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1 Kor J Oral Maxillofac Pathol 2013;37(6): 흡연이구강병관련세균과 Human Papillomavirus 의검출에미치는영향 <Abstract> 손화경 1), 김주영 2), 박정란 1), 김진 2) * 백석대학교치위생학과 1), 연세대학교치과대학구강병리학교실 구강종양연구소 2) The Impact of Smoking in Detection of Bacteria Related to Oral Disease and Human Papillomavirus Hwa kyung Son 1), Ju Young Kim 2), Jeong Ran Park 1), Jin Kim 2) Department of Dental Hygiene, Baekseok University Devision of Health science, Cheonan, Korea 1), Department of Oral pathology, Oral Cancer Research Institute, Yonsei University College of Dentistry 2) Smoking is a risk factor for oral leukoplakia and oral cancer, as well as lung cancer, cardiovascular diseases and many other systemic diseases. Smoking is considered increasing factor of some oral diseases involved indigenous bacteria. In addition, a relationship between smoking and infection of Human papillomavirus (HPV), which is associated with oropharyngeal cancer, remains unclear. The aim of this study is to assess whether smoking has an impact on increase of bacteria inducing oral disease such as dental caries and periodontitis, and HPV infection. DNA of saliva gathered from smokers and non-smokers, consisted of men and women, was analyzed using PCR. Oral disease-causing bacteria were more detected in men smokers than men non-smokers and HPV was most found in women non-smokers. Taken together, this study suggests smoking is related with variation of oral microorganism existence in some way. Key words:smoking, HPV, S.mutans, P. gingivalis Ⅰ. 서론 흡연은폐암과심장질환뿐아니라, 호흡기감염과치주질환, 그리고세균성수막염과같은미생물감염의위험요인을증가시킨다 1). 흡연은담배연기의높은열과타르, 일산화탄소, 니코틴등의해로운화학물질에의해구강병을일으키는중요한위험요인으로서경조직뿐아니라연조직의손상에심각한영향을주는것으로알려져있다 2). 흡연과치아우식증과의관계는논란이있지만, 보고되어온많은연구들에서흡연이치아우식증을증가시키는중요한위험요소라는것을보여준다 3,4). * Correspondence: Jin Kim, Department of Oral Pathology, Oral Cancer Research Institute, Yonsei University College of Dentistry,134 Shinchon-Dong, Seoul , Republic of Korea. Tel: , Fax: , jink@yuhs.ac Received: Nov 18, 2013; Revised: Nov 20, 2013; Accepted: Nov 27, 2013 또한흡연은치주조직에서염증의진행과부착의소실, 치은퇴축과같은치주병을일으키는원인으로서보고되었다 5-7). 흡연의또다른위험성은전암병소인백반증과구강암의유발에있다 8,9). 흡연은암억제자인 p53 단백질의돌연변이를일으켜서, 손상된 DNA 가세포내에축적되어결과적으로암의발생에중요한역할을한다고보고되어있다 10). 또한, 흡연은인체의면역시스템을저해하여다양한구강병을일으키는세균이나바이러스의증식에기여할수있다고알려져있다 11). 최근의연구에서, 흡연은비인두체계에서상재세균의수를감소시켜서병원균의증가를야기시킨다고보고되었다 12,13). 치아우식증을유발하는대표적인미생물인 Streptococcus mutans (S. mutans) 는통성혐기성그람양성세균으로서, 주로소와와열구의우식을유발한다 14). S. mutans는구강내상재세균이지만, 특별한상황에서기회균으로전환하여병을일으킨다 15). S. mutans 는설탕을젖산으로대사하면서생성한

