Attenuation of Cisplatin Ototoxicity by Otoprotective ...

Otology & Neurotology 00:00Y00 Ó 2014, Otology & Neurotology, Inc.

Attenuation of Cisplatin Ototoxicity by Otoprotective Effects of Nanoencapsulated Curcumin and Dexamethasone in a Guinea Pig Model *Pezhman Salehi, *Olubunmi Victoria Akinpelu, *Sofia Waissbluth, *Emilia Peleva, †Brian Meehan, †Janusz Rak, and *‡Sam J. Daniel *McGill Auditory Sciences Laboratory, ÞMontreal Children’s Hospital Research Institute, Cancer and Angiogenesis Laboratory, McGill University; and þDepartment of Otolaryngology, The Montreal Children’s Hospital, Montreal, Quebec, Canada

Objectives: Cisplatin, one of the most effective and widely used chemotherapeutic agents in the treatment of head and neck malignancies, has severe dose-limiting side effects including ototoxicity. This study evaluates the effectiveness of nanoencapsulated curcumin and dexamethasone in preventing degenerative changes in inner ear cells caused by cisplatin. Study Design: Prospective study, animal experiment. Methods: Cultured auditory cells [House Ear Institute Organ of Corti-1 (HEI-OC1)] and a guinea pig model were used for in vitro and in vivo experiments, respectively. Cell viability assays were conducted to compare the direct toxicity of cisplatin against auditory cells in the presence or absence of pretreatment with nanoencapsulated curcumin and dexamethasone. To recapitulate these effects in vivo, 68 guinea pigs received cisplatin either alone, or along with dexamethasone, nanoencapsulated curcumin, or the combination of both products. Outcome measures included auditory brainstem response, cochlear morphology under both light and scanning electron microscopy, and antioxidant enzyme assays.

Results: Pretreatment of auditory cells with naonoencapsulated curcumin and dexamethasone resulted in significant attenuation of cisplatin toxicity. Similarly, in the corresponding animal model (guinea pig), cisplatin caused an average hearing loss of 50 dB, which was attenuated by nanoencapsulated curcumin and dexamethasone across all of the hearing frequencies. There was also greater preservation of histologic structures in this group. Superoxide dismutase and catalase activities were increased in cisplatin-treated animals, whereas the nanoencapsulated curcumin with dexamethasone led to a diminution of this effect. Conclusion: Nanoencapsulated curcumin administered in combination with dexamethasone provides a partial but marked protection against cisplatin-induced hearing loss, likely because of reduced toxic damage to auditory cells. Key Words: CisplatinVCurcuminVDexamethasoneVNanoencapsulated— Ototoxicity. Otol Neurotol 00:00Y00, 2014.

Platinum-based chemotherapy, such as cisplatin, provides an effective treatment for a variety of malignancies (1,2); however, its cytotoxic effects also lead to doselimiting side effects including ototoxicity, nephrotoxicity, and neurotoxicity. Approximately 60% to 80% of the patients treated with cisplatin develop hearing loss, which is usually permanent, bilateral, and progressive (2Y4). Curcumin, a polyphenol isolated from turmeric (Curcuma longa), has received increased attention over the last few decades because of its antioxidant, anti-inflammatory, and antineoplastic activities (5). Regardless of the administration

route, the bioavailability of curcumin is minimal because of hydrolytic degradation, chemical instability in alkaline pH, and rapid intestinal and hepatic metabolism/elimination (6,7). Recent nanoencapsulation of curcumin has provided a strategy for increasing its solubility and stability in aqueous media. Because of these properties, it is plausible that nanoencapsulated curcumin (NCUR) could reach many cellular compartments, including inner ear cells, which are the main target of cisplatin ototoxicity. It is known that cisplatin can elicit an inflammatory response in the inner ear leading to cellular injury (2). Corticosteroids have been evaluated as a potential otoprotective measure, in this regard, on the principle of their anti-inflammatory activities. Dexamethasone, one of the most potent corticosteroids, has shown evidence of partial protection against cisplatin-induced morphologic changes in the ear, especially in the stria vascularis (8).

Address correspondence and reprint requests to Sam J. Daniel, M.D., M.Sc., FRCSC, Department of Otolaryngology, Head and Neck Surgery, McGill University, The Montreal Children’s Hospital, 2300 Tupper Avenue, Montreal, Quebec, Canada, H3H 1P3; E-mail: [email protected] The authors disclose no conflicts of interest. Supplemental digital content is available in the text.

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Copyright © 2014 Otology & Neurotology, Inc. Unauthorized reproduction of this article is prohibited.

