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

基因毒物的高通量筛选和高信息后续测试

• 批准号: 10576559
• 负责人: Jeffrey C Bemis
• 金额: $ 94.51万
• 依托单位: LITRON LABORATORIES, LTD.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-08 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10576559
• 关键词: Address; Animal Testing; Biological Assay; Biological Markers; Buffers; Canada; Cell Line; Cell Nucleus; Cells; Characteristics; Chemicals; Code; Collection; DNA Damage; DNA Double Strand Break; DNA Repair; Data; Data Analyses; Data Set; Development; Disease; Dose; Elements; End Point Assay; Environment; Exhibits; Experimental Designs; Flow Cytometry; Formulation; Frequencies; Goals; Health; Histone H3; Human; In Vitro; Industry; Logistics; Malignant Neoplasms; Measurement; Measures; Metabolic Activation; Methods; Microspheres; Miniaturization; Mitotic; Modeling; Modernization; Mutagenicity Tests; Mutagens; National Toxicology Program; Nuclear; Performance; Phase; Process; Protease Inhibitor; Reagent; Recommendation; Reporting; Risk Assessment; Sampling; Scheme; Sensitivity and Specificity; Speed; Statistical Data Interpretation; System; TP53 gene; Techniques; Temperature; Testing; Time; Toxicogenetics; Toxicology; Training; Validation; Work; base; blind; cell type; climate change; computerized tools; cytotoxicity; design; experimental study; follow-up; genotoxicity; high throughput screening; improved; innovation; instrumentation; machine learning model; micronucleus; phosphatase inhibitor; prevent; programs; prototype; response; response biomarker; screening; testing services

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.

项目概要 目前的遗传毒理学检测方法存在几个关键缺陷：首先，通量。 体外遗传毒性试验能力较低，不能满足当前的需要，特别是早期、大批量的试验 其次，筛选需要优先考虑化学品以进行进一步测试和/或开发的环境。 传统的检测提供了简单的二元分析，即基因毒性或非基因毒性。在该方案中，几乎没有或没有基因毒性。 没有提供有关遗传毒性作用模式的信息，这是严重限制的，因为它不产生关键。 确定化学品优先顺序以进行进一步测试、指导后续分析所需的信息 最后，大多数当前的分析都没有进行终点/实验设计或进行风险评估。 必要地强调剂量反应关系，因此不要将结果与 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。 开发、优化和验证基于多重 DNA 损伤的两层测试策略 第一层侧重于通量，是响应性生物标志物和高速流式细胞术分析。 用于对可能的遗传毒物进行优先排序，以便在第二级测试中进行更全面的分析。 涉及几种多重生物标志物的集合，这些标志物将用于识别可能的基因毒性剂和 提供基因毒性作用模式的初步评估 gH2AX 生物标志物检测 DNA 双。 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，以及细胞核与 微球计数提供有关治疗相关细胞毒性的信息。 信息内容并考虑更多浓度以及其他生物标志物，包括 总的来说，第二层结果提供了有关测试化学品的明确预测。 在这个项目的过程中，我们将研究 3,000 多种潜在的遗传毒性、作用方式和效力。 不同的化学品，以了解两层的性能特征和普遍性 测试策略将使用原型检测试剂盒进行，以评估可转移性。 这些方法的最终目标是为国家提供商用试剂盒和测试 服务。