Intracochlear administration of thiourea protects against cisplatin-induced outer hair cell loss in the guinea pig

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Hearing Research 181 (2003) 109^115 www.elsevier.com/locate/heares

Intracochlear administration of thiourea protects against cisplatin-induced outer hair cell loss in the guinea pig A. Ekborn b

a;

, G. Laurell a , H. Ehrsson b , J. Miller

c

a Department of Otorhinolaryngology, Karolinska Hospital, 17176 Stockholm, Sweden Karolinska Pharmacy and Department of Oncology^Pathology, Karolinska Institutet, Stockholm, Sweden c Kresge Hearing Research Institute, University of Michigan, Ann Arbor, MI, USA

Received 19 March 2003; accepted 14 May 2003

Abstract Amelioration of cisplatin-induced side-effects is of great clinical importance. Local administration of a cytoprotective agent to the inner ear offers a possibility to prevent cisplatin-induced ototoxicity without risk of interference with the antitumour effect. The ideal substance for local administration has yet to be identified. Thiourea (TU) has unique properties that make it an interesting candidate. This study was initiated to test the hypothesis that TU given by local administration protects against cisplatin ototoxicity in the guinea pig. After baseline auditory brainstem response (ABR) assessment, the left cochlea was implanted with a microtip catheter connected to an osmotic pump filled with either 27 mg/ml TU in artificial perilymph (AP), or AP administered for the full duration of the study. Three days post-implant, animals with normal ABRs received an intravenous injection of 8 mg/ kg body-weight cisplatin. Five days after the cisplatin treatment ABRs were reassessed, animals decapitated and bilateral cytocochleograms prepared. TU-treated ears demonstrated significantly lower outer hair cell (OHC) loss as compared to contralateral untreated ears, and significantly lower OHC loss compared to AP-treated ears. ABR threshold shift did not differ significantly between the two groups. It can be postulated that TU demonstrates partial protection against cisplatin-induced ototoxicity. 9 2003 Elsevier Science B.V. All rights reserved. Key words: Cisplatin; Ototoxicity; Protection; Thiourea; Intracochlear administration

1. Introduction Cisplatin has been used in the treatment of neoplastic disease for 30 years, often in combination chemotherapy and recently as a part of chemoradiotherapy, e.g. in cervix, oesophageal and rectal cancers (Cooper et al., 1999; Grigsby and Herzog, 2001; Valentini et al., 2001). Although newer platinum compounds have been developed, cisplatin can still be considered the

* Corresponding author. Tel.: +46 (8) 51776359; Fax: +46 (8) 51776267. E-mail address: [email protected] (A. Ekborn). Abbreviations: ABR, auditory brainstem response; AP, arti¢cial perilymph; EP, endocochlear potential; GP, guinea pig; HEPES, hydroxy-etyl-piperacin-etan-sulfonic acid; IHC, inner hair cell; OHC, outer hair cell; PBS, phosphate-bu¡ered saline; PFA, paraformaldehyde in phosphate bu¡er; TU, thiourea

most versatile platinum-containing antineoplastic drug (Lokich, 2001). Ototoxicity is one major dose-limiting side-e¡ect of the drug (Laurell and Jungnelius, 1990; Piccart et al., 2001). Cytoprotection of the cochlea and other organ systems during treatment with cisplatin-based chemotherapy could o¡er the prospect of safer and more intense and e¡ective treatment. A wide variety of substances have been evaluated for this purpose, e.g. thiosulphate, diethyldithiocarbamate, silibinin, ebselen, methylthiobenzoate, lipoate, phosphomycin, D-methionine and WR2721 (Berry et al., 1990; Bokemeyer et al., 1996; Campbell et al., 1996; Kaltenbach et al., 1997; Otto et al., 1988; Rybak et al., 1999, 2000; Saito et al., 1997). A reduction in the antitumour e¡ect has been shown in animal models with systemically administered sodium thiosulphate (Aamdal et al., 1987, 1988). Systemic administration of D-methionine lowers the amount of free

0378-5955 / 03 / $ ^ see front matter 9 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-5955(03)00181-3

