DEVELOPMENT AND APPLICATIONS OF INTEGRATED CELL BASED BIOASSAYS

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Hazardous waste sites contain complex mixtures of a wide variety of toxic chemicals. Unfortunately, development of a rapid and inexpensive detection of specific chemicals or chemical classes in environmental and biological samples has been hampered by the lack of available specific bioassay/biomarker systems. Accordingly, the overall goals of this project are to develop and validate a series of mechanistically-based cell bioassays/biomarkers that have application for chemical detection and screening. Since effective development and application of bioassays/biomarkers is greatly facilitated by an understanding of the specific response of a cell to a given toxicant of class of toxicants, each of the four proposed approaches will exploit information derived from an analysis of the basic molecular mechanisms by which chemicals affect cellular receptors, signal transduction pathways, and cell functions. In Aim 1, stably transfected cell lines will be developed which respond to one or more hormones (steroid, thyroid and retinoic acid) with the induction of receptor/dependent expression of firefly luciferase or green fluorescent protein receptor genes. The endocrine disrupting activity of a given chemical(s), or complex chemical mixtures could rapidly be determined by examining their ability to activate/inhibit induction of reporter gene expression. In Aim 2, measurement of perturbations in gene expression by dioxin, arsenic and chromium in human keratinocytes, using subtractive hybridization and DNA array techniques will allow identification of a series of gene products (i.e. potential biomarkers of effect) whose expression is altered by these chemicals. In Aim 3, validation of soluble epoxide hydrolase (sEH)-generated oxylipin metabolites as relevant biomarkers as relevant biomarkers of exposure/effect resulting from exposure to hazardous environmental chemicals which alter sEH activity will be evaluated using cells and mice. DNA array and metanomics approaches will also be developed and employed for identifying genetic and metabolic biomarkers of exposure to sEH inhibitors. In Aim 4, a series of transgenic skeletal myotubule cell lines (wild-type and mutant) will be developed as biomarkers of normal and heightened susceptibility to environmental chemicals causing dysfunction in Ca2+-regulation and signaling and as bioassays for the identification of environmental toxicants which influence calcium signaling. Overall, these approaches not only take advantage of the ability of a chemical to activate/inhibit intracellular receptor mediated signaling events and gene expression, but these bioassays and biomarkers provide rapid mechanistically-based screening systems to detect toxicants and toxicant exposure.