Single strand DNA breaks in rat brain cells exposed to microwave radiation

Mutation Research 596 (2006) 76–80

Single strand DNA breaks in rat brain cells exposed to microwave radiation R. Paulraj, J. Behari ∗ School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India Received 7 May 2005; received in revised form 9 December 2005; accepted 22 December 2005 Available online 2 February 2006

Abstract This investigation concerns with the effect of low intensity microwave (2.45 and 16.5 GHz, SAR 1.0 and 2.01 W/kg, respectively) radiation on developing rat brain. Wistar rats (35 days old, male, six rats in each group) were selected for this study. These animals were exposed for 35 days at the above mentioned frequencies separately in two different exposure systems. After the exposure period, the rats were sacrificed and the whole brain tissue was dissected and used for study of single strand DNA breaks by micro gel electrophoresis (comet assay). Single strand DNA breaks were measured as tail length of comet. Fifty cells from each slide and two slides per animal were observed. One-way ANOVA method was adopted for statistical analysis. This study shows that the chronic exposure to these radiations cause statistically significant (p < 0.001) increase in DNA single strand breaks in brain cells of rat. © 2006 Elsevier B.V. All rights reserved. Keywords: Microwave radiation; DNA strand breaks; Single gel electrophoresis

1. Introduction Radio frequency (RF) and microwave radiation are considered as a type of non-ionizing electromagnetic radiations present in the environment and are perceived as health risk. Increased exposition of RF electromagnetic field (EMF) produced by the appliances used in the telecommunications, industry and medicine may lead to biological effects in more individuals. Non-ionizing radiation has a significant and positive impact on modern society through a number of uses. There has been a growing concern among the public regarding the poten-

∗ Corresponding author. Tel.: +91 11 2670 4323; fax: +91 11 2671 7586x7502. E-mail addresses: [email protected], [email protected] (J. Behari).

0027-5107/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.12.006

tial human health hazard of exposure to these frequencies by these appliances [1]. Potential health effects of radio frequency and microwave radiation generated by telecommunication, industry or other emitters, are reported [2]. In ICNRIP report [3] on possible effects of long term exposure to RF radiation state that there is no convincing evidence of causal relation between RF and any adverse health effects. Epidemiological studies reported that an increased risk of brain tumors among analogue cellular phone users [4]. In contrary, Heynick et al. [5] report on epidemiological studies showed no indication of in vivo or in vitro exposures of RF field and cancer. Cleary [6] reported that RF radiation at 2.45 GHz and 27 MHz increase cell proliferation. Continuous wave and pulsed RF field have been reported to affect a variety of ion channel properties, such as decreased rate of channel

R. Paulraj, J. Behari / Mutation Research 596 (2006) 76–80

protein formation, decreased frequency of single channel opening and increased rate of rapid burst like firing [7]. Earlier, we have found an increase in the calcium ion efflux in rat brain exposed to 2.45 GHz radiation [8]. Microwaves have been reported to affect the kinetics of conformational changes of proteins (betalactoglobulin) [9]. Increased blood brain barrier permeability has been reported in rats to rhodomine-ferritin complex at 2.45 GHz [10]. 2.45 GHz radiation causes significant increase in ornithine decarboxylase activity and a decrease in protein kinase activity in rat brain after chronic microwave exposure [8]. Studies on central nervous system (CNS) showed that, exposure to electromagnetic field resulted in depressed activities of protein kinase C [11] and different types of neurotransmitter systems, such as acetylcholine esterase, acetylcholine, dopamine, serotonin and amino acids in the developing rat brain [12,13]. Structural and genomic changes have been reported in the brain and testis of rat exposed to 2.45 GHz radiation [14]. Lai and Singh [15] also reported that rats exposed to pulse or continuous wave of 2.45 GHz field for 2 h, resulted in an increase of single and double stand breaks in the DNA of brain cells. Different kinds of DNA damage may occur due to the non-ionizing radiation exposure, which appear to be influenced by higher order chromatin structure. Differences in DNA repair capacity have become the accepted explanation for this range of intrinsic radio sensitivities, since it is generally believed that a subset of the DNA breaks is lethal if unrepaired [16]. However, McNamee et al. [17] reported that, human blood cells, exposed to 1.9 GHz (both continuous and pulsed waves) for 24 h, did not result in any genotoxic effects. Malyapa et al. [18,19] reported that no DNA damage in cells of the rat cerebral cortex or the hippocampus after

