Mechanisms of dioxin insensitivity in developing frogs

描述（由申请人提供）：非洲爪蛙（Xenopus laevis）的胚胎代表了脊椎动物发育的长期模式。它们也用于FETAX（青蛙胚胎畸形试验-爪蟾）和类似的化学物质和环境样品的发育毒性试验。2,3,7,8四氯二苯并-对二恶英（TCDD）是大多数脊椎动物的一种强大的发育毒性物质。然而，青蛙通常对TCDD毒性不敏感，特别是在生命早期阶段。因此，FETAX和其他青蛙胚胎毒性试验可能不太适合确定含有二恶英样化合物的样品的发育毒性。本研究旨在描述蛙类TCDD不敏感的分子机制，以X. laevis为模型系统。在之前的资助期间，我们克隆了编码两种不同的野田鼠芳烃受体AHR1A和AHR1B的cdna，这两种受体在体外对TCDD的亲和力都非常低，并且在发育过程中可检测的信号活性异常晚起。该项目将阐明这两种ahr的功能及其在胚胎和蝌蚪阶段的活性调控。具体来说，我们将(1)使用瞬时转染的细胞在报告基因检测中剖析AHR1A和AHR1B的转录特性；(2)确定发育过程中AHR蛋白表达和功能性AHR信号的起始时间；(3)确定AHR阻遏蛋白在早期胚胎AHR信号衰减中的作用；(4)比较tcdd诱导蝌蚪和早期胚胎的致死率和消除率。了解不同生命阶段TCDD不敏感的分子机制对于确定青蛙胚胎毒性试验与人类健康的相关性非常重要。此外，青蛙AHR功能和表达的独特特征可能为研究AHR表达、AHR活性、TCDD发育毒性机制以及脊椎动物发育过程中AHR内源性功能之间的关系提供了新的视角。

DESCRIPTION (provided by applicant): Embryos of the African clawed frog, Xenopus laevis, represent a long-standing model of vertebrate development. They are also used in FETAX (Frog Embryos Teratogenesis Assay-Xenopus) and similar assays of the developmental toxicity of chemicals and environmental samples. 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is a potent developmental toxicant in most vertebrates. However, frogs are generally insensitive to TCDD toxicity, especially at early life stages. Thus, FETAX and other frog embryo toxicity tests may be poorly suited for determining the developmental toxicity of samples containing dioxin-like compounds. This research seeks to delineate the molecular mechanisms underlying TCDD insensitivity in frogs, using X. laevis as a model system. During the previous grant period, we cloned cDNAs encoding two distinct X. laevis aryl hydrocarbon receptors, AHR1A and AHR1B both of which exhibit extraordinarily low affinity for TCDD in vitro and unusually late onset of detectable signaling activity during development. The proposed project will elucidate the functions of the two AHRs and the regulation of their activity in embryo and tadpole stages. Specifically, we will (1) Dissect the transcriptional properties of AHR1A and AHR1B in reporter gene assays using transiently transfected cells; (2) Determine the timing of onset of AHR protein expression and functional AHR signaling during development; (3) Determine the role of AHR Repressor protein in the attenuation of AHR signaling in early embryos, and (4) Compare TCDD-induced lethality and elimination rates in tadpoles and early embryos. Understanding the molecular mechanisms of TCDD insensitivity in different life stages is important for determining the human health relevance of frog embryo toxicity assays. Moreover, the unique features of frog AHR function and expression may provide a novel perspective on the relationship between AHR expression, AHR activity, mechanisms developmental TCDD toxicity, and the endogenous functions of AHRs during vertebrate development.