- Email: [email protected]
Quercetine attenuates the gentamicin-induced ototoxicity in a rat model
International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Quercetine attenuates the gentamicin-induced ototoxicity in a rat model§ Mustafa Sagit a,*, Ferhat Korkmaz b, Seren Gulsen Gu¨rgen c, Ramazan Gundogdu a, Alper Akcadag d, Ibrahim Ozcan a a
Kayseri Training and Research Hospital, Department of ENT, Kayseri, Turkey Sanliurfa Training and Research Hospital, Department of ENT, S¸anlıurfa, Turkey c Celal Bayar University, School of Vocational Health Service, Department of Histology and Embryology, Manisa, Turkey d Kayseri Training and Research Hospital, Subdepartment of Audiology, Kayseri, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2015 Received in revised form 8 September 2015 Accepted 18 September 2015 Available online 28 September 2015
Objectives: The aim of this study is to evaluate the protective role of quercetin in gentamicin-induced ototoxicity through an auditory brainstem response (ABR) test and a histopathological evaluation of the cochlea. Methods: In this study, 48 female adult Sprague–Dawley rats aged 20–22 weeks and weighing 200–250 g were used. An ABR test was carried out on all rats prior to drug administration, after which, the rats were divided into four groups of 12 animals each. Drug administration was gentamicin 120 mg/ kg plus ethanol in group one; gentamicin 120 mg/kg plus quercetin 15 mg/kg in group two; quercetin 15 mg/kg in group three; and ethanol in group four. The drugs were administered intraperitoneally once a day for two weeks, and the ABR test was repeated after drug administration. Subsequently, the rats were sacrificed and their cochleae were dissected and examined histopathologically. Results: There was no significant difference between the pre-treatment ABR measurement values of the groups. However, a significant increase was detected in the ABR values in the group of rats that were administered gentamicin plus ethanol, while no statistically significant increase was found in the ABR values in the groups administered with gentamicin plus quercetin; quercetin alone; and ethanol alone. The number of TUNEL positive cells in the inner and outer hair cells in the Corti organ was found to be fewer, and Caspase 3 and 9 expressions were found to be weaker in the group receiving gentamicin plus quercetin than in the group receiving gentamicin plus ethanol. Conclusions: Auditory function was detected to be significantly protected and apoptotic cells were found to be decreased when quercetin was administered together with gentamicin. From these results it was concluded that quercetin, a powerful antioxidant, attenuates ABR thresholds and histopathological lesions in the cochlea in gentamicin-induced ototoxicity in rats. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Quercetin Gentamicin Auditory brainstem response
1. Introduction Ototoxicity is a clinical condition related to hearing loss due to damaged inner ear structures secondary to drugs, chemical material and external stimuli such as noise and infection, and leads to such symptoms as balance disorder and tinnitus [1]. The aminoglycoside group of antibiotics is in wide use around the
§ This study was presented in ‘3rd Turkish National Otology and Neurootology Congress’ between May 1 and 4, 2014 Antalya/Turkey. * Corresponding author. Tel.: +90 352 336 88 84; fax: +90 352 320 73 13. E-mail address: [email protected] (M. Sagit).
http://dx.doi.org/10.1016/j.ijporl.2015.09.023 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
world, having been developed to tackle tuberculosis and advanced bacterial infections, intratympanic gentamicin therapy is also used for Meniere’s disease, although their use is limited due to major side effects of ototoxicity and nephrotoxicity [2,3]. Ototoxicity secondary to gentamicin use is frequently bilateral, symmetrical and irreversible. Hearing loss starts initially in the high frequencies; however other frequencies are also affected with continued exposure. The frequency of ototoxicity due to gentamicin varies between 2% and 25% [4,5]. Many clinical and experimental studies have been made with the aim of protecting the inner ear from the potential toxic effects of gentamicin, in which agents such as iron chelators (deferoxamine and dihydroxybenzoate), glutathione, alpha-tocopherol,
2110
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
alpha lipoic acid, D-methionine, dexamethasone, trimetazidine, geranylgeranyl acetone, N-acetylcysteine, estradiol (E2) and thymoquinone have demonstrated a protective role against the ototoxicity of gentamicin [6–17]. Flavonoids are compounds with useful biochemical and antioxidant activity that are widely and abundantly present in vegetarian food, being found mainly in vegetables, fruits, tea, onions and legumes, and give color to most flowers and fruits. One of the most important effects of flavonoids is the function of removing free radicals, while the best-defined characteristic of almost all flavonoid groups is their anti-oxidant capacity [18]. Quercetin (3,5,7,30 ,40 -pentahydroxyflavone) is an main member of flavonoids, having a powerful antioxidant efficacy compared to others. Quercetin has various biological effects, being antiinflammatory, anti-proliferative, anti-viral, anti-allergic, antithrombotic, anti-atherosclerotic and anti-tumoral, in addition to its anti-oxidant features [19–23]. Quercetin has been demonstrated to have hepatoprotective, neuroprotective and nephroprotective functions in many studies, although it is unknown whether quercetin has an otoprotective effect when used alone [24–26]. In the present study, it was aimed to evaluate the possible protective role of quercetin in gentamicin-induced ototoxicity using an auditory brainstem response (ABR) test and a histopathological evaluation of the cochlea.
Drugs were administered once daily to all groups according to the above mentioned protocol for two weeks. At the 14th day, ABR measurements were repeated after drug administration under general anesthesia, and the rats were subsequently sacrificed. The temporal bullae of the rats were bilaterally dissected and stored in 10% formaldehyde for histopathological examination. The ABR values before and after drug administration were compared; thus cochlear toxicity was examined both electrophysiologically and histomorphologically. The individuals carrying out the ABR measurements and histopathological examinations were blind to the experiment groups. 2.3. ABR test
This study was approved by the Ethics Committee on Animal Experiment and Research of Erciyes University (13/59). The study was carried out at the Laboratory for Experimental Animals of Erciyes University.
ABR measurement was carried out in a quiet room in both ears of the anesthetized rats using an interacoustics EP-25 equipment (Interacoustics, Denmark, 2001) and ABR 3A ear phones. ABR responses were recorded using subdermal needle electrodes (Technomed Europe, Holland). Active electrodes were placed on the vertex, the ground electrode on the contralateral mastoid and the reference electrode on the ipsilateral mastoid. Click stimulus was used as the auditory stimulant. For the click stimulus, a 100–3000 Hz band-pass filter at a repeat rate of 21 s1 was set. Normal hearing was recorded when a normal ABR configuration was detected at 10 dBnHL. Hearing thresholds were defined starting from 70 dBnHL, decreasing in 20 dB increments each time. When a behavior was not achieved at the level of 70 dBnHL, the level of stimulus was set to 90 dBnHL. Behavior repeatability was tested by repeating the measurement at least twice and the threshold was elicited. The ABR threshold was defined as the lowest level of intensity at which a V wave was observed.
2.1. Animals
2.4. Histomorphological examination
The study was performed on 48 (96 ears) female adult Sprague– Dawley rats, produced in the Experimental Clinical Research Center of Erciyes University, aged 20–22 weeks and weighing 200–250 g. The study animals were accommodated in safe cages with no limitations in food (pellet and water) at a constant temperature of 21 8C on 12-h day/12-h night cycle.
Tissues extracted were stored in neutral formalin for 24 h and fixed, after which they were stored in an EDTA solution of 0.1 mol/l for three weeks for the decalcification of bone tissues. Tissues were washed under running water for 24 h following this procedure, and then dehydrated using an ethanol series classified according to the routine protocol. The tissues were then cleared in xylene and dried for placement in paraffin wax.
