SNAPSHOT

Created at: 2017-02-10 10:36

AOP ID and Title:


AOP 21: Activation of the aryl hydrocarbon receptor (AhR) leading to early life stage mortality
Short Title: AhR activation leading to early life stage toxicity in fish

Authors


Jon Doering, Ph.D., National Research Council Postdoctoral Toxicologist, US EPA Mid-Continent Ecology Division, Duluth, MN, USA,   doering.jonathan[at]epa.gov
 
Prof. Markus Hecker, Ph.D. University of Saskatchewan, Saskatoon, Saskatchewan, Canada, (markus.hecker[at]usask.ca
 
Dan Villeneuve, Ph.D., US EPA Mid-Continent Ecology Division, Duluth, MN, USA (villeneuve.dan[at]epa.gov)


Status

Author status OECD status OECD project SAAOP status
Open for comment. Do not cite EAGMST Under Review 1.7 Included in OECD work plan

Abstract


This adverse outcome pathway details the linkage between activation of the aryl hydrocarbon receptor (AhR) and early life stage mortality in fishes. Fishes represent the most sensitive taxa to adverse effects resulting from activation of the AhR. Embryos are more sensitive than juvenile or adult fishes based on endpoints that could be of significance to population trends. The AhR is critically involved in development of the heart and vasculature. Activation of the AhR can result in altered cardiovascular development and function in early life stages. Initial development of this AOP draws heavily on evidence collected from zebrafish (Danio rerio) through mechanistic investigations by use of targeted knockdown of genes and through use of selective agonists and antagonists. However, comparable manifestations of activation of the AhR have been recorded across freshwater and marine teleost and non-teleost fishes, as well as birds. Therefore, this AOP might be broadly applicable across oviparous vertebrate taxa. Despite this conservation, great differences in sensitivity exist both among and within taxonomic groups. Therefore, this AOP has utility in support of application toward the mechanistic understanding of adverse effects of chemicals that act as agonists of the AhR, particularly with regard to cross-species, cross-chemical, and cross-taxa extrapolation. 


Background


  • The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family of proteins (Okey 2007). The AhR is a highly conserved and ancient protein with homologs having been identified in most major animal groups, apart from the most ancient lineages, such as sponges (Porifera) (Hahn et al 2002).
  • Investigations of invertebrates possessing early homologs of the AhR suggest that the AhR evolutionarily functioned in regulation of the cell cycle, cellular proliferation and differentiation, and cell-to-cell communications (Hahn et al 2002). However, critical functions in angiogenesis, regulation of the immune system, neuronal processes, metabolism, development of the heart and other organ systems, and detoxification have emerged sometime in early vertebrate evolution (Duncan et al 1998; Lahvis and Bradfield 1998; Emmons et al 1999).
  • Activation of the AhR by anthropogenic pollutants that act as agonists can result in a range of adverse biological effects. These effects can include hepatotoxicity, histological lesions, hemopoiesis, suppression of immune responses and healing, impaired reproductive and endocrine processes, teratogenesis, carcinogenesis, wasting syndrome, and mortality (Kleeman et al 1988; Spitsbergen et al 1986; Walter et al 2000; Giesy et al 2002; Spitsbergen et al 1988a; 1988b).
  • Despite the AhR being a highly conserved protein, differences in relative sensitivity to adverse effects both among and within vertebrate taxa are greater than 1000-fold (Cohen-Barnhouse et al 2011; Doering et al 2013; Hengstler et al 1999; Korkalainen et al 2001).
  • Differences in binding affinity and transactivation of the AhR have been implicated as a key mechanism contributing to differences in sensitivity to agonists of the AhR among species and taxa. However, the precise mechanisms are not fully understood for all taxa.
  • High-throughput, next-generation ‘OMICs’ technologies have identified hundreds to thousands of different genes that are regulated, either directly or indirectly, by the AhR (Brinkmann et al 2016; Doering et al 2016; Huang et al 2014; Li et al 2013; Whitehead et al 2010). These genes include Phase I and Phase II biotransformation enzymes, such as cytochrome P450 1A (CYP1A). Expressions and activities of CYP1A are routinely used as biomarkers of exposure to anthropogenic pollutants that act as agonists of the AhR (Whyte et al 2008).
  • Exposure to mixtures of agonists of the AhR during the 1950’s, 1960’s, and 1970’s has been implicated in early life stage mortality of Lake Ontario lake trout (Salvelinus namaycush) leading to population collapse (Cook et al 2003). However, populations of mummichog (Fundulus heteroclitus) and Atlantic tomcod (Microgadus tomcod) exposed to lethal concentrations of agonists of the AhR have evolved tolerance through several mechanisms which has protected against population collapse (Nacci et al 2010; Wirgin et al 2011).

Summary of the AOP


Stressors


Name Evidence
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) Strong
Polycyclic aromatic hydrocarbons (PAHs) Strong

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

  • The AhR is activated by numerous planar aromatic hydrocarbons. Specifically, chemicals with molecular dimensions of 12 Å x 14 Å x 5 Å are potential ligands of the AhR (Waller & McKinney 1992; 1995).
  • However, the AhR is best known as the molecular target of dioxin-like compounds (DLCs). DLCs include polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and planar polychlorinated biphenyls (PCBs). A total of seven PCDDs, ten PCDFs, and 12 PCBs are considered 'dioxin-like' because they bind to the AhR with relatively great affinity (Denison & Heath-Pagliuso 1998; Van den Berg et al 1998).
  • The prototypical and most potent DLC is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Molecular Initiating Event

Title Short name
Activation, AHR Activation, AHR

18: Activation, AHR

Short Name: Activation, AHR

Stressors

Name
Benzidine
Dibenzo-p-dioxin
Polychlorinated biphenyl
Polychlorinated dibenzofurans
Hexachlorobenzene

Evidence for Perturbation of this Molecular Initiating Event by Stressor

The AHR can be activated by several structurally diverse chemicals, but binds preferentially to planar halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. Dioxin-like compounds (DLCs), which include polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and certain polychlorinated biphenyls (PCBs), are among the most potent AHR ligands[38]. Only a subset of PCDD, PCDF and PCB congeners has been shown to bind to the AHR and cause toxic effects to those elicited by TCDD. Until recently, TCDD was considered to be the most potent DLC in birds[39]; however, recent reports indicate that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) is more potent than TCDD in some species of birds.[40][13][41][21][42][43] When screened for their ability to induce aryl hydrocarbon hydroxylase (AHH) activity, dioxins with chlorine atoms at a minimum of three out of the four lateral ring positions, and with at least one non-chlorinated ring position are the most active[44]. Of the dioxin-like PCBs, non-ortho congeners are the most toxicologically active, while mono-ortho PCBs are generally less potent[45][9]. Chlorine substitution at ortho positions increases the energetic costs of assuming the coplanar conformation required for binding to the AHR [45]. Thus, a smaller proportion of mono-ortho PCB molecules are able to bind to the AHR and elicit toxic effects, resulting in reduced potency of these congeners. Other PCB congeners, such as di-ortho substituted PCBs, are very weak AHR agonists and do not likely contribute to dioxin-like effects [9].

  • Contrary to studies of birds and mammals, even the most potent mono-ortho PCBs bind to AhRs of fishes with very low affinity, if at all (Abnet et al 1999; Doering et al 2014; 2015; Eisner et al 2016; Van den Berg et al 1998).

The role of the AHR in mediating the toxic effects of planar hydrophobic contaminants has been well studied, however the endogenous role of the AHR is less clear [1]. Some endogenous and natural substances, including prostaglandin PGG2 and the tryptophan derivatives indole-3-carbinol, 6-formylindolo[3,2-b]carbazole (FICZ) and kynurenic acid can bind to and activate the AHR. [6][46][47][48][49] The AHR is thought to have important endogenous roles in reproduction, liver and heart development, cardiovascular function, immune function and cell cycle regulation [50][38][51][52][53][54][46][55][56][57] and activation of the AHR by DLCs may therefore adversely affect these processes.



Biological Organization

Level of Biological Organization
Molecular

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
zebra danio Danio rerio Strong NCBI
Fundulus heteroclitus Fundulus heteroclitus Strong NCBI
Gallus gallus Gallus gallus Strong NCBI
Life Stage Applicability
Life Stage Evidence
Embryo Strong
development Strong
Sex Applicability
Sex Evidence
Unspecific Strong

The AHR structure has been shown to contribute to differences in species sensitivity to DLCs in several animal models. In 1976, a 10-fold difference was reported between two strains of mice (non-responsive DBA/2 mouse, and responsive C57BL/6 14 mouse) in CYP1A induction, lethality and teratogenicity following TCDD exposure[3]. This difference in dioxin sensitivity was later attributed to a single nucleotide polymorphism at position 375 (the equivalent position of amino acid residue 380 in chicken) in the AHR LBD[30][19][31]. Several other studies reported the importance of this amino acid in birds and mammals[32][30][22][33][34][35][31][36]. It has also been shown that the amino acid at position 319 (equivalent to 324 in chicken) plays an important role in ligand-binding affinity to the AHR and transactivation ability of the AHR, due to its involvement in LBD cavity volume and its steric effect[35]. Mutation at position 319 in the mouse eliminated AHR DNA binding[35].

The first study that attempted to elucidate the role of avian AHR1 domains and key amino acids within avian AHR1 in avian differential sensitivity was performed by Karchner et al.[22]. Using chimeric AHR1 constructs combining three AHR1 domains (DBD, LBD and TAD) from the chicken (highly sensitive to DLC toxicity) and common tern (resistant to DLC toxicity), Karchner and colleagues[22], showed that amino acid differences within the LBD were responsible for differences in TCDD sensitivity between the chicken and common tern. More specifically, the amino acid residues found at positions 324 and 380 in the AHR1 LBD were associated with differences in TCDD binding affinity and transactivation between the chicken (Ile324_Ser380) and common tern (Val324_Ala380) receptors[22]. Since the Karchner et al. (2006) study was conducted, the predicted AHR1 LBD amino acid sequences were been obtained for over 85 species of birds and 6 amino acid residues differed among species[14][37] . However, only the amino acids at positions 324 and 380 in the AHR1 LBD were associated with differences in DLC toxicity in ovo and AHR1-mediated gene expression in vitro[14][37][16]. These results indicate that avian species can be divided into one of three AHR1 types based on the amino acids found at positions 324 and 380 of the AHR1 LBD: type 1 (Ile324_Ser380), type 2 (Ile324_Ala380) and type 3 (Val324_Ala380)[14][37][16].

  • Little is known about differences in binding affinity of AhRs and how this relates to sensitivity in non-avian taxa.
  • Low binding affinity for DLCs of AhR1s of African clawed frog (Xenopus laevis) and axolotl (Ambystoma mexicanum) has been suggested as a mechanism for tolerance of these amphibians to DLCs (Lavine et al 2005; Shoots et al 2015).
  • Among reptiles, only AhRs of American alligator (Alligator mississippiensis) have been investigated and little is known about the sensitivity of American alligator or other reptiles to DLCs (Oka et al 2016).
  • Among fishes, great differences in sensitivity to DLCs are known both for AhRs and for embryos among species that have been tested (Doering et al 2013; 2014).
  • Differences in binding affinity of the AhR2 have been demonstrated to explain differences in sensitivity to DLCs between sensitive and tolerant populations of Atlantic Tomcod (Microgadus tomcod) (Wirgin et al 2011).
  • Information is not yet available regarding whether differences in binding affinity of AhRs of fishes are predictive of differences in sensitivity of embryos, juveniles, or adults (Doering et al 2013).

How this Key Event Works

The AHR Receptor

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that belongs to the basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) superfamily and consists of three domains: the DNA-binding domain (DBD), ligand binding domain (LBD) and transactivation domain (TAD)[1]. Other members of this superfamily include the AHR nuclear translocator (ARNT), which acts as a dimerization partner of the AHR [2][3]; Per, a circadian transcription factor; and Sim, the “single-minded” protein involved in neuronal development [4][5]. This group of proteins shares a highly conserved PAS domain and is involved in the detection of and adaptation to environmental change[4].

  • Investigations of invertebrates possessing early homologs of the AhR suggest that the AhR evolutionarily functioned in regulation of the cell cycle, cellular proliferation and differentiation, and cell-to-cell communications (Hahn et al 2002).
  • However, critical functions in angiogenesis, regulation of the immune system, neuronal processes, metabolism, development of the heart and other organ systems, and detoxification have emerged sometime in early vertebrate evolution (Duncan et al., 1998; Emmons et al., 1999; Lahvis and Bradfield, 1998).

The molecular Initiating Event

The molecular mechanism of activation of gene expression by AHR1.

The molecular mechanism for AHR-mediated activation of gene expression is presented in the figure to the right. In its unliganded form, the AHR is part of a cytosolic complex containing heat shock protein 90 (HSP90), the HSP90 co-chaperone p23 and AHR-interacting protein (AIP)[6]. Upon ligand binding, the AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with ARNT[7]. The AHR-ARNT complex then binds to a xenobiotic response element (XRE) found in the promoter of an AHR-regulated gene and recruits co-regulators such as CREB binding protein/p300, steroid receptor co-activator (SRC) 1, SRC-2, SRC-3 and nuclear receptor interacting protein 1, leading to induction or repression of gene expression[6]. Expression levels of several genes, including phase I (e.g. cytochrome P450 (CYP) 1A, CYP1B, CYP2A) and phase II enzymes (e.g. uridine diphosphate glucuronosyl transferase (UDP-GT), glutathione S-transferases (GSTs)), as well as genes involved in cell proliferation (transforming growth factor-beta, interleukin-1 beta), cell cycle regulation (p27, jun-B) and apoptosis (Bax), are regulated through this mechanism [6][8][7][9].

AHR Isoforms

  • Over time the AhR has undergone gene duplication and diversification in vertebrates, which has resulted in multiple clades of AhR, namely AhR1, AhR2, and AhR3 (Hahn 2002).
  • Fishes and birds express AhR1s and AhR2s, while mammals express a single AhR that is homologous to the AhR1 (Hahn 2002; Hahn et al 2006).
  • The AhR3 is poorly understood and known only from some cartilaginous fishes (Hahn 2002).
  • Little is known about diversity of AhRs in reptiles and amphibians (Hahn et al 2002).
  • In some taxa, subsequent genome duplication events have further led to multiple isoforms of AhRs in some species, with up to four isoforms of the AhR (α, β, δ, γ) having been identified in Atlantic salmon (Salmo salar) (Hansson et al 2004).
  • Although homologs of the AhR have been identified in some invertebrates, compared to vertebrates these AhRs have differences in binding of ligands in the species investigated to date (Hahn 2002; Hahn et al 1994). 

Two AHR isoforms (AHR1 and AHR2) have been identified in the black-footed albatross (Phoebastria nigripes), great cormorant (Phalacrocorax carbo) and domestic chicken (Gallus gallus domesticus)[10]. AHR1 mRNA levels were similar in the kidney, heart, lung, spleen, brain, gonad and intestine from the great cormorant but were lower in muscle and pancreas. AHR2 expression was mainly observed in the liver, but was also detected in gonad, brain and intestine. AHR1 levels represented a greater proportion (80%) of total AHR levels than AHR2 in the cormorant liver[10], and while both AHR isoforms bound to TCDD, AHR2 was less effective at inducing TCDD-dependent transactivation compared to AHR1 in black-footed albatross, great cormorant and domestic chicken[11][10].

