International Journal of

Environmental Research
and Public Health

Article
Early Diagnosis of Oral Mucosal Alterations in Smokers and
E-Cigarette Users Based on Micronuclei Count: A
Cross-Sectional Study among Dental Students
Anca Maria Pop 1 , Raluca Coros, 2 , Alexandra Mihaela Stoica 3 and Monica Monea 3, *

1 Faculty of Medicine, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of
Târgu Mures, , 540139 Tirgu Mures, Romania; ancapop98@yahoo.com
2 Faculty of Dental Medicine, George Emil Palade University of Medicine, Pharmacy, Science, and Technology
of Târgu Mures, , 540139 Tirgu Mures, Romania; raluca_coros@yahoo.com
3 Department of Odontology and Oral Pathology, George Emil Palade University of Medicine, Pharmacy,
Science, and Technology of Târgu Mures, , 540139 Tirgu Mures, Romania; alexandra.stoica@umfst.ro
* Correspondence: monica.monea@umfst.ro

Abstract: The presence of micronuclei in oral epithelial cells is considered a marker of genotoxicity,
which can be identified using exfoliative cytology. The aim of this study was to investigate cytotoxic
damage through the evaluation of micronuclei in the oral mucosa of smokers and e-cigarette users
compared to nonsmokers. We obtained smears from the buccal mucosa of 68 participants divided in





 3 groups (smokers, e-cigarette users and nonsmokers), which were further processed with Papanico-
laou stain. The frequencies of micronuclei and micronucleated cells were recorded and statistically
Citation: Pop, A.M.; Coros, , R.; Stoica,
analyzed at a level of significance of p < 0.05. The mean micronuclei values per 1000 cells were
A.M.; Monea, M. Early Diagnosis of
3.6 ± 1.08 for smokers, 3.21 ± 1.12 for e-cigarette users and 1.95 ± 1.05 for nonsmokers. The mean
Oral Mucosal Alterations in Smokers
and E-Cigarette Users Based on
values of micronucleated cells per 1000 cells were 2.48 ± 0.91 for smokers, 2.39 ± 1.07 for e-cigarette
Micronuclei Count: A Cross-Sectional users and 1.4 ± 0.68 for nonsmokers. Smokers and e-cigarette users had significantly higher values
Study among Dental Students. Int. J. of micronuclei and micronucleated cells compared to nonsmokers, but there were no significant
Environ. Res. Public Health 2021, 18, differences between smokers and e-cigarette users. We concluded that the micronuclei count can
13246. https://doi.org/ be used as an early indicator for alterations of oral mucosa and exfoliative cytology represents an
10.3390/ijerph182413246 accessible tool which could be applied for mass screening.

Academic Editors: E. Melinda Keywords: cytodiagnosis; oral health; micronucleus assay; cigarette smoking; e-cigarette
Mahabee-Gittens and Ashley
L. Merianos

Received: 8 November 2021

1. Introduction
Accepted: 14 December 2021
Published: 16 December 2021
Smoking represents the most common cause for oral cancer and was identified as the
second most significant risk factor responsible for global death; its by-products such as
Publisher’s Note: MDPI stays neutral
polycyclic hydrocarbons and nitrosamine have mutagenic and carcinogenic properties, as
with regard to jurisdictional claims in
these compounds infiltrate into the oral mucosal cells and might induce cellular changes [1].
published maps and institutional affil- Adolescence has been associated with a high risk of substance use, among which tobacco
iations. smoking is the most frequent. The majority of smokers had their first cigarette or were
already addicted before they turned 18 and compared to adults, young people need fewer
cigarettes and less time to become nicotine addicted [2]. E-cigarettes were developed in
2003 in an effort to assist smoking cessation and to reduce the harm effects of tobacco and
Copyright: © 2021 by the authors.
other substances present in conventional cigarettes [3]. These devices consist of an atomizer
Licensee MDPI, Basel, Switzerland.
for heat generation, a battery with fixed voltage and a liquid reservoir containing a mixture
This article is an open access article
of volatile compounds alongside with different amounts of nicotine, propylene glycol,
distributed under the terms and glycerol, water, flavoring agents and dyes [4–6]. There are limited data regarding the
conditions of the Creative Commons impact of e-cigarettes on adolescents and young adults, but until now it is clear that their
Attribution (CC BY) license (https:// use is not restricted to current smokers and due to high youth awareness, these devices are
creativecommons.org/licenses/by/ becoming more popular [7]. In USA, between 2010–2013 the use of e-cigarettes in adults
4.0/). raised from 1.8% to 13% [8]; the highest prevalence was reported among young adults

