Metabolic activation of nitroarenes and Nrf2-Keap1

硝基芳烃和 Nrf2-Keap1 的代谢激活

• 批准号: 10394949
• 负责人: KARAM E EL-BAYOUMY
• 金额: $ 46.89万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-08 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394949
• 关键词: 1-Nitropyrene; A549; AKR1C1; Air Pollutants; Air Pollution; Amines; Aromatic Polycyclic Hydrocarbons; Benzo(a)pyrene; Biological Assay; Cancer Etiology; Carcinogens; Cells; Cessation of life; Chemopreventive Agent; Clustered Regularly Interspaced Short Palindromic Repeats; Code; DNA Adduction; DNA Adducts; Data; Diesel Exhaust; Electrons; Environmental Pollutants; Enzymes; Epigenetic Process; Epithelial Cells; Exhibits; Exposure to; Gene Silencing; Genes; Genotype; Human; Hyperactivity; Imidazole; Incidence; Individual; International Agency for Research on Cancer; Liquid Chromatography; Lung; Lung Adenocarcinoma; Malignant Neoplasms; Malignant neoplasm of lung; Mass Fragmentography; Measures; Mediating; Metabolic; Metabolic Activation; Molecular Structure; Mus; Mutagens; NADP; Nitrogen Dioxide; Nitroreductases; Oxides; Oxidoreductase; Pathway interactions; Pharmaceutical Preparations; Play; Predisposition; Process; Property; Proteins; Quinones; Reactive Oxygen Species; Recombinants; Risk; Rodent; Role; Side; Signal Pathway; Single Nucleotide Polymorphism; Sulforaphane; Testing; Time; Tumorigenicity; Up-Regulation; World Health Organization; biological adaptation to stress; bronchial epithelium; cancer initiation; carcinogenesis; cigarette smoke; epigenetic regulation; experimental study; exposure pathway; gene environment interaction; gene function; gene induction; genetic approach; genetic variant; genotoxicity; inhibitor; innovation; knock-down; lung cancer prevention; lung cancer screening; promoter; risk stratification; stable isotope; tandem mass spectrometry; transcriptomics

ABSTRACT METABOLIC ACTIVATION OF NITROARENES AND NRF2-KEAP1 Diesel exhaust contains unique nitroarenes (NO2-PAH) that contribute to the incidence of lung cancer caused by air pollution. Only a subset of individuals exposed to air pollutants succumbs to lung cancer suggesting that a significant gene-environment interaction exists. Individual susceptibility to NO2-PAH may be driven by intrinsic genotypes, non-synonymous single nucleotide polymorphisms (nsSNPs) that change the function of genes involved in their metabolic activation, while acquired susceptibility may be driven by persistent (transcriptomic) changes in the expression of the same genes. Identification of these genes is thus of paramount importance to risk stratify individuals for lung cancer screening. Genes most implicated in the metabolic activation of NO2-PAH are those that exhibit nitroreductase activity e.g. NADPH-Quinone Oxidoreductase (NQO1). However, our preliminary data shows that human Aldo-Keto Reductases (AKR1C1-AKR1C3) exhibit nitroreductase activity and may be involved in the metabolic activation of NO2-PAH. NQO1 is a prototypic Nrf2-regulated gene, but the AKR1C1 genes are among the most upregulated by Nrf2-Keap1 in humans yet the least studied. Induction of these genes by air pollutants, and by the electrophiles and reactive oxygen species they generate, could create a vicious circle whereby NO2-PAH exacerbate their own genotoxicity. Our hypothesis is that AKRs are involved in the metabolic activation of NO2-PAH; that the induction of NQO1 and AKR1C genes by Nrf2 increases NO2-PAH derived DNA adduct formation; that intrinsic susceptibility to NO2-PAH is dependent upon common AKR1C nsSNPs and that acquired susceptibility to NO2-PAH is determined by a hyperactive Nrf2-Keap1 pathway. Our hypothesis will be tested as follows: In Aim 1, we will test whether AKRs catalyze the nitroreduction of a panel of nitroarenes found in diesel exhaust e.g. 3-nitrobenzanthrone (3-NBA), 6-nitrochrysene (6-NC), 1-nitropyrene and 1,8-dinitropyrene. Product profiling will be performed by UPLC-ion trap mass spectrometry. In Aim 2, we will test whether upregulation of NQO1 and AKRs by Nrf2 activators and air pollutants exacerbate NO2-PAH activation in normal human bronchial epithelial (HBEC-kt) cells. We will determine whether increases in 3-NBA and 6-NC derived DNA adducts, measured by stable-isotope dilution liquid chromatography tandem mass spectrometry, are mediated by NQO1 and AKR1C induction. In Aim 3, we will determine: a) whether common allelic variants in AKR1C genes result in reduced nitroreductase activity to lower intrinsic susceptibility to NO2-PAH; b) whether acquired susceptibility to NO2-PAH is dependent upon hyperactive Nrf2 by measuring NO2-PAH derived DNA adducts in human lung adenocarcinoma (A549) cells in which the KEAP1 promoter is hypermethylated, and in A549 cells in which Nrf2 has been knocked-down by CRISPR/ Cas9; and c) whether HBEC-kt cells that are sensitive to Nrf2 activators produce significantly less NO2- PAH derived DNA adducts after Nrf2 gene-silencing. Collectively, the results of our experiments would demonstrate that acquired sensitivity to NO2-PAH is dependent upon high levels of Nrf2 that are epigenetically regulated. The impact of our studies would reveal for the first time a “dark-side” to Nrf2 activation in the context of lung cancer initiation by environmental pollutants.

