Auditory Brainstem Response Changes during ...

Original Paper Audiology Neurotology

Received: April 19, 2010 Accepted after revision: September 17, 2010 Published online: November 16, 2010

Audiol Neurotol 2011;16:270–276 DOI: 10.1159/000321337

Auditory Brainstem Response Changes during Exposure to GSM-900 Radiation: An Experimental Study Antigoni E. Kaprana a Theognosia S. Chimona a Chariton E. Papadakis a Stylianos G. Velegrakis c Ioannis O. Vardiambasis b Georgios Adamidis b George A. Velegrakis c

a

ENT Department, Chania General Hospital, Mournies, b Electronics Department, Microwave Communications and Electromagnetic Applications Lab, Technological Educational Institute of Crete, Chania Branch, Chania, and c ENT Department, University Hospital of Crete, Heraklion, Greece

Key Words Auditory brainstem response ⴢ Rabbits ⴢ Mobile phones ⴢ Electromagnetic fields

Abstract The objective of the present study was to investigate the possible electrophysiological time-related changes in auditory pathway during mobile phone electromagnetic field exposure. Thirty healthy rabbits were enrolled in an experimental study of exposure to GSM-900 radiation for 60 min and auditory brainstem responses (ABRs) were recorded at regular time-intervals during exposure. The study subjects were radiated via an adjustable power and frequency radio transmitter for GSM-900 mobile phone emission simulation, designed and manufactured according to the needs of the experiment. The mean absolute latency of waves III–V showed a statistically significant delay (p ! 0.05) after 60, 45 and 15 min of exposure to electromagnetic radiation of 900 MHz, respectively. Interwave latency I–III was found to be prolonged after 60 min of radiation exposure in correspondence to wave III absolute latency delay. Interwave latencies I–V and III–V were found with a statistically significant delay

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(p ! 0.05) after 30 min of radiation. No statistically significant delay was found for the same ABR parameters in recordings from the ear contralateral to the radiation source at 60 min radiation exposure compared with baseline ABR. The ABR measurements returned to baseline recordings 24 h after the exposure to electromagnetic radiation of 900 MHz. The prolongation of interval latencies I–V and III–V indicates that exposure to electromagnetic fields emitted by mobile phone can affect the normal electrophysiological activity of the auditory system, and these findings fit the pattern of general responses to a stressor. Copyright © 2010 S. Karger AG, Basel

Introduction

Exposure to electromagnetic fields (EMFs) created from electrical sources has risen exponentially, due to the soaring popularity of wireless technologies such as mobile phones. International scientific research confirms that EMFs are biologically active in animals and humans, and in some cases can cause discomfort and disease [Kaprana et al., 2008]. Thus, exposure to electromagnetic Antigoni E. Kaprana ENT Department, Chania General Hospital GR–73100 Mournies (Greece) Tel. +30 28 2102 2797, Fax +30 28 2104 4693 E-Mail antinik @ yahoo.com

radiation (EMR) has been linked to different forms of cancer, neurologic diseases, asthma and allergy [Mora et al., 2006]. Radiofrequency radiation (RFR) from mobile phone use has been associated with an increased risk for brain tumors in many studies. For every year of mobile phone use, the risk of brain cancer increases by 8% and the use of a mobile phone on 1 side of the head for 110 years increases the risk of a brain tumor to 470% [Hansson et al., 2007; Hardell et al., 2009]. Of all anatomical structures, the ear is closest to the mobile phone during its use and a number of studies have been carried out investigating the effect of mobile phone radiation on the auditory system. In most of them, the effect of EMR emitted by mobile phone use was investigated by recording possible psychoacoustical or electrophysiological changes in the auditory system. Mild highfrequency hearing loss has been associated with long-term exposure to EMF generated by mobile phones [Kellenyi et al., 1999; Callejo et al., 2005; Oktay and Dasdag, 2006]. On the other hand, no harmful effects of mobile phone usage have been reported regarding hearing, tinnitus and balance in a student population detected by a self-report method [Davidson and Lutman, 2007]. After Grisanti’s first reported changes in distortion product otoacoustic emissions attributed to mobile phone use, many researchers failed to find any significant effect of mobiles on distortion product otoacoustic emissions [Grisanti et al., 1998; Aran et al., 2004; Monnery et al., 2004; Galloni et al., 2005; Janssen et al., 2005; Parazzini et al., 2005; Mora et al., 2006]. Recently, in a case-control study, no significant transient evoked otoacoustic emission changes from baseline to postexposure recording for any of the subjects, nor any significant differences in the transient evoked otoacoustic emission change from baseline to postexposure between cases and controls were reported [Bamiou et al., 2008]. As of yet, there is no clear evidence of mobile phone radiation effects on auditory brainstem and cochlear responses [Arai et al., 2003; Aran et al., 2004; Sievert et al., 2005]. To our knowledge, no records of electrophysiological changes in the auditory system during the exposure to EMR of mobile phones have been performed. The purpose of the present experimental animal study was to investigate the possible electrophysiological timerelated changes in auditory pathway during mobile phone EMF exposure.

