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

来自以前使用的防御场所的 INBRE-2 内分泌干扰化学品

• 批准号: 8167411
• 负责人: Frank Arthur von Hippel
• 金额: $ 14.19万
• 依托单位: UNIVERSITY OF ALASKA FAIRBANKS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167411
• 关键词: 11-ketotestosterone; Address; Adverse effects; Androgens; Automobile Driving; Biological Assay; Computer Retrieval of Information on Scientific Projects Database; Country; Data; Development; Disease; Dose; Emerging Technologies; Endocrine; Endocrine Disruptors; Endocrinology; Epidemic; Equipment; Estradiol; Female; Fishes; Food; Funding; Gasterosteidae; Genes; Genotype; Glues; Grant; Histology; Hormones; Human; Human Pathology; Hydrocortisone; Institution; Laboratories; Life; Link; Mediating; Modeling; Pathway interactions; Perchlorates; Phase; Phenotype; Preparation; Process; Production; Proteins; Published Comment; Reproductive Health; Research; Research Personnel; Resources; Role; Sample Size; Sampling; Science; Sex Differentiation; Sexual Development; Site; Slide; Source; Sperm Count Procedure; Testing; Thyroid Gland; Thyroxine; Time; Tissues; United States; United States National Institutes of Health; Validation; Vertebrates; Vitellogenins; Water; developmental genetics; genetic analysis; genome-wide; genotypic sex; innovation; male; paralogous gene; reproductive; research study; response; toxicant

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 A. 具体目标 我们该项目这一阶段的目标是：1) 完成升档-降档实验，以确定高氯酸盐扰乱刺鱼性发育的关键窗口，2) 完成剂量反应实验，以确定最低的不利影响水平和高氯酸盐破坏性腺发育的最高无副作用水平，3) 使用刺鱼模型验证内分泌测定，以及 4) 测试高氯酸盐是否诱导 spiggin（雄性胶蛋白）雌性的生产，这表明雄激素介导的发育破坏机制。 B. 研究和结果 目标 1（升档-降档实验）：2009 年 6 月完成了在不同的高氯酸盐浓度和不同的暴露时间范围内生长刺鱼（当时鱼龄为 11 个月）。 此时，固定来自每种处理条件的鱼的子样本用于组织学分析，并准备匹配的组织用于基因型性别的遗传分析。 剩余的鱼在其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，可以从每次处理中获得更多样本。组织学载玻片的制备正在进行中，预计将于 2010 年 1 月完成。甲状腺组织的初步结果显示明显异常，但性腺组织尚未检查。 目标 2（剂量反应实验）：剂量反应实验中，在不同高氯酸盐浓度下生长刺鱼 11 个月，于 2009 年 6 月完成。此时，固定所有处理的鱼的代表性样品进行组织学分析，同时其余部分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，预计将于 2010 年 1 月完成。 目标 3（内分泌检测的验证）：我们开发并优化了卵黄蛋白原、11-酮睾酮、17b-雌二醇和 spiggin 的检测，甲状腺素和皮质醇的验证过程正在进行中。 内分泌实验室迁入新的康菲石油综合科学大楼 (CPISB)，所有移动设备的功能均已在适用的情况下进行了验证和校准。 目标 4（暴露于高氯酸盐的雌性中的 spiggin 产生测试）：我们已进入 spiggin 分析的“生产模式”，该分析已对所有剩余的剂量反应鱼完成，使我们成为美国唯一的实验室目前正在进行 spiggin 分析。 暴露于高氯酸盐的雌性没有 spiggin 诱导，因此高氯酸盐引起的雄性化似乎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 实验必需的背景数据来实现新资助的 RO1 提案的目标。 RO1 实验将解决高氯酸盐对性别分化的影响是否是通过甲状腺介导的问题，或者它们是否是由不依赖甲状腺的机制产生的（RO1 目标 1）。这些实验还将专门测试高氯酸盐的假定唯一靶标 NIS 及其旁系同源物在高氯酸盐引发的性腺表型中的作用（RO1 目标 2）。最后，实验将采用公正的全基因组视角，创新性地使用新兴技术来识别与性别分化相关的新基因和基因途径，这些基因和基因途径会因高氯酸盐暴露而受到干扰（RO1 目标 3）。由于所有骨脊椎动物的内分泌、遗传和发育机制都具有很强的保守性，我们的研究结果将增进我们对人类病理和疾病状态的理解，这些病理和疾病状态可能会因高氯酸盐（一种普遍存在于我们的水和食物中的有毒物质）而在敏感基因型中加剧。由于接触高氯酸盐可能会导致生殖疾病的日益流行，从而导致生活在美国和其他工业国家的人们的精子数量减少并增加睾丸发育不全的发生率，因此拟议实验的结果将与人类生殖健康密切相关。 D、计划 我们预计将于 2010 年 1 月完成升档-降档研究和剂量反应研究的组织学载玻片制备。 升档-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破坏性腺发育所需的最小剂量。我们计划在未来 2 个月内开始建立新的 CPISB 饲养场空间，以满足我们的鱼类饲养需求。此外，我们计划通过优化和验证皮质醇和甲状腺素的检测来继续并最终确定实验室内分泌学，就像其他激素和蛋白质的检测一样。最后，我们将开始将 spigin 分析的测定结果与高氯酸盐暴露的浓度和持续时间联系起来。在这一轮 INBRE 支持结束时，我们相信，随着 NIH 资金的到位，我们将做好充分准备，开始 RO1 实验。