Static magnetic field of 6 mT induces apoptosis and alters cell cycle in p53 mutant Jurkat cells Mohammad Reza Ahmadianpour 1 , Parviz Abdolmaleki 1 , Seyed Javad Mowla 2 & Saman Hosseinkhani 3 1 Department of Biophysics, Tarbiat Modares University, Tehran, Iran, 2 Molecular Genetics Department, Tarbiat Modares University, Tehran, Iran, and 3 Department of Biochemistry, Tarbiat Modares University, Tehran, Iran This study aimed to investigate the effect of 6 milliTesla (mT) static magnetic field (SMF) on apoptosis induction and cell cycle alteration in T-lymphoblastoid Jurkat E6.1 cells. Exposure of human p53 mutant Jurkat cells to 6 mT SMF resulted in apoptosis, which was detected by luminometric and flow cytometric analysis also, phosphorylated ATM and E2F1 proteins were detected by western blot analysis. Based on luminescence detection data, apoptosis initiated 36 h after exposure to 6 mT SMF. Apoptosis also reached its maximum rate 48 h after treatment. Flow cytometric analysis revealed a temporary G2 arrest after exposure to 6 mT SMF. Indeed, cellular population of S and G2 phases was increased. Based on reports of other investigations on the effect of magnetic fields on Ca2 þ flux changes in cell membranes and the effect of MFs on free radical formation, it can be suggested that the magnetic fields may induce the apoptosis and alter the cell population in different cell cycle phases of Jurkat cells via changing the Ca2 þ fluxes through cell membranes and playing a role in free radical formation. Western blot analysis showed that the amount of phosphorylated ATM and E2F1 proteins were increased in treated cells. The results of luminometric and flow cytometric detection did not show a significant difference in the apoptosis rate between 6 h-treated and 24 h-treated cells by 6 mT SMF. Thus, 6 mT SMF can induce apoptosis and alter cell cycle in Jurkat cells via a p53-independent pathway. Keywords Apoptosis, Luminescence, Static Magnetic Field, Cell cycle, Jurkat Cell INTRODUCTION Apoptosis is part of normal cell physiology, as are proliferation and differentiation, and is a major component of both normal cell development and disease (Guimaraes and Linden, 2004; Moreira and Barcinski, 2004; Dini and Abbro, 2005). Apoptosis, spontaneous and induced, has been reported to be influenced by SMFs (Fanelli et al., 1999; Teodori et al., 2002a; Chionna et al., 2003, 2005; Dini and Abbro, 2005). Ca 2þ ions as mediators of intracellular signalling are crucial for the development of apoptosis: more frequently, an increase of [Ca 2þ ]i due to emptying of intracellular [Ca 2þ ]i stores and to [Ca 2þ ]i influx from the extracellular medium, commits to apoptosis, independent of the apoptotic stimulus (Bian et al., 1997). The unbalance of the apoptotic process could be linked to Ca 2þ fluxes that are, in turn, dependent Address correspondence to Parviz Abdolmaleki, Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. E-mail: [email protected]Electromagnetic Biology and Medicine, 32(1): 9–19, 2013 Copyright Q Informa Healthcare USA, Inc. ISSN: 1536-8378 print / 1536-8386 online DOI: 10.3109/15368378.2012.692748 9 Electromagn Biol Med Downloaded from informahealthcare.com by Monash University on 08/04/13 For personal use only.
