Archives of Oral Biology 126 (2021) 105127

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Archives of Oral Biology

journal homepage: www.elsevier.com/locate/archoralbio

Oral cytological changes in young adults related to alcohol consumption

Merlyn dos Santos Maidana a, Antônio Sergio Varela Junior b, Carine Dahl Corcini c,
Jessica Ribeiro Pereira b, Diego Martins Pires c, Ronan Adler Tavella a, b,
Caroline Lopes Feijo Fernandes a, b, Marina dos Santos a, b, Edariane Menestrino Garcia a, d,
Flavio Manoel Rodrigues da Silva Júnior a, b, d, *
a
Programa de Pós graduação em Ciências da Saúde, Faculdade de Medicina, Universidade Federal do Rio Grande, Rio Grande, RS, Brazil
b
Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, Rio Grande, RS, Brazil
c
Faculdade de Veterinária, Universidade Federal de Pelotas, Pelotas, RS, Brazil
d
Centro Regional de Estudos, Prevenção e Recuperação de Dependentes Químicos - CENPRE Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, Rio
Grande, RS, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Objective: The study aimed to investigate the oral cytological changes in young adults with recent history of
Flow cytometry alcohol consumption, as well as its relation with the consumption of alcohol.
Micronucleus test Design: The sample included 67 young adults, who performed a smear of the oral mucosa and answered a
Oral mucosa
questionnaire about recent and lifetime consumption of alcohol and other drugs. The methods used were sen­
Illicit drugs
Ethanol
sitive to show the association between alcohol consumption and other drugs with the damage to oral cavity cells.
Results: DNA fragmentation index, mitochondrial functionality and cell viability, showed a significant difference
between alcohol users and nonusers. However, there was no distinction between these parameters and different
frequency consumption. Alcohol consumption, economic class and use of illicit drugs were related to the cyto­
logical parameters evaluated.
Conclusions: This result shows the existence of cell damages among the evaluated students and can direct future
studies towards more in-depth investigations of the mechanisms involved.

1. Introduction been associated with increased risk of pre-neoplastic lesions including

The use of alcohol, tobacco and other drugs is common worldwide
and are related to a series of health problems (UNODC, 2017). Alcohol
consumption may affect negatively liver (Maddrey, 2000), cardiovas­
cular (Gardner & Mouton, 2015; Whitman et al., 2019) and endocrine
system (Rachdaoui & Sarkar, 2017). Alcohol-related health problems
also include metabolic syndrome (Åberg et al., 2018), behavioral
problems (Ellickson et al., 2003) and different types of cancer (The
Lancet, 2017).
The relationship between drinking alcohol and cancer risk has been
evaluated extensively in epidemiologic studies. The IARC classified
alcohol as a group 1 carcinogen. Approximately, 5.8 % of all cancer
deaths worldwide were estimated to be attributable to alcohol (LoConte
et al., 2018). Several pathogenic mechanisms contribute to
alcohol-induced carcinogenesis (Varela-Rey et al., 2012). Cancer is
related to accumulation of mutations and mutagenicity biomarkers have
oral cavity lesions (Bremmer et al., 2008).
The application of cytological techniques for evaluation of oral
health is extremely broad (Alberti et al., 2003; Busamia et al., 2018).
However, the use of oral cytological and cytogenetic techniques to
investigate damages from drugs and other chemical substances is
limited. Abdelaziz and Osman (Abdelaziz & Osman, 2011) found that
alcohol and tobacco consumption is a risk factor for oral atypical cellular
changes and possibly of oral infection. Among the parameters evaluated
by them there were presence of bacteria and fungi, disturbed chromatin
distribution, alteration in nuclear size, increased nuclear DNA content,
prominent nuclei, multinucleation, abnormal nuclear line, hyperkera­
tosis and cytoplasmic vacuolations.
Genetic markers are efficient methods for detecting precancerous
lesions, indicating the time of progression (Bremmer et al., 2008) and
the micronucleus frequency, which has been related to alcohol, tobacco
and crack use (Wagh et al., 2019; Webber et al., 2016). However, the

* Corresponding author at: Programa de Pós graduação em Ciências da Saúde, Faculdade de Medicina, Universidade Federal do Rio Grande, Rio Grande, RS, Brazil.
E-mail address: f.m.r.silvajunior@gmail.com (F.M.R. da Silva Júnior).

