Mechanisms of cisplatin-induced ototoxicity and prevention

Hearing Research

Hearing Research 226 (2007) 157–167

www.elsevier.com/locate/heares

Review article

Mechanisms of cisplatin-induced ototoxicity and prevention Leonard P. Rybak a

a,b,*

, Craig A. Whitworth b, Debashree Mukherjea b, Vickram Ramkumar b

Department of Surgery, Division of Otolaryngology, Southern Illinois University, School of Medicine, P.O. Box 19653, Springfield, IL 62794-9653, USA b Department of Pharmacology, Southern Illinois University, School of Medicine, Springfield, IL, USA Received 4 April 2006; received in revised form 9 September 2006; accepted 24 September 2006 Available online 17 November 2006

Abstract Cisplatin is a widely used chemotherapeutic agent to treat malignant disease. Unfortunately, ototoxicity occurs in a large percentage of patients treated with higher dose regimens. In animal studies and in human temporal bone investigations, several areas of the cochlea are damaged, including outer hair cells in the basal turn, spiral ganglion cells and the stria vascularis, resulting in hearing impairment. The mechanisms appear to involve the production of reactive oxygen species (ROS), which can trigger cell death. Approaches to chemoprevention include the administration of antioxidants to protect against ROS at an early stage in the ototoxic pathways and the application of agents that act further downstream in the cell death cascade to prevent apoptosis and hearing loss. This review summarizes recent data that shed new light on the mechanisms of cisplatin ototoxicity and its prevention. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Cisplatin; Reactive oxygen species; Apoptosis; NADPH; Oxidase

1. Introduction Cisplatin is a highly effective chemotherapeutic agent that is widely used to treat a variety of soft tissue neoplasms, including ovarian, testicular, cervical, head and neck, lung and bladder cancer. Serious side effects include nephrotoxicity, neurotoxicity and ototoxicity. In order to effect cures, the dosing of cisplatin has been increased in recent treatment protocols. Some audiometric studies have reported elevated hearing thresholds in 75–100% of Abbreviations: AAV, adeno-associated virus; ABR, auditory brainstem response; AUC, area under the curve; BK, big conductance potassium; EP, endocochlear potential; CAP, compound action potential; CM, cochlear microphonic; GFP, green fluorescent protein; HMG1, high mobility group; iNOS, inducible nitric oxide synthase; KIM-1, kidney injury molecule; NOX-3, isoform of NADPH oxidase; ROS, reactive oxygen species; TUNEL, terminal nucleotidyl transferase-mediated dUTP-biotin nicke end-labelling; XIAP, the X-linked inhibitor of apoptosis protein * Corresponding author. Tel.: +1 217 545 2598; fax: +1 217 545 2588. E-mail addresses: [email protected] (L.P. Rybak), [email protected] (C.A. Whitworth), [email protected] (D. Mukherjea), [email protected] (V. Ramkumar). 0378-5955/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.09.015

patients treated with cisplatin (McKeage, 1995). This is particularly problematic in children receiving cisplatin. Risk factors that increase the risk for ototoxicity from cisplatin in children include: younger age, larger cumulative doses, pre-existing hearing loss and renal disease (Li et al., 2004; Knight et al., 2005) and irradiation of the brain or skull base (Chen et al., 2006). Ototoxicity of cisplatin can be reduced by various protective agents. This paper reviews recent research findings that provide new insights into the mechanisms for cisplatin ototoxicity and the effects of various protective agents that may ameliorate cisplatin ototoxicity. 2. Effects on cochlear function Adverse effects of cisplatin on auditory function have been documented in numerous reports. Ototoxicity has been demonstrated in animal experiments by reduction of the endocochlear potential (EP) (Ravi et al., 1995; Tsukasaki et al., 2000; Klis et al., 2000) and elevation of the thresholds for both the compound action potential (CAP)

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and cochlear microphonic (CM) after ototoxic doses of cisplatin. The CAP amplitude is reduced to a greater extent than the (CM) amplitude. The greater effect of cisplatin on the CAP threshold may result from several changes induced within the cochlea. These include detachment of the myelin sheath of spiral ganglion cells, alteration of hair cell function and reduction of the EP. This would explain why cisplatin-treated guinea pigs can exhibit a large discrepancy between the elevation of CAP threshold in the presence of low CM thresholds. Thus, the CAP threshold shift cannot be explained completely by the changes in CM threshold (Van Ruijven et al., 2005a). Distortion product otoacoustic emissions are diminished in cisplatin-treated gerbils (Alam et al., 2000). Auditory brainstem responses in cisplatin-treated animals demonstrate increased thresholds, with greatest effects in the higher frequencies (Rybak et al., 2000). 3. Effects on cochlear morphology Cisplatin ototoxicity has been shown to have at least three major tissue targets in the cochlea: organ of Corti, spiral ganglion cells and lateral wall (stria vascularis and spiral ligament). Studies in guinea pig reveal that cisplatin affects both the organ of Corti (primarily the outer hair cells) and the spiral ganglion cells (Van Ruijven et al., 2005a). Type I spiral ganglion cells undergo detachment of their myelin sheaths. The time sequence of damage to spiral ganglion cells and outer hair cells in guinea pig follow a similar time course, suggesting that injury to both cochlear areas occurred in parallel, rather than sequentially (Van Ruijven et al., 2005a). Cisplatin ototoxicity in rats was manifested by deleterious effects on the basal turn stria vascularis, including strial edema, bulging, rupture and compression of the marginal cells and depletion of organelles from the cytoplasm (Meech et al., 1998). Guinea pigs allowed to recover for more than four weeks after cisplatin administration demonstrated shrinkage in the area of the stria, even though the EP had recovered by this time. This shrinkage was caused by a decrease in the intermediate cell area and, to a lesser extent, by a decrease in the marginal cell area (Sluyter et al., 2003). TUNEL staining was used to detect DNA damage (breaks in double-stranded DNA) in the cochlea of cisplatin-treated gerbils. TUNEL is an acronym for terminal nucleotidyl transferase-mediated dUTP-biotin nick end-labelling and represents the in situ end-labelling or transfer of biotinylated nucleotide to the 3’’-OH end of DNA. It is frequently used as a marker for apoptotic cell death. Positive TUNEL staining indicates that the cell nucleus is undergoing degeneration and likely undergoing apoptosis. Additional staining with Hoechst 33432 is used to identify cells undergoing apoptosis. Cell stained with the latter that demonstrate pyknotic and condensed nuclei are classified as apoptotic (Alam et al., 2000). Apoptosis of cells in the organ of Corti, primarily the