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cisplatin in blood (Ekborn et al., 2002) and may thus lower the therapeutic e⁄cacy. By local administration of a protective agent to the inner ear, systemic interaction may be avoided, but little work has been done on this therapeutic strategy. For local drug delivery to the inner ear, two methods of inner ear administration have been studied, direct intracochlear administration (Brown et al., 1993; Prieskorn and Miller, 2000) and middle ear administration (Harner et al., 2001; Kohonen and Tarkkanen, 1969). It has been shown that administration to the round window membrane through the middle ear of L-methionine protects from cisplatininduced ototoxicity in the rat with retention of the antineoplastic e¡ect in a tumour model (Li et al., 2001). While middle ear administration has the clear clinical advantage of less invasiveness, there is less control of the amount of agent delivered to the inner ear. Although administration of protective substances to the middle ear of rodents can be e¡ective, similar administration in humans may prove to be more di⁄cult, due to the protected location of the inner ear behind the thick human round window membrane (Goycoolea, 2001). This prompts a search for the optimal substance, which ideally should be both highly reactive and di¡usible, i.e. small and uncharged. Thiourea (TU, CS(NH2 )2 ) is a small (molecular weight 76), uncharged nucleophile, with antioxidant properties, that rapidly reacts in vitro with cisplatin. It is more reactive with cisplatin in vitro than sodium thiosulphate (Riley et al., 1982). It inactivates cisplatin DNA monoadducts and increases cell survival when post-incubated with cisplatin-treated cells (FichtingerSchepman et al., 1995). Furthermore, TU acts as an antioxidant, as shown by its ability to decrease mutagenesis caused by oxidative stress in an experimental system (Yonezawa et al., 2001). TU reaches the inner ear via the round window membrane in the rat (Laurell et al., 2002). All these properties make TU a good otoprotector candidate which may decrease cisplatin ototoxicity by interacting directly with cisplatin, cisplatin^DNA adducts or by aiding the inner ear cellular antioxidant systems. The aim of this study was to test the hypothesis that TU administered directly to the perilymphatic compartment is protective against cisplatin-induced ototoxicity in vivo.

2. Materials and methods 2.1. Study design This study was performed in two parts. Part 1, dose titration. The e¡ect of scala tympani administration of either one of three solutions with dif-

ferent concentrations ^ 9, 27 and 90 mg/ml (a saturated solution of TU in water) ^ was evaluated in a total of nine guinea pigs (GPs). 9 and 27 mg/ml TU were dissolved in arti¢cial perilymph (AP) and 90 mg/ml TU was dissolved in sterile water. After baseline auditory brainstem response (ABR) measurement, animals were implanted with a microcannula osmotic pump containing the selected solution. After 7 days a second ABR was obtained, the middle ears examined for abnormalities and both cochleae removed and prepared for histological assessment. Based on this study the concentration of 27 mg/ml TU was deemed the most appropriate for the otoprotection study. Part 2, ototoxic evaluation. Twenty GPs with normal ABRs were implanted with a microcannula and an osmotic pump infusing either AP or 27 mg/ml TU in AP to the scala tympani for the full duration of the study. Ten GPs received a TU infusion and 10 GPs served as controls and were administered AP. In all cases the left ear was implanted, while the contralateral ear was available for further comparison. Inspection of the tympanic membrane and a second ABR assessment were performed 3 days after the pump implant. Only animals with normal-appearing tympanic membranes and ABRs were continued in the study. In these GPs cisplatin at 8 mg/kg body weight (b.w.) was administered as a bolus injection. After a further 5 days, a third ABR measurement was performed, and the animals were sacri¢ced. The middle ears were examined immediately post-mortem after removal of the tympanic membrane and both cochleae were prepared for histology. 2.2. General procedures 2.2.1. Animals Pigmented GPs (240^340 g, 285 M 7 g) (mean M S.E.M.) of both sexes from a local breeder (Elm Hill Breeding Laboratory, Chelmsford, MA, USA) were used. Water and standard GP food was provided ad libitum. Throughout the study the subjects were maintained in AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care International) approved facilities, under supervision of a certi¢ed veterinarian. During all measurements and surgical procedures they were anaesthetised with 40 mg/kg b.w. Ketamine (Ketalar, Parke-Davis) and 10 mg/kg b.w. Xylazine (Rompun Vet, Bayer AG); additional doses were provided as needed. All animals were allowed to fully recover and resume normal activity under a heating lamp before being returned to the cage in the animal room. All animals included in the study demonstrated baseline ABRs within normal limits and normal tympanic membranes at the start of the study. The general status of the animals was monitored daily and animals deteriorating in general health were

