<!-- Evidence for Perturbation of This Event by Stressors -->
<h3>Evidence for Perturbation by Stressor</h3>
<hr>
<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>
<br>
<br>
<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. <em>Toxicol.Sci.</em> <strong>124</strong>, 1-22.</p>
</p>
<br>
<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> </p>
<p>Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. <em>Critical Reviews in Toxicology</em> <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. <em>Environ.Health Perspect</em>. <strong>81</strong>, 225-239.</p>
</p>
<br>
<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. <em>Toxicol.Sci.</em> <strong>124</strong>, 1-22.</p>
</p>
<br>
<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. <em>Br. J Dermatol. </em><strong>111</strong> (4), 413-422.</p>
</p>
<br>
<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> </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>
<br>
<!-- end Evidence for Perturbation of This Event by Stressors -->
<h4>Domain of Applicability</h4>
<br>
<!-- loop to find taxonomic applicability under event -->
<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>
<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 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:"Times New Roman",serif">The AhR is a very conserved and ancient protein (95) and the AhR is present in human and mice (96–98). </span></span><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The AhR is present in human physiology and pathology. T</span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",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). 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:"Times New Roman",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:"Times New Roman",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:"Times New Roman",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:"Times New Roman",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:"Times New Roman",serif">Subramaniam, Barhoover</span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">). </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",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:"Times New Roman",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:"Times New Roman",serif">Rothhammer</span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">). </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Indoles, such as indole-3-carbinol or one of its secondary metabolites, 3-3'- 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> </p>
<p> </p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<h3>The AHR Receptor</h3>
<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 “single-minded” 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>
<div>Figure 1: The molecular mechanism of activation of gene expression by AHR.</div>
<div> </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 (α, β, δ, γ) 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> </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 & 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>
<p><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Role in mammals</span></span></em></p>
<p><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">AhR expression is essentially ubiquitous in mammals consistent with a broad-spectrum homeostatic role, however expression levels varying widely across tissues with the liver, thymus, lung, kidney, spleen, and placenta exhibiting greatest expression (Harper PA). Additionally, AhR expression is developmentally regulated, and more recent evidence indicates a role for the AhR in developmental process affecting hematopoiesis, immune system biology, neural differentiation, and liver architecture (Wright E J) . AHR is involved in regulating the rate of apoptosis of oocytes in germ cell nests during embryonic life and in regulating survival of oocytes in the fetal and neonatal ovary. Specifically, studies have shown that ovaries obtained from AHRKO mice on ED13.5 and cultured for 72 h in the absence of hormonal support with the aim of inducing apoptosis, contained higher numbers of non-apoptotic germ cells compared to wild-type (WT) ovaries cultured in the same conditions (Hernández-Ochoa)</span></span></em></p>
<p> </p>
<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>
<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>
<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 (<em>Homo sapiens</em>) (Abnet et al 1999) </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>) (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 AhR activation has been investigated (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 & 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. 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>
<br>
<h4>References</h4>
<ol>
<h4>References</h4>
<ol>
<li>↑ <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">↑</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>↑ <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>↑ <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">↑</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>↑ <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>↑ <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">↑</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>
<li>↑ <sup><a href="#cite_ref-Safe1994_9-0">9.0</a></sup> <sup><a href="#cite_ref-Safe1994_9-1">9.1</a></sup> <sup><a href="#cite_ref-Safe1994_9-2">9.2</a></sup> Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. <em>Critical Reviews in Toxicology</em> <strong>24</strong>, 87-149.</li>
<li>↑ <sup><a href="#cite_ref-Yasui2007_10-0">10.0</a></sup> <sup><a href="#cite_ref-Yasui2007_10-1">10.1</a></sup> <sup><a href="#cite_ref-Yasui2007_10-2">10.2</a></sup> Yasui, T., Kim, E. Y., Iwata, H., Franks, D. G., Karchner, S. I., Hahn, M. E., and Tanabe, S. (2007). Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. <em>Toxicol.Sci</em>. <strong>99</strong>, 101-117.</li>
<li>↑ <sup><a href="#cite_ref-Lee2009_11-0">11.0</a></sup> <sup><a href="#cite_ref-Lee2009_11-1">11.1</a></sup> Lee, J. S., Kim, E. Y., and Iwata, H. (2009). Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): association with aryl hydrocarbon receptor 1 and 2 isoforms. <em>Toxicol.Appl.Pharmacol</em>. <strong>234</strong>, 1-13.</li>
