Genotoxic profile among e-cigarette consumers, cigarette smokers, and nonsmokers

ABSTRACT

It is widely believed that e-cigarettes are less harmful than conventional cigarettes because they have a lower nicotine content. In contrast to this notion, several in vitro studies have evaluated and demonstrated the genotoxicity associated with e-cigarette smoking. However, there is a lack of human studies on the genotoxicity of e-cigarettes. This pilot study evaluated and compared indicators of genotoxicity in e-cigarette users, cigarette smokers, and nonsmokers. A total of 84 healthy participants, including 20 e-cigarette users, 31 cigarette smokers, and 33 nonsmokers, were recruited. Genotoxicity was evaluated by measuring tail moment (TM), tail length (TL), and % tail DNA intensity (%T) using the comet assay as an indicator of DNA damage in blood and detecting micronuclei in buccal cells with the buccal micronucleus (MN) cytome assay. Bivariate analyses showed that there was no significant difference in TM and TL between e-cigarette users and cigarette smokers, but in both groups, the three parameters were significantly higher than that in nonsmokers (p<0.02). In contrast, the frequency of micronuclei in e-cigarette users (40%) was higher than that in cigarette smokers (27.5%). Our findings indicate that e-cigarettes have a similar genotoxic effect to regular cigarettes and, therefore, contradict the notion that e-cigarettes are safer than regular cigarettes.

DNA damage in the blood of nonsmokers (TM=1.96), cigarette smokers (TM=16.3), and e-cigarette users (TM=10)

INTRODUCTION

The use of e-cigarettes is becoming increasingly common, especially among young adults and teenagers (Chapman and Wu, 2014; Chadi et al., 2019; Miech et al., 2019). The popularity of e-cigarettes is attributable to the perception that these cigarettes are less harmful than traditional cigarettes, their easy availability, cheaper price, the spread of vaping as a trend, and the variety of flavors available (de Andrade et al., 2015; Tomashefski, 2016; Collins et al., 2019). However, several studies have reported that, like conventional cigarettes, waterpipe smoking also has genotoxic effects (Al-Amrah et al., 2014; Katarkar et al., 2014; DehghanNezhad et al., 2020).

The oral cavity might be a good indicator of an individual’s health because the oral mucosa is the first tissue to come into contact with pollutants and often reflects pathogenic processes in the body (Bonassi et al., 2011; Kashyap and Reddy, 2012; Torres-Bugarin et al., 2014). Many studies have shown that oral habits, such as smoking and drinking alcohol, can induce changes in normal buccal mucosal cells that may lead to carcinomatous changes (Jindal et al., 2013; Zamora-Perez et al., 2013; Pradeep et al., 2014). Such changes in oral exfoliated buccal cells can be examined using the micronucleus (MN) assay, which is a simple, reliable, cheap, and noninvasive method for evaluating early genotoxic damage. It has been reported that a significant increase in the frequency of micronuclei in buccal cells is an indicator of oral cancer (Tak et al., 2014; Shashikala et al., 2015). In addition, the comet assay has frequently been used in combination with the MN assay in human biomonitoring studies on exposure to potentially genotoxic agents to detect DNA damage as an early biological effect of exposure (Kang et al., 2013; Bausinger and Speit, 2014). Peripheral blood is a convenient source of cells. However, most human biomonitoring studies use whole blood samples instead of peripheral blood mononuclear cells because they are easy to obtain and process (Beyls et al., 2021). There are several in vitro studies on the cytotoxicity and genotoxicity of the chemical constituents and/or aerosol condensate of e-cigarettes (Al-Saleh et al., 2020; Tellez et al., 2021), but there are very few human studies. The prevalence of smoking among Saudi Arabia residents is high at 49.2% (Abdelwahab et al., 2016). Therefore, the Saudi Arabian government has been making significant efforts to ban smoking in most public places and implement measures related to tobacco prices (Monshi and Ibrahim, 2021). In particular, the widespread perception that e-cigarettes are less harmful than conventional cigarettes, the wide range of available flavors, the ease of purchasing them online, and promotional offers have led to high levels of consumption among the youth (Khanagar et al., 2019; Habib et al., 2020). Despite this alarming trend, to our knowledge, no study has so far investigated e-cigarette use and health behaviors among citizens and Saudi Arabian residents. Therefore, this pilot study was conducted to evaluate whether e-cigarette use increases DNA strand breaks in blood and induces micronucleation in buccal cells. These genotoxic markers were compared between e-cigarette users, regular smokers, and nonsmokers.

