Quantitative analysis of AgNOR counts and pap stain in exfoliative cytology specimens of oral mucosa in bidi smokers and nonsmokers.

Background: Bidi smoking is a serious health hazard which is common throughout South Asia and parts of the Middle East. It has been strongly implicated to various benign and malignant lesions of oral cavity and oropharynx. These tobacco-filled leaves deliver more than three times the amount of nicotine, carbon monoxide, and tar as cigarettes which exert injurious effects on cells reflected in terms of accelerated proliferative activity in normal oral mucosal cells. Aim: This study aimed to compare the exfoliated cells from the oral mucosa of bidi smokers and nonsmokers, with emphasis on proliferative activity. Materials and
Methods: Exfoliative smears were obtained from the oral mucosa of forty participants (twenty nonsmokers and twenty smokers) with age group ranging from 30-80 years, in and around Barwala (Haryana). The cytologic smears were evaluated using Papanicolaou (PAP) stain and AgNOR in order to evaluate the presence of cytological alterations suggestive of inflammation, dysplasia, keratinization, and proliferative activity of epithelial cells. Only PAP Class I and Class II smears were observed.
Results: Comparison of the mean number of AgNORs showed a significant difference between nonsmokers and smokers. Inflammatory alterations were found in 70% of smokers and 55% of nonsmokers. A significant difference in proliferative activity was observed between smokers and nonsmokers classified as PAP Class II.
Conclusion: A significant difference of AgNORs/nucleus was observed between bidi smokers and nonsmokers.

INTRODUCTION

Bidi smoking is a traditional method of tobacco use throughout South Asia and parts of the Middle East. Today, bidis are popular and inexpensive in India. Their consumption outpaces that of conventional cigarettes. Bidis accounted for 48% of Indian tobacco consumption in 2008. These tobacco-filled leaves deliver more than three times the amount of nicotine, carbon monoxide, and tar as cigarettes and carry a greater risk of oral cancers.[1]

Oral squamous cell carcinomas (OSCCs) currently hold the sixth position in the worldwide cancer statistics, with a dismal 5-year survival rate, except when diagnosed in the early stages.[2] Hence, there is a need to promote early diagnosis of oral cancers. Biopsy is considered as gold standard, which is carried out only when the lesions become symptomatic, i.e., in the late/advanced stages.

Many problems arise microscopically in differentiating the malignant aberrations from the benign ones and the routine histopathological techniques do not reveal all the features which are of diagnostic and prognostic significance.[3] Therefore, it is eminent to develop adjunct procedures which can predict conversion to malignancy at the earliest and with accuracy. Studies have revealed the correlation between nucleolar function, size, and the cell doubling time in human cancer cell lines, which has stimulated a revolution of the importance of the nucleus in tumor pathology.

Exfoliative cytology might be a useful tool for the detection and monitoring of initial cellular alterations, which reflects the essential role of nucleus in the control of proliferation and protein synthesis. AgNOR correlates with the rate of proliferation, as can be estimated by Ki-67 and the percentages of the S-phase cells and the mitotic cells. Hence, the nucleolar organizer regions (NORs) are loops of ribosomal DNA which occur in the nucleoli of the cells on the short arms of the acrocentric chromosomes 13, 14, 15, 21, and 22.[4]

The interphasic NORs can be clearly visualized at the light microscopical level using a silver reaction which stains the acidic proteins of the NORs (RNA polymerase 1 upstream binding factor, topoisomerase 1, nucleolin, fibrillin, C23 protein, and B23 protein) on routinely prepared histopathological and cytological samples. After silver staining, the AgNORs can be identified as black dots throughout the nucleolar area. In quantitative terms, the number of AgNORs per nucleus suggests it to be a marker of the proliferative activity of the cell.[4]

It is a complementary diagnostic method which presents several advantages such as rapid and easy execution, low cost, diagnostic safety, efficacy, and noninvasiveness, and can be repeated several times. Papanicolaou (PAP) staining is highly effective for screening only. It is not diagnostic. It only identifies those at risk of dysplasia or cancer.

AgNOR counts can act as a marker of premalignant or malignant changes in oral mucosa.

MATERIALS AND METHODS

Exfoliative smears were obtained from the buccal mucosa using wooden spatula in forty participants (twenty bidi smokers and twenty nonsmokers) with age ranging from 30 to 80 years, in and around Barwala (Haryana). The following inclusion criteria were applied: nonalcohol users, absence of any oral lesion, and with no prior or present history of benign or malignant oral neoplasms. Two groups were analyzed: smokers and nonsmokers. Smokers were defined as those individuals that smoked over 15 bidis or more per day for at least 15 years. Cigarette, pipe smokers, or consumers of tobacco in other ways were not included. Nonsmokers were defined as people who have never smoked. The cytologic smears were stained using PAP stain and AgNOR and observed at ×40 and ×100, respectively.

