Guglielmo d'Ambrosio

Non‐thermal effects of microwaves on proteins: thermophilic enzymes as model system

Two thermophilic and thermostable enzymes, isolated from Sulfolobus solfataricus, S‐adenosylhomocysteine hydrolase and 5′‐methylthioadenosine phosphorylase, were exposed to 10.4 GHz microwave radiation in order to discriminate between thermal and non‐thermal microwave effects. The exposure causes a non‐thermal, irreversible and time‐dependent inactivation of both enzymes; the inactivation rate is related to the energy absorbed and is independent of the enzyme concentration. The influence of salts on enzyme inactivation has also been investigated. Conformational changes of S‐adenosylhomocysteine hydrolase, detected by fluorescence and circular dichroism techniques, suggest that microwaves induce protein structural rearrangements not related to temperature.

Electromagnetic Fields at Mobile Phone Frequency Induce Apoptosis and Inactivation of the Multi-chaperone Complex in Human Epidermoid Cancer Cells

The exposure to non‐thermal microwave electromagnetic field (MW‐EMF) at 1.95 MHz, a frequency used in mobile communication, affects the refolding kinetics of eukaryotic proteins (Mancinelli et al., 2004 ). On these basis we have evaluated the in vivo effect of MW‐EMF in human epidermoid cancer KB cells. We have found that MW‐EMF induces time‐dependent apoptosis (45% after 3 h) that is paralleled by an about 2.5‐fold decrease of the expression of ras and Raf‐1 and of the activity of ras and Erk‐1/2. Although also the expression of Akt was reduced its activity was unchanged likely as a consequence of the increased expression of its upstream activator PI3K. In the same experimental conditions an about 2.5‐fold increase of the ubiquitination of ras and Raf‐1 was also found and the addition for 12 h of proteasome inhibitor lactacystin at 10 μM caused an accumulation of the ubiquitinated isoforms of ras and Raf‐1 and counteracted the effects of MW‐EMF on ras and Raf‐1 expression suggesting an increased proteasome‐dependent degradation induced by MW‐EMF. The exposure of KB cells to MW‐EMF induced a differential activation of stress‐dependent pathway with an increase of JNK‐1 activity and HSP70 and 27 expression and with a reduction of p38 kinase activity and HSP90 expression. The overexpression of HSP90 induced by transfection of KB cells with a plasmid encoding for the factor completely antagonized the apoptosis and the inactivation of the ras → Erk‐dependent survival signal induced by MW‐EMF. Conversely, the inhibition of Erk activity induced by 12 h exposure to 10 mM Mek‐1 inhibitor U0126 antagonized the effects induced by HSP90 transfection on apoptosis caused by MW‐EMF. In conclusion, these results demonstrate for the first time that MW‐EMF induces apoptosis through the inactivation of the ras → Erk survival signaling due to enhanced degradation of ras and Raf‐1 determined by decreased expression of HSP90 and the consequent increase of proteasome dependent degradation.

Non‐thermal effects of electromagnetic fields at mobile phone frequency on the refolding of an intracellular protein: Myoglobin

Non‐thermal effects induced by exposure to microwave electromagnetic field (MW‐EMF) at 1.95 MHz, a frequency used in mobile communication, have been observed on the refolding kinetics of the heme binding site in an intracellular protein: tuna myoglobin, starting from acidic conditions. We have selected myoglobin because it can be considered a good model to study protein interactions with MW‐EMF for its well‐known high‐resolution crystallographic structure. Myoglobin solutions at pH 3.0 were subjected to 3 h exposure to microwave field (with a specific absorption rate of 51 ± 1 mW/g); the heme site refolding has been followed by measuring the molecular absorption in the Soret spectral region and the data were fitted to a bi‐exponential model. The kinetics of exposed samples appear to be slowered by MW‐EMF action. Moreover, the tryptophanyl lifetime distribution of the exposed protein, as deduced by the analysis of the fluorescence emission decay from its single tryptophan, appears sharper if compared to non‐exposed protein samples. This observation suggests that the presence of MW‐EMF could affect the propensity of protein molecules to populate specific conformational substates among which myoglobin molecules fluctuate at acidic pH. Changes in the structural fluctuation caused by MW perturbation can affect differently the aggregation process that occurs competitively during the protein folding, so representing a potential risk for protein “misfolding.” These data suggest that MW‐EMF could have also biochemical and, consequently, biological effects on eukaryotic cells that are still under investigation.

Genotoxic Effects of Amplitude-Modulated Microwaves on Human Lymphocytes Exposed in Vitro under Controlled Conditions

Peripheral human blood from 23 healthy donors aged between 23 and 95 years was exposed to continuous wave (CW) or 50 Hz amplitude modulated (AM) microwave radiation and was cultured for 72 h. Other exposure parameters were: frequency 9 GHz, specific absorption rate (SAR) 90 mW/g, exposure duration 10 min. The possible genotoxic effect was evaluated by means of cytokinesis-block micronucleus method. A significant (p < 0.05) increase in micronuclei was found following AM microwave exposure.

