Metabolic activation of nitroarenes and Nrf2-Keap1

摘要 硝基芳烃和NRF2-Keap1的代谢活化 柴油废气中含有独特的硝基芳烃(NO2-PAH)，会导致肺癌的发生。 受到空气污染的影响。只有一部分暴露在空气污染物中的人会死于肺癌，这表明 存在显著的基因-环境交互作用。个体对NO2-PAH的易感性可能是由内在因素驱动的 改变基因功能的非同义单核苷酸多态(NsSNPs) 参与它们的代谢激活，而获得性敏感性可能是由持久性(转录)驱动的 相同基因表达的变化。因此，识别这些基因对 肺癌筛查的风险分层。与NO2-PAH代谢激活有关的基因 是那些表现出硝基还原酶活性的酶，例如NADPH-Quinone氧化还原酶(NQO1)。然而，我们的 初步数据显示，人醛酮还原酶(AKR1C1-AKR1C3)具有硝基还原酶活性 可能参与了NO2-PAH的代谢活化。NQO1是一个典型的Nrf2调控基因，但 AKR1C1基因是Nrf2-Keap1在人类中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性和活性氧物种，可以创造 NO2-PAH加剧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；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 nsSNPs上，对NO2-PAH的获得性敏感性由 过度活跃的Nrf2-Keap1通路。我们的假设将被检验如下：在目标1中，我们将检验AKRs 催化柴油废气中的一组硝基芳烃的硝化还原，例如3-硝基苯甲酮(3-NBA)， 6-硝基菊烯(6-NC)、1-硝基和1，8-二硝基(1，8-DN)。产品分析将由UPLC-ION执行 陷阱质谱仪。在目标2中，我们将测试NRF2激活剂和AKRs是否上调NQO1和AKRs 空气污染物会加剧正常人支气管上皮细胞(HBEC-kt)中NO2-PAH的活性。我们会 通过稳定同位素稀释法确定3-NBA和6-NC衍生的DNA加合物是否增加 经NQO1和AKR1C诱导。在目标3中，我们 将确定：a)AKR1C基因中常见的等位基因变异是否导致硝酸还原酶活性降低 对NO2-PAH的固有敏感性较低；b)对NO2-PAH的获得性敏感性是否取决于 人肺腺癌细胞(A549)中NO2-PAH衍生DNA加合物测定的高活性NRF2 在Keap1启动子高甲基化的情况下，以及在Nrf2被敲除的A549细胞中 CRISPR/Cas9；以及c)对Nrf2激活剂敏感的HBEC-kt细胞产生的NO2- Nrf2基因沉默后PAH衍生的DNA加合物。总而言之，我们的实验结果将 证明对NO2-PAH的获得性敏感性依赖于表观遗传的高水平Nrf2 受监管的。我们研究的影响将首次揭示Nrf2在上下文中激活的“阴暗面” 由环境污染物引发的肺癌。

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.