The use of electronic cigarettes is increasing, with the widely held concept that EC are safer than smoking cigarettes [1,2,3,4, 6,7,8,9,10,11]. Despite the limited data on the health effects of EC on the human lung, the organ that takes the brunt of exposure to inhaled EC aerosol, the Royal College of Surgeons has recommended that “in the interest of public health, it is important to promote the use of e-cigarettes … as widely as possible as a substitute for smoking in the UK” [3]. This concept is supported by Public Health England [2]. While long term studies may eventually demonstrate that smoking EC is safer than smoking traditional cigarettes, this prompts the question: do EC aerosols have an adverse effect on the human lung? To begin to assess this question, we evaluated the biology of lung cells of healthy never smokers before and then after a brief, acute exposure to EC aerosols that is approximately equivalent in nicotine delivery to smoking 2 cigarettes. Even in this limited cohort study, we observed that acute exposure of EC aerosols to healthy naïve individuals disorders biology of at least 3 lung cell populations, including: the small airway epithelium, the initial site of cigarette smoking-induced lung abnormalities [17, 19], alveolar macrophages, the mononuclear phagocyte “defender” of the lower respiratory tract [20, 21] and, indirectly by assessment of circulating endothelial microparticles, a biomarker for health of the pulmonary capillary endothelium of the alveolar vascular bed [22, 23]. While larger studies will be required to determine if these biologic changes translates into an increased risk for lung disease, the data suggests that EC aerosols are not benign. These observations raise the concern as to whether it is premature for the medical community to proactively recommend EC use as a cigarette smoking alternative until more studies are carried out.

Components in EC aerosols possibly relevant to lung health

EC aerosols contain nicotine and a variety of other chemicals. Nicotine is capable of evoking extensive cellular changes in cells including proliferation, cell growth and apoptosis via activation of intracellular kinase signaling pathways [16]. Nicotine displaces the local cyto-transmitter acetylcholine (Ach) from nicotinic ACh receptors (nAChRs) which are composed of 5 subunits that form hetero- or homomeric pentamer channels made of either 5 identical α subunits or combinations of α and β subunits [25]. Nine different types of α subunits (α2– α10) and 3 types of β subunits (β2– β4) have been identified. Both the human airway epithelium and AM express multiple nAChR subunits, and it is likely that the effects of nicotine exposure on the epithelium and AM occur, at a minimum, in a nAChR-dependent manner.

The finding from our study that EC use significantly altered expression of multiple genes in the nicotine receptor pathway in the small airway epithelium further supports this concept. However, nicotine exposure may have biological effects on the lung independent of nACR-mediated signaling. A recent study by Lee et al. [26] demonstrated that exposure of mice to e-cigarette smoke for 12 wk. induced DNA damage in multiple organs including the lung via the production of DNA damaging agents following nitrosation and subsequent metabolizing of nicotine. These data suggest that long-term exposure to nicotine containing e-cigarettes in humans may have similar consequences.

In addition to possible harmful effects of nicotine per se, there is a growing body of literature documenting the presence of harmful chemical constituents in EC aerosols [5]. The liquids used in EC typically contain variable ratios of vegetable glycerin, propylene glycol (PG), and nicotine and flavoring chemicals. Formaldehyde, a known degradation product of PG, reacts with PG and glycerol during vaporization to produce hemiacetals. Assessment of 42 different brands of e-liquids found formaldehyde in all 42 samples at concentrations between 0.02–10.09 mg/L [27, 28]. Other contaminants, such as limonene and various hydrocarbons [alpha-pinene, beta-pinene, gamma-terpinene, and benzene 1-methyl-4-(1-methlethyl) (para-cymene)] have been detected in some but not all e-liquids at levels higher than the recommended exposure limits [28]. Emission of aldehydes from EC has also been reported following heating and oxidation of the e-liquid main components, vegetable glycerin and propylene glycol [29]. Our data from EC users without nicotine demonstrates multiple gene expression changes in both the SAE and AM following acute EC exposure. Therefore, these data suggest that non-nicotine derived chemicals present in EC aerosols can induce molecular changes in cell populations critical to lung health which in the long term may lead to harmful effects.

Evidence that EC aerosols modify the biology of lung cells

Consistent with the in vivo human data in the present study, there is in vitro and experimental animal evidence that EC aerosols modify lung cell biology. Exposure of cell lines from skin and lung to EC aerosols led to cytotoxic effects [12]. Exposure of human airway epithelial cells in vitro and mice in vivo to EC aerosols led to oxidative stress, low levels of inflammatory cell recruitment, delayed clearance of pathogens and other defects in host response [13, 15]. In addition, EC smoke exposure damages DNA and reduces repair activity in mouse lung and human lung cells in vitro [26]. Primary lung microvascular endothelial cells treated with e-liquid or condensed EC aerosol ± nicotine, resulted in increased endothelial permeability [14], and intra-tracheal administration of e-liquid to mice sensitized to ovalbumin aggravated allergen-induced airway inflammation and hyper-responsiveness [30].

