cell biochemistry and function Cell Biochem Funct 2002; 20: 279–283. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cbf.976
Effects of electromagnetic radiation from a cellular telephone on the oxidant and antioxidant levels in rabbits M. Kemal Irmak1, Ersin Fadllo glu2, Mukaddes Gu¨lec¸1, Hasan Erdogan2, Murat Yagmurca3 ,1 ¨ and Omer Akyol* 1
Department of Biochemistry, Faculty of Medicine, Inonu University, Malatya, Turkey Department of Physiology, Faculty of Medicine, Inonu University, Malatya, Turkey 3 Department of Histology and Embryology, Faculty of Medicine, Inonu University, Malatya, Turkey 2
The number of reports on the effects induced by electromagnetic radiation (EMR) in various cellular systems is still increasing. Until now no satisfactory mechanism has been proposed to explain the biological effects of this radiation. Oxygen free radicals may play a role in mechanisms of adverse effects of EMR. This study was undertaken to investigate the influence of electromagnetic radiation of a digital GSM mobile telephone (900 MHz) on oxidant and antioxidant levels in rabbits. Adenosine deaminase, xanthine oxidase, catalase, myeloperoxidase, superoxide dismutase (SOD) and glutathione peroxidase activities as well as nitric oxide (NO) and malondialdehyde levels were measured in sera and brains of EMR-exposed and sham-exposed rabbits. Serum SOD activity increased, and serum NO levels decreased in EMR-exposed animals compared to the sham group. Other parameters were not changed in either group. This finding may indicate the possible role of increased oxidative stress in the pathophysiology of adverse effect of EMR. Decreased NO levels may also suggest a probable role of NO in the adverse effect. Copyright # 2002 John Wiley & Sons, Ltd. key words — cellphone; oxidant; antioxidant
INTRODUCTION Cellular telephones and their base stations produce electromagnetic radiation (EMR), the effect of which on the body depends on its frequency and power. Frequency is the rate at which electromagnetic fields change direction, and is measured in Hertz (Hz). One megahertz (MHz) is one million cycles per second. Analogue telephones use frequencies between 800 and 900 MHz; and digital telephones use frequencies between 1850 and 1990 MHz, while microwave ovens use a frequency of 2450 MHz. Today’s mobile telephones, with a total power output of about 2 W, are estimated to produce insignificant local heating, which is unlikely to produce any deleterious effects.
* Correspondence to: Dr O. Akyol, Inonu University, Faculty of Medicine, Department of Biochemistry, 44069, Malatya, Turkey. Tel: þ90 422 341 06 60/3304. Fax: þ90 422 341 0728. E-mail:
[email protected]
Copyright # 2002 John Wiley & Sons, Ltd.
Recent research from many countries suggests, however, that there are ‘non-thermal’ effects on living tissue, ranging from changes in the permeability of the blood–brain barrier, to changes in encephalogram and blood pressure.1 The greatest mystery about the nonthermal effects is their lack of a theoretical basis. Biological systems may interact resonantly with EMR but there is as yet no robust evidence to support this suggestion. Reactive oxygen species (ROS) have been implicated in tissue injury. The main ROS that have to be considered are superoxide anion (O 2 ), which is predominantly generated by the mitochondria; hydrogen peroxide (H2O2) produced from O 2 by the action of superoxide dismutase (SOD), and peroxynitrite (ONOO), generated by the reaction of O 2 with nitric oxide (NO). Many prooxidant enzymes are known to participate in the production of ROS (Figure 1). Based on oxidant products, three major classes of prooxidant enzymes can be designated: (1) nitric oxide synthases (NOS) produce NO as an oxidant product; (2) Received 9 October 2001 Accepted 6 December 2001
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m. k. irmak et al. Levels of these endogenous indices of oxidative stress have not yet been reported in EMR-exposed animals. Therefore, the aims of the current study were (i) to investigate whether EMR induces an imbalance in oxidants and antioxidants in rabbits, (ii) to investigate the role of NO in the EMR exposure, and (iii) to assess the possible relationships between MDA, NO, adenosine deaminase (ADA), XO, MPO, SOD, CAT and GSH-Px in EMR-exposed rabbits. MATERIALS AND METHODS
