Mechanism of action of aryl hydrocarbon receptor antagonists: inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 gene expression.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 gene expression as determined by increased CYP1A1 mRNA levels and ethoxyresorufin O-deethylase (EROD) activity in mouse Hepa 1c1c7, rat hepatoma H-4II E and human Hep G2 cancer cell lines. In contrast, treatment of these cell lines with either alpha-naphthoflavone (alpha NF) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) at concentrations as high as 10(-6) M resulted in only minimal induction of CYP1A1 mRNA levels or EROD activity. Cotreatment of the cells with 10(-9) M TCDD plus different concentrations (10(-8)-10(-6) M) of MCDF or alpha NF resulted in a concentration-dependent decrease in TCDD-induced CYP1A1 mRNA levels and EROD activity in the three cell lines. Moreover, using 10(-9) M [3H]TCDD, it was shown that the alpha NF- and MCDF-mediated antagonism of TCDD-induced CYP1A1 gene expression was paralleled by a decrease in levels of the nuclear [3H]TCDD-Ah receptor complex as determined by velocity sedimentation analysis of the nuclear extracts. The binding of nuclear extracts from the treated cells to a synthetic consensus dioxin responsive element (DRE) (a 26-mer) was determined by gel retardation studies using 32P-DRE. In cells treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M alpha NF, the concentration-dependent decrease in TCDD-induced CYP1A1 gene expression by alpha NF was also paralleled by decreased levels of a retarded band associated with the nuclear Ah receptor-DRE complex. In contrast, the results of the gel shift assay of nuclear extracts treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M MCDF indicated that there were relatively high levels of nuclear MCDF-Ah receptor complex in the cells co-treated with TCDD plus the antagonist but this was not accompanied by induced CYP1A1 gene expression. The results suggest that alpha NF and possibly MCDF compete with TCDD for cytosolic Ah receptor binding sites; however, MCDF may also inhibit the induction response by competing for and/or partially inactivating genomic binding sites.
Merchant
M
M
Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4466.
Morrison
V
V
Santostefano
M
M
Safe
S
S
D000818:Animals; D001483:Base Sequence; D001571:Benzoflavones / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008114:Liver Neoplasms, Experimental; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010088:Oxidoreductases / Q000378:metabolism*; D000072317:Polychlorinated Dibenzodioxins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D001571:Benzoflavones; D009838:Oligodeoxyribonucleotides; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1