, the skin reaction was no different from that of the group receiving X rays alone. Hyperthermic temperatures of 41 and 43 degrees C resulted in a thermal enhancement of 1.16 and 1.36 for the acute skin reactions. From Day 50 to Day 600 after treatment, the skin reactions showed regular fluctuations with a 150-day periodicity. Following a fractionated schedule of combined hyperthermia and X rays, late damage to the leg was less than that following X irradiation alone. Mice subjected to X rays and hyperthermic temperatures of 41 and 43 degrees C had a lower median survival time than the mice treated with hyperthermia alone. This effect was not associated with tumor incidence.     

Sensitization of low-dose-rate irradiation by nonlethal hyperthermia

To assess whether hyperthermia could radiosensitize cells irradiated at a low dose rate, Chinese hamster V79 cells were simultaneously heated and irradiated at 0.86 Gy/h. The data showed that heat treatments at 39 and 40 degrees C, which did not induce heat killing alone or high-dose-rate radiosensitization, resulted in enhanced cell killing with low-dose-rate irradiation. The dose-modification factor (ratio of the slopes of the curves for low dose rate and high dose rate) was reduced to 1.8 at 39 degrees C and 1.4 at 40 degrees C, compared to a value of 2.1 at 37 degrees C. These data indicate that nonlethal heat treatments can cause enhanced radiosensitization under low-dose-rate conditions. The implications of these results for interstitial thermoradiotherapy are discussed.     

Metabolic and vasomotor responses of rhesus monkeys exposed to 225-MHz radiofrequency energy

A previous study showed a substantial increase in the colonic temperature of rhesus monkeys (Macaca mulatta) exposed to radiofrequency (RF) fields at a frequency near whole-body resonance and specific absorption rates (SAR) of 2-3 W/kg. The present experiments were conducted to determine the metabolic and vasomotor responses during exposures to similar RF fields. We exposed five adult male rhesus monkeys to 225 MHz radiation (E orientation) in an anechoic chamber. Oxygen consumption and carbon dioxide production were measured before, during, and after RF exposure. Colonic, tail and leg skin temperatures were continuously monitored with RF-nonperturbing probes. The monkeys were irradiated at two carefully-controlled ambient temperatures, either cool (20 degrees C) or thermoneutral (26 degrees C). Power densities ranged from 0 (sham) to 10.0 mW/cm2 with an average whole-body SAR of 0.285 (W/kg)/(mW/cm2). We used two experimental protocols, each of which began with a 120-min pre-exposure equilibration period. One protocol involved repetitive 10-min RF exposures at successively higher power densities with a recovery period between exposures. In the second protocol, a 120-min RF exposure permitted the measurement of steady-state thermoregulatory responses. Metabolic and vasomotor adjustments in the rhesus monkey exposed to 225 MHz occurred during brief or sustained exposures at SARs at or above 1.4 W/kg. The SAR required to produce a given response varied with ambient temperature. Metabolic and vasomotor responses were coordinated effectively to produce a stable deep body temperature. The results show that the thermoregulatory response of the rhesus monkey to an RF exposure at a resonant frequency limits storage of heat in the body. However, substantial increases in colonic temperature were not prevented by such responses, even in a cool environment.     

Progenitor cell injury after irradiation to the developing brain can be modulated by mild hypothermia or hyperthermia

Ionizing radiation induced acute cell death in the dentate gyrus subgranular zone (SGZ) and the subventricular zone (SVZ). Hypomyelination was also observed. The effects of mild hypothermia and hyperthermia for 4 h after irradiation (IR) were studied in postnatal day 9 rats. One hemisphere was irradiated with a single dose of 8 Gy and animals were randomized to normothermia (rectal temperature 36 degrees C for 4 h), hypothermia (32 degrees C for 4 h) or hyperthermia (39 degrees C for 4 h). Cellular injury, e.g. chromatin condensation and nitrotyrosine formation, appeared to proceed faster when the body temperature was higher. Caspase-3 activation was more pronounced in the hyperthermia group and nuclear translocation of p53 was less pronounced in the hypothermia group 6 h after IR. In the SVZ the loss of nestin-positive progenitors was more pronounced (48%) and the size was smaller (45%) in the hyperthermia group 7 days post-IR. Myelination was not different after hypo- or hyperthermia. This is the first report to demonstrate that hypothermia may be beneficial and that hyperthermia may aggravate the adverse side-effects after radiation therapy to the developing brain.     

