Inhibition, Na+/I- symporter (NIS)

14797-73-0

VLTRZXGMWDSKGL-UHFFFAOYSA-M

Perchlorate

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

DTXSID6024252

14797-55-8

NHNBFGGVMKEFGY-UHFFFAOYSA-N

Nitrate

Nitrate (NO3-) Nitrate ion Nitrate ion (NO3-) Nitrate ion(1-) Nitrate(1-) Nitrates/nitrites Nitrato Nitric acid, ion(1-)

DTXSID5024217

302-04-5

ZMZDMBWJUHKJPS-UHFFFAOYSA-M

Thiocyanate

Thiocyanates Isothiocyanic acid, ion(1-) Rhodanide Thiocyanate (NCS1-) Thiocyanate anion Thiocyanate ion Thiocyanic acid, ion(1-) Thiocyanide

DTXSID8047763

51-52-5

KNAHARQHSZJURB-UHFFFAOYSA-N

6-Propyl-2-thiouracil

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

DTXSID5021209

60-56-0

PMRYVIKBURPHAH-UHFFFAOYSA-N

Methimazole

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

DTXSID4020820

PR:000015171

PR sodium/iodide cotransporter

CHEBI:60311

CHEBI thyroid hormone

CHEBI:16382

CHEBI iodide

CHEBI:30660

CHEBI thyroxine

UBERON:0002421

UBERON hippocampal formation

GO:0045202

GO synapse

GO:0008507

GO sodium:iodide symporter activity

GO:0006590

GO thyroid hormone generation

GO:0015705

GO iodide transport

MP:0005475

MP abnormal circulating thyroxine level

GO:0010817

GO regulation of hormone levels

GO:0010468

GO regulation of gene expression

GO:0007420

GO brain development

GO:0007268

GO chemical synaptic transmission

GO:0007611

GO learning or memory

GO:0050890

GO cognition

WIKI decreased

WIKI abnormal

WIKI morphological change

Perchlorate

2016-11-29T18:42:26

Nitrate

2016-11-29T18:42:26

Thiocyanate

2016-11-29T18:42:26

Dysidenin

2016-11-29T18:42:26

Aryltrifluoroborates

2016-11-29T18:42:26

Econazole

2016-12-07T11:06:26

5-(N,N-hexamethylene) amiloride (HMA)

2016-12-07T11:06:59

Small molecules: ITB3, ITB4, ITB5, ITB9

2016-12-07T11:16:09

Propylthiouracil

2016-11-29T18:42:22

Methimazole

2016-11-29T18:42:19

Iodine deficiency

2017-03-26T11:37:44

WCS_9606

common toxicological species human

10116

NCBI rat

10090

NCBI mouse

WikiUser_24

Wikiuser:Migration Pig

7955

NCBI zebra fish

224340

NCBI Xenopus (Silurana) epitropicalis

WCS_8355

common ecological species African clawed frog

WCS_8355

common ecological species Xenopus laevis

WCS_7955

common ecological species zebrafish

WCS_90988

common ecological species fathead minnow

9823

NCBI Sus scrofa

451443

NCBI Xenopus (Silurana) n. sp. tetraploid-1

WCS_9031

common ecological species chicken

10116

NCBI rats

9606

NCBI Homo sapiens

443947

NCBI Xenopus laevis laevis

WCS_9606

common toxicological species humans

Inhibition, Na+/I- symporter (NIS)

Molecular

<h4>Evidence for Perturbation by Stressor</h4> <h5>Overview for Molecular Initiating Event</h5> Thyroid Disrupting Chemicals (TDCs) are defined as the xenobiotics that interfere with the thyroid axis with different outcomes for the organism. A very well-studied mechanism of action of the TDCs is the reduction of the circulating levels of THs by inhibiting hormone synthesis in the thyroid gland. For example, perchlorate is a very potent inhibitor of iodide uptake through the sodium/iodide symporter (Tonacchera et al., 2004). Perchlorate has been detected in human breast milk ranging from 1.4 to 92.2 mg μl–1 (10.5 μg l–1 mean) in 18 US states (Kirk et al. 2005), and 1.3 to 411 μg l–1 (9.1 μg l–1 median) in the Boston area, United States (Pearce et al. 2007). Perchlorate has also been detected in human colostrum of 46 women in the Boston area (from < 0.05 to 187.2 μmol l–1 (Leung et al. 2009)). The mechanism of perchlorate action is quite simple, as it is believed to be mediated only by the NIS inhibition (Dohan et al., 2007; Wolff, 1998). Additionally, thiocyanate and nitrate are two known inhibitors that have been found to reduce circulating TH levels (Blount et al., 2006; Steinhaus et al., 2007), but they are both less potent than perchlorate (Tonacchera et al., 2004). However, there are also contradictory results from other studies that showed no correlation between thyroid parameters and perchlorate levels in humans (Pearce et al., 2010; Amitai et al., 2007; Tellez et al., 2005). Co-occurrence of perchlorate, nitrate, and thiocyanate can alter thyroid function in pregnant women. Horton et al. (2015) have shown positive associations between the weighted sum of urinary concentrations of these three analytes and increased TSH, with perchlorate showing the largest weight in the index. Interestingly, De Groef et al. 2006 showed that nitrate and thiocyanate, acquired through drinking water or food, account for a much larger proportion of iodine uptake inhibition than perchlorate, suggesting that NIS inhibition and any potential downstream effect by perchlorate are highly dependent on the presence of other environmental NIS inhibitors and iodine intake itself (Leung et al., 2010). In particular, Tonacchera et al. (2004) showed that the relative potency of perchlorate to inhibit radioactive I− uptake by NIS is 15, 30 and 240 times that of thiocyanate, iodide, and nitrate respectively on a molar concentration basis. These data are in line with earlier studies in rats (Alexander and Wolff, 1996; Greer et al. 1966). Contradictory findings in these studies may therefore be a result of the confounding mixtures in the environment, masking the primary effect of perchlorate. Decreased iodine intake can decrease TH production, and therefore exposure to perchlorate might be particularly detrimental in iodine-deficient individuals (Leung et al. 2010). Moreover, biologically based dose-response modeling of the relationships among iodide status (e.g., dietary iodine levels), perchlorate dose, and TH production in pregnant women has shown that iodide intake has a profound effect on the likelihood that exposure to goitrogens will produce hypothyroxinemia (Lewandowski et al. 2015). During pregnancy TH requirements increase, particularly during the first trimester (Alexander et al. 2004; Leung et al. 2010), due to higher concentrations of thyroxine-binding globulin, placental T4 inner-ring deiodination leading to the inactive reverse T3 (rT3), and transfer of small amounts of T4 to the foetus (during the first trimester foetal thyroid function is absent). Moreover, glomerular filtration rate and clearance of proteins and other molecules are both increased during pregnancy, possibly causing increased renal iodide clearance and a decreased of circulating plasma iodine (Glinoer, 1997). Thus, even though the foetal thyroid can trap iodide by about 12 week of gestation (Fisher and Klein, 1981), high concentrations of maternal perchlorate may potentially decrease thyroidal iodine available to the foetus by inhibiting placental NIS (Leung et al. 2010). Consequences of TH deficiency depend on the developmental timing of the deficiency (Zoeller and Rovet, 2004). For instance, if the TH deficiency occurs during early pregnancy, offspring show visual attention, visual processing and gross motor skills deficits, while if it occurs later, offspring may show subnormal visual and visuospatial skills, along with slower response speeds and motor deficits. If TH insufficiency occurs after birth, language and memory skills are most predominantly affected (Zoeller and Rovet, 2004). Along this line, age and developmental stage are crucial in determining sensitivity to NIS inhibitors (e.g., perchlorate, thiocyanate, and nitrate). In this regard, McMullen et al. (2017) have shown that adolescent boys and girls, more than adults, represent vulnerable subpopulations to NIS symporter inhibitors. Altogether these studies indicate that age, gender, developmental stage, and dietary iodine levels can affect the impact of NIS inhibitors. Finally, ten more small simple-structured molecules were identified in a large screening study (Lecat-Guillet et al., 2008b) that could block iodide uptake by specifically disrupting NIS in a dose-dependent manner. These molecules were named Iodide Transport Blockers (ITBs). There are few organic molecules that lead to NIS inhibition but no direct interaction with NIS has been determined (Gerard et al., 1994; Kaminsky et al., 1991, Lindenthal et al., 2009). Up to date, only dysidenin, a toxin isolated from the marine sponge Dysidea herbacea, has been reported to specifically inhibit NIS (Van Sande et al., 2003). Finally, the aryltrifluoroborates were found to inhibit iodide uptake with an IC50 value of 0.4 μM on rat-derived thyroid cells (Lecat-Guillet et al., 2008a). The biological activity is rationalized by the presence of the BF3− ion as a minimal binding motif for substrate recognition at the iodide binding site. It has been also shown that many anions, such as ClO3-, SCN-, NO3-, ReO4-, TcO4- and in a lower extent Br- and BF4-, are also acting as NIS substrates and they enter the cell by the same transporter mechanism (Van Sande et al., 2003). It has been also shown that ClO4- is transferred by NIS with high affinity and is considered as one of its most potent inhibitors (Dohan et al., 2007). Most recently, the aryltrifluoroborates were also shown to inhibit NIS function (Lecat-Guillet et al., 2008a). A library of 17,020 compounds was tested by a radioactive screening method with high specificity using transfected mammalian cells (Lecat-Guillet et al., 2008b; 2007) for NIS inhibition evaluation. Further studies with the most powerful iodide transport blockers showed a high diversity in their structure and mode of action (Lindenthal et al., 2009). Apart from the human, functional NIS protein has been also identified in different species, including the rat (Dai et al., 1996), the mouse (Perron et al., 2001), the pig (Selmi-Ruby et al., 2003), zebrafish (Thienpont et al., 2011) and xenopus (amphibian) (Lindenthal et al., 2009). Mouse and rat NIS proteins contain 618 amino acid residues, while the human and pig variants contain 643. There are several NIS variants that produce three active proteins in the pig due to alternative splicing at mRNA sites that are not present on the other species (Selmi-Ruby et al., 2003). NIS orthologs are discussed in the review by Darrouzet's group (Darrouzet et al., 2014). Interestingly, functional differences have been identified between mouse or rat NIS (mNIS or rNIS, respectively) and human NIS (hNIS). The rat and themouse orthologs were shown to accumulate radioisotopes more efficiently than the human protein (Dayem et al., 2008; Heltemes et al., 2003). The molecular basis of these functional differences could be helpful for further characterization of NIS. Zhang and collaborators showed that rNIS is localized in a higher proportion at the plasma membrane than hNIS and the N-terminal region up to putative transmembrane helix TM7 appears to be involved in this difference (Zhang et al., 2005). These authors also reported differences in the kinetics of the Na+ binding, implicating the region spanning from TM4 to TM6 and Ser200 of hNIS. They, thus, proposed that this region could be involved in sodium binding (Zhang et al., 2005). In our laboratory, it was shown that the Vmax of the mouse protein is four times higher than the Vmax of the human protein when expressed in the same cell line (HEK-293) (Dayem et al., 2008; Darrouzet et al., 2014). The KmI value determined for hNIS (9.0 ± 0.8 μM) was significantly lower than the KmI for the mouse protein (26.4 ± 3.5 μM) whereas the KmNa values were not significantly different indicating that mNIS has a lower iodide affinity than hNIS. Similarly to the rat protein, mNIS is predominantly localized in the plasma membrane whereas the human ortholog is detected intracellularly in 40% of the cells in which it is expressed (Darrouzet et al., 2014). However, the difference in the Vmax values does not only seem to be related to the higher intracellular localization of hNIS. Using chimeric proteins between human and mouse NIS, we showed that the N-terminal region up to TM8 is most probably involved in iodide binding, and that the region from TM5 to the C terminus could play an important role in targeting the protein to the plasma membrane (Dayem et al., 2008). One of the long-term goals of these studies is the engineering of a chimeric NIS protein most suitable for gene therapy, i.e. preserving regions responsible for the high turnover rate and the efficient plasma membrane localization of the mouse proteinwhile replacing the immunogenic extracellular regions with those of the human ortholog. The porcine NIS gene gives rise to splice variants leading to three active NIS proteins with differences in their C-terminal extremities [4]. However, it is not known if these differences lead to distinct properties (Darrouzet et al., 2014). There is evidence that the MIE (NIS inhibition) is of relevance also for fish as an expression of the slc5a5 transcript (sodium/iodide co-transporter) has been described by various publications for the zebrafish embryo (see <a href="http://www.zfin.org/">www.zfin.org</a>). It has been demonstrated that NIS inhibitors in zebra fish lead also to a strong repression of thyroid hormone levels (Thienpont et al., 2011) and in xenopus (amphibian) to  inhibition of the iodide-induced current  (Lindenthal et al., 2009). Biological state: Sodium/Iodide symporter (NIS) is a key protein in the thyroid function and its role has been thoroughly investigated after the determination of its molecular identity a few decades ago (Dai et al., 1996). NIS is an intrinsic membrane glycoprotein and it belongs to the superfamily of sodium /solute symporters (SSS) and to the family of human transporters SLC5 (De La Vieja, 2000; Jung, 2002). Its molecular weight is 87 kDa and it contains 13 transmembrane domains that transport 2 sodium cations (Na+) for each iodide anion (I-) into the follicular thyroid cell (Dohan et al., 2003). The regulation of NIS protein function is usually cell- and tissue-specific (Hingorani et al., 2010) and it is done at the transcriptional and posttranslational levels, including epigenetic regulation (Darrouzet et al., 2014; Russo et al., 2011a). One of the major NIS regulators is the thyroid stimulating hormone (TSH), which has been shown to enhance NIS mRNA and protein expression, therefore it can contribute to restore and maintain iodide uptake activity (Saito et al., 1997; Kogai et al., 2000). At the posttranslational level TSH also contributes to NIS regulation but the specific mechanisms that underlie these effects are still under investigation (Riedel et al., 2001). Biological compartments: NIS protein is mainly found at the basolateral plasma membrane of the thyroid follicular cells (Dai et al., 1996), where it actively mediates the accumulation of iodide that is the main component of thyroid hormone synthesis and therefore is considered as a major regulator of thyroid homeostasis. NIS also mediates active I- transport in extrathyroidal tissues but it is commonly agreed that is regulated and processed differently in each tissue. Functional NIS protein has been found in salivary gland ductal cells (Jhiang et al., 1998; La Perle et al., 2013), in the mammary gland during lactation (Perron et al., 2001; Cho et al., 2000), lung epithelial cells (Fragoso et la., 2004), intestinal enterocytes (Nicola et al., 2009), stomach cells (Kotani et al., 1998), placenta (Bidart et al., 2000) and testicular cells (Russo et al. 2011b). Additionally, contradictory results have been obtained regarding the NIS expression in human kidney tissue (Lacroix et al., 2001; Spitzweg et al., 2001). In the case of the lactating breast, it is suggested that NIS serves the transfer of iodide in the cells and its subsequent accumulation in the milk, thereby supplying newborns with this component during this sensitive developmental period (Tazebay et al., 2000). Additionally, NIS mRNA has been detected in various other tissues, such as colon, ovaries, uterus, and spleen (Perron et al., 2001; Spitzweg et al., 1998; Vayre et al., 1999), but the functional NIS protein and the site of its localization has not been verified. General role in biology: The NIS is known in the field of thyroidology because of its ability to mediate the active transport of I- into the thyrocytes, which is the first and most crucial step for T3 and T4 biosynthesis (Dohan et al., 2000). NIS is located on the basolateral membrane of the thyrocytes and co-transports 2 sodium ions along with 1 iodide (2:1 stoichiometry). The electrochemical gradient of sodium serves as the driving force for iodide uptake and it is generated and maintained by the Na+/K+ ATPase pump, which is located in the same membrane of the thyrocytes. The iodide molecules, after their active transport in the cytoplasm, are passively translocated in the follicular lumen via the transporter protein pendrin and possibly other unknown efflux proteins that are located on the apical membrane (Bizhanova and Kopp, 2009). Subsequently, the thyroid hormones are synthesized in the follicular lumen by incorporating the accumulated iodide, a process which is significantly suppressed in case of NIS dysfunction or inhibition (reviewed in Spitzweg and Morris, 2010). NIS is the last thyroid-related component to be expressed during development at the 10th gestational week, which temporaly coincides with the onset of thyroid function and hormonogenesis (Szinnai et al., 2007). Albeit the localization of NIS is not fully completed at this stage, the iodide accumulation has already started. Mutations of NIS gene (SLCA5A) cause expression of non-functional NIS molecule leading to inability of the thyrocyte to accumulate iodide (Matsuda and Koshugi, 1997; Pohlenz et al., 1998), a condition called iodide transport defect (ITD). This is a rear autosomic recessive disease, which if not properly treated is clinically identified by congenital hypothyroidism, goiter, low I- uptake, low saliva/plasma I- ratio and mental impairment of varying degrees (Dohan et al., 2003). Up to date 13 mutations have been described in the NIS gene (Spitzweg and Morris, 2010) and each one of them produces mutants with different structure but in all cases non-functional. The extensive study after NIS molecular characterization and the numerous findings have convinced the scientists that is one of the most crucial components of the entire thyroid system. Additionally, after the realization that NIS could be also used as diagnostic and therapeutic tool for thyroid and non-thyroid cancers (Portulano et al., 2013) a new research activity concerning this specific mechanism has been initiated.

