Metabolic activation of nitroarenes and Nrf2-Keap1

硝基芳烃和 Nrf2-Keap1 的代谢激活

• 批准号: 9927641
• 负责人: KARAM E EL-BAYOUMY
• 金额: $ 46.8万
• 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/9927641
• 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; Hyperactive behavior; 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; 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/antagonist; innovation; knock-down; lung cancer prevention; lung cancer screening; promoter; 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.

摘要 硝基芳烃和NRF 2-KEAP 1的代谢活化 柴油机废气中含有独特的硝基芳烃（NO2-PAH），可导致肺癌的发生 空气污染。只有一部分暴露于空气污染物的人会患肺癌，这表明， 存在显著的基因-环境相互作用。个体对NO2-PAH的易感性可能是由内在的 基因型，改变基因功能的非同义单核苷酸多态性（nsSNP） 参与其代谢活化，而获得性易感性可能由持续（转录组）驱动 相同基因表达的变化。因此，识别这些基因对于 对个人进行肺癌筛查的风险分层。与NO2-PAH代谢活化最相关的基因 是表现出硝基还原酶活性的那些，例如NADPH-醌氧化还原酶（NQO 1）。但我们的 初步数据显示人醛酮还原酶（AKR 1C 1-AKR 1C 3）表现出硝基还原酶活性 可能参与NO2-PAH的代谢活化。NQO 1是一个原型的Nrf 2调控基因，但NQO 1是一个典型的Nrf 2调控基因。 AKR 1C 1基因是人类中Nrf 2-Keap 1上调最多的基因之一，但研究最少。诱导 这些基因通过空气污染物，以及它们产生的亲电体和活性氧， NO2-PAH加剧其自身遗传毒性的恶性循环。我们的假设是AKR是 参与NO2-PAH的代谢活化; Nrf 2诱导NQO 1和AKR 1C基因 增加NO2-PAH衍生的DNA加合物的形成;对NO2-PAH的固有易感性依赖于 在常见的AKR 1C nsSNP上，获得性对NO2-PAH的易感性是由 过度活跃的Nrf 2-Keap 1通路。我们的假设将被测试如下：在目标1中，我们将测试AKR是否 催化柴油机废气中发现的一组硝基芳烃如3-硝基苯蒽酮（3-NBA）的硝基还原， 6-硝基芘（6-NC）、1-硝基芘和1，8-二硝基芘。将通过UPLC-离子色谱法进行产品分析 阱质谱法在目标2中，我们将测试Nrf 2激活剂和NQO 1/AKR的表达是否上调。 空气污染物加剧了正常人支气管上皮细胞（HBEC-kt）中NO2-PAH的活化。我们将 确定通过稳定同位素稀释测量的3-NBA和6-NC衍生的DNA加合物的增加是否 液相色谱串联质谱法，介导的NQO 1和AKR 1C诱导。在目标3中，我们 a）AKR 1C基因中常见的等位基因变体是否导致硝基还原酶活性降低， 对NO2-PAH的固有易感性较低; B）对NO2-PAH的获得性易感性是否依赖于 通过测量人肺腺癌（A549）细胞中NO2-PAH衍生的DNA加合物， 其中KEAP 1启动子被高甲基化，以及在A549细胞中，Nrf 2已被 c）对Nrf 2激活剂敏感的HBEC-kt细胞是否产生显著更少的NO2-。 Nrf 2基因沉默后PAH衍生的DNA加合物。总的来说，我们的实验结果 表明获得性对NO2-PAH的敏感性依赖于高水平的Nrf 2， 监管.我们研究的影响将首次揭示Nrf 2激活的“黑暗面”， 环境污染物引发肺癌的可能性。