Non-thermal effects of 2.45 GHz microwaves on spindle assembly, mitotic cells and viability of Chinese hamster V-79 cells

Mutation Research 716 (2011) 1–9

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Non-thermal effects of 2.45 GHz microwaves on spindle assembly, mitotic cells and viability of Chinese hamster V-79 cells Michela Ballardin a,1 , Ignazia Tusa a,1 , Nunzia Fontana b , Agostino Monorchio b , Chiara Pelletti b , Alessandro Rogovich b , Roberto Barale a,∗ , Roberto Scarpato a a b

Dipartimento di Biologia, University of Pisa, via Derna 1 56100 Pisa, Italy Dipartimento di Ingegneria dell’ Informazione: Elettronica, Informatica, Telecomunicazioni, University of Pisa, Pisa, Italy

a r t i c l e

i n f o

Article history: Received 25 October 2010 Received in revised form 15 July 2011 Accepted 22 July 2011 Available online 30 July 2011 Keywords: Non thermal effects Microwaves Spindle aberration Apoptosis V79 cells

a b s t r a c t The production of mitotic spindle disturbances and activation of the apoptosis pathway in V79 Chinese hamster cells by continuous 2.45 GHz microwaves exposure were studied, in order to investigate possible non-thermal cell damage. We demonstrated that microwave (MW) exposure at the water resonance frequency was able to induce alteration of the mitotic apparatus and apoptosis as a function of the applied power densities (5 and 10 mW/cm2 ), together with a moderate reduction in the rate of cell division. After an exposure time of 15 min the proportion of aberrant spindles and of apoptotic cells was significantly increased, while the mitotic index decreased as well, as compared to the untreated V79 cells. Additionally, in order to understand if the observed effects were due to RF exposure per se or to a thermal effect, V79 cells were also treated in thermostatic bath mimicking the same temperature increase recorded during microwave emission. The effect of temperature on the correct assembly of mitotic spindles was negligible up to 41 ◦ C, while apoptosis was induced only when the medium temperature achieved 40 ◦ C, thus exceeding the maximum value registered during MW exposure. We hypothesise that short-time MW exposures at the water resonance frequency cause, in V79 cells, reversible alterations of the mitotic spindle, this representing, in turn, a pro-apoptotic signal for the cell line. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Radiofrequencies and microwaves (RF/MW) are classified as non-ionising electromagnetic radiation (NIR) in the frequency range up to 300 GHz. Usually, MW radiation is considered a subset of RF radiation, although an alternative convention treats RF and MW radiation as two distinct spectral regions. It is known that the absorption of RF/MW energy varies with wave frequency. A multitude of devices that emits radiofrequency radiation (RFR) are used in medicine, in industry for heating, welding and sealing of plastics and metals, and for a variety of military purposes. A large increase in the number of people who are potentially exposed to RF radiation occurred with the introduction of household microwave ovens, which work predominantly at 2450 MHz, and wireless communication systems (handheld mobile phones as well as the newer personal communication devices that deliver voice, data and images) that operate below to 2000 MHz [1,2]. With the increasing use of consumer devices that emit RF radiation, several investigations have dealt with possible adverse effects on

∗ Corresponding author. Tel.: +39 050 2111505; fax: +39 050 2211527. E-mail address: [email protected] (R. Barale). 1 Contributed equally to the work. 0027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2011.07.009

human health [3–6]. In particular, many researchers have recently employed ad-hoc experimental techniques as well as classical cytogenetic methods to study the induction of genetic damage after in vitro and in vivo exposure of prokaryotic and eukaryotic cells to RF radiation. It is commonly assumed that RF radiation does not induce genetic damage even though some studies showed the presence of RFR-induced genotoxic damage in various cell systems exposed to different conditions [7–10]. There was also a number of reports indicating that RF radiation, while being not genotoxic itself, can enhance the effect of a chemical mutagen [11,12]. Thus, it could be interesting to investigate the possibility that MW exposure can lead to cell damage via an epigenetic mechanism. Indeed, literature reports suggest that microwave may affect biological structures of the cell, including microtubules, through the excitation of elastic resonances, or induce collagen matrix disruption, which can elicit spindle disturbances at the following mitosis [13–15]. Another relevant issue lies in the fact that, according to the literature, two types of effects can be ascribed to microwaves, i.e. thermal and non-thermal [16–19]. Biological alterations in the absence of a significant temperature increase represent a non thermal effect, while a thermal effect is due to transformation of electromagnetic energy into heat. In particular, it is related to the heat generated by the absorption of microwave energy by the water medium or by organic complex systems, both characterized by a