<li>↑ <sup><a href="#cite_ref-Raucy2010_12-0">12.0</a></sup> <sup><a href="#cite_ref-Raucy2010_12-1">12.1</a></sup> <sup><a href="#cite_ref-Raucy2010_12-2">12.2</a></sup> <sup><a href="#cite_ref-Raucy2010_12-3">12.3</a></sup> <sup><a href="#cite_ref-Raucy2010_12-4">12.4</a></sup> <sup><a href="#cite_ref-Raucy2010_12-5">12.5</a></sup> Raucy, J. L., and Lasker, J. M. (2010). Current in vitro high throughput screening approaches to assess nuclear receptor activation. <em>Curr. Drug Metab</em> <strong>11</strong> (9), 806-814.</li>
<li>↑ <sup><a href="#cite_ref-Farmahin2012_13-0">13.0</a></sup> <sup><a href="#cite_ref-Farmahin2012_13-1">13.1</a></sup> <sup><a href="#cite_ref-Farmahin2012_13-2">13.2</a></sup> Farmahin, R., Wu, D., Crump, D., Hervé, J. C., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., and Kennedy, S. W. (2012). Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Environ.Sci.Technol. 46, 2967-2975.</li>
<li>↑ <sup><a href="#cite_ref-Farmahin2013b_14-0">14.0</a></sup> <sup><a href="#cite_ref-Farmahin2013b_14-1">14.1</a></sup> <sup><a href="#cite_ref-Farmahin2013b_14-2">14.2</a></sup> <sup><a href="#cite_ref-Farmahin2013b_14-3">14.3</a></sup> Farmahin, R., Manning, G. E., Crump, D., Wu, D., Mundy, L. J., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., Fredricks, T. B., and Kennedy, S. W. (2013b). Amino acid sequence of the ligand binding domain of the aryl hydrocarbon receptor 1 (AHR1) predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol.Sci. 131, 139-152.</li>
<li><a href="#cite_ref-Fujisawa2012_15-0">↑</a> Fujisawa, N., Ikenaka, Y., Kim, E. Y., Lee, J. S., Iwata, H., and Ishizuka, M. (2012). Molecular evidence predicts aryl hydrocarbon receptor ligand insensitivity in the peregrine falcon (Falco peregrines). European Journal of Wildlife Research 58, 167-175.</li>
<li>↑ <sup><a href="#cite_ref-Manning2012_16-0">16.0</a></sup> <sup><a href="#cite_ref-Manning2012_16-1">16.1</a></sup> <sup><a href="#cite_ref-Manning2012_16-2">16.2</a></sup> Manning, G. E., Farmahin, R., Crump, D., Jones, S. P., Klein, J., Konstantinov, A., Potter, D., and Kennedy, S. W. (2012). A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the embryolethality of polychlorinated biphenyls in avian species. Toxicol.Appl.Pharmacol. 263, 390-399.</li>
<li><a href="#cite_ref-Mol2012_17-0">↑</a> Mol, T. L., Kim, E. Y., Ishibashi, H., and Iwata, H. (2012). In vitro transactivation potencies of black-footed albatross (Phoebastria nigripes) AHR1 and AHR2 by dioxins to predict CYP1A expression in the wild population. Environ.Sci.Technol. 46, 525-533.</li>
<li><a href="#cite_ref-Yueh2005_18-0">↑</a> Yueh, M. F., Kawahara, M., and Raucy, J. (2005). Cell-based high-throughput bioassays to assess induction and inhibition of CYP1A enzymes. <em>Toxicol. In Vitro</em> <strong>19</strong> (2), 275-287.</li>
<li>↑ <sup><a href="#cite_ref-Poland1982_19-0">19.0</a></sup> <sup><a href="#cite_ref-Poland1982_19-1">19.1</a></sup> 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. <em>Annu. Rev. Pharmacol. Toxicol. </em> <strong>22</strong>, 517-554.</li>
<li>↑ <sup><a href="#cite_ref-Hesterman2000_20-0">20.0</a></sup> <sup><a href="#cite_ref-Hesterman2000_20-1">20.1</a></sup> Hestermann, E. V., Stegeman, J. J., and Hahn, M. E. (2000). Relative contributions of affinity and intrinsic efficacy to aryl hydrocarbon receptor ligand potency. <em>Toxicol. Appl. Pharmacol </em> <strong>168</strong> (2), 160-172.</li>
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<li>↑ <sup><a href="#cite_ref-Karchner2006_22-0">22.0</a></sup> <sup><a href="#cite_ref-Karchner2006_22-1">22.1</a></sup> <sup><a href="#cite_ref-Karchner2006_22-2">22.2</a></sup> <sup><a href="#cite_ref-Karchner2006_22-3">22.3</a></sup> <sup><a href="#cite_ref-Karchner2006_22-4">22.4</a></sup> <sup><a href="#cite_ref-Karchner2006_22-5">22.5</a></sup> <sup><a href="#cite_ref-Karchner2006_22-6">22.6</a></sup> 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. <em>Proc. Natl. Acad. Sci. U. S. A</em> <strong>103</strong> (16), 6252-6257.</li>
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<div>Hirano, M.; Hwang, JH; Park, HJ; Bak, SM; Iwata, H. and Kim, EY (2015) In Silico Analysis of the Interaction of Avian Aryl Hydrocarbon Receptors and Dioxins to Decipher Isoform-, Ligand-, and Species-Specific Activations.<cite> Environmental Science & Technology</cite> <strong>49 </strong>(6): 3795-3804.DOI: 10.1021/es505733f</div>
<div> </div>
<p>Bonati, L.; Corrada, D.; Tagliabue, S.G.; Motta, S. (2017) Molecular modeling of the AhR structure and interactions can shed light on ligand-dependent activation and transformation mechanisms. <em>Current Opinion in Toxicology </em><strong>2</strong>: 42-49. https://doi.org/10.1016/j.cotox.2017.01.011.</p>
<p>Sovadinová, I. , Bláha, L. , Janošek, J. , Hilscherová, K. , Giesy, J. P., Jones, P. D. and Holoubek, I. (2006), Cytotoxicity and aryl hydrocarbon receptor‐mediated activity of N‐heterocyclic polycyclic aromatic hydrocarbons: Structure‐activity relationships. <em>Environmental Toxicology and Chemistry</em>, <strong>25</strong>: 1291-1297. doi:<a href="https://doi.org/10.1897/05-388R.1">10.1897/05-388R.1</a></p>
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<p style="text-align:justify"> </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:"Times New Roman",serif">Wakx A, Nedder M, Tomkiewicz-Raulet C, Dalmasso J, Chissey A, et al. 2018. Expression,</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:"Times New Roman",serif">Localization, and Activity of the Aryl Hydrocarbon Receptor in the Human Placenta. Int J Mol Sci. 19(12)</span></span></span></span></p>
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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:"Times New Roman",serif">Ema M, Ohe N, Suzuki M, Mimura J, Sogawa K, et al. 1994. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors.</span></span></span></span></p>
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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:"Times New Roman",serif">Kall MA, Vang O, Clausen J. 1996. Effects of dietary broccoli on human in vivo drug metabolizing enzymes:</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:"Times New Roman",serif">evaluation of caffeine, oestrone and chlorzoxazone metabolism. Carcinogenesis. 17(4):793–99</span></span></span></span></p>
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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:"Times New Roman",serif">Miller CA. 1997. Expression of the human aryl hydrocarbon receptor complex in yeast. Activation of transcription by indole compounds. J. Biol. Chem. 272(52):32824–29</span></span></span></span></p>