Real-Time Effect of Mobile Radiation on Auditory System

Materials and Methods Subjects Thirty healthy New Zealand rabbits (Oryctolagus cuniculus), 8 months of age and weighing 4.5–5.0 kg, were enrolled in the study. They were housed individually in wire cages under controlled environmental conditions (20–22 ° C room temperature, 50–60% relative humidity, 12 h light/12 h dark cycle) at the animal vivarium of the University of Crete, School of Medicine. The project was approved by the Animal Care and Use Committee of the University of Crete, School of Medicine. Food and water were provided ad libitum throughout the study. Before any intervention, all ears were found to be normal upon otoscopy by microscope. The subjects’ hearing integrity was confirmed with baseline auditory brainstem response (ABR) recordings. Two animals were excluded from the study due to unilateral and bilateral hearing loss, respectively. Twenty-eight rabbits were exposed to EMR similar to that emitted by mobile phones for 1 h. ABRs were recorded during radiation (real-time measurements) at regular time intervals (see below ‘ABR Measurements’). Otoscopy as well as all ABR measurements were performed under general anesthesia with intramuscular injection of ketamine (Ketalar, 90 mg/kg) and xylazine (Rompun, 2 mg/kg). Repeated doses of ketamine were used if needed according to the recommended methods of anesthesia for common laboratory animals of the Hellenic National Bioethics Commission. The facilities and the experimental design were in compliance with Directive 86/609/EEC [Louhimies, 2002].

Radiation System The animals were radiated via the power- and frequency-adjustable radio transmitter (fig. 1a), which was designed and manufactured according to the needs of the experiment for GSM-900 mobile phone emission simulation. The transmitter produces a single nonmodulated continuouswave radio-carrier signal at 903 MHz, which is an almost pure (sinusoidal) radio-frequency tone, having only low-power spurious content at 1806 MHz (253 times below carrier) and at 2709 MHz (12167 times below carrier). Figure 2 shows the frequency spectrum of the transmitted signal (as measured using an HP8592B spectrum analyzer). The main peak represents the average transmitted power (0.219 W) at the carrier frequency (903 MHz), while the other 2 peaks represent the average transmitted power at the 2nd- (1806 MHz) and 3rd- (2709 MHz) order harmonics. Thus, the total average output power of the transmitter is about 0.22 W, which is a typical value of the average power transmitted by a cell phone operating under favorable conditions near to the base station. The output power of the transmitter is fed to a ␭/4 monopole antenna, which is a tuned wire antenna with a length equal to 8 cm (the quarter of the wavelength at the carrier frequency). During the experiments the transmitter was placed near the ear of each subject and the antenna was placed in the entrance of the external auditory bony canal (fig. 1b). ABR Measurements ABR measurements were performed by using the EP25Eclipse platform (Interacoustics). Stainless steel needle electrodes (Medtronic Sensory Needle electrodes 30 ! 0.35 mm – 28 G) were connected to the preamplifier. The noninverting (positive) elec-

Audiol Neurotol 2011;16:270–276

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Carrier f = 903 MHz P = 219 mW

20

Power (dBm)

10

a

1000 100 f = 1806 MHz P = 0.865 mW

0

10 f = 2709 MHz P = 0.018 mW

–10

0 0.1

Power (mW)

30

–20

0.01

–30

1 × 10–3

–40

1 × 10–4 1 × 10–5

–50 900

1200

1500 1800 2100 Frequency (MHz)

