Establishment of transactivation assay systems using fish, amphibian, reptilian and human thyroid hormone receptors

Abstract

Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRβ) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3 : 10(-11) M in TRα and 10(-10) M in TRβ) and thyroxine (T4 : 10(-9) M in TRα and 10(-8) M in TRβ). Analogs of thyroid hormone (3,5,3',-triiodothyroacetic acid and 3,3',5,5'-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4 . These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3 -stimulated transcriptional activity of the O. latipes TRα was inhibited by 10(-5) M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10(-5) M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation.

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

“…In addition, one in vitro study using GH3.TRE-Luc cell lines also revealed a non-monotonic dose-dependent TH signaling disrupting action of TBBPA ranging from 100 nmol/L-10 μmol/L in the presence of T3 (Freitas et al, 2011). However, other in vitro reporter gene assays could not detect non-monotonic dose-dependent effects of TBBPA on TH signaling, even when a wide concentration range of TBBPA was tested (Hofmann et al, 2009;Kudo et al, 2006;Oka et al, 2012). It is known that non-monotonic dose-response for endocrine disrupting chemicals is observed frequently (Beausoleil et al, 2013;Vandenberg, 2014).…”

Molecular mechanisms and tissue targets of brominated flame retardants, BDE-47 and TBBPA, in embryo-larval life stages of zebrafish (Danio rerio)

“…Since several in vitro studies have demonstrated that BFRs have limited agonistic effects on TR-mediated gene transcription [69][70][71], it seems plausible that the increases in thraa and thrb transcription following TBBPA and BDE-47 exposures were a result of increased local T3 levels.…”

Development of a common carp (Cyprinus carpio) pregnane X receptor (cPXR) transactivation reporter assay and its activation by azole fungicides and pharmaceutical chemicals

“…g . Bury et al, 2003, Lange et al, 2012, Oka et al, 2013, Todo et al, 1999) and applied to establish the roles of specific nuclear receptors in hormone signalling in fish. They have also been used as efficient chemical screening systems.…”