Luminescent radio frequency radiation dosimetry.

Thermoluminescent dosimetry has been the industry standard for ionizing radiation dosimetry because it is inexpensive, sensitive, and accurate. No such system exists for radio frequency radiation. This paper describes the state of the art of efforts toward developing such a system. Thermochemiluminescent (TCL) dosimetry, first reported in 1991, is a first step toward achieving this goal. However, it has had problems in the production of TCL materials and in conversion of the luminescent signal into specific absorption rate (SAR). The former problem has been solved by the development of a genetically engineered Escherichia coli bacterium (JM 109/plC20RNR1.1), described herein, that produces the TCL material in a fermentation process. The latter problem stems from the difficulty in determining the structure of the currently best TCL material diazoluminomelanin. A theoretical approach for the solution of this problem has been achieved by combining equations for delayed fluorescence, temperature determination by TCL, and the free energy equation for equilibrium reactions. It has led to an explanation for the stable display of steady-state energy disposition, illustrated by TCL, in phantoms without the expected disruption by thermal conduction or convection, at frequencies ranging from 2.06 GHz to 35 GHz.

Database created from magnetic resonance images of a Sprague-Dawley rat, rhesus monkey, and pigmy goat.

To obtain a database of accurate anatomical images onto which dosimetry data of electromagnetic fields could be mapped, a healthy Sprague-Dawley rat, rhesus monkey, and pigmy goat were scanned using magnetic resonance imaging (MRI). Axial sections throughout the length of the animals were collected. Sections were 3 mm thick for the rat and 5 mm thick for the monkey and goat. Sagittal sections (2 mm thick) of the rat head were also scanned. Images were recorded on magnetic tape and transferred to computer disk for image enhancement and network distribution. Images are available in 16 bit Big Endian signed or 8 bit TIFF formats. This is the first database of contiguous MRI axial scans of rat, monkey, and goat available for distribution via magnetic tape (4 mm DDS) or Internet file transfer protocol. Digital transfer of the data was selected to preserve the integrity of each image, circumventing the need for the user to scan the images back into a digital format for use with their software. These images should be useful to physiologists, neuroscientists, veterinarians, anatomists, and teachers. Reconstructing these 2-dimensional images into 3-dimensional structures is an effective media for conveying spatial anatomical information in a quick and comprehensive manner.

Absence of a synergistic effect between moderate-power radio-frequency electromagnetic radiation and adriamycin on cell-cycle progression and sister-chromatid exchange.

In our laboratories we are conducting investigations of potential interactions between radio-frequency electromagnetic radiation (RFR) and chemicals that are toxic by different mechanisms to mammalian cells. The RFR is being tested at frequencies in the microwave range and at different power levels. We report here on the 1) ability of simultaneous RFR exposures to alter the distribution of cells in first and second mitoses from that after treatment by adriamycin alone, and 2) on the ability of simultaneous RFR exposure to alter the extent of sister chromatid exchanges (SCEs) induced by adriamycin alone. This chemical was selected because of its reported mechanism of action and because it is of interest in the treatment of cancer. In our studies, Chinese hamster ovary (CHO) cells were exposed for 2 h simultaneously to adriamycin and pulsed RFR at a frequency of 2,450 MHz and a specific absorption rate of 33.8 W/Kg. The maximal temperature (in the tissue-culture medium) was 39.7 +/- 0.2 degrees C. The experiments were controlled for chemical and RFR exposures, as well as for temperature. Verified statistically, the data indicate that the RFR did not affect changes in cell progression caused by adriamycin, and the RFR did not change the number of SCEs that were induced by the adriamycin, which adriamycin is known to affect cells by damaging their membranes and DNA.

Influence of radiofrequency radiation on chromosome aberrations in CHO cells and its interaction with DNA-damaging agents.