Transcript
Static magnetic field of 6mT induces apoptosis andalters cell cycle in p53 mutant Jurkat cells
Mohammad Reza Ahmadianpour1, Parviz Abdolmaleki1,Seyed Javad Mowla2 & Saman Hosseinkhani3
1Department of Biophysics, Tarbiat Modares University, Tehran, Iran, 2Molecular GeneticsDepartment, Tarbiat Modares University, Tehran, Iran, and 3Department of Biochemistry,Tarbiat Modares University, Tehran, Iran
This study aimed to investigate the effect of 6 milliTesla (mT) static magnetic field (SMF) onapoptosis induction and cell cycle alteration in T-lymphoblastoid Jurkat E6.1 cells. Exposure ofhuman p53 mutant Jurkat cells to 6mT SMF resulted in apoptosis, which was detected byluminometric and flow cytometric analysis also, phosphorylated ATM and E2F1 proteins weredetected by western blot analysis.Based on luminescence detection data, apoptosis initiated 36 h after exposure to 6mT SMF.Apoptosis also reached its maximum rate 48 h after treatment. Flow cytometric analysis revealeda temporary G2 arrest after exposure to 6mT SMF. Indeed, cellular population of S and G2 phaseswas increased. Based on reports of other investigations on the effect of magnetic fields onCa2 þ flux changes in cell membranes and the effect of MFs on free radical formation, it can besuggested that the magnetic fields may induce the apoptosis and alter the cell population indifferent cell cycle phases of Jurkat cells via changing the Ca2 þ fluxes through cell membranesand playing a role in free radical formation. Western blot analysis showed that the amount ofphosphorylated ATM and E2F1 proteins were increased in treated cells. The results ofluminometric and flow cytometric detection did not show a significant difference in theapoptosis rate between 6 h-treated and 24 h-treated cells by 6mT SMF. Thus, 6mT SMF caninduce apoptosis and alter cell cycle in Jurkat cells via a p53-independent pathway.
Keywords Apoptosis, Luminescence, Static Magnetic Field, Cell cycle, Jurkat Cell
INTRODUCTION
Apoptosis is part of normal cell physiology, as are proliferation and differentiation,and is a major component of both normal cell development and disease (Guimaraesand Linden, 2004; Moreira and Barcinski, 2004; Dini and Abbro, 2005). Apoptosis,spontaneous and induced, has been reported to be influenced by SMFs (Fanelli et al.,1999; Teodori et al., 2002a; Chionna et al., 2003, 2005; Dini and Abbro, 2005).Ca2þ ions as mediators of intracellular signalling are crucial for the development ofapoptosis: more frequently, an increase of [Ca2þ]i due to emptying of intracellular[Ca2þ]i stores and to [Ca2þ]i influx from the extracellular medium, commits toapoptosis, independent of the apoptotic stimulus (Bian et al., 1997). The unbalanceof the apoptotic process could be linked to Ca2þ fluxes that are, in turn, dependent
Address correspondence to Parviz Abdolmaleki, Department of Biophysics, Faculty of BiologicalSciences, Tarbiat Modares University, Tehran, Iran. E-mail: [email protected]
Electromagnetic Biology and Medicine, 32(1): 9–19, 2013Copyright Q Informa Healthcare USA, Inc.ISSN: 1536-8378 print / 1536-8386 onlineDOI: 10.3109/15368378.2012.692748
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on the effect on the plasma membrane exerted by SMFs. In particular, it hasbeen suggested that SMFs alter the function of the organism’s transmembranecalcium flux in diverse experimental models (Rosen, 1990; Fanelli et al., 1999).However, the role of [Ca2þ]i elevation in the development of apoptosis is ambiguousbecause it exerts different effects in different cell systems (Magnelli et al.,1994). It may act as a trigger for cell death in some well-studied cells such as ratthymocytes (McConkey et al., 1989) or may be a defense mechanism againstapoptosis in other cell systems, which are less studied concerning apoptosis, suchas nerve cells (Galli et al., 1995).
Up until now, few studies have investigated the effects of SMF on expression ofgenes, especially those involved in apoptosis. Previous research has shown that SMFexposure alone extensively modulates the expression of bcl-2, bax, p53, and hsp70genes (Tenuzzo et al., 2009).
The present study aims to investigate the effect of 6 mT SMF alone on theinduction of apoptosis, the cell cycle alteration, and increasing expression of ATMand E2F1 genes which contribute to the apoptosis initiation, in p53 mutant Jurkatcells, after exposure to SMF for 6 and 24 h.
MATERIALS AND METHODS
Cell CultureThe human T lymphoblastoid Jurkat cell line E6.1 (C121) was obtained from Iran’sPasteur institute (Iran, Tehran). The cells were cultured in RPMI-1640 medium(GIBCO) supplemented with 10% fetal bovine serum (FBS), 150 UI/ml penicillin,50mg/ml streptomycin and kept at 378C in a humidified atmosphere of 5% CO2 inair. The cultures were split every other day by dilution to a concentration of2 £ 105 cells/ml. The cell lines in the maximal range of up to 20 passages wereused for this study. Also, exponentially growing cells were used throughout thestudy. The cell counts were performed (both static magnetic field-exposed andcontrol samples) with a hemocytometer at the start and the end of eachexperiment, and the cell membrane integrity was determined using the Trypanblue exclusion technique.
