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PM16 Molecular Indicators for Ecological Exposure
(PM265) Modulation of T3-induced thyroid receptor gene expression as a biomarker of endocrine disruption in an amphibian, Rana pipiens.
Gallant, N1, Werry, K1, Trudeau, V1, 1 University of Ottawa, Ottawa, ON, Canada
ABSTRACT- Many endocrine disrupting chemicals in the aquatic environment can upset amphibian development but their mechanisms of action are often unknown. Thyroid hormones control amphibian metamorphosis by acting through the thyroid receptors (TRs) which we hypothesize are targets for endocrine disruptors. We cloned a full length TR- sequence for R. pipens that is 99% and 86% identical to R. catesbeiana and X. laevis TR-, respectively. Primers and probes were then designed for a multiplex QPCR assay to assess levels of TR- and TR- transcripts in R. pipiens tissues. Tadpoles (Gosner stage 33) were exposed to triiodothyronine (T3; 0.5nM, 5nM, 50nM) for 48 hours. Brain and tail were dissected and RNA was isolated for synthesis into cDNA. T3 induced a dose-dependent increase in TR- to a maximum of 5-fold in brain and 10-fold in tail. TR- increased 2-fold after exposure to 50nM T3. Results were normalized using ribosomal protein L8 as an internal control. This is the first demonstration of TR- autoinduction in the brain of a non-Xenopus amphibian, and is the basis of a new assay to determine the effects of endocrine disrupting chemicals on the thyroid axis. We have demonstrated that chronic waterborne ethinylestradiol (EE2) exposure increases tadpole size and alters the rate of metamorphosis suggesting that estrogen may affect the thyroid axis. Tadpoles were pre-exposed to EE2 (5 nM; 48hrs) and then to T3 (5 and 50nM; 48hrs). EE2 did not affect T3-induced TR/ expression which suggests that EE2 does not affect amphibian development through direct TR mechanisms. We are currently screening other potential endocrine disruptors for their ability to modulate TR-expression. Supported by CNTC and NSERC.
Key words: thyroid receptors, endocrine disruptors, QPCR, gene expression
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