A limited number of contradictory reports have appeared in the literature about the ability of radiofrequency (rf) radiation to induce chromosome aberrations in different biological systems. The technical documentation associated with such reports is often absent or deficient. In addition, no information is available as to whether any additional genotoxic hazard would result from a simultaneous exposure of mammalian cells to rf radiation and a chemical which (by itself) induces chromosome aberrations. In the work described, we have therefore tested two hypotheses. The first is that rf radiation by itself, at power densities and exposure conditions which are higher than is consistent with accepted safety guidelines, can induce chromosome aberrations in mammalian cells. The second is that, during a simultaneous exposure to a chemical known to be genotoxic, rf radiation can affect molecules, biochemical processes, or cellular organelles, and thus result in an increase or decrease in chromosome aberrations. Mitomycin C (MMC) and Adriamycin (ADR) were selected because they act by different mechanisms, and because they might put normal cells at risk during combined-modality rf radiation (hyperthermia)-chemotherapy treatment of cancer. The studies were performed with suitable 37 degrees C and equivalent convection heating-temperature controls in a manner designed to discriminate between any thermal and possible nonthermal action. Radiofrequency exposures were conducted for 2 h under conditions resulting in measurable heating (a maximum increase of 3.2 degrees C), with pulsed-wave rf radiation at a frequency of 2450 MHz and an average net forward power of 600 W, resulting in an SAR of 33.8 W/kg. Treatments with MMC or ADR were for a total of 2.5 h and encompassed the 2-h rf radiation exposure period. The CHO cells from each of the conditions were subsequently analyzed for chromosome aberrations. In cells exposed to rf radiation alone, and where a maximum temperature of approximately 40 degrees C was achieved in the tissue culture medium, no alteration in the frequency from 37 degrees C control levels was observed. Relative to the chemical treatment with MMC alone at 37 degrees C, for two different concentrations, no alteration was observed in the extent of chromosome aberrations induced by either simultaneous rf radiation exposure or convection heating to equivalent temperatures. At the ADR concentration that was used, most of the indices of chromosome aberrations which were scored indicated a similar result.(ABSTRACT TRUNCATED AT 400 WORDS)

Proflavin and microwave radiation: absence of a mutagenic interaction.

The potential ability of radiofrequency electromagnetic radiation (RFR) in the microwave range to induce mutagenesis, chromosomal aberrations, and sister chromatid exchanges in mammalian cells is being explored in our laboratories. In addition, we have also been examining the ability of simultaneous exposure to RFR and chemical mutagens to alter the genotoxic damage induced by chemical mutagens acting alone. We have performed experiments to determine whether there is an interaction between 2.45-GHz, pulsed-wave, RFR and proflavin, a DNA-intercalating drug. The endpoint studied was forward mutation at the thymidine kinase locus in L5178Y mouse leukemic cells. Any effect on the size distribution of the resulting colonies of mutated cells was also examined. The exposures were performed at net forward powers of 500 or 600 W, resulting in a specific absorption rate (SAR) of approximately 40 W/kg. The culture-medium temperature reached a 3 degrees C maximal increase during the 4-h exposure; appropriate 37 degrees C and convection-heating temperature controls (TC) were performed. In no case was there any indication of a statistically significant increase in the induced mutant frequency due to the simultaneous exposure to RFR and proflavin, as compared with the proflavin exposures alone. There was also no indication of any change in the colony-size distribution of the resulting mutant colonies, neither, and there was no evidence in these experiments of any mutagenic action by the RFR exposure alone.

Interactions of methemoglobin and green hemoprotein in chemiluminescent gels.

Peroxidation of luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) catalyzed by human green hemoprotein (GHP) and bovine methemoglobin (MetHb) in gels composed of cross-linked bovine serum albumin was examined. The chemiluminescence (CL) was followed with a low-light intensity video camera and imaging system attached to a circularly polarized microwave guide (2450 MHz) for heating the samples from 24 degrees C to 37 degrees C. Steady-state CL was maintained in the gels for 10 min. The intensity of the CL varied with temperature. When combined with MetHb in the same gel, GHP inhibited CL of MetHb from 83.6% to 98.2% over a fiftyfold concentration range of GHP. Although MetHb/GHP combination gels were inhibited, they generated a 6.12-fold CL per degree C change compared to a 0.19-fold per degree C change for MetHb gels and a 0.31-fold per degree C change for GHP gels. The data suggest an interaction between GHP and MetHb that inhibits the CL reaction, is not interfered with by large amounts of albumin, and is partially reversed by heating.

Absence of mutagenic interaction between microwaves and mitomycin C in mammalian cells.