There are several methods that are used nowadays to detect the functionality of NIS but none of these methods is OECD validated (OECD Scoping document, 2017). The most well established methods are the following: 1. Measurement of radioiodide uptake (125I-) in NIS expressing cells. For this method the FRTL5 cell line is the most commonly used, as it endogenously express the NIS protein, but also NIS transfected cell lines have been successfully implemented in many cases (Lecat-Guillet et al., 2007; 2008b; Lindenthal et al., 2009). Once inhibitory activity is identified for a compound then further tests are performed in order to verify that the observed effect is specific due to NIS inhibition. This method has been also adapted in a high throughput format and has been already used for the screening of a chemical library of 17.020 compounds (Lecat-Guillet et al., 2008b). 2. More recently a non-radioactive method has been developed, which has been also adapted in a high throughput format (Waltz et al., 2010). It is a simple spectrophotometric assay for the determination of iodide uptake  using  rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell-Kolthoff reaction). The assay is fast, highly reproducible and equally sensitive with the radioiodine detection method. 3. A fluorescence-based method has been developed, which uses the variant  YFP-H148Q/I152L of the Yellow Fluorescent Protein (YFP) in order to detect the efflux of iodide into the rat FRTL5 cells. As a positive control perchlorate is used  as it is a well known  competitive inhibitor of iodide transport by NIS. Fluorescence of recombinant YFP-H148Q/I152L is  suppressed by perchlorate and iodide with similar affinities. Fluorescence changes in FRTL-5 cells are  Na+-dependent, consistent with the Na+-dependence of NIS activity.   It is supposed to be an innovative approach to detect the cellular uptake of perchlorate and characterize the kinetics of transport by NIS. This method needs further optimization, as YFP is not specific for iodide and thus binding of other ionic molecules could affect the results of the assay (Cianchetta et al., 2010; Rhoden et al., 2008; Di Bernarde et al., 2011). 4. In vivo 125I uptake assays is based on  immunofluorescence analyses of thyroid glands after the treatment of rat with excess I−,  injected with Ci Na125I  as previously described by Ferreira et al., 2005. Then the thyroid glands are  removed and weighed, and the amount of 125I in the thyroid gland is  measured in a γ-counter (PerkinElmer; model Wizard). The counts per minute in the thyroid gland are used to calculate the percentage of 125I in the thyroid gland, having in account that 100% corresponded to the counts per minute injected I− into the rat (Arriagada et al., 2015). 5. The U.S. EPA's Endocrine Disruptor Screening Program aims to use high-throughput assays and computational toxicology models to screen and prioritize chemicals that may disrupt the thyroid signaling pathway. Thyroid hormone biosynthesis requires active iodide uptake mediated by the sodium/iodide symporter (NIS). Monovalent anions, such as the environmental contaminant perchlorate, are competitive inhibitors of NIS, yet limited information exists for more structurally diverse chemicals. A novel cell line expressing human NIS, hNIS-HEK293TEPA, was used in a radioactive iodide uptake (RAIU) assay to identify inhibitors of NIS-mediated iodide uptake. The RAIU assay was optimized and performance evaluated with 12 reference chemicals comprising known NIS inhibitors and inactive compounds. An additional 39 chemicals including environmental contaminants were evaluated, with 28 inhibiting RAIU over 20% of that observed for solvent controls. Cell viability assays were performed to assess any confounding effects of cytotoxicity. RAIU and cytotoxic responses were used to calculate selectivity scores to group chemicals based on their potential to affect NIS. RAIU IC50 values were also determined for chemicals that displayed concentration-dependent inhibition of RAIU (≥50%) without cytotoxicity. Strong assay performance and highly reproducible results support the utilization of this approach to screen large chemical libraries for inhibitors of NIS-mediated iodide uptake (Hallinger et al., 2017). 6. This study (Wang et al., 2018) applied a previously validated high-throughput approach to screen for NIS inhibitors in the ToxCast phase I library, representing 293 important environmental chemicals. Here 310 blinded samples were screened in a tiered-approach using an initial single-concentration (100 μM) radioactive-iodide uptake (RAIU) assay, followed by 169 samples further evaluated in multi-concentration (0.001 μM−100 μM) testing in parallel RAIU and cell viability assays. A novel chemical ranking system that incorporates multi-concentration RAIU and cytotoxicity responses was also developed as a standardized method for chemical prioritization in current and future screenings. Representative chemical responses and thyroid effects of high-ranking chemicals are further discussed. This study significantly expands current knowledge of NIS inhibition potential in environmental chemicals and provides critical support to U.S. EPA’s Endocrine Disruptor Screening Program (EDSP) initiative to expand coverage of thyroid molecular targets, as well as the development of thyroid adverse outcome pathways (AOPs).

Apart from the human, functional NIS protein has been also identified in different species, including  the rat (Dai et al., 1996), the mouse (Perron et al., 2001), the pig (Selmi-Ruby et al., 2003), zebrafish (Thienpont et al., 2011) and in xenopus (amphibian)  (Lindenthal et al., 2009). Mouse and rat contain 618 amino acid residues, while the human and pig contain 643. There are several NIS variants that produce three active proteins in the pig due to alternative splicing at mRNA sites that are not present on the other species (Selmi-Ruby et al., 2003). NIS orthologs are discussed in the review by Darrouzet's group ( Darrouzet et al., 2014). Interestingly, functional differences have been identified between mouse or rat NIS (mNIS or rNIS, respectively) and human NIS (hNIS). The rat and themouse orthologs were shown to accumulate radioisotopes more efficiently than the human protein (Dayem et al., 2008; Heltemes et al., 2003). The molecular basis of these functional differences could be helpful for further characterization of NIS. Zhang and collaborators showed that rNIS is localized in a higher proportion at the plasma membrane than hNIS and the N-terminal region up to putative TM7 appears to be involved in this difference (Zhang et al., 2005). These authors also reported differences in the kinetics of the Na+ binding, implicating the region spanning from TM4 to TM6 and Ser200 of hNIS. They, thus, proposed that this region could be involved in sodium binding (Zhang et al., 2005). In our laboratory, it was shown that the Vmax of the mouse protein is four times higher than the Vmax of the human protein when expressed in the same cell line (HEK-293) (Dayem et al., 2008; Darrouzet et al., 2014). The KmI value determined for hNIS (9.0 ± 0.8 μM) was significantly lower than the KmI for the mouse protein (26.4 ± 3.5 μM) whereas the KmNa values were not significantly different. Similarly to the rat protein, mNIS is predominantly localized in the plasma membrane whereas the human ortholog is detected intracellularly in 40% of the cells in which it is expressed (Darrouzet et al., 2014). However, the difference in the Vmax values does not only seem to be related to the higher intracellular localization of hNIS. Using chimeric proteins between human and mouse NIS, we showed that the N-terminal region up to TM8 is most probably involved in iodide binding, and that the region from TM5 to the C terminus could play an important role in targeting the protein to the plasma membrane (Dayem et al., 2008). One of the long-term goals of these studies is the engineering of a chimeric NIS protein most suitable for gene therapy, i.e. preserving regions responsible for the high turnover rate and the efficient plasma membrane localization of the mouse proteinwhile replacing the immunogenic extracellular regions with those of the human ortholog. The porcine NIS gene gives rise to splice variants leading to three active NIS proteins with differences in their C-terminal extremities [4]. However, it is not known if these differences lead to distinct properties (Darrouzet et al., 2014). There is evidence that the MIE (NIS inhibition) is of relevance also for fish as an expression of the slc5a5 transcript (sodium/iodide co-transporter) has been described by various publications for the zebrafish embryo (see <a href="http://www.zfin.org">www.zfin.org</a>). It has been demonstrated that NIS inhibitors in zebra fish lead also to a strong repression of thyroid hormone levels (Thienpont et al., 2011) and in xenopus (amphibian) to  inhibition of the iodide-induced current  (Lindenthal et al., 2009).

CL:0002258

CL thyroid follicular cell

High Mixed

High

Pregnancy

High

Birth to < 1 month

High

During brain development

High High High High High Moderate Not Specified

Alexander EK, Marqusee E, Lawrence J, Jarolim P, Fischer GA, Larsen PR (2004). Timing and magnitude of increases in levothyroxine requirements during pregnancy in women with hypothyroidism. N Engl J Med. Jul 15; 351(3):241-9. Alexander WD and Wolff J (1996). Thyroidal iodide transport VIII, Relation between transport goitrogenic and antigoitrogenic properties of certain anions. Endocrinology 78 581–590. Amitai Y, Winston G, Sack J, Wasser J, Lewis M, Blount BC, Valentin-Blasini L, Fisher N, Israeli A, Leventhal A. (2007). Gestational exposure to high perchlorate concentrations in drinking water and neonatal thyroxine levels. Thyroid. 17:843-850. Arriagada A.A, Eduardo Albornoz, Ma. Cecilia Opazo, Alvaro Becerra, Gonzalo Vidal, Carlos Fardella, Luis Michea, Nancy Carrasco, Felipe Simon, Alvaro A. Elorza, Susan M. Bueno, Alexis M. Kalergis, and Claudia A.  (2015).Excess Iodide Induces an Acute Inhibition of the Sodium/Iodide Symporter in Thyroid Male Rat Cells by Increasing Reactive Oxygen Species. Endocrinology. 2015 Apr; 156(4): 1540–1551. Bidart JM, Lacroix L, Evain-Brion D, Caillou B, Lazar V, Frydman R, Bellet D, Filetti S, Schlumberger M. (2000). Expression of Na+/I− symporter and Pendred syndrome genes in trophoblast cells. J Clin Endocrinol Metab 85:4367–4372. Bizhanova A, Kopp P. (2009). The sodium-iodide symporter NIS and pendrin in iodide homeostasis of the thyroid. Endocrinol 150:1084-1090. Blount BC, Pirkle JL, Osterloh JD, Valentin-Blasini L, Caldwell KL. (2006). Urinary perchlorate and thyroid hormone levels in adolescent and adult men and women living in the United States. Env Health Persp. 114:1865-1871. Cho JY, Leveille R, Kao R, Rousset B, Parlow AF, Burak WE Jr, Mazzaferri EL, Jhiang SM. (2000). Hormonal regulation of radioiodide uptake activity and Na+/I− symporter expression in mammary glands. J Clin Endocrinol Metab 85:2936–2943. Cianchetta S, di Bernardo J, Romeo G, Rhoden KJ. (2010). Perchlorate transport and ihnibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L. Toxicol App Pharmacol. 243:372-380. Dai G, Levy O, Carrasco N. (1996). Cloning and characterization of the thyroid iodide transporter. Nature 379:458–460. Darrouzet E, Lindenthal S, Marcellin D, Pellequer J, Pourcher T. (2014). The sodium/iodide symporter: state of the art of its molecular characterization. Biochim Biophys Acta 1838:244-253. Dayem M, Basquin C, Navarro V, Carrier P, Marsault R, Chang P, Huc S, Darrouzet E, Lindenthal S, Pourcher T. (2008). Comparison of expressed human and mouse sodium/iodide symporters reveals differences in transport properties and subcellular localization. J Endocrinol. 197:95–109. 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Characterization of small-molecule inhibitors of the sodium iodide symporter. J Endocrinol 200:357–365. Matsuda A, Kosugi S. (1997). A homozygous missense mutation of the sodium/iodide symporter gene causing iodide transport defect. J Clin Endocrinol Metab 82:3966–3971. McMullen J, Ghassabian A, Kohn B, Trasande L (2017). Identifying Subpopulations Vulnerable to the Thyroid-Blocking Effects of Perchlorate and Thiocyanate. J Clin Endocrinol Metab. Jul 1;102(7):2637-2645. Nicola JP, Basquin C, Portulano C, Reyna-Neyra A, Paroder M, Carrasco N. (2009). The Na+/I− symporter mediates active iodide uptake in the intestine. Am J Physiol Cell Physiol 296:C654–C662. OECD Series on Testing and Assessment (2017). New Scoping Document on in vitro and ex vivo Assays for the Identification of Modulators of Thyroid Hormone Signalling (page 36 - 38). Pearce EN, Lazarus JH, Smyth PPA, He X, Dall'amico D, Parkes AB, Burns R, Smith DF, Maina A, Bestwick JP, Jooman M, Leung AM, Braverman LE. (2010). 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2019-04-04T08:52:00

Thyroid hormone synthesis, Decreased

TH synthesis, Decreased

Cellular

The thyroid hormones (TH), triiodothyronine (T3) and thyroxine (T4) are thyrosine-based hormones. Synthesis of THs is regulated by thyroid-stimulating hormone (TSH) binding to its receptor and thyroidal availability of iodine via the sodium iodide symporter (NIS). Other proteins contributing to TH production in the thyroid gland, including thyroperoxidase (TPO), dual oxidase enzymes (DUOX), and the transport protein pendrin are also necessary for iodothyronine production (Zoeller et al., 2007). The production of THs in the thyroid gland and resulting serum concentrations are controlled by a negatively regulated feedback mechanism. Decreased T4 and T3 serum concentrations activates the hypothalamus-pituitary-thyroid (HPT) axis which upregulates thyroid-stimulating hormone (TSH) that acts to increase production of additional THs (Zoeller and Tan, 2007). This regulatory system includes: 1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH); 2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary; 3) hormonal transport by the plasma binding proteins; 4) cellular uptake mechanisms at the tissue level; 5) intracellular control of TH concentrations by deiodinating mechanisms; 6) transcriptional function of the nuclear TH receptor; and 7) in the fetus, the transplacental passage of T4 and T3 (Zoeller et al., 2007). TRH and the TSH primarily regulate the production of T4, often considered a “pro-hormone,” and to a lesser extent of T3, the transcriptionally active TH. Most of the hormone released from the thyroid gland into circulation is in the form of T4, while peripheral deiodination of T4 is responsible for the majority of circulating T3. Outer ring deiodination of T4 to T3 is catalyzed by the deiodinases 1 and 2 (DIO1 and DIO2), with DIO1 expressed mainly in liver and kidney, and DIO2 expressed in several tissues including the brain (Bianco et al., 2006). Conversion of T4 to T3 takes place mainly in the liver and kidney, but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009).  In mammals, most evidence for the ontogeny of TH synthesis comes from measurements of serum hormone concentrations. And, importantly, the impact of xenobiotics on fetal hormones must include the influence of the maternal compartment since a majority of fetal THs are derived from maternal blood early in fetal life, with a transition during mid-late gestation to fetal production of THs that is still supplemented by maternal THs. In humans, THs can be found in the fetus as early as gestational weeks 10-12, and concentations rise continuously until birth. At term, fetal T4 is similar to maternal levels, but T3 remains 2-3 fold lower than maternal levels. In rats, THs can be detected in the fetus as early as the second gestational week, but fetal synthesis does not start until gestational day 17 with birth at gestational day 22-23. Maternal THs continue to supplement fetal production until parturition. (see Howdeshell, 2002; Santisteban and Bernal, 2005 for review). Due to the maternal factor, the life stage specific impact of TPO inhibition after exposure to environmental chemicals is complex (Ramhoj et al., 2022). Decreased TH synthesis in the thyroid gland may result from several possible molecular-initiating events (MIEs) including: 1) Disruption of key catalytic enzymes or cofactors needed for TH synthesis, including TPO, NIS, or dietary iodine insufficiency. Theoretically, decreased synthesis of Tg could also affect TH production (Kessler et al., 2008; Yi et al., 1997). Mutations in genes that encode requisite proteins in the thyroid may also lead to impaired TH synthesis, including mutations in pendrin associated with Pendred Syndrome (Dossena et al., 2011), mutations in TPO and Tg (Huang and Jap 2015), and mutations in NIS (Spitzweg and Morris, 2010). 2) Decreased TH synthesis in cases of clinical hypothyroidism may be due to Hashimoto's thyroiditis or other forms of thyroiditis, or physical destruction of the thyroid gland as in radioablation or surgical treatment of thyroid lymphoma. 3) It is possible that TH synthesis may also be reduced subsequent to disruption of the negative feedback mechanism governing TH homeostasis, e.g. pituitary gland dysfunction may result in a decreased TSH signal with concomitant T3 and T4 decreases. 4) More rarely, hypothalamic dysfunction can result in decreased TH synthesis.  Increased fetal TH levels are also possible. Maternal Graves disease, which results in fetal thyrotoxicosis (hyperthyroidism and increased serum T4 levels), has been successfully treated by maternal administration of TPO inhibitors (c.f., Sato et al., 2014).   It should be noted that different species and different life stages store different amounts of TH precursors and iodine within the thyroid gland. Thus, decreased TH synthesis via transient iodine insufficiency or inhibition of TPO may not affect TH release from the thyroid gland until depletion of stored iodinated Tg. Adult humans may store sufficient Tg-DIT residues to serve for several months to a year of TH demand (Greer et al., 2002; Zoeller, 2004). Neonates and infants have a much more limited supply of less than a week. While the TH system is highly conserved across vertebrates, there are some taxon-specific considerations. Zebrafish and fathead minnows are oviparous fish species in which maternal THs are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic TH synthesis is initiated. Maternal transfer of THs to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016). Decreases in TH synthesis can only occur after initiation of embryonic TH synthesis. The components of the TH system responsible for TH synthesis are highly conserved across vertebrates and therefore interference with the same molecular targets compared to mammals can lead to decreased TH synthesis (TPO, NIS, etc.) in fish. Endogenous transcription profiles of thyroid-related genes in zebrafish and fathead minnow showed that mRNA coding for these genes is also maternally transferred and increasing expression of most transcripts during hatching and embryo-larval transition indicates a fully functional HPT axis in larvae (Vergauwen et al., 2018). Although the HPT axis is highly conserved, there are some differences between fish and mammals (Blanton and Specker, 2007; Deal and Volkoff, 2020). For example, in fish, corticotropin releasing hormone (CRH) often plays a more important role in regulating thyrotropin (TSH) secretion by the pituitary and thus TH synthesis compared to TSH-releasing hormone (TRH). Also, in most fish species thyroid follicles are more diffusely located in the pharyngeal region rather than encapsulated in a gland.

