Ototoxic Insults Research Articles

Aminoglycosides Inhibit KCNQ4 Channels in Cochlear Outer Hair Cells via Depletion of Phosphatidylinositol(4,5)bisphosphate

Aminoglycoside antibiotics (AGs) are severely ototoxic. AGs cause degeneration of outer hair cells (OHCs), leading to profound and irreversible hearing loss. The underlying mechanisms are not fully understood. OHC survival critically depends on a specific K+ conductance (I(K,n)) mediated by KCNQ4 (Kv7.4) channels. Dysfunction or genetic ablation of KCNQ4 results in OHC degeneration and deafness in mouse and humans. As a common hallmark of all KCNQ isoforms, channel activity requires phosphatidylinositol(4,5)bisphosphate [PI(4,5)P₂]. Because AGs are known to reduce PI(4,5)P₂ availability by sequestration, inhibition of KCNQ4 may be involved in the action of AGs on OHCs. Using whole-cell patch-clamp recordings from rat OHCs, we found that intracellularly applied AGs inhibit I(K,n). The inhibition results from PI(4,5)P₂ depletion indicated by fluorescence imaging of cellular PI(4,5)P₂ and the dependence of inhibition on PI(4,5)P₂ availability and on PI(4,5)P₂ affinity of recombinant KCNQ channels. Likewise, extracellularly applied AGs inhibited I(K,n) and caused substantial depolarization of OHCs, after rapid accumulation in OHCs via a hair cell-specific apical entry pathway. The potency for PI(4,5)P₂ sequestration, strength of I(K,n) inhibition, and resulting depolarization correlated with the known ototoxic potential of the different AGs. Thus, the inhibition of I(K,n) via PI(4,5)P₂ depletion and the resulting depolarization may contribute to AG-induced OHC degeneration. The KCNQ channel openers retigabine and zinc pyrithione rescued KCNQ4/I(K,n) activity from AG-induced inhibition. Pharmacological enhancement of KCNQ4 may thus offer a protective strategy against AG-induced ototoxicity and possibly other ototoxic insults.

An otoprotective role for the apoptosis inhibitor protein survivin

Hearing impairment caused by ototoxic insults, such as noise or gentamicin is a worldwide health problem. As the molecular circuitries involved are not yet resolved, current otoprotective therapies are rather empirical than rational. Here, immunohistochemistry and western blotting showed that the cytoprotective protein survivin is expressed in the human and guinea pig cochlea. In the guinea pig model, moderate noise exposure causing only a temporary hearing impairment transiently evoked survivin expression in the spiral ligament, nerve fibers and the organ of Corti. Mechanistically, survivin upregulation may involve nitric oxide (NO)-induced Akt signaling, as enhanced expression of the endothelial NO synthase and phosphorylated Akt were detectable in some surviving-positive cell types. In contrast, intratympanic gentamicin injection inducing cell damage and permanent hearing loss correlated with attenuated survivin levels in the cochlea. Subsequently, the protective activity of the human and the guinea pig survivin orthologs against the ototoxin gentamicin was demonstrated by ectopic overexpression and RNAi-mediated depletion studies in auditory cells in vitro. These data suggest that survivin represents an innate cytoprotective resistor against stress conditions in the auditory system. The pharmacogenetic modulation of survivin may thus provide the conceptual basis for the rational design of novel therapeutic otoprotective strategies.

PACAP ameliorates oxidative stress in the chicken inner ear: An in vitro study

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide. Numerous studies prove that PACAP has neuroprotective effects in diverse neuronal systems in vitro and in vivo. The involvement of PACAP in visual and olfactory sensory processing has also been documented, but little is known about its effects in the auditory system. The presence of PACAP and its receptor, the specific PAC1 receptor, has been shown in the cochlea and in brain structures involved in auditory pathways. The aim of the present study was to investigate whether PACAP is protective in cochlear oxidative stress-induced cell death, which is known to play a role in several ototoxic insults. Chicken cochlear cells were exposed to 1 mM H 2O 2, which resulted in a marked reduction of cell viability and a parallel increase of apoptotic and necrotic cells assessed by MTT test, annexin V/propidium iodide flow cytometry and JC-1 apoptosis assay. Co-incubation with 100 nM PACAP increased cell viability and reduced the percentage of apoptotic cells. Furthermore, oxidative stress increased the activation of caspase-3, while simultaneous PACAP treatment reduced it. In summary, our present results demonstrate that PACAP effectively protects cochlear cells against oxidative stress-induced apoptotic cell death.

