Protective effect of gallic acid against cisplatin-induced ototoxicity in rats

Introduction Cisplatin is an antineoplastic agent widely used in the treatment of a variety of cancers. Ototoxicity is one of the main side-effects restricting the use of cisplatin. Objective The purpose of this study was to investigate the protective efficacy of gallic acid, in biochemical, functional and histopathological terms, against ototoxicity induced by cisplatin. Methods Twenty-eight female Sprague Dawley rats were included. Rats were randomly assigned into four groups of seven animals each. Cisplatin group received a single intraperitoneal dose of 15 mg/kg cisplatin. Gallic acid group received intraperitoneal gallic acid at 100 mg/kg for five consecutive days. Cisplatin + gallic acid group received intraperitoneal gallic acid at 100 mg/kg for five consecutive days and a single intraperitoneal dose of 15 mg/kg cisplatin at 3rd day. A control group received 1 mL intraperitoneal saline solution for five consecutive days. Prior to drug administration, all rats were exposed to the distortion product otoacoustic emissions test. The test was repeated on the 6th day of the study. All rats were then sacrificed; the cochleas were removed and set aside for biochemical and histopathological analyses. Results In cisplatin group, Day 6 signal noise ratio values were significantly lower than those of the other groups. Also, malondialdehyde levels in cochlear tissues were significantly higher, superoxide dismutase and glutathione peroxidase activities were significantly lower compared to the control group. Histopathologic evaluation revealed erosion in the stria vascularis, degeneration and edema in the connective tissue layer in endothelial cells, impairment of outer hair cells and a decrease in the number of these calls. In the cisplatin + gallic acid group, this biochemical, histopathological and functional changes were reversed. Conclusion In the light of our findings, we think that gallic acid may have played a protective role against cisplatin-induced ototoxicity in rats, as indicated by the distortion product otoacoustic emissions test results, biochemical findings and immunohistochemical analyses.


Conclusion:
In the light of our findings, we think that gallic acid may have played a protective role against cisplatin-induced ototoxicity in rats, as indicated by the distortion product otoacoustic emissions test results, biochemical findings and immunohistochemical analyses. © 2018 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

Introduction
Cis-diammineedichloroplatinum (cisplatin) is a chemotherapeutic agent widely used in the treatment of cancer since being approved by the Federal Drug Administration in 1978. The importance of long-term complications developing in association with chemotherapeutics is growing as survival rates in cancer patients improve. Side-effects such as ototoxicity, nephrotoxicity, and neurotoxicity are seen following cisplatin chemotherapy. Hearing losses at rates between 30% and 100% have been reported in association with cisplatin ototoxicity. 1 This hearing loss results in multifaceted decreases in quality of life and can affect social and academic development, particularly in children. 2 Cisplatin-induced ototoxicity involves high-frequency bilateral and symmetrical hearing loss. Such losses may be permanent and irreversible and are frequently associated with tinnitus. 3 The severity of this hearing loss is associated with the frequency of drug application and the dosage concerned. No methods other than reducing the cisplatin dosage or modification to chemotherapeutic regimens not containing cisplatin have to date been discovered for the purpose of reducing cisplatinrelated ototoxicity. These two methods may also have adverse impacts on treatment outcomes. Various studies have therefore investigated agents capable of preventing cisplatin-induced ototoxicity. Several antioxidant agents including d-methionine, allopurinol, flunarizine, curcumin and lutein have been employed for this purpose in experimental studies. 4 ---8 Gallic acid is a trihydroxybenzoic acid, a phenolic acid found in numerous medicinal plants and food materials. It has long been known to exhibit powerful antioxidant effects. 9 Phenolic acids have been reported to exhibit their antioxidant effects by directly scavenging free radicals, by inactivating enzymes responsible for Reactive Oxygen Species (ROS) production and increasing the activation of antioxidant enzymes. 10 To the best of our knowledge, no previous studies have investigated the protective effect of gallic acid against cisplatin-induced ototoxicity. The purpose of this study was to investigate the protective efficacy of gallic acid in rats, in biochemical, functional and histopathological terms, against ototoxicity induced by cisplatin.

