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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 A.具体目标 我们在项目这一阶段的目标是：1）完成了高氯酸盐干扰棘鱼性腺发育的上移-下移实验，确定了高氯酸盐干扰棘鱼性腺发育的临界窗口; 2）完成了高氯酸盐干扰棘鱼性腺发育的剂量-反应实验，确定了高氯酸盐干扰性腺发育的最低不良效应水平和最高无不良效应水平; 3）用棘鱼模型验证了内分泌测定，以及4）测试高氯酸盐是否诱导雌性中spiggin（雄性胶蛋白）的产生，这将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）：在2009年6月（鱼11个月大时）完成了在各种高氯酸盐浓度和各种暴露时间窗口中的棘鱼生长。 此时，将来自每个处理条件的鱼的子样品固定用于组织学，制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，可以从每个处理中获得更多样本。组织学切片的制备正在进行中，应于2010年1月完成。 甲状腺组织的初步结果显示明显异常，但性腺组织尚未检查。 目的2（剂量反应实验）：在2009年6月完成了在不同高氯酸盐浓度下生长棘鱼11个月的剂量反应实验。 在这一点上，从所有治疗的鱼的代表性样品固定的组织学，而其余的被保存用于内分泌分析。 组织学切片的制备正在进行中，应于2010年1月完成。 目标3（内分泌测定的验证）：我们已经开发和优化了卵黄原蛋白，11-酮睾酮，17 b-雌二醇和spiggin的测定，甲状腺素和皮质醇的验证过程正在进行中。 内分泌实验室已移至新的康菲石油综合科学大楼（CPISB），所有搬迁设备的功能均已经过验证和校准（如适用）。 目标4（测试暴露于高氯酸盐的雌性鱼的spiggin产量）：我们已经进入spiggin分析的“生产模式”，对所有剩余的剂量反应鱼已经完成，使我们成为美国目前唯一进行spiggin分析的实验室。 暴露于高氯酸盐的雌性动物没有spiggin诱导，因此似乎高氯酸盐导致的雄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RO 1实验所必需的背景数据来实现我们新资助的RO 1提案的目标。 RO 1实验将解决高氯酸盐对性分化的影响是否通过甲状腺介导的问题，或者它们是否是由一种不依赖甲状腺的机制产生的（RO 1目的1）。实验还将专门测试高氯酸盐，NIS，以及它的旁系同源物，在性腺表型引起高氯酸盐（RO 1目的2）的假定的唯一目标的作用。最后，实验将采取一个公正的全基因组的观点，在一个创新的新兴技术的使用，以确定新的基因和基因通路参与性别分化被破坏高氯酸盐暴露（RO 1目标3）。由于所有骨脊椎动物的内分泌，遗传和发育机制的强烈保护，我们的研究结果将促进我们对人类病理和疾病状态的理解，这些疾病状态可能在敏感基因型中因高氯酸盐而加剧，高氯酸盐是一种普遍存在于我们的水和食物中的有毒物质。由于高氯酸盐暴露可能会导致生殖疾病的流行，这会降低美国和其他工业国家的精子数量并增加睾丸发育不全的发病率，因此拟议实验的结果将与人类生殖健康密切相关。 D.计划 我们预计在2010年1月完成组织学切片制备，用于升档-降档研究和剂量-反应研究。 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，以满足我们的鱼类养殖需求。此外，我们计划通过优化和验证皮质醇和甲状腺素的测定，继续并完成实验室内分泌学，其他激素和蛋白质的测定已经完成。最后，我们将开始将spiggin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。在这一轮的INBRE支持结束时，我们相信，随着NIH资金的到位，我们将做好充分的准备，开始RO 1实验。