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permanent or induced polarization. Undoubtedly, the amount of produced heat will depend also on frequency and power of the applied field, and on the duration of the exposure. Currently, very little is known about the molecular mechanisms involved in the putative non-thermal effects which might involve direct energy transfer from the electromagnetic field to the vibrational modes of macromolecules altering their conformation [20,21]. The aim of this study was therefore to analyze mitotic spindle alterations and induced lethal events occurring in V79 Chinese hamster cells exposed to continuous MW radiation at a frequency of 2.45 GHz and power densities of 5 or 10 mW/cm2 . In addition, in order to understand if the observed effects were due to RF exposure per se or to a thermal effect, V79 cells were also treated in thermostatic bath mimicking the same temperature increase recorded during microwave emissions. 2. Materials and methods 2.1. Cell culture, MW exposure and influence of thermal aspects In this study, we decided to use a stable continuous line of V79 because the status of this established line is well-known and commonly used in the cyto-toxicology studies. These cells have a modal chromosome number equal to 21, as previously determined by Sciandrello et al. [22] with a doubling time of 12–15 h. V79 cells were also used because they are fibroblast cells that grow in culture by attachment to the surface of the culture vessel, unlike lymphoblastoid cells which grow in suspension in the culture medium. This character is important in the study of the fidelity of mitosis when it is essential that the dividing cells are physically disturbed as little as possible. The cells were cultured directly on 26 mm × 76 mm stove-sterilized glass microscope slides (Menzel-Gläser, Germany) in the centre area of a culture dish so they can be exposed to the test compound and prepared for microscopic examination with minimal disturbance. The sterile glass microscope slides were placed in 90 mm Petri dishes on which V79 cells were seeded at approximately 8 × 105 cells/ml and grown 18 h prior to MW exposure in fresh medium consisting of Dulbecco’s Modified Eagles Medium (DMEM) without phenol red (Gibco-Invitrogen, Paisley, UK), and supplemented with 5% foetal calf serum (FBS) (Gibco-Invitrogen, Paisley, UK), penicillin (100 U/ml) and streptomycin (100 ␮g/ml). Cultures were maintained at 37 ◦ C in a 5% CO2 humidified incubator. The stability of the electromagnetic field used for cells exposure is a very important issue for the experiment. As shown in Fig. 1, this requirement is met by using a loop controlled system that impinges an e.m. field in a Gigahertz Transverse Electromagnetic Cell (GTEM – Shaffner mod 250 operating from DC to 20 GHz) with a

fixed power (additional details are reported in Appendix). Thus, simultaneously to each MW experiment, cell samples were placed in water-bath by increasing temperature from 38 ◦ C to 41 ◦ C for 15 min. All the experiments were made in triplicate. Moreover negative control cells were kept in the same experimental conditions, but they were not exposed to MW.

2.2. Indirect immunostaining, determination of mitotic index and incidence of aberrant spindles Both analysis of aberrant spindles and evaluation of mitotic index (MI) in cell population were performed immediately after MW exposure. For the microtubule analysis, after the MW exposure, cells were prefixed at room temperature for 6 min with methanol added to the culture medium in the ratio 1:1; then the cells were fixed with pure methanol for 8 min. After washing in PBS and rinsing three times in PBS + 1% BSA (Bovine Serum Albumin, Sigma) with 0.1% Triton X-100 (Sigma), cells were incubated overnight at 4 ◦ C with mouse anti-␤-tubulin antibody (Sigma, Saint Louis, Missouri), diluted 1:100 in PBS + 1% BSA (Bovine Serum Albumin) with 0.1% Triton X-100 [23]. After three rinses in the same washing solution, cells were incubated in a humidified chamber for 30 min at 37 ◦ C with fluorescein (FITC)-conjugated anti-mouse IgG (Sigma, Saint Louis, Missouri) diluted 1:28. After three rinses, the preparations were mounted in antifade solution (1 mg/ml p-phenylenediamine dihydrochloride in 1 part PBS and 9 parts 87% glycerol, pH 8.0). The slides were then observed under a Nikon fluorescence photomicroscope equipped with a HBO 100 W mercury lamp and a suitable filter. The antibody-treated cells were then classified according to the types of spindles observed as normal, bipolar and aberrant tripolar, tetrapolar or multipolar structures (Fig. 2). At least 100 cells per experiment were viewed to determine the incidence of aberrant spindles (i.e. 300 cells in total). The MI is a measure of the proliferation of a cell population. It is defined as the ratio between the number of cells in mitosis and the total number of cells. A cell population grows as cells pass through mitosis to complete the cell cycle. Many cells lose the capacity to divide, if their maturation or proliferation is affected by internal or external reasons. In this study, MI was obtained by scoring 1000 cells per slide using a fluorescence microscope.

2.3. Apoptosis assay Cells were washed and fixed as reported above and 24 h after the MW exposure, they were assayed for apoptotic chromatin condensation. The apoptotic cells were detected using a TdT-FragEL kit (Oncogene, USA and Canada), according to the recommendations of the manufacturer. At least 200 cells were scored in each sample and three independent experiments were performed to determine the frequencies of apoptotic cells. This assay allowed us to distinguish apoptotic cells, coloured dark brown, from normal cells, coloured blue–green. Under the microscope, the

Fig. 1. The loop controlled system used during microwave exposure.

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Fig. 2. The antibody-treated V79 cells were classified according to the types of spindles observed: (A) bipolar (normal) spindle with one ␤-tubulin-containing spot localized at each pole of the spindle, (B) tripolar, (C) tetrapolar and (D) multipolar spindles with ␤-tubulin-containing spots localized at each pole of the spindle.

2.4. Statistical analysis Data were statistically elaborated by means of ANOVA in order to assess the effect of MW exposure (time on aberrant spindles or apoptotic cells). Dunnett’s test was used to analyze differences between each experimental point and the baseline level [24,25]. All data were presented as mean ± standard deviation of three independent experiments. A difference at p < 0.05 was considered statistically significant. All statistical calculations were carried out using the Statgraphics Plus version 5.1 (Statistical Graphics Corporation, 2001, Rockville, USA) software package.

3. Results 3.1. Immunostaining and mitotic index The results of analysis of variance (ANOVA) on spindle formation after exposing cells to MW radiation for two different controlled temperatures are reported in Figs. 3 and 4, respectively. Compared to the control value (20.44 ± 3.12%), a significant increase (p < 0.001, Dunnett’s test) in the number of cells with aberrant spindles was detected as a function of power density: at 5 or 10 mW/cm2 , in fact, the average frequencies increased up to 30.2 ± 6.91% or 35.6 ± 3.36%, respectively (Fig. 3). At variance, we did not observe statistically significant differences in the proportion of aberrant cells between control (20.44 ± 3.12%) and the 38 ◦ C- (20.40 ± 4.34%), 39 ◦ C- (22.06 ± 4.83%), 40 ◦ C- (22.5 ± 2.66%), or 41 ◦ C-treated V79 cells (19.6 ± 3.29%) (Fig. 4).

