Electronic cigarette and carcinogenesis: A narrative reviewAbstract
Electronic Nicotine Delivery Devices (ENDS), commonly known as e-cigarettes, have become increasingly popular. However, despite the well-established human health risks, the mechanisms of carcinogenesis remain unclear. The key characteristics of carcinogens provide an efficient strategy for identifying and organizing data from mechanistic studies. We aimed to identify, organize, and synthesize the biological evidence indicating the carcinogenic potential of these devices, categorizing them into five key characteristics potentially involved in cancer initiation and/or promotion: immunosuppression, inflammation, genotoxicity, oxidative stress, and epigenetic alterations. A narrative review was conducted through a bibliographic search of articles published in English over the past ten years (2015-2025) in the following scientific databases PubMed, Scopus, EMBASE, and Lilacs, using an extensive combination of exposure and effect descriptors. The analysis of the selected studies suggests that e-cigarettes cause toxic effects on the immune system (immunosuppressive and pro-inflammatory activity), on DNA (genotoxicity and oxidative stress effects), and act as a disruptor of gene expression.
Keywords:
Vaping; Toxicity; Neoplasms; Cancer.
Resumo
Dispositivos eletrônicos de administração de nicotina (ENDS, na sigla em inglês), comumente conhecidos como cigarros eletrônicos, tornaram-se cada vez mais populares. No entanto, apesar dos riscos bem estabelecidos para a saúde humana, os mecanismos de carcinogênese permanecem obscuros. As principais características dos carcinógenos fornecem uma estratégia eficiente para identificar e organizar dados de estudos mecanísticos. Nosso objetivo foi identificar, organizar e sintetizar as evidências biológicas que indicam o potencial carcinogênico desses dispositivos, categorizando-as em cinco características principais potencialmente envolvidas na iniciação e/ou promoção do câncer: imunossupressão, inflamação, genotoxicidade, estresse oxidativo e alterações epigenéticas. Uma revisão narrativa foi conduzida por meio de uma busca bibliográfica de artigos publicados em inglês nos últimos dez anos (2015-2025) nas bases de dados científicas PubMed, Scopus, EMBASE e Lilacs, utilizando uma ampla combinação de descritores de exposição e efeito. A análise dos estudos selecionados sugere que os cigarros eletrônicos causam efeitos tóxicos no sistema imunológico (atividade imunossupressora e pró-inflamatória), no DNA (efeitos de genotoxicidade e estresse oxidativo) e atuam como disruptores da expressão gênica.
Palavras-chave:
Vaporização; Toxicidade; Neoplasias; Câncer.
Introduction
Electronic smoking devices (ENDS), known as e-cigarettes, are liquid-heating systems that create aerosols to be inhaled by the user, without tobacco combustion. Most of these ENDS are nicotine delivery systems, although nicotine-free versions exist. These liquids contain additives with varied and attractive flavors, as well as toxic substances, many of which appear to exceed the limits considered safe (Bernal et al., 2023). Although traditional smoking is associated with the risk of cancer, the contribution of ENDS to carcinogenesis is still unknown. Added to this is the risk of concomitant use with conventional cigarettes and the possible synergistic effects.
The key characteristics of carcinogenesis mechanisms published by the International Agency for Research on Cancer (Smith et al., 2015) are an efficient strategy for identifying and organizing data from studies on carcinogenesis mechanisms. By this method, all known human carcinogens possess at least one of these properties: i) it is electrophilic or can be metabolically activated to electrophilic; ii) it is genotoxic; iii) it alters DNA repair or causes genomic instability; iv) it induces epigenetic alterations; v) it induces oxidative stress; vi) it induces chronic inflammation; vii) it is immunosuppressive; viii) it modulates receptor-mediated effects; ix) it causes immortalization; and x) it alters proliferation, cell death, or nutrient supply (Smith et al., 2015).
Data from the literature from experimental studies in murine models have provided consistent evidence on the deleterious effects of ENDS on multiple biological systems, revealing carcinogenicity mechanisms that deserve detailed investigation in human populations. In this sense, it was observed that:
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i) Exposure to ENDS significantly decreased the ability to defend against infections in mice, reducing phagocytosis by alveolar macrophages and increasing susceptibility to bacterial and viral infections, with greater morbidity and mortality (Sussan et al., 2015; Hwang et al., 2016; Serpa et al., 2020);
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ii) ENDS induced complex inflammatory patterns, altering the distribution of macrophages and neutrophils, modifying the cytokine secretion profile and cellular infiltration in bronchoalveolar lavage (BAL), with specific responses dependent on the aromas used (Lerner et al., 2015; Hwang et al., 2016; Muthumalage et al., 2020; Been et al., 2022);
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iii) Multiple studies have demonstrated a consistent elevation of oxidative stress markers (MDA and 8-OHdG) after exposure to ENDS, indicating systemic cellular damage, regardless of the type of flavoring used (Been et al., 2022);
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iv) Exposure to ENDS caused a reduction in serum IgM levels, alterations in the frequency of B and T lymphocytes, and redistribution of helper T cells (CD4+), in addition to promoting the expansion of IL-10-producing immunosuppressive cells in specific models (Lechasseur et al., 2020; Scieszka et al., 2023; Kastratovic et al., 2025); and
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v) Studies in Sprague Dawley rats have demonstrated genotoxic and mutagenic effects of ENDS vapors, including damage to leukocyte DNA, increased frequency of micronucleated reticulocytes, myelosuppression, and pro-mutagenic effects of urinary metabolites, suggesting an increase in point mutations and frameshifting (Canistro; Vivarelli; Cirillo, 2017).
