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  • <h1>SNAPSHOT</h1>
  • <h4>Created at: 2017-02-10 10:36</h4>
  • </div>
  • <!-- Title Section, includes id, name and short name -->
  • <div id="title">
  • <h2>AOP ID and Title:</h2>
  • <hr>
  • <div class="title">
  • AOP 21: Activation of the aryl hydrocarbon receptor (AhR) leading to early life stage mortality
  • </div>
  • <strong>Short Title: AhR activation leading to early life stage toxicity in fish</strong>
  • <br>
  • <div class="title">AOP 21: Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</div>
  • <strong>Short Title: AhR mediated mortality, via COX-2</strong>
  • </div>
  • <!-- Author section, includes text of author names as they have been entered by the user -->
  • <h2>Graphical Representation</h2>
  • <img src="https://aopwiki.org/system/dragonfly/production/2017/04/18/3gysyjyu9l_AhR_agonism_embry_toxicity_figure_002_.pptx" height="500" width="700" alt=""/>
  • <div id="authors">
  • <h2>Authors</h2>
  • <hr>
  • <p>Jon Doering, Ph.D., National Research Council Postdoctoral Toxicologist, US EPA Mid-Continent Ecology Division, Duluth, MN, USA,&nbsp;&nbsp; doering.jonathan[at]epa.gov<br />
  • &nbsp;<br />
  • Prof. Markus Hecker, Ph.D. University of Saskatchewan, Saskatoon, Saskatchewan, Canada, (markus.hecker[at]usask.ca<br />
  • &nbsp;<br />
  • Dan Villeneuve, Ph.D., US EPA Mid-Continent Ecology Division, Duluth, MN, USA (villeneuve.dan[at]epa.gov)</p>
  • <p>Jon Doering, Ph.D., National Research Council,&nbsp;US EPA Mid-Continent Ecology Division, Duluth, MN, USA, (doering.jonathon[at]epa.gov)</p>
  • <p>Prof. Markus Hecker, Ph.D. University of Saskatchewan, Saskatoon, Saskatchewan, Canada, (markus.hecker[at]usask.ca)</p>
  • <p>Dan Villeneuve, Ph.D., US EPA Mid-Continent Ecology Division, Duluth, MN, USA (villeneuve.dan[at]epa.gov)</p>
  • <p>Prof. Xiaowei Zhang, Ph.D., Nanjing University, School of the Environment, Nanjing, China (Zhangxw[at]nju.edu.cn)</p>
  • <br>
  • </div>
  • <!-- Status Section, lists status of aop -->
  • <div id="status">
  • <h2>Status</h2>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <th>Author status</th>
  • <th>OECD status</th>
  • <th>OECD project</th>
  • <th>SAAOP status</th>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Author status</th>
  • <th scope="col">OECD status</th>
  • <th scope="col">OECD project</th>
  • <th scope="col">SAAOP status</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Open for comment. Do not cite</td>
  • <td>EAGMST Under Review</td>
  • <td>1.7</td>
  • <td>Included in OECD work plan</td>
  • <td>Open for citation &amp; comment</td>
  • <td>WPHA/WNT Endorsed</td>
  • <td>1.27</td>
  • <td>Included in OECD Work Plan</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- Abstract Section, text as generated by author -->
  • <div id="abstract">
  • <h2>Abstract</h2>
  • <p>This adverse outcome pathway details the linkage between activation of the aryl hydrocarbon receptor (AhR) and early life stage mortality in oviparous vertebrates.&nbsp;This AOP can be initiated by a range of planar aromatic hydrocarbons, but is best known as the target of dioxin-like compounds (DLCs). These planar compounds are able to bind to the AhR causing&nbsp;heterodimerization with the aryl hydrocarbon nuclear translocator (ARNT) and interaction with dioxin-responsive elements on the DNA causing an up-regulation in dioxin responsive genes. Hundreds to thousands of genes are regulated, either directly or indirectly, by the AhR. One dioxin-responsive gene is cyclooxygenase 2 (COX-2) which has roles in development of the cardiovascular system. Up-regulation in expression of COX-2 causes alteration in cardiovascular development and function which results in reduced heart pumping efficiency, reduced blood flow, and eventual cardiac collapse and death. Comparable apical 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 conservation in&nbsp;the AOP across taxa, great differences in sensitivity to perturbation 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-chemical, cross-species, and cross-taxa extrapolation.&nbsp;</p>
  • <p>In general, biological plausibility of this AOP is strong based&nbsp;heavily on evidence collected from zebrafish (<em>Danio rerio</em>) through mechanistic investigations by use of targeted knockdown of AhR, ARNT, or COX-2 and through use of selective agonists and antagonists of COX-2. Quantitative understanding is largely limited to the indirect KER between&nbsp;AhR activation and early life stage mortality.</p>
  • <p>Activation of the AhR causes pleotropic responses, including interaction with multiple potential target genes such as CYP1A, Sox9b, and HIF1a/VEGF. Therefore, it is a challenge to elucidate the precise series of key events which link activation of the AhR to early life stage mortality. Because of this uncertainty, other AOPs, such as through the HIF1a/VEGF signalling pathway (<a href="https://aopwiki.org/aops/150">AOP 150</a>), have also been developed. These other AOPs likely occur simultaneously with COX-2 to cause altered cardiovascular development and function leading to early life stage mortality.</p>
  • <h2>Abstract</h2>
  • <hr>
  • <p>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 (<em>Danio rerio</em>) 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.&nbsp;</p>
  • <br>
  • </div>
  • <!-- Background Section, text as generated by author -->
  • <div id="background">
  • <h3>Background</h3>
  • <hr>
  • <ul>
  • <h3>Background</h3>
  • <ul>
  • <li>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).</li>
  • <li>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&nbsp;1998; Lahvis and Bradfield&nbsp;1998; Emmons et al&nbsp;1999).</li>
  • <li>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).</li>
  • <li>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).</li>
  • <li>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.</li>
  • <li>High-throughput, next-generation &lsquo;OMICs&rsquo; 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).</li>
  • <li>One gene which is regulated by AhR is cyclooxygenase-2 (COX-2) which is known to have roles in development of the heart in vertebrates (Dong et al 2010; Teraoka et al 2008; 2014). AhR-mediated dysregulation of COX-2 is associated with altered cardiovascular development, decreased blood flow, and cardiac failure causing mortality in early life stages of fish and birds&nbsp;(Dong et al 2010; Teraoka et al 2008; 2014).</li>
  • <li>Exposure to mixtures of agonists of the AhR during the 1950&rsquo;s, 1960&rsquo;s, and 1970&rsquo;s has been implicated in early life stage mortality of Lake Ontario lake trout (<em>Salvelinus namaycush</em>) leading to population collapse (Cook et al 2003). However, populations of mummichog (<em>Fundulus heteroclitus</em>) and Atlantic tomcod (<em>Microgadus tomcod</em>) 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).</li>
  • </ul>
  • <br>
  • </div>
  • <!-- AOP summary, includes summary of each of the events associated with this aop -->
  • <div id="aop_summary">
  • <h2>Summary of the AOP</h2>
  • <hr>
  • <!-- stressor table -->
  • <h3>Stressors</h3>
  • <br>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <h3>Events</h3>
  • <h3>Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)</h3>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sequence</th>
  • <th scope="col">Type</th>
  • <th scope="col">Event ID</th>
  • <th scope="col">Title</th>
  • <th scope="col">Short name</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <th>Name</th>
  • <th>Evidence</th>
  • <td>1</td>
  • <td>MIE</td>
  • <td>18</td>
  • <td><a href="/events/18">Activation, AhR</a></td>
  • <td>Activation, AhR</td>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)</td>
  • <td>Strong</td>
  • </tr>
  • <tr>
  • <td>Polycyclic aromatic hydrocarbons (PAHs)</td>
  • <td>Strong</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)</h4>
  • <ul>
  • <li>The AhR is activated by numerous planar aromatic hydrocarbons. Specifically, chemicals with molecular dimensions of 12 &Aring; x 14 &Aring; x 5 &Aring; are potential ligands of the AhR (Waller &amp; McKinney 1992; 1995).</li>
  • <li>However, the AhR is best known as the molecular target of dioxin-like compounds (DLCs). DLCs include polychlorinated dibenzo-<em>p</em>-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and planar polychlorinated biphenyls (PCBs). A total of seven PCDDs, ten PCDFs, and 12 PCBs are considered &#39;dioxin-like&#39; because they bind to the AhR with relatively great affinity (Denison &amp; Heath-Pagliuso 1998; Van den Berg et al 1998).</li>
  • <li>The prototypical and most potent DLC is 2,3,7,8-tetrachlorodibenzo-<em>p</em>-dioxin (TCDD).</li>
  • </ul>
  • <!-- mie table -->
  • <h3>Molecular Initiating Event</h3>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr><td></td><td></td><td></td><td></td><td></td></tr>
  • <tr>
  • <th>Title</th>
  • <th>Short name</th>
  • <td>2</td>
  • <td>KE</td>
  • <td>944</td>
  • <td><a href="/events/944">dimerization, AHR/ARNT</a></td>
  • <td>dimerization, AHR/ARNT</td>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td><a href="/events/18">Activation, AHR</a></td>
  • <td>Activation, AHR</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <div>
  • <div>
  • <h4><a href="/events/18">18: Activation, AHR</a><br></h4>
  • <h5>Short Name: Activation, AHR</h5>
  • <tr>
  • <td>3</td>
  • <td>KE</td>
  • <td>1269</td>
  • <td><a href="/events/1269">Increase, COX-2 expression</a></td>
  • <td>Increase, COX-2 expression</td>
  • </tr>
  • <tr>
  • <td>4</td>
  • <td>KE</td>
  • <td>317</td>
  • <td><a href="/events/317">Altered, Cardiovascular development/function</a></td>
  • <td>Altered, Cardiovascular development/function</td>
  • </tr>
  • <tr>
  • <td>5</td>
  • <td>KE</td>
  • <td>947</td>
  • <td><a href="/events/947">Increase, Early Life Stage Mortality</a></td>
  • <td>Increase, Early Life Stage Mortality</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <div>
  • <!-- loop to find all aops that use this event -->
  • <h4>AOPs Including This Key Event</h4>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <h3>Key Event Relationships</h3>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Upstream Event</th>
  • <th scope="col">Relationship Type</th>
  • <th scope="col">Downstream Event</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Quantitative Understanding</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <th>AOP ID and Name</th>
  • <th>Event Type</th>
  • <td><a href="/relationships/972">Activation, AhR</a></td>
  • <td>adjacent</td>
  • <td>dimerization, AHR/ARNT</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td><a href="/aops/21">21: Activation of the aryl hydrocarbon receptor (AhR) leading to early life stage mortality</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/57">57: AhR activation leading to hepatic steatosis</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/131">131: AhR activation leading to uroporphyria</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/150">150: Aryl hydrocarbon receptor activation leading to embryolethality via cardiotoxicty</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <tr>
  • <td><a href="/relationships/1350">dimerization, AHR/ARNT</a></td>
  • <td>adjacent</td>
  • <td>Increase, COX-2 expression</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1351">Increase, COX-2 expression</a></td>
  • <td>adjacent</td>
  • <td>Altered, Cardiovascular development/function</td>
  • <td>Moderate</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1567">Altered, Cardiovascular development/function</a></td>
  • <td>adjacent</td>
  • <td>Increase, Early Life Stage Mortality</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td></td>
  • <td></td>
  • <td></td>
  • <td></td>
  • <td></td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/984">Activation, AhR</a></td>
  • <td>non-adjacent</td>
  • <td>Increase, Early Life Stage Mortality</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- loop to find stressors under event -->
  • <div>
  • <h4>Stressors</h4>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>Name</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Benzidine</td>
  • </tr>
  • <tr>
  • <td>Dibenzo-p-dioxin</td>
  • </tr>
  • <tr>
  • <td>Polychlorinated biphenyl</td>
  • </tr>
  • <tr>
  • <td>Polychlorinated dibenzofurans</td>
  • </tr>
  • <tr>
  • <td>Hexachlorobenzene</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Evidence for Perturbation of this Molecular Initiating Event by Stressor</h4>
  • <p>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<sup><a href="#cite_note-Denison2011-38">[38]</a></sup>. 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<sup><a href="#cite_note-Van1998-39">[39]</a></sup>; however, recent reports indicate that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) is more potent than TCDD in some species of birds.<sup><a href="#cite_note-Cohen2011b-40">[40]</a></sup><sup><a href="#cite_note-Farmahin2012-13">[13]</a></sup><sup><a href="#cite_note-Farmahin2013a-41">[41]</a></sup><sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup><sup><a href="#cite_note-Herve2010a-42">[42]</a></sup><sup><a href="#cite_note-Herve2010b-43">[43]</a></sup> 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<sup><a href="#cite_note-Poland1973-44">[44]</a></sup>. Of the dioxin-like PCBs, non-ortho congeners are the most toxicologically active, while mono-ortho PCBs are generally less potent<sup><a href="#cite_note-McFarland1989-45">[45]</a></sup><sup><a href="#cite_note-Safe1994-9">[9]</a></sup>. Chlorine substitution at ortho positions increases the energetic costs of assuming the coplanar conformation required for binding to the AHR <sup><a href="#cite_note-McFarland1989-45">[45]</a></sup>. 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 <sup><a href="#cite_note-Safe1994-9">[9]</a></sup>.</p>
  • <ul>
  • <li>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).</li>
  • </ul>
  • <p>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 <sup><a href="#cite_note-Okey2007-1">[1]</a></sup>. 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. <sup><a href="#cite_note-Fujii2010-6">[6]</a></sup><sup><a href="#cite_note-Omie2011-46">[46]</a></sup><sup><a href="#cite_note-Swed2010-47">[47]</a></sup><sup><a href="#cite_note-Diani2011-48">[48]</a></sup><sup><a href="#cite_note-Wincent2012-49">[49]</a></sup> The AHR is thought to have important endogenous roles in reproduction, liver and heart development, cardiovascular function, immune function and cell cycle regulation <sup><a href="#cite_note-Baba2005-50">[50]</a></sup><sup><a href="#cite_note-Denison2011-38">[38]</a></sup><sup><a href="#cite_note-Fernandez1995-51">[51]</a></sup><sup><a href="#cite_note-Ichihara2007-52">[52]</a></sup><sup><a href="#cite_note-Lahvis2000-53">[53]</a></sup><sup><a href="#cite_note-Mimura1997-54">[54]</a></sup><sup><a href="#cite_note-Omie2011-46">[46]</a></sup><sup><a href="#cite_note-Schmidt1996-55">[55]</a></sup><sup><a href="#cite_note-Thack2002-56">[56]</a></sup><sup><a href="#cite_note-Zhang2010-57">[57]</a></sup> and activation of the AHR by DLCs may therefore adversely affect these processes.</p>
  • <br>
  • <h3>Stressors</h3>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Name</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <br>
  • <!-- biological organization -->
  • <div>
  • <h4>Biological Organization</h4>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>Level of Biological Organization</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Molecular</td>
  • </tr>
  • <h4>2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)</h4>
  • <ul>
  • <li>The AhR is activated by numerous planar aromatic hydrocarbons. Specifically, chemicals with molecular dimensions of 12 &Aring; x 14 &Aring; x 5 &Aring; are potential ligands of the AhR (Waller &amp; McKinney 1992; 1995).</li>
  • <li>However, the AhR is best known as the molecular target of dioxin-like compounds (DLCs). DLCs include polychlorinated dibenzo-<em>p</em>-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and planar polychlorinated biphenyls (PCBs). A total of seven PCDDs, ten PCDFs, and 12 PCBs are considered &#39;dioxin-like&#39; because they bind to the AhR with relatively great affinity (Denison &amp; Heath-Pagliuso 1998; Van den Berg et al 1998).</li>
  • <li>The prototypical and among the most potent of the DLCs is 2,3,7,8-tetrachlorodibenzo-<em>p</em>-dioxin (TCDD).</li>
  • </ul>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end of bio org -->
  • <!-- cell term -->
  • <div>
  • </div>
  • <!-- end of cell term -->
  • </div>
  • <!-- organ term -->
  • <div>
  • </div>
  • <!-- end of organ term -->
  • <div id="overall_assessment">
  • <h2>Overall Assessment of the AOP</h2>
  • <p><strong>Assessment of WoE calls:</strong></p>
  • <p><strong>Activation, AhR leads to dimerization, AHR/ARNT</strong>: High</p>
  • <p>Rationale: The call of &#39;High&#39; is based on overwhelming empirical&nbsp;evidence in numerous species of mammals, birds, amphibians, and fishes. Further, because of overwhelming evidence of essentiality based on targeted knockdown/knockout studies. No uncertainties or inconsistencies are known which affect the WoE call.</p>
  • <p><strong>Dimerization, AHR/ARNT leads to increase, COX-2 expression</strong>: High</p>
  • <p>Rationale: The call of &quot;High&quot; is based on convincing empirical evidence in three species (two fish and one bird). Further, because of convincing biological plausibility based on identification of dioxin-response elements in the promoter region of COX-2. Uncertainties and inconsistencies are only related to lack of any information on species outside of the three model species that have been investigated.</p>
  • <p><strong>Increase, COX-2 expression leads to altered, cardiovascular development/function</strong>: Moderate</p>
  • <p>Rationale: The call of &quot;Moderate&quot; is based on overwhelming empirical&nbsp;evidence and evidence of essentiality&nbsp;in three species (two fish and one bird) based on studies using targeted knockdown of genes and selective agonists/antagonists. However, a lack of information on the role of COX-2 in cardiovascular development/function makes biological plausibility questionable at this time. Further, there is some uncertainty associated with pleiotropic effects of AhR activation and the high probability of multiple mechanisms acting concurrently to cause altered cardiovascular development/function.</p>
  • <p><strong>Altered, cardiovascular development/function leads to increase, early life stage mortality</strong>; High</p>
  • <p>Rationale: The call of &quot;High&quot; is based on overwhelming empirical evidence and biological plausibility in numerous species of mammals, birds, and fish. There are no known uncertainties or inconsistencies at this time.</p>
  • <p><strong>Activation, AhR leads to increase, early life stage mortality</strong>: High</p>
  • <p>Rationale: The call of &quot;High&quot; is based on overwhelming empirical&nbsp;evidence and evidence of essentiality in numerous species of mammals, birds, amphibians, and fishes using regression analysis and targeted knockdown/knockout of AhR. There are no known uncertainties of inconsistences at this time.</p>
  • <h4>Evidence Supporting Applicability of this Event</h4>
  • <br>
  • <!-- loop to find taxonomic applicability under event -->
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <h3>Domain of Applicability</h3>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Term</th>
  • <th>Scientific Term</th>
  • <th>Evidence</th>
  • <th>Links</th>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>zebra danio</td>
  • <td>Danio rerio</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Fundulus heteroclitus</td>
  • <td>Fundulus heteroclitus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8078" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>Development</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for taxons -->
  • <!-- life stages -->
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>Life Stage</th>
  • <th>Evidence</th>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Embryo</td>
  • <td>Strong</td>
  • </tr>
  • <tr>
  • <td>development</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>zebrafish</td>
  • <td>Danio rerio</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>medaka</td>
  • <td>Oryzias latipes</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end life stages -->
  • <!-- sex terms -->
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Sex</th>
  • <th>Evidence</th>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Unspecific</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end sex terms -->
  • <div>
  • <p>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<sup><a href="#cite_note-Poland1976-3">[3]</a></sup>. 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<sup><a href="#cite_note-Ema1994-30">[30]</a></sup><sup><a href="#cite_note-Poland1982-19">[19]</a></sup><sup><a href="#cite_note-Poland1994-31">[31]</a></sup>. Several other studies reported the importance of this amino acid in birds and mammals<sup><a href="#cite_note-Backlund2004-32">[32]</a></sup><sup><a href="#cite_note-Ema1994-30">[30]</a></sup><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup><sup><a href="#cite_note-Murray2005-33">[33]</a></sup><sup><a href="#cite_note-Pandini2007-34">[34]</a></sup><sup><a href="#cite_note-Pandini2009-35">[35]</a></sup><sup><a href="#cite_note-Poland1994-31">[31]</a></sup><sup><a href="#cite_note-Ramadoss2004-36">[36]</a></sup>. 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<sup><a href="#cite_note-Pandini2009-35">[35]</a></sup>. Mutation at position 319 in the mouse eliminated AHR DNA binding<sup><a href="#cite_note-Pandini2009-35">[35]</a></sup>.</p>
  • <p><strong>Sex: </strong>This AOP is only applicable to early life stages prior to sexual differentiation.</p>
  • <p>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 <em>et al.</em><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>. 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<sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>, 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<sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>. 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<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Head2008-37">[37]</a></sup> . 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<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Head2008-37">[37]</a></sup><sup><a href="#cite_note-Manning2012-16">[16]</a></sup>. 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)<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Head2008-37">[37]</a></sup><sup><a href="#cite_note-Manning2012-16">[16]</a></sup>.</p>
  • <p><strong>Life stages: </strong>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 &amp; Stegeman 2008).</p>
  • <ul>
  • <li>Little is known about differences in binding affinity of AhRs and how this relates to sensitivity in non-avian taxa.</li>
  • <li>Low binding affinity for DLCs of AhR1s of African clawed frog (<em>Xenopus laevis</em>) and axolotl (<em>Ambystoma mexicanum</em>) has been suggested as a mechanism for tolerance of these amphibians to DLCs (Lavine et al 2005; Shoots et al 2015).</li>