Potentiation of Chemical Ototoxicity by Noise

High-intensity and/or prolonged exposure to noise causes temporary or permanent threshold shifts in auditory perception. Occupational exposure to solvents or administration of clinically important drugs, such as aminoglycoside antibiotics and cisplatin, also can induce permanent hearing loss. The mechanisms by which these ototoxic insults cause auditory dysfunction are still being unraveled, yet they share common sequelae, particularly generation of reactive oxygen species, that ultimately lead to hearing loss and deafness. Individuals are frequently exposed to ototoxic chemical contaminants (e.g., fuel) and noise simultaneously in a variety of work and recreational environments. Does simultaneous exposure to chemical ototoxins and noise potentiate auditory dysfunction? Exposure to solvent vapor in noisy environments potentiates the permanent threshold shifts induced by noise alone. Moderate noise levels potentiate both aminoglycoside- and cisplatin-induced ototoxicity in both rate of onset and in severity of auditory dysfunction. Thus, simultaneous exposure to chemical ototoxins and moderate levels of noise can potentiate auditory dysfunction. Preventing the ototoxic synergy of noise and chemical ototoxins requires removing exposure to ototoxins and/or attenuating noise exposure levels when chemical ototoxins are present.

Adeno-associated virus-mediated Bcl-xL prevents aminoglycoside-induced hearing loss in mice

Recent studies showed that aminoglycosides destroyed the cochlear cells and induced ototoxicity by producing reactive oxygen species, including free radicals in the mitochondria, damaging the membrane of mitochondria and resulting in apoptotic cell death. Bcl-x(L) is a well characterized anti-apoptotic member of the Bcl-2 family. The aim of this study was to determine the potential cochlear protective effect of Bcl-x(L) as a therapeutic agent in the murine model of aminoglycoside ototoxicity. Serotype 2 of adeno-associated virus (AAV2) as a vector encoding the mouse Bcl-x(L) gene was injected into mice cochleae prior to injection of kanamycin. Bcl-x(L) expression in vitro and in vivo was examined with Western blotting and immunohistochemistry separately. Cochlear dissection and auditory steady state responses were checked to evaluate the cochlear structure and function. The animals in the AAV2-Bcl-x(L)/kanamycin group displayed better auditory steady state responses hearing thresholds and cochlear structure than those in the artificial perilymph/kanamycin or AAV2-enhanced humanized green fluorescent protein/kanamycin control group at all tested frequencies. The auditory steady state responses hearing thresholds and cochlear structure in the inoculated side were better than that in the contralateral side. AAV2-Bcl-x(L) afforded significant preservation of the cochlear hair cells against ototoxic insults and protected the cochlear function. AAV2-mediated Bcl-x(L) might be an approach with respect to potential therapeutic application in the cochlear degeneration.

A protein derived from the fusion of TAT peptide and FNK, a Bcl‐xL derivative, prevents cochlear hair cell death from aminoglycoside ototoxicity in vivo

