Methodology of detailed dosimetry and treatment of uncertainty and variations for in vivo studies

Detailed and accurate dosimetric information is a basic precondition for acquiring adequate interpretations and valuations of in vivo studies testing radiofrequency (RF) electromagnetic fields (EMF). Instantaneous locally induced fields depend on many parameters, for example, orientation of the animal with respect to the incident field, animal size and posture, and tissue distribution. These parameters are often constrained, resulting in significant uncertainties in the dosimetric assessment of the exposure, averaged over all animals and the entire experimental phase, as well as in significant variations of the local exposures during the experiment. A sufficient analysis should therefore include (1) average and peak spatial specific absorption rate (SAR) values for the whole body and specific organs, (2) the uncertainty of each assessed SAR value, and (3) the short term and long term SAR variations between the tissues of individual animals. A methodology to obtain this pertinent information is developed and proposed in this paper. Using this methodology the dosimetry of a rat exposure apparatus operating at the carrier frequency of 1747 MHz, previously developed for a 2-year bioassay study within the European Union project PERFORM, was obtained. We have demonstrated that comprehensive dosimetric data can be obtained with reasonable effort using the proposed method, providing that the exposure setup is soundly formulated.     

Behavioral evaluation of microwave irradiation

Establishing safe exposure levels for microwave irradiation is important since new, more powerful emitters are developed and the potential for accidental exposure is increasing. Analysis of the behavior of exposed laboratory animals has proven to be an accurate and repeatable metric for assessing the effects of microwave irradiation. Determining the specific absorption rate (SAR) at which an animal will cease an ongoing behavior has proven useful in the development of safe exposure levels for humans. Behaviors that have been used are simple tasks, and the point at which behavior changes significantly or ceases has often been referred to as “work stoppage.” The tasks have been used to evaluate the overwhelming effects of heating produced by microwave irradiation. Both whole‐body exposures and partial‐body exposures with hotspots have been evaluated. Recent studies have suggested that microwave effects on specific cognitive aspects of behavior such as attention, learning, memory, discrimination, and time perception may occur at SAR levels far below the SARs needed to cause work stoppage. New research studies are underway to evaluate microwave‐induced cognitive effects. Bioelectromagnetics 20:64–74, 1999. Published 1999 Wiley‐Liss, Inc.     

Single strand DNA breaks in rat brain cells exposed to microwave radiation

This investigation concerns with the effect of low intensity microwave (2.45 and 16.5 GHz, SAR 1.0 and 2.01 W/kg, respectively) radiation on developing rat brain. Wistar rats (35 days old, male, six rats in each group) were selected for this study. These animals were exposed for 35 days at the above mentioned frequencies separately in two different exposure systems. After the exposure period, the rats were sacrificed and the whole brain tissue was dissected and used for study of single strand DNA breaks by micro gel electrophoresis (comet assay). Single strand DNA breaks were measured as tail length of comet. Fifty cells from each slide and two slides per animal were observed. One-way ANOVA method was adopted for statistical analysis. This study shows that the chronic exposure to these radiations cause statistically significant (p<0.001) increase in DNA single strand breaks in brain cells of rat.     

A study of absorption of energy of the extremely high frequency electromagnetic radiation in the rat skin by various dosimetric methods and approaches

Using experimental and theoretical methods of dosimetry, the energy absorption of extremely high-frequency electromagnetic radiation (EHF EMR) in the skin of laboratory rats was analyzed. Specific absorption rate (SAR) in the skin was determined on the basis of both microthermometric measurements of initial rates of temperature rise in rat skin induced by the exposure and microcalorimetric measurements of specific heat of the skin. Theoretical calculations of SAR in the skin were performed with consideration for dielectric parameters of rat skin obtained from the measurements of the standing wave ratio upon reflection of electromagnetic waves from the skin surface and for the effective area of stationary overheating measured by infrared thermography. A numerical method was developed to determine electromagnetic wave energy reflected, absorbed, and transmitted in the model of flat layers. The algorithm of the method was realized in a computer program and used to calculate SAR in the skin on the basis of the complex dielectric constant of rat skin. The SAR values obtained from experimental measurements, theoretical calculations and numerical analysis are in good mutual correspondence and make about 220-280 W/kg at a frequency of 42.25 GHz and a power of 20 mW at the radiator output. The results obtained can be used for dosimetric supply of biomedical experiments on studying the physicochemical mechanisms of the biological effects of EHF EMR.     

