PROJECT SUMMARY – TEEGUARDEN PROJECT Lack of understanding the molecular basis for susceptibility across toxicity test systems is a long-standing barrier to improved risk assessment, improved cleanup decisions for SRP sites, and successful health mitigations. Our objective is to develop transformative, activity-based proteomic probes and multi-modal chemical microscopy to measure the metabolic and distributional processes that contribute to differential susceptibility to PAH exposure. We hypothesize that measurable but commonly ignored differences in tissue distribution and metabolic capacity to activate and/or detoxify PAHs contributes to differential susceptibility. To test our hypothesis, we will complete three aims: 1. Identify the complete set of active P450, glutathione-S- transferase and UDP-glucuronosyltransferase enzymes that metabolize PAHs & PAH metabolites. We will incubate activity-based probes in tissue samples to enrich and identify active enzymes using our proteomics platform. 2. Develop a single assay to determine both PAH metabolic rates and metabolic susceptibility by relating PAH metabolic rates to global measures of enzyme activity. We will incubate activity-based probes in tissues while conducting standard PAH metabolism assays to determine substrate specificity and metabolic constants for active enzymes. 3. Determine how exposure susceptibility across test systems and humans depends upon PAH physicochemical properties, tissue composition, and body composition. We will measure tissue composition across our systems and measure and predict the disposition of PAHs across these systems, and humans. The products of our research directly enhance the relevance and impact of each project and core for stakeholders. Our innovations (ABPP and multi-modal chemical microscopy) will open new scientific horizons in the understanding of two key molecular determinants of differential susceptibility. The work will satisfy explicit expectations for SRP by a) demonstrating “that the hazardous substance, its dose, exposure pathway and model organisms are considered within the context of timing, prevalence, and detection of exposure” and by b) assuring that the “work is contextualized in terms of its relevance to human exposure.” Our research results will directly inform and improve human health assessment for PAHs at Superfund sites.