Fig. 5 shows that the mitotic index of V79 cells exposed to the power density of 10 mW/cm2 (29.20 ± 6.02‰) differed significantly (p < 0.01, Dunnett’s test) from the mitotic index of control cells (44.38 ± 8.69‰), unlike those exposed to power density of 5 mW/cm2 . We also calculated the frequency of cells with aberrant spindles after correcting them for their corresponding MI values. As graphically visualized in Fig. 6, the average values did not decrease as a function of MW power density (9.07 ± 0.27‰, 10.01 ± 0.50‰

No. of aberrant spindles (%)

TdT-positive cells appeared round and small, with condensed chromatin often arranged to resemble a metaphase plate.

50 40 30 20 10 0 0

5

10

Power density (mW/cm^2) Fig. 3. Induction of aberrant spindles in control and microwave treated V79 cells by box and whisker plot. Box represents the interquartile range, and whiskers represent the lower and upper quartiles. Asterisk and horizontal line within each box are the mean and the median value, respectively.

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80

50

70 40

60

Mitotic index

No. of aberrant spindles (%)

4

30 20

50 40 30 20

10

10 0

0 37

38

39

40

37

41

38

Temperature (°C) Fig. 4. Induction of aberrant spindles in control and temperature treated V79 cells by box and whisker plot. Box represents the interquartile range, and whiskers represent the lower and upper quartiles. Asterisk and horizontal line within each box are the mean and the median value, respectively.

40

41

Fig. 7. Course of aberrant spindles corrected for the corresponding mitotic index in control and temperature treated V79 cells by box and whisker plot. Box represents the interquartile range, and whiskers represent the lower and upper quartiles. Asterisk and horizontal line within each box are the mean and the median value, respectively.

80

20

Apoptotic cells (%)

70

Mitotic index

39

Temperature (°C)

60 50 40 30 20 10

16 12 8 4

0 0

5

0

10

0

Power density (mW/cm^2) Fig. 5. Course of mitotic index in control and microwave treated V79 cells by box and whisker plot. Box represents the interquartile range, and whiskers represent the lower and upper quartiles. Asterisk and horizontal line within each box are the mean and the median value, respectively.

and 10.42 ± 0.20‰ at O, 5 and 10 mW/cm2 , respectively); on the contrary, they tended slightly to increase (R2 of the regression line = 0.951). When V79 cells were exposed to increasing temperatures, the number of dividing cells did not significantly vary as compared to the control value; only mitotic index of 40 ◦ C-treated V79 cells (29.33 ± 7.71‰) differed significantly (p < 0.01, Dunnett’s test) from the mitotic index of control cells (44.38 ± 8.69‰) (Fig. 7).

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Fig. 8. Induction of apoptosis in control and microwave treated V79 cells by box and whisker plot. Box represents the interquartile range, and whiskers represent the lower and upper quartiles. Asterisk and horizontal line within each box are the mean and the median value, respectively.

3.2. Apoptotic cells Figs. 8 and 9 graphically visualize the results of analysis of variance (ANOVA) on the apoptosis assay after exposing cells to MW radiation or to different controlled temperatures. Once again, the frequency of apoptotic cells significantly increased with increasing power density: the mean values obtained at 5 mW/cm2 (4.04 ± 2.56%) or 10 mW/cm2 (8.69 ± 3.45%) were

18 20 15

Apoptotic cells (%)

No of aberrant spindles corrected for MI (%)

5

Power density (mW/cm^2)

12 9 6 3

16 12 8 4

0 0

5

10

Power density (mW/cm^2)

0 37

38

39

40

41

Temperature (°C) Fig. 6. Course of mitotic index in control and microwave treated V79 cells by box and whisker plot. Box represents the interquartile range, and whiskers represent the lower and upper quartiles. Asterisk and horizontal line within each box are the mean and the median value, respectively. Equation and R2 of the regression line describing the dose–response relationship is also reported: y = 8.48 + 0.68x; R2 = 0.951.

Fig. 9. Induction of apoptosis in control and temperature treated V79 cells by box and whisker plot. Box represents the interquartile range, and whiskers represent the lower and upper quartiles. Asterisk and horizontal line within each box are the mean and the median value, respectively.

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significantly higher (p < 0.001, Dunnett’s test) than the control (1.88 ± 1.45%) (Fig. 8). No significant increase in the spontaneous level of apoptotic cells was observed when V79 cells were heated for 15 min at 38 ◦ C (2.00 ± 1.91%) or 39 ◦ C (1.74 ± 1.53%). The effect of temperature on the rate of apoptosis occurred only when the cells were treated at 40 ◦ C (8.25 ± 3.72%) and 41 ◦ C (10 ± 5.29%) (Fig. 9).