Acute effects of pulsed microwaves and 3-nitropropionic acid on neuronal ultrastructure in the rat caudate-putamen

Ultrastructure of the medium sized "spiny" neuron in rat dorsal-lateral caudate-putamen was assessed after administration of 3-nitropropionic acid (3-NP) and exposure to pulsed microwaves. Sprague-Dawley male rats were given two daily intraperitoneal doses of 0 or 10 mg/kg 3-NP and 1.5 h after each dose were exposed to microwave radiation at a whole body averaged specific absorption rate (SAR) of 0 (sham exposure), 0.6, or 6 W/kg for 30 min. Microwave exposure consisted of 1.25 GHz radiation delivered as 5.9 micros pulses with repetition frequency 10 Hz. Tissue samples taken 2-3 h after the second sham or microwave exposure showed no injury with light microscope methods. Blinded qualitative assessment of ultrastructure of randomly selected neurons from the same samples did reveal differences. Subsequent detailed, quantitative measurements showed that, when followed by sham exposure, administration of 3-NP significantly increased endoplasmic reticulum (ER) intracisternal width, ER area density, and nuclear envelope thickness. Microwave exposure at 6 W/kg alone also significantly increased these measures. Exposure of 3-NP treated animals at 6 W/kg significantly increased effects of 3-NP on ultrastructure. Although exposure at 0.6 W/kg alone did not affect ultrastructure measures, exposure of 3-NP treated animals at 0.6 W/kg reduced the effects of 3-NP. We concluded that 3-NP changed neuronal ultrastructure and that the microwave exposures used here changed neuronal ultrastructure in ways that depended on microwave SAR and neuron metabolic status. The apparent cancellation of 3-NP induced changes by exposure to pulsed microwaves at 0.6 W/kg indicated the possibility that such exposure can protect against the effects of mitochondrial toxins on the nervous system.     

Microwave irradiation and instrumental behavior in rats: Unitized irradiation and behavioral evaluation facility

A facility for the exposure of small animals to pulse-modulated microwave radiation ( PM MWR ) concurrent with their performance of operant behavioral tasks is described. The computer-managed facility comprises an array of 32 individual waveguide exposure cells, each enclosing instrumental conditioning apparatus within a plastic subhousing. The distribution of the microwave electric field intensity within the waveguide was measured by a nonperturbing probe and the modifications induced by the behavioral apparatus and animal within the waveguide determined. Input and interior voltage standing wave ratios are presented to characterize the design of the chambers and to demonstrate the suitability of the chambers for whole-body irradiation of rat. The specific absorption rate (SAR) is presented utilizing data derived from incremental thermometric examination of saline loads and of selected sites in rat carcasses. This is compared with the whole-body SAR derived from the input/ output energy balance equation for the waveguide. The results of continuous monitoring of the SAR by the latter method, while unrestrained rats were engaged in operant and exploratory behavior within the waveguide, are utilized to derive a relationship between chamber input power and the dose rate for adult rats behaviorally active within the waveguide. From these data, we conclude that the experimental array provides a practical method for exposing a large number of animals to PM MWR for long periods of time and coincident with the establishment and/or performance of complex operant behavior.     

