Effects of Electronic Nicotine Delivery System on Larynx: Experimental Study lar C¸akır, *G€ € nnetc¸i, *Yavuz Atar, *Tolgar Lu € tfi Kumral, *G€ *Ziya Salturk, †C¸ag urcan Su uven Yıldırım, *G€ uler Berkiten, and *Yavuz Uyar, *yIstanbul, Turkey Summary: Objective. We aimed to assess the effects of electronic nicotine delivery system (ENDS) or also termed electronic cigarette vapor on the laryngeal mucosa of rats. Materials and Methods. Sixteen female Wistar albino rats were divided into two groups. The study group was exposed to ENDS vapor for 1 hour/day for 4 weeks. The control group was not subjected to any chemical or physical stimulus. The vocal folds of the study and control group rats were evaluated histopathologically by hematoxylin and eosin staining and immunohistochemically by Ki67 staining. Epithelial distribution, inflammation, hyperplasia, and metaplasia were evaluated. Results. Epithelial distribution and inflammation did not differ between the two groups. Two cases of hyperplasia were detected in the study group but there was no hyperplasia in the control group. Four cases of metaplasia were detected in the study group and one case in the control group. Statistical analysis revealed no significant difference between the study and control groups (P ¼ 0.131 and 0.106, respectively). Conclusions. Exposure to ENDS for 4 weeks caused hyperplasia and metaplasia of the laryngeal mucosa of rats but this was not significant statistically. These results implemented that further studies with larger cohort and longer duration are required to evaluate long-term effects. Key Words: Electronic nicotine delivery system–Electronic cigarette–Larynx–Histopathology–Metaplasia. INTRODUCTION Nicotine is a widely used addictive psychoactive drug.1 Biobehavioral addiction renders it difficult to quit, and withdrawal symptoms include anxiety and irritability.2 Electronic nicotine delivery systems (ENDS), also termed electronic cigarettes (e-cigarettes), are being used in efforts to quit smoking because of their physical resemblance to a standard tobacco cigarette. Vapor is generated electronically.3 Although ENDS vapor contains 9- to 450-fold lower levels of toxic and carcinogenic compounds (carbonyls, volatile organic compounds, nitrosamines, and heavy metals) than do tobacco cigarettes,4 the particles therein are comparable with those of nicotine-containing cigarettes.5 In addition, one report found metal and silica nanoparticles6 that may damage mucosal surfaces. Although it is accepted that ENDS use reduces the harmful effects of smoking,4 it was concluded that they contain toxic and carcinogenic materials.7 Effects of regular smoking have been widely studied in humans and rats. Cadaveric studies showed that smoking causes epithelial changes, including precancerous lesions, which are related to the duration of smoking.8,9 Similarly, clinical studies showed associations of smoking and Reinke’s edema, metaplasia, and keratotic and erythroplastic lesions with atypia.10–12 Animal studies also revealed vocal fold changes, including hyperplasia, metaplasia, and keratinization, triggered by regular smoking.13–15
Accepted for publication October 23, 2014. This study has not been published totally or partially before. From the *Okmeydanı Training and Research Hospital, ENT Clinic, Istanbul, Turkey; and the yOkmeydanı Training and Research Hospital, Pathology Clinic, Istanbul, Turkey. Address correspondence and reprint requests to Ziya Salturk, Okmeydanı Training and _ Research Hospital ENT Clinic, Darulaceze cad., S¸is¸li, Istanbul, Turkey. E-mail: [email protected] Journal of Voice, Vol. -, No. -, pp. 1-4 0892-1997/$36.00 Ó 2015 The Voice Foundation http://dx.doi.org/10.1016/j.jvoice.2014.10.013
The vocal fold is the most common site of laryngeal carcinoma,16 and it is vulnerable to vapor produced by ENDS because the cord lies directly in the upper respiratory tract. We sought to evaluate the histopathologic effects of 4 weeks of exposure to vapor created by ENDS on the vocal fold mucosa of rats. MATERIALS AND METHODS Institutional Review Board approval was obtained from the Istanbul University Experimental Animal Research Ethics Committee. Sixteen healthy, adult, female Wistar albino rats (200–250 g; 7–8 months old) were used. A smoke drum designed by C.F. Palmer Ltd. (London, United Kingdom) was used to expose the rats to vapor.17 The machine contains two holes, one for inspiration and the other for expiration. The cigarette was placed in the inspiration hole, smoke was drawn off by vacuuming, and the smoke was conducted via a 1.5-m polyethylene tube to the exit. The cabin was 30 3 40 3 50 cm in dimension and made of plexiglass. There were 10 holes affording aeration, each 2 cm in diameter. The machine yielded 200 mL/minute of smoke. Ego T (China) was the ENDS used and was filled with a solution of 0.9% (wt/ vol) nicotine. The vapor was distributed through the cabin after 20 min. The study rats were treated for 60 minutes/day for 4 weeks. The 16 rats were divided into two groups. Eight rats were exposed to vapor for 60 minutes/day over 4 weeks. Rats had free access to food and water and were kept under a 12-h:12h light-dark cycle. The mean room temperature was 25 C. The other eight rats were not exposed to vapor and were housed identically. At the end of 4 weeks, rats were sacrificed under high-dose ketamine anesthesia. Each vocal fold was removed intact and stored in 10% (vol/vol) buffered formalin solution. Specimens were cut into 5-mm-thick sections. Standard tissue-processing methods were applied, and the sections were stained with
