Citation:

Hassan, I., H. El-Masri, J. Ford, A. Brennan, S. Handa, AND M. Gilbert. In vitro to In vivo Extrapolation of High-throughput Screening Assay for Thyroperoxidase Inhibition. Society of Toxicology, Baltimore, Maryland, March 10 - 14, 2019.

Impact/Purpose:

United States Environmental Protection Agency Toxicology in the 21st century program has been established to develop methods to rapidly determine the ability of chemicals to interfere with biological processes, including the thyroid hormone axis. As a result, a rat microsomal based in vitro assay, Amplex-UltraRed (AUR)-TPO inhibition assay, was developed to assess xenobiotic-induced inhibition of thyroperoxidase (TPO), an important enzyme in thyroid hormone synthesis. Although TPO inhibition HTP screening assay has increased the number of chemicals assessed, the in vitro responses need to be extrapolated to in vivo measures before it can be applied to risk assessment. This study used known drugs that inhibit, methimazole (MMI) and 6-propylthiouracil (PTU) to conduct in vitro to in vivo extrapolation (IVIVE). Adult male rats were exposed to PTU and MMI in drinking water. Serum and thyroid gland levels of hormone were significantly reduced by exposure to these chemicals. Microsomes were prepared from thyroid gland of untreated, as well as PTU and MMI and AUR-TPO inhibition assay was conducted. Results of the TPO inhibition assay showed that in vitro exposure to PTU and MMI was much more potent than in dosed animals, whereas pharmacokinetic modeling resulted in in vivo IC50 that was closely aligned with in vitro IC50. In vitro results were then extrapolated to in vivo responses for the interpretation of AUR-TPO inhibition assay. These data provide an in vivo based anchor to serve as a reference for estimates derived from HTP outputs of thyroid disruption.

Description:

High-throughput (HTP) assays are being used to rapidly assess the potential of chemicals to interfere with the thyroid system. Amplex-UltraRed (AUR)-thyroperoxidase (TPO) inhibition assay is a rat microsomal HTP assay developed to screen the thyroid synthesis enzyme, TPO. Although AUR-TPO assay has accelerated chemical testing, translation of in vitro outputs to in vivo measures of thyroid dysfunction remain an obstacle to their use in risk assessment. Our goal was to explore in vitro to in vivo extrapolation (IVIVE) approaches to aid in interpretation of AUR data. Adult rats were exposed to varying doses of two known TPO inhibitors, methimazole (MMI) and 6-propylthiouracil (PTU) via drinking water for 4, 7, or 14 days. We first compared in vitro TPO inhibition to ex vivo TPO inhibition using thyroid gland microsomes prepared from the glands of PTU and MMI exposed animals. A second approach was to introduce chemical and glandular hormone levels obtained in vivo to a one-compartment pharmacokinetic (PK) model to derive an in vivo IC50. Serum and gland PTU and MMI levels increased in a dose- and time- dependent manner. Similarly, PTU and MMI reduced serum and gland triiodothyronine (T3) and thyroxine (T4) and increased serum thyroid stimulating hormone (TSH) in a dose- and time-dependent manner. Estimates of IC50 for TPO inhibition from the ex vivo study ranged from 108- 169 µM and were higher than those from in vitro exposure in microsomes from naïve glands (IC50=0.1-1.2 µM). Ex vivo estimates were well correlated with serum T4 at 4-day exposures but were inconsistent at later timepoints. The second approach to IVIVE incorporated the measured values of thyroid gland T4, MMI, and PTU into a PK model. Excellent fits of the 4-day PTU data were achieved with an estimation of an in vivo derived TPO inhibition of 6.2 µM, a value 5-fold higher than the in vitro estimate of 1.2 µM. Assessment of 7 and 14- day exposures for PTU and for MMI are underway. In summary, ex vivo measures of TPO inhibition using the AUR assay were highly correlated with serum thyroid hormones at short exposures durations, but were inconsistent at longer exposures, and IC50s were much higher than in vitro estimates. PK derived in vivo estimates of TPO inhibition with short exposures to PTU were more closely aligned to in vitro estimates. Our results provide an in vivo based anchor to aid in interpretation of HTP outputs of thyroid disruption. Does not reflect US EPA policy.

Record Details: