Extrapolating In Vitro Screening Assay Data for Thyroperoxidase Inhibition to Predict Serum Thyroid Hormones in the Rat

Thyroperoxidase (TPO) is an enzyme essential for thyroid hormone (TH) synthesis and a target site for a number of xenobiotics that disrupt TH homeostasis. An in vitro high-throughput screening assay for TPO inhibition, the Amplex UltraRed-TPO (AUR-TPO), has been used to screen the ToxCast chemical libraries for this action. Output from this assay would be most useful if it could be readily translated into an in vivo response, namely a reduction of TH in serum. To this end, the relationship between TPO inhibition in vitro and serum TH decreases was examined in rats exposed to 2 classic TPO inhibitors, propylthiouracil (PTU) and methimazole (MMI). Serum and gland PTU, MMI, and TH levels were quantified using tandem liquid chromatography mass spectrometry. Thyroperoxidase activity was determined in thyroid gland microsomes treated with PTU or MMI in vitro and ex vivo from thyroid gland microsomes prepared from exposed animals. A quantitative model was constructed by contrasting in vitro and ex vivo AUR-TPO results and the in vivo time-course and dose-response analysis. In vitro:ex vivo correlations of AUR-TPO outputs indicated that less than 30% inhibition of TPO in vitro was sufficient to reduce serum T4 by 20%, a degree of regulatory significance. Although further testing of model estimates using other TPO inhibitors is essential for verification of these initial findings, the results of this study provide a means to translate in vitro screening assay results into predictions of in vivo serum T4 changes to inform risk assessment.

Impact/Purpose

This paper developed a quantitative model in aid of interpretation of the inhibition constant (IC50) from of AmplexUltra Red (AUR) -thyroperoxidase (TPO) in vitro assay to thyroid hormone decrements in serum. AUR-TPO inhibition assay is a high-throughput assay used to assess chemicals that inhibit TPO, an enzyme important in the synthesis of thyroid hormones. In vitro assays have accelerated the rate of chemical testing and have provided IC50 as a measure of observed biological response. However, regulatory agencies are currently utilizing in vivo endpoints or serum decrements of T4 in risk evaluations. Therefore, the goal of the current study was to address the gap between in vitro assays and in vivo effects. Dose-dependent decrements in gland and serum thyroid hormones and internal doses of propylthiouracil and methimazole in the serum and thyroid gland, and in vitro IC50 were used to develop a computational model. The model links in vitro assay output of synthesis inhibition to serum hormone reductions in vivo. This study showed that in vitro and ex vivo measures of TPO activity are linearly correlated and when extrapolated to predict serum T4, we observed that 30% TPO inhibition corresponded with a 20% decrease in serum T4. Although future testing of other TPO inhibitors is required to fully evaluate the generalizability of this model to other chemicals, the approach provides a simple scheme to incorporate chemical potency factors derived from in vitro assays to make predictions of an in vivo endpoint. This work developed a methodology important for extrapolating in vitro assay data to in vivo effects and demonstrates quantitative linkages from the molecular initiating event, to an adverse outcome to aid in regulatory decision making.

Citation

Hassan, I., H. El-Masri, J. Ford, A. Brennan, S. Handa, K. Friedman, AND M. Gilbert. Extrapolating In Vitro Screening Assay Data for Thyroperoxidase Inhibition to Predict Serum Thyroid Hormones in the Rat. Society of Toxicology, RESTON, VA, 173(2):280-292, (2020). [DOI: 10.1093/toxsci/kfz227]

DOI: Extrapolating In Vitro Screening Assay Data for Thyroperoxidase Inhibition to Predict Serum Thyroid Hormones in the Rat