Time response of carboplatin-induced hearing loss in rat

Hearing Research 191 (2004) 110–118 www.elsevier.com/locate/heares Time response of carboplatin-induced hearing loss in rat K. Husain *, B. Scott, C. Whitworth, L. P. Rybak Department of Surgery, Southern Illinois University School of Medicine, Springfield, IL 62794, USA Received 30 October 2003; accepted 8 January 2004 Abstract Carboplatin is currently being used as an anticancer drug against human cancers. However, high dose of carboplatin chemotherapy resulted in hearing loss in cancer patients. We have shown that carboplatin-induced hearing loss was related to dosedependent oxidative injury to the cochlea in rat model. However, the time response of ototoxic dose of carboplatin on hearing loss and oxidative injury to cochlea has not been explored. The aim of the study was to evaluate the time response of carboplatin-induced hearing loss and oxidative injury to the cochlea of the rat. Male Wistar rats were divided into two groups of 30 animals each and treated as follows: (1) control (normal saline, i.p.) and (2) carboplatin (256 mg/kg, a single i.p. bolus injection). Auditory brainevoked responses (ABRs) were recorded before and 1–5 days after treatments. The animals (n ¼ 6) from each group were sacrificed on day 1, 2, 3, 4, and 5 and cochleae were isolated and analyzed. Carboplatin significantly elevated the hearing thresholds to clicks and to 2, 4, 8, 16, and 32 kHz tone burst stimuli only 3–5 days post-treatment. Carboplatin significantly increased nitric oxide (NO), malondialdehyde (MDA) levels and manganese superoxide dismutase (Mn-SOD) activity in the cochlea 4–5 and 3–5 days posttreatment, respectively, indicating enhanced influx of free radicals and oxidative injury to the cochlea. Carboplatin significantly depressed the reduced to oxidized glutathione (GSH/GSSG) ratio, antioxidant enzyme activities such as copper/zinc-superoxide dismutase (CuZn-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) as well as enzyme protein expressions in the cochlea 3–5 days after treatment. The data suggest that carboplatin-induced hearing loss involves oxidative injury to the cochlea of the rat in a time-dependent manner. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Carboplatin; Oxidative injury; Hearing loss; Cochlea; Rat 1. Introduction Carboplatin [cis-diamine (1,1-cyclobutanedicarboxylate) platinum (II)] is being used in the clinic as an alternative anti-cancer drug for the treatment of a variety * Corresponding author. Present address: Department of Pharmacology and Toxicology, Ponce School of Medicine, P.O. Box 7004, Ponce, PR 00732-7004, USA. Tel.: +1-787-840-2575x2192; fax: +1787-259-7085. E-mail address: [email protected] (K. Husain). Abbreviations: ABR, Auditory brainstem-evoked response; ANOVA, Analysis of variance; BSO, Buthionine sulfoximine; CAT, Catalase; CuZn-SOD, Copper zinc-superoxide dismutase; dB, Decibel; EDTA, Ethylenediamine tetraacertic acid; ELISA, Enzyme linked immunosorbent assay; GSH, Glutathione reduced; GSH-Px, Glutathione peroxidase; GSSG, Glutathione oxidized; HPLC, High performance liquid chromatography; iNOS, Inducible nitric oxide synthase; kHz, Kilo hertz; MDA, Malondialdehyde; Mn-SOD, Manganesesuperoxide dismutase; NADPH, Nicotinamide adenine di-nucleotide phosphate reduced; NO, Nitric oxide; ROS, Reactive oxygen species 0378-5955/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2004.01.011 of cancers such as small-cell lung cancer, ovarian cancer, carcinomas of head and neck as well as other types of cancers (Harper, 2003; Fujiwara et al., 2003; Steiner et al., 2002; Chamberlain, 2002; Meyer et al., 2001; Ettinger, 1998). The anti-tumor action of carboplatin is mediated by alkylation of DNA followed by killing of the cancerous cells (Los et al., 1993). Carboplatin displays less toxicity than its analog cisplatin, but antitumor activity is equivalent to that of cisplatin (Meyer et al., 2001; DeLauretis et al., 1999; Alberts, 1995). However, optimal use of carboplatin is limited in the clinic by a profound hearing loss in cancer patients (Montaguti et al., 2003; DeLauretis et al., 1999; Obermair et al., 1998; Neuwelt et al., 1998; Cavaletti et al., 1998; Kennedy et al., 1990). Carboplatin-induced hearing impairment has also been demonstrated