Binding, Transthyretin in serum

The key event that initiates this AOP (i.e. the MIE) is binding of a xenobiotic to the thyroxine (T4)-binding site of transthyretin (TTR), displacing T4 from the binding site(s) of TTR and adding this to the pool of free T4 in serum, which is normally roughly 0.02% (20 pM) of total T4 (100 nM) [in CSF, free T4 is roughly 1.4% (70 pM) of total T4 (2-3 nM)](Schweizer and Kohrle 2013).

TTR has been found to bind with a number of ligands, including pharmacologic agents as well as flavonoids and halogenated aromatic compounds, with differing strengths with some xenobiotics found to be possessing a binding affinity equal to or stronger than T4 (i.e. "competitive binder").  Studies with other xenobiotics with similar structural characteristics have found that a higher degree of halogen substitution, as well as placement on the ring relative to the hydroxyl group, increases binding affinity (Chauhan et al. 2000, Lans et al. 1993, Lans et al. 1994, Ren and Guo 2012, van den Berg 1990, van den Berg et al. 1991, Weiss et al 2015). Weiss et al (2015) compiled a database of almost 150 compounds found to bind to TTR and 52 of these were found to have higher affinity than T4 and thus, could be considered competitive binders in serum.

The function of the serum protein TTR is to deliver T4 to target cells in the liver, tight junctions, etc. where it is facilitated across the membrane via specific receptors and converted to the active form T3, where it can activate nuclear receptors. TTR facilitates passage across key tight junctions, such as the blood-brain barrier, the CSF barrier and transplacentally, and the interruption of thyroid serum protein-assisted transport during certain developmental windows can lead to profound developmental neurotoxicity (i.e. cretinism).  It should be noted, though, that in mammals with all three functional serum transport proteins (TTR, albumin and TBG), substantial reductions in total T4 can be observed with little to no adverse effect due to overall redundancy of this system. In this scenario, roughly 75% of serum T4 is bound to TBG, 15% to TTR and up to 5% for to albumin (OECD DRP 2012).  That being said, TTR is the sole transport protein in the CSF and T4 is not biosynthesized by the fetus until the second trimester - thus, the mother is the sole source of T4 for the fetus during early gestation and this appears to be the developmental window of greatest concern to human physiology (for example, see Loubiere et al 2010).

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Transthyretin is a 55-kDa tetramer protein composed of two identical monomer subunits, each of which is formed by two four-stranded beta sheets, which are assembled around the central channel of the protein (Blake et al. 1978). This central channel contains two identical binding sites; however, there is a 100-fold difference in binding constants between the first and second T4 molecule bound and this "negative cooperativity" results in a 1:1 relationship between T4 and TTR in terms of protein transport in serum (Ferguson et al. 1975). The phenolic hydroxyl and iodine substitutions on the phenolic ring of T4 are important structural characteristics that differentiate ability to bind to TTR, as opposed to other serum binding proteins like TBG (Andrea et al. 1980). T4 binds to TTR in one of two ways: "forward" and "reverse" mode. In forward mode, the phenolic ring of T4 is buried deeply in the TTR binding site while this ring interacts with residues at the entrance of the binding channel in reverse mode (Lans et al 1993). Detailed structural analysis of the protein is available from X-ray crystallography (Rerat and Schwick 1967; Blake et al 1977).

There are three main in vitro approaches to measuring binding affinity between TTR and its ligands: radioligand-binding assays (RLBAs), surface plasmon resonance (SPR) and fluorescence displacement. Biochemical (in chemico) approaches using GC/MS or LC/MS also exist that use recombinant (rTTR) and display limits of detection in the low nM range, including a multi-mode, ultra-high-performance LC method and anon-radioactive label, [¹³C₆]-T4 (Aqai et al 2012).