In situ real‐time study of crosslinking kinetics in thermal and microwave fields

A comparative investigation has been conducted of the mechanisms and rate of chemical reactions in thermal and microwave fields. A number of nonpolymer-forming and polymer-forming mixtures of different functionality and molecular architecture were prepared and investigated. The advancement of reactions in thermal and microwave fields was monitored in real time by in situ remote near-infrared spectroscopy. The principal finding was that the use of microwaves in lieu of thermal heating had no effect on the mechanism or kinetics during the isothermal cure of various epoxies, polyimides and bismaleimides. No “microwave effect” was observed and it was concluded that the claims of “accelerated kinetics” in the microwave field are unfounded. However, a comparison between thermal and microwave cure assumes a whole new dimension when the temperature distribution inside the sample is considered, and that constitutes a scientifically challenging area that warrants further research.

A high-efficiency waveguide applicator for in vitro exposure of mammalian cells at 1.95 GHz

A standardized exposure device for a set of in vitro bioelectromagnetic experiments, to be carried out in different laboratories under strictly standardized exposure conditions, was designed and tested on the basis of efficiency and field uniformity criteria. In particular, a WR-430 waveguide was chosen in order to expose mammalian cells cultures at 1.95 GHz. The cells could be exposed in either one, two, or four 35-mm Petri dishes filled with 3-mL samples. Numerical and experimental dosimetry was carried out in order to assess the optimum orientation and positioning of the circular Petri dishes inside the simple waveguide chamber, obtaining very satisfactory performances of the system when the electric field is parallel to the sample. The efficiency and the nonuniformity degree simulated were successfully verified through measurements of the scattering parameters and local temperature increases.

Different Effects of Microwave Energy and Conventional Heat on the Activity of a Thermophilic ß-Galactosidase From Bacillus acidocaldarius

The effect of microwave (f = 10.4 GHz) irradiation on a thermostable enzyme was experimentally tested, showing that irreversible inactivation is obtained. Enzymatic solutions (500 microliters, with concentrations between 10-100 micrograms/ml) were exposed at 70 degrees C, at SAR levels of 1.1 and 1.7 W/g for 15, 30, 45, or 60 min, and their activity was compared to that of a sample heated in a water bath at the same temperature. The residual activity of the exposed samples depends on enzyme concentration, microwave power level, and exposure time; activity was reduced to 10% in 10 micrograms/ml solutions treated at 1.7 W/g for 60 min. Microwave effects disappeared at concentrations above 50 micrograms/ml. These results were not found following water bath heating at the same temperature and durations.

Evaluation of Cytotoxic and Genotoxic Effects in Human Peripheral Blood Leukocytes Following Exposure to 1950-MHz Modulated Signal

Human peripheral blood leukocytes from six volunteers were exposed to a Universal Mobile Telecommunication System (UMTS) signal (frequency carrier of 1950 MHz) for 24 h. The exposures were carried out in a waveguide system at specific absorption rates (SAR) of 0.5 and 2.0 W/kg, and for each blood donor, sham-exposed samples were also set up. The alkaline comet assay was used to quantify DNA damage, while cytotoxicity was determined by the Trypan blue exclusion method. The results obtained indicate the absence of genotoxic and cytotoxic effects at both SAR levels investigated, as assessed by comparing sham-exposed and exposed samples. Therefore, the findings indicate that, in the experimental conditions adopted, 24-h in vitro exposure to 1950-MHz radio-frequency radiation (UMTS signal) does not induce DNA damage in human leukocytes

Genotoxic Effects of Mitomycin-C and Microwave Radiation on Bovine Lymphocytes

The aim of this work was to ascertain whether microwave radiation (frequency 9 GHz; specific absorption rate 70 mW/gr, exposure duration 10 min) may produce genotoxic effects, assessed by cytokinesis-block micro-nucleus (MN) assay, in bovine (Bos taurus L.) peripheral blood lymphocyte cultures. As positive control for the MN induction, mitomycin-C (MMC) was used: it is known that this alkylating agent has a potent genotoxic (radiomimetic) action. To evaluate possible cooperative effects, some cultures from microwave-exposed samples were treated with MMC. The results obtained indicate that the optimal dose of MMC to induce MN frequency increase in this species is 0.044 µg/ml and that microwave radiation induces a statistically significant increase of MN frequency both with and without MMC.

SAR and efficiency evaluation of a 900 MHz waveguide chamber for cell exposure.

In this work we present the results of numerical and experimental dosimetry carried out for an in vitro exposure device to irradiate sample groups at 900 MHz. The cells are kept in 8 and 15 ml cell cultures, contained, respectively in T25 and T75 rectangular flasks. The dosimetric assessment of the distribution of the specific absorption rate (SAR) is performed for both the bottom of the flask and the whole volume of the sample to provide results for experiments on either the cell layer or the cell suspension. The irradiating chamber is a rectangular waveguide (WG). Different configurations are considered to assess the optimum orientation and positioning of the cell cultures inside the WG. The system performance is optimal when the electric field is parallel to the sample and the WG is terminated by a matched load. In this condition two 15 or four 8 ml cells cultures can be exposed. The efficiency (ratio between the power absorbed by the sample and the incident power) and the non-uniformity degree (ratio between the standard deviation of SAR values and the average SAR over the sample) are calculated and successfully verified through measurements of the scattering parameters and local temperature increases. In the chosen exposure configuration, the efficiency is 0.40 and the non-uniformity degree is 39% for the 15 ml samples. For the 8 ml samples, the efficiency is 0.19 and a low non-uniformity degree (15%) is found.