Hazardous waste sites contain complex mixtures of a wide variety of toxic chemicals. Unfortunately, development of 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 and in vitro 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 or class of toxicants, each of the four proposed approaches will exploit information derived from an analysis of the basic molecular mechanisms by which selected chemicals affect cellular receptors, signal transduction pathways and cellular/enzyme functions.
In Aim 1, stably transfected cell lines will be developed which respond to dioxin-like chemicals, steroid/thyroid hormones or hormone-like chemicals with the induction or inhibition of receptor-dependent expression of firefly luciferase or green fluorescent protein reporter genes. We will also develop a novel portable in vitro AhR-based bioassay for detection of dioxin-like chemicals.
In Aim 2, human keratinocytes will be used to examine genomic, proteomic and metabolomic effects following exposure to metals/metalloids and chemicals that produce oxidative stress to identify potential biomarkers that are specifically altered by these chemicals.
In Aim 3 in vitro and cell based bioassay systems will be used to examine the influence of superfund chemicals on regulatory lipids that control inflammation and to identify xenobiotics that alter expression and activity of soluble epoxide hydrolase.
Aim 4 proposes to develop and validate ryanodine receptor-based in vitro and intact cell bioassays to identify and characterize non-coplanar halogenated persistent organic pollutants that can affect calcium signaling pathways. In the final Aim, these assay systems will be integrated and optimized and then used in a series of validation studies for the detection and relative quantitation of toxic chemicals present in complex mixtures of chemicals extracted from a variety of matrices. Overall, the proposed studies will not only increase our basic knowledge of the biological and toxicological effects of a variety of Superfund priority chemicals, but the resulting specific bioassays and biomarkers that will be developed will provide rapid mechanistically-based screening systems for the detection of toxicants and toxicant exposure.
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