J. Nihon Univ. Sch. Dent., Vol. 39, No. 4, 202-210, 1997

A study of effects of mouthwash on the human oral mucosae: With special references to sites, sex differences and smoking

Kayo Kuyama1 and Hirotsugu Yamamoto2

Departments of Public Health1 and Pathology2, Nihon University School of Dentistry at Matsudo

(Received8 Septemberand accepted20 September1997)

Abstract : In recent years, the use of mouthwash an ingredientof almost all mouthwashesat zero to 23.0 % has become widespread as a part of routine oral (9, 10), was discussed in particular (4, 6-8). In this hygiene. However, there have been no fundamental connection,Gagari et al. (1) pointed out that the exposure studies on the influence of mouthwashes on the human time of ethanol to the oral mucosae by mouthwashing oral mucosae. One hundred and twenty-five subjects was probably longer than that provided by drinking an (50 males and 75 females) were selected for this study. alcoholic beverage. The inflammation and/or The effects of mouthwash was assessed with the use of hyperkeratosis of the hamster cheek pouch caused by exfoliative cytological and cytomorphometric analyses exposure to a commerciallyavailable mouthwash with a of smears obtained from clinically normal upper high ethanol content were examined (11, 12). In a study labium and cheek mucosae before mouthwashing, 30 of human oral mucosae, epithelial peeling, ulceration, s, 10 min and 1 h after mouthwashing. The inflammationand other miscellaneouschanges occurred independent variables examined were oral site, sex in the mucosae as a result of mouthwashing with high- and smoking (smokers versus never-smokers). In all alcoholproducts (13). subjects and sites, the appearance rate of exfoliated At present, 12 manufacturersare producing28 kinds of cells stained by light green SFY decreased just after mouthwash products in Japan, and the sale of these mouthwashing, and the rate after 1 h was lower than mouthwashes comprises 14.6 % of the total sales of that of the untreated controls. The oral mucosae of dental and oral products in the domestic market (14). smokers were more irritated action by mouthwashing Nevertheless, there seems to be almost no fundamental than that of the never-smokers. Even after 1 h, studies of the effects of various mouthwashes on the decreases in the nuclear and cytoplasmic areas of cells human oral mucosae. and increases in inflammatory cells were observed. In In the present study, cytologicaland cytomorphometric conclusion, the use of mouthwash was so inflamed the methods were used to clarify the influence of human oral mucosae that more attention should be mouthwashingon the human oral mucosae in relation to paid when it is used daily in oral hygiene. several internal factors, such as sex differences, sites of mucosa, age and menstrual cycle, and external factors, Key words: mouthwash; oral mucosae; exfoliative such as smokingor never-smokingstatus. cytology; smoking; cytomorphometry. Materials and Methods 1. Subjects Introduction The criteria for enrollment in the study were no oral During the past two decades, the use of mouthwash mucosal disease, no dentures, non-pregnant, a stable has become quite widespread, and with the recent menstrual cycle, not medically compromised, no habitual proliferation of the varieties of mouthwash (1), its alcoholic drinking, and no habitual mouthwashing. unrestricted use by the general public has increased Numerous subjects participated in the questionnaire and substantially. The increased use of various mouthwashes interviews about these criteria. One hundred and twenty- has also resulted in a number of reports of mouthwash- five subjects (50 males and 75 females; age range 18 to associated antimicrobial and antiplaque effects (2, 3). In 30 years old) were selected. All gave informed consent to spite of this increased usage, systematic studies of the participate in the study. A profile of the subjects and effects of mouthwashing on the human oral mucosae experiments is given in Table 1. have not been performed. All prior studies concerning this topic were based on an epidemiological approach. 2. Test solution and application conditions A number of epidemiological studies examined the The test solution, a typical antiseptic mouthwash relationship between the use of mouthwash and the risk formula, is obtainable in the Japanese domestic market of oral cancer (1, 3-8). The influence of ethanol, which is and worldwide. All ingredients of this solution are shown in Table 2 (14, 15). According to the instructions on the label of the mouthwash, as reported as the standard (4, Correspondence to Dr. Kayo Kuyama, Department of Public Health, Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho- 14), the recommended use of the mouthwash was as Nishi, Matsudo, Chiba 271, Japan follows: 20 ml of the product was measured out from the 203

