A Summary of In Vitro and In Vivo Studies Evaluating the Impact of E-Cigarette Exposure on Living Organisms and the Environment

Worldwide use of electronic cigarettes has been rapidly expanding over recent years, but the long-term effect of e-cigarette vapor exposure on human health and environment is not well established; however, its mechanism of action entails the production of reactive oxygen species and trace metals, and the exacerbation of inflammation, which are associated with potential cytotoxicity and genotoxicity. The present study examines the effects of selected liquid chemicals used in e-cigarettes, such as propylene glycol/vegetable glycerin, nicotine and flavorings, on living organisms; the data collected indicates that exposure to e-cigarette liquid has potentially detrimental effects on cells in vitro, and on animals and humans in vivo. While e-liquid exposure can adversely influence the physiology of living organisms, vaping is recommended as an alternative for tobacco smoking. The study also compares the impact of e-cigarette liquid exposure and traditional cigarette smoke on organisms and the environmental impact. The environmental influence of e-cigarette use is closely connected with the emission of airborne particulate matter, suggesting the possibility of passive smoking. The obtained data provides an insight into the impact of nicotine delivery systems on living organisms and the environment.


Introduction
Although their initial inception dates back to 1963, the design of e-cigarettes in 2003 by Hon Lik, has spurred increasing global sales, which now show profits worth billions of dollars. Their popularity among the smoking community has also grown exponentially to the extent that e-cigarettes have become a leading product among the various alternatives to traditional cigarettes [1][2][3][4]. They comprise a battery-charged system containing a power source, a heater and a cartridge containing a liquid which transforms into vapor. The vapor produced from the liquid, usually containing propylene glycol (PG)/vegetable glycerin (VG), nicotine (N) and flavorings (F) [2], is inhaled by the user. By contrast, a list of chemical compounds in conventional cigarettes and cigarette smoke includes more than 7000 items of which 69 are known carcinogens [5]. E-cigarettes are widely regarded as a safer alternative to smoking, a known cause of significant morbidity and mortality accounting for more than seven million deaths annually [6]. Although they are also considered as a tool for harm reduction and smoking cessation [7,8], the effects of both short-term and long-term exposure remain largely unknown.
While smoking cigarettes is a well-known and major risk factor for lung cancer [9], recent studies suggest that e-cigarette smoking may also induce the signaling pathways associated with tumor development. Carcinogenesis, a process characterized by an abnormal proliferation of cells, involves several stages: its onset is characterized by genetic alteration and tumor initiation, which is followed by accelerated division of cells, leading to the outgrowth and accumulation of abnormalities associated with the unregulated cellular signaling pathways [10]. Lung cancer is both the most common cancer (2.1 million cases) and the most common cause of cancer death (1.8 million deaths) worldwide [11]. Its etiology is based on the occurrence of mutations in oncogenes leading to excessive proliferation of mutated cells and tumor formation [12,13]. One risk factor increasing the possibility of lung cancer is chronic obstructive pulmonary disease (COPD) [14,15], a progressive aggravation of lung function [15]. Exposure to e-cigarette vapor may trigger inflammation in the airways, which is a risk factor for the development of lung cancer; both COPD and lung cancer share an etiology of oxidative stress associated with inflammation [14]. Reactive oxygen (ROS) and nitrogen species (RNS) induce endothelial dysfunction and tissue injury by stimulating the production of chemical mediators of inflammation [16]. In addition, ROS also upregulate the expression of numerous genes involved in immune and inflammatory processes by activating nuclear factor κB (NF-κB), a member of the transcription factor protein family [17].
This review examines the literature concerning the mechanisms of action of e-cigarettes, the effect of exposure to e-liquid in vitro and in vivo and the environmental impact of electronic nicotine delivery system.

The Mechanisms of E-Cigarette Action
A typical constituent of e-liquid is nicotine. Consumption is known to increase the risk of adverse cardiovascular events, and absorbed nicotine may release catecholamine by the activation of nAChRs localized on sympathetic nerve endings; subsequent activation in vascular smooth muscle cells contracts the vascular tissues and elevates blood pressure. Nicotine may also exert a direct effect on vascular smooth muscle cells to induce vascular relaxation or contraction, thus altering cardiovascular function, both directly and indirectly via nicotine-induced adrenergic stimulation. The cellular mechanisms of nicotine action may be related to its ability to prolong action potentials and depolarize membrane potential [18]. However, the electronic nicotine delivery system poses a lower cardiovascular risk than tobacco smoking [19].
