Mitochondrial DNA mutations associated with aminoglycoside induced ototoxicity

Aminoglycosides(Am An) are widely used for their great efficiency against gram-negative bacterial infections. However, they can also induce ototoxic hearing loss, which has affected millions of people around the world. As previously reported, individuals bearing mitochondrial DNA mutations in the 12 S rRNA gene, such as m.1555A>G and m.1494C>T, are more prone to Am An-induced ototoxicity. These mutations cause human mitochondrial ribosomes to more closely resemble bacterial ribosomes and enable a stronger aminoglycoside interaction. Consequently,exposure to Am An can induce or worsen hearing loss in these individuals. Furthermore, a wide range of severity and penetrance of hearing loss was observed among families carrying these mutations. Studies have revealed that these mitochondria mutations are the primary molecular mechanism of genetic susceptibility to Am An ototoxicity, though nuclear modifier genes and mitochondrial haplotypes are known to modulate the phenotypic manifestation.

Gene expression in cisplatin ototoxicity and protection with p53 inhibitor

Cisplatin damages cochlear hair cells and spiral ganglion neurons through cell death signaling pathways that are not fully understood. We used focused apoptosis gene microarrays to study early changes in gene expres- sion in cochlear cultures from P3 neonatal rats treated with cisplatin (0.2 mM). After 12 hours of cisplatin treat- ment, more than 50% of the 96 genes on the array showed a significant decrease in expression, consistent with widespread cell death. However, after 3 hours of cisplatin treatment, 10 genes showed significant increase in ex- pression in total cochlear tissue. In experiments with subsets of cochlear tissues, at 3h, cisplatin induced increased expression of 12 genes in the cochlear sensory epithelium (basilar membrane) and 11 genes in the spiral ganglion (tissue of Rosenthal’s canal, containing the spiral ganglion). These included pro- and anti-apoptotic genes in- volved in the p53 signaling pathway, TNF receptor family, NF-kappaB pathway, death domain family, death effec- tor domain family, Bcl-2 family, CARD family, TRAF family, and GTP signal transduction. Although the changes in gene expression showed an overlap between basilar membrane and spiral ganglion, other changes, which may reflect the unique response of each tissue, were also observed. Pifithrin-α blocked cisplatin-induced up-regulation of genes in the p53 signaling pathway when assayed by both superarray and real time PCR. The data add to our understanding of the involvement of p53 in cisplatin-induced ototoxicity and otoprotection, conferred by the p53 inhibitor Pifithrin-α.

Calpain mediated cisplatin-induced ototoxicity in mice

Ototoxic drug-induced apoptosis of inner ear cells has been shown to be associated with calpain expression. Cisplatin has severe ototoxicity, and can induce cochlear cell apoptosis. This study assumed that cisplatin activated calpain expression in apoptotic cochlear cells. A mouse model of cisplatin-induced ototoxicity was established by intraperitoneal injection with cisplatin （2.5, 3.5, 4.5, 5.5 mg/kg）. Immunofluorescence staining, image analysis and western blotting were used to detect the expression of calpain 1 and calpain 2 in the mouse cochlea. At the same time, the auditory brainstem response was measured to observe the change in hearing. Results revealed that after intraperitoneal injection with cisplatin for 5 days, the auditory brainstem response threshold shifts increased in mice. Calpain 1 and calpain 2 expression significantly increased in outer hair cells, the spiral ganglion and stria vascularis. Calpain 2 protein expression markedly increased with an increased dose of cisplatin. Results suggested that calpain 1 and calpain 2 mediated cisplatin-induced ototoxicity in BALB/c mice. During this process, calpain 2 plays a leading role.

