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The protective effect of intratympanic dexamethasone on cisplatin-induced ototoxicity in guinea pigs
Otolaryngology–Head and Neck Surgery (2007) 137, 747-752
ORIGINAL RESEARCH—OTOLOGY AND NEUROTOLOGY
The protective effect of intratympanic dexamethasone on cisplatin-induced ototoxicity in guinea pigs Alper Daldal, MD, Onur Odabasi, MD, and Bulent Serbetcioglu, MD, PhD, Denizli, Aydin, and Izmir, Turkey OBJECTIVE: The purpose of this study was to investigate the effectiveness of intratympanic dexamethasone injection as a protection agent against cisplatin-induced ototoxicity. STUDY DESIGN AND SETTING: The four groups of guinea pigs were injected as follows: 1) cisplatin, 2) intratympanic dexamethasone, 3) cisplatin following intratympanic dexamethasone, and 4) cisplatin after intratympanic saline. Before and 3 days following injections, the ototoxic effect was measured with distortion product otoacoustic emissions (DPOAEs). RESULTS: The DPOAEs amplitudes and signal-to-noise ratio (SNR) values at 1 to 6 kHz frequencies for group 1 animals after injections significantly decreased over those before injections (P ⬍ 0.05). In group 2, there were no significant differences in DPOAE amplitude and SNR values between before and after intratympanic dexamethasone injections (P ⬎ 0.05). Considering group 3, there were also no significant differences in DPOAEs amplitudes and SNR values before and after of dexamethasone and cisplatin injections (P ⬎ 0.05). CONCLUSIONS: Intratympanic dexamethasone injection did not cause any ototoxic effect; in contrast, it might have a significant protective effect after cisplatin injection. © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
C
isplatin (cis-diamminedichloroplatinum II) is a potent antineoplastic drug and is widely used to treat head and neck squamous cell carcinoma: solid tumors of the testis, ovary, bladder, prostate, and cervix; and non–small cell carcinoma of the lung.1 However, severe side effects such as nephrotoxicity, myelotoxicity, gastrointestinal toxicity, ototoxicity, and peripheral neuropathy limit the clinical use of cisplatin. In particular, nephrotoxicity and ototoxicity are dose-limiting side effects. Nephrotoxicity can be effectively ameliorated with forced diuresis with hypertonic saline and diuretic agents. Although this method will increase the antitumor dosage of cisplatin, it does not affect the incidence or severity of ototoxicity.2 The ototoxic effect of cisplatin is characterized by irreversible, progressive, bilateral, high -frequency, sensorineural hearing loss associated with tinnitus. Factors that affect the incidence of ototoxicity include the administration
route, cumulative dose, age, dietary factors, serum protein level, genetic factors, and cranial radiotherapy history.1,2 Cisplatin destroys the outer hair cells (OHCs) in the cochlea in a progressive manner, from the base to the apex. In addition, there is sporadic destruction of inner hair cells (IHCs). The ototoxicity of cisplatin is not limited to hair cells but also includes atrophy of the stria vascularis, collapse of Reissner’s membrane, and damage to the cells supporting the organ of Corti.3 Although the mechanism of the antineoplastic effect of cisplatin has been well described, the cellular and molecular mechanisms of cisplatin-induced ototoxicity are not well understood. Increasing evidence indicates that the accumulation of reactive oxygen species mediates cisplatin ototoxicity. It has been postulated that these toxic effects arise via the stimulation of nitric oxide (NO) synthesis.4 NO is a free radical with important roles in physiologic and pathophysiologic processes. In the inner ear, inducible nitric oxide synthase (NOS) is also expressed under pathologic conditions such as lipopolysaccharide inoculation, gentamicin and cisplatin ototoxicity, noise exposure, and endolymphatic hydrops.4,5 As a result, the stimulation of continuous production resulting in large quantities of NO forms the main mechanism of toxicity. Various chemoprotective antioxidant agents have been reported to ameliorate cisplatin ototoxicity; these include sodium thiosulfate, diethyldithiocarbamate, 4-methylthiobenzoic acid, D- and L-methionine, N-acetylcysteine, and glutathione ester.6-9 This study investigated the otoprotective effect of intratympanic dexamethasone given before exposure to toxic doses of cisplatin on the auditory hair cells.
MATERIALS AND METHODS Animals and Anesthesia The experimental animals were 24 adult, female, albino guinea pigs, that weighed 400 to 750 g. Guinea pigs were studied because they are similar to human beings with
Received November 29, 2006; revised May 26, 2007; accepted May 31, 2007.
0194-5998/$32.00 © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2007.05.068
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respect to their well-defined temporal bone anatomy, hearing physiology, and ototoxic response to drugs. After purchase, the animals were kept in the animal laboratory at Adnan Menderes University, School of Veterinary, for at least 1 week. The guinea pigs had free access to water and commercial food and were housed in temperature-controlled rooms with a 12-hour light/dark cycle. This study was approved by the Committee for Ethics in Animal Experiments of the Current University School of Veterinary Medicine. The animals were anesthetized with 30 mg/kg ketamine hydrochloride (Ketalar, Eczacibasi Ilac Sanayi ve Ticaret A.S, Luleburgaz, Turkey) and 4 mg/kg xylazine (Alfazyne 2%, Alfasan International B.V, Woerden, The Netherlands) given as an intraperitoneal infusion before cisplatin administration and testing. The depth of anesthesia was determined with the pedal reflex. To maintain anesthesia during testing, half-doses of the xylazine/ketamine were administered as needed.
