Molecular Mechanisms of Atrazine in Altering the Endocrine Gene Network

阿特拉津改变内分泌基因网络的分子机制

• 批准号: 7902731
• 负责人: HOLLY A. INGRAHAM
• 金额: $ 4.25万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2010-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7902731
• 关键词: Address; Adrenal Glands; Affect; Amphibia; Aromatase; Atrazine; Binding; Biochemical; Candidate Disease Gene; Categories; Cell Line; Cells; Classification; Country; Cultured Cells; Cyclic AMP; DNA Binding; Data; Endocrine; Endocrine Disruptors; Endocrine disruption; Endocrine system; Estrogens; Event; FSTL3 gene; Female; Fishes; G Protein-Coupled Receptor Genes; Gene Targeting; Genes; Genome; Health; Hepatocyte; Herbicides; Human; Human Chorionic Gonadotropin; Lead; Libraries; Life; Link; Mammalian Cell; Mediating; Molecular; Molecular Target; Nuclear Receptors; Orthologous Gene; Phosphorylation; Phosphotransferases; Physiology; Proteins; Reporter; Reproduction; Reproductive Health; SF1; Signal Transduction; Small Interfering RNA; Specificity; Steroids; Surveys; Testing; United States; Work; Zebrafish; cellular targeting; cofactor; environmental chemical; gain of function; high risk; insight; knock-down; loss of function; male; member; phosphoric diester hydrolase; public health relevance; receptor; reproductive; reproductive development; response

DESCRIPTION (provided by applicant): Molecular Mechanisms of Atrazine in Altering the Endocrine Gene Network Atrazine (ATR) is widely used as a broadleaf herbicide in the United States and world. While toxic levels of ATR are linked to reproductive abnormalities in wild life, effects in humans remain controversial. Moreover, the molecular and cellular targets of ATR are still unknown. We and others have found that ATR stimulates the nuclear receptor steroidogenic factor 1 (SF-1, NR5A1). In the last decade our lab has worked extensively to define the molecular mechanisms regulating members of subfamily V (NR5A) receptors that includes SF-1, LRH-1, Ftz-f1, ff1a-d. As such, we have begun to ask how ATR functions in mammalian endocrine cell lines and in zebrafish to affect NR5A receptor activity. Our preliminary data show that environmentally relevant ATR concentrations increased the zebrafish (z)Cyp19A1 expression encoding aromatase, and increased the ratio of female to male fish. ZCyp19A1 is a known target of the nuclear receptor SF-1 (NR5A1). By contrast, the second zebrafish gene encoding aromatase zCyp19A2 was unchanged after ATR treatment. Remarkably, in mammalian cells ATR functions in a cell-specific manner to upregulate SF-1 targets and other genes critical for steroid synthesis and reproduction, including Cyp19A1, StAR, Cyp11A1, hCG, FSTL3, LHss, INH1, 1GSU, and 11ss-HSD2. Further cellular studies suggest that ATR increases the activity of mammalian and zebrafish NR5A receptor orthologs by receptor phosphorylation, and amplification of cAMP and investigator3K signaling. Importantly, our data show that ATR does not directly affect SF-1 DNA-binding, nor does ATR appear to directly bind SF-1. Instead, we hypothesize that this environmental herbicide potentially influences endocrine gene networks by indirectly activating NR5A receptors as well as other cAMP-mediated transcriptional events. The precise molecular targets of ATR that result in cell-specific stimulation of NR5A receptors remain to be defined. In this narrowly focused R21 application, two aims are planned to identify the molecular targets of ATR. In our first aim, a systematic genome siRNA screen will be carried out on ATR-responsive reporter cell lines. In our second aim, we will test different categories of candidate targets of ATR. Identifying the underlying mechanism(s) of ATR stimulation of NR5A receptors will begin to address the potentially adverse endocrine disrupting effects of this pervasive and persistent environmental chemical. PUBLIC HEALTH RELEVANCE: Endocrine disrupting chemicals (EDCs) such as the herbicide atrazine (ATR) are widely used and are proposed to adversely affect the endocrine system. Although the consequences of EDCs on the reproductive health and viability of some wild life are established, the precise effects on human health remain controversial. In the United States, use of ATR is heavy in many agriculturally settings, especially in the Midwest where corn is grown, however this herbicide is banned in most EU countries. In this application we seek to define the precise molecular targets of ATR using mammalian culture cells. Our proposed experimental plan to systematically define the molecular targets of ATR is an important step in addressing the potential impact of ATR on wild life and human health and the controversy surrounding its continued use in the US. These studies will also shed new insight into the activation of NR5A receptors as important regulators in reproductive development and physiology.