Ligustrazine facilitates hair cell regeneration in the cochlea following gentamicin ototoxicity

BACKGROUND： Ligustrazine （tetramethylpyrazine） decreases ototoxicity induced by gentamicin and facilitates hair cell regeneration and repair, but the precise mechanisms remain controversial.OBJECTIVE： To explore the protective effects of ligustrazine on gentamicin ototoxicity by determining heat shock protein 70 mRNA and protein expression in the cochlear stria vascularis of guinea pigs at different time points.DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Laboratory of Physiology, Shenyang Medical College of China in 2007.MATERIALS： Ligustrazine parenteral solution （Qiqihar Pharmaceutical Factory, China） and gentamicin sulfate （Shenyang First Pharmaceutical Factory, China） were used in this experiment.METHODS： White guinea pigs with red eyes were randomly intraperitoneally administered gentamicin sulfate injection ＋ saline, gentamicin sulfate injection ＋ ligustrazine, and ligustrazine ＋ saline, respectively.MAIN OUTCOME MEASURES： Auditory brains tern response threshold was measured. Immunohistochemistry, in situ hybridization, and image analyzing techniques were utilized to determine heat shock protein 70 mRNA and protein expression in cochlear stria vascularis of guinea pigs.RESULTS： Following gentamicin ototoxicity, the auditory brainstem response threshold increased, peaked on day 3, and then decreased with increased time after drug withdrawal. The auditory brainstem response threshold was significantly diminished following ligustrazine intervention, but recovered to normal on day 30 （P〉0.05）. Heat shock protein 70 expression also increased, peaked on day 3, and then decreased in the cochlear stria vascularis of guinea pigs following gentamicin ototoxicity. Ligustrazine intervention resulted in decreased heat shock protein 70 expression in the cochlear stria vascularis, which recovered to normal on day 14. Heat shock protein 70 mRNA expression increased in the cochlear stria vascularis following gentamicin ototoxicity, but ligustrazine intervention resulted in decreased levels. CONCLUSION： Ligustrazine significantly ameliorated gentamicin ototoxicity by reducing heat shock protein 70 mRNA and protein expression in the cochlear stria vascularis.

Partial Protection by Lipoic Acid Against Carboplantin-induced Ototoxicity in Rats

Objective To investigate the alterations in auditory brainstem evoked responses (ABRs) and the changes of carboplatin-induced ototoxicity in the cochlear oxidant/antioxidant systems and otoprotection by an antioxidant lipoate. Methods Male wistar rats were divided into four groups and treated as follows: 1) vehicle (saline) control, 2) carboplatin (256 mg/kg, i.p.), 3) lipoate (100 mg/kg, i.p.), 4) lipoate + carboplatin. Post-treatment ABRs were performed after four days and rats were sacrificed with their cochleae harvested and analyzed. Results Carboplatin significantly elevated ABR threshold above the pretreatment thresholds. Lipoate+carboplatin treated rats showed decreased elevation of hearing threshold. Carboplatin significantly depleted cochlear reduced to oxizized glutathione (GSH/GSSG) ratio, whereas lipoate+carboplatin treatment increased GSH/GSSG ratio. Carboplatin significantly decreased cochlear copper zinc-superoxide dismutase (CuZn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and glutathione-S-transferase (GST) activities and enzyme protein expressions and a significant increase in Mn-SOD activity, protein expression and malondialdehyde (MDA) level. Cochlear antioxidant enzyme activities, enzyme protein expressions and MDA level were partially restored in lipoate+carboplatin treated rats, compared to carboplatin alone. Conclusion Carboplatin-induced ototoxicity is related to impairment of cochlear antioxidant system and otoprotection conferred by lipoate is associated with partial sparing of the cochlear antioxidant defense system.

Mitochondrial DNA Mutations Associated with Aminoglycoside Ototoxicity

The mitochondrial 12S rRNA has been shown to be the hot spot for mutations associated with both aminoglycoside-induced and non-syndromic hearing loss. Of all the mutations, the homoplasmic A1555G and C1494T mutations at a highly conserved decoding region in the 12S rRNA have been associated with aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. The A1555G or C1494T mutation is expected to form novel 1494C-G1555 or 1494U-A1555 base-pair at the highly conserved A-site of 12S rRNA. These transitions make the secondary structure of this RNA more closely resemble the corresponding region of bacterial 16S rRNA. Thus, the new U-A or G-C pair in 12S rRNA created by the C1494T or A1555G transition facilitates the binding of aminoglycosides, thereby accounting for the fact that the exposure to aminoglycosides can induce or worsen hearing loss in individuals carrying these mutations. Furthermore, the growth defect and impairment of mitochondrial translation were observed in cell lines carrying the A1555G or C1494T mutation in the presence of high concentration of aminoglycosides. In addition, nuclear modifier genes and mitochondrial haplotypes modulate the phenotypic manifestation of the A1555G and C1494T mutations. These observations provide the direct genetic and biochemical evidences that the A1555G or C1494T mutation is a pathogenic mtDNA mutation associated with aminoglycoside-induced and nonsyndromic hearing loss. Therefore, these data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