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In this study, we present a novel experimental approach to protect the inner ear against the damaging effects of cisplatin by using a combination therapy of NCUR and dexamethasone. MATERIALS AND METHODS Cell Culture and Viability HEI-OC1 cells (a generous gift from Dr. Kalinec, House Research Institute) were maintained in high-glucose Dulbecco’s modified Eagle medium (Life Technologies, Canada) supplemented with 10% fetal bovine serum (Wisent, Canada) at 33-C and 10% CO2. Cells were seeded at 3,000 cells/well and left to attach for 24 hours. HEI-OC1 cells were exposed to cisplatin for 8 hours to determine the lethal dose 50% (LD50). Cisplatin at an LD50 concentration of 70 KM (8 hr exposure) was selected for subsequent experiments. Culture cells were treated with 200 nM dexamethasone as Rinehart et al. (9) showed dexamethasone dose at 80 ng/ml (,200 nM) achieved desired anti-inflammatory effects. To evaluate the cytotoxic effect of the NCUR, HEI-OC1 cells were exposed to various concentrations of the drug ranging from 5 to 80 Kg/ml for 48 hours. NCUR nanoparticles were synthesized using a previously described method (Materials, Supplemental Digital Content 1, http://links.lww.com/MAO/A218) (10). The cell viability was determined using (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, salt) (MTS) assay. MTS assay is considered to be a surrogate of cell viability, unless there is increased/decreased activity of the mitochondria. The cells received NCUR and dexamethasone 24 hours before cisplatin exposure. The MTS solution (25 KL/well) (Promega) was added (24 hr after cisplatin exposure), and the cells were incubated for 90 minutes. The absorbance at a wavelength of 490 nm was measured using a microplate reader (Biotek, USA). The viability of the cells was calculated using the following formula: % control ¼

Mean optical density of the experimental group 100 Mean optical density of the controls

transducers and presented separately to each ear through plastic tubes connected to the earphones that were inserted into the external ear canal of the animals. Acoustic signals were emitted at 8-, 16-, 20-, and 25-kHz tone bursts (rise-fall time of 5 ms, 44 Blackman envelope) at a rate of 39.1 bursts per second after 1,600 sweeps. Threshold recording started with administration of a 100 dB SPL signal for the click and 8- and 16-kHz stimuli and an 85 dB SPL signal for the 20- and 25-kHz tone bursts. Evoked responses were recorded using 2 subdermal needle electrodes positioned at the pinna of the test ear. The analysis time window for each response was 16 milliseconds. The evoked potentials were filtered with a bandpass filter between 100 and 1,500 Hz. The ABR thresholds were recorded as the lowest sound wave to which a visible, repeatable wave V could be identified. All thresholds were determined based on subjective judgment by an experimenter who was blinded to the treatments. Hearing assessments were performed before treatment (baseline measurement) and 24 hours after the last cisplatin injection (post measurement). The average hearing thresholds of the right and left ear were compared to obtain a threshold change for each of the tested frequencies.

Light Microscopy A qualitative study using light microscopy (LM) was undertaken on the cochleae of the guinea pigs. Three randomly selected cochleae from each group were fixed in 10% formalin, decalcified with 10% ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (pH 7.4) and then taken through graded alcohol dehydration. Eight micron thick midmodiolar sections were obtained from paraffin blocks and stained with hematoxylin and eosin. The sections were visualized under a light microscope (Carl Zeiss, Germany) at 5 to 40. The organ of Corti was considered to have ‘‘preserved architecture’’ if we could clearly observe the presence of 3 rows of outer hair cells (OHCs), 1 row of inner hair cells, supporting cells, inner and outer pillar cells and intact tunnel of Corti bound by the inner and outer pillar cells. The stria vascularis (SV) makes up part of the lateral wall of the cochlea, and its morphology was considered as preserved if its 3-layered epithelium was well highlighted demonstrating the marginal, intermediate, and basal cells. The histologic changes of the spiral ganglion cells (SGCs) within the Rosenthal’s canal were visually assessed.

Animals Sixty-eight female Hartley guinea pigs (500Y600 g) were randomly divided into 7 groups (Table 2): cisplatin (Group 1), dexamethasone (Group 2), NCUR (Group 3), cisplatin/nonloaded polymer (Group 4), cisplatin/dexamethasone (Group 5), cisplatin/ NCUR (Group 6), and cisplatin/NCUR/dexamethasone (Group 7). Cisplatin was administered intraperitoneally (IP) over 7 consecutive days for a total dose of 13 mg/kg. Dexamethasone and NCUR (0.5 mg/ml) were administered IP over 9 consecutive days, beginning 1 day before the first cisplatin injection and ended 1 day after the last cisplatin injection, at doses of 10 and 20 mg/kg per day, respectively. The dose of NCUR was selected based on the maximum-tolerated volume of IP injections for guinea pigs (11). NCUR was injected 5 hours before cisplatin administration each day based on the maximum detected level of curcumin in plasma at 5 hours postinjection reported by Bisht et al. (12). Dexamethasone dosage was selected based on previously tested, nontoxic high doses in a guinea pig animal model (8).

Scanning Electron Microscopy Scanning electron microscopy (SEM) was performed on 4 randomly selected animals in each of the following groups: Group 1 (cisplatin), Group 5 (cisplatin/dexamethasone), Group 6 (cisplatin/NCUR), Group 7 (cisplatin/NCUR/dexamethasone), and controls to evaluate the appearance of the outer hair cells (OHCs). Using a modified version of the method described by Saito et al., the OHCs were counted at a magnification of 1000 across the basal turn of the cochlea at 3 corresponding sites (13,14). OHCs with intact V- or W-shaped stereocilia bundles were considered as normal, OHCs with damaged stereocilia or loss of the normal V- or W-shaped stereocilia were considered abnormal, and the total absence of stereocilia and rupture of the cuticular plate was referred to as absent OHCs. The results in each group were presented as mean percentages of the total counted cells in each visual field.

