In Vitro Assays for Thyroid Disruption
Both the EFSA (Plant Protection Products) and ECHA (Biocides) introduced endocrine disruption criteria in their regulations, from 2018 onwards, meaning that EATS-mediated (Estrogen, Androgen, Thyroid, Steroidogenesis) adversity needs to be fully investigated. Endocrine disruption assessment requires the following 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.
Based on OECD Document 207 and our ongoing efforts in the EU-NETVAL validation of these assays, we’ve implemented the following three in vitro thyroid assays to assess the potential for endocrine disruption.
Thyroid Receptor Transactivation (TRβ-CALUX)
The TRβ-CALUX assay investigates binding of test items to the thyroid receptor. The TRβ-CALUX is comprised of a human bone marrow cell line incorporating the firefly luciferase gene coupled to thyroid responsive elements (TREs). Once thyroid-like compounds bind to the cytosolic thyroid receptor, the ligand-receptor complex binds to TRE and, by adding an appropriate substrate for luciferase, light is emitted. The amount of light produced is proportional to the amount of ligand-specific receptor binding, which is benchmarked against the relevant reference compound T3.
Thyroid Peroxidase (TPO) Inhibition
The thyroperoxidase (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 thyroglobulin bound T3 and T4.
Inhibition of TPO is a well-documented MIE in the thyroid hormone AOP, displaying a clear relationship between chemically induced TPO inhibition – decreased TH synthesis and decreased systemic TH levels in humans and animals (OECD, 2014). The TPO inhibition assay implemented here evaluates both effects on TPO peroxidative activity, as well as TPO iodination activity. Thyroid microsomes of humans, as well as other species like minipigs and rats, can be used as a TPO source. In addition, human cell lines can be used as a source of TPO. The assay uses L-tyrosine as a substrate and the formation of the metabolite MIT is measured by LC-MS. A decrease in the formation by the test item indicates the inhibition of the TPO enzyme.
Sodium-Iodine Symporter (NIS) Assay
The final developed assay investigates transport of iodine via the NIS into the thyroid follicular cells. Chemicals can inhibit iodine uptake by competing with iodine for binding to the NIS transport protein or down regulating expression of NIS. In both cases, this results in decreased synthesis of T3 and T4. The developed assay by Solvo used HEK293 cells overexpressing the hrNIS. The influx of iodine is detected with the Sandell-Kolthoff reaction.
Frequently Asked Questions (FAQs) for In Vitro Thyroid
Which in vitro thyroid assays can be used to assess the potential for endocrine disruption?
Based on OECD Document 207 and our ongoing efforts as part of EU-NETVAL’s validation of these assays, we’ve implemented the following three in vitro thyroid assays to assess the potential for endocrine disruption.
- Thyroid Receptor Transactivation assay (TRβ-CALUX)
- Thyroid peroxidase (TPO) inhibition in thyroid microsomes from humans and other species as well as a human cell line
- Sodium Iodide Symporter (NIS) assay.