MATERIALS AND METHODS

SUBJECTS

This study included 84 healthy volunteers (51 males and 28 females) aged 19–59 years who were enrolled between 2018 and 2020. The volunteers comprised three groups: (1) e-cigarette consumers (n=20), who had used an e-cigarette regularly for almost 1 year and did not smoke regular cigarettes; (2) current cigarettes smokers (n=31), who had been smoking more than one cigarette daily for more than 2 years and had never used an e-cigarette or other tobacco products; and (3) nonsmokers (n=33), who had never smoked or used a regular cigarette, e-cigarette, or any other type of tobacco product. The study was approved by the King Faisal Specialist Hospital and Research Centre Research Ethics Committee (RAC #2170007), and each volunteer signed an informed consent form prior to the commencement of the study. Each participant was interviewed to fill in a detailed questionnaire regarding smoking habits, age, and health conditions. General and oral health conditions were good in all participants. We excluded participants who (1) were undergoing smoking cessation therapy; (2) had oral lesions; (3) had dental implants; (4) had undergone periodontal treatment in the last year; (5) were under regular medication and/or treatment; and (6) had chronic health problems such as heart disease, diabetes, hypertension, and mental health issues.

SAMPLE COLLECTION

BUCCAL CELLS

Exfoliated buccal cells were collected by rotating a cytobrush against the inside of each cheek wall in a circular motion. The brush was then inserted in a 15-ml conical tube containing 10 ml of buccal cell buffer (0.1 M EDTA tetrasodium salt, 0.01 M Tris-HCl, and 0.02 M NaCl [pH 7.0]).

BLOOD SAMPLES

Venous blood (5 ml) was collected in an EDTA vacutainer tube. For genotoxicity testing, four aliquots of whole blood (volume, 50 μl) were stored at −80°C for more than 3 months before the comet assay was performed.

GENOTOXICITY BIOMARKERS

COMET ASSAY

Peripheral blood lymphocytes were collected for the comet assay, which was performed according to a standard protocol (Singh et al., 1988) with minor modifications. For each participant, 50 cells from two slides (25 cells per slide) were assessed using the computer image analysis software Comet Assay IV with a monochrome CCD IEEE1394 FireWire video camera (Perceptive Instruments, Halstead, UK). Image analysis was performed at a magnification of 20× with a fluorescence optical microscope (Eclipse Ti-E; Nikon, Japan) equipped with excitation (465 nm) and barrier (595 nm) filters. Five parameters were measured, including the head length (HL), tail length (TL), head intensity (TI), % tail DNA (%T), and tail moment (TM). TM was chosen as a measure of DNA damage.

MN CYTOME ASSAY

This assay was performed on buccal cells with a modified standard protocol (Thomas et al., 2009). The samples of exfoliated buccal cells were washed three times, resuspended in fresh buccal cell buffer and transferred onto microscope slides using a cytocentrifuge. The slide-staining process has been described by Thomas et al. (2009). As previously described, the slides were scored using a Nikon Ti2 transmitted light and fluorescence microscope with a far-red filter at 1,000× magnification with immersion oil (Fenech et al., 2003; Thomas et al., 2009). Compared to normal nuclei, micronuclei are oval or round in shape and are smaller (<1/3^rd or <1/16^th the normal size of a nucleus), but they have similar staining intensity and texture. For each participant, 1,000 buccal cells were screened for nucleated cells with well-preserved cytoplasm. Cells containing micronuclei on a bright field were confirmed by examination under far-red fluorescence.

Five participants were excluded from this analysis because their buccal sample did not contain a sufficient number of cells. Therefore, the MN assay included only 79 participants (20 e-cigarette users, 29 cigarette smokers, and 30 nonsmokers).