2. Material and methods
2.2. Study design and experiment groups Animals were sedated using an intraperitoneal (i.p.) combination of ketamine hydrochloride 40 mg/kg (Ketalar, Eczacıbasi, Turkey) and xylazine 10 mg/kg (Rompun, Bayer, Germany). The external ear canals and tympanic membranes of all rats were examined microscopically at the initiation of the study, and any debris and/or earwax found present in the external ear canal were removed. At the beginning of the study totally three rats that were found to have serous otitis media (2 rats) and tympanic membrane perforation (1 rat) were excluded from the study. Following an otomicroscopic examination of all rats, the presence of normal hearing was analyzed by measuring ABR thresholds in bilateral ears. Included in the study were 96 ears of 48 rats that were found to have a normal hearing threshold in ABR measurements, and these 48 rats were subsequently divided randomly into four groups. Group 1 (n = 12) received i.p. gentamicin 120 mg/kg (Genta 40 mg ampule, I.E Ulagay, Turkey) plus i.p. 1 ml 20% ethanol solution; group 2 (n = 12) received i.p. gentamicin 120 mg/kg plus quercetin 15 mg/kg (Sigma-Aldrich Chemical Co., St. Louis, MO, USA; dissolved in 1 ml 20% ethanol solution); group 3 (n = 12) received i.p. quercetin 15 mg/kg and group 4 (n = 12) received i.p. 1 ml 20% ethanol solution.
2.4.1. TUNEL method Terminal deoxynucleotidyl nick-end labeling (TUNEL) stain is the method that shows the last stage of the nuclear fragmentation of apoptosis. The cytoplasm of cells underwent apoptosis are observed reduced, membrane vesicles and brown like as apoptotic bodies with TUNEL method. Sections that were deparafinized and rehydrated as stated above were stained using a commercial kit (Apoptag, S7101, Chemicon, CA, USA) according to the manufacturer’s instructions. The sections that were stained using the TUNEL technique were evaluated using a CX41 radiant field microscope (Olympus, Tokyo, Japan). TUNEL scoring was made by two independent investigators who were blind to the experiment information. The number of positive immune reactive cells was analyzed, starting from the apical region and ending at the basal region. The mean number of apoptotic cells was defined by counting the TUNEL positive cells in randomly selected areas for each case. In each case, the number of TUNEL positive or negative cells was calculated so that the total number would be 100 cells, and TUNEL positive cells were expressed in percentages. Cells in necrotic areas, in areas with weak morphology or at the borders of the sections were excluded. Figures were obtained from the basal turn of the cochlea.
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
2.4.2. Immunohistochemical method Sections of 5 mm were obtained from the tissue paraffin blocks and were prepared for immunohistochemical staining. The prepared slides were stained with the primary antibodies of Caspase-3 (1:100, LabVision, USA) and Caspase-9 (1:100, LabVision, USA) using immunohistochemical methods. Tagging and background staining was carried out using DAB and Mayer’s hematoxylene dye, respectively. An analysis of the sections was made under an Olympus (CX41, Tokyo, Japan) light microscope, starting from the apical region and ending at the basal region, and figures were obtained from the basal turn of the cochlea. The control samples were processed using the same procedures; however, the duration of incubation with the primary antibody was omitted. The staining intensity of the preparates with immunohistochemical protocol was evaluated as semi-qualitative and an HSCORE was calculated using the following equation: HSCORE = SPi(i + 1); i whose staining intensity is expressed as 1, 2 or 3 (weak, moderate and strong, respectively) and percentage of cells for each intensity is expressed as Pi, changing from 0% to 100%.
2111
in the ABR threshold value post-drug administration in group 1 when compared to the other groups (Table 1). 3.2. Histomorphological results It was noted that intense TUNEL positive cells were found in the outer hair cells of the corti organ of group 1, and that the number of TUNEL positive cells was lower in the corti organ of group 2. The number of TUNEL positive cells was found to be low in the corti organ of groups 3 and 4, and the number of TUNEL positive cells was statistically significantly lower in group 2 when compared to group 1 (p < 0.001) (Fig. 1). Strong caspase-3 and caspase-9 expressions was observed in all corti organs in group 1, while caspase-3 and caspase-9 expressions were found with moderate intensity in the inner and outer hair cells of group 2. Generally, weak caspase-3 and caspase-9 expressions were observed in especially the inner and outer hair cells of the corti organ of groups 3 and 4 (Figs. 2 and 3). Caspase-3 and caspase-9 expressions were found to be statistically weaker in group 2 when compared to group 1 (p < 0.001).
2.5. Statistical analysis 4. Discussion A statistical analysis to evaluate the findings of the study was made using an SPSS version 16.0 package program (SPSS Inc., Chicago, Illinois, USA). One-way Anova test was used to compare the ABR test results between the groups before and after drug administration. A Bonferroni posthoc test was used to detect the group causing the difference, and intra-group comparisons of the ABR test results were made using a Student’s t-test. Betweengroups comparisons of the number of TUNEL positive cells and Caspase 3 and 9 expressions were made using an independent samples t-test, with p < 0.05 accepted as statistically significant. 3. Results In the course of the study, two rats from group 1 and one rat each from the groups 3 and 4 died under anesthesia, and so the study was completed with 44 rats. Auditory and histomorphological evaluations were made in 88 ears of 44 rats. 3.1. ABR test results No statistically significant differences in the ABR threshold values were found between the groups prior to drug administration. A statistically significant increase was detected in the ABR threshold values post-drug administration compared to the predrug administration in group 1, while no statistically significant differences were found in the ABR threshold levels pre- and postdrug administration in groups 2, 3 and 4 (Table 1). When the ABR threshold values of the groups were compared following treatment, a statistically significant difference was identified between the groups, with a significant increase found
Table 1 Auditory brainstem response thresholds before and after application of drugs. Group Gentamicin plus etanol Gentamicin plus quercetin Quercetin Etanol p (ANOVA)
Pretreatment 14.00 5.02 15.00 5.10 15.90 5.90 16.36 4.92 0.481
Post-treatment 47.50 15.85 16.66 6.37 18.18 9.57 16.81 7.16 p < 0.001
**, +,8
p <0.001* 0.162 0.171 0.789
8 p < 0.001 (post hoc Bonferroni) compared with group etanol. * Statistical analysis is performed with paired samples t test p < 0.05. ** p < 0.001 (post hoc Bonferroni) compared with group gentamicin plus quercetin. + p < 0.001 (Post hoc Bonferroni) compared with group quercetin.