outer hair cells, and spiral ganglion cells in the basal turn of the gerbil cochlea occurred after cisplatin administration. On the other hand, the stria vascularis demonstrated TUNEL-positive staining in all three turns (Alam et al., 2000). The ototoxic effect of cisplatin on lateral wall tissues in the gerbil was further substantiated with in vitro studies of the spiral ligament. Gerbil type I spiral ligament cells also undergo significant apoptosis after cisplatin exposure in cell culture. This was caused by cisplatin blocking BK channels (Liang et al., 2005). Administration of a marginally ototoxic dose of cisplatin (10 mg/kg) resulted in positive TUNEL staining in the stria vascularis of guinea pigs; however, no TUNEL staining was detected in hair cells. This dose of cisplatin was probably not sufficient to cause hair cell death (Watanabe et al., 2003). Platinated DNA was immunolocalized to the nuclei of outer hair cells, supporting cells of the organ of Corti, marginal cells of the stria vascularis and the cells in the spiral ligament of the basal turn. This was demonstrated using a polyclonal rabbit serum containing antibodies that recognize cisplatin–DNA adducts. These antibodies also stained samples of renal cortex, which served as positive controls (Van Ruijven et al., 2005b). This finding confirms that these cells are targets for cisplatin toxicity. However, a more recent study by Thomas et al. (2006) demonstrated immunostaining for platinum–DNA adducts primarily in the marginal cells of the stria vascularis. In contrast to the study by Van Ruijven et al. (2005b), the investigation by Thomas et al. (2006) showed no specific accumulation of cisplatin–DNA adducts. The discrepancies between the findings in these two studies in the guinea pig could be attributed to the dosing protocol used, the antibodies used to detect platinated DNA in the tissues and the quality of tissue preservation after cisplatin. Van Ruijven et al. (2005b) employed albino Dunkin– Hartley guinea pigs and administered cisplatin at a dose of 2 mg/kg by intraperitoneal (i.p.) injection five times per week for a period of two weeks, resulting in a cumulative dose of 20 mg/kg, and sacrificed the animals 48 h after the last dose. Thomas et al. (2006) treated pigmented guinea pigs of the same strain with 12.5 mg/kg i.p. and sacrificed the animals at 8, 24 or 48 h after cisplatin injection. A polyclonal antibody was used by Van Ruijven et al. (2005b) as opposed to a monoclonal antibody directed against platinum–guanine–guanine intrastrand crosslinks. The polyclonal antiserum also contained antibodies that react with other nuclear antigens besides cisplatin– DNA adducts. The polyclonal antiserum was preabsorbed with a homogenate of non-treated guinea pig kidneys prior to immunostaining the cochlea (Van Ruijven et al., 2005b). Because of mild fixation and extensive proteolytic digestion, the hair cells were less well preserved in the study by Thomas et al. (2006) preventing a systematic evaluation of these cells. These studies should be repeated using monoclonal antibodies with better tissue preservation of the organ of Corti.

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4. Biochemical and molecular effects Cisplatin interacts with cochlear tissue explants to generate reactive oxygen species (ROS) (Clerici et al., 1995, 1996; Kopke et al., 1997) such as superoxide anion (Dehne et al., 2001; Banfi et al., 2004). Cochlear tissues from animals receiving ototoxic doses of cisplatin were depleted of glutathione and antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase and glutathione reductase) with a reciprocal increase in malondialdehyde levels, an indicator of lipid peroxidation (Rybak et al., 2000). The lipid peroxidation may be a consequence of impaired activities of antioxidant enzymes and glutathione reductase as well as depletion of glutathione in cochlear tissues (Fig. 1). Depletion of cochlear antioxidant enzyme activities may result from: (1) direct binding of cisplatin to essential sulfhydryl groups within the enzymes; (2) depletion of copper and selenium, which are essential for superoxide dismutase and glutathione peroxidase activities (DeWoskin and Riviere, 1992); (3) increased ROS and organic peroxides which inactivate antioxidant enzymes (Pigeolet et al., 1990); and (4) depletion of glutathione and the cofactor NADPH, which are essential for the activities of glutathione peroxidase and glutathione reductase activities (Somani et al., 2001). Inhibition of antioxidant enzyme activity by cisplatin can allow ROS, such as superoxide and hydrogen peroxide, and toxic lipid peroxides to increase within the cochlea. This can lead to calcium influx within cochlear cells, leading to apoptosis (Clerici et al., 1995; Ikeda et al., 1993). Cisplatin induces apoptosis in spiral ligament fibrocytes of the lateral wall by activation of the potassium channels, leading to potassium efflux, reducing intracellular ionic and osmotic strength, which can trigger apoptosis by activating pro-apoptotic enzymes, such as caspases and pro-apoptotic nucleases. The resulting apoptosis causes cellular losses in the spiral ligament, leading to a reduction of potassium transport and diminution of EP generation in the stria vascularis. The average voltage activation threshold of whole cell current was sharply shifted to 40 mV in the cisplatin-treated cells as compared with a value of 40 mV in CISPLATIN DEPLETES ANTIOXIDANT SYSTEM IN COCHLEA SOD

Lipid Peroxidation

CAT GSH

CISPLATIN GSH-Px

GSSG

GR

Cochlea Fig. 1. Cisplatin depletes the antioxidant system in the cochlea. Cisplatin treatment leads to the depletion of glutathione and the antioxidant enzymes, allowing an increase in lipid peroxidation (Somani et al., 2001).

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control cells. The average whole-cell current of cisplatintreated cells induced by a depolarization voltage step from 80 to 10 mV was increased to 1.2 ± 0.4 nA as compared to 0.08 ± 0.1 nA in control cells. Co-incubation with tetraethylammonium and cisplatin retained the whole cell current in the normal range. The cisplatin-activated whole cell current in these cells is highly sensitive to iberiotoxin. This apoptosis is reduced by application of inhibitors of the calcium- and voltage-dependent big conductance potassium (BK) channels. Inhibition of the BK channels by tetraethylammonium significantly reduced cisplatin-induced apoptosis in these cells (Liang et al., 2005). Persistent activation of potassium conductance would lead to potassium efflux, resulting in a reduction of intracellular potassium. This would cause a decrease in the intracellular osmotic and ionic strength, triggering pro-apoptotic enzymes, leading to cell death (Bortner and Cidlowski, 1998). Low intracellular potassium concentration facilitates the proteolytic activity of caspases and pro-apoptotic nucleases. On the other hand, physiological intracellular potassium levels inhibit their activities (Hughes et al., 1997; Liang et al., 2005). Potassium channel blockers significantly reduce apoptotic cell shrinkage, caspase-3 activation, cytochrome c release, DNA laddering and apoptotic morphological changes in various cells in vitro (Maeno et al., 2000; Liang et al., 2005). It remains to be demonstrated whether membrane potassium conductance is involved in apoptosis of the inner ear cell types injured by cisplatin (Liang et al., 2005). RT-PCR has been used to demonstrate that NOX-3, an isoform of NADPH oxidase, is highly expressed in the mammalian inner ear. This enzyme generates low levels of ROS in a subunit independent manner, but high levels in the presence of activator and organizer subunits. RNA for this enzyme was demonstrated using in situ hybridization in the mouse cochlea. When the enzyme was expressed in embryonic kidney cells, and the cells were exposed to cisplatin, there was a marked increase in superoxide production (Banfi et al., 2004). Our laboratory has demonstrated the presence of NOX-3 mRNA in the cochlea of rats and in three hair cell lines derived from the Immortomouse (OC-k3, HEI-OC1 and UB/OC-1 cells). This enzyme was up-regulated following systemic cisplatin administration in the rat (Fig. 2) and after in vitro cisplatin application in these hair cell lines. Induction of NOX-3 increases superoxide production in the cochlea and forms part of the pathway leading to cisplatin-mediated damage (Mukherjea et al., 2006). Superoxide anion may be generated from other sources bedsides NOX-3 or other NADPH oxidase isoforms. Superoxide radicals generated by cisplatin exposure (Dehne et al., 2001) lead to the formation of hydrogen peroxide. Hydrogen peroxide is catalyzed by iron to form the highly reactive hydroxyl radical, which interacts with polyunsaturated fatty acids in membranes to form the highly toxic aldehyde, 4-hydroxynonenal. Superoxide reacts with nitric oxide to form peroxynitrite, which in turn reacts with proteins to form

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these aldehydes, 4-hydroxynonenal, functions as a mediator of apoptosis for both auditory neurons and hair cells (Huang et al., 2000). In the stria of cisplatin-treated ani-

Nox-3 expression in Cisplatin treated rat cochlea Fold increase

3 2.5 2 1.5 1 0.5 0 Control

24 hr

48 hr

72 hr

Fig. 2. Changes in the expression of NOX-3 in the cochlea of cisplatintreated rats. Cochleae were removed at 24, 48 and 72 h and compared to control NOX-3 expression using RT-PCR. A marked increase in the expression of this enzyme was noted at each time point, with the peak expression occurring 48 h after cisplatin administration (Mukherjea et al., 2006).

