92-87-5HFACYLZERDEVSX-UHFFFAOYSA-NHFACYLZERDEVSX-UHFFFAOYSA-N
C.I. Azoic Diazo Component 1124-(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
DTXSID20206821403-66-3GentamicinGentacycol
Gentalline
gentamicina
gentamicine
GENTAMYCIN
Gentavet
Lyramycin
Oksitselanim
Septigen
Centicin
Gentamycins
DTXSID503464210028-15-6CBENFWSGALASAD-UHFFFAOYSA-NCBENFWSGALASAD-UHFFFAOYSA-N
OzoneAtmospheric ozone
Healozone
Oxygen, mol.
Ozone(16O16O16O)
Triatomic oxygen
DTXSID002109810102-43-9MWUXSHHQAYIFBG-UHFFFAOYSA-NMWUXSHHQAYIFBG-UHFFFAOYSA-N
Nitric oxideNitric oxide (NO)
Amidogen, oxo-
monoxido de nitrogeno
Monoxyde d'azote
Nitric oxide trimer
Nitrogen monooxide
nitrogen monoxide
Nitrogen(II) oxide
Nitrosyl radical
Oxido nitrico
Stickstoffmonoxid
UN 1660
DTXSID1020938NOCASCigarette smokeCS
DTXSID5035038NOCASDiesel engine exhaustDiesel Exhaust
DE
DTXSID1024043PR:000003858aryl hydrocarbon receptorCHEBI:26523reactive oxygen speciesGO:0004874aryl hydrocarbon receptor activityGO:1903409reactive oxygen species biosynthetic processVT:0002327respiratory function trait1increased2decreasedBenzidine2016-11-29T18:42:262016-11-29T18:42:26Dibenzo-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)2017-02-09T15:43:002017-02-09T15:43:00Food deprivation2021-09-06T07:33:542021-09-06T07:33:54Gentamicin2017-10-25T08:30:152017-10-25T08:30:15Ozone2021-07-21T10:18:562021-09-28T08:26:52Nitric oxide2021-07-22T09:57:382021-07-22T09:57:38Cigarette smoke2021-06-24T07:10:582021-09-28T09:07:54Diesel engine exhaust2021-08-06T08:41:152021-09-28T08:55:47PM102021-07-22T09:54:462021-07-22T09:54:467955zebra danioWCS_9031Gallus gallus143350Pagrus major7904Acipenser transmontanus41871Acipenser fulvescensWCS_8022rainbow trout8030Salmo salarWCS_8355Xenopus laevis8296Ambystoma mexicanumWCS_9054Phasianus colchicusWCS_93934Coturnix japonica10090mouse10116ratWCS_9606human34823Microgadus tomcod9606Homo sapiensWikiUser_28VertebratesWCS_7955zebrafishActivation, 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><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The AhR is present in human physiology and pathology. T</span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">he AhR is highly expressed at several important physiological barriers such as the placenta, lung, gastrointestinal system, and liver in human (Wakx, Marinelli, Watanabe). In these tissues, the AhR is involved in both detoxication processes involving xenobiotic metabolizing enzymes such as cytochromes P450, and in immune functions translating chemical signals into immune defence pathways (Marinelli, Stobbe). Moreover, it has a regulatory role in human dendritic cells and myelination (Kado, Shackleford).</span></span> <span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The lung constitutes another barrier exposed to components of air pollution such as particles and hydrocarbons (air pollution, cigarette smoke). The AhR detects such hydrocarbons and protects the pulmonary cells from their deleterious effects through metabolization.</span></span> <span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The regulatory effect on blood cells of the AhR, balancing different related cell types, can be extended to the megakaryocytes and their precursors; indeed, StemRegenin 1 (SR1), an antagonist of the AhR increases the human population of CD34+CD41low cells, a fraction of very efficient precursors of proplatelets (Bock).</span></span> <span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The occurrence of a nystagmus has been subsequently diagnosed in humans bearing a AhR mutation (Borovok).</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">In human cancer, the AhR has either a pro or con tumor effect depending on the tissue, the ligand, and the duration of the activation (Zudaire, Chang, Litzenburg, Gramatzki, Lin, Wang). In human breast cancer, the AhR is thoughts to be responsible of its progression (Goode, Kanno, Optiz, Novikov, Hall, </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Subramaniam, Barhoover</span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">). </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">In human mammary benign cells, Brooks et al. noted that a high level of AhR was associated with a modified cell cycle (with a 50% increase in population doubling time in cells expressing the AhR by more than 3-fold) and EMT including increased cell migration. Narasimnhan et al. found that suppression of the AhR pathway had a pro-tumorigenic effect in vitro (EMT, tumor migration) in triple negative breast cancer.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Many endogenous and exogenous ligands are present for the AhR in human (Optiz, Adachi, Schroeder, </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Rothhammer</span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">). </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Indoles, such as indole-3-carbinol or one of its secondary metabolites, 3-3'- Diindolylmethane, are degradation products found in cruciferous vegetables and characterized as AhR ligands (Ema, Kall, Miller) they are also inducers of the human and rat CYP1A1 (Optiz). FICZ is the most potent AhR ligand known to date: it has a stronger affinity than TCDD for the human AhR (TCDD Kd=0.48 nM/FICZ Kd=0.07 nM) (Coumoul).</span></span></span></span></p>
<p> </p>
<p> </p>
HighUnspecificHighEmbryoHighDevelopmentHighAll life stagesHighHighHighHighHighHighHighHighHighHighHighHighHighHighHighNot Specified<ol>
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<li><a href="#cite_ref-Hoffman1991_2-0">↑</a> Hoffman, E. C., Reyes, H., Chu, F. F., Sander, F., Conley, L. H., Brooks, B. A., and Hankinson, O. (1991). Cloning of a factor required for activity of the Ah (dioxin) receptor. <em>Science</em> <strong>252</strong>, 954-958.</li>
<li>↑ <sup><a href="#cite_ref-Poland1976_3-0">3.0</a></sup> <sup><a href="#cite_ref-Poland1976_3-1">3.1</a></sup> Poland, A., Glover, E., and Kende, A. S. (1976). Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. <em>J.Biol.Chem.</em> <strong>251</strong>, 4936-4946.</li>
<li>↑ <sup><a href="#cite_ref-Gu2000_4-0">4.0</a></sup> <sup><a href="#cite_ref-Gu2000_4-1">4.1</a></sup> Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily: sensors of environmental and developmental signals. <em>Annu.Rev.Pharmacol.Toxicol.</em> <strong>40</strong>, 519-561.</li>
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<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-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>
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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-Murray2005_33-0">↑</a> Murray, I. A., Reen, R. K., Leathery, N., Ramadoss, P., Bonati, L., Gonzalez, F. J., Peters, J. M., and Perdew, G. H. (2005). Evidence that ligand binding is a key determinant of Ah receptor-mediated transcriptional activity. Arch.Biochem.Biophys. 442, 59-71.</li>
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<li>↑ <sup><a href="#cite_ref-Pandini2009_35-0">35.0</a></sup> <sup><a href="#cite_ref-Pandini2009_35-1">35.1</a></sup> <sup><a href="#cite_ref-Pandini2009_35-2">35.2</a></sup> Pandini, A., Soshilov, A. A., Song, Y., Zhao, J., Bonati, L., and Denison, M. S. (2009). Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis. Biochemistry 48, 5972-5983.</li>
