AHR2 mediates cardiac teratogenesis of polycyclic aromatic hydrocarbons and PCB-126 in Atlantic killifish (Fundulus heteroclitus)
Introduction
The Atlantic killifish or mummichog (Fundulus heteroclitus), found in estuaries along the Atlantic coast of North America from Newfoundland to Florida (Shute, 1980), has a variety of attributes that make it an important field and laboratory toxicology model (Burnett et al., 2007). Fundulus are tolerant of significant variation in environmental conditions, including salinity, temperature, oxygen, and pH (Dunson et al., 1993, Wood and Marshall, 1994, Smith and Able, 2003, Stierhoff et al., 2003, Nordlie, 2006). Because of their wide distribution and abundance, Fundulus are important components of estuarine ecosystems (Meredith and Lotrich, 1979, Kneib, 1986). However, despite wide distribution, individual Fundulus have relatively small home ranges (Lotrich, 1975) and are ideal for studying the impacts of local contamination and other stressors (Burnett et al., 2007). Fundulus have been used in studies of a wide variety of contaminants including toxicity of polycyclic aromatic hydrocarbons (PAHs) (e.g. Wassenberg and Di Giulio, 2004), polychlorinated biphenyls (PCBs) (e.g. Nacci et al., 1999, Jonsson et al., 2007), metals (e.g. Roling et al., 2006), and pesticides (e.g. Fortin et al., 2008); Fundulus have also been used to study processes such as endocrine disruption (e.g. Kelly and Di Giulio, 2000), environmental carcinogenesis (e.g. Vogelbein et al., 1990, Wills et al., in press), and evolutionary adaptation (e.g. Schulte et al., 2000). Additionally, individual Fundulus populations have been identified that are resistant to a number of anthropogenic stressors, including mercury (Weis and Weis, 1984), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Prince and Cooper, 1995b), PCBs (Nacci et al., 1999, Meyer and Di Giulio, 2002), and PAHs (Meyer et al., 2002, Ownby et al., 2002).
Of the Fundulus populations adapted to environmental contaminants, most are resistant to the acute toxicity and teratogenic effects caused by PCBs and PAHS, which are known to interact with the aryl hydrocarbon receptor (AHR) pathway. PCBs and dioxin have long been known to cause a suite of deformities collectively known as “blue sac disease” (reviewed in Peterson et al., 1993) and some PAHs and PAH mixtures also induce similar cardiac deformities (Billiard et al., 1999, Wassenberg and Di Giulio, 2004). Furthermore, alteration of the AHR pathway appears to be an important component of adaptation to these contaminants. Notably, these populations exhibit a marked recalcitrance to induction of cytochrome P4501A (CYP1A) in response to exposure to a variety of AHR agonists (Prince and Cooper, 1995a, Van Veld and Westbrook, 1995, Elskus et al., 1999, Nacci et al., 1999, Bello et al., 2001, Arzuaga and Elskus, 2002, Meyer et al., 2002, Wills et al., 2010).
The AHR mediates responses to halogenated hydrocarbons and PAHs, such as CYP1A induction, by recognizing and binding these ligands and driving gene expression (Schmidt and Bradfield, 1996). Following activation by ligand binding, the AHR dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT); this complex associates with specific DNA sequences known as xenobiotic response elements (XREs) or AHR response elements (AHREs) to induce transcription of a number of genes, including components of Phase I and II metabolism (i.e. CYP1A, 1B1, 1C1; some glutathione S-transferases (GSTs); NADP(H):oxidoreductase; UDP-glucuronosyltransferase (UGT) (Nebert et al., 2000). The AHR complex also induces transcription of a factor called the aryl hydrocarbon receptor repressor (AHRR), which down-regulates the pathway by competing with the AHR-ARNT complex for XREs (Karchner et al., 2002).
