International Journal of Pediatric Otorhinolaryngology 78 (2014) 663–669
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Protective effect of trimetazidine on amikacin-induced ototoxicity in rats Fadlullah Aksoy a, Remzi Dogan a,*, Orhan Ozturan a, Sabri Baki Eren a, Bayram Veyseller a, ¨ nder Hu¨seyinbas c Alev Pektas b, O a b c
Bezmialem Vakif University, Department of Otorhinolaryngology, Fatih, Istanbul, Turkey Bezmialem Vakif University, Faculty of Health Sciences, Department of Audiology, Fatih, Istanbul, Turkey Bezmialem Vakif University, Research Center, Fatih, Istanbul, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 September 2013 Received in revised form 20 January 2014 Accepted 22 January 2014 Available online 5 February 2014
Objective: Aminoglycoside antibiotics are known to have ototoxic effects and may induce sensorineural hearing loss. This study investigated the protective effect of trimetazidine, which has antioxidant and cytoprotective properties, against amikacin ototoxicity. Methods: Thirty-two male rats were divided into four groups – amikacin, amikacin + trimetazidine, trimetazidine, and control groups. Trimetazidine, 10 mg/kg per day, was given for 14 days by oral gavage. Amikacin, 600 mg/kg per day, was also given for 14 days, by the intramuscular route. Distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) tests were applied to the rats for hearing assessment. At the termination of the study, the biochemical parameters were calculated to evaluate the oxidative status. Results: The DPOAE values of the amikacin group were significantly lower on the 7th and 14th days than those of the trimetazidine + amikacin group and there was an increase in the ABR thresholds. The ABR thresholds for the amikacin group on the 7th and 14th days were significantly higher than the levels on the first day of the study, while there was no significant increase in those values in the trimetazidine + amikacin group. The total oxidant status (TOS) and oxidant status index (OSI) values of the amikacin group were significantly higher than those of the trimetazidine + amikacin group. The total antioxidant status (TAS) values of the amikacin group were lower than those of the trimetazidine + amikacin group. Conclusions: The audiologic tests and biochemical parameters investigated in this study both point to the protective effect of trimetazidine against amikacin-induced ototoxicity. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: ABR Amikacin DPOAE Oxidative status Trimetazidine
1. Introduction Ototoxicity is a clinical condition, usually brought about by the detrimental effects of some chemical agent on the auditory and balance functions of the ear [1]. More than 130 agents are known to have ototoxic effects, with aminoglycoside (AG) and macrolide antibiotics, loop diuretics, NSAIDs, antineoplastic and antimalarial drugs taking the lead [1]. While amikacin (AK), kanamycin and neomycin cause damage primarily to the cochlea, streptomycin and gentamicin primarily cause vestibular damage [2]. Cochlear damage may lead to permanent hearing loss, and vestibular damage may lead to vertigo, ataxia and/or nystagmus [3].
* Corresponding author at: Bezmialem Vakif University, Medical Faculty, Department of Otorhinolaryngology, Fatih, Istanbul, Turkey. Tel.: +90 505 7915844; fax: +90 212 533 2326. E-mail address: [email protected] (R. Dogan). 0165-5876/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2014.01.031
AK is a semi-synthetic AG, produced by the acetylation of kanamycin A. The specific properties of its structure make it resistant to bacterial enzymes which can inactivate natural AGs such as gentamicin, kanamycin and tobramycin, thus it has the broadest spectrum among all AGs. AK is a frequently preferred antibiotic due to its rapid action, broad spectrum, lower bacterial resistance, its synergetic effects with beta-lactam antibiotics, and its lower cost [4]. However, 10–80% of patients who are treated with AK are reported to suffer from its ototoxic effects. The hearing loss is typically neurosensorial, nonsyndromic, bilateral, progressive, and is a high-frequency hearing loss [5]. Since the hair cells of the cochlea cannot regenerate, the hearing loss is irreversible [6]. AK ototoxicity is brought about by the drug’s excitotoxic effects, caused by impairment of mitochondrial protein synthesis and overactivation of glutamatergic receptors (N-methyl-D-aspartate), which increase the formation of free radicals and induce apoptosis [7,8]. There have been numerous studies of oxidative stress leading to sensory neural hearing loss [9–11]. Experimental studies have shown that antioxidant agents may prevent AG ototoxicity [12–16].
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Trimetazidine (TRM) is a selective mitochondrial 3-ketoacyl coenzyme A thiolase (3-KAT) inhibitor. Thus, it decreases free fatty acid (FFA) metabolism and regulates cardiac metabolism. It controls oxidative stress, preserves mitochondrial respiration in ischemia–reperfusion injury, and prevents cardiac ischemia [17,18]. TRM has also been shown to have gastroprotective, hepatoprotective, anti-inflammatory, antinociceptive and anti-apoptotic properties [18,19]. Previous studies have shown that TRM’s cytoprotective effects are brought about by preventing injury in neurosensorial tissue secondary to overstimulation of the cochleovestibular system by glutamate, and by its antioxidant effects [20,21]. In experimental animal studies, it was reported that the ototoxic effect induced by gentamicin and neomycin is prevented by TRM [22,23]. The fields of use of TRM in otorhinolaryngology include the reduction in vertigo duration and frequency [24], treatment of Me´nie`re’s disease related cochleovestibular conditions [25], correction of inner ear ischemia [26], correction of isolated tinnitus [27], and correction of hypoacusis [28]. Clinical studies report that TRM has excellent tolerability [21]. Oxidative stress means disruption of the balance between pro-oxidants and antioxidants in favor of the pro-oxidants [29]. Measuring the amount of different antioxidants individually is difficult, requires a significant amount of time, increases laboratory burden, is high cost and is a complicated technique. Furthermore, there will be complicated interactions among different antioxidants in the serum, therefore, these measurements may not be sufficiently objective. The recently developed method of measuring the total antioxidant status (TAS) enables all antioxidants to be recorded at a very low cost in a short time as a single parameter by a simple measurement in serum [30,31]. Similarly, there are no practical methods for the measurement of individual pro-oxidant molecules, but again, a single parameter, the total oxidant status (TOS), can be measured in serum instead [32]. The purpose of this study was to investigate the protective effect of TRM, which has antioxidant and cytoprotective properties, against AK ototoxicity.
2. Materials and methods 2.1. Animals After obtaining permission for experimental studies from the Local Ethics Committee (2011/39), 32 healthy female Wistar Albino rats weighing 200–240 g were included in the study. Those rats which had a negative Preyer response (when clapping, a rapid movement clearly seen in the bodies of animals indicates a positive Preyer reflex) were examined, and/or those which had ear pathologies (cerumen, otitis media with effusion, acute otitis media, etc.) seen in endoscopic ear examinations were not included in the study. The rats were placed in an environment which was illuminated for 12 h and dark for 12 h, had a temperature of 21 1 8C, with free access to food and water, and with a background noise level of under 50 dB. The animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals [33]. 2.2. Anesthesia Before distortion product otoacoustic emissions (DPOAE) and auditory brainstem responses (ABR) were recorded, all of the rats were anesthetized intraperitoneally with ketamine hydrochloride (45 mg/kg) and xylazine (5 mg/kg).