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<li>↑ <sup><a href="#cite_ref-Head2008_37-0">37.0</a></sup> <sup><a href="#cite_ref-Head2008_37-1">37.1</a></sup> <sup><a href="#cite_ref-Head2008_37-2">37.2</a></sup> Head, J. A., Hahn, M. E., and Kennedy, S. W. (2008). Key amino acids in the aryl hydrocarbon receptor predict dioxin sensitivity in avian species. <em>Environ.Sci.Technol. </em> <strong>42</strong>, 7535-7541.</li>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Kanno Y, Takane Y, Izawa T, Nakahama T, Inouye Y. 2006. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">The inhibitory effect of aryl hydrocarbon receptor repressor (AhRR) on the growth of human breast cancer MCF-7 cells. Biol Pharm Bull. 29(6):1254–57</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Goode G, Pratap S, Eltom SE. 2014. Depletion of the aryl hydrocarbon receptor in MDA-MB- 231 human breast cancer cells altered the expression of genes in key regulatory pathways of cancer. PLoS One. 9(6):e100103</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Opitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, et al. 2011. An endogenous tumour promoting ligand of the human aryl hydrocarbon receptor. Nature. 478(7368):197–203</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Novikov O, Wang Z, Stanford EA, Parks AJ, Ramirez-Cardenas A, et al. 2016. An Aryl Hydrocarbon Receptor-Mediated Amplification Loop That Enforces Cell Migration in ER-/PR-/Her2- Human Breast Cancer Cells. Mol Pharmacol. 90(5):674–88</span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Litzenburger UM, Opitz CA, Sahm F, Rauschenbach KJ, Trump S, et al. 2014. Constitutive IDO expression in human cancer is sustained by an autocrine signaling loop involving IL-6, STAT3 and the AHR. Oncotarget. 5(4):1038–51</span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Hall JM, Barhoover MA, Kazmin D, McDonnell DP, Greenlee WF, Thomas RS. 2010. Activation of the aryl-hydrocarbon receptor inhibits invasive and metastatic features of human breast cancer cells and promotes breast cancer cell differentiation. Mol Endocrinol. 24(2):359–69</span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Gramatzki D, Pantazis G, Schittenhelm J, Tabatabai G, K.hle C, et al. 2009. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Aryl hydrocarbon receptor inhibition downregulates the TGF-beta/Smad pathway in human glioblastoma cells. Oncogene. 28(28):2593–2605</span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Subramaniam V, Ace O, Prud’homme GJ, Jothy S. 2011. Tranilast treatment decreases cell growth, migration and inhibits colony formation of human breast cancer cells. Exp Mol Pathol. 90(1):116–22</span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Rothhammer V, Borucki DM, Kenison JE, Hewson P, Wang Z, et al. 2018. Detection of aryl hydrocarbon receptor agonists in human samples. Sci Rep. 8(1):4970</span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Lin P, Chang H, Tsai W-T, Wu M-H, Liao Y-S, et al. 2003. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Overexpression of aryl hydrocarbon receptor in human lung carcinomas. Toxicol Pathol. 31(1):22–30</span></span></span></span></p>
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<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Barhoover MA, Hall JM, Greenlee WF, Thomas RS. 2010. Aryl hydrocarbon receptor regulates cell cycle progression in human breast cancer cells via a functional interaction with cyclin-dependent kinase 4. Mol Pharmacol. 77(2):195–201</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"> </span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Wang K, Li Y, Jiang Y-Z, Dai C-F, Patankar MS, et al. 2013. </span></span><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells. Cancer Lett. 340(1):63–71</span></span></span></span></p>
<p style="text-align:justify"> </p>
<p><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-size:10.0pt"><span style="font-family:"Times New Roman",serif">Borovok N, Weiss C, Sharkia R, Reichenstein M, Wissinger B, et al. 2020. Gene and Protein Expression in Subjects With a Nystagmus-Associated AHR Mutation. Front Genet. 11:582796</span></span></span></span></p>
2016-11-29T18:41:222024-02-28T05:12:53Altered gene expression, AHR nuclear translocator (ARNT)-dependent pathwayAltered expression of AHR/ARNT pathway-dependent genesMolecular<p>Sustained AHR/ARNT dimerization induced by DLCs may sequester ARNT from its other dimerization partners at inappropriate times during embryonic cardiomorphogenesis, disrupting ARNT-dependent cellular functions<sup><a href="#cite_note-Heid2001-1">[1]</a></sup><sup><a href="#cite_note-Walker1997-2">[2]</a></sup>. ARNT serves as a dimerization partner for hypoxia inducible factor 1&alph; (HIF-1α), and this complex is involved in mediating physiological responses to hypoxia. Dimerization between ARNT and HIF-1α forms a transcription factor complex (HIF-1) that binds to hypoxia response enhancer sequences on DNA to activate the expression of genes such as vascular endothelial growth factor (VEGF), which is involved in angiogenesis<sup><a href="#cite_note-Forsythe1996-3">[3]</a></sup><sup><a href="#cite_note-Goldberg1994-4">[4]</a></sup><sup><a href="#cite_note-Jiang1996-5">[5]</a></sup><sup><a href="#cite_note-Maxwell1997-6">[6]</a></sup><sup><a href="#cite_note-Shweiki1992-7">[7]</a></sup>.</p>
<ol>
<li><a href="#cite_ref-Heid2001_1-0">↑</a> Heid, S. E., Walker, M. K., and Swanson, H. I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol.Sci 61, 187-196.</li>
<li><a href="#cite_ref-Walker1997_2-0">↑</a> Walker, M. K., Pollenz, R. S., and Smith, S. M. (1997). Expression of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator during chick cardiogenesis is consistent with 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced heart defects. Toxicol.Appl.Pharmacol. 143, 407-419.</li>
<li><a href="#cite_ref-Forsythe1996_3-0">↑</a> Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., and Semenza, G. L. (1996). Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol.Cell Biol. 16, 4604-4613.</li>
<li><a href="#cite_ref-Goldberg1994_4-0">↑</a> Goldberg, M. A., and Schneider, T. J. (1994). Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J.Biol.Chem. 269, 4355-4359.</li>
<li><a href="#cite_ref-Jiang1996_5-0">↑</a> Jiang, B. H., Rue, E., Wang, G. L., Roe, R., and Semenza, G. L. (1996). Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J.Biol.Chem. 271, 17771-17778.</li>
<li><a href="#cite_ref-Maxwell1997_6-0">↑</a> Maxwell, P. H., Dachs, G. U., Gleadle, J. M., Nicholls, L. G., Harris, A. L., Stratford, I. J., Hankinson, O., Pugh, C. W., and Ratcliffe, P. J. (1997). Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc.Natl.Acad.Sci U.S.A 94, 8104-8109.</li>
<li><a href="#cite_ref-Shweiki1992_7-0">↑</a> Shweiki, D., Itin, A., Soffer, D., and Keshet, E. (1992). Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359, 843-845.</li>
</ol>
2016-11-29T18:41:222021-08-19T07:32:30Increased, Reactive oxygen speciesIncreased, Reactive oxygen speciesCellular<p>Biological State: increased reactive oxygen species (ROS)</p>
<p>Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.</p>
<p>Reactive oxygen species (ROS) are O2- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). <br />
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). </p>
<p>Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies.</p>
