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Carboplatin-induced oxidative stress in rat cochlea
Hearing Research 158 (2001) 14^22 www.elsevier.com/locate/heares
Carboplatin-induced oxidative stress in rat cochlea K. Husain a
a;
*, C. Whitworth b , S.M. Somani a , L.P. Rybak
b
Department of Pharmacology, Southern Illinois University School of Medicine, 801 North Rutledge, Spring¢eld, IL 62794-9629, USA b Department of Surgery, Southern Illinois University School of Medicine, Spring¢eld, IL 62794-9629, USA Received 12 February 2001; accepted 30 April 2001
Abstract Carboplatin is currently being used in the clinic against a variety of human cancers. However, high dose carboplatin chemotherapy resulted in ototoxicity in cancer patients. This is the first study to show carboplatin-induced oxidative stress response in the cochlea of rat. Male Wistar rats were divided into two groups of six animals each and treated as follows: (1) control (normal saline, i.p.) and (2) carboplatin (256 mg/kg, i.p.). Animals in both groups were sedated with ketamine/xylazine and auditory brainstem-evoked responses were recorded before and 4 days after treatments. The animals were sacrificed on the fourth day and cochleae were harvested and analyzed. A significant elevation of the hearing threshold shifts was noted at clicks, 8, 16, and 32 kHz tone burst stimuli following carboplatin administration. Carboplatin significantly increased nitric oxide and malondialdehyde levels, xanthine oxidase and manganese-superoxide dismutase activities in the cochlea indicating enhanced flux of free radicals. Cochlear glutathione levels, antioxidant enzyme activities such as copper zinc-superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase and enzyme protein levels were significantly depleted 4 days after carboplatin treatment. The data suggest that carboplatin induced free radical generation and antioxidant depletion, and caused oxidative injury in the cochleae of rats. ß 2001 Published by Elsevier Science B.V. Key words: Carboplatin; Oxidative stress; Cochlear antioxidant; Lipid peroxidation; Nitric oxide
1. Introduction Carboplatin [cis-diammine(1,1-cyclobutanedicarboxylate)platinum(II)] is a second generation platinum-containing anticancer drug. It is currently being used in the clinic against a variety of cancers such as small-cell lung cancer, ovarian cancer, and carcinomas of head and * Corresponding author. Tel.: +1 (217) 785-2202; Fax: +1 (217) 524-0145. E-mail address: [email protected] (K. Husain). Abbreviations: ABR, auditory brainstem-evoked response; ABTS, 2,2-azino-di-(3-ethylbenzthiazoline-6-sulfonate); CAT, catalase; CDNB, 1-chloro-2,4-dinitrobenzene; EDTA, ethylenediamine tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; GR, glutathione reductase; GSH, reduced glutathione; GSH-Px, glutathione peroxidase; GSSG, oxidized glutathione; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PBS, phosphate-bu¡ered saline; ROS, reactive oxygen species; SOD, superoxide dismutase; XO, xanthine oxidase
neck (Gridelli et al., 2001; Bolis et al., 2001; Meyer et al., 2001; Pivot et al., 2001 ; Ettinger, 1998; Cavaletti et al., 1998). The identi¢cation of dose escalation of carboplatin is an important factor in achieving optimal antineoplastic e¡ects (Bohm et al., 1999 ; Wandt et al., 1999). 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). A single high dose or repeated doses of carboplatin chemotherapy have been shown to produce ototoxicity as a side e¡ect in cancer patients (DeLauretis et al., 1999 ; Obermair et al., 1998 ; Neuwelt et al., 1998; Cavaletti et al., 1998 ; Kennedy et al., 1990). Carboplatin-induced ototoxicity has also been demonstrated in experimental animals such as guinea pigs and chinchillas (Hofstetter et al., 2000 ; Maldoon et al., 2000 ; Hu et al., 1999; Mount et al., 1995; Taudy et al., 1992). These investigators have demonstrated the changes in cochlear morphology, cochlear potential and auditory brainstem-evoked responses (ABR) following carboplatin administration. We have recently reported
0378-5955 / 01 / $ ^ see front matter ß 2001 Published by Elsevier Science B.V. PII: S 0 3 7 8 - 5 9 5 5 ( 0 1 ) 0 0 3 0 6 - 9
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the dose response of carboplatin-induced hearing loss in a rat model (Husain et al., 2001). However, the biochemical mechanism of carboplatin-induced hearing loss and oxidative injury in the cochlea of rats is not well understood. It is hypothesized that carboplatin induces excess amounts of NO and reactive oxygen species (ROS) generation which in turn causes oxidative impairment of the cochlea leading to hearing loss. Therefore, this study was undertaken in order to evaluate the hearing loss and mechanism of oxidative injury in the cochleae of rats treated with an ototoxic dose of carboplatin. 2. Methods 2.1. Chemicals Chemicals such as reduced (GSH) and oxidized glutathione (GSSG), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and Q-glutamyl glutamate ; enzymes (CuZn-superoxide dismutase (SOD), Mn-SOD, catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione S-transferase (GST)), carboplatin, 2,2-azino-di-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 1-chloro-2,4-dinitrobenzene (CDNB), 1,1,1,1-tetraethoxy-propane, monoclonal antibody for CuZn-SOD, and GST, peroxidase-conjugated secondary antibody were purchased from Sigma Chemicals (St. Louis, MO, USA). Monoclonal antibodies for Mn-SOD, GSH-Px and CAT were purchased from Biodesign, Kennebunk, ME, USA and Oxis Health Products, Portland, OR, USA, respectively. Coomassie protein assay reagent was purchased from Pierce (Rockford, IL, USA). 2.2. Animals Male Wistar rats (250^300 g) were obtained from Charles River (Wilmington, MA, USA) and divided into two groups of six animals and treated as follows: (1) control vehicle-treated (rats were treated with a single bolus administration of normal saline (1 ml/kg) i.p.) and (2) carboplatin-treated (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. Posttreatment ABRs were performed 4 days later and the data were compared to the pretreatment ABRs for changes in thresholds. Thus, each animal served as its own control for the ABRs. The rats in all the groups were sacri¢ced 4 days after treatment. The selection of the carboplatin dose was based on our previous study (Husain et al., 2001). How-
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ever, the selection of the 4-day time period was based on our time^response study of carboplatin-induced hearing loss in rats. We did not observe a signi¢cant ABR threshold shift on 1, 2 or 3 days post carboplatin treatment in rats (unpublished observations). The heads were collected in ice water, the temporal bones were dissected, the bullae opened, and the cochleae isolated carefully. The isolated cochleae were frozen in liquid nitrogen and stored at 380³C until biochemical analysis could be completed. Cochleae were homogenized in bu¡er 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 Medicine's Laboratory Animal Care and Use Committee (LACUC) and as per the guidelines of NIH. The approved grant title and number are Carboplatin-induced hearing loss in a rat model and # CRC-98-99 and NOHR-99-00. 2.3. ABRs Rats were sedated with a Rompun cocktail (xylazine, ketamine: 3.4 mg/kg, 172.4 mg/kg). Control ABRs were measured using a DEC PDP 11/73 (Digital Equipment Corporation, NH, USA)-based signal generating/averaging system in response to 100 Ws clicks and tone pips at 8, 16, and 32 kHz, which were of 10 ms plateau with a 1 ms rise/fall time. The stimuli were presented inside a double wall radio frequency-shielded sound booth using an Etymotic ER-2 earphone placed directly into the ear canal. Clicks and tone pips were presented at a rate of 5/s. Stimulus intensities were measured using a Bruel and Kjaer sound pressure level meter (model 2209) with a 1/4Q microphone (model 4136) inside an arti¢cial ear canal (RE : 20 WPa). Intensities were expressed in dB sound pressure level (SPL) peak equivalent, based on the calibration. Animals were presented with a stimulus intensity series, which was initiated at 10 dB SPL and reached a maximum of 90 dB SPL. Stimulus intensity was progressively increased in 10 dB increments and the resulting ABRs were observed on a video monitor. Intensities that appeared to be near threshold were repeated. Threshold was de¢ned as the lowest intensity capable of producing a visually detectable, reproducible response. Threshold responses typically displayed wave IV and/or a wave II/III complex. There was some variation due to electrode placement and stimulus frequency. The voltage associated with threshold was 0.5 WV. Sub-dermal electrodes were used to record brain potentials di¡erentially. The active lead was positioned at the vertex and referred to the second electrode at the tip of the nose. The ground electrode was located over the neck muscles. Potentials were ampli¢ed 1000 times inside the sound attenuation booth (bandwidth, 0.1 Hz^ 10 kHz) and signals were further ampli¢ed to produce