AgNOR staining procedure

The alcohol-fixed smears were immersed in 95% absolute ethanol followed by progressive rehydration and washing in distilled water. AgNORs were stained according to the method recommended by the International Committee on AgNOR Quantitation, revised by Trerè, with pre-staining fixation for 30 min in Carnoy's solution (acetic acid: absolute ethanol, 1:3). NORs were directly counted under a light microscope according to the parameters established by Crocker et al., i.e., well-defined black dots in the nucleus were counted, with aggregations (overlapping or fused black dots) being considered a single structure [Figure 1].[5] AgNOR in 50 cells were counted.

Figure 1

More than three AgNOR dots in each of the squamous cells of normal mucosa in smokers (AgNOR, ×100)

Papanicolaou staining

The PAP test has been widely applied all over the world in the diagnosis of precancerous lesions and cancer. Evaluation of PAP-stained smears was carried out at ×40.

The presence of two or more of the following features indicated the presence of epithelial atypia: nuclear enlargement associated with increased nuclear-cytoplasmic ratio, hyperchromatism, chromatin clumping with moderately prominent nucleolation and irregular nuclear borders, bi- or multi-nucleation, increased keratinization and scantiness of the cytoplasm, and variations in size and/or shape of the cells and nuclei.

The Papanicolaou classification

Class I: Absence of atypical or abnormal cells

Class II: Atypical cytology but no evidence of malignancy

Class III: Cytology suggestive of, but not conclusive of, malignancy

Class IV: Cytology strongly suggestive of malignancy

Class V: Cytology conclusive of malignancy.

RESULTS

The mean age of the population was 45 years for smokers and 25 years for nonsmokers.

Comparison of the mean number of AgNORs showed a significant difference between nonsmokers (1.82±0.34 AgNORs/nucleus) and smokers (4.23±0.33 AgNORs/nucleus) [Table 1].

Table 1

Statistical analysis of AgNOR dots in normal buccal mucosa cytology of smokers and nonsmokers

Study group	Number of cases	Mean of AgNOR±SD	Mean percentage of cells with 3 or >3 AgNOR dots
Nonsmokers	20	1.82±0.34	5.6
Smokers	20	4.23±0.32	80.4

SD=Standard deviation

To evaluate the influence of the smoking habit on the number of AgNORs, smoking patients were divided into three groups according to the number of pack-years (pack-years = number of bidis × number of years smoked/number of bidis in 1 bundle): Group A: 25-35 pack-years; Group B: 45-64 pack-years; and Group C: 60–79 pack-years. The mean AgNOR number and respective standard deviation were determined in each group [Table 2]. No significant difference in the mean number of AgNORs per nucleus was observed between Group A and Group B (t = 0.790, P > 0.01); Group B and Group C (t = 0.761, P > 0.01); and Group A and Group C (t = 0.772, P > 0.01).

Table 2

Mean±standard deviation of AgNORs per nucleus in 50 cells in 20 groups of smokers divided according to the pack-years

Group	Pack-years	Patients (20)	AgNORs/nucleus (mean±SD)
A	25-35	12	3.85±0.35
B	45-64	3	2.98±0.0721
C	60-79	5	4.12±0.22

SD=Standard deviation

PAP stained smears in smokers were classified as class II (80%) and class I (20%). While in non smokers 70% smears were class II and 30 % class I [Figure 2].

Figure 2

Class I PAP smear in smokers

The number of AgNORs was also determined in smokers and nonsmokers divided according to the PAP classification [Table 3]. A significant difference in proliferative activity was observed between smokers and nonsmokers classified as PAP Class II.

Table 3

Number of AgNORs per nucleus in smears from smoking and nonsmoking patients divided according to the cytologic evaluation

Patients	PAP Class I (mean number of AgNOR/nucleus±SD)	PAP Class II (mean number of AgNOR/nucleus±SD)
Smokers (n)	4.50±0.36	5.06±0.46
Nonsmokers (n)	1.33±0.221	2.012±1.22

SD=Standard deviation

DISCUSSION

A strong correlation between AgNOR count and bidi smoking was proved in this study. Heat and the chemical products in tobacco increase the proliferative capability of oral mucosal epithelial cells. Thus, smokers have higher potential to develop oral SCC. This proliferation is observable with AgNOR staining before any clinical symptoms appear. Our results also revealed a significant correlation between bidi smokers and the mean number of AgNOR/nucleus.

