J Periodontol • May 2005

737

* Department of Prosthodontics, Baskent University, Faculty of Dentistry, Ankara, Turkey.† Department of Oral and Maxillofacial Surgery, Baskent University, Faculty of Dentistry.‡ Department of Periodontology, Baskent University, Faculty of Dentistry.§ Department of Biochemistry, Baskent University, Faculty of Medicine.� Department of Pediatric Oncology, Hacettepe University, Faculty of Medicine, Ankara,

Turkey.

Apoptosis or programmed celldeath was first introduced in 1972by Kerr et al.1 as an active bio-

energy saving cell-elimination mechanismby which aged, unwanted, or sublethaldamaged cells are abolished and theircontents with precious caloric value areutilized again by macrophages or byphagocytosing adjacent cells.2 Apoptosisis believed to account for numerousphysiological and pathologic events.3

Nuclear matrix proteins (NMP) makeup the internal structural framework ofthe nucleus4 and they are associated withfunctions as DNA replication,5 RNA syn-thesis,4 and hormone receptor binding.6

NMPs are released from dead and dyingcells and can be used as a marker todetect cell death. Although the nuclearmatrix has been shown to be highlyinsoluble in vitro, it is now known thatcell death releases soluble nuclear matrixproteins that can be detected in culturesupernatant and other fluids containingdead and dying cells.7

Tobacco smoking is an important riskfactor for precancerous lesions like leuko-plakia and oral cancers.8 Epithelialhyperplasia, increase in thickness of theoverlying orthokeratin and moderate lev-els of acanthosis are believed to be themajor causes of these pathologies.9,10

The decreased epithelial apoptosismay contribute to the epithelial hyper-plasia developing in response to cigarettesmoking. Detection of apoptotic abnor-malities before the consequences becomeclinically or histologically detectable willgreatly enhance the potential for earlydiagnosis of diseases such as cancer.Saliva is a readily obtained body fluidthat contains exfoliated epithelial cellsfrom the mucosal lining of the mouth

Cigarette Smoking and ApoptosisP. Imirzalioglu,* S. Uckan,† E.E. Alaaddinoglu,‡ A. Haberal,§ and D. Uckan�

Background: Epithelial cell hyperplasia and significant increasein thickness of the overlying orthokeratin layer are characteristicfindings noted in the oral cavity of subjects who smoke. Increasedproliferation of epithelial cells or defective apoptosis may play arole in the development of epithelial hyperplasia. Thus we ana-lyzed soluble Fas and nuclear matrix protein (NMP) levels in thesaliva of smokers (N = 13) and non-smokers (N = 14) to assessapoptosis.

Methods: Ten ml of unstimulated saliva samples was obtainedfrom 14 non-smoker and 13 smoker subjects with the spittingmethod. These samples were analyzed by using an immuno-assay kit to detect soluble human APO-1/Fas and cell deathdetection enzyme-linked immunosorbent assay (ELISA) kit basedon nuclear matrix protein 41/7 qualification.

Results: The mean soluble Fas levels were 153.8 ± 290 pg/mland 315.4 ± 490 pg/ml and NMP levels were 21.81 ± 10.70 U/mland 30.31 ± 19.86 U/ml, respectively, in smokers and non-smokers. The difference between NMP levels of smoker andnon-smoker groups was statistically significant (P = 0.05).

Conclusion: The results of the present study suggest thatsmoking may induce anti-apoptotic mechanism in the oral cav-ity. J Periodontol 2005;76:737-739.

KEY WORDSApoptosis; saliva/analysis; smoking/adverse effects.

40149.qxd 5/6/05 3:07 PM Page 737

738

Cigarette Smoking and Apoptosis Volume 76 • Number 5

and throat which could be used to diagnose oralmalignancies.11

The aim of the present study was to speculate onthe possible role of dysregulated apoptosis of epithelialcells in the pathogenesis of smoking related oralchanges.

MATERIALS AND METHODSStudy PopulationThe study population consisted of 27 systemicallyhealthy patients diagnosed with chronic gingivitis. Thenature and the purpose of the study were briefly out-lined to each subject. The subjects were divided intotwo groups according to their smoking habits. Personswho had never smoked were considered non-smokers(N = 14; eight women, six men; mean age 24.4 ± 2.2years). Subjects smoking one or more packs ofcigarettes daily for at least 2 years before the studywere defined as smokers (N = 13; 10 women, threemen; mean age 23.6 ± 1.2 years).

Clinical EvaluationOne examiner performed the oral examination usinga manual North Carolina probe. Gingival and plaqueindices and probing depths were recorded for eachsubject (Table 1).12,13 Patients with significant orallesions were excluded from the study.

