Endocrine Abstracts

Developmental toxicity tests have been made by embryonic stem cell tests at the European Centre for the Validation of Alternative Methods or by embryonic body test in our laboratory. However, no neuronal-specific developmental toxicity test has been made yet. Therefore, this study was carried out using a 46C cell line, mouse embryonic stem cells with an endogenous Sox1-GFP reporter, to exploit the developmental neurotoxicity test. The expression of Sox1, a marker for neural progenitor, can be detected by green fluorescence and the fluorescence density is a critical factor to achieve neuronal differentiation. 46C cells were treated for 24 hours with 5-fluorouracil, hydroxyurea, chlorpyrifos, clioquinol, diazinon, nicotine and lead acetate as developmental neurotoxicants, or saccharin, sodium bicarbonate, sodium gluconate, and penicillin G as non-neurotoxicants. CCK-8 assays were performed to determine IC₅₀ values after 48 hours of chemical treatment. The fluorescence intensity of GFP was measured after 4 days of treatment with cells using an automated digital microscope. Through CCK-8 assay, IC₅₀ values of developmental neurotoxicant chemicals were obtained, whereas non-neurotoxicant chemicals showed low effects. In addition, the fluorescence intensity of GFP was not decreased with non-neurotoxicants. However, neurotoxicants decreased the fluorescence intensity of GFP at higher concentrations. This decrease of fluorescence intensity indicates that the neuronal differentiation of 46C cells is inhibited by the chemicals. Taken together, this study produced a model of the developmental neurotoxicity tests used embryonic stem cells that may use to evaluate the toxicity of new chemicals or new candidate drugs.