<p>Yuan, Yan, et al., (2013) described ROS monitoring by using H<sub>2</sub>-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H<sub>2</sub>-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm.</p>
<p>Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013).</p>
<p>Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS.</p>
<p> </p>
<p>ROS is a normal constituent found in all organisms.</p>
HighUnspecificHighAll life stagesHigh<p>B.H. Park, S.M. Fikrig, E.M. Smithwick Infection and nitroblue tetrazolium reduction by neutrophils: a diagnostic aid Lancet, 2 (1968), pp. 532-534</p>
<p>Bedard, Karen, and Karl-Heinz Krause. 2007. “The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology.” Physiological Reviews 87 (1): 245–313.</p>
<p>Bisht, Shilpa, Muneeb Faiq, Madhuri Tolahunase, and Rima Dada. 2017. “Oxidative Stress and Male Infertility.” Nature Reviews. Urology 14 (8): 470–85.</p>
<p>Brieger, K., S. Schiavone, F. J. Miller Jr, and K-H Krause. 2012. “Reactive Oxygen Species: From Health to Disease.” Swiss Medical Weekly 142 (August): w13659.</p>
<p>Chattopadhyay, Sukumar, et al. "Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants." Toxicology letters 136.1 (2002): 65-76.</p>
<p>Drew, Barry, and Christiaan Leeuwenburgh. 2002. “Aging and the Role of Reactive Nitrogen Species.” Annals of the New York Academy of Sciences 959 (April): 66–81.</p>
<p>Goud, Anuradha P., Pravin T. Goud, Michael P. Diamond, Bernard Gonik, and Husam M. Abu-Soud. 2008. “Reactive Oxygen Species and Oocyte Aging: Role of Superoxide, Hydrogen Peroxide, and Hypochlorous Acid.” Free Radical Biology & Medicine 44 (7): 1295–1304.</p>
<p>Griendling, Kathy K., Rhian M. Touyz, Jay L. Zweier, Sergey Dikalov, William Chilian, Yeong-Renn Chen, David G. Harrison, Aruni Bhatnagar, and American Heart Association Council on Basic Cardiovascular Sciences. 2016. “Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association.” Circulation Research 119 (5): e39–75.</p>
<p>Ozcan, Ayla, and Metin Ogun. 2015. “Biochemistry of Reactive Oxygen and Nitrogen Species.” In Basic Principles and Clinical Significance of Oxidative Stress, edited by Sivakumar Joghi Thatha Gowder. Rijeka: IntechOpen.</p>
<p>Parrish, A. R. 2010. “2.27 - Hypoxia/Ischemia Signaling.” In Comprehensive Toxicology (Second Edition), edited by Charlene A. McQueen, 529–42. Oxford: Elsevier.</p>
<p>Sharma, Gunjan, Nishant Kumar Rana, Priya Singh, Pradeep Dubey, Daya Shankar Pandey, and Biplob Koch. 2017. “p53 Dependent Apoptosis and Cell Cycle Delay Induced by Heteroleptic Complexes in Human Cervical Cancer Cells.” Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie 88 (April): 218–31.</p>
<p>Yen, Cheng Chien, et al. "Inorganic arsenic causes cell apoptosis in mouse cerebrum through an oxidative stress-regulated signaling pathway." Archives of toxicology 85 (2011): 565-575.</p>
<p>Yuan, Yan, et al. "Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway." PloS one 8.5 (2013): e64330.</p>
2016-11-29T18:41:292023-07-26T14:34:09Increase, Cell deathIncrease, Cell deathCellular<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Cell death is part of normal development and maturation cycle, and is the component of many response patterns of living tissues to xenobiotic agents (i.e.. micro organisms and chemicals) and to endogenous modulations, such as inflammation and disturbed blood supply (Kanduc et al., 2002). Many physiological processes require cell death for their function (e.g.., embryonal development and immune selection of B and T cells) (Bertheloot et al., 2021). Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer (Crowley et al., 2016). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Cell death can be </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">divided into: programmed cell death (cell death as a normal component of development) and non-programmed cell death (uncontrolled death of the cell). Although this simplistic view has blurred the intricate mechanisms separating these forms of cell death, studies have and will uncover new effectors, cell death pathways and reveal a more complex and intertwined landscape of processes involving cell death </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Bertheloot et al., 2021).</span></span></p>
<p><span style="font-size:18px"><em><span style="font-family:"Calibri",sans-serif">Programmed cell death:</span></em></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> is a form of cell death in which the dying cell plays an active part in its own demise </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Cotter & Al-Rubeai, 1995)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Apoptosis</u></strong> At a morphological level, it is characterized by cell shrinkage rather than the swelling seen in necrotic cell death. It is characterized by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme‐dependent biochemical processes. The result of it being the clearance of cells from the body, with minimal damage to surrounding tissues. An essential feature of apoptosis is the release of cytochrome c from mitochondria, regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7). Apoptosis culminates in the breakdown of the nuclear membrane by caspase-6, the cleavage of many intracellular proteins (e.g., PARP and lamin), membrane blebbing, and the breakdown of genomic DNA into nucleosomal structures (Bertheloot et al., 2021). Mechanistically, two main pathways contribute to the caspase activation cascade downstream of mitochondrial cytochrome c release: </span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><u>Intrinsic pathway</u> is triggered by dysregulation of or imbalance in intracellular homeostasis by toxic agents or DNA damage. It is characterized by mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome c into the cytosol.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><u>Extrinsic pathway</u> is initiated by activation of cell surface death receptors. Proapoptotic death receptors include TNFR1/2, Fas and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5.</span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><u>Other pathways of programmed cell death are called »non-apoptotic programmed cell-death« or »caspase-independent programmed cell-death« </u>(Blank & Shiloh, 2007)<u>.</u></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Necroptosis:</u></strong> This type of regulated cell death, occurs following the activation of the tumor necrosis receptor (TNFR1) by TNFα. Activation of other cellular receptors triggers necroptosis. These receptors include death receptors (i.e., Fas/FasL), Toll-like receptors (TLR4 and TLR3) and cytosolic nucleic acid sensors such as RIG-I and STING, which induce type I interferon (IFN-I) and TNFα production and thus promote necroptosis in an autocrine feedback loop. Most of these pathways trigger NFκB- dependent proinflammatory and prosurvival signals. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Pyroptosis</u></strong> is a type of cell death culminating in the loss of plasma membrane integrity and induced by activation of so-called inflammasome sensors. These include the Nod-like receptor (NLR) family, the DNA receptor Absent in Melanoma 2 (AIM2) and the Pyrin receptor.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Autophagy:</u></strong> is a process where cellular components such as macro proteins or even whole organelles are sequestered into lysosomes for degradation (Mizushima et al., 2008; Shintani & Klionsky, 2004). The lysosomes are then able to digest these substrates, the components of which can either be recycled to create new cellular structures and/or organelles or alternatively can be further processed and used as a source of energy (D’Arcy, 2019).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Anoikis</u></strong> is apoptosis induced by loss of attachment to substrate or to other cells (anoikis). Anoikis overlaps with apoptosis in molecular terms, but is classified as a separate entity because of its specific form od induction (Blank & Shiloh, 2007). Induction of anoikis occurs when cells lose attachment to ECM, or adhere to an inappropriate type of ECM, the latter being the more relevant <em>in vivo </em>(Gilmore, 2005).</span></span></p>
