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Alexandra Rolaki, Sharon Munn and Anna Bal-Price* (*corresponding author)
European Commission Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy
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The thyroid hormones (TH) are essential for brain development, maturation, and function as they regulate the early key developmental processes such as neurogenesis, cell migration, proliferation, myelination and neuronal and glial differentiation. Normal brain development and cognitive function in mammals relays on sufficient production of TH during the perinatal period. The function of Na+/I- symporter (NIS) is critical for the physiological production of TH levels in the serum, as it is a membrane bound glycoprotein that mediates the transport of iodide form the bloodstream into the thyroid cells, and this constitutes the initial step for TH synthesis. NIS is a well-studied target of chemicals, and its inhibition results in decreased hormone synthesis and secretion into blood leading to subsequent TH insufficiency in the brain with detrimental effects in neurocognitive function in children. The present AOP describes developmental neurotoxicity (DNT) effects induced by the decreased levels of TH in the blood and consequently in the brain, as a result of NIS inhibition. Many environmental chemicals have been reported to disrupt iodide uptake, but the studies that have been focused on NIS inhibition are mainly restricted to perchlorate and some small ionic or drug-like molecules. Perchlorate, which is the most potent inhibitor of NIS, has been associated with reduced TH production and also with cognitive deficits in animals and human.
Summary of the AOP
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Molecular Initiating Event
|Molecular Initiating Event||Support for Essentiality|
|Na+/I- symporter (NIS), Inhibition||Strong|
|Learning and memory, Impairment|
Relationships Among Key Events and the Adverse Outcome
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Life Stage Applicability
|Homo sapiens||Homo sapiens||Strong||NCBI|
|Rattus sp.||Rattus sp.||Strong||NCBI|
Overall Assessment of the AOP
Domain of Applicability
Life Stage Applicability,
Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains.
Essentiality of the Key Events
Molecular Initiating Event Summary,
Key Event Summary
The essentiality of each one of the key events in this AOP was supported by introducing a recovery period in the exposure experiments with NIS inhibitors, mainly with perchlorate. Greer et al., 2002, showed that after a recovery period of 15 days the inhibitory effect of perchlorate was eliminated, almost completely, as the measurements of iodide uptake were indistinguishable from their respective baseline values. Similar results were produced in other studies after a longer recovery period of 30 days, in which the iodide uptake as well as the serum TH levels returned to their baseline values (Siglin et al., 2000). The essential effect of NIS inhibition to thyroidal iodide uptake was also shown with the use of cells that did not endogenously express the NIS transfer protein (Cianchetta et al., 2010). In those experiments iodide was not transferred through the cellular membrane unless the cells were previously transfected with hNIS.
Weight of Evidence Summary
Biological Plausibility: The functional relationship between NIS and thyroidal iodide uptake is well established but the structural is still lacking supporting evidence. In the human, NIS mutations are associated with congenital iodide transport defect, a condition characterized by low iodide uptake, hypothyroidism and goiter (Bizhanova and Kopp, 2009; De La Vieja et al., 2000; Pohlenz and Refetoff, 1999). The same is true for the relationship between iodide uptake and serum TH concentration, as it is known that Iodide Deficient (ID) suffer also by low thyroid levels in the blood (Wolff, 1998; DeLange, 2000). The correlation of serum and brain concentrations of TH are supported by a smaller amount of quantitative data but the biological plausibility of this connection is mainly based on the number of studies that show that the brain TH is proportional to the serum TH (Broedel et al., 2003). BDNF is thought to underlie the effects of developmental hypothyroidism but this notion is based mainly on their common physiological role during brain development rather than on solid experimental evidence (Gilbert and Lasley, 2013). On the other hand, the role of BDNF on the GABAergic interneurons development and function is well established, as many experimental data have been produced the last decades in support to this relationship (Woo and Lu, 2006; Palizvan et al., 2004; Patz et al., 2004). It is also widely accepted that the GABAergic signalling and therefore the proper function of GABAergic interneurons is fundamental for the normal synapse formation, which in turn controls the neuronal network formation, maturation and function. Numerous studies have shown that the depolarizing GABA signalling is controlled by the intracellular Cl- concentration in the postsynaptic cells and is the first drive for synapse formation (Wang and Kriegstein, 2008; Cancedda et al., 2007; Ge et al., 2006; Chudotvorova et al., 2005; Akerman and Cline, 2006). This early synaptogenesis period is critical for the establishment of the basic neuronal circuitry, despite the fact that synaptogenesis is a continuous process throughout life (Rodier, 1995).
