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Evaluation of Genotoxic Effects in Human Fibroblasts after Intermittent Exposure to 50 Hz Electromagnetic Fields: A Confirmatory Study Author(s): Maria Rosaria Scarfí, Anna Sannino, Alessandro Perrotta, Maurizio Sarti, Pietro Mesirca and Ferdinando Bersani Source: Radiation Research, Vol. 164, No. 3 (Sep., 2005), pp. 270-276Published by: Radiation Research SocietyStable URL: http://www.jstor.org/stable/3581476Accessed: 25-07-2015 13:10 UTC

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RADIATION RESEARCH 164, 270-276 (2005) 0033-7587/05 $15.00 C 2005 by Radiation Research Society. All rights of reproduction in any form reserved.

Evaluation of Genotoxic Effects in Human Fibroblasts after Intermittent Exposure to 50 Hz Electromagnetic Fields: A Confirmatory Study

Maria Rosaria ScarfPfa, Anna Sannino,a Alessandro Perrotta,a Maurizio Sarti,a Pietro Mesircab and Ferdinando Bersanib

a CNR-Institute for Electromagnetic Sensing of Environment (IREA), 80124 Napoli, Italy; and b Department of Physics, University of Bologna, 40127 Bologna, Italy

Scarfi, M. R., Sannino, A., Perrotta, A., Sarti, M., Mesirca, P. and Bersani, F. Evaluation of Genotoxic Effects in Human Fibroblasts after Intermittent Exposure to 50 Hz Electromag- netic Fields: A Confirmatory Study. Radiat. Res. 164, 270-276 (2005).

The aim of this investigation was to confirm the main results reported in recent studies on the induction of genotoxic effects in human fibroblasts exposed to 50 Hz intermittent (5 min field on/10 min field off) sinusoidal electromagnetic fields. For this purpose, the induction of DNA single-strand breaks was evaluated by applying the alkaline single-cell gel electrophoresis (SCGE)/comet assay. To extend the study and validate the results, in the same experimental conditions, the potential genotoxicity was also tested by exposing the cells to a 50 Hz powerline signal (50 Hz frequency plus its harmonics). The cytokinesis-block micronucleus assay was applied after 24 h intermittent exposure to both sinusoidal and powerline signals to obtain information on cell cycle kinetics. The experiments were carried out on human diploid fibroblasts (ES-1). For each experimental run, exposed and sham-exposed samples were set up; positive controls were also provided by treating cells with hydrogen peroxide or mitomycin C for the comet or micronucleus assay, respectively. No statistically significant difference was detected in exposed compared to sham- exposed samples in any of the experimental conditions tested (P > 0.05). In contrast, the positive controls showed a statistically significant increase in DNA damage in all cases, as expected. Accordingly, our findings do not confirm the results reported previously for either comet induction or an increase in micronucleus frequency. ? 2005 by Radiation Research Society

INTRODUCTION

Some epidemiological studies have suggested a moderate association between residential or occupational exposures to extremely low-frequency (ELF) electromagnetic fields (EMFs) and the incidence of cancer, such as childhood leu- kemia, brain tumors and breast tumors (1-7). Because of the close correlation between DNA damage and cancer oc-

currence, the genotoxic potential of EMFs has been investigated in humans and animals both in vivo and in vitro.

In vitro, several conditions were investigated by varying experimental protocols, exposure parameters, exposure duration, cell types and cytogenetic tests. The results in the literature have been reviewed by several authors (8-11), and the prevalent opinion is that they do not support the hypothesis that ELF EMF exposure induces genotoxic effects. Nevertheless, since some studies reported the induction of DNA damage, it is crucial to continue the investigation to clarify the possible role of ELF EMFs in the induction of genotoxic effects.

Recently, in the framework of the EU Project REFLEX ("Risk evaluation of potential environmental hazard from low energy EMF exposure using sensitive in vitro methods"), Ivancsits et al. reported induction of DNA damage after intermittent exposure to 50 Hz sinusoidal EMFs in different experimental conditions (cell type, intermittent cycle duration, exposure duration, field strength, etc.). The greatest effect was found in human fibroblasts after an intermittent exposure of 5 min field on/10 min field off, while the continuous exposure did not produce genotoxic effects. The cytogenetic test employed was the neutral and alkaline comet assay (12, 13). These results raised a public concern and a debate in the scientific community (14).