Animals
Approval for the study was granted by the Ataturk University Animal Studies Local Ethical Committee with number E.1700305147-11:145. Twenty-eight female Sprague Dawley rats weighing 220---240 g were included. The study was conducted in accordance with the Care and Use of Laboratory Animals Guide. All rats were housed in special cages with ad libitum access to chow and water in a stable temperature of 22 • ± 1 • C and humidity of 45% ± 2%. The lighting system was adjusted to a 12 h light/12 h dark cycle.

Experimental protocol
The gallic acid used in the study was obtained from the Sigma-Aldrich Chemical Company, and cisplatin from Kocak Pharma Co. (Kocak, Istanbul, Turkey). Rats were randomly assigned into four groups of seven animals each. Prior to drug administration, all rats underwent otoscopic examination under 40 mg/kg ketamine and 10 mg/kg xylazine anesthesia. All rats were then exposed to the Distortion Product Otoacoustic Emissions (DPOAE) test. The DPOAE test was repeated on the 6th day of the study under the same anesthesia protocol. All rats were then sacrificed, and the temporal bones were dissected. The cochleas were removed and right cochlea was set aside for biochemical analyses while the left cochlea was set aside for histopathological analyses.

DPOAE procedure
DPOAE measurements was conducted with an Otometrics Madsen Capella device. This device was calibrated before each measurement. Before measurement, all rats were anesthetized with 40 mg/kg ketamine hydrochloride (Ketasol 10%, Richter Pharma Ag, Wels, Austria) and 10 mg/kg xylazine (Alfazyne 2%, Alfasan International BV, Voerden, the Netherlands). Following anesthesia, rats were subjected to otoscopic examination. Appropriate probes were placed on both right and left outer ear channels of the rats to perform DPOAE measurements in a silent room. Measurements were taken in the form of DPgrams. The difference between the levels of L1 and L2 was kept at 10 dB SPL (sound pressure level) (L1 = 65 dB SPL, L2 = 55 dB SPL). In order to obtain the most powerful response, the test was set to two separate frequencies, f1/f2 = 1.22. DPgram measurements were performed at frequencies of 2002, 2500, 3154, 4004, 5000, 6299, 7998 and 10,000 Hz. SNR values of 3 dB or above were regarded as positive. The Signal Noise Ratio (SNR) values of all groups obtained during the DPOAE test were compared within and between the groups.

Immunohistochemical examination
All sections placed onto adhesive slides (poly-l-Lysine) for immunoperoxidase examination were passed through xylol and alcohol series, deparaffinized and dehydrated. The procedure was performed according to the guideline of Ab80436 kit. Caspase-3 was used as primary antibody. Following the washing procedure, 3-3 ′ Diaminobenzidine (DAB) chromogen was dropped onto the section and allowed to stand for 5---10 min to absorb the chromogen. After emersion in Mayer's hematoxylin for 1 ---2 min for background staining, specimens were washed under tap water. The slides were then passed through alcohol and xylol series, covered, and examined under a light microscope (Leica DM 1000). Sections' immune positivity was graded as none (−), slight (+), moderate (++) or severe (+++).

Biochemical analysis
The cochleas set aside for biochemical examination were washed with physiological saline solution immediately after excision and then frozen at −80 • C. On the day of study, they were removed from the freezer, thawed, and homogenized with a 9 mL per gram 1.15% KCl solution. The homogenates were centrifuged at 10,000 rpm for 30 min for supernatant separation.
The spectrophotometric method described by Sun et al. was employed for Superoxide Dismutase (SOD) analysis. 11 This technique is based on the reduction of Nitroblue Tetrazolium (NBT) by the superoxide producer xanthine---xanthine oxidase at 560 nm. SOD activity was expressed as U/L. Malondialdehyde (MDA) analysis was performed using the Thiobarbituric Acid (TBA) technique described by Ohkawa and Yagi. 12 This is based on spectrophotometric measurement of the pink product resulting from the reaction between MDA and TBA. The results were expressed as nmoL/mL.
Glutathione peroxidase (GPx) was measured using a modification of the method described by Paglia and Valentine. 13 GPx is an enzyme that produces glutathione oxidation in a reaction in which hydrogen peroxide is reduced to water. Oxidized glutathione is again reduced to glutathione with the enzyme glutathione reductase in the presence of NADPH. The method is based on spectrophotometric measurement of the decrease in NADPH absorbance at 340 nm. GPx activity was expressed as IU/L.

