Ultrasound-Mediated Lysozyme Microbubbles Targeting NOX4 Knockdown Alleviate Cisplatin-Exposed Cochlear Hair Cell Ototoxicity.

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.

Protective effects of Y-27632 against cisplatin-induced ototoxicity: A zebrafish model Y-27632 and cisplatin-induced ototoxicity.

Cisplatin is an effective chemotherapy agent against various solid malignancies; however, it is associated with irreversible bilateral sensorineural hearing loss, emphasizing the need for drug development to prevent this complication, with the current options being very limited. Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine protein kinase involved in various cellular processes, including apoptosis regulation. In this study, we used a transgenic zebrafish model (Brn3C: EGFP) in which hair cells within neuromasts are observed in green under fluorescent microscopy without the need for staining. Zebrafish larvae were exposed to cisplatin alone or in combination with various concentrations of Y-27632, a potent ROCK inhibitor. Hair cell counts, apoptosis assessments using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay, FM1-43FX labeling assay and behavioral analyses (startle response and rheotaxis) were performed to evaluate the protective effects of Y-27632 against cisplatin-induced ototoxicity. Cisplatin treatment reduced the number of hair cells in neuromasts, induced apoptosis, and impaired zebrafish larval behaviors. Y-27632 demonstrated a dose-dependent protective effect against cisplatin-induced hair cell loss and apoptosis. These findings suggest that Y-27632, as a ROCK inhibitor, mitigates cisplatin-induced hair cell loss and associated ototoxicity in zebrafish.

The miR-182-5p/GPX4 Pathway Contributes to Sevoflurane-Induced Ototoxicity via Ferroptosis.

Our study aimed to investigate the role of ferroptosis in sevoflurane-induced hearing impairment and explore the mechanism of the microRNA-182-5p (miR-182-5p)/Glutathione Peroxidase 4 (GPX4) pathway in sevoflurane-induced ototoxicity. Immunofluorescence staining was performed using myosin 7a and CtBP2. Cell viability was assessed using the CCK-8 kit. Fe2+ concentration was measured using FerroOrange and Mi-to-FerroGreen fluorescent probes. The lipid peroxide level was assessed using BODIPY 581/591 C11 and MitoSOX fluorescent probes. The auditory brainstem response (ABR) test was conducted to evaluate the hearing status. Bioinformatics tools and dual luciferase gene reporter analysis were used to confirm the direct targeting of miR-182-5p on GPX4 mRNA. GPX4 and miR-182-5p expression in cells was assessed by qRT-PCR and Western blot. Ferrostatin-1 (Fer-1) pretreatment significantly improved hearing impairment and damage to ribbon synapses in mice caused by sevoflurane exposure. Immunofluorescence staining revealed that Fer-1 pretreatment reduced intracellular and mitochondrial iron overload, as well as lipid peroxide accumulation. Our findings indicated that miR-182-5p was upregulated in sevoflurane-exposed HEI-OC1 cells, and miR-182-5p regulated GPX4 expression by binding to the 3'UTR of GPX4 mRNA. The inhibition of miR-182-5p attenuated sevoflurane-induced iron overload and lipid peroxide accumulation. Our study elucidated that the miR-182-5p/GPX4 pathway was implicated in sevoflurane-induced ototoxicity by promoting ferroptosis.

Quantitative profiling of cochlear synaptosomal proteins in cisplatin-induced synaptic dysfunction.

The disruption of ribbon synapses in the cochlea impairs the transmission of auditory signals from the cochlear sensory receptor cells to the auditory cortex. Although cisplatin-induced loss of ribbon synapses is well-documented, and studies have reported nitration of cochlear proteins after cisplatin treatment, yet the underlying mechanism of cochlear synaptopathy is not fully understood. This study tests the hypothesis that cisplatin treatment alters the abundance of cochlear synaptosomal proteins, and selective targeting of nitrative stress prevents the associated synaptic dysfunction. Auditory brainstem responses of mice treated with cisplatin showed a reduction in amplitude and an increase in latency of wave I, indicating cisplatin-induced synaptic dysfunction. The mass spectrometry analysis of cochlear synaptosomal proteins identified 102 proteins that decreased in abundance and 249 that increased in abundance after cisplatin treatment. Pathway analysis suggested that the dysregulated proteins were involved in calcium binding, calcium ion regulation, synapses, and endocytosis pathways. Inhibition of nitrative stress by co-treatment with MnTBAP, a peroxynitrite scavenger, attenuated cisplatin-induced changes in the abundance of 27 proteins. Furthermore, MnTBAP co-treatment prevented the cisplatin-induced decrease in the amplitude and increase in the latency of wave I. Together, these findings suggest a potential role of oxidative/nitrative stress in cisplatin-induced cochlear synaptic dysfunction.