Ototoxicity induced by kanamycin in iron-deficient young rats

Using auditory electrophysiological,histopathological,enzymehistochemical and scanning electron microscopic techniques,we observed thechanges of function and structure of cochlea in iron-deficient growing rats receiv-ing kanamycin intramuscularly.The results showed that at an equal dose ofkanamycin,hearing loss and hair cell damage were more serious,and incidence ofhearing loss was much higher in iron-deficient rats than in normal rats.Theinhibition of succinate dehydrogenase activity of cochlea induced by interaction ofkanamycin and iron deficiency was more obvious than that by either one of them.Iron deficiency can increase the sensitivity of cochlea to kanamycin.The resultsindicated that iron deficiency may be one of the reasons for high incidence ofototoxic deafness in children,and it should be cautious when using such drugs inchildren with iron-deficient anemia.

Ototoxicity of styrene

Styrene is extensively used in industry,but its ototoxicity,in particular in the pregnant female and the offspring,is still not well understood.In the current study,young adult male rats and pregnant female rats were exposed to styrene by gavage at different doses.The young adult male rats received a total of 12g/kg styrene within different periods (800 mg/kg/day for 5 days/week for 3 weeks,400 mg/kg/day for 5 days/week for 6 weeks,200 mg/kg/day for 5 days/week for 12 weeks,and 100 mg/kg/day for 5 days/week for 24 weeks) and the pregnant female rats received styrene at a dose of 400 mg/kg/day for 5 days/week for 6 weeks starting from the gestation day-4.Hearing loss and hair cell loss were assessed 5 days after the styrene treatment in the young adult male rats and in the mother rats.The cochlear impairments in the rat pups were examined 2 months after their birth.The styrene exposure caused hearing loss and hair cell loss starting from the mid-frequency region in the third row of outer hair cells (OHCs) and the impairments appeared to be related to the dosing level in each single day.Significantly,the styrene exposure to the pregnant rats interfered with auditory functional development of their fetus,leading to a deficit of cochlear amplification,although the OHCs appeared to develop well.The results indicate that a short-term high-level styrene exposure may be more ototoxic than a long-term low-level exposure for a similar total styrene dose and the styrene in the pregnant woman’s body may interfere with auditory development of their fetus.

Ototoxicity of povidone-iodine——A case report

Objective: The ototoxicity of povidone-iodine has been documented in animal studies. However, there is limited evidence of these ototoxic effects in humans. This is the first report to show the ototoxic effects of povidoneiodine in a human subject.Patient: A 36-year-old woman came to our hospital complaining of left unilateral persistent hearing loss. One month before presentation, her child had accidentally struck her on her left ear. She applied approximately three drops of povidone-iodine(10% weight/volume) into her left auditory canal. Immediately after application, she felt severe pain and vertigo. An audiogram revealed severe left unilateral sensorineural hearing loss. Magnetic resonance imaging showed mild enhancement of the left vestibule and basal turn of the left cochlea.Conclusions: Even a single application of povidone-iodine could cause significant hearing loss and disequilibrium. It should, therefore, be used with caution.