Electronic cigarettes (e-cigarettes; ECDs) are a public health challenge, especially for young people, with their popularization based on a supposed benignity compared to conventional cigarettes, sometimes being considered a safe alternative, even as a strategy for smoking cessation. Thus, this review aims to identify, organize, and synthesize the biological evidence that points to the carcinogenic potential of these devices, with the intention of supporting health protection measures. For this work, five key characteristics were selected, namely: immunosuppression, induction of inflammation, genotoxicity, induction of oxidative stress, and epigenetic alterations.
Methods
This is a narrative review, for which a bibliographic search of articles published in English (2015 to 2025) was carried out in the scientific databases PubMed, Scopus, EMBASE and Lilacs.1
To identify the search terms, the controlled vocabularies of the health area DeCs (Descriptors in Health Sciences), MeSH (Medical Subject Headings) and EmTREE (Embase Subject Headings) were consulted, using the following combination of descriptors:
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i) exposition: Electronic cigarette; e-cigarette; Electronic Cigarette Vapor; electronic nicotine delivery systems; Nicotine Replacement Therapy; vaping; Vape; Heat-not-burn product; E-cigarette; ECIG; E-cigarette/electronic vapor delivery systems; E-cigs;
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ii) genotoxic effects: micronucleus; micronucleus test; Micronucleus Assay; chromosomal aberration; chromosomal aberrations; comet assay; comet test; DNA double-stranded breaks; DNA Breaks, Double-Stranded; DNA damage; Damage, DNA; DNA Injuries; DNA Injury; DNA Lesion; Genotoxic Stress; Injuries, DNA; Injury, DNA; Stress, Genotoxic; oxidative stress; Oxidative DNA damage; apoptosis; cytotoxicity; genotoxicity; Mutagenicity Tests; Ames assay; Telomere Shortening; Pig-a gene mutation assay; In vivo Pig-a gene mutation assay; PIG-A; GPI anchor, Paroxysmal nocturnal hemoglobinuria; CD55; CD59; CD14; CD16;
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iii) immunotoxic effects: immune; Immunity; Immune System; immunosuppression; immune response; immune cells; immunophenotyping; cytometry; flow cytometry; hemogram; blood count; complete blood count; monocyte; macrophage; macrophage activation; lymphocyte; leukocytes, NK cells; natural killer cells; granulocyte; cytokine; cytokines; antibody; antibodies; nitric oxide; c-reactive protein; inflammation; inflammatory diseases, inflammatory response, auto-immunity; allergy.
The most relevant studies that examined the five key characteristics of carcinogenesis mechanisms chosen for inclusion in this narrative review were selected for discussion (Figure 1; Table 1), with funding from institutions linked to the tobacco industry being the exclusion criterion, regardless of conflict of interest declarations.
Key characteristics of carcinogens, according to the International Agency for Research on Cancer (IARC), highlighting the characteristics reviewed in the present study (Smith et al., 2015). Figure created by the authors using vector images from free databases
Results and Discussion
Immunosuppression
Immunological surveillance is an important extrinsic antitumor mechanism, with persistent immunosuppression frequently associated with an increased risk of cancer, especially lymphomas. While exposure to conventional cigarettes is known to induce a state of chronic immunosuppression, the impact of ENDS is still poorly understood.
Innate immunity constitutes the front line of antitumor control, and there is evidence that EDSs can compromise this arm of immunity, with several authors highlighting the cytotoxic effect in these cellular compartments. Human alveolar macrophages obtained from non-smokers and the human monocyte cell line (THP-1) induced to differentiate into macrophages in vitro, cultured and exposed to electronic fluids or electronic fluid vapor condensate showed lower cell viability, both by apoptosis and necrosis, with the condensate being more cytotoxic than the liquid. The authors also reported that both significantly inhibited phagocytosis, while vapor also induced increased generation of reactive oxygen species (ROS), and inflammatory cytokines and chemokines, suggesting that vaporized ENDS is cytotoxic, pro-inflammatory, and inhibits phagocytosis in alveolar macrophages (Scott et al., 2018).
Bradley et al. (2025) showed that immortalized human macrophage cell lines from the central nervous system (DBTRG-05MG) and peripheral nervous system (KG-1), exposed to electronic liquids, especially flavored ones, exhibited reduced cell viability and phagocytic activity, along with increased ROS and pro-inflammatory cytokines. When evaluating human macrophages exposed to ENDS aerosol, with or without nicotine, a significant reduction in the phagocytosis of Escherichia coli by these innate cells was observed (Rahman et al., 2023). In an ex vivo culture model of normal lung tissue, exposure of this tissue to e-juices was able to exacerbate Influenza A infection by dysregulating TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) signaling (Agraval et al., 2023).
Proteomics data from saliva of African Americans who use e-juices show that these individuals exhibit impaired oral innate response, with decreased activity in several areas: gamma receptor-mediated phagocytosis, NK cell function, leukocyte migration, and phagosomal function, with reduced defense against bacterial invasion due to loss of mucosal barrier integrity (He et al., 2025).
Finally, the pulmonary epithelium is also an important innate mechanism of protection against infections. In vitro exposure of human respiratory epithelium to menthol and cinnamon flavorings in ENDS induced impairment of the respiratory tract epithelial barrier and aggravated the cytopathic effects of respiratory syncytial virus (RSV) infection (Raduka et al., 2023). Barrier dysfunction was also demonstrated in vitro in the 16-HBE cell line (Muthumalage et al., 2019) and in the Calu-3 and Beas-2B cell lines of human bronchial epithelial cells, the latter accompanied by cell damage resulting from SARS-CoV-2 infection intensified by exposure to ENDS (Tsou et al., 2022).
A comparative in vitro study on the effects of cigarette smoke versus heated tobacco vapor on immortalized human CD4+ T lymphocytes (Jurkat cell line) demonstrated that both resulted in increased cell death, with smoke being more cytotoxic than vapor. Furthermore, smoking induced an increase in the production of ROS, nitric oxide (NO), and inflammatory cytokines, while heated vapor had more moderate effects. Both types of exposure reduced phorbol 12-myristate 13-acetate (PMA)-induced interleukin-2 (IL-2) secretion and reduced cell proliferation (Scharf et al., 2021). Although the Jurkat cell line originates from acute leukemia (therefore, from T precursors) and has differences compared to primary CD4+ T lymphocytes, this cell line has a behavior similar to primary cells and, therefore, is a valid model for in vitro studies (Carrasco-Padilla et al., 2023).