in experimental animals such as guinea pigs and chinchillas (Hofstetter et al., 2000; Hu et al., 1999; Mount et al., 1995; Taudy et al., 1992). We have reported the K. Husain et al. / Hearing Research 191 (2004) 110–118 dose–response of carboplatin-induced hearing loss in a rat model (Husain et al., 2001a), which was related to oxidative injury to both the cochlea and inferior colliculus (Husain et al., 2001b, Husain et al., 2003). However, time–response of carboplatin-induced hearing loss and oxidative injury to the auditory system has not been explored. Hence, this study investigated the time–response of carboplatin-induced hearing loss (ABR threshold shifts), oxidative stress (oxidant/antioxidant imbalance) and NO production in the cochlea of the rat. 2. Methods 2.1. Chemicals Chemicals such as GSH, oxidized glutathione (GSSG), NADPH, and c-glutamyl glutamate; enzymes (CuZn-SOD, Mn-SOD, CAT, GSH-Px), carboplatin, 1,1,1,1-tetraethoxy-propane, monoclonal antibody for CuZn-SOD and peroxidase-conjugated secondary antibody were purchased from Sigma Chemicals (St. Louis, MO). Monoclonal antibodies for Mn-SOD, GSH-Px and CAT were purchased from Biodesign Int., Kennebunk, ME and Oxis Health Products Inc., Portland, OR, respectively. Coomassie protein assay reagent was purchased from Pierce Company (Rockford, IL). 2.2. Animals Male Wistar rats (250–300 g) were obtained from Charles River (Wilmington, MA) and divided into two groups and treated as follows: (1) control vehicle-treated rats (n ¼ 6) were treated with single bolus administration of normal saline (1 ml/kg) intraperitoneally (i.p.); (2) carboplatin-treated (n ¼ 30) rats were treated with a single bolus administration of carboplatin at a dose of 256 mg/kg (i.p.). Pretreatment ABRs were performed in rats from all groups while they were under xylazine: ketamine sedation, which were followed by the drug treatment described above. Post-treatment ABRs were performed 1–5 days later and the data were compared to the pretreatment ABRs for changes in thresholds. The rats in all the groups were sacrificed after post-treatment ABRs recordings. The selection of carboplatin dose was based on our previous studies (Husain et al., 2001a,b; Husain et al., 2003). The heads were collected in ice water, the temporal bones were dissected, the bulla opened and cochleae isolated carefully. The isolated cochlea contained the bone of the external capsule and modiolus, plus spiral ganglion and organ of Corti. The isolated cochleae were frozen in liquid nitrogen and stored at )80 °C until biochemical analysis could be 111 completed. The tissues were homogenized in 50 mM phosphate buffer (pH 7.0) and homogenate was used for biochemical assays. The care and use of the animals reported on in this study were approved by SIU School of MedicineÕs Laboratory Animal Care and Use Committee (LACUC) and as per the guidelines of NIH. 2.3. Auditory brain stem-evoked responses Rats were sedated with xylazine and ketamine (3.4 and 172.4 mg/kg). The ABRs in control and carboplatin-treated rats were recorded in an electrically shielded, double-walled, radiofrequency shielded sound booth in response to 10 ms tone burst at clicks, and at 2, 4, 8, 16 and 32 kHz. Intensities were expressed in decibels sound pressure level peak equivalent (dB SPL pe). Auditory stimuli were presented at a rate of 5 per second in 10 dB steps between 0 and 100 dB SPL pe. Responses were amplified 1000 times by a pre-amplifier and an additional 100 times by the averaging system, for a total amplification of 100,000 times. Twenty millisecond responses were recorded on a PC-based, signal averaging system (Tucker-Davis Technologies, Alachua, FL). The responses were synchronized with the onset of the stimulus, with a 1-ms delay to compensate for the length of the earphone (Etymotic ER-2) sound delivery tube. Spectrum analyses have been conducted for our Etymotic earphone using our stimulus waveform (10 ms envelope with a 1-ms rise/fall time). For 16 and 32 kHz stimuli, there was a minimum attenuation of )30 dB between the desired frequency and any resonant frequencies. Each ABR waveform consisted of 512 averaged responses. The sampling rate of the D/A processor was 500 kHz. A 40-kHz antialiasing filter was used for the stimuli. This filter has an attenuation of at