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Using the mouse embryonic stem cell test (EST) to evaluate the embryotoxicity of haloacetic acids
Citation:
BARRIER, M., S. C. JEFFAY, H. P. NICHOLS, K. SLENTZ-KESLER, AND E. S. HUNTER. Using the mouse embryonic stem cell test (EST) to evaluate the embryotoxicity of haloacetic acids. Presented at International Society for Stem Cell Research, Philadelphia, PA, June 11 - 14, 2008.
Impact/Purpose:
Presentation @ International Society for Stem Cell Research
Description:
The Embryonic Stem Cell Test (EST) is used to predict the embryotoxic potential of a test compound by combining the data from cytotoxicity assays in undifferentiated mouse embryonic stem (mES) cells and differentiated mouse cells with the data from a differentiation assay in mES cells. The prediction model used in the EST classifies a test compound into one of three categories of embryotoxic potential: Strong, Weak, and None. The predictive model of the EST was evaluated by the European Centre for the Validation of Alternative Methods (ECVAM) using a set of 20 well-characterized reference compounds. The differentiation assay involves the formation of embryoid bodies (EBs), which are multicellular aggregates formed in a suspension culture of mES cells. These EBs are plated and the characteristics of the cell outgrowths are evaluated for the inhibition of differentiation after 10 days of treatment with the test compound. In the original EST, a qualitative analysis of differentiation was used by monitoring the presence of beating cardiomyocytes. To better quantify the effects of xenobiotics, quantitative analyses of gene and protein markers of stem and differentiated cells have been used. We have used real time RT-PCR to quantitatively assess the expression of a panel of marker genes during the differentiation of D3 mES cells to determine the utility of these genes in evaluating differentiation. Additionally, we are determining whether an “in-cell western” analysis (using Oct-4, Troponin-I, Troponin-T, and Myosin Heavy Chain as stem or cardiac-specific protein markers) can be used for high throughput analysis. We have evaluated the cytotoxic effects of 12 different haloacetic acids in D3 ES cells. In addition, the effects of several haloacetic acids on differentiation have been determined using a combination of beating cardiomyocyte, RT-PCR, and in-cell western analyses. We are examining the assay data for each compound to see how the concentration that perturbs differentiation of mES cells compares to the concentration producing cytotoxicity. For example, for dichloroacetic acid, the concentration that perturbs differentiation is less than that producing cytotoxicity, and for bromodichloroacetic acid, the concentrations are similar. Thus, the ability of the halaoacetic acids to perturb development varies based on the chemical characteristics of the compound.