nitrotyrosine. Cisplatin-treated mice show 4-hydroxynonenal and peroxynitrite immunoreactivity in the cochlea. Cochlear hair cells demonstrated immunoreactivity for 4hydroxynonenal but not for nitrotyrosine, whereas auditory neurons immunostained for both nitrotyrosine and 4-hydroxynonenal after cisplatin administration. These findings indicate that the hydroxyl radical plays a critical role in cisplatin-induced outer hair cell degeneration and hearing loss (Lee et al., 2004a). Generation of excessive ROS would overwhelm the antioxidant defense mechanisms of the cochlea, causing a cascade resulting in apoptosis of hair cells. Activation and redistribution of Bax in the cytosol leads to the release of cytochrome c from injured mitochondria. Cytochrome c can then activate caspase-9 and caspase-3; resulting in the breakdown of DNA by caspase-activated deoxyribonuclease (Watanabe et al., 2003); and the cleavage of fodrin within the cuticular plate of injured hair cells by caspase-3 (Wang et al., 2004). Hair cell death would follow (Fig. 3a). The interaction of reactive oxygen species and other free radicals with membrane phospholipids of auditory sensory cells creates aldehydic lipid peroxidation products. One of

c Fig. 3. Schematic representation of cisplatin in various portions of the cochlea. (a) Outer hair cells. Cisplatin triggers an increase in the expression of NOX-3, generating superoxide radicals (Mukherjea et al., 2006). Superoxide can then form hydrogen peroxide which reacts with iron to form the highly reactive hydroxyl radical. The latter can then react with polyunsaturated fatty acids (PUFA) in the cell membrane to generate the highly toxic aldehyde 4-hydroxynonenal (4-HNE). The latter can then trigger an apoptotic cascade resulting in cell death Lee et al., 2004a,b. (b) Cisplatin interaction with the tissues of the lateral wall leads to activation of NFkB. This transcription factor can induce the formation of nitric oxide (NO) by iNOS. NO can then react with superoxide to form the highly toxic peroxynitrite radical, which can activate caspase-3. The latter enzyme can, in turn, activate caspase-activated deoxyribonuclease in the stria vascularis and spiral ligament, leading to apoptosis of cells in these tissues (Watanabe et al., 2003; Lee et al., 2004b; Liang et al., 2005). (c) Interaction of cisplatin with spiral ganglion cells. Cisplatin increases the expression of the high mobility group (HMG1) protein, which induces iNOS, leading to NO production, and subsequent cell death. ROS can react with PUFA to generate 4-HNE, leading to cell death in this tissue (Lee et al., 2004a; Li et al., 2006).

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mals, the onset of apoptosis coincided with increased immunolabelling of 4-hydroxynonenal, nitrotyrosine and inducible nitric oxide synthase (iNOS) (Lee et al., 2004b). Reactive nitrogen species, such as NO, may also play a role in cisplatin ototoxicity. Increased NO levels have been found in rat whole cochlear extracts after treatment with cisplatin (Kelly et al., 2003). However, other studies utilized cochlear fractions to more closely examine the role of NO in cisplatin-induced ototoxicity. Cells in the stria vascularis appear to respond differently to cisplatin than do outer hair cells. Cisplatin-treated guinea pigs displayed immunostaining for caspase-3 and caspase-activated deoxyribonuclease in the stria vascularis and spiral ligament but not in the hair cells (Watanabe et al., 2003) (Fig. 3b). Cisplatin-treated mice show increased immunolabelling for nuclear factor kappa B (NF-kB) and iNOS in the stria vascularis and spiral ligament. One would predict that up-regulation of NF-kB would induce iNOS expression in these tissues, leading to NO production and auditory dysfunction. However, in auditory hair cells, NF-kB promotes cell survival during development (Nagy et al., 2005). Adult rodents pretreated with protective agents prior to aminoglycoside exposure exhibited increased expression of NF-kB in basal turn outer hair cells, accompanied by increased survival of these cells (Jiang et al., 2006). NF-kB also protects Type II spiral ganglion cells from ouabain-induced apoptosis (Lang et al., 2005). Type II spiral ganglion neurons displayed high levels of NFkB immunoreactivity. These neurons have been shown to have some selective survival advantage over the Type I neurons in the cat after ototoxic lesions of the organ of Corti (Lang et al., 2005). NFkB also prevents auditory nerve degeneration in noise-exposed and aging mice (Lang et al., 2006). Mice deficient in the p50 subunit demonstrated increased sensitivity to low level noise exposure and accelerated hearing loss with age (Lang et al., 2006). Only the stria vascularis demonstrated immunoreactivity for single-stranded DNA. Thus, apoptosis in the stria vascularis was triggered by NO generated by iNOS and this enzyme appears to mediate otototoxicity of cisplatin in the stria vascularis (Watanabe et al., 2002). Binding of high mobility group (HMG1) protein to DNA is associated with the antitumor actions of cisplatin (Ohndorf et al., 1999). Increased expression of HMG1 and binding of this protein to DNA in target tissues sensitizes them to cisplatin cytotoxicity (He et al., 2000; Li et al., 2006). HMG1 expression in modiolar extracts from untreated control rats containing spiral ganglion cells demonstrated a much greater level of HMG1 protein expression on Western blot analysis compared with cardiac tissue. However, kidney tissue from untreated control rats had even greater levels of HMG1 protein than did modiolar extracts (Li et al., 2006). Cisplatin-treated rats displayed marked up-regulation of HMG1 in basal turn spiral ganglion cells and in kidney tubules but not in cardiac tissues (Li et al., 2006). It would

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be interesting to determine whether HMG1 is expressed in the cells of the organ of Corti, particularly in the outer hair cells, since these cells are the predominant targets of cisplatin-induced ototoxicity. However, in the study Li et al. (2006) only kidney, cardiac tissue and the spiral ganglion cells in the cochlea were studied for HMG1 expression in control and cisplatin-treated rats. It would be very interesting to examine the organ of Corti and the lateral wall tissues to see whether HMG1 is expressed and whether this protein is up-regulated following cisplatin treatment. This is particularly relevant, since the spiral ganglion cells are more resistant to cisplatin-induced cell death in vivo than are the outer hair cells, stria vascularis and spiral ligament (Van Ruijven et al., 2005b). Until the hair cells and cells of the lateral wall of the cochlea are tested for the expression of HMG1 in control and cisplatin-treated animals, the relevance of these findings are of questionable value in determining the relative sensitivity of the cells of the cochlea to this ototoxic agent. Western blot analysis of spiral ganglion cells demonstrated an increase in iNOS protein levels 1 day later. Pretreatment with the protective agent L-methionine prevented these changes (Li et al., 2006). It is apparent that different tissue compartments within the cochlea respond differentially to cisplatin. Molecules such as NF-kB promote cell survival in hair cells and spiral ganglion neurons, whereas in the lateral wall tissues it may promote apoptosis. 5. Prevention of ototoxicity The cochlea has endogenous mechanisms to deal with oxidative stress caused by agents like cisplatin. Protective molecules include: glutathione and the antioxidant enzymes, heat shock proteins, adenosine A1 receptors, NRF2 and heme-oxygenase-1, and the kidney injury molecule (KIM-1) (Mukherjea et al., 2006). Although these cytoprotective molecules are expressed in the cochlea, and are up-regulated following oxidative stress, oxidative stress imposed by cisplatin can overwhelm these intrinsic putative defense mechanisms. Antioxidants provide upstream protection of the cochlea to prevent initiation of cell death. Several thiol antioxidants protect against cisplatin ototoxicity. The rationale for the use of these agents is the high affinity of sulfur for platinum. Most thiols are electrophilic and can act as free radical scavengers. Such thiols include: sodium thiosulfate, diethyldithiocarbamate, D- or L-methionine, methylthiobenzoic acid, lipoic acid, N-acetylcysteine, tiopronin, glutathione ester and amifostine (Rybak and Whitworth, 2005). Certain thiols such as sodium thiosulfate and mesna (2-mercapto-ethane sulphonate) are directly incompatible with cisplatin (Boven et al., 2002). Both mesna and sodium thiosulfate neutralize cisplatin by forming a complex that is excreted by the kidney, reducing both its ototoxic activity and antineoplastic potency (Boven et al., 2002; Wimmer et al., 2004). Other thiols, such as D-methionine did not interfere with the tumoricidal action of cis-

