Evaluation of keratinization and AgNORs count in exfoliative cytology of normal oral mucosa from smokers and non-smokers

ORELLANA-BUSTOS AI, ESPINOZA-SANTANDER IL, FRANCO-MARTÍNEZ E, LOBOS-JAMES-FREYRE N, ORTEGA-PINTO AV. EVALUATION OF KERATINIZATION AND AGNORS COUNT IN EXFO-LIATIVE CYTOLOGY OF NORMAL ORAL MUCOSA FROM SMOKERS AND NON-SMOKERS. MED ORAL 2004;9:197-203.

SUMMARY

Objetive.In smokers with clinically normal buccal mucosa, cytological changes such as increased keratinization, and higher nucleolar activity have been observed. In these studies the cells for cytological smears were obtained with a wooden spatula.
Our objectives were to evaluate the depth of cytological smears of oral mucosa obtained with both a brush (endobrush) and a wooden spatula, and to compare the degree of keratinization and the nucleolar activity in smokers and non-smokers.
Design. We obtained cytological smears of clinically normal lateral tongues of 30 smokers and 30 non-smokers using both a wooden spatula and endobrush. The samples were dyed with Papanicolaou and the AgNORs.
Results. With the wooden spatula we found a greater percentage of enucleated superficial epithelial cells (P = 0.016) and deeper cells were obtained with an endobrush (intermediate cells) (P = 0.035). The smokers showed a greater percentage of enucleated superficial cells with both techniques, however this difference was significantly greater with Endobrush (P=0.005). The average of AgNORs in the nucleated cells was greater in smokers (3.83) than in non-smokers (2.79) (P=0.003).
Conclusion. The Endobrush allows the clinician to obtain deeper cells of buccal mucosa. Smokers with clinically normal mucosa show a greater percentage of keratinized cells and a greater nucleolar activity, suggesting that cigarette smoking influences the cellular activity of the mucosa of the lateral tongue.

Key words: AgNOR, nucleolar organizer region, oral cytology, oral mucosa, smoking.

INTRODUCTION

One of the most important risk factors of oral cancer is tobacco smoking (1-5). This risk is associated with the number of cigarettes smoked and the length of time of the habit (2,6). In a survey made three years ago in Chile, it was observed that 48.7% of urban people between ages 12 and 64 consumed tobacco (7).

The most common site for intraoral squamous cell carcinoma is the tongue usually the lateral and ventral surfaces (8,9).

In smokers with clinically healthy oral mucosa it has been determined some changes such as: a higher rate of proliferation of epithelial cells (10), nuclear and cytoplasm alterations (11-13) as well as an increase in the number of keratinized cells (14) and the AgNORs count (Nucleolar Organizer Regions affinity by Silver) (15,16).

The NORs or Nucleolar Organizer Regions are loops of DNA located in chromosomes 13-15 and 21. They are responsible for the ribosomic RNA copy (17,18). This ribosomic DNA is associated with nonhistone acidic proteins with silver affinity called AgNORs. The number of AgNORs per nucleus has been correlated with the rate of transcription of the ribosomal ARN, proliferative activity and DNA ploidy (19,20).

The silver staining technique for NORs has long been used in the cytology of salivary glands and mesothelial cells (21,22). Recently, De Castro Sampaio et al. (15) and Cançado et al. (16) have used wooden spatulas to obtain smears in order to study the effect of the tobacco in normal buccal mucosa from smokers.

For some years, the effectiveness of brush biopsy has been emphasized as a method of obtaining deeper cells, from oral mucosa (23-26) and gynecologic mucosa (27).

The purpose of this article is to compare the percentage of keratinized cells and the nucleolar activity observed in the lateral tongue mucosa from smokers and non-smokers. The smears were obtained with wooden spatula and Endobrush.

MATERIALS AND METHODS

The sample for this study comprises 60 individuals, 30 smokers and 30 non-smokers. Smokers were determined by those who smoked over 10 cigarettes per day over 10 years. Both groups were free from systemic diseases, were non-alcoholic and non-users of mouth rinses containing alcohol. The smokers were between 29 and 57 (average of 39.5 years old) and the non-smokers were between 30 and 58 (average of 40.5 years old). The ratio male/female was 12/18 and of 11/19, respectively. Following informed consent, lingual smears were obtained: one with a wooden spatula and two with Endobrush, and fixed immediately in Citofix ^R spray.

All smears obtained with the wooden spatula and one of the smears obtained with Endobrush received Papanicolaou's method to determine the number of enucleated superficial cells, nucleated superficial cells and intermediate cells (14) using an 400x magnification. The second smears obtained with Endobrush were stained with the silver soaking technique adapted by Ploton et al. (28). The staining procedure was a fresh solution of Part A (2 g. gelatin in 100 ml. of water containing 2% formic acid) and of Part B (50% silver nitrate). They were mixed (1:2 v/v) in a dark room (28). The smears were soaked in this solution and incubated 25 min. at 45° C. The nucleuses from the first 100 cells were included in the counting. Cells without nucleus and the nucleated cells with keratohialine granules that might produce errors were not included. The number of AgNORs per cell was counted according to the Howat's method (29).

The smears were observed at x1000 magnification under an oil immersion in a Nikon Microphot-FXA microscope by an examiner who did not know the origin of the samples. The first 10 slides were counted non-consecutively three times to calibrate the examiner. The average number of AgNORs obtained per slide were compared in both groups; smokers and non-smokers. The cytological differences between smokers and non-smokers were evaluated by a chi-square (X²) test and a Student t - test with PRIMER statistical software. Statistical significance was defined as P≤0.05.