  • <li>Among reptiles, only AhRs of American alligator (<em>Alligator mississippiensis</em>) have been investigated and little is known about the sensitivity of American alligator or other reptiles to DLCs (Oka et al 2016).</li>
  • <li>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).</li>
  • <li>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 (<em>Microgadus tomcod</em>) (Wirgin et al 2011).</li>
  • <li>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).</li>
  • </ul>
  • <br>
  • </div>
  • <!-- event text -->
  • <h4>How this Key Event Works</h4>
  • <h3>The AHR Receptor</h3>
  • <p><strong>Taxonomic:</strong> The specific characteristics of altered cardiovascular development and function vary to some degree among taxonomic groups of vertebrates.</p>
  • <p>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)<sup><a href="#cite_note-Okey2007-1">[1]</a></sup>. Other members of this superfamily include the AHR nuclear translocator (ARNT), which acts as a dimerization partner of the AHR <sup><a href="#cite_note-Hoffman1991-2">[2]</a></sup><sup><a href="#cite_note-Poland1976-3">[3]</a></sup>; Per, a circadian transcription factor; and Sim, the &ldquo;single-minded&rdquo; protein involved in neuronal development <sup><a href="#cite_note-Gu2000-4">[4]</a></sup><sup><a href="#cite_note-Kewley2004-5">[5]</a></sup>. This group of proteins shares a highly conserved PAS domain and is involved in the detection of and adaptation to environmental change<sup><a href="#cite_note-Gu2000-4">[4]</a></sup>.</p>
  • <p>This AOP is applicable to:</p>
  • <ul>
  • <li>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).</li>
  • <li>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).</li>
  • <li>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).</li>
  • <li>All birds (Canga et al 1993; Cohen-Barnhouse et al 2011; Fujisawa et al 2014; Heid et al 2001; Ivnitski et al 2001; Walker &amp; 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 (<em>Gallus gallus</em>) (Fujisawa et al 2014).</li>
  • <li>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.</li>
  • <li>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.</li>
  • </ul>
  • <h3>The molecular Initiating Event</h3>
  • <div>
  • <div><a class="image" href="/wiki/index.php/File:AHR_mechanism.jpeg"><img alt="" class="thumbimage" src="/wiki/images/thumb/6/6e/AHR_mechanism.jpeg/450px-AHR_mechanism.jpeg" style="height:331px; width:450px" /></a>
  • <div>
  • <div><a class="internal" href="/wiki/index.php/File:AHR_mechanism.jpeg" title="Enlarge"><img alt="" src="/wiki/skins/common/images/magnify-clip.png" style="height:11px; width:15px" /></a></div>
  • The molecular mechanism of activation of gene expression by AHR1.</div>
  • </div>
  • </div>
  • <p>This AOP is not applicable to:</p>
  • <p>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)<sup><a href="#cite_note-Fujii2010-6">[6]</a></sup>. Upon ligand binding, the AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with ARNT<sup><a href="#cite_note-Mimura2003-7">[7]</a></sup>. 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<sup><a href="#cite_note-Fujii2010-6">[6]</a></sup>. 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 <sup><a href="#cite_note-Fujii2010-6">[6]</a></sup><sup><a href="#cite_note-Giesy2006-8">[8]</a></sup><sup><a href="#cite_note-Mimura2003-7">[7]</a></sup><sup><a href="#cite_note-Safe1994-9">[9]</a></sup>.</p>
  • <ul>
  • <li>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 &amp; Walker 2009). Further, studies of CYP1A1 and CYP1A2 null mice (<em>Mus musculus</em>) demonstrate that wasting syndrome and mortality are mediated by CYP1A1 in mammals (Uno et al 2004).</li>
  • <li>Invertebrates because AhRs of invertebrates have less diverse functionalities relative to vertebrates, AhRs of most invertebrates likely do not&nbsp;bind agonists that represent anthropogenic pollutants, and no AhR-mediated, critical adverse effects are known in invertebrates as a result of exposure to AhR agonists&nbsp;(Hahn 2002; Hahn et al 1994).</li>
  • <li>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.</li>
  • </ul>
  • <h3>Essentiality of the Key Events</h3>
  • <p>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.</p>
  • <h3>AHR Isoforms</h3>
  • <p>&nbsp;Rationale for essentiality calls:</p>
  • <ul>
  • <li>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).</li>
  • <li>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).</li>
  • <li>The AhR3 is poorly understood and known only from some cartilaginous fishes (Hahn 2002).</li>
  • <li>Little is known about diversity of AhRs in reptiles and amphibians (Hahn et al 2002).</li>
  • <li>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 (&alpha;, &beta;, &delta;, &gamma;) having been identified in Atlantic salmon (<em>Salmo salar</em>) (Hansson et al 2004).</li>
  • <li>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).&nbsp;</li>
  • <li>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 &amp; Di Giulio 2011).</li>
  • </ul>
  • <p>Two AHR isoforms (AHR1 and AHR2) have been identified in the black-footed albatross (<em>Phoebastria nigripes</em>), great cormorant (<em>Phalacrocorax carbo</em>) and domestic chicken (<em>Gallus gallus domesticus</em>)<sup><a href="#cite_note-Yasui2007-10">[10]</a></sup>. 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<sup><a href="#cite_note-Yasui2007-10">[10]</a></sup>, 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<sup><a href="#cite_note-Lee2009-11">[11]</a></sup><sup><a href="#cite_note-Yasui2007-10">[10]</a></sup>.</p>
  • <p>Roles of isoforms in Birds:</p>
  • <p>&nbsp;</p>
  • <ul>
  • <li>AhR1 and AhR2 both bind and are activated by TCDD <em>in vitro</em> (Yasui et al 2007).</li>
  • <li>AhR1 has greater binding affinity and sensitivity to activation by TCDD relative to AhR2 (Yasui et al 2007).</li>
  • <li>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).</li>
  • <li>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).</li>
  • </ul>
  • <p>Roles of isoforms in fishes:</p>
  • <p>&nbsp;</p>
  • <ul>
  • <li>AhR1 and AhR2 both bind and are activated by TCDD <em>in vitro</em> (Bak et al 2013; Doering et al 2014; 2015; Karchner et al 1999; 2005).</li>
  • <li>AhR1 has greater sensitivity to activation by TCDD than AhR2 in red seabream (<em>Pagrus major</em>), white sturgeon (<em>Acipenser transmontanus</em>), and lake sturgeon (<em>Acipenser fulvescens</em>) (Bak et al 2013; Doering et al 2014; 2015)</li>
  • <li>AhR2 has greater binding affinity or activation by TCDD than AhR1 in zebrafish (<em>Danio rerio</em>) and mummichog (<em>Fundulus heteroclitus</em>) (Karchner et al 1999; 2005).</li>
  • <li>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 (<em>Danio rerio</em>) and mummichog (<em>Fundulus heteroclitus</em>), while knockdown of AhR1 offers no protection (Clark et al 2010; Prasch et al 2003; Van Tiem &amp; Di Giulio 2011).</li>
  • <li>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).</li>
  • </ul>
  • <p>Roles of isoforms in amphibians and reptiles:</p>
  • <p>&nbsp;</p>
  • <ul>
  • <li>Less is known about AhRs of amphibians or reptiles.</li>
  • <li>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).</li>
  • <li>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.</li>
  • <li>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. Concentrations of DLCs tested in amphibians studied to date were not sufficient to cause altered cardiovascular development or function and no increase in mortality was observed (Jung et al 1997).</li>
  • </ul>
  • <br>
  • <h4>How it is Measured or Detected</h4>
  • <p><em>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? </em></p>
  • <h3>Transactivation Reporter Gene Assays (recommended approach)</h3>
  • <h3>Weight of Evidence Summary</h3>
  • <p><strong>Biological Plausibility: </strong></p>
  • <h4>Transient transfection transactivation</h4>
  • <ul>
  • <li>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 strong.</li>
  • <li>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.</li>
  • <li>The prostaglandin synthesis pathway, of which COX-2 is a rate limiting step, is known to have roles in development of the heart (Delgado et al 2004; Gullestad &amp; Aukrust 2005; Hocherl et al 2002; Huang et al 2007; Wong et al 1998;&nbsp;Dong et al 2010; Huang et al 2007; Teraoka et al 2008; 2014).</li>
  • <li>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.</li>
  • </ul>
  • <p>Transient transfection transactivation is the most common method for evaluating nuclear receptor activation<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. 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)<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. 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<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>.</p>
  • <p>&nbsp;</p>
  • <h5>Luciferase reporter gene (LRG) assay</h5>
  • <p><strong>Concordance of dose-response relationships: </strong></p>
  • <ul>
  • <li>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 &amp; Hahn 2008; Karchner et al 1999; Tanguay et al 1999; Wirgin et al 2011).</li>
  • <li>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 &amp; 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).</li>
  • <li>Less is known regarding concordance of dose-response relationships for COX-2. In Japanese medaka (<em>Oryzias latipes</em>), 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).</li>
  • </ul>
  • <p>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 <em>Renilla</em> 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 <em>Renilla</em> luciferase units <sup><a href="#cite_note-Farmahin2012-13">[13]</a></sup>. 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).<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Farmahin2012-13">[13]</a></sup><sup><a href="#cite_note-Fujisawa2012-15">[15]</a></sup><sup><a href="#cite_note-Lee2009-11">[11]</a></sup><sup><a href="#cite_note-Manning2012-16">[16]</a></sup><sup><a href="#cite_note-Mol2012-17">[17]</a></sup></p>
  • <h4>Transactivation in stable cell lines</h4>
  • <p>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 <sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. 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<sup><a href="#cite_note-Yueh2005-18">[18]</a></sup>. An added benefit of this model is the potential to multiplex 3 assays in a single well: receptor activation, cell viability and enzyme activity<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. Such assays are used extensively in drug discovery due to their high throughput efficiency, and may serve just as useful for risk assessment purposes.</p>
  • <p>&nbsp;</p>
  • <h3>Ligand-Binding Assays</h3>
  • <p><strong>Temporal concordance among the key events and adverse effect:</strong></p>
  • <p>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<sup><a href="#cite_note-Poland1982-19">[19]</a></sup> and can explain differences in species sensitivities to DLCs<sup><a href="#cite_note-Hesterman2000-20">[20]</a></sup><sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>; they are therefore worth mentioning. Binding affinity and efficacy have been used to develop structure-activity relationships for AHR disruption<sup><a href="#cite_note-Hesterman2000-20">[20]</a></sup><sup><a href="#cite_note-Lee2015-23">[23]</a></sup> 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<sup><a href="#cite_note-Jones2003-24">[24]</a></sup><sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. 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.</p>
  • <ul>
  • <li>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 &amp; Stegeman 2006).</li>
  • <li>AhR transcript and protein is first detectable in zebrafish around 24 hpf (Tanguay et al 1999).</li>
  • <li>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).</li>
  • <li>Therefore, there is a general temporal concordance in this AOP.</li>
  • <li>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.</li>
  • </ul>
  • <h4>Hydroxyapatite (HAP) binding assay</h4>
  • <p>&nbsp;</p>
  • <p>The HAP binding assay makes use of an <em>in vitro</em> 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)<sup><a href="#cite_note-Gasiewicz1982-25">[25]</a></sup><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>. This assay is simple, repeatable and reproducible; however, it is insensitive to weak ligand-receptor interactions<sup><a href="#cite_note-Karchner2006-22">[22]</a></sup><sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup><sup><a href="#cite_note-Nakai1995-26">[26]</a></sup>.</p>
  • <p><strong>Consistency:</strong></p>
  • <h4>Whole cell filtration binding assay</h4>
  • <ul>
  • <li>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.</li>
  • <li>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.</li>
  • <li>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).</li>
  • <li>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.</li>
  • </ul>
  • <p>Dold and Greenlee<sup><a href="#cite_note-Dold1990-27">[27]</a></sup> developed a method to detect specific binding of TCDD to whole mammalian cells in culture and was later modified by Farmahin et al.<sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup> 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<sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup>.</p>
  • <p>&nbsp;</p>
  • <h3>Protein-DNA Interaction Assays</h3>
  • <p><strong>Uncertainties, inconsistencies, and data gaps:</strong></p>
  • <p>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<sup><a href="#cite_note-Perez2007-28">[28]</a></sup>. Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions <em>in vivo</em>. 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 <em>in vivo</em>. 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<sup><a href="#cite_note-Heid2001-29">[29]</a></sup>.</p>
  • <br>
  • <ul>
  • <li>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 &amp; Stegeman 2008), and AhR cross-talk with hypoxia inducible factor 1&alpha; (HIF1&alpha;) causing reduced transcription of vascular endothelial growth factor (VEGF) (<a href="https://aopwiki.org/aops/150">AOP 150</a>).</li>
  • <li>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). Specifically, knockdown or knockout of Sox9b is associated with mild pericardial edema, unlooping, loss of proepicardium, and failure to form epicardium and endocardial cushions, but does not result in typical TCDD-mediated effects of a compacted ventricle or an&nbsp;elongated string-like atrium (Hofsteen et al 2013). Altered cardiovascular development as a result of complete knockout of&nbsp;Sox9b is not severe enough to cause complete cardiac failure and early life stage mortality in zebrafish (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).Considering,&nbsp;TCDD is only able to decrease expression of Sox9b in the heart by up to about 50% and complete knockout of&nbsp;Sox9b expression is not lethal suggests that Sox9b is not essential to TCDD-mediated alteration in cardiovascular development and function&nbsp;(Hofsteen et al 2013).</li>
  • <li>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 &amp; 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).</li>
  • <li>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.</li>
  • <li>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.</li>
  • <li>Despite these uncertainties, the strong, quantitative link between activation of the AhR and early life stage mortality means that elucidating the&nbsp;precise series of key events is less critical. Therefore, the evidence suggesting COX-2 as a primary mechanism&nbsp;might be all that is necessary, although multiple mechanisms acting together is the most likely true mechanism.</li>
  • </ul>
  • <h3>Quantitative Consideration</h3>
  • <ul>
  • <li>The majority of the quantitative understanding and the strongest quantitative understanding is for the indirect relationship between activation of the AhR and early life stage mortality.</li>
  • <li>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).</li>
  • <li>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).</li>
  • <li>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 (R<sup>2</sup> = 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.</li>
  • <li>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.</li>
  • </ul>
  • <ul>
  • </ul>
  • </div>
  • <div id="considerations_for_potential_applicaitons">
  • <h2>Considerations for Potential Applications of the AOP (optional)</h2>
  • <p>There are great differences in sensitivity to agonists of the AhR among species and among taxa and &nbsp;great differences in potency among agonists of the AhR. Therefore, this AOP has utility towards the mechanistic understanding of adverse effects of agonists of the AhR with regard to cross-chemical, cross-species, and cross-taxa extrapolations. This utility has led to the development of a qAOP that has demonstrated utility in guiding more objective ecological risk assessments of native species to agonists of the AhR, particularly assessments of threatened or endangered species that often cannot be investigated in laboratory toxicity testing (Doering et al 2018).</p>
  • </div>
  • <div id="references">
  • <h2>References</h2>
  • <p>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<em>.</em> 159, 41-51.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>Belair, C.D.; Peterson, R.E.; Heideman, W. (2001). Disruption of erythropoiesis by dioxin in the zebrafish. Dev. Dyn. 222 (4), 581-594.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>Buckler J.; Candrl, J.S.; McKee, M.J.; Papoulias, D.M.; Tillitt, D.E.; Galat, D.L. Sensitivity of shovelnose sturgeon (<em>Scaphirhynchus platorynchus</em>) and pallid sturgeon (<em>S. albus</em>) early life stages to PCB-126 and 2,3,7,8-TCDD exposure. <em>Enviro. Toxicol. Chem. </em><strong>2015</strong>, 34(6), 1417-1424.</p>
  • <p>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.&nbsp; 44, 1142&ndash;1151.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>Chen, G.; Bunce, N.J. (2003). Polybrominated diphenyl ethers as Ah receptor agonists and antagonists. Toxicol. Sci. 76 (2), 310-320.</p>
  • <p>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 (<em>Fundulus heteroclitus</em>). Aquat. Toxicol. 99, 232-240.</p>
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  • </div>
  • <div id="appendicies">
  • <h2>Appendix 1</h2>
  • <h3>List of MIEs in this AOP</h3>
  • <h4><a href="/events/18">Event: 18: Activation, AhR</a></h4>
  • <h5>Short Name: Activation, AhR</h5>
  • <h4>Key Event Component</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Process</th>
  • <th scope="col">Object</th>
  • <th scope="col">Action</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>aryl hydrocarbon receptor activity</td>
  • <td>aryl hydrocarbon receptor</td>
  • <td>increased</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/21">Aop:21 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/57">Aop:57 - AhR activation leading to hepatic steatosis</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/131">Aop:131 - Aryl hydrocarbon receptor activation leading to uroporphyria</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/150">Aop:150 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via reduced VEGF</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/310">Aop:310 - Embryonic Activation of the AHR leading to Reproductive failure, via epigenetic down-regulation of GnRHR</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/151">Aop:151 - AhR activation leading to preeclampsia</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/414">Aop:414 - Aryl hydrocarbon receptor activation leading to lung fibrosis through TGF-β dependent fibrosis toxicity pathway</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/415">Aop:415 - Aryl hydrocarbon receptor activation leading to lung fibrosis through IL-6 toxicity pathway</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/416">Aop:416 - Aryl hydrocarbon receptor activation leading to lung cancer through IL-6 toxicity pathway</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/417">Aop:417 - Aryl hydrocarbon receptor activation leading to lung cancer through AHR-ARNT toxicity pathway</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/418">Aop:418 - Aryl hydrocarbon receptor activation leading to impaired lung function through AHR-ARNT toxicity pathway</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/419">Aop:419 - Aryl hydrocarbon receptor activation leading to impaired lung function through P53 toxicity pathway</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/420">Aop:420 - Aryl hydrocarbon receptor activation leading to lung cancer through sustained NRF2 toxicity pathway</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/439">Aop:439 - Activation of the AhR leading to metastatic breast cancer </a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/455">Aop:455 - Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced impeded craniofacial development</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/456">Aop:456 - Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/458">Aop:458 - AhR activation in the liver leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/494">Aop:494 - AhR activation leading to liver fibrosis </a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/459">Aop:459 - AhR activation in the thyroid leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>Benzidine</td></tr>
  • <tr><td>Dibenzo-p-dioxin</td></tr>
  • <tr><td>Polychlorinated biphenyl</td></tr>
  • <tr><td>Polychlorinated dibenzofurans</td></tr>
  • <tr><td>Hexachlorobenzene</td></tr>
  • <tr><td>Polycyclic aromatic hydrocarbons (PAHs)</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Molecular</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h3>Evidence for Perturbation by Stressor</h3>
  • <h4>Overview for Molecular Initiating Event</h4>
  • <p>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<sup><a href="#cite_note-Denison2011-38">[38]</a></sup>. 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<sup><a href="#cite_note-Van1998-39">[39]</a></sup>; however, recent reports indicate that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) is more potent than TCDD in some species of birds.<sup><a href="#cite_note-Cohen2011b-40">[40]</a></sup><sup><a href="#cite_note-Farmahin2012-13">[13]</a></sup><sup><a href="#cite_note-Farmahin2013a-41">[41]</a></sup><sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup><sup><a href="#cite_note-Herve2010a-42">[42]</a></sup><sup><a href="#cite_note-Herve2010b-43">[43]</a></sup> 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<sup><a href="#cite_note-Poland1973-44">[44]</a></sup>. Of the dioxin-like PCBs, non-ortho congeners are the most toxicologically active, while mono-ortho PCBs are generally less potent<sup><a href="#cite_note-McFarland1989-45">[45]</a></sup><sup><a href="#cite_note-Safe1994-9">[9]</a></sup>. Chlorine substitution at ortho positions increases the energetic costs of assuming the coplanar conformation required for binding to the AHR <sup><a href="#cite_note-McFarland1989-45">[45]</a></sup>. 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 <sup><a href="#cite_note-Safe1994-9">[9]</a></sup>.</p>
  • <ul>
  • <li>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).</li>
  • </ul>
