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Evaluation of the possible protective role of naringenin on gentamicin-induced ototoxicity: A preliminary study
Accepted Manuscript Evaluation of the possible protective role of naringenin on gentamicin-induced ototoxicity: A preliminary study İ. Koçak, S. Sarac, E. Aydogan, E. Şentürk, D. Akakın, K. Koroglu, Ö.F. Özer PII:
S0165-5876(17)30321-X
DOI:
10.1016/j.ijporl.2017.07.008
Reference:
PEDOT 8612
To appear in:
International Journal of Pediatric Otorhinolaryngology
Received Date: 27 February 2017 Revised Date:
16 June 2017
Accepted Date: 7 July 2017
Please cite this article as: İ Koçak, S Sarac, E Aydogan, E Şentürk, D Akakın, K Koroglu, Ö. Özer, Evaluation of the possible protective role of naringenin on gentamicin-induced ototoxicity: A preliminary study, International Journal of Pediatric Otorhinolaryngology (2017), doi: 10.1016/j.ijporl.2017.07.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Evaluation of the Possible Protective Role of Naringenin on Gentamicin-Induced
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Ototoxicity: A Preliminary Study
Koçak İ1, Sarac S1 ,Aydogan E1, Şentürk E2 , Akakın D3, Koroglu K3, Özer ÖF4
Department of Otolaringology, Koç University Hospital, Istanbul, Turkey.
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Department of Otolaringology, Alaca State Hospital, Çorum, Turkey.
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Department of Histology and Embryology, Marmara University School of Medicine, Istanbul
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Department of Biochemistry, Bezmialem Vakif University, Medical Faculty, Istanbul, Turkey
Compliance with Ethical Standards
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Ethical Procedure • The research involving animals and meets all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. • As an expert scientist and along with co-authors of concerned field, the paper has been submitted with full responsibility, following due ethical procedure, and there is no duplicate publication, fraud, plagiarism, or concerns about animal or human experimentation.
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A Disclosure / Conflict Of Interest Statement • None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. • It is to specifically state that “no competing interests are at stake and there is no conflict of interest” with other people or organizations that could inappropriately influence or bias the content of the paper.
Most sincerely,
Corresponding Author: İlker Koçak, M.D.
Koc University Hospital, Department of Otolaringology Davutpasa Cad. No: 4, Topkapi, 34010, Istanbul, Turkey E-mail: [email protected]
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Phone: +905557116870
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Evaluation of the Possible Protective Role of Naringenin on Gentamicin-Induced Ototoxicity: A Preliminary Study Introduction
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Aminoglycoside antibiotics such as gentamicin are widely used around the world to treat gram-negative bacterial infections and tuberculosis, despite well-known side effects, such as ototoxicity and nephrotoxicity (1,2) In addition, intratympanic gentamicin is an important treatment modality in the management of Meniere’s disease(3). Gentamicin-induced ototoxicity is dependent on dose and duration, and may frequently be bilaterally symmetric and irreversible. Hearing loss initially affects high frequencies and, in long-term exposure to gentamicin all frequencies.
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In aminoglycoside ototoxicity, target structures are the organ of Corti and the hair cells. The most widely proposed pathophysiologic mechanism is the creation of reactive oxygen species accumulating in the cochlea, causing apoptosis and cell death (4,5).
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A number of experimental and clinical studies into the reduction of gentamicin-induced ototoxicity have been carried out, showing that substances such as thymoquinone, quercetin, trimetazidine, alpha lipoic acid, Nacetylcysteine, and vitamin A are effective against gentamicin ototoxicity, thanks to their antioxidant properties (6-11).
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Flavonoids are phenolic compounds widely present in fruit and vegetables that exhibit powerful antioxidant activity because of their ability to reduce free radical formation. Naringenin is a bioflavonoid highly enriched in tomatoes, cocoa and citrus fruits such as grapefruit, lemon and orange. It was reported that naringenin has antioxidant(12,13), anti-inflammatory (14,15) and free radical scavenging effects (16), and also inhibits lipid peroxidation(17). Past studies demonstrated that naringenin has anti-tumoral(18,19), hepatoprotective(20) and nephroprotective(21) features. It was also shown that naringenin treatment significantly protected against gentamicin-induced nephrotoxicity in rats (22). However, the potential protective effect of naringenin on gentamicininduced ototoxicity has not been investigated.
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The aim of this study was to evaluate the protective effect of naringenin against gentamicin ototoxicity with audiological tests, and biochemical, and histopathological parameters.
Material and methods
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The protocol of this study was approved by the Ethics Committee on Animal Research of Istanbul University. The study was performed at the Experimental Animal Studies Laboratory of Istanbul University.
Animals
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Thirty-two adult male Sprague Dawley rats weighing 250 to 300 g were obtained at the Experimental Animal Studies Laboratory of Istanbul University for study. Rats with a positive Preyer reflex were chosen and a 2.7mm 0o endoscope was used to examine the tympanic membranes and external ear canals of all rats. Rats with cerumen in their ear canals and those showing signs of otitis media or tympanic membrane perforation were excluded from the study. All rats had free access to food and water, and were maintained in an environment with controlled temperature (25 °C), under a 12-hour light/dark cycle.
Drugs and Chemicals
Gentamicin (Genta) was obtained from İ.E. Ulagay İlaç Sanayii Türk A.Ş, Istanbul, Turkey. 0.5% Carboxymethyl cellulose and 98% naringenin were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO, USA (98% naringenin dissolved in 0.5% carboxymethyl cellulose). Xylazine hydrochloride (Rompun) was purchased from Bayer, Istanbul, Turkey. And ketamine hydrochloride (Ketalar) was from Eczacibasi, Istanbul, Turkey. Experimental groups
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The 32 rats were randomly divided into four groups. Each group contained eight rats: Group 1 (n = 8) received 0.5% carboxymethyl cellulose perorally (p.o) daily for 14 days.
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Group 2 (n = 8) received gentamicin 120 mg/kg intraperitoneally (i.p.) and 0.5% carboxymethyl cellulose p.o. daily for 14 days. Group 3 (n=8) received gentamicin 120 mg/kg i.p. and naringenin 50 mg/kg p.o. daily for 14 days.
