Literature Review on Nicotine’s Role in Human Health

Relevant articles were retrieved from various databases, including: ○

PubMed

Recent reviews and monographs dealing with the topic of interest were evaluated, including: ○

US Surgeon General Report of 2014 “The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General” (24)

Reference lists of recent suitable articles from the above sources were screened for further relevant articles on the topic of interest.

In total, about 500 articles were retrieved for more detailed screening by applying the inclusion criteria described in the following section.

Human studies considered for evaluation must have investigated users of the NGPs electronic cigarettes (ECs), heated tobacco products (HTPs), nicotine pouches (NPs), nicotine replacement therapy (NRT) products (skin patches, chewing gum, lozenges, inhaler), smokeless tobacco (SLT) products (snus, oral snuff). Furthermore, at least one biological endpoint (disease, disorder, tissue or cell damage, physiological changes, biomarkers of effect or potential harm) must have been studied. Observed changes related to product use must be compared to at least one of the comparator conditions: non-users (NU), smokers of combustible cigarettes (CC), baseline (BL) prior to NGP use. Animal (in vivo) and in vitro studies were only considered for evaluation in this review, if the study design allows to allocate the contribution of nicotine to an observed effect with relevance to understand human effects.

Three aspects were considered in order to evaluate the role of nicotine in effects observed in human users of nicotine products: (i) sufficient scientific evidence that nicotine directly participates in a physiological (or pathophysiological) mechanism (e.g., by acting via a nicotine-specific receptor), (ii) evidence cited by the authors of a study showing that nicotine is likely (or unlikely) to be involved, (iii) judgement by the authors of this review considering (i), (ii) and the presented study data, using a simple coding system (see explanation in the following section).

The human studies considered in this review are briefly summarized in Tables 1–8. All tables are of the same structure and provide the following information:

Tables 1–8 are divided in seven subsections of various diseases, disorders and biological markers. The results of which are summarized below. The vast majority of the studies on CVD, but also for the other diseases discussed in this review, have been performed with ECs. However, with respect to the participation of nicotine in pathogenesis, most of the findings are assumed to be valid also for the other nicotine-containing NGPs.

Cardiovascular diseases (CVD) are responsible for a significant proportion of (premature) deaths worldwide. Atherosclerosis is a key factor in the development of CVD, and it primarily develops in vascular regions with disturbed blood flow (25). Oxidative stress, endothelial cell activation, and inhibited release of endothelial nitric oxide (NO) are key molecular events that contribute to atherosclerosis (29). Cigarette smoking plays a significant role in all stages of plaque formation in atherosclerosis. Smoking causes oxidative stress, upregulation of inflammatory cytokines, endothelial dysfunction, and reduces the bioavailability of NO (30). This leads to the formation of vulnerable plaques, platelet activation, stimulation of the coagulation cascade, and impaired anti-coagulative fibrinolysis. Nicotine is involved in almost all steps of this process except fibrinolysis. Smoking causes vascular dysfunction by reducing NO bioavailability, which is one of the first steps initiated by smoking in the pathogenesis to CVD. Smoking also leads to the formation of foam cells (macrophages, which are loaded up with oxLDL), which is another step in early atherogenesis (31, 32).

Smoking also has a number of cardiovascular effects, including acute ischemic events and more chronic atherogenesis-related effects, such as systemic hemodynamic effects, coronary blood flow, myocardial remodeling, arrhythmogenesis, thrombogenesis, endothelial dysfunction, inflammation, angiogenesis, dyslipidemia, hypertension, and insulin resistance (25, 33,34,35,36). Acute smoking increases plasma norepinephrine and epinephrine, as well as heart rate, blood pressure (BP), blood glycerol, and the blood lactate/pyruvate ratio. These effects can be regarded as inherent nicotine effects, mediated by nicotine-specific receptors (nAChRs) (37). The dose-response between cigarettes per day and the risk of CVD is reported to be nonlinear (25).

A key role in pathogenesis of atherosclerosis is assigned to the vascular extracellular matrix (ECM) (38). Of importance are matrix metalloproteases (MMPs), which upon activation mediate degradation of ECM of the atherosclerotic plaque, inflammation and proliferation of smooth muscle cells, all of which exacerbate atherosclerosis and MI (38).

Smoke constituents involved in CVD include nicotine, CO, and particulate matter. Smoking is a major risk factor for CVD and is found to be responsible for 15–20% of all CVD cases. Smoking cessation can reverse most of the steps in the development of CVD, opening the chance that switching from smoking to a harm-reduced product could be beneficial.

Table 1 contains short summaries and evaluations of human studies on NGP use and CVD endpoints (see page 42).

In the following, results of animal studies were briefly summarized, which try to work out the effect of either pure nicotine or nicotine as a constituent of NGPs (most frequently ECs with and without nicotine) on the development of CVD.

A mouse model (ApoE^−/− mice) was reported to show nicotine and cotinine plasma levels after intermittent exposure to EC aerosol very similar to human vapers, thus representing a suitable tool for in vivo animal studies for investigating vaping effects on CNS, CVD, metabolism and carcinogenesis (125). In this study, EC-exposed mice (2.4% nicotine, 12 weeks) showed reduced body weight and food intake as well as increased locomotion compared to saline controls.

Rats were treated with nicotine (0.6 mg/kg, i.p.) for 28 h (126). Compared to a saline control, chronic nicotine administration impaired aortic reactivity, probably via redox imbalance (increased MDA, decreased SOD and GSH) and vascular remodeling mechanism.

In a long-term (60 weeks) study with mice exposed to EC aerosol derived from e-liquid with 0, 6 and 24 mg/mL nicotine, impaired endothelium-dependent and endothelium-independent vasodilation were observed with nicotine-containing exposure occurring earlier and more severe than without nicotine. The effects were similar to those found with smoke (CC) exposure (127). The authors concluded that long-term vaping can induce CVD similar to smoking. The same mouse model and exposure regime was applied in another study to further elucidate the mechanism how EC aerosol induced vascular epithelial dysfunction (VED) (128). According to the authors’ interpretation, EC aerosol activates NADPH oxidase and uncouples eNOS, causing superoxide generation and vascular oxidant stress that triggers VED and hypertension with predisposition to other CVD. The observed effects were nicotine dose-dependent, with the zero nicotine dose still having detrimental effects to the air exposure control group.

A main issue when comparing exposure effects, particularly those related to CVD, observed in animal studies with those measured in human vapers or users of other NGPs is that the treatment of animals is usually associated with stress responses which are also risk factors for CVD (1).

In a 12-week study with ApoE knock-out mice exposed to EC aerosol with 0 and 2.4% nicotine and saline (control), the nicotine group but not the nicotine-free and saline groups showed detrimental changes such as decreased left ventricular fractional shortening and ejection fraction, increase in serum FFA and cardiac MDA as well as atherosclerotic lesions (129, 130). The authors concluded that these results indicate profound adverse effects of e-cigarettes with nicotine on the heart in obese mice and raise questions about the safety of the nicotine e-cigarettes use (129, 130).

A 6-month study with ApoE knock-out mice exposed to CC smoke, PG/VG aerosol with (ECN) and without nicotine (EC0) revealed that ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged following EC0 exposure, while ECN caused an increase in isovolumic relaxation time similar to CC smoke exposure (131). ECN also increased PWV and arterial stiffness, but to a significantly lower extent than CC smoke did. The authors conclude that EC aerosol exposure induce substantially lower biological responses associated with CVD compared to CC smoke. The contribution of nicotine in the EC aerosol to the observed effects appear to be medium to small.

Espinoza et al. (132) reviewed mostly animal study-based effects of EC aerosol exposure and came to the conclusion that mechanisms such as oxidative stress, inflammation, lipid accumulation, and sympathetic dominance relevant for the generation of CVD may be aggravated by vaping and that nicotine plays a detrimental role in this process.

Mice treated with nicotine for 14 days were found to have profound aggravation of the immune response after ischemia/reperfusion injury (133). According to the authors, these observations are not only relevant for stroke occurrence but also for nicotine-related inflammatory responses in other organs.

In an attempt to identify which smoke or aerosol component might be responsible for the acute impairment of the endothelial function (measure as a decrease in FMD in rats), nicotine (as high and low levels in CC smoke), particles (as inert carbon particles), acrolein and acetaldehyde were measured (134). All agents tested showed similar reductions of FMD compared to air. The effect was prevented by bilateral vagotomy. The authors conclude that acute endothelial dysfunction by disparate inhaled products is caused by vagus nerve-signaling initiated by airway irritation.

Wu et al. (135) showed in a mouse model that nicotine exerts its pro-atherosclerotic property via a pyroptosis mechanism mediated by ROS-stimulated inflammation processes.

In a mouse model (ApoE^−/−), exposure to EC aerosol (2 h/d, 5 d/week, 16 weeks; e-liquid with 2.4% nicotine) was found to increase level of damaged mitochondrial DNA in circulating blood and induce the expression of TLR9, which in turn elevates the release of pro-inflammatory cytokines (IL-6, TNF-α) in monocytes/macrophages and consequently lead to atherosclerosis (136). In addition, the authors report enhanced TLR9-expression in human femoral artery atherosclerotic plaques from EC users and a significant increase of oxidative mitochondria DNA lesions (8-OHdG) in the plasma of EC-exposed mice. Unfortunately, the authors did not include a nicotine-free EC group or discuss the potential role of nicotine in the observed effects. Rats were exposed to EC aerosol with and without nicotine, CC smoke and fresh air (137). E-cig exposure did not increase myocardial infarct size or worsen the no-reflow phenomenon, but EC aerosol with nicotine (not so without nicotine, CC smoke or fresh air) induced deleterious changes in LV structure leading to cardiovascular dysfunction and increased systemic arterial resistance after coronary artery occlusion followed by reperfusion.

In summary, animal studies have limitations when intended to extrapolate to human nicotine product users since animal treatment (particularly by inhalation) can be associated with stress responses, which are also risk factors for CVD. In mice and rats, chronic nicotine administration impaired aortic reactivity, probably via redox imbalance, and vascular remodeling mechanisms. Long-term vaping in mice exposed to EC aerosol derived from e-liquid with nicotine showed impaired endothelium-dependent and endothelium-independent vasodilation (similar to smoking) and increased the risk of CVD. Nicotine played a detrimental role in the process of oxidative stress, inflammation, lipid accumulation, and sympathetic dominance. However, the contribution of nicotine in EC aerosol to the observed effects appears to be medium to small. In mice treated with nicotine, there was profound aggravation of the immune response after ischemia/reperfusion injury, a process important for stroke.

Overall, animal studies suggest that nicotine is involved in a number of patho-physiological processes related to the development of CVD.

In the following, some findings of in vitro studies on nicotine’s role in CVD-related processes are briefly described.

Nicotine was reported to stimulate DNA synthesis and proliferation of vascular endothelial cells in vitro (138), even at concentrations significantly lower than smokers’ nicotine plasma levels (< 10⁻⁸ M). The author suggests that the results may be important in tumor angiogenesis, atherogenesis and vasculargenesis.

In vitro studies with aortic smooth muscle cells exposed to EC aerosol condensate showed no influence of nicotine on cytotoxicity, LDH, ROS and IL-8 release. In contrast, high EC power and cinnamon flavor increased pro-inflammatory effects (139).

In in vitro and in vivo (mice) models, it was shown that acute and chronic exposure to nicotine can lead to edema formation in the brain, most probably mediated by nAChRs (140). The authors conclude that these findings support the paradigm that nicotine products not only increase the incidence of stroke but also have the potential to worsen stroke outcome by increased edema formation.

Kaisar et al. (141) reported dose- and time-dependent effects of nicotine on the blood-brain barrier (BBB) endothelium of mice consisting of loss of BBB integrity and vascular inflammation and promotion of the onset of stroke. In conclusion, in vitro studies suggest that nicotine can stimulate proliferation of vascular endothelial cells, thus potentially contributing to atherogenesis. However, in vitro studies with aortic smooth muscle cells showed no influence of nicotine on cytotoxicity and ROS formation. Nicotine exposure has been linked to edema formation in the brain and can also have effects on the BBB, thus promoting the onset of stroke.

A series of reviews and monographs dealing with the CVD risks of nicotine products (with particular focus on ECs) are available. A brief summary is provided below.

Benowitz and Gourlay (142) evaluated the safety of NRT products in terms of cardiovascular effects of nicotine and came to the conclusion that the risk of NRT for smokers, even for those with CVD are small and substantially outweighed by the potential benefits of smoking cessation. The EU-based Scientific Committee on Health, Environmental and Emerging Risks (SCHEER) (143) reviewed the most recent scientific and technical information on ECs and concluded for CVD: “The overall weight of evidence for risks of long-term systemic effects on the cardiovascular system is moderate.” However, SCHEER evaluated the evidence that nicotine is involved in the development to CVD as “strong”.

In a recent review, Buchanan et al. (144) evaluated pre-clinical and clinical studies on EC aerosol exposure and cardiovascular risk. The authors came to the conclusion that the impact of chronic EC exposure is essentially unstudied. The available, mostly acute studies suggest that exposure to EC could be a potential cardiovascular health concern. Further, mostly long-term studies were needed before considering ECs safe alternatives to CC. The role of nicotine was related to its activation of the sympathetic nervous system causing increased HR, BP, and myocardial contractility.

The NASEM report of 2018 (6) came to the following conclusions with respect of the association between EC use and CVD: ○

No evidence for an association between vaping and clinical cardiovascular outcomes (CVD, stroke, peripheral artery disease);

According to the National Academies of Sciences, Engineering, and Medicine (NASEM) (6), while nicotine can induce a more atherogenic lipid profile, cessation studies using nicotine replacement therapy (NRT) found improvements in high-density lipoprotein (HDL)/low-density lipoprotein (LDL) ratios and reduced dyslipidemia. NASEM also suggests that nicotine-induced vasoconstriction may play a role in the progression of chronic hypertension to malignant hypertension. Nicotine also appears to be responsible for increased insulin resistance in smokers, as it releases several hormones that are insulin antagonists and activates certain receptors in adipose tissue. NASEM notes that exposure to nicotine from e-cigarettes likely elevates the risk of cardiovascular disease in people with preexisting cardiovascular diseases, but the risk in people without cardiovascular disease is uncertain.

The Committee on Toxicity of Chemicals in Food (COT) stated that no long-term studies with inhaled nicotine or vaping products are as yet available (7). Studies on CVD and NRT use as an aid to quitting cigarette smoking, were, according to COT, “mostly of inadequate quality to draw clear conclusions but have not shown evidence of serious cardiovascular events”. The committee further stated that the risk related to nicotine when switching from CC to EC use would not be expected to increase, however, there are currently no data on adverse effects in NU who switch to NGPs containing nicotine.

