An assay for screening xenobiotics for inhibition of rat thyroid gland peroxidase activity

Abstract

1. A number of chemicals have been shown to produce disruption of the thyroid gland, resulting in reduced thyroid hormone synthesis, by a mechanism involving inhibition of thyroid peroxidase (TPO) activity (EC 1.11.1.8).

2. An assay was developed for rat thyroid gland microsomal TPO activity, employing L-tyrosine as the physiological substrate, with analysis of the formation of the 3-iodo-L-tyrosine (3MIT) metabolite by ultra-performance liquid chromatography-mass spectrometry-mass spectrometry.

3. Formation of 3MIT was linear with respect to both rat thyroid gland microsomal protein concentration and incubation time, whereas only small quantities of 3,5-diodo-L-tyrosine were formed.

4. Studies were performed with nine known TPO inhibitors. The most potent inhibitors were 3-amino-1,2,4-triazole, ethylene thiourea, methimazole and 6-propyl-2-thiouracil which had IC₅₀ values (i.e. concentration to produce a 50% inhibition of enzyme activity) of 0.059, 0.791, 1.07 and 1.96 μM, respectively, whereas the least potent inhibitor was sodium perchlorate which had an IC₅₀ value of 13,800 µM.

5. For five inhibitors, where literature data were available, the observed IC₅₀ values obtained in this study employing rat thyroid gland microsomes and L-tyrosine as substrate were similar to those previously reported using the spectrophotometric guaiacol oxidation assay.

Keywords:

• Thyroid gland
• thyroid peroxidase
• thyroid peroxidase inhibitors
• rat thyroid gland microsomes
• iodination of L-tyrosine
• mass spectrometric analysis

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

This study was funded by LFR Molecular Sciences.