Ototoxicity in Immune Checkpoint Inhibitors Therapy.

<b><br>Introduction:</b> Immune checkpoint inhibitors (ICIs) and T-cell therapies are a modern, well-established cancer treatment. The priority of oncological treatment is to cure cancer. However, treatment-related toxicities, i.e. immune-related adverse events (irAEs), continue to emerge and are not that well understood yet. ICIs can cause profound, multiple, and diverse irAEs - the sequelae of unknown mechanisms. One of the organs susceptible to collateral damage is the hearing organ. Complications related to hearing, tinnitus, and balance disorders are extremely burdensome and significantly impair many aspects of the quality of life of patients and survivors.</br> <b><br>Aim:</b> The aim of the work is to review the literature in the area of ototoxicity of ICIs.</br> <b><br>Materials and method:</b> A systematic search of the Web of Science, PubMed, and Embase databases for studies published until 1 March 2022 was conducted.</br> <b><br>Results:</b> Reported clinical symptoms ranged from sudden bilateral hearing loss and imbalance to mild hearing loss or tinnitus with preserved hearing. It was found that the median time from ICI initiation to hearing loss development was 3 months. The hearing impairment was secondary to bilateral sensorineural hearing loss in the majority of patients (>60%), and at least one other irAE accompanied the hearing loss in 2/3 of patients. Hearing loss significantly improved in 45.7% of the patients.</br> <b><br>Conclusions:</b> The majority of cases of ICI-related hearing loss presented in the literature were reversible. Therefore, it is important to develop and implement routine therapeutic algorithms. Further research is needed to define the true prevalence of ICI-related hearing loss, optimal diagnostics, and management.</br>.

Susceptibility of mouse cochlear hair cells to cisplatin ototoxicity largely depends on sensory mechanoelectrical transduction channels both Ex Vivo and In Vivo.

Cisplatin, a highly effective chemotherapeutic drug for various human cancers, induces irreversible sensorineural hearing loss as a side effect. Currently there are no highly effective clinical strategies for the prevention of cisplatin-induced ototoxicity. Previous studies have indicated that short-term cisplatin ototoxicity primarily affects the outer hair cells of the cochlea. Therefore, preventing the entry of cisplatin into hair cells may be a promising strategy to prevent cisplatin ototoxicity. This study aimed to investigate the entry route of cisplatin into mouse cochlear hair cells. The competitive inhibitor of organic cation transporter 2 (OCT2), cimetidine, and the sensory mechanoelectrical transduction (MET) channel blocker benzamil, demonstrated a protective effect against cisplatin toxicity in hair cells in cochlear explants. Sensory MET-deficient hair cells explanted from Tmc1Δ;Tmc2Δ mice were resistant to cisplatin toxicity. Cimetidine showed an additive protective effect against cisplatin toxicity in sensory MET-deficient hair cells. However, in the apical turn, cimetidine, benzamil, or genetic ablation of sensory MET channels showed limited protective effects, implying the presence of other entry routes for cisplatin to enter the hair cells in the apical turn. Systemic administration of cimetidine failed to protect cochlear hair cells from ototoxicity caused by systemically administered cisplatin. Notably, outer hair cells in MET-deficient mice exhibited no apparent deterioration after systemic administration of cisplatin, whereas the outer hair cells in wild-type mice showed remarkable deterioration. The susceptibility of mouse cochlear hair cells to cisplatin ototoxicity largely depends on the sensory MET channel both ex vivo and in vivo. This result justifies the development of new pharmaceuticals, such as a specific antagonists for sensory MET channels or custom-designed cisplatin analogs which are impermeable to sensory MET channels.

Risk factors associated with ototoxicity in long-term nasopharyngeal carcinoma survivors.

