Extremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells
Cosmin Teodor Miha, Gabriela Vochita, Florin Brinza, Pincu Rotinberg
aa r X i v : . [ q - b i o . S C ] J a n Extremely low-frequency electromagnetic fieldscause DNA strand breaks in normal Vero cells
Cosmin Teodor Mihai ∗ , Gabriela Vochita , Florin Brinza ,Pincu Rotinberg ∗ Department of Biology, ”Alexandru Ioan Cuza” University of Iasi,Bd. Carol I, nr. 20A, 700505, Iasi, RomaniaE-mail: [email protected] National Institute of Research and Development for Biological Sciences, branchInstitute of Biological Research Iasi,Str. Lascar Catargi, nr. 47, 700107,Iasi, Romania Faculty of Physics, Alexandru Ioan Cuza UniversityBd. Carol I, nr. 11, 700506, Iasi, Romania
Abstract.
Extremely low frequency electromagnetic fields aren’t considered as a realcarcinogenic agent despite the fact that some studies have showed impairment of theDNA integrity in different cells lines.The aim of this study was evaluation of the late effects of a 100 Hz and 5.6 mTelectromagnetic field, applied continuously or discontinuously, on the DNA integrityof Vero cells assessed by alkaline Comet assay and by cell cycle analysis. Normal Verocells were exposed to extremely low frequency electromagnetic fields (100 Hz, 5.6 mT)for 45 minutes. The Comet assay and cell cycle analysis were performed 48 hours afterthe treatment.Exposed samples presented an increase of the number of cells with high damagedDNA as compared with non-exposed cells. Quantitative evaluation of the comet assayshowed a significantly ( < Keywords : ELF-EMF; 5.6 mT and 100 Hz; continuous and discontinuous exposure;Comet assay; cell cycle analysis; DNA damage xtremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells
1. Introduction
The extremely low frequency electromagnetic fields (ELF-EMF) are omnipresent in hu-man life, being generated by common appliances electrical conductors that cross thepopulated areas or the walls of houses, medical devices used in the treatment of dif-ferent illness, electrical cars (used in the public transportation systems or as privatecars) and electrical trains (underground or suburban electrical trains). Both in the caseof the medical devices (mainly used in the physiotherapy) and of the electrical compo-nents of the cars and trains, the common generated frequency is that of 100 Hz [1]. Eventhough the patients or passengers are exposed for a short time, the deserving personnelare subject to prolonged exposure. Also, the combined treatment of 100 Hz magneticfield and X-rays has increased the survival time of hepatoma-implanted Balb/c mice ascompared to magnetic field or X-rays treated groups [2], suggesting the possible use inoncotherapy.The exposure of different cell lines or organisms to electromagnetic fields have produceda bulk of data that were sometimes contradictory and didn’t allow a concise and clearconclusion about the effects of electromagnetic fields on biological systems [3, 4].The International Agency for Research on Cancer (IARC) has evaluated the scientificdata and has classified ELF magnetic fields as being ”possibly carcinogenic” to human[5].The worrying but contradictory epidemiologic or experimental data about the possiblegenotoxic effects of this kind of electromagnetic fields, suggesting the possible carcino-genetic effect, requires an enrichment of the pool of experimental information and thedeciphering of its action mechanisms.Carcinogenic processes have three developmental stages: initiation, promotion and pro-gression. The first stage, tumour initiation, begins when the DNA in a cell or populationof cells is damaged by exposure to exogenous or endogenous carcinogens. If this damageis not repaired, it can lead to genetic mutations. The responsiveness of the mutatedcells to their microenvironment can be altered and may give them a growth advantagerelative to normal cells [6, 7, 8, 9].The possible carcinogenetic effect of the low frequency and intensity electromagneticfields are still under debate, the data being controversial. Studies in this field suggestedthat exposure to low frequency and intensity electromagnetic fields could alter the DNAintegrity, which could trigger the initiation of carcinogenetic processes or could accel-erate the development or spreading of already present cancers [10, 11]. Also, it wassuggested that chronic exposure to the ELF could be involved in the development ofsome neurodegenerative diseases by production of reactive oxygen species [12].Contrary, other researches identified no effects on the integrity of the DNA in the con-ditions of exposure to the electromagnetic fields [13, 14, 15, 16, 17].The aim of the study was to test if, in effect, there are any differences between con-tinuous or discontinuous extremely low frequency electromagnetic fields on the DNAintegrity of normal Vero cells, in order to evaluate the possible disruptions that could xtremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells
2. Materials and methods
Vero cells (ECACC 88020401) are adherent to substratum with a fibroblast-like mor-phology. The cells were cultivated in a DMEM medium (Dulbeco’s Modified EaglesMedium, Biochrom AG, Germany, FG 0415), supplemented with 2.0% foetal bovineserum (Sigma, Germany, F9665) and 100 µ g/mL streptomycin (Biochrom AG, Ger-many, A 331-26), 100 IU/mL penicillin (Biochrom AG, Germany, A 321-44). The cellcultures were seeded at a density of 5 x 10 cells in 25 cm flask (TPP Techno PlasticProducts AG, Trasadingen, Switzerland) and maintained in a CO incubator (Binder CB150, Tuttlingen, Germany), at 37 o C. When the cells reached confluence in the monolayerstage, the cultures were divided into control and electromagnetic treated cell cultures.