https://doi.org/10.1016/j.archoralbio.2021.105127
Received 28 February 2021; Received in revised form 30 March 2021; Accepted 14 April 2021
Available online 16 April 2021
0003-9969/© 2021 Elsevier Ltd. This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/).
M. dos Santos Maidana et al.
majority of studies have been performed with heavy users of substances.
According to American Society of Clinical Oncology (ASCO) the greatest
cancer risks are concentrated in the heavy and moderate drinker cate­
gories. Nevertheless, some cancer risk persists even at low levels of
consumption (LoConte et al., 2018).
This study aimed to evaluate oral cytological changes in young adults
with recent history of alcohol consumption, as well as, its relation to
alcohol consumption.
2. Material and methods
2.1. Study population and data collection
The study included 67 young adults, who were students of Medicine,
Nursing and Biological Sciences courses of the Federal University of Rio
Grande, Rio Grande, Brazil, during the year of 2016. Participants were
randomly chosen. Those who accepted and signed the informed consent
form were asked to answer a semi-structured questionnaire about
alcohol consumption and other psychoactive substances, as well as,
socioeconomic and demographic questions and health outcomes.
The students were divided in three groups based on self-report of
alcohol consumption: no recent use (n = 23), recent use (n = 20) and
frequent use (n = 24). Subjects who reported not consuming alcohol in
the last 30 days were classified as “no recent use”; subjects who reported
having consumed alcohol only once in the last 30 days, but did not
consume in the last seven days, were classified as “recent use”; and
subjects who reported having consumed alcohol at least once a week in
the last 30 days were classified as “frequent use”.
Smears from buccal cells were performed in all subjects using a
Cytobrush® cells collectors after hygiene with alcohol-free mouthwash.
The material was immediately refrigerated and transported for cyto­
logical and cytogenetic analysis.
2.2. Cytological measurements by flow cytometry
The Attune Acoustic Focusing® equipment (Life Technologies) was
used for flow cytometric analysis using the blue (Argon 488 nm) and
violet (UV 405 nm) lasers. The latter was used for the analysis of the
following cellular structures: DNA Fragmentation Index (DFI), cellular
viability, reactive oxygen species (ROS), fluorescence intensity of the
fluidity, and mitochondrial. The results were obtained using the soft­
ware version 2.1 (Life Technologies). The Hoechst 33,342 dye at 16.2
mM was used for all flow cytometric analysis except for DNA fragmen­
tation. The cells were stained with fluorophores that had been added to
calcium-free PBS and a total of 20,000 cells were counted per analysis
(excluding debris).
To verify the percentage of cellular viability, the cells that were
Propidium Iodide (PI) negative were classified as viable where as those
that were PI positive were regarded as ruptured.
The chloromethyl 2′ ,7′ -dichlorodihydrofluorescein diacetate (CM-
H2DCFDA) probe (Invitrogen, Spain), a derivative of fluorescein, was
used for detection of reactive oxygen species (ROS). This fluorescence
probe was combined with PI (Sigma-Aldrich, USA). Only intact cells (PI
negative) were selected and classified. ROS production was obtained by
the median of the green fluorescence intensity.
The DNA fragmentation index was assessed by the chromatin
structure assay. In this assay 10 μL of suspension of collected oral cells
were added to 5 μL of TNE (0.01 M Tris− HCl, 0.15 M NaCl, 0.001 M
EDTA, pH 7.2) and 10 μLof 1X Triton (Triton X–100, 1%) (V/V) at 30 s
intervals. Acridine orange dye was added to the solution followed by a
short incubation period of between 30 s to 2 min after which the results
were analyzed. Oral cells with fragmented DNA presented with red
fluorescence, while those with intact DNA exhibited green fluorescence.
The DNA fragmentation rate index (DFI%) was calculated as follows:
