Human fibroblasts and 900 MHz radiofrequency radiation: evaluation of DNA damage after exposure and co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5h)-furanone (MX).

The aim of this study was to investigate DNA damage in human dermal fibroblasts from a healthy subject and from a subject affected by Turner's syndrome that were exposed for 24 h to radiofrequency (RF) radiation at 900 MHz. The RF-radiation exposure was carried out alone or in combination with 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a well-known environmental mutagen and carcinogen produced during the chlorination of drinking water. Turner's syndrome fibroblasts were also exposed for a shorter time (1 h). A signal similar to that emitted by Global System for Mobile Communications (GSM) mobile phones was used at a specific absorption rate of 1 W/kg under strictly controlled conditions of temperature and dosimetry. To evaluate DNA damage after RF-radiation exposure alone, the alkaline comet assay and the cytokinesis-block micronucleus assay were used. In the combined-exposure experiments, MX was given at a concentration of 25 microM for 1 h immediately after the RF-radiation exposure, and the effects were evaluated by the alkaline comet assay. The results revealed no genotoxic and cytotoxic effects from RF radiation alone in either cell line. As expected, MX treatment induced an increase in DNA migration in the comet assay, but no enhancement of the MX-induced DNA damage was observed in the cells exposed to RF radiation.

Evaluation of genotoxic effects in human leukocytes after in vitro exposure to 1950 MHz UMTS radiofrequency field.

In the present study the third generation wireless technology of the Universal Mobile Telecommunication System (UMTS) signal was investigated for the induction of genotoxic effects in human leukocytes. Peripheral blood from six healthy donors was used and, for each donor, intermittent exposures (6 min RF on, 2 h RF off) at the frequency of 1950 MHz were conducted at a specific absorption rate of 2.2 W/kg. The exposures were performed in a transverse electro magnetic (TEM) cell hosted in an incubator under strictly controlled conditions of temperature and dosimetry. Following long duration intermittent RF exposures (from 24 to 68 h) in different stages of the cell cycle, micronucleus formation was evaluated by applying the cytokinesis block micronucleus assay, which also provides information on cell division kinetics. Primary DNA damage (strand breaks/alkali labile sites) was also investigated following 24 h of intermittent RF exposures, by applying the alkaline single cell gel electrophoresis (SCG)/comet assay. Positive controls were included by treating cell cultures with Mitomycin-C and methylmethanesulfonate for micronucleus and comet assays, respectively. The results obtained indicate that intermittent exposures of human lymphocytes in different stages of cell cycle do not induce either an increase in micronucleated cells, or change in cell cycle kinetics; moreover, 24 h intermittent exposures also fail to affect DNA structure of human leukocytes soon after the exposures, likely indicating that repairable DNA damage was not induced.

Cytogenetic observations in human peripheral blood leukocytes following in vitro exposure to THz radiation: a pilot study.

Emerging technologies are considering the possible use of Terahertz radiation in different fields ranging from telecommunications to biology and biomedicine. The study of the potential effects of Terahertz radiation on biological systems is therefore an important issue in order to safely develop a variety of applications. This paper describes a pilot study devoted to determine if Terahertz radiation could induce genotoxic effects in human peripheral blood leukocytes. For this purpose, human whole blood samples from healthy donors were exposed for 20 min to Terahertz radiation. Since, to our knowledge, this is the first study devoted to the evaluation of possible genotoxic effects of such radiation, different electromagnetic conditions were considered. In particular, the frequencies of 120 and 130 GHz were chosen: the first one was tested at a specific absorption rate (SAR) of 0.4 mW g-1, while the second one was tested at SAR levels of 0.24, 1.4, and 2 mW g-1. Chromosomal damage was evaluated by means of the cytokinesis block micronucleus technique, which also gives information on cell cycle kinetics. Moreover, human whole blood samples exposed to 130 GHz at SAR levels of 1.4 and 2 mW g-1 were also tested for primary DNA damage by applying the alkaline comet assay immediately after exposure. The results obtained indicate that THz exposure, in the explored electromagnetic conditions, is not able to induce either genotoxicity or alteration of cell cycle kinetics in human blood cells from healthy subjects.

Evaluation of genotoxic effects in human peripheral blood leukocytes following an acute in vitro exposure to 900 MHz radiofrequency fields.

Human peripheral blood leukocytes from healthy volunteers have been employed to investigate the induction of genotoxic effects following 2 h exposure to 900 MHz radiofrequency radiation. The GSM signal has been studied at specific absorption rates (SAR) of 0.3 and 1 W/kg. The exposures were carried out in a waveguide system under strictly controlled conditions of both dosimetry and temperature. The same temperature conditions (37.0 +/- 0.1 degrees C) were realized in a second waveguide, employed to perform sham exposures. The induction of DNA damage was evaluated in leukocytes by applying the alkaline single cell gel electrophoresis (SCGE)/comet assay, while structural chromosome aberrations and sister chromatid exchanges were evaluated in lymphocytes stimulated with phytohemagglutinin. Alterations in kinetics of cell proliferation were determined by calculating the mitotic index. Positive controls were also provided by using methyl methanesulfonate (MMS) for comet assay and mitomycin-C (MMC), for chromosome aberration, or sister chromatid exchange tests. No statistically significant differences were detected in exposed samples in comparison with sham exposed ones for all the parameters investigated. On the contrary, the positive controls gave a statistically significant increase in DNA damage in all cases, as expected. Thus the results obtained in our experimental conditions do not support the hypothesis that 900 MHz radiofrequency field exposure induces DNA damage in human peripheral blood leukocytes in this range of SAR.

