Chiu Lynn L, Cunningham Lisa L, Raible David W, Rubel Edwin W, Ou Henry C
Department of Otolaryngology-Head and Neck Surgery, University of Washington, Box 356515, Seattle, WA 98195, USA.
J Assoc Res Otolaryngol. 2008 Jun;9(2):178-90. doi: 10.1007/s10162-008-0118-y. Epub 2008 Apr 12.
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.
斑马鱼是研究毛细胞发育、结构、遗传学和行为的宝贵模型。斑马鱼和其他水生脊椎动物在其体表有毛细胞，这些毛细胞组成了一个称为侧线的感觉系统。这些毛细胞极易获取，使用荧光染料就能轻松观察到。其与哺乳动物内耳毛细胞在形态和功能上的相似性，使得斑马鱼成为研究毛细胞毒性的有力实验对象。药物的耳毒性潜力以往是通过轶事报告发现的，这些报告促使了更正式的研究。目前，药物研发中不存在耳毒性的标准筛选方法。因此，对于绝大多数美国食品药品监督管理局（FDA）批准的药物，其耳毒性潜力仍不为人知。在本研究中，我们使用5日龄的斑马鱼幼体，对1040种FDA批准的药物和生物活性物质（美国国立神经疾病和中风研究所定制文库II）进行筛选，以检测其对侧线毛细胞的耳毒性作用。使用一种荧光活性染料对毛细胞核进行选择性标记。在初步筛选中，将鱼暴露于文库中的药物，浓度为100 μM，在96孔组织培养板中孵育1小时。使用荧光显微镜在体内评估毛细胞活力。快速筛选了1040种药物的耳毒性作用。文库中包含的7种已知耳毒性药物，包括新霉素和顺铂，通过这些方法得到了阳性鉴定，作为概念验证。发现14种先前未知耳毒性的化合物对毛细胞具有选择性毒性。通过量化毛细胞存活率作为药物浓度的函数，确定了所有21种耳毒性化合物的剂量反应曲线。然后使用成年小鼠椭圆囊的体外制剂，研究了两种未知耳毒性的化合物（乙磺半胱氨酸和溴丙胺太林）在哺乳动物内耳中的剂量反应关系。两种化合物在小鼠椭圆囊中均显示出显著的剂量依赖性毛细胞损失。这项研究是验证将斑马鱼侧线用作筛选潜在耳毒性药物工具的重要一步。