E-cigarette liquid formulations also contain a wide variety of flavoring agents [20]. Flavors may influence nicotine absorption by altering pH [21]. Although none have been tested so far, viz. acetylpyridine, diacetyl, dimethylpyrazine, cinnamaldehyde, eucalyptol, eugenol, isoamylacetate, menthol and vanillin, have been found to cause cultured endothelial cell dysfunction (porcine aortic endothelial cells) [22]; studies on cellular models suggest that they may exert a toxic effect on respiratory cells based on oxidative stress and inflammatory processes [23][24][25][26][27][28]. Contact with such flavorant agents results in elevated level of ROS production and excessive IL-8 secretion in airway epithelium cells, with both mechanisms leading to cell cytotoxicity: oxidative stress is a cause of DNA damage [29]. In both cases, various molecular cell signaling cascades are activated, followed by inflammatory responses in lung cells [30].
ROS are highly reactive chemicals possessing unpaired electrons in the outer shell. They can initiate oxidative stress followed by other pathological processes, and have been found to damage proteins, lipids and DNA and sustain proinflammatory responses. E-cigarette vapor has been found to contain 7 × 10 11 free radicals per puff compared to 10 14 in cigarette smoke [31,32]. ROS production has been found to be provoked by the inhalation of toxic agents, and to stimulate inflammatory signaling pathways [33]. One such stimulant of ROS release is exposure to the vapor from e-cigarettes [34,35]. report any inflammatory responses in mice exposed to e-cigarettes PG vapor for two hours per day for eight weeks [65]. Additionally, Cotta indicates that PG does not appear to display toxicity effects via inhalation and does not regard it as a significant hazard [66].
Another important component of e-liquid vapor is nicotine, a highly addictive compound whose concentration ranges from 0 mg to 36 mg/mL [67]. Nicotine exerts its effects on the brain by modulating the activity of certain ligand-gated ion channels known as nicotinic acetylcholine receptors (nAChRs) [68]; these comprise junctions of individual subunits activated by the binding of endogenous neurotransmitter acetylcholine (Ach) and other biologically active compounds, including nicotine. Nicotine itself poses numerous health hazards by influencing several processes, such as cell proliferation, apoptosis and the immune response; however, it also contributes to oxidative stress and consequent DNA mutations, which can lead to cancer. Nicotine exposure is associated with an increased probability of tumor proliferation, metastasis, and can also increase the likelihood of treatment failure by reducing the effectiveness of chemotherapy and radiotherapy [69]. Nicotine promotes lung cancer development and accelerates its proliferation, angiogenesis, migration, invasion and EMT, via its influence on nAChRs receptors, whose presence has been confirmed in lung cancer cells [70].
Nicotine is predominantly metabolized in vivo into cotinine [71]; although its serum level constitutes a predictor of lung cancer risk [72], it is nevertheless believed to be nontoxic and noncancerogenic [73]. Nicotine is also metabolized into nitrosamines, chemicals with proven carcinogenicity, which lead to the occurrence of methylation intermediates in DNA [74]. It has been established that e-cigarettes induce O6-methyl-deoxuguanosine adducts and aldehyde-derived cyclic 1,N2-propano-dG adducts in lung, heart, liver, and bladder tissues in a mouse model, with the lungs demonstrating higher levels of adducts than the bladder or heart [75]. The covalent binding of carcinogens to DNA is believed to contribute to the induction of tumorigenesis by preventing DNA repair [76]. Nitrosamines are metabolized into N-nitrosonornicotine (NNN) and nicotine-derived nitrosamine ketone (NNK). NNK can be further metabolized into methyldiazohydroxide, pyridyl-butyl derivatives, and formaldehyde, and NNN degrades into hydroxyl or keto pyridyl-butyl [75]. In fact, e-cigarette vapor becomes toxic through degradation into potent carcinogenic nitrosamines, the induction of DNA damage in vitro [76] and subsequent tumors in various organs, as observed in in vivo animal models [77,78]. NNK and nicotine activate the serine/threonine kinase Akt taking part in key cellular processes such as glucose metabolism, cell cycle progression and apoptosis and were found to stimulate Akt signal transduction in normal human bronchial epithelial cells [NHBE cells] and small airway epithelial cells, which serve as precursors to squamous cell carcinomas and adenocarcinomas, respectively. Even small doses of nicotine may trigger the activation of Akt A processes, which in turn may result in increased cell growth and apoptosis [79]. Nevertheless, it is difficult to establish which effects are a result of tobacco smoking or e-cigarette smoking, particularly among smokers who switch from one to the other [80]. The respiratory system is extremely sensitive to foreign bodies, such as pathogens or chemical compounds. The inhalation of potentially harmful substances can be countered by responses such as coughing and sneezing, and the presence of irritant compounds in the airway can stimulate the secretion of profuse amounts of mucus, and induce pain, as protective responses. Inhaled substances may injure the pulmonary epithelium, leading to a wide range of respiratory tract disorders [64]. E-liquids often contain flavoring agents (1-4%), most of which include a significant number of aldehydes [81]. Although the diversity of flavorings has been increasing rapidly, the most commonly used types are menthol, diacetyl and aldehydes, 2,3-pentanedione, acetoin, 2,5-dimethypyrazine, 3,4-dimethoxybenzaldehyde, vanillin, cinnamaldehyde, 2-methoxycinnamaldehyde and maltol [30]. The most prevalent aldehydes are acetaldehyde and formaldehyde while the most prevalent nonaldehydes are acetoin and diacetyl [82].