AgNOR technique has been successfully used in various studies that included cigarette smokers without any oral lesions like in our study that included bidi smokers, showing higher cellular proliferation in smokers as compared to nonsmokers. Salehinejad et al. evaluated cigarette smokers’ oral mucosal cells and found an increased number of AgNORs/nucleus in smokers (3.6) than in nonsmokers (1.96).[6] Patricia Campos Fontes et al. studied AgNOR count of the tongue in cigarette smokers and nonsmokers on the basis of number of cigarettes consumed per day and the duration of smoking. Tobacco smokers were found to be at major risk to develop premalignant lesions.[7]

Ahmed et al. evaluated cytological atypical changes in apparently healthy oral mucosa exposed to smoking, alcohol, hot meals, and pepper using the AgNOR and PAP methods. Statistical analyses revealed a greater mean number of AgNORs per nucleus in smokers (3.68) followed by (2.82) alcohol consumers, compared to the habitual peppers and hot meal consumers (2.28) and the nonexposed group (2.00), whereas in case of PAP method, increased keratinization was detected among 45% of the smokers, 32.7% of the pepper and hot meal consumers, 11.8% of the alcohol consumers, and among 3.7% of the nonexposed group.[8]

The present study includes only bidi smokers as opposed to cigarette smokers in previous studies. In our study, a statistically significant difference in the mean number of AgNORs per nucleus in 50 cells was observed between bidi smokers and nonsmokers. The mean number of AgNORs per nucleus was highest in Group C (3.8) including bidi smokers, with the largest number of pack-years ranging from 60 to 79. Almost an equal number of AgNORs per nucleus was observed in Group A (2.995) including bidi smokers which had a range of 25-35 pack-years compared to Group B (2.98) with a range of 45-64 pack-years. Although there was a trend for increase in AgNORs with increasing pack-years, our data did not reach statistical significance. Comparison of the mean number of AgNORs per nucleus according to the duration of smoking revealed no significant difference, a result also observed by Cançado et al.[9] The mean percentage of cells with three or more than three AgNORs per nucleus was significantly higher in bidi smokers (73.6%) when compared to nonsmokers (4.4%), again demonstrating a higher proliferative activity in the group of bidi-smoking patients.

Cançado et al. collected cytologic smears from two anatomic sites, mouth floor and tongue border, with the purpose of relating the frequency of smoking with the quantitative analyses of the AgNORs. This study showed that the number of cigarettes per day had a significant correlation to the amount of AgNORs per nucleus in the mouth floor, but that was not true for the tongue border smears.[10]

In the normal individuals, the epithelial cells revealed 2.07–3.05 NORs per nucleus. In malignancies, the number of NORs per epithelial cell ranged from 4.83 to 6.09. The cutoff value to differentiate between normal and malignant cells is considered to be 4.[11]

It is important to consider that our patient inclusion criteria included nonalcohol use and absence of any clinically evident oral lesion, with no previous or present history of benign or malignant neoplasms.

Cytological alterations (increased nuclear-cytoplasmic ratio, hyperchromatism, chromatin clumping with moderately prominent nucleolation and irregular nuclear borders, bi- or multi-nucleation, increased keratinization and scantiness of the cytoplasm, and variations in size and/or shape of the cells and nuclei) were observed both in bidi smokers and nonsmokers, even when there are no clinical manifestations, which may indicate epithelial modifications in response to a physiochemical environment caused by the substances present in bidis.

Although the PAP classification alone was not sufficient enough, it gained meaning when correlated with the results of AgNOR quantification. Comparison of the mean number of AgNORs per nucleus in smears classified as Class II PAP smears showed a significant difference between smokers and nonsmokers.

The effect of smoking was demonstrated by a significantly larger number of AgNORs per nucleus in mucosal cells of the buccal mucosa, indicating a higher proliferative activity of these cells. Sampaio HC, using the histochemical AgNOR method, suggested that smoking influences the proliferative activity of cells.[12]

The present results suggest that bidi smoking produces alterations in the mechanisms of cell growth control. Tobacco has been considered to be an initiating factor in the process of oral carcinogenesis, which is frequently associated with alcohol as a promoting factor. In addition, the results suggest that the oral mucosa is susceptible to the effects caused by bidi smoking, responding with an increase in cell proliferation which corroborates similar findings in cigarette smokers.

The results of this study indicate higher proliferative activity in bidi smokers compared to nonsmokers, even in the absence of clinical lesions.

CONCLUSION

PAP staining in combination with the AgNOR technique act as a reliable indicator to measure the effects of smoking on oral mucosa. Other proliferative indices such as Ki-67 may be used for comparison with the AgNOR method as well

It was also concluded that bidi smokers show higher cellular proliferation as compared to nonsmokers, which is similar to results obtained for cigarette smokers. However, it is not clear whether bidi smokers are at a greater risk of malignant transformation to OSCC than cigarette smokers. Further long-term studies which include large sample sizes comparing bidi and cigarette smokers need to be carried out to determine this.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.