Activation, AhR

92-87-5

HFACYLZERDEVSX-UHFFFAOYSA-N

C.I. Azoic Diazo Component 112

4-(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

DTXSID2020137

262-12-4

NFBOHOGPQUYFRF-UHFFFAOYSA-N

Dibenzo-p-dioxin

Dibenzo[b,e][1,4]dioxin Dibenzo[1,4]dioxin dibenzo-p-dioxina dibenzo-p-dioxinne Diphenylene dioxide Oxanthrene Phenodioxin

DTXSID8020410

118-74-1

CKAPSXZOOQJIBF-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

DTXSID2020682

51-52-5

KNAHARQHSZJURB-UHFFFAOYSA-N

6-Propyl-2-thiouracil

6-Propyl-2 thiouracil (PTU) 4(1H)-Pyrimidinone, 2,3-dihydro-6-propyl-2-thioxo- 2,3-Dihydro-6-propyl-2-thioxo-4(1H)-pyrimidinone 2-Mercapto-4-hydroxy-6-n-propylpyrimidine 2-Mercapto-4-hydroxy-6-propylpyrimidine 2-Mercapto-6-propylpyrimidin-4-ol 2-Thio-4-oxo-6-propyl-1,3-pyrimidine 2-Thio-6-propyl-1,3-pyrimidin-4-one 6-n-Propyl-2-thiouracil 6-n-Propylthiouracil 6-Propyl-2-thio-2,4(1H,3H)pyrimidinedione 6-Propylthiouracil NSC 6498 NSC 70461 Procasil Propacil propiltiouracilo Propycil Propyl-Thiorist Propylthiorit propylthiouracil Propylthiouracile Propyl-Thyracil Prothiucil Prothiurone Prothycil Prothyran Protiural Thiuragyl Thyreostat II URACIL, 4-PROPYL-2-THIO- Uracil, 6-propyl-2-thio-

DTXSID5021209

60-56-0

PMRYVIKBURPHAH-UHFFFAOYSA-N

Methimazole

2H-Imidazole-2-thione, 1,3-dihydro-1-methyl- 1,3-Dihydro-1-methyl-2H-imidazole-2-thione 1-Methyl-1,3-dihydroimidazole-2-thione 1-Methyl-1H-imidazole-2-thiol 1-Methyl-2-mercapto-1H-imidazole 1-Methyl-2-mercaptoimidazole 1-Methyl-4-imidazoline-2-thione 1-Methylimidazole-2(3H)-thione 1-Methylimidazole-2-thiol 1-Methylimidazole-2-thione 2-Mercapto-1-methyl-1H-imidazole 2-Mercapto-1-methylimidazole 2-Mercapto-N-methylimidazole 4-Imidazoline-2-thione, 1-methyl- Basolan Danantizol Favistan Frentirox Imidazole-2-thiol, 1-methyl- Mercaptazole Mercazole Mercazolyl Metazolo Methimazol Methylmercaptoimidazole Metothyrin Metothyrine Metotirin N-Methyl-2-mercaptoimidazole N-Methylimidazolethiol NSC 38608 Strumazol Tapazole Thacapzol Thiamazol thiamazole Thycapzol Thymidazol Thymidazole tiamazol

DTXSID4020820

14797-73-0

VLTRZXGMWDSKGL-UHFFFAOYSA-M

Perchlorate

Perchlorate ion Perchlorate ion (ClO41-) Perchlorate ion(1-) Perchlorate(1-) Perchloric acid, ion(1-)

DTXSID6024252

PR:000003858

PR aryl hydrocarbon receptor

PR:000001325

PR beta-1,3-glucuronyltransferase

CHEBI:30660

CHEBI thyroxine

UBERON:0002421

UBERON hippocampal formation

GO:0045202

GO synapse

GO:0004874

GO aryl hydrocarbon receptor activity

GO:0017162

GO aryl hydrocarbon receptor binding

GO:0015020

GO glucuronosyltransferase activity

GO:0003824

GO catalytic activity

GO:0008152

GO metabolic process

MP:0005475

MP abnormal circulating thyroxine level

GO:0010817

GO regulation of hormone levels

GO:0010468

GO regulation of gene expression

GO:0007268

GO chemical synaptic transmission

GO:0007611

GO learning or memory

GO:0050890

GO cognition

WIKI increased

WIKI decreased

WIKI abnormal

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

2017-02-09T14:32:32

Polychlorinated biphenyl

2016-11-29T18:42:27

PCB 126

2023-04-20T20:20:56

Benzidine

2016-11-29T18:42:26

Dibenzo-p-dioxin

2016-11-29T18:42:27

Polychlorinated dibenzofurans

2016-11-29T18:42:27

Hexachlorobenzene

2016-11-29T18:42:27

Polycyclic aromatic hydrocarbons (PAHs)

2017-02-09T15:43:00

Propylthiouracil

2016-11-29T18:42:22

Methimazole

2016-11-29T18:42:19

Perchlorate

2016-11-29T18:42:26

Iodine deficiency

2017-03-26T11:37:44

7955

NCBI zebra danio

WCS_9031

common ecological species Gallus gallus

143350

NCBI Pagrus major

7904

NCBI Acipenser transmontanus

41871

NCBI Acipenser fulvescens

WCS_8022

common ecological species rainbow trout

8030

NCBI Salmo salar

WCS_8355

common ecological species Xenopus laevis

8296

NCBI Ambystoma mexicanum

WCS_9054

common ecological species Phasianus colchicus

WCS_93934

common ecological species Coturnix japonica

10090

NCBI mouse

10116

NCBI rat

WCS_9606

common toxicological species human

34823

NCBI Microgadus tomcod

9606

NCBI Homo sapiens

WCS_8355

common ecological species African clawed frog

10116

NCBI Rattus norvegicus

10118

NCBI Rattus sp.

WCS_9031

common ecological species chicken

WCS_7955

common ecological species zebrafish

WCS_90988

common ecological species fathead minnow

9823

NCBI Sus scrofa

10116

NCBI rats

WikiUser_14

Wikiuser: Ftschudi Monkey

WikiUser_17

mammals

WikiUser_6

ApacheUser fish

8292

NCBI Amphibia

8782

NCBI Aves

WCS_9606

common toxicological species humans

Activation, AhR

Molecular

<h3>The AHR Receptor</h3> 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)<a href="#cite_note-Okey2007-1">[1]</a>. Other members of this superfamily include the AHR nuclear translocator (ARNT), which acts as a dimerization partner of the AHR <a href="#cite_note-Hoffman1991-2">[2]</a><a href="#cite_note-Poland1976-3">[3]</a>; Per, a circadian transcription factor; and Sim, the “single-minded” protein involved in neuronal development <a href="#cite_note-Gu2000-4">[4]</a><a href="#cite_note-Kewley2004-5">[5]</a>. This group of proteins shares a highly conserved PAS domain and is involved in the detection of and adaptation to environmental change<a href="#cite_note-Gu2000-4">[4]</a>. 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). <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> 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)<a href="#cite_note-Fujii2010-6">[6]</a>. Upon ligand binding, the AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with ARNT<a href="#cite_note-Mimura2003-7">[7]</a>. 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<a href="#cite_note-Fujii2010-6">[6]</a>. 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 <a href="#cite_note-Fujii2010-6">[6]</a><a href="#cite_note-Giesy2006-8">[8]</a><a href="#cite_note-Mimura2003-7">[7]</a><a href="#cite_note-Safe1994-9">[9]</a>. <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 (Salmo salar) (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>   Roles of isoforms in birds: Two AHR isoforms (AHR1 and AHR2) have been identified in the black-footed albatross (Phoebastria nigripes), great cormorant (Phalacrocorax carbo) and domestic chicken (Gallus gallus domesticus)<a href="#cite_note-Yasui2007-10">[10]</a>. 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<a href="#cite_note-Yasui2007-10">[10]</a>, 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<a href="#cite_note-Lee2009-11">[11]</a><a href="#cite_note-Yasui2007-10">[10]</a>. <ul> <li>AhR1 and AhR2 both bind and are activated by TCDD in vitro (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> Roles of isoforms in fishes: <ul> <li>AhR1 and AhR2 both bind and are activated by TCDD in vitro (Bak et al 2013; Doering et al 2014; 2015; Karchner et al 1999; 2005).</li> <li>AhR1 has greater sensitivity to activation by TCDD than AhR2 in red seabream (Pagrus major), white sturgeon (Acipenser transmontanus), and lake sturgeon (Acipenser fulvescens) (Bak et al 2013; Doering et al 2014; 2015)</li> <li>AhR2 has greater binding affinity or activation by TCDD than AhR1 in zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus) (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 (Danio rerio) and mummichog (Fundulus heteroclitus), while knockdown of AhR1 offers no protection (Clark et al 2010; Prasch et al 2003; Van Tiem & Di Giulio 2011).</li> </ul> Roles of isoforms in amphibians and reptiles: <ul> <li>Less is known about AhRs of amphibians or reptiles.</li> <li>AhR1 is believed to mediate toxicities in amphibians (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015). However, all AhRs of amphibians that have been investigated have very low affinity for TCDD (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015).</li> <li>Both AhR1s and AhR2 of American alligator (Alligator mississippiensis) are activated by agonists with comparable sensitivities (Oka et al 2016). AhRs of no other reptiles have been investigated.</li> </ul> Role in mammals AhR expression is essentially ubiquitous in mammals consistent with a broad-spectrum homeostatic role, however expression levels varying widely across tissues with the liver, thymus, lung, kidney, spleen, and placenta exhibiting greatest expression (Harper PA). Additionally, AhR expression is developmentally regulated, and more recent evidence indicates a role for the AhR in developmental process affecting hematopoiesis, immune system biology, neural differentiation, and liver architecture (Wright E J) . AHR is involved in regulating the rate of apoptosis of oocytes in germ cell nests during embryonic life and in regulating survival of oocytes in the fetal and neonatal ovary. Specifically, studies have shown that ovaries obtained from AHRKO mice on ED13.5 and cultured for 72 h in the absence of hormonal support with the aim of inducing apoptosis, contained higher numbers of non-apoptotic germ cells compared to wild-type (WT) ovaries cultured in the same conditions (Hernández-Ochoa)

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? <h3>Transactivation Reporter Gene Assays (recommended approach)</h3> <h4>Transient transfection transactivation</h4> Transient transfection transactivation is the most common method for evaluating nuclear receptor activation<a href="#cite_note-Raucy2010-12">[12]</a>. 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)<a href="#cite_note-Raucy2010-12">[12]</a>. 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<a href="#cite_note-Raucy2010-12">[12]</a>. <h5>Luciferase reporter gene (LRG) assay</h5> The described luciferase reporter gene (LRG) assays have been used to investigate activation of AhRs of: <ul> <li>Humans (Homo sapiens) (Abnet et al 1999) </li> <li>Species of birds, namely chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica), and common tern (Sterna hirundo) (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 (Phoebastria nigripes) and common cormorant (Phalacrocorax carbo) (Yasio et al 2007).</li> <li>American alligator (Alligator mississippiensis) 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 (Xenopus laevis) and salamander (Ambystoma mexicanum) (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 (Salmo salar), Atlantic tomcod (Microgadus tomcod), white sturgeon (Acipenser transmontanus), rainbow trout (Onchorhynchys mykiss), red seabream (Pagrus major), lake sturgeon (Acipenser fulvescens), and zebrafish (Danio rerio) (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> 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 Renilla luciferase vector to control for transfection efficiency. After seeding, the cells were exposed to DLC and luciferase activity was measured using a luminometer. Luminescence, which is proportional to the extent of AHR activation, is expressed as the ratio of firefly luciferase units to Renilla luciferase units <a href="#cite_note-Farmahin2012-13">[13]</a>. 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).<a href="#cite_note-Farmahin2013b-14">[14]</a><a href="#cite_note-Farmahin2012-13">[13]</a><a href="#cite_note-Fujisawa2012-15">[15]</a><a href="#cite_note-Lee2009-11">[11]</a><a href="#cite_note-Manning2012-16">[16]</a><a href="#cite_note-Mol2012-17">[17]</a> <h4>Transactivation in stable cell lines</h4> 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 <a href="#cite_note-Raucy2010-12">[12]</a>. 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<a href="#cite_note-Yueh2005-18">[18]</a>. An added benefit of this model is the potential to multiplex 3 assays in a single well: receptor activation, cell viability and enzyme activity<a href="#cite_note-Raucy2010-12">[12]</a>. Such assays are used extensively in drug discovery due to their high throughput efficiency, and may serve just as useful for risk assessment purposes. <h3>Ligand-Binding Assays</h3> 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<a href="#cite_note-Poland1982-19">[19]</a> and can explain differences in species sensitivities to DLCs<a href="#cite_note-Hesterman2000-20">[20]</a><a href="#cite_note-Farmahin2014-21">[21]</a><a href="#cite_note-Karchner2006-22">[22]</a>; they are therefore worth mentioning. Binding affinity and efficacy have been used to develop structure-activity relationships for AHR disruption<a href="#cite_note-Hesterman2000-20">[20]</a><a href="#cite_note-Lee2015-23">[23]</a> 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<a href="#cite_note-Jones2003-24">[24]</a><a href="#cite_note-Raucy2010-12">[12]</a>. 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. <h4>Hydroxyapatite (HAP) binding assay</h4> The HAP binding assay makes use of an in vitro transcription/translation method to synthesize the AHR protein, which is then incubated with radiolabeled TDCPP and a HAP pellet. The occupied protein adsorbs to the HAP and the radioactivity is measured to determine saturation binding. An additional ligand can also be included in the mixture in order to determine its binding affinity relative to TCDD (competitive binding)<a href="#cite_note-Gasiewicz1982-25">[25]</a><a href="#cite_note-Karchner2006-22">[22]</a>. This assay is simple, repeatable and reproducible; however, it is insensitive to weak ligand-receptor interactions<a href="#cite_note-Karchner2006-22">[22]</a><a href="#cite_note-Farmahin2014-21">[21]</a><a href="#cite_note-Nakai1995-26">[26]</a>. <h4>Whole cell filtration binding assay</h4> Dold and Greenlee<a href="#cite_note-Dold1990-27">[27]</a> developed a method to detect specific binding of TCDD to whole mammalian cells in culture and was later modified by Farmahin et al.<a href="#cite_note-Farmahin2014-21">[21]</a> 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<a href="#cite_note-Farmahin2014-21">[21]</a>. <h3>Protein-DNA Interaction Assays</h3> 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<a href="#cite_note-Perez2007-28">[28]</a>. Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions in vivo. It involves the treatment of cells with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. Nuclear fractions are isolated, the genomic DNA is sheared, and nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. After reversal of the crosslinking, the associated DNA fragments are sequenced. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. Electrophoretic mobility shift assay (EMSA) provides a rapid method to study DNA-binding protein interactions in vitro. This relies on the fact that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments. The protein-DNA complex components are then identified with appropriate antibodies. The EMSA assay was found to be consistent with the LRG assay in chicken hepatoma cells dosed with dioxin-like compounds<a href="#cite_note-Heid2001-29">[29]</a>. <h3>In silico Approaches</h3> 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).

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<a href="#cite_note-Poland1976-3">[3]</a>. 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<a href="#cite_note-Ema1994-30">[30]</a><a href="#cite_note-Poland1982-19">[19]</a><a href="#cite_note-Poland1994-31">[31]</a>. Several other studies reported the importance of this amino acid in birds and mammals<a href="#cite_note-Backlund2004-32">[32]</a><a href="#cite_note-Ema1994-30">[30]</a><a href="#cite_note-Karchner2006-22">[22]</a><a href="#cite_note-Murray2005-33">[33]</a><a href="#cite_note-Pandini2007-34">[34]</a><a href="#cite_note-Pandini2009-35">[35]</a><a href="#cite_note-Poland1994-31">[31]</a><a href="#cite_note-Ramadoss2004-36">[36]</a>. 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<a href="#cite_note-Pandini2009-35">[35]</a>. Mutation at position 319 in the mouse eliminated AHR DNA binding<a href="#cite_note-Pandini2009-35">[35]</a>. The first study that attempted to elucidate the role of avian AHR1 domains and key amino acids within avian AHR1 in avian differential sensitivity was performed by Karchner et al.<a href="#cite_note-Karchner2006-22">[22]</a>. 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<a href="#cite_note-Karchner2006-22">[22]</a>, 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<a href="#cite_note-Karchner2006-22">[22]</a>. 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<a href="#cite_note-Farmahin2013b-14">[14]</a><a href="#cite_note-Head2008-37">[37]</a> . 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<a href="#cite_note-Farmahin2013b-14">[14]</a><a href="#cite_note-Head2008-37">[37]</a><a href="#cite_note-Manning2012-16">[16]</a>. 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)<a href="#cite_note-Farmahin2013b-14">[14]</a><a href="#cite_note-Head2008-37">[37]</a><a href="#cite_note-Manning2012-16">[16]</a>. <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 (Xenopus laevis) and axolotl (Ambystoma mexicanum) has been suggested as a mechanism for tolerance of these amphibians to DLCs (Lavine et al 2005; Shoots et al 2015).</li> <li>Among reptiles, only AhRs of American alligator (Alligator mississippiensis) have been investigated and little is known about the sensitivity of American alligator or other reptiles to DLCs (Oka et al 2016).</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 (Microgadus tomcod) (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> The AhR is a very conserved and ancient protein (95) and the AhR is present  in human and mice (96–98). The AhR is present in human physiology and pathology. The 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). 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. 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). The occurrence of a nystagmus has been subsequently diagnosed in humans bearing a AhR mutation (Borovok). 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, Subramaniam, Barhoover). 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. Many endogenous and exogenous ligands are present for the AhR in human (Optiz, Adachi, Schroeder, Rothhammer). 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).

High Unspecific

High

Embryo

High

Development

High

All life stages

High High High High High High High High High High High High High High High Not Specified

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Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. J. Biol. Chem. 1992, 267 (19), 13728-13734   Prasch, A.L.; Teraoka, H.; Carney, S.A.; Dong, W.; Hiraga, T.; Stegeman, J.J.; Heideman, W.; Peterson, R.E. 2003. Toxicol. Sci. Aryl hydrocarbon receptor 2 mediated 2,3,7,8-tetrachlorodibenzo-p-dioxin developmental toxicity in zebrafish. 76 (1), 138-150.   Shoots, J.; Fraccalvieri, D.; Franks, D.G.; Denison, M.S.; Hahn, M.E.; Bonati, L.; Powell, W.H. 2015. An aryl hydrocarbon receptor from the salamander Ambystoma mexicanum exhibits low sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enviro. Sci. Technol. 49, 6993-7001.   Tanguay, R.L.; Abnet, C.C.; Heideman, W. Peterson, R.E. (1999). Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor1. Biochimica et Biophysica Act 1444, 35-48.   Van den Berg, M.; Birnbaum, L.; Bosveld, A.T.C.; Brunstrom, B.; Cook, P.; Feeley, M.; Giesy, J.P.; Hanberg, A.; Hasegawa, R.; Kennedy, S.W.; Kubiak, T.; Larsen, J.C.; van Leeuwen, R.X.R.; Liem, A.K.D.; Nolt, C.; Peterson, R.E.; Poellinger, L.; Safe, S.; Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PECDFs for human and wildlife. Enviro. Hlth. Persp. 1998, 106, 775-792.   Van Tiem, L.A.; Di Giulio, R.T. 2011. AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (Danio rerio). Toxicol. Appl. Pharmacol. 254 (3), 280-287.   Whitlock, J.P.; Okino, S.T.; Dong, L.Q.; Ko, H.S.P.; Clarke Katzenberg, R.; Qiang, M.; Li, W. 1996. Induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. Faseb. J. 10, 809-818.   Wirgin, I.; Roy, N.K.; Loftus, M.; Chambers, R.C.; Franks, D.G.; Hahn, M.E. 2011. Mechanistic basis of resistance to PCBs in Atlantic tomcod from the Hudson River. Science. 331, 1322-1324.   Yamauchi, M.; Kim, E.Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryos: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. Aquat. Toxicol. 2006, 16, 166-179.   Yasui, T.; Kim, E.Y.; Iawata, H.; Franks, D.G.; Karchner, S.I.; Hahn, M.E.; Tanabe, S. 2007. Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. Toxicol. Sci. 99 (1), 101-117. <div>Hirano, M.; Hwang, JH; Park, HJ; Bak, SM; Iwata, H. and Kim, EY (2015) In Silico Analysis of the Interaction of Avian Aryl Hydrocarbon Receptors and Dioxins to Decipher Isoform-, Ligand-, and Species-Specific Activations.<cite> Environmental Science & Technology</cite> 49 (6): 3795-3804.DOI: 10.1021/es505733f</div> <div> </div> Bonati, L.; Corrada, D.; Tagliabue, S.G.; Motta, S. (2017) Molecular modeling of the AhR structure and interactions can shed light on ligand-dependent activation and transformation mechanisms. Current Opinion in Toxicology 2: 42-49. https://doi.org/10.1016/j.cotox.2017.01.011. Sovadinová, I. , Bláha, L. , Jano&scaron;ek, J. , Hilscherová, K. , Giesy, J. P., Jones, P. D. and Holoubek, I. (2006), Cytotoxicity and aryl hydrocarbon receptor‐mediated activity of N‐heterocyclic polycyclic aromatic hydrocarbons: Structure‐activity relationships. Environmental Toxicology and Chemistry, 25: 1291-1297. doi:<a href="https://doi.org/10.1897/05-388R.1">10.1897/05-388R.1</a>     Wakx A, Nedder M, Tomkiewicz-Raulet C, Dalmasso J, Chissey A, et al. 2018. Expression, Localization, and Activity of the Aryl Hydrocarbon Receptor in the Human Placenta. Int J Mol Sci. 19(12)   Ema M, Ohe N, Suzuki M, Mimura J, Sogawa K, et al. 1994. Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors.   Kall MA, Vang O, Clausen J. 1996. Effects of dietary broccoli on human in vivo drug metabolizing enzymes: evaluation of caffeine, oestrone and chlorzoxazone metabolism. Carcinogenesis. 17(4):793–99   Miller CA. 1997. Expression of the human aryl hydrocarbon receptor complex in yeast. Activation of transcription by indole compounds. J. Biol. Chem. 272(52):32824–29   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   Adachi J, Mori Y, Matsui S, Takigami H, Fujino J, et al. 2001. Indirubin and indigo are potent aryl hydrocarbon receptor ligands present in human urine. J. Biol. Chem. 276(34):31475–78   Marinelli L, Martin-Gallausiaux C, Bourhis J-M, B.guet-Crespel F, Blotti.re HM, Lapaque N. 2019. Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells. Sci Rep. 9(1):643   Stobbe-Maicherski N, Wolff S, Wolff C, Abel J, Sydlik U, et al. 2013. The interleukin-6-type cytokine oncostatin M induces aryl hydrocarbon receptor expression in a STAT3-dependent manner in human HepG2 hepatoma cells. FEBS J. 280(24):6681–90   Kado S, Chang WLW, Chi AN, Wolny M, Shepherd DM, Vogel CFA. 2017. Aryl hydrocarbon receptor signaling modifies Toll-like receptor-regulated responses in human dendritic cells. Arch Toxicol. 91(5):2209–21   Bock KW. 2019. Human AHR functions in vascular tissue: Pro- and anti-inflammatory responses of AHR agonists in atherosclerosis. Biochem Pharmacol. 159:116–20   Schroeder JC, Dinatale BC, Murray IA, Flaveny CA, Liu Q, et al. 2010. The uremic toxin 3- indoxyl sulfate is a potent endogenous agonist for the human aryl hydrocarbon receptor. Biochemistry. 49(2):393–400   Watanabe I, Tatebe J, Namba S, Koizumi M, Yamazaki J, Morita T. 2013. Activation of aryl hydrocarbon receptor mediates indoxyl sulfate-induced monocyte chemoattractant protein-1 expression in human umbilical vein endothelial cells. Circ J. 77(1):224–30   Shackleford G, Sampathkumar NK, Hichor M, Weill L, Meffre D, et al. 2018. Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis. Proc Natl Acad Sci U S 115(6):E1319–28     Zudaire E, Cuesta N, Murty V, Woodson K, Adams L, et al. 2008. The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers. J Clin Invest. 118(2):640–50   Goode GD, Ballard BR, Manning HC, Freeman ML, Kang Y, Eltom SE. 2013. Knockdown of aberrantly upregulated aryl hydrocarbon receptor reduces tumor growth and metastasis of MDA-MB- 231 human breast cancer cell line. Int J Cancer. 133(12):2769–80   Chang JT, Chang H, Chen P-H, Lin S-L, Lin P. 2007. Requirement of aryl hydrocarbon receptor overexpression for CYP1B1 up-regulation and cell growth in human lung adenocarcinomas. Clin Cancer Res. 13(1):38–45   Kanno Y, Takane Y, Izawa T, Nakahama T, Inouye Y. 2006. 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   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   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   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   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   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   Gramatzki D, Pantazis G, Schittenhelm J, Tabatabai G, K.hle C, et al. 2009. 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An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells. Cancer Lett. 340(1):63–71   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 Harper PA, Riddick DS, Okey AB. Regulating the regulator: factors that control levels and activity of the aryl hydrocarbon receptor. Biochem Pharmacol. 2006;72(3):267-79. Wright EJ, De Castro KP, Joshi AD, Elferink CJ. Canonical and non-canonical aryl hydrocarbon receptor signaling pathways. Curr Opin Toxicol. 2017;2:87-92. Hernández-Ochoa I, Karman BN, Flaws JA. The role of the aryl hydrocarbon receptor in the female reproductive system. Biochem Pharmacol. 2009;77(4):547-59.     AOPWiki 2016-11-29T18:41:22

2025-05-31T07:56:20

Induction, Upregulation of glucuronyltransferase activity

Molecular

CL:0000255

CL eukaryotic cell

Not Specified Not Specified

AOPWiki 2016-11-29T18:41:23

2017-09-16T10:14:22

Increased, Clearance of thyroxine from serum

Tissue

Thyroxin (T4) and T3 are metabolized and cleared from tissues in a number of ways: inner ring or outer ring deiodination via specific enzymes, conjugation (glucuronidation or sulfation), oxidative deamination and ether-linked cleavage (Zoeller et al 2007). Deiodination: There are three types of deiodinase enzymes. D1 and D2 convert T4 to T3 by removing an iodine atom from the outer ring while D3 removes an iodine atom from the inner ring, converting T4 to reverse T3. Differential expression of these enzymes during brain development are critical to the functionality of thyroid hormone in different areas of the fetal brain. Much of the T4 is carried to the liver, where it is transported across the cellular membrane, converted into T3 via deiodination as mediated by deiodinase enzymes, and it is this T3 that triggers the TH receptors found in the nucleus. Roughly 80% of the T3 needed is produced via outer-ring deiodination of T4, which "activates" T4 to T3 (as opposed to inner-ring deiodination, which "degrades" T4 to reverse T3 which is eliminated). About 30% of the T4 produced daily (~ 130 nmol) is converted to roughly 40 nmol of T3 (Visser 2012) via enzyme D1 (liver, kidney) while conversion to rT3 accounts for roughly 40% of T4 turnover and is mediated via enzyme D3 (brain, placenta, fetus). Conjugation: Glucuronidation and sulfation of T4 accounts for the rest of the metabolized T4 and leads to rapid elimination through bile. It is thought that 20% of daily T4 production is eliminated through biliary excretion of glucuronide conjugates. Glucuronidation is carried out by UDP-glucuronoyltransferase (UGT) enzymes (Hood and Klaassen 2000a, 2000b) and appears to be more important in murine species than in man (Henneman and Visser 1997) and sulfation of T4 is done largely through an initial inner ring deiodination step (via D3). Circulating levels of THs in serum can be affected by compounds that induce the activity of UDP-UGT enzymes. Uptake into the liver involves "high affinity, low capacity" and "low affinity, high capacity" processes with Km values in the nano- to micro-molar range (as opposed to the free T3 and T4 concentrations, which are in the picomolar range) (Henneman et al 2001 from Visser 2010). Both MCT8 and MCT 10 can transport THs; however, MCT8 is expressed in human liver where MCT10 is not and MCT8 display higher efficacy of cellular uptake and efflux relative to T3 (Ref 12 in Visser 2010).         Data in animals for PCBs Previous reports have been made showing serum TH decreases in rats and mice in response to PCBs, PCB congeners and TCDD and these decreases have been thought to be driven by UDP-UGT (particularly 1A1 and 1A6)(Barter and Klaassen 1994, Schuur et al 1997, Van Birgelen et al 1995, Visser 1996) Hallgren et al 2001, Hallgren and Darnerud 2002 showed that both T4 and T3 are significantly decreased following exposure to PCB mixtures or individual congeners. Kato et al 2003 (and Kato et al 2002) showed for the first time that a commercial PCB mixture (Kanechlor-500, KC500) decreased serum TH without an increase in glucuronidation of T4. Male Wistar rats and ddy mice were given a single ip injection of 100 mg/kg and 4 days later, organ weights were measured and microsomal enzymes measured.  Significant increases were noted for both endpoints in both species; however, treatment with PCBs led to significant increases in UDP-UGT activity in rats but not mice. Gene expression of UDP-UGTs was also examined and, again, rats (but not mice) displayed time-dependent increases in levels of UGT1A1 and UGT1A6 following treatment with PCBs.  This agrees with past reports showing that clofibrate, phenobarbital, pregnenolone-16-alpha-carbonitrile and beta-naphthoflavone decrease serum TH and increase hepatic UDP-UGT activity in rats but not mice (Viollon-Abadie et al 1999).   This implies that mice may reduce serum TH through a mechanism that does not involve increased glucuronidation, but may involve a TTR-associated pathway (as hydroxylated metabolites of PCBs have been displayed high affinity for TTR in in vitro studies).  Kanechlor-500 does not display any appreciable amount of outer ring deiodination activity (which would convert serum T4 to T3) and treatment with the mixture did not significantly change TSH levels (indicating there is no induction of the thyroid feedback loop from the measured decreases in serum TH).  Kato et al 2004 performed the same experiment next with Wistar and Gunn rats, the latter species being a Wistar mutant strain that lacks UGT1A isoforms.  Both species showed serum T4 (free and total) decrease after single injection of either KC500 or pentaCB and only Wistar rats showed an associated increase in UDP-UGT activity. Significant decrease in type I deiodinase was observed in both rats in addition to detection of hydroxylated PCB metabolites bound to TTR.  Gunn rats treated with clofibrate also showed decreased serum T4 without an increase in UDP-UGT activity (Visser et al 1993).  These results imply that decreases of serum TH by PCB or pentaCB were managed by formation of OH-PCB metabolites that were then transported by TTR.  This is supported by the fact that the main metabolite found in KC500-treated rats was 4-OH-2,3,3’,4’,5-pentachlorobiphenyl, which displays a binding affinity towards TTR that exceeds that of T4 by more than 3-fold (Meerts et al 2002).  In fact, the dihydrohxylated PCBs show several fold higher affinity than the monohydroxylated PCBs (Lans et al 1993).  It should be noted that an increase in sulfation via SULT enzymes may also offer an esxplanation for the observed results. Kato et al 2007 treated Wistar and Gunn rats with KC500 at a lower dose (10 mg/kg) once daily for 10 days, noting decrease in total and free serum T4 as well as the differential UDP-UGT response across the different strains.  Clearance of [125I]T4 from serum was higher in both species treated with KC500 and accumulation in several tissues, particularly the liver, was observed.  These data imply that reduction of serum TH from exposure to KC500 would be mediated through accumulation in the tissues and not through an increase in glucuronidation.  In addition, competitive inhibition by PCB or its metabolites with serum transport proteins (like TTR) could also decrease serum T4 by inducing a change in tissue distribution, especially the liver where more than 40% of[125I]T4 accumulated following treatment. Kato et al 2009 treated C57BL/6 and DBA/2 mice with the heptaCB metabolite 4-OH-CB187, decreasing free and total serum T4 with no observed UDP-UGT activity or effect on TSH.  A number of OH-PCBs have been identified in human serum, including 4-OH-CB107, 3-OH-CB153, 4-OH-CB146, 3’-OH-CB138 and 4-OH-CB187 (which specifically has a 5-fold higher affinity for TTR relative to T4)(Hovander et al 2002).  Levels of [125I]T4-TTR were decreased with accompanying increases in binding to TBG and albumin in both strains of mice. Finally, T4 levels increased in tissues, particularly the liver and kidney.  Decreases in total and free serum T4 mediated by 4-OH-CB187 were observed in wild-type and TTR-heterozygous mice but not in TTR-deficient mice, with heterozygous mice displaying a smaller decrease in T4 relative to TTR-deficient mice.  In both strains of mice, treatment with 4-OH-CB187 promoted clearance of [125I]T4 from serum relative to controls and serum pharmacokinetic data were estimated, along with tissue-to-serum (Kp value) concentration ratios and [125I]T4 tissue distribution levels. These data imply that 4-OH-CB187 inhibits formation of the [125I]T4-TTR complex, which may lead to a change in tissue distribution, with accumulation in the liver and kidney mainly. Kato et al 2012 treated C57BL/6 (wild type) and TTR-null mice with single ip injections of pentaCB at 112 mg/kg, noting significant decreases in total serum T4 and T4-TTR complex and measuring [125I]T4 clearance from serum and accumulation in tissues.  Treatment with pentaCB resulted in decrease of [125I]T4-TTR and increase in [125I]T4-albumin and [125I]T4-TBG complexes in wild type mice, but not in TTR-deficient mice, although liver accumulation was noted in both strains independent of UDP-UGT activity.  These data imply that penta-CB mediated increases in T4 liver concentration occurs mainly through inhibition of efflux of T4 and/or promotion on influx of T4 into hepatic cells (which is a receptor mediated process independent of TTR transport at the liver). Kato et al 2013 treated C57BL/6 and DBA/2 mice with 50 mg/kg CB118 (pentaCB) in a single ip injection for 5 days, noting decreased serum T4 in both strains and decrease in TSH for the DBA/2 mice but not C57BL/6.   CB118-mediated changes in [125I]T4 complexes with TBG, albumin and TTR were only observed in C57BL/6 mice (and not DBA/2), despite [125I]T4 accumulation in the liver of both strains. It is thought that the strain differences are dependent on differences in induction of CYP1A enzymes responsible for the hydroxylation of PCBs (creating metabolites that display far greater affinity for TTR than the natural T4 ligand). Martin and Klaassen 2010 treated male Sprague Dawley rats with Aroclors 1242 and 1254; PCBs 95, 99, 118, 126 or TCDD at 4 doses via gavage daily for 7 days, then measured serum TH via radioimmunoassay and induction of hepatic Cyp1a and Cyp2b.  This study was the first to examine all three classes of PCB congeners: TCDD-type (no chlorine substitutions in ortho position, high affinity for arylhydrocarbon receptor, induce Cyp1a, PCBs 77 and 126), PB-type (at least 2 ortho substitutions, low affinity for AhR, induce Cyp2b, PCBs 28, 95, 99, 101 and 153) or mixed type (1 ortho substitution, low affinity for AhR, induce both Cyp1a and Cyp2b, Aroclors and PCB 118).  This study showed that PB-type and mixed type PCB congeners are more effective than TCDD type in reducing serum T4, with Aroclor 1254 (mixed) and PCBs 99 (PB) and 118 (mixed) producing the greatest reduction in serum T4 (as well as T3).  Serum TSH was not affected by any compound.  Total and free serum T4 was decreased by all treatments in a dose-dependent manner; however marked reduction were noted following treatment with Aroclor 1254, PCB 99 and PCB 118. PCB 118 and 126 caused significant increase in Cyp1a activity while Aroclor 1254 and PCBs 99 and 118 significantly induced Cyp2b.  Thus, it appears TCDD type congeners induce CYP1A2 (EROD) activity and UGT-UDP activity in the liver (associated wth binding at AhR) while PB type congeners induce CYP1B2 (PROD) activity and do not induce UGT-UDPs in the liver (associated with increased tissue uptake). The PB type congeners may induce Oatp1a4 activity to increase clearance from plasma and enhance tissue uptake. Guo et al 2002 reported increase of Oatp1a4 following treatment with PCB 99 (and a decrease following treatment with PCB 126, a TCDD type congener). There are also reports of PB type congeners that accumulate in the liver with little to no increase in glucuronidation or biliary excretion and no changes in serum binding proteins, such as PCB 153, which implies a possible induction of OATP hepatic cellular transport proteins (Kato et al 2011). Martin et al 2012 treated male Wistar rats with Aroclors 1242 and 1254, PCBs 95, 99, 118 and 126 and TCDD via gavage one per day for 7 days, followed 24 hours later with injection of [125I]T4 and collection of urine, blood, bile and urine. No treatments increased urinary excretion of [125I]T4, but serum T4 was reduced in all treatments and biliary excretion increased following treatment of Aroclor 1254, PCBs 118 and 126, and TCDD as measured by induction of UDP-UGT activity in the liver. PCBs 95 and 99 (PB type congeners) did not induce UGT-UDP activity despite very large and rapid decrease of serum [125I]T4 by PCB 99.  These data imply that increased tissue uptake (perhaps through increased TH transport across cell membranes) is another mechanism by which serum T4 can be reduced.  Kato et al 2013 showed that PCB 118 (mixed type) mediated changes in tissue distribution and transport proteins in C57BL/6 mice, but not DBA/2 mice.  Kato et al 2012 showed the same with synthesized 2,2’,4,5,5’-pentaCB (PCB 101, PB type). Kato et al 2014 showed that PCB 77 (TCDD type) mediated changes in tissue distribution and transport proteins in DBA/2 mice, but not C57BL/6 mice.   Erratico et al 2012 used pooled and single-donor human liver microsomes, human recombinant cytochrome P450 (CYP) enzymes and CYP-specific antibodies to evaluate the oxidative metabolism of BDE-99.  Ten (10) hydroxylated metabolites were produced by human microsomes and identified via HPLC-MS/MS and rates of formation were determined, including several that are much more potent than the natural ligand.  All ten were found to be catalyzed solely by CYP2B6.  Previous studies had also shown formation of hydroxylated metabolites of BDE-99 by human hepatic preparations (Lupton et al 2009, 2010; Stapleton et al 2009); however, fewer OH-PBDEs and additional CYP enzymes were found in similar work done with rat microsomes (Erratico et al 2011). Feo et al 2013 incubated BDE-47 and recombinant CYPs, measuring the metabolites via GC-MS/MS, as well as specific kinetic studies with BDE-47, CYP2B6 and pooled human liver microsomes.  Six (6) OH-PBDEs were found to be catalyzed by CYP2B6 and additional metabolites were identified upon GC-MS/MS (including the novel finding of dihydroxylated metabolites) and these metabolites have been previously found in human serum (Athanasiadou et al 2008; Qui et al 2009).  The kinetic studies showed that hydroxylation can occur at low concentrations and that CYPT2B6 has high affinity for BDE-47.  CYP2C19 and CYP3A4 were also suggested to play minor roles in the formation of OH-PBDEs.