Experimental Protocol and Groups Initially, the normal cochlear function of the animals was confirmed by measuring the distortion product otoacoustic emissions (DPOAEs). There was no interaural differences in DP amplitudes for each animal before the data from the both ears were combined. The DP amplitudes from the both ears were compared for each frequency. The cisplatin-induced ototoxity starts in hours and stabilized in 3 days, and it continues its effect up to 7 days of high dose cisplatin application. The dexametasone rises its maximum concentration in inner ear fluid in about 60 hours of intratympanic application. The studies that investigated the effect of protective agents on cisplatin-induced ototoxitiy used the 3 to 5 day period for such measurement.3 Based on these literature findings, we also planned to obtain such measurements on day 3. In this study, the randomization was established by animals and not by ears. Therefore, the 24 animals were randomly assigned to four groups of 6 animals (12 ears) each. Group 1 (cisplatin only). Cisplatin 12 mg/kg (Cisplatin DBL, Faulding Pharmaceuticals, Warwickshire, UK) was administered intraperitoneally as a slow infusion. Group 2 (intratympanic dexamethasone only). Under an operating microscope, an intratympanic injection of dexamethasone at 4 mg/mL (Dekort, Deva Holding, Istanbul, Turkey) was given slowly through a myringotomy in the anterosuperior quadrant, with a 28-gauge dental needle to fill the middle ear cavity (approximately 0.1 to 0.3 mL).10 After keeping the animal in the same position for 30 minutes, the procedure was performed in the other ear. Group 3 (cisplatin and intratympanic dexamethasone). The procedure described for group 2 was performed. Then, 30 minutes after the dexamethasone injection, cisplatin 12
mg/kg was administered intraperitoneally as a slow infusion. Group 4 (cisplatin and intratympanic saline). The procedure described for group 2 was performed in both middle ear cavities except an infusion of 0.9% saline was used instead of dexamethasone.
DPOAE Measurements The animals were anesthetized with ketamine hydrochloride and xylazine before testing. Before the DPOAE was measured, the ear was examined under an operating microscope to assess the external auditory canal, tympanic membrane, and signs of otitis media. The DPOAE was measured at the beginning of treatment and after 3 days of drug injections with an Otodynamics Echoport USB cochlear emissions analyzer and Otodynamics ILO version 6.0 software (Otodynamics, London, UK). A probe with a microphone assembly and two transducer tubes was acoustically coupled to the external ear canal of the test animal. The DPOAE was measured as the sound pressure level (SPL) with stimuli at constant intensity and different frequencies. The stimulus intensity of f1 and f2 was 65 dB SPL. The f2/f1 ratio was set at 1.22, and 2f1-f2 was recorded at four points per octave over the frequency range of f2 (1001 to 6165 Hz). The amplitude of the DPOAE above the noise floor (signal-tonoise ratio) was noted for each of the six test frequencies. The upper frequency limit of the distortion product otoacoustic emissions is considered to be 8 kHz. However, the use of relative operating characteristic (ROC) curve to describe the test performance of DPOAE signal-to-noise ratio (DPOAE/noise) was optimal for 4 kHz, but the performance was poorer at 8 kHz. The upper limit of distortion product otoacoustic emissions is 8 kHz, cochlear responses obtained from basiler membrane that has characteristic frequency higher than 6 kHz may not correlate with valid data.11
Statistical Analysis An initial pilot study with four animals gave an alpha error of 0.05 and power of 0.95, which suggested that a sample size of 20 animals would be sufficient for statistical significance. Considering possible unforeseen events resulting in the loss of animals during the study, we used 24 animals, in four groups of 6, ie, 24 animals were randomly assigned to four groups, each group including 12 ears. The data were analyzed using the Wilcoxon paired 2-sample test, Mann-Whitney U test, and Kruskal Wallis variance analysis in SPSS 11.0 for Windows. Statistical significance was set at P ⬍ 0.05.
RESULTS In group 1, the DPOAE amplitudes and SNR values decreased significantly at frequencies of 1 to 6 kHz at 3 days after intraperitoneal cisplatin injection (both P ⫽ 0.002; Fig 1). In group 2, there were no significant differences in the
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The protective effect of intratympanic dexamethasone . . .
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Figure 1 DPOAE amplitudes and SNR (signal-to-noise ratio) values in guinea pigs before and after intraperitoneal cisplatin injection (Wilcoxon paired 2-sample test).
DPOAE amplitudes or SNR values between before and after intratympanic dexamethasone injection (P ⬎ 0.05; Fig 2), suggesting that intratympanic dexamethasone injection had no toxic effect on cochlear emissions. Similarly, in group 3 (Fig 3) as well as in group 4 (Fig 4), which underwent intratympanic saline injection, there were no significant differences in the DPOAE amplitudes or SNR values between before and after the treatment (P ⬎ 0.05 for each). Figure 5 shows a comparison between the pre- and postinjection DPOAE amplitudes and SNR values for all groups. The postinjection DPOAE amplitudes and SNR values were significantly lower than the preinjection measurements in group 1 compared with the differences in group 3 (P ⫽ 0.000 for each), suggesting that intratympanic dexamethasone injection had an otoprotective effect in subjects given a single high dose of cisplatin. In addition, no tympanic membrane injury, such as perforation, was observed in any test animal. Therefore, the intratympanic injection technique is safe and does not interfere with the measurement of DPOAE.