Int. J. Environ. Res. Public Health 2021, 18, 13246. https://doi.org/10.3390/ijerph182413246 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2021, 18, 13246 2 of 10

aged 18–24 years old and among former smokers but also individuals who never smoked
before [9]. However, in spite of the continuous increase in the use of e-cigarette among
young people, information about its possible negative consequences on the oral and overall
health is still limited [10].
It is well known that smoking conventional cigarettes is associated with poor dental
health, with inflammation of oral tissues leading to gingival and periodontal disease [4,11],
but only few studies focused on the correlations between e-cigarettes and oral health
status. An association between e-cigarette use and tooth fractures in adolescents has been
suggested [12] but also the observations that in comparison to smokers, individuals that
use e-cigarettes and those who never smoked have less periodontal inflammation [11,13].
A recent review [14] showed that some studies reported that e-cigarette users exhibit higher
levels of proinflammatory cytokines, higher plaque index and more frequent periodontal
disease compared to nonsmokers [15,16], but others did not confirm this observation [17,18].
Moreover, most of the studies focusing on the effects of e-cigarettes either compared their
users with conventional smokers, or with nonsmokers and only a few studies compared
these two types of nicotine users with controls (nonsmokers) [19,20]. Even so, studies who
included all the three groups mentioned above selected patients older than 45 years, with
long-term exposure to nicotine [19] or with already present clinical alterations or lesions of
the oral mucosa [20].
The conflicting data raised many concerns upon the effects of these devices on the oral
health [21]. The association between smoking and alcohol increases the risk of occurrence
for both oral premalignant and cancerous lesions, as the cariogenic compounds present
in the smoke are dissolved in alcohol, which also determines vasodilation and enhances
the absorption of these substances by the oral mucosa [22,23]. All forms of tobacco use
can cause cancer, therefore early detection and treatment of oral premalignant lesions are
of utmost importance in reducing the morbidity, mortality and costs associated with the
treatment of oral malignancies [22].
Exfoliative cytology allows the early identification of morphological changes in the
oral mucosa; it is a simple and non-invasive technique considered to be a valuable adjuvant
in the diagnosis of premalignant lesions, allowing appropriate preventive measures against
the development of oral cancer [24]. It is also suitable for the follow-up of premalignant
lesions, because it avoids the use of repeated biopsies, which present disadvantages such as
the invasive character, esthetic concerns, risk of infection or damage to normal tissues [25].
The advancements in this method facilitated the evaluation of genotoxic exposure using
different parameters such as cellular and nuclear diameters, nuclear shape and disconti-
nuity or assessment of micronuclei (MN). The latter are extracellular cytoplasmic bodies
that are formed during anaphase from chromosomal fragments. Several studies showed
that there is a correlation between the frequency of MN and severity of the genotoxic
damage, these being further used for grading the lesion [26,27]. Therefore, MN count is
regarded as an indicator of chromosomal aberrations and is useful in detecting early-stage
carcinogenesis [28,29].
As there are limited data regarding the presence of MN in clinically normal oral
mucosa in different types of young nicotine users compared to nonsmokers, the aim of
our study was to evaluate based on exfoliative cytology, in a group of young adults, the
frequency of MN in the oral epithelial cells of smokers, e-cigarette users and never smokers.

2. Materials and Methods

2.1. Study Participants and Selection Criteria
After the approval of the study by the Ethics Committee of our university, all students
from the Faculty of Dental Medicine were invited to participate in this research. An e-mail
was sent in February 2021 containing the aim of the study and a questionnaire with inclu-
sion and exclusion criteria. It was mentioned that the enrollment is voluntary and the data
will remain anonymous. Participants were defined as nonsmokers if they had no history of
smoking, as smokers if they consumed at least 10 conventional cigarettes/day in the past
Int. J. Environ. Res. Public Health 2021, 18, 13246 3 of 10

12 months and as e-cigarette users if they recalled current consumption of e-cigarettes daily
in the past 12 months, with no conventional cigarette smoking in the last 3 months. As
exclusion criteria we used the following conditions: consumption of other forms of tobacco
(hand-made cigarettes, country-style cigarettes, cigars and pipes or tobacco chewing), dual
users (current simultaneous use of conventional and e-cigarettes), chronic use of alcohol
(>7 units/week for women and > 14 units/week for men) or other drugs, orthodontic treat-
ment in progress, alteration of the oral mucosa or previous diagnosis of oral premalignant
lesions, systemic diseases (diabetes, autoimmune diseases, hematological disorders). A
total of 68 participants, who agreed to take part in the research and met the inclusion and
exclusion criteria, were enrolled in three groups: A-25 conventional cigarette smokers, B-23
e-cigarette users and C-20 nonsmokers.