In the present study, the possible protective role of quercetin in ototoxicity due to gentamicin use was examined. A significant increase was observed following drug administration in the ABR threshold values of the group that received gentamicin plus ethanol. The cochlear histomorphological findings were parallel to these results, thus confirming ototoxicity. In the group receiving gentamicin plus quercetin, on the other hand, the ABR threshold values were found to be preserved. Upon a histomorphological evaluation, the number of TUNEL positive cells was observed to be lower in the group receiving gentamicin plus quercetin when compared to the group receiving gentamicin plus ethanol. In addition, caspase-3 and caspase-9 expressions in the inner and outer hair cells in the group receiving gentamicin plus quercetin were found to be weaker when compared to the group receiving gentamicin plus ethanol. In conclusion, the animals treated with gentamisin plus quercetin showed less deterioration in the ABR thresholds and less histopathological lesions in the cochlea when compared with the animals treated with gentamicin plus ethanol. Aminoglycosides are aminoglycosidic aminocyclitols that were developed for the treatment of tuberculosis and advanced bacterial infections, although their use is limited due to the major side effects of ototoxicity. The first localization of the ototoxic effects of these antibiotics is the outer hair cells localized in the basal turn of the cochlea. According to histopathological studies, outer hair cells are affected first, followed by the inner hair cells. Although the first place of involvement is the basal turn of the cochlea, involvement advances through the apical turn, and even the stria vascularis may be affected. Degeneration may also be seen in the ganglion cells in high doses [13,27]. In this study, the observation of intense TUNEL positive cells in the outer hair cells in the corti organ and strong caspase-3 and caspase-9 expression in the group receiving gentamicin plus ethanol once again demonstrates the ototoxic effect of gentamicin. The mechanism of the ototoxicity secondary to gentamicin use is not yet completely known. Aminoglycosides increase free radical production in cochlea through various mechanisms, and cause cell death. Since aminoglycosides are positively charged, they are easily bound to cells with a negative electrical load and mitochondrial membranes, which occurs through a reaction with the phosphatidyl inositol in the cell and the mitochondrial membrane. Consequently, the permeability of the cell membrane is increased, and the cell loses magnesium. Magnesium loss halts the oxidative enzymatic reactions in which magnesium plays role
2112
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
Fig. 1. Tunel staining method in the cochlear sections of the rat groups. TUNEL positive inner and outer hair cells were significantly increased treatment with gentamicin plus ethanol (A) when compared with gentamicin plus quercetine (B), quercetine (C) and ethanol (D) groups.!: Inner hair cells, F: Outer hair cells, SL: Spiral limbus, BM: Basilar membrane. 1000. Bar = 5 mm.
as a cofactor, and thus free oxygen radicals are increased. The caspase enzyme system (caspase 3, 8 and 9) is activated through free oxygen radicals, the mitochondrial membrane becomes damaged and the cell goes into apoptosis, resulting in cell death. Eventually, aminoglycoside group antibiotics may be suggested as causing ototoxicity through free radicals [27,28], and this study also supports the finding that this was the active pathway of the
gentamicin toxicity, since strong caspase-3 and caspase-9 expression was observed in spiral limbus and in the inner and outer hair cells in the group receiving gentamicin plus ethanol. Oxidative stress is balanced by the antioxidants that are present in physiological circumstances. Antioxidants protect cells against the unwanted direct or indirect effects of drugs, carcinogens and toxic radical reactions. They inhibit the peroxidation chain reaction
Fig. 2. Caspase-3 immunostaining in cochlear sections of the rat groups. Strong caspase-3 immunostaining intensity was seen in all inner and outer hair cells cytoplasm of the gentamicin plus ethanol (A) group. There was moderate immunostaining intensity in the gentamicin plus quercetine (B) and weak immunstaining in the quercetine (C) and ethanol (D) group.!: Inner hair cells, F: Outer hair cells, SL: Spiral limbus, BM: Basilar membrane. 1000. Bar = 5 mm.
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
2113
Fig. 3. Caspase-9 immunostaining in cochlear sections of the rat groups. Very strong caspase-9 immunostaining intensity was seen in all inner and outer hair cells cytoplasm of the gentamicin plus ethanol (A) group. There was moderate immunostaining intensity in the gentamicin plus quercetine (B) and weak immunstaining in the quercetine (C) and ethanol (D) group.!: Inner hair cells, F: Outer hair cells, SL: Spiral limbus, BM: Basilar membrane. 1000. Bar = 5 mm.
and collect the reactive oxygen species, and thus inhibit lipid peroxidation. In order to limit to a minimum or eliminate altogether the damage caused by free oxygen radicals to the organism, it is necessary to avoid the oxidant increasing effects, inhibit the triggered biochemical events and neutralize the cells that release free oxygen radicals, or treat with antioxidants. The most important and popular of these methods is antioxidant use. Many studies have been published using various antioxidant materials to prevent the ototoxicity caused by gentamicin. Agents such as iron chelators (deferoxamine and dihydroxybenzoate), glutathione, alpha-tocopherol, alpha lipoic acid, D-methionine, dexamethasone, trimetazidine, geranylgeranyl acetone, N-acetylcysteine, estradiol (E2) and thymoquinon have been demonstrated as having a protective role against gentamicin-induced ototoxicity [6–17]. That said, there is currently no agent that is used and accepted in clinical practice in gentamicin-induced ototoxicity. Quercetin, due to its powerful antioxidant activity, is an important agent in toxicity studies, and is known to exert its antioxidant activity through various mechanisms. It has the potential to decrease the toxic effects of free oxygen radicals by clearing hydrogen peroxide, superoxide, singlet oxygen and alkoxyl and hydroxyl radicals, inhibiting superoxide anion production through xanthine oxidase, breaking the lipid peroxyl chain, and chelating such transition metals as iron and copper, decreasing calcium entry into the cells [29,30]. In their study of the Kuppfer cells of the liver, Kawada et al. [31] demonstrated the liver-protecting effect of quercetin by inhibiting the liver nitric oxide synthase enzyme and decreasing NO production. Quercetin also has a neuroprotective effect. Selvakumar et al. [32], in their study on a rat model of the hippocampal oxidative stress produced by polychlorinated biphenyl, demonstrated the protection of hippocampus through the use of quercetin. Schu¨ltke et al. [33,34] in two separate studies demonstrated neuroprotective effects of quercetin in both acute spinal cord injury and acute brain trauma. In a study of Unsal et al. [25], the neuroprotective effect of quercetin was demonstrated in brain damage resulting from
cadmium. It has been demonstrated that EGb 761 (ginkgo biloba) use in gentamicin ototoxicity is protective against cochleotoxicity, while quercetin, bilobalide, ginkgolide A and ginkgolide B that are present in EGb 761 are protective against hair cell damage in rat cochlea cultures [35]. To the best of our knowledge, there has to date been no study demonstrating the possible protective effect of quercetin use alone against ototoxicity due to gentamicin. Lee et al. [36] in their study on a zebrafish embryo model, demonstrated that quercetin plays a protective role against hair cell damage in cisplatin-induced ototoxicity. In the present study, the fewer TUNEL positive cells in the inner and outer hair cells in the Corti organ in the group of rats receiving quercetin plus gentamicin, and the detection of weaker Caspase 3 and 9 expressions, suggests that quercetin protects cochlear cells against apoptosis in gentamicininduced ototoxicity.
5. Conclusion This study is the first study evaluating the effect of quercetin usage in ototoxicity due to gentamicin. Mean ABR threshold values and apoptotic cell numbers in the group receiving gentamicin plus quercetin were found to be decreased compared to the group receiving gentamicin plus ethanol. In the light of these findings, it can be concluded that quercetin attenuates ABR thresholds and histopathological lesions in the cochlea in gentamicin-induced ototoxicity in rats. It is necessary to evaluate further the protective role of quercetin in gentamicin-induced ototoxicity through further studies of electrophysiology and histomorphology. References [1] J. Xie, A.E. Talaska, J. Schacht, New developments in aminoglycoside therapy and ototoxicity, Hear. Res. 281 (2011) 28–37. [2] A.P. Casani, N. Cerchiai, E. Navari, I. Dallan, P. Piaggi, S. Sellari-Franceschini, Intratympanic gentamicin for Meniere’s disease: short- and long-term follow-up of two regimens of treatment, Otolaryngol. Head Neck Surg. 150 (2014) 847–852.
