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The Virtual Embryo Project (v-Embryo™):
A computational framework for developmental toxicity
Experimental Approaches
Traditional prenatal studies
EPA’s Toxicology Reference Database (ToxRefDB) is being developed to house reference in vivo toxicology data. This relational database holds conventional toxicity data from chronic, subchronic, reproductive and developmental animal studies. Its primary goal is to provide traditional toxicological data in a searchable format, primarily from EPA Pesticide Data Evaluation Records (DERs) for hundreds of registered pesticidal active ingredients that make up a significant fraction of the 320 phase I chemicals of ToxCast™. Those records contain data on all toxicologically significant effects from legacy toxicity studies in rats, mice, rabbits among other test species. The infrastructure of v-Embryo™ includes a searchable document repositorythat builds on ToxRefDB complete with text-mining tools for semi-automated feature extraction.
Developmental pathways
Self-regulating genetic regulatory networks that pattern early development provide important information on the flow of molecular regulatory information at key stages of embryogenesis. Such information can be captured from public databases and literature resources using a combination of data-mining and text-mining tools. BioTapestry, an interactive web resource developed at the Institute for Systems Biology and California Institute of Technology, can be used to build these developmental gene regulatory networks and visualize them as standardized and extensible computational models or ‘wiring diagrams’. In a dynamic model, the relationships among factors in the wiring diagram can be made explicit by defining the relationships in terms of rates, quantities, or state changes.
ToxCast™
EPA’s ToxCast™ research program addresses chemical prioritization and predictive testing that will assist EPA in the management and regulation of environmental contaminants. ToxCast™ applies the pharmaceutical industry’s experience in the use of state-of-the-art high-throughput screening, toxicogenomics, and computational chemistry tools to environmental toxicology. Bioactivity profiling data from the ToxCast™ assays is managed through the Aggregated Computational Toxicology Resource (ACToR). Proof-of-concept is underway (Phase I) to test the hypothesis that bioactivity signatures correlated with endpoints from traditional animal testing can be used to predict adverse outcome. It is envisaged that once trained and validated the program will enable in silico prediction for experimental outcomes on new chemicals.
Embryonic stem cell tests
Pluripotent embryonic stem (ES) cells derived from the inner cell mass of early mouse embryos can be triggered to differentiate into any cell of the body. ES cells have the capability to recapitulate many signaling pathways of an intact embryo. As such, they have become a tool for developmental biologists to evaluate process, gene function, cell-cell interactions and other facets of cellular function. Developmental toxicologists have also taken advantage of stem cell differentiation to provide an insight into the potential for chemicals to disrupt embryological pathways.
In an ES test, pluripotent murine D3 ES cells are placed into hanging drops and the cells allowed to aggregate into balls called embryoid bodies (EB). After three days in hanging drops the EB are then transferred into a static culture but not allowed to attach to the culture dish. After 2 days in static culture, one EB is transferred to each well of a 24-well plate, where it attaches and grows for 5 more days. Finally, the number of wells containing beating heart cells is counted as an assessment of normal differentiation. To evaluate xenobiotics, mESC are exposed to 5-10 concentrations of each chemical throughout the 10 day culture and the number of wells containing beating heart cells is determined. The concentration that produces a 50% decrease (IC50) in differentiation is determined. In addition to the effects on differentiation, the induction of cell death and alteration in proliferation are assessed in mESC and mouse embryonic fibroblasts (3T3) cells using a standard cell viability analysis. In summary, 3 endpoints are determined for each chemical: the IC50 for differentiation, IC50 for mESC cell death, and an IC50 for 3T3 cell death. These values are placed into "predictive model" equations that then classify each compound as a non-, weak- or strong-embryotoxicant.
There is a growing literature basis for understanding stem cell differentiation and the processes that it represents during embryogenesis. By combining the information generated regarding the developmental biology of stem cell differentiation and the effects of xenobiotics on stem cell differentiation, there is a great opportunity to better define the gene networks and signaling pathways involved in differentiation and the ability of compounds to disrupt those pathways and produce dysdifferentiation.
Zebrafish embryos
Free living zebrafish embryos hold promise for rapid screening of chemicals based on the potential to directly perturb developmental processes. The small eggs of zebrafish fit comfortably into a 96 or 384-well microtiter plate, enabling robotic handling and analyses of the developing embryo. Because zebrafish embryos are transparent the sequence of development may be directly observed without disturbing the embryo and consequently followed through precisely timed stages as the embryo advances from fertilized egg to a swimming fish larva. This progression occurs rapidly, in just 5 days, and recapitulates many of the same anatomical features, morphogenetic processes and cell signaling pathways used by the early human embryo. As such, Zebrafish embryos provide a powerful alternative to mammalian animal models in research aimed to document and classify the potential developmental toxicity of environmental chemicals and to model these dynamical processes in silico.