Research Project 1: In vitro and in vivo assessment of flavorant toxicity

本项目涉及烟草制品中心（CTP）在毒性下的研究重点 RFA-OD-17-006中定义的科学领域，以及该领域的总体综合科学主题 TCORS聚结用于测试烟草制品中香料的效果并表征 肺毒性新兴烟草产品（电子尼古丁输送系统ENDS/电子烟， 小雪茄，水烟），通常以调味品种出售，代表了一个重要的和不断增长的比例， 烟草消费在美国。调味化学品通常用于ENDS产品， 小雪茄和水烟通常销售的口味包括烟草，薄荷/薄荷醇，水果/糖果 （葡萄，芒果，甜瓜，菠萝，苹果，桃子，香蕉），咖啡/茶，巧克力，浆果（草莓和 蓝莓）、奶油/黄油、肉桂/肉桂和酒精饮料。这些味道的基础是化学物质 调味剂，其中一些具有已知的呼吸毒性（例如，二乙酰、肉桂醛）。的发展 比较毒性数据--基于暴露导致的氧化应激、DNA损伤和炎症 在烟草产品中使用不同的调味剂进行气溶胶/烟雾的方法是迫切需要的。我们假设 在新兴的调味烟草产品（电子烟/ ENDS，小雪茄， 水管）在氧化、DNA损伤、上皮屏障功能障碍方面差异性地影响毒性， 炎症反应，并影响体外和体内（肺）毒性，其强度和持续时间不同， exposure.我们的目标是确定和比较各种香料在新兴烟草产品中的效果 （电子烟，小雪茄和水管）对毒理学和免疫炎症反应的影响。我们特别 目的1）确定使用气溶胶的所选烟草产品调味剂的比较体外毒性 用于无细胞活性氧物质反应性的暴露系统，以及通过 气液界面系统，并在三维培养系统中，目的2）确定比较氧化，DNA损伤 在常用的小鼠品系C57 BL/6 J中对烟草制品调味剂的免疫炎症反应 (Th1应答）和BalB/c（Th 2应答），并使用最先进的报告模型（NF- B荧光素酶 和DNA修复/NHEJ报告小鼠），以确定不良反应的调味剂毒性的预测性质。 调味品的呼吸健康结果，目的3）确定比较表观基因组学（DNA甲基化 和转录组学）-响应烟草产品调味剂的表观遗传生物标志物。这将决定 通过在毒性、生物标志物和 调味品的危害等级。监管科学的结局：同类药物的毒性评估 在体外和体内研究中，不同类型的烟草制品的香料将提供至关重要的 与毒性/危险等级（从高到低）和与下列物质相关的不良健康后果有关的信息 这将直接支持FDA的监管工作。

SUMMARY This project addresses the Center for Tobacco Products (CTP) research priorities under Toxicity scientific domain as defined in RFA-OD-17-006, and the overarching integrative scientific theme of this TCORS coalesce for testing the effects of flavorings in tobacco products and characterize the pulmonary toxicity. Emerging tobacco products (Electronic Nicotine Delivery Systems ENDS/e-cigarettes, cigarillos, waterpipe tobacco), often sold in flavored varieties, represent a significant and increasing proportion of tobacco consumption in the United States. Flavoring chemicals are commonly used in ENDS products, cigarillos and waterpipe tobacco. Commonly marketed flavors include tobacco, mint/menthol, fruits/candy (grapes, mango, melon, pineapple, apple, peach, banana), coffee/tea, chocolate, berries (strawberry and blueberry), crème/butter, clove/cinnamon, and alcoholic beverages. Underlying these flavors are chemical flavorings, some of which have known respiratory toxicity (e.g., diacetyl, cinnamaldehyde). The development of comparative toxicity data -- based on oxidative stress, DNA damage, and inflammation resulting from exposure to aerosol/smoke with different flavorings in tobacco products- is urgently needed. We hypothesize that different chemical flavorings used in emerging flavored tobacco products (e-cigarettes/ ENDS, cigarillos, and waterpipe) differentially influence toxicity in terms of oxidative, DNA damage, epithelial barrier dysfunction, and inflammatory responses, and influence toxicity in vitro and in vivo (lungs) with varying intensity and duration of exposure. Our goal is to determine and compare the effects of various flavorings in emerging tobacco products (e-cigarettes, cigarillos, and waterpipe) on toxicological and immune-inflammatory responses. Specifically, we will: Aim 1) Determine comparative in vitro toxicity of selected tobacco product flavorings using the aerosol exposure system for cell-free reactive oxygen species reactivity, and exposure to human lung epithelial cells by air-liquid interface system, and in a 3D culture system, Aim 2) Determine comparative oxidative, DNA damage and immune-inflammatory responses to tobacco product flavorings in commonly used mouse strains C57BL/6J (Th1 response) and Balb/c (Th2 response), and using the state-of-the-art reporter models (NF- B luciferase and DNA repair/NHEJ reporter mouse) to determine the predictive nature of flavoring toxicity for adverse respiratory health outcomes of flavorings, and Aim 3) Determine comparative epigenomics (DNA methylation and transcriptomics)- epigenetic biomarkers in response to tobacco product flavorings. This will determine the biological effects on respiratory health by flavoring tobacco products in terms of toxicity, biomarkers, and hazard ranking of flavorings. Outcomes for Regulatory Science: Assessment of toxicity of the same class of flavorings across the different types of tobacco products in in vitro and in vivo studies will provide crucial information related to toxicity/hazard ranking (higher to lower) and adverse health outcomes associated with specific flavorings, which would directly support the FDA’s regulatory efforts.