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Effects of GSM microwaves, pulsed magnetic field, and temperature on fractal dimension of brain tumors

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Effects of GSM microwaves, pulsed magnetic field, and temperature on fractal dimension of brain tumors
  Effects of GSM microwaves, pulsed magnetic field,and temperature on fractal dimension of brain tumors M. Babincov  aa  a,* , P. Sourivong  b , D. Leszczynska  c , P. Babinec  a a Department of Biophysics and Chemical Physics, Comenius University, Mlynsk   aa  dolina F1, 842 48 Bratislava, Slovakia b Department of Radiation Oncology, 987521 Nebraska Medical Center, Omaha, NE 68198, USA c Department of Civil Engineering, FAMU-FSU College of Engineering, Tallahassee, FL 32310, USA Accepted 19 September 2003 Abstract Fractal dimension of a two-dimensional C-6 rat glioma tumors growing in the microwave field generated by signalsimulation of the Global System for Mobile communications (GSM) with frequency 960 MHz was found significantlyenhanced as compared with field free tumors growing at different temperatures and on the other hand a strong pulsedmagnetic field lowered fractal dimension of tumors.   2003 Elsevier Ltd. All rights reserved. 1. Introduction In several studies by Sedivy, Landini, Rippin, Waliszewski, and others [1–3], the usefulness of fractal parameters intumor pathology has been emphasized. The fractal dimension of a tumor could be understood as a measure of ir-regularity [4], which serves as an additional morphometric parameter in surgical pathology and is specific for a giventumor. Many epidemiological studies have implicated environmental and residential exposure to electromagnetic fieldas a possible factor in the development of certain human cancers [5–7]. Extremely low frequency electromagnetic ra-diation has been reported to affect a wide range of basic cellular functions, including cell proliferation [8] gene tran-scription and expression [9] on transport [10], protein kinase C activation [11] and cell morphology [12]. Although thedetailed mechanism of the influence of a weak electromagnetic field on cellular processes is still unknown [13],electrostimulation of the proliferation of   Saccharomyces cerevisiae  [14] or  Pseudomonas stutzeri   [15], in a weak lowfrequency field have already direct biotechnological implications.Recently media attention has focused on claims for damages due to alleged brain tumors, in particular glioblas-tomas, caused by mobile phone usage. The aim of the present communication is to study the influence of an alternatingmagnetic fields on rat C-6 glioma cell line growing in monolayer. Glioblastomas are high-grade malignant neuroepi-thelial tumors having a median survival time of 8 months. These tumors have such a grim outcome in part due to theirrapid volumetric growth, but also because the tumor has already grossly invaded the surrounding brain tissue longbefore it can be diagnosed.As has been recently shown [16] this brain tumor model has super-rough fractal contour and therefore tumor growthmay be more susceptible to external influences than ordinary three-dimensional tumors. Moreover tumor interfacespeed growth is in this two-dimensional case linear and not exponential [17,18] and may be characterized by one-parameter. During tumor growth and evolution, mutations continue and cells behave differently from the normal cells * Corresponding author. Tel.: +421-2-6029-5685; fax: +421-2-6541-2305. E-mail address: (M. Babincov  aa).0960-0779/$ - see front matter    2003 Elsevier Ltd. All rights reseved.doi:10.1016/j.chaos.2003.09.020Chaos, Solitons and Fractals 20 (2004) 1041–  of the tissue where they appeared. In advanced stages of cancer, cells start to detach from the tumor and invade theblood stream or lymphatic system. They can be carried to other body parts producing new metastatic tumors. Thedetachment and invasion of other tissues result in part from the incorrect expression of adhesion molecules on the cellsurface for the mutated genome. This process causes a decrease in cellular adhesion between cells with additionalconsequences such as an increase in mobility of the cells on the surface of the tumor. As a result, the boundaries of thetumor become very irregular. This change on tumor morphology, associated with additional information, help phy-sicians to diagnose cancer stage of development. Recent studies indicate that the fractal dimension of tumors is useful asan indicative of malignancy. 