Statistical analysis
Statistical analysis was performed on SPSS 17.0 software. One-Way ANOVA was used for statistical analysis of normally distributed parameters, and the post hoc Tukey test was employed to identify sources of variation. The non-parametric Kruskal---Wallis test was used to analyze variations among data obtained using the semi-quantitative method at histopathological examination. Analyses between two groups were performed using the Mann---Whitney U test. p < 0.05 was regarded as statistically significant.

Results
No significant difference was observed between the groups in terms of SNR values in the DPOAE results on the first day of the study. Also there was no difference between the right and left ear SNR values. When DPOAE results on Day 6 were compared with the findings from Day 1, no significant variation was observed in SNR values in Groups 2, 3 or 4. In Group 1, SNR values on Day 1 were significantly higher than those on Day 6 at all studied frequencies. This shows that cisplatin exerts an adverse effect on hearing functions by affecting the cochlea.
When the DPOAE test results on Day 6 were compared among themselves, SNR values in Group 1 were significantly lower than those of the other groups. No significant variation was determined between SNR values in Groups 2, 3 and 4. These findings show that the use of gallic acid with cisplatin prevented impairment in SNR values and exhibited functional protection against the ototoxic effect of cisplatin ( Figs. 1 and 2).
The results of biochemical analysis of rat cochleas are shown in Fig. 3. MDA levels in cochlear tissues in Group 1 were significantly higher compared to the control group. At the same time, SOD and GPx activities in cochlear tissues decreased significantly compared to the control group. This shows that oxidative stress occurred in the cochleas of rats receiving cisplatin, and that this stress was manifested with an increase in amounts of free oxygen radicals emerging with a rise in lipid peroxidation, and also with a decrease in antioxidant enzyme activities. No significant variation was observed between MDA levels, SOD and GPx activities in rats from Groups 2, 3 and 4. Additionally, MDA levels in the cochleas of rats from Group 3, those receiving gallic acid together with cisplatin, were significantly lower than those of rats in Group 1, those receiving cisplatin only, while SOD and GPx activities were significantly higher. This shows that gallic acid also protects against oxidative stress caused by the ototoxic effect of cisplatin.
In histopathologic examination, the cochleas in the control and gallic acid groups exhibited a normal histological appearance (Fig. 4A--  degeneration and edema in the connective tissue layer in endothelial cells. The structure of outer hair cells was impaired, and a decrease in these cells was also observed (Fig. 4B). Examination of the cochleas in the Cis + gallic acid group revealed mild hyperemia in the stria vascularis, degeneration in epithelial cells and very mild degeneration in the spinal ganglia (Fig. 4D). These findings decreased in a statistically significant manner in the Cis + gallic group compared to the cisplatin group (p < 0.05). Histopathological cochlea findings are summarized in Table 1. Caspase-3 staining was performed to determine apoptosis in cells. No Caspase-3 immunopositivity was observed at cochlear staining in the control or gallic acid groups (Fig. 5A---C). In the cisplatin group, severe intraplasmic immunopositivity was observed in outer hair cells (Fig. 5B). In the Cis + gallic acid group, mild intraplasmic Caspase-3 expression was determined in outer hair cells (Fig. 5D). A statistically significant decrease was determined compared to the cisplatin group (p < 0.05). Immunohistochemical findings are summarized in Table 1.

