A State-of-Art NMR technique to Investigate Biologicals Effects of Electronic Nicotine Delivery Systems

项目摘要/摘要 吸烟是已知的肺癌风险因素，但在肺癌患者中也很常见。 电子尼古丁输送系统(“Ends”)，即“电子烟”，作为一种更安全的 然而，作为香烟的替代品，“更安全”并不一定意味着“安全”。 在我们对组成末端气溶胶和气溶胶的分子结构的理解上存在着知识鸿沟 END气溶胶的生物后果。这一信息对于发展早期的 了解END对人类健康的潜在不利影响。为了解决这一知识鸿沟， 提出了以下两个具体目标。具体目标1：探讨其形成机制 在不同的温度下结束气溶胶，这将通过使用我们最新开发的现场(几个 KHz)魔角旋转(MAS)技术，能够在 样品含有气体、液体和固体的混合物，温度显著升高(从0到&gt； 250C)和压力(低于1巴至&gt；100巴)。这项独特的技术是确定敏感度的理想方法 不同的E-液体组分作为温度的函数而被雾化，尤其包括 有毒的化学物质，如醛，可由高温下的末端溶剂裂解而产生 并确定在不同温度下产生的气雾剂的分子结构。特定的 目的2：应用非破坏性Slow-MAS核磁共振代谢组学平台确定动力学 肺组织培养物对末梢气溶胶的反应。Slow-MAS核磁共振显著提高光谱 对完整的生物组织和细胞的分辨率，允许检测到比可能的更多的代谢物特征 用常规的核磁共振仪器进行解析。Slow-MAS核磁共振(40-100赫兹)的无损能力也是 唯一适用于远优于破坏性方法的扩展活细胞/组织测量 检查单个时间点。非破坏性检测的特征特别有利，因为一些 代谢物只存在于活的生物系统中。我们已经开发了一个肺器官型培养平台， 使我们能够研究单细胞群体(例如，正常细胞与癌细胞)以及混合细胞群体 (例如，正常/癌细胞共培养)。我们将使用这个系统来定义正常人的基线代谢组 肺上皮细胞、肺癌细胞及其混合物的共培养及其动态变化 实验组端诱导产生不同温度的气溶胶。潜在毒性指数 (细胞活性、应激反应基因表达)将在相同的条件下定义。我们的目标是 确定在不同温度下生产末端是否会改变组成和/或分子结构 通过发现和利用新陈代谢来研究末端气溶胶及其对人体细胞系统的潜在毒性 签名。

PROJECT SUMMARY/ABSTRACT Cigarette smoking is a known risk factor for lung cancer, yet is also common among lung cancer patients. Electronic nicotine delivery systems (“ENDS”), i.e., “E-cigarettes”, are rapidly growing in popularity as a safer alternative to cigarettes to aid in tobacco cessation efforts, however, “safer” does not necessarily mean “safe”. There is a knowledge gap in our understanding of the molecular structures that comprise ENDS aerosols and the biological consequences of ENDS aerosols. This information is critically needed to develop an early understanding of the potential adverse health effects of ENDS in humans. To address this knowledge gap, the following two Specific Aims are proposed. Specific Aim 1: Investigation of the formation mechanism of ENDS aerosols at different temperatures, which will be enabled by using our recently developed in situ (a few kHz) magic angle spinning (MAS) technique that is capable of generating high resolution NMR spectra on samples containing a mixture of gases, liquids, and solids at significantly elevated temperature (from 0 to > 250C) and pressure (below 1 bar to >100 bars). This unique technique is ideal to determine how sensitive different E-liquid components are to aerosolization as a function of the temperature, including in particular the highly toxic chemicals such as aldehydes that can be generated by pyrolysis of ENDS solvents at high temperatures, and to determine the molecular structure of aerosols produced at different temperatures. Specific Aim 2: Application of non-destructive slow-MAS NMR metabolomics platform to define the dynamic response of lung organotypic cultures to ENDS aerosols. Slow-MAS NMR dramatically increases spectral resolution on intact biological tissues and cells, allowing detection of more metabolite features than can be resolved by conventional NMR instruments. The non-destructive capability of slow-MAS NMR (40-100Hz) is also uniquely suited for extended live cell/tissue measurements which is far superior to destructive approaches examining single time points. The feature of non-destructive detection is particularly advantageous as some metabolites only exist in live biological systems. We have developed a lung organotypic culture platform that enables us to investigate single cell populations (e.g. normal vs cancer cells), as well as mixed cell populations (e.g. normal/cancer cell co-cultures). We will use this system to define the baseline metabolome of normal human lung epithelial cells, lung cancer cells and their mixture as cocultures, as well as dynamic changes in these experimental groups induced by ENDS aerosols generated at different temperatures. Indices of toxic potential (cell viability, stress-responsive gene expression) will be defined under identical conditions. The goal is to determine if the production of ENDS at different temperatures alters the constituents and/or molecular structure of ENDS aerosols and their toxic potential on human cell systems by discovering and utilizing metabolic signatures.