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platin (Jones and Basinger, 1989; Melvik and Peterson, 1987; Cloven et al., 2000). Perilymphatic perfusion of sodium thiosulfate in guinea pigs prevents cisplatin ototoxicity (Wang et al., 2003). It is unlikely that this highly invasive route would be considered in humans. On the other hand, when this thiol was applied to the round window membrane using an osmotic minipump, it was not effective in preventing cisplatin ototoxicity (Wimmer et al., 2004). There is a clear discrepancy between the results of the study by Wang et al. (2003) and those of Wimmer et al. (2004). Both studies utilized guinea pigs as subjects. In the former study, sodium thiosulfate was perfused as a 10 mM solution perilymphatically starting 2 days before cisplatin administration and continued for a total of 7 days. Cisplatin was administered by intraperitoneal injection at a dose of 2 mg/kg per day for five consecutive days. However, the perfusion rate with an osmotic mini-pump was not specified. In the study by Wimmer et al. (2004) sodium thiosulfate was applied to the round window membrane using an osmotic mini-pump attached to a catheter. The delivery rate of sodium thiosulfate solution (15 mg/ml) was specified as 1 ll/h for 7 days. Cisplatin was injected in a higher dose that in the study by Wang et al. (2003). The study by Wimmer et al. (2004) utilized 3 mg/kg of cisplatin daily for 5 consecutive days. In the latter study, no protection against cisplatin ototoxicity was observed. This could have been caused by several factors: (1) improper placement or dislodgement of the round window catheter in the latter study; (2) inadequate dose of sodium thiosulfate delivery relative to the higher systemic dose of cisplatin in the Wimmer study. This study should be repeated with measurements of perilymphatic concentrations of sodium thiosulfate to ensure adequate delivery of the protectant across the round window membrane. It should be possible to deliver sodium thiosulfate to the inner ear fluids and tissues via the round window membrane. N-Acetylcysteine protects against cisplatin ototoxicity whether it is administered systemically or transtympanically. Intravenous N-acetylcysteine administered before intra-arterial cisplatin in rats provided significant preservation of auditory brainstem response thresholds (Dickey et al., 2004). Transtympanic injection of N-acetylcysteine in saline preserves distortion product otoacoustic emissions after intraperitoneal injection of cisplatin (Choe et al., 2004). Sodium thiosulfate and N-acetylcysteine are able to covalently bind to platinum, producing an inactive complex (Dickey et al., 2005). They could displace cisplatin after it is bound to target molecules (Schweitzer, 1993). This raises the intriguing possibility of a time window after cisplatin administration during which these agents could be administered as rescue agents. Efficacy of rescue would be provided by competing with proteins for binding cisplatin, protecting the protein from disruption, whereas cisplatin bound to DNA would presumably already exerted its antitumor action.

Although amifostine was found to protect against peripheral ototoxicity in the hamster, central auditory conduction times were prolonged. These findings were consistent with neurotoxicity (Church et al., 2004). Clinical trials testing amifostine as a protective agent have shown that it was not effective in preventing cisplatin-induced hearing loss. Adult patients with metastatic melanoma, experienced unacceptable ototoxicity following cisplatin infusion despite amifostine pretreatment (Ekborn et al., 2004). Children with germ cell tumors treated with amifostine in combination with cisplatin, etoposide and bleomycin had no protection against ototoxicity(Marina et al., 2005; Sastry and Kellie, 2005). D- and L-methionine are sulfur-containing antioxidants that may act as platinum chelators. As such, one would think that they may interfere with the antitumor effects of cisplatin. However, cisplatin-methionine complexes retain significant cytotoxic activity against tumors while being free from nephrotoxicity (Deegan et al., 1994). Methionine compounds also act as free radical scavengers or as agents that prevent induction of c-myc (Tao et al., 2000). The latter effect prevents up-regulation of the downstream gene product HMG and the subsequent induction of iNOS (Li et al., 2006). Radiolabelled Dmethionine and thiourea each were found to quickly and readily pass through the round window membrane into the cochlea. Autoradiography demonstrated that the most intense labelling was in the lateral wall of the cochlea, but there was also uptake in the organ of Corti. These agents achieved high local concentrations in rat scala tympani perilymph (Laurell et al., 2002). Round window application of D-methionine in the chinchilla prevents ototoxicity of topically applied cisplatin (Korver et al., 2002). D-methionine also prevented depletion of antioxidant enzymes and elevation of malondialdehyde in the cochlea of rats treated with cisplatin (Campbell et al., 2003). Other antioxidant agents that are free radical scavengers that protect the cochlea from cisplatin damage and hearing loss in experimental animals include: alpha-tocopherol (alone or in combination with tiopronin), aminoguanidine, sodium salicylate and ebselen (alone or in combination with allopurinol, which is an inhibitor of xanthine oxidase) (Lynch et al., 2005a,b; Rybak and Whitworth, 2005). Ebselen is a glutathione peroxidase mimic, but also is an excellent scavenger of peroxynitrite radicals. Thus it is able to protect against lipid peroxidation in the presence of glutathione and other thiols (Lynch et al., 2005a,b; Sies and Arteel, 2000). Antioxidant defense mechanisms in the cochlea may be mediated in part by the function of adenosine receptors. Adenosine A1 receptor agonists applied on the round window protect against cisplatin-induced auditory brainstem threshold elevation in the chinchilla and protection against hair cell loss from damage by cisplatin. These agonists also reduced the content of malondialdehyde in the cochlea from cisplatin, suggesting that the antioxidant defense

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mechanisms in the cochlea were increased by this treatment. Local application of the A1 adenosine receptor agonist, R-PIA, resulted in increases in the activity of cochlear glutathione peroxidase and superoxide dismutase within 90 min (Ford et al., 1997). This could explain the protective effect of the locally applied adenosine A1 receptor agonists against cisplatin ototoxicity. This protective effect was lost when the antagonist of the adenosine A1 receptor was applied to the round window before the adenosine A1 receptor agonist. By contrast, an adenosine A2 receptor agonist increased the ototoxicity of cisplatin (Whitworth et al., 2004). Neurotrophins such as neurotrophin-3 can protect against cytotoxic damage from cisplatin in vitro. A herpes simplex virus (HSV) amplicon vector was used to deliver the gene for expression of NT-3 within the cochlea. Mouse cochlear explants transduced with the HSV-NT-3 vector were protected against cisplatin-induced degeneration (Chen et al., 2001). Aged mice pretreated with this vector prior to cisplatin had greater numbers of spiral ganglion neurons and fewer cells undergoing cisplatin-induced apoptosis or necrosis compared to those treated with control virus (Bowers et al., 2002). In vitro experiments with an organ of Corti-derived cell line, HEI-OC1 demonstrated decreased viability associated with an increase in ROS production, lipid peroxidation (malondialdehyde) and DNA fragmentation after cisplatin exposure. Flunarizine, a calcium channel blocker, restored cell viability with reduced lipid peroxidation in the presence of cisplatin, despite no change in the generation of ROS. Flunarizine co-treatment prevented mitochondrial permeability transition and cytosolic release of cytochrome c in these cells, as indicated by rhodamine 123 fluorescence and immunoblotting for cytochrome c in cytosolic and mitochondrial fractions. This protective effect was not mediated by modulation of intracellular calcium levels. Complete protection against cisplatin-induced apoptosis in neonatal rat organ of Corti explants was also observed (So et al., 2005). Protection against cisplatin ototoxicity by flunarizine was associated with up-regulation of the endogenous protective genes NRF2 and heme-oxygenase1. siRNA experiments revealed that both molecules must be expressed in order for flunarizine to prevent cisplatin ototoxicity (So et al., 2006). Intracochlear perfusion of inhibitors of caspase-3 and caspase-9 provide downstream protection against cisplatin ototoxicity. These agents dramatically reduced the severity of hearing loss and apoptosis of hair cells following cisplatin administration in the guinea pig (Wang et al., 2004). This treatment, however, is too invasive to use in patients. Furthermore, acute treatment with caspase inhibitors is too short-acting for therapeutic use (Cooper et al., 2006). Gene therapy could provide a specific and chronic means of delivering potential therapeutic agents into the cochlea. Introduction of an antiapoptotic gene into the cochlea could provide long-term prophylaxis against oto-