The setup for electromagnetic exposure consisted in a Helmholtz pair of coils connectedin parallel to a magnetodiaflux (IBF, Romania) device, that generates a pulse electro-magnetic field (PEMF) having a frequency of 100 pulses/second.The 29 cm diameter coils were made of copper wires with 620 turns. The coils were setat a distance equal with their radius (14.5 cm) which assured a central homogeneousmagnetic field (5.6 mT). The unpowered coils presented a magnetic field with a 0.021mT intensity, similar to the registered magnetic field background. The magnetodiafluxdevice delivered to the two coils a pulsating direct current (PDC), obtained by con-verting and rectifying the 220 V/50 Hz alternative current. The PDC had a 100 Hzfrequency and the peak voltage variation, measured with an oscilloscope (Tektronix,Guernsey, Channel Islands), was of 42 volts and 2.0 Ampers.The coils were housed in the cell incubator, the temperature being constant and uni-formly distributed all the time of the exposure (37 ± o C), as monitored by a ther-mocouple thermometer (Hanna Instruments, Italy). The homogeneity of the magneticfield produced by the coils in the area of exposure is shown in the figure 1, image beinggenerated by Vizimag ver.3.193 software ( c (cid:13)
J. Beeteson 1999-2009), using the providedcharacteristics of the coils and current.
The magnetic flux density measurement in-side of Helmholtz coils system was performed using LakeShore 421 Gaussmeter, havingvalid NIST certificates. xtremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells Figure 1.
Magnetic flux density (left image) and flux lines distribution (right image)in the area of exposure of the cells when the Helmholtz coils were powered with a directcurrent with the frequency of 100 Hz and an intensity of 2.0 Amperes.
Both axial and transverse probes were used for the measurements. Before the measure-ment process, each probe was calibrated in a zero-field chamber. For the characterizationof the uniformity in the samples volume, experimental measurements and software simu-lation were used. The measurements were performed in equidistantly distributed pointsinside of coils. In each point, axial (using axial probe) and radial (using transverseprobe) values of the magnetic flux density were tested. The experimentally detectedvalues were compared to the calculated ones.The field lines distribution and the magnetic induction values in the centre of the coilssystem were calculated using a Vizimag ver.3.193 software. In Figure 1, the field linesdistribution and the field lines density for our coils are presented. The experimentalmeasured values of the magnetic flux density are in good accordance with calculatedones. The conclusion is that the whole volume of cell culture container is subjected to auniform magnetic induction value. The low values of magnetic susceptibility of the bio-logical samples ensure a uniform value of the magnetic field inside the container volume.
