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

本项目旨在解决烟草制品中心(CTP)在毒性方面的研究重点 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联合用于测试烟草产品中调味料的效果并表征 肺部毒性。新兴烟草产品(电子尼古丁传递系统结束/电子烟， 雪茄、水烟)，通常以加味品种出售，占相当大的比例，而且还在不断增加。 美国烟草消费的最大增长。调味剂通常用于最终产品， 雪茄和水烟。通常销售的口味包括烟草、薄荷/薄荷、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、丁香/肉桂和酒精饮料。这些味道的背后是化学物质 调味品，其中一些已知有呼吸道毒性(如双乙酰、肉桂醛)。的发展。 比较毒性数据--基于暴露引起的氧化应激、DNA损伤和炎症 迫切需要在烟草产品中使用不同香料的气雾剂/烟雾。我们假设 用于新兴加味烟草产品的不同化学调味料(电子烟/烟头、雪茄和 水管)在氧化、DNA损伤、上皮屏障功能障碍方面对毒性有不同的影响，以及 炎症反应，并影响不同强度和持续时间的体外和体内(肺)毒性 曝光。我们的目标是确定和比较各种调味料在新兴烟草产品中的作用。 (电子烟、雪茄和水烟)对毒理和免疫炎症反应的影响。具体来说，我们 目的：1)用气雾剂测定所选烟草产品调味料的体外比较毒性 无细胞活性氧物种反应的暴露系统，以及通过 空气-液体界面系统，以及在3D培养系统中，目的2)确定相对氧化，DNA损伤 以及常用小鼠品系C57BL/6J对烟草制品调味品的免疫炎症反应 (Th1应答)和Balb/c(Th2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)以确定对不良反应的调味剂毒性的预测性 调味品的呼吸健康结果，以及目的3)决定比较表观基因组学(DNA甲基化 和转录组)--对烟草产品调味品作出反应的表观遗传生物标记物。这将决定 烟草产品调味对呼吸健康的生物影响从毒性、生物标志物和 调味品的危害等级。监管科学的成果：同一类别的毒性评估 不同类型烟草产品的调味料在体外和体内的研究将提供至关重要的 与毒性/危险等级(从高到低)和与以下相关的不良健康后果有关的信息 具体的调味品，这将直接支持FDA的监管努力。

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.