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The role of free oxygen radicals in noise induced hearing loss: effects of melatonin and methylprednisolone
Auris Nasus Larynx 29 (2002) 147– 152 www.elsevier.com/locate/anl
The role of free oxygen radicals in noise induced hearing loss: effects of melatonin and methylprednisolone Turgut Karlidag˘ a,*, S¸inasi Yalc¸in a, Ahmet O8 ztu¨rk a, Bilal U8 stu¨ndag˘ b, U8 zeyir Go¨k a, I: rfan Kaygusuz a, Nihat Susaman a a
Department of Otorhinolaryngology, Medical School, Fırat Uni6ersity, Tıp Faku¨ltesi, KBB Anabilim Dalı, 23119 Elazıg˘, Turkey b Department of Biochemistry, Medical School, Firat Uni6ersity, Elazıg˘, Turkey Received 11 May 2001; received in revised form 30 July 2001; accepted 21 September 2001
Abstract The aim of this study was to investigate the role of cochlear damage caused by free oxygen radicals occuring as a result of exposure to noise and to determine the prophylactic effects of melatonin and methylprednisolone. Fifty male albino guinea pigs were randomly divided into five groups. All groups were exposed to 60 h of continuous wide band noise at 100 9 2 dB, except group I. Group I was not exposed to noise or treated with drugs. Group II was exposed to noise and not treated with drugs. Group III was exposed to noise and treated with melatonin. Group IV was exposed to noise and treated with methylprednisolone. Group V was exposed to noise and treated with melatonin and methylprednisolone. A high dose of 40 mg/kg methylprednisolone and/or 20 mg/kg melatonin were administered intramuscularly 24 h before exposure to noise, immediately before noise exposure and once a day until noise exposure was completed. Just after the noise ended, guinea pigs were decapitated. Venous blood was obtained into tubes with EDTA and it was used to measure activity levels of plasma malondialdehyde, erythrocyte glutathione peroxidase and the cochlear tissue malondialdehyde. After the noise ended, in comparison group II with I; it was found that the malondialdehyde activity of the plasma and tissue had increased, the erythrocyte glutathione peroxidase activity levels had decreased and consequently, hearing thresholds had increased (PB0.01). A significant difference was found in the malondialdehyde and erythrocyte glutathione peroxidase activity levels between groups II and III (PB 0.01) and the hearing thresholds exhibited a parallel trend (PB0.05). The hearing threshold and malondialdehyde activity levels obtained from groups IV and V were found to be similar to those of group II (P\ 0.05). As a conclusion, we suggest that the use of methlyprednisolone in order to prevent the cochlear damage caused by noise does not provide sufficient prophylaxy, however the use of melatonin provides a more effective prophylaxy, thus being a promising alternative. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Melatonin; Methylprednisolone; Cochlea; Free oxygen radicals; Noise induced hearing loss
1. Introduction Living in a noisy environment has become a necessity in our day due to the changing means of communication and increased mechanisation in parallel with the fast growing technology and industry. Noise causes damage of the auditory system leading to hearing loss, depending on its intensity, frequency, nature and of the the sensitivity individual [1].
* Corresponding author. Tel.: + 90-424-233-3555; fax: + 90-424238-7688. E-mail address: turgut – [email protected] (T. Karlidag˘).
Noise induced hearing loss (NIHL), is an important problem still awaiting to be solved as one of the occupational diseases. Noise-related injuries are among the top 10 leading work-related injuries and it has been reported that in the USA 7.4 –10.2 million industrial workers are under the risk of occupational induced hearing loss [2]. It was reported that during or after exposure to noise, vasoconstriction occurs in the cochlear vascular system and cochlear microcirculation decreases, leading to hearing loss [3]. In recent years, free oxygen radicals (FORs) are accepted as one of the causes of NIHL. Following blast trauma, it is reported that hearing thresholds and the malondialdehyde (MDA) levels of the guinea pigs increase [4].
0385-8146/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 8 5 - 8 1 4 6 ( 0 1 ) 0 0 1 3 7 - 7
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The melatonin excreted from the pineal glands in a circadian rhythm has a direct antioxidant and is a strong scavenger of FORs as well as increasing the activity of glutathione peroxidase (GSH-Px), which is an antioxidant enzyme [5]. High doses of methylprednisolone applied in vitro prevent the damage caused by the FORs in the lipid membranes and have antioxidant properties [6]. It has also been demonstrated that the same drug prevents tissue damage after ischemiareperfusion by decreasing the level of MDA significantly [7]. The present study was designed to investigate the role of FORs in noise induced cochlear damage and whether melatonin and methylprednisolone have any protective effects in the prophylactic treatment of hearing loss.