UBERON:0002107

UBERON liver

Not Specified Not Specified Not Specified

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AOPWiki 2016-11-29T18:41:28

2021-01-26T10:41:19

Thyroxine (T4) in serum, Decreased

T4 in serum, Decreased

Tissue

All iodothyronines are derived from the modification of tyrosine molecules (Taurog, 2000). There are two biologically active thyroid hormones (THs) in serum, triiodothyronine (T3) and T4, and a few less active iodothyronines, reverse T3 (rT3),  and 3,3'-Diiodothyronine (3,5-T2). T4 is the predominant TH in circulation, comprising approximately 80% of the TH excreted from the thyroid gland in mammals and is the pool from which the majority of T3 in serum is generated (Zoeller et al., 2007). As such, serum T4 changes usually precede changes in other serum THs. Decreased thyroxine (T4) in serum results from one or more MIEs upstream and is considered a key biomarker of altered TH homeostasis (DeVito et al., 1999). Serum T4 is used as a biomarker of TH status because the circulatory system serves as the major transport and delivery system for TH delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In serum, it is the unbound, or ‘free’ form of the hormone that is thought to be available for transport into tissues. Free hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. There are major species differences in the predominant binding proteins and their affinities for THs (see below). However, there is broad agreement that changes in serum concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis across vertebrates (DeVito et al., 1999; Miller et al., 2009; Zoeller et al., 2007; Carr and Patiño, 2011). Normal serum T4 reference ranges can be species and lifestage specific. In rodents, serum THs are low in the fetal circulation, increasing as the fetal thyroid gland becomes functional on gestational day 17, just a few days prior to birth. After birth serum hormones increase steadily, peaking at two weeks, and falling slightly to adult levels by postnatal day 21 (Walker et al., 1980; Harris et al., 1978; Goldey et al., 1995; Lau et al., 2003). Similarly, in humans, adult reference ranges for THs do not reflect the normal ranges for children at different developmental stages, with TH concentrations highest in infants, still increased in childhood, prior to a decline to adult levels coincident with pubertal development (Corcoran et al. 1977; Kapelari et al., 2008). In some frog species, there is an analogous peak in THs in tadpoles that starts around embryonic NF stage 56, peaks at stage 62 and the declines to lower levels by stage 56 (Sternberg et al., 2011; Leloup and Buscaglia, 1977).  Additionally, ample evidence is available from studies investigating responses to inhibitors of TH synthesis in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.

Serum T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone concentrations are clinically considered more direct indicators of T4 and T3 activities in the body, but in animal studies, total T3 and T4 are typically measured. Historically, the most widely used method in toxicology is the radioimmunoassay (RIA). The method is routinely used in rodent endocrine and toxicity studies. The ELISA method is commonly used as a human clinical test method. Analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates, through methods employing HPLC, liquid chromatography, immuno luminescence, and mass spectrometry are less common, but are becoming increasingly available (Hornung et al., 2015; DeVito et al., 1999; Baret and Fert, 1989; Spencer, 2013; Samanidou V.F et al., 2000; Rathmann D. et al., 2015 ). In fish early life stages most evidence for the ontogeny of thyroid hormone synthesis comes from measurements of whole body thyroid hormone levels using LC-MS techniques (Hornung et al., 2015) which are increasingly used to accurately quantify whole body thyroid hormone levels as a proxy for serum thyroid hormone levels (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). It is important to note that thyroid hormones concentrations can be influenced by a number of intrinsic and extrinsic factors (e.g., circadian rhythms, stress, food intake, housing, noise) (see for example, Döhler et al., 1979). Any of these measurements should be evaluated for the relationship to the actual endpoint of interest, repeatability, reproducibility, and lower limits of quantification using a fit-for-purpose approach. This is of particular significance when assessing the very low levels of TH present in fetal serum. Detection limits of the assay must be compatible with the levels in the biological sample. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by an indirect methodology, whereas analytical determination is the most direct measurement available. All these methods, particularly RIA, are repeatable and reproducible.

Taxonomic: This KE is plausibly applicable across vertebrates and the overall evidence supporting taxonomic applicability is strong. THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in zebrafish development, embryo-to-larval transition and larval-to-juvenile transition (Thienpont et al., 2011; Liu and Chan, 2002), and amphibian and lamprey metamorphoses is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990; Furlow and Neff, 2006). Their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of THs in the different species depends on the expression and function of specific proteins (e.g receptors or enzymes) under TH control and may vary across species and tissues. As such, extrapolation regarding TH action across species and developmental stages should be done with caution. With few exceptions, vertebrate species have circulating T4 (and T3) that are bound to transport proteins in blood. Clear species differences exist in serum transport proteins (Dohler et al., 1979; Yamauchi and Isihara, 2009). There are three major transport proteins in mammals; thyroid binding globulin (TBG), transthyretin (TTR), and albumin. In adult humans, the percent bound to these proteins is about 75, 15 and 10 percent, respectively (Schussler 2000).  In contrast, in adult rats the majority of THs are bound to TTR. Thyroid- binding proteins are developmentally regulated in rats. TBG is expressed in rats until approximately postnatal day (PND) 60, with peak expression occurring during weaning (Savu et al., 1989). However, low levels of TBG persist into adult ages in rats and can be experimentally induced by hypothyroidism, malnutrition, or caloric restriction (Rouaze-Romet et al., 1992). While these species differences impact TH half-life (Capen, 1997) and possibly regulatory feedback mechanisms, there is little information on quantitative dose-response relationships of binding proteins and serum hormones during development across different species. Serum THs are still regarded as the most robust measurable key event causally linked to downstream adverse outcomes. Life stage: The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, T4 levels are not expected to decrease in response to exposure to inhibitors of TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH system disruptors. Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

UBERON:0001977

UBERON serum

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Axelrad DA, Baetcke K, Dockins C, Griffiths CW, Hill RN, Murphy PA, Owens N, Simon NB, Teuschler LK. Risk assessment for benefits analysis: framework for analysis of a thyroid-disrupting chemical. J Toxicol Environ Health A. 2005 68(11-12):837-55. Baret A. and Fert V.  T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay. J Biolumin Chemilumin. 1989. 4(1):149-153 Bartalena L, Robbins J. Thyroid hormone transport proteins. Clin Lab Med. 1993 Sep;13(3):583-98. Bassett JH, Harvey CB, Williams GR. (2003). Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11. Capen CC. Mechanistic data and risk assessment of selected toxic end points of the thyroid gland. Toxicol Pathol. 1997 25(1):39-48. Carr JA, Patino R. 2011. The hypothalamus-pituitary-thyroid axis in teleosts and amphibians: Endocrine disruption and its consequences to natural populations. General and Comparative Endocrinology. 170(2):299-312. Chang J, Wang M, Gui W, Zhao Y, Yu L, Zhu G. 2012. Changes in thyroid hormone levels during zebrafish development. Zoological Science. 29(3):181-184. Cope RB, Kacew S, Dourson M. A reproductive, developmental and neurobehavioral study following oral exposure of tetrabromobisphenol A on Sprague-Dawley rats. Toxicology. 2015 329:49-59. Corcoran JM, Eastman CJ, Carter JN, Lazarus L. (1977). Circulating thyroid hormone levels in children. Arch Dis Child. 52: 716-720. Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences. 93(2):278-285. Crofton KM. Developmental disruption of thyroid hormone: correlations with hearing dysfunction in rats. Risk Anal. 2004 Dec;24(6):1665-71. DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, Meier C, Miller R, Tietge J, Tyl R. (1999). Screening methods for thyroid hormone disruptors. Environ Health Perspect. 107:407-415. Döhler KD, Wong CC, von zur Mühlen A (1979).   The rat as model for the study of drug effects on thyroid function: consideration of methodological problems.  Pharmacol Ther B. 5:305-18. Eales JG. (1997). Iodine metabolism and thyroid related functions in organisms lacking thyroid follicles: Are thyroid hormones also vitaminsProc Soc Exp Biol Med. 214:302-317. Furlow JD, Neff ES. (2006). A developmental switch induced by thyroid hormone: Xenopus laevis metamorphosis. Trends Endocrinol Metab. 17:40–47. Goldey ES, Crofton KM. Thyroxine replacement attenuates hypothyroxinemia, hearing loss, and motor deficits following developmental exposure to Aroclor 1254 in rats. Toxicol Sci. 1998 45(1):94-10 Goldey ES, Kehn LS, Lau C, Rehnberg GL, Crofton KM.  Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Tox Appl Pharmacol. 1995 135(1):77-88. Harris AR, Fang SL, Prosky J, Braverman LE, Vagenakis AG.  Decreased outer ring monodeiodination of thyroxine and reverse triiodothyronine in the fetal and neonatal rat.  Endocrinology. 1978 Dec;103(6):2216-22 Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604. Heyland A, Hodin J. (2004). Heterochronic developmental shift caused by thyroid hormone in larval sand dollars and its implications for phenotypic plasticity and the evolution of non-feeding development. Evolution. 58: 524-538. Heyland A, Moroz LL. (2005). Cross-kingdom hormonal signaling: an insight from thyroid hormone functions in marine larvae. J Exp Biol. 208:4355-4361. Hill RN, Crisp TM, Hurley PM, Rosenthal SL, Singh DV. Risk assessment of thyroid follicular cell tumors.  Environ Health Perspect. 1998 Aug;106(8):447-57. Hornung MW, Kosian P, Haselman J, Korte J, Challis K, Macherla C, Nevalainen E, Degitz S (2015) In vitro, ex vivo and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicol Sci 146:254-264. Hulbert AJ. Thyroid hormones and their effects: a new perspective. Biol Rev Camb Philos Soc. 2000 Nov;75(4):519-631. Review. Kapelari K, Kirchlechner C, Högler W, Schweitzer K, Virgolini I, Moncayo R. 2008. Pediatric reference intervals for thyroid hormone levels from birth to adulthood: a retrospective study. BMC Endocr Disord. 8: 15. Lau C, Thibodeaux JR, Hanson RG, Rogers JM, Grey BE, Stanton ME, Butenhoff JL, Stevenson LA.  Exposure to perfluorooctane sulfonate during pregnancy in rat and mouse. II: postnatal evaluation.  Toxicol Sci. 2003 Aug;74(2):382-92. Leloup, J., and M. Buscaglia. La triiodothyronine: hormone de la métamorphose des amphibiens. CR Acad Sci 284 (1977): 2261-2263. Liu J, Liu Y, Barter RA, Klaassen CD.: Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study. J Pharmacol Exp Ther 273, 977-85, 1994 Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32. Liu YW, Chan WK. 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation. 70(1):36-45. Manzon RG, Youson JH. (1997). The effects of exogenous thyroxine (T4) or triiodothyronine (T3), in the presence and absence of potassium perchlorate, on the incidence of metamorphosis and on serum T4 and T3 concentrations in larval sea lampreys (Petromyzon marinus L.). Gen Comp Endocrinol. 106:211-220.  McClain RM. Mechanistic considerations for the relevance of animal data on thyroid neoplasia to human risk assessment. Mutat Res. 1995 Dec;333(1-2):131-42 Miller MD, Crofton KM, Rice DC, Zoeller RT.  Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes. Environ Health Perspect. 2009 117(7):1033-41 Morse DC, Wehler EK, Wesseling W, Koeman JH, Brouwer A. Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). Toxicol Appl Pharmacol. 1996 Feb;136(2):269-79. Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203. NTP National Toxicology Program.: NTP toxicology and carcinogenesis studies of 3,3'-dimethylbenzidine dihydrochloride (CAS no. 612-82-8) in F344/N rats (drinking water studies). Natl Toxicol Program Tech Rep Ser 390, 1-238, 1991. O'Connor, J. C., J. C. Cook, et al. (1998). "An ongoing validation of a Tier I screening battery for detecting endocrine-active compounds (EACs)." Toxicol Sci 46(1): 45-60. O'Connor, J. C., L. G. Davis, et al. (2000). "Detection of dopaminergic modulators in a tier I screening battery for identifying endocrine-active compounds (EACs)." Reprod Toxicol 14(3): 193-205. Opitz R, Maquet E, Zoenen M, Dadhich R, Costagliola S. 2011. Tsh receptor function is required for normal thyroid differentiation in zebrafish. Molecular Endocrinology. 25(9):1579-1599. Power DM, Llewellyn L, Faustino M, Nowell MA, Bjornsson BT, Einarsdottir IE, Canario AV, Sweeney GE. 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 130(4):447-459. Rathmann D, Rijntjes E, Lietzow J, Köhrle J. (2015) Quantitative Analysis of Thyroid Hormone Metabolites in Cell Culture Samples Using LC-MS/MS. Eur Thyroid J. Sep;4(Suppl 1):51-8. Rouaze-Romet M, Savu L, Vranckx R, Bleiberg-Daniel F, Le Moullac B, Gouache P, Nunez EA. 1992. Reexpression of thyroxine-binding globulin in postweaning rats during protein or energy malnutrition. Acta Endocrinol (Copenh).127:441-448. Samanidou VF, Kourti PV. (2009) Rapid HPLC method for the simultaneous monitoring of duloxetine, venlaflaxine, fluoxetine and paroxetine in biofluids. Bioanalysis. 2009 Aug;1(5):905-17. Savu L, Vranckx R, Maya M, Gripois D, Blouquit MF, Nunez EA. 1989. Thyroxine-binding globulin and thyroxinebinding prealbumin in hypothyroid and hyperthyroid developing rats. BiochimBiophys Acta. 992:379-384. Schneider S, Kaufmann W, Strauss V, van Ravenzwaay B.    Vinclozolin: a feasibility and sensitivity study of the ILSI-HESI F1-extended one-generation rat reproduction protocol. Regul Toxicol Pharmacol. 2011 Feb;59(1):91-100. Schussler, G.C. (2000). The thyroxine-binding proteins. Thyroid 10:141–149. Spencer, CA. (2013). Assay of thyroid hormone and related substances. In De Groot, LJ et al. (Eds). Endotext. South Dartmouth, MA Sternberg RM, Thoemke KR, Korte JJ, Moen SM, Olson JM, Korte L, Tietge JE, Degitz SJ Jr. Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis. Gen Comp Endocrinol. 2011. 173(3):428-37 Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223. Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217. Taurog A. 2005. Hormone synthesis. In: Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (Braverman LE, Utiger RD, eds). Philadelphia:Lippincott, Williams and Wilkins, 47–81Walker P, Dubois JD, Dussault JH.  Free thyroid hormone concentrations during postnatal development in the rat.  Pediatr Res. 1980 Mar;14(3):247-9. Thienpont B, Tingaud-Sequeira A, Prats E, Barata C, Babin PJ, Raldúa D. Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. Environ Sci Technol. 2011 Sep 1;45(17):7525-32. Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331. Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32. Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347. Yamauchi K1, Ishihara A. Evolutionary changes to transthyretin: developmentally regulated and tissue-specific gene expression. FEBS J. 2009. 276(19):5357-66. Yaoita Y, Brown DD. (1990). A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis. Genes Dev. 4:1917-1924. Yen PM. (2001). Physiological and molecular basis of thyroid hormone action. Physiol Rev. 81:1097-1142. Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Crit Rev Toxicol. 2007 Jan-Feb;37(1-2):11-53 Zoeller, R. T., R. Bansal, et al. (2005). "Bisphenol-A, an environmental contaminant that acts as a thyroid hormone receptor antagonist in vitro, increases serum thyroxine, and alters RC3/neurogranin expression in the developing rat brain." Endocrinology 146(2): 607-612. AOPWiki 2016-11-29T18:41:23

2022-10-10T08:52:30

Thyroxine (T4) in neuronal tissue, Decreased

T4 in neuronal tissue, Decreased

Organ

Thyroid hormones (TH) are present in brain tissue of most vertebrate species, and thyroxine (T4) is converted to triiodothyronine (T3) locally in this tissue.  The amount of THs in brain is known to vary during development and to differ among brain regions (Calvo et al., 1990; Kester et al., 2004; Tu et al., 1999). In human cerebral cortex, T3 increases steadily from 13-weeks, reaching adult levels by 20 weeks post conception. This occurs despite very low and unchanging levels in fetal serum T3, when fetal serum T4 increases 3-fold over the same period. This indicates that T3 in fetal brain is locally generated from serum-derived T4 via the activity of deiodinases, primarily DIO2. DIO2 serves to convert T4 to T3. During this time in fetal development DIO3 activity, which converts T3 to the inactive reverse T3 (rT3), remains very low in cortex.  In contrast, in other brain regions including hippocampus and cerebellum, T3 remains low throughout early and mid-gestation and corresponds with high activity of DIO3 in these brain regions. In late gestation and after birth, DIO3 levels drop in hippocampus and cerebellum with a corresponding increase in T3 concentrations (Kester et al., 2004).  A similar spatial and temporal profile of deiodinase activity and corresponding brain hormone concentrations has been observed in rodent brain (Calvo et al., 1990; Tu et al., 1999). In the rat, either whole brain or cortex have been preferentially assessed due to the low levels of hormones present and the small tissue volumes make quantitification difficult. Brain T3 and T4 rise in parallel from gestational day 10 to gestational day 20 in rat. They are typically both quite low until gestational 17 with steep increases between GD18 and GD20 corresponding to the onset of fetal thyroid function (Calvo et al., 1990; Ruiz de Ono et al., 1988; Obergon et al., 1981). Just before birth, brain T3 and T4 concentrations are about one-third to one-half that of adult brain. Brain development in the early postnatal period in rat is roughly equivalent to the 3rd trimester in humans such that adult levels of T3 and T4 in brain are not reached in rodents until the 2nd-3rd postnatal week. For THs to gain access to brain tissue they need to cross the blood brain barrier (BBB) which regulates the active transport of TH into neurons. Many transporter proteins have been identified, and the monocarboxylate transporters (Mct8, Mct10) and anion-transporting polypeptide (OATP1c1) show the highest degree of affinity towards TH and are prevalent in brain (Jansen et al., 2007; Mayer et al., 2014).  Transporters express a distinct distribution pattern that varies by tissue and age (Friesema et al., 2005; Henneman et al., 2001; Visser et al., 2007; Heuer et al., 2005; Muller and Heuer, 2007). Although several transporters have been identified, current knowledge of cell specific profile of transporters is limited.  Most of the hormone transported across the blood brain barrier is in the form of T4, primarily through the cellular membrane transporters (e.g., OATP1c1 transporter) into the astrocyte (Visser and Visser, 2012; Sugiyama et al., 2003; Tohyama et al., 2004). Within the astrocyte, T4 is converted into T3 via the local activity of deiodinase 2 (DIO2) (Guadano-Ferraz et al., 1997).  A small amount of T3 may cross the blood brain barrier directly via the T3-specific transporter, MCT8 (Heuer et al., 2005). Although in mature brain T3 derives partially from the circulation and from the deiodination of T4, in the fetal brain T3 is exclusively a product of T4 deiodination (Calvo et al., 1990; Grijota-Martinez et al., 2011). In both cases, only the required amount of T3 is utilized in neurons and the excess is degraded by the neuron-specific deiodinase DIO3 (Tu et al., 1999; St. Germain et al., 2009; Hernandez et al., 2010). Both deiodinase and transporter expression in brain peak in different brain regions at different times in fetal and neonatal life (Kester et al., 2004; Bates et al., 1999; Muller and Heuer, 2014; Heuer, 2007). Collectively, these spatial and temporal patterns of transporter expression and deiodinase activity provide exquisite control of brain T3 available for nuclear receptor activation and regulated gene expression.

Radioimmunoassays (RIAs) are commonly used to detect TH in the brain (e.g., Obregon et al., 1982; Calvo et al., 1990; Morse et al., 1996; Bansal et al., 2005; Gilbert et al., 2013). The method (and minor variants) is well established in the published literature. However, it is not available in a simple 'kit' and requires technical knowledge of RIAs, thus has not been used in most routine toxicology studies. Evaluations in neuronal tissue are complicated by the difficulty of the fatty matrix, heterogeneity of regions within the brain, and low tissue concentrations and small tissue amounts especially in immature brain. Most often whole brain homogenates are assessed, obfuscating the known temporal and regional differences in brain hormone present. Two analytical techniques, LC- and HPLC-inductively coupled plasma–mass spectrometry have recently been used to measure brain concentrations of TH. These techniques have proven capable of measuring very low levels in whole-body homogenates of frog tadpoles at different developmental stages (e.g., Simon et al., 2002; Tietge et al., 2010). The assay detects I–, MIT, DIT, T4, T3, and rT3. More recently, Wang and Stapleton (2010) and Donzelli et al. (2016) used liquid chromatography-tandem mass spectrometry for the simultaneous analysis of five THs including thyroxine (T4), 3,3′,5-triidothyronine (T3), 3,3′,5′-triiodothyronine (rT3; reverse T3), 3,3′-diiodothyronine (3,3′-T2), and 3,5-diiodothyronine (3,5-T2) in serum and a variety of tissues including brain. These analytical methods require expensive equipment and technical expertise and as such are not routinely used.

THs are critical for normal brain development in most vertebrates, primarily documented empirically in mammalian species (Bernal, 2013).  However, there is compelling data that demonstrates the need for TH in brain development for many other taxa, including: birds, fish and frogs (Van Herck et al., 2013; Denver, 1998; Power et al., 2001). The most well known non-mammalian action of TH is to induce metamorphosis in amphibians and some fish species. However, there is a fundamental difference in the mechanisms by which T3 affects amphibian metamorphosis vs its role in mammalian brain development (Galton, 1983). In the rat, brain development proceeds, even if defective, despite the absence of TH. By contrast, TH administration to tadpoles induces early metamorphosis, whereas in its absence, tadpoles grow to extremely large size, but the metamorphosis program is never activated (Galton, 1983).

UBERON:0000955

UBERON brain

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Bansal R, You SH, Herzig CT, Zoeller RT (2005). Maternal thyroid hormone increases HES expression in the fetal rat brain: an effect mimicked by exposure to a mixture of polychlorinated biphenyls (PCBs). Brain Res Dev Brain Res 156:13-22. Bates JM, St Germain DL, Galton VA. Expression profiles of the three iodothyronine deiodinases, D1, D2, and D3, in the developing rat. Endocrinology. 1999 Feb;140(2):844-51. Bernal J. (2013). Thyroid Hormones in Brain Development and Function.  In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2015. www.thyroidmanager.org Calvo R, Obregón MJ, Ruiz de Oña C, Escobar del Rey F, Morreale de Escobar G. (1990). Congenital hypothyroidism, as studied in rats. Crucial role of maternal thyroxine but not of 3,5,3′-triiodothyronine in the protection of the fetal brain. J. Clin. 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Visser WE, Friesema EC, Jansen J, Visser TJ. (2007). Thyroid hormone transport by monocarboxylate transporters. Best Pract Res Clin Endocrinol Metab. 21:223–236. Wang, D. and Stapleton, HM. (2010) Analysis of thyroid hormones in serum by liquid chromatography -tandem mass spectrometry. Anal Bioanal Chem. 2010 Jul; 397(5): 1831–1839 AOPWiki 2016-11-29T18:41:23

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Hippocampal gene expression, Altered

