Advanced in vitro exposure systems.

The comparative in vitro assessment of e-cigarette and cigarette smoke aerosols using the gH2AX assay and applied dose measurements

7. Dec. 2016

David Thorne, Sophie Larard, Andrew Baxter, Clive Meredith, Marianna Gaҫa

British American Tobacco, R&D Centre, Southampton, Hampshire, SO15 8TL, UK


Cigarette smoke generates a dose dependent increase in double strand breaks.
E-cigarette aerosols did not generate any increases in double strand breaks above control levels.
E-cigarette exposures were approximately 28-fold greater than that of cigarette smoke exposures.
In vitro dosimetry ensures accurate interpretation of the data, and confirms delivery of test agent.

In vitro dosimetry allows cross-product, study and system comparisons.


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 Vitrocell1 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 120 min exposure (diluting airflow, 0.25–1 L/min). Following exposure, cells were immediately fixed, incubated with primary (0.1% gH2AX antibody in PBS) and secondary antibodies (DyLightTM 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 (mg/cm2) 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.

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