Identification of Free Radical Induced Biomarkers of Exposure to Electronic Cigarette Aerosol

项目摘要/摘要：电子烟(e-cig)的使用率正在上升，特别是在年轻人中； 然而，它们的潜在危害尚不清楚，使知情监管战略的制定复杂化。 缺乏有关电子烟相关危害的数据，部分原因是缺乏暴露于电子烟的特定生物标志物。 气雾剂。我们发现，e-cig气雾剂含有高度活性的自由基，可导致氧化损伤 用户。自由基可以破坏许多细胞通路，可能导致血管紧张素转换酶 癌症和其他疾病。电子CIG自由基的检测可以通过自旋陷阱捕获来完成 (例如，DMPO)并通过电子顺磁共振(EPR)光谱分析。初步研究与 EPR显示，e-cig气雾剂中的自由基是由e-液体溶剂、丙二醇(PG)和 甘油(Glycerin，Gly)是所有电子烟的共同成分，具有独特的结构特征。我们的目标是确定这些 自由基结构，并利用其独特的结构来开发暴露的e-CIG特异性生物标记物。这个 拟议研究的具体目标是：(目标1)确定产生的自由基的结构 在电子烟中测定PG和GLY；(目标2)确定自由基形成的主要靶点和加合物 电子烟暴露对啮齿动物模型组织的攻击以及这些自由基可能形成的代谢物 电子烟暴露鼠血清中的加合物。该项目代表了一个重要的研究方向，其中 化学/生物方法可以为烟草管理科学提供信息。因此，这其中的一个重要方面 应用它来扩展我在翻译科学相关领域的背景，除了提供具体的 培训与生物标记物相关的新研究领域，包括代谢组学和自由基结构 身份证明。为此，我将通过一系列课程进行培训，相关辅导和实践 经验，每一次都能确保我过渡到生物标记物领域的独立研究人员 发展与监管科学。课程、指导、会议参与和实践 培训/体验将在K99阶段完成。在这一阶段，研究计划的目标1将 完成后，将启动目标2(在颁奖的R00阶段完成)。要完成以下任务 研究目的、先进的脉冲EPR和质谱学方法将用于产生的自由基 由PG和GLY在e-cig气雾剂中。在小鼠暴露模型中，自由基暴露和攻击目标 将使用一种新的体内DMPO/抗DMPO抗体方法来确定肺。这将允许 飞行时间-质谱法鉴定特定蛋白质和DNA加合物DMPO后的暴露研究将包括 用非靶向成对代谢组学方法观察e-cig前后代谢物的变化 曝光。通过利用e-cig的独特结构产生的自由基及其在 肺，暴露于e-cig气雾剂的生物标志物可以被识别并用于制定监管 旨在减少电子烟暴露危害的战略。

Project Summary/Abstract: Electronic cigarette (e-cig) usage is on the rise, particularly among youths; however, their potential for harm is not understood, complicating development of informed regulatory strategies. The lack of data on e-cig related harm is, in part, due to the lack of specific biomarkers for exposure to e-cig aerosols. We found that e-cig aerosol contains highly reactive free radicals that can cause oxidative damage to the user. Free radicals can damage numerous cellular pathways possibly contributing to the progression of cancers and other diseases. Detection of e-cig free radicals can be accomplished by trapping with spin traps (e.g., DMPO) and analysis by electron paramagnetic resonance (EPR) spectroscopy. Preliminary research with EPR shows that free radicals produced in the e-cig aerosol by e-liquid solvents, propylene glycol (PG) and glycerin (GLY), common to all e-cigs, display unique structural characteristics. Our objective is to identify these free radical structures and utilize their unique structure to develop an e-cig specific biomarker of exposure. The specific aims of the proposed research are: (Aim 1) To determine the structures of the free radicals produced by PG and GLY in e-cigs; (Aim 2) To determine the primary targets of and adducts formed from free radical assault in the tissue of e-cig exposures in rodent models and possible metabolites formed from the these radical adducts in the serum of e-cig exposed rodent. This project represents an important research direction where a chemical/biological approach can inform tobacco regulatory science. As such, an important aspect of this application it to extend my background in areas relevant to translational science in addition to providing specific training in new biomarker-relevant research areas including metabolomics and free radical structural identification. To this end, my training will occur through a series of courses, relevant mentorship, and practical experience, each geared to ensure my transition to an independent researcher in the fields of biomarker development and regulatory science. Coursework, mentorship, conference participation, and practical training/experience will be completed during the K99 phase. During this phase, Aim 1 of the research plan will be completed and Aim 2 will be initiated (for completion during the R00 phase of the award). To accomplish the research aims, advanced pulsed EPR and mass spectroscopy approaches will be utilized for radicals produced by PG and GLY in e-cig aerosols. In a mouse exposure model, free radical exposure and targets of attack in the lung will be determined using a novel in vivo DMPO/anti-DMPO antibody approach. This will allow for the identification of specific protein and DNA adducts by TOF-MS. A post-DMPO exposure study will consist of an untargeted pairwise metabolomics approach to look at changes in metabolite profiles before and after e-cig exposure. By leveraging the unique structures of e-cig produced free radicals and their targets of attack in the lung, biomarkers of exposure specific to e-cig aerosols can be identified and used to develop regulatory strategies aimed at reducing harm from e-cig exposure.