2.3. Experimental design The 32 rats included in the study were divided into four groups, with eight rats in each group: Group 1 (AK), Group 2 (AK + TRM), Group 3 (TRM), and Group 4 (control group, no active treatment (NAT)). TRM was given to Groups 2 and 3 for 14 days by oral gavage, at a dose of 10 mg/kg per day. Rats in Groups 1 and 2 were administered daily intramuscular injections of AK for 14 days, at a dose of 600 mg/kg per day. Rats in Group 4 were given daily intraperitoneal shots of 1 ml saline solution for 14 days. At the very beginning of the study, and also on the 7th and 14th days, DPOAE and ABR tests were performed on all of the rats. On the 14th day, intracardiac blood samples were drawn from all of the rats to calculate the biochemical parameters. 2.4. DPOAE measurements The GSI Audera device was used for DPOAE measurements in the assessment of the rats’ peripheral hearing system. The smallest sized rubber tip of the tympanometer probe was used for the tests. The emissions were measured in General Diagnostic mode, both as distortion product diagrams (DPgram) and as input/output (I/O) measurements. Otoacoustic emissions were measured using stimuli of different frequencies and intensities. The intensity level of the primary stimuli was adjusted to 65 dB (L1 = L2). Two different frequencies (f1 and f2) were chosen with a ratio of f2/ f1 = 1:10. DPgram measurements were taken at 3000, 4008, 5004, 6000, 6996, 8004, 9012, 10008, 11004 and 12000 Hz frequencies. DPOAEs 3 dB over the 2f1–f2 frequency noise intensity during measurements were accepted as positive. 2.5. ABR measurements ABR measurements were carried out in a silent room, with a Viasys Medelec Synergy instrument, using subcutaneous needle electrodes (Technomed Europe). Stimuli were delivered in alternating polarities with ER-3A insert earphones, using 8 kHz tone-burst stimuli. The filter was set at 30–1500 Hz bandwidth, the repetition rate was set at 21/s, and the time window was set as 25 ms; 1024 samples were obtained for signal averaging. Stimuli were delivered at an intensity level of 80 dB nHL, and were reduced in 20 dB steps until the intensity level approached threshold values. Nearing the threshold, intensity steps were reduced to 10 dB, and the threshold value was determined. A minimum of two tracks were generated for each measurement to check for reproducibility, and for confirmation of the threshold value. The ABR threshold was defined as the minimum intensity level where an ABR wave III was observed. 2.6. TAS, TOS, and OSI measurements Measuring the total antioxidant status (TAS), the combined activity of all antioxidants, provides an evaluation of the overall antioxidant status [30]. Total oxidant status (TOS) is an indicator of the overall oxidant status of the patients [32]. The oxidant status index (OSI) is the ratio of TOS/TAS, and is a means to calculate oxidative stress in the body – comparing TAS to TOS is thought to be a better indicator of the overall oxidative stress. To measure these parameters, blood samples taken from all rats in all groups were centrifuged at 3000 rpm for 15 min, the serum was separated and stored at 80 8C. After all samples had been prepared in this way, TAS and TOS values were calculated with the relevant kit (Rel Assay Diagnostics), and OSI values were calculated with the relevant formula (OSI: TOS/TAS 100).
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Fig. 1. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 1 (AK) at different time points.
2.7. Statistical analysis Statistical analysis was carried out using the Statistical Package for the Social Sciences version 13.0 software for Windows (SPSS Inc., Chicago, IL, USA). All quantitative variables were estimated using measures of central location (i.e. mean and median) and measures of dispersion (i.e. standard deviation (SD)). Data normality was checked using the Kolmogorov–Smirnov tests of normality. 2.7.1. Assessment of DPOAE and ABR results For the comparison within group, the Repeated ANOVA test was applied (the difference within group was considered to be statistically significant if p < 0.05). To determine the days between which there were differences, the Bonferroni test was administered as a post hoc test. Since this was a multiple comparison, the Bonferroni correction was applied and p < 0.016 was accepted as statistically significant. For the comparison between groups, the one-way analysis of variance (ANOVA) test was applied (the difference between groups was considered to be statistically significant if p < 0.05). Tukey’s HSD was administered as a post hoc test to identify between-group differences. Since this was a multiple comparison, the Bonferroni correction was applied and p < 0.008 was accepted as statistically significant. 2.7.2. Assessment of biochemical results One-way of analysis (ANOVA) was used for comparing the data between groups. Tukey’s HSD was administered as a post hoc test. Since this was a multiple comparison, the Bonferroni correction was applied and p < 0.008 was accepted as statistically significant.
3 (TRM) and 4 (NAT) (n = 8 and 16 ears in each group) (p > 0.016) (Figs. 2–4). Comparisons between groups showed that there were no statistically significant differences in the baseline values of DPOAE (p < 0.05), while on the 7th and 14th days of the study, there were statistically significant differences between Group 1 (AK) and the other three groups (p < 0.008). 3.2. ABR A comparison of ABR thresholds of all the groups is included in Table 1. In Group 1 (AK), there were statistically significant differences between the baseline ABR threshold values and those of the 7th and 14th days, and also between the ABR threshold values of the 7th and 14th days (p < 0.016) (Table 1) (Fig. 5). When the ABR threshold values of Groups 2 (AK + TRM), 3 (TRM) and 4 (NAT) were compared within each group, no statistically significant differences were found between the baseline, 7th day and 14th day values (p > 0.05) (Table 1) (Fig. 5). In comparing the ABR threshold values between groups, while no statistically significant differences were found between groups for baseline values (p > 0.05), there were statistically significant differences in those values on days 7 and 15 (p = 0.002 and p = 0.001, respectively) (Table 1) (Fig. 5). The ABR threshold values on days 7 and 15 did not show any statistically significant difference between Groups 2 (AK + TRM), 3 (TRM) and 4 (NAT) (p > 0.05). There were statistically significant differences between the 7th and 14th day ABR threshold values of Group 1 (AK) (p < 0.05) (Table 1) (Fig. 5). 3.3. Biochemical parameters
3. Results 3.1. DPOAE There were statistically significant differences between the baseline values of DPOAE and the 7th and 14th day values in Group 1 (AK) (n = 8, 16 ears) (p < 0.016) (Fig. 1). There were no statistically significant differences between the baseline values of DPOAE and the 7th and 14th day values in Groups 2 (AK + TRM),
3.3.1. TOS (total oxidant status) The highest TOS values were measured in Group 1 (AK) (7.19 0.68 mmol H2O2 equiv./L) (Table 2). The TOS values measured in Groups 2 (AK + TRM), 3 (TRM) and 4 (NAT) were significantly lower than those in Group 1 (p < 0.08). TOS values in Groups 2 (AK + TRM), and 3 (TRM) were lower than those in Group 4 (NAT), yet the difference was not statistically significant (p > 0.05) (Table 2).
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Fig. 2. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 2 (AK + TRM) at different time points.
Fig. 3. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 3 (TRM) at different time points.
Fig. 4. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 4 (control group, no active treatment (NAT)) at different time points.
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Table 1 Comparison of ABR thresholds within groups and between groups.
3.3.2. TAS (total antioxidant status) The highest TAS values were encountered in Group 3 (TRM) (1.91 0.079 mmol Trolox equiv./L), and the lowest TAS values were found in Group 1 (AK) (1.43 0.14 mmol Trolox equiv./L) (Table 2). There was a statistically significant difference between TAS values in Groups 1 and 3 (p < 0.08). TAS values in Groups 2 (AK + TRM) and 4 (NAT) were higher than those in Group 1 (AK) but the difference was not statistically significant (p > 0.08) (Table 2). 3.3.3. OSI (oxidant status index) Group 1 (AK) had the highest OSI values (0.047 0.013) (Table 2). OSI values in Groups 2 (AK + TRM) and 3 (TRM) were statistically significantly lower than those in Group 1 (AK) (p < 0.08) (Table 2). OSI values in Groups 2 (AK + TRM) and 3 (TRM) were also statistically significantly lower than those in the control group (Group 4) (p < 0.08).
4. Discussion Among other agents, AG antibiotics are the most frequently used drugs with ototoxic properties [34]. Amikacin is prescribed, especially to children, for febrile neutropenia [35], prophylaxis, sepsis, meningitis, bacteremia [36], urinary infections [37], respiratory tract infections [38] and particularly for microorganisms that are resistant to other aminoglycoside antibiotics [39]. We aimed to use AK in this study since it has such a wide degree of effect. The ear injury caused by AK begins at the base of the cochlea and progresses toward the apex. The injury can progress even further and may damage the stria vascularis and the 8th nerve [40]. The specific injury is brought about through free oxygen radicals generated by AK, and their interaction with phospholipids, membrane proteins and DNA, which results in irreversible damage
Fig. 5. Variations in amplitudes of ABR threshold values in all groups at different time points.