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Group 4 (n = 8) received naringenin 50 mg/kg and 0.5% carboxymethyl cellulose p.o. daily for 14 days.
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At the beginning of the study and on day 14, all rats were anesthetized by intra-peritoneal 5 mg/kg xylazine hydrochloride and 40 mg/kg ketamine hydrochloride. DPOAE and ABR measurements were performed after anesthesia. On the 14th day, intracardiac blood samples were taken from all rats to calculate the biochemical parameters. Again on the 14th day, their cochleae were bilaterally harvested for histopathologic observations, and sent for pathological examination in 10 % buffered formaldehyde.
Audiological evaluation
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Distortion product otoacoustic emission(DPOAE) A Neurosoft Neuro-audio device (Ivanova, Russia) was used to measure DPOAEs. Measurements were performed in a silent room. The smallest size tympanometry probe was placed into the external ear canal and measurements were performed on both ears. DPgram measurements were performed at frequencies of 988, 2222, 3200, 4444, 5000, 6154, 8000, 8889, 10000 and 11429 Hz. DPOAE values 6 dBSPL above the signal to noise ratio were accepted as positive. Auditory brainstem response(ABR)
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ABR measurements were conducted in a silent room with Neurosoft Neuro-audio device (Ivanova, Russia), using subdermal needle electrodes. An ER-2 insert ear phone was used to provide click stimuli in alternating polarities. A tone-burst stimulus of 16 kHz with a 30-2000 Hz band-pass filter at a repeat rate of 21/ second was used as the auditory stimulant. The threshold was determined by starting at 100 dB SPL , then decreasing by increments of 10 dB until the threshold was reached. Repeatability was confirmed, and the threshold determination was developed over two tests. The ABR threshold was defined as the lowest intensity level where the fifth wave was observed.
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Biochemical evaluation
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For biochemical analyses, blood samples taken from the rats were centrifuged for 15 minutes in 3,000 rpm., and the serum was isolated and kept at –80°C until blood specimens from all the rats were prepared.
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Oxidative stress means that the balance between pro- and antioxidants is skewed towards pro-oxidants (23). It is difficult to measure different antioxidants individually because of time consumption and cost. The recently developed method of measuring the total antioxidant status (TAS) allows all antioxidants to be recorded at a very low cost in a short time as a single parameter by a simple measurement in the serum (24). TAS measures the combined activity of antioxidants and the total antioxidant level, therefore evaluating the comprehensive antioxidant status. However, there are no practical methods for the measurement of individual pro-oxidant molecules, although, a single parameter, total oxidant status (TOS), can be measured in the serum instead(25). TOS is an indicator of the total oxidant levels. The oxidative stress index (OSI) provides a more accurate index for oxidative stress in the body, since this ratio takes into account the sum total of all oxidant and antioxidant activities. TAS and TOS were measured by the Rel Assay Diagnostics kit (Mega Tip San ve Tic Ltd Sti, Gaziantep, Turkey), and the OSI was calculated from the TAS and TOS (OSI: TOS/TASX100). Histopathologic examination
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The cochleae were dissected under a dissecting microscope and fixed with 10 % buffered formalin. Subsequently, they were placed in an EDTA solution for the decalcification of osseous tissues, and washed overnight under a water flow. Tissues were dehydrated in an ethanol series of graded concentrations and cleared in xylene. The samples were then embedded in paraffin. Longitudinal 5 µm thick sections passing parallel to the modiolus were deparaffinized, hydrated and stained with the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) method for determining apoptotic cells.
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TUNEL method
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TUNEL staining was used to evaluate the extent of apoptosis in the organ of Corti and stria vascularis. The TUNEL staining assay kit was used in accordance with the user's manual from the manufacturer (Millipore ApopTag Plus Peroxidase In Situ Apoptosis Kit S7101, Temecula, CA, USA). After staining with Mayer's hematoxylin, sections were coverslipped with Entellan mounting medium. TUNEL-positive cells in the organ of Corti and stria vascularis were counted in randomly chosen fields for each case under a light microscope (Olympus BX51, Tokyo, Japan) with a 40x objective lens and photographed with a digital camera (Olympus DP72, Tokyo, Japan). Three of the 5 cochlear sections (2 basal turns, 2 medial turns and 1 apical turn) were evaluated in each slide and the average number of TUNEL-positive cells in the stria vascularis and organ of Corti were recorded for each slide.
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Two histologists, who were blinded to the animal grouping, independently evaluated the TUNEL scores.
Statistical analysis
Statistical analysis was carried out using SPSS ver. 22.0 (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). Data normality was checked using the Kolmogorov-Smirnov tests of normality.
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One-way ANOVA was used for intergroup comparisons of DPOAE and ABR values (the differences among groups were considered statistically significant at p<0.05). The Tukey’s (HSD) post-hoc test was used for determining the differences between the groups.
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The paired samples t test was used for within-group comparisons of DPOAE and ABR values. (the difference within group was considered statistically significant at p < 0.05).
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One-way ANOVA was used in the evaluation of biochemical parameters between groups (the differences between groups were considered statistically significant at p<0.05). The Tukey’s HSD post-hoc test was used for determining which groups differed.
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RESULTS
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The Kruskal-Wallis test was used to compare the number of TUNELpositive cells between groups (the differences between groups were considered statistically significant at p<0.05). The Mann-Whitney U test was used for determining the differences between the groups.
Distortion product otoacoustic emission
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No significant differences were observed between the baseline DPOAE SNR levels in any of the groups (p >0.05).
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In group 2 (gentamicin) on day 14, DPOAE SNR levels significantly decreased as compared to their baseline levels (at all frequencies, except 998 and 2222 Hz.) (p < 0.05) (Figure 1) In group 1(control), group 3 (gentamicin+naringenin) and group 4(naringenin) there were no significant differences of the DPOAE SNR levels between days 0 and 14 (p>0.05) (Figure 1). On day 14, the DPOAE SNR levels of group 2 (gentamicin) were significantly lower than those of groups 1, 3 and 4 (at all frequencies, except 998 and 2222 Hz.) (p < 0.008) (Figure 2).
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Also on day 14, there were no significant differences of the DPOAE SNR levels between group 3 (gentamicin+naringenin) and group 1(control) (p >0.05) (Figure 2).
