A high throughput screening assay for human Thyroperoxidase inhibitors.

Abstract

Rapid, human relevant assays are needed to assess potential hazards of the many chemicals in commerce. An assay of thyroid peroxidase (TPO) inhibition, using the substrate Amplex Ultra Red, was recently adapted for human TPO (AUR-hTPO). We tested a large number (788) of chemicals through this AUR-hTPO assay and compared performance with published results from an assay using enzyme from rat thyroid microsomes (AUR-rTPO). Coded chemicals, from the US EPA ToxCast Inventory, were tested in a tiered approach: 1) Initial screening at a single concentration; 2) Potency estimation for active chemicals with multiple concentrations; 3) Screening active chemicals for the non-specific activity. The assay gave consistent results for positive chemical methimazole and several positive and negative reference chemicals. hTPO inhibition was observed for 190 chemicals reported as positive in rTPO. Of these, 158 showed no confounding activity (interference due to fluorescence or non-specific protein inhibition). Comparison of all result with rTPO data and with evidence of TPO inhibition found in the literature suggest that the current assay has a higher rate of false negative but a much lower rate of false positive compared with the rTPO screen. These findings underscore the effectiveness of the AUR assay, using hTPO enzyme from engineered cell lines, to identify moderate to strong inhibitors but some improvements may be needed to detect weak TPO inhibitors.