An umbrella review of Peruzzi et al. (145) on vaping and CVD evaluating 7 systematic reviews suggests that EC use, and likely also HTP use, despite clearly causing an increase in overall cardiovascular risk, may represent a temporary lesser evil than CC. The authors mention that the role of nicotine was mostly not clearly disentangled. As key adverse cardiovascular effects of ECs and HTPs and probably also other nicotine-containing products, the authors named acute myocardial infarction, hypertension, oxidative stress, endothelial dysfunction, arterial stiffness, and thrombosis. According to the authors an involvement of nicotine appears likely. Similar effects were also discussed in another recent review (146).

A recent review investigated the impact of ECs on CV health with a special focus on causal pathways and public health implications (147). The authors stated that additives in the e-liquid such as nicotine and flavors are mostly responsible for the effects, which include prolonged sympathoexcitatory CV autonomic effects such as increased HR and BP as well as decreased oxygen saturation. Vaping is therefore a risk factor for developing atherosclerosis, hypertension, arrhythmia, MI and heart failure. According to the authors, studies on the long-term effects of EC use are urgently needed (147).

With respect to the urgent requirement of long-term studies our review comes to the same conclusions as the previously cited review (147). However, in our opinion it is not justified to state that vapers are at increased risk of developing various CVDs as Critselis and Panagiotakos (147) did. Our literature evaluation with special focus on the role of nicotine in the development of CVD, basically came to the same conclusions. The use of NGPs such as ECs, HTPs, nicotine pouches, snus, and NRT products has gained increasing popularity over the years. The potential risks associated with the use of these products in relation to CVD have been a topic of concern. This chapter aimed to explore the involvement of nicotine in the pathogenesis of CVD and the role of nicotine in contributing to CVD.

Quite a number of human, animal, and in vitro studies have been conducted to investigate the effects of nicotine on the cardiovascular system. Nicotine has been found to participate in acute effects such as an increase in HR and BP as well as a decrease in NO production. Also decrease in FMD of arteries was frequently reported upon use of nicotine-containing products. Moreover, nicotine may also be involved in more chronic processes such as atherogenic plaque formation, disturbance of lipid metabolism, oxidative stress, inflammation, and thrombogenesis. Although the evidence for a nicotine involvement in these processes is much weaker.

Smoking CC is reported to be responsible for 15–20% of the total population CVD. Epidemiology shows that there is no linear dose-response relationship between smoking (CC) and CVD. Smoking-related effects on the cardiovascular system are mostly reversible, this means that switching from CC to NGPs could be beneficial.

Human studies on HR and BP revealed acute increases in NGP users, but lower than in smokers (CC). Causal participation of nicotine through its sympathomimetic properties is plausible.

Use of SLT products (including snus), NRT products, and ECs showed partly inconsistent results and partly significant increased risks for MI, stroke, atherosclerotic changes, and hypertension. The role of nicotine in the development of these CVD is unclear. In cross-sectional studies, there are issues with causality, temporality as well as misclassification in product use (false self-reports and vague recollection of product use, particularly in exclusive NGP user groups).

Arterial stiffness is an important predictor of CVD. Acute studies showed an increase in PWV and AI compared to sham use, the effect, however, was found to be lower than smoking (CC). An involvement of nicotine appears to be possible. The effect of chronic NGP use was as yet hardly investigated and results so far are inconclusive. Long-term studies are required to determine the involvement of nicotine, if any, in the development of arterial stiffness.

BOBEs have been used to assess the potential risks associated with the use of NGPs. Some improvements (indicating lower CVD risk) have been observed in medium-term (90–360 days) and long-term (24 months) studies in subjects who switched from CCs to NGPs (ECs or HTPs). No clear evidence for the participation of nicotine is reported or can be deduced from the data. More long-term studies are required to determine the involvement of nicotine in BOBEs.

Animal and in vitro studies provide some direct evidence that nicotine (either as a pure compound or released from NGPs) is involved in a number of pathogenic pathways leading to CVD. However, limitations in terms of transferability to humans and the adequate nicotine doses have to be considered.

In conclusion, the use of NGPs has been associated with both acute and chronic effects on the cardiovascular system. While nicotine has been implicated in acute effects, its role in chronic processes leading to CVD is still unclear. Long-term studies are required to better understand the effects of NGPs on the cardiovascular system and the involvement of nicotine in these effects. Epidemiological and field studies may potentially be biased by misclassification of products user groups (particularly NGP only users). An objective assessment of long-term product use would be of major importance.

Tobacco smoking can be involved in carcinogenesis by various mechanisms (25). CC smoking is associated with the uptake of more than 70 carcinogens, which, either as un-metabolized parent compound or after metabolic activation, can form DNA adducts. DNA adducts, as a rule, are repaired, but upon persistence, they can form the origin of mutations. If mutations occur in genes which are critical for tumorgenesis such as oncogenes or tumor-suppressor genes, a loss of normal cell growth can be the consequence, which finally may end up in cancer. Apart from tumor initiators, tobacco smoke also contains cocarcinogens (e.g., catechol, alkyl catechols) and tumor promotors, which primarily do not interact with DNA but stimulate cell proliferation and thus tumor growth. An important difference between genotoxic tumor initiators and cocarcinogens and tumor promotors is that effects of the latter two are reversible. Furthermore, it is scientifically accepted that no threshold dose exists for carcinogen, below which no cancer risk can be assumed (151). The fact that smoking cessation decreases the cancer risk, indicates that cocarcinogens and promotors play an important role in smoking-related carcinogenesis (25). Furthermore, epigenetic effects, which impact gene-regulation by either increasing or decreasing DNA methylation can significantly influence tumorigenesis.

Also epigenetic effects are supposed to be mainly reversible. Other possible mechanisms how smoking can have an impact on cancer development include induction of phase 1 enzymes (e.g., cytochrome P450 enzymes) responsible for the formation of the ultimate carcinogens, inhibition of phase 2 enzymes (responsible for conjugation and detoxifications of carcinogens), inhibition of repair enzymes, impairment of the immune system and stimulation of tumor-angiogenesis.

A simplified scheme of the described mechanistic pathways to smoking-related cancers (in general chemical carcinogenesis) is shown in Figure 1 (modified from (25)).

Figure 1.

In quite a large number of studies, the exposure to carcinogens has been investigated in users of NGPs, usually compared to smokers (CC) and NU (e.g., (152,153,154,155,156,157,158)), for review see (16). The aspect of exposure to toxicants, including carcinogens, is not discussed in this review. Overall reduction in the product use-related exposure to carcinogens is 95% in NGP users compared to smokers (CC). An exception are tobacco-specific nitrosamines (TSNAs) in SLT (excluding Swedish snus) which might release similar or even higher amounts of TSNAs compared to CC. This is mentioned several times throughout this review.

Braznell et al. (159) tried to estimate the lung cancer risk in HTP users on the basis of 16 biomarkers (7 BOEs and 9 BOBEs) which are known to correlate with lung cancer risk in smokers. From their results, the authors concluded that the appropriateness of this approach is limited.

Cameron et al. (160) summarized the latest evidence about the deleterious effects of vaping on oral health and the risks of oral cancer and came to the conclusion that e-cigarette use is not risk-free.

Table 2 contains 10 studies, 4 of them investigated cancer as an endpoint (56, 161,162,163), whereas the other studies investigated epigenetic effects (mainly DNA methylation) in blood (164, 165) or saliva (166) or in epithelial cells from the oral cavity (167) and the lung (168) (see page 62). One cancer study (169) was not a study with a classical epidemiological approach (prospective or case-control), but was a longitudinal observational study with a limited number of participants (in total 912 subjects: smokers, vapers, dual users).

All cancers as endpoints were evaluated in studies with SLT (56, 161, 162), NRT (nicotine gum) (163) and ECs (169). None of these studies showed significant associations between NGP use and cancer risk. Only in three studies (56, 161, 169), the overall cancer risk of smoking (CC) was investigated, one showed a significant increase (56).

No significant relationship between SLT use and pancreatic cancer was found, although a significant trend with increasing consumption of SLT was observed (162). Cigarette smoking (CC) was not investigated in this study.

No significant association between NRT use and cancer of the lung and the gastrointestinal (GI) tract was reported (163). CC use was found to significantly increase the risk of lung cancer, but not of GI cancer.

DNA methylation and implicated gene regulations were investigated in the 5 studies of Table 2. In all of them, EC use compared to smoking (CC) was in the focus of the study. One study also included SLT users (165). The provided data show clear evidence that EC use (and probably also use of other NGPs) are distinct from CC users and NU with some overlap. An increase in genotoxicity levels associated with vaping habits was also deduced from a recent study of peripheral blood samples from vapers (EC), smokers (CC) and NU (170). Vapers showed changes at the epigenetic level specifically associated with the loss of methylation of the LINE-1, which were reflected in its representative RNA expression detected in vapers.

In terms of biological effects, changes, perturbations, physiological malfunctions, disorders or even cancer formation, there are too many gaps for any reasonable predictions.

With respect to an involvement of nicotine, the evaluated studies show either no evidence for a participation of nicotine in tumorigenesis and/or a role of nicotine cannot be deduced from the provided data (0/?). There might be some weak evidence that TSNAs from SLT are involved in the development of pancreatic cancer (162). The carcinogenic potential of smokeless tobacco (SLT) has been reviewed by Hecht et al. (171). The TSNAs NNN and NNK were considered to be the most potent carcinogens in SLT.

There are a number of epidemiological studies on cancer and use of oral tobacco with controversial results, depending on the point in time of study and the geographical region (e.g., USA, Scandinavia, India) where the study was conducted (172, 173). Older SLT products could have high TSNA levels resulting in significantly higher NNK and NNN exposure of the users than exhibited by smokers (CC). Therefore, these SLT products cannot be regarded as NGPs suitable for tobacco harm reduction, at least not for cancer endpoints. With respect to the focus of this review (role of nicotine in the development of diseases), cancer studies with older SLT products will not be included in the evaluation. As a result, we decided to only consider SLTs with very low levels of TSNAs as NGPs.

The potential carcinogenicity of chronic NGP use and the avertable role of nicotine was evaluated in a number of recent monographs and reviews.

The NASEM (6) concluded that nicotine probably does not increase the risk of cancer. This statement is mainly based on the Lung Health Study (163), which followed users of NRT products for 7.5 years, found no evidence for an increased cancer risk. The NASEM further stated that nicotine might be a tumor promotor, but it would be unlikely to increase the human cancer risk (6). This evaluation is also shared by COT (7).

The EU-based Scientific Committee on Health, Environmental and Emerging Risks (SCHEER) (143) reviewed the most recent scientific and technical information on ECs and concluded for cancer in the respiratory tract: “The overall weight of evidence for risks of carcinogenicity of the respiratory tract due to long-term, cumulative exposure to nitrosamines and due to exposure to acetaldehyde and formaldehyde is weak to moderate. The weight of evidence for risks of adverse effects, specifically carcinogenicity, due to metals in aerosols is weak”.

Travis et al. (174) performed an umbrella review on cardiopulmonary and carcinogenic risks of EC use. The authors state that there are some acute cardiopulmonary risks for vapers, although lower than in smokers. It is expected that in the long-term switcher from CC to EC will probably benefit, however, studies to show this are as yet lacking.

In a model calculation, the following order of life-time cancer risks was reported (175): combustible cigarettes (CC) o heat-not-burn (HTP) o e-cigarettes (EC) $ nicotine inhaler (NRT), with EC use representing < 1% of the cancer risk associated with CC use.

In a recent review by Maan et al. (176), molecular insights of EC use on oral carcinogenesis was investigated. The authors provided a summary of the effects of EC usage on cancer therapy resistance, cancer stem cells (CSCs), immune evasion, and microbiome dysbiosis, all of which may lead to increased tumor malignancy and poorer patient prognosis. It was concluded that ECs may not be as safe as they are perceived to be, however further research is needed to definitively determine their oncogenic potential. EC use was also shown to lead to transcriptomic changes in both blood and sputum, which was only partly overlapping with corresponding changes in smokers (CC) (177). Observed transcriptomic alterations were stronger in CC than in EC users. The biological impact of these changes, however, are as yet unknown.

Taken together, the presented evidence suggests that chronic use of NGPs has as yet not shown to increase the cancer risk. Although theoretical possible, there is also no evidence that long-term use of nicotine is involved in human carcinogenesis. However, for obvious reasons no long-term cancer studies with NGPs are currently available.

As mentioned above, nicotine can exert quite a number of effects in the mammalian organism, impacting the brain, cardiovascular system, lung and many others. Nicotine may be involved in the modulation of many physiological processes such as the activity of certain ligand-gated ion channels known as nicotinic acetylcholine receptors (nAChRs) modulating cell proliferation, apoptosis, immune response, oxidative stress, tumor proliferation, metastasis, promotion of lung cancer, cell proliferation, angiogenesis, migration and invasion (178). Several of these are directly or indirectly associated with cancer development.

In the following, evidence is presented from animal and in vitro studies, which investigated the role of either pure nicotine or NGP exposure with and without nicotine in tumorigenesis.

Maier et al. (179) reported no effect of nicotine in a mutant K-Ras1-driven mouse model for lung cancer as well as in mice treated with NNK and additionally with oral nicotine. The authors conclude that nicotine, applied in doses similar to those in NRT use, does not enhance lung tumorigenesis.

In similar experiments, Murphy et al. (180) found no enhancing effect of nicotine administered 2 weeks prior to and 44 weeks after NNK treatment of mice on multiplicity of lung tumors. Nicotine alone over 46 weeks did not increase the lung tumor multiplicity. The authors concluded that these results question the interpretation of in vitro studies suggesting that nicotine stimulates cancer cell growth.

In a study with mice exposed to EC aerosol, Lee et al. (181) found O⁶-medG and γ-OH-PdG elevated in lung, heart and bladder. DNA repair activity and proteins were reduced in the lung. Nicotine and NNK were reported to have similar effects in human lung and bladder cells in vitro. The authors suggest that EC use may contribute to lung and bladder cancer as well as heart disease through the nitrosation of nicotine (181). The same working group (182) exposed mice (FVB/N strain, highly susceptible to tumor induction) to EC aerosol, PG/VG aerosol (without nicotine) and fresh air for 54 weeks. The EC group developed lung adenocarcinomas (9 of 40 mice) and bladder urothelial hyperplasia (23/40), whereas these lesions were extremely rare in the PG/VG and fresh air group. According to the authors, these results together with previous findings (181) suggest that EC aerosol is a lung and potential bladder carcinogen for mice, whether it is also a risk factor for human vapers needs to be investigated.

Exposure of male ApoE^−/− mice to air, EC aerosol derived from e-liquids with 6 and 36 mg nicotine/mL showed nicotine dose-dependent increases in epigenomic DNA methylation in WBC as well as an increase in plasma mitochondrial DNA and 8-OHdG levels (183). The authors concluded that the epigenomic-wide CpG site methylation pattern overlaps with previously published methylation sites in vapers/smokers and that the methylation pattern correlates well with enhanced systematic inflammation reported in animal models and human vapers.

