The data presented here show that to provide an estimate of the relative cytotoxicity and therefore potency of e-cigarettes, undiluted aerosol techniques can be used. With the emergence of electronic nicotine delivery systems, fit-for-purpose in vitro screening methods are required. Reconstituted 3D human airway epithelium, was exposed to undiluted aerosols at the air-liquid interface, using a Vitrocell VC 10. TEER, cilia beat frequency and cytotoxic responses were assessed. Using two smoking regimes (ISO and HCI) a 3R4F reference cigarette, produced ICs of 5.2 and 2.1 min, 1458 ng/mL and 1640 ng/mL nicotine respectively. Using an open tank e-cigarette device, a full cytotoxicity dose-response curve was obtained giving an IC of 30 min with corresponding nicotine of 10,957 ng/mL, 6-14 times less cytotoxic than cigarette smoke. A commonly used e-liquid flavourant cinnamaldehyde and known skin sensitizer was added to the standard e-liquid formulation and used as an aerosolised positive control, at 0.1, 0.025, 0.01 and 0%, demonstrating a full dose response. The delivery of undiluted aerosols in vitro has resulted in increased method sensitivity, throughput and quantitative e-cigarette comparisons. A positive control aerosol generated from a 'safe' e-liquid benchmark can inform risk assessments on supportable levels of flavour ingredients.

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This study assessed the toxicological and biological responses of aerosols from a novel hybrid tobacco product. Toxicological responses from the hybrid tobacco product were compared to those from a commercially available Tobacco Heating Product (c-THP), a prototype THP (p-THP) and a 3R4F reference cigarette, using in vitro test methods which were outlined as part of a framework to substantiate the risk reduction potential of novel tobacco and nicotine products. Exposure matrices used included total particulate matter (TPM), whole aerosol (WA), and aqueous aerosol extracts (AqE) obtained after machine-puffing the test products under the Health Canada Intense smoking regime. Levels of carbonyls and nicotine in these matrices were measured to understand the aerosol dosimetry of the products. The hybrid tobacco product tested negative across the in vitro assays including mutagenicity, genotoxicity, cytotoxicity, tumour promotion, oxidative stress and endothelial dysfunction. All the THPs tested demonstrated significantly reduced responses in these in vitro assays when compared to 3R4F. The findings suggest these products have the potential for reduced health risks. Further pre-clinical and clinical assessments are required to substantiate the risk reduction of these novel products at individual and population levels.

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BackgroundCigarette smoking is a leading cause of mortality and morbidity and is associated with cardiovascular disease via contributory processes such as endothelial dysfunction, inflammation and thrombosis. Cigarette smoke both contains and stimulates the production of cellular oxidants and it may also promote vascular inflammation. Osteopontin is a non-collagenous matrix protein first identified in bone and there is increasing evidence for its role in inflammation and cardiovascular disease via its action as a soluble cytokine.MethodsIn this study we have examined the mechanisms underlying the expression of osteopontin in human vascular endothelial cells in vitro following exposure to cigarette smoke particulate matter (PM), using PCR, electrochemiluminescence, immunostaining and Western blotting. We further determined if serum osteopontin levels changed in humans who quit smoking.ResultsNon-cytotoxic concentrations of PM increased osteopontin levels in cultured human endothelial cells and this effect was reduced in the presence of ascorbate, suggesting a role for oxidants in the response to PM. However, oxidant production played no role in the PM-evoked induction MMP-3, an enzyme which cleaves osteopontin. In smokers who quit smoking for 5 days, serum osteopontin levels were significantly lowered compared to those measured prior to smoking cessation.ConclusionsIn vitro cigarette smoke extract exposure induced osteopontin expression in human endothelial cells in an oxidative stress-dependent manner, which may involve MMP-3 cleavage. In humans, serum osteopontin was decreased with short-term smoking cessation. Endothelial-derived osteopontin may contribute to inflammation in smokers, and may also contribute to atherosclerosis and cardiovascular disease-related processes.

Endothelial damage plays a key role in atherosclerosis and this is impacted upon by numerous risk factors including cigarette smoking. A potential measure to reduce the cardiovascular burden associated with smoking is to reduce smoke toxicant exposure. In an in vitro endothelial damage repair assay, endothelial cell migration was inhibited by cigarette smoke particulate matter (PM) generated from several cigarette types. This inhibition was reduced when cells were exposed to PM from an experimental cigarette with reduced smoke toxicant levels. As a number of toxicants induce oxidative stress and since oxidative stress may link cigarette smoke and endothelial damage, we hypothesized that PM effects were dependent on elevated cellular oxidants. However, although PM-induced cellular oxidant production could be inhibited by ascorbic acid or n-acetylcysteine, both these antioxidants were without effect on migration responses to PM. Furthermore, reactive oxygen species production, as indicated by dihydroethidium fluorescence, was not different in cells exposed to smoke from cigarettes with different toxicant levels. In summary, our data demonstrate that a cardiovascular disease-related biological response may be modified when cells are exposed to smoke containing different levels of toxicants. This appeared independent of the induction of oxidative stress.

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In vitro aerosol systems offer advantages in generating aerosols and exposing cell cultures at the air-liquid interface (ALI), mimicking human exposure. However, dilution technologies, principles, and characteristics of exposure chambers differ between systems. In addition, various cell types and biological end points can be assessed, meaning that in vitro aerosol data are presented in different ways, so researchers are unable to compare data between systems, studies, and laboratories. In this case study we present the comparison of in vitro data across multiple aerosol exposure studies using a 3R4F reference tobacco product. The first aim assessed whether a consistent in vitro dosimetry approach could gain insight into the characterization of ALI aerosol exposure systems. The second aim assessed whether cytotoxicity data sets generated on contrasting exposure systems with different experimental setups could be directly compared. Cytotoxicity data generated from five independent studies were compared. When plotted on the x-axis as a function of dilution principle, the ''standard way'' to present whole aerosol data, there was little read across between the data sets. Expressing each data set as a function of particulate dose (lg/cm 2) and nicotine concentration (mg), however, allowed comparisons between all data sets and with human daily exposure estimates. Furthermore, compared as a function of dose, the data set showed remarkable commonalities between themselves despite clear study differences. The data demonstrate that in vitro dosimetry techniques can align data between contrasting setups and experimental protocols to facilitate comparisons and provide a link between in vitro, in vivo, and human dosimetry studies.