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
Studies and Results
Fundulus Transcriptome and Genome
Previously, using 454-based RNA-seq of killifish embryos after exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB-126), Dr. Hahn and his research team demonstrated broad de-sensitization of the AHR signaling pathway in NBH fish (Oleksiak et al. 2011). RNA-seq also revealed novel PCB-regulated genes in each population. The research team has begun to confirm some of these by qRT-PCR. An uncharacterized transcript (isogroup3702) and jagged1b were among the transcripts that had many more reads in the SC-PCB library as compared to the NBH-PCB library; the SC PCB/DMSO ratio was 150-fold and 75-fold higher than the NBH PCB/DMSO ratio for isogroup3702 and jagged1b, respectively. The qRT-PCR results for isogroup3702 agreed with the 454 data, whereas that of jagged1b did not. Future experiments will examine the possibility of premature termination of cDNA synthesis due to secondary structure in the 3'UTR of jagged1b as suggested by truncated 454 sequence reads.
Targeted mutation of AHR genes in killifish
To test hypotheses about the relative roles of the four AHR genes, the researchers are using new gene-targeting approaches to generate null alleles at each of the four killifish AHRs. Previously, the team had used the zinc-finger nuclease (ZFN) approach to target AHR2a; fish raised from ZFN-injected embryos are now breeding and are being screened for founders, in collaboration with scientists at the U.S. Environmental Protection Agency. This year the researchers used a newer approach, CRISPR-Cas RNA-guided targeting (described in Hwang, et al. (2013) Nature Biotechnology 31, 227), to mutate killifish AHR2b. Screening of a subset of the injected embryos demonstrated the effectiveness of the technique in mutating the target site; the remaining embryos are being raised to adulthood. The research team also assessed off-target mutations by sequencing the target-homologous region of the other three AHRs (AHR2a, AHR1a, AHR1b); no off-target mutations were identified.
Knock-down of AHR genes in killifish
Because generation of germline AHR-null fish is a long-term effort, the team is also using transient knock-down of AHR expression in developing embryos to better understand the function of each AHR. They designed and used a splice-blocking morpholino-modified oligonucleotide (MO) to knock down AHR2b. The MO was effective, causing retention of an intron, which is predicted to lead to a truncated and non-functional AHR2b protein. In this initial experiment, knock-down of AHR2b had no effect on survival of embryos or on induction of CYP1A mRNA by PCB-126. Interestingly, PCB-126 induced expression of AHR2b transcripts in both control-MO and AHR2b-MO-injected embryos. Additional gene expression analysis is underway.
Sensitivity of PCB-resistant fish to ortho-substituted PCBs
Killifish from NBH are resistant to effects of the dioxin-like (non-ortho-substituted) PCBs, but whether they are also resistant to ortho-substituted PCBs--which comprise the majority of PCBs at the site--is not known. To begin to assess the sensitivity of NBH and reference (SC) fish to ortho-substituted PCBs, in collaboration with scientists at the U.S. EPA. Dr. Hahn and his research team exposed F2 embryos from both sites to the most abundant ortho-substituted PCB, PCB-153 (2,2',4,4',5,5'-hexachlorobiphenyl) and measured embryotoxicity and altered gene expression. Embryos from NBH were less sensitive than SC embryos to the embryotoxic effects of PCB-153. Induction of CYP1A (a marker of exposure to dioxin-like PCBs) was not seen in either population, demonstrating that the effects of PCB-153 were not due to trace contamination with dioxin-like PCBs. The results suggest that NBH killifish have evolved resistance to both dioxin-like and ortho-substituted PCBs.
This year the research team also began a collaboration with colleagues at the University of Rhode Island in which passive samplers are being used to isolate the bioavailable fraction of hydrophobic contaminants from NBH and Smith College. Water and air samplers were deployed for several weeks and recovered. After chemical analysis of the extracts, the team will expose killifish embryos to the extracts and perform gene expression analysis to explore patterns of gene expression that may reveal interactive effects of these mixtures.
Research Translation and Training Activities
Interviewed by Smith College undergraduate class on environmental influences on embryonic development. Interview incorporated into videos, which can be found online. Interviewed for article about PCB-resistant fish in New Bedford Standard Times. Interview on local NPR station regarding the researchers' studies on PCB-resistant fish, as part of a series on New England Fisheries (WCAI) (article).
This research explores how natural populations of animals respond to prolonged, high-level exposure to contaminants. The research team uses fish as models to investigate the mechanisms underlying differential sensitivity to the developmental toxicity of dioxin-like chemicals. The existence of PCB-sensitive and PCB-resistant populations of killifish provides a unique opportunity to understand the molecular mechanisms of differential PCB sensitivity and the impact of evolved resistance on the sensitivity of fish to other environmental stressors. The research addresses a key question concerning the extent to which changes in the sensitivity to one class of chemicals have far-reaching effects on the ability of animals to respond to other types of chemicals or environmental stressors. The results will have relevance for ecological risk assessment at Superfund sites and other contaminated sites, and will contribute to the fundamental understanding of cross-talk among signaling pathways and its role in the response to chemical exposure.