2 산성환경을이용하여고도로광화된법랑치질을부식할수있게만든다 16). S. mutans는또한설탕을이용하여끈적끈적한다당을생산하여치태를형성하면서다른세균들과함께응집된다 17). 치주질환과관련이깊은 Porphyromonas gingivalis (P. gingivalis) 는혐기성그람음성병원균으로서구강뿐아니라, 위장관과호흡기, 결장에서도관찰된다 18). 치주질환에서보여지는교원질의분해는부분적으로 P. gingivalis 의교원질분해효소에의한결과이며, P. gingivalis는사람의치은섬유모세포를침범하며, 상당한농도의항생제에서도살아남을수있다 19). Human papillomavirus (HPV) 는피부의각질과점막에만감염하는 DNA virus 이다. 대다수의알려진종류의 HPV 는대부분의사람에서별다른증상을나타내지않지만, 어떤종류의 HPV 는사마귀를일으키거나, 생식기와구강인두에서암을일으킨다. 총 120여종류이상의 HPV 중에서타입 16, 18, 31, 45를포함한 12개종류의 HPV 는자궁경부암과생식기암 을일으키는고위험군으로일컫는다 20). 그중에서특히타입 16은 HPV 양성구강인두암과관련되어발견된다 21). 흡연과구강의 HPV 감염과의연관성에대해서는많은논란이있다. 흡연이구강에서 HPV 감염의지속성을증가시킨다는보고가있었고 22), 흡연은 HPV가감염된상황에서두경부암의진행을위한추가적위험요인이라는보고가있었는가하면 23), 어떤연구자들은두경부에서흡연과 HPV 감염과의연관성이없다고보고하기도하였다 24,25). 본연구는실제로흡연이구강병유발세균의증식과두경부편평세포암관련바이러스의감염에영향을주는요인인지를확인하기위하여흡연자들과비흡연자들에서치아우식증과치주병을일으키는대표적인세균과 HPV의 DNA를검출하여비교함으로써흡연과구강질환을유발시키는미생물들과의연관성을알아보고자하였다. Table 1. Nucleotide sequences and 5 positions of PCR primers GENE S. mutans P. gingivalis HPV 16 primer sequence Forward 5 - TGG GAC GCA AGG GAA CAC A -3 Reverse 5 - GCG GCG TTG CTC GGT CAG A -3 Forward 5 - CGC AGC CTA CGA TAA CAT TT -3 Reverse 5 - TCC GTT TTT CTG AAG TTT GC -3 Forward 5 - ATG TTT CAG GAC CCG CAG GAG CGA TTA CAG CTG GGT TTC TCT ACG TG -3 Figure 1. Detection of S. mutans DNA in saliva by PCR Subject s saliva samples are classified according to the presence of smoking and gender. The DNA product was amplified by PCR using AccuPower HotStart PCR PreMix. MN1, 3, 4, 5, 6, 9, MS1, 2, 3, 4, 6, 7 and FS1, 2, 4, 6, 9 are positive for S. mutans. M; Male, F; Female, MS Male smoker, MN; Male nonsmoker, FS; Female smoker, FN; Female nonsmoker 304

3 Ⅱ. 연구대상및방법 1. 연구대상본연구는 2013년 6월 15일부터 6월 30일까지천안지역일부대학교에재학중인학생들을대상으로타액을채취하여사용하였다. 대상자로부터연구목적에대한인지를확인한후에실시하였고, 흡연을시작한후 3년이상경과된남녀흡연자각 25명과흡연경험이없는남녀비흡연자각 25명을대상으로하였고, 채취된타액중검출가능한농도의 DNA 를함유한시료만을선택하여연구하였다. 2. 연구방법대상자의타액으로부터 QIAamp DNA mini kit (Qiagen, Hilden, Germany) 을이용하여 DNA를추출하였고, AccuPower HotStart PCR PreMix (Bioneer, Daejeon, South Korea) tube에 DNA 100 ng, forward 와 reverse primer를각 10 pmole씩첨가하고멸균된증류수로최종시료의양이 20 μl가되도록하였다. primer 염기서열은다음과같다 (Table 1). PCR 조건은 94 에서 5분간변성시키고이어서 초, 58 30초, 72 30초로 30주기를순환시킨후, 마지막으로 72 에서 10분간처리하는과정을거쳤다. 모든 PCR 산물은 1% agarose gel로확인하였다. 3. 