2 hematoxylin and eosin before analysis by light microscopy. An expert pathologist blinded to group assignment evaluated epithelial distribution, inflammation, hyperplasia, and metaplasia. Inflammation was graded based on cellular density: grade 1 represented scattered inflammatory cells, grade 3 represented inflammatory cell density that forms lymphoid follicles, and grade 2 represented cellular density between grades 1 and 3. Mucosal cellular reactivities were assessed immunohistochemically by staining for Ki67, which is expressed during all phases of the cell cycle except for G0. Expression of Ki67 closely parallels [3H]-thymidine incorporation, which is a standard method of measuring cell proliferation rate. Immunostaining of normal tissue for the Ki67 antigen reveals nuclear reactivity in cells of the germinal centers of cortical follicules, cortical thymocytes, and neck cells of the gastrointestinal mucosa. Resting cells such as hepatocytes, renal cells, and Paneth’s cells of the gastrointestinal mucosa do not show staining. For immunohistochemical evaluation, formalin-fixed paraffin-embedded tissue blocks were cut at a thickness of 5 mm and mounted on poly L-lysine coated slides, followed by staining with an anti-Ki67 antibody (BioGenex, San Ramon, CA; clone: BGX-Ki67, 1:100 dilution). All procedures were performed using the standard protocol of a Leica Bond Max autostainer. Positive and negative controls were run. Any identifiable nuclear staining, regardless of intensity, was recorded as positive. Distribution of Ki67 in the basal and parabasal areas of the laryngeal mucosa was evaluated. Statistical analysis was performed using the aid of SPSS software, version 17. Pearson’s chi-squared test was used for data analysis. Because the epithelial distributions and cellular reactivities of the mucosa were identical in both groups, these data were not analyzed. P < 0.05 was accepted as significant.
RESULTS No macroscopic difference was evident between larynges of the study and control groups. Microscopically, three types of epithelial cells were detected in the control group. The ventral part of the glottis was composed of striated ciliary epithelium adjacent to loose connective tissue. The free margins of the vocal folds and adjacent areas were covered by striated nonciliated columnar epithelium. The arytenoid region and adjacent areas were covered by striated squamous epithelium, which was thinner on the arytenoid cartilages. The study group exhibited similar histopathologic features. Both groups exhibited typical epithelial linings and basal membranes. The extent of inflammation did not differ between the two groups (Figure 1; Table 1). Squamous metaplasia was detected in four of the eight rats in the study group but in only one rat in the control group (Figure 2; Table 2); this difference was not significant (P ¼ 0.106). Two larynges in the study group developed hyperplasia, compared with none in the control group (Table 3). There was also no statistically significant difference (P ¼ 0.131). Ki67 expression levels were examined to evaluate cellular reactivities. All larynges revealed a basal proliferation pattern (Figure 3); no between-group difference was apparent.
Journal of Voice, Vol. -, No. -, 2015
FIGURE 1. Normal vocal fold epithelia and mild subepithelial inflammation.
DISCUSSION Smoking is the leading cause of preventable deaths in the United States, and the harmful effects thereof can be reduced by quitting.18,19 Many techniques aiding quitting have been developed, but they are not very effective, even when combined with counseling programs, because smoking is affected by many factors including age, sex, time of onset of smoking, housing conditions, and health status.20–24 E-cigarettes deliver nicotine and mimic the action of smoking. Therefore, e-smoking satisfies the behavioral, sensory, and physical components of smoking.2,25 Thus, the quitting success rate using this technique may be higher than that achieved using other methods. ENDS safety has been widely discussed, and e-cigarettes are forbidden in some countries. Studies on the health effects of ENDS have appeared.26–29 The health risk was higher than those of various nicotine replacement therapies, but lower than that of smoking.30,31 Smoking is the leading cause of laryngeal cancer, the risk of which is 35-fold higher in smokers than nonsmokers. The type of tobacco used, the number of cigarettes smoked, the duration of smoking history, and passive exposure are all independent risk factors.16 In addition, smoking causes Reinke’s edema, chronic laryngitis, and diffuse vocal fold polyps.32 Smoking also triggers reflux affecting the larynx.32,33 Cadaveric studies of histopathologic changes caused by smoking in the human larynx found epithelial changes.8,9 Aurbach and Hammond8 performed a histologic study of the effects of smoking on the larynx using cadavers and concluded that the true vocal fold was the main site affected by smoking.
TABLE 1. Status of Inflammation Inflammation
1+
2+
3+
Total
Control group 1 (12.5%) 5 (62.5%) 2 (25%) 8 (100%) Study group 3 (37.5%) 3 (37.5%) 2 (25%) 8 (100%) Total 4 (25%) 8 (50%) 4 (25%) 16 (100%) Notes: Pearson chi-squared value (P) ¼ 1.500.