Nicotine has also been suspected to be a precursor for carcinogenic metabolites such as NNK and NNN. Hecht et al. (184) showed with human liver microsomes containing CYP 2A6 that nicotine is 2’-hydroxylated yielding the ring-opened aminoketone, which, upon nitrosation, could form the lung carcinogen NNK. The final step, which according to the authors would have important toxicological consequences, however, has not been observed until now in humans.

Tang et al. (185) stated that EC aerosol is carcinogenic in mice. As possible mechanism these authors propose that nicotine can be nitrosated to NNK and NNN, which, upon further metabolism, form ultimate carcinogens and both groups of agents can form DNA adducts. Additionally, formaldehyde is a potential inhibitor of DNA repair, thus further accelerating the carcinogenic process. The authors provide some evidence from the literature which should support their hypothesis.

Already in 2011, Shields (186) emphasized that, despite of the fact that animal studies provide no cause for concern (179, 180), long-term cancer studies with NRT users are urgently needed to show that nicotine does not promote the carcinogenesis. More than 10 years later, the situation has not much changed with respect to human cancer studies. In terms of evidence derived from animal and in vitro studies, it can be noted that the investigations of Lee et al. (181) and Tang et al. (182) give cause for concern that nicotine in EC aerosol might be involved in lung and bladder carcinogenesis. Whether these findings are transferable to human vapers (or in general NGP users) remains questionable.

Based on various human, animal and in vitro studies, NASEM came to the following role of nicotine in the process of cancer induction (6): “When the evidence is viewed in total, while there is a biological rationale for how nicotine could potentially act as a carcinogen in humans, there is no human evidence to support the hypothesis that nicotine is a human carcinogen. While it is biologically plausible that nicotine can act as a tumor promoter, the existing body of evidence indicates this is unlikely to translate into increased risk of human cancer”.

Cigarette smoking is an established risk factor for cancer of various organs, including lung, bladder, kidneys and pancreas. Tobacco smoke contains more than 70 carcinogens which are involved in the turmorigenesis by acting as initiators, cocarcinogens and promotors. Epigenetic effects mediated by the DNA methylation grade leading to up- and downregulation of genes possibly involved in tumorigenesis gained increasing interest and was also shown to be significantly altered by smoke exposure.

A participation of nicotine has long been assumed and could occur on a number of different mechanisms, including metabolism to carcinogens (NNK, NNN), epigenetic effects, angiogenesis, cell growth stimulation.

For obvious reasons, only a few human cancer studies with NGPs (including SLTs, NRT products and ECs) are available. There is no evidence that these products significantly increase the cancer risk, suggesting that nicotine is not a driving factor in carcinogenesis. The results of long-term animal studies with cancer endpoints are as yet inconclusive.

Human and animal studies reveal that exposure to EC aerosols (other NGPs have not yet been investigated) significantly change the epigenetic DNA methylation profile in cells of target organs (lung, oral cavity) or blood cells. Overlap with smokers or NU is reported to be low, suggesting that the methylation profiles are rather specific for the exposure. Consequences in terms of disorders, diseases or even cancer are currently unknown.

Overall, while the studies presented suggest that NGPs may be a less harmful alternative to smoking in terms of cancer risk, more research is needed to understand the long-term effects of nicotine and other components of these products on cancer development.

As major mechanisms for effects in the respiratory tract, which are common in users of inhalable nicotine products (CCs, ECs, HTPs), Davis et al. named dysregulated inflammation and decreased pathogen resistance in their recent review. In addition, dysregulated lipid processing might be unique to vaping (187). The authors relate the latter effect mainly to the humectants PG and VG in the EC aerosol. The impacted processes in the lung after aerosol exposure accordingly would comprise: (i)

dysregulated mucin expression followed by impaired ciliary clearance

Smoking-related (nonmalignant) respiratory diseases include chronic bronchitis, COPD, emphysema and the worsening of asthma. According to the US Surgeon General Report of 2010 (25), definitions of these RD are as follows:

Tobacco smoke toxicants such as acrolein and formaldehyde are regarded to be responsible for initiating oxidative stress (e.g., by the formation of ROS), inflammatory processes and a proteinase/anti-proteinase imbalance. These processes are key players in the development of respiratory diseases in smokers (188, 189).

Intact lung defense systems, including nasal hair, convoluted passages of nasal sinuses, coughing, sneezing, swallowing, mucociliary cells, normal flora, inflammatory cells, alveolar macrophages represent the first barrier against the inhalable toxicants (25). Impairment of these defenses for example by smoking are the first step for the development of various respiratory diseases (RDs).

A decline in lung function is also a typical consequence of long-term smoking. Spirometrically determined parameters such as forced expired volume in 1 second (FEV1), forced vital capacity (FVC) and forced expired flow at 25–75% (Figure 2) can be measured non-invasively and, therefore, are frequently applied in clinical and epidemiological studies.

Figure 2.

As in smoking, the aerosol of inhalable NGPs (ECs, HTPs) may affect various areas of the lungs by interaction with the epithelial cells of the trachea, bronchial tubes and branchings as well as the alveoli (191, 192).

In principle, vaping and use of other inhalable NGPs can induce respiratory diseases by similar mechanisms as for smokers (CC). Inflammatory processes, which can be monitored by a series of biomarkers are suggested to be in the focus (193, 194).

It is almost self-evident that oral NGPs such as snus or nicotine pouches (NPs) as well as nicotine gums (NGs) have no direct detrimental effects on the respirtory tract. The same applies for nicotine taken up with these products. NASEM stated three pathways, how nicotine could be involved in respiratory diseases in vapers: (i)

decreases viral and bacterial clearance

From two short-term studies, the NASEM (6) and the COT (7) concluded that using ECs with nicotine but not without nicotine could impair the mucociliary clearance process in the lung.

Overall, the NASEM report stated only limited to moderate evidence for lung damaging effects of vaping (6).

The majority of vapers are former smokers and thus could bear pre-existing lung damages which could be aggravated by vaping and, therefore, would lead to a confounded evaluation of the risks associated with EC (1) or HTP use. Mir et al. summarized the EC effects on the respiratory tract as follows (53): “Increased susceptibility to respiratory infections, case of e-cigarette related organizing pneumonia, pro-inflammatory effects via oxidative stress, increased risk of developing COPD, diffuse alveolar damage, mucociliary dysfunction, fluctuations in surfactant composition lead to gas exchange abnormalities, lung damage and cell death, reduced functional residual capacity.”

Table 3 contains 40 entries with short descriptions of investigations on respiratory tract effects of NGP use (101, 119, 195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232) (see page 65). In all studies EC use was compared to either smoking (CC) or NU (or both). In only one study, an HTP was investigated (215). Studies are mostly of cross-sectional type (21 studies). However, also 5 longitudinal studies with observational periods between 1 and 5 years were included (196,197,198,199, 223). It is noteworthy that there are 5 studies (201, 208, 209, 222, 226), all of the cross-over type, in which the effects of ECs with and without nicotine were compared. These studies would provide direct evidence on the role of nicotine in the observed effects. Finally, Table 3 also contains two meta-analyses comprising 13 (231) and 11 cross-sectional studies (232).

Both meta-analyses investigated the association between vaping and asthma and found a significantly increased risk (odds ratio (OR) = 1.2–1.3), which is only slightly lower than that observed for CC or dual (CC/EC) users (231, 232). These finding were in accordance with two EC studies in Table 3 (200, 212) and one HTP study (215) but opposed to another EC study (216). All asthma studies have a number of limitations, which are mentioned in Table 3.

Lung function parameters such as FEV1 and FVC were most frequently determined in the studies listed in Table 3. Reported results are heterogeneous, comprising impaired, unchanged and increased values of spirometric variables. Consistent results in terms of lung function and COPD (measured as CAT score) were reported by POLOSA and coworkers (198, 199, 223): in a longitudinal study lasting for up to 5 years, smokers (CC) who switched to EC showed no change or slightly improved lung function values and CAT scores compared to subjects who continued to use CCs. The evaluation of 3 cross-sectional studies revealed no increased risk for COPD and other respiratory diseases such as chronic bronchitis, emphysema and wheezing in vapers (216), whereas Perez et al. 2019 (213) reported an increased risk of COPD in EC users.

Polosa et al. (233) found that smokers with COPD who switched to HTPs over a period of 3 years showed consistent improvements in respiratory symptoms, exercise tolerance, quality of life, and rate of disease exacerbations compared to smokers with COPD who continued to use CCs.

FeNO, an acute BOBE of respiratory inflammation as well as CVD showed heterogeneous changes (unchanged, decreased and increased levels) upon vaping in several studies (see Table 3). In a long-term study over one year, with those subjects who quit smoking and used ECs for staying abstinent, an increase compared to baseline levels was reported (196). An increased risk in vapers for chronic bronchitis was found in a longitudinal study over 1 year (197). Various BOBEs were measured in either BAL, EBC or sputum (Table 3). The obtained results do not allow to draw clear conclusions, whether vaping is associated with detrimental effects in the respiratory tract.

The presence or absence of nicotine in e-liquids of ECs was found to have no acute effect on lung functions (FEV1 etc.), FeNO (208, 209, 222), while acute increase in airway resistance (208), EMP, changes in the transcriptome in small airway epithel (SAE) and upregulation of inflammasome genes were reported to be nicotine-dependent (201, 226).

Overall, findings on respiratory effects of ECs described in Table 3 are partly contradictory and as yet inconclusive. The same is true for the possible involvement of nicotine on these effects. More research in this field, particularly on the effects of long-term NGP use is required in order to draw solid conclusions.

Other recent monographs and reviews in this area of research are in accordance with these conclusions:

The EU-based Scientific Committee on Health, Environmental and Emerging Risks (SCHEER) (143) reviewed the most recent scientific and technical information on ECs and concluded for respiratory diseases: “The overall weight of evidence is moderate for risks of local irritative damage to the respiratory tract of users of electronic cigarette due to the cumulative exposure to polyols, aldehydes and nicotine. However, the overall reported incidence is low.”

Wills et al. (234) conducted an integrative review on EC use and respiratory disorders covering epidemiological and animal studies. Meta-analysis of 15 epidemiological studies revealed significantly increased risks for asthma and COPD in vapers compared to NU. According to the authors, these observations were consistent with evidence from animal and in vitro studies which show an impact of EC aerosol on cytotoxicity, oxidative stress, inflammation, gene changes in the immune system of lung cells and tissues. The authors cite evidence that these effects were both dependent and independent of nicotine in the aerosol.

Results from the National Health and Nutrition Examination Survey (NHANES) showed higher prevalence of asthma and COPD in EC and dual users (EC + CC) than in smokers (CC only) and NU (235). Vapers with asthma were significantly younger than any of the other groups with this disease, suggesting that other factors may play a role as well. A possible reason for this observation could be that young subjects with asthma symptoms start with ECs, assuming that these are less detrimental for them.

In a recent state-of-the-art review on respiratory effects of vaping (236), it was found that human, animal and in vitro studies show measurable adverse biologic effects, which are similar to or different from those of CC. The authors assume that nicotine exposure will likely have pharmacologic effects in any organ where nAChR are expressed, thus having impact on inflammation in the airways, susceptibility to infection and the risk of developing COPD or lung cancer. The authors suggest that sufficient information on chronic effects of EC use will not be available before the middle of this century.

Based on more or less the same available evidence as the previously cited reviews, Bravo-Gutiérrez et al. (237) in their systematic review came to the (strong) conclusion that “ECs and HTPs use is involved in damage related to the development of pulmonary diseases. The evidence available so far is significant enough for physicians, researchers, and public health policymakers to address these devices as an emerging public health problem that needs regulation” (237). The authors cite extended evidence, particularly from animal and in vitro studies showing that damaging effects of EC and HTP aerosols were found to be both dependent and independent on the presence of nicotine.

In a recent state-of-the-art review, Jonas (191) concluded that relatively little is known about the potential effects of chronic vaping on the respiratory system, and a growing body of literature supports the notion that vaping is not without risk. Furthermore, the author emphasized that in particular long-term effects on the lungs are unknown. The same applies for the other inhalable NGP, HTP.

The literature on health impact of using ECs or HTPs in COPD patients was evaluated in a review (238). The authors conclude that, while ECs and HTPs may offer some benefits in reducing harm from CC, their long-term effects on COPD are still unclear.

Chronic exposure of mice to EC aerosol revealed that in the presence of nicotine, increased airway hyper-reactivity, distal airspace enlargement, mucin production, cytokine and protease expression was observed. These changes were not found when nicotine was absent (239). Experiments with normal human bronchial epithelial cells showed impaired ciliary beat frequency, airway surface liquid volume, as well as increased IL-6 and IL-8 secretion with nicotine, but not without nicotine (239).

In a study with mice exposed to PG and VG aerosol, with and without nicotine, detrimental effects in terms of lung inflammation and function were observed, independent of the presence of nicotine (240).

In a review on EC use and lung diseases, Rowell and Tarran (241) cite evidence from mostly murine studies that nicotine might be involved in various lung diseases by proliferating and inflammatory effects on various lung cells. Acute exposure (3 d, 2 h/d) of mice to EC aerosol with nicotine was reported to increase sex-specifically the inflammatory response in the lungs (242). No effect was observed with nicotine-free aerosols.

The same working group (243) reported that sub-chronic EC exposure of mice (2 h/d, 5 d/week, over 30 d) with or without nicotine affected lung inflammation and repair responses/extracellular matrix remodeling, which were mediated by the nicotinic receptor nAChRα7 in a sex-dependent manner. The authors speculate that, as an agonist, nicotine might have pro- and anti-inflammatory roles, which might explain these results (243). In this case, it has to be assumed that other EC aerosol constituents (e.g., aldehydes) can have similar detrimental effects in the lung, however acting via a different mechanism than nicotine.

Glynos et al. (244) concluded from the results of their 4-week mice inhalation study that all major constituents of EC aerosol (PG, VG, N and flavors, tested separately or in combination) can negatively impact lung function and inflammation. Nicotine was found to exert an exaggerating, ameliorating or indifferent role in terms of lung damage. In another study, mice were exposed to EC aerosol (with/without nicotine; 2 × 30 min/d, 21 d) (245). The exposure regime induced airway inflammation, impairment of the lung function and increase in ACE-2 expression, to a greater degree in males exposed to EC aerosol with nicotine. Increase in IL-6 was independent of sex and nicotine. The authors conclude that vaping may facilitate the infection with SARS-CoV-2.

In a mice inhalation study with various aerosols, including ECs with and without different flavor and nicotine (2 × 30 min/d, 6 d/week, 0–18 d), it was found that ECs with nicotine suppressed airway inflammation, but did not alter airway hyper-responsiveness and remodeling (246).

