Thorne David, Larard Sophie, Baxter Andrew, Meredith Clive, Gaҫa Marianna
British American Tobacco, R&D Centre, Southampton, Hampshire, SO15 8TL, UK.
British American Tobacco, R&D Centre, Southampton, Hampshire, SO15 8TL, UK.
Toxicol Lett. 2017 Jan 4;265:170-178. doi: 10.1016/j.toxlet.2016.12.006. Epub 2016 Dec 10.
DNA damage can be caused by a variety of external and internal factors and together with cellular responses, can establish genomic instability through multiple pathways. DNA damage therefore, is considered to play an important role in the aetiology and early stages of carcinogenesis. The DNA-damage inducing potential of tobacco smoke aerosols in vitro has been extensively investigated; however, the ability of e-cigarette aerosols to induce DNA damage has not been extensively investigated. E-cigarette use has grown globally in recent years and the health implications of long term e-cigarette use are still unclear. Therefore, this study has assessed the induction of double-strand DNA damage in vitro using human lung epithelial cells to e-cigarette aerosols from two different product variants (a "cigalike" and a closed "modular" system) and cigarette smoke. A Vitrocell VC 10 aerosol exposure system was used to generate and dilute cigarette smoke and e-cigarette aerosols, which were delivered to human bronchial epithelial cells (BEAS-2Bs) housed at the air-liquid-interface (ALI) for up to 120min exposure (diluting airflow, 0.25-1L/min). Following exposure, cells were immediately fixed, incubated with primary (0.1% γH2AX antibody in PBS) and secondary antibodies (DyLight™ 549 conjugated goat anti-mouse IgG) containing Hoechst dye DNA staining solution (0.2% secondary antibody and 0.01% Hoechst in PBS), and finally screened using the Cellomics Arrayscan VTI platform. The results from this study demonstrate a clear DNA damage-induced dose response with increasing smoke concentrations up to cytotoxic levels. In contrast, e-cigarette aerosols from two product variants did not induce DNA damage at equivalent to or greater than doses of cigarette smoke aerosol. In this study dosimetry approaches were used to contextualize exposure, define exposure conditions and facilitate comparisons between cigarette smoke and e-cigarette aerosols. Quartz crystal microbalance (QCM) technology and quantified nicotine delivery were both assessed at the exposure interface. Nicotine was eluted from the QCM surface to give a quantifiable measure of exposure to support deposited mass. Dose measured as deposited mass (μg/cm) and nicotine (ng/mL) demonstrated that in vitro e-cigarette exposures were conducted at doses up to 12-28 fold to that of cigarette smoke and demonstrated a consistent negative finding.
DNA损伤可由多种外部和内部因素引起，并且与细胞反应一起，可通过多种途径导致基因组不稳定。因此，DNA损伤被认为在致癌作用的病因学和早期阶段发挥重要作用。烟草烟雾气溶胶在体外诱导DNA损伤的潜力已得到广泛研究；然而，电子烟气溶胶诱导DNA损伤的能力尚未得到广泛研究。近年来，电子烟的使用在全球范围内不断增加，长期使用电子烟对健康的影响仍不明确。因此，本研究评估了使用人肺上皮细胞对来自两种不同产品变体（一种“香烟型”和一种封闭式“模块化”系统）的电子烟气溶胶和香烟烟雾进行体外双链DNA损伤诱导情况。使用Vitrocell VC 10气溶胶暴露系统生成和稀释香烟烟雾和电子烟气溶胶，将其输送到置于气液界面（ALI）的人支气管上皮细胞（BEAS-2B）中，暴露长达120分钟（稀释气流，0.25 - 1L/分钟）。暴露后，立即固定细胞，用含有Hoechst染料DNA染色溶液（0.2%二抗和0.01% Hoechst于PBS中）的一抗（0.1%γH2AX抗体于PBS中）和二抗（DyLight™ 549缀合山羊抗小鼠IgG）孵育，最后使用Cellomics Arrayscan VTI平台进行筛选。本研究结果表明，随着烟雾浓度增加直至细胞毒性水平，存在明显的DNA损伤诱导剂量反应。相比之下，来自两种产品变体的电子烟气溶胶在等同于或高于香烟烟雾气溶胶剂量时未诱导DNA损伤。在本研究中，使用剂量测定方法来确定暴露情况、定义暴露条件并便于比较香烟烟雾和电子烟气溶胶。在暴露界面评估了石英晶体微天平（QCM）技术和定量尼古丁递送情况。从QCM表面洗脱尼古丁以给出可量化的暴露量度，以支持沉积质量。以沉积质量（μg/cm）和尼古丁（ng/mL）测量的剂量表明，体外电子烟暴露的剂量比香烟烟雾高12 - 28倍，并且呈现出一致的阴性结果。