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Auditory brainstem response There were no significant differences in any group between the baseline ABR thresholds (p > 0.05) (Table 1).
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In group 2 (gentamicin) on days 14, the ABR threshold significantly increased as compared to the baseline level (p<0.001) (Table 1).
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In group 1(control), group 3 (gentamicin+naringenin) and group 4(naringenin) there were no significant differences in the ABR thresholds between days 0 and 14 (p >0.05) (Table 1). On day 14, the ABR threshold of group 2 (gentamicin) was significantly higher than those of groups 1, 3 and 4 (p < 0.001) (Table 1).
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Also on day 14, no significant differences were observed in the ABR thresholds between group 3 (gentamicin+naringenin) and group 1 (control) (p >0.05) (Table 1).
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Biochemical evaluation
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The TOS and OSI values of group 2 were significantly higher than the values of the other groups (p<0.05) (Table 2). No significant differences were observed between the TOS and OSI values of group 1, group 3 and group 4 (p>0.05) (Table 2). The TAS values of groups 3 and 4 were significantly higher than those of group 2 (p<0.05) (Table 2).
Histopathologic results
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The numbers of TUNEL-positive cells are shown in Table 3. Fewer TUNELpositive cells were observed in group 1(control) and group 4(naringenin). Relative to group 1, group 3(naringenin+gentamicin) and group 4, TUNELpositive cells in the organ of Corti and stria vascularis were significantly increased in group 2(gentamicin) (p<0.001). However, the number of TUNELpositive cells in group 3 was significantly lower than in group 2 (p<0.05).
Discussion
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There is a wide range of use for aminoglycoside antibiotics, however, this is limited by the ototoxic effects of these drugs. This study has researched the potential protective effect of naringenin against gentamicin ototoxicity. We observed a decrease in DPOAE values and an increase in ABR thresholds in the group that had been administered gentamicin. Biochemical and histopathologic findings confirmed ototoxicity. We have seen that DPOAE values and ABR thresholds were preserved in the group being given naringenin+gentamicin. In the biochemical evaluation, we found that oxidative parameters in the naringenin+gentamicin group were lower and, the antioxidative parameters higher than in the gentamicin group. In addition, histopathologic evaluation showed that the number of TUNEL-positive cells in the naringenin+gentamicin group was lower than in the gentamicin group. Ultimately, the protective effect of naringenin in gentamicin-induced ototoxicity was shown through audiological, biochemical and histopathologic evaluations.
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Gentamicin damages the outer hair cells beginning from the basal region of the cochlea, then progressing to the middle and apical regions. Thus, the hearing loss that first involved only higher frequencies extends to lower frequencies. The damage to outer hair cells can be evaluated by DPOAE testing. ABR testing is an objective method for evaluating the auditory pathways proximal to distal(26). In our study, DPOAE values on day 14 in the gentamicin group were lower than initial values and ABR thresholds were higher than initially. These findings show that gentamicin damages outer hair cells and has a toxic effect on the auditory pathways. Further, we detected no significant difference in the DPOAE values and ABR thresholds between day 14 and initial
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values in the naringenin+gentamicin group, which demonstrates the protective effect of naringenin against gentamicin ototoxicity.
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The mechanism of gentamicin-induced ototoxicity is not entirely understood. It is assumed that the production of free oxygen radicals in the inner ear because of aminoglycosides causes cell death (27). Free radicals are attacking cell membranes, proteins, and DNA, leading to lipid peroxidation causing irreparable cell damage and cell death. In addition, by producing an excess of free oxygen radicals, gentamicin leads to a reduction of antioxidant enzymes and a disturbance of the balance between oxidants and antioxidants. Various studies have reported that gentamicin increased oxidative stress, thus causing a toxic effect(4,5,9). In our study, to measure the total oxidative/antioxidative balance, we used objective and sensitive biochemical parameters (TOS, TAS, and OSI). TOS and OSI values, being oxidative stress parameters, were significantly higher in group 2 (gentamicin) compared to the other groups (Table 2). This finding supports the result that gentamicin produces free oxygen radicals.
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Numerous studies have investigated the effectiveness of various antioxidant agents against gentamicin ototoxicity (6-11). However, there is still no agent being approved for clinical application against gentamicin ototoxicity.
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Naringenin is the major bioactive flavonoid compound derived from citrus fruits and a strong antioxidant agent (12,13). It manifests its antioxidant activity through different mechanisms, such as scavenging free oxygen radicals and inhibiting lipid peroxidation (16,17). In addition, previous studies have shown a naringenin to have a protective effect against oxidative cell damage through an increase of antioxidant enzymes such as GSH (glutathione) or SOD (superoxide dismutase) (28). In our study, we found the value of the antioxidant parameter TAS in groups 3 (gentamicin+naringenin) and 4 (naringenin) to be significantly higher than in group 2 (gentamicin) (Table 2). This finding supports previous reports that naringenin increases antioxidant activity.
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Nitric oxide (NO), is a free radical that also plays an important role in hair cell injury and gentamicin-induced ototoxicity (11,29). Gentamicin stimulates inducible nitric oxide synthase (iNOS), leading to an increased production of NO, which reacts with superoxide radicals to form a cytotoxic peroxynitrite radical (30). Peroxynitrite reacts with the cell membrane and cellular proteins, leading to cell death (31). There are studies showing that naringenin inhibits iNOS activity and reduces NO synthesis, thus exhibiting a protective effect against oxidative stress in hepatocytes (32). It has also been reported that naringenin provides a protective effect against gentamicin-induced nephrotoxicity by reducing iNOS synthesis (22).
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Another mechanism being proposed for gentamicin ototoxicity is the activation of the nuclear factor-kappa B (NF-κB) signal pathway and damage to outer hair cells caused by inflammatory cytokines (33). Previous studies have demonstrated that naringenin provides a cytoprotective effect by suppressing the NF-κB pathway (34,35). These findings support the observation that naringenin has a protective effect against gentamicin-induced ototoxicity.