통계학적분석구강병유발세균이나 HPV의검출비율과흡연유무와의유의성을검증하기위해대표적으로 chi-square test 를시행하였다. 수집된자료와측정값분석은 SPSS 19.0 (SPSS Inc, IBM, Chicago, USA) 을이용하였으며, p < 0.05 일때유의한것으로판단하였다. Table 2. Detection of bacteria according to the presence of smoking and gender 흡연자 비흡연자 남 (%) 여 (%) 남 (%) 여 (%) S. mutans 10/15(66.7) 11/19(57.9) 12/22(54.6) 6/19(31.58) Total 21/34(61.8) 18/41(43.9) P. gingivalis 1/15(6.7) 2/19(10.5) 1/22(4.5) 2/19(10.5) Total 3/34(8.8) 3/41(7.3) Figure 2. Detection of P. gingivalis DNA in saliva by PCR Subject s saliva samples are classified according to the presence of smoking and gender. The reaction mixture was subjected to 30 cycles of 40 s at 94 C, 40 s at 58 C and 40 s at 72 C. MN4, MS4, FN3 and FS7 are positive for P. gingivalis. M; Male, F; Female, MS Male smoker, MN; Male nonsmoker, FS; Female smoker, FN; Female nonsmoker 305

Ⅲ. 결과 1. 연구대상자의일반적특성총 100개의타액중에서검출가능한농도의 DNA 를함유한타액을기증한연구대상자의일반적특성을빈도분석한결과, 성별은남자 49.3%, 여자가 50.7% 로비슷하였다. 흡연유무를보면흡연을하는경우는 45.3%, 흡연을하지않는경우는 54.7% 였다. 남자중흡연자는 40.5%, 비흡연자는 59.

4 Ⅲ. 결과 1. 연구대상자의일반적특성총 100개의타액중에서검출가능한농도의 DNA 를함유한타액을기증한연구대상자의일반적특성을빈도분석한결과, 성별은남자 49.3%, 여자가 50.7% 로비슷하였다. 흡연유무를보면흡연을하는경우는 45.3%, 흡연을하지않는경우는 54.7% 였다. 남자중흡연자는 40.5%, 비흡연자는 59.5% 이었으며, 여자중흡연자는 50%, 비흡연자는 50% 였다. 흡연과성별에따른타액에서의세균검출빈도는다음과같다 (Table 2). 2. 타액에서 S. mutans와흡연과의관계분석본연구의대상자들의타액으로부터 S. mutans의 DNA를검출한대표적결과를 Figure 1에나타내었다. 남자흡연자의 66.7% 와, 여자흡연자의 57.9% 에서 S. mutans의 DNA가검출되어, 남자흡연자가여자흡연자보다검출비율이약 9% 가높았으며, 남자비흡연자의 54.6%, 여자비흡연자의 31.58% 에서 S. mutans의 DNA가검출되어, 남자비흡연자가여자 비흡연자에비해약 23% 높은검출비율을나타냈다. 또한남자흡연자가남자비흡연자에비해 S. mutans DNA의검출비율이약12% 높았으며, 여자흡연자는여자비흡연자보다약 26% 높은검출비율을나타냈다. 전체적으로흡연자는 61.8% 에서, 비흡연자는 43.9% 에서 S. mutans의 DNA가검출됨으로서, 흡연자는비흡연자보다 S. mutans DNA의검출비율이 17.9% 높았다. 3. 타액에서 P. gingivalis와흡연과의관계분석타액에서 P. gingivalis의 DNA를검출한결과, 남자흡연자가 6.7%, 여자흡연자가 10.5% 였고, 남자비흡연자가 4.5%, 여자비흡연자가 10.5% 로서, S. mutans의 DNA검출비율에비해 1/5~1/10 정도의검출비율을나타냈지만, S. mutans의 DNA 검출의경우처럼남자흡연자가남자비흡연자보다더높은검출비율을나타내었다. 전체적으로흡연자는 8.8%, 비흡연자는 7.3% 가검출됨으로써흡연자가비흡연자보다 P. gingivalis의 DNA의검출비율이높았다 (Figure 2). 4. 타액에서 HPV 16과흡연과의관계분석 Table 3. Detection of HPV 16 according to the presence of smoking and gender 남 (%) 여 (%) Total(%) 흡연자 5/15(33.3) 2/19(10.5) 7/34(20.6) 비흡연자 8/22(36.4) 10/19(52.6) 18/41((43.9) Figure 3. Detection of HPV DNA in saliva by PCR Subject s saliva samples are classified according to to the presence of smoking and gender. HPV16 DNA was detected by PCR in subject s saliva. MN4, 5, 7, 9, MS4, FN2, 3, 6, 8, 9 and FS4, 5, 6 are positive for HPV. M; Male, F; Female, MS Male smoker, MN; Male nonsmoker, FS; Female smoker, FN; Female nonsmoker 306