<p><strong><u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Cornification</span></span></u></strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">: is programmed cell death of keratinocytes. Cell death in the context of cornification involves distinct enzyme classes such as transglutaminases, proteases, DNases and others </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Eckhart et al., 2013)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:18px"><em>Non-programmed cell death:</em></span> occurs accidentally in an unplanned manner.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Necrosis</u></strong> is generally characterized to be the uncontrolled death of the cell, usually following a severe insult, resulting in spillage of the contents of the cell into surrounding tissues and subsequent damage thereof (D’Arcy, 2019).</span></span></p>
<p> </p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Assays for Quantitating Cell Death:</strong></span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Cell death can be measured by staining a sample of cells with trypan blue, assay is described in protocol: Measuring Cell Death by Trypan Blue Uptake and Light Microscopy (Crowley, Marfell, Christensen, et al., 2015d). Or with propidium Iodide, assay is described in protocol: Measuring Cell Death by Propidium Iodide (PI) Uptake and Flow Cytometry (Crowley & Waterhouse, 2015a) </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TUNEL technique: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Uribe et al., 2013).</span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Assays for Quantitating Cell Survival </strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Colony-forming assay can be used to define the number of cells in a population that are capable of proliferating and forming large groups of cells. Described in Protocol: Measuring Survival of Adherent Cells with the Colony-Forming Assay (Crowley, Christensen, & Waterhouse, 2015c); Measuring Survival of Hematopoietic Cancer Cells with the Colony-Forming Assay in Soft Agar (Crowley & Waterhouse, 2015b).</span></span></p>
<p><em><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>ASSAYS TO DISTINGUISH APOPTOSIS FROM NECROSIS AND OTHER DEATH MODALITIES</strong></span></span></em></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Detecting Nuclear Condensation:</u></strong> The nucleus is generally round in healthy cells but fragmented in apoptotic cells. Dyes such as Giemsa or hematoxylin, which are purple in color and therefore easily viewed using light microscopy, are commonly used to stain the nucleus. Other features of apoptosis and necrosis, such as plasma membrane blebbing or rupture, can be identified by staining the cytoplasm with eosin. Eosin is pinkish in color and can also be viewed using light microscopy. Hematoxylin and eosin are, therefore, commonly used together to stain cells. Assay is described in Protocol: Morphological Analysis of Cell Death by Cytospinning Followed by Rapid Staining (Crowley, Marfell, & Waterhouse, 2015c); Analyzing Cell Death by Nuclear Staining with Hoechst 33342 (Crowley, Marfell, & Waterhouse, 2015a).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Detection of DNA Fragmentation: </u></strong>Apoptotic cells with fragmented DNA can be identified and distinguished from live cells by staining with Propidium Iodide (PI) and measuring DNA content by flow cytometry. This assay is described in Protocol: Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry (Crowley, Chojnowski, & Waterhouse, 2015a).<strong><u> TUNEL technique </u></strong>can also be used<strong>:</strong> in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Crowley, Marfell, & Waterhouse, 2015b; Uribe et al., 2013).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Detecting Phosphatidylserine Exposure:</u></strong> Apoptosis is also characterized by exposure of phosphatidylserine (PS) on the outside of apoptotic cells, which acts as a signal that triggers removal of the dying cell by phagocytosis. Annexin V, can selectively bind to PS to label apoptotic cells in which PS is exposed. Purified annexin V can be conjugated to various fluorochromes, which can then be visualized by fluorescence microscopy or detected by flow cytometry. This assay is described in protocol: Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry (Crowley, Marfell, Scott, et al., 2015e). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><u>Detecting Caspase Activity:</u></strong> antibodies that specifically recognize the cleaved fragments of caspases and their substrates can be used to specifically detect caspase activity in apoptotic cells by immunocytochemistry. Flow cytometry (using primary antibodies conjugated to fluorescent molecules, or by counter staining with fluorescently labeled antibodies against the primary antibody) can then be used to quantitate the number of apoptotic cells. This assay is described in protocol: Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry (Crowley & Waterhouse, 2015a).</span></span></p>
<p><strong><u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Detecting Mitochondrial Damage:</span></span></u></strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> flow cytometry can be used to quantitate the number of cells that have reduced mitochondrial transmembrane potential, which is commonly associated with cytochrome c release during apoptosis. For this assay see protocol: Measuring Mitochondrial Transmembrane Potential by TMRE Staining </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Crowley, Christensen, & Waterhouse, 2015b)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p> </p>
<p> </p>
<p><span style="font-size:11px"><span style="color:#e74c3c">Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed. </span></span></p>
<p> </p>
<p><span style="display:none"> </span><span style="font-size:11px"><span style="color:#e74c3c">Measures of apoptotic cytomorphological alterations: </span></span></p>