Magnetic Field ExposureExposure to MF was performed using a locally designed SMF generator (Fig. 1). Theelectrical power was provided using a power supply working in range of 0–50 V and0–20 A with a maximum power of 1 kW. This system was consisted of a 40 cm-longsolenoid (1800 loops of 2.5 mm coated copper wire) equipped with an includedincubator inside the solenoid (a copper container with 40 cm length, 8 cm diameter).Using three different sensors the controller system was able to control thetemperature, humidity, and CO2 pressure. Heat was efficiently removed by a gas-cooled system using tetrafluoroethane. Circulation system consisted of a condenser,refrigerator engine as well as a heat-exchanging pipes network of copper with 8 mmthickness which turned around both inner and outer sides of solenoid. This systemdesigned to generate SMF in the range of 0.5 mT to 90 mT with stable conditions.An electronic board was used to stabilize the system so that we always got a uniformSMF inside the exposure unit. Calibration of the system as well as tests for theaccuracy and uniformity of the MFs were performed by a teslameter (13610.93,PHYWE, Gottingen, Germany) with a probe type of Hall Sound. The accuracy of theteslameter was ^ 0.1% for MF and the range of measurements was 3mT –30 mT.Presence of any pulsation in the efferent current was tested by an oscilloscope(40 MHz, model 8040, Leader, Japan). The predefined SMF of 6 mT was generated by
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passing a 1.5 Amperes DC current according to calibration data. Temperature wasroutinely checked before and after all field and control exposures. Three flasks wereplaced at the center of the incubator (10 cm distance from the center in each side)within solenoid with a homogenous magnetic field, in each exposure. The durationof exposures was 6 and 24 h. Before each exposure, the MF intensity was set to 6 mTusing a teslameter.
Luminometric AnalysisIn order to detect apoptosis, the caspase-Glo3/7 assay kit and the Berthold detectionsystem (GmbH, Pforzheim, Germany) apparatus were used. The Caspase-Glo3/7assay is a homogeneous, luminescent assay that measures caspase-3 and -7activities. These members of the cysteine aspartic acid-specific protease (caspase)family play key effector roles in apoptosis in mammalian cells (Nicholson andThornberry, 1997). After adding the Caspase-Glo3/7 reagent in cell lysis, caspasecleaves the substrate. This liberates free aminoluciferin, which is consumed by the
FIGURE 1 Photograph showing the apparatus used to generate static magnetic field (a) the wholebody of the system (b) the incubator within solenoid.
Static magnetic field of 6mT induces apoptosis in Jurkat cells 11
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luciferase, generating a “glow-type” luminescent signal. The obtained signal isproportional to the amount of caspase activity present.
Luminescence emission spectrum was measured with a Berthold detectionsystem (GmbH, Pforzheim, Germany) apparatus. Jurkat cells were plated in 96-wellplates at , 2 £ 104 cells per well before irradiation. Then, at various times afterthe irradiation, a volume of 100ml of Caspase-Glo3/7 reagent was added to 100ml ofblank, negative control cells or treated cells in culture medium, and the sampleswere incubated for 1 h at 228C. The luminescence emission spectra were thenread with spectrophotometer and data were collected over a wavelength range of400 –700 nm. The spectra were automatically corrected for the photosensitivity ofthe equipment.
Flow Cytometric AnalysisUntreated control and irradiated cells were centrifuged at 1300 rpm for 8 min at roomtemperature; the supernatant (culture medium and FBS) was decanted. The cellswere suspended in phosphate saline buffer (PBS) and re-centrifuged at 1300 rpmfor 8 min at room temperature. The supernatant was decanted and the cells gentlyre-suspended in 0.5 ml PBS. Then, the cells were fixed by adding 4.5 ml cold ethanol(70%). After 24 h, fixed cells were again centrifuged as above, washed once andre-centrifuged with cold PBS. Centrifuged cells were re-suspended in stainingsolution containing 0.5 ml PBS, 10ml of 10 mg/ml ribonuclease A, 1ml triton-x-100and 10ml of 1 mg/ml propidium iodide stock solution, and incubated at 378Cfor 30 min. Flow cytometric measurements were performed using a LSR II flowcytometer (Becton-Dickinson).
