芳烃受体拮抗剂的作用机制：抑制2,3,7,8-四氯二苯并对二恶英诱导的CYP1A1基因表达。

Mechanism of action of aryl hydrocarbon receptor antagonists: inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 gene expression.

作者信息

Merchant M, Morrison V, Santostefano M, Safe S

机构信息

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4466.

出版信息

Arch Biochem Biophys. 1992 Nov 1;298(2):389-94. doi: 10.1016/0003-9861(92)90426-w.

DOI:10.1016/0003-9861(92)90426-w

PMID:1329656

Abstract

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.

摘要

2,3,7,8-四氯二苯并-对-二恶英（TCDD）可诱导CYP1A1基因表达，这在小鼠Hepa 1c1c7、大鼠肝癌H-4II E和人肝癌Hep G2细胞系中通过CYP1A1 mRNA水平升高和乙氧基异吩恶唑酮O-脱乙基酶（EROD）活性得以确定。相比之下，用浓度高达10⁻⁶ M的α-萘黄酮（α NF）或6-甲基-1,3,8-三氯二苯并呋喃（MCDF）处理这些细胞系，仅导致CYP1A1 mRNA水平或EROD活性的最小诱导。用10⁻⁹ M TCDD与不同浓度（10⁻⁸ - 10⁻⁶ M）的MCDF或α NF共同处理细胞，导致三种细胞系中TCDD诱导的CYP1A1 mRNA水平和EROD活性呈浓度依赖性下降。此外，使用10⁻⁹ M [³H]TCDD表明，α NF和MCDF介导的对TCDD诱导的CYP1A1基因表达的拮抗作用与核提取物的速度沉降分析所确定的核[³H]TCDD-Ah受体复合物水平降低平行。通过使用³²P-DRE的凝胶阻滞研究确定处理细胞的核提取物与合成的共有二恶英反应元件（DRE）（一个26聚体）的结合。在用10⁻⁹ M TCDD或TCDD加10⁻⁸ - 10⁻⁶ M α NF处理的细胞中，α NF对TCDD诱导的CYP1A1基因表达的浓度依赖性降低也与与核Ah受体-DRE复合物相关的阻滞带水平降低平行。相比之下，用10⁻⁹ M TCDD或TCDD加10⁻⁸ - 10⁻⁶ M MCDF处理的核提取物的凝胶迁移分析结果表明，在与TCDD加拮抗剂共同处理的细胞中有相对高水平的核MCDF-Ah受体复合物，但这并未伴随诱导的CYP1A1基因表达。结果表明，α NF以及可能的MCDF与TCDD竞争胞质Ah受体结合位点；然而，MCDF也可能通过竞争和/或部分使基因组结合位点失活来抑制诱导反应。