EFFECT OF LONG TERM MOBILE PHONE EXPOSURE ON OXIDATIVE- ANTIOXIDATIVE PROCESSES AND NITRIC OXIDE IN RATS

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1 Articles MB EFFECT OF LONG TERM MOBILE PHONE EXPOSURE ON OXIDATIVE- ANTIOXIDATIVE PROCESSES AND NITRIC OXIDE IN RATS S. Dasdag 1, H.M. Bilgin 2, M.Z. Akdag 1, H. Celik 3, F. Aksen 1 University of Dicle, Faculty of Medicine, Department of Biophysics, Diyarbakir, Turkey 1 University of Dicle, Faculty of Medicine, Department of Physiology, Diyarbakir, Turkey 2 University of Harran, Faculty of Medicine, Department of Biochemistry, Urfa, Turkey 3 Correspondence to: Suleyman Dasdag dasdag@dicle.edu.tr ABSTRACT The effects of radiation emitted from cellular phones on humans are an emerging area of investigation. The previous studies have shown the role of oxidative stress and NO at radiofrequency exposed rats. Therefore, the purpose of this study was to investigate the effect of microwave /radiofrequency emitted from mobile phones and its possible oxidative damage. Wistar Albino rats were divided into three groups as exposure, sham and cage control. Rats in exposure group were exposed to 900 MHz microwave radiation ( mw / cm 2 ) in a carousel for 2 hours/7 days in a week during ten months. The same process was applied to sham group but the generator was turned off. Rats in cage control group were kept in cage only during the study. The levels of catalase (CAT), myeloperoxidase (MPO), malondialdehyde (MDA), total antioxidant capacity (TAC), total oxidant status (TOS), oxidative stress index (OSI) in liver and nitric oxide (NO) levels in serum were determined to demonstrate the role of oxidative mechanisms. The increase of nitric oxide levels in exposure and sham groups were found significant compared to cage control group (p<0.01, p<0.05). Although serum NO levels increased in exposed rats, the difference between exposure and sham groups was not significant (p>0.05). MDA and TOS levels in liver tissue were found higher in exposed group compared to sham and cage control group (p<0.05). However, no significant alterations were found in other endpoints such as CAT, MPO, TAC and OSI. In this study we showed that 900 MHz radiofrequency radiation emitted from mobile phones exhibited an increase on MDA and TOS levels. Therefore, RF radiation emitted from GSM cellular phone may play a role to induce oxidative damage by increasing lipid peroxidation and oxidative stress. Keywords: mobile phones, nitric oxide, oxidative stress, lipid peroxidation Introduction Public and scientific awareness of questions about cell phone safety has been increased greatly in the last few years by media reports of potential adverse health effects for humans exposed to radiation emitted from cellular phones. Although some epidemiologic research was conducted several decades ago on radiofrequency (RF) fields in occupational settings, in general the effects of RF in humans are an emerging area of investigation. Furthermore, the results of studies about mobile phone risks have received widespread public attention but their interpretation is not straightforward because of methodological difficulties (1). Reactive oxygen species (ROS) induced by oxidative stress may be an important factor in tissue injury resulted from radiation. Therefore, if ROS are not scavenged, these species may lead to widespread lipid, protein and DNA damage (16). Several antioxidant systems have different activities to protect tissues. SOD, the first line of defense against ROS, catalyzes the dismutation of the superoxide anion into hydrogen peroxide. Hydrogen peroxide can then be transformed into H 2 O and by CAT. In general, plasma lipid peroxidation (LPO) was found to be increased because of the reduced antioxidant defense system (27). Malondialdehyde (MDA) is the breakdown product of the major chain reactions leading to oxidation of polyunsaturated fatty acids and thus serves as a reliable marker of oxidative stress mediated lipid peroxidation in tissues (28). Myeloperoxidase (MPO) is used as a standard oxidative parameter to evaluate neutrophil activation in tissues and has been involved in the pathogenesis of several diseases through excessive production of ROS (3). The number of different antioxidant components in tissues makes it relatively difficult to measure each antioxidant component separately. In addition, since there is cooperation between various antioxidants, looking at one in isolation from the rest may not accurately