DISCUSSION Despite the development of new regimens and dosage limits, the ototoxic effect of cisplatin treatment is still unavoid-
able. A single injection of cisplatin 12 to 16 mg/kg rapidly causes ototoxicity with a high incidence.8 Ototoxic agents primarily affect the OHCs before affecting other cochlear elements. OAE measurements are ideal for monitoring cochlear function in drug-induced ototoxicity. Evoked OAEs, especially DPOAEs due to frequency specificity, were shown to be more sensitive for evaluating OHCs than were conventional audiometry, ultra high frequency audiometry, and auditory brainstem response (ABR).12 Yilmaz et al13 reported that intratympanic dexamethasone injection had no significant negative effect on the accuracy of transient evoked otoacoustic emissions (TEOAEs). The DPOAE measurements in our control group (group 3) concurred with those results. Various antioxidant agents ameliorate cisplatin ototoxicity. Rybak et al8 reported that 4-methylthiobenzoic acid protected against cisplatin-induced changes in cochlear function and in the antioxidant system of rats. In addition, recent studies have shown that D-methionine, 4-methylthiobenzoic acid, and N-acetylcysteine provides complete protection from cisplatin ototoxicity.6,9 Furthermore, the administration of other antioxidant agents, including diethyldithiocarbamate,7 sodium thiosulfate,6 and glutathione ester, reduced cisplatin ototoxicity. The pathophysiology of cisplatin-induced ototoxicity is not completely understood. Currently, it appears that cispla-
Figure 2 DPOAE amplitudes and SNR (signal-to-noise ratio) values in guinea pigs before and after intratympanic dexamethasone injection (Wilcoxon paired 2-sample test).
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Figure 3 DPOAE amplitudes and SNR (signal-to-noise ratio) values in guinea pigs before and after intratympanic dexamethasone injection followed by intraperitoneal cisplatin injection (Wilcoxon paired 2-sample test).
tin causes ototoxicity by increasing reactive oxygen species and altering the antioxidant defense system of the cochlea.8 Some authors4,5 suggest that the principle mechanism of ototoxicity is related to the production of NO, which is induced by the production of reactive oxygen species and the over-induction of inducible NOS (NOS II) synthesis. In the early phase of inner ear damage, constitutive NOS (NOS I and NOS III) is synthesized, whereas NOS II is more commonly synthesized during the late phase of inner ear damage and excessively increases the amount of available NO.4 The major toxicity of NO is modest but is greatly potentiated by its reaction with superoxide to form peroxynitrite. Peroxynitrite has direct negative effects on proteins, lipids, and DNA, and the resulting increase in protein oxidation and lipid peroxidation may damage DNA and cause apoptotic changes.14 Watanabe et al4 investigated the effect of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on cisplatin-induced ototoxicity in the guinea pig. They reported that the ABR threshold shift was minimized and that immunohistological observations of the cochlea revealed decreased NO levels, possibly caused by a reduction in NOS II activity. Steroids, especially dexamethasone, are used to treat many conditions, including sudden hearing loss, noise-induced hearing loss, Ménière’s disease, salicylate ototox-
icity, and aminoglycoside ototoxicity.15-17 Glucocorticoids confer protection by up-regulating antioxidant enzyme activity either before or during hypoxia-ischemia. The protection afforded to H2O2-treated cells by hydrocortisone suggests that the steroid protects against drug toxicity by decreasing cell destruction caused by free radicals.18 Palmer et al19 also showed that dexamethasone and hydrocortisone decreased NO-induced endothelial cell damage. These studies have documented that steroids inhibit both the increase of NOS mRNA and the release of reactive nitrogen intermediates. The inhibitor effect of steroids on the NOS increase was also supported by the findings of Himeno et al.17 Our study suggests that dexamethasone also has a protective effect against cisplatin-induced ototoxicity, possibly by affecting NO synthesis. An exhaustive review of the English-language literature failed to find any previous report on the protective effect of dexamethasone against cisplatin-induced ototoxicity. There have been a few reports16,17 on the protective effect of the systemic or local (through the round window membrane) application of dexamethasone against salicylate- or aminoglycoside-induced ototoxicity. Silverstein et al20 reported that the intratympanic application of glucocorticoids had no protective effect and showed that glucocorticoids increased the auditory
Figure 4 DPOAE amplitudes and SNR (signal-to-noise ratio) values in guinea pigs before and after intratympanic saline injection followed by intraperitoneal cisplatin injection (Wilcoxon paired 2-sample test).
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Figure 5 Differences in the pre- and postinjection DPOAE amplitudes (above) and SNR (signal-to-noise ratio) values (bottom) for all groups (Mann-Whitney U test and Kruskal-Wallis variance analysis).