2114
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
[3] V. Palomar Garcı´a, F. Abdulghani Martı´nez, E. Bodet Agustı´, L. Andreu Mencı´a, V. Palomar Asenjo, Drug-induced otoxicity: current status, Acta Otolaryngol. 121 (2001) 569–570. [4] J. Schacht, Aminoglycoside ototoxicity: prevention in sight? Otolaryngol. Head Neck Surg. 118 (1998) 674–677. [5] J. Schacht, Biochemical basis of aminoglycoside ototoxicity, Otolaryngol. Clin. North Am. 26 (1993) 845–856. [6] B.E. Mostafa, S. Tawfik, N.G. Hefnawi, M.A. Hassan, F.A. Ismail, The role of deferoxamine in the prevention of gentamicin ototoxicity: a histological and audiological study in guinea pigs, Acta Otolaryngol. 127 (2007) 234–239. [7] B.B. Song, D.J. Anderson, J. Schacht, Protection from gentamicin ototoxicity by iron chelators in guinea pig in vivo, J. Pharmacol. Exp. Ther. 282 (1997) 369–377. [8] J. Lautermann, J. McLaren, J. Schacht, Glutathione protection against gentamicin ototoxicity depends on nutritional status, Hear. Res. 86 (1995) 15–24. [9] A.R. Fetoni, B. Sergi, E. Scarano, G. Paludetti, A. Ferraresi, D. Troiani, Protective effects of alpha-tocopherol against gentamicin-induced oto-vestibulo toxicity: an experimental study, Acta Otolaryngol. 123 (2003) 192–197. [10] 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. [11] K.C. Campbell, R.P. Meech, J.J. Klemens, M.T. Gerberi, S.S. Dyrstad, D. Larsen, et al., Prevention of noise- and drug-induced hearing loss with D-methionine, Hear. Res. 226 (2007) 92–103. [12] S.K. Park, D. Choi, P. Russell, E.O. John, T.T. Jung, Protective effect of corticosteroid against the cytotoxicity of aminoglycoside otic drops on isolated cochlear outer hair cells, Laryngoscope 114 (2004) 768–771. [13] O.F. Unal, S.M. Ghoreishi, A. Atas¸, N. Akyu¨rek, G. Akyol, B. Gu¨rsel, Prevention of gentamicin induced ototoxicity by trimetazidine in animal model, Int. J. Pediatr. Otorhinolaryngol. 69 (2005) 193–199. [14] H. Sano, S. Yoneda, H. Iwase, A. Itoh, D. Hashimoto, M. Okamoto, Effect of geranylgeranylacetone on gentamycin ototoxicity in rat cochlea culture, Auris Nasus Larynx 34 (2007) 1–4. [15] L. Feldman, S. Efrati, E. Eviatar, R. Abramsohn, I. Yarovoy, E. Gersch, et al., Gentamicin-induced ototoxicity in hemodialysis patients is ameliorated by N-acetylcysteine, Kidney Int. 72 (2007) 359–363. [16] M. Nakamagoe, K. Tabuchi, I. Uemaetomari, B. Nishimura, A. Hara, Estradiol protects the cochlea against gentamicin ototoxicity through inhibition of the JNK pathway, Hear. Res. 261 (2010) 67–74. [17] M. Sagit, F. Korkmaz, S.G. Gu¨rgen, M. Kaya, A. Akcadag, I. Ozcan, The protective role of thymoquinone in the prevention of gentamicin ototoxicity, Am. J. Otolaryngol. 35 (2014) 603–609. [18] R.J. Nijveldt, E. van Nood, D.E. van Hoorn, P.G. Boelens, K. van Norren, P.A. van Leeuwen, Flavonoids: a review of probable mechanisms of action and potential applications, Am. J. Clin. Nutr. 74 (2001) 418–425. [19] R. Kleemann, L. Verschuren, M. Morrison, S. Zadelaar, M.J. van Erk, P.Y. Wielinga, et al., Anti-inflammatory, anti-proliferative and anti-atherosclerotic effects of quercetin in human in vitro and in vivo models, Atherosclerosis 218 (2011) 44–52.
[20] A.W. Boots, G.R. Haenen, A. Bast, Health effects of quercetin: from antioxidant to nutraceutical, Eur. J. Pharmacol. 585 (2008) 325–337. [21] S.C. Bischoff, Quercetin: potentials in the prevention and therapy of disease, Curr. Opin. Clin. Nutr. Metab. Care 11 (2008) 733–740. [22] K.V. Hirpara, P. Aggarwal, A.J. Mukherjee, N. Joshi, A.C. Burman, Quercetin and its derivatives: synthesis, pharmacological uses with special emphasis on antitumor properties and prodrug with enhanced bio-availability, Anticancer Agents Med. Chem. 9 (2009) 138–161. [23] A. Murakami, H. Ashida, J. Terao, Multitargeted cancer prevention by quercetin, Cancer Lett. 269 (2008) 315–325. [24] C.M. Liu, Y.L. Zheng, J. Lu, Z.F. Zhang, S.H. Fan, D.M. Wu, et al., Quercetin protects rat liver against lead-induced oxidative stress and apoptosis, Environ. Toxicol. Pharmacol. 29 (2010) 158–166. [25] C. Unsal, M. Kanter, C. Aktas, M. Erboga, Role of quercetin in cadmium-induced oxidative stress, neuronal damage, and apoptosis in rats, Toxicol. Ind. Health (May) (2013), http://dx.doi.org/10.1177/0748233713486960. [26] C.M. Liu, J.Q. Ma, Y.Z. Sun, Quercetin protects the rat kidney against oxidative stress-mediated DNA damage and apoptosis induced by lead, Environ. Toxicol. Pharmacol. 30 (2010) 264–271. [27] L.P. Rybak, V. Ramkumar, Ototoxicity, Kidney Int. 72 (2007) 931–935. [28] T. Karasawa, P.S. Steyger, Intracellular mechanisms of aminoglycoside-induced cytotoxicity, Integr. Biol. (Camb.) 3 (2011) 879–886. [29] R. Casagrande, S.R. Georgetti, W.A. Verri, J.R. Jabor, A.C. Santos, M.J. Fonseca, Evaluation of functional stability of quercetin as a raw material and in different topical formulations by its antilipoperoxidative activity, AAPS. PharmSciTech. 7 (2006) E10. [30] J.Q. Moskaug, H. Carlsen, M. Myhrstad, R. Blomhoff, Molecular imaging of the biological effects of quercetin and quercetin-rich foods, Mech. Ageing Dev. 125 (2004) 315–324. [31] N. Kawada, S. Seki, M. Inoue, T. Kuroki, Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and kupffer cells, Hepatology 27 (1998) 1265–1274. [32] K. Selvakumar, S. Bavithra, G. Krishnamoorthy, P. Venkataraman, J. Arunakaran, Polychlorinated biphenyls-induced oxidative stress on rat hippocampus: a neuroprotective role of quercetin, Sci. World J. 980 (2012) 314–324. [33] E. Schu¨ltke, H. Kamencic, M. Zhao, G.F. Tian, A.J. Baker, R.W. Griebel, et al., Neuroprotection following fluid percussion brain trauma: a pilot study using quercetin, J. Neurotrauma 22 (2005) 1475–1484. [34] E. Schu¨ltke, E. Kendall, H. Kamencic, Z. Ghong, R.W. Griebel, B.H. Juurlink, Quercetin promotes functional recovery following acute spinal cord injury, J. Neurotrauma 20 (2003) 583–591. [35] T.H. Yang, Y.H. Young, S.H. Liu, EGb 761 (Ginkgo biloba) protects cochlear hair cells against ototoxicity induced by gentamicin via reducing reactive oxygen species and nitric oxide-related apoptosis, J. Nutr. Biochem. 22 (2011) 886–894. [36] S. Lee, K. Oh, A. Chung, H. Park, S. Lee, S. Kwon, et al., Protective role of quercetin against cisplatin-induced hair cell damage in zebrafish embryos, Hum. Exp. Toxicol. (Jan) (2015), http://dx.doi.org/10.1177/0960327114567766.