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Apoptosis, a kind of controlled cell death, occurs for a variety of reasons during normal growth and development. Apoptosis may be the primary result of gentamicin ototoxicity. Experimental studies show apoptosis occurring in the cochlea after gentamicin use (6,7,11). The TUNEL method was described by Gavrieli and accepted as a marker of apoptosis(36). TUNEL-positive staining indicates fragmented DNA chains and apoptosis. In our study, we exhibited apoptosis in the organ of Corti and stria vascularis through TUNEL staining; the number of apoptotic cells was significantly greater in the gentamicin group than in the other groups (Figure 3). In addition, the number of TUNEL-positive cells was significantly decreased in the naringenin+gentamicin group compared to the gentamicin-only group. These findings demonstrate that naringenin prevented apoptosis in gentamicin-induced ototoxicity. It has been demonstrated that naringenin plays a role in protecting against gentamicin nephrotoxicity as a result of its antioxidant and cytoprotective effects(22). Also, Sahu et al. has reported that naringin, which is the precursor of naringenin, ameliorates gentamicin-induced
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nephrotoxicity(37). To the best of our knowledge, the possible protective effect of naringenin against gentamicin ototoxicity has not been previously demonstrated. In this study we were able to show that naringenin was effective against gentamicin-induced ototoxicity. In addition, Li et al.(38) has documented that long-term and high-dose naringenin exposure does not lead to any lethality or toxic clinical symptoms in rats, thus naringenin can be used safely in gentamicin ototoxicity.
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The strengths of our study included the use of DPOAE and ABR tests in the total audiological evaluation, the use of biochemical parameters in the assessment of oxidative stress, and histopathologic evaluation.
Conclusion
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By way of limitations, we did not assess the effectiveness of different doses, the number of animals in each group, though statistically sufficient, was small. Although dose response studies of naringenin efficacy have been documented in the literature (37,38), we did not perform the dose comparison for naringenin effect. In our study, naringenin dose was selected on the basis of previously published reports (20-22,28). This may be a limitation of our research. Additionally, we did not investigate the effect of naringenin on the antimicrobial activity of gentamicin, this is another limitation of this study. Further studies are required to evaluate the effectiveness of different doses and the efficacy of naringenin on the antimicrobial activity of gentamicin.
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This is the first study evaluating the effect of naringenin usage in gentamicin-induced ototoxicity. Our study demonstrated with audiological (DPOAE and ABR), and biochemical measurements and histopathological evaluation that the ototoxic effects of gentamicin could be limited by the concurrent use of naringenin. Further experimental and prospective randomized clinical studies are needed to confirm our findings. Funding: None Conflict of interest: No conflict of interest was declared by the authors.
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Ethical approval: All applicable international, national and/or institutional guidelines for care and use of animals were followed. Acknowledgments: None.
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35. Raza SS, Khan MM, Ahmad A, Ashafaq M, Islam F, Wagner AP, Safhi MM, Islam F. Neuroprotective effect of naringenin is mediated through suppression of NF-κB signaling pathway in experimental stroke. Neuroscience. 2013 Jan 29;230:157-71.
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36. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119: 493-501. 37. Sahu BD, Tatireddy S, Koneru M, Borkar RM, Kumar JM, Kuncha M, Srinivas R, Shyam Sunder R, Sistla R. Naringin ameliorates gentamicin-induced nephrotoxicity and associated mitochondrial dysfunction, apoptosis and inflammation in rats: possible mechanism of nephroprotection. Toxicol Appl Pharmacol. 2014 May 15;277(1):8-20
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38. Li P, Wang S, Guan X, Cen X, Hu C, Peng W, Wang Y, Su W. Six months chronic toxicological evaluation of naringin in Sprague-Dawley rats. Food Chem Toxicol. 2014 Apr;66:65-75.
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Baseline
14th Day
Paired T Test
Group 1
47,5 ± 4,4
48,7 ± 3,4
p=0.43
Group 2
48,1 ± 4,0
68,7 ± 8,0 *
p<0.001
Group 3
45,6 ± 5,1
49,3 ± 5,7 **
Group 4
44,3 ± 5,1
46,8 ± 4,7
One Way ANOVA
P=0.1
p=0.001
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Groups
p=0.08
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p=0.16
* p<0.001 compared with group 1, 3 and 4 ** p=0.4 compared with group 1
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Table 1: Comparison of ABR thresholds between-groups . For the comparison within-group, the paired T test was applied (P < 0.05 was accepted as statistically significant). For the comparison between groups, the One-way ANOVA test was applied (P < 0.05, statistically significant). The Tukey’s HSD post-hoc test was applied to identify within-group differences.
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TAS (μmol Troloxequiv./L)
TOS (μmol H2O2 equiv./L) 5,29 ± 1,54
OSI (TOS/TASX100)
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Group 1 1,23 ± 0,32 0,047 ± 0,02 (control) Group 2 1,06 ± 0,35 9,34 ±2,09 0,10 ± 0,05 (gentamicin) Group 3 1,66 ± 0,45 4,75 ± 1,51 0,029 ± 0,011 (gentamicin+naringenin) Group 4 1,81 ± 0,28 5,73 ± 1,29 0,032 ± 0,009 (naringenin) p(ANOVA) P<0.05 P<0.05 P<0.05 G2 vs G1 ¶ ¶ G2 vs G3 ¶ ¶ ¶ G2 vs G4 ¶ ¶ ¶ TOS; total oxidant status, TAS; total antioxidant status, OSI; oxidative stress index. p<0.05, significance level obtained between groups, one-way analysis of variance (ANOVA) p<0.008, significance level obtained (Tukey honest significant difference post hoc test)
Table 2. Biochemical parameters
Group 2
Group 3
Group 4
p value
*
** 9.7±2.9
3.3±1.7
P<0.001
**
4.5±1.5
P<0.001
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Group 1 2.4±1.0
22.8±3.8
Number of TUNEL-positive cells in Stria vascularis
3.2±1.3
27.1±3.0
*
11.1±2.5
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Number of TUNEL-positive cells in organ of Corti
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* p <0.05; comparison of group 2 and group 1 ** p<0.05; comparison of group 2 and group 3
Table 3. Number of TUNEL-positive cells of groups. For the comparison between groups, Kruskal–Wallis test was applied (P<0.05 was accepted as statistically significant). Mann-Whitney U test was used to compare the subgroups.
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Figure 1: Comparison of DPOAE SNR levels within-group. For the comparison within- group, the paired T test was applied (P < 0.05 was accepted as statistically significant).