Tissue

Thyroid hormones control genes in the developing brain by classical ligand (T3) activation of thyroid receptors which leads to DNA binding and subsequent transcription and translation (for a review of TH rols in brain development see, Bernal 2015). Gene expression profiles have been published for the developing human and rodent hippocampus (Zhang et al., 2002; Mody et al., 2001). In both humans and rodents, the hippocampus undergoes typical stages of neurodevelopment found in most brain regions, including: cell proliferation, migration, differentiation, synapse formation, and the maturation of synaptic function. In the rodent, peak windows during pre- and post-natal periods have been identified during which major cellular and physiological events occur (see Figure 1). Each window expresses distinct patterns of gene transcription and clusters of genes increase their expression corresponding to the progression of events of hippocampal ontogeny (see Mody et al., 2001).  Tables of gene clusters associated with these phases can be found in Supplementary Tables of Mody et al. (2001). <img alt="" 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cHxGu86juOsra0JIRoaGjg+foHGAJMnT87Nzc3Pzy8qKqqoqLC0tCSEiIqKwtIVuHDhAl6qxgRcNq3GDBgwgOO43bt3w0dTU1Mw9eTn56urq0OPf/Xq1fv374U1BmsuLi5++fJl4WYh1GjbeI3BG5VeavD5fGgcHo+HC8RNmzZhhsjISBgdunTpUlJSUudZ0xrTAI3XGGlpaZwZ3L17FwOjJ02aBIm0xvTv3//9+/ccx5WXl2PIv4KCQlpaGsdxaWlpaOFZsWIFfV6oDRs2bIDE0aNHQ4qBgQEurAsLC3ft2gU1LygouHTpksC+URyXp06diq2KGgMWUUhHPy0hxNzcHAL9X79+jeaXuXPnQk5aY5SVla9evQrpx48fRysQXqYXL17cuHEDDVMcx9XU1ODYeuLECcz2lRpz69YtrD+cVFFREUzmWrVqdePGDcyJPX/w4MEcx1VXV5uYmEDKnDlzsJK4Ct+yZQvHcTweD68gXhSOkqhOnTrBvSmsMQUFBTo6OpA4btw4/G1KSgoKHl4gWmNcXV1hklFRUTFo0CBIlJeXT01N5f6uMW3atDl9+jRcX2woQoi1tTV0tuTkZDzWkiVL4FiJiYmwKFFSUnr9+jVWDFctK1euhBTa9jNnzhwYJ+/cuSMrKwuJv/76K/4cZ04wz0YqKiouX75MH4jjuLVr10JmJycnHAz/cxojzLRp0yCPsMZglD1tBoVppqysLPquv0xj+vTpgyrFcVxMTAzMPeXk5J4/f15cXFxYWPjp06fIyEi4G9XU1ARiCuqkaTUGXKavXr2CwVdUVBRU4fjx43ixOY5LT08X1hg0LPTp04cunL6LAgMDIbHxGoOLqo4dOx49elQgyvzt27dKSkpQ1Xv37n369KmwsLC4uPjFixdwCtLS0gIdF2lyjRHo9IMHD4Z0W1tbSEGNUVNTox3CN2/exPnyzZs3OY47e/YsfFRRURFwHeO+ruHDh3Mc9+nTJ2zerVu3NnCVaXg83oIFC+BXuHiiNYb2QOBKUU5Ojt51izaoUaNGQQqtMQEBAfQRzczMIH3kyJH11aqkpASXj9u3b4fEr9eY27dv49WEJdedO3dg3q2hoQGO8cLCwpKSkitXrkC2rl27wpiAFh4jIyPwksbFxYHBFufydCcMDQ0V7oRSUlLQhYQ1JiIiAu53KSkp2pjMUZaDXr16QQpqjKqqKu1Rv3XrFvYfkExaY9atW4c5z58/D4kCBigPDw9Ix42N6Ojt06dPSUnJx48fCwsLy8rKcKzw8PCAnKgx4AJAcIGFusVx3JQpU/6xGwB8Ph99ot27d0cX9X9OY+Tk5FxcXPpRnD9/HvIIawxeAGtrawiI2rp1K4i5oaEhysaXaczatWvpGuLiWkxMTFdXV09Pr3379vr6+hoaGpjemJ3kzaExHMc5OztDCkzf0Dq/Z88ejuNSUlKENQancgLXns/n4yiMQfGN15hLly7RvgQ9Pb1ly5Zhsz979gzSRUREtLW1oRn19PS0tbXxV/Xt4KE1xtfX99KlS+f+x59//onztcZrjIBPG4enTp06QX9AjenYsSN9sV68eIHG6DNnznDUZbW0tBSIL0K7f8+ePTmOy8jIQEsUHSghAI/HCwoKmjVr1oABAywtLQ0MDPDcMYqU1pi9e/fib3FEVlFRoQV71qxZkBlVitYYmDsjKAOOjo6YmJeXd/DgwXHjxjk5OVlYWOjr66PRD6cjX68xx44dw/5z/fp1jrKetWrVCjpM+/btO3TogNN5RUVF6GP37t2Dc5eQkIAdUehGsrOzg0WYQCfU19cX7oTh4eFcXRpz+vRpSNHW1hbwHeIOrfbt25eXl3OUxnTo0IGegL58+RJts2AdpTWGnjFfv34dEjU0NGhfMvZ2XF7Mnj0bUmRlZeGMoInatm0L6VZWVuB3QI0R2FSHVtalS5di4uTJkyHR29tb+ErdvXt30aJFgwcPtra2NjQ0RIOblZXVf1djGohdFtYYPp+Pkbvi4uL0hnbanEVrDB3d1LDG4NISwL4lDPRLExOTxjydqZk05sCBA5BiYGAQHh4OZp+2bdvCYq5OjUG/wsKFCwXKx0bAboEaQ7s069QYjuNOnTpFm4wJId26dQOzPnpf60NXVzczM7POs6Y15tq1awLf4jDUeI0R8P2sW7cO6wAmR9QYfX192nKVlpbWrl07+sRxkWFnZydQMZQu6NsvX75ERyv6qAQoLS2ljSQC1KkxO3bswJ+jxrRr146OIsPBBUugNYa+ZTjK4Na7d29IefnypYWFRX21akKNQVWWkZEBUxJO0oWBM7W3t4dLVllZ2bVrV/gK7GA438IpxT92Qh0dHbhxhDVmx44dkNKhQweBsOD9+/fDV+rq6vAVaoyenh4dgpyeno6uCzAG0hpDh4fg1FZdXZ3eVDBx4kRIHz16NKQIbPEWQFxcHC142A3QGwRgrEFjNKampmbp0qX1He4/rTENxC4La0xiYiKYHZSVlaGrSUpKGhkZCYTV16cxuLJrjMZcvnwZ0uXk5P7444+IiIhwinv37tHbcRqgmTTm1atXMLOWkpLq1q0btAYOJXVqDA6148ePpwuvqqrCgM7NmzcLZKY1pqysDOKayN81huO4vLy8Q4cO0e5xmHA9fvwYPoqKih49elS4GYXDMZEv2B/TsMZs3LiRLgFNzz169IAZH72OoTc0PHnyBOfvMNFGr7KxsbHA1gcsdsiQIXAtMNbu7NmzdZ4plqaiohIQEHDr1q3IyEiMgmsmjRF4dAI6nCFzVVUVzjMsLS2PHTt29+7d8PBwVJ2m0pgPHz6g36h79+4wGhw6dAhS1NTUgoOD6W4TFhYWFhaGkWAcJVF9+/Z99+4drBgkJCTQ1vTXX39hJzxy5EgDnVBYY3AyRwdxABgL0KNHD6g2vY6hA8CePn2K/QdmS1+vMTNmzIAUa2vryMhI+ozCwsIiIyPRzflZGoMLJgFbGa41paWlV65cef369cjISOy3/3WNqW9vkbDGgHRLSEj89ddf8fHx9+7de/HihfD2q/DwcPihlJQUhnxw1Mk3RmOePn0KQ4OEhISAHfaz2Lp1K5Svq6tLu0+Bjx8/3r9//9GjR/QuDWGENYaj7P4Imhnr1BicqHbp0oX2PCUkJKD3GwOO0VFPu/5CQ0Nxq4SAxgC1tbW459TY2JjH471//76B+OOGaXKN6d+/P3a2iooKnP+iKQk1Rk5ODsMfOI7bvHmzQI/CmEM6rIjjuJqaGvRCg5m7oKAA57AYXADk5+eDjQUH7sOHD+O3eNGbSWNwExjHce/evQOPBflftHp6ejosv6SlpemdIuh++zKN6d+/P51eUFBAx6phLA8E2sGFaGAWAoSFhYHLRF1dfd68edAUdnZ2eK0zMzOxI2HcWp0IawzGI4iJiQl0YGwKnPKjxsjJyd25cwdz4lAmKSkJ4ctfrzFYZteuXRtun8/SGJQumCEheJnoZ8xcunQJEr8HjQEh6dOnDyFERERkwoQJwcHBDx8+DA0NjY6OFtgZm5iYiDE/gwYNyszMfPnyJW2LaIzGlJSUYOy5vb09bc8pLS0NCQmBLZb1kZGRERMTk5iYiE9mU1dXf/DgwfPnz1NSUuB0ysvL4TKLiIgsW7asgdLq1Bjaik0I0dLSwnaoU2PQX0oIWbFiRWlpKZ/Pz8zMRNeOsrIyRFJxlD9TTk7u3LlzhYWFp0+fhtA1ADSGx+Pt37+f3jWCM1ArK6va2tra2los38TEhPZglZeXh4eHQ9xknTS5xsBtlpaWlp+fTwfb4D5TOq5MXV09KCiouLj45s2bKBLW1tYwG3j9+jUavt3c3LKysvh8fmlp6cKFC7GE27dvcxzH5/PR9966devjx4+XlJR8+vTp/PnzBgYGIOqodliTtLQ0FJ5m0hhCyKxZs7Kzs9+8eYM7UURERGBwjI+PhxRpaWncs0I3xZdpTM+ePRMSEhISEiIiInbt2oWBBtBhcKGfl5eHO7Hc3d3pVUtxcXFwcDDdkSoqKnBXEJolad9bdXU1rtKMjIxoySwvL4+IiMB7RFhjCgsL0T5sbW2dkpJSW1tbUVEREBCAbvwjR47Az+m4MnV19YsXLxYXF9+6dQut+lZWVtB/vl5joqOj8TlVixcvhskKdJLs7OygoCA04XyWxvj7+0OilpZWUlJSfn4+rN4wNA63ZObn52Pif1Fj8FaE2W6deVBjbGxsIM9vv/2GgaGIuLi4srLy5MmTUQZ4PJ6joyNmkJOTQ8mBLkhrDOakI1CBkydPYiGamprDhw+fPn36wIED4S6lL4wAubm5+vr6kpKS0tLS2A8IIZKSklJSUvLy8jDzffv2LYYPYmhTneCqnJ4GZmZmos+NEDJ58mT8KiUlBRqK1piamho6AL9Tp06Ojo60T4sONKLDSQkhOJ6i+xo0prS0VEFBoVWrVkOGDPHz85s2bRr6LTBYBU2XhBBlZeWhQ4fOmDFj6NChBgYGIiIiAiGSNPHx8bhsEtaYPXv2wFeysrKftT+mTZs29Fl37NiR3upM/o66ujoG9RIqYJfjuDVr1mC6iopK3759cTAihAwbNgyXrc+fP0dTCSGkffv2OIBCM/r4+MBHNTW1VatWLVmyBM1H5O8ag4NyYzQG56R1+vwBJSUlvLiEkJ9++gmqXVJSglslLC0tf/nll4kTJ9JN8Vkag3sYRUREpKSkpKSkcIAGDA0NBZ6tvm3bNvxWX19/1KhR06ZN+/nnn2ETMR4dEIhTFRcXp7cnc3/vhEpKSgKdEO1Cde6PobfvyMvL29vb4+4uQkjv3r3RTyOwP4YI9R/0gVVXV6MuNkZj0AhB3y90zLqpqemECRMmTZrUt29fuAdx+z12A4w0A3A0oOPKMGCSEKKoqNi2bdtBgwZx1I54GRmZ+fPnr1mzBtfr5O8a85977nKHDh3q0xg8K1zrZGVl4RZI+sYA7O3t8TwfPHhAT7oJIRoaGrt27YItct26dUONQRcCvaEdWbFiBd1FaOg9OgKkpKTU+RMEnm+Rl5eHcTIDBw5soLkwDv2nn36i0+nH2dJ70JKTkzGdfoLFixcv6I3BSNu2bXfu3EmXzOPxcOqEjBs3Di/cb7/9xnFcTU1Nz549hQvs168f7TPfvn07qqkADSzg4uLicCQS9mTs3LkTC8HnJkVGRmIijhGoMX369MEwYjxxevsFaoympqbwHv6xY8fS1s6SkpI6HycsIiIyY8YMgU0/e/bsoWUGUFVVhaHwxYsX9NN9AHd3d7DWwsZbjuP4fD7KWJ0TAklJSfqBaWhbx96FGiMiIuLs7CwQGq6rq0vb/c6fPy9QZ2VlZfSoo+suKSkJM9S3Kh03bpxwQwEaGhrz58+nVyoAj8fDwVEYgf6Au7MBtK7T7Nixo75OiIMszlEIIbh6q62tpffb0gwfPpyOXEWN0dDQEO4/Y8aMwf5TXV2NT9zAHYEc5QaWlZWlQwRxKUzvAs7Ly6vzcQ/k7yqL3QB21yH29vaQjksTjuOKi4vpTaPkf8+sysnJEb7T+/fvD+varl274tiLNvxv/P6Y8PBwb29vT0/PXbt2CTtUgKioqBEjRmCe8vJyEF4xMbHNmze/efMmKSnp6dOnS5YswbUC/Tj3+Pj40aNH9+rVq0+fPrNmzYLdTKtXr/b09Ny+fTse9PDhw56enp6envXF/ERERCxdutTDw6NPnz62trbOzs4zZsy4dOkSLk6Fqays3LBhw7x58xYK4evru3z5cuiXfD7/1KlTnp6eI0aMaMBkxHHc48ePR44c6enpiRu2gbi4uLFjx3p6eq5atYoe/kpLS/38/Dw9PWfNmiVw9xYXF+/fv3/QoEG9evWys7Pr27fv3LlzBSZ9AJ/PDwwMdHJy6tmzp6urKxgEnj175unpOWrUKNxFnJGRsXr1aldXV1tb2z59+gwZMuTo0aPCTwB89uyZv7//8OHDHRwcbG1tHR0dJ0+efPbs2QbeqVNUVLRixQpPT88pU6ZAuBFNUlLSxIkTPX1tvYkAACAASURBVD09V6xYgYf78OHDtGnTPD09fX19sWTUmM2bN2dkZEydOrV37969evWaOHEibeXjKI3p2rVrbm7uvn374PRdXFwOHDggHJzC4/H+/PPPUaNG9enTx87Ork+fPmPGjAkODq7zdKKioqZOnWpvb29ra+vk5LRq1Srok0BmZqavry8cbsCAAX/++SePx1u8eLGnpycdALZ9+3ZPT89hw4bRz5d78+bNlClTPD09lyxZQrfn9evXoW9jmDK9jrlw4UJMTIy3tzfU3NfXV3ijUkRExOjRo+3t7e3s7Hx8fOLj49+/fw9dEXvsx48f586d6+npOW3atPqeDPTkyZP58+cvWLAA7oIFCxYsWbJk586dISEhdISuMLdu3VqwYIG7u3vv3r1tbW379es3b968mzdv0g5FjuNqamoCAgLgZH18fCAQWZh/7ITZ2dnTp0/39PScN2+eQIROSEjIpEmToCl69eo1bNgw4XkPakyXLl1yc3P3799P9x9a9vh8/pYtW+Dep+++V69eTZo0SaBXcxwXFBQEZyfw4L7q6upz587NmDHDzc3Nzs7Ozs5uwIABK1eu/Ouvv3CIu3btmkA3AI4fP+7p6enl5UW7jjiOy8nJmT9/PtzRbm5u+G1RUdGaNWtcXV179uzp7Oy8b9++mpoa6JBbtmzBw505cwYOJxwL+mU077uWaUJDQ/H+p9OLi4vRokI//w6ora2tT8O+gAYefdri4PF4jTwd4VCF+gpsZFM3ssCmQjh2GRxFwjlRY/T09HDcaUxt+Xx+U51+sz7fntYYtPs1pht8855fW1vbtHX44k7I4/HqC4VFjWnfvj1K1L/W2xt/RzeS+mr+L7+C4d/TGDQIwKMXcnJyioqK0tLS5syZA8ZoZWXlFvcqM8a/QH37Y4Spb3/Md0MD+2MYX099+2MYX8O/pzF5eXm4OQNmCt26daOfOi5gR2IwANy3KxA6KMzvv/8OOXV0dL7LMaKsrAwdP+BRYzQhOEfR1tZm892m4t/TGI7jkpOTR40aJeDtl5WVHThwYANP6WD84OA+BoE9mMJcvHgRcnbu3LkxD6NrcZSXl2OcdH0bQhlfTFBQELRtx44dBd5zyPhi/lWNAd68eXPt2rVjx44dOXLkzJkzP+YrURmNJzEx8ejRo6dPnxZ4bZownz59CgoKOnToUH0xuN8BT548OXTo0MWLFxuItmB8Gdh/6OAjxlfyDTSGwWAwGD8ITGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYxn+CrKysgICAVatWXbhwAZ9NGRQU5O/vHxAQ8ObNmyY5Sk5Ozt69e3fu3AlvXK6P3NzcHTt2+Pv7//777//yA0AZLYKcnJy3b99+61q0DL4TjYmNjT158uQvv/yycePGM2fOCD9J/nsiPz//+vXr+/btW7169f79+2/fvi38KP4WB75bqV27dvAYj4qKCnhdECHk6NGjTXKUEydO4APNBN4QQxMcHAzZpKWlG352fVORnJx84cKF7du3r1mzJjAw8Ny5cw2/p/W/QFFRUWho6O3bt+/8nZCQkPv37zf89vEWzf79+zU0NFRVVceNG/cdn2ZT0eI1Jjs7e/z48fhCe0BZWdnPz49+gnd5eXl6ejq+iriFUl5evn79enzPG2JkZHTu3LnmPvr79+/T0tIaeO/O14Aag4+8LS8vx2cMN5XG4CsR6bdnCoMao6Ki0lRLqPqIjIwcMmSIkpKSwDWVk5OztbWFdzn/N6HfSy2AmJhYne80+j7A97tLSUnhK1wZ9dGyNaakpARfBieAhoZGYWEhZKuqqho4cKCKioquri79usmWRVFR0YABA+q7q9u2bSv8LsIm5M8//9TV1VVRUaFfQtyE4GPV8bH85eXl+LbgpnrG8LFjx6DAzp07C7zDiubGjRuQTVVVtVlNIhs2bMA32NeJoqJic4vcF1PfOxyBJ0+efOsKNhczZ86EczQ2NmaPZ/5HWrbGbNq0CS62uLj4smXLwsLCQkNDd+zYYWxs3LVrVxxEeDyehoYG5Gyhb92ora3F17XC2DdhwoRt27atXbvWxcUFXsATHx/ffBXAVyN369atOcr/ATXm119/pQdlDw+P48ePX7x48bfffvPz88M34+I7g/9ruLu7Qw2NjIx27ty5bdu2rVu3bt26NSAgYM+ePd/lc6+Bjx8/BgcHX79+nS1iGkPL1hh857azszOdXlJS8vTpU3AdV1dXZ2Zm4ls39u7dW1BQIPzg7vz8/LS0tOjo6MzMzAZuDx6Pl52dnZGRkZWVVVBQUFBQkJ+fjwsmpLKyMiMjIzo6Oi0trYGxLCsrKy4u7sWLF/W94xa5cuUKDkaGhoZxcXH0t0FBQT4+PgJTKh6P9+bNm+jo6Li4uHfv3tVZ7MePH+nKZ2RkxMfHCz/euLi4GOXcxMTk/fv3eXl5+Lrc6urqnJwcfB/4ixcv4uPjBezUfD7/7du3MTExsbGxdY7aX6Axb9++jY2NffnyZQOviqmsrExLS0tMTIRrii+YaaTGqKioQOEfPnyIjY1NTU0VWMOVlpbm5ubW6dr5+PFjbm6uwEuFkbi4OElJSVSyM2fOCOe5evXq9OnT6RfOA9iY9Z17WVlZbm4uvlQxJycnPj7+zZs3DbxmsaKiAntsI5/ojBpDv6NegE+fPuXl5QlXsqioKCcnp7S0FD7W1NTk5OSgGbaysjI5Ofn58+d1Ls2LiorwBXTFxcUxMTHCNvDs7OyEhAR4t3R9dSssLExNTYUOWefS/MOHD4mJiYmJicKXgKv/1aKlpaUZGRl//fVXcnJynfXn8/l5eXnYZ3g8XnJycmJi4nf5Vj2upWuMra0t9PKePXvWl8fX11dTU1NcXBxytmvXTk9Pr1OnTmA04/P5V65cGT16tI6OjpiYGCFESkrKwMBg2bJlwjfGyZMn+/bt265dO1FRUQUFhQ4dOujp6bVv375jx470a7oPHTrUs2dPsIGIiYl169Ztx44dAkWFhoa6ubnJy8tDrTQ0NGbOnNnAqDd48GDIKSYmFhoa2nCz8Hi8AwcO/PTTTzIyMiIiIqKiovLy8u7u7gLBVDdu3OjcuXOHDh3CwsLS0tJGjhzZunVrMTExNTW1UaNGoa87NjbW0NBQUVERKtCqVav27dtra2uPGDECVHzMmDEaGhre3t4FBQVz5sxp1aqVmJjYpk2b8EDHjh1zdnZu3bo1VKZ169YDBgwICgqiK/NZGnP58mVXV9fWrVsTQkRERHR1dRcvXizceleuXOnZs6eEhISYmJiBgcGBAwcOHz4Ma75GaoyOjs6zZ88WLFgAF11cXNzBweHZs2eQLS0tzdLSUlNT09jYOCYmhi4hPDy8c+fOmpqa2EoCTJ48GScNn+VL+/PPP52dnWVlZQkhoqKi7du3X7ZsGS1yGRkZ3bt319LSCgwMLC4uXr58uYaGhpiYmLy8fN++ff/66y/hMvfv329jY4M91sjIaNeuXf9YE9QYd3f3OjN8/Pixb9++2traurq669atw/SbN2927dpVQ0Nj4sSJkDJz5kx1dXUo5/LlyzY2NuLi4qKiourq6qtXr6YbMDw83MDAQE9P79atW48fPzY0NBQVFdXW1saJ0bNnz0aOHKmiogJ1U1JSGj58+MuXL+mKFRYWLl++vGPHjqKiooQQGRkZe3v78PBwzBAdHT18+HBlZWUoRFlZ2dvbGydqp0+f7ty5s5aW1pw5c+i65ebmrlq1ytzcXEJCghAiLi4O5ygQvrF582YdHR1LS8usrKywsLC+ffuKi4uLiYnp6+uvWLGivklJy6Vla8zo0aPxRp08ebJwCFBtba2JiQmpC3jh1YULF+r8lhAyePBgejK+Zs2a+nISQhYvXgyHQ1utAN7e3th7wsPDYXwUICwsrM7TzMrKUlNTgzy9e/du+LXz5eXlI0aMqLMOIiIiBw8exJxo/ho6dKi+vr5A5t69e8M0E0d/AVq1agUvBgfPvJaWloeHB32+HMfV1NRMmjSpvkYLCAjAyjReY3bs2FFnadbW1vS08dy5czBpoNHW1oZhpZEaIy8vL9wympqaT58+5TguOzsbX7g3ffp0uoSpU6diMwqXX1hY2KFDB8jg7Ozc8AWl2bJlS53nbmdnh1OiJ0+eQKKtra2wt1JZWfnFixdYII/HmzZtWp1l+vj4NPzid9SYYcOG1ZmBx+ONGTMG8sjIyIAtt6SkxNzcHBK3bdsGOS0tLQkhampqs2bNEq7J7NmzsUy0dg4ePFhXVxfzgPDfu3cP1UXgkj148ABKKCsrGzRokHCeoUOHQoYXL15oaWkJZ8BOuGLFCkixsrLCa5eZmYnnJYCKigr9Qhq4PUVFRUeOHCktLS2Qee7cuY3rCy2Glq0x+N5DQFtb29/fX8BIGhAQ8PPPP8O8jxBiYWExdOhQd3f3yMhIjuO2b99OCOnatauvr+/BgwePHj1KD9D4ds6YmBgYsFRVVY8ePRoVFbVgwQJI0dDQmD59enR0NMdxv/32G/xQWVl5x44dUVFRe/fuRXk4ffo0lObm5gYp48ePj4yMvHfv3rp166ytret7s1ZYWBjMvgkhS5cubbhNVq9ejfV3dXX19/efPXu2qqoqVgx9yPv376dbT1dX18PDAx1XhJDff/+d47iXL1+OGDHCyMgIEuXl5d3c3AYNGrR48WI+n8/n80HFsYaamppt2rSBcSEwMBBL69u3r5+fn6+vr7a2NqTIycnhBLORGvPo0SOYJLZq1WrZsmWRkZGnT5/u1q2bQOOUl5djhSUkJNzd3XFABBqpMYCqqurIkSNhHARw5o5jor6+PppYS0pKUJloUUeePXuG+rdv3z5M//Tp07t377IoaPmJiIgAgZSSkvLz84uMjDx58mSXLl2gnFWrVkG26OhoyAbIysq6u7vTM61Zs2ZhmQcPHoREFRWV3bt3R0VF7d69G4fpCxcuNNDTsElNTU33Uhw4cADNPpmZmbgaGDVqFEeF9llaWmLMfe/eveku5ODg4OnpieOviIgIjtFo7cSLq6Oj06ZNm4yMjKKiIlTuESNGhISEBAcHY2CCvb09SObly5fxkl24cOHx48cnTpwYMGDA/PnzBa6pm5vb3bt3IyMjt27damdnd/HiRciwdu1aLBMN8nggCQmJ0aNHr169etSoURjQYW9vjxPWcePG0SdrYWExaNAgGRkZ+CgpKZmQkNBAs7c4WrbGcBw3b9488ndUVFTWrl1LG1irqqowChbGTSQ1NfXUqVMCDpUePXpA5i1btkDK7t27IWXt2rWYDSx1qqqqMN+vrKyEYUhUVPTw4cOY7fDhw/BbcBrxeDx0Dj18+BCzlZeX1xevha+AJYTs3r27gdbIycnBAWLy5MmYfv/+fVRZf39/SKQ1xsHB4fXr1xzHJSQkoMyMHz8eS9izZw8ODXRQOI/Ho8evGTNmfPjw4fXr1/n5+cXFxRhmPWLECPxVTEwMTv/nzJkDiY3UmPHjxwv8kOO4x48fgy1UV1cXpvPnz5+HbCIiIjiI//HHHzjeNV5jLC0tweteWlqKFktZWVlYDYSFhaFaXLlyBX5+69YtGEGUlJQyMzOFyw8JCcHyr127BokfP37s06ePmpqaOoWbmxvUk8/njxw5En4Ci2a8slCBTp06gZeR1hhtbe2IiAgo3MHBARJNTExgvKuoqDAzM4Mee+LECSxz3759kLN///71NRFHaYwwOBxz1IgsIyNz48aN7t27w3Whw7JBYwghEhISW7duBVdHUFAQygwuZWiNUVNTu3nzZnZ2NkwrDx06BOl2dnZYcmlpKei9iIgITCvXr18P2YYPH06fDjqHsDL0WFFVVYV2CGGNuXfvHtZq7969+Cs0FdAXGjQGmD59OloLUJDoacd3QIvXGI7jtm3bpqOjI9DLx44di0N2ZWUlTnBOnTr1jwUOHz5c4GZetWoVpOzfvx+z9evXjxAiLi4Ow010dDR4cRUVFVNTU6uqqiorK2tqah48eAD3vK6uLsykcEbco0eP69ev/+M2rtOnT+N5HTlypIGcuLFDTk5OIB4JDVmDBg2CFNQYHR0dOihgwoQJkO7m5oaJtMbQWkhrTL9+/eh594MHD9DFJfDyWpQKBwcHSGmMxnz8+BFFKzg4mMfjVVZWVlVV5eTkwEJNVFQU5oALFiyAbObm5nSVtm7dCumNt5XRjpaIiAh07MEcv6amxsLCAlLGjRsH2XDjyMCBA+ss/88//8QLisH0GRkZGAWAiIuLw1IvLy8PDTihoaE1NTWVlZXV1dVZWVmwt0ZCQiIlJYWjNEZcXPzGjRt4UFyyaGlpwZLr8ePHMK4pKSm9evUKe2xERARopL6+fgOdEzVGVFRUSkpKUlJSUlKyVatWUlJSaADgOK6wsBBneGgidnJyoovCYX3KlCl0OtxidD9BjZGWlhZw6eEMYPXq1RzHVVZWVlZW8vl8FxcXSIddVgcOHMDKrF+/Xjg2bMiQIdhJTp8+LdxPaI0RSDEyMqINjGVlZdiHcXqKGuPk5ISdk8fjgd4TQmjf1XfA96AxHMe9e/du48aNuD4AcDguKytDjaHna0B1dfXly5cXLVrk7e3t5ORkZWWF/m3UGDTK2djYJCYm5uXlnThxAlYGurq6MBO5dOkS5GnVqpWxsbGZmZmpqamFhUXnzp3xrkhPT+eoXg5YWFgEBgbiNEoY2iRIL6SEwRmogYGBQLAT+pPMzMxgSYEaY2pqSt8YdQ6Ru3btwtrS4w6tMQcOHKCPiNsqtbW1BQLn0IbWoUMHKK0xGpOamooz9C5dupibm5uampqZmRkZGYEBjRACcQ04S5g6dSp93M+NK1NTU6Oj7DIzM9HyiZPNjRs3Qoq6ujosiNFMV9/W0bt37+IFRYd/VVXVihUrhg4d6u3tbWNjA9+2adMmOTmZ47iEhAT8iYGBAZy7ubm5oaEhyt6dO3c4SmOkpKRo1wu6HrW1tUFjMKW+HisrK5uRkVFfK6HGODg4xMTEREdHR0dHP3v2LCYmRsBxfeLECdo3JicnJzDnQI355Zdf6PQlS5ZAeteuXWFxg1ewU6dO9OMtamtr0WSqra1tYWFhamoKTYSRNStXruQ47v3797SPTU1NbfLkybRb/vr164TC0NBw3bp1dASQsMaMHTsWUsANSYOG8UmTJkEKagyGPADoPFu/fn19bd4S+U40BsjJyZk5cyYaOvv16wfpDWhMQUFBA0t+1Bgej4dx0rKysjgvI5TfEr2R9dGtWzewZtTW1m7ZskVga7e9vb1w0DBw9+5dPKkJEyY00AK0kAhY3nA3RseOHeEr1BhjY2P6dp0/fz6kf67G7Nmzhz4iGgo6duwoIHh4aHV1dfiqMRqD3uz6kJOTg3UMXqwFCxbQx/3K/TG5ubk4PAUGBkJiUlISTs+vXr368uVLuFjKysr17a2Jj4+XkpKCn8DAJwC2BmpMZGRkw+euoKCA62nUGHqzPZaJGoPGpfowMjJqoJXwxvH09KwvDxAbG0tvNVVRURFoGdQYgSkU9mc9PT3otLTG0HsMaB9YnYiLi+PeuJiYGLQcYjufP38eSztx4gQdUEAIMTQ0RMEW1pj+/ftDioBscJT9AE1zqDFjxozBbHw+v0+fPpDONOa/jp2dHd4hMGEvKyvD/nfy5Ek6M9qFNDU1N23a9Mcff1y7dg37H2pMZmYmzJLQNUcI0dLS2rJlC6520QegoKBw8uTJq1evXqG4dOlSWloafejU1NT169fjnJEQsnDhwjrPKDs7W11dHfJ07NixAfMFzqkF7kCO45YuXQpfOTo6QspnaQwKhqWlJT1LpTVGwFeEiyoNDQ2BHQa428bKygquUWM0JjExEdvq119/FWjhoKAgiLzgOG7o0KF13vO4tGqkxqirq9MrsMzMTHR30TtacIiZMmUKXgIPD4/6yi8rK8O1TufOnYX3o5w6dQq+RY2Jjo7Gc9+2bZvwueNMvPEag+O1oqLi77//Tpd5+fLly5cvw7K7Pv4xdhkRDnScN28enQE15tdff6XTp0+fDuk2NjZwo9EaQ+9yq6mpwVtp4sSJ165dE2if8PBwerFeU1MTFBTk7u6OC6x27drRk7z379/v3LmTDvTACyqsMQMHDoQUYbnFB88sWbIEUpjGtBhqamrqdKhigIeFhQX0y/Lycux/tDe+qKgI/NsiIiL03hEMt0WN8fX1hWwhISEPHz68ePFiVFSUwCCFfhcBG0XDfPz40dvbGw5nZ2dX38YuT09P7Ot1znwfPHhQW1uLY6iUlNTjx4/pDNiD0Xz0WRqzd+9eSDQyMqpvHSOgMWhzkJCQoA30HGVAGDFiBKQ0RmNyc3NxiG94TwlGkHft2pUeWTBCpJEaIyMjc/fuXUzHSqIDGTh58iSkKygo4GxG2CpLs2zZMrygY8eOFXiqKVpHUWOysrLQhHv58uUGSm68xmC8ooyMjMAEqDGgxjSgphzH3b59G2MycSyWlpam9xGjxtBjdHV1NSoxbvOsT2M4juvbty98Rcc6/yNBQUG4DBXopRzHVVZWoulYT08P2k1YY2bPng0p7du3p5fs79+/x5BOHHmYxrQYkpOTtbS0vL294cHDPB6voqJi//79GIuCwYi1tbX4ZA5vb+/q6uqCgoLy8vKcnBwFBQVCiKioKIbPJyUloSChxoDbUEREZObMmZGRkYmJic+ePUtNTaUfEPnx40ccEwcNGkTv2uXxeFFRUTBYVFZWnjp1ijZz44aPvn370iFbNHfv3qUDUseOHXv79u309PSUlJRz584NGjSodevWGRkZdFyZm5sb7Pzn8/kBAQFobTt79iyU+Vkag+olJycXERFRXV0NE3x6B5KAxuTl5aHBwcHBAXdcHzhwAF0IGEPRGI3h8Xjo1zU0NKSHRT6fHxcXh8/IwjUlIWTZsmXl5eWfPn3y8/PDxMbHlbVv3z44OLimpiYuLg4Dhbt160b/PDc3V2BHhaqqap2bw5EPHz7Qzza1tbW9cOFCSkpKWVlZamrq3LlzIR01pqamxtXVFRLNzc0hCBDPPTY2FneGNl5jCgoKUBGHDh1KR1fW1NQ8fPgQggjqAzXG2dk5MzMz/X+kpaWhKayqqqpXr16Qzd/f/8OHDxhSSO+qQY0hhEyePDknJ6esrGzx4sWYiP2kAY3ZsGEDfCUvLy+gFm/evLl37x7cXPfv36dDIYqLi9FIADF458+fpx/LhLHOnTt3BqepsMbcunULq7p8+XKY1hQXF+MGPklJSQzTZxrTYoiPj4dxU0REREtLy8TEhDbIKisr0yt9jPskhHTp0kVdXf3YsWMcx1lbW0OimZnZoUOH/P39cd5BKI05dOgQGtAJIWJiYrBfXUdHx9fXF0dPDHEmhBgYGEybNm3FihU+Pj5mZmatWrVas2YNx3F5eXmtWrVSVlaeMmVKYGDgokWL8IgQD1Mf6P8EREREFBUVQSMBWDxhaCYhREdHx93dHUOx4a5AOfksjaF9IUpKSh06dLCwsIBVV30aI9AgGhoagwYNwkczEEIsLS1xXGtk7DLtmlJXVx8zZszKlSsnTZrUs2dPWVnZIUOGQLaSkhLaCNmxY0eMB/msPZjY1F26dEHXMaGcMciUKVPon4wcObKBSwlEREQI+OQUFBS0tLToa9qqVSscm+haaWpqjhs3buXKlRMnTrSxsZGRkcEVYeM1hvvf/jCgW7du06dPX758uY+Pj6mpqYSEBOxTrg/cySghIaGkpKT4PxQUFNq1awfxeMePH4c8KioqILoYokk/sYLWGLiy9L3cvn17nLE1oDFZWVm49UpKSsrd3X358uWzZ892cnJSUlLS0dGBTg7bY52dnTds2BAQEICrH21tbTBa6urqtm7d2sfHZ9u2bf7+/ujKRUkQjl3m8/m4RCOEmJiYDB06FGckhBBfX1+sJ9OYFsOnT58cHR1JXRgaGgo8cCUsLEzgAbcwTFy7dk14qy16jHHP7cePHw0MDLD7CueHp3XV1tbiBm9hdu7cyXFcaWkp9loaJyenhh9YxOfz/fz8cJuLAJ6enjDJordxCODh4UFPrtGNr6enR2sMGprQcwOn5uXlRZfWtm1b0BgcDrZv3y5Q5+rqarQECtC/f396Mo5jBz4irLy8HCeY9E7Gbdu2Ccf4AnRARFBQkMCVkpCQWLRoEcRraGhoNPBUuqtXr2KrYuQr4uLiIqxP4eHhuDgjf/fWNMDTp0+dnZ3rPBfAwcGBXl788ssvGEEnwIwZM7BMTKQDr7GFFRQUcHTm8XgTJ06s7+h0pL4w6PSqk3v37hUVFeGqGg28tHPRwsICLAGoMdbW1gK6KysrS++kwagNjOKjuXXrFgb+CWBoaAh+RPQF0sjJyaEFUiAcADAxMcEoZ9zn3717d3THvnjxwtDQUPiHEhISixcvpl2YGPRIP+eNz+fDziFC7WD7PmjBGsNxXFFR0ZEjR4YOHWpubm5sbGxtbT1w4MADBw7UOXzcuHGjf//+ENTo6emJq5z79+97enpaWloaGxs7OTkFBwfn5eV5eXk5OjpC566pqcFnHi9cuDApKenRo0d3796dM2cODiuwygYuX748ZcqUvn37mpmZGRsb9+jRY9SoUSdPnkTXbmxs7IwZM3r06GFsbGxubu7o6Lhnz55GPogwNjbW39/fyckJCre1tZ08efK1a9cE9pyeOnVq8ODBZmZmJiYmpqamP//88/HjxwUMcY8ePRowYICjo6O/vz/91bVr1xwdHR0dHendZNDaixYt6tWrl5GRUY8ePTCKbPXq1Y6Ojv369RMISAVqa2vPnz/v4eFhbm4OlXFxcTlw4IDAc0ri4uIGDRrk6Oi4cOFCOJfa2tply5Y5Ojr+/PPP6MwHHj58OH/+fFdXVwsLC2NjY0tLy6FDh+7evVsg7r03NAAAIABJREFUMO/evXtDhw41NTU1MTEZPHgweFbWrl3r6Oi4aNGiBh4QmZKSMnDgQEdHxzt37pSUlKxevdrKysrIyMja2nrdunV1PgGTz+ejk0lVVfUfH3JKt8+NGzdmzpzZo0cPExMTExMTMzMze3v7WbNm/fHHH8IPb71///68efP69esH5969e3cPD4+9e/fiEbOzs0eNGuXo6Dh69Gg64vbly5fQwgsWLBCIOQwKCpo8efJPP/1kamoKPdbHx+fUqVMNvMaN47g7d+6MHz9+4sSJk/7OhAkTpk2blp+fn5GR4eTk5OjoOH78ePpEzp8/7+zs7OjoOGzYMFBr1JiAgID4+HgvLy+IPPby8rp//z590NjYWLg0S5YsqXPP8qtXr9atWzd48GC4v8zMzFxcXNauXYsu0qKioh07djg7O8PJ2tnZTZ8+nXYOpaenL1682NbW1tjY2NTU1MHBYdOmTfRjikJCQlxcXBwdHXGPNpCfn79t27affvrJxMQEuuXIkSOFny546tQpuL8EPHbbtm2D++j27dsNNHuLo2VrDFJVVVVaWtqY5z5VVVXV+Zat6urq+naoPH78GG4APT09gZ+0a9cOvrp69arwDysqKhrY9cJxXGlp6Re/R+8fC4c8ZWVlTf5WsYaHnvqorKxs8srAVWv4fTbl5eVf/57Q2trakpKSBjpYdXU1DpS4GfOz4PP5ZWVlZWVl+ATrhoE+37Tv8mlMp2oOsOlw+2F5eflXdhU+n19aWtpAY5aXl//j7flldSgrKystLW34aW8/Dt+JxjQrYWFhcAO0adMmKCiouLi4srIyJydn3bp1EDAjLS39n32RFOPf4d69e+goavgxXwxh6tsfw/gOYBrzzxQUFNAOZCMjIxsbG3zVPKG2YTJ+WDCCSFVVVdhPwGgYjARpOOyF0RJhGtMo4uPjBw8eTG/AJISIiora2dlhKDDjh+X9+/foqRZ4yD+jMWBI1YYNG751XRhNDNOYz+Dly5dnzpzZvn371q1b9+7dGxUVxUyuDI7jysvLf//9982bN587d66BkGhGfTx+/Hjr1q379u1rvjdbM74VTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXX6gxOZ+qN914t+TP14svpLG/7+Bv2cV0/8uvlvzx7WvC/tgf+/uMvz/SVl3/cCepsGmFoQn5Qo2JSi+a9ntaVHpxVHoR+/sO/v54ljv2aNJDdkHZH/traX877n5Yfy2jaYWhCflCjXmYXrTsYlrTVoXxDckoqJh7NuVb14LBYHw21+PyN17/7jQmKr1oyR9MY74fXmaXzTmTUsv/1vVgMBifyaWYPKYxjP86TGMYjBYK0xhGC4BpDIPRQmEaw2gBMI1hMFooTGMYLQCmMQxGC4VpDKMFwDSGwWihMI1pkbx79y4mJqa6uvpbV+Rf4ofVmLS0tMTERD7/xztzxvfCj6gxfD4/Pj7+5s2b169fj4iISE1NbXGD9eTJkwkhaWnfs47S/KPGfPjw4c6dO8HBwSEhIQkJCfn5+f9i7ZoROzs7NTW1kpKSb10RBuML+RE15s2bN4qKioTCxsbm1q1bX1rJJiA5OTkhIaHx09Xx48cTQpKTk5u1Vv8d/lFjvL296Quqpqb2yy+/fP30/9WrV9HR0bW1tV9ZDlJQUPD48ePGa0bPnj1VVFQ+fvzYVBVgMP5lfkSNSUpKIoQ4OTmFhITcuHFj/fr1kpKSMjIyDx8+/NJ6fi29e/c2MDAoLy9vZP6JEycSQlJSfpSt7/+oMX369JGTk7tw4cLt27d/++03S0tLQoifn99XHtfLy0tRUbGsrOwry0GOHDkiLi4eERHRyPx2dnbq6upFRUVNVQEG41/mR9SYly9fEkKmTp2KKSdPniSEjBs3Tjiz8LjP4/F4PJ5wTpw119bW1jfz5fF4NTU1wvPr7t276+jolJaWCv+kpqZG+HBMYwSwt7fX0tKqqamBj7m5uZqamq1btxZYAdR37QDhph48eLC0tHSd1wULbHxpHMft37+fEBIWFtbI/ExjGC2dH1djJkyYgCmZmZkSEhJ2dnbw0c/Pb+TIkcnJyfb29goKCmvWrIH0pKSk4cOH6+joaGtrDxky5MmTJ1jCnj17XFxc3r17t2TJEl1dXS0trblz5xYUFGCGhw8fDhs2TENDQ0NDQ19ff9WqVTByhYWFde/eXUZGRlJS0sLCwszM7MyZMxzHlZaW7t6929LSUkNDQ01NzcHB4ebNm1ga0xgBHBwcNDQ0Cgv///GuQ4cOJYS8fPkSPj579mzgwIGqqqqqqqo2NjZHjx6lf37p0qUePXpoaWnBhauqqkpNTbWxsVFQUBATE4Prsn37do7jMjIyzMzMQkJCrl+/bmNjo6qq6uLicv/+fSwqOzt72bJlXbp00dDQUFdXHzFiRGJiIsdxPB7Py8tLS0uLEGJgYGBhYTF27FiO4/Lz81etWtWtWze40B4eHrGxsVga0xhGS4dpDMdxXHx8vIiIyIgRI+DjsGHD2rZt2759+y5dugwZMiQgIIDjuNjY2LZt2+rp6W3ZsmXHjh2dO3eWk5N7+vQp/GThwoUSEhJdunRp37790qVL+/fvTwjx8fGBBU1hYWGXLl1MTEz8/f13797t6upKCFm2bBkUO3bsWCUlJTk5uVGjRo0ePTo0NJTjuJMnT8rIyHh7e+/YsWPNmjVKSkoyMjLPnz+HwzGNEUBYY3r27CkhIQHO/5CQEBkZGXV19YULFy5atMjExIQQsnr1ash57949ERERNze306dPr1q1ysHBISsrKzs7e9KkSdra2hISEqNGjfLx8blw4QLHcc+fPyeEmJiYqKioTJw4cc6cOa1atVJSUkLf2PLlyxUVFadOnbpnz55FixaJiop269atpKSEz+evXLnS2tqaEOLq6jp69OiNGzdyHLdx40Z5eflJkybt2bNn6dKlrVq16tChA85OmMYwWjo/rsZMmzYNPhYWFnp4eBBCLl68CCkQtdWrVy80ktTW1rq4uKirq+PN/+HDBxkZGQ8PD/i4atUqQoitrW1eXh6k9O3bV0xM7PXr1xzHVVVVxcbG0ga0Dh066OrqYoqtrW3Hjh3pSr579y41NRU/3rp1ixCybds2+Mg0RgCwlUF78vn83bt3E0K8vLz4fH55eXm3bt2UlZUzMzMhc2VlpZ2dnYSEBKxy5s6dSwjJycmBb/l8PsYZDhs2rE2bNvSBEhISREVF5eTkbty4ASmHDx+mL01KSsqHDx8w/7p16wghd+7cgY8nTpwghDx+/BgzpKWlvXv3Dj9u27aNEHL58mX4yDSG0dL5cTVGQ0PDycnJ0dFRXV2dELJgwQJ0k4wbN44QEhMTgz/Jzs4mhIwdO7asrKywsLCwsLC0tLRXr14qKirgsFm5ciUh5MGDB/iTzZs3E0JwoYPweLzS0lJXV1eMF+Lz+T169NDX1//06VOdFa6oqHj69CkhZOnSpZDCNEaAfv36SUpKOjg4ODo6GhsbE0KsrKxAVMLDwwkhvr6+dH4Y6w8fPsz9z0eyYMEC4YjnIUOGyMnJ0dclISGBEDJr1ixMSUpKEhUVnTlzpnCtSktLQYFOnToFKQcOHCCE3Lt3r86zKCsrO3XqFCFk//79kMI0htHS+XE1RkFBwdjY2M7Obtq0aTgnBcaMGSMqKvrmzZv/LzAqSlRUVEtLq2fPnt27d+/evXuPHj2UlZX19fUhDnXFihUC89MdO3bQKRUVFXv27HF2djY3Nzc0NJSWltbT0wPbTn0aExcXN2vWLBsbGzMzMz09PULIihUr4CumMQK4urqKiIh07drV0tLSy8trz5492Ji///47IeT48eN0/rCwMELIypUrOY4rLS0dPXo0IURFRWXlypX0KqQ+jZk/fz6mvHr1SkZGZsqUKZgSGho6cuRIa2trY2NjNTU1QsjJkyfhqzo1JiIiYsyYMdbW1iYmJjDjYRrD+G74cTWG9scIABqTnp6OKQ8ePCCEeHt73759++bNmzdv3rxx40ZoaGh8fDxkAI2JiorCnwQGBhJCIC6gpKTE1dVVVFR0zJgxW7Zs+e2330xMTDQ1NVFjrK2tO3ToQG+bCA0NVVJS6tSp0/Lly/ft27dx40Z04XBMY4RwcHBQV1evcx9JAxoDHhHg6tWrgwYNIoR06dIFLJwcxw0ZMqRNmzb0dQGNmTdvHqakp6fLyMhgmOLhw4chfmTNmjWHDh0Cu6vAOiY8PBx/furUKSkpKSsrq9WrVx88eHDmzJlMYxjfEz+uxkycOLG+DMIaA7ay8ePH1/eT+jQGbGU3btwQGNFGjBihrKyMGmNra6urq0uPZW5ubuLi4mip//jxIyFkyZIl8JFpjADg8y8uLhb+KiIiQkAVuP/Zx65cuSKQ+ciRI4SQ9evXw8chQ4ZISkrSxTasMZWVldra2mZmZvhtcHCw8Drm9u3b8LGmpqZz586dO3fGqGsQv3379sFHpjGMls6PqzGftY6pra0Fiz+tIgUFBehGblhjzp49SwjZu3cvfBUdHa2mpqalpYVxUIMGDRITE6OPaG9v37p1a8ywevVqZiv7rLgypKSkpGPHjgoKChBDzHFcRkZGx44ddXV1IX9MTAyqyMOHDwkhmzZtgo9gQ4uOjsbSGtaYT58+ycjI2NjYwFelpaUQToLrmDNnzhBCAgMD4WNFRYWSkpKxsTF+9PHxIYQcOHAAUpjGMFo6P6LGwD5/jFQWxtPTkxBCh3VxHBcbG6uhoSEmJubh4bFgwYIhQ4YoKiqiXX7BggWEkMjISMz/66+/EkIePXrEcdyHDx90dXVFRERGjRo1ZswYLS2tdu3aKSkpYZTazp07CSFGRkaurq6HDh3i/jeh7tKly+zZsx0cHDQ1NemhbeTIkfTmj++ef9SY7t27t27dmt6QRBMcHNymTRtZWdnBgwe7u7vLy8srKiri04OcnJzU1dWnT58+c+ZMZWVlNTU1fBDcb7/9RgjR09MbMGDAhg0bOI6Li4sT2MCbmppKCBkzZgx8nDNnDkQYzp0719jYWFtbmxBy5MgR+DYpKQn2Qg0ePHj06NF8Ph9mJ1ZWVnPnzjU3N9fR0SGE7NixA/KbmprKysqyZ8kwWi4/osbk5OS4ubmhyVuYPXv2eHl5CQ9Yr1698vX17dmzp4mJib29/YoVK9Bwf/78+YEDB+JHjuOCg4NdXV1fvXoFH5OSkry9vS0tLfv163f9+vXLly+PHTu2oqICvq2oqFizZo2VlVX37t0xzvXw4cO9evUyNzefMWNGWlrarFmzfvvtN/jqwIEDHh4eGCf93fOPGuPv7z9+/PgGHsYTExMzZcoUKysrMzOzsWPH0vL85MmTMWPGmJqaCn9VU1Ozfft2Gxsbc3Pzc+fOcRz3/v37gQMHnjhxAvPk5+d7enrCzIDjuIqKirVr1/bo0cPKymrVqlWpqane3t70xv4bN244OzubmprCqrSqqmrTpk02NjaWlpbLli1LT0/38fHB/bYrV66cNGlSVVXVZzYYg/Ff4UfUGEaL44d9tj+D0dJhGsNoATCNYTBaKExjGC0ApjEMRguFaQyjBcA0hsFooTCNYbQAmMYwGC0UpjGMFgDTGAajhcI0htECYBrDYLRQvk+NeZhWtPSP1H/Ox2ghvMqvmHuWXVAGo+Vx7Xnehu9SY2adSXvxoYz9fR9/NxMKJh57kZhV+s1rwv7YH/v7rL8DEdnrrr5uUl1oSr5QY9Lzyhf/mTHvbNrcMyns7zv48z2buuB86ryzqd+8JuyP/bG/z/pbejX7/JOcphWGJuQLNQbgcxyfz/7YH/tjf+zv2/01lRo0D1+lMQwGg8FgNADTGAaDwWA0F0xjGAwGg9FcMI1hMBgMRnPBNIbBYDAYzQXTGAaDwWD8H3v3HdfU1f8B/PpYFRcOEI0IKFQFRMGNgrNOqq1Fq1VmUMHViiLVaoH2cSu4cLPCCEtx4AYVF4qAoCIZQBYJO8wQAmSc3x/HJz+qdmlCQL7v1/flixxubk4umA93naMukDEAAADUBTIGAACAukDGAAAAUBfIGAAA+KzQiuqTGXXPOXWNzXJN9wUyBgAAPiOPmNWu4QVrLpS7RHL8rvEyuXWa7Q9kDAAAfCYamuXb4vPJVL5D0BvHYLprJNeZkn/yQQlXKNFUlyBjAADgM1ErkW6i5rmEsxyCcnE5hjDIVMHaqIKY9IpqcXPrdwkyBgAAPh+UJyVr4sudwvKUMeMQlOsUlu8WLdhygZ1Mq2qWtepIzZAxAADw+ZA0yyOflZIpeWRqoWMIvWXSuESwXSK5vyVys3itd5IGMgYAAD43eaXiw3cELuEsl0iuQxDt/w+dBdNco3iOIYyT9wWFla1xkgYyBgAAPk/P2XW/XuWRqQJnSkHLHRrHEAY5mu8emReXUV7ToN6TNJAxAADwOUum13hd5DiFMlvu0DgE5TqH5ZOpfO+L7Ed5NQqFuk7SQMYAAMC/IG6S1zfKPr3ETTJRo6ykpqmourGopulTq7qxqLqRWSrOLapvWcxSMbuiIYVe5X2B9c6FAP87ScNxieAcuC2gFYvVsbkgY0D71iyTN8sUzTL5J5ZUrmiWKSrrm4WiZmH9J5eoWShq5lU2sssb2BWSTyxOhYRdIcktrn8tqH/FF6mknuTXJOdW3qWpoFIY1YkvhdHPy2LTVVBx6WVhT4qP3+WfUEUF3uMfvs3zvcrxucJWSe26zNoal78lLn9LbN4nFl6PRwTDPYLhoYpyj2A4h9De2VnB5RZGdwujv9/+v5M0dNconislPzS1rLS2SbX/Q1WcMQoVFUJIrkD1jTKRSkoiEzXKSmubSmpUUKW1zaW1TRyhpKBcUlDWoJLK4tWl5tc8LVBBpbFqk3OrrmSXX31Z8el17WVFbHpp0KOi4MfFn16hT0pOpwj23+Ltv8nbf5P76bX3OndnAmvnJRXUrkusnZdYP0Xn/RjN3ET91PoxOu/H6DxyKN05hOaikgqlOVMKXKN4rpFcKM2WSzjbJZzlEs5WRbGcw/KdVFihTMcQxocr+E8z5m3ShDLI0YKNMeyEFxVCkcpO0nx8xtzMEZ56VNHyT4YjSYW7r3F2X+fsvvaptec65/dEjveFgm3x+dsuFHxqxed7xee7hzPWhDPWqqbojsE053CWM6VANRXOclFhRbDx/q9KyjWS6xrFc41UUUXxyNRCVZVrVKEK36lLBEdlP1BKgTOlwCmUqcJyDKY7BNEcglVUb//apamo/urDC6od1erYktVxZRcyyzSfMQducz2u1LlFC5RFjua7RvFUVpFcl3C2Cj98ncLyVFiOIXTHYJoKS2WfHcpPEBV/GEFBQeF693/EJ/3f/7Pdjo+qv/obJeRv9mOcKflkKv/Xq7zn7LommcoG0/z4jDl2t9A1qlDTP2yoDl8qzuZ3V+4YTP/4UunHx19/gvzbcg7L/1c7ZA5Bb1pulncWcIlgq+xIVCTHNYpHpvJVVtECt9gSldTq2BK3mCKnUKbDeeXWeOMYTP+EIwdsfBRLBRVCdwymO4fQXENpLn8s11CaRwRjTTj9A7/hQbkOb8eb4XtE5V/OrqhvlKkqXTCVZ4yqdr1V+pd4MO0vPize3+7/9CequnIKZTpT8p3DVFThLPzr+8nFcQxh/P+Hy/k3TqFMMpX/8Ue3ogrJVL5bTLHKKrpIlcfZIznv/DI4hTKdwvI++uC445/8l/64cgrOVc2pnRCaSyhtQxRza1y+Z+zf15a4/HURjP//KziY5hSc6xmb7xmThxfYHJO38xJrz3Xuf69xVFHcg7d4p+4LTqqiAu/xzz0sin5eppKKSS+Pzyjbe53jEsHGW8OZUrA5Nj8pt+rOm39dt99UJr2pzODUZvFELz69uHVZPFFBeQNPKOG2KH5VY1ltUwandtdlllPoe9eVBdNcI3lOocxTKUWFlY0qjBalTzhWdovrcaWOHC3A5RYtIEcL3h64V82xMp6Kzqq9Laew/A8c9QplOoYw3v3P/I+Olanor49gumMIY3UYfUMUc0NU3qfX+kjm1rj8Xy+zd11ifWL9epm1Npyu3D5OoXmbqMwjSYWHb/M+rg7d4h67y1fJ5QPBj4tDnpRQUksuZ1dcziq/nFXxiXUluyI+o4wcSlO+X5cINiW15FlBzZP8f12P82ue5Ne8KapnlNQzSsSfWPTienpJfVF1I75cTSVV2yD9p9fgNslvva50iy5SfqpujGJWiaXKBUSNssZmuUyuUFGp8PNNLeIzytziSvDWcI0q/P0aR9M9+isKhSIpt3JLXME7t2E6BOU6U1hkaqHvVW4mt1Z9Hfj4jLn+uuLUI+Hxu3xcJ+4JTqUIop6VUtNKqWlln14xz8tuvxEm5VaqpJJzKzM5tdm8uqw/Vg5fFP281DWSq/y7dXUY/Rmr5mWhKOu9hVsWp0IiqGpUTVU3ldU2VdY3V4pUUfXSOomsUSqXNH9qNUnl/rd5ZCofbxy36KKT9/gq/OVraxQKtC6C4RSWj98vOaYok6PhuTfaiAf0areYYmXG/BjNlKvtlr22j5pW6hb7dmu4RvJ8r7I13aM/lVUo8rtWSI4qdG4xErNDUK5jKJMcLfgxln39dWWjVL2pDvfHoHR2rVtM0f/2YPLXRTBk8o77/+cdx5ILydGCDpIx9Y0yj5YZEy14WqDGv+/akXu0qpYZs4nKVPcHU1vWLjJGUNV47lGpMyXPNZLX8ggwHq/MJSwvOLWsrK41hvqHjEGp+TVu//sYdQrL94hgqPysV/t1NOkPGRMIGdMhQca01MYzRqZQJL6sWB/DJkcL3jkR4BLOco3k7b7Be80XtVp/IGMgY/4KZIymO9UmQMa01MYz5nJWuVtMsXP4H86+4Kt1NkXn33lT1dy6p7wgYyBj/gpkjKY71SZAxrTUljOmSabYfqHANYrX8ipZMrXQJZQR8ri4olUOjr0DMgYy5q9Axmi6U20CZExLbTljpHKF3xX22xPMwTR839KBW3x6Sb2mugQZAxnzVyBjNN2pNgEypqW2nDEIoTRW7ZooNjla4BrJ+/kS71FejVyjFzFBxkDG/BXIGE13qk2AjGmpjWcMQii/TByfWX6PUVMnkWq6L5AxkDF/CTJG051qEyBjWmr7GdOmQMZAxvyVI3cK3WJL8GBZq2NLT9z9zDNmbTjdOZyN369bbEkqZAxCCKF7tKrVcWV4s7hEcDZEMTpyxkQ9K1kd/3ZrkKmCX6+wNN2jNg0yBjLmrxxJKnSLKXYIpjsE091iSk587vsxa8MZzhTW/95vcWo+ZAxCCN3NrXKLLcWbxTmcvSGqQ+/HRKWVro57uzXIUXyfK7Af81cgY9CTvJrVMcV46DBnCss9nA4Zo8QVSrJ4olf8+lf8+iyeiFcp0XSP1EimQLlF4peF//9+q8QauNazDaqsb37BrcOb5SVfRCsWa/Y0smaV1DRl/W9rZPNE+WUNmu5RmwYZgx4xq90TqvC4mWSqwDWUJmoDJ8oAAOAzABmDymqbk3OrUxjVKYzqe/SqR3k10g78NxoAAKgQZAwAAAB1gYwBAACgLpAxAAAA1AUyBgAAgLpAxgAAAFAXyBgAAADqAhkDAABAXSBjAAAAqAtkDAAAAHWBjAEAAKAukDEAgA8oLi7esGHDvHnzKioqPriASCRydXWdO3cuhUJp5b6BdgQyBgDwB1Kp9Pz58wMGDCAIgiAINvvDY9f7+fnhBX744YdW7iFoRzpixsTHx0dFRX3wW3V1ddevX9+7d6+fn9+lS5fq6upauW/tQkPD5zmYOYPBuHr1amIL165du3HjRnZ2tqa71qq2bNlCEISXl9fXX3/duXNnLpf7/jJ0Ol1LS2vBggU9e/Z0cHBo/U62pidPnvz222/r16/fu3dvZmbmO98ViUTh4eGenp4bNmw4efKkQCDQSCfbrI6VMa9fv/7qq68Ighg8eLBM9u4kMbdu3Ro5ciRBENra2n369PnPf/4THByskX62WTk5OYsXL545c+Zn+R9p+/btxB917tyZIIivvvpK011rVefOnbt79y5CaPXq1QRBfDBjvv76axMTk9TU1K5du65atarV+9hKZDKZi4sLQRBdu3bt1asXQRBaWlqhoaHKBVgs1rhx4zp16jRq1CgzMzOCIIyMjF68eKHBPrc1HShjjh492qNHj7FjxxoYGLyfMW/evOnTp4+RkVFSUlJNTY1YLE5LS8vPz9dUb9uaxsbGgwcP9u7dmyCIbt265ebmarpHqsflcu/cuZOcnHz37t27d++mpKQEBAQQBLFp0yZNd00znJycPpgxN27cIAji+vXrPB6PIIjPOGNEItFXX3116NAhLpfb0NBw+fLlvn379u7dW/k3Ft5EN2/elMlkMpns2rVrBEHY2dkpFDA/yFsdJWOkUunXX3+9Z88eqVQ6ffr0/v37v5MxK1asIAiiox0V+efWrFmjpaUVEBBllQvbAAAgAElEQVSwYMGC7t27MxgMTfeoNXh4eBAEkZWVpemOaMYHM0YikRgbG8+aNQshlJOT83lnjFwul0r/MGPhxo0bCYK4ffs2fmhsbGxkZNRygSFDhowYMeL9wyQdVkfJGIVCoTyLMHnyZB0dnZa/BCUlJb169Vq8eLGGetcOXLhwAQfw0qVLv/jii46QMSwWS0tLa/78+ZruiMZ8MGP27dtHEMTLly8RQtnZ2Z93xrzv119/JQgiKSkJP5w3b94XX3yhPEnz8uVLgiBcXFw01r+2p6NkTEvvZ8zjx48Jgjh16lRhYWFcXFxMTExaWhr8JfJB3377bZcuXTpCxuDTM9euXdN0RzTm/YxhsVja2tq+vr7KhwRBuLq6aqiDGmBra6ulpVVaWoofpqam9unTx9DQ8NatW48fPzYxMbGysuLz+ZrtZJsCGYMQQhcuXCAIYsyYMXp6et27d8fne+fNmwe/K+/rIBlTUVExYMCAkSNHNjU1abovGvN+xri5uf3nP/+JiYlJTk5OSko6e/YsQRCzZ89OTk5Wfux+xm7evEkQxObNm1s20mg0Q0ND/KFhZWUlEok01b22CTIGIYQiIiIIghg3blxMTExBQUFubu4vv/xCEMSKFSvg3N07OkjGnDx5Eu/aarojmvR+xkybNo34E+fPn9dgV1tBaWnp0KFDTUxMqqqqlI1yufzo0aMkEmnBggWTJk3S0tJav359TU2NBvvZ1kDGIIRQTEwMQRDv3DRjaWnZt2/fjvDX2b/SETKmsbFxxIgR/fv3Ly8v13RfNIlMJhME0XIj5OTk3L59Ozk5GV99d+7cOYIgZs2adefOnbKyMg12Vd3EYvGsWbO++OKL1NRUZaNcLl+/fj1BEMeOHUMISSQSX19fgiAWLVokkUg019m2BTIGIYSSkpIIgjhy5EjLxZYsWdK9e/c/u8m5w+oIGXPp0iV8E6KmO6IZHA7n0aNH6enpc+fOJQjiwoULqampH/yJ5+fnEwTh6OjY+p1sTU1NTfb29gRBXLx4sWX78+fPCYJwc3Nr2ejm5kYQxMOHD1u3j21XR8wYa2trHR2dli15eXmdO3e2s7Nr2WhmZmZoaCgWi1u3d21dR8iY6dOnd+3alUajabojmrF58+auXbt27969a9euXbp06datW7du3ezt7d9fMicnp2vXrs7Ozq3fyVYjkUiWL19OEERsbOw730pISCAI4sSJEy0b8VHW5OTkVuxjm9aBMkahUEilUqlUOmnSpP79+zc2NiqvfJfL5TY2NgRB3L9/H7ccP36cIIidO3dqrr9t1Pfff9+lS5fP+MDIkydPCIJYsmSJpjuiMXw+PzU19dmzZ+np6RkZGc+ePUtNTf3g/chNTU0vXrzg8Xit38nW0dDQ4ODgQBDE9u3bKyoquFwuh8NhsVhCoRAhlJub27lzZzMzMxaLhZdns9lmZmba2trFxcUa7Xgb0oEyZtWqVbq6urq6uvgUpa6uro6Ozr59+/B3Hz9+3LdvX4IgbGxsRo0aRRDE119/XV1drdk+tx1paWnBwcFBQUEWFhYEQfz3v/8NDg5+/PixpvulYgqF4ptvviEIAg+mAjq4zMxM/HHRr1+/Xr16de/evXv37lpaWnPmzGlubkYIHT9+vHPnzr17954wYcKECRO0tbX79u37ziG1Dq4DZcyJEydcXFzWrFmzadOmn376ac2aNc7OzgkJCcoFcnJyPD09Z82atXLlypCQEPw7BDB8lJkgiC5duvTs2RN/vWzZMk33S8XkcvmuXbv27Nkjl8s13RegedXV1adPn/b39z9w4MC+ffv279+/f//+3bt3U6lU5W9Idnb2/v373dzcPDw8jhw5wuFwNNrlNqcDZQz4FKWlpdnZ2TktZGdnf5YjYwIAVAgyBgAAgLpAxgAAAFAXyBgAAADqAhkDAABAXSBjAAAAqAtkDAAAAHWBjAEAAKAukDEAAADUBTIGAACAukDGAAAAUBfIGAAAAOoCGQMAAEBdIGMAAACoC2QMAAAAdYGMAQAAoC6QMQAAANQFMgYAAIC6QMYAAABQF8gYAAAA6gIZAwAAQF0gYwAAAKgLZAwAAAB1UW/G8Ksk7PIGtb4EaGVcoeQZp6GyvlnTHQEAtAPqypg6iTQmo3xtVAE5gv2AWa2mVwGtjF/V6BGZtzqubHMc+/abymaZQtM9AgC0aarPGJlccZ9ete0ih0zlO4XluydU+d8pVPmrAI1glIhdIzkuEWzncJZrJM/3KieLV6fpTgEA2i4VZ0yOQLTnBs8lguMSznYKzSNHCzbHsWnFYtW+CtAUuQJdflG+OjyPTOU7hjJco3iOocyT9wWFVY2a7hoAoC1SWcaU1jadf1TsFMIgRxU6htBdo3iuYczwp6VCUZOqXgK0EezyhuN3BU6hTJdwlmMIgxwt8IjKT3hRUd8o1XTXAABtiwoyRtwku/aqcmN0AZnKdwplOgTTXEIZAUmF7ArJp68ctFmZXJFvIs8plOkQlOtMySdT+Tsuc58U1Gq6XwCANuRTMyaDU+ubWEim8p0pBQ5BuQ5BuY4hjA1RzKBHRRFPS4IeFZ9/VNQh6mHRwVu8Pde5eztGHbjJO5rM97nCcgqhOwTTHIJyHYJoLhEclwj24SRBXikcHQUAIPQpGVNY2Xg4qcglnO0ayfnfp8z/KpjmEsF2jeJ1rIrkukRyXDtSOYXl/eHnHpTrGEx3iylyieRGPy9VwEVnAHR4H58xvMrGg3cELhFs10juuxkTRHMJ73gZE8VzjSrsOOUSwXEMpr/zc3eN5DpTCvyTBLnF9ZAxAIBPPVb2nF3re5VHjip0DmfhDxqnUObGKGbEs5L4zLKotNLIZx2iIp6WXs0uv50jvNUB6naO8B696i6t8r/XOE4hTPxzd6YUkKn8Xy5xnrFqVPKrCQD4DKjgnL+kWX7tpXBjdAE5+u05f+cQ+pFkPgvu8P98ldU2BT0uWROR5xhMdwxlkqP566LyLmWV1zfKNN01AEAborJrl8tqm84/KnIOobtGFTqGMFyjeK5hjLDU0rJauHb5s1LfKL/wosI96u1lhK5RPKdQ5qkUQVE1XEYIAHiXiu/BpBXX77/Fd4lgu0SwnUKZbtGCn+LYb4rqVfsqQFOqG6Q+V9huscVOYfn4Vv89N/ivBfDzBQB8mOrHkpErFE8Kardf4pKj+c6UAveEqsO3eSp/FfWRy+Wa7kLbRSuuJ1P5ZCqfHC3YEs++x6iWymBzAQD+lLrGxKxvlCZkCT2oLLdIVlsbE/PIkSOOjo7vt9fW1trb2xsZGfn5+bV6p9qHhibZ6RTBzsSiqLTyKjEMvQwA+BvqHdu/tLapsFL1Z/43bdpkaWlpZmZmZma2bdu2ysrKf/X0bdu2TZ48GSEklUrnz5+/f/9+3O7r62tiYpKcnMxgMFTYWzc3Nw8PDxWuUONEEhgzBgDwj7S/OcoUCsW4cePs7e0vXboUHR09ePDgGTNmNDb+izEZd+7cOWPGDISQVCpdunTp0aNHcfv8+fM9PT0/vYd+fn7Hjx9XPty0adOWLVs+fbUAANDutL+Mkcvl1tbWhw4dwg9v3LhBEERGRoZCoWCxWAqFgslkvn79Wip9+7d2dnZ2ZmYml8tVrkGZMQihsrKy6upqhBCNRhs3bpyLi0tmZiZuEYvFmZmZL168UD6xqqqqqKhIJpO9ePGCz+cjhIqKivAy9fVvz3szGIzx48fPmzcvMzOzqKgIIVRZWVleXq5ciUgkyszMzMzMVD5FJpOxWCyEUGlp6YsXL0pLS9Ww2QAAQAPaZcZMmTJl7969+OHjx4+1tLRoNFpDQ8P48eMdHR2NjIysrKyEQmFFRcWSJUt0dHT09fUHDRp05MgR/JSWGfPVV1/t2rULITR37txu3br17t1bX1//6dOn2dnZVlZW+vr6AwYMmD9/fnFxMUIoIiIC70KRSCQfH5+goCBTU1O8cktLSxqNhhD67rvvtLS0evbsqa+vj4NwzZo19vb2+OWSkpIsLCz09fUHDx5sbm7+5MkThFBtbe3kyZOdnZ0nT55MIpEGDx789OnT1tykAACgJu0yY6ZOnbp9+/bS0tKcnBwzM7OJEyc2NTXV1dUZGhoOHz48JydHLBYrFIrvv//ewsKCzWaLxeLw8HCCIJKTk9EfM2bWrFne3t4IIYlEYmtr6+npKRaLq6urJ0yYcPjwYYSQTCabNm2au7s7Qig+Pp4gCHd395qamsbGxosXL2ZkZOD1TJs2bdWqVXg9dnZ2rq6uYrG4ubkZIUQmk5csWYIQKi4uHjhw4ObNm8VicV1d3bJly4yNjevq6hobG4cPHz5y5MisrKza2lpbW9sFCxa0/oYFAACVa38ZgxCaM2dOr169Bg0a1K9fv0WLFrHZbIRQRUWFiYlJXFwcXqa4uFhXV/fy5cvKZ82cORPHyTv7MT///LNyge3btyOEUlJSevfu7eXl9csvv+zatWvq1KlmZmYIISqVOmLECOUxLoRQdnb2L7/8snPnzokTJy5fvhw3fvPNNziTsNWrV+P9mIiIiEGDBonFb8ckLigoGDhwYFpamlwuNzU1pVAouP3UqVNmZmYyGdwwDwBo99pfxsjlchsbG09Pz4KCAnwaA6uoqBg+fHhCQgJ+yGAwhgwZ0vKg05IlS9auXYv+PGNmzJiBQyg2NrZHjx6LFi1asmTJkiVLHBwc8HG2iIiIUaNG1da+nSLlwIEDJBIJL2NsbKy8Hnrx4sUfzJjjx4+PGzdOef+NQCAwMDC4c+cOQsjc3DwiIgK3BwUFDR06tKkJxkcAALR77TJjpkyZorzgWAlnTHx8PH4oFApJJFJISAh+KJVKDQwM/P390T/ImPv372tra5eVlb3zEhQKxdzcHF8RUFlZ2b9/f7xChJCHhwc+IIYQWrhwoZubm/JZyoy5dOmSjo5OSUkJbk9KSurVq1dBQUFzc7O5ublyP+bcuXPGxsaQMQCAz0C7zBhLS0t8or6l8vJyEolEpVKVLVu2bOnevXtQUFBCQsLixYuNjIzwhV5eXl4TJ07Ey0yZMmXz5s3464kTJ/70008IoaamptmzZ48ePTouLi4hIWH79u34WuTg4GAjI6OqqiqEUENDw+jRo+fNm5eQkLBr166uXbsqj5WtW7du4MCBFy5cwNekOTg4LFy4ECFUVVVlbm5uY2OTkJAQHh4+aNAgFxcXhFBtba2RkVFQUBB++smTJ/X09CBjAACfgXaZMW5ubsHBwe+0V1dXL1269O7du8qW5ubmvXv3mpqampqarly5Unlg7fTp08qbIjds2BAYGIi/9vDwOHHiBP5aKBSuW7cOP3fJkiVv3rxBCF2/fn358uV1dXV4mZycnPnz55uamjo5Of3222++vr64nc/nL1q0yNTUFK95z549yl0lPp/v4OCAV7t79+6GhgaEUH19/fLly69fv46XuXTp0pIlS/D1AgAA0K61v4wBAADQXkDGAAAAUBfIGAAAAOoCGQMAAEBdIGMAAACoC2QMAAAAdYGMAQAAoC7tOGNevnz55MmTnJwcTXcEAADAh7XLjJFKpVu3bu3fv7+RkZG2travr69yELCWFAoFj8fLy8t7/7sKhSI/P18gEOCHJSUl6pi1RSAQCIVCla8WAADai3aZMQkJCV26dElJSWlubj558mS3bt1aDo6pJBaLJ0yYQBCE8u59paNHjxIEMXfuXPxw3Lhxs2bN+vSOCYVC5ajMTU1NytFiAACgY2qXGRMQEDBkyBC8d5KQkNC3b188vP87RCLRpEmTzM3NR48eLRKJlO11dXUTJkwwNTWdO3cuXsmFCxeuXbv26R2bOXPm2bNn8ddyuTw8PDwlJeXTVwsAAO1Uu8wYJpPZv3//4ODglJQUHR2d33777YOLiUSicePG+fn5mZiYREVFKdvDw8Otra19fHxmzJiBp2lJT09PTU3F3xWLxQEBAX5+focPHz5y5AgeZDMtLe3BgwdZWVl+fn54xpfg4GA/P7///ve/dDodISSRSI4dO6arqztr1iw/P7+srCyE0KNHj/AX2I0bN/z8/H7//Xc8+hlCqKqqKjY2VigUUiiU33///eHDh2rZXgAAoCHtMmMQQlu3biUIYsKECXjaFT6fHx8f/85YxSKRyMLCIi4uzsvLy9bWFseJTCaztbU9fvz4qVOnbGxs8H6Mvb09nnqyurp6zpw5lpaWCxcu7N69O0EQfn5+CCFfX189PT3cHhcX5+7uPnnyZDs7Oxsbm4EDB6anp0skEjKZrK2tPWLEiIULFyYlJSGErK2t16xZgxCSSqUeHh4kEmnhwoXTpk3r0aPHxYsXEUJ5eXkDBgwYPXr0ggULbG1tO3fufPv27VbekgAAoD7tMmMSExMtLS0tLCy+/PJLPJtLZGRkz549a2pqWi4mEolGjRoVExNDo9G0tLRevnyJELp//76urm5VVdWRI0emTp2KM8bFxQWPzB8XF9e/f3+pVIoQolKpxsbGeIH9+/f37t0bJwdCKC8vT/kqkyZNwjMC4K/xLADYvHnzPD09cYe7deuWlpaG2zds2DB8+PDm5maBQKCtrb1+/XrcPnPmzBUrVqhhg6mSVPaByytAK5PLFZruAgD/SPvLGDab3adPnxMnTojFYisrq7Fjx0okkt27d9vY2CgUf/iPhzMGT1M2fvx4PE/MypUrf/jhB4TQvn373s8YGo02bNiwI0eO3Lp1a/HixRMmTJBIJAghX1/f2bNnK9csFou9vb3Hjh07fvz4fv36/frrr7h93LhxLSdPmzdv3pYtWxBC27Zts7W1Vbanp6fr6+vzeDyBQDB06NDnz5/jdm9v72nTpqljo6lEbpHo4G2+zzX+y8L6v18aqA2jROybyN17k5dVKPr7pQHQqPaXMRERET169MATHguFwilTpowfP97IyOj06dPvLIkz5vz58wihM2fOGBsbp6amGhoaPnjwACG0d+/e9zMmKyvL2tp67NixFhYWK1eu5PF4eFV+fn7Tp0/HGVZbWzt16tRp06ZRKBQqlWppaamcHmbs2LGHDh1SdkCZMV5eXvPnz1e2P336VE9Pj8vlFhUVGRkZKa8L+Pnnn6dMmaKGbfapyuqaQlNLXcPzyVS+e0LVrssfuIoPtJqT9wUel2vJ1EKn0LzTD4r5VY2a7hEAf6r9ZcyDBw8IgoiOjsYPaTQaQRAEQdy5c+edJVtmTFlZmZGRkYGBwezZs/GJmQ9mzKFDhyZMmMBkMvPy8rhcrnJVLTMmJyeHIAgcVAihCRMmbNu2DX9tYWHRcoJO5bGy0NDQ3r17K1f4448/jhgxorm5mcPhtMwYb2/vqVOnqnJjfbJGqfxmTtWmWDaZyncKzXMJZ7tFC+Iz3p2FGrSmFEa1czjLNZLnFMokU/nrqAWXsoT1jVJN9wuAD2h/GSOXy9evX9+rV69Vq1Y5OTmNGzfuhx9+WLt2ra6u7sGDB1suWVdXRyKRlDfHbNy4kSCIyMhI/HDXrl3m5ubvnPMvKCjQ1dXV09PT19cfNGjQ3Llz8akXb2/vMWPG4IwRiURTpkwxNzd3cnKysbHp3LmzMmM8PDz69Onj5OSEz9xMnjwZn/OvqamZNWuWsbGxk5OTnZ1d3759L1++jBBiMBi9evVSnubZsGGDubm52rfgP5bOqfv1KpdM5TtTCpzD8slUvvdFzpP8GgTnAjTtBbfO91qha1Th2x9NtGDHJd5TVp2m+wXAu9pfxmAJCQnu7u7u7u5XrlzBLRQKJSAgoOUyTU1Nx48fz8jIwA+ZTObu3burqqrwwwcPHpw7dw7HxsWLF2NiYhBCZ8+eXbBgAZ/Pr66uFgqF48aNW7lyJULo3r17QUFByvM9QqHw119/dXd3T0hIuHbt2q1bt3B7bW3t77//7u7unp6ejhAKCwu7ceMG/pZMJjty5Ii7u/uGDRtoNJpyPYcPH1be3HPr1q1z586pY3P9WwXl4qPJfGdKvmsk1yGY5hzK2HaRfetNlab7Bf7gYV7Nzis8lzCmQzDNJYLjEsE5nCSgl4g13S8A/l97zRg1mTVrFt6hQQiVl5ebmZkpL/rqICrrm6OelZLDGGQq3zGE4RCU6xhCJ4fRQx4XP8qrfsCsvkurSqZVQmm27tKrUuhVT/KqqWmlayOYb39SwXRyFN85lBH8uKistunvf9gAqB9kzB9cu3Zt+PDhQ4YMGTp06ODBg+3t7UtKSjTdqVYiapTdfF25JZ5FpvKdwvIcgnJblks42y26yC2mGKotVZFLBMchmNbyJ+UUyiRHCzZG51/OqqhpgJM0QMMgY94lFAofPHiQkpLy7NkzTfelVRWUNXjG5q29KHSmFDgEvWnxsfXGMYThGsl1jeJBtbmK5DmFMt/5eTmH5a+5ULGemkcvhuNmQMMgY8D/EzXKErIq1lPz3aIFjqEM/JnlGMJwj2Dsv8Hdc53zWyJUG6rd1zkHb3HXR709VoZ/WGQq3z2yICa9vFoMOzFA8yBjwLuKqhvPPSpxpuS7RvEcgmmOIYyN0ayErMqGZpmmuwb+QCqT36FVb45jO4YwHIJprpFcZ0rB8XtFPKFE010D4C3IGPBhr/mi3Tf4rlE8Z0qBUyjTlVroncB5mFfzzmAKQFPS2LW7rnDJUYVOYXnOlAJXauGvV3kZXLjzH7QtkDHgT0lliqTcKs94tlu0wCkszyWc7RrF23uDx6+EP5M1qbS2KSCJ7xLOcongOIUyydH8DdH5114JJc0wlBxocyBjwN8QiprDn5a6hTPJ1ELHEMbaC8IDt7h//zSgNiGPi90TqpxCma5RPKcQ+vlHxUXVMJwMaKMgY8A/wigRH7jFcwlnrYkvP50i0HR3OrT4jLLVcWUuEZz/XuO+5sPBMdCmQcaAf0qBUDqn7j6jVtQIJ/81qaFZ9jCv5hmrTgoj/IM2DzIGAACAukDGAAAAUBfIGAAAAOoCGQPaFjw9KL4LRyaTcbncsjKYrgaA9goyBqiXQqEQiURVVVWVlZWVlZVNTX8YD7ipqam+/v9nbvby8urbt+/EiRPr6+sbGhqWLFmio6ODp49rBfX19c3Nzf98ealUWl9fDzelAvAXIGOAeolEounTpw8cOJBEIpFIpFGjRlEoFOV3f/75Z2NjY7FYjBB68uRJnz59kpOTS0tL5XL5+fPnSSQSjUarqKhQU9+kUqkyIcrKyvT09Pz9/f9ieblcLpX+