Effects of hepatic enzyme inducers on NIS, TPO and DIO1-2-3 activities

Effects of hepatic enzyme inducers on in vitro NIS, TPO, and DIO1-2-3 activities

Audrey Baze1, Laure Asselin1, Betty Ory1, Lucille Wiss1, Amélie Schäfer1, Cassandra Chanel1, Klaus Biemel2 and Lysiane Richert3

1 KaLy-Cell, Plobsheim, France

2 Pharmacelsus, Saarbrücken, Germany

3 Zylan, Obernai, France

Purpose: Thyroid hormones (THs) regulate metabolism, growth, and neurodevelopment. Disruption of TH homeostasis by xenobiotics is an increasing concern, as environmental chemicals can interfere with the thyroid axis via distinct molecular-initiating events (MIEs), ultimately reducing circulating TH levels. In response, Regulation (EU) 2018/605 and joint guidance from the European Chemicals Agency (ECHA) and the European Food Safety Authority (EFSA) mandate the evaluation of endocrine-disrupting potential of all active substances undergoing (re-)registration. Key MIEs of TH synthesis and metabolism include inhibition of the sodium/iodide symporter (NIS), thyroid peroxidase (TPO), and deiodinases (DIO1, DIO2, DIO3), which alter iodide uptake, TH synthesis, and peripheral conversion of thyroxine (T4) into active triiodothyronine (T3) or inactive reverse T3 (rT3). The activation of hepatic nuclear receptors such as constitutive androstane receptor (CAR) and pregnane X receptor (PXR) leads to induction of enzymes such as phase I CYPs and phase II UGTs that could contribute to increased hepatic conjugated TH clearance in humans. This study aims to assess whether the reduction of TH levels in humans reported for some compounds known as CAR/PXR activators might be the consequence of alternative MoAs, such as NIS, TPO, and/or DIO inhibition.

Methods: NIS activity was assessed in HEK293 cells stably expressing human NIS using the Sandell-Kolthoff reaction. TPO activity was measured in human thyroid microsomes using the Amplex UltraRed method. DIO activities were assessed in HEK293 cell pellets expressing each human DIO isoform, with T3 and rT3 quantified by LC-MS following T4 incubation. Positive and negative controls were included to ensure assay specificity and reproducibility.

Results: Preliminary results show that rifampicin and phenobarbital, both known as CAR/PXR activators, inducing hepatic enzymes in humans, also act as inhibitors of TPO (mild for phenobarbital, potent for rifampicin) and of DIO1 (mild for phenobarbital) in vitro. These findings suggest that direct inhibitory effects on TH synthesis (TPO inhibition) and metabolism (DIO1 inhibition) may, rather than liver-mediated mechanisms of enzyme induction, largely contribute to the reduced TH levels in vivo reported in humans with these compounds.

Conclusion: These findings highlight that some compounds may display various MoAs in humans, making it difficult to identify those leading to disruption of TH levels. Ongoing work includes the testing of additional compounds identified in the literature, to further explore their potential effects on TH synthesis (via NIS and TPO) and metabolism (via DIO1–3).

Keywords: Thyroid hormones (THs); endocrine disruption; Mechanism of action (MoA); New Approach Methodologies (NAMs); deiodinase (DIO); thyroid peroxidase (TPO); sodium–iodide symporter (NIS)