(red fluorescence)/[total fluorescence intensity (red + green)].
The Mitochondrial membrane potential was verified using 10 μL of
2
Archives of Oral Biology 126 (2021) 105127
sample incubated with 3.1 μM Rhodamine 123 (green fluorescence) and
7.5 μM IP for 10 min. Only viable cells (PI negative) were selected and
then were analyzed for fluorescence median intensity by Rhodamine
accumulation. The mitochondrial functionality rate was calculated by
the following formula: [(number of oral cells with high mitochondrial
membrane potential)/(high oral cells count potential of mitochondrial
membrane + oral cells with low mitochondrial membrane potential)] ×
100
The intensity of membrane fluidity (membrane disorder) was per­
formed by incubation 10 μL of sample in 2.7 μM of hydrophobic mer­
ocyanin 540 (M540) dye and 0.1 μM of YO PRO–1 (Invitrogen-Eugene,
OR, USA) for 10 min. Intensity of membrane fluidity by concentration of
M540 cells were evaluated only for the presence of intact oral cells (YO-
PRO negative). The membrane flow rate was calculated using the
following formula: [(number of oral cells with high fluidity)/(number of
oral cells with high fluidity) + (oral cells with low fluidity)] × 100.
2.3. Micronucleus test
Mutagenicity was evaluated via the micronucleus test of the oral
mucosa. It was performed according to Pinto et al. (2017). Smears from
exfoliated cells of the oral mucosa were applied on slides and these were
fixated with absolute methanol and stained with Eosin methylene blue.
2.4. Data analysis
The descriptive analysis was performed using Fisher’s exact test or
the Chi-square test considering a critical p < 0.05. The means of cyto­
logical parameters were compared using one-way analysis of variance,
followed by Tukey’s posterior test, considering a critical p value <0.05
Associated factors to cell damage were evaluated through Poisson
regression analysis (bi- and multivariate). The dependent variable ” cell
damage " was based on the three cytological parameters (DNA frag­
mentation, mitochondrial functionality and cell viability) that were
sensitive to alcohol consumption (significant difference). The subjects
were classified as “cell damage” group when the average DNA frag­
mentation value was above the median of the studied population and the
average value of mitochondrial functionality and cell viability was
below the median of the population. In this way, only subjects who had
damage in all three parameters were classified in this group. The
multivariate Poisson regression analysis was performed based on the
construction of a hierarchical model between the independent variables.
The first level included socioeconomic and demographic variables: age,
sex (male/female), skin color (white/not white), income and economic
class (higher/average: A + B+C and bottom: D + E). The second level
included habits such as daily coffee consumption (yes/no), recent to­
bacco use (yes/no) and illicit drugs (yes/no). The third level included
health conditions: chronic non-communicable diseases (yes/no) and
continuous-use medication (yes/no). The variables with p < 0.2
remained in the model, as it is considered that they can influence the
other variables in the model, but the significant critical p was considered
p < 0.05.
The sample power was calculated by comparing the means and
standard deviations of each parameter in the groups (Not use, Recent use
and Frequent use), considering a level of significance of 5% in the
ANOVA model. The calculations were made using the website
http://powerandsamplesize.com/ and the highest power values were
presented in the comparison between the groups.
3. Ethical aspects
The study respected the ethical principles of Resolution 466/12 of
the National Health Council of the Ministry of Health and was approved
by the Research Ethics Committee in the Health Area of the Federal
University of Rio Grande (CEPAS / FURG), reference number 195/2015.
M. dos Santos Maidana et al. Archives of Oral Biology 126 (2021) 105127

