2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds elicit a broad spectrum of species and tissue-specific toxic and biologic effects in laboratory animals and mammalian cells in culture. We have recently shown that 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) antagonizes the effects of TCDD and this project will probe the mechanism of this process. MCDF, like some hormone antagonists, may act, in part, by modulating nuclear and/or cytosolic Ah receptor levels. Therefore, the proposed studies (experiment I) will investigate the effects of MCDF on the levels of nuclear [3H]-TCDD-Ah receptor complex, the stability and rate of processing of the nuclear receptor complex and the effects of MCDF on the rate of replenishment of cytosolic Ah receptor. Both of the studies will be carried out using ED80-100 doses of TCDD (for AHH induction) and the maximum inhibitory levels of MCDF in male Long Evans rats and rat hepatoma H-4-II E cells in culture). It is proposed that the observed decrease in the inductions of AHH (by TCDD) is associated with a concurrent decrease in TCDD-induced transcription. Therefore, experiment 2 will determine the effects of MCDF on TCDD- induced transcription of cytochrome p-4501A1 and P-4501A2 mRNAs is male rats and cytochrome P-4501A1 mRNA in rate hepatoma H-4-II E cells using cDNA probes which are available in this laboratory. Preliminary studies have demonstrated that the 8-iodo analog of MCDF (i.e., I-MCDF) exhibits comparable properties as a TCDD antagonist. Experiment 3 will fully characterize the activity of I-MCDF as an antagonist of the induction of the transcription and translation of the cytochrome P-4501A1 and P-4501A2 genes by TCDD. The second phase of this study will (a) determine the molecular properties of the 125I-MCDF- Ah receptor complex and utilize the radiolabled antagonist to detect the formation of nuclear antagonist receptor complexes. The final study will more fully investigate the molecular mechanism of action of TCDD antagonists using the plasmid pMcat5.12 which contains a chloramphenicol acetyl transferase (CAT) gene, the MMTV promoter and an 82 bp fragment designated DRE 3 (dioxin regulatory element). Following transfection into Hepa 1c1c7 cells, the effect of TCDD, MCDF and TCDD plus MCDF on transcriptional activation will be determined. The effects of MCDF on the interaction of the TCDD-Ah receptor complex with DRE 3 and the direct interaction of 125I-MCDF-receptor complexes with this proposed nuclear binding site will be determined by gel retardation assays using the appropriate nuclear extracts, the 82 bp polynucleotide fragment and 32P-labeled DNA and 125I-labeled agonist or antagonist. These proposed studies are designed to delineate the mechanisms of action associated with the antagonism of the induction of cytochrome P-450 gene transcription by TCDD.
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