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euthanised. Experiments were performed in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 0-23 (revised 1996)). The animal procedures were reviewed and approved by the University of Michigan Unit for Laboratory Medicine. Ethical approval was obtained both from the University of Michigan and the Karolinska Institute animal care and use committee (Norra djurfo«rso«ksetiska na«mnden No. 137/99). 2.2.2. Pump implant All pumps were prepared according to manufacturers’ instructions under aseptic conditions using Millipore (Millex GV, 0.22 Wm) ¢ltered £uids. The pumps were incubated overnight in normal saline at 37‡C. TU (Sigma Chemicals) was dissolved in AP (9 or 27 mg/ml) or sterile water (90 mg/ml). AP was composed of 145 mM NaCl, 2.7 mM KCl, 2 mM MgSO4 , 1.2 mM CaCl2 and 5.0 mM HEPES (hydroxy-etyl-piperacinetan-sulfonic acid) in water. One dose of 30 mg/kg b.w. chloramphenicol was administered s.c. as prophylaxis against infection at induction of anaesthesia before pump implant. The animals were shaved and supplemental local anaesthesia with lidocaine was administered. During surgery, body temperature was maintained by a homeothermic pad. After careful surgical scrub and draping the skin was incised, a hole was drilled in the vertex region and a stainless steel screw placed in dural contact. The technique for local intracochlear administration used in the study, preparation of the microcannula^osmotic pump system and its implantation have been described (Brown et al., 1993; Prieskorn and Miller, 2000). Brie£y, under aseptic conditions the left cochlea was exposed via a post-auricular approach through a hole in the bulla. A hole was handdrilled in the otic capsule in the basal turn of the cochlea and a microtip catheter was inserted in scala tympani. The cannula was pre-¢lled with £uid of the same composition as in the primed pump. The microtip cathether was ¢xed in place with cyanoacrylate glue at the bulla defect, which was then sealed with dental cement. The catheter, secured to the vertex screw with methacrylate cement, was connected to an Alzet0 (Alza Corp.) (0.5 Wl/h, 14 days) miniosmotic pump, implanted subcutaneously in the intrascapular region, and the skin subsequently closed. 2.2.3. Cisplatin administration Anaesthetised GPs were examined by otoscopy and ABR before being placed on their backs on the homeothermic pad. The internal jugular vein was exposed and cannulated. 1 mg/ml cisplatin (Platinol, Bristol Myers Squibb), 8 mg/kg b.w., was administered as a 15 s bolus injection. All animals received 15 ml of normal saline

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subcutaneously immediately after cisplatin administration to decrease the possibility of renal damage. 2.3. Ototoxic evaluation 2.3.1. ABR ABR measurements were performed with a TDT system II (Tucker Davies Technologies, Gainesville, FL, USA) standard modular system using TDT BIO SIG software. The stimuli consisted of 80 ms sine wave tone bursts at 4, 12, and 16 kHz, cosine-gated and presented at a rate of 10 s31 through a speculum in the left ear canal with the animal placed in a soundproof room. Bioelectric signals were obtained through the vertex epidural screw and through one stainless steel needle inserted subcutaneously in the left infra-auricular region and one reference needle electrode inserted over the bridge of the nose. The sampling period was 15 ms and 1024 signals were averaged. The signals were lowand high-pass (3 and 0.3 kHz) ¢ltered, ampli¢ed 105 times, digitally averaged and then presented on the computer monitor for comparison. The threshold was de¢ned as the lowest sound intensity level where a visually reproducible waveform with appropriate peak latencies was evident. The stimulus was varied in 5 dB sound pressure level (peak equivalent) increments around the electrophysiological hearing threshold. 2.3.2. Cytocochleograms The temporal bones were removed after euthanasia and the bulla opened under an operating microscope. A qualitative assessment was made of the mucosa and associated parts of the middle ear. The cochleae were perfused with 4% paraformaldehyde in phosphate bu¡er (PFA) through a perforation in the apex and through the opened oval and round windows. They were post¢xed in 4% PFA overnight and stored in bu¡ered 0.5% PFA at 4‡C. Paraformaldehyde was removed by rinsing in phosphate-bu¡ered saline (PBS) and the bone was dissected to free the modiolus. The cells were permeabilised in 0.3% Triton X-100 in PBS for 10 min, followed by careful rinsing in PBS and subsequent staining with 1% TRITC in PBS (tetrametylrhodamine B isothiocyanate, phalloidin, Molecular Probes) for 30 min and one more PBS rinsing. The organ of Corti was dissected free and mounted in Citi-£uor mounting medium. In a Leitz £uorescence microscope, inner (IHC) and outer hair cells (OHC) were counted in discrete steps of 0.19 mm tissue from apex to base. Missing IHCs or OHCs were apparent as dark spots or as typical phalangeal scars formed by the supporting cells. The percentage loss of IHCs and OHCs per 0.19 mm counted distance was calculated by comparison to a previous normative laboratory database. Having obtained the percentage of OHCs lost per row per counted