An inhalation study with rats exposed for 28 d to nicotine-free aerosol generated at 1.5 and 0.25 Ω enhanced xanthine oxidase and P450 enzymes to a higher degree at the low resistance condition (247). A nicotine-containing EC aerosol was not tested.

In a mice study, it was also observed that nicotine-free EC aerosol exposure (2 h/d, 6 d/week, 8 weeks) increased airway resistance and affected how the lungs react to tobacco cigarette smoke exposure in dual users (248). A nicotine-containing EC aerosol was not tested.

Exposure of mice to PG aerosols with and without nicotine (2 h/d, 3 d) were reported to alter the circadian molecular clock genes in the lung in only the nicotine-exposed animals, which, according to the authors, may have consequences for the lung cellular and biological functions (249). Mice exposed to PG/VG aerosol with and without nicotine over 4 months did not develop pulmonary inflammation or emphysema in contrast to smoke- (CC) exposed mice (250). EC-exposed animals were found to exert disruption of pulmonary lipid homeostasis and immune impairment independent of nicotine in the aerosol.

Sun et al. (251) investigated biomarkers of oxidative stress (8-OHdG), inflammation (CRP) and tissue injury on lungs of mice exposed (2 h/d, 5 d/week, 8 weeks) to PG/VG aerosols (0, 12, 24 mg/mL nicotine), smoke (CC, 0.7 mg nicotine/cig) and air. CC exposure led to the highest biomarker levels in plasma and lung, except for CRP in plasma for which EC aerosol without nicotine was highest. Increasing nicotine levels in EC appear to suppress 8-OHdG in lung and plasma as well as CRP in plasma. The authors conclude that EC exposure can lead to lung damage.

Female mice were exposed (2 h/d, 6 weeks) to PG/VG aerosol with and without vanilla flavor (252). EC vehicle exposure was found to disrupt immune homeostasis, irrespective of the presence of vanilla flavor. No EC aerosol with nicotine was tested.

In an inhalation study with ApoE-deficient mice (3 h/d, 5 d/week, 6 months), Lavrynenko et al. (253) observed that HTP and EC aerosols in contrast to smoke (CC) did not induce significant changes in the ceramide profiles (a lipid class dysregulated in CVD and respiratory disorders) and associated enzymes. All aerosols contained nicotine, therefore, the role of nicotine cannot be deduced from this study. Exposure to various EC aerosols (PG/VG, PG/VG/N, PG/VG/N/F (flavor)) showed a slight impact on lung inflammation and epithelial irritation compared to sham exposure (254). However, no differences were observed for EC aerosols with and without nicotine, suggesting that nicotine is not responsible for the observed effects.

Chung et al. (255) found in in vitro (human lung epithelial cells) and in vivo (sheep) experiments that EC aerosols with nicotine (0, 10, 20 mg) impaired dose-dependently the mucociliary clearance. The main nicotine effect could be suppressed by a TRPA1 (transient receptor potential ankyrin 1) inhibitor. The authors concluded that nicotine exerted its effect via the TRPA1 and not via nicotinic acetylcholine receptors.

Ma et al. (256) investigated the cell toxicity of EC aerosols with and without nicotine as well as the NF-κB-mediated oxidative stress in a mouse model. It was found that the cytotoxicity in various cell systems was independent of nicotine and mostly caused by aldehydes in the aerosol. In vivo mouse exposure with the EC aerosols showed elevated expression of NF-κB and heme oxygenase-1, irrespective of the presence of nicotine. The authors suggest that oxidative stress, pro-inflammatory, NF-κB pathway activation, and cell death are involved in EC exposure induced acute lung inflammation.

Mice were exposed to EC aerosols with nicotine and various flavors as well as to a PG/VG aerosol (257). Exposure dose-dependent increases in total cells, macrophages and neutrophils in BAL were observed with all aerosol, irrespective of the nicotine content. Oxidative stress markers in blood (8-OHdG, MDA) were also found to be increased, dose-dependently and independent of nicotine. This was also true for some inflammatory response on the mRNA level.

Mice exposed to smoke (CC) or EC aerosol derived from e-liquid with 6 and 12 mg nicotine over 10 weeks showed the highest detrimental effects in terms of impaired lung function, elevated inflammation markers and severe inflammation proteome network perturbations after CC exposure (258). Effects (if any) of EC aerosol exposure were much smaller and independent of the nicotine level. The authors suggest that in this animal model, EC aerosol is less harmful to the respiratory system than cigarette smoke at the same dose of nicotine.

Maishan et al. (259) exposed mice to an EC aerosol with and without nicotine for 1 h/d over 9 d. From the results it was concluded that acute exposure to aerosolized nicotine can impair clearance of viral infection and exacerbate lung injury.

Based on the studies reviewed, the role of nicotine in the various effects of electronic cigarettes and HTPs on the lung can be summarized as follows: ○

Nicotine appears to be involved in airway hyper-reactivity, mucin production, cytokine and protease expression, and impairment of ciliary beat frequency and airway surface liquid volume;

In vitro investigations with aerosols from ECs and HTPs suggest that nicotine might be at least partly responsible for the observed cytotoxicity (260,261,262,263).

EC aerosols with and without nicotine stimulated the expression of MUC5AC in human bronchial and nasal epithelial cells (205, 264), suggesting that aerosol constituents other than nicotine are involved in the impairment of the mucociliary transport system of users, which could lead to the various respiratory diseases, including COPD.

Studies with human alveolar macrophages revealed that EC aerosol is cytotoxic, proinflammatory and inhibits phagocytosis (265). These effects were found to be partly dependent on nicotine.

A study with EC aerosol derived from VG (no nicotine, no flavors) including human volunteers, animals (sheep) and in vitro experiments with primary human bronchial epithelial cells (HBECs) found that VG aerosols can potentially cause harm in the airway by inducing inflammation and ion channel dysfunction with consequent mucus hyper-concentration (266). The authors cite evidence from other studies (mostly mice and in vitro) showing that the EC aerosol in the absence of nicotine can induce airway inflammation and high mucus load. In similar experiments with PG aerosols, it was also found that mucus hyper-concentrations were induced in sheep (in vivo) and in HBECs (in vitro) (267). Furthermore, metabolism of PG to methylglyoxal (MGO) was reported for airway epithelial cells (267).

Nicotine was found to significantly contribute to the disruption of the lung epithelial barrier function both when present in condensate of CCs and ECs (268). The authors suggest that another constituent in EC aerosol causing this effect was acrolein.

In vitro experiments, PG/VG aerosols (+/− nicotine and +/− WS-23 (a synthetic menthol-like cooling agent)) and airway endothelial cells (AECs) revealed suppressing effects on IL-6 and s-ICAM-1 mRNA as well as enhancing effects on MUC5AC mRNA of the nicotine- and WS-23-containing aerosols compared to the sole PG/VG aerosols (269). The authors conclude that WS-23 and nicotine aerosols modulate the AEC responses and induce goblet cell hyperplasia, which could impact the airway physiology and susceptibility to respiratory diseases.

Mori et al. (270) compared mitochondrial DNA copy number (mtCN) in lung biospecimen from smokers (CC), vapers (EC) and never-smokers (NU) and found significant ly higher mtCN in CC compared to NU, with EC in between. Further evaluations comprised associations of mtCN with immune response markers in BAL and gene methylations. From their results, the authors conclude that smoking may elicit a lung toxic effect through mtCN, while the impact of EC is less clear and the observed associations suggest exposure may not be harmless.

Adverse pulmonary and systemic effects of a nicotine aerosol were shown in a rat (in vivo) model and in cell culture experiments with normal human bronchial epithelial cells (271). From their results, the authors deduced an action scheme of inhaled nicotine, according to which the alkaloid acts via nAChR to cause release HMGB, caspases, E-cadherin and many other cellular mediators. As a result, nicotine stimulates inflammation, cell death and increased lung epithelial permeability.

Cinnamaldehyde, an EC flavor agent, was found to dose-dependently decrease the ciliary beat frequency in human bronchial epithelial cells via inhibition of mitochondrial energy supply (272). Addition of nicotine had no effect. The authors conclude that inhalation of cinnamaldehyde with EC vapor may increase the risk of respiratory infections. Diacetyl and 2,3-pentanedione, two EC flavor compounds, were shown by transcriptomic studies with normal human bronchial epithelial cells (NHBEC) to impair the cilia function and likely contribute to the adverse effects of ECs in the lung (273). The role of nicotine in this system was not investigated.

The EC aerosol matrix compounds PG/VG have been shown in in vitro experiments with HBEC to decrease the glucose uptake and metabolism (274). The results suggest that PG/VG could reduce the cell volume and membrane fluidity, with further consequences on epithelial barrier function.

Human nasal epithelial cells derived from smokers and non-smokers were exposed to PG/VG without and with nicotine (as salt or free base) (275). Changes in MUC5AC and pro-inflammatory cytokines were used as biological endpoints. Observed effects were dependent on the type of nicotine as well as on the source of the cells (smokers or non-smokers). The authors conclude that it is important to understand that the biological effects of ECs use are likely dependent on prior cigarette smoke exposure.

The in vitro evidence reviewed above can be shortly summarized as follows: ○

In vitro studies suggest that nicotine is at least partly responsible for the cytotoxicity and pro-inflammatory effects of electronic cigarette aerosols on lung cells, including alveolar macrophages and airway endothelial cells;

Cigarette smoking (CC) is an established risk factor for non-malignant lung diseases such as chronic bronchitis, COPD, emphysema and asthma. Impairment of various lung defense systems including mucociliary clearance and innate immunity as well as activation of inflammatory processes are first steps in the smoking-related pathogenesis. Smoking is significantly associated with a decline in spirometrically determined lung function as well as FeNO.

Numerous studies with vapers (EC) (and to a much lower extent also with HTP users) have investigated acute effects of product use on the respiratory system with as yet inclusive results. A limited number of long-term studies (observation periods of > 1 year) with vapers suggest that detrimental effects on the respiratory system, if any, are much smaller than in smokers (CC). No involvement of nicotine in the pathogenesis can be deduced from human studies. A few studies reported a possible role of nicotine in the gene regulation of lung cells. Consequences of these observations in terms of risk for lung diseases are as yet unknown. In vivo animal and in vitro studies found that nicotine may play a role in some pathways of respiratory tract pathogenesis, for example mucociliary clearance, cell proliferating and inflammatory effects. Almost all those effects have been also observed for the aerosols without nicotine.

In conclusion, presently there is no sufficient evidence that nicotine in NGPs has any detrimental effects leading to an increase in non-malignant lung diseases for the users. Well-designed long-term studies are urgently required to fill this gap of knowledge.

The US Surgeon General Report on the health consequences of SLT use concluded that an early effect is leukoplakia formation with the possibility of transformation to dysplastic lesions and finally (after decades of use) to oral cancer (276). Suspected, but still not confirmed at that time, were detrimental effects of SLT use on gingivitis, periodontitis, damage of the salivary glands and negative effects on teeth. It should be mentioned, however, that the Surgeon General’s conclusions are based on findings from various countries including those where more harmful oral tobacco products were sold than for example in Scandinavia and the USA.

In general, the lack of dental hygiene is a potential confounder in oral health effects (277).

Delayed healing after oral surgery or tooth extraction is another negative health effect of tobacco use. Less a health effect but rather a social problem could be bad breath (278) and dental staining (279) which is frequently observed in chronic smokers.

Major oral diseases associated with tobacco use and smoking are periodontal diseases and oral cancer (280). Involved mechanisms for periodontal diseases are the promotion by the invasion of pathogenic bacteria, inhibiting auto-immune defense, aggravating the inflammatory reaction, and aggravating the marginal bone loss (MBL). According to the authors, the link between periodontal disease and oral cancer is limited and needs further research. Evidence is provided that nicotine has a damaging effect on periodontal cells and alveolar bone (alveolar process).

Indicators of periodontitis risk such as increased plaque index (PI), probing depth (PD), clinical attachment loss (CAL) and decreased bleeding on probing (BOP) are more frequently observed in smokers than NU (281).

The oral microbiome of smokers (CC) is significantly different from that of non-smokers, creating an “at-risk-for-harm” oral environment (282, 283). CC use was demonstrated to create greater abundance of Parvimonas, Fusobacterium, Campylobacter, Bacteroides, and Treponema and lower levels of Veillonella, Neisseria, and Streptococcus (284).

Medical investigations of the oral cavity and gum use a number of diagnostic measures to evaluate oral health, here are short definitions of the most important ones: ○

PI (plaque index): A measure of the amount of plaque on teeth and gums;

A systematic review on the effects of EC use in the oral cavity comprising 8 studies was published in 2019 (285). The major lesions and markers of interest were plaque index (PI), clinical attachment loss (CAL), probing depth (PD), marginal bone loss (MBL), bleeding on probing (BOP) and pro-inflammatory cytokine levels. All parameters (except for BOP which decreased with exposure), were significantly elevated in EC users compared to NU, but lower than observed in smokers (CC). Additionally, 9 different types of lesions of the oral mucosa were detected, with nicotinic stomatitis, hairy tongue and angular cheilitis being more prevalent in vapers. The authors cite evidence that the decrease in BOP is a nicotine effect (vasoconstriction).

NASEM (6) reviewed 4 human and 5 in vitro studies on vaping and oral health. The authors found limited evidence suggesting that switching to ECs would improve periodontal disease in smokers and concluded that there was limited evidence suggesting that nicotine or other constituents of EC aerosol could adversely affect cell viability and cause cell damage in oral tissue in NU.

In a recent systematic review by Yang et al. (286), seven categories of oral damages were identified to be of interest: mouth effects, throat effects, periodontal effects, dental effects, cytotoxic/genotoxic/oncologic effects, oral microbiome effects, and traumatic/accidental injury. The evaluation of 99 studies revealed that the symptoms associated with vaping were minor and temporary. In switchers (CC to EC), a mitigation of oral symptoms was usually reported. The authors further conclude that well-designed, long-term studies on oral health are needed. Very similar conclusions were also drawn in another review (287).

In a large population-based cross-sectional questionnaire study in the USA with > 450,000 adults, daily EC use was associated with poor oral health (adjusted OR (CI) = 1.78 (1.39–2.30), daily EC users versus NU) (288).

From a systematic review of the literature on the impact of vaping on periodontitis (289), the authors conclude that the available results point to increased destruction of the periodontium leading to the development of the periodontitis. The authors cite evidence that nicotine might play a role in reduction of BOP by vasoconstriction in the gingival tissue. For a systematic review on vaping and periodontal indices (290), 5 studies with 512 subjects were identified. Smokers (CC) had the worst values for PD and PI, non-users (NU) the best. The BOP parameter was equally reduced in CC and EC compared to NU. The latter finding is consistent with a reduced microcirculation in gingival tissues caused by nicotine.

