60-56-0PMRYVIKBURPHAH-UHFFFAOYSA-NPMRYVIKBURPHAH-UHFFFAOYSA-N
Methimazole2H-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
DTXSID402082051-52-5KNAHARQHSZJURB-UHFFFAOYSA-NKNAHARQHSZJURB-UHFFFAOYSA-N
6-Propyl-2-thiouracil6-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-
DTXSID502120914797-73-0VLTRZXGMWDSKGL-UHFFFAOYSA-MVLTRZXGMWDSKGL-UHFFFAOYSA-M
PerchloratePerchlorate ion
Perchlorate ion (ClO41-)
Perchlorate ion(1-)
Perchlorate(1-)
Perchloric acid, ion(1-)
DTXSID6024252PR:000011397nuclear receptor subfamily 1 group I member 2CHEBI:30660thyroxinePR:000001325beta-1,3-glucuronyltransferaseUBERON:0002421hippocampal formationGO:0045202synapseGO:0023052signalingMP:0000032cochlear degenerationGO:0010817regulation of hormone levelsMP:0005475abnormal circulating thyroxine levelGO:0015020glucuronosyltransferase activityGO:0003824catalytic activityGO:0007420brain developmentGO:0007268chemical synaptic transmissionGO:0010468regulation of gene expressionGO:0008152metabolic process1increased2decreased8morphological change4abnormalMethimazole2016-11-29T18:42:192016-11-29T18:42:19Propylthiouracil2016-11-29T18:42:222016-11-29T18:42:22Perchlorate2016-11-29T18:42:262016-11-29T18:42:26Iodine deficiency2017-03-26T11:37:442017-03-26T11:37:4410116Rattus norvegicus10090Mus musculus9606Homo sapiens10116ratWCS_9606humanWCS_9031chicken10090mouseWCS_8355Xenopus laevisWCS_7955zebrafishWCS_90988fathead minnow9823Sus scrofaWCS_8355African clawed frog10116rats10118Rattus sp.Activation, Pregnane-X receptor, NR1l2Activation, Pregnane-X receptor, NR1l2Molecular<p><em>Pregnane X receptor (PXR), also known as steroid NR subfamily 1, group I, member 2 (NR1I2), is a nuclear receptor that can be activated by pregnenolone and progesterone and many chemicals due to low specificity; once activated it binds to Retinoid X receptor (RXR) and increases upregulation of target genes (Mellor et al. 2016). PXR has an important role in lipid homeostasis by regulating the rate of lipid intake particularly through activation of cluster of differentiation 36, also known as platelet glycoprotein 4 [(CD36) (Zhou et al. 2006; Gwag et al. 2019)].</em></p>
<p><em>Pregnane X receptor (PXR) is measured by changes in gene expression and protein levels. Effects of PXR on expression of downstream genes can be investigating using metabolomics and RT-qPCR approaches. In addition, targeted ToxCast assays using SeqAPASS evaluations can evaluate gene expression changes from chemical exposure for model species (NCBI Accession Number NP_071285.1 for NR1I2 in Lalone et al. 2018).</em></p>
<p><em>Life Stage: Older individuals are more likely to manifest this key event (adults > juveniles) due to increased opportunity to upregulate gene expression.</em></p>
<p><em>Sex: Applies to both males and females.</em></p>
<p><em>Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).</em></p>
CL:0000255eukaryotic cellHighUnspecificHighAdultModerateJuvenileHighHighModerate<p><em>Gwag, T., Meng, Z., Sui, Y., Helsley, R.N., Park, S.-H., Wang, S., Greenberg, R.N., and Zhou, C. 2019. Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis. Journal of Hepatology 70: 930–940.</em></p>
<p><em>LaLone, C.A., Villeneuve, D.L., Doering, J.A., Blackwell, B.R., Transue, T.R., Simmons, C.W., Swintek, J., Degitz, S.J., Williams, A.J., and Ankley, G.T. 2018. Evidence for Cross Species Extrapolation of Mammalian-Based High-Throughput Screening Assay Results. Environmental Science and Technology 52: 13960−13971.</em></p>
<p><em>Mellor, C.L., Steinmetz, F.P., and Cronin, T.D. 2016. The identification of nuclear receptors associated with hepatic steatosis to develop and extend adverse outcome pathways. Critical Reviews in Toxicology 46(2): 138-152. DOI:10.3109/10408444.2015.1089471</em></p>
<p><em>Zhou, J., Zhai, Y., Mu, Y., Gong, H., Uppal, H., Toma, D., Ren, S., Evans, R.M., and Xie, W. 2006. A Novel Pregnane X Receptor-mediated and Sterol Regulatory Element-binding Protein-independent lipogenic pathway. The Journal of Biological Chemistry 281(21): 15013–15020.</em></p>
<p><em>NOTE: Italics symbolize edits from John Frisch</em><br />
</p>
2016-11-29T18:41:232024-03-26T10:34:27Loss, Cochlear functionLoss, Cochlear functionOrganUBERON:0001690earNot Specified2016-11-29T18:41:242017-09-16T10:14:23Thyroxine (T4) in neuronal tissue, Decreased T4 in neuronal tissue, DecreasedOrgan<p>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). </p>
<p>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 3<sup>rd</sup> trimester in humans such that adult levels of T3 and T4 in brain are not reached in rodents until the 2<sup>nd</sup>-3<sup>rd</sup> postnatal week.</p>
<p>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. </p>
<p>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).</p>
<p>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.</p>
<p>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.</p>
<p>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).</p>
UBERON:0000955brainHighFemaleHighMaleHighDuring brain developmentHighHighLow<p>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.</p>
<p>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.</p>
<p>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</p>
<p>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. Invest. 86:889-899.</p>
<p>Chatonnet F., Picou F., Fauquier T., and Flamant F., (2011). Thyroid Hormone Action in Cerebellum and Cerebral Cortex Development, Journal of Thyroid Research, Volume 2011, Article ID 145762, 8 pages http://dx.doi.org/10.4061/2011/145762)</p>
<p>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.</p>
<p>Donzelli R, Colligiani D, Kusmic C, Sabatini M, Lorenzini L, Accorroni A, Nannipieri M, Saba A, Iervasi G, Zucchi R. Effect of Hypothyroidism and Hyperthyroidism on Tissue Thyroid Hormone Concentrations in Rat. Eur Thyroid J. 2016 Mar;5(1):27-34.</p>
<p>Friesema EC, Jansen J, Milici C, Visser TJ (2005) Thyroid hormone transporters. Vitam Horm 70:137-167.</p>
<p>Galton VH 1983 Thyroid hormone action in amphibian metamorphosis. In: Oppenheimer JH, Samuels HH (eds) Molecular Basis of Thyroid Hormone Action. Academic Press, New York, pp 445–483.</p>
<p>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.</p>
<p>Grijota-Martinez C, Diez D, Morreale de Escobar G, Bernal J, Morte B. (2011). Lack of action of exogenously administered T3 on the fetal rat brain despite expression of the monocarboxylate transporter 8. Endocrinology. 152:1713-1721.</p>
<p>Guadano-Ferraz A, Obregon MJ, St Germain DL, Bernal J. (1997). The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain. Proc Natl Acad Sci USA. 94: 10391–10396.</p>
<p>Hennemann G, Docter R, Friesema EC, de Jong M, Krenning EP, Visser TJ. (2001). Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocr Rev. 22:451-476.</p>
<p>Hernandez A, Quignodon L, Martinez ME, Flamant F, St Germain DL. Type 3 deiodinase deficiency causes spatial and temporal alterations in brain T3 signaling that are dissociated from serum thyroid hormone levels. Endocrinology. 2010 Nov;151(11):5550-8.</p>
<p>Heuer H. (2007). The importance of thyroid hormone transporters for brain development and function. Best Pract Res Clin Endocrinol Metab. 21:265–276.</p>
<p>Heuer H, Maier MK, Iden S, Mittag J, Friesema EC, Visser TJ, Bauer K. (2005). The monocarboxylate transporter 8 linked to human psychomotor retardation is highly expressed in thyroid hormone-sensitive neuron populations. Endocrinology 146:1701–1706.</p>
<p>Jansen J, Friesema EC, Kester MH, Milici C, Reeser M, Gruters A, Barrett TG, Mancilla EE, Svensson J, Wemeau JL, Busi da Silva Canalli MH, Lundgren J, McEntagart ME, Hopper N, Arts WF, Visser TJ (2007) Functional analysis of monocarboxylate transporter 8 mutations identified in patients with X-linked psychomotor retardation and elevated serum triiodothyronine. J Clin Endocrinol Metab 92:2378-2381.</p>
<p>Kester MH, Martinez de Mena R, Obregon MJ, Marinkovic D, Howatson A, Visser TJ, Hume R, Morreale de Escobar G. (2004). Iodothyronine levels in the human developing brain: major regulatory roles of iodothyronine deiodinases in different areas. J Clin Endocrinol Metab 89:3117–3128.</p>
<p>Mayer 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 1;124(5):1987-99.</p>
<p>Moog N.K., Entringer S., Heim Ch., Wadhwa PD., Kathmann N., Buss C. (2017). Influence of maternal thyroid hormones during gestation on fetal brain development. Neuroscience, 2017, 342: 68–100. doi:10.1016/j.neuroscience.2015.09.0</p>
<p>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</p>
<p>Müller J, Heuer H. Expression pattern of thyroid hormone transporters in the postnatal mouse brain. Front Endocrinol (Lausanne). 2014 Jun 18:5:92.</p>
<p>Obregon MJ, Mallol J, Escobar del Rey F, Morreale de Escobar G. (1981). Presence of l-thyroxine and 3,5,3-triiodo-l-thyronine in tissues from thyroidectomised rats. Endocrinology 109:908-913.</p>