4. Discussion The present study has dealt with the possible cell damage, expressed as mitotic spindle disturbance or activation of the apoptosis pathway, following by a continuous 2.45 GHz MW exposure of V79 cells. We demonstrated that a 5 mW/cm2 and 10 mW/cm2 MW exposure, respectively, for 15 min was able to induce alteration of the mitotic apparatus and apoptosis as a function of the applied power densities of the incident e.m. field. Moreover, we noted a moderate reduction in the rate of cell division by observing the proportion of aberrant spindles and apoptotic cells or the mitotic index. Mitotic index includes all cells in different stages of division, from prophase to telophase. If the percentage of cells harbouring aberrant spindles were normalized for the MI (this would represent the fraction of cells with an altered microtubule apparatus among the total population of dividing cells), then it emerged that MW exposure affects cell proliferation by increasing the frequency of these cells. This poses the question of what could happen after further 24 h culture to treated cells with aberrant spindles. It is likely to suppose that a fraction of these cells may rapidly die or progress to micronucleated or aneuploid cells. However, data from our group (unpublished data) showed that human lymphocytes exposed to the same conditions of MW irradiation used in the present study, did not induce micronuclei 24 h after the end of treatment, unless MW exposure were prolonged up to 1 h. Thus, even though we did not show data on this, it is our opinion that no appreciable induction of micronuclei could be observed, mainly due to the already small difference in the initial frequency of mitotically disturbed cells (cells fixed immediately after the exit from GTEM) between MW and sham exposed cultures, and also because the cells could have undergone a non programmed (early at the end of treatment) or an apoptotic (24 h later) death. In order to study if the observed effects were the consequence of cell culture heating, we exposed cells to thermostatically controlled conditions that reproduced the medium temperature increase in the GTEM cell after their MW exposure. The effect of temperature on the correct assembly of mitotic spindles was negligible up to 41 ◦ C, while apoptosis was induced only when the medium temperature achieved 40 ◦ C, thus exceeding the maximum value registered during MW radiation. However, it should be stressed that no sign of apoptosis was seen unless cells were allowed to proliferate further 24 h after the cessation of exposure. Strong experimental reports show that extremely lowfrequency (ELF) magnetic fields can affect cells under specific combinations of frequency, magnetic flux density of alternating and static magnetic fields [26–33]. Different biological endpoints were affected, such as gene expression, chromatin conformation, calcium efflux and enzymatic activity [34–41]. Usually, investigations on possible mutagenic effects of ELF provided negative results [42], however for most of these studies, the conditions of exposure were not optimised and experimental evidence for mutagenic effects was obtained only at specific combinations of frequency and intensities [43,44]. Spindle disturbances may result from damage to the functioning of a variety of cellular targets. These include targets common to mitotic cell division such as highly dynamic microtubules, microtubule-based motors, microtubule-associated proteins, and

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condensed chromosomes as important structural components of the spindle morphogenesis. The very complex protein superstructure of the spindle [45] is based on a bipolar array of microtubules, each of which is a polarised protein polymer with a plus and a minus end. It is known that any defects in spindle bipolarity lead to potential errors in chromosome segregation and that microtubule attachment to the kinetochores can be disrupted by chemical agents, chromosome micromanipulation or antibodies to centromere proteins [46]. Missegregation of chromosomes during mitosis can cause numerical chromosome aberrations giving rise to aneuploidy in daughter cells which can lead to severe adverse effects in humans including cancer [47]. One mechanism by which aneuploidy occurs is via centrosome amplification. During mitosis, centrosomes, the major microtubule-organizing centers exert an important function by formation of the spindle poles. These organelles are crucial for the number of spindle poles formed during mitosis [48]. The duplication of centrosomes is strictly regulated and tightly correlated to the DNA content of the cell to maintain accurate chromosome segregation to the daughter cells. Aberrant centrosome numbers can cause multipolar spindle formation and unequal pulling of the chromosomes resulting in chromosome missegregation (chromosome loss and non-disjunction) and in the subsequent production of aneuploid progeny aneuploidy. Observation of multipolar spindle structures in MW-exposed cells, in fact, suggests that the radiation may interact with components of the centrosomes which make up the poles of the mitotic spindle. The interactions of MW with the centrosome may result in the production of mitotic cells with deviations from the normal bipolar structure. A number of authors have demonstrated that the correct functioning and the number of centrosomes is critical to maintaining kariotype integrity [49] and that abnormal centrosome function is important in cancer development and progression [50–52]. Another mechanism that could be involved in the formation of aberrant spindles concerns the polar protein structures. Microtubule fibers represent extremely dynamic structure which functioning dependent on dynamical instability, that is continuously binding and releasing of free tubule proteins. It is reasonably to assume that the external EM radiation might interact with polar cytoskeletal structure. This structure greatly contributes to the balance between internal physiological electrostatic forces. Depending on the dipole moment, an external electric field could theoretically disturb the equilibrium [53] and increase the number of free cytoplasmatic tubulin proteins following a mechanism of depolarization of tubulins involved in the formation of the mitotic apparatus. Recently, it has been shown that electromagnetic field (90 V/m at 835 MHz) exposure can induce significant increases of mitotic disturbances in human–hamster hybrid cells [44]. Apoptosis, or programmed cell death, is an important biological phenomenon because it can provide protection in response to injury to minimize further damage initiated by the injury itself. A deregulation of apoptosis may be involved in different pathologies such as neurodegenerative diseases, in the case of a high rate of apoptosis, or cancers, in the case of a low rate of apoptosis. Apoptosis is characterized by a number of cytological alterations, including chromatin condensation, DNA fragmentation [54] and activation of cysteinyl aspartate-specific proteinases: the caspases [55]. The pathways leading to apoptosis may be dependent on or independent of caspases. Caspase-dependent apoptosis was described and studied first, but caspase-independent apoptosis is now a widely recognized phenomenon [56]. Few data are available concerning the interactions of RF fields with the process that leads to apoptosis. In vitro studies conducted on different types of cells gave different results, indicating that sensitivity to EMFs may differ according to the cell type. For example, apoptosis was not induced by RF fields in human peripheral blood mononuclear cells [57,58], or lymphoblastoid cell [59], whereas in human colon