<p><span style="display:none"> </span><span style="font-size:11px"><span style="color:#e74c3c">Apoptotic cells exhibit electron dense nuclei, nuclear fragmentation, intact cell membrane up to the disintegration phase, disorganized cytoplasmic organelles, large clear vacuoles, blebs at cell surface, and apoptotic bodies, which can be visualized with various methods. (Elmore, 2007; Watanabe et al., 2002) </span></span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Method of Measurement </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Reference </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Description </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">OECD Approved Assay </span></strong></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Transmission electron microscopy (TEM) / Scanning electron microscopy (SEM)/ Fluorescence microscopy </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Martinez, Reif, and Pappas, 2010; Watanabe et al., 2002 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">TEM and SEM can image the cytomorphological alterations caused by apoptosis. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td colspan="3">
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Stains: </span></strong></span></p>
</td>
<td>
<p> </p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Hematoxylin with eosin </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Elmore, 2007 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Hematoxylin stains nuclei blue and eosin stains the cytoplasm/extracellular matrix pink, allowing for the visualization of the cytomorphological alterations of cells. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Toluidine blue or methylene blue </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Watanabe et al., 2002 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Toluidine blue stains cellular nuclei, and identifies malignant tissue, which has an increased DNA content and a higher nuclear-to-cytoplasmic ratio. </span></span></p>
<p><span style="font-size:11px"><span style="color:#e74c3c">Methylene blue stain applied to a healthy cell sample results in a colorless stain. This is due to the cell's enzymes, which reduce the methylene blue, thereby, reducing its color. Methylene blue stain applied to a dead cell sample turns blue. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">DAPI </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Crowley, Marfell, and Waterhouse, 2016 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Binds strongly to adenine–thymine-rich regions in the DNA. DAPI can stain live and fixed cells. It passes less efficiently through the membrane in live cells. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Yes </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Hoescht 33342 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Crowley, Marfell, and Waterhouse, 2016 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Binds to DNA in live and fixed cells, used to measure DNA condensation. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Yes </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Acridine Orange (AO) </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Watanabe et al., 2002 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Interacts with DNA/RNA through intercalation/electrostatic interaction, is able to penetrate cell membranes. Stains live cells green and dead cells red. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Nile blue sulfate </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Watanabe et al., 2002 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Stains cell nuclei and lysosomes, indicating apoptotic bodies. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Neutral red </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Watanabe et al., 2002 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Measures lysosomal membrane integrity </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">LysoTracker Red </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Watanabe et al., 2002 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Measures phagolysosomal activity that occurs due to the engulfment of apoptotic bodies. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><span style="font-size:11px"><span style="color:#e74c3c">DNA damage/fragmentation assays: </span></span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Assay </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Reference </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Description </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">OECD Approved Assay </span></strong></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Kressel and Groscurth, 1994 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Apoptosis is detected with the TUNEL method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Yes </span></span></p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Nicoletti Assay (SubG1 cell fragment measurement) </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Nicoletti et al., 1991 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Measures DNA content in nuclei at the pre-G1 phase of the cell cycle (apoptotic nuclei have less DNA than nuclei in healthy cells). </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Cell Death Detection ELISA kit </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Parajuli, 2014 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Apoptotic nucleosomes are detected using the Cell Death Detection ELISA kit, which were calculated as absorbance subtraction at 405 nm and 490 nm. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><span style="font-size:11px"><span style="color:#e74c3c">Measurement of apoptotic markers through immunochemistry: </span></span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Method of Measurement </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Reference </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Description </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">OECD Approved Assay </span></strong></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Western blot / immunofluorescence microscopy / immunohistochemistry </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Elmore 2007; Martinez, Reif, and Pappas, 2010; Parajuli et al, 2014 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Apoptosis can be detected with the expression of various apoptotic markers by western blotting using antibodies. Markers can include: cytosolic cytochrome-c; caspases 2, 3, 6, 7, 8, 9, 10; Bax; Bcl-2 (apoptosis inhibitor); BIRC2; BIRC3; GAPDH; PARP; CDK2; CDK4; cyclin D1; p53; p63; p73; cytokeratin-18 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><span style="font-size:11px"><span style="color:#e74c3c">Measures of altered caspase activity: </span></span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Method of Measurement </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Reference </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Description </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">OECD Approved Assay </span></strong></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c"> Wu, 2016 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Visualizes caspase-3 and caspase-9 activity </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">PhiPhiLux Assay </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Watanabe et al., 2002 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">The PhiPhiLux molecule becomes fluorescent once it is cleaved by caspase-3, indicating caspase activity. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Ferrocene reporter </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Martinez, Reif, and Pappas, 2010 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">An electrochemical method to detect apoptosis. Ferrocene is attached to a peptide. The peptide sequence is a caspase 3 cleavage site and the ferrocene acts as the electrochemical reporter. The more caspase cleavage that occurs, the more ferrocene molecules are cleaved, the stronger the signal. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Self-assembled monolayers for matrix assisted laser desorption ionization time-of-flight mass spectrometry (SAMDI-MS) assay </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Martinez, Reif, and Pappas, 2010 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">This assay detects caspase activity. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><span style="font-size:11px"><span style="color:#e74c3c">Measures of altered mitochondrial physiology: </span></span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Method of Measurement </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Reference </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Description </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">OECD Approved Assay </span></strong></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Laser scanning confocal microscopy (LSCM) </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Watanabe et al., 2002 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">LCSM can monitor many mitochondrial events following staining of cells, such as: mitochondrial permeability transition, depolarization of the inner mitochondrial membrane, which may be indicative of apoptosis. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
<p> </p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Fluorescent, cationic, lipophilic mitochondrial dyes, such as: JC-1 dye, Rhodamine, DiOC6, Mitotracker red </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Martinez, Reif, and Pappas, 2010; Sivandzade, Bhalerao, and Cucullo, 2019 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">These mitochondrial dyes can indicate disintegration of the mitochondrial outer membrane’s electrochemical gradient, as different fluorescence is observed between healthy and apoptotic cells. In healthy cells the dye accumulates in aggregates, but in apoptotic cells missing the electrochemical membrane, the dye will spread out into the cytoplasm providing different fluorescent signals. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><span style="font-size:11px"><span style="color:#e74c3c">Other measures: </span></span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Method of measurement </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Reference </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">Description </span></strong></span></p>
</td>
<td>
<p><span style="font-size:11px"><strong><span style="color:#e74c3c">OECD Approved Assay </span></strong></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Apoptosis PCR microarray </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Elmore, 2007 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">A method to profile the gene expression of many apoptotic-related genes, for example: ligands, receptors, intracellular modulators, and transcription factors. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Fluorescence correlation spectroscopy (FCS) or dual-colour fluorescence cross-correlation spectroscopy (dcFCCS) </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Martinez, Reif, and Pappas, 2010 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Used to measure protease activity. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">No </span></span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Apoptosis is measured with Annexin V-FITC probes </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Elmore, 2007; Wu et al., 2016 </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">A measure of apoptotic membrane alterations. Annexin-V detects externalized phosphatidylserine residues, a result of apoptosis. Can be conducted in conjunction with propidium iodide (PI) staining. The relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry. </span></span></p>
</td>
<td>
<p><span style="font-size:11px"><span style="color:#e74c3c">Yes </span></span><span style="display:none"> </span><span style="display:none"> </span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The process of cell death is highly conserved within multi‐cellular organisms. </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Lockshin & Zakeri, 2004)</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p> </p>
<p><span style="font-size:11px"><span style="color:#e74c3c"><strong>Taxonomic applicability</strong>: Increased cell death is applicable to all animals. This includes vertebrates such as humans, mice and rats (Alberts et al., 2002). </span></span></p>
<p><span style="font-size:11px"><span style="color:#e74c3c"><strong>Life stage applicability</strong>: There is insufficient data on life stage applicability of this KE. </span></span></p>
<p><span style="font-size:11px"><span style="color:#e74c3c"><strong>Sex applicability</strong>: This key event is not sex specific (Forger and de Vries, 2010; Ortona Matarrese, and Malorni, 2014). </span></span></p>
<p><span style="font-size:11px"><span style="color:#e74c3c"><strong>Evidence for perturbation by a stressor</strong>: Multiple studies show that cell death can be increased or disrupted by many types of stressors including ionizing radiation and altered gravity (Zhu et al., 2016). </span></span></p>
UBERON:0000062organCL:0000000cellHighUnspecificHighAll life stagesHighHighHighHigh<p style="margin-left:32px"><span style="font-size:11px"><span style="color:#e74c3c">Alberts, B. et al. (2002), “Programmed Cell Death (Apoptosis)”, in Molecular Biology of the Cell. 4th edition, Garland Science, New York, https://www.ncbi.nlm.nih.gov/books/NBK26873/ </span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bertheloot, D., Latz, E., & Franklin, B. S. (2021). Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. <em>Cellular & Molecular Immunology</em>, <em>18</em>, 1106–1121. https://doi.org/10.1038/s41423-020-00630-3</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Bever, M. M., & Fekete, D. M. (1999). Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears. <em>Journal of Neurocytology</em>, <em>28</em>(10–11), 781–793. https://doi.org/10.1023/a:1007005702187</span></span></p>
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<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Cotter, T. G., & Al-Rubeai, M. (1995). Cell death (apoptosis) in cell culture systems. <em>Trends in Biotechnology</em>, <em>13</em>(4), 150–155. https://doi.org/10.1016/S0167-7799(00)88926-X</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Chojnowski, G., & Waterhouse, N. J. (2015a). Measuring the DNA content of cells in apoptosis and at different cell-cycle stages by propidium iodide staining and flow cytometry. <em>Cold Spring Harbor Protocols</em>, <em>10</em>, 905–910. https://doi.org/10.1101/pdb.prot087247</span></span></p>