<p>Power DM, Llewellyn L, Faustino M, Nowell MA, Björnsson BT, Einarsdottir IE, Canario AV, Sweeney GE. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 2001 Dec;130(4):447-59.</p>
<p>Ruiz de Oña C, Obregón MJ, Escobar del Rey F, Morreale de Escobar G. Developmental changes in rat brain 5'-deiodinase and thyroid hormones during the fetal period: the effects of fetal hypothyroidism and maternal thyroid hormones. Pediatr Res. 1988 Nov;24(5):588-94.</p>
<p>Simon R, Tietge JE, Michalke B, Degitz S, Schramm KW. Iodine species and the endocrine system: thyroid hormone levels in adult Danio rerio and developing Xenopus laevis. Anal Bioanal Chem. 2002 Feb;372(3):481-5.</p>
<p>St Germain DL, Galton VA, Hernandez A. (2009). Minireview: Defining the roles of the iodothyronine deiodinases: current concepts and challenges. Endocrinology. 150:1097-107.</p>
<p>Sugiyama D, Kusuhara H, Taniguchi H, Ishikawa S, Nozaki Y, Aburatani H, Sugiyama Y. (2003). Functional characterization of rat brain-specific organic anion transporter (Oatp14) at the blood–brain barrier: high affinity transporter for thyroxine. J Biol Chem. 278:43489–43495.</p>
<p>Tietge JE, Butterworth BC, Haselman JT, Holcombe GW, Hornung MW, Korte JJ, Kosian PA, Wolfe M, Degitz SJ. Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis. Aquat Toxicol. 2010 Jun 1;98(1):44-50.</p>
<p>Tohyama K, Kusuhara H, Sugiyama Y. (2004). Involvement of multispecific organic anion transporter, Oatp14 (Slc21a14), in the transport of thyroxine across the blood-brain barrier. Endocrinology. 145: 4384–4391.</p>
<p>Tu HM, Legradi G, Bartha T, Salvatore D, Lechan RM, Larsen PR. (1999). Regional expression of the type 3 iodothyronine deiodinase messenger ribonucleic acid in the rat central nervous system and its regulation by thyroid hormone. Endocrinology. 140: 784–790.</p>
<p>Van Herck SL, Geysens S, Delbaere J, Darras VM. Regulators of thyroid hormone availability and action in embryonic chicken brain development. Gen Comp Endocrinol. 2013.190:96-104.</p>
<p>Visser EW, Visser TJ. (2012). Finding the way into the brain without MCT8. J Clin Enodcrinol Metab. 97:4362-4365.</p>
<p>Visser WE, Friesema EC, Jansen J, Visser TJ. (2007). Thyroid hormone transport by monocarboxylate transporters. Best Pract Res Clin Endocrinol Metab. 21:223–236.</p>
<p>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</p>
2016-11-29T18:41:232019-04-04T09:13:27 Thyroxine (T4) in serum, DecreasedT4 in serum, DecreasedTissue<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">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).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">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).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Normal serum T4 reference ranges can be species and lifestage specific. In <strong>rodents</strong>, 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 <strong>humans</strong>, 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).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In some <strong>frog </strong>species, there is an analogous peak in </span><span style="color:black">THs </span><span style="color:black">in tadpoles that starts around embryonic NF stage 56, peaks at </span><span style="color:black">s</span><span style="color:black">tage 62 and the declines to lower levels by </span><span style="color:black">s</span><span style="color:black">tage 56 (Sternberg et al., 2011; Leloup and Buscaglia, 1977). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Additionally, ample evidence is available from studies investigating responses to inhibitors of </span><span style="color:black">TH </span><span style="color:black">synthesis in <strong>fish</strong>. 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.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">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).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">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.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: 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). </span><span style="color:black">T</span><span style="color:black">heir 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 TH</span><span style="color:black">s</span><span style="color:black"> 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</span><span style="color:black">,</span><span style="color:black"> extrapolation regarding TH action across species and developmental stages should be done with caution.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">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</span><span style="color:black">-</span> <span style="color:black">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.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: 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 </span><span style="color:black">TH </span><span style="color:black">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 </span><span style="color:black">system </span><span style="color:black">disruptors.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex</span></strong><span style="color:black">: The KE is plausibly applicable to both sexes. </span><span style="color:black">THs</span> <span style="color:black">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 </span><span style="color:black">TH</span> <span style="color:black">levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (HernandezāMariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in </span><span style="color:black">TH</span> <span style="color:black">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.</span></span></span></span></p>
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2016-11-29T18:41:232022-10-10T08:52:30Induction, Upregulation of glucuronyltransferase activityInduction, Upregulation of glucuronyltransferase activityMolecularCL:0000255eukaryotic cellNot SpecifiedNot Specified2016-11-29T18:41:232017-09-16T10:14:22Hippocampal anatomy, Altered Hippocampal anatomy, Altered Tissue<p>The hippocampus is a major brain region located in the medial temporal lobe in humans and other mammals (West, 1990). Developmentally it is derived from neuronal and glial cells in the neural tube and differentiates in the proencephalon and telencephalon. The hippocampus is a cortical structure, but only contains 3-layers, distinct from the 6-layered neocortical structures. For this reason, it is known as archicortex or paleocortex meaning old cortex. Within humans, the structure is identified as early as fetal week 13 and matures rapidly until 2 to 3 years of age (Kier et al 1997), with continuing slow growth thereafter until adult ages (Utsunomiya et al., 1999). In rodents, the hippocampus begins to form in midgestation, with the CA fields forming in advance of the dentate gyrus. Dentate gyrus forms in late gestation with most of its development occurring in the first 2-3 postnatal weeks (Altman and Bayer, 1990a; 1990b).</p>
<p>The structure of the hippocampus has been divided into regions that include CA1 through CA4 and the dentate gyrus. The principal cell bodies of the CA field are pyramidal neurons, those of the dentate gyrus are granule cells. The dentate gyrus forms later in development than the CA fields of the hippocampus. These regions are generally found in all mammalian hippocampi.</p>
<p>The major input pathway to the hippocampus is from the layer 2 neurons of the entorhinal cortex to the dentate gyrus via the perforant path forming the first connection of the trisynaptic loop of the hippocampal circuit. Direct afferents from the dentate gyrus (mossy fibers) then synapse on CA3 pyramidal cells which in turn send their axons (Schaeffer Collaterals) to CA1 neurons to complete the trisynaptic circuit (Figure 1). From the CA fields information then passes through the subiculum entering the fiber pathways of the alveus, fimbria, and fornix and it routed to other areas of the brain (Amaral and Lavenex, 2006). Through the interconnectivity within the hippocampus and its connections to amygdala, septum and cortex, the hippocampus plays a pivotal role in several learning and memory processes, including spatial behaviors. The primary input pathway to the CA regions of the hippocampus is from the septum by way of the fornix and direct input from the amygdala. Reciprocal outputs from the hippocampus back to these regions and beyond also exist.</p>
<p><strong> </strong></p>
<p><strong>Trisynaptic Hippocampal Circuitry</strong></p>
<p><img alt="" 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" /></p>
<p style="text-align:justify"> </p>
