Electronic cigarettes (E-cigarettes) are a potential means of addressing the harm to public health caused by tobacco smoking by offering smokers a less harmful means of receiving nicotine. As e-cigarettes are a relatively new phenomenon, there are limited scientific data on the longer-term health effects of their use. This study describes a robust in vitro method for assessing the cytotoxic response of e-cigarette aerosols that can be effectively compared with conventional cigarette smoke. This was measured using the regulatory accepted Neutral Red Uptake assay modified for air–liquid interface (ALI) exposures. An exposure system, comprising a smoking machine, traditionally used for in vitro tobacco smoke exposure assessments, was adapted for use with e-cigarettes to expose human lung epithelial cells at the ALI. Dosimetric analysis methods using real-time quartz crystal microbalances for mass, and post-exposure chemical analysis for nicotine, were employed to detect/distinguish aerosol dilutions from a reference Kentucky 3R4F cigarette and two commercially available e-cigarettes (Vype eStick and ePen). ePen aerosol induced 97%, 94% and 70% less cytotoxicity than 3R4F cigarette smoke based on matched EC50 values at different dilutions (1:5 vs. 1:153 vol:vol), mass (52.1 vs. 3.1 μg/cm2) and nicotine (0.89 vs. 0.27 μg/cm2), respectively. Test doses where cigarette smoke and e-cigarette aerosol cytotoxicity were observed are comparable with calculated daily doses in consumers. Such experiments could form the basis of a larger package of work including chemical analyses, in vitro toxicology tests and clinical studies, to help assess the safety of current and next generation nicotine and tobacco products.
BackgroundThere have been many recent developments of in vitro cigarette smoke systems closely replicating in vivo exposures. The Borgwaldt RM20S smoking machine (RM20S) enables the serial dilution and delivery of cigarette smoke to exposure chambers for in vitro analyses. In this study we have demonstrated reliability and robustness testing of the RM20S in delivering smoke to in vitro cultures using an in-house designed whole smoke exposure chamber.ResultsThe syringe precision and accuracy of smoke dose generated by the RM20S was assessed using a methane gas standard and resulted in a repeatability error of ≤9%. Differential electrical mobility particle spectrometry (DMS) measured smoke particles generated from reference 3R4F cigarettes at points along the RM20S. 53% ± 5.9% of particles by mass reached the chamber, the remainder deposited in the syringe or connecting tubing and ~16% deposited in the chamber. Spectrofluorometric quantification of particle deposition within chambers indicated a positive correlation between smoke concentration and particle deposition. In vitro air-liquid interface (ALI) cultures (H292 lung epithelial cells), exposed to whole smoke (1:60 dilution (smoke:air, equivalent to ~5 μg/cm2)) demonstrated uniform smoke delivery within the chamber.ConclusionsThese results suggest this smoke exposure system is a reliable and repeatable method of generating and exposing ALI in vitro cultures to cigarette smoke. This system will enable the evaluation of future tobacco products and individual components of cigarette smoke and may be used as an alternative in vitro tool for evaluating other aerosols and gaseous mixtures such as air pollutants, inhaled pharmaceuticals and cosmetics.
BackgroundRecently there has been a rapid increase in approaches to assess the effects of cigarette smoke in vitro. Despite a range of gravimetric and chemical methods, there is a requirement to identify simpler and more reliable methods to quantify in vitro whole smoke dose, to support extrapolation and comparisons to human/in vivo dose. We have previously characterised an in vitro exposure system using a Borgwaldt RM20S smoking machine and a chamber exposing cellular cultures to whole smoke at the air-liquid interface. In this study we demonstrate the utility of a quartz crystal microbalance (QCM), using this exposure system, to assess real-time cigarette smoke particulate deposition during a 30 minute smoke exposure. Smoke was generated at various dilutions (1:5–1:400, smoke:air) using two cigarette products, 3R4F Kentucky reference and 1 mg commercially available cigarettes. The QCM, integrated into the chamber, assessed particulate deposition and data generated were compared to traditional chemical spectrofluorometric analysis.ResultsThe QCM chamber was able to detect mass differences between the different products within the nanogram range. 3R4F reference cigarette smoke deposition ranged from 25.75 ±2.30 μg/cm2 (1:5) to 0.22 ±0.03 μg/cm2 (1:400). 1 mg cigarette smoke deposition was less and ranged from 1.42 ±0.26 μg/cm2 (1:5), to 0.13 ±0.02 μg/cm2 (1:100). Spectrofluorometric analysis demonstrated statistically significant correlation of particulate deposition with the QCM (p < 0.05), and regression R2 value were 97.4 %. The fitted equation for the linear model which describes the relationship is: QCM = −0.6796 + 0.9744 chemical spectrofluorescence.ConclusionsWe suggest the QCM is a reliable, effective and simple tool that can be used to quantify smoke particulate deposition in real-time, in vitro and can be used to quantify other aerosols delivered to our chamber for assessment.
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