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<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., Christensen, M. E., & Waterhouse, N. J. (2015d). Measuring cell death by trypan blue uptake and light microscopy. <em>Cold Spring Harbor Protocols</em>, <em>7</em>, 643–646. https://doi.org/10.1101/pdb.prot087155</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., Scott, A. P., Boughaba, J. A., Chojnowski, G., Christensen, M. E., & Waterhouse, N. J. (2016). Dead cert: Measuring cell death. <em>Cold Spring Harbor Protocols</em>, <em>2016</em>(12), 1064–1072. https://doi.org/10.1101/pdb.top070318</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., Scott, A. P., & Waterhouse, N. J. (2015e). Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. <em>Cold Spring Harbor Protocols</em>, <em>11</em>, 953–957. https://doi.org/10.1101/pdb.prot087288</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015a). Analyzing cell death by nuclear staining with Hoechst 33342. <em>Cold Spring Harbor Protocols</em>, <em>9</em>, 778–781. https://doi.org/10.1101/pdb.prot087205</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015b). Detection of DNA fragmentation in apoptotic cells by TUNEL. <em>Cold Spring Harbor Protocols</em>, <em>10</em>, 900–905. https://doi.org/10.1101/pdb.prot087221</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015c). Morphological analysis of cell death by cytospinning followed by rapid staining. <em>Cold Spring Harbor Protocols</em>, <em>9</em>, 773–777. https://doi.org/10.1101/pdb.prot087197</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Crowley, L. C., & Waterhouse, N. J. (2015a). Detecting cleaved caspase-3 in apoptotic cells by flow cytometry. <em>Cold Spring Harbor Protocols</em>, <em>11</em>, 958–962. https://doi.org/10.1101/pdb.prot087312</span></span></p>
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2020-12-04T15:13:072023-03-22T11:07:45Decrease, Lung functionDecreased lung functionIndividual<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Segoe UI",sans-serif">Lung function is a clinical term referring to the physiological functioning of the lungs, most often in association with the tests used to assess it. Lung function loss can be caused by acute or chronic exposure to airborne toxicants or by an intrinsic disease of the respiratory system. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Although signs of cellular injury are typically exhibited first in the nose and larynx, alveolar-capillary barrier breakdown may ultimately arise and result in local edema (Miller and Chang, 2003). Clinically, bronchoconstriction and hypoxia are seen in the acute phase, with affected subjects exhibiting shortness of breath (dyspnea) and low blood oxygen saturation, and with reduced lung function indices of airflow, lung volume and gas exchange (Hert and Albert, 1994; and How it is Measured or Detected;). When alveolar damage is extensive, the reduced lung function can develop into acute respiratory distress syndrome (ARDS). This severe compromise of lung function is reflected by decreased gas exchange indices (PaO<sub>2</sub>/FIO<sub>2</sub> ≤200 mmHg, due to hypoxemia and impaired excretion of carbon dioxide), increased pulmonary dead space and decreased respiratory compliance (Matthay et al., 2019). Acute inhalation exposures to chemical irritants such as ammonia, hydrogen chloride, nitrogen oxides and ozone typically cause local edema that manifests as dyspnea and hypoxia. In cases where a breakdown of the alveolar capillary function ensues, ARDS develops. ARDS has a particularly high risk of mortality, estimated to be 30-40% (Gorguner and Akgun, 2010; Matthay et al., 2018; Reilly et al., 2019).</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Lung function decrease due to reduction in lung volume is seen in pulmonary fibrosis, which can be linked to chronic exposures to e.g. silica, asbestos, metals, agricultural and animal dusts (Meltzer and Noble, 2008; Cheresh et al., 2013; Cosgrove, 2015; Trethewey and Walters, 2018). Additionally. decreased lung function occurs in pleural disease, chest wall and neuromuscular disorders, because of obesity and following pneumectomy (Moore, 2012). Decreased lung function can also be a result of narrowing of the airways by inflammation and mucus plugging resulting in airflow limitation. Decreased lung function is a feature of obstructive pulmonary diseases (e.g. asthma, COPD) and linked to a multitude of causes, including chronic exposure to cigarette smoke, dust, metals, organic solvents, asbestos, pathogens or genetic factors. </span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Pulmonary function tests are a group of tests that evaluate several parameters indicative of lung size, air flow and gas exchange. Decreased lung function can manifest in different ways, and individual circumstances, including potential exposure scenarios, determine which test is used. The section outlines the tests used to evaluate lung function in humans (https://www.nhlbi.nih.gov/health-topics/pulmonary-function-tests, accessed 22 March 2021) and in experimental animals.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Lung function tests used to evaluate human lung function</span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">The most common (“gold standard”) lung function test in human subjects is spirometry. Spirometry results are primarily used for diagnostic purposes, e.g. to discriminate between obstructive and restrictive lung diseases, and for determining the degree of lung function impairment. Specific criteria for spirometry tests have been outlined in the American Thoracic Society (ATS) and the European Respiratory Society (ERS) Task Force guidelines (Graham et al., 2019). These guidelines consist of detailed recommendations for the preparation and conduct of the test, instruction of the person tested, as well as indications and contraindications, and are complemented by additional guidance documents on how to interpret and report the test results (Pellegrino et al., 2005; Culver et al., 2017).</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Spirometry measures several different parameters during forceful exhalation, including:</span></span></span></span></span></p>
<ul>
<li style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Forced expiratory volume in 1 s (FEV1), the maximum volume of air that can forcibly be exhaled during the first second following maximal inhalation</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Forced vital capacity (FVC), the maximum volume of air that can forcibly be exhaled following maximal inhalation </span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Vital capacity (VC), the maximum volume of air that can be exhaled when exhaling as fast as possible</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">FEV1/FVC ratio</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Peak expiratory flow (PEF), the maximal flow that can be exhaled when exhaling at a steady rate</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Forced expiratory flow, also known as mid-expiratory flow; the rates at 25%, 50% and 75% FVC are given</span></span></span></span></span></li>
<li style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Inspiratory vital capacity (IVC), the maximum volume of air that can be inhaled after a full expiration</span></span></span></span></span></li>
