Abstract
Introduction:
A proper thyroid hormone homeostasis plays a crucial role in the human development and basal metabolic rate and disruption of thyroid hormone homeostasis may lead to severe neurodevelopmental and physical adverse effects. To improve identification of thyroid hormone system disruptors, the validation of multiple fast, low-cost, robust, sensitive, animal-free, and human-relevant thyroid in vitro assays was coordinated by the European Union Reference Laboratory for alternatives to animal testing. The thyroid peroxidase (TPO) enzyme plays a critical role in the thyroid hormone synthesis and is a potential target of xenobiotics. In this study, the development and in house validation of an in vitro assay for evaluation of chemicals for the potential interference with TPO-catalyzed iodination is reported.
Methods:
First, an analytical method to quantify in vitro monoiodotyrosine formation by Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry was validated. Assay reproducibility was assessed using six chemicals in at least five valid runs. Next, 30 blinded chemicals were tested to evaluate the performance of the method to identify potential TPO disruptors. The chemicals were unblinded after experimental completion and compared to previously reported data.
Results and Discussion:
24 out of 30 test chemicals inhibited TPO by >20% (i.e., TPO-mediated iodination disruptors). Seventeen test chemicals produced a maximum TPO inhibition of >90%. Seven test chemicals produced a maximum TPO inhibition between 20 and 90%. Six test chemicals produced a maximum TPO inhibition below 20% (i.e., non-disruptors). Interestingly, this assay identified four chemicals as TPO inhibitors that were previously classified as non-inhibitors based on assays that only evaluate TPO-catalyzed oxidation, suggesting specific disruption of TPO-mediated iodination.
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