We constructed a powerful artificial cytoprotective protein, FNK, from an antiapoptotic member of the BCL-2 family, Bcl-x(L). To test the efficacy of FNK in protecting cochlear hair cells (HCs) from aminoglycoside-induced cell death in vivo, we fused FNK with protein transduction domain, TAT, of the HIV/Tat protein to construct a fusion protein of TAT-FNK. We demonstrated that, after an intraperitoneal administration to guinea pigs, TAT-myc-FNK protein was diffusely distributed in the cochlea, most prominently in the HCs and supporting cells, followed by the spiral ganglion cells, 3 hr after the injection. We next demonstrated that TAT-FNK attenuated cochlear damage induced by an ototoxic combination of kanamycin sulfate (KM) and ethacrynic acid (EA) administered at 2 different dosages: 400 mg/kg KM + 50 mg/kg EA and 200 mg/kg KM + 40 mg/kg EA. TAT-FNK or vehicle was intraperitoneally injected from 3 hr before through 5 hr after inducing the ototoxic insults, 14 days after which auditory brainstem response (ABR) and HC loss were evaluated. In comparison with vehicle-administered controls, the TAT-FNK protein significantly attenuated ototoxic drug-induced ABR threshold shifts and the extent of HC death at either dosage. The TAT-FNK protein also significantly reduced the amount of cleaved poly-(ADP-ribose) polymerase-positive HCs 8 hr after the ototoxic insults compared with that in the vehicle-administered controls. These findings indicate that systemically administered TAT-FNK was successfully delivered to the guinea pig cochlea and effectively prevented apoptotic cell death of the cochlear HCs induced by KM and EA.

Prevention of noise- and drug-induced hearing loss with d-methionine

A number of otoprotective agents are currently being investigated. Various types of agents have been found in animal studies to protect against hearing loss induced by cisplatin, carboplatin, aminoglycosides, or noise exposure. For over a decade we have been investigating d-methionine ( d-met) as an otoprotective agent. Studies in our laboratory and others around the world have documented d-met’s otoprotective action, in a variety of species, against a variety of ototoxic insults including cisplatin-, carboplatin-, aminoglycoside- and noise-induced auditory threshold elevations and cochlear hair cell loss. For cisplatin-induced ototoxicity, protection of the stria vascularis has also been documented. Further d-met has an excellent safety profile. d-Met may act as both a direct and indirect antioxidant. In this report, we provide the results of three experiments, expanding findings in d-met protection in three of our translational research areas: protection from platinum based chemotherapy-, aminoglycoside- and noise-induced hearing loss. These experiments demonstrate oral d-met protection against cisplatin-induced ototoxicity, d-met protection against amikacin-induced ototoxicity, and d-met rescue from permanent noise-induced hearing loss when d-met is initiated 1 h after noise exposure. These studies demonstrate some of the animal experiments needed as steps to translate a protective agent from bench to bedside.

The fate of outer hair cells after acoustic or ototoxic insults

In epithelial sheets, clearance of dead cells may occur by one of several routes, including extrusion into the lumen, phagocytic clearance by invading lymphocytes, or phagocytosis by neighboring cells. The fate of dead cochlear outer hair cells is unclear. We investigated the fate of the “corpses” of dead outer hair cells in guinea pigs and mice following drug or noise exposure. We examined whole mounts and plastic sections of normal and lesioned organ of Corti for the presence of prestin, a protein unique to outer hair cells. Supporting cells, which are devoid of prestin in the normal ear, contained clumps of prestin in areas of hair cell loss. The data show that cochlear supporting cells surround the corpses and/or debris of degenerated outer hair cells, and suggest that outer hair cell remains are phagocytosed by supporting cells within the epithelium.

Mechanisms of hair cell death and protection

Sensory hair cells are mechanotransducers of the inner ear that are essential for hearing and balance. Hair cell death commonly occurs following acoustic trauma or exposure to ototoxins, such as the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Loss of these inner ear sensory cells can lead to permanent sensorineural hearing loss, balance disturbance, or both. Currently, the only effective clinical intervention is prevention from exposure to known ototoxic insults. To help improve therapeutic strategies, a better understanding of the molecular mechanisms underlying hair cell degeneration is required. Current knowledge of these cell death mechanisms and potential therapeutic targets are discussed in this review. Studies have shown that caspase-9 and caspase-3 are key mediators of hair cell death induced by noise, aminoglycosides, and cisplatin. The Bcl-2 family consists of a group of proapoptotic and antiapoptotic molecules that act upstream of and regulate caspase activation. Recent studies have shed light on the roles of molecules acting more upstream, including mitogen-activated protein kinases and p53. The mechanisms of sensory hair cell degeneration in response to different ototoxic stimuli share a final common pathway: caspase activation. Inhibition of caspases prevents or delays hair cell death and may preserve hearing/balance function. Inhibition of mitogen-activated protein kinases protects against noise-induced and aminoglycoside-induced but not cisplatin-induced hair cell death, which suggests divergent upstream regulatory mechanisms.