Some chemicals in e-liquids exert a harmful effect on the lungs [83]. Diacetyl and 2,3-pentanedione impair cilia function in NHBE cells by downregulating the genes taking part in cilia biogenesis [84]. Recent studies have highlighted the role of flavoring agents on airway tract cells and the consequence of inhaling them on ROS release, cytotoxic response, immune-mediated responses, DNA damage and mucociliary clearance [30]. In addition, food flavorings are also commonly used as e-liquid additives, one example being diacetyl, which imparts a buttery flavor to food. However, while being safe for consumption in food, the inhalation of high doses of diacetyl was found to be associated with the incidence of bronchiolitis obliterans, or "popcorn lung", a severe irreversible lung disease, in microwave popcorn production workers [85]. Diacetyl is present in e-liquid above the laboratory limit of detection [86]. The buttery flavor in e-liquid is also supported by the presence of acetoin, which may further contribute to the toxicity of the vapor [87]. Sweet-flavored e-liquids also often contain diacetyl and acetyl propionyl at concentrations exceeding safety limits [88]. Among the aldehyde components, acetaldehyde and formaldehyde can react with DNA, lipids and proteins and can create adducts [89]. These carbonyls are formed in significant amounts, with similar levels of formaldehyde being detected both in e-liquid vapor and tobacco smoke [23]. They may also play an important role in the progression of COPD and lung disease [90] and take part in the pathogenesis of lung cancer [91]. Particles containing formaldehyde, acetylaldehyde and ROS can form deposits in the bronchioles or alveoli and cause inflammatory damage of the pulmonary epithelium [92].

In Vitro Studies on E-Liquid Exposure to Cells
Cells have been commonly used to analyze the biological effects of e-cigarette liquid e-liquid. Such studies have assessed cell viability, DNA damage, ROS levels, inflammatory mediator production, changes in cell morphology and function, dysregulation of gene expression and pro-tumor events after vapor exposure using a variety of approaches and cell lines. E-cigarette usage has been found to trigger cell cytotoxicity and genotoxicity, and vapor exposure is also related to oxidative stress, with a strong connection being found between vapor exposure and overproduction of proinflammatory agents.
The studies performed on human epithelial normal bronchial cell (Nuli1), human premalignant dysplastic oral mucosal keratinocyte (POE9n) and human oral squamous cell carcinoma (UM-SCC-1) lines revealed that e-cigarette aerosols induce a dose-dependent and nicotine-independent increase of DNA damage. Exposure to vapor is characterized by elevated ROS level and increased presence of 8-oxo-7,8-dihydro-2 -deoxyguanosine (8-oxo-dG), the most abundant oxidative stress-induced DNA lesions [93]. 8-oxo-dG lesions are removed from DNA by base excision repair (BER) [94]. Polymorphisms in the enzyme involved in the BER process may affect the efficiency of repair [95]; in addition, chronic oxidative stress may induce mutations in tumor suppressor genes such as p53 [96] and proto-oncogenes such as Ras [97]. This combination of impaired BER activity and accumulated DNA damage is associated with increased cancer risk.