Western Blot AnalysisAt various times after exposure (1, 2, 4, 6, 8, 12, 16, and 24 h) to 6 mT SMF, thecontrol and treated Jurkat cells were washed out with PBS and lysed. Whole cellextracts were prepared by lysis in 700ml of lysis RIPA buffer. The lysatescontaining equal amounts of protein (30mg) were then loaded onto a 7.5 or 12.5%SDS- polyacrylamide gel. After electrophoresis, proteins were transferred to aPVDF membrane. Once transferred, the PVDF membrane was incubatedtwo hours with blocking buffer containing 5% BSA in TBST (0.605 g Tris, 1.875 gNaCl, 250ml Tween 20 (Merck); final volume of 500 ml, adjusted to pH 8.0 with4 mol/l HCl).
After the blocking step, PVDF membrane was incubated overnight with a 1:200dilution of the primary antibodies in 2% BSA in TBST buffer: anti-p-ATM (Ser-1981),anti-E2F1 (C-7), and Actin (MM2/193), from Santa Cruz Biotechnology, USA. Oncethe primary antibodies were removed and washed, a secondary antibody (goat anti-mouse IgG-HRP; Sc-2005, and goat anti-mouse IgM-HRP; Sc-2064, Santa CruzBiotechnology, USA) was added (2 h, dilution 1:5000 in 2% BSA-TBST buffer). Finally,the blots were washed (3 £ /10 min) with TBST. During the last washing step, theband visualization solution was prepared by diluting 25 mg of 3,3 -DAB (D-5637,Sigma) in 2 ml of bidistilled water (Anderson, 1996). This solution was mixed with48 ml phosphate buffered saline containing 45ml hydrogen peroxide (H-1009,Sigma). Blots were incubated with this solution for 10–60 minutes. The reaction wasstopped with tap water, and the blots were dried with paper towels and stored awayfrom light at room temperature, sometimes the blots were incubated with asecondary peroxidase-conjugated antibody (goat anti-mouse IgG-HRP; Sc-2005,Santa Cruz Biotechnology, USA) and the signal was developed with achemi-luminescence (ECL) detection kit (Amersham) by exposure to an X-ray film(Foma, Hradec Kralove, CZ).
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In each step of the experiments, we had concurrent controls, i.e., control flasks ofunexposed cells, as many as the number of MF-exposed cell flasks. Concurrentcontrol flasks were placed into an incubator at another room with identicaltemperature, humidity and CO2 conditions to the exposed cells. Unexposed cellswere also analyzed through luminometry, flow cytometry, and Western blottingtechniques, like the exposed ones.
Statistical AnalysisAll the results were reported as the mean ^ SD of at least four independentexperiments. Statistically significant differences were carried out using two-wayANOVA (SPSS 15; and Minitab 16). The P values less than 0.05 were consideredstatistically significant, as compared to the unexposed control group.
RESULTS
Induction of Apoptosis in Jurkat Cells Exposure to SMFIn the present study, luminometric assay was used to measure the rate of early andlate apoptosis. As shown in Fig. 2a, 36 h after treating the Jurkat cells with 6 mT staticmagnetic field, the rate of apoptosis significantly increased in comparison withcontrol cell (p , 0.03). Apoptosis also reached to its maximum rate 48 h aftertreatment, so that it was 31.2% in cells treated for 6 h (p , 0.02) and 35.5% in cells
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FIGURE 2 The rate of induction of apoptosis after exposure to 6 mT static magnetic field.(a) Luminometric detection of rate of apoptotic cells. (b) Flow cytometric detection of percentage ofapoptotic cells. Each point represents the average of four independent experiments (mean ^ SD).Values after 36 h are less than significant level (p , 0.05).
Static magnetic field of 6mT induces apoptosis in Jurkat cells 13
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treated for 24 h by 6 mT SMF (p , 0.005), respectively. However, 72 h after treatmentthe apoptosis rate decreased (Table 1).
Flow cytometric determination of DNA content corresponding to the sub-G1 peakof apoptotic cells was then employed to determine early apoptosis. As illustrated inFig. 2b, early apoptosis was occurred in treated cells with a low rate. The firstsignificant difference between treated and control cells can be observedapproximately 36 h after exposure to 6 mT SMF (p , 0.001). The rate of apoptoticcells increased to 6% in cells treated for 6 h and to 8% in cells treated for 24 h, 72 hafter treatment (p , 0.001).