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Quercetine attenuates the gentamicin-induced ototoxicity in a rat model§ Mustafa Sagit a,*, Ferhat Korkmaz b, Seren Gulsen Gu¨rgen c, Ramazan Gundogdu a, Alper Akcadag d, Ibrahim Ozcan a a
Kayseri Training and Research Hospital, Department of ENT, Kayseri, Turkey Sanliurfa Training and Research Hospital, Department of ENT, S¸anlıurfa, Turkey c Celal Bayar University, School of Vocational Health Service, Department of Histology and Embryology, Manisa, Turkey d Kayseri Training and Research Hospital, Subdepartment of Audiology, Kayseri, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2015 Received in revised form 8 September 2015 Accepted 18 September 2015 Available online 28 September 2015
Objectives: The aim of this study is to evaluate the protective role of quercetin in gentamicin-induced ototoxicity through an auditory brainstem response (ABR) test and a histopathological evaluation of the cochlea. Methods: In this study, 48 female adult Sprague–Dawley rats aged 20–22 weeks and weighing 200–250 g were used. An ABR test was carried out on all rats prior to drug administration, after which, the rats were divided into four groups of 12 animals each. Drug administration was gentamicin 120 mg/ kg plus ethanol in group one; gentamicin 120 mg/kg plus quercetin 15 mg/kg in group two; quercetin 15 mg/kg in group three; and ethanol in group four. The drugs were administered intraperitoneally once a day for two weeks, and the ABR test was repeated after drug administration. Subsequently, the rats were sacrificed and their cochleae were dissected and examined histopathologically. Results: There was no significant difference between the pre-treatment ABR measurement values of the groups. However, a significant increase was detected in the ABR values in the group of rats that were administered gentamicin plus ethanol, while no statistically significant increase was found in the ABR values in the groups administered with gentamicin plus quercetin; quercetin alone; and ethanol alone. The number of TUNEL positive cells in the inner and outer hair cells in the Corti organ was found to be fewer, and Caspase 3 and 9 expressions were found to be weaker in the group receiving gentamicin plus quercetin than in the group receiving gentamicin plus ethanol. Conclusions: Auditory function was detected to be significantly protected and apoptotic cells were found to be decreased when quercetin was administered together with gentamicin. From these results it was concluded that quercetin, a powerful antioxidant, attenuates ABR thresholds and histopathological lesions in the cochlea in gentamicin-induced ototoxicity in rats. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Quercetin Gentamicin Auditory brainstem response
1. Introduction Ototoxicity is a clinical condition related to hearing loss due to damaged inner ear structures secondary to drugs, chemical material and external stimuli such as noise and infection, and leads to such symptoms as balance disorder and tinnitus [1]. The aminoglycoside group of antibiotics is in wide use around the
§ This study was presented in ‘3rd Turkish National Otology and Neurootology Congress’ between May 1 and 4, 2014 Antalya/Turkey. * Corresponding author. Tel.: +90 352 336 88 84; fax: +90 352 320 73 13. E-mail address: [email protected] (M. Sagit).
http://dx.doi.org/10.1016/j.ijporl.2015.09.023 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
world, having been developed to tackle tuberculosis and advanced bacterial infections, intratympanic gentamicin therapy is also used for Meniere’s disease, although their use is limited due to major side effects of ototoxicity and nephrotoxicity [2,3]. Ototoxicity secondary to gentamicin use is frequently bilateral, symmetrical and irreversible. Hearing loss starts initially in the high frequencies; however other frequencies are also affected with continued exposure. The frequency of ototoxicity due to gentamicin varies between 2% and 25% [4,5]. Many clinical and experimental studies have been made with the aim of protecting the inner ear from the potential toxic effects of gentamicin, in which agents such as iron chelators (deferoxamine and dihydroxybenzoate), glutathione, alpha-tocopherol,
2110
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
alpha lipoic acid, D-methionine, dexamethasone, trimetazidine, geranylgeranyl acetone, N-acetylcysteine, estradiol (E2) and thymoquinone have demonstrated a protective role against the ototoxicity of gentamicin [6–17]. Flavonoids are compounds with useful biochemical and antioxidant activity that are widely and abundantly present in vegetarian food, being found mainly in vegetables, fruits, tea, onions and legumes, and give color to most flowers and fruits. One of the most important effects of flavonoids is the function of removing free radicals, while the best-defined characteristic of almost all flavonoid groups is their anti-oxidant capacity [18]. Quercetin (3,5,7,30 ,40 -pentahydroxyflavone) is an main member of flavonoids, having a powerful antioxidant efficacy compared to others. Quercetin has various biological effects, being antiinflammatory, anti-proliferative, anti-viral, anti-allergic, antithrombotic, anti-atherosclerotic and anti-tumoral, in addition to its anti-oxidant features [19–23]. Quercetin has been demonstrated to have hepatoprotective, neuroprotective and nephroprotective functions in many studies, although it is unknown whether quercetin has an otoprotective effect when used alone [24–26]. In the present study, it was aimed to evaluate the possible protective role of quercetin in gentamicin-induced ototoxicity using an auditory brainstem response (ABR) test and a histopathological evaluation of the cochlea.
Drugs were administered once daily to all groups according to the above mentioned protocol for two weeks. At the 14th day, ABR measurements were repeated after drug administration under general anesthesia, and the rats were subsequently sacrificed. The temporal bullae of the rats were bilaterally dissected and stored in 10% formaldehyde for histopathological examination. The ABR values before and after drug administration were compared; thus cochlear toxicity was examined both electrophysiologically and histomorphologically. The individuals carrying out the ABR measurements and histopathological examinations were blind to the experiment groups. 2.3. ABR test
This study was approved by the Ethics Committee on Animal Experiment and Research of Erciyes University (13/59). The study was carried out at the Laboratory for Experimental Animals of Erciyes University.
ABR measurement was carried out in a quiet room in both ears of the anesthetized rats using an interacoustics EP-25 equipment (Interacoustics, Denmark, 2001) and ABR 3A ear phones. ABR responses were recorded using subdermal needle electrodes (Technomed Europe, Holland). Active electrodes were placed on the vertex, the ground electrode on the contralateral mastoid and the reference electrode on the ipsilateral mastoid. Click stimulus was used as the auditory stimulant. For the click stimulus, a 100–3000 Hz band-pass filter at a repeat rate of 21 s1 was set. Normal hearing was recorded when a normal ABR configuration was detected at 10 dBnHL. Hearing thresholds were defined starting from 70 dBnHL, decreasing in 20 dB increments each time. When a behavior was not achieved at the level of 70 dBnHL, the level of stimulus was set to 90 dBnHL. Behavior repeatability was tested by repeating the measurement at least twice and the threshold was elicited. The ABR threshold was defined as the lowest level of intensity at which a V wave was observed.
2.1. Animals
2.4. Histomorphological examination
The study was performed on 48 (96 ears) female adult Sprague– Dawley rats, produced in the Experimental Clinical Research Center of Erciyes University, aged 20–22 weeks and weighing 200–250 g. The study animals were accommodated in safe cages with no limitations in food (pellet and water) at a constant temperature of 21 8C on 12-h day/12-h night cycle.
Tissues extracted were stored in neutral formalin for 24 h and fixed, after which they were stored in an EDTA solution of 0.1 mol/l for three weeks for the decalcification of bone tissues. Tissues were washed under running water for 24 h following this procedure, and then dehydrated using an ethanol series classified according to the routine protocol. The tissues were then cleared in xylene and dried for placement in paraffin wax.