Ziya Salturk, et al
3
Effects of electronic cigarette on larynx
TABLE 3. Number of Hyperplasia in Study and Control Groups Hyperplasia Control group Study group Total
Negative
Positive
Total
8 (100%) 6 (75%) 14 (87.5%)
0 (0%) 2 (25%) 2 (12.5%)
8 (100%) 8 (100%) 16 (100%)
Notes: Pearson chi-squared value (P) ¼ 0.131.
FIGURE 2. Metaplasia in the study group. To a lesser degree, the false vocal fold and the area of the larynx above the vocal fold were affected. Comparison of smokers’ and nonsmokers’ larynxes revealed that histologic changes (including cells atypical nuclei, carcinoma in situ, and early invasive carcinoma) were much greater in cigarette smokers; they also increased in proportion to the number of cigarettes smoked per day before death and were seen to a greater degree in cigarette smokers than in cigar and pipe smokers. Similarly, M€ uller and Khron9 concluded that smoking caused precancerous lesions in humans. These findings were also supported by clinical studies.10,12 Zietel10 suggested that geographic localization of keratotic and erythroplastic lesions on the superior/ ventricular surface of the musculomembranous vocal fold are likely to contain atypia in smokers. It was also shown that metaplasia in the supraglottic region increased with aging and with tobacco and alcohol consumption.12 Animal studies have confirmed the effects of smoking on the laryngeal mucosa and have enabled an understanding of the developmental stages that precede cancer. However, the ENDS is a recent technological innovation, and no research on the use of ENDS has yet been published. We did not detect chronic inflammation or allergic reactions. Epithelial responses, including hyperplasia and metaplasia, were evident but not significant. Although we did not detect any dysplasia, we suggest that further studies with longer follow-up periods and higher nicotine dosages be performed. Duarte et al13 evaluated the outcomes of passive smoke inhalation of 10 cigarettes daily at 25, 50, and 75 days. Histopathologic evaluation revealed hyperplasia and metaplasia. Is¸ık
et al14 evaluated the vocal folds of experimental animals by electron microscopy after the animals smoked for 2 hours/day for 60 days and found that the epithelial layer was disturbed. Loss of desmosomes and widening of the distances between cells were detected. In addition, cells on the luminal surface became keratinized. Smith et al15 detected squamous metaplasia of the larynx and trachea by electron microscopy after exposure of animals to smoking for 3–4 weeks. These studies all concluded that the short-term detrimental effects of regular cigarette smoking were evident in rats. Although no study has evaluated the relevance of ENDS smoke exposure of rats to exposure in humans, this comparison was made for regular smoking via carboxyhemoglobin (CoHb), serum nicotine level, and urine cotinine level.34 Studies revealed that CoHb levels measured immediately after exposure to regular smoking were between 11% and 19%, which was above the level in human smokers who smoked 30 cigarettes/day. In addition, higher morning blood nicotine level was detected compared with humans. The cotinine/creatinine level was comparable with those in humans who smoked 20 cigarettes/day. This study involved 1 hour of smoke exposure twice daily on weekdays and once daily on weekends. We did not take these measurements because no study performed in humans has been published. No prior study has evaluated the effects of ENDS on the laryngeal mucosa. We found changes in laryngeal mucosa exposed to 0.9% (wt/vol) nicotine for 1 hour/day over 4 weeks, but these changes were not significant. Our study suggested that ENDS might have effects on the vocal fold epithelium although the reduction in particle numbers and the use of vaporization rather than combustion seem to render ENDS safer than regular
TABLE 2. Number of Metaplasia in Study and Control Groups Metaplasia Control group Study group Total
Negative
Positive
Total
7 (87.5%) 4 (50%) 11 (68.8%)
1 (12.5%) 4 (50%) 5 (31.3%)
8 (100%) 8 (100%) 16 (100%)
Notes: Pearson chi-squared value (P) ¼ 0.106.
FIGURE 3. Normal epithelial distribution with Ki67 staining.