<p>Data in support of this key event have been collected using a wide variety of standard biochemical, histological and anatomical methods (e.g., morphometrics, immunohistochemical staining, in situ hybridization and imaging procedures). Many of methods applied to reveal anatomical abnormalities are routine neurohistopathology procedures similar to those recommended in EPA and OECD developmental neurotoxicity guidelines (US EPA, 1998; OCED, 2007). Subtle cytoarchitectural features depend on more specialized birth dating procedures and staining techniques. It is essential to consider the timing of events during development for detection to occur, as well as the timing for detection (Hevner, 2007; Garman et al., 2001; Zgraggen et al., 2012). Similar techniques used in rodent stydies have been applied to postmortem tissue in humans. </p>
<p>In humans, structural neuroimaging techniques are used to assess hippocampal volume with an analysis technique known as voxel-based morphometry (VBM). Volume of brain regions is measured by drawing regions of interest (ROIs) on images from brain scans obtained from magnetic resonance imaging (MRI) or positron emission tomography (PET) scans and calculating the volume enclosed. (Mechelli et al., 2005). Similar imaging techniques can be applied in rodent models (Powell et al., 2009; Hasegawa et al., 2010; Pirko et al., 2005; Pirko and Johnson, 2008).</p>
<p>The hippocampus is generally similar in structure function across most mammalian species (West, 1990). The vast majority of information on the structure of the hippocampus is from mice, rats and primates including humans.</p>
UBERON:0000955brainHighMaleHighFemaleHighDuring brain developmentHighHighHigh<p>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.</p>
<p>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.</p>
<p>Amaral D, Lavenex P (2006). "Ch 3. Hippocampal Neuroanatomy". In Andersen P, Morris R, Amaral D, Bliss T, O'Keefe J. The Hippocampus Book. Oxford University Press. ISBN 978-0-19-510027-3.</p>
<p>Garman RH, Fix AS, Jortner BS, Jensen KF, Hardisty JF, Claudio L, Ferenc S. Methods to identify and characterize developmental neurotoxicity for human health risk assessment. II: neuropathology. Environ Health Perspect. 2001 Mar;109 Suppl 1:93-100.</p>
<p>Hasegawa M, Kida I, Wada H. A volumetric analysis of the brain and hippocampus of rats rendered perinatal hypothyroid. Neurosci Lett. 2010 Aug 2;479(3):240-4.</p>
<p>Hevner RF. Layer-specific markers as probes for neuron type identity in human neocortex and malformations of cortical development. J Neuropathol Exp Neurol. 2007 66(2):101-9.</p>
<p>Kier, EL, Kim, JH, Fulbright, K, Bronen, RA. Embryology of the human fetal hippocampus: MR imaging, anatomy, and histology. AJNR Am J Neuroradiol: 1997, 18(3);525-32.</p>
<p>Mechelli A, Price C, Friston K, Ashburner J (2005) Voxel-Based Morphometry of the Human Brain: Methods and Applications. Curr Med Imaging Rev 1:105-113.</p>
<p>OECD. 2007. OECD guidelines for the testing of chemicals/ section 4: Health effects. Test no. 426: Developmental neurotoxicity study. http://www.oecd.org/dataoecd/20/52/37622194.</p>
<p>Pirko I, Fricke ST, Johnson AJ, Rodriguez M, Macura SI. Magnetic resonance imaging, microscopy, and spectroscopy of the central nervous system in experimental animals. NeuroRx. 2005 Apr;2(2):250-64.</p>
<p>Pirko I, Johnson AJ. Neuroimaging of demyelination and remyelination models. Curr Top Microbiol Immunol. 2008; 318:241-66.</p>
<p>Powell MH, Nguyen HV, Gilbert M, Parekh M, Colon-Perez LM, Mareci TH, Montie E. Magnetic resonance imaging and volumetric analysis: novel tools to study the effects of thyroid hormone disruption on white matter development. Neurotoxicology. 2012 Oct;33(5):1322-9.</p>
<p>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.</p>
<p>Utsunomiya, H., K Takano, M Okazaki, A Mitsudome Development of the temporal lobe in infants and children: analysis by MR-based volumetry. AJNR Am J Neuroradiol: 1999, 20(4);717-23.</p>
<p>West MJ (1990). "Stereological studies of the hippocampus: a comparison of the hippocampal subdivisions of diverse species including hedgehogs, laboratory rodents, wild mice and men". Progress in Brain Research. Progress in Brain Research 83: 13–36.</p>
<p>Zgraggen E, Boitard M, Roman I, Kanemitsu M, Potter G, Salmon P, Vutskits L, Dayer AG, Kiss JZ. Early postnatal migration and development of layer II pyramidal neurons in the rodent cingulate/retrosplenial cortex. Cereb Cortex. 2012 Jan;22(1):144-57. </p>
<p> </p>
2016-11-29T18:41:262022-05-20T05:45:05Hippocampal Physiology, AlteredHippocampal Physiology, AlteredTissue<p>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).</p>
<p>During development (see KE: 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 brought about by activity-dependent synaptic pruning and synapse elimination. This is a classic case of the interaction between physiological and anatomical development, where anatomy develops first, and can be ‘reshaped’ by physiological function (Kutsarova et al., 2017).</p>
<p>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 areaCA1s is first seen on postnatal day 5 and increases 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. </p>
<p>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 from slices taken from naive or exposed animals. Field potentials 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., iodine deficiency, thyroidectomy, 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 function of the hippocampus are excitatory synaptic transmission, inhibitory synaptic transmission, and synaptic plasticity in the form of long-term potentiation (LTP).</p>
<p>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).</p>
<p>Inhibitory Synaptic Transmission: Pairs of stimulus pulses delivered in close temporal proximity is 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).</p>
<p>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 maintanence (Bramham and Messaoudi, 2005; Kandell et al., 2012; Malenka and Bear, 2004).</p>
<p>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).</p>
<p>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.</p>
<p>The majority of evidence for this key event come 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).</p>
UBERON:0000955brainHighFemaleHighMaleHighDuring brain developmentModerateHighHigh<p>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-</p>
<p>Bramham CR, Messaoudi E (2005) BDNF function in adult synaptic plasticity: the synaptic consolidation hypothesis. Prog Neurobiol 76:99-125.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>Harris KM, Teyler TJ. Developmental onset of long-term potentiation in area CA1 of the rat hippocampus. J Physiol. 1984. 346:27-48.</p>
<p>Kandell, E., Schwartz, J., Siegelbaum, A. and Hudspeth, A.J. (2012) Principles of Neural Science, 5<sup>th</sup> Edition. Elsevier, North Holland.</p>
<p>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.</p>
<p>Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44:5-21.</p>
<p>McCarthy, G. (1995) Review: Functional Neuroimaging and Memory. The Neuroscientist, 1:155-163.</p>
<p>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.</p>
<p>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.</p>
<p>Smith, E and Jonides, J. (1997). Working Memory: A View from Neuroimaging. Cognitive Psychology, 33:5-42.</p>
<p>Tietze, KJ. (2012). Review of Laboratory and Diagnostic Tests- Positron Emission Tomography. In Clinical Sills for Pharmacists, 3rd Edition, pp 86-122. </p>
<p>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.</p>
<p>Willoughby KA, McAndrews MP, Rovet JF (2014) Effects of maternal hypothyroidism on offspring hippocampus and memory. Thyroid 24:576-584.</p>
2016-11-29T18:41:262018-08-11T09:41:37Hippocampal gene expression, Altered Hippocampal gene expression, Altered Tissue<p>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).</p>
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" /></p>
<p> </p>
<p>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.</p>
<p>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.</p>
<p>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).</p>
<p>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.</p>
UBERON:0000955brainHighFemaleHighMaleHighDuring brain developmentHighHighModerate<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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..</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p style="margin-left:13.5pt !msorm">Kempermann G. New neurons for 'survival of the fittest'. Nat Rev Neurosci. 2012 Oct;13(10):727-36.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
2016-11-29T18:41:262018-08-11T09:26:56Increased, Clearance of thyroxine from serumIncreased, Clearance of thyroxine from serumTissue<p>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).</p>
<p>Deiodination:</p>
<p>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.</p>
<p>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).</p>
<p>Conjugation:</p>
<p>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.</p>
