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Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure Meric Arda Esmekayaa,* , Mehmet Zahid Tuysuza , Arın Tomruka , Ayse G. Cansevena , Engin Yücelb , Zuhal Aktunac , Semih Keskild, Nesrin Seyhana a
Department of Biophysics, Gazi University, Ankara, Turkey Department of Neurosurgery, Baskent University, Alanya Training and Research Hospital, Antalya, TURKEY Department of Medical Pharmacology, Kırıkkale University, Kırıkkale, TURKEY d Department of Neurosurgery, Kırıkkale University, Kırıkkale, TURKEY b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 July 2015 Accepted 28 January 2016 Available online xxx
The objective of the this study was to evaluate the effects of cellular phone radiation on oxidative stress parameters and oxide levels in mouse brain during pentylenetetrazole (PTZ) induced epileptic seizure. Eight weeks old mice were used in the study. Animals were distributed in the following groups: Group I: Control group treated with PTZ, Group II: 15 min cellular phone radiation + PTZ treatment + 30 min cellular phone radiation, Group III: 30 min cellular phone radiation + PTZ treatment + 30 min cellular phone radiation. The RF radiation was produced by a 900 MHz cellular phone. Lipid peroxidation, which is the indicator of oxidative stress was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). The glutathione (GSH) levels were determined by the Ellman method. Tissue total nitric oxide (NOx) levels were obtained using the Griess assay. Lipid peroxidation and NOx levels of brain tissue increased significantly in group II and III compared to group I. On the contrary, GSH levels were significantly lower in group II and III than group I. However, no statistically significant alterations in any of the endpoints were noted between group II and Group III. Overall, the experimental findings demonstrated that cellular phone radiation may increase the oxidative damage and NOx level during epileptic activity in mouse brain. ã 2016 Elsevier B.V. All rights reserved.
Keywords: Seizure Pentylenetetrazole Mice Cell phone Radiation
1. Introduction Epilepsy is one of the most common neurologic disorder in which the patient experiences chronic abnormal bursts of electrical discharge in the brain. About 0.8% of the world’s population suffer from epilepsy. Severe and continuous seizure activities may cause a variety of symptoms such as loss of speech, uncontrollable motor behavior, and/or unusual sensory experiences, complete or partial loss of consciousness in individuals with epilepsy. It may be caused by either brain injuries or chemical imbalances and it is associated with ongoing neuronal damage. Some chemical or physical agents have been used in in-vivo models (e.g. picrotoxin (PTX) induced epilepsy, pentylenetetrazol (PTZ) induced epilepsy, maximal electro shock (MES) induced epilepsy and etc.) to induce some kinds of epileptic seizures so far.
* Corresponding author. E-mail address: [email protected] (M.A. Esmekaya).
Due to brain’s limited antioxidant capacity, its high lipid content and higher energy requirement it is highly vulnerable to oxidative stress. The brain makes up about 2% of body mass but consumes 20% of the metabolic oxygen (Juurlink and Paterson,1998). There is equilibrium between the generation of reactive oxygen species (ROS) and the antioxidant activity in healthy neuronal cells, thus the ROS generation in neurons is under homeostatic control. However, when the cells are exposed to chemical or physical agents such as radiation, it may exceed the antioxidant levels resulting in oxidative damage and cause necrotic cell death (Kannan and Jain, 2001). The decreased antioxidant activity makes the brain more susceptible to oxidative stress, which is shown as higher lipid peroxidation. Glutathione (GSH) functions as a major antioxidant and plays a key role in defense against free radicals in the brain. Prolonged depletion of GSH in the brain is associated with oxidative neuronal death (Regan and Guo, 2001). Oxidative damage is emerging as a mechanism that may play an important role in the etiology and progression of epilepsy. The levels of superoxide anions and hydroxyl radicals increase during seizures in new born pigs. The excessive levels of free
http://dx.doi.org/10.1016/j.jchemneu.2016.01.011 0891-0618/ ã 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: M.A. Esmekaya, et al., Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure, J. Chem. Neuroanat. (2016), http://dx.doi.org/10.1016/j.jchemneu.2016.01.011