The aim of the present study was to confirm the main results of Ivancsits et al. by employing the same cells (human diploid fibroblasts, ES-1), the same operational procedures, and the same type of exposure system. Moreover, we extended the study by evaluating the DNA damage with the cytokinesis-block micronucleus (MN) assay. In addition, the effect of a powerline signal (50 Hz frequency plus its harmonics) in the same experimental conditions was investigated.

MATERIAL AND METHODS

Chemicals

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), L-glutamine, trypsin EDTA and penicillin/streptomycin were from Gibco (Milan, Italy). Cytochalasin-B, Triton X-100, Hepes, N-lauryl sarcosine, mitomycin C and hydrogen peroxide were from Sigma (St. Louis,

'Address for correspondence: CNR-Institute for Electromagnetic Sens- ing of Environment, Via Diocleziano, 328-80124 Naples, Italy; e-mail: scarfi.mr@ irea.cnr.it.

270



50 Hz EMFs DO NOT INDUCE GENOTOXIC EFFECTS IN HUMAN FIBROBLASTS 271

MO). Dimethyl sulfoxide, NaOH, Na2EDTA, Na2HPO4, KC1, methanol and Giemsa were from Baker (Deventer, The Netherlands). Tris was from BDH (Poole, England); NaC1 was from Carlo Erba (Milan, Italy). Nor- mal-melting-point agarose, low-melting-point agarose and ethidium bromide were from Bio-Rad Laboratories (GmbH, Munich, Germany).

Exposure System

To expose cell cultures to the same experimental conditions used by Ivancsits et al. (12, 13), an identical exposure system was provided by IT'IS-foundation (Zurich, Switzerland), within the framework of the RE- FLEX project, thanks to the support of the VERUM Foundation (Munich, Germany). The characteristics of the system have been described in detail elsewhere (15). Briefly, the exposure system consisted of two identical apparatuses made up of four coaxial square coils (side length 20 cm), placed horizontally, and thus parallel to the surface of the culture plates, each of them housed inside a pi-metal shielding box. The geometry of the coil system was calculated numerically to optimize the extension of the uniformity of the magnetic field; this was then verified experimentally. The vertical distances from the center to the inner and outer coils were 3 and 9 cm, respectively. Under to these conditions, the field uniformity was better than 1% within a region of approximately 12 x 12 x 20 cm3 and better than 5% within a region of about 15 x 15 x 20 cm3. A dish holder was used to keep petri dishes inside the region of uniformity.

Coils were wound by a pair of parallel wires, inner coils 50 turns and outer coils 56 turns, so that, depending on the different connections, the current could flow in either the same ("wound configuration") or the opposite direction ("counter-wound configuration"). In the latter case, the magnetic fields produced by counter-wound coils cancelled each other, allowing a "true" sham system, in which current and power dissipation were the same as in the wound configuration, but the magnetic flux den- sity was zero in practice (16). Both systems were placed inside a com- mercial CO2 incubator (Heraeus, D6450); two fans for each

px-metal box

ensured atmospheric exchange between the boxes and the incubator. The air temperature was inside the boxes monitored continuously by Pt 100 probes. The exposure setup was controlled by a PC that decided randomly which system of coils was "active" or "sham" to guarantee blind exposure conditions. The exposure parameters (magnetic-field intensity, temperatures, fan currents, etc.) were registered continuously in an en- crypted file. The exposure files were decoded by IT'IS Foundation after all the slides had been scored. The experiments were carried out at 50 Hz, 1 mTs,,, using two different kind of signals: a pure sinusoidal signal (MF-Sin) and a signal composed by a 50 Hz basic frequency plus its harmonics, called the powerline signal (MF-Pl), corresponding to the maximum accepted distortion for low- to medium-voltage power systems by the International Electrotechnical Commission (17). The details of the setup and signals can be found elsewhere (15).