2. Materials and methods
2.1. Subjects and preparations The protocol of the study was approved by local ethics committee of Medical Faculty of Fırat University (Protocol No: 2017). Fifty male adult albino guinea pigs were obtained from animal breeding unit in Elazıg˘ Animal Diseases Research Central of Agricultural Ministry of Turkey. Guinea pigs weighing 400–600 g with normal Preyer reflex were used. Animals were randomly divided into five groups: Group I (Control group) was not exposed to noise or treated with drugs. Group II (Noise group) was exposed to noise and not treated with drugs. Group III (Melatonin group) was exposed to noise and treated with melatonin (Melatonin, SIGMA Chemical Co., St. Louis, USA). Group IV (Methylprednisolone group) was exposed to noise and treated with methylprednisolone (Methylprednisolone, Prednol-L, Mustafa Nevzat, Turkey). Group V (Melatonin+Methylprednisolone group) was exposed to noise and treated with melatonin and Methylprednisolone. A high dose of 40 mg/kg methylprednisolone and/or 20 mg/kg melatonin were administered intramuscularly 24 h before noise exposure, immediately before noise exposure and once a day until noise exposure was completed.
2.2. Noise exposure Subjects were exposed to noise in cage within a sound exposure chamber with dimensions of 1× 0.5× 0.5 m3 that allows free access to food and water [8]. For noise exposure, the wide band noise (250– 10,000
Hz) produced by Interacoustics Clinical Computer Audiometer Model AC5 (Interacoustics Co., Denmark) was recorded to device by direct connection from the audiometer output. The level of noise obtained from the recording device was measured by the noise meter CEL-254 (Lucas CEL Institute Ltd., Hitchin, Herts, England). Measurements were taken at several points of the cage, the noise level was standardised to approximately 1009 2 dB, and noise was applied continuously for 60 h [9]. Just after the noise ended, ECochG was performed in order to determine the compound action potential (CAP) shifts in all groups.
2.3. Preparations of the subjects for the test Following the exposure to noise, all guinea pigs were intramuscularly received a combination of 2% xylazine hydrochloride (Rompum, Bayer, Turkey) 5 mg/kg and ketamine hydrochloride (Ketelar, Eczacıbas¸ı, Turkey) 60 mg/kg in order to achieve anaesthesia. Body temperature was maintained at 389 1 °C using a heating blanket and monitered by means of a rectal probe. Three silver wire electrode were used for the ECochG recordings. These electrodes were isolated except a piece of 1 cm from the end. The electrode was positioned with a micromanipulator by means of an operating microscope (Zeiss, OpMi-1). In each animal, the active electrode was placed in the posterior–inferior quadrant of ear canal, the reference electrode was placed in the neck muscles on the same side and the earthing electrode was placed in the neck muscles on the opposite side [10]. Resistance between the electrodes was reduced below 2000 V.
2.4. Electrophysiologic measurements For ECochG recordings Medelec Audiostar Electric Response Audiometer (Vickers Healtcare Co., England) was used. Three stimuli were used for the ECochG recordings as click stimuli and two tone bursts with frequencies of 4 and 8 kHz. Tone burst were used with a 2 ms plateau, 2 ms rise and fall times. Rarefaction clicks (filter setting of 30 Hz–3 kHz) were presented at a rate of 10/s and were 100 ms fixed duration. Average responses from 1024 stimuli were obtained at the 10 dB intervals near threshold of CAP. The hearing threshold obtained from the group that was subjected to neither noise nor drug was taken as the normative levels. Just after the noise ended, the threshold was measured again in all groups using the same stimuli. The presence of the CAP response was determined using a visual detection criterion. CAP threshold was defined as a just noticeable deflection (N1 wave) on the oscilloscope, equivalent to about 1 mV [9].
T. Karlidag˘ et al. / Auris Nasus Larynx 29 (2002) 147–152
2.5. Measurement of free radicals Just after the hearing threshold was determined, the subjects were decapitated after giving ketamine hydrochloride. Following decapitation, the blood taken in tubes with EDTA were centrifuged for 10 min at 3000 rpm. For the measurement of MDA activity from separated plasma and GSH-Px activity from erythrocytes, samples were kept at −20 °C. After blood was taken, lateral wall of cochlea was opened by postauricular approach and the cochlear tissue including the membranous cochlea and the modiolus were removed by microsurgical technique. The dissected tissue was immediately rinsed with 300 ml of 0.9% NaCl to remove the blood and kept at −20 °C until the MDA measurements were taken.
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Paglia et al. [15] based on the oxidation of NADPH by glutathione reductase enzyme. Erythrocyte GSH-Px activity levels were expressed in U/mg Hbg.
2.6. Statistical analysis Comparisons among groups were used by Mann– Whitney U-test, one-way Kruskal–Wallis Variance Analysis (ANOVA) and postANOVA Tukey-B test. Differences at the level of 5% were considered statistically significant.
