The goal of the basic research proposed here is to understand the effects of long-term, multi-generational exposure to high levels of contaminants on natural populations of animals inhabiting Superfund sites. The research addresses a key question concerning the extent to which adaptive changes in the sensitivity to one class of chemicals may have far-reaching effects on the ability of animals to respond to other types of chemicals or environmental stressors. The research applies innovative molecular approaches in an ecological context to investigate mechanisms of cross-talk among signaling pathways involved in the response to many Superfund chemicals. The studies will be performed in the Atlantic killifish Fundulus heterociitus, a unique model system for integrated investigation of ecological and mechanistic questions concerning the impact of chemicals at Superfund sites. At several locations along the Atlantic coast, populations of killifish have evolved resistance to planar (dioxin-like, non-ortho-subsituted) polychlorinated biphenyls (PCBs) and other chemicals that act through the aryl hydrocarbon receptor (AHR). The central hypothesis is that diminished AHR-dependent signaling in dioxin/PCB-resistant fish impairs the ability of these fish to respond to environmental chemicals and stressors acting through other signaling pathways. To test this hypothesis, in Aim 1 we will measure the sensitivity of PCB-sensitive and PCB-resistant killifish to a suite of environmental stressors including hypoxia, a pro-oxidant chemical (NRF2 activator), an orthosubstituted, non-planar PCB (PXR agonist), and an estrogenic compound (ER agonist). In Aim 2, we will characterize two new AHRs and use morpholino anti-sense and zinc finger nuclease (ZFN) technologies to generate AHR-null and compound AHR-null killifish and use them to investigate the role of each AHR in regulating the response to these stressors. The proposed experiments represent a unique opportunity to obtain new insight on AHR function and the effects of long-term exposure to AHR agonists by utilizing naturally occurring populations exhibiting differences in chemical sensitivity, together with engineered null mutants and duplicated AHR genes that will allow pleiotropic functions of the AHR to be studied in isolation.