Cell Cultures and Exposure Conditions

Human diploid fibroblasts (ES-1, derived from a 6-year-old male) were kindly provided by Prof. Riidiger's group. The cells were cultured in DMEM containing 10% FBS, 20 mM Hepes buffer, 2 mM L-glutamine, 100 IU/ml penicillin and 100 ig/ml streptomycin at 370C in a humidified atmosphere of 95% air/5% CO2. Cells were supplied with fresh culture medium every 48 h and split once a week. Cells were received at passage 6, and a master bank was established with cells at passage 8. For experiments, cells from passages 10-16 were used. The induction of comets was evaluated by applying both MF-Sin and MF-PI for several exposure durations. The MF-Sin was applied for 15 or 24 h (three independent experiments for each exposure duration), while the MF-PI was applied for 15, 18 or 24 h (three, three and five experiments, respectively). The exposures were intermittent, with the field on for 5 min followed by 10 min field off, according to the protocol of Ivancsits et al., who found this condition to be the most efficient of the intermittencies tested (12). Sev- enteen independent experiments were carried out by setting up a total of eight cultures for each experimental run: four for exposure and four for

sham exposure. To investigate the induction of micronuclei, cell cultures were exposed or sham-exposed for 24 h: Three experiments were carried out to evaluate the effect of MF-Sin and four experiments to evaluate the MF-Pl, and for each experimental run four cultures were set up: two for exposure and two for sham exposure. To validate cell sensitivity to well- known DNA damage inducers, positive controls were also provided. For this purpose, in preliminary experiments, dose-response curves were established by using hydrogen peroxide and mitomycin C for the comet and MN assays, respectively, to assess the experimental conditions. In- creasing doses of hydrogen peroxide (H202, 25-100 pIM final concentration) were tested for 30 min to induce comets, while MMC at final concentrations ranging from 0.005 to 0.025 pLg/ml was used to induce micronuclei. The optimal conditions to induce reproducible DNA damage were 50 pLM H202 for 30 min and 0.025 pLg/ml MMC added at the start of culture and left throughout the whole culture period for the induction of comets and micronuclei, respectively.

Cytogenetic Analysis

1. Alkaline comet assay

Twenty-four hours before the experiments, 5 X 104 cells/3 ml complete medium were seeded into 35-mm petri dishes (Corning, catalog no. 430165). After field exposure, cultures were processed for the comet assay as described in detail elsewhere (12). After trypsinization, cells were collected and centrifuged for 10 min at 1200 rpm, the supernatant was discarded, and cells were resuspended in 100 p1 low-melting-point agarose (0.6% w/v; 370C) and sandwiched between a lower layer of 1.5% normal-melting agarose (1.5% w/v) and an upper layer of low-melting- point agarose (0.6% w/v) on microscope slides. The slides were then immersed for 90 min in freshly prepared cold lysis solution (2.5 M NaC1, 100 mM Na2EDTA, 10 mM Tris, pH 10, 1% N-lauryl sarcosine) with 1% Triton X-100 and 10% DMSO added just before use at 40C. At the end of lysis treatment, slides were drained and placed in a horizontal gel electrophoresis tank with fresh alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH > 13) and left in the solution for 40 min at 4oC to allow the equilibration and the unwinding of DNA to express the alkali-labile damage. Using the same buffer, electrophoresis was carried out at 4'C for 20 min at 25 V using an Amersham Pharmacia Biotech power supply and adjusting the current to 300 mA by modulating the buffer level. At the end of this treatment, slides were rinsed three times with Tris (400 mM, pH 7.5) and air-dried.

Slides were stained just before analysis with ethidium bromide (12 [Lg/ ml), and images of 1000 randomly selected cells (250 from each of four replicated cultures) were analyzed with a computerized image analysis system (Delta Sistemi, Rome, Italy) fitted with a Leica DM BL fluores- cence microscope at 250x magnification. This system acquires images, computes the integrated intensity profile for each cell, estimates the comet cell components, head and tail, and evaluates a range of derived parameters. DNA damage was evaluated taking into account tail moment, comet moment and percentage of DNA in the tail. To ensure a direct comparison with the data of Ivancsits et al., the comet tail factor was calculated on the basis of the amount of DNA in the tail as determined by the software as described in detail elsewhere (18).

