Cells to Systems Screening for Thyroid Hormone Disrupting Chemicals in the Brain

Abstract

Abstract Precisely timed and regulated thyroid hormone (TH) levels are essential for vertebrate development and metabolism. To detect environmental chemicals that disrupt TH action, we developed an integrated luciferase reporter cell line in rat pituitary GH3 cells that is TH responsive through activation of endogenous thyroid hormone receptors (TRs). High throughput screening as part of the Tox21 program identified several non-TH related positive hits as RXR agonists, a supposed “silent” heterodimer partner for TRs. These data showed RXR agonists can affect TR signaling at least in pituitary cells. We extended these results to our sensitive and specific in vivo model for TH action, Xenopus laevis metamorphosis. We previously demonstrated that RXR agonists like organotins and the pharmaceutical bexarotene, strongly potentiate (and RXR antagonists inhibit) TH induced metamorphic programs, at the morphological, cellular (e.g. apoptosis or proliferation), transgenic reporter gene levels, and transcriptomic responses in tadpole tails. We have now extended this analysis to include RNA-Seq experiments over distinct time points in the tadpole brain, a common target of TH in humans and frogs, revealing specific gene sets particularly affected by TH and RXR ligands working in concert. Very few genes were affected by RXR ligands alone. The remarkable overlap between the environmental toxicant tributyltin and the synthetic and specific RXR ligand bexarotene regulated transcriptomes provides strong evidence that they have a common molecular target in multiple tissues. We have also created germline mutations in all RXR family members in Xenopus tropicalis (alpha, beta and gamma), and both copies of the duplicated TR beta gene in Xenopus laevis via genome editing approaches. This will allow us to further investigate TR-RXR heterodimer function across tissues and developmental timeframes, and in response to known and suspected TR and RXR ligands. Our studies revealed an unanticipated degree of TR and RXR ligand interactions in vitro and in vivo, highlighting a surprising role of RXRs as avenues for TH endocrine disruption, including the brain.