Effects of ligand structure on the in vitro transformation of the rat cytosolic aryl hydrocarbon receptor.
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abstract
Incubation of radiolabeled, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF),1,2,3,7,8-pentachlorodibenzo-p-dioxin(PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), 1,2,7,8-TCDF, and 2,3,7-trichlorodibenzo-p-dioxin (TrCDD) with rat hepatic cytosol for 2 h at 0 degrees C gave liganded aryl hydrocarbon (Ah) receptor complexes which were indistinguishable as determined by velocity sedimentation analysis and DNA-Sepharose column chromatography. Incubation of the cytosol plus the different radioligands for 2 h at 20 degrees C resulted in the formation of Ah receptor complexes which exhibited increased retention times on DNA-Sepharose columns. It was apparent that the amount of specifically bound Ah receptor complex or the levels of the transformed Ah receptor complex which eluted from the column with 0.2-0.3 M salt were dependent on the structure of the radioligand. For example, after incubation for 2 h at 20 degrees C the overall yields of the specifically bound transformed Ah receptor complex were 3.4, 2.0, 1.2, 1.9, 0.3, and 0.1%, respectively, using 2,3,7,8-TCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDD, 1,2,3,7,8-PeCDF, 1,2,7,8-TCDF, and 2,3,7,8-TrCDD as radioligands. A more quantitative measure of the structure-dependent transformation of the liganded cytosolic Ah receptor complex was determined using a gel retardation assay with a consensus synthetic dioxin-responsive element (DRE) (26-mer, duplex). The EC50 values obtained for the concentration-dependent formation of the retarded DRE-Ah receptor complex using 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,7-TrCDD, and 1,2,7,8-TCDF as ligands were 0.26, 0.35, 0.78, 1.75, 27.0, and 220 nM, respectively. The structure-dependent differences in these values were similar to their different potencies as Ah receptor agonists and these data suggest that the structure-dependent transformation of the liganded cytosolic Ah receptor may significantly contribute to the structure-activity relationships observed for 2,3,7,8-TCDD and related compounds.