77

a 2 or 4 h exposure to 2450 MHz (CW). Above experimental results refer to acute exposure. In the present investigation, it is intended to carry out the effects of chronic exposure (35 days) on developing rats brain. Thirty-five days old rats were selected because during this period the brain developments take place in rat. A choice of 2.45 GHz is made because of its wide spread use in radar, industry, scientific research and medicine, and hence the probability of its leakage in the environment is possible. A choice of second frequency and dose was made to see if the effect is of general nature. Also no data at frequency 16.5 GHz are reported in the literature. 2. Materials and methods 2.1. Materials Low melting point agarose, normal melting point agarose, ethidium bromide and DNAse free proteinase K were procured from Sigma Chemical Co., USA. Rest of the chemicals was purchased from local firms. 2.2. Animals Male Wistar rats of 35-day-old (60–70 g) were obtained from Jawaharlal Nehru University animal facility. Animals were supplied with food and water ad libitum and were grouped in to two (control and experimental). Each group consists of six rats (n = 6). 2.3. Exposure chamber The chamber is a tapered one and specially designed for the frequency varying from 2 to 10 GHz [20]. Source of radiation is magnetron (Fig. 1). Similarly for 16.5 GHz exposure a gunn diode source using a separate setup and kept sepa-

Fig. 1. Schematic diagram of anechoic chamber with animal cage indicating individual animal’s place.

78

R. Paulraj, J. Behari / Mutation Research 596 (2006) 76–80

rately (Senor Microwaves, New Delhi, India). These chambers are made up of wood. The inner lining of the chamber is pyramid shaped black radar absorbing material (40 dB). Proper ventilation arrangements have been made with windows and exhaust fan. Absolute calibration was made with an identical horn antenna of the same aperture, which was connected to the power meter through a wave-guide. The power at the receiving end was measured and the power density computed. 2.45 GHz irradiation, six rats were kept in a plexi glass cage (43 cm × 27 cm × 15 cm). Small circular holes (1 cm2 ) were drilled at equidistance in the cage to keep the animal aerated during the exposure. Each animal was kept in a pre-specified compartment of the cage throughout the exposure period. The dimension of the cage was made (according to the animal size) in such a way that animals remain restrained. The animals are restrained in the cage during the exposure period. Each animal was thus irradiated homogeneously at the same power level. Temperature in the chamber was maintained at 30 ◦ C throughout the period. The cage was constantly aerated to avoid the possibility of any increase in temperature. The cage was placed symmetrically along the midline of the pyramidal horn antenna, having dimensions 13 cm × 9.8 cm. Six rats were kept simultaneously in a cage and placed inside the chamber. Everyday the cage was placed in the same position facing the horn antenna and same number of rat positions was filled. Exposure was given for 2 h per day for a total period of 35 days (excluding the weekends) at power density of 0.344 mW/cm2 . Sham exposure (control group, n = 6) was performed similarly as the exposed group but without power input. We have adopted the specific absorption rate (SAR) value from the theoretical estimations of Durney et al. [21]. For a small rat, where the E field is parallel to the body of the animal, SAR for the above radiation intensity turns out to be 1.0 W/kg. Another set of animals were treated similarly but at different frequency, i.e. 16.5 GHz amplitude modulated frequency at power density 1.0 mW/cm2 (SAR 2.01 W/kg). These rats were housed in a rectangular plexi glass cage having dimensions 32 cm × 10 cm × 9 cm. Plexi glass has a low dielectric constant (∼2.0) so as to simulate the electric characteristics as close to those of free space. Two rats were kept simultaneously in a cage and placed inside the chamber. Exposure was given for 2 h per day for 35 days. Control animals were kept in the same way without power input (sham irradiation). In total six animals were used in each category. 2.4. DNA strand breaks In the present investigations, comet assay (also referred as single cell gel electrophoresis) is used to determine DNA damage in terms of DNA strand breaks. Immediately after the exposure period, one rat at a time was anesthetized by placing it in a glass jar containing cotton dipped in anesthetic ether. The rat was then decapitated and its brain was dissected out immediately for DNA strand break assay. To allow time for tissue processing, there was