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an overall gain of approximately 100 000 and viewed on an oscilloscope. Care was taken to ensure that the band pass of the entire system included those frequencies that represent the ABR. The ABRs were sampled for 20.5 ms following stimulus onset. Stimuli were repeated 5U/ s and a total of 512 trials were averaged using an analog to digital converting system. Evoked potentials were recorded before drug administration and 3 days post administration. The ABR measurement in control rats proved to be highly reproducible in the retest schedule, indicating high intertest reliability (Ravi et al., 1995 ; Rybak et al., 1995). 2.4. Determination of GSH and its disul¢de (GSSG) by high pressure liquid chromatography (HPLC) The concentrations of GSH and GSSG were determined in the cochlea by a modi¢ed HPLC method of Fariss and Reed (1987). 250 Wl of the tissue-acid extract containing internal standard (Q-glutamyl glutamate) was mixed with 100 Wl 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 Wl 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 Q-glutamyl glutamate occurred soon after the change in pH. N-Dinitrophenyl derivatization of the samples was obtained by incubation for 12 h at 4³C in the presence of 1% 1-£uoro-dinitrobenzene. 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 pmol/injection volume for GSSG. 2.5. Enzyme assays SOD activity was determined at room temperature according to the method of Misra and Fridovich (1972). 10 Wl of tissue extract was added to 970 Wl (0.05 M, pH 1:0.2, 0.1 mM ethylenediamine tetraacetic acid (EDTA)) carbonate bu¡er. 20 Wl 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 at 90^100 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 Wl of 20 mM NaCN to inhibit CuZn-SOD activity. CuZn-SOD activity was determined by subtracting the Mn-SOD from total SOD activity. CAT activity was determined at room temperature by
a slight modi¢cation of a method of Aebi (1984). 10 Wl ethanol was added per 100 Wl of tissue extract (dissolved in 0.5 M, pH 7.0, 0.1 mM EDTA, phosphate bu¡er), and then placed in an ice bath for 30 min. Then 10 Wl of Triton X-100 RS was added per 100 Wl of the tissue extract. 10 Wl of tissue extract was added in a cuvette containing 240 Wl phosphate bu¡er and 250 Wl (0.066 M) H2 O2 (dissolved in phosphate bu¡er) and measured at 240 nm for 30 s. The molar extinction coe¤cient of 43.6 mM cm31 was used to determine CAT activity. One unit of CAT activity was de¢ned as mmol of H2 O2 degraded/min/mg protein. GSH-Px activity was determined by a method of Flohe and Gunzler (1984) at 37³C. All reaction mixtures were dissolved in 0.05 M, pH 7.0, 0.1 mM EDTA phosphate bu¡er. A reaction mixture consisted of 500 Wl phosphate bu¡er, 100 Wl 0.01 M glutathione (GSH), 100 Wl 1.5 mM NADPH, and 100 Wl GR (0.24 U). 100 Wl of the tissue extract was added to the reaction mixture and incubated at 37³C for 10 min. Then 50 Wl of 12 mM t-butyl hydroperoxide was added to the tissue reaction mixture and measured at 340 nm for 180 s. The millimolar extinction coe¤cient of 6.22 mM cm31 was used to determine the activity of GSH-Px. One unit of activity was de¢ned as mmol of NADPH oxidized/min/mg protein. GR activity was determined by the method of Carlberg and Mannervik (1985) at 37³C. 50 Wl of NADPH (2 mM) in 10 mM Tris^HCl bu¡er (pH 7.0) added in a cuvette containing 50 Wl of GSSG (20 mM) in phosphate bu¡er (0.5 M, pH 7.0, 0.1 mM EDTA), and 800 Wl of phosphate bu¡er were incubated at 37³C for 10 min. 100 Wl of tissue extract was added to the NADPH-GSSG bu¡ered solution and measured at 340 nm for 3 min. The millimolar extinction coe¤cient of 6.22 cm31 was used to determine the activity of GR. One unit of GR activity was de¢ned as mmol of NADPH oxidized/min/mg protein. GST activity was assayed by the method of Habig et al. (1974) using 10 mM CDNB as substrate. 50 Wl of tissue homogenate was added to 750 Wl 0.1 M phosphate bu¡er containing 0.1 mM EDTA and 100 Wl of 10 mM GSH. 100 Wl of CDNB was added to start the reaction. The changes in optical density were recorded at 340 nm for 3 min. The enzyme activity was calculated using an extinction coe¤cient of 9.6 mM cm31 and expressed as Wmol of CDNB utilized/min/mg protein. Xanthine oxidase (XO) activity was assayed as per the modi¢ed method of Singh et al. (1987) using ABTS as chromogen. In a tube containing 0.5 ml of substrate^bu¡er solution (10 mmol/l hypoxanthine in potassium phosphate bu¡er pH 7.9, 20 mmol/l NaN3 ), 2.5 Wl of uricase and 25 Wl of tissue extract were mixed and incubated for 10 min at 37³C. 500 Wl
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of reagent solution (2 mmol/l ABTS and 2500 U/l peroxidase in 1000 ml of phosphate bu¡er pH 7.9) was added, vortexed and 0.5 ml of 2 mol/l perchloric acid immediately added. Tubes were vortexed and centrifuged for 5 min at 3000 rpm and absorbance of the supernatant was read at 410 nm. The enzyme activity was expressed as Wmol of hypoxanthine oxidized/min/ mg protein. 2.6. Enzyme protein levels by enzyme-linked immunosorbent assay (ELISA) The antioxidant enzyme (CuZn-SOD, Mn-SOD, CAT, GSH-Px and GST) protein levels were determined using the ELISA technique. Tissue extracts (0.05 ml) prepared in phosphate-bu¡ered saline (PBS) (10 mM phosphate bu¡er, pH 7.4, 150 mM NaCl and 0.1% sodium azide) were pipetted into each well of a polyvinyl microtiter plate and incubated overnight at 4³C. Coating solution was removed and washed three times with washing bu¡er (10 mM phosphate bu¡er, pH 7.4, 150 mM NaCl, 0.05% Tween 20) and distilled water. 100 Wl of monoclonal antibody (CuZn-SOD) (Sigma, St. Louis, MO, USA) diluted in PBS (1:300) or other diluted (1:300) antibodies, viz. anti-Mn-SOD, anti-catalase, anti-GSH-Px, and anti-GST, were added to each well, incubated at room temperature for 2 h, and washed three times as before. 100 Wl of peroxidaseconjugated secondary antibody diluted in PBS (1:100) was added to each well, incubated for 2 h and washed three times as before. 100 Wl of substrate (1% H2 O2 and 1 mg/ml 5-aminosalicylic acid) in reaction bu¡er (0.02 M phosphate bu¡er, 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, BioTek Instruments, Winooski, VT, USA).
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2.8. NO assay NO levels in the cochlea were determined by the NO assay kit (Cayman Chemical, Ann Arbor, MI, USA). 50 Wl of tissue extract was added to the wells of the ELISA microplate followed by 10 Wl of enzyme cofactors and 10 Wl of nitrate reductase mixture. The plate was covered and incubated for 1 h at room temperature. After incubation, 50 Wl of Griess reagent 1 was added followed immediately by 50 Wl of Griess reagent 2. The plate was allowed to develop the color for 10 min at room temperature and absorbance was read at 540 nm using an ELISA plate reader (Automated Microplate Reader, Model EL311, Bio-Tek Instruments). 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 are expressed as mean þ S.E.M. The data for biochemical parameters such as GSH, CuZn-SOD, Mn-SOD, CAT, GSH-Px, GR, GST, XO, NO and MDA were analyzed statistically using one-way analysis of variance followed by Duncan's multiple range test using the SAS statistical software package (SAS Institute, Cary, NC, USA) for comparison of the carbopla-
2.7. Lipid peroxidation assay The end product of lipid peroxidation (malondialdehyde (MDA)) was estimated by the method of Ohkawa et al. (1979). 100 Wl of tissue homogenate was added to 50 Wl of 8.1% sodium dodecyl sulfate, vortexed and incubated for 10 min at room temperature. 375 Wl of 20% acetic acid and 375 Wl of thiobarbituric acid (0.6%) were added and placed in a boiling water bath in sealed tubes for 60 min. The samples were allowed to cool at room temperature. 1.25 ml of n-butanol:pyridine (15:1) was added, vortexed and centrifuged at 1000 rpm for 5 min. 500 Wl of the colored pink layer was measured at 532 nm on a spectrophotometer using 1,1,3,3-tetraethoxypropane as standard. MDA concentration was expressed as nmol/mg protein.
