Microwave-induced changes in nerve cells: effects of modulation and temperature

Helix aspersa neurons were irradiated with continuous-wave (CW) and noise-amplitude-modulated microwaves (carrier frequency 2450 MHz, 20% AM, 2 Hz-20 kHz) in a specially designed waveguide exposure system. Continuous-wave microwave irradiations were conducted at 8 degrees, 21 degrees, and 28 degrees C, while noise-modulated irradiation was performed at 21 degrees C. The results showed that exposure of snail neurons to CW microwaves for 60 min at 12.9 W/kg inhibited spontaneous activity and reduced input resistance at 8 degrees and 21 degrees C but not at 28 degrees C. The relative decrease in resistance at 21 degrees C was half that at 8 degrees C. Exposure of neurons to noise-modulated microwaves at 6.8 and 14.4 W/kg predominately caused excitatory responses characterized by augmented membrane resistance and the appearance of greater activity. The effect differed qualitatively from the inhibition observed with continuous, unmodulated microwave irradiation.     

Analysis of chromate-induced DNA-protein crosslinks with the comet assay

Modifications of the comet assay have been introduced to measure crosslinks by determining the reduction of induced DNA migration. Our previous results indicated that the modified protocol of the alkaline comet assay is a sensitive tool for the detection of formaldehyde-induced DNA-protein crosslinks. But results for mitomycin C and cisplatin suggested that the modified protocol is not well suited for the evaluation of DNA-DNA crosslinkers. We now used the comet assay to investigate in V79 cells the effect of potassium chromate (K(2)CrO(4)), another DNA-protein crosslinker, to see whether the results obtained for formaldehyde can be generalized. However, chromate did not reduce spontaneous or radiation-induced DNA migration in the alkaline (pH 13) comet assay but led to a small but significant induction of DNA migration. A crosslinking effect of chromate could also not be detected with the alkaline comet assay after postincubation of cells in normal medium after chromate treatment to enable repair of other (migration-inducing) lesions that might mask the crosslinking effect. Exposure of slides to proteinase K further increased DNA migration of chromate-treated cells, thus indicating the presence of DNA-protein crosslinks. In contrast to the alkaline comet assay, a "neutral" version at pH 9 was suited to demonstrate reduced induction of DNA migration after gamma-irradiation of chromate-treated cells. The crosslinking effect was seen immediately at the end of the chromate treatment as well as after a 3h postincubation period. Using the "neutral" protocol in combination with proteinase K, we were able to demonstrate the presence of DNA-protein crosslinks as the probable cause for the migration-reducing effect. Further investigations will have to show whether this protocol can be recommended as a universal approach for the detection of DNA-protein crosslinks and also of DNA-DNA crosslinks with the comet assay.     

Combined action of gamma and UHF radiation on conditioned reflex behavior of rats

Dynamics of the conditioned behaviour of rats in a shuttle box was studied after combined exposure to Cs-gamma radiation (1 Gy, 1.32 Gy/min) and microwave radiation (2450 MHz, 1 mW/cm2, 3 h). The number of conditioned and interstimulus responses was found to decrease on day 5 after microwave + gamma irradiation and to increase on day 30 after gamma + microwave irradiation.     

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.     

Acute low-intensity microwave exposure increases DNA single-strand breaks in rat brain cells

Levels of DNA single-strand break were assayed in brain cells from rats acutely exposed to low-intensity 2450 MHz microwaves using an alkaline microgel electrophoresis method. Immediately after 2 h of exposure to pulsed (2 μs width, 500 pulses/s) microwaves, no significant effect was observed, whereas a dose rate-dependent [0.6 and 1.2 W/kg whole body specific absorption rate (SAR)] increase in DNA single-strand breaks was found in brain cells of rats at 4 h postexposure. Furthermore, in rats exposed for 2 h to continuous-wave 2450 MHz microwaves (SAR 1.2 W/kg), increases in brain cell DNA single-strand breaks were observed immediately as well as at 4 h postexposure. © 1995 Wiley-Liss, Inc.     

Transbilayer movement of 24Na in sonicated phosphatidylcholine vesicles exposed to frequency-modulated microwave radiation

Sonicated egg phosphatidylcholine vesicles loaded with ²⁴Na⁺ were exposed at 20mW to a frequency-modulated (3 Hz) microwave field in the range of 2350 to 2550 MHz, or at 80 mW to a 2450-MHz CW (continuous wave) field, in a waveguide. The vesicle suspension absorbed microwaves at about 1 mW/ml and 25 mW/ml (CW experiment). The average temperature change of the irradiated suspension was < 0.1 °C from ambient. Leakage of ²⁴Na⁺ from the vesicles for up to 19 hours was measured. No difference was noted in the movement of ²⁴Na⁺ from the vesicles in the irradiated and control dispersions.     

