Zinc protoporphyin (ZnPP) is a naturally occurring metalloporphyrin (Mp) that is formed endogenously during heme biosynthesis. This compound is synthesized at higher levels in conditions of iron deficiency anemia and lead intoxication. Under these conditions, red blood cell turnover allows for deposition of ZnPP in tissues such as the liver, spleen. Animal experiments suggest that ZnPP is an effective inhibitor of bilirubin production. However, other studies demonstrate that ZnPP is an inhibitor of bone marrow cell proliferation and that it results in apoptosis in hamster fibroblasts ceils in culture. Therefore, it may be that ZnPP plays a role in cell cycle regulation. The Specific Aims of this proposal are to: 1) describe the effect of ZnPP on cell cycle regulation in vitro, 2) describe the effect of ZnPP on cell cycle regulation in vitro and 3) elucidate the mechanisms by which ZnPP mediates cell cycle regulation in vitro. Using primary hepatocytes in culture, we will evaluate the effect of exogenous ZnPP on cell cycle progression as well as the expression of cyclins and other proteins that regulate the cell cycle. Neonatal mice will be injected with ZnPP at various concentrations. Tissues will be evaluated for ZnPP incorporation. Tissues demonstrating ZnPP deposition will be further examined for markers of cell proliferation and apoptosis and for cell cycle protein expression. The amount of ZnPP binding to HO-1 and HO-2 proteins will be determined by immunoprecipitation techniques and Western analysis. To document ZnPP-DNA binding, we will detect radiolabeling of nuclear DNA using DNAse footprinting after incubation with radiolabeled 65ZnPP. This will allow us to determine whether there are direct or indirect effects of ZnPP on gene transcription. We will also evaluate how incubation with ZnPP specifically alters the cell cycle. We hypothesize that ZnPP mediates its effects, in part, through induction of early growth response transcription factor (Egr-1) and through Egr-l-mediated gene transcription. Using hepatocytes derived from Egr-1 and p53 null mutants, we will specifically address whether ZnPP-mediated changes in cell cycle occur through Egr-1 and/or p53 dependent pathways. These experiments will allow us to determine specifically how ZnPP serves as a signaling molecule to suppress cell proliferation. A better understanding of the role of ZnPP in cell cycle regulation will determine whether ZnPP functions as a modifier of the cell cycle. This may also lead to therapeutic strategies to modify ZnPP so as to prevent abnormal proliferation in cells.