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GENOMIC ADAPTATION OF THE EMBRYONIC STEM CELL TEST (EST) FOR A TOXICOLOGICAL STUDY OF DRINKING WATER DISINFECTION BY-PRODUCTS
Citation:
SLENTZ-KESLER, K., C. R. WOOD, M. R. BLANTON, AND E. S. HUNTER. GENOMIC ADAPTATION OF THE EMBRYONIC STEM CELL TEST (EST) FOR A TOXICOLOGICAL STUDY OF DRINKING WATER DISINFECTION BY-PRODUCTS. Presented at Keystone Symposium: Stem Cells, Whistler, BC, CANADA, March 27 - April 01, 2006.
Description:
Among the many promised and potential applications of embryonic stem cells, in vitro toxicology is one area in which ES cells have already proven their utility. In 2003, the Embryonic Stem Cell Test (EST) protocol was validated in Europe as an in vitro alternative to live animal testing for developmental toxicology studies. In this assay, pluripotent mouse ES cells are allowed to differentiate in the presence of various toxicants. After 10 days, inhibition of spontaneous cardiomyocyte development is visually assessed to gauge the embryotoxicity of each chemical. These data are analyzed along with 7-day cytotoxicity profiles generated from both mouse ES cells and 3T3 mouse fibroblast cells in order to classify the chemicals as non-toxic, weakly toxic, or strongly toxic. Recent improvements to the EST have included more sensitive quantitative measurements of cardiomyocyte differentiation using FACS or quantitative RT-PCR (q-PCR), as well as the assessment of other somatic cell types such as osteoblast, chondrocytic, and neuronal cells. The EST may prove quite useful to scientists at the US Environmental Protection Agency, who are faced with ever-lengthening lists of putative environmental toxicants that must be classified and prioritized. For example, we are investigating compounds that appear in drinking water as unwanted by-products of various disinfection treatments. The Haloacetic Acids (HAAs) form one class of these disinfection by-products which are generated when disinfectants such as chlorine or ozone react with naturally occurring organic matter in raw water sources. Rats exposed to HAAs in drinking water experienced specific toxicities and adverse effects to pregnancy outcome including birth defects and fetal death. Also, recent studies from our lab using whole embryo culture of mice and rats have shown that HAAs are developmental dysmorphogens. Currently we are adapting the Embryonic Stem Cell Test as a method to be tested in parallel with whole embryo culture for assessing the relative potential of HAAs (and other xenobiotics) to disrupt development. Specifically, we have assembled a panel of q-PCR probes which are used to measure gene expression markers of pluripotency and differentiation along multiple cell lineages, both before and after the ES cells are exposed to test chemicals. This panel was also used to characterize different mESC lines, optimize pluripotency maintenance in culture, and guide our method development for embryoid body (EB) formation (?hanging drop? versus stirred suspension), and EB outgrowth (suspension versus adherent outgrowth).
(Disclaimer: This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.