Metabolic activation of nitroarenes and Nrf2-Keap1

硝基芳烃和 Nrf2-Keap1 的代谢激活

基本信息

批准号：
10617646
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
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-08 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10617646
关键词：
1-Nitropyrene A549 AKR1C1 Air Pollutants Air Pollution Ames Assay Amines Aromatic Polycyclic Hydrocarbons Benzo(a)pyrene Biological Assay Cancer Etiology Carcinogens Cells Cessation of life Chemopreventive Agent Classification Clustered Regularly Interspaced Short Palindromic Repeats Code DNA Adduction DNA Adducts Darkness Data Diesel Exhaust Electrons Environmental Pollutants Enzymes Epigenetic Process Epithelial Cells Exhibits Exposure to Gene Silencing Genes Genotype Human Hyperactivity Hypermethylation Imidazole Incidence Individual International Agency for Research on Cancer Intrinsic drive Liquid Chromatography Lung Lung Adenocarcinoma Malignant Neoplasms Malignant neoplasm of lung Mass Fragmentography Measures Mediating Metabolic Metabolic Activation Molecular Structure Mus Mutagens NADP Nitroreductases Oxidoreductase Pathway interactions Pharmaceutical Preparations Physical condensation Play Predisposition Prevention strategy Process Property Proteins Quinones Reactive Oxygen Species Recombinants Risk Rodent Role Side Signal Pathway Single Nucleotide Polymorphism Sudden infant death syndrome Sulforaphane Terpenes Testing Therapeutic 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 prototype risk stratification stable isotope tandem mass spectrometry transcriptomics

项目摘要

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.

抽象的硝基芳烃和 NRF2-KEAP1 的代谢激活柴油机尾气中含有独特的硝基芳烃 (NO2-PAH)，会导致肺癌的发生空气污染造成的。只有一部分暴露于空气污染物的人会死于肺癌，这表明存在显着的基因-环境相互作用，个体对 NO2-PAH 的易感性可能是由内在驱动的。基因型、改变基因功能的非同义单核苷酸多态性 (nsSNP) 参与其代谢激活，而获得性易感性可能是由持久性（转录组）驱动的因此，识别这些基因对于相同基因的表达至关重要。对个体进行肺癌筛查的风险分层。与 NO2-PAH 代谢激活最相关的基因。是那些表现出硝基还原酶活性的酶，例如 NADPH-醌氧化还原酶 (NQO1)。初步数据显示人类醛酮还原酶 (AKR1C1-AKR1C3) 具有硝基还原酶活性 NQO1 可能参与 NO2-PAH 的代谢激活，是一个原型 Nrf2 调节基因，但 AKR1C1 基因是人类中 Nrf2-Keap1 上调程度最高的基因之一，但对诱导作用的研究却最少。这些基因通过空气污染物以及它们产生的亲电子试剂和活性氧，可以产生 NO2-PAH 会加剧其自身的遗传毒性，形成恶性循环。我们的假设是 AKR 是一种恶性循环。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 加合物形成；对 NO2-PAH 的内在敏感性是依赖的基于常见的 AKR1C nsSNP，并且对 NO2-PAH 的获得性敏感性由我们的假设将被检验如下：在目标 1 中，我们将检验 AKR 是否活跃。催化柴油机尾气中的一组硝基芳烃的硝基还原，例如 3-硝基苯并酮 (3-NBA)， 6-硝基 (6-NC)、1-硝基芘和 1,8-二硝基芘将通过 UPLC-ion 进行产品分析。在目标 2 中，我们将测试 Nrf2 激活剂是否上调 NQO1 和 AKR。空气污染物会加剧正常人支气管上皮 (HBEC-kt) 细胞中的 NO2-PAH 活化。确定 3-NBA 和 6-NC 衍生的 DNA 加合物是否增加，通过稳定同位素稀释测量在目标 3 中，我们通过 NQO1 和 AKR1C 诱导介导了液相色谱串联质谱法。将确定：a) AKR1C 基因中的常见等位基因变异是否会导致硝基还原酶活性降低对 NO2-PAH 的内在敏感性较低；b) 对 NO2-PAH 的获得性敏感性是否取决于通过测量人肺腺癌细胞 (A549) 中 NO2-PAH 衍生的 DNA 加合物来检测过度活跃的 Nrf2 其中 KEAP1 启动子被高甲基化，并且在 A549 细胞中，Nrf2 已被敲低 CRISPR/ Cas9；以及 c) 对 Nrf2 激活剂敏感的 HBEC-kt 细胞是否产生显着较少的 NO2- Nrf2 基因沉默后的 PAH 衍生 DNA 加合物，我们的实验结果将是这样的。证明对 NO2-PAH 的获得性敏感性取决于表观遗传上高水平的 Nrf2 我们的研究的影响将首次揭示 Nrf2 激活的“黑暗面”。环境污染物诱发肺癌。