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.