reflect their combined action. Therefore, the measurement of total antioxidant capacity (TAC) and total oxidant status (TOS) developed by Erel (10, 11) seems to represent a suitable biochemical parameter for evaluating the overall oxidant and antioxidant status resulting from antioxidant intake or production and their consumption by the increasing levels of oxidative stress. The interaction mechanisms of RF radiation emitted by mobile phone on liver tissue is not well understood, although there are some studies, which use 900 MHz RF radiation for 7 days to 3 months (30 min/day to 1 h/day), in relation to lipid peroxidation and free radical formation (14, 23, 25). Previous studies suggested that RF radiation (900 MHz, 945 MHz and 1800 MHz, 30 min to 7 h a day for 8 to 60 days) can induce 992 Biotechnol. & Biotechnol. Eq. 22/2008/4

2 oxidative damage by increasing MDA and changing antioxidant enzyme activities such as CAT and SOD (2, 17, 21, 24, 33). Moustafa et al. (19) also indicated that acute exposure (1, 2 and 4 h) to radiofrequency fields of commercially available cellular phones may modulate the oxidative stress of free radicals. However, other studies reported that 834 MHz, MHz RF radiation (20 min/day-7.5 h/day for 6-30 days exposure time) do not affect lipid peroxidation and free radical formation (6, 12). These authors indicated that more tests using a longer period of exposure are necessary to ensure that there is no health risk associated with the use of mobile phones. Nitric oxide (NO) has been demonstrated to play a role in a variety of biological processes including neurotransmission, immune defense and the regulation of cell death (apoptosis). It is also recognized as a free radical which is generated in cells from L-arginine by nitric oxide synthase (NOS). Exposure to EMF released by mobile phones (900 MHz, 30 min, 30 min/ day for 2 weeks) has been shown to increase NO levels in the sinus and nasal mucosa (26, 32). Radiation emitted from 900 MHz (exposure time: 7 days to 3 months (30 min/day to 1 h/ day) mobile phone was shown to increase NO levels in heart, renal tissue and brain (14, 23, 25). On the other hand, nitric oxide levels were used as a marker of retinal oxidative stress in rats following the use of EMR (30 min/day for 60 days) emitted from 900 MHz mobile phone and the authors found that the retinal levels of NO and MDA were increased (24). On the contrary of the studies mentioned above, a significant decrease in serum NO levels after exposure to radiation emitted from mobile phones (900 MHz, 30 min/day for 7 days) was found in another study (15). However, the authors stated that electromagnetic radiation may destroy NO by generation of superoxide anion and/or radiation emitted from mobile phones which causes a reduction in production of NO. Biochemical mediated MW (microwave)-induced oxidative stress is probably detoxified in human liver, although hepatoxicity rarely occurs (17). Since very little is known about the mechanism of RF-induced liver damage, determining the activities of TAC, TOS, MDA, CAT and MPO in liver tissue may help to explain free radical formation after RF-induced toxicity. The present study was intended to determine the oxidative damage, LPO activation in liver tissue and NO alterations in rats exposed to radiation emitted from mobile phones. Materials and Methods Subjects and animal care The experiments were performed on thirty one male Wistar Albino rats obtained from Medical Science Application and Research Center of Dicle University, aged 4 months at the beginning of the study, weighing 267 g (± 15 g), and fed with standard pelleted food (TAVAS Inc. Adana, Turkey). The rats were divided into three groups: exposure (n:14), sham (n:7) and cage control (n:10). The animals were kept in 14/10h light/ dark environment at constant temperature of 22 ± 3 C, 45 ± Biotechnol. & Biotechnol. Eq. 22/2008/4 10% humidity. This protocol was approved by the local ethics committee. Production and measurement of radiation 900 MHz experimental phone exposure generator (GSM Simulator 900PM10 type Everest Comp., Adapazarı, Turkey) was used to expose rats. Power output of the generator was fixed to 2W during the study to simulate maximum output of mobile phones. Antenna of the generator was placed at the center of Plexiglas carousel to provide ideal exposure (Fig. 1). Distance of antenna to head of the rats was 1.5 cm. Power Density inside Plexiglas carousel cages was measured by EMR 300 (NARDA, Pfullingen, Germany). Average power density at the center of the carousel cages was measured as mw / cm 2. However, average power density at the area of rat tails was measured as mw / cm 2. Exposure of rats The rats were confined in a Plexiglas carousel (Fig. 1). The heads of the rats in the carousel were exposed to 900 MHz microwave exposure. For the experimental group, rats were exposed to radiation emitted from the generator for 2 hours/ day (7 days in a week) during 10 months. The sham group was placed into the carousel and same procedure was applied but the generator was turned off. Rats in cage control were only kept in a cage during the study. After 10 months of exposure periods, blood of the animals was collected by cardiac puncture under ketamine anesthesia (100 mg/kg, intramuscularly) to measure the levels of NO. Fig. 1. Exposure System Determination of NO Since NO is a very labile molecule, its direct measurement in the biological samples is very difficult. Therefore, the stable oxidation and products of NO, nitrite (N ) and nitrate (NO 3 ) can be readily measured in biological fluids and have been used in vitro and in vivo as indicators of NO production. Serum nitrite levels were measured by Griess reaction (5). Reduction of nitrate to nitrite was accomplished by catalytic reaction using cadmium. Absorbance of this complex was measured at 545 nm. A standard curve was established with a set of serial 993

3 dilutions ( mol/l) of sodium nitrite. Linear regression was made by using the peak area from the nitrite standard. The resulting equation was then used to calculate the unknown sample concentrations. Results were expressed as micromoles per liter serum. Tissue Sampling and Homogenization On the last day of the study, immediately after the euthanization, liver tissues of rats were removed to determine the levels of CAT, MDA, TAC, TOS, OSI, MPO. Before biochemical assays, all tissues were weighed and placed in empty glass tubes. 10 ml of 140 mm KCl solution per 1 gram of tissue were added to each tube containing tissue samples, then the tissues were homogenized in a motor-driven homogenizer. The homogenate was centrifuged at 2800 g for 10 min at 4 C, placed in labeled vials and stored deep-frozen at -80 C. Microprotein level was measured by Lowry method (18). The determination of myeloperoxidase The method described by Wei and Frenkel (30) was used for tissue MPO activity assay, which is a lysosomal oxidative enzyme that is found in WBC. Data are expressed as U/g protein. Measurement of total antioxidative capacity The TAC levels of all tissues were measured using a novel automated colorimetric measurement method developed by Erel (10, 11). In this method, the hydroxyl radical, the most potent biological radical, is produced by the Fenton reaction and reacts with the colorless substrate O-dianisidine to produce the dianisyl radical, which is bright yellowish-brown in color. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals present in the reaction mix are suppressed by the antioxidant components of all the homogenates, preventing the color change and thereby providing an effective measure of the total antioxidant capacity of the plasma. The assay has excellent precision values, which are lower than 3%. The results are expressed as µmol Trolox equiv./gr protein. Measurement of total oxidant status TOS levels of all tissues were determined using a novel automated measurement method, developed by Erel (9). In this method, oxidants present in the sample oxidize the ferrous iono-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide, and the results are expressed in terms of micromolar hydrogen peroxide equivalent per g protein (µmol H 2 equiv/gr protein). Oxidative stress index The percent ratio of the TOS to the TAC gave the oxidative stress index (OSI), an indicator of the degree of oxidative stress (9). The OSI value was calculated according to the formula: OSI=[(TOS, µmol H 2 Eqiv./gr protein) / (TAC, µmol Trolox equiv./gr protein)]. Determination of catalase activity Catalase (CAT) activity in supernatant of tissues was assayed by a method described by Goth (13). 