5 대상자의타액에서검출된 HPV 16을성별과흡연여부에따라분류하였다 (Table 3). 타액에서 HPV16의 DNA 검출유무에대한결과는 Figure 3과같다. HPV 16은성별에따른유의성을보이지는않았지만흡연여부로분류하였을때비흡연자에서의검출비율이흡연자의검출비율보다통계학적으로유의하게높은것을알수있었다 (p<0.05). 성별에따라흡연여부를분류하였을때에는남자흡연자와남자비흡연자간에는유의하지않았지만여자비흡연자가여자흡연자보다 HPV 16의 DNA 검출비율이통계학적으로유의하게높았다 (p<0.05). IV. 고찰구강병을일으키는구강미생물의증식과흡연과의연관성에관해서많은연구가보고되어왔다. 가장공통적인의견은, 흡연이인체의면역반응을저해시켜서숙주가다양한세균과진균, 바이러스에쉽게감염될수있다는것이다 26). 그래서흡연자는타액의유출량이감소하여저하된타액의완충작용은 lactobacilli나 S. mutans 와같은치아우식균의증식을도울수있다 27). 또한, 최근의연구에서, 흡연자의금연은치주병원균의수준을감소시키면서치주치료에따른미생물의재집락화의패턴을변화시킨다고보고하였다 28). 또다른의견은, 담배의타르성분이음식물이라는세균의영양분과세균자신이구강내치아나치주조직에오랫동안붙어서활동할수있는환경을만들어준다는것이다. 다시말해서, 타르에의해치아나치주조직에붙게된세균들은치태형성을돕고, 형성된치태는수일내에집락특징이변화되는데, 초기에는통기성세균인치아우식균이집락하다가 4~5일이후에는혐기성치주질환균이득세하게된다. 이후치태는타르에의해석회작용이더욱빨리진행될수있다는것이다 29). 물론모든질병의진행은개인의건강과면역상태에따라결과가달라지겠지만, 본연구에서는흡연자체의발암성분이, 구강병을유발할수있는잠재성을가지고있는치아우식균과치주병균이나 HPV를구강내에오래머물러있게할수있을것이라는것에초점을맞추고진행하였기때문에, 위에서언급한흡연과구강미생물의증식과관련된연구중후자의내용과관련이깊다. 본연구의 Figure 1에서, 남녀흡연자간, 남녀비흡연자간에따른검출비율의차이는구강위생의관심도의차이에기인한다고생각할수있는데, S. mutans 의 DNA 검출비율이남자비흡연자보다여자흡연자가더높았고, 여자비흡연자에비해여자흡연자에서검출비율이 26.3% 가높음으로써흡연에의한타르등의발암성분이 S. mutans 와결합하여흡연자의구강내에서 S. mutans 가더오래남아있게하였을것으로생각된다. P. gingivalis 는혐기성그람음성세균으로서, 주로염증성치주낭에서균총을형성하므로타액에서는 DNA의검출비율이낮았지만, 남자흡연자가남자비흡연자보다높은검출비율을나타냄으로써, S. mutans DNA 의검출에서와유사한결과를나타내었다 (Figure 2). 미국의 1988년연구에따르면두경부편평세포암의 75% 의원인이흡연과음주에있다고보고한바있다 30). 두경부편평세포암의또다른위험요인으로서 HPV 16이있으며, 혈청반응에서 HPV 16 양성의결과는음성과비교해서두경부편평세포암의위험요인으로서약 4배나높다고보고되었다 31). 그러나, 2001년의한역학연구는흡연자나음주자보다는비흡연자와비음주자의경우에더욱 HPV 16에양성인종양을갖는다는결과를제안하였다 32). 최근의연구에서, 혈청검사에서 HPV 16 양성인두암의위험요인의증가는비흡연그룹에서흡연그룹보다 30배더높았다는결과가있었고, HPV 16이어떤방식으로든흡연의영향을막는것같다고보고하였다 33). 본연구에서는흡연자보다비흡연자에서, HPV 16의검출이통계적으로유의하게높은결과를나타내었고, 여자비흡연자에서가장높은 HPV 16의검출비율을나타내었다. 본연구가대상자의구강종양과관련된 HPV 연구는아니었지만, 흡연자가비흡연자에비해통계학적으로낮은 HPV 16을가지고있다는것은, 구강세균의분포와는반대로, 흡연이 HPV 16의지속적감염을막는역할을했다고볼수있다. 본연구는대상자의혈청학적검사없이구강타액에서만 HPV 16을검출하였기때문에, 민감도가떨어지는결과를나타낼수있다고할수도있겠지만 34), HPV 항체의위치는바이러스에노출된위치에특이성이없어서 HPV가호흡기관에감염되었는지의여부를판단할수가없기때문에, HPV의혈청검사도 HPV 의구강감염과흡연과같은다른위험요인과의 307

