Polycyclic aromatic hydrocarbons (PAHs) disrupt hematopoietic development in the bone marrow (BM). This presents human health risk because certain foods and cigarettes contain PAHs. Immunosuppression, leukemias, and lymphomas are potential outcomes. PAH toxicity in mouse BM requires metabolism by cytochrome P450 1B1 (CYP1B1). PAHs elevate CYP1B1 in BM, and related CYPs in liver, by activating the Ah receptor (AhR). CYP1B1 is expressed in both mouse BM and human hematopoietic progenitor cells, which are highly sensitive to PAHs. Human exposures to PAHs readily achieve levels that activate liver AhR. The goal of this study is to determine the mechanism of generation of reactive PAH metabolites in BM, and how this alters hematopoiesis. Experiments will be carried out in vivo and in cultured BM cells using mice with mechanism-based gene deletions. BM toxicity of benzo(a)pyrene (BP) and 7,12-dimethylbenz [a]anthracene (DMBA) depends on tumor necrosis factor (TNF) and on the stress response regulators, p53 and PKR. We hypothesize that reactive metabolites formed by CYP1B1 in the BM synergize with TNF to target hematopoietic cells. PAHs are metabolized to dihydrodiols (PAHDH) and then to highly reactive dihydrodiol epoxides (PAHDE). We will use mice deficient in liver CYP oxidoreductase (OxR), which prevents liver CYP-mediated metabolism to test the role of liver in delivery of PAHDH to blood and then to BM. Intraperitoneal and intragastric administration of DMBA and BP will be compared. We expect to show that CYP1B1 converts PAHDH to PAHDE directly in BM, where myeloperoxidase (MPO) converts PAHs to quinone oxidants. We will use mice with gene deletions of CYP1B1 or MPO to dissect these processes. Activation of the AhR by BP diminishes BM toxicity. We will test whether AhR induction of UDP glucuronosyl transferases enhances removal of PAHDH and quinones. Stromal cells release cytokines that modulate lineage-specific commitment of hematopoietic progenitors. We hypothesize that PAH treatment in vivo affects both stromal and progenitor cells and that TNF activation of PKR plays an essential role in attenuating progenitor progression. We will use ex vivo and in vitro models to test the effects of reactive metabolites on both stromal and progenitor cells. We will separate different BM cell types to localize CYP1B1 and MPO expression in relation to changes in TNF and PKR. Deletions of TNF, TNFR1, and PKR will be used to dissect their contributions to stromal and progenitor activities. We expect to demonstrate selective effects of reactive PAH metabolites on several steps in hematopoiesis, which will identify susceptibility factors for equivalent processes in human BM. ? ? ? ? ? ? ?
