API

Event: 424

Key Event Title

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Inhibition, Na+/I- symporter (NIS)

Short name

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Inhibition, Na+/I- symporter (NIS)

Key Event Component

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Process Object Action
sodium:iodide symporter activity sodium/iodide cotransporter decreased

Key Event Overview


AOPs Including This Key Event

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Stressors

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Level of Biological Organization

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Biological Organization
Molecular

Cell term

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Cell term
thyroid follicular cell


Organ term

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Taxonomic Applicability

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Term Scientific Term Evidence Link
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
mouse Mus musculus Strong NCBI
Pig Pig Strong NCBI
zebra fish Danio rerio Strong NCBI
Xenopus (Silurana) epitropicalis Xenopus (Silurana) epitropicalis Moderate NCBI

Life Stage Applicability

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Life stage Evidence
Pregnancy Moderate
Birth to < 1 month Moderate
During brain development Moderate

Sex Applicability

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Term Evidence
Mixed Strong

How This Key Event Works

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Biological state: Sodium/Iodide symporter (NIS) is a key protein in the thyroid function and its role has been thoroughly investigated after the determination of its molecular identity a few decades ago (Dai et al., 1996). NIS is an intrinsic membrane glycoprotein and it belongs to the superfamily of sodium /solute symporters (SSS) and to the family of human transporters SLC5 (De La Vieja, 2000; Jung, 2002). Its molecular weight is 87 kDa and it contains 13 transmembrane domains tha transport 2 sodium cations (Na+) for each iodide anion (I-) into the follicular thyroid cell (Dohan et al., 2003). The regulation of NIS protein function is usually cell- and tissue-specific (Hingorani et al., 2010) and it is done at the transcriptional and posttranslational levels, including epigenetic regulation (Darrouzet et al., 2014; Russo et al., 2011a). One of the major NIS regulators is the thyroid stimulating hormone (TSH), which has been shown to enhance NIS mRNA and protein expression, therefore it can contribute to restore and maintain iodide uptake activity (Saito et al., 1997; Kogai et al., 2000). At the posttranslational level TSH also contributes to NIS regulation but the specific mechanisms that underlie these effects are still under investigation (Riedel et al., 2001).

Biological compartments: NIS protein is mainly found at the basolateral plasma membrane of the thyroid follicular cells (Dai et al., 1996), where it actively mediates the accumulation of iodide that is the main component of thyroid hormone synthesis and therefore is considered as a major regulator of thyroid homeostasis. NIS also mediates active I- transport in extrathyroidal tissues but it is commonly agreed that is regulated and processed differently in each tissue. Functional NIS protein has been found in salivary gland ductal cells (Jhiang et al., 1998; La Perle et al., 2013), in the mammary gland during lactation (Perron et al., 2001; Cho et al., 2000), lung epithelial cells (Fragoso et la., 2004), intestinal enterocytes (Nicola et al., 2009), stomach cells (Kotani et al., 1998), placenta (Bidart et al., 2000) and testicular cells (Russo et al. 2011b). Additionally, contradictory results have been obtained regarding the NIS expression in human kidney tissue (Lacroix et al., 2001; Spitzweg et al., 2001). In the case of the lactating breast, it is suggested that NIS serves the transfer of iodide in the cells and it subsequent accumulation in the milk, thereby supplying newborns with this component during this sensitive developmental period (Tazebay et al., 2000). Additionally, NIS mRNA has been detected in various other tissues, such as colon, ovaries , uterus, and spleen (Perron et al., 2001; Spitzweg et al., 1998; Vayre et al., 1999), but the functional NIS protein and the site of its localization has not been verified.