Fig. 1. E¡ects of carboplatin (256 mg/kg, i.p.) on ABR threshold changes at click, 8, 16, and 32 kHz tone burst stimuli in rats 4 days after treatment. Signi¢cance: *P 6 0.05 as compared to control; **P 6 0.01 as compared to control; ***P 6 0.001 as compared to control.
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tin-treated group with the saline control group. The data of ABR were subjected to statistical analysis using a two-tailed t-test. The 0.05 level of probability was used as the criterion for statistical signi¢cance. 3. Results The changes in ABR thresholds in control and carboplatin-treated rats are depicted in Fig. 1. Carboplatin (256 mg/kg) signi¢cantly elevated the hearing thresholds (9.28 þ 2.21 dB (P 6 0.001), 9.09 þ 3.14 dB (P 6 0.01), 10.00 þ 3.56 dB (P 6 0.05) and 15.45 þ 4.92 dB (P 6 0.05)) for clicks, 8, 16, and 32 kHz tone burst stimuli, respectively, 4 days post treatment, compared to saline controls. The changes in thresholds of ABR suggest carboplatin-induced hearing loss at higher frequencies. The changes in NO, GSH and MDA contents in the cochleae of control and carboplatin-treated rats are shown in Table 1. Cochlear NO concentration signi¢cantly (P 6 0.05) increased (153% of control) in rats 4 days after carboplatin treatment. Cochlear GSH concentrations signi¢cantly (P 6 0.01) decreased (63% of control) 4 days after carboplatin administration in rats. Carboplatin signi¢cantly (P 6 0.001) increased cochlear MDA concentration (146% of control) indicating elevation of cochlear membrane lipid peroxidation. The changes in antioxidant enzyme activities in the cochleae of control and carboplatin-treated rats are presented in Table 2. CuZn-SOD activity signi¢cantly (P 6 0.05) decreased (54% of control) in the cochleae of rats 4 days after carboplatin treatment, whereas cochlear Mn-SOD activity signi¢cantly (P 6 0.01) increased (198% of control) following carboplatin administration in rats. Cochlear CAT activity signi¢cantly (P 6 0.01) decreased (70% of control) in rats 4 days after carboplatin treatment. Cochlear GSH-Px activity signi¢cantly (P 6 0.01) decreased (79% of control) after carboplatin 4 days post treatment. GR activity signi¢cantly (P 6 0.001) decreased (58% of control) in the cochleae of rats 4 days post treatment. Cochlear GST activity signi¢cantly (P 6 0.001) decreased (51% of conTable 1 E¡ects of carboplatin on cochlear NO, GSH and MDA concentrations in rats 4 days after treatment Treatment
Control (saline)
Carboplatin (256 mg/kg, i.p.)
NO (nmol/mg protein) GSH (nmol/mg protein) MDA (nmol/mg protein)
707 þ 40.2 6 þ 0.4 8 þ 0.4
1083 þ 160.2* 3 þ 0.5** 11 þ 0.5***
Each value represents the mean þ S.E.M. (n = 6). *P 6 0.05 compared to control; **P 6 0.01 compared to control; ***P 6 0.001 compared to control.
Table 2 E¡ects of carboplatin on antioxidant enzymes, GR, GST and XO activities in the cochleae of rats 4 days post treatment Enzyme activitiesa Control (saline) Carboplatin (256 mg/kg, i.p.) CuZn-SOD Mn-SOD CAT GSH-Px GR GST XO
39.8 þ 4 5.9 þ 0.7 51 þ 3 78 þ 4 26 þ 2 15 þ 1 52 þ 3
21.3 þ 3* 11.8 þ 1** 36 þ 3** 61 þ 3** 15 þ 1*** 8 þ 1** 75 þ 4***
Each value represents the mean þ S.E.M. (n = 6). *P 6 0.05 compared to control; **P 6 0.01 compared to control; ***P 6 0.001 compared to control. a Enzyme activities are expressed as units/mg protein.
trol) 4 days following carboplatin administration in rats. Cochlear XO activity signi¢cantly (P 6 0.001) increased (144% of control) 4 days following carboplatin administration in rats indicating enhanced production of superoxide anions. The e¡ects of carboplatin on antioxidant enzyme protein expression in the cochleae of rats are depicted in Table 3. CuZn-SOD protein level signi¢cantly (P 6 0.001) decreased (39% of control) in the cochleae of rats 4 days after carboplatin administration. However, Mn-SOD protein levels signi¢cantly (P 6 0.02) increased (182% of control) 4 days after carboplatin treatment. Cochlear CAT protein levels signi¢cantly (P 6 0.02) decreased (58% of control) in rats 4 days after carboplatin treatment. GSH-Px protein levels signi¢cantly (P 6 0.02) decreased (63% of control) in the cochleae of rats treated with carboplatin. Cochlear GST enzyme protein levels signi¢cantly (P 6 0.05) decreased (74% of control) in rats 4 days after carboplatin administration. 4. Discussion This study addressed the changes in ABR relationship with the changes in cochlear NO and GSH concentrations, antioxidant enzyme activity and enzyme Table 3 E¡ects of carboplatin on antioxidant enzyme protein levels (Wg/mg protein) in the cochleae of rats 4 days post treatment Enzyme proteins
Control (saline) Carboplatin (256 mg/kg, i.p.)
CuZn-SOD Mn-SOD CAT GSH-Px GST
1.5 þ 0.1 0.5 þ 0.08 4.5 þ 0.5 6.6 þ 0.5 4.4 þ 0.4
0.6 þ 0.09*** 0.9 þ 0.02* 2.6 þ 0.4** 4.1 þ 0.7** 3.3 þ 0.2*
Each value represents the mean þ S.E.M. (n = 6). *P 6 0.05 compared to control; **P 6 0.01 compared to control; ***P 6 0.001 compared to control.