When evaluating patients with ulcerative colitis who used nicotine ENDS, a significant increase in the number of immunosuppressive regulatory T cells (CD4+/FoxP3+) (producing IL-10, TGF-β, and IL-35) was observed in peripheral blood. In contrast, patients who used conventional cigarettes exhibited a reduction in three inflammatory cell compartments: i) T helper 1 Tbx21+ cells, producing TNF-α and IFN-γ; ii) Th2 Gata3+ cells, producing IL-4; and iii) Th17 IL-23R+/γT+ cells, producing IL-17 and IL-22 (Kastratovic et al., 2025).
Nicotine-induced immunosuppression is well known (Geng et al., 1995; White et al., 2024) and may partly explain the observed results; however, the authors consider that, since the amount of nicotine in ENDS is similar to conventional cigarettes, the differences observed between the two groups must be due to differences in the composition of toxic agents in the aerosols of these two products (Kastratovic et al., 2025).
ENDS use has also been associated with increased activation of human B cells, accompanied by functional changes such as increased class switching, clonal expansion, and a greater tendency to produce IgA (Moll et al., 2025). Finally, the low frequency of studies on the effects on acquired immunity and the need for more observational studies in human populations using ENDS, with special emphasis on the detection of immunosuppression, were identified.
Inflammation induction
Inflammation is a potent cancer-promoting factor, contributing to the survival, proliferation, and expansion of neoplastic cells. Thus, cancer-promoting agents, by acting reversibly and representing a limiting step in tumor growth, can contribute decisively to the primary prevention of cancer. In general, the data presented here indicate that exposure, both short-term and habitual, to the different aerosols available for endocrine disruptors induces a significant modification of cellular and humoral composition compatible with local and systemic pro-inflammatory processes, which can potentiate the development of chronic inflammatory diseases and initial carcinogenic events.
In vitro studies in human cells
To evaluate the inflammatory response and barrier dysfunction caused by different flavoring chemicals in ENDS, Gerloff et al. (2017) demonstrated that acetoin, diacetyl, pentanedione, maltol, and ortho-vanillin induced IL-8 release in Beas-2B cells and human lung fibroblasts (HFL-1) in a dose-dependent manner. Similarly, when evaluating exposure to flavorings in two human monocytic cell lines - U937 monocytic cells (human pleural tissue) and MonoMac 6 human monocyte-macrophage cell line (established from the peripheral blood of a patient with monoblastic leukemia) - it was confirmed that most flavorings increased IL-8 in monocytic cells, with diacetyl, pentanedione, o-vanillin, maltol, and coumarin demonstrating dose-dependent pro-inflammatory effects (Muthumalage et al., 2018).
Exposure of human respiratory tract epithelial cells to tobacco-flavored aerosol resulted in increased secretion of the inflammatory cytokines IL-6, which plays a central role in regulating the immune response and inflammation, and IL-8, a potent inducer of neutrophil chemotaxis. In lung fibroblasts, only the cinnamon-flavored liquid stimulated IL-8 secretion, while the tobacco-flavored liquid without nicotine did not produce an increase, but in the presence of nicotine it was able to induce IL-8 (Lerner et al., 2015). On the other hand, Wang et al. (2024) demonstrated that exposure of the HFL-1 cell line and Beas-2B cells to tobacco-flavored aerosol without nicotine also increased IL-8 levels, but the menthol flavor did not induce changes (Wang et al., 2024).
Sundar et al. (2016) demonstrated that human periodontal ligament fibroblasts (HPdLFs) and human gingival epithelial progenitor cells (HGEPp) exposed to e-cigarette vapor (with and without nicotine; different exposure durations) showed increased inflammatory responses, comparable to conventional cigarettes. The authors observed a significant increase in IL-8 and PGE2 in cell culture supernatant, as well as an elevation of COX-2 and S100A8 protein, both related to the inflammatory response, as well as the exacerbated expression of the RAGE receptor, involved in chronic inflammatory processes, which was significantly increased with flavored ENDS (menthol). When evaluating HGFs regarding the effects of ENDS liquids, Sancilio et al. (2016) observed cytotoxicity, suggesting a potential role in the pathogenesis of periodontitis.
When evaluating the effect of flavorings on the function of human aortic endothelial cells (HAECs), it was observed that flavorings such as vanilla, cinnamaldehyde, eugenol, and acetylpyridine present in ENDS increased the expression of IL-6, even at low concentrations (0.001-0.01 mmol/L). Furthermore, vanilla also increased the expression of intercellular adhesion molecule-1 (ICAM-1), revealing that, even at low concentrations, flavorings can induce inflammation and cellular dysfunction (Fetterman et al., 2018).
Using a physiologically relevant model of the human airways, composed of co-cultured Calu-3 cells and pulmonary fibroblasts (MRC-5), Vasanthi Bathrinarayanan et al. (2018) demonstrated a clear increase in IL-6 and IL-8 production after prolonged exposure to ENDS vapor. In a comparative model using an airway device coated with live human bronchiolar epithelium from normal patients or those with chronic obstructive pulmonary disease (COPD), a significant increase in IL-8 secretion was identified in the epithelium of patients with COPD, but not in healthy epithelium (Benam et al., 2016).
It is important to highlight that the possibility of using unregulated products can lead to exacerbated toxic concentrations. In a toxicological analysis of a panel of ENDS seized in the United Kingdom, Guraka et al. (2024) demonstrated that ENDS caused a dose-dependent increase in cell death and inflammation, manifested by increased release of IL-1β and IL-6.
