Citation:

Thomas, R., M. Hawks, J. Ford, AND M. Gilbert. Deiodinase metabolism of thyroid hormones as measured by LC-MS/MS is altered by exposure to iopanoic acid in a rodent model. Society of Toxicology, Salt Lake City, UT, March 10 - 14, 2024.

Impact/Purpose:

Environmental contaminants can interfere with thyroid hormone homeostasis in a variety of ways. Deiodinases (DIO) are a family of three metabolizing enzymes (DIO1, DIO2, DIO3) by activating or deactivating TH action. Little information available on outcomes of chemically-induced interference of this important thyroid regulatory system. We demonstrate alterations in serum hormone profiles can be seen with in vivo administration of a deiodinase inhibitor, iopanoic acid (IOP) and directly tie these serum changes to deiodination in the liver in ex vivo samples. This required the development of a sensitive mass spectrometry based assay.  Translation of in vitro based deiodinase activity to in vivo response is an essential step in the validation and use of high throughput assays for thyroid disruption. Furthermore, as deiodinase inhibition can have deleterious effects on brain development, analytical methods developed here will be essential to characterize these actions in brain tissue, the topic on ongoing research. Ultimately these efforts will contribute to our understanding of thyroid hormone regulation and its broader health implications. 

Description:

Thyroid hormones (TH) are highly iodinated molecules that regulate many aspects of mammalian physiology and are essential for normal growth and development. Deiodinases (DIO) are a family of three metabolizing enzymes (DIO1, DIO2, DIO3) by activating or deactivating TH action. The physiological effects of alterations in DIOs have largely been characterized in genetically-modified mouse models, with little information available on outcomes of chemically-induced interference of this important thyroid regulatory system. To determine if chemical interference with deiodinases would be reflected in serum TH profiles in a rodent model, we investigated iopanoic acid (IOP), a potent in vitro inhibitor of all three DIOs that is relatively unexplored in intact mammalian systems. Adult Long Evans female rats administered IOP (0, 1, 5, 50 and 100mg/kg/day) for 14 days exhibited an increase in serum thyroxine (T4) and reverse T3 (rT3). To verify these in vivo serum TH effects were induced by IOP action on deiodination pathways, we examined deiodinase activity ex vivo in liver of exposed animals. To achieve this goal, liquid chromatography mass spectrometry (LCMS) was used to quantify thyroid hormone metabolic products as readouts of deiodination. In hepatic microsomes prepared from naïve rats, 10uL of 20 mg/mL microsomal protein were placed in 96-well plates, spiked with 2uL of 10uM T3, T4, or rT3, in a total reagent volume of 200uL, incubated for 1 hour, and TH analytes measured by LCMS. These values were compared to samples spiked in the presence of a high concentration of a DIO1 inhibitor (100uM propylthiouracil, PTU), DIO3 inhibitor (300uM Xanthohumol, Xn), or the Dio1/Dio2/Dio3 pan-inhibitor IOP (800uM), concentrations derived from inhibition potency curves for each inhibitor dissolved in 1% DMSO. Negative controls included wells containing denatured microsomes and microsomes that did not receive the hormone spike. All samples were run in triplicate and the limit of detection of the TH analytes was 0.01ng/ml. Deiodination of rT3 in liver results from the activation of Dio1 and Dio3 to produce 3,3’T2. We found complete inhibition of Dio1 with PTU (2.2 +/- 0.1 ng/ml 3,3’-T2), and of Dio1/2/3 with IOP (2.6 +/- 1.2ng/ml) relative to control incubations with DMSO (448.9 +/- 16.0 ng/ml). To directly assess Dio3 inhibition, we examined microsomes prepared from placental tissue where Dio3 is highly expressed. These samples were incubated with T4 to monitor the conversion to 3,3’-T2 by Dio3 and Xn significantly decreased its production (56.67+/-14.5 vs 5.08 +/- 0.6 ng/ml in DMSO vs Xn incubations, respectively). In both liver and placenta, as expected, denatured microsomes exhibited no conversion to 3’-3-T2 (all values < 3.5 ng/ml). These data confirm the performance of the deiodinase assay with known inhibitors allowing us to differentiate the impact of chemical exposure on the function of each of these enzymes. In liver microsomes derived from IOP-exposed animals, Dio1 activity was isolated by blocking Dio3 withd with Xn, and dose-dependent reductions in conversion of rT3 to 3’3-T2 were evident at the lowest dose of IOP tested (51% +/- 1.7  in the 1 mg/kg dose group) . These findings were paralleled by high levels of rT3 at the higher IOP doses (e.g., mean=106-112ng/ml), comparable to those observed with denatured microsomes (mean=110ng/ml) where no conversion takes place, confirming IOP-induced inhibition of DIO1 activity in an in vivo rodent model. They are consistent with increases in T4 in in serum, confirming inhibition of Dio1 likely underlies the in vivo effects we observed. As deiodinase inhibition can have deleterious effect on brain development, analytical methods developed here will be essential to characterize these actions in brain tissue, ultimately contributing to our understanding of thyroid hormone regulation and its broader health implications. Does not reflect EPA policy.

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