简介（申请人提供）：阿特拉津改变内分泌基因网络的分子机制阿特拉津（Atrazine， ATR）是美国及世界上广泛使用的阔叶除草剂。虽然ATR的毒性水平与野生动物的生殖异常有关，但对人类的影响仍存在争议。此外，ATR的分子和细胞靶点仍然未知。我们和其他人发现ATR刺激核受体甾体生成因子1 （SF-1, NR5A1）。在过去的十年中，我们的实验室广泛地研究了调节V亚家族（NR5A）受体成员的分子机制，包括SF-1， LRH-1, Ftz-f1, ff1a-d。因此，我们开始研究ATR在哺乳动物内分泌细胞系和斑马鱼中如何影响NR5A受体活性。我们的初步数据表明，环境相关的ATR浓度增加了斑马鱼(z)Cyp19A1编码芳香酶的表达，并增加了雌鱼与雄鱼的比例。ZCyp19A1是已知的核受体SF-1 （NR5A1）的靶标。相比之下，编码芳香化酶zCyp19A2的第二个斑马鱼基因在ATR处理后没有变化。值得注意的是，在哺乳动物细胞中，ATR以细胞特异性的方式起作用，上调SF-1靶点和其他对类固醇合成和繁殖至关重要的基因，包括Cyp19A1、StAR、Cyp11A1、hCG、FSTL3、LHss、INH1、1GSU和11ss-HSD2。进一步的细胞研究表明，ATR通过受体磷酸化、cAMP和investigator3K信号的扩增，增加哺乳动物和斑马鱼NR5A受体同源物的活性。重要的是，我们的数据显示ATR不直接影响SF-1的dna结合，也不显示ATR直接结合SF-1。相反，我们假设这种环境除草剂可能通过间接激活NR5A受体以及其他camp介导的转录事件来影响内分泌基因网络。ATR导致NR5A受体细胞特异性刺激的精确分子靶点仍有待确定。在这个狭窄的R21应用中，计划有两个目标来确定ATR的分子靶点。在我们的第一个目标中，系统的基因组siRNA筛选将在atr应答报告细胞系上进行。在我们的第二个目标中，我们将测试不同类别的ATR候选目标。确定ATR刺激NR5A受体的潜在机制将开始解决这种普遍和持久的环境化学物质的潜在不利内分泌干扰效应。公共卫生相关性：内分泌干扰化学品（EDCs）如除草剂阿特拉津（ATR）被广泛使用，并被认为对内分泌系统产生不利影响。虽然已确定了EDCs对某些野生动物生殖健康和生存能力的影响，但对人类健康的确切影响仍有争议。在美国，ATR在许多农业环境中使用大量，特别是在种植玉米的中西部地区，然而这种除草剂在大多数欧盟国家是被禁止的。在这个应用中，我们试图用哺乳动物培养细胞来定义ATR的精确分子靶标。我们提出的实验计划系统地确定ATR的分子靶点，是解决ATR对野生动物和人类健康的潜在影响以及围绕其在美国继续使用的争议的重要一步。这些研究也将对NR5A受体在生殖发育和生理中的重要调控作用提供新的认识。