Additionally, a strong connection has been reported between vapor exposure and inflammation. Lerner found exposure to e-cigarette aerosol secretion by airway epithelial cells (H292) and human fetal lung fibroblasts (HFL1) increased the levels of IL-6 and IL-8 and exhibited stress followed by morphological changes [98]. Elsewhere, exposure to vapor has been found to accelerate the cytotoxic effect and stimulate ROS and inflammatory mediator production, thus triggering the inflammatory state, in human alveolar macrophages obtained from never-smokers [99]; in addition, normal human bronchial epithelial (NHBE) cells collected from a human donor exposed to vaporized e-liquid containing different concentrations of nicotine displayed excessive cytokine production [100,101]. Inflammation can act synergistically with DNA damage to induce mutations [102] which in turn may give rise to altered proteins, thus hastening the induction of human cancer [103]. E-cigarettes have also been found to induce adverse effects on the metabolomic status of NHBEcells comparable to those seen with tobacco smoke, with these being mediated by oxidative stress [104].
The treatment of cells with e-liquid without nicotine also caused certain toxic effects; this has been attributed to the presence of flavoring chemicals. The data indicate that diacetyl, cinnamaldehyde, acetoin, pentanedione, o-vanillin, maltol and coumarin, administered at different concentrations, trigger an inflammatory response and IL-8 secretion mediated by ROS production [105]. Additionally, cinnamaldehyde has been found to impair mitochondrial function and reduce ATP levels, which correlates with the suppression of ciliary beat frequency [106]. Exposure to e-cigarette vapor was found to induce significant phenotypical changes in the immortalized NHBE-CL1548 cell line [107].
In contrast, inhalation of flavorless nicotine-containing e-cigarette vapor induces several adverse effects such as dysregulation in the oxidative stress response [108] and the phospholipid and fatty acid triacylglycerol metabolism pathways [109]; it has also been connected with gene dysregulation, DNA damage, apoptosis [110] and inflammatory protein release [100,111]. Nicotine inhalation has profound effects upon mitochondrial [112] and lung functions by the dysregulation of fluid homeostasis [113] and mucociliary function [114,115]. Exposure to nicotine-containing e-cigarette fluids has been found to increase mutational susceptibility [75] and phenotypical changes [107]. Moreover, the presence of nicotine in e-cigarette vapor mostly elicits a cellular response. It would appear that the inhalation of nicotine-containing vapor during e-cigarette use may have potentially dangerous effects.
While most in vitro studies suggest that e-cigarettes have a negative impact on cells, the body of evidence indicates that e-cigarette aerosol has a limited mutagenic effect on transgenic mouse fibroblast or human fibroblast cells [116]. A study conducted on human lung epithelial cells (A549) exposed to 0-20 mg/mL e-liquid for 24 h found no cytotoxic, genotoxic or inflammatory effects [117].
Similarly, there is a lack of consensus on the possible harmful effects of e-cigarette vapor on a 3D differentiated co-culture model of human airways. The study, conducted on a multicellular model of human cells comprising human bronchial epithelial cells (CALU-3) and pulmonary fibroblasts (MRC-5), indicate that exposure to e-liquid ingredients including PG:VG, 16 mg/mL nicotine and proprietary flavors significantly impact cell viability, proinflammatory mediator production and oxidative stress [118]. In turn, a study on EpiAirway tissue, a human airway culture derived from primary human tracheal/bronchial epithelial cells, demonstrated similar cell viability to untreated controls following exposure to commercially available e-cigarette aerosols, with and without menthol flavoring, containing 4.5% and 3% nicotine by volume [119].
The adverse impact of exposure to e-cigarette vapor on different cell lines is summarized in Table 1. Table 1. The impact of e-cigarette liquid exposure to various cell models (in vitro study).

Type of Cells
Characteristic of E-Liquid Action Ref.

In Vivo Studies on E-Cigarette Liquid Exposure in Animal Models
Most animal models used for testing have been based on mice, but some rat models have been used. The animals are typically exposed to an e-cigarette liquid consisting of a base containing various concentrations of nicotine and different types of flavors. The following indicators have been determined after vapor exposure: respiratory system mechanics, mitochondrial function, oxidative stress, DNA damage, inflammation, changes in tissue or organ morphology and function, behavioral changes, dysregulation of gene expression, changes in maternal and offspring metabolism and pro-tumor events. It was observed that e-cigarette liquid alters the physiology of the lung, induces mitochondrial dysfunction and can elevate oxidative stress. E-cigarette vapor has a negative effect on the health of the animal, induces DNA damage and can trigger inflammatory responses. Therefore, it is suggested that vaping has a harmful impact on the functioning of the heart and liver. The mice have also been found to demonstrate changes in behavior and gene expression following after exposure to e-liquid vapor. Maternal e-cigarette use during pregnancy was found to have a significant impact on offspring health. In addition, collected data indicates that inhaled e-liquid enhances various types of cancer event.