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radicals may induce epileptic seizure either by inactivation of glutamine synthase or by inhibition of glutamate decarboxylase (Sudha et al., 2011). Nitric oxide (NO) is formed by NO synthase (NOS) following nerve stimulation in neurons and glial cells and transforms Larginine into NO and citrulline. NO is a neurotransmitter molecule in the central nervous system (CNS) and regulates several physiological functions (i.e. noradrenaline, dopamine release and etc.) (Kato et al., 1994). Furthermore, overproduction of NO may play a role in chronic neurological disorders, as many studies suggested a direct relation between NO and neurotoxicity (Brune et al., 1995; Dawson et al., 1993). NO has also been implicated in the pathology of epilepsy (Gonzalez et al., 2000). It plays a key role in the development of brain excitability, including triggering of the seizure activity. NO synthase (NOS) may worsen epileptic activity (Rondouin et al., 1993). It has been showed that NOS inhibitor treatment may inhibit experimentally induced seizures (Osonoe et al., 1994). The toxicity of NO may be increased by superoxide radicals. NO is reacted with superoxide (O 2) to form peroxynitrite (ONOO ) that is an extremely potent cellular oxidant and oxidizes sulfhydryl groups (Clelland et al., 1996). The present study was undertaken to research the effects of cellular phone radio frequency radiation (RFR) on lipid peroxidation, GSH and total NO (NOx) levels on mouse brain during epileptic seizure which was induced by PTZ treatment. 2. Material and methods 2.1. Experimental animals The study was performed on adult female Swiss albino mice (25–35 g) according to the rules established by the Ethical Committee on Animal Research of Gazi University in accordance with national and international laws. All animals were acclimatized to the laboratory conditions for two weeks before the experiment. The animals were maintained under controlled temperature (22 C 1) and humidity (50% 10) conditions with light/dark cycles of 12 h with lights on from 07:00 to 19:00 h and had free access to food and drinking water. At the end of the study, animals were sacrificed by injection of ketamine and xylazine combination. 2.2. PTZ treatment and experimental groups
2.2.3. Group 3: RF on + PTZ treatment + RF on (RF +/+) (n = 9) Each mouse was exposed to RFR for 30 min. Then the mouse was removed from the glass cage, which was still under exposure. After being injected with PTZ, the mouse was returned to the cage, and exposed to RFR for another 30 min. 2.3. RFR exposure setup Global system for mobile (GSM) 900 MHz dual-band mobile phone was used as RFR source. The phone was placed in the center of the standard glass cage which allowed observation and exposure to RFR (Fig. 1). SAR resulting from mobile phone exposure was determined by the SEMCAD-X software. SEMCAD X (Schmid & Partner Engineering AG, Switzerland) commercial 3-D full-wave simulation software, which uses The finite-difference time-domain (FDTD) method, was used for SAR simulations. FDTD method is based on a spatial and temporal discretization of Maxwell’s equations (Yee, 1966). Generic mobile phone model with l/4 monopole antenna which is accepted by the mobile manufacturers forum (MMF) and simplified mouse phantom were used to assess peak SAR values averaged over 10 g of tissue (Beard et al., 2006). The mouse was considered as a non-homogeneous simplified phantom, consisting of a cone and cylinder as shown in Fig. 1. Head was modeled as a cone having the dimensions of minimum diameter 0.6 cm, maximum diameter 3.0 cm and height 3.5 cm. A cylinder having the diameter of 3.0 cm and height of 6.0 cm was used as the trunk of mouse (Lopresto et al., 2007). Numerical simulations were performed on a simplified model of mouse contained in a square shaped glass cage and Generic Mobile Phone located 1 cm below the cage (Fig. 1). The dielectrical properties (relative permittivity-er, electric conductivity-s ) of mouse, cage and generic mobile phone are given in Table 1. Grading mesh algorithm was used in the simulation. The smallest voxel size was 0.2 0.2 0.1 mm3. The largest voxel size 17 mm was used far from the generic mobile phone and mice phantom. The simulation consist of 15.5 million voxels. SAR values were obtained by normalizing antenna input power to 0.250 W. 10 g peak spatial average SARs at 900 MHz were 0.301 W/kg in the head and 0.367 W/kg trunk, respectively. During each RFR exposure (communication mode), the magnitude of electric (E) field and magnetic (B) field strength were
Seizures were induced in female mice by the injection of PTZ (Sigma, USA) dissolved in sterile isotonic saline and administered intraperitoneally (i.p.) at a dose of 60 mg/kg in 0.1 ml (Löscher and Fiedler, 1996; Ossenkopp and Cain, 1991). This is the submaximal dose, between the 50% effective dose (ED50) of 33 mg/kg and the median effective lethal dose (LD50) of 75 mg/kg. These pretreatment times were chosen based on information about their biological activity from the literature and own previous studies (Keskil et al., 2001). 2.2.1. Group 1 (controls): RF off + PTZ treatment + RF off (RF / ) (n = 9) Each mouse was kept in the glass cage under sham exposure for 30 min. Then each mouse was removed from the glass cage and, after being injected with PTZ, returned to the cage under sham exposure for 30 min observation. 2.2.2. Group 2: RF on + PTZ treatment + RF on (RF +/+) (n = 9) Each mouse was exposed to radio frequency radiation (RFR) for 15 min. Then the mouse was removed from the glass cage, which was still under exposure. After being injected with PTZ, the mouse was returned to the cage, and exposed to RFR for another 30 min.
Fig. 1. The exposure setup.
