92-87-5HFACYLZERDEVSX-UHFFFAOYSA-NHFACYLZERDEVSX-UHFFFAOYSA-N
Benzidine4-(4-Aminophenyl)aniline
[1,1'-Biphenyl]-4,4'-diamine
(1,1'-Biphenyl)-4,4'-diamine
4,4'-Bianiline
4,4'-Biphenyldiamine
4,4'-Diamino-1,1'-biphenyl
4,4'-Diaminobiphenyl
4,4'-Diaminodiphenyl
4,4'-Diphenylenediamine
4'-Amino-[1,1'-biphenyl]-4-ylamine
bencidina
Benzidin
C.I. Azoic Diazo Component 112
Fast Corinth Base B
NSC 146476
p,p'-Bianiline
p,p'-Diaminobiphenyl
p-Diaminodiphenyl
UN 1885
DTXSID2020137262-12-4NFBOHOGPQUYFRF-UHFFFAOYSA-NNFBOHOGPQUYFRF-UHFFFAOYSA-N
Dibenzo-p-dioxinDibenzo[b,e][1,4]dioxin
Dibenzo[1,4]dioxin
dibenzo-p-dioxina
dibenzo-p-dioxinne
Diphenylene dioxide
Oxanthrene
Phenodioxin
DTXSID8020410118-74-1CKAPSXZOOQJIBF-UHFFFAOYSA-NCKAPSXZOOQJIBF-UHFFFAOYSA-N
Hexachlorobenzene(HCB
Benzene, hexachloro-
Anticarie
Benzene, 1,2,3,4,5,6-hexachloro-
Benzenehexachloride
Bunt-cure
Bunt-no-more
Co-op Hexa
Hexachlorbenzol
hexaclorobenceno
Julin's carbon chloride
No Bunt
No Bunt Liquid
NSC 9243
Pentachlorophenyl chloride
Perchlorobenzene
Sanocide
Snieciotox
UN 2729
Zaprawa nasienna sneciotox
1,2,3,4,5,6-Hexachloro-benzene
DTXSID2020682PR:000003858aryl hydrocarbon receptorCL:0000066epithelial cellD001943Breast NeoplasmsGO:0004874aryl hydrocarbon receptor activityGO:0006954inflammatory responseGO:0006915apoptotic processGO:0048870cell motilityGO:0043542endothelial cell migration1increased11pathological2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)2017-02-09T14:32:322017-02-09T14:32:32Benzidine2016-11-29T18:42:262016-11-29T18:42:26Dibenzo-p-dioxin2016-11-29T18:42:272016-11-29T18:42:27Polychlorinated biphenyl2016-11-29T18:42:272016-11-29T18:42:27Polychlorinated dibenzofurans2016-11-29T18:42:272016-11-29T18:42:27Hexachlorobenzene2016-11-29T18:42:272016-11-29T18:42:27Polycyclic aromatic hydrocarbons (PAHs)2017-02-09T15:43:002017-02-09T15:43:007955zebra danioWCS_9031Gallus gallus143350Pagrus major7904Acipenser transmontanus41871Acipenser fulvescensWCS_8022rainbow trout8030Salmo salarWCS_8355Xenopus laevis8296Ambystoma mexicanumWCS_9054Phasianus colchicusWCS_93934Coturnix japonica10090mouse10116ratWCS_9606human34823Microgadus tomcod9606Homo sapiens10090Mus musculus10116Rattus norvegicus6239Caenorhabditis elegansWikiUser_17mammalsWCS_9606humans10095miceActivation, AhRActivation, AhRMolecular<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>
<h3>The molecular Initiating Event</h3>
<div>
<div><a class="image" href="/wiki/index.php/File:AHR_mechanism.jpeg"><img alt="" class="thumbimage" src="/wiki/images/thumb/6/6e/AHR_mechanism.jpeg/450px-AHR_mechanism.jpeg" style="height:331px; width:450px" /></a>
<div>Figure 1: The molecular mechanism of activation of gene expression by AHR.</div>
<div> </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>Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? </em></p>
<h3>Transactivation Reporter Gene Assays (recommended approach)</h3>
<h4>Transient transfection transactivation</h4>
<p>Transient transfection transactivation is the most common method for evaluating nuclear receptor activation<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. Full-length AHR cDNAs are cloned into an expression vector along with a reporter gene construct (chimeric luciferase, P-lactamase or CAT reporter vectors containing the appropriate response elements for the gene of interest). There are a number of commercially available cell lines that can serve as recipients for these vectors (CV-1, HuH7, FLC-7, LS174T, LS180 MCF-7, HEC1, LLC-PK1, HEK293, HepG2, and Caco-2 cells)<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>. The greatest advantage of using transfected cells, rather than primary cell cultures, is the assurance that the nuclear receptor of interest is responsible for the observed induction. This would not be possible in a primary cell culture due to the co-regulation of different receptors for the same target genes. This model makes it easy to compare the responsiveness of the AHR across multiple species under the same conditions simply by switching out the AHR clone. One disadvantage to the transient transfection assay is the inherent variability associated with transfection efficiency, leading to a movement towards the use of stable cell lines containing the nuclear receptor and reporter gene linked to the appropriate response elements<sup><a href="#cite_note-Raucy2010-12">[12]</a></sup>.</p>
<h5>Luciferase reporter gene (LRG) assay</h5>
<p>The described luciferase reporter gene (LRG) assays have been used to investigate activation of AhRs of:</p>
<ul>
<li>Humans (<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>
<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></p>
HighUnspecificHighEmbryoHighDevelopmentHighAll life stagesHighHighHighHighHighHighHighHighHighHighHighHighHighHighHighNot Specified<ol>
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<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>
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<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>
<li>↑ <sup><a href="#cite_ref-Farmahin2014_21-0">21.0</a></sup> <sup><a href="#cite_ref-Farmahin2014_21-1">21.1</a></sup> <sup><a href="#cite_ref-Farmahin2014_21-2">21.2</a></sup> <sup><a href="#cite_ref-Farmahin2014_21-3">21.3</a></sup> <sup><a href="#cite_ref-Farmahin2014_21-4">21.4</a></sup> Farmahin, R., Jones, S. P., Crump, D., Hahn, M. E., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., and Kennedy, S. W. (2014). Species-specific relative AHR1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency. <em>Comp Biochem. Physiol C. Toxicol. Pharmacol.</em> <strong>161C</strong>, 21-25.</li>
<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>
<li><a href="#cite_ref-Lee2015_23-0">↑</a> Lee, S., Shin, W. H., Hong, S., Kang, H., Jung, D., Yim, U. H., Shim, W. J., Khim, J. S., Seok, C., Giesy, J. P., and Choi, K. (2015). Measured and predicted affinities of binding and relative potencies to activate the AhR of PAHs and their alkylated analogues. <em>Chemosphere</em> <strong>139</strong>, 23-29.</li>
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<li><a href="#cite_ref-Gasiewicz1982_25-0">↑</a> Gasiewicz, T. A., and Neal, R. A. (1982). The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxylapatite. <em>Anal. Biochem. </em> <strong>124</strong> (1), 1-11.</li>
<li><a href="#cite_ref-Nakai1995_26-0">↑</a> Nakai, J. S., and Bunce, N. J. (1995). Characterization of the Ah receptor from human placental tissue. <em>J Biochem. Toxicol. </em> <strong>10</strong> (3), 151-159.</li>
<li><a href="#cite_ref-Dold1990_27-0">↑</a> Dold, K. M., and Greenlee, W. F. (1990). Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture. <em>Anal. Biochem. </em> <strong>184</strong> (1), 67-73.</li>
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<li>↑ <sup><a href="#cite_ref-Ema1994_30-0">30.0</a></sup> <sup><a href="#cite_ref-Ema1994_30-1">30.1</a></sup> Ema, M., Ohe, N., Suzuki, M., Mimura, J., Sogawa, K., Ikawa, S., and Fujii-Kuriyama, Y. (1994). Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors. J.Biol.Chem. 269, 27337-27343.</li>
<li>↑ <sup><a href="#cite_ref-Poland1994_31-0">31.0</a></sup> <sup><a href="#cite_ref-Poland1994_31-1">31.1</a></sup> Poland, A., Palen, D., and Glover, E. (1994). Analysis of the four alleles of the murine aryl hydrocarbon receptor. Mol.Pharmacol. 46, 915-921.</li>
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<li><a href="#cite_ref-Thack2002_56-0">↑</a> Thackaberry, E. A., Gabaldon, D. M., Walker, M. K., and Smith, S. M. (2002). Aryl hydrocarbon receptor null mice develop cardiac hypertrophy and increased hypoxia-inducible factor-1alpha in the absence of cardiac hypoxia. <em>Cardiovasc.Toxicol.</em> <strong>2</strong>, 263-274.</li>
<li><a href="#cite_ref-Zhang2010_57-0">↑</a> Zhang, N., Agbor, L. N., Scott, J. A., Zalobowski, T., Elased, K. M., Trujillo, A., Duke, M. S., Wolf, V., Walsh, M. T., Born, J. L., Felton, L. A., Wang, J., Wang, W., Kanagy, N. L., and Walker, M. K. (2010). An activated renin-angiotensin system maintains normal blood pressure in aryl hydrocarbon receptor heterozygous mice but not in null mice. <em>Biochem.Pharmacol.</em> <strong>80</strong>, 197-2040.</li>
</ol>
<p>Abnet, C.C.; Tanguay, R.L.; Heideman, W.; Peterson, R.E. 1999. Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: Greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners. Toxicol. Appl. Pharmacol<em>.</em> 159, 41-51.</p>
<p> </p>
<p>Andreasen, E.A.; Tanguay, R.L.; Peterson, R.E.; Heideman, W. 2002. Identification of a critical amino acid in the aryl hydrocarbon receptor. J. Biol. Chem. 277 (15), 13210-13218.</p>
<p> </p>
<p>Bak, S.M.; Lida, M.; Hirano, M.; Iwata, H.; Kim, E.Y. 2013. Potencies of red seabream AHR1- and AHR2-mediated transactivation by dioxins: implications of both AHRs in dioxin toxicity. Environ. Sci. Technol. 47 (6), 2877-2885.</p>
<p> </p>
<p>Clark, B.W.; Matson, C.W.; Jung, D.; Di Giulio, R.T. 2010. AHR2 mediates cardiac teratogenesis of polycyclic aromatic hydrocarbons and PCB-126 in Atlantic killifish (<em>Fundulus heteroclitus</em>). Aquat. Toxicol. 99, 232-240.</p>
<p> </p>
<p>Doering, J.A.; Farmahin, R.; Wiseman, S.; Beitel, S.C.; Kennedy, S.W.; Giesy, J.P.; Hecker, M. 2015. Differences in activation of aryl hydrocarbon receptors of white sturgeon relative to lake sturgeon are predicted by identities of key amino acids in the ligand binding domain. Enviro. Sci. Technol. 49, 4681-4689.</p>
<p> </p>
<p>Doering, J.A.; Farmahin, R.; Wiseman, S.; Kennedy, S.; Giesy J.P.; Hecker, M. 2014. Functionality of aryl hydrocarbon receptors (AhR1 and AhR2) of white sturgeon (Acipenser transmontanus) and implications for the risk assessment of dioxin-like compounds. Enviro. Sci. Technol. 48, 8219-8226.</p>
<p> </p>
<p>Doering, J.A.; Giesy, J.P.; Wiseman, S.; Hecker, M. Predicting the sensitivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain. <em>Environ. Sci. Pollut. Res. Int.</em> <strong>2013</strong>, 20(3), 1219-1224.</p>
<p> </p>
<p>Doering, J.A.; Wiseman, S; Beitel, S.C.; Giesy, J.P.; Hecker, M. 2014. Identification and expression of aryl hydrocarbon receptors (AhR1 and AhR2) provide insight in an evolutionary context regarding sensitivity of white sturgeon (<em>Acipenser transmontanus</em>) to dioxin-like compounds. Aquat. Toxicol. 150, 27-35.</p>
<p> </p>
<p>Duncan, D.M.; Burgess, E.A.; Duncan, I. 1998. Control of distal antennal identity and tarsal development in Drosophila by spineless-aristapedia, a homolog of the mammalian dioxin receptor. Genes Dev. 12, 1290-1303.</p>
<p> </p>
<p>Eisner, B.K.; Doering, J.A.; Beitel, S.C.; Wiseman, S.; Raine, J.C.; Hecker, M. 2016. Cross-species comparison of relative potencies and relative sensitivities of fishes to dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls in vitro. Enviro. Toxicol. Chem. 35 (1), 173-181.</p>
<p> </p>
<p>Emmons, R.B.; Duncan, D.; Estes, P.A.; Kiefel, P.; Mosher, J.T.; Sonnenfeld, M.; Ward, M.P.; Duncan, I.; Crews, S.T. 1999. The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila. Development. 126, 3937-3945.</p>
<p> </p>
<p>Evans, B.R.; Karchner, S.I.; Franks, D.G.; Hahn, M.E. 2005. Duplicate aryl hydrocarbon receptor repressor genes (ahrr1 and ahrr2) in the zebrafish <em>Danio rerio</em>: structure, function, evolution, and AHR-dependent regulation <em>in vivo</em>. Arch. Biochem. Biophys. 441, 151-167.</p>
<p> </p>
<p>Hahn, M.E. 2002. Aryl hydrocarbon receptors: diversity and evolution. Chemico-Biol. Interact. 141, 131-160.</p>
<p> </p>
<p>Hahn, M.E.; Karchner, S.I.; Evans, B.R.; Franks, D.G.; Merson, R.R.; Lapseritis, J.M. 2006. Unexpected diversity of aryl hydrocarbon receptors in non-mammalian vertebrates: Insights from comparative genomics. J. Exp. Zool. A. Comp. Exp. Biol. 305, 693-706.</p>
<p> </p>
<p>Hahn, M.E.; Poland, A.; Glover, E.; Stegeman, J.J. 1994. Photoaffinity labeling of the Ah receptor: phylogenetic survey of diverse vertebrate and invertebrate species. Arch. Biochem. Biophys. 310, 218-228.</p>
<p> </p>
<p>Hansson, M.C.; Hahn, M.E. 2008. Functional properties of the four Atlantic salmon (<em>Salmo salar</em>) aryl hydrocarbon receptor type 2 (AHR2) isoforms. Aquat. Toxicol. 86, 121-130.</p>
<p> </p>
<p>Hansson, M.C.; Wittzell, H.; Persson, K.; von Schantz, T. 2004. Unprecedented genomic diversity of AhR1 and AhR2 genes in Atlantic salmon (<em>Salmo salar </em>L.). Aquat. Toxicol. 68 (3), 219-232.</p>
<p> </p>
<p>Karchner, S.I.; Franks, D.G.; Hahn, M.E. (2005). AHR1B, a new functional aryl hydrocarbon receptor in zebrafish: tandem arrangement of ahr1b and ahr2 genes. Biochem. J. 392 (1), 153-161.</p>
<p> </p>
<p>Karchner, S.I.; Powell, W.H.; Hahn, M.E. 1999. Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the Teleost <em>Fundulus heteroclitus</em>. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors. J. Biol. Chem. 274, 33814-33824.</p>
<p> </p>
<p>Lahvis, G.P.; Bradfield, C.A. 1998. Ahr null alleles: distinctive or different? Biochem. Pharmacol. 56, 781-787.</p>
<p> </p>
<p>Lavine, J.A.; Rowatt, A.J.; Klimova, T.; Whitington, A.J.; Dengler, E.; Beck, C.; Powell, W.H. 2005. Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Sci. 88 (1), 60-72.</p>
<p> </p>
<p>Oka, K.; Kohno, S.; Ohta, Y.; Guillette, L.J.; Iguchi, T.; Katsu, Y. (2016). Molecular cloning and characterization of the aryl hydrocarbon receptors and aryl hydrocarbon receptor nuclear translocators in the American alligator. Gen. Comp. Endo. 238, 13-22.</p>
<p> </p>
<p>Pongratz, I.; Mason, G.G.; Poellinger, L. Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. J. Biol. Chem. 1992, 267 (19), 13728-13734</p>
<p> </p>
<p>Prasch, A.L.; Teraoka, H.; Carney, S.A.; Dong, W.; Hiraga, T.; Stegeman, J.J.; Heideman, W.; Peterson, R.E. 2003. Toxicol. Sci. Aryl hydrocarbon receptor 2 mediated 2,3,7,8-tetrachlorodibenzo-<em>p</em>-dioxin developmental toxicity in zebrafish. 76 (1), 138-150.</p>
<p> </p>
<p>Shoots, J.; Fraccalvieri, D.; Franks, D.G.; Denison, M.S.; Hahn, M.E.; Bonati, L.; Powell, W.H. 2015. An aryl hydrocarbon receptor from the salamander Ambystoma mexicanum exhibits low sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enviro. Sci. Technol<em>. </em>49, 6993-7001.</p>
<p> </p>
<p>Tanguay, R.L.; Abnet, C.C.; Heideman, W. Peterson, R.E. (1999). Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor1. Biochimica et Biophysica Act 1444, 35-48.</p>
<p> </p>
<p>Van den Berg, M.; Birnbaum, L.; Bosveld, A.T.C.; Brunstrom, B.; Cook, P.; Feeley, M.; Giesy, J.P.; Hanberg, A.; Hasegawa, R.; Kennedy, S.W.; Kubiak, T.; Larsen, J.C.; van Leeuwen, R.X.R.; Liem, A.K.D.; Nolt, C.; Peterson, R.E.; Poellinger, L.; Safe, S.; Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PECDFs for human and wildlife. Enviro. Hlth. Persp. <strong>1998</strong>, 106, 775-792.</p>
<p> </p>
<p>Van Tiem, L.A.; Di Giulio, R.T. 2011. AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (<em>Danio rerio</em>). Toxicol. Appl. Pharmacol. 254 (3), 280-287.</p>
<p> </p>
<p>Whitlock, J.P.; Okino, S.T.; Dong, L.Q.; Ko, H.S.P.; Clarke Katzenberg, R.; Qiang, M.; Li, W. 1996. Induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. Faseb. J. 10, 809-818.</p>
<p> </p>
<p>Wirgin, I.; Roy, N.K.; Loftus, M.; Chambers, R.C.; Franks, D.G.; Hahn, M.E. 2011. Mechanistic basis of resistance to PCBs in Atlantic tomcod from the Hudson River. Science. 331, 1322-1324.</p>
<p> </p>
<p>Yamauchi, M.; Kim, E.Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2,3,7,8-tetrachlorodibenzo-<em>p</em>-dioxin (TCDD) in developing red seabream (<em>Pagrus major</em>) embryos: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. <em>Aquat. Toxicol.</em> <strong>2006</strong>, 16, 166-179.</p>
<p> </p>