PURPOSE: To investigate patient-reported outcomes among long-term survivors and to analyze their associated risk factors to provide better treatment and symptom management for nasopharyngeal carcinoma patients. MATERIALS AND METHODS: This retrospective study collected patients diagnosed with nasopharyngeal carcinoma who received radical intensity-modulated radiotherapy in our hospital from June 2009 to June 2016. The patients' disease status and patient-reported outcomes were analyzed by follow-up. The ototoxicity was graded according to CTCAE 5.0. RESULTS: A total of 223 patients were included in the study. Among the enrolled patients, the median follow-up time was 8.4 (6.0-13.0) years. Based on the patient-reported outcomes, ototoxicity was the most common symptom (52.9 %). After univariable and multivariable logistic regression, age ≥ 50 years old (OR, 4.066; 95 % CI, 1.799-9.190; P = .001), diabetes (OR, 3.520; 95 % CI, 1.442-8.591; P = .006), D2 ≥ 69 Gy (OR, 3.715; 95 % CI, 1.064-12.969; P = . 040) and V35 ≥ 91.5 % (OR, 3.398; 95 % CI, 1.113-10.372; P = .032) were associated with a higher incidence of grade 3-4 ototoxicity. Then, we constructed the individual nomogram and the C index of the graph was 0.815. By univariable logistic regression, we found that grade 3-4 ototoxicity was associated with an increased risk of multiple other symptoms, dysmasesia, tongue dysfunction, hoarseness, dysphagia and ocular toxicity. CONCLUSION: In long-term survivors of nasopharyngeal carcinoma patients receiving IMRT, the most common patient-reported outcome was ototoxicity. Age ≥ 50 years, diabetes, ear exposure dose of D2 ≥ 69 Gy and V35 ≥ 91.5 % are independent risk factors for grade 3-4 ototoxicity.

Suppression of the TGF-ß signaling exacerbates degeneration of auditory neurons in kanamycin-induced ototoxicity in mice.

Transforming growth factor-ß (TGF-ß) signaling plays a significant role in multiple biological processes, including inflammation, immunity, and cell death. However, its specific impact on the cochlea remains unclear. In this study, we aimed to investigate the effects of TGF-ß signaling suppression on auditory function and cochlear pathology in mice with kanamycin-induced ototoxicity. Kanamycin and furosemide (KM-FS) were systemically administered to 8-week-old C57/BL6 mice, followed by immediate topical application of a TGF-ß receptor inhibitor (TGF-ßRI) onto the round window membrane. Results showed significant TGF-ß receptor upregulation in spiral ganglion neurons (SGNs) after KM-FA ototoxicity, whereas expression levels in the TGF-ßRI treated group remained unchanged. Interestingly, despite no significant change in cochlear TGF-ß expression after KM-FS ototoxicity, TGF-ßRI treatment resulted in a significant decrease in TGF-ß signaling. Regarding auditory function, TGF-ßRI treatment offered no therapeutic effects on hearing thresholds and hair cell survival following KM-FS ototoxicity. However, SGN loss and macrophage infiltration were significantly increased with TGF-ßRI treatment. These results imply that inhibition of TGF-ß signaling after KM-FS ototoxicity promotes cochlear inflammation and SGN degeneration.

SERCA2 protects against cisplatin-induced damage of auditory cells: Possible relation with alleviation of ER stress.

AIMS: SERCA2, one of the P-type pumps encoded by gene ATP2A2, is the only calcium reflux channel of the endoplasmic reticulum (ER) and participates in maintaining calcium homeostasis. The present study was designed to explore SERCA2 expression pattern in auditory hair cells and the possible mechanism underlying the effects of SERCA2 on cisplatin-induced ototoxicity. MAIN METHODS: The SERCA2 expression pattern in cochlea hair cells and HEI-OC1 cells was measured by Western blot (WB) and immunofluorescence staining. The apoptosis and its related factors were detected by TUNEL assay and WB. The expression levels of ER stress-related factors, ATF6, PERK, IRE1α, and GRP78, were measured via WB. As for the determination of SERCA2 overexpression and knockdown, plasmids and lentiviral vectors were constructed, respectively. KEY FINDINGS: We found that SERCA2 was highly expressed in cochlea hair cells and HEI-OC1 cells. Of note, the level of SERCA2 expression in neonatal mice was remarkably higher than that in adult mice. Under the exposure of 30 µM cisplatin, SERCA2 was down-regulated significantly compared with the control group. In addition, cisplatin administration triggered the occurrence of ER stress and apoptosis. Those events were reversed by overexpressing SERCA2. On the contrary, SERCA2 knockdown could aggravate the above processes. SIGNIFICANCE: The findings from the present study disclose, for the first time, that SERCA2 is abundantly expressed in cochlea hair cells, and the suppression of SERCA2 caused by cisplatin could trigger ER homeostasis disruption, thereby implying that SERCA2 might be a promising target to prevent cisplatin-induced cytotoxicity of hair cells.