Please cite this article in press as: M.A. Esmekaya, et al., Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure, J. Chem. Neuroanat. (2016), http://dx.doi.org/10.1016/j.jchemneu.2016.01.011
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Component of modelling
er
s (S/m)
Head (grey matter) Trunk (muscle) Glass Plastic chassis of generic mobile phone Monopole antenna covered plastic
52.73 55.03 4.2 4 2.5
0.94 0.94 0 0.04 0.005
measured to be E = 14,310 3,3897 V/m and H = 0,0386 0,012 A/ m respectively. The background electric (E) field and magnetic (B) field strength levels were E = 0,225 0,0325 V/m and B = 0,010 0,001 A/m. 2.4. Measurement of tissue lipid peroxidation (LPO) and GSH levels Tissue samples were frozen immediately in liquid nitrogen and then stored in a 70 C deep-freezer until the assay. Lipid peroxidation was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). Samples were homogenized in ice-cold trichloroacetic acid in a tissue homogenizer (Heideloph Diax 900, Germany). Following centrifugation of the homogenate at 3000 rpm for 10 min (Hermle Z 323 K, Germany), 750 ml of supernatant was added to an equal volume of 0.67% (m/v) thiobarbituric acid and heated at 100 C for 15 min. The absorbance of the samples were measured at 535 nm. Lipid peroxide levels are expressed in terms of Malondialdehyde (MDA) equivalents using an extinction coefficient of 1.56 105 mol 1 cm 1. The GSH levels were determined by Ellman method with some modifications. Briefly, after centrifugation of the homogenates at 3000 rpm for 10 min. 0.5 ml of supernatant was added to 2 ml of 0.3 M Na2HPO4 2 H2O solution. A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml 1% sodium citrate) was added and after mixing, the absorbance at 412 nm was measured using a spectrophotometer (UV 1208, Shimadsu, Japan) at room temperature immediately. The GSH levels were calculated using an extinction coefficient of 13,600 mol–1 cm 1 (Aykaç et al., 1985; Nakamura and Omeya, 2004). 2.5. Determination of NOx (Griess assay) NOx levels were obtained from Elisa reader by vanadium chlorure (VCl3)/Griess assay. Before NOx determination, tissues were homogenized in five volumes of phosphate buffer saline (pH 7) and centrifuged at 2000 g for 5 min. After centrifugation, 0.25 ml 0.3 M NaOH were added to 0.5 ml supernatant. The incubation of the samples for 5 min at room temperature was followed by addition of 0.25 ml of 5% (w/v) ZnSO4 for deproteinization. This mixture was then centrifuged at 3000 g for 20 min and supernatants were used for the assays. Nitrate standard solution was serially diluted and the plates were loaded with samples (100 ml). Then Vanadium III chloride (VCl3) (100 ml) and Griess reagents sulphanilamide (SULF) (50 ml) and N-(1-naphtyl) ethylenediamide dihyrochloride (NEDD) (50 ml) were added to each well. After incubation in 37 C for 45 min, samples were measured at 540 nm using an ELISA reader (Miranda et al., 2001).
set at p < 0.05. Data were analyzed with the statistical package SPSS version 13.0. 3. Results Fig. 2 shows tissue levels of MDA levels in each group. Total brain MDA levels of the mice were significantly higher in both PTZ treated and RFR exposed group than PTZ treated group (p < 0.05). However, tissue levels of MDA were not significantly different between (p > 0.05) RFR exposed groups (Fig. 2). In contrast to brain MDA levels, GSH levels of the mice exhibited a significant reduction following RFR in exposed groups when compared to the control group (p > 0.05). No significant changes were detected between the RFR exposed groups (p > 0.05) (Fig. 3). As seen in Fig. 4, NOx levels were increased significantly in RFR exposed brain tissues of the mice (p < 0.05) as compared to the brain tissues of the mice those only experienced PTZ-induced brief seizures. However, there was no significant difference between the RFR exposed groups (p > 0.05) (Fig. 4). 4. Discussion The results presented in this study provide an evidence that cellular phone radiation may induce oxidative damage in mouse brain in PTZ induced seizure model. The oxidative damage was mediated by increased lipid peroxidation and decreased antioxidant (GSH) levels. We observed significant changes between control and RFR groups in terms of MDA levels. Similarly GSH levels were significantly different in exposed groups than control group. However we observed no significant changes between RFR groups which were exposed to cellular phone radiation at exposure durations of either 15 or 30 min. Similar to our study, researchers have reported increased lipid peroxidation and decreased antioxidant levels in brain tissues of different animals due to RFR exposure. Meral et al. (2007) investigated the effects of 890–915 MHz RFR emitted from cellular phone with a modulation frequency of 217 Hz on oxidant and antioxidant levels in brain tissues of male guinea pigs. They have exposed the animals to RFR for 12 h/day (11-h 45-min stand-by and 15-min speaking mode) for a month and at the end of the study they observed increased MDA level and decreased GSH level and CAT enzyme activity in the brain tissues of RF-exposed guinea pigs. Dasdag et al. (2004) and Sokolovic et al. (2008) [23] reported increased lipid peroxidation in brain tissues of the rats due to RFR
8 7 6 MDA levels nmol/g
Table 1 Dielectric properties for modelling components (mouse, cage and generic mobile phone).