In an in vivo study, C57BL6 male mice were exposed to air, cigarette smoke and e-cigarette vapor consisting of PG:VG or PG:VG with 18 mg/mL nicotine (PG:VG-N) or PG:VG-N with flavor (PG:VG-N+F). The exposure time was set at three days or four weeks; after which, oxidative stress, inflammatory markers and pulmonary mechanics were measured. It was observed that PG:VG-N+F increased bronchoalveolar lavage fluid (BALF) cellularity and Muc5ac production, and BALF and lung oxidative stress markers were found to be at a comparable or even higher level than in the case of cigarette smoke. BALF composition correlates with lung function parameters, while the production of one of the mucins, Muc5ac, indicates airway hyperreactivity. The results clearly indicate that e-cigarette vapor has a negative effect on respiratory system mechanisms, can trigger inflammatory responses and that both e-cigarette vaping and cigarette smoking have a harmful impact on lung function [140].
An evaluation of the biological impact of the nicotine delivery system on the lungs of mice exposed to vapor for one hour daily for four months found that inhaled nicotine enhances airspace enlargement, mucous cell hypertrophy and the release of inflammatory mediators, processes correlated with the pathogenesis and progression of COPD [100]. In other studies, mice exposed to e-cigarette aerosols demonstrated increased proinflammatory cytokine levels [98], as well as excessive production of ROS and airway inflammation [141]. E-cigarette vapor exposure was associated with a significant increase of cytochrome P450 enzyme activity, considerable amounts of free-radicals in the lungs, reduced antioxidant capacity and extensive DNA damage in a rat lung model [142]. Salturk et al. report hyperplasia and metaplasia of the laryngeal mucosa of Wistar albino rats following exposure; however, these findings were not statistically significant [143]. In addition, exposure to e-cigarette vapor has been linked with disturbed lung development [144] and cognitive and epigenetic changes in neonatal mice [145]. It appears that e-cigarette exposure influences the overall homeostasis of the organism by altering the physiology of the lung. It is not recommended that e-cigarettes be used during pregnancy.
Although some e-liquids do not contain nicotine, all those used in the selected in vivo studies did, and the nicotine contents of e-cigarettes are variable. Flavorless e-liquids were used for many of these studies ( Table 2).
The adverse impact of exposure to e-cigarette vapor in animals is summarized in Table 2.

In Vivo Studies on E-Cigarette Liquid Exposure on Human Organisms
The effects of e-cigarette on human organisms are largely unexplored. It is difficult to establish which effects are the results of conventional smoking and which are those triggered by e-cigarette use [64]. The literature on the subject both confirms that e-cigarette aerosol has harmful effects on human health while also indicating potential benefits.
Exposure to e-cigarette vapor in human model may impact pulmonary and vascular functions, dysregulate lung homeostasis, increase cardiac risk, and elevated oxidative stress and inflammation. It has also been found that gene expression and molecular pathways regulation were impaired by e-cigarette use.