Roles of isoforms in Birds:

  • AhR1 and AhR2 both bind and are activated by TCDD in vitro (Yasui et al 2007).
  • AhR1 has greater binding affinity and sensitivity to activation by TCDD relative to AhR2 (Yasui et al 2007).
  • AhR1 is believed to mediate toxicities of DLCs, while AhR2 has no known role in toxicities (Farmahin et al 2012; Farmahin et al 2013; Manning et al 2012).

Roles of isoforms in fishes:

  • AhR1 and AhR2 both bind and are activated by TCDD in vitro (Bak et al 2013; Doering et al 2014; 2015; Karchner et al 1999; 2005).
  • AhR1 has greater sensitivity to activation by TCDD than AhR2 in red seabream (Pagrus major), white sturgeon (Acipenser transmontanus), and lake sturgeon (Acipenser fulvescens) (Bak et al 2013; Doering et al 2014; 2015)
  • AhR2 has greater binding affinity or activation by TCDD than AhR1 in zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus) (Karchner et al 1999; 2005).
  • AhR2 is believed to mediate toxicities in fishes, while AhR1 has no known role in toxicities. Specifically, knockdown of AhR2 protects against toxicities of dioxin-like compounds (DLCs) and polycyclic aromatic hydrocarbons (PAHs) in zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus), while knockdown of AhR1 offers no protection (Clark et al 2010; Prasch et al 2003; Van Tiem & Di Giulio 2011).

Roles of isoforms in amphibians and reptiles:

  • Less is known about AhRs of amphibians or reptiles.
  • AhR1 is believed to mediate toxicities in amphibians (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015). However, all AhRs of amphibians that have been investigated have very low affinity for TCDD (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015).
  • Both AhR1s and AhR2 of American alligator (Alligator mississippiensis) are activated by agonists with comparable sensitivities (Oka et al 2016). AhRs of no other reptiles have been investigated.

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Transactivation Reporter Gene Assays (recommended approach)

Transient transfection transactivation

Transient transfection transactivation is the most common method for evaluating nuclear receptor activation[12]. Full-length AHR cDNAs are cloned into an expression vector along with a luminescent reporter gene construct (chimeric luciferase, P-lactamase or CAT reporter vectors containing the appropriate response elements for the gene of interest). There are a number of commercially available cell lines that can serve as recipients for these vectors (CV-1, HuH7, FLC-7, LS174T, LS180 MCF-7, HEC1, LLC-PK1, HEK293, HepG2, and Caco-2 cells)[12]. The greatest advantage of using transfected cells, rather than primary cell cultures, is the assurance that the nuclear receptor of interest is responsible for the observed induction. This would not be possible in a primary cell culture due to the co-regulation of different receptors for the same target genes. This model makes it easy to compare the responsiveness of the AHR across multiple species under the same conditions simply by switching out the AHR clone. One disadvantage to the transient transfection assay is the inherent variability associated with transfection efficiency, leading to a movement towards the use of stable cell lines containing the nuclear receptor and reporter gene linked to the appropriate response elements[12].

Luciferase reporter gene (LRG) assay
  • Luciferase reporter gene (LRG) assays have been used to investigate activation of AhRs of mammals (Abnet et al 1999), birds (Farmahin et al 2012; 2013; Manning et al 2013), reptiles (Oka et al 2016), amphibians (Lavine et al 2005; Shoots et al 2015; Ohi et al 2003), and fishes (Andreasen et al 2002; Abnet et al 1999; Bak et al 2013; Doering et al 2014; 2015; Evans et al 2005; Hansson & Hahn 2008; Karchner et al 1999; Tanguay et al 1999; Wirgin et al 2011).

For demonstrative purposes, a luciferase reporter gene assay used to measure AHR1-mediated transactivation for avian species is described here. A monkey kidney cell line (Cos-7) that has low endogenous AHR1 expression was transfected with the appropriate avian AHR1 clone, cormorant ARNT1, a CYP1A5 firefly luciferase reporter construct and a Renilla luciferase vector to control for transfection efficiency. After seeding, the cells were exposed to DLC and luciferase activity was measured using a luminometer. Luminescence, which is proportional to the extent of AHR activation, is expressed as the ratio of firefly luciferase units to Renilla luciferase units [13]. This particular assay was modified from its original version to increase throughput efficiency; (a) cells were seeded in 96-well plates rather than Petri dishes or 48- well plates, (b) DLCs were added directly to the wells without changing the cell culture medium, and (c) the same 96-well plates were used to measure luminescence without lysing the cells and transferring to another plate. Similar reporter gene assays have been used to measure AHR1 activation in domestic and wild species of birds, including the chicken, ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica), great cormorant, black-footed albatross and peregrine falcon (Falco peregrinus).[14][13][15][11][16][17]

Transactivation in stable cell lines

Stable cell lines have been developed and purified to the extent that each cell contains both the nuclear receptor and appropriate reporter vector, eliminating the variability associated with transfection [12]. A stable human cell line containing a luciferase reporter driven by multiple dioxin response elements has been developed that is useful in identifying AhR agonists and antagonists[18]. An added benefit of this model is the potential to multiplex 3 assays in a single well: receptor activation, cell viability and enzyme activity[12]. Such assays are used extensively in drug discovery due to their high throughput efficiency, and may serve just as useful for risk assessment purposes.

Ligand-Binding Assays

Ligand binding assays measure the ability of a test compound to compete with a labeled, high-affinity reference ligand for the LBD of a nuclear receptor. It is important to note that ligand binding does not necessitate receptor activation and therefore cannot distinguish between agonists and antagonists; however, binding affinities of AHR ligands are highly correlated with chemical potencies[19] and can explain differences in species sensitivities to DLCs[20][21][22]; they are therefore worth mentioning. Binding affinity and efficacy have been used to develop structure-activity relationships for AHR disruption[20][23] that are potentially useful in risk-assessment. There has been tremendous progress in the development of ligand-binding assays for nuclear receptors that use homogenous assay formats (no wash steps) allowing for the detection of low-affinity ligands, many of which do not require a radiolabel and are amenable to high throughput screening[24][12]. This author however was unable to find specific examples of such assays in the context of AHR binding and therefore some classic radioligand assays are described instead.

Hydroxyapatite (HAP) binding assay

The HAP binding assay makes use of an in vitro transcription/translation method to synthesize the AHR protein, which is then incubated with radiolabeled TDCPP and a HAP pellet. The occupied protein adsorbs to the HAP and the radioactivity is measured to determine saturation binding. An additional ligand can also be included in the mixture in order to determine its binding affinity relative to TCDD (competitive binding)[25][22]. This assay is simple, repeatable and reproducible; however, it is insensitive to weak ligand-receptor interactions[22][21][26].

Whole cell filtration binding assay

Dold and Greenlee[27] developed a method to detect specific binding of TCDD to whole mammalian cells in culture and was later modified by Farmahin et al.[21] for avian species. The cultured cells are incubated with radiolabeled TCDD with or without the presence of a competing ligand and filtered. The occupied protein adsorbs onto the filter and the radioactivity is measured to determine saturation binging and/or competitive binding. This assay is able to detect weak ligand-receptor interactions that are below the detection limit of the HAP assay[21].

Protein-DNA Interaction Assays

The active AHR complexed with ARNT can be measured using protein-DNA interaction assays. Two methods are described in detail by Perez-Romero and Imperiale[28]. Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions in vivo. It involves the treatment of cells with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. Nuclear fractions are isolated, the genomic DNA is sheared, and nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. After reversal of the crosslinking, the associated DNA fragments are sequenced. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. Electrophoretic mobility shift assay (EMSA) provides a rapid method to study DNA-binding protein interactions in vitro. This relies on the fact that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments. The protein-DNA complex components are then identified with appropriate antibodies. The EMSA assay was found to be consistent with the LRG assay in chicken hepatoma cells dosed with dioxin-like compounds[29].


References

  1. 1.0 1.1 Okey, A. B. (2007). An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XI. Toxicol.Sci. 98, 5-38.
  2. Hoffman, E. C., Reyes, H., Chu, F. F., Sander, F., Conley, L. H., Brooks, B. A., and Hankinson, O. (1991). Cloning of a factor required for activity of the Ah (dioxin) receptor. Science 252, 954-958.
  3. 3.0 3.1 Poland, A., Glover, E., and Kende, A. S. (1976). Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. J.Biol.Chem. 251, 4936-4946.
  4. 4.0 4.1 Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily: sensors of environmental and developmental signals. Annu.Rev.Pharmacol.Toxicol. 40, 519-561.
  5. Kewley, R. J., Whitelaw, M. L., and Chapman-Smith, A. (2004). The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int.J.Biochem.Cell Biol. 36, 189-204.
  6. 6.0 6.1 6.2 6.3 Fujii-Kuriyama, Y., and Kawajiri, K. (2010). Molecular mechanisms of the physiological functions of the aryl hydrocarbon (dioxin) receptor, a multifunctional regulator that senses and responds to environmental stimuli. Proc.Jpn.Acad.Ser.B Phys.Biol.Sci. 86, 40-53.
  7. 7.0 7.1 Mimura, J., and Fujii-Kuriyama, Y. (2003). Functional role of AhR in the expression of toxic effects by TCDD. Biochimica et Biophysica Acta - General Subjects 1619, 263-268.
  8. Giesy, J. P., Kannan, K., Blankenship, A. L., Jones, P. D., and Newsted, J. L. (2006). Toxicology of PCBs and related compounds. In Endocrine Disruption Biological Bases for Health Effects in Wildlife and Humans (D. O. Norris, and J. A. Carr, Eds.), pp. 245-331. Oxford University Press, New York.
  9. 9.0 9.1 9.2 Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. Critical Reviews in Toxicology 24, 87-149.
  10. 10.0 10.1 10.2 Yasui, T., Kim, E. Y., Iwata, H., Franks, D. G., Karchner, S. I., Hahn, M. E., and Tanabe, S. (2007). Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. Toxicol.Sci. 99, 101-117.
  11. 11.0 11.1 Lee, J. S., Kim, E. Y., and Iwata, H. (2009). Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): association with aryl hydrocarbon receptor 1 and 2 isoforms. Toxicol.Appl.Pharmacol. 234, 1-13.
  12. 12.0 12.1 12.2 12.3 12.4 12.5 Raucy, J. L., and Lasker, J. M. (2010). Current in vitro high throughput screening approaches to assess nuclear receptor activation. Curr. Drug Metab 11 (9), 806-814.
  13. 13.0 13.1 13.2 Farmahin, R., Wu, D., Crump, D., Hervé, J. C., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., and Kennedy, S. W. (2012). Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Environ.Sci.Technol. 46, 2967-2975.
  14. 14.0 14.1 14.2 14.3 Farmahin, R., Manning, G. E., Crump, D., Wu, D., Mundy, L. J., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., Fredricks, T. B., and Kennedy, S. W. (2013b). Amino acid sequence of the ligand binding domain of the aryl hydrocarbon receptor 1 (AHR1) predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol.Sci. 131, 139-152.
  15. Fujisawa, N., Ikenaka, Y., Kim, E. Y., Lee, J. S., Iwata, H., and Ishizuka, M. (2012). Molecular evidence predicts aryl hydrocarbon receptor ligand insensitivity in the peregrine falcon (Falco peregrines). European Journal of Wildlife Research 58, 167-175.
  16. 16.0 16.1 16.2 Manning, G. E., Farmahin, R., Crump, D., Jones, S. P., Klein, J., Konstantinov, A., Potter, D., and Kennedy, S. W. (2012). A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the embryolethality of polychlorinated biphenyls in avian species. Toxicol.Appl.Pharmacol. 263, 390-399.
  17. Mol, T. L., Kim, E. Y., Ishibashi, H., and Iwata, H. (2012). In vitro transactivation potencies of black-footed albatross (Phoebastria nigripes) AHR1 and AHR2 by dioxins to predict CYP1A expression in the wild population. Environ.Sci.Technol. 46, 525-533.
  18. Yueh, M. F., Kawahara, M., and Raucy, J. (2005). Cell-based high-throughput bioassays to assess induction and inhibition of CYP1A enzymes. Toxicol. In Vitro 19 (2), 275-287.
  19. 19.0 19.1 Poland, A., and Knutson, J. C. (1982). 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517-554.
  20. 20.0 20.1 Hestermann, E. V., Stegeman, J. J., and Hahn, M. E. (2000). Relative contributions of affinity and intrinsic efficacy to aryl hydrocarbon receptor ligand potency. Toxicol. Appl. Pharmacol 168 (2), 160-172.
  21. 21.0 21.1 21.2 21.3 21.4 Farmahin, R., Jones, S. P., Crump, D., Hahn, M. E., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., and Kennedy, S. W. (2014). Species-specific relative AHR1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency. Comp Biochem. Physiol C. Toxicol. Pharmacol. 161C, 21-25.
  22. 22.0 22.1 22.2 22.3 22.4 22.5 22.6 Karchner, S. I., Franks, D. G., Kennedy, S. W., and Hahn, M. E. (2006). The molecular basis for differential dioxin sensitivity in birds: Role of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U. S. A 103 (16), 6252-6257.
  23. Lee, S., Shin, W. H., Hong, S., Kang, H., Jung, D., Yim, U. H., Shim, W. J., Khim, J. S., Seok, C., Giesy, J. P., and Choi, K. (2015). Measured and predicted affinities of binding and relative potencies to activate the AhR of PAHs and their alkylated analogues. Chemosphere 139, 23-29.
  24. Jones, S. A., Parks, D. J., and Kliewer, S. A. (2003). Cell-free ligand binding assays for nuclear receptors. Methods Enzymol. 364, 53-71.
  25. Gasiewicz, T. A., and Neal, R. A. (1982). The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxylapatite. Anal. Biochem. 124 (1), 1-11.
  26. Nakai, J. S., and Bunce, N. J. (1995). Characterization of the Ah receptor from human placental tissue. J Biochem. Toxicol. 10 (3), 151-159.
  27. Dold, K. M., and Greenlee, W. F. (1990). Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture. Anal. Biochem. 184 (1), 67-73.
  28. Perez-Romero, P., and Imperiale, M. J. (2007). Assaying protein-DNA interactions in vivo and in vitro using chromatin immunoprecipitation and electrophoretic mobility shift assays. Methods Mol. Med. 131, 123-139.
  29. Heid, S. E., Walker, M. K., and Swanson, H. I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci 61 (1), 187-196.
  30. 30.0 30.1 Ema, M., Ohe, N., Suzuki, M., Mimura, J., Sogawa, K., Ikawa, S., and Fujii-Kuriyama, Y. (1994). Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors. J.Biol.Chem. 269, 27337-27343.
  31. 31.0 31.1 Poland, A., Palen, D., and Glover, E. (1994). Analysis of the four alleles of the murine aryl hydrocarbon receptor. Mol.Pharmacol. 46, 915-921.
  32. Backlund, M., and Ingelman-Sundberg, M. (2004). Different structural requirements of the ligand binding domain of the aryl hydrocarbon receptor for high- and low-affinity ligand binding and receptor activation. Mol.Pharmacol. 65, 416-425.
  33. Murray, I. A., Reen, R. K., Leathery, N., Ramadoss, P., Bonati, L., Gonzalez, F. J., Peters, J. M., and Perdew, G. H. (2005). Evidence that ligand binding is a key determinant of Ah receptor-mediated transcriptional activity. Arch.Biochem.Biophys. 442, 59-71.
  34. Pandini, A., Denison, M. S., Song, Y., Soshilov, A. A., and Bonati, L. (2007). Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modeling and mutational analysis. Biochemistry 46, 696-708.
  35. 35.0 35.1 35.2 Pandini, A., Soshilov, A. A., Song, Y., Zhao, J., Bonati, L., and Denison, M. S. (2009). Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis. Biochemistry 48, 5972-5983.
  36. Ramadoss, P., and Perdew, G. H. (2004). Use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin as a probe to determine the relative ligand affinity of human versus mouse aryl hydrocarbon receptor in cultured cells. Mol.Pharmacol. 66, 129-136.
  37. 37.0 37.1 37.2 Head, J. A., Hahn, M. E., and Kennedy, S. W. (2008). Key amino acids in the aryl hydrocarbon receptor predict dioxin sensitivity in avian species. Environ.Sci.Technol. 42, 7535-7541.
  38. 38.0 38.1 Denison, M. S., Soshilov, A. A., He, G., DeGroot, D. E., and Zhao, B. (2011). Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor. Toxicol.Sci. 124, 1-22.
  39. van den Berg, M., Birnbaum, L. S., Bosveld, A. T., Brunström, B., Cook, P., Feeley, M., Giesy, J. P., Hanberg, A., Hasegawa, R., Kennedy, S. W., Kubiak, T. J., Larsen, J. C., Van Leeuwen, F. X. R., Liem, A. K. D., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D. E., Tysklind, M., Younes, M., Wærn, F., and Zacharewski, T. R. (1998). Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ.Health Perspect. 106, 775-792.
  40. Cohen-Barnhouse, A. M., Zwiernik, M. J., Link, J. E., Fitzgerald, S. D., Kennedy, S. W., Hervé, J. C., Giesy, J. P., Wiseman, S. B., Yang, Y., Jones, P. D., Wan, Y., Collins, B., Newsted, J. L., Kay, D. P., and Bursian, S. J. (2011b). Sensitivity of Japanese quail (Coturnix japonica), Common pheasant (Phasianus colchicus), and White Leghorn chicken (Gallus gallus domesticus) embryos to in ovo exposure to TCDD, PeCDF, and TCDF. Toxicol.Sci. 119, 93-103.
  41. Farmahin, R., Crump, D., Jones, S. P., Mundy, L. J., and Kennedy, S. W. (2013a). Cytochrome P4501A induction in primary cultures of embryonic European starling hepatocytes exposed to TCDD, PeCDF and TCDF. Ecotoxicology 22(4), 731-739.
  42. Hervé, J. C., Crump, D., Jones, S. P., Mundy, L. J., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., Jones, P. D., Wiseman, S. B., Wan, Y., and Kennedy, S. W. (2010a). Cytochrome P4501A induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and two chlorinated dibenzofurans in primary hepatocyte cultures of three avian species. Toxicol. Sci. 113(2), 380-391.
  43. Hervé, J. C., Crump, D. L., McLaren, K. K., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., and Kennedy, S. W. (2010b). 2,3,4,7,8-pentachlorodibenzofuran is a more potent cytochrome P4501A inducer than 2,3,7,8-tetrachlorodibenzo-p-dioxin in herring gull hepatocyte cultures. Environ. Toxicol. Chem. 29(9), 2088-2095.
  44. Poland, A., and Glover, E. (1973). Studies on the mechanism of toxicity of the chlorinated dibenzo-p-dioxins. Environ.Health Perspect. 5, 245-251.
  45. 45.0 45.1 McFarland, V. A., and Clarke, J. U. (1989). Environmental occurrence, abundance, and potential toxicity of polychlorinated biphenyl congeners: Considerations for a congener-specific analysis. Environ.Health Perspect. 81, 225-239.
  46. 46.0 46.1 Omiecinski, C. J., Vanden Heuvel, J. P., Perdew, G. H., and Peters, J. M. (2011). Xenobiotic metabolism, disposition, and regulation by receptors: from biochemical phenomenon to predictors of major toxicities. Toxicol.Sci. 120 Suppl 1, S49-S75.
  47. Swedenborg, E., and Pongratz, I. (2010). AhR and ARNT modulate ER signaling. Toxicology 268, 132-138.
  48. Diani-Moore, S., Ma, Y., Labitzke, E., Tao, H., David, W. J., Anderson, J., Chen, Q., Gross, S. S., and Rifkind, A. B. (2011). Discovery and biological characterization of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole as an aryl hydrocarbon receptor activator generated by photoactivation of tryptophan by sunlight. Chem. Biol. Interact. 193(2), 119-128.
  49. Wincent, E., Bengtsson, J., Mohammadi, B. A., Alsberg, T., Luecke, S., Rannug, U., and Rannug, A. (2012). Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U. S. A 109(12), 4479-4484.
  50. Baba, T., Mimura, J., Nakamura, N., Harada, N., Yamamoto, M., Morohashi, K., and Fujii-Kuriyama, Y. (2005). Intrinsic function of the aryl hydrocarbon (dioxin) receptor as a key factor in female reproduction. Mol.Cell Biol. 25, 10040-10051.
  51. Fernandez-Salguero, P. M., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S., Kimura, S., Nebert, D. W., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1995). Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268, 722-726.
  52. Ichihara, S., Yamada, Y., Ichihara, G., Nakajima, T., Li, P., Kondo, T., Gonzalez, F. J., and Murohara, T. (2007). A role for the aryl hydrocarbon receptor in regulation of ischemia-induced angiogenesis. Arterioscler.Thromb.Vasc.Biol. 27, 1297-1304.
  53. Lahvis, G. P., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J., and Bradfield, C. A. (2000). Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice. Proc.Natl.Acad.Sci U.S.A 97, 10442-10447.
  54. Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., and Fujii-Kuriyama, Y. (1997). Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2, 645-654.
  55. Schmidt, J. V., Su, G. H., Reddy, J. K., Simon, M. C., and Bradfield, C. A. (1996). Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc.Natl.Acad.Sci U.S.A 93, 6731-6736.
  56. Thackaberry, E. A., Gabaldon, D. M., Walker, M. K., and Smith, S. M. (2002). Aryl hydrocarbon receptor null mice develop cardiac hypertrophy and increased hypoxia-inducible factor-1alpha in the absence of cardiac hypoxia. Cardiovasc.Toxicol. 2, 263-274.
  57. Zhang, N., Agbor, L. N., Scott, J. A., Zalobowski, T., Elased, K. M., Trujillo, A., Duke, M. S., Wolf, V., Walsh, M. T., Born, J. L., Felton, L. A., Wang, J., Wang, W., Kanagy, N. L., and Walker, M. K. (2010). An activated renin-angiotensin system maintains normal blood pressure in aryl hydrocarbon receptor heterozygous mice but not in null mice. Biochem.Pharmacol. 80, 197-2040.

