Objective

To analyze cytologically the buccal mucosa of smoking andnonsmoking volunteers to determine what cellular changesare induced by cigarettes and alcohol consumption.

Study Design

In order to evaluate cellularchanges induced by smokingand alcohol consumption, exfo-liative cytology was used forthe analysis of mucosal smearsobtained from the buccal mu-cosa of 25 smokers and 25nonsmokers. The number ofcigarettes consumed, durationof smoking, presence or absenceof alcohol ingestion, ingestedalcohol dose and frequency ofconsumption, and most frequently used type of alcoholic bev-erage were determined using a questionnaire. Three smearsfrom each individual stained by the Papanicolaou methodwere analyzed quantitatively and qualitatively under alight microscope by 2 experienced examiners in terms of in-flammatory and dysplastic alterations and of the degree ofepithelial maturation.

Results

Although numerous alterations were observed in smokers

they corresponded up to only Papanicolaou class II and were not significantly different from nonsmokers (Mann-Whitney and χ2 tests, p < 0.05). A higher proportion of in-flammatory cells (polymorphonuclear and mononuclearcells) were obtained from smokers as compared to nonsmok-ers, while the proportion of bacteria was similar in the 2

groups.

Conclusion

The findings indicate thateven after a short period ofcigarette use and alcohol con-sumption, inflammatory al-terations were detectable onexfoliative cytology of thebuccal mucosa in a younggroup, demonstrating theusefulness of cytology for early

detection in smokers. (Acta Cytol 2006;50:435–440)

Keywords: tobacco, alcohol consumption, buccalmucosa.

Exfoliative cytology has been studied since the 19thcentury. In 1941, Papanicolaou and Traut1 per-

formed studies on the uterine cervix, and in 1946 Pa-panicolaou2 reported its application to secretions ob-tained from various other parts of the body.

Even with a short period ofcigarette use and alcohol

consumption, inflammatoryalterations were detectable on

exfoliative cytology of the buccalmucosa in a young group....

Cytologic Analysis of Alterations Induced bySmoking and by Alcohol Consumption

Marcella Batista Pavanello, D.D.S., Fernanda Almeida Prado, Ivan Balducci, M.Sc.,D.D.S., Adriana Aigotti Haberbeck Brandão, Ph.D., M.Sc., D.D.S., andJanete Dias Almeida, Ph.D., M.Sc., D.D.S.

From the Department of Social Science and Pediatric Dentistry and of Bioscience and Oral Diagnosis, São José Campos Dental School, SãoPaulo State University, São José dos Campos, São Paulo, Brazil.

Dr. Batista Pavanello is Dentist.

Dr. Almeida Prado is Graduate Student.

Dr. Balducci is Professor, Department of Social Science and Pediatric Dentistry.

Drs. Haberbeck Brandão and Dias Almeida are Professors, Department of Bioscience and Oral Diagnosis.

Supported by Fundacão de Amparo à Pesquisa do estado de São Paulio (process no. 02/07083-4).

Address correspondence to: Janete Dias Almeida, Ph.D., M.Sc., D.D.S., Faculdade de Odontologia de São José dos Campos, Departamento deBiociências e Diagnóstico Bucal, Av. Francisco José Longo, 777, 12245-000, São José dos Campos, São Paulo, Brazil (janete@fosjc.unesp.br).

Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

Received for publication April 8, 2005.

Accepted for publication December 27, 2005.

Nongynecologic Cytopathology

0001-5547/06/5004-0435/$19.00/0 © The International Academy of Cytology ACTA CYTOLOGICA 435

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The first cytologic study of the oral mucosa wasconducted in 1951 by Montgomery.3 In 1953, Pomer-anz and Stahl4 correlated exfoliative cytology andbiopsy and showed that the former can be used as anexamination complementary to biopsy, as later report-ed by several other investigators.5-11 In 1967, Stahl etal,12 in comparing the cytologic and histologic resultson 2,297 specimens obtained from various regions ofthe oral mucosa, concluded that cytology is a comple-mentary examination of great value in the early diag-nosis of oral cancer as well as premalignant lesions.