Dose-response concordance: Multiple events were considered together in only limited number of studies. There is overwhelming evidence that supports the concordance of NIS inhibition with the decrease of thyroidal iodide uptake or the lower levels of serum TH but these two events have rarely been tested together. However, in the few cases that the levels of thyroidal iodide and the serum TH levels are measured in the same study the results are mostly conflicting, mainly due to the well-developed compensatory mechanisms that exist to maintain the TH levels in the body. That means that the effects of NIS inhibitors might not be detectable in short-term or low-dose experiments. Perchlorate is a well-described NIS inhibitor and the interpretation of related studies is straightforward because thyroid is considered the critical effect organ of perchlorate toxicity (National Research Council 2005); thus, any effects of perchlorate on the nervous system are necessarily interpreted to be subsequent to inhibition of iodide uptake by the thyroid gland and to a reduction in serum THs. Indeed, the use of potassium or sodium perchlorate has contributed to the identification of a dose-response relationships between NIS inhibition and thyroidal iodide uptake (Greer et al., 2002; Tonacchera et al., 2004; Cianchetta et al., 2010; Waltz et al., 2010; Lecat-Guillet et al., 2007; 2008) but the respective concordance with serum TH was not shown in most of these studies. On the other hand, in the human and animal studies that revealed a strong dose-dependent association between perchlorate exposure and circulating levels of TH (Blount et al., 2006; Cao et al., 2010; Suh et al., 2013; Steinmaus et al., 2007; Steinmaus et al., 2013; Siglin et al., 2000; Caldwell et al., 1995; Argus research laboratories 2001; York et al., 2003; York et al., 2004), the decrease of thyroidal iodide was not investigated. The downstream effects of TH insufficiency are better understood and documented but the majority of the dose-response data are derived from hypothyroid rodents after exposure with propylthiouracil (PTU) and methimazole (MMI), which is the most common used chemicals for the production of hypothyroid state to animals. Those types of experiments give information on the mechanisms through which TH insufficiency leads to neurodevelopmental deficits, but this pathway cannot be connected with NIS inhibition as data on specific NIS inhibitors is still lacking. In regards to the downstream events in the pathway, there is a strong correlation between each KE but the majority of the studies have been performed under severe hypothyroid conditions (high doses of PTU and/or MMI, thyroidectomies); therefore it is difficult to establish the dose-response relationships in each one of them. The association between serum TH levels and BDNF protein in the brain is very well documented but with the exception of few cases (Chakraborty et al., 2012; Blanco et al., 2013) no dose-response experiments are available. The same problem is also encountered in the relationship between BDNF levels and the GABAergic function, as there is only one recent study (Westerholz et al., 2013) that describes a correlation between these two events, but the results are described on the basis of T3 presence or complete absence in the cultures, which does not allow the establishment of dose-response evaluation. However, a dose-response relationship has been shown in earlier studies between the T3 hormone and the density of synapses in cortical cultures, an effect which was paralleled with the electrical activity of the network (Westerholz et al., 2010; Hosoda et al., 2003). More recently, a model of low level TH disruption has been developed, in which different concentrations of PTU have been tested and the subsequent dose-response relationships with GABAergic interneurons expression, synaptogenesis and learning and memory deficits were established (Sui and Gilbert, 2003; Gilbert and Sui, 2006; Gilbert, 2011; Gilbert et al, 2006; Berbel et al., 1996). Additionally, results from animal studies with perchlorate have also shown a dose-dependent reduction in excitatory and inhibitory synaptic function leading to learning and memory impairments (Gilbert and Sui, 2008). In contrast, there is only limited data in support to the correlation between TH insufficiency and the neuronal network function, and no dose-response relationship can be established.
Temporal concordance: In regards to temporality, the concordance between the KEs from the NIS inhibition until the TH levels in the brain is well-established. It is widely accepted that the most important role of iodine is the formation of the thyroid hormones (T4 and T3) and that iodine deficiency early in development can cause severe hypothyroidism leading to irreversible neurocognitive impairments (DeLange, 2000; Zimmermann et al., 2006). The majority of the data on TH insufficiency is derived from studies performed in different developmental stages and this study design facilitates the establishment of temporal concordance between the downstream KEs in the AOP. In general, TH insufficiency during the prenatal and early post-natal period is correlated with deficits in GABAergic morphology and function, especially of PV-positive interneurons (Berbel et al., 1996; Gilbert et al., 2007; Westerholz et al., 2010; 2013), with the decrease of active synapses and of synchronized electrical activity in cortical networks (Westerholz et al., 2010; Hosoda et al., 2003). This developmental window is known to be critical for the brain development and therefore TH deficits during this period has been correlated with mental retardation and other neurological impairments in children, which in some cases are irreversible (Mirabella et al., 2000; Porterfield and Hendrich, 1993). In at least two studies multiple KEs have been considered together and provide important information on the temporality of the AOP. Westerholz et al., 2010 and 2013 have shown that TH insufficiency during the first two postnatal weeks may cause alterations in the morphology and function of PV-positive GABAergic interneurons, with subsequent effects on the number of active synapses and the electrical activity of the neuronal network. During the same period the inhibition of BDNF function was shown to be also involved in the formation of synaptic connections (Westerholz et al., 2013). Further investigation of the mediating mechanisms revealed that a critical function in the above mentioned cascade was the timely shift of GABA signalling from depolarization to hyperpolarization, a milestone in brain development. The GABA switch takes place at the end of the second postnatal week in rodents, and thus we can conclude that all the KEs are performed during the perinatal period up to 14 days postnatal, which fits in the overall AOP, as this is the critical period for synaptogenesis and subsequently for the proper development of learning and memory functions.
Provide an overall discussion of the quantitative information available for this AOP. Support calls for the individual relationships can be included in the Key Event Relationship table above.
Considerations for Potential Applications of the AOP (optional)
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