Serum Prestin After Ototoxin Exposure Is Not Dependent on Outer Hair Cell Loss.

HYPOTHESIS: Cyclodextrin (CDX)-induced serum prestin burst is not dependent on outer hair cell (OHC) loss. BACKGROUND: Serum prestin has been proposed as a biomarker for ototoxicity. We recently used an automated Western approach to quantify serum prestin changes in a newly introduced model of CDX ototoxicity. To gain insights into prestin as a biomarker, here we further characterize serum prestin in the CDX model. METHODS: Guinea pigs were treated with 750, 3,000, or 4,000 mg/kg CDX, and serum samples were obtained through up to 15 weeks after exposure. Serum prestin levels were quantified using automated Western, and hair cell counts were obtained. RESULTS: All three doses induced an N -glycosylated ~134-kDa prestin burst; however, only the 3,000 and 4,000 mg/kg resulted in robust OHC loss. Prestin levels returned to baseline where they remained up to 15 weeks in the absence of OHCs. CONCLUSION: The ~134-kDa prestin burst induced after CDX administration is N -glycosylated, representing a posttranslational modification of prestin. Serum prestin seems to be a promising biomarker when using therapeutics with ototoxic properties because it is not dependent on OHC loss as a necessary event, thus affording the opportunity for early detection and intervention.

Calcium channel inhibitor and extracellular calcium improve aminoglycoside-induced hair cell loss in zebrafish.

Aminoglycosides are commonly used antibiotics for treatment of gram-negative bacterial infections, however, they might act on inner ear, leading to hair-cell death and hearing loss. Currently, there is no targeted therapy for aminoglycoside ototoxicity, since the underlying mechanisms of aminoglycoside-induced hearing impairments are not fully defined. This study aimed to investigate whether the calcium channel blocker verapamil and changes in intracellular & extracellular calcium could ameliorate aminoglycoside-induced ototoxicity in zebrafish. The present findings showed that a significant decreased number of neuromasts in the lateral lines of zebrafish larvae at 5 days' post fertilization after neomycin (20 µM) and gentamicin (20 mg/mL) exposure, which was prevented by verapamil. Moreover, verapamil (10-100 µM) attenuated aminoglycoside-induced toxic response in different external calcium concentrations (33-3300 µM). The increasing extracellular calcium reduced hair cell loss from aminoglycoside exposure, while lower calcium facilitated hair cell death. In contrast, calcium channel activator Bay K8644 (20 µM) enhanced aminoglycoside-induced ototoxicity and reversed the protective action of higher external calcium on hair cell loss. However, neomycin-elicited hair cell death was not altered by caffeine, ryanodine receptor (RyR) agonist, and RyR antagonists, including thapsigargin, ryanodine, and ruthenium red. The uptake of neomycin into hair cells was attenuated by verapamil and under high external calcium concentration. Consistently, the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin was also reduced by verapamil and high external calcium. Significantly, zebrafish larvae when exposed to neomycin exhibited decreased swimming distances in reaction to droplet stimulus when compared to the control group. Verapamil and elevated external calcium effectively protected the impaired swimming ability of zebrafish larvae induced by neomycin. These data imply that prevention of hair cell damage correlated with swimming behavior against aminoglycoside ototoxicity by verapamil and higher external calcium might be associated with inhibition of excessive ROS production and aminoglycoside uptake through cation channels. These findings indicate that calcium channel blocker and higher external calcium could be applied to protect aminoglycoside-induced listening impairments.

Hesperidin activates Nrf2 to protect cochlear hair cells from cisplatin-induced damage.

