Quinolyl nitrone derivative QN23 protects from auditory cell oxidative injury and noise-induced hearing loss

FIGURE 1_DATASET and TABLE 1_DATASET: HEI-OC1 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco-Thermo Fisher Scientific, Waltham, MA, USA) and 2.5ug/mL Amphotericin B (A2942, Sigma-Aldrich, Saint Louis, MO, USA). Nitrones QN23, F2 and TNs were dissolved in absolute EtOH ≥ 99.5% (#107017; Merck, Kenilworth, NJ, USA) at 20, 5 or 10 mM (stock solution), respectively, and sonicated 10 minutes at 37ºC in a Branson Bransonic® M Mechanical Bath (Emerson Electric). Then, they were tested first for ototoxicity at different concentrations in DMEM without FBS, in the absence or presence of H2O2 (range 1-1.4 mM) and subsequently their efficacy. Cell viability was evaluated by the XTT method by following the manufacturer´s instructions (Cell Proliferation Kit II, Roche Molecular Systems). Absorbance measurements at 450 and 660 nm were taken with the VERSAmax plate reader (Molecular Devices, San Jose, CA, USA) at 4, 6 and 23 hours after the addition of XTT. All reading times rendered similar results. Cell viability was calculated as follows: Specific absorbance = [Abs450 nm (test) - Abs450 nm (mock)] - Abs660 nm (test). Control wells without seeded cells were used as reference. FIGURE 2_DATASET: Hearing was assessed by recording auditory brainstem evoked potentials (ABR) before (baseline) and 1, 14 and 28 days after noise exposure. Click and tone burst stimuli (4, 8, 16, 24 and 32 kHz) were presented with an MF1 magnetic speaker (TDT) from 90 to 20 dB SPL in 5–10 dB SPL steps. Click stimuli were 0.1 ms and tone burst stimuli were 5 ms in duration (2.5 ms each for rise and decay, without plateau). The threshold of click-evoked and tone-evoked ABRs, peak latencies, and amplitudes were determined. Mice were exposed to 2–20 kHz, 105 dB SPL noise for 30 minutes. FIGURE 4_DATASET and FIGURE 5_DATASET: Cochlear RNA was extracted with the RNeasy kit (QIAGEN, Hilden, Germany). Quality and quantity of RNA were determined with an Agilent Bioanalyzer 2100 (Agilent Technologies Santa Clara, CA, USA). RNA from three animals per experimental group was pooled. cDNA was then generated by reverse transcription (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems, Foster City, CA, USA) and gene expression was analyzed in triplicate by qPCR on Applied Biosystems 7900 HT using TaqMan Gene Expression Assays (Applied Biosystems) for Nrf2, Hmox1, Nqo1, Nox4, Nlrp3, Tnfa, Tgfb, Il1b, Il6, Il10, Kim1 and Rplp0 was used as endogenous housekeeping control gene Dusp1 (Supplementary Table 1). Gene expression was calculated as 2−ΔΔCt (RQ).