Riddick D S, Huang Y, Harper P A, Okey A B
Department of Pharmacology, University of Toronto, Ontario, Canada.
J Biol Chem. 1994 Apr 22;269(16):12118-28.
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.
卤代芳烃如2,3,7,8-四氯二苯并-对-二噁英（TCDD）和多环芳烃如3-甲基胆蒽（MC）通过与芳烃（Ah）受体相互作用导致CYP1A1基因的转录激活。直接放射性配体结合和竞争性结合研究表明，来自小鼠肝癌细胞系Hepa-1的胞质Ah受体与TCDD结合的亲和力比对MC的亲和力大约高3-4倍。然而，在培养的Hepa-1细胞中，以暴露于诱导剂后14小时评估，TCDD作为CYP1A1介导的芳烃羟化酶活性的诱导剂比MC的效力大约高1000倍。为了理解Ah受体结合亲和力与CYP1A1诱导效力之间这种定量差异的基础，我们系统地比较了TCDD和MC在激活CYP1A1诱导机制中初始结合胞质Ah受体后发生的一系列事件的能力。使用凝胶阻滞试验，TCDD和MC在使胞质Ah受体体外转化为其DNA结合形式方面显示出同等效力。此外，当MC是配体时与当TCDD是配体时，转化的Ah受体以相同的表观亲和力结合到特定的二噁英反应性增强子序列上。在CYP1A1诱导过程的早期时间点（即2小时），TCDD在刺激配体-Ah受体复合物的核摄取方面仅比MC强约4-25倍，并且这两种配体在CYP1A1 mRNA诱导效力方面显示出相对较小的差异（≥10倍）。当在配体处理后4小时评估时，TCDD作为芳烃羟化酶诱导剂仅比MC强约10倍，表明在完整细胞中MC的效力存在时间依赖性降低。将Hepa-1细胞暴露于MC 16小时导致这些细胞将[3H]MC转化为碱可提取代谢物的能力增加。我们的数据与TCDD和MC在激活Ah受体介导事件的内在能力方面显示出相对较小差异的观点一致。在完整细胞和整体动物中观察到的MC生物学效力降低至少部分是由于与代谢缓慢的TCDD相比，这种配体的代谢失活更快。由此推断，TCDD的极高毒性可能不能仅由其对胞质Ah受体的高亲和力来解释。