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cancer cells [60] or human epidermoid cancer cells [61] apoptosis can be induced by EMF exposure. An interesting study [62] in acute T-lymphoblastoid leukemia cells (CCRF-CEM), known to be particularly susceptible to high frequency EMF exposure, showed that short time exposure (2 h at 900 MHz) induced an increase of the percentage of cells undergoing apoptosis, due to the early activation of both p53-dependent and independent apoptotic pathways. At longer times of exposure (24 h and 48 h), an increase in DNA synthesis occurred, with activation of pro-survival pathways. More likely, our data indicate that V79 cells are sufficiently sensitive to transform a low MW irradiation stimulus, that initially increased the number of aberrant mitotic cells, in a signal for triggering either a non programmed cell death or the apoptosis pathway. 5. Conclusions We can reasonably attribute the effects we observed in V79 cells to the action of MW exposure per se, thus excluding the presence of a thermal effect. It is also worth noting that both aberrant mitotic spindles and apoptotic cells were induced after a very short exposure. Due to the different designed experimental conditions (V79 cells are processed immediately or 24 h after cessation of treatment for immunostaining or the apoptosis protocol), it remains questionable if the early appearance of alteration of the mitotic apparatus passes through a cell damage such as chromosome missegregation before cells undergone apoptosis 24 h later. However, as available literature data reports a general lack of genotoxicity by MW in different cell systems, it is reasonable to speculate that the observed spindle alterations belong to a non permanent effect type and/or that the length of exposure was insufficient to render them irreversible. To this aim, we note that several chemical substances, including anticancer drugs, affect microtubule assembling of proliferating cells in a non or reversible manner. Irrespective of the modality of action of the chemical, mechanical perturbation of the mitotic apparatus will generally result in an altered chromosome distribution leading to formation of micronuclei or, via mitotic slippage, of tetraploid and aneuploid cells [63]. We therefore hypothesise that short-time MW exposures at the water resonance frequency cause, in V79 cells, other than cell death due to mitotic arrest, also reversible alterations of the mitotic spindle, allowing cells to re-enter the cell cycle. This hypothesis is also supported by the finding that, based on the present experimental conditions, the frequencies of mitotic cells with abnormal spin-

dles increased with increasing the potency of MW treatment. The generation of micronuclei from cells with altered spindles would represent, in turn, a further pro-apoptotic signal for the cell line. Conflict of interest The authors have nothing to declare. Appendix A. MW exposure set up and dosimetry In this section, a detailed description of the microwave exposure set up is provided together with an evaluation of exposure doses. The exposure chain system is controlled through a specific graphical user interface residing in a PC (Fig. 1), allowing to define the required power and to register the temperature during the MW exposure both in the experimental room and GTEM cell. A system composed by three heaters is electronically controlled for the maintenance of a 37 ◦ C temperature in the exposure room that plays a crucial role for the experiment. A LabVIEW developed control software allows the power system to produce a continuous wave at a fixed amplitude and frequency. The functioning is mainly detailed in Fig. 10, where the imposed amplitude forces a Rhode & Schwarz generator (mod. SMR20 operating from 1 GHz to 20 GHz) to produce a signal with associated power PG. The required exposure power PI comes from a Logimetrics amplifier (TWT A600/S operating from 2 to 4 GHz) that supplies a level PO. The GTEM cell is fed through a directional coupler (EMCC-SN 29196) introducing an attenuation AdBm (this term accounting also for the insertion loss of the coupler and the cables attenuation). Moreover, a −80 dB fraction of the emitted signal and a −40 dB sample of the reflected one by the GTEM cell are compared by using an oscilloscope. A −40 dB high power fixed coaxial attenuator (Aeroflex Weinshel 40–40–43, 150 Win) provides a correct amplitude into the oscilloscope dynamic range. In order to implement the loop power control, a sample of the signal delivered by the amplifier (power PPM ) is measured by a power meter (Boonton 4200 RF operating from 100 kHz to 110 GHz) and compared with the reference value. An isotropic field probe EP600 is used to define a correspondence lookup table between the generator amplitude and the power density into the GTEM cell. A PMM 8053A field meter equipped with an EP408 probe and later with the smaller EP183 probe has been used to verify the consistency of the previous measurements. In order to obtain a power density in the equipment under test (EUT) region of the GTEM cell [64] equal to 5 mW/cm2 (or 10 mW/cm2 ), a PPM equal to −5.29 dBm (or −2.8 dBm) should be

Fig. 10. Power generation and measurement chain.

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Fig. 11. Culture medium temperature profiles for 5 mW/cm2 and 10 mW/cm2 power densities. (Inset: specific absorption rate statistics and distribution on the surface of the glass slide.)

set, corresponding to a rms electric field strength equal to 137 V/m (or 194 V/m) as measured by the EP183. These results are consistent with the previous measurements made by employing the field probe EP600. In order to exclude thermal effects during exposure, the temperature is monitored by using an acquisition software that stores samples with a 1 s intervals. A digital fluoroptical thermometer (Luxtron – 712) with two optical fiber probes (STF type with 250 ms response time) provides the measured data. In our experiment cell samples were exposed to 2.45 GHz continuous wave radiation with a 5 mW/cm2 and 10 mW/cm2 power density for 15 min, respectively. The values of power density S were



