7,12-Dimethylbenzo[a]anthracene has been studied extensively as a model for carcinogenic and immuno-suppressive polycyclic aromatic hydrocarbons. Most of the biological effects of DMBA require activation by some member of the cytochrome P450 family. One of the most striking effects of DMBA on the immune system is bone marrow toxicity, resulting in severe depletion of progenitor B- lymphocytes. The long-term goal of this project is to identify the mechanisms involved in the bone marrow toxicity of DMBA. In preliminary studies, the investigators found that bone marrow stromal cells have high constitutive levels of cytochrome P450lBl (CYPlBl) that metabolizes DMBA to carcinogenic metabolites in vitro. The investigators also observed that progenitor B- lymphocytes undergo apoptosis when co-cultured with DMBA and bone marrow stromal cells in vitro, but not when incubated with DMBA alone. The central hypothesis is that DMBA metabolism by CYP1B1 in bone marrow stromal cells results in release of metabolites that, directly or indirectly, cause apoptosis in progenitor B- lymphocytes. The rationale for this study is based on: i) the critical importance of stromal cell-B cell interactions in normal B cell development; ii) the high constitutive levels of CYPlBl in stromal cells and its absence in pre B cells; and iii) the ability of DMBA to cause apoptosis in pre-B cells only when co-cultured with bone marrow stromal cells. Death of progenitor B cells might result from: DMBA-DNA adduct formation in pre B cells; loss of an obligatory growth signal from DMBA-treated bone marrow stromal cells; or the release of a toxic moiety for pre B cells by DMBA- treated bone marrow stromal cells. To resolve these possibilities, the investigators will pursue to following three specific aims: 1) Use gene knockout mice in a series of in vivo and in vitro experiments to delineate the roles of CYP1B1, and the AhR, in DMBA-induced pre B cell apoptosis. 2) Distinguish possible mechanisms (i.e., DNA-adduct formation, loss of growth signals, release of Fas ligand or other mediators) that could be responsible for the ability of DMBA-treated bone marrow stromal cells to cause apoptosis in pre-B cells; 3) Identify growth factors and associated regulatory mechanisms that affect CYP1B1 expression in bone marrow stromal cells. At the completion of this project, the investigators expect to have evaluated the role of bone marrow stromal cell CYP1B1, and resulting effector mechanisms, in DMBA-mediated apoptosis in progenitor B cells. These findings could lead to new insights into the mechanisms by which the toxicants cause bone marrow toxicity.
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