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a

b 80 Outer hair cell loss, %

Outer hair cell loss, %

80 60 TU treated left ear Untreated right ear

40 20 0

60 AP treated left ear Untreated right ear

40 20 0

0

5 10 15 Distance from apex, mm

20

0

5 10 15 Distance from apex, mm

20

Fig. 1. Cytocochleograms showing left and right ears from TU-treated animals (a) and AP-treated animals (b). Percentage OHC loss per 0.19 mm (mean of rows 1^3 from all cochleae in each group) as a function of counted distance from apex.

distance, the means for rows 1^3 per 0.19 mm counted distance were calculated. After this, the means for the group were calculated per 0.19 mm counted distance. Cytocochleograms were prepared displaying the mean percentage loss of IHCs and OHCs in rows 1^3 per 0.19 mm for all animals in each group as a function of counted distance from the apex. Percentage values were plotted at a distance corresponding to the end of each reticule. The result for the ¢rst 0.19 mm was plotted at 0.19 mm and then successively by 0.19 mm increments towards the base. 2.4. Statistics The Mann^Whitney Rank Sum test was used to compare ABR data at each frequency between groups as well as for the inter-group comparison of weight change. The Wilcoxon Signed Rank test was used to test for signi¢cant weight loss within each group. The signi¢cance of the di¡erence in percentage of missing IHCs and OHCs observed between the ears of each treatment group and between treated ears of the two groups were evaluated using an unpaired t-test. P 6 0.05 was considered signi¢cant. All statistic calculations were made with Sigma Stat1 software (Jandel Corp.).

3. Results 3.1. Dose titration (Part 1) All animals in the group receiving 90 mg/ml TU demonstrated in£ammation of the middle ear mucosa, pos-

sibly caused by TU administered in a concentrated solution. One of the animals receiving 9 mg/ml TU had a minor middle ear reaction. The animals receiving 27 mg/ml TU showed normal middle ear mucosa after 7 days of administration. There was a signi¢cant highfrequency ABR threshold shift in one animal from each group, the highest loss was seen in one animal in the 9 mg/ml TU group. The cytocochleograms from these animals supported the conclusion that the observed ABR threshold shifts were caused by a surgical trauma. A concentration of 27 mg/ml was chosen for the otoprotection study, as this was the highest administered dose, which did not cause observable adverse reactions. 3.2. Otoprotection (Part 2) 3.2.1. General Thirteen out of 20 animals could be evaluated for otoprotection. In the AP group, one animal was excluded due to deterioration in general condition and one had a middle ear infection. Five animals were excluded in the TU group, two animals because of deterioration in general condition and three as they showed ABR threshold elevations following microcannulation of the inner ear. The cytocochleograms from these three animals showed a pattern typical for cochlear lesions from surgical trauma, with abrupt and complete loss of OHCs in the basal region. There was no di¡erence in weight change between the two groups and no di¡erence within groups as compared to baseline. The weight gain 120 h after cisplatin administration was 2 M 12.9 g and 2 M 11.2 g (mean M S.E.M.) for the TU and AP group, respectively.