It appears that short term-exposure to e-cigarette vapor in human model may increase total respiratory impedance, flow respiratory resistance and peripheral airway resistance, and decrease the level of exhaled nitric oxide. E-cigarettes were found to exert a harmful physiological effect on the respiratory system comparable to that of tobacco smoking in an experimental group of healthy smokers [175]. Another study also found that parameters of lung function were impaired in e-cigarette users as compared to a control group of healthy adults [176]. Exposure to e-cigarette vapor has also been found to lead to elevated levels of oxidative stress and inflammation [177] as well as increased cardiac risk [178,179]. Thus, e-cigarette inhalation appears to have the potential to drive lung and vascular pathologies. It is important to note that the concentration of nicotine and the presence of flavors are not given in all studies reviewed herein. However, on the other hand, findings suggest that some e-cigarette components are of good quality. PG/VG and nicotine are mostly of pharmaceutical-grade quality, and the flavorings are the same as those used in food products. Additionally, the emission of acrolein and nitrosamines is significantly lower than in cigarette smoke, metals are present only in trace amounts, acetaldehyde and formaldehyde levels are lower than in the case of exposure to other air pollutants, and solid particles or carbon monoxide are absent [180]. A 42-month observation of e-cigarette users who had never smoked tobacco provided no evidence for impaired lung and heart parameters. It is again important to note that e-cigarettes are not intended as a product for nonsmokers; rather, they can offer some beneficial effects and protect against the detrimental consequences of tobacco smoke for ex-smokers. In addition, their potential to reduce the urge to smoke allows them to offer effective support in reducing nicotine usage and enhance smoking cessation [181]. E-cigarette use has greater success rates than nicotine replacement therapy [182], with many ex-smokers demonstrating decreased biomarkers for toxicity and an improvement in several clinical parameters following the switch from smoking to vaping [183,184]. Three-year studies on COPD patients indicate that e-cigarette use mostly brings benefits or even reverses some harmful effects of cigarette smoking on health: switching to e-cigarettes has been associated with exacerbations being reduced by half and deterioration in respiratory physiology being prevented [185]. In patients with asthma, significant improvements of lung function have also been observed [186].
The adverse impact of e-cigarettes vapor exposure on humans is summarized in Table 3. Table 3. The impact of e-cigarette liquid exposure on e-cigarette users (in vivo studies).

Patients
Characteristic of E-Liquid Action Ref.
Healthy never-smokers (before and after e-cigarettes usage) Unknown Increased: plasma endothelial microparticle levels Altered: transcriptomes of small airway epithelium and alveolar macrophages [187] 3. Healthy never-smokers (before and after e-cigarettes usage) 4.
Healthy e-cigarette users (divided into groups either exposed or not exposed to e-cigarette vapor) Increased: heart rate and arterial stiffness, flow resistance, blood pressure [190] 9.
Healthy sporadic smokers (divided into two groups either exposed or not exposed to e-cigarette vapor)

E-Cigarette Vapor vs. Cigarette Smoke
Tobacco use kills more than eight million people worldwide each year; this number comprises about seven million as a result of direct tobacco use and about 1.2 million passive smokers [192]. Cigarette smoking causes numerous adverse health effects which can be organized in order of time of appearance, i.e., immediate effects, intermediate and long-term effects. The immediate effects include physiological markers of diminished health status, such as increased oxidative stress, depletion of circulating antioxidant micronutrient concentrations, increased inflammation, impaired immune status and altered lipid profiles, in addition to poorer self-rated health status, respiratory symptoms such as coughing, phlegm production, wheezing and dyspnea, as well as nicotine addiction. The intermediate-term effects include absenteeism, increased utilization of medical services, subclinical atherosclerosis, impaired lung development and accelerated decline in function, increased susceptibility to infectious lung diseases, diabetes, periodontitis and asthma exacerbation, as well as a higher chance of various adverse surgical outcomes: wound healing and respiratory complications. Finally, the long-term morbidity effects include cancer, vascular disease, COPD, eye diseases such as age-related macular degeneration and nuclear cataracts, rheumatoid arthritis, as well as impaired bone health, such as a greater chance of hip fractures and reduced bone density [193].
Although e-cigarettes are considered as alternatives to conventional tobacco products, the rapid increase in the use of electronic nicotine delivery systems has brought the need to analyze the effects of nicotine addiction and vaping-related disease [194]. Regarding e-cigarette use, the World Health Organization (WHO) has advised that products containing nicotine are unsafe for young people and pregnant women, exposure to e-liquid may increase the incidence of cancer, cardiovascular and pulmonary disease, and bystanders are exposed to nicotine and toxic substances exhaled by e-cigarette users [192]. Although vaping offers benefits for former smokers, the long-term effects of e-cigarette use remain unclear, and further research is needed to understand them better.