2. Material and methods
2.2. Study design and experiment groups Animals were sedated using an intraperitoneal (i.p.) combination of ketamine hydrochloride 40 mg/kg (Ketalar, Eczacıbasi, Turkey) and xylazine 10 mg/kg (Rompun, Bayer, Germany). The external ear canals and tympanic membranes of all rats were examined microscopically at the initiation of the study, and any debris and/or earwax found present in the external ear canal were removed. At the beginning of the study totally three rats that were found to have serous otitis media (2 rats) and tympanic membrane perforation (1 rat) were excluded from the study. Following an otomicroscopic examination of all rats, the presence of normal hearing was analyzed by measuring ABR thresholds in bilateral ears. Included in the study were 96 ears of 48 rats that were found to have a normal hearing threshold in ABR measurements, and these 48 rats were subsequently divided randomly into four groups. Group 1 (n = 12) received i.p. gentamicin 120 mg/kg (Genta 40 mg ampule, I.E Ulagay, Turkey) plus i.p. 1 ml 20% ethanol solution; group 2 (n = 12) received i.p. gentamicin 120 mg/kg plus quercetin 15 mg/kg (Sigma-Aldrich Chemical Co., St. Louis, MO, USA; dissolved in 1 ml 20% ethanol solution); group 3 (n = 12) received i.p. quercetin 15 mg/kg and group 4 (n = 12) received i.p. 1 ml 20% ethanol solution.
2.4.1. TUNEL method Terminal deoxynucleotidyl nick-end labeling (TUNEL) stain is the method that shows the last stage of the nuclear fragmentation of apoptosis. The cytoplasm of cells underwent apoptosis are observed reduced, membrane vesicles and brown like as apoptotic bodies with TUNEL method. Sections that were deparafinized and rehydrated as stated above were stained using a commercial kit (Apoptag, S7101, Chemicon, CA, USA) according to the manufacturer’s instructions. The sections that were stained using the TUNEL technique were evaluated using a CX41 radiant field microscope (Olympus, Tokyo, Japan). TUNEL scoring was made by two independent investigators who were blind to the experiment information. The number of positive immune reactive cells was analyzed, starting from the apical region and ending at the basal region. The mean number of apoptotic cells was defined by counting the TUNEL positive cells in randomly selected areas for each case. In each case, the number of TUNEL positive or negative cells was calculated so that the total number would be 100 cells, and TUNEL positive cells were expressed in percentages. Cells in necrotic areas, in areas with weak morphology or at the borders of the sections were excluded. Figures were obtained from the basal turn of the cochlea.
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
2.4.2. Immunohistochemical method Sections of 5 mm were obtained from the tissue paraffin blocks and were prepared for immunohistochemical staining. The prepared slides were stained with the primary antibodies of Caspase-3 (1:100, LabVision, USA) and Caspase-9 (1:100, LabVision, USA) using immunohistochemical methods. Tagging and background staining was carried out using DAB and Mayer’s hematoxylene dye, respectively. An analysis of the sections was made under an Olympus (CX41, Tokyo, Japan) light microscope, starting from the apical region and ending at the basal region, and figures were obtained from the basal turn of the cochlea. The control samples were processed using the same procedures; however, the duration of incubation with the primary antibody was omitted. The staining intensity of the preparates with immunohistochemical protocol was evaluated as semi-qualitative and an HSCORE was calculated using the following equation: HSCORE = SPi(i + 1); i whose staining intensity is expressed as 1, 2 or 3 (weak, moderate and strong, respectively) and percentage of cells for each intensity is expressed as Pi, changing from 0% to 100%.
2111
in the ABR threshold value post-drug administration in group 1 when compared to the other groups (Table 1). 3.2. Histomorphological results It was noted that intense TUNEL positive cells were found in the outer hair cells of the corti organ of group 1, and that the number of TUNEL positive cells was lower in the corti organ of group 2. The number of TUNEL positive cells was found to be low in the corti organ of groups 3 and 4, and the number of TUNEL positive cells was statistically significantly lower in group 2 when compared to group 1 (p < 0.001) (Fig. 1). Strong caspase-3 and caspase-9 expressions was observed in all corti organs in group 1, while caspase-3 and caspase-9 expressions were found with moderate intensity in the inner and outer hair cells of group 2. Generally, weak caspase-3 and caspase-9 expressions were observed in especially the inner and outer hair cells of the corti organ of groups 3 and 4 (Figs. 2 and 3). Caspase-3 and caspase-9 expressions were found to be statistically weaker in group 2 when compared to group 1 (p < 0.001).
2.5. Statistical analysis 4. Discussion A statistical analysis to evaluate the findings of the study was made using an SPSS version 16.0 package program (SPSS Inc., Chicago, Illinois, USA). One-way Anova test was used to compare the ABR test results between the groups before and after drug administration. A Bonferroni posthoc test was used to detect the group causing the difference, and intra-group comparisons of the ABR test results were made using a Student’s t-test. Betweengroups comparisons of the number of TUNEL positive cells and Caspase 3 and 9 expressions were made using an independent samples t-test, with p < 0.05 accepted as statistically significant. 3. Results In the course of the study, two rats from group 1 and one rat each from the groups 3 and 4 died under anesthesia, and so the study was completed with 44 rats. Auditory and histomorphological evaluations were made in 88 ears of 44 rats. 3.1. ABR test results No statistically significant differences in the ABR threshold values were found between the groups prior to drug administration. A statistically significant increase was detected in the ABR threshold values post-drug administration compared to the predrug administration in group 1, while no statistically significant differences were found in the ABR threshold levels pre- and postdrug administration in groups 2, 3 and 4 (Table 1). When the ABR threshold values of the groups were compared following treatment, a statistically significant difference was identified between the groups, with a significant increase found
Table 1 Auditory brainstem response thresholds before and after application of drugs. Group Gentamicin plus etanol Gentamicin plus quercetin Quercetin Etanol p (ANOVA)
Pretreatment 14.00 5.02 15.00 5.10 15.90 5.90 16.36 4.92 0.481
Post-treatment 47.50 15.85 16.66 6.37 18.18 9.57 16.81 7.16 p < 0.001
**, +,8
p <0.001* 0.162 0.171 0.789
8 p < 0.001 (post hoc Bonferroni) compared with group etanol. * Statistical analysis is performed with paired samples t test p < 0.05. ** p < 0.001 (post hoc Bonferroni) compared with group gentamicin plus quercetin. + p < 0.001 (Post hoc Bonferroni) compared with group quercetin.
