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

项目总结/摘要 吸烟是肺癌的已知危险因素，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”，作为一种更安全的香烟， 然而，“更安全”并不一定意味着“安全”。 我们对构成ENDS气溶胶的分子结构的理解存在知识差距， ENDS气溶胶的生物后果。这些信息是迫切需要制定一个早期的 了解ENDS对人类健康的潜在不良影响。为了弥补这一知识差距， 提出了以下两个具体目标。具体目标1：研究 ENDS气溶胶在不同的温度下，这将是通过使用我们最近开发的原位（几个 kHz）魔角旋转（MAS）技术，该技术能够产生高分辨率的NMR谱， 含有气体、液体和固体混合物的样品在显著升高的温度下（从0到> 250 ℃）和压力（低于1巴至>100巴）。这种独特的技术是理想的，以确定如何敏感 不同的电子烟油组分作为温度的函数而雾化，特别包括 高毒性化学品，如醛，可通过ENDS溶剂在高温下热解产生， 在不同温度下产生的气溶胶的分子结构。具体 目的2：应用非破坏性慢MAS NMR代谢组学平台， 肺器官型培养物对ENDS气溶胶的反应。慢MAS NMR显著增加光谱 在完整的生物组织和细胞上的分辨率，允许检测更多的代谢物特征， 通过常规NMR仪器解析。慢MAS NMR（40- 100 Hz）的非破坏性能力也是 独特地适用于扩展的活细胞/组织测量，其远优于破坏性方法上级 检查单个时间点。非破坏性检测的特征是特别有利的，因为一些非破坏性检测的特征可以是非破坏性的。 代谢物只存在于活的生物系统中。我们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 (e.g.正常/癌细胞共培养物）。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作为共培养物，以及这些共培养物中的动态变化。 实验组诱导ENDS气溶胶在不同温度下产生。潜在毒性指数 (cell存活力、胁迫响应基因表达）将在相同条件下定义。目标是 确定在不同温度下产生的ENDS是否改变了组分和/或分子结构 的ENDS气溶胶及其对人体细胞系统的毒性潜力，通过发现和利用代谢 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.