Interaction of ethanol and microwaves on the blood-brain barrier of rats

The combined effects of ethanol and microwaves on the permeation of Evans blue dye through the mammalian blood-brain barrier was studied in male Wistar rats. Anesthetized rats were infused through a cannula in the left femoral vein with 0.1, 0.3, 0.5 or 0.7 grams of absolute ethanol per kilogram of body mass. A control group was given 0.7 g/kg of isotonic saline. The left hemisphere of the brain was irradiated by 3.15-GHz microwave energy at 3.0 W/cm2 rms for 15 min. The rat's rectal temperature was maintained at 37.0 degrees C. Immediately after irradiation, 2% Evans blue dye in saline (2.0 ml/kg body mass) was injected through the cannula. The results show that as the quantity of alcohol was increased, the degree of staining was decreased or eliminated. The temperature of the irradiated area of the brain increased for the first 4 to 5 minutes of irradiation and then stabilized for the remainder of the irradiation period. The steady-state temperature was highest in animals receiving saline or the smallest dose of alcohol. As the quantity of alcohol was increased, the steady-state temperature was reduced. These results indicate that ethanol inhibits microwave-induced permeation of the blood-brain barrier through reduced heating of the brain.     

Sensitivity of mice to systemic hyperthermia: effect of ascites tumor cell transplants

The effect of a transplantation of mastocytoma cells in the abdominal cavity on the sensitivity of mice to a systemic hyperthermia was studied. The systemic hyperthermia was induced by exposing whole-body of animals to 2,450 MHz waves under anesthesia. Core body temperature was raised up to 42.0 +/- 0.2 degrees C in 15 min and maintained constant at the temperature for variable length of time. Thermosensitivity of animal was expressed with LD50, 42 degrees which was the length of heating time at the temperature of 42 degrees C lethal for 50% of the animals examined. The transplants were mastocytoma FMA3 cells. They were transplanted at a dose of 10(5) cells per mouse. The LD50, 42 degrees observed 3, 12 hrs, 1, 2, 3 and 6 days after the transplantation was 33, 23, 17, 24 and 35 min, respectively. In mice without tumor it was 43 min.     

Hyperthermia effect on lipolytic processes in rat blood and adipose tissue

Rats anaesthetized with Brevinarcon were placed in a high-temperature chamber (air temperature 50 degrees C, relative humidity 50%) for induction of hyperthermia (rectal temperature 41.0 +/- +/- 0.5 degrees C). The control group comprised rats anaesthetized in the same way but kept at room temperature. In the serum in both groups glucose, free fatty acids, immunoreactive insulin, lipolytic activity and ability to mobilize lipids in vitro were determined. It was shown that the glucose and free fatty acid levels and the activity mobilizing serum lipids in vitro in the rats subjected to hyperthermia were lower than in the control group by 12%, 23% and 150% respectively. The lipolytic activity of the serum of rats subjected to hyperthermia was 42% higher, and the level of immunoreactive insulin rose by about 224% in relation to the control group. These results point to inhibition of lipolysis in the adipose tissue with simultaneous activation of intravascular lipolysis during hyperthermia in rats.     

Modification of radiation damage in the canine kidney by hyperthermia: a histologic and functional study

The purpose of this study was to evaluate the effect of hyperthermia on the histologic and functional response of the canine kidney, a late-responding normal tissue, to irradiation. Both kidneys were irradiated. Radiation was delivered in single doses of 0, 10, or 15 Gy. Whole-body hyperthermia was used to produce renal kidney temperatures approximating 42.0 degrees C for 60 min. Thirty-six beagles were placed randomly in the following six treatment groups: control, whole-body hyperthermia alone, 10 Gy alone, 10 Gy + whole-body hyperthermia, 15 Gy alone, and 15 Gy + whole-body hyperthermia. Renal histologic and functional changes were assessed at 1 to 9 months after therapy. No changes were seen in glomerular filtration rate or renal tissue volumes in control or hyperthermia alone groups. Renal vascular and glomerular volumes were not affected significantly by any combination of hyperthermia and/or radiation. In all groups receiving radiation, glomerular filtration rate decreased, percentage renal tubular volume decreased, and interstitial volume increased significantly after therapy. The magnitude of these changes in the functional and histologic response of the kidney and the latent period before expression of this damage were dependent on radiation dose. However, hyperthermia did not modify expression of radiation damage in the kidney based on glomerular filtration rate and histologic quantification of renal tissue components.     

