John McAughey

Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke

Abstract 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.

Assessment of an in vitro whole cigarette smoke exposure system: The Borgwaldt RM20S 8-syringe smoking machine

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.

Real-time assessment of cigarette smoke particle deposition in vitro

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.

Methodologies for the quantitative estimation of toxicant dose to cigarette smokers using physical, chemical and bioanalytical data

Abstract Methodologies have been developed, described and demonstrated that convert mouth exposure estimates of cigarette smoke constituents to dose by accounting for smoke spilled from the mouth prior to inhalation (mouth-spill (MS)) and the respiratory retention (RR) during the inhalation cycle. The methodologies are applicable to just about any chemical compound in cigarette smoke that can be measured analytically and can be used with ambulatory population studies. Conversion of exposure to dose improves the relevancy for risk assessment paradigms. Except for urinary nicotine plus metabolites, biomarkers generally do not provide quantitative exposure or dose estimates. In addition, many smoke constituents have no reliable biomarkers. We describe methods to estimate the RR of chemical compounds in smoke based on their vapor pressure (VP) and to estimate the MS for a given subject. Data from two clinical studies were used to demonstrate dose estimation for 13 compounds, of which only 3 have urinary biomarkers. Compounds with VP > 10−5 Pa generally have RRs of 88% or greater, which do not vary appreciably with inhalation volume (IV). Compounds with VP < 10−7 Pa generally have RRs dependent on IV and lung exposure time. For MS, mean subject values from both studies were slightly greater than 30%. For constituents with urinary biomarkers, correlations with the calculated dose were significantly improved over correlations with mouth exposure. Of toxicological importance is that the dose correlations provide an estimate of the metabolic conversion of a constituent to its respective biomarker.

Quantification of Cigarette Smoke Particle Deposition In Vitro Using a Triplicate Quartz Crystal Microbalance Exposure Chamber

There are a variety of smoke exposure systems available to the tobacco industry and respiratory toxicology research groups, each with their own way of diluting/delivering smoke to cell cultures. Thus a simple technique to measure dose in vitro needs to be utilised. Dosimetry—assessment of dose—is a key element in linking the biological effects of smoke generated by various exposure systems. Microbalance technology is presented as a dosimetry tool and a way of measuring whole smoke dose. Described here is a new tool to quantify diluted smoke particulate deposition in vitro. The triplicate quartz crystal microbalance (QCM) chamber measured real-time deposition of smoke at a range of dilutions 1 : 5–1 : 400 (smoke : air). Mass was read in triplicate by 3 identical QCMs installed into one in vitro exposure chamber, each in the location in which a cell culture would be exposed to smoke at the air-liquid interface. This resulted in quantification of deposited particulate matter in the range 0.21–28.00 μg/cm2. Results demonstrated that the QCM could discriminate mass between dilutions and was able to give information of regional deposition where cell cultures would usually be exposed within the chamber. Our aim is to use the QCM to support the preclinical (in vitro) evaluation of tobacco products.

Component-specific, cigarette particle deposition modeling in the human respiratory tract

Abstract Inhalation of cigarette smoke particles (CSP) leads to adverse health effects in smokers. Determination of the localized dose to the lung of the inhaled smoke aids in determining vulnerable sites, and identifying components of the smoke that may be responsible for the adverse effects; thus providing a roadmap for harm reduction of cigarette smoking. A particle deposition model specific to CSP was developed for the oral cavity and the lung by accounting for cigarette particle size growth by hygroscopicity, phase change and coagulation. In addition, since the cigarette puff enters the respiratory tract as a dense cloud, the cloud effect on particle drag and deposition was accounted for in the deposition model. Models of particle losses in the oral cavities were developed during puff drawing and subsequent mouth-hold. Cigarette particles were found to grow by hygroscopicity and coagulation, but to shrink as a result of nicotine evaporation. The particle size reached a plateau beyond which any disturbances in the environmental conditions caused the various mechanisms to balance each other out and the particle size remain stable. Predicted particle deposition considering the cloud effects was greater than when treated as a collection of non-interacting particles (i.e. no cloud effects). Accounting for cloud movement provided the necessary physical mechanism to explain the greater than expected, experimentally observed and particle deposition. The deposition model for CSP can provide the necessary input to determine the fate of inhaled CSP in the lung. The knowledge of deposition will be helpful for health assessment and identification and reduction of harmful components of CSP.