Exfoliative cytology is a simple and rapid laborato-ry examination based on the scraping of desquamativesuperficial cells of the skin and mucosa and subsequentmicroscopic analysis. The use of cytodiagnosis hasbeen increasing, and the method is currently appliednot only as a complementary examination in the de-tection of preneoplastic lesions and neoplasms but alsoin the diagnosis of autoimmune, viral and fungal dis-eases.6,8,11

The use of exfoliative cytology in the evaluation oftobacco-induced cellular alterations in the oral mu-cosa has been rarely reported in the literature, al-though the mouth is one of the sites most susceptibleto cellular changes, and different pathologies havebeen associated with the use of tobacco.13,14 Since the18th century, oral cancer has been one of the most fre-quent diseases observed among smokers.15

According to the Brazilian National Cancer Insti-tute,16 cigarette use is the most devastating preventa-ble cause of diseases and premature death in the histo-ry of humanity. Tobacco consumption has reachedthe proportion of a global epidemic, causing the deathof millions of people every year worldwide.

About 95% of malignant neoplasms of the mouthoriginate in the lining mucosal epithelium. In malig-nant lesions and some benign processes, the cohesionof epithelial cells becomes fragile, with the cells easilyremoved by scraping. In addition, malignant cells pos-sess characteristic alterations that can be detected bycytologic analysis.17

Since the oral mucosa is one of the sites affected byalcohol and smoking, Hillman and Kissin13 analyzedbuccal and tongue samples from alcoholics and smok-ers in order to find possible cellular alterations andchanges in the microflora and leukocytes of these re-gions and demonstrated that tobacco was able to in-duce cellular changes. Balaéz et al18 compared the oralmucosa of smokers and nonsmokers and observed sig-nificantly greater exfoliation of the palate and buccalmucosa in smokers, accompanied by a large number ofkeratinized cells without a nucleus, with the degree ofkeratinization depending on the duration of the habit.Ogden et al14 analyzed the size of the nucleus and cy-toplasm of cells collected from the normal oral mu-

cosa of individuals smoking cigarettes, cigars, pipes ora combination of the three and compared the resultsto those obtained for nonsmokers. Significant alter-ations were observed only regarding the cell nucleus.Since 1972, Trujillo19 has recommended periodic ex-foliative cytologic examinations in smokers based onevidence of alterations produced by tobacco in epithe-lial cells lining the mouth, pharynx, trachea andbronchi.

The objective of the present study was to analyzecytologically the buccal mucosa of smoking and non-smoking volunteers to determine whether cellularchanges were induced by cigarette use and alcoholconsumption.

Materials and Methods

Fifty students ranging in age from 19 to 27 years (me-dian, 22) underwent exfoliative cytology of the buccalmucosa. The study was approved by the researchethics committee of São José dos Campos DentalSchool.

Each student filled out a questionnaire containinginformation regarding the subject of the study, gener-al health and personal data, and signed an informedconsent form. The number of cigarettes consumed,duration of smoking, presence or absence of alcoholingestion, ingested alcohol dose and frequency of con-sumption, and most frequently used type of alcoholicbeverage were determined by the questionnaire. Thestudents were screened by complete stomatologicclinical examination to select individuals with no visi-ble clinical alterations in the region of the oral mucosaand were divided into 2 groups, a nonsmoking, controlgroup and a smoking group, consisting of 25 studentseach.

The material was collected with a metal spatulafrom the right buccal mucosa (region inferior and an-terior to the parotid duct) with no previous rinsing ofthe mouth. Three smears were prepared on glassslides for each participant. The slides were fixed in a1:1 ether:alcohol solution and stained by the Papani-colaou method. After processing, the slides were ex-amined under a light microscope by 2 experienced ex-aminers, and means were analyzed statistically by thepaired Student t test, with the level of significance setat 5%.

For quantitative evaluation of epithelial maturation(cytologic criteria of maturation), 100 well-distendedand isolated cells randomly selected from among the 3smears from each student were counted.