4 cigarette smoking.4,26 Regarding the main limitation of our study—the limited number of samples—further studies with longer duration and larger cohorts are required. Additional electron microscopic studies might also reveal further changes. Furthermore, we used a 0.9% (wt/vol) nicotine solution. As there exists a wide range of solutions containing different materials that have not been standardized, various liquids differing in nicotine content should also be evaluated. CONCLUSIONS Although we found that ENDS administration for 4 weeks did not cause inflammation or any anomalies in epithelial distribution. Hyperplasia and metaplasia were not significant. Studies of longer duration and with larger cohorts are required to evaluate effects of ENDS on vocal folds. REFERENCES 1. US Centers for Disease Control: How tobacco smoke causes disease—the biology and behavioral basis for smoking-attributable disease fact sheet. Available at: http://www.surgeongeneral.gov/library/tobaccosmoke/ factsheet.html. 2. Buchhalter AR, Acosta MC, Evans SE, Breland AB, Eissenberg T. Tobacco abstinence symptom suppression: the role played by the smoking-related stimuli that are delivered by denicotinized cigarettes. Addiction. 2005; 100:550–559. 3. Bullen C, Williman J, Howe C, Laugesen M, McRobbie H, Parag V, Walker N. Study protocol for a randomised controlled trial of electronic cigarettes versus nicotine patch for smoking cessation. BMC Public Health. 2013;13:210. 4. Goniewicz ML, Knysak J, Gawron M, et al. Levels of selected carcinogens and toxicants in vapour from electronic cigarettes. Tob Control. 2014;23:133–139. _ 5. Ingebrethsen BJ, Cole SK, Alderman SL. Electronic cigarette aerosol particle size distribution measurements. Inhal Toxicol. 2012;24:976–984. 6. William M, Villareal A, Bozhilov K, Lin S, Talbot P. Metal and silicate particles including nanoparticles are present in electronic cigarette cartomizer fluid and aerosol. PLoS One. 2013;8. 7. Kosmider L, Sobczak A, Fik M, Knysak J, Zaciera M, Kurek J, Goniewicz ML. Carbonyl compounds in electronic cigarette vaporseffects of nicotine solvent and battery output voltage. Nicotine Tob Res. 2014;16:1319–1326. 8. Auerbach O, Hammond EC. Histologic changes in the larynx in relation to smoking habits. Cancer. 1970;25:92–104. 9. Muller KM, Khron BR. Smoking habits and their relationship to precancerous lesions of the larynx. J Cancer Res Clin Oncol. 1980;96:211–217. 10. Zietel SM. Premalignant epithelium and microinvasive cancer of the vocal fold: the evolution of phonomicrosurgical management. Laryngoscope. 1995;45:1–44. 11. Zietel SM, Hilmann RE, Bunting GW, Vaughn T. Reinke’s edema: phonatory mechanisms and management strategies. Ann Otol Rhinol Laryngol. 1997;106:533–543. 12. Hirabayashi H, Koshii K, Uno K, et al. Laryngeal epithelial changes on effects of smoking and drinking. Auris Nasus Larynx. 1990;17:105–114. 13. Duarte JL, de Faria FA, Ceolin DS, Cestari TM, de Assis GF. Effects of passive smoke inhalation on the vocal folds of rats. Braz J Otorhinolaryngol. 2006;72:210–216.
Journal of Voice, Vol. -, No. -, 2015 14. Is¸ik AC, Kalender Y, Yardimci S, Erg€un A. Environmental tobacco smoke in rats. J Otolaryngol. 2004;33:382–386. 15. Smith G, Wilton LV, Binns R. Sequential changes in the structure of the rat respiratory system during and after exposure to cigarette smoke. Toxicol Appl Pharmacol. 1978;46:579–591. 16. Saydam L, Bozkurt MK. Larenks Kanserleri. In: Koc¸, ed. Kulak Burun Bogaz Hastalıkları Bas¸ ve Boyun Cerrahisi. 2nd ed. Ankara, Turkey: G€unes¸ Tıp Kitabevleri; 2013:1105–1113. 17. Khilnani G, Thaddanee R, Khilnani AK. The smoked drum. Indian J Pharmacol. 2013;45:643–645. 18. U.S. Dept. of Health and Human Services (USDHHS). Women and Smoking. A report of the Surgeon General. Rockville, MD: U.S. Dept of Health and Human Services, Public Health Service, Office of the Surgeon General; 2001. 19. Kenfield SA, Stampfer MJ, Rosner BA, Colditz GA. Smoking and smoking cessation in relation to mortality in women. JAMA. 2008;299:2037–2047. 20. Freund KM, D’Agostino RB, Belanger AJ, Kannel WB, Stokes J 3rd. Predictors of smoking cessation: the Framingham study. Am J Epidemiol. 1992;135:957–964. 21. Campbell J, Tønnsen P, Gustavsson G, Morera J. Sociodemographic predictors of success in smoking intervention. Tob Control. 2001;10:165–169. 22. Abdolahinia A, Sadr M, Hessami Z. Correlation between the age of smoking initiation and maintaining continuous abstinence for 5 years after quitting. Acta Med Iran. 2012;50:755–759. 23. Glasheen C, Hedden SL, Forman-Hoffman VL, Colpe LJ. Cigarette smoking behaviors among adults with serious mental illness in a nationally representative sample. Ann Epidemiol. 2014;24:776–780. 24. Philbrick AM, Newkirk EN, Farris KB, McDanel DL, Horner KE. Effect of a pharmacist managed smoking cessation clinic on quit rates. Pharm Pract (Granada). 2009;7:150–156. 25. Bullen C, McRobbie H, Thornley S, Glover M, Lin R, Laugesen M. Effect of an electronic nicotine delivery device (e cigarette) on desire to smoke and withdrawal, user preferences and nicotine delivery: randomised crossover trial. Tob Control. 2010;19:98–103. 26. Lee S, Kimm H, Yun JE, Jee SH. Public health challenges of electronic cigarettes in South Korea. J Prev Med Public Health. 2011;44:235–241. 27. Williams M, Villareal A, Bozhilov K, Lin S, Talbot P. Metal and silicate particles are present in electronic cigarette cartomizer fluid and aerosol. PLoS One. 2013;8. 28. Manzoli L, La Vecchia C, Flacco ME, et al. Multicentric cohort study on the long term efficacy and safety of electronic cigarettes: study design and methodology. BMC Public Health. 2013;13:883–889. 29. Farsolinos KE, Romagna G, Allifranchini E, Ripamonti E, Bochiotto E, Todeschi S. Comparision of cytotoxic potential of cigarette smoke and electronic cigarette vapour extract on cultured myocardial cells. Int J Environ Res Public Health. 2013;10:5146–5162. 30. World Health Organization, Study Group on Tobacco Regulation. Report on the scientific basis of tobacco product regulation: third report of a WHO study group. Geneva, Switzerland: World Health Organization; 2010. 31. Westenberger BJ. US Food and Drug Administration evaluation of e-cigarettes. Available at: http://www.fda.gov/downloads/Drugs/Science Research/ UCM173250.pdf. € g€ut F, Kılıc¸ MA. Larinksin Benin Lezyonları. In: Koc¸, ed. Kulak Burun 32. O Bogaz Hastalıkları Bas¸ ve Boyun Cerrahisi. 2nd ed. Ankara, Turkey: G€unes¸ Tıp Kitabevleri; 2013:1053–1059. 33. Stanciu C, Bennett JR. Smoking and gastro-oesophageal reflux. Br Med J. 1972;3:793–795. 34. Getryn-Nielsen MJ, Top EV, Preheim LC. A rat model to determine the biomedical consequences of concurrent ethanol ingestion and cigarette smoke exposure. Alcohol Clin Exp Res. 2004;28:1120–1128.