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Table 2 Biochemical parameters (mean standard deviation).
to the outer hair cells, impairing their function and finally leading them to apoptose [6]. Several agents have recently been tested to prevent ototoxicity caused by AK [11,13,41–47]. Even though there has been an increase in research on this topic in the last 10 years, there are still no products that have entered general use. TRM is being used for the treatment of cochleovestibular disturbances as the drug has cytoprotective properties and is an inhibitor of oxygen radicals [48]. AK damages the outer hair cells beginning from the basal region of the cochlea, and as it progresses to the middle and apical regions, lower frequencies are affected; this may lead to permanent damage in speech discrimination scores (SDS) [41]. The damage to outer hair cells can be evaluated by DPOAE testing [24]. In our study, rats that were given AK had significantly lower DPOAE values on the 7th and 14th days compared to their baseline values (p < 0.016) (Fig. 1). This finding is indicative of AK’s deleterious effect on outer hair cells. Also, the finding that there was a significant difference in DPOAE values of the TRM + AK group compared with the AK group on days 7 and 15 (p < 0.008) is indicative of the protective effect that TRM exerts on AK ototoxicity. AK injury to the ear may progress to disturb the 8th nerve. ABR testing is an objective method for evaluating the patency of the auditory pathways proximal to the 8th nerve and the 8th nerve itself [49]. In our study, the 7th and 14th day ABR thresholds were significantly higher than the baseline values in the AK group (p < 0.016) (Table 1). This finding demonstrates the toxic effect of AK on auditory pathways proximal to the cochlea. We have not encountered any clinical signs related to the vestibulotoxic effect. We believe that we did not see any vestibulotoxic effects in the experimental process since AK is primarily cochleotoxic. If we had continued to administer AK, we would probably have encountered potentially vestibulotoxic effects at later stages. The 7th and 14th day ABR values showed a significant difference among the TRM + AK and AK groups (p < 0.008) (Fig. 5); this finding is indicative of the protective effect of TRM against AK ototoxicity. However, a lack of histologic examination, preferably as a histocochleogram under electron microscopy, is a shortcoming of this study. AK-induced free oxygen molecules cause damage to the inner ear [50]. TRM inhibits the production of free oxygen radicals, thus decreasing membrane lipid peroxidation levels [51]. Previous studies have reported a significant correlation between AKinduced hearing loss and antioxidant enzyme activity [52]. In this study, we used objective and sensitive biochemical parameters
that measured the total oxidative/antioxidative balance. No other study in the medical literature has so far evaluated the ototoxic effects of AK biochemically, investigating the TAS, TOS and OSI values. The finding that the TOS values for the TRM + AK group were significantly lower than those of the AK group (p < 0.008) (Table 2) indicates that TRM decreases the oxidative stress caused by AK. TAS values in the TRM group were significantly higher than those in the AK group (p < 0.008); this finding supports previous reports that TRM increases antioxidant activity. TAS values in the TRM + AK group were higher than those in the AK group, but no statistically significant difference was observed between these two groups (p > 0.008) (Table 2). These findings show that TRM by itself does increase antioxidant activity strongly, but since AK increases oxidative stress, TAS values in the TRM + AK group were lower than those in the TRM group. Further studies are needed to see if an increase in TRM dosage may overcome this situation. OSI values in the AK group were higher than in all the other groups (0.047 0.005). OSI values in Groups 2 (AK + TRM) and 3 (TRM) were significantly lower than those in Group 4 (control) (p < 0.008) (Table 2). Various studies have reported that AGs increased oxidative stress, thus causing a toxic effect [12–15]. Our study is the first to introduce a correlation between OSI and AK. AK blocks the calcium-activated potassium channels in the vestibular system [15]. AK acts like an N-methyl-D-aspartate (NMDA) agonist which is a subtype of glutamate receptors, and has cytotoxic effects [15]. TRM inhibits excess accumulation of Na+ and Ca2+ inside cells and prevents K+ escape, which results in a decrease in intracellular edema [53]. TRM exerts its cytoprotective effects through preventing the overstimulation of glutamate, thus preventing its detrimental effects on neurosensorial tissues [48]. These data also support the protective effect of TRM against AK ototoxicity. It has been demonstrated that TRM plays a role in protecting against the ototoxicity of gentamicin and neomycin as a result of its antioxidant and cytoprotective effects [22,23]. The study we conducted demonstrated that TRM was effective against AK ototoxicity and this finding is in line with the literature. Judging by these findings, we are of the opinion that TRM may be effective in the treatment of chemicals, acoustic traumas, and autoimmune events, which may have an ototoxic effect on the inner ear owing to its antioxidant and cytoprotective effects. Currently, there is no drug clinically in use for preventing AKinduced ototoxicity. Our study experimentally demonstrated that TRM could prevent AK ototoxicity. Large prospective randomized studies are needed to confirm the beneficial effects of TRM in this regard and before implementing TRM in routine clinical usage.
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5. Conclusion Numerous agents have been studied previously in the search for protection against AK ototoxicity but TRM was not one of them. This study is the first to investigate TRM’s otoprotective effects, and shows that TRM does exert such an effect. However, additional electron microscopic studies are needed to determine and confirm the relevant histopathologic changes taking place. Since AK is a drug with a high tolerability and minimum side effects, in the light of the findings of this study, it may be beneficial to add TRM to the treatment protocols of AK-prescribed patients. References [1] World Health Organization, Report of an Informal Consultation on Strategies for Prevention of Hearing Impairment from Ototoxic Drugs, vol. 95, WHO/PDH, 1994p. 2. [2] E. Selimoglu, S. Kalkandelen, F. Erdogan, Comparative vestibulotoxicity of different aminoglycosides in the Guinea pigs, Yonsei Med. J. 44 (2003) 517–522. [3] J.A. Segal, P. Skolnick, Polyamine-like actions of aminoglycosides and aminoglycoside derivatives at NMDA receptors, Eur. J. Pharmacol. 347 (1998) 311–317. [4] E.J. Begg, M.L. Barclay, Aminoglycosides – 50 years on, Br. J. Clin. Pharmacol. 39 (1995) 597–603. [5] S. Murillo-Cuesta, J. Contreras, R. Cediel, I. Varela-Nieto, Comparison of different aminoglycoside antibiotic treatments to refine ototoxicity studies in adult mice, Lab. Anim. 44 (2010) 124–131. [6] R.M. Meyers, Ototoxic effects of gentamicin, Arch. Otolaryngol. 95 (1970) 160– 162. [7] M. Strupp, V. Arbusow, Acute vestibulopathy, Curr. Opin. Neurol. 14 (2001) 11–20. [8] M.D. Seidman, R.T. Standring, Noise and quality of life, Int. J. Environ. Res. Public Health 7 (10) (2010) 3730–3738. [9] J. Maruyama, T. Yamagata, M. Ulfendahl, G. Bredberg, R.A. Altschuler, J.M. Miller, Effects of antioxidants on auditory nerve function and survival in deafened guinea pigs, Neurobiol. Dis. 25 (2) (2007) 309–318. [10] J.K. Coleman, R. Kopke, J. Liu, X. Ge, E. Harper, G. Jones, et al., Pharmacological rescue of noise induced hearing loss using N-acetylcysteine and acetyl-L-carnitine, Hear Res. 226 (2007) 104–113. [11] V. Nekrassov, M. Sitges, Vinpocetine protects from aminoglycoside antibioticinduced hearing loss in guinea pig in vivo, Brain Res. 868 (2000) 222–229. [12] A.S. Basile, J.M. Huang, C. Xie, D. Webster, C. Berlin, P. Skolnick, N-methyl-Daspartate antagonists limit aminoglycoside antibiotic-induced hearing loss, Nat. Med. 2 (1996) 1338–1343. [13] B.J. Conlon, J.M. Aran, J.P. Erre, D.W. Smith, Attenuation of aminoglycosideinduced cochlear damage with the metabolic antioxidant alpha-lipoic acid, Hear Res. 128 (1999) 40–44. [14] S.L. Garetz, R.A. Altschuler, J. Schacht, Attenuation of gentamicin ototoxicity by glutathione in the guinea pig in vivo, Hear Res. 77 (1994) 81–87. [15] M. Sharifzadeh, M. Abdollahi, H. Behrooz, B. Minaii, A. Kebriaeezadeh, M.R. Kashani, et al., Effects of chronic lithium on ototoxicity induced by gentamicin and amikacin in guinea pigs, Pharmacol. Toxicol. 83 (1998) 220–224. [16] P.F. Kantor, A. Lucien, R. Kozak, G.D. Lopaschuk, The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase, Circ. Res. 86 (2000) 580–588. [17] C. Guarnieri, C. Muscari, Effect of trimetazidine on mitochondrial function and oxidative damage during reperfusion of ischemic hypertrophied rat myocardium, Pharmacology 46 (1993) 324–331. [18] R.X. Yin, W.W. Liang, T.W. Liu, X.Z. Tao, L.G. Zhu, R. Al-Ghazali, Inhibitory effect of trimetazidine on cardiac myocyte apoptosis in rabbit model of ischemia–reperfusion, Chin. Med. Sci. J. 19 (2004) 242. [19] J. Brucker, Trimetazidine: efficacy in ENT pathology, Gazz. Med. Fr. 89 (1982) 904– 911. [20] V. Dhote, R. Balaraman, Anti-oxidant activity mediated neuroprotective potential of trimetazidine on focal cerebral ischaemia–reperfusion injury in rats, Clin. Exp. Pharmacol. Physiol. 35 (2008) 630–637. [21] M. Marzilli, W.W. Klein, Efficacy and tolerability of trimetazidine in stable angina: a meta-analysis of randomized, double-blind, controlled trials, Coron. Artery Dis. 14 (2003) 171–179. [22] O.F. Unal, S.M. Ghoreishi, A. Atas, N. Akyurek, G. Akyol, B. Gursel, Prevention of gentamicin induced ototoxicity by trimetazidine in animal model, Int. J. Pediatr. Otorhinolaryngol. 69 (2) (2005) 193–199. [23] J. Chang, G.J. Im, S.W. Chae, S.H. Lee, S.Y. Kwon, H.H. Jung, et al., Protective role of trimetazidine against neomycin induced hair cell damage in zebrafish, Clin. Exp. Otorhinolaryngol. 6 (4) (2013) 219–225. [24] P. Klyskens, P. Lambert, D. D’Hooge, Trimetazidine contre be´tahistine dans la maladie de Meniere: Etude a` double insu, Ann. Otolaryngol. Paris 107 (1990) 11–19.
