Pathways of PAH activation in human lung cells

描述（由申请人提供）：多环芳烃（PAHs）是被世界卫生组织、国际癌症研究机构（IARC）升级为人类致癌物的环境污染物。多环芳烃是一种空气污染物，存在于化石燃料燃烧产生的烟雾、汽车尾气、细颗粒物（PM2.5）以及一手和二手烟草烟雾中。吸入毒性可能导致肺癌和气道癌。多环芳烃需要代谢激活才能发挥其有害作用，然而，多环芳烃在致癌物靶细胞——正常人支气管上皮细胞（NHBE）中激活的主要途径尚不确定。目前提出了三种途径：自由基阳离子途径（p450 -过氧化物酶依赖）产生脱氧dna加合物；二醇环氧化物途径（p450依赖），产生稳定的dma加合物；以及反应性和氧化还原活性的邻醌（依赖于醛酮还原酶（AKR））的形成，这可以引起共价DMA加合物和DMA的氧化损伤。基于AKR亚型的一致过表达和肺癌患者hOGG1（人氧鸟嘌呤糖基化酶，从DMA中去除8-氧- dguo的碱基切除修复酶）的一个等位基因缺失，我们假设AKR激活人支气管上皮（HBE）细胞中的PAHs，从而导致DNA氧化损伤增加。为了验证这一假设，提出了四个目标。在Aim# 1中，在存在和不存在（P450和AKR）诱导的情况下，将在亲代H358细胞（转化的HBE细胞）中测量苯并[a]芘（BP）的代谢，并通过LC- ms定量和鉴定自由基正离子代谢物（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）的水平。建立以[13C]- bp代谢物为内标物监测bp代谢组的稳定同位素稀释方法。在Aim#3中，用cdk4和人类端粒酶永生化的NHBE细胞将被用于在P450和AKR诱导剂存在和不存在的情况下，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细胞中检测BP， BP-7,8-二氢二醇（AKR底物）和BP-7,8-二酮（AKR产物）导致AKR依赖性8-氧- dguo形成的能力。这些研究将确定NHBE细胞中的主要bp代谢物，这些代谢物可用于生物监测人类多环芳烃暴露，并验证遗传易感性研究的候选基因。

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.