The use of E-cigarettes as a risk factor for oral potentially malignant disorders and oral cancer: a rapid review of clinical evidence

Background The popularity of e-cigarettes has increased rapidly in the last decade, particularly among teens and young adults, being advertised as a less harmful alternative to conventional tobacco products. However, in vitro and in vivo studies have evidenced a variable quantity of potentially harmful components and some recognized carcinogens which may cause DNA damage in oral cells. Additionally, evidence suggests that e-cigarettes may play active roles in the pathogenesis of other malignancies, such as lung and bladder cancers. Therefore, this rapid review aimed to assess the available clinical evidence about using e-cigarettes as a risk factor for oral potentially malignant disorders (OPMD) and oral cancer. Material and Methods A systematic search for English language articles published was performed in PubMed (MEDLINE), Embase, Scopus, and Web of Science. After the study selection process, the authors included twelve clinical studies about OPMD and oral cancer risk in e-cigarette users. Results The main findings showed the presence of carcinogenic compounds in saliva and morphologic changes, DNA damage, and molecular pathways related to carcinogenesis in the oral cells of e-cigarette users. However, results were inconsistent compared to tobacco smokers and control groups. Conclusions the current clinical evidence on this topic is limited and insufficient to support using e-cigarettes as a risk factor for OPMD and oral cancer. Nevertheless, dental care professionals should advise patients responsibly about the potentially harmful effects of e-cigarettes on the oral mucosa cells. Future long-term and well-designed clinical studies are needed. Key words:Oral potentially malignant disorders, oral cancer, e-cigarettes, tobacco.


Introduction
Oral cancer represents the 18th most common malignancy worldwide, with 377,713 new cases and 177,757 deaths estimated for 2020 (1).In addition, it is considered a significant public health problem because many patients are diagnosed with late-stage disease, which contributes to high morbidity and mortality rates (1,2).Approximately 90% of malignant tumors affecting the oral cavity are oral squamous cell carcinomas (OSCCs) (3).These neoplasms may arise de novo or be preceded by oral potentially malignant disorders (OPMD), defined as conditions with an increased risk of malignancy (3,4).However, the risk of malignant transformation to carcinoma varies depending on patient-or lesion-related factors (4,5).Tobacco, either in smoked or smokeless forms of consumption, is a wellestablished risk factor for oral cancer and is associated with an increased risk of malignant transformation of OPMD (3,5).In the early 2000s, in response to the substantial evidence of cancer risk and general morbidities associated with tobacco consumption, Electronic Nicotine Delivery Systems (ENDS), also referred to as electronic cigarettes (e-cigarettes), e-hookahs, hookah pens, or vapor pens, emerged as safer alternatives to conventional tobacco products or smoking cessation tools and since their commercialization its popularity has been increased among adolescents, and young adults worldwide (6,7).Therefore, the short and long-term effects, including the carcinogenic potential of ENDS, remain an active area of concern and scientific research (7).There is emerging evidence about the potentially harmful effects of ENDS, including potential oral health consequences related to periodontal tissue, dental problems, oral microbiota alterations, intraoral explosion injuries, and a variety of symptoms like xerostomia, burning, and irritation observed in ecigarettes users (6)(7)(8).In addition, in vitro and in vivo studies have reported the molecular changes induced by ENDS on the oral cells ranging from reduced antioxidant levels and gene dysregulations to DNA strand breaks (9).Therefore, using e-cigarettes as a harm-reduction tool remains controversial.Many health professionals, including dental clinicians, should provide advice based on scientific evidence, especially to implement preventive measures related to oral cancer and risk factors (8).Thus, this rapid systematic review aims to assess the available clinical evidence about using ENDS as a risk factor for OPMD and oral cancer.

