Project 2: E-cigarette Smoke Induced Bladder Carcinogenesis and Invasive Cancer Development

项目2摘要 烟草烟雾（TS）是膀胱癌的主要原因。除了尼古丁，高度上瘾的兴奋剂 在为吸烟者提供即时满足感的TS中，TS含有许多在吸烟过程中产生的致癌物质。 烟叶的烘烤和燃烧。然而，电子烟（E-cigs）的设计是为了提供电子烟烟雾 (ECS)其仅包含雾化气相溶剂中的尼古丁。电子烟不使用烟叶， 并且电子烟（ECS）的产生不涉及燃烧。因此，ECS仅含有尼古丁， 气相溶剂电子烟被宣传为提供TS“高”，而没有TS的负面影响。鉴于 随着电子烟使用者数量的迅速增加，ECS对健康的影响-特别是其致癌性-值得关注。 仔细检查。众所周知，在烟草烘烤过程中，尼古丁的亚硝化作用会产生烟草- 特定的亚硝胺，包括致癌的N ′-亚硝基去甲烟碱（NNN）和4-（甲基亚硝胺）-1-（3- 吡啶基）-1-丁酮（NNK）。研究ECS致癌性的一个重要因素是确定 尼古丁是否会在体内发生亚硝化。然而，很难测量NNN和NNK 在人类和啮齿类动物中的浓度，因为两者都有多种细胞色素p450， 和NNN转化为甲基重氮基氢氧化物（MDOH）、甲醛（FAL）和吡啶基-丁基-衍生物（PBD）。作为 结果，由吸入尼古丁的亚硝化产生的NNK和NNN水平在任何给定时间都非常低。 为了避免这一点，我们确定尼古丁诱导的DNA损伤，而不是直接测量尼古丁 代谢物NNK和NNN。通过我们的新方法，应该可以检测由DNA损伤引起的DNA损伤。 NNK和NNN代谢物，因为哺乳动物细胞修复DNA损伤需要数小时至数天。 我们最近的研究结果表明，在人尿路上皮细胞（HUCs）中，尼古丁处理：1）诱导致突变性O 6- 甲基-脱氧鸟苷和环状1，N2-γ-羟基-丙-dG加合物; 2）降低DNA修复能力; 3） 下调DNA修复基因;和4）增强HUC的突变和致瘤转化。我们也 发现ECS在小鼠膀胱粘膜中诱导与PdG和O 6-medG交叉反应的DNA加合物 抗体的基于这些结果，我们假设ECS是一种膀胱致癌物， 致癌作用通过尼古丁亚硝化成NNN和NNK，代谢产物可以反过来损害 DNA，修饰DNA修复蛋白，并增强突变易感性和致瘤性转化。我们将 通过使用小鼠模型来测试这一假设并确定分子机制，以确定：1）ECS的 对小鼠膀胱粘膜DNA修复的影响; 2）ECS诱导的DNA加合物的化学性质; 3） ECS诱导的小鼠膀胱组织中的分子改变;和4）ECS膀胱致癌性。这些 研究将有助于消除未经证实的声称，即ECS是安全的，并将阻止个人吸烟 电子烟因此，我们建议进行的研究非常切合时宜，对公众健康亦有极为重要的影响。

Project 2 Summary Tobacco smoke (TS) is a major cause of bladder cancer. In addition to nicotine, the highly addictive stimulant in TS that provides smokers with instant gratification, TS contains many carcinogens generated during the curing and burning of tobacco leaves. However, E-cigarettes (E-cigs) were designed to deliver E-cig smoke (ECS), which comprises only nicotine in aerosolized gas phase solvents. E-cigs do not use tobacco leaves, and the generation of E-cig smoke (ECS) does not involve burning. Hence, ECS contains only nicotine and the gas phase solvents. E-cigs are promoted as delivering a TS `high' without the negative effects of TS. Given the rapid rise in the number of E-cig users, the health effects of ECS—particularly its carcinogenicity—deserves careful scrutiny. It is well established that during tobacco curing, the nitrosation of nicotine generates tobacco- specific nitrosamines including carcinogenic N'-nitrosonornicotine (NNN) and 4-(methylnitrosamine)-1-(3- pyridyl)-1-butanone (NNK). An important factor when investigating the carcinogenicity of ECS is determining whether nicotine nitrosation can occur in vivo. However, it can be difficult to measure NNN and NNK concentrations in humans and rodents as both have multiple cytochrome p450s that quickly transform NNK and NNN into methyldiazohydroxide (MDOH), formaldehyde (FAL), and pyridyl-butyl-derivatives (PBDs). As a result, the levels of NNK and NNN resulting from nitrosation of inhaled nicotine are very low at any given time. To circumvent this, we determine nicotine-induced DNA damage, rather than directly measuring the nicotine metabolites NNK and NNN. With our novel approach, it should be possible to detect DNA damage induced by the NNK and NNN metabolites, given that it takes hours to days for mammalian cells to repair DNA damage. Our recent results show that in human urothelial cells (HUCs), nicotine treatment: 1) induces mutagenic O6- methyl-deoxyguanosine and cyclic 1,N2-gamma-hydroxy-propano-dG adducts; 2) reduces DNA repair capacity; 3) downregulates DNA repair genes; and 4) enhances HUC's mutation and tumorigenic transformation. We also found that ECS induces DNA adducts in mouse bladder mucosa which cross react with PdG and O6-medG antibodies. Based on these results, we hypothesize that ECS is a bladder carcinogen that causes carcinogenesis through nitrosation of nicotine into NNN and NNK, and the metabolites can, in turn, damage DNA, modify DNA repair proteins, and enhance mutation susceptibility and tumorigenic transformation. We will test this hypothesis and determine the molecular mechanisms by using mouse models to determine: 1) ECS's effect on DNA repair in mouse bladder mucosa; 2) the chemical nature of ECS-induced DNA adducts; 3) the ECS-induced molecular alterations in mouse bladder tissues; and 4) the ECS bladder carcinogenicity. These studies will help dispel the unsubstantiated claim that ECS is safe and will discourage individuals from smoking E-cigs. Our proposed studies therefore are very timely and have extremely important public health implications.