Hearing loss affects more than 28 million Americans, including approximately 50% of people more than the age of 75 years and 2% of children (National Institute on Deafness and Other Communication Disorders, NIDCD 2007
). Known etiologies of hearing loss include genetic factors, acoustic injury, mechanical trauma, medications, infection, and age-related changes. Most cases of peripheral sensorineural auditory pathology involve some form of cochlear abnormality that disrupts sensory transduction at the level of the auditory nerve, stria vascularis, or hair cell. Hair cells within the organ of Corti in the inner ear are highly metabolic and are particularly vulnerable to noxious insults like noise or chemicals. Thus, one of the most common histopathological findings in hearing loss is hair cell loss (Schuknecht 1993
One common form of ototoxicity is the tendency of drugs and environmental toxins to cause damage to the receptor epithelia of hearing and/or balance. Seligmann et al. (1996
) listed more than 130 drugs and chemicals reported to be potentially ototoxic, with the major classes of known ototoxic compounds being the aminoglycosides and other antimicrobials, anti-inflammatory agents, diuretics, antimalarial drugs, antineoplastic agents, and some topically administered agents. Most of these drugs were initially recognized as being ototoxic after anecdotal reports of hearing loss, tinnitus, or balance impairment. These reports subsequently prompted controlled studies in humans or laboratory animals on the ototoxicity of individual drugs. Without the initial anecdotal reports, further research into ototoxicity would not have been conducted, since screening for ototoxicity is not generally included in drug development protocols. At present, there is no standardized screening process for ototoxicity in drug development, and the ototoxic potential of the vast majority of Food and Drug Association (FDA)-approved drugs remains unknown.
Despite a large research effort into the mechanisms and etiologies underlying hearing loss, it is likely that some idiopathic sensorineural hearing loss is secondary to exposure to drugs or chemicals that are not known to be ototoxic. According to the NIDCD, less than 15% of patients with sudden deafness know the causes of their hearing loss (NIDCD 2007
). The etiology of sensorineural hearing loss in children is unknown 37.7% of the time (Morzaria et al. 2004
) and due to unknown environmental or nongenetic causes 22% of the time (Gurtler and Lalwani 2002
). It is reasonable to presume that hearing loss from ototoxic medications is underestimated, especially in pediatric patients, in whom hearing loss is more difficult to detect. Children often may be unable to recognize or communicate the presence of newly acquired hearing loss. Furthermore, ototoxic effects are likely to be misattributed to presbycusis in older patients, who are more likely to be receiving multiple medications. Another contributing factor to the underestimation of sensorineural hearing loss due to ototoxicity is the phenomenon of hair cell loss with an absence of detectable hearing loss on conventional audiometry. For example, conventional pure-tone audiometry can fail to detect hearing loss in children being treated with chemotherapeutic cisplatin derivatives (Stavroulaki et al. 2001
; Dhooge et al. 2006
; Knight et al. 2007
). In summary, it is probable that there is a subset of drugs currently used in practice that have occult ototoxic effects. To address this issue, we have begun to develop a standard screen for ototoxicity that can be used in drug development and drug safety analyses.
The zebrafish (Danio rerio
) is increasingly recognized as a powerful model system for studying disease and for in vivo drug discovery. For example, the zebrafish has been used to identify compounds that can correct genetic heart defects (Peterson and Fishman 2004
), suppress cancer genes (Stern et al. 2005
), and promote hematopoiesis (North et al. 2007
). The lateral line organ of zebrafish demonstrates unique advantages that make it useful for investigating hair cell toxicity. The hair cells of the lateral line reside in groups called neuromasts that are located in stereotyped positions on the surface of the head and body, making the hair cells easily accessible for exposure to chemicals (Fig. ). The hair cells share both morphological and functional similarity to those of the mammalian inner ear. In addition, the zebrafish larva is optically transparent, and the hair cells of the lateral line readily take up fluorescent dyes, such as YO-PRO1. These two factors allow rapid examination of hair cells in vivo using fluorescence microscopy. Finally, the zebrafish has high fecundity, with usual clutch sizes greater than 100 in number (Hertog 2005
). Our high throughput screening protocol takes advantage of these large numbers of animals by screening large numbers of chemicals in a relatively short amount of time using a single clutch of animals.
FIG. 1. A Live preparation of fluorescently labeled zebrafish larva 5 dpf (Harris et al. 2003). Neuromasts of the lateral line are stained with YO-PRO1. Scale bar=0.5 mm. B Schematic illustration of a neuromast. Hair cells are (more ...)
Aminoglycoside and cisplatin-induced hair cell death in the zebrafish lateral line has been studied in detail (Harris et al. 2003
; Murakami et al. 2003
; Ton and Parng 2005
; Santos et al. 2006
; Owens et al. 2007a
; Ou et al. 2007
). All of these studies have helped to validate the use of zebrafish as a screening tool for ototoxicity, but they have focused largely on the realm of known ototoxic agents. This study focuses on the detection of unknown ototoxic agents from a large library of compounds. We used the zebrafish lateral line to screen a library of 1,040 FDA approved compounds and bioactives for ototoxic effects. Twenty-one compounds were identified as selectively toxic to zebrafish hair cells. Dose–response relationships were examined for all 21 compounds. As proof of concept that these findings may be applicable to mammals, the ototoxic effects of two drugs identified in the zebrafish screen were confirmed in mature mouse utricular explants.