Decreased TH synthesis is often implied by measurement of TPO and NIS inhibition measured clinically and in laboratory models as these enzymes are essential for TH synthesis. Rarely is decreased TH synthesis measured directly, but rather the impact of chemicals on the quantity of T4 produced in the thyroid gland, or the amount of T4 present in serum is used as a marker of decreased T4 release from the thyroid gland (e.g., Romaldini et al., 1988). Methods used to assess TH synthesis include, incorporation of radiolabeled tracer compounds, radioimmunoassay, ELISA, and analytical detection.    Recently, amphibian thyroid explant cultures have been used to demonstrate direct effects of chemicals on TH synthesis, as this model contains all necessary synthesis enzymes including TPO and NIS (Hornung et al., 2010). For this work THs was measured by HPLC/ICP-mass spectometry. Decreased TH synthesis and release, using T4 release as the endpoint, has been shown for thiouracil antihyperthyroidism drugs including MMI, PTU, and the NIS inhibitor perchlorate (Hornung et al., 2010). Techniques for in vivo analysis of TH system disruption among other drug-related effects in fish were reviewed by Raldua and Piña (2014). TIQDT (Thyroxine-immunofluorescence quantitative disruption test) is a method that provides an immunofluorescent based estimate of thyroxine in the gland of zebrafish (Raldua and Babin, 2009; Thienpont et al., 2011; Jomaa et al., 2014; Rehberger et al., 2018).  Thienpont used this method with ~25 xenobiotics (e.g., amitrole, perchlorate, methimazole, PTU, DDT, PCBs). The method detected changes for all chemicals known to directly impact TH synthesis in the thyroid gland (e.g., NIS and TPO inhibitors), but not those that upregulate hepatic catabolism of T4. Rehberger et al. (2018) updated the method to enable simultaneous semi-quantitative visualization of intrafollicular T3 and T4 levels. Most often, whole body TH level measurements in fish early life stages are used as indirect evidence of decreased TH synthesis (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). Analytical determination of TH levels by LC-MS is becoming increasingly available (Hornung et al., 2015). More recently, transgenic zebrafish with fluorescent thyroid follicles are being used to visualize the compensatory proliferation of the thyroid follicles following inhibition of TH synthesis among others (Opitz et al., 2012).

Taxonomic: This KE is plausibly applicable across vertebrates. Decreased TH synthesis resulting from TPO or NIS inhibition is conserved across vertebrate taxa, with in vivo evidence from humans, rats, amphibians, some fish species, and birds, and in vitro evidence from rat and porcine microsomes. Indeed, TPO and NIS mutations result in congenital hypothyroidism in humans (Bakker et al., 2000; Spitzweg and Morris, 2010), demonstrating the essentiality of TPO and NIS function toward maintaining euthyroid status. Though decreased serum T4 is used as a surrogate measure to indicate chemical-mediated decreases in TH synthesis, clinical and veterinary management of hyperthyroidism and Graves’ disease using propylthiouracil and methimazole, known to decrease TH synthesis, indicates strong evidence for chemical inhibition of TPO (Zoeller and Crofton, 2005). Life stage: Applicability to certain life stages may depend on the species and their dependence on maternally transferred THs during the earliest phases of development. The earliest life stages of teleost fish (e.g., fathead minnow, zebrafish) rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). In externally developing fish species, decreases in TH synthesis can only occur after initiation of embryonic TH synthesis. 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. TPO inhibition in a homozygous knockout line abolished the T4 production in thyroid follicles of mutant zebrafish with phenotypic abnormalities occurring from 20 dpf onwards but not before 10 dpf (Fang et al., 2022). Therefore, it is still uncertain when exactly embryonic TH synthesis is activated and thus when exactly this process becomes sensitive to disruption. In fathead minnows, a significant increase of whole body TH levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It currently remains unclear when exactly embryonic TH production is initiated in zebrafish. Sex: The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.

UBERON:0002046

UBERON thyroid gland

CL:0002258

CL thyroid follicular cell

High Male High Female

High

All life stages

High High Moderate High Moderate High

Bakker B, Bikker H, Vulsma T, de Randamie JS, Wiedijk BM, De Vijlder JJ. 2000. Two decades of screening for congenital hypothyroidism in The Netherlands: TPO gene mutations in total iodide organification defects (an update). The Journal of clinical endocrinology and metabolism.  85:3708-3712. Bianco AC, Kim BW. (2006). Deiodinases: implications of the local control of thyroid hormone action. J Clin Invest. 116: 2571–2579. Blanton ML, Specker JL. 2007. The hypothalamic-pituitary-thyroid (hpt) axis in fish and its role in fish development and reproduction. Crit Rev Toxicol. 37(1-2):97-115. Campinho MA, Saraiva J, Florindo C, Power DM. 2014. Maternal thyroid hormones are essential for neural development in zebrafish. Molecular Endocrinology. 28(7):1136-1149. Chang J, Wang M, Gui W, Zhao Y, Yu L, Zhu G. 2012. Changes in thyroid hormone levels during zebrafish development. Zoological Science. 29(3):181-184. Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2004. Developmental changes of thyroid hormones in the fathead minnow, pimephales promelas. General and Comparative Endocrinology. 139(1):55-60. Deal CK, Volkoff H. 2020. The role of the thyroid axis in fish. Frontiers in Endocrinology. 11. Dossena S, Nofziger C, Brownstein Z, Kanaan M, Avraham KB, Paulmichl M. (2011). Functional characterization of pendrin mutations found in the Israeli and Palestinian populations. Cell Physiol Biochem. 28: 477-484.Gereben B, Zavacki AM, Ribich S, Kim BW, Huang SA, Simonides WS, Zeöld A, Bianco AC. (2008). Cellular and molecular basis of deiodinase-regulated thyroid hormone signalling. Endocr Rev. 29:898–938. Fang, Y., Wan, J. P., Zhang, R. J., Sun, F., Yang, L., Zhao, S. X., Dong, M., & Song, H. D. (2022). Tpo knockout in zebrafish partially recapitulates clinical manifestations of congenital hypothyroidism and reveals the involvement of TH in proper development of glucose homeostasis. General and Comparative Endocrinology, 323–324. https://doi.org/10.1016/j.ygcen.2022.114033 Gereben B, Zeöld A, Dentice M, Salvatore D, Bianco AC.  Activation and inactivation of thyroid hormone by deiodinases: local action with general consequences.  Cell Mol Life Sci. 2008 Feb;65(4):570-90 Greer MA, Goodman G, Pleus RC, Greer SE. Health effects assessment for environmental perchlorate contamination: the dose response for inhibition of thyroidal radioiodine uptake in humans. Environ Health Perspect. 2002. 110:927-937. Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604. Hornung MW, Degitz SJ, Korte LM, Olson JM, Kosian PA, Linnum AL, Tietge JE. 2010. Inhibition of thyroid hormone release from cultured amphibian thyroid glands by methimazole, 6-propylthiouracil, and perchlorate. Toxicol Sci 118:42-51. Hornung MW, Kosian PA, Haselman JT, Korte JJ, Challis K, Macherla C, Nevalainen E, Degitz SJ. 2015. In vitro, ex vivo, and in vivo determination of thyroid hormone modulating activity of benzothiazoles. Toxicological Sciences. 146(2):254-264. Howdeshell KL. 2002. A model of the development of the brain as a construct of the thyroid system. Environ Health Perspect. 110 Suppl 3:337-48. Huang CJ and Jap TS. 2015. A systematic review of genetic studies of thyroid disorders in Taiwan. J Chin Med Assoc. 78: 145-153. Jomaa B, Hermsen SAB, Kessels MY, van den Berg JHJ, Peijnenburg AACM, Aarts JMMJG, Piersma AH, Rietjens IMCM. 2014. Developmental toxicity of thyroid-active compounds in a zebrafish embryotoxicity test. Altex-Alternatives to Animal Experimentation. 31(3):303-317. Kessler J, Obinger C, Eales G. Factors influencing the study of peroxidase-generated iodine species and implications for thyroglobulin synthesis. Thyroid. 2008 Jul;18(7):769-74. doi: 10.1089/thy.2007.0310 Larsen PR. (2009). Type 2 iodothyronine deiodinase in human skeletal muscle: new insights into its physiological role and regulation. J Clin Endocrinol Metab. 94:1893-1895. Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32. Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203. Opitz R, Maquet E, Huisken J, Antonica F, Trubiroha A, Pottier G, Janssens V, Costagliola S. 2012. Transgenic zebrafish illuminate the dynamics of thyroid morphogenesis and its relationship to cardiovascular development. Developmental Biology. 372(2):203-216. Opitz R, Maquet E, Zoenen M, Dadhich R, Costagliola S. 2011. Tsh receptor function is required for normal thyroid differentiation in zebrafish. Molecular Endocrinology. 25(9):1579-1599. Power DM, Llewellyn L, Faustino M, Nowell MA, Bjornsson BT, Einarsdottir IE, Canario AV, Sweeney GE. 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 130(4):447-459. Raldua D, Babin PJ. 2009. Simple, rapid zebrafish larva bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. Environmental Science & Technology. 43(17):6844-6850. Raldua D, Pina B. 2014. In vivo zebrafish assays for analyzing drug toxicity. Expert Opinion on Drug Metabolism & Toxicology. 10(5):685-697. Ramhoj, L., Svingen, T., Fradrich, C., Rijntjes, E., Wirth, E.K., Pedersen, K., Kohrle, J., Axelstad, M., 2022. Perinatal exposure to the thyroperoxidase inhibitors methimazole and amitrole perturbs thyroid hormone system signaling and alters motor activity in rat offspring. Toxicology Letters 354, 44-55. Rehberger K, Baumann L, Hecker M, Braunbeck T. 2018. Intrafollicular thyroid hormone staining in whole-mount zebrafish (danio rerio) embryos for the detection of thyroid hormone synthesis disruption. Fish Physiology and Biochemistry. 44(3):997-1010. Romaldini JH, Farah CS, Werner RS, Dall'Antonia Júnior RP, Camargo RS. 1988.  "In vitro" study on release of cyclic AMP and thyroid hormone in autonomously functioning thyroid nodules.  Horm Metab Res.20:510-2. Ruuskanen S, Hsu BY. 2018. Maternal thyroid hormones: An unexplored mechanism underlying maternal effects in an ecological framework. Physiological and Biochemical Zoology. 91(3):904-916. Santisteban P, Bernal J. Thyroid development and effect on the nervous system. Rev Endocr Metab Disord. 2005 Aug;6(3):217-28. Spitzweg C, Morris JC. 2010. Genetics and phenomics of hypothyroidism and goiter due to NIS mutations. Molecular and cellular endocrinology. 322:56-63. Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223. Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217. Thienpont B, Tingaud-Sequeira A, Prats E, Barata C, Babin PJ, Raldúa D.  Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis.  Environ Sci Technol. 2011. 45(17):7525-32. Vergauwen L, Cavallin JE, Ankley GT, Bars C, Gabriels IJ, Michiels EDG, Fitzpatrick KR, Periz-Stanacev J, Randolph EC, Robinson SL et al. 2018. Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish. General and Comparative Endocrinology. 266:87-100. Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331. Walpita CN, Van der Geyten S, Rurangwa E, Darras VM. 2007. The effect of 3,5,3'-triiodothyronine supplementation on zebrafish (danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors. Gen Comp Endocrinol. 152(2-3):206-214. Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32. Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347. Yi X, Yamamoto K, Shu L, Katoh R, Kawaoi A. Effects of Propyithiouracil (PTU) Administration on the Synthesis and Secretion of Thyroglobulin in the Rat Thyroid Gland: A Quantitative Immuno-electron Microscopic Study Using Immunogold Technique. Endocr Pathol. 1997 Winter;8(4):315-325. Zoeller RT, Crofton KM. 2005.  Mode of action: developmental thyroid hormone insufficiency--neurological abnormalities resulting from exposure to propylthiouracil. Crit Rev Toxicol. 35:771-81 Zoeller RT, Tan SW, Tyl RW. 2007. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical reviews in toxicology.  37:11-53. Zoeller RT.  Interspecies differences in susceptibility to perturbation of thyroid hormone homeostasis requires a definition of "sensitivity" that is informative for risk analysis. Regul Toxicol Pharmacol. 2004 Dec;40(3):380.   AOPWiki 2016-11-29T18:41:23

2022-11-04T09:25:39

Decrease of Thyroidal iodide

Thyroidal Iodide, Decreased

Cellular

Biological state: Iodine (I2) is a non-metallic chemical element which is required for the normal cellular metabolism. It is one of the essential components of the TH, comprising 65% and 58% of T4's and T3's weight, respectively and therefore it is crucial for the normal thyroid function. It is a trace element and a healthy human body contains 15-20 mg of iodine, most of which is concentrated in the thyroid gland (Dunn, 1998). Iodide (I-) that enters the thyroid gland remains in the free state only briefly and subsequently it bounds to the tyrosine residues of thyroglobulin to form the precursors of the thyroid hormones mono-iodinated tyrosine (MIT) or di-iodinated tyrosine (DIT) (Berson and Yalow, 1955). The bounding rate of iodide is 50-100% of the intra-thyroidal iodide pool, meaning that only a very small proportion of this element is free in the thyroid and this comes mainly by the deiodination of MIT and DIT. The body is not able to produce or make iodine, thus the diet is the only source of this element. Iodine is found in nature in various forms, such as inorganic sodium and potassium salts (iodides and iodates), inorganic diatomic iodine and organic monoatomic iodine (Patrick, 2008). Thus, it is widely distributed in the environment but in many regions of the world the soil's iodine has been depleted due to different environmental phenomena. In these regions, the incidence of iodine deficiency is greatly increased (Ahad and Ganie, 2010). The daily iodine intake of adult humans varies greatly due to the different dietary habits between the different regions on earth (Dunn, 1993). In any case, the ingested iodine is absorbed through the intestine and transported into the plasma to reach the thyroid gland. However, thyroid is not the only organ of the body that concentrates iodide. It has been shown that other tissues have also the ability of iodide concentration, such as the salivary glands, the gastric mucosa, the mammary glands and the choroid plexus, all of which express NIS, the iodine transporter protein (Jhiang et al., 1998; Cho et al., 2000). Biological compartments: A sodium-iodide (Na/I) symporter pumps iodide (IO) actively into the cell, which previously has crossed the endothelium by largely unknown mechanisms. This iodide enters the follicular lumen from the cytoplasm by the transporter pendrin, in a purportedly passive manner. In the colloid, iodide (I−) is oxidized to iodine (I0) by an enzyme called thyroid peroxidase (TPO). IO is very reactive and iodinates the thyroglobulin at tyrosyl residues in its protein chain. In conjugation, adjacent tyrosyl residues are paired together. Thyroglobulin binds the megalin receptor for endocytosis back into the follicular cell. Proteolysis by various proteases liberates thyroxine (T4) and triiodothyronine molecules (T3), which enter the bloodstream where they are bound to thyroid hormone binding proteins, mainly thyroxin binding globulin (TBG) which accounts for about 75% of the bound hormone. The adult thyroid absorbs 60-80 μg of iodide per day to maintain the thyroid homeostasis (Degroot, 1966). Inadequate amount of iodide results to deficient production of thyroid hormones, which consequently leads to an increase of TSH secretion and goiter, as compensating effect (Delange, 2000). On the other hand, excess iodide could also inhibit TH synthesis (Wolff and Chaikoff, 1948). The proposed mechanism for this latter effect is the possible formation of 2-iodohexadecanal that inhibits the generation of H2O2 and the subsequent oxidation of iodide in the thyroid follicular cells. The lack of oxidized free radicals of iodide affects the reaction with the tyrosine residues of Thyroglobulin (Tg) (Panneels et al., 1994). During pregnancy, the organism of the mother is also supporting the needs of the foetus and therefore the iodide requirements are greatly increased (Glinoer, 1997). Additionally, small iodine concentrations have been found to have significant antioxidant effects that resembles to ascorbic acid (Smyth, 2003). General role in biology: The most important role of iodine is the formation of the thyroid hormones (T4 and T3). The thyroid actively concentrates the circulating iodide through the basolateral membrane of the thyrocytes by the sodium/iodide symporter protein (NIS). The concentrated thyroid-iodine is oxidized in the follicular cells of the gland and consequently binds to tyrosines to form mono- or di-iodotyrosines (MIT and DIT respectively), being incorporated into thyroglobulin. This newly formed iodothyroglobulin forms one of the most important constituents of the colloid material, present in the follicle of the thyroid unit. If two di-iodotyrosine molecules couple together, the result is the formation of thyroxin (T4). If a di-iodotyrosine and a mono-iodotyrosine are coupled together, the result is the formation of tri-iodothyronine (T3). From the perspective of the formation of thyroid hormone, the major coupling reaction is the di-iodotyrosine coupling to produce T4.

