Using the zebrafish lateral line to screen for ototoxicity.

Department of Otolaryngology-Head and Neck Surgery, University of Washington, Box 356515, Seattle, WA 98195, USA.
Journal of the Association for Research in Otolaryngology (Impact Factor: 2.55). 07/2008; 9(2):178-90. DOI: 10.1007/s10162-008-0118-y
Source: PubMed

ABSTRACT The zebrafish is a valuable model for studying hair cell development, structure, genetics, and behavior. Zebrafish and other aquatic vertebrates have hair cells on their body surface organized into a sensory system called the lateral line. These hair cells are highly accessible and easily visualized using fluorescent dyes. Morphological and functional similarities to mammalian hair cells of the inner ear make the zebrafish a powerful preparation for studying hair cell toxicity. The ototoxic potential of drugs has historically been uncovered by anecdotal reports that have led to more formal investigation. Currently, no standard screen for ototoxicity exists in drug development. Thus, for the vast majority of Food and Drug Association (FDA)-approved drugs, the ototoxic potential remains unknown. In this study, we used 5-day-old zebrafish larvae to screen a library of 1,040 FDA-approved drugs and bioactives (NINDS Custom Collection II) for ototoxic effects in hair cells of the lateral line. Hair cell nuclei were selectively labeled using a fluorescent vital dye. For the initial screen, fish were exposed to drugs from the library at a 100-muM concentration for 1 h in 96-well tissue culture plates. Hair cell viability was assessed in vivo using fluorescence microscopy. One thousand forty drugs were rapidly screened for ototoxic effects. Seven known ototoxic drugs included in the library, including neomycin and cisplatin, were positively identified using these methods, as proof of concept. Fourteen compounds without previously known ototoxicity were discovered to be selectively toxic to hair cells. Dose-response curves for all 21 ototoxic compounds were determined by quantifying hair cell survival as a function of drug concentration. Dose-response relationships in the mammalian inner ear for two of the compounds without known ototoxicity, pentamidine isethionate and propantheline bromide, were then examined using in vitro preparations of the adult mouse utricle. Significant dose-dependent hair cell loss in the mouse utricle was demonstrated for both compounds. This study represents an important step in validating the use of the zebrafish lateral line as a screening tool for the identification of potentially ototoxic drugs.

  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mechanosensory hair cells are vulnerable to environmental insult, resulting in hearing and balance disorders. We demonstrate that directional compartmental flow of intracellular Ca(2+) underlies death in zebrafish lateral line hair cells after exposure to aminoglycoside antibiotics, a well characterized hair cell toxin. Ca(2+) is mobilized from the ER and transferred to mitochondria via IP3 channels with little cytoplasmic leakage. Pharmacological agents that shunt ER-derived Ca(2+) directly to cytoplasm mitigate toxicity, indicating that high cytoplasmic Ca(2+) levels alone are not cytotoxic. Inhibition of the mitochondrial transition pore sensitizes hair cells to the toxic effects of aminoglycosides, contrasting with current models of excitotoxicity. Hair cells display efficient ER-mitochondrial Ca(2+) flow, suggesting that tight coupling of these organelles drives mitochondrial activity under physiological conditions at the cost of increased susceptibility to toxins.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 07/2014; 34(29):9703-19. DOI:10.1523/JNEUROSCI.0281-14.2014 · 6.75 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objective: Caffeic acid phenethyl ester (CAPE) is known to reduce the generation of oxygen-derived free radicals, which is a major mechanism of aminoglycoside-induced ototoxicity. The objective of the present study was to evaluate the effects of CAPE on neomycin-induced ototoxicity in zebrafish (Brn3c: EGFP). Methods: Five-day post-fertilization zebrafish larvae (n = 10) were exposed to 125 mu M neomycin and one of the following CAPE concentrations for 1 h: 50, 100, 250, 500, or 1000 mu M. Ultrastructural changes were evaluated using scanning electron microscopy (SEM). The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) assay and 2-[4-(dimethylamino)styryll-N-ethyl-pyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. Results: CAPE decreased neomycin-induced hair cell loss in the neuromasts (500 mu M CAPE: 12.7 +/- 1.1 cells, 125 mu M neomycin only: 6.3 1.1 cells; n = 10, P < 0.05). In the ultrastructural analysis, structures of mitochondria and hair cells were preserved when exposed to 125 mu M neomycin and 500 mu M CAPE. CAPE decreased apoptosis and mitochondrial damage. Conclusion: In the present study, CAPE attenuated neomycin-induced hair cell damage in zebrafish. The results of the current study suggest that neomycin induces apoptosis, and the apoptotic cell death can be prevented by treatment with CAPE in zebrafish.
    International Journal of Pediatric Otorhinolaryngology 05/2014; 78(8). DOI:10.1016/j.ijporl.2014.05.018 · 1.32 Impact Factor

Full-text (2 Sources)

Available from
May 15, 2014