Determination of Antioxidant Enzyme Activity Hearing Assessment Auditory brainstem response (ABR) tests were performed using the Smart EP Device (Intelligent Hearing Systems, Miami, FL, USA). Acoustic stimuli were generated by IHS-4745 high-frequency

Five animals per group were used to compare the local antioxidant enzyme activity in cisplatin, cisplatin/dexamethasone, cisplatin/NCUR, and cisplatin/dexamethasone/NCUR groups relative to nontreated controls. The bony shell of the cochlea

Otology & Neurotology, Vol. 00, No. 00, 2014


ATTENUATION OF CISPLATIN OTOTOXICITY was removed, and the cochlear tissues (wet weight ranging from 5 to 15 mg) were extracted, pooled (the right and the left ear for each animal separately), and homogenized in 100 Kl of 50 mM phosphate buffer (pH 7) containing 1 mM EDTA. Superoxide dismutase (SOD) assay for 5 samples per group was performed using the SOD assay kit-WST (Dojindo, USA). The catalase assay was similarly conducted using the catalase assay kit from Cayman Chemical (USA).

Statistical Analysis Statistical analysis of the cell viability assay and ABR experiments were performed using 1-way analysis of variance (ANOVA). The Tukey-Kramer multiple comparisons test was subsequently performed for further analysis between 2 groups. Frequency was not considered as a covariate in this study because we investigated the effect of the drugs at each individual frequency separately. The Kruskal-Wallis and Dunn’s multiple comparison tests were conducted to compare the antioxidant enzyme activities and the SEM cell counts between experimental groups. Three cochleae for LM, 4 cochleae for SEM, and 10 cochleae per group for enzyme assays were selected as a practical sample number that were manageable within the budget of the study. Data are reported as mean and standard error of the mean unless otherwise stated. Statistical significance was set at a p e 0.05.

RESULTS NCUR Combined With Dexamethasone Improves the Viability of Auditory Cells in Vitro (MTS Assay) The MTS assay results are presented as mean percentages of the controls, calculated from mean optical densities, which corresponds to the changes in the number of viable cells in each group as compared with the controls (Table 1). Because we consider the untreated control cells fully viable, the mean percentage of the controls is calculated as 100%. When HEI-OC1 cells were treated with 70 KM cisplatin for 8 hours, MTS assay showed 50% reduction of viable cells. Upon treatment with dexamethasone at concentrations ranging from 50 to 900 nM, the MTS assay did not show any statistically significant difference between viability of the treated cells and controls (p 9 0.05, data not shown). When cells were treated with NCUR, MTS assay showed no drug toxicity at concentrations of 5, 10, 20, and 40 Kg/ml (48-hr exposure). However, NCUR at TABLE 1.

concentrations of 60 and 80 Kg/ml showed statistically significant reduction in viability of the treated cells as compared with the controls (p G 0.01). Interestingly, addition of NCUR at concentrations ranging from 5 to 80 Kg/ml did not significantly protect HEI-OC1 cells against cisplatin cytotoxicity. A protective effect was observed with the NCUR/dexamethasone combination at NCUR concentrations of 5, 10, 20, 40, and 60 Kg/ml (p G 0.01). Pretreatments of the cells with dexamethasone or dexamethasone/ nonloaded polymer (0.8 mg/ml) had no protective effect against cisplatin cytotoxicity (data not shown). NCUR Combined With Dexamethasone Protects Hearing Function in Vivo (ABR) The results of the ABR test indicate partial protection by NCUR and dexamethasone against cisplatin-induced hearing loss (Table 2). The cisplatin-treated group showed average thresholds, which correspond to mild to moderately severe hearing loss in humans (15). The average threshold shifts, below 10 dB, were observed in Groups 2 (dexamethasone) and 3 (NCUR). Groups 4 (cisplatin/nonloaded polymer), 5 (cisplatin/dexamethasone), and 6 (cisplatin/ NCUR) exhibited similar threshold elevations as Group 1 (cisplatin). However, in cisplatin/NCUR/dexamethasone group, a statistically significant reduction in ABR threshold elevations were observed across all frequencies tested as compared with the cisplatin group (p G 0.01). NCUR and Dexamethasone Pretreatment Preserve Structural Integrity of the Inner Ear (LM and SEM Evaluation) LM of the cochleae obtained from the control (nontreated) animals showed preserved morphology of the organ of Corti, the SV, the spiral ligament, and the SGCs. A variety of morphologic changes including vacuolization and edema and bulging of the marginal cells were observed in the SV of Groups 1 (cisplatin) and 4 (cisplatin/nonloaded polymer) (see Figure, Supplemental Digital Content 2, http://links.lww.com/MAO/A219). The groups receiving dexamethasone or NCUR only (Groups 2 and 3) showed normal SV morphology as in the control group. Also, the group receiving cisplatin/dexamethasone (Group 5),

Cell viability (MTS) assay of the HEI-OC1 cells pretreated with NCUR and NCUR/dexamethasone before cisplatin exposure