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protein expression and lipid peroxidation 4 days following carboplatin administration in rats. Earlier studies had shown that carboplatin is ototoxic in guinea pigs and chinchillas at 50^150 mg/kg i.p. (Hu et al., 1999 ; Taudy et al., 1992). These doses are equivalent to the clinical therapeutic doses of carboplatin in cancer patients (Maldoon et al., 2000; MacDonald et al., 1994), although general toxicity-inducing doses of carboplatin in rats have been reported to be 40^180 mg/kg i.p. (Cavaletti et al., 1998; Blommaert et al., 1996; Nonclercq et al., 1989). However, the acute ototoxic dose of carboplatin in rats has been reported to be 192^256 mg/ kg i.p. (Husain et al., 2001). Higher doses of carboplatin are being tried clinically and our treatment protocol corresponds to the higher doses used (Wandt et al., 1999 ; Bishop, 1992). In patients, a cumulative dose of 1.6 g/m2 and greater was associated with hearing loss (Wandt et al., 1999; Obermair et al., 1998; Bishop, 1992). The data show that carboplatin at a dose of 256 mg/ kg (i.p.) signi¢cantly elevated the ABR threshold at higher frequencies (8^32 kHz) 4 days after treatment. Carboplatin-induced high frequency hearing loss has also been reported in clinical studies (Neuwelt et al., 1998 ; MacDonald et al., 1994; Bauer et al., 1992; Kennedy et al., 1990) and in experimental studies in guinea pig and chinchilla (Hu et al., 1999; Taudy et al., 1992). In the present study, we observed that ABR threshold changes were accompanied by NO elevation and GSH depletion in the cochleae of rats treated with carboplatin. Evidence for the involvement of NO in cytotoxicity and apoptosis in the cochlea has been reported in animals treated with the platinum-containing anticancer drug cisplatin (Srivastava et al., 1996; Watanabe et al., 2000a). NO and NO donor compounds have been shown to enhance the cytotoxicity of cisplatin (Wink et al., 1997). However, NO synthase inhibitor suppresses the ototoxic side e¡ects of cisplatin (Watanabe et al., 2000b). High amounts of NO are produced by enhanced activity of inducible NO synthase (iNOS) and may react with ROS which have direct cytotoxicity. Thus, carboplatin-induced ototoxicity is related to elevated NO levels which may be due to enhanced iNOS activity in the cochlea. In the present study, the concentration of cochlear GSH in rats is comparable to the concentration reported earlier (Edkins et al., 1992; Ravi et al., 1995 ; Lautermann et al., 1997). The depletion of cochlear GSH in rats treated with carboplatin may be due to loss of GSH through platinum complex formation or NO complex formation followed by metabolism and/or excretion. The inhibition of GST activity and suppression of enzyme protein synthesis suggest that the metabolism of carboplatin through GST is impaired in the cochlea of rat. The depletion of GSH by buthionine sulfoximine resulted in potentiation of ototoxicity
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of carboplatin and cisplatin in vitro as well as in vivo (Hu et al., 1999; Ho¡man et al., 1988). Depletion of tissue GSH is a prime factor which can impair the cell's defense against the toxic actions of ROS and may lead to peroxidative cell injury (Deleve and Kaplowitz, 1990 ; Younes and Siegers, 1981). The absence of GSSG concentrations in cochlear samples suggests that lipid peroxidation may be secondary to the inhibition of antioxidant enzyme activity, enzyme protein expression, and/or due to the generation of ROS by carboplatin (Hu et al., 1999 ; Smith and Mitchell, 1989). The generation of ROS has been reported to be associated with GSSG e¥ux for other alkylating agents (Ishikawa, 1992). It is possible that other events may be contributing to the removal of the GSSG formed in the cochlea. Clinical and experimental studies have demonstrated the importance of intracellular GSH for protection against cisplatin- as well as carboplatin-induced toxicity (Bohm et al., 1999 ; Hu et al., 1999; Hamers et al., 1993; Anderson et al., 1990). Carboplatin-induced ototoxicity may be related to an enhanced £ux of free radicals in the cochlea as evidenced by enhanced NO levels, XO, and Mn-SOD activities and increased lipid peroxidation as a consequence of impaired cochlear antioxidant enzyme activities, suppression of antioxidant enzyme protein expression and GSH depletion. In the present study, the data of cochlear antioxidant enzyme activities in rats are comparable to those reported earlier in the literature (Pierson and Gray, 1982 ; Farms et al., 1993 ; Lautermann et al., 1997). The cochlear CuZnSOD, CAT, GSH-Px, GR and GST activities in the carboplatin-treated groups were signi¢cantly inhibited as compared to the control group. The inhibition of antioxidant enzyme activities increases the endogenous superoxide anion, H2 O2 , and lipid peroxides in subcellular compartments which leads to Ca2 in£ux and pathological changes in the cochlea (Ikeda et al., 1993 ; Clerici et al., 1995, 1996). The impaired antioxidant enzyme activities and enzyme protein synthesis in the cochlea may cause an enhanced ROS-induced membrane lipid peroxidation. The inhibition of cochlear antioxidant enzyme activities in carboplatin-treated rats may be due to (1) oxidative inactivation of enzyme proteins; (2) depletion of copper, zinc, manganese and selenium which are essential for CuZn-SOD, Mn-SOD and GSH-Px activities (DeWoskin and Riviere, 1992); (3) increased NO, ROS and organic peroxides which impair antioxidant enzyme protein synthesis (Clerici et al., 1996 ; Pigeolet et al., 1990); and/or (4) depletion of GSH and NADPH which are essential for GSH-Px, GR and GST enzymes. The inhibition of cochlear antioxidant enzyme activities, de novo synthesis of enzyme proteins and depletion of GSH might be associated with the increase in ABR threshold in rats treated
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with carboplatin. Carboplatin-induced production of superoxide ions (Hu et al., 1999) and NO may lead to pathological changes in the acoustic transduction by modulating hair cell motility such as by changing the shape of the cells (Clerici et al., 1995), pathological changes in the organ of Corti, the stria vascularis and spiral ganglion cells (Watanabe et al., 2000a,b), ultimately resulting in NO-mediated apoptotic/necrotic cell death (Ranjan et al., 1998). Administration of the superoxide scavenging enzyme SOD has prevented the superoxide-induced Ca2 in£ux in the isolated cochlear hair cells (Ikeda et al., 1993; Clerici et al., 1996). Exogenous administration of free radical scavengers, antioxidants and NO synthase inhibitors such as the hydroperoxide scavenging enzyme GSH-Px (ebselen), WR2721, K-lipoic acid, vitamin E and N-nitro-L-arginine methyl ester has also been shown to attenuate hearing loss caused by ototoxic drugs in laboratory animals (Teranishi et al., 2001; Watanabe et al., 2000a,b; Rybak et al., 1999; Conlon et al., 1999; Husain et al., 1998 ; Song and Schacht, 1996; Church et al., 1995). These reports further support the role of NO/ free radicals and endogenous antioxidants in carboplatin-induced hearing loss in rats. In summary, carboplatin induced high frequency hearing loss which was associated with a depletion of GSH, inhibition of antioxidant enzyme activities, depletion of enzyme protein levels and increased XO and Mn-SOD activities and enhanced lipid peroxidation in the cochleae of rats 4 days after treatment. The data suggest that carboplatin enhanced free radical production, depleted antioxidants by inhibiting de novo synthesis of enzyme proteins and induced oxidative injury in the cochleae of rats. Acknowledgements This work was supported in part by a National Organization of Hearing Research (NOHR) grant and the Central Research Committee, Southern Illinois University School of Medicine.