Van den Berg et al (1991) used an in vitro competitive radioligand binding assay with gel-filtration procedure modified from Somack et al  (1982) as well to screen 65 compounds from 12 chemical classes for evidence of interference with the T4 binding site of TTR using a radioligand binding assay and reported results semi-quantitatively, while previous work using the same method on chlorinated phenols found that affinity rose with degree of chlorination (pentachlorophenol displayed an IC50 of 2.3 x 10⁸ M; derived K_a = 1.2 x 10⁸ M^-1) versus the natural ligand (T4; 4 x 10⁸ M)(Van Den Berg, 1990).  

Lans et al (1993, 1994) used purified TTR and T4 [¹²⁵I] as a displaceable radioligand and gel-filtration procedure modified from Somack et al (1982) to study hydroxylated PCBs PCDDs and PCDFs and found many competitive binders in all classes; again, dependent on degree of chlorination, presence and placement of hydroxyl atom relative to chlorine placement (IC50 in 6.5-25 nM range; K_a = 0.78-3.95 x 10⁸ M^-1) versus the natural ligand (T4, K_a = 2.05 x 10⁷ M^-1).  Meerts et al (2000) studied PBDEs and related compounds (pentabromophenol, Tetrabromobisphenol A) and found multiple competitive binders (IC50 range 7.7 - 67 nM; Ka = 4.3 - 38 x 10⁷ M^-1) versus the natural ligand (T4; IC50 = 80.7 nM; Ka = 3.5 x 10⁷ M^-1).

Cao et al (2010) used a novel fluorescence displacement method using a protein-binding probe that fluoresces when bound, and the intensity of which dips following displacement for a ligand. Analyte titration curves are used to derive IC50 and binding constant values. They used an ANSA probe to screen 14 hydroxylated PBDEs and found competitive binding with TTR (K_a = 1.4 x 10⁷ M^-1 to 6.9 x 10⁸ M^-1). Molecular docking analysis revealed a ligand-binding channel in TTR for OH-PBDE binding. Ren and Guo (2012) reported use of a non-radioactive, fluorescin-T4 conjugate designed as a fluorescent probe (vs ANSA probe which is less TTR-specific) to study the binding interaction of multiple hydroxylated PBDEs with differing degrees of bromination and differing hydroxy positions and TTR.   Competitive binders were found (IC50 range = 110-219 nM) relative to the natural ligand (T4; IC50 = 260 nM) and Kd values for the competitive binders ranged from 101-210 nM versus T4 (Kd = 239 nM). Weiss et al (2009) initially determined the competitive binding of TTR with PFCs via radioligand-binding assay of Hamers et al (2006) and Lans et al (1993).  Ren et al (2016) used a fluorescence displacement assay to study 16 PFCs and found T4 to have an IC50 of 31 nM (Kd = 5 nM), as compared to PFOS (the strongest binder) TC50 of 130 nM (Kd = 20 nM).

The radioligand methods have been criticized for use of hazardous and costly radio-labeled material and physical separation of bound and free T4-[¹²⁵I] is required.  Marchesini et al (2006, 2008) reported a surface plasmon resonance biosensor-based method using rTTR, a surface-based measurement which may not fully characterize the binding reaction of a homogenous solution or matrix. Optimized SPR is more sensitive, amenable to high-throughput screening and easier to perform than RLBAs.  Furthermore, hydroxy-metabolites do not endure destructive clean-up/extraction methods and differences in physicochemical qualities from parent compounds complicates separation.

Montano et al (2012) introduced a new method (using a modified method of Murk et al 1994 for in vitro bioactivation of PCBs and PBDEs with an ANSA-TTR displacement assay) to selectively extract metabolites (and limit co-extraction of interferants, like free fatty acids) and derive an IC50 dose-response curve for OH-PBDEs, OH-PCBs, etc. using bioactivated parent compound in a 96-well plate system.  IC50 for the natural ligand (T4) was 0.26 uM while the IC50s for the hydroxy metabolites ranged from 0.23-0.63 uM.