2.2. Exfoliative Cytology Sampling Technique

The participants were asked to rinse the oral cavity with tap water and the excess
of saliva and debris were gently wiped off with a cotton pellet. A thorough examination
was performed by a single investigator, who was not aware to which study group the
participant belonged to. Buccal cells were collected using a wooden spatula and then
transferred on glass slides. For each participant two glass slides (one from each cheek)
were prepared and fixed with 95% ethanol. The specimens were further processed for
histopathological evaluation using Papanicolaou (Pap) stain.

2.3. Histopathological Protocol

For the Pap stain, the slides were introduced in 1% acetic acid (10 dips) and then
treated with Hematoxylin preheated 60 ◦ C. They were washed in tap water and dipped in
1% acetic acid. They were then treated with Orange Green-6, followed by 1% acetic acid
(10 dips) and in Eosin Azure-50. Again, they were dipped by 1% acetic acid, methanol
(10 dips), and xylene (10 dips). The slides were coded, then randomized and scored by one
examiner in a single-blind manner (the examiner was not aware to which group the slides
belonged).

2.4. Micronucleus Analysis

All slides were viewed under an optical microscope (Zeiss, Oberkochen, Germany)
at 10x magnification for screening and 40x magnification for MN counting. For each
participant, 1000 cells were evaluated and the frequency of MN and micronucleated
cells (MNC) per 1000 cells were recorded by a single examiner, who demonstrated good
intraobserver reliability, with an intraclass correlation coefficient (ICC) of 0.84 (0.82–0.87).
Only cells which met the following criteria were included in the analysis: presence of intact
cytoplasm and relative flap cell position on the slide, little or no overlap with adjacent cells,
little or no debris, normal and intact nucleus with smooth and distinct nuclear perimeter
without nuclear overlapping.
The previously published criteria by Tolbert et al [30] used for identifying MN were
applied: texture similar to the nucleus, normal smooth perimeter, less than 1/3 the diameter
of the nucleus, absence of overlap or bridge to the nucleus. The criteria for excluding cells
for MN assessment were also followed: cells with two nuclei, degenerating or dead cells,
presence of MN-like structure connected with the main nucleus with a bridge.

2.5. Statistical Analysis

Descriptive statistics was obtained using Microsoft Excel (Microsoft, Redmond, WA,
USA) and ICC with 95% confidence interval was calculated by SPSS Statistics version 25
(IBM Corporation, Armonk, NY, USA). Data were statistically analyzed using GraphPad
Prism 7 (GraphPad Software, San Diego, CA, USA) and were summarized as mean ± stan-
dard deviation (SD) for continuous variables and as absolute numbers and percentages for
categorical variables. Kolmogorov-Smirnov test was used for assessing normal distribution,
and then data were compared by one-way analysis of variance (ANOVA) or Kruskal-Wallis
 Int. J. Environ. Res. Public Health 2021, 18, x 4 of 10