检测DNA-蛋白质交联的新方法

将彗星实验进行改进以用于DNA-蛋白质交联作用的检测。利用甲醛对受试动物肝细胞的影响来判定此法是否适用于检测DNA-蛋白质交联。由于在实验中添加一定量的蛋白酶K, 可使单细胞在电泳时产生更大的迁移, 因此可以利用添加蛋白酶K前后的彗星尾距比来判断外来化合物对生物机体产生DNA损伤效应的时候是否有出现DNA-蛋白质交联作用。结果表明, 该方法快速、经济、灵敏度较高, 可以在单细胞水平对甲醛等强交联剂引发的不同组织的DNA-蛋白质交联效应进行检测, 希望该方法能成为指示DNA交联能力的有用工具。     

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

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

The induction of DNA-protein crosslinks in hypoxic cells and their possible contribution to cell lethality

The induction of single-strand breaks (SSBs) in the DNA of Chinese hamster ovary cells by X rays under different irradiation conditions was measured by the alkaline elution technique. The oxygen enhancement ratio (OER) for SSB induction determined for cells irradiated in air versus irradiation of cells made hypoxic by metabolic depletion of O2 was 9.7. However, when proteinase K was included in the cell lysis solution the OER was reduced to 4.2. The proteinase affected the elution rate only of the cells irradiated under hypoxic conditions, suggesting that DNA-protein crosslinks (DPCs) are preferentially produced in hypoxic cells by radiation. The ability to repair these DPCs was compared in two cell lines: the wild-type AA8 line and an excision-repair-deficient mutant line, UV-41. The AA8 line removed about 80% of the DPCs induced by radiation under hypoxic conditions within a 24-h repair incubation. The UV-41 line, on the other hand, removed only about 20% of the DPCs in the same time. The OERs for cell survival of these two lines are 3.1 for AA8 but only 1.9 for UV-41, suggesting that the DPCs preferentially induced in the DNA of cells irradiated under hypoxic conditions may contribute to cell killing when the normal DNA-repair mechanisms are compromised.     

Primary DNA damage in human blood lymphocytes exposed in vitro to 2450 MHz radiofrequency radiation

Human peripheral blood samples collected from three healthy human volunteers were exposed in vitro to pulsed-wave 2450 MHz radiofrequency (RF) radiation for 2 h. The RF radiation was generated with a net forward power of 21 W and transmitted from a standard gain rectangular antenna horn in a vertically downward direction. The average power density at the position of the cells in the flask was 5 mW/cm(2). The mean specific absorption rate, calculated by finite difference time domain analysis, was 2.135 (+/-0.005 SE) W/kg. Aliquots of whole blood that were sham-exposed or exposed in vitro to 50 cGy of ionizing radiation from a (137)Cs gamma-ray source were used as controls. The lymphocytes were examined to determine the extent of primary DNA damage (single-strand breaks and alkali-labile lesions) using the alkaline comet assay with three different slide-processing schedules. The assay was performed on the cells immediately after the exposures and at 4 h after incubation of the exposed blood at 37 +/- 1 degrees C to allow time for rejoining of any strand breaks present immediately after exposure, i.e. to assess the capacity of the lymphocytes to repair this type of DNA damage. At either time, the data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to the comet tail length, fluorescence intensity of the migrated DNA in the tail, and tail moment. The conclusions were similar for each of the three different comet assay slide-processing schedules examined. In contrast, the response of lymphocytes exposed to ionizing radiation was significantly different from RF-radiation- and sham-exposed cells. Thus, under the experimental conditions tested, there is no evidence for induction of DNA single-strand breaks and alkali-labile lesions in human blood lymphocytes exposed in vitro to pulsed-wave 2450 MHz radiofrequency radiation, either immediately or at 4 h after exposure.     

The effect of 2450-MHz microwave radiation during microtubular polymerization in vitro

Exposure to 2450-MHz (cw) microwave radiation causes inhibition of cell division in intact cells and varied in vivo biological effects in both avian and mammalian species. Because these reported effects may result from alterations in the dynamics of microtubule formation, we studied the effects of simultaneous microwave exposure (2450 MHz, cw) during each of the three critical stages of the intracellar polymerization cycle. In addition, using circular dichroism spectroscopy, we studied the effect of microwave irradiation on the secondary structure of purified tubulin polypeptides. These studies were accomplished using specially constructed exposure systems that permit the continuous recording of turbidometric or circular dichroism measurements during simultaneous exposure to microwaves. The baseline turbidity of microtubular protein did not change under the influence of microwave radiation (20 or 200 mW/g SAR) and irradiation had no effect on the light-scattering properties of the depolymerized protein. EGTA-induced polymerization and cold-induced depolymerization patterns were also similar for both control and microwave-irradiated samples. The circular dichroism spectrum of purified tubulin also did not appear to be influenced by microwave irradiation, indicating a lack of effect on the protein secondary structure. The data suggest that the cellular effects of microwaves are not due to changes in microtubular proteins or their rate of polymerization.     