2. Micronucleus assay

From preliminary experiments, the cell cycle duration of ES-1 cells, which was assessed to determine the right time for addition of cytochalasin B, was found to be 28 h (data not shown).

A total of 5 X 104 cells/5 ml complete medium were seeded in slide flasks (Nunclon, cod. 170920; 9 cm2 growth area) and left for 24 h at 37?C in a CO2 incubator. Then cell cultures were placed in the exposure system, and the intermittent magnetic-field exposure was carried out for 24 h. To block cytokinesis, 4 h before the end of the first cycle (48 h after seeding), cytochalasin B (3 pLg/ml final concentration) was added, and at the end of the second replication cycle (80 h after seeding), cells



272 SCARFI ET AL.

were incubated for 30 min at 370C in hypotonic solution (KC1, 0.075 M) and fixed for 10 min (80% methanol in distilled water). Air-dried slides were stained for 8 min (10% Giemsa in phosphate buffer, pH 6.8).

Micronuclei were scored in binucleated cytokinesis-blocked cells with well-preserved cytoplasm using a light microscope, and their frequency was evaluated in 2000 cells (1000 cells for each duplicate slide). The morphological criteria adopted for MN scoring in binucleated cells were similar to those used by Fenech for human lymphocytes (19). The results were expressed as micronucleated binucleated cells per 2000 binucleated cells.

On the same slides, by classifying 1000 cells according to the number of nuclei, the binucleate cell index and the cytokinesis-block proliferation index were also evaluated for each culture, and the cell cycle kinetics was determined. The binucleate cell index is the number of binucleated cells per 1000 cells scored, expressed as a percentage, and provides an index of cytotoxicity and responsiveness to the mitogen (18). The cytokinesis-block proliferation index is defined as [Ml + 2M2 + 3(M3 + M4)]/N, where Ml to M4 represent the number of cells with one to four nuclei, respectively, and N is the total number of scored cells. This index represents the average number of cell cycles undergone by a cell population and can be considered as an index of cell kinetics or average cell division with some similarities to the proliferation rate used for meta- phases in cultures treated with bromodeoxyuridine (20, 21).

Statistical Analysis

Groups of sham-exposed samples, exposed samples and positive controls (Tables 1 and 3) were tested by analysis of variance. P values lower than 0.05 were considered statistically significant. To identify statistically significant differences among groups, a multiple comparison test (Tukey's honestly significant difference test) was performed.

For the remaining groups that comprise sham-exposed and exposed samples (Tables 2 and 4), the two-tailed paired Student's t test (P < 0.05) was used.

All the statistical tests were performed with MATLAB software.

RESULTS

Positive Controls

Preliminary experiments were conducted to set up dose- response curves for positive controls. For the comet assay, when increasing doses of H202 were tested for 30 min, an approximately three times increase in all the comet parameters investigated was detected after treatments at 25 pM final concentration, while 50 VpM produced an increase of almost eight times and was used as a positive control. High- er doses (75 and 100 pM) resulted in complete DNA frag- mentation (data not shown).

The dose-response curve for MMC was determined by treating cells at concentrations ranging from 0.005 to 0.05 pg/ml. The lower doses resulted in a slight increase in MN formation, while doses higher than 0.025 .g/ml strongly affected cell proliferation and were discarded. The best concentration was found to be 0.025 pg/ml, which induced about a four times increase in MN formation, together with a slight decrease in cell proliferation; this concentration was used as a positive control.