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Obermair, A., Speiser, P., Thoma, M., Kaider, A., Salzer, H., Dittrich, C., Sevelda, R., 1998. Prediction of toxicity but not of clinical course by determining carboplatin exposure in patients with epithelial ovarian cancer treated with a combination of carboplatin and cisplatin. Int. J. Oncol. 13, 1023^1030. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animals and tissue by thiobarbituric acid reaction. Anal. Biochem. 95, 351^358. Pierson, M.G., Gray, B.H., 1982. Superoxide dismutase activity in cochlea. Hear. Res. 6, 141^151. Pigeolet, E., Corbisier, P., Houbion, A., Lambert, D., Michiels, C., Raes, M., Zachary, M.D., Ramacle, J., 1990. Glutathione peroxidase, superoxide dismutase and catalase inactivation by peroxides and oxygen derived free radicals. Mech. Ageing Dev. 51, 283^ 297. Pivot, X., Cals, L., Cupissol, D., Guardiola, E., Tchiknavorian, X., Guerrier, P., Merad, L., Wendling, J.L., Barnouin, L., Savary, J., Thyss, A., Schneider, M., 2001. Phase II trial of a paclitaxel-carboplatin combination in recurrent squamous cell carcinoma of the head and neck. Oncology 60, 66^71. Ranjan, P., Sodhi, A., Singh, S.M., 1998. Murine peritoneal macrophages treated with cisplatin and interferon-gamma undergo NOmediated apoptosis via activation of an endonuclease. Anticancer Drugs 9, 333^341. Ravi, R., Somani, S.M., Rybak, L.P., 1995. Mechanism of cisplatin ototoxicity: Antioxidant system. Pharmacol. Toxicol. 76, 386^394. Read, S.M., Northcole, D.H., 1981. Minimization of variation in the response to di¡erent protein of the coomassie blue G dye-binding assay for protein. Anal. Biochem. 116, 53^64. Rybak, L.P., Husain, K., Whitworth, C., Somani, S.M., 1999. Dose dependent protection by lipoic acid against cisplatin-induced ototoxicity in rats: antioxidant defense system. Toxicol. Sci. 47, 195^ 202. Rybak, L.P., Ravi, R., Somani, S.M., 1995. Mechanism of protection by diethyldithiocarbamate against cisplatin ototoxicity: Antioxidant system. Fund. Appl. Toxicol. 26, 293^300. Singh, N.M., Bogavac, L., Kalimanovska, V., Jelic, Z., Spasic, S., 1987. Spectrophotometric assay of xanthine oxidase with 2,2-azino-di(3-methylbenzthiazoline-6-sulphonate) (ABTS) as chromogen. Clin. Chim. Acta 162, 29^36. Smith, V.C., Mitchell, J.R., 1989. Pharmacological aspects of glutathione in drug metabolism. In: Dolphin, D., Avramovibc, O., Poulson, R. (Eds.), Glutathione: Chemical, Biochemical, and Medical Aspects. Wiley, New York, pp. 141^154. Song, B.B., Schacht, J., 1996. Variable e¤ciency of radical scavengers and iron chelators to attenuate gentamicin ototoxicity in guinea pig in vivo. Hear. Res. 94, 87^93. Srivastava, R.C., Farookh, A., Ahmad, N., Misra, M., Hasan, S.K., Husain, M.M., 1996. Evidence for the involvement of nitric oxide in cisplatin-induced toxicity in rat. Biometals 9, 139^142. Taudy, M., Syka, J., Popelar, J., Ulehlova, L., 1992. Carboplatin and cisplatin ototoxicity in guinea pigs. Audiology 31, 293^299. Teranishi, M., Nakashima, T., Wakabayashi, T., 2001. E¡ects of alpha-tocopherol on cisplatin-induced ototoxicity in guinea pigs. Hear. Res. 151, 61^70. Wandt, H., Birkmann, J., Denzel, T., Schafer, K., Schwab, G., Pilz, D., Egger, H., Both, A., Gallmeir, W.M., 1999. Sequential cycles of high dose chemotherapy with dose escalation carboplatin with or without paclitaxel supported by G-CSF mobilized peripheral blood progenitor cells: a phase I/II study in advanced ovarian cancer. Bone Marrow Transplant. 23, 763^770. Watanabe, K., Hess, A., Michel, O., Yagi, T., 2000a. Nitric oxide synthase inhibitor reduces the apoptotic changes in the cisplatin-treated cochlea of guinea pigs. Anticancer Drugs 11, 731^ 735.
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Carboplatin-induced oxidative stress in rat cochlea K. Husain a
a;
*, C. Whitworth b , S.M. Somani a , L.P. Rybak
b
Department of Pharmacology, Southern Illinois University School of Medicine, 801 North Rutledge, Spring¢eld, IL 62794-9629, USA b Department of Surgery, Southern Illinois University School of Medicine, Spring¢eld, IL 62794-9629, USA Received 12 February 2001; accepted 30 April 2001
Abstract Carboplatin is currently being used in the clinic against a variety of human cancers. However, high dose carboplatin chemotherapy resulted in ototoxicity in cancer patients. This is the first study to show carboplatin-induced oxidative stress response in the cochlea of rat. Male Wistar rats were divided into two groups of six animals each and treated as follows: (1) control (normal saline, i.p.) and (2) carboplatin (256 mg/kg, i.p.). Animals in both groups were sedated with ketamine/xylazine and auditory brainstem-evoked responses were recorded before and 4 days after treatments. The animals were sacrificed on the fourth day and cochleae were harvested and analyzed. A significant elevation of the hearing threshold shifts was noted at clicks, 8, 16, and 32 kHz tone burst stimuli following carboplatin administration. Carboplatin significantly increased nitric oxide and malondialdehyde levels, xanthine oxidase and manganese-superoxide dismutase activities in the cochlea indicating enhanced flux of free radicals. Cochlear glutathione levels, antioxidant enzyme activities such as copper zinc-superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase and enzyme protein levels were significantly depleted 4 days after carboplatin treatment. The data suggest that carboplatin induced free radical generation and antioxidant depletion, and caused oxidative injury in the cochleae of rats. ß 2001 Published by Elsevier Science B.V. Key words: Carboplatin; Oxidative stress; Cochlear antioxidant; Lipid peroxidation; Nitric oxide
1. Introduction Carboplatin [cis-diammine(1,1-cyclobutanedicarboxylate)platinum(II)] is a second generation platinum-containing anticancer drug. It is currently being used in the clinic against a variety of cancers such as small-cell lung cancer, ovarian cancer, and carcinomas of head and * Corresponding author. Tel.: +1 (217) 785-2202; Fax: +1 (217) 524-0145. E-mail address: [email protected] (K. Husain). Abbreviations: ABR, auditory brainstem-evoked response; ABTS, 2,2-azino-di-(3-ethylbenzthiazoline-6-sulfonate); CAT, catalase; CDNB, 1-chloro-2,4-dinitrobenzene; EDTA, ethylenediamine tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; GR, glutathione reductase; GSH, reduced glutathione; GSH-Px, glutathione peroxidase; GSSG, oxidized glutathione; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PBS, phosphate-bu¡ered saline; ROS, reactive oxygen species; SOD, superoxide dismutase; XO, xanthine oxidase
neck (Gridelli et al., 2001; Bolis et al., 2001; Meyer et al., 2001; Pivot et al., 2001 ; Ettinger, 1998; Cavaletti et al., 1998). The identi¢cation of dose escalation of carboplatin is an important factor in achieving optimal antineoplastic e¡ects (Bohm et al., 1999 ; Wandt et al., 1999). 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). A single high dose or repeated doses of carboplatin chemotherapy have been shown to produce ototoxicity as a side e¡ect in cancer patients (DeLauretis et al., 1999 ; Obermair et al., 1998 ; Neuwelt et al., 1998; Cavaletti et al., 1998 ; Kennedy et al., 1990). Carboplatin-induced ototoxicity has also been demonstrated in experimental animals such as guinea pigs and chinchillas (Hofstetter et al., 2000 ; Maldoon et al., 2000 ; Hu et al., 1999; Mount et al., 1995; Taudy et al., 1992). These investigators have demonstrated the changes in cochlear morphology, cochlear potential and auditory brainstem-evoked responses (ABR) following carboplatin administration. We have recently reported
0378-5955 / 01 / $ ^ see front matter ß 2001 Published by Elsevier Science B.V. PII: S 0 3 7 8 - 5 9 5 5 ( 0 1 ) 0 0 3 0 6 - 9
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the dose response of carboplatin-induced hearing loss in a rat model (Husain et al., 2001). However, the biochemical mechanism of carboplatin-induced hearing loss and oxidative injury in the cochlea of rats is not well understood. It is hypothesized that carboplatin induces excess amounts of NO and reactive oxygen species (ROS) generation which in turn causes oxidative impairment of the cochlea leading to hearing loss. Therefore, this study was undertaken in order to evaluate the hearing loss and mechanism of oxidative injury in the cochleae of rats treated with an ototoxic dose of carboplatin. 2. Methods 2.1. Chemicals Chemicals such as reduced (GSH) and oxidized glutathione (GSSG), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and Q-glutamyl glutamate ; enzymes (CuZn-superoxide dismutase (SOD), Mn-SOD, catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione S-transferase (GST)), carboplatin, 2,2-azino-di-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 1-chloro-2,4-dinitrobenzene (CDNB), 1,1,1,1-tetraethoxy-propane, monoclonal antibody for CuZn-SOD, and GST, peroxidase-conjugated secondary antibody were purchased from Sigma Chemicals (St. Louis, MO, USA). Monoclonal antibodies for Mn-SOD, GSH-Px and CAT were purchased from Biodesign, Kennebunk, ME, USA and Oxis Health Products, Portland, OR, USA, respectively. Coomassie protein assay reagent was purchased from Pierce (Rockford, IL, USA). 2.2. Animals Male Wistar rats (250^300 g) were obtained from Charles River (Wilmington, MA, USA) and divided into two groups of six animals and treated as follows: (1) control vehicle-treated (rats were treated with a single bolus administration of normal saline (1 ml/kg) i.p.) and (2) carboplatin-treated (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. Posttreatment ABRs were performed 4 days later and the data were compared to the pretreatment ABRs for changes in thresholds. Thus, each animal served as its own control for the ABRs. The rats in all the groups were sacri¢ced 4 days after treatment. The selection of the carboplatin dose was based on our previous study (Husain et al., 2001). How-