/yBgJ06c0NHRKSoqUlP3APgMQMYA9aqurjY2Nt63bx+dTqfT6fv27evRo8ePP/6Iv3vp0iUfHx+8cxMTEzNixAjlE7dt26acy0dNZs6cee/ePfx1bW2tt7f33bt3/2L5xMTEuXPnKh/ev39/27ZtNTU1au0kAO0aZAxQr+rq6pEjR8bGxipb4uPjCYK4c+cOQqi0tDQ7Oxsh9OrVK0dHR319/eDg4LS0tEePHs2YMcPKyopCoeTm5iKEKisrw8PDKRQKh8PB6xEKhY8ePaqtrY2MjFTOV81msykUSkREhHLCUx6Pl5aWJpFIYmNjqVRqZWUlQqiuri44OLhXr15r166lUCjFxcUIoZcvXyp3SgoLCykUCoVCefDgAW55+fKlk5NT3759z58/j6c3ra6uzszMVB5ea2hoiIqKolAoKSkpyjebmZlZUFBQXFxMoVASExPVsYUBaMsgY4B64YyJiIhQtigUCmNjYw8PD4RQQECAsbExPjI2ZMgQLS2tMWPG7N+/f+fOnTo6Otra2paWlvHx8a9fvx41apSFhcWYMWP09fVv376NEEpNTR00aJCtre2IESPIZDJC6OrVq4aGhlZWViNGjDAzM8vJyUEIUSgUfX39OXPmTJo0SUdHZ8KECXV1dQKBYMqUKV27djUyMrK0tHz+/LlcLjcxMTl48CBC6O7du+bm5lZWVlZWVgMGDNi/f79CoTh//ryBgUG3bt3GjBnj5OSEEIqPj9fV1cX59OrVqzFjxgwbNszS0lJXV3fTpk34qJqLi8uYMWNmzpxpaWnZpUuXTZs2yeUwqhjoQCBjgHq9nzEIoa+//nrJkiUIoZMnT44ZM6axsREhFBQUZGFhoVxm48aNixcvxl/PmDHDz88Pf+3r6ztu3DiZTPb69esvvvjCxcUFn1MpLy9vucP0/fff29vbI4QSEhK++OKLQ4cOIYRYLJa2tjbuTFNTk7GxcUJCAl5eIpFYWVkdO3YMIcThcPDeFUIoPDxcT08P7/2Eh4ebmpoqe5iQkGBsbFxRUaFQKGxsbBYsWIDfyP379zt16nTlyhWE0Lp16/r37//w4UOEUFhYWO/evVksluq2LgBtHWQMUK8PZszMmTOXLl2K/pgxp0+fNjc3V154tm7dOjs7O4RQQUGBrq7urFmz7Ozs7OzsJkyY0KtXL7FY/PLlS0NDw4KCArz8nTt3evTo8fXXX9vZ2S1atGj48OEjR45ECEVHR5ubm0skb8eKnjRpEo6rurq6YcOGxcTE4PaWGYMQotFoS5cutbOzs7a2xkGCEAoKChoxYoRyVThjamtri4qKdHV1W57LmTNnjpeXF0LI1dV11apVuJHD4QwYMCA1NVWlGxiANg0yBqjX+xlTW1vbv3//vXv3IoQCAwOVGXPq1Clzc3P8NULIw8MDZ0xWVlafPn3c3d19fX19fHz27t0bEREhk8nS09MNDQ3fvHmDl4+Li9PW1vb29vbx8fHx8Tlw4MDly5cRQlFRUWZmZsrTM5MnT96xYwfuxrBhw+Li4nB7y4y5ePGijo7OunXrfH19V61apcyYc+fOmZqaKnuIM6auro7NZuvr66enpyvf44IFCzZu3IgQIpPJK1euxI1cLldHR0d5ggeAjgAyBqgXzhj8cY/9+OOPWlpaeP/jn2RMaWmprq5ufHz8O2t++vSpoaEhPumCEEpPT+/Vq5cycpQiIyPNzMzwwS6E0KRJk3bu3IkQqq2tJZFIkZGRuDMBF38AACAASURBVL2hocHKyur48eMIoUWLFs2aNQu3x8bGGhgY4Iw5e/bskCFDlGvGGVNVVdXQ0KCvr3/48GHcXlZW1qdPn7CwMIQQmUz+4YcfcDubzdbR0cHHzQDoICBjgHpVV1cPHjzYzs7Oz8/Pz89v3rx5enp6+FwFQujw4cOGhob46FNAQMDgwYOVR6IcHR1tbW3x13v37tXS0vL09PTz83Nzc8O7CKmpqb1793758qXytchk8qBBg3bt2uXn5/f999/v378fIRQSEkIikYRCIV5m5MiRnp6eCCG5XG5jYzNq1Cg/Pz86nd7c3Dxs2LADBw4ghA4ePNizZ08fHx9vb+9+/foNGDCgvLwcIfTw4cNOnTqtXr365MmTCKG4uLj+/fuXlJTgp3zxxRe4h5aWljY2NnV1dQihZcuWLVq0CL90QUFBp06d/vryaAA+M5AxQL0kEskvv/zyzTffzJ8/f/78+Tt27Gh502JSUtIvv/yCL8FKTk7++eeflTc5hoSEKPcMEELR0dELFiyYP38+mUzG+0D5+fk//fQTn89XLiOTyY4fP45faOvWrXgQmidPnvz8888i0dt5Vnx9fZW7RHQ6ffny5fPnz8c7Qzt37sRXrDU3N+P1ODg43Lp1y8/PD98Eo1AogoKC7Ozstm7dihDKzMzcsmWL8v6Y+Ph43MPff/8d31WKEDp37hwOJIRQeXm5h4cHnU5X7RYGoC2DjAEAAKAukDEAAADUBTIGAACAukDGAAAAUBfIGAAAAOoCGQMAAEBdIGMAAACoC2QMaA0SiSQsLOzMmTM3b97UdF8AAK0HMgaonUAgsLW1NTQ0nDhxYs+ePbdv3/5n8xMrFIpr165duHChww7qJZFI7t+/j+cLAOAzABkD1M7Pz09fX7+2thYh5O/vr6Ojoxw9rKWSkpIFCxYMGTLEzMyMRCKdOHFC3R0rKSl5/fr1nwWeRjAYDIIg8LBpAHwGIGOA2nl7e48aNQp/7eXlZWZmVl9f//5i27dvHzRoEI6i/Pz858+fq7tjZ86cMTc3V/er/CsNDQ0PHz7EY6AB8BmAjAFql5OTo6Ojc+7cuQMHDvTp0wfPsvy+devWDR8+vGVLUVHRxo0blTPE1NfXe3t7Z2Rk5OTkHD169OXLl87OzitWrLh//75yAQqFYm9vb29vv2fPHuXQZ8ePH3/16lVMTIy9vf2PP/6IYywyMnL06NG9e/desmSJr68vXpLP53t4eNjb269Zs4bNZit7EhUVZW9vv2zZsrVr165atSotLQ23nzlzxt7efvny5ZmZmbjlzp07586de/bs2ffff79y5UrlXGcIITab7eLiYm9vv2/fPuUMza9evfrhhx/s7e3xTMwSieTo0aMvXrxACCkUipCQEHt7+9WrV7cclg2AdgQyBrSG7777jiCI2bNn5+bmIoRSU1MDAgLeWSY1NbVv377Tpk1LSUnBLXV1dXp6engASoTQnTt3CIIoKChITEzs0qWLpaWlt7f3/PnzlUP6U6lUa2vr7du3b9++/csvv3R1dcXHwWxsbExNTRctWrR161YjI6OJEyfW19ffuHFj5syZurq6np6ep0+fRghlZ2cbGhriIS/nzZtHIpFwbwMDA0kk0tatW1evXk0QxPTp0/EYmuvXr7ewsNi+fTuZTNbT08PnkPz9/bt27Tp58mQvL6/JkycPHjwYn1zJzs42MTFxcnLavn27paXl8uXLFQoFi8UyMTEhk8leXl5kMlkqlVZXV+vp6Z0/fx4hdOzYMQMDg59//nnVqlVnzpxphR8TACoHGQPUbseOHRMnTpw7d66pqSkeY9/Hx2fo0KHvL/n48eOpU6d26tRp8eLFhYWFCKE9e/YYGBg0NDQghNasWYPHyb9y5Yq2tjYeJF8mk5mYmPj7+yOElHNoIoTOnj1rZGSEZ6OxsbFZuHChXC5HCGVnZ3fv3j05ORkhFBQUNHbsWOVTFi9ePHfuXOXDSZMmrVmzBiE0ZcqULVu24MYJEyb8/vvvCKGMjIzBgwfjEEIILV269Ntvv0UIBQYG6urq4swrKyvT1dW9cOECQmj58uVOTk544czMzH79+r169SolJaV79+7KM/wKhaKysnLkyJF4VpulS5cq+9PyrQHQjkDGAPVKSkrq3Lnzixcv5HL5uHHjxowZU15evmLFCjKZ/GdPSUxMHDBgwOzZs5ubm5XzejU0NCinRo6JiTE3N1cO1z9t2rRt27YhhBobG/fu3TtkyJAhQ4bo6uqOGTMGfzTb2toeOnQILyyVSseOHYvH2z969Ojo0aPxITWJRDJq1KiWJ9t9fHxmzpyJEDpy5MjgwYMPHTq0c+dObW1tPDVASEhIt27dhg0bNmTIEAMDgz59+uD8CwgImDJlivK1Ro4cee7cOYTQ+PHj9fT0DAwMcPc6d+6ckpLS2Ni4atWq/v37e3p6crlchJBQKBw5cmR4eDhCKC0tzdTU1NLSMiwsrE1dmADAPwcZA9TLz8/PwMAAf11bW/vVV18ZGxv/7bT2kZGRBEHweDyE0KJFi9zc3C5duqTcoYmNjR05ciSeNwwhZGtrizNm27Ztenp6d+7cycrKOnDggJmZGZ7xzMbGBk8+hhBqaGgwMTEJCgpCCAUEBFhaWuKP76amJktLy5YZs3Hjxjlz5iCEvLy8vvrqq2+++ebbb79VzmJ5+vRpfX39Z8+eZWRkZGRk0Ol03B9/f/+JEyfibGtubh4xYgQ+zGVmZrZ58+aXL19mZGRkZWWx2WzlHDMPHjyYMGGCqalpWVlZfX29MmMQQpWVlWfOnNHT01PuSAHQvkDGAPW6fv06QRBRUVH44aVLlwiCIAgCH61q6eHDh8rrzdzc3IYNG4ankrx06ZKhoeG4ceOUn7PvZ8zPP/+MEJo+ffo333yDG9etWzdixAh8rGz69OlWVlZ4bjQKhdK5c2cajYYQOnHihJ6ennLmzR9++MHKygp/9PN4PB0dHbz3M3nyZG9v75SUlJSUlIyMDLxwRkZGz549r1+/jh9WVlbiRGyZMU1NTSNGjMD7TCtWrFBO64kQotPpcrmcxWLhy7gLCgoIgnj69Cne9YmIiEAIvXr1Ci/s7e1tbGwMuzKgPYKMAeolk8m8vLz69u1rbW1tbW1tbm6+f/9+Hx+fvn377tu3r+WSdnZ2xsbG1tbWFhYWOjo6ly5dwu319fXm5uYEQSinVY6IiBg4cGBpaSl+OGbMmPXr1yOEIiMj+/XrZ21tPXnyZENDw5EjR+L8WLBggbm5+aRJk6ytrXv27IlPqCCEsrOzdXR0zMzM8HkXDodjaWk5bNgwa2trAwODZcuW4czbt29fp06dTE1NTU1N9fX13d3d8Wr37t3bv39/5fsKCwtDCO3Zs2fkyJHKjNHT08NBxePxxo4dO3ToUGtr63Hjxi1YsKC5uTkyMhK/5S+//PK7774Ti8U1NTUDBw4MDQ1FCH3//fejR4+2trYeMmQInPMH7RRkDGgNjx49CgwMDAwM5HA4uOX27dtJSUktl6mvrw8NDQ0MDDx16hQ+4a/k7u4+duxYmUyGH3K53MTEROX+x507d/DFvgihtLS0wMDAuLi4srKypKQkfK7F1tb28OHDT58+DQwMfGf/KTs7+/Tp08rdkfr6+qCgoMDAwMuXL+OWsrKyiRMn4guLEUI5OTmdO3dW9hyvMzAwUDkwAY1Gu3XrFr6+QC6XX716lcFg4G+JxeLg4ODAwMCzZ89WVVXhBS5evBgYGBgREYHfXWNjY2JiIovFQggVFxefOnUqMDDw8ePHH7fZAdA4yBjQ1tXU1BgbGx87duyj1zB58mQ/P7+Pey6dTicI4vz583w+n8/n+/r66ujo0On0j+4MAB0KZAxo6y5evKivr4//tP848+bNU57z/7dkMhk+vT9o0KBBgwZNmjQJhvUE4J+DjAFtXXV1dUVFxaesoaqq6oOj1/xzlZWVAoFAIBDAfSoA/CuQMQAAANQFMgYAAIC6QMYAAABQF8gYAAAA6gIZAwAAQF0gYwAAAKgLZAwAAAB1gYwBAACgLpAxAAAA1AUyBgAAgLpAxgAAAFAXyBgAPge8Sgmz5JPGZAOfvWaZIvJZyeG7ZY/zalptxjvIGADat2qxNCqtbHVEgTOFdY9WpenugLYrnVPnFlO8OrbEJZy9/yaPVtwaf5RAxgDQXsnlivuMGq8LHDJV4BSWv/Zi5bG7hX//NNBRFZQ3rA5nukUXOYUyXamFLqHM0CclpbXqHUocMgaAdim3WLzvFt8lkusSznYKyydHC7wTuMxSsab7Bdo0erF4/61Clwi2SwTbKTSPHC3YFMO69kooaZap6RUhYwBoZ4qrG88+KHIOZbpG8RxDGK5RvLVR+THp5bXiZk13DbQPGZy6364XulDyHIJpzpQCMrXQJ5GXzqlTx2tBxgDQbjQ0y6+9Eq6LyiNHC5xCmY7BNJcwZuD9Il6lRNNdUz2FQlHfJGtokmuymuW1DdLimibNVklNU1F1I6NUTCuupxeLP7GYpQ1coSSTK9pzg+cclucQlOsQTHON5DpTCo7eK+IIVfy7BBkDQPuQya3zucolU/nOlAKHoFyHoFzHEIZHBOPkPcH5R8Un7wkCVVEn7wn23+D6JXJ9r7I1W78kFGyNy/eK12Rti8/3jM3ziGS4RzA8IjVXEQz3CIZjcK7D+Vz8o//0IofSV4fRHVu0OIbQydH8tRH5Ec/KiqobVfV7CxkDQFtXWNnon1TkHM5yjeQ6BNPe+bBwCWe5RnJVWrw2UKp9R59ULm2kIjgqLNdIrjMl/73soeELz+IyylT12wsZA0Bbx6ts9E/GGcN7J2Mcg+mukVxytECF5RZT5BZTrOGKLXGN5DqGMBxDmRosp9A8hyCaWxjdLQz/q7FaTaFviGJuouZtpDI/vTZFM7fF52+IYji2+HVyDme5RvF+u16YzROp8O4ZyBgA2ofsQtHv1wtdo3gu4ay3ARPK/DEmn5pWevVlRXxGWczzUpXU9VfCZFqlZusurfpRXvULbl0mV6TByuDUveKL2BUSdnkDu0KiqWKVS7hCSZVYWi2WVombP6WqxdL6Rll9oyyDU7vzEtspNM8hKNcplEmm8jdFF9zMqWySylX7ewsZA0C70SSV335T6RnPJkcLnELzHIJpZEqef5Igr6xB010D7UZtgzT6edmaiDxnSoFjMM01qtApmB70qLhMPTfKQMYA0M4IRc3hT8vIFCaZyncMYbhGcp1CmWceFAmqPsOry4BqpTCqPOPZZKrAKTTPJYLtGsnde7PwTZEab/iHjAGgXWKWiv2T+M6UApwx5GjB+mhWOqdW0/0CbRe9ROwUlucayXUKyyNHC7YlcO4zamRy9Y5cBhkDQDuWml+zI4FFjip0Dme5X6red4Or6R6BtoteInYKYayOLVkTmR+dUVHbIG2FF4WMAaB9a2iSXXtV+WMsa30sN4VRrenugLZLoVA8ZFZT04UF5a13Ag8yBoDPgVDULKhS2X1zAKgKZAwAAAB1gYwBAACgLpAxAAAA1AUyBgAAgLpAxgAAAFAXyBgAAADqAhkDAFCxqKiosWPHWlpaLl26lMfjabo7GpCbm2tra5uamqrpjmgeZAwAQJXOnj3bo0ePw4cPUygUFxeXsLAwdb8ilUr18fFR96v8K69evRo7duyjR4803RHNg4wBAKjS3Llz7ezsWrbU1dVxOBzlQ4VCwePxRCJRXV2dUChECDGZzLy8vJZPqa+vp9PpdDq9ru7tJPNyubyoqAghJBaL6XR6aWkpbi8uLl65cqWpqSmdTscLYAUFBXQ6XSAQvL9aJpPJZrOZTGZDw9vb3aurq+l0ekFBgXJJoVCIX5pOp7NYrHfeI5PJpNPpIpGoZSODwaDT6TKZDCEkk8lKSkoaG9/eFVtbW0un0/l8/j/Yfp8byBgAgCp5eXn16dPnyZMnypbs7Ow+ffo8fvwYP0xLS+vXrx+TyQwLC5s2bdrq1asNDAy0tLQ2btyoUCgQQomJiePHjyeRSCQSycLCIi0tDSEkEonmzJnz+++/z5w5k0Qi6enpXblyBSHk6enZs2fPrl27kkikdevWIYTq6+s3bNigo6NDIpEGDRp06NAh/LpZWVljx44lkUh9+/bt1KnT4MGD09PTEUI3btwwMzMjkUi6urpeXl5SqRQh9MsvvyxcuHDVqlUkEql79+67d+/GK6mvr1+zZs2AAQNIJNLo0aMfPnyIEKqurnZ2dh4wYMDAgQO3b9+OEOJyuV9++eXdu3cRQqmpqVZWViQSaejQoQ8ePGidn0LbARkDAFClsrKyefPmEQSxbNmyrKwshFBzc7O1tfWqVavwAt7e3uPHj0cIBQUFEQSxZcsWoVAYGxv7n//8B39kX79+PSEhAS/8ww8/TJs2DSHU2Nhobm5uYmJy8+bNysrK1atXa2trV1RU1NfX//jjjzY2NpWVlXjHwtPTU19fPyMjo7KyMjY2tlOnTjiNbG1tnZ2da2pqmEzmgAEDTp06JZVK8/PzjY2NL168iBDicrlGRkZnz55FCO3atatTp04HDhyorKw8cuSIlpYWg8FACG3btm3GjBnV1dUIIR8fn1GjRonFYiqVqqOjU11dXVlZmZiYiBBis9mDBw/GGTNr1iwHBweZTPb8+XOclx0KZAwAQMXkcjmVSh09enTPnj1xWpw9e3bQoEEikUgul1tYWJw4cQIhdPz48dGjR+OnyGSyESNGnDlzBj8sLy8/ePDggQMHFi1aNHXqVISQRCIxNzc/ffo0XoDH4w0ePDg5ORkhtGPHjlmzZuH2urq6oUOHBgYGKjuzePHiNWvWIIRmz569detWhJBQKOzbt29QUBBC6NSpUyQSKSAg4MCBA/7+/l9++aW9vT1CyMvLa+bMmcrOkEikpKQkhUJhbm6+YsWKQ4cOHThw4KeffiIIgsFgPHnyREdHx8fHp7b27dwKBQUFhoaGOGPWrVtnYWFx6dIl9W3wtgwyBgCgFlKpdNGiRUZGRs3NzdXV1fr6+levXn306BGJRCorK0MIHTt2bOLEic3NzQghuVxuZmYWEBCAELpw4YK+vv706dPnzp07YsQInB8SiWTMmDERERF45UKh0NjYOD4+HiHk7e2tzIOSkhJjY+Nr164pu7FmzZrvvvsOIbRv374uXboMHTpUV1d35cqV+FSQj49Pv379FixYMHfu3Llz5y5duhRfpLB161blWaWqqioSiXTt2rWGhgYDA4Nx48bhhe3s7MhkMj7lk5CQMHnyZLx7hBBis9mGhoZJSUkIoaamph07dowcOdLa2jo3N7cVtnybAhkDAFCXAwcO9O/fXywWI4TIZPLXX3/t5ua2dOlS/N1jx46NHz8enxiXSqWmpqZ4/8PCwsLV1RUvExAQMGnSJIRQY2OjmZnZnj17cHt6enqvXr0yMjIQQlu2bMHH0xBCYrF4+PDhynMwCCFLS8sdO3Y0NjZaWVmFhIS8ePHi5cuXyu8eOXLE2Nj4/Z5v3bp1wYIF+GuhUEgikRITE2UymZGR0blz5/7s/R49erRHjx4CgaCkpESZMVhzc/PMmTPnz5//77Zg+wcZAwBQmfr6+mXLloWFhSUnJ4eFhfXp02fbtm34W0+ePOnevXu3bt3u3buHWw4fPmxubq7MGENDQ39/f4TQvHnzJkyYcPfu3bNnz/br18/a2hoh1NTUZGVl1bdv32PHjiUnJ48ePdrW1hZfxBUQENC1a9eLFy/iyPHz8+vWrRuFQklOTvbw8NDW1sanUpYsWTJixIgZM2bMmDHj+++/x1eR4WNuK1asSE5OTk5O9vT0xPtAmzZtUu4bVVRUaGtr432m3bt39+7dOzQ0NDk5mUql/vTTTwqF4uzZs0ePHr13796uXbtIJJJQKOTz+bq6unfv3pVKpVu3bk1MTHz48KGlpaUyOzsOyBgAgMpIpVJ8VsPExGTYsGH79u1TXh8sl8snT55sZGSkvKI3MjJy+fLlTU1NCCGZTLZ48eLw8HCEEIvFWrx4sYmJydy5c319fT08PBBCDQ0NEyZM2LZt27fffmtiYmJvb19cXIzXIxQKly9fbmxsvHnzZvxCBw4cGDZsGF7DixcvEEKNjY2rVq1auXJlcHBwcHCwg4PDl19+ideQm5s7f/58ExMTExMTR0dHfKXyoUOH8OsihKqrq2fOnInP/cjl8kOHDhkbG5uYmIwePRofu3v48KGVlZWJicnkyZPxfZd8Pn/OnDn4DL+fnx9enkwm4wN0HQpkDACglUydOvWXX375uOfW19dbWFhERkZ+3NMfPHigra2tvKPlwYMH3bp1a3nQDKgJZAwAoDVcuXKFIIhXr1593NNFItGQIUPwBWkfIS8vz9jYeMqUKWQymUwmGxkZrV69Gh9qA2oFGQMAaA2JiYkHDx786I/1pqamo0ePPn/+/KM7UFxcvGXLFpwxFy5c+Oj1gH8FMgYAAIC6QMYAAABQF8gYAAAA6gIZAwAAQF0gYwAAavHq1auXL19WVlZquiNAkyBjAAAqxuFw5syZo6urSyKRTExMUlJSPrhYU1NTUVHRB29LbGxsLCoqemeCltZUV1dXUVGhqVf/nEDGAABUzM3NzdjYmM/n19bW2trazp49+4OLpaam9uzZc/DgwXisFyW5XL548WItLa1jx479xatIpdLa2lq5XK6SPkskkpaRtnr1aiMjIzwGAfgUkDEAABWzsbHBw+kjhBwdHZUD77/j3r17gwcPNjAw2LRpU8v2+/fvGxgYGBkZKUfA/CA6nT5q1Cg8lcun27dvn3KwToTQjRs3Tp8+DTdpfjoNZIxcLr9x44aaph2Vy+U3b97k8XjvfysuLs7f3185RWsb0QrTFmVlZf3ZwQoA1OHo0aN6enpsNtvf3793797379//4GJ4aEsfHx8DA4Py8nJl+4oVK9asWfPNN9/89ttvuKWoqCggIMDf3z88PBzPlclkMjds2NCtW7cdO3acPXu2ubmZz+dTqVTl3Mz379/Hk06WlpbevHmzrKzs6NGjeBKXp0+f+vv7+/v7K2elvHXrlo2NDR6U8/r16wghDofz+PFj/FoIITabfeTIEX9//5bv5fbt22w2OyMjw9/fXzmpGniHujKGwWCYmZmdP3/+/W81NTWNHj368uXL/3xtxcXFKSkpeA7UvyaVSq2srOLi4t5pP3PmTK9evaZNm6baEYpCQkLMzc3xjBFKcXFxZmZmeDa9v+Xu7u7m5vYRL/3o0aOWc6T/BS8vr4ULF37wWzQazdXV1cTExNzcHE8FiBCSy+VHjx7FQwTu2LFDIpEghKRS6YEDB0xMTIyNjX/77Tc85wdCqLCw0MnJaeXKlR/xFsBnzNjYuG/fvnPnzsWTLmdmZj579uydZZKTk01MTHJycoYPH378+HHcyGKxDAwMMjMz7ezs/Pz8EEIMBmPMmDFTp06dNm2agYHBsmXLxGJxSkrKmDH/1965BzV1bX88nSloioEJgRi1IqQ8RAKEl3LyKgHUQR5GfGCM0ajFNoFgQUVAeSnlIT44CFEGrHUk4gjYYkVA2hEItFXboghqtVLqiK3WB/JSQbLuH3vumVy58/v5h8ztY3/+OqwTds5kzjnftddaey8Pc3NzX19fmUz27NmzqqoqGo128+ZNNM6CBQvCwsIAwGAwMJlMb29vb2/vrVu3Hjx40NXVVSwWi8ViFouFHt6srKwZM2ZYWVmJxWIkbJmZmU5OTmgeU1lZyWaz/fz8xGIxk8mktlwLCAjg8XgikUgsFr/99ts7d+6c+N/1r8dEaUxycjKDwVi8eDHlCJgikUiQs/CanDlzxsXF5TUDr1KpdHzLuaVLl1I+0RskLi6OTqeLxWJqc1mj0bh06VI6nR4TE/M6IyQmJr4SKHhNfHx8ysrKXueTqampK1as+K+nysvLMzMz6+rqUlJSLC0tOzs7AUCn01lbW1dXV586dYrFYqWlpQFAfn4+m82uqak5efKklZVVXl4eAFy8eDEsLEwmk0kkkjcVFsf8DcjKyvL09JwyZYpGo0GW8PBwquUXRWNjo52dXV9f37Zt2zw8PJBx06ZNKH8jlUqRxvT19V27dg2d7erqsrCw+P777wGgvb19+vTpqN0ZAFRWVlpbW6NdkwFg6dKlyPW5dOmSmZkZ6oAJAN3d3VQyPzU1lcfjoeOUlJTg4GDq2vLy8nx9fQHgyZMn06ZNo3Tl5MmTZmZm6AKCg4Pd3d17e3sBYNeuXXZ2dtQsCkMxIRrT39/v7+9fUVHh6+uLdroGgIGBgejoaIIg9u3bJ5FI6uvrAeDcuXPBwcEEQSQmJiJ/uba2liTJQ4cOEQTxwQcfjIyMdHR0uLq6WlhYzJ07Fzk7Fy5cCAwMJAhi9+7dSMOGhoY+/PBDgiD27t0bEBBQU1Njej1ZWVksFsvR0VGpVL58+fLAgQMlJSXh4eFJSUkA8OTJE5VKRRBERETErVu3AODBgwfp6ekVFRVSqTQsLOzOnTvV1dUEQaxfv76vr890ZK1Wu3jxYolEQqlaW1tbaGjounXrPvroI2Q5ffq0UCgkCCI7O5uaihUXFwsEgvj4eJVKlZyc3NPTEx0d/fjxY3R2x44dr4QXjh07RhCEQCBA2pyQkMBgMLhcbnh4OLXDOQC8ePFCr9cLhUKBQLB3717khf0fGmOKm5vbiRMnAEAikVAtb8vKyng83sDAgFAopFoQFhQUeHp6jo2NdXd3//zzz/X19VKpFEeuMYhjx45NmjTpxo0bLS0tNBpt165dAEAQRGZm5iufbGxsnDlz5sOHD7u6uhgMxqVLl0ZGRhwcHCoqKgBAIpEgjQGA7u7uJUuWCAQCX19fKyurH3/8EQAMBgOHw0HPLABUVlYymczxGtPW1mZvb9/T00N9b3V1NXpGHB0dRSIRMpo2VwYTjWlubra1taViBi9fvnR3d0dRCqlUSnmuzc3NhwtP/AAAC05JREFUbDZ7glIAf2kmRGPq6uqCgoLGxsYUCgVy0o1Go0Kh8PLyIkkyJCTknXfeMRgMY2NjUVFReXl5JSUlDg4OqampAKDT6SwsLJRKJUmSjo6OsbGxDx482Lhx47vvvpubm2swGDo7O0UiUWlpqV6v9/f3R29DlUrl6elJkmRoaCidTjdtPwcA586d8/LyioyMPHr06MjIiEwmo9PpiYmJZ8+effHixfz58wMDA0mSVCqVLi4u9+/ff/TokY2NDZfL3bdvX2Bg4KxZs4KCgkiStLe3p5QDoVar1Wp1dnb2smXLkEWlUuXk5KSmpkZHRwPA+fPnmUzmjh07SJK0s7NLSUkBAL1eb21tvWfPnvj4eDqdnpaWNjw87ODggITqxo0bs2fPvn79OvUtSO30en1JSQmfz//uu+9Onz7N5XKjoqLKysqoFuIA0NPTEx4eXlxcXFRUZGNjg7pxvI7GdHZ2crnc7u7u4eFhLpfb0tKC7K2trbNnz75y5YqTkxPy3QCgoaHB0dGRctkqKyuxxmAoIiIi5s+fj46/+OILBoMRGRnp4OBgeksjkMag97JUKtVoNOXl5c7OzsjdpDSmtbWVw+Go1WqSJNPS0mxtbVFLGNS2ubu7G41WXV1ta2tL5WLlcvnKlSsBoK2tbdasWdRMKD09ncViZWdnHzhwYNmyZQRBIHtCQoJp/RulMV999ZWdnR3lyY2MjMyZM6eoqAgAAgMD0RONLsba2vo1w9f/KCZEY2JiYlAYp6Ghwc/Pb3R09N69e25ubigXMjQ05ObmdvbsWfThkZGR4eHh+Pj4yMhIACgqKhIKhejUmTNnUBvUlpYWPz8/ZExPT3/vvfdiYmK0Wu2cOXMWL17c19fH4/HQG/D58+fu7u6vzGMAYM2aNaWlpeg4JCQE9TICgNraWldXV6pmkc/nl5aWPnv2jMvlom593377LYvFQk6QTqd7//33TYdVq9UbNmzo7u5GlZr9/f1z5869c+fOtm3bNm7cCAAymQzNlgDg888/d3JyGhoaWr58+SeffIKMSqUSyXBMTMyqVasAgCRJ1H6cIjQ01N/fX6vVajQaS0vLnJwcAECq819//+fPnw8NDclkMtSC8HU0JiIiIj4+HgDu3r07a9YsSk5++OGH2bNnNzY2Ojs7U0+pwWBwcHD47bff0J9YYzCmaLVaNptNrXrZvXs3jUbjcrnj64CRxiBV0Ov1lpaWbDabqiWjNCYpKWnmzJnIeOHCBWtra9Tv0mAwTJkyBbWzBICWlhYrKyvUO+DevXsODg7r1q2D/9SYsbExFxcXKo6dnJzs7e2NjuPj4318fKhrozTml19+YTAYx48fR/bz58+bm5tfvHgR/lNjmpqaqBcFxpQ3rzEPHjywt7dftGiRRqNRKBTm5uZXrlzp7Oz09fWlAk2BgYFIYwoLC+3s7DgcDpPJ3LBhA7JQL8QbN264u7sPDw/X1tZ6eXkhx3nt2rV8Pl+r1cbGxiYlJdXV1XV0dPj4+FDLiRcsWDA+HxMVFUXV2i9atIjqQlFaWkp17UYfy8/PHxwc9Pb2RnPwb775xsPDA3XuO3LkSGhoqOmwarVaoVAAwMKFCz/99FO9Xi+TyQAgLi4ONdGTSqXULuLXr1/n8/m9vb1BQUFUOiolJUWr1QKAwWDw9PTs6+uTyWSmtRJGo5HP54eGhsbGxsbGxu7YsaO9vd1oNIpEovElFc+fP09OTuZwOBwOx9LSEinZ/6sxarU6KCgICe2TJ08cHR2bm5vRqaamJmdn566uLhcXF/RgA0BtbS2Xyx0cHER/Yo3BmPLrr7+6urryeDyNRqNSqVxcXFAc29vbm6rjQtTW1lpYWCDfv7+/n8Vi0Wg0Ktzk4eGxdetWAGhoaJg0aZJcLo+JibG3t6fRaOhW7O3tnTp1qlgsTkhIePjw4dOnT728vJydnTUajVQqZbFYyGlrampiMBhXr15Fw3788cc2NjYajSYqKsrCwsLLywvF248fP06j0dasWYOqADIyMhwdHVGWccuWLQwGY+PGjRqNZurUqVR/TB8fH8pbbWxspNFoVKQOQ/HmNaaysnLatGkqlUoulyuVSjc3t5iYmPv37zs7O3d0dADA4OCgq6trc3PzzZs32Wx2a2vr0NBQUlISuiGKiorEYjEaqqamhsfjjY6O1tTU8Pl8ZNy8eXN4eLjpN96+fdvJyam9vR0Anj17xuPxxs9jXtEY1DYcAKqqqpycnKjXpY+Pj16vHxgY4PP5XV1dANDa2urm5obev2VlZa/kLdVqNYr5Hj161MfHZ968eaikTavVohtx4cKFVLawqqqKz+cPDAyEhYWhuQgAKBQKyqsKDg5OS0uTSCSveENSqTQ3N9fUYjQa582bh0Jhppw6dWrGjBm3b9/u7++Xy+XIyTLVmPElGGlpaSEhIZR9dHTU29sbpfQBIDMzUygUDgwMeHp6ovgAACQmJkokEupf6uvrFyxYABjMv+nv79++fbtcLl+3bh2aEw8ODsbHx1PRC8RPP/2Unp5OpSH1er3posvCwsK6ujp03NDQsHr1aqVSaTAYSJKkHpCWlhaVShUbG4sixr29vZs2bZLL5W1tbV9++SVqmtnd3Z2RkUEtWhgdHS0oKJDL5WlpaU1NTcXFxUhIxsbGdDqdQqFA4fevv/56z549VCVLRUUFatWMckWIoqIiylm8detWcnIy3jhnPG9YY4xGY3Bw8ObNmylLdXU1k8l8/PjxqlWrhEIhSZLh4eFmZmYtLS09PT0zZsxIT08/dOgQh8NBC6A+++yzyZMnq1QqlMBARU2XL1+2sbFJTk5ua2u7fPkyk8mMjY0lSTI6Oho5HVFRUf7+/iRJymQyMzOz8UVrYWFh1HtTIpFQ8/G+vj5fX1+UbomMjPTz8xsaGrp37x6Xy0WK2NTUNHPmTDSFKi4ulkgkpsOuXbs2IiICAP744w8GgzF9+nR0r2/YsAFJZlVVlYWFRWpqakFBwbRp09AjdPjwYVtbW5IkExIS6HQ65Qrt37//rbfeQkE2U0pKSqZMmbJz506SJJcvX44Wu6xYsUIgEBw+fNi0lKW+vp7NZpMkuWfPHnNzcxSm27JlC5p+nT171snJyXTx0L59+2g0WnJy8sGDB0mSRIUGOp2OTqfn5OTk5uYymUz0UOXn51tYWOTn52dlZTGZTKTid+/eLSwsVCgUHA5n//79VBYHg8FgEG9YYwYHB9VqNRVUAYDHjx+vXr26o6Pj6dOnSqWSIIijR49mZ2cj76ayslIkEoWFhWVnZyO10Ol0ISEh2dnZBEFs376dKsTKzs4WCoUoKnrhwgVUjbZ+/XpUOPjo0aO1a9cSBHHkyJGcnJzxqxrz8vKoyU1GRoZpMO3333+Xy+UEQahUKrQQ7OHDh3FxcXfu3AGAa9euaTQalIQ8d+7cKwXQOp0uPz8fHefm5lKefklJCRWOO3HiBCpiOXz4MLK8fPkyLy+PIIi0tLTS0tIjR44g+9WrVydPnvyKrwcARqOxvLxcIBAQBJGRkYFk7PLly4GBgStXrjTdAMNoNBYWFgoEglWrVu3cuRPJQ3l5OdLXtra2JUuWUHkUACgoKAgJCQkICCAIAtXpUdcvEAgEAgH1QxmNRpIkBQKBSCSqra1Fxvb2dpFIFBAQsHDhQoFAcODAAcBgMBgT/nR7yeTn54+vo/+HUFlZ6enpSYUOMBgM5q/On05jysvL4+Li/tdX8b9Bq9VSMwkMBoP5G/Cn05h/MnipPAaD+ZuBNQaDwWAwEwXWGAwGg8FMFFhjMBgMBjNRYI3BYDAYzESBNQaDwWAwEwXWGAwGg8FMFFhjMBgMBjNRYI3BYDAYzESBNQaDwWAwE8W/AFbfItbSZ003AAAAAElFTkSuQmCC" />   During the very early prenatal period, genes corresponding to general cellular function are prominent (Mody et al., 2001).  These are followed in time by genes regulating neuronal differentiation and migration in the mid to late gestational period. From late gestation (gestational day 15) until birth almost all the cells in the CA fields switch from a highly active proliferation state to a postmitotic state, and then undergo differentiation and migration. Expression of proliferative genes involved in cell cycle progression are highly expressed at gestational day 16, then subsequently are silent immediately after birth when genes directing neuronal growth switch on. The pyramidal neurons of the CA fields in the hippocampus proper develop in advance of the granule cells that comprise the principal cells of the dentate gyrus. As such, the genes controlling the distinct phases of neurodevelopment are expressed at different times in these two hippocampal subregions (Altman and Bayer, 1990a; b). In both subregions, however, many phenotypic changes within the hippocampal neuron occur in the period immediately after birth (postnatal day 1 to 7). Almost all neurons show extensive growth and differentiation during the first postnatal week. These cellular changes are marked by rapid cytoskeletal changes, production of cell adhesion molecules, and extracellular matrix formation. The gene families involved in these processes include actins, tubulins, and chaperonin proteins essential for promoting correct protein folding of cytoskeletal components. Cell adhesion and extracellular matrix proteins are also upregulated during this period as these genes are critical for differentiation and synaptogenesis. During late postnatal hippocampal development (postnatal day 16-30), hippocampal circuits become more active and exhibit increased synaptic plasticity. Many genes upregulated during this phase of development are involved in synaptic function and include genes regulating vesicle associated proteins and calcium-mediated transmitter release, neurotrophins, and neurotransmitter receptors. Efficient energy utilization is essential during this period of increased synaptic activity, events mirrored by an upregulation of enzymes involved in glucose and oxidative metabolism.