  • <p>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 <sup><a href="#cite_note-Okey2007-1">[1]</a></sup>. 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. <sup><a href="#cite_note-Fujii2010-6">[6]</a></sup><sup><a href="#cite_note-Omie2011-46">[46]</a></sup><sup><a href="#cite_note-Swed2010-47">[47]</a></sup><sup><a href="#cite_note-Diani2011-48">[48]</a></sup><sup><a href="#cite_note-Wincent2012-49">[49]</a></sup> The AHR is thought to have important endogenous roles in reproduction, liver and heart development, cardiovascular function, immune function and cell cycle regulation <sup><a href="#cite_note-Baba2005-50">[50]</a></sup><sup><a href="#cite_note-Denison2011-38">[38]</a></sup><sup><a href="#cite_note-Fernandez1995-51">[51]</a></sup><sup><a href="#cite_note-Ichihara2007-52">[52]</a></sup><sup><a href="#cite_note-Lahvis2000-53">[53]</a></sup><sup><a href="#cite_note-Mimura1997-54">[54]</a></sup><sup><a href="#cite_note-Omie2011-46">[46]</a></sup><sup><a href="#cite_note-Schmidt1996-55">[55]</a></sup><sup><a href="#cite_note-Thack2002-56">[56]</a></sup><sup><a href="#cite_note-Zhang2010-57">[57]</a></sup> and activation of the AHR by DLCs may therefore adversely affect these processes.</p>
  • <h4>Dibenzo-p-dioxin</h4>
  • <p><p>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.&nbsp;<em>Toxicol.Sci.</em>&nbsp;<strong>124</strong>, 1-22.</p>
  • </p>
  • <h4>Polychlorinated biphenyl</h4>
  • <p><p>Of the dioxin-like PCBs, non-ortho congeners are the most toxicologically active, while mono-ortho PCBs are generally less potent (McFarland and Clarke 1989; Safe 1994). Chlorine substitution at ortho positions increases the energetic costs of assuming the coplanar conformation required for binding to the AHR (McFarland and Clarke 1989). 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 (Safe 1994).</p>
  • <p>&nbsp;</p>
  • <p>Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment.&nbsp;<em>Critical Reviews in Toxicology</em>&nbsp;<strong>24</strong>, 87-149.</p>
  • <p>McFarland, V. A., and Clarke, J. U. (1989). Environmental occurrence, abundance, and potential toxicity of polychlorinated biphenyl congeners: Considerations for a congener-specific analysis.&nbsp;<em>Environ.Health Perspect</em>.&nbsp;<strong>81</strong>, 225-239.</p>
  • </p>
  • <h4>Polychlorinated dibenzofurans</h4>
  • <p><p>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.&nbsp;<em>Toxicol.Sci.</em>&nbsp;<strong>124</strong>, 1-22.</p>
  • </p>
  • <h4>Hexachlorobenzene</h4>
  • <p><p>Cripps, D. J., Peters, H. A., Gocmen, A., and Dogramici, I. (1984) Porphyria turcica due to hexachlorobenzene: a 20 to 30 year follow-up study on 204 patients.&nbsp;<em>Br. J Dermatol.&nbsp;</em><strong>111</strong>&nbsp;(4), 413-422.</p>
  • </p>
  • <h4>Polycyclic aromatic hydrocarbons (PAHs)</h4>
  • <p><p>PAHs are pontent AHR agonists, but due to their rapid metabolism, they cause a transient alteration in AHR-mediated gene expression; this property results in a very different toxicity profile relative to persistent AHR-agonists such as dioxin-like compounds (Denison et al. 2011).</p>
  • <p>&nbsp;</p>
  • <p>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. <em>Toxicol.Sci.</em> <strong>124</strong>, 1-22.</p>
  • </p>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>zebra danio</td>
  • <td>Danio rerio</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Pagrus major</td>
  • <td>Pagrus major</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=143350" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Acipenser transmontanus</td>
  • <td>Acipenser transmontanus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7904" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Acipenser fulvescens</td>
  • <td>Acipenser fulvescens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=41871" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rainbow trout</td>
  • <td>Oncorhynchus mykiss</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8022" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Salmo salar</td>
  • <td>Salmo salar</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8030" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Xenopus laevis</td>
  • <td>Xenopus laevis</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Ambystoma mexicanum</td>
  • <td>Ambystoma mexicanum</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8296" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Phasianus colchicus</td>
  • <td>Phasianus colchicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9054" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Coturnix japonica</td>
  • <td>Coturnix japonica</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=93934" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Microgadus tomcod</td>
  • <td>Microgadus tomcod</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34823" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Homo sapiens</td>
  • <td>Homo sapiens</td>
  • <td></td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>Development</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p>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<sup><a href="#cite_note-Poland1976-3">[3]</a></sup>. 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<sup><a href="#cite_note-Ema1994-30">[30]</a></sup><sup><a href="#cite_note-Poland1982-19">[19]</a></sup><sup><a href="#cite_note-Poland1994-31">[31]</a></sup>. Several other studies reported the importance of this amino acid in birds and mammals<sup><a href="#cite_note-Backlund2004-32">[32]</a></sup><sup><a href="#cite_note-Ema1994-30">[30]</a></sup><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup><sup><a href="#cite_note-Murray2005-33">[33]</a></sup><sup><a href="#cite_note-Pandini2007-34">[34]</a></sup><sup><a href="#cite_note-Pandini2009-35">[35]</a></sup><sup><a href="#cite_note-Poland1994-31">[31]</a></sup><sup><a href="#cite_note-Ramadoss2004-36">[36]</a></sup>. 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<sup><a href="#cite_note-Pandini2009-35">[35]</a></sup>. Mutation at position 319 in the mouse eliminated AHR DNA binding<sup><a href="#cite_note-Pandini2009-35">[35]</a></sup>.</p>
  • <p>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 <em>et al.</em><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>. 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<sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>, 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<sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>. 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<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Head2008-37">[37]</a></sup> . 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<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Head2008-37">[37]</a></sup><sup><a href="#cite_note-Manning2012-16">[16]</a></sup>. 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)<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Head2008-37">[37]</a></sup><sup><a href="#cite_note-Manning2012-16">[16]</a></sup>.</p>
  • <ul>
  • <li>Little is known about differences in binding affinity of AhRs and how this relates to sensitivity in non-avian taxa.</li>
  • <li>Low binding affinity for DLCs of AhR1s of African clawed frog (<em>Xenopus laevis</em>) and axolotl (<em>Ambystoma mexicanum</em>) has been suggested as a mechanism for tolerance of these amphibians to DLCs (Lavine et al 2005; Shoots et al 2015).</li>
  • <li>Among reptiles, only AhRs of American alligator (<em>Alligator mississippiensis</em>) have been investigated and little is known about the sensitivity of American alligator or other reptiles to DLCs (Oka et al 2016).</li>
  • <li>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).</li>
  • <li>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 (<em>Microgadus tomcod</em>) (Wirgin et al 2011).
  • <ul>
  • <li>This was attributed to the rapid evolution of populations in highly contaminated areas of the Hudson River, resulting in a 6-base pair deletion in the AHR sequence (outside the LBD) and reduced ligand binding affinity, due to reduces AHR protein stability.</li>
  • </ul>
  • </li>
  • <li>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).</li>
  • </ul>
  • <p><span style="font-size:12pt"><span style="font-family:&quot;Times New Roman&quot;,serif">The AhR is a very conserved and ancient protein (95) and the AhR is present &nbsp;in human and mice (96&ndash;98).&nbsp;</span></span><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">The AhR is present in human physiology and pathology. T</span></span><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">he AhR is highly expressed at several important physiological barriers such as the placenta, lung, gastrointestinal system, and liver in human (Wakx, Marinelli, Watanabe). &nbsp;In these tissues, the AhR is involved in both detoxication processes involving xenobiotic metabolizing enzymes such as cytochromes P450, and in immune functions translating chemical signals into immune defence pathways (Marinelli, Stobbe). Moreover, it has a regulatory role in human dendritic cells and myelination (Kado, Shackleford).</span></span> <span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">The lung constitutes another barrier exposed to components of air pollution such as particles and hydrocarbons (air pollution, cigarette smoke). The AhR detects such hydrocarbons and protects the pulmonary cells from their deleterious effects through metabolization.</span></span> <span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">The regulatory effect on blood cells of the AhR, balancing different related cell types, can be extended to the megakaryocytes and their precursors; indeed, StemRegenin 1 (SR1), an antagonist of the AhR increases the human population of CD34+CD41low cells, a fraction of very efficient precursors of proplatelets (Bock).</span></span> <span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">The occurrence of a nystagmus has been subsequently diagnosed in humans bearing a AhR mutation (Borovok).</span></span></span></span></p>
  • <p><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">In human cancer, the AhR has either a pro or con tumor effect depending on the tissue, the ligand, and the duration of the activation (Zudaire, Chang, Litzenburg, Gramatzki, Lin, Wang). In human breast cancer, the AhR is thoughts to be responsible of its progression (Goode, Kanno, Optiz, Novikov, Hall, </span></span><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Subramaniam, Barhoover</span></span><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">). </span></span><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">In human mammary benign cells, Brooks et al. noted that a high level of AhR was associated with a modified cell cycle (with a 50% increase in population doubling time in cells expressing the AhR by more than 3-fold) and EMT including increased cell migration. Narasimnhan et al. found that suppression of the AhR pathway had a pro-tumorigenic effect in vitro (EMT, tumor migration) in triple negative breast cancer.</span></span></span></span></p>
  • <p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Many endogenous and exogenous ligands are present for the AhR in human (Optiz, Adachi, Schroeder, </span></span><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Rothhammer</span></span><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">). </span></span><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Indoles, such as indole-3-carbinol or one of its secondary metabolites, 3-3&#39;- Diindolylmethane, are degradation products found in cruciferous vegetables and characterized as AhR ligands (Ema, Kall, Miller) they are also inducers of the human and rat CYP1A1 (Optiz). FICZ is the most potent AhR ligand known to date: it has a stronger affinity than TCDD for the human AhR (TCDD Kd=0.48 nM/FICZ Kd=0.07 nM) (Coumoul).</span></span></span></span></p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <h4>Key Event Description</h4>
  • <h3>The AHR Receptor</h3>
  • <p>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)<sup><a href="#cite_note-Okey2007-1">[1]</a></sup>. Other members of this superfamily include the AHR nuclear translocator (ARNT), which acts as a dimerization partner of the AHR <sup><a href="#cite_note-Hoffman1991-2">[2]</a></sup><sup><a href="#cite_note-Poland1976-3">[3]</a></sup>; Per, a circadian transcription factor; and Sim, the &ldquo;single-minded&rdquo; protein involved in neuronal development <sup><a href="#cite_note-Gu2000-4">[4]</a></sup><sup><a href="#cite_note-Kewley2004-5">[5]</a></sup>. This group of proteins shares a highly conserved PAS domain and is involved in the detection of and adaptation to environmental change<sup><a href="#cite_note-Gu2000-4">[4]</a></sup>.</p>
  • <p>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).</p>
  • <h3>The molecular Initiating Event</h3>
  • <div>
  • <div><a class="image" href="/wiki/index.php/File:AHR_mechanism.jpeg"><img alt="" class="thumbimage" src="/wiki/images/thumb/6/6e/AHR_mechanism.jpeg/450px-AHR_mechanism.jpeg" style="height:331px; width:450px" /></a>
  • <div>Figure 1: The molecular mechanism of activation of gene expression by AHR.</div>
  • <div>&nbsp;</div>
  • </div>
  • </div>
  • <p>The molecular mechanism for AHR-mediated activation of gene expression is presented in Figure 1. 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)<sup><a href="#cite_note-Fujii2010-6">[6]</a></sup>. Upon ligand binding, the AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with ARNT<sup><a href="#cite_note-Mimura2003-7">[7]</a></sup>. 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<sup><a href="#cite_note-Fujii2010-6">[6]</a></sup>. 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 <sup><a href="#cite_note-Fujii2010-6">[6]</a></sup><sup><a href="#cite_note-Giesy2006-8">[8]</a></sup><sup><a href="#cite_note-Mimura2003-7">[7]</a></sup><sup><a href="#cite_note-Safe1994-9">[9]</a></sup>.</p>
  • <h3>AHR Isoforms</h3>
  • <ul>
  • <li>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).</li>
  • <li>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).</li>
  • <li>The AhR3 is poorly understood and known only from some cartilaginous fishes (Hahn 2002).</li>
  • <li>Little is known about diversity of AhRs in reptiles and amphibians (Hahn et al 2002).</li>
  • <li>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 (&alpha;, &beta;, &delta;, &gamma;) having been identified in Atlantic salmon (<em>Salmo salar</em>) (Hansson et al 2004).</li>
  • <li>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).</li>
  • </ul>
  • <p>&nbsp;</p>
  • <p>Roles of isoforms in birds:</p>
  • <p>Two AHR isoforms (AHR1 and AHR2) have been identified in the black-footed albatross (<em>Phoebastria nigripes</em>), great cormorant (<em>Phalacrocorax carbo</em>) and domestic chicken (<em>Gallus gallus domesticus</em>)<sup><a href="#cite_note-Yasui2007-10">[10]</a></sup>. 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<sup><a href="#cite_note-Yasui2007-10">[10]</a></sup>, 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<sup><a href="#cite_note-Lee2009-11">[11]</a></sup><sup><a href="#cite_note-Yasui2007-10">[10]</a></sup>.</p>
  • <ul>
  • <li>AhR1 and AhR2 both bind and are activated by TCDD <em>in vitro</em> (Yasui et al 2007).</li>
  • <li>AhR1 has greater binding affinity and sensitivity to activation by TCDD relative to AhR2 (Yasui et al 2007).</li>
  • <li>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).</li>
  • </ul>
  • <p>Roles of isoforms in fishes:</p>
  • <ul>
  • <li>AhR1 and AhR2 both bind and are activated by TCDD <em>in vitro</em> (Bak et al 2013; Doering et al 2014; 2015; Karchner et al 1999; 2005).</li>
  • <li>AhR1 has greater sensitivity to activation by TCDD than AhR2 in red seabream (<em>Pagrus major</em>), white sturgeon (<em>Acipenser transmontanus</em>), and lake sturgeon (<em>Acipenser fulvescens</em>) (Bak et al 2013; Doering et al 2014; 2015)</li>
  • <li>AhR2 has greater binding affinity or activation by TCDD than AhR1 in zebrafish (<em>Danio rerio</em>) and mummichog (<em>Fundulus heteroclitus</em>) (Karchner et al 1999; 2005).</li>
  • <li>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 (<em>Danio rerio</em>) and mummichog (<em>Fundulus heteroclitus</em>), while knockdown of AhR1 offers no protection (Clark et al 2010; Prasch et al 2003; Van Tiem &amp; Di Giulio 2011).</li>
  • </ul>
  • <p>Roles of isoforms in amphibians and reptiles:</p>
  • <ul>
  • <li>Less is known about AhRs of amphibians or reptiles.</li>
  • <li>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).</li>
  • <li>Both AhR1s and AhR2 of American alligator (<em>Alligator mississippiensis</em>) are activated by agonists with comparable sensitivities (Oka et al 2016). AhRs of no other reptiles have been investigated.</li>
  • </ul>
  • <h4>How it is Measured or Detected</h4>
  • <p><em>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? </em></p>
  • <h3>Transactivation Reporter Gene Assays (recommended approach)</h3>
  • <h4>Transient transfection transactivation</h4>
  • <p>Transient transfection transactivation is the most common method for evaluating nuclear receptor activation<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. Full-length AHR cDNAs are cloned into an expression vector along with a 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)<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. 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<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>.</p>
  • <h5>Luciferase reporter gene (LRG) assay</h5>
  • <p>The described luciferase reporter gene (LRG) assays have been used to investigate activation of AhRs of:</p>
  • <ul>
  • <li>Humans&nbsp;(<em>Homo sapiens</em>)&nbsp;(Abnet et al 1999)&nbsp;</li>
  • <li>Species of birds, namely chicken (<em>Gallus gallus</em>), ring-necked pheasant (<em>Phasianus colchicus</em>), Japanese quail (<em>Coturnix japonica</em>), and common tern (<em>Sterna hirundo</em>)&nbsp;(Farmahin et al 2012; Manning et al 2013), Mutant AhR1s with ligand binding domains resembling those of at least 86 avian species have also been investigated (Farmahin et al 2013). AhR2s of birds have only been investigated in black-footed albatross (<em>Phoebastria nigripes</em>) and common cormorant (<em>Phalacrocorax carbo</em>) (Yasio et al 2007).</li>
  • <li>American alligator (<em>Alligator mississippiensis</em>) is the only reptile for which&nbsp;AhR activation&nbsp;has been investigated&nbsp;(Oka et al 2016), AhR1A, AhR1B, and AhR2 of American alligator were assayed (Oka et al 2016).</li>
  • <li>AhR1 of two amphibians have been investigated, namely African clawed frog (<em>Xenopus laevis</em>) and salamander (<em>Ambystoma mexicanum</em>) (Lavine et al 2005; Shoots et al 2015; Ohi et al 2003),</li>
  • <li>AhR1s and AhR2s of several species of fish have been investigated, namely Atlantic salmon (<em>Salmo salar</em>), Atlantic tomcod (<em>Microgadus tomcod</em>), white sturgeon (<em>Acipenser transmontanus</em>), rainbow trout (<em>Onchorhynchys mykiss</em>), red seabream (<em>Pagrus major</em>), lake sturgeon (<em>Acipenser fulvescens</em>), and zebrafish (<em>Danio rerio</em>) (Andreasen et al 2002; Abnet et al 1999; Bak et al 2013; Doering et al 2014; 2015; Evans et al 2005; Hansson &amp; Hahn 2008; Karchner et al 1999; Tanguay et al 1999; Wirgin et al 2011).</li>
  • </ul>
  • <p>For demonstrative purposes, a luciferase reporter gene assay used to measure AHR1-mediated transactivation for avian species is described here. However, comparable assays are utilized for investigating AHR1s and AHR2s of all taxa. 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 <em>Renilla</em> 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 <em>Renilla</em> luciferase units <sup><a href="#cite_note-Farmahin2012-13">[13]</a></sup>. 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).<sup><a href="#cite_note-Farmahin2013b-14">[14]</a></sup><sup><a href="#cite_note-Farmahin2012-13">[13]</a></sup><sup><a href="#cite_note-Fujisawa2012-15">[15]</a></sup><sup><a href="#cite_note-Lee2009-11">[11]</a></sup><sup><a href="#cite_note-Manning2012-16">[16]</a></sup><sup><a href="#cite_note-Mol2012-17">[17]</a></sup></p>
  • <h4>Transactivation in stable cell lines</h4>
  • <p>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 <sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. 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<sup><a href="#cite_note-Yueh2005-18">[18]</a></sup>. An added benefit of this model is the potential to multiplex 3 assays in a single well: receptor activation, cell viability and enzyme activity<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. Such assays are used extensively in drug discovery due to their high throughput efficiency, and may serve just as useful for risk assessment purposes.</p>
  • <h3>Ligand-Binding Assays</h3>
  • <p>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<sup><a href="#cite_note-Poland1982-19">[19]</a></sup> and can explain differences in species sensitivities to DLCs<sup><a href="#cite_note-Hesterman2000-20">[20]</a></sup><sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>; they are therefore worth mentioning. Binding affinity and efficacy have been used to develop structure-activity relationships for AHR disruption<sup><a href="#cite_note-Hesterman2000-20">[20]</a></sup><sup><a href="#cite_note-Lee2015-23">[23]</a></sup> 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<sup><a href="#cite_note-Jones2003-24">[24]</a></sup><sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. 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.</p>
  • <h4>Hydroxyapatite (HAP) binding assay</h4>
  • <p>The HAP binding assay makes use of an <em>in vitro</em> 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)<sup><a href="#cite_note-Gasiewicz1982-25">[25]</a></sup><sup><a href="#cite_note-Karchner2006-22">[22]</a></sup>. This assay is simple, repeatable and reproducible; however, it is insensitive to weak ligand-receptor interactions<sup><a href="#cite_note-Karchner2006-22">[22]</a></sup><sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup><sup><a href="#cite_note-Nakai1995-26">[26]</a></sup>.</p>
  • <h4>Whole cell filtration binding assay</h4>
  • <p>Dold and Greenlee<sup><a href="#cite_note-Dold1990-27">[27]</a></sup> developed a method to detect specific binding of TCDD to whole mammalian cells in culture and was later modified by Farmahin et al.<sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup> 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<sup><a href="#cite_note-Farmahin2014-21">[21]</a></sup>.</p>
  • <h3>Protein-DNA Interaction Assays</h3>
  • <p>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<sup><a href="#cite_note-Perez2007-28">[28]</a></sup>. Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions <em>in vivo</em>. 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 <em>in vivo</em>. 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<sup><a href="#cite_note-Heid2001-29">[29]</a></sup>.</p>
  • <h3>In silico Approaches</h3>
  • <p>In silico homology modeling of the ligand binding domain of the AHR in combination with molecular docking simulations can provide valuable insight into the transactivation-potential of a diverse array of AHR ligands.&nbsp; Such models have been developed for multiple AHR isoforms and ligands (high/low affinity, endogenous and synthetic, agonists and antagonists), and can accurately predict ligand potency based on their structure and physicochemical properties (Bonati et al 2017; Hirano et al 2015; Sovadinova et al 2006).</p>
