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 analytical methods which facilitate the study of mutagenesis, as well as the identification of chemical or physical agents that can mutate DNA. Methods for measuring in vivo mutation currently exist, each with their own advantages and limitations. While some are based on colony formation and require tissue culture work, others rely on expensive, proprietary trangenic 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 nonfunctional GPI anchors prevent certain proteins from being expressed on the cell surface, and this represents a phenotype which can be measured by 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, including humans. As was the case for Phase I, the proposed Phase II experiments will focus on two readily obtained cell populations for the determination of GPI-anchor deficiency: peripheral blood erythrocytes (total) and an immature fraction of erythrocytes (reticulocytes). Whereas Phase I feasibility studies were conducted strictly with mice, these experiments will consider exposures of both mice and rats to each of six prototypical mutagens. The treatment schedule and the blood harvest times will be varied in order to determine the most appropriate experimental designs. Other experiments will involve isolation and DNA sequencing of GPI-anchor deficient erythroid progenitors from mutagen-treated mice. The presumptive Pig-a mutant colonies will be sequenced to provide mutation spectra data that helps validate the system as an effective mutagenesis assay. Upon successful completion of these proposed experiments, society will benefit as pharmaceutical and chemical companies eliminate genotoxicants from their new product development processes more efficiently. Furthermore, if the system proves compatible with human blood specimens, myriad other research activities will benefit as data generated in laboratory animal models are easily extended to include real-world human exposure scenarios, including: 1) clinical trials, 2) post-market survallience of drugs, 3) environmental exposures, and 4) occupational exposures.
It is well known that DNA damage is a precursor to the development of cancer and other significant diseases. It is, therefore, in the interest of public health to reduce the occurrence of mutagenic chemicals in the environment, in our drugs, and from our workplaces. This research project will refine and validate a powerful new method for detecting mutagenic agents, thereby enhancing the nation's ability to effectively reduce exposure to these toxic compounds.