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Figure 2: Comparison of DPOAE SNR levels between-groups. For the comparison between groups, the One-way ANOVA test was applied (P < 0.05, statistically significant). The Tukey’s HSD post-hoc test was applied to identify within-group differences.
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Figure 3: Histological micrographs of organ of Corti (a-d) and stria vascularis (eh) in the cochlear sections of study groups. a,e: Control group. b,f: Gentamicin group. c,g: Naringenin-Gentamicin group. d,h: Naringenin group. Arrows indicate TUNEL-positive cells. In group 2 showed increased TUNEL-positive cells compared to group 1, 3 and 4. Fewer TUNEL-positive cells were observed in group 3 compared to group 2. (TUNEL staining method, Bar: 20 µm).
S0165-5876(17)30321-X
DOI:
10.1016/j.ijporl.2017.07.008
Reference:
PEDOT 8612
To appear in:
International Journal of Pediatric Otorhinolaryngology
Received Date: 27 February 2017 Revised Date:
16 June 2017
Accepted Date: 7 July 2017
Please cite this article as: İ Koçak, S Sarac, E Aydogan, E Şentürk, D Akakın, K Koroglu, Ö. Özer, Evaluation of the possible protective role of naringenin on gentamicin-induced ototoxicity: A preliminary study, International Journal of Pediatric Otorhinolaryngology (2017), doi: 10.1016/j.ijporl.2017.07.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Evaluation of the Possible Protective Role of Naringenin on Gentamicin-Induced
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Ototoxicity: A Preliminary Study
Koçak İ1, Sarac S1 ,Aydogan E1, Şentürk E2 , Akakın D3, Koroglu K3, Özer ÖF4
Department of Otolaringology, Koç University Hospital, Istanbul, Turkey.
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Department of Otolaringology, Alaca State Hospital, Çorum, Turkey.
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Department of Histology and Embryology, Marmara University School of Medicine, Istanbul
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Department of Biochemistry, Bezmialem Vakif University, Medical Faculty, Istanbul, Turkey
Compliance with Ethical Standards
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Ethical Procedure • The research involving animals and meets all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. • As an expert scientist and along with co-authors of concerned field, the paper has been submitted with full responsibility, following due ethical procedure, and there is no duplicate publication, fraud, plagiarism, or concerns about animal or human experimentation.
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A Disclosure / Conflict Of Interest Statement • None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. • It is to specifically state that “no competing interests are at stake and there is no conflict of interest” with other people or organizations that could inappropriately influence or bias the content of the paper.
Most sincerely,
Corresponding Author: İlker Koçak, M.D.
Koc University Hospital, Department of Otolaringology Davutpasa Cad. No: 4, Topkapi, 34010, Istanbul, Turkey E-mail: [email protected]
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Phone: +905557116870
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Evaluation of the Possible Protective Role of Naringenin on Gentamicin-Induced Ototoxicity: A Preliminary Study Introduction
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Aminoglycoside antibiotics such as gentamicin are widely used around the world to treat gram-negative bacterial infections and tuberculosis, despite well-known side effects, such as ototoxicity and nephrotoxicity (1,2) In addition, intratympanic gentamicin is an important treatment modality in the management of Meniere’s disease(3). Gentamicin-induced ototoxicity is dependent on dose and duration, and may frequently be bilaterally symmetric and irreversible. Hearing loss initially affects high frequencies and, in long-term exposure to gentamicin all frequencies.
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In aminoglycoside ototoxicity, target structures are the organ of Corti and the hair cells. The most widely proposed pathophysiologic mechanism is the creation of reactive oxygen species accumulating in the cochlea, causing apoptosis and cell death (4,5).
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A number of experimental and clinical studies into the reduction of gentamicin-induced ototoxicity have been carried out, showing that substances such as thymoquinone, quercetin, trimetazidine, alpha lipoic acid, Nacetylcysteine, and vitamin A are effective against gentamicin ototoxicity, thanks to their antioxidant properties (6-11).
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Flavonoids are phenolic compounds widely present in fruit and vegetables that exhibit powerful antioxidant activity because of their ability to reduce free radical formation. Naringenin is a bioflavonoid highly enriched in tomatoes, cocoa and citrus fruits such as grapefruit, lemon and orange. It was reported that naringenin has antioxidant(12,13), anti-inflammatory (14,15) and free radical scavenging effects (16), and also inhibits lipid peroxidation(17). Past studies demonstrated that naringenin has anti-tumoral(18,19), hepatoprotective(20) and nephroprotective(21) features. It was also shown that naringenin treatment significantly protected against gentamicin-induced nephrotoxicity in rats (22). However, the potential protective effect of naringenin on gentamicininduced ototoxicity has not been investigated.
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The aim of this study was to evaluate the protective effect of naringenin against gentamicin ototoxicity with audiological tests, and biochemical, and histopathological parameters.
Material and methods
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The protocol of this study was approved by the Ethics Committee on Animal Research of Istanbul University. The study was performed at the Experimental Animal Studies Laboratory of Istanbul University.
Animals
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Thirty-two adult male Sprague Dawley rats weighing 250 to 300 g were obtained at the Experimental Animal Studies Laboratory of Istanbul University for study. Rats with a positive Preyer reflex were chosen and a 2.7mm 0o endoscope was used to examine the tympanic membranes and external ear canals of all rats. Rats with cerumen in their ear canals and those showing signs of otitis media or tympanic membrane perforation were excluded from the study. All rats had free access to food and water, and were maintained in an environment with controlled temperature (25 °C), under a 12-hour light/dark cycle.
Drugs and Chemicals
Gentamicin (Genta) was obtained from İ.E. Ulagay İlaç Sanayii Türk A.Ş, Istanbul, Turkey. 0.5% Carboxymethyl cellulose and 98% naringenin were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO, USA (98% naringenin dissolved in 0.5% carboxymethyl cellulose). Xylazine hydrochloride (Rompun) was purchased from Bayer, Istanbul, Turkey. And ketamine hydrochloride (Ketalar) was from Eczacibasi, Istanbul, Turkey. Experimental groups
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The 32 rats were randomly divided into four groups. Each group contained eight rats: Group 1 (n = 8) received 0.5% carboxymethyl cellulose perorally (p.o) daily for 14 days.