In order to further increase the utility of Fundulus for the study of aryl hydrocarbon toxicity and adaptation, better understanding of the components of the molecular pathways governing PAH response in Fundulus is crucial. The AHR pathway appears to be well-conserved among mammals and lower vertebrates (Hahn, 2002, Hahn et al., 2006) and a variety of genes in the AHR pathway have been characterized in Fundulus. Due to gene and genome duplication events, multiple AHRs have arisen and been identified in fish, including two in Fundulus, AHR1 and AHR2 (Karchner et al., 1999, Tanguay et al., 1999, Andreasen et al., 2002). ARNT and the AHRR have also been identified in Fundulus and other fish (Powell et al., 1999, Andreasen et al., 2002, Karchner et al., 2002, Evans et al., 2005). In zebrafish, AHR2 mediates teratogenesis induced by PAH mixtures (Billiard et al., 2006), 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126) (Jonsson et al., 2007), and TCDD (Prasch et al., 2003, Teraoka et al., 2003). Currently, it is unclear whether AHR1, AHR2, or both perform this role in Fundulus. In vitro, both AHR1 and AHR2 have been demonstrated to bind TCDD and the model PAH β-naphthoflavone (BNF) and to activate a luciferase reporter upon TCDD binding (Karchner et al., 1999, Karchner et al., 2002).
In the current study, we used a morpholino gene knockdown approach to investigate the roles of AHR1 and AHR2 in mediating cardiac teratogenesis induced by PAHs and PCB-126 in F. heteroclitus. We confirmed knockdown of AHR1 and AHR2 protein by Western blot analysis. Our findings lead us to conclude that AHR2 and not AHR1 is the primary mediator of this response in Fundulus. We also verified the lack of effect of AHR1 knockdown using a second morpholino directed at a splice junction, which allowed confirmation of knockdown via RT-PCR.
Section snippets
Fish
Adult killifish were collected with minnow traps on King's Creek, a relatively-uncontaminated tributary of the Severn River which feeds into the lower Chesapeake Bay in southeastern Virginia (37°17′52.4″N, 76°25′31.4″W). In the lab, fish were maintained at 23–25 °C, in 20‰ artificial sea water (ASW; Instant Ocean, Foster & Smith, Rhinelander, WI, USA), with a 14:10 light:dark cycle, and were fed pelleted fish feed (Aquamax® Fingerling Starter 300, PMI Nutritional International, LLC, Brentwood,
Deformity assessment
Targeted morpholino knockdown of AHR1, AHR2, or the combination was used to determine the role of each of the Fundulus AHRs in mediating the cardiac deformities induced by PAHs and PCB-126. Morpholino-injected and non-injected embryos were exposed to BNF, BkF, and PCB-126 as described previously and the effect of gene knockdown on response was assessed.
We have previously demonstrated that 10 μg/L of the model PAH BNF induces cardiac deformities of mild to intermediate severity in naïve Fundulus (
Discussion
Fundulus are an important and highly-studied model in environmental toxicology, in part due to identification of multiple populations adapted to contamination from the AHR agonists PAHs, PCBs, and dioxin. Because populations adapted to these contaminants demonstrate a recalcitrant AHR pathway, it is logical to suspect that alteration of the AHR pathway plays a crucial role in circumventing toxicity. However, it was previously unclear what role AHR1 and AHR2 played in mediating the toxic effects
Acknowledgements
We thank laboratory members Lauren Wills, Lindsey Van Tiem, and Carrie Fleming for advice and assistance conducting morpholino injection and deformity screening and Autumn Bernal for assistance in developing Western blot methodology. Antibodies were generously provided by Dr. Mark Hahn (AHR1 and AHR2) and Dr. John Stegeman (CYP1A) of the Woods Hole Oceanographic Institution. This work was funded by the NIEHS-supported Duke University Superfund Basic Research Program (P42ES010356) and Duke
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Present address: Department of Physiology, Dartmouth Medical School, Hanover, NH 03755, USA.