Development of physiologically relevant test methods to analyse potential irritant effects to the respiratory tract caused by e-cigarette aerosols is required. This paper reports the method development and optimisation of an acute in vitro MTT cytotoxicity assay using human 3D reconstructed airway tissues and an aerosol exposure system. The EpiAirway™ tissue is a highly differentiated in vitro human airway culture derived from primary human tracheal/bronchial epithelial cells grown at the air-liquid interface, which can be exposed to aerosols generated by the VITROCELL® smoking robot. Method development was supported by understanding the compatibility of these tissues within the VITROCELL® system, in terms of airflow (L/min), vacuum rate (mL/min) and exposure time. Dosimetry tools (QCM) were used to measure deposited mass, to confirm the provision of e-cigarette aerosol to the tissues. EpiAirway™ tissues were exposed to cigarette smoke and aerosol generated from two commercial e-cigarettes for up to 6 h. Cigarette smoke reduced cell viability in a time dependent manner to 12% at 6 h. E-cigarette aerosol showed no such decrease in cell viability and displayed similar results to that of the untreated air controls. Applicability of the EpiAirway™ model and exposure system was demonstrated, showing little cytotoxicity from e-cigarette aerosol and different aerosol formulations when compared directly with reference cigarette smoke, over the same exposure time.

There is evidence that, following exposure to crystalline silica, the release of several proinflammatory cytokines contributes to the induction of unbalanced inflammatory reaction leading to lung fibrosis. We have examined the potential contribution of interleukin-10 (IL-10), an anti-inflammatory cytokine, in the development of silicosis. In a mouse model of inflammatory lung reaction induced by intratracheal instillation of silica (0.5 mg and 5 mg DQ12/mouse), the levels of IL-10 protein (determined by ELISA) both in cells obtained after bronchoalveolar lavage (BAL) and in lung tissue homogenates were significantly increased when compared with controls. After in vitro lipopolysaccharide (LPS) stimulation (1 microg/ml), BAL cells obtained from silica-treated animals produced significantly more IL-10 protein and mRNA than cells obtained from control animals. To examine the role of IL-10 in the lung reaction induced by silica, IL-10-deficient animals were instilled with 5 mg of silica. Twenty-four hours after treatment, the amplitude of the inflammatory response (lactate dehydrogenase [LDH], protein and number of inflammatory cells in BAL) was significantly greater in IL-10-deficient animals than in the wild type. In contrast, the fibrotic response, evaluated by measuring lung hydroxyproline content and by histopathologic analysis 30 days after silica, was significantly less important in IL-10-deficient than in wild-type mice. Together, these data suggest that increased IL-10 synthesis induced by silica can limit the amplitude of the inflammatory reaction, but also contributes to amplify the lung fibrotic response.

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Salmonella typhimurium strains TA98 and TA100 were used to assess the mutagenic potential of the aerosol from a commercially available, rechargeable, closed system electronic-cigarette. Results obtained were compared to those for the mainstream smoke from a Kentucky reference (3R4F) cigarette. Two different test matrices were assessed. Aerosol generated from the e-cigarette was trapped on a Cambridge filter pad, eluted in DMSO and compared to cigarette smoke total particulate matter (TPM), which was generated in the same manner for mutagenicity assessment in the Salmonella assay. Fresh e-cigarette and cigarette smoke aerosols were generated on the Vitrocell VC 10 smoking robot and compared using a modified scaled-down 35mm air agar interface (AAI) methodology. E-cigarette aerosol collected matter (ACM) was found to be non-mutagenic in the 85mm plate incorporation Ames assay in strains TA98 and TA100 conducted in accordance with OECD 471, when tested up to 2400μg/plate. Freshly generated e-cigarette aerosol was also found to be negative in both strains after an AAI aerosol exposure, when tested up to a 1L/min dilution for up to 3h. Positive control responses were observed in both strains, using benzo[a]pyrene, 2-nitrofluorene, sodium azide and 2-aminoanthracene in TA98 and TA100 in the presence and absence of metabolic activation respectively. In contrast, cigarette smoke TPM and aerosol from 3R4F reference cigarettes were found to be mutagenic in both tester strains, under comparable test conditions to that of e-cigarette exposure. Limited information exists on the mutagenic activity of captured e-cigarette particulates and whole aerosol AAI approaches. With the lower toxicant burden of e-cigarette aerosols compared to cigarette smoke, it is clear that a more comprehensive Ames package of data should be generated when assessing e-cigarettes, consisting of the standard OECD-five, TA98, TA100, TA1535, TA1537 (or TA97) and E. coli (or TA102). In addition, TA104 which is more sensitive to the carbonyl based compounds found in e-cigarette aerosols under dry-wicking conditions may also prove a useful addition in a testing battery. Regulatory standard product testing approaches as used in this study will become important when determining whether e-cigarette aerosols are in fact less biologically active than cigarette smoke, as this study suggests. Future studies should be supported by in vitro dosimetry approaches to draw more accurate comparisons between cigarette smoke, e-cigarette aerosol exposure and human use.

The bioactivation of pro-toxicants is the biological process through which some chemicals are metabolized into reactive metabolites. Therefore, in vitro toxicological evaluation should ideally be conducted in cell systems retaining adequate metabolic competency and relevant to the route of exposure. The respiratory tract is the primary route of exposure to inhaled pro-toxicants and lung-derived BEAS-2B cell line has been considered as a potentially suitable model for in vitro toxicology testing. However, its metabolic activity has not been characterized. We performed a gene expression analysis for 41 metabolism-related genes and compared the profile with liver- and lung-derived cell lines (HepaRG, HepG2 and A549). To confirm that mRNA expression was associated with the corresponding enzyme activity, we used a series of metabolic substrates of CYPs (CYP1A1/1B1, CYP1A2, CYP2A6/2A13 and CYP2E1) known to bioactivate inhaled pro-toxicants. CYP activities were compared between BEAS-2B, HepaRG, HepG2, and A549 cells and published literature on primary bronchial epithelium cells (HBEC). We found that in contrast to HBEC, BEAS-2B and A549 have limited CYP activity which was in agreement with their CYP gene expression profile. Control cell lines such as HepG2 and HepaRG were metabolically active for the tested CYPs. We recommend that similar strategies can be used to select suitable cell systems in the context of pro-toxicant assessment.