Induction of oxidative stress in murine cell lines by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX).

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the potent bacterial mutagen produced during chlorination of drinking water, was tested for the induction of oxidative stress in two murine cell lines: NIH 3T3 (fibroblasts) and L929 (fibrosarcoma cells). Following 1 h MX treatment at concentrations between 100 and 1000 microM, cellular stress conditions were monitored by measuring reactive oxygen species formation (ROS) and reduced glutathione levels (GSH). The kinetics of ROS formation and GSH depletion was investigated from 10 min to 1 h. MX caused detachment of cells at 1000 microM in L929 cells and at 300 microM in NIH 3T3 cells but the viability of the cells, measured by the trypan blue assay, decreased only by 20 and 7%, respectively, in 1h. MX increased ROS production in L929 cells in a dose-dependent manner, by 120% at 500 microM of MX in 1 h. The maximum ROS production was attained already in 10min. In NIH 3T3 cells, the ROS production was slightly, but not statistically significantly stimulated at 200 microM between 20 and 60 min. Concomitantly, MX decreased the intracellular content of GSH dose-dependently in both cell lines, by 48% in L929 cells at 500 microM of MX and 32% in NIH 3T3 cells at 200 microM of MX in one hour. The majority of this GSH depletion had occurred in 10 min. These findings indicate that MX induces oxidative stress in mammalian cells in vitro though the sensitivity of cells may differ for this effect.

Lack of genotoxic effects (micronucleus induction) in human lymphocytes exposed in vitro to 900 MHz electromagnetic fields.

In the present study, we investigated the induction of genotoxic effects in human peripheral blood lymphocytes after exposure to electromagnetic fields used in mobile communication systems (frequency 900 MHz). For this purpose, the incidence of micronuclei was evaluated by applying the cytokinesis-block micronucleus assay. Cytotoxicity was also investigated using the cytokinesis-block proliferation index. The experiments were performed on peripheral blood from 20 healthy donors, and several conditions were tested by varying the duration of exposure, the specific absorption rate (SAR), and the signal [continuous-wave (CW) or GSM (Global System of Mobile Communication) modulated signal]. The following exposures were carried out: (1) CW intermittent exposure (SAR = 1.6 W/kg) for 6 min followed by a 3-h pause (14 on/off cycles); (2) GSM signal, intermittent exposure as described in (1); (3) GSM signal, intermittent exposure as described in (1) 24 h before stimulation with phytohemagglutinin (8 on/off cycles); (4) GSM signal, intermittent exposure (SAR = 0.2 W/kg) 1 h per day for 3 days. The SARs were estimated numerically. No statistically significant differences were detected in any case in terms of either micronucleus frequency or cell cycle kinetics.

THz Exposure of Whole Blood for the Study of Biological Effects on Human Lymphocytes.

The aim of the present study is toinvestigate the genotoxic effect of THzradiation in human peripheral bloodlymphocytes following 20 minutes exposureto 1 mW average power Free Electron Laserradiation in the frequency range 120-140GHz. For this purpose 9 healthy donors wereemployed and cytokinesis block techniquewas applied to study micronucleusfrequency and cell proliferation. Theresults obtained indicate that all theelectromagnetic conditions adopted so far do not alter the investigated parameters,suggesting absence of direct chromosomaldamage and alteration of cell cyclekinetics (two tailed paired Student's test:p> 0.05 in all cases).

Micronucleus frequency and cell proliferation in human lymphocytes exposed to 50 Hz sinusoidal magnetic fields.

In the present study micronucleus induction and cell proliferation in human peripheral blood lymphocytes cultured in vitro and exposed to 50 Hz sinusoidal magnetic fields for 72 h at different intensities (1.0, 0.75, 0.5, 0.25, and 0.05 mT rms) were investigated. The results obtained from 42 healthy donors aged between 26 and 54 y indicate that, for the field intensities tested, no genotoxic effects were found, as assessed by the cytokinesis-block micronucleus assay. On the contrary, cell proliferation, evaluated by the cytokinesis-block proliferation index, was slightly affected by the field at the intensities tested.

Genotoxicity and oxidative stress induced by pesticide exposure in bovine lymphocyte cultures in vitro.

The genotoxic activity of the pesticides gliphosate, vinclozolin and DPX-E9636 was studied in in vitro cultures of bovine lymphocytes, using chromosome aberration (CA) and sister chromatid exchange (SCE) frequencies as genetic end-points and a variation of glucose 6-phosphate dehydrogenase (G6PD) enzyme activity as a marker of changes in the normal cell redox state. Results indicated a statistically significant increase of structural aberrations, sister chromatid exchanges and G6PD activity, suggesting that the pesticides tested induce either oxidative stress or a mutagenic effect in this species. The evaluation of both mitotic index and cell viability, after pesticide exposure, demonstrates a high cytotoxic effect which is always associated with the observed genotoxic effect.

