A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling.
A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling.
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DOI：
10.1016/j.aquatox.2017.06.019
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发表时间：
2017-09
期刊：
影响因子：
--
通讯作者：
Furlow JD
中科院分区：
文献类型：
--
作者：
Mengeling BJ;Wei Y;Dobrawa LN;Streekstra M;Louisse J;Singh V;Singh L;Lein PJ;Wulff H;Murk AJ;Furlow JD
甲状腺激素（TH）信号在哺乳动物发育中的重要作用保证了人造化学品对其干扰的检查。在脊椎动物物种中，TH信号传导的分子组分是高度保守的，包括甲状腺激素受体（TR）、它们的异二聚体结合配偶体类维生素A-X受体（RXR）以及它们的DNA识别序列（TREs）。这种分子保守性允许检查潜在的TH破坏在易处理的，在体内模型系统的两栖动物变态。变态需要TH信号的煽动和进展，它提供了戏剧性的和充分表征的表型，涉及不同的细胞命运。在这里，我们描述了一个定量的，早熟变态试验套件，我们开发了一个星期后受精（PF）非洲爪蟾蝌蚪，以评估TH信号中断。在这个发育阶段的蝌蚪（Nieuwkoop-Faber（NF）-48）有能力对TH激素做出反应，尽管还没有产生TH，但它们沿着沿着许多变质途径，并且它们的大小是一致的。这使我们能够量化处理5天后与自然变态相关的形态学变化（例如鳃和尾吸收、脑扩张和颅面重塑）。使用相同的蝌蚪从形态学测量，我们量化了20倍的增加TH诱导的细胞增殖的喙头区域的整装免疫细胞化学。在分子水平上，我们使用来自转基因X的F3代蝌蚪。laevis细胞系，其在天然TRE的控制下表达荧光素酶，以评估化合物破坏TR功能的能力。荧光素酶报告显示超过10倍的激活生理浓度的TH。我们使用合成的TR拮抗剂NH-3来证明我们的测定套件在受体水平上测量TH活性抑制的可行性。最后，我们评估了可疑的TH干扰化学品四溴二苯醚47（BDE-47）和四溴双酚A（TBBPA）的能力。我们发现，BDE-47显示一般毒性，而不是TH破坏，因为它没有增加大脑宽度，也不影响TRE-fluorescence报告。然而，TBBPA，一个可疑的TR拮抗剂，虽然不能有效地拮抗细胞增殖，显着抑制的TRE-luciferase报告，这表明它作为一个TH破坏者值得密切关注。总的来说，该试验套件具有重要的优势，在经典的蝌蚪变态试验的动物大小的均匀性，小的测试体积，重现性，和短的测试周期。在内源性TH产生和自由饲喂开始之前进行测定，这进一步降低了复杂性和可变性。
The essential role of thyroid hormone (TH) signaling in mammalian development warrants the examination of man-made chemicals for its disruption. Among vertebrate species, the molecular components of TH signaling are highly conserved, including the thyroid hormone receptors (TRs), their heterodimer binding partners the retinoid-X receptors (RXRs), and their DNA recognition sequences (TREs). This molecular conservation allows examination of potential TH disruption in the tractable, in vivo model system of amphibian metamorphosis. Metamorphosis requires TH signaling for both instigation and progression, and it provides dramatic and well-characterized phenotypes involving different cell fates. Here we describe a quantitative, precocious-metamorphosis assay suite we developed using one-week post-fertilization (PF) Xenopus laevis tadpoles in order to assess disruption of TH signaling. Tadpoles at this developmental stage (Nieuwkoop-Faber (NF)-48) are competent to respond to TH hormone, although not yet producing TH, along many metamorphic pathways, and they are uniform in size. This allowed us to quantify changes in morphology associated with natural metamorphosis (e.g. gill and tail resorption, brain expansion, and craniofacial remodeling) after five days of treatment. Using the same tadpoles from morphological measurements, we quantified a 20-fold increase in TH-induced cellular proliferation in the rostral head region by whole-mount immunocytochemistry. At the molecular level, we used F3-generation tadpoles from a transgenic X. laevis line, which expresses luciferase under the control of a native TRE, to assess the ability of compounds to disrupt TR function. The luciferase reporter showed over 10-fold activation by physiologic concentrations of TH. We used the synthetic TR antagonist NH-3 to demonstrate the feasibility of our assay suite to measure inhibition of TH activity at the level of the receptor. Finally, we assessed the capabilities of suspected TH-disrupting chemicals tetrabrominated diphenyl ether 47 (BDE-47) and tetrabromobisphenol A (TBBPA). We found that BDE-47 displays general toxicity rather than TH disruption, as it did not increase brain width nor affect the TRE-luciferase reporter. However, TBBPA, a suspected TR antagonist, although not effective in antagonizing cell proliferation, significantly inhibited the TRE-luciferase reporter, suggesting that it bears closer scrutiny as a TH disruptor. Overall the assay suite has important advantages over the classical tadpole metamorphosis assays with respect to the uniformity of animal size, small test volume, reproducibility, and short test period. The assays are performed before endogenous TH production and free feeding start, which further reduces complexity and variability.
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