<p>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).</p>
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<p> </p>
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<p>Data in animals for PCBs</p>
<p>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)</p>
<p>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.</p>
<p>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). </p>
<p>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 <em>in vitro</em> 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). </p>
<p>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.</p>
<p>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 [<sup>125</sup>I]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[<sup>125</sup>I]T4 accumulated following treatment.</p>
<p>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 [<sup>125</sup>I]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 [<sup>125</sup>I]T4 from serum relative to controls and serum pharmacokinetic data were estimated, along with tissue-to-serum (K<sub>p</sub> value) concentration ratios and [<sup>125</sup>I]T4 tissue distribution levels. These data imply that 4-OH-CB187 inhibits formation of the [<sup>125</sup>I]T4-TTR complex, which may lead to a change in tissue distribution, with accumulation in the liver and kidney mainly.</p>
<p>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 [<sup>125</sup>I]T4 clearance from serum and accumulation in tissues. Treatment with pentaCB resulted in decrease of [<sup>125</sup>I]T4-TTR and increase in [<sup>125</sup>I]T4-albumin and [<sup>125</sup>I]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).</p>
<p>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 [<sup>125</sup>I]T4 complexes with TBG, albumin and TTR were only observed in C57BL/6 mice (and not DBA/2), despite [<sup>125</sup>I]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).</p>
<p>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).</p>
<p>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).</p>
<p>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 [<sup>125</sup>I]T4 and collection of urine, blood, bile and urine. No treatments increased urinary excretion of [<sup>125</sup>I]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 [<sup>125</sup>I]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. </p>
<p>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.</p>
<p> </p>
<p>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).</p>
<p>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.</p>
<p><em>Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? </em></p>
<p>Thyroid hormone uptake into human tissues has been measured by analyzing the rate of disappearance of radiolabeled TH from plasma into rapidly and slowly equilibrating tissue compartments (Visser 2010).</p>
<p>Measuring the rate of T4 glucuronidation and sulfation as well as biliary excretion informs the mechanism of action of thyroid system modulation. Studies involving knock/out mice and thyroidectomized rats also inform this mechanism.</p>
<p> </p>
<p>Total T4 is most often measured using human serum based diagnostic kits, but free T4 (and T3) is only directly measured through equilibrium dialysis and ultrafiltration (Midgley 2001). Large volumes of serum must be used due to the very low concentrations of free T4 normally found (0.1% of total T4), which requires pooling of samples taken from fetus or pup. Some researchers have tried to “micronize” this process through combining RIA to measure total TH and dialysis to estimate the free fraction (Zoeller et al 2007). Extracted materials can also be quantified by HPLC. The reference range for free T4 is 9.8 to 18.8 pM/L (Dirinck et al 2016).</p>
<p>T3 is found in similar plasma concentrations to T4 (i.e. 5-10 pM) with < 0.4% being in the unbound state. Measuring free serum T3 is labor intensive and requires equipment not available in many clinical reference laboratories and thus ultrafiltration is often used (Abdalla and Bianco 2014). Immunoassays and MS/MS are also used.</p>
<p>Measuring displacement of T4 from serum transport proteins is done mainly via one of three <em>in vitro</em> methods: radioligand binding assay, plasmon resonance-based biosensor, or fluorescence displacement.</p>
<p>Radioligand binding assays, using [<sup>125</sup>I]-T4 as a label, were developed to demonstrate affinity for xenobiotics to human or rat TTR and TBG (Brouwer and van den Berg 1986, Lans et al 1994). The most commonly used method was first published by Somack et al 1982 and adapted by Hamers et al 2006, Lans et al 1993 and Ucan-Marin et al 2010. Similar assays have been developed using [<sup>125</sup>I]-T3 as a label for affinity to chicken and bullfrog TTR (Yamauchi et al 2003). Radioligand methods suffer from having to use heavily regulated isotopes and lower throughput to provide free T4 measurements (due to the extra wash/separation procedure needed). The most well-known protocol uses TTR purified from human serum (which may not be as stable as recombinant) and performed in a pure aqueous solution, which may not be as stable for lipophilic compounds (Chauhan et al 2000 is an example using PCBs).</p>
<p>Purkey et al 2001 published a binding assay using polyclonal TTR antibodies covalently bound to sepharose resin which is then mixed with plasma pre-treated with compound of interest, washed and analyzed via HPLC.</p>
<p>Marchesini et al 2006 reported on the development of two surface plasmon resonance(SPR)-based biosensor assays using recombinant TTR and TBG, validated with known thyroid disruptors and structurally related compounds including halogenated phenols, polychlorinated biphenyls, bisphenols and a hydroxylated PCB metabolite (4-OH-CB 14). TH is covalently bound to a gold-layered chip and a mixture of the compound of interest and transport protein are injected in a flow cell passing over the bound TH. The authors found that these biosensor methods were more sensitive (IC50 of 8.6 ± 0.7 nM for rTTR), easier to perform and more rapid that radioligand binding assays and immunoprecipitation-HPLC.</p>
<p>Marchesini et al 2008 applied their biosensor-based screen to 62 chemicals of public health concern and found that hydroxylated metabolites of PCBs (particularly para-hydroxylated ones) and PBDEs (BDEs 47, 49 and 99) displayed the most potent binding to TBG and TTR, confirming many other previous studies. The authors conclude their optimized assays are suitable for high-throughput screening for potential thyroid disruption.</p>
<p>Cao et al 2010, Cao et al 2011 and Ren and Guo 2012 developed the FLU-TTR, based on a protein-binding fluorescent probe (ANSA, or 8-anilo-1-naphthalenesulfonic acid ammonium salt) that becomes highly fluorescent after binding to T4. When the compound of interest is introduced and displaces the ANSA-thyroxine probe, this fluorescence is reduced. This allows generation of binding constant (K) data as opposed to past efforts that generated IC<sub>50</sub> values. Cao et al 2011 developed a fluorescent microtiter method for pTTR and TBG tested with bisphenol A.</p>
<p>Montano et al 2012 developed a competitive T4-TTR fluorescence displacement assay in a 96-well format, modified from the original method (Nilsson and Petersen 1975) and using a new selective method to extract hydroxylated metabolites while reducing fatty acid interference (modified from Hovander et al 2000).</p>
<p>Aqai et al 2012 described a rapid and isotope-free (<sup>13</sup>C<sub>6</sub>-T4) screening of thyroid transport protein ligands, using a competitive binding assay for rTTR using fast ultrahigh performance LC-electrospray ionization triple-quadrupole MS. The method involves the use of immunomagnetic beads followed by screening with flow cytometry and UPLC-MS. The high-throughput screening mode is capable of detecting T4 in water at the part-per-trillion level and in the part-per-billion level in urine.</p>
<p>Relevant Phase II enzymes that are responsible for TH metabolism include UGT1A1, UGT1A6 and SULT2A1 while relevant cellular import/export transport proteins include MCT8, OATP1A4 and MRP2. All contribute towards systemic clearance of TH and conjugates from serum whether increasing biliary excretion or moving TH into tissues and across the placenta and BBB. Enzyme induction can only be measured via in vitro cell-based assays and since these enzymes are all controlled by specific nuclear receptors, assays targeting these receptors might act as surrogate measurement (Murk et al 2013). Several methods measuring expression of UGT or SULT mRNA have been published; however, there have been limited efforts to develop higher-throughput methods. The EPA ToxCast Phase I efforts used quantitative nuclease protection assays (qNPA) to screen several hundred chemicals for UGT1A1 and SULT2A1 (Rotroff et al 2010, Sinz et al 2006).</p>