0.2 ml homogenate was incubated in 1.0 ml substrate (65 μmol per H 2 in 60 mmol/l sodium potassium phosphate buffer, ph 7.4) at 37 C for 60 s. The enzymatic reaction was stopped with 1.0 ml of 32.4 mmol/l ammonium molybdate ((NH 4 ) 6 Mo 7 4.4H 2 O) and the yellow complex of molybdate. H 2 was measured at 405 nm against blank 3. Homogenate CAT activity is linear up to 100 ku/l. When the CAT activity exceeded 100 ku/l, the homogenate was diluted with the phosphate buffer (2- to 10- fold) and the assay was repeated. One unit of CAT decomposes 1 μmol of H 2 /L min under these conditions. Results were expressed as ku/gr protein. Determination of malondialdehyde levels Lipid peroxidation (LPO) of all tissues was evaluated by the fluorometric method based on the reaction between MDA and thiobarbutiric acid (4). Briefly, 50 μl of supernatant of tissues was added to 1 ml of 10 mmol/l diethylthiobarbutiric acid (DETBA) reagent in a phosphate buffer (0.1mol/L, ph 3). The mixture was mixed for 5 sec and incubated for 60 min at 95 C. Samples were placed on ice for 5 min, and then 5 ml of butanol was added. The mixture was shaken for 1 min to extract the DETBA-MDA adduct and then centrifuged at 1500 g for 10 min at 4 C. Fluorescence of the butanol extract was measured at an excitation wavelength of 539 nm and emission wavelength of 553 nm. 1,1,3,3,Tetraethoxypropane (Sigma) was used as the standard solution and the values were presented as μmol/gr protein. Statistical Analysis All experiments were performed comparing 900 MHz exposed samples with the unexposed sham and cage control samples. Mean values and standard deviations were calculated, and any statistical significance of the differences between exposed samples and controls was evaluated. A computer program (SPSS 10.0; SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Data were analyzed by Kruskal-Wallis one-way analysis of variance ANOVA on ranks and post hoc multiple comparisons Dunnett test. Pearson correlation coefficients were used to assess the relationship between NO parameters. All hypotheses were tested by using a criterion level of p=0.05. Results and Discussion As seen in Table 1, serum NO levels of exposure group were found higher than those of the sham and cage control groups. The increase observed in nitric oxide levels of exposed rats compared to the cage control rats was found statistically significant (p<0.01) while the increase of NO in exposed rats was not significant in comparison to sham group (p>0.05). 994 Biotechnol. & Biotechnol. Eq. 22/2008/4

4 However, NO levels of sham group were higher than the cage control group (p<0.05). No significant correlation was also determined between the exposed and sham groups (r = ; p>0.05). However, there was a significant correlation between exposed and cage control groups (r = 0.612; p<0.01). This result may suggest that increased NO levels in sham group may be due to a little stress during sham exposure. Table 1 Statistical analyzes of nitric oxide levels. Results are expressed as mean ± standard deviation. Groups NO (Mean±S.D) Compared groups P Exposed group ±5.044 Exposed - Sham >0.05 Sham ±3.426 Sham-Cage control Cage Control 7.642±2.880 Exposed- Cage control <0.05 <0.01 The increase of MDA levels, an index of lipid peroxidation, in liver tissue of exposed group were determined statistically significant in comparison to sham and cage control groups (p<0.05) (Table 2). TOS levels in exposure group were also found higher than sham and cage control groups (p<0.05). No difference was found between the groups in terms of CAT, MPO, TAC and OSI parameters (p > 0.05). The use of mobile phones operating in the 900 and 1800 MHz frequency bands is widespread and increasing rapidly. The question of whether microwave (MW) at these frequencies could cause some adverse biological effects is of public interest (20). Mobile phone induction of free radical formation in some tissues has been reported (14, 15, 23). In biological systems affected by magnetic fields, the mechanisms of tissue damage are thought to involve ROS (16). The close proximity of the antenna of mobile phone to the abdominal organs has raised concerns about the biological interactions between RF radiation and the liver. The direct biological effects of exposure to 900- MHz RF radiation have not been studied extensively on liver tissue. We found that MDA and TOS levels were increased compared to sham and cage-control groups in rats liver after long-term exposure to 900 MHz RF radiation. Therefore, it was suggested that ROS were generated under the experimental conditions in liver tissue. The increasing MDA levels in our study seem to support the results of some previously mentioned studies about RF exposure emitted from cellular phone (2, 14, 17, 19, 23, 25, 33). It can be suggested that long-term 900 MHz