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
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 assess the sensitivity of NBH and SC fish to ortho-substituted PCBs, in collaboration with scientists at the U.S. E.P.A. Hahn and Karchner and their 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. RNA-seq analysis (collaboration with the Bioinformatics and Molecular Modeling Core) showed that PCB-153 exposure caused mostly down-regulation of gene expression, and that embryos from NBH were less sensitive than SC embryos to effects on gene expression, as they were to the embryotoxic effects of PCB-153. Additional experiments were initiated to examine the effects of another non-ortho PCB, PCB-95 (2,2',3,5',6-pentachlorobiphenyl), which is being studied by the Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures project because of its ability to interact with the ryanodine receptor (RyR).
Functional and structural characterization of four killifish AHRs
Through deep sequencing of the killifish transcriptome, the researchers recently identified two new AHR genes (AHR1b and AHR2b) that may be involved in the mechanism of resistance. To assess the functional properties of these new AHR proteins in relation to the two AHRs that they characterized previously, researchers expressed all four AHR proteins by in vitro transcription and translation and used velocity sedimentation on sucrose gradients to measure their ability to bind radiolabeled TCDD. AHR1a, AHR1b, and AHR2a exhibited high-affinity binding of TCDD, whereas AHR2b did not. Multiple allelic variants of AHR2b were identified, and some of these differed in residues in the ligand-binding domain that might affect the ability to bind ligand. Functional characterization of these AHR2b variants is underway. In addition, the research team has begun working with the Bioinformatics and Molecular Modeling Core to generate homology models of these AHRs and test hypotheses about specific residues involved in determining ligand affinity and specificity.
Targeted mutation of AHR genes in killifish
To test hypotheses about the relative roles of the four AHR genes, the research team is using new gene-targeting approaches to generate null alleles at each of the four killifish AHRs using either the zinc-finger nuclease (ZFN) approach or CRISPR-Cas RNA-guided targeting. Additional targeting experiments involving AHR2a and AHR2b were performed this year, and fish are being raised to adulthood, after which they will be screened to identify founders. A direct comparison of ZFN and CRISPR-Cas targeting of the same gene (AHR2a) showed greater efficiency and higher frequency of mutations with CRISPR-Cas9 compared to ZFN. The research team is especially interested in identifying bi-allelic mutant founders after CRISPR-Cas targeting, which will permit earlier generation of homozygous null offspring. A manuscript describing their initial results from ZFN and CRISPR-Cas targeting of killifish AHRs has recently been published (Aluru et al., 2015, Aquatic Toxicology).
Characterization of killifish PXR
This year the research team published a paper describing their cloning of a killifish PXR cDNA and studies (in collaboration with the Developmental Toxicity of non-Dioxin-like PCBs and Chemical Mixtures Project) to determine the role of PXR in the response to PCBs in resistant and sensitive populations of killifish (Gräns et al., 2015, Aquatic Toxicology). The results suggest a difference between the two populations in the functioning of the PXR signaling pathway.
This year the research team also began collaborating with scientists at the U.S. E.P.A. to examine the possible role of the gene AIP (AHR-Interacting Protein; also known as XAP-2 or Ara9) in the resistance to dioxin-like PCBs. The AIP protein is an immunophilin homolog that acts as a chaperone for AHR proteins, enhancing their stability and modulating nuclear translocation. Multiple lines of emerging evidence are beginning to implicate AIP in the mechanism of resistance. The researchers are in the process of exploring the expression of AIP and possible splice variants in NBH and SC fish, using data from the RNA-seq experiment described above.