Polyhalogenated and polycyclic aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene, are ubiquitous, persistent environmental contaminants causing toxic responses in humans and wildlife. Most of the toxic effects induced by these compounds are mediated by the aryl hydrocarbon receptor (AhR). Upon binding to ligands, the AhR translocates into the nucleus and binds to enhancer sequences causing alterations of gene expression. However, it is not known what connects this action to the rather broad range, yet distinct toxic responses induced by TCDD and related compounds. The investigator's preliminary studies demonstrated physical association and functional, mutual repression between AhR and NF-kB. Because NF-kB is a pleiotropic transcription factor involved in many physiological functions that are known to be affected by TCDD, the AhR-mediated suppression of NF-kB offers a mechanism for hitherto poorly understood TCDD toxicity. Remarkably, the suppression of AhR by NF-kB also offers an underlying mechanism for cytokine-induced suppression of cytochrome P450s (such as CYP1A1, 1A2). To further investigate the AhR/NF-kB interaction, the investigator proposes three specific aims: (1) to define the interacting domains of the AhR and NF-kB; (2) to analyze the functional interaction between AhR and NF-kB, and modulation of this interaction by other protein factors such as ARNT and transcription coactivators, such as p300/CBP; (3) to use the AhR (-/-) mouse and dominant negative suppressor of the NF-KB to examine the physiological effects of AhR/NF-kB interaction on the programmed cell death which leads to TCDD-induced thymus atrophy, as well as to examine cytokine-induced suppression of cytochrome P450s, which leads to decreased ability to metabolize xenobiotics. The functional significance of AhR/NF-KB interaction will be tested by the ability of proteins to interfere with each other in promoter binding. This will be tested by electrophoretic mobility shift assays and transient transfection assays. AhR and NF-kB proteins will be labeled with green fluorescence protein to detect effects of AhR/NF-kB interaction on their respective nuclear localization. The importance of this proposed research is further accentuated by the finding that many commonly consumed vegetables contain organic compounds, such as indole-3-carbinol and its derivatives, which also bind to AhR with high affinity. The physiological effects of these compounds, whether harmful or beneficial, are almost completely unknown. The studies proposed will increase our understanding of the role AhR plays in normal physiology, as well as in pathology induced by TCDD and like compounds.
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