Using the Zebrafish Lateral Line to Screen for Ototoxicity

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-μM 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.

Hair Cell Toxicity in Anti-cancer Drugs: Evaluating an Anti-cancer Drug Library for Independent and Synergistic Toxic Effects on Hair Cells Using the Zebrafish Lateral Line

“…To account for this variability, the anti-cancer drug library was screened at multiple concentrations and durations. The general zebrafish screening protocol has been described previously (Chiu et al 2008). Briefly, one YOPRO-1 labeled 5 dpf zebrafish was placed into each well of a 96-well optical plate (NUNC) with in 150 μL of embryo media.…”

“…The zebrafish lateral line is a powerful system for identifying and evaluating drugs that damage mechanoreceptive hair cells, such as those in the inner ear of mammals (Ton and Parng 2005;Chiu et al 2008). Due to the small size and optically clear body of larval zebrafish, hair cell damage can be rapidly assessed.…”

“…The zebrafish has been used effectively to examine hair cell toxicity and elucidate doseresponse relationships for aminoglycosides and cisplatin (Wiliams and Holder 2000;Harris et al 2003;Ton and Parng 2005;Owens et al 2009;Ou et al 2007). In addition, it has been used to screen for toxicity to hair cells in a library of FDA-approved drugs and bioactives (Chiu et al 2008).…”

“…This collection of FDA-approved drugs contains a representative sample of older cytotoxic drugs as well as new targeted therapies. To improve the sensitivity of our screen in comparison to our previous toxicity screens (Chiu et al 2008), we screened the library at multiple drug concentrations and at multiple exposure durations. Screening in this fashion, we successfully identified four out of five known ototoxins and identified multiple other drugs as potential novel ototoxins.…”

Early Development of Hearing in Zebrafish

“…Finally, the zebrafish genome has been sequenced, and many human genes are conserved in zebrafish. Particularly, the zebrafish has been used as a unique vertebrate model for studying hearing and balance disorders (Nicolson et al 1998;Ernest et al 2000;Whitfield 2002;Söllner et al 2004;Kappler et al 2004;Nicolson 2005;Shen et al 2008;Gleason et al 2009;Phillips et al 2011;Yariz et al 2012), with its externally exposed hair cells of the lateral line being used for study in hair cell death and regeneration, ototoxicity, and drug screens (Chiu et al 2008;López-Schier et al 2004;Owens et al 2009;Coffin et al 2010;Buck et al 2012).…”