Reeve et al. (291) investigated mRNA and miRNA in the buccal mucosa of vapers (EC) and non-users (NU). From their findings (evaluation of 5 cross-sectional studies, all conducted in Saudi Arabia), the authors conclude that an average history of 2 years of EC use results in no detectable histologic or transcriptome abnormalities in the buccal mucosa. Robbins and Ali (292) commented on this review by stating that large-scale, multi-centered, randomized controlled trials would be preferable, however, probably unethical (because of a CC group). A better control for confounding factors in cross-sectional studies, according to the commenters is essential.

A systemic review comprising 14 studies evaluated xerostomia in vapers (EC) and smokers (CC) (293). The prevalence was 33 and 24% in users of ECs and CCs, respectively. The difference, however, was not significant. Park et al. (294) compared several sets of the oral microbiome in EC users and NU by analyzing saliva and gingival fluid samples. A large degree of disparity was observed. According to the authors, the results suggest that the observed changes may increase inflammatory processes and lead to periodontal disease.

In a review of the literature about the effects of vaping on oral health, it was found the at EC use might be implicated with less marked detrimental effects on the buccal mucosa and gingival tissues than smoking (CC) (295). In terms of nicotine’s role in oral health, the authors conclude that the alkaloid may be involved in migration inhibition, cyto-skeleton alterations, and extracellular matrix remodeling in human gingival fibroblasts and increase the amount of pro-inflammatory cytokines secreted in cultured gingival keratinocytes and fibroblasts. Furthermore, e-liquids, irrespective of the presence of nicotine, may induce oxidative stress buccal mucosa cells.

Mir et al. (53) summarized the EC effects in the oral cavity as follows: “Increased pro-inflammatory markers in gingival epithelial cells, increased pro-senescence response in periodontal cells, heightened capacity for Staphylococcus aureus colonization of oral epithelial cells and biofilm formation, higher carriage of oral Candida albicans.”

In Table 4 (see page 76), 23 studies, 18 of the cross-sectional and 5 of the longitudinal type are briefly described and evaluated (281, 296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317). All longitudinal studies (281, 301, 305, 314, 315), that investigated the effects of ECs, comprised observation periods between 6 months and > 1 year and reported somewhat impaired oral health in vapers compared to NU. The effects were significantly lower than those found in smokers (CC). In one longitudinal study (315), acrolein-derived DNA adducts were reported to be elevated in buccal cells of EC users compared to NU, but lower than in smokers (CC).

Of special interest is also the finding that PCR-determined DNA lesions in special genes (e.g., HPRT) in vapers (EC) were similar to smokers (CC) but significantly elevated compared to NU (317).

In one study of Table 4, pro-inflammatory cytokines in saliva of snus (including nicotine pouches) users were investigated (316). Levels were reported to be higher in snus and nicotine pouch users than in smokers (CC) and vapers (EC), which in turn were higher than in NU.

8-OHdG adducts (a marker for oxidative stress) were not found to be different between buccal cells of NU, EC and CC users (308). In contrast, myeloperoxidase (MPO), another oxidative stress biomarker, was reported to be higher in EC, CC and dual users compared to NU (309).

In almost all studies of Table 4, inflammation markers were found to be highest in smokers (CC) and lowest in NU, with EC and snus users in between. The same is true for the oral/tooth health indicators PI, PD, BOP, MBL and CAL. BOP was consistently reported to decrease nicotine-dependently in several studies, which is interpreted as a nicotine-related vasoconstriction in the gingival tissue (281, 296, 299, 300, 303,304,305, 311). For other biological effects listed in Table 4, either no involvement of nicotine can be stated (0) or a participation of nicotine is not deducible from the reported data (?).

The oral microbiome was found to differ significantly between users of CCs, ECs and NU, with only some overlap (312, 314). Consequences in terms of oral health risk can, as yet, not be inferred.

The same might be true for the reported dysregulation of genes in the oral cavity (306, 317), although the authors interpret the changes as indication for a higher oral health risk.

Taken together, there is (although as yet) limited evidence from human studies showing that use of NGPs can deteriorate oral health markers such as PI, PD, BOP, MBL, CAL and inflammation markers compared to NU. The reported effects are in general, however, significantly lower than in smokers (CC). The oral microbiome as well as the gene regulation in buccal cells was found to be different between smokers (CC), vapers (EC) and NU. The impact or changes in the oral microbiome for oral health is as yet unknown. Of interest is the vaping-dependent increase in acrolein-derived DNA adducts in buccal cells, which certainly requires further investigations. For most of the reported effects there is no, or only questionable evidence, for an involvement of nicotine. An exception is the oral health marker BOP, which is consistently found to be decreased in users of nicotine-containing products, most probably due to the vasoconstriction effect of nicotine in gingival tissue.

No in vivo animal studies for the purpose of interest could be identified.

An in vitro model for oral mucosa tissue found that pro-inflammatory responses (release of MMPs and LDH) was higher for nicotine-rich compared to nicotine-free EC aerosols, both, however were lower than for CC smoke (318).

The oral cavity is the primary target for all tobacco and nicotine product habits. The use of conventional tobacco products can lead to non-malignant disorders such as leukoplakia, gingivitis, periodontitis, salivary gland and teeth damage, delayed healing, bad breath, and dental staining.

Most human studies in this field investigated EC use and reported impairments in the clinical oral health indicators PI, BOP, CAL, PD, MBL, as well as in pro-inflammatory cytokine levels. Effects are usually slightly higher than in NU, but significantly lower than in smokers (CC). The oral microbiome and gene regulation in buccal cells is found to significantly differ between EC and CC users and NU. Whether this has implications for oral health risk is not yet known. DNA lesions in buccal mucosa cells, as found to be elevated in smokers (CC), were not increased in vapers (EC). In a very recent study, EC use was reported to increase acrolein-derived DNA adducts in buccal cells.

A participation of nicotine in the development of the reported oral health disorders was not unequivocally observed with the exception of BOP. The decrease in BOP was found to be dependent on the presence of nicotine, most probably through its vasoconstriction effect in the gum. Whether this has detrimental effects upon chronic use of NGP is not known.

Smoking is associated with an increase in oxidative stress, which is thought to be a major mechanism underlying the pathogenesis of smoking-related diseases (25, 319).

Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS), which are formed by subsequent addition of electrons to molecular oxygen (O₂), yielding: superoxide radical anions (O₂^•−), hydrogen peroxide (H₂O₂), hydroxyl radicals (OH^•) and water (H₂O). The highly reactive superoxide radical anion and the hydroxyl radical can initiate lipid peroxidation to form lipid peroxides (ROO^•). The body has the ability to detoxify these molecules by various processes, including: ○

Antioxidant enzymes (superoxide dismutase (SOD), catalase, glutathione peroxidase)

Smoking disturbs the oxidant/antioxidant balance so that ROS can cause damage to cellular components, including DNA, proteins, and lipids.

Cigarette smoke contains a complex mixture of more than 7,000 chemicals, including free radicals and other ROS. These ROS can directly damage cellular components, including DNA, proteins, and lipids, leading to oxidative stress. In addition, cigarette smoke can also activate immune cells, such as neutrophils and macrophages, which produce additional ROS and other inflammatory molecules. This creates a cycle of oxidative stress and inflammation that can contribute to the pathogenesis of smoking-related diseases.

One of the key mechanisms by which smoking causes oxidative stress is through the activation of the nicotin-amide adenine dinucleotide phosphate (NADPH) oxidase system. NADPH oxidase is an enzyme complex that produces ROS as part of the normal immune response. However, cigarette smoke can activate this system in a way that leads to excessive production of ROS, contributing to oxidative stress. In addition, cigarette smoke can also impair the function of SOD and other antioxidant enzymes, further exacerbating oxidative stress.

Finally, smoking can also lead to oxidative stress through the depletion of antioxidants. Cigarette smoke contains a number of chemicals that can deplete antioxidants, including ascorbate and glutathione (GSH). As a physiological response (compensation), GSH in various cells and tissues of smokers is elevated compared to that of non-smokers.

In summary, smoking is associated with an increase in oxidative stress, which is thought to be a major mechanism underlying the pathogenesis of smoking-related diseases. Smoking can cause oxidative stress through a number of mechanisms, including the activation of the NADPH oxidase system.

Nicotine has not been reported to be a cause or otherwise involved in the smoking-related oxidative stress, for which a number of chemicals in tobacco including aldehydes, radicals, quinones and many others were found to be responsible (320). It is also probable that nicotine is not causally involved in inflammatory processes (321).

Another important process how smoking is causally involved in the development of various diseases is the stimulation of inflammatory processes (25, 193). Inflammation is a complex process involving the immune system, which aims to protect the body from harmful stimuli, including pathogens, damaged cells, and irritants. When the immune system detects such stimuli, it triggers a cascade of molecular and cellular events, including the release of cytokines, chemokines, and reactive oxygen species (ROS), the recruitment of immune cells such as neutrophils and macrophages, as well as the activation of inflammatory signaling pathways. Smoking can trigger inflammation through several mechanisms.

Firstly, cigarette smoke contains numerous harmful chemicals, including “tar”, carbon monoxide, and nicotine, which can directly damage cells and tissues and trigger an immune response. For example, “tar” can induce DNA damage and oxidative stress, which can activate the nuclear factor kappa B (NF-κB) pathway, a key regulator of inflammation. Additionally, cigarette smoke can increase the production of ROS, which can cause oxidative damage and trigger inflammation by activating redox-sensitive signaling pathways.

Secondly, smoking can alter the composition of the microbiome in the respiratory tract or oral cavity, leading to dysbiosis and inflammation. The respiratory tract is normally colonized by a diverse community of microbes, which can play a role in immune regulation and protection against pathogens. However, smoking can disrupt this balance by reducing the diversity of the microbiome and promoting the growth of pathogenic bacteria, thus initiating inflammatory processes.

Thirdly, smoking can activate immune cells and promote the release of pro-inflammatory mediators. For example, smoking can stimulate neutrophil activation and recruitment, leading to the release of cytokines such as interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α). Additionally, smoking can activate macrophages and dendritic cells, which can present antigens to T cells and trigger an adaptive immune response.

Fourthly, smoking can alter epigenetic modifications, which can lead to persistent changes in gene expression and inflammation. Epigenetic modifications, such as DNA methylation and histone acetylation, can regulate gene expression by altering the accessibility of chromatin to transcription factors. Smoking has been shown to alter DNA methylation patterns in immune cells, leading to changes in the expression of genes involved in inflammation and immunity.

Overall, smoking can trigger inflammation through multiple mechanisms, including direct damage to cells and tissues, dysbiosis of the microbiome, activation of immune cells, and alterations in epigenetic modifications. These inflammatory processes can contribute to the development and progression of smoking-related diseases, such as cardiovascular disease, chronic obstructive pulmonary disease, and cancer.

Emma et al. (319) summarized the knowledge derived from human, animal and in vitro studies on the impact of CC, EC and HTP use on oxidative stress. The overall conclusion of the authors is that, although NGPs release significantly lower amounts of toxicants compared to CCs, the new products can also induce oxidative stress in various organ systems, consequently, harm is reduced but not eliminated. With respect to the dependence of oxidative stress on the presence of nicotine, the authors presented evidence for both, dependence and independence of nicotine. Not unexpectedly, nicotine-dependence of effects was observed more frequently in cardiovascular system.

A cross-sectional study by Carnevale and others (322) compared changes in oxidative stress markers after ad libitum vaping or smoking for a week. The study found increased levels of soluble NOX-2-derived peptide and 8-iso-prostaglandin F2α and significantly decreased nitric oxide and vitamin E levels. Furthermore, there was a statistically significant reduction in flow-mediated dilation function after participants vaped or smoked for a week. The authors concluded that both smoking and vaping induced the oxidative stress, but the use of vaping products appeared to produce a less pronounced effect on levels of soluble NOX-2-derived peptide, 8-iso-prostaglandin F2α and nitric oxide than cigarette smoking. Both human studies explored a relatively short exposure to vaping effects, and Carnevale and others (322) noted that future research should clarify the chronic vascular effects of vaping product use. In Table 5 (see page 82), 11 studies investigating the effect of NGP use (9 with EC, 1 with NRT product and 1 with nicotine pouch users) on oxidative stress and inflammation biomarkers are briefly described (323,324,325,326,327,328,329,330,331,332,333). Study types were cross-sectional (9 ×) and cross-over (2 ×). The most frequently applied oxidative stress marker was 8-epi-PGF_2α, whereas CRP and s-ICAM were most often used as inflammation marker.

Overall, the studies listed in Table 5 suggest that use of NGPs (mostly EC use was investigated) leads to no change or to an increase in the markers for oxidative stress or inflammation compared to NU. Increases, however, were mostly found to be significantly lower than in smokers. Principal weaknesses in terms of current and former use of CCs in cross-sectional studies have to be considered (see comments in Table 5). Another general issue is that the duration of use of NGPs is too short for evaluating the long-term effects of NGP use.

The role of nicotine in oxidative stress or inflammation in general was not deducible from the study data. A study with long-term nicotine pouch or snus users showed no significant increase in oxidative stress or inflammation markers, suggesting that nicotine might play no role (0) in these processes (333).

NASEM stated that comparisons between cell studies were difficult due to different cell cultures used, varying exposure methods (for example, cells were exposed to vaping e-liquids, aerosol extract or aerosol generated directly by vaping products), and different lengths of exposure (6). Based mainly on findings from cell and animal studies, NASEM concluded that “there is substantial evidence that components of e-cigarette aerosols can promote formation of reactive oxygen species/oxidative stress. Although this supports the biological plausibility of tissue injury and disease from long-term exposure to e-cigarette aerosols, generation of reactive oxygen species and oxidative stress induction are generally lower from e-cigarettes than from combustible tobacco cigarette smoke”.

Rats exposed to nicotine-free EC aerosols showed increased oxidative stress (ROS, lipid peroxidation, protein carbonylation) in the testis (334). The authors suggested that human vapers may also develop gonads dysfunction. Kuntic et al. (75) reported that in mice, EC vapor without nicotine had more detrimental effects on endothelial function, markers of oxidative stress, inflammation, and lipid peroxidation than vapor containing nicotine. The authors conclude that EC aerosol exposure increases vascular, cerebral, and pulmonary oxidative stress via a NOX-2-dependent mechanism and that the EC vapor constituent acrolein might act as a key mediator of the observed adverse vascular consequences.

In vitro experiments with mouse epithelial lung cells showed that the presence of nicotine in the EC aerosol had no influence or even reduced the extent of oxidative stress implicated with the exposure (335).

In vitro stem cell studies with EC aerosol revealed that mitochondria were nicotine-dependently stressed (including oxidative stress) combined with autophagy dysfunction to clear damaged mitochondria leading to faulty stem cell populations, resulting in cellular aging (336).