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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:"Times New Roman",serif">Opitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, et al. 2011. An endogenous tumour promoting ligand of the human aryl hydrocarbon receptor. Nature. 478(7368):197–203</span></span></span></span></p>
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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:"Times New Roman",serif">Adachi J, Mori Y, Matsui S, Takigami H, Fujino J, et al. 2001. Indirubin and indigo are potent aryl hydrocarbon receptor ligands present in human urine. J. Biol. Chem. 276(34):31475–78</span></span></span></span></p>
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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:"Times New Roman",serif">Marinelli L, Martin-Gallausiaux C, Bourhis J-M, B.guet-Crespel F, Blotti.re HM, Lapaque N. 2019. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells. Sci</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:"Times New Roman",serif">Rep. 9(1):643</span></span></span></span></p>
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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:"Times New Roman",serif">Stobbe-Maicherski N, Wolff S, Wolff C, Abel J, Sydlik U, et al. 2013. The interleukin-6-type cytokine oncostatin M induces aryl hydrocarbon receptor expression in a STAT3-dependent manner in human HepG2 hepatoma cells. FEBS J. 280(24):6681–90</span></span></span></span></p>
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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:"Times New Roman",serif">Kado S, Chang WLW, Chi AN, Wolny M, Shepherd DM, Vogel CFA. 2017. Aryl hydrocarbon receptor signaling modifies Toll-like receptor-regulated responses in human dendritic cells. Arch Toxicol. 91(5):2209–21</span></span></span></span></p>
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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:"Times New Roman",serif">Bock KW. 2019. Human AHR functions in vascular tissue: Pro- and anti-inflammatory responses of AHR agonists in atherosclerosis. Biochem Pharmacol. 159:116–20</span></span></span></span></p>
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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:"Times New Roman",serif">Schroeder JC, Dinatale BC, Murray IA, Flaveny CA, Liu Q, et al. 2010. The uremic toxin 3- indoxyl sulfate is a potent endogenous agonist for the human aryl hydrocarbon receptor. Biochemistry. 49(2):393–400</span></span></span></span></p>
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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:"Times New Roman",serif">Watanabe I, Tatebe J, Namba S, Koizumi M, Yamazaki J, Morita T. 2013. Activation of aryl hydrocarbon receptor mediates indoxyl sulfate-induced monocyte chemoattractant protein-1 expression in human umbilical vein endothelial cells. Circ J. 77(1):224–30</span></span></span></span></p>
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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:"Times New Roman",serif">Shackleford G, Sampathkumar NK, Hichor M, Weill L, Meffre D, et al. 2018. Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis. Proc Natl Acad Sci U S 115(6):E1319–28</span></span></span></span></p>
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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:"Times New Roman",serif">Zudaire E, Cuesta N, Murty V, Woodson K, Adams L, et al. 2008. The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers. J Clin Invest. 118(2):640–50</span></span></span></span></p>
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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:"Times New Roman",serif">Goode GD, Ballard BR, Manning HC, Freeman ML, Kang Y, Eltom SE. 2013. Knockdown of aberrantly upregulated aryl hydrocarbon receptor reduces tumor growth and metastasis of MDA-MB- 231 human breast cancer cell line. Int J Cancer. 133(12):2769–80</span></span></span></span></p>
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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:"Times New Roman",serif">Chang JT, Chang H, Chen P-H, Lin S-L, Lin P. 2007. Requirement of aryl hydrocarbon receptor overexpression for CYP1B1 up-regulation and cell growth in human lung adenocarcinomas. Clin 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:"Times New Roman",serif">Res. 13(1):38–45</span></span></span></span></p>
<p style="text-align:justify"> </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:"Times New Roman",serif">Kanno Y, Takane Y, Izawa T, Nakahama T, Inouye Y. 2006. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The inhibitory effect of aryl hydrocarbon receptor repressor (AhRR) on the growth of human breast cancer MCF-7 cells. Biol Pharm Bull. 29(6):1254–57</span></span></span></span></p>
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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:"Times New Roman",serif">Goode G, Pratap S, Eltom SE. 2014. Depletion of the aryl hydrocarbon receptor in MDA-MB- 231 human breast cancer cells altered the expression of genes in key regulatory pathways of cancer. PLoS One. 9(6):e100103</span></span></span></span></p>
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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:"Times New Roman",serif">Opitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, et al. 2011. An endogenous tumour promoting ligand of the human aryl hydrocarbon receptor. Nature. 478(7368):197–203</span></span></span></span></p>
<p style="text-align:justify"> </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:"Times New Roman",serif">Novikov O, Wang Z, Stanford EA, Parks AJ, Ramirez-Cardenas A, et al. 2016. An Aryl Hydrocarbon Receptor-Mediated Amplification Loop That Enforces Cell Migration in ER-/PR-/Her2- Human Breast Cancer Cells. Mol Pharmacol. 90(5):674–88</span></span></span></span></p>
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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:"Times New Roman",serif">Litzenburger UM, Opitz CA, Sahm F, Rauschenbach KJ, Trump S, et al. 2014. Constitutive IDO expression in human cancer is sustained by an autocrine signaling loop involving IL-6, STAT3 and the AHR. Oncotarget. 5(4):1038–51</span></span></span></span></p>