<p>Yasui, T.; Kim, E.Y.; Iawata, H.; Franks, D.G.; Karchner, S.I.; Hahn, M.E.; Tanabe, S. 2007. Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. Toxicol. Sci. 99 (1), 101-117.</p>
<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>
2016-11-29T18:41:222022-12-20T08:29:48Increase, InflammationIncrease, InflammationCellular<p>Inflammation is compex to define.</p>
<p>In cancer, is a cascade of events created by the host in response to the spread of the cancer (Coussens). In response to an injury or the presence of cancer, the host heals itself through inflammation. Indeed, the activation and the migration of leukocytes (neutrophils, monocytes and eosinophils) to the wound induces the healing process. These inflammatory cells provide an extracellular matrix that forms upon which fibroblast and endothelial cells proliferate and migrate in order to re create a normal environnement. In cancer, this inflammatory state induces cell proliferation, increases the production of reactive oxygen species leading to oxidative DNA damage, and reduces DNA repair (Coussens)</p>
<p>Damage of the epithelial layer lining the airways initiate inflammatory reactions.</p>
<ul>
<li>Activation of the innate immune response and the release of various inflammatory cytokines (Flake and Morgan, 2017).</li>
<li><span style="font-size:8.0pt"><span style="font-family:"Times New Roman",serif">Cytokines dosage</span></span></li>
<li><span style="font-size:8.0pt"><span style="font-family:"Times New Roman",serif">Chemokine dosage</span></span></li>
<li><span style="font-size:8.0pt"><span style="font-family:"Times New Roman",serif">Metalloprotesae</span></span></li>
</ul>
<p>Breast cancer cell lines</p>
<p> </p>
CL:0000255eukaryotic cellNot SpecifiedFemaleNot SpecifiedAdultNot Specified<p>Flake, G. P., & Morgan, D. L. (2017). Pathology of diacetyl and 2,3-pentanedione airway lesions in a rat model of obliterative bronchiolitis. <em>Toxicology</em>, <em>388</em>, 40–47. <a href="https://doi.org/10.1016/j.tox.2016.10.013"><u>https://doi.org/10.1016/j.tox.2016.10.013</u></a></p>
<p>Palmer, S. M., Flake, G. P., Kelly, F. L., Zhang, H. L., Nugent, J. L., Kirby, P. J., … Morgan, D. L. (2011). Severe airway epithelial injury, aberrant repair and Bronchiolitis obliterans develops after diacetyl instillation in rats. <em>PLoS ONE</em>, <em>6</em>(3). <a href="https://doi.org/10.1371/journal.pone.0017644"><u>https://doi.org/10.1371/journal.pone.0017644</u></a></p>
<p>Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002 Dec 19-26;420(6917):860-7. doi: 10.1038/nature01322. PMID: 12490959; PMCID: PMC2803035.</p>
2016-11-29T18:41:232022-12-20T08:53:06ApoptosisApoptosisCellular<p>Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Elmore, 2007]. Apoptosis, also called “physiological cell death”, is involved in cell turnover, physiological involution, and atrophy of various tissues and organs [Kerr et al., 1972]. The formation of apoptotic bodies involves marked condensation of both nucleus and cytoplasm, nuclear fragmentation, and separation of protuberances [Kerr et al., 1972]. Apoptosis is characterized by DNA ladder and chromatin condensation. Several stimuli such as hypoxia, nucleotides deprivation, chemotherapeutical drugs, DNA damage, and mitotic spindle damage induce p53 activation, leading to p21 activation and cell cycle arrest [Pucci et al., 2000]. The SAHA or TSA treatment on neonatal human dermal fibroblasts (NHDFs) for 24 or 72 hrs inhibited proliferation of the NHDF cells [Glaser et al., 2003]. Considering that the acetylation of histone H4 was increased by the treatment of SAHA for 4 hrs, histone deacetylase inhibition may be involved in the inhibition of the cell proliferation [Glaser et al., 2003]. The impaired proliferation was observed in HDAC1<sup>-/-</sup> ES cells, which was rescued with the reintroduction of HDAC1 [Zupkovitz et al., 2010]. The present AOP focuses on the p21 pathway leading to apoptosis, however, alternative pathways such as NF-kappaB signaling pathways may be involved in the apoptosis of spermatocytes [Wang et al., 2017].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Apoptosis is characterized by many morphological and biochemical changes <span style="color:black">such as homogenous condensation of chromatin to one side or the periphery of the nuclei, membrane blebbing and formation of apoptotic bodies with fragmented nuclei, DNA fragmentation, enzymatic activation of pro-caspases, or phosphatidylserine translocation that can be measured using electron and cytochemical optical microscopy, proteomic and genomic methods, and spectroscopic techniques [Archana et al., 2013; Martinez et al., 2010; Taatjes et al., 2008; Yasuhara et al., 2003].</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・<span style="color:black">DNA fragmentation can be quantified with comet assay using electrophoresis, where the tail length, head size, tail intensity, and head intensity of the comet are measured [Yasuhara et al., 2003].</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Apoptotic nucleosomes are detected using Cell Death Detection ELISA kit, which was calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Cleavage of PARP is detected with Western blotting [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm [Wu<span style="color:black"> et al.</span>, 2016].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Apoptosis is measured with Annexin V-FITC probes, and the relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry [Wu et al., 2016].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Apoptosis is detected with the Terminal dUTP Nick End-Labeling (TUNEL) method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks [Kressel and Groscurth, 1994].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・For the detection of apoptosis, the testes are fixed in neutral buffered formalin and embedded in paraffin. Germ cell death is visualized in testis sections by Terminal dUTP Nick End-Labeling (TUNEL) staining method [Wade et al., 2008]. The incidence of TUNEL-positive cells is expressed as the number of positive cells per tubule examined for one entire testis section per animal [Wade et al., 2008].</span></span></p>
<ul>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Apoptosis is detected with the </span></span><span style="font-size:8.0pt"><span style="font-family:"Times New Roman",serif">Annexin V test</span></span></li>
</ul>
<p>・Apoptosis is induced in human prostate cancer cell lines (<em>Homo sapiens</em>) [Parajuli et al., 2014].</p>
<p>・Apoptosis occurs in B6C3F1 mouse (<em>Mus musculus</em>) [Elmore, 2007].</p>
<p>・Apoptosis occurs in Sprague-Dawley rat (<em>Rattus norvegicus</em>) [Elmore, 2007].</p>
<p>・Apoptosis occurs in the nematode (<em>Caenorhabditis elegans</em>) [Elmore, 2007].</p>
<ul>
<li>Apoptosis occurs in breast cancer cells, human and mouse</li>
</ul>
<p> </p>
<p> </p>
UBERON:0000062organCL:0000000cellHighUnspecificHighNot Otherwise SpecifiedHighHighHighHigh<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Archana, M. et al. (2013), "Various methods available for detection of apoptotic cells", Indian J Cancer 50:274-283</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Elmore, S. (2007), "Apoptosis: a review of programmed cell death", Toxicol Pathol 35:495-516</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Kerr, J.F.R. et al. (1972), "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer 26:239-257</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Kressel, M. and Groscurth, P. (1994), "Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA", Cell Tissue Res 278:549-556</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Martinez, M.M. et al. (2010), "Detection of apoptosis: A review of conventioinal and novel techniques", Anal Methods 2:996-1004</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Pucci, B. et al. (2000), "Cell cycle and apoptosis", Neoplasia 2:291-299</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Taatjes, D.J. et al. (2008), "Morphological and cytochemical determination of cell death by apoptosis", Histochem Cell Biol 129:33-43</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wu, R. et al. (2016), "microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs", PLoS One 11:e0167052</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">Yasuhara, S. et al. (2003), </span>"<span style="color:black">Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis</span>"<span style="color:black">, J Histochem Cytochem 51:873-885</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181</span></span></p>
2017-02-07T13:21:502022-12-20T08:33:23Increased, MotilityIncreased, MotilityCellular<p>Cell motility is the capacity of cells to translocate onto a solid substratum. It is especially important in cancer. Indeed, cell motilityt promotes cancer agressivity. Inorder to metastise the cell must breach the basal membrane, escape from the primary tumour, migration to blood and lymphatic vessels, intravasation and extravasation and movement into distant organs (Stuleten). Cell motility is therefore essential in these steps.</p>
<p>Technics of measurement (Justus)</p>
<ul>
<li><span style="font-size:8.0pt"><span style="font-family:"Times New Roman",serif">Scratch wound</span></span></li>
<li><span style="font-size:8.0pt"><span style="font-family:"Times New Roman",serif">Boyden chamber</span></span></li>
<li><span style="font-size:8.0pt"><span style="font-family:"Times New Roman",serif">Organoid branching</span></span></li>
</ul>
<ul>
<li>Human breast cancer cell lines</li>
<li>Mice</li>
<li>Fish</li>
</ul>
CL:0000255eukaryotic cellNot SpecifiedFemaleNot SpecifiedAdultNot Specified<p>Stuelten, C., Parent, C. & Montell, D. Cell motility in cancer invasion and metastasis: insights from simple model organisms. <em>Nat Rev Cancer</em> <strong>18</strong>, 296–312 (2018). https://doi.org/10.1038/nrc.2018.15</p>
<p>Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV. In vitro cell migration and invasion assays. J Vis Exp. 2014 Jun 1;(88):51046. doi: 10.3791/51046. PMID: 24962652; PMCID: PMC4186330.</p>
2016-11-29T18:41:312022-12-20T08:43:45Increased, Migration (Endothelial Cells)Increased, Migration (Endothelial Cells)Cellular<p>This key event is defined by the migration of endothelial cells during neo angiogenesis in cancer (Mierke). It is a key step in metastasis</p>
<p>It is measured: </p>
<ul>
<li><em><span style="font-size:8.0pt"><span style="font-family:"Times New Roman",serif">In vivo through the measure of vessel density in fat pads</span></span></em></li>
<li><span style="font-size:8.0pt"><span style="font-family:"Times New Roman",serif">Matrigel</span></span></li>
</ul>
<p>Human, breast cancer cell lines</p>
<p>Mice</p>
CL:0000115endothelial cellNot SpecifiedMixedNot SpecifiedAdultNot Specified<p>Mierke CT. Role of the endothelium during tumor cell metastasis: is the endothelium a barrier or a promoter for cell invasion and metastasis? J Biophys. 2008;2008:183516. doi: 10.1155/2008/183516. Epub 2009 Mar 5. PMID: 20107573; PMCID: PMC2809021.</p>
2016-11-29T18:41:302022-12-20T09:00:35Increased, InvasionIncreased, InvasionCellular<p>In cancer, invasion is defined as the degradation and the passing of the basal membrane by cancer cells. It is a marker of poor prognosis in cancer.</p>
<p>It can be measured: </p>
<ul>
<li>Matrigel test</li>
<li>In vivo, through the passing of the basal membrane</li>
</ul>
<p>Human breast cancer cell lines</p>
Not SpecifiedMixedNot SpecifiedAdultsNot Specified<p>Martin TA, Ye L, Sanders AJ, et al. Cancer Invasion and Metastasis: Molecular and Cellular Perspective. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013. Available from: https://www.ncbi.nlm.nih.gov/books/NBK164700/</p>
2016-11-29T18:41:302022-12-20T09:06:16Increase, angiogenesisIncrease, angiogenesisTissue2017-04-17T10:26:192017-04-17T10:26:19N/A, Breast CancerN/A, Breast CancerIndividual<p>Cancers are thought to arise from a collection of permissive factors which interact within and between different cells of a tissue or tumor to promote tumor growth and invasive characteristics (Sonnenschein and Soto 1999; Hanahan and Weinberg 2011; Floor, Dumont et al. 2012; Goodson, Lowe et al. 2015; Schwarzman, Ackerman et al. 2015; Smith, Guyton et al. 2016; Grashow, De La Rosa et al. 2018). Permissive factors or hallmarks include changes to the cell’s dependence on growth signals, proliferation, metabolism, apoptosis, senescence, angiogenesis, and invasion and metastasis. These hallmarks are modified by other factors including growth factors, inflammation, oxidative stress, changes to the microenvironment, DNA damage, and changes in gene expression.</p>
<p>The mammary gland is a hormone responsive organ with multiple phases of development from embryogenesis into adulthood. Consequently, certain hallmarks and contributing factors including proliferative response to growth signals, growth factors, changes to the microenvironment, and changes in gene expression play a larger role in this organ, and the importance of various factors shifts depending on developmental stage (Rudel, Fenton et al. 2011). Established risk factors of breast cancer extend beyond genetic contributors (principally alterations in DNA damage response genes) and DNA damaging environmental agents to include exposure to pharmaceutical hormones, timing of puberty and first birth, and lifetime exposure to estrogen and progesterone ((IOM) Institute of Medicine 2012). </p>
<p>Hormonal and other environmental influences during proliferation and differentiation alter the pace and structure of cellular or mammary gland development to leave tissue in the adult gland more susceptible to cancer. In addition, the elevated hormone concentrations associated with the menstrual cycle and pregnancy provide a regular proliferative stimulus to any pre-cancerous cells present in the breast (Rudel, Fenton et al. 2011). A substantial majority of breast cancers express hormone receptors, and these cancers are particularly responsive to hormones (Badowska-Kozakiewicz, Patera et al. 2015).</p>
<p>Consistent with the importance of growth factors and DNA damage in the development of cancer, driver mutations (mutations that favor the success of the nascent cancer cells and are therefore selected) commonly appear in the growth factor related signaling pathways (BRAF, EGRF, RAS, PI3K, STK11) and in DNA damage response and cell cycle checkpoint signal pathways (ATM, TP53, CHEK2, CDKN2B (P15), CDK4) (Greenman, Stephens et al. 2007; Croce 2008; Kaufmann, Nevis et al. 2008; Stratton, Campbell et al. 2009; Vandin, Upfal et al. 2012). These and other mutations are acquired over the development of a cancer and contribute to the evolution of the cancer (Wang, Waters et al. 2014; Yates, Gerstung et al. 2015; Begg, Ostrovnaya et al. 2016).</p>
<p>In breast cancer, TP53, PI3K and GATA3 are each mutated in more than 10% of cancers, amplification or mutation of the RB1 pathway are common, and HER2 (an EGFR receptor) is amplified in HER2 type cancers (CGAN 2012). EGFR, HER2, BRAF, RAS, and PI3K participate in the EGFR (growth factor) signaling pathway. Activating mutations in PI3K generate growth factor independent proliferation of mammary epithelial cells, possibly via the RB1 pathway (Gustin, Karakas et al. 2009). GATA is a transcription factor that maintains luminal epithelial cell differentiation and suppresses proliferation, and mutation results in the proliferation of undifferentiated cells (Kouros-Mehr, Slorach et al. 2006; Shahi, Wang et al. 2017).</p>
