Assessment of chemicals' potential to cause chromosomal damage is an established and important part of preclinical genotoxicity safety testing for many consumer products, industrial chemicals, and all pharmaceutical agents. Currently the mammalian erythrocyte micronucleus test is the most commonly employed assay for in vivo assessment of chromosomal damage, but this assay reports specifically on genotoxicity that occurs in the bone marrow. In order to obtain a more comprehensive understanding of potential genotoxicity, testing guidance's recommend evaluation of a second tissue. The liver, the site of metabolism and in many cases activation of genotoxicants, is usually regarded as the preferred second tissue. Even so, there is a lack of efficient and effective tools for studying liver genotoxicity. The Comet assay and transgenic rodent mutation models can be employed to study the liver, but these assays suffer from methodological and cost issues that limit their utility. Another important consideration is that these assays are not highly amenable to integration with on-going toxicology studies, meaning additional animals are required for the liver genotoxicity assessment. One alternative approach is to examine liver hepatocytes for the formation of micronuclei, an established indicator of chromosomal damage. However existing methods for examining liver micronuclei are still emerging and currently based on a multi-step sample processing scheme followed by manual scoring by microscopy. This approach is subjective and labor- intensive, and results in too few cells being scored for reliable enumeration of micronucleated hepatocytes, a situation that diminishes the ability of the test to detect weakly genotoxic agents. We will overcome these deficiencies by combining simple, rapid tissue processing and staining with high-speed flow cytometric analysis to greatly improve the execution of liver micronucleus scoring. Furthermore, we will multiplex several cytotoxicity measurements into the liver micronucleus assay, thereby providing information that we predict will be important for interpreting the genotoxicity results. The methodology will be reduced to practice in the form of commercially available kits, and will contribute to the reduction and refinement of animal testing, as it will make it feasible to integrate a liver genotoxicity assay ito ongoing toxicology studies. Overall, this project will meet a critical need in the practice of genetic toxicology by improving chemical safety assessments in several meaningful ways.
Public Health Relevance Statement: Public Health Relevance: The assessment of chemical-induced chromosomal damage in the liver is an important component of genetic toxicology safety testing. Current methods are tedious, costly, and not amenable to integration with on-going toxicology studies. We propose to develop an easy, efficient, and automated method for processing and analyzing liver tissue for the frequency of micronucleated hepatocyte, an indicator of chromosomal damage. This method will substantially improve existing approaches for evaluating drugs and other chemicals for their ability to cause DNA damage to the liver. Besides genetic toxicity, our assay will provide concurrent assessments of overt toxicity, information that is critical for interpreting DNA damage results. The methodology will be made available through commercial kits, and will contribute to the reduction and refinement of animal testing, as it will make it feasible to integrate a liver genotoxicity assay into ongoing toxicology studies.
Project Terms: Affect; Age; age group; Animal Testing; Animals; Antibodies; Apoptotic; Automation; base; Benchmarking; Biological Assay; Bone Marrow; Cell Nucleus; Cells; Chemicals; Comet Assay; consumer product; cost; cytotoxicity; Data; design; Detergents; Development; Diethylnitrosamine; DNA; DNA Damage; DNA Double Strand Break; Dose; Dyes; Erythrocytes; Evaluation; Experimental Designs; Exposure to; Family suidae; Female; Flow Cytometry; Fluorescence Microscopy; Frequencies (time pattern); Genetic; genotoxicity; Goals; Harvest; Hepatocyte; high throughput screening; Histopathology; improved; in vivo; indexing; Japan; Kinetics; Label; Laboratories; Liver; male; Manuals; Measurement; Measures; meetings; Metabolism; method development; Methodology; Methods; micronucleus; Micronucleus Tests; Microscopy; Mitotic; Modeling; Mutagenicity Tests; Mutagens; Mutation; Organ; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Play; pre-clinical; Procedures; Process; prototype; public health relevance; quinoline; Rattus; Reagent; Reporting; Research Design; Rodent; Safety; safety testing; Sampling; Schedule; Scheme; Scoring Method; sex; Site; Solutions; Speed (motion); Temperature; Testing; Time; Tissue Harvesting; tissue processing; Tissue Stains; Tissues; tool; Toxic effect; Toxicogenetics; Toxicology; Transgenic Organisms; treatment duration; treatment group; Validation