Measurement of genomic profiles in developing brain use methods that are well established and accepted in the published literature.  Microarray studies with expression profile analyses have been conducted in cortex and hippocampus of humans (Zhang et al., 2002), non-human primates, and rodent brains of various ages (Mody et al., 2001; Royland et al., 2008; Dong et al., 2015). More commonly, quantitative rtPCR or in situ hybridization have been used to probe individual gene transcripts (Dowling et al., 2000, Morte et al., 2010) or their protein products (Alvarez-Dolado et al., 1994; Gilbert et al., 2007). Recently RNA-Seq technology was applied to T3-treated primary mouse cortical cells and gene targets enriched in astrocytes and neurons to identify TH-responsive genes (Gil-Ibanez et al, 2015).

Gene expression in the developing brain in general is analogous across most mammalian species (Kempermann, 2012). Most of the empirical data on gene expression in hippocampus is from rat, mouse and human studies.

UBERON:0000955

UBERON brain

High Female High Male

High

During brain development

High High Moderate

Altman J, Bayer SA. Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J Comp Neurol. 1990a Nov 15;301(3):365-81. Altman J, Bayer SA. Prolonged sojourn of developing pyramidal cells in the intermediate zone of the hippocampus and their settling in the stratum pyramidale. J Comp Neurol. 1990b Nov 15;301(3):343-64. Alvarez-Dolado M, Ruiz M, Del Rio JA, Alcantara S, Burgaya F, Sheldon M, Nakajima K, Bernal J, Howell BW, Curran T, Soriano E, Munoz A (1999) Thyroid hormone regulates reelin and dab1 expression during brain development. J Neurosci 19:6979-6993. Bernal J. (2105)  Thyroid Hormones in Brain Development and Function. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.. Dong H, You SH, Williams A, Wade MG, Yauk CL, Thomas Zoeller R (2015) Transient Maternal Hypothyroxinemia Potentiates the Transcriptional Response to Exogenous Thyroid Hormone in the Fetal Cerebral Cortex Before the Onset of Fetal Thyroid Function: A Messenger and MicroRNA Profiling Study. Cereb Cortex 25:1735-1745. Dowling AL, Zoeller RT. 2000. Thyroid hormone of maternal origin regulates the expression of RC3/neurogranin mRNA in the fetal rat brain. Brain Res: Molec Brain Res.  82:126-132. Gilbert ME, Sui L, Walker MJ, Anderson W, Thomas S, Smoller SN, Schon JP, Phani S, Goodman JH (2007) Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology 148:92-102. Gil-Ibanez P, Garcia-Garcia F, Dopazo J, Bernal J, Morte B. 2015. Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types. Cerebral cortex. Nov 2. Kempermann G.  New neurons for 'survival of the fittest'.  Nat Rev Neurosci. 2012 Oct;13(10):727-36. Mody M, Cao Y, Cui Z, Tay KY, Shyong A, Shimizu E, Pham K, Schultz P, Welsh D, Tsien JZ. Genome-wide gene expression profiles of the developing mouse hippocampus. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8862-7. Morte B, Ceballos A, Diez D, Grijota-Martinez C, Dumitrescu AM, Di Cosmo C, Galton VA, Refetoff S, Bernal J.  Thyroid hormone-regulated mouse cerebral cortex genes are differentially dependent on the source of the hormone: a study in monocarboxylate transporter-8- and deiodinase-2-deficient mice. Endocrinology. 2010. 151:2381-2387. Royland JE, Parker JS, Gilbert ME. A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. J Neuroendocrinol. 2008 Dec;20(12):1319-38. Zhang Y, Mei P, Lou R, Zhang MQ, Wu G, Qiang B, Zhang Z, Shen Y. Gene expression profiling in developing human hippocampus. J Neurosci Res. 2002 Oct 15;70(2):200-8. AOPWiki 2016-11-29T18:41:26

2018-08-11T09:26:56

Hippocampal Physiology, Altered

Tissue

The hippocampus functions as a highly integrated and organized communication and information processing network with millions of interconnections among its constitutive neurons. Neurons in the hippocampus and throughout the brain transmit and receive information largely through chemical transmission across the synaptic cleft, the space where the specialized ending of the presynaptic axon terminus of the transmitting neuron meets the specialized postsynaptic region of the neuron that is receiving that information (Kandell et al., 2012). During development (see KE 657: Hippocampal anatomy, Altered), as neurons reach their final destination and extend axonal processes, early patterns of electrical synaptic activity emerge in the hippocampus. These are large fields of axonal innervation of broad synaptic target sites that are replaced by more elaborate, but highly targeted and refined axonal projections and synaptic connectivity brought about by activity-dependent synaptic stabilization, pruning, or synapse elimination.  This is a classic case of the interaction between physiological and anatomical development, where anatomy develops first, and is ‘reshaped’ by physiological function (Kutsarova et al., 2017). In the rat, excitatory processes are fully mature in area CA1 of hippocampus within 2 weeks of birth with inhibitory processes lagging begin by several weeks (Muller et al., 1989; Michelson and Lothman, 1988; Harris and Teyler, 1984). In hippocampal slices, inhibitory function in area CA1 field is first seen on postnatal day 5, increasing in strength at postnatal day 12 through 15.  In vivo studies fail to detect inhibition until postnatal day 18 with steady increase thereafter to adult levels by postnatal day 28. Synaptic plasticity in the form of long-term potentiation (LTP) is absent in the very young animal, only emerging about postnatal day 14, appearing to require the stability of both excitatory and inhibitory function to be established (Muller et al., 1989; Bekenstein and Lothman, 1991). These features of the maturation of hippocampal physiology are paralleled in dentate gyrus, but as with anatomical indices in the rat, the development of these physiological parameters lag behind the CA1 by about 1 week. As described in structural development in KE4, a very similar pattern of maturation occurs in the human developing hippocampus, but with a trajectory that is largely complete before birth.

In animals, synaptic function in the hippocampus has been examined with imaging techniques, but more routinely, electrical field potentials recorded in two subregions of the hippocampus, area CA1 and dentate gyrus, have been assessed in vivo or in vitro. Field potentials recorded in both regions of the hippocampus reflect the summed synaptic response of a population of neurons following direct stimulation of input pathways across a monosynaptic connection. Changes in response amplitude due to chemical perturbations and other stressors (e.g., chemical exposures, nutritional deficits, gene knockouts) is evidence of altered synaptic function. This can be measured in vitro, in vivo, or in hippocampal slices taken from treated animals (Gilbert and Burdette, 1995). The most common physiological measurements used to assess the function of the hippocampus are excitatory synaptic transmission, inhibitory synaptic transmission, and synaptic plasticity in the form of LTP. Excitatory Synaptic Transmission: Two measures, the excitatory postsynaptic potential (EPSP) and the population spike are derived from the compound field potential at increasing stimulus strengths. The function described by the relationship of current strength (input, I) and evoked response (output, O), the I-O curve is the measure of excitatory synaptic transmission (Gilbert and Burdette, 1995). Inhibitory Synaptic Transmission: Pairs of stimulus pulses delivered in close temporal proximity are used to probe the integrity of inhibitory synaptic transmission. The response evoked by the second pulse of the pair at brief intervals (<30 msec) arrives during the activation of feedback inhibitory loops in the hippocampus. An alteration in the degree of suppression to the 2nd pulse of the pair reflects altered inhibitory synaptic function (Gilbert and Burdette, 1995). Long Term Potentiation (LTP): LTP is widely accepted to be a major component of the cellular processes that underlie learning and memory (Malenka and Bear, 2004; Bramham and Messaoudi, 2005). LTP represents, at the synapse and molecular level, the coincident firing of large numbers of neurons that are engaged during a learning event. The persistence of LTP emulates the duration of the memory. Synaptic plasticity in the form of LTP is assessed by delivering trains of high frequency stimulation to induce a prolonged augmentation of synaptic response. Probe stimuli at midrange stimulus strengths are delivered before and after application of LTP-inducing trains. The degree of increase in EPSP and PS amplitude to the probe stimulus after train application, and the duration of the induced synaptic enhancement are metrics of LTP. Additionally, contrasting I-O functions of excitatory synaptic transmission before and after (hours to days) LTP is induced is also a common measure of LTP maintenance (Bramham and Messaoudi, 2005; Kandell et al., 2012; Malenka and Bear, 2004). LTP has been assessed also using in vitro neuronal networks (Odawara et al., 2016; Pre et al., 2022). Excitatory and inhibitory synaptic currents (EPSCs and IPSCs) can also be measured in single cells, mostly ex vivo within slices of hippocampus using intracellular and patch clamp techniques as described in previous KEs. These same outputs can evaluate the integrity of synaptic transmission and synaptic plasticity. Synaptic function in the human hippocampus has been assessed using electroencephalography (EEG) and functional neuroimaging techniques (Clapp et al., 2012). EEG is a measure of electrical activity over many brain regions but primarily from the cortex using small flat metal discs (electrodes) placed over the surface of the skull. It is a readily available test that provides evidence of how the brain functions over time. Functional magnetic resonance imaging or functional MRI (fMRI) uses MRI technology to measure brain activity by detecting associated changes in blood flow. This technique relies on the fact that cerebral blood flow and neuronal activation are coupled. Positron emission tomography (PET) is a functional imaging technique that detects pairs of gamma rays emitted indirectly by a radionuclide (tracer) injected into the body (Tietze, 2012; McCarthy, 1995). Like fMRI, PET scans indirectly measure blood flow to different parts of the brain – the higher the blood flow, the greater the activation (McCarthy, 1995). These techniques have been widely applied in clinical and research settings to assess learning and memory in humans and can provide information targeted to hippocampal functionality (McCarthy, 1995; Smith and Jonides, 1997; Willoughby et al., 2014; Wheeler et al., 2015; Gilbert et al., 1998). Assays of this type are fit for purpose, have been well accepted in the literature, and are reproducible across laboratories. The assay directly measures the key event of altered neurophysiological function.

Most of the evidence for this key event comes from work in rodent species (i.e., rat, mouse). There is a moderate amount of evidence from other species, including humans (Clapp et al., 2012).

UBERON:0000955

UBERON brain

High Female High Male

High

During brain development

Moderate High High

Bekenstein JW, Lothman EW. An in vivo study of the ontogeny of long-term potentiation (LTP) in the CA1 region and in the dentate gyrus of the rat hippocampal formation. Brain Res Dev Brain Res. 1991 Nov 19;63(1-2):245- Bramham CR, Messaoudi E (2005) BDNF function in adult synaptic plasticity: the synaptic consolidation hypothesis. Prog Neurobiol 76:99-125. Clapp WC, Hamm JP, Kirk IJ, Teyler TJ. Translating long-term potentiation from animals to humans: a novel method for noninvasive assessment of cortical plasticity. Biol Psychiatry. 2012 Mar 15;71(6):496-502. Gilbert, M.E. and Burdette, L.J. (1995). Hippocampal Field Potentials: A Model System to Characterize Neurotoxicity. In Neurotoxicology: Approaches and Methods. L.W Chang and W. Slikker (Eds). Academic Press:New York, 183-204. Gilbert ME, Mack CM. Chronic lead exposure accelerates decay of long-term potentiation in rat dentate gyrus in vivo. Brain Res. 1998 Apr 6;789(1):139-49. Harris KM, Teyler TJ. Developmental onset of long-term potentiation in area CA1 of the rat hippocampus. J Physiol. 1984. 346:27-48. Kandell, E., Schwartz, J., Siegelbaum, A. and Hudspeth, A.J.  (2012) Principles of Neural Science, 5th Edition.  Elsevier, North Holland. Kutsarova E, Munz M, Ruthazer ES.  Rules for Shaping Neural Connections in the Developing Brain.  Front Neural Circuits. 2017 Jan 10;10:111. doi: 10.3389/fncir.2016.00111. Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44:5-21. McCarthy, G. (1995) Review: Functional Neuroimaging and Memory. The Neuroscientist, 1:155-163. Michelson HB, Lothman EW. An in vivo electrophysiological study of the ontogeny of excitatory and inhibitory processes in the rat hippocampus. Brain Res Dev Brain Res. 1989 May 1;47(1):113-22. Muller D, Oliver M, Lynch G. Developmental changes in synaptic properties in hippocampus of neonatal rats. Brain Res Dev Brain Res. 1989 Sep 1;49(1):105-14. OECD Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity (DNT) In-Vitro Testing Battery; Series on Testing and Assessment No. 377. 2023. Available at: https://one.oecd.org/document/ENV/CBC/MONO(2023)13/en/pdf Smith, E and Jonides, J. (1997). Working Memory: A View from Neuroimaging. Cognitive Psychology, 33:5-42. Tietze, KJ. (2012). Review of Laboratory and Diagnostic Tests- Positron Emission Tomography. In Clinical Sills for Pharmacists, 3rd Edition, pp 86-122.  Wheeler SM, McLelland VC, Sheard E, McAndrews MP, Rovet JF (2015) Hippocampal Functioning and Verbal Associative Memory in Adolescents with Congenital Hypothyroidism. Front Endocrinol (Lausanne) 6:163. Willoughby KA, McAndrews MP, Rovet JF (2014) Effects of maternal hypothyroidism on offspring hippocampus and memory. Thyroid 24:576-584. AOPWiki 2016-11-29T18:41:26

2024-07-24T23:15:42

Cognitive function, decreased

Individual

Learning and memory depend upon the coordinated action of different brain regions and neurotransmitter systems constituting functionally integrated neural networks (D’Hooge and DeDeyn, 2001). Among the many brain areas engaged in the acquisition or retrieval of a learned event, the hippocampal-based memory systems have received the most study. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990; Squire, 2004; Gilbert., 2006).  In humans, the hippocampus is involved in recollection of an event’s rich spatial-temporal contexts and distinguished from simple semantic memory which is memory of a list of facts (Burgess et al., 2000). Hemispheric specialization has occurred in humans, with the left hippocampus specializing in verbal and narrative memories (i.e., context-dependent episodic or autobiographical memory) and the right hippocampus, more prominently engaged in visuo-spatial memory (i.e., memory for locations within an environment). The hippocampus is particularly critical for the formation of episodic memory, and autobiographical memory tasks have been developed to specifically probe these functions (Eichenbaun, 2000; Willoughby et al., 2014). In rodents, there is obviously no verbal component in hippocampal memory, but reliance on the hippocampus for spatial, temporal and contextual memory function has been well documented. Spatial memory deficits and fear-based context learning paradigms engage the hippocampus, amygdala, and prefrontal cortex (Eichenbaum, 2000; Shors et al., 2001; Samuels et al., 2011; Vorhees and Williams, 2014; D’Hooge and DeDeyn, 2001; Lynch, 2004; O’Keefe and Nadal, 1978). These tasks are impaired in animals with hippocampal dysfunction (O’Keefe and Nadal, 1978; Morris and Frey, 1987; Gilbert et al., 2016).

In rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, and most commonly, the Morris water maze (MWM). Tests such as novel object recognition, and fear-based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. The text below provides brief descriptions of the most used tasks. <ol> <li>RAM, Barnes Maze, and MWM are examples of spatial tasks in which animals are required to learn: the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze); or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014). </li> <li>Novel Object Recognition (NOR) and its variants are widely used in neuroscience, although their suitability for safety assessment remains unclear (Vorhees and Williams, 2024). NOR and novel place recognition (NPR) are examples of ‘incidental learning’ and rely on the dorsal hippocampus. They are simple tasks and are used to probe recognition memory. Two objects are presented to animals in an open field on trial 1, and animals are allowed time to briefly explore them. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention (i.e., one of these objects is familiar, the other is novel (Cohen and Stackman, 2015). In novel place recognition, the objects are shifted to a location within the arena. Compared to tests of spatial learning, the learning event is transient, the results often variable, and the test has a very narrow dynamic range.</li> <li>Contextual Fear Conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event (unconditional stimulus, US). The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009). </li> <li>Trace Fear Conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, e.g., a light or a tone) and an aversive stimulus (US, e.g., a foot shock). The unconditioned response (CRUR) that is elicited upon delivery of the foot shock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2004). </li> </ol> Most methods used in animals are well established in the published literature, and many have been engaged to evaluate the effects of developmental neurotoxicants. The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 or OECD 426) both require testing of learning and memory (USEPA, 1998; OECD, 2007). These DNT Guidelines have been deemed valid to identify DNT and adverse neurodevelopmental outcomes (Makris et al., 2009).  A variety of standardized learning and memory tests have been developed for human neuropsychological testing. These include episodic autobiographical memory, word pair recognition memory; object location recognition memory. Some components of these tests have been incorporated in general tests of adult intelligence (IQ) such as the Wechsler Adult Intelligence Scale (WAIS) which calculates four composite scores that examine various domains within an individual’s overall cognitive ability: Verbal Comprehension Index (VCI), Perceptual Reasoning Index (PRI), Working Memory Index (WMI), and Processing Speed Index (PSI) (Climie and Rostad, 2011). Modifications have been made and norms developed for incorporating tests of learning and memory in children. Examples of some of these tests include:  <ol> <li>Rey Osterieth Complex Figure (RCFT) which probes a variety of functions including visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006). </li> <li>Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1994; Talley, 1986).  </li> <li>Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994). </li> <li>Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neuropsychological test designed to measure memory functions (Lezak, 1994; Talley, 1986). </li> <li>Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2011). </li> <li>Staged AM Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buying lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014). </li> </ol>

Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans.Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans.

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Alexander RD (1990) Epigenetic rules and Darwinian algorithms: The adaptive study of learning and development. Ethology and Sociobiology 11:241-303.  Bellinger DC (2012) A strategy for comparing the contributions of environmental chemicals and other risk factors to neurodevelopment of children. Environ Health Perspect 120:501-507.  Burgess N (2002) The hippocampus, space, and viewpoints in episodic memory. Q J Exp Psychol A 55:1057-1080.  Climie, E. A., & Rostad, K. (2011). Test Review: Wechsler Adult Intelligence Scale. Journal of Psychoeducational Assessment, 29(6), 581–586. https://doi.org/10.1177/0734282911408707  Cohen, SJ and Stackman, RW. (2015). Assessing rodent hippocampal involvement in the novel object recognition task. A review. Behav. Brain Res. 285: 105-1176.  Curzon P, Rustay NR, Browman KE. Cued and Contextual Fear Conditioning for Rodents. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2009  D'Hooge R, De Deyn PP (2001) Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev 36:60-90.  Eichenbaum H (2000) A cortical-hippocampal system for declarative memory. Nat Rev Neurosci 1:41-50.  Fivush R. The development of autobiographical memory. Annu Rev Psychol. 2011. 62:559-82.  Gilbert ME, Sanchez-Huerta K, Wood C (2016) Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787.  Gilbert ME, Sui L (2006) Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22.  Herold, C, Lässer, MM, Schmid, LA, Seidl, U, Kong, L, Fellhauer, I, Thomann, PA, Essig, M and Schröder, J. (2015). Neuropsychology, Autobiographical Memory, and Hippocampal Volume in “Younger” and “Older” Patients with Chronic Schizophrenia. Front. Psychiatry, 6: 53.  Lezak MD (1984) Neuropsychological assessment in behavioral toxicology--developing techniques and interpretative issues. Scand J Work Environ Health 10 Suppl 1:25-29.  Lezak MD (1994) Domains of behavior from a neuropsychological perspective: the whole story. Nebr Symp Motiv 41:23-55.  Lynch, M.A. (2004). Long-Term Potentiation and Memory. Physiological Reviews. 84:87-136.  Makris SL, Raffaele K, Allen S, Bowers WJ, Hass U, Alleva E, Calamandrei G, Sheets L, Amcoff P, Delrue N, Crofton KM. A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426. Environ Health Perspect. 2009 Jan;117(1):17-25.  Morris RG, Frey U. Hippocampal synaptic plasticity: role in spatial learning or the automaticrecording of attended experience? Philos Trans R Soc Lond B Biol Sci. 1997 Oct 29;352(1360):1489-503. Review  O’Keefe, J. and Nadel, L. (1978). The Hippocampus as a Cognitive Map. Oxford: Oxford University Press.  OECD. 2007. OECD guidelines for the testing of chemicals/ section 4: Health effects. Test no. 426: Developmental neurotoxicity study.  www.Oecd.Org/dataoecd/20/52/37622194.Pdf [accessed May 21, 2012].  OECD Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity (DNT) In-Vitro Testing Battery; Series on Testing and Assessment No. 377. 2023. Available at: https://one.oecd.org/document/ENV/CBC/MONO(2023)13/en/pdf Samuels BA, Hen R (2011) Neurogenesis and affective disorders. Eur J Neurosci 33:1152-1159.  Shin, MS, Park, SY, Park, SR, Oeol, SH and Kwon, JS. (2006). Clinical and empirical appliations fo the Rey-Osterrieth complex figure test. Nature Protocols, 1: 892-899.  Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (2001) Neurogenesis in the adult is involved in the formation of trace memories. Nature 410:372-376. Squire LR (2004) Memory systems of the brain: a brief history and current perspective. Neurobiol Learn Mem 82:171-177.  Stanton ME, Spear LP (1990) Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group I report: comparability of measures of developmental neurotoxicity in humans and laboratory animals. Neurotoxicol Teratol 12:261-267.  Talley, JL. (1986). Memory in learning disabled children: Digit span and eh Rey Auditory verbal learning test. Archives of Clinical Neuropsychology, Elseiver.  U.S.EPA. 1998. Health effects guidelines OPPTS 870.6300 developmental neurotoxicity study. EPA Document 712-C-98-239.Office of Prevention Pesticides and Toxic Substances.  Vorhees CV, Williams MT (2014) Assessing spatial learning and memory in rodents. ILAR J 55:310-332.  Vorhees, C and Williams M. Tests for Learning and Memory in Rodent Regulatory Studies. Current Research in Toxicology, 2024, Curr Res Toxicol. 2024; 6: 100151. doi: 10.1016/j.crtox.2024.100151 Willoughby KA, McAndrews MP, Rovet JF. Accuracy of episodic autobiographical memory in children with early thyroid hormone deficiency using a staged event. Dev Cogn Neurosci. 2014. 9:1-11.  AOPWiki 2016-11-29T18:41:24

2024-07-25T17:23:57

<upstream-id>6767a62d-514c-4b44-913c-00a0fa252d5b</upstream-id> <downstream-id>726fa2fd-e318-4d33-ba39-fef3dc708eb6</downstream-id>

AOPWiki 2023-04-12T11:25:14

2023-04-12T11:25:14

<upstream-id>d69be190-b213-448a-ba05-bdfa8e99ac98</upstream-id> <downstream-id>6e5c1096-220b-44b4-8ff1-90bb18b7aece</downstream-id> Thyroid hormones (TH) are critical for normal development of the structure and function of the brain, including hippocampal development and cognitive function (Anderson et al., 2003; Bernal, 2007; Willoughby et al., 2014).   Brain concentrations of T4 are dependent on transfer of T4 from serum, through the vascular endothelia, into astrocytes.  In astrocytes, T4 is converted to T3 by deiodinase and subsequently transferred to neurons cellular membrane transporters. In the brain T3 controls transcription and translation of genes responsible for normal hippocampal structural and functional development. Clearly the brain circuitry controlling cognitive function is complex and is not solely accomplished by the functionality of the hippocampus. However, it is well documented that normal hippocampal structure and physiology are critical for the development of cognitive function. Thus, there is an indisputable indirect link between serum T4 and cognitive function.

The weight of evidence for this indirect relationship is strong. Alterations in serum TH concentrations are very well correlated with adverse impacts on cognitive behaviors such as learning and memory. This includes a large amount of literature, from more than four decades of research, that links hypothyroidism and/or hypothyroxinemia with alterations in spatial cognitive function, a hippocampal dependent behavior. A number of reviews are cited below that are primarily from humans and rodents, but this indirect relationship has also been shown for a number of other species. In humans, severe serum TH reductions that accompany congenital hypothyroidism dramatically impair brain function and lead to severe mental retardation. Lower global IQ scores, language delays and weak verbal skills, motor weakness, attentional deficits and learning impairments accompany low serum TH in children (Derksen-Lubsen and Verkerk 1996). Standard tests of IQ function in children born to mothers with even marginal hypothyoidism during pregnancy or in children with a defective thyroid gland who are then treated remain approximately 6 points below expected values. Selective deficits on visual spatial, motor, language, memory and attention tests are observed, the exact phenotype largely dependent on the developmental window over which the insufficiency occurred and the severity of the hormone deficit (Mirabella et al. 2000; Rovet 2002; Zoeller and Rovet 2004; Willoughby et al 2014). Indeed, this link is recognized as being so clinically important that T4 and TSH are monitored in all newborns in the US.    In rodent models, reductions in serum TH induced by TPO inhibitors such as MMI and PTU, when induced during development, lead to a variety of neurobehavioral impairments. These impairments can occur in the sensory, motor, and cognitive domains. The specific phenotype is dependent on both the window of exposure, the duration of exposure, and the severity of the hormone reduction (Zoeller and Rovet, 2004).  This includes more than four decades of work linking serum TH changes to alterations in hippocampal-dependent spatial behaviors (Akaike et al., 2004; Axelstand etal., 2008; Brosvic et al; Kawada et al, 1988; Friedhoff et al, 2000; Gilbert and Sui, 2006; Gilbert et al., 2016; Gilbert, 2011).

The biological plausibility of this KER is rated as strong. The relationship is consistent with the known biology of how the relationship between serum TH concentrations, brain TH concentrations, and TH control of brain development.

Empirical support for this KER is rated as strong. Empirical data from studies that measure serum TH concentrations and then assess alterations in cognitive function, including hippocampal dependent behaviors, is vast. The qualitative relationship between reduced serum hormone levels and adverse cognitive outcomes is well accepted in endocrinology, as well as developmental neuroendocrinology. Indeed, the relationship between serum T4 and T3 levels and adverse neurodevelopmental outcomes (e.g., IQ loss in children) is beyond reproach. Temporal Evidence: The temporal nature of this KER is developmental (Seed et al., 2005). It is a well-recognized fact that there are critical developmental windows for disruption of the serum THs that result in cognitive function.  In humans, hormone insufficiency that occurs in mid-pregnancy due to maternal drops in serum hormone, and that which occurs in late pregnancy due to disruptions in the fetal thyroid gland lead to different patterns of cognitive impairment (Zoeller and Rovet, 2004). In animal models, deficits in hippocampal-dependent cognitive tasks result from developmental, but not adult hormone deprivation (Gilbert and Sui, 2006; Gilbert et al., 2016; Axelstad et al, 2009; Gilbert, 2011; Opazo et al., 2008). Replacement studies have demonstrated that varying adverse neurobehavioral outcomes, including cognitive function, can be reduced or eliminated if T4 (and/or T3) treatment is given during the critical windows (e.g., Kawada et al., 1988; Goldey and Crofton, 1998; Reid et al., 2007). Dose-Response Evidence: An increasing amount of literature is now available that provides clear evidence of the ‘dose-response’ nature of this KER.  Most research over that last 40 years has employed high doses of chemicals, or chemicals plus thyroidectomies, that results in severe depletion of circulating thyroid hormones. More recently, researchers produced graded degrees of TH insufficiency in dams and pups by administering varying doses of chemicals and have correlated them to the dose-dependency of the observed effects.  This work has provided increased confidence in the relationship between serum TH decrements and a variety of neurodevelopmental impairments, and also to the specificity of the observed effects on brain development that is directly mediated by TH insufficiency (Goldey et al., 1995; Crofton, 2004; Gilbert and Sui, 2006; Gilbert, 2011; Bastian et al., 2014; Royland et al., 2008; Sharlin et al., 2008).

There are no inconsistencies in this KER, but there are some remaining uncertainties. It is widely accepted that changes in serum THs during development will result in alterations in behavior controlled by the hippocampus. This has been repeatedly demonstrated in animal models and in humans. A major uncertainty is the precise relationship between the degree, timing and duration of serum TH changes that leads to these behavioral deficits. Inconsistencies may also exist for chemicals other than classical TPO inhibitors that may reduce serum TH and induce impairments in cognitive function, but through action on other endocrine systems, or via direct action on the brain in the absence of an intervening endocrine action.

Except for a quantitative relationship between serum T4 and hearing loss in rodents (Crofton, 2004), there are no other reports of development of quantitative predictive models linking serum TH and adverse neurological outcomes. Insufficient data exist to develop a quantitative predictive model of adverse cognitive outcomes from serum TH concentrations. However, evidence from human studies suggests that decreases as low as 25% in serum T4 in pregnant women will yield small decrements in IQ in children (e.g., Haddow et al., 1995). Since publication of this seminal paper, several reports have appeared providing supportive if not direct confirmatory data on the association of reductions in maternal or early postnatal serum TH and adverse neurodevelopmental outcomes (e.g., Rovet and Willoughby, 2010, Wheeler et al., 2011, Willoughby et al., 2014, Wheeler et al., 2015; Pop et al., 1999, Pop et al., 2003, Kooistra et al., 2006, Henrichs et al., 2010, Korevaar et al., 2016). Based on these data, regulatory authorities have used 10 and/or 20% changes in serum T4 as a point of departure for hazard assessments in rodent studies (EPA, 2011).

