2,3,7,8-Tetrachlorodibenzo-p-dioxin versus 3-methylcholanthrene: comparative studies of Ah receptor binding, transformation, and induction of CYP1A1.
Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC) cause transcriptional activation of the CYP1A1 gene via their interaction with the aromatic hydrocarbon (Ah) receptor. Direct radioligand binding and competitive binding studies demonstrated that the cytosolic Ah receptor from the mouse hepatoma cell line Hepa-1 bound TCDD with an affinity approximately 3-4-fold greater than that for MC. However, TCDD was approximately 1,000-fold more potent than MC as an inducer of CYP1A1-mediated aryl hydrocarbon hydroxylase activity in cultured Hepa-1 cells as assessed at 14 h following exposure to inducer. To understand the basis for this quantitative discrepancy between Ah receptor binding affinity and CYP1A1 induction potency, we systematically compared TCDD and MC for their abilities to activate sequential events in the CYP1A1 induction mechanism that occur subsequent to initial binding to the cytosolic Ah receptor. Using a gel retardation assay, TCDD and MC were shown to be equipotent in causing in vitro transformation of the cytosolic Ah receptor to its DNA-binding form. In addition, the transformed Ah receptor bound to a specific dioxin-responsive enhancer sequence with the same apparent affinity when MC was the ligand as when TCDD was the ligand. At an early time point (i.e. 2 h) in the CYP1A1 induction process, TCDD was only approximately 4-25-fold more potent than MC in stimulating the nuclear uptake of the ligand-Ah receptor complex, and the two ligands displayed a relatively small difference (> or = 10-fold) in CYP1A1 mRNA induction potency. When assessed at 4 h following ligand treatment, TCDD was only approximately 10-fold more potent than MC as an aryl hydrocarbon hydroxylase inducer, suggesting a time-dependent reduction in the potency of MC in intact cells. Exposure of Hepa-1 cells to MC over a 16-h time course resulted in an increased ability of these cells to convert [3H]MC to alkali-extractable metabolites. Our data are consistent with the idea that TCDD and MC display relatively small differences in their intrinsic abilities to activate Ah receptor-mediated events. The reduced biological potency of MC observed in intact cells and whole animals is at least partially due to the more rapid metabolic inactivation of this ligand compared with the poorly metabolized TCDD. By extension, the extraordinary toxicity of TCDD may not be explained solely by its high affinity for the cytosolic Ah receptor.
Riddick
D S
DS
Department of Pharmacology, University of Toronto, Ontario, Canada.
Huang
Y
Y
Harper
P A
PA
Okey
A B
AB
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D003600:Cytosol / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D008748:Methylcholanthrene / Q000378:metabolism* / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism* / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011869:Radioligand Assay; D018336:Receptors, Aryl Hydrocarbon / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014316:Tritium; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D014316:Tritium; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D015500:Chloramphenicol O-Acetyltransferase