Equilin does not affect thyroid hormone signaling in the developing Xenopus laevis tadpole brain

Toxcast/Tox21 is a massive federally run research effort dedicated to better understanding the potential toxicity of thousands of compounds in a high throughput manner. Among this list of compounds is equilin, an estrogen-like compound that was flagged as a potential thyroid hormone agonist. Here we examine if equilin acts like a thyroid hormone agonist on cellular and molecular mechanisms of brain development in Xenopus laevis tadpoles. To examine the effect of equilin, tadpoles were divided into eight groups and received 4 days of exposure. The experimental groups were as follows: 1 L, 10 L, and 100 L of equilin, 1 L, 10M, and 100 M of 17-{beta} estradiol as an estrogen control, 15 g/mL thyroxine (T4) as a thyroid hormone control, and a no-exposure control. After 4 days of treatment, animals were treated with CldU to label dividing cells for 2hr and then euthanized in MS-222. After fixation, body length was measured and the brains dissected out. IHC was performed on brains for CldU to label proliferating neural progenitor cells. Brains were then whole-mounted and analyzed using confocal microscopy. We found that equilin did not increase the number of dividing progenitor cells in a T4-like manner. Instead, equilin decreased proliferation in a dose-dependent manner, as did estradiol. The same paradigm was performed separately staining for caspase-3 and h2ax, finding that equilin increased cell death in contrast to CNTL and T4. In another experiment, RNA was extracted from tadpole brains in each group and qPCR was performed to assess change in expression of thyroid hormone-sensitive genes, Equilin did not affect gene expression in a thyroid hormone-like manner. Our data indicate that equilin does not act as a thyroid hormone agonist in the Xenopus laevis nervous system but instead acts similarly to estradiol. Our data strongly suggest that equilin is not a TH disruptor, contrary to the findings of the ToxCast/Tox21 dataset.