# In vitro thyroperoxidase inhibition assessment by LC-ICP-MS-based L-tyrosine iodination assay: comparison with Amplex Ultrared assay and its modifications

## TL;DR

This study compares two in vitro assays for measuring thyroid peroxidase inhibition, finding that one is more sensitive and captures more steps of the enzyme's activity.

## Contribution

The study introduces a more sensitive Tyr-I assay using HPLC-ICP-MS for thyroid peroxidase inhibition assessment compared to the AUR assay.

## Key findings

- The Tyr-I assay demonstrated superior sensitivity in detecting TPO inhibition by measuring both peroxidation and iodination steps.
- Fourteen out of twenty-one tested chemicals showed TPO-inhibition potential, with some effective concentrations as low as 15–74 nM.
- Adding sodium iodide improved the AUR assay's sensitivity but still did not match the Tyr-I assay's performance.

## Abstract

Disruption of the thyroid hormone (TH) system by environmental chemicals poses significant risks to human and wildlife health. Reliable in vitro assays are essential for assessing thyroid peroxidase (TPO) inhibition, which disrupts a key step in thyroid hormone (TH) synthesis, while adhering to 3R principles. This study aimed to compare the sensitivity and specificity of two types of in vitro assays, tyrosine iodination (Tyr-I) detected by HPLC-ICP-MS and Amplex UltraRed (AUR) and its modifications, using human HEK-TPOA7 cells and rat thyroid microsomes. It involved the evaluation of TPO inhibition by 21 chemicals (concentration range 0.002–200 µM) from diverse use categories, including industrial pollutants, pesticides, and pharmaceuticals. TPO-inhibition potential was indicated for 14 compounds. The Tyr-I assay, which measures the conversion of L-tyrosine to monoiodotyrosine, demonstrated superior sensitivity by capturing both the peroxidation and iodination steps of TPO activity, with effective concentrations for some human exposure-relevant chemicals (benzophenone 2, resorcinol) in the 15–74 nM range confirmed with both human and rat TPO sources. In contrast, the AUR assay detects only the peroxidation step, limiting its ability to fully assess TPO inhibition. The inclusion of sodium iodide (NaI) in the AUR assay significantly enhanced its sensitivity (though it was still lower than in Tyr-I assay), while adding L-tyrosine together with NaI did not. The study documents the advantages and limitations, as well as the application and interpretation potential of the different assays´ variants. It provides valuable information and scientifically sound methodology to support the development of efficient testing strategies for the assessment of thyroid hormone system-disrupting chemicals.

The online version contains supplementary material available at 10.1007/s00204-025-04258-y.