Liver Vs Bone PAH Metabolism: Synergy with TNF in Bone

描述(申请人提供)：多环芳烃(PAHs)扰乱骨髓(BM)的造血发育。这会给人类健康带来风险，因为某些食品和香烟含有多环芳烃。免疫抑制、白血病和淋巴瘤是潜在的结果。多环芳烃在小鼠骨髓中的毒性需要细胞色素P450 1b1(CyP1B1)的代谢。多环芳烃通过激活AH受体(AhR)，使BM中的细胞色素P1B1水平升高，肝脏中与之相关的细胞色素P450水平升高。在对多环芳烃高度敏感的小鼠骨髓和人类造血祖细胞中都表达了细胞色素P1B1。人类暴露于多环芳烃很容易达到激活肝脏AhR的水平。这项研究的目的是确定骨髓中反应性多环芳烃代谢产物的产生机制，以及这是如何改变造血的。实验将在体内和培养的BM细胞中进行，使用基于机制的基因缺失的小鼠。苯并(A)芘(BP)和7，12-二甲基苯并[a]菲(DMBA)的BM毒性依赖于肿瘤坏死因子(TNF)和应激反应调节因子P53和PKR。我们假设在骨髓中由细胞色素P1B1形成的反应性代谢物与肿瘤坏死因子协同作用，以靶向造血细胞。多环芳烃被代谢成二氢二醇(PAHDH)，然后生成高活性的二氢二醇环氧化物(PAHDE)。我们将使用肝脏CYP氧化还原酶(OxR)缺陷的小鼠来测试肝脏在将PAHDH输送到血液中然后运送到骨髓中的作用。将比较DMBA和BP的腹膜内和胃内给药情况。我们希望证明CYP1B1在BM中直接将PAHDH转化为PAHDE，而髓过氧化物酶(MPO)将PAHs转化为苯醌氧化剂。我们将使用带有CYP1B1或MPO基因缺失的小鼠来剖析这些过程。BP激活AhR可降低BM的毒性。我们将测试AhR对UDP葡萄糖醛酸基转移酶的诱导是否增强了PAHDH和苯二酚的去除。基质细胞释放细胞因子，调节造血祖细胞的谱系特异性承诺。我们假设，体内PAH治疗同时影响基质细胞和祖细胞，并且PKR的肿瘤坏死因子激活在抑制祖细胞进展中起重要作用。我们将使用体外和体外模型来测试反应性代谢物对基质细胞和祖细胞的影响。我们将分离不同的骨髓细胞类型，以定位与肿瘤坏死因子和蛋白激酶受体变化相关的CYP1B1和MPO的表达。肿瘤坏死因子、肿瘤坏死因子受体和蛋白激酶受体的缺失将被用来分析它们对基质和祖细胞活动的贡献。我们希望证明反应性多环芳烃代谢产物在造血过程中的几个步骤上的选择性作用，这将确定人类骨髓中同等过程的易感因素。

DESCRIPTION (provided by applicant): 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.