In vivo mutation assay based on pig-a locus

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DESCRIPTION (provided by applicant): Mutation to DNA is a primary mechanism by which cancers arise. These events have also been implicated in diseases such as atherosclerosis, and processes such as aging. Therefore, there is an important need for sensitive methods which are capable of identifying chemical or physical agents that can mutate DNA. Methods for measuring in vivo mutation currently exist, each with advantages and limitations. While some are based on colony formation and require tissue culture work, others rely on proprietary rodents. The in vivo mutation assay that is proposed herein is based on the pig-a locus. The pig-a gene product is essential for the biosynthesis of glycosyl-phosphatidylinositol (GPI) anchors. Mutations giving rise to non-functional GPI anchors prevent certain proteins from being expressed on the cell surface, and this represents a phenotype which can be measured by high speed flow cytometry. Importantly, harvested cells are not cultured before analysis, thus the need for costly- and labor-intensive tissue culture work is eliminated. Furthermore, since pig-a is an endogenous gene located on the X-chromosome, it is likely that this mutation scoring system will be applicable to any mammalian species of toxicologic interest. Phase I experiments will focus on evaluating the feasibility of enumerating in vivo pig-a mutation with a single-laser flow cytometer. The animal model for these initial investigations is the Sprague-Dawley rat, and the target cells will be peripheral blood erythrocytes. Blood from vehicle control and genotoxicant-treated animals will be used in Phase I to identify fluorescent staining techniques which provide for resolution of GPI-competent and GPI-deficient erythrocytes, and also to assess experimental parameters such as cell harvest time.