Cisplatin is widely employed in clinical oncology as an anticancer chemotherapy drug in clinical practice and is known for its severe ototoxic side effects. Prior research indicates that the accumulation of reactive oxygen species (ROS) plays a pivotal role in cisplatin's inner ear toxicity. Hesperidin is a flavanone glycoside extracted from citrus fruits that has anti-inflammatory and antioxidant effects. Nonetheless, the specific pharmacological actions of hesperidin in alleviating cisplatin-induced ototoxicity remain elusive. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical mediator of the cellular oxidative stress response, is influenced by hesperidin. Activation of Nrf2 was shown to have a protective effect against cisplatin-induced ototoxicity. The potential of hesperidin to stimulate Nrf2 in attenuating cisplatin's adverse effects on the inner ear warrants further investigation. This study employs both in vivo and in vitro models of cisplatin ototoxicity to explore this possibility. Our results reveal that hesperidin mitigates cisplatin-induced ototoxicity by activating the Nrf2/NQO1 pathway in sensory hair cells, thereby reducing ROS accumulation, preventing hair cell apoptosis, and alleviating hearing loss.

FDA-Approved Tedizolid Phosphate Prevents Cisplatin-Induced Hearing Loss Without Decreasing Its Anti-tumor Effect.

PURPOSE: Cisplatin is a low-cost clinical anti-tumor drug widely used to treat solid tumors. However, its use could damage cochlear hair cells, leading to irreversible hearing loss. Currently, there appears one drug approved in clinic only used for reducing ototoxicity associated with cisplatin in pediatric patients, which needs to further explore other candidate drugs. METHODS: Here, by screening 1967 FDA-approved drugs to protect cochlear hair cell line (HEI-OC1) from cisplatin damage, we found that Tedizolid Phosphate (Ted), a drug indicated for the treatment of acute infections, had the best protective effect. Further, we evaluated the protective effect of Ted against ototoxicity in mouse cochlear explants, zebrafish, and adult mice. The mechanism of action of Ted was further explored using RNA sequencing analysis and verified. Meanwhile, we also observed the effect of Ted on the anti-tumor effect of cisplatin. RESULTS: Ted had a strong protective effect on hair cell (HC) loss induced by cisplatin in zebrafish and mouse cochlear explants. In addition, when administered systemically, it protected mice from cisplatin-induced hearing loss. Moreover, antitumor studies showed that Ted had no effect on the antitumor activity of cisplatin both in vitro and in vivo. RNA sequencing analysis showed that the otoprotective effect of Ted was mainly achieved by inhibiting phosphorylation of ERK. Consistently, ERK activator aggravated the damage of cisplatin to HCs. CONCLUSION: Collectively, these results showed that FDA-approved Ted protected HCs from cisplatin-induced HC loss by inhibiting ERK phosphorylation, indicating its potential as a candidate for preventing cisplatin ototoxicity in clinical settings.

Ototoxicity among cisplatin, carboplatin, and oxaliplatin in zebrafish model.

Platinum-based antineoplastic drugs, including cisplatin, carboplatin, and oxaliplatin, are widely used in the treatment of various cancers. Ototoxicity is a common adverse effect of platinum-based drugs. Ototoxicity leads to irreversible hearing impairment. We hypothesize that different platinum-based drugs exhibit varying ototoxic concentrations, time effects, and ototoxic mechanisms. We tested this hypothesis by using a zebrafish model (pvalb3b: TagGFP) to assess the viability of hair cells collected from zebrafish larvae. Cisplatin, carboplatin, and oxaliplatin were administered at dosages of 100, 200, or 400 µM, and the ototoxic effects of these drugs were assessed 1, 2, or 3 h after administration. Fm4-64 and a TUNEL assay were used to label the membranes of living hair cells and to detect cell apoptosis, respectively. We observed that >50% of hair cells were damaged at 1 h after cisplatin (100 µM) exposure, and this ototoxic effect increased at higher dosages and over time. Owing to the smaller ototoxic effects of carboplatin and oxaliplatin, we conducted higher-strength and longer-duration experiments with these drugs. Neither carboplatin nor oxaliplatin was obviously ototoxic, even at 1600 µM and after 6 h. Moreover, only cisplatin damaged the membranes of the hair cells. Cell apoptosis and significantly increased antioxidant gene expression were observed in only the cisplatin group. In conclusion, cisplatin significantly damages sensory hair cells and has notable dosage and time effects. Carboplatin and oxaliplatin are less ototoxic than cisplatin, likely due to having different ototoxic mechanisms than cisplatin.

Pharmacogenetics of aminoglycoside-related ototoxicity: a systematic review.