Real-time measurement of inhaled and exhaled cigarette smoke: Implications for dose

Inhalation of tobacco smoke aerosol is a two-step process involving puffing followed by inhalation. Measured smoke deposition efficiencies in the lung (20-70%) are greater than expected for smoke particles of 150 -- 250 nm count median diameter (CMD). Various mechanisms have been put forward to explain this enhanced deposition pattern, including coagulation, hygroscopic growth, condensation and evaporation, changes in composition, or changes in inhalation behaviour. This paper represents one of a series of studies seeking to better quantify smoke chemistry, inhalation behaviour and cumulative particle growth. The studies have been conducted to better understand smoke dosimetry and links to disease as part of a wider programme defining risk and potential harm reduction. In this study, the average CMD of inhaled smoke was 160 nm while the average CMD of exhaled smoke was 239 nm with an average growth factor of 1.5.

Puffing and inhalation behaviour in cigarette smoking: Implications for particle diameter and dose

Inhalation of tobacco smoke aerosol is a two-step process involving puffing followed by inhalation. Measured smoke deposition efficiencies in the lung (20-70%) are greater than expected for smoke particles of diameter 150 -- 250 nm CMD. Various mechanisms have been put forward to explain this enhanced deposition pattern, including coagulation, hygroscopic growth, condensation and evaporation, changes in composition, or changes in inhalation behaviour. This paper represents one of a series of studies seeking to better quantify smoke chemistry, inhalation behaviour and cumulative particle growth. The studies have been conducted to better understand smoke dosimetry and links to disease as part of a wider programme defining risk and potential harm reduction. In this study, it was noted that particle deposition increased with increasing inhalation depth, and that smoke inhalation volumes were generally greater than normal tidal breathing volumes. A weak association was observed between particle diameter and puff flow, but no strong association between particle diameter and retention efficiency.

Assessment of tobacco heating product THP1.0. Part 4: Characterisation of indoor air quality and odour

ABSTRACT The tobacco heating product THP1.0, which heats but does not burn tobacco, was tested as part of a modified‐risk tobacco product assessment framework for its impacts on indoor air quality and residual tobacco smoke odour. THP1.0 heats the tobacco to less than 240 °C ± 5 °C during puffs. An environmentally controlled room was used to simulate ventilation conditions corresponding to residential, office and hospitality environments. An analysis of known tobacco smoke constituents, included CO, CO2, NO, NO2, nicotine, glycerol, 3‐ethenyl pyridine, sixteen polycyclic aromatic hydrocarbons, eight volatile organic compounds, four carbonyls, four tobacco‐specific nitrosamines and total aerosol particulate matter. Significant emissions reductions in comparison to conventional cigarettes were measured for THP1.0. Levels of nicotine, acetaldehyde, formaldehyde and particulate matter emitted from THP1.0 exceeded ambient air measurements, but were more than 90% reduced relative to cigarette smoke emissions within the laboratory conditions defined Residual tobacco smoke odour was assessed by trained sensory panels after exposure of cloth, hair and skin to both mainstream and environmental emissions from the test products. Residual tobacco smoke odour was significantly lower from THP1.0 than from a conventional cigarette. These data show that using THP1.0 has the potential to result in considerably reduced environmental emissions that affect indoor air quality relative to conventional cigarettes. HighlightsTHP emissions are reduced in indoor air versus conventional cigarettes.Tobacco combustion markers were not observed for THP emissions.Residual tobacco odour post THP exposure was less than cigarettes.First reported high‐resolution time‐resolved particle size and mass measurements.

Assessment of tobacco heating product THP1.0. Part 9: The placement of a range of next-generation products on an emissions continuum relative to cigarettes via pre-clinical assessment studies

ABSTRACT This series of nine papers described the operation and pre‐clinical assessment of a tobacco heating product THP1.0. This last paper contextualises the pre‐clinical assessment data on THP1.0 with data from other next generation products relative to cigarette smoke. The tobacco and nicotine risk continuum is a concept that ranks products according to their potential harm, with cigarettes at the highest risk extreme and Nicotine Replacement Therapy at the least risky extreme. Data generated in pre‐clinical studies on THP1.0 and a range of Next Generation Products (NGPs) may provide some initial indication of potential ranking of these products, although importantly, data from such studies are limited and cannot take into consideration several important aspects for risk such as long term product use patterns. In each of the studies, the responses to the emissions from THP1.0 were substantially reduced relative to cigarette smoke. Additionally, responses from THP1.0 were very similar to those from the other NGP emissions. A comparison of the results clearly showed the emissions from all the NGPs were considerably lower than those from cigarettes and all in around the same emissions level. These results show that THP1.0 could have the potential to be a reduced risk product compared to cigarettes, though further studies assessing the exposure, individual and population risk reduction profile would be required to substantiate this potential. HighlightsComparison of pre‐clinical data and a range of next generation products (NGPs).A tobacco heating product, THP1.0, showed substantially reduced responses in pre‐clinical tests in comparison to cigarettes.All tested NGPs have substantially reduced responses in pre‐clinical assessment studies in comparison to cigarettes.