General role in biology: The NIS is known in the field of thyroidology because of its ability to mediate the active transport of I- into the thyrocytes, which is the first and most crucial step for T3 and T4 biosynthesis (Dohan et al., 2000). NIS is located on the basolateral membrane of the thyrocytes and co-transports 2 sodium ions along with 1 iodide (2:1 stoichiometry). The electrochemical gradient of sodium serves as the driving force for iodide uptake and it is generated and maintained by the Na+/K+ ATPase pump, which is located in the same membrane of the thyrocytes. The iodide molecules, after their active transport in the cytoplasm, are passively translocated in the follicular lumen via the transporter protein pendrin and possibly other unknown efflux proteins that are located on the apical membrane (Bizhanova and Kopp, 2009). Subsequently, the thyroid hormones are synthesized in the follicular lumen by incorporating the accumulated iodide, a process which is significantly suppressed in case of NIS dysfunction or inhibition (reviewed in Spitzweg and Morris, 2010). NIS is the last thyroid-related component to be expressed during development at the 10th gestational week, which temporaly coincides with the onset of thyroid function and hormonogenesis (Szinnai et al., 2007). Albeit the localization of NIS is not fully completed at this stage, the iodide accumulation has already started. Mutations of NIS gene (SLCA5A) cause expression of non-functional NIS molecule leading to inability of the thyrocyte to accumulate iodide (Matsuda and Koshugi, 1997; Pohlenz et al., 1998), a condition called iodide transport defect (ITD). This is a rear autosomic recessive disease, which if not properly treated is clinically identified by congenital hypothyroidism, goiter, low I- uptake, low saliva/plasma I- ratio and mental impairment of varying degrees (Dohan et al., 2003). Up to date 13 mutations have been described in the NIS gene (Spitzweg and Morris, 2010) and each one of them produces mutants with different structure but in all cases non-functional. The extensive study after NIS molecular characterization and the numerous findings have convinced the scientists that is one of the most crucial components of the entire thyroid system. Additionally, after the realization that NIS could be also used as diagnostic and therapeutic tool for thyroid and non-thyroid cancers (Portulano et al., 2013) a new research activity concerning this specific mechanism has been initiated.


How It Is Measured or Detected

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There are several methods that are used nowadays to detect the functionality of NIS but none of these methods is OECD validated. The most well established methods are the following:

1. Measurement of radioiodide uptake (125I-) in NIS expressing cells. For this method the FRTL5 cell line is the most commonly used, as it endogenously express the NIS protein, but also NIS transfected cell lines have been successfully implemented in many cases (Lecat-Guillet et al., 2007; 2008b; Lindenthal et al., 2009). Once inhibitory activity is identified for a compound then further tests are performed in order to verify that the observed effect is specific due to NIS inhibition. This method has been also adapted in a high throughput format and has been already used for the screening of a chemical library of 17.020 compounds (Lecat-Guillet et al., 2008b).

2. More recently a non-radioactive method has been developed, which has been also adapted in a high throughput format (Waltz et al., 2010). It is a simple spectrophotometric assay for the determination of iodide uptake  using  rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell-Kolthoff reaction). The assay is fast, highly reproducible and equally sensitive with the radioiodine detection method.

3. A fluorescence-based method has been developed, which uses the variant  YFP-H148Q/I152L of the Yellow Fluorescent Protein (YFP) in order to detect the efflux of iodide into the rat FRTL5 cells. As a positive control perchlorate is used  as it is a well known  competitive inhibitor of iodide transport by NIS. Fluorescence of recombinant YFP-H148Q/I152L is  suppressed by perchlorate and iodide with similar affinities. Fluorescence changes in FRTL-5 cells are  Na+-dependent, consistent with the Na+-dependence of NIS activity.   It is supposed to be an innovative approach to detect the cellular uptake of perchlorate and characterize the kinetics of transport by NIS. This method needs further optimization, as YFP is not specific for iodide and thus binding of other ionic molecules could affect the results of the assay (Cianchetta et al., 2010; Rhoden et al., 2008; Di Bernarde et al., 2011).

4. In vivo 125I uptake assays is based on  immunofluorescence analyses of thyroid glands after the treatment of rat with excess I,  injected with Ci Na125I  as previously described by Ferreira et al., 2005. Then the thyroid glands are  removed and weighed, and the amount of 125I in the thyroid gland is  measured in a γ-counter (PerkinElmer; model Wizard). The counts per minute in the thyroid gland are used to calculate the percentage of 125I in the thyroid gland, having in account that 100% corresponded to the counts per minute injected I into the rat (Arriagada et al., 2015).