Comet Assay After 15 h intermittent exposure (5 min on/10 min off)

to MF-Sin, there were no statistically significant differences

in any of the parameters investigated when sham-exposed cells were compared to exposed cells (P > 0.05 in all cases). Similar results were obtained when cells that were exposed or sham-exposed for 24 h were examined (P > 0.05 in all cases). In contrast, when sham-exposed cultures were compared to their positive controls (50 pVM H202), a statistically significant increase in all the parameters investigated was detected (P < 0.05). The results of three independent experiments for each exposure duration investigated are reported in Table 1; 1000 cells were examined (250 from each of four replicate cultures), except for positive controls, where 500 cells were examined (250 from duplicate cultures). Similar results were obtained when intermittent exposures were carried out with the MF-Pl, as shown in Table 2. After 15 (three experiments), 18 (three experiments) and 24 h (five experiments) exposure or sham exposure, no var- iation was detected in all the parameters investigated (P > 0.05), while H202 treatments resulted in significant damage, as expected. In this case, positive controls were used in only three out of 11 experiments (one experiment for each exposure duration), as shown in Table 2.

Micronucleus Assay

When MN formation after 24 h intermittent exposure was compared to that in sham-exposed cultures, no statistically significant difference was detected for either the MF- Sin or the MF-P1 signal (P > 0.05 in both cases). No effect was found on cell proliferation, as assessed by evaluating the cytokinesis-block proliferation index and the binucleate cell index (Tables 3 and 4). This is at variance of MMC- treated cultures, where all the parameters investigated were significantly different from those in both sham-exposed and exposed cultures (P < 0.05), with an increase in micronuclei of about four times and a slight decrease in cell proliferation. The data in Tables 3 and 4 are for 2000 randomly selected binucleated cells/treatment examined for MN frequency (1000 cells from two slides from duplicate cultures) and 1000 cells examined for cytokinesis-block proliferation index and binucleate cell index analysis (500 from each slide).

DISCUSSION

This study was designed to confirm the induction of DNA damage found by Ivancsits et al. in cultured human fibroblasts after intermittent exposure to 50 Hz sinusoidal EMFs, as assessed by the alkaline SCGE/comet assay (12, 13). We also extended the investigation by testing a powerline signal and by employing a cytogenetic technique with different sensitivity, the cytokinesis-block MN assay. The latter provides information to complement that obtained with the comet assay; the MN test detects lesions that survive at least one mitotic cycle (22), whereas the comet assay may be considered as a more sensitive method to assess changes in DNA damage and/or DNA confor-




TABLE 1 DNA Migration in ES-1 Human Fibroblasts Exposed to Intermittent 1 mT, 50 Hz

Sinusoidal Magnetic Field (MF-Sin) Parameter investigated

Experiment Treatment Tail factor Percentage DNA Tail moment Comet moment 15 h

Exp. 1 MF-Sin 6.74 4.64 1.45 2.18 Sham 6.53 4.55 1.41 2.11 H202 31.87 28.39 9.25 11.62

Exp. 2 MF-Sin 5.83 3.75 1.14 1.90 Sham 5.50 3.38 1.03 1.77 H202 23.58 22.25 7.64 10.64

Exp. 3 MF-Sin 5.16 3.13 0.94 1.73 Sham 4.44 2.27 0.66 1.40 H202 33.64 31.05 12.53 15.79

Mean ? SE MF-Sin 5.9 ? 0.5 3.8 ? 0.4 1.2 _

0.1 1.9 ? 0.1 Sham 5.5 ? 0.6 3.4 ? 0.7 1.0 ? 0.2 1.8

_ 0.2

H202 30 ? 3 27 ? 3 10 _

1 13 ? 2 24 h

Exp. 1 MF-Sin 3.39 1.05 0.31 0.79 Sham 4.24 2.11 0.63 1.36 H202 29.49 27.74 10.70 14.26

Exp. 2 MF-Sin 3.83 1.61 0.47 1.22 Sham 3.45 1.21 0.34 1.18 H202 22.51 21.84 7.79 11.10

Exp. 3 MF-Sin 3.16 0.91 0.26 0.96 Sham 3.11 0.92 0.24 0.90 H202 24.64 22.66 7.51 10.17

Mean ? SE MF-Sin 3.5 ? 0.2 1.2 ? 0.2 0.35 ? 0.06 1.0 ? 0.1 Sham 3.6 ? 0.3 1.4 ? 0.4 0.4 ? 0.1 1.1 ? 0.1 H202 26 + 2 24 2 9 + 1 12 ? 1

Notes: The alkaline comet assay was performed immediately after the exposure, and DNA damage was evaluated by means of tail factor, tail DNA percentage, tail moment and comet moment. For each experiment/treatment 1000 cells were examined from four replicate cultures, and each data point is the mean of the four replicate cultures. H202: positive control (50 pM for 30 min).

mation by genotoxic agents and identifies repairable DNA lesions or alkali-labile sites.

