This Phase I application extends our previous research by investigating whether modifications to the automated mouse micronucleus (MN) assay can provide more mechanistic information regarding a chemical's genotoxic action. The in vivo identification of aneugens is an important phase of genotoxicity evaluation. Aneuploidy inducing agents (aneugens) are a potential source of human disease and reproductive failure. Evidence suggests that abnormal chromosomes and the events giving rise to aneuploidy may be mechanistically involved in the process of in vivo tumor formation and in vitro cell transformation. Therefore, the detection and evaluation of aneugenic chemicals is of extreme importance. Since micronuclei may arise from acentric chromosome fragments or from lagging whole chromosomes, this endpoint is capable of detecting clastogens as well as aneugens. By discriminating MN comprised of whole chromosomes from fragments, it will be possible to distinguish between the two. An automated method of detecting aneugenic activity would represent a significant advance over existing methods in terms of turnaround-time, cost and reliability. With the support of this SBIR funding, we intend to develop a reliable method to discriminate clastogen versus aneugen induced MN. Preliminary experiments suggest that the DNA content of MN may be used to distinguish clastogens from aneugens. This method is somewhat limited in that we cannot distinguish MN with centromeres from those without, thus making a definitive indication of aneuploidy impossible. The recent development of anti-kinetochore antibodies, and centromeric and telomeric probes, makes the positive identification of aneugenic potential possible utilizing flow cytometry technology. During Phase I, we propose to investigate the feasibility of utilizing FCM in conjunction with anti-kinetochore antibodies, or centromeric and/or telomeric probes, to identify chemicals that induce aneuploidy. These procedures will be used with fixing and staining methods previously developed at our laboratories for the detection of micronuclei.
An automated method of detecting aneugenic activity would represent a significant advance over existing methods in terms of turnaround-time, cost and reliability. The experiments proposed are intended to develop such an automated test system, and establish Litron Laboratories as an expert testing facility which provides both industry and government with highly reliable genotoxicity assays. Additionally, by packaging the reagents in kit form, another commercial outlet may be realized.
