2-Methoxyethanol metabolism, embryonic distribution, and macromolecular adduct formation in the rat: the effect of radiofrequency radiation-induced hyperthermia.

Exposure of pregnant rats to the solvent 2-methoxyethanol (2ME) and radiofrequency (RF) radiation results in greater than additive fetal malformations (Nelson, B.K., Conover, D.L., Brightwell, W.S., Shaw, P.B., Werren, D.W., Edwards, R.M., Lary, J.M., 1991. Marked increase in the teratogenicity of the combined administration of the industrial solvent 2-methoxyethanol and radiofrequency radiation in rats. Teratology 43, 621-34; Nelson, B.K., Conover, D.L., Shaw, P.B., Werren, D.W., Edwards, R.M., Hoberman, A.M., 1994. Interactive developmental toxicity of radiofrequency radiation and 2-methoxyethanol in rats. Teratology 50, 275-93). The current study evaluated the metabolism of 14C-labeled 2ME and the distribution of methoxyacetic acid (MAA) in maternal and embryonic tissues of pregnant Sprague-Dawley rats either exposed to 10 MHz RF radiation or sham conditions. Additionally, adduct formation for both plasma and embryonic protein was tested as a possible biomarker for the observed 2ME/RF teratogenicity. Rats were administered [ethanol-1,2-(14)C]-2ME (150 mg/kg, 161 microCi/rat average) by gavage on gestation day 13 immediately before RF radiation sufficient to elevate body temperature to 42 degrees C for 30 min. Concurrent sham- and RF-exposed rats were sacrificed at 3, 6, 24 or 48 h for harvest of maternal blood, urine, embryos and extra-embryonic fluid. Tissues were either digested for determination of radioactivity or deproteinized with TCA and analyzed by HPLC for quantification of 2ME metabolites. Results show the presence of 2ME and seven metabolites, with the major metabolite, MAA, peaking at 6 h in the tissues tested. MAA, the proximal teratogen, was detectable in maternal serum, urine, embryo and extraembryonic fluid 48 h after dosing. Clearance of total body 14C was significantly reduced for the RF-exposed animals (P<0.05) for the 24-48 h period, but MAA values for serum, embryos and extraembryonic fluid were similar for both sham- and RF-exposed rats. Additionally, no difference was noted for 2ME metabolite profiles in urine or tissue for sham- or RF-exposed rats, thus eliminating an effect of RF radiation on MAA production as a possible explanation for the reported RF-2ME synergism. Subsequently, serum and embryo protein-bound adducts were evaluated by analysis of covalently bound radioactivity. Serum protein binding was significantly higher for sham than RF rats at 3- and 6-h - highest for sham rats at 6 h (519+/-95 microg as parent 2ME/g of protein) whereas RF serum values were highest at 24 h (266+/-79 microg/g protein). Embryonic protein binding was significantly higher for sham rats at 6 h, but binding was highest for both groups at 24 h (sham=229+/-71 microg/g, RF=185+/-48 microg/g). Formation of protein adducts after 2ME is thought to be related to levels of methoxyacetaldehyde, a reactive intermediate in the formation of MAA. These results suggest that no direct relationship exists for covalent binding in the embryo which would explain RF-2ME synergistic malformations. In comparison with urinary metabolites, the relatively slow elimination of adducted serum 2ME indicates that analysis of protein-bound concentrations could be a potential tool for long- term biomonitoring of worker exposure.

Photochemical oxygen consumption, oxygen evolution and spectral changes during UVA irradiation of EMT6 spheroids.

Remarkable rates of oxygen consumption are observed via microelectrode measurements immediately upon the onset of 325 nm irradiation of multicell tumor spheroids. Consumption is irradiance dependent over the range 20-200 mW cm-2, and its magnitude is comparable to that observed previously in the same system using exogenous photosensitizers. Oscillations in the oxygen concentrations suggest that oxygen is also being evolved during irradiation. Oxygen evolution is likely the result of enzymatic dissociation of hydrogen peroxide, which is formed through UV-induced photochemistry. Irradiation of spheroids at 442 and at 514 nm produces a much more modest but detectable oxygen consumption. The dynamics of oxygen concentration changes are quite different at these wavelengths, suggesting a different photochemical mechanism. In these cases, initial oxygen depletion is followed immediately by a more gradual, monotonic increase in the oxygen concentration, consistent with irreversible photobleaching. No oscillations in the oxygen concentration are detectable. At 662 nm, no oxygen consumption was observed over the range of irradiances studied. Fluorescence spectra of cells prior to irradiation include contributions from anthranilic acid and reduced nicotinamide adenine dinucleotide (NADH). During 325 nm irradiation, anthranilic acid is rapidly and irreversibly bleached, while NADH emission undergoes only modest reduction.