669
[25] M. Wayoff, Etude a` double insu contre place´bo de l’activite´ de la trime´tazidine dans les atteintes cochle´o-vestibulaires, Sem. Hopitaux Paris 60 (1984) 2729– 2739. [26] A. Coyas, The efficacy of trimetazidine in cochleovestibular disorders of ischemic origin: a crossover control versus placebo trial, Ann. Otolaryngol. Chir Cervicofac 107 (1990) 82–87. [27] A. Morgon, A. Pech, P. Labaeye, M. Wayoff, Y. Lacomme, J.P. Be´be´ar, A two-month multicenter double blind placebo controlled study of trimetazidine in tinnitus, Ann. Otolaryngol. Paris 107 (1990) 66–77. [28] J.P. Haguenauer, Trimetazidine in degenerative hypoacusis: effects on hearing and integration, Drugs Today 26 (1990) 59–65. [29] H. Sies, Oxidative stress: introductory remarks, in: H. Sies (Ed.), Oxidative Stress, Academic Press, London, 1985, pp. 1–8. [30] O. Erel, A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation, Clin. Biochem. 37 (2004) 277–285. [31] G. Cao, R.L. Prior, Comparison of different analytical methods for assessing total antioxidant capacity of human serum, Clin. Chem. 44 (1998) 1309–1315. [32] O. Erel, A new automated colorimetric method for measuring total oxidant status, Clin. Biochem. 38 (2005) 1103–1111. [33] Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council, The Guide for the Care and Use of Laboratory Animals, 7th ed., National Academy of Sciences, Washington, DC, 1996. [34] J.A.A. Oliveira, T.M.O. Bernal, Ototoxicidade de Aminoglicı´deos e Otoprotec¸a˜o, in: C.A.H. Campos, H.O.O. Costa (Eds.), Tratado de Otorrinolaringologia, vol. II, Editora Roca, Sa˜o Paulo, 2003, pp. 148–167. [35] K.S. Chen, A. Bach, A. Shoup, N.J. Winick, Hearing loss and vestibular dysfunction among children with cancer after receiving aminoglycosides, Pediatr. Blood Cancer 60 (11) (2013) 1772–1777. [36] P. Jyothi, M.C. Basavaraj, P.V. Basavaraj, Bacteriological profile of neonatal septicemia and antibiotic susceptibility pattern of the isolates, J. Nat. Sci. Biol. Med. 4 (2) (2013) 306–309. [37] M. Dash, S. Padhi, I. Mohanty, P. Panda, B. Parida, Antimicrobial resistance in pathogens causing urinary tract infections in a rural community of Odisha, India, J. Family Community Med. 20 (1) (2013) 20–26. [38] S. Antoniu, D. Azoicai, Novel amikacin inhaled formulation for the treatment of lower respiratory tract infections, Drugs Today (Barc.) 49 (11) (2013) 683–692. [39] D. Wrzes´niok, A. Beberok, M. Otre˛ba, E. Buszman, Modulation of melanogenesis and antioxidant defense system in melanocytes by amikacin, Toxicol. In Vitro 27 (3) (2013) 1102–1108. [40] A. Fausti, A. Henry, I. Schaffer, D.J. Olson, R.H. Frey, W.J. McDonald, High frequency audiometric monitoring for early detection of aminoglycoside ototoxicity, J. Infect. Dis. 165 (1992) 1026–1032. [41] M.C. Miman, O. Ozturan, M. Iraz, T. Erdem, E. Olmez, Amikacin ototoxicity enhanced by Ginkgo biloba extract (EGb 761), Hear Res. 169 (2002) 121–129. [42] T. Erdem, O. Ozturan, M. Iraz, M.C. Miman, E. Olmez, Dose-dependent dual effect of melatonin on ototoxicity induced by amikacin in adult rats, Eur. Arch. Otorhinolaryngol. 262 (2005) 314–321. [43] E. Lecain, B. Omri, F. Behar-Cohen, P. Tran Ba Huy, P. Crisanti, The role of PKCzeta in amikacin-induced apoptosis in the cochlea: prevention by aspirin, Apoptosis 12 (2) (2007) 333–342. [44] G. Berkiten, Z. Salturk, I. Topalog˘lu, H. Ug˘ras¸, Protective effect of pentoxifylline on amikacin-induced ototoxicity in rats, Am. J. Otolaryngol. 33 (6) (2012) 689–692. [45] A. Amora Lde, A. Murashima Ade, M. Rossato, M.B. Moreira, Hyppolito MAˆ, D.J. Fagundes, The effects of hyperbaric oxygen therapy upon ototoxic injuries produced by amikacin in guinea pigs, Braz. J. Otorhinolaryngol. 79 (3) (2013) 342–348. [46] T. Bayindir, A. Filiz, M. Iraz, S. Kaya, M. Tan, M.T. Kalcioglu, Evaluation of the protective effect of beta glucan on amikacin ototoxicity using distortion product otoacoustic emission measurements in rats, Clin. Exp. Otorhinolaryngol. 6 (1) (2013) 1–6. [47] P. Pavlidis, J. Maurer, E. Apostolidou, G. Kekes, D. Kouvelas, Memantine’s action against aminoglycoside-induced ototoxicity, Eur. Arch. Otorhinolaryngol. (2013)[Epub ahead of print]. [48] P. Gil-Loyzaga, E. Herna´ndez, F. Carricondo, F. Simo´n, J. Poch-Broto, Trimetazidine prevents cochlear lesions induced by intraperitoneal and perilymphatic administration of kainic acid, Brain Res. 826 (1999) 95–103. [49] J. Schacht, Aminoglycoside ototoxicity: prevention in sight? Otolaryngol. Head Neck Surg. 118 (1998) 674–677. [50] G.J. Matz, Aminoglycoside cochlear toxicity, Otolaryngol. Clin. North Am. 26 (1993) 705–712. [51] I. Maridonneau-Parini, C. Harpey, Effect of trimetazidine on membrane damage induced by oxygen free radicals in human red cells, Br. J. Clin. Pharmacol. 20 (2) (1985) 148–151. [52] J.J. Klemens, R.P. Meech, L.F. Hughes, S. Somani, K.C. Campbell, Antioxidant enzyme levels inversely covary with hearing loss after amikacin treatment, J. Am. Acad. Audiol. 14 (2003) 134–143. [53] K.J.A. Davies, S.W. Lin, R.E. Pacifici, Protein damage and degradation by oxygen radicals: IV. Degradation of denatured protein, J. Biol. Chem. 262 (1987) 9914–9920.