  • <h4>References</h4>
  • <ol>
  • <h4>References</h4>
  • <ol>
  • <li>&uarr; <sup><a href="#cite_ref-Okey2007_1-0">1.0</a></sup> <sup><a href="#cite_ref-Okey2007_1-1">1.1</a></sup> Okey, A. B. (2007). An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XI. <em>Toxicol.Sci.</em> <strong>98</strong>, 5-38.</li>
  • <li><a href="#cite_ref-Hoffman1991_2-0">&uarr;</a> 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. <em>Science</em> <strong>252</strong>, 954-958.</li>
  • <li>&uarr; <sup><a href="#cite_ref-Poland1976_3-0">3.0</a></sup> <sup><a href="#cite_ref-Poland1976_3-1">3.1</a></sup> 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. <em>J.Biol.Chem.</em> <strong>251</strong>, 4936-4946.</li>
  • <li>&uarr; <sup><a href="#cite_ref-Gu2000_4-0">4.0</a></sup> <sup><a href="#cite_ref-Gu2000_4-1">4.1</a></sup> Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily: sensors of environmental and developmental signals. <em>Annu.Rev.Pharmacol.Toxicol.</em> <strong>40</strong>, 519-561.</li>
  • <li><a href="#cite_ref-Kewley2004_5-0">&uarr;</a> Kewley, R. J., Whitelaw, M. L., and Chapman-Smith, A. (2004). The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. <em>Int.J.Biochem.Cell Biol.</em> <strong>36</strong>, 189-204.</li>
  • <li>&uarr; <sup><a href="#cite_ref-Fujii2010_6-0">6.0</a></sup> <sup><a href="#cite_ref-Fujii2010_6-1">6.1</a></sup> <sup><a href="#cite_ref-Fujii2010_6-2">6.2</a></sup> <sup><a href="#cite_ref-Fujii2010_6-3">6.3</a></sup> 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. <em>Proc.Jpn.Acad.Ser.B Phys.Biol.Sci.</em> <strong>86</strong>, 40-53.</li>
  • <li>&uarr; <sup><a href="#cite_ref-Mimura2003_7-0">7.0</a></sup> <sup><a href="#cite_ref-Mimura2003_7-1">7.1</a></sup> Mimura, J., and Fujii-Kuriyama, Y. (2003). Functional role of AhR in the expression of toxic effects by TCDD. <em>Biochimica et Biophysica Acta - General Subjects</em> <strong>1619</strong>, 263-268.</li>
  • <li><a href="#cite_ref-Giesy2006_8-0">&uarr;</a> 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.</li>
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  • <p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Barhoover MA, Hall JM, Greenlee WF, Thomas RS. 2010. Aryl hydrocarbon receptor regulates cell cycle progression in human breast cancer cells via a functional interaction with cyclin-dependent kinase 4. Mol Pharmacol. 77(2):195&ndash;201</span></span></span></span></p>
  • <p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif">&nbsp;</span></span></p>
  • <p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Wang K, Li Y, Jiang Y-Z, Dai C-F, Patankar MS, et al. 2013. </span></span><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells. Cancer Lett. 340(1):63&ndash;71</span></span></span></span></p>
  • <p style="text-align:justify">&nbsp;</p>
  • <p><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Borovok N, Weiss C, Sharkia R, Reichenstein M, Wissinger B, et al. 2020. Gene and Protein Expression in Subjects With a Nystagmus-Associated AHR Mutation. Front Genet. 11:582796</span></span></span></span></p>
  • <br>
  • <!-- end event text -->
  • </div>
  • <!-- ke table -->
  • <h3>Key Events</h3>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <h3>List of Key Events in the AOP</h3>
  • <h4><a href="/events/944">Event: 944: dimerization, AHR/ARNT</a></h4>
  • <h5>Short Name: dimerization, AHR/ARNT</h5>
  • <h4>Key Event Component</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Process</th>
  • <th scope="col">Object</th>
  • <th scope="col">Action</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <th>Title</th>
  • <th>Short name</th>
  • <td>protein dimerization activity</td>
  • <td>aryl hydrocarbon receptor</td>
  • <td>increased</td>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td><a href="/events/944">dimerization, AHR/ARNT</a></td>
  • <td>dimerization, AHR/ARNT</td>
  • </tr>
  • <tr>
  • <td><a href="/events/1269">Increase, COX-2 expression</a></td>
  • <td>Increase, COX-2 expression</td>
  • </tr>
  • <tr>
  • <td><a href="/events/317">Altered, Cardiovascular development/function</a></td>
  • <td>Altered, Cardiovascular development/function</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <div>
  • <div>
  • <h4><a href="/events/944">944: dimerization, AHR/ARNT</a><br></h4>
  • <h5>Short Name: dimerization, AHR/ARNT</h5>
  • </div>
  • <div>
  • <!-- loop to find all aops that use this event -->
  • <h4>AOPs Including This Key Event</h4>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>AOP ID and Name</th>
  • <th>Event Type</th>
  • <td>protein dimerization activity</td>
  • <td>aryl hydrocarbon receptor nuclear translocator</td>
  • <td>increased</td>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td><a href="/aops/150">150: Aryl hydrocarbon receptor activation leading to embryolethality via cardiotoxicty</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/21">21: Activation of the aryl hydrocarbon receptor (AhR) leading to early life stage mortality</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- loop to find stressors under event -->
  • <div>
  • </tbody>
  • </table>
  • </div>
  • <br>
  • <!-- biological organization -->
  • <div>
  • <h4>Biological Organization</h4>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>Level of Biological Organization</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Molecular</td>
  • </tr>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/150">Aop:150 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via reduced VEGF</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/21">Aop:21 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/310">Aop:310 - Embryonic Activation of the AHR leading to Reproductive failure, via epigenetic down-regulation of GnRHR</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/151">Aop:151 - AhR activation leading to preeclampsia</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/455">Aop:455 - Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced impeded craniofacial development</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/456">Aop:456 - Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)</td></tr>
  • <tr><td>Stressor:147 Dibenzo-p-dioxin</td></tr>
  • <tr><td>Polychlorinated biphenyl</td></tr>
  • <tr><td>Polychlorinated dibenzofurans</td></tr>
  • <tr><td>Polycyclic aromatic hydrocarbons</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of bio org -->
  • <!-- cell term -->
  • <div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Molecular</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of cell term -->
  • <!-- organ term -->
  • <div>
  • <h4>Cell term</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Cell term</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>eukaryotic cell</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of organ term -->
  • <h4>Evidence Supporting Applicability of this Event</h4>
  • <br>
  • <!-- loop to find taxonomic applicability under event -->
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>Term</th>
  • <th>Scientific Term</th>
  • <th>Evidence</th>
  • <th>Links</th>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>chicken</td>
  • <td>Gallus gallus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>zebrafish</td>
  • <td>Danio rerio</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>chicken</td>
  • <td>Gallus gallus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>zebrafish</td>
  • <td>Danio rerio</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Coturnix japonica</td>
  • <td>Coturnix japonica</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=93934" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Phasianus colchicus</td>
  • <td>Phasianus colchicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9054" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rainbow trout</td>
  • <td>Oncorhynchus mykiss</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8022" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Pagrus major</td>
  • <td>Pagrus major</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=143350" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Acipenser fulvescens</td>
  • <td>Acipenser fulvescens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=41871" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Acipenser transmontanus</td>
  • <td>Acipenser transmontanus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7904" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Salmo salar</td>
  • <td>Salmo salar</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8030" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Xenopus laevis</td>
  • <td>Xenopus laevis</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Ambystoma mexicanum</td>
  • <td>Ambystoma mexicanum</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8296" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Microgadus tomcod</td>
  • <td>Microgadus tomcod</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34823" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for taxons -->
  • <!-- life stages -->
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Life Stage</th>
  • <th>Evidence</th>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Embryo</td>
  • <td>Strong</td>
  • </tr>
  • <tr>
  • <td>development</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>Development</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end life stages -->
  • <!-- sex terms -->
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Sex</th>
  • <th>Evidence</th>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Unspecific</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end sex terms -->
  • <div>
  • <ul>
  • <p>Taxonomic Presence of ARNT genes:</p>
  • <ul>
  • <li>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).</li>
  • <li>ARNTs have been identified in a great phylogenetic diversity of fishes, including early fishes (Doering et al 2014; 2016).</li>
  • <li>ARNT has been identified in investigated invertebrates (Powell-Coffman et al 1998).</li>
  • </ul>
  • <p>Taxonomic Applicability of Heterodimerization of ARNT isoforms with AhR isoforms:</p>
  • <ul>
  • <li>&nbsp;In mouse (<em>Mus mus</em>) and chicken (<em>Gallus gallus</em>) both the ARNT1 and ARNT2 were capable of heterdimerizing with AHR and interacting with dioxin-responsive elements on the DNA <em>in vitro</em> (Hirose et al 1996; Lee et al 2007; Lee et al 2011; Prasch et al 2004). However, no studies have yet confirmed involvement of both ARNT1 and ARNT2 <em>in vivo</em>.</li>
  • <li>In zebrafish, all adverse effects of DLCs so far examined <em>in vivo</em> are mediated solely by ARNT1 based on knockdown studies, although ARNT2 is capable of heterodimerizing with AHR2 and interacting with dioxin-responsive elements on the DNA <em>in vitro</em> (Prasch et al 2004; Prasch et al 2006).&nbsp;In addition to AHRs of zebrafish, AHRs of Atlantic salmon (<em>Salmo salar</em>), Atlantic tomcod (<em>Microgadus tomcod</em>), mummichog, rainbow trout, and red seabream (<em>Pagrus major</em>) have been demonstrated to heterodimerize with ARNT1 <em>in vitro</em> (Abnet et al 1999; Bak et al 2013; Hansson &amp; Hahn 2008; Karchner et al 1999; Wirgin et al 2011), while AHRs of white sturgeon (<em>Acipenser transmontanus</em>), and lake sturgeon (<em>Acipenser fulvescens</em>) have been demonstrated to heterodimerize with ARNT2 <em>in vitro</em> (Doering et al 2014b; 2015b; Prasch et al 2004; 2006).&nbsp;</li>
  • </ul>
  • <p>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).</p>
  • <br>
  • </div>
  • <!-- event text -->
  • <h4>How this Key Event Works</h4>
  • <p><strong>Structure and Function of ARNT</strong></p>
  • <h4>Key Event Description</h4>
  • <p><strong>Structure and Function of ARNT</strong></p>
  • <ul>
  • <li>The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the Per-Arnt-Sim (PAS) family of proteins (Gu et al 2000).</li>
  • <li>PAS proteins share highly conserved PAS domains (Gu et al 2000).</li>
  • <li>PAS proteins act as transcriptional regulators in response to environmental and physiological cues (Gu et al 2000).</li>
  • <li>ARNTs have numerous key roles in vertebrates related to responses to developmental and environmental cues.</li>
  • </ul>
  • <p>Isoforms of ARNT:</p>
  • <ul>
  • <li>Over time ARNT has undergone gene duplication and diversification in vertebrates, which has resulted in three clades of ARNT, namely ARNT1, ARNT2, and ARNT3.</li>
  • <li>Each clade can include multiple isoforms and splice variants (Hill et al 2009; Lee et al 2007; Lee et al 2011; Powel &amp; Hahn 2000; Tanguay et al 2000).</li>
  • <li>ARNT1s have been demonstrated to function predominantly through heterodimerization with the aryl hydrocarbon receptor (AhR) and hypoxia inducible factor 1 &alpha; (HIF1&alpha;) (Prasch et al 2004; 2006; Wang et al 1995).</li>
  • <li>ARNT2s are believed to function predominantly through heterodimerization with Single Minded (SIM) (Hirose et al 1996).</li>
  • <li>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).</li>
  • <li>ARNTs have numerous key roles in vertebrates related to responses to developmental and environmental cues.</li>
  • </ul>
  • <p>Roles of ARNTs in mammals:</p>
  • <ul>
  • <li>ARNT1 functions in normal vascular and hematopoietic development (Kozak et al 1997; Maltepe et al 1997; Abbott &amp; Buckalew 2000).</li>
  • <li>ARNT2 functions in development of the hypothalamus and nervous system (Hosoya et al 2001; Keith et al 2001).</li>
  • <li>ARNT3 functions in biological rhythms (Gekakis et al 1998).</li>
  • </ul>
  • <p>Roles of ARNTs in other taxa:</p>
  • <ul>
  • <li>ARNTs have been demonstrated to have roles in development of the heart, brain, liver, and possibly the peripheral nervous system in zebrafish (<em>Danio rerio</em>) (Hill et al 2009).</li>
  • <li>Roles of ARNTs in other taxa have not been sufficiently investigated to date.</li>
  • </ul>
  • <p><strong>Interaction with AHR</strong></p>
  • <ul>
  • <li>Both ARNT1s and ARNT2s are able to heterodimerize with AhR and interact with dioxin-responsive elements on the DNA in<em> in vitro</em> systems (Hirose et al 1996; Lee et al 2007; Lee et al 2011; Prasch et al 2004).</li>
  • <li>Selective knockdown of ARNTs in zebrafish (<em>Danio rerio</em>) demonstrates that ARNT1s, but not ARNT2s, are required for activation of the AhR<em> in vivo </em>(Prasch et al 2004; 2006).</li>
  • <li>In limited investigations ARNT3 has not been demonstrated to interact with the AHR either <em>in vivo</em> or <em>in vitro</em> (Jain et al 1998).&nbsp;</li>
  • </ul>
  • <p>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).</p>
  • <br>
  • <h4>How it is Measured or Detected</h4>
  • <p>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).</p>
  • <br>
  • <h4>How it is Measured or Detected</h4>
  • <p>AhR/ARNT heterodimerization can be measured in several ways:</p>
  • <p>1) 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).</p>
  • <p>2) Species-specific differences in dimerization and differences in dimerization between ARNT isoform&nbsp;and AhR isoform combinations have been assessed through luciferase reporter gene (LRG) assays utilizing&nbsp;COS-7 cells transfected with expression constructs of AhR and ARNT isoforms of mammals, birds, and fishes (Abnet et al 1999; Bak et al 2013; Doering et al 2014; 2015; Hansson &amp; Hahn 2008;&nbsp;Hirose et al 1996;&nbsp;Karchner et al 1999;&nbsp;Lee et al 2007; Lee et al 2011; Prasch et al 2004;&nbsp;Wirgin et al 2011). However, this method is indirect as it also includes binding of a ligand to the AhR, and interaction of the AhR/ARNT heterodimer with dioxin-responsive elements on the DNA.</p>
  • <h4>References</h4>
  • <p>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.</p>
  • <h4>References</h4>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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<em>.</em> 159, 41-51.</p>
  • <p>&nbsp;</p>
  • <p>Andreasen, E.A.; Hahn, M.E.; Heideman, W.; Peterson, R.E.; Tanguay, R.L. 2002. The zebrafish (<em>Danio rerio</em>) aryl hydrocarbon receptor type 1 is a novel vertebrate receptor. Molec. Pharmacol. 62, 234-249.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Chen, G.; Bunce, N.J. (2003). Polybrominated diphenyl ethers as Ah receptor agonists and antagonists. Toxicol. Sci. 76 (2), 310-320.</p>
  • <p>&nbsp;</p>
  • <p>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. <strong>1998</strong>, 61 (5), 557-568.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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 (<em>Acipenser transmontanus</em>) to dioxin-like compounds. Aquat. Toxicol. 150, 27-35.</p>
  • <p>&nbsp;</p>
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  • <p>&nbsp;</p>
  • <p>Farmahin, R.; Crump, D.; O&rsquo;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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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 <em>in vitro </em>function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict <em>in vivo </em>sensitivity to dioxins. Enviro. Sci. Toxicol. 46 (5), 2967-2975.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Hansson, M.C.; Hahn, M.E. 2008. Functional properties of the four Atlantic salmon (<em>Salmo salar</em>) aryl hydrocarbon receptor type 2 (AHR2) isoforms. Aquat. Toxicol. 86, 121-130.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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 <em>Fundulus heteroclitus</em>. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors. J. Biol. Chem. 274, 33814-33824.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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 (<em>Phalacrocorax carbo</em>). Comp. Biochem. Physiol. C. 145, 379-393.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
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  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
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  • <p>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-<em>p</em>-dioxin developmental toxicity in zebrafish. 76 (1), 138-150.</p>
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  • <p>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<em>. </em>49, 6993-7001.</p>
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  • <p>Tanguay, R.L.; Abnett, C.C.; Heideman, W.; Peterson, R.E. 1999. Cloning and characterization of the zebrafish (<em>Danio rerio</em>) aryl hydrocarbon receptor. Biochem. Biophys. Acta. 1444, 35-48.</p>
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  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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. <strong>1998</strong>, 106, 775-792.</p>
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  • <p>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.</p>
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  • <p>Jain, S.; Maltepe, E.; Lu, M.M.; Simon, C.; Bradfield, C.A. 1998. Expression of ARNT, ARNT2, HIF1 alpha, HIF2 alpha, and Ah receptor mRNAs in the developing mouse. Mech. Dev. 73, 117-123.</p>
  • <br>
  • <!-- end event text -->
  • </div>
  • <div>
  • <div>
  • <h4><a href="/events/1269">1269: Increase, COX-2 expression</a><br></h4>
  • <h5>Short Name: Increase, COX-2 expression</h5>
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  • <h4>AOPs Including This Key Event</h4>
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  • <h4><a href="/events/1269">Event: 1269: Increase, COX-2 expression</a></h4>
  • <h5>Short Name: Increase, COX-2 expression</h5>
  • <h4>Key Event Component</h4>
  • <div class="table-responsive">
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  • <th scope="col">Process</th>
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  • <th scope="col">Action</th>
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  • <tr>
  • <th>AOP ID and Name</th>
  • <th>Event Type</th>
  • <td>gene expression</td>
  • <td>prostaglandin G/H synthase 2</td>
  • <td>increased</td>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td><a href="/aops/21">21: Activation of the aryl hydrocarbon receptor (AhR) leading to early life stage mortality</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
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  • <h4>Biological Organization</h4>
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  • <th>Level of Biological Organization</th>
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  • <td>Molecular</td>
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  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
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  • <thead class="thead-light">
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  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
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  • <td><a href="/aops/21">Aop:21 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </tbody>
  • </table>
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  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
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  • <tr><th scope="col">Name</th></tr>
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  • <tr><td>2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)</td></tr>
  • <tr><td>Aristolochic acid</td></tr>
  • <tr><td>Doxorubicin</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of bio org -->
  • <!-- cell term -->
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  • <h4>Biological Context</h4>
  • <div class="table-responsive">
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  • <tr><th scope="col">Level of Biological Organization</th></tr>
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  • <tr><td>Molecular</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of cell term -->
  • <!-- organ term -->
  • <div>
  • <h4>Cell term</h4>
  • <div class="table-responsive">
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  • <thead class="thead-light">
  • <tr><th scope="col">Cell term</th></tr>
  • </thead>
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  • <tr><td>eukaryotic cell</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of organ term -->
  • <h4>Evidence Supporting Applicability of this Event</h4>
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  • <!-- loop to find taxonomic applicability under event -->
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  • <strong>Taxonomic Applicability</strong>
  • <div class="panel panel-default">
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  • <thead>
  • <tr>
  • <th>Term</th>
  • <th>Scientific Term</th>
  • <th>Evidence</th>
  • <th>Links</th>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Danio rerio</td>
  • <td>Danio rerio</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Oryzias latipes</td>
  • <td>Oryzias latipes</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8090" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Danio rerio</td>
  • <td>Danio rerio</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Oryzias latipes</td>
  • <td>Oryzias latipes</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for taxons -->
  • <!-- life stages -->
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Life Stage</th>
  • <th>Evidence</th>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Embryo</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end life stages -->
  • <!-- sex terms -->
