Cigarette smoke but not novel tobacco vapor product cause epigenetic disruption and cell apoptosis.

Novel tobacco vapor product, generating vapor without combusting tobacco leaves, has been developed expecting the number and quantity of chemicals in the vapor of these products to be reduced compared to conventional combustible cigarettes. However, if the lower chemical levels correlate with lower toxicity remained to be clarified. Here we examined the difference of conventional cigarette smoke (CS) and novel tobacco vapor product (NTV) using cultured cancer cell line A549 and normal bronchial epithelium cell line BEAS-2B. 0.5% of 3R4F which is conventional CS markedly decreased cell proliferation of both A549 and BEAS-2B, however Ploom TECH or Ploom TECH+ which are commercially available NTV did not affect cell growth. To clarify the cause of decreased cell proliferation, Tunnel assay was performed and clarified that apoptosis was observed in both A549 and BEAS-2B after 24hours after exposure to 3R4F. To further explore the effect of CS to epigenetics, we performed western blotting Histone H2A phosphorylation which is known to correlate transcriptional regulation. Only 3R4F decreased histone H2A phosphorylation of both A549 and BEAS-2B. Then we examined alterations of gene expression after 3R4F treatment of A549 cell. 339, 107, 103 genes which were upregulated more than 2fold were observed in 3R4F, Ploom TECH or Ploom TECH+ treated A549 cell, respectively. Among 339 genes which was upregulated 3R4F, we focused EGR1, FOS, and FOSB gene since they were upregulated more than100 fold. We confirmed this upregulation using RTqPCR. These data suggest that Cigarette smoke but not novel tobacco vapor product cause epigenetic disruption and cell apoptosis possibly by elevating genes such as EGR1. Overall design: A549 cell mRNA profile of adding DMSO control, cigarette smoke (3R4F) and novel tobacco vapor product (Ploom TECH, and Ploom TECH plus) were generated by deep sequencing in duplicate, using Illumina MiSeq.

无需燃烧烟叶即可产生蒸汽的新型烟草蒸汽产品（novel tobacco vapor product, NTV）已被开发，其研发初衷为相较传统可燃卷烟，该类产品蒸汽中的化学物质种类与含量均能得到降低。然而，更低的化学物质水平是否对应更低的毒性，仍有待阐明。

本研究利用体外培养的肺癌细胞系A549与正常支气管上皮细胞系BEAS-2B，对比分析了传统卷烟烟气（conventional cigarette smoke, CS）与新型烟草蒸汽产品的差异。0.5%体积比的标准参比卷烟烟气3R4F可显著抑制A549与BEAS-2B两种细胞的增殖，而市售新型烟草蒸汽产品Ploom TECH及Ploom TECH+则对细胞生长无明显影响。为阐明细胞增殖受抑的机制，本研究开展了TUNEL测定法（Tunnel assay），结果显示，经3R4F处理24小时后，A549与BEAS-2B细胞均出现了细胞凋亡。

为进一步探究卷烟烟气对表观遗传学的影响，本研究针对已知与转录调控相关的组蛋白H2A磷酸化水平，开展了蛋白质印迹（western blotting）实验。仅3R4F处理可降低A549与BEAS-2B细胞的组蛋白H2A磷酸化水平。随后，本研究分析了3R4F处理A549细胞后的基因表达变化。经3R4F、Ploom TECH及Ploom TECH+处理的A549细胞中，分别有339、107、103个基因的表达上调幅度超过2倍。在3R4F处理后上调的339个基因中，本研究重点关注了EGR1、FOS及FOSB基因，因其上调幅度超过100倍。本研究通过实时定量聚合酶链式反应（RT-qPCR）验证了上述基因的表达上调现象。

上述实验结果表明，卷烟烟气而非新型烟草蒸汽产品，或可通过上调EGR1等基因的表达，引发表观遗传紊乱与细胞凋亡。

实验整体设计：以二甲基亚砜（Dimethyl sulfoxide, DMSO）为溶剂对照，分别采用标准参比卷烟烟气3R4F与新型烟草蒸汽产品（Ploom TECH、Ploom TECH+）处理A549细胞，通过Illumina MiSeq平台开展双重复深度测序，以获取细胞的mRNA表达谱。