2400

2700

Fig. 2. The average power of the continuous-wave signal transmitted from the manufactured GSM-900 mobile phone emission simulation radiator versus frequency, as measured with a spectrum analyzer. Note that the signal at 903 MHz is almost sinusoidal, having only low-power spurious content at 1806 MHz (2ndorder harmonic; 253 times below carrier) and at 2709 MHz (3rdorder harmonic; 12167 times below carrier).

b

Fig. 1. a The adjustable power and frequency radio transmitter for GSM-900 mobile phone emission simulation. b One of the subjects anesthesized with the needle electrodes, the inserted earphones and the radio transmitter in place.

wave latency as well as interwave latency data were measured at 1, 15, 30, 45 and 60 min. The ear exposed to radiation was selected randomly in each animal and ABR recordings were obtained both ipsilaterally and contralaterally to the exposed ear. ABR waveforms were examined, with at least 2 trial sequences being repeated on baseline ABR in order to confirm the reliability of responses, whereas the recordings during animal radiation were performed once. The temperature was maintained at 39 8 0.5 ° C using a rectal probe thermostatically coupled to a heating pad. During radiation exposure, the transformer was covered with a lead box in order to eliminate the noise of EMF emitted by the transmission system during its operation. ABR recordings were also performed in the study subjects 24 h after exposure to EMR of 900 MHz.

trode was placed subcutaneously on the vertex, while the inverting (negative) electrode was placed on the retroauricular area of the ear. A ground electrode was positioned subcutaneously in the lower back (fig. 1). Electrode impedances up to 4 k⍀ were accepted. If the values exceeded these predefined limits, the electrode position was corrected or the electrodes were replaced with new ones. Acoustic signals were directed from the Etymotic Research transducer (fig. 1) through a tube to an inserted earphone covered by a foam plug that was positioned deep into the rabbit’s external ear canal. Stimuli were clicks of 0.1 ms duration with alternating polarity and a rate of 20.1/s (2000 sweeps). A bandpass filter was set at 100–3000 Hz (filter slope = 12 dB/oct), and artifact rejection was set at 8 40 ␮V. The nontested ear was masked with 50 dB to counteract acoustic crossover. Hearing threshold, absolute wave latency and interwave latency data were assessed on baseline ABR recordings. Every baseline recording started with a stimulus level of 80 dBnHL and continued with gradually lower intensity of 20- or 10-dBnHL steps, until the hearing threshold was found. These recordings were used as controls for comparison with the following ABRs performed during radiation exposure. For radiation exposure recordings only a suprathreshold stimulus (80 dBnHL) was used and absolute

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Statistical Analysis Data were analyzed with the statistical software package SPSS v.16.0. The mean absolute latencies of waves I–V and interwave latencies I–III, I–V and III–V of baseline ABR recordings and ABR recordings during radiation exposure, at a stimulus level of 80 dBnHL, were compared using 2-way ANOVA. A p value of !0.05 was considered statistically significant.

Results

Four positive waveforms (waves I–IV) were clearly detectable at baseline ABR recordings within the first 10 ms after the stimulation onset (figure 3), whereas wave V was Kaprana et al.

Fig. 3. Example of baseline ABR recording. R = Response obtained from right ear; L = response obtained from

left ear.

detected less frequently and only at higher stimulus levels (80 dBnHL). All 5 waves differed in amplitude and wave I proved dominant in most cases. Baseline mean absolute latency values of ABR waves, obtained with alternating click stimuli at 80 dBnHL, were found as follows: I = 1.413 ms (8 0.14), II = 2.103 ms (8 0.17), III = 3.558 ms (8 0.13), IV = 4.820 ms (8 0.12) and V = 5.875 ms (8 0.17). Consequently, the mean interwave latency values of I–III, I–V and III–V were 1.903 (8 0.16), 4.321 (8 0.16) and 2.418 (8 0.14) ms, respectively. Table 1 presents the mean absolute wave latencies and mean interwave latencies of the ipsilateral to radiation side at baseline ABR recordings as well as at different times of radiation exposure, evoked by an alternating click stimulus of 80 dBnHL. Compared to baseline recordings, statistically significant delay in mean absolute wave latencies was found for wave III (3.8 8 0.14 ms), wave IV (5.15 8 0.16 ms) and wave V (6.250 8 0.14 ms) at 60, 45 and 15 min of exposure to EMR of 900 MHz, respectively. Mean interwave latency I–III was found to be prolonged after 60 min of radiation exposure, in accordance with wave III delay. Mean interwave latencies Real-Time Effect of Mobile Radiation on Auditory System