a 5 min time gap between consecutive animals was given. Whole brain was washed four times with phosphate buffered saline (PBS) (1.37 mM NaCl, 4.3 mM Na2 HPO4 , 2.7 mM KCl, 1.4 mM KH2 PO4 , pH 7.4) to remove red blood cells (RBC). These were minced in to small pieces by using a tissue press (Biospec Products Inc., USA) and a single cell suspension was made using a 5 ml pipette. From the cell suspension, 10 ␮l of its suspension was mixed with 0.2 ml, 0.5% agarose. Agarose was suspended in phosphate buffered saline (PBS) (3:1 agarose higher resolution) and was kept at 37 ◦ C to maintain physiological condition [22]. The mixture was pipetted out and poured on to a fully frosted slide, immediately covered with coverglass (24 mm × 50 mm). These slides were kept in an ice-cold steel tray on ice for 1 min to allow the agarose to gel. Again a layer was made over the gel with 100 ␮l of agarose as before, after removing the coverglass [15,23]. These slides were immersed in ice-cold lysing solution (2.5 M NaCl, 100 mM EDTA disodium salt, 1% laurylsarcosine sodium salt, 10 mM Tris–HCl, pH 10) and kept overnight at 4 ◦ C. After lysing overnight the slides were treated with DNAase free proteinase K (1 mg/ml) put on the horizontal slab of an electrophoretic assembly. One litre of electrophoresis buffer (300 mM NaOH, 0.1% 8-hydroxyquinoline, 2% dimethyl sulfoxide (DMSO), 100 mM Tris and 10 mM tetrasodium EDTA, pH 13) was gently poured into the assembly. After 20 min to allow for DNA unwinding, electrophoresis was started at 250 mA (25 V) for 60 min. The slides were removed from the electrophoretic apparatus and placed in Coplin jar containing 0.4 mol dm−3 Tris, pH 7.4 to neutralise NaOH in microgels. After 30 min, the slides were transferred to another jar of Tris for 15 min. After one more change of 15 min, slides were immersed in absolute ethanol for 30 min to precipitate the DNA and dehydrate the gels. Slides were left vertical at room temperature to dry and stained with 50 ␮l of 1 mmol dm−3 solution of YOYO-1 [benzoxazolium-4-quinolium oxalo yellow dimer] covered with a 24 mm × 50 mm coverglass. Microscopic slides (two slides per animal) were prepared with each individual animal separately [15,23]. Slides were coded before analysis and examined and analyzed with a Reichert vertical fluorescent microscope equipped with a filter combination for fluorescence isothyocyanate (excitation at 490 nm, emission filter at 515 nm and dichromatic filter at 500 nm). DNA damage was quantitative as length of the comet tail with the help of an ocular micrometer. The least count of the micrometer is 0.5 ␮m, while the average tail minimum length is 19.97 ␮m. Earlier studies by Lai and Singh [22] measured the length of DNA migration by ocular micrometer mounted in the eyepiece of the microscope and produced accurate result. In the present study, we obtained similar accuracy. 2.5. Comet scoring Two slides were assayed for single strand DNA breaks. Fifty representative cells were scored from each slide. Therefore, from each animal, 100 cells each were scored. Tail migration length (in ␮m) from the beginning of the nuclear area to the last five pixels of DNA perpendicular to the direction of migra-

R. Paulraj, J. Behari / Mutation Research 596 (2006) 76–80

tion at the leading edge was measured. The migration length is used as the index of DNA strand breaks. Tail length of individual cells was measured, cells which are overlapped are not counted. 2.6. Data analysis Statistical analysis was performed with SX package. All data are presented as mean ± standard deviation (S.D.). The difference between exposed and control groups were tested for significance by using one-way ANOVA with Bonferroni correction for comparison of means. A difference at p < 0.05 was considered statistically significant.

3. Results and discussion Our results show, that the prolonged chronic exposure to 2.45 and 16.5 GHz separately causes reproducible increase in single strand DNA breaks in brain cells of rat in all the exposed group animals. A significant increase in the length of DNA migration was observed in rat brain exposed to 2.45 and 16.5 GHz radiation. A corresponding no change in tail length was observed in all the control animals. We did not find any cell death by treating the field mentioned above. The average values of DNA migration of rat brain cells exposed to 2.45 GHz continuous wave is given in Table 1. It shows that there is a significant increase in the tail length of DNA as compared to the control group. For the control group the average value comes out to be 24.11 ± 4.47 ␮m whereas for the exposed group it was 41.011 ± 4.625 ␮m, which were found to be statistically significant (p < 0.001). A statistically significant increase in the DNA migration was also observed for cells exposed to 16.5 GHz radiation. For control group, it was 20.46 ± 3.58 ␮m, whereas for the exposed group it was 31.147 ± 4.66 ␮m (p < 0.001) (Table 2). In our present study was aimed to find out the effect of chronic exposure of radiation. Data so obtained show that prolonged (35 days) exposure to microwave radiation (2.45 and 16.5 GHz) causes single strand DNA Table 1 DNA migration (in ␮m) of individual animals exposed to 2.45 GHz radiation Animals

Control

1 2 3 4 5 6

24.87 24.65 24.77 24.40 22.98 23.01

± ± ± ± ± ±

Exposed 4.50 3.59 4.76 4.38 5.52 4.17

The values are average (±S.D.) of 100 cells.