Material and Methods
Rapid systematic reviews are a type of knowledge synthesis in which components of the systematic review process are simplified or omitted to produce informed evidence and support decision-making in health policy and practice in a short period (10).Following the PRIS-MA statement, this rapid review was conducted using a systematic review methodology (11) but with some modifications like a shorter search strategy, faster data extraction, and primarily qualitative synthesis.A systematic review protocol was registered in PROSPERO under the registration number CRD42023391709.Despite previous in vitro and in vivo studies reported DNA damage in oral cells (9) there is still a debate regarding the harmful effects of e-cigarette use which may lead healthcare professionals, including dental practitioners, without confidence when advising patients about using these products.Thus, this rapid review aims to summarize the current clinical findings about using e-cigarettes as a risk factor for OPMD and oral cancer.
-Search strategy A specific search strategy was designed for this rapid review (Supplement 1).The literature search was undertaken on January 13, 2023 in Embase, PubMed, Scopus, and Web of Science.Rayyan Qatar Computing Research Institute (QCRI) software was used to remove duplicate references.In addition, reference lists of included studies were reviewed for citations not captured by the database search.
-Screening and selection of the papers Considering that there are recently published systematic reviews focused on head and neck cancer risk and cell damage in e-cig users based on detailed analysis of in vitro and in vivo studies (9,12,13), only clinical evidence was included for this rapid review.We defined clinical evidence as all those studies that included samples from the oral mucosa of electronic cigarette users.The inclusion and exclusion criteria for the study selection are detailed in Table 1.Two reviewers completed the selection in a two-phase process.In phase 1, articles were assessed by title and abstract screening.In phase 2, the studies were selected by full-text reading.In cases of discrepancy, the two authors discussed, and in inconclusive cases, a third author was consulted.Summary information and characteristics of the included studies were extracted: author and year; country; study design; objectives; methods; sample; main results, and authors' conclusions.Data were extracted by one reviewer and checked by a second reviewer for consistency and correctness.The quality of included studies was assessed using The Joanna Briggs Institute Critical Appraisal Tools (© JBI), and the checklist used varied on the study design.Finally, a narrative synthesis was performed by describing and comparing data reported in the included studies.
Observational studies presenting variable study designs were assessed as clinical evidence regarding using ecigarettes and OPMD/oral cancer risk.Two were cohort studies (20,21), three were reported cases (14,16,22), and seven were analytical cross-sectional studies (15,(17)(18)(19)(23)(24)(25).Among the last group, one article was published as a prospective case-control study (18).Despite including a control group and the sampling being done over two years, data were collected at a single point in time, which is why we classified it as an analytical cross-sectional study.All included studies were published in English between

Results
Two-hundred and thirty-eight articles were identified through the electronic literature search.After duplicate remotion, 143 studies were screened by title and abstract, and 101 were excluded.Subsequently, 42 articles were assessed by full-text reading, and 34 records were removed based on exclusion criteria (Supplement 2).Additionally, four studies were identified from the references lists (14)(15)(16)(17), all meeting the inclusion criteria.Thus, 12 studies (14-25) were included for qualitative synthesis.The flowchart showing the selection process is detailed in Fig. 1.  2016 and 2022.Six were conducted in the United States (16,19,(20)(21)(22)(23), two in Italy (15,18), and one each in Brazil (17), Malaysia (25), Romania (24), and the United Kingdom (14).The risk of bias assessment for the individual studies is summarized in Table 2, and the independently answered questions for each study type are detailed in Supplement 3. Six studies (14,15,18,19,21,23) were classified with moderate risk of bias, four (17,22,24,25) presented with low risk, and two (16,20) with a high risk of bias.The strategies to deal with confounding factors were unclear or not stated in most studies classified with moderate or high risk of bias.The summarized information of the included articles is detailed in Table 3.In general, nine clinical studies aimed to analyze the carcinogenic effects of e-cigarettes on the oral mucosa.Therefore, the researches were designed as observational studies; most developed a cross-sectional analysis (15,(17)(18)(19)(23)(24)(25), and two were conducted as cohort studies (20,21) with 3-6 months of follow-up.However, to describe the current clinical evidence on this topic, we also included reported OPMDs and oral cancer cases identified in ENDS users (14,16,22).A total of 255 ENDS users were identified among the included studies of this rapid review.The main analyzed materials were the oral cells obtained from mouth brushings and saliva (Table 3).Eight studies (15,17,(19)(20)(21)(23)(24)(25) included smokers and non-smokers individuals as comparison groups.An additional group of former smokers was identified in the studies conducted by Schwarzmeier et al. (17) and Bardellini et al. (18).Saliva samples were evaluated in two studies (Table 3).First, Bustamante et al. 2018 (19) demonstrated the endogenous formation of the carcinogen N´nitrosonornicotine (NNN) in e-cigarette users.However, compared with smokers, the overall exposure to NNN in e-cigarette users was dramatically lower.Later, Pandarathodiyil et al., 2021 (25) reported that the smoker and vaper groups' lactate dehydrogenase (LDH) enzyme levels were significantly higher than in the control group.Nevertheless, no difference in salivary LDH activity level was observed in vapers compared to smokers.A significant difference was observed for micronuclei frequency only between the smoker and control groups.However, the e-cig group showed nuclear features of genotoxicity and cytotoxicity represented by significantly higher number of broken eggs than the smoker group and more karyolysis, binucleation, broken eggs, and nuclear buds than the former smoker and control groups.