threshold. Conversely, more recent studies have proved that the instillation of glucocorticoids in inner ear disease is safe and has no adverse effects on hearing.15 Our well-designed, controlled study suggests that dexamethasone does not affect cochlear function, and our findings concur with those of recent studies. Intratympanic injection for the treatment of ear diseases is a newly developed drug application modality. Since its first use by Schuknecht in 1956 for Ménière’s disease, it has been applied for the treatment of many other diseases, including sudden hearing loss, tinnitus, and autoimmune inner ear disease.15,16,20 The intratympanic administration has two theoretical advantages: the potential for steroid uptake through the round window membrane, which results in higher perilymph levels, and the possible reduction of systemic steroid absorption and toxicity.15,17 Corticosteroids, administered by either a systemic or intratympanic route, cross the blood-labyrinthine barrier and are detected in both the perilymph and endolymph. Dexamethasone, hydrocortisone, and methylprednisolone all achieved higher perilymphatic-endolymphatic concentrations when administered by intratympanic injection compared with systemic administration.10 Moreover, a recent study reported that the systemic administration of steroids minimized chemotherapy-induced adverse effects and reduced the tumoricidal activity of chemotherapy.21 This will likely limit the future systemic application of steroids and favor the local application of such drugs. We used local application to exclude
possible adverse effects of systemic application and to more rapidly achieve a higher concentration of dexamethasone in the cochlear fluid. In our study, there were no significant differences in the pre- and post– drug injection measurements of DPOAE or SNR in group 3. This suggests that intratympanic dexamethasone injection selectively inhibited NOS II, possibly by preventing iNOS-mRNA synthesis and thereby preventing late-phase NO synthesis, without affecting cochlear blood flow or cochlear vascular tonus. Intratympanic dexamethasone may minimize inner ear damage induced by cisplatin. We acknowledge the limitations of this study, which specifically include the small number of subjects in each group and the lack of electron microscopic and histologic examinations of the hair cells before and after cisplatin application. In addition, as DPOAE specifically reflects the functioning of the outer hair cells, which are the principal target of cisplatin ototoxicity, the functioning of other inner cells, which may also be susceptible to cisplatin ototoxicity, was not measured in this study. We also acknowledge the methodologic limitation of our study, since intratympanic drug injection was used and it was assumed that the injected volume (0.1 to 0.3 mL) was enough to cover the round window in an animal, although sampling from inner ear for the measurement of the diffused drug concentration was not made.
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CONCLUSION We studied the protective effect of intratympanic dexamethasone against cisplatin-induced ototoxicity in guinea pigs. Intratympanic dexamethasone had a significant protective effect on OHCs, which are damaged by cisplatin. In addition, intratympanic dexamethasone had no ototoxic or systemic side effects. This can easily be monitored with DPOAE measurements. However, well-designed, placebocontrolled human studies are needed to confirm our results and to determine the best dexamethasone regimen for preventing cisplatin-induced ototoxicity. Our study provides the initial work for developing a new otoprotective protocol and a new indication for intratympanic steroid injections.
ACKNOWLEDGEMENT We thank Feray Gursoy MD and M. Nil Kaan MD, Department of Anesthesiology, for helpful advice on the statistical methods, support, and helpful discussions.
AUTHOR INFORMATION From the Department of Otorhinolaryngology (Dr Daldal), Acipayam Goverment Hospital, Denizli, Turkey; the Department of Otorhinolaryngology (Dr Odabasi), Adnan Menderes University, School of Medicine, Aydin, Turkey; and the Department of Otorhinolaryngology (Dr Serbetcioglu), Dokuz Eylul University, School of Medicine, Izmir, Turkey. Corresponding author: Onur Odabasi, MD, Associate Professor, Department of Otorhinolaryngology, Adnan Menderes University, School of Medicine, Aydin, Turkey. E-mail address: [email protected], [email protected].
AUTHOR CONTRIBUTIONS Onur Odabasi, study design, writer; Alper Daldal, study design, writer, data collection, conducting the study; Bulent Serbetcioglu, audiologic evaluation, study design.
FINANCIAL DISCLOSURE None.
REFERENCES 1. Sakamoto M, Kaga K, Kamio T. Extended high-frequency ototoxicity induced by the first administration of cisplatin. Otolaryngol Head Neck Surg 2000;122:828 –33.
2. Cooley ME, Davis L, Abrahm J. Cisplatin: a clinical review. Part II– Nursing assessment and management of side effects of cisplatin. Cancer Nurs 1994;17:283–93. 3. Laurell G, Bagger-Sjoback D. Dose-dependent inner ear changes after i.v. administration of cisplatin. J Otolaryngol 1991;20:158 – 67. 4. Watanabe K, Hess A, Michel O, et al. Nitric oxide synthase inhibitor reduces the apoptotic change in the cisplatin-treated cochlea of guinea pigs. Anticancer Drugs 2000;11:731–5. 5. Takumida M, Anniko M. Nitric oxide in guinea pig vestibular sensory cells following gentamicin exposure in vitro. Acta Otolaryngol 2001; 121:346 –50. 6. Wimmer C, Mees K, Stumpf P, et al. Round window application of D-methionine, sodium thiosulfate, brain-derived neurotrophic factor, and fibroblast growth factor-2 in cisplatin-induced ototoxicity. Otol Neurotol 2004;25:33– 40. 7. Rybak LP, Ravi R, Somani SM. Mechanism of protection by diethyldithiocarbamate against cisplatin ototoxicity: antioxidant system. Fundam Appl Toxicol 1995;26:293–300. 8. Rybak LP, Husain K, Evenson L, et al. Protection by 4-methylthiobenzoic acid against cisplatin-induced ototoxicity: antioxidant system. Pharmacol Toxicol 1997;81:173–9. 9. Kopke RD, Liu W, Gabaizadeh R, et al. Use of organotypic cultures of Corti’s organ to study the protective effects of antioxidant molecules on cisplatin-induced damage of auditory hair cells. Am J Otol 1997; 18:559 –71. 