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.
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