The cytotoxic, oxidative stress and inflammatory effects (release of IL-8) of the e-liquid flavors diacetyl, cinnamon aldehyde, acetoin, pentanedione, o-vanillin, maltol and coumarin were tested in the absence of nicotine in vitro in two human monocyte cell types (337). Dose-dependent increases were found for some of the chemicals. Mixture of the flavors showed larger effects. The authors conclude that flavorings used in e-liquids can trigger an inflammatory response mediated by ROS production leading to potential pulmonary toxicity and tissue damage in vapers.

In an ex vivo (healthy human NU)/in vitro study, it was shown that EC aerosols cause pro-inflammatory responses in neutrophils, which are independent of the nicotine content (0, 16, 24 mg/mL) (338).

Su et al. (339) investigated the impact of smoke (CC) and EC aerosols with increasing nicotine content (0.0313, 0.125, 0.5, 2, 8, or 32 μg/mL in e-liquid) on HUVEC. Smoke (CC) was found to have the highest impact on cell viability, apoptosis, adhesion molecules (ICAM, VCAM), inflammation cytokines (IL-6, IL-8, IL-1ß, TNF-α) and ROS. Effects of EC aerosols were much smaller (or absent) and showed no dependence on the nicotine dose. The authors concluded that ECs are not harmless at all, but could represent a good alternative to CCs.

Giebe et al. (340) exposed human monocytes with aqueous extracts of CC, EC, HTP and pure nicotine (as a control). As a general conclusion the authors stated that anti-oxidative and pro-inflammatory processes were activated by all products. NGPs (EC, HTP) overall showed lower responses relative to controls (nicotine) than CC extracts.

Smoking cigarettes is causally associated with oxidative stress and inflammation. Both processes are first steps in the pathogenesis of many smoking-related diseases.

Human studies on users of electronic cigarettes (ECs, other NGPs have been investigated distinctly less frequently) showed mixed results. Some studies suggest that ECs and other new nicotine products may also contribute to oxidative stress and inflammation. The extent of both effects, if any, was found to be significantly smaller than in smokers (CC). The role of nicotine in these processes is unclear. In general, cross-sectional studies may suffer from bias and confounding factors, which have to be considered when the results are interpreted.

Animal studies showed that exposure to releases of ECs and other new nicotine products can lead to oxidative stress and inflammation. Nicotine seems to play a role in this process, as animal studies have shown that nicotine alone or EC aerosols with nicotine compared to those without can cause oxidative stress and inflammation.

In vitro (cell) studies have also shown that exposure to ECs and other new nicotine products can cause oxidative stress and inflammation in various cell types. Nicotine has been shown to play a role in this process, but the exact mechanisms are still not fully understood. Extrapolation of the results of in vitro and animal studies to human users is limited due to distinct differences in applied models and exposure conditions (dose, time).

Overall, there is evidence to suggest that using ECs and other NGPs can contribute to oxidative stress and inflammation but to a much smaller extent than smoking (CC). There is insufficient evidence that nicotine may play a role in this process, but further research is needed to fully understand the mechanisms involved. In particular, long-term human studies are required to find out whether NGP use is associated with oxidative stress and inflammation in various organ systems.

Cigarette smoking is a known risk factor for metabolic syndrome. Smoking increases insulin resistance, a condition in which the body becomes less responsive to the hormone insulin, which regulates blood sugar levels. Insulin resistance can lead to high blood sugar levels and eventually to type 2 diabetes (25).

Smoking also increases the risk of developing abdominal obesity, one of the key components of metabolic syndrome. The toxins in cigarette smoke promote the accumulation of fat in the abdomen, which increases the risk of developing insulin resistance, high blood pressure, and abnormal cholesterol or triglyceride levels.

There are several biomarkers that can be used to early detect metabolic syndrome, including: ○

Fasting glucose: A blood test that measures the amount of glucose in blood after an overnight fast. Elevated levels of fasting glucose can indicate insulin resistance and prediabetes, both of which are risk factors for metabolic syndrome;

Overall, a combination of these biomarkers can be used to early detect metabolic syndrome.

Tweed et al. (341) reviewed the effects of smoking (CC) and the involvement of nicotine on the endocrine system. The authors provide suggestions for mechanisms how acute and chronic nicotine exposure might be involved in insulin resistance, weight gain and other hormone-controlled processes.

In an acute intravenous nicotine infusion study, Axelsson et al. (342) found that in patients with type 2 diabetes, but not in healthy subjects, the treatment induced a reduction in insulin sensitivity. The authors conclude that diabetic subjects are particularly susceptible to the detrimental effects of nicotine.

Hod et al. (343) reviewed the literature on EC use and weight gain in human, animal and in vitro studies. According to the authors, results from human studies (most of them were cross-sectional) were inconclusive, partly due to severe limitations in the study design. A high prevalence of EC use in overweight and obese subjects was noted. The role of nicotine was not clear. In contrast, most animal studies found reduced weight gain upon EC exposure with nicotine partly found to play a significant role. Results of in vitro studies with adipocytes were inconclusive in terms of explaining the influence of EC aerosol or nicotine on weight gain. The authors emphasize the need for further research including human studies with nicotine-free ECs. Evaluation of data from 5121 adults from the NHANES study, including TGs, HDL, fasting blood glucose and elevated blood pressure led the authors conclude that EC use or dual use is associated with MetS.

Table 6 (see page 85) summarizes 7 studies on the association between nicotine product use and the risk for metabolic syndrome (determined by various biomarkers or self-reported pre-diabetes), including 5 cross-sectional (113, 121, 334,335,336), one cross-over (347) and one pooled study consisting of 5 cohort studies (348). The nicotine products investigated included NRTs (gum (345), patch (347)), SLT products (113, 348), ECs (344, 346) and dual use of EC + CC (121).

All but one study (346) showed at least some evidence that product use might be associated with insulin resistance (Table 6). The risk for metabolic syndrome, however, was lower than observed for smokers (CC).

Three studies (345, 347, 348) provide evidence that nicotine might be involved in the development of metabolic syndrome, while one study (346) suggests that an involvement of nicotine is unlikely.

There have been animal studies that suggest nicotine may have an impact on metabolic syndrome, although the exact mechanisms are not yet fully understood.

In a recent study, Duncan et al. (349) reported that nicotine elevates blood glucose levels through a Tcf7l2-dependent stimulatory action on the medial habenula in rats and mice. The authors suggest that Tcf7l2 regulates the stimulatory actions of nicotine on a habenula-pancreas axis that links the addictive properties of nicotine to its diabetes-promoting actions.

Long-term oral nicotine exposure of obese rats was found to reduce insulin resistance through reduced hepatic glucose release and thus contributes to lowering the blood glucose level (350).

In another rat study, 6 weeks of nicotine exposure resulted in reduced weight gain, blood insulin and TG (351). No effect was observed on blood glucose. Further experiments with antagonists suggest that the nicotine-related enhanced insulin sensitivity is mediated via the α-nAChR.

Mice exposed for 12 weeks to EC aerosol, smoke from CCs or fresh air were not reported to differ in terms of insulin resistance and glucose tolerance (346).

Experiments with white adipose tissues of smokers and non-smokers showed that nicotine increases lipolysis, which results in body weight reduction (a typical effect of CC use), but this increase also elevates the levels of circulating FFA and thus causes insulin resistance in insulin-sensitive tissues (352). These mechanisms would explain the controversial effects of nicotine in smokers: reduction in weight gain and increase in insulin resistance. Nicotine was shown to induce insulin resistance in cardiomyocytes of mice via downregulation of Nrf2 (353).

Smoking cigarettes is a well-established risk factor for metabolic syndrome, which is a cluster of conditions including high blood pressure, high blood sugar, excess body fat, and abnormal cholesterol and/or triglyceride levels. Studies have shown that smoking cigarettes is associated with an increased risk of metabolic syndrome and its individual components, even after accounting for other risk factors such as age, sex, and body mass index (BMI). The mechanisms underlying this association are not fully understood, but it is thought that smoking may contribute to metabolic dysfunction through inflammation, oxidative stress, and impaired insulin sensitivity.

There is limited research on the association between NGP use and metabolic syndrome, and the available studies have produced mixed results. Some studies have suggested that NGP use may be associated with metabolic dysfunction, such as increased insulin resistance and impaired glucose tolerance, while others have found no significant association. The role of nicotine in these effects is unclear, as some studies have suggested that nicotine alone may have metabolic effects similar to those of smoking, while others suggested that the effects of ECs on metabolism may be due to other factors such as flavorings or other additives.

Animal studies have provided evidence that chronic exposure to nicotine has no or even beneficial effects in terms of insulin resistance and glucose tolerance. These observations would be in disagreement with most of the human studies.

On the other hand, evidence from in vitro studies suggest that nicotine is involved in the development of metabolic syndrome and provide some plausible mechanisms.

Overall, the available evidence suggests that smoking cigarettes and using NGPs can contribute to the development of metabolic syndrome. While there is still limited research on the specific effects of ECs and other NGPs on metabolism, some studies suggested that these products may have similar effects to smoking, although to a significantly lower extent. The effects may be due, in part, to nicotine. Further research, in particular long-term studies with human NGP users is needed to prove or disprove an association between NGP use and metabolic syndrome.

The US Surgeon General Reports of 2010 (25) and of 2014 (24) gave overviews of the detrimental effects of smoking during pregnancy and the possible effects on the offspring. Accordingly, in males, smoking can lead to a decrease in sperm count, motility, and morphology. In females, it can affect ovulation and reduce the chances of conception. Smoking during pregnancy can increase the risk of miscarriage, premature birth, and low birthweight. It can also increase the risk of stillbirth and sudden infant death syndrome (SIDS). Smoking can accelerate the loss of fertility in women, leading to earlier menopause. It can also decrease the effectiveness of assisted reproductive technologies, such as in vitro fertilization (IVF). Quitting smoking can improve fertility and reproductive outcomes.

The Report of the US Surgeon General of 2016 on EC use among youth and young adults (354) concludes that nicotine can cross the placenta and has known effects on fetal and postnatal development. Nicotine exposure during pregnancy can result in multiple adverse consequences, including SIDS, altered corpus callosum, deficits in auditory processing, and obesity.

The EU-based Scientific Committee on Health, Environmental and Emerging Risks (SCHEER) (143) reviewed the most recent scientific and technical information on ECs and concluded for CNS and reproductive disorders: “The overall weight of evidence for risks of other long-term adverse health effects, such as pulmonary disease, CNS and reprotoxic effects based on the hazard identification and human evidence, is weak, and further consistent data are needed”.

There were only few human studies on the use of NGPs during pregnancy and its impact on mother and baby. Previous reviews came to the conclusion that, in particular, vaping during pregnancy does not reduce the birthweight and that there is insufficient evidence for detrimental effects on fetal development (6, 7, 355). Also, no particular involvement of nicotine was identified. Based on animal studies, the COT (7) concluded with respect to effects on the developing lungs, there “is good biological plausibility for an effect of nicotine on development”. However, COT cautioned to simply transfer the effects of nicotine in animal studies to humans, given the unclear relationship of dosing to human exposures (7).

In a review on vaping during pregnancy, Calder et al. (356) found that prevalence was about 1–7%. Most of the women used ECs in order to reduce or quit CC smoking.

Another review on possible detrimental effects of vaping on reproduction came to the conclusion that human studies are scarce and that the effects observed in animal models suggest that caution should be taken when vaping and that more research is required to identify its potential adverse effects on fertility (357).

McNeill et al. expressed concern about nicotine use and fetal development and cognitive deficits in adolescents (358). The authors concluded that the limited literature suggests that vaping in pregnancy has little or no effect on birthweight.

In Table 7 (see page 87), five studies with users of NGPs are summarized (359,360,361,362,363). All studies were of the cross-sectional type and included ECs as NGP, in one study snuff use was investigated (360), in two studies (359, 361) also dual users (CC + EC) were evaluated.

Results for EC users were inconsistent: No impact of vaping during pregnancy on birthweight was found in one study (361), whereas decreased birthweight was reported in daily EC users in another (363). Reduced birthweights in offspring were, however, observed for women who were dual or CC users during pregnancy (361). Surprisingly, smallness for gestational age (SGA) in offspring was observed when mothers vaped during pregnancy, but not for CC and dual users (359). Fertility was not found to be diminished by either vaping (EC) or smoking (CC) (362). Total sperm count was found to be significantly decreased in daily users of CC or EC, not so in snuff users (360). The authors concede that dual use (CC + EC) could not be excluded in their study population.

In a large study with 24,904 reproductive-aged women, a significantly elevated risk for disability in reproduction was reported for vapers (similar to smokers and dual users) compared to NU (364).

None of the study results in Table 7 shows evidence for a contribution of nicotine on detrimental effects of human reproduction.

Taken together, inconsistent results were found for electronic cigarette (EC) users during pregnancy, with some studies reporting no impact on birthweight and others reporting decreased birthweight or smallness for gestational age in offspring. None of the study results show evidence for a contribution of nicotine on detrimental effects of human reproduction.

Gravid rats were exposed to an aerosol derived from ECs with 0 and 18 mg/mL nicotine in the liquid as well as to fresh air (365). Weight at birth was not different between the groups. However, cerebrovascular dysfunction was observed in the offspring of EC-exposed rats, which was independent of nicotine content. The authors state that their data show some evidence for an effect of nicotine in the very early phase of life (1 month old offspring).

Gravid mice were exposed to EC aerosol with and without nicotine (366). Since the exposure was detrimental to maternal and offspring lung health, irrespective of the presence of nicotine, the authors concluded that the effects are likely due to by-products of vaporization rather than nicotine.

Wang et al. (367) reported that adult mice exposed prenatally to EC aerosols +/− nicotine could be predisposed to developing pulmonary disease later in life. Some changes in the lungs of the offspring, such as MMP-9 downregulation, were primarily found after exposure with nicotine-containing aerosol. The authors conclude that vaping during pregnancy is unsafe and increases the propensity for later-life interstitial lung diseases.

Offspring of mice exposed to EC aerosol with and without nicotine during gravity showed, regardless of nicotine presence, lung dysfunction and structural impairments that persists to adulthood (368).

Rehan et al. (369) reported asthma phenotype in terms of lung function impairments in the 1. (F1) and 2. generation (F2) of offspring from rats exposed to nicotine (1 mg/kg, s.c.) during gravity. The authors conclude that germline epigenetic marks imposed by exposure to nicotine during pregnancy can become permanently programmed and transferred through the germline to subsequent generations. Taken together, animal studies have shown that exposure to e-cigarette aerosol during gravity can lead to detrimental effects on offspring's health, including cerebrovascular dysfunction and lung structural impairments. The detrimental effects observed in offspring were not solely due to the presence of nicotine, as exposure to e-cigarette aerosol without nicotine also led to adverse health effects. The studies further suggest that exposure to nicotine during gravity can lead to permanent germline epigenetic marks, which can be transferred through generations and increase the propensity for later-life interstitial lung diseases in offspring.