<p>Environmental factors contribute significantly to the total number of breast cancers. Women exposed to the synthetic hormone DES or the pesticide DDT in utero are up to two to four times more likely to be diagnosed with breast cancer in their fifties (Palmer, Wise et al. 2006; Cohn, La Merrill et al. 2015). A study in 2002 found that recipients of hormone replacement therapy (HRT) around menopause are 26% more likely to be diagnosed with breast cancer (Narod 2011). When prescriptions of HRT began to fall in response to the study, so did cancer diagnoses. Over the next few years, approximately 5% fewer cancers were diagnosed in women over 45 (Glass, Lacey et al. 2007) with an estimated 126,000 fewer cases of breast cancer over the next ten years (Roth, Etzioni et al. 2014).</p>
<p>In rodent bioassays, tumors can be detected via visual observation or palpation of live animals, necropsy of dead animals, and via microscopic examination of tissue. Malignant tumors including carcinomas in situ are distinguishable from benign tumors on the basis of the thickness or shape of the epithelial cell layer, regularity of the lumen or the presence of cribiform luminae, inflammation or desmoplastic reaction of the stroma, dominance of a less differentiated cell type, and larger nuclei, while diagnosis of invasiveness depends on the identification of metastases or invasion of neoplastic cells into surrounding tissue (Russo and Russo 2000).</p>
<p>In humans, lumps are commonly detected by palpation or mammogram. Further imaging, biopsy, and/or surgical excision of the affected tissue are used to differentiate benign, cancerous, and invasive tumors (McDonald, Clark et al. 2016).</p>
<p> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Interest is growing on the role of the AhR in breast cancer. First, the AhR is often overexpressed in different breast cancer cell lines (7–9). Interestingly, the level of expression can be correlated to the stage or the molecular sub-type of the disease (7,10). Second, the AhR pathway has been associated with different pro-metastatic features in breast cancer, such as resistance to apoptosis, invasiveness, modified cell cycle, migration and proliferation (7,11,12). Triple negative cell lines, breast cancer cell lines with the worse prognosis (not over-expressing Her2 receptor or hormonal receptors), over-expressing the AhR seem to develop stem-like characteristics, favoring epithelial-mesenchymal transition (EMT) and thus metastasis (13). Thirdly, the AhR could be involved in the resistance of breast cancer to treatments (11,14): after AhR knockout, Goode <em>et al.</em> found enhanced sensitivity of paclitaxel (a drug targeting cancer cells) in triple negative breast cancer, a cancer particularly difficult to treat (14). Breast cancer patients expressing estrogen receptors (ER-positive) in their cancer cells, can benefit from an efficient endocrine therapy, which greatly improves their survival. Activation of the AhR can lead to the loss of expression of the ER alpha and therefore to the loss of a potential therapeutic target (15).</span></span></p>
<p>This can be applied to adult women and men and mice.</p>
HighMixedNot SpecifiedAdultHighHigh<p><a name="_ENREF_1">(IOM) Institute of Medicine (2012). Breast Cancer and the Environment: A Life Course Approach. Washington, DC, The National Academies Press.</a></p>
<p><a name="_ENREF_2">Badowska-Kozakiewicz, A. M., J. Patera, et al. (2015). "The role of oestrogen and progesterone receptors in breast cancer - immunohistochemical evaluation of oestrogen and progesterone receptor expression in invasive breast cancer in women." Contemp Oncol (Pozn) 19(3): 220-225.</a></p>
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<p><a name="_ENREF_4">CGAN (Cancer Genome Atlas Network) (2012). "Comprehensive molecular portraits of human breast tumours." Nature 490(7418): 61-70.</a></p>
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<p><a name="_ENREF_6">Cohn, B. A., M. La Merrill, et al. (2015). "DDT Exposure in Utero and Breast Cancer." J Clin Endocrinol Metab 100(8): 2865-2872.</a></p>
<p><a name="_ENREF_7">Croce, C. M. (2008). "Oncogenes and cancer." The New England journal of medicine 358(5): 502-511.</a></p>
<p><a name="_ENREF_8">EPA (Environmental Protection Agency) (2005). Guidelines for carcinogen risk assessment. Washington, DC, U.S. Environmental Protection Agency, Risk Assessment Forum: 1-166.</a></p>
<p><a name="_ENREF_9">FDA (Food and Drug Administration) (2007). Redbook 2000: Guidance for industry and other stakeholders. Toxicological principles for the safety assessment of food ingredients. Silver Spring, MD, U.S. Department of Health and Human Services, Food and Drug Administration.</a></p>
<p><a name="_ENREF_10">Floor, S. L., J. E. Dumont, et al. (2012). "Hallmarks of cancer: of all cancer cells, all the time?" Trends Mol Med 18(9): 509-515.</a></p>
<p><a name="_ENREF_11">Glass, A. G., J. V. Lacey, Jr., et al. (2007). "Breast cancer incidence, 1980-2006: combined roles of menopausal hormone therapy, screening mammography, and estrogen receptor status." Journal of the National Cancer Institute 99(15): 1152-1161.</a></p>
<p><a name="_ENREF_12">Goodson, W. H., 3rd, L. Lowe, et al. (2015). "Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead." Carcinogenesis 36 Suppl 1: S254-296.</a></p>
<p><a name="_ENREF_13">Grashow, R. G., V. Y. De La Rosa, et al. (2018). "BCScreen: A gene panel to test for breast carcinogenesis in chemical safety screening." Computational Toxicology 5: 16-24.</a></p>
<p><a name="_ENREF_14">Greenman, C., P. Stephens, et al. (2007). "Patterns of somatic mutation in human cancer genomes." Nature 446(7132): 153-158.</a></p>
<p><a name="_ENREF_15">Gustin, J. P., B. Karakas, et al. (2009). "Knockin of mutant PIK3CA activates multiple oncogenic pathways." Proceedings of the National Academy of Sciences of the United States of America 106(8): 2835-2840.</a></p>
<p><a name="_ENREF_16">Hanahan, D. and R. A. Weinberg (2011). "Hallmarks of cancer: the next generation." Cell 144(5): 646-674.</a></p>
<p><a name="_ENREF_17">Haseman, J. K., E. Young, et al. (1997). "Body weight-tumor incidence correlations in long-term rodent carcinogenicity studies." Toxicologic pathology 25(3): 256-263.</a></p>
<p><a name="_ENREF_18">Imaoka, T., M. Nishimura, et al. (2013). "Influence of age on the relative biological effectiveness of carbon ion radiation for induction of rat mammary carcinoma." International journal of radiation oncology, biology, physics 85(4): 1134-1140.</a></p>
<p><a name="_ENREF_19">Kaufmann, W. K., K. R. Nevis, et al. (2008). "Defective cell cycle checkpoint functions in melanoma are associated with altered patterns of gene expression." J Invest Dermatol 128(1): 175-187.</a></p>
<p><a name="_ENREF_20">Kouros-Mehr, H., E. M. Slorach, et al. (2006). "GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland." Cell 127(5): 1041-1055.</a></p>
<p><a name="_ENREF_21">McDonald, E. S., A. S. Clark, et al. (2016). "Clinical Diagnosis and Management of Breast Cancer." J Nucl Med 57 Suppl 1: 9S-16S.</a></p>
<p><a name="_ENREF_22">Narod, S. A. (2011). "Hormone replacement therapy and the risk of breast cancer." Nature reviews. Clinical oncology 8(11): 669-676.</a></p>
<p><a name="_ENREF_23">OECD (2009). Test No. 451: Carcinogenicity Studies.</a></p>
<p><a name="_ENREF_24">OECD (2009). Test No. 453: Combined Chronic Toxicity/Carcinogenicity Studies.</a></p>
<p><a name="_ENREF_25">OECD (Organisation for Economic Cooperation and Development) (2018). OECD guidelines for the testing of chemicals Section 4. Paris, OECD.</a></p>
<p><a name="_ENREF_26">Palmer, J. R., L. A. Wise, et al. (2006). "Prenatal diethylstilbestrol exposure and risk of breast cancer." Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 15(8): 1509-1514.</a></p>
<p><a name="_ENREF_27">Roth, J. A., R. Etzioni, et al. (2014). "Economic return from the Women's Health Initiative estrogen plus progestin clinical trial: a modeling study." Ann Intern Med 160(9): 594-602.</a></p>
<p><a name="_ENREF_28">Rudel, R. A., K. R. Attfield, et al. (2007). "Chemicals causing mammary gland tumors in animals signal new directions for epidemiology, chemicals testing, and risk assessment for breast cancer prevention." Cancer 109(12 Suppl): 2635-2666.</a></p>
<p><a name="_ENREF_29">Rudel, R. A., S. E. Fenton, et al. (2011). "Environmental exposures and mammary gland development: state of the science, public health implications, and research recommendations." Environmental health perspectives 119(8): 1053-1061.</a></p>
<p><a name="_ENREF_30">Russo, J. (2015). "Significance of rat mammary tumors for human risk assessment." Toxicologic pathology 43(2): 145-170.</a></p>
<p><a name="_ENREF_31">Russo, J. and I. H. Russo (2000). "Atlas and histologic classification of tumors of the rat mammary gland." J Mammary Gland Biol Neoplasia 5(2): 187-200.</a></p>
<p><a name="_ENREF_32">Schwarzman, M. R., J. M. Ackerman, et al. (2015). "Screening for Chemical Contributions to Breast Cancer Risk: A Case Study for Chemical Safety Evaluation." Environmental health perspectives 123(12): 1255-1264.</a></p>
<p><a name="_ENREF_33">Shahi, P., C. Y. Wang, et al. (2017). "GATA3 targets semaphorin 3B in mammary epithelial cells to suppress breast cancer progression and metastasis." Oncogene 36(40): 5567-5575.</a></p>
<p><a name="_ENREF_34">Smith, M. T., K. Z. Guyton, et al. (2016). "Key Characteristics of Carcinogens as a Basis for Organizing Data on Mechanisms of Carcinogenesis." Environmental health perspectives 124(6): 713-721.</a></p>
<p><a name="_ENREF_35">Sonnenschein, C. and A. M. Soto (1999). The society of cells : cancer control of cell proliferation. Oxford New York, Bios Scientific Publishers ;Springer.</a></p>
<p><a name="_ENREF_36">Stratton, M. R., P. J. Campbell, et al. (2009). "The cancer genome." Nature 458(7239): 719-724.</a></p>
<p><a name="_ENREF_37">Tucker, D. K., J. F. Foley, et al. (2017). "Sectioning Mammary Gland Whole Mounts for Lesion Identification." Journal of visualized experiments : JoVE(125).</a></p>
<p><a name="_ENREF_38">Vandin, F., E. Upfal, et al. (2012). "De novo discovery of mutated driver pathways in cancer." Genome research 22(2): 375-385.</a></p>
<p><a name="_ENREF_39">Wang, Y., J. Waters, et al. (2014). "Clonal evolution in breast cancer revealed by single nucleus genome sequencing." Nature 512(7513): 155-160.</a></p>
<p><a name="_ENREF_40">Yates, L. R., M. Gerstung, et al. (2015). "Subclonal diversification of primary breast cancer revealed by multiregion sequencing." Nat Med 21(7): 751-759.</a></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Zudaire E, Cuesta N, Murty V, Woodson K, Adams L, Gonzalez N, et al. The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers. J Clin Invest. 2008 Feb;118(2):640–50. </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">8. Kim DW, Gazourian L, Quadri SA, Romieu-Mourez R, Sherr DH, Sonenshein GE. The RelA NF-kappaB subunit and the aryl hydrocarbon receptor (AhR) cooperate to transactivate the c-myc promoter in mammary cells. Oncogene. 2000 Nov 16;19(48):5498–506. </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">9. Li ZD, Wang K, Yang XW, Zhuang ZG, Wang JJ, Tong XW. Expression of aryl hydrocarbon receptor in relation to p53 status and clinicopathological parameters in breast cancer. Int J Clin Exp Pathol. 2014;7(11):7931–7. </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">10. Zhao S, Ohara S, Kanno Y, Midorikawa Y, Nakayama M, Makimura M, et al. HER2 overexpression-mediated inflammatory signaling enhances mammosphere formation through up-regulation of aryl hydrocarbon receptor transcription. Cancer Lett. 2013 Mar 1;330(1):41–8. </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">11. Goode GD, Ballard BR, Manning HC, Freeman ML, Kang Y, Eltom SE. Knockdown of aberrantly upregulated aryl hydrocarbon receptor reduces tumor growth and metastasis of MDA-MB-231 human breast cancer cell line. Int J Cancer. 2013 Dec 15;133(12):2769–80. </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">12. Kanno Y, Takane Y, Izawa T, Nakahama T, Inouye Y. The inhibitory effect of aryl hydrocarbon receptor repressor (AhRR) on the growth of human breast cancer MCF-7 cells. Biol Pharm Bull. 2006 Jun;29(6):1254–7. </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">13. Stanford EA, Wang Z, Novikov O, Mulas F, Landesman-Bollag E, Monti S, et al. The role of the aryl hydrocarbon receptor in the development of cells with the molecular and functional characteristics of cancer stem-like cells. BMC Biol. 2016 Mar 16;14:20. </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">14. Goode G, Pratap S, Eltom SE. 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. 2014;9(6):e100103. </span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">15. Safe S, Wormke M, Samudio I. Mechanisms of inhibitory aryl hydrocarbon receptor-estrogen receptor crosstalk in human breast cancer cells. J Mammary Gland Biol Neoplasia. 2000 Jul;5(3):295–306.</span></span></p>
2016-11-29T18:41:302022-12-20T09:12:03Increased, tumor growthtumor growthOrgan<p>This key event is defined by the increase in tumor size, notable in breast cancer.</p>
<p>It can be measured: </p>
<ul>
<li>in vivo, through the size of tumor</li>
<li>Clinically in patients</li>
</ul>
<p>Human breast cancer cell lines</p>
Not SpecifiedMixedNot SpecifiedAdultsNot Specified2022-02-15T15:16:252022-12-20T09:01:53544e3b42-d4e6-4ea0-b6a9-1f20d174495844af7a24-6e7f-4e95-88d0-07400dbf05d3<p>In triple negative breast cell lines (MDA-MB436, MDA-MB-231) and ER-positive cell lines, it has been shown that the activation of the AhR can lead to an increase in inflammation. (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0695" name="bb0695">Yamashita et al., 2018 May 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0105" name="bb0105">Degner et al., 2009 Jan</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0655" name="bb0655">Vogel et al., 2011 Aug 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0295" name="bb0295">Kolasa et al., 2013 Apr 25</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0630" name="bb0630">Vacher et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0390" name="bb0390">Malik et al., 2019 Oct</a>). The stressors mainly used to activate the AhR were TCDD followed by benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo [4, 5-b] <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/pyridine" title="Learn more about pyridine from ScienceDirect's AI-generated Topic Pages">pyridine</a> (PhiP). After AhR inhibition (KO or antagonists), a decrease in inflammation biomarkers was found (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0695" name="bb0695">Yamashita et al., 2018 May 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0105" name="bb0105">Degner et al., 2009 Jan</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0655" name="bb0655">Vogel et al., 2011 Aug 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0295" name="bb0295">Kolasa et al., 2013 Apr 25</a>). Assays evaluating cell inflammation were quantitative dosages of IL-6, IL-8 and Cox2 activity/expression. Cox-2 and IL-8 were amongst the top “gene concepts” retrieved by the PubTator Central tool, likewise, “inflammation” was frequently found as a disease concept. The most consensual pathway linking the AhR activation to cell inflammation was the NF-kB pathway (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0655" name="bb0655">Vogel et al., 2011 Aug 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0295" name="bb0295">Kolasa et al., 2013 Apr 25</a>). Only half of the studies found a dose–response relationship (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0295" name="bb0295">Kolasa et al., 2013 Apr 25</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0390" name="bb0390">Malik et al., 2019 Oct</a>). No studies were carried out <em>in vivo</em> for breast cancer and therefore the concordance and evidence were classified as “moderate”.</p>