I–V and III–V were found to be significantly prolonged (p ! 0.05) after 30 min of radiation with mean values of 4.749 (8 0.17) and 2.729 (8 0.12) ms, respectively. No statistically significant delay was found for the mean absolute wave latencies or the mean interwave latencies in ABR recordings from the ear contralateral to the radiation source, at 60 min of radiation exposure compared with baseline recordings (p 1 0.05; table 1). ABR measurements performed in the study subjects 24 h after exposure to EMR of 900 MHz returned to normal compared to the baseline recordings.

Discussion

Mobile telephony has shown enormous growth since 1995. The mobile phone penetration rate has been reported up to 106% among EU-27 households [Fickinger and Lumio, 2007]. A relatively high energy deposition around the dominant ear region compared to other body parts has been connected with the mobile usage. In the last decade, a number of studies have been set up and several Audiol Neurotol 2011;16:270–276

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Fig. 4. ABR recording at 60 min radiation exposure time on the right ear (same subject as in fig. 3). In comparison with baseline recording (fig. 3) there is a significant delay of ABR waveform on the right side. Upper curve: response obtained from the right ear (ipsilateral to radiation exposure); lower curve: response obtained from the left ear (contralateral to radiation exposure).

Table 1. Mean values of ABR absolute latencies of waves I–V, and interwave latencies I–III, I–V, III–V of ipsilateral and contralateral

ears before radiation (baseline recording) and at different times of radiation exposure (stimulus: click alternating, 80 dBnHL) ABR waves and interwave intervals

Ipsilateral ear ABR absolute latencies and interwave latencies, ms

Contralateral ear ABR absolute latencies and interwave latencies ms

before radiation (baseline recording)

radiation exposure time 1 min





before radiation (baseline recording)


I II III IV V I–III I–V III–V

1.41380.14 2.10380.17 3.55880.13 4.82080.12 5.87580.17 1.90380.16 4.32180.16 2.41880.14

1.43480.13 2.11380.17 3.60480.15 4.85780.13 6.04880.12 1.93380.13 4.47980.12 2.54680.12

1.47580.14 2.14280.18 3.65280.16 4.93180.13 6.25080.14 1.92780.16 4.41980.14 2.55380.17

1.45780.13 2.16980.19 3.67780.12 5.08580.14 6.38880.13 1.98280.14 4.58780.12 2.60680.14

1.46580.16 2.18680.14 3.70080.17 5.14880.16 6.58880.17 1.99680.16 4.58080.15 2.66480.14

1.48280.15 2.21880.15 3.80880.14 5.27080.16 6.77080.18 2.03080.15 4.74980.17 2.72980.12

1.45580.08 2.29280.19 3.60780.21 4.77880.50 5.86180.47 2.15280.22 4.40680.46 2.25380.37

1.57780.19 2.3480.18 3.67080.22 4.86480.52 6.19580.50 2.09280.37 4.61880.55 2.52580.39

Figures are means 8 standard deviation.

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exposure systems have been designed in an effort to evaluate the possible biologic adverse effects of the exposure to EMFs. Although an increasing number of people own a handheld mobile phone, it is difficult to achieve a study participation of 170% [Olsen, 2009]. In this experimental animal study we investigated the changes in rabbit ABRs during exposure to GSM-900 radiation. In the present study, the first 4 ABR waves were constantly present. Wave V, in baseline recordings as well as in the recordings during radiation exposure, was more variable in appearance (and sometimes presented as 1 wave with wave IV), but was always detectable at a stimulus level of 80 dBnHL. Consequently, the early delay of wave V could not be consulted as an independent value and changes in absolute wave latencies are considered statistically significant only after 30 min of radiation exposure. Statistical analysis showed that there is no relation between exposure time and delay of ABR waves. Interwave latencies I–V and III–V showed statistically significant delay (p ! 0.05) after 30 min of radiation. The prolongation of interval latencies I–V and III–V indicates that exposure to EMF emitted by mobile phone can affect the normal electrophysiological activity of the auditory system. This effect becomes detectable by ABR, which can provide reliable information for both peripheral and central auditory function. No statistically significant difference in ABR parameters on the contralateral ear after 60 min of radiation exposure was found. This may be explained by a decrease in EMF intensity with distance from the antenna. The in-air intensity will decrease by the inverse square law, but the decrease in intensity with distance is more complicated in tissues and depends on structural specifics that include the type of cell and tissue. Probably, achievement of the same effect on the contralateral side needs longer exposure time. Our baseline latency values are consistent with those reported by other investigators [Stieve et al., 2006]. Evidence obtained from studies in adult rabbits has indicated that wave I is generated in the auditory nerve, subsequent waves arise in the auditory nuclei of the brainstem and particularly waves III and V are associated with neural activity in the upper pons and the inferior colliculus, respectively [Inagaki et al., 1997]. The interwave latencies of ABR seem to be the most reliable values to measure and the interwave latency of I–V is used as a value of the central conduction time [Inagaki et al., 1997]. According to the authors’ opinion the major strength of this study is that ABR recordings where performed during radiation exposure. Most of the studies carried out so far were short-term exposure experiments, and possible