3
5 4 3 2
2.6. Statistical analysis
1
All data were presented as mean values Standard Deviation (SD). Differences among three groups were analyzed by one way analysis of variance (ANOVA). Student’s t test was used for pairwise comparisons among groups. The accepted level of significance was
0 Group I
Group II
Group III
Fig. 2. Mean brain tissue levels of MDA in control (group I) and RFR exposed (group II and III) groups.
Please cite this article in press as: M.A. Esmekaya, et al., Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure, J. Chem. Neuroanat. (2016), http://dx.doi.org/10.1016/j.jchemneu.2016.01.011
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14 12
GSH levels
10 8 6 4 2 0 Group I
Group II
Group III
Fig. 3. Mean brain tissue levels of GSH (mmol/g) in control (group I) and RFR exposed (group II and III) Groups.
exposure. Moreover, some authors like Ilhan et al. (2004)showed that oxidative damage induced by RFR in the brain may be inhibited by antioxidants like Ginkgo Biloba (EGb-761). Due to brain’s high lipid content and higher energy requirement, it is susceptible to oxidative damage more than any other tissues. Brain has an high amount of unsaturated acids and these account for more than 20% of the total fatty acids of the brain. They are accompanied by relatively low levels of antioxidant enzymes. GSH reductase maintains approximately 98% of intracellular GSH in the reduced thiol form, GSH. Moreover it is known that oxidative stress deplates GSH reserves and this kind of depletion may cause mitochondrial damage in brain (Choi, 1993; Floyd et al., 1984; Maellaro et al., 1990). Hereby, the observed reduction in GSH levels in mobile phone radiation exposed mouse brain tissues in our study may be due to increased lipid peroxidation which is a marker of oxidative damage. Some researchers reported a direct linkage between epilepsy and ROS production in some experimental models of epilepsy in brain tissue. Lipid peroxidation level may increase in brain during epileptic seizures (Garey et al., 1994) and the role of free radicals during seizures may be a result of the anticonvulsant activity of antioxidants (Kabuto et al., 1998). Sudha
Acknowledgement EM Field measurements were performed with the devices purchased by a grant from the Gazi University Research Foundation Project No: 01/2003-62. References
30
25
NOx levels (umol/g)
et al. (2001) observed reduced antioxidant levels in blood of epileptic patients. The data obtained from this study also showed that cellular phone radiation leaded to an increase in NOx levels in mouse brain during epileptic seizure. NO may be released by cytotoxic macrophages in brain and controlled with the stimulation by glutamate of cyclic GMP formation in the cerebellum (Hibbs et al., 1987). Some previous studies suggested that NO production is involved in the pathophysiology of epilepsy and it has a significant effect on the PTZ-induced seizure. NO generation is involved in the regulation of seizure threshold by decreasing PTZ-induced seizure threshold (Nidhi et al., 1999). It has also been reported that NO may be responsible in the regulation of cyclooxygenase (COX) enzyme activity. COX is an enzyme that is responsible for the conversion of polyunsaturated substrates such as arachidonic acid (AA, an v-6 PUFA) to prostaglandins which are derived from fatty acids and play a major role as mediators of the inflammatory response (Dubois et al., 1998). Activation of inflammatory signals in microglial cells are considered to be responsible for inflammatory responses in the brain. Boje and Arora (1982) suggested that microglial-produced NO and reactive nitrogen oxides as a neurotoxic agent play a key role in neurodegenerative disease states. These substances may lead to neuronal damage (Gonzalez-Scarano and Baltuch, 1999). NADPH oxidase which generates reactive-oxygen-intermediates in microglial cells transfers an electron from a NADPH molecule to an oxygen molecule and produces superoxide anion (O 2) and its metabolite hydrogen peroxide (H2O2). This may lead to oxidization of proteins and lipids in neighboring cells (Weiss, 1989). In summary, our experiments provided evidence that mobile phone radiation exposure may induce oxidative damage mediated by increased lipid peroxidation and decreased antioxidant level during epileptic activity in mouse brain. The study also gave evidence that NOx levels may be increased due to mobile phone radiation exposure during epileptic seizure. Long term effects of RFR on kindling like models are needed to understand the role of free radicals in RFR induced biological effects better.
20
15
10
5
0 Group I
Group II
Group III
Fig. 4. Mean brain tissue levels of NOx in control (group I) and RFR exposed (group II and III) groups.