least )60 dB at 46 kHz and above. The intensity series recorded included a sub-threshold response and increased to 30 dB supra-threshold for each stimulus. Two averaged responses were recorded for each stimulus intensity. Each intensity series was observed to determine the threshold response based on the growth pattern of the waveform amplitude and shortening of wave latency with increasing stimulus intensity. Threshold was defined as the lowest intensity that displays a replicable waveform, with two distinct waves and a minimum amplitude of 0.5 lV. Threshold responses typically displayed wave IV and/or a wave II/ III complex as reported earlier (Rybak et al., 1997). There was some variation due to electrode placement and stimulus frequency. The ABR measurement in control rats proved to be highly reproducible in the retest schedule, indicating high inter-test reliability. The ABR values were expressed as standard error of the mean. 112 K. Husain et al. / Hearing Research 191 (2004) 110–118 2.4. Determination of GSH and its disulfide by high pressure liquid chromatography The ratio of GSH to GSSG in the cells is a good marker of oxidative stress. Under normal physiological conditions, the cells keep the ratio high but it is depressed under oxidative stress conditions. Therefore, concentrations of GSH and GSSG were determined in the tissues by a modified HPLC method of Fariss and Reed (1987). Two hundred and fifty microliters of the tissue-acid extract containing internal standard (c-glutamyl glutamate) was mixed with 100 ll of 100 mM iodoacetic acid in a 0.2 mM m-cresol purple solution. This acidic solution was brought to basic conditions (pH 8.9) by the addition of approximately 400 ll of 2 M KOH–2.4 M KHCO3 . The sample was placed in the dark at room temperature for 1 h. Rapid S-carboxymethyl derivatization of GSH, GSSG and c-glutamyl glutamate occurred soon after the change in pH. NDinitrophenyl derivatization of the samples was obtained by incubation for 12 h at 4 °C in the presence of 1% 1-fluoro-di-nitrobenzene. Multiple samples were analyzed using the ISCO auto sampler controlled by ISCO Chemical research program. The sensitivity of the HPLC for GSH was 50 pmol/injection volume and 25 picomol/injection volume for GSSG. 2.5. Enzyme assays Antioxidant enzyme SOD is the first line of defense to scavenge superoxide anions generated in cytosolic and mitochondrial compartment of the cell. Under oxidative stress conditions CuZn-SOD activity in the cytosol is depressed due to inactivation by ROS (Pigeolet et al., 1981). Whereas, Mn-SOD activity in the mitochondria is enhanced due to induction by cytokines, glucocorticoids, tissue oxygenation and ROS (Perera et al., 1995; Marklund, 1992). Therefore, SOD activity was determined at room temperature according to the method of Misra and Fridovich (1972). Ten microliters of tissue extract was added to 970 ll (0.05 M, pH 1:0.2, 0.1 mM EDTA) carbonate buffer. Twenty microliters of 30 mM epinephrine (dissolved in 0.05% acetic acid) was added to the mixture and SOD was measured at 480 nm for 4 min on a Hitachi U-2000 Spectrophotometer. The rate of the reaction was calculated where linearity occurred, usually between 90 and 180 s. SOD activity was expressed as the amount of enzyme that inhibits the oxidation of epinephrine by 50%, which is equal to 1 U. Mn-SOD activity was determined by adding 100 ll of 20 mM NaCN to inhibit CuZn-SOD activity. CuZn-SOD activity was determined by subtracting Mn-SOD activity from total SOD activity. Antioxidant enzyme CAT catalyzes the degradation of ROS (hydrogen peroxide) in the cell. Under oxidative stress conditions CAT activity is depressed due to inactivation by ROS (Pigeolet et al., 1981). Therefore, CAT activity was determined in tissues at room temperature by a slight modification of a method of Aebi (1984). Ten microliters of ethanol was added per 100 ll of tissue extract (dissolved in 0.5 M, pH 7.0, 0.1 mM EDTA, phosphate buffer), and then placed in an ice bath for 30 min. Then 10 ll of Triton X-100 RS was added per 100 ll of the tissue extract. Ten microliters of tissue extract was added in a cuvette containing 240 ll phosphate buffer and 250 ll (0.066 M) H2 O2 (dissolved in phosphate buffer) and measured at 240 nm for 30 s. The molar extinction coefficient of 43.6 mM cm