15
ever, the selection of the 4-day time period was based on our time^response study of carboplatin-induced hearing loss in rats. We did not observe a signi¢cant ABR threshold shift on 1, 2 or 3 days post carboplatin treatment in rats (unpublished observations). The heads were collected in ice water, the temporal bones were dissected, the bullae opened, and the cochleae isolated carefully. The isolated cochleae were frozen in liquid nitrogen and stored at 380³C until biochemical analysis could be completed. Cochleae were homogenized in bu¡er 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 Medicine's Laboratory Animal Care and Use Committee (LACUC) and as per the guidelines of NIH. The approved grant title and number are Carboplatin-induced hearing loss in a rat model and # CRC-98-99 and NOHR-99-00. 2.3. ABRs Rats were sedated with a Rompun cocktail (xylazine, ketamine: 3.4 mg/kg, 172.4 mg/kg). Control ABRs were measured using a DEC PDP 11/73 (Digital Equipment Corporation, NH, USA)-based signal generating/averaging system in response to 100 Ws clicks and tone pips at 8, 16, and 32 kHz, which were of 10 ms plateau with a 1 ms rise/fall time. The stimuli were presented inside a double wall radio frequency-shielded sound booth using an Etymotic ER-2 earphone placed directly into the ear canal. Clicks and tone pips were presented at a rate of 5/s. Stimulus intensities were measured using a Bruel and Kjaer sound pressure level meter (model 2209) with a 1/4Q microphone (model 4136) inside an arti¢cial ear canal (RE : 20 WPa). Intensities were expressed in dB sound pressure level (SPL) peak equivalent, based on the calibration. Animals were presented with a stimulus intensity series, which was initiated at 10 dB SPL and reached a maximum of 90 dB SPL. Stimulus intensity was progressively increased in 10 dB increments and the resulting ABRs were observed on a video monitor. Intensities that appeared to be near threshold were repeated. Threshold was de¢ned as the lowest intensity capable of producing a visually detectable, reproducible response. Threshold responses typically displayed wave IV and/or a wave II/III complex. There was some variation due to electrode placement and stimulus frequency. The voltage associated with threshold was 0.5 WV. Sub-dermal electrodes were used to record brain potentials di¡erentially. The active lead was positioned at the vertex and referred to the second electrode at the tip of the nose. The ground electrode was located over the neck muscles. Potentials were ampli¢ed 1000 times inside the sound attenuation booth (bandwidth, 0.1 Hz^ 10 kHz) and signals were further ampli¢ed to produce
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an overall gain of approximately 100 000 and viewed on an oscilloscope. Care was taken to ensure that the band pass of the entire system included those frequencies that represent the ABR. The ABRs were sampled for 20.5 ms following stimulus onset. Stimuli were repeated 5U/ s and a total of 512 trials were averaged using an analog to digital converting system. Evoked potentials were recorded before drug administration and 3 days post administration. The ABR measurement in control rats proved to be highly reproducible in the retest schedule, indicating high intertest reliability (Ravi et al., 1995 ; Rybak et al., 1995). 2.4. Determination of GSH and its disul¢de (GSSG) by high pressure liquid chromatography (HPLC) The concentrations of GSH and GSSG were determined in the cochlea by a modi¢ed HPLC method of Fariss and Reed (1987). 250 Wl of the tissue-acid extract containing internal standard (Q-glutamyl glutamate) was mixed with 100 Wl 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 Wl 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 Q-glutamyl glutamate occurred soon after the change in pH. N-Dinitrophenyl derivatization of the samples was obtained by incubation for 12 h at 4³C in the presence of 1% 1-£uoro-dinitrobenzene. 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 pmol/injection volume for GSSG. 2.5. Enzyme assays SOD activity was determined at room temperature according to the method of Misra and Fridovich (1972). 10 Wl of tissue extract was added to 970 Wl (0.05 M, pH 1:0.2, 0.1 mM ethylenediamine tetraacetic acid (EDTA)) carbonate bu¡er. 20 Wl 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 at 90^100 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 Wl of 20 mM NaCN to inhibit CuZn-SOD activity. CuZn-SOD activity was determined by subtracting the Mn-SOD from total SOD activity. CAT activity was determined at room temperature by
a slight modi¢cation of a method of Aebi (1984). 10 Wl ethanol was added per 100 Wl of tissue extract (dissolved in 0.5 M, pH 7.0, 0.1 mM EDTA, phosphate bu¡er), and then placed in an ice bath for 30 min. Then 10 Wl of Triton X-100 RS was added per 100 Wl of the tissue extract. 10 Wl of tissue extract was added in a cuvette containing 240 Wl phosphate bu¡er and 250 Wl (0.066 M) H2 O2 (dissolved in phosphate bu¡er) and measured at 240 nm for 30 s. The molar extinction coe¤cient of 43.6 mM cm31 was used to determine CAT activity. One unit of CAT activity was de¢ned as mmol of H2 O2 degraded/min/mg protein. GSH-Px activity was determined by a method of Flohe and Gunzler (1984) at 37³C. All reaction mixtures were dissolved in 0.05 M, pH 7.0, 0.1 mM EDTA phosphate bu¡er. A reaction mixture consisted of 500 Wl phosphate bu¡er, 100 Wl 0.01 M glutathione (GSH), 100 Wl 1.5 mM NADPH, and 100 Wl GR (0.24 U). 100 Wl of the tissue extract was added to the reaction mixture and incubated at 37³C for 10 min. Then 50 Wl of 12 mM t-butyl hydroperoxide was added to the tissue reaction mixture and measured at 340 nm for 180 s. The millimolar extinction coe¤cient of 6.22 mM cm31 was used to determine the activity of GSH-Px. One unit of activity was de¢ned as mmol of NADPH oxidized/min/mg protein. GR activity was determined by the method of Carlberg and Mannervik (1985) at 37³C. 50 Wl of NADPH (2 mM) in 10 mM Tris^HCl bu¡er (pH 7.0) added in a cuvette containing 50 Wl of GSSG (20 mM) in phosphate bu¡er (0.5 M, pH 7.0, 0.1 mM EDTA), and 800 Wl of phosphate bu¡er were incubated at 37³C for 10 min. 100 Wl of tissue extract was added to the NADPH-GSSG bu¡ered solution and measured at 340 nm for 3 min. The millimolar extinction coe¤cient of 6.22 cm31 was used to determine the activity of GR. One unit of GR activity was de¢ned as mmol of NADPH oxidized/min/mg protein. GST activity was assayed by the method of Habig et al. (1974) using 10 mM CDNB as substrate. 50 Wl of tissue homogenate was added to 750 Wl 0.1 M phosphate bu¡er containing 0.1 mM EDTA and 100 Wl of 10 mM GSH. 100 Wl of CDNB was added to start the reaction. The changes in optical density were recorded at 340 nm for 3 min. The enzyme activity was calculated using an extinction coe¤cient of 9.6 mM cm31 and expressed as Wmol of CDNB utilized/min/mg protein. Xanthine oxidase (XO) activity was assayed as per the modi¢ed method of Singh et al. (1987) using ABTS as chromogen. In a tube containing 0.5 ml of substrate^bu¡er solution (10 mmol/l hypoxanthine in potassium phosphate bu¡er pH 7.9, 20 mmol/l NaN3 ), 2.5 Wl of uricase and 25 Wl of tissue extract were mixed and incubated for 10 min at 37³C. 500 Wl
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of reagent solution (2 mmol/l ABTS and 2500 U/l peroxidase in 1000 ml of phosphate bu¡er pH 7.9) was added, vortexed and 0.5 ml of 2 mol/l perchloric acid immediately added. Tubes were vortexed and centrifuged for 5 min at 3000 rpm and absorbance of the supernatant was read at 410 nm. The enzyme activity was expressed as Wmol of hypoxanthine oxidized/min/ mg protein. 2.6. Enzyme protein levels by enzyme-linked immunosorbent assay (ELISA) The antioxidant enzyme (CuZn-SOD, Mn-SOD, CAT, GSH-Px and GST) protein levels were determined using the ELISA technique. Tissue extracts (0.05 ml) prepared in phosphate-bu¡ered saline (PBS) (10 mM phosphate bu¡er, pH 7.4, 150 mM NaCl and 0.1% sodium azide) were pipetted into each well of a polyvinyl microtiter plate and incubated overnight at 4³C. Coating solution was removed and washed three times with washing bu¡er (10 mM phosphate bu¡er, pH 7.4, 150 mM NaCl, 0.05% Tween 20) and distilled water. 100 Wl of monoclonal antibody (CuZn-SOD) (Sigma, St. Louis, MO, USA) diluted in PBS (1:300) or other diluted (1:300) antibodies, viz. anti-Mn-SOD, anti-catalase, anti-GSH-Px, and anti-GST, were added to each well, incubated at room temperature for 2 h, and washed three times as before. 100 Wl of peroxidaseconjugated secondary antibody diluted in PBS (1:100) was added to each well, incubated for 2 h and washed three times as before. 100 Wl of substrate (1% H2 O2 and 1 mg/ml 5-aminosalicylic acid) in reaction bu¡er (0.02 M phosphate bu¡er, 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, BioTek Instruments, Winooski, VT, USA).
