Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists: characterization of 6-[125I]methyl-8-iodo-1,3-dichlorodibenzofuran-Ah receptor complexes.
6-Methyl-8-iodo-1,3,-dichlorodibenzofuran (I-MCDF) and its radiolabeled analog [125I]MCDF have been synthesized and used to investigate the mechanism of action of 1,3,6,8-substituted dibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonists. Like 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), I-MCDF partially antagonized the induction by TCDD of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities in rat hepatoma H-4-II E cells and male Long-Evans rat liver. Incubation of rat liver cytosol with [125I]MCDF followed by velocity sedimentation analysis on sucrose gradients gave a specifically bound peak which sedimented at 9.6 S. This radioactive peak was displaced by coincubation with a 200-fold excess of unlabeled I-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and benzo [a]pyrene. Based on the velocity sedimentation results and the elution profile from a Sephacryl S-300 gel permeation column, the Stokes radius and apparent molecular weights of the cytosolic [125I]MCDF-Ah receptor complex were 6.5 nm and 259,200, respectively. In addition, the nuclear [125I]MCDF-receptor complex eluted at a salt concentration of 0.29 M KCl from a DNA-Sepharose column. Velocity sediment analysis of the nuclear [125I]MCDF-Ah receptor complex from rat hepatoma H-4-II E cells gave a specifically bound peak at 5.6 +/- 0.8 S. All of these properties were similar to those observed using [3H]TCDD as the radioligand. In addition, there were several ligand-dependent differences observed in the properties of the I-MCDF and TCDD receptor complexes; for example, the [125I]MCDF rat cytosolic receptor complex was unstable in high salt buffer and was poorly transformed into a form with increased binding affinity on DNA-Sepharose columns; Scatchard plot analysis of the saturation binding of [3H]TCDD and [125I]MCDF with rat hepatic cytosol gave KD values of 1.07 and 0.13 nM and Bmax values of 137 and 2.05 fmol/mg protein, respectively. The nuclear extract from rat hepatoma H-4-II E cells treated with I-MCDF or TCDD interacted with a dioxin-responsive element in a gel retardation assay. These results suggest that the mechanism of antagonism may be associated with competition of the antagonist receptor complex for nuclear binding sites.
Piskorska-Pliszczynska
J
J
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station 77843-4466.
Astroff
B
B
Zacharewski
T
T
Harris
M
M
Rosengren
R
R
Morrison
V
V
Safe
L
L
Safe
S
S
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001483:Base Sequence; D001572:Benzofurans / Q000378:metabolism*; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*
D001572:Benzofurans; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D014157:Transcription Factors; C054923:6-methyl-1,3,8-trichlorodibenzofuran; C066925:6-methyl-8-iodo-1,3-dichlordibenzofuran; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1