Microwave radiation (2.45 GHz)-induced oxidative stress: Whole-body exposure effect on histopathology of Wistar rats

Man-made microwave and radiofrequency (RF) radiation technologies have been steadily increasing with the growing demand of electronic appliances such as microwave oven and cell phones. These appliances affect biological systems by increasing free radicals, thus leading to oxidative damage. The aim of this study was to explore the effect of 2.45 GHz microwave radiation on histology and the level of lipid peroxide (LPO) in Wistar rats. Sixty-day-old male Wistar rats with 180 ± 10 g body weight were used for this study. Animals were divided into two groups: sham exposed (control) and microwave exposed. These animals were exposed for 2 h a day for 35 d to 2.45 GHz microwave radiation (power density, 0.2 mW/cm²). The whole-body specific absorption rate (SAR) was estimated to be 0.14 W/kg. After completion of the exposure period, rats were sacrificed, and brain, liver, kidney, testis and spleen were stored/preserved for determination of LPO and histological parameters. Significantly high level of LPO was observed in the liver (p < 0.001), brain (p < 0.004) and spleen (p < 0.006) in samples from rats exposed to microwave radiation. Also histological changes were observed in the brain, liver, testis, kidney and spleen after whole-body microwave exposure, compared to the control group.

Based on the results obtained in this study, we conclude that exposure to microwave radiation 2 h a day for 35 d can potentially cause histopathology and oxidative changes in Wistar rats. These results indicate possible implications of such exposure on human health.     

The Effects of Single and Repeated Exposure to 2.45 GHz Radiofrequency Fields on c-Fos Protein Expression in the Paraventricular Nucleus of Rat Hypothalamus

This study investigated the effects of microwave radiation on the PVN of the hypothalamus, extracted from rat brains. Expression of c-Fos was used to study the pattern of cellular activation in rats exposed once or repeatedly (ten times in 2 weeks) to 2.45 GHz radiation in a GTEM cell. The power intensities used were 3 and 12 W and the Finite Difference Time Domain calculation was used to determine the specific absorption rate (SAR). High SAR triggered an increase of the c-Fos marker 90 min or 24 h after radiation, and low SAR resulted in c-Fos counts higher than in control rats after 24 h. Repeated irradiation at 3 W increased cellular activation of PVN by more than 100% compared to animals subjected to acute irradiation and to repeated non-radiated repeated session control animals. The results suggest that PVN is sensitive to 2.45 GHz microwave radiation at non-thermal SAR levels.     

Microwave radiation energy: a probe for the neurobiologist

Microwave energy radiation is widely used as a method for rapidly sacrificing small laboratory animals so that measurements of endogenous levels of labile neurochemical substances can be assessed after various drug treatments or pharmacological maneuvers. Several factors are important to insure that microwave energy is efficiently coupled to the rodent brain, including: the frequency of microwave radiation, the size of the waveguide for the propagation of the energy, the tuning of the waveguide system, the placement of the animal at the point of maximum power within the waveguide, the amount of power which is delivered, the time during which the power is delivered, and whether the animal is restrained during the microwave protocol.     

Radio frequency electromagnetic exposure: Tutorial review on experimental dosimetry

Radio frequency (RF) dosimetry is the quantification of the magnitude and distribution of absorbed electromagnetic energy within biological objects that are exposed to RF fields. At RF, the dosimetric quantity, which is called the specific absorption rate (SAR), is defined as the rate at which energy is absorbed per unit mass. The SAR is determined not only by the incident electromagnetic waves but also by the electrical and geometric characteristics of the irradiated subject and nearby objects. It is related to the internal electric field strength (E) as well as to the electric conductivity and the density of tissues; therefore, it is a suitable dosimetric parameter, even when a mechanism is determined to be “athermal.” SAR distributions are usually determined from measurements in human models, in animal tissues, or from calculations. This tutorial describes experimental techniques that are used commonly to determine SAR distributions along with the SAR limitations and unresolved problems. The methods discussed to obtain point, planar, or whole-body averaged SARs include the use of small E-field probes or measurement of initial rate of temperature rise in an irradiated object. © 1996 Wiley-Liss, Inc.     

Immediate post-exposure effects of high-peak-power microwave pulses on operant behavior of Wistar rats.