2

calculated by the equation S = Erms  /0 , being  0 the characteristic impedance of the free space, and Erms the rms value of the electric field. In order to limit the growing of the samples temperature that comes from the MW radiation, a preliminary investigation defines the exposure times. Fig. 11 shows the culture medium temperature profile for 5 mW/cm2 and 10 mW/cm2 . In particular, the sample temperature is monitored during the exposure. In order

to avoid cells contamination, the temperature is measured inside a dedicated dish placed into the GTEM cell that contains only culture medium. Three samples were treated to measure the incidence of either aberrant spindles or apoptosis. By prior measurements through the electric field probe EP600, we ensured that all the four dishes are placed within to the EUT region of the GTEM cell (see Fig. 12). It is well known that, if not properly designed, electromagnetic field exposures might produce RF hot spots in the medium containing the cells; to this aim, we have extensively studied [65] the interactions between the polarization of the incident plane wave and the glass slide into the culture medium. According to the requirements for in vitro exposure setups [66], in order to guarantee a satisfactory homogeneity of the induced electric field into the cells into the GTEM cell, we numerically evaluated the statistics of the electric field strength and the specific absorption rate (SAR) on the surface of the glass slide: region of interest (ROI) (inset of Fig. 11). In subsequent studies, a configuration constituted

Fig. 12. Local arrangement of the dishes 2: (a) Longitudinal side arrangement. (b) Transverse side arrangement.

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by two couples of Petri dishes was considered. The two couples were parallel one each other and the effects of the E polarization (E vector orthogonal to the glass slide) of the incident plane wave were analyzed. From the numerical analysis with 5 mW/cm2 (or 10 mW/cm2 ) incident power density, we obtained that the living cells were exposed to a minimum value of the electric field equal to 4.36 V/m (or 6.18 V/m) and to a maximum value of the electric field equal to 14.4 V/m (or 20.34 V/m) on the glass slide considered as reference. On the other glass slides, we numerically obtained a variation of the mean electric field lower than +8.72% compared to the mean electric field strength on the reference glass slide. Moreover, in terms of SAR we obtained a minimum value equal to 0.04 W/kg (or 0.09 W/kg) and a maximum value equal to 0.51 W/kg (or 1.03 W/kg) for 5 mW/cm2 (or 10 mW/cm2 ) incident power density. On the other glass slides, we numerically obtained a variation of the mean SAR on the ROIs lower than +25% compared to the mean SAR on the reference glass slide. It is worth observing that the use of glass slide, where cells are attached, provides a better SAR uniformity with respect to the case of cells in suspension [65]. References [1] Research and Regulatory Efforts on Mobile Phone Health Issues, Report GAO-01-545 to Congressional Requesters, U.S. General Accounting Office, Washington, DC, 2001. [2] Vijayalaxmi, G. Obe, Controversial cytogenetic observations in mammalian somatic cells exposed to radiofrequency radiation, Radiat. Res. 162 (2004) 481–496. [3] Vijayalaxmi, G. Obe, Controversial cytogenetic observations in mammalian somatic cells exposed to extremely low frequency electromagnetic radiation: a review and future research recommendations, Bioelectromagnetics 26 (2005) 412–430. [4] M. Röösli, Radiofrequency electromagnetic field exposure and non-specific symptoms of ill health: a systematic review, Environ. Res. 107 (2008) 277–287. [5] L. Hardell, C. Sage, Biological effects from electromagnetic field exposure and public exposure standards, Biomed. Pharmacother. 62 (2008) 104–109. [6] H.W. Ruediger, Genotoxic effects of radiofrequency electromagnetic fields, Pathophysiology 16 (2009) 89–102. [7] D. Brusick, R. Albertini, D. McRee, D. Peterson, G. Williams, P. Hanawalt, J. Preston, Genotoxicity of radiofrequency radiation, Environ. Mol. Mutagen. 32 (1998) 1–16. [8] M.L. Meltz, Radiofrequency exposure and mammalian cell toxicity genotoxicity, and transformation, Bioelectromagnectics (Suppl. 6) (2003) S196–S213. [9] L. Verschaeve, A. Maes, Genetic, carcinogenic and teratogenic effects of radiofrequency fields, Mutat. Res. 410 (1998) 141–165. [10] L. Verschaeve, Genetic effects of radiofrequency radiation (RFR), Toxicol. Appl. Pharmacol. 207 (2005) 336–341. [11] A. Maes, M. Collier, D. Slaets, L. Verschaeve, 954 MHz microwaves enhance the mutagenic properties of mitomycin C, Environ. Mol. Mutagen. 28 (1996) 26–30. [12] M.B. Zhang, J.L. He, L.F. Jin, D.Q. Lu, Study of low-intensity 2450-MHz microwave exposure enhancing the genotoxic effects of mitomycin C using micronucleus test and comet assay in vitro, Biomed. Environ. Sci. 15 (2002) 283–290. [13] R.K. Adair, Biophysical limits on athermal effects of RF and microwave radiation, Bioelectromagnetics 24 (2003) 39–48. [14] A.R. Sheppard, M.L. Swicord, Q. Balzano, Quantitative evaluations of mechanisms of radiofrequency interactions with biological molecules and processes, Health Phys. 95 (2008) 365–395. ´ D. Havelka, O. Kuˇcera, Electric field generated by axial [15] M. Cifra, J. Pokorny, longitudinal vibration modes of microtubule, BioSystems 100 (2010) 122–131. [16] M. Porcelli, G. Cacciapuoti, S. Fusco, R. Massa, G. D’Ambrosio, C. Bertoldo, M. De Rosa, V. Zappia, Non-thermal effects of microwaves on proteins: thermophylic enzymes as model system, FEBS Lett. 402 (1997) 102–106. [17] H. Fröhlich, The biological effects of microwaves and related questions, in: P.W. Hawkes (Ed.), Advances in Electronics and Electron Physics, 53, Elsevier Inc., Department of Physics University of Liverpool, Liverpool, England, 1980, pp. 85–152. [18] E. Marani, H.K.P. Feirabend, Future perspectives in microwave applications in life sciences, Eur. J. Morphol. 32 (1994) 330–334. [19] J.L. Kirschvink, Microwave absorption by magnetite: a possible mechanism for coupling nonthermal levels of radiation to biological systems, Bioelectromagnetics 17 (1996) 187–194. [20] L.S. Taylor, The mechanisms of athermal microwave biological effects, Bioelectromagnetics 2 (1981) 259–267. [21] E.E. Fesenko, V.I. Geletyuk, V.N. Kazachenko, N.K. Chemeris, Preliminary microwave irradiation of water solutions changes their channel-modifying activity, FEBS Lett. 366 (1995) 49–52. [22] G. Sciandrello, F. Caradonna, G. Barbata, Karyotype abnormalities in a variant Chinese hamster cell line resistant to methyl methanesulphonate, Hereditas 24 (1996) 39–46.