Abnet, C.C.; Tanguay, R.L.; Heideman, W.; Peterson, R.E. 1999. Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: Greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners. Toxicol. Appl. Pharmacol. 159, 41-51.

 

Andreasen, E.A.; Tanguay, R.L.; Peterson, R.E.; Heideman, W. 2002. Identification of a critical amino acid in the aryl hydrocarbon receptor. J. Biol. Chem. 277 (15), 13210-13218.

 

Bak, S.M.; Lida, M.; Hirano, M.; Iwata, H.; Kim, E.Y. 2013. Potencies of red seabream AHR1- and AHR2-mediated transactivation by dioxins: implications of both AHRs in dioxin toxicity. Environ. Sci. Technol. 47 (6), 2877-2885.

 

Clark, B.W.; Matson, C.W.; Jung, D.; Di Giulio, R.T. 2010. AHR2 mediates cardiac teratogenesis of polycyclic aromatic hydrocarbons and PCB-126 in Atlantic killifish (Fundulus heteroclitus). Aquat. Toxicol. 99, 232-240.

 

Doering, J.A.; Farmahin, R.; Wiseman, S.; Beitel, S.C.; Kennedy, S.W.; Giesy, J.P.; Hecker, M. 2015. Differences in activation of aryl hydrocarbon receptors of white sturgeon relative to lake sturgeon are predicted by identities of key amino acids in the ligand binding domain. Enviro. Sci. Technol. 49, 4681-4689.

 

Doering, J.A.; Farmahin, R.; Wiseman, S.; Kennedy, S.; Giesy J.P.; Hecker, M. 2014. Functionality of aryl hydrocarbon receptors (AhR1 and AhR2) of white sturgeon (Acipenser transmontanus) and implications for the risk assessment of dioxin-like compounds. Enviro. Sci. Technol. 48, 8219-8226.

 

Doering, J.A.; Giesy, J.P.; Wiseman, S.; Hecker, M. Predicting the sensitivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain. Environ. Sci. Pollut. Res. Int. 2013, 20(3), 1219-1224.

 

Doering, J.A.; Wiseman, S; Beitel, S.C.; Giesy, J.P.; Hecker, M. 2014. Identification and expression of aryl hydrocarbon receptors (AhR1 and AhR2) provide insight in an evolutionary context regarding sensitivity of white sturgeon (Acipenser transmontanus) to dioxin-like compounds. Aquat. Toxicol. 150, 27-35.

 

Duncan, D.M.; Burgess, E.A.; Duncan, I. 1998. Control of distal antennal identity and tarsal development in Drosophila by spineless-aristapedia, a homolog of the mammalian dioxin receptor. Genes Dev. 12, 1290-1303.

 

Eisner, B.K.; Doering, J.A.; Beitel, S.C.; Wiseman, S.; Raine, J.C.; Hecker, M. 2016. Cross-species comparison of relative potencies and relative sensitivities of fishes to dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls in vitro. Enviro. Toxicol. Chem. 35 (1), 173-181.

 

Emmons, R.B.; Duncan, D.; Estes, P.A.; Kiefel, P.; Mosher, J.T.; Sonnenfeld, M.; Ward, M.P.; Duncan, I.; Crews, S.T. 1999. The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila. Development. 126, 3937-3945.

 

Evans, B.R.; Karchner, S.I.; Franks, D.G.; Hahn, M.E. 2005. Duplicate aryl hydrocarbon receptor repressor genes (ahrr1 and ahrr2) in the zebrafish Danio rerio: structure, function, evolution, and AHR-dependent regulation in vivo. Arch. Biochem. Biophys. 441, 151-167.

 

Hahn, M.E. 2002. Aryl hydrocarbon receptors: diversity and evolution. Chemico-Biol. Interact. 141, 131-160.

 

Hahn, M.E.; Karchner, S.I.; Evans, B.R.; Franks, D.G.; Merson, R.R.; Lapseritis, J.M. 2006. Unexpected diversity of aryl hydrocarbon receptors in non-mammalian vertebrates: Insights from comparative genomics. J. Exp. Zool. A. Comp. Exp. Biol. 305, 693-706.

 

Hahn, M.E.; Poland, A.; Glover, E.; Stegeman, J.J. 1994. Photoaffinity labeling of the Ah receptor: phylogenetic survey of diverse vertebrate and invertebrate species. Arch. Biochem. Biophys. 310, 218-228.

 

Hansson, M.C.; Hahn, M.E. 2008. Functional properties of the four Atlantic salmon (Salmo salar) aryl hydrocarbon receptor type 2 (AHR2) isoforms. Aquat. Toxicol. 86, 121-130.

 

Hansson, M.C.; Wittzell, H.; Persson, K.; von Schantz, T. 2004. Unprecedented genomic diversity of AhR1 and AhR2 genes in Atlantic salmon (Salmo salar L.). Aquat. Toxicol. 68 (3), 219-232.

 

Karchner, S.I.; Franks, D.G.; Hahn, M.E. (2005). AHR1B, a new functional aryl hydrocarbon receptor in zebrafish: tandem arrangement of ahr1b and ahr2 genes. Biochem. J. 392 (1), 153-161.

 

Karchner, S.I.; Powell, W.H.; Hahn, M.E. 1999. Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the Teleost Fundulus heteroclitus. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors. J. Biol. Chem. 274, 33814-33824.

 

Lahvis, G.P.; Bradfield, C.A. 1998. Ahr null alleles: distinctive or different? Biochem. Pharmacol. 56, 781-787.

 

Lavine, J.A.; Rowatt, A.J.; Klimova, T.; Whitington, A.J.; Dengler, E.; Beck, C.; Powell, W.H. 2005. Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Sci. 88 (1), 60-72.

 

Oka, K.; Kohno, S.; Ohta, Y.; Guillette, L.J.; Iguchi, T.; Katsu, Y. (2016). Molecular cloning and characterization of the aryl hydrocarbon receptors and aryl hydrocarbon receptor nuclear translocators in the American alligator. Gen. Comp. Endo. 238, 13-22.

 

Pongratz, I.; Mason, G.G.; Poellinger, L. Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. J. Biol. Chem. 1992, 267 (19), 13728-13734

 

Prasch, A.L.; Teraoka, H.; Carney, S.A.; Dong, W.; Hiraga, T.; Stegeman, J.J.; Heideman, W.; Peterson, R.E. 2003. Toxicol. Sci. Aryl hydrocarbon receptor 2 mediated 2,3,7,8-tetrachlorodibenzo-p-dioxin developmental toxicity in zebrafish. 76 (1), 138-150.

 

Shoots, J.; Fraccalvieri, D.; Franks, D.G.; Denison, M.S.; Hahn, M.E.; Bonati, L.; Powell, W.H. 2015. An aryl hydrocarbon receptor from the salamander Ambystoma mexicanum exhibits low sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enviro. Sci. Technol. 49, 6993-7001.

 

Tanguay, R.L.; Abnet, C.C.; Heideman, W. Peterson, R.E. (1999). Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor1. Biochimica et Biophysica Act 1444, 35-48.

 

Van den Berg, M.; Birnbaum, L.; Bosveld, A.T.C.; Brunstrom, B.; Cook, P.; Feeley, M.; Giesy, J.P.; Hanberg, A.; Hasegawa, R.; Kennedy, S.W.; Kubiak, T.; Larsen, J.C.; van Leeuwen, R.X.R.; Liem, A.K.D.; Nolt, C.; Peterson, R.E.; Poellinger, L.; Safe, S.; Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PECDFs for human and wildlife. Enviro. Hlth. Persp. 1998, 106, 775-792.

 

Van Tiem, L.A.; Di Giulio, R.T. 2011. AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (Danio rerio). Toxicol. Appl. Pharmacol. 254 (3), 280-287.

 

Whitlock, J.P.; Okino, S.T.; Dong, L.Q.; Ko, H.S.P.; Clarke Katzenberg, R.; Qiang, M.; Li, W. 1996. Induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. Faseb. J. 10, 809-818.

 

Wirgin, I.; Roy, N.K.; Loftus, M.; Chambers, R.C.; Franks, D.G.; Hahn, M.E. 2011. Mechanistic basis of resistance to PCBs in Atlantic tomcod from the Hudson River. Science. 331, 1322-1324.

 

Yamauchi, M.; Kim, E.Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryos: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. Aquat. Toxicol. 2006, 16, 166-179.

