Mechanisms of Perchlorate-Induced Disruption of Sexual Differentiation

描述（由申请人提供）：最近生殖疾病发生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（ClO 4-）是一种持久性的氯化水溶性污染物，在美国普遍存在。作为一种有毒物质，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风险。高氯酸盐是一种已知的内分泌干扰物，竞争性地抑制甲状腺中钠碘同向转运体（NIS）的碘摄取，从而阻碍甲状腺激素的合成。然而，研究表明，高氯酸盐暴露使雌性和雄性棘鱼（Gasterosteus aculeatus）雄性化，导致雌雄同体的雌性和雄性睾丸肥大，这不是简单的直接甲状腺破坏机制所预测的结果。本项目的目标是调和高氯酸盐行动的主导范式-完全由甲状腺中的NIS破坏-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先前未被怀疑的途径。我们的工作假设是高氯酸盐通过独立于甲状腺的作用破坏性腺发育。目的1将确定是否所有观察到的表型反应高氯酸盐暴露在刺鱼介导的甲状腺通过挽救甲状腺激素水平高氯酸盐暴露的鱼。目的2将使用原位杂交定位mRNA（Aim 2a）、使用吗啉代反义寡核苷酸敲低NIS和NIS旁系同源物表达的功能丧失实验和使用锌指核酸酶诱导突变（Aim 2b）来确定NIS和NIS旁系同源物在高氯酸盐破坏性腺发育中的功能作用，和通过过表达NIS和NIS旁系同源物进行的功能获得实验（Aim 2c）。目的3将确定高氯酸盐改变性别分化的机制，使用全基因组转录谱，以确定哪些基因是早期反应高氯酸盐暴露，哪些可能是下游基因，以及是否响应基因与甲状腺或性腺发育。定量PCR（qPCR）和原位杂交将验证表达谱结果。意义拟议的实验将确定高氯酸盐破坏性腺发育的分子和生理途径，无论是通过甲状腺中的NIS还是通过其他机制。因为高氯酸盐在美国是一种普遍的污染物，我们提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。 公共卫生相关性：高氯酸盐是一种持久性的水溶性污染物，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，而且改变棘鱼的性发育，棘鱼是遗传研究中常用的模式生物。最近人类生殖疾病发病率的急剧增加和地理差异可能是由于环境的变化，包括高氯酸盐暴露。拟议的实验将确定基因和基因功能，在高氯酸盐污染的侮辱，破坏男性和女性性腺的正常发育。实验还将确定将遗传变化转化为性腺发育障碍的激素机制。这项研究将促进我们对人类甲状腺疾病和最近流行的人类生殖健康受损的理解。

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.