[23] E. Schiebel, Tubulin complexes: binding to the centrosome, regulatin and microtubule nucleation, Curr. Opin. Cell Biol. 12 (2000) 113–118. [24] D.J. Kirkland, Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge, UK, 1989. [25] A. Dardano, M. Ballardin, M. Ferdeghini, E. Lazzeri, C. Traino, N. Caraccio, G. Mariani, R. Barale, F. Monzani, Anticlastogenic effect of Ginkgo biloba extract in Grave’s disease patients receiving radioiodine therapy, J. Clin. Endocrinol. Metab. 92 (2007) 4286–4292. [26] R.J. Fitzsimmons, J.T. Ryaby, S. Mohan, F.P. Magee, D.J. Baylink, Combined magnetic fields increase insulin-like growth factor-II in TE-85 human osteosarcoma bone cell cultures, Endocrinology 136 (1995) 3100–3106. [27] I.Y. Belyaev, S. Eriksson, J. Nygren, J. Torudd, M. Harms-Ringdahl, Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis, Biochim. Biophys. Acta 1428 (1999) 348–356. [28] C.F. Blackman, S.G. Benane, J.R. Rabinowitz, D.E. House, W.T. Joines, A role for the magnetic field in the radiation-induced efflux of calcium ions from brain tissue in vitro, Bioelectromagnetics 6 (1985) 327–337. [29] S.D. Smith, B.R. McLeod, A.R. Liboff, Calcium cyclotron resonance and diatom mobility, Bioelectromagnetics 8 (1987) 215–227. [30] F.S. Prato, J.J. Carson, K.P. Ossenkopp, M. Kavaliers, Possible mechanisms by which extremely low frequency magnetic fields affect opioid function, FASEB J. 9 (1995) 807–814. [31] Y.D. Alipov, I.Y. Belyaev, O.A. Aizenberg, Systemic reaction of E. coli cells to weak electromagnetic fields of extremely low frequency, Bioelectrochem. Bioenerg. 34 (1994) 5–12. [32] Y.D. Alipov, I.Y. Belyaev, Difference in frequency spectrum of ELF effect on the genome conformational state of AB1157 and EMG2 E. coli cells, Bioelectromagnetics 17 (1996) 384–387. [33] I.Y. Belyaev, Y.D. Alipov, A.Y. Matronchik, S.P. Radko, Cooperativity in E. coli cell response to resonance effect of weak extremely low frequency electromagnetic field, Bioelectrochem. Bioenerg. 37 (1995) 85–90. [34] C.F. Blackman, D.E. House, S.G. Benane, W.T. Joines, R.J. Spiegel, Effects of ambient levels of power-line-frequency electric fields on a developing vertebrate, Bioelectromagnetics 9 (1988) 129–140. [35] E.M. Goodman, B. Greenebaum, M.T. Marron, Effects of electromagnetic fields on molecules and cells, Int. Rev. Cytol. 158 (1995) 279–338. [36] H. Lin, R. Goodman, A. Shirley-Henderson, Specific region of the c-myc promoter is responsive to electric and magnetic fields, J. Cell. Biochem. 54 (1994) 281–288. [37] J.L. Phillips, W. Haggren, W.J. Thomas, T. Ishida-Jones, W.R. Adey, Magnetic field-induced changes in specific gene transcription, Biochim. Biophys. Acta 1132 (1992) 140–144. [38] E. Lindstrom, A. Berglund, K.H. Mild, P. Lindstrom, E. Lundgren, CD45 phosphatase in Jurkat cells is necessary for response to applied ELF magnetic fields, FEBS Lett. 370 (1995) 118–122. [39] M. Niehaus, H. Bruggemeyer, H.M. Behre, A. Lerchl, Growth retardation, testicular stimulation, and increased melatonin synthesis by weak magnetic fields (50 Hz) in Djungarian hamsters, Phodopus sungorus, Biochem. Biophys. Res. Commun. 234 (1997) 707–711. [40] S. Roy, Y. Noda, V. Eckert, M.G. Traber, A. Mori, R. Liburdy, L. Packer, The phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst in rat peritoneal neutrophils is increased by a 0.1 mT (60 Hz) magnetic field, FEBS Lett. 376 (1995) 164–166. [41] F.M. Uckun, T. Kurosaki, J. Jin, X. Jun, A. Morgan, M. Takata, J. Bolen, R. Luben, Exposure of B-lineage lymphoid cells to low energy electromagnetic fields stimulates Lyn kinase, J. Biol. Chem. 270 (1995) 27666–27670. [42] J. McCann, F. Dietrich, C. Rafferty, The genotoxic potential of electric and magnetic fields: an update, Mutat. Res. 411 (1998) 45–86. [43] S. Tofani, A. Ferrara, L. Anglesio, G. Gilli, Evidence of genotoxic effects of resonant ELF magnetic fields, Bioelectrochem. Bioenerg. 36 (1995) 9–13. [44] T. Schrader, K. Munter, T. Kleine-Ostmann, E. Schmid, Spindle disturbances in human–hamster hybrid (AL) cells induced by mobile communication frequency range signals, Bioelectromagnetics 29 (2008) 626–639. [45] D.A. Compton, Spindle assembly in animal cells, Annu. Rev. Biochem. 69 (2000) 95–114. [46] S. Inoue, E.D. Salmon, Force generation by microtubule assembly/disassembly in mitosis and related movements, Mol. Biol. Cell 6 (1995) 1619–1640. [47] P. Duesberg, R. Slindl, R. Hehlmann, Explaining the high mutation rates of cancer cells in drug and multidrug resistance by chromosome reassortment that are catalysed by aneuploidy, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 14295–14300. [48] R. Heald, R. Tournebize, A. Habermann, E. Karsenti, A. Hyman, Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule selforganization, J. Cell Biol. 138 (1997) 615–628. [49] A. Khodjakov, C. Rieder, Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression, J. Cell Biol. 153 (2001) 237–242. [50] A.F. Pluta, A.M. Mackay, A.M. Ainsztein, I.G. Goldberg, W.C. Earnshaw, The centromere: hub of chromosomal activities, Science 270 (1995) 1591–1594. [51] G.A. Pihan, A. Purohit, J. Wallace, H. Knecht, B. Woda, P. Quesenberry, S.J. Doxsey, Centrosome defects and genetic instability in malignant tumours, Cancer Res. 58 (1998) 3974–3985. [52] B.R. Brinkley, Managing the centrosome numbers game: from chaos to stability in cancer cell division, Trends Cell Biol. 11 (2001) 18–21. [53] M.J. Ortner, M.J. Galvin, R.D. Irwin, The effect of 2450-MHz microwave radiation during microtubular polymerization in vitro, Radiat. Res. 93 (1983) 353–363.