The criteria proposed by Silva and Rados8 withmodifications were used to evaluate the presence of al-terations suggestive of inflammation, malignancy anddegree of epithelial maturation. The following itemswere considered: anucleated superficial cells, superfi-

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cial cells with a pyknotic nucleus, intermediate cells,keratohyaline granules, irregular staining, vacuolesand lysis.

The data were analyzed statistically using theMann-Whitney and χ2 tests, with the level of signifi-cance set at 5%.

Results

The results of quantitative and qualitative analyses ob-tained by both examiners were compared by thepaired Student t test to verify the evaluation by the ex-aminers. It was verified that the divergences were notstatistically significant.

Analysis of the questionnaires showed that smokersused on average of 239 cigarettes per month, corre-sponding to 8 cigarettes per day in 4 years; 96.15% ofthe smokers used cigarettes and alcohol simultaneous-ly, and 92.30% reported that they used more ciga-rettes when drinking. In the nonsmoking group, 80%

consumed alcohol. Beer was the beverage most citedby smokers (51.21%) and nonsmokers (53.33%) whoingested alcoholic beverages.

Smokers drank alcoholic beverages more frequent-ly (median, 2 or 3 times per month) than nonsmokers(median, ≤ 1 time per month). Distribution values dif-fered statistically by the Mann-Whitney test (p =0.0001).

Smokers consumed more doses of alcoholic bever-ages (median, 3 or 4 doses) than nonsmokers (median,1 or 2 doses). Distribution of numbers of doses dif-fered statistically by the Mann-Whitney test (p =0.0002).

To evaluate distribution values of alterations sug-gestive of inflammation, malignancy and epithelialmaturation in both groups, the Mann-Whitney testwas applied. As shown in Table I, no significant dif-ferences in cellular changes were observed betweenthe 2 groups.

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Table I Cellular Alterations: Descriptive Statistics and Results of the Mann-Whitney Test for Comparison of 25 Smokers and 25 Nonsmokers

Smokers NonsmokersMann-Whitney

Variable Mean SD Median Mean SD Median (p value)

Anucleated superficial cells 2.40 2.75 1.00 2.80 3.07 2.00 0.46

Superficial cell with pyknotic nucleus 8.30 10.50 5.00 8.32 9.16 6.00 0.77

Intermediate cells 89.30 11.10 94.00 89.04 9.21 91.00 0.52

Keratohyaline granules 33.90 14.60 35.00 29.60 13.60 27.00 0.31

Irregular staining 11.24 9.89 9.00 16.7 16.0 11.1 0.073

Vacuole 1.36 2.20 1.00 1.36 1.00 1.66 1.0

Lysis 2.32 2.32 1.00 1.92 2.00 2.25 0.63

α = 5%.

Figure 1 Comparison of the

prevalence of inflammatory cells

(polymorphonuclear and

mononuclear) and bacteria in

smokers vs. nonsmokers (p

value by χ2 test).

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Based on Papanicolaou’s cytologic classificationsystem, the smears were classified as I and II, with nosmear showing dysplastic alterations.

A higher proportion of inflammatory cells (poly-morphonuclear and mononuclear) were obtained forsmokers as compared to nonsmokers, while the pro-portion of bacteria was similar in the 2 groups (Figure1). It was possible to consider, by χ2 test, that the cellproportion between groups was no significantly dif-ferent for polymorphonuclear cells (80% vs. 60%,χ2 = 2.381, df = 1, p = 0.123), for mononuclear cells(52% vs. 28%, χ2 = 3.000, df = 1, p = 0.083) and for bac-teria (84% vs. 84%, χ2 = 0.000, df = 1, p = 1.000).

Discussion

Exfoliative cytology is a simple, rapid and low-costmethod that is very useful as a complementary exami-nation in the diagnosis of alterations that occur in theoral mucosa and in the follow-up of treated areas of

cancer.10,20

The present results were similar to those reportedby Meneguzzi et al,21 who performed oral exfoliativecytology at different sites in smokers and nonsmokersand did not observe significant alterations.

Brown and Young20 reported higher rates of cellmaturation in male smokers older than 60 years ascompared to younger smokers.