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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:"Times New Roman",serif">Hall JM, Barhoover MA, Kazmin D, McDonnell DP, Greenlee WF, Thomas RS. 2010. Activation of the aryl-hydrocarbon receptor inhibits invasive and metastatic features of human breast cancer cells and promotes breast cancer cell differentiation. Mol Endocrinol. 24(2):359–69</span></span></span></span></p>
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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:"Times New Roman",serif">Gramatzki D, Pantazis G, Schittenhelm J, Tabatabai G, K.hle C, et al. 2009. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Aryl hydrocarbon receptor inhibition downregulates the TGF-beta/Smad pathway in human glioblastoma cells. Oncogene. 28(28):2593–2605</span></span></span></span></p>
<p style="text-align:justify"> </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:"Times New Roman",serif">Subramaniam V, Ace O, Prud’homme GJ, Jothy S. 2011. Tranilast treatment decreases cell growth, migration and inhibits colony formation of human breast cancer cells. Exp Mol Pathol. 90(1):116–22</span></span></span></span></p>
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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:"Times New Roman",serif">Rothhammer V, Borucki DM, Kenison JE, Hewson P, Wang Z, et al. 2018. Detection of aryl hydrocarbon receptor agonists in human samples. Sci Rep. 8(1):4970</span></span></span></span></p>
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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:"Times New Roman",serif">Lin P, Chang H, Tsai W-T, Wu M-H, Liao Y-S, et al. 2003. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Overexpression of aryl hydrocarbon receptor in human lung carcinomas. Toxicol Pathol. 31(1):22–30</span></span></span></span></p>
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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:"Times New Roman",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):195–201</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:"Times New Roman",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:"Times New Roman",serif">An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells. Cancer Lett. 340(1):63–71</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",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>
<p><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Harper PA, Riddick DS, Okey AB. Regulating the regulator: factors that control levels and activity of the aryl hydrocarbon receptor. Biochem Pharmacol. 2006;72(3):267-79.</span></span></em></p>
<p><em><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:14px">Hernández-Ochoa I, Karman BN, Flaws JA. The role of the aryl hydrocarbon receptor in the female reproductive system. Biochem Pharmacol. 2009;77(4):547-59.</span></span></em></p>
<td><a href="/aops/36">Aop:36 - Peroxisomal Fatty Acid Beta-Oxidation Inhibition Leading to Steatosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/57">Aop:57 - AhR activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/58">Aop:58 - NR1I3 (CAR) suppression leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/60">Aop:60 - NR1I2 (Pregnane X Receptor, PXR) activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/529">Aop:529 - Xenobiotic binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism leading to liver steatosis</a></td>
<td><a href="/aops/34">Aop:34 - LXR activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/57">Aop:57 - AhR activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/318">Aop:318 - Glucocorticoid Receptor activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/517">Aop:517 - Pregnane X Receptor (PXR) activation leads to liver steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/518">Aop:518 - Liver X Receptor (LXR) activation leads to liver steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/529">Aop:529 - Xenobiotic binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism leading to liver steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/580">Aop:580 - Mineralocorticoid Receptor Activation Leading to Increased Body Mass Index</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/591">Aop:591 - DBDPE-induced DNA damage increase in liver leading to Non-alcoholic fatty liver disease via liver steatosis and inhibition of regeneration</a></td>
<p><em>Life Stage: Older individuals are more likely to manifest this key event (adults > juveniles) due to accumulation of triglycerides.</em></p>
<p><em>Sex: Applies to both males and females.</em></p>
<p><em>Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats). Likely pervasive in many animal taxa.</em></p>
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<h4>Key Event Description</h4>
<p>Leads to Fatty Liver Cells.
</p>
<br>
<h4>Key Event Description</h4>
<p><em>Triglycerides are important building blocks for a wide variety of compounds found in organisms, with cellular concentrations reflecting the relative rate of influx and efflux, as well as the relative rate of synthesis and breakdown. However, excess accumulation </em>leads to Fatty Liver Cells <em>and steatosis</em>.</p>
<p><br />
<em>In this key event we focus on excessive accumulation of triglycerides in mammalian systems. Four major pathways for triglyceride accumulation are: 1. Increased fatty acid uptake; 2. Increased De Novo FA and Lipid Synthesis; 3. Decreased FA Oxidation; 4. Decreased Lipid Efflux (Angrish et al. 2016). Chemical stressors can increase gene expression of key genes involving these pathways, leading to increased accumulation of triglycerides (Aguayo-Orozco et al. 2018). In addition, excessive dietary compounds of fatty compounds can also increase likelihood of accumulation of triglycerides (Nguyen et al. 2008). Nuclear receptors that have been implicated in causing excessive accumulation of triglycerides leading to steatosis, when overexpressed, include (Mellor et al. 2016): Aryl hydrocarbon receptor (AHR), Constitutive androstane receptor (CAR), Oestrogen receptor (ER), Farnesoid X receptor (FXR), Glucocorticoid receptor (GXR), Liver X receptor (LXR), Peroxisome proliferator-activated receptor (PPAR), Pregnane X receptor (PXR), and Retinoic acid receptor (RAR or RXR). </em><br />
</p>
<p> </p>
<p> </p>
<h4>How it is Measured or Detected</h4>