The cell culture flasks with the cells were placed in the central region of the Helmholtzcoils, perpendicular to the magnetic field lines. The cells were exposed once for 45minutes and were returned to the incubator after the treatment. The exposure of thecells was performed in a continuous (cEMF, permanent exposure to the magnetic fieldduring the 45 minutes of the treatment) or in a discontinuous manner (dcEMF, cyclesof one second on and three seconds off). Sham-exposed cells were put into the sameexperimental conditions as the treated samples but without energizing the coils, theEMF background remaining practically unchanged. xtremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells
We followed the technique described by Ostling and Johanson [18] with minor modifi-cations by Singh et al. [19], as presented in [20].The 50 L of ELF-exposed and the sham-exposed cells (20,000 cells) were mixed with150 µ L of low-melting agarose (0.8%, 37 o C), and this cell suspension was pipetted onto1% normal-melting agarose pre-coated slides, spread with a cover slip, and kept on acold flat tray for approximately 10 minutes to solidify.The slides were then immersed in freshly prepared cold lysis solution (2.5 mol/l NaCl,100 mmol/l Na EDTA, 10 mmol/l Tris, pH 10, 1% sodium sarcosinate, 1% Triton X-100,10% DMSO, pH 10) and lysed overnight at 4 o C. Subsequently, the slides were drainedand placed in a horizontal gel electrophoresis tank, side by side and very close to theanode. The tank was filled with fresh electrophoresis buffer (1 mmol/l Na EDTA, 300mmol/l NaOH, pH >
13 to a level approximately 0.4 cm above the slides. The slides wereleft in the solution for 40 minutes, to allow equilibration and unwinding of the DNAbefore electrophoresis.The electrophoresis was conducted at 25 V, 300 mA, 4 o C, 20 min, field strength 0.8V/cm. All steps were performed under dimmed light to prevent the occurrence of ad-ditional DNA damage. After electrophoresis, the slides were washed three times withTris buffer (0.4 mol/l Tris, pH 7.5), to be neutralized, then air-dried and stored untilneeded for analysis. Comets were visualized by ethidium bromide staining (20 µ g/ml,30 s) and examined at 200 magnification with a fluorescence microscope (Nikon Eclipse600, Nikon corp., Japan). After the electromagnetic treatment cells were harvested from the surface of cultureflasks by trypsinization, they were resuspended in a complete medium and then pelletedby centrifugation. The cells were washed twice in cold PBS. The cell pellet was resus-pended in NIM-DAPI (Beckman Coulter, USA) and were stained overnight at 4 o C. For xtremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells
All of the experiments were carried out with at least three independent repetitionsand all data were expressed as the mean value and standard error of mean (SEM).The statistical analysis was performed using Student’s t test and the differences wereexpressed as significant at the level of p <
3. Results
The qualitative analysis of the cellular damage determined by electromagnetic fields wasevaluated by the extent of the damage and graded according to [20], the results beingpresented in table 1.
Table 1.
Absolute frequencies of Comet types found in control group and in samplestreated with cEMF and dcEMF (n = 400 cells) for 45 minutes once and evaluated byComet assay after 48 hours from the treatment.
Control cEMF 100 Hz dcEMF 100 Hz%Mean ± SEM %Mean ± SEM p %Mean ± SEM pA ( < ± ± < ± − ± ± < ± < − ± ± < ± < − ± ± < ± < > ± ± < ± < < xtremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells µ m ± µ m ± µ m ± < < ± ± ± Figure 2.
Tail length (left figure) and % content in DNA (right figure) of the taildetermined in Vero cells at 48 hours after the exposure to extremely low frequencyelectromagnetic fields (100 Hz, 5.6 mT, 45 minutes). *** = < Table 2.
Impact of the extremely low frequency electromagnetic field (100 Hz, 5.6mT, 45 minutes once) on comet assay indices (tail moment and Olive tail moment) inVero cell lines, after 48 hours from the moment of the treatment.
Tail Moment Olive TailMomentMean( µ m) ± SEM p Mean ( µ m) ± SEM pControl cells 8.26 ± ± ± < ± < ± < ± < µ m ± µ m ± µ m ± xtremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells µ m ± µ m ± µ m ± Table 3.
Impact of the extremely low frequency electromagnetic field (100 Hz, 5.6mT, 45 minutes once) on comet assay indices (tail moment and Olive tail moment) inVero cell lines, after 48 hours from the moment of the treatment.
Cell cycle distribution (%)G0/G1 stage S stage G2/M stageControl cells 74.94 6.01 18.86cEMF 58.74 * (-21.62%) 22.42 *** (+273.04%) 18.68 (-0.95%)dcEMF 68.00 (-9.26%) 14.45 *** (+140.43%) 17.31 (-8.22%)Data are means of three experiments, SD being > > ∗ p < ∗ ∗ ∗ p < ′ s t testCompared to controls, ELF-EMF-exposure caused a blockage of the cells in the Sstage of the cell cycle. After 48 hours, the highest percentage of cells blocked in the Sphase was registered in the case of cEMF followed by dcEMF, as it can be seen in table3.
4. Discussions
The general assumption is that extremely low frequency electromagnetic fields arentgenotoxic and do not affect the integrity of the DNA molecule, but the scientific dataare still controversial and supplementary evidence is necessary to consider this physicalagent as a real carcinogenic factor.Other aspects that need to be clarified are the differences either in effect or in themagnitude of the effect, in respect to the use of continuous and intermittent electro-magnetic fields. Ivancsits [20, 23, 24] reported that intermittent electromagnetic fieldscaused DNA damage of the human diploid fibroblasts, while continuous electromagnetic xtremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells xtremely low-frequency electromagnetic fields cause DNA strand breaks in normal Vero cells
Acknowledgements
This study was possible with financial support from the SectoralOperational Programme for Human Resources Development, project Developing theinnovation capacity and improving the impact of research through post-doctoralprogrammes, POSDRU/89/1.5/S/49944
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