 

Yasui, T.; Kim, E.Y.; Iawata, H.; Franks, D.G.; Karchner, S.I.; Hahn, M.E.; Tanabe, S. 2007. Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. Toxicol. Sci. 99 (1), 101-117.


Key Events

Title Short name
dimerization, AHR/ARNT dimerization, AHR/ARNT
Increase, COX-2 expression Increase, COX-2 expression
Altered, Cardiovascular development/function Altered, Cardiovascular development/function

944: dimerization, AHR/ARNT

Short Name: dimerization, AHR/ARNT

Biological Organization

Level of Biological Organization
Molecular

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
chicken Gallus gallus Strong NCBI
zebrafish Danio rerio Strong NCBI
mouse Mus musculus Strong NCBI
Life Stage Applicability
Life Stage Evidence
Embryo Strong
development Strong
Sex Applicability
Sex Evidence
Unspecific Strong
  • ARNTs have been identified in all tetrapods investigated to date (Drutel et al 1996; Hirose et al 1996; Hoffman et al 1991; Lee et al 2007; Lee et al 2011).
  • ARNTs have been identified in a great phylogenetic diversity of fishes, including early fishes (Doering et al 2014; 2016).
  • ARNT has been identified in investigated invertebrates (Powell-Coffman et al 1998).

This mechanism is conserved across species. Mammals possess a single AHR, whereas birds and fish express multiple isoforms, and all three express multiple ARNT isoforms. Not all of the isoforms identified are functionally active. For example, killifish AHR1 and AHR2 are active and display different transcription profiles, whereas zebrafish AHR2 and ARNT2 are active in mediating xenobiotic-mediated toxicity and AHR1 is inactive (Hahn et al. 2006; Prasch et al. 2006).


How this Key Event Works

Structure and Function of ARNT

  • The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the Per-Arnt-Sim (PAS) family of proteins (Gu et al 2000).
  • PAS proteins share highly conserved PAS domains (Gu et al 2000).
  • PAS proteins act as transcriptional regulators in response to environmental and physiological cues (Gu et al 2000).

Isoforms of ARNT:

  • Over time ARNT has undergone gene duplication and diversification in vertebrates, which has resulted in three clades of ARNT, namely ARNT1, ARNT2, and ARNT3.
  • Each clade can include multiple isoforms and splice variants (Hill et al 2009; Lee et al 2007; Lee et al 2011; Powel & Hahn 2000; Tanguay et al 2000).
  • ARNT1s have been demonstrated to function predominantly through heterodimerization with the aryl hydrocarbon receptor (AhR) and hypoxia inducible factor 1 α (HIF1α) (Prasch et al 2004; 2006; Wang et al 1995).
  • ARNT2s are believed to function predominantly through heterodimerization with Single Minded (SIM) (Hirose et al 1996).
  • ARNT3s, which are also known as ARNT-like (ARNTL), Brain and Muscle ARNT-like-1 (BMAL1), or Morphine Preference 3 (MOP3), are believed to function predominantly through heterodimerization with Circadian Locomotor Output Cycles Kaput (CLOCK) (Gekakis et al 1998).
  • ARNTs have numerous key roles in vertebrates related to responses to developmental and environmental cues.

Roles of ARNTs in mammals:

  • ARNT1 functions in normal vascular and hematopoietic development (Kozak et al 1997; Maltepe et al 1997; Abbott & Buckalew 2000).
  • ARNT2 functions in development of the hypothalamus and nervous system (Hosoya et al 2001; Keith et al 2001).
  • ARNT3 functions in biological rhythms (Gekakis et al 1998).

Roles of ARNTs in other taxa:

  • ARNTs have been demonstrated to have roles in development of the heart, brain, liver, and possibly the peripheral nervous system in zebrafish (Danio rerio) (Hill et al 2009).
  • Roles of ARNTs in other taxa have not been sufficiently investigated to date.

Interaction with AHR

Upon ligand binding, the aryl hydrocarbon receptor (AHR) migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with AHR nuclear translocator (ARNT) (Mimura and Fujii-Kuriyama 2003). The AHR-ARNT complex then binds to a xenobiotic response element (XRE) found in the promoter of an AHR-regulated gene and recruits co-regulators such as CREB binding protein/p300, steroid receptor co-activator (SRC) 1, SRC-2, SRC-3 and nuclear receptor interacting protein 1, leading to induction or repression of gene expression (Fujii-Kuriyama and Kawajiri 2010). Expression levels of several genes, including phase I (e.g. cytochrome P450 (CYP) 1A, CYP1B, CYP2A) and phase II enzymes (e.g. uridine diphosphate glucuronosyl transferase (UDP-GT), glutathione S-transferases (GSTs)), as well as genes involved in cell proliferation (transforming growth factor-beta, interleukin-1 beta), cell cycle regulation (p27, jun-B) and apoptosis (Bax), are regulated through this mechanism (Fujii-Kuriyama and Kawajiri 2010; Giesy et al. 2006; Mimura and Fujii-Kuriyama 2003; Safe 1994).


How it is Measured or Detected

The active AHR complexed with ARNT can be measured using protein-DNA interaction assays. Two methods are described in detail by Perez-Romero and Imperiale (Perez-Romero and Imperiale 2007). Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions in vivo. It involves the treatment of cells with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. Nuclear fractions are isolated, the genomic DNA is sheared, and nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. After reversal of the crosslinking, the associated DNA fragments are sequenced. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. Electrophoretic mobility shift assay (EMSA) provides a rapid method to study DNA-binding protein interactions in vitro. This relies on the fact that complexes of protein and DNA migrate through a non-denaturing polyacrylamide gel more slowly than free DNA fragments. The protein-DNA complex components are then identified with appropriate antibodies. The EMSA assay was found to be consistent with the luciferase reporter gene assay (in chicken hepatoma cells dosed with dioxin-like compounds (Heid et al. 2001).


References

1. Fujii-Kuriyama, Y., and Kawajiri, K. (2010). Molecular mechanisms of the physiological functions of the aryl hydrocarbon (dioxin) receptor, a multifunctional regulator that senses and responds to environmental stimuli. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86(1), 40-53.

2. Giesy, J. P., Kannan, K., Blankenship, A. L., Jones, P. D., and Newsted, J. L. (2006). Toxicology of PCBs and related compounds. In Endocrine Disruption Biological Bases for Health Effects in Wildlife and Humans (D.O.Norris and J.A.Carr, Eds.), pp. 245-331. Oxford University Press, New York.

3. Hahn, M. E., Karchner, S. I., Evans, B. R., Franks, D. G., Merson, R. R., and Lapseritis, J. M. (2006). Unexpected diversity of aryl hydrocarbon receptors in non-mammalian vertebrates: insights from comparative genomics. J. Exp. Zool. A Comp Exp. Biol. 305(9), 693-706.

4. Heid, S. E., Walker, M. K., and Swanson, H. I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci 61(1), 187-196.

5. Mimura, J., and Fujii-Kuriyama, Y. (2003). Functional role of AhR in the expression of toxic effects by TCDD. Biochimica et Biophysica Acta - General Subjects 1619(3), 263-268.

6. Perez-Romero, P., and Imperiale, M. J. (2007). Assaying protein-DNA interactions in vivo and in vitro using chromatin immunoprecipitation and electrophoretic mobility shift assays. Methods Mol. Med. 131, 123-139.

7. Prasch, A. L., Tanguay, R. L., Mehta, V., Heideman, W., and Peterson, R. E. (2006). Identification of zebrafish ARNT1 homologs: 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish requires ARNT1. Mol. Pharmacol. 69(3), 776-787.

8. Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. Critical Reviews in Toxicology 24(2), 87-149.

Abnet, C.C.; Tanguay, R.L.; Heideman, W.; Peterson, R.E. 1999. Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: Greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners. Toxicol. Appl. Pharmacol. 159, 41-51.

 

Andreasen, E.A.; Hahn, M.E.; Heideman, W.; Peterson, R.E.; Tanguay, R.L. 2002. The zebrafish (Danio rerio) aryl hydrocarbon receptor type 1 is a novel vertebrate receptor. Molec. Pharmacol. 62, 234-249.

 

Andreasen, E.A.; Tanguay, R.L.; Peterson, R.E.; Heideman, W. 2002. Identification of a critical amino acid in the aryl hydrocarbon receptor. J. Biol. Chem. 277 (15), 13210-13218.

 

Antkiewicz, D.S.; Burns, C.G.; Carney, S.A.; Peterson, R.E.; Heideman, W. 2005. Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. 84, 368-377.

 

Bak, S.M.; Lida, M.; Hirano, M.; Iwata, H.; Kim, E.Y. 2013. Potencies of red seabream AHR1- and AHR2-mediated transactivation by dioxins: implications of both AHRs in dioxin toxicity. Environ. Sci. Technol. 47 (6), 2877-2885.

 

Billiard, S.M.; Hahn, M.E.; Franks, D.G.; Peterson, R.E.; Bols, N.C.; Hodson, P.V. (2002). Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs). Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 133 (1), 55-68.

 

Chen, G.; Bunce, N.J. (2003). Polybrominated diphenyl ethers as Ah receptor agonists and antagonists. Toxicol. Sci. 76 (2), 310-320.

 

Denison, M.S.; Heath-Pagliuso, S. The Ah receptor: a regulator of the biochemical and toxicological actions of structurally diverse chemicals. Bull. Environ. Contam. Toxicol. 1998, 61 (5), 557-568.

 

Doering, J.A.; Tang, S.; Peng, H.; Eisner, B.K.; Sun, J.; Giesy, J.P.; Wiseman, S.; Hecker, M. 2016. High conservation in transcriptomic and proteomic response of white sturgeon to equipotent concentrations of 2,3,7,8-TCDD, PCB 77, and benzo[a]pyrene. Enviro. Sci. Technol. 50 (9), 4826-4835.

 

Doering, J.A.; Farmahin, R.; Wiseman, S.; Kennedy, S.; Giesy J.P.; Hecker, M. 2014. Functionality of aryl hydrocarbon receptors (AhR1 and AhR2) of white sturgeon (Acipenser transmontanus) and implications for the risk assessment of dioxin-like compounds. Enviro. Sci. Technol. 48, 8219-8226.

 

Doering, J.A.; Farmahin, R.; Wiseman, S.; Beitel, S.C.; Kennedy, S.W.; Giesy, J.P.; Hecker, M. 2015. Differences in activation of aryl hydrocarbon receptors of white sturgeon relative to lake sturgeon are predicted by identities of key amino acids in the ligand binding domain. Enviro. Sci. Technol. 49, 4681-4689.

 

Doering, J.A.; Wiseman, S; Beitel, S.C.; Giesy, J.P.; Hecker, M. 2014b. Identification and expression of aryl hydrocarbon receptors (AhR1 and AhR2) provide insight in an evolutionary context regarding sensitivity of white sturgeon (Acipenser transmontanus) to dioxin-like compounds. Aquat. Toxicol. 150, 27-35.

 

Drutel, G.; Kathmann, M.; Heron, A.; Schwartz, J.; Arrang, J. (1996). Cloning and selective expression in brain and kidney of ARNT2 homologous to the Ah receptor nuclear translocator (ARNT). Biochem. Biophys. Res. Comm. 225 (2), 333-339.

 

Farmahin, R.; Crump, D.; O’Brien, J.M.; Jones, S.P.; Kennedy, S.W. (2016). Time-dependent transcriptomic and biochemical responses of 6-formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are explained by AHR activation time. Biochem. Pharmacol. 115 (1), 134-143.

 

Farmahin, R.; Manning, G.E.; Crump, D.; Wu, D.; Mundy, L.J.; Jones, S.P.; Hahn, M.E.; Karchner, S.I.; Giesy, J.P.; Bursian, S.J.; Zwiernik, M.J.; Fredricks, T.B.; Kennedy, S.W. 2013. Amino acid sequence of the ligand-binding domain of the aryl hydrocarbon receptor 1 predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol. Sci. 131 (1), 139-152.

 

Farmahin, R.; Wu, D.; Crump, D.; Herve, J.C.; Jones, S.P.; Hahn, M.E.; Karchner, S.I.; Giesy, J.P.; Bursian, S.J.; Zwiernik, M.J.; Kennedy, S.W. 2012. Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Enviro. Sci. Toxicol. 46 (5), 2967-2975.

 

Gekakis, N., Staknis, D., Nguyen, H.B., Davis, F.C., Wilsbacher, L.D., King, D.P., Takahashi, J.S., Weitz, C.J. 1998. Role of the CLOCK protein in the mammalian circadian mechanism. Science. 280, 1564-1569.

 

Gu, Y.; Hogenesch, J.B.; Bradfield, C.A. 2000. The PAS superfamily: Sensors of environmental and developmental signals. Annu. Rev. Pharmacol. Toxicol. 40, 519-561.

 

Hansson, M.C.; Hahn, M.E. 2008. Functional properties of the four Atlantic salmon (Salmo salar) aryl hydrocarbon receptor type 2 (AHR2) isoforms. Aquat. Toxicol. 86, 121-130.

 

Hill, A.J.; King-Heiden, T.C.; Heideman, W.; Peterson, R.E. (2009). Potential roles of Arnt2 in zebrafish larval development. Zebrafish. 6 (1), 79-91.

 

Hirose, K., Morita, M., Ema, M., Mimura, J., Hamada, H., Fujii, H., Saijo, Y., Gotoh, O., Sogawa, K., Fujii-Kuriyama, Y. 1996. cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/ PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon nuclear translocator (Arnt). Mol. Cell. Biol. 16, 1706-1713.

 

Hoffman, E.C., Reyes, H., Chu, F.F., Sander, F., Conley, L.H., Brooks, B.A., Hankinson, O. 1991. Cloning of a factor required for activity of the Ah (dioxin) receptor. Science. 252, 954-958.

 

Karchner, S.I.; Powell, W.H.; Hahn, M.E. 1999. Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the Teleost Fundulus heteroclitus. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors. J. Biol. Chem. 274, 33814-33824.

 

Lavine, J.A.; Rowatt, A.J.; Klimova, T.; Whitington, A.J.; Dengler, E.; Beck, C.; Powell, W.H. 2005. Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Sci. 88 (1), 60-72.

 

Lee, J., Kim, E., Iwata, H., Tanabe, S. 2007. Molecular characterization and tissue distribution of aryl hydrocarbon receptor nuclear translocator isoforms, ARNT1 and ARNT2, and identification of novel splice variants in common cormorant (Phalacrocorax carbo). Comp. Biochem. Physiol. C. 145, 379-393.

 

Lee, J., Kim, E., Iwabuchi, H., Iwata, H. (2011). Molecular and functional characterization of aryl hydrocarbon receptor nuclear translocator 1 (ARNT1) and ARNT2 in chicken (Gallus gallus). Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 153 (3), 269-279.

 

Mandl, M.; Depping, R. (2014). Hypoxia-inducible aryl hydrocarbon receptor nuclear translocator (ARNT) (HIF-1B): Is it a rare exception? Mol. Med. 20 (1), 215-220.

 

Murk, A.J.; Legler, J.; Denison, M.S.; Giesy, J.P.; Van De Guchte, C.; Brouwer, A. (1996). Chemical-activated luciferase gene expression (CALUX): A novel in vitro bioassay for Ah receptor active compounds in sediments and pore water. Toxicol. Sci. 33 (1), 149-160.