Figure 1. Shematic representation of the relationships between free oxygen radical formation, antioxidant system and lipid peroxidation. ADA, adenosine deaminase; XO, xanthine oxidase; COX, cyclooxygenases; MPO, myeloperoxidase; O 2 , superoxide anion radical; HOCl, hypochlorous acid; SOD, superoxide dismutase; MAO, monoamine oxidase; CAT, catalase; H2O2, hydrogen peroxide; GSH-Px, glutathione peroxidase; OH, hydroxyl ion; OH, hydroxyl radical (the most potent oxygen free radical); NOS, nitric oxide synthases; NO, nitric oxide radical; ONOO, peroxynitrite; MDA, malondialdehyde; PUFA, polyunsaturated fatty acid
cyclooxygenases (COX), xanthine oxidase (XO) and NADPH oxidase generate O 2 as the main oxidant in various cell types; and (3) myeloperoxidase (MPO) and monoamine oxidase (MAO) generate hypochlorous acid (HOCl) and H2O2 as main oxidants in leukocytes and in parenchymal cells respectively.2 These continuously-produced ROS are scavenged by SOD, glutathione peroxidase (GSH-Px) and catalase (CAT). Under some circumstances, these endogenous antioxidative defences are likely to be perturbed as a result of overproduction of oxygen radicals, inactivation of detoxification systems, consumption of antioxidants, and failure to adequately replenish antioxidants in tissue. It has been demonstrated in numerous studies that ROS are directly involved in oxidative damage of cellular macromolecules such as lipids, proteins, and nucleic acids in tissues. 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.3 Copyright # 2002 John Wiley & Sons, Ltd.
Sixteen adult male albino (New Zealand type) rabbits were divided into two groups of eight rabbits each: exposure and sham exposure. The rabbits in the exposure group, were exposed to a commercially available cellular telephone of the GSM 900 type (Global System for Mobile communication at 900 MHz, 2 W peak power, average power density 0.02 mW cm2) for 30 min day1, for 7 days. Another group of eight rabbits was sham-exposed under the same environmental conditions as the exposure group. The telephones were positioned in close contact with the rabbits. Two situations were evaluated: in the first experimental group, cellular telephones were turned to the speech position (established contact) for 30 min, and in the second experimental group, telephones were in standby position for 30 min. After the last exposure on day 7, blood samples were collected through a cardiac puncture and brains were removed after decapitation. Right hemispheres were immediately frozen in liquid nitrogen and stored at 80 C until biochemical analysis. Serum was separated and stored at 80 C until analysis. After weighing the right hemispheres, homogenate, supernatant and extracted samples were prepared as described previously,4 and the following determinations were made on the samples using products supplied by Sigma. Malondialdehyde3 and NO5 levels and MPO activity6 were determined in the homogenate, ADA,7 XO,8 CAT,9 and GSH-Px10 activities in the supernatant and SOD activity in the extracted samples11,12 according to the methods described elsewhere. Protein measurements were made at all stages according to the method explained elsewhere.13 All determinations except CAT and MPO activities were also performed on the sera of the rabbits. Data were expressed as mean standard deviation. All statistical analyses were carried out using SPSS statistical software (SPSS for Windows; Chicago, IL, USA). Parametric analyses with Student’s t-test were performed on the data of the biochemical variables to examine differences between the groups. Cell Biochem Funct 2002; 20: 279–283.
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effects of emr on rabbits
Table 1. Brain oxidant and antioxidant levels in rabbits exposed and sham-exposed to electromagnetic radiation. Results are expressed as mean standard deviation BRAIN
ADA (U mg1 pr.)
XO (U mg1 pr.)
MPO (U g1 pr.)
SOD (U mg1 pr.)
GSH-Px (U mg1 pr.)
CAT (k g1 pr.)
MDA (nmol g1 pr.)
NO (nmol mg1 pr.)