STATISTICAL ANALYSIS

The results are presented as mean±standard deviation (SD) for quantitative variables and frequency (percentage) for categorical variables. All tested parameters were log-transformed to obtain a normal distribution. Pearson’s correlation analysis was used to test the associations between pairs of continuous variables. One-way analysis of variance (ANOVA) was applied to compare the three groups. A pairwise significance comparison between three groups was conducted using Dunnett’s post-hoc t-test. Due to the relatively low detection frequency (<50%) of micronuclei in buccal cells, this parameter was not included in ANOVA.

Statistical analyses were conducted using IBM SPSS Statistics for Windows, version 20.0 (IBM Corp., Armonk, USA). A p-value pf <0.05 was considered to indicate statistical significance.

RESULTS

All participants were healthy at the time of the study. Notably, 11 participants (4 nonsmokers, 1 cigarette smoker, and 6 e-cigarette users) had a history of heart surgery, allergy, asthma, high cholesterol, epilepsy, hemophilia, high glucose, and kidney atrophy, but they were healthy at the time of recruitment and were not under any medication. They were, therefore, included in the analysis. Most participants were Saudi (69%), whereas the rest were from different nationalities (31%). The majority lived in Riyadh (95.2%), and only 4 participants were from other parts of the Kingdom. Of the 84 participants, 2, 63, and 19 described their areas of residence as polluted, clean, and mixed, respectively. Further, the majority were unmarried (64.3%) and working (77.4%).

Of the 20 e-cigarette users who were vaping for 1–6 years, 18 reported using e-cigarettes daily and 2 used them once a week. When the e-cigarette users were asked about their reasons for vaping, 16 (80%) replied that it helped them quit cigarettes or shisha. The other reasons cited included better odor, weight loss, and less harm than regular smoking. Our 31 regular smokers were smoking ≥1 cigarette daily for more than 2 years. Of the 33 nonsmokers, 15 and 5 were living and socializing with smokers, respectively.

Table 1 presents the descriptive statistics for the five comet assay parameters and MN values in the three groups of participants. %T, TM, and TL were selected for evaluation of DNA damage because they were widely used in human biomonitoring studies on account of their accuracy in detecting DNA damage under different exposure conditions (Azqueta et al., 2020; Milić et al., 2021). Strong correlations were observed between %T and TM (r=0.974), %T and TL (r=0.714), and TM and TL (r=0.844) (p<0.001 for each correlation). ANOVA showed significant differences in TM, %T, and TL (p<0.001 for each) among the three groups of participants. As shown in Fig. 1, no significant differences were detected between e-cigarette users and cigarette smokers in TM and TL. However, %T was significantly higher in cigarette smokers than in e-cigarette users (p=0.026). In contrast, the levels of the three parameters were significantly higher in e-cigarette users than in nonsmokers (p<0.001 for TM and %T, and p=0.012 for TL). Because micronuclei were detected only in 19 (24%) of our participants, we could not examine the significance of differences among the three groups. While only one micronucleus was detected in the buccal cells of 3 (10%) nonsmokers, one to three micronuclei were observed in 8 cigarette smokers (27.6%) and 8 e-cigarette users (40%). Fig. 2 displays the findings for the number of buccal cells with one or two micronuclei in smokers and e-cigarette users.

Table 1 Descriptive statistics of comet parameters and MN among the three groups of participants

		Comet parameters					MN
		HL	TL	HI	%T	TM
Nonsmokers	N	33	33	33	33	33	30
	Mean	91.785	63.256	89.412	10.588	3.636	0.100
	SD	16.203	12.633	4.198	4.198	1.696	0.305
	Minimum	66.680	39.960	78.855	5.171	1.525	0.000
	Maximum	136.120	96.560	94.829	21.145	8.528	1.000
Cigarette smokers	N	31	31	31	31	31	29
	Mean	87.498	84.099	74.453	25.547	10.911	0.379
	SD	11.660	23.917	10.011	10.011	6.609	0.728
	Minimum	69.400	52.920	58.421	8.138	3.076	0.000
	Maximum	115.240	135.680	91.862	41.579	24.122	3.000
E-cigarette smokers	N	20	20	20	20	20	20
	Mean	94.512	76.198	80.760	19.240	8.061	0.500
	SD	12.034	16.173	10.803	10.803	6.330	0.761
	Minimum	74.120	50.280	43.761	9.791	3.433	0.000
	Maximum	117.240	119.600	90.209	56.239	31.585	3.000
All participants	N	84	84	84	84	84	79
	Mean	90.852	74.030	81.831	18.169	7.374	0.304
	SD	13.831	20.330	10.639	10.639	6.034	0.627
	Minimum	66.680	39.960	43.761	5.171	1.525	0.000
	Maximum	136.120	135.680	94.829	56.239	31.585	3.000