10. Parnes LS, Sun AH, Freeman DJ. Corticosteroid pharmacokinetics in the inner ear fluids: an animal study followed by clinical application. Laryngoscope 1999;109:1–17. 11. Gorga MP, Neely ST, Dorn PA. Distortion product otoacoustic emissions in relation to hearing loss. In: Robinette RM, Glattke T, editors. Otoacoustic emissions: clinical applications, 2nd ed. New York: Thieme; 2002. p. 243–72.) 12. Ress BD, Sridhar KS, Balkany TJ, et al. Effects of cis-platinum chemotherapy on otoacoustic emissions: the development of an objective screening protocol. Third place–Resident Clinical Science Award 1998. Otolaryngol Head Neck Surg 1999;121:693–701. 13. Yilmaz I, Yilmazer C, Erkan AN, et al. Intratympanic dexamethasone injection effects on transient-evoked otoacoustic emission. Am J Otolaryngol 2005;26:113–7. 14. Takumida M, Anniko M, Popa R, et al. Pharmacological models for inner ear therapy with emphasis on nitric oxide. Acta Otolaryngol 2001;121:16 –20. 15. Chandrasekhar SS. Intratympanic dexamethasone for sudden sensorineural hearing loss: clinical and laboratory evaluation. Otol Neurotol 2001;22:18 –23. 16. Silverstein H, Choo D, Rosenberg SI, et al. Intratympanic steroid treatment of inner ear disease and tinnitus (preliminary report). Ear Nose Throat J 1996;75:468 –71, 474, 476. 17. Himeno C, Komeda M, Izumikawa M, et al. Intra-cochlear administration of dexamethasone attenuates aminoglycoside ototoxicity in the guinea pig. Hear Res 2002;167:61–70. 18. Tuor UI. Glucocorticoids and the prevention of hypoxic-ischemic brain damage. Neurosci Biobehav Rev 1997;21(2):175–9. 19. Palmer RM, Bridge L, Foxwell NA, et al. The role of nitric oxide in endothelial cell damage and its inhibition by glucocorticoids. Br J Pharmacol 1992;105:11–2. 20. Silverstein H, Isaacson JE, Olds MJ, et al. Dexamethasone inner ear perfusion for the treatment of Meniere’s disease: a prospective, randomized, double-blind, crossover trial. Am J Otol 1998;19:196 –201. 21. Zhang C, Marme A, Wenger T, et al. Glucocorticoid-mediated inhibition of chemotherapy in ovarian carcinomas. Int J Oncol 2006;28: 551– 8.
ORIGINAL RESEARCH—OTOLOGY AND NEUROTOLOGY
The protective effect of intratympanic dexamethasone on cisplatin-induced ototoxicity in guinea pigs Alper Daldal, MD, Onur Odabasi, MD, and Bulent Serbetcioglu, MD, PhD, Denizli, Aydin, and Izmir, Turkey OBJECTIVE: The purpose of this study was to investigate the effectiveness of intratympanic dexamethasone injection as a protection agent against cisplatin-induced ototoxicity. STUDY DESIGN AND SETTING: The four groups of guinea pigs were injected as follows: 1) cisplatin, 2) intratympanic dexamethasone, 3) cisplatin following intratympanic dexamethasone, and 4) cisplatin after intratympanic saline. Before and 3 days following injections, the ototoxic effect was measured with distortion product otoacoustic emissions (DPOAEs). RESULTS: The DPOAEs amplitudes and signal-to-noise ratio (SNR) values at 1 to 6 kHz frequencies for group 1 animals after injections significantly decreased over those before injections (P ⬍ 0.05). In group 2, there were no significant differences in DPOAE amplitude and SNR values between before and after intratympanic dexamethasone injections (P ⬎ 0.05). Considering group 3, there were also no significant differences in DPOAEs amplitudes and SNR values before and after of dexamethasone and cisplatin injections (P ⬎ 0.05). CONCLUSIONS: Intratympanic dexamethasone injection did not cause any ototoxic effect; in contrast, it might have a significant protective effect after cisplatin injection. © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
C
isplatin (cis-diamminedichloroplatinum II) is a potent antineoplastic drug and is widely used to treat head and neck squamous cell carcinoma: solid tumors of the testis, ovary, bladder, prostate, and cervix; and non–small cell carcinoma of the lung.1 However, severe side effects such as nephrotoxicity, myelotoxicity, gastrointestinal toxicity, ototoxicity, and peripheral neuropathy limit the clinical use of cisplatin. In particular, nephrotoxicity and ototoxicity are dose-limiting side effects. Nephrotoxicity can be effectively ameliorated with forced diuresis with hypertonic saline and diuretic agents. Although this method will increase the antitumor dosage of cisplatin, it does not affect the incidence or severity of ototoxicity.2 The ototoxic effect of cisplatin is characterized by irreversible, progressive, bilateral, high -frequency, sensorineural hearing loss associated with tinnitus. Factors that affect the incidence of ototoxicity include the administration
route, cumulative dose, age, dietary factors, serum protein level, genetic factors, and cranial radiotherapy history.1,2 Cisplatin destroys the outer hair cells (OHCs) in the cochlea in a progressive manner, from the base to the apex. In addition, there is sporadic destruction of inner hair cells (IHCs). The ototoxicity of cisplatin is not limited to hair cells but also includes atrophy of the stria vascularis, collapse of Reissner’s membrane, and damage to the cells supporting the organ of Corti.3 Although the mechanism of the antineoplastic effect of cisplatin has been well described, the cellular and molecular mechanisms of cisplatin-induced ototoxicity are not well understood. Increasing evidence indicates that the accumulation of reactive oxygen species mediates cisplatin ototoxicity. It has been postulated that these toxic effects arise via the stimulation of nitric oxide (NO) synthesis.4 NO is a free radical with important roles in physiologic and pathophysiologic processes. In the inner ear, inducible nitric oxide synthase (NOS) is also expressed under pathologic conditions such as lipopolysaccharide inoculation, gentamicin and cisplatin ototoxicity, noise exposure, and endolymphatic hydrops.4,5 As a result, the stimulation of continuous production resulting in large quantities of NO forms the main mechanism of toxicity. Various chemoprotective antioxidant agents have been reported to ameliorate cisplatin ototoxicity; these include sodium thiosulfate, diethyldithiocarbamate, 4-methylthiobenzoic acid, D- and L-methionine, N-acetylcysteine, and glutathione ester.6-9 This study investigated the otoprotective effect of intratympanic dexamethasone given before exposure to toxic doses of cisplatin on the auditory hair cells.
