Basic science of electronic cigarettes: assessment in cell culture and in vivo models

An increasing number of studies is reporting on effects of ECIGs on cultured cells, studies that were initiated to gain insight into the biological and toxicological effects of ECIGs. Different approaches were used to investigate these effects, and both effects of ECIG-liquids, as well as effects of vapour generated by ECIGs and inhaled by users, were investigated. Effects of ECIGs were evaluated using a wide range of target cells. Some studies focussed on the cells that are in direct contact with the inhaled ECIG-vapour, such as airway epithelial cells [8, 13‐20]. This is highly relevant, since the airway epithelium requires specific attention since it is the first and largest body surface exposed to smoke derived from an ECIG or tobacco cigarette. In these studies, discussed in detail in the next paragraphs, both ECIG vapour, ECIG liquid and ECIG vapour extracts were used. The potential systemic and other consequences of ECIGs were investigated by studying the effect of ECIG-vapour or liquid on a broader range of cell types including human fibroblasts [8, 21, 22], murine fibroblasts [23], endothelial cells [24], vascular smooth muscle cells [25], rat Kupffer cells [26], human embryonic stem cells [21], neutrophils [27], and murine neural stem cells [21].

There are also major differences between studies in the use of tumour cell lines, immortalized cell lines and primary cell lines. This is especially important when studying exposures of airway epithelial cells that are well differentiated and composed of various cell types including basal cells, mucus-producing goblet cells, ciliated cells and club cells [28]. Primary airway epithelial cells show this differentiation when cultured at the air-liquid interface, whereas most immortalized or tumour cell lines do not. Therefore, it can be argued that for studying the effect of aerosols on epithelial cells, the use of primary airway epithelial cells and air-liquid interface (ALI) culture and exposure systems is best suited. Nevertheless, in inhalation toxicology the use on non-differentiated tumour of immortalized cell lines is widespread because these cells are easier to handle, do not show inter-donor differences (because they are derived from one donor), and have an extended life span, thus increasing their availability.

Thus far, only one study has used primary human airway epithelial cells that were differentiated at the ALI and exposed to ECIG-vapour at the ALI [29]. This study from British American Tobacco, a company that produces both tobacco cigarettes and ECIGs, showed that ECIG-vapour exposure did not result in cytotoxicity or decrease in epithelial barrier activity as assessed by transepithelial electrical resistance (TEER), in contrast to exposure to whole cigarette smoke. Two other studies investigated the effect of ECIG-vapour on airway epithelial cells using non-differentiated primary airway epithelial cells, showing reduced viability [14, 15] and increased oxidative stress [15], whereas Lerner et al. showed that exposure of the airway epithelial tumour cell line NCI-H292 causes increased production of IL-6 and IL-8 [8]. In one of these studies, the effect of ECIG-vapour on non-differentiated primary bronchial epithelial cells, a new immortalized bronchial epithelial cell line with differentiation potential (CL-1548), and the A549 cell line was compared [14]. The results showed that A549 were least susceptible to the aerosol when using cell viability as a read-out, whereas primary bronchial epithelial cells were most susceptible and CL-1548 showed intermediate sensitivity. Interestingly, despite the fact that primary bronchial epithelial cells and CL-1548 showed apparent comparable differentiation capacity, for the exposure experiments non-differentiated cultures were used. This study also confirmed that the toxicity resulting from ECIG vapour exposure was markedly lower than that resulting from tobacco smoke. This was also the conclusion from another study using exposure of the A549 alveolar epithelial tumour cell line to ECIG-vapour using cell viability and pro-inflammatory cytokine release as a read-out [19].

Three studies reported on the use of an aqueous ECIG-vapour extract. One study used the immortalized bronchial epithelial cell line BEAS-2B and showed that ECIG-vapour extract causes protein aggregation due to inhibition of autophagy, resulting in oxidative stress, apoptosis and senescence [17]. This mechanism has been proposed to contribute to COPD development and progression, and thus may also contribute to adverse health effects of EC. Another study used the A549 cell line, and showed that ECIG-extract decreased cell viability but to a far lesser extent than cigarette smoke extract [20]. Finally, an ECIG-vapour extract caused reduced cell viability and DNA strand breaks in the keratinocyte cell line HaCaT and in head and neck squamous cell carcinoma cell lines [18].

