Embryotoxicity assessment of developmental neurotoxicants using a neuronal endpoint in the embryonic stem cell test

• Published in: Journal of applied toxicology Vol. 32; no. 8; pp. 617 - 626
• Main Authors: Baek, Dae Hyun, Kim, Tae Gyun, Lim, Hwa Kyung, Kang, Jin Wook, Seong, Su Kyoung, Choi, Seung Eun, Lim, So Yun, Park, Sung Hee, Nam, Bong-hyun, Kim, Eun Hee, Kim, Mun Sin, Park, Kui Lea
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01-08-2012; Wiley; Wiley Subscription Services, Inc
• Subjects: Animals; Arsenates - toxicity; arsenic; Arsenites - toxicity; BALB 3T3 Cells; Biocompatibility; Biological and medical sciences; Biomedical materials; Cadmium compounds; Carcinogenesis, carcinogens and anticarcinogens; Cell Differentiation; Cell Survival - drug effects; Chemical agents; Chemical and industrial products toxicology. Toxic occupational diseases; developmental neurotoxicity; Differentiation; Dose-Response Relationship, Drug; embryonic stem cell test; Embryonic Stem Cells - drug effects; Embryos; Endpoint Determination; Heavy metals; In vitro testing; Lethal Dose 50; Medical sciences; Metals and various inorganic compounds; methylmercury; Methylmercury Compounds - toxicity; Mice; Neurons - drug effects; Neurotoxicity; Reproducibility of Results; Screens; Sodium Compounds - toxicity; Stem cells; Surgical implants; Toxicity Tests - methods; Toxicology; Tumors; valproic acid; Valproic Acid - toxicity; Embryo; Arsenic; Toxicity; Stem cell; Nervous system; Anticonvulsant; methylmercury; Neurotoxicity; Mood stabilizer; Development; Mercury Organic compounds; Evaluation; Nervous system diseases; 4-Aminobutyrate transaminase; Enzyme; Transferases; Enzyme inhibitor; Valproic acid; developmental neurotoxicity; embryonic stem cell test; Heavy metal; Carcinogen; Histone deacetylase inhibitor; Neuron; Embryonic cell; Transaminases
• ISSN: 0260-437X; 1099-1263
• DOI: 10.1002/jat.1747

ABSTRACT
The embryonic stem cell test (EST) is a validated in vitro embryotoxicity test; however, as the inhibition of cardiac differentiation alone is used as a differentiation endpoint in the EST, it may not be a useful test to screen embryotoxic chemicals that affect the differentiation of noncardiac tissues. Previously, methylmercury (MeHg), cadmium and arsenic compounds, which are heavy metals that induce developmental neurotoxicity in vivo, were misclassified as nonembryotoxic with the EST. The aim of this study was to improve the EST to correctly screen such developmental neurotoxicants. We developed a neuronal endpoint (Tuj‐1 ID50) using flow cytometry analysis of Tuj‐1‐positive cells to screen developmental neurotoxicants (MeHg, valproic acid, sodium arsenate and sodium arsenite) correctly using an adherent monoculture differentiation method. Using Tuj‐1 ID50 in the EST instead of cardiac ID50, all of the tested chemicals were classified as embryotoxic, while the negative controls were correctly classified as nonembryotoxic. To support the validity of Tuj‐1 ID50, we compared the results from two experimenters who independently tested MeHg using our modified EST. An additional neuronal endpoint (MAP2 ID50), obtained by analyzing the relative quantity of MAP2 mRNA, was used to classify the same chemicals. There were no significant differences in the three endpoint values of the two experimenters or in the classification results, except for isoniazid. In conclusion, our results indicate that Tuj‐1 ID50 can be used as a surrogate endpoint of the traditional EST to screen developmental neurotoxicants correctly and it can also be applied to other chemicals. Copyright © 2011 John Wiley & Sons, Ltd.
The embryonic stem cell test (EST) is a validated in vitro embryotoxicity test; however, as the inhibition of cardiac differentiation alone is used as a differentiation endpoint in the EST, it may not be a useful test to screen embryotoxic chemicals that affect the differentiation of noncardiac tissues. Previously, methylmercury (MeHg), cadmium and arsenic compounds, which are heavy metals that induce developmental neurotoxicity in vivo, were misclassified as nonembryotoxic with the EST.