Population studies
According to Singh et al. (2019), users of endocrine disruptors (EDs) showed elevated levels of inflammatory mediators IL-1β, IL-6, IL-8, IL-13, and interferon (IFN-γ) in plasma, increased IFN-γ in urine, and increased IL-1β in saliva, when compared to non-users. In the plasma of users, an increase in endothelial growth factors (EGF), vascular growth factors (VEGF), platelet-derived growth factors (PDGF), hepatocyte growth factors (HGF), stem cell growth factors (SCF), and placental growth factors (PGF) was also observed.
A study conducted by the Population Assessment of Tobacco and Health (PATH) initiative, a collaboration between the U.S. National Institutes of Health (NIH) and the U.S. Food and Drug Administration (FDA), investigated the association between different patterns of tobacco product use (conventional cigarettes and ENDS) and inflammation biomarkers, revealing that: i) exclusive cigarette users and dual users had significantly higher levels of all inflammatory biomarkers (hsCRP, IL-6, sICAM, fibrinogen) compared to non-users; ii) exclusive ENDS users did not show a significant difference in levels of hsCRP, IL-6, sICAM, and fibrinogen compared to non-users; and iii) exclusive ENDS users had significantly lower levels of almost all inflammatory biomarkers compared to exclusive cigarette users (Stokes et al., 2021).
In a comparative analysis of BAL material and bronchial brushings, Song et al. (2020) evaluated the effects of exposure on the lungs of young adults who used traditional cigarettes and e-cigarettes, and of people who had never smoked. For inflammatory cells, the authors demonstrated that conventional cigarette smokers had higher total cell counts, macrophages, and neutrophils in BAL, while e-cigarette users showed intermediate values, generally closer to non-smokers. Similarly, e-cigarette users also had intermediate levels of inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8, IFN-γ), suggesting less damaging potential compared to conventional smoking.
In another study evaluating BAL material from young adult ENDE users, conventional cigarette smokers, and never smokers, associations were observed between the number of mitochondrial DNA copies (mtCN) and immune responses, which revealed significant associations between IL-2 and IL-4 with mtCN, only in the ENDE user group, demonstrating a unique and specific inflammatory profile for this group (Mori et al., 2022).
Pozuelos et al. (2022) observed persistence of the inflammatory response when evaluating nasal epithelium samples from female volunteers who were former smokers and had switched to ENDEs for at least six months. Through transcriptome comparison, genes involved in inflammatory responses were overexpressed, such as CXCL8, CXCL13, and IL-36γ, which led to the production of proteins (cytokines/chemokines) IL-8, CXCL13, and IL-36γ at high concentrations. Inflammation did not revert to non-smoker levels, with persistent elevation of the inflammatory regulator MMP9 (an inflammatory regulator released by neutrophils and macrophages), consistent with findings for elevated MMP9 and neutrophilic granular enzymes in the sputum of e-cigarette users evaluated by Reidel et al. (2018). When assessing the pro-inflammatory effects on alveolar macrophage function, Scott et al. (2018) demonstrated that e-cigarette vapor condensate also significantly increased MMP-9 secretion, as well as the pro-inflammatory cytokines IL-6, tumor necrosis factor alpha (TNF-α), chemokine (CXCL8), and monocyte chemoattractant protein 1 (MCP-1), being significantly more toxic than non-vaporized liquid.
Genotoxicity
Chemical agents capable of binding to DNA or chromosomes are considered genotoxic and therefore potentially carcinogenic. The literature review provides evidence of the genotoxic potential of ENDS.
Lerner et al. (2016) performed an in vitro test using the alkaline comet assay as a method and observed significant increases in DNA fragmentation of HFL-1 fibroblasts after longer exposure (10 and 15 min) to ENDS aerosols, compared to the control group exposed to air. Similarly, Guraka et al. (2024) tested ENDS of five different flavors and observed that only the Tigerblood flavor caused dose-dependent DNA damage in co-culture of A549 cells (human alveolar basal epithelial adenocarcinoma cells) and THP-1 cells (4:1 ratio). Exposure to the other vaporizers resulted in a small increase in the percentage of DNA in the tail, but without statistical significance. The authors discuss the fact that this flavor produced vapors with a high concentration of lead in the air, reaching 70 times above the safe limit, having strongly contributed to the observed toxic effects (Guraka et al., 2024). In fact, the high concentration of lead produced in Tigerblood vapor plays an important role, but this does not prevent synergistic action with the other components present in this flavor.
In another experiment, this time performing the neutral comet assay (less sensitive as it only detects single and double strand breaks in DNA) with representative cells of normal human epithelium (HaCaT - spontaneously transformed immortal keratinocytes) and two human cancer cells - HN30 (derived from a primary laryngeal tumor) and UMSCC10B (derived from a metastatic lymph node) -, it was possible to observe in all cell lines that ENDS vapor results in a significant increase, up to 1.5 times, in DNA strand breaks compared to the untreated control (Yu et al., 2016). This damage was aggravated in ENDS samples with nicotine.
In contrast, an experimental model used Beas-2B cells exposed to fourth-generation ENDS vapor extract, with and without nicotine (24 mg/mL), and conventional cigarette smoke extract for 24 hours, all at concentrations of 1.25%, 5%, and 20% v/v. The results showed high cytotoxicity in the conventional cigarette treatment and a consequent increase in DNA damage (verified by the increase in Olive Tail Moment, OTM), when compared to the control, but without statistical significance. In the case of vapor produced by fourth-generation ENDS, the increase in OTM was dose-dependent, but with statistical significance only for the highest dose (20%) with nicotine, when compared to the control (Rankin et al., 2019). The authors conclude that, although ENDS aerosol is less toxic than conventional cigarettes, the accumulation of evidence shows that it is also not benign and can alter cellular function, with impacts that are still unknown.