The radioactive iodine uptake test, or RAIU test, is a type of scan used in the diagnosis of thyroid gland dysfunction (<a href="http://www.thyca.org/pap-fol/rai/">http://www.thyca.org/pap-fol/rai/</a>; Kwee, et al., 2007). The patient swallows radioactive iodine in the form of capsule or fluid, and its absorption by the thyroid is studied after 4–6 hours and after 24 hours with the aid of a gamma scintillation counter. The percentage of RAIU 24 hours after the administration of radioiodide is the most useful, since this is the time when the thyroid gland has reached the plateau of isotope accumulation, and because it has been shown that at this time, the best separation between high, normal, and low uptake is obtained. The test does not measure hormone production and release but merely the avidity of the thyroid gland for iodide and its rate of clearance relative to the kidney.

Various species express functional NIS  encoded by the following genes: Human SLC5A5 (6528), Mouse Slc5a5 (114479), Rat Slc5a5 (114613), Zebrafish slc5a5 (561445), chicken SLC5A5 (431544), domestic cat SLC5A5 (101092587), dog SLC5A5 (484830), domestic guinea pig Slc5a5 (100714457), naked mole-rat Slc5a5 (101701995), cow SLC5A5 (505310), sheep SLC5A5 (101112315). The encoded protein is responsible for the uptake of iodine in tissues such as the thyroid and lactating breast tissue. The iodine taken up by the thyroid is incorporated into the metabolic regulators triiodothyronine (T3) and tetraiodothyronine (T4). Mutations in this gene are associated with thyroid dyshormonogenesis that significantly influences phenotypic expressions such as severity of hypothyroidism, goiter rates, and familial clustering demonstrating essentiality of NIS function to maintain TH status (Bakker et al., 2000; Spitzweg and Morris, 2010; Ramesh et al., 2016) . Animal studies have also proven that iodine normalizes elevated adrenal corticosteroid hormone secretion and has the ability to reverse the effects of hypothyroidism in the ovaries, testicles and thymus in thyroidectomized rats (Nolan et al., 2000).

UBERON:0002046

UBERON thyroid gland

CL:0002258

CL thyroid follicular cell

Moderate Mixed

Moderate

Birth to < 1 month

Moderate

Pregnancy

Moderate

During brain development

High High High High High Moderate Not Specified

Ahad F, Ganie SA. (2010). Iodine, iodine metabolism and iodine deficiency disorders revisited. Indian J Endocrinol Metab. 14: 13-17. Bakker B, Bikker H, Vulsma T, de Randamie JS, Wiedijk BM, De Vijlder JJ. 2000. Two decades of screening for congenital hypothyroidism in The Netherlands: TPO gene mutations in total iodide organification defects (an update). The Journal of clinical endocrinology and metabolism. Oct;85:3708-3712. Berson SA, Yalow RS. (1955). The iodide trapping and binding functions of the thyroid. J Clin Invest. 34: 186-204. Cho JY, Leveille R, Kao R, Rousset B, Parlow AF, Burak WE Jr, Mazzaferri EL, Jhiang SM.(2000). Hormonal regulation of radioiodide uptake activity and Na+/I- symporter expression in mammary glands. J Clin Endocrinol Metab. 85:2936-2943. Degroot LJ.(1966). Kinetic analysis of iodine metabolism. J Clin Endocrinol Metab. 26: 149-173. Delange F. (2000). Iodine deficiency. In: Braverman L, Utiger R, editors. Werner and Ingbar's the thyroid: a fundamental and clinical text. Philadelphia: JD Lippincott. pp 295-316. Dunn JT. (1993). Sources of dietary iodine in industrialized countries. In: Delange F, Dunn JT, Glinoer D, editors. Iodine deficiency in Europe. A continuing concern. New York: Plenum press. pp 17-21. Dunn JT. (1998). What's happening to our iodine? J Clin Endocrinol Metab. 83: 3398-3400. Glinoer D. (1997). The regulation of thyroid function in pregnancy: pathways of endocrine adaptation from physiology to pathology. Endocr Rev. 18: 404-433. <a href="http://www.thyca.org/pap-fol/rai/">http://www.thyca.org/pap-fol/rai/</a>: Thyroid Cancer Survivors' Association, Inc.,Radioactive Iodine (RAI) Jhiang SM, Cho JY, Ryu KY, DeYoung BR, Smanik PA, McGaughy VR, Fischer AH, Mazzaferri EL.(1998). An immunohistochemical study of Na+/I- symporter in human thyroid tissues and salivary gland tissues. Endocrinology. 139:4416-4419. Kwee, Sandi A.; Coel, Marc N.; Fitz-Patrick, David (2007). Eary, Janet F.; Brenner, Winfried, eds. "Iodine-131 Radiotherapy for Benign Thyroid Disease". Nuclear Medicine Therapy. CRC Press: 172. <a href="https://en.wikipedia.org/wiki/International_Standard_Book_Number" title="International Standard Book Number">ISBN</a> <a href="https://en.wikipedia.org/wiki/Special:BookSources/978-0-8247-2876-2" title="Special:BookSources/978-0-8247-2876-2">978-0-8247-2876-2</a>. Nolan LA, Windle RJ, Wood SA, Kershaw YM, Lunness HR, Lightman SL, Ingram CD, Levy A. (2000). Chronic iodine deprivation attenuates stress-induced and diurnal variation in corticosterone secretion in female Wistar rats. J Neuroendocrinol. 12:1149-1159. Panneels V, Van den Bergen H, Jacoby C, Braekman JC, Van Sande J, Dumont JE, Boeynaems JM. (1994). Inhibition of H2O2 production by iodoaldehydes in cultured dog thyroid cells. Mol Cell Endocrinol. 102:167-176. Patrick L. (2008).Iodine:Deficiency and therapeutic considerations. Altern MedRev. 13:166-127. Ramesh BG, Bhargav PR, Rajesh BG, Devi NV, Vijayaraghavan R, Varma BA.(2016). Genotype‑phenotype correlations of dyshormonogenetic goiter in children and adolescents from South India . I J Endocrinol and Metab. 20: 816-824. Smyth PA. (2003). Role of iodine in antioxidant defense in thyroid and breast disease. Biofactors. 19:121-130. Spitzweg C, Morris JC. 2010. Genetics and phenomics of hypothyroidism and goiter due to NIS mutations. Molecular and cellular endocrinology. Jun 30;322:56-63. Wolff J, Chaikoff IL. (1948). Plasma inorganic iodide as a homeostatic regulator of thyroid function. J Biol Chem. 174: 555-564. AOPWiki 2016-11-29T18:41:24

2019-04-04T09:00:11

Thyroxine (T4) in serum, Decreased

T4 in serum, Decreased

Tissue

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

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

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

UBERON:0001977

UBERON serum

High Female High Male

High

All life stages

High High High Moderate Moderate High High High

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

2022-10-10T08:52:30

Thyroxine (T4) in neuronal tissue, Decreased

T4 in neuronal tissue, Decreased

Organ

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

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

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

UBERON:0000955

UBERON brain

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Bansal R, You SH, Herzig CT, Zoeller RT (2005). Maternal thyroid hormone increases HES expression in the fetal rat brain: an effect mimicked by exposure to a mixture of polychlorinated biphenyls (PCBs). Brain Res Dev Brain Res 156:13-22. Bates JM, St Germain DL, Galton VA. Expression profiles of the three iodothyronine deiodinases, D1, D2, and D3, in the developing rat. Endocrinology. 1999 Feb;140(2):844-51. Bernal J. (2013). Thyroid Hormones in Brain Development and Function.  In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2015. www.thyroidmanager.org Calvo R, Obregón MJ, Ruiz de Oña C, Escobar del Rey F, Morreale de Escobar G. (1990). Congenital hypothyroidism, as studied in rats. Crucial role of maternal thyroxine but not of 3,5,3′-triiodothyronine in the protection of the fetal brain. J. Clin. Invest. 86:889-899. 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) 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. 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. Friesema EC, Jansen J, Milici C, Visser TJ (2005) Thyroid hormone transporters. Vitam Horm 70:137-167. 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. 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. 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. 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. 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. 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. Heuer H. (2007). The importance of thyroid hormone transporters for brain development and function. Best Pract Res Clin Endocrinol Metab. 21:265–276. 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. 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. 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. 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. 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 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 Müller J, Heuer H. Expression pattern of thyroid hormone transporters in the postnatal mouse brain. Front Endocrinol (Lausanne). 2014 Jun 18:5:92. 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. 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. 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. 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. St Germain DL, Galton VA, Hernandez A. (2009). Minireview: Defining the roles of the iodothyronine deiodinases: current concepts and challenges. Endocrinology. 150:1097-107. 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. 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. 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. 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. 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. Visser EW, Visser TJ. (2012). Finding the way into the brain without MCT8. J Clin Enodcrinol Metab. 97:4362-4365. Visser WE, Friesema EC, Jansen J, Visser TJ. (2007). Thyroid hormone transport by monocarboxylate transporters. Best Pract Res Clin Endocrinol Metab. 21:223–236. Wang, D. and Stapleton, HM. (2010) Analysis of thyroid hormones in serum by liquid chromatography -tandem mass spectrometry. Anal Bioanal Chem. 2010 Jul; 397(5): 1831–1839 AOPWiki 2016-11-29T18:41:23

2019-04-04T09:13:27

Hippocampal gene expression, Altered

Tissue

Thyroid hormones control genes in the developing brain by classical ligand (T3) activation of thyroid receptors which leads to DNA binding and subsequent transcription and translation (for a review of TH rols in brain development see, Bernal 2015). Gene expression profiles have been published for the developing human and rodent hippocampus (Zhang et al., 2002; Mody et al., 2001). In both humans and rodents, the hippocampus undergoes typical stages of neurodevelopment found in most brain regions, including: cell proliferation, migration, differentiation, synapse formation, and the maturation of synaptic function. In the rodent, peak windows during pre- and post-natal periods have been identified during which major cellular and physiological events occur (see Figure 1). Each window expresses distinct patterns of gene transcription and clusters of genes increase their expression corresponding to the progression of events of hippocampal ontogeny (see Mody et al., 2001).  Tables of gene clusters associated with these phases can be found in Supplementary Tables of Mody et al. (2001). <img alt="" 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" />   During the very early prenatal period, genes corresponding to general cellular function are prominent (Mody et al., 2001).  These are followed in time by genes regulating neuronal differentiation and migration in the mid to late gestational period. From late gestation (gestational day 15) until birth almost all the cells in the CA fields switch from a highly active proliferation state to a postmitotic state, and then undergo differentiation and migration. Expression of proliferative genes involved in cell cycle progression are highly expressed at gestational day 16, then subsequently are silent immediately after birth when genes directing neuronal growth switch on. The pyramidal neurons of the CA fields in the hippocampus proper develop in advance of the granule cells that comprise the principal cells of the dentate gyrus. As such, the genes controlling the distinct phases of neurodevelopment are expressed at different times in these two hippocampal subregions (Altman and Bayer, 1990a; b). In both subregions, however, many phenotypic changes within the hippocampal neuron occur in the period immediately after birth (postnatal day 1 to 7). Almost all neurons show extensive growth and differentiation during the first postnatal week. These cellular changes are marked by rapid cytoskeletal changes, production of cell adhesion molecules, and extracellular matrix formation. The gene families involved in these processes include actins, tubulins, and chaperonin proteins essential for promoting correct protein folding of cytoskeletal components. Cell adhesion and extracellular matrix proteins are also upregulated during this period as these genes are critical for differentiation and synaptogenesis. During late postnatal hippocampal development (postnatal day 16-30), hippocampal circuits become more active and exhibit increased synaptic plasticity. Many genes upregulated during this phase of development are involved in synaptic function and include genes regulating vesicle associated proteins and calcium-mediated transmitter release, neurotrophins, and neurotransmitter receptors. Efficient energy utilization is essential during this period of increased synaptic activity, events mirrored by an upregulation of enzymes involved in glucose and oxidative metabolism.

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

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

UBERON:0000955

UBERON brain

High Female High Male

High

During brain development

High High Moderate

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

2018-08-11T09:26:56

Hippocampal anatomy, Altered

Tissue

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). 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. 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.   Trisynaptic Hippocampal Circuitry <img alt="" 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" />

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.  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).

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.

UBERON:0000955

UBERON brain

High Male High Female

High

During brain development

High High High

Altman J, Bayer SA. Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J Comp Neurol. 1990a Nov 15;301(3):365-81. Altman J, Bayer SA. Prolonged sojourn of developing pyramidal cells in the intermediate zone of the hippocampus and their settling in the stratum pyramidale. J Comp Neurol. 1990b Nov 15;301(3):343-64. 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. 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. 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. 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. 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. 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. 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. 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. Pirko I, Johnson AJ. Neuroimaging of demyelination and remyelination models. Curr Top Microbiol Immunol. 2008; 318:241-66. 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. 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. 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. 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. 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.    AOPWiki 2016-11-29T18:41:26

2022-05-20T05:45:05

Hippocampal Physiology, Altered

Tissue

The hippocampus functions as a highly integrated and organized communication and information processing network with millions of interconnections among its constitutive neurons. Neurons in the hippocampus and throughout the brain transmit and receive information largely through chemical transmission across the synaptic cleft, the space where the specialized ending of the presynaptic axon terminus of the transmitting neuron meets the specialized postsynaptic region of the neuron that is receiving that information (Kandell et al., 2012). During development (see KE: 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). 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.

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). Excitatory Synaptic Transmission: Two measures, the excitatory postsynaptic potential (EPSP) and the population spike are derived from the compound field potential at increasing stimulus strengths. The function described by the relationship of current strength (input, I) and evoked response (output, O), the I-O curve is the measure of excitatory synaptic transmission (Gilbert and Burdette, 1995). Inhibitory Synaptic Transmission: Pairs of stimulus pulses delivered in close temporal proximity 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). 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). Synaptic function in the human hippocampus has been assessed using electroencephalography (EEG) and functional neuroimaging techniques (Clapp et al., 2012). EEG is a measure of electrical activity over many brain regions but primarily from the cortex using small flat metal discs (electrodes) placed over the surface of the skull. It is a readily available test that provides evidence of how the brain functions over time. Functional magnetic resonance imaging or functional MRI (fMRI) uses MRI technology to measure brain activity by detecting associated changes in blood flow. This technique relies on the fact that cerebral blood flow and neuronal activation are coupled. Positron emission tomography (PET) is a functional imaging technique that detects pairs of gamma rays emitted indirectly by a radionuclide (tracer) injected into the body (Tietze, 2012; McCarthy, 1995). Like fMRI, PET scans indirectly measure blood flow to different parts of the brain – the higher the blood flow, the greater the activation (McCarthy, 1995). These techniques have been widely applied in clinical and research settings to assess learning and memory in humans and can provide information targeted to hippocampal functionality (McCarthy, 1995; Smith and Jonides, 1997; Willoughby et al., 2014; Wheeler et al., 2015; Gilbert et al., 1998). Assays of this type are fit for purpose, have been well accepted in the literature, and are reproducible across laboratories. The assay directly measures the key event of altered neurophysiological function.

