Liver Vs Bone PAH Metabolism: Synergy with TNF in Bone

描述（由申请人提供）：多环芳烃（PAH）破坏骨髓（BM）中的造血发育。这对人类健康构成威胁，因为某些食物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。小鼠BM中的PAH毒性需要通过细胞色素P450 1B 1（CYP 1B 1）代谢。多环芳烃通过激活Ah受体（AhR）升高BM中的CYP 1B 1和肝脏中的相关CYP。CYP 1B 1在小鼠骨髓和人类造血祖细胞中表达，这对多环芳烃高度敏感。人类暴露于多环芳烃很容易达到激活肝脏AhR的水平。本研究的目的是确定BM中反应性PAH代谢产物的产生机制，以及这如何改变造血。将使用具有基于机制的基因缺失的小鼠在体内和培养的BM细胞中进行实验。苯并（a）芘（BP）和7，12-二甲基苯并[a]蒽（DMBA）的骨髓毒性取决于肿瘤坏死因子（TNF）和应激反应调节因子p53和PKR。我们推测，骨髓中CYP 1B 1形成的反应性代谢产物与TNF协同作用靶向造血细胞。多环芳烃被代谢成二氢二醇（PAHDH），然后代谢成高活性的二氢二醇环氧化物（PAHDE）。我们将使用肝脏β-氧化还原酶（OxR）缺陷的小鼠来测试肝脏在将PAHDH输送到血液然后输送到BM中的作用，所述肝脏β-氧化还原酶（OxR）阻止肝脏CYP介导的代谢。将比较DMBA和BP的腹膜内和胃内给药。我们希望表明，CYP 1B 1转换PAHDH PAHDE直接在BM中，髓过氧化物酶（MPO）转换多环芳烃醌氧化剂。我们将使用CYP 1B 1或MPO基因缺失的小鼠来剖析这些过程。BP激活AhR可降低BM毒性。我们将测试UDP葡萄糖醛酸转移酶的AhR诱导是否增强PAHDH和醌类的去除。基质细胞释放调节造血祖细胞谱系特异性定型的细胞因子。我们假设PAH治疗在体内同时影响基质细胞和祖细胞，并且TNF激活PKR在减弱祖细胞进展中起重要作用。我们将使用离体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BM细胞类型，以定位与TNF和PKR变化相关的CYP 1B 1和MPO表达。TNF、TNFR 1和PKR的缺失将用于剖析它们对基质和祖细胞活性的贡献。我们预计将证明反应性多环芳烃代谢物对造血过程中的几个步骤的选择性影响，这将确定人类骨髓中等效过程的易感因素。

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.