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Journal of Toxicology
Volume 2011 (2011), Article ID 286034, 8 pages
Research Article

Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation

Unidad de Toxicología y Seguridad Química, Instituto de Bioingeniería, Universidad Miguel Hernández de Elche, Avenida de la Universidad s/n, 03202 Elche, Spain

Received 11 May 2011; Revised 30 June 2011; Accepted 1 July 2011

Academic Editor: Yujian James Kang

Copyright © 2011 Andrea C. Romero et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicity in vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 μg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes: Pnpla6, Afp, Hdac7, Vegfa, and Nes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression of Nes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting for in vitro risk assessment embryotoxicity.