Changes in otoacoustic emissions and auditory brain stem response after cis-platinum exposure in gerbils.

Ototoxicity associated with cis-platinum administration commonly presents as hearing loss and tinnitus. The hearing loss is usually an irreversible, high-frequency sensorineural loss. Histologic studies in humans and animals suggest that the outer hair cells (OHCs) are most susceptible to cis-platinum. Evoked otoacoustic emissions (EOAE), as a measure of outer hair cell function, are potentially useful in following ototoxic insults involving OHCs. Distortion-product otoacoustic emissions (DPOAE) test frequency-specific regions of the cochlea and therefore may be particularly well suited for monitoring ototoxic injuries. We measured distortion product otoacoustic emissions, at f2 = 2, 4, 6, 8, 10, and 12 kHz, in gerbils after a single large dose of cis-platinum. Animals treated with saline served as controls. The findings were compared to auditory brain stem evoked response (ABR) thresholds, using tone pips of the same frequencies. The DPOAE and ABR thresholds were measured before treatment and again 2, 5, and 14 days after drug administration. The changes in DPOAE were compared with the changes in ABR. No treatment effect was noted in the 2-day group. Animals treated with cis-platinum demonstrated significant elevation of DPOAE and ABR thresholds compared with control animals at 5 and 14 days. There was no significant difference between the threshold changes in the 5- and 14-day groups.

Drug delivery to the cochlea after implantation: consideration of the risk factors

As cochlear implant technology continues to evolve, general outcomes improve, but some implant users still suffer from other symptoms of their underlying condition, such as tinnitus and vertigo. Furthermore, the ongoing neural degeneration characteristic of hearing loss may not be fully prevented by electrical stimulation, which implies the possibility of some degree of performance degradation after several decades of implant use. As criteria for implantation expand, ears with progressively more residual hearing are implanted, and often the better-hearing ear is chosen. Minimal trauma electrode designs and surgical approaches have allowed the benefits of combined electric and acoustic stimulation to be demonstrated, which will increase user expectations for protection of residual hearing. Current advances in molecular and inner-ear biology and genetics hint at new possibilities for enhancement of general outcome through application of drugs or biologics. For example, increased understanding of the molecular mechanisms of cell death after ototoxic and acoustic insults allows the investigation of a number of possible therapies to ameliorate the effects of trauma on neural tissue. Those currently under investigation generally belong to the families of neurotrophic factors, antioxidants or anti-apoptotic factors (Raphael, 2002). Additionally, the fields of gene therapy and stem cell transplantation, though still in their infancy, may in time offer long-lasting solutions to some underlying cochlear insufficiencies. Finally, regrowth of peripheral processes of the auditory neurons can be achieved using neurotrophic factors. If certain hurdles can be overcome, this technology might eventually allow closer coupling between implant and nerve, which might have the potential to reduce stimulation currents and increase the number of useful information channels. Each possible therapy identified through basic research requires a delivery solution capable of accurate dosage to the target tissues. Possible delivery mechanisms include systemic, round window (diffusion or injection), depot substances, including coatings applied to a substrate, and delivery in fluid form through a catheter integrated into the implant electrode. Proteins, peptides, liposomes and biologics require parenteral delivery. For many drugs it is anticipated that local delivery will be preferable, to avoid systemic effects and allow accurate dosage, and in some cases this may be necessary due to the blood–perilymph barrier. Round-window delivery does not allow accurate dosage and tends to restrict treatment to the base of the cochlea (Salt and Ma, 2001). Biodegradeable coatings may offer certain advantages (such as ease of use and spatially uniform delivery), but products of breakdown may be toxic, delivery rate is not precisely defined and drug application cannot be terminated without explantation. Prieskorn and Miller (2000) have demonstrated the feasibility of delivery of large proteins in solution to the modiolus through scala tympani application, using an implantable pump. The cochlear implant electrode array is inserted deep into the perilymph of the scala tympani. If used as a conduit, it offers the potential for safe delivery of an accurate and controllable dose of drug to the cochlea. The cochlea is, however, a delicate and highly structured organ offering its own unique risks and challenges. The venture requires extensive multidisciplinary collaboration, and the following are essential criteria for the therapy.

Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio).

Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 microM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24-48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.

Inhibition of Caspases Prevents Ototoxic and Ongoing Hair Cell Death

Sensory hair cells die after acoustic trauma or ototoxic insults, but the signal transduction pathways that mediate hair cell death are not known. Here we identify several important signaling events that regulate the death of vestibular hair cells. Chick utricles were cultured in media supplemented with the ototoxic antibiotic neomycin and selected pharmacological agents that influence signaling molecules in cell death pathways. Hair cells that were treated with neomycin exhibited classically defined apoptotic morphologies such as condensed nuclei and fragmented DNA. Inhibition of protein synthesis (via treatment with cycloheximide) increased hair cell survival after treatment with neomycin, suggesting that hair cell death requires de novo protein synthesis. Finally, the inhibition of caspases promoted hair cell survival after neomycin treatment. Sensory hair cells in avian vestibular organs also undergo continual cell death and replacement throughout mature life. It is unclear whether the loss of hair cells stimulates the proliferation of supporting cells or whether the production of new cells triggers the death of hair cells. We examined the effects of caspase inhibition on spontaneous hair cell death in the chick utricle. Caspase inhibitors reduced the amount of ongoing hair cell death and ongoing supporting cell proliferation in a dose-dependent manner. In isolated sensory epithelia, however, caspase inhibitors did not affect supporting cell proliferation directly. Our data indicate that ongoing hair cell death stimulates supporting cell proliferation in the mature utricle.

Effect of transgenic GDNF expression on gentamicin-induced cochlear and vestibular toxicity.

Gentamicin administration often results in cochlear and/or vestibular hair cell loss and hearing and balance impairment. It has been demonstrated that adenovirus-mediated overexpression of glial cell line-derived neurotrophic factor (GDNF) can protect cochlear hair cells against ototoxic injury. In this study, we evaluated the protective effects of adenovirus-mediated overexpression of GDNF against gentamicin ototoxicity. An adenovirus vector expressing the human GDNF gene (Ad.GDNF) was administered into the scala vestibuli as a rescue agent at the same time as gentamicin, or as a protective agent, 7 days before gentamicin administration. Animals in the Rescue group displayed hearing thresholds that were significantly better than those measured in the Gentamicin or Ad.LacZ/Gentamicin groups. In the Protection group, Ad.GDNF afforded significant preservation of utricular hair cells. The data demonstrated protection of the inner ear structure, and rescue of the inner ear structure and function against ototoxic insults. These experiments suggest that inner ear gene therapy may be developed as a clinical tool for protecting the ear against environmentally induced insults.

Otoacoustic emissions — an approach for monitoring aminoglycoside induced ototoxicity in children