The damage caused by heated tobacco products (HTPs) and conventional cigarette smoke (CC) in the outermost layer of the epidermis was evaluated by Morishita et al. (2022). The authors selected primary human oral keratinocytes (HOKs) to assess DNA damage, quantifying γH2AX foci, as they function as a sensitive marker for DNA double-strand breaks (Khanna; Jackson, 2001). An increase in the formation of γH2AX foci was observed in a dose-dependent manner, and these were greater in the CC and HTP groups than in the controls (p < 0.05), leading the authors to conclude that HTP and CC cause similar damage to the oral mucosa (Morishita et al., 2022).
When evaluating users of e-cigarettes or conventional cigarettes, compared to non-smokers, smokers had a higher frequency of genotoxic effects as determined by the micronucleus test with cytokinesis blockade, in a peripheral blood sample (Bernal et al., 2023). The authors report that some volunteers used both conventional cigarettes and e-cigarettes (37.5%) but found no difference in genotoxic effects when these were compared. Logistic regression analysis was also performed to identify factors that increase the risk of genotoxic effects, and it was observed that being an e-cigarette user increases the risk of genotoxic effects by 3.69 times, with this risk being greater when nicotine is added (OR=3.73).
In an effort to assess whether flavorings and other compounds found in ENDS increase the risk of cancer, Hung et al. (2020) conducted a combined approach using a rapid, multiplex, high-information in vitro screening assay and in silico Quantitative Structure-Activity Relationship (QSAR in silico) assessments to evaluate the genotoxic potential and mechanism of action of 150 different compounds, primarily flavorings used in ENDS liquids. The in vitro assay was performed on human TK6 lymphoblastoid cells (p53-competent), treated with test compounds, with and without metabolic activation. For the QSAR in silico assessments, the predicted endpoints were mutagenicity (bacterial tests, expert opinion); clastogenicity (chromosomal aberrations in vitro, micronucleus in vivo); and carcinogenicity in rodents (mice/females/rats). The results showed that 25 (17%) were considered potentially genotoxic in in vitro tests, with 10% having a clastogenic effect, 5.3% considered aneugenic, and 1.3% (2/150) showing both types of damage. The in silico QSAR assessment showed that 46 compounds (31%) were predicted as positive, with 15% being mutagens, 25% clastogenic, and 23% carcinogenic to rodents. When comparing the in vitro and in silico studies, the authors concluded that there was good agreement between the results of the laboratory tests and the predictions of the computer models, with up to 83% agreement in some cases, in which the chemical classes frequently identified were aldehydes and alcohols.
Oxidative stress
A recurring theme in the scientific publications analyzed is the central role of oxidative stress as a pathophysiological mechanism underlying the harmful effects of the use of ENDS. Oxidative stress is characterized by an imbalance between the generation of reactive oxygen species (ROS - free radicals such as the superoxide anion [O₂•-], hydroxyl radical [•OH] and non-radical molecules such as hydrogen peroxide [H₂O₂]) and the body's antioxidant capacity (Halliwell; Gutteridge, 2015). Thus, when ROS production exceeds the antioxidant system's ability to neutralize them, cellular and molecular damage occurs, as well as metabolic dysfunctions. These alterations reflect damage to the lipid membrane via lipid peroxidation, modifications in proteins and alterations in DNA, including strand breaks, point mutations and base modifications, which in turn can result in the activation of proto-oncogenes or inactivation of tumor suppressor genes.
The body possesses enzymatic antioxidant systems, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, and non-enzymatic systems, such as vitamins C and E and reduced glutathione, which act in the neutralization of ROS (Sies, 1997). Since the enzymatic pathway is susceptible to saturation, such damage is associated with the development and progression of various pathologies, including neurodegenerative diseases, cardiovascular diseases, cancer, and aging processes.
Production of ROS and activation of NADPH oxidase
Several studies have identified that the aerosol from e-cigarettes promotes a significant increase in the production of ROS, including hydrogen peroxide (H₂O₂) and superoxide anion, in different cell types such as: A549, periodontal fibroblasts, oral keratinocytes, and circulating immune cells (PBMCs). The activation of the NADPH oxidase enzyme (a multiprotein enzyme that plays a central role in ROS production), especially its components NCF1, NCF2, and NCF4, was highlighted by Pozuelos et al. (2022) and Halstead et al. (2023) as one of the main sources of ROS after chronic use of e-cigarettes.
The study conducted by Pozuelos et al. (2022) investigated the effects of replacing conventional cigarettes with e-cigarettes on the integrity of the respiratory epithelium, with a special focus on oxidative stress processes, inflammation, and ciliary regeneration. Through transcriptomic analysis, the authors compared the gene expression of nasal mucosal cells from three groups: active smokers (CS), non-smokers (NS), and ex-smokers who exclusively used e-cigarettes for at least six months (EC). The results demonstrated that the EC group presented more differentially expressed genes compared to the NS group than the CS group, suggesting that replacing traditional cigarettes with e-cigarettes does not reverse the transcriptomic alterations induced by smoking. One of the main findings was the activation of the NADPH oxidase pathway, strongly associated with increased ROS production, indicating increased oxidative stress in the EC group. The NCF1, NCF2, and NCF4 genes, fundamental cytoplasmic components for NADPH oxidase activation, were significantly overexpressed in this group. Thus, although e-cigarettes are frequently promoted as a less harmful alternative to tobacco, their continuous use does not restore the respiratory epithelium to the healthy state observed in non-smokers, maintaining an environment of elevated oxidative stress and persistent inflammation, which may favor the progression of various respiratory diseases.
