Adult and Transgenerational Toxicity Due to Developmental TCDD Exposure

描述(由申请人提供)：改善人类健康的一个重要目标是了解在怀孕和儿童时期暴露于环境毒物如何导致成年后的疾病。确定早期接触单一毒物是否会影响后代人的健康也是至关重要的。了解和识别这些风险将对人类健康产生积极影响。我的研究重点是用斑马鱼来理解在发育过程中暴露到2，3，7，8四氯二苯并对二恶英(TCDD，二恶英)的成年人的两种毒性反应，以百万分之几(Ppt)的水平。我们已发表的工作和我的初步结果表明，发育过程中的一次接触会导致成人的骨骼畸形和生殖毒性，并在随后的F1和F2代中造成各种形式的毒性。F0成鱼毒性包括明显的雄性雌性化，其中具有雌性第二性特征的鱼有睾丸。一个目标是确定TCDD是否改变了性腺的命运，将卵巢改变为睾丸，或者使男性的身体女性化。另一个主要的毒性迹象是 在发育早期暴露的成人是类似于人类脊柱侧弯的轴骨畸形。我将通过染色和测量基因表达的变化来确定骨骼发育中这些变化的来源，检查骨骼和胶原蛋白。Sox9b基因下调是TCDD诱导斑马鱼颌骨畸形的原因。人类Sox9基因是骨骼形成和性别指定的主要调节者：Sox9被认为是男性Y染色体sry基因的靶标。我还观察到sox9b在斑马鱼脊椎发育部位的表达。因此，我将研究人Sox9的两个斑马鱼同源物：Sox9a和sox9b在TCDD处理的斑马鱼发育中的骨和性腺中的表达。我在暴露的F0鱼的F1和F2后代中发现了可复制的毒性 只是在开发过程中短暂的。我将更好地描述这种跨代毒性，测量每一代受影响组织中全球基因表达的变化，并将使用基因特异性和全局技术来检测TCDD暴露引起的DNA甲基化和染色质结构的变化，并将其传播到F1和F2鱼类。在这个项目的第三年，我计划将这项工作的结果作为初步数据来申请R21或R03拨款。跨代研究，即使是在斑马鱼身上，也需要几年的时间才能成熟，所以拟议的工作将允许未来的拨款专注于推动我的结果的机制。作为K01获奖者，我将遵循职业发展计划，为每个奖项年度制定非常具体的研究和开发里程碑。我将定期与我的导师和咨询委员会会面，以加强我将翻译研究和机械研究联系起来的能力。我将在翻译研究领域获得成功所需的经验培训，包括项目规划、手稿和拨款撰写，以及识别和掌握推动我的研究向前发展所需的细胞和分子技术。我有临床背景，重点是研究。我将在机械论、假设检验驱动的研究方面获得经验。这项计划将扩展我的研究专业知识，并加强我在发育毒理学研究方面的培训。威斯康星大学有许多由NIH赞助的培训项目、核心设施和资助的研究人员，这使它成为我作为一名独立科学家发展的理想培训环境。

DESCRIPTION (provided by applicant): An important goal in improving human health is to understand how exposure to environmental toxicants during gestation and childhood leads to disease later in adulthood. It is also vital to determine whether a single early toxicant exposure can affect the health of subsequent generations. Understanding and identifying these risks will positively impact human health. My research focus is to use zebrafish to understand two toxic responses seen in adults exposed during development to 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD, dioxin), at the parts per trillion (ppt) level. Our published work and my preliminary results indicate that a single exposure during development can cause skeletal malformation and reproductive toxicity in the adults, and various forms of toxicity in the subsequent F1 and F2 generations. F0 adult toxicity includes an apparent male feminization in which fish with female secondary sex characteristics have testes. A goal is to determine whether TCDD alters gonadal fate, changing ovaries to testes, or feminizes the body of males. Another major sign of toxicity in adults exposed during early development is malformations in the axial skeleton resembling human scoliosis. I will identify the source of these changes in skeletal development examining bone and collagen by staining and measuring alterations in gene expression. Down-regulation of the sox9b gene is the cause of TCDD-induced jaw malformation in zebrafish. The human sox9 gene is a master regulator of both skeletal formation and sex specification: sox9 is thought to be the target of the male Y-chromosome sry gene. I have also observed sox9b expression at the site of vertebral development in zebrafish. Therefore, I will examine the expression of both zebrafish homologs of human sox9: sox9a and sox9b in developing bone and gonad of TCDD-treated zebrafish. I find reproducible toxicity in F1 and F2 offspring of F0 fish that were exposed only briefly during development. I will better characterize this transgenerational toxicity, measuring changes in global gene expression in affected tissues from each generation and will use both gene- specific and global techniques to examine alterations in DNA methylation and chromatin structure induced by TCDD exposure and transmitted into F1 and F2 fish. In year 3 of this project, I plan to use the results from this work as preliminary data to apply for an R21 or R03 grant. Transgenerational work, even in zebrafish, takes several years to mature, so the proposed work would allow a future grant to focus on mechanisms driving my results. As a K01 recipient, I will follow a career development plan with very specific research and development milestones for each award year. I will meet regularly with my mentors and advisory committee to strengthen my ability to link translational and mechanistic research. I will obtain experiential training in areas needed for a successful career in translational research, including project planning, manuscript and grant writing, and identification and mastery of cellular and molecular techniques needed to move my research forward. I have a clinical background with a research emphasis. I will gain experience in mechanistic, hypothesis testing driven research. This plan will stretch my research expertise and enhance my training in developmental toxicology research. The University of Wisconsin has numerous NIH-sponsored training programs, core facilities and funded researchers making it an ideal training environment for me to develop as an independent scientist.