Analysis and Effect of Persistent Ah Receptor Activation

我们研究的总体目标是了解卤代芳香烃(HAHS)、多环芳烃(PAHs)及其相关化学物质与ah受体(AhR)相互作用的分子机制，以改变细胞和动物对这些诱导剂的基因表达和反应。AhR是一种配体依赖的转录因子，介导了HAHS和PAHs的大部分生物学和毒理作用。据报道，在接触HAHS和PAHs后观察到的毒性和生物反应的光谱以及产生这些反应所需的化学品浓度方面存在显著差异，HAHS的效力明显高于PAHs。尽管HAHS和PAHs对HAHS和PAHs的不同反应可能是由于靶细胞的各种生化和生理特性造成的，但人们普遍认为HAHS更大的毒理和生物学效力是因为它们显著更高的AhR结合亲和力和对新陈代谢的抵抗力。最近的证据表明，HAHS和PAHs作为AhR依赖的基因表达的激活剂的效力和有效性存在差异，与它们在细胞中的持久性和代谢稳定性直接相关的激活剂是不同的。我们假设，PAHs和HAHS在效力和生物反应方面的一些差异直接与AhR蛋白和/或AhR蛋白复合体结构的配体特异性差异有关，这种差异改变了AhR的功能及其对DNA/染色质的相对亲和力/特异性。因此，我们建议进行详细的比较研究，以表征在体外和在培养细胞中所选择的HAHS和PAHs在小鼠AhR的配体激活和持久性以及DNA/染色质结合方面的异同，并鉴定和表征AhR结构的配体依赖变化。DR基因的核苷酸 HAHS结合的AhR复合体转录激活基因表达的特异性 和多环芳烃，以及HAH和PAH诱导基因表达的异同，使用微阵列在细胞培养和CYP缺失小鼠中进行评估。最后，将产生表达持续活性(例如，非配体依赖)的AhR复合体的转基因动物，以检查AhR和配体对与这种持续的AhR激活相关的不良影响的贡献。总体而言，这些研究将深入了解HAHS和PAHs激活AhR的能力在物种和配体方面的不同，导致这种激活持续的机制，以及它在HAHS和PAHs的毒性和生物效应中所起的作用。

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.