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toxic effects of cisplatin. XIAP, the X-linked inhibitor of apoptosis protein, is a human protein that blocks several effectors of apoptosis, including caspases 3, 7 and 9 as well as other mediators of the cell death pathways (Deveraux et al., 1997). Two groups of rats were treated with unilateral injection through the round window membrane into scala tympani with adeno-associated virus (AAV) containing a gene encoding either XIAP or green fluorescent protein (GFP). After at least two months of gene expression, ABR threshold shifts and outer-hair-cell (OHC) number were measured in these two groups of animals after 72hour treatment with cisplatin. Uninjected and AAV. GFP-injected ears displayed profound ABR threshold elevations and OHC loss after cisplatin treatment. Ears that had been injected with AAV encoding XIAP, however, were significantly protected from these effects. Cisplatininduced ABR-threshold shift and hair-cell loss were attenuated by as much as 78% and 45%, respectively, as compared with contralateral (untreated) ears. Thus, XIAP delivery to the cochlea can protect against the audiometric changes and hair-cell loss associated with cisplatin ototoxicity. The efficacy, specificity, and duration of the protective effects make this a potentially attractive therapeutic method (Cooper et al., 2006). However, the need to penetrate the round window membrane in order to introduce this gene raises concerns about clinical applicability. If the gene could be inserted into cochlear tissues without violation of the round window membrane, this could be a clinically viable preventative method. Application of the p53 inhibitor, pifithrin-alpha, to organotypic organ of Corti cultures exposed to cisplatin prevented hair cell damage. The protection correlated with reduction in p53 expression and caspase activation. These findings are consistent with a role of p53 in triggering apoptotic cell death in cochlear hair cells (Zhang et al., 2003). However, it is questionable whether the efficacy of pifithrin to block cisplatin ototoxicity in immature cultures of the organ of Corti will translate into safe and effective treatments in humans. Blocking the effect of p53 by systemic pifithrin could result in a reduction of tumor cell killing mechanisms in patients. As demonstrated in the above discussion, the study of downstream agents to block cisplatin ototoxicity have largely been performed in vitro or have required invasive approaches to deliver the agent into the inner ear. These factors raise significant concerns about whether these therapies can be effective and be delivered in a safe and relatively non-invasive manner in patients. 6. Interference with cisplatin therapy Authors have expressed concerns about protective agents interfering with the antitumor effect of cisplatin. They have suggested caution in the use of these drugs in patients receiving cisplatin (Blakley et al., 2001). Sodium thiosulfate interferes with the antitumor effects of cisplatin. Simultaneous application of sodium thiosulfate and cis-

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platin to a human squamous cell carcinoma line in vitro reduced the tumoricidal activity of cisplatin (Viallet et al., 2006). There appears to be no differential protective effect for sodium thiosulfate in normal cells compared to tumor cells (Dickey et al., 2005). This problem of interference with the antitumor efficacy of cisplatin depends on the timing of administration of sodium thiosulfate in relation to cisplatin injection. Delayed administration of sodium thiosulfate after cisplatin reduces ototoxicity in guinea pigs at times and concentrations which do not reduce antitumor activity (Muldoon et al., 2000). Coadministration of N-acetylcysteine reversed the cytotoxic and apoptotic effects of cisplatin in small cell lung carcinoma and ovarian cancer cell lines (Wu et al., 2005). This raises concerns about the risks of interference with the antitumor effects of cisplatin in cancer chemotherapy when N-acetylcysteine is considered as a protective agent. Recent studies have confirmed that the chemoprotection route and timing can be adjusted to maintain the efficacy of cisplatin therapy by separating the thiol (sodium thiosulfate or N-acetylcysteine) from the administration of cisplatin in time and space (Dickey et al., 2005). The area under the curve (AUC) for cisplatin in blood was about 30% lower in guinea pigs pretreated with Dmethionine 0, 10 or 60 min before cisplatin compared to control animals (Ekborn et al., 2002). On the other hand, D-methionine provided cytoprotection against cisplatin toxicity without significant compromise of antitumor activity in a rat model for ovarian cancer (Cloven et al., 2000). Amifostine lowered the dose-normalized AUC for cisplatin and its monohydrated complex in adults with metastatic melanoma treated with combined therapy without lowering the incidence of ototoxicity (Ekborn et al., 2004). Thus, not only did amifostine adversely affect cisplatin pharmacokinetics, but it also failed to provide the desired protection against cisplatin ototoxicity in patients. The potential for interference with the therapeutic effects of cisplatin may be obviated by intratympanic or round window administration of the protective agent. L-methionine applied to the round window protected against ototoxicity but did not compromise antitumor efficacy of cisplatin in tumor-bearing rats (Li et al., 2001). As discussed above, D-methionine and N-acetylcysteine applied to the round window membrane prevented cisplatin ototoxicity in rodent models. Systemic sodium salicylate protected against cisplatin ototoxicity and nephrotoxicity without altering its antineoplastic efficacy in tumor-bearing rats (Li et al., 2002). Combined oral administration of ebselen and allopurinol in rat breast and ovarian cancer models not only preserved the antitumor activity of cisplatin, it actually enhanced it. These rats had significant protection against auditory brainstem threshold shift, myelotoxicity, nephrotoxicity and hepatotoxicity (Lynch et al., 2005a,b). Sodium butyrate is a histone deacetylase inhibitor with anticancer activity. It is also considered to be neuroprotective. Treatment of guinea pigs with sodium butyrate 7 days before and 5 days after cisplatin gave almost complete protection

against cisplatin ototoxicity in guinea pigs (Drottar et al., 2006). 7. Conclusions Cisplatin ototoxicity appears to be triggered by ROS that initiate a cascade of molecular events that lead to apoptosis of outer hair cells, resulting in loss of DPOAEs and elevation of high frequency thresholds for CAP and ABR. Ototoxic effects on the stria vascularis are transient, resulting in temporary reduction of EP associated with strial edema. The EP recovers but residual shrinkage of cells in the stria persist. Spiral ganglion cells are the least affected. The effects of cisplatin on frequency selectivity and intensity coding are not known. Upstream protection with antioxidant compounds may prevent the generation of ROS and thus block the downstream cascade that leads to cell death. Downstream protection with inhibitors of molecules involved in the cell death pathway, such as p53 and caspases 3 and 9 may also prevent cell death and hearing loss. The efficacy of CDDP in the presence of L- or DMET was evaluated in vitro using cultures of MTLN-3 breast tumor cell lines, and in vivo using implanted MTLN-3 tumors. Both L- and D-MET reduced the ability of CDDP to kill tumor cells in vitro and in vivo, however, our data suggest that a higher proportion of the antineoplastic activity of CDDP is retained in the presence of LMET (Reser et al., 1999). Future experimental studies should provide opportunities for therapeutic intervention to preserve auditory structure and function while preserving the intended antitumor effect of cisplatin. 8. Future directions To date, the Food and Drug Administration in the US has not approved any drugs as protective agents against cisplatin ototoxicity. However, it is likely that in the near future clinical trials will be initiated to study potential drugs to prevent cisplatin ototoxicity. From this review of the literature, several ideas for prevention of cisplatin ototoxicity can be gleaned. (1) Thiols are highly effective in antagonizing cisplatin ototoxicity. Unfortunately, some thiols also reduce its antitumor efficacy. The safest method for using thiols to provide upstream protection would be to administer them: (a) by intratympanic injection; or (b) by delayed systemic administration, either orally or intravenously. Provided that the proper timing could be worked out, the delayed use of systemic sodium thiosulfate could be useful. Unfortunately, it is not effective when administered intratympanically. (2) Adenosine A1 receptor agonists are effective protective agents. They have only been tested with round window application. Administration of these agents by this route would avoid potential interference with antineoplastic effects of cisplatin.

