Bacterial Biosensors for Endocrine Disrupting Compounds

描述（由申请人提供）：截至1998年，超过80，000种化学品被EPA注册为在美国商业化。在每年进口或制造超过100万磅的近3000种化学品中，43%没有可用的毒理学数据，只有7%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，并与乳腺和其他肿瘤以及人类和动物群体生育能力的降低有关。尽管有这些发现，但在NIH TOXNET数据库中列出的近10万种化学品中，只有一小部分被充分表征为内分泌干扰作用，主要是由于准确分类所需的测定方法的复杂性。因此，一个关键的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，该生物传感器用于快速可靠地检测孤立化学品和复杂混合物中的内分泌干扰效应。这种传感器依赖于一种工程化的四域融合蛋白，它包括通过一个小的稳定结构域与报告酶连接的给定核激素受体的配体结合结构域。当在适当的大肠杆菌宿主菌株中表达时，该蛋白质产生酶敏感表型，其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。该系统已被证明是能够检测各种各样的雌激素化合物，包括植物雌激素，外源性雌激素和雌激素类固醇，它可以可靠地区分激动和拮抗行为。此外，传感器的模块化设计促进了用于甲状腺激素样化合物甚至昆虫激素样化合物的附加传感器的构建。在我们提出的工作中，我们将把这种传感器开发成一种经过充分验证的、功能齐全的一线工具，用于识别潜在的内分泌干扰物。具体目标包括其正式验证雌激素化合物鉴定，沿着通过配体结合域交换生成和验证额外的传感器。还建议增强系统读出，以促进基于高通量技术的未来应用。预计该系统最终将作为系统发现和分类内分泌干扰化合物的多层次计划的一部分，具有重要的实用价值。在过去的几十年里，几个令人不安的健康趋势，包括乳腺癌，自闭症率和生育问题的增加，都与被称为内分泌干扰物的模仿激素的化合物有关。本提案中描述的工作旨在开发一种独特的生物传感器系统，其中细菌细胞通过简单、廉价和可靠的生长/无生长测定来指示内分泌干扰化合物的存在。该系统与传统方法相比具有许多优点，并且可以提供天然和人工化合物单独和混合物对特定激素受体靶标的活性的有效第一线评价。

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.