The present sample consisted of individuals with amean age of 22 years, with this young age perhaps ex-plaining the low rates of cellular changes observed.

The influence of smoking on the oral mucosa wasobserved as a higher number of keratohyaline granulesand the presence of an intense inflammatory infiltrate,although not statistically significant in the smokinggroup. The most frequent cytologic alterations seen inthe smokers was: presence of keratohyaline granulesand irregular staining, alterations in cell format (Fig-ure 2), binucleation (Figure 2A) and cytoplasmic vac-

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Figure 2 Cytologic aspects of smears obtained from smokers. (A) Binucleation. (B) Increased keratinization, lysis and cytoplasmic

vacuolization. (C) Cytoplasmic border lysis, leukocytes and filamentous mucus. (D) neutrophils and lymphocytes surrounding and destroying an

epithelial cell. Note the increased keratinization (Papanicolaou stain, × 630).

A B

C D

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uolization (Figure 2B).The frequency and duration of use of tobacco and

alcohol are important factors in the interpretation ofthe data obtained in this study.

Smokers consumed on average of 8 cigarettes perday in 4 years. According to the questionnaire, almost100% of smokers used alcohol at least once a week,and the majority reported that they consumed morecigarettes when drinking.

Silva and Rados,8 performing exfoliative cytologyon 50 male nonsmokers and 50 male smokers with aminimum smoking habit duration of 10 years, ob-served a larger number of inflammatory alterationsand an increase in the velocity of cell maturation insmokers.

The results of a cytologic study conducted by Sam-paio et al22 on the normal oral mucosa of smokersusing the AgNOR method suggested that cigaretteshave an influence on the proliferative activity of cells.

Silva et al,23 analyzing the cellular changes thatoccur in the labial semimucosa of smokers and pa-tients exposed to solar radiation, observed that thecombination of smoking and solar exposure causescellular alterations and that the persistence of insultsmight induce cancerous disorders and cancer of thelower lip.

Silva and Rados,8 in studying the influence of smok-ing on the oral mucosa, also observed inflammatoryalterations and changes in the cell maturation processof the oral mucosa, once again demonstrating that to-bacco alters the cytologic pattern of the oral mucosa.These alterations, even in the absence of clinical man-ifestations, might be understood as a modification inthe epithelium to withstand the physicochemical dam-age provoked by smoke.

According to McCance and Huether,24 the inci-dence of oral, laryngeal, esophageal and liver cancer ishigher in individuals simultaneously consuming to-bacco and alcohol. This finding can be explained bythe fact that alcohol promotes carcinogenesis becauseit acts as a solvent for the carcinogenic products pres-ent in cigarettes. The frequency of cigarette con-sumption and its association with alcohol for pro-longed periods of time increase by > 15 times the riskof contracting oral cancer.25 Alcohol increases thepenetration of carcinogenic agents into the oral mu-cosa by increasing mucosal permeability and by in-creasing the solubility of carcinogenic substances thatcome in contact with the mucosa.26

Hilman and Kissin,13 subjecting smokers and alco-holics to exfoliative cytology of the buccal and tonguemucosa, observed alterations in the size of the cell nu-cleus, numerous keratinized cells, a low concentrationof bacteria and the presence of filamentous fungi andleukocytes. These alterations were even more evident

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when smoking was associated with alcohol.The present study showed the importance of estab-

lishing inclusion criteria related to age, time of con-sumption and association with other habits and exclu-sion criteria for future studies applying exfoliativecytology to the smoking population in general.

Our findings indicate that even with a short periodof cigarette use and alcohol consumption, inflamma-tory alterations were detectable on exfoliative cytol-ogy of the buccal mucosa in a young group, demon-strating the usefulness of cytology for early detectionin smokers.