Induction of neoplastic transformation in C3H/10T1/2 cells by 2.45-GHz microwaves and phorbol ester

C3H/10T1/2 cells were exposed to 2.45-GHz microwaves for 24 h and/or 1.5 Gy of 238-kVp X rays at 3.75 Gy/min. Transformation frequency and cell survival were measured with or without postirradiation addition of the tumor promoter tetradecanoyl-phorbol-13-acetate (TPA) at 0.1 microgram/ml. We previously reported (Carcinogenesis 6,859-864, 1985) an enhancement of transformation frequency when 10T1/2 cells exposed to a special sequence of microwaves and X rays were subsequently cultured in TPA. The same sequence of microwaves and X rays without promotion resulted in a transformation response similar to that induced by X rays alone. We now report statistically significant (at P greater than 0.999) enhancement of transformation response by TPA in cells exposed to 2.45-GHz microwaves (SAR = 4.4 W/kg). Microwaves alone had no effect on transformation. Plating efficiency and cell survival were not affected by TPA or microwave treatments.     

Noise-modulated-microwave-induced response in snail neurons

Helix aspersa neurons were irradiated with noise-amplitude-modulated microwaves (carrier frequency 2450 MHz, 20% AM, 0-20 kHz, specific absorption rate 6.8 and 14.4 mW/g). It was found that such an exposure caused an appearance of high frequency bursts and a rise in membrane resistance.     

A novel method for the study of fluorescent probes in biological material during exposure to microwave radiation

Instrumentation has been developed which allows the monitoring of fluorescnece in erythrocyte ghost membranes before, during, and after exposure to microwave radiation. Using non-fluorescent, UV-transmitting transmitting fiber optic cables, excitation light of specific wavelengths was delivered to a stirred sample undergoing irradiation (2450 MHz, CW) within a fluid-filled, temperature-controlled waveguide. Fluorescence was collected using an identical cable and transferred through appropriate filters to standard detecting, amplification and recording devices. We have used the fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS)_to monitor the effect of mirowave radiation on the binding of calcium to erthrocyte ghosts. Microwave radiation at specific absorption rates of 10 and 200 mW/g had no effect on the binding of ANS to the membranes. Dose-responses curves also showed no influence of microwaves on calcium binding between 2.0 and 10.0 · 10^?4 M. In addition, experiments studying fluorescence energy transfer between intrinsic tryptophan residues and membrane bound ANS showed that intermolecular distance between donor and acceptor are also unaffected by microwave radiation. We have thus shown that 2450 MHz microwve radition at the specific absorption rates studies rates used does not interfere with the binding of calcium to erythrocyte ghosts or alter intermolecular distances intrinsic molecules and bound ANS.     

Effect of 2450 MHz microwave exposure on behavioural thermoregulation in mice

1. 1.|The purpose of this study was to determine the threshold specific absorption rate (SAR) during exposure to 2450 MHz continuous wave (CW) microwaves that affected thermoregulatory behaviour in mice.

2. 2.|A Plexiglas shuttle box was placed inside a waveguide imposed with a temperature gradient. The temperature gradient allowed the mice to select a particular section of the shuttle box which was, presumably, related to their state of thermal comfort. Exposing the mice to 2450 MHz inside the waveguide at SARs of 0–5.3 W kg⁻¹ for 1 h caused no significant change in their preferred ambient temperature.

3. 3.|Increasing SAR from 5.3 to 18.1 W kg⁻¹ caused the animals to shift their position to the cooler end of the shuttle box.

4. 4.|Following termination of microwave exposure animals that had selected a cool ambient temperature returned to the warm side of the shuttle box.

5. 5.|It is concluded that for mice exposed to radiation at 2450 MHz the thermoregulatory behaviour is significantly affected at SARs of 5.3 to 9.9 W kg⁻¹.

Induction and rejoining of large DNA fragments after ion irradiation.