An evaluation of near infrared spectroscopy and cryospectrophotometry estimates of haemoglobin oxygen saturation in a rodent mammary tumour model.

Haemoglobin oxygen saturation in subcutaneous rat mammary tumours was measured using near infrared spectroscopy (NIRS) in vivo and in rapidly frozen sections from the same tumours using cryospectrophotometry, which reports oxygen saturation in individual blood vessels to depths of 4 mm from the tissue surface. Measurements were performed on tumours while animals breathed either room air or carbogen. In five of nine tumours, the average saturation calculated from cryospectrophotometric measurements agreed with that determined from NIRS to within 13%, and in four of these five tumours agreement was 5% or better. In the remaining four of nine tumours, where agreement was poor, the volume-averaged saturations estimated from NIRS were consistently higher than those calculated from cryospectrophotometry. Monte Carlo simulations demonstrated that the depth of tissue probed by NIRS was significantly greater than that sampled by cryospectrophotometry. Analysis of the frequency of severely hypoxic vessels showed that when NIRS reported a saturation of approximately 70% or higher, the fraction of tumour vessels with saturations less than 10% was limited to 0.06 or less. Sensitivity and specificity analysis suggests that NIRS and NIRS imaging may identify clinically relevant hypoxia, even when its spatial extent is below the resolution limit of the NIRS technique.

Carbogen-induced changes in rat mammary tumour oxygenation reported by near infrared spectroscopy.

We have evaluated the ability of steady-state, radially-resolved, broad-band near infrared diffuse reflectance spectroscopy to measure carbogen-induced changes in haemoglobin oxygen saturation (SO2) and total haemoglobin concentration in a rat R3230 mammary adenocarcinoma model in vivo. Detectable shifts toward higher saturations were evident in all tumours (n = 16) immediately after the onset of carbogen breathing. The SO2 reached a new equilibrium within 1 min and remained approximately constant during 200-300 s of administration. The return to baseline saturation was more gradual when carbogen delivery was stopped. The degree to which carbogen increased SO2 was variable among tumours, with a tendency for tumours with lower initial SO2 to exhibit larger changes. Tumour haemoglobin concentrations at the time of peak enhancement were also variable. In the majority of cases, haemoglobin concentration decreased in response to carbogen, indicating that increased tumour blood volume was not responsible for the observed elevation in SO2. We observed no apparent relationship between the extent of the change in tumour haemoglobin concentration and the magnitude of the change in the saturation. Near infrared diffuse reflectance spectroscopy provides a rapid, non-invasive means of monitoring spatially averaged changes in tumour haemoglobin oxygen saturation induced by oxygen modifiers.

Effect of environmental temperature on the interactive developmental toxicity of radiofrequency radiation and 2-methoxyethanol in rats.

OBJECTIVE: This research was conducted to determine if altered environmental temperatures would affect the interactive developmental toxicity of radiofrequency (RF) radiation and the industrial solvent, 2-methoxyethanol (2ME). This is important because RF radiation is used in a variety of workplaces that have poorly controlled environmental temperatures, and many workers are concurrently exposed to various chemicals. Furthermore, we have previously demonstrated that combined exposure to RF radiation (10 MHz) and 2ME produces enhanced teratogenicity in rats. METHODS: RF radiation sufficient to maintain colonic temperatures at the control value (38degrees ), 39.0degrees or 40.0 degrees C for 2 or 4 h combined with either 0 or 100 mg/ kg 2ME at environmental temperatures of 18 degrees , 24 degrees and 30 degrees C (65 degrees , 75 degrees , and 85 degrees F) were given on gestation day 13 to Sprague-Dawley rats. Dams were killed on gestation day 20, and the fetuses were examined for external malformations. RESULTS AND CONCLUSIONS: Environmental temperature does affect the specific absorption rate (SAR) necessary to maintain a specific colonic temperature but does not affect the interactive developmental toxicity of RF radiation and 2ME in rats. These results, consistent with the literature, add to the evidence that the developmental toxicity of RF radiation (combined or alone) is associated with colonic temperature, not with SAR.

Electric field-induced changes in agonist-stimulated calcium fluxes of human HL-60 leukemia cells.

