ECE2021 Audio Eposter Presentations Environmental Endocrinology (2 abstracts)
Development of in vitro rat, dog and human deiodinase inhibition assays with LC-MS/MS analysis for the identification of endocrine disruptors
Dongtao Lee 1 , Kenneth Macleod 2 , Larry Higgins 3 , Rachel McBrinn 4 , Robin Dickinson 1 & Claudia McGinnis 1
1Concept Life Sciences, Endocrine Toxicology, Dundee, United Kingdom; 2University of Dundee, School of Life Sciences, Dundee, United Kingdom; 3H-Toxicology Consulting, Cupar, United Kingdom; 4University of Dundee School of Medicine, Systems Medicine, Dundee, United Kingdom
Thyroid hormones are important regulators of metabolism and development. Iodothyronine selenodeiodinases can both activate and inactivate thyroid hormones (Bianco et al. 2002) thus in vitro assays to quantify deiodinase inhibition are considered to be a valuable tool for studying effects on thyroid hormone metabolism by potential endocrine disruptors. The prohormone thyroxine (T4) is converted to 3, 5, 3’-triiodothyronine (T3) by outer-ring deiodination, or to 3, 3’, 5’-triiodothyronine (reverse T3/rT3) by inner ring deiodination. These compounds can then be further de-iodinated to diiodothyronine (T2). This work describes the development of screening in vitro methodology for the analysis of thyroid hormone metabolism by LC-MS/MS. Recombinant deiodinase enzymes D1, D2 and D3 for rat, dog and human were ectopically expressed in HEK293 cells and used to set up species-specific and isoform -specific incubation conditions to determine optimal protein and incubation kinetics for each enzyme. The conversion of T4 or rT3 by each respective recombinant deiodinase was assessed, and reactions were monitored by LC-MS/MS assay for formation of T2 or T3 for D1, T3 for D2 and rT3 for D3. Using known inhibitors of deiodinases, 6-propyl-2-thiouracil (PTU) for D1 and aurothioglucose (ATG) for D2 and D3, we demonstrate that the rat, dog and human deiodinase assays generate robust and reproducible data. These deiodinase assays are now routinely performed to assess the potential endocrine disrupting properties of small molecules.
Volume 73
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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