SNAPSHOT

424: Inhibition, Na+/I- symporter (NIS)

Short Name: Inhibition, Na+/I- symporter (NIS)

AOPs Including This Key Event

Stressors

Evidence for Perturbation of this Molecular Initiating Event by Stressor

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). 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). 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.

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Apart from the human, functional NIS protein has been also identified in 3 other species, namely the rat (Dai et al., 1996), the mouse (Perron et al., 2001) and the pig (Selmi-Ruby et al., 2003). 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).

425: Decreased, Thyroidal iodide uptake

Short Name: Decreased, Thyroidal iodide uptake

AOPs Including This Key Event

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

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).

 

280: Decreased, Thyroxine (T4) in neuronal tissue

Short Name: Decreased, Thyroxine (T4) in neuronal tissue

AOPs Including This Key Event

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

THs are critical for normal brain development in most vertebrates, primarily documented empirically in mammalian species. 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).

381: Reduced, Release of BDNF

Short Name: Reduced, Release of BDNF

AOPs Including This Key Event

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

BDNF plays a critical role in normal brain development in most vertebrates, primarily documented empirically in mammalian species. Klein et al. (2011) examined blood, serum, plasma and brain-tissue and measured BDNF levels in three different mammalian species: rat, pig, and mouse, using an ELISA method (Aid et al., 2007), whereas Trajkovska et al. 2007 determined BDNF levels in human blood.

There is compelling data that demonstrates the role  of  BDNF  in brain development for many other taxa, including fish where it acts as neurotrophic factor in controlling cell proliferation (D'Angelo L et al., 2014; Heinrich and Pagtakhan, 2004) and  birds where BDNF influences development of the brain area that involved in the song control (Brenowitz 2013) and  the addition of new neurons to a cortical nucleus in adults . In the Xenopus visual system, BDNF acts as neurotrophic factor that mediates synaptic differentiation and maturation of the retinotectal circuit through cell autonomous TrkB signaling on retinal ganglion cells (Sanchez et al., 2006; Marshak et al., 2007).

385: Decreased, Synaptogenesis

Short Name: Decreased, Synaptogenesis

AOPs Including This Key Event

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

The mechanisms governing synapse formation is considered conserved among both vertebrates and invertebrates (Munno and Syed, 2003). Invertebrates have served as simple animal models to study synapse formation. Indeed, Colón-Ramos (2009) has recently reviewed the early developmental events that take place in the process of synaptogenesis pointing out the importance of this process in neural network formation and function. The experimental evaluation of synaptogenesis has been performed using invertebrates and in particular C. elegans and Drosophila as well as vertebrates (Colón-Ramos, 2009).

This vulnerable period of synaptogenesis appears to happen in different developmental stages across species. For example, in rodents primarily synaptogenesis occurs during the first two weeks after birth (Bai et al., 2013). For rhesus monkeys, this period ranges from approximately 115-day gestation up to PND 60 (Bai et al., 2013). In humans, it starts from the third trimester of pregnancy and continues 2-3 years following birth (Bai et al., 2013).

281: Decreased, Thyroxin (T4) in serum

Short Name: Decreased, Thyroxin (T4) in serum

AOPs Including This Key Event

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

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 amphibian and larbean metamorphoses is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990; Furlow and Neff, 2006). Their existence and importance has also been described in many different animal and plant kingdoms (Eales, 1997; Heyland and Moroz, 2005), while 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 TH 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 should be done with caution.

With few exceptions, vertebrate species have circulating T3 and T4 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 in adult humans is about 75, 15 and 10 percent, respectively (Schussler 2000). In contrast, the majority of THs are bound to TTR in adult rats. And thyroid binding proteins are developmentally regulated in rats. Thyroxine binding globulin is expressed in rats until approximately postnatal day (PND) 60, with peak expression occurring during weaning (Savu et al., 1989). Low 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 hormone half-life (Capen, 1997) and possibly regulatory feedback mechanisms, there is little information on quantitative dose-response relationships. SerumTHs are still regarded as the most robusts measurable key event causally linked to downstream adverse outcomes.

851: Altered, GABAergic interneurons morphology and function

Short Name: Altered, GABAergic interneurons morphology and function

AOPs Including This Key Event

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

GABAergic interneurons play a vital role in the wiring and circuitry of the developing nervous system of all organisms, both invertebrates and vertebrates (Hensch, 2005; Owens and Kriegstein, 2002; Wang et al., 2004). However, restricted expression of GABA in a considerable population of neurons is observed in the non-vertebrate animals. A nematode Caenorhabditis elegans has 302 neurons, among them, 26 cells are GABAergic (Sternberg and Horvitz, 1984; McIntire et al., 1993). Another nematode Ascaris has 26 GABAergic neurons (Obata, 2013). GAD, VGAT, GABA receptors and GABA-system-specific molecules are analogous to those of vertebrates. Except for one interneuron, GABAergic neurons are connected with muscle cells and exert direct inhibitory, sometimes excitatory, control on locomotion, defecation and foraging. The muscle innervation of both excitatory and inhibitory axons is maintained also in Crustacea (Obata, 2013).   

386: Decreased, Neuronal network function in developing brain

Short Name: Decreased, Neuronal network function in developing brain

AOPs Including This Key Event

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

In vitro studies in brain slices applying electrophysiological techniques showed significant variability among species (immature rats, rabbits and kittens) related to synaptic latency, duration, amplitude and efficacy in spike initiation (reviewed in Erecinska et al., 2004).

277: Decreased, Thyroid hormone synthesis

Short Name: Decreased, Thyroid hormone synthesis

AOPs Including This Key Event

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Decreased TH synthesis resulting from TPO or NIS inhibition is conserved across taxa, with in vivo evidence from humans, rats, amphibians, 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.

Typically decreased serum thyroxine (T4) is used as a surrogate measure to indicate chemical-mediated decreases in TH synthesis. However, clinical and veterinary management of hyperthyroidism and Grave's disease involves administration of drugs including propylthiouracil and methimazole, known to decrease TH synthesis, indicating strong medical evidence for chemical initiation of this event (Zoeller and Crofton, 2005).

341: Impairment, Learning and memory

Short Name: Impairment, Learning and memory

AOPs Including This Key Event

Biological Organization

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

Learning and memory have been studied in invertebrates such as gastropod molluscs and drosophila and vertebrates such as rodents and primates. Recently, larval zebrafish has also been suggested as a model for the study of learning and memory (Roberts et al., 2013).

Reduced, Release of BDNF leads to Decreased, Synaptogenesis

Evidence Supporting Applicability of this Relationship

Empirical evidence comes from work with laboratory rodent-derived cells and brain slices, and rodent in vivo studies.

How Does This Key Event Relationship Work

Disruption of BDNF signaling during development was shown to interfere with synaptogenesis in the hippocampus (Sanchez-Martin et al., 2013; Neal et al., 2010; Stansfiled et al., 2012). In the adult brain, BDNF is involved in synaptic plasticity (Lu et al., 2013; Leal et al., 2014). Synaptic dysfunction is a key pathophysiological hallmark in neurodegenerative disorders, including Alzheimer's disease, and synaptic repair therapies based on the use of trophic factors, such as BDNF, are currently under consideration (Lu et al., 2013).

BDNF is released by the BDNF-producing neurons of the CNS and binds to Trk-B of the PV-interneurons, an interaction necessary for the subsequent developmental effects of this neurotrophin (Polleux et al., 2002; Jin et al., 2003; Rico et al., 2002; Aguado et al., 2003). BDNF promotes the morphological and neurochemical maturation of hippocampal and neocortical interneurons and promotes GABAergic synaptogenesis (Danglot et al., 2006; Hu and Russek, 2008).

BDNF plays an important role in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development (Chapleau et al., 2009). Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, as well as in mouse models of these disorders (Chapleau et al., 2009).

Weight of Evidence

Biological Plausibility

NMDAR activity has been linked to the signaling of the trans-synaptic neurotorophin BDNF (Neal et al., 2010). BDNF, in addition to its pro-survival effects, has powerful synaptic effects, promoting synaptic transmission, synaptic plasticity and synaptogenesis (Lu et al., 2013; Sanchez-Martin et al., 2013; Neal et al., 2010; Stansfiled et al., 2012; Danglot et al., 2006; Hu and Russek, 2008). Use of selective agonist or antagonist of BDNF receptor TrkB demonstrates the contribution of BDNF in synaptogenesis in adult-generated neurons in the rat dentate gyrus (Ambrogini et al., 2013). In this regard, exogenous application of BDNF significantly increased the number of functional synapses in culture (Vicario-Abejon et al., 1998; Marty et al., 2000), while blocking of BDNF with antibodies greatly reduced the formation of inhibitory synapses (Seil and Drake-Baumann, 2000). Similar results were described also in an in vivo study on mutant mice characterized by deletion of the trkB gene in cerebellar precursors (obtained by Wnt1-driven Cre--mediated recombination). TrkB mutant mice showed reduced amounts of GABAergic markers and develop reduced numbers of GABAergic boutons and synaptic specializations, whilst granule and Purkinje cell dendrites appeared normal and the former presented typical numbers of excitatory synapses. This study demonstrated that TrkB is essential to the development of GABAergic neurons and the regulation of synapse formation (Rico et al., 2002). BDNF is also a potent regulator of spontaneous neuronal activity (Aguado et al., 2003), a critical function of the developing hippocampus and an important functional feature of the CNS.

Empirical Support for Linkage

- Westerholz et al., 2013 In recent in vitro studies with rat T3-deficient cultures of cortical GABAergic PV⁺ interneurons, which are subject to BDNF regulation, it was shown that the number of synaptic boutons (i.e., presynaptic terminals containing the presynaptic marker synaptophysin) was reduced, an effect that was abolished after exogenous BDNF application. Additionally, inhibition of BDNF by K252a (a TrKB antagonist) in cultures containing T3 resulted also in decreased number of synaptic boutons, as in the T3-deprived cultures. These results indicate that BDNF signaling promotes the formation of synaptic boutons and that this function is mediated by THs (T3 and T4). Additionally, T3-related increase of spontaneous network activity was remarkably reduced after addition of K252a, and also upon inhibition of mTOR pathway (with rapamycin), a pathway known to control synaptogenesis (Buckmaster et al., 2009).

- Sato et al., 2007 This study on rat cultured hippocampal slices showed that beta-estradiol (E2) induced synaptogenesis between mossy fibers (one of the major inputs to cerebellum) and hippocampal CA3 neurons by enhancing BDNF release from dentate gyrus (DG) granule cells, by increasing the expression of PSD95, a postsynaptic marker. E2 effects on in hippocampal slice cultures and subregional neuron cultures were completely inhibited by blocking the BDNF receptor (TrkB) with K252a (200 nM) or by using a function-blocking antibody to BDNF (10 μg/ml), which inhibited the expression of PSD95 induced by E2. Both K252a and the antibody anti-BDNF elicited ~ 60-70% decrease of spine density, and ~ 55% decrease of presynaptic sites (measured as number of puncta/neuron).

- Schjetnan and Escobar, 2012 In this study, intrahippocampal microinfusion of BDNF (3 µg/3 µl; 0.2 µl/min,) in adult rats modified the ability of the hippocampal mossy fiber pathway to present long-term potentiation (LTP, i.e., a persistent strengthening of synapses based on recent patterns of activity) by high frequency stimulation (HFS). This indicates that BDNF initiates the metaplastic mechanisms that allow the modifications of the ability of the mossy fiber pathway to present LTP induced by subsequent HFS. On the contrary, microinfusion of K252a (administered in combination with BDNF: 3 μg of BDNF/3 μl of K252a 20 μM; 0.2 μl/min) blocked the functional and morphological effects produced by BDNF (shown by densitometric analysis on synaptic reorganization: ~ 30% reduction of the relative area of the dorsal hippocampus in the contralateral side of HFS, and ~ 70% reduction in the ipsilateral side of HFS, compared to BDNF administered alone), supporting the role of BDNF in the regulation of synaptic plasticity.

- Schildt et al., 2013 Using field potential recordings in CA3 of adult heterozygous BDNF knockout (BDNF+/-) mice, an impairment of NMDAR-independent mossy fiber (MF)-LTP (~ 50% decrease) was observed. Additionally, inhibition of TrkB/BDNF with K252a (slices preincubated for 3 hr with 100 nM), or with the selective BDNF scavenger TrkB-Fc (slices preincubated for 3 hr with 5 μg/ml), both inhibited MF-LTP to the same extent as observed in BDNF+/- mice (K252a: ~ 60% decrease vs control slices; TrkB-Fc: ~ 50% decrease vs control slices).

- Cortés et al., 2012 Adult male Sprague-Dawley rats were treated with 6-propyl-2-thiouracil (PTU, a TPO inhibitor) (0.05% in drinking water) for 20 days to induce hypothyroidism. PTU-treated rats showed decrease serum fT4 (~ 70% decrease vs control) and tT3 (~ 45% decrease vs control) levels, and increased TSH levels (~ 9.5-fold increase over control). The hippocampus of hypothyroid adult rats displayed increased apoptosis levels in neurons and astrocyte and reactive gliosis compared with controls. The glutamatergic synapses from the stratum radiatum of CA3 from hypothyroid rats, contained lower postsynaptic density (PSD) than control rats (~ 25% lower PSD than control). This observation was in agreement with a reduced content of NMDAR subunits (NR1 and NR2A/B subunits, both subunits: ~ 25% decrease vs control) at the PSD in hypothyroid animals. Additionally, the hippocampal amount of BDNF mRNA (assessed by in situ hybridization) was higher (~ 4.8-fold increase over control) of hypothyroid rats, while the content of TrkB protein (BDNF receptor) was reduced (~ 30% decrease vs control) at the PSD of the CA3 region of hypothyroid rats, compared with controls.

 

KEs proceeding the AO (decreased cognition), such as "Reduced BDNF Release" and "Decreased synaptogenesis" are also common to the AOP 13, entitled "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" (https://aopwiki.org/aops/13). In this AOP 13, data on lead (Pb) exposure, as a reference chemical, are reported. These studies do not refer to TH disruption; however, they provide empirical support for this KER (Reduced release of BDNF leads to decreased synaptogenesis).

Synaptic structural plasticity was shown to be modified by Pb treatment during early (pre-weaning) or late (post-weaning) brain development in rats exposed to 2 mM Pb in drinking water for 3 weeks (Xiao et al., 2014). An iron chelator (clioquinol) can rescue the Pb-induced impaiment of synaptic plasticity in hippocampus (Chen et al., 2007), showing that Pb can affect synaptogenesis and synaptic plasticity. Primary hippocampal neurons obtained from ED18 rat pups and treated with Pb (1, 2 microM) for 5 days exhibited pre-synaptic deficits due to disruption of NMDAR-dependent BDNF signaling (Neal et al., 2010; Stansfield et al., 2012). A decrease in bdnf expression was observed in mouse embryonic stem cells differentiated into neurons, if they were exposed to Pb 0.1 microM throughout the whole differentiation process (Sanchez-Martin et al., 2013). Similar alterations in gene expression patterns of neural markers (synapsin 1), neurotrophins (bdnf), transcription factors and glutamate-related genes were found in mice, when their mothers were exposed to 0-3 ppm of Pb in drinking water from 8 weeks prior to mating, through gestation and until postnatal day 10 (Sanchez-Martin et al., 2013).