1 was used to determine CAT activity. One unit of CAT activity was defined as the millimoles of H2 O2 degraded/ min/mg protein. Antioxidant enzyme GSH-Px catalyzes the degradation of ROS (hydrogen peroxide, lipid peroxides and lipid hydroperoxides) in the cell. Under oxidative stress conditions GSH-Px activity is depressed either due to inactivation by ROS or depletion of selenium which is essential for enzyme activity (Pigeolet et al., 1981). Therefore, GSH-Px activity was determined in tissues by a method of Flohe and Gunzler (1984) at 37 °C. All reaction mixtures were dissolved in 0.05 M, pH 7.0, 0.1 mM EDTA phosphate buffer. A reaction mixture consisted of 500 ll phosphate buffer, 100 ll of 0.01 M glutathione (GSH), 100 ll of 1.5 mM NADPH, and 100 ll glutathione reductase (0.24 U). One hundred microliters of the tissue extract was added to the reaction mixture and incubated at 37 °C for 10 min. Then 50 ll of 12 mM t-butyl hydroperoxide was added to the tissue reaction mixture and measured at 340 nm for 180 s. The millimolar extinction coefficient of 6.22 mM cm
1 was used to determine the activity of GSH-Px. One unit of activity was equal to the millimoles of NADPH oxidized/min/mg protein. 2.6. Enzyme protein levels By ELISA The antioxidant enzyme protein expression is a measure of gene expression. Under oxidative stress conditions, the de novo synthesis of enzyme proteins is suppressed. Hence, antioxidant enzyme (CuZn-SOD, Mn-SOD, CAT, and GSH-Px) protein levels were determined using enzyme linked immunosorbent assay (ELISA) technique (Husain et al., 2001b). Tissue extracts (0.05 ml) prepared in phosphate-buffered saline (PBS) (10 mM phosphate buffer, pH 7.4, 150 mM NaCl and 0.1% sodium azide) were pipetted into each well of polyvinyl microtiter plate and incubated overnight at 4 °C. Coating solution was removed and washed 3 times with washing buffer (10 mM phosphate buffer, pH 7.4, 150 mM NaCl, 0.05% Tween 20) and distilled water. One-hundred microliters of monoclonal antibody (CuZn-SOD) (Sigma Chemical Co., MO) diluted in PBS (1:300) or other diluted (1:300) antibodies viz. anti-Mn-SOD, anti-catalase, 113 K. Husain et al. / Hearing Research 191 (2004) 110–118 and anti-glutathione peroxidase, respectively, were added to each well, incubated at room temperature for 2 h, and washed three times as before. One-hundred microliters of peroxidase conjugated secondary antibody diluted in PBS (1:100) was added to each well, incubated for 2 h and washed three times as before. One-hundred microliters of substrate (1% H2 O2 and 1 mg/ml 5-amino salicylic acid) in reaction buffer (0.02 M phosphate buffer, pH 6.8) was added to each well and incubated for 30 min. The reaction was stopped by adding 0.1 ml of 3 N NaOH and absorption of the microtiter wells read at 450 nm using an ELISA reader (Automated Microplate Reader, Model EL311, Bio-Tek Instruments, Inc., Winooski, VT). 2.7. Lipid peroxidation assay Malondialdehyde (MDA) is an end product of membrane lipid peroxidation. The enhanced production of MDA in tissues is an index of oxidative stress. Therefore, MDA concentration was estimated by the method of Ohkawa et al. (1979). One hundred microliters of tissue homogenate was added to 50 ll of 8.1% sodium dodecyl sulfate, vortexed and incubated for 10 min at room temperature. Three hundred and seventyfive microliters of 20% acetic acid and 375 ll of thiobarbituric acid (0.6%) were added and placed in boiling water bath in sealed tubes for 60 min. The samples were allowed to cool at room temperature. n-Butanol(1.25 ml):pyridine (15:1) was added, vortexed and centrifuged at 1000 rpm for 5 min. Five hundred microliters of the colored pink layer was measured at 532 nm on spectrophotometer using 1,1,3,3-tetra-ethoxypropane as standard. MDA concentration was expressed as nmol/ mg protein. 540 nm using ELISA plate reader (Automated Microplate Reader, Model EL311, Bio-Tek Instruments, Inc., Winooski, VT). 2.9. Protein assay Protein concentration was estimated according to the method of Read and Northcole (1981) using Coomassie protein assay dye and bovine serum albumin as a standard. 