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The overall objectives of this project are to gain further insights into the molecular mechanisms of toxicity of dioxin-like chemicals and endocrine disruptor chemicals. Additionally, we will take advantage of advances in the Ah (dioxin) and estrogen receptor (AhR and ER, respectively)-dependent mechanisms of action of these chemicals to develop high-throughput recombinant cell-based screening bioassays for rapid and sensitive detection of dioxins and related chemicals (i.e. polychlorinated dioxins, furans and biphenyls, as well as carcinogenic polycyclic aromatic hydrocarbons) as well as endocrine disruptors (particularly that of xenoestrogens) that can be used for screening of environmental, biological and food/feed samples. To accomplish these goals, we propose three specific aims. <P> 1) Determine structure-function relationships of the AhR ligand binding domain in order to understand both the molecular mechanisms by which structurally diverse ligands bind to and activate the AhR and to develop novel ligand-selective AhRs by site-directed mutagenesis that can be used to develop chemical specific bioassay systems. <P> 2) Develop a series of novel recombinant amplified AhR- and ER-based cell bioassay systems for detection of ultra low levels of endocrine disruptor chemicals that exert their action through these nuclear receptor signaling pathways.<P> 3) Utilize the newly developed bioassay systems for screening of extracts of environmental, biological and food samples as well as pure chemicals to identify those that contain chemicals that can affect the AhR an ER signaling pathways. <P> Not only do we expect to gain further insights into the basic molecular mechanisms by which dioxin- and estrogen-like chemicals can produce adverse health effects, but we will develop, optimize and utilize produce novel enhanced cell bioassays particularly useful for detection of low level contamination by these two classes of contaminant.
Non-Technical Summary: <BR>Humans and animals are exposed to contaminants from a wide variety of sources including air, water, soil, food and commercial and consumer products and these contaminants exist as a complex mixture of chemicals whose individual components and concentration can vary dramatically, complicating their detection and hazard assessment. While there are many mechanisms by which these chemicals can produce their adverse effects, their ability to modulate the function of intracellular receptor-mediated signaling pathways is one mechanism by which diverse chemicals can produce a common set of responses. Decreased reproductive success and feminization of males of several wildlife species, combined with reported reductions in sperm counts in men and significant increases in hormone-dependent breast and prostate cancer, are responses that may result from exposure to chemicals that adversely affect steroid hormone action. Numerous environmental contaminants that act as endocrine disrupting chemicals (EDCs) have been identified and shown to dramatically affect the function of such nuclear hormone receptors. Although the effects of many of these chemicals on human health remains controversial, since clear evidence exists for their adverse effects in many wildlife populations, at sufficient levels of EDCs would also produce toxic effects in human. This project will focus on the molecular mechanisms by which EDCs disrupt normal physiological processes, particularly receptors that mediate the action of dioxin-like chemicals and those that mediate the action of estrogen (specifically the Ah (dioxin) receptor (AhR) and estrogen receptor, respectively). A continuing emphasis will be on the analysis of the AhR-dependent molecular mechanisms by which dioxin-like chemicals produce their biological and toxicological effects. These chemicals represent a group of highly toxic and persistent environmental contaminants present in relatively high concentrations not only in the state of California, but also throughout the US and the rest of the world and they represent a significant risk to human and animal health. Therefore, increasing our understanding of the basic molecular mechanism by which these chemicals can bind to and activate the AhR will not only allow us to understand the early signaling events produced by these chemicals, but to potentially identify targets for the development of protective measures against HAH toxicity. Additionally, we will continue our application of recombinant DNA technologies to develop, improve and evaluate/validate several simple AhR- and ER-based cell bioassay systems for use in biomonitoring, detection and characterization of dioxins and EDCs in environmental, agricultural, biological, food and feed samples and to identify novel chemicals or classes of chemicals that can modulate the activity of their respective receptors. The identification of natural and/or synthetic chemicals that can affect or interfere with endocrine action is an important first step in exposure assessment and evaluation of the potential risks/effects of materials that contain these chemicals. <P> Approach: <BR> In Aim 1 we will use information derived from our homology model of the AhR ligand binding domain to generate a large series of targeted mutations of amino acids within the AhR ligand binding domain and to use these mutant AhRs to identify those that are selectively activated by dioxin-like chemicals as well as other classes of AhR ligands. Not only will these basic mechanistic studies provide insights into how these toxic chemicals can bind to and activate the AhR, the protein that mediates their toxicity, but we hope to be able to develop chemical-specific AhRs that can be used to generate chemical-selective bioassay systems. Stable co-transfection of these chemical selective AhRs into AhR deficient cells along with an AhR-responsive DRE-luciferase reporter gene to generate chemical-specific cell bioassays and these will be optimized and characterized as we have done in previous studies. If we are successful, samples for bioassay analysis would not only require minimal clean-up (making the analysis rapid and very inexpensive), but it would generate a chemical specific bioassay with new applications for both field and laboratory use. In Aim 2 we will improve existing bioassays for dioxin- and estrogen-receptor-based cell bioassays to create novel assays with significantly lower limits of detection and greater overall response. We will examine the effect of increasing the number of responsive elements (i.e. DNA binding sites for the AhR and ER) in a given reporter gene plasmid (preliminary results suggest that this will enhance our bioassay response) and we will also examine the effect of amplification of the number of responsive plasmids per cell. Both of these approaches can easily be accomplished using one of several well-documented approaches. TCDD or hormone responsiveness of each stable cell line generated using the above approaches will be examined in dose response experiments as we have carried out in our previous studies and we will identify those which have a significantly lower limit of TCDD or estrogen detection and/or a significantly greater overall induction response. The availability of these novel cell bioassays will greatly facilitate large scale screening studies with large sample numbers but limited sample size and/or ultra low levels of dioxin-like chemicals and endocrine disruptors. Finally, in Aim 3 we will optimize and validate these novel bioassays for the detection of their respective ligands using pure chemicals, chemical mixtures and unknown chemical mixtures. The optimized bioassay systems will also be adapted for use in high throughput screening analysis of extracts from various environmental, biological, food and feed samples and commercial/consumer products obtained from ongoing studies. Positive extracts will be subjected to bioassay-directed fractionation and instrumental analysis to identify the responsible chemical(s). These approaches will allow us to rapidly screen extracts of diverse matrices for the presence of dioxin-like chemicals and endocrine disruptors and should allow us to identify new chemicals or classes of chemicals that can affect these receptor systems.