Ototoxicity of vancomycin in guinea pigs

Objective: To study the effect of vancomycin (V) with multiple intravenous injections on the inner ear of albino guinea pigs. Methods: Three groups of animals were injected with vancomycin hydrochloride (54, 108, 216 mg/kg respectively once a day for 14 d). Two groups were treated with gentamycin (GM) (80 mg·kg -1 ·d -1 ) and saline respectively as control groups. Auditory brainstem responses (ABR) and the duration of post-rotatory nystagmus (PRN) were measured before and after administration. Surface preparation and scanning electron microscopy (SEM) of the cochlea were performed for histological examination. Results: In V 54, 108 mg/kg group, similar to saline control, there was 0-1.1 dB of threshold shift. In V 216 mg/kg group, average hearing loss was 1.0-5.7 dB immediately after administration and 1.3-3.8 dB after 14 d, which was significantly lower than those in GM control group. As the saline control, no significant difference was found in PRN in all V groups before and after treatment; while in the GM group, PRN decreased significantly after treatment. Morphological evaluation demonstrated that in all V and saline animals there was no obvious missing of outer and inner hair cells and SEM showed normal surface morphology. In the GM group, there was 10%-30% of outer and inner hair cells lost in the basal turn. Conclusion: The ototoxicity of vancomycin is absent or minimal after multiple introvenous administration within this dose range.

ULTRASTRUCTURAL OBSERVATIONS ON REGENERATING HAIR CELLS IN THE CHICK BASILAR PAPILLA FOLLOWING AMINOGLYCOSIDE OTOTOXICITY

Newly hatched chicks were injected with so mg/(kg' d) of gentamycin sulfate (GM )for 10 days. After 18st injection, animals were killed on survival days 3,6 .9 and 12. The chick basilar papillae (BP) were observed by transmission electron microscopy (TEM ). The results sho've'Ithat regenerated hair cells were erupted to the hasal membrane surrace in the early phase. The cytoplasm of these regenerated hair cells was heavily stained an contained numerous mitochondria. Afterseveral days, stereocilia appeared. When stereocilis bundles were rormed and cuticular layer was integrated, hasal nerve terminals were round. The results suggested that regenerating hair cells 'veredirectly produced on the hasal membrane surface following gentamycin induced ototoxlcity and thenI,roliferated and developed Into mature normal hair cells.

PGC-1α-mediated imbalance of mitochondria-lipid droplet homeostasis in neomycin-induced ototoxicity and nephrotoxicity

Ototoxicity and nephrotoxicity are the most prevalent side effects of aminoglycoside antibiotics(gentamicin,amikacin,neomycin)and platinum anti-tumor drugs(cisplatin,carboplatin).The inner ear and kidney share similarities in drug deposition and toxicity,but the underlying pathophysiological mechanisms remain unclear.Investigating the shared mechanisms and metabolic alterations in these distinct organs will provide valuable insights for clinical therapy.A strong correlation has been identified between the spatiotemporal accumulation patterns of neomycin and the specific occurrence of lipid metabolism disorders in these two organs.The primary allocation of neomycin to mitochondria results in a notable escalation in the accumulation of lipid droplets(LDs)and more interactions between mitochondria and LDs,leading to a sequence of disturbances in lipid metabolism,such as increased lipid ROS and the blocked transfer of fatty acids from LDs to mitochondria.PGC-1αdeficiency worsens the neomycin-induced disorders in lipid metabolism and intensifies the pathological interactions between mitochondria and LDs,as indicated by the exacerbated disturbance of dynamic LD turnover,increased level of oxidized lipids and decreased use of fatty acids.This investigation provides a fresh perspective on the lipid metabolic dysfunction related to mitochondria-LD interactions in drug-induced ototoxicity and nephrotoxicity,potentially providing novel avenues for intervention strategies.

Glycodiversification of gentamicins through in vivo glycosyltransferase swapping enabled the creation of novel hybrid aminoglycoside antibiotics with potent activity and low ototoxicity