 

Oka, K.; Kohno, S.; Ohta, Y.; Guillette, L.J.; Iguchi, T.; Katsu, Y. (2016). Molecular cloning and characterization of the aryl hydrocarbon receptors and aryl hydrocarbon receptor nuclear translocators in the American alligator. Gen. Comp. Endo. 238, 13-22.

 

Powell, W.H.; Hahn, M.E. (2002). Identification and functional characterization of hypoxia-inducible factor 2a from the estuarine teleost, Fundulus heteroclitus: Interaction of HIF-2a with two ARNT2 splice variants. J. Exp. Zoo. A. 294 (1), 17-29.

 

Prasch, A.L.; Tanguay, R.L.; Mehta, V.; Heideman, W.; Peterson, R.E. (2006). Identification of zebrafish ARNT1 homologs: 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish requires ARNT1. Mol. Pharmacol. 69 (3), 776-787.

 

Prasch, A.L.; Teraoka, H.; Carney, S.A.; Dong, W.; Hiraga, T.; Stegeman, J.J.; Heideman, W.; Peterson, R.E. 2003. Toxicol. Sci. Aryl hydrocarbon receptor 2 mediated 2,3,7,8-tetrachlorodibenzo-p-dioxin developmental toxicity in zebrafish. 76 (1), 138-150.

 

Shoots, J.; Fraccalvieri, D.; Franks, D.G.; Denison, M.S.; Hahn, M.E.; Bonati, L.; Powell, W.H. 2015. An aryl hydrocarbon receptor from the salamander Ambystoma mexicanum exhibits low sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enviro. Sci. Technol. 49, 6993-7001.

 

Tanguay, R.L.; Abnett, C.C.; Heideman, W.; Peterson, R.E. 1999. Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor. Biochem. Biophys. Acta. 1444, 35-48.

 

Tanguay, R.L.; Andreasen, E.; Heideman, W.; Peterson, R.E. (2000). Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions. BBA. 1494 (1-2), 117-128.

 

Van den Berg, M.; Birnbaum, L.; Bosveld, A.T.C.; Brunstrom, B.; Cook, P.; Feeley, M.; Giesy, J.P.; Hanberg, A.; Hasegawa, R.; Kennedy, S.W.; Kubiak, T.; Larsen, J.C.; van Leeuwen, R.X.R.; Liem, A.K.D.; Nolt, C.; Peterson, R.E.; Poellinger, L.; Safe, S.; Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PECDFs for human and wildlife. Enviro. Hlth. Persp. 1998, 106, 775-792.

 

Van den Berg, M.; Birnbaum, L.S.; Dension, M.; De Vito, M.; Farland, W.; Feeley, M.; Fiedler, H.; Hakansson, H.; Hanberg, A.; Haws, L.; Rose, M.; Safe, S.; Schrenk, D.; Tohyama, C.; Tritscher, A.; Tuomisto, J.; Tysklind, M.; Walker, N.; Peterson, R.E. 2006. The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. 93 (2), 223-241.

 

Waller, C.L.; McKinney, J.D. (1992). Comparative molecular field analysis of polyhalogenated dibenzo-p-dioixns, dibenzofurans, and biphenyls. J. Med. Chem. 35, 3660-2666.

 

Waller, C.; McKinney, J. (1995). Three-dimensional quantitative structure-activity relationships of dioxins and dioxin-like compounds: model validation and Ah receptor characterization. Chem. Res. Toxicol. 8, 847-858.

 

Wang, G.L., Jiang, B.H., Rue, E.A., Semenza, G.L. 1995. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. U.S.A. 92, 5510-5514.

 

Whitlock, J.P.; Okino, S.T.; Dong, L.Q.; Ko, H.S.P.; Clarke Katzenberg, R.; Qiang, M.; Li, W. 1996. Induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. Faseb. J. 10, 809-818.

 

Whyte, J.J.; Jung, R.E.; Schmitt, C.J.; Tillitt, D.E. (2008). Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit. Rev. Toxicol. 30 (4), 347-570.

 

Wirgin, I.; Roy, N.K.; Loftus, M.; Chambers, R.C.; Franks, D.G.; Hahn, M.E. 2011. Mechanistic basis of resistance to PCBs in Atlantic tomcod from the Hudson River. Science. 331, 1322-1324.


1269: Increase, COX-2 expression

Short Name: Increase, COX-2 expression

AOPs Including This Key Event


Biological Organization

Level of Biological Organization
Molecular

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Danio rerio Danio rerio Strong NCBI
Oryzias latipes Oryzias latipes Strong NCBI
Gallus gallus Gallus gallus Strong NCBI
Life Stage Applicability
Life Stage Evidence
Embryo Strong
Sex Applicability
Sex Evidence
Unspecific Strong
  • There is a high level of conservation of COX-2, as well as its function, especially across vertebrates (Havird et al 2008; 2015), indicating that numerous vertebrate taxa might be susceptible to up-regulation in COX-2.
  • Typically, teleost fish genomes contain more than one COX -2 gene, likely a result of genome duplication after divergence of teleosts from tetrapods (Ishikawa et al 2007; Havird et al 2015). In zebrafish there is COX-2a and COX-2b (Teraoka et al 2014).
  • In invertebrates, COX is found in most crustaceans, the majority of molluscs, but only in specific lineages within Cnidaria and Annelida. COX genes are not found in Hemichordata, Echinodermata, or Platyhelminthes. Insecta COX genes lack in homology, but might function as COX enzymes based on structural analyses (Havird et al 2015).

How this Key Event Works

  • Prostaglandin-endoperoxide synthase (PTGS; KEGG ID E.C. 1.14.99.1) is an enzyme that has two catalytic sites.
  • Cyclooxygenase site (COX) catalyzes conversion of arachidonic acid into endoperoxide prostaglandin G2 (Simmons et al 2004).
  • Peroxidase active site converts PGG2 to PGH2 (KEGG reactions 1599, 1590). PGH2 is a precursor for synthesis of other prostaglandins (PGEs, PGFs), prostacyclin, and thromboxanes (Simmons et al 2004; Botting & Botting 2011).
  • There are two isoforms, COX-1 and COX-2
  • COX-2 is inducible by certain chemical exposures, inflammation, during discrete stages of gamete maturation, and more (Green et al 2012). 

How it is Measured or Detected

  • COX-2 has been measured as mRNA by use of quantitative real-time polymerase chain reaction (q-RT PCR) (Dong et al 2010; Fujisawa et al 2014; Teraoka et al 2008; 2014).

References

Bacchi, S., Palumbo, P., Sponta, A., & Coppolino, M. F. (2012). Clinical pharmacology of non-steroidal anti-inflammatory drugs: a review. Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Inflammatory and Anti-Allergy Agents), 11(1), 52-64.

 

Botting, R. M., & Botting, J. H. (2011). C14 Non-steroidal anti-inflammatory drugs. In Principles of Immunopharmacology (pp. 573-584). Birkhäuser Basel.

 

Chandrasekharan, N. V., Dai, H., Roos, K. L. T., Evanson, N. K., Tomsik, J., Elton, T. S., & Simmons, D. L. (2002). COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proceedings of the National Academy of Sciences,99(21), 13926-13931.

 

Crofford, L.J. (1997). COX-1 and COX-2 tissue expression: implications and predictions. J. Rheumatol. Suppl. 49, 15-90.

 

Degner, S.C.; Kemp, M.Q.; Hockings, J.K.; Romagnolo, D.F. (2007). Cyclooxygenase-2 promoter activation by the aromatic hydrocarbon receptor in breast cancer MCF-7 cells: Repressive effects of conjugated linoleic acid. Nutri. Canc. 56 (2), 248-257.

 

Dong, W.; Matsumura, F.; Kullman, S.W. (2010). TCDD induced pericardial edema and relative COX-2 expression in medaka (Oryzias latipes) embryos. Toxicol. Sci. 118 (1), 213-223.

 

Fujisaw, N.; Nakayama, S.M.M.; Ikenaka, Y.; Ishizuka, M. 2014. TCDD-induced chick cardiotoxicity is abolished by a selective cyclooxygenase-2 (COX-2) inhibitor NS398. Arch. Toxicol. 88, 1739-1748.

 

Havird, J. C., Kocot, K. M., Brannock, P. M., Cannon, J. T., Waits, D. S., Weese, D. A., ... & Halanych, K. M. (2015). Reconstruction of Cyclooxygenase Evolution in Animals Suggests Variable, Lineage-Specific Duplications, and Homologs with Low Sequence Identity. Journal of molecular evolution, 1-16.

 

Havird, J. C., Miyamoto, M. M., Choe, K. P., & Evans, D. H. (2008). Gene duplications and losses within the cyclooxygenase family of teleosts and other chordates. Molecular biology and evolution, 25(11), 2349-2359.

 

Ishikawa, T. O., Griffin, K. J., Banerjee, U., & Herschman, H. R. (2007). The zebrafish genome contains two inducible, functional cyclooxygenase-2 genes.Biochemical and biophysical research communications, 352(1), 181-187.

 

Jonsson, M.E.; Kubota, A.; Timme-Laragy, A.R.; Woodin, B.; Stegeman, J.J. (2012). Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish. Toxicol. Appl. Pharmacol. 265 (2), 166-174.

 

Picot, D.; Loll, P.J.; Garavito, R.M. (1994). The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature. 367 (6460), 243-290.

 

Simmons, D. L., Botting, R. M., & Hla, T. (2004). Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacological reviews,56(3), 387-437.

 

Teraoka, H.; Kubota, A.; Kawai, Y.; Hiraga, T. (2008). Prostanoid signaling mediates circulation failure caused by TCDD in developing zebrafish. Interdis. Studies Environ. Chem. Biol. Resp. Chem. Pollut. 61-80.

 

Teraoka, H.; Okuno, Y.; Nijoukubo, D.; Yamakoshi, A.; Peterson, R.E.; Stegeman, J.J.; Kitazawa, T.; Hiraga, T.; Kubota, A. (2014). Involvement of COX2-thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish. Aquat. Toxicol. 154, 19-25.


317: Altered, Cardiovascular development/function

Short Name: Altered, Cardiovascular development/function

Biological Organization

Level of Biological Organization
Organ

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
chicken Gallus gallus Strong NCBI
mouse Mus musculus Strong NCBI
zebrafish Danio rerio Strong NCBI
Life Stage Applicability
Life Stage Evidence
Embryo Strong
development Strong
Sex Applicability
Sex Evidence
Unspecific Strong
  • Some form of cardiovascular system is present in members of the clade Bilateria (Bishopric 2005). This clade includes most animal phyla, except for sponges (Porifera), jellyfishes and corals (Cnidaria), placozoans (Placozoa), and comb jellies (Ctenophora).
  • Differences in cardiovascular systems are present among taxa. Vertebrates have closed circulatory systems, while some invertebrate taxa have open circulatory systems (Kardong 2006).

Birds, fish and mammals are all susceptible to cardiotoxicity following embryonic chemical exposure.

 

Cardiovascular development and function altered by dioxin-like compounds (DLCs):

  • Applicable to all teleost and non-teleost fishes so far investigated (Buckler et al 2015; Elonen et al 1998; Huang et al 2012; Johnson et al 1998; Park et al 2014; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Yamauchi et al 2006; Zabel et al 1995). However, altered cardiovascular development and function has not been investigated in jawless fishes, such as lamprey (Petromyzontiformes) and hagfish (Myxiniformes), or in cartilaginous fishes (Chondrichthyes).
  • Applicable to birds (Heid et al 2001).
  • Altered cardiovascular development and function by DLCs has not been sufficiently investigated in amphibians, reptiles, or invertebrates that possess a cardiovascular system.

How this Key Event Works

This key event applies to the disruption of cardiogenesis early enough in embryogenesis to result in gross morphological alterations leading to reduced cardiac function.

With respect to dioxin-like compounds that are strong AHR agonists, the malformations that have been observed following embryonic exposure are summarized in table 1.

Table 1: Cardiotoxic effects of strong AHR-agonists

Zebrafish Embryo Chicken Embryo Mouse
  • Reduced extension of common cardinal vein
  • Reduced blood flow
  • Reduced heart rate
  • Disrupted erythropoiesis
  • Decreased heart volume
  • Pericardial edema
  • Overt heart malformations
  • Enlarged left ventricle
  • Increased heart rate
  • Increased myosin content
  • Reduced β-adrenergic responsiveness
  • Increased ANF mRNA
  • Arrhythmia
  • Increased apoptosis
  • Reduced myocyte proliferation
  • Pericardial edema
  • Overt heart malformations
Embryo/Fetus
  • Reduced heart-to-body weight
  • Reduced myocyte proliferation
  • Vascular remodeling

21 Days old

  • Increased heart-to-body weight
  • Increased left ventricle weight
  • Reduced heart rate
  • Cardiac hypertrophy
  • Increased ANF mRNA
  • Increased risk of heart disease

ANF= cardiac atrial natriuretic factor; an indicator of cardiac stress. Source: (Kopf and Walker 2009)


References


1. Carro, T., Dean, K., and Ottinger, M. A. (2013a). Effects of an environmentally relevant polychlorinated biphenyl (PCB) mixture on embryonic survival and cardiac development in the domestic chicken. Environ. Toxicol. Chem. 23(6), 1325-1331.

2. Carro, T., Taneyhill, L. A., and Ottinger, M. A. (2013b). The effects of an environmentally relevant 58 congener polychlorinated biphenyl (PCB) mixture on cardiac development in the chick embryo. Environ. Toxicol. Chem.

3. DeWitt, J. C., Millsap, D. S., Yeager, R. L., Heise, S. S., Sparks, D. W., and Henshel, D. S. (2006). External heart deformities in passerine birds exposed to environmental mixtures of polychlorinated biphenyls during development. Environ. Toxicol. Chem. 25(2), 541-551.

4. Heid, S. E., Walker, M. K., and Swanson, H. I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci 61(1), 187-196.

5. Walker, M. K., and Catron, T. F. (2000). Characterization of cardiotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related chemicals during early chick embryo development. Toxicol. Appl. Pharmacol. 167(3), 210-221.

6. Walker, M. K., Pollenz, R. S., and Smith, S. M. (1997). Expression of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator during chick cardiogenesis is consistent with 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced heart defects. Toxicol. Appl. Pharmacol. 143(2), 407-419.

7. Kopf, P. G., and Walker, M. K. (2009). Overview of developmental heart defects by dioxins, PCBs, and pesticides. J. Environ. Sci. Health C. Environ. Carcinog. Ecotoxicol. Rev. 27(4), 276-285.

Bishopric, N.H. (2005). Evolution of the heart from bacteria to man. Ann. N. Y. Acad. Sci. 1047, 13-29.

 

Buckler J.; Candrl, J.S.; McKee, M.J.; Papoulias, D.M.; Tillitt, D.E.; Galat, D.L. Sensitivity of shovelnose sturgeon (Scaphirhynchus platorynchus) and pallid sturgeon (S. albus) early life stages to PCB-126 and 2,3,7,8-TCDD exposure. Enviro. Toxicol. Chem. 2015, 34(6), 1417-1424.

 

Carney, S.A.; Prasch, A.L.; Heideman, W.; Peterson, R.E. 2006. Understanding dioxin developmental toxicity using the zebrafish model. Birth Defects Research. A. 76, 7-18.