<p>AOP 21 also found the association between AhR activation and inflammation <em>via</em> COX 2 (Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2) with a weight of evidence classified as “high”. Indeed, the AhR/ARNT heterodimer links to the dioxin responsive elements which in turn up-regulates COX-2 (66,67].</p>
HighMixedHighAdultsHigh2022-02-15T15:22:522022-12-20T09:24:55544e3b42-d4e6-4ea0-b6a9-1f20d174495841f61035-1d50-4907-beb8-f3a26899e167<h4>KER 2569 Activation of the AhR leads to decreased apoptosis</h4>
<p>Several studies have found that the activation of the AhR by stressors such as TCDD, can promote a decrease in apoptosis (KER1), which is a deleterious event with regards to cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0005" name="bb0005">Al-Dhfyan et al., 2017 Jan 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>). Additionally, an increase in cell death was found when blocking the AhR pathway using AhR silencing (RNA interference or knock-out), knockout cell lines or antagonists (CH223191 or alpha-naphthoflavone) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0005" name="bb0005">Al-Dhfyan et al., 2017 Jan 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>). The most frequently used assay to evaluate apoptosis was <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/cytometry" title="Learn more about cytometry from ScienceDirect's AI-generated Topic Pages">cytometry</a> with the use of Annexin V: this was performed with ER-positive cells lines (MCF-7, T-47D), triple negative cell lines (MDA-MB-231, HS 578), cells over-expressing the Her2 (SK-BR-3) and cells lines derived from cancer samples from patients (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0005" name="bb0005">Al-Dhfyan et al., 2017 Jan 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0145" name="bb0145">Fujisawa et al., 2011</a>).</p>
<p>The concordance of the evidence was classified as “moderate” since the aim of most studies was to evaluate the capacity to survive in an apoptosis-promoting environment (i.e., chemotherapeutic drugs). Indeed, they assessed the resistance to chemotherapy agents such as doxorubicin and paclitaxel and found that the concomitant inactivation of the AhR pathway could decrease the resistance to these chemotherapy agents through an increase in cell death when compared to cells with a functional (or expressed at sufficient levels) AhR (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0005" name="bb0005">Al-Dhfyan et al., 2017 Jan 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0145" name="bb0145">Fujisawa et al., 2011</a>). Since the environment was modified by the presence of chemotherapy, the hypothesis of an alternative pathway cannot be completely discarded. It must be noticed that the exact biological mechanisms linking the activation of the AhR to the decrease in apoptosis remains unclear. Indeed, Anderson <em>et al</em>. suggested that the AhR interacts with the <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/glucocorticoid" title="Learn more about glucocorticoid from ScienceDirect's AI-generated Topic Pages">glucocorticoid</a> receptor (GR) and the hypoxia inducible factor-2α (HIF-2α) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>). The presence of the GR is associated with a poor prognosis, notably in triple negative breast cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0455" name="bb0455">Pan et al., 2011</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0420" name="bb0420">Moran et al., 2000 Feb 15</a>). Indeed, this receptor is involved in survival and resistance to chemotherapy through up-regulation of c-myc, Bcl2 and Kruppel-like factor 5 (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0455" name="bb0455">Pan et al., 2011</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0685" name="bb0685">Wu et al., 2004</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0350" name="bb0350">Li et al., 2017</a>). Both GR and HIF 2α could be up regulated by the AhR. They then activate Brk (also known as PTK6), a ligand of EGFR (epidermal growth factor receptor), involved in the inhibition of apoptosis (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0355" name="bb0355">Li et al., 2012</a>). Another possible mechanism suggested by Bekki et al. is that the decrease in apoptosis was caused by the induction of cyclooxygenase 2 (COX-2) and the NF-κB subunit RelB (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>). They both prevent apoptosis through induction of Bcl2, an anti-apoptotic factor (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0615" name="bb0615">Tsujii and DuBois, 1995</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0650" name="bb0650">Vogel et al., 2007</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0605" name="bb0605">Thomas et al., 2020</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0025" name="bb0025">Baud and Jacque, 2008 Dec</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0110" name="bb0110">Demicco et al., 2005 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0660" name="bb0660">Wang et al., 2007 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0375" name="bb0375">Liu et al., 2001 May 25</a>).</p>
HighMixedNot SpecifiedAdultsHigh2022-02-15T15:23:112022-12-20T09:18:02544e3b42-d4e6-4ea0-b6a9-1f20d17449581ce263a8-fa14-448b-8e56-1045cf036b35<p>The activation of the AhR can modulate cell motility in different types of breast cancers such as: ER-positive cells lines (MCF-7, T-47D, ZR-75–1), triple negative (MDA-MB-231, MDA-MB-435, HS-578-T, SUM149), and cells overexpressing the Her2 (SK-BR-3) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0460" name="bb0460">Parks et al., 2014 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0525" name="bb0525">Qin et al., 2011 Oct 20</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0435" name="bb0435">Nguyen et al., 2016 Nov 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0445" name="bb0445">Novikov et al., 2016 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0130" name="bb0130">Dwyer et al., 2021 Feb</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0235" name="bb0235">Hsieh et al., 2012 Feb</a>). Activation of the AhR with TCDD, butyl-benzyl <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/phthalate" title="Learn more about phthalate from ScienceDirect's AI-generated Topic Pages">phthalate</a>, di-n-butyl phthalate, hexachlorobenzene, and benzo[a]pyrene can promote cell migration in different assays (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0460" name="bb0460">Parks et al., 2014 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0525" name="bb0525">Qin et al., 2011 Oct 20</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0445" name="bb0445">Novikov et al., 2016 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0235" name="bb0235">Hsieh et al., 2012 Feb</a>). On the other hand, the use of AhR antagonists, AhR silencing or AhR knockout reversed this effect (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0460" name="bb0460">Parks et al., 2014 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0525" name="bb0525">Qin et al., 2011 Oct 20</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0445" name="bb0445">Novikov et al., 2016 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0235" name="bb0235">Hsieh et al., 2012 Feb</a>). The most frequently used assays for evaluating cell migration were the scratch wound assay and the transwell chamber assay. Only three works evaluated the dose–response concordance of AhR activation with stressors and cell migration (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>). The evidence was therefore classified as “moderate”.</p>
HighMixedNot SpecifiedAdultsHigh2022-02-15T15:23:242022-12-20T09:20:14544e3b42-d4e6-4ea0-b6a9-1f20d17449584948d904-079a-45e9-acc1-0cb3bd978b4e2022-02-15T15:23:402022-02-15T15:23:40544e3b42-d4e6-4ea0-b6a9-1f20d1744958f1f45ddf-25ff-456f-87aa-6d4aaaba0986<p>Due to the extensive robust and concordant literature of the link between activation of the AhR-increased cell motility-increased invasion-breast cancer progression, the confidence in these key events was rated as high. However, due to the use of ligands to activate the AhR, it cannot be completely ruled out that alternative pathways (independent of the AhR) can also contribute to these features. For instance, 2 main pathways seem to explain this increase in migration and invasion: the c-Src/HER1/STAT5b, and ERK1/2 pathways. Yet, these pathways seem only to explain the relation between the AhR activation and cell migration / invasion, when the ligand used is hexachlorobenzene, an organochlorinated pesticide (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0510" name="bb0510">Pontillo et al., 2013 May 1</a>). Even though alternative mechanisms may present themselves, all studies blocked the AhR pathway and found a decrease in cell migration/invasion. The evidence for alternative mechanisms was therefore classified as “moderate” and the biological plausibility of KER was also classified as “moderate”.</p>
Not SpecifiedMixedNot SpecifiedAdultsHigh2022-02-15T15:24:052022-12-20T09:24:3244af7a24-6e7f-4e95-88d0-07400dbf05d3f1f45ddf-25ff-456f-87aa-6d4aaaba0986<p>In the specific setting of AhR activation, only 2 studies showed the continuum between AhR activation – increased inflammation – increased invasion (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0695" name="bb0695">Yamashita et al., 2018 May 1</a>). However, in general, there is extensive knowledge on the relationship between cell inflammation and organ invasion. First, COX-2 is expressed at higher levels in triple negative invasive breast cancers than in less aggressive ER-positive cancers (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0175" name="bb0175">Gilhooly and Rose, 1999 Aug</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0380" name="bb0380">Liu and Rose, 1996 Nov 15</a>). COX-2 catalyzes the conversion of arachidonic acid into <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/prostaglandin" title="Learn more about prostaglandin from ScienceDirect's AI-generated Topic Pages">prostaglandin</a> H2, a pro-inflammatory factor, and is therefore considered as a prognosis factor in breast cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0540" name="bb0540">Ristimäki et al., 2002 Feb 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0465" name="bb0465">Parrett et al., 1997 Mar</a>). Transfection with COX-2 triple negative MDA-MB-435 cells increased cell migration 2-fold compared to control cells in a transwell-Matrigel® assay. Antagonism of COX-2 through an inhibitor (NS-398) reversed this action in a dose-dependent way (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0580" name="bb0580">Singh et al., 2005 May</a>). Second, <em>in vivo</em>, the use of anti-inflammatory treatments such as celecoxib (COX-2 inhibitor) can reduce tumor growth and spread (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0215" name="bb0215">Harris et al., 2000 Apr 15</a>). Finally, epidemiologic evidence suggests that inflammatory breast cancers have the worse prognosis. Indeed, the median overall survival of patients with inflammatory breast cancer compared with those with non-inflammatory breast cancer tumors is 4.75 years <em>versus</em> 13.40 years for stage III disease and 2.27 years <em>versus</em> 3.40 years for stage IV disease (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0560" name="bb0560">Schlichting et al., 2012 Aug</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0140" name="bb0140">Fouad et al., 2017 Apr</a>).</p>
<p>The mechanism of action of COX-2 are consensual. COX-2 promotes cell invasion through upregulation of MMPs (notably 2 and 9) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0600" name="bb0600">Takahashi et al., 1999 Oct 22</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0585" name="bb0585">Sivula et al., 2005 Feb</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0340" name="bb0340">Larkins et al., 2006 Jul</a>). Moreover, COX-2 could also activate the urokinase plasminogen activator (uPA) which degrades the basal membrane of epithelia (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0580" name="bb0580">Singh et al., 2005 May</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0600" name="bb0600">Takahashi et al., 1999 Oct 22</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0340" name="bb0340">Larkins et al., 2006 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0200" name="bb0200">Guyton et al., 2000 Mar</a>).</p>
<p>The relationship between inflammation and invasion is well document therefore the evidence was classified as “strong”.</p>
HighMixedHighAdultHigh2022-02-15T15:24:442022-12-20T09:25:5744af7a24-6e7f-4e95-88d0-07400dbf05d3d068c332-ee2b-43c9-b0d1-18c01ca9a8d3<p>Likewise, two studies evaluated the specific continuum AhR activation – increased inflammation – increased <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/angiogenesis" title="Learn more about angiogenesis from ScienceDirect's AI-generated Topic Pages">angiogenesis</a> (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0705" name="bb0705">Zárate et al., 2020 Aug</a>). As previously mentioned, the AhR activation increases inflammation, notably through an increase in COX 2 (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0105" name="bb0105">Degner et al., 2009 Jan</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0705" name="bb0705">Zárate et al., 2020 Aug</a>).</p>
<p>COX-2 can promote angiogenesis through an increase in VEGF (Vascular endothelial growth factor) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0220" name="bb0220">Harris et al., 2014 Oct 10</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0290" name="bb0290">Kirkpatrick et al., 2002</a>)<em>.</em> In a pathologic study characterizing 46 breast cancer specimen using immunochemistry, it was found that the density of microvessels was significantly higher in patients with COX-2 expression than in those without expression (p = 0.03) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0085" name="bb0085">Costa et al., 2002 Jun</a>). The relationship between COX-2 and angiogenesis has also been shown in gastric and colorectal cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0620" name="bb0620">Tsujii et al., 1998 May 29</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0625" name="bb0625">Uefuji et al., 2000 Jan</a>). Indeed, colon carcinoma cells overexpressing COX-2 produce proangiogenic factors (VEGF, bFGF, TBF-β, PDGF, and endothelin-1), and stimulate endothelial migration and the formation of tube vessels. These effects were reversed by an inhibitor (NS-398). <em>In vivo</em>, Diclofenac, a COX-2 inhibitor, decreased angiogenesis in mice presenting a colorectal cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0565" name="bb0565">Seed et al., 1997 May 1</a>). Likewise, in a murine model of breast cancer, celecoxib (a selective COX-2 inhibitor) reduced metastasis and tumor burden through a decrease of micro vessel density and VEGF (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0700" name="bb0700">Yoshinaka et al., 2006 Dec</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0715" name="bb0715">Zhang et al., 2004 Sep</a>). In clinical studies, patients with inflammatory breast cancers have increased levels of genes involved in angiogenesis such as VEGF (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0640" name="bb0640">Van der Auwera et al., 2004 Dec 1</a>). Patients with an inflammatory breast cancer benefit the most from anti-angiogenic treatment bevacizumab (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0485" name="bb0485">Pierga et al., 2012 Apr</a>).</p>
