INBRE-2 ENDOCRINE-DISRUPTING CHEMICALS FROM FORMERLY USED DEFENCE SITES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 A.具体目标 本项目这一阶段的目标是：1)完成升降档实验，以确定高氯酸盐干扰刺背鱼性发育的临界窗口；2)完成剂量-反应实验，以确定高氯酸盐扰乱性腺发育的最低不良反应水平和最高无不良影响水平；3)用刺背模型验证内分泌分析；4)测试高氯酸盐是否诱导雌性鱼体产生spiggin(雄性胶蛋白)，这将表明高氯酸盐干扰发育的机制。 B.研究和成果 目标1(升档-降档实验)：在不同的高氯酸盐浓度下，在不同的暴露时间窗口内生长刺鱼于2009年6月完成(当时鱼11个月大)。在这一点上，来自每种处理条件的鱼的一个子样本被固定用于组织学，匹配的组织准备用于基因分型性别的遗传分析。剩余的鱼在处理条件下保持存活，以便在需要增加样本量或需要检查其他组织时，从每种处理中获得更多的样本。组织学载玻片的准备工作正在进行中，应于2010年1月完成。甲状腺组织的初步结果显示明显异常，但性腺组织尚未进行检查。 目的2(剂量-反应实验)：在2009年6月完成的剂量-反应实验中，刺鱼在不同浓度的高氯酸盐中生长11个月。在这一点上，来自所有处理的有代表性的鱼样本被固定用于组织学，而其余的被保存用于内分泌分析。组织学载玻片的准备工作正在进行中，应于2010年1月完成。 目标3(内分泌分析的验证)：我们已经开发和优化了卵黄蛋白原、11-酮-睾酮、17b-雌二醇和spiggin的分析，甲状腺激素和皮质醇的验证过程正在进行中。内分泌学实验室已迁至新的康菲石油综合科学大楼(康菲石油综合科学大楼)，所有已搬迁设备的功能已得到验证和校准，如果适用的话。 目标4(高氯酸盐暴露的女性的排泄物生产测试)：我们已经进入了排泄物分析的“生产模式”，对所有剩余的剂量反应鱼进行了排泄物分析，使我们的实验室成为美国目前唯一进行排泄物分析的实验室。暴露在高氯酸盐中的雌性没有的诱导，所以似乎她们由于高氯酸盐而形成的男性化不是由雄激素直接介导的。 C.意义 实现上述目标使我们能够通过提供RO1实验所必需的背景数据来实现我们新资助的RO1计划的目标。RO1实验将解决这个问题，即高氯酸盐对性分化的影响是通过甲状腺介导的，还是由一种不依赖甲状腺的机制产生的(RO1目标1)。这些实验还将具体测试假定的高氯酸盐唯一靶标NIS及其类似物在高氯酸盐引起的性腺表型中的作用(RO1目标2)。最后，实验将采取公正的全基因组观点，创新地使用新兴技术来识别与性别分化有关的新基因和基因途径，这些基因和基因途径会被高氯酸盐破坏(RO1目标3)。由于所有骨质脊椎动物的内分泌、遗传和发育机制都非常保守，我们的结果将促进我们对人类病理和疾病状态的理解，这些疾病和疾病可能会因高氯酸盐敏感基因而加剧，高氯酸盐是一种在我们的水和食物中普遍存在的有毒物质。由于接触高氯酸盐可能会导致生殖疾病的日益流行，这种疾病正在降低美国和其他工业国家居民的精子数量，增加睾丸发育不全的比率，因此拟议中的实验结果将与人类生殖健康密切相关。 D.计划 我们预计在2010年1月完成组织学载玻片的准备工作，包括升降档研究和剂量反应研究。升降档结果将提供暴露时间所需的数据，而剂量反应研究将提供扰乱性腺发育所需的最小剂量。我们计划在未来两个月内开始建立新的CPISB Varium空间，以满足我们的养鱼需求。此外，我们计划通过优化和验证皮质醇和甲状腺素的分析来继续并最终完成实验室内分泌学，就像已经完成的其他激素和蛋白质的分析一样。最后，我们将开始将唾液分析的检测结果与高氯酸盐暴露的浓度和持续时间联系起来。到本轮INBRE支持结束时，我们有信心当NIH的资金到位时，我们将为开始RO1实验做好充分准备。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims Our Aims for this phase of the project were to: 1) complete the upshift-downshift experiment to define the critical window when perchlorate disrupts sexual development of stickleback fish, 2) complete the dose-response experiment to determine the lowest adverse effect level and the highest no adverse effect level for disruption of gonadal development by perchlorate, 3) validate endocrine assays with the stickleback model, and 4) test whether perchlorate induces spiggin (the male glue protein) production in females, which would indicate an androgen-mediated mechanism of developmental disruption. B. Studies and Results Aim 1 (upshift-downshift experiment): Growing stickleback in the various perchlorate concentrations over the various time windows of exposure was completed in June, 2009 (when the fish were 11 months old). At that point, a subsample of fish from each treatment condition was fixed for histology, with matched tissues prepared for genetic analysis of genotypic sex. The remaining fish have been maintained alive in their treatment condition so that further samples are available from each treatment should sample sizes need to be increased or other tissues need to be examined. The preparation of histological slides is on-going, and should be complete by January, 2010. Preliminary results of thyroid tissue showed marked abnormalities, but gonadal tissue has not yet been examined. Aim 2 (dose-response experiment): Growing stickleback in the various perchlorate concentrations for 11 months in the dose-response experiment was completed in June, 2009. At that point, representative samples of fish from all treatments were fixed for histology, while the remainder were preserved for endocrine