NCUR concentration (Kg/ml) 5 10 20 40 60 80

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NCUR-treated cells

Cis-NCURYtreated cells

1.86 T 0.1(111%) 1.68 T 0.1(100%) 1.87 T 0.1(111%) 1.74 T 0.1(103%) 1.56 T 0.1(92%) 1.41 T 0.07(84%)

0.84 T 0.05(50%) 0.89 T 0.07(53%) 0.91 T 0.08(54%) 0.90 T 0.08(54%) 0.84 T 0.05(50%) 0.80 T 0.03(48%)

Cis-NCUR-DexYtreated cells 0.79 T 0.81 T 0.84 T 0.88 T 0.80 T 0.75 T

0.08(56%)* 0.07(58%)* 0.06(59%)* 0.07(62%)* 0.06(56%)* 0.07(53%)

NCUR and NCUR/dexamethasone were kept at culture medium of the cisplatin-treated cells to the end of the experiment. Cells receiving cisplatin/ NCUR/dexamethasone exhibited greater survival as compared with the group receiving cisplatin only. Significant differences were observed for NCUR concentrations G80 Kg/ml (p G 0.05). No significant differences were observed between the cells receiving cisplatin/NCUR and cells receiving cisplatin only (p 9 0.05). The mean optical densities of the controls for NCUR-, Cis-NCURY, and Cis-NCUR-DexYtreated cells were 1.68 T 0.1, 1.68 T 0.1, and 1.41 T 0.2, respectively. Results are given as mean optical density T standard deviation (mean percentage of the control). *p G 0.05. Cis: cisplatin, Dex: dexamethasone, SD: standard deviation. MTS: Mean absorbance 490 nm T SD (mean percentages of the control). Otology & Neurotology, Vol. 00, No. 00, 2014


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P. SALEHI ET AL. TABLE 2.

Group

N

ABR measurements 1 14 2 8 3 8 4 8 5 8 6 8 7 14 ABR measurements 1 14 2 8 3 8 4 8 5 8 6 8 7 14 ABR measurements 1 14 2 8 3 8 4 8 5 8 6 8 7 14 ABR measurements 1 14 2 8 3 8 4 8 5 8 6 8 7 14

Administration

Mean ABR threshold shifts in decibels for all groups Baseline-measurement (SD)

Postmeasurement (SD)

Baseline-post difference (SD)

43.9 (15.5) 46.9 (8.7) 31.9 (18.3) 43.8 (15.4) 40.6 (20.8) 48.8 (7.2) 52.1 (9.9)

108.5 (12.8) 51.3 (8.9) 40.0 (21.6) 109.3 (9.0) 106.8 (9.1) 103.6 (14.1) 91.7 (16.2)

64.5 (12.7) 4.4 (10.3) 8.1 (24.3) 65.5 (18.6) 66.1 (22.5) 54.8 (17.6) 39.6 (18.2)*

36.8 (16.6) 48.1 (8.3) 34.4 (10.3) 34.4 (7.3) 34.4 (10.3) 35.6 (10.9) 43.6 (13.4)

99.8 (12.2) 45.6 (10.9) 36.3 (10.9) 96.8 (9.4) 92.4 (5.7) 93.5 (15.8) 83.2 (12.2)

63.0 (17.5) j2.5 (9.3) 1.9 (11.1) 62.4 (11.8) 58.1 (11.6) 57.9 (19.3) 39.6 (16.9)*

at 8 kHz Cis Dex NCUR Cis-nonloaded Polymer Cis-Dex Cis-NCUR Cis-NCUR-Dex at 16 kHz Cis Dex NCUR Cis-nonloaded Polymer Cis-Dex Cis-NCUR Cis-NCUR-Dex at 20 kHz Cis Dex NCUR Cis-nonloaded Polymer Cis-Dex Cis-NCUR Cis-NCUR- Dex at 25 kHz Cis Dex NCUR Cis-nonloaded Polymer Cis-Dex Cis-NCUR Cis-NCUR- Dex

27.5 (8.9) 31.3 (10.9) 28.8 (8.9) 27.5 (7.7) 33.1 (10.1) 35.0 (11.5) 38.6 (14.8)

Q78.2 (5.1) 35.0 (8.2) 26.9 (6.0) Q79.6 (5.6) Q74.4 (6.7) Q78.5 (7.4) Q69.5 (9.5)

Q50.7 (9.5) 3.8 (10.2) j1.9 (9.1) Q52.1 (8.9) Q41.3 (12.5) Q43.5 (12.7) Q31.0 (17.0)*

44.1 (12.6) 40.6 (10.0) 41.3 (10.2) 36.3 (11.5) 39.4 (7.7) 48.8 (11.5) 53.2 (11.2)

Q79.6 (5.6) 41.3 (9.6) 38.1 (8.3) Q80.6 (11.7) Q74.4 (10.0) Q79.4 (7.0) Q73.6 (12.5)

Q35.5 (12.8) 0.6 (13.4) j3.1 (8.7) Q44.4 (15.8) Q35.5 (12.6) Q30.6 (10.1) Q20.4 (13.2)*

Statistically significant differences were observed across all frequencies in Group 7 (cisplatin/NCUR/dexamethasone) as compared with Group 1 (cisplatin) (p G 0.01). ABR results are given as mean T standard deviation. The calibrated smart-EP device has an upper limit when testing 20 and 25 kHz. We use the sign ‘‘Q’’ when the threshold shifts are above the limit; therefore, the actual threshold elevations might be greater than what is reported in this table. Cis: cisplatin, Dex: dexamethasone.