Smoking cigarettes (CC) is an established risk factor for impairing female and male fertility. Smoking during pregnancy also increases the risk for low birthweight and has detrimental effects on the offspring.

Studies on new NGPs, particularly ECs, are limited, but previous reviews suggest that vaping during pregnancy does not reduce birthweight, nor is there sufficient evidence for detrimental effects on fetal development. Animal studies suggest that nicotine can have an effect on lung development, but it is unclear if this can be translated to humans. Gravid rats exposed to an aerosol derived from electronic cigarettes with nicotine showed a decreased number of offspring and a delay in fetal development.

Overall, inconsistent results were found for electronic cigarette (EC) users during pregnancy, with some studies reporting no impact on birthweight and others reporting decreased birthweight or smallness for gestational age (SGA) in offspring. None of the study results show convincing evidence for a contribution of nicotine on detrimental effects of human reproduction. Some animal studies suggest that nicotine exposure can affect reproduction. Further research is certainly needed, in particular human long-term studies, to clarify the somewhat controversial results between human and animal studies with respect to the role of nicotine.

Cigarette smoking can have several detrimental effects on the eyes (370), including: ○

Increased risk of age-related macular degeneration (AMD). Smoking was found to increase the risk of developing AMD by up to four times;

The exact mechanisms by which smoking causes these detrimental effects are not fully understood. However, it is thought that smoking may cause oxidative stress and inflammation in the eyes, leading to damage to the ocular cells and tissues.

Nicotine is known to constrict blood vessels, which can decrease blood flow to the eyes and other parts of the body. This can contribute to the development of AMD and other eye diseases. Additionally, nicotine has been shown to have a detrimental effect on the tear film, which can contribute to the development of dry eye syndrome.

In Table 8, four studies on EC use and detrimental ocular effects are briefly described (371,372,373,374). The rather small studies investigated both acute (371, 374) and chronic effects (1 to > 3 years of EC use) (372, 373). No acute effects on tear film quality (371) or on choroid thickness (CT) and centralfoveal thickness (CFT) (374) were reported. Long-term use (a few years) was found to be associated with detrimental effects on tear film quality (372) and foveal vision (373). In the latter study, nicotine-related vasoconstriction in the retina was suggested. From all other studies no involvement of nicotine can be deduced. The chronic studies were cross-sectional studies and may suffer from misclassification of product use.

Cigarette smoking can have several detrimental effects on the bones (375), including: ○

Increased risk of osteoporosis, which causes bones to become weak and brittle, making them more susceptible to fractures. Smoking can contribute to osteoporosis by reducing bone density and interfering with the body’s ability to absorb calcium.

The exact mechanisms by which smoking causes these detrimental effects are not fully understood. However, it is thought that smoking may cause oxidative stress and inflammation in the bones, leading to damage to the cells and tissues that make up the bones.

Nicotine may have detrimental effects on bone health, for example by interfering with the body's ability to absorb calcium, which is essential for healthy bones. Additionally, nicotine has been shown to reduce bone density and bone mineral content, which can contribute to the development of osteoporosis (376).

Nicholson and coworkers (377, 378) evaluated the literature for possible detrimental effects of vaping on bone health. The authors stated that long-term studies in humans are lacking. However, the fact that nicotine receptors are expressed in human osteoblasts and osteoclasts suggests, according to the authors, that use of nicotine products could have an impact on bone health, particular on bone healing following orthopedic surgery and injury. Evidence is discussed showing that nicotine and other constituents of EC aerosols, such as carbonyl compounds and metal can significantly impair osteoblast function, suggesting the EC use may be detrimental to bone health. In vitro experiments of the Nicholson group (379) with EC condensate demonstrated nicotine-dependent reduction of viability and impaired function of human osteoblasts.

Kallala et al. (376) reviewed the nicotine-related effects of smoking in humans, animals and in vitro systems. Nicotine is found to have effects on osteoneogenesis, osseointegration and steady-state skeletal bone. High nicotine doses mostly exhibit negative effects, whereas low doses have stimulating effects. The authors also mention emerging evidence of nicotine’s potential future therapeutic role in bone augmentation and reconstructive surgeries.

Reumann et al. (380) exposed Apo-E^−/− mice to EC aerosol with and without nicotine. In contrast to CC smoke and similar to sham exposure, EC aerosol exposure did not change bone structure and integrity, irrespective of the nicotine content.

In Table 8, two cross-sectional studies were briefly reviewed, one suggested that vaping (EC) could increase the risk of bone fragility (381), the other (382) implies that EC use is a risk factor for arthritis. An involvement of nicotine in inflammatory processes such as arthritis cannot be excluded. The weaknesses of cross-sectional studies have to be considered when these results are interpreted.

Cigarette smoking can have impairing effects on physical performance (383, 384), including: ○

Decreased lung function: Smoking can damage the lungs and reduce lung capacity, making it more difficult to breathe during exercise. This can result in reduced endurance and performance.

Smoking-related oxidative stress and inflammation in the body lead to damage of lung cells and tissues as well as blood vessels and muscles.

Nicotine can have a stimulant (sympathomimetic) effect on the body, which can increase heart rate and blood pressure. However, nicotine can also constrict blood vessels, thus reducing blood flow to the muscles and impairing physical performance. Additionally, nicotine can interfere with the body’s ability to transport and use oxygen, which can contribute to reduced endurance. Overall, it has to be assumed that the negative effects of smoking on physical performance outweigh any potential benefits from nicotine’s stimulant effects.

Physical performance in EC-exposed female mice (measured by the grip strength, swimming time and glycogen content in liver and muscles) decreased with increasing nicotine content in the e-liquid (385). The authors do not provide a plausible explanation for this observation.

Impairment of skeleton muscle force and regeneration was compared in male mice exposed to air, PG/VG +/− nicotine aerosols (386). Impairment was found after exposure to sole PG/VG aerosol, however, effects were stronger with the nicotine-containing aerosol. The nicotine aerosol also decreased the adrenal and increased the blood epinephrine and norepinephrine levels. Furthermore, the glycogen stores in the muscles and the liver were elevated after exposure to nicotine aerosol.

In Table 8, a short description of a study is listed, which investigated the physical performance of long-term smokers (CC, 28 years use duration) and SLT users (25 years of use) in comparison to NU (387). While for smokers a significantly lower VO_2max and workload was found, no decrease was seen in SLT users. The results suggest that nicotine might not be responsible for an impairment of physical performance.

Cigarette smoking was reported to have a range of detrimental effects on brain and the mood, including depression, anxiety disorders, schizophrenia, and substance use disorders (388, 389).

Studies have shown that cigarette smoking can increase the risk of developing depression and anxiety disorders, and can worsen the symptoms of these disorders in people who are already affected. Observations are controversial, while smoking was found to worsen the symptoms of schizophrenia and impair cognition and memory, other studies reported improving effects of smoking on these traits (see also next chapter).

Several hypotheses have been proposed with respect to the mechanism how smoking may affect the brain (390). One possibility is that the nicotine in cigarettes can affect the release of various neurotransmitters in the brain, including dopamine and serotonin, which are involved in mood regulation. Nicotine can also activate the hypothalamic-pituitary-adrenal (HPA) axis, leading to increased levels of stress hormones, which can exacerbate anxiety and other mental health problems.

Additionally, smoking is associated with chronic inflammation, which may play a role in the development of mental disorders. Studies have shown that people with mental illnesses often have higher levels of inflammation biomarkers in their bodies, and smoking may contribute to this by increasing levels of pro-inflammatory cytokines.

Overall, while nicotine may play a role in both detrimental and beneficial effects of cigarette smoking on mental health, the exact mechanisms are likely complex and multifaceted.

The Committee on Toxicity of Chemicals in Food (COT) (7) also reviewed nicotine effects in adolescents and young adults but found no data on the direct effects of nicotine in humans to examine. COT commented that while brain development in humans continues to around 25 years of age, there was the potential for nicotine to have adverse neurodevelopment effects as well as negative mental health effects.

In a state-of-the-art review, Ruszkiewicz et al. (391) stated that vaping can cause the following effects in the human brain: calcium dyshomeostasis, epigenetic changes, impaired autophagy, impaired neurotransmission, mitochondrial dysfunction, neuroinflammation, and oxidative stress. As possibly involved agents in the EC aerosol, nanoparticles, free radicals, heavy metals, flavor chemicals, PG/VG and nicotine were named. The authors cite evidence that nicotine can be rapidly delivered to and accumulate in the brain by use of ECs and other products, similar to CC use. Interestingly, quite a number of animal studies were cited, which show adverse brain effects of EC aerosols also in the absence of nicotine. On the other hand, developmental neurotoxicity of nicotine is, according to the authors, well established, as well as all effects related to cholinergic stimulation related to dependence formation and withdrawal effects. In this review also a section on neuroprotective effects of nicotine is included comprising its (mostly beneficial) role in Parkinson’s disease, Alzheimer’s disease, schizophrenia and depression. Furthermore, it is mentioned that nicotine has positive cognitive effects. The authors, however, add for consideration that these beneficial effects of nicotine may be nullified by other harmful and neurotoxic compounds in the EC aerosol (391).

A vast amount of literature shows that smoking-derived nicotine exacerbates ischemic brain damage and induction of stroke (392). Siegel et al. (392) listed 4 pathways, how nicotine may exert adverse effects on the brain: (i)

inhibition of aromatase enzyme activation thus abolishing the de novo synthesis of 17β-estradiol (E2);

In a literature review, Raval provides evidence that nicotine inhibits estrogen signaling. According to the authors this may make women’s brains more susceptible to ischemic damage (393).

Ren et al. (394) reviews the effects of nicotine exerted via the nAChRs in the brain across the life-span. Evidence from in vivo (animal) and human studies, according to the authors reveal that nicotine exposure during the perinatal period disrupts general growth, cardiovascular and lung function, the endocrine system, motor function, reward, and attention.

Nicotine exposure during adolescence may enhance susceptibility to addiction, impulsivity, and mood disorders, while during adulthood nicotine may not have the apparent adverse consequences on the brain. The authors finally point to potential neuroprotective effects (in e.g., Alzheimer’s and Parkinson’s diseases, see also chapter on beneficial effects of this review) of nicotine in senescence, which might comprise an interesting field of research to explore further.

In a recent review, Leslie (395) emphasized that nicotine can have particularly detrimental effect on adolescent brains. Most of the evidence comes from animal studies, however, human studies on adolescent brain showed that there is an increased number and activity of nAChRs in brain regions that are important for reward and an increase in nicotine-induced dopamine release in limbic regions. Some effects, such as nicotine reward, are greater in adolescents than adults, whereas other behavioral effects, such as aversion, are greater in adults. The author emphasized the need for further longitudinal studies in teen and young adult EC users.

The Report of the US Surgeon General of 2016 on EC use among youth and young adults (354) concludes that nicotine exposure during adolescence can cause addiction and can harm the developing adolescent brain.

In in vitro and in vivo/ex vivo experiments with mouse brain cells, it was found that nicotine and EC aerosol can induce glucose deprivation which could lead to enhanced ischemic brain injury and stroke risk (396).

In Table 8, two relatively large cross-sectional studies on the use NGPs and mental disorders/mood are listed (397, 398). In both studies, an association between EC use and depression as well as suicidality was observed. An involvement of nicotine cannot directly be deduced from the results, but is supposed by the authors (397, 398).

In the same section of Table 8, a meta-analysis comprising 31 placebo-controlled clinical trials with nicotine patches and their acute impact on cognitive functions, attention and memory is reviewed (399). A significant improvement in cognitive function and attention was observed upon nicotine patch use.

There is some evidence to suggest that nicotine may have some cognitive benefits. Research suggests that nicotine can improve attention, working memory, and other cognitive functions, particularly in people who are abstinent from smoking or have a genetic predisposition to cognitive deficits. The former observation would suggest that nicotine acts primarily by eliminating withdrawal effects of abstinence, which has impaired cognitive capabilities. Nicotine appears to work by increasing the release of neurotransmitters such as dopamine and norepinephrine, which are involved in attention and memory processes.

In a nicotine replacement study (abstinent smokers used nicotine lozenges), it was shown by EEC measurement that the treatment directly affected brain neuronal activity modulating the cognitive network (403).

Comparing the cognitive performance in subjects smoking nicotine-containing and de-nicotinized (placebo) cigarettes suggests that response expectancies can be experimentally manipulated and can influence perceived rewarding effects of smoking, but do not affect the actual cognitive performance when smoking nicotine cigarettes (404).

Newhouse et al. (405) stressed the fact that the effect of nicotine on cognitive performance depends on the starting level. Normal individuals are unlikely to show cognitive benefits after nicotinic stimulation except under extreme task conditions, individuals with mental diseases such as Parkinson’s or Alzheimer’s disease or schizophrenia may benefit from nicotine.

A meta-analysis comprising 31 studies revealed that transdermal nicotine had statistically significant positive effects on attention, and non-significant effects on memory, in healthy non-smoking adults (399).

Nicotine can improve some of the cognitive deficits associated with schizophrenia, such as attention, working memory, and sensory processing. The same mechanisms as described in the previous section are involved.

In addition, some studies have found that smoking may have a protective effect against the development of schizophrenia, and that people with schizophrenia who smoke may have better cognitive function and fewer negative symptoms than those who do not smoke. Smoking rate in schizophrenic patients is significantly higher than in the general population, suggesting that schizophrenics may use nicotine products for self-medication (406, 407).

It was suggested that also other patients with psychiatric disorders may benefit from the pharmacological effects of nicotine on cognitive functions (408). Therefore, various groups of patients with psychiatric diseases may particularly benefit from harm-reduced NGPs.

In a review of 2017 (409), 9 relevant studies were identified which investigated the replacement of CC with EC in patients with mental illnesses. It was found that some studies suggest that combustible tobacco product use is decreased among these patients after initiating EC use, other studies imply that rates of dual use are higher than in the general population. The authors conclude that more research is required on the potential benefit or harm for subjects with mental illness when they switch from CC to EC.

Another review on EC use in mentally ill adolescents and young adults (12–26 years old) identified 40 (mostly) studies and found a significant relationship between vaping and mental illness compared with NU. The mental illness included depression, anxiety, suicidality, eating disorders, post-traumatic stress disorder, externalizing disorders (attention-deficit/hyperactivity disorder and conduct disorder), and transdiagnostic concepts (impulsivity and perceived stress). Notably, the authors name the association between the illnesses and EC use ‘comorbidities’. However, they concede significant methodological limitations, e.g., directionality in cross-sectional studies.