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Journal of Chemical Neuroanatomy xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
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Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure Meric Arda Esmekayaa,* , Mehmet Zahid Tuysuza , Arın Tomruka , Ayse G. Cansevena , Engin Yücelb , Zuhal Aktunac , Semih Keskild, Nesrin Seyhana a
Department of Biophysics, Gazi University, Ankara, Turkey Department of Neurosurgery, Baskent University, Alanya Training and Research Hospital, Antalya, TURKEY Department of Medical Pharmacology, Kırıkkale University, Kırıkkale, TURKEY d Department of Neurosurgery, Kırıkkale University, Kırıkkale, TURKEY b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 July 2015 Accepted 28 January 2016 Available online xxx
The objective of the this study was to evaluate the effects of cellular phone radiation on oxidative stress parameters and oxide levels in mouse brain during pentylenetetrazole (PTZ) induced epileptic seizure. Eight weeks old mice were used in the study. Animals were distributed in the following groups: Group I: Control group treated with PTZ, Group II: 15 min cellular phone radiation + PTZ treatment + 30 min cellular phone radiation, Group III: 30 min cellular phone radiation + PTZ treatment + 30 min cellular phone radiation. The RF radiation was produced by a 900 MHz cellular phone. Lipid peroxidation, which is the indicator of oxidative stress was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). The glutathione (GSH) levels were determined by the Ellman method. Tissue total nitric oxide (NOx) levels were obtained using the Griess assay. Lipid peroxidation and NOx levels of brain tissue increased significantly in group II and III compared to group I. On the contrary, GSH levels were significantly lower in group II and III than group I. However, no statistically significant alterations in any of the endpoints were noted between group II and Group III. Overall, the experimental findings demonstrated that cellular phone radiation may increase the oxidative damage and NOx level during epileptic activity in mouse brain. ã 2016 Elsevier B.V. All rights reserved.
Keywords: Seizure Pentylenetetrazole Mice Cell phone Radiation
1. Introduction Epilepsy is one of the most common neurologic disorder in which the patient experiences chronic abnormal bursts of electrical discharge in the brain. About 0.8% of the world’s population suffer from epilepsy. Severe and continuous seizure activities may cause a variety of symptoms such as loss of speech, uncontrollable motor behavior, and/or unusual sensory experiences, complete or partial loss of consciousness in individuals with epilepsy. It may be caused by either brain injuries or chemical imbalances and it is associated with ongoing neuronal damage. Some chemical or physical agents have been used in in-vivo models (e.g. picrotoxin (PTX) induced epilepsy, pentylenetetrazol (PTZ) induced epilepsy, maximal electro shock (MES) induced epilepsy and etc.) to induce some kinds of epileptic seizures so far.
* Corresponding author. E-mail address: [email protected] (M.A. Esmekaya).
Due to brain’s limited antioxidant capacity, its high lipid content and higher energy requirement it is highly vulnerable to oxidative stress. The brain makes up about 2% of body mass but consumes 20% of the metabolic oxygen (Juurlink and Paterson,1998). There is equilibrium between the generation of reactive oxygen species (ROS) and the antioxidant activity in healthy neuronal cells, thus the ROS generation in neurons is under homeostatic control. However, when the cells are exposed to chemical or physical agents such as radiation, it may exceed the antioxidant levels resulting in oxidative damage and cause necrotic cell death (Kannan and Jain, 2001). The decreased antioxidant activity makes the brain more susceptible to oxidative stress, which is shown as higher lipid peroxidation. Glutathione (GSH) functions as a major antioxidant and plays a key role in defense against free radicals in the brain. Prolonged depletion of GSH in the brain is associated with oxidative neuronal death (Regan and Guo, 2001). Oxidative damage is emerging as a mechanism that may play an important role in the etiology and progression of epilepsy. The levels of superoxide anions and hydroxyl radicals increase during seizures in new born pigs. The excessive levels of free
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Please cite this article in press as: M.A. Esmekaya, et al., Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure, J. Chem. Neuroanat. (2016), http://dx.doi.org/10.1016/j.jchemneu.2016.01.011
