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

 描述（申请人提供）：在人细胞系中表达细菌生物发光：工程自动生物发光报告细胞筛选内分泌干扰物化学品项目摘要这个小企业创新研究第二阶段项目提出建立在我们成功的第一阶段示范自主内分泌干扰物化学品（EDC）检测使用人类细胞系工程新的第1层筛选测定低成本，高通量检测多种人体组织类型中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）对优先级和安全性评估的改进测试系统的要求。目前的1级EDC筛查方法需要使用非人类细胞系，这些细胞系可能会掩盖生物利用度数据，使用需要专门使用区域和经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，从而无法进行高通量检测。此外，在最初的第1级筛选之后，那些可表达内分泌干扰活性的化学品仍必须针对第2级动物模型进行筛选，以验证结果。目前，仅在美国就有超过500家合同测试服务公司为化学、农药和个人护理产品行业提供Tier 1风格的分析，每次分析的平均成本为1，000美元。然而，随着需要检测的潜在内分泌干扰物数量的增加，以及许多州现在通过立法开始筛查所有市政供水中的内分泌干扰物，预计到2016年，该行业的市场增长将以13.5%的年增长率增长至接近360亿美元，这清楚地表明需要新的检测方法，可以降低筛查成本，增加筛查通量，并提供更相关的人体生物利用度数据，而无需使用动物模型。这种EDC敏感、无基质、490 BioTech在我们的I期工作中开发的自生物发光报告细胞系证明了在使用人细胞培养物时与现有的1级筛选测定类似地检测EDC的能力。该模型显著降低了测试的成本和人员工作量，同时在报告细胞的生命周期内提供不间断的视觉数据流，因为它与细胞相互作用。对EDC治疗有反应在这一提议中， 我们将利用这项技术开发完全独立的、基于人体细胞的高通量筛选试验，提供关于EDC生物利用度和对人体健康影响的更准确和真实的信息。随着这些检测方法的应用，第1层EDC筛查的成本降低了100倍以上，每个化合物不到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，并提高我们对新型化合物内分泌干扰物活性的理解，因为它涉及公共卫生和消费者安全。

 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.