3. Results
3.1. Electrophysiological results 2.5.1. Plasma malondialdehyde measurement Determination of MDA, which is the final product of lipid peroxidation was made spectrophotometrically (Schimadzu UV-1201 spectrophotometer, Schimadzu Corp., Japan) using a method modified from Satoh [11] and Yagi [12]. The measured plasma MDA levels were expressed as nmol/ml. 2.5.2. Tissue malondialdehyde measurement Homogenate preparation, after the dissected cochlea tissue was thoroughly washed with cold 0.9% NaCl and dried, the tissue samples were weighed. During the work homogeneat was prepared in Elvenjem– Potter (Du pont Instruments, Sorwall Homogenizer, USA) homogeniser using teflon lead, such that 9 ml of 1.15% KCl was used for 1 g of tissue. Determination of MDA, which is one of the last products of lipid peroxidation in the homogeneates, was made spectrophotometrically using a method defined by Ohkawa [13]. Homogeneates were properly diluted, protein was measured by Lowry method [14] and the results were expressed as nmol/mg per protein. 2.5.3. Erythrocyte glutathione peroxidase (GSH-Px) measurement GSH-Px activity was measured using the method of
The mean hearing threshold in groups I and II were measured using ECochG with click, 4 and 8 kHz tone burst stimuli. In group II, it was found that following exposure to noise, hearing threshold increased for each of the three test stimuli compared to group I and the difference between two groups was significant (PB 0.01) (Table 1). Among the mean hearing threshold obtained using click, 4 and 8 kHz tone burst stimuli in group II and groups receiving both noise and drugs, the slightest threshold shift was seen in group III. The statistical comparison of the group II and the groups receiving both noise and drugs revealed that the mean hearing threshold obtained with each of the three test stimuli had significant differences between groups II and III (PB 0.05), whereas no significant difference was found among the other groups (P\ 0.05) (Table 2).
3.2. Biochemical results It was found that plasma MDA activity in group II significantly increased compared to group I (PB0.01). Similarly, in group II, the MDA activity levels in the cochlear tissue increased to about four times the levels of group I and that this increase was statistically signifi-
Table 1 Hearing threshold means 9 standard deviations and statistical significance levels obtained at click, 4 and 8 kHz tone bursts in the groups I and II
Mann–Whitney U-Test (PB0.01).
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Table 2 The changes of hearing threshold means obtained at click, 4 and 8 kHz tone burst in the group II and the groups receiving both noise and drugs
One-way Kruskal–Wallis Variance Analysis (ANOVA) (PB0.001), Tukey-B test (PB0.05). Table 3 Means9standard deviations of the plasma and tissue malondialdehyde activity levels and erythrocyte GSH-Px activity levels obtained for the groups I and II
Mann–Whitney U-Test (PB0.01). MDA, malondialdehyde; GSH-Px, glutathione peroxidase.
cant (PB0.001). Erythrocyte GSH-Px activity in group II was decreased compared to group I (P B 0.01) (Table 3). Among the groups which were exposed to noise and the groups receiving both noise and drugs, the highest increase in plasma MDA activity levels was found in group II whereas the lowest increase in group III. The statistical comparison between the noise group and the groups receiving both noise and drugs revealed significant difference (PB0.01) between groups II and III, whereas no significant difference was found among the other groups (P \0.05) (Fig. 1). In all of the groups exposed only to noise and to groups receiving both noise and drugs, the increase in cochlear tissue MDA activity levels were parallel to plasma MDA activity levels. The statistical comparison between the noise group and the groups receiving both noise and drugs revealed significant difference between groups II and III (PB0.01), whereas no significant difference was found between group II and the other groups (groups IV and V) (P \ 0.05). The statistical comparison revealed significant difference between groups III and IV as well as group V (P B 0.05) (Fig. 2). Erythrocyte GSH-Px activity levels were found to be higher in all of the groups receiving both noise and drugs than the group II. This difference was found to be significant in favour of the groups receiving both noise and drugs (PB 0.01) (Fig. 3).
4. Discussion Except for the hearing loss induced with presbyacusis, the most common cause of sensory–neural hearing loss is noise [16]. Several theories have been formulated in the literature concerning the basic mechanism that leads to NIHL. Although the structural changes occurring in the cochlea due to exposure to extreme noise have already been described, the mechanisms leading to tissue damage has not been clearly identified yet [9,17]. In cochlea, the FORs being produced in response to the reperfusion after hypoxia or ischemic period cause
Fig. 1. Plasma malondialdehyde (nmol MDA/ml) activity levels.
T. Karlidag˘ et al. / Auris Nasus Larynx 29 (2002) 147–152
Fig. 2. Cochlear tissue malondialdehyde (nmol MDA/mg protein) levels.
Fig. 3. Erythrocyte GSH-Px activity (U/mg Hbg) levels.
localised damage in the cochlear sensorial epithelium. In NIHL in the postscemic period, lipid peroxidation, being a result of the cochlear damage, has made FORs a factor to be investigated [4,6,18– 20]. Because noise-induced hearing loss cannot be treated medically or by surgery, the main aim is directed to prevent cochlear damage caused by noise and to protect hearing ability. For this purpose many alternative treatments are carried out [9,16,21]. Yamane et al. [18] have discovered that superoxide anion radical is formed in the luminal membranes of the stria vascularis marginal cells of guinea pigs which were subjected to 120– 125 dB noise for 3 h. Similarly Liu [4] reported that cochlear tissue MDA level started to increase in the initial hours after blast trauma, reached its peak on the 3rd and 6th days, was reduced to a normal level on the 8th day, it made a second peak on the 12th day, and the hearing thresholds were parallel to the MDA levels. Based on these findings, he claimed that increased FORs reactions were responsible for the hearing loss induced with blast trauma.
