Mechanisms of Perchlorate-Induced Disruption of Sexual Differentiation

描述(由申请人提供)：最近生殖疾病发病率的急剧增加和地理差异可能受到环境变化的影响，包括高氯酸盐暴露。高氯酸盐(ClO4-)是一种持久性氯化水溶性污染物，在美国普遍存在。作为一种毒物，高氯酸盐通过摄入受污染的水、食物和母乳对人类健康构成重大威胁。高氯酸盐是一种已知的内分泌干扰物，它竞争性地抑制甲状腺钠碘转运体(NIS)的碘摄取，从而阻碍甲状腺激素的合成。然而，研究表明，接触高氯酸盐会使雌性和雄性刺鱼(尖吻刺鱼)雄性化，导致两性雌性和雄性睾丸肥大，这是简单、直接的甲状腺干扰机制无法预测的结果。这个项目的目标是调和高氯酸盐作用的主导范例-完全通过破坏甲状腺中的NIS-与刺鱼的行为、生理和形态的男性化。该项目的目标是确定高氯酸盐可能影响人类生殖健康的以前不为人知的途径。我们的工作假设是，高氯酸盐通过独立于甲状腺发挥作用来扰乱性腺发育。目的1将确定是否所有观察到的刺鱼对高氯酸盐暴露的表型反应都是由甲状腺通过挽救高氯酸盐暴露的鱼类的甲状腺激素水平来调节的。目的2将确定NIS和NIS-paralog在高氯酸盐通过原位杂交定位mRNA扰乱性腺发育中的功能作用(Aim 2a)，用吗啉反义寡核苷酸抑制NIS和NIS-paralog表达的功能丧失实验和使用锌指核酸酶诱导突变(Aim 2b)，以及通过过表达NIS和NIS-paralog获得功能实验(Aim 2c)。目的3将通过全基因组转录图谱确定高氯酸盐改变性别分化的机制，以确定哪些基因是高氯酸盐暴露的早期反应基因，哪些可能是下游基因，以及反应基因是否与甲状腺或性腺发育有关。定量聚合酶链式反应(QPCR)和原位杂交将验证表达谱结果。意义重大。拟议的实验将确定高氯酸盐扰乱性腺发育的分子和生理途径，无论是仅通过甲状腺中的NIS还是通过其他机制。由于高氯酸盐在美国是一种普遍存在的污染物，我们拟议的工作对人类健康有直接影响，特别是在甲状腺疾病和性发育障碍方面。 与公共健康相关：高氯酸盐是一种持久性、水溶性污染物，在美国普遍存在，通过摄入受污染的水、食物和牛奶对人类健康构成重大风险。高氯酸盐不仅抑制甲状腺活动，还改变刺鱼的性发育，刺鱼是遗传学研究中常用的模式生物。最近人类生殖障碍频率的急剧增加和地理差异很可能是由于环境的变化，包括高氯酸盐暴露。拟议中的实验将确定在高氯酸盐污染的侮辱下，扰乱男性和女性性腺正常发育的基因和基因功能。实验还将确定将遗传变化转化为性腺发育障碍的荷尔蒙机制。这项研究将促进我们对人类甲状腺疾病和最近流行的损害人类生殖健康的疾病的了解。

DESCRIPTION (provided by applicant): The recent dramatic increase and geographic differences in frequency of reproductive diseases are likely influenced by changes in the environment, including perchlorate exposure. Perchlorate (ClO4-) is a persistent, chlorinated water-soluble contaminant that is pervasive in the United States. As a toxicant, perchlorate poses a major risk to human health through ingestion of contaminated water, food, and breast milk. Perchlorate is a known endocrine disruptor that competitively inhibits iodide uptake at the Sodium-Iodide Symporter (NIS) in the thyroid, thus hindering thyroid hormone synthesis. Studies demonstrate, however, that perchlorate exposure masculinizes both female and male stickleback fish (Gasterosteus aculeatus), leading to hermaphroditic females and males with testicular hypertrophy, results that are not predicted by a simple, direct thyroid- disruption mechanism. The goal of this project is to reconcile the dominant paradigm of perchlorate action - exclusively by disruption of NIS in the thyroid - with masculinization of behavior, physiology, and morphology in stickleback. The project's goal is to identify previously unsuspected pathways by which perchlorate may impact human reproductive health. Our working hypothesis is that perchlorate disrupts gonadal development by acting independently of the thyroid. Aim 1 will determine whether all observed phenotypic responses to perchlorate exposure in stickleback are mediated by the thyroid by rescuing thyroid hormone levels in perchlorate-exposed fish. Aim 2 will define the functional roles of NIS and NIS-paralogs in disruption of gonadal development by perchlorate using in situ hybridization to localize mRNA (Aim 2a), loss-of-function experiments to knock down expression of NIS and NIS-paralogs with morpholino anti-sense oligonucleotides and induced mutations using zinc finger nucleases (Aim 2b), and gain-of-function experiments by over- expressing the NIS and NIS-paralogs (Aim 2c). Aim 3 will determine the mechanism by which perchlorate alters sex differentiation using whole genome transcription profiling to determine which genes are early responders to perchlorate exposure, which are likely to be downstream genes, and whether responding genes are related to thyroid or gonad development. Quantitative PCR (qPCR) and in situ hybridization will verify expression profiling results. Significance. The proposed experiments will identify molecular and physiological pathways by which perchlorate disrupts gonadal development, whether solely via NIS in the thyroid or by other mechanisms. Because perchlorate is a pervasive contaminant in the U.S., our proposed work has direct implications for human health, particularly regarding thyroid diseases and disorders of sexual development. PUBLIC HEALTH RELEVANCE: Perchlorate is a persistent, water-soluble contaminant that is pervasive in the United States and poses a major risk to human health through ingestion of contaminated water, food and milk. Perchlorate not only inhibits thyroid activity, but also alters sexual development in stickleback fish, a commonly used model organism in genetic studies. The recent dramatic increase and geographic differences in frequency of human reproductive disorders are likely due to changes in the environment, including perchlorate exposure. Proposed experiments will identify genes and gene functions that, under the insult of perchlorate contamination, disrupt normal development of male and female gonads. Experiments will also determine the hormonal mechanisms that translate genetic changes into developmental disorders of the gonads. This research will advance our understanding of human thyroid diseases and the recent epidemic of impaired human reproductive health.