Expression data for hindlimb and tail samples in a metamorphosis assay for thyroid axis disruption - inhibitors

Thyroid hormones (TH), thyroxine (T4) and 3, 5, 3’-triiodothyronine (T3), play crucial roles in regulation of growth, development and metabolism in vertebrates and are targets for endocrine disruptive agents. Perturbations in TH action can contribute to the development of disease states and the U.S. Environmental Protection Agency is developing a high throughput screen using TH-dependent metamorphosis of the Xenopus laevis tadpole as an assay platform. Currently this methodology relies on external morphological endpoints and changes in central thyroid axis parameters. However, exposure-related changes in gene expression in TH-sensitive tissue types that occur over shorter time frames have the potential to augment this screen. Using a combination of cDNA array and real time quantitative polymerase chain reaction (QPCR) analyses, this study identifies molecular markers in tissues peripheral to the central thyroid axis. We examine the hindlimb and tail of tadpoles up to 96 hours of continuous exposure to T3, T4, methimazole, propylthiouracil, or perchlorate. Several novel biomarker candidates are indicated that include transcripts encoding importin, RNA helicase II/Gu, and defender against death protein, DAD1. In combination with previously-identified biomarker candidates, these transcripts will greatly augment the predictive and diagnostic power of the Xenopus metamorphosis assay for perturbation of TH action. Keywords: time course Twenty-eight early prometamorphic (NF stage 54;(Nieuwkoop and Faber, 1956; Shi, 2000)) tadpoles were continuously exposed to three separate T4 concentrations (10, 20.1, and 40.3 nM), and a LSW control in one exposure set, or five different T3 concentrations (0.48, 0.97, 1.92, 3.84, and 7.68 nM), and a LSW control in the second exposure set as described in detail in Zhang et al (2006). Each chemical exposure concentration was replicated twice along with the associated LSW control. At 24 h, 48 h and 96 h two animals per exposure replicate (four animals total per each individual treatment) were randomly selected, euthanized in MS-222, and preserved in RNAlater (Ambion Inc., Austin, Texas, USA) for analysis of gene expression. On exposure day 14, all remaining organisms were euthanized in MS-222, weighed, and developmentally staged in a blind evaluation. Animals exposed to either chemical showed an acceleration of metamorphosis which was published previously (Zhang et al., 2006). NF stage 54 tadpoles were continuously exposed to a single concentration of PTU (20 mg/L), MMI (100 mg/L) or PER (4 mg/L). We have previously shown that exposure to these concentrations resulted in an increase in thyroid gland size at day 8 and significantly delayed metamorphosis at 14 days (Degitz et al., 2005; Tietge et al., 2005). The exposure regimen details are recorded elsewhere (Zhang et al., 2006). Briefly, tadpoles were randomly placed into 24 tanks (20 tadpoles/tank) and exposed (six tanks/chemical) to a single concentration of each chemical. At 24 h, 48 h and 96 h 5 tadpoles from two of the 6 tanks (10 tadpoles per each individual treatment) were randomly selected, euthanized in MS-222, and preserved in RNAlater (Ambion Inc., Austin, Texas, USA) for analysis of gene expression.