Analysis and Effect of Persistent Ah Receptor Activation

Ah受体持续激活的分析及效果

• 批准号: 6867595
• 负责人: MICHAEL STEVEN DENISON
• 金额: $ 24.54万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6867595
• 关键词: DNA; DNA footprinting; acetylcholine; carbopolycyclic compound; chemical binding; cholinergic receptors; chromatin; chromatin immunoprecipitation; gene expression; gene induction /repression; genetically modified animals; heterocyclic polycyclic compound; laboratory mouse; microarray technology; molecular cloning; polymerase chain reaction; protein structure; receptor expression; tissue /cell culture; toxicant interaction

The overall goal of our research is to understand the molecular mechanism by which halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs) and related chemicals interact with the Ah receptor (AhR) to alter gene expression and responses of cells and animals to these inducers. The AhR is a ligand-dependent transcription factor that mediates the majority of the biological and toxicological actions of HAHs and PAHs. Significant species, tissue- and ligand-specific differences have been reported in the spectrum of toxic and biological responses observed following exposure to HAHs and PAHs but also in the concentration of chemicals needed to produce these responses, with HAHs being significantly more potent than PAHs. Although differential responsiveness to HAHs and PAHs can result from a variety of biochemical and physiological characteristics in target cells, it is generally accepted that the greater toxicological and biological potency of HAHs results from their significantly higher AhR binding affinity and resistance to metabolism. Recent evidence has demonstrated that differences exist in the potency and efficacy of HAHs and PAHs as activators of AhR-dependent gene expression that are separate from those directly related to their persistence and metabolic stability in the cell. We hypothesize that some of the differences in the potency and biological responses produced by PAHs and HAHs are directly related to ligand-specific differences in the structure of the AhR protein and/or AhR protein complex that alters the functionality of the AhR and its relative affinity/specificity for DNA/chromatin. Accordingly, here we propose to conduct detailed comparative studies to characterize the similarities and differences in the activation and persistence of ligand and DNA/chromatin binding of mouse AhR occupied by selected HAHs and PAHs in vitro and in cells in culture and to identify and characterize ligand-dependent changes in AhR structure. The DREnucleotide specificity for transcriptional activation of gene expression by AhR complexes bound by HAHs and PAHs and similarities and differences in HAH- and PAH-induction of gene expression assessed in cells culture and CYP null mice using microarrays. Finally, transgenic animals expressing a consitutively active (e.g., ligand-independent) AhR complex will be generated to examine contributions of the AhR and the ligand to the adverse effects associated with this persistent AhR activation. Overall, these studies will provide insights into the species- and ligand-specific differences in the ability of HAHs and PAHs to activate the AhR, the mechanisms responsible for the persistence of this activation and the role that it plays in the toxic and biological effects of HAHs and PAHs.

我们研究的总体目标是了解卤代芳烃（HAHs），多环芳烃（PAH）和相关化学物质与Ah受体（AhR）相互作用以改变细胞和动物对这些诱导剂的基因表达和反应的分子机制。AhR是一种配体依赖性转录因子，介导了多环芳烃和多环芳烃的大部分生物学和毒理学作用。据报道，在暴露于多环芳烃和多环芳烃后观察到的毒性和生物反应谱中，以及在产生这些反应所需的化学品浓度中，存在显著的物种、组织和配体特异性差异，其中多环芳烃的效力明显高于多环芳烃。虽然不同的反应，多环芳烃和多环芳烃可能会导致在靶细胞的各种生化和生理特性，它是普遍接受的，更大的毒理学和生物学效力的多环芳烃的结果，从他们显着更高的AhR结合亲和力和耐代谢。最近的证据表明，存在差异的效力和功效的多环芳烃和多环芳烃作为激活剂的AhR依赖的基因表达是独立的那些直接相关的持久性和代谢稳定性在细胞中。我们推测，一些由多环芳烃和多环芳烃产生的效力和生物反应的差异直接与AhR蛋白和/或AhR蛋白复合物的结构中的配体特异性差异有关，该结构改变了AhR的功能及其对DNA/染色质的相对亲和力/特异性。因此，在这里，我们建议进行详细的比较研究，以表征的相似性和差异的激活和持久性的配体和DNA/染色质结合的小鼠AhR所占据的选定的多环芳烃和多环芳烃在体外和细胞培养，并确定和表征配体依赖的AhR结构的变化。DR核苷酸 通过与HAH结合的AhR复合物对基因表达的转录激活的特异性 和多环芳烃和相似性和差异HAH-和PAH-诱导基因表达的细胞培养物和基因敲除小鼠使用微阵列评估。最后，表达结构活性（例如，将产生配体非依赖性）AhR复合物，以检查AhR和配体对与这种持续AhR活化相关的不良作用的贡献。总的来说，这些研究将提供深入了解的物种和配体的具体差异的能力，多环芳烃和多环芳烃激活的AhR，机制负责这种激活的持久性和作用，它发挥的毒性和生物效应的多环芳烃和多环芳烃。