standard deviation (SD) for continuous variables and as absolute numbers and percent-
Int. J. Environ. Res. Public Health 2021, 18, 13246 4 of 10
ages for categorical variables. Kolmogorov-Smirnov test was used for assessing normal
distribution, and then data were compared by one-way analysis of variance (ANOVA) or
Kruskal-Wallis test, as appropriate. Post-hoc analysis was conducted using Tukey Kramer
or Dunn’s
test, multiplePost-hoc
as appropriate. comparison test, was
analysis as appropriate
conducted and
usingalso in particular
Tukey Kramer orsituations with
Dunn’s mul-
Mann-Whitney test for assessing trends. Categorical variables were analyzed
tiple comparison test, as appropriate and also in particular situations with Mann-Whitneyusing Chi-
square
test test. The level
for assessing of Categorical
trends. statistical significance was set
variables were at a value
analyzed of pChi-square
using < 0.05 (two-tailed).
test. The
level of statistical significance was set at a value of p < 0.05 (two-tailed).
3. Results
3. Results
The demographic data of the participants are summarized in Table 1. The groups
wereThe demographic
considered data of theasparticipants
homogenous, arestatistically
there were no summarizedsignificant
in Table 1.differences
The groupsregard-
were
considered
ing genderhomogenous, as age
(p = 0.4119) and there(p were no statistically
= 0.1553) distribution.significant differences regarding
gender (p = 0.4119) and age (p = 0.1553) distribution.
Table 1. Demographic characteristics of the study groups.
Table 1. Demographic characteristics of the study groups.
Gender
Age
Study Group n (%)
Gender
Mean
Age ± SD
Study Group Male n (%) Female
Mean ± SD
Group A 14Male
(60.41%) 11 (39.59%)
Female 22.36 ± 1.41
Group B 15 (65.2%) 8(34.8%) 21.52 ± 1.65
Group A 14 (60.41%) 11 (39.59%) 22.36 ± 1.41
Group C 9 (45%) 11 (55%) 21.7 ± 1.59
Group B 15 (65.2%) 8(34.8%) 21.52 ± 1.65
p value
Group C 9 (45%) 0.4119 * 11 (55%) 0.1553
21.7 **
± 1.59
* No statistically
p value significant difference based0.4119
on Chi-square
* test. ** no statistically0.1553
significant
** dif-
ference
* No basedsignificant
statistically on ANOVA.difference based on Chi-square test. ** no statistically significant difference based on
ANOVA.
Examples of representative histopathological aspects are presented in Figures 1 and
Examples of representative histopathological aspects are presented in Figures 1 and 2.
2.

Figure 1. Specimen from conventional cigarette smoker. One cell with 4 MN was observed in a
Figure 1. Specimen from conventional cigarette smoker. One cell with 4 MN was observed in a
field with 15 epithelial cells. MN were identified based on their well-defined shape, size (less than
field with 15 epithelial cells. MN were identified based on their well-defined shape, size (less than
1/3 of the nucleus), similar color and refractility with the nucleus and absence of any connection to
1/3 of the nucleus),
it (bridge) similarMicroorganisms
(black arrow). color and refractility with the nucleus
are recognizable andnumerous
as small, absence ofbodies
any connection
inside andto
itbetween
(bridge)epithelial
(black arrow). Microorganisms are recognizable
cells (blue arrow). (Pap stain, x20). as small, numerous bodies inside and
between epithelial cells (blue arrow). (Pap stain, ×20).
Int. J. Environ. Res. Public Health 2021, 18, 13246 5 of 10
Int.J.J.Environ.
Int. Environ.Res.
Res.Public
PublicHealth
Health2021,
2021,18,
18,xx 5 5ofof1010

Figure2.
Figure
Figure 2.2.Epithelial
Epithelialcells
Epithelial cellscontaining
cells containing111MN
containing MN(black
MN (blackarrows)
(black arrows)from
arrows) from(a)
from (a)e-cigarette
(a) e-cigaretteuser
e-cigarette user (Pap
user(Pap stain,
(Papstain, ×40)
stain,×40)
×40)
and(b)
and (b)nonsmoker
nonsmoker(Pap (Papstain,
stain,×20).
×20).Beside
Besidemicroorganisms,
microorganisms,dye
dyegranules
granulescan
canbe
bedistinguished
distinguished
and (b) nonsmoker (Pap stain, ×20). Beside microorganisms, dye granules can be distinguished due
duetotointense
due intensestaining
staining (redarrow).
arrow).
to intense staining (red (red
arrow).

Thedistribution
The
The distributionofofof
distribution MNMNMN and
and
and MNC MNC
MNC values,
values,
values, expressed
expressed
expressed asasmedian
median
as median andinterquartile
and interquartile
and interquartile range
range
range in
in the
the three
three study
study groups
groups is
is presented
presented in
in the three study groups is presented in Figure 3. in Figure
Figure 3.
3.