References

1. Papanicolaou GN, Traut HF: The diagnostic value of vaginalsmears in carcinoma of the uterus. Am J Obstet Gynecol 1941;42:193–206

2. Papanicolaou GN: Diagnostic value of exfoliative cells fromcancerous tissues. JAMA 1946;131:372–378

3. Montgomery PW: A study of exfoliative cytology of normalhuman oral mucosa. J Dental Res 1951;30:12–18

4. Pomeranz MJ, Stahl SS: A correlative study of cytodiagnosisand biopsy. Oral Surg Oral Med Oral Pathol 1953;6:1026–1031

5. Almeida JD, Cabral LAG: Diagnóstico do carcinoma bucal:Uso da citologia esfoliativa como método auxiliar. Rev GaúchaOdontol 1992; 40:167–170

6. Marcucco G, Araújo NS: Citologia esfoliativa. In Câncer Bucal.Edited by AF Tommasi, V Garrafa. São Paulo, Medisa 1980, pp395–407

7. Martinelli C, Castro AL, Pinto RS: Correlação Histocitológicade lesões da cavidade bucal. Ars Cvr Odontol 1979;6:29–32

8. Silva MCA, Rados PV: Citopatologia: Um recurso auxiliar naprevenção de câncer bucal em pacientes do sexo masculino. RevFac Odontol Porto Alegre 1997;32:3–10

9. Tommasi AF: Diagnóstico em patologia bucal. São Paulo, ArtesMédicas, 1982, pp 41–45

10. Almeida JD, Cabral LAG, Brandão AAH: Exfoliative cytologyas a diagnostic method in estomatology. J Dent Res 1994;73:765

11. Almeida JD, Cabral LAG: Citologia esfoliativa. In DiagnósticoBucal. Edited by S Kignel. São Paulo, Robe Editorial, 1997, pp41–49

12. Stahl SS, Koss LG, Brown RC Jr, Murray D: Oral cytologicscreening in a large metropolitan area. JADA 1967;75:1385–1388

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14. Ogden GR, Cowpe JG, Green MW: Quantitative exfoliativecytology of normal buccal mucosa: Effect of smoking. J OralPathol Med 1990;19:53–55

15. Silverman S Jr, Shillitoe EJ: Oral Cancer. Hamilton, BC Deck-er, 1998, pp 7–24

16. Ministério da Saúde, Instituto Nacional de Câncer, Coorde-nação Nacional de Controle de Tabagismo e PrevençãoPrimária: Falando sobre tabagismo, 1996, Rio de Janeiro,CONTAPP, 1996

17. Folsom TC, White CP, Bromer L, Canby HF, Garrington GE:

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Oral exfoliative study: Review of literature and report of a threeyear study. Oral Surg 1972;33:61–74

18. Baláez AB, Diaz EM, Peres HR: Modificaciones de los indicesde exfoliacion celular en la mucosa del paladar y carrillo de fu-madores de tabacos (puros). Rev Cub Estomatol 1989;23:177–18

19. Trujillo FR: Citologia esfoliativa en estomatología: comentarioal trabajo del Dr. Valenzuela Quezada Titulado. Leido a ser in-gresso a la Academia Nacional de México, 1972, pp 126–140

20. Brown AM, Young A: The effects of age and smoking on thematuration of oral mucosa. Acta Cytol 1970;14:566–569

21. Meneguzzi R, Braga FL, Paiva R, Rados PV: Avaliação histopa-tológica da mucosa bucal de fumantes e não fumantes. PesquiOdontol Bras 2002;16:59

22. Sampaio H de C, Braga FL, Paiva R, Rados PV: AgNOR countin exfoliative cytology of normal buccal mucosa: Effect ofsmoking. Acta Cytol 1999; 43:117–120

23. Silva JBP, Sobrinho JA, Boraks S, Galvão MAL, Rapoport A:Alterações citológicas da semi-mucosa do lábio inferior em pa-cientes expostos às radiações solares e o uso do fumo. Rev BrasOtorrinolaringol 2000;66:494–498

24. McCance KL, Huether SE: Pathophysiology. St Louis, Mosby,1998, pp 304–349

25. Neville BW, Damm DD, Allen CM, Bouquot JE: Oral andMaxillofacial Pathology. Philadelphia, WB Saunders, 1995

26. Ogden GR, Wight AJ, Cowpe JG: Quantitative oral exfoliativecytology: Effect of alcohol on normal buccal mucosa. AnalQuant Cytol Histol 1999;21:126–130

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