Objectives: the early detection of hearing impairment caused by ototoxic drugs, such as aminoglycosides, has been the aim of research world-wide. Histopathological studies have shown that the outer hair cells are the most susceptible cochlear components to injury from ototoxic drugs like aminoglycosides. Otoacoustic emissions reflect the functional status of the outer hair cells and constitute the only non-invasive means of objective cochlear investigation. The aim of this study was to evaluate the potential of otoacoustic emissions in early identification of aminoglycoside-induced cochlear dysfunction. In addition, a comparison with pure-tone audiometry or auditory brainstem responses was performed in order to determine if this test might provide a more reliable method of monitoring early ototoxic insults to the cochlea. Methods: twenty four children receiving gentamicin (4 mg/kg once daily) for 6–29 days were included in the study. Eleven children received gentamicin for up to 7 days (group A), while 13 underwent longer-term therapy lasting 8–29 days (group B). Hearing was serially monitored using transient evoked otoacoustic emissions and pure-tone audiometry (0.25–12 kHz) or auditory brainstem responses for younger or uncooperative children. Transient evoked otoacoustic emissions data were analysed in terms of emission amplitude and response reproducibility as a function of frequency. Results: all patients yielded a normal baseline audiometric assessment upon hospital admission. For group A patients no significant changes in hearing levels were observed either by pure-tone audiometry ( P=0.2), auditory brainstem responses ( P=0.3) or transient evoked otoacoustic emissions (mean response: P=0.06, reproducibility by frequency: P>0.05). For group B patients no significant changes in hearing levels measured by pure-tone audiometry ( P=0.1) or auditory brainstem responses ( P=0.4) were observed. Transient evoked otoacoustic emissions however revealed a statistically significant decrease in the mean response level ( P=0.017) and in the reproducibility over the whole frequency spectrum (l kHz: P=0.0057, 2 kHz: P=0.0247, 3 kHz: P=0.0134, 4 kHz: P=0.0049, 5 kHz: P=0.0019). Conclusions: the findings suggest that transient evoked otoacoustic emissions are an extremely sensitive measure of the early effects of aminoglycoside-induced injury to the peripheral auditory system. Therefore, their use is recommended for regular monitoring of cochlear function, in the presence of potentially toxic factors, aiming at prevention of permanent damage.

Changes in otoacoustic emissions and auditory brain stem response after cis-platinum exposure in gerbils

Ototoxicity associated with cis -platinum administration commonly presents as hearing loss and tinnitus. The hearing loss is usually an irreversible, high-frequency sensorineural loss. Histologic studies in humans and animals suggest that the outer hair cells (OHCs) are most susceptible to cis -platinum. Evoked otoacoustic emissions (EOAE), as a measure of outer hair cell function, are potentially useful in following ototoxic insults involving OHCs. Distortion-product otoacoustic emission (DPOAE) test frequency-specific regions of the cochlea and therefore may be particularly well suited for monitoring ototoxic injuries. We measured distortion product otoacoustic emissions, at f2 = 2, 4, 6, 8, 10, and 12 kHz, in gerbils after a single large dose of cis -platinum. Animals treated with saline served as controls. The findings were compared to auditory brain stem evoked response (ABR) thresholds, using tone pipe of the same frequencies. The DPOAE and ABR thresholds were measured before treatment and again 2, 5, and 14 days after drug administration. The changes in DPOAE were compared with the changes in ABR. No treatment effect was noted in the 2-day group. Animals treated with cis -platinum demonstrated significant elevation of DPOAE and ABR thresholds compared with control animals at 5 and 14 days. There was no significant difference between the threshold changes in the 5-and 14-day groups. (Otolaryngol Head Neck Surg 1997; 116:585-92.)

A sensitive animal model to assess acute and chronic ototoxic effects.

Ototoxic side effects of aminoglycosides or cisplatin are strongly influenced by the nutritional state of the experimental animal. We previously demonstrated that guinea pigs fed a low-protein diet are a sensitive model to investigate chronic ototoxic effects of drugs. We now demonstrate that this model is equally sensitive to acute ototoxic insults. Guinea pigs were fed either a full-protein (18.5% protein) or a low-protein (7% protein) diet. Gentamicin sulfate was given as a single injection (100 mg/kg of body weight subcutaneously), followed 90 minutes later by varying concentrations of ethacrynic acid intravenously. Auditory function was assessed by auditory brain-stem response and morphologic characteristics in surface preparations of the organ of Corti. In animals fed a full-protein diet, 35 mg of ethacrynic acid per kilogram of body weight was required for a significant threshold shift at all frequencies tested (3, 8, and 18 kHz). In animals fed a low-protein diet, 15 mg of ethacrynic acid per kilogram significantly elevated threshold at all frequencies, and 20 mg/kg caused a profound threshold shift (> 90 dB). Hair cell loss in the organ of Corti paralleled the pathophysiological findings. A metabolically challenged animal may better represent the situation of a severely ill patient undergoing drug therapy. Therefore, studies in diet-restricted animals may be more appropriated than studies in healthy animals for an assessment of potential adverse therapeutic side effects.