<p>The evidence was classified as “moderate” due to the lack of dose response studies.</p>
HighMixedHighAdultsHigh2022-02-15T15:25:002022-12-20T09:27:151ce263a8-fa14-448b-8e56-1045cf036b35f1f45ddf-25ff-456f-87aa-6d4aaaba0986<p>The relation between cell migration and organ invasion has already been shown (KER-1306, <a href="https://aopwiki.org/relationships/1306" rel="noreferrer noopener" target="_blank">https://aopwiki.org/relationships/1306</a>). Since the 2 are closely linked, most articles studied both cell migration (chemo-tactic) and the capacity to invade the extra-cellular matrix. Cell invasion is indeed defined as the capacity of a cell to migrate and degrade/invade the extracellular matrix. <em>In vitro</em>, this process was evaluated mostly using transwell chamber with Matrigel® and the presence of matrix metalloproteinases (MMP). This effect was found in ER-positive cells, triple negative cell lines and cells overexpressing the Her2.</p>
HighMixedHighAdultsHigh2016-11-29T18:41:372022-12-20T09:22:344948d904-079a-45e9-acc1-0cb3bd978b4ed068c332-ee2b-43c9-b0d1-18c01ca9a8d32022-02-15T15:26:062022-02-15T15:26:06f1f45ddf-25ff-456f-87aa-6d4aaaba0986406fd43e-d7ef-40df-aca9-1bf82dadc59c2022-02-15T15:26:422022-02-15T15:26:4241f61035-1d50-4907-beb8-f3a26899e16798ffc486-813b-4011-a272-201792438290<p>For KER 2, <em>in vivo</em>, Goode et al. showed that the knockout of the AhR in mice reduced tumor growth through an increase of cell apoptosis (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>).</p>
<p>The relationship between decreased apoptosis and increase in tumor growth (KER 2) is not detailed here due to extensive evidence in the scientific literature (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0210" name="bb0210">Hanahan and Weinberg, 2011 Mar 4</a>).</p>
Not SpecifiedMixedNot SpecifiedAdultHigh2022-02-15T15:26:582022-12-20T09:19:10d068c332-ee2b-43c9-b0d1-18c01ca9a8d3406fd43e-d7ef-40df-aca9-1bf82dadc59c2022-02-15T15:27:242022-02-15T15:27:2498ffc486-813b-4011-a272-201792438290406fd43e-d7ef-40df-aca9-1bf82dadc59c2022-02-15T15:27:452022-02-15T15:27:45Activation of the AhR leading to breast cancer AhR activation to breast cancer<p>Xavier Coumoul</p>
<p>Robert Barouki</p>
<p>Meriem Koual</p>
<p>Karine Audouze</p>
<p>Celine Tomkiewicz</p>
Under Development: Contributions and Comments WelcomeUnder DevelopmentIncluded in OECD Work Plan1.105<p>Breast cancer is the deadliest cancer in women with a poor prognosis in case of metastatic breast cancer. The role of the environments in the formation of metastasis has been suggested. We hypothesized that activation of the AhR (MIE), a xenobiotic receptor, could lead to breast cancer metastasis (AO), through different KEs, constituting a new AOP.</p>
<p>An artificial intelligence tool (AOP-helpfinder), which screens the available literature, was used to collect all existing scientific abstracts to build a novel AOP, using a list of key words. Four hundred and seven abstracts were found containing at least a word from our MIE list and either one word from our AO or KE list. A manual curation retained 113 pertinent articles, which were also screened using PubTator. <span style="font-size:12pt"><span style="font-family:Arial,sans-serif">From these analyses, an AOP was created linking the activation of the AhR to breast cancer related death through decreased apoptosis, inflammation, endothelial cell migration, and increased mortality. These KEs promote an increased tumor growth, angiogenesis and invasion<ins> </ins>which leads to breast cance metastasis.</span></span></p>
<p>The evidence of the proposed AOP was weighted using the tailored Bradford Hill criteria and the OECD guidelines (from the OECD user handbook, https://www.oecd.org/gov/Handbook.pdf). The confidence in our AOP and the biological plausibility was considered strong. Indeed, <em>in vitro</em> and i<em>n vivo</em> findings on multiple types of breast cancers (with or without oesrtogen receptors, for instanace) supported our proposed AOP. An <em>in vitro</em> validation must be carried out, but our review proposes a strong relationship between AhR activation and breast metastasis with an innovative use of an artificial intelligence literature search.</p>
<p> </p>
<p>This work was published in Envionnmental International: <a href="https://doi.org/10.1016/j.envint.2022.107323" rel="noreferrer noopener" target="_blank" title="Persistent link using digital object identifier">https://doi.org/10.1016/j.envint.2022.107323</a></p>
<p>Breast cancer is a frequent disease, responsible of 2 262 419 new cases and 684 996 deaths in 2020 in the world, making it the deadliest female cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0045" name="bb0045">Bray et al., 2018</a>). In 70% of cases, the disease is localized, and the prognosis is favorable with a 5-year survival of 99%. However, once the disease spreads (lymph nodes, metastasis), survival is severely altered with a 5-year survival rate of 26% in case of metastasis (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0225" name="bb0225">Henley et al., 2020</a>). It is therefore of paramount importance to understand the mechanisms of metastasis in breast cancer.</p>
<p>Amongst risk factors clearly established, including obesity, genetic mutations and hormonal exposure, the importance of the role of the environment is currently emerging (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0315" name="bb0315">Koual et al., 2020 Nov 17</a>). In an epidemiologic study, we found a positive association between the concentrations of 2.3.7.8-TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxine) in the <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/adipose-tissue" title="Learn more about adipose tissue from ScienceDirect's AI-generated Topic Pages">adipose tissue</a> surrounding the tumors, and breast cancer metastasis in overweight and obese patients (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0310" name="bb0310">Koual et al., 2019</a>). Moreover, we have shown that, using both <em>in vivo</em> and <em>in vitro</em> models, <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/tcdd" title="Learn more about TCDD from ScienceDirect's AI-generated Topic Pages">TCDD</a> exposure could promote an aggressive phenotype to breast cancer cells, thus favoring the formation of metastatic cells (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0320" name="bb0320">Koual et al., 2021</a>). TCDD is a potent ligand of the aryl hydrocarbon receptor (AhR), a transcriptional factor involved notably in the metabolism of <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/xenobiotic" title="Learn more about xenobiotics from ScienceDirect's AI-generated Topic Pages">xenobiotics</a> (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0335" name="bb0335">Larigot et al., 2022</a>). Hence, the impact of the environment on breast cancer aggressiveness could be mediated by the activation of the AhR.</p>
<p>Interest is growing on the role of the AhR in breast cancer. First, the AhR is often overexpressed in different breast cancer cell lines (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0730" name="bb0730">Zudaire et al., 2008</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0285" name="bb0285">Kim et al., 2000 Nov 16</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0360" name="bb0360">Li et al., 2014</a>). Interestingly, the level of expression can be correlated to the stage or the molecular sub-type of the disease (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0730" name="bb0730">Zudaire et al., 2008</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0725" name="bb0725">Zhao et al., 2013</a>). Second, the AhR pathway has been associated with different pro-metastatic features in breast cancer, such as resistance to apoptosis, <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/invasiveness" title="Learn more about invasiveness from ScienceDirect's AI-generated Topic Pages">invasiveness</a>, modified cell cycle, migration and proliferation (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0730" name="bb0730">Zudaire et al., 2008</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0275" name="bb0275">Kanno et al., 2006</a>). Triple negative cell lines, breast cancer cell lines with the worse prognosis (not over-expressing Her2 receptor or hormonal receptors), over-expressing the AhR seem to develop stem-like characteristics, favoring epithelial-mesenchymal transition (EMT) and thus metastasis (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0590" name="bb0590">Stanford et al., 2016</a>). Thirdly, the AhR could be involved in the resistance of breast cancer to treatments (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0185" name="bb0185">Goode et al., 2014</a>): after AhR knockout, Goode <em>et al.</em> found enhanced sensitivity of paclitaxel (a drug targeting cancer cells) in triple negative breast cancer, a cancer particularly difficult to treat (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0185" name="bb0185">Goode et al., 2014</a>). Breast cancer patients expressing estrogen receptors (ER-positive) in their cancer cells, can benefit from an efficient endocrine therapy, which greatly improves their survival. Activation of the AhR can lead to the loss of expression of the ER alpha and therefore to the loss of a potential therapeutic target (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0555" name="bb0555">Safe et al., 2000 Jul</a>).</p>
<p>The mechanisms linking the activation of the AhR to breast cancer aggressiveness are still unclear. Based on the AOP-wiki database (<a href="https://aopwiki.org/" rel="noreferrer noopener" target="_blank"><u>https://aopwiki.org/</u></a>, last accessed March 2022), the central repository for AOPs, the AhR has already been proposed in several AOPs, but never in one characterized by the AO breast cancer metastasis. Likewise, an AOP linking an MIE to breast cancer aggressiveness has never been proposed. From our expertise and available knowledge, we hypothesize that the activation of the AhR could be a MIE leading to breast cancer related death (AO) through different KEs and KERs.</p>
<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>
<p>Because of the long latency of mammary tumors, the two-year rodent carcinogenicity bioassay is the primary assay for this adverse outcome. The assay is included in the OECD Test No. 451 and 453 for carcinogenicity and combined toxicity and carcinogenicity (OECD 2009; OECD 2009), and is also used by the US National Toxicology program (Chhabra, Huff et al. 1990), and the FDA (FDA (Food and Drug Administration) 2007), and referenced by the EPA (EPA (Environmental Protection Agency) 2005) in guidelines for risk assessments. Other assays from short term (2-4 weeks) and subchronic (90 day) to chronic (1 year) toxicity also call for the documentation of mammary tumors (FDA (Food and Drug Administration) 2007; OECD (Organisation for Economic Cooperation and Development) 2018), so these assays could capture the early onset of tumors, and could be modified to report earlier key events like proliferation and inflammation.</p>
<p>Several characteristics of classic cancer bioassays limit the sensitivity of these assays to mammary gland carcinogens. First, no assays require prenatal or early post-natal exposures for carcinogenicity testing. The US NIH’s National Toxicology Program assays start exposures at five to six weeks of age and OECD regulatory assay exposures suggest (but do not require) exposures beginning after weaning and before eight weeks of age. Assays initiating exposures at later ages have diminished sensitivity to agents that affect breast development and increase future susceptibility to cancer, such as estrogenic hormones, DDT and dioxins (EPA (Environmental Protection Agency) 2005; Rudel, Fenton et al. 2011). Agents with similar activity to ionizing radiation and DNA damaging chemicals may not be fully captured in some of these assays, since sensitivity appears to peak around or before week seven for these agents (around puberty) (Imaoka, Nishimura et al. 2013). Second, carcinogenicity assay guidelines do not require the best methods for detecting tumors in mammary gland: whole mount preparations of mammary gland coupled with longitudinal sections (dorsoventral sections parallel to the body) of mammary gland for histology (Tucker, Foley et al. 2017). Palpation and transverse sections of mammary gland can easily miss tumors or lesions of interest. Interestingly the NTP reproductive toxicity guidelines do specify these preferable methods for mammary gland analysis.</p>
<p>Two additional factors affect the sensitivity of standard carcinogenicity assays. First, benign tumors are not always considered to be an indicator of carcinogenicity, leading to a possible underestimation of risk. NTP and EPA guidance suggest that benign tumors provide additional weight of evidence if malignant tumors are also present or if studies suggest benign tumors can progress to carcinogenicity. In a short-term study, benign tumors may indicate a need for a longer-term study. However, benign mammary tumors (fibroadenomas) almost always coincide with carcinogenic tumors in mammary gland or other organs, and carcinomas sometimes grow from fibroadnomas (Rudel, Attfield et al. 2007; Russo 2015) suggesting that benign tumors may be an underutilized indicator of carcinogenicity.</p>
<p>Finally, the dose selection guidance in carcinogenicity testing typically calls for a high dose that is sufficiently toxic to suppress body weight (OECD 2009). However, body weight interacts with risk of breast cancer (Haseman, Young et al. 1997; Rudel, Attfield et al. 2007), reducing the sensitivity of the upper end of the dose range and the likelihood of a positive dose-response.</p>
adjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedModerateadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHighadjacentNot SpecifiedHigh<p> </p>
<table border="1" cellpadding="1" cellspacing="1" style="width:500px">
<tbody>
<tr>
<td>KEY EVENT</td>
<td>LEVEL OF ESSENTIALITY</td>
<td>EVIDENCE</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 1262 : decreased apoptosis </strong></td>
<td>Strong</td>
<td>