Cytogenetic damage and induction of pro-oxidant state in human lymphocytes exposed in vitro to gliphosate, vinclozolin, atrazine, and DPX-E9636.

We analyzed chromosome aberrations (CAs), sister chromatid exchanges (SCEs), mitotic index (MI), and glucose 6-phosphate dehydrogenase (G6PD) enzyme activity in human peripheral lymphocytes from three healthy donors exposed in vitro to different concentrations of gliphosate, vinclozolin, atrazine, and DPX-E9636. The pesticides gliphosate, vinclozolin, and atrazine have been studied in a broad range of genetic tests with predominantly conflicting or negative results, whereas little is known about the genotoxicity of DPX-E9636. In our experimental conditions, each chemical compound tested produced a dose-related increase in the percent of aberrant cells and an increase of SCE/cell. Furthermore, at the highest concentrations of vinclozolin, atrazine, and DPX-E9636, we observed a significant reduction of the mitotic index. The increase of G6PD activity in exposed lymphocyte cultures strongly indicated an induction of a pro-oxidant state of the cells as an initial response to pesticide exposure.

Spontaneous and mitomycin-C-induced micronuclei in lymphocytes from subjects affected by Turner's syndrome.

Peripheral blood lymphocytes from 15 subjects affected by Turner's syndrome (TS) and aged between 2 and 24 years (mean age 10.40 +/- 6.25) were tested to evaluate the spontaneous and Mitomycin-C-induced (MMC) micronucleus (MN) frequency. A group of 15 healthy subjects, in the same range of age (mean age 14.67 +/- 8.30), was also tested as control. As expected, statistically significant differences between spontaneous and MMC-induced MN were found either in TS and in healthy subjects. Unexpectedly, when the two groups of donors were compared, TS subjects showed a lower spontaneous and MMC-induced MN frequency, in comparison with healthy subjects. Cell proliferation kinetic and cytotoxicity were also measured applying the cytokinesis-block proliferation index (CBPI): the results show that MMC, at the employed concentration, does not induce cell cycle delay both in healthy and in TS donors. Whereas, when CBPI from TS and healthy donors were compared, a faster proliferation was found in TS patients in both untreated and MMC-treated cultures.

Lack of chromosomal aberration and micronucleus induction in human lymphocytes exposed to pulsed magnetic fields.

We exposed human peripheral blood lymphocyte cultures to 50 Hz pulsed magnetic fields (PMFs) in order to evaluate a possible genotoxic effect of such non-ionizing radiation. The genotoxic effect was evaluated in terms of both micronucleus (MN) induction and classical chromosomal aberrations (CA); the mitotic index (MI) was also calculated. Khalil and Qassem (1991) found chromosomal and chromatid breaks and mitotic delay in human lymphocytes exposed for 24, 48 and 72 h to a field with characteristics similar to those used in our laboratory. These data are in contrast with our results previously reported in terms of MN induction using the cytokinesis block method (Scarfi et al., 1991). In this study lymphocytes from five healthy human donors were examined with the above mentioned tests. No genotoxic effects and increased MI were found in exposed samples compared to the control ones, in agreement with our previous results.

Measurement of micronuclei by cytokinesis-block method in bovine lymphocytes.

The present study was carried out in order to set up a standardized quantitative assay for spontaneous micronuclei in bovine lymphocytes. For this purpose the cytokinesis-block micronucleus (MN) method, originally proposed by Fenech and Morley (1985) for human lymphocytes, was applied to peripheral blood lymphocytes of 20 healthy cows of Italian Friesian breed. The results demonstrate that the optimal concentration of cytochalasin B to obtain the highest frequency of binucleated cells (mean = 400.26 +/- 23.76/1000 cells scored) was 6 micrograms/ml. The baseline frequency of spontaneous MN formation in 500 binucleated cells was 12.3 +/- 4.1, i.e., 3 times higher than that reported in human lymphocytes (Fenech and Morley, 1985; Scarfi et al., 1991). The possible reason(s) for this difference (sensitivity to cytochalasin B, chromosome number, environmental genotoxic pollutants) is discussed.

50 Hz AC sinusoidal electric fields do not exert genotoxic effects (micronucleus formation) in human lymphocytes.

Electromagnetic fields produce a variety of effects in several biological systems, including human peripheral blood lymphocytes. A great concern exists regarding possible carcinogenic effects in subjects exposed environmentally to such fields in the vicinity of power lines and electric domestic appliances. Using human lymphocytes from 33 healthy donors and a sensitive cytogenetic method, the cytokinesis-block micronucleus assay, we have demonstrated that the exposure to 50-Hz AC sinusoidal electric fields over a wide range of intensities (0.5-10 kV/m in air) does not increase the spontaneous frequency of micronuclei. Moreover, these fields did not affect the mitomycin-C-induced micronucleus formation, suggesting that they did not exert any synergistic or antagonistic effect.