High Male High Female

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During brain development

High High High High High Moderate Moderate

Taxonomic: There is a plethora of data supporting this KER in rats, mice, and humans. According to the evaluation of the empirical taxonomic domain of applicability (tDOA) of an adverse outcome pathway network for thyroid hormone system disruption (THSD) by Haigis et al., 2023, the level of confidence for a linkage between THSD and developmental neurotoxicity (DNT) in general was considered high for mammals and fish and moderate for amphibians and birds. However, evidence specifically linking THSD to decreased cognitive function in nonmammalian vertebrates is generally missing. No empirical evidence linking THSD to decreased cognitive function was found for reptiles, but this taxon is considered part of the plausible tDOA based on the evaluation by Haigis et al., 2023. Scientific inference and the presence of comparable neurological structures indicate that decreased cognitive function can plausibly be measured and is plausibly linked to THSD (Hussan et al., 2022, Naumann et al., 2015, Rivera and Lock, 2008).

Akaike M, Kato N, Ohno H, Kobayashi T (1991) Hyperactivity and spatial maze learning impairment of adult rats with temporary neonatal hypothyroidism. Neurotoxicol Teratol 13:317-322. Anderson GW, Schoonover CM, Jones SA (2003) Control of thyroid hormone action in the developing rat brain. Thyroid 13:1039-56. Axelstad M, Hansen PR, Boberg J, Bonnichsen M, Nellemann C, Lund SP, Hougaard KS, Hass U. Developmental neurotoxicity of propylthiouracil (PTU) in rats: relationship between transient hypothyroxinemia during development and long-lasting behavioural and functional changes. Toxicol Appl Pharmacol. 2008 Oct 1;232(1):1-13 Bastian TW, Prohaska JR, Georgieff MK, Anderson GW (2014) Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression. Endocrinology 155:1157-1167. Bernal J. 2007. Thyroid hormone receptors in brain development and function. Nature clinical practice Endocrinology & metabolism. 3:249-259. Brosvic GM, Taylor JN, Dihoff RE. (2002). Influences of early thyroid hormone manipulations: delays in pup motor and exploratory behavior are evident in adult operant performance. Physiol Behav. Apr 15;75(5):697-715. Crofton KM. Developmental disruption of thyroid hormone: correlations with hearing dysfunction in rats. Risk Anal. 2004 Dec;24(6):1665-71. Derksen-Lubsen, G. and P. H. Verkerk (1996). "Neuropsychologic development in early treated congenital hypothyroidism: analysis of literature data." Pediatr Res 39(3): 561-6. EPA (2011) FIFRA Scientific Advisory Panel Consultation, Integrated Approaches to Testing and Assessment Strategy:Use of New Computational and Molecular Tools, US Environmental Protection Agency, Office of Pesticide Programs, Washington DC May 24-26, 2011. Friedhoff AJ, Miller JC, Armour M, Schweitzer JW, Mohan S. Role of maternal biochemistry in fetal brain development: effect of maternal thyroidectomy on behaviour and biogenic amine metabolism in rat progeny. Int J Neuropsychopharmacol. 2000 Jun;3(2):89-97. Gilbert ME. Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci. 2011;124(2):432-445. Gilbert ME, Sui L. Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res. 2006 Jan 19;1069(1):10-2 Gilbert ME, Sanchez-Huerta K, Wood C (2016) Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787. Goldey ES, Kehn LS, Rehnberg GL, Crofton KM. Effects of developmental hypothyroidism on auditory and motor function in the rat. Toxicol Appl Pharmacol. 1995 Nov;135(1):67-76. Goldey ES, Crofton KM. (1998) Thyroxine replacement attenuates hypothyroxinemia, hearing loss, and motor deficits following developmental exposure to Aroclor 1254 in rats.  Toxicol Sci. 45:94-105. Haddow, J. E., G. E. Palomaki, et al. (1999). "Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child." N Engl J Med 341(8): 549-55. Haigis A-C., Vergauwen L., LaLone C.A., Villeneuve D.L., O'Brien J.M., Knapen D. (2023). Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption. Toxicol Sci. 195, 1-27. Henrichs J, Bongers-Schokking JJ, Schenk JJ, Ghassabian A, Schmidt HG, Visser TJ, Hooijkaas H, de Muinck Keizer-Schrama SM, Hofman A, Jaddoe VV, Visser W, Steegers EA, Verhulst FC, de Rijke YB, Tiemeier H (2010) Maternal thyroid function during early pregnancy and cognitive functioning in early childhood: the generation R study. J Clin Endocrinol Metab 95:4227-4234. Hussan, M. T., Sakai, A., and Matsui, H. (2022). Glutamatergic pathways in the brains of turtles: A comparative perspective among reptiles, birds, and mammals. Front. Neuroana. 16, 937504. Kawada J, Mino H, Nishida M, Yoshimura Y. (1988) An appropriate model for congenital hypothyroidism in the rat induced by neonatal treatment with propylthiouracil and surgical thyroidectomy: studies on learning ability and biochemical parameters.  Neuroendocrinology. 47:424-30. Kooistra L, Crawford S, van Baar AL, Brouwers EP, Pop VJ (2006) Neonatal effects of maternal hypothyroxinemia during early pregnancy. Pediatrics 117:161-167. Korevaar TI, Muetzel R, Medici M, Chaker L, Jaddoe VW, de Rijke YB, Steegers EA, Visser TJ, White T, Tiemeier H, Peeters RP (2016) Association of maternal thyroid function during early pregnancy with offspring IQ and brain morphology in childhood: a population-based prospective cohort study. Lancet Diabetes Endocrinol 4:35-43. Mirabella, G., D. Feig, et al. (2000). "The effect of abnormal intrauterine thyroid hormone economies on infant cognitive abilities." J Pediatr Endocrinol Metab 13(2): 191-4. Naumann, R. K., Ondracek, J. M., Reiter, S., Shein-Idelson, M., Tosches, M. A., Yamawaki, T. M., and Laurent, G. (2015). The reptilian brain. Curr. Biol. 25, R317–R321. Opazo MC, Gianini A, Pancetti F, Azkcona G, Alarcón L, Lizana R, Noches V, Gonzalez PA, Marassi MP, Mora S, Rosenthal D, Eugenin E, Naranjo D, Bueno SM, Kalergis AM, Riedel CA (2008), Maternal hypothyroxinemia impairs spatial learning and synaptic nature and function in the offspring. Endocrinology 149:5097-5106. Pop VJ, Brouwers EP, Vader HL, Vulsma T, van Baar AL, de Vijlder JJ (2003) Maternal hypothyroxinaemia during early pregnancy and subsequent child development: a 3-year follow-up study. Clin Endocrinol (Oxf) 59:282-288. Pop VJ, Kuijpens JL, van Baar AL, Verkerk G, van Son MM, de Vijlder JJ, Vulsma T, Wiersinga WM, Drexhage HA, Vader HL (1999) Low maternal free thyroxine concentrations during early pregnancy are associated with impaired psychomotor development in infancy. Clin Endocrinol (Oxf) 50:149-155 Reid RE, Kim EM, Page D, O'Mara SM, O'Hare E. Thyroxine replacement in an animal model of congenital hypothyroidism.Physiol Behav. 2007 91(2-3):299-303. Epub 2007 Mar 15. Rivera, S., and Lock, B. (2008). The reptilian thyroid and parathyroid glands. Vet. Clin. North Am. Exot. Anim. Pract. 11, 163–175, viii. Rovet, J. F. (2002). Congenital hypothyroidism: an analysis of persisting deficits and associated factors." Child Neuropsychol 8(3): 150-62. Royland JE, Parker JS, Gilbert ME. 2008a. A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. Journal of neuroendocrinology. Dec;20:1319-1338. Sharlin DS, Tighe D, Gilbert ME, Zoeller RT (2008) The balance between oligodendrocyte and astrocyte production in major white matter tracts is linearly related to serum total thyroxine. Endocrinology 149:2527-2536. Willoughby KA, McAndrews MP, Rovet J. Effects of maternal hypothyroidism on offspring hippocampus and memory. Thyroid, 2014;24:576-584. Zoeller RT, Rovet J. Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings. J Neuroendocrinol. 2004 Oct;16(10):809-18.   AOPWiki 2016-11-29T18:41:34

2026-04-27T05:59:04

<upstream-id>726fa2fd-e318-4d33-ba39-fef3dc708eb6</upstream-id> <downstream-id>32fa070f-b0ac-44ed-bea0-0efbe536afe3</downstream-id>

Not Specified Not Specified Not Specified

AOPWiki 2021-09-10T08:32:29

2021-09-10T08:32:29

<upstream-id>32fa070f-b0ac-44ed-bea0-0efbe536afe3</upstream-id> <downstream-id>d69be190-b213-448a-ba05-bdfa8e99ac98</downstream-id>

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AOPWiki 2021-01-26T10:42:59

2021-01-26T10:42:59

<upstream-id>d69be190-b213-448a-ba05-bdfa8e99ac98</upstream-id> <downstream-id>9844a59e-a802-436d-8a8e-2d3a91514c91</downstream-id> In mammals, thyroxine (T4) in brain tissue is derived almost entirely from the circulating pool of T4 in blood. Transfer of free T4 (and to a lesser extent, T3) from serum binding proteins (thyroid binding globulin (TBG), transthyretin (TTR) and albumin; see McLean et al., 2017, for a recent review) into the brain requires transport across the blood brain barrier (BBB) and /or indirect transport from the cerebral spinal fluid (CSF) into the brain through the blood-CSF-barrier.  The blood vessels in rodents and humans expresses the main T4 transporter, MCT8, (Roberts et al. 2008), as does the choroid plexus which also expresses TTR and secretes the protein into the CSF (Alshehri et al. 2015). T4 entering the brain through the BBB is taken up into astrocytes via cell membrane iodothyronine transporters (e.g., organic anion-transporting polypeptides OATP), monocarboxylate transporter 8 (MCT8) (Visser et al., 2011).  In astrocytes, T4 is then deiodinated by Type II deiodinase to triiodothyronine (T3) (St Germain and Galton, 1997), which is then transported via other iodothyronine transporters (MCT8) into neurons (Visser et al., 2011). While some circulating T3 may be taken up into brain tissue directly from blood (Dratman et al., 1991), the majority of neuronal T3 comes from deiodination of T4 in astrocytes. Decreases in circulating T4 will eventually result in decreased brain T3 tissue concentrations. It is also known that Type II deiodinase can be up-regulated in response to decreased T4 concentrations to maintain tissue concentrations of T3 (Pedraza et al., 2007; Lavado-Autric et al., 2013; Morse et al., 1986), except in tanycytes of the paraventricular nucleus (Fekete and Lechan, 2014).

The weight of evidence linking reductions in circulating serum TH and reduced brain concentrations of TH is moderate. Many studies support this basic linkage. However, there are compensatory mechanisms (e.g., upregulation of deiodinases, transporters) that may alter the relationship between hormones in the periphery and hormone concentrations in the brain. There is limited information available on the quantitative relationship between circulating levels of TH, these compensatory processes, and neuronal T4 concentrations, especially during development. Furthermore, in certain conditions, such as iodine deficiency, the decreases in circulating hormone might have greater impacts on tissue levels of TH (see for instance, Escobar del Rey, et al., 1989).

The biological relationship between these two KEs is strong as it is well accepted dogma within the scientific community. There is no doubt that decreased circulating T4 leads to declines in tissue concentrations of T4 and T3 in a variety of tissues, including brain. However, compensatory mechanisms (e.g., increased expression of Type 2 deiodinase) may differ during different lifestages and across different tissues, especially in different brain regions.  Similarly, the degree to which serum TH must drop to overwhelm these compensatory responses has not been established.

Several studies have shown that tissue levels, including brain, of TH are proportional to serum hormone level (Oppenheimer, 1983; Morreale de Escobar et al., 1987; 1990; Calvo et al., 1992; Porterfield and Hendrich, 1992, 1993; Broedel et al., 2003). In thyroidectomized rats, brain concentrations of T4 were decreased and Type II deiodinase (DII) activity was increased. Both brain T3 and T4 as well as DII activity returned to normal following infusion of T4 (Escobar-Morreale et al., 1995; 1997). Animals treated with PTU, MMI, or iodine deficiency during development demonstrate both lower serum and lower brain TH concentrations (Escobar-Morreale et al 1995; 1997; Taylor et al., 2008; Bastian et al., 2012; 2014; Gilbert et al., 2013).  Compared to the wildtype, a mouse MCT8 knockout model has was shown to have decreased plasma T4, decreased uptake of T4 into the brain, and decreased brian T3 concentrations, as well as increased cortical diodinase Type 2 activity and increased plasma T3 concentrations (Mayerl et al., 2014; Barez-Lopez et al., 2016).  Temporal Evidence: The temporal relationship between serum T4 and T4 in growing neuronal tissue described in this KER is dependent on the developmental stage (Seed et al., 2005).  While all brain regions will be impacted by changes in serum hormones, brain concentrations will be a function of development stage and brain region. Data are available from thyroid hormone replacement studies that demonstrate recovery of fetal brain T3 and T4 levels (following low iodine diets or MMI exposure) to control levels after maternal thyroid hormone replacement or iodine supplementation (e.g., Calvo et al.,1990; Obregon et al., 1991). For example, Calvo et al. (1990) carried out a detailed study of the effects of TPO inhibition on serum and tissues levels of TH in gestating rats. Clear dose-dependent effects of T4 replacement, but not T3 replacement were seen in all maternal tissues. However, for fetal tissues, neither T4 nor T3, at any dose, could completely restore tissue TH levels to control levels. Dose-Response Evidence:  There is good evidence, albeit from a limited number of studies of the correlative relationship between circulating thyroid hormone concentrations and brain tissue concentrations during fetal and early postnatal development following maternal iodine deficient diets or chemical treatments that depress serum THs (c.f., Calvo et al., 1990; Obregon et al., 1991; Morse et al.,1996).

The fact that decreased serum TH results in lower brain TH concentrations is well accepted.  However, the ability of the developing brain to compensate for insuffiencies in serum TH has not been well studied.  Limited data is available that demonstrates that changes in local deiodination in the developing brain can compensate for chemical-induced alterations in TH concentrations (e.g., Calvo et al., 1990; Morse et al., 1996; Sharlin et al., 2010). And, there are likely different quantitative relationships between these two KEs depending on the compensatory ability based on both developmental stage and specific brain region (Sharlin et al., 2010). For these reasons, the empirical support for this linkage is rated as moderate The role of cellular transporters represents an additional uncertainly. In addition, future work on cellular transport mechanisms and deiodinase activity is likley to inform addition of new KEs and KERs between serum and brain T4.

While it is well established that decreased in serum TH levels result in decreased brain TH concentrations, particularly fetal brain concentrations, a major gap is the lack of empirical data that allow direct quantification of this relationship (Hassan et al., 2018). Recently, serum TH and brain TH were measured in fetal cortex and postnatal day 14 offspring following graded degrees of hypothyroidism induced by PTU (O’Shaughnessy et al., 2018). Results showed that brain levels TH levels at both ages were quantitatively related to serum T4 levels. Additional dose-response information is necessary to confirm these findings, and standardization of analysis for the measurements in these distinct matrices is crucial to allow comparisons to be made between independent experiments.

Moderate Male Moderate Female

High

During brain development

Moderate

All life stages

Moderate Moderate Moderate

The majority of the information on this KER comes from in vivo studies with rodents (mainly MCT8 knock-out mice and thyroidectomized rats) and histopathological analyses of human brain tissues derived from patients affected by AHDS (Allan-Herndon-Dudley syndrome). The evoluationary conservation of the transport of TH from circulation to the developing brain suggests, with some uncertainty, that this KER is also applicable to other mammalian species.

Alshehri B, D'Souza DG, Lee JY, Petratos S, Richardson SJ.  The diversity of mechanisms influenced by transthyretin in neurobiology: development, disease and endocrine disruption. J Neuroendocrinol. 2015 May;27(5):303- 23. Bárez-López S, Obregon MJ, Martínez-de-Mena R, Bernal J, Guadaño-Ferraz A, Morte B.  Effect of Triiodothyroacetic Acid Treatment in Mct8 Deficiency: A Word of Caution. Thyroid. 2016 May;26(5):618-26. Bastian TW, Anderson JA, Fretham SJ, Prohaska JR, Georgieff MK, Anderson GW (2012), Fetal and neonatal iron deficiency reduces thyroid hormone-responsive gene mRNA levels in the neonatal rat hippocampus and cerebral cortex. Endocrinology 153:5668-5680. Bastian TW, Prohaska JR, Georgieff MK, Anderson GW (2014) Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression. Endocrinology 55:1157-1167. Broedel, O., Eravci, M., Fuxius, S., Smolarz, T., Jeitner, A., Grau, H., Stoltenburg-Didinger, G., Plueckhan, H., Meinhold, H., and Baumgartner, A. (2003). Effects of hyper and hypothyroidism on thyroid hormone concentrations in regions of the rat brain. Am. J. Physiol. Endocrinol. Metab. 285:E470–480. Calvo, R., Obregon, M.J., Escobar del Rey, F., and Morreale de Escobar, G. (1992). The rat placenta and the transfer of thyroid hormones from the mother to the fetus. Effects of maternal thyroid status. Endocrinology 131:357–365. Calvo R, Obregon MJ, Ruiz de Ona C, Escobar del Rey F, Morreale de Escobar G 1990 Congenital hypothyroidism, as studied in rats. Crucial role of maternal thyroxine but not of 3,5,3’-triiodothyronine in the protection of the fetal brain. J Clin Invest 86:889–899. Denver, RJ. (1998). The molecular basis of thyroid hormone-dependent central nervous system remodeling during amphibian metamorphosis. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 119:219-228. Dratman MB, Crutchfield FL, Schoenhoff MB. Transport of iodothyronines from bloodstream to brain: contributions by blood:brain and choroid plexus:cerebrospinal fluid barriers.  Brain Res. 1991 Jul 19;554(1-2):229-36. Gilbert ME, Hedge JM, Valentin-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW (2013) An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome. Toxicol Sci 132:177-195. Escobar del Rey F, Ruiz de Oña C, Bernal J, Obregón MJ, Morreale de Escobar G. Generalized deficiency of 3,5,3'-triiodo-L-thyronine (T3) in tissues from rats on a low iodine intake, despite normal circulating T3 levels. Acta Endocrinol (Copenh). 1989 Apr;120(4):490-8. Escobar-Morreale HF, Obregón MJ, Escobar del Rey F, Morreale de Escobar G. Replacement therapy for hypothyroidism with thyroxine alone does not ensure euthyroidism in all tissues, as studied in thyroidectomized rats. J Clin Invest. 1995 Dec;96(6):2828-38. Escobar-Morreale HF1, Obregón MJ, Hernandez A, Escobar del Rey F, Morreale de Escobar G. Regulation of iodothyronine deiodinase activity as studied in thyroidectomized rats infused with thyroxine or triiodothyronine. Endocrinology. 1997 Jun;138(6):2559-68. Fekete C, Lechan RM.  Central regulation of hypothalamic-pituitary-thyroid axis under physiological and pathophysiological conditions.  Endocr Rev. 2014 Apr;35(2):159-94 Lavado-Autric R, Calvo RM, de Mena RM, de Escobar GM, Obregon MJ. Deiodinase activities in thyroids and tissues of iodine-deficient female rats. Endocrinology. 2013 Jan;154(1):529-36. Mayerl S, Müller J, Bauer R, Richert S, Kassmann CM, Darras VM, Buder K, Boelen A, Visser TJ, Heuer H. Transporters MCT8 and OATP1C1 maintain murine brain thyroid hormone homeostasis.  J Clin Invest. 2014 May;124(5):1987-99. McLean TR, Rank MM, Smooker PM, Richardson SJ.  Evolution of thyroid hormone distributor proteins. Mol Cell Endocrinol. 2017 Feb 27. pii: S0303-7207(17)30151-X. doi: 10.1016/j.mce.2017.02.038. [Epub ahead of print] Morreale de Escobar, G., Obregon, M.J., and Escobar del Ray, F. (1987). Fetal and maternal thyroid hormones. Hormone Res. 26:12–27. Morreale de Escobar, G., Calvo, R., Obregon, M.J., and Escobar del Rey, F. (1990). Contribution of maternal thyroxine to fetal thyroxine pools in normal rats near term. Endocrinology 126:2765–2767. Morse DC, Wehler EK, Wesseling W, Koeman JH, Brouwer A. Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). Toxicol Appl Pharmacol. 1996 Feb;136(2):269-79 Obregon MJ, Ruiz de Ona C, Calvo R, Escobar del Rey F, Morreale de Escobar G 1991 Outer ring iodothyronine deiodinases and thyroid hormone economy: Responses to iodine deficiency in the rat fetus and neonate. Endocrinology 129:2663–2673. Oppenheimer, J.H. (1983). The nuclear Receptor-triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism, and biological action. In: Molecular Basis of Thyroid Hormone Action, eds. J.H. Oppenheimer and H.H. Samuels, pp. 1–35. New York: Academic Press. O'Shaughnessy KL, Wood, C, Ford RL, Kosian, PA, Hotchkiss, MG, Degitz SJ, Gilbert ME. Thyroid hormone disruption in the fetal and neonatal rat: Predictive hormone measures and bioindicators of hormone action in the developing cortex. Toxicol Sci. 2018 Aug 6. doi: 10.1093/toxsci/kfy190.  [Epub ahead of print] Pedraza PE, Obregon MJ, Escobar-Morreale HF, del Rey FE, de Escobar GM (2006) Mechanisms of adaptation to iodine deficiency in rats: thyroid status is tissue specific. Its relevance for man. Endocrinology 147:2098-2108. Porterfield, S.P. and Hendrich, C.E. (1992). Tissue iodothyronine levels in fetuses of control and hypothyroid rats at 13 and 16 days gestation. Endocrinology 131:195–200. Porterfield, S.P. and Hendrich, C.E. (1993). The role of thyroid hormones in prenatal neonatal neurological development-current perspectives. Endocrine Rev. 14:94–106. Power DM, Llewellyn L, Faustino M, Nowell MA, Björnsson BT, Einarsdottir IE, Canario AV, Sweeney GE. (2001). Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. Dec;130(4):447-59. Roberts LM, Woodford K, Zhou M, Black DS, Haggerty JE, Tate EH, Grindstaff KK, Mengesha W, Raman C, Zerangue N.  Expression of the thyroid hormone transporters monocarboxylate transporter-8 (SLC16A2) and organic ion transporter-14 (SLCO1C1) at the blood-brain barrier. Endocrinology. 2008 Dec;149(12):6251-61. Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72. Sharlin DS, Gilbert ME, Taylor MA, Ferguson DC, Zoeller RT. (2010).The nature of the compensatory response to low thyroid hormone in the developing brain. J Neuroendocrinol.  Mar;22(3):153-65. St. Germain, D.L. and Galton, V.A. (1997). The deiodinase family of selenoproteins. Thyroid 7:655–668. Taylor MA, Swant J, Wagner JJ, Fisher JW, Ferguson DC (2008) Lower thyroid compensatory reserve of rat pups after maternal hypothyroidism: correlation of thyroid, hepatic, and cerebrocortical biomarkers with hippocampal neurophysiology. Endocrinology 149:3521-3530. Van Herck SL, Geysens S, Delbaere J, Darras VM. (2013). Regulators of thyroid hormone availability and action in embryonic chicken brain development. Gen Comp Endocrinol.190:96-104. Visser WE, Friesema EC, Visser TJ.  Minireview: thyroid hormone transporters: the knowns and the unknowns.  Mol Endocrinol. 2011 Jan;25(1):1-14. AOPWiki 2016-11-29T18:41:33

2019-04-04T10:50:44

<upstream-id>9844a59e-a802-436d-8a8e-2d3a91514c91</upstream-id> <downstream-id>e07e2a31-57ae-4b17-80fa-b8b0b4fb894e</downstream-id> Many cellular and biochemical effects of thyroid hormones (TH) are mediated through regulation of gene expression (Oppenheimer, 1983; Bernal, 2007).  Thyroxine (T4) is transferred from the serum to the brain (see KER: Thyroxine (T4) in Serum, Decreased leads to Thyroxine (T4) in Neuronal Tissue, Decreased), where it converted to triiodothyronine (T3), the level of which is highly controlled by deiodinases. T3 binds to thyroid receptors (TR) in the nucleus of neuronal and glial cells to control gene expression. It is generally accepted that the modulation of TR gene expression in the hippocampus, or any other brain region, must therefore depend on the presence of hormone in these tissues.

The weight of evidence is moderate for TH concentrations affecting gene expression in the developing brain is (Oppenheimer and Schwartz, 1997; Oppenheimer, 1983; Bernal, 2007; Morte et al., 2010a; 2010b; Williams, 2008). Direct measurement of TH in brain tissue, and in hippocampus in particular, has shown correlations with gene expression. Therefore, it is assumed that reductions in TH-responsive genes in the hippocampus stem from reduced availability of hormone in the brain from the serum. However, studies in which there are simultaneous assessments of hippocampal concentrations of thyroid hormone and hippocampal gene expression is limited.

The biological relationship between these two KEs is strong. It is a generally accepted fact that TH produce their actions on brain development by binding to nuclear receptors to affect gene transcription. See KER (1387): "T4 in serum, Decreased leads (non-adjacently) to Hippocampal gene expression, Altered" for more information on TR regulated genes. As the primary means whereby TH promotes its action is by binding to TR in brain, TH must be present in brain to affect this action. Circulating levels of T4 represent the primary source of T4 in the brain, which is then converted to the active hormone T3 by deiodinases within neuronal tissue.

The empirical support for this KER is moderate. Many in vitro studies have demonstrated a relationship between hormone concentraionsTH and the induction of gene expression in brain cells, including hippocampal neurons in culture (Gil-Ibanez et al., 2015; Morte et al., 2010b). However, there are a limited number of studies investigating TH concentrations in the hippocampus and hippocampal gene expression. This is the case because thyroid hormone is difficult to measure in hippocampus and TH-induced gene expression changes can be subtle. We are aware of only four in vivo studies in which both thyroid hormones in the brain and gene expression in brain were simultaneously measured (Bastian et al., 2012; 2014; Hernandez et al., 2010; Sharlin et al., 2008). Only two of these reports, stemming from the same laboratory, specifically assessed thyroid hormone and gene expression in hippocampus. In these studies, Bastian et al., (2012; 2014) measured decrements in hippocampal T3 using RIA and correlated these reductions with alterations in the expression of myelin associated genes (Mbp, Plp), the neurotrophin, Ngf, the calcium binding protein Parv, a TH-dependent transcription factor, Hr, and Agt. Temporal Evidence: The temporal nature of this KER on TH dependent gene regulation is developmental (Seed et al., 2005). The impact of brain TH concentrations on regulation of TR regulated genes is age-dependent for a number of genes critical for normal hippocampal development. It is widely accepted that different genes are altered dependent upon the window of exposure in the fetal, neonatal or adult brain (c.f., Pathak et al, 2011; Mohan et al., 2012; Quignodon et al., 2004; Williams, 2008). Thyroid hormone supplementation has been shown to reverse some of the effects on gene expression (Mohan et al., 2012; Liu et al., 2010; Pathak et al., 2011). Dose-Response Evidence:  Dose-response data exists but is limited to a small number of studies and a small number of genes (Bastian et al., 2012; 2014; Sharlin et al., 2008).

There are no inconsistencies in this KER, but there are uncertainties. Uncertainties remain in the relationship of neuronal TH concentrations and gene expression in the brain because of the lack of studies simultaneously examining brain hormone and gene expression in the same study. This stems from the technological challenges associated with measuring brain hormone and the sometimes-subtle changes in brain gene expression induced by manipulations of the thyroid system. In addition, there are also some physiological actions of T4 that are mediated non-genomically at the cell membrane (Davis et al., 2016).  However, the exact role for the non-genomic effects is not well accepted or understood (Galton, 2017).

There is only one study available to date that provides empirical data on both TH concentrations and measures of gene expression changes in brain.  O'Shaughnessy et al (2018) demostrates dose-response relationships between brain T4 and T3 concentrations and changes in a variety of genes (e.g., Parv, Col11a2, Hr, Ngf) that were "statistically significant at doses that decreased brain t4 and/or T3".  There was no quantitation of this relationship reported.

High Male High Female

High

During brain development

Moderate Moderate

Most of the data available has come from rodent models. The evolutionary conservation of thyroid receptors (Holzer et al., 2017) coupled with their role in TR regulated gene transcription in neurodevelopment, suggests that this KER may also be applicable to other species (see text above).

Bastian TW, Anderson JA, Fretham SJ, Prohaska JR, Georgieff MK, Anderson GW (2012), Fetal and neonatal iron deficiency reduces thyroid hormone-responsive gene mRNA levels in the neonatal rat hippocampus and cerebral cortex. Endocrinology 153:5668-5680. Bastian TW, Prohaska JR, Georgieff MK, Anderson GW (2014) Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression. Endocrinology 155:1157-1167. Bernal J (2007) Thyroid hormone receptors in brain development and function. Nat Clin Pract Endocrinol Metab 3:249-259. Davis, P.J., Goglia, F., Leonard, J.L., 2016. Nongenomic actions of thyroid hormone. Nat. Rev. Endocrinol. 12, 111-121. Galton VA. The ups and downs of the thyroxine pro-hormone hypothesis. Mol Cell Endocrinol. 2017 Jan 24. pii: S0303-7207(17)30042-4. doi: 10.1016/j.mce.2017.01.029. Gil-Ibañez P, García-García F, Dopazo J, Bernal J, Morte B.  Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types.  Cereb Cortex. 2017 Jan 1;27(1):706-717. Hernandez A, Quignodon L, Martinez ME, Flamant F, St Germain DL (2010), Type 3 deiodinase deficiency causes spatial and temporal alterations in brain T3 signaling that are dissociated from serum thyroid hormone levels. Endocrinology 151:5550-5558. Holzer G, Roux N, Laudet V. Evolution of ligands, receptors and metabolizing enzymes of thyroid signaling. Mol Cell Endocrinol. 2017 Mar 22. pii: S0303-7207(17)30191-0. doi: 10.1016/j.mce.2017.03.021. Liu D, Teng W, Shan Z, Yu X, Gao Y, Wang S, Fan C, Wang H, Zhang H. The effect of maternal subclinical hypothyroidism during pregnancy on brain development in rat offspring.  Thyroid. 2010 Aug;20(8):909-15. Mohan V, Sinha RA, Pathak A, Rastogi L, Kumar P, Pal A, Godbole MM (2012) Maternal thyroid hormone deficiency affects the fetal neocorticogenesis by reducing the proliferating pool, rate of neurogenesis and indirect neurogenesis. Exp Neurol 237:477-488. Morte B, Diez D, Auso E, Belinchon MM, Gil-Ibanez P, Grijota-Martinez C, Navarro D, de Escobar GM, Berbel P, Bernal J (2010a) Thyroid hormone regulation of gene expression in the developing rat fetal cerebral cortex: prominent role of the Ca2+/calmodulin-dependent protein kinase IV pathway. Endocrinology 151:810-820. Morte B, Ceballos A, Diez D, Grijota-Martínez C, Dumitrescu AM, Di Cosmo C, Galton VA, Refetoff S, Bernal J. (2010b) Thyroid hormone-regulated mouse cerebral cortex genes are differentially dependent on the source of the hormone: a study in monocarboxylate transporter-8- and deiodinase-2-deficient mice.  Endocrinology. 2010 May;151(5):2381-7 Oppenheimer, J. (1983). The nuclear-receptor-triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism, and biological action. Molecular Basis of Thyroid Hormone Action. J. O. a. H. Samuels. New York, Academic Press: 1-34. Oppenheimer, J. H. and H. L. Schwartz (1997). "Molecular basis of thyroid hormone-dependent brain development." Endocr Rev 18(4): 462-75. O'Shaughnessy KL, Wood, C, Ford RL, Kosian, PA, Hotchkiss, MG, Degitz SJ, Gilbert ME. Thyroid hormone disruption in the fetal and neonatal rat: Predictive hormone measures and bioindicators of hormone action in the developing cortex. Toxicol Sci. 2018 Aug 6. doi: 10.1093/toxsci/kfy190.  [Epub ahead of print] Pathak A, Sinha RA, Mohan V, Mitra K, Godbole MM. 2011. Maternal thyroid hormone before the onset of fetal thyroid function regulates reelin and downstream signaling cascade affecting neocortical neuronal migration. Cerebral cortex. 21:11-21. Quignodon L, Legrand C, Allioli N, Guadano-Ferraz A, Bernal J, Samarut J, Flamant F (2004) Thyroid hormone signaling is highly heterogeneous during pre- and postnatal brain development. J Mol Endocrinol 33:467-476. Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE.  Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72. Sharlin DS, Tighe D, Gilbert ME, Zoeller RT (2008) The balance between oligodendrocyte and astrocyte production in major white matter tracts is linearly related to serum total thyroxine. Endocrinology 149:2527-2536. Williams GR (2008), Neurodevelopmental and neurophysiological actions of thyroid hormone. J Neuroendocrinol. 2008 Jun;20(6):784-94 AOPWiki 2016-11-29T18:41:35

2018-08-11T19:18:16

<upstream-id>e07e2a31-57ae-4b17-80fa-b8b0b4fb894e</upstream-id> <downstream-id>ea1f772c-7295-49e5-ad1e-7ccbb2a2d3fc</downstream-id>

AOPWiki 2023-04-12T11:28:06

2023-04-12T11:28:06

<upstream-id>ea1f772c-7295-49e5-ad1e-7ccbb2a2d3fc</upstream-id> <downstream-id>6e5c1096-220b-44b4-8ff1-90bb18b7aece</downstream-id> It is a well-accepted assertion that hippocampal synaptic integrity and plasticity are essential for spatial information processing in animals and spatial and episodic memory in humans (Burgess, 2002; Martin et al., 2000; Sweatt, 2016). Many studies with a variety of techniques and approaches (e.g. nutritional and chemical stressors, gene knockouts) have linked hippocampal functional deficits to decreased spatial ability, context learning, and fear learning. Study of human disease states and conditions where hippocampal function is impaired (i.e., brain trauma, Alzheimer’s disease, temporal lobe epilepsy, Down’s Syndrome), and imaging studies of hippocampal activation during memory challenge, makes irrefutable that the hippocampus is essential for specific types of cognitive abilities. Decades of animal research has reinforced this assertion. There are many forms of synaptic plasticity and numerous ways in which physiological function of neural circuits can be assessed. Similarly, there are many forms of learning and memory relying on different brain regions and and multiple tasks and specifics associated with these tasks that vary from laboratory to laboratory. An emerging field of computational cognitive neuroscience lies at the intersection of computational neuroscience, machine learning and neural network theory. These computational and theoretical frameworks support the participation of hippocampal synaptic transmission and plasticity in learning and memory in animals and humans (for review see: Ashby and Helie, 2012).

Most of the text in this KER was reviewed and revised as part of the OECD AOP project and approved in 2019. This text was developed using standard literature search procedures set to identify any links between anatomical changes in the hippocampus and subsequent changes in hippocampal physiology. The most important resulting papers were identified as having measured both KE5 and KE6. This was updated by a recent systematic search conducted by EFSA in 2021 and 2022 (see Appendix B of EFSA technical report, 2024). Additional papers, including those using gene models, were identified, and added to the text.