</ul>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">A reduced FEV1, with normal or reduced VC, normal or reduced FVC, and a reduced FEV1/FVC ratio are indices of airflow limitation, i.e., airway obstruction as seen in COPD (Moore, 2012). In contrast, airway restriction is demonstrated by a reduction in FVC, normal or increased FEV1/FVC ratio, a normal spirometry trace and potentially a high PEF (Moore, 2012).</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Lung capacity or lung volumes can be measured using one of three basic techniques: 1) plethysmography, 2) nitrogen washout, or 3) helium dilution. Plethysmography consists of a series of sequential measurements in a body plethysmograph, starting with the measurement of functional residual capacity (FRC),</span></span> <span style="font-family:"Segoe UI",sans-serif"><span style="color:black">the volume of gas present in the lung at end-expiration during tidal breathing. Once the FRC is known, expiratory reserve volume (ERV; the volume of gas that can be maximally exhaled from the end-expiratory level during tidal breathing, i.e., the FRC), vital capacity (VC; the volume change at the mouth between the positions of full inspiration and complete expiration), and inspiratory capacity (IC; the maximum volume of air that can be inhaled from FRC) are determined, and total lung capacity (TLC;</span></span> <span style="font-family:"Segoe UI",sans-serif"><span style="color:black">the volume of gas in the lungs after maximal inspiration, or the sum of all volume compartments) and residual volume (RV; the volume of gas remaining in the lung after maximal exhalation) are calculated (Weinstock and McCannon, 2017).</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">The other two techniques used to measure lung volumes—helium dilution and nitrogen washout—are based on the principle of conservation of mass: [initial gas concentration] x [initial volume of the system] = [final gas concentration] x [final volume of the system]. The nitrogen washout method is based on the fact that nitrogen is present in the air, at a relatively constant amount. The subject is given 100% oxygen to breathe, and the expired gas, which contains nitrogen in the lung at the beginning of the test, is collected. When no more nitrogen is noted in the expirate, the volume of air expired and the entire amount of nitrogen in that volume are measured, and the initial volume of the system (FRC) can be calculated. In the helium dilution method, a known volume and concentration of helium is inhaled by the subject. Helium, an inert gas that is not absorbed significantly from the lungs, is diluted in proportion to the lung volume to which it is added. The final concentration of helium is then measured and FRC calculated (Weinstock and McCannon, 2017).</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Measurements of lung volumes in humans are technically more challenging than spirometry. However, they complement spirometry (which cannot determine lung volumes) and may be a preferred means of lung function assessment when subject compliance cannot be reasonably expected (e.g. in pediatric subjects) or where forced expiratory maneuvers are not possible (e.g. in patients with advanced pulmonary fibrosis). There are recommended standards for lung volume measurements and their interpretation in clinical practice, issued by the ATS/ERS Task Force (Wanger et al., 2005; Criée et al., 2011). </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Finally, indices of gas exchange across the alveolar-capillary barrier are tested by diffusion capacity of carbon monoxide (DLCO) studies (also referred to as transfer capacity of carbon monoxide, TLCO). The principle of the test is the increased affinity of hemoglobin to preferentially bind carbon monoxide over oxygen (Weinstock and McCannon, 2017). Complementary to spirometry and lung volume measurements, DLCO provides information about the lung surface area available for gas diffusion. Therefore, it is sensitive to any structural changes affecting the alveoli, such as those accompanying emphysema, pulmonary fibrosis, pulmonary edema, and ARDS. Recommendations for the standardization of the test and its evaluation have been outlined by the ATS/ERS Task Force (Graham et al., 2017). An isolated reduction in DLCO with normal spirometry and in absence of anemia suggests an injury to the alveolar-capillary barrier, as for example seen in the presence of pulmonary emboli or in patients with pulmonary hypertension (Weinstock and McCannon, 2017; Lettieri et al., 2006; Seeger et al., 2013). Reduced DLCO together with airflow obstruction (i.e., reduced FEV1) indicates lung parenchymal damage and is commonly observed in smokers and in COPD patients (Matheson et al., 2007; Harvey et al., 2016), whereas reduced DLCO with airflow restriction is seen in patients with interstitial lung diseases (Dias et al., 2014; Kandhare et al., 2016).</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Lung function tests used to evaluate experimental animal lung function</span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Because spirometry requires active participation and compliance of the subject, it is not commonly used in animal studies. However, specialized equipment such as the flexiVent system (SCIREQ<sup>®</sup>) are available for measuring FEV, FVC and PEF in anesthetized and tracheotomized small laboratory animals. Other techniques such as plethysmography or forced oscillation are increasingly preferred for lung function assessment in small laboratory animals (McGovern et al., 2013; Bates, 2017). </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">In small laboratory animals, plethysmography can be used to determine respiratory physiology parameters (minute volume, respiratory rate, time of pause and time of break), lung volume and airway resistance of conscious animals. Both whole body and head-out plethysmography can be applied, although there is a preference for the latter in the context of inhalation toxicity studies, because of its higher accuracy and reliability (OECD, 2018a; Hoymann, 2012).</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Gas diffusion tests are not frequently performed in animals, because reproducible samplings of alveolar gas are difficult and technically challenging (Reinhard et al., 2002; Fallica et al., 2011). Modifications to the procedure employed in humans have, however, open possibilities to obtain a human-equivalent DLCO measure or the diffusion factor for carbon monoxide (DFCO)—a variable closely related to DLCO, which can inform on potential structural changes in the lungs that have an effect on gas exchange indices (Takezawa et al., 1980; Dalbey et al., 1987; Fallica et al., 2011; Limjunyawong et al., 2015).</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:black">Pulmonary function tests reflect the physiological working of the lungs. Therefore, the AO is applicable to a variety of species, including (but not limited to) rodents, rabbits, pigs, cats, dogs, horses and humans, independent of life stage and gender.</span></span></span></span></span></p>
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2016-11-29T18:41:312021-09-08T04:54:28Aryl hydrocarbon receptor activation leading to impaired lung function through AHR-ARNT toxicity pathwayAHR activation decreasing lung function via AHR-ARNT tox path<p>Dianke Yu</p>
<p>Department of Toxicology, School of Public Health, Qingdao University, Qingdao, China</p>
<p> </p>
Under development: Not open for comment. Do not cite2021-08-14T01:58:132023-04-29T16:03:05