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(3) The use of pifithrin or caspase inhibitors may cause unintended side effects, such as enhancement of tumor growth. (4) Combined oral administration of ebselen and allopurinol protects against cisplatin ototoxicity while not only preserving, but actually enhancing antitumor activity of cisplatin in tumor bearing animals. This would be an ideal route because it is non-invasive. This combination seems to have the greatest clinical promise. If clinical trials show that this combination is effective in protecting patients against cisplatininduced hearing loss, it would be a great advance. (5) Sodium butyrate appears to be an effective protective agent with anticancer activity of its own. It is also very promising. (6) Salicylates are highly effective against cisplatin in animals. They have been successfully employed in a recent clinical trial in China to prevent aminoglycoside ototoxicity. Potential side effects would include gastrointestinal upset and possible bleeding secondary to platelet depletion by cisplatin. Exciting and potentially useful treatments for the prevention of cisplatin ototoxicity await further study and eventual clinical trials. Gene therapy with antiapoptotic genes is very promising but novel delivery systems that are non-invasive would need to be developed. These could include such techniques as nanotechnology to allow cochlear penetration of antiapoptotic genes. Intratympanic drug therapy of inner ear disease is widely used to treat Meniere disease and sudden deafness. The intratympanic application of adenosine agonists seems to hold great potential. Other methods of up-regulating endogenous protective molecules using conditioning sound exposure may stimulate the production of protective molecules in the cochlea. The future holds great promise for clinically relevant protection against cisplatin ototoxicity. Acknowledgements Hair cell lines derived from the Immortomouse (OC-k3 and HEI-OC1) were obtained from Dr. Federico Kalinec at the House Ear Institute. UB/OC-1 cells were provided by Dr. Matthew Holley at the Institute of Molecular Physiology in Sheffield, UK. The authors acknowledge the support of the National Institutes of Health, NIDCD, grant # R01-DC 02396. References Alam, S.A., Ikeda, K., Oshima, T., Suzuki, M., Kawase, T., Kikuchi, T., Takasaka, T., 2000. Cisplatin-induced apoptotic cell death in Mongolian gerbil cochlea. Hear. Res. 141, 28–38. Banfi, B., Malgrange, B., Knisz, J., Steger, K., Dubois-Dauphin, M., Crause, K.-H., 2004. Nox3, a superoxide-generating NADPH oxidase of the inner ear. J. Biol. Chem. 277, 39739–39748. Blakley, B.W., Cohen, J.I., Doolittle, N.D., Muldoon, L.L., Campbell, K.C., Dickey, D.T., Neuwelt, E.A., 2001. Strategies for prevention of

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toxicity caused by platinum-based chemotherapy: review and summary of the annual meeting of the Blood–Brain Barrier Disruption Program, Gleneden Beach, Oregon, March 10, 2001. Laryngoscope 112, 1997– 2001. Bortner, C.D., Cidlowski, J.A., 1998. A necessary role for cell shrinkage in apoptosis. Biochem. Pharmacol. 56, 1549–1559. Boven, E., Verschraagen, M., Hulscher, T.M., Erkelens, C.A.M., Hausheer, F.H., Pinedo, H.M., van der Vijgh, W.J.F., 2002. BNP7787, a novel protector against platinum-related toxicities, does not affect the efficacy of cisplatin or carboplatin in human tumour xenografts. Eur. J. Cancer 38, 1148–1156. Bowers, W.J., Chen, X., Guo, H., Frisina, D.R., Federoff, H.J., Frisina, R.D., 2002. Neurotrophin-3 transduction attenuates cisplatin spiral ganglion neuron ototoxicity in the cochlea. Mol. Ther. 6, 12–18. Campbell, K.C., Meech, R.P., Rybak, L.P., Hughes, L.F., 2003. The effect of D-methionine on cochlear oxidative state with and without cisplatin administration: mechanisms of otoprotection. J. Am. Acad. Audiol. 14, 144–156. Chen, X., Frisina, R.D., Bowers, W.J., Frisina, D.R., Federoff, H.J., 2001. HSV amplicon-mediated neurotrophin-3 expression protects murine spiral ganglion neurons from cisplatin-induced damage. Mol. Ther. 3, 958–963. Chen, W.C., Jackson, A., Budnick, A.S., Pfister, D.G., Kraus, D.H., Hunt, M.A., Stambuk, H., Levegrun, S., Wolden, S.L., 2006. Sensorineural hearing loss in combined modality treatment of nasopharyngeal carcinoma. Cancer 106, 820–829. Choe, W.T., Chinosornvatana, N., Chang, K.W., 2004. Prevention of cisplatin ototoxicity using transtympanic N-acetylcysteine and lactate. Otol. Neurotol. 25, 910–915. Church, M.W., Blakley, B.W., Burgio, D.L., Gupta, A.K., 2004. WR2721 (amifostine) ameliorates cisplatin-induced hearing loss but causes neurotoxicity in hamsters. J. Assoc. Res. Otolaryngol. 5, 227–237. Clerici, W.J., DiMartino, D.L., Prasad, M.R., 1995. Direct effect of reactive oxygen species on cochlear outer hair cells. Hear. Res. 84, 30–40. Clerici, W.J., Hensley, K., DiMartino, D.L., Butterfield, D.A., 1996. Direct detection of ototoxicant-induced reactive oxygen species generation in cochlear explants. Hear. Res. 98, 116–124. Cloven, N.G., Re, A., McHale, M.T., Burger, R.A., DiSaia, P.J., Rose, G.S., Campbell, K.C., Fan, H., 2000. Evaluation of D-methionine as a cytoprotectant in cisplatin treatment of an animal model for ovarian cancer. Anticancer Res. 20, 4205–4209. Cooper, L.B., Chan, D.K., Roediger, F.C., Shaffer, B.R., Fraser, J.F., Musatov, S., Selesnick, S.H., Kaplitt, M.G., 2006. AAV-mediated delivery of the caspase inhibitor XIAP protects against cisplatin ototoxicity. Otol. Neurotol. 27, 484–490. Deegan, P.M., Pratt, I.S., Ryan, M.P., 1994. The nephrotoxicity, cytotoxicity and handling of a cisplatin-methionine complex in male Wistar rats. Toxicology 89, 1–14. Dehne, N., Lautermann, J., Petrat, F., Rauen, U., de Groot, H., 2001. Cisplatin ototoxicity: involvement of iron and enhanced formation of superoxide anion radicals. Toxicol. Appl. Pharmacol. 174, 27–34. Deveraux, Q.L., Takahashi, R., Salvesen, G.S., Reed, J.C., 1997. X-linked IAP is a direct inhibitor of cell death proteases. Nature 388, 300–304. DeWoskin, R.S., Riviere, J.E., 1992. Cisplatin-induced loss of kidney copper and nephrotoxicity is ameliorated by a single dose of diethyldithiocarbamate but not mesna. Toxicol. Appl. Pharmacol. 112, 182–189. Dickey, T.D., Muldoon, L.L., Kraemer, D.F., Neuwelt, E.A., 2004. Protection against cisplatin ototoxicity by N-acetylcysteine in a rat model. Hear. Res. 193, 25–30. Dickey, D.T., Wu, Y.J., Muldoon, L.L., Neuwelt, E.A., 2005. Protection against cisplatin-induced toxicities by N-acetylcysteine and sodium thiosulfate as assessed at the molecular, cellular and in vivo levels. J. Pharmacol. Exp. Ther. 314, 1052–1058. Drottar, M., Liberman, M.C., Ratan, R.R., Roberson, D.W., 2006. The histone deacetylase inhibitor sodium butyrate protects against cisplatin-induced hearing loss in guinea pigs. Laryngoscope 116, 292–296.

