Comparison of in vitro and in vivo bioassays to measure thyroid hormone disrupting activity in water extracts

Abstract

Environmental chemicals can induce thyroid disruption through a number of mechanisms including altered thyroid hormone biosynthesis and transport, as well as activation and inhibition of the thyroid receptor. In the current study six in vitro bioassays indicative of different mechanisms of thyroid disruption and one whole animal in vivo assay were applied to 9 model compounds and 4 different water samples (treated wastewater, surface water, drinking water and ultra-pure lab water; both unspiked and spiked with model compounds) to determine their ability to detect thyroid active compounds. Most assays correctly identified and quantified the model compounds as agonists or antagonists, with the reporter gene assays being the most sensitive. However, the reporter gene assays did not detect significant thyroid activity in any of the water samples, suggesting that activation or inhibition of the thyroid hormone receptor is not a relevant mode of action for thyroid endocrine disruptors in water. The thyroperoxidase (TPO) inhibition assay and transthyretin (TTR) displacement assay (FITC) detected activity in the surface water and treated wastewater samples, but more work is required to assess if this activity is a true measure of thyroid activity or matrix interference. The whole animal Xenopus Embryonic Thyroid Assay (XETA) detected some activity in the unspiked surface water and treated wastewater extracts, but not in unspiked drinking water, and appears to be a suitable assay to detect thyroid activity in environmental waters.

Transthyretin-Binding Activity of Complex Mixtures Representing the Composition of Thyroid-Hormone Disrupting Contaminants in House Dust and Human Serum

“…The TTR-binding potency of the individual compounds, the reconstituted mixtures, and the SRM2585 extract was tested in a competitive binding assay making use of a fluorescent conjugate of T 4 and fluorescein 5-isothiocyanate (FITC). The method originally developed by Ren and Guo (2012) was previously transformed into a 96-well plate format (Ouyang et al 2017) and further modified and optimized (Leusch et al 2018) in our laboratory. The FITC-T 4 conjugate shows high fluorescence when its T 4 group is bound to TTR.…”

Thyroid Disrupting Activities in Wastewater and Surface Waters: A Review of Their <i>in Vitro</i> Analyses

“…Interestingly, these chemicals have been shown to interfere with the endocrine system through multiple pathways [15], and it is therefore less likely that TR agonist activity is not environmentally relevant as performance limitation of reporter gene assays based on cell line type is increasingly becoming more probable [6].…”

“…The hypothalamic-pituitary-gonad axis, and the hypothalamic-pituitary-thyroid axis are two major axes of the endocrine system that are particularly vulnerable to exogenous chemical assault owing to their delicate intricate cascade of cellular and molecular regulation of physiological functions [3] [4]. However, while the effects of EDCs on the HPG axis have been considerably investigated and comprehensively described, a lot remains to be clarified on the effect of EDCs on the HPT axis, principally involving the thyroid hormone system [5] [6].…”

“…However, with the in vitro detection approach, it appears that a possible major mode of actions of TDCs, thyroid receptor activation, is not environmentally relevant. Further, while the reason for this is not yet clear, and particularly challenging, performance limitation of in vitro assays is highly probable, given the inherent cell type-induced variations in the sensitivity of the widely used in vitro assays to potential TDCs [6] [11].…”

“…Thus, the ligand binding assays and the reporter gene assays both measure TR-mediated disruption of TH signaling. Studies show that TR-mediated disruption of TH signaling is a major mode of action of TDCs in wastewater and surface waters, and TDCs with potential to interfere with TRs can either act as antagonists, repressing the receptor upon binding M. E. Imiuwa, A. O. Ekperusi DOI: 10.4236/jep.2019.109067 1135 Journal of Environmental Protectionto it or as agonists when they are able to activate the receptor upon binding the receptor[6]. However, while TR binding assays and reporter gene assays can both detect TDCs with potential to interfere with TR, only reporter gene assays can discriminate between agonist and antagonist activities.…”