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<p>Multiple UDP-glucuronyltransferases for the glucuronidation of thyroid hormone with preference for 3,3',5'-triiodothyronine (reverse T3). FEBS Lett. 315(1): 65-68.</p>
<p>Weiss, J.M., P.L. Andersson, M.H. Lamoree, P.E. Leonards, S.P. van Leeuwen and T. Hamers. (2009) Competitive binding of poly- and perfluorinated compounds to the thyroid hormone transport protein transthyretin. Toxicol. Sci. 109(2): 206-216.</p>
<p>Weiss, J. M., Andersson, P. L., Zhang, J., Simon, E., Leonards, P. E. G., Hamers, T., & Lamoree, M. H. (2015). Tracing thyroid hormone-disrupting compounds: database compilation and structure-activity evaluation for an effect-directed analysis of sediment. Analytical and Bioanalytical Chemistry, 5625–5634. <a href="http://doi.org/10.1007/s00216-015-8736-9" target="_blank">http://doi.org/10.1007/s00216-015-8736-9</a></p>
<p>Yamauchi, K., A. Ishihara, H. Fukazawa and Y. Terao. (2003) Competitive interactions of chlorinated phenol compounds with 3,3',5-triiodothyronine binding to transthyretin: detection of possible thyroid-disrupting chemicals in environmental waste water. Toxicol. Appl. Pharmacol. 187(2): 110-117.</p>
<p>Yen, P. M. (2001). Physiological and molecular basis of thyroid hormone action. Physiological Reviews, 81(3), 1097–1142.</p>
<p>Zhang, J., J.H. Kamstra, M. Ghorbanzadeh, J.M. Weiss, T. Hamers and P.L. Andersson. (2015) In Silico Approach To Identify Potential Thyroid Hormone Disruptors among Currently Known Dust Contaminants and Their Metabolites. Environ. Sci. Technol. 49(16): 10099-10107.</p>
<p>Zoeller, R. T., Tan, S. W., & Tyl, R. W. (2007). General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical Reviews in Toxicology, 37(1-2), 11–53.</p>
<p>Zoeller, R.T. and J. Rovet. (2004) Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings. J. Neuroendocrinol. 16(10): 809-818.</p>
2016-11-29T18:41:282021-01-26T10:41:19831a5862-56b8-48e0-9f45-13ca2e58c1a3a7f2f946-b935-470b-a5c4-c0348e7d8f4aNot Specified2021-09-14T12:29:422021-09-14T12:29:42a7f2f946-b935-470b-a5c4-c0348e7d8f4ae172c1d7-325f-40d4-b5e1-b6776e30e561Not SpecifiedNot SpecifiedNot Specified2021-09-10T08:32:292021-09-10T08:32:29e172c1d7-325f-40d4-b5e1-b6776e30e5612134baa0-ef7e-4892-ad71-6a3a7fcd0ec3Not SpecifiedNot SpecifiedNot Specified2021-01-26T10:42:592021-01-26T10:42:592134baa0-ef7e-4892-ad71-6a3a7fcd0ec3d0138474-31a3-4480-b70d-b4db64f1b3e4<p>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).</p>
<p>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).</p>
<p>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).</p>
<p>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.</p>
<p>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). </p>
<p style="margin-left:40px"><em>Temporal Evidence: </em>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.</p>
<p style="margin-left:40px"><em>Dose-Response Evidence</em>: 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).</p>
<p>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</p>
<p>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.</p>
ModerateMaleModerateFemaleHighDuring brain developmentModerateAll life stagesModerateModerateModerate<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>Fekete C, Lechan RM. Central regulation of hypothalamic-pituitary-thyroid axis under physiological and pathophysiological conditions. Endocr Rev. 2014 Apr;35(2):159-94</p>
<p>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.</p>
<p>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.</p>
<p>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]</p>
<p>Morreale de Escobar, G., Obregon, M.J., and Escobar del Ray, F. (1987). Fetal and maternal thyroid hormones. Hormone Res. 26:12–27.</p>
<p>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.</p>
<p>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</p>
<p>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.</p>
<p>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.</p>
<p>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]</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>St. Germain, D.L. and Galton, V.A. (1997). The deiodinase family of selenoproteins. Thyroid 7:655–668.</p>
<p>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.</p>
<p>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.</p>
<p>Visser WE, Friesema EC, Visser TJ. Minireview: thyroid hormone transporters: the knowns and the unknowns. Mol Endocrinol. 2011 Jan;25(1):1-14.</p>
2016-11-29T18:41:332019-04-04T10:50:44d0138474-31a3-4480-b70d-b4db64f1b3e42d0e0b41-5eda-4f23-900f-5e95105708e8<p>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. </p>
<p>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. </p>
<p>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 <em>(non-adjacently)</em> 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. </p>
<p>The empirical support for this KER is moderate<em>.</em> 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.</p>
<p style="margin-left:.5in"><em>Temporal Evidence: </em>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).</p>
<p style="margin-left:.5in"><em>Dose-Response Evidence: </em>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).</p>
<p>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).</p>
HighMaleHighFemaleHighDuring brain developmentModerateModerate<p>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).</p>
<p>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.</p>
<p>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.</p>
<p>Bernal J (2007) Thyroid hormone receptors in brain development and function. Nat Clin Pract Endocrinol Metab 3:249-259.</p>
<p>Davis, P.J., Goglia, F., Leonard, J.L., 2016. Nongenomic actions of thyroid hormone. Nat. Rev. Endocrinol. 12, 111-121.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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</p>
<p>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.</p>
<p>Oppenheimer, J. H. and H. L. Schwartz (1997). "Molecular basis of thyroid hormone-dependent brain development." Endocr Rev 18(4): 462-75.</p>
<p>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]</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>Williams GR (2008), Neurodevelopmental and neurophysiological actions of thyroid hormone. J Neuroendocrinol. 2008 Jun;20(6):784-94</p>
2016-11-29T18:41:352018-08-11T19:18:162d0e0b41-5eda-4f23-900f-5e95105708e892792a9d-c1da-480b-bbda-6e3e94e37a8f<p>The basic biological processes that link gene regulation in the structural formation and function of all organs of the body are similar throughout the developing organism. In the developing brain, genes encode proteins critical for developmental events intrinsic to structural development (e.g., neurogenesis, neuronal migration, synaptogenesis, myelination). The development of the hippocampus is no exception to this general rule of biology.</p>
<p>The overall weight of evidence is moderate for a direct linkage between perturbation of the expression of genes in brain (and in hippocampus specifically) and neuroanatomical abnormalities. It is widely acknowledged that the development of the structure of the hippocampus is under the control of hippocampal gene expression. However, while an extensive body of literature exists linking some genes to hippocampal structure, there is no complete compendium on the total number of genes involved, nor direct causative links between the myriad of genes and the intricate development (both timing and location) of the majority of hippocampal structure. </p>
<p><span style="color:black">The biological plausibility of this KER is rated as strong.</span><span style="color:red"> </span>It is well established that gene regulation controls brain development. This also applies to the development of the hippocampus, where nuclear thyroid receptors that regulate gene transcription, directly or indirectly via transcription factor regulation, to control translation.</p>
<p>Empirical support for this KER is rated as strong<em>. </em>The number of publications in this area is extensive. A few examples are: Strange et al. (2014); Takei et al. (2016); and Shin et al. (2015). Work supporting the relationship includes use of a variety of animal models (i.e., nutritional deficiencies, chromosome abnormalities, gene deletions, knock out animals, toxicant exposures and developmental hormonal imbalance) (e.g., Frotscher, 2010; Castren and Castren, 2014; Spilker et al., 2016; Skucas et al., 2011; Lessman et al., 2011). Mutant mouse lines generated for genes involved in human cortical malformations such as doublecortin, reelin, Lis1 and Tuba1a also show gross disorganization within the hippocampus (Khalaf-Nazzal et al., 2013). Collectively, data from these studies clearly support the link between alterations in hippocampal gene expression and structural changes in hippocampal volume, cell number, and/or cytoarchitecture. A direct linkage between some specific gene targets and structural change in the hippocampus has been demonstrated using knock out and mutant mouse models (e.g., Grant et al., 1992; Lee et al., 2000; Frotscher, 2010; Castren and Castren, 2014; Spilker et al., 2016; Skucas et al., 2011; Lessman et al., 2011; Khalaf-Nazzal et al., 2013).</p>