Control Exposure
625 357 547 333
0.23 0.16 0.12 0.06
0.70 0.96 1.30 0.83
0.41 0.05 0.35 0.07
0.23 0.04 0.21 0.04
6.37 1.27 6.45 1.38
580 248 439 220
231.9 56 204.8 57
P values
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n ¼ 8. n.s., not significant; pr., protein; ADA, adenosine deaminase; XO, xanthine oxidase; MPO, myeloperoxidase; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase; MDA, malondialdehyde; NO, nitric oxide.
Pearson’s correlation coefficients were calculated to test the relationships between parameters studied. P values less than 0.05 were considered to be significant. RESULTS AND DISCUSSION Exposure to EMR did not significantly change the levels of any parameters in brains of rabbits (p > 0.05). However, exposure led to significantly elevated activity of SOD (p ¼ 0.021) and significantly reduced levels of NO (p ¼ 0.001) in the sera of EMR-exposed rabbits compared to the sham exposure group. The results are summarized in Tables 1 and 2. In correlation analyses of exposure group, there was significant positive correlation between NO and MDA (r ¼ 0.698, p ¼ 0.025); XO and SOD (r ¼ 0.904, p ¼ 0.002) in brains and SOD and GSHPx (r ¼ 0.767, p ¼ 0.026) in sera of rabbits, while we found a negative correlation between GSH-Px and MDA (r ¼ 0.767, p ¼ 0.035) in the sera of rabbits. No correlation was observed between other parameters (p > 0.05). The present study has shown that exposure to EMR with a frequency of 900 MHz has no significant effect on rabbit brain, suggesting that oxygen free radicals were not generated under the experimental conditions employed. But, we observed a significant increase in serum SOD activity in the exposed group, and
SOD activity was positively associated with GSH-Px activity. These results suggest that EMR induces an oxidative stress within the blood vessels of rabbits. The change in SOD activity may be regarded as an indicator of increased ROS production occurring during the exposure period and may reflect the pathophysiological process of the exposure. There are several reports which indicate that free radicals are involved in EMRinduced tissue injury. First, microwave cooking was shown to increase MDA concentrations in fat from meat.14 EMR exposure in rats also resulted in the augmentation in levels of free radicals and decreased the serum levels of melatonin which is an efficient free radical scavenger. This fall was explained by an increased uptake of melatonin by tissues that were experiencing oxidant stress.15 In addition, treatment of rats before and after EMR exposure with melatonin16 and vitamin E17 was found to block the adverse effect of EMR, possibly by affecting the lifetime of the radicals. Indication of oxidative stress observed in sera but not in brain seems paradoxical since neuronal cells are well known to have a higher rate of oxidative metabolic activity, and possess higher concentrations of readily oxidizable membrane polyunsaturated fatty acids than other organs.18 However, the skull seems to protect this sensitive organ from adverse effects of EMR. These data are in good
Table 2. Serum oxidant and antioxidant levels in rabbits exposed and sham-exposed to electromagnetic radiation. Results are expressed as mean standard deviation SERUM
ADA (U l1)
XO (U ml1)
SOD (U ml1)
GSH-Px (U dl1)
MDA (mmol l1)
NO (mmol ml1)
Control Exposure
154 166 227 152
0.173 0.13 0.171 0.12
14.74 1.69 16.23 0.98
109.2 25 88.86 36
0.580 0.28 0.748 0.18
49.90 23.9 19.54 9.05
P values
n.s.
n.s.
0.004
n.s.
0.042
n.s.
n ¼ 8. n.s., not significant; ADA, adenosine deaminase; XO, xanthine oxidase; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; NO, nitric oxide.
Copyright # 2002 John Wiley & Sons, Ltd.
