Taylor Mark, Carr Tony, Oke Oluwatobiloba, Jaunky Tomasz, Breheny Damien, Lowe Frazer, Gaça Marianna
a Research and Development Center, British American Tobacco Plc , Southampton , UK.
Toxicol Mech Methods. 2016 Jul;26(6):465-476. doi: 10.1080/15376516.2016.1222473.
Tobacco smoking is a risk factor for various diseases. The underlying cellular mechanisms are not fully characterized, but include oxidative stress, apoptosis, and necrosis. Electronic-cigarettes (e-cigarettes) have emerged as an alternative to and a possible means to reduce harm from tobacco smoking. E-cigarette vapor contains significantly lower levels of toxicants than cigarette smoke, but standardized methods to assess cellular responses to exposure are not well established. We investigated whether an in vitro model of the airway epithelium (human bronchial epithelial cells) and commercially available assays could differentiate cellular stress responses to aqueous aerosol extracts (AqE) generated from cigarette smoke and e-cigarette aerosols. After exposure to AqE concentrations of 0.063-0.500 puffs/mL, we measured the intracellular glutathione ratio (GSH:GSSG), intracellular generation of oxidant species, and activation of the nuclear factor erythroid-related factor 2 (Nrf2)-controlled antioxidant response elements (ARE) to characterize oxidative stress. Apoptotic and necrotic responses were characterized by increases in caspase 3/7 activity and reductions in viable cell protease activities. Concentration-dependent responses indicative of oxidative stress were obtained for all endpoints following exposure to cigarette smoke AqE: intracellular generation of oxidant species increased by up to 83%, GSH:GSSG reduced by 98.6% and transcriptional activation of ARE increased by up to 335%. Caspase 3/7 activity was increased by up to 37% and the viable cell population declined by up to 76%. No cellular stress responses were detected following exposure to e-cigarette AqE. The methods used were suitably sensitive to be employed for comparative studies of tobacco and nicotine products.
吸烟是多种疾病的风险因素。其潜在的细胞机制尚未完全明确，但包括氧化应激、细胞凋亡和坏死。电子烟已成为一种替代吸烟的方式，并且可能是减少吸烟危害的一种手段。电子烟烟雾中有毒物质的含量明显低于香烟烟雾，但评估细胞对暴露反应的标准化方法尚未完善。我们研究了气道上皮细胞（人支气管上皮细胞）的体外模型和市售检测方法能否区分细胞对香烟烟雾和电子烟烟雾产生的水性气溶胶提取物（AqE）的应激反应。在暴露于浓度为0.063 - 0.500口/毫升的AqE后，我们测量了细胞内谷胱甘肽比率（GSH:GSSG）、细胞内氧化剂的生成以及核因子红细胞相关因子2（Nrf2）控制的抗氧化反应元件（ARE）的激活，以表征氧化应激。细胞凋亡和坏死反应通过半胱天冬酶3/7活性的增加和活细胞蛋白酶活性的降低来表征。暴露于香烟烟雾AqE后，所有终点均获得了表明氧化应激的浓度依赖性反应：细胞内氧化剂的生成增加高达83%，GSH:GSSG降低98.6%，ARE的转录激活增加高达335%。半胱天冬酶3/7活性增加高达37%，活细胞数量减少高达76%。暴露于电子烟AqE后未检测到细胞应激反应。所使用的方法具有足够的敏感性，可用于烟草和尼古丁产品的比较研究。