2.10. Statistical analysis The data were expressed as mean  SEM. The cochlear data for biochemical parameters such as GSH/ GSSG ratio, CuZn-SOD, Mn-SOD, CAT, GSH-Px, NO and MDA were analyzed statistically by two independent one-way analysis of variances (ANOVAs). DuncanÕs multiple range test was used from SAS statistical software package (SAS Institute, Cary, NC) for comparison of carboplatin-treated group at different time points with saline control group. The data of ABR were subjected to statistical analysis using two-tailed t-test. The 0.05 level of probability was used as the criterion for statistical significance. 3. Results The changes in ABR thresholds in control and carboplatin-treated rats at different time points are depicted in Fig. 1. Carboplatin did not significantly alter the hearing thresholds for clicks, 2, 4, 8, 16 and 32 kHz tone 30 2.8. Nitric oxide assay Control Carboplatin (day 1) Carboplatin (day 2) Carboplatin (day 3) Carboplatin (day 4) Carboplatin (day 5) Nitric oxide (NO) is generated in the tissues by constitutive enzymes (neuronal NO synthase, endothelial NO synthase) and inducible NO synthase. Excess production of NO in the auditory system of cisplatin-treated animals has been demonstrated through inducible NO synthase (Kelly et al., 2003; Watanabe et al., 2000). NO reacts with proteins and generates nitrotyrosine under oxidative stress conditions. Hence, NO levels in tissues were determined by NO assay kit (Cayman Chemical, Ann Arbor, MI) as described earlier (Husain et al., 2001b). Fifty microliters of tissue extract was added to wells of an ELISA plate followed by 10 ll of enzyme-cofactors and 10 ll of nitrate reductase mixture. The plate was covered and incubated for 1 h at room temperature. After incubation, 50 ll of Griess reagent 1 followed immediately by 50 ll of Griess reagent 2 was added. The plate was allowed to develop the color for 10 min at room temperature and absorbance was read at ABRThreshold Changes (dB) 25 20 *** *** 15 10 ** * * * 4kHz 8kHz 16kHz M U L ** * * ** * 5 0 Click (S T 2kHz I 32kHz U S) Fig. 1. Effects of carboplatin on auditory brain stem-evoked response (ABR) threshold changes at click, 2, 4, 8, 16, and 32 kHz tone burst stimuli in rats at different time points. The ABR values are expressed as standard error of the mean. Significant *p < 0:05 as compared to control; **p < 0:01 as compared to control; ***p < 0:001 as compared to control. 114 K. Husain et al. / Hearing Research 191 (2004) 110–118 1–3 days post-treatment but it significantly increased cochlear NO concentration (187% of control, p < 0:01 and 128% of control, p < 0:05) 4 and 5 days posttreatment. Carboplatin did not significantly alter cochlear MDA content 1–2 days post-treatment but it significantly increased cochlear MDA concentration (127% of control, p < 0:05), (146% of control, p < 0:01), and (184% of control, p < 0:01), 3–5 days post-exposure, respectively, indicating time-dependent oxidative injury to the inner ear. Carboplatin did not significantly alter cochlear GSH/GSSG ratio 1–2 days post-treatment. However, it significantly decreased cochlear GSH/ GSSG ratio (74% of control, p < 0:05), (52% of control, p < 0:01) and 45% of control, p < 0:001, 3–5 days posttreatment, respectively, indicating a time-dependent susceptibility of cochlea to oxidative injury. The changes in antioxidant enzyme activities in the cochlea of control and carboplatin-treated rats at different time points are presented in Table 2. No significant changes in cochlear CuZn-SOD and Mn-SOD activities were observed 1–3 days following carboplatin administration. Carboplatin significantly decreased CuZn-SOD activity (72% of control, p < 0:01) and (62% of control, p < 0:01) 4 and 5 days post-exposure, respectively. Whereas, Mn-SOD activity significantly increased (145% of control, p < 0:05) and (153% of control, p < 0:05) in the cochlea of the rat 4 and 5 days following carboplatin burst stimuli, respectively, 1–2 days post-treatment, compared to saline-treated controls. However, it elevated ABR thresholds 6.02  2.10, 1.16  0.90, 1.20  0.77, 2.40  0.85, 6.11  2.30 (p < 0:05) and 3.5  1.41 dB for clicks, 2, 4, 8, 16 and 32 kHz tone burst stimuli, respectively, 3 days post-treatment, compared to saline-treated controls. Carboplatin significantly elevated ABR thresholds 10.80  2.11 dB (p < 0:05), 4.33  1.70, 2.50  0.95, 6.66  2.50 dB (p < 0:05), 9.66  3.33 dB (p < 0:05) and 12.50  3.50 dB (p < 0:01) for clicks, 2, 4, 8, 16 