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Sex</th>
  • <th>Evidence</th>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Unspecific</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end sex terms -->
  • <div>
  • <ul>
  • <p><strong>COX-2 Structure and Function:</strong></p>
  • <ul>
  • <li>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.</li>
  • <li>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).</li>
  • <li>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 are two isoforms,&nbsp;COX-2a and COX-2b (Teraoka et al 2014).</li>
  • <li>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).</li>
  • </ul>
  • <br>
  • </div>
  • <!-- event text -->
  • <h4>How this Key Event Works</h4>
  • <ul>
  • <h4>Key Event Description</h4>
  • <p><strong>COX Pathway:</strong></p>
  • <p><strong><a href="https://aopwiki.org/system/dragonfly/production/2017/05/08/7vmvnr8r73_COX_pathway.pdf">https://aopwiki.org/system/dragonfly/production/2017/05/08/7vmvnr8r73_COX_pathway.pdf</a></strong></p>
  • <ul>
  • <li>Prostaglandin-endoperoxide synthase (PTGS; KEGG ID E.C. 1.14.99.1) is an enzyme that has two catalytic sites.</li>
  • <li>Cyclooxygenase site (COX) catalyzes conversion of arachidonic acid into endoperoxide prostaglandin G2 (<a href="https://aopwiki.org/wiki/index.php?title=Simmons_et_al.,_2004&amp;action=edit&amp;redlink=1" title="Simmons et al., 2004 (page does not exist)">Simmons et al 2004</a>).</li>
  • <li>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 &amp; Botting 2011).</li>
  • <li>There are two isoforms, COX-1 and COX-2</li>
  • <li>COX-2 is inducible by certain chemical exposures, inflammation, during discrete stages of gamete maturation, and more (Green et al 2012).&nbsp;</li>
  • <li>COX-2 is inducible by certain chemical exposures, inflammation, during discrete stages of gamete maturation, and more (Green et al 2012).</li>
  • <li>However, COX biology is complex and important details of the pathway remain unknown (Grosser 2006).</li>
  • </ul>
  • <br>
  • <h4>How it is Measured or Detected</h4>
  • <ul>
  • <li>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).</li>
  • <p><strong>COX Cardiovascular Roles:</strong></p>
  • <ul>
  • <li>Prostaglandins which are catalyzed by COX and have roles in cellular homeostasis and in promoting inflammatory responses (Chien et al 2015; Smith et al 2000; Tilley et al 2001; Vane et al 1994).</li>
  • <li>Significant&nbsp;evidence suggests a link between COX-2 mediated inflammatory responses and progression of alterations in&nbsp;cardiovascular development and function in murine&nbsp;models, humans, and zebrafish (<em>Danio rerio</em>)&nbsp;(Delgado et al 2004; Gullestad &amp; Aukrust 2005; Hocherl et al 2002; Huang et al 2007; Wong et al 1998 ).</li>
  • <li>However, the precise mechanism by which prostaglandins produce alterations in cardiovascular development have not been clearly elucidated (Hocherl &amp; Dreher 2002).</li>
  • </ul>
  • <br>
  • <h4>How it is Measured or Detected</h4>
  • <ul>
  • <li>COX-2 can be&nbsp;measured as abundance of transcript by use of quantitative real-time polymerase chain reaction (q-RT PCR). Transcript abundance of COX-2 has been measured in whole embryos of fishes (Dong et al 2010; Huang et al 2007; Teraoka et al 2008; 2014) and&nbsp;embryonic hepatic and&nbsp;cardiac tissue of birds (Fujisawa et al 2014).</li>
  • <li>COX-2 could be measured by use of ELISA or Western Blot, but commercial kits are not currently available for fishes or birds.</li>
  • </ul>
  • <h4>References</h4>
  • <p>Bacchi, S., Palumbo, P., Sponta, A., &amp; Coppolino, M. F. (2012). Clinical pharmacology of non-steroidal anti-inflammatory drugs: a review. Anti-Inflammatory &amp; Anti-Allergy Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Inflammatory and Anti-Allergy Agents), 11(1), 52-64.</p>
  • <h4>References</h4>
  • <p>Bacchi, S., Palumbo, P., Sponta, A., &amp; Coppolino, M. F. (2012). Clinical pharmacology of non-steroidal anti-inflammatory drugs: a review. Anti-Inflammatory &amp; Anti-Allergy Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Inflammatory and Anti-Allergy Agents), 11(1), 52-64.</p>
  • <p>&nbsp;</p>
  • <p>Botting, R. M., &amp; Botting, J. H. (2011). C14 Non-steroidal anti-inflammatory drugs. In Principles of Immunopharmacology (pp. 573-584). Birkh&auml;user Basel.</p>
  • <p>&nbsp;</p>
  • <p>Chien, P.; Lin, C.; Hsiao, L.; Yang, C. (2015). c-SRC/Pyk2/EGFR/PI3K/Akt/CREB-activated pathway contributes to human cardiomyocyte hypertrophy: Role of COX-2 induction. Mol. Cell. Endocrin. 409. 59-72.</p>
  • <p>&nbsp;</p>
  • <p>Chandrasekharan, N. V., Dai, H., Roos, K. L. T., Evanson, N. K., Tomsik, J., Elton, T. S., &amp; 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.</p>
  • <p>&nbsp;</p>
  • <p>Crofford, L.J. (1997). COX-1 and COX-2 tissue expression: implications and predictions. J. Rheumatol. Suppl. 49, 15-90.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Delgado R.; Newar, M.; Zewail, A.; Kar, B.; Vaughn, W.; Wu, K.; Aleksic, N,; Sivasubramanian, N.; McKay, K.; Mann, D. (2004). Cyclooxygenase-2 inhibitor treatment improves left ventricle function and mortality in a murine model of doxorubicin-induced heart failure. Circulation. 109, 1428-1433.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Gullestad, L.; Aukrust, P. (2005). Review of trials in chronic heart failure showing broad-spectrum anti-inflammatory approaches. Am. J. Cardiol. 95, 17C-23C; discussion 38C-40C.</p>
  • <p>&nbsp;</p>
  • <p>Havird, J. C., Kocot, K. M., Brannock, P. M., Cannon, J. T., Waits, D. S., Weese, D. A., ... &amp; 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.</p>
  • <p>&nbsp;</p>
  • <p>Havird, J. C., Miyamoto, M. M., Choe, K. P., &amp; 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.</p>
  • <p>&nbsp;</p>
  • <p>Hocherl, K.; Dreher, F.; Kurtz, A.; Bucher, M. (2002). Cyclooxygenase-2 inhibition attenuates liposaccaride-induced cardiovascular failure. Hypertension. 40, 947-953.</p>
  • <p>&nbsp;</p>
  • <p>Huang, C.; Chen, P., Huang, C.; Yu J. (2007). Aristolochic acid induces heart failure in zebrafish embryos that is mediated by inflammation. Toxicol, Sci. 100 (2), 486-494.</p>
  • <p>&nbsp;</p>
  • <p>Ishikawa, T. O., Griffin, K. J., Banerjee, U., &amp; Herschman, H. R. (2007). The zebrafish genome contains two inducible, functional cyclooxygenase-2 genes.Biochemical and biophysical research communications, 352(1), 181-187.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Simmons, D. L., Botting, R. M., &amp; Hla, T. (2004). Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacological reviews,56(3), 387-437.</p>
  • <p>&nbsp;</p>
  • <p>Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: structural, cellular, and molecular biology.&nbsp;Annu Rev Biochem<em>.&nbsp;</em>2000;&nbsp;69:&nbsp;145&ndash;182.</p>
  • <p>&nbsp;</p>
  • <p>Streicher, J.M.; Kamei, K.; Ishikawa, T.; Herschman, H.; Wang, Y. (2010). Compensatory hypertrophy induced by ventricular cardiomyocyte specific COX-2 expression in mice. J. Mol. Cell. Cardiol. 49 (1), 88-94.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Tilley SL, Coffman TM, Koller BH. Mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes.&nbsp;J Clin Invest<em>.&nbsp;</em>2001;&nbsp;108:&nbsp;15&ndash;23.</p>
  • <p>&nbsp;</p>
  • <p>Vane JR, Mitchell JA, Appleton I, Tomlinson A, Bishop-Bailey D, Croxtall J, Willoughby DA. Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation.&nbsp;Proc Natl Acad Sci U S A<em>.&nbsp;</em>1994;91:&nbsp;2046&ndash;2050.</p>
  • <p>&nbsp;</p>
  • <p>Wong, S.; Fukuchi, M.; Melnyk, P.; Rodger, I.; Giaid, A. (1998). Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure. Circulation, 98, 100-103.</p>
  • <br>
  • <!-- end event text -->
  • </div>
  • <div>
  • <div>
  • <h4><a href="/events/317">317: Altered, Cardiovascular development/function</a><br></h4>
  • <h5>Short Name: Altered, Cardiovascular development/function</h5>
  • </div>
  • <div>
  • <!-- loop to find all aops that use this event -->
  • <h4>AOPs Including This Key Event</h4>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <h4><a href="/events/317">Event: 317: Altered, Cardiovascular development/function</a></h4>
  • <h5>Short Name: Altered, Cardiovascular development/function</h5>
  • <h4>Key Event Component</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Process</th>
  • <th scope="col">Object</th>
  • <th scope="col">Action</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <th>AOP ID and Name</th>
  • <th>Event Type</th>
  • <td>abnormal cardiovascular system physiology</td>
  • <td></td>
  • <td>morphological change</td>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td><a href="/aops/150">150: Aryl hydrocarbon receptor activation leading to embryolethality via cardiotoxicty</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/21">21: Activation of the aryl hydrocarbon receptor (AhR) leading to early life stage mortality</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- loop to find stressors under event -->
  • <div>
  • <tr>
  • <td>cardiovascular system development</td>
  • <td>cardiovascular system</td>
  • <td>abnormal</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <br>
  • <!-- biological organization -->
  • <div>
  • <h4>Biological Organization</h4>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>Level of Biological Organization</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Organ</td>
  • </tr>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/150">Aop:150 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via reduced VEGF</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/21">Aop:21 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/456">Aop:456 - Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of bio org -->
  • <!-- cell term -->
  • <div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Organ</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of cell term -->
  • <!-- organ term -->
  • <div>
  • <h4>Organ term</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Organ term</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>heart</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of organ term -->
  • <h4>Evidence Supporting Applicability of this Event</h4>
  • <br>
  • <!-- loop to find taxonomic applicability under event -->
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>Term</th>
  • <th>Scientific Term</th>
  • <th>Evidence</th>
  • <th>Links</th>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>chicken</td>
  • <td>Gallus gallus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>zebrafish</td>
  • <td>Danio rerio</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Vertebrates</td>
  • <td>Vertebrates</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Invertebrates</td>
  • <td>Invertebrates</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for taxons -->
  • <!-- life stages -->
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Life Stage</th>
  • <th>Evidence</th>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Embryo</td>
  • <td>Strong</td>
  • </tr>
  • <tr>
  • <td>development</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end life stages -->
  • <!-- sex terms -->
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Sex</th>
  • <th>Evidence</th>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Unspecific</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end sex terms -->
  • <div>
  • <ul>
  • <ul>
  • <li>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).</li>
  • <li>Differences in cardiovascular systems are present among taxa. Vertebrates have closed circulatory systems, while some invertebrate taxa have open circulatory systems (Kardong 2006).</li>
  • </ul>
  • <h4>Key Event Description</h4>
  • <p>This key event applies to the disruption of cardiogenesis early enough in embryogenesis to result in gross morphological alterations leading to reduced cardiac function.</p>
  • <h4>How it is Measured or Detected</h4>
  • <p>Altered cardiovascular development/function can be measured in numerous ways:</p>
  • <p>Birds, fish and mammals are all susceptible to cardiotoxicity following embryonic chemical exposure.</p>
  • <p>1) As blood flow in the mesencephalic vein by use of time-lapse recording using a digital video camera (Teraoka et al 2008;&nbsp;2014). Blood flow is measured as the number of red blood cells passing the mesencephalic vein per second (Teraoka et al 2008; 2014). This method is described in detail by Teraoka et al (2002). However, some studies have assessed blood flow through visualized scoring techniques by use of a microscope as (1) same rate&nbsp;as control, (2) slower rate than control, or (3) no flow (Henry et al 1997).</p>
  • <p>&nbsp;</p>
  • <p>2) As heart area, pericardial edema area, or yolk sac edema area quantified with&nbsp;area analysis by use of a microscope linked digital camera and conventional image software&nbsp;(Dong et al 2010; Teraoka et al 2008; 2014; Yamauchi et al 2006). Images at the same magnification are used to obtain the&nbsp;area measured as number of pixels (Teraoka et al 2008; 2014). This method can use either live individuals or histologic samples. This method is described in detail by Teraoka et al (2003).</p>
  • <p>Cardiovascular development and function altered by dioxin-like compounds (DLCs):</p>
  • <p>3) As basic physical measurements such as heart weight, heart aspect ratio (horizontal length versus vertical length), heart weight to body weight ratio (Fujisawa et al 2014).</p>
  • <ul>
  • <li>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).</li>
  • <li>Applicable to birds (Heid et al 2001).</li>
  • <li>Altered cardiovascular development and function by DLCs has not been sufficiently investigated in amphibians, reptiles, or invertebrates that possess a cardiovascular system.</li>
  • </ul>
  • <br>
  • </div>
  • <!-- event text -->
  • <h4>How this Key Event Works</h4>
  • <p>This key event applies to the disruption of cardiogenesis early enough in embryogenesis to result in gross morphological alterations leading to reduced cardiac function.</p>
  • <p>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.</p>
  • <p><strong>Table 1: Cardiotoxic effects of strong AHR-agonists</strong></p>
  • <table border="1" class="table" style="text-align:left">
  • <tbody>
  • <tr>
  • <th>Zebrafish Embryo</th>
  • <th>Chicken Embryo</th>
  • <th>Mouse</th>
  • </tr>
  • <tr>
  • <td>
  • <ul>
  • <li>Reduced extension of common cardinal vein</li>
  • <li>Reduced blood flow</li>
  • <li>Reduced heart rate</li>
  • <li>Disrupted erythropoiesis</li>
  • <li>Decreased heart volume</li>
  • <li>Pericardial edema</li>
  • <li>Overt heart malformations</li>
  • </ul>
  • </td>
  • <td>
  • <ul>
  • <li>Enlarged left ventricle</li>
  • <li>Increased heart rate</li>
  • <li>Increased myosin content</li>
  • <li>Reduced &beta;-adrenergic responsiveness</li>
  • <li>Increased ANF mRNA</li>
  • <li>Arrhythmia</li>
  • <li>Increased apoptosis</li>
  • <li>Reduced myocyte proliferation</li>
  • <li>Pericardial edema</li>
  • <li>Overt heart malformations</li>
  • </ul>
  • </td>
  • <td>Embryo/Fetus
  • <ul>
  • <li>Reduced heart-to-body weight</li>
  • <li>Reduced myocyte proliferation</li>
  • <li>Vascular remodeling</li>
  • </ul>
  • <p>21 Days old</p>
  • <ul>
  • <li>Increased heart-to-body weight</li>
  • <li>Increased left ventricle weight</li>
  • <li>Reduced heart rate</li>
  • <li>Cardiac hypertrophy</li>
  • <li>Increased ANF mRNA</li>
  • <li>Increased risk of heart disease</li>
  • </ul>
  • </td>
  • </tr>
  • </tbody>
  • </table>
  • <p>4) As incidence of malformation&nbsp;measured as percent occurrence among individuals (Buckler et al 2015; Dong et al 2010; Park et al 2014; Yamauchi et al 2006).This method is described in detail by Dong et al (2010).</p>
  • <p>ANF= cardiac atrial natriuretic factor; an indicator of cardiac stress. Source: (Kopf and Walker 2009)</p>
  • <br>
  • <p>5) As heartbeat rate measured by direct observation by use of a microscope (Park et al 2014). This method is described in detail by Park et al (2014).</p>
  • <h4>References</h4>
  • <p><br />
  • <h4>References</h4>
  • <p><br />
  • 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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>Bishopric, N.H. (2005). Evolution of the heart from bacteria to man. Ann. N. Y. Acad. Sci. 1047, 13-29.</p>
  • <p>&nbsp;</p>
  • <p>Buckler J.; Candrl, J.S.; McKee, M.J.; Papoulias, D.M.; Tillitt, D.E.; Galat, D.L. Sensitivity of shovelnose sturgeon (<em>Scaphirhynchus platorynchus</em>) and pallid sturgeon (<em>S. albus</em>) early life stages to PCB-126 and 2,3,7,8-TCDD exposure. <em>Enviro. Toxicol. Chem. </em><strong>2015</strong>, 34(6), 1417-1424.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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 (<em>Coturnix japonica</em>), common pheasant (<em>Phasianus colchicus</em>), and white leghorn chicken (<em>Gallus gallus domesticus</em>) embryos to <em>in ovo</em> exposure to TCDD, PeCDF, and TCDF. Toxicol. Sci. 119, 93-102.</p>
  • <p>&nbsp;</p>
  • <p>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-<em>p</em>-dioxin to seven freshwater fish species during early life-stage development.<em> Enviro. Toxico. Chem. </em><strong>1998</strong>, 17, 472-483.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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-<em>p</em>-dioxin to early life stage brooke trout (<em>Salvelinus fontinalis</em>) following parental dietary exposure. <em>Enviro. Toxicol. Chem.</em> <strong>1998</strong>, 17 (12), 2408-2421.</p>
  • <p>&nbsp;</p>
  • <p>Kardong, K.V. (2006). Vertebrates: comparative anatomy, function, evolution. McGraw-Hill Higher Eduction. Boston, USA.</p>
  • <p>&nbsp;</p>
  • <p>Lemly, A.D. (2002). Symptoms and implications of selenium toxicity in fish: the Belews Lake case example. Aquat. Toxicol. 57 (1-2), 39-49.</p>
  • <p>&nbsp;</p>
  • <p>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. <em>Sci. Totl. Enviro.</em> <strong>2014</strong>, 491-492, 271-278.</p>
  • <p>Teraoka, H.; Dong, W.; Hiraga, T. (2003). Zebrafish as a novel experimental model for development toxicology. Congenit. Anom. 43, 123-132.</p>
  • <p>&nbsp;</p>
  • <p>Teraoka, T.; Dong, W.; Ogawa, S.; Tsukiyama, S.; Okuhara, Y.; Niiyama, M.; Ueno, N.; Peterson, R.E. (2002). 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: Altered regional blood flow and impaired lower jaw development. Toxicol. Sci. 65, 192-199.</p>
  • <p>&nbsp;</p>
  • <p>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&rsquo;,4,4&rsquo;,5-pentachlorobiphenyl. 2015. Enviro. Toxicol. Chem. DOI: 10.1002/etc.3614.</p>
  • <p>&nbsp;</p>
  • <p>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 <em>Fundulus heteroclitus</em> embryos. <em>Aquat. Toxicol.</em> <strong>2001</strong>, 53, 127-138.</p>
  • <p>&nbsp;</p>
  • <p>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 (<em>Salvelinus namaycush</em>). <em>Canad. J. Fisheries Aqua. Sci.</em> <strong>1991</strong>, 48, 875-883.</p>
  • <p>&nbsp;</p>
  • <p>Yamauchi, M.; Kim, E.Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2,3,7,8-tetrachlorodibenzo-<em>p</em>-dioxin (TCDD) in developing red seabream (<em>Pagrus major</em>) embryos: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. <em>Aquat. Toxicol.</em> <strong>2006</strong>, 16, 166-179.</p>
  • <p>&nbsp;</p>
  • <p>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. <strong>1995</strong>. 31, 315-328.</p>
  • <br>
  • <!-- end event text -->
  • </div>
  • <!-- ao table -->
  • <h3>Adverse Outcomes</h3>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <h4><a href="/events/947">Event: 947: Increase, Early Life Stage Mortality</a></h4>
  • <h5>Short Name: Increase, Early Life Stage Mortality</h5>
  • <h4>Key Event Component</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Process</th>
  • <th scope="col">Object</th>
  • <th scope="col">Action</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <th>Title</th>
  • <th>Short name</th>
  • <td>embryonic lethality</td>
  • <td></td>
  • <td>increased</td>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td><a href="/events/350">Increase, Mortality</a></td>
  • <td>Increase, Mortality</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <div>
  • <div>
  • <h4><a href="/events/350">350: Increase, Mortality</a><br></h4>
  • <h5>Short Name: Increase, Mortality</h5>
  • </div>
  • <div>
  • <!-- loop to find all aops that use this event -->
  • <h4>AOPs Including This Key Event</h4>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>AOP ID and Name</th>
  • <th>Event Type</th>
  • <td>mortality</td>
  • <td></td>
  • <td>increased</td>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td><a href="/aops/21">21: Activation of the aryl hydrocarbon receptor (AhR) leading to early life stage mortality</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- loop to find stressors under event -->
  • <div>
  • </div>
  • <br>
  • <!-- biological organization -->
  • <div>
  • <h4>Biological Organization</h4>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>Level of Biological Organization</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Individual</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end of bio org -->
  • <!-- cell term -->
  • <div>
  • </div>
  • <!-- end of cell term -->
  • <!-- organ term -->
  • <div>
  • </tbody>
  • </table>
  • </div>
  • <!-- end of organ term -->
  • <!-- loop to find taxonomic applicability under event -->
  • <div>
  • </div>
  • <!-- end loop for taxons -->
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/150">Aop:150 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via reduced VEGF</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/21">Aop:21 - Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/455">Aop:455 - Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced impeded craniofacial development</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/456">Aop:456 - Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- life stages -->
  • <div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end life stages -->
  • <!-- sex terms -->
  • <div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Individual</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end sex terms -->
  • <h3>Evidence for Perturbation by Stressor</h3>
  • <h4>2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)</h4>
  • <p><p>Exposure of embryos to 2,3,7,8-TCDD causes early life stage mortality in all studied species of fishes (Doering et al 2013).</p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • </p>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Vertebrates</td>
  • <td>Vertebrates</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>Foetal</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>Development</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p>All members of the subphylum vertebrata are susceptible to early life stage death (<span style="font-family:calibri,sans-serif; font-size:11.0pt">Weinstein 1999).</span></p>
  • <div>
  • </div>
  • <h4>Key Event Description</h4>
  • <p>Increased early life stage mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.</p>
  • <p>In Birds:</p>
  • <p>Early life stage mortality occurs&nbsp;at any stage in development prior to birth/hatch and is considered embryolethal.</p>
  • <p>In Fishes:</p>