Discussion
Our study clearly demonstrated the protective efficacy of gallic acid against cisplatin-induced ototoxicity. Gallic acid was determined, both biochemically and histopathologically, to prevent oxidative stress arising in the cochlea following cisplatin administration. The DPOAE findings also showed that gallic acid exerts a protective effect against hearing loss associated with cisplatin-induced ototoxicity.
Cisplatin is an antineoplastic agent widely used in the treatment of a variety of cancers. The main side-effects of this agent are nephrotoxicity, neurotoxicity and ototoxicity. While nephrotoxicity can be controlled with hydration therapy, ototoxicity remains the most significant side-effect of cisplatin use. 8 The mechanism by which cisplatin leads to ototoxicity is not yet fully understood. Previous studies have shown that cisplatin causes hearing losses by impacting on various areas of the cochlea. The histopathological finding that has attracted the greatest interest is hair cell degeneration. Damage beginning in the tip links in the stereocilia   14,15 In our study, histopathologic examination of the cochlea in the cisplatin group revealed erosion in the stria vascularis, degeneration and edema in the connective tissue layer in endothelial cells, impairment of the structure of outer hair cells and a decrease in the number of these cells. The pathology responsible for this damage in the cochlea at the molecular and cellular level is not yet fully understood, although oxidative stress is believed to be the main agent. Cisplatin initiates numerous pathophysiological processes in the cochlea by increasing ROS production and suppressing antioxidant enzyme systems. Increased oxidative stress in the cochlea increases the peroxidation of lipid membranes, inactivates important cellular enzymes and compromises ion channel functions. Finally, increased ROS production leads to histopathological processes and functional impairments in the cochlea by causing apoptosis and necrotic cell death. 16 In our study, Caspase-3 staining was performed to determine apoptosis in cells. Severe intraplasmic immunopositivity was observed in outer hair cells of the rats in the cisplatin group.
The cochlea has an effective antioxidant defense mechanism containing antioxidant such as vitamin C, vitamin E and Glutathione (GSH) and enzymes such as SOD, GPx and Catalase (CAT). 17  and maybe certain other peroxides. Additionally, GPx also accelerates the conversion of reduced GSH to its oxidized form (GSSG) during the detoxification of H2O2. 16 If this ROS elimination system is compromised, ROS lead to lipid peroxidation and cellular damage. This manifests itself with elevated MDA levels. Levels of MDA were significantly higher in the cochleas of rats receiving cisplatin compared to the control group in our study. However, SOD and GPx activities in the cochlear tissues of rats receiving cisplatin were significantly lower than those of the control group. These findings reveal that oxidative stress is involved in cochlear damage occurring in cisplatin-induced ototoxicity.
In the light of these physiological processes, it has been hypothesized that reducing ROS levels and increasing levels of antioxidant enzymes can be protective against cisplatininduced ototoxicity. Various antioxidant agents have been investigated in cisplatin ototoxicity. The purpose of our study was to determine whether gallic acid, a powerful antioxidant, would exhibit a protective effect against cisplatin-induced ototoxicity.
Gallic acid is a poly-hydroxyphenolic compound found in numerous plants, fruits and foodstuffs. It also occurs naturally in a number of terrestrial and aquatic plants. 18 It exhibits powerful anti-carcinogenic, anti-mutagenic and anti-inflammatory effects. 19 Gallic acid and the derivatives thereof are powerful antioxidants capable of scavenging free radicals, such as superoxide anions, hydrogen peroxide (H 2 O 2 ), hydroxyl radicals and hypochlorous acid. 19 It has been suggested that this free radical scavenging activity may account for the ameliorative effect of gallic acid on oxidative stress.
Oyagbemi et al. showed that gallic acid ameliorates oxidative stress by suppressing ROS production in the rat brain and improving antioxidant status. 20 Prince et al. reported that gallic acid increased the activity of SOD, CAT and GSH dependent enzymes in diabetic brain damage induced with streptozocin and prevented the formation of free radicals. 21 Omobowale et al. also reported that gallic acid exhibited a protective effect against hepatic damage in doxorubicin-induced hepatotoxicity. 22 Li et al. investigated the antioxidant substances that best prevented oxidative stress experimentally induced in ovarian tissue. They concluded that gallic acid functioned as a powerful antioxidant by reducing ROS production and by activating the antioxidant enzyme system. 23 It was suggested in our study that, administration of gallic acid together with cisplatin showed a significant biochemical and histopathologic protection against cisplatin induced ototoxicity by acting as a powerful antioxidant.
The DPOAE test is an objective method that elicits functional outcomes of damage occurring in the cochlea in a rapid, inexpensive and non-invasive manner. Based on our DPOAE responses, significant impairment was observed in SNR values at high frequencies in rats receiving cisplatin compared to those in the control group. In addition, we documented significantly higher SNR values in rats administered gallic acid together with cisplatin compared to the cisplatin only group. This shows that gallic acid also exhibits a functional protective effect against ototoxicity induced with cisplatin.

Conclusion
In the light of our findings, we think that gallic acid may have played a protective role against cisplatin-induced ototoxicity in rats, as indicated by the DPOAE results, biochemical findings and immunohistochemical analyses. Although our results do indicate that gallic acid can reduce cisplatininduced ototoxicity in rats, its usefulness in the clinical setting remains uncertain. Further studies are now needed to determine the appropriate indications and dosages of gallic acid before clinical use against cisplatin-induced ototoxicity can be recommended.