Table 1 Summary of subjects and items of experiments

a : preliminary study, b : the microscopic analysis (composition of exfoliated cells), c : the frequency of inflammatory cells, d : the quantitative cytomorphometric analysis The term "smoker" means who are smoking habitually. "Female never-smoker" is consist of two groups : around ovulatory phase group (menstrual cycle between 10-15 days) and another phase group. The latter group participated in preliminary study.

Table 2 The formula and pH value of the test solution mouthwashing, therefore no brushing was done before this sampling. Smear collections were performed with a Cytobrush, and the range and direction of collection were fixed as follows: the upper labium mucosa, the area between the right and left upper canine teeth, the right cheek mucosa, and the area between the first and second premolars.

4. Observations of the exfoliated cells 1) Microscopic observation. Exfoliated cell samples were surveyed with a light microscope (OLYMPUS, VANOX-S, AH-2) at 100-400 magnification. Five regions with cells of homogeneous distribution were selected randomly, and from each selected region 20 cells bottle just before mouthwashing, kept in the mouth for 30 were closely investigated and classified by staining with s of washing, then spit out completely. This procedure Orange G, Eosin Y and light green SFY mainly in the was followed in the present experiment. cytoplasms (19). The appearance rate of each type of cell was also measured. In addition, all inflammatory cells, 3. Sampling method of smears mainly lymphocytes and neutrophils, in the same 5 Smears were taken from the tissue by stroking the regions of all samples were counted (Table 1). surface of the mucosa membrane with a Cytobrush (16). The smear samples were then immediately transferred to 2) Quantitative cytomorphometric analysis. In the glass slides by spreading them on the surface while cytomorphometric analysis of the effects of mouthwash, turning the slide in a clockwise direction. The slides were the nuclear (NA) and cytoplasmic (CA) areas of cells then fixed with 95 % ethanol and stained with standard within each smear sample were measured using a semi- Papanicolaou stain (17). automatic image analysis system (the NIH Image Smears were collected according to the following program, Table 1). At least 100 cells per sample were protocol. measured. The data obtained by this method were in satisfactory agreement with those obtained by the 1) Sample collecting times. Samples were collected microscopic observation. just before mouthwashing (control), 30 s after, 10 min after and 1 h after mouthwashing. 5. Statistical analysis Simple descriptive statistics were used to evaluate the 2) Sites and other factors. The collection sites were distribution of the study subjects according to sites, sex, the upper labium and right cheek. These areas were smoking status and menstrual cycle. The appearance rate characterized as lining mucosae by their distention ability of each classified cell in relation to other factors such as and nonkeratinized nature (18). From the time of site, sex differences and smoking status was examined mouthwashing until after the final sampling (1 h after), mainly by Wilcoxon's U test. Significance of differences drinking, eating, cigarette smoking and other between/among studied data were examined by Student's mouthwashing were absolutely prohibited. The purpose t test. Probability ("p") values less than 0.05 were of this study was to examine the chemical effect of considered statistically significant. 204

Results The appearance rate of exfoliated cells from the oral 1. Preliminary examination mucosae for all subjects are given in Table 3. The smears As a preliminary examination, samples for the from the upper labium and cheek mucosae were occupied cytological procedure were obtained from 50 of the by the cells stained by light green SFY at 32.0 % and subjects without their use of the mouthwash product 54.0 %, respectively, which are significantly different (p (Table 1). To investigate the effects of taking samples < 0.01). The female subjects showed a significantly with the Cytobrush and of mouthwashing with saline on higher rate of keratinization (cells stained by Orange G the composition of the exfoliated cells, smear samples (17)) in comparison with male subjects (p < 0.01). The were collected and studied cytologically. No significant keratinization rates in the upper labium and cheek effects of the Cytobrush use or of the saline on the mucosae of the male smokers were 2.5- and 2.9- fold of composition of exfoliated cells and frequency of the male never-smokers (p < 0.01, Figs. 3-a, 4-a). The inflammatory cells were observed (p > 0.01). keratinization rates at both sites were more influenced by menstrual cycle than smoking status (p < 0.01) in the 2. Differences in the components of exfoliated females (Fig. 5-a). cells from the oral mucosae in controls