<p><em>Concentrations of triglycerides, cholesterols, fatty acids, and related compounds are measured biochemically to assess levels in control versus potentially affected individuals; common techniques include high throughput enzymatic analyses, analytical ultracentrifuging, gradient gel electrophoresis, Nuclear Magnetic Resonance, lipidomics, and other direct assessment techniques (Schaefer et al. 2016; Yang and Han 2016). Analysis is often performed to look at gene expression levels to see which pathway(s) have increased expression levels, to attribute plausibility to changes in influx, eflux, synthesis, and/or breakdown pathways (Nguyen et al. 2008; Mellor et al. 2016, Aguayo-Orozco et al. 2018). Assessment of cellular components including mitochondria and membrane integrity can also be used as evidence of alteration of normal function within cells.</em></p>
<h4>References</h4>
<p><em>Aguayo-Orozco, A.A., Bois, F.Y., Brunak, S., and Taboureau, O. 2018. Analysis of Time-Series Gene Expression Data to Explore Mechanisms of Chemical-Induced Hepatic Steatosis Toxicity. Frontiers in Genetics 9(Article 396): 1-15.</em></p>
<p><em>Angrish, M.M., Kaiser, J.P., McQueen, C.A., and Chorley, B.N. 2016. Tipping the Balance: Hepatotoxicity and the 4 Apical Key Events of Hepatic Steatosis. Toxicological Sciences 150(2): 261–268.</em></p>
<p><br />
<em>Mellor, C.L., Steinmetz, F.P., and Cronin, T.D. 2016. The identification of nuclear receptors associated with hepatic steatosis to develop and extend adverse outcome pathways. Critical Reviews in Toxicology, 46(2): 138-152.</em></p>
<p><br />
<em>Nguyen, P., Leray, V., Diez, M., Serisier, S., Le Bloc’h, J., Siliart, B., and Dumon, H. 2008. Liver lipid metabolism. Journal of Animal Physiology and Animal Nutrition 92: 272–283.</em></p>
<p><em>Schaefer EJ, Tsunoda F, Diffenderfer M, Polisecki, E., Thai, N., and Astalos, B. The Measurement of Lipids, Lipoproteins, Apolipoproteins, Fatty Acids, and Sterols, and Next Generation Sequencing for the Diagnosis and Treatment of Lipid Disorders. [Updated 2016 Mar 29]. In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK355892/</em></p>
<p><em>Yang, K. and Han, X. 2016. Lipidomics: Techniques, applications, and outcomes related to biomedical sciences. Trends in Biochemical Sciences 2016 November ; 41(11): 954–969.</em></p>
<p><em>NOTE: Italics symbolize edits from John Frisch</em></p>
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<div>
<h4><a href="/events/54">Event: 54: Up Regulation, CD36</a><br></h4>
<!-- Evidence for Perturbation of This Event by Stressors -->
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<!-- event text -->
<h4>KeyEvent Description</h4>
<p>Fatty acid translocase CD36 (FAT/CD36) is a scavenger protein mediating uptake and intracellular transport of long-chain fatty acids (FA) in diverse cell types <sup id="cite_ref-1" class="reference"><a href="#cite_note-1">[1]</a></sup>, <sup id="cite_ref-2" class="reference"><a href="#cite_note-2">[2]</a></sup>. In addition, CD36 can bind a variety of molecules including acetylated low density lipoproteins (LDL), collagen and phospholipids <sup id="cite_ref-3" class="reference"><a href="#cite_note-3">[3]</a></sup>. CD36 has been shown to be expressed in liver tissue <sup id="cite_ref-4" class="reference"><a href="#cite_note-4">[4]</a></sup>, <sup id="cite_ref-5" class="reference"><a href="#cite_note-5">[5]</a></sup>. It is located in lipid rafts and non-raft domains of the cellular plasma membrane and most likely facilitates LCFA transport by accumulating LCFA on the outer surface <sup id="cite_ref-6" class="reference"><a href="#cite_note-6">[6]</a></sup>, <sup id="cite_ref-7" class="reference"><a href="#cite_note-7">[7]</a></sup>, <sup id="cite_ref-8" class="reference"><a href="#cite_note-8">[8]</a></sup>.
</p><p>FAT/CD36 gene is a liver specific target of LXR activation <sup id="cite_ref-9" class="reference"><a href="#cite_note-9">[9]</a></sup>. Studies have confirmed that the lipogenic effect of LXR and activation of FAT/CD36 was not a simple association, since the effect of LXR agonists on increasing hepatic and circulating levels of triglycerides and free fatty acids (FFAs) was largely abolished in FAT/CD36 knockout mice suggesting that intact expression and/or activation of FAT/CD36 is required for the steatotic effect of LXR agonists <sup id="cite_ref-10" class="reference"><a href="#cite_note-10">[10]</a></sup>, <sup id="cite_ref-11" class="reference"><a href="#cite_note-11">[11]</a></sup>. In addition to the well-defined pathogenic role of FAT/CD36 in hepatic steatosis in rodents the human up-regulation of the FAT/CD36 in NASH patients is confirmed <sup id="cite_ref-12" class="reference"><a href="#cite_note-12">[12]</a></sup>. There are now findings that can accelerate the translation of FAT/CD36 metabolic functions determined in rodents to humans <sup id="cite_ref-13" class="reference"><a href="#cite_note-13">[13]</a></sup> and suggest that the translocation of this fatty acid transporter to the plasma membrane of hepatocytes may contribute to liver fat accumulation in patients with NAFLD and HCV <sup id="cite_ref-14" class="reference"><a href="#cite_note-14">[14]</a></sup>. In addition, hepatic FAT/CD36 up-regulation is significantly associated with insulin resistance, hyperinsulinaemia and increased steatosis in patients with NASH and HCV G1 (Hepatitis C Virus Genotype1) with fatty liver. Recent data show that CD36 is also increased in the liver of morbidly obese patients and correlated to free FA levels <sup id="cite_ref-15" class="reference"><a href="#cite_note-15">[15]</a></sup>.
</p>
<br>
<h4>References</h4>
<ol class="references">
<li id="cite_note-1"><span class="mw-cite-backlink"><a href="#cite_ref-1">↑</a></span> <span class="reference-text">Su & Abumrad 2009</span>
</li>
<li id="cite_note-2"><span class="mw-cite-backlink"><a href="#cite_ref-2">↑</a></span> <span class="reference-text">He et al. 2011</span>
<p><em>Life Stage: Older individuals are more likely to manifest this key event (adults > juveniles) due to increased opportunity to upregulate gene expression.</em></p>
<p><em>Sex: Applies to both males and females.</em></p>
<p><em>Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</em></p>