<p style="margin-left:.5in"><em>Temporal Evidence</em>: 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 TR-regulated genes and subsequent formation of the anatomy of the hippocampus. This has been demonstrated in multiple studies. Many of the gene-anatomy relationships critical to brain development only exist during development, or exist only to a very limited extent in the adult brain. For example, genes controlling neuronal proliferation and migration are critically essential in hippocampal development, and their disruption results in abnormal hippocampal anatomy. Whereas, in the adult brain the genes are largely without effect as these processes are completed in the early neonatal period. In support of this, a limited number of studies have defined critical periods for the interaction of some genes and resulting neuroanatomical organization of the hippocampus (Lee et al., 2015; Favaro et al., 2009; Lee et al., 2000). In addition, there are some ‘rescue’ experiments for a select number of genes (eg., Lee et al., 2015; Spilker et al., 2016). Several examples are described below:</p>
<p style="margin-left:.5in">In the Jacob/Nsfm knockout model, hippocampal dysplasia is seen in hippocampal areas CA1 and CA3, characterized by reduced complexity of the synapto-dendritic cytoarchitecture, shorter dendrites and fewer branches (Spilker et al., 2016). Simplified dendritic trees and reduced synaptogenesis were also observed in hippocampal primary neurons cultured from these knock out mice relative to cultures from wild type mice. The protein product of Jacob/Nsfm regulates activity-dependent brain-derived neurotrophic factor (<em>Bdnf</em>) transcription. Lower BDNF levels were seen in area CA1 of knock out mice on postnatal day 10. The dysplasia seen in hippocampal neuronal cultures from knock mice could be reversed by BDNF supplementation if administered in early (2-4 days in vitro) but not later (15 days in vitro) in development.</p>
<p style="margin-left:.5in">Neuroregluin-2 (<em>Nrg2</em>) contributes to synaptogenesis of the granule cell layer of the hippocampus. In hippocampal slice cultures, inducible microRNA targeting strategies have demonstrated early suppression of <em>Nrg2</em> (4 days in vitro) but not late suppression (7 days in vitro) reduced synaptogenesis of inhibitory neurons. On the other hand, late treatment impaired the dendritic outgrowth of excitatory synaptic connections. These effects could be eliminated with overexpression of <em>Nrg2</em> (Lee et al., 2015).</p>
<p style="margin-left:.5in">Many of the gene-regulated processes involved in hippocampal development are also present in the developing cortex. In models of prenatal hypothyroidism, altered expression patterns of many genes involved in neuronal migration and apoptosis are associated with disruptions in hippocampal organization and cytoarchitecture of the cerebral cortex (Pathak et al, 2011; Mohan et al., 2012; Lui et al., 2010). Structural changes in hippocampus and cerebral cortex are dependent on time of exposure (Auso et al., 2003; Berbel et al., 2010; Pathak et al., 2011) and can be reversed with TH supplementation (Mohan et al., 2012; Pathak et al., 2011; Berbel et al., 2010).</p>
<p style="margin-left:.5in"><em>Dose-Response Evidence:</em> Dose-response data is lacking for this KER. Papers that utilize knock-out and mutant models do not provide ‘dose-response’ information for gene-anatomy relationships. Studies in which genes and anatomy were reported following developmental hypothyroidism were single high-dose studies that focused on varying the developmental window of exposure, but not necessarily the dose.</p>
<p>There are no inconsistencies in this KER, but there are some uncertainties. Few studies exist that report both gene expression changes and structural changes in the hippocampus in same study to provide direct causative evidence for this KER. Lacking also is the specific suite of genes that are altered in the hippocampus at particular developmental times that are causal to the structural defects reported. For future research, it is critical to generate data in which the upstream KE is modulated in a ‘dose-response’ manner to better support the causative relationship. Significant data gaps also exist for basic fetal hippocampal development.</p>
HighMaleHighFemaleModerateDuring brain developmentModerateHigh<p>The majority of data in support of this KER is 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. </p>
<p>Auso E, Lavado-Autric R, Cuevas E, Del Rey FE, Morreale De Escobar G, Berbel P (2004) A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration. Endocrinology 145:4037-4047.</p>
<p>Berbel P, Navarro D, Ausó E, Varea E, Rodríguez AE, Ballesta JJ, Salinas M, Flores E, Faura CC, de Escobar GM. Role of late maternal thyroid hormones in cerebral cortex development: an experimental model for human prematurity. Cereb Cortex. 2010 20(6):1462-75.</p>
<p>Castrén ML, Castrén E. BDNF in fragile X syndrome. Neuropharmacology. 2014 76:729-36.</p>
<p>Favaro R, Valotta M, Ferri AL, Latorre E, Mariani J, Giachino C, Lancini C, Tosetti V, Ottolenghi S, Taylor V, Nicolis SK. Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh. Nat Neurosci. 2009 12(10):1248-56.</p>
<p>Frotscher M. Role for Reelin in stabilizing cortical architecture. Trends Neurosci. 2010 Sep;33(9):407-14.</p>
<p>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.</p>
<p>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. [Epub ahead of print]</p>
<p>Khalaf-Nazzal R, Bruel-Jungerman E, Rio JP, Bureau J, Irinopoulou T, Sumia I, Roumegous A, Martin E, Olaso R, Parras C, Cifuentes-Diaz C, Francis F. Organelle and cellular abnormalities associated with hippocampal heterotopia in neonatal doublecortin knockout mice. PLoS One. 2013 Sep 2;8(9):e72622.</p>
<p>Lee KH, Lee H, Yang CH, Ko JS, Park CH, Woo RS, Kim JY, Sun W, Kim JH, Ho WK, Lee SH. Bidirectional Signaling of Neuregulin-2 Mediates Formation of GABAergicSynapses and Maturation of Glutamatergic Synapses in Newborn Granule Cells ofPostnatal Hippocampus. J Neurosci. 2015 Dec 16;35(50):16479-93.</p>
<p>Lee SM, Tole S, Grove E, McMahon AP. A local Wnt-3a signal is required fordevelopment of the mammalian hippocampus. Development. 2000 Feb;127(3):457-67.</p>
<p>Lessmann V, Stroh-Kaffei S, Steinbrecher V, Edelmann E, Brigadski T, Kilb W, Luhmann HJ. The expression mechanism of the residual LTP in the CA1 region of BDNF k.o. mice is insensitive to NO synthase inhibition. Brain Res. 2011 1391:14-23.</p>
<p>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.</p>
<p>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.</p>
<p>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.11-21.</p>
<p>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(8-9):664-72.</p>
<p>Shin JH, Kim YN, Kim IY, Choi DH, Yi SS, Seong JK. Increased Cell Proliferations and Neurogenesis in the Hippocampal Dentate Gyrus of Ahnak Deficient Mice. Neurochem Res. 2015 Jul;40(7):1457-62.</p>
<p>Skucas VA, Mathews IB, Yang J, Cheng Q, Treister A, Duffy AM, Verkman AS, Hempstead BL, Wood MA, Binder DK, Scharfman HE. Impairment of select forms ofspatial memory and neurotrophin-dependent synaptic plasticity by deletion ofglial aquaporin-4. J Neurosci. 2011 31(17):6392-7.</p>
<p>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, MikhaylovaM, Budinger E, Ohl FW, Stork O, Zenclussen AC, Karpova A, Schwegler H, Kreutz MR.A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impared BDNFSignaling in Dendritogenesis. PLoS Genet. 2016 Mar 15;12(3):e1005907</p>
<p>Strange BA, Witter MP, Lein ES, Moser EI. Functional organization of the hippocampal longitudinal axis. Nat Rev Neurosci. 2014 Oct;15(10):655-69.</p>
<p>Takei Y, Kikkawa YS, Atapour N, Hensch TK, Hirokawa N. Defects in Synaptic Plasticity, Reduced NMDA-Receptor Transport, and Instability of Postsynaptic Density Proteins in Mice Lacking Microtubule-Associated Protein 1A. J Neurosci. 2015 35(47):15539-54.</p>