Figure
Figure3. (a)
(a)Boxplot
Boxplotillustrating
illustratingthe distribution of of
MN among thethe
three study groups; the the
median of Group A was identical
Figure 3.3.(a) Boxplot illustrating the
the distribution
distribution of MNMN among
among three
the three study
study groups;
groups; median
the median ofofGroup
Group AAwas
was iden-
iden-
with
ticalthe value
with the of quartile
value of 3; (b)
quartileBoxplot
3; (b) illustrating
Boxplot the distribution
illustrating the of MNC
distribution among
of MNC the three
among study
the groups;
three
tical with the value of quartile 3; (b) Boxplot illustrating the distribution of MNC among the three study groups; the the
study median
groups; of
the
Group
median A was
of identical
Group A with
was the value
identical of
with quartile
the value 1.
median of Group A was identical with the value of quartile 1.of quartile 1.

There
Therewas
There wasaaastatistically
was statisticallysignificant
statistically difference
significant
significant between
difference
difference thethe
between
between three groups
thethree
three regarding
groups
groups MN
regarding
regarding
(pMN
< 0.0001) and MNC count (p = 0.0007). (Table 2)
MN (p(p<<0.0001)
0.0001)and
andMNC
MNCcountcount(p(p==0.0007).
0.0007).(Table
(Table2)2)
Table
Table2. Mean
Meanvalues
valuesof the
theMN
MNand
andMNC
MNCcount
countin the
thestudy
studygroups.
Table 2.2.Mean values ofofthe MN and MNC count ininthe study groups.
groups.
MN MN Count
Count MNC MNC Count
Count Number of Samples
Number
Group
Group MN Count MNC Count Number ofofSamples
Samples
Group Mean ±
Mean±±SDSDSD Mean ±
Mean±SD ±SDSD Containing MNC
Mean Mean ContainingMNC
Containing MNC
Group
GroupA
Group AA 3.6 ±±±1.08
3.6
3.6 1.08
1.08 2.48 ± ±0.91
2.48
2.48 ±0.91
0.91 25 25
25
Group B 3.21 ± 1.12 2.39 ± 1.07 23
GroupBB
Group 3.21±±1.12
3.21 1.12 2.39±±1.07
2.39 1.07 23
23
Group C 1.95 ± 1.05 1.4 ± 0.68 18
pGroup
valueCC
Group 1.95±±1.05
1.95
<0.0001 1.05
* 1.4±±0.68
1.4
0.0007 *0.68 18
18
p p value
value <0.0001
<0.0001 * *
* Statistically significant difference based on Kruskal-Wallis test. 0.0007
0.0007 **
* *Statistically
Statisticallysignificant
significantdifference
differencebased
basedon onKruskal-Wallis
Kruskal-Wallistest.
test.
The results of the post-hoc Dunn’s analysis are presented in Table 3. The values
The
The
of both resultsof
results
smokers ofthe
thepost-hoc
(Group post-hoc Dunn’sanalysis
A) and Dunn’s analysis
e-cigarette are
usersare presented
presented
(Group ininTable
B) were Table3.3.The
Thevalues
significantly valuesofof
different
both smokers (Group A) and e-cigarette users (Group B) were significantly different
both smokers (Group A) and e-cigarette users (Group B) were significantly different from from
Int. J. Environ. Res. Public Health 2021, 18, 13246 6 of 10

from the values of nonsmokers (Group C) regarding MN and MNC count. Conversely,
between smokers (Group A) and e-cigarette users (Group B) these two parameters showed
no statistically significant differences. However, in order to obtain the exact value of p
regarding the comparison between Groups A and B we also performed the Mann-Whitney
test as post-hoc analysis and obtained a p value of 0.2207 for MN count and 0.7953 for MNC
count, respectively. These values were interpreted as non-suggestive for trends (p > 0.1).

Table 3. Results of the Dunn’s multiple comparison test.

MN Count MNC Count

Comparison Mean Rank Mean Rank
p Value p Value
Difference Difference
Group A vs. B 6.453 >0.05 2.327 >0.05
Group A vs. C 25.015 <0.001 * 20.24 <0.01 *
Group B vs. C 18.562 <0.01 * 17.913 <0.01 *
* Statistically significant difference based on the adjusted value of p according to Dunn’s multiple comparison test.