RF exposure can affect LPO in rats. Numerous studies demonstrated that MDA and NO levels increased while SOD and CAT activities decreased in some tissues of EMR and RF exposed animals (14, 23, 25). In another study, (15), serum SOD activity increased and NO levels decreased in EMR-exposed rabbits compared to the sham group while MDA levels did not show a significant change in both groups. It was reported that the levels of CAT and LPO in liver tissue increased at 1800 MHz MW exposed group compared with the control group (17). Yürekli et al. (33) reported that MDA levels increased and GSH (reduced glutathione) concentration decreased significantly at 900 MHz RF radiation. Ferreira et al. (12) suggested that acute ultra high frequency electromagnetic field ( MHz) exposure is not able to produce detectable oxidative stress in rats from any age tested. Balci et al. (2) found that MDA level and CAT activity significantly increased in the mobile phone group compared with the mobile phone plus vitamin C group and with the control in rats after exposure to 900 MHz RF radiation in corneal tissue, while it was demonstrated that MDA levels of renal tissue increased but SOD and CAT activities were reduced after exposure to 900 MHz RF radiation (21). Moustafa et al. (19) showed that the plasma level of lipid peroxide in healthy adult male volunteers was significantly increased after 1, 2 and 4 h of exposure to radiofrequency fields of the cellular phone in standby position. They indicated that acute exposure to radiofrequency fields of commercially available cellular phones may modulate the oxidative stress of free radicals by enhancing lipid peroxidation and reducing the activation of SOD. MDA is a major oxidative degradation product of membrane unsaturated fatty acid and has been shown to be biologically Table 2 Liver miyeloperoxidase (MPO), catalase (CAT), malondialdehyde (MDA), total antioxidant capacity (TAC), total oxidant status (TOS) and Oxidant Stress Index (OSI) in rats exposed, sham and cage control group. Parameters Exposed group Sham Cage Control MPO (U/gr protein) 3.015± ± ±1.696 CAT (ku/gr protein) ± ± ± MDA (μmol/gr protein) 4.400±0.875 a.b 3.439± ±0.664 TAC (µmoltroloxeqv./gr protein) ± ± ±0.556 TOS (µmolh 2 equiv/grprotein) 7.714±1.415 a.b 5.994± ±0.876 OSI (AU) 0.612± ± ± The values represent the mean ±S.D a Statistically significant, compares sham group (p<0.05). b Statistically significant, compares cage control group (p<0.05). Biotechnol. & Biotechnol. Eq. 22/2008/4 995

5 active with hepatotoxic and genotoxic properties (17). In the present study, exposure to 900 MHz RF radiation affected lipid peroxidation in liver. This could be due to the cytochrome P 450 -mediated metabolism of the organic hydroperoxide to active alkoxyl radicals that initiated LPO and led to liver damage as suggested by Koyu et al. (17). This metabolic pathway can increase cellular free radicals, which may attack phospholipids, proteins, and nucleic acid. Thus antioxidant activity and the inhibition of ROS generation are important in protecting the liver from MW-induced damage. According to the MDA results, we can state that the increased MDA levels are related to the 900 MHz RF radiation. It may be suggested that 900 MHz RF radiation can induce lipid peroxidation by affecting metabolic pathway in rats liver. We may indicate that long term 900 MHz RF radiation exposure may modulate the oxidative stress of free radicals by enhancing lipid peroxidation as suggested by Moustafa et al. (19). TOS levels of our study are in agreement with the data mentioned above which indicate that RF radiation emitted from cellular phone can induce oxidative stress or damage (2, 14, 17, 23, 25, 33).The significant difference in relation to TOS levels between exposure group and sham group demonstrate that free radical formation is altered after long-term 900 MHz RF exposure. Such long-term 900 MHz RF exposure can be explained with an increase of the oxidative stress and induction of oxidative damage. NO plays an important role for the biological systems but only few data about NO levels in human beings or animals after exposure to mobile phone have been published. Most of the studies related to NO and mobile phones are based on short term effects and these studies have been usually stated that radiation emitted from mobile phones alter the NO levels. Ozguner et al. (23) exposed rats to 900 MHz radiation emitted from mobile phone for 30 min/day during 10 days and