2016-11-29T18:41:352018-08-11T19:05:2992792a9d-c1da-480b-bbda-6e3e94e37a8f1ba1bd15-817e-4295-9a7e-38d0f409f68f<p>The hippocampus is a highly integrated and organized communication and information processing network with millions of interconnections among its constitutive neurons (see Andersen et al, 2006). The neuronal spine is the primary site of action for synaptic interface between neurons. Although difficult to measure due to their small size, large number and variable shapes, changes in the frequency and structure of dendritic spines of hippocampal neurons has dramatic effects on synaptic physiology and plasticity (Harris et al., 1992). Anatomical integrity at a more macro-level is also essential for physiological function. The connectivity of axons emanating from one set of cells that synapse on the dendrites of the receiving cells must be intact for effective communication between neurons to be possible. Synaptogenesis is a critical step for neurons to be integrated into neural networks during development. Changes in the placement of cells within the network due to delays or alterations in neuronal migration, the absence of a full proliferation of dendritic arbors and spine upon which synaptic contacts are made, and the lagging of transmission of electrical impulses due to insufficient myelination will independently and cumulatively impair synaptic function.</p>
<p>The weight of evidence supporting the relationship between structural abnormalities in brain induced and altered synaptic function is moderate. There is no doubt that altered structure can lead to altered function. Many examples from knock out models, genetic mutations, prenatal alcohol, nutritional deficits demonstrate a correlative link between altered structure and impaired synaptic function within the hippocampus (Gil-Mohapel et al., 2010; Berman and Hannigan, 2000; Grant et al., 1992; Palop et al., 2010; Ieraci and Herrera, 2007). However, the scientific understanding of the causative and quantitative relationship between the two KEs is incomplete. </p>
<p>The biological plausibility of alterations in hippocampal structure having an impact on synaptic function and plasticity in brain is strong. Because synaptic transmission in the hippocampus relies on the integrity of contacts and the reliability of electrical and chemical transmission between pre- and post-synaptic neurons, it is well accepted that interference on the anatomical levels will very much impact the functional output on the neurophysiological level (Knowles, 1992; Schultz and Engelhardt, 2014). </p>
<p>Empirical support for this KER is rated as moderate. Numerous examples of a direct linkage between hippocampal anatomy and hippocampal physiology are evident in knock out or transgenic mouse models (eg., Lessman et al., 2011). Other data is derived from nutritional deficiencies, alcohol exposure, and hippocampal slice culture models (Berman and Hannigan, 2000; Ieraci and Herrera, 2007; Gilbert et al., 2016). Although several examples are evident to demonstrate direct linkages between alterations in hippocampal anatomy and disruptions in hippocampal physiology, there is not a mechanism, anatomical insult, or signature pattern of synaptic impairment that accompanies each of these treatments.</p>
<p>Below are a few examples where direct linkages have been reported and they serve to bear witness to a direct relationship between altered hippocampal anatomy and altered hippocampal physiology.</p>
<p>Fyn is a tyrosine kinase gene involved in synaptic plasticity. Mutations of this gene lead to a lack of expression during development and result in an increase in the number of neurons in the dentate gyrus and CA subfields of the hippocampus. Fyn mutant mice also exhibited impairments in long term potentiation in hippocampal CA1 whereas two other forms of short-term plasticity remained intact (Grant et al., 1992).</p>
<p>Neuroregluin-2 (NRG2) is a growth factor and is highly expressed in the hippocampal dentate where it contributes to synaptogenesis of newborn granule cells. In hippocampal slice cultures, inducible microRNA targeting strategies have demonstrated suppression of NRG2 reduced synaptogenesis of inhibitory neurons and impaired dendritic outgrowth and maturation of glutamatergic synapses. These anatomical alterations were accompanied by reductions in the amplitude of excitatory synaptic currents. The magnitude of the impairment was dependent on the timing of the infection and could be eliminated with overexpression of NRG2 in this in vitro model (Lee et al., 2015).</p>
<p>Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal synapogenesis, dendrite formation, and synaptic plasticity in the developing and adult nervous system (Lessmann et al., 2011; Panja and Bramham, 2014). Jacob is a protein that translocates to the nucleus upon activation of BDNF-dependent pathways and is involved in both neuronal plasticity and neurodegeneration. Hippocampal neurons in culture derived from Jacob/Nsmf knockout mice exhibit shorter neurite length, reduced branching, and a few synaptic contacts. This effect was specific to hippocampal neurons, as cortical cells derived from the same animals did not display these abnormalities. In vivo, these animals exhibited a reduction of dendritic complexity of CA1 neurons, lower number of branches, decreased spine density. Deficits in synaptic plasticity in the form of LTP accompanied these structural impairments (Spilker et al., 2016).</p>
<p>In Alzheimer’s Disease, amyloid-b protein accumulates in the hippocampus and leads to the formation of amyloid plaques, neuritic dystrophy and aberrant sprouting of axon terminals of the hippocampus. In a developmental germ-line knockout mouse model, high levels of amyloid-b induced aberrant neuronal network excitability and altered innervation of inhibitory interneurons. Deficits in hippocampal plasticity were seen in the dentate gyrus without change in basal levels of synaptic transmission. In contrast, in area CA1, synaptic transmission was impaired while measures of synaptic plasticity remained intact (Palop et al., 2007).</p>
<p>Other evidence for a direct linkage between hippocampal anatomy and hippocampal physiology comes from the area of adult neurogenesis. The neurogenesis process refers to the acquisition of new neurons on the hippocampus of the adult brain and is associated with enhanced hippocampal synaptic function and learning ability (Deng et al., 2010). Manipulations such as caloric restriction, exercise and hormones can enhance neurogenesis and increase synaptic transmission and plasticity (Kapoor et al., 2015; Trivino-Paredes et al., 2016; Deng et al., 2010). A reciprocal relationship also exists whereby increases in hippocampal neural activity serves to increase neurogenesis (Bruel-Jungerman et al., 2007, Bruel-Jungerman et al., 2009, Kameda et al., 2012). Manipulations that decrease hippocampal neurogenesis including exposure to antidepressants, hormone disruption, stress, and alcohol are associated with impaired synaptic function (Herrera et al., 2003; Saxe et al., 2006; Gilbert et al., 2016; Montero-Pedrazuela et al., 2006; Gil-Mohapel et al., 2006; Sofroniew et al., 2006).</p>
<p style="margin-left:.5in"><em>Temporal Evidence:</em> The temporal nature of this KER is developmental (Seed et al., 2005). This has been demonstrated in multiple studies. A few examples detailed above defined critical periods for the manipulation that alters the structural development of the hippocampus that persists to adulthood to disrupt the synaptic physiology measured in the hippocampus in adulthood (Lee et al., 2015; Grant et al., 1992). A more limited number of ‘rescue’ experiments have been reported. Lee et al (2015), using an in vitro model, demonstrated impaired synaptogenesis that was dependent on the timing of the infection and could be eliminated with overexpression of NRG2. In Spliker et al (2016), BDNF application rescued the morphological deficits in hippocampal pyramidal neurons from Jacob/Nsmf mice.</p>
<p style="margin-left:.5in"><em>Dose-Response Evidence:</em> Dose-response data is lacking for this KER. For future research, it is critical to generate data in which the upstream KE is modulated in a ‘dose-response’ manner to better support the causative relationship.</p>
<p>There are no inconsistencies in this KER, but there are uncertainties. Although several examples are evident to demonstrate direct linkages between alterations in hippocampal anatomy and disruptions in hippocampal physiology, there is not a common cellular mechanism, anatomical insult, or signature pattern of synaptic impairment that defines a common anatomically driven physiological phenotype. In addition, it is also known that there is an interaction between physiological and anatomical development, where anatomy develops first, and can be ‘reshaped’ by the ongoing maturation of physiological function (e.g., Kutsarova et al., 2017)</p>
HighMaleHighFemaleHighDuring brain developmentModerateModerateNot Specified<p>The majority of data in support of this KER is from rodent models. The evolutionary conservation of hippocampal anatomy in mammals, birds, and reptiles (see Hevner, 2016; Streidter, 2015) suggests, with some uncertainty, that this KER is also applicable to multiple species.</p>
