Abstract
A simple and rapid luminometric assay for the detection of chemical inhibitors of human thyroid peroxidase (hTPO) activity was developed and validated with 10 model compounds. hTPO was derived from the human thyroid follicular cell line Nthy-ori 3-1 and its activity was quantified by measuring the oxidation of luminol in the presence of hydrogen peroxide (H2O2), which results in the emission of light at 428 nm. In this assay,hTPO activity was shown to be inhibited by 5 known TPO inhibitors and not inhibited by 5 non-inhibitors. Similar results were obtained with porcine TPO (pTPO).The inhibition of hTPO by the model compounds was also tested with guaiacol and Ampliflu Red as alternative indicator substrates. While all substrates allowed the detection of pTPO activity and its inhibition, only the Ampliflu Red and luminol-based methods were sensitive enough to allow the quantification of hTPO activity from Nthy-ori 3-1 cell lysates. Moreover, luminol gave results with a narrower 95% confidence interval and therefore more reliable data.Whole extracts of fast-growing Nthy-ori 3-1 cells circumvent the need for animal-derived thyroid organs,thereby reducing costs, eliminating potential contamination and providing the possibility to study human instead of porcine TPO. Overall, the application of luminol and Nthy-ori 3-1 cell lysate for the detection of the disruption of hTPO activity was found to represent a valuable in vitro alternative and a possible candidate for inclusion within a high throughput integrated testing strategy for the detection of compounds that potentially interfere with normal thyroid function in vivo.
Highlights
Thyroid hormones are essential for normal development and the regulation of basal metabolism
Five thyroidactive compounds that interfere with the thyroid hormone system through a non-thyroid peroxidase (TPO) mode of action were selected, i.e., T3, T4, benzyl butyl phthalate (BBP), OUB and amiodarone hydrochloride (AMI)
Tab. 1: Selected model compounds Test compounds used in this study with a brief description of their use or nature and their chemical structure
Summary
Thyroid hormones are essential for normal development and the regulation of basal metabolism. A deficiency in these hormones due to a lack of iodine during the first two trimesters of pregnancy is prevalent in landlocked areas and leads to cretinism (Cao et al, 1994). Thyroid hormone deficiency or excess lead to goitre, which is a swelling of the neck due to the enlargement of the thyroid gland. Addressing endocrine disruption, including the disruption of the thyroid hormone system, has been mandatory in the US since 1996 and the EU since 2006 (USEPA, 1996a,b; European Commission, 2006). Testing for thyroid hormone disrupters has been challenging due to the long duration, costs and unethical nature of the animal experiments involved. This has prompted the Organisation for Economic Co-operation
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