Table 3 Results of all subjects about component differences of exfoliated cells from the oral mucosae

Table 4 Cytomophometric analysis of average NA and CA (pixcels) and NA/CA ratio before mouthwashing , after 30 s, 10min and lh

**P<0 .01, *P<0.05 205

Fig.1 A time series and transition of component of exfoliated cells after mouthwashing. ** (p < 0.01) 0; cells stained by Orange G, E; cells stained by Eosin Y, L; cells stained by light green SFY 206

Fig. 2 Number of leukocytes (neutrophils > lymphocytes), before mouthwashing, and 30 s, 10 min and lh after mouthwashing.

a b c d

Fig. 3-a Smear preparation of the upper labium in a male never-smoker before mouthwashing. Cells stained mainly by light green SFY and Eosin Y. (Pap. stain, x100) Fig. 3-b Smear preparation of the upper labium in a male never-smoker 30 s after mouthwashing. A shrinkage of the cytoplasm was observed. (Pap. stain, x100) Fig. 3-c Smear preparation of the upper labium in a male never-smoker 10 min after mouthwashing . A few leukocytes were found. (Pap. stain, x100) Fig. 3-d Smear preparation of the upper labium in a male never-smoker 1 h after mouthwashing . Shrinkage of the cytoplasm was recovered relative to fig. 3-c, after 10 min. (Pap. stain, x100) 207

a b c d

Fig. 4-a Smear preparation of the upper labium in a male smoker before mouthwashing. Consists of mainly two kinds ofcells stained by light green and Eosin Y. There are more keratinized cells than in the never-smoker depicted in fig. 3-a. (Pap. stain, x100) Fig. 4-b Smear preparation of the upper labium in a male smoker 30 s after mouthwashing. The shrinkage of cytoplasmsand some leukocytes were apparent. (Pap. stain, x100) Fig. 4-c Smear preparation of the upper labium in a male smoker 10 min after mouthwashing. Consists of three kinds of cells stained by light green, Eosin Y and Orange G. Fairly large number of scattered leukocytes were observed.(Pap. stain, x100) Fig. 4-d Smear preparation of the upper labium in a male smoker mouthwashing 1 h after mouthwashing. Numerous leukocytes were found in the field. (Pap. stain, x100)

a b c d

Fig. 5-a Smear preparation of the upper labium in a female never-smoker in the ovulatory phase before mouthwashing. Consists of mainly three kinds of cells stained by light green and Eosin Y and Orange G. (Pap. stain, x100) Fig. 5-b Smear preparation of the upper labium in a female never-smoker in the ovulatory phase 30 s after mouthwashing. Many non- nucleated cells stained by Orange G and the shrinkage of cytoplasm were observed in the field. (Pap. stain, x100) Fig. 5-c Smear preparation of the upper labium in a female never-smoker in the ovulatory phase 10 min after mouthwashing. The keratinization rate was increased compared with fig. 5-a: control. (Pap. stain, x100) Fig. 5-d Smear preparation of the upper labium in a female never-smoker in the ovulatory phase 1 h after mouthwashing. The keratinization rate was recovered compared with fig. 5-c: after 10 min. (Pap. stain, x100)

Fig. 7 Smear preparation of the cheek in a male smoker 1 h Fig. 6 Smear preparation of the cheek in a male never-smoker after mouthwashing. A Large number of scattered 30 s after mouthwashing. The shrinkage of cytoplasm was observed clearly. (Pap. stain, x200) leukocytes were seen (Pap. stain, x200) 208