Accepted for publication October 23, 2014. This study has not been published totally or partially before. From the *Okmeydanı Training and Research Hospital, ENT Clinic, Istanbul, Turkey; and the yOkmeydanı Training and Research Hospital, Pathology Clinic, Istanbul, Turkey. Address correspondence and reprint requests to Ziya Salturk, Okmeydanı Training and _ Research Hospital ENT Clinic, Darulaceze cad., S¸is¸li, Istanbul, Turkey. E-mail: [email protected] Journal of Voice, Vol. -, No. -, pp. 1-4 0892-1997/$36.00 Ó 2015 The Voice Foundation http://dx.doi.org/10.1016/j.jvoice.2014.10.013
The vocal fold is the most common site of laryngeal carcinoma,16 and it is vulnerable to vapor produced by ENDS because the cord lies directly in the upper respiratory tract. We sought to evaluate the histopathologic effects of 4 weeks of exposure to vapor created by ENDS on the vocal fold mucosa of rats. MATERIALS AND METHODS Institutional Review Board approval was obtained from the Istanbul University Experimental Animal Research Ethics Committee. Sixteen healthy, adult, female Wistar albino rats (200–250 g; 7–8 months old) were used. A smoke drum designed by C.F. Palmer Ltd. (London, United Kingdom) was used to expose the rats to vapor.17 The machine contains two holes, one for inspiration and the other for expiration. The cigarette was placed in the inspiration hole, smoke was drawn off by vacuuming, and the smoke was conducted via a 1.5-m polyethylene tube to the exit. The cabin was 30 3 40 3 50 cm in dimension and made of plexiglass. There were 10 holes affording aeration, each 2 cm in diameter. The machine yielded 200 mL/minute of smoke. Ego T (China) was the ENDS used and was filled with a solution of 0.9% (wt/ vol) nicotine. The vapor was distributed through the cabin after 20 min. The study rats were treated for 60 minutes/day for 4 weeks. The 16 rats were divided into two groups. Eight rats were exposed to vapor for 60 minutes/day over 4 weeks. Rats had free access to food and water and were kept under a 12-h:12h light-dark cycle. The mean room temperature was 25 C. The other eight rats were not exposed to vapor and were housed identically. At the end of 4 weeks, rats were sacrificed under high-dose ketamine anesthesia. Each vocal fold was removed intact and stored in 10% (vol/vol) buffered formalin solution. Specimens were cut into 5-mm-thick sections. Standard tissue-processing methods were applied, and the sections were stained with
2 hematoxylin and eosin before analysis by light microscopy. An expert pathologist blinded to group assignment evaluated epithelial distribution, inflammation, hyperplasia, and metaplasia. Inflammation was graded based on cellular density: grade 1 represented scattered inflammatory cells, grade 3 represented inflammatory cell density that forms lymphoid follicles, and grade 2 represented cellular density between grades 1 and 3. Mucosal cellular reactivities were assessed immunohistochemically by staining for Ki67, which is expressed during all phases of the cell cycle except for G0. Expression of Ki67 closely parallels [3H]-thymidine incorporation, which is a standard method of measuring cell proliferation rate. Immunostaining of normal tissue for the Ki67 antigen reveals nuclear reactivity in cells of the germinal centers of cortical follicules, cortical thymocytes, and neck cells of the gastrointestinal mucosa. Resting cells such as hepatocytes, renal cells, and Paneth’s cells of the gastrointestinal mucosa do not show staining. For immunohistochemical evaluation, formalin-fixed paraffin-embedded tissue blocks were cut at a thickness of 5 mm and mounted on poly L-lysine coated slides, followed by staining with an anti-Ki67 antibody (BioGenex, San Ramon, CA; clone: BGX-Ki67, 1:100 dilution). All procedures were performed using the standard protocol of a Leica Bond Max autostainer. Positive and negative controls were run. Any identifiable nuclear staining, regardless of intensity, was recorded as positive. Distribution of Ki67 in the basal and parabasal areas of the laryngeal mucosa was evaluated. Statistical analysis was performed using the aid of SPSS software, version 17. Pearson’s chi-squared test was used for data analysis. Because the epithelial distributions and cellular reactivities of the mucosa were identical in both groups, these data were not analyzed. P < 0.05 was accepted as significant.
