Pathways of PAH activation in human lung cells

人肺细胞中 PAH 激活途径

• 批准号: 7478339
• 负责人: Trevor M Penning
• 金额: $ 47.65万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-03 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7478339
• 关键词: 8-Oxo-2' -Deoxyguanosine; A549; Adenocarcinoma Cell; Alleles; Antibodies; Aromatic Hydrocarbons; Aromatic Polycyclic Hydrocarbons; Aryl Hydrocarbon Receptor; Attenuated; Benzo(a)pyrene; Binding; Biological Assay; Biological Monitoring; Breathing; Cancer Patient; Candidate Disease Gene; Carcinogens; Cations; Cell Line; Cell model; Cells; Collaborations; Coupled; Cytochrome P450; DNA; DNA Adducts; DNA Damage; Data; Deoxyguanosine; Environmental Pollutants; Environmental Tobacco Smoke; Enzyme Induction; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Epithelial; Epithelial Cells; Epoxy Compounds; Excision; Fossil Fuels; Genes; Genetic Predisposition to Disease; Genetic Transcription; Glycol; Hand; Human; In Vitro; Institutes; International Agency for Research on Cancer; Label; Laboratories; Link; Liquid Chromatography; Lung; Malignant Epithelial Cell; Malignant neoplasm of lung; Measurement; Measures; Mediating; Metabolic; Metabolic Activation; Metabolism; Methodology; Methods; Modeling; Monitor; Normal Cell; Oxidation-Reduction; Particulate Matter; Pathway interactions; Patients; Pennsylvania; Peroxidase; Peroxidases; Phenotype; Polycyclic Hydrocarbons; Predisposition; Protein Isoforms; Protocols documentation; Quinones; Radioisotope Dilution Technique; Reaction; Receptor Activation; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Route; Small Interfering RNA; Smoke; Standards of Weights and Measures; Sulforaphane; Telomerase; Testing; Tetrachlorodibenzodioxin; Time; Tobacco smoke; Toxic effect; Universities; World Health Organization; adduct; anticancer research; base; benzo(a)pyrene tetrol; benzoquinone; carbonyl reductase (NADPH); cell transformation; exhaust; immortalized cell; in vivo; inhibitor/antagonist; knock-down; oxidative DNA damage; pollutant; programs; repair enzyme; stable isotope; tandem mass spectrometry

DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that have been upgraded to human carcinogens by the WHO, International Agency for Research on Cancer (IARC). PAH are airborne pollutants found in smoke from fossil-fuel combustion, car-exhaust, fine-particulate matter (PM2.5), and first and second hand tobacco smoke. Inhalation toxicity likely results in cancer of the lung and airway. PAH require metabolic activation to exert their deleterious effects, however, the major pathway of PAH activation in the carcinogen target cell, normal human bronchial epithelial cells (NHBE) is uncertain. Three pathways have been proposed: the radical cation pathway (P450-peroxidase dependent) which produces depurinating-DNA adducts; the diol-epoxide pathway (P450-dependent) which produces stable-DMA adducts; and the formation of reactive and redox-active o-quinones (aldo-keto reductase (AKR) dependent), which can give rise to covalent DMA adducts and oxidative damage of DMA. Based on the consistent over-expression of AKR isoforms and the loss of one allele of hOGG1 (human oxoguanine glycosylase the base excision repair enzyme specific for removal 8-oxo-dGuo from DMA) in patients with lung cancer, we hypothesize that AKRs activate PAHs in human bronchial epithelial (HBE) cells and this results in increased oxidative DNA damage. Four aims are proposed to test this hypothesis. In Aim #1, the metabolism of benzo[a]pyrene (BP) will be measured in parental H358 cells (transformed HBE cells) in the presence and absence of (P450 and AKR) induction and the levels of radical cation metabolites (BP-1,6-, 3,6- and 6,12-diones), diol-epoxide metabolites (BP-tetrols) and o-quinone metabolites (BP-7,8-dione) will be quantified and identified by LC- MS. In Aim#2, a stable isotope dilution method using [13C]-BP-metabolites as internal standards to monitor the BP-metabolome will be established. In Aim#3, NHBE cells immortalized with cdk4 and human telomerase will be used to monitor the BP-metabolome by LC-MS stable isotope dilution methodology in the presence and absence of P450 and AKR inducers. In Aim#4, the ability of BP, BP-7,8-dihydrodiol (AKR substrate) and BP-7,8-dione (AKR product) to cause AKR-dependent 8-oxo-dGuo formation will be examined in NHBE cells. These studies will identify major BP-metabolites in NHBE cells which could be used to biomonitor human PAH exposure, and validate candidate genes for genetic predisposition studies.

描述（申请人提供）：多环芳烃（PAH）是环境污染物，已被世界卫生组织、国际癌症研究机构（IARC）升级为人类致癌物。 PAH 是在化石燃料燃烧、汽车尾气、细颗粒物 (PM2.5) 以及一手和二手烟草烟雾产生的烟雾中发现的空气污染物。吸入毒性可能导致肺癌和气道癌。 PAH需要代谢激活才能发挥其有害作用，然而，PAH在致癌物靶细胞——正常人支气管上皮细胞（NHBE）中激活的主要途径尚不确定。已经提出了三种途径： 产生脱嘌呤 DNA 加合物的自由基阳离子途径（P450 过氧化物酶依赖性）；二醇-环氧化物途径（P450依赖性）产生稳定的DMA加合物；反应性和氧化还原活性邻醌的形成（醛酮还原酶 (AKR) 依赖性），可产生共价 DMA 加合物和 DMA 氧化损伤。基于肺癌患者中 AKR 同工型的一致过度表达以及 hOGG1（人氧代鸟嘌呤糖基化酶，一种碱基切除修复酶，专门用于从 DMA 中去除 8-oxo-dGuo）的一个等位基因的缺失，我们假设 AKR 会激活 PAH在人支气管上皮 (HBE) 细胞中，这会导致 DNA 氧化损伤增加。提出了四个目标来检验这一假设。在目标#1中，将在存在和不存在（P450和AKR）诱导的情况下测量亲本H358细胞（转化的HBE细胞）中苯并[a]芘（BP）的代谢以及自由基阳离子代谢物（BP- 1,6-、3,6- 和 6,12-二酮）、二醇环氧化物代谢物（BP-四醇）和邻醌代谢物(BP-7,8-二酮) 将通过 LC-MS 进行定量和鉴定。在目标#2中，将建立一种使用[13C]-BP代谢物作为内标来监测BP代谢组的稳定同位素稀释方法。在 Aim#3 中，在存在和不存在 P450 和 AKR 诱导剂的情况下，用 cdk4 和人端粒酶永生化的 NHBE 细胞将用于通过 LC-MS 稳定同位素稀释方法监测 BP 代谢组。在 Aim#4 中，将在 NHBE 细胞中检查 BP、BP-7,8-二氢二醇（AKR 底物）和 BP-7,8-二酮（AKR 产物）引起 AKR 依赖性 8-oxo-dGuo 形成的能力。这些研究将鉴定 NHBE 细胞中的主要 BP 代谢物，可用于生物监测人类 PAH 暴露，并验证遗传易感性研究的候选基因。