The mechanism of biological effects of extremely-low-frequency electric and magnetic fields may involve induced changes of Ca2+ transport through plasma membrane ion channels. In this study we investigated the effects of externally applied, low-intensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m2) on the agonist-induced Ca2+ fluxes of HL-60 leukemia cells. The suspensions of HL-60 cells received E-field or sham exposure for 60 min and were simultaneously stimulated either by 1 microM ATP or by 100 microM histamine or were not stimulated at all. After E-field or sham exposure, the responses of the intracellular calcium levels of the cells to different concentrations of ATP (0.2-100 microM) were assessed. Compared with control cells, exposure of ATP-activated cells to an E-field resulted in a 20-30% decrease in the magnitude of [Ca2+]i elevation induced by a low concentration of ATP (<1 microM). In contrast, exposure of histamine-activated HL-60 cells resulted in a 20-40% increase of ATP-induced elevation of [Ca2+]i. E-field exposure had no effect on non-activated cells. Kinetic analysis of concentration-response plots also showed that compared with control cells, exposure to the E-field resulted in increases of the Michaelis constant, Km, value in ATP-treated cells and of the maximal [Ca2+]i peak rise in histamine-treated HL-60 cells. The observed effects were reversible, indicating the absence of permanent structural damages induced by acute 60 min exposure to electric fields. These results demonstrate that low-intensity electric fields can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca2+ and/or ion channels.

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 degrees 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 degrees 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.

Interactions of radiofrequency radiation on 2-methoxyethanol teratogenicity in rats.

Concurrent exposures to chemical and physical agents occur in the workplace; exposed workers include those involved with microelectronics industry, plastic sealers and electrosurgical units. 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 radiofrequency (r.f.; 10 MHz) radiation, which also induces hyperthermia and is teratogenic to exposed animals, and the industrial solvent 2-methoxyethanol (2ME) produces enhanced teratogenicity in rats. A subsequent study replicated and extended that research by investigating the interactive dose-related teratogenicity of r.f. radiation (sham exposure or maintaining colonic temperatures at 42.0 degrees C for 0, 10, 20 or 30 min by r.f. radiation absorption) and 2ME (0, 75, 100, 125 or 150 mg/kg) on gestation days 9 or 13 of rats. The purpose of the present research is to determine the effects of r.f. radiation (sufficient to maintain colonic temperatures at 42.0 degrees C for 10 min) on a range of doses of 2ME (0, 20, 40, 60, 80, 100, 120 and 140 mg kg-1) 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. Dose-related developmental toxicity was observed for 2ME both in the presence and absence of r.f. radiation. However, concurrent RF radiation exposure changed the shape of the dose-effect curve of 2ME. These data indicate that combined exposure effects should be considered when developing exposure guidelines and intervention strategies.

Interactive developmental toxicity of radiofrequency radiation and 2-methoxyethanol in rats.

Concurrent exposures to chemical and physical agents occur in the workplace; exposed workers include those involved with the microelectronics industry, plastic sealers, and electrosurgical units. 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 radiofrequency (RF; 10 MHz) radiation, which also induces hyperthermia and is teratogenic to exposed animals, and the industrial solvent, 2-methoxyethanol (2ME), produces enhanced teratogenicity in rats. The present study replicates and extends the previous research investigating the enhanced teratogenicity of combined RF radiation and 2ME exposures. The interactive dose-related teratogenicity of RF radiation (sham exposure or maintaining colonic temperatures at 42.0 degrees C for 0, 10, 20, or 30 min) and 2ME (0, 75, 100, 125, or 150 mg/kg) was investigated by administering various combinations of RF radiation and 2ME to groups of rats on gestation days 9 or 13; gestation-day 20 fetuses were examined for external, skeletal, and visceral malformations. The results are consistent with and extend our previous research findings. Synergism was observed between RF radiation and 2ME for some treatment combinations, but not for others. The study also clarified which gestational periods, RF radiation exposure durations, and 2ME doses would be most informative in future interaction studies to determine the lowest interactive effect level. Day 9 exposures generally evidenced little effect by 2ME, either by itself or in combination with RF radiation. In contrast, day 13 exposures resulted in highly significant effects from 2ME and RF radiation. The structures showing strong evidence of effects from both 2ME and RF radiation after exposure on gestation day 13 were the forepaw digits, forepaw phalanges, hindpaw digits, hindpaw phalanges, hind limbs, metacarpals, and metatarsals. Statistical analyses did not show a global synergistic effect, but did show evidence for a synergistic effect at intermediate levels of the dose ranges. Future research will address potential interactions at lower doses.