2. Material and methods  2.1. Exposure systems The microwave field was generated by signal simulation of the Global System for Mobile communications (GSM), a960 MHz carrier amplitude modulated with a 217 Hz square pulse of duty cycle 12%. The experimental apparatusconsisted of a pair of horizontal rectangular (25 cm · 15 cm) coils, maintained in a pseudo-Helmholtz configuration(distance between the coils was 15 cm) and powered by AC generator. Field intensities were measured using a Hall effectprobe magnetometer (Model 9640, FW Bell, Orlando, FL USA).The strong pulsed magnetic field was generated between two air-gap water cooled coils placed in the Helmholtzposition with16 cm inner diameter separated by 8 cm. The two coils, wired in parallel, had resistance of 2.7  X . Themaximum achieved intensity was 0.2 T, corresponding to 175 A current produced by power supply. A square-wavesignal was sent to this power supply in order to pulse-modulate its output. The frequency of square pulses was   1 Hz.The cell cultures were placed in the middle part between the coils and were exposed to magnetic fields and cells wereallowed to grow for another 24 h in field free box. The whole system was placed in at thermostated box kept at37.0±0.1   C (in some field free experiments we have also used temperature 40.0±0.1   C). The temperature of theculture medium was monitored by using a nonabsorbing fluoroptic thermometry system (Luxtron 3000, MountainView, CA, USA) and no relevant heating of medium was observed during the experiments. 5% CO 2  inflowA/D CONVERTERCOMPUTER VCRINTERFACEOUTPUTsettingfocuspoweredstageobjectivecontrastphaseCCDcamerapHlight sourceglasselectrodetemperature INCUBATOR phase ringheatingsilica gel Fig. 1. Experimental setup for the study of two-dimensional tumor growth.1042  M. Babincov  aa  et al. / Chaos, Solitons and Fractals 20 (2004) 1041–1045   2.2. Cell culture 10 4 dissociated C-6 glioblastoma cells, cloned srcinally from rat glioma [19] and obtained from The American TypeCulture Collection (Rockville, MD, USA), were plated in a 5  l l of Dulbecco’s modified Eagle’s medium containing 10%(v/v) horse serum and 2.5% (v/v) fetal-calf serum on 35 mm Petri dishes. After cells attachment 2  l l of medium wasadded to growing culture, which allowed tumors to grow mainly on plate surface. In a regular time intervals control andexposed tumors were photographed under the inverted microscope with a coupled digital camera (Fig. 1).  2.3. Determination of fractal dimension The photographs were analyzed in a computer and tumor images were analyzed using program HarFA [20] based onthe improved box counting method where binary images of tumors were covered with different grids (box length  e ), andthe number of boxes  N  ð e Þ  required to cover the structures of the nuclei was recorded. If an object is fractal,  N  ð e Þ  in-creases according to the relation  N  ð e Þ ¼  C  e  D ; where  D  is fractal dimension and  C   is a constant. From this equation the fractal dimension can be obtained as  D  ¼  lim e ! 0 f log ð  N  ð e ÞÞ = log ð e Þg :  2.4. Statistical analysis Statistical evaluation of the experimental data was performed with two-tailed Student’s  t  -test with  p   <  0 : 05 as theminimum level of significance. 3. Results and discussion Fractal dimensions of tumor boundaries are shown in Fig. 2. For the comparison we have determined fractal di-mension of tumors growing at 37   C (control), tumors growing in microwave field (GSM), tumors growing in pulsedmagnetic field (pulse), and tumors growing at temperature 40   C (heat).As is clearly seen the fractal dimension of ‘‘GSM’’ tumors is significantly higher than in the other groups, whichdemonstrate for the first time the new effect mediated by mobile phones. It should be stressed that the intensity of usedGSM microwave radiation is about 50 fold higher than the intensity which is generated by mobile phones during theircommon use. Nevertheless these results indicates possible role of GSM radiation not in initiation but in acceleration of brain tumor growth and metastasing activity, which is probably higher in tumors with enhanced fractal dimension. Onthe other hand a strong pulsed magnetic field slightly lowered fractal dimension of tumors, which may be yet anotherreason for the applications of such a fields for the treatment of cancer [21].In conclusion, our results gives evidence of altered cellular reactions responsible for tumor cells proliferation bymicrowaves used in mobile communication. Because the fractal dimension of tumors growing at enhanced temperature40   C was not significantly different from the control growth, we can only hypothesize that observed increase in tumorgrowth in GSM field is due to some previously suggested nonthermal mechanisms