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In the literature, use of FORs scavenger melatonin and methylprednisolone to prevent noise induced cochlear damage is scarcely reported. Melatonin stimulates antioxidant enzymes such as glutathione peroxidase, glutathione reductase and superoxide dismutase and Methylprednisolone prevents lipid peroxidation, acting as a PAF (plateled activiting factor) and MIF (macrophage inhibiting factor) inhibitor [7,22,23]. Melchiori et al. [24] have reported that in the rats which were subjected to high doses of paraquat and were given melatonin, the mortality rate in 24 h decreased to one-thirds, the MDA levels were reduced and that the level of oxidised glutathione increased. It was assumed to be the effect of melatonin inhibiting lipid peroxidation, thus preventing cell damage. In a research aiming to find out the effects of methylprednisolone on lipid peroxidation, anaesthetised rats have been given methylprednisolone, trilasade mesylate and vitamin E using experimental spinal cord compression model, and this treatment has demonstrated that all three types of medicine effectively inhibits lipid peroxidation, thus minimising tissue damage and decreasing MDA levels [7]. The glutathione enzyme system acts as a protective mechanism against NIHL. The research has shown that, after exposure to noise, while the glutathione level in the cochlear tissue increased, the degree of damage in the Corti organ sensorial epithelium decreased [7,25,26]. In our study, melatonin aplication during noise exposure both led to an increase in the erythrocyte GSH-Px level and helped to keep the hearing threshold constant. Ising [27], reported that noise caused an increase in the level of acute and chronic hormones and consequently elevated levels of free oxygen radicals was observed. Similarly, it was shown that the level of these oxidant agents can also be measured in several other organs, i.e. liver. Herken et al. [28] studied schizophrenic patients whereas and Mihailovich et al. [29] examined patients with arterial hypertension and chronic heart failure, respectively. They measured the level of activity of GSH-Px in peripheral blood and determined a correlation between the level of GSH-Px and these diseases. The method used in the present study and the results were well correlated with the literature in this aspect. When we examined the results of our study; we found that in group III MDA activity levels were increased minimally (PB 0.01) and erythrocyte GSHPx activity levels were close to the normal levels (group I). However, it was observed that hearing thresholds at click, 4 and 8 kHz tone burst stimuli were considerably preserved (PB 0.05). On the basis of these findings it can be concluded that melatonin may play an effective role in preventing noise induced cochlear damage. Hearing thresholds and MDA activity levels obtained in groups IV and V are similar to those obtained in
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group II (P \ 0.05). Since methylprednisolone or melatonin and methylprednisolone together use are effective only on erythrocyte GSH-Px activities (P B 0.01), enough cochlear protection cannot be achieved. One of the important finding of this study is, although using only melatonin had an effective role on the prevention of noise induced cochlear damage caused by the free oxygen radicals, using melatonin and methylprednisolone together did not potentialise each other’s effect. We do not know the reason of that. The detailed studies concerning administration of melatonin and methylprednisolone together are required for clarifying this condition.
5. Conclusion The administration of melatonin prevented the increase of MDA activity levels and the decrease of erythrocyte GSH-Px activity levels, thus protected the hearing thresholds. Since melatonin provides partial protection in the cochlear sensorial epithelium against damage from FORs, it can be a new, effective and reliable alternative treatment for the prevention of NIHL.
Acknowledgements The present study was financially supported by the Research Fund of Fırat University (FUNAF). Contract grant number: FUNAF- 374.
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[9] Seidman MD, Shivapuja BG, Quirk WS. The protective effects of allopurinol and superoxide dismutase on noise-induced cochlear damage. Otolarygol Head Neck Surg 1993;109:1052 – 6. [10] Walsted A, Nilsson P, Gerlif J. Cerebrospinal fluid loss and threshold changes. 2. Electrocochleographic changes of the compound action potential after CSF aspiration: an experimental study. Audiol Neurootol 1996;1(5):256 – 64. [11] Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colometric methods. Clin Chim Acta 1978;90:37 – 43. [12] Yagi K. Assay of lipid peroxidation in blood plasma or serum. Methods Enzymol 1984;105:328 – 63. [13] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbutiric acid relations. Anal Biochem 1979;95:351 – 8. [14] Lowry OH, Rosebrough NJ, Fair AL, et al. Protein measurement with the folin phenol reagent. J Biol Chem 1951:265 – 75. [15] Paglia D, Valentine W. Studies on the quantitative and qualitative characterization of erythtrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158 – 69. [16] Dobie RA. Prevention of noise-induced hearing loss. Arch Otolaryngol Head Neck Surg 1995;121:385 – 91. [17] Lonsbury-Martin BL, Martin GK. Auditory dysfunction from excessive sound stimulation. In: Cummigs CW, Fredrickson JM, Harker LA, Krause CJ, Schuller