cisplatin/NCUR (Group 6), and cisplatin/NCUR/dexamethasone (Group 7) exhibited SV morphology, which was comparable to the untreated control group (Fig. 1). OHCs are known to be greatly affected by cisplatin and were analyzed in more detail. In cisplatin-treated animals, morphologic changes included complete destruction of the OHCs, gross collapse of the tunnel of Corti, and destruction of the supporting cells. The apical turns had preserved OHCs morphology, whereas the basal turn suffered the most damage in cisplatin-treated animals. Less severe damage to the organ of Corti was observed in Groups 5 (cisplatin/ dexamethasone), 6 (cisplatin/NCUR), and 7 (cisplatin/ NCUR/dexamethasone) as compared with Groups 1 (cisplatin) and 4 (cisplatin/nonloaded polymer). Group 7 (cisplatin/NCUR/dexamethasone) showed the presence of the organ of Corti with some distortion in shape, evidenced by distorted tunnel of Corti, collapse of the supporting cells leading to abnormal orientation of the OHCs. In Groups 5 (cisplatin/dexamethasone) and 6 (cisplatin/NCUR), the supporting cells were partly destroyed leading to collapse (misalignment) of the OHCs and distortion of the tunnel of Corti. There was degeneration of SGCs as evidenced by apparent cell loss in Groups 1 (cisplatin) and 4 (cisplatin/ nonloaded polymer). The SGCs in the remaining 5 groups exhibited very similar features (Fig. 1).

Morphologic assessment of the OHCs with SEM was completed for Groups 1 (cisplatin), 5 (cisplatin/dexamethasone), 6 (cisplatin/NCUR), 7 (cisplatin/NCUR/dexamethasone), and for nontreated animals (controls). The SEM findings in Group 1 (cisplatin) demonstrated greater damage to the OHCs at the basal turn of the cochlea as compared with Groups 5 (cisplatin/dexamethasone), 6 (cisplatin/NCUR), and 7 (cisplatin/NCUR/dexamethasone), in which most of the cells exhibited deformed stereocilia or completely ruptured cuticular plates (Fig. 2). The results of the OHC count are presented in Table 3. The OHCs of the control group (nontreated) were 100% intact. The cochleae from Group 1 (cisplatin) showed 6.8% of the cells with normal stereocilia, 62.5% of cells with abnormal stereocilia, and 30% absent cells. The cisplatin treatment caused a significant change in the OHC counts, as compared with the nontreated group (p G 0.001). In Group 5 (cisplatin/dexamethasone), 55.6% abnormal stereocilia, 19.5% absent cells, and 24.9% showed normal stereocilia. In Group 6 (cisplatin/NCUR), 59.5% abnormal stereocilia, 14.6% absent cells, and 25.5% showed normal stereocilia (p G 0.05). Interestingly, the combination treatment group (Group 7) did provide significant protection against cisplatin as evidenced by 40.3% of the cells considered as normal,



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FIG. 1. LM of the basal turn of the cochleae showed preserved morphology of the SV, the organ of Corti, and the SGCs in control (nontreated) animals. Intact marginal cells of the SV (A), inner hair cells (B), OHCs (C), tunnel of Corti (D), and supporting cells (E) are shown in control group. Gross morphologic changes of the SV including extensive vacuolization of the intermediate layer (F) and destruction of the marginal cells (G), total destruction of the organ of Corti (H), loss of all OHCs, destruction of tunnel of Corti, and degenerated SGCs (I) were observed in Group 1 (cisplatin). Lower level of vacuolization of the intermediate layer and less destruction of the marginal cells of the SV (J, K), less structural damage of organ of Corti (L, M), and less SGCs degeneration (N, O) were observed in Groups 5 (cisplatin/dexamethasone) and 6 (cisplatin/NCUR) as compared with Group 1 (cisplatin). A decreased level of the damage to the SV (P), the organ of Corti (Q), and the SGCs (R) was observed in Group 7 (cisplatin/NCUR/dexamethasone) as compared with Groups 1 (cisplatin) and 6 (cisplatin/NCUR). Detailed qualitative assessment of the SV, SGCs, and OHCs was completed at 40. Bar equals 30 Km. Cis: cisplatin, Dex: dexamethasone. Otology & Neurotology, Vol. 00, No. 00, 2014


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FIG. 2. SEM of the basal turn of the organ of Corti. A, Nontreated control showing 3 intact rows of OHCs. B, Severe damage of all rows of OHCs in cisplatin-treated animals. Severe damage to the OHCs was observed in cisplatin/dexamethasoneY (C) and cisplatin/ NCURYtreated (D) animals, which was comparable to cisplatin onlyYtreated animals. E, SEM of the animals pretreated with dexamethasone and NCUR showing less damage to the OHCs as compared with the cisplatin-treated guinea pigs. Bar equals 100 Km.

TABLE 3.