There is some evidence to suggest that smoking and nicotine may have beneficial effects for people with ulcerative colitis (UC), a type of inflammatory bowel disease. Research suggests that smoking may decrease the risk of developing UC, and that people with UC who smoke may have less severe symptoms and a lower risk of requiring surgery than non-smokers with UC (410, 411).

Possible mechanisms responsible for this remain purely speculative but may involve mucosal eicosanoids and intestinal mucus. It is also not clear whether nicotine by binding to the nACHR is the major component in smoke responsible for the beneficial effects in UC (412, 413).

As to the role for nicotine, some studies have found that nicotine replacement therapy (NRT, usually nicotine patches) may improve symptoms in people with UC who do not smoke. A review comprising 22 studies came to the conclusion that transdermal nicotine in combination with conventional therapy was more beneficial than individual treatment with either (414).

In a case control study, no association between vaping (EC) and the treatment outcome of UC was observed compared to NU (415). However, the study was rather small (47 UC cases using ECs).

Taken together, there is supportive evidence that smoking (CC) could be beneficial for UC, but there is as yet inconclusive evidence that nicotine and NGPs can have such a beneficial effect.

There is epidemiological evidence to suggest that smoking (CC) may have beneficial effects on the development and severity of Parkinson’s disease (PD) (416). The author states that the frequently discussed ‘pre-Parkinson’s personality’ appears unlikely as an explanation for the association. Research suggests that nicotine can improve some of the motor and cognitive symptoms associated with PD, such as tremors, rigidity, and attention. Nicotine appears to work by increasing the release of dopamine and other neurotransmitters that are involved in motor and cognitive processes. A recent investigation with neurons of Drosophila brain indicates that both the serotonergic and dopaminergic systems contribute to different aspects of PD symptomatology and that nicotine has beneficial effects on specific symptoms (417).

Quik et al. (418) suggest that beneficial nicotine effects may work by several mechanisms: (i)

protective action against nigrostriatal damage;

It was suggested that nicotine gum could be a promising tool against various brain disorders, including PD and Alzheimer’s disease (AD).

Various research suggests that nicotine may have beneficial effects for people with Alzheimer’s disease (AD) by improving memory performance and cognitive deficits (419, 420). Nicotine appears to work by increasing the release of dopamine that is involved in cognitive processes. In addition, some studies have found that smoking may have a protective effect against the development of AD, and that people with AD who smoke may have slower cognitive decline than non-smokers with AD.

Possible mechanisms for the beneficial effects of nicotine in AD but also in PD and in cognitive function in healthy individuals have recently been reviewed (421). The mechanisms involved are not yet clear. Stimulation of the nicotinic receptors in the brain is hypothesized. However, anti-inflammatory, anti-oxidant effects as well as neuro-protective effects by enhancing the survival of certain types of neurons are also discussed.

Tourette’s syndrome (TS) is a neuropsychiatric disorder characterized by motor and vocal tics, obsessions, compulsions and visual-motor deficits (422). It has been shown that administration of nicotine (patches or gums) can potentiate the efficacy of treatment with neuroleptics (422, 423). The effect was observed to persist for days, weeks or even months without further nicotine administration.

It has been proposed that TS is caused by excessive striatal dopamine release and/or dopamine receptor hypersensitivity. Nicotine is known to pre-synaptically rapid release of several neurotransmitters, such as acetylcholine, γ-aminobutyric acid, norepinephrine, dopamine, and serotonin, followed by a prolonged desensitization of the receptors. The latter effect might be responsible for the persistent effect of nicotine in TS (422).

It is well established that smoking cessation is associated with a significant weight gain (424,425,426,427).

The mechanism of this phenomenon is not exactly known, various possibilities are discussed: (i)

increased food intake;

The role of nicotine is discussed controversially. In a long-term smoking cessation study, nicotine gum users gained significantly less weight than NU (428). A similar effect was observed, when nasal nicotine spray was applied as smoking cessation aid (429). In a smoking cessation study over 12 months in Japan, it was found that when Varenicline was used as a quitting aid, weight gain was lower than when using nicotine patches (0.94 versus 2.78 kg) (430). In a 12-month smoking cessation study with postmenopausal women, weight gain in a nicotine and a placebo patch group was similar after 1 year, despite the fact that after 12 weeks (duration of patch use) the nicotine group consumed significantly more calories (431).

Another nicotine patch study compared standard treatment duration (8 weeks) versus extended treatment (24 weeks) (432). After 24 weeks, the extended-treatment group gained significantly less weight than the standard-treatment group. In a systematic review on the association between body weight and EC use, 13 studies were evaluated, comprising human, animal (in vivo) and in vitro studies (343). The authors found a high prevalence of EC use among an obese population. From that, however, the authors concede that no causal inference can be concluded, as the majority of the human studies were cross-sectional. The reviewed animal studies consistently suggest that EC use may cause weight decrease. However, these observations were not supported by the reviewed in vitro data. The authors conclude that the effect of vaping on body weight changes, require further investigations (343).

Nicotine can act as an appetite suppressant, reducing feelings of hunger and increasing basic metabolism. Nicotine appears to work by stimulating the release of adrenaline and other neurotransmitters that increase energy expenditure and reduce food intake (433).

Long-term epidemiological studies, which investigate chronic effects of NGP use for 10 or more years are by now virtually available only for products such as SLT (e.g., snus) or NRT products by which the class of NGPs was extended. Endpoints include myocardial infarction (MI) (39,40,41,42), heart failure (43), stroke (42, 48), cardiovascular diseases (CVD) (56), hypertension (HT) (87, 88), various cancers (56, 161,162,163), type 2 diabetes (348).

In a series of human studies the long-term use of ECs were investigated. The duration of use is frequently not well defined, but can be assumed to be less than 10 years, due to the market availability of these products since about 2006. Endpoints of these studies include stroke (52), hypertension (89, 90), asthma (212, 231, 232), COPD and various respiratory impairments (119, 198, 199, 213, 216, 223), oral health risks (301), metabolic syndrome and diabetes (344, 346), arthritis and bone impairments (381, 382), low birthweight delivery (363), mental health symptoms and depression (397, 398).

Brief study descriptions, results, role of nicotine and limitations of these long-term studies are presented in Tables 1–4 and 6–8.

Results of the long-term studies cited above are very inconsistent. Chronic use of NGPs ranged from lowering the risk to levels comparable to NU risk to increasing the risk close to or even higher than that of cigarette smoking (CC).

All studies have a number of weaknesses and limitations noted in the summary tables. Apart from the fact that all chronic EC studies presently available, cover by far a too short time period of EC use (significantly lower than 10 years) in order to draw any firm conclusions with respect to causing chronic detrimental effects or diseases, there are at least two major issues, which could erroneously increase the risk of NGP use determined in long-term studies, particularly cross-sectional studies: ○

Misclassification of NGP use: It is highly likely that subjects defined as NGP only users are actually, at least for some time periods, dual users of NGPs with most frequently combustible cigarettes (CC). Questionnaires and short-term biomarkers of exposure to CC (e.g., COHb/COex, but also cotinine) are in general not suitable to eliminate this confounder (16, 434). Almost all studies have shown that dual use is implicated with health risks similar or close to that of CC only use (see Tables 1–8 in this review). A suitable measure to avoid or at least reduce the confounder of misclassification would be to apply specific long-term biomarkers of exposure to CC, such as 2-cyanoethyl-valine hemoglobin adducts (CEVal), which can indicate CC use of the last 3–4 months (123, 333, 435).

The possible involvement or non-involvement of nicotine in the pathogenesis of the investigated diseases or disorders would also have to be evaluated by considering the general study limitations described above.

On the other hand, the evaluation ‘0’ (meaning that nicotine is not involved, e.g., in (39, 40, 161, 163, 199)) could also be wrong for reasons discussed in the following.

First, the duration of EC use could be too short for a profound statement in the implicated risk. Second, other weaknesses might be too small group sizes and very heterogeneous product designs preventing clear study outcomes. Third, nicotine delivery of the first generation of ECs was rather low compared to CCs and the newer generations of ECs (97, 105, 209), so that the alkaloid could not unfold an effect. Forth, the same could be true with the low nicotine deliveries of NRT products investigated in long-term (163).

The involvement of nicotine in the pathogenesis of various diseases, disorders or any physiological changes upon chronic and acute use of NGPs has been evaluated in the presented human studies (Tables 1–8) as described in the methodological sections 2.3 and 2.4. The results of this evaluation were further condensed to an even simpler system, comprising 3 classes for describing the probability of an involvement of nicotine in detrimental effects in NGP users: ○

Nicotine is unlikely to be involved in the pathogenesis. Class I comprises the codes 0 and 0/? used in Tables 1–8).

Table 9 summarizes the results of Tables 1–8 according to this classification system (see page 90).

From the number of evaluable observations in human studies, it is obvious that for most of the single diseases of disorders the number of studies is too low for any strong conclusions. For all disorders, Class II (involvement of nicotine cannot be deduced, but can also not be ruled out) is found to have the highest frequency (50%). In disorders with at least 10 observations, Class I (involvement of nicotine rather unlikely), cancer shows the highest frequency (40%).

CVD-related endpoints were most frequently investigated in our review (75 observations), followed by RD-related (43 observations) and oral health-related endpoints (23 observations). Although we are well aware of the fact that the relative distributions cannot be representative for any disease or product investigated, the described classification for the role of nicotine may provide some valid indications in which diseases or detrimental effects the participation of nicotine is possible or unlikely.

In all CVD-related effects in NGP users presented in Table 1, 49% were assigned to Class III (evidence for participation of nicotine). Some disorders in the CVD category showed exceptionally high percentages for Class III, namely risk for hypertension (100%), acute increase in heart rate and blood pressure (72%) and arterial stiffness (47%) (Table 9). An involvement of nicotine in CVD-related disorders was also reported in long-term animal studies (most frequently using mice as a model) (127,128,129,130,131). As possible mechanisms for the involvement of nicotine in CVD development in mice the stimulation of oxidative stress, inflammatory processes in the vascular endothelium, lipid accumulation and sympathetic dominance were discussed (132). In vitro studies showed that nicotine can stimulate vascular endothelial cell proliferation and inflammation and thus contribute to atherosclerosis (138, 141).

The role of nicotine in respiratory disease- (RD) related endpoints was evaluated in 43 human studies with NGPs (Table 3). A participation of nicotine could not be deduced from the presented study data (but also not excluded) in 65% or the observations (Class II), while the percentages for an unlikely (Class I) and a possible involvement of nicotine (Class II) in the pathogenesis was 16 and 19%, respectively (Table 9). In other words, for this category of disorders, the uncertainty of the role of nicotine is higher compared to CVD-related disorders. In considering the literature on human studies with NGPs and the effects on the respiratory tract (see Section 5.2), the overall conclusion with respect to the role of nicotine was that there is inconsistent evidence for a participation of nicotine in the development of RD. A controversial picture of the participation of nicotine in RD-related effects of EC and HTP aerosols must be also deduced from animal and in vitro studies reviewed in Sections 5.3 and 5.4, respectively. In quite a number of both types of studies, detrimental effects on respiratory tract and lung epithelial cells were also observed in the absence of nicotine.

Damaging effects on the oral cavity and buccal cells were investigated in 23 human studies (Table 4). In the majority of observations (57%) a participation of nicotine cannot be deduced from the data, but also not completely ruled out (Class II). A participation of nicotine in the observed effect of NGP use was unlikely in 9% of the studies (Class I), whereas a contribution of nicotine to detrimental effect appears possible in 35% of the observations (Class III). A similar conclusion in terms of the participation of nicotine in oral health risks can be drawn from various reviews considered in Section 6.2. A consistent finding is that nicotine is causally associated with a decrease in BOP, due to its vasoconstriction effect in gingival tissue. No animal studies on the role of nicotine in oral mucosa damage are available. An in vitro model showed that nicotine may induce inflammatory processes in oral mucosa tissue (318).

Not unexpectedly (chemical carcinogenesis typically requires decades in humans), there are only a limited number of human cancer studies available which allow an evaluation of the role of nicotine in the development of this class of disease (10 studies are summarized in Table 2). The Class I/II/III distribution for the probability of a participation of nicotine in cancer induction was found to be 40/50/10% (Table 9), with a high degree of uncertainty, given the low number of studies and the extent of bias and confounding factors in human long-term studies, as discussed earlier in this review. The role of nicotine in carcinogenesis is discussed controversially since at least the first US Surgeon General Report on Smoking and Health (8). In none of the recent evaluations of the cancer risk in NGP users (particularly vapers) presented in Section 4.2, nicotine is genuinely considered as a product constituent responsible for increasing the cancer risk (6), although the possibility that nicotine can be a precursor for the human carcinogens NNN and also (much less likely) NNK has to be kept in mind (184, 185). In long-term animal studies, nicotine was not found to increase the lung cancer rate in mice (179, 180). On the other hand, EC aerosol without nicotine was reported to increase the lung and bladder tumor rate in mice (30, 181, 182). To sum up the potential role of nicotine in cancer risk of NGP users, it appears safe to state that there is presently no evidence that nicotine might increase the cancer risk compared to NU. The data available rather give cause to assume that cancer risk in NGP users is reduced compared to smokers of CC.

There is at least some evidence that nicotine might be involved in the development of a metabolic syndrome in NGP users (7 studies, Tables 6 and 9). Evidence from animal studies (Section 8.3) and in vitro studies (Section 8.4) show contradictory effects of nicotine with respect to insulin resistance, glucose tolerance and weight gain. Clarification or the association between chronic NGP use and metabolic syndrome is certainly an interesting field of research for the future.

Five human studies on use of NGPs and reproduction were evaluated for a possible role of nicotine (Table 7). None of the studies shows evidence for a participation of nicotine in generation of detrimental effects to the offspring. A rat study (reviewed in Section 9.3) suggests that nicotine may have detrimental effects in lung and CNS development in the offspring (367). However, no nicotine-dependency of these endpoints were reported in other studies (365, 366, 368).

Too few observations for a meaningful evaluation of the role of nicotine in other disorders such as detrimental effects on the eyes, bones, brain or physical performance are available (Table 9). In terms of mental disorders, the considerations provided in Section 10.4 suggest that nicotine may have both detrimental effects as well as beneficial effects on the brain. For the developing brain (up to 25 years of age in humans), the Committee on Toxi-City of Chemicals in Food (COT) (7) suggested that there is the potential for nicotine to have adverse neuro-development effects negatively influencing mental health.

In Tables 1–8, which review potential risks for diseases and disorders of NGP users, gaps (“G”), limitations (“L”), potential bias and confounding factors are briefly mentioned. Also, for promising approaches, possible research proposals (“P”) are mentioned, which we think would broaden our knowledge in the field of NGPs and health risks. Further valuable information on nicotine effects could be gained from medium- to long-term studies with NRT products such as nicotine nasal spray, gum or patches.