Uncertainties or Inconsistencies

Alterations of BDNF signaling is probably not the only mechanism leading to impaired synaptogenesis and synaptic plasticity. Indeed NMDAR activity can also modulate nitric oxide (NO) signaling. Exogenous NO addition during Pb exposure results in complete recovery of whole-cell synaptophysin levels and partial recovery of synaptophysin and synaptobrevin in synapses in Pb-exposed neurons (Neal et al., 2012). In addition, in Wistar rats, the anti-oxidant and radical scavenger quercetin was able to relieve the impairment of synaptic plasticity induced by chronic Pb exposure (from parturition through adulthood (PND 60); 0.2% Pb in drinking water of mothers and post-weaning pups) (Hu et al., 2008), suggesting that oxidative stress can also interfere with synapse formation.

Additionally, while PTU (a TPO inhibitor) has been shown to decrease brain BDNF levels and expression in offspring born from PTU-treated rat dams (Shafiee et al. 2016; Chakraborty et al., 2012; Gilbert et al. 2016), in the study from Cortés and colleagues (Cortés et al., 2012), treatment of adult male Sprague-Dawley rats with PTU induced an increase in the amount of BDNF mRNA in the hippocampus, while the content of TrkB protein, the BDNF receptor, resulted reduced at the PSD of the CA3 region compared with controls. Treated rats presented also thinner PSD than control rats, and a reduced content of NMDAr subunits (NR1 and NR2A/B subunits) at the PSD. These indicate differential effects elicited by PTU (i.e., TPO inhibition) on BDNF expression/regulation comparing the adult vs foetal brain. Therefore, results variability from study to study is due to different experimental study designs, accounting for differences in brain development stages (PND vs adult), times of exposures to chemicals, and regional brain differences.

Finally, there are no studies directly showing that inhibition of thyroid NIS expression, functional activity or protein level are linked to a reduction of BDNF and/or synaptogenesis.

Quantitative Understanding of the Linkage

There is a lack of studies directly linking BDNF levels (gene and/or protein) with the quantitative analysis of synaptogenesis induced by decreased TH levels, and therefore no robust quantitative information can be provided. However, in the AOP 13 ("Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" https://aopwiki.org/aops/13) direct associations between Pb-induced decrease of BDNF (protein and/or mRNA) and decrease of pre- and post-synaptic proteins are discussed, supported also by quantitative analyses of spines and dendrite morphology.

References

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Ambrogini P, Lattanzi D, Ciuffoli S, Betti M, Fanelli M, Cuppini R. (2013). Physical exercise and environment exploration affect synaptogenesis in adult-generated neurons in the rat dentate gyrus: possible role of BDNF. Brain Res 1534: 1-12.

Buckmaster PS, Ingram EA, Wen X. (2009). Inhibition of the mammalian target of rapamycin signaling pathway suppresses dentate granule cell axon sprouting in a rodent model of temporal lobe epilepsy. J Neurosci. Jun 24; 29(25):8259-69.

Chakraborty G, Magagna-Poveda A, Parratt C, Umans JG, MacLusky NJ, Scharfman HE. (2012). Reduced hippocampal brain-derived neurotrophic factor (BDNF) in neonatal rats after prenatal exposure to propylthiouracil (PTU). Endocrinology 153:1311–1316.

Chapleau CA, Larimore JL, Theibert A, Pozzo-Miller L. (2009). Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism. J Neurodev Disord;1:185–196.

Chen WH, Wang M, Yu SS, Su L, Zhu DM, She JQ, et al. (2007). Clioquinol and vitamin B12 (cobalamin) synergistically rescue the lead-induced impairments of synaptic plasticity in hippocampal dentate gyrus area of the anesthetized rats in vivo. Neuroscience 147(3): 853-864.

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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, Feb;157(2):774-87

Hu Y, Russek SJ. (2008). BDNF and the diseased nervous system: a delicate balance between adaptive and pathological processes of gene regulation. J Neurochem. 105:1-17.

Hu P, Wang M, Chen WH, Liu J, Chen L, Yin ST, et al. (2008). Quercetin relieves chronic lead exposure-induced impairment of synaptic plasticity in rat dentate gyrus in vivo. Naunyn Schmiedebergs Arch Pharmacol. Jul;378(1):43-51.

Jin X, Hu H, Mathers PH, Agmon A. (2003). Brain-derived neurotrophic factor mediates activity-dependent dendritic growth in nonpyramidal neocortical interneurons in developing organotypic cultures. J Neurosci 23:5662–5673.

Leal G, Comprido D, Duarte CB. (2014). BDNF-induced local protein synthesis and synaptic plasticity. Neuropharmacology 76 Pt C: 639-656.

Lu B, Nagappan G, Guan X, Nathan PJ, Wren P. (2013). BDNF-based synaptic repair as a disease-modifying strategy for neurodegenerative diseases. Nat Rev Neurosci 14(6): 401-416.

Marty S, Wehrle R, Sotelo C. (2000). Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus. J Neurosci 20: 8087–8095.

Neal AP, Stansfield KH, Worley PF, Thompson RE, Guilarte TR. (2010). Lead exposure during synaptogenesis alters vesicular proteins and impairs vesicular release: potential role of NMDA receptor-dependent BDNF signaling. Toxicol Sci 116(1): 249-263.

Neal AP, Stansfield KH, Guilarte TR. (2012). Enhanced nitric oxide production during lead (Pb(2)(+)) exposure recovers protein expression but not presynaptic localization of synaptic proteins in developing hippocampal neurons. Brain Res 1439: 88-95.

Polleux F, Whitford KL, Dijkhuizen PA, Vitalis T, Ghosh A. (2002). Control of cortical interneuron migration by neurotrophins and PI3-kinase signaling. Development 129:3147–60.

Rico B, Xu B, Reichardt LF. (2002). TrkB receptor signaling is required for establishment of GABAergic synapses in the cerebellum. Nat Neurosci 5:225–233.

Sanchez-Martin FJ, Fan Y, Lindquist DM, Xia Y, Puga A. (2013). Lead induces similar gene expression changes in brains of gestationally exposed adult mice and in neurons differentiated from mouse embryonic stem cells. PLoS One 8(11): e80558.

Sato K, Akaishi T, Matsuki N, Ohno Y, Nakazawa K. (2007). beta-Estradiol induces synaptogenesis in the hippocampus by enhancing brain-derived neurotrophic factor release from dentate gyrus granule cells. Brain Res. May 30;1150:108-20.

Schildt S, Endres T, Lessmann V, Edelmann E. (2013). Acute and chronic interference with BDNF/TrkB-signaling impair LTP selectively at mossy fiber synapses in the CA3 region of mouse hippocampus. Neuropharmacology. Aug;71:247-54.

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Reduced, Release of BDNF leads to Altered, GABAergic interneurons morphology and function

Evidence Supporting Applicability of this Relationship

Empirical evidence comes from work with laboratory rodents (rats and mice).

How Does This Key Event Relationship Work

GABAergic interneurons are remarkably diverse and complex in nature and they are believed to play a key role in numerous neurodevelopmental processes (Southwell et al., 2014). Among them, those that express parvalbumin (PV) as their calcium-binding protein are the ones subject to regulations by neurotrophins and BDNF specifically (Woo and Lu, 2006). These neurons do not express the BDNF protein but its functional receptor, namely the Trk-B (Cellerino et al., 1996; Marty et al., 1996; Gorba and Wahle, 1999). BDNF is released by the BDNF-producing neurons of the CNS and binds to Trk-B of the PV-interneurons, an interaction necessary for the subsequent developmental effects of this neurotrophin (Polleux et al., 2002; Jin et al., 2003; Rico et al., 2002; Aguado et al., 2003). BDNF promotes the morphological and neurochemical maturation of hippocampal and neocortical interneurons and promotes GABAergic synaptogenesis (Danglot et al., 2006 and Hu and Russek, 2008). BDNF also regulates the expression of the neuron-specific K(+)/Cl(-) co-transporter, KCC2, which is responsible for switching of GABA action from excitatory to inhibitory, and consequently determines the nature of GABA-induced responses in developing neurons (Blaesse et al., 2009).

Weight of Evidence

Biological Plausibility

Proper function of the Central Nervous System (CNS) results from the closely regulated development and function of the neuronal cells and is driven by the overall balance between excitation and inhibition. In the cerebral cortex the synaptic inhibition is mediated by the GABAergic interneurons, which regulate also the neuronal excitability and thereby the function and maturation of the neuronal networks (Voigt et al., 2001; Cherubini et al., 2011).

Many trophic factors have been plausibly implicated in the regulation of these processes but among them BDNF stands out as the prime candidate due to do its effects on interneuron development (Palizvan et al., 2004; Patz et al., 2004; Woo and Lu, 2006; Huang et al., 2007; Huang, 2009). Exogenous application of BDNF in developing neocortical and hippocampal interneurons has demonstrated an enhanced dendritic elongation and branching in cultures (Jin et al., 2003; Vicario-Abejon et al., 1998). Interneuron differentiation was also affected by endogenous BDNF, as the length and branching of BDNF interneurons was promoted only when they were innervated by BDNF-releasing interneurons (Kohara et al., 2003). Due to these dendritic effects of BDNF on GABAergic interneurons, this neurotrophin was suggested to promote also the formation of inhibitory synapses, which was further supported by several in vitro studies. Exogenous application of BDNF significantly increased the number of functional synapses in culture (Vicario-Abejon et al., 1998; Marty et al., 2000), while blocking BDNF with antibodies greatly reduced the formation of inhibitory synapses (Seil and Drake-Baumann, 2000). Similar results were exerted also by in vivo cerebellar studies, in which Trk-b receptor was found to be the prerequisite for inhibitory synapses formation (Rico et al., 2002). Additionally, BDNF was reported to elicit presynaptic changes in GABAergic interneurons, as several presynaptic proteins were up-regulated after BDNF application (Yamada et al., 2002; Berghuis et al., 2004), while a significant increase of GABAA receptor was observed in cultured hippocampus-derived neurons after treatment with BDNF (Yamada et al., 2003).

BDNF is also a potent regulator of spontaneous neuronal activity (Aguado et al., 2003; Carmona et al., 2006), a major milestone of the developing hippocampus and an important feature of the CNS. Further supporting studies have shown that it has the ability to depolarize cortical neurons in culture (Kafitz et al., 1999), an effect which has been linked to the developmentally regulated spontaneous network activity (Feller, 1999; O'Donovan, 1999).

The spontaneous neuronal activity early in development is also closely related to Cl-homeostasis, which is developmentally regulated by KCC2, the main K+ Cl- co-transporter in the brain (Rivera et al., 1999). Because neuronal expression of KCC2 is low during early development, the intracellular [Cl-] cannot be extruded leading to the depolarizing effect of GABA during this period (Ben-Ari et al., 2004). Taking these under consideration, it was assumed that the effects of BDNF on neuronal activity was mediated by the KCC2 regulation, as observed in several in vitro and in vivo studies (Ludwig et al., 2011a and 2011b; Yeo et al., 2009; Aguado et al., 2003; Carmona et al., 2006).

In support to this relationship, recent studies have demonstrated that injured hippocampal neurons can survive and be regenerated through the same mechanism (Shulga et al., 2013). Indeed, after mature nerve injury, KCC2 is down-regulated and the GABA responses switch to depolarization in a way similar to the early developmental stages. The rescue and re-generation of these neurons requires the switch of GABA from depolarization to hyperpolarization, a process driven by BDNF and the subsequent KCC2 up-regulation in hippocampal neurons (Shulga et al., 2009).

Empirical Support for Linkage

It is widely accepted that BDNF expression is regulated by TH (Koibuchi et al., 1999; 2001; Chakraborty et al., 2012). Deregulation of BDNF signaling has been shown to decrease cortical GABA interneuron markers (Kelsom and Lu, 2013; Fiumelli et al., 2000; Arenas et al., 1996; Jones et al., 1994).

- Westerholz et al., 2013 In recent in vitro studies with rat T3-deficient cultures of cortical PV+ interneurons, it was shown that the number of synaptic boutons was reduced, an effect that was abolished after exogenous BDNF application. Additionally, inhibition of BDNF by K252a (a TrK antagonist) in cultures containing T3 resulted also in decreased number of synaptic boutons, as in the T3-deprived cultures. These results suggest that BDNF signaling promotes the formation of synaptic boutons and that this function is mediated by TH (T3 and T4).

- Chen et al., 2016 BDNF-Val66Met knock-in mice (BDNF^Met/Met) are known for reduction in the activity-dependent BDNF secretion and elevated anxiety-like behaviors. This study showed that GABAergic innervations of pyramidal neurons of BDNF^Met/Met mice are reduced at distal dendrites in hippocampal CA1 and medial prefrontal cortex, compared to wild type mice.

- Kong et al., 2014 This study showed that chronic seizure rats 6 months after treatment with cyclothiazide (CTZ, a seizure inducer), underwent decrease of both GAD (from 75.2 ± 13.0 in CA1, 79.7 ± 9.7 in CA3, and 251.5 ± 4.3 in DG, respectively, to 3.0 ± 0.5 in CA1, 3.6 ± 0.9 in CA3, and 5.3 ± 1.8 in DG) and GAT-1 (from 60.7 ± 3.0 in CA1, 55.7 ± 9.1 in CA3, and 212.3 ± 11.3 in DG, respectively, to 20.7 ± 8.6 in CA1, 24.3 ± 3.4 in CA3, and 24.7 ± 13.3 in DG) across CA1, CA3, and dentate gyrus area of the hippocampus. Also, hippocampal decrease of both BDNF⁺ cells (from 70.7 ± 9.0 in CA1, 72.2 ± 3,7 in CA3, and 138.3 ± 15.9 in DG, respectively, to 4.1 ± 1.0 in CA1, 2.9 ± 0.1 in CA3, and 21.2 ± 16.2 in DG) and TrkB⁺ (BDNF receptor) cells (from 126.7 ± 7.2 in CA1, 275.7 ± 56.3 in CA3, and 399.2 ± 22.4 in DG, respectively, to 64.7 ± 16.2 in CA1, 158.3 ± 41.7 in CA3, and 250.3 ± 46.8 in DG) was observed.

- Sawano et al., 2013 This study investigated the effects methimazole (MMI, a TPO inhibitor) on the developing rat hippocampus, one of the brain regions most sensitive to TH status. MMI was administered at the concentration of 0.025% in drinking water to pregnant dams from gestational day 15 until 4 weeks postpartum. Looking at the pre- and post-synaptic components of the GABAergic system, the level of glutamic acid decarboxylase 65 (GAD65) protein was reduced to less than 50% of control in the hippocampus of hypothyroid rats, and recovered to control levels by daily thyroxine-replacement after birth. Reduction in GAD65 protein was correlated immunohistochemically with a 37% reduction in the number of GAD65⁺ cells, as well as a reduction in GAD65⁺ processes. In contrast, GAD67 was not affected by MMI treatment. A subpopulation of GABAergic neurons containing PV was also confirmed to be highly dependent on TH status (with a 33% reduction in total PV⁺ neurons compared with the control). Moreover, the physiologically occurring transient rise of KCC2 expression observed at PND 10 (followed by a large increase in KCC2 protein at PND 15) in the euthyroid hippocampus, was completely suppressed by MMI (~ 80% reduction in KCC2 protein at PND 15 vs control). While it should be considered that BDNF brain levels were not assessed in this study, it is plausible that in these experimental settings BDNF may result downregulated, since KCC2 expression is known to be up-regulated by BDNF during development (Aguado et al., 2003; Blaesse et al., 2009), and KCC2 resulted downregulated in Sawano et al., 2013 study. Conversely, the effects of BDNF on KCC2 expression in the hippocampus encompass down-regulation in mature (adult) neurons (Rivera et al., 2002, 2004; Wake et al., 2007).

