In 2018, both the European Food Safety Authority (EFSA) and European Chemicals Agency (ECHA) introduced criteria for the identification of endocrine disruptors in the context of their regulations, as well as a guidance document for the identification of endocrine disruptors. The latter document mainly focuses on EATS-modalities (estrogen, androgen, thyroid and steroidogenesis), making use of tests as described in the OECD conceptual framework for endocrine disruptor testing. The OECD conceptual framework describes different levels of testing:
- Level 1 testing requires reviewing existing data and performing in silico modelling.
- Level 2 testing consists of a battery of in vitro assays.
- Levels 3, 4, and 5 all consist of in vivo assays in mammalian or non-mammalian test systems.
With respect to in vitro assays, guidelines for the EAS modalities have already been established for several years and these assays are readily available at Charles River Laboratories. However, there are no guidelines for investigating the thyroid modality in vitro. OECD scoping document 207 describes possible sites of action of environmental contaminants on the HPT axis, and possible in vitro tests that can investigate this. Based on this document our team has recently implemented four in vitro assays to assess the effects of test substances on thyroid receptor binding and transactivation (TRβ-CALUX® assay), thyroid peroxidase (TPO) (tyrosine iodination assay), iodine uptake (NIS assay), and UDPGT induction.
The TRβ-CALUX assay investigates the test items’ potential to interact with the thyroid receptor. The TRβ-CALUX is based on a human U2OS bone marrow cell line that is stably transfected to express the thyroid receptor β (TRβ), and consists of a firefly luciferase gene coupled to thyroid responsive elements (TREs). Upon binding of thyroid-like compounds to the cytosolic thyroid receptor, the ligand-receptor complex binds to the TREs and luciferase is expressed. Upon addition of substrate for luciferase, light is emitted, which is proportional to the amount of ligand-specific receptor binding. Using this method, the TR agonistic as well as the TR antagonistic activity of a test item can be determined.
TPO Inhibition Assay
The thyro peroxidase (TPO) enzyme is responsible for the oxidation of iodide, iodination of tyrosyl residues of thyroglobulin and coupling of monoiodotyrosine (MIT) and/or diiodothyronine (DIT), resulting in the active T3 and T4 hormones. Our TPO inhibition assay evaluates effects on TPO iodination activity using the tyrosine iodination assay. Hereby, potential TPO inhibition caused by a test item is evaluated by the measuring the formation of MIT upon incubation of the substrate tyrosine, potassium iodide, and TPO together with different concentrations of test item. In case the test item causes inhibition of TPO, the formation of MIT will decrease as a function of the test item concentration. Thyroid microsomes of humans, as well as other species like rats and minipigs, can be used as TPO source.
The transport of iodine into the thyroid follicular cells occurs via the sodium-iodine symporter (NIS). Chemicals can inhibit iodine uptake by 1) competing with iodine for binding to the NIS transport protein or 2) by down- regulating expression of NIS. In both cases, this results in decreased synthesis of T3 and T4. Implemented by Solvo (a Charles River company), the in vitro assay evaluates effects on the NIS making use of HEK293 cells overexpressing the human recombinant (hr) NIS. In this assay, the effect of a test item on the influx of iodine into the cells is determined by measuring iodine levels using the Sandell-Kolthoff reaction.
UDPGT Induction Assay
The final developed assay, UDPGT induction, investigates the test item’s potential to induce hepatic UDPGT enzymes. An important clearance pathway for both T3 and T4 is glucuronidation by UDPGT enzymes. Induction of these enzymes may result in a higher clearance of T3 and T4 and a decrease in circulating thyroid hormone plasma levels. An in vitro model in cryopreserved hepatocytes is established whereby induction is evaluated by measuring the formation of T4-glucuronide upon incubation with the substrate T4, after the hepatocytes have been exposed to the test item for 72 hours. In addition, the expression of mRNA levels of the different UDPGT (and CYP) genes can be evaluated as well.
Taken together, these four assays are valuable additional tools in assessing the effects of test items on thyroid modalities, as required in the new EU regulations.