Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, ON, Canada K1A 0K9.
Department of Biochemistry and Molecular Biology, Medical Center of Postgraduate Education, Warsaw, Poland.
Toxicol In Vitro. 2020 Feb;62:104662. doi: 10.1016/j.tiv.2019.104662. Epub 2019 Oct 16.
Impaired synthesis or action of thyroid hormones (THs) during critically sensitive periods of development can have long term adverse effects on health. Development of rapid assays to identify chemicals that impair THs physiology is an important goal for reducing risks from chemical use. Thyroid peroxidase (TPO) is a key enzyme regulating THs synthesis in thyroid gland and a vulnerable target for chemicals that disrupt THs synthesis. To develop a human-relevant, rapid assay for TPO inhibition, we have engineered two cell lines (CHO and LentiX- 293) to express active human TPO (hTPO) enzyme and applied them in a recently-described assay using a stable fluorescent product (Amplex UltraRed). Assay performance was assessed by comparing activity of 19 reference chemicals with known strong, weak or no TPO inhibitory activity. The assay using hTPO from either cell line consistently identified the relative potency of strong to moderate inhibitors and chemicals known to be inactive. Results were less consistent for chemicals reported to be weak inhibitors of rodent TPO, possibly suggesting some species specificity. Our studies support the use of hTPO from stably transfected cell lines to substitute for animal-derived thyroid microsomes for rapid high throughput screening assays to identify and characterize TPO inhibitors.
在发育的关键敏感时期，甲状腺激素（THs）的合成或作用受损可能对健康产生长期的不良影响。开发快速检测方法以鉴定影响 THs 生理功能的化学物质是减少化学物质使用风险的重要目标。甲状腺过氧化物酶（TPO）是调节甲状腺中 THs 合成的关键酶，也是破坏 THs 合成的化学物质的脆弱靶标。为了开发与人相关的、快速的 TPO 抑制测定法，我们已经设计了两种细胞系（CHO 和 LentiX-293）来表达活性人 TPO（hTPO）酶，并将其应用于最近描述的使用稳定荧光产物（Amplex UltraRed）的测定法中。通过比较 19 种参考化学物质的活性，评估了测定法的性能，这些化学物质具有已知的强、弱或无 TPO 抑制活性。该测定法使用来自任一种细胞系的 hTPO 均能一致地确定强至中度抑制剂以及已知无活性的化学物质的相对效力。对于报告为弱抑制啮齿动物 TPO 的化学物质，结果不太一致，这可能表明存在一些物种特异性。我们的研究支持使用稳定转染的细胞系中的 hTPO 替代动物源性甲状腺微粒体，用于快速高通量筛选测定法，以鉴定和表征 TPO 抑制剂。
