2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the prototype for a family of highly toxic halogenated aromatic compounds that are widely dispersed in the environment. We propose that estimating the hazard posed by these compounds is dependent upon a thorough understanding of the molecular basis of toxicity and knowledge of how this mechanism differs among animal models. It is the underlying hypothesis of our research that interspecies differences in TCDD toxicity result from differences in the primary structure of the Ah receptor (AHR), differences in the expression of other components of the signaling pathway or differences in the battery of regulated genes. In an effort to test this hypothesis, we propose two lines of experimentation. First, we will complete a detailed molecular analysis of those proteins already known to play important roles in TCDD signaling, i.e., the AHR and the Ah receptor nuclear translocator (ARNT). These efforts will focus upon the characterization of functional domains and on the identification of species specific differences in these domains that may relate to altered responses to agonists like TCDD. In a parallel series of experiments, we will attempt to expand upon our overly simplistic model of the TCDD signaling pathway. To this end, we will employ a series of promising genetic, biochemical and computer driven approaches to clone and characterize additional components of this pathway, as well as provide the tools to identify novel regulated endpoints that may be intimately involved in toxicity. We propose that integration of these data will provide a model that describes the complete pathway of TCDDs effects, from receptor binding to toxicity and that such information will provide a sound mechanistic basis for TCDD risk analysis. To accomplish these broad objectives, we have proposed the following specific aims.
Aim 8 : Generate a high resolution functional domain maps of the AHR and ARNT Aim 9: Complete the cloning and basic structural/functional comparisons of the AHR and ARNT cDNAs from important animal models and target species.
Aim 1 0: Clone and analyze novel bHLH-PAS cDNAs that interact with components of the TCDD signaling pathway.
Aim 1 1: Determine the Pairing rules, DNA half-site specificities and functional domain maps of the AHR, ARNT and novel bHLH-PAS proteins.
Aim l2: Identify coactivators and additional regulators of the TCDD signaling pathway.
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