In the present study, the possible protective role of quercetin in ototoxicity due to gentamicin use was examined. A significant increase was observed following drug administration in the ABR threshold values of the group that received gentamicin plus ethanol. The cochlear histomorphological findings were parallel to these results, thus confirming ototoxicity. In the group receiving gentamicin plus quercetin, on the other hand, the ABR threshold values were found to be preserved. Upon a histomorphological evaluation, the number of TUNEL positive cells was observed to be lower in the group receiving gentamicin plus quercetin when compared to the group receiving gentamicin plus ethanol. In addition, caspase-3 and caspase-9 expressions in the inner and outer hair cells in the group receiving gentamicin plus quercetin were found to be weaker when compared to the group receiving gentamicin plus ethanol. In conclusion, the animals treated with gentamisin plus quercetin showed less deterioration in the ABR thresholds and less histopathological lesions in the cochlea when compared with the animals treated with gentamicin plus ethanol. Aminoglycosides are aminoglycosidic aminocyclitols that were developed for the treatment of tuberculosis and advanced bacterial infections, although their use is limited due to the major side effects of ototoxicity. The first localization of the ototoxic effects of these antibiotics is the outer hair cells localized in the basal turn of the cochlea. According to histopathological studies, outer hair cells are affected first, followed by the inner hair cells. Although the first place of involvement is the basal turn of the cochlea, involvement advances through the apical turn, and even the stria vascularis may be affected. Degeneration may also be seen in the ganglion cells in high doses [13,27]. In this study, the observation of intense TUNEL positive cells in the outer hair cells in the corti organ and strong caspase-3 and caspase-9 expression in the group receiving gentamicin plus ethanol once again demonstrates the ototoxic effect of gentamicin. The mechanism of the ototoxicity secondary to gentamicin use is not yet completely known. Aminoglycosides increase free radical production in cochlea through various mechanisms, and cause cell death. Since aminoglycosides are positively charged, they are easily bound to cells with a negative electrical load and mitochondrial membranes, which occurs through a reaction with the phosphatidyl inositol in the cell and the mitochondrial membrane. Consequently, the permeability of the cell membrane is increased, and the cell loses magnesium. Magnesium loss halts the oxidative enzymatic reactions in which magnesium plays role
2112
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
Fig. 1. Tunel staining method in the cochlear sections of the rat groups. TUNEL positive inner and outer hair cells were significantly increased treatment with gentamicin plus ethanol (A) when compared with gentamicin plus quercetine (B), quercetine (C) and ethanol (D) groups.!: Inner hair cells, F: Outer hair cells, SL: Spiral limbus, BM: Basilar membrane. 1000. Bar = 5 mm.
as a cofactor, and thus free oxygen radicals are increased. The caspase enzyme system (caspase 3, 8 and 9) is activated through free oxygen radicals, the mitochondrial membrane becomes damaged and the cell goes into apoptosis, resulting in cell death. Eventually, aminoglycoside group antibiotics may be suggested as causing ototoxicity through free radicals [27,28], and this study also supports the finding that this was the active pathway of the
gentamicin toxicity, since strong caspase-3 and caspase-9 expression was observed in spiral limbus and in the inner and outer hair cells in the group receiving gentamicin plus ethanol. Oxidative stress is balanced by the antioxidants that are present in physiological circumstances. Antioxidants protect cells against the unwanted direct or indirect effects of drugs, carcinogens and toxic radical reactions. They inhibit the peroxidation chain reaction
Fig. 2. Caspase-3 immunostaining in cochlear sections of the rat groups. Strong caspase-3 immunostaining intensity was seen in all inner and outer hair cells cytoplasm of the gentamicin plus ethanol (A) group. There was moderate immunostaining intensity in the gentamicin plus quercetine (B) and weak immunstaining in the quercetine (C) and ethanol (D) group.!: Inner hair cells, F: Outer hair cells, SL: Spiral limbus, BM: Basilar membrane. 1000. Bar = 5 mm.
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
2113
Fig. 3. Caspase-9 immunostaining in cochlear sections of the rat groups. Very strong caspase-9 immunostaining intensity was seen in all inner and outer hair cells cytoplasm of the gentamicin plus ethanol (A) group. There was moderate immunostaining intensity in the gentamicin plus quercetine (B) and weak immunstaining in the quercetine (C) and ethanol (D) group.!: Inner hair cells, F: Outer hair cells, SL: Spiral limbus, BM: Basilar membrane. 1000. Bar = 5 mm.
and collect the reactive oxygen species, and thus inhibit lipid peroxidation. In order to limit to a minimum or eliminate altogether the damage caused by free oxygen radicals to the organism, it is necessary to avoid the oxidant increasing effects, inhibit the triggered biochemical events and neutralize the cells that release free oxygen radicals, or treat with antioxidants. The most important and popular of these methods is antioxidant use. Many studies have been published using various antioxidant materials to prevent the ototoxicity caused by gentamicin. Agents such as iron chelators (deferoxamine and dihydroxybenzoate), glutathione, alpha-tocopherol, alpha lipoic acid, D-methionine, dexamethasone, trimetazidine, geranylgeranyl acetone, N-acetylcysteine, estradiol (E2) and thymoquinon have been demonstrated as having a protective role against gentamicin-induced ototoxicity [6–17]. That said, there is currently no agent that is used and accepted in clinical practice in gentamicin-induced ototoxicity. Quercetin, due to its powerful antioxidant activity, is an important agent in toxicity studies, and is known to exert its antioxidant activity through various mechanisms. It has the potential to decrease the toxic effects of free oxygen radicals by clearing hydrogen peroxide, superoxide, singlet oxygen and alkoxyl and hydroxyl radicals, inhibiting superoxide anion production through xanthine oxidase, breaking the lipid peroxyl chain, and chelating such transition metals as iron and copper, decreasing calcium entry into the cells [29,30]. In their study of the Kuppfer cells of the liver, Kawada et al. [31] demonstrated the liver-protecting effect of quercetin by inhibiting the liver nitric oxide synthase enzyme and decreasing NO production. Quercetin also has a neuroprotective effect. Selvakumar et al. [32], in their study on a rat model of the hippocampal oxidative stress produced by polychlorinated biphenyl, demonstrated the protection of hippocampus through the use of quercetin. Schu¨ltke et al. [33,34] in two separate studies demonstrated neuroprotective effects of quercetin in both acute spinal cord injury and acute brain trauma. In a study of Unsal et al. [25], the neuroprotective effect of quercetin was demonstrated in brain damage resulting from
cadmium. It has been demonstrated that EGb 761 (ginkgo biloba) use in gentamicin ototoxicity is protective against cochleotoxicity, while quercetin, bilobalide, ginkgolide A and ginkgolide B that are present in EGb 761 are protective against hair cell damage in rat cochlea cultures [35]. To the best of our knowledge, there has to date been no study demonstrating the possible protective effect of quercetin use alone against ototoxicity due to gentamicin. Lee et al. [36] in their study on a zebrafish embryo model, demonstrated that quercetin plays a protective role against hair cell damage in cisplatin-induced ototoxicity. In the present study, the fewer TUNEL positive cells in the inner and outer hair cells in the Corti organ in the group of rats receiving quercetin plus gentamicin, and the detection of weaker Caspase 3 and 9 expressions, suggests that quercetin protects cochlear cells against apoptosis in gentamicininduced ototoxicity.
5. Conclusion This study is the first study evaluating the effect of quercetin usage in ototoxicity due to gentamicin. Mean ABR threshold values and apoptotic cell numbers in the group receiving gentamicin plus quercetin were found to be decreased compared to the group receiving gentamicin plus ethanol. In the light of these findings, it can be concluded that quercetin attenuates ABR thresholds and histopathological lesions in the cochlea in gentamicin-induced ototoxicity in rats. It is necessary to evaluate further the protective role of quercetin in gentamicin-induced ototoxicity through further studies of electrophysiology and histomorphology. References [1] J. Xie, A.E. Talaska, J. Schacht, New developments in aminoglycoside therapy and ototoxicity, Hear. Res. 281 (2011) 28–37. [2] A.P. Casani, N. Cerchiai, E. Navari, I. Dallan, P. Piaggi, S. Sellari-Franceschini, Intratympanic gentamicin for Meniere’s disease: short- and long-term follow-up of two regimens of treatment, Otolaryngol. Head Neck Surg. 150 (2014) 847–852.
