Humans are exposed to toxic chemical and physical agents through a variety of sources. Of extreme concern are agents which can damage DNA, as genotoxicity is associated with cancer, atherosclerosis, and birth defects. The research effort proposed herein is aimed at developing murine ex vivo culture system that is capable of detecting agents which damage chromosomes through clastogenic and aneugenic activity. Currently, chemical-induced chromosome damage is evaluated by scoring chromosome abberations or micronucleus formation in cell lines or in laboratory rodents. By developing an ex vivo erythropoiesis system, reticulocytes can be generated in culture which serve as source material for micronucleus scoring. The primary cultures should provide more relevant toxicity information compared to immortilized cell lines. Furthermore, the feasibility of developing a flow cytometric scoring technique to enumerate micronuclei in ex vivo-derived reticulocytes is high, and overcomes artifacts associated with automated scoring of the standard in vitro assay. Relative to rodent tests, the proposed primary culture system will require few animals. To investigate the feasibility and utility of a genetic toxicity screening system based on ex vivo erythropoiesis, we will: 1. EVALUATE reticulocyte cell production by ex vivo culture(s). 2. MEASURE the frequency of micronuclei in ex vivo derived reticulocytes. 3. DETERMINE the responsiveness of the system to known clastogens and a model aneugen.