<p>Fatty acid translocase CD36 (FAT/CD36) is a scavenger protein mediating uptake and intracellular transport of long-chain fatty acids (FA) in diverse cell types <sup><a href="#cite_note-1">[1]</a></sup>, <sup><a href="#cite_note-2">[2]</a></sup>. In addition, CD36 can bind a variety of molecules including acetylated low density lipoproteins (LDL), collagen and phospholipids <sup><a href="#cite_note-3">[3]</a></sup>. CD36 has been shown to be expressed in liver tissue <sup><a href="#cite_note-4">[4]</a></sup>, <sup><a href="#cite_note-5">[5]</a></sup>. It is located in lipid rafts and non-raft domains of the cellular plasma membrane and most likely facilitates LCFA transport by accumulating LCFA on the outer surface <sup><a href="#cite_note-6">[6]</a></sup>, <sup><a href="#cite_note-7">[7]</a></sup>, <sup><a href="#cite_note-8">[8]</a></sup>.</p>
<p>FAT/CD36 gene is a liver specific target of LXR activation <sup><a href="#cite_note-9">[9]</a></sup>. Studies have confirmed that the lipogenic effect of LXR and activation of FAT/CD36 was not a simple association, since the effect of LXR agonists on increasing hepatic and circulating levels of triglycerides and free fatty acids (FFAs) was largely abolished in FAT/CD36 knockout mice suggesting that intact expression and/or activation of FAT/CD36 is required for the steatotic effect of LXR agonists <sup><a href="#cite_note-10">[10]</a></sup>, <sup><a href="#cite_note-11">[11]</a></sup>. In addition to the well-defined pathogenic role of FAT/CD36 in hepatic steatosis in rodents the human up-regulation of the FAT/CD36 in NASH patients is confirmed <sup><a href="#cite_note-12">[12]</a></sup>. There are now findings that can accelerate the translation of FAT/CD36 metabolic functions determined in rodents to humans <sup><a href="#cite_note-13">[13]</a></sup> and suggest that the translocation of this fatty acid transporter to the plasma membrane of hepatocytes may contribute to liver fat accumulation in patients with NAFLD and HCV <sup><a href="#cite_note-14">[14]</a></sup>. In addition, hepatic FAT/CD36 up-regulation is significantly associated with insulin resistance, hyperinsulinaemia and increased steatosis in patients with NASH and HCV G1 (Hepatitis C Virus Genotype1) with fatty liver. Recent data show that CD36 is also increased in the liver of morbidly obese patients and correlated to free FA levels <sup><a href="#cite_note-15">[15]</a></sup>.</p>
<h4>How it is Measured or Detected</h4>
<p><em>CD36 is measured by changes in gene expression and protein levels. </em></p>
<h4>References</h4>
<ol>
<li><a href="#cite_ref-1">↑</a> Su & Abumrad 2009 - Su X., Abumrad N.A., Cellular fatty acid uptake: a pathway under construction. Trends<br />
Endocrinol. Metab., 20 (No 2), 72-77, 2009</li>
<li><a href="#cite_ref-2">↑</a> He et al. 2011 - He J. et al, The emerging roles of fatty acid translocase/CD36 and the aryl hydrocarbon<br />
receptor in fatty liver disease, Exp. Med. And Biology, 236, 1116-1121, 2011</li>
<li><a href="#cite_ref-3">↑</a> Krammer 2011 - Krammer J. et al, Overexpression of CD36 and Acyl-CoA Synthetases FATP2, FATP4<br />
and ACSL1 Increases Fatty Acid Uptake in Human Hepatoma Cells, Int. J. Med. Sci.,<br />
8(7), 599-614, 2011</li>
<li><a href="#cite_ref-4">↑</a> Pohl et al. 2005 - Pohl J., et al, FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires<br />
<li><a href="#cite_ref-5">↑</a> Cheung et al. 2007 - Cheung L., et al, Hormonal and nutritional regulation of alternative CD36 transcripts<br />
in rat liver--a role for growth hormone in alternative exon usage, BMC Mol. Biol., 8, 60,<br />
2007</li>
<li><a href="#cite_ref-6">↑</a> Ehehalt et al. 2008 - Ehehalt R., et al, Uptake of long chain fatty acids is regulated by dynamic interaction<br />
of FAT/CD36 with cholesterol/sphingolipid enriched microdomains (lipid rafts). BMC<br />
Cell. Biol., 9, 45, 2008</li>
<li><a href="#cite_ref-7">↑</a> Pohl et al. 2005 - Pohl J., et al, FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires<br />
<li><a href="#cite_ref-8">↑</a> Krammer 2011 - Krammer J. et al, Overexpression of CD36 and Acyl-CoA Synthetases FATP2, FATP4<br />
and ACSL1 Increases Fatty Acid Uptake in Human Hepatoma Cells, Int. J. Med. Sci.,<br />
8(7), 599-614, 2011</li>
<li><a href="#cite_ref-9">↑</a> Zhou 2008 - Zhou J., Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and<br />
PPAR gamma in promoting steatosis, Gastroenterology, 134 (No 2),556-567, 2008</li>
<li><a href="#cite_ref-10">↑</a> Febbraio et al. 1999 - Febbraio M., et al, A null mutation in murine CD36 reveals an important role in fatty<br />
acid and lipoprotein metabolism, J Biol Chem, 274, 19055–19062, 1999</li>
<li><a href="#cite_ref-11">↑</a> Lee et al. 2008 - Lee J.H., et al, PRX and LXR in hepatic Steatosis: a new dog and an old dog with new<br />
tricks, Mol. Pharm., 5(No 1),60-66, 2008</li>
<li><a href="#cite_ref-12">↑</a> Zhu et al. 2011 - Zhu L., et al, Lipid in the livers of adolescents with non-alcoholic steatohepatitis:<br />
combined effects of pathways on steatosis, Metabolism Clinical and experimental, 30,<br />
1001-1011, 2011</li>
<li><a href="#cite_ref-13">↑</a> Love-Gregory et al. 2011 - Love-Gregory L., Abumrad N.A., CD36 genetics and the metabolic complications of<br />
obesity, Current Opinions in Clinical Nutition and Metabolic Care, 14 (No 6), 527-534,<br />
2011</li>
<li><a href="#cite_ref-14">↑</a> Miquilena-Colina et al. 2011 - Miquilena-Colina M.E., et al, Hepatic fatty acid translocase CD36 upregulation is<br />
associated with insulin resistance, hyperinsulinaemia and increased steatosis in nonalcoholic<br />
steatohepatitis and chronic hepatitis C, Gut., 60 (No 10), 1394-1402 , 2011</li>
<li><a href="#cite_ref-15">↑</a> Bechmann et al. 2010 - Bechmann L.P., et al, Apoptosis is associated with CD36/fatty acid translocase<br />
upregulation in non-alcoholic steatohepatitis, Liver Int., 30 (No 6), 850-859, 2010 </li>
</ol>
<p><em>NOTE: Italics symbolize edits from John Frisch</em></p>
<td><a href="/aops/57">Aop:57 - AhR activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/60">Aop:60 - NR1I2 (Pregnane X Receptor, PXR) activation leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/58">Aop:58 - NR1I3 (CAR) suppression leading to hepatic steatosis</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/624">Aop:624 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated mitochondrial dysfunction</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/625">Aop:625 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated oxidative stress</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/626">Aop:626 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated endoplasmic reticulum stress</a></td>