Saliva Collection and ApoptosisThe subjects were instructed to sit upright, lean slightlyforward, tilt their jaw down, keep their eyes open, andclear their mouth of saliva just before the beginning ofcollection. Patients were then instructed to allow salivato accumulate in the floor of the mouth and to expec-torate 10 ml of the accumulated saliva into a graduatedtest tube as described by Navazesh and Christensen.14

Patients were asked not to smoke, brush or floss theirteeth, eat, or drink for 1 hour prior to collection. Col-lected saliva samples were immediately centrifuged(2,000 rpm for 10 minutes) and the supernatants werestored at −20°C until assay.

An immunoassay kit¶ was used to detect solublehuman APO-1/Fas and an ELISA kit# based on nuclearmatrix protein 41/7 qualification was used for celldeath. Methods were followed according to the manu-facturer’s instructions.

Briefly, diluted saliva samples were added on thewells coated with antibody specific for hAPO-1/Fas.Then samples were incubated with biotinylated secondantibody followed by streptavidin-peroxidase incubationand the intensity of colored product was measured.

The levels of NMP released from dead and dyingcells was determined as a measure of apoptosis in thesaliva by cell death detection ELISA kit that is a sand-wich enzyme immunoassay employing mouse mono-clonal antibodies for measurement of soluble NMP. A

mouse monoclonal antibody specific for human NMP41/7 is used. One hundred µl of samples were addedon the wells and incubated at room temperature.Detector antibody incubation was followed by incuba-tion with HRP-SAD reagent substrate. After adding stopsolution, the samples were read at 450/595 nm wave-length. The mean absorbance values for sFas and NMPwere calculated.

The Student t test was used to determine thedifferences in apoptosis between the smoker and thenon-smoker groups. A P value <0.05 was consideredstatistically significant.

RESULTSThe mean soluble Fas levels were 153.8 ± 290 pg/mland 315.4 ± 490 pg/ml, respectively, in smokers andnon-smokers. NMP levels were 21.81 ± 10.7 U/ml forsmokers and 30.31 ± 19.86 U/ml for non-smokers (P =0.05) (Table 2).

The mean clinical indices were not significantly dif-ferent for the two groups (Table 1).

DISCUSSIONApoptosis is an active bioenergy-saving cell elimina-tion mechanism by which aged, injured, or sub-lethallydamaged cells are destroyed and their valuable con-tents re-used by macrophages or by adjacent cells that

Table 1.

Clinical Characteristics of Smokers andNon-Smokers

Probing GingivalGroup N Depth (mm) Index Plaque Index

Smoker 13 2.56 ± 0.69 1.2 ± 0.23 1.8 ± 0.49

Non-smoker 14 2.37 ± 0.21 1.6 ± 0.44 1.2 ± 0.31

Table 2.

Apoptotic Epithelial Cells in Smokers andNon-Smokers

Group N Mean ± SD P

Apo/Fas (pg/ml) Smoker 13 153.8 ± 290 0.156Non-smoker 14 315.4 ± 490

NMP U/ml Smoker 13 21.81 ± 10.70 0.050Non-smoker 14 30.31 ± 19.86

¶ Biosource International, Nivelles, Belgium.# Oncogene, San Diego, CA.

40149.qxd 5/6/05 3:07 PM Page 738

739

J Periodontol • May 2005 Imirzalioglu, Uckan, Alaaddinoglu, Haberal, Uckan

phagocytose them.1,2 Fas is an apoptosis inducingmolecule that requires interaction with its ligand Fasligand (FasL) or an antibody in order to trigger celldeath. Fas and FasL are typical members of the tumornecrosis factor (TNF) receptor and TNF ligand fam-ily, respectively, with a pivotal role in the regulation ofapoptotic processes including activation-inducedimmune privilege and tumor surveillance. Elevatedlevels of Fas expression on cells indicate increasedsensitivity for apoptosis.15 In contrast, a soluble iso-form (sFas) derived by alternative splicing from thesame mRNA as a membrane form seems to play animportant role in signaling, modifies ligand concen-tration, downregulates membrane receptor numbersand specifically inhibits ligand-receptor association inthe extracellular space, thus preventing the inductionof apoptosis.16 Increased sFas has been shown to blockapoptosis in cells by inhibiting the binding of FasL toFas on cell membrane.17,18

Aps et al. demonstrated that the cellular componentsof human saliva were composed of erythrocytes, leuko-cytes, epithelial cells, and bacteria.19 In our study mostof the cells detected in saliva samples were similar totypical epithelial cells of oral mucosa, just as in a recentstudy investigating apoptotic cells in whole saliva ofpatients with oral premalignant and malignant lesions.11

In the present study, soluble Fas and NMP levelswhich correlate with the number of dying or dead cellswere determined in saliva. No significant differencesbetween the two groups for sFas levels could be deter-mined. In comparison to non-smokers, smokersshowed a decrease of the NMP levels, which is believedto be an antiapoptotic indicator.