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Group 2 (n = 8) received gentamicin 120 mg/kg intraperitoneally (i.p.) and 0.5% carboxymethyl cellulose p.o. daily for 14 days. Group 3 (n=8) received gentamicin 120 mg/kg i.p. and naringenin 50 mg/kg p.o. daily for 14 days.
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Group 4 (n = 8) received naringenin 50 mg/kg and 0.5% carboxymethyl cellulose p.o. daily for 14 days.
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At the beginning of the study and on day 14, all rats were anesthetized by intra-peritoneal 5 mg/kg xylazine hydrochloride and 40 mg/kg ketamine hydrochloride. DPOAE and ABR measurements were performed after anesthesia. On the 14th day, intracardiac blood samples were taken from all rats to calculate the biochemical parameters. Again on the 14th day, their cochleae were bilaterally harvested for histopathologic observations, and sent for pathological examination in 10 % buffered formaldehyde.
Audiological evaluation
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Distortion product otoacoustic emission(DPOAE) A Neurosoft Neuro-audio device (Ivanova, Russia) was used to measure DPOAEs. Measurements were performed in a silent room. The smallest size tympanometry probe was placed into the external ear canal and measurements were performed on both ears. DPgram measurements were performed at frequencies of 988, 2222, 3200, 4444, 5000, 6154, 8000, 8889, 10000 and 11429 Hz. DPOAE values 6 dBSPL above the signal to noise ratio were accepted as positive. Auditory brainstem response(ABR)
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ABR measurements were conducted in a silent room with Neurosoft Neuro-audio device (Ivanova, Russia), using subdermal needle electrodes. An ER-2 insert ear phone was used to provide click stimuli in alternating polarities. A tone-burst stimulus of 16 kHz with a 30-2000 Hz band-pass filter at a repeat rate of 21/ second was used as the auditory stimulant. The threshold was determined by starting at 100 dB SPL , then decreasing by increments of 10 dB until the threshold was reached. Repeatability was confirmed, and the threshold determination was developed over two tests. The ABR threshold was defined as the lowest intensity level where the fifth wave was observed.
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Biochemical evaluation
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For biochemical analyses, blood samples taken from the rats were centrifuged for 15 minutes in 3,000 rpm., and the serum was isolated and kept at –80°C until blood specimens from all the rats were prepared.
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Oxidative stress means that the balance between pro- and antioxidants is skewed towards pro-oxidants (23). It is difficult to measure different antioxidants individually because of time consumption and cost. The recently developed method of measuring the total antioxidant status (TAS) allows all antioxidants to be recorded at a very low cost in a short time as a single parameter by a simple measurement in the serum (24). TAS measures the combined activity of antioxidants and the total antioxidant level, therefore evaluating the comprehensive antioxidant status. However, there are no practical methods for the measurement of individual pro-oxidant molecules, although, a single parameter, total oxidant status (TOS), can be measured in the serum instead(25). TOS is an indicator of the total oxidant levels. The oxidative stress index (OSI) provides a more accurate index for oxidative stress in the body, since this ratio takes into account the sum total of all oxidant and antioxidant activities. TAS and TOS were measured by the Rel Assay Diagnostics kit (Mega Tip San ve Tic Ltd Sti, Gaziantep, Turkey), and the OSI was calculated from the TAS and TOS (OSI: TOS/TASX100). Histopathologic examination
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The cochleae were dissected under a dissecting microscope and fixed with 10 % buffered formalin. Subsequently, they were placed in an EDTA solution for the decalcification of osseous tissues, and washed overnight under a water flow. Tissues were dehydrated in an ethanol series of graded concentrations and cleared in xylene. The samples were then embedded in paraffin. Longitudinal 5 µm thick sections passing parallel to the modiolus were deparaffinized, hydrated and stained with the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) method for determining apoptotic cells.
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TUNEL method
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TUNEL staining was used to evaluate the extent of apoptosis in the organ of Corti and stria vascularis. The TUNEL staining assay kit was used in accordance with the user's manual from the manufacturer (Millipore ApopTag Plus Peroxidase In Situ Apoptosis Kit S7101, Temecula, CA, USA). After staining with Mayer's hematoxylin, sections were coverslipped with Entellan mounting medium. TUNEL-positive cells in the organ of Corti and stria vascularis were counted in randomly chosen fields for each case under a light microscope (Olympus BX51, Tokyo, Japan) with a 40x objective lens and photographed with a digital camera (Olympus DP72, Tokyo, Japan). Three of the 5 cochlear sections (2 basal turns, 2 medial turns and 1 apical turn) were evaluated in each slide and the average number of TUNEL-positive cells in the stria vascularis and organ of Corti were recorded for each slide.
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Two histologists, who were blinded to the animal grouping, independently evaluated the TUNEL scores.
Statistical analysis
Statistical analysis was carried out using SPSS ver. 22.0 (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). Data normality was checked using the Kolmogorov-Smirnov tests of normality.
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One-way ANOVA was used for intergroup comparisons of DPOAE and ABR values (the differences among groups were considered statistically significant at p<0.05). The Tukey’s (HSD) post-hoc test was used for determining the differences between the groups.
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The paired samples t test was used for within-group comparisons of DPOAE and ABR values. (the difference within group was considered statistically significant at p < 0.05).
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One-way ANOVA was used in the evaluation of biochemical parameters between groups (the differences between groups were considered statistically significant at p<0.05). The Tukey’s HSD post-hoc test was used for determining which groups differed.
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RESULTS
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The Kruskal-Wallis test was used to compare the number of TUNELpositive cells between groups (the differences between groups were considered statistically significant at p<0.05). The Mann-Whitney U test was used for determining the differences between the groups.
Distortion product otoacoustic emission
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No significant differences were observed between the baseline DPOAE SNR levels in any of the groups (p >0.05).
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In group 2 (gentamicin) on day 14, DPOAE SNR levels significantly decreased as compared to their baseline levels (at all frequencies, except 998 and 2222 Hz.) (p < 0.05) (Figure 1) In group 1(control), group 3 (gentamicin+naringenin) and group 4(naringenin) there were no significant differences of the DPOAE SNR levels between days 0 and 14 (p>0.05) (Figure 1). On day 14, the DPOAE SNR levels of group 2 (gentamicin) were significantly lower than those of groups 1, 3 and 4 (at all frequencies, except 998 and 2222 Hz.) (p < 0.008) (Figure 2).