4. Results fragmentation index and lower mitochondrial functionality and cell

viability. However, there was no distinction between these parameters
The majority of students were female (64 %), self-reported white skin between recent users and frequent users (Fig. 1a–e).
color (70 %) and were single (95 %). The mean age was 22 (18− 48) In Poisson regression (bi- and multivariate analysis) only economic
years old. Most students were economic class C and average family in­ class and recent use of illicit drugs were significant. The lower economic
come per month was R$ 6,300 (equivalent to US $ 1,500). The variables classes (D and E) had a prevalence ratio (RR) 4.6 (IC 95 % 1.5–14.1) for
that showed a significant difference between the 3 groups were sex, risk of cell damage, while the recent nonuse of illicit drugs has an RR of
marital status, economic class, recent cigarette use and chronic non- 0.07 (IC 95 % 0.02− 0.21), representing an association to cell damage
communicable disease. Male sex had higher prevalence among recent (Table 2).
or frequent use of alcohol, while female sex was predominantly
composed by no recent users. All frequent alcohol consumers were 5. Discussion
single. Students from economic classes C had higher prevalence of
frequent use. Moreover, participants with a higher frequency of alcohol As proposed, through our study it was possible to observe oral
use have more cigarette users (Table 1). cytological changes in young adults with a recent history of alcohol
For the analysis of the different biomarkers evaluated, the powers of consumption. Among the main findings are the negative outcomes
the samples were 0.99 (DNA fragmentation), 0.93 (membrane fluidity), observed in the cytological parameters of alcohol users, with higher
0.99 (mitochondrial functionality), 0.66 (micronuclei), 0.99 (cell DNA fragmentation index, lower mitochondrial functionality and
viability) and 0.07 (ROS intensity). Among the six cytological parame­ decreased cell viability. Furthermore, among alcohol users it was
ters evaluated in the present study, three (DNA fragmentation index, possible to ascertain that lower economic classes and the use of drugs
mitochondrial functionality and cell viability) showed a significant had a significant relationship with the analyzed outcome (cell damage,
difference between alcohol users and nonusers. Students who reported based on parameters defined in this study).
recent and frequent use of alcohol had significant higher DNA Alcohol consumption is related to age, gender, health status, eco­
nomic conditions, among others (World Health Organization, 2018).
The study population was composed by young adults, a group vulner­
Table 1 able to the effects of alcohol on health. In this study, as observed
Socioeconomic and demographic characteristics of students according to alcohol worldwide, female sex are more often abstainers than male (World
consumption. Health Organization, 2018). Moreover, as observed by other studies, a
Variable Total No Recent Frequent p value high prevalence of alcohol use was observed among the upper and
(67) recent use use middle economic classes, as they may have greater commercial avail­
use (20) (24)
ability of alcohol, which can further normalize alcohol use (PAHO,
(23)
2020).
Age 22 22 22 22 >0.05 According to the World Health Organization (2018), there is a
(18− 48) (18− 37) (18− 48) (18− 40)
Sex 0.001
relation between alcohol use and cancer development. Alcohol has been
Male 24 (36) 5 (22) 8 (40) 11 (46) shown to damage permanently the DNA strands in cells, and to inhibit
Female 43 (64) 18 (78) 12 (60) 13 (54) DNA repair processes. This substance can affect proliferative cells
Skin color >0.05 through intracellular and intercellular pathways (Ogden, 2005).
White 47 (70) 17 (74) 14 (70) 16 (67)
Different studies have linked alcohol consumption to cytological ab­
Non-white 20 (30) 6 (26) 6 (30) 8 (33)
Marital status 0.01 normalities such as increased nuclear area and mitotic figures,
Single 64 (95) 21 (91) 19 (95) 24 (100) dysplastic changes with keratosis, decreased basal cell size and cyto­
Married 3 (5) 2 (9) 1 (5) 0 (0) plasmic area (Feng & Wang, 2013; Maier et al., 1994). Corroborating
Economic Class* <0.001 with our findings, alcohol consumption has already been associated with
A-B 12 (18) 3 (13) 6 (30) 3 (13)
C 51 (76) 19 (83) 12 (60) 20 (83)
the presence of polyploid DNA and an increase in the frequency of
D-E 4 (6) 1 (4) 2 (10) 1 (4) micronuclei (Maier et al., 1994; Ogden, 2005).
Income 6,304 5,507 6,100 7,239 >0.05 The relationship between oral health damage and use of alcohol and
(400- (400- (400- (600- other drugs is well established (Alsarraf et al., 2018; Johnson, 2001; van
30,000) 20,000) 30,000) 30,000)
Zyl, 2014). Lesions that precede cancer could be intraepithelial lesion,
Daily coffee >0.05
consumption low- and high-grade squamous, among others (Pandey et al., 2018).
Yes 45 (67) 14 (61) 13 (65) 18 (75) Thus, markers of integrity, functionality and cell viability, as well as
No 22 (33) 9 (39) 7 (35) 6 (25) markers of genetic damage, such as DNA index, performed through
Recent cigarette <0.0001 cytopathology, and used in our research, are highly correlated with
use
Yes 11 (16) 1 (4) 2 (10) 8 (33)
carcinogenicity processes and tumor progression stage (Bremmer et al.,
No 56 (84) 22 (96) 18 (90) 16 (67) 2008; Pandey et al., 2018).
Recent use of >0.05 Levels of alcohol consumption can be measured using several in­
illicit drugs dicators. The prevalence (or the number) of current drinkers or ab­
Yes 12 (18) 3 (13) 3 (15) 6 (25)
stainers, is defined by WHO as the percentage of those in the population
No 55 (82) 20 (87) 17 (85) 18 (75)
Chronic Non- <0.0001 aged 15 years and older who have consumed alcoholic beverages in the
Communicable previous 12-month period (World Health Organization, 2018). In our
Disease study, alcohol use was assessed in a period of 30 days to detect a more
Yes 11 (16) 3 (13) 6 (30) 2 (8) recent consumption pattern, mainly because the magnitude of
No 56 (84) 20 (87) 14 (70) 22 (92)
Continuous 0.03
alcohol-induced changes and damages are correlated with the amount
medications and time of alcohol consumption (Tapia-Rojas et al., 2017).
Yes 8 (12) 4 (17) 1 (5) 3 (13) It is difficult to define what is light, moderate, or heavy alcohol
No 59 (88) 19 (83) 19 (95) 21 (87) consumption. According to the Report on Alcohol and Health of Pan
All variables were present in absolute frequency = n (relative frequency (%), American Health Organization (PAHO, 2020) in 2016, 46.1 % of the
excepted age and income present in median (min-max). * The descending order adult population drank in the past year, however, the rates of heavy
of Economy is A, B, C, D and E. episodic drinking among drinkers increased by 12 %. Binge-drinking