DE, editors. Otolaryngology Head-Neck Surgery. St. Louis: Mosby Year Book, 1993:2885 – 900. [18] Yamene H, Nakai Y, Takayama M, Iguchi H, Nakagawa T, Kojima A. Appearange of free radicals in the guinea pig inner ear after noise induced acoustic trauma. Eur Arch Otorhinolaryngol 1995;252:504 – 8. [19] Yamene H, Nakai Y, Takayama M, Konishi K, Iguchi H, Nakagawa T, et al. The emergence of free radicals after acoustic travma and strial blood flow. Acta Otolarygol Suppl (Stockh) 1995;519:87 – 92. [20] Yamasoba T, Schacht J, Shoji F, Miller JM. Attenuation of cochlear damage from noise trauma by iron chelator, a free radical scavenger and glia cell line-derived neurotrophic factor in vivo. Brain Res 1999;815:317 – 25. [21] Takahashi K, Kusakari J, Kimura S, Wada T, Hara A. The effect of methylprednisolone on acoustic trauma. Acta Otolarygol 1996;116:209 – 12. [22] Reiter RJ, Tan DX, Cabrera J, D’Arpa D, Sainz RM, Mayo JC, Ramos S. The oxidant/antioxidant network: role of melatonin. Biol Signals Recept 1999;8:56 – 63. [23] Reiter RJ, Tan DX, Poeggeler A, Menendez Pelaez A, Chen LD, Saarela S. Melatonin as a free radical scavenger: implications for aging and age related disease. Ann N Y Acad Sci 1994;31:1 –12. [24] Melchiorri D, Reiter RJ, Attia AM, Hara M, Burgos A, Nistico G. Potent protective effect of melatonin on in vivo paraquat-induced oxidative damage in rats. Life Sci 1995;56:83 – 9. [25] Yamasoba T, Harris C, Schoji F, Lee RJ, Nuttall AL, Miller JM. Influence of intense sound exposure on glutathione synthesis in the cochlea. Brain Res 1998;804:72 – 8. [26] Jacono AA, Hu B, Kopke RD, Henderson D, Van De Wetar TR, Steinman HM. Changes in cochlear antioxidant enzyme activity after sound conditioning and noise exposure in the chinchilla. Hear Res 1998;117:31 – 8. [27] Ising H. Acute and chronic stress hormone increases in noise exposure. Schriftenr Ver Wasser Boden Lufthyg 2000;106:169 –77. [28] Herken H, Uz E, Ozyurt H, Sogut S, Virit O, Akyol O. Evidence that the activites of erythrocyte free radical scavenging enzymes and the products of lipid peroxidation are increased in different forms of schizophrenia. Mol Psychiatry 2001;6:66 – 73. [29] Mihailovic MB, Avramovic DM, Jovanovic IB, Pesut OJ, Matic DP, Stojanov VJ. Blood and plasma selenium levels and GSH-Px activites in patients with arterial hypertension and chronic heart disease. J Environ Pathol Toxicol Oncol 1998;17:285 – 9.
The role of free oxygen radicals in noise induced hearing loss: effects of melatonin and methylprednisolone Turgut Karlidag˘ a,*, S¸inasi Yalc¸in a, Ahmet O8 ztu¨rk a, Bilal U8 stu¨ndag˘ b, U8 zeyir Go¨k a, I: rfan Kaygusuz a, Nihat Susaman a a
Department of Otorhinolaryngology, Medical School, Fırat Uni6ersity, Tıp Faku¨ltesi, KBB Anabilim Dalı, 23119 Elazıg˘, Turkey b Department of Biochemistry, Medical School, Firat Uni6ersity, Elazıg˘, Turkey Received 11 May 2001; received in revised form 30 July 2001; accepted 21 September 2001
Abstract The aim of this study was to investigate the role of cochlear damage caused by free oxygen radicals occuring as a result of exposure to noise and to determine the prophylactic effects of melatonin and methylprednisolone. Fifty male albino guinea pigs were randomly divided into five groups. All groups were exposed to 60 h of continuous wide band noise at 100 9 2 dB, except group I. Group I was not exposed to noise or treated with drugs. Group II was exposed to noise and not treated with drugs. Group III was exposed to noise and treated with melatonin. Group IV was exposed to noise and treated with methylprednisolone. Group V was exposed to noise and treated with melatonin and methylprednisolone. A high dose of 40 mg/kg methylprednisolone and/or 20 mg/kg melatonin were administered intramuscularly 24 h before exposure to noise, immediately before noise exposure and once a day until noise exposure was completed. Just after the noise ended, guinea pigs were decapitated. Venous blood was obtained into tubes with EDTA and it was used to measure activity levels of plasma malondialdehyde, erythrocyte glutathione peroxidase and the cochlear tissue malondialdehyde. After the noise ended, in comparison group II with I; it was found that the malondialdehyde activity of the plasma and tissue had increased, the erythrocyte glutathione peroxidase activity levels had decreased and consequently, hearing thresholds had increased (PB0.01). A significant difference was found in the malondialdehyde and erythrocyte glutathione peroxidase activity levels between groups II and III (PB 0.01) and the hearing thresholds exhibited a parallel trend (PB0.05). The hearing threshold and malondialdehyde activity levels obtained from groups IV and V were found to be similar to those of group II (P\ 0.05). As a conclusion, we suggest that the use of methlyprednisolone in order to prevent the cochlear damage caused by noise does not provide sufficient prophylaxy, however the use of melatonin provides a more effective prophylaxy, thus being a promising alternative. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Melatonin; Methylprednisolone; Cochlea; Free oxygen radicals; Noise induced hearing loss
1. Introduction Living in a noisy environment has become a necessity in our day due to the changing means of communication and increased mechanisation in parallel with the fast growing technology and industry. Noise causes damage of the auditory system leading to hearing loss, depending on its intensity, frequency, nature and of the the sensitivity individual [1].