The results reported here do not support the hypothesis that intermittent exposures to 50 Hz induce genotoxic effects in ES-1 cells at either signal investigated.

Concerning the alkaline comet assay, our replicate experiments refer to the conditions in which Ivancsits et al., by applying a sinusoidal field, found a statistically significant increase in the tail factor after 24 (12) and 15 h exposure (13). We also repeated the experiments using a MF- P1 signal; in this case, 18-h exposures were also carried out. It is interesting to note that in the first study (12) the authors found about a doubling of the tail factor in the exposed cultures. This increase was not reported in the second paper (13), where the difference between sham-exposed and exposed samples appears very small, if not negligible. Be- cause the increase in the tail factor in the second paper peaked around 15-18 h, we also tested an 18-h exposure duration.

The results reported here were obtained using experimental conditions similar to those of Ivancsits et al. It is

difficult to explain why the genotoxic effect was not con- firmed. As stated in the Materials and Methods, we strictly followed the operational procedures for cell growth and the comet assay provided by the authors. Moreover, an identical exposure system was employed, and the same exposure protocol was followed.

To make direct comparisons between our data and those reported in refs. (12, 13), the extent of DNA damage was evaluated using the comet tail factor, expressed as a percentage, on 1000 randomly selected DNA spots. It consists of classifying cells in five groups according to the amount of DNA in the tail and of calculating the average extent of DNA migration among cells (18). The only difference in the method is that we classified cells in different groups by using the parameters obtained by the image analysis instead of a classification by eye. In principle, the parameters obtained with imaging software should be as precise as or more precise than classification by eye (23). Moreover, because of the lack of agreement on a single appropriate comet parameter capable of adequately describing DNA damage (24, 25), we also measured other parameters, simulta-



274 SCARFI ET AL.

TABLE 2 DNA Migration in ES-I Human Fibroblasts Exposed to Intermittent 1 mT, 50 Hz

Powerline Magnetic Field (MF-Pl) Parameter investigated

Experiment Treatment Tail factor Percentage DNA Tail moment Comet moment

15 h

Exp. 1 MF-PI 6.46 4.45 1.18 2.06 Sham 5.76 3.65 0.96 1.91 H202 37.79 34.36 14.35 17.32

Exp. 2 MF-PI 4.53 2.35 0.78 1.50 Sham 5.01 2.89 0.85 1.77

Exp. 3 MF-PI 4.62 2.48 0.69 1.49 Sham 4.93 2.68 0.88 1.61

Mean ? SE MF-PI 5.2 ? 0.6 3.1 _ 0.7 0.9 ? 0.1 1.7 ? 0.2

Sham 5.2 ? 0.3 3.1 ? 0.3 0.90 ? 0.03 1.76 ? 0.09

18 h Exp. 1 MF-PI 5.80 3.93 1.42 2.20

Sham 5.94 4.10 1.50 2.27 H202 26.74 25.07 8.15 10.68

Exp. 2 MF-PI 6.42 4.48 1.67 2.30 Sham 6.55 4.64 1.62 2.36

Exp. 3 MF-PI 6.00 3.84 1.24 1.91 Sham 7.60 5.60 1.86 2.79

Mean ? SE MF-PI 6.1 ? 0.2 4.1 ? 0.2 1.4 ? 0.1 2.1 ? 0.1 Sham 6.7 ? 0.5 4.8 _ 0.4 1.7 ? 0.1 2.5 ? 0.2