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).

UBERON:0000955

UBERON brain

High Female High Male

High

During brain development

Moderate High High

Bekenstein JW, Lothman EW. An in vivo study of the ontogeny of long-term potentiation (LTP) in the CA1 region and in the dentate gyrus of the rat hippocampal formation. Brain Res Dev Brain Res. 1991 Nov 19;63(1-2):245- Bramham CR, Messaoudi E (2005) BDNF function in adult synaptic plasticity: the synaptic consolidation hypothesis. Prog Neurobiol 76:99-125. Clapp WC, Hamm JP, Kirk IJ, Teyler TJ. Translating long-term potentiation from animals to humans: a novel method for noninvasive assessment of cortical plasticity. Biol Psychiatry. 2012 Mar 15;71(6):496-502. Gilbert, M.E. and Burdette, L.J. (1995). Hippocampal Field Potentials: A Model System to Characterize Neurotoxicity. In Neurotoxicology: Approaches and Methods. L.W Chang and W. Slikker (Eds). Academic Press:New York, 183-204. Gilbert ME, Mack CM. Chronic lead exposure accelerates decay of long-term potentiation in rat dentate gyrus in vivo. Brain Res. 1998 Apr 6;789(1):139-49. Harris KM, Teyler TJ. Developmental onset of long-term potentiation in area CA1 of the rat hippocampus. J Physiol. 1984. 346:27-48. Kandell, E., Schwartz, J., Siegelbaum, A. and Hudspeth, A.J.  (2012) Principles of Neural Science, 5th Edition.  Elsevier, North Holland. Kutsarova E, Munz M, Ruthazer ES.  Rules for Shaping Neural Connections in the Developing Brain.  Front Neural Circuits. 2017 Jan 10;10:111. doi: 10.3389/fncir.2016.00111. Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44:5-21. McCarthy, G. (1995) Review: Functional Neuroimaging and Memory. The Neuroscientist, 1:155-163. Michelson HB, Lothman EW. An in vivo electrophysiological study of the ontogeny of excitatory and inhibitory processes in the rat hippocampus. Brain Res Dev Brain Res. 1989 May 1;47(1):113-22. Muller D, Oliver M, Lynch G. Developmental changes in synaptic properties in hippocampus of neonatal rats. Brain Res Dev Brain Res. 1989 Sep 1;49(1):105-14. Smith, E and Jonides, J. (1997). Working Memory: A View from Neuroimaging. Cognitive Psychology, 33:5-42. Tietze, KJ. (2012). Review of Laboratory and Diagnostic Tests- Positron Emission Tomography. In Clinical Sills for Pharmacists, 3rd Edition, pp 86-122.  Wheeler SM, McLelland VC, Sheard E, McAndrews MP, Rovet JF (2015) Hippocampal Functioning and Verbal Associative Memory in Adolescents with Congenital Hypothyroidism. Front Endocrinol (Lausanne) 6:163. Willoughby KA, McAndrews MP, Rovet JF (2014) Effects of maternal hypothyroidism on offspring hippocampus and memory. Thyroid 24:576-584. AOPWiki 2016-11-29T18:41:26

2018-08-11T09:41:37

Cognitive Function, Decreased

Individual

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

In rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, and most commonly, the Morris water maze (MWM). Test of novelty such as novel object recognition, and fear based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. A brief description of these tasks follows. 1) RAM, Barnes, MWM are examples of spatial tasks in which animals are required to learn: the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze); or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014). 2) Novel Object recognition. This is a simpler task that can be used to probe recognition memory. Two objects are presented to animal in an open field on trial 1, and these are explored. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention (i.e., I have seen one of these objects before, but not this one. Cohen and Stackman, 2015). 3) Contextual Fear conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event. The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009). 4) Trace fear conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, a light or a tone) and an aversive stimulus (US, a footshock). The unconditioned response (CR) that is elicited upon delivery of the footshock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2004). Most methods are well established in the published literature and many have been engaged to evaluate the effects of developmental thyroid disruption. The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 or OECD 426) both require testing of learning and memory (USEPA, 1998; OECD, 2007). These DNT Guidelines have been deemed valid to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009). A variety of standardized learning and memory tests have been developed for human neuropsychological testing. These include episodic autobiographical memory, word pair recognition memory; object location recognition memory. Some components of these tests have been incorporated in general tests of adult intelligence (IQ) such as the WAIS and the Wechsler. Modifications have been made and norms developed for incorporating of tests of learning and memory in children. Examples of some of these tests include: 1) Rey Osterieth Complex Figure (RCFT) which probes a variety of functions including as visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006). 2) Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1995; Talley, 1986).  3) Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994). 4) Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neurospychological test designed to measure memory functions (Lezak, 1994; Talley, 1986). 5) Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2015). 6) Staged Autobiographical Memory Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buy lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014).

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

High Male High Female

High

During brain development

High High High

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

2018-08-09T11:55:05

<upstream-id>ef657918-c27f-4074-863f-2abef3523de1</upstream-id> <downstream-id>21e6c233-1d52-42e8-ac24-8dc1575f8db3</downstream-id> NIS is a membrane protein implicated in iodide uptake into the follicular cells of the thyroid. Other large anions can be also bound by NIS and inhibit accumulation of iodide into the thyroid by competing binding with iodide (Wolff, 1964).

NIS is a membrane bound glycoprotein and its main physiological function is to transport one iodide ion along with two sodium ions across the basolateral membrane of thyroid follicular cells. It uses the sodium gradient generated by the Na+/K+ ATPase for the active transport of iodide into the thyrocytes (Eskandari et al., 1997). Extensive studies on NIS protein have identified 14 different mutations and each one of them is related to Iodine Transport Deficiencies (ITD) (reviewed in Spitzweg and Morris, 2010). Most of these mutations have been characterized and it is well known that they even lead to the synthesis of truncated protein (Pohlenz et al., 1997; Pohlenz et al., 1998), partial deletions (Kosugi et al., 2002; Tonacchera et al., 2003; Montanelli et al., 2009) or substitutions of amino acids (Matsuda and Kosugi, 1997; Kosugi et al., 1999; Szinnai et al., 2006) that eventually result in total or partial NIS dysfunction. While most of the NIS mutants have been further investigated and the functional relationship between the NIS dysfunction and ITD is well established (reviewed in Darrouzet et al., 2014; Portulano et al., 2014), the exact structural relationship between mutated NIS and ITD still needs to be elucidated and the molecular modelling of the protein would greatly benefit these studies. At the same time, causative link between  iodide deficiency, thyroid hormones, and neurodevelopment deffects is well documented (Gilbert et al., 2009). Recent revision of the affinity constant for perchlorate binding to the NIS symporter based on in vitro and human in vivo data, performed by refitting published in vitro data, in which perchlorate-induced inhibition of iodide uptake via the NIS was measured, yielding a Michaelis-Menten kinetic constant (Km) of 1.5 μm, showed that a 60% lower value for the Km, equal to 0.59 μm. Substituting this value into the PBPK model for an average adult human significantly improved model agreement with the human RAIU data for exposures <100 μg kg-1 day-1 (Schlosser PM, 2016). The effects of maternal hypothyroidism could also contribute to this KER.  During pregnancy TH requirements increase, particularly during the first trimester (Alexander et al. 2004; Leung et al. 2010), due to higher concentrations of thyroxine-binding globulin, placental T4 inner-ring deiodination leading to the inactive reverse T3 (rT3), and transfer of small amounts of T4 to the foetus (during the first trimester foetal thyroid function is absent). Moreover, glomerular filtration rate and clearance of proteins and other molecules are both increased during pregnancy, possibly causing increased renal iodide clearance and a decreased of circulating plasma iodine (Glinoer, 1997). Thus, even though the foetal thyroid can trap iodide by about 12 week of gestation (Fisher and Klein, 1981), high concentrations of maternal perchlorate may potentially decrease thyroidal iodine available to the foetus by inhibiting placental NIS (Leung et al. 2010). Consequences of TH deficiency depend on the developmental timing of the deficiency (Zoeller and Rovet, 2004). For instance, if the TH deficiency occurs during early pregnancy, offspring show visual attention, visual processing and gross motor skills deficits, while if it occurs later, offspring may show subnormal visual and visuospatial skills, along with slower response speeds and motor deficits. If TH insufficiency occurs after birth, language and memory skills are most predominantly affected (Zoeller and Rovet, 2004). There are limited data regarding low-level environmental perchlorate exposure and maternal thyroid function during pregnancy. A Chilean study found no increases in TSH or decreases in free thyroxine or urinary iodine concentrations in pregnant women living in three areas (all of which had more than adequate mean urinary iodine levels) with long-term environmental perchlorate exposure (Téllez Téllez et al. 2005). A follow-up analysis of this cohort also confirmed the lack of association between individual urinary iodide or perchlorate concentrations and thyroid function in the pregnant women (Gibbs and Van Landingham, 2008). Studies of large cohorts of first-trimester pregnant women from the U.S., Europe and Argentina found that environmental perchlorate exposure did not affect maternal thyroid function (Pearce et al. 2009).

Many studies have shown inhibition of radioactive iodide uptake by using different cell models and assays. However, there have been identified only few specific NIS inhibitors up to date, while all the others are thought to act through different inhibitory mechanisms. Monovalent anions, others than iodide, are also transported by NIS but Nitrate (NO3-), thiocyanate (SCN-), perchlorate (ClO-4), dysidenin and aryltrifluoroborates are of particular dietary and environmental importance (Jones et al., 1996; Tonacchera et al., 2004; De Groef et al., 2006). Recent revision of the affinity constant for perchlorate binding to the NIS symporter based on in vitro and human in vivo data, performed by refitting published in vitro data, in which perchlorate-induced inhibition of iodide uptake via the NIS was measured, yielding a Michaelis-Menten kinetic constant (Km) of 1.5 μm, showed that a 60% lower value for the Km, equal to 0.59 μm. Substituting this value into the PBPK model for an average adult human significantly improved model agreement with the human RAIU data for exposures <100 μg kg-1 day-1 (Schlosser PM, 2016). There are many studies showing the effect of inhibition of NIS on thyroidal iodide uptake: - Cianchetta et al., 2010 For this study the rat FRTL5 thyroid cell line endogenously expressing NIS, and the monkey kidney fibroblast-like cells (COS-7) transfected with hNIS were used. NIS functionality was assessed with the use of the Yellow Fluorescent Protein (YFP) variant YFP-H148Q/I152L, a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Decrease of YFP-H148Q/I152L fluorescence in FRTL-5 cells occurs as a result of NIS-mediated uptake and binding to the intracellular fluorochrome (Rhoden et al., 2007). The biosensor was used to compare the kinetics of iodide and perchlorate transport by NIS, and to assess the ability of perchlorate to inhibit iodide transport. Additionally, perchlorate was shown to inhibit NIS function (competitive inhibition) by preventing iodide-induced changes in fluorescence of FRTL5 cells. Perchlorate caused a concentration-dependent inhibition of iodide uptake in the initial influx rate (IC50=1.6μM) and in the intracellular concentration of iodide (IC50=1.1μM). Also, both perchlorate and iodide (1–1000 μM) induced concentration-dependent decreases in YFP-H148Q/I152L fluorescence in COS-7 cells expressing hNIS, but had no effect (< 2%) in COS-7 cells lacking hNIS. Additionally, perchlorate induced a significantly smaller decrease in fluorescence (10.6% at 1 mM) than iodide (31.8% at 1 mM iodide). Thus, it was confirmed that the reduction of fluorescence was due to NIS-mediated anion transport into the cells, excluding non-specific effects. - Tonacchera et al., 2004 Chinese hamster ovary (CHO) cell line had been stably transfected with human NIS and the measurement of iodide uptake was performed with the use of radioactive iodide uptake (RAIU) method. It was shown that the inhibition of iodide uptake was dose-dependent when using the known NIS inhibitors (ClO-4, NO3-, SCN-). Additionally, unlabeled I- (non 125I) was used to investigate the inhibition level of radioiodide uptake and to compare it with the potency of the other monoions, which are known NIS inhibitors. The IC50 values for the studied monoions were the following: ClO4-: IC50 was 1.22 μΜ; SCN-: IC50 was 18.7 μΜ; NO3-: IC50 was 293 μΜ; I-: IC50 was 36.6 μΜ. Finally, the present study investigated the joint effects of simultaneous exposure to multiple RAIU inhibitors, by generating multiple dose-response curves in the presence of fixed concentrations of inhibitors. The results of those experiments indicated a competition between the four anions with similar size for access to the binding sites of the NIS. The prediction model developed in this study, actually suggests that thyroidal iodide uptake is approximately proportional to iodide nutrition for any fixed inhibitor concentration, answering to the question whether dietary iodide can modulate the inhibitory effects of the known environmental goitrogens. - Waltz et al., 2010 Measurement of iodide uptake was performed with a non-radioactive method. By using the rat thyroid low –serum 5 (FRTL5) cells, which endogenously express NIS, a spectrophotometric assay was developed and the iodide accumulation was determined based on the catalytic reduction of yellow cerium to colorless cerium in the presence of arsenious acid (Sandell-Kolthoff reaction). A dose-dependent inhibition of iodide uptake was shown. The IC50 values for the studied compounds were the following: Sodium perchlorate (NaClO4): IC50 was 0.1 μΜ Sodium thiocyanate (NaSCN): IC50 was 12 μΜ Sodium nitrate (NaNO3): IC50 was 800 μΜ Sodium Tetrafluoroborate (NaBF4): IC50 was 1.2 μΜ - Lecat-Guillet et al., 2007; 2008a A fully automated radioiodide uptake assay was developed and some known NIS inhibitors were tested. A dose-dependent inhibition of iodide uptake was shown. The IC50 values for the studied compounds were the following: Sodium perchlorate (NaClO4): IC50 was 1 μΜ; Sodium thiocyanate (NaSCN): IC50 was 14 μΜ; Sodium nitrate (NaNO3): IC50 was 250 μΜ; Sodium Tetrafluoroborate (NaBF4): IC50 was 0.75 μΜ.  Additionally, a library of 17020 compounds was screened for the identification of new human NIS inhibitors. The identification was based on the magnitude of changes in iodide uptake using Human Embryonic Kidney 293 (HEK293) cells, stably transfected with the hNIS. The same experiments and with similar results were also performed in rat thyroid derived cells (FRTL5), which endogenously express NIS. Compounds that inhibited iodide uptake in a time-dependent manner were considered to act through direct NIS inhibition. In contrast, those compounds that had a delayed effect on iodide uptake were thought to act through a sodium gradient disruption system resulting in indirect inhibition of iodide transport. Perchlorate was used as a positive control in these experiments and, as expected, it blocked iodide uptake immediately and totally throughout the experiment. Dysidenin was also used as a control and the IC50 value identified was 2 μΜ. All the compounds that were used for these experiments were small drug-like molecules that have not been detected in the environment and they were named as ITBs (Iodide Transport Blockers). - Lecat-Guillet et al., 2008b With the same fully automated radioiodide uptake assay, as described above, new NIS inhibitors were also identified. The organotrifluoroborate (BF3−) was found to inhibit iodide uptake with an IC50 value of 0.4 μM using rat-derived thyroid cells (FRTL5). The biological activity is rationalized by the presence of the ion BF3− as a minimal binding motif for substrate recognition at the iodide binding site. - Lindenthal et al., 2009 With the use of a patch-clamp technique an analysis of the NIS inhibitors identified by Lecat-Guillet et al., 2008 (named ITB-1 to ITB-10 for "Iodide Transport Blockers") was evaluated in Xenopus oocytes expressing NIS to further assess the inhibitory effect of those molecules specifically on NIS activity. Four of those molecules (ITB-3, ITB-9, ITB-5 and ITB-4) were identified as the most potent, non-competitive NIS inhibitors. The effects of dysidenin were also analyzed with the same technique, as it had been reported to be a specific inhibitor of NIS (Vroye et al., 1998). It was found that dysidenin (50 μM) induced a rapid and reversible inhibition of the iodide (about 40%) of induced current in mNIS-expressing oocytes, but did not evoke any currents in the absence of iodide, suggesting that this effect was due to the inhibition of NIS activity. - Greer et al., 2002 In human studies, potassium perchlorate was used to predict inhibition of thyroidal iodide uptake by applying the RAIU method. Greer et al., tested body weight adjusted doses of potassium perchlorate and an assessment of RAIU uptake was performed on day 2 and day 14 of treatment and 24 h following treatment termination (on day 15). The NOEL value for inhibition of thyroidal uptake was 0.007 mg/kg-day, while the true NEL value was estimated to be 0.0052 and 0.0064 mg/kg-day. According to the dose-response inhibition of iodide uptake the maximum percentage of iodide inhibition at the doses of 0.0052 and 0.0064 mg/kg-day is 8.3-9.5%, which is physiologically insignificant for a person with dietary sufficient iodine intake. - Wen et al., 2016 By using human MCF-7 cells, a breast adenocarcinoma cell line, which express inducible NIS in the presence of all-trans retinoic acid (ATRA) it has been shown that inhibition of sterol regulatory element-binding proteins (SREBP) maturation by treatment with 25-hydroxycholesterol (5 µM) for 48 hr reduced ATRA (1 µM)-induced mRNA concentration of NIS and decreased iodide uptake by approximately 20%. This study showed for the first time that the NIS gene and iodide uptake are regulated by SREBP in cultured human mammary epithelial cells. - Arriagada et al. 2015 This study showed that 2 hr or 5 hr exposure to excess I- (100 μM) respectively in FRTL-5 cells and in ex-vivo rat thyroid gland (removed after single in vivo i.p. injection of 100 μg of I− in 500 μL of distilled water, and analysis of 125I thyroid uptake), induced inhibition of I- uptake through the NIS (~ 30% uptake inhibition after 5 hr in vivo), a process known as the Wolff-Chaikoff effect, which was not associated with a decrease of NIS expression or a change in NIS localization. Incubation of FRTL-5 cells with excess I- for 2 hr increased hydrogen peroxide generation. Also incubation with hydrogen peroxide (100 μM) decreased NIS-mediated I- transport, effect that was reverted by ROS scavengers.

