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Event Title

Serum thyroxine (T4) from transthyretin, Displacement

Key Event Overview

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AOPs Including This Key Event

AOP Name Event Type Essentiality
Interference with thyroid serum binding protein transthyretin and subsequent adverse human neurodevelopmental toxicity KE

Taxonomic Applicability

Name Scientific Name Evidence Links

Level of Biological Organization

Biological Organization

How this Key Event works

Despite the two binding sites for T4 on the TTR serum binding protein, each molecule of TTR only carries a single T4 molecule due to the negative cooperativity displayed by these binding sites (Ferguson et al 1975). As such, xenobiotics and pharmacologic agents can displace T4 from TTR and early on, this was demonstrated for ethnacrynic acid, salicylates, penicillin and 2,4-dinitrophenol (Munro et al 1989). More recently, work with the flavonoid EMD 21388 (and other compounds structurally similar to thyroxine) showed competitive binding and displacement of T4 from the TTR carrier protein.

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 of hydroxyl chlorine atoms in the ortho position. {insert Figure 2/Van den Berg 1990}

Kohrle et al (1989) showed complete displacement (via gel electrophoresis) of radiolabeled T3 and T4 by EMD 21388 in pooled rat serum followed by increase in the percent free TH as measured by equilibrium dialysis. Complete inhibition occurred at 10 umol and displaced T4 from TTR to serum albumin and thyroxine-binding globulin (TBG), which normally serve a lesser role in thyroid hormone transport in humans. {insert Figure 2}

Kohrle et al (1989) administered EMD 21388 via ip route to rats at 2 umol/100 g BW and observed displacement of radiolabeled T3 and T4 from TTR followed by a decrease of T3 and T4 in serum while the percent free TH remained unchanged. {insert Figures 3-5}

Lueprasitsakul et al (1990) repeated this protocol and found that inhibition of binding occurred within 3 minutes followed by a decrease in serum T4 concentration and an increase in both serum percent free T4 as well serum total T4. {insert Figures 1 and 4}

Mendel et al (1992) demonstrated in rats dosed via IP with EMD 21388 (2 μmol/100 g BW) both displacement of radiolabeled T4 from TTR (as assessed via electrophoresis of serum proteins) and susbsequent increase of free T4 in serum.

To initially evaluate the impact of EMD 21388 on maternal/fetal hormones, Pedraza et al (1996) administered 2.5 mg 21388/day subcutaneously in pregnant female rats which led to displacement of T4 from TTR, reduced total T4 and increased free T4 in maternal circulation.

How it is Measured or Detected

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?

In humans, approximately 0.03% of total serum T4 is present in unbound/free condition (Refetoff et al 1970). Of the bound T4, approximately 75% is bound to TBG, approximately 20% to TTR and the remainder to ALB and some high density lipoprotein carriers. ALB is present at roughly 100-fold the molar concentration of TTR and roughly 2,000-fold higher than TBG; however, the affinity of T4 to TBG is 50-fold higher than TTR and 7,000-fold higher than ALB (Refetoff 2015). TTR binds roughly 80% of the T4 circulating in ventricular CSF although it constitutes only 25% of protein found there (Herbert et al 1986). In serum, only about 0.5% of circulating TTR is bound to T4, average serum concentration is 25 mg/dL (which can bind up to 300 ug T4/dL (Refetoff 2015).

TTR can be measured by densitometry after its separation from other serum proteins via electrophoresis, hormone saturation and/or immunoassays (Refetoff 2015).

Evidence Supporting Taxonomic Applicability


Brouwer, a, & van den Berg, K. J. (1986). Binding of a metabolite of 3,4,3’,4'-tetrachlorobiphenyl to transthyretin reduces serum vitamin A transport by inhibiting the formation of the protein complex carrying both retinol and thyroxin. Toxicology and Applied Pharmacology, 85(3), 301–312. http://doi.org/10.1016/0041-008X(86)90337-6

Ferguson, R.N., H. Edelhoch, H.A. Saroff, J. Robbins and H.J. Cahnmann.(1975). Negative cooperativity in the binding of thyroxine to human serum prealbumin. Preparation of tritium-labeled 8-anilino-1-naphthalenesulfonic acid. Biochemistry 28;14(2):282-9.

Kohrle, J., S.L. Fang, Y. Yang, K. Irmscher, R.D. Hesch, S. Pino, S. Alex, and L.E. Braverman. (1989). Rapid effects of the flavonoid EMD 21388 on serum thyroid hormone binding and thyrotropin regulation in the rat. Endocrinology 125: 532-537

Lueprasitsakul, W., Alex, S., Fang, S. L., Pino, S., Irmscher, K., Köhrle, J., & Braverman, L. E. (1990). Flavonoid administration immediately displaces thyroxine (T4) from serum transthyretin, increases serum free T4, and decreases serum thyrotropin in the rat. Endocrinology. 126: 2890-2895

Mendel, C. M., Cavalieri, R. R., & Kohrle, J. (1992). Thyroxine (T4) transport and distribution in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from transthyretin. Endocrinology, 130(3), 1525–1532.

Munro SL, Lim CF, Hall JG, Barlow JW, Craik DJ, Topliss DJ, Stockigt JR. Drug competition for thyroxine binding to transthyretin (Prealbumin): Comparison with effects on thyroxine-binding globulin. J Clin Endocrinol Metab 1989; 68:1141-1147

Pedraza, P., Calvo, R., Obregón, M. J., Asuncion, M., Escobar Del Rey, F., & Morreale De Escobar, G. (1996). Displacement of T4 from transthyretin by the synthetic flavonoid EMD 21388 results in increased production of T3 from T4 in rat dams and fetuses. Endocrinology, 137(11), 4902–4914. http://doi.org/10.1210/en.137.11.4902

Refetoff, S. (2015). Thyroid hormone serum transport proteins.

Rickenbacher, U., McKinney, J. D., Oatley, S. J., & Blake, C. C. (1986). Structurally specific binding of halogenated biphenyls to thyroxine transport protein. Journal of Medicinal Chemistry, 29(5), 641–648.

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