BACKGROUND: Aminoglycosides (AGs) are important antibiotics in the treatment of Gram-negative sepsis. However, they are associated with the risk of irreversible sensorineural hearing loss (SNHL). Several genetic variants have been implicated in the development of ototoxicity. OBJECTIVES: To evaluate the pharmacogenetic determinants of AG-related ototoxicity. METHODS: This study followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses and was registered on Prospero (CRD42022337769). In Dec 2022, PubMed, Cochrane Library, Embase and MEDLINE were searched. Included studies were those reporting original data on the effect of the AG-exposed patient's genome on the development of ototoxicity. RESULTS: Of 10 202 studies, 31 met the inclusion criteria. Twenty-nine studies focused on the mitochondrial genome, while two studied the nuclear genome. One study of neonates found that 30% of those with the m.1555A > G variant failed hearing screening after AG exposure (level 2 evidence). Seventeen additional studies found the m.1555A > G variant was associated with high penetrance (up to 100%) of SNHL after AG exposure (level 3-4 evidence). Nine studies of m.1494C > T found the penetrance of AG-related SNHL to be up to 40%; however, this variant was also identified in those with SNHL without AG exposure (level 3-4 evidence). The variants m.1005T > C and m.1095T > C may be associated with AG-related SNHL; however, further studies are needed. CONCLUSIONS: This review found that the m.1555A > G and m.1494C > T variants in the MT-RNR1 gene have the strongest evidence in the development of AG-related SNHL, although study quality was limited (level 2-4). These variants were associated with high penetrance of a SNHL phenotype following AG exposure.

Ivacaftor attenuates gentamicin-induced ototoxicity through the CFTR-Nrf2-HO1/NQO1 pathway.

OBJECTIVES: Gentamicin is one of the most common ototoxic drugs that can lower patients' quality of life. Oxidative stress is a key factors inducing sensory hair cell death during gentamicin administration. So far, there are no effective drugs to prevent or treat gentamicin- induced hearing loss. A recent study found cystic fibrosis transmembrane conductance regulator (CFTR) as a new target to modulate cellular oxidative balance. The objective of this study was to estimate the effect of the CFTR activator ivacaftor on gentamicin-induced ototoxicity and determine its mechanism. METHODS: The hair cell count was analyzed by Myosin 7a staining. Apoptosis was analyzed by TUNEL Apoptosis Kit. Cellular reactive oxygen species (ROS) level was detected by DCFH-DA probes. The Nrf2 related proteins expression levels were analyzed by western blot. RESULTS: An in vitro cochlear explant model showed that gentamicin caused ROS accumulation in sensory hair cells and induced apoptosis, and this effect was alleviated by pretreatment with ivacaftor. Western blotting showed that ivacaftor administration markedly increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO1), and NAD(P)H:quinone oxidoreductase 1 (NQO1). The protective effect of ivacaftor was abolished by the Nrf2 inhibitor ML385. DISCUSSION: Our results indicate the protective role of the CFTR-Nrf2-HO1/NQO1 pathway in gentamicin-induced ototoxicity. Ivacaftor may be repositioned or repurposed towards aminoglycosides-induced hearing loss.

Nobiletin alleviates cisplatin-induced ototoxicity via activating autophagy and inhibiting NRF2/GPX4-mediated ferroptosis.

Nobiletin, a citrus polymethoxy flavonoid with antiapoptotic and antioxidative properties, could safeguard against cisplatin-induced nephrotoxicity and neurotoxicity. Cisplatin, as the pioneer of anti-cancer drug, the severe ototoxicity limits its clinical applications, while the effect of nobiletin on cisplatin-induced ototoxicity has not been identified. The current study investigated the alleviating effect of nobiletin on cisplatin-induced ototoxicity and the underlying mechanisms. Apoptosis and ROS formation were evaluated using the CCK-8 assay, Western blotting, and immunofluorescence, indicating that nobiletin attenuated cisplatin-induced apoptosis and oxidative stress. LC3B and SQSTM1/p62 were determined by Western blotting, qPCR, and immunofluorescence, indicating that nobiletin significantly activated autophagy. Nobiletin promoted the nuclear translocation of NRF2 and the transcription of its target genes, including Hmox1, Nqo1, and ferroptosis markers (Gpx4, Slc7a11, Fth, and Ftl), thereby inhibiting ferroptosis. Furthermore, RNA sequencing analysis verified that autophagy, ferroptosis, and the NRF2 signaling pathway served as crucial points for the protection of nobiletin against ototoxicity caused by cisplatin. Collectively, these results indicated, for the first time, that nobiletin alleviated cisplatin-elicited ototoxicity through suppressing apoptosis and oxidative stress, which were attributed to the activation of autophagy and the inhibition of NRF2/GPX4-mediated ferroptosis. Our study suggested that nobiletin could be a prospective agent for preventing cisplatin-induced hearing loss.