6 관계를판단하는데있어서유일한방법이라고할수는없다 33). 흡연이구강인두암의강력한위험요인임은중요한사실이기때문에, 흡연이오히려구강인두암의또다른위험인자인 HPV 16의감염을방해한다는본연구의결과는더욱더많은집단에서의재확인이필요하며, 대상자에대한더욱더구체적인흡연경향과흡연특성에대한연구가필요하다고판단되며, 향후이연구는 HPV 16 양성구강인두암의재발과대상자의생존에있어서흡연의역할과치료의표적을결정하는데있어서중요한기초자료가될것이라사료된다. V. 참고문헌 1. Doll R. Peto R: Mortality in relation to smoking: 20 years observations on male British doctors. BMJ 1976; 2: Van Winkelhoff AJ, CJ Bosch-Tijhof, EG Winkel, WA Van der Reijden: Smoking affects the subgingival microflora in periodontitis. J. Periodontol 2001;72: Axelsson P, Paulander J, Lindhe J: Relationship between smoking and dental status in 35, 50, 65, and 75-year-old individuals. J Clin Periodontol 1998;5: Bruno-Ambrosius K, Swanholm G, Twetman S: Eating habits, smoking and toothbrushing in relation to dental caries: a 3-year study in Swedish female teenagers. Int J Paediatr Dent 2005;15: Genco RJ: Current view of risk factors for periodontal diseases. J Periodontol 1996;67: Muller HP, Stadermann S, Heinecke A: Gingival recession in smokers and nonsmokers with minimal periodontal disease. J Clin Periodontol 2002;29: Razali M, Palmer RM, Coward P, Wilson RF: A retrospective study of periodontal disease severity in smokers and non-smokers. British Dent Journal 2005;198: Banoczy J, Gintner Z, Dombi C: Tobacco use and oral leukoplakia. J Dent Educ 2001;65: Williams SA, Kwan SY, Parsons S: Parental smoking practices and caries experience in pre-school children. Caries Res 2000;34: Nylander K, Dabelsteen E, Hall PA: The p53 molecule and its prognostic role in squamous cell carcinomas of the head and neck. J Oral Pathol Med 2000;29: King TE Jr, Savici D, Campbell PA: Phagocytosis and killing of Listeria monocytogenes by alveolar macrophages: smokers versus nonsmokers. J Infect Dis 1988;158: Brook I, AE Gober: Recovery of potential pathogens and interfering bacteria in the nasopharynx of smokers and nonsmokers. Chest 2005;127: Charlson ES, et al: Disordered microbial communities in the upper respiratory tract of cigarette smokers. PLoS One 2010;5:e Ikeda T, Sandham HJ: Prevalence of Streptococcus mutans on various tooth surfaces in negro children. Archives of Oral Biology 1971;16: Nicolas GG, Lavoie MC: Streptococcus mutans and oral streptococci in dental plaque. Can J Microbiol 2011;57: Jeon EH, Han JH, Ahn TY: Comparison of bacterial composition between human saliva and dental unit water system. J Microbiol 2007;45: Mukasa H, Slade HD: Mechanism of Adherence of Streptococcus mutans to Smooth Surfaces. Infect Immun 1973;8: Naito M, Hirakawa H, Yamashita A, et al: Determination of the Genome Sequence of Porphyromonas gingivalis Strain ATCC and Genomic Comparison with Strain W83 Revealed Extensive Genome Rearrangements in P. gingivalis. DNA Res 2008;15: Irshad M, van der Reijden WA, Crielaard W, Laine ML: In vitro invasion and survival of Porphyromonas gingivalis in gingival fibroblasts; role of the capsule. 308

