Metabolic activation of nitroarenes and Nrf2-Keap1

硝基芳烃和 Nrf2-Keap1 的代谢激活

• 批准号: 10178027
• 负责人: KARAM E EL-BAYOUMY
• 金额: $ 46.88万
• 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/10178027
• 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/antagonist; 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.

摘要