  • <p>Early Life Stage Mortality refers to death prior to yolk sac adsorption and swim-up.</p>
  • <!-- event text -->
  • <h4>How it is Measured or Detected</h4>
  • <p>In birds it may be identified as failure to hatch or lack of movement within the egg when candled; heartbeat monitors are available for identifying viable avian and reptillian eggs (ex. Avitronic&#39;s Buddy monitor). In mammals, stillborn or mummified offspring, or an increased rate of resorptions early in pregnancy are all considered embryolethal, and can be detected using ultra-high frequency ultrasound (30-70 MHz; a.k.a. ultrasound biomicroscopy) (Flores <em>et al. </em>2014). In fishes, mortality is typically measured by observation. Lack of any heart beat, gill movement, and&nbsp;body movement are typical signs of death used in the evaluation of mortality.</p>
  • <h4>Regulatory Significance of the AO</h4>
  • <p>Poor early life stage survival is an endpoint of major relevance to environmental regulators, as it is likely to lead to population decline.&nbsp; Early-life stage, acute and chronic test guidelines have been established by the Organisation for Economic Co-operation and Development (OECD), U.S. Environmental Protection Agency (EPA) and Environment and Climate Change Canada (ECCC), and are currently used in risk assessments to set limits for safe exposures.&nbsp; Aquatic test guidlines are most prevalent and include OECD210, OECD229, EPA850.1400 and ECCC&nbsp; EPS 1/RM/28 for fish and OECD241 for frogs.</p>
  • <h4>References</h4>
  • <p>1. Flores, L.E., Hildebrandt, T.B., Kuhl, A.A., and Drews, B. (2014) Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy. <em>Reproductive Biology and Endocrinology</em> <strong>12</strong>(38). DOI: 10.1186/1477-7827-12-38</p>
  • <p>2. <span style="font-family:calibri,sans-serif; font-size:11.0pt">Weinstein, B. M. (1999). What guides early embryonic blood vessel formation? <em>Dev. Dyn.</em> <strong>215</strong>(1), 2-11.</span></p>
  • <p>Doering, J.A.; Giesy, J.P.; Wiseman S.; Hecker, M. (2013). Predicting the sensivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain. Environmental Science and Pollution Research. 20 (3), 1219-1224.</p>
  • <!-- end event text -->
  • </div>
  • <!-- rel table -->
  • <h3>Scientific evidence supporting the linkages in the AOP</h3>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <tr>
  • <th>Upstream Event</th>
  • <th>Relationship Type</th>
  • <th>Downstream Event</th>
  • <th>Evidence</th>
  • <th>Quantitative Understanding</th>
  • <h2>Appendix 2</h2>
  • <h2>List of Key Event Relationships in the AOP</h2>
  • <div id="evidence_supporting_links">
  • <h3>List of Adjacent Key Event Relationships</h3>
  • <div>
  • <h4><a href="/relationships/972">Relationship: 972: Activation, AhR leads to dimerization, AHR/ARNT</a></h4>
  • <h4>AOPs Referencing Relationship</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP Name</th>
  • <th scope="col">Adjacency</th>
  • <th scope="col">Weight of Evidence</th>
  • <th scope="col">Quantitative Understanding</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Activation, AHR</td>
  • <td>directly leads to</td>
  • <td>dimerization, AHR/ARNT</td>
  • <td>Strong</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td>dimerization, AHR/ARNT</td>
  • <td>directly leads to</td>
  • <td>Increase, COX-2 expression</td>
  • <td>Strong</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td>Increase, COX-2 expression</td>
  • <td>directly leads to</td>
  • <td>Altered, Cardiovascular development/function</td>
  • <td>Moderate</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td>Altered, Cardiovascular development/function</td>
  • <td>directly leads to</td>
  • <td>Increase, Mortality</td>
  • <td>Moderate</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/150">Aryl hydrocarbon receptor activation leading to early life stage mortality, via reduced VEGF</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/21">Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/310">Embryonic Activation of the AHR leading to Reproductive failure, via epigenetic down-regulation of GnRHR</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/151">AhR activation leading to preeclampsia</a></td>
  • <td>adjacent</td>
  • <td></td>
  • <td></td>
  • </tr>
  • <tr>
  • <td><a href="/aops/455">Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced impeded craniofacial development</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/456">Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- Evidence for relationship links section, this lists the relationships and then supports them -->
  • <div id="evidence_supporting_links">
  • <hr>
  • <div>
  • <h4><a href="/relationships/972">Activation, AHR leads to dimerization, AHR/ARNT</a></h4>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <br>
  • <!-- loop to find taxonomic applicability under relationship -->
  • <div>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Term</th>
  • <th>Scientific Term</th>
  • <th>Evidence</th>
  • <th>Links</th>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Mus musculus</td>
  • <td>Mus musculus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Danio rerio</td>
  • <td>Danio rerio</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Mus musculus</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Danio rerio</td>
  • <td>Danio rerio</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rainbow trout</td>
  • <td>Oncorhynchus mykiss</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8022" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Pagrus major</td>
  • <td>Pagrus major</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=143350" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Acipenser fulvescens</td>
  • <td>Acipenser fulvescens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=41871" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Salmo salar</td>
  • <td>Salmo salar</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8030" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Acipenser transmontanus</td>
  • <td>Acipenser transmontanus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7904" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Xenopus laevis</td>
  • <td>Xenopus laevis</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Ambystoma mexicanum</td>
  • <td>Ambystoma mexicanum</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8296" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Microgadus tomcod</td>
  • <td>Microgadus tomcod</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34823" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Phasianus colchicus</td>
  • <td>Phasianus colchicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9054" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Coturnix japonica</td>
  • <td>Coturnix japonica</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=93934" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for taxons -->
  • <!-- loop to find life stages under relationship -->
  • <div>
  • </div>
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Life Stage</th>
  • <th>Evidence</th>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>all life stages</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for life stages -->
  • <!-- sex terms -->
  • <div>
  • </div>
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Sex</th>
  • <th>Evidence</th>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Unspecific</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end sex terms -->
  • </div>
  • <ul>
  • <li>The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) are highly conserved and ancient proteins with homologs having been identified in most major animal groups, apart from the most ancient lineages, such as sponges (Porifera) (Hahn et al 2002).&nbsp;</li>
  • <li><em>In vitro</em> dimerization of AhRs and ARNTs have been demonstrated in mammals, birds, reptiles, amphibians, teleost and non-teleost fishes, and some invertebrates (Butler et al 2001; Emmons et al 1999; Hahn et al 2002; Powell-Coffman et al 1998).</li>
  • </ul>
  • <h4>How Does This Key Event Relationship Work</h4>
  • <p>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).</p>
  • <p>Roles of AhR and ARNT isoforms:</p>
  • <h4>Key Event Relationship Description</h4>
  • <p>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) (Fujii-Kuriyama <em>et al. </em>2010).&nbsp; 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).</p>
  • <ul>
  • <li>AhRs can heterodimerize with ARNT1 and ARNT2 isoforms in order to activate reporter constructs in transfected cells and recognize response elements in gel shift assays in all investigated vertebrates, including birds, fishes, and reptiles (Abnet et al 1999; Andreasen et al 2002a; 2002b; Bak et al 2013; Doering et al 2014; Doering et al 2015; Farmahin et al 2012; 2013; Hansson &amp; Hahn 2008; Karchner et al 1999; 2006; Lavine et al 2005; Oka et al 2016; Shoots et al 2015; Tanguay et al 1999; 2000; Wirgin et al 2011).&nbsp;</li>
  • </ul>
  • <p>AhRs can heterodimerize with ARNT1 and ARNT2 isoforms in order to activate reporter constructs in transfected cells and recognize response elements in gel shift assays in all investigated vertebrates, including birds, fishes, and reptiles (Abnet et al 1999; Andreasen et al 2002a; 2002b; Bak et al 2013; Doering et al 2014; Doering et al 2015; Farmahin et al 2012; 2013; Hansson &amp; Hahn 2008; Karchner et al 1999; 2006; Lavine et al 2005; Shoots et al 2015; Tanguay et al 1999; 2000; Wirgin et al 2011).&nbsp;</p>
  • <!-- if nothing shows up in any of these fields, then weight of evidence will not be displayed -->
  • <h4>Weight of Evidence</h4>
  • <strong>Biological Plausibility</strong>
  • <p>The mechanism of AHR-mediated transcriptional regulation is well understood (Fujii-Kuriyama and Kawajiri 2010).</p>
  • <h4>Evidence Supporting this KER</h4>
  • <strong>Biological Plausibility</strong>
  • <p>The mechanism of AHR-mediated transcriptional regulation is well understood (Fujii-Kuriyama and Kawajiri 2010).</p>
  • <p>&bull; Numerous PAS proteins are known to interact with each other in response to environmental and developmental cues through dimerization at their PAS domains (Pohjanvirta 2012).</p>
  • <p>Numerous PAS proteins are known to interact with each other in response to environmental and developmental cues through dimerization at their PAS domains (Pohjanvirta 2012).</p>
  • <strong>Empirical Support for Linkage</strong>
  • <ul>
  • <strong>Empirical Evidence</strong>
  • <p>ARNT is a necessary dimerization partner for the transcriptional activation of AHR regulated genes (Hoffman et al. 1991; Poland et al. 1976). The AHR/ARNT complex was confirmed following in vitro exposure to halogenated aromatic hydrocarbons using an electrophoretic mobility shift assay; a dose-dependent supershift in DNA-binding was observed using specific antibodies in chicken and human cell lines (Heid et al. 2001).</p>
  • <ul>
  • <li>Unliganded AhR exists as a cytosolic 9S form, while in the presence of a ligand the AhR exists as a nuclear 6S form. ARNT exists as a nuclear 6S form (Okey 2007).</li>
  • <li>The 6S form of AhR is approximately 210 kDa. Ligated AhR is approximately 100 kDa and ARNT is approximately 110 kDa (Elferink et al 1990; Swanson et al 1993).</li>
  • <li>Dimerization of AhRs with ARNTs has been demonstrated in all invertebrate and vertebrate species so far investigated (Butler et al 2001; Emmons et al 1999; Hahn et al 2002; Powell-Coffman et al 1998).</li>
  • <li>Heterodimers are not formed on response elements in gel shift assays in the absence of AhR and/or ARNT (Tanguay et al 2000).&nbsp;</li>
  • </ul>
  • <p>ARNT is a necessary dimerization partner for the transcriptional activation of AHR regulated genes (Hoffman et al. 1991; Poland et al. 1976). The AHR/ARNT complex was confirmed following in vitro exposure to halogenated aromatic hydrocarbons using an electrophoretic mobility shift assay; a dose-dependent supershift in DNA-binding was observed using specific antibodies in chicken and human cell lines (Heid et al. 2001).</p>
  • <p>&nbsp;</p>
  • <strong>Uncertainties or Inconsistencies</strong>
  • <ul>
  • <strong>Uncertainties and Inconsistencies</strong>
  • <ul>
  • <li>There are uncertainties in the precise physiological and toxicological roles of different AhR clades (AhR1, AhR2, AhR3) and isoforms (&alpha;, &beta;, &delta;, &gamma;).</li>
  • <li>There are uncertainties in the precise physiological and toxicological roles of different ARNT clades (ARNT1, ARNT2, ARNT3) and isoforms (a, b, c).</li>
  • <li>Nothing is known about differences in binding affinity of AhR for ARNT and of the AhR/ARNT heterodimer for DNA among species and taxa.</li>
  • </ul>
  • <strong>Quantitative Understanding of the Linkage</strong>
  • <ul>
  • <li>Strong quantitative relationships are known for exposure to ligands and interaction with DREs on the DNA by use of transfected COS-7 cells and gel shift assays (Abnet et al 1999; Andreasen et al 2002a; 2002b; Bak et al 2013; Doering et al 2014; Doering et al 2015; Farmahin et al 2012; 2013; Hansson &amp; Hahn 2008; Karchner et al 1999; 2006; Lavine et al 2005; Manning et al 2012; Oka et al 2016; Shoots et al 2015; Tanguay et al 1999; 2000; Wirgin et al 2011).</li>
  • <li>Specifically, greater concentrations of ligands or greater potency ligands cause greater interaction with DREs on the DNA.</li>
  • <li>Numerous ligands of the AhR are rapidly metabolized and only cause transient activation of the AhR. These ligands do not result in sustained interaction with DREs and do not cause downstream effects (Farmhin et al 2016).</li>
  • <li>There is uncertainty in whether anthropogenic contaminants that act as ligands of the AhR and lead to dimerization of AhR with ARNT in vertebrates also act as ligands in invertebrates.</li>
  • </ul>
  • <!--<!% unless aop_rel.relationship.relationship_taxons.blank? %>-->
  • <!--<!%= render 'snapshot_taxons', taxons: aop_rel.relationship.relationship_taxons %>-->
  • <!--<!% unless aop_rel.relationship.taxon_evidence.blank? %>-->
  • <!--<h3>Evidence Supporting Taxonomic Applicability</h3>-->
  • <!--<!%== aop_rel.relationship.taxon_evidence %>-->
  • <!--<!% end %>-->
  • <!--<!% end %>-->
  • <h4>References</h4>
  • <p><br />
  • 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.</p>
  • <h4>References</h4>
  • <p>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.</p>
  • <p>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.</p>
  • <p>3. 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.</p>
  • <p>4. 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(5008), 954-958.</p>
  • <p>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.</p>
  • <p>6. 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(16), 4936-4946.</p>
  • <p>7. 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.</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Butler, R.A.; Kelley, M.L.; Powell, W.H.; Hahn, M.E.; Van Beneden, R.J. (2001). An aryl hydrocarbon receptor (AHR) homologue from the soft-shelled clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone binding. Gene. 278, 223-234.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Elfrink, C.; Gasiewicz, T.; Whitlock, J. (1990). Protein-DNA interactions at a dioxin-responsive enhancer. Evidence that the transformed Ah receptor is heteromeric. J. Biol. Chem. 265, 20708-20712.</p>
  • <p>&nbsp;</p>
  • <p>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 and control antennal and tarsal development in Drosophilia. Dev. 126, 3937-3945.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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 <em>in vitro</em> function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict <em>in vivo</em> sensitivity to dioxins. Enviro. Sci. Toxicol. 46 (5), 2967-2975.</p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <p>Farmahin, R.; Crump, D.; O&rsquo;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.</p>
  • <p>&nbsp;</p>
  • <p>Hahn, M.E. 2002. Aryl hydrocarbon receptors: diversity and evolution. Chemico-Biol. Interact. 141, 131-160.</p>
  • <p>&nbsp;</p>
  • <p>Hansson, M.C.; Hahn, M.E. 2008. Functional properties of the four Atlantic salmon (<em>Salmo salar</em>) aryl hydrocarbon receptor type 2 (AHR2) isoforms. Aquat. Toxicol. 86, 121-130.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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 <em>Fundulus heteroclitus</em>. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors. J. Biol. Chem. 274, 33814-33824.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Powell-Coffman, J.A.; Bradfield, C.A.; Wood, W.B. (1998). Caenorhabditis elgans orthologs of the aryl hydrocarbon receptor and its dimerization partner the aryl hydrocarbon receptor nuclear translocator. Proceedings of the National Academy of Sciences of the United States of America. 95, 2844-2449.</p>
  • <p>&nbsp;</p>
  • <p>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<em>. </em>49, 6993-7001.</p>
  • <p>&nbsp;</p>
  • <p>Swanson, H.; Tullis, K.; Denison, M. (1993). Binding of transformed Ah receptor complex to a dioxin responsive transcriptional enhancer: evidence for two distinct heterodimeric DNA-binding forms. Biochem. 32, 12841-12849.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • <p>&nbsp;</p>
  • <p>Okey, A. (2007). An aryl hydrocarbon receptor odyssey to the shores of toxicology: the deichmann Lecture, International Congress of Toxicology-XI. Toxicol. Sci. 98, 5-38.</p>
  • <p>&nbsp;</p>
  • <p>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.</p>
  • </div>
  • <br>
  • <div>
  • <h4><a href="/relationships/1350">dimerization, AHR/ARNT leads to Increase, COX-2 expression</a></h4>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <br>
  • <!-- loop to find taxonomic applicability under relationship -->
  • <div>
  • <div>
  • <h4><a href="/relationships/1350">Relationship: 1350: dimerization, AHR/ARNT leads to Increase, COX-2 expression</a></h4>
  • <h4>AOPs Referencing Relationship</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP Name</th>
  • <th scope="col">Adjacency</th>
  • <th scope="col">Weight of Evidence</th>
  • <th scope="col">Quantitative Understanding</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/21">Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Term</th>
  • <th>Scientific Term</th>
  • <th>Evidence</th>
  • <th>Links</th>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Danio rerio</td>
  • <td>Danio rerio</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Oryzias latipes</td>
  • <td>Oryzias latipes</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8090" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>Weak</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Danio rerio</td>
  • <td>Danio rerio</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Oryzias latipes</td>
  • <td>Oryzias latipes</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>Low</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for taxons -->
  • <!-- loop to find life stages under relationship -->
  • <div>
  • </div>
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Life Stage</th>
  • <th>Evidence</th>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Embryo</td>
  • <td>Strong</td>
  • </tr>
  • <tr>
  • <td>development</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>Development</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for life stages -->
  • <!-- sex terms -->
  • <div>
  • </div>
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Sex</th>
  • <th>Evidence</th>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Unspecific</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end sex terms -->
  • </div>
  • <ul>
  • <li>Dimerization of AhR/ARNT leading to increased expression of COX-2 has only been investigated in zebrafish, Japanese medaka, and chicken (Dong et al 2010; Teraoka et al 2008; 2014; Fugisawa et al 2014).</li>
  • <li>Due to the presence of a functional AhR/ARNT pathway and COX-2 genes among all vertebrate taxa, it is acknowledged that this key event relationship is likely applicable to vertebrates in general and possibly some invertebrates.</li>
  • </ul>
  • <h4>How Does This Key Event Relationship Work</h4>
  • <ul>
  • <h4>Key Event Relationship Description</h4>
  • <ul>
  • <li>The AhR/ARNT heterodimer is able to interact with dioxin-responsive elements (DREs) on the DNA causing the up-regulation in dioxin-responsive genes (Whitlock et al 1996).</li>
  • <li>DREs in the promoter region of COX-2 allow the AhR/ARNT heterodimer to up-regulate expression of COX-2 (Degner et al 2007; Jonsson et al 2012).&nbsp;</li>
  • </ul>
  • <!-- if nothing shows up in any of these fields, then weight of evidence will not be displayed -->
  • <h4>Weight of Evidence</h4>
  • <strong>Biological Plausibility</strong>
  • <ul>
  • <h4>Evidence Supporting this KER</h4>
  • <strong>Biological Plausibility</strong>
  • <ul>
  • <li>Putative DREs have been identified in the promoter region of COX-2 in zebrafish and presumably exist in other species and taxa (Degner et al 2007; Jonsson et al 2012).</li>
  • </ul>
  • <ul>
  • <li>DREs are well characterized and numerous other genes that have DREs in their promoter region are known to be up-regulated by the AhR/ARNT heterodimer (Denison et al 1988).</li>
  • </ul>
  • <strong>Empirical Support for Linkage</strong>
  • <ul>
  • <strong>Empirical Evidence</strong>
  • <ul>
  • <li>Expression of COX-2 is up-regulated in response to exposure to ligands that activate AhR causing dimerization with ARNT (Dong et al 2010; Teraoka et al 2008; 2014).</li>
  • <li>Knockdown of ARNT1 prevents interaction of AhR with DREs and the up-regulation in dioxin-responsive genes (Antkiewicz et al 2006; Prasch et al 2004).</li>
  • <li>Depletion of ARNT lessens or prevents interaction of AhR with DREs and the up-regulation in dioxin-responsive genes (Prasch et al 2004).</li>
  • <li>However, expressions of&nbsp;COX-2 have not yet been investigated following targeted knockdown of either AhR or ARNT1 preventing dimerization and interaction with DREs.</li>
  • </ul>
  • <strong>Uncertainties or Inconsistencies</strong>
  • <ul>
  • <strong>Uncertainties and Inconsistencies</strong>
  • <ul>
  • <li>In chicken (<em>Gallus gallus</em>), and presumably other species of birds, COX-2 is believed to be up-regulated by the AhR through non-genomic mechanisms that are independent of the AhR/ARNT heterodimer (Fujisawa et al 2014). DREs are not believed to be present in the promoter region of COX-2 in chicken (Fujisawa et al 2014).</li>
  • <li>However, nothing is known regarding the presence or absence of DREs in the promoter region of COX-2 in species or taxa other than zebrafish.</li>
  • <li>Amounts of COX-2 protein have not been investigated and therefore only increases in expressions of transcript are known.</li>
  • </ul>
  • <strong>Quantitative Understanding of the Linkage</strong>
  • <ul>
  • <li>Limited dose-response information is available regarding dimerization of AhR/ARNT leading to increased expression of COX-2.</li>
  • <li>In Japanese medaka (<em>Oryzias latipes</em>), abundance of transcript of COX-2 followed a dose-dependent increase to waterborne concentrations of the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Dong et al 2010).</li>
  • </ul>
  • <!--<!% unless aop_rel.relationship.relationship_taxons.blank? %>-->