4. Discussion
The aim of this study was to determine whether smokers or e-cigarette users exhibit
cytotoxic damage of the buccal mucosa in the absence of any significant clinical manifesta-
tion noticeable to the individual, which could be a false indicator of wellbeing. The early
diagnosis of oral lesions has become an important objective, as it has an immense impact
on the successful treatment of these patients [31]. We selected students without chronic
use of alcohol, dual use of conventional and e-cigarettes or systemic diseases which are
known to influence the oral mucosa. In the e-cigarette users group we included students
who previously smoked conventional cigarettes but replaced them with e-cigarettes for at
least 3 months, as there were no participants who never experienced conventional smoking
before.
The frequency of MN was proved to be an accurate hallmark of carcinogenesis, having
a higher value in cancerous compared to precancerous lesions [32]. Their quantitative
assessment can be considered an indicator of genetic damage, because in the majority of
cases cells with MN which enter mitosis produce daughter cells with higher number of
MN [23,33]. Moreover, the evaluation of other parameters gave contradictory results, as
nuclear abnormalities specific for apoptosis (condensed chromatin, karyorrhexis and py-
knosis) were identified less frequently in tumoral compared to normal cells [32]. However,
as most of the research conducted focused on patients with oral lesions or with long-term
exposure to genotoxic agents, the novelty of our study is represented by the investigation
regarding the presence of MN in clinically normal oral mucosa and in a young age group
with relative short exposure to carcinogens. In apparently healthy smokers, several studies
have reported morphological alterations such as high rate of epithelial cell proliferation,
the presence of MN, increasing number of keratinized cells and an altered ratio between
nuclear and cytoplasmic volumes [34,35].
Based on a recent Cochrane analysis [36], oral cytology showed 90% sensitivity and
94% specificity in detecting oral lesions. Its performance was better in comparison with
vital staining and light-based techniques, as these can erroneously provide more false
positive results due to lower specificity and are difficult to interpretate by the non-expert
clinician. Although the diagnosis of any oral lesion still needs a biopsy for confirmation,
oral cytology represents a promising technique based on the following arguments: the high
sensitivity supports its application for the screening of small, homogenous lesions, which
do not exhibit clinical features suggestive for malignancy, non-invasive character, low cost
and ability to provide a quantitative analysis of reliable genotoxicity. [36,37] Therefore,
oral cytology was found to be the most precise method for oral lesions diagnosis, after the
surgical biopsy, which make it a large-scale applicable technique for screening and early
diagnosis of malignant lesions [36] In our study we selected the buccal mucosa for collection
site, as cells located in this area are suitable for the assessment of any changes developed
Int. J. Environ. Res. Public Health 2021, 18, 13246 7 of 10

in relation to smoking. Several collection instruments have been proposed for harvesting
oral epithelial cells, such as wooden or metal spatula and different types of cytobrushes. In
our study we used wooden spatulas, as they are efficient and easily accessible, although
cytobrushes have been considered to provide better homogeneity of the smears and to
facilitate cell collection from all layers of the mucosa [38]. In the aforementioned Cochrane
meta-analysis the sensitivity in detecting oral mucosal abnormalities reported values of 93%
for smears collected by scraping (spatula) and 91% for brush, respectively. The specificity
showed a value of 92% for scraping and 94% for brush [36].
Among commonly used stains for MN such as Pap, Feulgen and Giemsa, we used Pap
stain, as it is considered the most effective staining technique for cytological assessment,
which enables the visualization of clear nuclear and cytoplasmatic characteristics and em-
phasizes differences between cells located in various layers of the epithelium [39]. However,
Feulgen stain was also proposed for assessing MN count due to its DNA specificity, but it
was regarded as expensive, with more complicated fixation and staining process and it also
requires additional compounds for the contouring of the cytoplasmatic borders. Therefore,
based on the acceptable accuracy regarding MN evaluation, Pap stain may be used for
routine screening and samples with potential abnormalities can be further processed with
DNA-specific stains [40].
However, there are several factors which can interfere with the correct estimation of
MN count, such as bacteria normally present in the oral cavity, but microorganisms usually
have smaller size, various shape and are located also between oral cells. Moreover, small
dye granules need to be differentiated from MN based on the more intense staining and
different refractility [41].
Several normal ranges for MN frequency have been previously proposed, be-
tween 0.05–11.5 MN/1000 cells, with the majority of studies reporting values between
0.5–2.5 MN/1000 cells [42]. The values of MN obtained in our study for the nonsmokers
group fall within the range of 0.5–2.5 MN/1000 cells, but the other groups exposed to
carcinogens exhibited higher values, however still fitting in the normal range. A possible
explanation for this can be the young age of the participants, the relative decreased number
of package-years in the group of conventional smokers and the fact that exfoliative cytology
can sometimes underestimate the MN count due to the time needed for the migration of
cells from basal to superficial layers of the oral epithelium. We found that more cells show-
ing MN were present in specimens from smokers and e-cigarettes users compared with
nonsmokers, similar to data reported by other studies [29,43]. Furthermore, a systematic
review of clinical studies published in 2018 found a higher frequency of MN in exfoliated
cells of smokers compared to non-smokers and concluded that this is associated with cyto-
toxic and genotoxic effects [44]. In an effort to exclude the bias caused by a normal aspect of
only occasionally exposed to carcinogens tissues, we selected participants with consistent,
continuous and recent smoking or e-cigarette use, because in the absence of a persistent
exposure only one sample may fail in detecting temporary cellular abnormalities [45].
Oral epithelial cells have a relatively decreased turnover [34]. However, in our study we
also found an increased turnover, which may indicate the toxicity of several compounds
released from tobacco. Smoking usually causes cellular irritation which predisposes to
accelerated proliferation and altered cellular morphology [22].
E-cigarettes have become more popular in the last years as an alternative to conven-
tional cigarettes and data show that the decrease of conventional smokers or dual users is
accompanied by an increase in exclusive e-cigarette users. Although considered safer in
comparison to conventional cigarettes, e-cigarettes have been associated with health risks,
including cardiac and pulmonary diseases, but also with nicotine addiction leading to a
tendency of experiencing other forms of tobacco [46,47]. While conventional smoking is
recognized as an important risk factor affecting the oral health, there is still a debate in the
scientific literature regarding the possible risks of e-cigarette use. Several studies reported
milder oral symptomatology in e-cigarette compared to conventional cigarette users [48],
but others found more frequent oral lesions, burns or inflammation compared to former or
Int. J. Environ. Res. Public Health 2021, 18, 13246 8 of 10