analyses. The preparation of histological slides is on-going, and should be complete by January, 2010. Aim 3 (validation of endocrine assays): We have developed and optimized assays of vitellogenin, 11-ketotestosterone, 17b-estradiol, and spiggin, and the validation process is underway for thyroxine and cortisol. The endocrinology lab was moved into the new Conoco-Phillips Integrated Science Building (CPISB), and the functioning of all moved equipment has been validated and calibrated, when applicable. Aim 4 (test for spiggin production in females exposed to perchlorate): We have moved into "production mode" for the spiggin analysis, which has been completed for all of the surplus dose-response fish, making ours the only laboratory in the United States currently performing spiggin analysis. Females exposed to perchlorate do not have spiggin induction, so it appears that their masculinization due to perchlorate is not mediated directly by androgens. C. Significance Achieving the aims outlined above allows us to pursue the Aims of our newly funded RO1 proposal by providing the background data essential for our RO1 experiments. The RO1 experiments will address the question of whether the effects of perchlorate on sexual differentiation are mediated through the thyroid, or whether they are generated by a mechanism that is thyroid-independent (RO1 Aim 1). The experiments will also test specifically the roles of the presumed sole target for perchlorate, NIS, as well as its paralogs, in the gonadal phenotype provoked by perchlorate (RO1 Aim 2). Finally, experiments will take an unbiased genome-wide viewpoint in an innovative use of emerging technology to identify new genes and gene pathways involved in sex differentiation that are disrupted by perchlorate exposure (RO1 Aim 3). Because of the strong conservation of endocrine, genetic, and developmental mechanisms among all bony vertebrates, our results will advance our understanding of human pathologies and disease states that may be exacerbated in sensitive genotypes by perchlorate, a toxicant ubiquitously found in our water and food. Because perchlorate exposure may contribute to the growing epidemic of reproductive disease which is driving down sperm counts and increasing rates of testicular dysgenesis in people living in the United States and other industrial countries, the results of the proposed experiments will be strongly relevant to human reproductive health. D. Plans We expect to complete histological slide preparation in January, 2010 for both the upshift-downshift study and the dose-response study. The upshift-downshift results will provide the data needed for the timing of exposure, while the dose-response study will provide the minimal doses needed for disruption of gonadal development. We plan to begin setting up the new CPISB Vivarium space over the next 2 months to accommodate our fish husbandry needs. In addition, we plan to continue and finalize the laboratory endocrinology by optimizing and validating assays for cortisol and thyroxine as has been completed for the other hormones and proteins. And finally, we will begin to link assay results from the spiggin analyses to concentrations and durations of perchlorate exposure. By the end of this round of INBRE support, we feel confident that we will be fully prepared to begin the RO1 experiments as the NIH funding becomes available.