  • <!--<!%= render 'snapshot_taxons', taxons: aop_rel.relationship.relationship_taxons %>-->
  • <!--<!% unless aop_rel.relationship.taxon_evidence.blank? %>-->
  • <!--<h3>Evidence Supporting Taxonomic Applicability</h3>-->
  • <!--<!%== aop_rel.relationship.taxon_evidence %>-->
  • <!--<!% end %>-->
  • <!--<!% end %>-->
  • <h4>References</h4>
  • <p>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.</p>
  • <h4>References</h4>
  • <p>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.</p>
  • <p>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.</p>
  • <p>Denison, M.S.; Fisher, J.M.; Whitlock, J.P. (1988). The DNA recognition site for the dioxin-Ah receptor complex, Nucleotide sequence and functional analysis. J. Biol. Chem. 263, 17221-17224.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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-<em>p</em>-dioxin developmental toxicity in zebrafish. 76 (1), 138-150.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • </div>
  • <br>
  • <div>
  • <h4><a href="/relationships/1351">Increase, COX-2 expression leads to Altered, Cardiovascular development/function</a></h4>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <br>
  • <!-- loop to find taxonomic applicability under relationship -->
  • <div>
  • <div>
  • <h4><a href="/relationships/1351">Relationship: 1351: Increase, COX-2 expression leads to Altered, Cardiovascular development/function</a></h4>
  • <h4>AOPs Referencing Relationship</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP Name</th>
  • <th scope="col">Adjacency</th>
  • <th scope="col">Weight of Evidence</th>
  • <th scope="col">Quantitative Understanding</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/21">Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>adjacent</td>
  • <td>Moderate</td>
  • <td>Moderate</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Term</th>
  • <th>Scientific Term</th>
  • <th>Evidence</th>
  • <th>Links</th>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Danio rerio</td>
  • <td>Danio rerio</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Oryzias latipes</td>
  • <td>Oryzias latipes</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8090" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Danio rerio</td>
  • <td>Danio rerio</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Oryzias latipes</td>
  • <td>Oryzias latipes</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for taxons -->
  • <!-- loop to find life stages under relationship -->
  • <div>
  • </div>
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Life Stage</th>
  • <th>Evidence</th>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Embryo</td>
  • <td>Strong</td>
  • </tr>
  • <tr>
  • <td>development</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>Development</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for life stages -->
  • <!-- sex terms -->
  • <div>
  • </div>
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Sex</th>
  • <th>Evidence</th>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Unspecific</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end sex terms -->
  • </div>
  • <ul>
  • <li>Links between induction of COX-2 and alteration of cardiovascular development and function has only been demonstrated in zebrafish, Japanese medaka, and chicken (Dong et al 2010; Teraoka et al 2008; 2014).</li>
  • <li>However, it is acknowledged that this key event relationship could be applicable to all vertebrate taxa and some invertebrate taxa based on presence of COX-2 genes and a cardiovascular system.</li>
  • </ul>
  • <h4>How Does This Key Event Relationship Work</h4>
  • <ul>
  • <h4>Key Event Relationship Description</h4>
  • <ul>
  • <li>The precise role that COX-2 plays in altered cardiovascular development/function has not been investigated. However, 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).</li>
  • </ul>
  • <!-- if nothing shows up in any of these fields, then weight of evidence will not be displayed -->
  • <h4>Weight of Evidence</h4>
  • <strong>Biological Plausibility</strong>
  • <ul>
  • <h4>Evidence Supporting this KER</h4>
  • <strong>Biological Plausibility</strong>
  • <ul>
  • <li>The prostaglandin synthesis pathway, of which COX-2 is a rate limiting step, is known to have roles in development of the heart and therefore altered levels of expression of COX-2 could be expected to result in altered development of the heart.</li>
  • </ul>
  • <strong>Empirical Support for Linkage</strong>
  • <ul>
  • <li>Blocking induction of COX-2 through knockdown of COX-2 or through selective antagonists of COX-2 prevents alteration in cardiovascular development and function in zebrafish, Japanese medaka (<em>Oryzias latipes</em>), and chicken (<em>Gallus gallus</em>) (Dong et al 2010; Teraoka et al 2008; 2014).</li>
  • <li>Knockdown of and selective antagonists of thromboxane A synthase 1 (CYP5A), which is down-stream of COX-2 in the prostaglandin synthesis pathway, prevents alteration in cardiovascular development and function by inducers of COX-2 (Teraoka et al 2008).</li>
  • <strong>Empirical Evidence</strong>
  • <ul>
  • <li>Blocking induction of COX-2 through knockdown of COX-2 or through selective antagonists of COX-2 &nbsp;in zebrafish, Japanese medaka (<em>Oryzias latipes</em>), and chicken (<em>Gallus gallus</em>)&nbsp;prevents alteration in cardiovascular development and function by 2,3,7,8-TCDD, including prevention of pericardial edema, changes in heart size, and reduction in blood blow (Dong et al 2010; Teraoka et al 2008; 2014).</li>
  • <li>Knockdown of and selective antagonists of thromboxane A synthase 1 (CYP5A), which is down-stream of COX-2 in the prostaglandin synthesis pathway, prevents alteration in cardiovascular development and function by 2,3,7,8-TCDD&nbsp;(Teraoka et al 2008).</li>
  • </ul>
  • <p><strong>COX-2 Cardiovascular Development Roles:</strong></p>
  • <ul>
  • <li>Transgenic mice (<em>Mus musculus</em>) that over express COX-2 display altered cardiac remodeling&nbsp;that results in cardiomyocyte hypertrophy&nbsp;(Streicher et al 2010). However, significantly impaired cardiac function was not observed in the strain of transgenic mice investigated in this study (Streicher et al 2010).</li>
  • <li>Embryos of zebrafish (<em>Danio rerio</em>) exposed to the COX-2 inducers, aristolochic acid and doxorubicin, develop hypertrophy of the heart, disorganization of cardiomyocytes, and loss of endocardium (Huang et al 2007). These&nbsp;effects result&nbsp;in reduced function of the heart and eventually cause death in zebrafish (Huang et al 2007).</li>
  • <li>COX-2 has also been demonstrated&nbsp;to have roles in cardiovascular development in humans, chicken (<em>Gallus gallus</em>), and Japanese medaka (<em>Oryzias latipes</em>) (Fujisawa et al 2014; Gullestad &amp; Aukrust 2005; Hocherl et al 2002; Huang et al 2007; Wong et al 1998).&nbsp;</li>
  • </ul>
  • <strong>Uncertainties or Inconsistencies</strong>
  • <ul>
  • <strong>Uncertainties and Inconsistencies</strong>
  • <ul>
  • <li>Intermediary steps between increased expression of COX-2 and altered heart development and function have not been investigated.</li>
  • <li>The precise role of COX-2 and the prostaglandin synthesis pathway in early development of the heart is not known.</li>
  • </ul>
  • <strong>Quantitative Understanding of the Linkage</strong>
  • <ul>
  • <li>There is an R<sup>2</sup> of 0.84 between relative COX-2 mRNA and altered heart development measured as pericardial area in embryos of Japanese medaka exposed to the COX-2 inducer, 2,3,7,8-teterachlorodibenzo-p-dioxin (TCDD) (Dong et al 2010).</li>
  • </ul>
  • <!--<!% unless aop_rel.relationship.relationship_taxons.blank? %>-->
  • <!--<!%= render 'snapshot_taxons', taxons: aop_rel.relationship.relationship_taxons %>-->
  • <!--<!% unless aop_rel.relationship.taxon_evidence.blank? %>-->
  • <!--<h3>Evidence Supporting Taxonomic Applicability</h3>-->
  • <!--<!%== aop_rel.relationship.taxon_evidence %>-->
  • <!--<!% end %>-->
  • <!--<!% end %>-->
  • <h4>References</h4>
  • <p>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.</p>
  • <h4>References</h4>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • <p>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.</p>
  • </div>
  • <br>
  • <div>
  • <h4><a href="/relationships/1352">Altered, Cardiovascular development/function leads to Increase, Mortality</a></h4>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <br>
  • <!-- loop to find taxonomic applicability under relationship -->
  • <div>
  • <div>
  • <h4><a href="/relationships/1567">Relationship: 1567: Altered, Cardiovascular development/function leads to Increase, Early Life Stage Mortality</a></h4>
  • <h4>AOPs Referencing Relationship</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP Name</th>
  • <th scope="col">Adjacency</th>
  • <th scope="col">Weight of Evidence</th>
  • <th scope="col">Quantitative Understanding</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/150">Aryl hydrocarbon receptor activation leading to early life stage mortality, via reduced VEGF</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/21">Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/456">Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Term</th>
  • <th>Scientific Term</th>
  • <th>Evidence</th>
  • <th>Links</th>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Danio rerio</td>
  • <td>Danio rerio</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>mammals</td>
  • <td>mammals</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>fish</td>
  • <td>fish</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>chicken</td>
  • <td>Gallus gallus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for taxons -->
  • <!-- loop to find life stages under relationship -->
  • <div>
  • </div>
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Life Stage</th>
  • <th>Evidence</th>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Embryo</td>
  • <td>Strong</td>
  • </tr>
  • <tr>
  • <td>development</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end loop for life stages -->
  • <!-- sex terms -->
  • <div>
  • </div>
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Sex</th>
  • <th>Evidence</th>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Unspecific</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <!-- end sex terms -->
  • <ul>
  • <li>Altered cardiovascular development and function leading to mortality has been extensively demonstrated in numerous teleost and non-teleost fishes and to a lesser extent in birds (Brunstrom 1990; Brunstrom &amp; Andersson 1988; Buckler et al 2015; Cohen-Barnhouse et al 2011; Elonen et al 1998; Hoffman et al 1996; 1998; Huang et al 2012; Johnson et al 1998; Park et al 2014; Powell et al 1998; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Yamauchi et al 2006; Zabel et al 1995).</li>
  • <li>Although limited studies are available, altered cardiac development and function leading to mortality is acknowledged to be applicable to reptiles, amphibians, and possibly some invertebrates based on presence of a cardiovascular system.</li>
  • </ul>
  • <h4>How Does This Key Event Relationship Work</h4>
  • <ul>
  • <li>The cardiovascular system functions in the transport of nutrients, oxygen, carbon dioxide, and hormones to all parts of the organism and is required for survival (Kardong 2006).</li>
  • </ul>
  • <!-- if nothing shows up in any of these fields, then weight of evidence will not be displayed -->
  • <h4>Weight of Evidence</h4>
  • <strong>Biological Plausibility</strong>
  • <ul>
  • <li>A properly functioning cardiovascular system is required for survival of vertebrates and some invertebrates. Therefore, altered development leading to dysfunction would be expected to cause mortality.</li>
  • </ul>
  • <strong>Empirical Support for Linkage</strong>
  • <ul>
  • <li>Pumping efficiency of the heart and measured blood cell perfusion rate decreases with increased severity of cardiovascular teratogenesis and increases percent mortality (Dong et al 2010; Teraoka et al 2008).</li>
  • <li>Severe hemorrhage is observable in mortalities as a result of complete cardiac collapse due to cardiovascular teratogenesis (Buckler et al 2015; 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).</li>
  • <li>Organisms are significantly less sensitive to mortality due to alteration in cardiovascular development and function post-heart developmental stages (Carney et al 2004; Lanham et al 2012).</li>
  • </ul>
  • </div>
  • <p>Cardiovasular remodelling and cardiac failure leading to embryo death has been observed in mammals (kopf and Walker 2009, Thakur et al.2013), fish (kopf and Walker 2009) and chickens (kopf and Walker 2009).&nbsp; Although the chick is preferenrially used as a lab model for developemental studies, this KER likely extends to other avian species aswell.</p>
  • <strong>Uncertainties or Inconsistencies</strong>
  • <ul>
  • <li>There are numerous different alterations in development or function that can result in mortality. The exact phenotypes that result in lethal versus sub-lethal outcomes is not well known.</li>
  • <li>Nothing is known about differences in altered cardiovascular development and function leading to mortality in invertebrates with open circulatory systems or closed circulatory systems.</li>
  • </ul>
  • <h4>Key Event Relationship Description</h4>
  • <p>Changes in heart morphology can result in decreased cardiac output and are associated with myocardial disease, abnormalities in cardiac loading, rhythm disorders, ischemia (restriction in blood supply to tissues, causing a shortage of oxygen and glucose needed for cellular metabolism), and cardiac compression. Severe cardiac dysfunction can result in congestive fetal heart failure (inability of the heart to deliver adequate blood flow to organs) leading to fluid build-up in tissues and cavities (edema and effusion, respectively). Fluid buildup exerts a positive pressure on fetal cardiac chambers, which further limits the diastolic ventricular filling reserve, potentiating the diminished cardiac output and leading to fetal death (Thakur et al. 2013).</p>
  • <p>It remains unclear whether edema plays an essential role in causing fetal death, or whether it simply accelerates the rate of deterioration; nonetheless, it is a reliable indicator of cardiotoxicity.</p>
  • <h4>Evidence Supporting this KER</h4>
  • <strong>Biological Plausibility</strong>
  • <p>The connection between altered cardiovascular developement during embryogenesis, diminished cardiac output and embryonic death have been well studied (Thakur et al. 2013; kopf and Walker 2009)</p>
  • <strong>Quantitative Understanding of the Linkage</strong>
  • <ul>
  • <li>There is strong quantitative understanding between incidence of and severity of cardiovascular teratogenicity and mortality (Buckler et al 2015; 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).</li>
  • <li>However, numerous different cardiovascular endpoints are investigated among studies making side-by-side comparisons difficult.</li>
  • <strong>Empirical Evidence</strong>
  • <ul>
  • <li>The most common cause of infant death due to birth defects is congenital cardiovascular malformation (Kopf and Walker 2009)</li>
  • <li>At low doses of dioxin-like compounds, disrupted heart looping (Henshel et al. 1993), congenital heart defects, (Cheung et al. 1981) and impaired contraction of cardiac myocytes (Canga et al. 1993) were observed in chick embryos without the onset of edema. Whereas at higher doses edema and embryo death are increased (Walker et al. 1997).</li>
  • <li>Changes in heart morphology consistent with dilated cardiomyopathy (decreased cardiac output and ventricular cavity expansion) were observed in chick embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) followed by progression to congestive heart failure.</li>
  • <li>Changes in heart morphology and decreases in cardiac output and peripheral blood flow precede heart failure in Zebrafish (Antkiewicz et al. 2005; Belair et al. 2001; Henry et al. 1997; Plavicki et al. 2013)</li>
  • <li>When mannitol is used as a protective agent against chemical-induced edema in zebrafish, cardiotoxic effects are still observed; therefore, edema is secondary to cardiotoxicity (Antkiewicz et al. 2005; Plavicki et al. 2013)</li>
  • <li>Edema is a hallmark sign of cardio-developmental toxicity in fish, chick, and mammalian species exposed to strong AHR agonists early in embryogenesis (Carney et al. 2006)
  • <ul>
  • <li>Note that it presents as pericardial and yolk sac edema in fish, pericardial, peritoneal and subcutaneous edema on chicks, and peritoneal and subcutaneous edema in mice.</li>
  • </ul>
  • </li>
  • </ul>
  • <strong>Uncertainties and Inconsistencies</strong>
  • <p>There is no doubt that severely altered cardiovascular development early in embryogenesis causes embryonic death, however the precise sequence of events leading to heart failure remains to be elucidated.</p>
  • <!--<!% unless aop_rel.relationship.relationship_taxons.blank? %>-->
  • <!--<!%= render 'snapshot_taxons', taxons: aop_rel.relationship.relationship_taxons %>-->
  • <!--<!% unless aop_rel.relationship.taxon_evidence.blank? %>-->
  • <!--<h3>Evidence Supporting Taxonomic Applicability</h3>-->
  • <!--<!%== aop_rel.relationship.taxon_evidence %>-->
  • <!--<!% end %>-->
  • <!--<!% end %>-->
  • <h4>References</h4>
  • <p>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.</p>
  • <p>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.</p>
  • <p>Buckler J.; Candrl, J.S.; McKee, M.J.; Papoulias, D.M.; Tillitt, D.E.; Galat, D.L. Sensitivity of shovelnose sturgeon (<em>Scaphirhynchus platorynchus</em>) and pallid sturgeon (<em>S. albus</em>) early life stages to PCB-126 and 2,3,7,8-TCDD exposure. <em>Enviro. Toxicol. Chem. </em><strong>2015</strong>, 34(6), 1417-1424.</p>
  • <p>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 (<em>Coturnix japonica</em>), common pheasant (<em>Phasianus colchicus</em>), and white leghorn chicken (<em>Gallus gallus domesticus</em>) embryos to <em>in ovo</em> exposure to TCDD, PeCDF, and TCDF. Toxicol. Sci. 119, 93-102.</p>
  • <p>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-<em>p</em>-dioxin to seven freshwater fish species during early life-stage development.<em> Enviro. Toxico. Chem. </em><strong>1998</strong>, 17, 472-483.</p>
  • <p>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.</p>
  • <p>Hoffman, D.J., Rice, C.P., Kubiak, T.J., 1996. PCBs and dioxins in birds. In: Beyer, W.N.,</p>
  • <p>Heinz, G.H., Redmon-Norwood, A.W. (Eds.), Environmental Contaminants in Wildlife:</p>
  • <p>Interpreting Tissue Concentrations. CRC Press, pp. 165&ndash;207.</p>
  • <p>Hoffman, D.J., Melancon, M.J., Klein, P.N., Eisemann, J.D., Spann, J.W., 1998. Comparative</p>
  • <p>developmental toxicity of planar polychlorinated biphenyl congeners in chickens,</p>
  • <h4>References</h4>
  • <p>1. Thakur, V., Fouron, J. C., Mertens, L., and Jaeggi, E. T. (2013). Diagnosis and management of fetal heart failure. Can. J Cardiol. 29(7), 759-767.</p>
  • <p>American kestrels, and common terns. Environ. Toxicol. Chem. 17, 747&ndash;757.</p>
  • <p>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.</p>
  • <p>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-<em>p</em>-dioxin to early life stage brooke trout (<em>Salvelinus fontinalis</em>) following parental dietary exposure. <em>Enviro. Toxicol. Chem.</em> <strong>1998</strong>, 17 (12), 2408-2421.</p>
  • <p>Kardong, K.V. (2006). Vertebrates: comparative anatomy, function, evolution. McGraw-Hill Higher Eduction. Boston, USA.</p>
  • <p>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. <em>Sci. Totl. Enviro.</em> <strong>2014</strong>, 491-492, 271-278.</p>
  • <p>2. 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.</p>
  • <p>Powell, D.C., Aulerich, R.J., Meadows, J.C., Tillitt, D.E., Kelly, M.E., Stromborg, K.L.,</p>
  • <p>3. Antkiewicz, D. S., Burns, C. G., Carney, S. A., Peterson, R. E., and Heideman, W. (2005). Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. 84(2), 368-377.</p>
  • <p>Melancon, M.J., Fitzgerald, S.D., Bursian, S.J., 1998. Effects of 3,3&prime;,4,4&prime;,5-</p>
  • <p>4. Belair, C. D., Peterson, R. E., and Heideman, W. (2001). Disruption of erythropoiesis by dioxin in the zebrafish. Dev. Dyn. 222(4), 581-594.</p>
  • <p>pentachlorobiphenyl and 2,3,7,8-tetrachlorodibenzo-p-dioxin injected into the</p>
  • <p>5. 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(6), 1142-1151.</p>
  • <p>yolks of double-crested cormorant (Phalacrocorax auritus) eggs prior to incubation.</p>
  • <p>6. Cheung, M. O., Gilbert, E. F., and Peterson, R. E. (1981). Cardiovascular teratogenicity of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin in the chick embryo. Toxicol. Appl. Pharmacol. 61(2), 197-204.</p>
  • <p>Environ. Toxicol. Chem. 17, 2035&ndash;2040.</p>
  • <p>7. Henry, T. R., Spitsbergen, J. M., Hornung, M. W., Abnet, C. C., and Peterson, R. E. (1997). Early life stage toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish (Danio rerio). Toxicol. Appl. Pharmacol. 142(1), 56-68.</p>
  • <p>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&rsquo;,4,4&rsquo;,5-pentachlorobiphenyl. 2015. Enviro. Toxicol. Chem. DOI: 10.1002/etc.3614.</p>
  • <p>8. Henshel, D. S., Hehn, B. M., Vo, M. T., and Steeves, J. D. (1993). A short-term test for dioxin teratogenicity using chicken embryos. In Environmental Toxicology and Risk Assessment: Volume 2 (J.W.Gorsuch, F.J.Dwyer, C.G.Ingersoll, and T.W.La Point, Eds.), pp. 159-174. American Society of Testing and materials, Philedalphia.</p>
  • <p>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 <em>Fundulus heteroclitus</em> embryos. <em>Aquat. Toxicol.</em> <strong>2001</strong>, 53, 127-138.</p>
  • <p>9. Plavicki, J., Hofsteen, P., Peterson, R. E., and Heideman, W. (2013). Dioxin inhibits zebrafish epicardium and proepicardium development. Toxicol. Sci. 131(2), 558-567.</p>
  • <p>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.</p>
  • <p>10. Carney, S. A., Prasch, A. L., Heideman, W., and Peterson, R. E. (2006). Understanding dioxin developmental toxicity using the zebrafish model. Birth Defects Res. A Clin Mol. Teratol. 76(1), 7-18.</p>
  • <p>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 (<em>Salvelinus namaycush</em>). <em>Canad. J. Fisheries Aqua. Sci.</em> <strong>1991</strong>, 48, 875-883.</p>
  • <p>11. 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.</p>
  • <p>Yamauchi, M.; Kim, E.Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2,3,7,8-tetrachlorodibenzo-<em>p</em>-dioxin (TCDD) in developing red seabream (<em>Pagrus major</em>) embryos: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. <em>Aquat. Toxicol.</em> <strong>2006</strong>, 16, 166-179.</p>
  • <p>12. 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.</p>
  • <p>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. <strong>1995</strong>. 31, 315-328.</p>
  • <p>&nbsp;</p>
  • </div>
  • <br>
  • <h3>List of Non Adjacent Key Event Relationships</h3>
  • <div>
  • <h4><a href="/relationships/984">Relationship: 984: Activation, AhR leads to Increase, Early Life Stage Mortality</a></h4>
  • <h4>AOPs Referencing Relationship</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP Name</th>
  • <th scope="col">Adjacency</th>
  • <th scope="col">Weight of Evidence</th>