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3.2.2. ABR Among animals receiving TU, the mean ABR threshold shift after cisplatin administration was 22 M 13 dB at 16 kHz, 14 M 9.9 dB at 12 kHz and 1 M 1 dB at 4 kHz (mean M S.E.M.). Among animals given AP, the average threshold shift was 33 M 9.4 dB at 16 kHz, 35 M 10 dB at 12 kHz and 14 M 5.7 dB at 4 kHz (mean M S.E.M.). While there was a tendency for a greater threshold shift in the APtreated animals, particularly at 4 kHz, the di¡erence between TU- and AP-treated ears in threshold shift did not reach the level of signi¢cance (P s 0.09). 3.2.3. Cytocochleograms Fig. 1a shows the mean cytocochleograms from TUtreated animals and Fig. 1b mean cytocochleograms from AP-treated animals. TU-treated ears demonstrated signi¢cantly (P 6 0.05) lower OHC loss compared to the untreated ears. Visually, there was a clear di¡erence in favour of the treated ear with almost no OHC loss on the treated side in four out of ¢ve animals. In the ¢fth animal the loss of OHCs was similar on both sides. Moreover, the TU-treated ears had signi¢cantly (P 6 0.05) less OHC loss compared to the APtreated ears. There was no signi¢cant di¡erence between treated and untreated ears in the AP group which had generally bilateral symmetric loss of OHCs, although seemingly somewhat greater in the AP-treated ears. Furthermore, there was no di¡erence between untreated ears in the TU and AP groups. In no ear did we ¢nd a signi¢cant loss of IHCs.

4. Discussion Several studies indicate that systemic administration of thiol-containing agents attenuate cisplatin-induced ototoxicity, administration of which, however, may lead to a partial reduction of the antineoplastic e¡ectiveness of cisplatin. Thus, it would appear that systemic protection, although e¡ective in preserving hearing, has a rather limited value in clinical oncology. Hence, a search for alternative techniques of pharmacologic otoprotection is warranted. In the present study, perilymphatic infusion of TU resulted in an attenuation of the ototoxicity of cisplatin, as demonstrated by the reduction in drug-induced OHC loss. TU did not eliminate the cisplatin-induced hearing loss, but there was a promising tendency for reduced ABR threshold shifts 5 days after cisplatin injection. The dose response study of TU showed that a TU concentration of 90 mg/ml produced a local in£ammatory reaction. This reaction was absent when lower concentrations were used. Although the in£ammatory reaction might be due to a speci¢c action of the

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compound at high concentrations on the middle ear, the reaction may also re£ect a general tissue reaction to extended exposure to a hypertonic solution. From previous studies, we know that cisplatin ototoxicity is clearly evident 4 days following cisplatin administration (Laurell and Bagger-Sjoback, 1991; Laurell and Engstrom, 1989). At this time permanent OHC damage has appeared, while mortality is still limited without nephroprotective measures (Ekborn et al., 2000). Pre-treatment infusion of TU was chosen to allow the TU concentration to reach a high and stable level in cochlear £uids and tissues prior to cisplatin administration. Moreover, by implanting the pump 3 days before cisplatin injection, ears with surgically induced hearing loss could be detected and excluded. By using hydration with normal saline to limit renal damage, animal mortality was reduced and the observation period could be extended to 5 days. The high-frequency loss and progressive bilateral symmetric loss of OHCs in the basal turn observed in unprotected ears demonstrated expected characteristics for ototoxic insult and were similar to what we have previously seen for 8 mg/kg b.w. cisplatin (Ekborn et al., 2002; Komune et al., 1981). In TU-protected ears there was almost no OHC damage. Although the OHCs were protected in these ears, cisplatin-induced elevations of electrophysiological hearing thresholds were found. It is possible that temporary damage to the stria vascularis may explain the lack of di¡erence in ABR thresholds between AP- and TU-protected ears. A cisplatin dose of 8 mg/kg b.w. does lead to depression of the endocochlear potential (EP) in the GP (Laurell and Engstrom, 1989). Partial recovery from ototoxicity induced by multiple low-dose cisplatin has been observed in the GPs and attributed to normalisation of the EP and strial function but limited by OHC loss (Klis et al., 2002). This indicates that the protection of OHCs observed in our study may prove clinically of major signi¢cance, even in the presence of short-term ABR depression. A future study should look at possible longterm recovery of hearing. Such a study would help to address the alternative interpretation of these results: that TU prevented OHC death, but that the protection was not su⁄cient to maintain normal OHC function. If TU provided protection, the selective protection of the OHCs by TU might be due to the method of administration. By round window administration TU is found to reach the stria vascularis and OHCs (Laurell et al., 2002). However, it can be speculated that the amount of TU reaching the stria vascularis by di¡usion from the perilymph will be small compared to the amount of cisplatin reaching the vascularised lateral wall through the circulation after i.v. administration, whereas the amounts reaching the OHCs may be comparable as both substances likely reach this target by di¡usion.