2114
M. Sagit et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 2109–2114
[3] V. Palomar Garcı´a, F. Abdulghani Martı´nez, E. Bodet Agustı´, L. Andreu Mencı´a, V. Palomar Asenjo, Drug-induced otoxicity: current status, Acta Otolaryngol. 121 (2001) 569–570. [4] J. Schacht, Aminoglycoside ototoxicity: prevention in sight? Otolaryngol. Head Neck Surg. 118 (1998) 674–677. [5] J. Schacht, Biochemical basis of aminoglycoside ototoxicity, Otolaryngol. Clin. North Am. 26 (1993) 845–856. [6] B.E. Mostafa, S. Tawfik, N.G. Hefnawi, M.A. Hassan, F.A. Ismail, The role of deferoxamine in the prevention of gentamicin ototoxicity: a histological and audiological study in guinea pigs, Acta Otolaryngol. 127 (2007) 234–239. [7] B.B. Song, D.J. Anderson, J. Schacht, Protection from gentamicin ototoxicity by iron chelators in guinea pig in vivo, J. Pharmacol. Exp. Ther. 282 (1997) 369–377. [8] J. Lautermann, J. McLaren, J. Schacht, Glutathione protection against gentamicin ototoxicity depends on nutritional status, Hear. Res. 86 (1995) 15–24. [9] A.R. Fetoni, B. Sergi, E. Scarano, G. Paludetti, A. Ferraresi, D. Troiani, Protective effects of alpha-tocopherol against gentamicin-induced oto-vestibulo toxicity: an experimental study, Acta Otolaryngol. 123 (2003) 192–197. [10] 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. [11] K.C. Campbell, R.P. Meech, J.J. Klemens, M.T. Gerberi, S.S. Dyrstad, D. Larsen, et al., Prevention of noise- and drug-induced hearing loss with D-methionine, Hear. Res. 226 (2007) 92–103. [12] S.K. Park, D. Choi, P. Russell, E.O. John, T.T. Jung, Protective effect of corticosteroid against the cytotoxicity of aminoglycoside otic drops on isolated cochlear outer hair cells, Laryngoscope 114 (2004) 768–771. [13] O.F. Unal, S.M. Ghoreishi, A. Atas¸, N. Akyu¨rek, G. Akyol, B. Gu¨rsel, Prevention of gentamicin induced ototoxicity by trimetazidine in animal model, Int. J. Pediatr. Otorhinolaryngol. 69 (2005) 193–199. [14] H. Sano, S. Yoneda, H. Iwase, A. Itoh, D. Hashimoto, M. Okamoto, Effect of geranylgeranylacetone on gentamycin ototoxicity in rat cochlea culture, Auris Nasus Larynx 34 (2007) 1–4. [15] L. Feldman, S. Efrati, E. Eviatar, R. Abramsohn, I. Yarovoy, E. Gersch, et al., Gentamicin-induced ototoxicity in hemodialysis patients is ameliorated by N-acetylcysteine, Kidney Int. 72 (2007) 359–363. [16] M. Nakamagoe, K. Tabuchi, I. Uemaetomari, B. Nishimura, A. Hara, Estradiol protects the cochlea against gentamicin ototoxicity through inhibition of the JNK pathway, Hear. Res. 261 (2010) 67–74. [17] M. Sagit, F. Korkmaz, S.G. Gu¨rgen, M. Kaya, A. Akcadag, I. Ozcan, The protective role of thymoquinone in the prevention of gentamicin ototoxicity, Am. J. Otolaryngol. 35 (2014) 603–609. [18] R.J. Nijveldt, E. van Nood, D.E. van Hoorn, P.G. Boelens, K. van Norren, P.A. van Leeuwen, Flavonoids: a review of probable mechanisms of action and potential applications, Am. J. Clin. Nutr. 74 (2001) 418–425. [19] R. Kleemann, L. Verschuren, M. Morrison, S. Zadelaar, M.J. van Erk, P.Y. Wielinga, et al., Anti-inflammatory, anti-proliferative and anti-atherosclerotic effects of quercetin in human in vitro and in vivo models, Atherosclerosis 218 (2011) 44–52.
[20] A.W. Boots, G.R. Haenen, A. Bast, Health effects of quercetin: from antioxidant to nutraceutical, Eur. J. Pharmacol. 585 (2008) 325–337. [21] S.C. Bischoff, Quercetin: potentials in the prevention and therapy of disease, Curr. Opin. Clin. Nutr. Metab. Care 11 (2008) 733–740. [22] K.V. Hirpara, P. Aggarwal, A.J. Mukherjee, N. Joshi, A.C. Burman, Quercetin and its derivatives: synthesis, pharmacological uses with special emphasis on antitumor properties and prodrug with enhanced bio-availability, Anticancer Agents Med. Chem. 9 (2009) 138–161. [23] A. Murakami, H. Ashida, J. Terao, Multitargeted cancer prevention by quercetin, Cancer Lett. 269 (2008) 315–325. [24] C.M. Liu, Y.L. Zheng, J. Lu, Z.F. Zhang, S.H. Fan, D.M. Wu, et al., Quercetin protects rat liver against lead-induced oxidative stress and apoptosis, Environ. Toxicol. Pharmacol. 29 (2010) 158–166. [25] C. Unsal, M. Kanter, C. Aktas, M. Erboga, Role of quercetin in cadmium-induced oxidative stress, neuronal damage, and apoptosis in rats, Toxicol. Ind. Health (May) (2013), http://dx.doi.org/10.1177/0748233713486960. [26] C.M. Liu, J.Q. Ma, Y.Z. Sun, Quercetin protects the rat kidney against oxidative stress-mediated DNA damage and apoptosis induced by lead, Environ. Toxicol. Pharmacol. 30 (2010) 264–271. [27] L.P. Rybak, V. Ramkumar, Ototoxicity, Kidney Int. 72 (2007) 931–935. [28] T. Karasawa, P.S. Steyger, Intracellular mechanisms of aminoglycoside-induced cytotoxicity, Integr. Biol. (Camb.) 3 (2011) 879–886. [29] R. Casagrande, S.R. Georgetti, W.A. Verri, J.R. Jabor, A.C. Santos, M.J. Fonseca, Evaluation of functional stability of quercetin as a raw material and in different topical formulations by its antilipoperoxidative activity, AAPS. PharmSciTech. 7 (2006) E10. [30] J.Q. Moskaug, H. Carlsen, M. Myhrstad, R. Blomhoff, Molecular imaging of the biological effects of quercetin and quercetin-rich foods, Mech. Ageing Dev. 125 (2004) 315–324. [31] N. Kawada, S. Seki, M. Inoue, T. Kuroki, Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and kupffer cells, Hepatology 27 (1998) 1265–1274. [32] K. Selvakumar, S. Bavithra, G. Krishnamoorthy, P. Venkataraman, J. Arunakaran, Polychlorinated biphenyls-induced oxidative stress on rat hippocampus: a neuroprotective role of quercetin, Sci. World J. 980 (2012) 314–324. [33] E. Schu¨ltke, H. Kamencic, M. Zhao, G.F. Tian, A.J. Baker, R.W. Griebel, et al., Neuroprotection following fluid percussion brain trauma: a pilot study using quercetin, J. Neurotrauma 22 (2005) 1475–1484. [34] E. Schu¨ltke, E. Kendall, H. Kamencic, Z. Ghong, R.W. Griebel, B.H. Juurlink, Quercetin promotes functional recovery following acute spinal cord injury, J. Neurotrauma 20 (2003) 583–591. [35] T.H. Yang, Y.H. Young, S.H. Liu, EGb 761 (Ginkgo biloba) protects cochlear hair cells against ototoxicity induced by gentamicin via reducing reactive oxygen species and nitric oxide-related apoptosis, J. Nutr. Biochem. 22 (2011) 886–894. [36] S. Lee, K. Oh, A. Chung, H. Park, S. Lee, S. Kwon, et al., Protective role of quercetin against cisplatin-induced hair cell damage in zebrafish embryos, Hum. Exp. Toxicol. (Jan) (2015), http://dx.doi.org/10.1177/0960327114567766.