Aminoglycosides(AGs)are a class of antibiotics with a broad spectrum of activity.However,their use is limited by safety concerns associated with nephrotoxicity and ototoxicity,as well as drug resistance.To address these issues,semi-synthetic approaches for modifying natural AGs have generated new generations of AGs,however,with limited types of modification due to significant challenges in synthesis.This study explores a novel approach that harness the bacterial biosynthetic machinery of gentamicins and kanamycins to create hybrid AGs.This was achieved by glycodiversification of gentamicins via swapping the glycosyltransferase(GT)in their producer with the GT from kanamycins biosynthetic pathway and resulted in the creation of a series of novel AGs,therefore referred to as genkamicins(GKs).The manipulation of the hybrid biosynthetic pathway enabled the targeted accumulation of different GK species and the isolation and characterization of six GK components.These compounds display retained antimicrobial activity against a panel of World Health Organization(WHO)critical priority pathogens,and GK-C2a,in particular,demonstrates low ototoxicity compared to clinical drugs in zebrafish embryos.This study provides a new strategy for diversifying the structure of AGs and a potential avenue for developing less toxic AG drugs to combat infectious diseases.

Effects of Erlong Zuoci Pill (耳聋左慈丸) and Its Disassembled Prescriptions on Gentamicin-induced Ototoxicity Model in vitro

Objective：To study the effects of Erlong Zuoci Pill（耳聋左慈丸,ELZCP）and its disassembled prescriptions on gentamicin（GM）-induced ototoxicity model in vitro.Methods：After the spiral organ of cochleae of newborn mice（postnatal days：2-3）cultured for 24 h,GM alone or combined with water extracting-alcohol precipitating solution of ELZCP or with its disassembled prescriptions was added.Hair cells were observed under a fluorescence microscope after TRITC-phalloidin staining,and the cochlear hair cell loss rate was calculated by counting the whole cochlear hair cells and analyzed by whole cochlear hair cells analyzing software.Results： GM induced cochlear outer hair cells（OHCs）and inner hair cells（IHCs）injuries in a dose-dependent manner, and they were significantly different as compared with those in the normal control group（P〈0.05,P〈0.01）. ELZCP at the concentration of 0.003-3 mg/mL could decrease the hair cells loss induced by the 0.3 mmol/L GM （P〈0.05,P〈0.01）,the effects was in a dose-dependent manner,and the concentration of 0.3 mg/mL showed the optimal protective effect.For the ELZCP disassembled prescriptions,Liuwei-Dihuang could decrease OHC loss rate than that in the 0.3 mmol/L GM model group（P〈0.05）,but the OHC loss rate was still higher than that in the ELZCP group（P〈0.01）,which indicated that the protective effect of hair cells by Liuwei-Dihuang was not better than that of ELZCP.Poria decreased OHC loss rate from 72.1%±3.7%to 58.8%±8.2%（P〈0.05）. Conclusions：ELZCP could play a role in antagonizing the injury of cochlear hair cells induced by GM ototoxicity, and its disassembled prescriptions,Liuwei-Dihuang was the main component to protect the cochlear hair cells from GM-induced ototoxicity,and Magnetitum combined with Radix Bupleurui could strengthen the action of the whole prescription; Poria could reduce GM-induced OHC loss.

Aminoglycoside ototoxicity in three murine strains and effects on NKCC1 of stria vascularis

Background After establishing a murine model of aminoglycoside antibiotic （AmAn） induced ototoxicity, the sensitivity of AmAn induced ototoxicity in three murine strains and the effect of kanamycin on the expression of Na-K-2Cl cotransporter-1 （NKCC1） in stria vascularis were investigated. Methods C57BL/6J, CBA/CaJ, NKCC1^＋/- mice （24 of each strain） were randomly divided into four experimental groups： A： kanamycin alone; B： kanamycin plus 2,3-dihydroxybenzoate; C： 2,3-dihydroxybenzoate alone; and D： control group. Mice were injected with kanamycin or/and 2,3-dihydroxybenzoate twice daily for 14 days. Auditory brainstem response （ABR） was measured and morphology of cochlea delineated with succinate dehydrogenase staining. Expression of NKCC1 in stria vascularis was detected immunohistochemically. Results All three strains in groups A and B developed significant ABR threshold shifts （P〈0.01）, which were accompanied by outer hair cell loss. NKCC 1 expression in stria vascularis was the weakest in group A （A cf D, P〈0.01） and the strongest in groups C and D （P〈0.05）. CBA/CaJ mice had the highest sensitivity to AmAn.Conclusions Administration of kanamycin established AmAn induced ototoxicity. Kanamycin inhibited the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate attenuated AmAn induced ototoxicity-possibly by enhancing the expression of NKCC 1. Age related hearing loss did not show additional sensitivity to AmAn induced ototoxicity in murine model.