<p>A decrease in apoptosis is an essentiel element in promoting tumor growth. Indeed, in case of a decrease in cell death, the tumor will continue to grow. However, cell proliferation is also an essentiel element in promoting tumor growth. <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Yet, due to the presence of diverging evidence on the activation of the AhR and cell proliferation, we chose not to include these in our AOP. Indeed, on one hand, activation of the AhR through ligands such as NK150460, ANI-7, emodine or derivates of revesterol decrease cell proliferation in ER-positive and ER-negative breast cancer cell lines. TCDD has been found to promote cell cycle arrest through phosphorylation of the retinoblastoma protein which binds to E2F. In ER-positive cell lines, beta-naphthoflavone mediated cell cycle arrest through an upregulation of P21. On the other hand, AhR activation could promote cell proliferation. Pearce et al. found that MCDF (6-methyl-1,3,8-trichlorodibenzofuran), an AhR agonist could stimulate cell proliferation with a dose-response concordance. Likewise, I3C, HCB, CPF and licorice could also promote cell proliferation. However, it seems that this cell proliferation is ER-dependent. Indeed, these ligands induced cell proliferation only in ER-positive cells lines with an effect dependent on the level of estrogen present in the medium. Whether this increase in ER-dependent cell proliferation can be independent of the AhR remains unclear. This increase in proliferation could also be mediated by the association of the RelA subunit of NF-kappaB with the AhR resulting in the activation of c-myc gene transcription in breast cancer cells. This would explain why Rodriguez <em>et al</em>. found that proliferation was modulated by the CYP1A1, independently of an exogenous ligand activation of the AhR. These complex effects, highly dependent on the context (cell types, medium content, type of ligand…) were therefore not included in our AOP despite the strong evidence. </span></span></p>
</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 1971 : tumor growth</strong></td>
<td>STRONG</td>
<td>An increase in tumor size is associated with breast cancer metastasis and is essential to the progression of the illness (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0210" name="bb0210">Hanahan and Weinberg, 2011 Mar 4</a>). Indeed, clinical evidence suggests that tumor size is directly correlated to the presence of metastasis (<span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Liu Y, He M, Zuo WJ, Hao S, Wang ZH, Shao ZM. Tumor Size Still Impacts Prognosis in Breast Cancer With Extensive Nodal Involvement. Front Oncol and Narod SA. Tumour size predicts long-term survival among women with lymph node-positive breast cancer. Curr Oncol.)</span></span></td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>
<p><strong>KE 1241 Increased cell motility </strong></p>
</td>
<td>MODERATE</td>
<td>The relation between cell migration and organ invasion is essntial and is already used in different AOPs (KER-1306, <a href="https://aopwiki.org/relationships/1306" rel="noreferrer noopener" target="_blank">https://aopwiki.org/relationships/1306</a>). Organ invasion can be promonted by cell migration, motility and inflammation. Therefore the essentilality of cell motility was classified as moderate since other factors can promote organ invasion</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>
<p><strong>KE 1196: organ invasion </strong></p>
</td>
<td>STRONG</td>
<td>Organ invasion is an essential step in promoting breast cancer agressivness and metastasis. Without invasion of the basal membrane, the cancer remains located in an in situ state and does not induce metastasis.</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 149 Increased inflammation </strong></td>
<td>MODERATE</td>
<td>
<p>Organ invasion can be promonted by cell migration, motility and inflammation. Therefore the essentilality of cell motility was classified as moderate since other factors can promote organ invasion.</p>
<p>In angiogenesis, however, increased inflammation is a key factor. Indeed, inflammation, through the secretion of growth factor promotes the creation of blood vessels.</p>
</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 1190 Increased endothelial migration </strong></td>
<td>STRONG</td>
<td>Endothelial cell migration is an essential key event in promoting angiogenesis. Extensive data exists on the essentialitty of this step (Franziska van Zijl, Georg Krupitza, Wolfgang Mikulits, Initial steps of metastasis: Cell invasion and endothelial transmigration, Mutation Research/Reviews in Mutation Research, Volume 728, Issues 1–2, 2011, Pages 23-34, ISSN 1383-5742, https://doi.org/10.1016/j.mrrev.2011.05.002.)</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 1213: angiogenesis</strong></td>
<td>STRONG</td>
<td>Without the creation of new vessels in order to receive nutrients and energy, the cancer cell cannot survive and create metastatis. It is an essential key event and considered as one of the hallmarks of cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0210" name="bb0210">Hanahan and Weinberg, 2011 Mar 4</a>). </td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>
<p> </p>
HighFemaleLowMaleHighAdultHighHigh<p>The <em>biological plausibility</em> of KERs is defined by the OECD as the « understanding of the fundamental biological processes involved and whether they are consistent with the causal relationship being proposed in the AOP ». The biological plausibility is strong due to the presence of overwhelming evidence present in different studies. A minor setback would be the difficulty to dismiss alternative mechanisms caused by the ligands used for AhR activation. This is detailed in the discussion.</p>
<p>The <em>essentiality of KEs</em> refers to « experimental data for whether or not downstream KEs or the AO are prevented or modified if an upstream event is blocked ». The essentiality of KEs is strong: most works use suppression or inhibition of the AhR (knock out, antagonists and/or silencing) with results coherent with our findings.</p>
<p>Finally, the <em>empirical support</em> of KERs, is often « based on toxicological data derived by one or more reference chemicals where dose–response and temporal concordance for the KE pair can be assessed ». The overall assessment of the empirical support of our KERs is also strong. There is evidence in human cell lines and mice showing a dose–response and temporal concordance for severity of our KE and the presence of metastasis.</p>
<p>The <em>biological applicability domain</em> of the putative AOP concerned mainly females of menstrual of post-menopausal age. Indeed, existing cell lines were derived from women of menstrual of post-menopausal age and <em>in vivo</em>, studies were performed on mice of reproductive age. Only one study used the zebra fish larvae (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>). However, it could be extrapolated to men. Indeed, breast cancers in men present similar tumor characteristics and no work has found diverging functions of the AhR between men and women. Moreover, no difference in AhR expression has been characterized between men and women. Furthermore, our AOP concerns ER-positive and triple negative cells lines.</p>
<p>Studies were carried out in humans, mice, and zebrafish (xenotransplant studies, no mammary gland) (i.e. PubTator results) and it can be hypothesized that this AOP is conserved across mammals. Indeed, the AhR is a very conserved and ancient protein (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0205" name="bb0205">Hahn, 2002 Sep 20</a>). However, since the sensitivity to adverse events are variable among taxa, we can only postulate this AOP in human and mice (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0305" name="bb0305">Korkalainen et al., 2001 Aug 3</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0080" name="bb0080">Cohen-Barnhouse et al., 2011 Jan</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0125" name="bb0125">Doering et al., 2013 Mar</a>).</p>
<p> </p>
<p>The AhR is a fascinating yet complex receptor since its activation is ligand and cell dependent. To avoid more bias, we decided to limit our AOP to breast cancer. First, this cancer is the most frequent female malignancy, which makes it a major public health concern. Second, this illness is hormonal-dependent and therefore the impact of the environment, through the AhR, can be strongly suggested. However, we have reasons to believe this AOP could be extrapolated to other cancers which share common regulatory pathways (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0335" name="bb0335">Larigot et al., 2022</a>). The AhR is overexpressed not only in breast cancer but also in lung, liver, stomach, head & neck, cervix, and ovarian cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0590" name="bb0590">Stanford et al., 2016</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0115" name="bb0115">DiNatale et al., 2010 Aug 6</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0385" name="bb0385">Liu et al., 2013 Aug</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0595" name="bb0595">Stanford et al., 2016 Aug</a>). Moreover, in these cancers, the level of expression is correlated to the stage of the disease (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0730" name="bb0730">Zudaire et al., 2008</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0300" name="bb0300">Koliopanos et al., 2002 Sep 5</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0060" name="bb0060">Chang et al., 2007 Jan 1</a>). Additionally, Moenniks <em>et al.</em> found that mice with constitutively active AhR had more liver tumors than wild type mice (55% <em>versus</em> 6%) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0415" name="bb0415">Moennikes et al., 2004 Jul 15</a>). <em>In vitro</em> evidence suggests that the AhR activation could promote a more aggressive phenotype to renal, lung, head and neck, and urothelial cancer through an increase in invasion, migration, and resistance to apoptosis which constitute representative key events of our AOP (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0730" name="bb0730">Zudaire et al., 2008</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0595" name="bb0595">Stanford et al., 2016 Aug</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0245" name="bb0245">Ishida et al., 2015 Jul 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0240" name="bb0240">Ishida et al., 2010 Feb</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0120" name="bb0120">Diry et al., 2006 Sep 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0250" name="bb0250">John et al., 2014 Oct</a>). Besides, an AOP associating AhR activation and lung cancer initiation is currently under development (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0010" name="bb0010">AOP, 2021</a>) (<a href="https://aopwiki.org/aops/417" rel="noreferrer noopener" target="_blank"><u>https://aopwiki.org/aops/417</u></a>, accessed May 2022).</p>
<p>Likewise, our AOP covers only breast cancer progression and not initiation. The mechanisms of breast cancer initiation are different from the metastatic pathway, but the AhR could also be involved in breast cancer initiation. <em>In vitro</em>, it was noted that human mammary benign cells with a high level of AhR had an increase in cell proliferation, and migration, and potentially display EMT-like features (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0050" name="bb0050">Brooks and Eltom, 2011 Jun</a>). <em>In vivo</em>, mice fed with 7,12-dimethylbenz[a]anthracene (DMBA, an AhR activator and a potent mutagen) had an increased risk of mammary tumors, with higher AhR expression (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0090" name="bb0090">Currier et al., 2005</a>). Strangely in regard of the deadly outcomes associated with aggressive breast tumors, the number of studies focusing on this specific aspect of mammary carcinogenesis is limited and therefore, epidemiological data on the effects of the exposome in breast cancer aggressiveness is scarce. Indeed, occupational exposure is difficult to quantify, and patients are usually exposed to a mixture of pollutants and not a single pollutant in a chronic way. A memory bias cannot be excluded since the half-life of TCDD, for instance, is 7–11 years (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0490" name="bb0490">Pirkle et al., 1989</a>). Industrial accidents, such as the Seveso incident, studied the increase in breast cancer incidence but did not record breast cancer aggressiveness since it is more complex to quantify. At an early stage, breast cancer has a favorable prognosis whereas the therapeutic challenge lies in the treatment of breast cancer metastases. Therefore, even though epidemiologic and cell evidence suggests that exposure to pollutants and Ahr activation could promote breast cancer initiation, we chose to study breast cancer progression, the most complex situation (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0475" name="bb0475">Pesatori et al., 2009 Sep</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0670" name="bb0670">Warner et al., 2002 Jul</a>).</p>
<p> </p>
<table border="1" cellpadding="1" cellspacing="1" style="width:500px">
<tbody>
<tr>
<td>KEY EVENT</td>
<td>LEVEL OF ESSENTIALITY</td>
<td>EVIDENCE</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 1262 : decreased apoptosis </strong></td>
<td>Strong</td>
<td>
<p>A decrease in apoptosis is an essentiel element in promoting tumor growth. Indeed, in case of a decrease in cell death, the tumor will continue to grow. However, cell proliferation is also an essentiel element in promoting tumor growth. <span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Yet, due to the presence of diverging evidence on the activation of the AhR and cell proliferation, we chose not to include these in our AOP. Indeed, on one hand, activation of the AhR through ligands such as NK150460, ANI-7, emodine or derivates of revesterol decrease cell proliferation in ER-positive and ER-negative breast cancer cell lines. TCDD has been found to promote cell cycle arrest through phosphorylation of the retinoblastoma protein which binds to E2F. In ER-positive cell lines, beta-naphthoflavone mediated cell cycle arrest through an upregulation of P21. On the other hand, AhR activation could promote cell proliferation. Pearce et al. found that MCDF (6-methyl-1,3,8-trichlorodibenzofuran), an AhR agonist could stimulate cell proliferation with a dose-response concordance. Likewise, I3C, HCB, CPF and licorice could also promote cell proliferation. However, it seems that this cell proliferation is ER-dependent. Indeed, these ligands induced cell proliferation only in ER-positive cells lines with an effect dependent on the level of estrogen present in the medium. Whether this increase in ER-dependent cell proliferation can be independent of the AhR remains unclear. This increase in proliferation could also be mediated by the association of the RelA subunit of NF-kappaB with the AhR resulting in the activation of c-myc gene transcription in breast cancer cells. This would explain why Rodriguez <em>et al</em>. found that proliferation was modulated by the CYP1A1, independently of an exogenous ligand activation of the AhR. These complex effects, highly dependent on the context (cell types, medium content, type of ligand…) were therefore not included in our AOP despite the strong evidence. </span></span></p>
</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 1971 : tumor growth</strong></td>
<td>STRONG</td>
<td>An increase in tumor size is associated with breast cancer metastasis and is essential to the progression of the illness (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0210" name="bb0210">Hanahan and Weinberg, 2011 Mar 4</a>). Indeed, clinical evidence suggests that tumor size is directly correlated to the presence of metastasis (<span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Liu Y, He M, Zuo WJ, Hao S, Wang ZH, Shao ZM. Tumor Size Still Impacts Prognosis in Breast Cancer With Extensive Nodal Involvement. Front Oncol and Narod SA. Tumour size predicts long-term survival among women with lymph node-positive breast cancer. Curr Oncol.)</span></span></td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>
<p><strong>KE 1241 Increased cell motility </strong></p>
</td>
<td>MODERATE</td>