3
M. dos Santos Maidana et al. Archives of Oral Biology 126 (2021) 105127

Fig. 1. Oral cytological parameters of students according to alcohol consumption. The bars represent means and error bars refer to the standard error. * means
significant difference (p < 0.05) in relation to the control group (without alcohol use). Biomarkers evaluated: (a) DNA fragmentation; (b) Membrane fluidity; (c)
Mitochondrial functionality; (d) Micronucleus; (e) Cell viability and (f) ROS intensity.

habit, drinking five or more alcoholic beverages, during consecutive
days followed by nondrinking days, intermittently tend to increase with
age (Merrill & Carey, 2016), reaching values of 10.8 % between 18 and
25 years old (Gass et al., 2014), which is a health concern between
young population such as students. Nevertheless, in our study it was not
observed differences in cytological damage and mutagenicity between
different frequencies of alcohol consumption (recent and frequent use).
Alcohol associated or not with other drugs is a known risk factor for
the development of oral and pharyngeal cancer and deficiencies in oral
health (Fioretti et al., 1999; Griswold et al., 2018; Khairnar et al., 2017;
Ogden, 2005). Our study showed that the recent use of illicit drugs was
associated with the development of cell damage among young adults
who consumed alcohol with certain frequency. Some malignancies are
causally linked to both alcohol drinking and cigarette smoking (LoConte

4
M. dos Santos Maidana et al. Archives of Oral Biology 126 (2021) 105127

Table 2 factor to cell damage. It is possible to confirm that there were temporary
Associated factors to cell damage. or permanent cell damages in the studied population. Thus, future
Level Variables Crude p Adjusted p value studies were encouraged to deeply evaluate alcohol consumption
analysis value analysis pattern, as well as, other illicit drugs that may influence on cell damage,
(IC 95) (IC 95 %) in order to direct future public policies for reducing the use of these
0.97 0.95 substances among students and prevent future public health problems.
Age 0.49 0.13
(0.90− 1.05) (0.89− 1.01)
Sex 0.97 0.92
Funding
1.02 1.06
Male
(0.33− 3.1) (0.34− 3.29)
Female 1 1 This study was financed in part by the Coordenação de Aperfeiçoa­
Skin color 0.84 0.68 mento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001,
1.13 1.25 and Conselho Nacional de Desenvolvimento Científico e Tecnológico.
1 White
(0.83− 3.84) (0.41− 3.86)
Non-White 1 1
Economic class 0.03 0.008 Declaration of Competing Interest
Higher/Average 1 1
3.5 4.6
Lower The authors declare that they have no known competing financial
(1.1− 13.07) (1.5− 14.1)
1.00 1.00 interests or personal relationships that could have appeared to influence
Income 0.02 0.07
(1.00− 1.00) (1.00− 1.00) the work reported in this paper.
Daily coffee
0.78 0.83
consumption
Yes 1 1 Acknowledgements
1.2 1.1
No
(0.38− 3.6) (0.47− 2.58) We would like to thank the Coordenação de Aperfeiçoamento de
Recent cigarette
0.86 0.06 Pessoal do Ensino Superior (CAPES, Brasilia, DF, Brazil) by post grad­
use
2 Yes 1 1 uate scholarships awarded to Merlyn dos Santos Maidana, Caroline
0.88 2.9 Lopes Feijo Fernandes, Ronan Adler Tavella, Jessica Ribeiro Pereira. A.
No
(0.22− 3.54) (0.95− 9.4) S. Varela Junior, C.D. Corcini and F. M.R Da Silva Júnior are research
Recent use of fellow from CNPq (310327/2018-0, 310203/2018-0 and 310856/2020-
0.001 <0.001
illicit drugs
Yes 1 1
5, respectively).
0.18 0.07
No
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