MATERIALS AND METHODS Animals and Anesthesia The experimental animals were 24 adult, female, albino guinea pigs, that weighed 400 to 750 g. Guinea pigs were studied because they are similar to human beings with
Received November 29, 2006; revised May 26, 2007; accepted May 31, 2007.
0194-5998/$32.00 © 2007 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2007.05.068
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Otolaryngology–Head and Neck Surgery, Vol 137, No 5, November 2007
respect to their well-defined temporal bone anatomy, hearing physiology, and ototoxic response to drugs. After purchase, the animals were kept in the animal laboratory at Adnan Menderes University, School of Veterinary, for at least 1 week. The guinea pigs had free access to water and commercial food and were housed in temperature-controlled rooms with a 12-hour light/dark cycle. This study was approved by the Committee for Ethics in Animal Experiments of the Current University School of Veterinary Medicine. The animals were anesthetized with 30 mg/kg ketamine hydrochloride (Ketalar, Eczacibasi Ilac Sanayi ve Ticaret A.S, Luleburgaz, Turkey) and 4 mg/kg xylazine (Alfazyne 2%, Alfasan International B.V, Woerden, The Netherlands) given as an intraperitoneal infusion before cisplatin administration and testing. The depth of anesthesia was determined with the pedal reflex. To maintain anesthesia during testing, half-doses of the xylazine/ketamine were administered as needed.
Experimental Protocol and Groups Initially, the normal cochlear function of the animals was confirmed by measuring the distortion product otoacoustic emissions (DPOAEs). There was no interaural differences in DP amplitudes for each animal before the data from the both ears were combined. The DP amplitudes from the both ears were compared for each frequency. The cisplatin-induced ototoxity starts in hours and stabilized in 3 days, and it continues its effect up to 7 days of high dose cisplatin application. The dexametasone rises its maximum concentration in inner ear fluid in about 60 hours of intratympanic application. The studies that investigated the effect of protective agents on cisplatin-induced ototoxitiy used the 3 to 5 day period for such measurement.3 Based on these literature findings, we also planned to obtain such measurements on day 3. In this study, the randomization was established by animals and not by ears. Therefore, the 24 animals were randomly assigned to four groups of 6 animals (12 ears) each. Group 1 (cisplatin only). Cisplatin 12 mg/kg (Cisplatin DBL, Faulding Pharmaceuticals, Warwickshire, UK) was administered intraperitoneally as a slow infusion. Group 2 (intratympanic dexamethasone only). Under an operating microscope, an intratympanic injection of dexamethasone at 4 mg/mL (Dekort, Deva Holding, Istanbul, Turkey) was given slowly through a myringotomy in the anterosuperior quadrant, with a 28-gauge dental needle to fill the middle ear cavity (approximately 0.1 to 0.3 mL).10 After keeping the animal in the same position for 30 minutes, the procedure was performed in the other ear. Group 3 (cisplatin and intratympanic dexamethasone). The procedure described for group 2 was performed. Then, 30 minutes after the dexamethasone injection, cisplatin 12
mg/kg was administered intraperitoneally as a slow infusion. Group 4 (cisplatin and intratympanic saline). The procedure described for group 2 was performed in both middle ear cavities except an infusion of 0.9% saline was used instead of dexamethasone.
DPOAE Measurements The animals were anesthetized with ketamine hydrochloride and xylazine before testing. Before the DPOAE was measured, the ear was examined under an operating microscope to assess the external auditory canal, tympanic membrane, and signs of otitis media. The DPOAE was measured at the beginning of treatment and after 3 days of drug injections with an Otodynamics Echoport USB cochlear emissions analyzer and Otodynamics ILO version 6.0 software (Otodynamics, London, UK). A probe with a microphone assembly and two transducer tubes was acoustically coupled to the external ear canal of the test animal. The DPOAE was measured as the sound pressure level (SPL) with stimuli at constant intensity and different frequencies. The stimulus intensity of f1 and f2 was 65 dB SPL. The f2/f1 ratio was set at 1.22, and 2f1-f2 was recorded at four points per octave over the frequency range of f2 (1001 to 6165 Hz). The amplitude of the DPOAE above the noise floor (signal-tonoise ratio) was noted for each of the six test frequencies. The upper frequency limit of the distortion product otoacoustic emissions is considered to be 8 kHz. However, the use of relative operating characteristic (ROC) curve to describe the test performance of DPOAE signal-to-noise ratio (DPOAE/noise) was optimal for 4 kHz, but the performance was poorer at 8 kHz. The upper limit of distortion product otoacoustic emissions is 8 kHz, cochlear responses obtained from basiler membrane that has characteristic frequency higher than 6 kHz may not correlate with valid data.11
Statistical Analysis An initial pilot study with four animals gave an alpha error of 0.05 and power of 0.95, which suggested that a sample size of 20 animals would be sufficient for statistical significance. Considering possible unforeseen events resulting in the loss of animals during the study, we used 24 animals, in four groups of 6, ie, 24 animals were randomly assigned to four groups, each group including 12 ears. The data were analyzed using the Wilcoxon paired 2-sample test, Mann-Whitney U test, and Kruskal Wallis variance analysis in SPSS 11.0 for Windows. Statistical significance was set at P ⬍ 0.05.