behind the cells growth duringelectrostimulation [13]. On the other hand, in contrast with electric fields, magnetic fields can interact with living matterin the absence of direct contact with electrodes. When living matter is exposed to time-varying magnetic fields, electriccurrents (eddy currents) are induced in the matter due to the small conductivity of living tissues (their conductivity  r  is  1 S/m, as compared with   10 7 S/m of copper). The circular current  j  is given by the equation:  j  ¼ ð r r  = 2 Þð d  B = d t  Þ ; where  r   is the radius of current loop, and  B  is magnetic induction. From this formula is clear that the largest electriccurrent will be found at the periphery of the tumor and at the center of tumor would not be induced current, which mayexplain the sensitivity of tumors boundaries (as reflected by their fractal dimensions) to external fields. M. Babincov  aa  et al. / Chaos, Solitons and Fractals 20 (2004) 1041–1045  1043  Acknowledgement This work was supported by VEGA grant 1/9179/02. References [1] Sedivy R. Fractal tumors: their real and virtual images. Wien Klin Wochenschr 1996;108:547–51.[2] Landini G, Rippin JW. Fractal dimensions of the epithelial-connective tissue interfaces in premalignant and malignant epitheliallesions of the floor of the mouth. Anal Quant Cytol Histol 1993;15:144–9.[3] Waliszewski P. Complexity, dynamic cellular network, and tumour ignesis. Pol J Pathol 1997;46:235–41.[4] Mandelbrot BB. How long is the coast of Britain? Statistical self-similarity and fractional dimension. Science 1967;156:636–8.[5] Sheikh K. Exposure to electromagnetic fields and the risk of leukemia. Archiv Environ Health 1986;41:56–63.[6] Ahlbom A. A review of the epidemiological literature on magnetic field and cancer. Scand J Work Environ Health 1988;14:337–43.[7] Szmigielski S. Cancer morbidity in subjects occupationally exposed to high frequency (radiofrequency and microwave)electromagnetic radiation. Sci Total Environ 1996;180:9–17.[8] Kwee S, Rasmark P. Changes in cell proliferation due to environmental non-ionizing radiation. 1. ELF electromagnetic fields.Bioelectrochem Bioenerg 1995;36:109–13.[9] Garcia-Sancho J, Montero M, Alvarez J, Fonteriz RI, Sanchez A. Effects of extremely-low-frequency electromagnetic fields on iontransport in several mammalian cells. Bioelectromagnetics 1994;15:579–83.[10] Monti MG, Pernecco L, Moruzzi MS, Battini R, Zaniol P, Barbiroli B. Effect of ELF pulsed electromagnetic fields on proteinkinase C activation process in HL-60 leukemic cells. J Bioelectr (USA) 1991;10:119–25.[11] French PW, Donnelan M, McKenzie DR. Electromagnetic radiation at 835 MHz changes the morphology and inhibitsproliferation of a human astrocytoma cell line. Biolectrochem Bioenerg 1997;43:13–8.[12] Berg H. Possibilities and problems of low frequency weak electromagnetic fields in cell biology. Bioelectrochem Bioenerg1995;38:153–9.[13] Berg H. Problems of weak electromagnetic field effects in cell biology. Bioelectrochem Bioenerg 1999;48:355–60. control pulse GSM heat0.    F  r  a  c   t  a   l   d   i  m  e  n  s   i  o  n Condition Fig. 2. Fractal dimensions of tumors boundaries at different conditions: tumors growing at 37   C (control); in microwave field (GSM);in pulsed magnetic field (pulse); and at temperature 40   C (heat). All data are results from seven independent experiments.1044  M. Babincov  aa  et al. / Chaos, Solitons and Fractals 20 (2004) 1041–1045  [14] Fiedler U, Grubner U, Berg H. Electrostimulation of yeast proliferation. Bielectrochem Bioenerg 1995;38:423–5.[15] Hones I, Pospichil A, Berg H. Electrostimulation of proliferation of the denitrifying bacterium Pseudomonas stutzeri.Bioelectrochem Bioenerg 1998;44:275–7.[16] Bru A, Pastor JM, Fernaud I, Bru I, Melle S, Berenguer C. Super-rough dynamics on tumor growth. Phys Rev Lett 1998;81:4008– 11.[17] Freshney RI. Animal cell culture. Oxford: IRL Press; 1986.[18] Babincova M, Leszczynska D, Sourivong P, Babinec P. Influence of alternating magnetic field on two-dimensional tumor growth.Electro- and Magnetobiology 2000;19:351–6.[19] Benda P, Lightbody J, Sato G, Levine L, Sweet W. Differentiated rat glial cell strain in tissue culture. Science 1968;161:370–2.[20] HarFA, Harmonic and Fractal Image Analyser. Software for determination of fractal dimension (2001). Available[21] Tuffet S, de Seze R, Moreau JM, Veyret B. Effects of a strong pulsed magnetic field on the proliferation of tumor cells in vitro.Bielectrochem Bioenerg 1993;30:151–60. M. Babincov  aa  et al. / Chaos, Solitons and Fractals 20 (2004) 1041–1045  1045
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