The Ah receptor is a ligand-activated transcription factor that controls the expression of cytochrome P4501A1 (CYP1A1) in mammals and is thought to mediate most of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) and related planar halogenated aromatic hydrocarbons. The overall objective of this research is to characterize the structure, function, regulation, and evolutionary relationships of the Ah receptor in non-mammalian species, particularly fish. The presence and properties of the Ah receptor in certain taxonomic groups could determine the susceptibility of animals in those groups to TCDD toxicity. Information on the mechanism of TCDD action in non-mammalian species is necessary for a meaningful assessment of the risk posed by the presence of these compounds in the environment. The knowledge gained by studying the Ah receptor in phylogenetically diverse species is important for establishing the fundamental properties of TCDD's mechanism of action and for validating the use of non-traditional species in toxicology. A phylogenetic approach may also enhance our understanding of the fundamental significance of the Ah receptor, providing clues to its original physiologic function, the identify of its """"""""endogenous"""""""" ligand, and the evolution of gene regulatory systems. In the proposed studies, the molecular properties of the Ah receptor in fish will be determined, and several ligand-binding assays will be compared and optimized. Transformation of the fish Ah receptor to its DNA-binding form will be examined, and its interaction with dioxin-response elements flanking the fish CYP1A1 gene will be characterized. The broad temperature tolerance of poikilothermic vertebrates will be exploited to dissect and dissociate the molecular events involved in Ah receptor transformation. The sequences of Ah receptor cDNAs will be obtained for teleost and elasmobranch fish using the polymerase chain reaction, and the evolutionary conservation of Ah receptor structure will be evaluated. Fish Ah receptor sequences will permit the production of probes for use in studies of Ah receptor regulation and evolution. The phylogenetic distribution of the Ah receptor will be established by investigating diverse vertebrate and invertebrate species for the presence of the Ah receptor and related genes. Additional studies will determine the cell- and tissue-specific expression of the Ah receptor in fish, using ligand- binding assays, amplification of messenger RNA, and in situ hybridization to fish-specific Ah receptor probes. The developmental regulation of Ah receptor expression will also be determined. The results of these studies will provide a greater understanding of the fundamental significance of the Ah receptor and its role in the mechanism of TCDD toxicity.
