Molecular Mechanisms of Aminoglycoside-Induced Ototoxicity in Murine Auditory Cells: Implications for Otoprotective Drug Development

  • Int J Mol Sci. 2025 Jul 13;26(14):6720. doi: 10.3390/ijms26146720.
Cheng-Yu Hsieh  1  2  3 ,  Jia-Ni Lin  1 ,  Yi-Fan Chou  1  2 ,  Chuan-Jen Hsu  1  4 ,  Peir-Rong Chen  2  5 ,  Yu-Hsuan Wen  2  5 ,  Chen-Chi Wu  3  4  6 ,  Chuan-Hung Sun  1  2
Affiliations
  • 1. Department of Otolaryngology, Head and Neck Surgery, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung 427213, Taiwan.
  • 2. School of Medicine, Tzu Chi University, Hualien 970374, Taiwan.
  • 3. Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei 100233, Taiwan.
  • 4. Department of Otolaryngology, National Taiwan University Hospital, Taipei 100225, Taiwan.
  • 5. Department of Otolaryngology, Head and Neck Surgery, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 970473, Taiwan.
  • 6. Department of Medical Research, National Taiwan University Hospital Hsin-Chu Branch, Hsinchu 300195, Taiwan.
PMID: 40724969 DOI: 10.3390/ijms26146720
Abstract

Aminoglycoside antibiotics are critical in clinical use for treating severe infections, but they can occasionally cause irreversible sensorineural hearing loss. To establish a rational pathway for otoprotectant discovery, we provide an integrated, three-tier methodology-comprising cell-model selection, transcriptomic analysis, and a gentamicin-Texas Red (GTTR) uptake assay-to guide the development of otoprotective strategies. We first utilized two murine auditory cell lines-UB/OC-2 and HEI-OC1. We focused on TMC1 and OCT2 and further explored the underlying mechanisms of ototoxicity. UB/OC-2 exhibited a higher sensitivity to gentamicin, which correlated with elevated OCT2 expression confirmed via RT-PCR and Western blot. Transcriptomic analysis revealed upregulation of PI3K-Akt, calcium, and GPCR-related stress pathways in gentamicin-treated HEI-OC1 cells. Protein-level analysis further confirmed that gentamicin suppressed phosphorylated Akt while upregulating ER stress markers (GRP78, CHOP) and apoptotic proteins (cleaved Caspase 3, PARP). Co-treatment with PI3K inhibitors (LY294002, wortmannin) further suppressed Akt phosphorylation, supporting the role of PI3K-Akt signaling in auditory cells. To visualize drug entry, we used GTTR to evaluate its applicability as a fluorescence-based uptake assay in these cell lines, which were previously employed mainly in cochlear explants. Sodium thiosulfate (STS) and N-acetylcysteine (NAC) significantly decreased GTTR uptake, suggesting a protective effect against gentamicin-induced hair cell damage. In conclusion, our findings showed a complex ototoxic cascade involving OCT2- and TMC1-mediated drug uptake, calcium imbalance, ER stress, and disruption of PI3K-Akt survival signaling. We believe that UB/OC-2 cells serve as a practical in vitro model for mechanistic investigations and screening of otoprotective compounds. Additionally, GTTR may be a simple, effective method for evaluating protective interventions in auditory cell lines. Overall, this study provides molecular-level insights into aminoglycoside-induced ototoxicity and introduces a platform for protective strategies.

Keywords
aminoglycosides; mouse auditory cell lines; ototoxicity; transcriptomic analysis.
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