- Shiraki et al., 2012 this study compared the differential effects of MMI (0, 50, 200 ppm in the drinking water) comparing the developmental and adult-stage, in particular comparing pregnant rats treated from gestation day 10 to PND 21 (i.e., developmental hypothyroidism) and adult male rats treated from PND 46 through to PND 77 (i.e., adult-stage hypothyroidism). With regard to precursor granule cells, a sustained reduction of Pax6⁺ stem or early progenitor cells and a transient reduction of doublecortin⁺ late-stage progenitor cells were observed after developmental hypothyroidism with MMI at 50 and 200 ppm. These cells were unchanged by adult-stage hypothyroidism. The number of PV⁺ cells (a GABAergic interneuron subpopulation in the dentate hilus) was decreased (~ 60% reduction at PND 21, with 200 ppm MMI) and the number of calretinin⁺ cells was increased (~ 85% increase at PND 21, with 200 ppm MMI) after both developmental and adult-stage hypothyroidism. BDNF brain levels were not assessed in this study.

- Gilbert et al., 2007 In this study pregnant rat dams were exposed to propylthiouracil (PTU, a TPO inhibitor, administered at 0, 3, 10 ppm in the drinking water, from gestational day 6 until PND 30). PTU decreased maternal serum T4 by ~ 50-75% and increased TSH. At weaning, T4 was reduced by approximately 70% in offspring in the low-dose group and fell below detectable levels in high-dose animals. PV⁺ cells were diminished in the hippocampus and neocortex of offspring sacrificed on PND 21 (~ 45% reduction in the cortex, and ~ 55% reduction in the dentate gyrus, with 3 ppm treatment), and altered staining persisted to adulthood despite the return of TH to control levels. Specifically, BDNF brain levels were not assessed in this study.

Uncertainties or Inconsistencies

Despite the well described relationship between BDNF and GABAergic interneurons morphology and function, there are several other conflicting studies. In a recent one (Puskarjov et al., 2015) BDNF-/- mice were utilized to show that in the absence of BDNF the seizure-induced up regulation of KCC2 was eliminated, but interestingly no change in early (P5-6) or later (P13-14) postnatal KCC2 expression was observed compared to the wild type littermates. However, neither the functionality of the protein was investigated, nor the ability of the neurons to extrude Cl^- in the absence of BDNF. Additionally, other studies have shown that the up-regulation of KCC2 via the transcription factor Egr4 is also regulated by a different neurotrophic factor, neurturin (Ludwig et al., 2011b). These results reveal that the same transcriptional pathways can be activated by different neurotrophic factors and might lead to the same outcome under different conditions. This hypothesis should be further investigated, as this could explain the compensation mechanisms that are activated in the total absence of BDNF, and which might be different from those that are triggered by a decrease of BDNF levels.

Furthermore, while some studies (Sawano et al., 2013; Shiraki et al., 2012) have shown a decrease of GABAergic cells upon induction of hypothyroidism, Saegusa and co-workers (Saegusa et al., 2010) reported about an increase of GABAergic interneurons. In Saegusa's study, rat dams were treated from gestation day 10 with either PTU at 3 or 12 ppm (0.57 or 1.97 mg/kg body weight/day) or MMI at 200 ppm (27.2 mg/kg body weight/day) in the drinking water, and male offspring were immunohistochemically examined on PND 20 and at the adult stage (i.e., 11-week-old). MMI and 12 ppm PTU caused in the offspring growth retardation, lasting into the adult stage. All exposure groups showed a sustained increase of Reelin (a secreted extracellular matrix glycoprotein that plays a critical role in neuronal migration and positioning during brain development in the process regulated by TH (Alvarez-Dolado et al., 1999)) in the dentate hilus (~ 75% increase after MMI treatment) until the adult stage, in parallel with Calbindin-D-28K⁺ cells at weaning (~ 170% increase after MMI treatment, at PND 20) and with GAD67⁺ cells in the adult stage (~ 90% increase in 11-week-old rats treated with12 ppm PTU), indicating an increase in GABAergic interneurons.

It should be noticed that in Saegusa et al., 2010 study, increase of GAD67⁺ cells was mainly observed in the adult stage (11-week-old rats) and analysis of GABAergic PV⁺ cells, which appear to be the GABAergic population most affected by TH dysregulation, was not evaluated. On the opposite, Sawano's and Shiraki's studies reported a decrease of GABAergic PV⁺ neurons at earlier stages, respectively on PND 15 and 21 (Sawano et al., 2013; Shiraki et al., 2012). Discrepancies in results are due to the fact that THs have effects on multiple components of the GABA system. For instance, in the developing brain, hypothyroidism generally decreases enzyme activities and GABA levels, whereas in adult brain, hypothyroidism generally increases enzyme activities and GABA levels. Additionally, hyperthyroidism does not always have the opposite effect, and there are conflicting results on effects of long term changes in TH levels on GABA reuptake. Therefore, results variability from study to study is due to different experimental study designs, accounting for differences in brain development stages (PND vs adult), times of exposures to chemicals, and regional brain differences (Wiens and Trudeau, 2006).

Finally, apart from indirect observations on the effects of hypothyroidism (e.g., as a consequence of chemically-induced TPO inhibition), there are no studies showing that inhibition of NIS expression, functional activity or protein levels are directly linked to a reduction of BDNF levels and/or GABAergic neuron morphological or functional alterations. Further studies are needed to confirm these associations.

Quantitative Understanding of the Linkage

There is a lack of studies linking BDNF levels (gene and/or protein) and the amount of changes (morphological and function) in GABAergic interneurons and therefore no robust quantitative information can be provided.

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Altered, GABAergic interneurons morphology and function leads to Decreased, Synaptogenesis

Evidence Supporting Applicability of this Relationship

Most of the available studies have been performed in rodent models and human cortical neurons, referenced in the "Biological plausibility" section.

The relationship between KCC2 and GABA signalling has been also demonstrated in the retinotectal circuit of Xenopus (Akerman and Cline, 2006).

How Does This Key Event Relationship Work

Early in cortical development, the GABAergic interneurons have been found to contribute to key aspects of the brain development. A precise balance between excitatory and inhibitory drives in cortical neurons is crucial for the formation and maturation of the neuronal connections and eventually the proper neural circuitry function. In the cerebral cortex, the young neurons first receive GABAergic depolarizing inputs before forming any synapses (Owens et al., 1999; Tyzio et al., 1999; Hennou et al., 2002), and thus the GABAergic system is believed to be the initial regulator of synaptogenesis. Indeed, initial depolarizing GABAergic transmission is required for the formation of the glutamatergic synapses and is therefore responsible for the regulation of the balance between excitation and inhibition in the developing cortex (Wang and Kriegstein, 2009; Owens et al., 1999; Tyzio et al., 1999; Hennou et al., 2002; Ben-Ari, 2006).

Weight of Evidence

Biological Plausibility

Early in the development of the neocortex, GABAergic interneurons are implicated in the emergence of a spontaneous synchronized activity, which has a fundamental role in the activation of glutamatergic synapses, the synchronization of synaptogenesis and the establishment of long–range cortico-cortical connections (Voigt et al., 2001; 2005). Increasing evidence suggests that GABAergic signaling is the main regulator of this early neuronal activity, as it is established before the glutamatergic one in the neocortex (Wang and Kriegstein, 2009; Owens et al., 1999; Tyzio et al., 1999; Hennou et al., 2002; Ben-Ari, 2006). Despite the fact that GABA is the main inhibitory neurotransmitter in the adult CNS, it exerts depolarizing actions in the immature brain (Ben-Ari et al., 2007), caused by the low levels of Cl^- concentration in the post-synaptic cells (Rivera et al., 1999; Ehrlich et al., 1999). K-Cl co-transporter 2 (KCC2) is the main Cl^- efflux mechanism with a developmentally-regulated expression profile in the brain and it is therefore thought to be the regulator of GABA signalling during early neuronal development. The effects of KCC2 on the levels of [Cl-]I in immature neurons and the subsequent effects on the shift of the GABA signaling has been extensively studied during the last decades:

• Existing data indicate that KCC2 expressed by GABA neurons is sufficient to induce the end of the depolarizing and excitatory period of GABA during cortical neuron development (Lee et al., 2005; Chudotvorova et al., 2005) and to effectively decrease the [Cl-]I in immature rat neurons (Chudotvorova et al., 2005).

• Transcriptional repression of KCC2 in rat cortical neurons delayed the GABA switch corresponding to significant changes of [Cl-]I in the same neurons (Yeo et al., 2009).

Several studies focused on the effects of GABA signaling on synaptogenesis and they all had convergent results leading to a strong biological plausibility of this key event relationship.

• The early shift of GABA-induced excitation-to-inhibition not only affects synaptic integration, but it also results in deficient circuitry development (Wang and Kriegstein, 2008). This has been demonstrated in rodents and mammals cortical neurons in culture.

• Premature GABA switch has also morphological effects in cortical neurons, as it has been shown to drive in fewer and shorter dendrites with defective effects in synaptic formation (Cancedda et al., 2007).

• In the dentate gyrus of the adult hippocampus, newborn granule cells are tonically activated by ambient GABA before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic Cl^- ion content (Ge et al., 2006).

• An early hyperpolarizing shift in Cl⁻ reversal potential, by premature expression of KCC2, has been shown to increase the ratio of inhibitory-to-excitatory inputs both in Xenopus tectal neurons and rat cortical neurons in vitro (Chudotvorova et al., 2005; Akerman and Cline, 2006).

The mechanistic details of this relationship are not entirely known, but the most possible mechanism entails a functional relationship between GABA and NMDA receptor activation (Wang and Kriegstein, 2008; Cserép et al., 2012). Cortical neurons begin to express functional NMDA receptors when they migrate to the cortical plate, but these initial glutamatergic synapses are “silent” because of the Mg²⁺ block of NMDA receptors at the resting membrane potential (LoTurco et al., 1991; Akerman and Cline, 2006). GABAergic depolarization can facilitate relief of this voltage-dependent Mg²⁺ block and allow Ca²⁺ entry to initiate intracellular signalling cascades (Leinekugel et al., 1997). This mechanism suggests that the initial depolarizing GABAergic transmission is required for the formation of the glutamatergic synapses and is therefore responsible for the regulation of the balance between excitation and inhibition in the developing cortex (Wang and Kriegstein, 2009). 

Empirical Support for Linkage

As described above, the correlation between the GABA function and synaptogenesis has been mainly studied through the developmental modifications of intracellular Cl^- gradient and the subsequent GABA switch. In all available cases, this is performed by disturbing KCC2 or NKCC1 expression with genetic or mechanical manipulations of the neuronal models. In regards to toxicological studies, bisphenol A (BPA), an environmental toxicant known to inhibit NIS-mediated iodide uptake (Wu Y et al., 2016), has also been found to delay and decrease both KCC2 expression and the developmental Cl^- shift (Yeo et al., 2013):

- Yeo et al., 2013 In primary rat cortical neurons and primary human cortical neurons (obtained from human fetal brain tissue specimens), 100 nM BPA caused decrease of KCC2 mRNA expression (≥ 25% decrease in rat cells at 4-5 DIV, ~ 70% decrease in human cells at 10 DIV) and attenuated [Cl⁻]_i shift in migrating cortical inhibitory precursor neurons.

The temporal concordance of GABA shift and synaptogenesis is extensively reviewed by Ben-Ari et al., 2007 and 2012. It is widely accepted that the first spontaneous synaptic activity in the cortex is driven by the GABA-mediated depolarization and it is necessary for the subsequent synapse formation in the brain. Furthermore, the absence of T3 in cultures of cortical GABAergic interneurons can delay the typical developmental KCC2 up-regulation and subsequently the GABA shift, with a profound decrease in the number of synapses (Westerholz et al., 2010; 2013).

- Westerholz et al., 2013 This study showed that in rat T3-deficient cultures of cortical GABAergic PV⁺ interneurons, the number of synaptic boutons (presynaptic terminals containing the presynaptic marker synaptophysin) was reduced, an effect that was abolished after exogenous BDNF application.

- Sawano et al., 2013 This study investigated the effects methimazole (MMI, a TPO inhibitor) on the developing rat hippocampus, one of the brain regions most sensitive to TH levels. MMI was administered at the concentration of 0.025% in drinking water to pregnant dams from gestational day 15 until 4 weeks postpartum. The level of glutamic acid decarboxylase 65 (GAD65) protein was reduced to less than 50% of control in the hippocampus of hypothyroid rats, and recovered to control levels by daily thyroxine-replacement after birth. This decrease correlated with a 37% reduction in the number of GAD65⁺ cells, as well as a reduction in GAD65⁺ processes, while GAD67 was not affected by MMI treatment. Moreover, the physiologically occurring transient rise of KCC2 expression observed at PND 10 (followed by a large increase in KCC2 protein at PND 15) in the euthyroid hippocampus, was completely suppressed by MMI (~ 80% reduction in KCC2 protein at PND 15 vs control).

These findings also concur with studies in other brain areas, such as the auditory brainstem and the hippocampus (Friauf et al., 2008; Hadjab-Lallemend et al., 2010), supporting correlation between KCC2 expression and GABA presence in the brain, and their implication in synaptogenesis.

Uncertainties or Inconsistencies

In vivo evidence for the role of GABA in synaptogenesis is controversial. Ji et al., 1999 have shown that in GAD65/67-deficient mice, in which the production of GABA was reduced to less than 5%, the development of brain morphology until birth was normal. These mice die at birth and therefore synaptogenesis and circuit development could not be controlled, however no morphological defects were detected in the neocortex, cerebellum and hippocampus of these animals by the time of their death. The authors of this study suggested that GABA may not be crucial for development. However, functional changes were not assessed in this study. One hypothesis is that glutamate, glycine and taurine could compensate for the lack of GABA (LoTurco et al., 1995; Flint et al., 1998).

In KCC2 knock out mice, apart from lung atelectasis, no other obvious histological changes in the brain were observed in neonatal mice (Hubner et al., 2001). Moreover, these mice died at birth, before the GABA switch takes place, and neuronal electrical activity or synaptogenesis were not evaluated.

Additionally, after premature expression of KCC2 transporter an increase of the excitatory synapses was observed, but the glutamatergic synapses were not affected (Chudotvorova et al., 2005), as in the case of NKCC1 knock out mice (Wang and Kriegstein, 2008). These contradictory results reveal the complexity of the developmental brain and suggest that many different mechanisms are involved in the regulation of the temporal profile of the two main neuronal co-transporters, namely the KNCC1 and KCC2. However, in all cases the importance of Cl^- homeostasis in the developmental cortex and its correlation with the proper synapse formation is demonstrated.

Quantitative Understanding of the Linkage

There is a lack of studies directly linking alteration of GABAergic interneurons (morphology and function) with quantitative analyses of synaptogenesis modifications, and therefore no robust quantitative information can be provided.

References

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Ben-Ari Y. (2006). Basic developmental rules and their implications for epilepsy in the immature brain. Epileptic Disord. Jun; 8(2):91-102.

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Decreased, Synaptogenesis leads to Decreased, Neuronal network function in developing brain

Evidence Supporting Applicability of this Relationship

Colón-Ramos has reviewed the early developmental events that take place during the process of synaptogenesis, pointing out the importance of this process in neuronal network formation and function. The experimental findings reviewed in this paper derive from knowledge acquired in the field of neuroscience using invertebrates, in particular C. elegans and Drosophila, at the same time emerging findings derived from vertebrates are also discussed (Colón-Ramos, 2009).