Some studies assess the harmfulness of exposure to e-cigarette liquid in relation to traditional cigarette smoke. One such study has found that exposure to e-cigarette aerosol elicited significantly less cytotoxicity among NHBE cells compared to tobacco smoke [195,196] and no cellular oxidative stress responses [197]. Another study also found 21 e-liquids to be less cytotoxic against cultured myocardial cells than tobacco smoke [198]. In addition, while exposure to cigarette smoke was found to be closely connected with dysregulated expression of numerous genes in NHBE cells, including those encoding proteins taking part in oxidative stress and cell death processes, exposure to e-cigarette vapor elicited minimal modulation of gene expression [199]. Additionally, such findings indicating the lower harm of e-cigarette aerosol has been confirmed in human respiratory 3D tissue models [200]. Another study on human volunteers suggests that e-cigarette smoking is significantly less harmful to lung physiology and may be recommended for tobacco smoking cessation [201]. Interestingly, the results of the in vitro cell transformation assays, useful in assessing the carcinogenic activity of particular chemicals and complex mixtures, suggest that e-cigarettes are a better alternative to traditional cigarettes [202], the compounds in the aerosol may potentiate the genotoxic effects of tobacco carcinogens when used simultaneously with conventional smoke [203]. One such study conducted on rats demonstrates that e-cigarette exposure has a detrimental effect on pulmonary structures comparable to tobacco smoke [204].
Briefly, studies comparing exposure to e-cigarettes vs air control, as in most studies described above, indicate a negative result for vaping, while those comparing them with tobacco smoke give a positive result.

Environmental Impact of E-Cigarettes
The impact of electronic cigarettes on public health includes a range of consequences for the environment, such as air quality effects, energy and materials used, issues related to environmentally responsible disposal and land-use decisions.
It has been estimated that cigarette smoke comprises 98 chemicals constituents listed as hazardous [205], and their impact on human health and the environment is well documented [193,[206][207][208]. Many of the toxic and carcinogenic agents in tobacco cigarette smoke, including polycyclic aromatic hydrocarbons, volatile organic compounds (VOCs), nitrosamines and carbon monoxide, arise as byproducts of combustion [209]. Because e-cigarettes do not have a combustion source, the health risks of vaping are believed to be significantly less than those associated with traditional cigarette smoking. E-cigarette vapor has been found to be less complex than cigarette smoke, with 16 chemicals being present at detectable levels: formaldehyde, acetaldehyde, acrolein, allyl alcohol, glyoxal, methylglyoxal, glycerol, propylene glycerol, chrysene, nicotine, myosmine, cotinine, NNN, chromium, acetone, butyraldehyde; in addition, the vapor typically contains chemicals with lower toxicities, and the emission of hazardous substances was found to be 82% to >99% lower [210].
On the one hand, research indicates that passive exposure to aerosol is not benign. are One of its main air pollutants is airborne particulate matter (PM), comprising small particles consisting of solid and liquid droplets; of these, the most adverse health effects are associated with the inhalation of those of the PM10 size fraction and fractions with lower aerodynamic diameters. It has been found that during use, e-cigarettes produce PMs and moreover PM10 was almost made of PM1 size fraction [3]. There confirm other findings suggesting that e-cigarettes emit PM10, VOCs, nicotine and definitely impair air quality [211]. Analyses of the health risk of e-cigarette use for bystanders, i.e., passive smoking, indicate that exposure may be associated with irritation of the respiratory tract, systemic effects of nicotine action and increased risk of tumors [212].
On the other hand, studies suggest that e-cigarette emission is composed mainly of PG and/or VG. Moreover, the emission profiles of the components differ between e-cigarettes and conventional cigarettes, with respective lifetimes of approximately 10-20 s and 1.4 h [213]. Exhaled particles and liquid droplets produced by e-cigarettes are smaller than for conventional tobacco and evaporate almost immediately after exhalation, unlike for conventional cigarettes [214]. Particularly noteworthy here is the problem of vaping in small rooms, in close proximity to other people, and around children [215].
Data gained by analyzing the environmental impact of e-cigarettes will serve to identify current and future environmental implications associated with the production, use and disposal of e-cigarettes; this will clearly be of value for regulatory authorities, e-cigarette producers and the public [216].

Conclusions
The present paper reviews the mechanisms of e-cigarette action, effects of exposure on e-liquid, the impact on living organisms and the environment. It summarizes evidence obtained from cell cultures, animal models and human subjects, compares vaping with smoking and assesses the impact of e-cigarettes on air quality as an environmental aspect connected with human health. Further studies should examine the effects of aerosols on living organisms more thoroughly; only relatively few studies have so far been conducted, particularly on humans, and the findings remain unclear and need further analysis. As the effects of e-cigarette use on health and the environment will probably only become apparent in the near future, it is important to provide accurate data to fully educate the public about their use.