* Corresponding author. Tel.: + 90-424-233-3555; fax: + 90-424238-7688. E-mail address: turgut – [email protected] (T. Karlidag˘).
Noise induced hearing loss (NIHL), is an important problem still awaiting to be solved as one of the occupational diseases. Noise-related injuries are among the top 10 leading work-related injuries and it has been reported that in the USA 7.4 –10.2 million industrial workers are under the risk of occupational induced hearing loss [2]. It was reported that during or after exposure to noise, vasoconstriction occurs in the cochlear vascular system and cochlear microcirculation decreases, leading to hearing loss [3]. In recent years, free oxygen radicals (FORs) are accepted as one of the causes of NIHL. Following blast trauma, it is reported that hearing thresholds and the malondialdehyde (MDA) levels of the guinea pigs increase [4].
0385-8146/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 8 5 - 8 1 4 6 ( 0 1 ) 0 0 1 3 7 - 7
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T. Karlidag˘ et al. / Auris Nasus Larynx 29 (2002) 147–152
The melatonin excreted from the pineal glands in a circadian rhythm has a direct antioxidant and is a strong scavenger of FORs as well as increasing the activity of glutathione peroxidase (GSH-Px), which is an antioxidant enzyme [5]. High doses of methylprednisolone applied in vitro prevent the damage caused by the FORs in the lipid membranes and have antioxidant properties [6]. It has also been demonstrated that the same drug prevents tissue damage after ischemiareperfusion by decreasing the level of MDA significantly [7]. The present study was designed to investigate the role of FORs in noise induced cochlear damage and whether melatonin and methylprednisolone have any protective effects in the prophylactic treatment of hearing loss.
2. Materials and methods
2.1. Subjects and preparations The protocol of the study was approved by local ethics committee of Medical Faculty of Fırat University (Protocol No: 2017). Fifty male adult albino guinea pigs were obtained from animal breeding unit in Elazıg˘ Animal Diseases Research Central of Agricultural Ministry of Turkey. Guinea pigs weighing 400–600 g with normal Preyer reflex were used. Animals were randomly divided into five groups: Group I (Control group) was not exposed to noise or treated with drugs. Group II (Noise group) was exposed to noise and not treated with drugs. Group III (Melatonin group) was exposed to noise and treated with melatonin (Melatonin, SIGMA Chemical Co., St. Louis, USA). Group IV (Methylprednisolone group) was exposed to noise and treated with methylprednisolone (Methylprednisolone, Prednol-L, Mustafa Nevzat, Turkey). Group V (Melatonin+Methylprednisolone group) was exposed to noise and treated with melatonin and Methylprednisolone. A high dose of 40 mg/kg methylprednisolone and/or 20 mg/kg melatonin were administered intramuscularly 24 h before noise exposure, immediately before noise exposure and once a day until noise exposure was completed.
2.2. Noise exposure Subjects were exposed to noise in cage within a sound exposure chamber with dimensions of 1× 0.5× 0.5 m3 that allows free access to food and water [8]. For noise exposure, the wide band noise (250– 10,000
Hz) produced by Interacoustics Clinical Computer Audiometer Model AC5 (Interacoustics Co., Denmark) was recorded to device by direct connection from the audiometer output. The level of noise obtained from the recording device was measured by the noise meter CEL-254 (Lucas CEL Institute Ltd., Hitchin, Herts, England). Measurements were taken at several points of the cage, the noise level was standardised to approximately 1009 2 dB, and noise was applied continuously for 60 h [9]. Just after the noise ended, ECochG was performed in order to determine the compound action potential (CAP) shifts in all groups.
2.3. Preparations of the subjects for the test Following the exposure to noise, all guinea pigs were intramuscularly received a combination of 2% xylazine hydrochloride (Rompum, Bayer, Turkey) 5 mg/kg and ketamine hydrochloride (Ketelar, Eczacıbas¸ı, Turkey) 60 mg/kg in order to achieve anaesthesia. Body temperature was maintained at 389 1 °C using a heating blanket and monitered by means of a rectal probe. Three silver wire electrode were used for the ECochG recordings. These electrodes were isolated except a piece of 1 cm from the end. The electrode was positioned with a micromanipulator by means of an operating microscope (Zeiss, OpMi-1). In each animal, the active electrode was placed in the posterior–inferior quadrant of ear canal, the reference electrode was placed in the neck muscles on the same side and the earthing electrode was placed in the neck muscles on the opposite side [10]. Resistance between the electrodes was reduced below 2000 V.
2.4. Electrophysiologic measurements For ECochG recordings Medelec Audiostar Electric Response Audiometer (Vickers Healtcare Co., England) was used. Three stimuli were used for the ECochG recordings as click stimuli and two tone bursts with frequencies of 4 and 8 kHz. Tone burst were used with a 2 ms plateau, 2 ms rise and fall times. Rarefaction clicks (filter setting of 30 Hz–3 kHz) were presented at a rate of 10/s and were 100 ms fixed duration. Average responses from 1024 stimuli were obtained at the 10 dB intervals near threshold of CAP. The hearing threshold obtained from the group that was subjected to neither noise nor drug was taken as the normative levels. Just after the noise ended, the threshold was measured again in all groups using the same stimuli. The presence of the CAP response was determined using a visual detection criterion. CAP threshold was defined as a just noticeable deflection (N1 wave) on the oscilloscope, equivalent to about 1 mV [9].