Other studies investigated the effect of ECIG-liquid on airway epithelial cells. Whereas application of ECIG-liquid to non-differentiated primary airway epithelial cells caused an increase in IL-6 production and rhinovirus infection, accompanied by a decrease in production of the innate host defense mediator SPLUNC1 [13], ECIG-liquid application to ALI-differentiated primary epithelial cells caused a shift in the metabolome [16]. The analysis of the effect of ECIG flavouring additives is complicated by the increasingly large number of companies that offer these liquids. A screening approach has been used to test multiple ECIG liquids on the epithelial cell line 16HBE14o- and subsequently in well-differentiated mouse epithelium [30]. A number of liquids with toxic potential were identified and the chocolate flavouring 2,5-dimethypyrazine was identified to activate CFTR in epithelial cells. Another study investigated the interaction between ECIG liquids and neutrophils and found that exposure of neutrophils to extracts of the ECIG vapour induced a pro-inflammatory response characterized by induction of CD11b, CD66b, MMP-9 and CXCL8 [27].

In summary, these studies that used a variety of approaches show adverse effects of ECIG vapour and liquid on primary airway epithelial cells and tumour cell lines, and other epithelial cell lines, that ranged from reducing viability, an increase in production of inflammatory mediators and oxidative stress, to reducing antimicrobial defences and pro-carcinogenic events. Only one study did not observe adverse effects, but only assessed cell viability and epithelial barrier function as read-out [29]. Interestingly, in four of the studies showing adverse effects, the specific contribution of nicotine to these effects was investigated, and it was demonstrated that these effects were not only mediated by nicotine and even some times largely independent of nicotine concentrations [13, 15, 18, 19]. This is in line with the results from a study on the effect of ECIG-liquid on human gingival fibroblasts [22].

These studies on epithelial cells and a variety of other cell types demonstrate that ECIG-vapour and ECIG-liquid may be less toxic than cigarette smoke, but do cause marked adverse effects on a variety of parameters in various relevant cell types including airway epithelial cells. The studies are somewhat difficult to compare because of differences in cell types, exposure systems and ECIG brands investigated. In addition, the lack of uniformity in generating EC aerosols also hampers interpretation of these studies [7]. Future studies are needed to harmonize approaches to investigate potential harmful effects on cell cultures.

Animal studies have been extensively used to study the effect of exposure to cigarette smoke in development of lung diseases such as chronic obstructive lung disease (COPD) or lung cancer [31]. While these models have expanded the knowledge about disease mechanisms, there also was criticisms whether these results can be translated into clinical practice [32]. It is also a challenge to compare the results between different species or experimental exposure systems used in various setups. Nevertheless, animal models might be a valuable tool to learn about the potential long term outcomes of the exposure to ECIGs. A few studies exist that applied ECIG solutions or aerosols to animals in experimental models.

Neonatal mice were exposed to ECIG for the first 10 days of their life and were found to have modestly impaired lung growth, alveolar cell proliferation, and total body weight [33]. The whole body exposure system comprised a commercial ECIG, from which an aerosol was generated by a pump. In a murine model of asthma, which was induced by systemic sensitization to ovalbumin, the application of diluted ECIG solution increased airway inflammation including an increase in eosinophils levels of Th1-cytokines IL-4, IL-5 and IL-13, and OVA-specific IgE, and worsened hyperresponsiveness [34]. In a recent paper, mice were exposed to aerosolized phosphate-buffered saline, nicotine-free or nicotine-containing ECIG solutions [35]. Exposure to inhaled nicotine-containing e-cigarette fluids triggered effects normally associated with the development of a COPD-like tissue damage in a nicotine-dependent manner.

Cigarette smoke is known to inhibit the innate host defense of the lung [36]. One study investigated the effect of exposure to ECIG vapour for two weeks and showed an increased susceptibility to infection with influenza A and Streptococcus pneumoniae [37]. The effect of ECIGs was linked the oxidative stress and impaired phagocytosis. In this study, the animals were whole-body exposed to an aerosol of commercial ECIGs applied to a classical smoking machine while monitoring aerosol exposure and cotinine levels in the animals. The generation of oxidative stress by ECIG was studied by exposing C57B/6 mice to aerosols from commercial ECIG devices using a standard smoke exposing system [8]. ECIG exposure resulted in increased levels (IL-6, MCP-1, IL-1α, IL-13) and decreased glutathione levels. There was no comparison to conventional cigarettes.

The psychological and behavioural effects of ECIGs were studied using whole-body exposure to cigarette smoke or ECIG vapour, followed by a series of biochemical and behavioural studies. The results showed that nicotine-containing ECIG vapour induces addiction-related neurochemical, physiological and behavioural changes [38]. The offspring of the pregnant mice, which were exposed to nicotine-containing ECIG liquid, showed significant behavioural alterations. This indicated that exposure to ECIG components in a susceptible time period of brain development could induce persistent behavioural changes [39].