3. Effects of mouthwashing on the oral mucosal pointed out the irritation of the oral mucosae caused by cells ethanol (1, 4, 6, 8). The test solution used in the present 1) Microscopic analysis of exfoliated cells. The study is more than 20.0 % ethanol. Its other ingredients exfoliated cell compositions at each time point after have been described and have been sold for at least the mouthwashing are shown in Fig.l. For all subjects and past five years in the domestic market. sites, just after mouthwashing, the keratinization rate of exfoliated cells was significantly increased compared to 2. Differences in the components of exfoliated the control values. At 1 h after mouthwashing, the cells from the oral mucosae keratinization rate was still increased (p < 0.01) compared The experimental procedure of the present study to the control values in the cheek mucosa of the male examined the influence of factors including sex, site, and subjects and to both sites of mucosae of the female smoking on the effects of mouthwash on the oral subjects (p < 0.01). A conspicuous increase in the mucosae. The component of exfoliated cells before keratinization rate after mouthwashing was seen in the mouthwashing, i.e., control value in the present study, upper labium mucosa of the female subjects (Fig. 5-b, c). revealed that these factors had an effect that was The neutrophil counts reached the maximum point 1 h consistent with those obtained in previous studies (19, after mouthwashing, as did lymphocytes in the male and 21-23). Miyashita et al. (19) investigated the composition female smokers in both the upper labium and the cheek of exfoliated cell samples of the oral mucosae, and their mucosae (p < 0.01, Fig. 4-d, 7). In contrast, an increase of observation of site-dependent differences was in neutrophils was observed in the never-smokers, until 10 agreement with histological findings of the same subjects. min after mouthwashing. At 1 h, recovery from The outer most layer was covered by superficial cells inflammation was observed. Lymphocytes remained at which had a loose connection of stratified squamous the same level at 10 min and 1 h after mouthwashing epithelium. Cytological investigations of the oral (Fig. 2). mucosae also revealed a sex-dependent difference which 2) Cytomorphometric analysis of exfoliated cells. suggested the existence of a connection to the increase/ Table 4 shows the mean NA and CA values. According to decrease of the estrogen/progesterone concentration in the cytomorphometric analysis (NIH Image), the NA of the blood (19, 21, 22). the smokers decreased to a minimum at 10 min, Few studies using exfoliative cytology revealed the significantly smaller than the control value (p < 0.01), effect of smoking on the oral mucosae. Brown et al. (23), and at 1 h revealed recovery signs at both sites. The NA who studied the hard palate and buccal mucosae, found of the smokers had decreased significantly at lh that the keratinization in smokers was increased compared to the control values (p < 0.01). In contrast, the compared with that of non-smokers. This difference NA of never-smokers recovered at 1 h. The CA of the might be due to the possible existence of the thicker smokers at the upper labium reached its minimum at 30 s keratinized layer of the oral mucosae and loose (Figs. 4-b, 6), and the CA value at the cheek reached its connection among stratified squamous epithelia caused minimum at 10 min; thereafter, no significant recovery by the chronic irritation of smoking (24). Furthermore, was seen at 1 h compared with the control values (both hyperkeratosis of the oral mucosae caused by the chronic sites: p < 0.01), whereas the CA of the never-smokers had irritation of smoking which is due to nicotine and related recovered by 1h. thermal decomposition products and/or heat stimuli, was reported (25). Meanwhile, Ogden et al. (26) described a Discussion slight increase in the NA value of exfoliated cells 1. Selection of the test method obtained from the oral mucosae of smokers compared To carry out a study such as the present investigation, with that of non-smokers in cancer-free patients. it is necessary to repeatedly collect smears from the same subjects according to the fixed procedure of sampling. It 3. Influence of mouthwashing on the oral is also important to avoid an invasive sampling procedure mucosae and to make sure each subject understands the test In addition to 20.0 % ethanol, the test solution contains procedure. Miyashita et al. (19) investigated the surfactants and other ingredients, and has a pH of 4.2. A consistency of cytological findings of exfoliated cells previous report (27) described