nonsmokers [20]. Therefore, recent data support the need for correct information about
health-related risks before the decision of using these products, which are highly prevalent
in the young population [47].
Several studies have observed that smoking initiation is common between 11 and
13 years of age, a time when adolescents may become very easily addicted to nicotine.
Moreover, it has also been reported that some electronic nicotine delivery devices may have
a more aggressive addictive potential compared to conventional cigarettes [49]. The trend
of e-cigarettes was noticed also in Romania, where students use these devices 2.4 times
more frequently than adults [50]. Due to the insufficient data regarding the full spectrum of
risks associated with e-cigarettes, their use should be monitored especially in young people.
Proper information is of utmost importance as preventive strategies need to be supported
by solid scientific evidence, which should be easily accessible and understandable by large
groups of the population.

5. Conclusions
Nicotine containing products have a negative influence on the oral health, which
emphasizes the importance of correct information regarding the side effects of both conven-
tional and e-cigarette use. Clinically healthy young tobacco smokers and e-cigarette users
presented an increased number of MN in the oral epithelial cells, compared to nonsmoking
individuals, suggesting the presence of cytologic changes. The MN count can be used
as an early indicator for susceptibility to alterations of oral mucosa and carcinogenesis.
Exfoliative cytology is a noninvasive, rapid and cost-effective method that could be used
for mass screening.

Author Contributions: Conceptualization, A.M.P., R.C. and M.M.; methodology, A.M.P. and M.M.;
software, A.M.P.; validation, A.M.S. and M.M.; formal analysis, M.M.; investigation, A.M.P., R.C.
and A.M.S.; resources, R.C.; data curation, R.C. and A.M.S.; writing—original draft preparation,
A.M.P. and R.C.; writing—review and editing, A.M.S. and M.M.; visualization, A.M.P. and M.M.;
supervision, M.M.; A.M.P. and R.C. contributed equally to this manuscript. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Ethics Committee of George Emil Palade University of
Medicine, Pharmacy, Science, and Technology of Târgu Mures, (protocol number 1243/14.01.2021).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement: All relevant data presented in this study are contained within the
article.
Acknowledgments: The authors thank Sabin Turdean from the Department of Pathology of the
Clinical County Hospital Mures, for his help in the cytological assessment of the smears and also
Peter Olah from the Department of Medical Informatics and Biostatistics of our university for his
advice regarding the statistical analysis.
Conflicts of Interest: The authors declare no conflict of interest.

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