Opitz Robert, Lutz Ilka, Nguyen Ngoc-Ha, Scanlan Thomas S, Kloas Werner
Department of Inland Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Mueggelseedamm 301, Berlin D-12587, Germany.
Toxicol Appl Pharmacol. 2006 Apr 1;212(1):1-13. doi: 10.1016/j.taap.2005.06.014. Epub 2005 Jul 22.
Amphibian metamorphosis represents a unique biological model to study thyroid hormone (TH) action in vivo. In this study, we examined the utility of thyroid hormone receptors alpha (TRalpha) and betaA (TRbetaA) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TRalpha gene expression whereas a marked up-regulation of TRbetaA mRNA occurred in hind limbs (prometamorphosis), head (late prometamorphosis), and tail tissue (metamorphic climax). Treatment of premetamorphic tadpoles with 1 nM 3,5,3'-triiodothyronine (T3) caused a rapid induction of TRbetaA mRNA in head and tail tissue within 6 to 12 h which was maintained for at least 72 h after initiation of T3 treatment. Developmental stage had a strong influence on the responsiveness of tadpole tissues to induce TRbetaA mRNA during 24 h treatment with thyroxine (0, 1, 5, 10 nM T4) or T3 (0, 1, 5, 10 nM). Premetamorphic tadpoles were highly sensitive in their response to T4 and T3 treatments, whereas sensitivity to TH was decreased in early prometamorphic tadpoles and strongly diminished in late prometamorphic tadpoles. To examine the utility of TRbetaA gene expression analysis for detection of agonistic and antagonistic effects on T3 action, mRNA expression was assessed in premetamorphic tadpoles after 48 h of treatment with the synthetic agonist GC-1 (0, 10, 50, 250 nM), the synthetic antagonist NH-3 (0, 40, 200, 1000 nM), and binary combinations of NH-3 (0, 40, 200, 1000 nM) and T3 (1 nM). All tested concentrations of GC-1 as well as the highest concentration of NH-3 caused an up-regulation of TRbetaA expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TRbetaA mRNA up-regulation. Results of this study suggest that TRbetaA mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in X. laevis tadpoles.
两栖动物变态是研究甲状腺激素（TH）体内作用的独特生物学模型。在本研究中，我们检测了非洲爪蟾蝌蚪中甲状腺激素受体α（TRα）和βA（TRβA）mRNA表达模式作为指示TH作用调节的分子标记的实用性。在自发变态过程中，TRα基因表达仅出现适度变化，而TRβA mRNA在 hind limbs（前变态期）、头部（前变态后期）和尾部组织（变态高峰期）中显著上调。用1 nM 3,5,3'-三碘甲状腺原氨酸（T3）处理前变态蝌蚪，在6至12小时内头部和尾部组织中TRβA mRNA迅速诱导，T3处理开始后至少维持72小时。发育阶段对蝌蚪组织在用甲状腺素（0、1、5、10 nM T4）或T3（0、1、5、10 nM）处理24小时期间诱导TRβA mRNA的反应性有强烈影响。前变态蝌蚪对T4和T3处理反应高度敏感，而早期前变态蝌蚪对TH的敏感性降低，晚期前变态蝌蚪则显著降低。为了检测TRβA基因表达分析在检测对T3作用的激动和拮抗效应方面的实用性，在用合成激动剂GC-1（0、10、50、250 nM）、合成拮抗剂NH-3（0、40、200、1000 nM）以及NH-3（0、40、200、1000 nM）和T3（1 nM）的二元组合处理48小时后，评估前变态蝌蚪中的mRNA表达。所有测试浓度的GC-1以及最高浓度的NH-3均导致TRβA表达上调。NH-3与T3共同处理显示NH-3对T3诱导的TRβA mRNA上调有强烈拮抗作用。本研究结果表明，TRβA mRNA表达分析可作为一种敏感的分子检测方法，用于研究环境化合物对非洲爪蟾蝌蚪甲状腺系统的影响。