 

Cohen-Barnhouse, A.M.; Zwiernik, M.J.; Link, J.E.; Fitzgerald, S.D.; Kennedy, S.W.; Herve, J.C.; Giesy, J.P.; Wiseman, S.; Yang, Y.; Jones, P.D.; Yi, W.; Collins, B.; Newsted, J.L.; Kay, D.; Bursian, S.J. 2011. Sensitivity of Japanese quail (Coturnix japonica), common pheasant (Phasianus colchicus), and white leghorn chicken (Gallus gallus domesticus) embryos to in ovo exposure to TCDD, PeCDF, and TCDF. Toxicol. Sci. 119, 93-102.

 

Elonen, G.E.; Spehar, R.L.; Holcombe, G.W.; Johnson, R.D.; Fernandez, J.D.; Erickson, R.J.; Tietge, J.E.; Cook, P.M. Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to seven freshwater fish species during early life-stage development. Enviro. Toxico. Chem. 1998, 17, 472-483.

 

Goldstone, H.M.H.; Stegeman, J.J. (2008). Molecular mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin cardiovascular embryotoxicity. Drug. Metab. Rev. 38, 261-289.

 

Heid, S.E.; Walker, M.K.; Swanson, H.I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci. 61 (1), 187-196.

 

Huang, L.; Wang, C.; Zhang, Y.; Li, J.; Zhong, Y.; Zhou, Y.; Chen, Y.; Zuo, Z. (2012). Benzo[a]pyrene exposure influences the cardiac development and the expression of cardiovascular relative genes in zebrafish (Daniorerio) embryos. Chemosphere. 87 (4), 369-375.

 

Johnson, R.D.; Tietge, J.E.; Jensen, K.M.; Fernandez, J.D.; Linnum, A.L.; Lothenbach, D.B.; Holcombe, G.W.; Cook, P.M.; Christ, S.A.; Lattier, D.L.; Gordon, D.A. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to early life stage brooke trout (Salvelinus fontinalis) following parental dietary exposure. Enviro. Toxicol. Chem. 1998, 17 (12), 2408-2421.

 

Kardong, K.V. (2006). Vertebrates: comparative anatomy, function, evolution. McGraw-Hill Higher Eduction. Boston, USA.

 

Lemly, A.D. (2002). Symptoms and implications of selenium toxicity in fish: the Belews Lake case example. Aquat. Toxicol. 57 (1-2), 39-49.

 

Park, Y.J.; Lee, M.J.; Kim, H.R.; Chung, K.H.; Oh, S.M. Developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in artificially fertilized crucian carp (Carassius auratus) embryo. Sci. Totl. Enviro. 2014, 491-492, 271-278.

 

Tillitt, D.E.; Buckler, J.A.; Nicks, D.K.; Candrl, J.S.; Claunch, R.A.; Gale, R.W.; Puglis, H.J.; Little, E.E.; Linbo, T.L.; Baker, M. Sensitivity of lake sturgeon (Acipenser fulvescens) early life stages to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,3’,4,4’,5-pentachlorobiphenyl. 2015. Enviro. Toxicol. Chem. DOI: 10.1002/etc.3614.

 

Toomey, B.H.; Bello, S.; Hahn, M.E.; Cantrell, S.; Wright, P.; Tillitt, D.; Di Giulio, R.T. TCDD induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos. Aquat. Toxicol. 2001, 53, 127-138.

 

Walker, M.K.; Spitsbergen, J.M.; Olson, J.R.; Peterson, R.E. 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) toxicity during early life stage development of lake trout (Salvelinus namaycush). Canad. J. Fisheries Aqua. Sci. 1991, 48, 875-883.

 

Yamauchi, M.; Kim, E.Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryos: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. Aquat. Toxicol. 2006, 16, 166-179.

 

Zabel, E.W; Cook, P.M.; Peterson, R.E. Toxic equivalency factors of polychlorinated dibenzo-p-dioxin, dibenzofuran and biphenyl congeners based on early-life stage mortality in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol. 1995. 31, 315-328.


Adverse Outcomes

Title Short name
Increase, Mortality Increase, Mortality

350: Increase, Mortality

Short Name: Increase, Mortality

AOPs Including This Key Event


Biological Organization

Level of Biological Organization
Individual

Scientific evidence supporting the linkages in the AOP

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Activation, AHR directly leads to dimerization, AHR/ARNT Strong Moderate
dimerization, AHR/ARNT directly leads to Increase, COX-2 expression Strong Moderate
Increase, COX-2 expression directly leads to Altered, Cardiovascular development/function Moderate Moderate
Altered, Cardiovascular development/function directly leads to Increase, Mortality Moderate Strong

Graphical Representation

Overall Assessment of the AOP

Consider the following criteria (may include references to KE Relationship pages): 1. concordance of dose-response relationships; 2. temporal concordance among the key events and adverse effect; 3. strength, consistency, and specificity of association of adverse effect and initiating event; 4. biological plausibility, coherence, and consistency of the experimental evidence; 5. alternative mechanisms that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP; 6. uncertainties, inconsistencies and data gaps.


Domain of Applicability

Life Stage Applicability
Life Stage Evidence
Embryo Strong
development Strong
Taxonomic Applicability
Term Scientific Term Evidence Links
Atlantic killifish Fundulus heteroclitus Moderate NCBI
zebrafish Danio rerio Strong NCBI
rainbow trout Oncorhynchus mykiss Moderate NCBI
medaka Oryzias latipes Strong NCBI
fathead minnow Pimephales promelas Moderate NCBI
channel catfish Ictalurus punctatus Moderate NCBI
Salvelinus namaycush Salvelinus namaycush Moderate NCBI
Salvelinus fontinalis Salvelinus fontinalis Moderate NCBI
Carassius carassius Carassius carassius Moderate NCBI
Pagrus major Pagrus major Moderate NCBI
Acipenser fulvescens Acipenser fulvescens Moderate NCBI
Coregonus artedi Coregonus artedi Moderate NCBI
Catostomus commersonii Catostomus commersonii Moderate NCBI
Esox lucius Esox lucius Moderate NCBI
Scaphirhynchus albus Scaphirhynchus albus Moderate NCBI
Scaphirhynchus platorynchus Scaphirhynchus platorynchus Moderate NCBI
Xenopus laevis laevis Xenopus laevis laevis Weak NCBI
Gallus gallus Gallus gallus Strong NCBI
Sex Applicability
Sex Evidence
Unspecific Strong

Sex: This AOP is only applicable to early life stages prior to sexual differentiation.

Life stages: This AOP is only applicable starting from embryonic development. In zebrafish, this critical window extends from fertilization to approximately 24 hours post fertilization (hpf) (Belair et al 2001; Goldstone & Stegeman 2008).

Taxonomic: The specific characteristics of altered cardiovascular development and function vary to some degree among taxonomic groups of vertebrates. However, reduction in myocyte proliferation and heart pumping efficiency is common to all investigated vertebrates (Antkiewicz et al 2005; Jones & Kennedy 2009; Thackaberry et al 2005).

This AOP is applicable to:

  • All teleost and non-teleost fishes that have been investigated as embryos so far (Buckler et al 2015; Doering et al 2013; Elonen et al 1998; Johnson et al 1998; Park et al 2014; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Yamauchi et al 2006; Zabel et al 1995).
  • All birds (Canga et al 1993; Cohen-Barnhouse et al 2011; Fujisawa et al 2014; Heid et al 2001; Ivnitski et al 2001; Walker & Catron 2000). However, some details of the AOP might be different in birds as cyclooxygenase-2 (COX-2) is believed to be up-regulated through non-genomic mechanisms in these taxa based on investigations in chicken (Gallus gallus) (Fujisawa et al 2014).
  • Amphibians and reptiles have insufficient mechanistic and early life stage mortality information to demonstrate applicability at this time. However, amphibians and reptiles express AhRs that are activated by agonists in a manner consistent with other vertebrates and express AhRs during embryonic development (Lavine et al 2005; Shoots et al 2015; Ohi et al 2003; Oka et al 2016). However, altered cardiovascular development and function and early life stage mortality have not been observed at any investigated concentration of DLC in amphibians studied to date (Jung et al 1997). This tolerance is believed to result from AhRs of amphibians having very low affinity for agonists (Lavine et al 2005; Shoots et al 2015). Therefore, it is acknowledged that this AOP is likely to be applicable to reptiles. However, it might not be applicable to amphibians due to their extreme tolerance to activation of the AhR.
  • Cartilaginous fishes (Chondrichthyes) have insufficient mechanistic and early life stage mortality information to demonstrate applicability at this time. However, sharks and rays are known to express AhRs that are structurally comparable to AhRs of teleost fishes (Hahn 2002). Sharks and rays have also been shown to respond to exposure to agonists of the AhR through responses that are comparable to teleost fishes, specifically through induction of CYP1A (Hahn et al 1998). Therefore, it is acknowledged that this AOP is likely to be applicable to Chondrichthyes.

This AOP is not applicable to:

  • Mammals because the cause of mortality of the young is primarily a result of wasting syndrome and not necessarily altered cardiovascular development and function (Kopf & Walker 2009). Further, studies of CYP1A1 and CYP1A2 null mice (Mus musculus) demonstrate that wasting syndrome and mortality are mediated by CYP1A1 in mammals (Uno et al 2004).
  • Invertebrates because AhRs of invertebrates have less diverse functionalities relative to vertebrates, AhRs of invertebrates are not known to bind agonists that represent anthropogenic pollutants, and no critical adverse effects are known in invertebrates as a result of activation of the AhR (Hahn 2002; Hahn et al 1994).
  • Jawless fishes, such as lamprey (Petromyzontiformes) and hagfish (Myxiniformes), because of a lack of measurable AhR-mediated responses (Hahn et al 1998). Although additional information is necessary for this taxa, it is currently acknowledged that this AOP is likely not applicable to jawless fishes.

Essentiality of the Key Events

Support for essentiality for key events in the AOP was provided by a series of knockdown and targeted agonist and antagonist experiments. These investigations were conducted mainly with zebrafish as the model species and TCDD as the model agonist of the AhR.

 Rationale for essentiality calls:

  • AhR, activation: [Strong] Knockdown of AhR prevents TCDD induced alteration in cardiovascular development and function (Clark et al 2010; Hanno et al 2010; Karchner et al 1999; Prasch et al 2003; Van Tiem & Di Giulio 2011).

 

  • AhR/ARNT, dimerization: [Strong] Knockdown of ARNT prevents TCDD induced alteration in cardiovascular development and function (Antkiewicz et al 2006; Prasch et al 2004). Depletion of ARNT lessens or prevents TCDD induced alteration in cardiovascular development and function (Prasch et al 2004).

 

  • COX-2, increase: [Strong] Knockdown of COX-2 and selective antagonists of COX-2 prevent TCDD induced alteration in cardiovascular development and function (Dong et al 2010; Teraoka et al 2008; 2014). COX-2 inducers that are not agonists of the AhR cause altered cardiovascular development and function that is consistent with activation of the AhR (Huang et al 2007). Knockdown of and selective antagonists of thromboxane A synthase 1 (CYP5A), which is down-stream of COX-2 in the prostaglandin synthesis pathway, prevents TCDD induced alteration in cardiovascular development and function (Teraoka et al 2008). Exposure to the substrate for COX-2, arachidonic acid, causes an up-regulation in COX-2 and altered cardiovascular development and function that is consistent with exposure to TCDD (Dong et al 2010).

 

  • Cardiovascular development and function, altered: [Strong] Isosmotic rearing solution prevents yolk sac edema, but has no effect on TCDD induced alteration in cardiovascular development and function or mortality (Hill et al 2004). This indicates that mortality is not caused by yolk sac edema. Knockdown of cytochrome P450 1A (CYP1A) or injection with antioxidants decreases oxidative stress but has no effect on TCDD induced alteration in cardiovascular development and function or mortality (Carney et al 2004; Scott et al 2011). This is suggestive that mortality is not caused by oxidative stress. Exposure to agonists of the AhR post-heart development lessens or prevents alteration in cardiovascular development, decreased blood flow, and cardiac failure (Carney et al 2004; Lanham et al 2012). Exposure to agonists of the AhR post-heart development dramatically reduces mortality (Carney et al 2004; Lanham et al 2012). This suggests that mortality is caused by circulatory failure as a result of cardiovascular teratogenenesis.

Weight of Evidence Summary

Biological Plausibility:

  • In general, the biological plausibility and coherence linking activation of the AhR through early life stage mortality from COX-2 induced alteration in cardiovascular development and function is very strong.
  • The AhR is known to have critical roles in development of the heart and therefore dysregulation of these roles would be expected to result in altered cardiac development.
  • The prostaglandin synthesis pathway, of which COX-2 is a rate limiting step, is known to have roles in development of the heart (Dong et al 2010; Huang et al 2007; Teraoka et al 2008; 2014).
  • A properly functioning circulatory system is widely acknowledged to be crucial for survival of vertebrates (Kardong 2006). General dysfunction of the heart or associated vasculature is widely documented to have the potential to result in mortality through cardiac failure, regardless of the mechanism of dysfunction.

 

Concordance of dose-response relationships:

  • There is significant evidence showing concordance of dose-response for incidence and severity of alteration in cardiovascular development and function and subsequently mortality across at least 16 different species of fish (Buckler et al 2015; Elonen et al 1998; Huang et al 2012; Johnson et al 1998; Park et al 2014; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Yamauchi et al 2006; Zabel et al 1995) and 8 different species of birds (Cohen-Barnhouse et al 2011; Brunstrom 1990; Brunstrom & Andersson 1988; Hoffman et al 1996; 1998; Powell et al 1998) for several PCDDs, PCDFs, planar PCBs, and PAHs. Concordance of dose-response has not been observed in amphibians studied to date because no elevated mortality or altered cardiovascular development and function was observed at any tested concentration of agonist (Jung et al 1997).
  • Less is known regarding concordance of dose-response relationships for COX-2. In Japanese medaka (Oryzias latipes), abundance of transcript of COX-2 is significantly greater than controls at concentrations of TCDD of 0.2 ppb and greater (Dong et al 2010). Likewise, incidence of cardiovascular development and heart area were both significantly different than controls at concentrations of TCDD of 0.2 ppb and greater (Dong et al 2010).

 

Temporal concordance among the key events and adverse effect:

  • Alterations in cardiovascular development or function is first observable in zebrafish around 48 hours post fertilization (hpf), while mortality does not begin to occur until around 86 hpf (Goldstone & Stegeman 2006).
  • AhR transcript and protein is first detectable in zebrafish around 24 hpf (Tanguay et al 1999).
  • COX-2 transcript is first detectable in zebrafish around 6 hpf (Teraoka et al 2008). Expression of COX-2 was investigated in zebrafish exposed to TCDD at 55 and 72 hpf (Teraoka et al 2014). At 55 hpf there was a trend towards up-regulation of COX-2 (~ 1.5-fold), while at 72 hpf there was a significant up-regulation of COX-2 (~ 4.5-fold) (Teraoka et al 2014).
  • Therefore, there is a general temporal concordance in this AOP.
  • However, there is some uncertainty in the early manifestations of altered cardiovascular development and up-regulation of COX-2. It is possible that the first manifestations of altered cardiovascular development result from mechanisms other than COX-2. For example, sex determining region Y-box-9b (Sox9b) is first expressed in zebrafish at around 24 hpf and has been known to cause some altered cardiovascular phenotypes (Hofsteen et al 2013; Li et al 2002). However, no studies have yet investigated temporal concordance of regulation of Sox9b by the AhR prior to 72 hpf (Hofsteen et al 2013). It is also possible that temporal concordance of early increases in COX-2 is obscured by the relatively little fold-changes observed for COX-2. Additional investigations into up-regulation of COX-2 by activation of the AhR across developmental stages is warranted.