Period
In summary, e-cig users presented genotoxicity and cytotoxicity in the oral mucosa cells.The damage cannot be attributed solely to the use of the e-cigs, since most vaporizers also consumed alcohol and had a history of using conventional cigarettes.Further studies are needed to assess the long-term effects of e-cigs.It is necessary to better characterize factors that contribute to the endogenous formation of NNN in saliva of e-cigarette users.This source of exposure to a human carcinogen should be taken into account for potential health impact in longterm e-cigarette users.

DNA adduct was observed in oral cells and urine of e-cigarette users.
The results showed that its levels were 9 times greater in e-cigarette users than in nonsmokers.However, conventional smokers presented higher exposure to acrolein when compared to e-cigarette users.
Oral cell DNA of e-cigarette users are exposed to significant levels of acrolein, a toxic and carcinogenic compound, when compared to non-users of these products.Further researches on the potential toxic and carcinogenic effects of ecigarettes are needed.RNA-seq analysis showed significant number of aberrantly expressed transcripts in both e-cig users and smokers relative to non-smokers.The deregulated transcripts in smokers were predominately from protein-coding genes and in vapers belonged to regulatory non-coding RNAs.Various molecular pathways identified in e-cig users are involved in tumorigenesis, particularly, in smoking-related cancers (lung, esophageal, bladder and ovarian cancer, head and neck squamous cell carcinoma and leukemia).
In summary, our whole transcriptome analysis of oral cells from exclusive e-cig users and smokers shows that vapers, similarly to smokers, have deregulation of key genes, the majority of which converging on cancer-related pathways and functions.The salivary LDH activity levels of smoker and vaper groups were significantly higher than in the control group.There was no significant difference of salivary LDH activity level in vapers when compared with smokers.
E-cigarettes release fewer toxins and carcinogens than conventional cigarettes, but high LDH levels were observed among vapers, confirming cytotoxic and harmful effects of e-cigarettes on the oral mucosa.As knowledge gaps still exist in terms of long-term exposure effects of vaping and its integration with toxicity assessments, more data is urgently needed to understand the potential health risks and public health impact of vaping.* The design of each study was established based on a critical analysis during the development of this rapid review.Differences could be encountered between the original publication and the summarized information in this Table.Six studies analyzed the oral cells samples of e-cigarette users (Table 3).Three of them (15,17,24) used the micronucleus assay test through microscopic evaluation of exfoliative cytology samples for analyzing the carcinogenic effects of e-cigarette exposure in oral cells.Franco et al. 2016 (15) identified a low prevalence of micronuclei in the e-cigarette users' group compared to smokers and described that these values were similar to nonsmokers, so they suggested that using ENDS seems to be safe for oral cells.In contrast, Pop et al., 2021 (24) found significantly higher values of micronuclei in ecigarette users and smokers compared to nonsmokers.However, they