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An additional future study should evaluate the distribution of TU throughout the various cochlear tissues when administered directly to the perilymph. Antioxidants are showing e⁄cacy in prevention of drug-induced hearing loss, both from cisplatin and gentamicin (Campbell et al., 1996; Sha and Schacht, 2000). Indeed, antioxidants have recently shown e⁄cacy in the attenuation of noise-induced hearing loss, signi¢cantly protecting OHCs and electrophysiological responsiveness (Miller et al., 2001). Cisplatin ototoxicity is associated with oxidative damage and generation of reactive oxygen species (Rybak and Somani, 1999). There is also a di¡erential susceptibility to oxidative damage in vitro among di¡erent OHC populations in the cochlea, which parallels the observed di¡erential sensitivity to cisplatin and other ototoxic agents (Sha et al., 2001), including damaging noise exposure (Ohinata et al., 2000), providing a rationale for the use of antioxidants such as TU for otoprotection. TU is not currently used in the clinic. One drawback is TU’s reported genotoxicity and it has been found to cause tumours of the thyroid, liver and the Zymbal glands of rats when administered in the diet for extended periods of time, although there are no adequate human data (IARC v.7, 1974). This dictates care in the use of TU for local otoprotection but does not per se exclude its future use. TU dosage levels, routes and times of administration could likely be entirely di¡erent in humans, and potentially structured to avoid systemic exposure. Moreover, the candidate patients already su¡er from a malignant disease and are receiving chemotherapy with an agent, which in itself is genotoxic and may cause malignancies. Local administration of an otoprotective substance o¡ers the tantalising prospect of avoiding the possible systemic interaction between drug and protector. The more protected position of the inner ear with a rather thick round window membrane in humans (Goycoolea, 2001) is an obstacle that has to be overcome to ensure the successful outcome of clinical trials with round window administration of protective substances. Two principal routes of administration exist for local treatment of the inner ear. Middle ear administration is already used in the clinic for the treatment of Meniere’s disease (Driscoll et al., 1997; Harner et al., 2001; Ho¡er et al., 2001). The other possibility is direct intracochlear administration, a method as yet unavailable in humans. This route of administration was used in the present study to ensure that TU reached the inner ear. However, a protective e¡ect may be overcome by damage to the inner ear associated with surgery of the kind used in the present study. Unless less traumatic surgical methods are developed, this mode of administration is less likely to be applied in clinical use. No matter which method is used, direct intracochlear administration or middle ear administration, the drug has to reach its

target by di¡usion favouring the use of a small uncharged molecule such as TU. Importantly, the antioxidant e⁄cacy of TU makes it of potential value in attenuating hearing impairment induced by a number of environmental and physiological stress factors, from drugs through noise to ageing, now thought to contribute to acquired deafness.

Acknowledgements Ms Nadine Brown is gratefully acknowledged for excellent technical help with the evaluation of cochlear pathology. This study was ¢nancially supported by: Helga Hjerpstedt Foundation, Foundation Tysta Skolan, Swedish council for working life and social research (FAS 2001-0171) and Swedish Foundation for International Cooperation in Research and Higher Education (STINT 98-905). J.M. was partially supported by General Motors and United Automotive Workers.

