Bacterial Biosensors for Endocrine Disrupting Compounds

用于内分泌干扰化合物的细菌生物传感器

• 批准号: 7451331
• 负责人: David W Wood
• 金额: $ 21.15万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7451331
• 关键词: Affect; Affinity; Animals; Autistic Disorder; Behavior; Binding; Biological Assay; Biosensor; Cells; Characteristics; Chemicals; Chimeric Proteins; Classification; Commerce; Complex; Complex Mixtures; Condition; Congresses; Cosmetics; Culture Media; Data; Databases; Detection; Development; Distress; Ecdysone; Endocrine; Endocrine Disruptors; Endocrine disruption; Endocrine system; Engineering; Environment; Environmental Health; Enzymes; Escherichia coli; Estrogens; Evaluation; Facility Construction Funding Category; Fertility; Fertility Rates; Fluorescence; Food; Future; Generations; Genistein; Goals; Growth; Health; Home environment; Hormone Receptor; Hormones; Household; Human; In Vitro; Industrial Waste; Insect Hormones; Libraries; Ligand Binding Domain; Ligands; Link; Malignant Neoplasms; Mammary gland; Measures; Methods; Nature; Nuclear Hormone Receptors; Numbers; Perfume; Phenotype; Phytoestrogens; Plant Extracts; Pliability; Population; Proteins; Public Health; Range; Rate; Regulation; Reporter; Risk; Screening procedure; Source; Steroids; System; Testing; Thyroid Gland; Thyroid Hormone Receptor; Thyroid Hormones; Toxicology; United States; United States Environmental Protection Agency; United States National Institutes of Health; Validation; Water; Work; Xenobiotics; base; bisphenol A; design; high throughput technology; malignant breast neoplasm; programs; receptor binding; research study; response; sensor; small molecule libraries; stem; tool; trend; tumor; xenoestrogen

DESCRIPTION (provided by applicant): As of 1998, more than 80,000 chemicals were registered by the EPA as being in commerce in the United States. Of the nearly 3000 chemicals that were imported or manufactured at a rate of more than one million pounds per year, 43% had no available toxicology data, and only 7% had full information on basic health and environmental effects. Some of these compounds are now classified as endocrine disruptors, and have been associated with mammary and other tumors, as well as decreases in fertility in human and animal populations. Despite these findings, only a small fraction of the close to 100,000 chemicals listed in the NIH TOXNET database have been fully characterized for endocrine disruptive effects, primarily due to the complexity of the assays required for accurate classification. Thus a critical goal is to develop quick and reliable methods to identify and characterize unknown compounds, so that risks and appropriate regulations can be determined. This proposal seeks to aid in this goal through the development of a bacterial biosensor for the rapid and reliable detection of endocrine disrupting effects in isolated chemicals and complex mixtures. This sensor relies on an engineered, four-domain fusion protein, which includes the ligand-binding domain of a given nuclear hormone receptor linked to a reporter enzyme through a small stabilization domain. When expressed in an appropriate Escherichia coli host strain, this protein generates a hormone-sensitive phenotype, which can be used to detect ligands that interact with the included nuclear hormone receptor domain. This system has been shown to be capable of detecting a wide variety of estrogenic compounds, including phytoestrogens, xenoestrogens, and estrogenic steroids, and it can reliably differentiate agonistic from antagonistic behaviors. Further, the modular design of the sensor has facilitated the construction of additional sensors for thyroid hormone-like compounds, and even insect-hormone like compounds. In our proposed work, we will develop this sensor into a fully validated and functional first-line tool for use in identifying potential endocrine disruptors. Specific aims include its formal validation for estrogenic compound identification, along with the generation and validation of additional sensors through ligand-binding domain swapping. Enhancement of the system readout is also suggested to facilitate future applications based on high-throughput technologies. It is anticipated that this system will ultimately hold significant utility as part of a multi-tiered program for their systematic discovery and classification of endocrine disrupting compounds. PUBLIC HEALTH RELEVANCE Over the last several decades, several distressing health trends, including increases in breast cancer, autism rates, and fertility problems have been linked to hormone-mimicking compounds known as endocrine disruptors. The work described in this proposal seeks to develop a unique biosensor system wherein bacterial cells indicate the presence of endocrine disrupting compounds through a simple, inexpensive and reliable growth/no growth assay. This system has many advantages over conventional approaches, and may provide an effective first-line evaluation of natural and artificial compounds, alone and in mixtures, for activity against specific hormone receptor targets.

描述（由申请人提供）：截至1998年，EPA注册了80,000多种化学药品，为美国的商业。在以每年超过一百万磅的速度进口或制造的近3000种化学药品中，有43％的化学物质没有可用的毒理学数据，只有7％的化学物质有关于基本健康和环境影响的完整信息。这些化合物中的一些现在被归类为内分泌干扰物，并且与乳腺和其他肿瘤有关，并且人类和动物种群的生育力降低。尽管有这些发现，但仅在NIH Toxnet数据库中列出的近100,000种化学物质中，只有一小部分是针对内分泌破坏性作用的完全表征，这主要是由于准确分类所需的测定的复杂性。因此，一个关键目标是开发快速可靠的方法来识别和表征未知化合物，以便可以确定风险和适当的法规。该提案旨在通过开发细菌生物传感器来帮助实现这一目标，以快速可靠地检测到孤立的化学物质和复杂混合物中内分泌破坏作用。该传感器依赖于一种设计的四域融合蛋白，其中包括通过小稳定域与报道酶相关的给定核激素受体的配体结合域。当在适当的大肠杆菌宿主菌株中表达时，该蛋白会产生一种激素敏感的表型，可用于检测与所包括的核激素受体结构域相互作用的配体。该系统已被证明能够检测到多种雌激素化合物，包括植物雌激素，异种雌激素和雌激素类固醇，并且可以可靠地将激动与拮抗行为区分开。此外，传感器的模块化设计有助于构建类似甲状腺激素的化合物的其他传感器，甚至是昆虫激素（如化合物）。在我们提出的工作中，我们将将该传感器开发为一个经过全面验证和功能的一线工具，用于识别潜在的内分泌干扰物。具体目的包括其对雌激素化合物识别的正式验证，以及通过配体结合域交换对其他传感器的产生和验证。还建议增强系统读数，以促进基于高通量技术的未来应用。可以预料，该系统最终将拥有大量的实用程序，作为多层程序的一部分，用于系统发现和对内分泌干扰化合物的分类。在过去的几十年中，公共卫生相关性，几种令人沮丧的健康趋势，包括乳腺癌的增加，自闭症率和生育问题与模仿激素的化合物（称为内分泌干扰物）有关。该提案中描述的工作旨在开发一个独特的生物传感器系统，其中细菌细胞表明通过简单，廉价且可靠的生长/无生长测定法表明内分泌破坏化合物的存在。该系统比常规方法具有许多优势，并且可以单独和混合物对自然和人工化合物进行有效的一线评估，以对特定的激素受体靶标进行活性。