Cell survival and division of exposed Jurkat cells showed a decrement compared,using trypan blue staining (data not shown).
Cell Cycle Alteration in exposed Jurkat cellsFlow cytometry was also employed to study the cell cycle of Jurkat cells. As shown inFig. 3, the population of S-phase and G2-phase cells exposed to SMF changed within24-h periods. Population of S and G2 cells decreased 24 h after treatment; however, itstarted to increment 30 and 36 h after treatment, which is accompanied with a slightdecrease in population of G1 cells. S-phase and G2-phase cells decreased 48 h aftertreatment with SMF, resulting in an increase in G1 population (p , 0.05). Also, anumber of cells died due to apoptosis and quitted their cell cycle. Such a procedurerepeated in the next 24-h period (i.e., 48–72 h after treatment).
Phosphorylation of ATM and E2F1 ProteinsWestern blot analysis was used to determine the changes in the amount ofphosphorylated ATM and E2F1 proteins in the treated cells. Our results illustratedthat the amount of phosphorylated forms of these two proteins increased in theexposed Jurkat cells to 6 mT SMF. As can be seen in Fig. 4a, 1 h after exposure to 6 mTSMF, the level of phosphorylated ATM (in Ser-1981) increases in treated cells andkeeps until 2, 4, and 6 h of magnetic field exposure, after that it starts to decrease.Concentration of phosphorylated E2F1 (in Ser-31) is markedly increased after 1 and2 h of exposure and keeps until 12 h of exposure, and then it decreases (Fig. 4b).
DISCUSSION
The human T-lymphoblastoid Jurkat cell line is widely used to investigate cell cyclearrest and apoptosis induced by external physical and chemical factors. In response
TABLE 1 Luminescence data of induced apoptosis by the 6 mT static magnetic field. Each numberrepresents the average of four independent experiments (mean ^ SD). Values after 36 h are lessthan significant level (p , 0.05).
to DNA damage, apoptosis signaling is triggered by the tumor suppressor p53, whichis a critical regulator of the apoptosis pathway and DNA repair signaling (Canmanet al., 1998; Powers et al., 2004). However, several mutations prevent the expressionof p53 in the human Jurkat cell line. Thus, Jurkat cells are an appropriate modelsystem to study molecular mechanisms of p53- independent apoptosis inT-lymphocytes. On the other hand, lacking the wild type p53 gene conferred ahigher resistance than cells with wild type p53 gene, against physical and chemicalfactors which induced single-strand or double-strand breaks in DNA molecule andpromoted the apoptosis process (Kim et al., 2010).
The data obtained in the present work highlights three main bioeffects of SMF ofmoderate intensity on human T-lymphoblastoid Jurkat cell line: (i) induction ofapoptosis; (ii) modulation of apoptosis-related gene expression; and (iii) temporarilyalteration in cell cycle.
According to past investigations, a general mechanism for the action of moderateintensity static MFs on biological systems would be by virtue of their effect onthe molecular structure of excitable membranes, an effect sufficient to modifythe function of embedded ion-specific channels (Fanelli et al., 1999; Teodori
FIGURE 3 Histograms obtained from flow cytometric analysis showing cell cycle in Jurkat cellsexposed to 6 mT static magnetic fields for 6 h (a) and 24 h (b). Apoptotic cells were identified as cellswith sub-diploid DNA content, i.e., sub-G1 peak.
Static magnetic field of 6mT induces apoptosis in Jurkat cells 15
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et al., 2002a,b; Rosen, 2003). The magnetic fields may decrease the concentration ofcalcium ion through influencing either the function of Ca2þ-ATPase or modifying thefunction of Ca2þ binding protein and the contiguous ion specic channels (Aldinucciet al., 2003). This hypothesis would explain virtually all of the bioeffects attributed tothese fields, including the modulation of apoptosis, and is testable using severaldifferent neurophysiological techniques (Rosen, 2003). Other possible effects ofstatic MFs leading to perturbation of the apoptotic rate (Fanelli et al., 1999; Jajte,2000; Chionna et al., 2003), such as alteration of the gene pattern expression(Marinelli et al., 2004; Tenuzzo et al., 2009) or increment of oxygen free radicals cannot be excluded. It is well known that free radicals are mediators of apoptosis (Jaiteet al., 2002; Brune, 2003; Lai and Singh, 2004). Interestingly, the concentration of freeradicals in transformed cells and tissues is higher than in their normal counterparts(Szatrowski and Nathan, 1991). In addition, concentration of free radicals has beendescribed as increasing in different conditions of exposure, from SMF to pulsed MFto EMF (Jaite et al., 2002; Stevens, 2004). Most likely, magnetic field exposure affectsiron homeostasis in certain cells, leading to an increase in free iron in the cytoplasmand nucleus, which in turn leads to an increase in hydroxyl radicals, via the catalyticreaction of the Fenton reaction, which damages DNA, lipids, and proteins (Lai andSingh, 2004). Damage to lipids (lipid peroxidation) in the cellular membrane in turnleads to an increase in calcium leakage from internal storage sites in the cell (Lai andSingh, 2004).