SEM: OHC counts at the basal turn

OHC counts at the basal turn Absent stereocilia T SD (%total count)

Administration Cis Cis-Dex Cis-NCUR Cis-NCUR- Dex

21.1 T 4.8 16.3 T 1.6 10.0 T 2.2 8.1 T 2.6

(30.3%) (21.2%) (14.6%)* (11.4%)*

Abnormal stereocilia T SD (%total count ) 43.4 T 5.0 45.5 T 5.6 40.7 T 1.4 34.8 T 3.0

(62.5%) (59.0%) (59.5%) (49.0%)*

Normal OHC (%total count ) 4.8 15.3 17.4 28.6

T 3.0 T 2.7 T 3.3 T 4.0

(6.8%) (19.8%) (25.5%) (40.3%)*

Total count (%total count ) 69.5 T 3.3 77.1 T 5.2 68.3 T 2.8 70.9 T 3.6

(100%) (100%) (100%) (100%)

This table represents the mean OHC count and the percentages of the counted cell to the total number of the cell counts per group. Significant differences were observed in all 3 OHC grades. A decreased quantity of absent cells was seen in Group 6 (cisplatin/NCUR) as compared with Group 1 (cisplatin) (p G 0.05). A greater percentage of normal cells and a lower percentage of cells with abnormal stereocilia or absent cells were observed in the group treated with cisplatin/NCUR/dexamethasone (Group 7) as compared with the cisplatin-treated animals. Total number of 63 T 4 cells per visual field was counted at a magnification of 1000. *p G 0.01, Cis: cisplatin, Dex: dexamethasone, SD: standard deviation. Otology & Neurotology, Vol. 00, No. 00, 2014


ATTENUATION OF CISPLATIN OTOTOXICITY 49% presenting abnormal stereocilia, and 11.4 % of the cells being absent (p G 0.01). Changes in Antioxidant Enzyme Activities in Tissues Exposed to Cisplatin, NCUR, and Dexamethasone SOD activity was calculated as % inhibition of formazan dye formation. As shown in Fig. 3, cisplatin group showed higher values of SOD activity in the cochlear tissue lysates as compared with controls; however, this change was not statistically significant (p 9 0.05). Cochlear SOD activity of the cisplatin and the cisplatin/dexamethasone group reached 48% and 45% as compared with nontreated controls (42%), whereas the cisplatin/NCUR and the cisplatin/ NCUR/dexamethasone group showed decreased SOD activity values, to 44% and 30%, respectively (p 9 0.05 for all reported changes as compared with the controls). The cisplatin/NCUR/dexamethasone group exhibited a significant decrease in the activity of SOD as compared with the cisplatin treated group (p G 0.05). Cochlear catalase activity values in Groups 1 (cisplatin) and 5 (cisplatin/ dexamethasone) increased to 6.08 and 6.41 KM/min per milligram of tissue, respectively (p 9 0.05). Groups 6 (cisplatin/NCUR) showed decreased activity values of the enzyme with 4.89 KM/min per milligram of tissue, whereas the only statistically significant change was observed when we compared the catalase activity in Group 7 (cisplatin/NCUR/dexamethasone) with controls (3.86 and 5.21 KM/min per milligram of tissue, respectively).

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inflammatory microenvironment and so are the countereffects of NCUR/dexamethasone. Thus, we observed that pretreatment of the HEI-OC1 cells with NCUR and dexamethasone before the cisplatin exposure led to improved cell viability, as compared with cells treated with cisplatin alone or pretreated with either NCUR or dexamethasone. Considering that an ideal antineoplastic agent acts against cancer cells with minimal side effects on normal cells, NCUR appears to be a promising candidate to be used in combination with other chemotherapeutic agents. The cell viability assay results in this study are consistent with the ABR results, which demonstrate that a combination therapy of NCUR and dexamethasone significantly

DISCUSSION During the last decade, many attempts have aimed at mopping up the generated reactive oxygen species or countering the intense inflammatory response, have not led to the development of a robust otoprotective strategy against cisplatin-induced ototoxicity; however, clinical trials are still in progress for several otoprotective agents like sodium thiosulfate, aspirin, SPI-1005, and dexamethasone (www.clinicaltrials.gov) (16Y18). Attenuation of cisplatin ototoxicity by various reported synthetic or phytochemical compounds like N-acetylcysteine, KR22335, micronized flavonoid fraction, ginkgo biloba, and resveratrol have been shown in several studies. Although a proper comparison between these agents and NCUR requires the same experimental setup, we believe that along with antioxidant properties, a possible antineoplastic effect of this agent makes it uniquely interesting when compared with the other protective agents evaluated thus far (19Y23). The multitherapeutic properties of curcumin, including its antioxidant, anti-inflammatory, and antineoplastic activities, make it a good candidate for further evaluation. The results from our experiments demonstrate that NCUR has potential of reducing the deleterious effects of cisplatin on the sensory cells of the cochlea and in hearing preservation. The results of the cell viability assay suggest that the toxic effects of cisplatin may include intrinsic damage to auditory cells rather than being solely dependent on the

FIG. 3. Antioxidant enzyme activity: (A) Catalase and (B) SOD activity. SOD activity is calculated based on the formation of formazan dye from tetrazolium salt upon reduction with a superoxide anion. The rate of the reduction with O2- is linearly related to the xanthine oxidase activity, which is inhibited by SOD. The catalase assay is based on the reaction of catalase with methanol in the presence of an optimal concentration of H2O2. The formaldehyde production was measured using a spectrophotometer at 540 nm. Significant differences were observed in the activity of both enzymes. Statistically significant decreases were detected for Group 7 (cisplatin/NCUR/dexamethasone) as compared with the cisplatin-treated group. *p G 0.05, n = 5 guinea pigs per group, results are given as means T standard error of the mean; Cis: cisplatin, Dex: dexamethasone. Otology & Neurotology, Vol. 00, No. 00, 2014