How Does This Key Event Relationship Work

The ability of a neuron to communicate is based on neural network formation that relies on functional synapse establishment (Colón-Ramos, 2009). The main roles of synapses are the regulation of intercellular communication in the nervous system, and the information flow within neural networks. The connectivity and functionality of neural networks depends on where and when synapses are formed. Therefore, the decreased synapse formation during the process of synaptogenesis may lead to decreased neural network formation and function in the adult brain.

Synaptic transmission and plasticity require the integrity of the anatomical substrate. The connectivity of axons emanating from one set of cells to synapse on the dendrites of the receiving cells must be intact for effective communication between neurons to be possible. Changes in the placement of cells within the network due to delays 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 individually and cumulatively impair synaptic function. These anatomical alterations are among a host of many structural anomalies reported in various regions of the brain following severe developmental hypothyroidism. Although the primary evidence of synaptic transmission impairments in hypothyroid models have come from studying the hippocampus, it is assumed that the role thyroid hormones play in these processes is likely similar across different brain regions. Altered hippocampal structure from TH modulation of the neurogenesis process in the developing hippocampus or cortex may contribute to deficits in synaptic function.

Weight of Evidence

Biological Plausibility

Neuronal network connections are established via the process of synaptogenesis. The developmental period of synaptogenesis is critical for the formation of the basic circuitry of the nervous system, although neurons are able to form new synapses throughout life (Rodier, 1995). The brain electrical activity dependence on synapse formation is critical for proper neuronal communication. Alterations in synaptic connectivity lead to refinement of neuronal networks during development (Cline and Haas, 2008). Indeed, knockdown of PSD95 arrests the functional and morphological development of glutamatergic synapses (Ehrlich et al., 2007).

The biological plausibility of the known effects of TH insufficiency on brain structure having an impact on synaptic function and plasticity in brain is strong. Reductions in myelination of axons, cell number, dendritic arborization, and synaptogenesis have been described in models of severe hormone deprivation. Because synaptic transmission relies on the integrity of synaptic contacts and the electrical and chemical transmission between pre- and post-synaptic neurons, it is well accepted that interference with the anatomical levels will very much impact the neural network function.

Empirical Support for Linkage

Most of the information on developmental hypothyroidism and altered synaptic function has been provided by study of the hippocampus. It is presumed that structural deficits at some level underlie functional deficits revealed in synaptic transmission and plasticity impairments (Vara et al., 2002, Sui and Gilbert, 2003, Gilbert, 2004, Dong et al., 2005, Sui et al., 2005), but the precise structural aberration is not known. Within the hippocampus, area CA1 has been investigated primarily with in vitro techniques, using slices of hippocampus from exposed animals and measuring synaptic function across CA1-pyramidal cell synapses (Vara et al., 2002, Sui and Gilbert, 2003, Gilbert, 2004, Taylor et al., 2008). Pyramidal neurons of hypothyroid animals have fewer synapses and an impoverished dendritic arbor (Rami et al., 1986a, Madeira et al., 1992).

The other major region in hippocampus investigated in hypothyroid models is the perforant path-dentate gyrus synapse (Gilbert, 2011). Granule cells are the principal cell type of the dentate gyrus region of the hippocampal formation and receive input from cortical neurons in the entorhinal cortex. TPO inhibitors like PTU and MMI decrease the volume of the granule cell layer, the density of cells within the layer, and estimates of total granule cell number (Madeira et al., 1991). Migration of granule cells from the proliferative zone to the granule cell layer is retarded by thyroid deficiency as is dendritic arborization and synaptogenesis assessed by immunohistochemistry for the synaptic protein, synaptophysin (Rami et al., 1986b, Rami and Rabie, 1990, Dong et al., 2005). Impairments in synaptic function from both rodent and human studies are summarized below.

Excitatory and inhibitory synaptic transmission is reduced in CA region of hippocampus in animals with TH insufficiencies in early life (Vara et al., 2002, Sui and Gilbert, 2003, Gilbert, 2004, Dong et al., 2005, Sui et al., 2005). Similarly, excitatory and inhibitory synaptic transmission is reduced in the CA1 and dentate gyrus regions of the hippocampus (Gilbert and Paczkowski, 2003, Gilbert and Sui, 2006, Gilbert, 2011, Gilbert et al., 2013) under decreased TH levels. Parvalbumin (PV) is a calcium binding protein expressed exclusively in GABA inhibitory neurons of the hippocampus. Impairments in inhibitory synaptic transmission are associated with reductions in the number of PV⁺ cells (Gilbert et al., 2007).

- Gilbert and Sui, 2006, Gilbert, 2011, Gilbert et al., 2016: Long-term potentiation (LTP) is a model of activity-dependent synaptic plasticity critical for learning and memory. LTP is impaired in both sub-regions of hippocampus under conditions of TH deficiency, and these impairments persist in the adult offspring on recovery of euthyroid status. LTP induces activation of neurotrophins, particularly BDNF and related signaling molecules. Induction of these pathways underlies the persistence of experience-dependent plasticity. Offspring of hypothyroid animals are deficient in LTP and in the induction of neurotrophin gene changes in response to neuronal activation. Similar plasticity is operative during early brain development, influencing synapse formation and formation of neural networks.

- Westerholz et al., 2010; 2013: In the developing cortex, spontaneous activity is characterized by synchronous bursts of action potentials in populations of glutamatergic and GABAergic neurons which propagate throughout developing neural networks. In cortical neurons in culture, T3 augments the density and growth of GABAergic neurons and accelerates the maturation of neural networks. BDNF and associated signaling molecules have been implicated in T3 modulation of network formation in vitro. In this manner, T3 modulation of activity-dependent processes represents one potential mechanism whereby thyroid hormones may refine and stabilize synaptic connectivity in the developing brain.

- Wheeler et al., 2011, 2012: In addition to the rodent data, functional magnetic resonance imaging (fMRI) data from humans also support the relationship between altered serum concentrations of TH, hippocampal structure, and synaptic function. Children of women with high TSH during pregnancy exhibit reduced hippocampal volumes (Wheeler et al., 2011) and altered patterns of hippocampal activation when engaged in memory tasks (Willoughby et al., 2013, Willoughby et al., 2014, Wheeler et al., 2015). An association between hippocampal volume and everyday memory function was observed in adolescent children born with congenital hypothyroidism (CH), despite remediation soon after birth (Wheeler et al., 2011). In a paired object recognition task, no differences were observed in task performance scores in control and CH children (Wheeler et al., 2012). However, an increased activation of hippocampus was seen in CH children engaged in this task relative to controls, and bilateral hippocampal activation was evident when in control children this was restricted to the left hemisphere. These data suggest differential synaptic activation in CH children to accomplish simple memory tasks.

 

KEs proceeding the AO (learning and memory deficits), such as "Decreased synaptogenesis" (KEup) and "Decreased Neural Network Function" (KEdown) are also common to the AOP 13, entitled "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" (https://aopwiki.org/aops/13). In this AOP 13, data on lead (Pb) exposure as reference chemical are reported. While these studies do not refer to TH disruption, they provide empirical support for the same KER (Decreased synaptogenesis leads to decreased neuronal network function in developing brain) described in the present AOP.

- Otto and Reiter, 1984: At low Pb²⁺ levels (less than 30 µg/dl), slow cortical potentials have been observed to be positive in children under five years old but negative in children over five years. However, age-related polarity reversal has been observed in children with higher Pb²⁺ levels.

- Kumar and Desiraju, 1992: In experiments carried out in Wistar rats that have been fed with lead acetate (400 µg/g body weight/day) from PND 2 until PND 60, EEG findings show statistically significant reduction in the delta, theta, alpha and beta band of EEG spectral power in motor cortex and hippocampus with the exception of the delta and beta bands power of motor cortex in wakeful state.

- McCarren and Eccles, 1983: Male Sprague-Dawley rats have been exposed to Pb²⁺ from parturition to weaning though their dams' milk (dams received drinking water containing 1.0, 2.5, or 5.0 mg/ml lead acetate). Starting from 15 weeks of age, the characteristics of the electrically elicited hippocampal after discharge (AD) and its alteration by phenytoin (PHT) showed significant increase in primary AD duration only in the animals exposed to the higher dose of Pb²⁺, whereas all groups responded to PHT with increases in primary AD duration.

Uncertainties or Inconsistencies

The exact mechanism by which a change in cell number, reduced dendritric arborization and synaptogenesis may lead to decreased neuronal network function has not been fully elucidated. Dose-dependent reductions in synaptic function in hippocampus have been demonstrated in models of moderate degrees of TH reduction, but studies of the anatomical integrity of the specific cell populations examined electrophysiologically have largely been evaluated in models of severe hypothyroidism and often in brain regions distinct from the hippocampus.

Quantitative Understanding of the Linkage

There are no data on the quantitative linkages between altered (hippocampal or cortical) synaptogenesis and impaired neuronal network function. Developmental window of exposure and duration of exposure can modulate response-response relationships.

References

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Dong J, Yin H, Liu W, Wang P, Jiang Y, Chen J. (2005). Congenital iodine deficiency and hypothyroidism impair LTP and decrease C-fos and C-jun expression in rat hippocampus. Neurotoxicology 26:417-426.

Ehrlich I, Klein M, Rumpel S, Malinow R. (2007). PSD-95 is required for activity-driven synapse stabilization. Proc Natl Acad Sci U S A. 104: 4176-4181.

Gilbert ME. (2004). Alterations in synaptic transmission and plasticity in area CA1 of adult hippocampus following developmental hypothyroidism. Brain Res Dev Brain Res 148:11-18.

Gilbert ME. (2011). Impact of low-level thyroid hormone disruption induced by propylthiouracil on brain development and function. Toxicol Sci 124:432-445.

Gilbert ME, 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.

Gilbert ME, Paczkowski C. (2003). Propylthiouracil (PTU)-induced hypothyroidism in the developing rat impairs synaptic transmission and plasticity in the dentate gyrus of the adult hippocampus. Brain Res Dev Brain Res 145:19-29.

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.

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.

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Sui L, Gilbert ME. (2003). Pre- and postnatal propylthiouracil-induced hypothyroidism impairs synaptic transmission and plasticity in area CA1 of the neonatal rat hippocampus. Endocrinology 144:4195-4203.

Taylor MA, Swant J, Wagner JJ, Fisher JW, Ferguson DC. (2008). Lower thyroid compensatory reserve of rat pups after maternal hypothyroidism: correlation of thyroid, hepatic, and cerebrocortical biomarkers with hippocampal neurophysiology. Endocrinology 149:3521-3530.

Vara H, Martinez B, Santos A, Colino A. (2002). Thyroid hormone regulates neurotransmitter release in neonatal rat hippocampus. Neuroscience 110:19-28.

Westerholz S, de Lima AD, Voigt T. (2010). Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone. Neuroscience 168:573-589.

Westerholz S, de Lima AD, Voigt T. (2013). Thyroid hormone-dependent development of early cortical networks: temporal specificity and the contribution of trkB and mTOR pathways. Front Cell Neurosci 7:121.

Wheeler SM, McAndrews MP, Sheard ED, Rovet J. (2012). Visuospatial associative memory and hippocampal functioning in congenital hypothyroidism. J Int Neuropsychol Soc 18:49-56.

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.

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Decreased, Neuronal network function in developing brain leads to Impairment, Learning and memory

Evidence Supporting Applicability of this Relationship

Synaptic transmission and plasticity are achieved via mechanisms common across taxonomies. LTP has been recorded in aplysia, lizards, turtles, birds, mice, guinea pigs, rabbits and rats. Deficiencies in hippocampally based learning and memory following developmental hypothyroidism have been documented mainly in rodents and humans.

How Does This Key Event Relationship Work

Learning and memory is one of the outcomes of the functional expression of neurons and neural networks from mammalian to invertebrates. Damage or destruction of neurons by chemical compounds during development when they are in the process of synapses formation, integration and formation of neural networks, will derange the organization and function of these networks, thereby setting the stage for subsequent impairment of learning and memory. Exposure to the potential developmental toxicants during neuronal differentiation and synaptogenesis will increase the risk of functional neuronal network damage leading to learning and memory impairment. Impairments in learning and memory are measured using behavioral techniques. It is well accepted that these alterations in behavior are the result of structural or functional changes in neurocircuitry. Functional impairments are often measured using field potentials of critical synaptic circuits in hippocampus and cortex. A number of studies have been performed in rodent models that reveal deficits in both excitatory and inhibitory synaptic transmission in the hippocampus as a result of developmental thyroid insufficiency. A well-established model of memory at the synaptic levels is known as long-term potentiation (LTP) (i.e., a persistent strengthening of synapses based on recent patterns of activity). Deficiencies in LTP are generally regarded as potential substrates of learning and memory impairments. In rodent models where synaptic function is impaired by TH deficiencies, deficits in hippocampus-mediated memory are also prevalent.

Weight of Evidence

Biological Plausibility

Long-term potentiation (LTP) is a long-lasting increase in synaptic efficacy (not always high frequency stimulation leads to LTP), and its discovery suggested that changes in synaptic strength could provide the substrate for learning and memory (reviewed in Lynch, 2004). Moreover, LTP is intimately related to the theta rhythm, an oscillation long associated with learning. Learning-induced enhancement in neuronal excitability, a measurement of neural network function, has also been shown in hippocampal neurons following classical conditioning in several experimental approaches (reviewed in Saar and Barkai, 2003).

On the other hand, memory requires the increase in magnitude of excitatory postsynaptic currents (EPSCs) to be developed quickly and to be persistent for few weeks at least without disturbing already potentiated contacts. Once again, a substantial body of evidence has demonstrated that tight connection between LTP and diverse instances of memory exist (reviewed in Lynch, 2004).

Moreover, it is well accepted that alterations in synaptic transmission and plasticity contribute to deficits in cognitive function. There are a number of studies that have linked exposure to TPO inhibitors (e.g., PTU, MMI), as well as iodine deficient diets, to changes in serum TH levels, which result in alterations in both synaptic function and cognitive behaviors (Akaike et al., 1991; Vara et al., 2002; Gilbert and Sui, 2006; Axelstad et al., 2008; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016).

Empirical Support for Linkage

Developmental hypothyroidism reduces the functional integrity in brain regions critical for learning and memory. Neurophysiological indices of synaptic transmission of excitatory and inhibitory circuitry are impaired in the hippocampus of hypothyroid animals. Both hippocampal regions (area CA1 and dentate gyrus) exhibit alterations in excitatory and inhibitory synaptic transmission following reductions in serum TH in the pre and early postnatal period (Vara et al., 2002; Sui and Gilbert, 2003; Sui et al., 2005; Gilbert and Sui, 2006; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016). These deficits persist into adulthood long after recovery to euthyroid status. The latter observation indicates that these alterations represent permanent changes in brain function induced from transient hormones insufficiencies induced during critical window of development.  

Because the adult hippocampus is involved in learning and memory, it is a brain region of remarkable plasticity. Use-dependent synaptic plasticity is critical during brain development for synaptogenesis and fine tuning of synaptic connectivity. In the adult brain, similar plasticity mechanisms underlie use-dependency that underlies learning and memory, as exhibited in LTP model of synaptic memory. Hypothyroidism during development reduces the capacity for synaptic plasticity in juvenile and adult offspring (Vara et al., 2002; Sui and Gilbert, 2003; Dong et al., 2005; Sui et al., 2005; Gilbert and Sui, 2006; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016). Impairments in synaptic function and plasticity are observed coincident with deficits in learning tasks that require the hippocampus.

- Davenport and Dorcey, 1972; Tamasy et al., 1986: Deficits in passive avoidance learning have been reported, but these early observations are often limited to animals suffering fairly severe hormonal deprivation.