exfoliation caused by the using histological findings, and found that the findings swelling of the upper layer of the oral mucosae after the were in agreement; other studies supported this use of a dentifrice. This oral mucosae exfoliation was conclusion (16, 20). Based on these considerations, the considered to be due mainly to the surfactants, but other exfoliative cytological method was used in the present components influence the exfoliation by their co- study. existence with surfactants. In recent years, many mouthwash products have been In the present study, keratinized and inflammatory put on the market. The descriptions of their benefits cells were increased for all subjects at 30 s, and they include the prevention of periodontitis, caries, halitosis already showed indications of recovery from and plaque control (14), however, very few studies (13) inflammation at 1 h, but it was not a complete recovery. of their influence on the human oral mucosae have been The significant increase in the frequency of leukocytes of conducted. The major ingredient of most mouthwashes, inflammation observed at both sites exposed to a after water, is ethanol (9, 10, 15), and several reports have mouthwash indicated the emigration caused by 209 leucocytosis-promoting factor (28). These reactions, reactions against chemicals and others caused by the observed in the present study, might indicate the typical smoking habit. acute inflammation of the oral mucosae . The results revealed that a high content of ethanol, low pH, and other 4. Roles of mouthwashes in oral hygiene ingredients constitute potential irritants, both individually All of the subjects of this study reported some pain and synergistically. The induction of oral mucosal induced by mouthwashing. Such pain has a relation to the reactions by a high-ethanol mouthwash is especially ethanol content and the duration of the mouthwashing interesting in view of the role of ethanol in the (34). In all of the present subjects, the connection of epidemiologic background of oral cancer. Decreases of keratinized cells of the oral mucosae became looser just NA and CA values were observed in all subjects in the after mouthwashing, and at 1 h a higher keratinization present study, suggesting a dehydration of the protein rate of mucosa, decreases of NA and CA and an increase hydrate of the cytoplasm caused by the 2.0 % ethanol of inflammatory cells were identified. These results contained in the mouthwash. These results call to mind indicate potentially important detriments of mouthwashes the finding that Saccharomyces were degenerated and as a daily-use agent in oral hygiene. In conclusion, our ceased fermentation activity when exposed to ethanol at study revealed clear influence of mouthwash on the 10 % or more (29). In any case, these results suggest that human oral mucosae by using exfoliative cytological and more attention should be paid to the customary use of cytomorphometric analyses. mouthwash. There was no sex difference in the effect of Acknowledgments mouthwash on the oral mucosae at both sites, but there The authors are indebted to Prof. S. Otake was a variance in microscopic results among the (Department of Clinical Pathology, Nihon University individual female subjects in this study. This finding School of Dentistry at Matsudo) and Prof. M. Morimoto suggests that the estrogen level might have a role in the (Research Institute, Nihon University) for their advice differentiation of epithelial cells (22). Several recent regarding the study and the manuscript, and appreciate epidemiological analyses of oral cancer have found that Prof. Y. Kozawa (Department of Anatomy, Nihon being female may be an important factor of oral cancer University School of Dentistry at Matsudo), Prof. H. caused by mouthwashing (6, 7, 30). However, the Nakura (Department of Oral Surgery, Nihon University information about the relationship between gender and School of Dentistry at Matsudo) and Prof. T. Maeda oral cancer is inadequate to draw any conclusions and (Department of Pedodontics, Nihon University School of further investigations of this problem are required. Dentistry at Matsudo) for reviewing the manuscript. The The speed of recovery from mucosal reactions, i.e., the authors also wish to thank Dr. T. Horiuchi (Department increase in the rate of keratinization, the frequency of of Public Health, Nihon University School of Dentistry at inflammatory cells and the shrinkage of the NA and CA Matsudo) for his advice during the conduct of this study, values of the smokers, were lower than those of the and to thank Mr. T. 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