Progress towards Development of an Amphibian-Based Thyroid Screening Assay Using Xenopus laevis. Organismal and Thyroidal Responses to the Model Compounds 6-Propylthiouracil, Methimazole, and Thyroxine

Abstract

In response to the initial Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. This research highlighted key limitations associated with relying on tail resorption as a measure of anti/thyroid activity. The most critical limitation being that tail tissues of tadpoles at metamorphic climax are insensitive to perturbation by thyroid axis agonists/antagonists. To improve upon the initial proposal, we have conducted experiments comparing the sensitivity of pre-metamorphic (stage 51) and pro-metamorphic (stage 54) larvae to the model thyroid axis disruptors methimazole (control, 6.25, 12.5, 25, 50, 100 mg/l), 6-propylthiouracil (PTU) (control, 1.25, 2.5, 5, 10, and 20 mg/l), and thyroxine (T4) (0.25, 0.5, 1, 2, 4 microg/l). Exposures were conducted using two different experimental designs. For experimental design 1, tadpoles were exposed to methimazole or PTU starting at either NF stage 51 or NF 54 for 14 days. For experimental design 2, tadpoles were exposed to PTU or T4 starting at NF stage 51 or NF 54 for 14 and 21 days, respectively. Methimazole and PTU, which are thyroid hormone synthesis inhibitors, both caused a concentration dependent delay in larval development. As determined from this endpoint, there were only minor differences in sensitivity observed among the two stages examined. Further, both compounds caused concentration dependent changes in thyroid gland morphology. These changes were characterized as reduced colloid, glandular hypertrophy, and cellular hyperplasia and hypertrophy. Treatment failed to negatively affect growth, even in tadpoles that experienced significant metamorphic inhibition. T4 treatment resulted in a concentration dependent increase in developmental rate, as would be expected. Similar to studies with methimazole, there were no differences in sensitivity among the two developmental stages examined. These results indicate that tadpoles in the early stages of metamorphosis are sensitive to thyroid axis disruption and that development of a short-term, diagnostic amphibian-based thyroid screening assay shows considerable promise.

Mast cell population in the frog brain: distribution and influence of thyroid status

“…By contrast, PTU treatment at pre-metamorphic stage (stages 26-27) blocks larval development but it seems to bear no significant effect on pro-metamorphic tadpoles (stages 29-30). This last result is consistent with recent observations in the African clawed frog in which it was demonstrated that late pro-metamorphic tadpoles are practically insensible to inhibitors of T 4 synthesis, such as thiourea or metilazolo (Degitz et al, 2005). However, either in pre-or in pro-metamorphosis, PTU injections lead to a significant increase (more than 2-fold) in the brain MC population.…”

Evaluation of the amphibian metamorphosis assay: Exposure to the goitrogen methimazole and the endogenous thyroid hormone L‐thyroxine

“…Methimazole (1-methyl-2-imidazolethiol) is a known inhibitor of thyroperoxidase, the enzyme responsible for synthesizing thyroid hormones. Methimazole has previously been shown to significantly inhibit the rate of X. laevis development and to affect thyroid gland histology at concentrations !12.5 mg/L [18,19]. L-thyroxine (T4) is the major thyroid hormone synthesized by the thyroid gland in vertebrates.…”

Molecular characterization and developmental expression patterns of thyroid hormone receptors (TRs) and their responsiveness to TR agonist and antagonist in Rana nigromaculata

“…In the literature, Xenopus laevis is the most-used model species for studying thyroid disrupting effects (Degitz et al, 2005;Heimeier et al, 2009). Given the obtainability of experimental animals, some native amphibian species, such as Rana catesbeiana and Rana pipiens (Helbing et al, 2006;Croteau et al, 2009), Rana temporaria (Arukwe and Jenssen, 2005;Gutleb et al, 2000) and Rana rugosa (Hanada et al, 2003;Oka et al, 2009), are used to assess effects of chemicals on the thyroid system.…”