The transthyretin protein has been found to be highly conserved among vertebrates, as supported by X-ray crystallography studies in man, rat, chicken and fish as well as comparison of amino acid sequence (Richardson 2007). Over time, evolution has driven the nature of the N-terminal region of TTR from a more hydrophobic and longer region (as found in fish and amphibians) to a shorter and more hydrophilic region (as found in the placental mammals). The consequence of this biochemical change in the TTR protein was to change the primary function of TTR from binding and carrying T3 in serum to carrying T4 in serum (as it does almost exclusively in rats, as rats lack TBG during adult life)(Alshehri et al. 2015). Thus, in the placental mammals, TTR operates to carry T4 to specific tissues, where it is displaced, transported across the cell membrane by a different ligand-mediated process, and then converted to T3 via deiodinase enzymes within the cell (where it activates the nuclear receptor to cause downstream effects).

TTR is encoded by a single gene on human chromosome 18 (18q11.2-12.1) (LeBeau and Geurts van Kessel 1991).

TTR is synthesized in the adult liver only by eutherians (birds and placental mammals) and herbivorous marsupials; however, it is also synthesized in the choroid plexus of reptiles, birds and mammals. TTR is one of three thyroid hormone serum transport proteins, along with albumin (ALB) and thyroid-binding globulin (TBG). Over evolutionary time, the N-terminal section of TTR has become shortened and more hydrophilic such that T3 is more tightly bound in birds and reptiles but T4 is more tightly bound to TTR in mammals (Schrieber 2002). They all differ in binding affinity and dissociation rate for both T4 and T3 and together form a buffered system that seeks to keep circulating T4 within a certain range: between the normal concentration of free T4 in serum (30 pM) and solubility limit in serum (2 uM) (Schreiber 2002). Given these differences, more T4 available for cellular uptake likely comes from ALB-bound T4 in capillaries with fast moving blood but TTR-bound T4 is likely more important in slow moving fluid (like CSF)(Schrieber 2002).

Mammalian TTR has a greater affinity for T4 (and lower affinity for T3) in mammals relative to birds and reptiles, neither of which express a high-affinity TBG protein in serum (Schweizer and Kohrle 2013); while Larsson reported the presence of TTR (i.e. thyroxine-binding prealbumin) in all vertebrate species investigated and failed to detect the presence of TBG in cat, rabbit, rat, chicken, frog and salmon.  Binding capacity of serum TTR in female rats is lower relative to males (Emerson et al 1990 in Zoeller et al 2007).

According to the evaluation of the empirical taxonomic domain of applicability (tDOA) of an adverse outcome pathway network for thyroid hormone system disruption (THSD) by Haigis et al., 2023, the level of confidence for a linkage between TTR binding in serum and altered thyroid hormone (TH) levels was considered high for mammals (Akiyoshi et al., 2012, Cao et al., 2011, Hallgren and Darnerud, 2002, Kato et al., 2009, Köhrle et al., 1989, Mendel et al., 1992, Monk et al., 2013, Ren et al., 2020, Ren and Guo, 2012, Ućan-Marin et al., 2010, Zhang et al., 2018), and moderate for fish (Akiyoshi et al., 2012, Morgado et al., 2007, Tu et al., 2016, Zhang et al., 2018). For birds and amphibians in vitro evidence of TTR binding is available, however mostly without a clear link to THSD in vivo (Akiyoshi et al., 2012, Eguchi et al., 2008, Ishihara et al., 2003a,b, Kudo et al., 2006, Kudo and Yamauchi, 2005, Ućan-Marin et al., 2010, Yamauchi et al., 2000, 2003). This was supported by structural protein conservation analysis by Haigis et al., 2023. Structural protein conservation of mammalian, fish, avian, amphibian and reptilian TTR was found compared to the human (Homo sapiens) protein target using the U.S. Environmental Protection Agency’s Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS v6.0; seqapass.epa.gov/seqapass/) tool, while acknowledging the potential existence of interspecies differences in conservation. However, a lower level of conservation was found in fish compared to the other vertebrates.

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