24 h Exp. 1 MF-PI 6.71 4.51 1.54 1.86

Sham 5.35 3.36 0.92 1.72 H202 37.00 31.70 10.35 12.55

Exp. 2 MF-PI 4.89 2.63 0.83 1.56 Sham 3.98 1.70 0.55 1.03

Exp. 3 MF-PI 9.00 7.13 2.06 3.48 Sham 7.80 5.83 1.68 2.96

Exp. 4 MF-Pl 3.56 1.30 0.30 0.84 Sham 4.26 2.26 0.49 1.06

Exp. 5 MF-PI 6.93 5.02 1.76 2.32 Sham 7.57 5.84 1.87 2.54

Mean ? SE MF-PI 6.2 ? 0.9 4 ? 1 1.3 ? 0.3 2.0 _ 0.4 Sham 5.8 ? 0.8 3.8 ? 0.9 1.1 ? 0.3 1.9 ? 0.4

Notes. The alkaline comet assay was performed immediately after the exposure and DNA damage was evaluated by means of tail factor, tail DNA percentage, tail moment and comet moment. For each experiment/treatment 1000 cells were examined from four replicated cultures, and each data point is the mean of the four replicate cultures. H202: positive control (50 pM for 30 min).

neously recorded for each comet, currently used in the literature: tail moment, percentage of DNA in the tail, and comet moment. In particular, the latter parameter has been suggested to give a more accurate evaluation than the tail moment (24). Also in these cases the analysis, as well as the related statistics, was carried out on 1000 cells (250 from each of four replicate cultures), which is a very high number, considering that most data reported in literature are for no more than 200 cells scored when imaging software is used.

For the induction of micronuclei, we found neither genotoxic nor cell cycle kinetics effects induced by 24 h exposure to 1 mT intermittent 50 Hz EMFs for either the MF- Sin or MF-Pl signal. Our study was carried out following

the guidelines suggested by the working group on MN experiments (21), namely demonstration of cell proliferation, provision of positive controls, and slide scoring criteria.

It is known that the background frequency of micronuclei can change depending on the cell type studied and may influence the sensitivity of the assay to detect genotoxic effects. The human fibroblasts used (ES-1) showed a very low and stable background frequency of micronuclei ranging in sham-exposed cultures from 6 to 12 cytokinesis- blocked cells containing micronuclei/2000 cytokinesis- blocked cells, as shown in Tables 3 and 4, thus satisfying the requirements for a correct application of the cytokinesis-block MN assay. Moreover, our protocol allows us to obtain the optimal culture conditions for cell proliferation.




TABLE 3 Incidence of Micronucleus Formation, Proliferation Index (CBPI), and Binucleate Cell Index (BCI) in ES-1 Human Fibroblasts Exposed for 24 h to Intermittent 1 mT, 50 Hz

Sinusoidal Magnetic Field (MF-Sin) Parameter investigated

Experiment Treatment CBMN CBPI BCI

1 MF-Sin 10 1.44 44.3 Sham 9 1.44 43.8 MMC 32 1.27 27.0

2 MF-Sin 9 1.40 39.9 Sham 11 1.42 42.6 MMC 44 1.28 28.2

3 MF-Sin 8 1.48 48.0 Sham 8 1.49 48.8 MMC 45 1.29 29.5

Mean ? SE MF-Sin 9.0 ? 0.6 1.44 ? 0.02 44 ? 2 Sham 9.3 ? 0.8 1.45 ? 0.02 45 ? 2 MMC 40 ? 4 1.27 _ 0.01 28.2 ? 0.7

Notes. The incidence of micronuclei (CBMN) was determined by examining a total of 2000 binucleated cells per treatment, i.e. 1000 cells from two slides derived from duplicate cultures. CBPI and BCI were determined from the analysis of 1000 cells (500 from each slide). MMC: positive control (0.025 pIg/ml final concentration).

Indeed, we found that the binucleate cell index in sham- exposed cultures ranged from 40 to 50%, which is in agreement with the suggestions of Fenech, who recommended 35-60% binucleated cells for human peripheral blood lymphocytes after 72 h PHA stimulation (26).