and 32 kHz tone burst stimuli, respectively, 4 days post-treatment, compared to saline treated controls. Carboplatin significantly elevated ABR thresholds 10.22  2.00 dB (p < 0:05), 10.66  2.90 dB (p < 0:01), 5.88  2.55 dB (p < 0:05), 10.00  3.51 dB (p < 0:01), 20.11  4.30 dB (p < 0:001) and 15.50  3.50 dB (p < 0:001) for clicks, 2, 4, 8, 16 and 32 kHz tone burst stimuli, respectively, 5 days post-treatment, compared to saline-treated controls. For saline control group the ABR threshold changes were 0.25  0.08, 0.45  0.10, 0.20  0.06, 0.25  0.08, 0.15  0.05, and 0.26  0.09 dB, respectively. The changes in ABR thresholds suggest that carboplatin induces hearing loss in rats. The changes in NO, MDA contents and GSH/GSSG ratio in the cochlea of control and carboplatin-treated rats at different time points are shown in Table 1. Carboplatin did not significantly alter cochlear NO content Table 1 Effects of carboplatin on nitric oxide (NO), reduced to oxidized glutathione (GSH/GSSG) ratio and malondialdehyde (MDA) concentrations in the cochlea of rats at different time points Treatments Control (saline) Carboplatin treated Day 1 NO (nmol/mg protein) GSH/GSSG ratio MDA (nmol/mg protein) 9.90  1.24 4.70  0.55 7.88  0.98 8.08  1.78 5.01  0.80 7.96  0.76 Day 2 9.09  2.05 4.50  0.76 8.76  0.87 Day 3 9.60  1.37 3.51  0.56 9.98  0.85 Day 4 Day 5  18.51  2.54 2.44  0.64 11.92  1.37 12.66  1.33 2.12  0.52 14.48  1.88 Each value represents mean  SEM (n ¼ 6). p < 0:05 compared to control. ** p < 0:01 compared to control. *** p < 0:001 compared to control. * Table 2 Effects of carboplatin on antioxidant enzyme activities in the cochlea of rats at different time points Enzyme activitiesa Control (saline) Carboplatin treated Day 1 Copper/zinc-superoxide dismutase (CuZn-SOD) Manganese-superoxide dismutase (Mn-SOD) Catalase (CAT) Glutathione peroxidase (GSH-Px) Day 3 Day 4  Day 5 25.40  4.12 40.71  3.50 41.05  4.44 39.81  3.98 35.05  2.88 29.15 13.52  2.22 12.64  2.50 13.95  2.60 15.82  2.61 19.56  2.25 20.65  3.02 55.65  5.42 76.81  6.91 51.61  4.99 65.02  5.25 51.20  4.85 68.01  5.33 45.75  3.55 69.00  6.12 35.60  3.80 60.66  5.50 34.33  3.36 56.90  5.29 Each value represents mean  SEM (n ¼ 6). Enzyme activities are expressed as units/mg protein. * p < 0:05 compared to control. ** p < 0:02 compared to control. *** p < 0:01 compared to control. a Day 2  3.99 115 K. Husain et al. / Hearing Research 191 (2004) 110–118 Table 3 Effects of carboplatin on antioxidant enzyme protein expressions in the cochlea of rats at different time points Enzyme activitiesa Copper/zinc-superoxide dismutase (CuZn-SOD) Manganese-superoxide dismutase (Mn-SOD) Catalase (CAT) Glutathione peroxidase (GSH-Px) Control (saline) Carboplatin treated Day 1 Day 2 Day 3 Day 4  Day 5 1.46  0.16 1.38  0.14 1.41  0.15 1.15  0.11 0.88  0.07 0.78  0.06 4.53  0.68 4.44  0.92 3.98  0.78 4.98  0.95 6.52  0.88 7.15  0.98 3.20  0.32 5.58  0.55 3.33  0.29 5.05  0.50 3.11  0.27 5.41  0.45 3.01  0.30 5.80  0.53 2.28  0.21 4.20  0.38 2.08  0.20 3.88  0.35 Each value represents mean  SEM (n ¼ 6). Enzyme protein levels are expressed as (lg/mg protein). * p < 0:05 compared to control. ** p < 0:01 compared to control. a administration. The data indicate a compensatory response to get rid of superoxide in the mitochondrial compartment of the cochlea. Carboplatin significantly decreased CAT activity (64% of control, p < 0:01) and (62% of control, p < 0:01) 4 and 5 days post-treatment. No significant change in cochlear GSH-Px activity was observed 1–3 days following carboplatin administration. Whereas, GSH-Px activity significantly decreased (79% of control, p < 0:05), and (74% of control, p < 0:05) in the cochlea rats 4 and 5 days after carboplatin treatment. The effects of carboplatin on antioxidant enzyme protein expression in the cochlea of the rat at different time points are depicted in Table 3. No significant changes in cochlear CuZn-SOD, Mn-SOD, CAT and GSH-Px enzyme protein expressions were observed 1–3 days following carboplatin administration. However, carboplatin administration significantly decreased CuZn-SOD protein levels (60% of control, p < 0:01), CAT protein levels (71% of control, p < 0:01), and GSHPx protein levels (75% of control, p < 0:05) in the cochlea of rats 4 days after treatment. The significant