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improved the hearing thresholds in cisplatin-treated animals. It is well known that systemic cisplatin administration causes greater damage at the basal turn of the cochlea, with a major impact on high-frequency hearing thresholds (24). As shown in Table 1, hearing threshold elevations at the 20 and 25 kHz frequencies were less than threshold shifts at lower frequencies. Although cisplatin damage is expected to be more severe at higher frequencies, our result does not accurately show this expected outcome because of limitation of the smart EP device in recording the hearing thresholds above 85 dB for 20 and 25 kHz frequencies. The actual threshold elevations at 20 and 25 kHz frequencies can be greater than the reported values in Table 1. The LM and SEM findings also demonstrated that OHCs are preserved better in the group receiving cisplatin/NCUR/dexamethasone as compared with the other groups. This may be related to the combined antioxidant properties of NCUR and the effects of dexamethasone because none of these 2 drugs had a protective effect against cisplatin ototoxicity when administered separately. At the same time, it is thought provoking that in our experiments in vitro dexamethasone added a level of cytoprotection to NCUR effects in the context of cisplatin toxicity against auditory cells. This suggests that there could be a direct manner, unrelated to interaction with inflammatory cells, in which corticosteroids may act on auditory cells, through gene expression changes or other mechanisms which still need to be identified. Still, the magnitude of the cytoprotective effects of NCUR/dexamethasone was far greater in vivo than in vitro, which suggests that indirect influences such as inflammation play a role in living organs. Previous studies investigating the effect of cisplatin on oxidative stress reported a decreased activity of antioxidant enzymes in the cochlea, kidney, and liver (25Y27); however, there are also reports of increased activity of antioxidant enzymes such as SOD and catalase in the inner ear following cisplatin administration (28,29). Zhang et al. stated that an increased activity of SOD and catalase caused by cisplatin is a nonspecific response to oxidative stress, which includes depletion of glutathione and lipid peroxidation. This study also demonstrated that higher doses of cisplatin cause greater SOD and catalase activities in vitro (29). Ravi et al. (23) indicated that the increased activity of SOD might be due to the inhibition of glutathione peroxidase or an increased generation of reactive oxygen species in the cochlea of the cisplatintreated animals. Our results are in keeping with and extend these findings. In our study, the activity of SOD and catalase showed a higher value as compared with the controls in the cochlear tissue of the cisplatin-treated animals; however, these changes were not statistically significant. Interestingly, the animals receiving cisplatin/NCUR/dexamethasone showed lower SOD and catalase activities as compared with animals receiving cisplatin only. The decreased activity of these antioxidant enzymes in the cochlea can be compensatory to the oxidative stress after cisplatin administration. These responses might be affected by the

cisplatin dose and duration of the treatment and needs further investigation. In conjunction with previous reports, our present study offers a possibility that curcumin derivatives, such as NCUR, in conjunction with dexamethasone may exert antithetical and advantageous effects in the context of cancer treatment. We suggest that these agents could simultaneously contribute to the antitumor activity while protecting certain normal tissues, such as cochlear epithelium. Finally, although no abnormality on histopathology of visceral tissues of NCUR treated mice was reported in previous studies, addressing the safety issues and biodegradation of the NCUR nanoparticles using different animal models is needed in future experiments (12). CONCLUSION NCUR appears to be a promising candidate in the prevention of cisplatin-induced ototoxicity. NCUR and dexamethasone in combination protects the auditory function and morphologic integrity of the inner ear, and attenuates the toxic effects of cisplatin against auditory cells. Although the exact mechanism for otoprotection and the interactions between the 2 drugs are currently unknown, our results clearly indicate that this combination markedly reduced the degree of cisplatin-induced hearing loss. Future research can focus on in vivo synergistic effects of NCUR with antineoplastic agents (including cisplatin) against cancer cells, the sensitization of resistant cancer cells to chemotherapy, as well as its antioxidant and anti-inflammatory properties. REFERENCES 1. Kelland LR, Farrell NP. Platinum-Based Drugs in Cancer Therapy. Totowa, NJ: Humana Press, 2000. 2. Rybak LP. Mechanisms of cisplatin ototoxicity and progress in otoprotection. Curr Opin Otolaryngol Head Neck Surg 2007;15: 364Y9. 3. Al-Khatib T, Cohen N, Carret AS, et al. Cisplatinum ototoxicity in children, long-term follow up. Int J Pediatr Otorhinolaryngol 2010;74:913Y9. 4. Kolinsky DC, Hayashi SS, Karzon R, et al. Late onset hearing loss: a significant complication of cancer survivors treated with Cisplatin containing chemotherapy regimens. J Pediatr Hematol Oncol 2010;32: 119Y23. 5. Aggarwal BB, Sung B. Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets. Trends Pharmacol Sci 2009;30:85Y94. 6. Mohanty C, Sahoo SK. The in vitro stability and in vivo pharmacokinetics of curcumin prepared as an aqueous nanoparticulate formulation. Biomaterials 2010;31:6597Y611. 7. Asai A, Miyazawa T. Occurrence of orally administered curcuminoid as glucuronide and glucuronide/sulfate conjugates in rat plasma. Life Sci 2000;67:2785Y93. 8. Waissbluth S, Salehi P, He X, et al. Systemic dexamethasone for the prevention of cisplatin-induced ototoxicity. Eur Arch Otorhinolaryngol 2013;270:1597Y605. 9. Rinehart J, Keville L, Neidhart J, et al. Hematopoietic protection by dexamethasone or granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients treated with carboplatin and ifosfamide. Am J Clin Oncol 2003;26:448Y58.