<td>The relation between cell migration and organ invasion is essntial and is already used in different AOPs (KER-1306, <a href="https://aopwiki.org/relationships/1306" rel="noreferrer noopener" target="_blank">https://aopwiki.org/relationships/1306</a>). Organ invasion can be promonted by cell migration, motility and inflammation. Therefore the essentilality of cell motility was classified as moderate since other factors can promote organ invasion</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>
<p><strong>KE 1196: organ invasion </strong></p>
</td>
<td>STRONG</td>
<td>Organ invasion is an essential step in promoting breast cancer agressivness and metastasis. Without invasion of the basal membrane, the cancer remains located in an in situ state and does not induce metastasis.</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 149 Increased inflammation </strong></td>
<td>MODERATE</td>
<td>
<p>Organ invasion can be promonted by cell migration, motility and inflammation. Therefore the essentilality of cell motility was classified as moderate since other factors can promote organ invasion.</p>
<p>In angiogenesis, however, increased inflammation is a key factor. Indeed, inflammation, through the secretion of growth factor promotes the creation of blood vessels.</p>
</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 1190 Increased endothelial migration </strong></td>
<td>STRONG</td>
<td>Endothelial cell migration is an essential key event in promoting angiogenesis. Extensive data exists on the essentialitty of this step (Franziska van Zijl, Georg Krupitza, Wolfgang Mikulits, Initial steps of metastasis: Cell invasion and endothelial transmigration, Mutation Research/Reviews in Mutation Research, Volume 728, Issues 1–2, 2011, Pages 23-34, ISSN 1383-5742, https://doi.org/10.1016/j.mrrev.2011.05.002.)</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td><strong>KE 1213: angiogenesis</strong></td>
<td>STRONG</td>
<td>Without the creation of new vessels in order to receive nutrients and energy, the cancer cell cannot survive and create metastatis. It is an essential key event and considered as one of the hallmarks of cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0210" name="bb0210">Hanahan and Weinberg, 2011 Mar 4</a>). </td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>
<p> </p>
<h4>KER 2569 Activation of the AhR leads to decreased apoptosis</h4>
<p>Several studies have found that the activation of the AhR by stressors such as TCDD, can promote a decrease in apoptosis (KER1), which is a deleterious event with regards to cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0005" name="bb0005">Al-Dhfyan et al., 2017 Jan 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>). Additionally, an increase in cell death was found when blocking the AhR pathway using AhR silencing (RNA interference or knock-out), knockout cell lines or antagonists (CH223191 or alpha-naphthoflavone) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0005" name="bb0005">Al-Dhfyan et al., 2017 Jan 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>). The most frequently used assay to evaluate apoptosis was <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/cytometry" title="Learn more about cytometry from ScienceDirect's AI-generated Topic Pages">cytometry</a> with the use of Annexin V: this was performed with ER-positive cells lines (MCF-7, T-47D), triple negative cell lines (MDA-MB-231, HS 578), cells over-expressing the Her2 (SK-BR-3) and cells lines derived from cancer samples from patients (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0005" name="bb0005">Al-Dhfyan et al., 2017 Jan 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0145" name="bb0145">Fujisawa et al., 2011</a>).</p>
<p>The concordance of the evidence was classified as “moderate” since the aim of most studies was to evaluate the capacity to survive in an apoptosis-promoting environment (i.e., chemotherapeutic drugs). Indeed, they assessed the resistance to chemotherapy agents such as doxorubicin and paclitaxel and found that the concomitant inactivation of the AhR pathway could decrease the resistance to these chemotherapy agents through an increase in cell death when compared to cells with a functional (or expressed at sufficient levels) AhR (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0005" name="bb0005">Al-Dhfyan et al., 2017 Jan 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0145" name="bb0145">Fujisawa et al., 2011</a>). Since the environment was modified by the presence of chemotherapy, the hypothesis of an alternative pathway cannot be completely discarded. It must be noticed that the exact biological mechanisms linking the activation of the AhR to the decrease in apoptosis remains unclear. Indeed, Anderson <em>et al</em>. suggested that the AhR interacts with the <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/glucocorticoid" title="Learn more about glucocorticoid from ScienceDirect's AI-generated Topic Pages">glucocorticoid</a> receptor (GR) and the hypoxia inducible factor-2α (HIF-2α) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>). The presence of the GR is associated with a poor prognosis, notably in triple negative breast cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0455" name="bb0455">Pan et al., 2011</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0420" name="bb0420">Moran et al., 2000 Feb 15</a>). Indeed, this receptor is involved in survival and resistance to chemotherapy through up-regulation of c-myc, Bcl2 and Kruppel-like factor 5 (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0455" name="bb0455">Pan et al., 2011</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0685" name="bb0685">Wu et al., 2004</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0350" name="bb0350">Li et al., 2017</a>). Both GR and HIF 2α could be up regulated by the AhR. They then activate Brk (also known as PTK6), a ligand of EGFR (epidermal growth factor receptor), involved in the inhibition of apoptosis (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0355" name="bb0355">Li et al., 2012</a>). Another possible mechanism suggested by Bekki et al. is that the decrease in apoptosis was caused by the induction of cyclooxygenase 2 (COX-2) and the NF-κB subunit RelB (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>). They both prevent apoptosis through induction of Bcl2, an anti-apoptotic factor (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0615" name="bb0615">Tsujii and DuBois, 1995</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0650" name="bb0650">Vogel et al., 2007</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0605" name="bb0605">Thomas et al., 2020</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0025" name="bb0025">Baud and Jacque, 2008 Dec</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0110" name="bb0110">Demicco et al., 2005 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0660" name="bb0660">Wang et al., 2007 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0375" name="bb0375">Liu et al., 2001 May 25</a>).</p>
<p><strong>KER 2577: Decreased apoptosis promotes tumor growth</strong></p>
<p>For KER 2, <em>in vivo</em>, Goode et al. showed that the knockout of the AhR in mice reduced tumor growth through an increase of cell apoptosis (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>).</p>
<p>The relationship between decreased apoptosis and increase in tumor growth (KER 2) is not detailed here due to extensive evidence in the scientific literature (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0210" name="bb0210">Hanahan and Weinberg, 2011 Mar 4</a>).</p>
<p><strong>KER 2570: Activation of the AhR leads to an increased cell motility</strong></p>
<p>The activation of the AhR can modulate cell motility in different types of breast cancers such as: ER-positive cells lines (MCF-7, T-47D, ZR-75–1), triple negative (MDA-MB-231, MDA-MB-435, HS-578-T, SUM149), and cells overexpressing the Her2 (SK-BR-3) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0460" name="bb0460">Parks et al., 2014 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0525" name="bb0525">Qin et al., 2011 Oct 20</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0435" name="bb0435">Nguyen et al., 2016 Nov 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0445" name="bb0445">Novikov et al., 2016 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0130" name="bb0130">Dwyer et al., 2021 Feb</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0235" name="bb0235">Hsieh et al., 2012 Feb</a>). Activation of the AhR with TCDD, butyl-benzyl <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/phthalate" title="Learn more about phthalate from ScienceDirect's AI-generated Topic Pages">phthalate</a>, di-n-butyl phthalate, hexachlorobenzene, and benzo[a]pyrene can promote cell migration in different assays (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0460" name="bb0460">Parks et al., 2014 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0525" name="bb0525">Qin et al., 2011 Oct 20</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0445" name="bb0445">Novikov et al., 2016 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0235" name="bb0235">Hsieh et al., 2012 Feb</a>). On the other hand, the use of AhR antagonists, AhR silencing or AhR knockout reversed this effect (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0180" name="bb0180">Goode et al., 2013 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0530" name="bb0530">Regan Anderson et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0460" name="bb0460">Parks et al., 2014 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0525" name="bb0525">Qin et al., 2011 Oct 20</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0445" name="bb0445">Novikov et al., 2016 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0235" name="bb0235">Hsieh et al., 2012 Feb</a>). The most frequently used assays for evaluating cell migration were the scratch wound assay and the transwell chamber assay. Only three works evaluated the dose–response concordance of AhR activation with stressors and cell migration (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>). The evidence was therefore classified as “moderate”.</p>
<p> <strong>KER 2572: Activation of the AhR leads to an increased invasion</strong></p>
<p>Due to the extensive robust and concordant literature of the link between activation of the AhR-increased cell motility-increased invasion-breast cancer progression, the confidence in these key events was rated as high. However, due to the use of ligands to activate the AhR, it cannot be completely ruled out that alternative pathways (independent of the AhR) can also contribute to these features. For instance, 2 main pathways seem to explain this increase in migration and invasion: the c-Src/HER1/STAT5b, and ERK1/2 pathways. Yet, these pathways seem only to explain the relation between the AhR activation and cell migration / invasion, when the ligand used is hexachlorobenzene, an organochlorinated pesticide (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0505" name="bb0505">Pontillo et al., 2011 Apr</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0510" name="bb0510">Pontillo et al., 2013 May 1</a>). Even though alternative mechanisms may present themselves, all studies blocked the AhR pathway and found a decrease in cell migration/invasion. The evidence for alternative mechanisms was therefore classified as “moderate” and the biological plausibility of KER was also classified as “moderate”.</p>
<p><strong>KER 1306: Increased cell motility promotes organ invasion</strong></p>
<p>The relation between cell migration and organ invasion has already been shown (KER-1306, <a href="https://aopwiki.org/relationships/1306" rel="noreferrer noopener" target="_blank">https://aopwiki.org/relationships/1306</a>). Since the 2 are closely linked, most articles studied both cell migration (chemo-tactic) and the capacity to invade the extra-cellular matrix. Cell invasion is indeed defined as the capacity of a cell to migrate and degrade/invade the extracellular matrix. <em>In vitro</em>, this process was evaluated mostly using transwell chamber with Matrigel® and the presence of matrix metalloproteinases (MMP). This effect was found in ER-positive cells, triple negative cell lines and cells overexpressing the Her2.</p>
<p><strong> KER 2572: Activation of the AhR leads to an increased invasion</strong></p>
<p>The activation of the AhR through the use of different ligands (benzophenone, butyl benzyl phthalate, di-n-butyl phthalate, hexachlorobenzene, <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/chlorpyrifos" title="Learn more about chlorpyrifos from ScienceDirect's AI-generated Topic Pages">chlorpyrifos</a>, TCDD) or the blockage of the AhR (silencing, KO or antagonism) increased or decreased cell invasion, respectively (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0460" name="bb0460">Parks et al., 2014 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0525" name="bb0525">Qin et al., 2011 Oct 20</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0435" name="bb0435">Nguyen et al., 2016 Nov 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0235" name="bb0235">Hsieh et al., 2012 Feb</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0510" name="bb0510">Pontillo et al., 2013 May 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0040" name="bb0040">Belguise et al., 2007 Dec 15</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0695" name="bb0695">Yamashita et al., 2018 May 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0410" name="bb0410">Miret et al., 2020 May</a>). The dose–response concordance for cell invasion was demonstrated using increasing doses of hexachlorobenzene, benzo[a]pyrene, chlorpyrifos and TCDD (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0405" name="bb0405">Miret et al., 2016 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0510" name="bb0510">Pontillo et al., 2013 May 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0410" name="bb0410">Miret et al., 2020 May</a>). To further explore cell invasion, Nguyen et al. created a model of a lymphatic barrier using a three-dimensional lymph endothelial cell as a monolayer co-cultured with <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/spheroid" title="Learn more about spheroids from ScienceDirect's AI-generated Topic Pages">spheroids</a> of MDA-MB231 cells (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0435" name="bb0435">Nguyen et al., 2016 Nov 15</a>). They found that silencing or antagonizing the AhR (DIM) or activating the AhR (FICZ) respectively decreased or increased invasion of the lymphatic barrier.</p>
<p>On an organ level, <em>in vivo</em>, an increase in metastasis has been found in mice and zebrafish after the activation of the AhR with different ligands (butyl benzyl phthalate, di-n-butyl phthalate, hexachlorobenzene, TCDD) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0185" name="bb0185">Goode et al., 2014</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0235" name="bb0235">Hsieh et al., 2012 Feb</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0510" name="bb0510">Pontillo et al., 2013 May 1</a>). In the zebrafish model, Narasimham et al. treated the animals either with triple negative MDA-MB-231 cells only (untreated) or with MDA-MB-231 cells treated with an AhR inhibitor (CB7993113 or CH22319) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>). Untreated fish had significantly more metastasis (OR = 9, IC95%=3–35). Similar results were found using mice models (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0185" name="bb0185">Goode et al., 2014</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0570" name="bb0570">Shan et al., 2020 Nov</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0430" name="bb0430">Narasimhan et al., 2018 May 7</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0235" name="bb0235">Hsieh et al., 2012 Feb</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0510" name="bb0510">Pontillo et al., 2013 May 1</a>).</p>