Evidence in the literature from in vitro and in vivo studies as to whether or not radiofrequency radiation (RFR) in the microwave range is mutagenic is predominantly negative, with some positive reports. No evidence is available as to whether RFR will alter the mutagenic activity of genotoxic chemicals during a simultaneous exposure, a likely real-life situation. Two hypotheses have been proposed: a) that RFR by itself can cause mutations in a mammalian cell in vitro assay system; and b) that a simultaneous exposure to RFR during a chemical treatment of the cells with a known genotoxic agent, mitomycin C (MMC), will alter the extent of mutagenesis induced by the treatment of the cells by the chemical alone. These studies were performed using the forward mutation assay at the thymidine kinase locus in L5178Y mouse leukemic cells. The pulsed wave RFR was broadcast from an antenna horn at a frequency of 2.45 GHz. The power density was 48.8 mW/cm2 and the measured specific absorption rate (SAR) in this system was 30 W/kg (600 W forward power), which is well above current safety guidelines. The conclusions from five different experiments, employing three different concentrations of MMC, were that a) RFR exposure alone, at moderate power levels which resulted in a temperature increase in the cell culture medium of less than 3 degrees C, is not mutagenic; and b) when cells are simultaneously treated with MMC and RFR at these same moderate power levels, the RFR does not affect either the inhibition of cell growth or the extent of mutagenesis resulting from the treatment with the chemical MMC alone.

Mechanisms of biological effects of radiofrequency electromagnetic fields: an overview.

Manmade sources of electromagnetic (EM) fields, and therefore human exposures to them, continue to increase. Public concerns stem from the effects reported in the literature, the visibility of the sources, and somewhat from confusion between EM fields and ionizing radiation. Protecting humans from the real hazards and allaying groundless fears requires a self-consistent body of scientific data concerning effects of the fields, levels of exposures which cause those effects, and which effects are deleterious (or beneficial or neutral). With that knowledge, appropriate guidelines for safety can be devised, while preserving the beneficial uses of radiofrequency radiation (RFR) energy for military or civilian purposes. The task is monumental because of the large and growing number of biological endpoints and the infinite array of RFR exposure conditions under which those endpoints might be examined. The only way to reach this goal is to understand the mechanisms by which EM fields interact with tissues. As in other fields of science, a mechanistic understanding of RFR effects will enable scientists to generalize from a selected few experiments to derive the "laws" of RFR bioeffects. This article gives an overview of present knowledge of those mechanisms and the part that the USAF School of Aerospace Medicine has played in expanding that knowledge.

Green hemoprotein of erythrocytes: methemoglobin superoxide transferase.

Influences of base (pH 10), heat (50 degrees C), microwave radiation (2450 MHz, 103 +/- 4 W/kg), and hydrogen peroxide (5.6 mM) generated by glucose oxidase on oxidation of human oxyhemoglobin to methemoglobin were examined. Conversion of oxyhemoglobin to methemoglobin was followed by the difference in absorbancy of 540 or 542 nm and 576 nm wavelength light versus time. Fresh basic hemolysates auto-oxidized on heating with a zero order rate constant, implying that hemoglobin or another protein saturated with oxyhemoglobin catalyzed the oxidation. Simultaneous microwave irradiation inhibited thermally induced auto-oxidation on the average by 28.6%. However, there was great variability among samples and a decrease in auto-oxidation with aging of individual samples. The auto-oxidation rate was independent of initial oxyhemoglobin concentration. Oxidation of partially purified oxyhemoglobin by hydrogen peroxide was not influenced by microwave irradiation. Adding green hemoprotein isolated from human erythrocytes to the oxyhemoglobin/glucose oxidase reaction mixture yielded absorption spectra (500-600 nm) that were a combination of oxyhemoglobin, deoxyhemoglobin, and methemoglobin spectra. Green hemoprotein was labile in hemolysates but stable in a partially purified ferric form. These results imply that thermally unstable reduced green hemoprotein can reverse oxidation of oxyhemoglobin by hydrogen peroxide and could mediate the thermally induced and microwave inhibited auto-oxidation of oxyhemoglobin.

Dosimetry considerations in far field microwave exposure of mammalian cells.

A circulating water bath exposure system has been designed for in vitro radiofrequency radiation (RFR) exposure studies in the 915 to 2450 MHz range. A Styrofoam float, in which 10 T-25 plastic tissue culture flasks are embedded, is rotated at approximately 20 rpm in a Plexiglas water bath at a distance beneath a rectangular horn. The continuous circular rotation of the flasks is designed to "average out" the heterogeneity present in stationary flask exposures. The rotation also serves to prevent the establishment of chemical gradients in the medium within the flasks. Several factors have been demonstrated to affect the specific absorption rate (SAR) measured in the medium in the exposed flasks. These factors include: 1) the position of the exposure flasks relative to the long axis of the antenna horn; 2) whether the flasks are exposed while stationary or in rotation; 3) the volume of the medium contained in the flask; and 4) the depth in the medium in the flask at which temperatures for SAR calculation are measured. The presence of cells in the exposure flask (as attached monolayer or cell suspension) did not result in an SAR different from that measured in the same volume of medium without cells present.

