Simple and rapid in vitro assay for detecting human thyroid peroxidase disruption

Area of Science:

• Biochemistry
• Toxicology
• Assay Development

Background:

• Thyroid peroxidase (TPO) is crucial for thyroid hormone synthesis.
• Assessing TPO inhibitors is vital for understanding thyroid function disruption.
• Existing methods for TPO inhibition detection can be costly or use animal-derived materials.

Purpose of the Study:

• To develop and validate a simple, rapid luminometric assay for detecting human TPO (hTPO) inhibitors.
• To establish an in vitro alternative using cell lysates instead of animal-derived TPO.
• To evaluate the assay's suitability for high-throughput screening of potential thyroid disruptors.

Main Methods:

• Human TPO (hTPO) was derived from the Nthy-ori 3-1 cell line.
• hTPO activity was quantified by luminol oxidation, measuring light emission at 428 nm.
• Assay validation involved 10 model compounds, including known TPO inhibitors and non-inhibitors, and comparison with alternative substrates (guaiacol, Ampliflu Red).

Main Results:

• The luminometric assay successfully detected inhibition of hTPO by known inhibitors and showed no inhibition by non-inhibitors.
• Similar results were observed using porcine TPO (pTPO).
• Luminol and Ampliflu Red substrates were sensitive for quantifying hTPO activity from cell lysates, with luminol providing more reliable data (narrower confidence interval).

Conclusions:

• The developed luminometric assay using luminol and Nthy-ori 3-1 cell lysate is a valuable in vitro method for detecting hTPO activity disruption.
• This approach offers a cost-effective, animal-free alternative for TPO inhibition testing.
• The assay is a potential candidate for high-throughput integrated testing strategies to identify compounds interfering with thyroid function.