Reduction in metabolic heat production during exposure to radio-frequency radiation in the rat

The purpose of this study was to assess the ability of the rat to reduce metabolic rate when exposed to deep-penetrating radio-frequency (RF) radiation. Male Sprague-Dawley rats were maintained at an ambient temperature (Ta) of 10 degrees C and exposed to 600-MHz radiation while metabolic rate (MR) was measured by indirect calorimetry. RF radiation exposures were made in a waveguide-type system that permitted the continuous control of specific absorption rate (SAR). SAR's of 2-5 W/kg led to significant reductions in MR when averaged from 30 to 60 min after the initiation of RF radiation exposure. The total decrease in MR during RF radiation exposure accounted for approximately 37% of the total RF heat load. Exposure of another group of rats to the same SAR's at a Ta of 10 degrees C resulted in a significant elevation in colonic temperature. Thus, despite the decrease in MR, heat gain still exceeded heat loss during RF radiation exposure, with a resultant elevation in deep body temperature. In conclusion, in a cold environment the rat exposed to RF radiation decreases its MR. However, the response time and efficiency of the response is not adequate to prevent an increase in body temperature.     

Erythrocyte hemolysis by radiofrequency fields

A field-strength-dependent hemolytic effect of continuous-wave radiofrequency (RF) exposure in vitro has been demonstrated. Erythrocytes in whole heparinized rabbit blood were hemolyzed by a 2-h exposure to 50- or 100-MHz RF fields at field strengths of greater than 4 V/cm. An effect of comparable magnitude resulted from exposure to 10-MHz RF at a field strength of 9 V/cm. Sample temperatures were maintained at 22.5 degrees +/- 0.2 degrees C. There was no apparent involvement of heating or temperature gradients, nor were there any RF exposure effects on cellular K+ or Na+ concentration, nor on pH. The mechanism of the hemolytic effect is not known. Since the percentage of lysed erythrocytes was less than 1% and there was an absence of effects on cellular cation concentrations, RF radiation may have irreversibly altered the plasma membrane permeability of a sensitive subpopulation of red cells (possibly aged cells) leading to osmotic lysis. RF radiation at these frequencies appears to affect red cells in a manner that is qualitatively and quantitatively different from microwave radiation.     

Hyperthermia in a differentiating murine erythroleukemia cell line: cell killing by heat and radiation

The survival response of Friend erythroleukemia cells (a differentiating cell system) to heat and radiation has been examined. The Friend erythroleukemia cells (FELC) were more heat and radiation sensitive than V79 cells, and the heat and radiation survival curves possessed shoulders, showing the ability of the cells to accumulate sublethal damage. Thermal tolerance was expressed after prolonged heating at 41.0-42.0 degrees C. Thermal radiosensitization by heating at 42.0 or 45.0 degrees C was greatest for simultaneous heat and radiation treatments, and recovery occurred when the cells were incubated at 37 degrees C between the heat and radiation or radiation and heat treatments. Arrhenius analysis of the FELC heat survival data showed that the curve for thermal inactivation possessed a break at about 43.0 degrees C and that the thermal inactivation energies above and below the break point were comparable to those for V79 cells and other cell lines reported in the literature.     

Effects of continuous and intermittent exposure to RF fields with a wide range of SARs on cell growth, survival, and cell cycle distribution

To examine the biological effects of radio frequency (RF) electromagnetic fields in vitro, we have examined the fundamental cellular responses, such as cell growth, survival, and cell cycle distribution, following exposure to a wide range of specific absorption rates (SAR). Furthermore, we compared the effects of continuous and intermittent exposure at high SARs. An RF electromagnetic field exposure unit operating at a frequency of 2.45 GHz was used to expose cells to SARs from 0.05 to 1500 W/kg. When cells were exposed to a continuous RF field at SARs from 0.05 to 100 W/kg for 2 h, cellular growth rate, survival, and cell cycle distribution were not affected. At 200 W/kg, the cell growth rate was suppressed and cell survival decreased. When the cells were exposed to an intermittent RF field at 300 W/kg(pk), 900 W/kg(pk) and 1500 W/kg(pk) (100 W/kg(mean)), no significant differences were observed between these conditions and intermittent wave exposure at 100 W/kg. When cells were exposed to a SAR of 50 W/kg for 2 h, the temperature of the medium around cells rose to 39.1 degrees C, 100 W/kg exposure increased the temperature to 41.0 degrees C, and 200 W/kg exposure increased the temperature to 44.1 degrees C. Exposure to RF radiation results in heating of the medium, and the thermal effect depends on the mean SAR. Hence, these results suggest that the proliferation disorder is caused by the thermal effect.