- Akaike, 1991; Axelstad et al., 2008: Radial arm maze deficits reported in adult offspring of rat dams treated with high doses of the TPO inhibitor, propylthiouracil (PTU), throughout gestation and lactation.

- Gilbert and Sui, 2006; Gilbert et al., 2016: Morris water maze acquisition and reversal deficits are seen in animals treated with more moderate doses of PTU and who also displayed synaptic transmission and LTP deficits in hippocampus.

- Gilbert, 2011; Gilbert et al., 2016: Trace fear conditioning deficits to context and to cue reported in animals treated with PTU and who also displayed synaptic transmission and LTP deficits in hippocampus.

BPA, an environmental toxicant known to inhibit NIS-mediated iodide uptake (Wu Y et al., 2016) has been found to cause learning and memory deficits in rodents:

- Wang et al., 2016 Pregnant Sprague-Dawley female rats were orally treated with either vehicle or BPA (0.05, 0.5, 5 or 50 mg/kg BW/day) during days 9-20 of gestation. Male offspring were tested on PND 21 with the object recognition task. Data revealed a decrease in BDNF (~ 38% decrease at 50 mg/kg BW/day vs control) in the hippocampus. BPA-exposed male offspring underwent memory and cognitive impariments: they not only spent more time (~ 43% more, at 1.5 hr after training) in exploring the familiar object at the highest dose than the control, but also displayed a significant decrease in the object recognition index (at 50 mg/kg BW/day, ~ 54% lower short term memory measured 1.5 hr after training).

- Jang et al., 2012 In this study pregnant female C57BL/6 mice (F0) were exposed to BPA (0.1-10 mg/kg) from gestation day 6 to 17, and female offspring (F2) from F1 generation mice were analysed. Exposure of F0 mice to BPA (10 mg/kg) decreased hippocampal neurogenesis (~ 30% decrease of hippocampal BrdU⁺ cells vs control) in F2 female mice. High-dose BPA (10 mg/kg) caused neurocognitive deficit (i.e., reduced memory retention) as shown by passive avoidance testing (~ 33% decrease vs control) in F2 mice. Furthermore, 10 mg/kg BPA decreased the hippocampal levels of BDNF (~ 35% lower vs control) in F2 mice. These results suggest that BPA exposure (causing also inhibition of NIS function) in pregnant mothers could adversely affect hippocampal neurogenesis and cognitive function in future generations.

In humans, the data linking these two specific KE are much more limited, but certainly clear reductions in IQ, with specific impairments in hippocampus-mediated functions have been observed.

- Oerbeck et al., 2003 This study reported visual and verbal memory deficits in congenitally hypothyroid children. All verbal memory tasks were impaired; visual memory domain gave less consistent findings.

- Wheeler et al., 2011 This study reported lower IQ scores, verbal learning deficits and everyday memory task deficits in congenitally hypothyroid children.

- Wheeler et al., 2015 This study reported paired word recognition deficits with no impairment on simple word lists. Word pairing associations, but not word list recall, are hippocampus-mediated.

- Willoughby et al., 2014 Autobiographical memory is also a memory function that is heavily dependent on the hippocampus (Willoughby et al., 2013). Congenitally hypothyroid children and children born to women with high TSH during pregnancy were weaker in recalling event and perceptual details from past naturally-occurring autobiographical events than age matched controls. They were also less accurate than controls in recalling event and perceptual details of a staged event.

- Wheeler et al., 2012 In this study hippocampal synaptic function based on fMRI was altered while subjects engaged in a memory task. Although no differences existed between control and congenital hypothyroid (CH) adolescents in task aptitude, clear differences in the degree of hippocampus activation were seen during task engagement. fMRI revealed increased bilateral hippocampal engagement with object pair recognition task and bilateral activation, when controls revealed only left hippocampal activation.

- Willoughby et al., 2013 Analogously, in this study, fMRI revealed increased hippocampus activation with word pair recognition task in CH and children born to women with hypothyroxinemia during midgestation. These differences in functional activation were not seen with single word recognition, but were revealed when retention of word pair associations was probed. The latter is a task requiring engagement of the hippocampus.

 

A series of important findings suggest that the biochemical changes that happen after induction of LTP also occur during memory acquisition, showing temporality between the two KEs (reviewed in Lynch, 2004).

- D'Hooge and De Deyn, 2001 A review on Morris water maze (MWM) as a tool to investigate spatial learning and memory in laboratory rats also pointed out that the disconnection between neuronal networks rather than the brain damage of certain regions is responsible for the impairment of MWM performance. Functional integrated neural networks that involve the coordination action of different brain regions are consequently important for spatial learning and MWM performance.

- Morris et al., 1986 This study found that blocking the NMDA receptor with AP5 inhibits spatial learning in rats. Most importantly, in the same study they measured brain electrical activity and recorded that this agent also inhibits LTP, however, they have not proven that spatial learning and LTP inhibition are causally related.

Since then a number of NMDA receptor antagonists have been studied towards their ability to induce impairment of learning and memory. It is worth mentioning that similar findings have been found in human subjects:

- Grunwald et al., 1999 By combining behavioural and electrophysiological data from patients with temporal lobe epilepsy exposed to ketamine, involvement of NMDA receptors in human memory processes was demonstrated.

Some epidemiological and in vivo studies have indicated associations between a lower iodide uptake, (e.g., as a consequence of exposure to perchlorate or other pollutants, such as BDE-47) and decreased cognition. For instance:

- Taylor et al., 2014 In this historical cohort study of 21,846 women in Cardiff, United Kingdom, and Turin, Italy, who were pregnant from 2002 to 2006, levels of urinary perchlorate (a NIS inhibitor) in the highest 10% were associated with a higher risk for having children with IQ scores in the lowest decile at age three, as described in 487 mother–child pairs in mothers who were hypothyroid/hypothyroxinemic during pregnancy.

- Chen et al., 2014 In this prospective birth cohort, maternal serum concentrations of BDE-47 and other PBDE congeners were measured in 309 women at 16 weeks of gestation, and associated with neurodevelopment in children. Importantly, BDE-47 and other chemicals, such as triclosan, triclocarban and BPA, have been reported to disturb TH homeostasis by inhibiting NIS-mediated iodide uptake and altering the expression of genes involved in TH synthesis in rat thyroid follicular FRTL-5 cells (Wu Y et al., 2016). A 10-fold increase in prenatal BDE-47 was associated with a 4.5-point decrease in Full-Scale IQ and a 3.3-point increase in the hyperactivity score at age 5 years in children.

- Roze et al., 2009 Similarly, this study assessed the level of several compounds, including BDE-47 (i.e., 2,2'-bis-(4 chlorophenyl)-1,1'-dichloroethene, pentachlorophenol (PCP), PCB-153, 4OH-CB-107, 4OH-CB-146, 4OH-CB-187, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, and hexabromocyclododecane), in 62 mothers during the 35^th week of pregnancy, and possible associations with the neuropsychological level in their children at 5-6 years of age. THs were determined in umbilical cord blood. Brominated flame retardants correlated with worse fine manipulative abilities, worse attention, better coordination, better visual perception, and better behavior. Chlorinated OHCs correlated with less choreiform dyskinesia. Hydroxylated polychlorinated biphenyls correlated with worse fine manipulative abilities, better attention, and better visual perception. The wood protective agent (PCP) correlated with worse coordination, less sensory integrity, worse attention, and worse visuomotor integration.

- van Wijk  et al., 2008 This in vivo study assessed the behavioural effects of perinatal and chronic hypothyroidism during development in both male and female offspring of hypothyroid rats. To induce hypothyroidism, dams and offspring were fed an iodide-poor diet and drinking water with 0.75% sodium perchlorate (NIS inhibitor). Treatment was started in dams 2 weeks prior to mating, and in pups either until the day of killing (i.e., chronic hypothyroidism) or only until weaning (i.e., perinatal hypothyroidism) to test for reversibility of the effects observed. Early neuromotor competence, as assessed in the grip test and balance beam test, was impaired by both chronic and perinatal hypothyroidism. The open field test, assessing locomotor activity, revealed hyperactive locomotor behavioural patterns in chronic hypothyroid animals only. The Morris water maze test, used to assess cognitive performance, showed that chronic hypothyroidism affected spatial memory in a negative manner. Perinatal hypothyroidism was found to impair spatial memory in female rats only. In general, the effects of chronic hypothyroidism on development were more pronounced than the effects of perinatal hypothyroidism. This suggests that the early effects of hypothyroidism on functional alterations of the developing brain may be partly reversible.

 

 

The last KE preceeding the AO (learning and memory deficits), i.e. "Decreased Neural Network Function", is also common to the AOP 13, entitled "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" (https://aopwiki.org/aops/13). In this AOP 13, data on lead (Pb) exposure as reference chemical are reported. While these studies do not refer to TH disruption, they provide empirical support for the same KER described in the present AOP.

Exposure to low levels of Pb²⁺ during early development has been implicated in long-lasting behavioural abnormalities and cognitive deficits in children (Needleman et al., 1975; Needleman and Gatsonis, 1990; Bellinger et al., 1991; 1992; Baghurst et al., 1992; Leviton et al., 1993; Needleman et al., 1996; Finkelstein et al., 1998; Lanphear et al., 2000; 2005; Canfield et al., 2003; Bellinger, 2004; Surkan et al., 2007; Jusko et al., 2008; Neal and Guilarte, 2010) and experimental animals (Brockel and Cory-Slechta, 1998; Murphy and Regan, 1999; Moreira et al., 2001). Multiple lines of evidence suggest that Pb²⁺ can impair hippocampus-mediated learning in animal models (reviewed in Toscano and Guilarte, 2005).

The majority of the studies addressing the effects of Pb²⁺ on hippocampal-associated spatial learning and memory processes have been carried out mainly in male rats (Cao et al., 2008, Gilbert et al., 2005); only a few studies have examined both sexes simultaneously (Jett et al., 1997, Xu et al., 2009).

- Jett et al., 1997 Female rats exposed to Pb²⁺ through gestation and lactation have shown more severe impairment of memory than male rats with similar Pb²⁺ exposures.

- De Souza Lisboa et al. 2005 This study reported that exposure to Pb²⁺ during both pregnancy and lactation caused depressive-like behaviour (detected in the forced swimming test) in female but not male rats.

- Anderson et al., 2012 This study investigated the neurobehavioral outcomes in Pb²⁺-exposed rats (250, 750 and 1500 ppm Pb²⁺ acetate in food) during gestation and through weaning and demonstrated that these outcomes are very much influenced by sex and rearing environment. In females, Pb²⁺ exposure lessened some of the benefits of enriched environment on learning, whereas, in males, enrichment does help to overcome detrimental effects of Pb²⁺ on learning. Regarding reference memory, environmental enrichment has not been beneficial in females when exposure to Pb²⁺ occurs, in contrast to males.

- Jaako-Movits et al., 2005 Wistar rat pups were exposed to 0.2% Pb²⁺ via their dams' drinking water from PND 1 to PND 21 and directly via drinking water from weaning until PND 30. At PND 60 and 80, the neurobehavioural assessment has revealed that developmental Pb²⁺ exposure induces persistent increase in the level of anxiety and inhibition of contextual fear conditioning. The same behavioural syndrome in rats has been described in Salinas and Huff, 2002.

- Finkelstein et al., 1998 These observations are in agreement with observations on humans, as children exposed to low levels of Pb²⁺ displayed attention deficit, increased emotional reactivity and impaired memory and learning.

- Kumar and Desiraju, 1992 In Wistar rats fed with lead acetate (400 µg/g body weight/day) from PND 2 until PND 60, EEG findings showed statistically significant reduction in the delta, theta, alpha and beta band EEG spectral power in motor cortex and hippocampus, but not in delta and beta bands power of motor cortex in wakeful state. After 40 days of recovery, animals were assessed for their neurobehaviour, and revealed that Pb²⁺ treated animals showed more time and sessions in attaining criterion of learning than controls.

Further data obtained using animal behavioral techniques demonstrate that NMDA mediated synaptic transmission is decreased by Pb²⁺ exposure (Cory-Slechta, 1995; Cohn and Cory-Slechta, 1993 and 1994).

- Xiao et al., 2014 Rat pups from parents exposed to 2 mM PbCl₂ three weeks before mating until their weaning (pre-weaning Pb²⁺) and weaned pups exposed to 2 mM PbCl₂ for nine weeks (post-weaning Pb²⁺) were assessed for their spatial learning and memory by MWM on PND 85-90. The study revealed that both rat pups in pre-weaning Pb²⁺ and post-weaning Pb²⁺ groups performed significantly worse than those in the control group. The number of synapses in pre-weaning Pb²⁺ group increased significantly, but it was still less than that of control group. The number of synapses in post-weaning Pb²⁺ group was also less than that of control group, although the number of synapses had no differences between post-weaning Pb²⁺ and control groups before MWM. In both pre-weaning Pb²⁺ and post-weaning Pb²⁺ groups, synaptic structural parameters such as thickness of postsynaptic density (PSD), length of synaptic active zone and synaptic curvature increased, whereas width of synaptic cleft decreased compared to controls.

Uncertainties or Inconsistencies

One of the most difficult issues for neuroscientists is to link neuronal network function to cognition, including learning and memory. It is still unclear what modifications of neuronal circuits need to occur in order to alter motor behaviour as it is recorded in a learning and memory test (Mayford et al., 2012), meaning that there is no clear understanding about how these two KEs are connected.

The direct relationship of alterations in neural network function and specific cognitive deficits is difficult to ascertain given the many forms that learning and memory can take and the complexity of synaptic interactions in even the simplest brain circuit. Linking of neurophysiological assessments to learning and memory processes have, by necessity, been made across simple monosynaptic connections and largely focused on the hippocampus. Alterations in synaptic function have been found in the absence of behavioral impairments. This may result from measuring only one component in the complex brain circuitry that underlies 'cognition', behavioral tests that are not sufficiently sensitive for the detection of subtle cognitive impairments, and behavioral plasticity whereby tasks are solved by the animal via different strategies developed as a consequence of developmental insult.

Moreover, single NIS mutations, causing decreased thyroidal iodide uptake, may not necessarily lead to cognitive disorders. In this regard, Nicola and coworkers (Nicola et al., 2015) recently identified a new NIS mutation (V270E) in a patient (full-term girl born to healthy, non-consanguineous Jamaican parents), who resulted to be heterozygous for this NIS mutation (R124H/V270E). The presence of the mutation V270E markedly reduces iodide uptake (5.4% 24 hours after the oral administration of 100 μCi ¹²³I− (normal range, 10–40%)) via a pronounced (but not total) impairment of the protein's plasma membrane targeting. However, the retaining of a minimal iodide uptake was enough to enable sufficient TH biosynthesis and prevent cognitive impairment.

Numerous studies reported that iodine deficiency in critical periods of brain development and growth causes severe and permanent growth and cognitive impairment (cretinism) (Pesce and Kopp, 2014; de Escobar et al., 2007; de Escobar et al., 2008; Zimmermann, 2007; Melse-Boonstra and Jaiswal, 2010; Horn and Heuer, 2010; Zimmermann, 2012; Zimmermann, 2011). However, direct quantitative correlation between decreased neural network function and decreased cognition, in support to this KER, were not assessed in these reports.

Finally, in order to provide empirical support for this KER, data on the effects of lead (Pb) exposure are reported. However, Pb exposure is not always associated with learning and memory impairment in children. In this regard, Koller's review has commented that in some occasions, low-level Pb dose and cognitive deficits of the subjects are negatively correlated, and this may be due to the high influence of social and parenting factors in cognitive ability, like learning and memory (Koller et al., 2004).