RESULTS No macroscopic difference was evident between larynges of the study and control groups. Microscopically, three types of epithelial cells were detected in the control group. The ventral part of the glottis was composed of striated ciliary epithelium adjacent to loose connective tissue. The free margins of the vocal folds and adjacent areas were covered by striated nonciliated columnar epithelium. The arytenoid region and adjacent areas were covered by striated squamous epithelium, which was thinner on the arytenoid cartilages. The study group exhibited similar histopathologic features. Both groups exhibited typical epithelial linings and basal membranes. The extent of inflammation did not differ between the two groups (Figure 1; Table 1). Squamous metaplasia was detected in four of the eight rats in the study group but in only one rat in the control group (Figure 2; Table 2); this difference was not significant (P ¼ 0.106). Two larynges in the study group developed hyperplasia, compared with none in the control group (Table 3). There was also no statistically significant difference (P ¼ 0.131). Ki67 expression levels were examined to evaluate cellular reactivities. All larynges revealed a basal proliferation pattern (Figure 3); no between-group difference was apparent.
Journal of Voice, Vol. -, No. -, 2015
FIGURE 1. Normal vocal fold epithelia and mild subepithelial inflammation.
DISCUSSION Smoking is the leading cause of preventable deaths in the United States, and the harmful effects thereof can be reduced by quitting.18,19 Many techniques aiding quitting have been developed, but they are not very effective, even when combined with counseling programs, because smoking is affected by many factors including age, sex, time of onset of smoking, housing conditions, and health status.20–24 E-cigarettes deliver nicotine and mimic the action of smoking. Therefore, e-smoking satisfies the behavioral, sensory, and physical components of smoking.2,25 Thus, the quitting success rate using this technique may be higher than that achieved using other methods. ENDS safety has been widely discussed, and e-cigarettes are forbidden in some countries. Studies on the health effects of ENDS have appeared.26–29 The health risk was higher than those of various nicotine replacement therapies, but lower than that of smoking.30,31 Smoking is the leading cause of laryngeal cancer, the risk of which is 35-fold higher in smokers than nonsmokers. The type of tobacco used, the number of cigarettes smoked, the duration of smoking history, and passive exposure are all independent risk factors.16 In addition, smoking causes Reinke’s edema, chronic laryngitis, and diffuse vocal fold polyps.32 Smoking also triggers reflux affecting the larynx.32,33 Cadaveric studies of histopathologic changes caused by smoking in the human larynx found epithelial changes.8,9 Aurbach and Hammond8 performed a histologic study of the effects of smoking on the larynx using cadavers and concluded that the true vocal fold was the main site affected by smoking.
TABLE 1. Status of Inflammation Inflammation
1+
2+
3+
Total
Control group 1 (12.5%) 5 (62.5%) 2 (25%) 8 (100%) Study group 3 (37.5%) 3 (37.5%) 2 (25%) 8 (100%) Total 4 (25%) 8 (50%) 4 (25%) 16 (100%) Notes: Pearson chi-squared value (P) ¼ 1.500.
Ziya Salturk, et al
3
Effects of electronic cigarette on larynx
TABLE 3. Number of Hyperplasia in Study and Control Groups Hyperplasia Control group Study group Total
Negative
Positive
Total
8 (100%) 6 (75%) 14 (87.5%)
0 (0%) 2 (25%) 2 (12.5%)
8 (100%) 8 (100%) 16 (100%)
Notes: Pearson chi-squared value (P) ¼ 0.131.