Combined genetic polymorphisms of the GSTT1 and NRF2 genes increase susceptibility to cisplatin-induced ototoxicity: A preliminary study.

OBJECTIVE: The genotype-phenotype relationship in cisplatin-induced ototoxicity remains unclear. By assessing early shifts in distortion product otoacoustic emission (DPOAE) levels after initial cisplatin administration, we aimed to discriminate patients' susceptibility to cisplatin-induced ototoxicity and elucidate their genetic background. STUDY DESIGN: A prospective cross-sectional study. SETTING: Tertiary referral hospital in Japan. PATIENTS: Twenty-six patients with head and neck cancer were undergoing chemoradiotherapy with three cycles of 100 mg/m2 cisplatin. INTERVENTIONS: Repetitive pure-tone audiometry and DPOAE measurements, and blood sampling for DNA extraction were performed. Patients were grouped into early ototoxicity presence or absence based on whether DPOAE level shifts exceeded the corresponding reference limits of the 21-day test interval. MAIN OUTCOME MEASURES: Hearing thresholds after each cisplatin cycle, severity of other adverse events, and polymorphisms in cisplatin-induced ototoxicity-associated genes were compared. RESULTS: Early ototoxicity was present in 14 and absent in 12 patients. Ototoxicity presence on DPOAEs was associated with greater progression of hearing loss in frequencies ≥2 kHz throughout therapy and with higher ototoxicity grades compared with ototoxicity absence. Ototoxicity was further associated with grade ≥2 nausea. Ototoxicity presence was genetically associated with the GSTT1 null genotype and G-allele of NFE2L2 rs6721961, whereas ototoxicity absence was associated with the GSTM1 null genotype. Dose-dependent progression of hearing loss was the greatest in the combined genotype pattern of GSTT1 null and the T/G or G/G variants of rs6721961. CONCLUSION: Early DPOAE changes reflected genetic vulnerability to cisplatin-induced ototoxicity. Hereditary insufficiency of the antioxidant defense system causes severe cisplatin-induced hearing loss and nausea.

Inhibition of CISD1 attenuates cisplatin-induced hearing loss in mice via the PI3K and MAPK pathways.

Cisplatin is an effective chemotherapeutic drug for different cancers, but it also causes severe and permanent hearing loss. Oxidative stress and mitochondrial dysfunction in cochlear hair cells (HCs) have been shown to be important in the pathogenesis of cisplatin-induced hearing loss (CIHL). CDGSH iron sulfur domain 1 (CISD1, also known as mitoNEET) plays a critical role in mitochondrial oxidative capacity and cellular bioenergetics. Targeting CISD1 may improve mitochondrial function in various diseases. However, the role of CISD1 in cisplatin-induced ototoxicity is unclear. Therefore, this study was performed to assess the role of CISD1 in cisplatin-induced ototoxicity. We found that CISD1 expression was significantly increased after cisplatin treatment in both HEI-OC1 cells and cochlear HCs. Moreover, pharmacological inhibition of CISD1 with NL-1 inhibited cell apoptosis and reduced mitochondrial reactive oxygen species accumulation in HEI-OC1 cells and cochlear explants. Inhibition of CISD1 with small interfering RNA in HEI-OC1 cells had similar protective effects. Furthermore, NL-1 protected against CIHL in adult C57 mice, as evaluated by the auditory brainstem response and immunofluorescent staining. Mechanistically, RNA sequencing revealed that NL-1 attenuated CIHL via the PI3K and MAPK pathways. Most importantly, NL-1 did not interfere with the antitumor efficacy of cisplatin. In conclusion, our study revealed that targeting CISD1 with NL-1 reduced reactive oxygen species accumulation, mitochondrial dysfunction, and apoptosis via the PI3K and MAPK pathways in HEI-OC1 cell lines and mouse cochlear explants in vitro, and it protected against CIHL in adult C57 mice. Our study suggests that CISD1 may serve as a novel target for the prevention of CIHL.