The thyroid system is quite complex and therefore some inconsistent results have been produced by recent studies. For example, it has been observed in healthy volunteers that a 6-month exposure to perchlorate at doses up to 3 mg/d (low doses) had no effect on thyroid function, including inhibition of thyroid iodide uptake as well as serum levels of thyroid hormones, TSH, and Tg (Braverman et al., 2006). However, this study was limited by the small sample size and is obviously underpowered. The review by Charnley (2008) examines a number of studies where association between perchlorate environmental (low) exposure and thyroid effects were analysed and many inconsistent conclusions have been drawn. For instance, no correlations were found between TH serum levels and urinary iodine concentrations among women exposed to perchlorate participating in the 2000-2001 National Health and Nutrition Examination Survey (NHANES). Available evidence does not support a causal relationship between changes in TH levels and current environmental levels of perchlorate exposure, but does support the conclusion that the US Environmental Protection Agency's reference dose (RfD) for perchlorate is conservatively health-protective. However, potential perchlorate risks are unlikely to be distinguishable from the ubiquitous background of naturally occurring substances present at much higher exposures that can affect the thyroid via the same biological mode of action as perchlorate, such as nitrate and thiocyanate. Therefore, risk management approaches that account for both aggregate and cumulative exposures and that consider the larger public health context in which exposures are occurring are desirable. Additionally, a more comprehensive study by Pearce et al. (2010) conducted during 2002-2006 on 22,000 women at less than 16-week gestation showed that while low-level perchlorate exposure was ubiquitous in these women (with a median urinary perchlorate concentration of 5 µg/liter in the Turin cohort and 2 µg/liter in the Cardiff cohort), no associations between urine perchlorate concentrations and serum TSH or free T4 in the individual euthyroid or hypothyroid/hypothyroxinemic cohorts were found. The data assessing the effect of maternal perchlorate exposure in neonates and children and thyroid function remain unclear (Leung et al., 2010). Decreased iodine intake can decrease TH production, and therefore exposure to perchlorate might be particularly detrimental in iodine-deficient individuals (Leung et al. 2010). Moreover, biologically based dose-response modeling of the relationships among iodide status (e.g., dietary iodine levels), perchlorate dose, and TH production in pregnant women has shown that iodide intake has a profound effect on the likelihood that exposure to goitrogens will produce hypothyroxinemia (Lewandowski et al. 2015). Consequences of TH deficiency depend on the developmental timing of the deficiency (Zoeller and Rovet, 2004). For instance, if the TH deficiency occurs during early pregnancy, offspring show problems in visual attention, visual processing and gross motor skills, while if it occurs later, offspring may show subnormal visual and visuospatial skills, slower response speeds and motor deficits. If TH insufficiency occurs after birth, language and memory skills are most predominantly affected (Zoeller and Rovet, 2004). Altogether these studies indicate that factors, such as age, gender, developmental stage, and iodide status can affect the impact of perchlorate and other NIS inhibitors. All these variables should be taken into account to explain possible inconsistencies in study findings.

For this relationship there is not enough data linking quantitatively the inhibition of NIS with the amount of thyroidal uptake. The NIS inhibition is possible to be directly measured by using the fact that the simultaneous transport of 2 Na+ and 1 I- generates a current, which could be easily measured with electrophysiological methods (Eskandari et al., 1997) or with patch clamp techniques (Van Sande et al., 2003). However, the exact stoichiometry of the molecules that are transferred is not yet known, meaning that in some cases it cannot be detected. For example, perchlorate does not cause depolarization of the cellular membrane, as it is thought to be transferred in 1 to 1 stoichiometry with the Na+ (Van Sande et al., 2003). However, I- uptake can also be measured in vivo, as shown in rats i.p. injected with 100 μg of I− in 500 μL of distilled water (known to cause an inhibition of NIS- mediated I- transport), followed by analysis of radioactive 125I thyroid uptake (Arriagada et al. 2015). Further studies are needed to support quantitative evaluation of this KER.

High Mixed

High

During brain development

High High High Moderate

Empirical evidence comes from in vitro works using rat follicular cells (Cianchetta et al., 2010; Waltz et al., 2010; Lecat-Guillet et al., 2007; 2008; Lecat-Guillet et al., 2008b), human in vitro cell models (Wen et al., 2016) and in vivo data (Arriagada et al. 2015), as well as Xenopus oocytes (Lindenthal et al., 2009) and Zebrafish (Thienpont et al., 2011).

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eaf34410> AOPWiki 2016-11-29T18:41:34

2018-05-29T07:24:34

<upstream-id>21e6c233-1d52-42e8-ac24-8dc1575f8db3</upstream-id> <downstream-id>9a9fc6b2-e4c4-4661-80e7-71cfc4b83349</downstream-id> Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are synthesized in the thyroid gland in the presence of functional NIS and thyroid peroxidase (TPO) as iodinated thyroglobulin (Tg), and stored in the colloid of thyroid follicles. NIS is a membrane bound glycoprotein whose main physiological function is to transport one iodide ion along with two sodium ions across the basolateral membrane of thyroid follicular cells. Extensive studies on NIS protein have identified 14 different mutations and each one of them is related to Iodine Transport Deficiencies (ITD) (Spitzweg and Morris, 2010). Once inside the follicular cells, the iodide diffuses to the apical membrane, where it is metabolically oxidized through the action of TPO to iodinium (I+), which in turn iodinates tyrosine residues of the Tg proteins in the follicle colloid. Therefore, NIS is essential for the synthesis of thyroid hormones (T3 and T4). TPO is a heme-containing apical membrane protein within the follicular lumen of thyrocytes that acts as the enzymatic catalyst for TH synthesis (Taurog, 2005). Propylthiouracil (PTU) and methimazole (MMI), are thioureylene drugs that are known to inhibit the ability of TPO to: a) activate iodine and transfer it to thyroglobulin (Tg) (Davidson et al., 1978) and, b) couple thyroglobulin (Tg)-bound iodotyrosyls to produce Tg-bound T3 and T4 (Taurog, 2005). PTU and MMI have been found to decrease also the expression of NIS mRNA and consequently iodide accumulation, as shown in FRTL-5 cells (Spitzweg et al. 1999). Other compounds, such as triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and bisphenol A (BPA) have been reported to decrease thyroid hormone (TH) levels by inducing an inhibition of NIS-mediated iodide uptake and altering the expression of genes involved in TH synthesis in rat thyroid follicular FRTL-5 cells, and on the activity of thyroid peroxidase (TPO), using rat thyroid microsomes (Wu Y et al. 2016). Perchlorate, thiocyanate, nitrate, and iodide, which are competitive inhibitors of iodide uptake, have been shown to inhibit radioactive iodide uptake by NIS (Tonacchera et al. 2004), consequentially resulting in inhibition of TH synthesis. In particular, perchlorate blocks iodide uptake into the thyroid through NIS inhibition and decreases the production of TH (Steinmaus, 2016a). More recent evidence also suggests that young children, pregnant women, foetuses, and people co-exposed to similarly acting agents may be especially susceptible to perchlorate-induced toxicity (Steinmaus et al., 2016b). Concern about environmental perchlorate exposure is focused on its inhibition of iodide uptake into the thyroid (MIE). Decreased iodine intake may decrease thyroid hormone production. Perchlorate exposure, therefore, might be particularly detrimental in iodine-deficient individuals. Median urinary iodine levels are used instead and reflect dietary iodine sufficiency across populations (International Council for the Control of Iodine Deficiency Disorders (ICCIDD); available from: <a href="http://www.iccidd.org">www.iccidd.org</a>). According to ICCIDD report Iodine deficiency continues to be an important global public health issue, with an estimated 2.2 million people (38% of the world's population) living in iodine-deficient areas. In 1990, the United Nations World Summit for Children set forth the goal of eliminating iodine deficiency worldwide (UNICEF World Summit for Children. Available from: <a href="http://www.unicef.org/wsc/declare.htm">http://www.unicef.org/wsc/declare.htm</a>; 1990).  Considerable progress has been achieved by programmes of universal salt iodisation (USI) in various countries, in line with the recommendations of the World Health Organization (WHO) (WHO, UNICEF, ICCIDD. A guide for programme managers. World Health Organization; Geneva: 2007. Assessment of the iodine deficiency disorders and monitoring their elimination.WHO/NHD/01.1). However, many countries remain iodine deficient (de Benoist et al., 2013; Lazarus and Delange, 2004). In the U.S., data from large population studies have shown that median urinary iodine levels decreased by approximately 50% between the early 1970s and the early 1990s, although the population overall remained iodine sufficient (Hollowell et al., 1998). Subsequent studies have shown that this decrease has stabilised (Caldwell et al., 2005). The WHO still considers iodine deficiency, which leads to hypothyroidism, the single most important preventable cause of brain damage worldwide (WHO/UNICEF/ICCIDD, 2007). The most vulnerable groups are pregnant and lactating women and their developing fetuses and neonates, given the crucial importance of iodine to ensure adequate levels of thyroid hormones for brain maturation. Iodine deficiency in pregnancy is a prevailing problem not only in developing countries, but also in western industrialized nations and other countries classified as free of iodine deficiency, and solution may be found in dietary changes (Moog et al., 2017).

The association between these two KEs is strong, and supported by in vitro, in vivo and epidemiological studies. Blocking iodide uptake into the thyroid follicular cells as a consequence of NIS inhibition or functional impairment, leads to reduced TH synthesis. Compounds that have been shown to inhibit NIS function (e.g., perchlorate, thiocyanate, nitrate, and iodide), has also been proven to decrease TH synthesis by inducing a downregulation of TPO gene expression and/or increase of TSH level, which are both indicative of a reduce TH biosynthesis. TSH receptor controls transcription and posttranslational modification of NIS (Dai et al., 1996). Stimulation of TSH receptor increases T3 and T4 production and secretion (Szkudlinski et al., 2002). NIS gene expression is suppressed by growth factors such as IGF-1 and TGF-β (the latter is induced by the BRAF-V600E oncogene), which prevent NIS to localize in the basolateral membrane (Riesco-Eizaguirre et al., 2009). The BRAF-V600E oncogene is also associated with downregulation TSH receptor (Kleiman et al. 2013). Altogether these studies support the association between NIS inhibition-induced decreased iodide uptake (KE up) and reduced TH synthesis (KE down).

Several in vitro and epidemiological studies have shown that iodide uptake blockade occurring as a consequence of NIS (and TPO) inhibition leads to reduced TH synthesis: - Spitzweg et al., 1999: In this in vitro study, a 48 hr treatment of FRTL-5 cells with MMI (100 µM), PTU (100 µM), and potassium iodide (40 µM) induced ~ 50% decrease of NIS mRNA steady-state levels. Incubation with MMI and PTU resulted in a 20% and 25% decrease of iodide accumulation, respectively, whereas potassium iodide suppressed iodide accumulation by approximately 50%. - Wu Y et al., 2016: This in vitro study showed that triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and bisphenol A (BPA) induced a concentration-dependent inhibition of NIS-mediated iodide uptake. Moreover,  triclosan or triclocarban did not affect the expression of genes involved in TH synthesis (Slc5a5, TPO, and Tgo) or thyroid transcription factors (Pax8, Foxe1, and Nkx2-1), BDE-47 decreased the level of TPO, while BPA altered the expression of all six genes, as shown in rat thyroid follicular FRTL-5 cells. At the same time, triclosan and triclocarban also inhibited the activity of TPO at 166 and >300 μM, respectively.  - Steinmaus et al., 2016b: In 1,880 pregnant women from San Diego County, California, during 2000–2003, it has been found that the presence of high level of perchlorate, thiocyanate, nitrate, and iodide in water supply induced a decrease of total thyroxine (T4) [regression coefficient (β) = –0.70; 95% CI: –1.06, –0.34], a decrease of free thyroxine (fT4) (β = –0.053; 95% CI: –0.092, –0.013), and an increase of thyroid-stimulating hormone (TSH), all indicators of reduced TH synthesis. - Horton et al., 2015: in this study TSH levels measured in blood samples of 284 pregnant women at 12 (± 2.8) weeks gestation were found to positively correlate with the levels of urinary concentrations of perchlorate, nitrate and thiocyanate (NIS inhibitors), but perchlorate had the largest weight in the index, indicating the largest contribution to the weighted quantile sum regression. This indicates a perchlorate-dependent alteration of maternal thyroid function, through NIS inhibition. - Brechner et al., 2000: Median newborn TSH levels in a city where drinking water supply was perchlorate-contaminated (from the Colorado River below Lake Mead) were significantly higher than those in a city totally supplied with non-perchlorate-contaminated drinking water, even after adjusting for factors known or suspected to elevate newborn TSH levels. - Charatcharoenwitthaya et al. 2014: this cross-sectional epidemiological study conducted in 200 pregnant Thai women with a gestational age of 14 weeks or less, showed that low-level exposure to perchlorate (i.e., 1.9 μg/L of urinary perchlorate) was positively associated with TSH and negatively associated with free T4 using multivariate analyses in first-trimester pregnant women. Low thiocyanate urinary levels (510.5 μg/L) were also positively associated with TSH in a subgroup of pregnant women with low iodine excretion (less than 100 μg/L). Several other studies have proven that NIS inhibitors lead to a decrease of thyroidal iodide uptake (Jones et al., 1996; Tonacchera et al., 2004; De Groef et al., 2006; Waltz et al., 2010), leading to a reduction of TH synthesis.