Quantitative Understanding of the Linkage

There is very limited information on the degree of quantitative change in neural network function required to alter cognitive behaviors. This is a result of the diversity of methods for measuring both physiology and cognitive function, which hamper cross-study analyses. This highlights the need to develop empirical data based models of this KER.

Temporal concordance of TH insufficiency and disrupted development, defined at many levels of biological organization, has repeatedly been documented by demonstrating that there are critical windows during development where permanent changes are affected. Hormone replacement studies have also demonstrated that structural alterations in brain are reduced or eliminated if T4 (and/or T3) treatment is given during the critical windows (Goodman and Gilbert., 2007; Auso et al., 2004; Lavado-Autric et al., 2003; Berbel et al., 2010; Koibuchi and Chin, 2000).

Similarly, temporal concordance of insufficient iodide uptake (as a consequence e.g., of exposure to perchlorate or other pollutants, such as BDE-47) and decreased cognitive functions has been documented by some epidemiological studies (Taylor et al., 2014; Chen et al., 2014; Roze et al. 2009) and have demonstrated that there are critical windows during gestation/development where permanent changes are affected.

Graded degrees of TH insufficiency have been also produced in dams and pups by administering varying doses of NIS inhibitor (e.g., sodium perchlorate) (van Wijk et al., 2008) or TPO inhibitors and assessing the dose-dependency of the observed effects on a variety of endpoints. In these low dose model studies, the absence of overt signs of toxicity to the dams or the nursing pups is taken as evidence of the temporal concordance of hormone insufficiency and neurodevelopmental impairment and the specificity of the observed effects on brain development to be mediated by TH insufficiency (van Wijk  et al., 2008; Gilbert and Sui, 2006; Sharlin et al., 2007; Axelstad et al., 2008; Sharlin et al., 2008; Babu et al., 2011; Gilbert, 2011; Powell et al., 2012; Gilbert et al., 2013; Bastian et al., 2014; Gilbert et al., 2014; Gilbert et al., 2016).

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Decreased, Thyroxine (T4) in neuronal tissue leads to Reduced, Release of BDNF

Evidence Supporting Applicability of this Relationship

The connection between TH levels and BDNF expression has been studied only in rodent models up to date (see above studies).

How Does This Key Event Relationship Work

It is widely accepted that the thyroid hormones (TH) have a prominent role in the development and function of the Central Nervous System (CNS) and their action has been closely linked to the cognitive function because of their importance in the neocortical development (Gilbert et al., 2012). During the early cortical network development TH has been shown to regulate the morphology and function of the GABAergic neurons (Westerholz et al., 2010). One of the mediators of this regulation has been suggested to be the brain derived neurotrophic factor (BDNF), whose role in brain development and function has been very well-documented (Binder and Scharfman, 2004) and which has been plausibly associated with TH (Gilbert and Lasley, 2013). Several studies have shown that TH can regulate BDNF expression in the brain (Koibuchi et al., 1999; Koibuchi and Chin, 2000; Sui and Li, 2010), with the subsequent neurodevelopmental consequences.

In view of the above evidence, it has been suggested that the thyroid insufficiency, which eventually results to lower TH levels in the brain, may lead to impairments of cognitive function by reducing the levels of BDNF mRNA or protein in the developmental brain.

Weight of Evidence

Biological Plausibility

The importance of thyroid hormones (TH) in brain development has been recognised and investigated for many decades (Bernal, 2011). Several human studies have shown that low levels of circulating maternal TH, even in the modest degree, can lead to neurophysiological deficits in the offspring, including learning and memory deficits, or even cretinism in most severe cases (Zoeller and Rovet, 2004; Henrichs et al., 2010). The levels of serum TH at birth are not always informative, as most of the neurological deficits are present despite the normal thyroid status of the newborn. That means that the cause of these impairments is rooted in the early stages of the neuronal development during the gestational period. The nature and the temporal occurrence of these defects suggest that TH may exert their effects through the neurotrophins, as they are the main regulators of neuronal system development (Lu and Figurov, 1997). Among them, BDNF represents the prime candidate because of its critical role in CNS development and its ability to regulate synaptic transmission, dendritic structure and synaptic plasticity in adulthood (Binder and Scharfman, 2004). Additionally, hippocampus and neocortex are two of the regions characterized by the highest BDNF expression (Kawamoto et al., 1996), and are also key brain areas for learning and memory functions.

Empirical Support for Linkage

Many in vivo studies have focused on the determination of the relationship between TH-mediated effects and BDNF expression in the brain. The majority of the work has been performed by evaluating the effects of TH insufficiency on BDNF developmental expression profile. The results, despite some differences, are showing a trend toward BDNF down-regulation.

Reductions in BDNF mRNA and protein were observed in hypothyroid rat models, created exposing them to the TPO inhibitors methimazole (MMI) or propylthiouracil (PTU), and perchlorate (NIS inhibitor) (Koibuchi et al., 1999; 2001; Sinha et al., 2009; Neveu and Arenas, 1996; Lasley and Gilbert, 2011). These studies supported direct associations between the level of TH and BDNF expression in the developmental cerebellum, hippocampus and cortex. The dose-response relationship could not be evaluated in these studies, as they were conducted in conditions of severe maternal hypothyroidism, namely after exposure to very high doses of the chemicals.

Compounds, such as 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and bisphenol A (BPA) have been reported to disturb TH homeostasis by inhibiting NIS-mediated iodide uptake and altering the expression of genes involved in TH synthesis (Wu Y et al., 2016).

- Byun et al., 2015 Perinatal exposure to BDE-47 (by gavaging Wistar rat dams with 0.002 and 0.2 mg/kg body weight, at gestation days 9 and 16, and at postnatal days (PND) 1, 8, and 15), elicited an decrease of Bdnf promoter methylation at some CpGs, which may underlie the development of cognitive deficits and perinatal hypothyroidism, as shown by analysis of 5-methylcytosine (5mC, used to measure DNA methylation) in frontal lobes from PND 41 offspring.

- Wang et al., 2011 This study assessed the effects of both PFOS and/or BDE-47 in Wistar rats, which were exposed to 3.2 and 32 mg/kg of PFOS or BDE-47 in their diet and co-exposed to a combination of each chemical (3.2 mg/kg) from gestational day 1 to PND 14. Chemicals adiministered in a combined manner synergistically downregulated BDNF expression in the cortex, in particular on PND 1.

- Wang et al., 2016 Pregnant Sprague-Dawley female rats were orally treated with either vehicle or BPA (0.05, 0.5, 5 or 50 mg/kg BW/day) during days 9-20 of gestation. Male offspring were tested on PND 21 with the object recognition task. Data revealed a decrease in Akt (~ 51% decrease at 50 mg/kg BW/day), phospho-Akt (~ 56% decrease at 50 mg/kg BW/day), p44/42 MAPK (~ 45% decrease at 50 mg/kg BW/day) and phospho-p44/42 MAPK (~ 22% decrease at 50 mg/kg BW/day) protein levels, compared to controls, and also lower levels of phospho-CREB (~ 58% decrease at 50 mg/kg BW/day) and BDNF (~ 38% decrease at 50 mg/kg BW/day) in the hippocampus. In addition, BPA-exposed male offspring underwent memory and cognitive impariments: they not only spent more time (~ 43% more, at 1.5 hr after training) in exploring the familiar object at the highest dose than the control, but also displayed a significant decrease in the object recognition index (at 50 mg/kg BW/day, ~ 54% lower short term memory measured 1.5 hr after training).

- Jang et al., 2012 In this study pregnant female C57BL/6 mice (F0) were exposed to BPA (0.1-10 mg/kg) from gestation day 6 to 17, and female offspring (F2) from F1 generation mice were analysed. Exposure of F0 mice to BPA (10 mg/kg) decreased hippocampal neurogenesis (~ 30% decrease of hippocampal BrdU⁺ cells vs control) in F2 female mice. High-dose BPA (10 mg/kg) caused neurocognitive deficit (i.e., reduced memory retention) as shown by passive avoidance testing (~ 33% decrease vs control) in F2 mice. Furthermore, 10 mg/kg BPA decreased the hippocampal levels of phospho-ERK (~ 70% lower vs control), BDNF (~ 35% lower vs control), and phospho-CREB in F2 mice. Also, high-dose BPA (10 mg/kg) increased DNA methylation of the CREB regulated transcription coactivator 1 (Crtc1) generated in F2 mice (16–33% of CpG sites found methylated in F2 mice, whereas few of them were found methylated in F2 controls). These results suggest that BPA exposure (causing also inhibition of NIS function) in pregnant mothers could adversely affect hippocampal neurogenesis and cognitive function in future generations.

- Koibuchi et al., 2001 Here newborn mice were rendered hypothyroid by administering MMI (TPO inhibitor) and perchlorate (NIS inhibitor) in drinking water to their mothers. Neurotrophin-3 (NT-3) and BDNF gene expression was depressed in the perinatal hypothyroid cerebellum. Furthermore, the expression of retinoid-receptor-related orphan nuclear hormone receptor-alpha (ROR-alpha), an orphan nuclear receptor that plays critical roles in Purkinje cell development, was also decreased. Morphologically, disappearance of the external granule cell layer was retarded and arborization of Purkinje cell dendrite was decreased, events that were also observed in hypothyroid rats.

- Chakraborty et al., 2012 PTU (TPO inhibitor) exposure in rat dams (4 ppm in drinking water) significantly decreased the levels of free T4 (~ 33% decrease vs control, at PND 7) and total T4 (~ 38% decrease vs control, at PND 7) in the offspring, and hippocampal BDNF protein levels in the offspring at 3 and 7 PNDs (~ 25% decrease of hippocampal BDNF, at PND 7, in female pups vs untreated female control). No significant BDNF reductions were observed in either the cerebellum or brain stem.

Additionally, in more complex models of maternal hypothyroidism a reduction of hippocampal and cortex BDNF expression was observed (Wang et al., 2012; Liu et al., 2010). In these latter cases, hypothyroidism was developed via thyroidectomies, and T4 supplementation was performed at specific stages during gestation.

- Blanco et al., 2013 A significant dose-dependent down-regulation of hippocampal BDNF mRNA (~ 32% decrease vs control) in combination with the dose-dependent reduction of plasma TH (T4: ~ 25% decrease vs control; T3: ~ 14% decrease vs control), was also shown in Sprague Dawley rats after exposure to BDE-99 (2 mg/kg/day, through gavage, from gestation day 6 to PND 21).

- Shafiee et al. 2016 This study investigated the effects of PTU (TPO inhibitor, 100 mg/L) in pregnant rats to evaluate the effects elicited by maternal hypothyroidism in the offspring. PTU was added to the drinking water from gestation day 6 to PND 21. Analysis of hippocampal BDNF levels, and learning and memory tests were performed on PNDs 45-52 on pups. These results indicated that hypothyroidism during the fetal period and the early postnatal period was associated with: (i) ~ 70% reduction of total serum T4, (ii) ~ 5-fold increase of total serum TSH levels (on PND 21; no significant differences could be found at the end of the behavioral testing, on PND 52), (iii) reduction of hippocampal BDNF protein levels (~ 8% decrease vs control), (iv) impairment of spatial learning and memory, in both male and female rat offspring. However, in this study measurement of TH levels specifically in the brain was not reported. 

- Gilbert et al. 2016 A similar study showed that exposure to PTU during development produced dose-dependent reductions in mRNA expression of nerve growth factor (Ngf) in whole hippocampus of neonates. These changes in basal expression persisted to adulthood despite the return to euthyroid conditions in blood. Developmental PTU treatment dramatically reduced the activity-dependent expression of neurotrophins and related genes (Bdnft, Bdnfiv, Arc, and Klf9) in adulthood and was accompanied by deficits in hippocampal-based learning.

- Abedelhaffez and Hassan, 2013 This study in rats reported that methimazole (MMI, a TPO inhibitor)-induced hypothyroidism reduced plasma free T3, free T4 and significantly increased TSH in the pups, showing also reduced hippocampal and cerebellar BDNF levels.

- da Conceição et al. 2016 Thyroidectomized (i.e,. hypothyroid) adult Wistar rats showed significant increase of serum TSH (~ 750% increase vs control rats), decrease of T4 (~ 80% decrease vs control) and T3 serum levels (~ 45% decrease vs control), together with a reduced hippocampal expression of MCT8 (~ 83% decrease vs control rats), TH receptor alfa (TRα1) (~ 77 % decrease vs control), deiodinase type 2 (DIO2) (~ 90% decrease vs control), and BDNF mRNA expression in hippocampus (~ 75% decrease vs control).

Uncertainties or Inconsistencies

Despite the fact that many in vivo studies have shown a correlation between hypothyroidism and BDNF expression in the brain, no clear consensus can be reached by the overall evaluation of the existing data. There are numerous conflicting studies showing no significant alterations in BDNF mRNA or protein levels (Alvarez-Dolado et al., 1994; Bastian et al., 2010; 2012; Royland et al., 2008; Lasley and Gilbert, 2011). However, the results of these studies cannot exclude the possibility of temporal- or region-specific BDNF effects as a consequence of foetal hypothyroidism. A transient TH-dependent BDNF reduction in early postnatal life can be followed by a period of normal BDNF levels or, on the contrary, normal BDNF expression in the early developmental stages is not predictive of equally normal BDNF expression throughout development. Moreover, significant differences in study design, the assessed brain regions, the age and the method of assessment in the existing studies, further complicate result interpretation.

While PTU (TPO inhibitor) has been shown to decrease brain BDNF levels and expression in offspring born from PTU-treated rat dams (Shafiee et al. 2016; Chakraborty et al., 2012; Gilbert et al. 2016), in Cortés et al., 2012 study, treatment of adult male Sprague-Dawley rats with PTU induced an increase in the amount of BDNF mRNA in the hippocampus, while the content of TrkB, the receptor for BDNF, resulted reduced at the postsynaptic density (PSD) of the CA3 region compared with controls. Treated rats presented also thinner PSD than control rats, and a reduced content of NMDAr subunits (NR1 and NR2A/B subunits) at the PSD in hypothyroid animals.

These indicate differential effects elicited by PTU (i.e., TPO inhibition) on BDNF expression/regulation comparing the adult vs foetal brain.

An additional consideration is that studies in support to this KER have mainly assedd the effects of TPO inhibitors (PTU and MMI) or chemicals known to reduce iodide uptake by NIS (such as BDE-47 and BPA), while, apart from Koibuchi et al., 2001 (showing in vivo downregulation of BDFN upon co-administration with MMI and the NIS inhibitor perchlorate), there are no other studies demonstrating direct associations between NIS inhibition, brain TH levels and BDND expression or protein levels.

Quantitative Understanding of the Linkage

There are no consistent quantitative data linking TH levels in the brain and the level of BDNF expression, due to differences in study designs, dose regimes and the methods used to assess the endpoints.

References

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Inhibition, Na+/I- symporter (NIS) leads to Decreased, Thyroidal iodide uptake

Evidence Supporting Applicability of this Relationship

Empirical evidence comes from in vitro works using rat follicular cells, human in vitro cell models and in vivo data as well as Xenopus oocytes (Lindenthal et al., 2009).

How Does This Key Event Relationship Work

NIS is a membrane protein responsible for iodide transport 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).

Weight of Evidence

Biological Plausibility

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 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 still needs to be elucidated and the molecular modelling of the protein would greatly benefit these studies.