166

L.P. Rybak et al. / Hearing Research 226 (2007) 157–167

Ekborn, A., Laurell, G., Johnstrom, P., Wallin, I., Eksborg, S., Ehrsson, H., 2002. D-methionine and cisplatin ototoxicity in the guinea pig: Dmethionine influences cisplatin pharmacokinetics. Hear. Res. 165, 53– 61. Ekborn, A., Hansson, J., Ehrsson, H., Eksborg, S., Wallin, I., Wagenius, G., Laurell, G., 2004. High dose cisplatin with amifostine: ototoxicity and pharmacokinetics. Laryngoscope 114, 1660–1667. Ford, M.S., Nie, Z., Whitworth, C., Rybak, L.P., Ramkumar, V., 1997. Up-regulation of adenosine receptors in the chinchilla cochlea by cisplatin. Hear. Res. 111, 143–152. He, Q., Ohndorf, U.M., Lippard, S.J., 2000. Intercalating residues determine the mode of HMG1 domains A and B binding to cisplatin-modified DNA. Biochemistry 39, 14426–14435. Huang, T., Cheng, A.G., Stupak, H., Liu, W., Kim, A., Staecker, H., Lefebvre, P.P., Malgrange, B., Kopke, R., Moonen, G., Van De Water, T.R., 2000. Oxidative stress-induced apoptosis of cochlear sensory cells: otoprotective strategies. Int. J. Dev. Neurosci. 18, 259–270. Hughes Jr., F.M., Bortner, C.D., Purdy, G.D., Cidlowski, J.A., 1997. Intracellular K + suppresses the activation of apoptosis in lymphocytes. J. Biol. Chem. 272, 30567–30576. Jiang, H., Sha, S.-H., Forge, A., Schacht, J., 2006. Caspase-independent pathways of hair cell death induced by kanamycin in vivo. Cell Death Differ. 13, 20–30. Jones, M.M., Basinger, M.A., 1989. Thiol and thioether suppression of cis-platinum-induced nephrotoxicity in rats bearing the Walker 256 carcinosarcoma. Anticancer Res. 9, 1937–1940. Kelly, T.C., Whitworth, C.A., Husain, K., Rybak, L.P., 2003. Aminoguanidine reduces cisplatin ototoxicity. Hear. Res. 186, 10–16. Klis, S.F., O’Leary, S.J., Hamers, F.P., De Groot, J.C., Smoorenburg, G.F., 2000. Reversible cisplatin ototoxicity in the albino guinea pig. Neuroreport 11, 623–626. Knight, K.R.G., Kraemer, D.F., Neuwelt, E.A., 2005. Ototoxicity in children receiving platinum chemotherapy: underestimating a commonly occurring toxicity that may influence academic and social development. J. Clin. Oncol. 23, 8588–8896. Kopke, R.D., Liu, W., Gabaizadeh, R., Jacono, A., Feghali, J., Spray, D., Garcia, P., Steinman, H., Malgrange, B., Ruben, R.J., Rybak, L., Van de Water, T., 1997. The 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. 18, 559–571. Korver, K., Rybak, L.P., Whitworth, C., Campbell, K.C.M., 2002. Round window application of D-methionine provides complete cisplatin otoprotection. Otolaryngol. Head Neck Surg. 126, 683–689. Lang, H., Schulte, B.A., Schmiedt, R.A., 2005. Ouabain induces apoptotic cell death Type I spiral ganglion neurons, but not Type II neurons. JARO, 63–74. Lang, H., Schulte, B.A., Zhou, D., Smythe, N., Spicer, S.S., Schmiedt, R.A., 2006. Nuclear factor kappaB deficiency is associated with auditory nerve degeneration and increased noise-induced hearing loss. J. Neurosci. 29, 3541–3550. 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. Hear. Res. 173, 198–209. Lee, J.E., Nakagawa, T., Kim, T.S., Endo, T., Shiga, A., Iguchi, F., Lee, S.H., Ito, J., 2004a. Role of reactive radicals in degeneration of the auditory system of mice following cisplatin treatment. Acta Otolaryngol. 124, 1131–1135. Lee, J.E., Nakagawa, T., Kita, T., Kim, T.S., Iguchi, F., Endo, T., Shiga, A., Lee, S.H., Ito, J., 2004b. Mechanisms of apoptosis induced by cisplatin in marginal cells in mouse stria vascularis. ORL 66, 111–118. Li, G., Frenz, D.A., Brahmblatt, S., Feghali, J.G., Ruben, R.J., Berggren, D., et al., 2001. Round window membrane delivery of L-methionine provides protection from cisplatin ototoxicity without compromising chemotherapeutic efficacy. Neurotoxicology 22, 163–176. Li, G., Sha, S.H., Zotova, E., Arezzo, J., Van De Water, T., Schacht, J., 2002. Salicylate protects hearing and kidney function from cisplatin toxicity without compromising its oncolytic action. Lab. Invest. 82, 585–596.

Li, G., Liu, W., Frenz, D., 2006. Cisplatin ototoxicity to the rat inner ear: a role for HMG1 and iNOS. Neurotoxicology 27, 22–30. Li, Y., Womer, R.B., Silber, J.H., 2004. Predicting cisplatin ototoxicity in children: the influence of age and the cumulative dose. Eur. J. Cancer 40, 2445–2451. Liang, F., Schulte, B.A., Qu, C., Hu, W., Shen, Z., 2005. Inhibition of the calcium- and voltage-dependent big conductance potassium channel ameliorates cisplatin-induced apoptosis in spiral ligament fibrocytes of the cochlea. Neuroscience 135, 263–271. Lynch, E.D., Gu, R., Pierce, C., Kil, J., 2005a. Combined oral delivery of ebselen and allopurinol reduces multiple cisplatin toxicities in rat breast and ovarian cancer models while enhancing anti-tumor activity. Anticancer Drugs 16, 569–579. Lynch, E.D., Gu, R., Pierce, C., Kil, J., 2005b. Reduction of acute cisplatin ototoxicity in rats by oral administration of allopurinol and ebselen. Hear. Res. 201, 81–89. Maeno, E., Ishizaki, Y., Kanaseki, T., Hazama, A., Okada, Y., 2000. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc. Natl. Acad. Sci. USA 97, 9487–9492. Marina, N., Chang, K.W., Malogolowkin, M., London, W.B., Frazier, A.L., Womer, R.B., Rescoria, F., Billmire, D.F., Davis, M.M., Perlman, E.J., Giller, R., Lauer, S.J., Olson, T.A., 2005. Amifostine does not protect against the ototoxicity of high-dose cisplatin combined with etoposide and bleomycin in pediatric germ-cell tumors: a Children’s Oncology Group study. Cancer 104, 841–847. McKeage, M.J., 1995. Comparative adverse effects of platinum drugs. Drug Safety 13, 228–244. Meech, R.P., Campbell, K.C., Hughes, L.F., Rybak, L.P., 1998. A semiquantitative analysis of the effects of cisplatin on the rat stria vascularis. Hear. Res. 124, 44–59. Melvik, J.E., Peterson, E.O., 1987. Reduction of cis-dichloro-diammineplatinum-induced cell inactivation by methionine. Inorgan. Chim. Acta 137, 115–118. Mukherjea, D., Whitworth, C.A., Nandish, S., Dunaway, G., Rybak, L.P., Ramkumar, V., 2006. Expression of the kidney injury molecule (KIM)1 in the rat cochlea and induction by cisplatin. Neuroscience 139, 733–740. Muldoon, L.L., Pagel, M.A., Kroll, R.A., Brummett, R.E., Doolittle, N.D., Zuhowski, E.G., Egorin, M.J., Neuwelt, E.A., 2000. Delayed administration of sodium thiosulfate in animal models reduces platinum ototoxicity without reduction of antitumor activity. Clin. Cancer Res. 6, 309–315. Nagy, I., Monge, A., Albinger-Hegyi, A., Schmid, S., Bodmer, D., 2005. NF-kappaB is required for survival of immature auditory hair cells in vitro. J. Assoc. Res. Otolaryngol. 6, 260–268. Ohndorf, U.-M., Rould, M.A., He, Q., Pabo, C.O., Lippard, S.J., 1999. Basis for recognition of cisplatin modified DNA by high-mobilitygroup proteins. Nature 399, 708–712. Pigeolet, E., Corbisier, P., Houbion, A., Lambert, D., Michiels, C., Raes, M., Zachary, M.D., Ramacle, J., 1990. Glutathione proxidase, superoxide dismutase and catalase inactivation by proxides and oxygen derived free radicals. Mech. Ageing Dev. 51, 283–297. Ravi, R., Somani, S.M., Rybak, L.P., 1995. Mechanism of cisplatin ototoxicity: antioxidant defense system. Pharmacol. Toxicol. 76, 386– 394. Reser, D., Rho, M., Dewan, D., Herbst, L., Li, G., Stupak, H., Zur, K., Romaine, J., Frenz, D., Goldbloom, L., Kopke, R., Arezzo, J., Van de Water, T.R., 1999. L- and D-methioniine provide long-term protection against CDDP-induced ototoxicity in vivo, with partial in vivo and in vitro retention of antineoplastic activity. Neurotoxicology 20, 731– 748. Rybak, L.P., Whitworth, C.A., 2005. Ototoxicity: therapeutic opportunities. Drug Discovery Today 10, 1313–1321. Rybak, L.P., Husain, K., Morris, C., Whitworth, C., Somani, S., 2000. Effect of protective agents against cisplatin ototoxicity. Am. J. Otol. 21, 513–520.