The results of the present study suggest that smok-ing may induce antiapoptic mechanism in the oral cav-ity. Diagnosis, predicting predisposition and prognosis,and monitoring disease progression according to theapoptotic changes in saliva by a non-invasive methodcould be a valuable tool for the dental team, especiallyfor periodontists who follow-up patients during long-term maintenance phase. However, these results mustbe confirmed in a wider population, and correlationbetween cancerous or precancerous lesions and thedegree of smoking must also be investigated.

REFERENCES1. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: A basic

biological phenomenon with wide-ranging implicationsin tissue kinetics. Br J Cancer 1972;26:239-257.

2. Wyllie AH, Kerr JFR, Currie AR. Cell death: The signifi-cance of apoptosis. Int Rev Cytol 1980;68:251-300.

3. Mariggio MA, Guida L, Laforgia A, Santacroce R, Curci E,

Montemurro P, Fumarulo R. Nicotine effects on poly-morphonuclear cell apoptosis and lipopolysaccharide-induced monocyte functions. A possible role in periodontaldisease? J Periodontal Res 2001;36:32-39.

4. Berezney R, Coffey DS. Identification of a nuclear proteinmatrix. Biochem Biophys Res Commun 1974;60:1410-1417.

5. Pardoll DM Pardoll DM, Vogelstein B, Coffey DS. A fixedsite of DNA replication in eukaryotic cells. Cell 1980;19:527-536.

6. Zeitlin S Zeitlin S, Parent A, Silverstein S, Efstratiadis A.Pre-mRNA splicing and the nuclear matrix. Mol Cell Biol1987;7:111-120.

7. Miller TE, Beausang LA, Winchell LF, Lidgard GP. Detec-tion of nuclear matrix proteins in serum from cancerpatients. Cancer Res 1992;52:422-427.

8. Bokor-Bratic M, Vuckovic N. Cigarette smoking as a riskfactor associated with oral leukoplakia. Arch Oncol2002;10:67-70.

9. Regezi JA, Sciubba J. Oral Pathology. Philadelphia: W.B.Saunders Co.; 1993:102-112.

10. Shafer WG, Hine MK, Levy BM: A Textbook of Oral Patho-logy. Philadelphia: W.B. Saunders Co.; 1983:92-104.

11. Cheng B, Rhodus NL, Williams B, Griffin RJ. Detectionof apoptotic cells in whole saliva of patients with oral pre-malignant and malignant lesions: A preliminary study.Oral Surg Oral Med Oral Pathol Oral Radiol Endod2004;97:465-470.

12. Löe H, Silness J. Periodontal disease in pregnancy. I.Prevalence and severity. Acta Odontol Scand 1963;21:533-551.

13. Silness J, Löe H. Periodontal disease in pregnancy. II.Correlation between oral hygiene and periodontal con-dition. Acta Odontol Scand 1964;22:121-135.

14. Navazesh M, Christensen CM. A comparison of wholemouth resting and stimulated salivary measurement pro-cedures. J Dent Res 1982;61:1158-1162.

15. Machner A, Bajer A, Willie A, et al. Higher susceptibilityto Fas ligand induced apoptosis and altered modulationof cell death by tumor necrosis factor in periarticulartenocytes from patients with knee joint osteoarthritis.Arthritis Res Ther 2003;5:253-261.

16. Heaney ML, Golde DW. Soluble cytokine receptors. Blood1996;87:847-857.

17. Hasunuma T, Kayagaki N, Asahara H, et al. Accumulationof soluble Fas in inflamed joints of patients with rheuma-toid arthritis. Arthritis Rheum 1997;40:80-86.

18. Nozawa K, Kayagaki N, Tokano Y, Yagita H, OkumuraK, Hasimoto H. Soluble Fas (APO-1, CD95) and solubleFas ligand in rheumatic diseases. Arthritis Rheum1997;40:1126-1129.

19. Aps JKM, van den Maagdenberg K, Delanghe JR,Martens LC. Flow cytometry as a new method to quan-tify the cellular content of human saliva and its relationto gingivitis. Clin Chim Acta 2002;321:35-41.

Correspondence: Dr. Pervin Imirzalioglu, Baskent Universitesi,Dis Hekimligi Fakultesi, 11. Sok. No: 26 06490, BahcelievlerAnkara, Turkey. Fax: 90-312-2152962; e-mail: pervini@baskent.edu.tr or pervini@superonline.com.

Accepted for publication September 13, 2004.

40149.qxd 5/6/05 3:07 PM Page 739