Project 4

Mechanisms and Impacts of Dioxin Resistance in Fish

Studying populations of fish that have evolved resistance to halogenated aromatic hydrocarbons (HAHs), polynuclear aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs).

scorton fish collection

Project Leaders

Mark E. Hahn, Project Leader
Woods Hole Oceanographic Institution

Sibel I. Karchner, Co-Leader
Woods Hole Oceanographic Institution

Project Description

The overall objective of Project 4 is to understand the effects of long-term, multi-generational exposure to high levels of contaminants on natural populations of animals inhabiting Superfund sites. We are studying populations of the fish model species, the Atlantic killifish Fundulus heteroclitus, that have evolved resistance to halogenated aromatic hydrocarbons (HAHs), polynuclear aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs), and are able to live in contaminated sites along the Atlantic coast. These chemicals act through the aryl hydrocarbon receptor (AHR), a class of intracellular receptors that regulate the metabolism of toxic substances and other cellular processes by turning specific genes on and off. 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. Our 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.

In the previous grant periods, we characterized a HAH- and PAH-resistant population of killifish from New Bedford Harbor (NBH), Massachusetts, a Superfund site that is highly contaminated with PCBs. We showed that NBH fish are approximately 14-fold less sensitive to effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) than fish from a clean site (Scorton Creek; SC) and that this diminished sensitivity occurs at the level of gene transcription and is heritable. We identified two distinct AHRs in killifish, including a novel AHR form (AHR2) that has since been identified in several other species of fish, but not in mammals. We identified an AHR repressor (AHRR) in killifish and showed that it is inducible via an AHR-dependent mechanism but its expression is neither elevated nor inducible in resistant NBH fish. The studies proposed here will build on these previous findings to define the genetic mechanisms involved in dioxin resistance and explore the broader implications of the dioxin-resistant phenotype involving interaction between the AHR pathway and other environmental signaling pathways. A growing literature provides compelling evidence of cross-talk between the AHR and several other transcription factors involved in mediating the response to other chemicals. This suggests that PCB-resistant fish may have indirect disruptions in other signaling pathways that interact with the AHR-signaling pathway. The proposed research will address the cost of this evolved resistance to PCBs on the animal’s sensitivity to other contaminants—including oxidants, xenoestrogens, and ortho-substituted PCBs—and environmental stressors such as hypoxia, and the implications for ecological risk assessments at Superfund sites. This study will help elucidate the role of AHR in living systems and the long-term ecological effects of Superfund chemicals.

Project 4 participates in our interdisciplinary work with the New Bedford Harbor and surrounding communities. Learn more about the history of the harbor and our work there.

Summer 2016 State of the Science Update

Project 4 studies the population of PCB-resistant killifish inhabiting the New Bedford Harbor Superfund site. This project seeks to elucidate the genetic mechanisms underlying this evolved resistance to dioxin-like PCBs (non-ortho PCBs) as well as the consequences or “costs” of the resistance in terms of altered sensitivity to other environmental chemicals (including ortho-PCBs) and stressors such as hypoxia and oxidative stress.

We have identified two new AHR genes (AHR1b and AHR2b, in addition to AHR1a and AHR2a identified earlier) that could be involved in the mechanism of resistance. Sequencing of the killifish genome confirmed all four AHR genes and showed that they are arranged as two tandem pairs (AHR1a and AHR2a; AHR1b and AHR2b). Functional characterization of these AHR proteins demonstrated differential sensitivity to ligand activation. We are using the CRISPR-Cas9 genome-editing approach to knock out each AHR and thus better understand their functions. We have demonstrated somatic frameshift mutations in AHR2a and AHR2b and potential founders are being raised. We also have begun collaborating with scientists at the USEPA to examine the possible role of the AHR-interacting protein (AIP) in the resistance to dioxin-like PCBs.

In experiments to explore the sensitivity of killifish embryos from New Bedford Harbor (NBH) and a reference site (Scorton Creek (SC)) to ortho-PCBs, RNA-seq analysis showed that exposure to the model ortho-PCB 2,2’,4,4’,5,5’-hexachlorobiphenyl (PCB-153) caused mostly down-regulation of gene expression and that NBH embryos were less sensitive than SC embryos, providing evidence that NBH fish have developed tolerance to ortho-PCBs in addition to the dioxin-like PCBs. In collaboration with Project 5, we cloned and characterized pregnane X receptor (PXR) in zebrafish and killifish and conducted studies to determine the role of killifish PXR in the response to PCBs in resistant and sensitive populations of killifish.  The results suggest a difference between the two populations in the functioning of the PXR signaling pathway.

The results of this project are increasing our understanding of the long-term impacts of chemical contamination at Superfund sites.

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