However, as shown here, upon SMF exposure, apoptosis can be modulated notonly by promoting the cytosolic release of [Ca2 þ ]i from the endoplasmic reticulumstore, or the influx into the cells (following increased permeability of the plasmamembrane), but also by the modulation of the apoptosis-related gene expression.Our results show that concentration of phosphorylated ATM (in Ser-1981) whichforms due to double-strand breaks in DNA, increases in Jurkat cells exposed to SMFfor 1 h. The active phosphorylated form of ATM protein induces phosphorylation ofother proteins which involve in DNA repair and/or apoptosis processes (Bakkenistand Kastan, 2003; Powers et al., 2004). E2F1 is one of the proteins which isphosphorylated (in Ser-31) and activated by ATM. Unlike other E2F proteins whichcontribute to stimulation or inhibition of the cell cycle, E2F1 promotes the apoptosisprocess. Phosphorylated form of E2F1 induces p53-dependent or p53-indepententapoptosis through enhancing the transcription of some genes or phosphorylation of
FIGURE 4 Western blot analysis of phosphorylated ATM (a) and E2F1 (b) proteins in exposed Jurkatcells to 6 mT SMF in different times (0, 1, 2, 4, 6, 8, 12, 18, and 24 h).
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such proteins as p53 and its homologs (p63 and p73) (Flores et al., 2002). Therefore,one possible mechanism for initiation of the p53-independent apoptosis in Jurkatcells is through the effect of phosphorylated E2F1 in enhancing the transcription ofp73 gene, because p73 protein can initiate apoptosis in cells through a pathwayindependent of p53 (Flores et al., 2002; Urist et al., 2004).
Six mT SMF may cause a delay in the cell cycle of Jurkat cells through direct orindirect effects on these cells, which temporarily trap the cells in G2 phase. This leads
FIGURE 5 Bar charts showing cell cycle phases of Jurkat cells at 24 (a), 48 (b), and 72 h (c) afterexposure to 6 mT static magnetic field.
Static magnetic field of 6mT induces apoptosis in Jurkat cells 17
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to an increase in cellular population in S and G2 phases. At the end of G2 phase,damaged cells which could repair their damages or those whose damages does notinduce the apoptosis, will divide and start a new cell cycle. Therefore, the decreasedpopulation of G1 cells begins to increment. However, cells with lethal damages, suchas un-repairable DNA double-strand breaks, will die through apoptosis process(Fig. 5).
Based on our obtained data from luminometric and flow cytometric detectionwe may propose that duration of exposure affected the apoptosis rate in treatedJurkat cells.
In summary, exposure to 6 mT SMF affects the apoptosis rate in Jurkat cells, theexpression and distribution of pro-apoptotic genes, and [Ca2 þ ]i. From our data, itcould be proposed that induction of apoptosis is the final result of multiple points’perturbation to Ca2 þ -regulated activities, to gene-related transcription factors andto components of the membranes, that altogether results in the induction ofapoptosis. Therefore, the ability of SMF to modulate the expression of genes involvedin the process of apoptosis, suggests the development of defined conditions,eventually at the clinical level, for eliminating specific unwanted cell populations byinducing them to initiate their own destruction.
ACKNOWLEDGEMENTS
This work was financially supported by grant No. 88000271 from Iran NationalScience Foundation (INSF) to PA on investigation about biological effect ofelectromagnetic radiation. The authors wish to express their gratitude to Miss Hayatfor her assistance in flow cytometry assays as well as Mr. Barzegari Asadabadi for hishelpful comments.
Declaration of interestHereby I will notify that there is no any conflict of interest regarding this paper.
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