Some studies have highlighted contradictory results in relation to response to chemicals. For instance, PTU and MMI have been shown to inhibit the activity of TPO in rats (Davidson et al., 1978), while inducing an increase of cellular TPO activity and TPO mRNA in cultured porcine thyroid follicles (Sugawara et al., 1999). PTU was also found to increase NIS gene expression, and the accumulation of 125I, as shown in in rat thyroid FRTL-5 cells, while MMI had no effect (Sue et al., 2012). Moreover, despite the well described effects of perchlorate, thiocyanate, nitrate, and iodide on iodide uptake into the thyroid, occupational and clinical dosing studies have not identified clear adverse effects, particularly in the case of perchlorate (Tarone et al. 2010). For instance, a longitudinal epidemiologic Chilean study found that there were no increases of thyroglobulin (Tg) or thyrotropin (TSH) levels, and no decreases of free T4 levels among either women during early pregnancy, late pregnancy, or the neonates at birth related to perchlorate in drinking water, suggesting that perchlorate in drinking water at 114 microg/L did not cause changes in neonatal thyroid function or fetal growth retardation (Téllez Téllez et al., 2005). Similarly, no associations between urine perchlorate concentrations and serum TSH or free T4 were found in individual euthyroid or hypothyroid/hypothyroxinemic cohorts of 261 hypothyroid/hypothyroxinemic and 526 euthyroid women from Turin and 374 hypothyroid/hypothyroxinemic and 480 euthyroid women from Cardiff (Pearce et al., 2010), suggesting that log perchlorate may not be a predictor of serum free T4 or TSH. However, it should be considered that these studies may be limited by short study durations, and the inclusion of mostly healthy adults (Steinmaus, 2016b). Charnley's (2008) review examines several studies pointing out a number of inconsistent conclusions regarding link between TH serum levels, urinary iodine concentrations, and environmental perchlorate exposure (Charnley et al. 2008). For instance, no correlations were found between TH serum levels and urinary iodine concentrations among women exposed to perchlorate participating in the 2000-2001 National Health and Nutrition Examination Survey (NHANES). Available evidence does not support a causal relationship between changes in TH levels and current environmental levels of perchlorate exposure, but does support the conclusion that the US EPA's reference dose (RfD) for perchlorate is conservatively health-protective. However, potential perchlorate risks are unlikely to be distinguishable from the ubiquitous background of naturally occurring substances present at much higher exposures that can affect the thyroid via the same biological mode of action as perchlorate, such as nitrate and thiocyanate. Therefore, risk management approaches that account for both aggregate and cumulative exposures and that consider the larger public health context in which exposures are occurring are desirable. In a cross-sectional analysis, McMullen et al. (2017) evaluated the exposure to perchlorate, thiocyanate, and nitrate in 3151 participants aged 12 to 80, to assess whether sensitivity  to perchlorate, thiocyanate, and nitrate (NIS inhibitors) could be a factor of age and sex. These results indicate that adolescent boys and girls represent the most vulnerable subpopulations to NIS symporter inhibitors. Therefore, discrepancies in results described in epidemiological studies may be due to difference in age of study participants.  Apart from age, relative source contribution of perchlorate exposure plays an important role in determining a significant reduction of serum TH levels. For instance, Lumen and George (2017) showed that there was no significant difference in geometric mean estimates of free T4 when perchlorate exposure from food only was compared to no perchlorate exposure in pregnant women. The reduction in maternal free T4 levels reached statistical significance when an added contribution from drinking water was assumed in addition to the 90th percentile of food intake for pregnant women. In particular, a daily intake of 0.45- 0.50μg/kg/day of perchlorate was necessary to produce results that were significantly different than those obtained from no perchlorate exposure. The authors comment that 'these modelling results can explain why findings from observational studies present inconsistent outcomes regarding the relationship between perchlorate exposure and thyroid hormone levels'."

In vitro and in vivo studies have specifically reported data supporting quantitative understanding of this KER. - Gilbert et al., 2011: This in vivo study examined the relationship between graded levels of iodine (ID) in rats and serum thyroid hormones levels, thyroid iodine content, and urinary iodide excretion. The study provided parametric and dose-response information for development of a quantitative model of the thyroid axis. Female Long Evans rats were fed casein-based diets containing varying iodine (I) concentrations for 8 weeks. Diets were created by adding 975, 200, 125, 25, or 0 μg/kg I to the base diet (~25 μg I/kg chow) to produce 5 nominal I levels, ranging from excess (basal+added I, Treatment 1: 1000 μg I/kg chow) to deficient (Treatment 5: 25 μg I/kg chow). Food intake and body weight were monitored throughout and on 2 consecutive days each week over the 8-week exposure period, animals were placed in metabolism cages to capture urine. Food, water intake, and body weight gain did not differ among treatment groups. Serum T4 was dose-dependently reduced relative to Treatment 1 with significant declines (19 and 48%) at the two lowest I groups, and no significant changes in serum T3 or TSH were detected. Increases in thyroid weight and decreases in thyroidal and urinary iodide content were observed as a function of decreasing ID in the diet. Data were compared with predictions from a published biologically based dose-response (BBDR) model for ID. These results challenged existing models and provide essential information for development of quantitative BBDR models for ID during pregnancy and lactation. - Spitzweg et al., 1999:  this in vitro study showed that inhibition of TH synthesis (induced by TPO specific inhibitors) decreases the expression of NIS. A 48 hr treatment of FRTL-5 cells with the TPO specific inhibitors MMI (100 µM), PTU (100 µM), and potassium iodide (40 µM), induced a ~ 50% decrease of NIS RNA steady-state levels. Incubation with MMI and PTU resulted in a 20% and 25% decrease of iodide accumulation, respectively, whereas potassium iodide suppressed iodide accumulation by approximately 50%. - Wu F et al., 2012: An in vivo study found that high dose of NIS inhibitor perchlorate (520 mg/kg b.wt.) in Sprague-Dawley rats (28-day old) caused a decrease of Tg (~ 50% lower than control), and TPO (~ 45% lower than control) gene expression, indicative of reduced TH biosynthesis, together with a decrease of free T3 (~ 50% lower than control) and free T4 levels (~ 50% lower than control), and a remarkable increase of TSH levels (125% higher than control) (Wu F et al. 2012). Additional studies with quantitative data for this KER are also described in Empirical Support for Linkage. However, further studies are needed in order to drive global conclusions about the magnitude of iodide uptake inhibition required to impact TH synthesis.

High Mixed

High

During brain development

High High Not Specified

Empirical evidence comes from in vivo studies in rats (Wu F et al., 2012; Davidson et al., 1978), in vitro studies using thyroid follicular rat cells (Spitzweg et al., 1999; Sue et al., 2012) and porcine thyroid follicles (Sugawara et al., 1999), and human epidemiological studies (Steinmaus et al., 2016b; Horton et al., 2015; Brechner et al., 2000)

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eafba740> AOPWiki 2016-11-29T18:41:35

2018-06-04T06:11:03

<upstream-id>9a9fc6b2-e4c4-4661-80e7-71cfc4b83349</upstream-id> <downstream-id>d4d962d7-b433-4111-b614-c759ed1bd785</downstream-id> Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are synthesized by NIS and TPO in the thyroid gland as iodinated thyroglobulin (Tg) and stored in the colloid of thyroid follicles across vertebrates. Secretion from the follicle into serum is a multi-step process. The first involves thyroid stimulating hormone (TSH) stimulation of the separation of the peptide linkage between Tg and TH. The next steps involve endocytosis of colloid, fusion of the endosome with the basolateral membrane of the thyrocyte, and finally release of TH into blood. More detailed descriptions of this process can be found in reviews by Braverman and Utiger (2012) and Zoeller et al. (2007).

The weight of evidence linking these two KEs of decreased TH synthesis and decreased T4 in serum is strong. It is commonly accepted dogma that decreased synthesis in the thyroid gland will result in decreased circulating TH (serum T4).

The biological relationship between two KEs in this KER is well understood and documented fact within the scientific community.

It is widely accepted that TPO inhibition leads to declines in serum T4 levels in adult mammals. This is due to the fact that the sole source for circulating T4 derives from hormone synthesis in the thyroid gland. Indeed, it has been known for decades that insufficient dietary iodine will lead to decreased serum TH concentrations due to inadequate synthesis. Strong qualitative and quantitative relationships exist between reduced TH synthesis and reduced serum T4 (Ekerot et al., 2013; Degon et al., 2008; Cooper et al., 1982; 1983; Leonard et al., 2016; Zoeller and Tan, 2007).  There is more limited evidence supporting the relationship between decreased TH synthesis and lowered circulating hormone levels during development.  Lu and Anderson (1994) followed the time course of TH synthesis, measured as thyroxine secretion rate, in non-treated pregnant rats and correlated it with serum T4 levels. Modeling of TH in the rat fetus demonstrates the quantitative relationship between TH synthesis and serum T4 concentrations (Hassan et al., 2017, 2020; Handa et al., 2021). Furthermore, a wide variety of drugs and chemicals that inhibit TPO are known to result in decreased release of TH from the thyroid gland, as well as decreased circulating TH concentrations. This is evidenced by a very large number of studies that employed a wide variety of techniques, including thyroid gland explant cultures, tracing organification of 131-I and in vivo treatment of a variety of animal species with known TPO inhibitors (King and May,1984; Atterwill et al., 1990; Brown et al., 1986; Brucker-Davis, 1998; Haselman et al., 2020; Hornung et al., 2010; Hurley et al., 1998; Kohrle, 2008; Tietge et al., 2010). Additionally, evidence is available from studies investigating responses to TPO inhibitors in fish. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. Several other studies have also shown that chemically induced Inhibition of TPO results in reduced TH synthesis in zebrafish (Van der Ven et al., 2006; Raldua and Babin, 2009; Liu et al., 2011; Thienpont et al., 2011; Rehberger et al., 2018). 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. Temporal Evidence: In mammals, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005).  There are currently no studies that measured both TPO synthesis and TH production during development. However, the impact of decreased TH synthesis on serum hormones is similar across all ages in mammals. Good evidence for the temporal relationship comes from thyroid system modeling of the impacts of iodine deficiency and NIS inhibition (e.g., Degon et al., 2008; Fisher et al., 2013). In addition, recovery experiments have demonstrated that serum thyroid hormones recovered in athyroid mice following grafting of in-vitro derived follicles (Antonica et al., 2012). In Xenopus, it has been shown that depression of TH synthesis in the thyroid gland precedes depression of circulating TH within 7 days of exposure during pro-metamorphosis (Haselman et al., 2020).    In oviparous fish such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal THs transferred to the eggs. Embryonic TH synthesis is activated later during embryo-larval development. (See Domain of applicability) Dose-response Evidence: Dose-response data is lacking from studies that include concurrent measures of both TH synthesis and serum TH concentrations. However, data is available demonstrating correlations between thyroidal TH and serum TH concentrations during gestation and lactation during development (Gilbert et al., 2013). This data was used to develop a rat quantitative biologically-based dose-response model for iodine deficiency (Fisher et al., 2013). In Xenopus, dose-responses were demonstrated in both thyroidal T4 and circulating T4 following exposure to three TPO inhibitors (Haselman et al., 2020).

There are no inconsistencies in this KER, but there are some uncertainties. The first uncertainty stems from the paucity of data for quantitative modeling of the relationship between the degree of synthesis decrease and resulting changes in circulating T4 concentrations. In addition, most of the data supporting this KER comes from inhibition of TPO, and there are a number of other processes (e.g., endocytosis, lysosomal fusion, basolateral fusion and release) that are not as well studied. For example, Kim et al. (2015) investigated the adverse effects of Triphenyl phosphate (TPP), a substance that disrupts the thyroid system. Therefore, Rat pituitary (GH3) and thyroid follicular cell lines (FRTL-5) were studied. In the GH3 cells, TPP led to an upregulation of the expression of important thyroid genes (tsh, tr alpha and tr beta) while T3, a positive control, downregulated the expression of these genes. In FRTL-5 cells, the expression of nis and tpo genes was significantly upregulated, suggesting that TPP stimulates TH synthesis in the thyroid gland. In zebrafish larvae at the age of 7 days post-fertilisation (dpf), TPP exposure resulted in a significant increase in T3 and T4 concentrations and the expression of genes involved in thyroid hormone synthesis. Exposure to TPP also significantly regulated the expression of genes involved in the metabolism (dio1), transport (ttr) and excretion (ugt1ab) of THs. The down-regulation of the crh and tsh genes in the zebrafish larvae suggests the activation of a central regulatory feedback mechanism that is triggered by the increased T3 levels in vivo. Taken together, these observations indicate that TPP increases TH concentrations in early life stages of zebrafish by disrupting central regulatory and hormone synthesis pathways.

During Xenopus metamorphosis, circulating T4 steadily increases to peak levels at metamorphic climax. Therefore, during Xenopus metamorphosis, this KER is operable at an increased rate as compared to a system that is maintaining steady circulating T4 levels through homeostatic control. In this case, developmental status is a modulating factor for the rates and trajectories of these KEs.

In rats, Hassan et al. (2020) demonstrated in vitro: ex vivo correlations of TPO inhibition using PTU and MMI and constructed a quantitative model relating level of TPO inhibition with changes in circulating T4 levels. They determined that 30% inhibition of TPO was sufficient to decrease circulating T4 levels by 20%. This is further supported by studies of Hassan et al. (2017) and Handa et al. (2021) In Xenopus, Haselman et al. (2020) collected temporal and dose-response data for both thyroidal and circulating T4 which showed strong qualitative concordance of the response-response relationship. A quantitative relationship exists there in, but is yet to be demonstrated mathematically in this species.

Fisher et al. (2013) published a quantitative biologically-based dose-response model for iodine deficiency in the rat. This model provides quantitative relationships for thyroidal T4 synthesis (iodine organification) and predictions of serum T4 concentrations in developing rats. There are other computational models that include thyroid hormone synthesis. Ekerot et al. (2012) modeled TPO, T3, T4 and TSH in dogs and humans based on exposure to myeloperoxidase inhibitors that also inhibit TPO.  This model was recently adapted for rats(Leonard et al., 2016) and Hassan et al (2017) have extended it to include the pregnant rat dam in response to TPO inhibition induced by PTU. While the original model predicted serum TH and TSH levels as a function of oral dose, it was not used to explicitly predict the relationship between serum hormones and TPO inhibition, or TH synthesis. Leonard et al. (2016) recently incorporated TPO inhibition into the model. Degon et al (2008) developed a human thyroid model that includes TPO, but does not make quantitative prediction of organification changes due to inhibition of the TPO enzyme. Further empirical support for the response-response relationship has been demonstrated in the amphibian model, Xenopus laevis, exposed to TPO inhibitors during pro-metamorphosis (Haselman et al., 2020) wherein temporal profiles were measured for both thyroidal and circulating T4.

Given that the thyroid gland contains follicular lumen space filled with stored thyroglobulin/T4, complete inhibition of thyroid hormone synthesis at a given point in time will not result in an instantaneous decrease in circulating T4. The system will be capable of maintaining sufficient circulating T4 levels until the gland stores are depleted. The time it takes to deplete stored hormone will greatly depend on species, developmental status and numerous other factors. In Xenopus, Haselman et al. (2020) demonstrated an approximately 5 day difference between a significant decrease in thyroidal T4 preceding a significant decrease in circulating T4 while exposed to a potent TPO inhibitor (MMI) continuously during pro-metamorphosis.

This KER is entirely influenced by the feedback loop between circulating T4 originating from the thyroid gland and circulating TSH originating from the pituitary. Intermediate biochemical processes exist within the hypothalamus to affirm feedback and coordinately release TSH from the pituitary. However, quantitative representations of these feedback processes are limited to models discussed previously. In Xenopus, circulating levels of T4 increase through pro-metamorphosis indicating a "release" of feedback to allow circulating levels of T4 to increase and drive metamorphic changes (Sternberg et al., 2011). This provides evidence that homeostatic control of feedback can be developmentally dependent, and likely species dependent.

High Male High Female

High

All life stages

High High High High Low Low

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eb030e68> AOPWiki 2016-11-29T18:41:33

2022-10-10T08:56:38

<upstream-id>d4d962d7-b433-4111-b614-c759ed1bd785</upstream-id> <downstream-id>16bb9262-e427-4c83-8639-53d035e2616f</downstream-id> In mammals, thyroxine (T4) in brain tissue is derived almost entirely from the circulating pool of T4 in blood. Transfer of free T4 (and to a lesser extent, T3) from serum binding proteins (thyroid binding globulin (TBG), transthyretin (TTR) and albumin; see McLean et al., 2017, for a recent review) into the brain requires transport across the blood brain barrier (BBB) and /or indirect transport from the cerebral spinal fluid (CSF) into the brain through the blood-CSF-barrier.  The blood vessels in rodents and humans expresses the main T4 transporter, MCT8, (Roberts et al. 2008), as does the choroid plexus which also expresses TTR and secretes the protein into the CSF (Alshehri et al. 2015). T4 entering the brain through the BBB is taken up into astrocytes via cell membrane iodothyronine transporters (e.g., organic anion-transporting polypeptides OATP), monocarboxylate transporter 8 (MCT8) (Visser et al., 2011).  In astrocytes, T4 is then deiodinated by Type II deiodinase to triiodothyronine (T3) (St Germain and Galton, 1997), which is then transported via other iodothyronine transporters (MCT8) into neurons (Visser et al., 2011). While some circulating T3 may be taken up into brain tissue directly from blood (Dratman et al., 1991), the majority of neuronal T3 comes from deiodination of T4 in astrocytes. Decreases in circulating T4 will eventually result in decreased brain T3 tissue concentrations. It is also known that Type II deiodinase can be up-regulated in response to decreased T4 concentrations to maintain tissue concentrations of T3 (Pedraza et al., 2007; Lavado-Autric et al., 2013; Morse et al., 1986), except in tanycytes of the paraventricular nucleus (Fekete and Lechan, 2014).

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

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

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

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

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

Moderate Male Moderate Female

High

During brain development

Moderate

All life stages

Moderate Moderate Moderate

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

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eb0a49a8> AOPWiki 2016-11-29T18:41:33

2019-04-04T10:50:44

<upstream-id>16bb9262-e427-4c83-8639-53d035e2616f</upstream-id> <downstream-id>5bd91265-2d91-4c82-8328-002699bcaa10</downstream-id> Many cellular and biochemical effects of thyroid hormones (TH) are mediated through regulation of gene expression (Oppenheimer, 1983; Bernal, 2007).  Thyroxine (T4) is transferred from the serum to the brain (see KER: Thyroxine (T4) in Serum, Decreased leads to Thyroxine (T4) in Neuronal Tissue, Decreased), where it converted to triiodothyronine (T3), the level of which is highly controlled by deiodinases. T3 binds to thyroid receptors (TR) in the nucleus of neuronal and glial cells to control gene expression. It is generally accepted that the modulation of TR gene expression in the hippocampus, or any other brain region, must therefore depend on the presence of hormone in these tissues.