Behavioral effects of high-peak-power microwave pulses on Wistar rats were studied by operant schedules. Each of twelve rats that had been trained to press a lever to receive food pellets was assigned randomly in groups of four to three different schedules of reinforcement: fixed-ratio (FR), variable-interval (VI), and differential-reinforcement-of-low-rates (DRL). After achieving a steady baseline performance, each animal was exposed for 10 min to 1.25-GHz microwave radiation at 1-MW peak-power (10-microseconds pulse width). Each pulse produced a peak whole-body SA and SAR of 2.1 J/kg and 0.21 MW/kg. Total doses (SAs) were set to 0.50, 1.5, 4.5, and 14 kJ/kg by adjusting the pulse-repetition rate. The corresponding time-averaged whole-body SARs were 0.84, 2.5, 7.6, and 23 W/kg. A microwave-transparent animal holder was used to keep the animal's body axis parallel to the E-field. Exposures at the highest dose caused an average colonic temperature rise of 2.5 degrees C and these animals failed to respond at all for about 13 minutes after the exposure. Their colonic temperatures had decreased to 1.1 degrees C, or less, above their pre-exposure (normal) temperature level when they began to respond. The FR and VI animals failed to reach their baseline levels of performance thereafter, while those on the DRL schedule displayed variable effects. No behavioral effects were found at the lower dose levels. It is concluded that the behavioral perturbations produced by pulsed microwave irradiation were thermal in nature.     

Effects of exposure of newborn patched1 heterozygous mice to GSM, 900 MHz

Patched1 heterozygous knockout mice (Ptc1+/-), an animal model of multiorgan tumorigenesis in which ionizing radiation dramatically accelerates tumor development, were used to study the potential tumorigenic effects of electromagnetic fields (EMFs) on neonatal mice. Two hundred Ptc1+/- mice and their wild-type siblings were enrolled in this study. Newborn mice were exposed to 900 MHz radiofrequency radiation (average SAR: 0.4 W/kg for 5 days, 0.5 h twice a day) or were sham exposed. We found that RF EMFs simulating the Global System for Mobile Communications (GSM) did not affect the survival of the mice, because no statistically significant differences in survival were found between exposed and sham-exposed animals. Also, no effects attributable to radiofrequency radiation were observed on the incidence and histology of Ptc1-associated cerebellar tumors. Moreover, the skin phenotype was analyzed to look for proliferative effects of RF EMFs on the epidermal basal layer and for acceleration of preneoplastic lesions typical of the basal cell carcinoma phenotype of this model. We found no evidence of proliferative or promotional effects in the skin from neonatal exposure to radiofrequency radiation. Furthermore, no difference in Ptc1-associated rhabdomyosarcomas was detected between sham-exposed and exposed mice. Thus, under the experimental conditions tested, there was no evidence of life shortening or tumorigenic effects of neonatal exposure to GSM RF radiation in a highly tumor-susceptible mouse model.     

Cognitive impairment in rats after long-term exposure to GSM-900 mobile phone radiation

Considering the frequent use of mobile phones, we have directed attention to possible implications on cognitive functions. In this study we investigated in a rat model the long-term effects of protracted exposure to Global System for Mobile Communication-900 MHz (GSM-900) radiation. Out of a total of 56 rats, 32 were exposed for 2 h each week for 55 weeks to radio-frequency electromagnetic radiation at different SAR levels (0.6 and 60 mW/kg at the initiation of the experimental period) emitted by a (GSM-900) test phone. Sixteen animals were sham exposed and eight animals were cage controls, which never left the animal house. After this protracted exposure, GSM-900 exposed rats were compared to sham exposed controls. Effects on exploratory behaviour were evaluated in the open-field test, in which no difference was seen. Effects on cognitive functions were evaluated in the episodic-like memory test. In our study, GSM exposed rats had impaired memory for objects and their temporal order of presentation, compared to sham exposed controls (P = 0.02). Detecting the place in which an object was presented was not affected by GSM exposure. Our results suggest significantly reduced memory functions in rats after GSM microwave exposure (P = 0.02).     