M. Ballardin et al. / Mutation Research 716 (2011) 1–9 [54] J.F.R. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics, Br. J. Cancer 26 (1972) 239–257. [55] D.W. Nicholson, N.A. Thornberry, Caspases: killer proteases, Trends Biochem. Sci. 22 (1997) 299–306. [56] I.S. Mathiasen, M. Jaattela, Triggering caspase-independent cell death to combat cancer, Trends Mol. Med. 8 (2002) 212–220. [57] M. Capri, E. Scarcella, E. Bianchi, C. Fumelli, P. Mesirca, P. Agostini, D. Remondini, J. Schuderer, N. Kuster, F. Bersani, 1800 MHz radiofrequency (mobile phones, different Global System for Mobile communication modulations) does not affect apoptosis and heat shock protein 70 level in peripheral blood mononuclear cells from young and old donors, Int. J. Radiat. Biol. 80 (2004) 389–397. [58] M. Capri, E. Scarcella, C. Fumelli, E. Bianchi, S. Salvioli, P. Mesirca, C. Agostini, A. Antolini, A. Schiavoni, C. Franceschi, In vitro exposure of human lymphocytes to 900 MHz CW and GSM modulated radiofrequency: studies of proliferation, apoptosis and mitochondrial membrane potential, Radiat. Res 162 (2004) 211–218. [59] G.J. Hook, P. Zhang, I. Lagroye, L. Li, R. Higashikubo, E.G. Moros, W.L. Straube, W.J. Pickard, J.D. Baty, J.L. Roti, Measurement of DNA damage and apoptosis in Molt-4 cells after in vitro exposure to radiofrequency radiation, Radiat. Res. 161 (2004) 193–200. [60] K. Maeda, T. Maeda, Y. Qi, In vitro and in vivo induction of human LoVo cells into apoptotic process by non-invasive microwave treatment: a potentially novel

[61]

[62]

[63]

[64] [65]

[66]

9

approach for physical therapy of human colorectal cancer, Oncol. Rep. 11 (2004) 771–775. M. Caraglia, M. Marra, F. Mancinelli, G. D’Ambrosio, R. Massa, A. Giordano, A. Budillon, A. Abruzzese, E. Bismuto, Electromagnetic fields at mobile phone frequency induce apoptosis and inactivation of the multi-chaperone complex in human epidermoid cancer cells, J. Cell. Physiol. 204 (2005) 539–548. F. Marinelli, D. La Sala, G. Cicciotti, L. Cattini, C. Trimarchi, S. Putti, A. Zamparelli, L. Giuliani, G. Tomassetti, C. Cinti, Exposure to 900 MHz electromagnetic field induces an unbalance between pro-apoptotic and pro-survival signals in Tlymphoblastoid leukemia CCRF-CEM cells, J. Cell. Physiol. 198 (2004) 324–332. C. Rosefort, E. Fauth, H. Zankl, Micronuclei induced by aneugens and clastogens in mononucleate and binucleate cells using the cytokinesis block assay, Mutagenesis 19 (2004) 277–284. GTEM test cells – test cells for EMC radiated & immunity testing DC to 20 GHz SCHAFFNER, pp. 2–5. N. Fontana, C. Pelletti, A. Rogovich, A. Monorchio, On the influence of a glass slide on the SAR distribution in Petri dishes for in vitro exposure to 2.45 GHz EM fields, in: IEEE International APS and USNC/URSI National Radio Science Meeting, 2010. N. Kuster, F. Schonborn, Recommended minimal requirements and development guidelines for exposure setups of bio-experiments addressing the health risk concern of wireless communications, Bioelectromagnetics 21 (2000) 508–514.