L.P. Rybak et al. / Hearing Research 226 (2007) 157–167 Sastry, J., Kellie, S.J., 2005. Severe neurotoxicity, ototoxicity and nephrotoxicity following high-dose cisplatin and amifostine. Pediatr. Hematol. Oncol. 22, 441–445. Schweitzer, V.G., 1993. Ototoxicity of chemotherapeutic agents. Otolaryngol. Clin. North Am. 26, 759–785. Sies, H., Arteel, G.E., 2000. Interaction of peroxynitrite with selenoproteins and glutathione peroxidase mimics. Free Radic. Biol. Med. 28, 1451–1455. Sluyter, S., Klis, S.F.L., de Groot, J.C.M.J., Smoorenburg, G., 2003. Alterations in the stria vascularis in relation to cisplatin ototoxicity and recovery. Hear. Res. 185, 49–56. So, H.-S., Park, C., Kim, H.-J., Lee, J.-H., Park, S.-Y., Lee, J.-H., Lee, Z.W., Kim, H.-M., Kalinec, F., Lim, D., Park, R., 2005. Protective effect of T-type calcium channel blocker flunarizine on cisplatin-induced death of auditory cells. Hear. Res. 204, 127–139. So, H.S., Kim, H.J., Lee, J.H., Lee, J.H., Park, S.Y., Park, C., Kim, Y.H., Kim, J.K., Lee, K.M., Kim, K.S., Chung, S.Y., Jang, W.C., Moon, S.K., Chung, H.T., Park, R.K., 2006. Flunarizine induces Nrf2mediated transcriptional activation of heme oxygenase-1 in protection of auditory cells from cisplatin. Cell Death Differ 13, 1763–1775. Somani, S.M., Husain, K., Jagannathan, R., Rybak, L.P., 2001. Amelioration of cisplatin-induced oto- and nephrotoxicity by protective agents. Ann. Neurosci. 8, 101–113. Tao, L., Yang, S., Xie, M., Kramer, P.M., Pereira, M.A., 2000. Effect of trichloroethylene and its metabolites, dichloroacetic acid and trichloroacetic acid on the methylation and expression of c-Jun and c-Myc protooncogenes in mouse liver: prevention by methionine. Toxicol. Sci. 54, 399–407. Thomas, J.P., Lautermann, J., Liedert, B., Seiler, F., Thomale, J., 2006. High accumulation of platinum–DNA adducts in strial marginal cells of the cochlea is an early event in cisplatin but not carboplatin ototoxicity. Mol. Pharm. 70, 23–29. Tsukasaki, N., Whitworth, C.A., Rybak, L.P., 2000. Acute changes in cochlear potentials due to cisplatin. Hear. Res. 149, 189–198. Van Ruijven, M.W.M., de Groot, J.C.M.J., Klis, S.F.L., Smoorenburg, G., 2005a. Cochlear targets of cisplatin: an electrophysiological and morphological time-sequence study. Hear. Res. 205, 241–248.

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Van Ruijven, M.W.M., de Groot, J.C.M.J., Hendriksen, F., Smoorenburg, G., 2005b. Immunohistochemical localization of platinated DNA in the cochlea of cisplatin-treated guinea pigs. Hear. Res. 203, 112–121. Viallet, N.R., Blakley, B.B., Begleiter, A., Leith, M.K., 2006. Sodium thiosulphate impairs the cytotoxic effects of cisplatin on FADU cells in culture. J. Otolaryngol. 35, 19–21. Wang, J., Faulconbridge, R.V.L., Fetoni, A., Guitton, M.J., Pujol, R., Puel, J.L., 2003. Local application of sodium thiosulfate prevents cisplatin-induced hearing loss in the guinea pig. Neuropharmacology 45, 380–393. Wang, J., Ladrech, S., Pujol, R., Brabet, P., Van De Water, T.R., Pujol, J.-L., 2004. Caspase inhibitors, but not c-Jun NH2-terminal kinase inhibitor treatment prevents cisplatin-induced hearing loss. Cancer Res. 64, 9217–9224. Watanabe, K., Inai, S., Jinnouchi, K., Bada, S., Hess, A., Michel, O., Yagi, T., 2002. Nuclear-factor kappa B (NF-kappa B)-inducible nitric oxide synthase (iNOS/NOS II) pathway damages the stria vascularis in cisplatin-treated mice. Anticancer Res. 22, 4081–4085. Watanabe, K., Inai, S., Jinnouchi, K., Baba, S., Yagi, T., 2003. Expression of caspase-activated deoxyribonuclease (CAD) and caspase-3 (CPP32) in the cochlea of cisplatin (CDDP)-treated guinea pigs. Auris Nasis Larynx 30, 219–225. Wimmer, C., Mees, K., Stumpf, P., Welsch, U., Reichel, O., Suckfull, M., 2004. Round window application of D-methionine, sodium thiosulfate, brain-derived neurotrophic factor and fibroblast growth factor-2 in cisplatin-induced ototoxicity. Otol. Neurotol. 25, 33–40. Whitworth, C.A., Ramkumar, V., Jones, B., Tsukasaki, N., Rybak, L.P., 2004. Protection against cisplatin ototoxicity by adenosine agonists. Biochem. Pharmacol. 67, 1801–1807. Wu, Y.J., Muldoon, L.L., Neuwelt, E.A., 2005. The chemoprotective agent N-acetylcysteine blocks cisplatin-induced apoptosis through caspase signaling pathway. J. Pharmacol. Exp. Ther. 312, 424–431. Zhang, M., Liu, W., Ding, D., Salvi, R., 2003. Pifithrin-alpha suppresses p53 and protects cochlear and vestibular hair cells from cisplatininduced apoptosis. Neuroscience 120, 191–205.