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2.8. NO assay NO levels in the cochlea were determined by the NO assay kit (Cayman Chemical, Ann Arbor, MI, USA). 50 Wl of tissue extract was added to the wells of the ELISA microplate followed by 10 Wl of enzyme cofactors and 10 Wl of nitrate reductase mixture. The plate was covered and incubated for 1 h at room temperature. After incubation, 50 Wl of Griess reagent 1 was added followed immediately by 50 Wl of Griess reagent 2. The plate was allowed to develop the color for 10 min at room temperature and absorbance was read at 540 nm using an ELISA plate reader (Automated Microplate Reader, Model EL311, Bio-Tek Instruments). 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 are expressed as mean þ S.E.M. The data for biochemical parameters such as GSH, CuZn-SOD, Mn-SOD, CAT, GSH-Px, GR, GST, XO, NO and MDA were analyzed statistically using one-way analysis of variance followed by Duncan's multiple range test using the SAS statistical software package (SAS Institute, Cary, NC, USA) for comparison of the carbopla-
2.7. Lipid peroxidation assay The end product of lipid peroxidation (malondialdehyde (MDA)) was estimated by the method of Ohkawa et al. (1979). 100 Wl of tissue homogenate was added to 50 Wl of 8.1% sodium dodecyl sulfate, vortexed and incubated for 10 min at room temperature. 375 Wl of 20% acetic acid and 375 Wl of thiobarbituric acid (0.6%) were added and placed in a boiling water bath in sealed tubes for 60 min. The samples were allowed to cool at room temperature. 1.25 ml of n-butanol:pyridine (15:1) was added, vortexed and centrifuged at 1000 rpm for 5 min. 500 Wl of the colored pink layer was measured at 532 nm on a spectrophotometer using 1,1,3,3-tetraethoxypropane as standard. MDA concentration was expressed as nmol/mg protein.
Fig. 1. E¡ects of carboplatin (256 mg/kg, i.p.) on ABR threshold changes at click, 8, 16, and 32 kHz tone burst stimuli in rats 4 days after treatment. Signi¢cance: *P 6 0.05 as compared to control; **P 6 0.01 as compared to control; ***P 6 0.001 as compared to control.
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tin-treated group with the saline control group. The data of ABR were subjected to statistical analysis using a two-tailed t-test. The 0.05 level of probability was used as the criterion for statistical signi¢cance. 3. Results The changes in ABR thresholds in control and carboplatin-treated rats are depicted in Fig. 1. Carboplatin (256 mg/kg) signi¢cantly elevated the hearing thresholds (9.28 þ 2.21 dB (P 6 0.001), 9.09 þ 3.14 dB (P 6 0.01), 10.00 þ 3.56 dB (P 6 0.05) and 15.45 þ 4.92 dB (P 6 0.05)) for clicks, 8, 16, and 32 kHz tone burst stimuli, respectively, 4 days post treatment, compared to saline controls. The changes in thresholds of ABR suggest carboplatin-induced hearing loss at higher frequencies. The changes in NO, GSH and MDA contents in the cochleae of control and carboplatin-treated rats are shown in Table 1. Cochlear NO concentration signi¢cantly (P 6 0.05) increased (153% of control) in rats 4 days after carboplatin treatment. Cochlear GSH concentrations signi¢cantly (P 6 0.01) decreased (63% of control) 4 days after carboplatin administration in rats. Carboplatin signi¢cantly (P 6 0.001) increased cochlear MDA concentration (146% of control) indicating elevation of cochlear membrane lipid peroxidation. The changes in antioxidant enzyme activities in the cochleae of control and carboplatin-treated rats are presented in Table 2. CuZn-SOD activity signi¢cantly (P 6 0.05) decreased (54% of control) in the cochleae of rats 4 days after carboplatin treatment, whereas cochlear Mn-SOD activity signi¢cantly (P 6 0.01) increased (198% of control) following carboplatin administration in rats. Cochlear CAT activity signi¢cantly (P 6 0.01) decreased (70% of control) in rats 4 days after carboplatin treatment. Cochlear GSH-Px activity signi¢cantly (P 6 0.01) decreased (79% of control) after carboplatin 4 days post treatment. GR activity signi¢cantly (P 6 0.001) decreased (58% of control) in the cochleae of rats 4 days post treatment. Cochlear GST activity signi¢cantly (P 6 0.001) decreased (51% of conTable 1 E¡ects of carboplatin on cochlear NO, GSH and MDA concentrations in rats 4 days after treatment Treatment
Control (saline)
Carboplatin (256 mg/kg, i.p.)
NO (nmol/mg protein) GSH (nmol/mg protein) MDA (nmol/mg protein)
707 þ 40.2 6 þ 0.4 8 þ 0.4
1083 þ 160.2* 3 þ 0.5** 11 þ 0.5***
Each value represents the mean þ S.E.M. (n = 6). *P 6 0.05 compared to control; **P 6 0.01 compared to control; ***P 6 0.001 compared to control.
Table 2 E¡ects of carboplatin on antioxidant enzymes, GR, GST and XO activities in the cochleae of rats 4 days post treatment Enzyme activitiesa Control (saline) Carboplatin (256 mg/kg, i.p.) CuZn-SOD Mn-SOD CAT GSH-Px GR GST XO
39.8 þ 4 5.9 þ 0.7 51 þ 3 78 þ 4 26 þ 2 15 þ 1 52 þ 3
21.3 þ 3* 11.8 þ 1** 36 þ 3** 61 þ 3** 15 þ 1*** 8 þ 1** 75 þ 4***
Each value represents the mean þ S.E.M. (n = 6). *P 6 0.05 compared to control; **P 6 0.01 compared to control; ***P 6 0.001 compared to control. a Enzyme activities are expressed as units/mg protein.
trol) 4 days following carboplatin administration in rats. Cochlear XO activity signi¢cantly (P 6 0.001) increased (144% of control) 4 days following carboplatin administration in rats indicating enhanced production of superoxide anions. The e¡ects of carboplatin on antioxidant enzyme protein expression in the cochleae of rats are depicted in Table 3. CuZn-SOD protein level signi¢cantly (P 6 0.001) decreased (39% of control) in the cochleae of rats 4 days after carboplatin administration. However, Mn-SOD protein levels signi¢cantly (P 6 0.02) increased (182% of control) 4 days after carboplatin treatment. Cochlear CAT protein levels signi¢cantly (P 6 0.02) decreased (58% of control) in rats 4 days after carboplatin treatment. GSH-Px protein levels signi¢cantly (P 6 0.02) decreased (63% of control) in the cochleae of rats treated with carboplatin. Cochlear GST enzyme protein levels signi¢cantly (P 6 0.05) decreased (74% of control) in rats 4 days after carboplatin administration. 4. Discussion This study addressed the changes in ABR relationship with the changes in cochlear NO and GSH concentrations, antioxidant enzyme activity and enzyme Table 3 E¡ects of carboplatin on antioxidant enzyme protein levels (Wg/mg protein) in the cochleae of rats 4 days post treatment Enzyme proteins
Control (saline) Carboplatin (256 mg/kg, i.p.)