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International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Protective effect of trimetazidine on amikacin-induced ototoxicity in rats Fadlullah Aksoy a, Remzi Dogan a,*, Orhan Ozturan a, Sabri Baki Eren a, Bayram Veyseller a, ¨ nder Hu¨seyinbas c Alev Pektas b, O a b c
Bezmialem Vakif University, Department of Otorhinolaryngology, Fatih, Istanbul, Turkey Bezmialem Vakif University, Faculty of Health Sciences, Department of Audiology, Fatih, Istanbul, Turkey Bezmialem Vakif University, Research Center, Fatih, Istanbul, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 September 2013 Received in revised form 20 January 2014 Accepted 22 January 2014 Available online 5 February 2014
Objective: Aminoglycoside antibiotics are known to have ototoxic effects and may induce sensorineural hearing loss. This study investigated the protective effect of trimetazidine, which has antioxidant and cytoprotective properties, against amikacin ototoxicity. Methods: Thirty-two male rats were divided into four groups – amikacin, amikacin + trimetazidine, trimetazidine, and control groups. Trimetazidine, 10 mg/kg per day, was given for 14 days by oral gavage. Amikacin, 600 mg/kg per day, was also given for 14 days, by the intramuscular route. Distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) tests were applied to the rats for hearing assessment. At the termination of the study, the biochemical parameters were calculated to evaluate the oxidative status. Results: The DPOAE values of the amikacin group were significantly lower on the 7th and 14th days than those of the trimetazidine + amikacin group and there was an increase in the ABR thresholds. The ABR thresholds for the amikacin group on the 7th and 14th days were significantly higher than the levels on the first day of the study, while there was no significant increase in those values in the trimetazidine + amikacin group. The total oxidant status (TOS) and oxidant status index (OSI) values of the amikacin group were significantly higher than those of the trimetazidine + amikacin group. The total antioxidant status (TAS) values of the amikacin group were lower than those of the trimetazidine + amikacin group. Conclusions: The audiologic tests and biochemical parameters investigated in this study both point to the protective effect of trimetazidine against amikacin-induced ototoxicity. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: ABR Amikacin DPOAE Oxidative status Trimetazidine
1. Introduction Ototoxicity is a clinical condition, usually brought about by the detrimental effects of some chemical agent on the auditory and balance functions of the ear [1]. More than 130 agents are known to have ototoxic effects, with aminoglycoside (AG) and macrolide antibiotics, loop diuretics, NSAIDs, antineoplastic and antimalarial drugs taking the lead [1]. While amikacin (AK), kanamycin and neomycin cause damage primarily to the cochlea, streptomycin and gentamicin primarily cause vestibular damage [2]. Cochlear damage may lead to permanent hearing loss, and vestibular damage may lead to vertigo, ataxia and/or nystagmus [3].
* Corresponding author at: Bezmialem Vakif University, Medical Faculty, Department of Otorhinolaryngology, Fatih, Istanbul, Turkey. Tel.: +90 505 7915844; fax: +90 212 533 2326. E-mail address: [email protected] (R. Dogan). 0165-5876/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2014.01.031
AK is a semi-synthetic AG, produced by the acetylation of kanamycin A. The specific properties of its structure make it resistant to bacterial enzymes which can inactivate natural AGs such as gentamicin, kanamycin and tobramycin, thus it has the broadest spectrum among all AGs. AK is a frequently preferred antibiotic due to its rapid action, broad spectrum, lower bacterial resistance, its synergetic effects with beta-lactam antibiotics, and its lower cost [4]. However, 10–80% of patients who are treated with AK are reported to suffer from its ototoxic effects. The hearing loss is typically neurosensorial, nonsyndromic, bilateral, progressive, and is a high-frequency hearing loss [5]. Since the hair cells of the cochlea cannot regenerate, the hearing loss is irreversible [6]. AK ototoxicity is brought about by the drug’s excitotoxic effects, caused by impairment of mitochondrial protein synthesis and overactivation of glutamatergic receptors (N-methyl-D-aspartate), which increase the formation of free radicals and induce apoptosis [7,8]. There have been numerous studies of oxidative stress leading to sensory neural hearing loss [9–11]. Experimental studies have shown that antioxidant agents may prevent AG ototoxicity [12–16].
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Trimetazidine (TRM) is a selective mitochondrial 3-ketoacyl coenzyme A thiolase (3-KAT) inhibitor. Thus, it decreases free fatty acid (FFA) metabolism and regulates cardiac metabolism. It controls oxidative stress, preserves mitochondrial respiration in ischemia–reperfusion injury, and prevents cardiac ischemia [17,18]. TRM has also been shown to have gastroprotective, hepatoprotective, anti-inflammatory, antinociceptive and anti-apoptotic properties [18,19]. Previous studies have shown that TRM’s cytoprotective effects are brought about by preventing injury in neurosensorial tissue secondary to overstimulation of the cochleovestibular system by glutamate, and by its antioxidant effects [20,21]. In experimental animal studies, it was reported that the ototoxic effect induced by gentamicin and neomycin is prevented by TRM [22,23]. The fields of use of TRM in otorhinolaryngology include the reduction in vertigo duration and frequency [24], treatment of Me´nie`re’s disease related cochleovestibular conditions [25], correction of inner ear ischemia [26], correction of isolated tinnitus [27], and correction of hypoacusis [28]. Clinical studies report that TRM has excellent tolerability [21]. Oxidative stress means disruption of the balance between pro-oxidants and antioxidants in favor of the pro-oxidants [29]. Measuring the amount of different antioxidants individually is difficult, requires a significant amount of time, increases laboratory burden, is high cost and is a complicated technique. Furthermore, there will be complicated interactions among different antioxidants in the serum, therefore, these measurements may not be sufficiently objective. The recently developed method of measuring the total antioxidant status (TAS) enables all antioxidants to be recorded at a very low cost in a short time as a single parameter by a simple measurement in serum [30,31]. Similarly, there are no practical methods for the measurement of individual pro-oxidant molecules, but again, a single parameter, the total oxidant status (TOS), can be measured in serum instead [32]. The purpose of this study was to investigate the protective effect of TRM, which has antioxidant and cytoprotective properties, against AK ototoxicity.
2. Materials and methods 2.1. Animals After obtaining permission for experimental studies from the Local Ethics Committee (2011/39), 32 healthy female Wistar Albino rats weighing 200–240 g were included in the study. Those rats which had a negative Preyer response (when clapping, a rapid movement clearly seen in the bodies of animals indicates a positive Preyer reflex) were examined, and/or those which had ear pathologies (cerumen, otitis media with effusion, acute otitis media, etc.) seen in endoscopic ear examinations were not included in the study. The rats were placed in an environment which was illuminated for 12 h and dark for 12 h, had a temperature of 21 1 8C, with free access to food and water, and with a background noise level of under 50 dB. The animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals [33]. 2.2. Anesthesia Before distortion product otoacoustic emissions (DPOAE) and auditory brainstem responses (ABR) were recorded, all of the rats were anesthetized intraperitoneally with ketamine hydrochloride (45 mg/kg) and xylazine (5 mg/kg).