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2.5. Measurement of free radicals Just after the hearing threshold was determined, the subjects were decapitated after giving ketamine hydrochloride. Following decapitation, the blood taken in tubes with EDTA were centrifuged for 10 min at 3000 rpm. For the measurement of MDA activity from separated plasma and GSH-Px activity from erythrocytes, samples were kept at −20 °C. After blood was taken, lateral wall of cochlea was opened by postauricular approach and the cochlear tissue including the membranous cochlea and the modiolus were removed by microsurgical technique. The dissected tissue was immediately rinsed with 300 ml of 0.9% NaCl to remove the blood and kept at −20 °C until the MDA measurements were taken.
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Paglia et al. [15] based on the oxidation of NADPH by glutathione reductase enzyme. Erythrocyte GSH-Px activity levels were expressed in U/mg Hbg.
2.6. Statistical analysis Comparisons among groups were used by Mann– Whitney U-test, one-way Kruskal–Wallis Variance Analysis (ANOVA) and postANOVA Tukey-B test. Differences at the level of 5% were considered statistically significant.
3. Results
3.1. Electrophysiological results 2.5.1. Plasma malondialdehyde measurement Determination of MDA, which is the final product of lipid peroxidation was made spectrophotometrically (Schimadzu UV-1201 spectrophotometer, Schimadzu Corp., Japan) using a method modified from Satoh [11] and Yagi [12]. The measured plasma MDA levels were expressed as nmol/ml. 2.5.2. Tissue malondialdehyde measurement Homogenate preparation, after the dissected cochlea tissue was thoroughly washed with cold 0.9% NaCl and dried, the tissue samples were weighed. During the work homogeneat was prepared in Elvenjem– Potter (Du pont Instruments, Sorwall Homogenizer, USA) homogeniser using teflon lead, such that 9 ml of 1.15% KCl was used for 1 g of tissue. Determination of MDA, which is one of the last products of lipid peroxidation in the homogeneates, was made spectrophotometrically using a method defined by Ohkawa [13]. Homogeneates were properly diluted, protein was measured by Lowry method [14] and the results were expressed as nmol/mg per protein. 2.5.3. Erythrocyte glutathione peroxidase (GSH-Px) measurement GSH-Px activity was measured using the method of
The mean hearing threshold in groups I and II were measured using ECochG with click, 4 and 8 kHz tone burst stimuli. In group II, it was found that following exposure to noise, hearing threshold increased for each of the three test stimuli compared to group I and the difference between two groups was significant (PB 0.01) (Table 1). Among the mean hearing threshold obtained using click, 4 and 8 kHz tone burst stimuli in group II and groups receiving both noise and drugs, the slightest threshold shift was seen in group III. The statistical comparison of the group II and the groups receiving both noise and drugs revealed that the mean hearing threshold obtained with each of the three test stimuli had significant differences between groups II and III (PB 0.05), whereas no significant difference was found among the other groups (P\ 0.05) (Table 2).
3.2. Biochemical results It was found that plasma MDA activity in group II significantly increased compared to group I (PB0.01). Similarly, in group II, the MDA activity levels in the cochlear tissue increased to about four times the levels of group I and that this increase was statistically signifi-
Table 1 Hearing threshold means 9 standard deviations and statistical significance levels obtained at click, 4 and 8 kHz tone bursts in the groups I and II
Mann–Whitney U-Test (PB0.01).
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Table 2 The changes of hearing threshold means obtained at click, 4 and 8 kHz tone burst in the group II and the groups receiving both noise and drugs
One-way Kruskal–Wallis Variance Analysis (ANOVA) (PB0.001), Tukey-B test (PB0.05). Table 3 Means9standard deviations of the plasma and tissue malondialdehyde activity levels and erythrocyte GSH-Px activity levels obtained for the groups I and II
Mann–Whitney U-Test (PB0.01). MDA, malondialdehyde; GSH-Px, glutathione peroxidase.
cant (PB0.001). Erythrocyte GSH-Px activity in group II was decreased compared to group I (P B 0.01) (Table 3). Among the groups which were exposed to noise and the groups receiving both noise and drugs, the highest increase in plasma MDA activity levels was found in group II whereas the lowest increase in group III. The statistical comparison between the noise group and the groups receiving both noise and drugs revealed significant difference (PB0.01) between groups II and III, whereas no significant difference was found among the other groups (P \0.05) (Fig. 1). In all of the groups exposed only to noise and to groups receiving both noise and drugs, the increase in cochlear tissue MDA activity levels were parallel to plasma MDA activity levels. The statistical comparison between the noise group and the groups receiving both noise and drugs revealed significant difference between groups II and III (PB0.01), whereas no significant difference was found between group II and the other groups (groups IV and V) (P \ 0.05). The statistical comparison revealed significant difference between groups III and IV as well as group V (P B 0.05) (Fig. 2). Erythrocyte GSH-Px activity levels were found to be higher in all of the groups receiving both noise and drugs than the group II. This difference was found to be significant in favour of the groups receiving both noise and drugs (PB 0.01) (Fig. 3).
