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Event: 957

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. 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. The short name should be less than 80 characters in length. More help
Binding, Transthyretin in serum

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.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. More help

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.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. More help
Organ term
serum

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). 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 signalling 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. 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 Allie Always (send email) Open for adoption Under Development
TH displacement from serum TTR leading to altered amphibian metamorphosis MolecularInitiatingEvent Brendan Ferreri-Hanberry (send email) Under development: Not open for comment. Do not cite

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. 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

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. 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. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. 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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. 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).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  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).

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

Rickenbacher et al (1986) provided initial direct evidence of competition for the T4 binding site using molecular modeling and binding assays using radiolabeled TH. Brouwer and van den Berg (1986) reported preferential binding of a metabolite of radiolabeled tetrachlorobiphenyl to TTR in rats (15 mg/kg, ip), using gel electrophoresis followed by HPLC analysis. Van den Berg (1990) used a competitive binding assay to assess the ability of hydroxylated chlorinated aromatic compounds to bind to radiolabeled T4. Van den Berg et al (1991) extended this work to 65 compounds from 12 different chemical groups in rats treated via a single ip dose and competitive binding assay. Chlorophenols were found to have higher affinity relative to other chlorinated aromatics, particular at higher levels of chlorination, and the combination and position of hydroxyl & chlorine atoms. {insert Figure 2/Van den Berg 1990}

Lans et al (1993) described the ability of hydroxylated metabolites of PCBs, PCDDs and PCDFs to act as competetive ligands at the TTR-T4 binding site using an in vitro binding assay. Many of the hydroxylated PCBs examined were more potent ligands than T4, as opposed to those PCDFs and PCDDs that lacked chlorine atoms substituted adjacent to hydroxyl groups. When the hydroxyl group was in the meta or para positions, a 35- to 136-fold greater potency was found relative to ortho substitutions. These results were confirmed through later competitive binding work published by Cheek et al (1999) and Chauhan et al (2000).

Weiss et al (2009) first confirmed competitive binding of TTR with perfluorinated compounds (PFCs)

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). 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