found that mobile phone exposure increased NO levels thus demonstrating the role of oxidative mechanisms in mobile phone-induced heart tissue damage. It was reported that exposure to EMF produced by a mobile phone may affect nasal NO level (26). Yariktas and colleagues (32) exposed rats to 900 MHz radiation for 30 minutes, five days a week, during two weeks. They stated that increased NO levels in the sinus and nasal mucosa may act as a defense mechanism and presumably related to tissue damage. Meanwhile, in a previous study (7) we exposed rats to 900 MHz microwave radiation for 2 hours/day during 10 months and did not find any pathology in nasal mucosa. Ilhan et al. (14) exposed rats to 900 MHz radiation emitted from mobile phones for 1 hour/day during a seven day period and demonstrated that mobile phone exposure increased NO levels in brain, claiming the role of reactive oxygen species in the mechanism that has been proposed to explain the biological side effects of mobile phones in brain tissue. Ozguner et al. (23) exposed rats to 900MHz radiation for 30 min/day during a 3 month period and showed that mobile phone exposure increased NO levels in renal tissue. They stated that an oxidative stress-induced impairment in renal tissue is available after mobile phone exposure. In another study, NO level was increased in retinal tissue of rats that were exposed to 900MHz radiation for 30 min/day for two months and it was stated that mobile phone exposure can cause retinal oxidative stress after long-term exposure (24). On the other hand, decreased NO levels were found in rats exposed to 900 MHz radiation emitted from mobile phones for 30 min /day, for 7 days (15). However, they claimed that this finding may indicate the possible role of increased oxidative stress in the pathophysiology of adverse effect of radiation emitted from mobile phones and decreased NO levels may also suggest a probable role of NO in the adverse effect. In our study, the duration period of mobile phone exposure was 2 hr/day for 10 months so this was a longer period of time than in other studies. Therefore, the aim of the study was to investigate the effect of long term and moderate exposure of 900 MHz radiation, which is emitted from mobile phones. In the present study measurements of NO were held in serum while other studies used tissue samples. Our results demonstrated that NO levels were found to be increased following the 900 MHz exposure. The increased NO levels in the exposure and sham groups may be related with the stress conditions formed by the radiation in order to scavenge - and may act as a defense mechanism presumably related to tissue damage. A connection of NO with the stress response has been proposed (23, 29, 31). Furthermore, mobile phones might induce vasodilatation and increase the production of NO as stated by Paredi et al (26) and Yariktas et al (32). The articles mentioned above mostly support our results. On the other hand, there is not an agreement between our study and Irmak et al (15). We believe that the controversy between these studies can be sourced from exposure circumstances as they exposed rats to the radiation emitted from mobile phones directly to observe the effect of mobile phone on NO. However, it is recently known that the researchers can not standardize experimental exposure circumstances by using mobile phones. The investigators can not fix the power density of mobile phones because of some technical features between mobile phones and base stations. Therefore, the power output given in their article is not fixed. In addition, Irmak et al (15) designed their study with only exposure and sham groups but without a cage control group. Finally, we believe that the controversy between the studies can be originated from the experimental design. Conclusions The increase observed in nitric oxide levels of exposed rats compared to the cage control rats was statistically significant while the increase of NO in exposed rats was not found significant compared to the sham group. However, NO levels of sham group were higher than the cage control group. These results demonstrate that the increase in NO levels is induced by stress as indicated in previous studies. Our results show that 2h/day (7days/week) exposure of 900 MHz radiation over a period of 10 months exhibited an increase on MDA and TOS levels in liver tissue. Therefore, RF 996 Biotechnol. & Biotechnol. Eq. 22/2008/4

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