RESULTS In group 1, the DPOAE amplitudes and SNR values decreased significantly at frequencies of 1 to 6 kHz at 3 days after intraperitoneal cisplatin injection (both P ⫽ 0.002; Fig 1). In group 2, there were no significant differences in the
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The protective effect of intratympanic dexamethasone . . .
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Figure 1 DPOAE amplitudes and SNR (signal-to-noise ratio) values in guinea pigs before and after intraperitoneal cisplatin injection (Wilcoxon paired 2-sample test).
DPOAE amplitudes or SNR values between before and after intratympanic dexamethasone injection (P ⬎ 0.05; Fig 2), suggesting that intratympanic dexamethasone injection had no toxic effect on cochlear emissions. Similarly, in group 3 (Fig 3) as well as in group 4 (Fig 4), which underwent intratympanic saline injection, there were no significant differences in the DPOAE amplitudes or SNR values between before and after the treatment (P ⬎ 0.05 for each). Figure 5 shows a comparison between the pre- and postinjection DPOAE amplitudes and SNR values for all groups. The postinjection DPOAE amplitudes and SNR values were significantly lower than the preinjection measurements in group 1 compared with the differences in group 3 (P ⫽ 0.000 for each), suggesting that intratympanic dexamethasone injection had an otoprotective effect in subjects given a single high dose of cisplatin. In addition, no tympanic membrane injury, such as perforation, was observed in any test animal. Therefore, the intratympanic injection technique is safe and does not interfere with the measurement of DPOAE.
DISCUSSION Despite the development of new regimens and dosage limits, the ototoxic effect of cisplatin treatment is still unavoid-
able. A single injection of cisplatin 12 to 16 mg/kg rapidly causes ototoxicity with a high incidence.8 Ototoxic agents primarily affect the OHCs before affecting other cochlear elements. OAE measurements are ideal for monitoring cochlear function in drug-induced ototoxicity. Evoked OAEs, especially DPOAEs due to frequency specificity, were shown to be more sensitive for evaluating OHCs than were conventional audiometry, ultra high frequency audiometry, and auditory brainstem response (ABR).12 Yilmaz et al13 reported that intratympanic dexamethasone injection had no significant negative effect on the accuracy of transient evoked otoacoustic emissions (TEOAEs). The DPOAE measurements in our control group (group 3) concurred with those results. Various antioxidant agents ameliorate cisplatin ototoxicity. Rybak et al8 reported that 4-methylthiobenzoic acid protected against cisplatin-induced changes in cochlear function and in the antioxidant system of rats. In addition, recent studies have shown that D-methionine, 4-methylthiobenzoic acid, and N-acetylcysteine provides complete protection from cisplatin ototoxicity.6,9 Furthermore, the administration of other antioxidant agents, including diethyldithiocarbamate,7 sodium thiosulfate,6 and glutathione ester, reduced cisplatin ototoxicity. The pathophysiology of cisplatin-induced ototoxicity is not completely understood. Currently, it appears that cispla-
Figure 2 DPOAE amplitudes and SNR (signal-to-noise ratio) values in guinea pigs before and after intratympanic dexamethasone injection (Wilcoxon paired 2-sample test).
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Figure 3 DPOAE amplitudes and SNR (signal-to-noise ratio) values in guinea pigs before and after intratympanic dexamethasone injection followed by intraperitoneal cisplatin injection (Wilcoxon paired 2-sample test).
tin causes ototoxicity by increasing reactive oxygen species and altering the antioxidant defense system of the cochlea.8 Some authors4,5 suggest that the principle mechanism of ototoxicity is related to the production of NO, which is induced by the production of reactive oxygen species and the over-induction of inducible NOS (NOS II) synthesis. In the early phase of inner ear damage, constitutive NOS (NOS I and NOS III) is synthesized, whereas NOS II is more commonly synthesized during the late phase of inner ear damage and excessively increases the amount of available NO.4 The major toxicity of NO is modest but is greatly potentiated by its reaction with superoxide to form peroxynitrite. Peroxynitrite has direct negative effects on proteins, lipids, and DNA, and the resulting increase in protein oxidation and lipid peroxidation may damage DNA and cause apoptotic changes.14 Watanabe et al4 investigated the effect of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on cisplatin-induced ototoxicity in the guinea pig. They reported that the ABR threshold shift was minimized and that immunohistological observations of the cochlea revealed decreased NO levels, possibly caused by a reduction in NOS II activity. Steroids, especially dexamethasone, are used to treat many conditions, including sudden hearing loss, noise-induced hearing loss, Ménière’s disease, salicylate ototox-
icity, and aminoglycoside ototoxicity.15-17 Glucocorticoids confer protection by up-regulating antioxidant enzyme activity either before or during hypoxia-ischemia. The protection afforded to H2O2-treated cells by hydrocortisone suggests that the steroid protects against drug toxicity by decreasing cell destruction caused by free radicals.18 Palmer et al19 also showed that dexamethasone and hydrocortisone decreased NO-induced endothelial cell damage. These studies have documented that steroids inhibit both the increase of NOS mRNA and the release of reactive nitrogen intermediates. The inhibitor effect of steroids on the NOS increase was also supported by the findings of Himeno et al.17 Our study suggests that dexamethasone also has a protective effect against cisplatin-induced ototoxicity, possibly by affecting NO synthesis. An exhaustive review of the English-language literature failed to find any previous report on the protective effect of dexamethasone against cisplatin-induced ototoxicity. There have been a few reports16,17 on the protective effect of the systemic or local (through the round window membrane) application of dexamethasone against salicylate- or aminoglycoside-induced ototoxicity. Silverstein et al20 reported that the intratympanic application of glucocorticoids had no protective effect and showed that glucocorticoids increased the auditory
Figure 4 DPOAE amplitudes and SNR (signal-to-noise ratio) values in guinea pigs before and after intratympanic saline injection followed by intraperitoneal cisplatin injection (Wilcoxon paired 2-sample test).