 

Consistency:

  • There are no known AhR-mediated effects that occur at concentrations below those that cause alteration in cardiovascular development and function that result in early life stage mortality in fishes, amphibians, reptiles, or birds.
  • There are no studies in which COX-2 and altered cardiovascular development or function were co-investigated in which altered cardiovascular development or function occurred without an up-regulation in COX-2.
  • In TCDD exposure groups, some individuals do not manifest alterations in cardiovascular development or function (Dong et al 2010). TCDD exposed individuals of Japanese medaka that did not manifest alterations in cardiovascular development or function had expression of COX-2 that was not statistically different than controls, while individuals that did manifest alterations in cardiovascular development or function had increased expression of COX-2 (Dong et al 2010).
  • There is also consistency in the TCDD-induced alterations in cardiovascular phenotype between distantly related oviparous taxa, namely fish and birds (Teraoka et al 2008; Fujisawa et al 2014). Likewise, COX-2 is known to be up-regulated in both these taxa (Teraoka et al 2008; Fujisawa et al 2014). Cardiovascular development and function and COX-2 have not been investigated in amphibians or reptiles.

 

Uncertainties, inconsistencies, and data gaps:

  • There are several other pathways by which activation of the AhR could result in altered cardiovascular development and function in developing embryos. These include, but are not limited to, down-regulation in Sox9b, BMP-4, and genes in the cell cycle gene cluster (Hofsteen et al 2013; Jonsson et al 2007), oxidative stress (Goldstone & Stegeman 2008), and AhR cross-talk with hypoxia inducible factor 1α (HIF1α) (Goldstone & Stegeman 2008).
  • However, investigations of knockdown and null strains for Sox9b in zebrafish do not result in the complete phenotype of altered cardiovascular development recorded in embryos following exposure to planar aromatic hydrocarbons (Hofsteen et al 2013). Injection of TCDD exposed embryos with Sox9b mRNA was able to prevent the Sox9b phenotype of cardiovascular development, however it did not prevent altered cardiovascular development altogether (Hofsteen et al 2013). Further, altered cardiovascular development as a result of down-regulation in Sox9b is not severe enough to cause complete cardiac failure and early life stage mortality in zebrafish (Hofsteen et al 2013).
  • Oxidative stress as a result of induction in CYP1A has commonly been proposed as the mechanism of altered cardiovascular development and function and CYP1A follows dose- and temporal concordance with mortality across numerous investigations in fishes and birds (Goldstone & Stegeman 2008). Early studies of CYP1A knockdown in zebrafish demonstrated protection against alteration in cardiovascular development and function induced by exposure to TCDD (Teraoka et al 2003). However, more recent investigations have observed no protection (Carney et al 2006). This inconsistency has been proposed to result from the earlier studies only recording alteration in cardiovascular development and function at early stages when adverse effects are difficult to accurately observe (Carney et al 2006). In birds, COX-2 inhibitors have no effect on expression of CYP1A but protect against TCDD induced alteration in cardiovascular development and function suggesting that CYP1A is not involved in toxicities (Fujisawa et al 2014).
  • For cross-species and cross-taxa extrapolation, there is uncertainty in whether COX-2 is up-regulated by AhR through genomic or non-genomic mechanisms. Specifically, there is no detailed analysis regarding how widespread COX-2 genes which contain DREs in the promoter region are among species and among taxa and whether non-genomic or genomic mechanisms of up-regulation in COX-2 are more ubiquitous.
  • All mechanistic investigations into mechanisms of AhR-mediated alteration in cardiovascular development and function have been conducted in zebrafish, Japanese medaka, and chicken. Therefore, no mechanistic information is available to conclude cross-species extrapolation outside of a shared phenotype of altered cardiovascular development and function. There is no information about AhR-mediated alteration in cardiovascular development or function or up-regulation of COX-2 in early fishes (Petromyzontiformes; Myxiniformes; Chondrichthyes), amphibians, or reptiles.

Quantitative Consideration

  • There is a strong quantitative understanding between quantitative structure-activity relationship (QSAR) or binding affinity for the AhR and potency among PCDDs, PCDFs, and planar PCBs (Van den Berg et al 1998; 2006). Specifically, these studies have demonstrated that congeners with greater binding affinity have greater potency (Van den Berg et al 1998; 2006). This has partially contributed to the successful development of the toxic equivalency factor (TEF) methodology in risk assessment (Van den Berg et al 1998; 2006).
  • There is also a strong quantitative understanding of differences in binding affinity of the AhR among species of birds and differences in sensitivity to early life stage mortality (Karchner et al 2006; Farmahin et al 2012; 2013; Manning et al 2013). Specifically, these studies demonstrate that species of birds with AhRs with greater affinity for DLCs have greater sensitivity than species with AhRs with lesser affinity for DLCs (Karchner et al 2006). These differences in sensitivity range by more than 40-fold for TCDD (Cohen-Barnhouse et al 2011). However, a quantitative understanding between differences in binding affinity of the AhR among species and differences in sensitivity to early life stage mortality is not yet available for other taxa (Doering et al 2013).
  • There is some quantitative understanding between up-regulation of COX-2 and incidence of and severity of cardiac deformities for medakafish exposed to TCDD (Dong et al 2010). This quantitative understanding includes a strong linear relationship (R2 = 0.88) between abundance of COX-2 transcript and heart area (Dong et al 2010). However, this information is not available with regards to multiple investigations, species, taxonomic groups, or chemicals.
  • There is strong quantitative understanding between incidence of and severity of cardiovascular deformities and mortality. However, numerous different cardiovascular endpoints are investigated among studies making side-by-side comparisons difficult.

References


Abnet, C.C.; Tanguay, R.L.; Heideman, W.; Peterson, R.E. 1999. Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: Greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners. Toxicol. Appl. Pharmacol. 159, 41-51.

Antkiewicz, D.S.; Burns, C.G.; Carney, S.A.; Peterson, R.E.; Heideman, W. 2005. Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. 84, 368-377.

Bak, S.M.; Lida, M.; Hirano, M.; Iwata, H.; Kim, E.Y. 2013. Potencies of red seabream AHR1- and AHR2-mediated transactivation by dioxins: implications of both AHRs in dioxin toxicity. Environ. Sci. Technol. 47 (6), 2877-2885.

Belair, C.D.; Peterson, R.E.; Heideman, W. (2001). Disruption of erythropoiesis by dioxin in the zebrafish. Dev. Dyn. 222 (4), 581-594.

Billiard, S.M.; Hahn, M.E.; Franks, D.G.; Peterson, R.E.; Bols, N.C.; Hodson, P.V. (2002). Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs). Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 133 (1), 55-68.

Brunstrom, B. (1990). Mono-ortho-chlorinated chlorobiphenyls: toxicity and induction of 7-ethoxyresorufin O-deethylase (EROD) activity in chick embryos. Arch. Toxicol. 64, 188-192.

Brunstrom, B.; Andersson, L. (1988). Toxicity and 7-ethoxyresorufin O-deethylase-inducing potency of coplanar polychlorinated biphenyls (PCBs) in chick embryos. Arch. Toxicol. 62, 263-266.

Buckler J.; Candrl, J.S.; McKee, M.J.; Papoulias, D.M.; Tillitt, D.E.; Galat, D.L. Sensitivity of shovelnose sturgeon (Scaphirhynchus platorynchus) and pallid sturgeon (S. albus) early life stages to PCB-126 and 2,3,7,8-TCDD exposure. Enviro. Toxicol. Chem. 2015, 34(6), 1417-1424.

Canga, L., Paroli, L., Blanck, T. J., Silver, R. B., and Rifkind, A. B. (1993). 2,3,7,8-Tetrachlorodibenzo-p-dioxin increases cardiac myocyte intracellular calcium and progressively impairs ventricular contractile responses to isoproterenol and to calcium in chick embryo hearts. Mol. Pharmacol.  44, 1142–1151.

Carney, S.A.; Peterson, R.E.; Heideman, W. 2004. 2,3,7,8-tetrachlorodibenzo-p-dioxin activation of aryl hydrocarbon receptors/aryl hydrocarbon receptor nuclear translocator pathway causes developmental toxicity through a CYP1A-independent mechanism in zebrafish. Mol. Pharmacol. 66 (2), 512-521.

Carney, S.A.; Prasch, A.L.; Heideman, W.; Peterson, R.E. 2006. Understanding dioxin developmental toxicity using the zebrafish model. Birth Defects Research. A. 76, 7-18.

Chen, G.; Bunce, N.J. (2003). Polybrominated diphenyl ethers as Ah receptor agonists and antagonists. Toxicol. Sci. 76 (2), 310-320.

Clark, B.W.; Matson, C.W.; Jung, D.; Di Giulio, R.T. 2010. AHR2 mediates cardiac teratogenesis of polycyclic aromatic hydrocarbons and PCB-126 in Atlantic killifish (Fundulus heteroclitus). Aquat. Toxicol. 99, 232-240.

Cohen-Barnhouse, A.M.; Zwiernik, M.J.; Link, J.E.; Fitzgerald, S.D.; Kennedy, S.W.; Herve, J.C.; Giesy, J.P.; Wiseman, S.; Yang, Y.; Jones, P.D.; Yi, W.; Collins, B.; Newsted, J.L.; Kay, D.; Bursian, S.J. 2011. Sensitivity of Japanese quail (Coturnix japonica), common pheasant (Phasianus colchicus), and white leghorn chicken (Gallus gallus domesticus) embryos to in ovo exposure to TCDD, PeCDF, and TCDF. Toxicol. Sci. 119, 93-102.

Cook, P.M.; Robbins, J.A.; Endicott, D.D.; Lodge, K.B.; Guiney, P.D.; Walker, M.K.; Zabel, E.W.; Peterson, R.E. 2003. Effects of aryl hydrocarbon receptor-mediated early life stage toxicity on lake trout populations in Lake Ontario during the 20th century. Enviro. Sci. Technol. 37 (17), 3864-3877.

Degner, S.C.; Kemp, M.Q.; Hockings, J.K.; Romagnolo, D.F. (2007). Cyclooxygenase-2 promoter activation by the aromatic hydrocarbon receptor in breast cancer MCF-7 cells: Repressive effects of conjugated linoleic acid. Nutri. Canc. 56 (2), 248-257.

Denison, M.S.; Heath-Pagliuso, S. The Ah receptor: a regulator of the biochemical and toxicological actions of structurally diverse chemicals. Bull. Environ. Contam. Toxicol. 1998, 61 (5), 557-568.

Doering, J.A.; Giesy, J.P.; Wiseman, S.; Hecker, M. Predicting the sensitivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain. Environ. Sci. Pollut. Res. Int. 2013, 20(3), 1219-1224.

Doering, J.A.; Wiseman, S; Beitel, S.C.; Giesy, J.P.; Hecker, M. 2014. Identification and expression of aryl hydrocarbon receptors (AhR1 and AhR2) provide insight in an evolutionary context regarding sensitivity of white sturgeon (Acipenser transmontanus) to dioxin-like compounds. Aquat. Toxicol. 150, 27-35.

Dong, W.; Matsumura, F.; Kullman, S.W. (2010). TCDD induced pericardial edema and relative COX-2 expression in medaka (Oryzias latipes) embryos. Toxicol. Sci. 118 (1), 213-223.

Duncan, D.M.; Burgess, E.A.; Duncan, I. 1998. Control of distal antennal identity and tarsal development in Drosophila by spineless-aristapedia, a homolog of the mammalian dioxin receptor. Genes Dev. 12, 1290-1303.

Elonen, G.E.; Spehar, R.L.; Holcombe, G.W.; Johnson, R.D.; Fernandez, J.D.; Erickson, R.J.; Tietge, J.E.; Cook, P.M. Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to seven freshwater fish species during early life-stage development. Enviro. Toxico. Chem. 1998, 17, 472-483.

Emmons, R.B.; Duncan, D.; Estes, P.A.; Kiefel, P.; Mosher, J.T.; Sonnenfeld, M.; Ward, M.P.; Duncan, I.; Crews, S.T. 1999. The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila. Development. 126, 3937-3945.

Farmahin, R.; Crump, D.; O’Brien, J.M.; Jones, S.P.; Kennedy, S.W. (2016). Time-dependent transcriptomic and biochemical responses of 6-formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are explained by AHR activation time. Biochem. Pharmacol. 115 (1), 134-143.

Farmahin, R.; Manning, G.E.; Crump, D.; Wu, D.; Mundy, L.J.; Jones, S.P.; Hahn, M.E.; Karchner, S.I.; Giesy, J.P.; Bursian, S.J.; Zwiernik, M.J.; Fredricks, T.B.; Kennedy, S.W. 2013. Amino acid sequence of the ligand-binding domain of the aryl hydrocarbon receptor 1 predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol. Sci. 131 (1), 139-152.

Farmahin, R.; Wu, D.; Crump, D.; Herve, J.C.; Jones, S.P.; Hahn, M.E.; Karchner, S.I.; Giesy, J.P.; Bursian, S.J.; Zwiernik, M.J.; Kennedy, S.W. 2012. Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Enviro. Sci. Toxicol. 46 (5), 2967-2975.

Fujisaw, N.; Nakayama, S.M.M.; Ikenaka, Y.; Ishizuka, M. 2014. TCDD-induced chick cardiotoxicity is abolished by a selective cyclooxygenase-2 (COX-2) inhibitor NS398. Arch. Toxicol. 88, 1739-1748.

Giesy, J.P.; Jones, P.D.; Kannan, K.; Newstead, J.L.; Tillitt, D.E.; Williams, L.L. Effects of chronic dietary exposure to environmentally relevant concentrations to 2,3,7,8-tetrachlorodibenzo-p-dioxin on survival, growth, reproduction and biochemical responses of female rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 2002, 59 (1-2), 35-53.

Goldstone, H.M.; Stegeman, J.J. 2008. Molecular mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin cardiovascular embryotoxicity. Drug. Metab. Rev. 38 (1), 261-289.

Hahn, M.E. 2002. Aryl hydrocarbon receptors: diversity and evolution. Chemico-Biol. Interact. 141, 131-160.

Hahn, M.E.; Karchner, S.I.; Evans, B.R.; Franks, D.G.; Merson, R.R.; Lapseritis, J.M. 2006. Unexpected diversity of aryl hydrocarbon receptors in non-mammalian vertebrates: Insights from comparative genomics. J. Exp. Zool. A. Comp. Exp. Biol. 305, 693-706.

Hahn, M.E.; Poland, A.; Glover, E.; Stegeman, J.J. 1994. Photoaffinity labeling of the Ah receptor: phylogenetic survey of diverse vertebrate and invertebrate species. Arch. Biochem. Biophys. 310, 218-228.

Hahn, M.E.; Woodlin, B.R.; Stegeman, J.J.; Tillitt, D.E. 1998. Aryl hydrocarbon receptor function in early vertebrates: Inducibility of cytochrome P450 1A in agnathan and elasmobranch fish. Comp. Biochem. Physiol. C. 120, 67-75.