In vivo evidence that EC aerosols maybe harmful to the human lung

To our knowledge, there have been no prior direct assessments of lung biology following acute exposure of EC aerosols to smoking-naive humans. However, consistent with the disordered lung biology observed in the present study, there is literature demonstrating clinical abnormalities associated with acute inhalation of EC, including cough, decreased fractional exhaled nitric oxide, increases respiratory impedance and increased respiratory flow resistance [2, 3, 31]. Furthermore, a recent study by Reidel et al. [32] using quantitative proteomics to compare induced sputum samples from cigarette smokers, e-cigarette users, and nonsmokers demonstrated e-cigarette use results in a unique innate immune response in the lung with increased neutrophilic activation and altered mucin secretions. Our study demonstrates that, in naïve individuals who have no prior history of EC or traditional tobacco product usage, acute exposure to EC aerosols results in transcriptome changes in SAE and AM. Transcriptome changes in the SAE were more robust compared to AM responses, as demonstrated by more genes showing differential expression in the SAE in response to EC exposure. In the SAE, alteration of several downstream targets of p53 are consistent with activation of p53-dependent signaling following EC exposure. The p53 signaling pathway plays a central role in regulating multiple cellular functions including apoptosis, cell cycle arrest, senescence and the DNA damage response [33,34,35]. Furthermore, p53 activation is critical to prevent development of tobacco smoke-induced lung cancer [36,37,38,39]. Based on the knowledge that EC aerosols contains multiple toxic chemicals [5, 27,28,29] and that nicotine-derived metabolites/breakdown products induce DNA damage in the lung [26], we hypothesize that altered expression of p53 downstream targets in the SAE is indicative of a cellular response to environmental stress and/or DNA damage. If true, these data further strengthen the argument that EC are not benign and even acute exposure to their aerosols induces harmful effects.

Standard pathways analysis did not identify a dominant pathway in the AM transcriptome data, but several individual genes known to be involved in macrophage physiology and host defense were affected by EC exposure including forkhead box M1 (FOXM1), coronin-1A (CORO1A) and prostaglandin E receptor 3 (PTGER3) suggesting an altered immune response. FOXM1 encodes a transcriptional activator that is known to regulate expression of cell cycle related genes and has a role in controlling cell proliferation [40]. Foxm1 was recently shown to regulate pulmonary inflammatory responses to hyperoxia in neonatal rodent lungs [25]. In addition, murine studies have shown it is required for macrophage recruitment during lung inflammation and tumor formation [41]. Based on the decreased expression of FOXM1 in AM in response to EC exposure, we can hypothesize that AM from EC users may have an impaired migratory and inflammatory response. CORO1A encodes coronin-1A, a member of the WD repeat (~ 40 amino acid conserved region that may facilitate protein-protein interactions) protein family, which has been shown to inhibit autophagosome formation around Mycobacterium tuberculosis-containing phagosomes in rodent macrophages in culture [26]. Therefore, decreased expression of CORO1A following EC exposure may impair the phagocytic capabilities of AM. PTGER3 encodes prostaglandin E receptor 3, which is a G-protein coupled receptor that is one of four known receptors for prostaglandin E2 (PGE2) [42]. Deletion of PTGER3 was shown to improve pulmonary host defense and protect mice from death following Streptococcus pneumoniae infection [27], and other studies suggest that prostaglandins may play key roles in pulmonary host defense [43,44,45]. Therefore, increased AM expression of PTGER3 following EC use may increase the susceptibility of EC users to Streptococcus pneumoniae infection. In conjunction with prior studies demonstrating EC use is associated with an altered lung immune response in both humans [32] and mice [15] our study further supports this claim and suggests EC-dependent transcriptome changes in AM are a contributing factor.

Consequences of disordered lung biology as a precursor to lung disease

The study of possible adverse effects of e-cigarette aerosols on lung health is complicated. There are many brands of e-cigarettes, with a variety of flavors and other additives in addition to nicotine [1,2,3,4]. Further, many studies to evaluate the consequences of e-cigarette aerosols are carried out in ex-cigarette smokers, where the lung has already been comprised to some degree [2, 3]. Because nicotine is addictive, it is not ethical to carry out studies exposing never smokers to long-term studies of chronic exposure to e-cigarette aerosols.