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Also on day 14, there were no significant differences of the DPOAE SNR levels between group 3 (gentamicin+naringenin) and group 1(control) (p >0.05) (Figure 2).
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Auditory brainstem response There were no significant differences in any group between the baseline ABR thresholds (p > 0.05) (Table 1).
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In group 2 (gentamicin) on days 14, the ABR threshold significantly increased as compared to the baseline level (p<0.001) (Table 1).
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In group 1(control), group 3 (gentamicin+naringenin) and group 4(naringenin) there were no significant differences in the ABR thresholds between days 0 and 14 (p >0.05) (Table 1). On day 14, the ABR threshold of group 2 (gentamicin) was significantly higher than those of groups 1, 3 and 4 (p < 0.001) (Table 1).
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Also on day 14, no significant differences were observed in the ABR thresholds between group 3 (gentamicin+naringenin) and group 1 (control) (p >0.05) (Table 1).
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Biochemical evaluation
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The TOS and OSI values of group 2 were significantly higher than the values of the other groups (p<0.05) (Table 2). No significant differences were observed between the TOS and OSI values of group 1, group 3 and group 4 (p>0.05) (Table 2). The TAS values of groups 3 and 4 were significantly higher than those of group 2 (p<0.05) (Table 2).
Histopathologic results
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The numbers of TUNEL-positive cells are shown in Table 3. Fewer TUNELpositive cells were observed in group 1(control) and group 4(naringenin). Relative to group 1, group 3(naringenin+gentamicin) and group 4, TUNELpositive cells in the organ of Corti and stria vascularis were significantly increased in group 2(gentamicin) (p<0.001). However, the number of TUNELpositive cells in group 3 was significantly lower than in group 2 (p<0.05).
Discussion
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There is a wide range of use for aminoglycoside antibiotics, however, this is limited by the ototoxic effects of these drugs. This study has researched the potential protective effect of naringenin against gentamicin ototoxicity. We observed a decrease in DPOAE values and an increase in ABR thresholds in the group that had been administered gentamicin. Biochemical and histopathologic findings confirmed ototoxicity. We have seen that DPOAE values and ABR thresholds were preserved in the group being given naringenin+gentamicin. In the biochemical evaluation, we found that oxidative parameters in the naringenin+gentamicin group were lower and, the antioxidative parameters higher than in the gentamicin group. In addition, histopathologic evaluation showed that the number of TUNEL-positive cells in the naringenin+gentamicin group was lower than in the gentamicin group. Ultimately, the protective effect of naringenin in gentamicin-induced ototoxicity was shown through audiological, biochemical and histopathologic evaluations.
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Gentamicin damages the outer hair cells beginning from the basal region of the cochlea, then progressing to the middle and apical regions. Thus, the hearing loss that first involved only higher frequencies extends to lower frequencies. The damage to outer hair cells can be evaluated by DPOAE testing. ABR testing is an objective method for evaluating the auditory pathways proximal to distal(26). In our study, DPOAE values on day 14 in the gentamicin group were lower than initial values and ABR thresholds were higher than initially. These findings show that gentamicin damages outer hair cells and has a toxic effect on the auditory pathways. Further, we detected no significant difference in the DPOAE values and ABR thresholds between day 14 and initial
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values in the naringenin+gentamicin group, which demonstrates the protective effect of naringenin against gentamicin ototoxicity.
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The mechanism of gentamicin-induced ototoxicity is not entirely understood. It is assumed that the production of free oxygen radicals in the inner ear because of aminoglycosides causes cell death (27). Free radicals are attacking cell membranes, proteins, and DNA, leading to lipid peroxidation causing irreparable cell damage and cell death. In addition, by producing an excess of free oxygen radicals, gentamicin leads to a reduction of antioxidant enzymes and a disturbance of the balance between oxidants and antioxidants. Various studies have reported that gentamicin increased oxidative stress, thus causing a toxic effect(4,5,9). In our study, to measure the total oxidative/antioxidative balance, we used objective and sensitive biochemical parameters (TOS, TAS, and OSI). TOS and OSI values, being oxidative stress parameters, were significantly higher in group 2 (gentamicin) compared to the other groups (Table 2). This finding supports the result that gentamicin produces free oxygen radicals.
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Numerous studies have investigated the effectiveness of various antioxidant agents against gentamicin ototoxicity (6-11). However, there is still no agent being approved for clinical application against gentamicin ototoxicity.
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Naringenin is the major bioactive flavonoid compound derived from citrus fruits and a strong antioxidant agent (12,13). It manifests its antioxidant activity through different mechanisms, such as scavenging free oxygen radicals and inhibiting lipid peroxidation (16,17). In addition, previous studies have shown a naringenin to have a protective effect against oxidative cell damage through an increase of antioxidant enzymes such as GSH (glutathione) or SOD (superoxide dismutase) (28). In our study, we found the value of the antioxidant parameter TAS in groups 3 (gentamicin+naringenin) and 4 (naringenin) to be significantly higher than in group 2 (gentamicin) (Table 2). This finding supports previous reports that naringenin increases antioxidant activity.
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Nitric oxide (NO), is a free radical that also plays an important role in hair cell injury and gentamicin-induced ototoxicity (11,29). Gentamicin stimulates inducible nitric oxide synthase (iNOS), leading to an increased production of NO, which reacts with superoxide radicals to form a cytotoxic peroxynitrite radical (30). Peroxynitrite reacts with the cell membrane and cellular proteins, leading to cell death (31). There are studies showing that naringenin inhibits iNOS activity and reduces NO synthesis, thus exhibiting a protective effect against oxidative stress in hepatocytes (32). It has also been reported that naringenin provides a protective effect against gentamicin-induced nephrotoxicity by reducing iNOS synthesis (22).
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Another mechanism being proposed for gentamicin ototoxicity is the activation of the nuclear factor-kappa B (NF-κB) signal pathway and damage to outer hair cells caused by inflammatory cytokines (33). Previous studies have demonstrated that naringenin provides a cytoprotective effect by suppressing the NF-κB pathway (34,35). These findings support the observation that naringenin has a protective effect against gentamicin-induced ototoxicity.