<td><a href="/aops/58">Aop:58 - NR1I3 (CAR) suppression leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/60">Aop:60 - NR1I2 (Pregnane X Receptor, PXR) activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/61">Aop:61 - NFE2L2/FXR activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/62">Aop:62 - AKT2 activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/36">Aop:36 - Peroxisomal Fatty Acid Beta-Oxidation Inhibition Leading to Steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/213">Aop:213 - Inhibition of fatty acid beta oxidation leading to nonalcoholic steatohepatitis (NASH)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/285">Aop:285 - Inhibition of N-linked glycosylation leads to liver injury</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/318">Aop:318 - Glucocorticoid Receptor activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/517">Aop:517 - Pregnane X Receptor (PXR) activation leads to liver steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/518">Aop:518 - Liver X Receptor (LXR) activation leads to liver steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/529">Aop:529 - Xenobiotic binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism leading to liver steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/232">Aop:232 - NFE2/Nrf2 repression to steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/57">Aop:57 - AhR activation leading to hepatic steatosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/494">Aop:494 - AhR activation leading to liver fibrosis </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/591">Aop:591 - DBDPE-induced DNA damage increase in liver leading to Non-alcoholic fatty liver disease via liver steatosis and inhibition of regeneration</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/624">Aop:624 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/625">Aop:625 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated oxidative stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/626">Aop:626 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via insulin resistance-associated endoplasmic reticulum stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/627">Aop:627 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/628">Aop:628 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated oxidative stress</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/629">Aop:629 - Increased 11β-Hydroxysteroid dehydrogenase type 1 activity leading to MASLD progression via lipogenesis-associated endoplasmic reticulum stress</a></td>
<p>Steatosis is the result of perturbations in well-known metabolic pathways that are well-studied and well-known in many taxa.</p>
<p><em>Life Stage: The life stage applicable to this key event is all life stages with a liver. Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides.</em></p>
<p><em>Sex: This key event applies to both males and females.</em></p>
<p><em>Taxonomic: This key event appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</em></p>
<h4>Key Event Description</h4>
<p>Biological state: liver steatosis is the inappropriate storage of fat in hepatocytes. <em>Four major pathways for triglyceride accumulation are: 1. Increased fatty acid uptake; 2. Increased De Novo FA and Lipid Synthesis; 3. Decreased FA Oxidation; 4. Decreased Lipid Efflux (Angrish et al. 2016). Chemical stressors can increase gene expression of key genes involving these pathways, leading to increased accumulation of triglycerides (Aguayo-Orozco et al. 2018). In addition, excessive dietary compounds of fatty compounds can also increase likelihood of accumulation of triglycerides (Nguyen et al. 2008). </em></p>
<p>Biological compartment: steatosis is generally an organ-level diagnosis; however, the pathology occurs within the hepatocytes.</p>
<p>Role in biology: steatosis is an adverse endpoint. </p>
<p><span style="color:#d35400"><strong>Consequences: Liver steatosis, or fatty liver, serves as a pivotal factor in the development of liver fibrosis by triggering a cascade of pathological events. According to the two-strikes hypothesis (Day and James, 1998), liver damage progresses in two stages: the first strike involves the accumulation of lipids in hepatocytes, often due to metabolic disturbances such as insulin resistance, excess free fatty acids, or oxidative stress. This stage, though asymptomatic, increases liver vulnerability by inducing mild oxidative stress and inflammation. The second strike introduces additional insults, such as inflammatory mediators or cellular damage, exacerbating liver injury and promoting fibrogenesis. The accumulation of fat sensitizes the liver to oxidative stress and triggers mechanisms like the activation of hepatic stellate cells (HSCs) and hepatocyte apoptosis or necrosis, central to the fibrotic process. While early-stage steatosis is reversible, chronic steatosis perpetuates a cycle of inflammation and fibrosis, creating a feedback loop that amplifies liver damage (Pafili K et al, 2021). Consequently, liver steatosis is not only a precursor but also a critical driver of fibrosis progression.</strong></span></p>
<p><span style="font-size:12px"><span style="color:#d35400"><strong>Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998 Apr;114(4):842-5. doi: 10.1016/s0016-5085(98)70599-2. PMID: 9547102.</strong></span></span></p>
<p><span style="font-size:12px"><span style="color:#d35400"><strong>Pafili K, Roden M. Nonalcoholic fatty liver disease (NAFLD) from pathogenesis to treatment concepts in humans. Mol Metab. 2021 Aug;50:101122. doi: 10.1016/j.molmet.2020.101122. Epub 2020 Nov 19. PMID: 33220492; PMCID: PMC8324683.</strong></span></span></p>
<p>Description from EU-ToxRisk:</p>
<p>Activation of stellate cells results in collagen accumulation and change in extracellular matrix composition in the liver causing fibrosis. (Landesmann, 2016; Koo et al 2016)</p>
<!-- event text -->
<h4>How it is Measured or Detected</h4>
<p>Steatosis is measured by lipidomics approaches<em> (e.g. Yang and Han 2016)</em> that measure lipid levels, or by histology. <em>Concentrations of triglycerides, cholesterols, fatty acids, and related compounds are measured biochemically include high throughput enzymatic analyses, analytical ultracentrifuging, gradient gel electrophoresis, Nuclear Magnetic Resonance, and other direct assessment techniques (Schaefer et al. 2016).</em></p>
<h4>Regulatory Significance of the AO</h4>
<p>Steatosis is a regulatory endpoint and has been used as an endpoint in many US EPA assessments, including IRIS assessments.</p>
