Expressing bacterial bioluminescence in human cell lines: Engineering autobioluminescent reporter cells to screen for endocrine disruptor chemicals

 描述(申请人提供)：在人类细胞系中表达细菌生物发光：通过工程设计自体生物发光报告细胞来筛选内分泌干扰物化学物质项目摘要这个小型企业创新研究第二阶段项目建议以我们成功的使用人类细胞系检测自主内分泌干扰物化学物质(EDC)的第一阶段演示为基础，设计一种新型的Tier 1筛选试验，用于低成本、高通量地检测多种人体组织类型的雌激素和雄激素活性化合物，以满足国家环境健康科学研究所(NIEHS)对改进的测试系统进行优先排序和安全评估的要求。目前的一级EDC筛查方法需要使用非人类细胞系，这可能会模糊生物利用度数据，使用需要专门使用区域和受过专门培训的人员的放射性材料，或者依赖于使用昂贵的分析设备来阻止高通量测试。此外，在最初的Tier 1筛查之后，那些假定表达内分泌干扰活动的化学物质仍然必须与Tier 2动物模型进行筛选以验证结果。目前，仅在美国就有500多家合同检测服务公司为化学、农药和个人护理产品行业提供一级检测，平均每份检测成本为1,000美元。然而，随着需要检测的潜在EDC数量的增加，以及许多州现在通过立法开始为EDC筛查所有市政供水，该行业的市场增长预计将以每年13.5%的速度增长，到2016年接近360亿美元，这清楚地表明，需要新的检测方法来降低筛查成本，增加筛查吞吐量，并在不需要使用动物模型的情况下提供更相关的人体生物利用度数据。在我们的第一阶段工作中，490生物技术公司开发的EDC响应型、无底物、自体生物发光报告细胞系展示了检测EDC的能力，其检测方法类似于现有的Tier 1筛查分析，同时采用基于人类细胞培养的模型，该模型显著降低了测试成本和人员工作量，同时在报告细胞与EDC治疗相互作用和反应时，在报告细胞的整个生命周期内提供不间断的视觉数据流。在这份提案中， 我们将利用这项技术开发完全独立的、基于人类细胞的高通量筛查分析，提供关于EDC生物利用度和对人类健康影响的更准确和更真实的信息。随着这些分析的应用，将一级EDC筛查的成本降低了100多倍，每种化合物的成本不到10美元，我们相信我们拥有一种能够显著影响EDC筛查市场的产品，并提高我们对新化合物内分泌干扰物活性的理解，因为它与公共健康和消费者安全有关。

 DESCRIPTION (provided by applicant): Expressing bacterial bioluminescence in human cell lines: Engineering autobioluminescent reporter cells to screen for endocrine disruptor chemicals Project Summary This Small Business Innovation Research Phase II project proposes to build upon our successful Phase I demonstration of autonomous endocrine disruptor chemical (EDC) detection using human cell lines to engineer a novel Tier 1 screening assay for the low cost, high-throughput detection of estrogenically- and androgenically-active compounds across multiple human tissue types to address the National Institute of Environmental Health Sciences (NIEHS) request for Improved Test Systems for Prioritization and Safety Evaluation. Current Tier 1 EDC screening approaches require the use of non-human cell lines that can obscure bioavailability data, employ radioactive materials that require dedicated use areas and specially trained personnel, or rely upon the use of expensive analytical equipment that prevents high-throughput testing. Furthermore, following this initial Tier 1 screen, those chemicals that putatively express endocrine disrupting activities must still be screened against Tier 2 animal models to validate the results. Currently, there are over 500 contract testing service companies in the U.S. alone that perform Tier 1 style assays for the chemical, pesticide, and personal care products industries at an average cost of $1,000 per assay. However, as the number of potential EDCs requiring testing expands, and with many states now adopting legislation to begin screening all municipal water supplies for EDCs, market growth within this sector is expected to increase at an annual rate of 13.5% to approach $36 billion by 2016, clearly demonstrating the need for new assays that can reduce screening costs, increase screening throughput, and provide more relevant human bioavailability data without necessitating the use of animal models. The EDC-responsive, substrate-free, autobioluminescent reporter cell lines developed by 490 BioTech under our Phase I efforts demonstrated the ability to detect EDCs similarly to existing Tier 1 screening assays while employing a human cell culture-based model that significantly reduced the cost and personnel effort of testing while simultaneously providing an uninterrupted stream of visual data over the lifetime of the reporter cell as it interacted with and reacted to EDC treatment. In this proposal, we will leverage this technology to develop fully self-contained, human cell-based, high- throughput screening assays that provide more accurate and realistic information in regards to EDC bioavailability and effects on human health. With application of these assays reducing the cost of Tier 1 EDC screening over 100-fold to less than $10 per compound, we believe we possess a product capable of significantly impacting the EDC screening market and advancing our understanding of novel compound endocrine disruptor activity as it pertains to public health and consumer safety.