FIGURE 2. Metaplasia in the study group. To a lesser degree, the false vocal fold and the area of the larynx above the vocal fold were affected. Comparison of smokers’ and nonsmokers’ larynxes revealed that histologic changes (including cells atypical nuclei, carcinoma in situ, and early invasive carcinoma) were much greater in cigarette smokers; they also increased in proportion to the number of cigarettes smoked per day before death and were seen to a greater degree in cigarette smokers than in cigar and pipe smokers. Similarly, M€ uller and Khron9 concluded that smoking caused precancerous lesions in humans. These findings were also supported by clinical studies.10,12 Zietel10 suggested that geographic localization of keratotic and erythroplastic lesions on the superior/ ventricular surface of the musculomembranous vocal fold are likely to contain atypia in smokers. It was also shown that metaplasia in the supraglottic region increased with aging and with tobacco and alcohol consumption.12 Animal studies have confirmed the effects of smoking on the laryngeal mucosa and have enabled an understanding of the developmental stages that precede cancer. However, the ENDS is a recent technological innovation, and no research on the use of ENDS has yet been published. We did not detect chronic inflammation or allergic reactions. Epithelial responses, including hyperplasia and metaplasia, were evident but not significant. Although we did not detect any dysplasia, we suggest that further studies with longer follow-up periods and higher nicotine dosages be performed. Duarte et al13 evaluated the outcomes of passive smoke inhalation of 10 cigarettes daily at 25, 50, and 75 days. Histopathologic evaluation revealed hyperplasia and metaplasia. Is¸ık
et al14 evaluated the vocal folds of experimental animals by electron microscopy after the animals smoked for 2 hours/day for 60 days and found that the epithelial layer was disturbed. Loss of desmosomes and widening of the distances between cells were detected. In addition, cells on the luminal surface became keratinized. Smith et al15 detected squamous metaplasia of the larynx and trachea by electron microscopy after exposure of animals to smoking for 3–4 weeks. These studies all concluded that the short-term detrimental effects of regular cigarette smoking were evident in rats. Although no study has evaluated the relevance of ENDS smoke exposure of rats to exposure in humans, this comparison was made for regular smoking via carboxyhemoglobin (CoHb), serum nicotine level, and urine cotinine level.34 Studies revealed that CoHb levels measured immediately after exposure to regular smoking were between 11% and 19%, which was above the level in human smokers who smoked 30 cigarettes/day. In addition, higher morning blood nicotine level was detected compared with humans. The cotinine/creatinine level was comparable with those in humans who smoked 20 cigarettes/day. This study involved 1 hour of smoke exposure twice daily on weekdays and once daily on weekends. We did not take these measurements because no study performed in humans has been published. No prior study has evaluated the effects of ENDS on the laryngeal mucosa. We found changes in laryngeal mucosa exposed to 0.9% (wt/vol) nicotine for 1 hour/day over 4 weeks, but these changes were not significant. Our study suggested that ENDS might have effects on the vocal fold epithelium although the reduction in particle numbers and the use of vaporization rather than combustion seem to render ENDS safer than regular
TABLE 2. Number of Metaplasia in Study and Control Groups Metaplasia Control group Study group Total
Negative
Positive
Total
7 (87.5%) 4 (50%) 11 (68.8%)
1 (12.5%) 4 (50%) 5 (31.3%)
8 (100%) 8 (100%) 16 (100%)
Notes: Pearson chi-squared value (P) ¼ 0.106.
FIGURE 3. Normal epithelial distribution with Ki67 staining.
4 cigarette smoking.4,26 Regarding the main limitation of our study—the limited number of samples—further studies with longer duration and larger cohorts are required. Additional electron microscopic studies might also reveal further changes. Furthermore, we used a 0.9% (wt/vol) nicotine solution. As there exists a wide range of solutions containing different materials that have not been standardized, various liquids differing in nicotine content should also be evaluated. CONCLUSIONS Although we found that ENDS administration for 4 weeks did not cause inflammation or any anomalies in epithelial distribution. Hyperplasia and metaplasia were not significant. Studies of longer duration and with larger cohorts are required to evaluate effects of ENDS on vocal folds. REFERENCES 1. US Centers for Disease Control: How tobacco smoke causes disease—the biology and behavioral basis for smoking-attributable disease fact sheet. Available at: http://www.surgeongeneral.gov/library/tobaccosmoke/ factsheet.html. 2. Buchhalter AR, Acosta MC, Evans SE, Breland AB, Eissenberg T. Tobacco abstinence symptom suppression: the role played by the smoking-related stimuli that are delivered by denicotinized cigarettes. Addiction. 2005; 100:550–559. 3. Bullen C, Williman J, Howe C, Laugesen M, McRobbie H, Parag V, Walker N. Study protocol for a randomised controlled trial of electronic cigarettes versus nicotine patch for smoking cessation. BMC Public Health. 2013;13:210. 4. Goniewicz ML, Knysak J, Gawron M, et al. Levels of selected carcinogens and toxicants in vapour from electronic cigarettes. Tob Control. 2014;23:133–139. _ 5. Ingebrethsen BJ, Cole SK, Alderman SL. Electronic cigarette aerosol particle size distribution measurements. Inhal Toxicol. 2012;24:976–984. 6. William M, Villareal A, Bozhilov K, Lin S, Talbot P. Metal and silicate particles including nanoparticles are present in electronic cigarette cartomizer fluid and aerosol. PLoS One. 2013;8. 7. Kosmider L, Sobczak A, Fik M, Knysak J, Zaciera M, Kurek J, Goniewicz ML. Carbonyl compounds in electronic cigarette vaporseffects of nicotine solvent and battery output voltage. Nicotine Tob Res. 2014;16:1319–1326. 8. Auerbach O, Hammond EC. Histologic changes in the larynx in relation to smoking habits. Cancer. 1970;25:92–104. 9. Muller KM, Khron BR. Smoking habits and their relationship to precancerous lesions of the larynx. J Cancer Res Clin Oncol. 1980;96:211–217. 10. Zietel SM. Premalignant epithelium and microinvasive cancer of the vocal fold: the evolution of phonomicrosurgical management. Laryngoscope. 1995;45:1–44. 11. Zietel SM, Hilmann RE, Bunting GW, Vaughn T. Reinke’s edema: phonatory mechanisms and management strategies. Ann Otol Rhinol Laryngol. 1997;106:533–543. 12. Hirabayashi H, Koshii K, Uno K, et al. Laryngeal epithelial changes on effects of smoking and drinking. Auris Nasus Larynx. 1990;17:105–114. 13. Duarte JL, de Faria FA, Ceolin DS, Cestari TM, de Assis GF. Effects of passive smoke inhalation on the vocal folds of rats. Braz J Otorhinolaryngol. 2006;72:210–216.