reported no significant differences between smokers and e-cigarette users.In the study by Schwarzmeier et al., 2021 (17), besides the micronuclei assay, they also evaluated the metanuclear anomalies of oral cells among e-cigarette users and compared results between smokers, former smokers, and nonsmokers.These study results showed a significant difference in micronuclei frequency between smokers and nonsmokers.However, the e-cigarette group showed more metanuclear anomalies than the smoker, former smoker, and control groups suggesting the genotoxicity and cytotoxicity in the oral mucosa cells of ENDS users.The reminded three studies (20-23) evaluated the DNA damage of oral cells and suggested the potential genotoxicity caused by e-cigarette exposure.The study conducted by Cheng et al. 2022 (20) showed higher levels of DNA adduct of acrolein in e-cigarette users than nonsmokers.However, smokers presented higher exposure to acrolein when compared to e-cigarette users.Guo et al. 2021 (21) analyzed the DNA exposure to mutagens in oral cells of vapers, smokers, and non-smokers based on identifying apurinic/apyrimidinic (AP) sites.They described that AP sites in e-cigarette users were significantly lower than in smokers and non-smokers and stated that propylene glycol in e-cigarettes might inhibit bacteria in the oral cavity, resulting in reduced inflammation and related effects, and consequently reduced AP site levels in e-cigarette users' DNA.Finally, Tommasi et al. 2019 (23), through RNA-seq analysis, showed increased numbers of deregulated transcripts in vapers.Additionally, the bioinformatic analysis identified that various molecular pathways presented in e-cigarette users are responsible for tumorigenesis in smoking-related cancers.Regarding OPMD in ENDS users, we identified one case report (14) of oral lichenoid reaction in a 55-yearold man with a history of e-cigarette use.According to this report, the white-striated lesions coincidently appeared when he started using an e-cigarette.Furthermore, the clinic-pathological correlation stated the diagnosis of lichenoid eruption related to e-cigarette use, which improved after removing the causative agent (ecigarette) and using a steroid mouthwash (Table 3).An analytical cross-sectional study by Bardellini et al. 2018 (18) aimed to describe the prevalence of oral lesions in e-cigarette and former smokers.They described various types of lesions in both groups and among e-cigarette users, only one patient presented leucoplakia (hyperkeratosis without dysplasia).In contrast, the oral lesions in former smokers included two oral lichen planus, two leucoplakias without dysplasia, and one OSCC.There are also reported cases of oral cancer in e-cigarette users (Table 3).For example, in 2019, Nguyen et al. (16) reported two basaloid squamous cell carcinoma cases in two male adults without any identifiable risk factors other than e-cigarette use.One of these cases affected the tongue, and the other, the lower lip.Later, in 2021, Klawinski et al. (22) described a case of HPVnegative OSCC in a young adult with a history of vaping.However, the patient mentioned a previous history of smoking (0.5 packs of cigarettes per day for one year) before using e-cigarettes.In both articles, the authors concluded that there might be a carcinogenic effect in e-cigarettes which may represent an increased risk of oral cancer.

