*Square brackets [--] indicate parts of the Research Training Plan that have been changed* Electronic cigarettes (EC) are nicotine delivery devices, often advertised for smoking cessation. Recently released into the consumer marketplace, EC devices are quickly becoming popular worldwide. EC parts include: a mouthpiece housing a cartridge of nicotine-containing fluid, an atomizer used for heating, and a battery. The cartridge fluids vary, typically containing some concentration of nicotine, a humectant, such as propylene glycol, and various flavorings. One visible trend in the EC community is to refill the nicotine solution by dripping refill fluid into an empty cartridge. In spite of EC growing popularity, few scientific studies have been conducted to assess possible health effects of EC aerosol on users or bystanders, and it remains unclear if EC pose a health concern. Flavorings used in EC are often food additives and perfumes, which are not intended to be heated and inhaled and which have unknown consequences on human health. My long-term objective is to characterize the effects of EC use on human health. This proposal specifically focuses on the cytotoxicity of EC refill fluid aerosol. I will first (Aim #1) use human embryonic stem cells (hESC) and human pulmonary fibroblasts (hPF) to evaluate the cytotoxicity of EC refill fluid aerosols from different manufacturers. Screening for cytotoxicity will be done using the MTT cell viability assay and a metabolizing assay. This first step will identify EC products that may produce adverse health effects in humans. Secondly (Aim #2), I will use gas chromatography and mass spectrometry to identify toxic chemicals in EC aerosols. Each chemical identified will then be purchased and analyzed using the MTT assay with hESC and hPF. Identification of toxicants in these products would allow manufacturers to remove them and make users aware of potentially dangerous products. I will then create a hierarchy of potency for EC refill fluid aerosols and the identified chemicals. [Finally, I will test the hypothesis that EC aerosols and the identified chemicals induce reactive oxygen species (ROS), which lead to DNA damage and apoptosis. To test this (Aim #3), the most cytotoxic aerosols and chemicals will be screened with and without vitamin E (a ROS inhibitor) to assess induction of ROS using time-lapse data and fluorescent ROS detectors. To determine if increased ROS leads to DNA damage, the alkaline comet assay and immunocytochemistry will be used to assess breaks in DNA. For aerosols and chemicals that induce oxidative stress and are genotoxic, time-lapse data will be analyzed for hESC and hPF in conjunction with apoptotic indicators. Western blotting and the Annexin V assays will be used to verify activation of the apoptotic pathway proteins.] This will be the first large-scale assessment of cytotoxicity in EC refill fluid aerosols. The proposed study will collect valuable data that wil provide insight into which of these products are unsafe, the doses that produce cytotoxic and genotoxic effects, the specific chemicals that are toxic, and finally, which products are likely to adversely affect the health of users.