7 Arch Immunol Ther Exp (Warsz) 2012;60: Parkin DM: The global health burden of infection-associated cancers in the year Int J Cancer 2006;118: D'Souza G, Kreimer AR, Viscidi R, et al: Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med 2007;356: Kero K, Rautava J, Syrjänen K, Willberg J, Grenman S, Syrjänen S: Smoking increases oral HPV persistence among men: 7-year follow-up study, Eur J Clin Microbiol Infect 2013; Sinha P, Logan HL, Mendenhall WM, Human papillomavirus, smoking, and head and neck cancer. Am J Otolaryngol 2012;33: Gillison ML, Koch WM, Capone RB, et al: Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000;92: Applebaum KM, Furniss CS, Zeka A, et al: Lack of association of alcohol and tobacco with HPV16- associated head and neck cancer. J Natl Cancer Inst 2007;99: Mohan Sopori: Effects of cigarette smoke on the immune system. Nature Reviews immunology 2002;2: Johnson NW, Bain CA: Tobacco and oral disease. EU-Working Group on Tobacco and Oral Health. Br Dent J 2000;189: Delima SL, RK McBride, PM Preshaw, PA Heasman, PS Kumar: Response of subgingival bacteria to smoking cessation. J Clin Microbiol 2010;48: César Neto JB, Rosa EF, Pannuti CM, Romito GA: Smoking and periodontal tissues. a revie, Braz Oral Res 2012;26: Blot WJ, McLaughlin JK, Winn DM, et al: Smoking and drinking in relation to oral and pharyngeal cancer. Cancer Res 1988;48: Furniss CS, McClean MD, Smith JF, et al: Human papillomavirus 16 and head and neck squamous cell carcinoma. Int J Cancer 2007;120: Lindel K, Beer KT, Laissue J, Greiner RH, Aebersold DM: Human papillomavirus positive squamous cell carcinoma of the oropharynx: a radiosensitive subgroup of head and neck carcinoma. Cancer 2001;92: Applebaum KM, Furniss CS, Zeka A, et al: Lack of association of alcohol and tobacco with HPV16-associated head and neck cancer. J Natl Cancer Inst2007;99: Castle PE: Human papillomavirus in oral exfoliated cells and risk of head and neck cancer. J Natl Cancer Inst 2004;96:

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