High Throughput Screen and High Information Follow-Up Tests for Genotoxicants

项目摘要 目前的遗传毒理学检测显示出几个严重的缺陷。首先，吞吐量 体外遗传毒性检测能力低，不能满足目前的需要，特别是早期、大批量 筛选需要优先处理化学品以进行进一步测试和/或开发的环境。第二, 传统的分析提供简单的二元呼叫，遗传毒性的或非遗传毒性的。在这个方案中，几乎没有或 没有提供有关遗传毒性作用模式的信息。这是一个严重的限制，因为它不生成密钥 为进一步测试确定化学品的优先顺序所需的信息，指导后续测试 终端/实验设计，或进行风险评估。最后，目前的大多数化验方法都没有 对剂量反应关系的必要强调，因此不将结果与以下方面联系起来 威力。这些缺陷阻碍了遗传毒性数据对现代风险评估的最佳贡献， 所有这些能力和高信息含量都是必不可少的。我们将通过以下方式解决这些问题 基于多重DNA损伤的两级测试策略的开发、优化和验证 响应性生物标志物和高速流式细胞仪分析。第一层侧重于吞吐量， 用于确定可能的遗传毒物的优先顺序，以便在第二级测试中进行更全面的分析。具体地说，它 涉及几个多路生物标记物的集合，这些生物标记物将用于识别可能的遗传毒性物质和 提供遗传毒性作用模式的初步评估。GH2 AX生物标记物检测DNA重复 链断裂，磷酸组蛋白H3识别有丝分裂细胞，核P53含量报告P53激活 对DNA损伤的反应，8n细胞的频率测量多倍化，以及核与 微球计数提供了与治疗相关的细胞毒性信息。第二层侧重于 信息含量，并考虑更多的浓度以及其他生物标志物，包括 微核形成。总而言之，第二级结果提供了关于测试化学品的明确预测 基因毒性潜力、作用方式和效力。在这个项目的过程中，我们将研究3000多个 不同的化学品，以了解两级的性能特点和通用性 测试策略。将用原型化验试剂盒进行实验室间试验，以评估可转移性。 这些方法的最终目标是向国家提供商业可用的试剂盒和测试 服务。

Project Summary Current batteries of genetic toxicology assays exhibit several critical deficiencies. First, the throughput capacity of in vitro genotoxicity tests is low, and does not meet current needs, especially for early, high volume screening environments that need to prioritize chemicals for further testing and/or development. Second, conventional assays provide simplistic binary calls, genotoxic or non-genotoxic. In this scheme there is little or no information provided about genotoxic mode of action. This is severely limiting, as it does not generate key information necessary for prioritizing chemicals for further testing, guiding subsequent assays’ endpoints/experimental designs, or conducting risk assessments. Finally, most current assays do not place requisite emphasis on dose response relationships, and therefore do not contextualize the results in terms of potency. These deficiencies prevent genotoxicity data from optimally contributing to modern risk assessments, where all of these capabilities and high information content are essential. We will solve these issues by developing, optimizing, and validating a two-tiered testing strategy based on multiplexed DNA damage responsive biomarkers and high-speed flow cytometric analysis. The first-tier focuses on throughput and is used to prioritize likely genotoxicants for more comprehensive analysis in second tier testing. Specifically, it involves a collection of several multiplexed biomarkers that will be used to identify likely genotoxic agents and provide a preliminary assessment of genotoxic mode of action. The gH2AX biomarker detects DNA double strand breaks, phospho-histone H3 identifies mitotic cells, nuclear p53 content reports on p53 activation in response to DNA damage, the frequency of 8n+ cells measure polyploidization, and the ratio of nuclei to microsphere counts provides information about treatment-related cytotoxicity. The second tier focuses on information content and considers many more concentrations as well as additional biomarkers, including micronucleus formation. Collectively, the tier two results provide definitive predictions about test chemicals’ genotoxic potential, mode of action, and potency. Over the course of this project we will study more than 3,000 diverse chemicals in order to understand the performance characteristics and generalizability of the two-tiered testing strategy. An interlaboratory trial will be conducted with prototype assay kits to assess the transferability of the methods, with the ultimate goal of providing the Nation with commercially available kits and testing services.