References Aamdal, S., Fodstad, O., Pihl, A., 1987. Some procedures to reduce cis-platinum toxicity reduce antitumour activity. Cancer Treat. Rev. 14, 389^395. Aamdal, S., Fodstad, O., Pihl, A., 1988. Sodium thiosulfate fails to increase the therapeutic index of intravenously administered cisdiamminedichloroplatinum (II) in mice bearing murine and human tumors. Cancer Chemother. Pharmacol. 21, 129^133. Berry, J.M., Jacobs, C., Sikic, B., Halsey, J., Borch, R.F., 1990. Modi¢cation of cisplatin toxicity with diethyldithiocarbamate. J. Clin. Oncol. 8, 1585^1590. Bokemeyer, C., Fels, L.M., Dunn, T., Voigt, W., Gaedeke, J., Schmoll, H.J., Stolte, H., Lentzen, H., 1996. Silibinin protects against cisplatin-induced nephrotoxicity without compromising cisplatin or ifosfamide anti-tumour activity. Br. J. Cancer 74, 2036^2041. Brown, J.N., Miller, J.M., Altschuler, R.A., Nutall, A.L., 1993. Osmotic pump implant for chronic infusion of drugs into the inner ear. Hear. Res. 70, 167^172. Campbell, K.C., Rybak, L.P., Meech, R.P., Hughes, L., 1996. D-methionine provides excellent protection from cisplatin ototoxicity in the rat. Hear. Res. 102, 90^98. Cooper, J.S., Guo, M.D., Herskovic, A., Macdonald, J.S., Martenson Jr, J.A., Al-Sarraf, M., Byhardt, R., Russel, A.H., Beitler, J.J., Spencer, S., Asbell, S.O., Graham, M.V., Leichman, L.L., 1999. Chemoradiotherapy of locally advanced esophageal cancer: longterm follow-up of a prospective randomized trial (Radiation Therapy Oncology Group RTOG 85-01). JAMA 281, 1623^1627. Driscoll, C.L., Kasperbauer, J.L., Facer, G.W., Harner, S.G., Beatty, C.W., 1997. Low-dose intratympanic gentamicin and the treatment of Meniere’s disease: preliminary results. Laryngoscope 107, 83^89. Ekborn, A., Laurell, G., Andersson, A., Wallin, I., Eksborg, S., Ehrsson, H., 2000. Cisplatin-induced hearing loss: in£uence of the mode of drug administration in the guinea pig. Hear. Res. 140, 38^44. Ekborn, A., Laurell, G., Johnstro«m, P., Wallin, I., Eksborg, S., Ehrsson, H., 2002. D-methionine and cisplatin ototoxicity in the guinea

HEARES 4722 26-6-03

A. Ekborn et al. / Hearing Research 181 (2003) 109^115 pig: D-methionine in£uences cisplatin pharmacokinetics. Hear. Res. 165, 53^61. Fichtinger-Schepman, A.M., van Dijk-Knijnenburg, H.C., Dijt, F.J., van der Velde-Visser, S.D., Berends, F., Baan, R.A., 1995. E¡ects of thiourea and ammonium bicarbonate on the formation and stability of bifunctional cisplatin-DNA adducts: consequences for the accurate quanti¢cation of adducts in (cellular) DNA. J. Inorg. Biochem. 58, 177^191. Goycoolea, M.V., 2001. Clinical aspects of round window membrane permeability under normal and pathological conditions. Acta Otolaryngol. (Stockh.) 121, 437^447. Grigsby, P.W., Herzog, T.J., 2001. Current management of patients with invasive cervical carcinoma. Clin. Obstet. Gynecol. 44, 531^ 537. Harner, S.G., Driscoll, C.L., Facer, G.W., Beatty, C.W., McDonald, T.J., 2001. Long-term follow-up of transtympanic gentamicin for Meniere’s syndrome. Otol. Neurotol. 22, 210^214. Ho¡er, M.E., Kopke, R.D., Weisskopf, P., Gottshall, K., Allen, K., Wester, D., Balban, C., 2001. Use of the round window microcatheter in the treatment of Meniere’s disease. Laryngoscope 111, 2046^2049. Kaltenbach, J.A., Church, M.W., Blakley, B.W., McCaslin, D.L., Burgio, D.L., 1997. Comparison of ¢ve agents in protecting the cochlea against the ototoxic e¡ects of cisplatin in the hamster. Otolaryngol. Head Neck Surg. 117, 493^500. Klis, S.F.L., O’Leary, S.J., Wijbenga, J., de Groot, J.C.M.J., Hamers, F.P.T., Smoorenburg, G.F., 2002. Partial recovery of cisplatininduced hearing loss in the albino guinea pig in relation to cisplatin dose. Hear. Res. 164, 138^146. Kohonen, A., Tarkkanen, J., 1969. Cochlear damage from ototoxic antibiotics by intratympanic application. Acta Otolaryngol. (Stockh.) 68, 90^97. Komune, S., Asakuma, S., Snow, J.B.J., 1981. Pathophysiology of the ototoxicity of cis-diamminedichloroplatinum. Otolaryngol. Head Neck Surg. 89, 275^282. Laurell, G., Bagger-Sjoback, D., 1991. Dose-dependent inner ear changes after i.v. administration of cisplatin. J. Otolaryngol. 20, 158^167. Laurell, G., Engstrom, B., 1989. The ototoxic e¡ect of cisplatin on guinea pigs in relation to dosage. Hear. Res. 38, 27^33. Laurell, G., Jungnelius, U., 1990. High-dose cisplatin treatment: hearing loss and plasma concentrations. Laryngoscope 100, 724^734. Laurell, G., Teixeira, M., Sterkers, O., Bagger-Sjoback, D., Eksborg, S., Lidman, O., Ferrary, E., 2002. Local administration of antioxidants to the inner ear. Kinetics and distribution (1). Hear. Res. 173, 198^209. Li, G., Frenz, D.A., Brahmblatt, S., Feghali, J.G., Ruben, R.J., Berggren, D., Arezzo, J., Van De Water, T.R., 2001. Round window membrane delivery of L-methionine provides protection from cisplatin ototoxicity without compromising chemotherapeutic e⁄cacy. Neurotoxicology 22, 163^176.