Halstead et al. (2023) examined 40 healthy young adults (18-24 years): 20 users of endocrine disruptors (10M/10F) with a history of chronic use ≥6 months and 20 healthy controls (10M/10F) with no history of use. Microvascular endothelium-dependent vasodilatory function was assessed in vivo by means of cutaneous vascular conductance. Endocrine disruptor users showed higher circulating measures of oxidative stress, and administration of the superoxide scavenger tempol increased endothelium-dependent dilation by increasing NO-dependent dilation, suggesting that oxidative stress, specifically the superoxide anion, contributes to microvascular endothelial dysfunction in healthy young adults using endocrine disruptors, potentially increasing long-term cardiovascular risk, especially in women.
Biochemical markers of oxidative stress
Classic biomarkers of oxidative stress have been observed in both acute (Chatterjee et al., 2021) and chronic models (Singh et al., 2019; He et al., 2025). The study by Chatterjee et al. (2021) investigated the acute effects of nicotine-free ENDS aerosol inhalation in healthy, non-smoking adults. The research combined blood analyses of inflammatory and oxidative stress biomarkers with vascular function assessments by magnetic resonance imaging (MRI), before and after a single vaping session. The results indicated that aerosol exposure promoted a significant increase in ROS in human pulmonary endothelial cells (HPMVECs) cultured with post-vaping serum, accompanied by a reduction in NO metabolite levels in the participants' blood. These findings demonstrate a state of acute oxidative stress, capable of compromising endothelial function.
The study by Singh et al. (2019) investigated the systemic toxic effects of e-cigarette use, with a special focus on oxidative stress. From the analysis of 48 individuals, including users and non-users of e-cigarettes, the authors identified significant increases in biomarkers of oxidative stress in users, mainly 8-isoprostane (in urine and exhaled air condensate - EBC) and 8-oxo-dG (in urine), both associated with lipid peroxidation and oxidative DNA damage, respectively. The body of evidence reinforces that e-cigarette use promotes a state of persistent oxidative stress, contributing to tissue damage and chronic inflammation, especially in the respiratory and cardiovascular systems.
The study by He et al. (2025) examined the effects of e-cigarette use on redox metabolism and oxidative stress in the oral health of African American individuals. Using omics approaches (metabolomics and proteomics) on saliva samples, the authors identified significant alterations in oxidative stress markers among ENDS users. Notable findings included increased malondialdehyde (MDA), indicative of lipid peroxidation, and a decreased GSH/GSSG ratio, reflecting an imbalance in the glutathione system, a fundamental axis of cellular antioxidant defense. This dysfunction was exacerbated by a reduction in the G6PD enzyme, crucial for NADPH regeneration and therefore for combating ROS accumulation. In summary, the study demonstrates that ENDS use in African Americans promotes exacerbated oxidative stress, with antioxidant imbalance.
Mitochondrial and metabolic dysfunction
The study by Assiri et al. (2024) investigated the toxic effects of ENDS vapor on A549 cells, with a special focus on metabolic alterations and the induction of oxidative stress. The results showed that exposure to ENDS vapor extract for 24 hours caused a significant increase in ROS production, in a dose-dependent manner depending on the concentration, peaking at 40%. At higher concentrations (80%), ROS generation decreased, possibly due to accentuated cell death. Metabolomic analysis revealed that ENDS vapor induced profound alterations in essential cellular metabolic pathways, including the glutathione pathway, pyruvate metabolism, fatty acid biosynthesis, and amino acid metabolism, all closely related to the control of cellular redox balance. In conclusion, the study demonstrated that exposure to ENDS vapor promotes cytotoxicity mediated by oxidative stress, associated with mitochondrial dysfunction and the collapse of fundamental metabolic pathways.
Regarding clinical and functional consequences, oxidative stress can cause microvascular endothelial dysfunction mediated by superoxide (Halstead et al., 2023); reduced glutathione peroxidase activity in gingival fluid, associated with periodontitis in vapers (He et al., 2025; Karaaslan et al., 2020). Therefore, the increasing use of electronic cigarettes, especially among adolescents, raises concerns about their adverse health effects, particularly when it comes to flavored liquids (E-liquids) that come into direct contact with the oral mucosa.
Recent studies have shown that these liquids, when vaporized, can induce oxidative stress in oral epithelial cells, negatively affecting their morphology, healing capacity, and functional integrity. Exposure to flavored e-liquids, particularly those with cinnamon (rich in cinnamaldehyde), has been shown to induce significant loss of intracellular GSH in OKF6/TERT-2 cells, an immortalized human oral keratinocyte cell line. This depletion is indicative of cytotoxicity mediated by oxidative stress, which may culminate in cell apoptosis (Shamim et al., 2025).
Higher oxidative stress and vascular damage have been observed, especially in users of oral contraceptives (Mastrangeli et al., 2018). The authors analyzed the acute effects of conventional cigarettes (CCs) and electronic cigarettes (ECs) on oxidative stress and endothelial dysfunction in healthy individuals. The research involved 20 smokers and 20 non-smokers exposed to controlled sessions of CC and EC use, focusing on serum biomarkers and vascular function. The results showed that both CCs and ECs increase oxidative stress, with reduced NO bioavailability and increased markers such as 8-iso-PGF2α-III and sNOX2-dp, indicating NADPH oxidase activation. ECs caused significant oxidative impacts, especially in non-smokers, who showed greater sensitivity. In addition, women using oral contraceptives showed more pronounced changes in endothelial function compared to others (Mastrangeli et al., 2018).
Finally, it is important to highlight that oxidative stress was observed even in the absence of nicotine, as demonstrated by Sundar et al. (2016) and Chatterjee et al. (2021). Sundar et al. (2016) investigated the cytotoxic and inflammatory effects of flavored e-cigarette aerosols on human periodontal cells, with emphasis on the mechanisms of oxidative stress and DNA damage. The study observed that flavored e-cigarettes without nicotine, such as menthol flavor, also produced relevant effects, suggesting that flavorings contribute to oxidative stress independently of the presence of nicotine, with a potential negative impact on tissue regeneration and periodontal health. Chatterjee et al. (2021) evaluated the acute effects of nicotine-free ENDS aerosol inhalation in healthy, non-smoking adults. The results showed that aerosol exposure increased ROS in pulmonary endothelial cells and reduced NO levels in the blood, indicating acute oxidative stress. This negatively affected endothelial function and suggested that, even without nicotine, e-cigarette use can trigger inflammation and impair vascular reactivity, with negative implications for cardiovascular health.