The weight of evidence for physiological hippocampal function and episodic memory in humans and the animal analogue, spatial and fear-based context learning, is strong. Seminal studies over the past 60 years firmly established the cellular basis of behavior with synaptic plasticity (long term potentiation and long-term depression, LTP and LTD respectively). Recent work has provided details on the local hippocampal circuitry needed for memory formation and behavioral change (Sweatt, 2016). In humans, virtual reality experiments in large-scale spatial contexts show the convergence of spatial memory performance in normal patients with fMRI of the hippocampus clearly demonstrating the essentiality of hippocampal function to spatial learning (Burgess, 2002). This assertion is consistent with a wealth of animal data on hippocampal learning and memory. In rodent models, functional impairment of the hippocampus assessed using electrophysiological techniques is correlated with deficits in spatial memory typically assessed using mazes, and memory for context often assessed in fear-based learning paradigms (O’Keefe and Nadel, 1978; Clark et al., 2000; Squire, 2004; Eichenbaum, 2000; Panja and Bramham, 2014).

The biological plausibility of the KER is rated as strong. It is well accepted that the normal hippocampal function is critical for the acquisition and memory of context and spatially mediated tasks in rodents and humans (Sweatt, 2016).

Empirical support for this KER is strong. The requisite of hippocampal integrity to optimal visuo-spatial context learning (i.e., episodic memory) in humans and spatial learning in rodents is well documented. In vivo recording in conscious behaving animals has demonstrated activity-dependent neural changes taking place in the hippocampus during spatial learning (Gruart and Delgado-Garcia, 2007). Impairments in hippocampal function induced by drugs, chemicals, lesions, nutritional deficiencies, mutant or knock out models that cause changes in synaptic transmission, plasticity, and hippocampal network activity, are coincident with deficits in spatial and context-based fear learning (O’Keefe and Nadel, 1978; Bannerman et al., 2014; Lynch, 2004; Verret et al., 2012). Similarly, treatments found to enhance or facilitate hippocampal synaptic transmission and plasticity are associated with improved learning and memory (Deng et al., 2010; Novkovic et al., 2015; Andrade et al., 2015; Trivino-Paredes et al., 2016). A few examples of a large literature are briefly summarized below. It is well known that n-Methyl-d-aspartate (NMDA)-mediated glutamatergic synaptic transmission is essential for the induction of hippocampal synaptic plasticity in the form of LTP. Blockade of this form of plasticity by selective NMDA-receptors blockers impairs LTP and hippocampal tests of learning and memory (reviewed in Sweatt, 2016). Perturbation of hippocampal plasticity and impaired spatial learning have been reported in adult offspring following prenatal ethanol exposure (An and Zhang, 2015). Developmental morphine exposure caused a decrease in the amplitude and slope of fEPSC sand inhibition of LTP in CA1 neurons fEPSPs that resulted in decreased maze performance (Aghighi et al., 2019). Developmental nutrition deficiency and hypoxic stress are both associated with, changes in synaptic structure, altered EPSPs, and hippocampal based cognitive behaviors (Dumets et al., 2020; Zhuravin et al., 2019).  Rodent models of developmental TH insufficiency are associated with, impairments in hippocampal synaptic transmission and plasticity and are coincident with deficits in learning tasks that require the hippocampus (Opazo et al., 2008; Gilbert and Sui, 2006, Gilbert, 2011, Gilbert et al., 2016). There are also several mutant mouse models that have linked changes in hippocampal physiology with alteration in cognitive behaviors.  The fyn mutant mouse (fyn is a tyrosine kinase pathway) displays impairments in hippocampal synaptic transmission and plasticity, as well as spatial learning deficits (Grant et al., 1992). Brain-derived neurotrophic factor (BDNF) knockout animals exhibit synaptic plasticity deficits and learning impairments (Aarse et al., 2016; Panja and Bramham, 2014). In the Jacob/Nfsm model which also exhibits pronounced alterations in BDNF-mediated signaling, hippocampal synaptic transmission and plasticity impairments were accompanied by deficits in contextual fear conditioning and novel location recognition tasks (Spilker et al., 2016). The aryl-hydrocarbon receptor (AhR) knockout was shown to decrease hippocampal mossy fibers and impair maze performance (Powers et al., 2005).  Knockout of SALM4/Lrfn3, a synaptic adhesion molecule that modulates NMDA receptor function, increases NMDA-mediated currents and enhances contextual fear memory. In this model, control level of performance could be restored via treatment with fluoxetine, a selective serotonin reuptake inhibitor (Lie et al.,2021). Finally, a knockout of LIMK-1, a kinase associated with actin dynamics, was shown to alter hippocampal spine morphology and LTP, with subsequent changes in fear behaviors and a spatial learning task (Meng et al., 2002). In humans, hippocampal physiology assessed using neuroimaging reveals activation of hippocampus upon engagement in spatial learning and episodic memory providing a direct linkage of these two specific KEs (Burgess, 2002). In fMRI studies of congenitally hypothyroid children, or children born to women with altered thyroid function during pregnancy, changes in hippocampal activity patterns during memory encoding and retention were observed and associated with memory impairments (Wheeler et al., 2012; 2015; Willoughby et al., 2013; 2014). <h4>Temporal Evidence</h4> The temporal nature of this KER is developmental (Seed et al., 2005). This has been demonstrated in multiple studies. It is well-recognized that there are critical developmental windows for disruption of the functional development of the hippocampus and the integrity of this structure is essential for later development of spatial ability, context learning, and fear learning. A wealth of studies has shown correlation between hippocampal LTP and spatial learning performance, as well as the role of glutamatergic synaptic transmission and BDNF-mediated signaling pathways in these processes (Bramham, 2007; Andero et al., 2014; Morris et al., 1986; Sweatt, 2016; Migaud et al., 1998). Although studies on reversibility are relatively rare, a few examples of deficits in hippocampal synaptic transmission and plasticity documented in knockout mouse models are described above. In addition, in slices from BDNF knockout animals, physiological function can be rescued with recombinant BDNF (Patterson et al., 1996).

There are no inconsistencies in this KER, but there are some uncertainties. It is a widely held assertion that synaptic transmission and plasticity in the hippocampus underlie spatial learning (Martin et al., 2000; Gruart and Delgado-Garcia, 2007; Bramham, 2007). However, the causative relationship of which specific alterations in synaptic function are associated with specific cognitive deficits is difficult to ascertain given the many forms of learning and memory, and the complexity of synaptic interactions in even the simplest brain circuit.

There are currently no known modulating factors.

Information does not exist to develop quantitative relationships between the KEs in this KER.

Limited dose-response information is available. Mutation and knockout mouse models are not conducive to examination of varying levels of impairment at the physiological or behavioral level. Studies have investigated dose-dependency of impairments in hippocampal electrophysiological and behavior have been reported in animals suffering from developmental TH insufficiency (e.g., Gilbert and Sui, 2006; Gilbert, 2011; Gilbert et al., 2016).

There are currently no known feedforward/Feedback loops influencing this KER.

High Male High Female

High

During brain development

High High High

Most work has been conducted with rodent models. Sex-specific differences in sensitivity to disruption and sex-dependent differences in behavioral performance of hippocampal tasks have been reported in both rodent models and human studies.

Aarse J, Herlitze S, Manahan-Vaughan D. The requirement of BDNF for hippocampal synaptic plasticity is experience-dependent. Hippocampus. 2016 Jun;26(6):739-51. Aghighi F, Mohammadifar M, Banafsheh HR, Salami M, Talaei SA. Behavioral and electrophysiological aspects of cognition in neonate rats lactated by morphine addicted mothers. Iran J Basic Med Sci 2019; 22:1059-1064. doi: 10.22038/ijbms.2019.36892.8789 An L, Zhang T. Prenatal ethanol exposure impairs spatial cognition and synaptic plasticity in female rats. Alcohol. 2015 Sep;49(6):581-8. Andero R, Choi DC, Ressler KJ. BDNF-TrkB receptor regulation of distributed adult neural plasticity, memory formation, and psychiatric disorders. Prog Mol Biol Transl Sci. 2014. 122:169-92. Andrade-Talavera Y, Benito I, Casañas JJ, Rodríguez-Moreno A, Montesinos ML. Rapamycin restores BDNF-LTP and the persistence of long-term memory in a model of Down's syndrome. Neurobiol Dis. 2015. 82:516-25 Ashby FG, Helie S. The Neurodynamics of Cognition: A Tutorial on Computational Cognitive Neuroscience. J Math Psychol. 2011 Aug 1;55(4):273-289. Bannerman DM, Sprengel R, Sanderson DJ, McHugh SB, Rawlins JNP, Monyer H, Seeburg PH (2014) Hippocampal synaptic plasticity, spatial memory and anxiety. Nat Rev Neurosci 15:181-192. Bramham CR. Control of synaptic consolidation in the dentate gyrus: mechanisms, functions, and therapeutic implications. Prog Brain Res. 2007. 163:453-71. Burgess N (2002) The hippocampus, space, and viewpoints in episodic memory. Q J Exp Psychol A 55:1057-1080. Clark RE, Zola SM, Squire LR. Impaired recognition memory in rats after damage to the hippocampus. J Neurosci. 2000 Dec 1;20(23):8853-60. Deng W, Aimone JB, Gage FH (2010) New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory Nat Rev Neurosci 11:339-350. Dumetz F, Ginieis R, Bure C, Marie A, Alfos S, Pallet V, Bosch-Bouju C. Neuronal morphology and synaptic plasticity in the hippocampus of vitamin A deficient rats. Nutr Neurosci. 2022 Apr;25(4):779-790. doi: 10.1080/1028415X.2020.1809877. Epub 2020 Sep 12. PMID: 32924835. Eichenbaum H. (2000). A cortical-hippocampal system for declarative memory. Nature reviews. Neuroscience, 1(1), 41–50. <a href="https://doi.org/10.1038/35036213">https://doi.org/10.1038/35036213</a> Gilbert ME (2011) Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci 124:432-445. Gilbert ME, Sanchez-Huerta K, Wood C (2016) Mild Thyroid Hormone Insufficiency During Development Compromises Activity- Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787. Gilbert ME, Sui L (2006) Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22. Grant SG, O'Dell TJ, Karl KA, Stein PL, Soriano P, Kandel ER. Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science. 1992 Dec 18;258(5090):1903-10. Gruart A, Delgado-García JM. Activity-dependent changes of the hippocampal CA3-CA1 synapse during the acquisition of associative learning in conscious mice. Genes Brain Behav. 2007 Jun;6 Suppl 1:24-31. Lie E, Yeo Y, Lee EJ, Shin W, Kim K, Han KA, Yang E, Choi TY, Bae M, Lee S, Um SM, Choi SY, Kim H, Ko J, Kim E. SALM4 negatively regulates NMDA receptor function and fear memory consolidation. Commun Biol. 2021 Sep 29;4(1):1138. doi: 10.1038/s42003-021-02656-3. PMID: 34588597; PMCID: PMC8481232. Lynch, M.A. (2004). Long-Term Potentiation and Memory. Physiological Reviews. 84:87-136. Martin SJ, Grimwood PD, Morris RG. Synaptic plasticity and memory: an evaluation of the hypothesis. Annu Rev Neurosci. 2000. 23:649-711. Meng Y, Zhang Y, Tregoubov V, Janus C, Cruz L, Jackson M, Lu WY, MacDonald JF, Wang JY, Falls DL, Jia Z. Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice. Neuron. 2002 Jul 3;35(1):121-33. doi: 10.1016/s0896-6273(02)00758-4. PMID: 12123613. Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O'Dell TJ, Grant SG. Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature. 1998 Dec 3;396(6710):433-9. Morris RG, Frey U. Hippocampal synaptic plasticity: role in spatial learning or the automatic recording of attended experience? Phylos Trans R Soc Lond B Biol Sci. 1997 Oct 29;352(1360):1489-503. Review Novkovic T, Mittmann T, Manahan-Vaughan D. BDNF contributes to the facilitation of hippocampal synaptic plasticity and learning enabled by environmental enrichment. Hippocampus. 2015 Jan;25(1):1-15. O’Keefe, J. and Nadel, L. (1978). The Hippocampus as a Cognitive Map. Oxford: Oxford University Press. Opazo MC, Gianini A, Pancetti F, Azkcona G, Alarcón L, Lizana R, Noches V, Gonzalez PA, Marassi MP, Mora S, Rosenthal D, Eugenin E, Naranjo D, Bueno SM, Kalergis AM, Riedel CA (2008), Maternal hypothyroxinemia impairs spatial learning and synaptic nature and function in the offspring. Endocrinology 149:5097-5106 Panja, D. and C. R. Bramham (2014). "BDNF mechanisms in late LTP formation: A synthesis and breakdown." Neuropharmacology 76 Pt C: 664-676. Patterson SL, Abel T, Deuel TA, Martin KC, Rose JC, Kandel ER. Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron. 1996 Jun;16(6):1137-45. Powers BE, Lin TM, Vanka A, Peterson RE, Juraska JM, Schantz SL. Tetrachlorodibenzo-p-dioxin exposure alters radial arm maze performance and hippocampal morphology in female AhR mice. Genes Brain Behav. 2005 Feb;4(1):51-9. doi: 10.1111/j.1601-183X.2004.00098.x. PMID: 15660668. Schultz C, Engelhardt M, Anatomy of the hippocampal formation. Front Neurol Neurosci. 2014. 34:6-17 Seed J, Carney EW, Corley RA, Crofton KM, DeSesso JM, Foster PM, Kavlock R, Kimmel G, Klaunig J, Meek ME, Preston RJ, Slikker W Jr, Tabacova S, Williams GM, Wiltse J, Zoeller RT, Fenner-Crisp P, Patton DE. Overview: Using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit Rev Toxicol. 2005 35:664-72. Spilker C, Nullmeier S, Grochowska KM, Schumacher A, Butnaru I, Macharadze T, Gomes GM, Yuanxiang P, Bayraktar G, Rodenstein C, Geiseler C, Kolodziej A, Lopez-Rojas J, Montag D, Angenstein F, Bär J, D'Hanis W, Roskoden T, Mikhaylova M, Budinger E, Ohl FW, Stork O, Zenclussen AC, Karpova A, Schwegler H, Kreutz MR. A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis. PLoS Genet. 2016 Mar 15;12(3):e1005907 Squire LR 2004. Memory systems of the brain: A brief history and current perspective. Neurobiology of Learning and Memory, 82: 171-177 Sweatt JD. Neural plasticity and behavior - sixty years of conceptual advances. J Neurochem. 2016 Oct;139 Suppl 2:179-199. doi: 10.1111/jnc.13580. Review. PubMed PMID: 26875778. Triviño-Paredes J, Patten AR, Gil-Mohapel J, Christie BR. The effects of hormones and physical exercise on hippocampal structural plasticity. Front Neuroendocrinol. 2016. 41:23-43. Verret L, Mann EO, Hang GB, Barth AM, Cobos I, Ho K, Devidze N, Masliah E, Kreitzer AC, Mody I, Mucke L, Palop JJ. Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell. 2012Apr 27;149(3):708-21. Wheeler SM, McAndrews MP, Sheard ED, Rovet J (2012) Visuospatial associative memory and hippocampal functioning in congenital hypothyroidism. J Int Neuropsychol Soc 18:49-56. 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2024-07-26T12:57:40

AhR activation in the liver leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals

AhR activation in the liver leading to Adverse Neurodevelopmental Outcomes in Mammals

Prakash Patel

Prakash Patel PhD; Mathematical Modeller, Cyprotex Discovery Ltd., An Evotec Company, Alderley Park, Cheshire, SK10 4TG, UK Simon Thomas PhD; Head of Modelling and Simulation, Cyprotex Discovery Ltd., An Evotec Company, Alderley Park, Cheshire, SK10 4TG, UK

Open for adoption

2.0

Polychlorinated biphenyls (PCBs) and dioxins are environmental contaminatants whose prenatal exposure in humans and exposure in breast milk is correlated to a specific set of mental impairments (Boucher et al. 2009, Boersma and Lanting 2000, Koopman-Esseboom et al. 1996). What the main mechanism(s) of causing these delays for the various PCBs and dioxins is unclear, but studies in rodents regarding 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and "dioxin-like" PCBs like PCB-126 that it could be caused by lowering maternal and subsequently foetal thyroxine (T4) levels in the brain. This AOP summarises the findings that chemicals that are AhR receptor agonists can reduce T4 levels in the plasma in rodents and possibly humans and lead to brain changes in neonates, via activation of liver enzymes that increase the metabolism of T4.  This AOP was created in order to collate and summarise information around the reduction of T4 levels in the plasma by endocrine disruptors, distinct from the reduction of binding to plasma proteins or interfering with thyroid hormone secretion. A short summary: <ol> <li>Chemical activates AhR</li> <li>UGT activity in the liver increases</li> <li>More T4 is metabolised and reduces plasma T4 levels</li> <li>Adverse outcomes (such as cognitive impairment)</li> </ol> The problem is that AhR agonists TCDD and dioxin-like PCBs (PCB-126, PCB-169) have multiple effects on several organs as AhR receptor is present in these organs whose influence cannot be ruled out (complementary to AOP 459: AhR activation in the thyroid leading to Subsequent Adverse Neurodevelopmental Outcomes in Mammals) but its effect on the liver has been relatively well-characterised. Also, one needs to understand that all AhR agonists do not produce toxic effects - some are found in vegetables, fruits and teas and are important for normal physiology. AhR -/- mice develop immune and liver defects (Quintana 2012). For the case of 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) which is a potent agonist of AhR in vitro and in vivo but does not cause toxic effects (Henry et al. 2006)       This AOP was developed as part of the ScreenED project, which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 825745

This AOP summarises the findings of Kohn et al. 1996 and other papers that corroborate its findings, whilst also summarising evidence of hippocampal and mental effects on rodents of TCDD and various PCBs, although their effects on the thyroid directly and other organs cannot be ruled out. Relevant publications were found by searching through PubMed for terms such as "AhR" "thyroxine" "TCDD" "T4".

The AHR can be activated by several structurally diverse chemicals, but binds preferentially to planar halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. Dioxin-like compounds (DLCs), which include polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and certain polychlorinated biphenyls (PCBs), are among the most potent AHR ligands<a href="#cite_note-Denison2011-38">[38]</a>. Only a subset of PCDD, PCDF and PCB congeners has been shown to bind to the AHR and cause toxic effects to those elicited by TCDD. Until recently, TCDD was considered to be the most potent DLC in birds<a href="#cite_note-Van1998-39">[39]</a>; however, recent reports indicate that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) is more potent than TCDD in some species of birds.<a href="#cite_note-Cohen2011b-40">[40]</a><a href="#cite_note-Farmahin2012-13">[13]</a><a href="#cite_note-Farmahin2013a-41">[41]</a><a href="#cite_note-Farmahin2014-21">[21]</a><a href="#cite_note-Herve2010a-42">[42]</a><a href="#cite_note-Herve2010b-43">[43]</a> When screened for their ability to induce aryl hydrocarbon hydroxylase (AHH) activity, dioxins with chlorine atoms at a minimum of three out of the four lateral ring positions, and with at least one non-chlorinated ring position are the most active<a href="#cite_note-Poland1973-44">[44]</a>. Of the dioxin-like PCBs, non-ortho congeners are the most toxicologically active, while mono-ortho PCBs are generally less potent<a href="#cite_note-McFarland1989-45">[45]</a><a href="#cite_note-Safe1994-9">[9]</a>. Chlorine substitution at ortho positions increases the energetic costs of assuming the coplanar conformation required for binding to the AHR <a href="#cite_note-McFarland1989-45">[45]</a>. Thus, a smaller proportion of mono-ortho PCB molecules are able to bind to the AHR and elicit toxic effects, resulting in reduced potency of these congeners. Other PCB congeners, such as di-ortho substituted PCBs, are very weak AHR agonists and do not likely contribute to dioxin-like effects <a href="#cite_note-Safe1994-9">[9]</a>. <ul> <li>Contrary to studies of birds and mammals, even the most potent mono-ortho PCBs bind to AhRs of fishes with very low affinity, if at all (Abnet et al 1999; Doering et al 2014; 2015; Eisner et al 2016; Van den Berg et al 1998).</li> </ul> The role of the AHR in mediating the toxic effects of planar hydrophobic contaminants has been well studied, however the endogenous role of the AHR is less clear <a href="#cite_note-Okey2007-1">[1]</a>. Some endogenous and natural substances, including prostaglandin PGG2 and the tryptophan derivatives indole-3-carbinol, 6-formylindolo[3,2-b]carbazole (FICZ) and kynurenic acid can bind to and activate the AHR. <a href="#cite_note-Fujii2010-6">[6]</a><a href="#cite_note-Omie2011-46">[46]</a><a href="#cite_note-Swed2010-47">[47]</a><a href="#cite_note-Diani2011-48">[48]</a><a href="#cite_note-Wincent2012-49">[49]</a> The AHR is thought to have important endogenous roles in reproduction, liver and heart development, cardiovascular function, immune function and cell cycle regulation <a href="#cite_note-Baba2005-50">[50]</a><a href="#cite_note-Denison2011-38">[38]</a><a href="#cite_note-Fernandez1995-51">[51]</a><a href="#cite_note-Ichihara2007-52">[52]</a><a href="#cite_note-Lahvis2000-53">[53]</a><a href="#cite_note-Mimura1997-54">[54]</a><a href="#cite_note-Omie2011-46">[46]</a><a href="#cite_note-Schmidt1996-55">[55]</a><a href="#cite_note-Thack2002-56">[56]</a><a href="#cite_note-Zhang2010-57">[57]</a> and activation of the AHR by DLCs may therefore adversely affect these processes.

A prime example of impairments in cognitive function as the adverse outcome for regulatory action is developmental lead exposure and IQ function in children (Bellinger, 2012). In addition, testing for the impact of chemical exposures on cognitive function, often including spatially-mediated behaviors, is an integral part of both EPA and OECD developmental neurotoxicity guidelines (USEPA, 1998; OECD, 2007).

adjacent

High

High adjacent

High

High adjacent

High

High adjacent

Moderate

High adjacent

Moderate

High adjacent

Low

Moderate adjacent

Low

Low non-adjacent

High

Moderate Mixed

Moderate

Embryo

Moderate

Foetal

Moderate

Birth to < 1 month

High High Low Low

Domain of Applicability <ul> <li>Chemicals: This AOP applies to a wide range of chemicals structures that activate AhR either in vivo or in vitro, but one needs to understand that not all AhR agonists do not produce toxic effects</li> <li>Sex: This AOP applies to males and females. Disruption of thyroid hormone regulation during foetal and early postnatal development, but the subsequent adverse impacts on nervous system development may differ and be more severe in males than females (Seo et al. 1999, Rice 1999). </li> <li>Life stages: The relevant life stages for this AOP are fetal and early postnatal ages during critical windows of nervous system development where thyroid hormones guide normal development of the brain. There are clear windows of developmental susceptibility and different brain regions show distinct ontogenetic profiles for TH requirements. Distinct phenotypes have been described in both humans and animal models for different periods of TH insufficiency. The influence of maternal thyroid status prior to onset of fetal thyroid function is an important consideration. This AOP does not apply to adult life states.</li> <li>Taxonomic: Based on the majority of the available evidence the taxonomic applicability domains of this AOP is mammals. Most evidence for this AOP has been gathered primarily from laboratory rodents and humans. </li> </ul>

<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:154px"> Key Events </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:154px"> Direct evidence </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:154px"> Indirect evidence </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:154px"> No or contradictory evidence </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> MIE 18 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> TCDD and various PCBs can be confirmed to be agonists of AhR from both the EROD (Petrulis et al. 1999) and CALUX assay (Murk et al. 1996) to estimate exposure and activation of the AhR-ARNT pathways.     </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> KE 295 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> TCDD – upregulation of UGT1A6 in mouse pups heterozygous for AhR +/- gene but not for AhR -/- gene whose mothers were administered 10 ug/kg TCDD on gestation day 12.5 (Nishimura et al. 2005) Induction of the UGT-1 gene in Holtzman rats (TCDD-sensitive strain) in pups whose mothers were given a single oral dose of 200 ng or 800 ng TCDD/kg on gestational day 15 (Nishimura et al. 2003) TCDD - liver UGT mRNA increased in female Sprague-Dawley rats compared to controls after a TCDD dose of 3.5 and 100 ng/kg/day for 31 weeks (Sewall et al. 1995) PCB-126 - increase in T4-glucuronide production in adult male SD rats compared to controls (Fisher et al. 2006) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">     </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> KE 961 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> TCDD - rats cleared more T4 via the biliary route (Bastomsky 1977) and more T4-glucuronide excreted (Bastomsky 1977, Henry and Gasiewicz 1987) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> KE 281 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> TCDD – reduction of serum T4 in pups heterozygous for AhR +/- gene but not for AhR -/- gene whose mothers were administered 10 ug/kg TCDD on gestation day 12.5 (Nishimura et al. 2005) Reduction in serum T4 in Holtzman rats (TCDD-sensitive strain) in pups whose mothers were given a single oral dose of 200 ng or 800 ng TCDD/kg on gestational day 15 (Nishimura et al. 2003) PCB-126:  Pregnant albino rats received PCB 126 (20 or 40μg/kgb.wt.) by oral gavage from gestation day (GD) 1 to 20.  Both administrations of PCB 126 elevated serum thyrotropin (TSH) concentration, and decreased free thyroxine (FT4) and free triiodothyronine (FT3) concentrations, resulting in a maternofetal hypothyroidism  (Ahmed et al. 2018). Arochlor 1254 - SD rat dams were dosed daily with 0, 1, or 4 mg/kg A1254 from gestational day 6 (GD6) until they were sacrificed on GD16. A1254 significantly reduced circulating levels of triiodothyronine(T3) and thyroxine (T4) in pregnant rats (Gauger et al. 2004) TCDD - Marked dose-dependent reduction in T4 recorded in rats (Kohn et al. 1996, National Toxicology Program 2006) PCB-126 - reductions in serum T4 on acute (Fisher et al. 2006) and chronic administration of PCB-126 (National Toxicology Program 2006)  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">     </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> KE 280 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> Several studies have demonstrated that fetal brain TH levels, previously decreased by maternal exposure to TH synthesis inhibitors or thyroidectomy, recovered following maternal supply of T4 (e.g., Calvo et al., 1990). However, there are no studies with direct infusion of T4 or T3 directly into brain. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> The upregulation of deiodinase has been shown to compensate for some loss of neuronal T3 (Escobar-Morreale et al. 1995; Escobar-Morreale et al. 1997). Indirect evidence shows that T4 replacement that brings circulating T4 concentration back to physiological levels normal, leads to recovery of brain TH and prevents downstream effects including alterations in cell morphology, differentiation and function.   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> PCB-118 acted via the TH pathway in the rat foetus whilst PCB-126 did not and acted mainly via reducing maternal thyroid hormone levels (Fritsche et al. 2005, Gauger et al. 2004) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> KE 759 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> Hippocampal Gene Expression, Altered: It is well established specific genomic pathways underlie the progression of a number of neurodevelopmental processes in the hippocampus. There is some evidence from ex vivo studies that administration of growth factors will reverse the hippocampal dysplasia seen in Jacob/Nsfm knockout mice (Spilker et al., 2016). Less is known about the impact of hormone replacement on TH-responsive gene expression and the qualitative and quantitative relationships between altered TH-dependent gene expression in this brain region and altered hippocampal cytoarchitectural anatomy. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> KE 758 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> Hippocampal anatomy, altered: It is well accepted that normal hippocampal anatomy is critical for hippocampal physiological function, and that alterations in anatomy lead to altered neuronal activity in the hippocampus (Lee et al., 2015; Grant et al., 1992; Spilker et al., 2016). Hippocampal physiology, altered: It is a well-accepted assertion that hippocampal synaptic integrity and neuronal plasticity are essential for spatial information processing in animals and spatial and episodic memory in humans. However, other brain regions also can influence these complex behaviors. Limited data from studies in BDNF knockout animals demonstrate that deficits in hippocampal synaptic transmission and plasticity, and downstream behaviors can be rescued with recombinant BDNF (Aarse et al. 2016; Andero et al. 2014).  The relative size of the IIP-MF in females and males. The IIP-MF was smaller in TCDD exposed female AhR+/- mice with respect to their genotype control group. (Powers et al. 2005) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> Pregnant Sprague-Dawley rats were given a consecutive daily dose of TCDD (200 or 800 ng/day/kg) or an equivalent volume of vehicle by gavage on gestational days 8-14 as the prenatal TCDD exposure model. When the male pups grew to adults, morphology and number of neurons in the hippocampus CA1 region was not affected, although the activity of astrocytes in the same region was significantly reduced (Zhang et al. 2018) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px">   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> AO 402 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> TCDD exposed female AhR+/- mice performed worse on the spatial water maze task than non exposed mice (Powers et al. 2005) Pregnant Sprague-Dawley rats were given a consecutive daily dose of TCDD (200 or 800 ng/day/kg) or an equivalent volume of vehicle by gavage on gestational days 8-14. The results of the behavioural tests showed that gestational TCDD exposure induced premature motor activity and earlier eyes-opening, but lead to serious deficits of spatial memory and learning ability in adult male offspring. (Zhang et al 2018) Monkeys exposed to TCDD perinatally exhibited retarded learning of shape reversals (Shantz and Bowman 1994) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> Pregnant Long-Evans rats were dosed by gavage with TCDD or at a dose of 0, 200, or 800 ng/kg on gestational day 15, and the offspring was tested during adulthood. Paired-associate learning was found to be impaired in the 200 ng/kg TCDD group, but not in either group exposed to 800 ng/kg TCDD (Kakeyama et al. 2014) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:154px"> Long-Evans dams dosed at 0, 0.25, or 1.0 ug/kg/day PCB-126 Monday to Friday beginning 5 weeks before and continuing through gestation and lactation. On the spatial delayed alternation task, there was no convincing evidence for impairment as a result of PCB exposure on adult male pups, as assessed by overall accuracy of performance and measures of perseverative and other types of inappropriate responding. (Rice 1999)   Time-mated Sprague–Dawley rats were gavaged with either TCDD (0.1 ug/kg/day) or corn oil vehicle on gestation days 10–16. On day 80, both male and female TCDD-exposed grown-up pups showed a deficit in learning on the visual partial discrimination-reversal learning task, but TCDD-exposed male rats displayed a pronounced decrease in errors relative to control males in the Morris Water Maze task. There was no difference in the performance of TCDD exposed rats in the radial arm maze task. (Seo et al. 1999) Pregnant Sprague–Dawley rats (10 per dose) received either 0 or 0.1 ug/kg TCDD orally in corn oil from GD 10 to GD 16. One male and one female from each litter were tested beginning at 100 days of age. the results of the current study underscore the fact that (1) alterations in cognitive function observed following early TCDD exposure are very subtle and (2) under some conditions, learning is actually facilitated, rather than impaired, in TCDD-exposed animals. (Widholm et al. 2003) Monkeys exposed to TCDD perinatally showed a slight facilitation of learning on both delayed spatial alternation and spatial reversal learning tasks (Seegal and Schantz 1994) </td> </tr> </tbody> </table>

Biological plausibility Biological plausibility refers to the structural or functional relationship between the key events based on our fundamental understanding of "normal biology". In general, the biological plausibility and coherence linking AhR activation by PCBs and dioxins to decreases in circulating concentrations of THs is great. The biological plausibility of decreases in circulating concentrations of THs, to adverse impacts in the developing hippocampus and subsequent cognitive behaviors is very solid (AOP 42). The problem is the interaction with PCBs and dioxins directly with the foetal brain can act like thyroid hormones due to its similar shape to these compounds, and may compensate for the reduction of THs there. Some of the adverse effects have more in common with congenital hyperthyroidism than hypothyroidism e.g. that gestational TCDD exposure induced premature motor activity in neonatal rats and earlier eyes-opening (Zhang et al. 2018). Concordance of dose-response relationships: There are several studies that include correlative evidence between exposure to AhR agonists TCDD (Kohn et al. 1996, NTP 2006) and PCB-126 (NTP, 2006) to downstream KEs up until the reduction of T4 in the plasma (KE 281). Referring to the AOPs 42 and 54, there is ample evidence the reduction of plasma T4 correlates to neurological deficits. What is unclear is the effect of dioxin or dioxin-like PCB effects directly on the brain itself which can activate the TH receptors mitigate the effects of low T4 (Gauger et al. 2004, Fritsche et al. 2005, Kitamura et al. 2005, Zhang et al. 2018), so the dose concordance is still unproven due to this. Temporal concordance among the key events and adverse effect: There are two aspects of the temporal concordance of the key events in a developmental AOP.  The first is the temporal concordance refers to the degree to which the data support the hypothesized sequence of the key events; i.e., the effect on KE1 is observed before the effect on KE2, which is observed before the effect on KE3, and so on. This translates to the temporal concordance of the AOP from AhR activation to decreased TH synthesis, reduced circulating TH concentrations, decreased nervous system TH, altered gene expression and anatomy in the hippocampus, and subsequent alterations in hippocampal physiology that result in decrements in cognition. The strength of the temporal concordance between these KEs varies from weak to strong. There is strong evidence for the early direct KEs from both empirical and modelling studies, and for many of the later KEs via the indirect KERs. The temporal concordance between AhR activation and TH synthesis is clearly evidenced by data from ex vivo and in vitro studies, as well as computational models (Kohn et al. 1996). Data supporting the temporal concordance for the later KEs, i.e., from serum TH to changes in hippocampal physiology are limited or lacking. The second aspect of temporal concordance for developmental AOPs is evidenced by demonstrations for critical windows of development where key events are perturbed, for which the effects are permanent and found during early development and throughout adulthood. It is a well-recognized fact that there are critical developmental windows for disruption of serum THs that result in subsequent alterations in all downstream KEs including the AO cognitive function later in development and adulthood.  Consistency:  Uncertainties, inconsistencies, and data gaps: There are several areas of uncertainty and data gaps in the current AOP, especially regarding the later KERs and AOs. The main uncertainty is that AhR agonists do not produce toxic effects and can’t be measured without in vivo testing. Also, hippocampal changes and effect on mental functions in mammals are hard to measure.

<table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td>TCDD</td> <td>May act as agonists to thyroid receptors in the brain to mitigate lower T4 levels (Seo et al. 1999, Widholm et al. 2003)</td> <td> KER 761 </td> </tr> <tr> <td>PCB-118</td> <td>May act as agonists to thyroid receptors in the brain to mitigate lower T4 levels (Gauger et al. 2007)</td> <td>KER 761</td> </tr> </tbody> </table>

Assessment of quantitative understanding of the AOP: Currently, there are quantitative models for the early KERs from AhR activation to serum hormone concentrations (Kohn et al. 1996), but none for later KERs. One could link the reduction of thyroid hormones to malformations in the brain, as in Hassan et al. (2017) quantitatively linked PTU-induced TH synthesis declines in the dam and the foetus to decrements in serum and brain TH concentrations to a structural malformation in the postnatal brain. At present, the overall quantitative understanding of the AOP is insufficient to directly link a measure of chemical potency as a TPO inhibitor to a quantitative prediction of effect on cognitive function (e.g., IQ in humans, learning deficits in rodents).

To determine whether a particular compound can potentially cause this AO, one must find out first if the compound is a activator of UGT isoforms in the liver that can glucuronidate T4. It is not sufficient to see whether it has a positive outcome from the EROD or CALUX assays as they only measure CYP inhibition and AhR nuclear activation, respectively.  This could possibly done in hepatocyte cell lines or primary hepatocytes, but it is uncertain what level of activation would cause T4 levels to go sufficiently down in the brain in vivo to counteract that some AhR agonists can also activate thyroid hormone receptors in the brain and can mitigate the AO.

Not Specified

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