Discussion
Considering the increasing global use of ENDS, most of the current research is focused on the role of e-cigarettes in cancer pathogenesis.Growing evidence suggests that they may play active roles in lung and bladder cancers (26,27).Therefore, several in vivo and in vitro studies are conducted to understand their short-and long-term effects on oral health, including oral carcinogenesis.The results described so far DNA damage in the oral cells of e-cigarette users (9).However, there is limited clinical evidence about e-cigarette use as a potential risk for OPMD and oral cancer.Thus, this rapid review assessed the published clinical studies in English and summarized the available data about ENDS users' risk for developing of OPMD or oral cancer.Most clinical studies included in this rapid review were designed as observational studies with cross-sectional analysis to understand the potential oral carcinogenic effects of e-cigarettes.However, to consider e-cigarettes an independent causative factor for a multifactorial disease like oral cancer, it is essential to identify the presence of confounding factors to make a reliable inference (28).Unfortunately, our results showed that most studies presented a moderate risk of bias due to unclear or negative answers regarding identifying or managing confounding factors.Hence, alcohol consumption, type, quantity, time of e-cigarette use, and the fact that e-cigarettes are mainly used by tobacco and former smokers (3,5,17,29) are essential confounding factors to consider in future studies.The remaining articles included were cohort studies with limited exposure time and small sample size, as well as case reports, which led to only low-level evidence (30) could be included in this rapid review.The studies described potential carcinogenic effects in ENDS users' saliva and oral cells.For example, high levels of the carcinogen NNN, LDH enzyme, and DNA adduct of acrolein (19,20,25) and increased numbers of deregulated transcripts and molecular pathways involved in tumorigenesis of smoking-related cancers were identified in e-cigarette users (23).However, these findings are inconsistent when compared with smokers and controls.In addition, three studies on micronuclei evidenced contradictory results when comparing e-cigarette users with non-users (15,17,24).One of them also reported more metanuclear anomalies in vapers suggesting its genotoxicity and cytotoxicity (17).Nevertheless, one study described the DNA damage in oral cells measured by AP sites and reported significantly lower levels in e-cigarette users than in smokers and non-smokers (21).Therefore, based on the mentioned findings, the available data might be critically analyzed before discussing with patients the "safeness" or "benefits" of e-cigarette use.First, however, it is essential to point out evidence of potential damage and risks related to its use due to the proven or potential carcinogenic agents in e-cigarettes components, including metals (cadmium, chromium, etc.), carbonyls (acrolein), propylene oxide, and especially flavoring additives (9,28).Few studies have characterized ENDS-related oral mucosal lesions and reported xerostomia, nicotine stomatitis, hairy tongue, angular cheilitis intra-oral explosion injuries, periodontal disease, and oral microbiome alterations were frequently found in e-cigarette users (7).However, there is scarce evidence about OPMD and oral cancer in ENDS users.We found a reported case of oral lichenoid eruption associated with e-cigarette use (14) and a cross-sectional study describing one case of oral leukoplakia without dysplasia among 45 e-cigarette users (18).Despite this, it is essential to highlight that lacking long-term clinical studies on using e-cigarettes leads to a scarcity of direct evidence for association of e-cigarettes with oral malignant transformation.However, persistent local irritation may result in pathological mucosal reactions that may predispose to the development of OPMD (28).Regarding oral cancer, there are 3 cases reported in the literature, and the presence of vaping history is a common finding in all cases (16,22).Nevertheless, isolated reported cases are not considered strong enough for a causal relationship between oral cancer and e-cigarette use.However, they help to generate a hypothesis for causal inference for future studies (26).It is important to highlight some limitations of this review, like the exclusion of non-English primary articles during the literature search, which might lead to pos-sible missed relevant publications.Additionally, the included studies presented different designs.Most were conducted without property identification of confounding variables such as type, time, and concentrations of e-cigarette liquid or vapor, exclusive e-cigarette use, or combination with conventional forms of tobacco and alcohol consumption.The sample selection and analysis variation among the cited studies is also a limitation.

Conclusions
In summary, there is limited and low-quality clinical evidence associating using ENDS with malignant transformation for OPMD and oral carcinogenesis.Therefore, it might be too soon to include e-cigarette use as a risk factor for OPMD and oral cancer.However, based on current findings, clinicians and dental-care professionals must carefully advise patients about the use of e-cigarettes especially considering that even lower dosages of potential carcinogens present in e-cigarettes, compared to conventional tobacco smokers, could induce molecular changes in the oral mucosa and DNA damage in oral cells with no apparent clinical change.Future research on this topic is needed especially longterm studies with larger sample sizes, including individuals that are exclusively e-cigarette users and considering that newer generations of e-cigarettes with different designs and patterns are being produced.

Fig. 1 :
Fig. 1: Flow diagram of literature search and selection criteria.

Table 1 :
Eligibility criteria considered for study selection process.

Table 2 :
Risk of bias assessed by The Joanna Briggs Institute Critical Appraisal Tools/ © JBI, 2020.

Table 3 :
Summarized information, results and conclusions of the included studies (n = 12).

Table 3 cont . :
Summarized information, results and conclusions of the included studies (n = 12).