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Apoptosis, a kind of controlled cell death, occurs for a variety of reasons during normal growth and development. Apoptosis may be the primary result of gentamicin ototoxicity. Experimental studies show apoptosis occurring in the cochlea after gentamicin use (6,7,11). The TUNEL method was described by Gavrieli and accepted as a marker of apoptosis(36). TUNEL-positive staining indicates fragmented DNA chains and apoptosis. In our study, we exhibited apoptosis in the organ of Corti and stria vascularis through TUNEL staining; the number of apoptotic cells was significantly greater in the gentamicin group than in the other groups (Figure 3). In addition, the number of TUNEL-positive cells was significantly decreased in the naringenin+gentamicin group compared to the gentamicin-only group. These findings demonstrate that naringenin prevented apoptosis in gentamicin-induced ototoxicity. It has been demonstrated that naringenin plays a role in protecting against gentamicin nephrotoxicity as a result of its antioxidant and cytoprotective effects(22). Also, Sahu et al. has reported that naringin, which is the precursor of naringenin, ameliorates gentamicin-induced
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nephrotoxicity(37). To the best of our knowledge, the possible protective effect of naringenin against gentamicin ototoxicity has not been previously demonstrated. In this study we were able to show that naringenin was effective against gentamicin-induced ototoxicity. In addition, Li et al.(38) has documented that long-term and high-dose naringenin exposure does not lead to any lethality or toxic clinical symptoms in rats, thus naringenin can be used safely in gentamicin ototoxicity.
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The strengths of our study included the use of DPOAE and ABR tests in the total audiological evaluation, the use of biochemical parameters in the assessment of oxidative stress, and histopathologic evaluation.
Conclusion
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By way of limitations, we did not assess the effectiveness of different doses, the number of animals in each group, though statistically sufficient, was small. Although dose response studies of naringenin efficacy have been documented in the literature (37,38), we did not perform the dose comparison for naringenin effect. In our study, naringenin dose was selected on the basis of previously published reports (20-22,28). This may be a limitation of our research. Additionally, we did not investigate the effect of naringenin on the antimicrobial activity of gentamicin, this is another limitation of this study. Further studies are required to evaluate the effectiveness of different doses and the efficacy of naringenin on the antimicrobial activity of gentamicin.
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This is the first study evaluating the effect of naringenin usage in gentamicin-induced ototoxicity. Our study demonstrated with audiological (DPOAE and ABR), and biochemical measurements and histopathological evaluation that the ototoxic effects of gentamicin could be limited by the concurrent use of naringenin. Further experimental and prospective randomized clinical studies are needed to confirm our findings. Funding: None Conflict of interest: No conflict of interest was declared by the authors.
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Ethical approval: All applicable international, national and/or institutional guidelines for care and use of animals were followed. Acknowledgments: None.
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Baseline
14th Day
Paired T Test
Group 1
47,5 ± 4,4
48,7 ± 3,4
p=0.43
Group 2
48,1 ± 4,0
68,7 ± 8,0 *
p<0.001
Group 3
45,6 ± 5,1
49,3 ± 5,7 **
Group 4
44,3 ± 5,1
46,8 ± 4,7
One Way ANOVA
P=0.1
p=0.001
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Groups
p=0.08
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p=0.16
* p<0.001 compared with group 1, 3 and 4 ** p=0.4 compared with group 1
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Tukey (HSD) posthoc test
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Table 1: Comparison of ABR thresholds between-groups . For the comparison within-group, the paired T test was applied (P < 0.05 was accepted as statistically significant). For the comparison between groups, the One-way ANOVA test was applied (P < 0.05, statistically significant). The Tukey’s HSD post-hoc test was applied to identify within-group differences.
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TAS (μmol Troloxequiv./L)
TOS (μmol H2O2 equiv./L) 5,29 ± 1,54
OSI (TOS/TASX100)
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Group 1 1,23 ± 0,32 0,047 ± 0,02 (control) Group 2 1,06 ± 0,35 9,34 ±2,09 0,10 ± 0,05 (gentamicin) Group 3 1,66 ± 0,45 4,75 ± 1,51 0,029 ± 0,011 (gentamicin+naringenin) Group 4 1,81 ± 0,28 5,73 ± 1,29 0,032 ± 0,009 (naringenin) p(ANOVA) P<0.05 P<0.05 P<0.05 G2 vs G1 ¶ ¶ G2 vs G3 ¶ ¶ ¶ G2 vs G4 ¶ ¶ ¶ TOS; total oxidant status, TAS; total antioxidant status, OSI; oxidative stress index. p<0.05, significance level obtained between groups, one-way analysis of variance (ANOVA) p<0.008, significance level obtained (Tukey honest significant difference post hoc test)
Table 2. Biochemical parameters
Group 2
Group 3
Group 4
p value
*
** 9.7±2.9
3.3±1.7
P<0.001
**
4.5±1.5
P<0.001
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Group 1 2.4±1.0
22.8±3.8
Number of TUNEL-positive cells in Stria vascularis
3.2±1.3
27.1±3.0
*
11.1±2.5
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Number of TUNEL-positive cells in organ of Corti
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* p <0.05; comparison of group 2 and group 1 ** p<0.05; comparison of group 2 and group 3
Table 3. Number of TUNEL-positive cells of groups. For the comparison between groups, Kruskal–Wallis test was applied (P<0.05 was accepted as statistically significant). Mann-Whitney U test was used to compare the subgroups.
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Figure 1: Comparison of DPOAE SNR levels within-group. For the comparison within- group, the paired T test was applied (P < 0.05 was accepted as statistically significant).
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Figure 2: Comparison of DPOAE SNR levels between-groups. For the comparison between groups, the One-way ANOVA test was applied (P < 0.05, statistically significant). The Tukey’s HSD post-hoc test was applied to identify within-group differences.
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Figure 3: Histological micrographs of organ of Corti (a-d) and stria vascularis (eh) in the cochlear sections of study groups. a,e: Control group. b,f: Gentamicin group. c,g: Naringenin-Gentamicin group. d,h: Naringenin group. Arrows indicate TUNEL-positive cells. In group 2 showed increased TUNEL-positive cells compared to group 1, 3 and 4. Fewer TUNEL-positive cells were observed in group 3 compared to group 2. (TUNEL staining method, Bar: 20 µm).