ATTENUATION OF CISPLATIN OTOTOXICITY 10. Salehi P, Makhoul G, Roy R, et al. Curcumin loaded NIPAAM/VP/ PEG-A nanoparticles: physicochemical and chemopreventive properties. J Biomater Sci Polym Ed 2013;24:574Y88. 11. Rice SA, Fish KJ. Aesthetic toxicity. New York, NY: Raven Press, 1994. 12. Bisht S, Mizuma M, Feldmann G, et al. Systemic administration of polymeric nanoparticle-encapsulated curcumin (NanoCurc) blocks tumor growth and metastases in preclinical models of pancreatic cancer. Mol Cancer Ther 2010;9:2255Y64. 13. Waissbluth S, Dupuis I, Daniel SJ. Protective effect of erdosteine against cisplatin-induced ototoxicity in a guinea pig model. Otolaryng Head Neck 2012;146:627Y32. 14. Saito T, Manabe Y, Honda N, et al. Semiquantitative analysis by scanning electron microscopy of cochlear hair cell damage by ototoxic drugs. Scanning Microsc 1995;9:271Y80. 15. Smith RJH, Shearer AE, Hildebrand MS, et al. Deafness and hereditary hearing loss overview. GeneReviews. Seattle 2013;1993Y2013. 16. Riga MG, Chelis L, Kakolyris S, et al. Transtympanic injections of N-acetylcysteine for the prevention of cisplatin-induced ototoxicity: a feasible method with promising efficacy. Am J Clin Oncol 2013;36:1Y6. 17. Rybak LP, Mukherjea D, Jajoo S, Ramkumar V. Cisplatin ototoxicity and protection: clinical and experimental studies. Tohoku J Exp Med 2009;219:177Y86. 18. Ekborn A, Hansson J, Ehrsson H, et al. High-dose Cisplatin with amifostine: ototoxicity and pharmacokinetics. Laryngoscope 2004;114:1660Y7. 19. Thomas Dickey D, Muldoon LL, Kraemer DF, Neuwelt EA. Protection against cisplatin-induced ototoxicity by N-acetylcysteine in a rat model. Hear Res 2004;193:25Y30. 20. Shin YS, Song SJ, Kang S, Hwang HS, Jung YS, Kim CH. Novel synthetic protective compound, KR-22335, against cisplatininduced auditory cell death. J Appl Toxicol 2014;34:191Y204.

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21. Bazo?lu MS, Eren E, Aslan H, Bingo¨lballN AG, Oztu¨rkcan S, KatNlmNz H. Prevention of cisplatin ototoxicity: efficacy of micronized flavonoid fraction in a guinea pig model. Int J Pediatr Otorhinolaryngol 2012;76:1343Y6. 22. Cakil B, Basar FS, Atmaca S, Cengel SK, Tekat A, Tanyeri Y. The protective effect of Ginkgo biloba extract against experimental cisplatin ototoxicity: animal research using distortion product otoacoustic emissions. J Laryngol Otol 2012;126:1097Y101. 23. Erdem T, Bayindir T, Filiz A, Iraz M, Selimoglu E. The effect of resveratrol on the prevention of cisplatin ototoxicity. Eur Arch Otorhinolaryngol 2012;269:2185Y8. 24. Fausti SA, Henry JA, Schaffer HI, et al. High-frequency monitoring for early detection of cisplatin ototoxicity. Arch Otolaryngol Head Neck Surg 1993;119:661Y6. 25. Rybak LP, Husain K, Evenson L, et al. Protection by 4-methylthiobenzoic acid against cisplatin-induced ototoxicity: antioxidant system. Pharmacol Toxicol 1997;81:173Y9. 26. Noori S, Mahboob T. Antioxidant effect of carnosine pretreatment on cisplatin-induced renal oxidative stress in rats. Ind. Clin Biochem 2010;25:86Y91. 27. Omar HM, Ahmed EA, Abdel-Ghafar S, et al. Hepatoprotective effects of vitamin C, DPPD, and L-cysteine against cisplatininduced oxidative stress in male rats. J Biol Earth Sci 2012;2: B28Y36. 28. Ravi R, Somani SM, Rybak LP. Mechanism of cisplatin ototoxicity: antioxidant system. Pharmacol Toxicol 1995;76:386Y94. 29. Zhang J G, Lindup WE. Differential effects of cisplatin on the production of NADH-dependent superoxide and the activity of antioxidant enzymes in rat renal cortical slices in vitro. Pharmacol Toxicol 1996;79:191Y8.