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agreement with the work of others who suggested that the brain could be at lower risk than other watery organs such as eyes, because it is well protected by the skull.19 The water content of eyes and plasma may make them more vulnerable by assisting the absorption of EMR. Superoxide dismutases are specific antioxidant enzymes that dismutate O 2 , forming H2O2, which is scavenged by peroxisomal CAT or GSH-Px. Three SODs, copper/zinc SOD (cytosolic SOD), manganese SOD (mitochondrial SOD), and extracellular SOD (ECSOD), are major antioxidant enzymes based on cellular distribution and localization. Of the three isoforms, ECSOD may be the most important in blood vessels, accounting for up to 70% of the total activity of SOD in blood vessels.20 The distribution of extracellular SOD in the vessel wall seems ideal for detoxifying superoxide anions produced in sera of EMRexposed rabbits. ATP catabolism results in local release of its metabolite adenosine which can then be further deaminated to inosine by the action of adenosine deaminase (ADA). Inosine is further metabolized to uric acid by XO. Xanthine oxidase is a major potential source of oxygen free radicals. The burst of XOmediated free radical generation in the tissue is triggered by a large increase in substrate formation, which occurs secondary to the degradation of adenine nucleotides. In the present study, we found that EMR did not affect the ADA and XO activities. But, this finding does not provide proof that ATP degradation did not occur. There is probably sufficient spare capacity of ADA and XO activity to cope with any additional ATP turnover without the requirement of induction of further enzyme levels. The extremely rapid interconversion of ROS (O 2 , H2O2, ONOO) within the cell can make it difficult to identify the originating species. The mitochondrial respiratory chain is the major site for the generation of 21 It is possible that EMR superoxide radicals (O 2 ). may affect the mitochondrial membranes to produce large amounts of oxygen radicals. Cyclooxygenase-2 (COX-2) activation and neutrophil infiltration may also be other sources of ROS under our experimental conditions.22,23 We found a significant decrease in serum NO levels after the exposure. This result may suggest that, EMR may destroy NO by generation of superoxide anion and/or EMR causes a reduction in production of NO. The positive correlation found between NO and MDA levels may also suggest that NO behaves as an oxidant radical. Nitric oxide reacts with superoxide anion at a rate that is three times faster than the dismuCopyright # 2002 John Wiley & Sons, Ltd.
tation of superoxide anion by SOD.24 Because of the efficiency of this reaction, the local concentration of SOD may be an important determinant of the biological half-life of NO. Although formation of peroxynitrite, from interaction of NO and superoxide has the potential to be cytotoxic, the inactivation of superoxide by NO also appears to be protective under the present conditions, since oxidative stress did not result in lipid peroxidation in rabbits. Immunocytochemistry shows that peroxynitrite is formed in the endothelium and macrophages25 and it was shown to inhibit platelet aggregation and leukocyte adhesion to endothelium (protective effects) with no evidence of cell injury.26,27 Based on these biochemical characteristics, we suggest that NO is a protective molecule in part because of its ability to react with and inactivate superoxide anion in EMR-exposed rabbits. On the other hand, NO-mediated dilatation of blood vessels may be impaired by excess generation of reactive oxygen species that destroy NO.28 In this respect, NO depletion could induce vasoconstriction which may be regarded as another adverse effect of EMR. In the present study, we used an analogue telephone with a frequency of 900 MHz. The radiation we used was indeed of very low intensity but an oscillatory similitude between this pulsed microwave radiation and certain electrochemical activities of the rabbit should prompt some concern. The current trend is towards greater use of digital technology and higher frequencies of transmission. Digital telephones operate at a lower average power than analogue telephones and we would not expect them to carry a higher risk, unless there is an important aspect of exposure other than the rate of energy deposition. In a case such as this, where the epidemiological evidence for a link between an agent and a disease is weak, laboratory evidence becomes critical for risk evaluation. Our results may indicate a probable role of ROS in the adverse effects of EMR from an analogue cellular telephone. However, we also know that it is difficult to extrapolate effects from rodents to humans because the entire body of a rabbit is exposed whereas for a person using a mobile telephone, only the small region of the head that is close to the telephone would be exposed. Our results do not support the view that exposure to EMR from hand-held, analogue cellular telephones causes significant ROS production in rabbit brains, but the exposure seems to induce an oxidative stress in vascular structures of rabbits. In conclusion, according to our findings in rabbits, oxidant and antioxidant imbalance may have a pathophysiological role in the adverse effects of cellular telephones. Cell Biochem Funct 2002; 20: 279–283.
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