Empirical Support for Linkage

Many studies have shown inhibition of radioactive iodide uptake by using different cell models and assays where NIS function was suppressed. 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 that iodide, are also transported by NIS but Nitrate (NO₃^-), thiocyanate (SCN^-), and perchlorate (ClO₄^-) are of particular dietary and environmental importance (Jones et al., 1996; Tonacchera et al., 2004; De Groef et al., 2006). There are many studies showing the effect of inhibition of NIS on thyroidal iodide uptake:

- Cianchetta et al., 2010 For this study, the FRTL5 thyroid cell line and 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), the fluorescence of which was suppressed following NIS inhibition. Iodide uptake was quantified with the use of fluorescent changes. Perchlorate was shown to inhibit NIS 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 = 1.6 μM) and in the intracellular concentration of iodide (IC₅₀ = 1.1 μM). Perchlorate and iodide had the same effect in COS-7 cells expressing hNIS, meaning that they caused a concentration- and time-dependent reduction of fluorescence to those cells, but they had no effect on COS-7 cells which were not stably transfected with the hNIS. Thus, it was confirmed that the reduction of fluorescence was due to NIS anion transfer 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 inhibitors. The IC₅₀ values for the studied compounds 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 other 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 used 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 iodide uptake in 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. The time-dependent inhibition of the compounds that had an immediate effect in iodide uptake, suggested that they acted 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 on 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 (ITBs) was performed in Xenopus oocytes expressing NIS. The aim of this analysis was to further assess the inhibitory effect of those molecules specifically on NIS activity. 4 of those molecules 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 in the past to be a specific inhibitor of NIS (Vroye et al., 1998). It was found that dysidenin induced a rapid and reversible inhibition of about 40% of the iodide induced current in mouse NIS-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 Human studies have also used potassium perchlorate to predict the perchlorate inhibition of thyroidal iodide uptake with the use of RAIU method. Greer and co-workers tested body weight adjusted doses of potassium perchlorate and an assessment of RAIU uptake was performed on day 2 and day 14 of the treatment and one more assessment on day 15 after the treatment's termination. The no-observed-effect level (NOEL) value for inhibition of thyroidal uptake was 0.007 mg/kg-day, while the true no-effect level (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 was 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 cell line, a breast adenocarcinoma cell line, which expresses inducible NIS by all-trans retinoic acid (ATRA) it has been shown that 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) in FRTL-5 cells and the rat thyroid gland (as measured in vivo by single i.p. injection of 100 μg of I⁻ in 500 μL of distilled water, and analysis of ¹²⁵I thyroid uptake), respectively, 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.

Uncertainties or Inconsistencies

The thyroid system is quite complex and therefore some inconsistent results have been produced by recent studies. For example, it has been observed 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 statistically underpowered.

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 (K_m) 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).

Quantitative Understanding of the Linkage

For this relationship there is not enough data to link quantitatively the inhibition of NIS with the amount of thyroidal uptake. NIS inhibition can be directly measured considering that he 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.

References

Arriagada AA, Albornoz E, Opazo MC, Becerra A, Vidal G, Fardella C, Michea L, Carrasco N, Simon F, Elorza AA, Bueno SM, Kalergis AM, Riedel CA. (2015). Excess iodide induces an acute inhibition of the sodium/iodide symporter in thyroid male rat cells by increasing reactive oxygen species. Endocrinology. Apr;156(4):1540-51.

Braverman LE, Pearce EN, He X, Pino S, Seeley M, Beck B, Magnani B, Blount BC, Firek A. (2006). Effects of six months of daily low-dose perchlorate exposure on thyroid function in healthy volunteers. J Clin Endocrinol Metab. 91:2721-2724.

Cianchetta S, di Bernardo J, Romeo G, Rhoden KJ (2010). Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L. Toxicol Appl Pharmacol. 243:372-380.

Darrouzet E, Lindenthal S, Marcellin D, Pellequer JL, Pourcher T. (2014). The sodium/iodide symporter: state of the art of its molecular characterization. Biocim Biophys Acta. 1838:244-253.

De Groef B, Decallonne BR, Van der Geyten S, Darras VM, Bouillon R. (2006). Perchlorate versus other environmental sodium/iodide symporter inhibitors: potential thyroid-related health effects. Europ J Endocr. 155:17-25.

Eskandari S, Loo DD, Dai G, Levy O, Wright M, Carrasco N. (1997). Thyroid Na+/I- symporter: mechanism, stoichiometry, and specificity. J Biol Chem 272: 27230-27238.

Greer MA, Goodman G, Pleus RC, Greer SE. (2002). Health effects assessment for environmental perchlorate contamination: the dose response for inhibition of thyroidal radioiodine uptake in humans. Environm Health Persp. 110: 927-937.

Jones PA, Pendlington RU, Earl LK, Sharma RK, Barrat MD. (1996). In vitro investigations of the direct effects of complex anions on thyroidal iodide uptake: identification of novel inhibitors. Toxicol. In Vitro. 10: 149-160.

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Kosugi S, Okamoto H, Tamada A, Sanchez-Franco F. (2002). A novel peculiar mutation in the sodium/iodide symporter gene in Spanish siblings with iodide transport defect. J Clin Endocrinol Metab. 87: 3830–3836.

Lecat-Guillet N, Merer G, Lopez R, Pourcher T, Rousseau B, Ambroise Y. (2008a). Small-molecule inhibitors of sodium iodide symporter function. Chembiochem 9:889–895.

Lecat-Guillet N, Ambroise Y. (2008b). Discovery of aryltrifluoroborates as potent sodium/iodide symporter (NIS) inhibitors. Chem Med Chem 3:1207–1209.

Lecat-Guillet N, Merer G, Lopez R, Pourcher T, Rousseau B, Ambroise Y. (2007). A 96-well automated radioiodide uptake assay for sodium/iodide symporter inhibitors. Assay Drug Dev Technol 5:535-540.

Lindenthal S, Lecat-Guillet N, Ondo-Mendez A, Ambroise Y, Rousseau B, Pourcher T. (2009). Characterization of small-molecule inhibitors of the sodium iodide symporter. J Endocrinol 200:357–365.

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Pohlenz J, Rosenthal IM, Weiss RE, Jhiang SM, Burant C, Refetoff S. (1998). Congenital hypothyroidism due to mutations in the sodium/iodide symporter. Identification of a nonsense mutation producing a downstream cryptic 3′ splice site. J Clin Invest. 101:1028-1035.

Pohlenz J, Medeiros-Neto G, Gross JL, Silveiro SP, Knobel M, Refetoff S. (1997). Hypothyroidism in a Brazilian kindred due to iodide trapping defect caused by a homozygous mutation in the sodium/iodide symporter gene. Biochem Biophys Res Commun. 240: 488-491.

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Schlosser PM. (2016). Revision of the affinity constant for perchlorate binding to the sodium-iodide symporter based on in vitro and human in vivo data. J Appl Toxicol. Dec;36(12):1531-1535.

Spitzweg C, Morris JC. (2010). Genetics and phenomics of hypothyroidism and goiter due to NIS mutations. Mol Cell Endocrinol. 322: 56-63.

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Decreased, Thyroxin (T4) in serum leads to Decreased, Thyroxine (T4) in neuronal tissue

Evidence Supporting Applicability of this Relationship

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.

How Does This Key Event Relationship Work

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. 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 the blood. More detailed descriptions of this process can be found in reviews by Braverman 2012, Utiger 2006, and Zoeller et al., 2007.

Monocarboxylate transporter 8 (MCT8) is a specific human transporter for the T4 and T3 that allows their entry in the brain and other organs. Mutations in MCT8 (Allan-Herndon-Dudley syndrome, AHDS) lead to a severe form of X-linked truncal hypotonia, spasticity, mental retardation, and are characterised by normal to high TSH, elevated plasma T3, low T4, and decreased TH signaling in discrete brain areas (McAninch and Bianco, 2014; Anık et al., 2014; López-Espíndola et al., 2014).

Other in vivo studies have proven direct associations between levels of serum T4 and the levels of TH-dependent signalling in the brain (i.e., brain TH levels). For instance, mice characterized by single MCT8 deficiency showed low serum T4, elevated serum TSH and T3, and decreased T3-dependent gene expression in both the hypothalamus and the cortex (Stohn et al., 2016). Analogously, another study reported that MCT8 knock-out mice were characterized by high serum T3, low serum T4, and decreased forebrain TH content (Müller et al., 2014). The hippocampus seems to be one of the brain regions mostly affected by low TH levels, as shown in thyroidectomized Wistar rats, which resulted affected by hippocampal hypothyroidism (da Conceição et al., 2016). These data altogether support direct associations between levels of serum T4 and levels of TH-dependent signalling in the brain (i.e., brain TH levels).

Weight of Evidence

Biological Plausibility

The biological relationship between these two KEs is a well accepted fact within the scientific community. There is no doubt that decreased circulating T4 leads to declines in tissue contractions of T4 and T3 in a variety of tissues, including the brain.

Empirical Support for Linkage

Several studies have shown that tissue levels, including brain, of TH are proportional to serum TH levels (Oppenheimer, 1983; Morreale de Escobar et al., 1987; 1990; Calvo et al., 1992; Porterfield and Hendrich, 1992, 1993; Porterfield, 1994; Broedel et al., 2003). Empirical support for the linkage comes from pathological analyses of human brain tissues and in vivo studies on MCT8 knock-out mice and thyroidectomized rats.

Four male patients affected by AHDS from two unrelated Turkish families (age from 1.5 to 11 years) presented high plasma T3, low plasma T4 and rT3, and normal/mildly elevated TSH levels. Functional analysis of a novel missense MCT8 mutation (p.G495A) revealed lowered TH transport (from 20 to 85%) depending on the cell/tissue context. Phenotypically, patients were characterized by severe psychomotor retardation, axial hypotonia, lack of speech, diminished muscle mass, increased muscle tone, hyperreflexia, myopathic facies (Anık et al., 2014).

Another study analyzed brain sections from a 30^th gestational week male fetus and an 11-year-old boy in comparison with  brain tissue from a 30^th and 28^th gestational week male and female fetuses, respectively, and a 10-year-old girl and a 12-year-old boy, as controls. The MCT8-deficient fetal cerebral cortex showed 50% reduction of TH (i.e., T4, T3, and rT3), while T3 and T4 levels were normal in the liver. This TH deficiency in the brain produced an expected increase in type 2 deiodinase and decrease in type 3 deiodinase mRNA expression. Also, MCT8-deficient fetus showed a delay in cortical and cerebellar development and myelination, loss of parvalbumin expression, abnormal calbindin-D28k content, impaired axonal maturation, and diminished biochemical differentiation of Purkinje cells. The 11-year-old boy displayed altered cerebellar structure, deficient myelination, deficient synaptophysin and parvalbumin expression, and abnormal calbindin-D28k expression (López-Espíndola et al., 2014).

Several in vivo studies have proven direct associations between levels of serum T4 and the levels of TH-dependent signalling in the brain (i.e., brain TH levels). For instance, mice characterized by single MCT8 deficiency showed low serum T4, elevated serum TSH and T3, and decreased T3-dependent gene expression in both the hypothalamus and the cortex (Stohn et al., 2016).

Analogously, another study reported that MCT8 knock-out mice were characterized by high serum T3, low serum T4, and decreased forebrain TH content, while TH concentrations in the liver, kidneys, and thyroid gland resulted increased (Müller et al., 2014).

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).

The hippocampus seems to be one of the brain regions mostly affected by low TH levels, as shown in thyroidectomized Wistar rats, which resulted affected by hippocampal hypothyroidism. Thyroidectomized rats were characterized by increased serum TSH, decreased T4 and T3 serum levels, and a reduced hippocampal expression of MCT8, thyroid hormone receptor alfa, deiodinase type 2, ectonucleotide pyrophosphatase/phosphodiesterase 2 and brain-derived neurotrophic factor (BDNF) genes (da Conceição et al., 2016). These data altogether support direct associations between levels of serum T4 and levels of TH-dependent signalling in the brain (i.e., brain TH levels).

Uncertainties or Inconsistencies

The ability of the developing brain to compensate for TH insufficiency is not well known. There may also be 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).

Quantitative Understanding of the Linkage

While it is a well-established fact decreased in serum TH levels result in decreased tissue TH levels, a major gap is the lack of empirical data that allow quantification of this relationship. There may also be different quantitative relationships between these two KEs depending on the compensatory ability of different developing brain regions (Sharlin et al., 2010).

- Fisher et al., (2013) recently published a quantitative biologically-based dose-response model (BBDR) for iodine deficiency in the rat. In particular, HPT axis adaptations to dietary iodide intake in euthyroid (4.1-39 µg iodide/day) and iodide-deficient (0.31 and 1.2 µg iodide/day) conditions were evaluated. In rat pups that were iodide deficient during gestation and lactation, decreases in serum T4 levels were associated with declines in TH levels in the fetal brain and a suppression of synaptic responses in the hippocampal region of the brain of the adult offspring (Gilbert et al., 2013).

- Anık et al., 2014 Four males with AHDS from two unrelated Turkish families (age from 1.5 to 11 years) presented high plasma T3, low plasma T4 and rT3, and normal/mildly elevated TSH levels. Functional analysis of a novel missense MCT8 mutation (p.G495A) revealed lowered TH transport (from 20 to 85%) depending on the cell/tissue context.

- López-Espíndola et al., 2014 This study analyzed brain sections from a 30^th gestational week male fetus and an 11-year-old boy in comparison with  brain tissue from a 30^th and 28^th gestational week male and female fetuses, respectively, and a 10-year-old girl and a 12-year-old boy, as controls. The MCT8-deficient fetal cerebral cortex showed 50% reduction of TH (i.e., T4, T3, and rT3), while T3 and T4 levels were normal in the liver. This TH deficiency in the brain produced an expected increase in type 2 deiodinase and decrease in type 3 deiodinase mRNA expression.

- Stohn et al., 2016 In this study, mice characterized by single MCT8 deficiency showed low serum T4 (~ 47% decrease vs control mice, at age P21), elevated serum TSH (~ 125% increase vs control mice, at age P21)  and T3 (~ 33% increase vs control mice, at age P21), and decreased T3-dependent gene expression in both the hypothalamus (~ 57% decrease vs control mice, at age P21) and the cortex (~ 40% decrease vs control mice, at age P21), as indicated by the lower expression of the rat hairless (hr) gene, which is highly up-regulated by TH in the developing CNS.

- Müller et al., 2014 This study reported that MCT8 knock-out mice were characterized by high serum T3 (~ 100% increase at P21, and ~ 300% increase at 2.5 months of age, compared to control mice), low serum T4 (~ 62% decrease at P21, and ~ 50% decrease at 2.5 months of age, compared to control mice), and decreased TH content in the forebrain (~ 43% decrease for both T3 and T4, at 2.5 months of age, compared to control mice), while TH concentrations in the liver, kidneys, and thyroid gland resulted increased.

- da Conceição et al., 2016 Thyroidectomized Wistar rats resulted affected by hippocampal hypothyroidism. Thyroidectomized rats were characterized by increased serum TSH (~ 750% increase vs control rats), decreased T4 (~ 80% decrease vs control rats) and T3 (~ 45% decrease vs control rats) serum levels, and a reduced hippocampal expression of MCT8 (~ 83% decrease vs control rats), thyroid hormone receptor alfa (Trα1, ~ 77% decrease vs control rats), deiodinase type 2 (Dio2, ~ 90% decrease vs control rats), ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2, ~ 80% decrease vs control rats) and brain-derived neurotrophic factor (BDNF, ~ 75% decrease vs control rats) mRNAs.

