Mechanisms of Perchlorate-Induced Disruption of Sexual Differentiation

描述（由申请人提供）：最近生殖疾病频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-）是一种持久性的、氯化的水溶性污染物，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，它竞争性地抑制甲状腺中碘化钠同体（NIS）对碘的吸收，从而阻碍甲状腺激素的合成。然而，研究表明，高氯酸盐暴露会使雌性和雄性棘鱼（Gasterosteus aculeatus）雄性化，导致雌性和雄性雌雄同体并伴有睾丸肥大，这一结果无法通过简单、直接的甲状腺破坏机制来预测。本项目的目标是调和高氯酸盐作用的主要范例-完全通过破坏甲状腺中的NIS -与棘鱼行为，生理和形态的雄性化。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将通过挽救高氯酸盐暴露鱼的甲状腺激素水平来确定是否所有观察到的棘鱼对高氯酸盐暴露的表型反应都是由甲状腺介导的。Aim 2将定义NIS和NIS- paroggs在高氯酸盐破坏性腺发育中的功能作用，使用原位杂交定位mRNA (Aim 2a)，使用morpholino反义寡核苷酸降低NIS和NIS- paroggs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- paroggs获得功能实验（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来确定哪些基因是高氯酸盐暴露的早期应答者，哪些可能是下游基因，以及应答基因是否与甲状腺或性腺发育有关。定量PCR （qPCR）和原位杂交将验证表达谱结果。的意义。所提出的实验将确定高氯酸盐破坏性腺发育的分子和生理途径，无论是仅仅通过甲状腺中的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，我们提出的工作对人类健康有直接的影响，特别是在甲状腺疾病和性发育障碍方面。

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.