Combined chemical, physical hazards make exposures harder to calculate. Studies show multiple exposures must be evaluated wholly rather than 'adding up' individual risks.

Clearly, current research adds to the mounting scientific evidence that combined exposures can produce greater toxicity than would be predicted using an additive model. Although there is not yet a quantitative method to determine the risks associated with multiple exposures, safety and health specialists must begin to consider the health effects from the entire work process, rather than individual chemical or physical agents. Moreover, organizations involved in risk assessment and standard-setting must evaluate the interactive effects of combined chemical and physical agent exposures. This will make personnel exposure standards comprehensive in addressing realistic occupational health risks.

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.

Absence of embryotoxic effects from low-level (nonthermal) exposure of rats to 100 MHz radiofrequency radiation.

Pregnant Sprague-Dawley rats were exposed to radio-frequency radiation at a frequency of 100 MHz and a power density of 25 mW/cm2 for 6 h 40 min daily on gestation days 6--11. The total exposure time was 40 h. The exposure resulted in a specific absorption rate of 0.4 W/kg. This value corresponds to the maximum permissible level for specific absorption rate in the 1982 American National Standards Institute (ANSI) standard for radiofrequency/microwave exposure. The exposure produced no increase in maternal colonic temperature. Irradiated rats did not differ from sham-irradiated rats with respect to the number of implantations per litter, percentage of implantations dead or resorbed, percentage of fetuses malformed. fetal weight, fetal crown-rump length, or fetal sex ratio. The irradiated fetuses had fewer minor skeletal variations than the controls. These results suggest that radiofrequency/microwave radiation is not teratogenic or embryotoxic for rats at the maximum permissible exposure level of the 1982 ANSI standard.

Teratogenicity of 27.12-MHz radiation in rats is related to duration of hyperthermic exposure.

Five groups of pregnant Sprague-Dawley rats were either sham exposed or were irradiated in a 27.12-MHz radiofrequency (RF) field at 55 A/m and 300 V/m on gestation day 9. The absorbed power (approximately 11 W/kg) caused a relatively rapid increase in the rat's colonic temperature. Rats in group I were sham irradiated for 2.5 h at 0 A/m, 0 V/m. In group II RF irradiation was terminated after the rat's colonic temperature reached 41.0 degrees C. In group III the 41.0- degrees C temperature was maintained an additional 2 h by manually varying the incident field strength. In group IV irradiation was terminated after the rat's colonic temperature reached 42.0 degrees C. In group V the 42.0- degrees C temperature was maintained an additional 15 min by varying the field strength. At both temperatures the teratogenic and embryotoxic effects of the RF-induced hyperthermia increased as the exposure duration increased, but the increase was especially noticeable at 42.0 degrees C. The results indicate that the teratogenic and embryotoxic effects of RF-induced hyperthermia are related to both the temperature of the dam during exposure and the length of time the dam's temperature remains elevated.

Teratogenic effects of 27.12 MHz radiofrequency radiation in rats.

High-intensity 27.12 MHz radiofrequency (RF) radiation was determined to be teratogenic in rats during most of the gestation period. Eight groups of pregnant rats were exposed to a magnetic field strength of 55 amps/meter and an electric field strength of 300 volts/meter on gestation days 1, 3, 5, 7, 9, 11, 13, or 15. Exposures ceased once the dam's colonic temperature reached 43.0 degrees C (about 20-40 minutes' duration). Eight matching control groups were sham-irradiated for 30 minutes at 0 amps/meter and 0 volts/meter. An additional group of pregnant rats received no treatment. With one exception, no significant differences occurred between sham-irradiated and untreated control groups. RF exposure, however, caused a significant incidence of fetal malformations throughout the postimplantation period (days 7 through 15). It also caused a low but significant incidence of preimplantation malformations. Fetal weight and crown-rump length were reduced in all postimplantation exposure groups but were not affected by preimplantation exposure. The incidence of dead or resorbed fetuses was significantly increased in rats irradiated on days 7 or 9. The effects observed appeared to be caused by RF-induced hyperthermia in the treated dams. Since a number of industrial, scientific, and medical devices operating at or near 27.12 MHz can cause hyperthermia in humans, women of childbearing age should avoid exposure to RF-radiation levels that exceed current US occupational standards.