References

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Decreased, Thyroidal iodide uptake leads to Decreased, Thyroid hormone synthesis

Evidence Supporting Applicability of this Relationship

Inhibition of NIS expression and/or activity, leading to reduction of iodide uptake is known to impact TH synthesis, and this may occur also via direct or indirect inhibition of TPO, as suggested by in vivo studies in rats and using thyroid follicular rat cells. On the other hand, TPO inhibitors, such as MMI and PTU have been found to decrease NIS expression and activity in rat follicular thyroid cells vitro (Spitzweg et al., 1999). Moreover, human epidemiological studies indicate that inhibitors of iodide uptake via NIS (e.g., perchlorate and thiocyanate) have been found to raise TSH levels (Steinmaus et al., 2016b; Horton et al., 2015; Brechner et al., 2000), indicating reduced TH synthesis. Also, perchlorate was found to decrease Tg and TPO gene expression in rats, indicating reduction of TH biosynthesis (Wu F et al., 2012).

How Does This Key Event Relationship Work

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 disturb thyroid hormone (TH) homeostasis, 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, these compounds elicit also 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).

Weight of Evidence

Biological Plausibility

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 (i.e. TSH receptor and TPO inhibition) (KE down).

Empirical Support for Linkage

- Spitzweg et al., 1999: 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 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 Y et al., 2016: This 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, with perchlorate (NIS inhibitors) having 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 whose drinking water supply was 100% perchlorate-contaminated water (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.

- Wu F et al., 2012: This study found that high dose 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 FT3 (~ 50% lower than control) and FT4 levels (~ 50% lower than control), and a remarkable increase of TSH levels (125% higher than control).

Several other studies have proven that NIS inhibitors lead to a decrease of thyroidal iodide uptake (as detailed in KER1) (Jones et al., 1996; Tonacchera et al., 2004; De Groef et al., 2006; Waltz et al., 2010), leading to a reduction of TH synthesis.

Uncertainties or Inconsistencies

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 small sample sizes, short study durations, and the inclusion of mostly healthy adults (Steinmaus, 2016b).

Quantitative Understanding of the Linkage

In vitro and in vivo studies have specifically reported data supporting quantitative understanding of this KER.

- Spitzweg et al., 1999:  this 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 are needed in order to drive global conclusions about the magnitude of iodide uptake inhibition required to impact TH synthesis.

References

Brechner RJ, Parkhurst GD, Humble WO, Brown MB, Herman WH. (2000). Ammonium perchlorate contamination of Colorado River drinking water is associated with abnormal thyroid function in newborns in Arizona. J Occup Environ Med. Aug;42(8):777-82.

Dai G, Levy O, Carrasco N. (1996). Cloning and characterization of the thyroid iodide transporter. Nature;379:458–460.

Davidson, B., Soodak, M., Neary, J.T., Strout, H.V., and Kieffer, J.D. (1978). The irreversible inactivation of thyroid peroxidase by methylmercaptoimidazole, thiouracil, and propylthiouracil in vitro and its relationship to in vivo findings. Endocrinology 103:871–882.

De Groef B, Decallonne BR, Van der Geyten S, Darras VM, Bouillon R. (2006). Perchlorate versus other environmental sodium/iodide symporter inhibitors: potential thyroid-related health effects. Europ J Endocr. 155:17-25.

Horton MK, Blount BC, Valentin-Blasini L, Wapner R, Whyatt R, Gennings C, Factor-Litvak P. (2015). CO-occurring exposure to perchlorate, nitrate and thiocyanate alters thyroid function in healthy pregnant women. Environ Res. Nov;143(Pt A):1-9.

Jones PA, Pendlington RU, Earl LK, Sharma RK, Barrat MD. (1996). In vitro investigations of the direct effects of complex anions on thyroidal iodide uptake: identification of novel inhibitors. Toxicol. In Vitro. 10: 149-160.

Kleiman DA, Buitrago D, Crowley MJ, Beninato T, Veach AJ, Zanzonico PB, Jin M, Fahey TJ 3rd, Zarnegar R. (2013). Thyroid stimulating hormone increases iodine uptake by thyroid cancer cells during BRAF silencing. J Surg Res. Jun 1;182(1):85-93.

Pearce EN, Lazarus JH, Smyth PP, He X, Dall'amico D, Parkes AB, Burns R, Smith DF, Maina A, Bestwick JP, Jooman M, Leung AM, Braverman LE. (2010). Perchlorate and thiocyanate exposure and thyroid function in first-trimester pregnant women. J Clin Endocrinol Metab. Jul;95(7):3207-15.

Riesco-Eizaguirre G, Rodríguez I, De la Vieja A, Costamagna E, Carrasco N, Nistal M, Santisteban P. (2009). The BRAFV600E oncogene induces transforming growth factor beta secretion leading to sodium iodide symporter repression and increased malignancy in thyroid cancer. Cancer Res. Nov 1;69(21):8317-25.

Spitzweg C, Morris JC. (2010). Genetics and phenomics of hypothyroidism and goiter due to NIS mutations. Mol Cell Endocrinol. 322: 56-63.

Spitzweg C, Joba W, Morris JC, Heufelder AE. (1999). Regulation of sodium iodide symporter gene expression in FRTL-5 rat thyroid cells. Thyroid. Aug;9(8):821-30.

Steinmaus CM. (2016a). Perchlorate in Water Supplies: Sources, Exposures, and Health Effects. Curr Environ Health Rep. Jun;3(2):136-43.

Steinmaus C, Pearl M, Kharrazi M, Blount BC, Miller MD, Pearce EN, Valentin-Blasini L, DeLorenze G, Hoofnagle AN, Liaw J. (2016b). Thyroid Hormones and Moderate Exposure to Perchlorate during Pregnancy in Women in Southern California. Environ Health Perspect. Jun;124(6):861-7.

Sue M, Akama T, Kawashima A, Nakamura H, Hara T, Tanigawa K, Wu H, Yoshihara A, Ishido Y, Hiroi N, Yoshino G, Kohn LD, Ishii N, Suzuki K.(2012). Propylthiouracil increases sodium/iodide symporter gene expression and iodide uptake in rat thyroid cells in the absence of TSH. Thyroid. 2012 Aug;22(8):844-52.

Sugawara M, Sugawara Y, Wen K. (1999). Methimazole and propylthiouracil increase cellular thyroid peroxidase activity and thyroid peroxidase mRNA in cultured porcine thyroid follicles. Thyroid. May;9(5):513-8.

Szkudlinski MW, Fremont V, Ronin C, Weintraub BD. (2002). Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiological Reviews. 82 (2): 473–502.

Tarone RE, Lipworth L, McLaughlin JK. (2010). The epidemiology of environmental perchlorate exposure and thyroid function: a comprehensive review. J Occup Environ Med. Jun;52(6):653-60.

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, 47–81.

Téllez Téllez R, Michaud Chacón P, Reyes Abarca C, Blount BC, Van Landingham CB, Crump KS, Gibbs JP. (2005). Long-term environmental exposure to perchlorate through drinking water and thyroid function during pregnancy and the neonatal period. Thyroid, Sep;15(9):963-75.

Tonacchera M, Pinchera A, Dimida A, Ferrarini E, Agretti P, Vitti P, Santini F, Crump K, Gibbs J. (2004). Relative potencies and additivity of perchlorate, thiocyanate, nitrate, and iodide on the inhibition of radioactive iodide uptake by the human sodium iodide symporter. Thyroid, Dec;14(12):1012-9.

Waltz F, Pillette L, Ambroise Y. (2010). A nonradioactive iodide uptake assay for sodium iodide symporter function. Anal Biochem. 396:91-95.

Wu F, Zhou X, Zhang R, Pan M, Peng KL. (2012). The effects of ammonium perchlorate on thyroid homeostasis and thyroid-specific gene expression in rat. Environ Toxicol. Aug;27(8):445-52.

Wu Y, Beland FA1, Fang JL. (2016). Effect of triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes involved in thyroid hormone synthesis. Toxicol In Vitro. Apr;32:310-9.

Decreased, Thyroid hormone synthesis leads to Decreased, Thyroxin (T4) in serum

Evidence Supporting Applicability of this Relationship

While the majority of the empirical evidence comes from work with laboratory rodents, there is a large amount of supporting data from humans (with anti-hyperthyroidism drugs including propylthiouracil and methimazole), some amphibian species (e.g., frog) (Hornung et al. 2010), and some avian species (e.g., chicken) (Van Herck et al. 2013).

Despite many physiological similarities, humans and rats exhibit significantly different susceptibilities to thyroid perturbation. For instance, dose-response data for changes in serum T3, T4, and TSH levels have been analysed from studies in humans, rats, mice, and rabbits. It was found that thyroid homeostasis in the rat appeared to be strikingly more sensitive to perchlorate (NIS inhibitor) than any of the other species. Therefore, data obtained from rat studies should be critically evaluated for their relevance to humans (Lewandowski et al., 2004).

How Does This Key Event Relationship Work

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. NIS main physiological function is to transport one iodide ion along with two sodium ions across the basolateral membrane of thyroid follicular cells. 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 and TPO are both essential for the synthesis of thyroid hormones (T3 and T4).

Compounds, such as triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and bisphenol A (BPA) have been reported to disturb TH homeostasis by inducing an inhibition of NIS-mediated iodide uptake and altering the expression of genes involved in TH synthesis, as shown in rat thyroid follicular FRTL-5 cells, and on the activity of TPO, using rat thyroid microsomes (Wu Y et al., 2016).

Perchlorate, thiocyanate, nitrate, and iodide, competitive inhibitors of iodide uptake, have been shown to inhibit radioactive iodide uptake by NIS (Tonacchera et al. 2004). Consequentially, these compounds seem to elicit also inhibition of TH synthesis. In particular, perchlorate blocks iodide uptake into the thyroid and decreases the production of TH (Steinmaus, 2016a).

Secretion of TH 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 THs into blood. More detailed descriptions of this process can be found in reviews by Braverman and Utiger, 2012 and Zoeller et al., 2007.

Weight of Evidence

Biological Plausibility

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

Empirical Support for Linkage

There is limited direct evidence supporting the relationship between decreased TH syntheses 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. So while empirical data is scarce, it is widely accepted fact the TPO inhibition leads to declines in serum TH levels. This is due to the fact that the sole source for circulating T4 is thyroid gland synthesis. Indeed, it has been known for decades that insufficient dietary iodine will lead to decreased serum TH concentrations due to inadequate synthesis. 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 ¹³¹I and in vivo treatment of a variety of species with known TPO inhibitors (Atterwill et al., 1990; Brown et al., 1986; Brucker-Davis, 1998; Hornung et al., 2010; Hurley et al., 1998; Kohrle, 2008).

Similarly, other studies have shown associations between NIS inhibition and decreased TH serum levels (Dong et al., 2017; Calil-Silveira et al., 2016; Tang et al., 2013; Liu et al., 2012; Pearce et al., 2012).

Temporal Evidence: there is a lack of studies that measured both TPO synthesis and TH production during development. Antonica and co-workers reported that a transient overexpression of the transcription factors NKX2-1 and PAX8 is sufficient to direct mouse embryonic stem-cell differentiation into thyroid follicular cells that organize into three-dimensional follicular structures when treated with thyrotropin. These in vitro-derived follicles showed appreciable iodide organification activity, and when grafted in vivo into athyroid mice, these follicles were able to rescue TH plasma levels, promoting subsequent symptomatic recovery (Antonica et al., 2012).

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 following development exposures (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).

Uncertainties or Inconsistencies

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, there are a number of other processes (e.g., endocytosis, lysosomal fusion, basolateral fusion and release) that are not as well studied.

Quantitative Understanding of the Linkage

- Fisher et al., 2013 recently published a quantitative biologically-based dose-response model (BBDR) 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.  In particular, HPT axis adaptations to dietary iodide intake in euthyroid (4.1-39 µg iodide/day) and iodide-deficient (0.31 and 1.2 µg iodide/day) conditions were evaluated. Alterations in T4 homeostasis were more apparent than for T3. In rat pups that were iodide deficient during gestation and lactation, decreases in serum T4 levels were associated with declines in TH levels in the fetal brain and a suppression of synaptic responses in the hippocampal region of the brain of the adult offspring (Gilbert et al., 2013).

There are also a few other computational models that include TH 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 and has recently adapted for rat (Leonard et al., 2016). 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 THs and TPO inhibition, or thyroidal hormone 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.

- Steinmaus et al., 2016b Authors of this study showed that the presence of high level of perchlorate, thiocyanate, nitrate, and iodide (NIS inhibitors) in water supply could induce a decrease of total 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 TSH, as shown in 1880 pregnant women from San Diego County, California, during 2000–2003.

- Wu F et al., 2012 Authors of this study found that high dose perchlorate (520 mg/kg body weight) 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).

- Dong et al., 2017 Wistar rats exposed (at 0, 150, 300, and 600 mg/kg/day for 3 and 6 months) to di-(2-ethylhexyl)phthalate (DEHP), a chemical known to elicit a reduction of serum TH levels, underwent decreased of serum TH levels (FT3, FT4 in the DEHP-dosed group were lower than those in the control (p < 0.05); with the increase of DEHP-treated dose, rats serum TT3, TT4 and TSH level in all groups of different DEHP doses were lower than the control group at 6 M (p < 0.05), as well as a perturbation (i.e., a general increase) of  the expression of thyrotropin releasing hormone receptor (TRHr), Deiodinases 1 (D1), thyroid stimulating hormone beta (TSHβ), NIS, thyroid stimulating hormone receptor (TSHr), TPO, thyroid transcription factor 1 (TTF-1), and thyroglobulin (TG) genes. In particular, NIS protein, after 3 month exposure to middle (300 mg/kg/day) and high-dose (600 mg/kg/day) DEHP resulted decreased (by ~ 30% with 300 mg/kg/day, and by ~ 85% with 600 mg/kg/day), whilst after 6 months a progressive protein increase was observed. Globally these data indicate that DEHP-dependent reduction of TH serum levels occurs via a strong perturbation of the HPT axis.

- Calil-Silveira et al., 2016 Male Wistar rats treated for two months with NaI (0.05% and 0.005%) or NaI⁺NaClO₄ (0.05%) (NIS inhibitors), underwent high levels of urine iodine, increased serum thyrotropin levels, slightly decreased serum TH levels, and a decreased expression of NIS, thyrotropin receptor, and TPO mRNA and protein, indicating a primary thyroid dysfunction.

- Tang et al., 2013 showed that 2,3',4,4',5-pentachlorobiphenyl (PCB118) (i.p. injected in male Wistar rats at 10, 100, or 1000 μg/kg/day, 5 days/week for 13 weeks) caused a progressive decrease of free T4, free T3 and TSH levels in serum (e.g., serum free T3, free T4 and TSH were reduced to 75%, 31% and 52%, respectively, at 1000 μg/kg/day PCB118), together with a downregulation of NIS (~ 30% at 1000 μg/kg/day PCB118) and Tg (~ 40% at 1000 μg/kg/day PCB118) mRNA expression. Moreover, PCB118 led to histopathological deterioration of the thyroid (i.e., follicular hyperplasia and expansion).

- Liu et al., 2012 This in vivo study reported that PCB153 (i.p. injected in Sprague-Dawley rats at 0, 4, 16 and 32 mg/kg/day for 5 consecutive days) caused a decrease of NIS, TPO and Tg, deiodinases, the receptors (TSHr and TRHr), together with a decrease of serum total T4, total T3, and thyrotropin releasing hormone (TRH).

- Pearce et al., 2012 A cross-sectional study conducted on 134 first-trimester pregnant women from Athens, Greece, showed an inverse correlations between the level of urinary perchlorate (NIS inhibitor) and free T3 and free T4 plasma values.

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