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 (DLCs), including certain polychlorinated biphenyls (PCBs). Dr. Mark Hahn's team is 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. NBH killifish are much less sensitive to DLCs than killifish from a reference site, Scorton Creek, in Sandwich, MA (SC). DLCs cause toxicity by activating aryl hydrocarbon receptor (AHR) proteins. Killifish have two AHRs (AHR1 and AHR2) and an AHR repressor (AHRR), a protein that inhibits AHRs. The specific objectives of the research are: 1) to understand the mechanisms by which NBH killifish are less sensitive to the developmental toxicity of DLCs that act through AHRs, and 2) to determine the impact of evolved dioxin resistance on the sensitivity to low oxygen (hypoxia).
This year the team has continued their studies of AHR variants encoded by polymorphic AHR alleles. They have identified two AHR2 variants that were more prevalent at NBH (resistant population) and two that were more prevalent at SC (sensitive fish). The team performed experiments to test the ability of each variant to activate transcription. Each variant was expressed in cell culture and the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was measured. All four variants were able to activate transcription. However, the two variants that are more prominent at NBH appeared to be less sensitive, requiring higher concentrations of TCDD to become active.
Dr. Hahn is working with Margie Oleksiak (BU and Duke SBRPs) and Rich DiGiulio (Duke SBRP) to develop microarrays for measuring transcriptional changes in killifish embryos co-exposed to chemicals and hypoxia. They continue to collect ESTs (expressed sequence tags) from cDNA libraries made from all 40 developmental stages and adult tissues from adults exposed to different stresses. ESTs will be used to print microarrays and perform gene expression profiling in embryos exposed to PCBs and hypoxia. These ESTs have been annotated and can be found on the Crawford Lab Marine Genomics web page RNA samples from all 40 stages of killifish development have been isolated, amplified and labeled to provide a statistical analysis of gene expression during development. They have optimized array-printing conditions and are working on optimizing hybridization conditions.
During the summer the researchers collected adult fish from NBH and SC, and used them to generate embryos. An experiment was performed in which embryos were co-exposed to hypoxia and PCB. Embryos were frozen for analysis by RT-PCR and microarray during the coming year.
This year they also worked to develop methods for isolation of primary hepatocytes from individual Fundulus heteroclitus livers, for use in characterizing the sensitivity of individual fish to PCBs. Hepatocytes were isolated using a modification of the protocol they described previously (Bello et al., Tox. Sci., 2001). Because the livers are quite small, the previous studies were done using hepatocytes pooled from multiple fish. In these studies, the team is seeking to modify the earlier methods so that hepatocytes from individual fish can be grown in 96-well plates and tested for PCB sensitivity. They have succeeded in getting the hepatocytes to grow and proliferate in the 96-well plates. Experiments to optimize their responsiveness to chemical exposure are in progress.
This research explores how natural populations of animals respond to prolonged, high-level exposure to contaminants. Fish are used 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.