2.3. Experimental design The 32 rats included in the study were divided into four groups, with eight rats in each group: Group 1 (AK), Group 2 (AK + TRM), Group 3 (TRM), and Group 4 (control group, no active treatment (NAT)). TRM was given to Groups 2 and 3 for 14 days by oral gavage, at a dose of 10 mg/kg per day. Rats in Groups 1 and 2 were administered daily intramuscular injections of AK for 14 days, at a dose of 600 mg/kg per day. Rats in Group 4 were given daily intraperitoneal shots of 1 ml saline solution for 14 days. At the very beginning of the study, and also on the 7th and 14th days, DPOAE and ABR tests were performed on all of the rats. On the 14th day, intracardiac blood samples were drawn from all of the rats to calculate the biochemical parameters. 2.4. DPOAE measurements The GSI Audera device was used for DPOAE measurements in the assessment of the rats’ peripheral hearing system. The smallest sized rubber tip of the tympanometer probe was used for the tests. The emissions were measured in General Diagnostic mode, both as distortion product diagrams (DPgram) and as input/output (I/O) measurements. Otoacoustic emissions were measured using stimuli of different frequencies and intensities. The intensity level of the primary stimuli was adjusted to 65 dB (L1 = L2). Two different frequencies (f1 and f2) were chosen with a ratio of f2/ f1 = 1:10. DPgram measurements were taken at 3000, 4008, 5004, 6000, 6996, 8004, 9012, 10008, 11004 and 12000 Hz frequencies. DPOAEs 3 dB over the 2f1–f2 frequency noise intensity during measurements were accepted as positive. 2.5. ABR measurements ABR measurements were carried out in a silent room, with a Viasys Medelec Synergy instrument, using subcutaneous needle electrodes (Technomed Europe). Stimuli were delivered in alternating polarities with ER-3A insert earphones, using 8 kHz tone-burst stimuli. The filter was set at 30–1500 Hz bandwidth, the repetition rate was set at 21/s, and the time window was set as 25 ms; 1024 samples were obtained for signal averaging. Stimuli were delivered at an intensity level of 80 dB nHL, and were reduced in 20 dB steps until the intensity level approached threshold values. Nearing the threshold, intensity steps were reduced to 10 dB, and the threshold value was determined. A minimum of two tracks were generated for each measurement to check for reproducibility, and for confirmation of the threshold value. The ABR threshold was defined as the minimum intensity level where an ABR wave III was observed. 2.6. TAS, TOS, and OSI measurements Measuring the total antioxidant status (TAS), the combined activity of all antioxidants, provides an evaluation of the overall antioxidant status [30]. Total oxidant status (TOS) is an indicator of the overall oxidant status of the patients [32]. The oxidant status index (OSI) is the ratio of TOS/TAS, and is a means to calculate oxidative stress in the body – comparing TAS to TOS is thought to be a better indicator of the overall oxidative stress. To measure these parameters, blood samples taken from all rats in all groups were centrifuged at 3000 rpm for 15 min, the serum was separated and stored at 80 8C. After all samples had been prepared in this way, TAS and TOS values were calculated with the relevant kit (Rel Assay Diagnostics), and OSI values were calculated with the relevant formula (OSI: TOS/TAS 100).
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Fig. 1. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 1 (AK) at different time points.
2.7. Statistical analysis Statistical analysis was carried out using the Statistical Package for the Social Sciences version 13.0 software for Windows (SPSS Inc., Chicago, IL, USA). All quantitative variables were estimated using measures of central location (i.e. mean and median) and measures of dispersion (i.e. standard deviation (SD)). Data normality was checked using the Kolmogorov–Smirnov tests of normality. 2.7.1. Assessment of DPOAE and ABR results For the comparison within group, the Repeated ANOVA test was applied (the difference within group was considered to be statistically significant if p < 0.05). To determine the days between which there were differences, the Bonferroni test was administered as a post hoc test. Since this was a multiple comparison, the Bonferroni correction was applied and p < 0.016 was accepted as statistically significant. For the comparison between groups, the one-way analysis of variance (ANOVA) test was applied (the difference between groups was considered to be statistically significant if p < 0.05). Tukey’s HSD was administered as a post hoc test to identify between-group differences. Since this was a multiple comparison, the Bonferroni correction was applied and p < 0.008 was accepted as statistically significant. 2.7.2. Assessment of biochemical results One-way of analysis (ANOVA) was used for comparing the data between groups. Tukey’s HSD was administered as a post hoc test. Since this was a multiple comparison, the Bonferroni correction was applied and p < 0.008 was accepted as statistically significant.
3 (TRM) and 4 (NAT) (n = 8 and 16 ears in each group) (p > 0.016) (Figs. 2–4). Comparisons between groups showed that there were no statistically significant differences in the baseline values of DPOAE (p < 0.05), while on the 7th and 14th days of the study, there were statistically significant differences between Group 1 (AK) and the other three groups (p < 0.008). 3.2. ABR A comparison of ABR thresholds of all the groups is included in Table 1. In Group 1 (AK), there were statistically significant differences between the baseline ABR threshold values and those of the 7th and 14th days, and also between the ABR threshold values of the 7th and 14th days (p < 0.016) (Table 1) (Fig. 5). When the ABR threshold values of Groups 2 (AK + TRM), 3 (TRM) and 4 (NAT) were compared within each group, no statistically significant differences were found between the baseline, 7th day and 14th day values (p > 0.05) (Table 1) (Fig. 5). In comparing the ABR threshold values between groups, while no statistically significant differences were found between groups for baseline values (p > 0.05), there were statistically significant differences in those values on days 7 and 15 (p = 0.002 and p = 0.001, respectively) (Table 1) (Fig. 5). The ABR threshold values on days 7 and 15 did not show any statistically significant difference between Groups 2 (AK + TRM), 3 (TRM) and 4 (NAT) (p > 0.05). There were statistically significant differences between the 7th and 14th day ABR threshold values of Group 1 (AK) (p < 0.05) (Table 1) (Fig. 5). 3.3. Biochemical parameters
3. Results 3.1. DPOAE There were statistically significant differences between the baseline values of DPOAE and the 7th and 14th day values in Group 1 (AK) (n = 8, 16 ears) (p < 0.016) (Fig. 1). There were no statistically significant differences between the baseline values of DPOAE and the 7th and 14th day values in Groups 2 (AK + TRM),
3.3.1. TOS (total oxidant status) The highest TOS values were measured in Group 1 (AK) (7.19 0.68 mmol H2O2 equiv./L) (Table 2). The TOS values measured in Groups 2 (AK + TRM), 3 (TRM) and 4 (NAT) were significantly lower than those in Group 1 (p < 0.08). TOS values in Groups 2 (AK + TRM), and 3 (TRM) were lower than those in Group 4 (NAT), yet the difference was not statistically significant (p > 0.05) (Table 2).
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Fig. 2. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 2 (AK + TRM) at different time points.
Fig. 3. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 3 (TRM) at different time points.
Fig. 4. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 4 (control group, no active treatment (NAT)) at different time points.
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Table 1 Comparison of ABR thresholds within groups and between groups.
3.3.2. TAS (total antioxidant status) The highest TAS values were encountered in Group 3 (TRM) (1.91 0.079 mmol Trolox equiv./L), and the lowest TAS values were found in Group 1 (AK) (1.43 0.14 mmol Trolox equiv./L) (Table 2). There was a statistically significant difference between TAS values in Groups 1 and 3 (p < 0.08). TAS values in Groups 2 (AK + TRM) and 4 (NAT) were higher than those in Group 1 (AK) but the difference was not statistically significant (p > 0.08) (Table 2). 3.3.3. OSI (oxidant status index) Group 1 (AK) had the highest OSI values (0.047 0.013) (Table 2). OSI values in Groups 2 (AK + TRM) and 3 (TRM) were statistically significantly lower than those in Group 1 (AK) (p < 0.08) (Table 2). OSI values in Groups 2 (AK + TRM) and 3 (TRM) were also statistically significantly lower than those in the control group (Group 4) (p < 0.08).
4. Discussion Among other agents, AG antibiotics are the most frequently used drugs with ototoxic properties [34]. Amikacin is prescribed, especially to children, for febrile neutropenia [35], prophylaxis, sepsis, meningitis, bacteremia [36], urinary infections [37], respiratory tract infections [38] and particularly for microorganisms that are resistant to other aminoglycoside antibiotics [39]. We aimed to use AK in this study since it has such a wide degree of effect. The ear injury caused by AK begins at the base of the cochlea and progresses toward the apex. The injury can progress even further and may damage the stria vascularis and the 8th nerve [40]. The specific injury is brought about through free oxygen radicals generated by AK, and their interaction with phospholipids, membrane proteins and DNA, which results in irreversible damage
Fig. 5. Variations in amplitudes of ABR threshold values in all groups at different time points.