effects were observed only after prolonged or repeated exposure to EMR of mobile phones. In some experiments, tests were made days after exposure and in most of them only electrophysiological changes have been reported without molecular and biochemical interpretation. In our study, ABR measurements, performed in the study subjects 24 h after exposure to EMR of 900 MHz, returned to normal compared to the baseline recordings. These responses fit the pattern of general responses to a stressor. Indeed, it has been proposed that EMR is a stressor [Joubert et al., 2006]. The stress response shows that cells react to EMFs as potentially harmful. Stress proteins help damaged proteins refold to regain their conformations and also act as attendant for transporting cellular proteins to their destinations in cells. The cellular response pathways to EMF have been found to share some of the characteristics of heat shock stress [Blank and Goodman, 2001]. In heat shock, the stress response is activated when extracellular signals affect receptors in the plasma membrane [Lin et al., 2001]. The cell-based signal transduction pathways of the heat shock response are involved in regulation of the EMF-stimulated process, probably through the feedback control mechanisms that respond to the stress proteins [Lin et al., 1997; Sanchez et al., 2006]. Repeated induction of the stress response in a cell seems to induce cytoprotection, and a reduced response is associated with restimulation [Blank and Goodman, 2001]. This feedback control mechanism can explain theoretically the recovery of ABR waveform that was observed in measurements 1 day after exposure. Some possible limitations of experimental animal studies in this field should also be discussed. First, it is unsafe to extrapolate neurological and behavioral data from nonhuman in vivo experiments to the effect of mobile phone use in humans. The structure and anatomy of animal brains are quite different from those of the human brain, thus, neurological data from human studies should be more reliable indicators for mobile phone effects. From the data available, hardly any dose-response study has been carried out and there is no consistent pattern presenting specific absorption rates of pulsed EMF lower than those of continuous EMF. This is an important consideration on the possible neurological effects of exposure to EMF during mobile phone use, since mobile phones emit waves of various forms and characteristics. Additionally, thermal effect cannot be discounted. Even in cases when no significant change in body or local tissue temperature was detected, thermal effect cannot be excluded. Secondly, though it is difficult to deny that RFR can affect the nervous system, the available data suggest a

Real-Time Effect of Mobile Radiation on Auditory System


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complex reaction of the nervous system to EMF. Exposure to RFR does produce various effects on the central nervous system. The response is not likely to be linear with respect to the intensity of the radiation. Other parameters of RFR exposure, such as frequency, duration, waveform, frequency and amplitude modulation, are important determinants of biological responses and affect the shape of the dose intensity-response relationship. In order to understand the possible health effects of exposure to RFR from mobile telephones, one first needs to understand the effects of these different parameters and how they interact with each other.

In conclusion, exposure to EMF emitted by mobile phones can affect the normal electrophysiological activity of the auditory system in rabbits. This effect becomes detectable by ABR recordings ipsilaterally to the side exposed to radiation. The main finding is prolongation of interval latencies I–V and III–V at as soon as 30 min of exposure time, while recordings from the contralateral side do not show this effect even after 60 min. Future experiments are expected to elicit more data about the effects of EMR exposure emitted by mobile phones as well as a single unifying neural mechanism accounting for the neurological effects.

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