Development and assessment of new risk assessment methods
Title of Research:
13_PT01-01

Development of in vivo fluorescent imaging of neuronaldifferentiation in zebrafish for developmental neurotoxicity testing
Principal Investigator:
Yuhei Nishimura (Department of Integrative Pharmacology, Mie University Graduate School ofMedicine)Collaborators:
Toshio Tanaka (Department of Systems Pharmacology, Mie University Graduate School ofMedicine)
Summary of Research:
The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicantsduring development has been implicated in various neuropsychiatric and neurological disorders,
including autism spectrum disorder, attention deficit hyperactive disorder, schizophrenia,
Parkinson’s disease, and Alzheimer’s disease. Although rodents have been widely used fordevelopmental neurotoxicity testing, experiments using large numbers of rodents are timeconsuming, expensive, and raise ethical concerns. Using alternative non-mammalian animalmodels may relieve some of these pressures by allowing testing of large numbers of subjects while
reducing expenses and minimizing the use of mammalian subjects. Recently, zebrafish has
emerged as an alternative non-mammalian animal model that allows testing of large numbers ofsubjects while reducing expenses and minimizing the use of mammalian subjects.
In this study, we have developed a transgenic zebrafish line whose neurons, astrocytes, andoligodendrocytes can be visualized using fluorescence stereomicroscope. Using the zebrafish, weevaluated the developmental neurotoxicity of 10 positive and 3 negative control chemicals. The
neuronal differentiation of the transgenic zebrafish was significantly affected by 9 out of the 10positive control chemicals. The 3 negative control chemicals did not show any significant effects on
the neuronal differentiation. Hierarchical clustering of the 13 chemicals based on the fluorescentsignals revealed the similarity among valproic acid, trichostatin A, and carbamazepine and thesimilarity between nicotine and chlorpyrifos. Valproic acid, trichostatin A, and carbamazepine are
histone deacetylase inhibitors. Nicotine and chlorpyrifos can disrupt acetylcholine signaling. These
results suggest that in vivo fluorescent imaging of neuronal differentiation in the transgenic
zebrafish developed in this study can be a useful tool not only to detect the developmental
neurotoxicity of various chemicals but also to reveal the adverse outcome pathways.
Timeline:
Nov 2013 ~ Feb 2018
Topics:Presented at TEST SMART DNT4 (2014) and ICCA-LRI and NIHS workshop（2016）
Publications:
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Nishimura Y, Murakami S, Ashikawa Y, Sasagawa S, Umemoto N, Shimada Y, Tanaka T:
Zebrafish as a systems toxicology model for developmental neurotoxicity testing.
Congenital anomalies 2015; 55(1):1-16.
Nishimura Y, Okabe S, Sasagawa S, Murakami S, Ashikawa Y, Yuge M, Kawaguchi K, KawaseR, Tanaka T: Pharmacological profiling of zebrafish behavior using chemical and geneticclassification of sleep-wake modifiers. Frontiers in pharmacology 2015; 6:257.
Nishimura Y, Inoue A, Sasagawa S, Koiwa J, Kawaguchi K, Kawase R, Maruyama T, Kim S,Tanaka T: Using zebrafish in systems toxicology for developmental toxicity testing.
Congenital anomalies 2016; 56(1):18-27.