Astaxanthine attenuates cisplatin ototoxicity in vitro and protects against cisplatin-induced hearing loss in vivo

Astaxanthine(AST) has important biological activities including antioxidant and antiinflammatory effects that could alleviate neurological and heart diseases, but its role in the prevention of cisplatin-induced hearing loss(CIHL) is not yet well understood. In our study, a steady interaction between AST and the E3 ligase adapter Kelch-like ECH-associated protein 1, a predominant repressor of nuclear factor erythroid 2-related factor 2(NRF2), was performed and tested via computer molecular docking and dynamics. AST protected against cisplatin-induced ototoxicity via NRF2 mediated pathwayusing quantitative PCR and Western blotting. The levels of reactive oxygen species(ROS) and mitochondrial membrane potential revealed that AST reduced ROS overexpression and mitochondrial dysfunction.Moreover, AST exerted anti-apoptosis effects in mouse cochlear explants using immunofluorescence staining and HEI-OC1 cell lines using quantitative PCR and Western blotting. Finally, AST combined with poloxamer was injected into the middle ear through the tympanum, and the protection against CIHL was evaluated using the acoustic brain stem test and immunofluorescent staining in adult mice. Our results suggest that AST reduced ROS overexpression, mitochondrial dysfunction, and apoptosis via NRF2-mediated pathway in cisplatin-exposed HEI-OC1 cell lines and mouse cochlear explants, finally promoting cell survival. Our study demonstrates that AST is a candidate therapeutic agent for CIHL.

Effect of Ligustrazine on Kanamycin Ototoxicity

Objective: To evaluate the effect of ligustrazine on kanamycin ototoxicity. Methods: By measuring superoxide dismutase (SOD) and malondialdehyde (MDA) in cochlear tissues, compound action potentials (CAP) and cochlear microphonics (CM) in guinea-pigs which were treated with kanamycin and ligustrazine. Results : Kanamycin produced a severe hearing loss and significant increase in SOD and MDA. Ligustrazine could reduce these changes significantly and improve hearing of animals. Conclusion: Lipid peroxidativereaction is correlated with kanamycin ototoxicity and ligustrazine could prevent and treat kanamycin ototoxicity by antioxidation.

Hidden Hearing Loss: Causes, Current Knowledge, and Future Directions

Introduction: Hidden hearing loss (HHL) is a type of auditory disorder that affects the auditory neural processing and hearing sensitivity in subjects with normal hearing thresholds. Unlike central auditory processing disorders, HHL happens when the cochlea (the peripheral auditory organ) is affected. There are several known risk factors to HHL which includes noise exposure, ototoxic drugs, and peripheral neuropathies, and age. Recent studies have shed light on this type of hearing loss, its etiology, prevalence, and how it can affect the auditory acuity in humans. Methods: This paper covers the current research regarding HHL, its causes, the different mechanisms involved in this disorder, and the diagnosis and potential treatments related to it. We will delve deeply into different researches concerning HHL. 4 articles from 285 were selected focusing on normal hearing individuals with bad speech intelligibility were discussed in this paper. In addition, articles discussing the effects of noise exposure on hearing impaired individuals were not considered as this study solely aims to focus on normal hearing sensitivity individuals with HHL, resulting in 4 articles from 285. Results: Numerous literatures over the decades have suggested that HHL is due to the degeneration of cochlear ribbon synapses, or hair cells synapses without hair cell damage. Their association with HHL was noted several times through this study, whether we were studying the effect of noise exposure, of age, or of ototoxicity. In all cases, no significant hair cell damage was observed, and normal thresholds were recovered. However, a decline in the amplitude of Auditory Brainstem Response (ABR) peak I from auditory nerve (AN) responses in noise exposed subjects and a decline in compound action potential (CAP) was measured when certain drugs were applied to the round window of Guinea pigs. Conclusion: Most studies, have proven that cochlear synaptophysin is the major contributor to noise induced, age, and ototoxic related HHL. There are several audiometric tests that were used to help identify HHL including Puretone audiometry in background noise, ABR, CAP, Distortion Product Otoacoustic Emission (DPOAE).

