To the extent possible under law, AOP-Wiki has waived all copyright and related or neighboring rights to KE:957

Event: 957

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Binding, Transthyretin in serum

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. More help
Binding, Transthyretin in serum
Explore in a Third Party Tool

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Organ term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Organ term
serum

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor).  Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description.  To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons.  If a desired term does not exist, a new term request may be made via Term Requests.  Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
binding transthyretin increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Transthyretin interference MolecularInitiatingEvent Kristie Sullivan (send email) Under Development: Contributions and Comments Welcome Under Development
TH displacement from serum TTR leading to altered amphibian metamorphosis MolecularInitiatingEvent Jonathan Haselman (send email) Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
Homo sapiens Homo sapiens High NCBI
Bubalus bubalis Bubalus bubalis High NCBI
Rattus norvegicus Rattus norvegicus High NCBI
Mus musculus Mus musculus High NCBI
Gallus gallus Gallus gallus High NCBI
Sus scrofa Sus scrofa High NCBI
Pan troglodytes Pan troglodytes High NCBI
Monodelphis domestica Monodelphis domestica High NCBI
Xenopus tropicalis Xenopus (Silurana) tropicalis High NCBI
Bos taurus Bos taurus High NCBI
Macaca mulatta Macaca mulatta High NCBI
Meleagris gallopavo Meleagris gallopavo High NCBI
Canis lupus familiaris Canis lupus familiaris High NCBI
Ovis aries Ovis aries High NCBI
Erinaceus europaeus Erinaceus europaeus High NCBI
Xenopus laevis Xenopus laevis NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help

Sex Applicability

An indication of the the relevant sex for this KE. More help

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

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).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

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 Ka = 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; Ka = 0.78-3.95 x 108 M-1) versus the natural ligand (T4, Ka = 2.05 x 10M-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 10M-1) versus the natural ligand (T4; IC50 = 80.7 nM; Ka = 3.5 x 10M-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 (Ka = 1.4 x 10M-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.

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

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).

References

List of the literature that was cited for this KE description. More help

Alshehri, B., D’Souza, D. G., Lee, J. Y., Petratos, S., & Richardson, S. J. (2015). The Diversity of Mechanisms Influenced by Transthyretin in Neurobiology: Development, Disease and Endocrine Disruption. Journal of Neuroendocrinology, 27(5), 303–323. http://doi.org/10.1111/jne.12271

Andrea et al. 1980

Blake et al 1978

Cao, J., Y. Lin, L.H. Guo, A.Q. Zhang, Y. Wei, and Y. Yang. 2010. Structure-based investigation on the binding interaction of hydroxylated polybrominated diphenyl ethers with thyroxine transport proteins. Toxicology 277(1-3):20-8.

Chauhan, K. R., Kodavanti, P. R. S., & McKinney, J. D. (2000). Assessing the Role of ortho-Substitution on Polychlorinated Biphenyl Binding to Transthyretin, a Thyroxine Transport Protein. Toxicology and Applied Pharmacology, 162(1), 10–21. http://doi.org/10.1006/taap.1999.8826

Ferguson et al. 1975

Lans, M. C., Klasson-Wehler, E., Willemsen, M., Meussen, E., Safe, S., & Brouwer, A. (1993). STRUCTURE-DEPENDENT, COMPETITIVE INTERACTION OF HYDROXY-POLYCHLOROBIPHENYLS, -DIBENZO-p-DIOXINS AND -DIBENZOFURANS WITH HUMAN TRANSTHYRETIN. Chemico-Biological Interactions, 88, 7–21.

Lans, M. C., Spiertz, C., Brouwer, a, & Koeman, J. H. (1994). Different competition of thyroxine binding to transthyretin and thyroxine-binding globulin by hydroxy-PCBs, PCDDs and PCDFs. European Journal of Pharmacology, 270(2-3), 129–136. http://doi.org/10.1016/0926-6917(94)90054-X

Loubiere et al 2010

Ren, X. M., & Guo, L. H. (2012). Assessment of the binding of hydroxylated polybrominated diphenyl ethers to thyroid hormone transport proteins using a site-specific fluorescence probe. Environmental Science and Technology, 46(8), 4633–4640. http://doi.org/10.1021/es2046074

Richardson, S. J. (2007). Cell and molecular biology of transthyretin and thyroid hormones. International Review of Cytology, 258(January), 137–93. http://doi.org/10.1016/S0074-7696(07)58003-4

Schreiber, G. (2002). The evolutionary and integrative roles of transthyrein in thyroid hormone homeostasis. Journal of Endocrinology, 175(1), 61–73. http://doi.org/10.1677/joe.0.1750061

Schweizer and Kohrle 2013

Van den Berg, K. J. (1990). Interaction of chlorinated phenols with thyroxine binding sites of human transthyretin, albumin and thyroid binding globulin. Chemico-Biological Interactions, 76(1), 63–75. http://doi.org/10.1016/0009-2797(90)90034-K

Van den Berg, K. J., Van Raaij, J. a G. M., Bragt, P. C., & Notten, W. R. F. (1991). Interactions of halogenated industrial chemicals with transthyretin and effects on thyroid hormone levels in vivo. Archives of Toxicology, 65(1), 15–19. http://doi.org/10.1007/BF01973497

Weiss et al 2009

Weiss, J. M., Andersson, P. L., Zhang, J., Simon, E., Leonards, P. E. G., Hamers, T., & Lamoree, M. H. (2015). Tracing thyroid hormone-disrupting compounds: database compilation and structure-activity evaluation for an effect-directed analysis of sediment. Analytical and Bioanalytical Chemistry, 5625–5634. http://doi.org/10.1007/s00216-015-8736-9