Superfund Research Program
Mechanisms and Impacts of PCB Resistant Fish
Project Leader: Mark E. Hahn (Woods Hole Oceanographic Institution)
Co-Investigators: Sibel I. Karchner (Woods Hole Oceanographic Institution), Neelakanteswar Aluru (Woods Hole Oceanographic Institution)
Grant Number: P42ES007381
Funding Period: 1995-2020
The overall objective of this project is to understand the impact of long-term, multi-generational exposure to high levels of dioxin-like chemicals, including certain polychlorinated biphenyls (PCBs). To address this problem, Dr. Hahn and his research team are studying a population of estuarine fish, Atlantic killifish (Fundulus heteroclitus), inhabiting New Bedford Harbor, MA (NBH), a Superfund site that is highly contaminated with PCBs and other chemicals. Previously, the researchers showed that NBH killifish are approximately 14-fold less sensitive to dioxin-like compounds than killifish from a reference site, Scorton Creek (SC), in Sandwich, MA, and that this "dioxin resistance" is heritable. Dioxin causes toxicity by activating a protein known as the aryl hydrocarbon receptor (AHR). The researchers have identified two killifish aryl hydrocarbon receptors (AHR1 and AHR2) and an AHR repressor (AHRR), a protein that inhibits effects occurring through the AHRs. The specific objectives of this research are: 1) to understand the mechanisms by which NBH killifish are less sensitive to the developmental toxicity of dioxin-like compounds that act through the AHR proteins, and 2) to determine the impact of evolved dioxin resistance on the sensitivity to other environmental stressors, such as low oxygen (hypoxia).
This year the researchers continued their studies of AHR gene polymorphisms (i.e. multiple forms of the gene that lead to variant AHR proteins) and their possible role in the mechanism of resistance. They identified two AHR2 haplotypes (sets of associated SNPs) that were more prevalent at NBH (resistant population) and two that were more prevalent at SC (sensitive fish). cDNAs for each of these four haplotypes were cloned into expression vectors and used in functional assays. Each variant exhibited high-affinity binding of [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In preliminary transient transfection assays, each variant was able to activate transcription. Quantitative dose-response experiments are in progress.
This year the research team also continued to develop genomic resources that will allow them to perform gene expression profiling, using microarrays, in killifish embryos exposed to PCBs and hypoxia. A workshop was held to discuss the development and distribution of killifish microarrays for use by several research groups using killifish in research. Within this project and in collaboration with Margie Oleksiak (BU and Duke SBRPs) and Rich DiGiulio (Duke SBRP), the isolation and sequencing of developmentally expressed genes continues, with sequencing of ESTs (expressed sequence tags) from developmental cDNA libraries. In preparation for gene expression profiling, a preliminary microarray study was performed to determine differences in gene expression of 384 metabolic genes during late organogenesis among 20 embryos of parents from a clean and polluted site. Most (> 90%) of the 384 metabolic genes were expressed during this developmental stage (stage 31) based on hybridization signal intensities that were above negative controls. Expression of 9% (11 / 115) of metabolic genes was significantly different (p < 0.01) during late organogenesis of Fundulus development.
Higher density arrays will be printed this fall using an inkjet printer (ArrayJet120) and used for gene expression analysis of exposed embryos. These arrays will contain 6,000 - 8,000 genes printed in triplicate.
This research explores how natural populations of animals respond to prolonged, high-level exposure to contaminants. Project researchers use fish as models to investigate the mechanisms underlying differential sensitivity to the developmental toxicity of dioxin-like chemicals. The existence of dioxin-sensitive and dioxin-resistant populations of killifish provides a unique opportunity to understand the molecular mechanisms of differential dioxin sensitivity and the impact of evolved resistance on the sensitivity of fish to other environmental stressors.