Key Event Overview
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AOPs Including This Key Event
The following are chemical initiators that operate directly through this Event:
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Level of Biological Organization
How this Key Event works
Disruption of the thyroid hormone system is increasingly being recognized as an important toxicity pathway, as it can cause many adverse outcomes. Thyroid hormones do not only play an important role in the adult individual, but they are also critical during embryonic development. Thyroid hormones (THs) play an important role in a wide range of biological processes in vertebrates including growth, development, reproduction, cardiac function, thermoregulation, response to injury, tissue repair and homeostasis. Numerous chemicals are known to disturb thyroid function, for example by inhibiting thyroperoxidase (TPO) or deiodinase (DIO), upregulating excretion pathways or modifying gene expression. The two major thyroid hormones are triiodothyronine (T3) and thyroxine (T4), both iodinated derivatives of tyrosine. The synthesis of the thyroid hormones is a process that involves several steps. Thyroglobulin, the thyroid hormone precursor, is produced by the thyroid epithelial cells and transported to the lumen via exocytosis. Then thyroperoxidase (TPO) plays an essential role in the production of mainly T4. The prohormone T4 is then released in the circulation under the influence of thyroid stimulating hormone (TSH), in order to be transported to the various tissues, including the liver, the kidneys and the heart. Most TH actions depend on the binding of T3 to its nuclear receptors. Active and inactive THs are tightly regulated by enzymes called iodothyronine deiodinases (DIO). The activation occurs via outer ring deiodination (ORD), i.e. removing iodine from the phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.
Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable to convert T4 into T3, as well as to convert rT3 to the inactive thyroid hormone 3,3’ T2, through outer ring deiodination. rT3 is the preferred substrate for DIO1 (Hennemann G, Visser TJ 1997). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate. DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively.
How it is Measured or Detected
At this time, there are no approved OECD or EPA guideline protocols for measurement of DIO inhibition. Deiodination is the major pathway regulating T3 bioavailability in mammalian tissues. The objective of this in vitro assay is to examine inhibition of deiodinase 2 (DIO2) activity upon exposure to thyroid disrupting compounds, using unexposed pig liver tissue. There are three types of deiodinase measurements available. A first in vitro assay measures deiodinase activities by quantifying the radioactive iodine release from iodine-labelled substrates, depending on the preferred substrates of the isoforms of deiodinases. A second assay is a chromatography-based method coupled to mass spectroscopy to measure products of thyroxin by deiodinase type-1 activity (Butt et al., 2010). Finally, a colorimetric method was developed (Renko et al., 2012) that measures the release of iodine from T4.
Although the radioactive based assays uses radioactivity to measure deiodinase activity, they provide a good balance between specificity and resources needed. The chromatography-based assay has a high sensitivity and specificity to measure all thyroid hormones metabolites, but a high degree of technical expertise and expensive instrumentation is required. Although the colorimetric method is a promising alternative, the sensitivity of this assay is still limited.
For all the reasons above, we chose to use the radioactive method. Since DIO1 and DIO2 prefer a different substrate to deiodinate, i.e. rT3 and T4 respectively, it is possible to quantify outer-ring deiodination using the specific enzymkinetics of both enzymes. This assay measures the amount of radioactive iodine that is released from 125I-labelled substrates by conversion of one of the substrates by the DIO enzymes. We used a pig liver homogenate preparation and reaction buffers containing DTT as co-substrate. Furthermore, a concentration range of potential thyroid-disrupting chemicals can be added to measure the inhibitory potencies of the chemicals the inhibit DIO enzyme activity. Enzym activity is expressed as picomoles or femtomoles of released radioactive iodine per minute per mg protein and if inhibition occurs, the half maximal inhibitory concentration (IC50) was determined.
Evidence Supporting Taxonomic Applicability
Deiodination by DIO enzymes is known to exist in a wide range of vertebrates and invertebrates.
Visser, T.J., Van Overmeeren, E., Fekkes, D., Docter, R., Hennemann, G. 1979. Inhibition of iodothyronine 5'-deiodinase by thioureylenes: structure-activity relationship. FEBS Letters, 103, 2.
Butt, C.M., Wang, D., Stapleton, H.M. 2011. Halogenated phenolic contaminants inhibit the in vitro activity of the thyroid-regulating deiodinases in human liver. Toxicological sciences 124: 339-347.
Renko, K., Hoefie, C.S., Hiller, F., Schomburg, L., Köhrle, J. 2012. Identification of Iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay. Endocrinology, 153: 2506-2513.