Pathways of PAH activation in human lung cells

描述（由申请人提供）：多环芳烃（PAH）是环境污染物，已被WHO、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）以及第一和第二手烟草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需要代谢活化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）中活化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）;产生稳定-DMA加合物的二醇-环氧化物途径（P450-依赖性）;以及反应性和氧化还原活性的邻醌的形成（醛酮还原酶（AKR）依赖性），其可以引起共价DMA加合物和DMA的氧化损伤。基于肺癌患者中AKR亚型的一致过表达和hOGG 1（人氧鸟嘌呤糖基化酶，特异性从DMA中去除8-oxo-dGuo的碱基切除修复酶）的一个等位基因的缺失，我们假设AKR激活人支气管上皮（HBE）细胞中的多环芳烃，这导致氧化DNA损伤增加。提出了四个目标来检验这一假设。在目标#1中，将在亲本H358细胞中测量苯并[a]芘（BP）的代谢（转化的HBE细胞）在存在和不存在（P450和AKR）诱导和自由基阳离子代谢产物水平（BP-1，6-、3，6-和6，12-二酮），二醇-环氧化物代谢物在目标#2中，将建立使用[13 C]-BP-代谢物作为内标物以监测BP-代谢物组的稳定同位素稀释方法。在目标#3中，在存在和不存在P450和AKR诱导剂的情况下，将用cdk 4和人端粒酶永生化的NHBE细胞用于通过LC-MS稳定同位素稀释方法监测BP-代谢物组。在目标#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-氧代-dGuo形成的能力。这些研究将识别NHBE细胞中的主要BP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.