Automation of a Liver Genotoxicity Assay

DESCRIPTIONprovided by applicantAssessment of chemicalspotential to cause chromosomal damage is an established and important part of preclinical genotoxicity safety testing for many consumer productsindustrial chemicalsand all pharmaceutical agentsCurrently the mammalian erythrocyte micronucleus test is the most commonly employed assay for in vivo assessment of chromosomal damagebut this assay reports specifically on genotoxicity that occurs in the bone marrowIn order to obtain a more comprehensive understanding of potential genotoxicitytesting guidance s recommend evaluation of a second tissueThe liverthe site of metabolism and in many cases activation of genotoxicantsis usually regarded as the preferred second tissueEven sothere is a lack of efficient and effective tools for studying liver genotoxicityThe Comet assay and transgenic rodent mutation models can be employed to study the liverbut these assays suffer from methodological and cost issues that limit their utilityAnother important consideration is that these assays are not highly amenable to integration with on going toxicology studiesmeaning additional animals are required for the liver genotoxicity assessmentOne alternative approach is to examine liver hepatocytes for the formation of micronucleian established indicator of chromosomal damageHowever existing methods for examining liver micronuclei are still emerging and currently based on a multi step sample processing scheme followed by manual scoring by microscopyThis approach is subjective and laborintensiveand results in too few cells being scored for reliable enumeration of micronucleated hepatocytesa situation that diminishes the ability of the test to detect weakly genotoxic agentsWe will overcome these deficiencies by combining simplerapid tissue processing and staining with high speed flow cytometric analysis to greatly improve the execution of liver micronucleus scoringFurthermorewe will multiplex several cytotoxicity measurements into the liver micronucleus assaythereby providing information that we predict will be important for interpreting the genotoxicity resultsThe methodology will be reduced to practice in the form of commercially available kitsand will contribute to the reduction and refinement of animal testingas it will make it feasible to integrate a liver genotoxicity assay ito ongoing toxicology studiesOverallthis project will meet a critical need in the practice of genetic toxicology by improving chemical safety assessments in several meaningful ways