The effect of chronic exposure to 835.62 MHz FDMA or 847.74 MHz CDMA radiofrequency radiation on the incidence of spontaneous tumors in rats

This study was designed to determine whether chronic exposure to radiofrequency (RF) radiation from cellular phones increased the incidence of spontaneous tumors in F344 rats. Eighty male and 80 female rats were randomly placed in each of three irradiation groups. The sham group received no irradiation; the Frequency Division Multiple Access (FDMA) group was exposed to 835.62 MHz FDMA RF radiation; and the Code Division Multiple Access (CDMA) group was exposed to 847.74 MHz CDMA RF radiation. Rats were irradiated 4 h per day, 5 days per week over 2 years. The nominal time-averaged specific absorption rate (SAR) in the brain for the irradiated animals was 0.85 +/- 0.34 W/kg (mean +/- SD) per time-averaged watt of antenna power. Antennas were driven with a time-averaged power of 1.50 +/- 0.25 W (range). That is, the nominal time-averaged brain SAR was 1.3 +/- 0.5 W/kg (mean +/- SD). This number was an average from several measurement locations inside the brain, and it takes into account changes in animal weight and head position during irradiation. All major organs were evaluated grossly and histologically. The number of tumors, tumor types and incidence of hyperplasia for each organ were recorded. There were no significant differences among final body weights or survival days for either males or females in any group. No significant differences were found between treated and sham-exposed animals for any tumor in any organ. We conclude that chronic exposure to 835.62 MHz FDMA or 847.74 MHz CDMA RF radiation had no significant effect on the incidence of spontaneous tumors in F344 rats.     

Compact shielded exposure system for the simultaneous long-term UHF irradiation of forty small mammals. II. Dosimetry

A four-antenna collinear array in an electromagnetically shielded chamber was designed and constructed to preferentially irradiate the brains of a large number of small mammals using cellular telephony microwave signals. Ten animals in special restrainers were positioned symmetrically around a centrally located antenna. These restrainers are resting on a circular structure made of acrylic plastic called a "carousel." Four carousels are stacked vertically, forming the array, inside a microwave anechoic chamber called a "chamberette." (Details of the design of this irradiator and of a 12-chamberette irradiation facility are given in a previous article.) In this article, the dosimetry on rats is reported. Both thermometric and thermographic measurements were performed. The average specific absorption rate (SAR) in brain tissue measured thermometrically was 0.85+/-0.34 W/kg per watt of net input power into the radiating antenna. This range agrees with the SAR levels reported in the literature for cellular telephones. Thermographic evaluation using splittable phantoms showed that most of the energy absorbed by the rats is concentrated in and around the brain. Moreover, it was found that the SAR in brain tissue can vary considerably for rats of similar weights, depending on position of the rats' heads inside the restrainers, and that there exists a significant dependence of SAR on animal weight. These variations may be of importance in the interpretation of results of lifelong studies. The data presented clearly show that the chamberette is, dosimetrically, a suitable irradiation system for electromagnetic bioeffects studies in the cellular communication frequency range, especially when a large number of laboratory animals is required.     

Influence of 2.45 GHz microwave radiation on enzyme activity

Summary The in vitro activity of acetylcholinesterase and creatine phosphokinase was determined during in vitro exposure to 2.45 GHz microwave radiation. The enzyme activities were examined during exposure to microwave radiation at specific absorption rates (SAR) of 1, 10, 50, and 100 mW/g. These specific absorption rates had no effect on the activity of either enzyme when the temperature of the control and exposed samples were similar. These data demonstrate that the activity of these two enzymes is not affected by microwave radiation at the SARs and frequency employed in this study.     

Radiofrequency radiation at 1950 MHz (UMTS) does not affect key cellular endpoints in neuron-like PC12 cells

In this study, rat pheochromocytoma (PC12) cells were exposed, as a model of neuron-like cells, to 1950 MHz radiofrequency (RF) radiation with a signal used by the 3G wireless technology of the Universal Mobile Telecommunications System (UMTS) to assess possible adverse effects. RF exposure for 24 h at a specific absorption rate (SAR) of 10 W/kg was carried out in a waveguide system under accurately controlled environmental and dosimetric parameters. DNA integrity, cell viability, and apoptosis were investigated as cellular endpoints relevant for carcinogenesis and other diseases of the central nervous system. Very sensitive biological assays were employed to assess the effects immediately after RF exposure and 24 h later, as demonstrated by the cellular response elicited in PC12 cells using positive control treatments provided for each assay. In our experimental conditions, 24 h of RF exposure at a carrier frequency and modulation scheme typical of a UMTS signal was not able to elicit any effect in the selected cellular endpoints in undifferentiated PC12 cells, despite the application of a higher SAR value than those applied in the majority of the studies reported in the literature.     