<h4>References</h4>
<p><em>Aguayo-Orozco, A.A., Bois, F.Y., Brunak, S., and Taboureau, O. 2018. Analysis of Time-Series Gene Expression Data to Explore Mechanisms of Chemical-Induced Hepatic Steatosis Toxicity. Frontiers in Genetics 9(Article 396): 1-15.</em></p>
<p><em>Angrish, M.M., Kaiser, J.P., McQueen, C.A., and Chorley, B.N. 2016. Tipping the Balance: Hepatotoxicity and the 4 Apical Key Events of Hepatic Steatosis. Toxicological Sciences 150(2): 261–268.</em></p>
<p>Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998 Apr;114(4):842-5. doi: 10.1016/s0016-5085(98)70599-2. PMID: 9547102.</p>
<p>Landesmann, B. (2016). Adverse Outcome Pathway on Protein Alkylation Leading to Liver Fibrosis, (2).</p>
<p>Koo, J. H., Lee, H. J., Kim, W., & Kim, S. G. (2016). Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2. <em>Gastroenterology</em>, <em>150</em>(1), 181–193.e8. https://doi.org/10.1053/j.gastro.2015.09.039</p>
<p><em>Nguyen, P., Leray, V., Diez, M., Serisier, S., Le Bloc’h, J., Siliart, B., and Dumon, H. 2008. Liver lipid metabolism. Journal of Animal Physiology and Animal Nutrition 92: 272–283. </em></p>
<p><em>Pafili K, Roden M. Nonalcoholic fatty liver disease (NAFLD) from pathogenesis to treatment concepts in humans. Mol Metab. 2021 Aug;50:101122. doi: 10.1016/j.molmet.2020.101122. Epub 2020 Nov 19. PMID: 33220492; PMCID: PMC8324683.</em></p>
<p><em>Schaefer EJ, Tsunoda F, Diffenderfer M, Polisecki, E., Thai, N., and Astalos, B. The Measurement of Lipids, Lipoproteins, Apolipoproteins, Fatty Acids, and Sterols, and Next Generation Sequencing for the Diagnosis and Treatment of Lipid Disorders. [Updated 2016 Mar 29]. In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK355892/</em></p>
<p><em>Yang, K. and Han, X. 2016. Lipidomics: Techniques, applications, and outcomes related to biomedical sciences. Trends in Biochemical Sciences 2016 November ; 41(11): 954–969.</em></p>
<p><em>NOTE: Italics symbolize edits from John Frisch</em></p>
<!-- end event text -->
</div>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<!-- Evidence for relationship links section, this lists the relationships and then supports them -->
<td><a href="/aops/517">Pregnane X Receptor (PXR) activation leads to liver steatosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/518">Liver X Receptor (LXR) activation leads to liver steatosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>Not Specified</td>
</tr>
<tr>
<td><a href="/aops/529">Xenobiotic binding to peroxisome proliferator-activated receptors (PPARs) causes dysregulation of lipid metabolism leading to liver steatosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</td>
</tr>
<tr>
<td><a href="/aops/57">AhR activation leading to hepatic steatosis</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/591">DBDPE-induced DNA damage increase in liver leading to Non-alcoholic fatty liver disease via liver steatosis and inhibition of regeneration</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p>Life Stage: All life stages with a liver. Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to accumulation of triglycerides.</p>
<p><br />
Sex: Applies to both males and females.</p>
<p><br />
Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).<br />
</p>
<h4>Key Event Relationship Description</h4>
<p>Steatosis is a key event representing increased accumulation of fat in liver cells. In this key event relationship we are focused on accumulation of triglycerides leading to steatosis. Increased accumulation of triglycerides in cells is evidence of imbalance in the influx and synthesis versus metabolism or breakdown of lipid compounds. Increased accumulation of triglycerides can be enhanced by chemical stressors, or alteration of regulation by gene expression. </p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The biological plausibility linking accumulation of triglycerides to steatosis is strong. Increased accumulation of triglycerides represents an imbalanced influx and synthesis of compounds versus normal function, resulting in liver steatosis.</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In vitro exposure of 20 mM amiodarone, 50 mM tetracycline.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">HepG2 human cells showed correlated increases in triglycerides and other lipid compounds and steatosis oxidation after 14 days of tetracycline exposure and after both 1 and 14 days of amiodarone exposure.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">In vitro exposure of at least 6 concentrations to 28 compounds selected for steatogenic potential.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Transgenic and wild-type mice with normal and high cholesterol diet.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Human subjects with liver steatosis had increased RBP4 gene expression. Transgenic mice with human RBP4 gene had correlated increases in triglycerides associated with steatosis, in comparison to wild-type mice.</span></span></p>
Antherieu, S., Rogue, A., Fromenty, B., Guillouzo, A., and Robin, M.-A. 2011. Induction of Vesicular Steatosis by Amiodarone and Tetracycline Is Associated with Up-regulation of Lipogenic Genes in HepaRG Cells. Hepatology 53:1895-1905.</p>
<p><br />
Donato, M.T., Martinez-Romero, A. Jimenez, N., Negro, A., Gerrerad, G., Castell, J.V., O’Connor, J.-E., and Gomez-Lechon, M.J. 2009. Cytometric analysis for drug-induced steatosis in HepG2 cells. Chemico-Biological Interactions 181: 417–423.</p>
<p><br />
Landesmann, B., Goumenou, M., Munn, S., and Whelan, M. 2012. Description of Prototype Modes-of-Action Related to Repeated Dose Toxicity. European Commission Report EUR 25631, 49 pages. https://op.europa.eu/en/publication-detail/-/publication/d2b09726-8267-42de-8093-8c8981201d65/language-en</p>
<p> </p>
<p>Liu, Y., Mu, D., Chen, H., Li, D., Song, J., Zhong, Y., and Xia, M. 2016. Retinol-Binding Protein 4 Induces Hepatic Mitochondrial Dysfunction and Promotes Hepatic Steatosis. The Journal of Clinical Endocrinology and Metabolism 101: 4338–4348.</p>
<p> </p>
<p>Negi, C.K., Bajard, L., Kohoutek, J., and Blaha, L. 2021. An adverse outcome pathway based in vitro characterization of novel flame retardants-induced hepatic steatosis. Environmental Pollution 289: 117855.</p>
<h4><a href="/relationships/499">Relationship: 499: Activation, AhR leads to Decreased, PCK1 expression (control point for glycolysis/gluconeogenesis pathway)</a></h4>