抽象的 硝酸和NRF2-KEAP1的代谢激活1 柴油排气含有独特的氮气（NO2-PAH），导致肺癌事件导致 通过空气污染。只有暴露于空气污染物的个体屈服于肺癌，这表明 存在重要的基因环境相互作用。个人对No2-Pah的敏感性可能由固有的驱动 基因型，非同义单核苷酸多态性（NSSNP），改变基因的功能 参与其代谢激活，而获得的易感性可能是由持续的（转录组）驱动的 相同基因表达的变化。因此，识别这些基因对 风险将肺癌筛查的个体分层。 NO2-PAH代谢激活最大的基因 是那些表现出硝酸还原酶活性的人，例如NADPH-夸酮氧化还原酶（NQO1）。但是，我们的 初步数据表明，人类Aldo-keto还原酶（AKR1C1-AKR1C3）展示了硝酸还原酶活性 并可能参与NO2-PAH的代谢激活。 NQO1是一种原型NRF2调节的基因，但该基因是 AKR1C1基因是人类中NRF2-KEAP1最新最新的基因之一，但研究最少。诱导 这些基因通过空气污染物以及它们产生的电力和活性氧的基因可能会产生 一个恶性循环，使No2-Pah加剧了自己的遗传毒性。我们的假设是AKR是 参与NO2-PAH的代谢激活； NRF2诱导NQO1和AKR1C基因 增加NO2-PAH衍生的DNA加合物的形成；对NO2-PAH的固有敏感性取决于 在常见的AKR1C NSSNP和对NO2-PAH的易感性的情况下，由A确定 多动NRF2-KEAP1途径。我们的假设将进行如下检验：在AIM 1中，我们将测试AKRS是否 催化在柴油机排气中发现的一组硝化碳的硝化作品，例如3-硝基苯甲酮（3-NBA）， 6-硝基苯乙烯（6-NC），1-硝基吡啶烯和1,8-二硝基吡啶。产品分析将由UPLC-ION进行 陷阱质谱法。在AIM 2中，我们将测试NRF2激活剂对NQO1和AKR的上调以及 空气污染物加剧了正常人支气管上皮（HBEC-KT）细胞中的NO2-PAH激活。我们将 确定通过稳定 - 异位稀释度测量的3-NBA和6-NC衍生的DNA加合物是否增加 液相色谱串联质谱法由NQO1和AKR1C诱导介导。在AIM 3中，我们 将确定：a）AKR1C基因中的常见等位基因变异是否导致硝化还原酶活性降低至 降低对No2-Pah的内在敏感性； b）获得对No2-Pah的易感性是否取决于 通过测量人类肺腺癌（A549）细胞中的NO2-PAH衍生的DNA加合物来激活NRF2 KEAP1启动子是高甲基化的，在A549细胞中，NRF2被击倒了 crispr/ cas9; c）对NRF2激活剂敏感的HBEC-KT细胞是否产生的NO2-显着少得多 NRF2基因分解后PAH衍生的DNA加合物。总体而言，我们的实验结果将 证明获得对NO2-PAH的敏感性取决于表观遗传上的高水平的NRF2 受监管。我们的研究的影响将首次揭示“深色侧”对NRF2在上下文中的激活 环境污染物的肺癌倡议。