37.47 41.23 42.20 40.28 43.42 41.46

± ± ± ± ± ±

5.07 4.21 4.84 5.08 3.74 4.80

79

Table 2 DNA migration (in ␮m) of individual animals exposed to 16.5 GHz radiation Animals

Control

1 2 3 4 5 6

20.14 19.97 20.54 20.41 20.89 20.82

± ± ± ± ± ±

Exposed 2.46 3.03 3.69 3.50 4.35 4.46

28.95 31.22 32.46 32.24 31.61 30.40

± ± ± ± ± ±

4.13 5.16 4.43 4.21 4.50 5.09

The values are average (±S.D.) of 100 cells.

breaks in brain cells of rat. Maes et al. [24] reported that acute 30–120 min exposure to 2450 MHz RFR at an SAR 75 W/kg and constant temperature, 36.1 ◦ C increased dicentric and acentric chromosomal fragments and micronuclei formation in human lymphocytes. Mitchell et al. [25] observed a decrease in motor activity in rats after 7 h of exposure to CW 2450MHz RFR (10 mW/cm2 , average SAR 2.7 W/kg). Lai and Singh [15,26] reported that acute exposure (2 h) to both pulsed and continuous wave (CW) 2.45 GHz radiation (power density 2 mW/cm2 , SAR 1.2 W/kg) produce a significant increase in the DNA single and double strand breaks in rat brain. The present study is in agreement with these studies showing a significant difference in DNA single strand breaks in the exposed group. DNA damage is closely related to human health risk. Particularly, DNA damage in brain cells could affect neurological functions and also possibly lead to neurodegenerative diseases [15]. The exact mechanism is yet to be elucidated, as to how DNA strand breaks occur due to RF radiation. In addition to direct damage to DNA by genotoxic chemicals and physical agents, DNA strand breaks are also generated as intermediate step during DNA repair, during repair of damages due to DNA–DNA and DNA–protein cross links and DNA adduct formation, etc. DNA single strand breaks are also produced when double strand breaks are repaired by recombination [15]. Using the comet assay a diverse types of DNA damage can be determined. Among those are DNA single strand breaks, bulky base modification and DNA double strand breaks. Since various tissues or cell types differ in their susceptibility towards EMF exposure, hence conflicting results among the mammalian cell types are also reported [27]. It is hoped that data presented here will help in identifying possible causal connections of exposure to electromagnetic fields and biological effects.

80

R. Paulraj, J. Behari / Mutation Research 596 (2006) 76–80

Acknowledgements Authors are thankful to Indian Council of Medical Research, New Delhi, for the financial assistance. Authors are thankful to Dr. N.P. Singh, University of Washington, USA, for providing the tissue press (Biospec products Inc., USA). References [1] J.E. Moulder, L.S. Erdreich, R.S. Malyapa, J. Merritt, W.F. Pickard, Vijayalaxmi, Cell phones and cancer: what is the evidence for connection? Radiat. Res. 151 (1999) 513–531. [2] D. Brusick, R. Albertini, D. McRee, D. Peterson, G. Williams, P. Hanawalt, J. Preston, Genotoxicity of radiofrequency radiation. DNA/Genetox Expert Panel, Environ. Mol. Mutagen. 32 (1998) 1–16. [3] ICNIRP Report, Epidemiology of health effects of radiofrequency, Environ. Health Perspect. 112 (17) (2004) 1741– 1754. [4] L. Hardell, K.H. Mild, M. Carlberg, Further aspects on cellular and cordless telephones and brain tumours, Int. J Oncol. 22 (2003) 399–407. [5] L.N. Heynick, S.A. Johnston, A. Patrick, P.A. Mason, Radio frequency electromagnetic fields: cancer, mutagenesis, and genotoxicity, Bioelctromagnetics 24 (2003) s74–s100. [6] S.F. Cleary, Biological effects of radiofrequency electromagnetic fields, in: O.P. Gandhi (Ed.), Biological effects and Medical Applications of Electromagnetic Energy, Prentice Hall, Englewood Cliffs, NJ, 1990, pp. 236–255. [7] M.H. Repacholi, Low level exposure to radiofrequency electromagnetic fields: health effects and research needs, Bioelectromagnetics 19 (1998) 1–19. [8] R. Paulraj, J. Behari, The effect of low level continuous 2.45 GHz wave on brain enzymes of developing rat brain, Electromag. Biol. Med. 21 (3) (2002) 231–241. [9] H. Bohr, J. Bohr, Microwave enhanced kinetics observed in ORD studies of protein, Bioelectromagnetics 21 (2000) 68–72. [10] C. Neubauer, A.M. Phelan, H. Kues, D.G. Lange, Microwave irradiation of rats at 2.45 GHz activates pinocytotic-like uptake of tracer by capillary endothelial cells of cerebral cortex, Bioelectromagnetics 11 (4) (1990) 261–268. [11] R. Paulraj, J. Behari, Radio frequency radiation effects on protein kinase C activity in rats’ brain, Mut. Res. 545 (2004) 127– 130. [12] K.K. Kunjilwar, J. Behari, Effect of amplitude modulated radiofrequency radiation on cholenergic system of developing rats, Brain Res. 601 (1993) 321.

