Analysis and Effect of Persistent Ah Receptor Activation

Ah受体持续激活的分析及效果

• 批准号: 6986219
• 负责人: MICHAEL STEVEN DENISON
• 金额: $ 24.27万
• 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/6986219
• 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香烃 (HAH)、多环芳香烃 (PAH) 和相关化学物质与 Ah 受体 (AhR) 相互作用以改变基因表达以及细胞和动物对这些物质的反应的分子机制。诱导剂。 AhR 是一种配体依赖性转录因子，介导 HAH 和 PAH 的大部分生物学和毒理学作用。据报道，在接触 HAH 和 PAH 后观察到的毒性和生物反应谱中存在显着的物种、组织和配体特异性差异，而且在产生这些反应所需的化学物质浓度方面也存在显着差异，其中 HAH 比 PAH 更有效。尽管靶细胞中的多种生化和生理特性可能导致对 HAH 和 PAH 的不同反应性，但人们普遍认为 HAH 更大的毒理学和生物学效力是由于其显着更高的 AhR 结合亲和力和对代谢的抵抗力。最近的证据表明，HAH 和 PAH 作为 AhR 依赖性基因表达激活剂的效力和功效存在差异，与其在细胞中的持久性和代谢稳定性直接相关。我们假设 PAH 和 HAH 产生的效力和生物反应的一些差异与 AhR 蛋白和/或 AhR 蛋白复合物结构中的配体特异性差异直接相关，这些差异改变了 AhR 的功能及其相对亲和力/DNA/染色质的特异性。因此，我们建议进行详细的比较研究，以表征体外和培养细胞中选定的 HAH 和 PAH 占据的小鼠 AhR 的配体激活和持久性以及 DNA/染色质结合的相似性和差异，并鉴定和表征配体AhR 结构的依赖性变化。 DRE核苷酸 HAH 结合的 AhR 复合物对基因表达转录激活的特异性 使用微阵列在细胞培养物和 CYP 缺失小鼠中评估 HAH 和 PAH 诱导基因表达的相似性和差异。最后，将产生表达组成型活性（例如，配体独立的）AhR复合物的转基因动物，以检查AhR和配体对与这种持续的AhR激活相关的不利影响的贡献。总体而言，这些研究将深入了解 HAH 和 PAH 激活 AhR 的能力的物种和配体特异性差异、导致这种激活持续存在的机制以及它在毒性和生物效应中所起的作用。 HAH 和 PAH。