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Table 2 Biochemical parameters (mean standard deviation).
to the outer hair cells, impairing their function and finally leading them to apoptose [6]. Several agents have recently been tested to prevent ototoxicity caused by AK [11,13,41–47]. Even though there has been an increase in research on this topic in the last 10 years, there are still no products that have entered general use. TRM is being used for the treatment of cochleovestibular disturbances as the drug has cytoprotective properties and is an inhibitor of oxygen radicals [48]. AK damages the outer hair cells beginning from the basal region of the cochlea, and as it progresses to the middle and apical regions, lower frequencies are affected; this may lead to permanent damage in speech discrimination scores (SDS) [41]. The damage to outer hair cells can be evaluated by DPOAE testing [24]. In our study, rats that were given AK had significantly lower DPOAE values on the 7th and 14th days compared to their baseline values (p < 0.016) (Fig. 1). This finding is indicative of AK’s deleterious effect on outer hair cells. Also, the finding that there was a significant difference in DPOAE values of the TRM + AK group compared with the AK group on days 7 and 15 (p < 0.008) is indicative of the protective effect that TRM exerts on AK ototoxicity. AK injury to the ear may progress to disturb the 8th nerve. ABR testing is an objective method for evaluating the patency of the auditory pathways proximal to the 8th nerve and the 8th nerve itself [49]. In our study, the 7th and 14th day ABR thresholds were significantly higher than the baseline values in the AK group (p < 0.016) (Table 1). This finding demonstrates the toxic effect of AK on auditory pathways proximal to the cochlea. We have not encountered any clinical signs related to the vestibulotoxic effect. We believe that we did not see any vestibulotoxic effects in the experimental process since AK is primarily cochleotoxic. If we had continued to administer AK, we would probably have encountered potentially vestibulotoxic effects at later stages. The 7th and 14th day ABR values showed a significant difference among the TRM + AK and AK groups (p < 0.008) (Fig. 5); this finding is indicative of the protective effect of TRM against AK ototoxicity. However, a lack of histologic examination, preferably as a histocochleogram under electron microscopy, is a shortcoming of this study. AK-induced free oxygen molecules cause damage to the inner ear [50]. TRM inhibits the production of free oxygen radicals, thus decreasing membrane lipid peroxidation levels [51]. Previous studies have reported a significant correlation between AKinduced hearing loss and antioxidant enzyme activity [52]. In this study, we used objective and sensitive biochemical parameters
that measured the total oxidative/antioxidative balance. No other study in the medical literature has so far evaluated the ototoxic effects of AK biochemically, investigating the TAS, TOS and OSI values. The finding that the TOS values for the TRM + AK group were significantly lower than those of the AK group (p < 0.008) (Table 2) indicates that TRM decreases the oxidative stress caused by AK. TAS values in the TRM group were significantly higher than those in the AK group (p < 0.008); this finding supports previous reports that TRM increases antioxidant activity. TAS values in the TRM + AK group were higher than those in the AK group, but no statistically significant difference was observed between these two groups (p > 0.008) (Table 2). These findings show that TRM by itself does increase antioxidant activity strongly, but since AK increases oxidative stress, TAS values in the TRM + AK group were lower than those in the TRM group. Further studies are needed to see if an increase in TRM dosage may overcome this situation. OSI values in the AK group were higher than in all the other groups (0.047 0.005). OSI values in Groups 2 (AK + TRM) and 3 (TRM) were significantly lower than those in Group 4 (control) (p < 0.008) (Table 2). Various studies have reported that AGs increased oxidative stress, thus causing a toxic effect [12–15]. Our study is the first to introduce a correlation between OSI and AK. AK blocks the calcium-activated potassium channels in the vestibular system [15]. AK acts like an N-methyl-D-aspartate (NMDA) agonist which is a subtype of glutamate receptors, and has cytotoxic effects [15]. TRM inhibits excess accumulation of Na+ and Ca2+ inside cells and prevents K+ escape, which results in a decrease in intracellular edema [53]. TRM exerts its cytoprotective effects through preventing the overstimulation of glutamate, thus preventing its detrimental effects on neurosensorial tissues [48]. These data also support the protective effect of TRM against AK ototoxicity. It has been demonstrated that TRM plays a role in protecting against the ototoxicity of gentamicin and neomycin as a result of its antioxidant and cytoprotective effects [22,23]. The study we conducted demonstrated that TRM was effective against AK ototoxicity and this finding is in line with the literature. Judging by these findings, we are of the opinion that TRM may be effective in the treatment of chemicals, acoustic traumas, and autoimmune events, which may have an ototoxic effect on the inner ear owing to its antioxidant and cytoprotective effects. Currently, there is no drug clinically in use for preventing AKinduced ototoxicity. Our study experimentally demonstrated that TRM could prevent AK ototoxicity. Large prospective randomized studies are needed to confirm the beneficial effects of TRM in this regard and before implementing TRM in routine clinical usage.
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5. Conclusion Numerous agents have been studied previously in the search for protection against AK ototoxicity but TRM was not one of them. This study is the first to investigate TRM’s otoprotective effects, and shows that TRM does exert such an effect. However, additional electron microscopic studies are needed to determine and confirm the relevant histopathologic changes taking place. Since AK is a drug with a high tolerability and minimum side effects, in the light of the findings of this study, it may be beneficial to add TRM to the treatment protocols of AK-prescribed patients. References [1] World Health Organization, Report of an Informal Consultation on Strategies for Prevention of Hearing Impairment from Ototoxic Drugs, vol. 95, WHO/PDH, 1994p. 2. [2] E. Selimoglu, S. Kalkandelen, F. Erdogan, Comparative vestibulotoxicity of different aminoglycosides in the Guinea pigs, Yonsei Med. J. 44 (2003) 517–522. [3] J.A. Segal, P. Skolnick, Polyamine-like actions of aminoglycosides and aminoglycoside derivatives at NMDA receptors, Eur. J. Pharmacol. 347 (1998) 311–317. [4] E.J. Begg, M.L. Barclay, Aminoglycosides – 50 years on, Br. J. Clin. Pharmacol. 39 (1995) 597–603. [5] S. Murillo-Cuesta, J. Contreras, R. Cediel, I. Varela-Nieto, Comparison of different aminoglycoside antibiotic treatments to refine ototoxicity studies in adult mice, Lab. Anim. 44 (2010) 124–131. [6] R.M. Meyers, Ototoxic effects of gentamicin, Arch. Otolaryngol. 95 (1970) 160– 162. [7] M. Strupp, V. Arbusow, Acute vestibulopathy, Curr. Opin. Neurol. 14 (2001) 11–20. [8] M.D. Seidman, R.T. Standring, Noise and quality of life, Int. J. Environ. Res. Public Health 7 (10) (2010) 3730–3738. [9] J. Maruyama, T. Yamagata, M. Ulfendahl, G. Bredberg, R.A. Altschuler, J.M. Miller, Effects of antioxidants on auditory nerve function and survival in deafened guinea pigs, Neurobiol. Dis. 25 (2) (2007) 309–318. [10] J.K. Coleman, R. Kopke, J. Liu, X. Ge, E. Harper, G. Jones, et al., Pharmacological rescue of noise induced hearing loss using N-acetylcysteine and acetyl-L-carnitine, Hear Res. 226 (2007) 104–113. [11] V. Nekrassov, M. Sitges, Vinpocetine protects from aminoglycoside antibioticinduced hearing loss in guinea pig in vivo, Brain Res. 868 (2000) 222–229. [12] A.S. Basile, J.M. Huang, C. Xie, D. Webster, C. Berlin, P. Skolnick, N-methyl-Daspartate antagonists limit aminoglycoside antibiotic-induced hearing loss, Nat. Med. 2 (1996) 1338–1343. [13] B.J. Conlon, J.M. Aran, J.P. Erre, D.W. Smith, Attenuation of aminoglycosideinduced cochlear damage with the metabolic antioxidant alpha-lipoic acid, Hear Res. 128 (1999) 40–44. [14] S.L. Garetz, R.A. Altschuler, J. Schacht, Attenuation of gentamicin ototoxicity by glutathione in the guinea pig in vivo, Hear Res. 77 (1994) 81–87. [15] M. Sharifzadeh, M. Abdollahi, H. Behrooz, B. Minaii, A. Kebriaeezadeh, M.R. Kashani, et al., Effects of chronic lithium on ototoxicity induced by gentamicin and amikacin in guinea pigs, Pharmacol. Toxicol. 83 (1998) 220–224. [16] P.F. Kantor, A. Lucien, R. Kozak, G.D. Lopaschuk, The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase, Circ. Res. 86 (2000) 580–588. [17] C. Guarnieri, C. Muscari, Effect of trimetazidine on mitochondrial function and oxidative damage during reperfusion of ischemic hypertrophied rat myocardium, Pharmacology 46 (1993) 324–331. [18] R.X. Yin, W.W. Liang, T.W. Liu, X.Z. Tao, L.G. Zhu, R. Al-Ghazali, Inhibitory effect of trimetazidine on cardiac myocyte apoptosis in rabbit model of ischemia–reperfusion, Chin. Med. Sci. J. 19 (2004) 242. [19] J. Brucker, Trimetazidine: efficacy in ENT pathology, Gazz. Med. Fr. 89 (1982) 904– 911. [20] V. Dhote, R. Balaraman, Anti-oxidant activity mediated neuroprotective potential of trimetazidine on focal cerebral ischaemia–reperfusion injury in rats, Clin. Exp. Pharmacol. Physiol. 35 (2008) 630–637. [21] M. Marzilli, W.W. Klein, Efficacy and tolerability of trimetazidine in stable angina: a meta-analysis of randomized, double-blind, controlled trials, Coron. Artery Dis. 14 (2003) 171–179. [22] O.F. Unal, S.M. Ghoreishi, A. Atas, N. Akyurek, G. Akyol, B. Gursel, Prevention of gentamicin induced ototoxicity by trimetazidine in animal model, Int. J. Pediatr. Otorhinolaryngol. 69 (2) (2005) 193–199. [23] J. Chang, G.J. Im, S.W. Chae, S.H. Lee, S.Y. Kwon, H.H. Jung, et al., Protective role of trimetazidine against neomycin induced hair cell damage in zebrafish, Clin. Exp. Otorhinolaryngol. 6 (4) (2013) 219–225. [24] P. Klyskens, P. Lambert, D. D’Hooge, Trimetazidine contre be´tahistine dans la maladie de Meniere: Etude a` double insu, Ann. Otolaryngol. Paris 107 (1990) 11–19.