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radicals may induce epileptic seizure either by inactivation of glutamine synthase or by inhibition of glutamate decarboxylase (Sudha et al., 2011). Nitric oxide (NO) is formed by NO synthase (NOS) following nerve stimulation in neurons and glial cells and transforms Larginine into NO and citrulline. NO is a neurotransmitter molecule in the central nervous system (CNS) and regulates several physiological functions (i.e. noradrenaline, dopamine release and etc.) (Kato et al., 1994). Furthermore, overproduction of NO may play a role in chronic neurological disorders, as many studies suggested a direct relation between NO and neurotoxicity (Brune et al., 1995; Dawson et al., 1993). NO has also been implicated in the pathology of epilepsy (Gonzalez et al., 2000). It plays a key role in the development of brain excitability, including triggering of the seizure activity. NO synthase (NOS) may worsen epileptic activity (Rondouin et al., 1993). It has been showed that NOS inhibitor treatment may inhibit experimentally induced seizures (Osonoe et al., 1994). The toxicity of NO may be increased by superoxide radicals. NO is reacted with superoxide (O 2) to form peroxynitrite (ONOO ) that is an extremely potent cellular oxidant and oxidizes sulfhydryl groups (Clelland et al., 1996). The present study was undertaken to research the effects of cellular phone radio frequency radiation (RFR) on lipid peroxidation, GSH and total NO (NOx) levels on mouse brain during epileptic seizure which was induced by PTZ treatment. 2. Material and methods 2.1. Experimental animals The study was performed on adult female Swiss albino mice (25–35 g) according to the rules established by the Ethical Committee on Animal Research of Gazi University in accordance with national and international laws. All animals were acclimatized to the laboratory conditions for two weeks before the experiment. The animals were maintained under controlled temperature (22 C 1) and humidity (50% 10) conditions with light/dark cycles of 12 h with lights on from 07:00 to 19:00 h and had free access to food and drinking water. At the end of the study, animals were sacrificed by injection of ketamine and xylazine combination. 2.2. PTZ treatment and experimental groups
2.2.3. Group 3: RF on + PTZ treatment + RF on (RF +/+) (n = 9) Each mouse was exposed to RFR for 30 min. Then the mouse was removed from the glass cage, which was still under exposure. After being injected with PTZ, the mouse was returned to the cage, and exposed to RFR for another 30 min. 2.3. RFR exposure setup Global system for mobile (GSM) 900 MHz dual-band mobile phone was used as RFR source. The phone was placed in the center of the standard glass cage which allowed observation and exposure to RFR (Fig. 1). SAR resulting from mobile phone exposure was determined by the SEMCAD-X software. SEMCAD X (Schmid & Partner Engineering AG, Switzerland) commercial 3-D full-wave simulation software, which uses The finite-difference time-domain (FDTD) method, was used for SAR simulations. FDTD method is based on a spatial and temporal discretization of Maxwell’s equations (Yee, 1966). Generic mobile phone model with l/4 monopole antenna which is accepted by the mobile manufacturers forum (MMF) and simplified mouse phantom were used to assess peak SAR values averaged over 10 g of tissue (Beard et al., 2006). The mouse was considered as a non-homogeneous simplified phantom, consisting of a cone and cylinder as shown in Fig. 1. Head was modeled as a cone having the dimensions of minimum diameter 0.6 cm, maximum diameter 3.0 cm and height 3.5 cm. A cylinder having the diameter of 3.0 cm and height of 6.0 cm was used as the trunk of mouse (Lopresto et al., 2007). Numerical simulations were performed on a simplified model of mouse contained in a square shaped glass cage and Generic Mobile Phone located 1 cm below the cage (Fig. 1). The dielectrical properties (relative permittivity-er, electric conductivity-s ) of mouse, cage and generic mobile phone are given in Table 1. Grading mesh algorithm was used in the simulation. The smallest voxel size was 0.2 0.2 0.1 mm3. The largest voxel size 17 mm was used far from the generic mobile phone and mice phantom. The simulation consist of 15.5 million voxels. SAR values were obtained by normalizing antenna input power to 0.250 W. 10 g peak spatial average SARs at 900 MHz were 0.301 W/kg in the head and 0.367 W/kg trunk, respectively. During each RFR exposure (communication mode), the magnitude of electric (E) field and magnetic (B) field strength were
Seizures were induced in female mice by the injection of PTZ (Sigma, USA) dissolved in sterile isotonic saline and administered intraperitoneally (i.p.) at a dose of 60 mg/kg in 0.1 ml (Löscher and Fiedler, 1996; Ossenkopp and Cain, 1991). This is the submaximal dose, between the 50% effective dose (ED50) of 33 mg/kg and the median effective lethal dose (LD50) of 75 mg/kg. These pretreatment times were chosen based on information about their biological activity from the literature and own previous studies (Keskil et al., 2001). 2.2.1. Group 1 (controls): RF off + PTZ treatment + RF off (RF / ) (n = 9) Each mouse was kept in the glass cage under sham exposure for 30 min. Then each mouse was removed from the glass cage and, after being injected with PTZ, returned to the cage under sham exposure for 30 min observation. 2.2.2. Group 2: RF on + PTZ treatment + RF on (RF +/+) (n = 9) Each mouse was exposed to radio frequency radiation (RFR) for 15 min. Then the mouse was removed from the glass cage, which was still under exposure. After being injected with PTZ, the mouse was returned to the cage, and exposed to RFR for another 30 min.
Fig. 1. The exposure setup.
