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

项目2摘要 烟草烟雾(TS)是导致膀胱癌的主要原因。除了尼古丁，这种高度上瘾的兴奋剂 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 烟叶的烘烤和焚烧。然而，电子烟(E-cig)是为传递电子烟而设计的 (ECS)，在气雾化气相溶剂中只包含尼古丁。电子烟不使用烟叶， 电子烟(ECS)的产生不涉及燃烧。因此，ECS只含有尼古丁和 气相溶剂。电子烟被宣传为提供了一种没有TS负面影响的TS“兴奋”。给定 电子烟使用者迅速增加，电子烟对健康的影响--尤其是其致癌作用--理应受到重视。 仔细检查。众所周知，在烟草烘烤过程中，尼古丁的亚硝化反应会产生烟草- 特定的亚硝胺，包括致癌的N‘-亚硝胺(NNN)和4-(甲基亚硝胺)-1-(3- 2)-1-丁酮(NNK)。研究ECS致癌性的一个重要因素是确定 尼古丁亚硝化是否可以在体内发生。然而，测量NNN和NNK可能很困难 在人类和啮齿动物体内的浓度都有多种细胞色素P450，可以快速转化NNK 和NNN生成甲基重氮氢氧化物(MDOH)、甲醛(FAL)和吡啶丁基衍生物(PBDS)。作为一名 结果吸入性尼古丁亚硝化所产生的NNK和NNN水平在任何时间都很低。 为了绕过这一点，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。使用我们的新方法，应该可以检测到由 NNK和NNN代谢物，因为哺乳动物细胞修复DNA损伤需要几个小时到几天的时间。 我们最近的研究结果表明，尼古丁对人尿路上皮细胞的作用：1)诱变O6- 甲基脱氧鸟苷和环1，2-羟丙基-DG加合物；2)降低DNA修复能力；3) 下调DNA修复基因；以及4)增强Huc的突变和致瘤转化。我们也 发现ECS诱导小鼠膀胱粘膜DNA加合物与PDG和O6-MedG交叉反应 抗体。根据这些结果，我们假设ECS是一种引起膀胱癌的致癌物 尼古丁通过亚硝化生成NNN和NNK而致癌，而代谢物反过来又能损害 DNA，修饰DNA修复蛋白，增强突变易感性和致癌转化。我们会 通过使用小鼠模型来验证这一假说并确定其分子机制：1)ECS 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的分子变化；4)ECS的膀胱癌致癌性。这些 研究将有助于消除未经证实的说法，即ECS是安全的，并将阻止个人吸烟 电子烟。因此，我们建议的研究非常及时，对公众健康具有极其重要的影响。

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.