Fig. 1

Participants’ mean TM, %T, and TL levels. Bars indicate the means±SEs of results obtained per group. The statistical differences were evaluated using one-way analysis of variance with Dunnett’s post-hoc test

Fig. 2

Buccal cell of (a) e-cigarette user with a single micronucleus; (b) e-cigarette user with two micronuclei; and (c) cigarette smoker with a single micronucleus. A fluorescence microscope captured all images, and micronuclei were marked with a yellow circle

DISCUSSION

The current comparative study provides evidence that the extent of DNA damage in e-cigarette users and cigarette smokers is similar; importantly, it is significantly higher among users of both types of cigarettes than among nonsmokers. A higher frequency of micronuclei was also observed in the buccal cells of e-cigarette users than in the buccal cells of nonsmokers. These findings contradict the popular notion that e-cigarettes are safer than regular cigarettes.

In the present study, a comet assay was used to detect DNA single-strand breaks with high sensitivity, and the parameters measured were TM, %T, and TL. The TM and TL values of the cigarette smokers and e-cigarette users in this study were relatively similar, whereas %T was more than one-fold higher in cigarette smokers than in e-cigarette users. TM was earlier considered a better marker of DNA migration than other DNA damage markers (Olive et al., 1990). However, over the years, there has been some debate over the use of TM because it can mask the induced effect in some cases (De Boeck et al., 2000). Instead, %T is currently preferred because it is normally distributed and has been found to increase proportionally with DNA migration in human studies (Møller et al., 2014a). TL is recommended as a marker only in cases with low levels of DNA damage because it does not change once the tail is established (Collins, 2004). The current findings indicate a significantly higher level of DNA migration in e-cigarette users and cigarette smokers than in nonsmokers. These findings suggest that the level of DNA damage caused by the inhalation of smoke from burned tobacco in regular cigarettes and the inhalation of nicotine-containing aerosols in e-cigarettes is similar. Thus, the genotoxic effect might be related to nicotine and other ingredients contained in the e-cigarette liquids. Our recent in vitro study tested 68 e-liquid refills from 33 brands with nicotine content ranging from 1 to 8 mg and showed that the majority induced substantial DNA damage (Al-Saleh et al., 2020). The migration of DNA damage induced by chemicals is an essential first step in carcinogenesis (Basu, 2018). Unfortunately, the cytotoxic and genotoxic effects of different compounds in e-liquids that can be carcinogenic are unknown (Armendáriz-Castillo et al., 2019) because manufacturers do not include their chemical composition or concentrations on labels. Tobacco smoke contains several harmful mutagenic/carcinogenic chemicals such as polycyclic aromatic hydrocarbons, N-nitrosamines, aromatic amines, aldehydes, volatile organic hydrocarbons, and metals, which have been associated with the etiology of human cancer (Centers for Disease Control and Prevention (US), 2010). Our findings are supported by several studies that have shown elevated DNA damage, as measured by the comet assay, in tobacco smokers (Dobrzynska et al., 2018). However, the mechanism by which different components of e-cigarette vapor induce DNA damage is unclear. Some mechanistic studies have proposed that e-cigarette vapor produces reactive oxygen species, which cause single- and double-strand breaks, lipid peroxidation, as well as oxidative DNA damage (Anderson et al., 2016; Kopa and Pawliczak, 2018; Zhao et al., 2018).