Radiofrequency (microwave) radiation exposure of mammalian cells during UV-induced DNA repair synthesis.

The effect of continuous-wave (CW) and pulsed-wave (PW) radiofrequency radiation (RFR) in the microwave range on UV-induced DNA repair has been investigated in MRC-5 normal human diploid fibroblasts. RFR exposure at power densities of 1 (or 5) and 10 mW/cm2 gave a maximum specific absorption rate (SAR) (at 10 mW/cm2) of 0.39 +/- 0.15 W/kg for 350 MHz RFR, 4.5 +/- 3.0 W/kg for 850 MHz RFR, and 2.7 +/- 1.6 W/kg for 1.2 GHz RFR. RFR exposures for 1 to 3 h at 37 degrees C, in either continuous-wave or pulsed-wave modes, had no effect on the rate of repair replication label incorporated into preexisting UV-damaged DNA. RFR exposures (PW), with a constant medium temperature of 39 degrees C at 350 and 850 MHz during the repair period after UV damage, also had no effect. Assay for induction of repair synthesis by RFR exposure alone in non-UV irradiated cells was negative for the 350-, 850-, and 1200-MHz CW and PW RFR at 37 degrees C and the 350- and 850-MHz PW RFR at 39 degrees C. RFR does not induce DNA repair under these exposure conditions. In preliminary experiments--with the tissue culture medium maintained at 39 degrees C and RFR exposures (PW) at the frequencies of 350, 850, and 1200 MHz--no effect on incorporation of [3H]thymidine into DNA undergoing semiconservative synthesis was observed.

Effects of radiofrequency radiation and simultaneous exposure with mitomycin C on the frequency of sister chromatid exchanges in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were exposed for 2 hr with and without mitomycin C (MMC) (1 X 10(-8)M) to pulsed wave radiofrequency radiation (RFR) at 2450 MHz. The repetition rate of 25,000 pulses per sec (pps), pulse width of 10 microseconds, and exposure geometry used, resulted in a specific absorption rate (SAR) of 33.8 W/kg. The following exposure regimens were used: a 37 degrees C water bath control; a water bath temperature control (TC) in which the continuously monitored medium temperature closely followed the temperature rise in the RFR-exposed flasks; and the RFR-exposed cells in a water bath set at 37 degrees C prior to exposure. RFR exposure resulted in a maximum cell culture medium temperature of 39.2 degrees C. In the absence of MMC, there was no significant increase in sister chromatid exchange (SCE) in the RFR-exposed or TC groups over that of the 37 degrees C control. When a simultaneous treatment of RFR and MMC occurred there was no statistical difference in SCE frequency from that caused by chemical treatment alone.

Physiologic aging of mature porcine erythrocytes: effects of various metabolites, antimetabolites, and physical stressors.

Red blood cells were collected from Landrace X Duroc pigs in pooled and single batches. The RBC were stored for 24 hours to 20 days and exposed to 1 or more chemical and physical stressors. The chemicals were pyruvate, lactate, inosine, concanavalin A-luminol-bovine serum albumin conjugate, hydrogen peroxide, L-mimosine, and 3-amino-L-tyrosine. Physical stressors included thermogenic microwave radiation (2,450 MHz, mean specific absorption rate of 91 W/kg) and conventional heating with hot air or hot-water bath. Heating erythrocytes to 43 C for 10 minutes with microwaves or hot air did not significantly (P greater than 0.05) increase hemolysis, compared with hemolysis of RBC at 4 C (controls). Pyruvate or lactate did not affect the RBC under these conditions. Hemolysis of cells coated with concanavalin A-luminol-bovine serum albumin and heated to 43 C for 10 minutes by use of microwaves or hot air was significantly (P less than 0.05 by Student's t test) greater (48%) than that of RBC at 4 C (controls). The method of heating or the presence of pyruvate, lactate, or inosine did not have a significant (P greater than 0.05) effect on hemolysis. Lysis of RBC (14 days after collection) coated with concanavalin A-luminol-bovine serum albumin and stored at 4 C was not significantly different (P greater than 0.05) than that of noncoated cells (6 days after collection) stored at 4 C (control). When RBC were heated 20 days after collection to 48 C for 30 minutes, using a hot water bath, hemolysis was 60.6% greater than that of control cells (4 C).(ABSTRACT TRUNCATED AT 250 WORDS)

Microwave radiation effects on the thermally driven oxidase of erythrocytes.