[13] H. Lai, M.A. Carino, A. Horita, A.W. Guy, Effects of a 60 Hz magnetic field on central cholinergic system of the rat, Bioelectromagnetics 14 (1993) 5–15. [14] S. Sarkar, S. Ali, J. Behari, Effect of low power microwave on the mouse genome: a direct DNA analysis, Mut. Res. 320 (1994) 141–147. [15] H. Lai, N.P. Singh, Single and double strand breaks in rats brain cells after acute exposure to radio frequency electromagnetic radiation, Int. J. Radiat. Biol. 69 (1996) 513–521. [16] G. Iliakis, The role of DNA double strand breaks in ionizing radiation induced killing of eukaryotic cells, Bio. Essays 13 (1991) 641–648. [17] J.P. McNamee, P.V. Bellier, G.B. Gajda, B.F. Lavall´ee, L. Marro, E. Lemay, A. Thansandote, No evidence for genotoxic effects from 24 h exposure of human leukocytes to 1.9 GHz radiofrequency fields, Radiat. Res. (2003) 693–697. [18] R.S. Malyapa, E.W. Ahern, Bi. Chen, W.L. Straube, M. LaRegina, W.F. Pickard, J.L. Roti Roti, DNA damage in rat brain cells after in vivo exposure to 2450 MHz electromagnetic radiation and various methods of Euthanasia, Radiat. Res. 149 (1998) 637–645. [19] R.S. Malyapa, E.W. Ahern, W.L. Straube, E.G. Moros, W.F. Pickard, J.L. Roti Roti, Measurement of DNA damage after exposure to 2450 MHz electromagnetic radiation, Radiat. Res. 148 (1997) 608–617. [20] S. Ray, J. Behari, Physiological changes in rats after exposure to low levels of microwaves, Radiat. Res. 125 (1990) 199–201. [21] Durney C.H., Johnson C.C., Barber P.W., Massoudi H., Iskander M.F., Lords J.L., Ryser D.K., Allen S.J., Mitchell J.C., Radiofrequency Radiation Dosimetry Handbook, Report SAM-TR-78-22, second ed., Brooks Air Force Base, USAF School of Aerospace Medicine, Texas 78235, 1978, p. 94. [22] H. Lai, N.P. Singh, Magnetic field-induced DNA strand breaks in brain cells of rat, Environ. Health Perspect. 112 (6) (2004) 687–694. [23] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J.-C. Ryu, Y.F. Sasaki, Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mut. 35 (2000) 206–221. [24] A. Maes, L. Verschaeve, A. Arroyo, C. DeWagter, L. Vercruyssen, In vitro cytogenetic effects of 2450 MHz waves on human peripheral blood lymphocytes, Bioelectromagnetics 14 (1993) 495–501. [25] C.L. Mitchell, D.J. McRee, N.J. Peterson, H.A. Tilson, Some behavioral effects of short-term exposure of rats to 2.45-GHz microwave radiation, Bioelectromagnetics 9 (1988) 259–268. [26] H. Lai, N.P. Singh, Acute low level microwave exposure increases DNA single strand breaks in rat brain cells, Bioelectromagnetics 16 (1995) 207–210. [27] Risk evaluation of potential environmental hazards from low energy electromagnetic field exposure using sensitive in vitro methods (REFLEX), final report, 2004, pp. 183–242.