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

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

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

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

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

High Male High Female

High

During brain development

Moderate Moderate

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

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eb119848> AOPWiki 2016-11-29T18:41:35

2018-08-11T19:18:16

<upstream-id>5bd91265-2d91-4c82-8328-002699bcaa10</upstream-id> <downstream-id>816d2cad-6e54-4b34-bda6-b324c327e0fc</downstream-id> 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.

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.

The biological plausibility of this KER is rated as strong. 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.

Empirical support for this KER is rated as strong. 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). Temporal Evidence: The temporal nature of this KER is developmental (Seed et al., 2005). It is a well-recognized fact that there are critical developmental windows for disruption of 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: 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 (Bdnf) 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. Neuroregluin-2 (Nrg2) contributes to synaptogenesis of the granule cell layer of the hippocampus. In hippocampal slice cultures, inducible microRNA targeting strategies have demonstrated early suppression of Nrg2 (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 Nrg2 (Lee et al., 2015). 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). Dose-Response Evidence: 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.

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.

There are no data on the quantitative linkages between gene expression changes and altered hippocampal anatomy.

High Male High Female

Moderate

During brain development

Moderate High

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.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eb1a66f8> AOPWiki 2016-11-29T18:41:35

2018-08-11T19:05:29

<upstream-id>816d2cad-6e54-4b34-bda6-b324c327e0fc</upstream-id> <downstream-id>fb190e44-8688-42a2-ae07-d00e1d0d48e8</downstream-id> 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.

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.

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).

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. 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. 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). 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). 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). 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). 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). Temporal Evidence: 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. Dose-Response Evidence: 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.

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)

Information does not exist to develop quantitative relationships between the KEs in this KER. Papers that utilize knock-out and mutant models have not provided ‘dose-response’ information for anatomy-physiology relationships.

High Male High Female

High

During brain development

Moderate Moderate Not Specified

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.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a0452560> AOPWiki 2016-11-29T18:41:35

2018-08-11T19:21:12

<upstream-id>fb190e44-8688-42a2-ae07-d00e1d0d48e8</upstream-id> <downstream-id>56a0929f-2ed1-4cb4-a594-59d042f5e464</downstream-id> It is a well-accepted assertion that hippocampal synaptic integrity and plasticity are essential for spatial information processing in animals and spatial and episodic memory in humans (Burgess, 2002; Martin et al., 2000; Sweatt, 2016). A large number of studies with a variety of techniques and approaches have linked hippocampal functional deficits to decreased spatial ability, context learning, and fear learning. Study of human disease states and conditions where hippocampal function is impaired (i.e., brain trauma, Alzheimer’s disease, temporal lobe epilepsy, Down’s Syndrome), and imaging studies of hippocampal activation during memory challenge, makes itirrefutable that the hippocampus is essential for specific types of cognition abilities. Decades of animal research has reinforced this assertion. There are many forms of synaptic plasticity and numerous ways in which physiological function of neural circuits can be assessed. Similarly, there are many forms of learning and memory and multiple tasks and specifics associated with these tasks that vary from laboratory to laboratory. An emerging field of computational cognitive neuroscience lies at the intersection of   computational neuroscience, machine learning and neural network theory. These computational and theoretical frameworks support the participation of the hippocampal synaptic transmission and plasticity in learning and memory in animals and humans (for review see: Ashby and Helie, 2012).

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

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

Empirical support for this KER is strong. The requisite of hippocampal integrity to optimal visuo-spatial context learning (i.e., episodic memory) in humans and spatial learning in rodents is well documented. In vivo recording in conscious behaving animals has demonstrated activity-dependent neural changes taking place in the hippocampus during spatial learning (Gruart and Delgado-Garcia, 2007). Impairments in hippocampal function induced by drugs, chemicals, lesions, mutant or knock out models that cause changes in synaptic transmission, plasticity, and hippocampal network activity, are coincident with deficits in spatial and context-based fear learning (O’Keefe and Nadel, 1978; Bannerman et al., 2014; Lynch, 2004; Verret et al., 2012). Similarly, treatments found to enhance or facilitate hippocampal synaptic transmission and plasticity are associated with improved learning and memory (Deng et al., 2010; Novkovic et al., 2015; Andrade et al., 2015; Trivino-Paredes et al., 2016).  For example, n-methyl-d-aspartate (NMDA)-mediated glutamatergic synaptic transmission is essential for the induction of hippocampal synaptic plasticity in the form of LTP. Blockade of this form of plasticity by selective NMDA-receptors blockers impairs LTP and hippocampal tests of learning and memory (reviewed in Sweatt, 2016). Perturbation of hippocampal plasticity and impaired spatial learning have been reported in adult offspring following prenatal ethanol exposure (An and Zhang, 2015).  The fyn mutant mouse (fyn is a tyrosine kinase pathway) displays impairments in hippocampal synaptic transmission and plasticity, as well asspatial learning deficits (Grant et al., 1992). Brain-derived neurotrophic factor (BDNF) knock out animals exhibit synaptic plasticity deficits and learning impairments (Aarse et al., 2016; Panja and Bramham, 2014). In the Jacob/Nfsm model which also exhibits pronounced alterations in BDNF-mediated signaling, hippocampal synaptic transmission and plasticity impairments were accompanied by deficits in contextual fear conditioning and novel location recognition tasks (Spilker et al., 2016).  Finally, in rodent models of developmental TH insufficiency, impairments in hippocampal synaptic transmission and plasticity are coincident with deficits in learning tasks that require the hippocampus (Opazo et al., 2008; Gilbert and Sui, 2006, Gilbert, 2011, Gilbert et al., 2016). In humans, hippocampal physiology assessed using neuroimaging reveals activation of hippocampus upon engagement in spatial learning and episodic memory providing a direct linkage of these two specific KEs (Burgess, 2002). In fMRI studies of congenitally hypothyroid children, or children born to women with altered thyroid function during pregnancy, changes in hippocampal activity patterns during memory encoding and retention were observed and associated with memory impairments (Wheeler et al., 2012; 2015; Willoughby et al., 2013; 2014). Temporal Evidence:  The temporal nature of this KER is developmental (Seed et al., 2005). This has been demonstrated in multiple studies.  It is well-recognized that there are critical developmental windows for disruption of the functional development of the hippocampus and the integrity of this structure is essential for later development of spatial ability, context learning, and fear learning. A wealth of studies have shown correlation between hippocampal LTP and spatial learning performance, as well as the role of glutamatergic synaptic transmission and BDNF-mediated signaling pathways in these processes (Bramham, 2007; Andero et al., 2014; Morris et al., 1986; Sweatt, 2016; Migaud et al., 1998). Although studies on reversibility are rare, deficits in hippocampal synaptic transmission and plasticity in slices from BDNF knockout animals can be rescued with recombinant BDNF (Patterson et al., 1996). Dose-Response Evidence: Limited dose-response information is available.  Studies have investigated dose-dependency of both electrophysiological and behavioral impairments in animals suffering from developmental TH insufficiency (e.g., Gilbert and Sui, 2006; Gilbert, 2011; Gilbert et al., 2016).

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

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

High Male High Female

High

During brain development

High High High

The majority of data in support of this KER is from rodent models. The evolutionary conservation of the role of the hippocampus in spatial cognitive functions suggests, with some uncertainty, that this KER is also applicable to other mammalian species.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a0ba3b70> AOPWiki 2016-11-29T18:41:35

2018-08-11T19:24:44

<upstream-id>d4d962d7-b433-4111-b614-c759ed1bd785</upstream-id> <downstream-id>56a0929f-2ed1-4cb4-a594-59d042f5e464</downstream-id> Thyroid hormones (TH) are critical for normal development of the structure and function of the brain, including hippocampal development and cognitive function (Anderson et al., 2003; Bernal, 2007; Willoughby et al., 2014).   Brain concentrations of T4 are dependent on transfer of T4 from serum, through the vascular endothelia, into astrocytes.  In astrocytes, T4 is converted to T3 by deiodinase and subsequently transferred to neurons cellular membrane transporters. In the brain T3 controls transcription and translation of genes responsible for normal hippocampal structural and functional development. Clearly the brain circuitry controlling cognitive function is complex and is not solely accomplished by the functionality of the hippocampus. However, it is well documented that normal hippocampal structure and physiology are critical for the development of cognitive function. Thus, there is an indisputable indirect link between serum T4 and cognitive function.

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

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

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

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

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

High Male High Female

High

During brain development

High High High

There is a plethora of data supporting this KER in rats, mice, and humans.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb8a0e108e0> AOPWiki 2016-11-29T18:41:34

2018-08-11T19:44:38

Sodium Iodide Symporter (NIS) Inhibition and Subsequent Adverse Neurodevelopmental Outcomes in Mammals

NIS and Cognitive Dysfunction

Evgeniia Kazymova

Mary Gilbert, National Health and Environmental Effects Research Laboratory, US EPA, RTP, NC USA <gilbert.mary@epa.gov> Anna Price, European Commission Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy <anna.price@jrc.ec.europa.eu> Kevin Crofton, National Center for Computational Toxicology, US EPA, RTP, NC USA <crofton.kevin@epa.gov>

BY-SA

1.29

1.0

This AOP describes one adverse outcome that results from the inhibition of the sodium iodine symporter (NIS) during mammalian development. Inhibition of NIS, the molecular-initiating event (MIE), results in decreased iodine uptake, decreased thyroidal iodine content, subsequent decreased synthesis of thyroid hormones (THs), and reduction in circulating levels of THs. THs are essential for normal human brain development, both prenatally and postnatally. Therefore, chemicals that interfere with TH synthesis have the potential to cause TH insufficiency that may result in adverse neurodevelopmental effects in offspring. This AOP includes changes in brain TH concentrations and subsequent impacts on hippocampal development that lead to declines in cognitive spatial behavior. The weight of evidence for this AOP is strong. And, while there are currently computational quantitative models that can predict serum TH levels from NIS inhibition, there is currently a lack of quantitative understanding of the degree of serum TH disruption that leads to the adverse outcome.

Thyroid Disrupting Chemicals (TDCs) are defined as the xenobiotics that interfere with the thyroid axis with different outcomes for the organism. A very well-studied mechanism of action of the TDCs is the reduction of the circulating levels of THs by inhibiting hormone synthesis in the thyroid gland. For example, perchlorate is a very potent inhibitor of iodide uptake through the sodium/iodide symporter (Tonacchera et al., 2004). Perchlorate has been detected in human breast milk ranging from 1.4 to 92.2 mg μl–1 (10.5 μg l–1 mean) in 18 US states (Kirk et al. 2005), and 1.3 to 411 μg l–1 (9.1 μg l–1 median) in the Boston area, United States (Pearce et al. 2007). Perchlorate has also been detected in human colostrum of 46 women in the Boston area (from < 0.05 to 187.2 μmol l–1 (Leung et al. 2009)). The mechanism of perchlorate action is quite simple, as it is believed to be mediated only by the NIS inhibition (Dohan et al., 2007; Wolff, 1998). Additionally, thiocyanate and nitrate are two known inhibitors that have been found to reduce circulating TH levels (Blount et al., 2006; Steinhaus et al., 2007), but they are both less potent than perchlorate (Tonacchera et al., 2004). However, there are also contradictory results from other studies that showed no correlation between thyroid parameters and perchlorate levels in humans (Pearce et al., 2010; Amitai et al., 2007; Tellez et al., 2005). Co-occurrence of perchlorate, nitrate, and thiocyanate can alter thyroid function in pregnant women. Horton et al. (2015) have shown positive associations between the weighted sum of urinary concentrations of these three analytes and increased TSH, with perchlorate showing the largest weight in the index. Interestingly, De Groef et al. 2006 showed that nitrate and thiocyanate, acquired through drinking water or food, account for a much larger proportion of iodine uptake inhibition than perchlorate, suggesting that NIS inhibition and any potential downstream effect by perchlorate are highly dependent on the presence of other environmental NIS inhibitors and iodine intake itself (Leung et al., 2010). In particular, Tonacchera et al. (2004) showed that the relative potency of perchlorate to inhibit radioactive I− uptake by NIS is 15, 30 and 240 times that of thiocyanate, iodide, and nitrate respectively on a molar concentration basis. These data are in line with earlier studies in rats (Alexander and Wolff, 1996; Greer et al. 1966). Contradictory findings in these studies may therefore be a result of the confounding mixtures in the environment, masking the primary effect of perchlorate. Decreased iodine intake can decrease TH production, and therefore exposure to perchlorate might be particularly detrimental in iodine-deficient individuals (Leung et al. 2010). Moreover, biologically based dose-response modeling of the relationships among iodide status (e.g., dietary iodine levels), perchlorate dose, and TH production in pregnant women has shown that iodide intake has a profound effect on the likelihood that exposure to goitrogens will produce hypothyroxinemia (Lewandowski et al. 2015). During pregnancy TH requirements increase, particularly during the first trimester (Alexander et al. 2004; Leung et al. 2010), due to higher concentrations of thyroxine-binding globulin, placental T4 inner-ring deiodination leading to the inactive reverse T3 (rT3), and transfer of small amounts of T4 to the foetus (during the first trimester foetal thyroid function is absent). Moreover, glomerular filtration rate and clearance of proteins and other molecules are both increased during pregnancy, possibly causing increased renal iodide clearance and a decreased of circulating plasma iodine (Glinoer, 1997). Thus, even though the foetal thyroid can trap iodide by about 12 week of gestation (Fisher and Klein, 1981), high concentrations of maternal perchlorate may potentially decrease thyroidal iodine available to the foetus by inhibiting placental NIS (Leung et al. 2010). Consequences of TH deficiency depend on the developmental timing of the deficiency (Zoeller and Rovet, 2004). For instance, if the TH deficiency occurs during early pregnancy, offspring show visual attention, visual processing and gross motor skills deficits, while if it occurs later, offspring may show subnormal visual and visuospatial skills, along with slower response speeds and motor deficits. If TH insufficiency occurs after birth, language and memory skills are most predominantly affected (Zoeller and Rovet, 2004). Along this line, age and developmental stage are crucial in determining sensitivity to NIS inhibitors (e.g., perchlorate, thiocyanate, and nitrate). In this regard, McMullen et al. (2017) have shown that adolescent boys and girls, more than adults, represent vulnerable subpopulations to NIS symporter inhibitors. Altogether these studies indicate that age, gender, developmental stage, and dietary iodine levels can affect the impact of NIS inhibitors. Finally, ten more small simple-structured molecules were identified in a large screening study (Lecat-Guillet et al., 2008b) that could block iodide uptake by specifically disrupting NIS in a dose-dependent manner. These molecules were named Iodide Transport Blockers (ITBs). There are few organic molecules that lead to NIS inhibition but no direct interaction with NIS has been determined (Gerard et al., 1994; Kaminsky et al., 1991). Up to date, only dysidenin, a toxin isolated from the marine sponge Dysidea herbacea, has been reported to specifically inhibit NIS (Van Sande et al., 2003). Finally, the aryltrifluoroborates were found to inhibit iodide uptake with an IC50 value of 0.4 μM on rat-derived thyroid cells (Lecat-Guillet et al., 2008a). The biological activity is rationalized by the presence of the BF3− ion as a minimal binding motif for substrate recognition at the iodide binding site. It has been also shown that many anions, such as ClO3-, SCN-, NO3-, ReO4-, TcO4- and in a lower extent Br- and BF4-, are also acting as NIS substrates and they enter the cell by the same transporter mechanism (Van Sande et al., 2003). It has been also shown that ClO4- is transferred by NIS with high affinity and is considered as one of its most potent inhibitors (Dohan et al., 2007). Most recently, the aryltrifluoroborates were also shown to inhibit NIS function (Lecat-Guillet et al., 2008a). A library of 17,020 compounds was tested by a radioactive screening method with high specificity using transfected mammalian cells (Lecat-Guillet et al., 2008b; 2007) for NIS inhibition evaluation. Further studies with the most powerful inhibitors showed a high diversity in their structure and mode of action (Lindenthal et al., 2009).

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

adjacent

High

High adjacent

High

High adjacent

High

High adjacent

Low

High adjacent

Low

Moderate adjacent

Low

Moderate adjacent

Low

Moderate adjacent

Low

Moderate non-adjacent

Low

High

High Male High Female

High

Perinatal

High High

AOPWiki 2016-11-29T18:41:16

2023-09-25T16:26:51