Epigenetic alterations
Exposure to toxic aerosols from ENDS, as well as conventional cigarettes containing a range of chemical components and flavorings, induces epigenetic changes, including modulation of DNA methylation, reduction in the activity of regulatory enzymes such as Histone Deacetylases (HDACs), alterations in the expression of microRNAs (miRNAs) and enhancer RNAs (eRNAs), and impairment of DNA repair pathways, in various cells and tissues, as detailed below.
Considered one of the main epigenetic mechanisms, DNA methylation is a biochemical process that regulates gene expression through the recruitment of proteins involved in transcriptional repression and the inhibition of transcription factor binding to DNA (Moore; Le; Fan, 2013). One of the processes of regulating transcriptional activity is the synergistic modifications of histones and miRNAs in an integrated regulatory network. In this context, HDACs are crucial enzymes in epigenetic regulation through histone deacetylation, the reduction of which leads to changes in expression, impairment of repair mechanisms, and contributes to the process of cellular senescence (Moore; Le; Fan, 2013).
Another resource to be observed is the activity of enhancers - regulatory DNA elements that amplify the expression of target genes regardless of location. The activity of enhancers is directly associated with their methylation status (Arnold; Wells; Li., 2020; Harrison; Bose., 2022; Li et al., 2023). The production of eRNAs, short non-coding transcriptional elements, correlates with the hypomethylation of CpGs, while hypermethylation suppresses their activity (Harrison; Bose, 2022). Bidirectional eRNA transcription is mediated by the recruitment of RNA Polymerase II to active enhancers (Arnold; Wells; Li, 2020; Li et al., 2023). This dynamic mechanism demonstrates how alterations in the DNA methylation pattern directly impact eRNA production and, consequently, gene expression (Harrison; Bose, 2022).
Finally, another marker for methylation assessments is the LINE-1 (Long Interspersed Element) retrotransposon, which is a mobile element whose expression is a characteristic of many types of malignancy (Ardeljan et al., 2017). Complete LINE-1 transcription is driven by an internal promoter rich in CpG dinucleotides. Some intact promoter sequences are found throughout the genome, and LINE-1 CpG methylation is considered a surrogate marker for genome-wide methylation levels (Yang et al., 2004; Ardeljan et al., 2017).
Changes in HDACs, eRNA, miRNAs, and post-transcriptional regulation
The first evidence of epigenetic alterations induced by ENDS was demonstrated by Sundar et al. (2016), who observed a trend of reduced HDAC2 levels in HPdLF and HGEPp cells exposed to vapors, particularly pronounced in groups exposed to menthol aroma. The authors further observed that the reduction in HDAC2 may be associated with RAGE receptor-dependent mechanisms, suggesting a specific pathway by which ENDS aerosols can compromise epigenetic regulation, demonstrating that the alterations induced by these devices encompass multiple regulatory mechanisms.
Patients diagnosed with squamous cell carcinoma of the lung with a proven history of tobacco use or exposure to ENDS vapor were investigated through transcriptomic analyses. Based on RNA-seq analyses of tumors and adjacent normal tissues, Tsai et al. (2021) identified 16 key eRNAs with significant correlations with eight clinical variables, associated with chromosomal alterations and reduced methylation at CpG sites. ENDS use promoted increased expression of these 16 key eRNAs (14 with oncogenic characteristics located near the GALNT6 oncogene, and two that exhibited tumor suppressor functions), suggesting a common mechanism between tobacco and ENDS vapor in promoting carcinoma.
RNA-seq approaches were also used by Li et al. (2025) to evaluate adults who exclusively use ENDS regarding the representation of plasma exosomal miRNAs. The authors identified four over-represented, but not significant, exosomal miRNAs (hsa-miR-100-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, and hsa-miR99a-5p) compared to the control group. Evaluating an ethnic-racial breakdown, the analysis of white participants identified four over-represented miRNAs (hsa-miR-100-5p, hsa-miR-125b-5p, hsa-miR-200b3p, and hsa-miR-99a-5p), with only hsa-miR-200b-3p being significant. Gene Ontology enrichment analysis indicated that these miRNAs are involved in processes such as transcription regulation and cellular protein modification.
Global genomic markers
Bernal et al. (2023) conducted the first assessment of the genetic and epigenetic impact of endocrine disruptors (EDs) through DNA methylation analyses in LINE-1 regions in blood samples. Using quantitative methylation-specific PCR (qMSP), groups of ED users, conventional cigarette smokers, and non-smokers were evaluated. The results indicated a significant decrease in LINE-1 methylation levels in ENDS users compared to the control group.
These changes in LINE-1 methylation patterns were reflected in the representative RNA expression detected in ENDS users, suggesting functional consequences of epigenetic modifications. The findings indicated that being an ENDS user increases the probability of having low LINE-1 methylation levels by 5.44 times, and that epigenetic modifications may explain the increase in transcriptional regulation processes observed.
Methylation and differential expression
Song et al. (2020) conducted a comparative analysis to assess possible epigenetic alterations among young adult users of conventional cigarettes, inhaled e-cigarettes, and people who have never smoked, using bronchoalveolar lavage (BAL) material and bronchial brushings. A subset of participants was analyzed, and the CpG sites were classified, allowing the identification of 451 differentially methylated sites, corresponding to 273 genes. Methylation alterations were correlated with changes in gene expression, suggesting a relevant biological effect, with the methylation profile of inhaled e-cigarette users being lower than in conventional smoking.