CuZn-SOD Mn-SOD CAT GSH-Px GST
1.5 þ 0.1 0.5 þ 0.08 4.5 þ 0.5 6.6 þ 0.5 4.4 þ 0.4
0.6 þ 0.09*** 0.9 þ 0.02* 2.6 þ 0.4** 4.1 þ 0.7** 3.3 þ 0.2*
Each value represents the mean þ S.E.M. (n = 6). *P 6 0.05 compared to control; **P 6 0.01 compared to control; ***P 6 0.001 compared to control.
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protein expression and lipid peroxidation 4 days following carboplatin administration in rats. Earlier studies had shown that carboplatin is ototoxic in guinea pigs and chinchillas at 50^150 mg/kg i.p. (Hu et al., 1999 ; Taudy et al., 1992). These doses are equivalent to the clinical therapeutic doses of carboplatin in cancer patients (Maldoon et al., 2000; MacDonald et al., 1994), although general toxicity-inducing doses of carboplatin in rats have been reported to be 40^180 mg/kg i.p. (Cavaletti et al., 1998; Blommaert et al., 1996; Nonclercq et al., 1989). However, the acute ototoxic dose of carboplatin in rats has been reported to be 192^256 mg/ kg i.p. (Husain et al., 2001). Higher doses of carboplatin are being tried clinically and our treatment protocol corresponds to the higher doses used (Wandt et al., 1999 ; Bishop, 1992). In patients, a cumulative dose of 1.6 g/m2 and greater was associated with hearing loss (Wandt et al., 1999; Obermair et al., 1998; Bishop, 1992). The data show that carboplatin at a dose of 256 mg/ kg (i.p.) signi¢cantly elevated the ABR threshold at higher frequencies (8^32 kHz) 4 days after treatment. Carboplatin-induced high frequency hearing loss has also been reported in clinical studies (Neuwelt et al., 1998 ; MacDonald et al., 1994; Bauer et al., 1992; Kennedy et al., 1990) and in experimental studies in guinea pig and chinchilla (Hu et al., 1999; Taudy et al., 1992). In the present study, we observed that ABR threshold changes were accompanied by NO elevation and GSH depletion in the cochleae of rats treated with carboplatin. Evidence for the involvement of NO in cytotoxicity and apoptosis in the cochlea has been reported in animals treated with the platinum-containing anticancer drug cisplatin (Srivastava et al., 1996; Watanabe et al., 2000a). NO and NO donor compounds have been shown to enhance the cytotoxicity of cisplatin (Wink et al., 1997). However, NO synthase inhibitor suppresses the ototoxic side e¡ects of cisplatin (Watanabe et al., 2000b). High amounts of NO are produced by enhanced activity of inducible NO synthase (iNOS) and may react with ROS which have direct cytotoxicity. Thus, carboplatin-induced ototoxicity is related to elevated NO levels which may be due to enhanced iNOS activity in the cochlea. In the present study, the concentration of cochlear GSH in rats is comparable to the concentration reported earlier (Edkins et al., 1992; Ravi et al., 1995 ; Lautermann et al., 1997). The depletion of cochlear GSH in rats treated with carboplatin may be due to loss of GSH through platinum complex formation or NO complex formation followed by metabolism and/or excretion. The inhibition of GST activity and suppression of enzyme protein synthesis suggest that the metabolism of carboplatin through GST is impaired in the cochlea of rat. The depletion of GSH by buthionine sulfoximine resulted in potentiation of ototoxicity
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of carboplatin and cisplatin in vitro as well as in vivo (Hu et al., 1999; Ho¡man et al., 1988). Depletion of tissue GSH is a prime factor which can impair the cell's defense against the toxic actions of ROS and may lead to peroxidative cell injury (Deleve and Kaplowitz, 1990 ; Younes and Siegers, 1981). The absence of GSSG concentrations in cochlear samples suggests that lipid peroxidation may be secondary to the inhibition of antioxidant enzyme activity, enzyme protein expression, and/or due to the generation of ROS by carboplatin (Hu et al., 1999 ; Smith and Mitchell, 1989). The generation of ROS has been reported to be associated with GSSG e¥ux for other alkylating agents (Ishikawa, 1992). It is possible that other events may be contributing to the removal of the GSSG formed in the cochlea. Clinical and experimental studies have demonstrated the importance of intracellular GSH for protection against cisplatin- as well as carboplatin-induced toxicity (Bohm et al., 1999 ; Hu et al., 1999; Hamers et al., 1993; Anderson et al., 1990). Carboplatin-induced ototoxicity may be related to an enhanced £ux of free radicals in the cochlea as evidenced by enhanced NO levels, XO, and Mn-SOD activities and increased lipid peroxidation as a consequence of impaired cochlear antioxidant enzyme activities, suppression of antioxidant enzyme protein expression and GSH depletion. In the present study, the data of cochlear antioxidant enzyme activities in rats are comparable to those reported earlier in the literature (Pierson and Gray, 1982 ; Farms et al., 1993 ; Lautermann et al., 1997). The cochlear CuZnSOD, CAT, GSH-Px, GR and GST activities in the carboplatin-treated groups were signi¢cantly inhibited as compared to the control group. The inhibition of antioxidant enzyme activities increases the endogenous superoxide anion, H2 O2 , and lipid peroxides in subcellular compartments which leads to Ca2 in£ux and pathological changes in the cochlea (Ikeda et al., 1993 ; Clerici et al., 1995, 1996). The impaired antioxidant enzyme activities and enzyme protein synthesis in the cochlea may cause an enhanced ROS-induced membrane lipid peroxidation. The inhibition of cochlear antioxidant enzyme activities in carboplatin-treated rats may be due to (1) oxidative inactivation of enzyme proteins; (2) depletion of copper, zinc, manganese and selenium which are essential for CuZn-SOD, Mn-SOD and GSH-Px activities (DeWoskin and Riviere, 1992); (3) increased NO, ROS and organic peroxides which impair antioxidant enzyme protein synthesis (Clerici et al., 1996 ; Pigeolet et al., 1990); and/or (4) depletion of GSH and NADPH which are essential for GSH-Px, GR and GST enzymes. The inhibition of cochlear antioxidant enzyme activities, de novo synthesis of enzyme proteins and depletion of GSH might be associated with the increase in ABR threshold in rats treated
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with carboplatin. Carboplatin-induced production of superoxide ions (Hu et al., 1999) and NO may lead to pathological changes in the acoustic transduction by modulating hair cell motility such as by changing the shape of the cells (Clerici et al., 1995), pathological changes in the organ of Corti, the stria vascularis and spiral ganglion cells (Watanabe et al., 2000a,b), ultimately resulting in NO-mediated apoptotic/necrotic cell death (Ranjan et al., 1998). Administration of the superoxide scavenging enzyme SOD has prevented the superoxide-induced Ca2 in£ux in the isolated cochlear hair cells (Ikeda et al., 1993; Clerici et al., 1996). Exogenous administration of free radical scavengers, antioxidants and NO synthase inhibitors such as the hydroperoxide scavenging enzyme GSH-Px (ebselen), WR2721, K-lipoic acid, vitamin E and N-nitro-L-arginine methyl ester has also been shown to attenuate hearing loss caused by ototoxic drugs in laboratory animals (Teranishi et al., 2001; Watanabe et al., 2000a,b; Rybak et al., 1999; Conlon et al., 1999; Husain et al., 1998 ; Song and Schacht, 1996; Church et al., 1995). These reports further support the role of NO/ free radicals and endogenous antioxidants in carboplatin-induced hearing loss in rats. In summary, carboplatin induced high frequency hearing loss which was associated with a depletion of GSH, inhibition of antioxidant enzyme activities, depletion of enzyme protein levels and increased XO and Mn-SOD activities and enhanced lipid peroxidation in the cochleae of rats 4 days after treatment. The data suggest that carboplatin enhanced free radical production, depleted antioxidants by inhibiting de novo synthesis of enzyme proteins and induced oxidative injury in the cochleae of rats. Acknowledgements This work was supported in part by a National Organization of Hearing Research (NOHR) grant and the Central Research Committee, Southern Illinois University School of Medicine.
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