Mechanisms of Cellular Injury: A Cellulomics Approach

本项目将开发和实施一种新的综合生物学方法来研究由选定的多环芳烃引起的细胞和组织水平的化学诱导应激和损伤机制。精确切割的肝脏和肾脏组织切片将被用于使用一种新的“细胞组学”方法进行非侵入性检测和分析精心选择的端点，以便开始弥合体外和体内模型之间的差距，以进行细胞损伤的机制分析。易感表型的动物模型，包括导致二恶英敏感（C57BL6J）和耐药（DBA2）表型的AhR多态性变异小鼠，具有人类AhR的转基因小鼠（hAhR； hAhR敲入）和AhR敲除小鼠，将用于确定多环芳烃对改变的细胞稳态的影响以及这可能如何促进疾病风险。虽然拟议的研究最初将集中在几个多环芳烃和两个器官系统上，但所开发的工具和方法将广泛适用于高通量器官和组织水平评估由模型多环芳烃和复杂混合物引起的细胞反应，以及新的和/或未经测试的化学品。本研究选择的模型多环芳烃化合物包括苯并[a]芘（BaP）、苯并[e]芘（BeP）、5-甲基蒽（5MCr）和3-甲基胆蒽（SMC）。利用多环芳烃的固有荧光来绘制BaP、BeP、5MCr和SMC在小鼠模型精确切割的肝脏和肾脏切片中的分布和原位代谢。细胞内稳态机制也将被评估，包括分析细胞内Ca2+内稳态和信号机制，作为细胞损伤的前哨和效应，以及其他功能内稳态的组合

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.