RESULTS

In smokers, the total distribution of observed cells was: 16.8% enucleated superficial cells, 29.6% nucleated superficial and 53.6% intermediate cells. In non-smokers, the total distribution of observed cells was: 9.3% enucleated superficial cells, 38.5% nucleated superficial and 52.2% intermediate cells. We observed a greater percentage of enucleated superficial cells with the wooden spatula (P = 0.016). Whereas with the Endobrush we obtained a greater percentage of deeper layer cells (type intermediate) (P = 0.035) (Table 1).

 Table 1. Distribution of the three cellular types obtained with wooden spatula and Endobrush.
Tabla 1. Distribución de los tres tipos celulares obtenidos con espátula de madera y endobrush.

 

When comparing smokers and non-smokers, we observed a greater percentage of enucleated superficial cells in smokers, a difference that was significantly different with the Endobrush (P = 0.005) (Table 2). 

Table 2. Distribution of the type of cells obtained with Endobrush in smokers and non smokers.
Tabla 2. Distribución del tipo de células obtenidas con endobrush en individuos fumadores y no fumadores.

 

The smokers had an average of 3.83 AgNORs per nucleus (Standard deviation of 0.68) and the non-smokers had an average of 2.79 AgNORs per nucleus (Standard deviation 0.62) (Figure 1, A and B). This difference in the number of AgNORs per nucleus between smokers and non-smokers was statistically significant (t = 3.488, P = 0.003). The difference between both averages was -1.04 with an Interval of Confidence of 95% which is between -1.67 and -0.41.

DISCUSSION

Because one limitation of exfoliation cytology is the obtaining of superficial cells, the use of brushes for the smears of the oral mucosa is being promoted (23-26). We observed that indeed Endobrush allows one to obtain a greater percentage of deeper layers cell, in this case intermediate cells, whereas with the wooden spatula a greater percentage of enucleated supericial cells are obtained. Ogden (23) described that they received a greater rate of dispersion and a greater amount of cells using brushes for smears.

In the lateral tongue mucosa, the intermediate cells were the most frequently observed, as much in smokers as in non-smokers, 55.5% and 60.4 %, respectively, which concurs with Varvaky et al (14). These authors found that the greater percentage of intermediate cells in lateral tongue and cheek (60% and 62% respectively), whereas the greater percentage of cells obtained from smears of lips corresponded to enucleated superficial cells.

In non-smokers the percentage of enucleated superficial cells of the lateral tongue was 9.3%, similar to 10% observed by Varvaky et al (14) who in their study excluded smokers. However, in the smokers of our study, the percentage of enucleated superficial cells of the lateral tongue was higher, 17%. This increase in the number of enucleated or orthokeratinizated superficial cells in oral mucosa of smokers has been observed by numerous authors (2,6,30-32). Nevertheless, this behaviour of the oral mucosa does not seem to happen in all anatomical sites of the oral cavity, Drouilly (30) did not observe significant differences in the types of epithelial cells obtained in smears of the mouth floor of smokers or non-smokers. It is postulated that this increase in the production of keratinizated cells would be caused by the direct stimulation of the heat of the cigarette and the chemical action of volatile products of the tobacco, with the purpose of protecting itself from these injurious agents (2,6). The importance of evaluating the cellular changes in the lateral tongue relates directly to the high frequency of the cancer in this part of the mouth in our country (33).

The technique of AgNORs was developed in the years 80´s and was quickly used in the study of biopsies of different neoplasm, and only recently has it been applied to cytology (15,16). In our study, the value average of AgNORs per nucleus, in smoker was of 3.83 and in the group of non-smokers was of 2.79, results that are similar to those described by De Castro Sampaio et al. (15) who observed an average per 3.4 AgNORs per nucleus in smokers, and of 2.6 in non-smokers. Recently, Cançado (16) also determined a greater count of AgNORs in smokers, but their values were smaller than those observed by De Castro Sampaio et al. (15) and by our study, 1.94 in smears of lateral tongue and 2.07 in smears of mouth floor.

Technically, the application times of silver solution were 30 minutes in the De Castro Sampaio et al. study (15), 25 minutes in our study and 20 minutes in the Cançado' study (16). Nevertheless like Cançado we do not think that this variation in the technique influences the count of the AgNORs.

These differences in the count of AgNORs could be due to different ages of the participants. For example, the Cançado study included participants between 50 and 70, and in our study between 29 and 57. It has been described that oral mucosa during the aging has a smaller proliferation activity and epithelial atrophy (34). Another possibly explanation of this higher count of AgNORs is the difference of the implements used to obtain smears because the cells that we obtained were deeper due to use of Endobrushes.

The count of AgNORs has been described as a good method to determine ploidy, proliferation activity (19,20), and metabolic cell activity not associated with proliferation capacity (35). In our study, the most common type of cells obtained in normal oral mucosa were intermediate layer cells, but not basal or parabasal layers cells. Therefore this increase of the count of AgNORs was associated with a promotion of the metabolic activity or protein synthesis in these cells.

The observation of smears can sometimes be difficult by what is believed to be a silver affinity to mucous, food residues and other debris (16). Therefore, we performed mouth rinsing with distilled water before collecting smears, since we previously observed (data not shown) that a great amount of background stains without rinsing make counting difficult.

The results obtained in this study suggest that tobacco smoking produces cellular alterations in clinically normal mucosa of the lateral tongue shown in exfoliation cytology. Because this method of using a brush to obtain deeper cells is a non-invasive technique and an easy procedure, the AgNORs count can be an auxiliary method to control risk groups for the prevention of oral cancer.

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