Evidence Supporting Taxonomic Applicability

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Apart from the human, functional NIS protein has been also identified in different species, including  the rat (Dai et al., 1996), the mouse (Perron et al., 2001), the pig (Selmi-Ruby et al., 2003), zebrafish (Thienpont et al., 2011) and in xenopus (amphibian)  (Lindenthal et al., 2009). Mouse and rat contain 618 amino acid residues, while the human and pig contain 643. There are several NIS variants that produce three active proteins in the pig due to alternative splicing at mRNA sites that are not present on the other species (Selmi-Ruby et al., 2003).

NIS orthologs are discussed in the review by Darrouzet's group ( Darrouzet et al., 2014). Interestingly, functional differences have been identified between mouse or rat NIS (mNIS or rNIS, respectively) and human NIS (hNIS). The rat and themouse orthologs were shown to accumulate radioisotopes more efficiently than the human protein (Dayem et al., 2008; Heltemes et al., 2003). The molecular basis of these functional differences could be helpful for further characterization of NIS. Zhang and collaborators showed that rNIS is localized in a higher proportion at the plasma membrane than hNIS and the N-terminal region up to putative TM7 appears to be involved in this difference (Zhang et al., 2005). These authors also reported differences in the kinetics of the Na+ binding, implicating the region spanning from TM4 to TM6 and Ser200 of hNIS. They, thus, proposed that this region could be involved in sodium binding (Zhang et al., 2005). In our laboratory, it was shown that the Vmax of the mouse protein is four times higher than the Vmax of the human protein when expressed in the same cell line (HEK-293) (Dayem et al., 2008; Darrouzet et al., 2014). The KmI value determined for hNIS (9.0 ± 0.8 μM) was significantly lower than the KmI for the mouse protein (26.4 ± 3.5 μM) whereas the KmNa values were not significantly different. Similarly to the rat protein, mNIS is predominantly localized in the plasma membrane whereas the human ortholog is detected intracellularly in 40% of the cells in which it is expressed (Darrouzet et al., 2014). However, the difference in the Vmax values does not only seem to be related to the higher intracellular localization of hNIS. Using chimeric proteins between human and mouse NIS, we showed that the N-terminal region up to TM8 is most probably involved in iodide binding, and that the region from TM5 to the C terminus could play an important role in targeting the protein to the plasma membrane (Dayem et al., 2008). One of the long-term goals of these studies is the engineering of a chimeric NIS protein most suitable for gene therapy, i.e. preserving regions responsible for the high turnover rate and the efficient plasma membrane localization of the mouse proteinwhile replacing the immunogenic extracellular regions with those of the human ortholog. The porcine NIS gene gives rise to splice variants leading to three active NIS proteins with differences in their C-terminal extremities [4]. However, it is not known if these differences lead to distinct properties (Darrouzet et al., 2014).

There is evidence that the MIE (NIS inhibition) is of relevance also for fish as an expression of the slc5a5 transcript (sodium/iodide co-transporter) has been described by various publications for the zebrafish embryo (see www.zfin.org). It has been demonstrated that NIS inhibitors in zebra fish lead also to a strong repression of thyroid hormone levels (Thienpont et al., 2011) and in xenopus (amphibian) to  inhibition of the iodide-induced current  (Lindenthal et al., 2009).


Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

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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.

It has been also shown that many anions, such as ClO3-, SCN-, NO3-, ReO4-, TcO4- and in a lower extent Br- and BF4-, are also acting as NIS substrates and they enter the cell by the same transporter mechanism (Van Sande et al., 2003). It has been also shown that ClO4- is transferred by NIS with high affinity and is considered as one of its most potent inhibitors (Dohan et al., 2007). Most recently, the aryltrifluoroborates were also shown to inhibit NIS function (Lecat-Guillet et al., 2008a). A library of 17,020 compounds was tested by a radioactive screening method with high specificity using transfected mammalian cells (Lecat-Guillet et al., 2008b; 2007) for NIS inhibition evaluation. Further studies with the most powerful inhibitors showed a high diversity in their structure and mode of action (Lindenthal et al., 2009).



References

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