Hansson, M.C.; Hahn, M.E. 2008. Functional properties of the four Atlantic salmon (Salmo salar) aryl hydrocarbon receptor type 2 (AHR2) isoforms. Aquat. Toxicol. 86, 121-130.

Hansson, M.C.; Wittzell, H.; Persson, K.; von Schantz, T. 2004. Unprecedented genomic diversity of AhR1 and AhR2 genes in Atlantic salmon (Salmo salar L.). Aquat. Toxicol. 68 (3), 219-232.

Heid, S. E., Walker, M. K., and Swanson, H. I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci.  61, 187–196.

Hengstler, J.G.; Van der Burg, B.; Steinberg, P.; Oesch, F. Interspecies differences in cancer susceptibility and toxicity. Drug. Metab. Rev. 1999, 31, 917-970.

Hill, A.J.; Bello, S.M.; Prasch, A.L.; Peterson, R.E.; Heideman, W. (2004). Water permeability and TCDD-induced edema in zebrafish early-life stages. Toxicol. Sci. 100, 486-494.

Hoffman, D.J., Rice, C.P., Kubiak, T.J., 1996. PCBs and dioxins in birds. In: Beyer, W.N.,

Heinz, G.H., Redmon-Norwood, A.W. (Eds.), Environmental Contaminants in Wildlife:

Interpreting Tissue Concentrations. CRC Press, pp. 165–207.

Hoffman, D.J., Melancon, M.J., Klein, P.N., Eisemann, J.D., Spann, J.W., 1998. Comparative

developmental toxicity of planar polychlorinated biphenyl congeners in chickens,

American kestrels, and common terns. Environ. Toxicol. Chem. 17, 747–757.

Hofsteen, P.; Plavicki, J.; Johnson, S.D.; Peterson, R.E.; Heideman, W. Sox9b is required for epicardium formation and plays a role in TCDD-induced heart malformation in zebrafish. Molec. Pharmacol. 2013, 84, 353-360.

Huang, C.C.; Chen, P.C.; Huang, C.W.; Yu, J. (2007). Aristolochic acid induces heart failure in zebrafish embryos that is mediated by inflammation. Toxicol. Sci. 100, 486-494.

Huang, L.; Wang, C.; Zhang, Y.; Li, J.; Zhong, Y.; Zhou, Y.; Chen, Y.; Zuo, Z. (2012). Benzo[a]pyrene exposure influences the cardiac development and the expression of cardiovascular relative genes in zebrafish (Daniorerio) embryos. Chemosphere. 87 (4), 369-375.

Ivnitski, I., Elmaoued, R., and Walker, M. K. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary development is preceded by a decrease in myocyte proliferation and an increase in cardiac apoptosis. Teratology  64, 201–212.

Johnson, R.D.; Tietge, J.E.; Jensen, K.M.; Fernandez, J.D.; Linnum, A.L.; Lothenbach, D.B.; Holcombe, G.W.; Cook, P.M.; Christ, S.A.; Lattier, D.L.; Gordon, D.A. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to early life stage brooke trout (Salvelinus fontinalis) following parental dietary exposure. Enviro. Toxicol. Chem. 1998, 17 (12), 2408-2421.

Jonsson, M.E.; Jenny, M.J.; Woodin, B.R.; Hahn, M.E.; Stegeman, J.J. (2007). Role of AHR2 in the expression of novel cytochrome P450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3’,4,4’,5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Sci. 100 (1), 180-193.

Jonsson, M.E.; Kubota, A.; Timme-Laragy, A.R.; Woodin, B.; Stegeman, J.J. (2012). Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish. Toxicol. Appl. Pharmacol. 265 (2), 166-174.

Jung, R.E.; Walker, M.K. (1997). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on development of anuran amphibians. Enviro. Toxicol. Chem. 16 (2), 230-240.

Karchner, S.I.; Franks, D.G.; Kennedy, S.W.; Hahn, M.E. 2006. The molecular basis for differential dioxin sensitivity in birds: Role of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. USA. 103, 6252-6257.

Karchner, S.I.; Powell, W.H.; Hahn, M.E. 1999. Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the Teleost Fundulus heteroclitus. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors. J. Biol. Chem. 274, 33814-33824.

Kardong, K.V. (2006). Vertebrates: comparative anatomy, function, evolution. McGraw-Hill Higher Eduction. Boston, USA.

Kleeman, J.M.; Olson, J.R.; Peterson, R.E. Species differences in 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity and biotransformation in fish. Fundam. Appl. Toxicol. 1988, 10(2), 206-213.

Kopf, P.G.; Walker, M.K. (2009). Overview of developmental heart defects by dioxins, PCBs, and pesticides. J. Environ. Sci. Health C. Environ. Carcinog. Ecotoxicol. Rev. 27 94), 276-285.

Korkalainen, M.; Tuomisto, J.; Pohjanvirta, R. The AH receptor of the most dioxin-sensitive specie, guinea pig, is highly homologous to the human AH receptor. Biochem. Biophys. Res. Commun. 2001, 285, 1121-1129.

Lahvis, G.P.; Bradfield, C.A. 1998. Ahr null alleles: distinctive or different? Biochem. Pharmacol. 56, 781-787.

Lavine, J.A.; Rowatt, A.J.; Klimova, T.; Whitington, A.J.; Dengler, E.; Beck, C.; Powell, W.H. 2005. Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Sci. 88 (1), 60-72.

Lanham, K.A.; Peterson, R.E.; Heideman, W. (2012). Sensitivity to dioxin decreases as zebrafish mature. Toxicol. Sci. 127 (2), 360-370.

Li, M.; Zhao, C.; Wang, Y.; Zhao, Z.; Meng, A. (2002). Zebrafish sox9b is an early neural crest marker. Dev. Genes Evol. 212, 203-206.

Manning G.E.; Farmahin, R.; Crump, D.; Jones, S.P.; Klein, J.; Konstantinov, A.; Potter, D.; Kennedy, S.W. 2012. A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD50 of polychlorinated biphenyls in avian species. Toxicol. Appl. Pharm. 263, 390-401.

Murk, A.J.; Legler, J.; Denison, M.S.; Giesy, J.P.; Van De Guchte, C.; Brouwer, A. (1996). Chemical-activated luciferase gene expression (CALUX): A novel in vitro bioassay for Ah receptor active compounds in sediments and pore water. Toxicol. Sci. 33 (1), 149-160.

Nacci, D.E. Champlin, D.; Jayaraman, S. (2010). Adaptation of the estuarine fish Fundulus heteroclitus (Atlantic killifish) to polychlorinated biphenyls (PCBs). Estuaries and Coasts. 33 (4), 853-864.

Ohi, H.; Fujita, Y.; Miyao, M.; Saguchi, K.; Murayama, N.; Higuchi, S. 2003. Molecular cloning and expression analysis of the aryl hydrocarbon receptor of Xenopus laevis. Biochem. Biophysic. Res. Comm. 307 (3), 595-599.

Okey, A.B. An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XI. Toxicol. Sci. 2007, 98, 5-38.

Park, Y.J.; Lee, M.J.; Kim, H.R.; Chung, K.H.; Oh, S.M. Developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in artificially fertilized crucian carp (Carassius auratus) embryo. Sci. Totl. Enviro. 2014, 491-492, 271-278.

Pongratz, I.; Mason, G.G.; Poellinger, L. Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. J. Biol. Chem. 1992, 267 (19), 13728-13734.

Powell, D.C., Aulerich, R.J., Meadows, J.C., Tillitt, D.E., Kelly, M.E., Stromborg, K.L.,

Melancon, M.J., Fitzgerald, S.D., Bursian, S.J., 1998. Effects of 3,3′,4,4′,5-

pentachlorobiphenyl and 2,3,7,8-tetrachlorodibenzo-p-dioxin injected into the

yolks of double-crested cormorant (Phalacrocorax auritus) eggs prior to incubation.

Environ. Toxicol. Chem. 17, 2035–2040.

Prasch, A.L.; Teraoka, H.; Carney, S.A.; Dong, W.; Hiraga, T.; Stegeman, J.J.; Heideman, W.; Peterson, R.E. 2003. Toxicol. Sci. Aryl hydrocarbon receptor 2 mediated 2,3,7,8-tetrachlorodibenzo-p-dioxin developmental toxicity in zebrafish. 76 (1), 138-150.

Scott, J.A.; Incardona, J.P.; Pelkki, K.; Shepardson, S.; Hodson, P.V. (2011). AhR2-mediated, CYP1A-independent cardiovascular toxicity in zebrafish (Danio rerio) embryos exposed to retene. Aquat. Toxicol. 101 (1), 165-174.

Shoots, J.; Fraccalvieri, D.; Franks, D.G.; Denison, M.S.; Hahn, M.E.; Bonati, L.; Powell, W.H. 2015. An aryl hydrocarbon receptor from the salamander Ambystoma mexicanum exhibits low sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enviro. Sci. Technol. 49, 6993-7001.

Spitsbergen, J.M.; Kleeman, J.M.; Peterson, R.E. 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in yellow perch (Perca flavescens). J. Toxicol. Environ. Health. 1988, 23, 359-383.

Spitsbergen, J.M.; Kleeman, J.M.; Peterson, R.E. Morphologic lesions and acute toxicity in rainbow trout (Salmo gairdneri) treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Toxicol. Environ. Health. 1988, 23, 333-358.

Spitsbergen, J.M.; Schat, K.A.; Kleeman, J.M.; Peterson, R.E. Interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with immune responses of rainbow trout. Vet. Immunol. Immunopathol. 1986, 12(1-4), 263-280.

Tanguay, R.L.; Abnet, C.C.; Heideman, W. Peterson, R.E. (1999). Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor1. Biochimica et Biophysica Act 1444, 35-48.

Teraoka, H.; Dong, W.; Tsujimoto, Y.; Iwasa, H.; Endoh, D.; Ueno, N.; Stegeman, J.J.; Peterson, R.E.; Hiraga, T. (2003). Induction of cytochrome P450 1A is required for circulatory failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish. Biochem. Biophys. Res. Commun. 304, 223-228.

Teraoka, H.; Kubota, A.; Kawai, Y.; Hiraga, T. (2008). Prostanoid signaling mediates circulation failure caused by TCDD in developing zebrafish. Interdis. Studies Environ. Chem. Biol. Resp. Chem. Pollut. 61-80.

Teraoka, H.; Okuno, Y.; Nijoukubo, D.; Yamakoshi, A.; Peterson, R.E.; Stegeman, J.J.; Kitazawa, T.; Hiraga, T.; Kubota, A. (2014). Involvement of COX2-thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish. Aquat. Toxicol. 154, 19-25.

Thackaberry, E.A.; Nunez, B.A.; Ivnitski-Steele, I.D. Friggins, M.; Walker, M.K. (2005). Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on murine heart development: Alteration in fetal and postnatal cardiac growth, and postnatal cardiac chronotropy. Toxicol. Sci. 88 (1), 242-249.

Tillitt, D.E.; Buckler, J.A.; Nicks, D.K.; Candrl, J.S.; Claunch, R.A.; Gale, R.W.; Puglis, H.J.; Little, E.E.; Linbo, T.L.; Baker, M. Sensitivity of lake sturgeon (Acipenser fulvescens) early life stages to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,3’,4,4’,5-pentachlorobiphenyl. 2015. Enviro. Toxicol. Chem. DOI: 10.1002/etc.3614.

Toomey, B.H.; Bello, S.; Hahn, M.E.; Cantrell, S.; Wright, P.; Tillitt, D.; Di Giulio, R.T. TCDD induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos. Aquat. Toxicol. 2001, 53, 127-138.

Uno, S.; Dalton, T.P.; Sinclair, P.R.; Gorman, N.; Wang, B.; Smith, A.G.; Miller, M.L.; Shertzer, H.G.; Nebert, D.W. (2004). Cyp1a1 (-/-) male mice: protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria. Toxicol. Appl. Pharmacol. 196 (3), 410-421.

Van den Berg, M.; Birnbaum, L.; Bosveld, A.T.C.; Brunstrom, B.; Cook, P.; Feeley, M.; Giesy, J.P.; Hanberg, A.; Hasegawa, R.; Kennedy, S.W.; Kubiak, T.; Larsen, J.C.; van Leeuwen, R.X.R.; Liem, A.K.D.; Nolt, C.; Peterson, R.E.; Poellinger, L.; Safe, S.; Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PECDFs for human and wildlife. Enviro. Hlth. Persp. 1998, 106, 775-792.

Van den Berg, M.; Birnbaum, L.S.; Dension, M.; De Vito, M.; Farland, W.; Feeley, M.; Fiedler, H.; Hakansson, H.; Hanberg, A.; Haws, L.; Rose, M.; Safe, S.; Schrenk, D.; Tohyama, C.; Tritscher, A.; Tuomisto, J.; Tysklind, M.; Walker, N.; Peterson, R.E. 2006. The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. 93 (2), 223-241.

Van Tiem, L.A.; Di Giulio, R.T. 2011. AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (Danio rerio). Toxicol. Appl. Pharmacol. 254 (3), 280-287.

Walker, M.K.; Catron, T.F. (2000). Characterization of cardiotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related chemicals during early chick embryo development. Toxicol. Appl. Pharmacol. 167 (3), 210-221.

Walker, M.K.; Spitsbergen, J.M.; Olson, J.R.; Peterson, R.E. 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) toxicity during early life stage development of lake trout (Salvelinus namaycush). Canad. J. Fisheries Aqua. Sci. 1991, 48, 875-883.

Waller, C.L.; McKinney, J.D. (1992). Comparative molecular field analysis of polyhalogenated dibenzo-p-dioixns, dibenzofurans, and biphenyls. J. Med. Chem. 35, 3660-2666.

Waller, C.; McKinney, J. (1995). Three-dimensional quantitative structure-activity relationships of dioxins and dioxin-like compounds: model validation and Ah receptor characterization. Chem. Res. Toxicol. 8, 847-858.

Walter, G.L.; Jones, P.D.; Giesy, J.P. Pathologic alterations in adult rainbow trout, Oncorhynchus mykiss, exposed to dietary 2,3,7,8-tetrachlorodibenzo-p-dioxin. Aquat. Toxicol. 2000, 50, 287-299.

Whitlock, J.P.; Okino, S.T.; Dong, L.Q.; Ko, H.S.P.; Clarke Katzenberg, R.; Qiang, M.; Li, W. 1996. Induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. Faseb. J. 10, 809-818.

Whyte, J.J.; Jung, R.E.; Schmitt, C.J.; Tillitt, D.E. (2008). Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit. Rev. Toxicol. 30 (4), 347-570.

Wirgin, I.; Roy, N.K.; Loftus, M.; Chambers, R.C.; Franks, D.G.; Hahn, M.E. 2011. Mechanistic basis of resistance to PCBs in Atlantic tomcod from the Hudson River. Science. 331, 1322-1324.

Yamauchi, M.; Kim, E.Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryos: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. Aquat. Toxicol. 2006, 16, 166-179.

Zabel, E.W; Cook, P.M.; Peterson, R.E. Toxic equivalency factors of polychlorinated dibenzo-p-dioxin, dibenzofuran and biphenyl congeners based on early-life stage mortality in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol. 1995. 31, 315-328.