depletion of CuZn-SOD protein levels (53% of control, p < 0:01), CAT protein levels (65% of control, p < 0:01), and GSHPx protein levels (70% of control, p < 0:05) in the cochlea were noted 5 days after carboplatin treatment. The data indicate that carboplatin down-regulated the de novo synthesis of antioxidant enzyme proteins in the inner ear of the rat. However, carboplatin significantly increased Mn-SOD protein expression (144% of control, p < 0:05) and (158% of control, p < 0:05) in the cochlea of rats 4 and 5 days after treatment, respectively. These results indicate that mitochondrial compartment adapted to scavenge excess superoxides through enhanced de novo synthesis of Mn-SOD proteins 4–5 days following carboplatin administration. 4. Discussion This study addressed the changes in hearing threshold (ABR) along with the changes in NO, GSH/GSSG ratio, antioxidant enzyme activities and enzyme protein expressions and lipid peroxidation in the cochlea of rats 1– 5 days following carboplatin administration. Our earlier studies demonstrated that carboplatin at higher doses significantly elevated the ABR threshold at higher frequencies (8–32 kHz) after 4 days post-treatment in a rat model (Husain et al., 2001b). The time response data of the present study show similar pattern of hearing loss after 3 days following carboplatin administration in rats. A recent report indicated that even moderate dose of carboplatin causes a significant ABR threshold shift at higher frequencies (10–30 kHz) 3 days post-administration to rats (Hatzopoulos et al., 2003). The present data show that high dose carboplatin administration to rats significantly elevated ABR threshold at lower frequencies (2–4 kHz) 4–5 days post-treatment. These observations suggest that the initial high frequency hearing loss spreads to lower frequency hearing loss in a time dependent manner. Carboplatin-induced hearing loss has been shown to be associated with loss of inner hair cells and type I spiral ganglion neurons in other animal models such as chinchilla and guinea pig (Wang et al., 2003; Hofstetter et al., 2000; Taudy et al., 1992). The loss of inner hair cells in the cochlea of chinchilla was observed after 1–2 days following carboplatin administration. However, we did not observe any significant ABR threshold shift 1–2 days after carboplatin administration to rats. It is likely that structural and physiological alterations in the cochlea of carboplatin-treated animals may vary depending upon the time of exposure and the animal species used. Carboplatin-induced hearing loss has also been reported in clinical studies using high doses (Montaguti et al., 2002; Neuwelt et al., 1998; Bauer et al., 1992; Kennedy et al., 1990). Interestingly, ABR threshold changes were accompanied by NO elevation in the cochlea of rats treated with carboplatin after 4–5 days. Evidence for the involvement of excess NO in the modulation of the auditory system has been reported in animals treated with platinum containing anticancer drugs 3–4 days post-treatment (Kelly et al., 2003; Husain et al., 2001b; Watanabe et al., 2000). 116 K. Husain et al. / Hearing Research 191 (2004) 110–118 However, this is the first report to show that carboplatin enhances NO production in the cochlea of rats not 1–3 days but 4–5 days post-treatment indicating the delayed induction of inducible nitric oxide synthase (iNOS). Direct round window exposure of guinea pig cochlea to a NO donor (sodium nitroprusside) has been implicated in a loss of cochlear hair cells as early as 1 day after treatment (Ruan et al., 1997). The non-selective inhibition of iNOS in cisplatin-treated animals reduced the hearing loss (Watanabe et al., 2000). We have recently reported that selective inhibition of iNOS reduced the cisplatininduced elevation in threshold shift for clicks and 16 kHz tone bursts in rats 3 days post-treatment (Kelly et al., 2003). The expression of iNOS has been demonstrated in the cochlea of cisplatin-treated animals 3 days post-exposure (Watanabe et al., 2000). It is likely that excess NO production may be due to the delayed induction of iNOS in the cochlea of carboplatin-treated rats. The oxidative injury to auditory system due to excess free radical or reactive oxygen species and antioxidant depletion may also play an