<p>Andersen, P., Morris,R., Amaral,D., Bliss,T., O'Keefe, J. (Editors). The Hippocampus Book. Oxford University Press, 2006. ISBN: 9780195100273</p>
<p>Berman RF, Hannigan JH. Effects of prenatal alcohol exposure on the hippocampus: spatial behavior, electrophysiology, and neuroanatomy. Hippocampus. 2000;10(1):94-110.</p>
<p>Bruel-Jungerman E, Davis S, Laroche S (2007) Brain plasticity mechanisms and memory: a party of four. Neuroscientist 13:492-505.</p>
<p>Bruel-Jungerman E, Veyrac A, Dufour F, Horwood J, Laroche S, Davis S (2009) Inhibition of PI3K-Akt signaling blocks exercise-mediated enhancement of adult neurogenesis and synaptic plasticity in the dentate gyrus. PLoS One 4:e7901.</p>
<p>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.</p>
<p>Gil-Mohapel J, Boehme F, Kainer L, Christie BR. Hippocampal cell loss and neurogenesis after fetal alcohol exposure: insights from different rodent models.Brain Res Rev. 2010 Sep 24;64(2):283-303.</p>
<p>Gilbert ME, Goodman JH, Gomez J, Johnstone AF, Ramos RL. Adult hippocampal neurogenesis is impaired by transient and moderate developmental thyroid hormone disruption. Neurotoxicology. 2016 Dec 31;59:9-21.</p>
<p>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.</p>
<p>Harris KM, Teyler TJ. Developmental onset of long-term potentiation in area CA1 of the rat hippocampus. J Physiol. 1984 Jan;346:27-48.</p>
<p>Herrera DG, Yague AG, Johnsen-Soriano S, Bosch-Morell F, Collado-Morente L, Muriach M, Romero FJ, Garcia-Verdugo JM (2003) Selective impairment of hippocampal neurogenesis by chronic alcoholism: protective effects of an antioxidant. Proc Natl Acad Sci U S A 100:7919-7924.</p>
<p>Hevner RF. Evolution of the mammalian dentate gyrus. J Comp Neurol. 2016 524(3):578-94.</p>
<p>Ieraci A, Herrera DG. Single alcohol exposure in early life damages hippocampal stem/progenitor cells and reduces adult neurogenesis. Neurobiol Dis. 2007 Jun;26(3):597-605.</p>
<p>Kameda M, Taylor CJ, Walker TL, Black DM, Abraham WC, Bartlett PF (2012) Activation of latent precursors in the hippocampus is dependent on long-term potentiation. Transl Psychiatry 2:e72.</p>
<p>Kapoor R, Fanibunda SE, Desouza LA, Guha SK, Vaidya VA (2015) Perspectives on thyroid hormone action in adult neurogenesis. J Neurochem 133:599-616.</p>
<p>Knowles WD, Normal anatomy and neurophysiology of the hippocampal formation. J Clin Neurophysiol. 1992 Apr;9(2):252-63.</p>
<p>Kutsarova E, Munz M, Ruthazer ES. Rules for Shaping Neural Connections in the Developing Brain. Front Neural Circuits. 2017. 10:111. doi: 10.3389/fncir.2016.00111.</p>
<p>Lee KH, Lee H, Yang CH, Ko JS, Park CH, Woo RS, Kim JY, Sun W, Kim JH, Ho WK, Lee SH. Bidirectional Signaling of Neuregulin-2 Mediates Formation of GABAergicSynapses and Maturation of Glutamatergic Synapses in Newborn Granule Cells ofPostnatal Hippocampus. J Neurosci. 2015 Dec 16;35(50):16479-93.</p>
<p>Lessmann V, Stroh-Kaffei S, Steinbrecher V, Edelmann E, Brigadski T, Kilb W, Luhmann HJ. The expression mechanism of the residual LTP in the CA1 region ofBDNF k.o. mice is insensitive to NO synthase inhibition. Brain Res. 2011. 1391:14-23.</p>
<p>Montero-Pedrazuela A, Venero C, Lavado-Autric R, Fernandez-Lamo I, Garcia-Verdugo JM, Bernal J, Guadano-Ferraz A (2006) Modulation of adult hippocampal neurogenesis by thyroid hormones: implications in depressive-like behavior. Mol Psychiatry 11:361-371.</p>
<p>Palop JJ, Chin J, Roberson ED, Wang J, Thwin MT, Bien-Ly N, Yoo J, Ho KO, Yu GQ, Kreitzer A, Finkbeiner S, Noebels JL, Mucke L. Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease. Neuron. 2007 Sep 6;55(5):697-711.</p>
<p>Panja, D. and C. R. Bramham (2014). "BDNF mechanisms in late LTP formation: A synthesis and breakdown." Neuropharmacology 76 Pt C: 664-676.Schultz C, Engelhardt M. Anatomy of the hippocampal formation. Front Neurol Neurosci. 2014. 34:6-17</p>
<p>Saxe MD, Battaglia F, Wang JW, Malleret G, David DJ, Monckton JE, Garcia AD,</p>
<p>Sofroniew MV, Kandel ER, Santarelli L, Hen R, Drew MR. Ablation of hippocampal neurogenesis impairs contextual fear conditioning and synaptic plasticity in the dentate gyrus. Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17501-6.</p>
<p>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.</p>
<p>Schultz C, Engelhardt M. Anatomy of the hippocampal formation. Front Neurol Neurosci. 2014. 4:6-17.</p>
<p>Sofroniew et al., 2006</p>
<p>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, MikhaylovaM, 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 BDNFSignaling in Dendritogenesis. PLoS Genet. 2016 Mar 15;12(3):e1005907</p>
<p>Striedter GF. Evolution of the hippocampus in reptiles and birds. J Comp Neurol. 2016 Feb 15;524(3):496-517</p>
<p>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.</p>
2016-11-29T18:41:352018-08-11T19:21:121ba1bd15-817e-4295-9a7e-38d0f409f68f89d276df-a8ad-4b27-a848-5eae65321d71Not Specified2021-09-14T12:35:062021-09-14T12:35:06Upregulation of Thyroid Hormone Catabolism via Activation of Hepatic Nuclear Receptors, and Subsequent Adverse Neurodevelopmental Outcomes in MammalsNuclear receptor induced TH Catabolism and Developmental Hearing Loss<p>Katie Paul Friedman, National Center for Computational Toxicology, US EPA, RTP, NC USA <paul-frieidman@epa.gov></p>
<p>Mary E. Gilbert, National Health and Environmental Effects Research Laboratory, US EPA, RTP, NC USA <gilbert.mary@epa.gov></p>
<p>Kevin M. Crofton, National Center for Computational Toxicology, US EPA, RTP, NC USA <crofton.kevin@epa.gov></p>
Open for adoptionUnder DevelopmentIncluded in OECD Work Plan1.9<p>Data from rodent studies demonstrate that thyroid hormone disruption during cochlear development culminates in ototoxicity. Developmental exposure of rats to polychlorinated biphenyls (PCBs) results in a low-frequency hearing loss in adult offspring (Goldey et al., 1995a; Herr et al., 1996; 2001; Crofton and Rice, 1999; Laskey et al., 2002). A body of work now supports the hypothesis that this ototoxicity results from PCB-induced hypothyroxinemia during a critical period of auditory development. Evidence for this hypothesis includes: a correlation between the severity of functional auditory impairment and the degree of thyroid hormone depletion (Goldey et al., 1995a; 1995b; Goldey and Crofton, 1998; Crofton, 2004), a cross-fostering study demonstrating that the critical exposure period is postnatal (Crofton et al., 2000a), and amelioration of the hearing loss following postnatal thyroxine replacement (Goldey and Crofton, 1998). Below an adverse outcome pathway is described for chemicals that activate xenobiotic nuclear receptors, including AhR, CAR, and PXR, leading to thyroid hormone disruption during cochlear development and resulting in permanent auditory loss.</p>
<p>This AOP is a revision and update of the original started on the Chemical Mode of Action wiki sponsored by WHO/IPCS. This MOA was described and published by Crofton and Zoeller 2005).</p>
<p>This AOP is an update of the WHO/IPCS MOA developed in 2005 by Crofton and Zoeller (Crit Rev Toxicol 2005).</p>
<p><em>Crofton KM, Zoeller RT. Mode of action: neurotoxicity induced by thyroid hormone disruption during development--hearing loss resulting from exposure to PHAHs. Crit Rev Toxicol. 2005 Oct-Nov;35(8-9):757-69. PMID: 6417043</em></p>
adjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedModerateFetal to ParturitionModerateNursing ChildNot Specified<p><a href="#Relationships_Among_Key_Events_and_the_Adverse_Outcome">Summary Table</a><br />
<em>Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages. </em></p>
<p><strong>Concordance of dose-response relationships</strong></p>
<p>Multiple studies provide limited (2-3 doses) dose-response data for many of the key events. These studies demonstrate similar magnitudes of effect on circulating hormones for doses of PCBs that are within an order of magnitude (3-25 mg/kg/day for Aroclor 1254) (e.g.,Morse et al., 1996; Goldey et al., 1998). Very limited data are available correlating any of the key events. One exception is the relationship between circulating serum T4 concentrations during development and the magnitude of hearing loss (Crofton, 2004). There is a very good correlation between total serum T4 concentrations on postnatal day (PND) 14 and hearing loss assessed in adult offspring of PCB exposed dams (Figure 2). All of these events occur within a 2-3 fold dose range.</p>
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Temporal concordance among the key events and the adverse outcome Strength, consistency, and specificity of association of adverse effect and initiating event Biological plausibility, coherence, and consistency of the experimental evidence Alternative mechanism(s) or MIE(s) that logically present themselves and the extent to which they may detract from the AOP Uncertainties, inconsistencies, and data gaps</p>
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