4. Discussion Except for the hearing loss induced with presbyacusis, the most common cause of sensory–neural hearing loss is noise [16]. Several theories have been formulated in the literature concerning the basic mechanism that leads to NIHL. Although the structural changes occurring in the cochlea due to exposure to extreme noise have already been described, the mechanisms leading to tissue damage has not been clearly identified yet [9,17]. In cochlea, the FORs being produced in response to the reperfusion after hypoxia or ischemic period cause
Fig. 1. Plasma malondialdehyde (nmol MDA/ml) activity levels.
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Fig. 2. Cochlear tissue malondialdehyde (nmol MDA/mg protein) levels.
Fig. 3. Erythrocyte GSH-Px activity (U/mg Hbg) levels.
localised damage in the cochlear sensorial epithelium. In NIHL in the postscemic period, lipid peroxidation, being a result of the cochlear damage, has made FORs a factor to be investigated [4,6,18– 20]. Because noise-induced hearing loss cannot be treated medically or by surgery, the main aim is directed to prevent cochlear damage caused by noise and to protect hearing ability. For this purpose many alternative treatments are carried out [9,16,21]. Yamane et al. [18] have discovered that superoxide anion radical is formed in the luminal membranes of the stria vascularis marginal cells of guinea pigs which were subjected to 120– 125 dB noise for 3 h. Similarly Liu [4] reported that cochlear tissue MDA level started to increase in the initial hours after blast trauma, reached its peak on the 3rd and 6th days, was reduced to a normal level on the 8th day, it made a second peak on the 12th day, and the hearing thresholds were parallel to the MDA levels. Based on these findings, he claimed that increased FORs reactions were responsible for the hearing loss induced with blast trauma.
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In the literature, use of FORs scavenger melatonin and methylprednisolone to prevent noise induced cochlear damage is scarcely reported. Melatonin stimulates antioxidant enzymes such as glutathione peroxidase, glutathione reductase and superoxide dismutase and Methylprednisolone prevents lipid peroxidation, acting as a PAF (plateled activiting factor) and MIF (macrophage inhibiting factor) inhibitor [7,22,23]. Melchiori et al. [24] have reported that in the rats which were subjected to high doses of paraquat and were given melatonin, the mortality rate in 24 h decreased to one-thirds, the MDA levels were reduced and that the level of oxidised glutathione increased. It was assumed to be the effect of melatonin inhibiting lipid peroxidation, thus preventing cell damage. In a research aiming to find out the effects of methylprednisolone on lipid peroxidation, anaesthetised rats have been given methylprednisolone, trilasade mesylate and vitamin E using experimental spinal cord compression model, and this treatment has demonstrated that all three types of medicine effectively inhibits lipid peroxidation, thus minimising tissue damage and decreasing MDA levels [7]. The glutathione enzyme system acts as a protective mechanism against NIHL. The research has shown that, after exposure to noise, while the glutathione level in the cochlear tissue increased, the degree of damage in the Corti organ sensorial epithelium decreased [7,25,26]. In our study, melatonin aplication during noise exposure both led to an increase in the erythrocyte GSH-Px level and helped to keep the hearing threshold constant. Ising [27], reported that noise caused an increase in the level of acute and chronic hormones and consequently elevated levels of free oxygen radicals was observed. Similarly, it was shown that the level of these oxidant agents can also be measured in several other organs, i.e. liver. Herken et al. [28] studied schizophrenic patients whereas and Mihailovich et al. [29] examined patients with arterial hypertension and chronic heart failure, respectively. They measured the level of activity of GSH-Px in peripheral blood and determined a correlation between the level of GSH-Px and these diseases. The method used in the present study and the results were well correlated with the literature in this aspect. When we examined the results of our study; we found that in group III MDA activity levels were increased minimally (PB 0.01) and erythrocyte GSHPx activity levels were close to the normal levels (group I). However, it was observed that hearing thresholds at click, 4 and 8 kHz tone burst stimuli were considerably preserved (PB 0.05). On the basis of these findings it can be concluded that melatonin may play an effective role in preventing noise induced cochlear damage. Hearing thresholds and MDA activity levels obtained in groups IV and V are similar to those obtained in
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group II (P \ 0.05). Since methylprednisolone or melatonin and methylprednisolone together use are effective only on erythrocyte GSH-Px activities (P B 0.01), enough cochlear protection cannot be achieved. One of the important finding of this study is, although using only melatonin had an effective role on the prevention of noise induced cochlear damage caused by the free oxygen radicals, using melatonin and methylprednisolone together did not potentialise each other’s effect. We do not know the reason of that. The detailed studies concerning administration of melatonin and methylprednisolone together are required for clarifying this condition.
5. Conclusion The administration of melatonin prevented the increase of MDA activity levels and the decrease of erythrocyte GSH-Px activity levels, thus protected the hearing thresholds. Since melatonin provides partial protection in the cochlear sensorial epithelium against damage from FORs, it can be a new, effective and reliable alternative treatment for the prevention of NIHL.
Acknowledgements The present study was financially supported by the Research Fund of Fırat University (FUNAF). Contract grant number: FUNAF- 374.
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