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DEVELOPMENT OF ZEBRAFISH-BASED ASSAYS FOR THE ASSESSMENT OF DNT CAUSED BY EXPOSURE TO CHEMICALS AT LOW DOSES
Yuhei Nishimura1,2,3,4, Shinichi Takeuchi1, Tomoko Misawa1, Tetsuo Kon1 and Toshio Tanaka1,2,3,4
1Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
2Mie University Medical Zebrafish Research Center, Tsu, Mie 514-8507, Japan
3Department of Omics Medicine, Mie University Industrial Technology Innovation Institute, Tsu, Mie 514-8507, Japan
4Department of Bioinformatics, Mie University Life Science Research Center, Tsu, Mie 514-8507, Japan
It is often difficult to detect the subtle effects of DNT caused by exposure to low concentrations of chemicals that do not induce any external malformation. Large studies in which specific functional domains are assessed using objective instruments are required to detect these subtle changes. Using alternative non-mammalian animal models allows for testing large numbers of individuals while reducing the expense and using fewer mammals. Zebrafish offer several advantages for studies of DNT, including their low cost of husbandry, high fecundity, their tendency to absorb a wide range of chemicals, and rapid ex utero development. In addition, zebrafish are transparent, which allows easy observation of morphological changes in the central and peripheral nervous system during earlier developmental stages by using fluorescence technology. We have identified novel fluorescent dyes that can non-invasively stain a range of neuronal cells, including retinal and hair cells. Use of these fluorescent dyes has enabled the development of an assay for the in vivo imaging of neuronal cells in zebrafish using 96-well plates. Zebrafish larvae also exhibit a number of simple and complex behavioral programs, including startle responses and non-associative learning. We have developed a video-tracking system using 96-well plates to monitor and quantify the behaviors of zebrafish larvae following exposure to chemicals. The morphological and behavioral assessment, combined with the ease of obtaining large numbers of zebrafish larvae, make it possible to undertake a systematic and objective analysis and to increase statistical power to detect subtle but significant DNT caused by exposure to chemicals at low doses. These assessments can be used to classify neurotoxicants based on morphological and behavioral phenotypes.