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Lokich, J., 2001. What is the ‘best’ platinum: cisplatin, carboplatin, or oxaliplatin? Cancer Invest. 19, 756^760. Miller, J., Schacht, J., Altschuler, R., 2001. Prevention of noise-induced hearing loss. In: Henderson, D., Prasher, D., Kopke, R., Salvi, R., Hamernik, R. (Eds.), Noise Induced Hearing Loss: Basic Mechanisms, Prevention and Control. Network Publications, London, pp. 215^230. Ohinata, Y., Miller, J.M., Altschuler, R.A., Schacht, J., 2000. Intense noise induces formation of vasoactive lipid peroxidation products in the cochlea. Brain Res. 878, 163^173. Otto, W.C., Brown, R.D., Gage-White, L., Kupetz, S., Anniko, M., Penny, J.E., Henley, C.M., 1988. E¡ects of cisplatin and thiosulfate upon auditory brainstem responses of guinea pigs. Hear. Res. 35, 79^85. Piccart, M.J., Lamb, H., Vermorken, J.B., 2001. Current and future potential roles of the platinum drugs in the treatment of ovarian cancer. Ann. Oncol. 12, 1195^1203. Prieskorn, D.M., Miller, J.M., 2000. Technical report: chronic and acute intracochlear infusion in rodents. Hear. Res. 140, 212^215. Riley, C.M., Sternson, L.A., Repta, A.J., Slyter, S.A., 1982. Reactivity of cis dichlorodiammineplatinum(II) (cisplatin) towards selected nucleophiles. Polyhedron 1, 201^202. Rybak, L.P., Somani, S., 1999. Ototoxicity. Amelioration by protective agents. Ann. N.Y. Acad. Sci. 884, 143^151. 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., Husain, K., Morris, C., Whitworth, C., Somani, S., 2000. E¡ect of protective agents against cisplatin ototoxicity. Am. J. Otol. 21, 513^520. Saito, T., Zhang, Z.J., Manabe, Y., Ohtsubo, T., Saito, H., 1997. The e¡ect of sodium thiosulfate on ototoxicity and pharmacokinetics after cisplatin treatment in guinea pigs. Eur. Arch. Otorhinolaryngol. 254, 281^286. Sha, S.H., Schacht, J., 2000. Antioxidants attenuate gentamicin-induced free radical formation in vitro and ototoxicity in vivo: Dmethionine is a potential protectant. Hear. Res. 142, 34^40. Sha, S.H., Taylor, R., Forge, A., Schacht, J., 2001. Di¡erential vulnerability of basal and apical hair cells is based on intrinsic susceptibility to free radicals. Hear. Res. 155, 1^8. Valentini, V., Coco, C., Cellini, N., Picciocchi, A., Fares, M.C., Rosetto, M.E., Mantini, G., Morganti, A.G., Barbaro, B., Cogliandolo, S., Nuzzo, G., Tedesco, M., Ambesi-Impiombato, F., Cosimelli, M., Rotman, M., 2001. Ten years of preoperative chemoradiation for extraperitoneal T3 rectal cancer: acute toxicity, tumor response, and sphincter preservation in three consecutive studies. Int. J. Radiat. Oncol. Biol. Phys. 51, 371^383. Yonezawa, Y., Kawamura, S., Yamoto, M., Nishioka, H., 2001. Muttest to detect substances suppressing spontaneous mutation due to oxidative damage. Mutat. Res. 490, 21^26.

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