Disproportionality analysis of ototoxicity in CDDP-treated patients using the US Food and Drug Administration adverse event reporting system (FAERS) database
We analyzed 44 years of data from 1978 to 2021 for the drugs listed in the FAERS database (https://www.fda.gov/drugs/questions-and-answers-fdas-adverse-event-reporting-system-faers/fda-adverse-event-reporting-system-faers-public-dashboard) and the Japanese Pharmacopoeia 18th Edition (https://www.pmda.go.jp/english/rs-sb-std/standards-development/jp/0029.html?print). For CDDP-induced ototoxicity, 12 preferred terms (PTs) related to hearing impairment were established according to the ICH International Medical Dictionary for Regulatory Activities/Japanese version 25.0 (Neurosensory hypoacusis, Deafness neurosensory, Mixed deafness, Hypoacusis, Sudden hearing loss, Conductive deafness, Deafness unilateral, Deafness bilateral, and Tinnitus), and Reporting Odds Ratios (RORs) were calculated based on a previous article [11].
Zebrafish strain and maintenance
Transgenic zebrafish Tg (cldn: gfp; atoh1a: rfp), which express red fluorescent protein (rfp) in hair cells and green fluorescent protein (gfp) in neuromasts were used [12]. The embryos were collected once a week and maintained at 29.5°C until 52 hpf before use in the experiment. This protocol was approved by the Institutional Animal Care and Use Committee of Iwate Medical University (approval number: 05–016).
RNA extraction and qRT–PCR
Total RNA was extracted from zebrafish larvae (n = 12) using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) based on the manufacturer’s instructions. Complementary DNA (cDNA) was prepared by reverse transcription using an RNA template after removing gDNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd., Osaka, Japan). Quantitative real-time polymerase chain reaction (qRT–PCR) was done using an Illumina Eco Real-Time PCR System (Illumina, Inc., San Diego, CA, USA) and THUNDERBIRD SYBR qPCR Mix (Toyobo Co., Ltd., Osaka, Japan). The specific primer pairs were synthesized (Eurofins Genomics K.K., Tokyo, Japan) and the reaction conditions are shown in Supplementary Table 1. The relative expression was analyzed as fold-change by the 2–∆∆CT method.
Drug treatment
CDDP (Sigma-Aldrich, St. Louis, MO, USA) was prepared as a 100 mM stock solution and DEX (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) was prepared as a 50 mM stock solution in DMSO (KOKUSAN CHEMICAL Co., Ltd., Tokyo, Japan), and stored at 30°C. The 52 hpf embryos were treated with 10 µM DEX for 8 h, followed by 0 to 400 µM CDDP, and incubated for 12 h at 29.5°C.
Gene editing by the CRISPR/Cas9 system
CrisprRNA (crRNA) was designed to target exon 1 of the zebrafish mt2 gene using the CRISPR Design Tool (horizon) and the tyrosinase (tyr) gene based on a previously reported sequence [13]. The crRNA and trans-activating crRNA (tracrRNA) were chemically synthesized (Fasmac Co., Ltd., Kanagawa, Japan) and the Cas9 nuclease protein was obtained from NIPPON GENE (Nippon Gene Co., Ltd., Tokyo, Japan). The crRNA and tracrRNA sequences are listed in Supplementary Table 2. The injection solution contained 50 ng/µL crRNA, 100 ng/µL tracrRNA, and 412.5 ng/µL Cas9 nuclease protein. Zebrafish embryos were microinjected at the 1-cell stage with a 1–2 nL injection solution.
Heteroduplex Mobility Assay (HMA)
The microinjected embryos were incubated for 52 hpf at 29.5°C. After drug treatment, genomic DNA was extracted using the REDExtract-N-Amp Tissue PCR Kit (Sigma-Aldrich, St. Louis, MO, USA) from a single individual. Primer pairs corresponding to the mt2 sequence were synthesized and used for PCR. The sequences of the primers are listed in Supplementary Table 3. The reaction product was incubated with T7 endonuclease I (New England BioLabs, Ipswich, MA, USA) at 37°C. Finally, we prepared a 15% polyacrylamide gel (Bio-Rad Laboratories, CA, USA) and analyzed the PCR products by electrophoresis.
Statistical analysis
The Bell Curve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan) was used for statistical analysis. Differences in mt2 mRNA expression were calculated by a one-way ANOVA, and the number of neuromasts between DMSO and 10 µM DEX was calculated using a two-way ANOVA and Tukey’s multiple comparisons tests. P-values < 0.05 were considered statistically significant.