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

项目摘要 目前的遗传毒理学试验组表现出几个严重的缺陷。第一，吞吐量 体外遗传毒性试验的能力较低，不能满足当前的需要，特别是对于早期、高容量 筛选需要优先考虑化学品以供进一步测试和/或开发的环境。第二、 常规测定提供了简单的遗传毒性或非遗传毒性的二元判定。在这个计划中， 未提供关于遗传毒性作用方式的信息。这是严重的限制，因为它不生成密钥。 确定化学品的优先顺序以进行进一步测试所需的信息， 终点/实验设计，或进行风险评估。最后，大多数当前的测定法不放置 必须强调剂量反应关系，因此不将结果置于 力量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。我们将解决这些问题， 开发、优化和验证基于多重DNA损伤的双层测试策略 响应性生物标志物和高速流式细胞术分析。第一层侧重于吞吐量， 用于优先考虑可能的遗传毒性物质，以便在二级检测中进行更全面的分析。具体 涉及几种多重生物标志物的集合，这些生物标志物将用于鉴定可能的遗传毒性剂， 提供遗传毒性作用模式的初步评估。gH 2AX生物标志物检测DNA双链 链断裂，磷酸化组蛋白H3鉴定有丝分裂细胞，细胞核p53含量报告p53激活， 对DNA损伤的反应，8 n+细胞的频率测量多倍化，以及核与核的比率。 微球计数提供了关于治疗相关细胞毒性的信息。第二层侧重于 信息内容，并考虑更多的浓度以及其他生物标志物，包括 微核形成。总的来说，二级结果提供了关于测试化学品的确定性预测。 遗传毒性潜力、作用方式和效力。在这个项目中，我们将研究3000多个 不同的化学品，以了解性能特点和两层的概括性 测试策略将使用原型检测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为全国提供市售试剂盒和检测 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.