In vitro assays for characterization of distinct multiple catalytic activities of thyroid peroxidase using LC-MS/MS

A diverse set of environmental contaminants have raised a concern about their potential adverse effects on endocrine signaling. Robust and widely accepted battery of in vitro assays is available to assess the disruption of androgenic and estrogenic pathways. However, such definitive systems to investigate effects on the disruption of thyroid pathways by the xenobiotics are not yet well established. One of the major “Molecular Initiating Events” (MIEs) in thyroid disruption involves targeting of thyroid peroxidase (TPO), a key enzyme involved in thyroid hormone synthesis. TPO catalyzes mono- and diiodination of L-Tyrosine (L-Tyr) to generate 3-Iodo-l-tyrosine (MIT) and 3,5-Diiodo-l-tyrosine (DIT), respectively, followed by the coupling of iodinated tyrosine rings to generate thyroid hormones, 3,3’5-Triiodo-l-thyronine (T3) and Levothyroxine (T4). We sought to develop a robust, sensitive, and rapid in vitro assay systems to evaluate the effects of test chemicals on the multiple catalytic activities of thyroid peroxidase. Simple in vitro assays were designed to study TPO mediated distinct reactions using a single LC-MS/MS method. Herein, we describe a battery of assays to investigate the iodination of L-Tyr to MIT and DIT, MIT to DIT as well as, T3 to T4 catalyzed by rat thyroid TPO. Importantly, two sequential reactions involving mono- and diiodination of L-Tyr could be analyzed in a single assay. The assay that monitors in vitro conversion of DIT to T4 was developed to study the coupling of tyrosine rings. Enzyme kinetics studies revealed distinct characteristics of multiple reactions catalyzed by TPO. Further, the known TPO inhibitors were used to assess their potency towards individual TPO substrates and reactions. The resultant half maximum inhibitory concentration (IC(50)) values highlighted differential targeting of TPO catalyzed reactions by the same inhibitor. Overall results underscore the need to develop more nuanced approaches that account for distinct multiple catalytic activities of TPO.