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 assaysÂ’ 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 chemicalsÂ’ 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. Public Health Relevance Statement Project Narrative Some chemicals in commercial use and in the environment can cause DNA damage and this damage can contribute to the development of cancer and other severe diseases. We will develop, optimize, and validate an improved testing strategy based on highly automated processes tracking several DNA damage biomarkers that can be analyzed without the need for animal testing. These methods will be configured into commercially available kits and testing services.
Project Terms: Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Buffers ; Canada ; Malignant Neoplasms ; Cancers ; Malignant Tumor ; malignancy ; neoplasm/cancer ; Cell Line ; CellLine ; Strains Cell Lines ; cultured cell line ; Cell Nucleus ; Nucleus ; Cells ; Cell Body ; Data Analyses ; Data Analysis ; data interpretation ; Statistical Data Interpretation ; Statistical Data Analyses ; Statistical Data Analysis ; statistical analysis ; Disease ; Disorder ; DNA Damage ; DNA Injury ; DNA Repair ; DNA Damage Repair ; Unscheduled DNA Synthesis ; Elements ; Environment ; Exhibits ; Experimental Designs ; Flow Cytometry ; Flow Cytofluorometries ; Flow Cytofluorometry ; Flow Microfluorimetry ; Flow Microfluorometry ; flow cytophotometry ; Goals ; Health ; Histone H3 ; Human ; Modern Man ; In Vitro ; Industry ; instrumentation ; Metabolic Activation ; Methods ; micronucleus ; Microspheres ; Microbeads ; Miniaturization ; Miniaturisations ; Modernization ; Mutagenicity Tests ; Genetic Toxicity Tests ; Genotoxicity Tests ; Mutagen Screening ; mutagen testing ; Mutagens ; Genotoxins ; genotoxic agent ; Protease Inhibitor ; Antiproteases ; Endopeptidase Inhibitors ; Peptidase Inhibitors ; Peptide Hydrolase Inhibitors ; Peptide Peptidohydrolase Inhibitors ; Protease Antagonists ; Proteinase Inhibitors ; Reagent ; Recommendation ; Sensitivity and Specificity ; Temperature ; Testing ; Time ; Toxicology ; Work ; Measures ; TP53 gene ; Antioncogene Protein p53 ; Cellular Tumor Antigen P53 ; Oncoprotein p53 ; P53 ; Phosphoprotein P53 ; Phosphoprotein pp53 ; Protein TP53 ; TP53 ; TRP53 ; Tumor Protein p53 ; Tumor Protein p53 Gene ; p53 Antigen ; p53 Genes ; p53 Tumor Suppressor ; protein p53 ; Risk Assessment ; Data Set ; Dataset ; base ; improved ; Phase ; Chemicals ; Training ; Logistics ; Measurement ; machine learned ; Machine Learning ; programs ; Frequencies ; cell type ; Techniques ; System ; Nuclear ; Lytotoxicity ; cytotoxicity ; Performance ; phosphatase inhibitor ; genotoxicity ; Speed ; Reporting ; Coding System ; Code ; Modeling ; Sampling ; response ; High Throughput Assay ; high throughput screening ; Endpoint Assays ; End Point Assay ; Genetic Toxicology ; Toxicology Genetics ; Toxicogenetics ; preventing ; prevent ; Address ; Dose ; Animal Testing ; DNA Double Strand Break ; Data ; Mitotic ; Collection ; Scheme ; Validation ; Characteristics ; Process ; follow-up ; Active Follow-up ; active followup ; follow up ; followed up ; followup ; Development ; developmental ; National Toxicology Program ; computerized tools ; computational tools ; design ; designing ; climate change ; global climate change ; blind ; innovation ; innovate ; innovative ; prototype ; Biological Markers ; bio-markers ; biologic marker ; biomarker ; screening ; Formulation ; response biomarker ; response markers ; experimental study ; experiment ; experimental research ; testing services ;