<p><strong>KER 2568: Activation of the AhR leads to an increased inflammation (existing in AOP 21)</strong></p>
<p>In triple negative breast cell lines (MDA-MB436, MDA-MB-231) and ER-positive cell lines, it has been shown that the activation of the AhR can lead to an increase in inflammation. (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0695" name="bb0695">Yamashita et al., 2018 May 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0105" name="bb0105">Degner et al., 2009 Jan</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0655" name="bb0655">Vogel et al., 2011 Aug 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0295" name="bb0295">Kolasa et al., 2013 Apr 25</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0630" name="bb0630">Vacher et al., 2018</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0390" name="bb0390">Malik et al., 2019 Oct</a>). The stressors mainly used to activate the AhR were TCDD followed by benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo [4, 5-b] <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/pyridine" title="Learn more about pyridine from ScienceDirect's AI-generated Topic Pages">pyridine</a> (PhiP). After AhR inhibition (KO or antagonists), a decrease in inflammation biomarkers was found (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0695" name="bb0695">Yamashita et al., 2018 May 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0105" name="bb0105">Degner et al., 2009 Jan</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0655" name="bb0655">Vogel et al., 2011 Aug 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0295" name="bb0295">Kolasa et al., 2013 Apr 25</a>). Assays evaluating cell inflammation were quantitative dosages of IL-6, IL-8 and Cox2 activity/expression. Cox-2 and IL-8 were amongst the top “gene concepts” retrieved by the PubTator Central tool, likewise, “inflammation” was frequently found as a disease concept. The most consensual pathway linking the AhR activation to cell inflammation was the NF-kB pathway (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0655" name="bb0655">Vogel et al., 2011 Aug 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0295" name="bb0295">Kolasa et al., 2013 Apr 25</a>). Only half of the studies found a dose–response relationship (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0295" name="bb0295">Kolasa et al., 2013 Apr 25</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0390" name="bb0390">Malik et al., 2019 Oct</a>). No studies were carried out <em>in vivo</em> for breast cancer and therefore the concordance and evidence were classified as “moderate”.</p>
<p>AOP 21 also found the association between AhR activation and inflammation <em>via</em> COX 2 (Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2) with a weight of evidence classified as “high”. Indeed, the AhR/ARNT heterodimer links to the dioxin responsive elements which in turn up-regulates COX-2 (66,67].</p>
<p><strong> KER 2573: Inflammation promotes organ invasion</strong></p>
<p>In the specific setting of AhR activation, only 2 studies showed the continuum between AhR activation – increased inflammation – increased invasion (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0695" name="bb0695">Yamashita et al., 2018 May 1</a>). However, in general, there is extensive knowledge on the relationship between cell inflammation and organ invasion. First, COX-2 is expressed at higher levels in triple negative invasive breast cancers than in less aggressive ER-positive cancers (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0175" name="bb0175">Gilhooly and Rose, 1999 Aug</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0380" name="bb0380">Liu and Rose, 1996 Nov 15</a>). COX-2 catalyzes the conversion of arachidonic acid into <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/prostaglandin" title="Learn more about prostaglandin from ScienceDirect's AI-generated Topic Pages">prostaglandin</a> H2, a pro-inflammatory factor, and is therefore considered as a prognosis factor in breast cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0540" name="bb0540">Ristimäki et al., 2002 Feb 1</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0465" name="bb0465">Parrett et al., 1997 Mar</a>). Transfection with COX-2 triple negative MDA-MB-435 cells increased cell migration 2-fold compared to control cells in a transwell-Matrigel® assay. Antagonism of COX-2 through an inhibitor (NS-398) reversed this action in a dose-dependent way (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0580" name="bb0580">Singh et al., 2005 May</a>). Second, <em>in vivo</em>, the use of anti-inflammatory treatments such as celecoxib (COX-2 inhibitor) can reduce tumor growth and spread (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0215" name="bb0215">Harris et al., 2000 Apr 15</a>). Finally, epidemiologic evidence suggests that inflammatory breast cancers have the worse prognosis. Indeed, the median overall survival of patients with inflammatory breast cancer compared with those with non-inflammatory breast cancer tumors is 4.75 years <em>versus</em> 13.40 years for stage III disease and 2.27 years <em>versus</em> 3.40 years for stage IV disease (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0560" name="bb0560">Schlichting et al., 2012 Aug</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0140" name="bb0140">Fouad et al., 2017 Apr</a>).</p>
<p>The mechanism of action of COX-2 are consensual. COX-2 promotes cell invasion through upregulation of MMPs (notably 2 and 9) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0600" name="bb0600">Takahashi et al., 1999 Oct 22</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0585" name="bb0585">Sivula et al., 2005 Feb</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0340" name="bb0340">Larkins et al., 2006 Jul</a>). Moreover, COX-2 could also activate the urokinase plasminogen activator (uPA) which degrades the basal membrane of epithelia (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0580" name="bb0580">Singh et al., 2005 May</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0600" name="bb0600">Takahashi et al., 1999 Oct 22</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0340" name="bb0340">Larkins et al., 2006 Jul</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0200" name="bb0200">Guyton et al., 2000 Mar</a>).</p>
<p>The relationship between inflammation and invasion is well document therefore the evidence was classified as “strong”.</p>
<p><strong> KER 2574: Inflammation promotes angiogenesis</strong></p>
<p>Likewise, two studies evaluated the specific continuum AhR activation – increased inflammation – increased <a href="https://www.sciencedirect.com/topics/earth-and-planetary-sciences/angiogenesis" title="Learn more about angiogenesis from ScienceDirect's AI-generated Topic Pages">angiogenesis</a> (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0705" name="bb0705">Zárate et al., 2020 Aug</a>). As previously mentioned, the AhR activation increases inflammation, notably through an increase in COX 2 (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0035" name="bb0035">Bekki et al., 2015</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0400" name="bb0400">Miller et al., 2005</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0105" name="bb0105">Degner et al., 2009 Jan</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0705" name="bb0705">Zárate et al., 2020 Aug</a>).</p>
<p>COX-2 can promote angiogenesis through an increase in VEGF (Vascular endothelial growth factor) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0220" name="bb0220">Harris et al., 2014 Oct 10</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0290" name="bb0290">Kirkpatrick et al., 2002</a>)<em>.</em> In a pathologic study characterizing 46 breast cancer specimen using immunochemistry, it was found that the density of microvessels was significantly higher in patients with COX-2 expression than in those without expression (p = 0.03) (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0085" name="bb0085">Costa et al., 2002 Jun</a>). The relationship between COX-2 and angiogenesis has also been shown in gastric and colorectal cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0620" name="bb0620">Tsujii et al., 1998 May 29</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0625" name="bb0625">Uefuji et al., 2000 Jan</a>). Indeed, colon carcinoma cells overexpressing COX-2 produce proangiogenic factors (VEGF, bFGF, TBF-β, PDGF, and endothelin-1), and stimulate endothelial migration and the formation of tube vessels. These effects were reversed by an inhibitor (NS-398). <em>In vivo</em>, Diclofenac, a COX-2 inhibitor, decreased angiogenesis in mice presenting a colorectal cancer (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0565" name="bb0565">Seed et al., 1997 May 1</a>). Likewise, in a murine model of breast cancer, celecoxib (a selective COX-2 inhibitor) reduced metastasis and tumor burden through a decrease of micro vessel density and VEGF (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0700" name="bb0700">Yoshinaka et al., 2006 Dec</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0715" name="bb0715">Zhang et al., 2004 Sep</a>). In clinical studies, patients with inflammatory breast cancers have increased levels of genes involved in angiogenesis such as VEGF (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0640" name="bb0640">Van der Auwera et al., 2004 Dec 1</a>). Patients with an inflammatory breast cancer benefit the most from anti-angiogenic treatment bevacizumab (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0485" name="bb0485">Pierga et al., 2012 Apr</a>).</p>
<p>The evidence was classified as “moderate” due to the lack of dose response studies.</p>
<p><strong><a href="https://aopwiki.org/relationships" rel="noreferrer noopener" target="_blank">KER 1266</a>: Activation of the AhR leads to an increased endothelial migration </strong></p>
<p>The activation of the AhR can lead to an increased endothelial cell migration. This was found when HMEC-1 or EA.hy926 cells were co-cultured with ER-positive MCF-7 cells and triple negative MDA-MB-231 cells (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0705" name="bb0705">Zárate et al., 2020 Aug</a>). The assay mainly used was the Matrigel® / tube formation assay. Only one study found an increase in endothelial cell proliferation and not migration, therefore it was not kept as a KE (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>). The main pathway explaining this relationship was again related to the activation of COX2 and subsequently to the increase in VEGF. The association between the activation of the AhR and endothelial cell migration was classified as “weak” since only 2 studies explored this feature, and both used hexachlorobenzene as a stressor. However, these works were robust with strong evidence, and both found a reversed association after AhR blockage. No contradicting results were found in the scientific literature.</p>
<p>As opposed to our work, another AOP displayed a link between AhR activation and angiogenesis (AOP 150) and found that activation of the receptor could decrease VEGF production with moderate evidence and quantitative understanding. It must be noted that these AOPs applied only to chicken, zebrafish, and certain rodents whereas our AOP concerns humans. As detailed further, the AhR presents a variability between species which must be considered.</p>
<p><strong>KER 1267: Increased endothelial migration promotes angiogenesis</strong></p>
<p>Pontillo et al. treated mice with increasing doses of hexachlorobenzene and then calculated the vessel density in mammary fat pads (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>). They found that mice treated with hexachlorobenzene had a higher vessel density with a dose–response concordance. Treatment by AhR antagonists completely reversed this association (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0500" name="bb0500">Pontillo et al., 2015 Nov 19</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0705" name="bb0705">Zárate et al., 2020 Aug</a>). The relationship between endothelial migration and angiogenesis was not detailed here since there is existing extensive knowledge (<a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0325" name="bb0325">Lamalice et al., 2007 Mar 30</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0440" name="bb0440">Norton and Popel, 2016 Nov 14</a>, <a href="https://www.sciencedirect.com/science/article/pii/S0160412022002501?via%3Dihub#b0015" name="bb0015">Ausprunk and Folkman, 1977 Jul 1</a>). The KER 12 was considered as “strong”.</p>
<p><strong> KER 1126, 1267 and 2578: Increased tumor growth, increased invasion, and increased angiogenesis lead to breast cancer metastasis</strong></p>
<p>Due to extensive data in the scientific literature and the empirical evidence in favor of these KERs, these KERs were not detailed here.</p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">We propose a simple and robust AOP associating activation of the AhR and breast cancer related death through migration, invasion, inflammation, and neo-angiogenesis. </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">One of the main limitations of our AOP is the existence of these diverse ligands and pathways, complexifying the definition of ‘AhR activation’ (6,54). Using PubTator, we found that TCDD was by far the most used chemical followed by I3C, alpha-naphthoflavone, polycyclic aromatic hydrocarbons and hexachlorobenzene, all ligands of the AhR. These ligands can activate different pathways after AhR binding and we therefore assumed that these compounds were AhR agonists. It can be difficult to dismiss alternative mechanisms caused by the ligands used for AhR activation. However, the AhR is the only characterized target of TCDD for example, and studies which use several ligands including TCDD, display similar results using the other modulators. Moreover, the concordance of studies using various ligands and the coherence with the AhR inhibition are in favor of the robustness of the proposed AOP. Indeed, to obtain the most accurate AOP possible, the KEs selected had to be present, no matter the ligand used by the study. </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Another minor setback of using the AhR, is that the dose response concordance is a non-monotonous curve for several ligands (122,123). Therefore, the tailored Bradford-Hill criteria could sometimes not be fulfilled. </span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Moreover, the originality of our work lies in the use of artificial intelligence too such as AOP-helpfinder, which enables a thoroughly search of existing knowledge in the PubMed database and PubTator (19–21). Therefore, our literature review was complete and evidence in favor of our proposed AOP was overwhelming. We plan to validate our proposed AOP in a quantitative <em>in vitro</em> work using Integrated Approaches to Testing and Assessment (IATA).</span></span></p>
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