essential role in carboplatininduced hearing loss. The data of the present study show that administration of carboplatin depressed GSH/ GSSG ratio and enhanced membrane lipid peroxidation in cochlea of rats 3–5 days post-treatment. The GSH/ GSSG ratio which is the sensitive index of oxidative stress reflects the oxidative injury to the auditory system. Glutathione play an important role in the preservation of hearing and cochlear function. The depletion of cochlear GSH by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, has been shown to potentiate carboplatin-induced hearing loss (Hu et al., 1999). Clinical and experimental studies have also demonstrated the protective role of glutathione against cisplatin as well as carboplatin-induced toxicities (Bohm et al., 1999; Hamers et al., 1993; Anderson et al., 1990). The depletion of tissue GSH/GSSG is a prime factor, which can impair the cellÕs defense against the toxic actions of free radicals/reactive oxygen species and may lead to membrane lipid peroxidation (Younes and Siegers, 1981). The endogenous superoxide anion, H2 O2 , and lipid peroxides in sub-cellular compartments of the cochlea may lead to Ca2þ influx (Ikeda et al., 1993) and activation of proteases (Ding et al., 2002) leading to the auditory impairment. In the present study, a significant increase in cochlear MDA level (an end product of lipid peroxidation) in rats treated with carboplatin is indicative of oxidative injury to the cochlear membrane following depressed glutathione levels. The membrane lipid peroxidation is a multistep process and MDA is the last end product before lipid hydroperoxide and conjugated diene (Husain and Somani, 1998). The delayed lipid peroxidation in the cochlear membrane of carboplatin-treated rats is evidenced by MDA production in a time-dependent manner followed by hearing impairment. Carboplatin-induced hearing loss may also be due to impaired antioxidant enzyme activities and suppression of antioxidant enzyme protein expressions in the cochlea of the rat. The antioxidant enzymes are the first line of defense against oxidative tissue injury and are well expressed in the cochlea (Husain et al., 2001b; Pierson and Gray, 1982). The data of antioxidant enzyme activities and protein expressions in the cochlea of the rat are comparable to those reported earlier in the literature (Lautermann et al., 1997; Farms et al., 1993; Pierson and Gray, 1982). The data of the present study show that the antioxidant enzyme activities such as CuZn-SOD, CAT, and GSH-Px significantly decreased in the cochlea of carboplatin-treated rats 4–5 days post-treatment. The impaired antioxidant enzyme activities and enzyme protein synthesis in the cochlea may cause an enhanced ROS-induced membrane lipid peroxidation leading to delayed apoptotic/necrotic cell death in the auditory system (Watanabe et al., 2000; Watanabe et al., 2002). Interestingly, Mn-SOD activity as well as protein expression significantly increased in the cochlea 4–5 days after carboplatin. The Mn-SOD activity is regulated by its biosynthesis, which is sensitive to tissue oxygenation, cytokines, tumor necrosis factor and corticosteroid hormones (Perera et al., 1995; Marklund, 1992). It is likely that carboplatin might have released these factors and thereby induced MnSOD activity in the cochlea. Moreover, ROS are generated after carboplatin administration and these are known to activate nuclear factor NF-kB and thereby induce transcription of Mn-SOD (Sen and Packer, 1996). The delayed alterations of antioxidant enzyme activities in the cochlea of carboplatin-treated rats may also be due to differences in pharmacokinetics of carboplatin in different cellular compartments (Los et al., 1993; Siddik et al., 1987). These reports further support the role of nitric oxide/free radicals and endogenous antioxidants in carboplatin-induced delayed hearing loss in rats. In summary, carboplatin-induced hearing loss (increased ABR threshold shift at higher and lower frequencies) was associated with a depletion of GSH/ GSSG ratio, inhibition of antioxidant enzyme activities, depression of enzyme protein expressions and increased Mn-SOD activity and enhanced lipid peroxidation in the cochlea of the rat 3–5 days after treatment. 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