Detection of apoptosis by RT-PCR array in mefloquine-induced cochlear damage

Objective To investigate the occurrence and possible mechanisms of apoptosis in cochlear epithelium and spiral ganglion neurons after mefloquine treatment. Methods We used quantitative RT-PCR apoptosis-focused gene arrays （96-well, 84 apoptosis related genes） to assess changes of gene expression in the cochlear basilar membrane （hair cells-supporting cells） and spiral ganglion neurons of rat cochlear organotypic cultures treated with 100 IxM mefloquine for 3 h. Results Significant up-or down-regulation in gene expression was detected in 23 genes in the cochlear basilar membrane, and in 32 genes in the spiral ganglion neurons compared with time-matched controls. The responding genes could be classified as pro-or anti-apoptotic, and were mainly implicated in the Bcl-2, Caspase, Card, IAP, TNF ligand / TNF receptor, Death domain / Death effector domain, DNA damage / p53, and NF-kappa B families. Synthetic analysis suggested that these families could be revised to two major pathways mainly involved in t]he death receptor-mediated signaling pathway and apoptotic mitochondrial pathway. In addition, it was found that numerous anti-apoptotic genes such as Bcl2al, Birclb, Birc3, Birc4, Bnipl, Cflar, II10, Lhx4, Mcll, Nfkbl, Prlr, Prok2, and TNF were greatly up-regulated in the cochlear tissue, which might imply the co-existence of protective response in the ceils at the early stage of mefloquine-induced damage.

OTOTOXIC EFFECTS OF CARBOPLATIN IN ORGANOTYPIC CULTURES IN CHINCHILLAS AND RATS

Carboplatin, a second-generation platinum chemotherapeutic drug, is considerably less ototoxic than cisplatin. While common laboratory species such as mice, guinea pigs and rats are highly resistant to carboplatin ototoxicity, the chinchilla stands out as highly susceptible. Moreover, carboplatin causes an unusual gradient of cell death in chinchillas. Moderate doses selectively damage type I spiral ganglion neurons (SGN) and inner hair cells (IHC) and the lesion tends to be relatively uniform along the length of the cochlea. Higher doses eventually damage outer hair cells (OHC), but the lesion follows the traditional gradient in which damage is more severe in the base than the apex. While carboplatin ototoxicity has been well documented in adult animals in vivo, little is known about its in vitro toxicity. To elucidate the ototoxic effects of carboplatin in vitro, we prepared cochlear and vestibular organotypic cultures from postnatal day 3 rats and adult chinchillas. Chinchilla cochlear and vestibular cultures were treated with carboplatin concentrations ranging from 50 μM to 10 mM for 48 h. Consistent with in vivo data, carboplatin selectively damaged IHC at low concentrations (50-100 μM). Surprisingly, IHC loss decreased at higher doses and IHC were intact at doses exceeding 500 μM. The mechanisms underlying this nonlinear response are unclear but could be related to a decrease in carboplatin uptake via active transport mechanisms (e.g., copper). Unlike the cochlea, the carboplatin dose-response function increased with dose with the highest dose destroying all chinchilla vestibular hair cells. Cochlear hair cells and auditory nerve fibers in rat cochlear organotypic cultures were unaffected by carboplatin concentrations <10 μM; however, the damage in OHC were more severe than IHC once the dose reached 100 μM. A dose at 500 μM destroyed all the cochlear hair cells, but hair cell loss decreased at high concentrations and nearly all the cochlear hair cells were present at the highest dose, 5 mM. Unlike the nonlinear dose-response seen with cochlear hair cells, rat auditory nerve fiber and spiral ganglion losses increased with doses above 50 μM with the highest dose destroying virtually all SGN. The remarkable species differences seen in vitro suggest that chinchilla IHC and type I SGN posse some unique biological mechanism that makes them especially vulnerable to carboplatin toxicity.