Daldal et al
The protective effect of intratympanic dexamethasone . . .
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Figure 5 Differences in the pre- and postinjection DPOAE amplitudes (above) and SNR (signal-to-noise ratio) values (bottom) for all groups (Mann-Whitney U test and Kruskal-Wallis variance analysis).
threshold. Conversely, more recent studies have proved that the instillation of glucocorticoids in inner ear disease is safe and has no adverse effects on hearing.15 Our well-designed, controlled study suggests that dexamethasone does not affect cochlear function, and our findings concur with those of recent studies. Intratympanic injection for the treatment of ear diseases is a newly developed drug application modality. Since its first use by Schuknecht in 1956 for Ménière’s disease, it has been applied for the treatment of many other diseases, including sudden hearing loss, tinnitus, and autoimmune inner ear disease.15,16,20 The intratympanic administration has two theoretical advantages: the potential for steroid uptake through the round window membrane, which results in higher perilymph levels, and the possible reduction of systemic steroid absorption and toxicity.15,17 Corticosteroids, administered by either a systemic or intratympanic route, cross the blood-labyrinthine barrier and are detected in both the perilymph and endolymph. Dexamethasone, hydrocortisone, and methylprednisolone all achieved higher perilymphatic-endolymphatic concentrations when administered by intratympanic injection compared with systemic administration.10 Moreover, a recent study reported that the systemic administration of steroids minimized chemotherapy-induced adverse effects and reduced the tumoricidal activity of chemotherapy.21 This will likely limit the future systemic application of steroids and favor the local application of such drugs. We used local application to exclude
possible adverse effects of systemic application and to more rapidly achieve a higher concentration of dexamethasone in the cochlear fluid. In our study, there were no significant differences in the pre- and post– drug injection measurements of DPOAE or SNR in group 3. This suggests that intratympanic dexamethasone injection selectively inhibited NOS II, possibly by preventing iNOS-mRNA synthesis and thereby preventing late-phase NO synthesis, without affecting cochlear blood flow or cochlear vascular tonus. Intratympanic dexamethasone may minimize inner ear damage induced by cisplatin. We acknowledge the limitations of this study, which specifically include the small number of subjects in each group and the lack of electron microscopic and histologic examinations of the hair cells before and after cisplatin application. In addition, as DPOAE specifically reflects the functioning of the outer hair cells, which are the principal target of cisplatin ototoxicity, the functioning of other inner cells, which may also be susceptible to cisplatin ototoxicity, was not measured in this study. We also acknowledge the methodologic limitation of our study, since intratympanic drug injection was used and it was assumed that the injected volume (0.1 to 0.3 mL) was enough to cover the round window in an animal, although sampling from inner ear for the measurement of the diffused drug concentration was not made.
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CONCLUSION We studied the protective effect of intratympanic dexamethasone against cisplatin-induced ototoxicity in guinea pigs. Intratympanic dexamethasone had a significant protective effect on OHCs, which are damaged by cisplatin. In addition, intratympanic dexamethasone had no ototoxic or systemic side effects. This can easily be monitored with DPOAE measurements. However, well-designed, placebocontrolled human studies are needed to confirm our results and to determine the best dexamethasone regimen for preventing cisplatin-induced ototoxicity. Our study provides the initial work for developing a new otoprotective protocol and a new indication for intratympanic steroid injections.
ACKNOWLEDGEMENT We thank Feray Gursoy MD and M. Nil Kaan MD, Department of Anesthesiology, for helpful advice on the statistical methods, support, and helpful discussions.
AUTHOR INFORMATION From the Department of Otorhinolaryngology (Dr Daldal), Acipayam Goverment Hospital, Denizli, Turkey; the Department of Otorhinolaryngology (Dr Odabasi), Adnan Menderes University, School of Medicine, Aydin, Turkey; and the Department of Otorhinolaryngology (Dr Serbetcioglu), Dokuz Eylul University, School of Medicine, Izmir, Turkey. Corresponding author: Onur Odabasi, MD, Associate Professor, Department of Otorhinolaryngology, Adnan Menderes University, School of Medicine, Aydin, Turkey. E-mail address: [email protected], [email protected].
AUTHOR CONTRIBUTIONS Onur Odabasi, study design, writer; Alper Daldal, study design, writer, data collection, conducting the study; Bulent Serbetcioglu, audiologic evaluation, study design.
FINANCIAL DISCLOSURE None.
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