Development of a Thyroperoxidase Inhibition Assay for High-Throughput Screening

Abstract

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including Z', dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z' score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2',4,4'-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach.

Simple and rapid in vitro assay for detecting human thyroid peroxidase disruption

“…It can be seen that the luminol assay detects the disruption of TPO activity by the 5 selected TPO inhibitors in a dose-dependent manner. Moreover, while the Ampliflu Red (Paul et al, 2014), guaiacol and luminol (Tab. 1) methods are all able to detect pTPO activity that is present in based activity in cells from non-thyroid origin.…”

Establishment of cumulative assessment groups of pesticides for their effects on the thyroid

“…by evaluating in vitro the potential for inhibition of the sodium-iodide symporter (NIS) (Cianchetta et al, 2010;Kogai and Brent, 2012;Hallinger et al, 2017) and thyroid peroxidase (TPO) (Kambe and Seo, 1997;Paul et al, 2014;Paul Friedman et al, 2016;Wu et al, 2016). by evaluating in vitro the potential for inhibition of the sodium-iodide symporter (NIS) (Cianchetta et al, 2010;Kogai and Brent, 2012;Hallinger et al, 2017) and thyroid peroxidase (TPO) (Kambe and Seo, 1997;Paul et al, 2014;Paul Friedman et al, 2016;Wu et al, 2016).…”

“…3) The presence of other possible thyroid-disrupting modes of action such as interference with TH synthesis should also be excluded, e.g. by evaluating in vitro the potential for inhibition of the sodium-iodide symporter (NIS) (Cianchetta et al, 2010;Kogai and Brent, 2012;Hallinger et al, 2017) and thyroid peroxidase (TPO) (Kambe and Seo, 1997;Paul et al, 2014;Paul Friedman et al, 2016;Wu et al, 2016). It must, however, be acknowledged that substances may interfere with the thyroid hormone system through many different mechanisms of action, and that currently validated/standardised in vitro assays do not exist to investigate all these different pathways and a reasonable effort is anticipated, based on available tools and current understanding of thyroid physiology'.…”

An exposure:activity profiling method for interpreting high-throughput screening data for estrogenic activity—Proof of concept

“…Previously, as an androgen reference material, we suggested investigating the possibility of using 3,3-diindolylmethane (formed during digestion of cruciferous vegetables), it is commercially available (e.g., SigmaAldrich) and has been described as a ''naturally occurring pure androgen antagonist'' (Le et al, 2003). To date, there are not enough HTS assay data to adequately classify putative thyrotoxicants, largely because not all modes of action for thyroid disruption are evaluated by current ToxCast™ or Tox21 assay tools (Rotroff et al, 2013a;Paul et al, 2014). For thyroid applications, a family of active compounds would likely be needed to address different molecular initiating events that result in thyroid perturbation, including thyroperoxidase inhibition, sodium-symporter inhibition, deiodinase inhibition, upregulation of hepatic catabolism, interference with serum binding proteins, and interactions with thyroid hormone receptors (Murk et al, 2013;Crofton, 2008).…”

“…It is evident from these early efforts to prioritize chemicals for further estrogen pathway-related effects that more exposure context needs to be provided to permit a transparent, science-based approach for identifying priority chemicals. There are even larger barriers, including a lack of appropriate HTS assays, for the use of HTS data to predict and prioritize substances for potential activity in thyroid and steroidogenesis pathways, (Rotroff et al, 2013a, Paul et al, 2014. Despite these limitations, such HTS methods hold great promise for improving the biological basis for priority setting and chemical screening, and may critically support targeted testing with a tiered, riskbased framework Pastoor et al, 2014).…”