Journal of Voice, Vol. -, No. -, 2015 14. Is¸ik AC, Kalender Y, Yardimci S, Erg€un A. Environmental tobacco smoke in rats. J Otolaryngol. 2004;33:382–386. 15. Smith G, Wilton LV, Binns R. Sequential changes in the structure of the rat respiratory system during and after exposure to cigarette smoke. Toxicol Appl Pharmacol. 1978;46:579–591. 16. Saydam L, Bozkurt MK. Larenks Kanserleri. In: Koc¸, ed. Kulak Burun Bogaz Hastalıkları Bas¸ ve Boyun Cerrahisi. 2nd ed. Ankara, Turkey: G€unes¸ Tıp Kitabevleri; 2013:1105–1113. 17. Khilnani G, Thaddanee R, Khilnani AK. The smoked drum. Indian J Pharmacol. 2013;45:643–645. 18. U.S. Dept. of Health and Human Services (USDHHS). Women and Smoking. A report of the Surgeon General. Rockville, MD: U.S. Dept of Health and Human Services, Public Health Service, Office of the Surgeon General; 2001. 19. Kenfield SA, Stampfer MJ, Rosner BA, Colditz GA. Smoking and smoking cessation in relation to mortality in women. JAMA. 2008;299:2037–2047. 20. Freund KM, D’Agostino RB, Belanger AJ, Kannel WB, Stokes J 3rd. Predictors of smoking cessation: the Framingham study. Am J Epidemiol. 1992;135:957–964. 21. Campbell J, Tønnsen P, Gustavsson G, Morera J. Sociodemographic predictors of success in smoking intervention. Tob Control. 2001;10:165–169. 22. Abdolahinia A, Sadr M, Hessami Z. Correlation between the age of smoking initiation and maintaining continuous abstinence for 5 years after quitting. Acta Med Iran. 2012;50:755–759. 23. Glasheen C, Hedden SL, Forman-Hoffman VL, Colpe LJ. Cigarette smoking behaviors among adults with serious mental illness in a nationally representative sample. Ann Epidemiol. 2014;24:776–780. 24. Philbrick AM, Newkirk EN, Farris KB, McDanel DL, Horner KE. Effect of a pharmacist managed smoking cessation clinic on quit rates. Pharm Pract (Granada). 2009;7:150–156. 25. Bullen C, McRobbie H, Thornley S, Glover M, Lin R, Laugesen M. Effect of an electronic nicotine delivery device (e cigarette) on desire to smoke and withdrawal, user preferences and nicotine delivery: randomised crossover trial. Tob Control. 2010;19:98–103. 26. Lee S, Kimm H, Yun JE, Jee SH. Public health challenges of electronic cigarettes in South Korea. J Prev Med Public Health. 2011;44:235–241. 27. Williams M, Villareal A, Bozhilov K, Lin S, Talbot P. Metal and silicate particles are present in electronic cigarette cartomizer fluid and aerosol. PLoS One. 2013;8. 28. Manzoli L, La Vecchia C, Flacco ME, et al. Multicentric cohort study on the long term efficacy and safety of electronic cigarettes: study design and methodology. BMC Public Health. 2013;13:883–889. 29. Farsolinos KE, Romagna G, Allifranchini E, Ripamonti E, Bochiotto E, Todeschi S. Comparision of cytotoxic potential of cigarette smoke and electronic cigarette vapour extract on cultured myocardial cells. Int J Environ Res Public Health. 2013;10:5146–5162. 30. World Health Organization, Study Group on Tobacco Regulation. Report on the scientific basis of tobacco product regulation: third report of a WHO study group. Geneva, Switzerland: World Health Organization; 2010. 31. Westenberger BJ. US Food and Drug Administration evaluation of e-cigarettes. Available at: http://www.fda.gov/downloads/Drugs/Science Research/ UCM173250.pdf. € g€ut F, Kılıc¸ MA. Larinksin Benin Lezyonları. In: Koc¸, ed. Kulak Burun 32. O Bogaz Hastalıkları Bas¸ ve Boyun Cerrahisi. 2nd ed. Ankara, Turkey: G€unes¸ Tıp Kitabevleri; 2013:1053–1059. 33. Stanciu C, Bennett JR. Smoking and gastro-oesophageal reflux. Br Med J. 1972;3:793–795. 34. Getryn-Nielsen MJ, Top EV, Preheim LC. A rat model to determine the biomedical consequences of concurrent ethanol ingestion and cigarette smoke exposure. Alcohol Clin Exp Res. 2004;28:1120–1128.
