Mechanisms of Cellular Injury: A Cellulomics Approach

该项目将开发和实施一种新的综合生物学方法，在细胞和组织水平上研究由选定的多环芳烃引起的化学诱导的应激和损伤机制。将利用肝和肾的精密切割组织切片，使用一种新的“细胞组学”方法，以非侵入性的方式检测和分析精选的终点，以便开始弥合体外和体内模型之间的差距，用于细胞损伤的机制分析。易感表型的动物模型包括具有导致二恶英敏感(C57BL6J)和抗性(DBA2)表型的AhR多态变异的小鼠、具有人AhR(HAHR；HAHR基因敲除)的转基因小鼠和AhR敲除小鼠，以确定多环芳烃对细胞内稳态改变的影响以及这可能如何促进疾病风险。虽然拟议的调查最初将侧重于几个多环芳烃和两个器官系统，但所开发的工具和方法将广泛适用于对模型多环芳烃和复杂混合物以及新的和/或未经测试的化学品造成的细胞反应的高通量器官和组织层面的评估。模型多环芳烃化合物包括苯并[a]芘(BaP)、苯并[e]芘(BEP)、5-甲基黄烷(5Mcr)和3-甲基胆菲(SMC)。多环芳烃的固有荧光将被用来定位BaP、BEP、5MCr和SMC在小鼠模型肝和肾切片中的分布和原位代谢。还将评估细胞内稳态机制，包括分析细胞内钙离子稳态，以及作为细胞损伤的哨兵和效应者的信号机制以及其他功能稳态的组合。 参数包括缝隙连接介导的细胞间通讯、活性氧和一氧化氮的产生、谷胱甘肽和谷胱甘肽-S转移酶活性、线粒体功能、脂质过氧化、细胞色素P450酶诱导、P-糖蛋白诱导和细胞凋亡/坏死。一旦优化，这些细胞组学研究将扩展到多环芳烃的二元和复杂混合物。

This project will develop and implement a novel integrative biology approach to investigate chemical-induced stress and injury mechanisms at the cell and tissue level caused by selected PAHs. Precision cut tissue slices of liver and kidney will be exploited to non-invasively detect and analyze well-chosen endpoints using a novel "cellulomics" approach in order to begin bridging the gap between in vitro and in vivo models for mechanistic analysis of cellular injury. Animal models of susceptible phenotypes including mice with polymorphic variations of the AhR that result in dioxin-sensitive (C57BL6J) and resistant (DBA2) phenotypes, a transgenic mouse possessing the human AhR (hAhR; the hAhR knock-in) and an AhR knock-out mouse will be utilized to identify PAH effects on altered cellular homeostasis and how this may contribute to disease risk. While the proposed investigations will initially focus on a few PAHs and two organ systems, the tools and approaches developed will have wide applicability to high throughput organ- and tissue-level assessment of cellular responses caused by model PAHs and complex mixtures as well as for new and/or untested chemicals. Model PAH compounds selected for this investigation include benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), 5-methylchrysene (5MCr), and 3-methylcholanthrene (SMC). The inherent fluorescence of PAHs will be utilized to map the distribution and in situ metabolism of BaP, BeP, 5MCr and SMC in the precision-cut liver and kidney slices of the mouse models. Cellular homeostasis mechanisms will also be assessed and include analysis of intracellular Ca2+ homeostasis and signaling mechanisms as both a sentinel and an effector of cellular injury along with a combination of other functional homeostasis parameters including gap junction mediated intercellular communication, reactive oxygen species and nitric oxide production, glutathione and glutathione-S-transferase activity, mitochondrial function, lipid peroxidation, cytochrome P450 enzyme induction, P-glycoprotein induction and apoptosis/necrosis. Once optimized, these cellulomics investigations will be extended to binary and complex mixtures of PAHs.