Another indicator of the cytotoxic and genotoxic effects of tobacco is the presence of micronuclei in exfoliated buccal cells collected from smokers (de Geus et al., 2018). Studies have shown that alterations in buccal cells associated with smoking are considered as early signs of oral cancer (Parmar et al., 2020). Despite the small number of e-cigarette users (N=20) recruited in the present study, one to three micronuclei were observed in the buccal cells of eight participants. In addition, increased DNA damage was detected in the blood of e-cigarette users based on a mean TM, %T, and TL of 7.5, 18.5, and 80, respectively. Among the 29 cigarette smokers included in the MN assay, 8 had one to three micronuclei, but their DNA damage parameters were higher than those of the e-cigarette users: 10.7 (TM), 25.4 (%T), and 83.5 (TL). In nonsmokers of the present study, only one micronucleus was observed in 3 of 30 participants, and the levels of DNA damage markers were lower than those in the other two groups: TM, 2.2; %T, 6.7; and TL, 58.3. The observed DNA damage may be attributable to their exposure to environmental pollutants (Samanta and Dey, 2012). The present findings are corroborated by the study of Franco et al. (2016), who found that the frequency of micronuclei in e-cigarette users was significantly lower than that in cigarette smokers. In addition, Pop et al. (2021) also reported that both cigarette smokers and e-cigarette users had a significantly higher micronucleus frequency than nonsmokers. Despite the similarities with previous studies, we must acknowledge that the sample size of our study is small and could have led to a bias in the results. However, the present findings are also supported by a few in vitro studies that demonstrated the effect of chemicals contained in e-liquids and/or e-cigarette vapors on micronucleus induction (Al-Saleh et al., 2020; Wieczorek et al., 2020). Moreover, a recent study showed that users of e-cigarettes containing nicotine had high micronuclei counts in buccal cells and salivary oxidative stress (Menicagli et al., 2020). Further, a recent study found that the level of DNA adducts of acroline (a genotoxic carcinogen usually found in e-cigarette vapor) in the oral cells of e-cigarette users was eight-folds higher than that in nonsmokers (Cheng et al., 2022). The TM, %T, and TL values in our e-cigarette users and cigarette smokers were higher than the baseline values of 3.96, 7.4, and 32.3, respectively, measured in 19,320 subjects from 27 countries (Milić et al., 2021). The authors observed that the level of DNA damage was not affected by age, gender, or smoking habits (Milić et al., 2021). Data from 13 studies found a correlation between arsenic exposure and DNA damage measured by TM, %T, and TL; however, the majority of the reported data for the exposed population were lower than those for e-cigarette users or cigarette smokers in the current study (Qian et al., 2021). Overall, our results demonstrate that e-cigarettes have a similar adverse genotoxic effect to cigarette smoking, but studies with a larger sample size are required to confirm our preliminary findings.

One of the limitations of this study is its pilot design; thus, the findings need to be confirmed in future research. Another limitation is the small sample size, which reduced the statistical power for some nonsignificant associations and limited the ability to adjust the analyses for specific confounders (Biau et al., 2008). Next, as the participants enrolled voluntarily, this may have led to a selection bias caused by arousal-seeking characteristics associated with exciting, demanding, or stressful situations (Guilbert et al., 2019). A bias might also have been caused by the self-administered questionnaires, leading to underestimation or overestimation of the answers depending on the participants’ subjective perceptions (Bauhoff, 2014). Another limitation is the lack of data on environmental exposure, which might be associated with oxidative stress and genotoxicity (Møller et al., 2014b), and the types of e-cigarettes used and their nicotine content, which could be related to the extent of DNA damage. Despite these limitations, our results provide valuable preliminary data regarding the potential harm of e-cigarettes.

CONCLUSION

This pilot study revealed that e-cigarettes are associated with increased DNA breakage in blood and micronucleus induction in buccal cells, with these effects being similar to those for regular cigarette smokers. This could mean that nicotine and other chemicals contained in vaping products induce oxidative stress and genotoxicity, as reported in several in vitro studies. Overall, our results highlight that e-cigarettes are not a safer alternative to regular cigarettes. However, large-scale, longitudinal research is required in the future to confirm the genotoxic effects of e-cigarettes and their potential long-term health effects.

ACKNOWLEDGMENTS

The authors acknowledge the assistance of (1) Dr. Patricia McWalter in recruiting volunteers from the Family Medicine and Polyclinics Department; (2) Mrs. Chaficia Eltabache for blood withdrawal; and Mr. Faisal Alabdulkarim in recruiting volunteers for their help at the beginning of the study.

DECLARATIONS

FUNDING

No funding was received.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Each patient signed an informed consent approved by the King Faisal Specialist Hospital and Research Centre Research Ethics Committee.

PATIENT CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

Not applicable.

COMPETING INTERESTS

The authors reported no potential conflict of interest.

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