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: 10605311
• 负责人: Jeffrey C Bemis
• 金额: $ 68.25万
• 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 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605311
• 关键词: Animal Testing; Biological Assay; Biological Markers; Buffers; Canada; Cell Line; Cell Nucleus; Cells; Characteristics; Chemicals; Code; Collection; DNA Damage; DNA Double Strand Break; Data; Data Analyses; Data Set; Development; Disease; Disparate; Dose; Elements; End Point Assay; Environment; Exhibits; Experimental Designs; Flow Cytometry; Formulation; Frequencies; Friends; Goals; Health; Histone H3; Human; In Vitro; Industry; Laboratories; Logistics; Malignant Neoplasms; Measurement; Measures; Metabolic Activation; Methods; Microspheres; Miniaturization; Mitotic; Modeling; Modernization; Mutagenicity Tests; Mutagens; National Toxicology Program; Nuclear; Performance; Phase; Polyploidy; Process; Protease Inhibitor; Reagent; Recommendation; Reporting; Risk Assessment; Sampling; Scheme; Sensitivity and Specificity; Specificity; Speed; Statistical Data Interpretation; System; TP53 gene; Techniques; Temperature; Testing; Time; Toxicogenetics; Toxicology; Training; Validation; Work; 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; repaired; 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.

项目总结