Sheep red blood cells (SRBCs) were labelled with a concanavalin A-luminol-bovine serum albumin conjugate specific for the transmembrane anion transport protein (Band 3) and exposed to 2450 MHz continuous wave microwave radiation at an average specific absorption rate of 91 W/kg for 10 min. The temperature was held constant at 25, 37, 40, 42 or 45 degrees C with an airflow heat exchange system. Following exposure to microwave or air heating, the decrease in residual base-activated chemiluminescence (CL) of the SRBCs was measured as an indication of infield oxidase activity. Air heating resulted in a significant decrease in residual CL at temperatures above 37 degrees C (74 per cent decrease at 45 degrees C). Microwave radiation inhibited the decline in residual CL above 37 degrees C. At 45 degrees C the inhibition was 40 per cent. The results suggest microwave radiation either reversibly altered the thermodynamics of oxygen binding to haemoglobin or failed to energize a significant portion of the haemoglobin molecules in each sample to the thermal threshold of haemoglobin autoxidation.

Metabolic effects of microwave radiation and convection heating on human mononuclear leukocytes.

The effects of microwave radiation (2450 MHz, continuous wave, mean specific absorption rate of 103.5 +/- 4.2 W/kg) and convection heating on the nonphosphorylating oxidative metabolism of human peripheral mononuclear leukocytes (96% lymphocytes, 4% monocytes) at 37 degrees C were investigated. Metabolic activity, determined by chemiluminescence (CL) of cells challenged with luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) linked to bovine serum albumin, was detected with a brightness photometer. A significant stimulation after microwave exposure (p less than 0.005) over total CL of matched 37 degrees C incubator controls was observed. A similar degree of stimulation compared to incubator controls was also detected after sham treatment. There was no significant difference between changes in total CL or stimulation indices of the microwave and sham exposed groups. It appears that exposure to microwave radiation, under normothermic (37 +/- 0.03 degrees C) conditions, has no effect on the oxidative metabolic activity of human peripheral mononuclear leukocytes. However, the significant differences between microwave or sham exposed cells and their respective incubator controls occurred because the temperature of the incubator controls did not exceed 35.9 degrees C and this temperature required 39 minutes to reach from 22 degrees C. Slow heating of incubator controls must be accounted for in thermal and radiofrequency radiation studies in vitro.

Thermochemiluminescent assay of porcine, rat, and human erythrocytes for antioxidative deficiencies.

The thermal induction of chemiluminescence of luminol-horseradish peroxidase-labeled erythrocytes from pigs, rats, and man was studied. The luminescent responses of rat, porcine, and human erythrocytes to heating were linear in respect to logs of counts per minute versus temperature. Landrace-Duroc crossbred pigs with a history of malignant hyperthermia (porcine stress syndrome) and Poland-China-miniature pigs inbred for malignant hyperthermia (MH) yielded erythrocytes with high-level thermochemiluminescence (TCL). Sprague-Dawley rat erythrocytes were intermediate in their production of TCL. Normal human and MH-resistant miniature swine erythrocytes produced low-level TCL. However, pretreatment of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) resulted in high-level TCL. Furthermore, halothane enhanced the TCL of CDNB-treated human erythrocytes and Landrace-Duroc porcine erythrocytes that were not treated with CDNB. Red blood cells from pigs susceptible to the porcine stress syndrome demonstrated a TCL response very similar to CDNB-treated erythrocytes.

Microwave and thermal interactions with oxidative hemolysis.

The influence of microwave radiation (2450 MHz, 3,333 pulses per second, duty factor of 0.02, and average specific absorption rate of 0.4 W/kg) on spontaneous hemolysis of human erythrocytes was examined. Cells were exposed to microwave radiation for 20 minutes at 37 degrees, 42 degrees, or 48 degrees C. Some of these cells were sensitized to oxidative damage by treatment with 1-chloro-2,4-dinitrobenzene (CDNB) and/or by coating with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) conjugate. Microwave radiation significantly decreased spontaneous hemolysis of untreated cells at 42 degrees C but had no effect at 37 degrees or 48 degrees C. Microwave exposure significantly enhanced a CDNB membrane stabilizing effect at 42 degrees C but had no effect at 37 or 48 degrees C. At 42 degrees C, microwave exposure increased hemolysis of WGA-HRP coated cells. Cells treated with both WGA-HRP and CDNB showed no change in fragility at 42 degrees C and increased fragility at 48 degrees C without a microwave effect. The microwave effects observed appear to involve perturbation of the thermal threshold for oxidative hyperthermic hemolysis.