Also evaluating gene expression, Ganguly et al. (2020) compared two popular e-cigarette liquids with mixed fruit flavors, with and without nicotine, in human lung mucosa models. The results pointed to significant differences in the expression of genes involved in epigenetic regulation, including DNMT1, DNMT3A, DNMT3B, HDAC1, HDAC2, HDAC3, SIRT1, SIRT6, EZH2, TET1, and TET2, which were evaluated in different types of exposure to e-cigarettes. These findings suggest lasting effects on gene expression and a possible impact on lung health.
Expanding on the analyses of BAL samples, Mori et al. (2022) investigated the relationship between mtCN and nuclear biomarkers in groups of e-cigarette users, conventional smokers, and individuals who had never smoked, to assess DNA methylation as well as genome-wide gene expression in the lung epithelium. Quantifications were associated with mtCN as an internal reference for relative quantification. The results showed that mtCN was higher in conventional smokers compared to non-smokers, while e-cigarette users did not show a statistically significant difference in relation to both groups (Mori et al., 2022). Approximately 10% of the CpGs analyzed (71,487 CpGs) and 321 transcripts were significantly associated with mtCN.
Impact on DNA repair mechanisms
Morishita et al. (2022) evaluated HOKs exposed to HTP products and conventional cigarettes using mRNA microarray, and the results were confirmed by quantitative RT-qPCR. The data showed significant reduction in the gene expression of MDC1 (Mediator of DNA Damage Checkpoint 1) and ATR (Ataxia Telangiectasia Related Protein and Rad3) compared to the unexposed control, indicating inhibition of DNA repair pathways in the oral mucosa. The results represent toxicogenomic effects that possibly result from epigenetic alterations induced by tobacco, such as potential DNA methylation at the MDC1/ATR promoters (Morishita et al., 2022).
Conclusions
The combined analysis of the studies shows that electronic cigarettes cause harm to health, as e-cigarettes modify the cellular and humoral distribution profile and the functionality of the immune system. In this context, a growing body of experimental evidence in animals and observational evidence in humans suggests that exposure to e-cigarette vapor induces a reduction in the phagocytic activity of macrophages, a lower response against infections, alterations in the distribution and functionality of lymphocytes, in the pattern of production of pro-inflammatory and/or anti-inflammatory cytokines, and in the levels of secreted immunoglobulin. This immune dysregulation can produce i) a state of immunosuppression and/or ii) a pattern of local and/or systemic inflammatory response that, together, can favor the development of tumors.
This analysis provides genotoxic mechanistic evidence from in vitro, in vivo, in silico, and human assays exposed to these devices. Exposure to e-cigarette e-liquids (EDs) showed increased levels of proteins associated with DNA damage, double-strand breaks, greater DNA fragmentation using the comet assay, increased micronucleus frequency, and other genotoxic effects identified in different assays. Thus, many of the compounds identified in e-cigarette liquids or aerosols proved to be genotoxic, with the possibility of clastogenic and aneugenic effects, and acting as mutagens and pro-mutagens.
Contributing to the genotoxic effect, oxidative stress identified in different studies is a key marker in vaping toxicology, with increased ROS generation, mitochondrial dysfunction, reduced antioxidant defenses, and multisystemic cellular damage, with important implications for vascular function, oral and respiratory health. The body of information shows that ENDS can induce oxidative DNA damage and contribute to genetic mutations, facilitating tumor initiation. Harmful effects are influenced by factors such as device type, presence of nicotine, type of flavoring, and individual characteristics (sex, hormones).
Although studies from an epigenetic perspective are still scarce, scientific evidence demonstrates that exposure to e-cigarettes induces significant and complex epigenetic changes that affect multiple levels of gene regulation. Although the epigenetic alteration profiles are quantitatively smaller when compared to conventional cigarette smokers, the evidence suggests that e-cigarettes are not safe. The changes observed in the most vulnerable tissues (pulmonary and oral epithelium) correlate directly with changes in gene expression, suggesting functional biological relevance and may have long-term implications for health. The impacts observed on DNA repair mechanisms are a concern of great magnitude, since the reduction in the expression of crucial genes, such as MDC1 and ATR, can compromise genomic integrity. The coordinated alteration of multiple genes involved in epigenetic regulation (DNMTs, HDACs, SIRTs, TET) indicates that ENDS promote comprehensive epigenetic reprogramming.
Although the findings presented here are of great relevance, our study has some limitations, such as the heterogeneity in the study design, the great variability of the substances and products analyzed, the restriction of the scope of the review to mechanistic evidence from five categories and, due to its narrative nature, potential selection biases.
Taken together, these results indicate that ENDS present risks to human health due to exposure to substances with carcinogenic potential, which justifies the maintenance of the prohibition on the marketing, importation and advertising in Brazil, according to Collegiate Board Resolution (RDC) 46/2009, ratified and expanded in 2024 to include the prohibition of the manufacture, distribution, storage and transport of ENDS.
Acknowledgements
This article was published with resources from the Sustentabilidade Project of PNCT, coordinated by the Divisão de Controle do Tabagismo (Tobacco Control Division - DITAB) of the Coordenação de Prevenção e Vigilância (Prevention and Surveillance Coordination) of the National Cancer Institute, with support from Vital Strategies, Bloomberg Philanthropies, and the Center for Studies, Research and Technological Development in Public Health (Centro de Estudos, Pesquisa e Desenvolvimento Tecnológico em Saúde Coletiva - Cepesc) of the State University of Rio de Janeiro (UERJ).
Notes
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The content of this document is the sole responsibility of the authors and should under no circumstances be interpreted as representing the position of Vital Strategies or Bloomberg Philanthropies.
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Reviewers:
Renata Santos e André Luiz Oliveira da Silva
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All research data are available in this article.
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Editor:
Jane Russo
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