Effects of microwave exposure and temperature on survival of mice infected with Streptococcus pneumoniae

Female CD-1 mice were injected with an LD₅₀ dose of Streptococcus pneumoniae and then exposed to 2.45 GHz (CW) microwave radiation at an incident power density of 10 mW/cm² (SAR = 6.8 W/kg), 4 h/d for 5 d at ambient temperatures of 19 °C, 22 °C, 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C. Four groups of 25 animals were exposed at each temperature with an equal number of animals concurrently sham-exposed. Survival was observed for a 10-d period after infection. Survival of the sham-exposed animals increased as ambient temperature increased from 19 °C–34 °C. At ambient temperatures at or above 37 °C the heat induced in the body exceeded the thermoregulatory capacity of the animals and deaths from hyperthermia occurred. Survival of the microwave-exposed animals was significantly greater than the shams (～20%) at each ambient temperature below 34 °C. Based on an analysis of the data it appears that the hyperthermia induced by microwave exposure may be more effective in increasing survival in infected mice than hyperthermia produced by conventional methods (ie, high ambient temperature). Microwave radiation may be beneficial to infected animals at low and moderate ambient temperatures, but it is detrimental when combined with high ambient temperatures.     

In utero exposure to microwave radiation and rat brain development

Timed-pregnancy rats were exposed in a circular waveguide system starting on day 2 of gestation. The system operated at 2,450 MHz (pulsed waves; 8 microseconds PW; 830 pps). Specific absorption rate (SAR) was maintained at 0.4 W/kg by increasing the input power as the animals grew in size. On day 18 of gestation the dams were removed from the waveguide cages and euthanized; the fetuses were removed and weighed. Fetal brains were excised and weighed, and brain RNA, DNA and protein were determined. Values for measured parameters of the radiated fetuses did not differ significantly from those of sham-exposed fetuses. A regression of brain weight on body weight showed no micrencephalous fetuses in the radiation group when using as a criterion a regression line based on two standard errors of the estimate of the sham-exposed group. In addition, metrics derived from brain DNA (ie, cell number and cell size) showed no significant differences when radiation was compared to sham exposure. We conclude that 2,450-MHz microwave radiation, at an SAR of 0.4 W/kg, did not produce significant alterations in brain organogenesis.     

Stress proteins are not induced in mammalian cells exposed to radiofrequency or microwave radiation

The induction of stress proteins in HeLa and CHO cells was investigated following a 2 h exposure to radiofrequency (RF) or microwave radiation. Cells were exposed or sham exposed in vitro under isothermal (37 ± 0.2 °C) conditions. HeLa cells were exposed to 27- or 2450 MHz continuous wave (CW) radiation at a specific absorption rate (SAR) of 25 W/kg. CHO cells were exposed to CW 27 MHz radiation at a SAR of 100 W/kg. Parallel positive control studies included 2 h exposure of HeLa or CHO cells to 40 °C or to 45 μM cadmium sulfate. Stress protein induction was assayed 24 h after treatment by electrophoresis of whole-cell extracted protein labeled with [³⁵S]-methionine. Both cell types exhibited well-characterized responses to the positive control stresses. Under these exposure conditions, neither microwave nor RF radiation had a detectable effect on stress protein induction as determined by either comparison of RF-exposed cells with sham-exposed cells or comparison with heat-stressed or Cd⁺⁺ positive control cells. Bioelectromagnetics 18:499–505, 1997. © 1997 Wiley-Liss, Inc.