As in the case of the comet assay, with the MN assay, the lack of genotoxic effects after exposures to 1 mT 50 Hz EMFs is supported by the comparison between sham- exposed cultures and positive controls, indicating that our biological model responded efficiently to stimuli of known

TABLE 4 Incidence of Micronucleus Formation, Proliferation Index (CBPI) and Binucleate Cell Index (BCI) in ES-1 Human Fibroblasts Exposed for 24 h to

Intermittent 1 mT, 50 Hz Powerline Magnetic Field (MF-PI)

Parameter investigated

Experiment Treatment CBMN CBPI BCI

1 MF-PI 8 1.41 41.0 Sham 8 1.43 43.0 MMC 28 1.30 29.7

2 MF-P1 10 1.51 50.0 Sham 11 1.53 53.0

3 MF-PI 5 1.42 42.0 Sham 6 1.40 40.0

4 MF-PI 13 1.47 47.0 Sham 12 1.48 48.0

Mean ? SE MF-PI 9 ? 2 1.45 ? 0.02 45 ? 2 Sham 9 ? 1 1.46 ? 0.03 46 + 3

Notes. The incidence of micronuclei (CBMN) was determined by examining a total of 2000 binucleated cells per treatment, i.e. 1000 cells from two slides derived from duplicate cultures. CBPI and BCI were determined from the analysis of 1000 cells (500 from each slide). MMC: positive control (0.025 pLg/ml final concentration).

genotoxic agents. We must point out here that even Rtidi- ger's group, in the framework of the REFLEX project, reported induction of micronuclei in ES-1 cells subjected to the same exposure conditions used in refs. (12, 13) for 24 h. These results were reported at the 25th Annual Meeting of the Bioelectromagnetics Society but, to the best of our knowledge, have not been published in peer-reviewed jour- nals.

On the whole, our findings are in agreement with most of the in vitro results reported in literature, which failed to demonstrate induction of genotoxic effects due to ELF EMF exposure. Nevertheless, in addition to the results obtained in Vienna, a number of positive results have been reported, often associated with the experimental conditions adopted (8-11). They deserve particular attention because of the close relationship between induction of DNA damage and cancer occurrence.

Regarding the comet assay, we would like to stress that our study is one of the few in bioelectromagnetic research that has attempted to replicate the conditions used in another laboratory as strictly as possible. In general, when a laboratory fails to replicate the results of another independent group, basically two hypotheses can be formulated: (a) There was a mistake in the experimental procedure adopted by one or both laboratories; (b) some subtle differences were present in the experimental procedures and/or in the environmental conditions that escaped the control of the experimenters. Apart from the trivial explanation of an experimental mistake, in general it is not easy to discover whether a subtle but relevant parameter was not controlled. In this context, the difference between the results obtained in our laboratory and those reported by Rtidiger's group requires further investigation to understand the reason of the discrepancy. This is even more important when taking



276 SCARFI ET AL.

into account that the Vienna group published several papers (12, 13, 27) containing a considerable amount of data on the genotoxic effects of 50 Hz EMFs as assessed by the comet assay, chromosomal aberrations and micronuclei.

In conclusion, in view of our present failure to explain the discrepancy in the results of the two groups, we wel- come any opportunity in the near future to carry out an extensive investigation on this point, including replications in other independent laboratories, possibly under an exter- nal "super partes" control.

ACKNOWLEDGMENTS

This work was funded by the Commission of the European Commu- nities, Program "Quality of life and management of living resources", QLK4-CT-1999-01574, Key Action 4 "Environment and Health" and by Italian Ministry of University and Scientific Research, Project "Salva- guardia dell'uomo e dell'ambiente dalle emissioni elettromagnetiche". We thank Professor Franz Adlkofer and Verum Foundation for their support, Professor Hugo Riidiger for sending the cells and the related experimental protocols, Professor Niels Kuster at IT'IS Foundation for hav- ing provided us the exposure system and the quality control, and Profes- sor Francesco Clementi and Dr. Diego Fornasari (University of Milano, Department of Pharmacology) for their assistance.

Received: March 10, 2005; accepted: May 10, 2005

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