669
[25] M. Wayoff, Etude a` double insu contre place´bo de l’activite´ de la trime´tazidine dans les atteintes cochle´o-vestibulaires, Sem. Hopitaux Paris 60 (1984) 2729– 2739. [26] A. Coyas, The efficacy of trimetazidine in cochleovestibular disorders of ischemic origin: a crossover control versus placebo trial, Ann. Otolaryngol. Chir Cervicofac 107 (1990) 82–87. [27] A. Morgon, A. Pech, P. Labaeye, M. Wayoff, Y. Lacomme, J.P. Be´be´ar, A two-month multicenter double blind placebo controlled study of trimetazidine in tinnitus, Ann. Otolaryngol. Paris 107 (1990) 66–77. [28] J.P. Haguenauer, Trimetazidine in degenerative hypoacusis: effects on hearing and integration, Drugs Today 26 (1990) 59–65. [29] H. Sies, Oxidative stress: introductory remarks, in: H. Sies (Ed.), Oxidative Stress, Academic Press, London, 1985, pp. 1–8. [30] O. Erel, A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation, Clin. Biochem. 37 (2004) 277–285. [31] G. Cao, R.L. Prior, Comparison of different analytical methods for assessing total antioxidant capacity of human serum, Clin. Chem. 44 (1998) 1309–1315. [32] O. Erel, A new automated colorimetric method for measuring total oxidant status, Clin. Biochem. 38 (2005) 1103–1111. [33] Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council, The Guide for the Care and Use of Laboratory Animals, 7th ed., National Academy of Sciences, Washington, DC, 1996. [34] J.A.A. Oliveira, T.M.O. Bernal, Ototoxicidade de Aminoglicı´deos e Otoprotec¸a˜o, in: C.A.H. Campos, H.O.O. Costa (Eds.), Tratado de Otorrinolaringologia, vol. II, Editora Roca, Sa˜o Paulo, 2003, pp. 148–167. [35] K.S. Chen, A. Bach, A. Shoup, N.J. Winick, Hearing loss and vestibular dysfunction among children with cancer after receiving aminoglycosides, Pediatr. Blood Cancer 60 (11) (2013) 1772–1777. [36] P. Jyothi, M.C. Basavaraj, P.V. Basavaraj, Bacteriological profile of neonatal septicemia and antibiotic susceptibility pattern of the isolates, J. Nat. Sci. Biol. Med. 4 (2) (2013) 306–309. [37] M. Dash, S. Padhi, I. Mohanty, P. Panda, B. Parida, Antimicrobial resistance in pathogens causing urinary tract infections in a rural community of Odisha, India, J. Family Community Med. 20 (1) (2013) 20–26. [38] S. Antoniu, D. Azoicai, Novel amikacin inhaled formulation for the treatment of lower respiratory tract infections, Drugs Today (Barc.) 49 (11) (2013) 683–692. [39] D. Wrzes´niok, A. Beberok, M. Otre˛ba, E. Buszman, Modulation of melanogenesis and antioxidant defense system in melanocytes by amikacin, Toxicol. In Vitro 27 (3) (2013) 1102–1108. [40] A. Fausti, A. Henry, I. Schaffer, D.J. Olson, R.H. Frey, W.J. McDonald, High frequency audiometric monitoring for early detection of aminoglycoside ototoxicity, J. Infect. Dis. 165 (1992) 1026–1032. [41] M.C. Miman, O. Ozturan, M. Iraz, T. Erdem, E. Olmez, Amikacin ototoxicity enhanced by Ginkgo biloba extract (EGb 761), Hear Res. 169 (2002) 121–129. [42] T. Erdem, O. Ozturan, M. Iraz, M.C. Miman, E. Olmez, Dose-dependent dual effect of melatonin on ototoxicity induced by amikacin in adult rats, Eur. Arch. Otorhinolaryngol. 262 (2005) 314–321. [43] E. Lecain, B. Omri, F. Behar-Cohen, P. Tran Ba Huy, P. Crisanti, The role of PKCzeta in amikacin-induced apoptosis in the cochlea: prevention by aspirin, Apoptosis 12 (2) (2007) 333–342. [44] G. Berkiten, Z. Salturk, I. Topalog˘lu, H. Ug˘ras¸, Protective effect of pentoxifylline on amikacin-induced ototoxicity in rats, Am. J. Otolaryngol. 33 (6) (2012) 689–692. [45] A. Amora Lde, A. Murashima Ade, M. Rossato, M.B. Moreira, Hyppolito MAˆ, D.J. Fagundes, The effects of hyperbaric oxygen therapy upon ototoxic injuries produced by amikacin in guinea pigs, Braz. J. Otorhinolaryngol. 79 (3) (2013) 342–348. [46] T. Bayindir, A. Filiz, M. Iraz, S. Kaya, M. Tan, M.T. Kalcioglu, Evaluation of the protective effect of beta glucan on amikacin ototoxicity using distortion product otoacoustic emission measurements in rats, Clin. Exp. Otorhinolaryngol. 6 (1) (2013) 1–6. [47] P. Pavlidis, J. Maurer, E. Apostolidou, G. Kekes, D. Kouvelas, Memantine’s action against aminoglycoside-induced ototoxicity, Eur. Arch. Otorhinolaryngol. (2013)[Epub ahead of print]. [48] P. Gil-Loyzaga, E. Herna´ndez, F. Carricondo, F. Simo´n, J. Poch-Broto, Trimetazidine prevents cochlear lesions induced by intraperitoneal and perilymphatic administration of kainic acid, Brain Res. 826 (1999) 95–103. [49] J. Schacht, Aminoglycoside ototoxicity: prevention in sight? Otolaryngol. Head Neck Surg. 118 (1998) 674–677. [50] G.J. Matz, Aminoglycoside cochlear toxicity, Otolaryngol. Clin. North Am. 26 (1993) 705–712. [51] I. Maridonneau-Parini, C. Harpey, Effect of trimetazidine on membrane damage induced by oxygen free radicals in human red cells, Br. J. Clin. Pharmacol. 20 (2) (1985) 148–151. [52] J.J. Klemens, R.P. Meech, L.F. Hughes, S. Somani, K.C. Campbell, Antioxidant enzyme levels inversely covary with hearing loss after amikacin treatment, J. Am. Acad. Audiol. 14 (2003) 134–143. [53] K.J.A. Davies, S.W. Lin, R.E. Pacifici, Protein damage and degradation by oxygen radicals: IV. Degradation of denatured protein, J. Biol. Chem. 262 (1987) 9914–9920.