Please cite this article in press as: M.A. Esmekaya, et al., Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure, J. Chem. Neuroanat. (2016), http://dx.doi.org/10.1016/j.jchemneu.2016.01.011
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Component of modelling
er
s (S/m)
Head (grey matter) Trunk (muscle) Glass Plastic chassis of generic mobile phone Monopole antenna covered plastic
52.73 55.03 4.2 4 2.5
0.94 0.94 0 0.04 0.005
measured to be E = 14,310 3,3897 V/m and H = 0,0386 0,012 A/ m respectively. The background electric (E) field and magnetic (B) field strength levels were E = 0,225 0,0325 V/m and B = 0,010 0,001 A/m. 2.4. Measurement of tissue lipid peroxidation (LPO) and GSH levels Tissue samples were frozen immediately in liquid nitrogen and then stored in a 70 C deep-freezer until the assay. Lipid peroxidation was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). Samples were homogenized in ice-cold trichloroacetic acid in a tissue homogenizer (Heideloph Diax 900, Germany). Following centrifugation of the homogenate at 3000 rpm for 10 min (Hermle Z 323 K, Germany), 750 ml of supernatant was added to an equal volume of 0.67% (m/v) thiobarbituric acid and heated at 100 C for 15 min. The absorbance of the samples were measured at 535 nm. Lipid peroxide levels are expressed in terms of Malondialdehyde (MDA) equivalents using an extinction coefficient of 1.56 105 mol 1 cm 1. The GSH levels were determined by Ellman method with some modifications. Briefly, after centrifugation of the homogenates at 3000 rpm for 10 min. 0.5 ml of supernatant was added to 2 ml of 0.3 M Na2HPO4 2 H2O solution. A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml 1% sodium citrate) was added and after mixing, the absorbance at 412 nm was measured using a spectrophotometer (UV 1208, Shimadsu, Japan) at room temperature immediately. The GSH levels were calculated using an extinction coefficient of 13,600 mol–1 cm 1 (Aykaç et al., 1985; Nakamura and Omeya, 2004). 2.5. Determination of NOx (Griess assay) NOx levels were obtained from Elisa reader by vanadium chlorure (VCl3)/Griess assay. Before NOx determination, tissues were homogenized in five volumes of phosphate buffer saline (pH 7) and centrifuged at 2000 g for 5 min. After centrifugation, 0.25 ml 0.3 M NaOH were added to 0.5 ml supernatant. The incubation of the samples for 5 min at room temperature was followed by addition of 0.25 ml of 5% (w/v) ZnSO4 for deproteinization. This mixture was then centrifuged at 3000 g for 20 min and supernatants were used for the assays. Nitrate standard solution was serially diluted and the plates were loaded with samples (100 ml). Then Vanadium III chloride (VCl3) (100 ml) and Griess reagents sulphanilamide (SULF) (50 ml) and N-(1-naphtyl) ethylenediamide dihyrochloride (NEDD) (50 ml) were added to each well. After incubation in 37 C for 45 min, samples were measured at 540 nm using an ELISA reader (Miranda et al., 2001).
set at p < 0.05. Data were analyzed with the statistical package SPSS version 13.0. 3. Results Fig. 2 shows tissue levels of MDA levels in each group. Total brain MDA levels of the mice were significantly higher in both PTZ treated and RFR exposed group than PTZ treated group (p < 0.05). However, tissue levels of MDA were not significantly different between (p > 0.05) RFR exposed groups (Fig. 2). In contrast to brain MDA levels, GSH levels of the mice exhibited a significant reduction following RFR in exposed groups when compared to the control group (p > 0.05). No significant changes were detected between the RFR exposed groups (p > 0.05) (Fig. 3). As seen in Fig. 4, NOx levels were increased significantly in RFR exposed brain tissues of the mice (p < 0.05) as compared to the brain tissues of the mice those only experienced PTZ-induced brief seizures. However, there was no significant difference between the RFR exposed groups (p > 0.05) (Fig. 4). 4. Discussion The results presented in this study provide an evidence that cellular phone radiation may induce oxidative damage in mouse brain in PTZ induced seizure model. The oxidative damage was mediated by increased lipid peroxidation and decreased antioxidant (GSH) levels. We observed significant changes between control and RFR groups in terms of MDA levels. Similarly GSH levels were significantly different in exposed groups than control group. However we observed no significant changes between RFR groups which were exposed to cellular phone radiation at exposure durations of either 15 or 30 min. Similar to our study, researchers have reported increased lipid peroxidation and decreased antioxidant levels in brain tissues of different animals due to RFR exposure. Meral et al. (2007) investigated the effects of 890–915 MHz RFR emitted from cellular phone with a modulation frequency of 217 Hz on oxidant and antioxidant levels in brain tissues of male guinea pigs. They have exposed the animals to RFR for 12 h/day (11-h 45-min stand-by and 15-min speaking mode) for a month and at the end of the study they observed increased MDA level and decreased GSH level and CAT enzyme activity in the brain tissues of RF-exposed guinea pigs. Dasdag et al. (2004) and Sokolovic et al. (2008) [23] reported increased lipid peroxidation in brain tissues of the rats due to RFR
8 7 6 MDA levels nmol/g
Table 1 Dielectric properties for modelling components (mouse, cage and generic mobile phone).