  • <th scope="col">Quantitative Understanding</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/150">Aryl hydrocarbon receptor activation leading to early life stage mortality, via reduced VEGF</a></td>
  • <td>non-adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/21">Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2</a></td>
  • <td>non-adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/456">Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity</a></td>
  • <td>non-adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/455">Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced impeded craniofacial development</a></td>
  • <td>non-adjacent</td>
  • <td>High</td>
  • <td>Moderate</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <!-- end relationship loop -->
  • <!-- graphical representation -->
  • <h2>Graphical Representation</h2>
  • <img src="http://aopwiki.ag.epa.gov/system/dragonfly/development/2017/02/09/3oieicx997_AhR_agonism_embry_toxicity_figure_002_.jpg" , height="500" , width="700"> </img>
  • <!-- end graphical representation -->
  • </div>
  • <!-- end summary -->
  • <!-- Overall assessment section, *** what is included here? *** -->
  • <div id="overall_assessment">
  • <h2>Overall Assessment of the AOP</h2>
  • <p><em>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. </em></p>
  • <hr>
  • <h3>Domain of Applicability</h3>
  • <strong>Life Stage Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Life Stage</th>
  • <th>Evidence</th>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Embryo</td>
  • <td>Strong</td>
  • </tr>
  • <tr>
  • <td>development</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>chicken</td>
  • <td>Gallus gallus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Japanese quail</td>
  • <td>Coturnix japonica</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=93934" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Ring-necked pheasant</td>
  • <td>Phasianus colchicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9054" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>turkey</td>
  • <td>Meleagris gallopavo</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9103" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>bobwhite quail</td>
  • <td>Colinus virginianus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9014" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>American kestrel</td>
  • <td>Falco sparverius</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=56350" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Double-crested cormorant</td>
  • <td>Double-crested cormorant</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Eastern bluebird</td>
  • <td>Eastern bluebird</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>zebrafish</td>
  • <td>Danio rerio</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Fundulus heteroclitus</td>
  • <td>Fundulus heteroclitus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8078" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Mus musculus</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Oncorhynchus mykiss</td>
  • <td>Oncorhynchus mykiss</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8022" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Xenopus laevis</td>
  • <td>Xenopus laevis</td>
  • <td>Low</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • </div>
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Term</th>
  • <th>Scientific Term</th>
  • <th>Evidence</th>
  • <th>Links</th>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Atlantic killifish</td>
  • <td>Fundulus heteroclitus</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8078" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>zebrafish</td>
  • <td>Danio rerio</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>rainbow trout</td>
  • <td>Oncorhynchus mykiss</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8022" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>medaka</td>
  • <td>Oryzias latipes</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8090" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>fathead minnow</td>
  • <td>Pimephales promelas</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>channel catfish</td>
  • <td>Ictalurus punctatus</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7998" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Salvelinus namaycush</td>
  • <td>Salvelinus namaycush</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8040" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Salvelinus fontinalis</td>
  • <td>Salvelinus fontinalis</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8038" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Carassius carassius</td>
  • <td>Carassius carassius</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=217509" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Pagrus major</td>
  • <td>Pagrus major</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=143350" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Acipenser fulvescens</td>
  • <td>Acipenser fulvescens</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=41871" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Coregonus artedi</td>
  • <td>Coregonus artedi</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=36181" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Catostomus commersonii</td>
  • <td>Catostomus commersonii</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7971" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Esox lucius</td>
  • <td>Esox lucius</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8010" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Scaphirhynchus albus</td>
  • <td>Scaphirhynchus albus</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=36178" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Scaphirhynchus platorynchus</td>
  • <td>Scaphirhynchus platorynchus</td>
  • <td>Moderate</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7910" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Xenopus laevis laevis</td>
  • <td>Xenopus laevis laevis</td>
  • <td>Weak</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=443947" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tr>
  • <td>Gallus gallus</td>
  • <td>Gallus gallus</td>
  • <td>Strong</td>
  • <td>
  • <a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" , target="_blank">NCBI</a>
  • </td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Embryo</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td>Development</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="panel panel-default">
  • <table class="table table-bordered table-striped">
  • <thead>
  • </div>
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th>Sex</th>
  • <th>Evidence</th>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>Unspecific</td>
  • <td>Strong</td>
  • </tr>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p><strong>Sex: </strong>This AOP is only applicable to early life stages prior to sexual differentiation.</p>
  • </div>
  • <ul>
  • <li>Overall, this KER is believed to be applicable to all&nbsp;vertebrates based on mortality as a result of exposure to known agonists of the AhR (Buckler et al 2015; Cohen-Barnhouse et al 2011; Elonen et al 1998; Johnson et al 1998; Jung et al 1997; Kopf &amp; Walker 2009; Park et al 2014; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Wang et al 2013; Yamauchi et al 2006; Zabel et al 1995).</li>
  • </ul>
  • <p><strong>Life stages: </strong>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 &amp; Stegeman 2008).</p>
  • <p>&nbsp;</p>
  • <ul>
  • <li>The correlation between AHR-mediated reporter gene activity and embryo death has been demonstrated in species of birds and fishes&nbsp;(Doernig et al 2018).</li>
  • <li>Less&nbsp;is known about differences in binding affinity of AhRs and how this relates to sensitivity in reptiles or amphibians.</li>
  • <li>Low binding affinity for DLCs of AhR1s of African clawed frog (<em>Xenopus laevis</em>) and axolotl (<em>Ambystoma mexicanum</em>) has been suggested as a mechanism for tolerance of these amphibians to DLCs (Lavine et al 2005; Shoots et al 2015).</li>
  • <li>Among reptiles, only AhRs of American alligator (<em>Alligator mississippiensis</em>) have been investigated and little is known about the sensitivity of American alligator or other reptiles to DLCs (Oka et al 2016).</li>
  • <li>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; 2018).</li>
  • <li>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 (<em>Microgadus tomcod</em>) (Wirgin et al 2011).</li>
  • </ul>
  • <h4>Key Event Relationship Description</h4>
  • <p>The aryl hydrocarbon receptor is commonly known for its involvement in xenobiotic metabolism and clearance, but it also regulates a number of endogenous processes including angiogenesis, immune responses, neuronal processes, metabolism, and development of numerous organ systems (Duncan et al., 1998; Emmons et al., 1999; Hahn et al 2002; Lahvis and Bradfield, 1998).&nbsp; Strong AHR agonists that cause <u>sustained</u> AHR activation interfere with the receptor&#39;s endogenous role in embryogenesis, which causes numerous developmental abnormalities and ultimately leads to embryonic death (Kopf and Walker 2009; Carreira et al 2015).</p>
  • <p><strong>Taxonomic:</strong> 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 &amp; Kennedy 2009; Thackaberry et al 2005).</p>
  • <p>It&#39;s important to note that his relationship only applies to AHR agonists that cause sustained AHR activation.&nbsp; Strong AHR agonists that are rapidly metabolized, such as polycyclic aromatic hydrocarbons, only cause transient AHR activation leading to an alternate mode of toxicity.</p>
  • <p>This AOP is applicable to:</p>
  • <p>This Key Event Relationship describes the indirect link between the Molecular Initiating Event (activation of the AhR) and the Adverse Outcome (increased early life stage mortality).</p>
  • <h4>Evidence Supporting this KER</h4>
  • <strong>Biological Plausibility</strong>
  • <p><em><strong>AHR Ligand Binding Domain</strong></em></p>
  • <ul>
  • <li>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).</li>
  • <li>All birds (Canga et al 1993; Cohen-Barnhouse et al 2011; Fujisawa et al 2014; Heid et al 2001; Ivnitski et al 2001; Walker &amp; 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 (<em>Gallus gallus</em>) (Fujisawa et al 2014).</li>
  • <li>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.</li>
  • <li>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.</li>
  • <li>Mammalian and avian sensitivity to DLCs ultimately comes down to the identity of two particular amino acids in the ligand binding domain (LBD) of the AHR: positions 375 and 319 in mice and 380 and 324 in birds.
  • <ul>
  • <li>A 10-fold difference 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 (Poland et al. 1976), was attributed to &nbsp;a single nucleotide polymorphism at position 375 (Ema et al. 1994; Poland et al. 1994; Poland and Knutson 1982).</li>
  • <li>Several other studies reported the importance of this amino acid in birds and mammals (Backlund and Ingelman-Sundberg 2004; Ema et al. 1994; Karchner et al. 2006; Murray et al. 2005; Pandini et al. 2007; Pandini et al. 2009; Poland et al. 1994; Ramadoss and Perdew 2004).</li>
  • </ul>
  • </li>
  • <li>The amino acid at position 319 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 (Pandini et al. 2009).
  • <ul>
  • <li>Mutation at position 319 in the mouse eliminated AHR DNA binding (Pandini et al. 2009).</li>
  • </ul>
  • </li>
  • </ul>
  • <p>This AOP is not applicable to:</p>
  • <p><em><strong>Using AHR LBD Constructs to Determine Avian Sensitivity</strong></em></p>
  • <ul>
  • <li>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 &amp; Walker 2009). Further, studies of CYP1A1 and CYP1A2 null mice (<em>Mus musculus</em>) demonstrate that wasting syndrome and mortality are mediated by CYP1A1 in mammals (Uno et al 2004).</li>
  • <li>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).</li>
  • <li>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.</li>
  • <li>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 (2006), showed that amino acid differences within the LBD were responsible for differences in TCDD sensitivity between the chicken and common tern.
  • <ul>
  • <li>They specifically attributed positions 324 and 380 with differences in TCDD binding affinity and transactivation between the chicken (Ile324_Ser380) and common tern (Val324_Ala380) receptors.</li>
  • </ul>
  • </li>
  • <li>The LBD of over 85 bird species have since been analyzed to find that 6 amino acid residues differed among species (Farmahin et al. 2013; Head et al. 2008), but only positions 324 and 380 in the AHR1 LBD were associated with differences in DLC toxicity in ovo and AHR1-mediated gene expression in vitro (Farmahin et al. 2013; Head et al. 2008; Manning et al. 2012).
  • <ul>
  • <li>Based on these results, 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; most sensitive), type 2 (Ile324_Ala380; moderately sensitive) and type 3 (Val324_Ala380; least sensitive) (Farmahin et al. 2013; Head et al. 2008; Manning et al. 2012).</li>
  • <li>A sampling of bird species and their AHR LBD category is described in table 1. A list of 86 species and their subtype can be found in Farmahin et al. (2013).</li>
  • </ul>
  • </li>
  • </ul>
  • <h3>Essentiality of the Key Events</h3>
  • <p>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.</p>
  • <p>&nbsp;Rationale for essentiality calls:</p>
  • <p><a class="image" href="/wiki/index.php/File:AHR1_LBD_Types.png"><img alt="AHR1 LBD Types.png" src="/wiki/images/6/68/AHR1_LBD_Types.png" style="height:487px; width:1088px" /></a></p>
  • <strong>Empirical Evidence</strong>
  • <p><strong>Mammals:</strong></p>
  • <ul>
  • <li>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 &amp; Di Giulio 2011).</li>
  • <li>AhR deficient strains of mice (<em>Mus musculus</em>)&nbsp;are unaffected by exposure to&nbsp;agonists of the AhR (Fernandez-Salguero et al 1996).</li>
  • <li>Strains of mice that express AhRs with lesser affinity for agonists are more tolerant to adverse effects of exposure relative to strains of mice that express AhRs with greater affinity for agonists (Bisson et al 2009; Ema et al 1993).</li>
  • </ul>
  • <p>&nbsp;</p>
  • <p><strong>Birds:</strong></p>
  • <p>Binding of dioxin-like compounds (DLCs) to avian AHR1 (Farmahin et al. 2014; Karchner et al. 2006) and AHR1-mediated transactivation measured using luciferase reporter gene (LRG) assays have been demonstrated in domestic and wild species of birds (Farmahin et al. 2012; Farmahin et al. 2013b; Fujisawa et al. 2012; Lee et al. 2009; Manning et al. 2012; Mol et al. 2012), and binding affinity was found to be strongly correlated with embryotoxicity (Manning et al. 2012) .</p>
  • <p><strong>Fish:</strong></p>
  • <ul>
  • <li>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).</li>
  • <li>Knockdown of the AhR2 prevents mortality following exposure to agonist of the AhR in fishes (Clark et al 2010; Hanno et al 2010; Prasch et al 2003; Van Tiem &amp; Di Giulio 2011). Relative potencies of dioxin-like compounds for activation of AHR2 alpha of rainbow trout (<em>Oncorhynchus mykiss</em>) is predictive of relative potencies for early life stage mortality (Abnet et al 1999).</li>
  • <li>AhR2-mediated transactivation measured using luciferase reproter gene (LRG) assays have been demonstrated in 8 species of freshwater and marine fishes to strongly correlate with early life stage mortality (Doering et al 2018). However, AhR1-mediated transactivation does not (Doering et al 2018). Further, the slope and y-intercept for the relationship between AhR2-mediated transactivation and early life stage mortality in fishes are not statistically different from the slope and y-intercept for the relatoinship between AhR1-mediated transactivatoin and embryotoxicity (Doering et al 2018).</li>
  • </ul>
  • <p>&nbsp;</p>
  • <p><strong>Amphibians:</strong></p>
  • <ul>
  • <li>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).</li>
  • <li>AhR1s of amphibians studied to date are insensitive to activation by dioxin-like compounds<em>&nbsp;in vitro</em>, while amphibians studies to date are extremely tolerant to adverse effects of exposure to dioxin-like compounds&nbsp;<em>in vivo</em>&nbsp;(Jung et al 1997; Lavine et al 2005; Shoots et al 2015).</li>
  • </ul>
  • <p>&nbsp;</p>
  • <p><strong>Invertebrates:</strong></p>
  • <ul>
  • <li>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.</li>
  • <li>Chemicals that activate the AhR of vertebrates are not known to bind AhRs of invertebrates and increased mortality in invertebrates&nbsp;has never been observed as a result of exposure&nbsp;to these agonists&nbsp;(Hahn 2002; Hahn et al 1994).</li>
  • </ul>
  • <h3>Weight of Evidence Summary</h3>
  • <p><strong>Biological Plausibility: </strong></p>
  • <strong>Uncertainties and Inconsistencies</strong>
  • <p>Interestingly, interference with endogenous AHR functions, <u>either by knock-out or by agonist exposure</u> during early development, causes similar cardiac abnormalities (Carreira et al 2015). Although this is counterintuitive, it demonstrates that the AHR has an optimal window of activity, and deviation either above or below this range results in toxicity.</p>
  • <p><strong>Uncertainites:</strong></p>
  • <ul>
  • <li>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.</li>
  • <li>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.</li>
  • <li>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).</li>
  • <li>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.</li>
  • <li>Only limited AhR activation information and mortality information is currently available for reptiles and amphibians.</li>
  • <li>Despite decades of research into the molecular initiating event (i.e., binding of&nbsp;chemicals to the AhR) and resulting adverse outcomes (i.e. mortality), less is known about the precise cascade of key events that link activation of the AhR to the adverse outcome (Doering et al 2016).</li>
  • <li>However, hundreds to thousands of different genes&nbsp;are regulated, either directly or indirectly, by activation of&nbsp;the AhR, which presents major uncertainties in the precise&nbsp;pathway of key events or whether perturbation to multiple&nbsp;pathways&nbsp;is the cause of mortality&nbsp;(Brinkmann et al 2016; Doering et al 2016; Huang et al 2014; Li et al 2013; Whitehead et al 2010).</li>
  • <li>Despite these uncertainties in the AOP, considerable research has investigated the indirect relationship between activation of the AhR and increased mortality among different chemicals, species, and taxa (Doering et al 2013).</li>
  • </ul>
  • <p>&nbsp;</p>
  • <p><strong>Concordance of dose-response relationships: </strong></p>
  • <p><strong>Inconsistencies:</strong></p>
  • <ul>
  • <li>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 &amp; 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).</li>
  • <li>Less is known regarding concordance of dose-response relationships for COX-2. In Japanese medaka (<em>Oryzias latipes</em>), 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).</li>
  • <li>There are no currently known inconsistencies between AhR activation and increased mortality among vertebrates.</li>
  • </ul>
  • <h4>References</h4>
  • <p><br />
  • 1. 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(2), 416-425.</p>
  • <p>&nbsp;</p>
  • <p>2. 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(44), 27337-27343.</p>
  • <p><strong>Temporal concordance among the key events and adverse effect:</strong></p>
  • <p>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. (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.</p>
  • <ul>
  • <li>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 &amp; Stegeman 2006).</li>
  • <li>AhR transcript and protein is first detectable in zebrafish around 24 hpf (Tanguay et al 1999).</li>
  • <li>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).</li>
  • <li>Therefore, there is a general temporal concordance in this AOP.</li>
  • <li>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.</li>
  • </ul>
  • <p>4. 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(19), 7535-7541.</p>
  • <p>&nbsp;</p>
  • <p>5. 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.</p>
  • <p><strong>Consistency:</strong></p>
  • <p>6. 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(3), 390-399.</p>
  • <ul>
  • <li>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.</li>
  • <li>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.</li>
  • <li>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).</li>
  • <li>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.</li>
  • </ul>
  • <p>7. 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(1), 59-71.</p>
  • <p>&nbsp;</p>
  • <p>8. 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(3), 696-708.</p>
  • <p><strong>Uncertainties, inconsistencies, and data gaps:</strong></p>
  • <p>9. 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(25), 5972-5983.</p>
  • <ul>
  • <li>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 &amp; Stegeman 2008), and AhR cross-talk with hypoxia inducible factor 1&alpha; (HIF1&alpha;) (Goldstone &amp; Stegeman 2008).</li>
  • <li>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).</li>
  • <li>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 &amp; 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).</li>
  • <li>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.</li>
  • <li>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.</li>
  • </ul>
  • <h3>Quantitative Consideration</h3>
  • <ul>
  • <li>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).</li>
  • <li>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).</li>
  • <li>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 (R<sup>2</sup> = 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.</li>
  • <li>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.</li>
  • </ul>
  • <p>10. 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(16), 4936-4946.</p>
  • <ul>
  • </ul>
  • </div>
  • <!-- potential consierations, text as entered by author -->
  • <div id="considerations_for_potential_applicaitons">
  • </div>
  • <!-- reference section, text as of right now but should be changed to be handled as table -->
  • <div id="references">
  • <h2>References</h2>
  • <hr>
  • <p>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<em>.</em> 159, 41-51.</p>
  • <p>11. 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. 12. Poland, A., Palen, D., and Glover, E. (1994). Analysis of the four alleles of the murine aryl hydrocarbon receptor. Mol. Pharmacol. 46(5), 915-921.</p>
  • <p>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.</p>
  • <p>13. 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(1), 129-136.</p>
  • <p>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.</p>
  • <p>14. Farmahin, R., Wu, D., Crump, D., Herv&eacute;, 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. Environ Sci Technol. 46(5), 2967-75.</p>
  • <p>Belair, C.D.; Peterson, R.E.; Heideman, W. (2001). Disruption of erythropoiesis by dioxin in the zebrafish. Dev. Dyn. 222 (4), 581-594.</p>
  • <p>15. 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.</p>
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