Radiation-induced DNA double-strand breaks (DSBs) were analyzed by separating large DNA fragments by pulsed-field gel electrophoresis. Human U-343MG glioma and K562 erythroleukemia cells were irradiated with 60Co gamma rays or nitrogen ions with high linear energy transfer (125 keV/microm). By comparing the fraction of DNA released into the gel below different size thresholds, corresponding to megabase-pair-sized DNA fragments, the relative effectiveness of the nitrogen ions was found to be dependent on both dose and the threshold size used in the evaluation. This dose dependence was most evident for the smallest threshold (6 Mbp) and was due to a linear dose response for release of the fragments for the ions compared to the curvilinear response for the gamma rays. The two curves intersected, and the relative yield of fragments (nitrogen ions/gamma rays) decreased from more than 3 below 1.5 Gy to 0.8 at 30 Gy. For the larger sizes (6-10.5 Mbp), the relative yield was constant at around 0.7. Thus the ion-induced fragments were shifted to smaller sizes compared to the 60Co gamma rays, and the data for nitrogen ions could not be fitted to random fragment distributions at doses < or =20 Gy. From these results, we conclude that a substantial fraction of the DSBs induced by heavy ions were nonrandomly distributed, correlated with DSBs within a region of < or =2 Mbp. After a dose of 20 Gy, the rejoining curves for ion-induced DSBs were different for each fragment size, resulting in different levels of unrejoined breaks after 6 h.     

Conformational properties of DNA after exposure to gamma rays and neutrons

DNA aqueous solutions were irradiated with 0-40 Gy of 60Co gamma rays and 0-1.5 Gy of (Pu-Be) neutrons. Thermal transition spectrophotometry (TTS) was used to trace the changes in the DNA conformation at the above doses. Previous results using the perturbed angular correlation (PAC) method were used to complement to the current analysis. The TTS and PAC methods are two different approaches to the study of the effects of radiation on DNA. Both showed that neutrons are more effective than gamma rays in inducing DNA damage. The TTS method showed that neutrons are 11 +/- 5 times more efficient than gamma rays, while the PAC method had shown this value to be 34 +/- 4. From the current study we deduced that the radiation damage to DNA is not a spontaneous effect but rather is an ensemble of damaging events that occur asynchronously. Any single method selected for the study of such damages can concentrate on only a part of the damage, leading to over- or underestimation of the relative effectiveness of the neutrons.     

DNA repair after gamma irradiation in lymphocytes exposed to low-frequency pulsed electromagnetic fields

The effect of exposure to extremely low-frequency pulsed electromagnetic fields (EMFs) on DNA repair capability and on cell survival in human lymphocytes damaged in vitro with gamma rays was studied by two different micromethods. In the first assay, which measures DNA repair synthesis (unscheduled DNA synthesis, UDS), lymphocyte cultures were stimulated with phytohemagglutinin (PHA) for 66 h and then treated with hydroxyurea (which blocks DNA replication), irradiated with 100 Gy of 60Co, pulsed with [3H]thymidine ([3H]TdR), and then exposed to pulsed EMFs for 6 h (the period in which cells repaired DNA damage). In the second assay, which measures cell survival after radiation or chemical damage, lymphocytes were first irradiated with graded doses of gamma rays or treated with diverse antiproliferative agents, and then stimulated with PHA, cultured for 72 h, and pulsed with [3H]TdR for the last 6 h of culture. In this case, immediately after the damage induced by either the radiation or chemicals, cultures were exposed to pulsed EMFs for 72 h, during which cell proliferation took place. Exposure to pulsed EMFs did not affect either UDS or cell survival, suggesting that this type of nonionizing radiation--to which humans may be exposed in the environment, and which is used for both diagnostic and therapeutic purposes--does not affect DNA repair mechanisms.     

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.     

TP53 tumor suppressor protein in normal human fibroblasts does not respond to 837 MHz microwave exposure.

The TP53 tumor suppressor protein (formerly known as p53) responds to a wide variety of environmental insults. To evaluate the safety of cellular telephones, TP53 responses in human fibroblast cells were studied after exposure to 837 MHz microwaves. Cells were exposed in a temperature-controlled transverse electromagnetic (TEM) chamber to a specific absorption rate (SAR) of 0.9 or 9.0 W/kg at 837 MHz continuous-wave (CW) microwave irradiation for 2 h. The TP53 protein levels were measured by Western blot at 2, 8, 24 and 48 h after treatment. The TP53 protein levels in microwave-treated cells, sham-treated cells, and untreated cells remained unchanged relative to each other at all times tested (Fisher test and Student-Newman-Keuls test, P > 0.05). No morphological alterations were observed in microwave-treated cells compared to sham-treated cells. We conclude that TP53 protein expression levels in cultured human fibroblast cells do not change significantly during a 48-h period after exposure to 837 MHz continuous microwaves for 2 h at SAR levels of 0.9 or 9.0 W/kg.