3
5 4 3 2
2.6. Statistical analysis
1
All data were presented as mean values Standard Deviation (SD). Differences among three groups were analyzed by one way analysis of variance (ANOVA). Student’s t test was used for pairwise comparisons among groups. The accepted level of significance was
0 Group I
Group II
Group III
Fig. 2. Mean brain tissue levels of MDA in control (group I) and RFR exposed (group II and III) groups.
Please cite this article in press as: M.A. Esmekaya, et al., Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure, J. Chem. Neuroanat. (2016), http://dx.doi.org/10.1016/j.jchemneu.2016.01.011
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14 12
GSH levels
10 8 6 4 2 0 Group I
Group II
Group III
Fig. 3. Mean brain tissue levels of GSH (mmol/g) in control (group I) and RFR exposed (group II and III) Groups.
exposure. Moreover, some authors like Ilhan et al. (2004)showed that oxidative damage induced by RFR in the brain may be inhibited by antioxidants like Ginkgo Biloba (EGb-761). Due to brain’s high lipid content and higher energy requirement, it is susceptible to oxidative damage more than any other tissues. Brain has an high amount of unsaturated acids and these account for more than 20% of the total fatty acids of the brain. They are accompanied by relatively low levels of antioxidant enzymes. GSH reductase maintains approximately 98% of intracellular GSH in the reduced thiol form, GSH. Moreover it is known that oxidative stress deplates GSH reserves and this kind of depletion may cause mitochondrial damage in brain (Choi, 1993; Floyd et al., 1984; Maellaro et al., 1990). Hereby, the observed reduction in GSH levels in mobile phone radiation exposed mouse brain tissues in our study may be due to increased lipid peroxidation which is a marker of oxidative damage. Some researchers reported a direct linkage between epilepsy and ROS production in some experimental models of epilepsy in brain tissue. Lipid peroxidation level may increase in brain during epileptic seizures (Garey et al., 1994) and the role of free radicals during seizures may be a result of the anticonvulsant activity of antioxidants (Kabuto et al., 1998). Sudha
Acknowledgement EM Field measurements were performed with the devices purchased by a grant from the Gazi University Research Foundation Project No: 01/2003-62. References
30
25
NOx levels (umol/g)
et al. (2001) observed reduced antioxidant levels in blood of epileptic patients. The data obtained from this study also showed that cellular phone radiation leaded to an increase in NOx levels in mouse brain during epileptic seizure. NO may be released by cytotoxic macrophages in brain and controlled with the stimulation by glutamate of cyclic GMP formation in the cerebellum (Hibbs et al., 1987). Some previous studies suggested that NO production is involved in the pathophysiology of epilepsy and it has a significant effect on the PTZ-induced seizure. NO generation is involved in the regulation of seizure threshold by decreasing PTZ-induced seizure threshold (Nidhi et al., 1999). It has also been reported that NO may be responsible in the regulation of cyclooxygenase (COX) enzyme activity. COX is an enzyme that is responsible for the conversion of polyunsaturated substrates such as arachidonic acid (AA, an v-6 PUFA) to prostaglandins which are derived from fatty acids and play a major role as mediators of the inflammatory response (Dubois et al., 1998). Activation of inflammatory signals in microglial cells are considered to be responsible for inflammatory responses in the brain. Boje and Arora (1982) suggested that microglial-produced NO and reactive nitrogen oxides as a neurotoxic agent play a key role in neurodegenerative disease states. These substances may lead to neuronal damage (Gonzalez-Scarano and Baltuch, 1999). NADPH oxidase which generates reactive-oxygen-intermediates in microglial cells transfers an electron from a NADPH molecule to an oxygen molecule and produces superoxide anion (O 2) and its metabolite hydrogen peroxide (H2O2). This may lead to oxidization of proteins and lipids in neighboring cells (Weiss, 1989). In summary, our experiments provided evidence that mobile phone radiation exposure may induce oxidative damage mediated by increased lipid peroxidation and decreased antioxidant level during epileptic activity in mouse brain. The study also gave evidence that NOx levels may be increased due to mobile phone radiation exposure during epileptic seizure. Long term effects of RFR on kindling like models are needed to understand the role of free radicals in RFR induced biological effects better.
20
15
10
5
0 Group I
Group II
Group III
Fig. 4. Mean brain tissue levels of NOx in control (group I) and RFR exposed (group II and III) groups.
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