Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2012  |  Volume : 55  |  Issue : 4  |  Page : 433--438

Micronucleus assay of buccal mucosa cells in smokers with the history of smoking less and more than 10 years


Noushin Jalayer Naderi, Sareh Farhadi, Samaneh Sarshar 
 Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Shahed University, Tehran, Iran

Correspondence Address:
Sareh Farhadi
Faculty of Dentistry, Department of Oral and Maxillofacial Pathology, Shahed University, between Vesal and Ghods, Italia street, Tehran
Iran

Abstract

Introduction and Aim: Cigarette smoking causes severe health problems such as cancer. Micronuclei are structures that present after genomic damages to the cells. The present study is aimed at evaluating the micronucleus assay of buccal mucosa cells in smokers who smoked less or more than 10 years. Materials and Methods: The present study has been a historical cohort study. The smokers were divided into two groups: First group include individuals with a smoking history less than 10 years (14 samples) and second group with the smoking history of more than 10 years (26 samples).The control group consisted of nonsmokers (23 samples). The exfoliated buccal mucosa cells were scrapped using spatula and were spread over the glass slide. Feulgen method was used for micronucleus staining. 500 cells per subjects were counted. The presence of micronucleus in all subjects and the mean percentage of micronucleus in nuclei were determined. Data were subjected to statistical analysis using T-test. Results: The mean number of micronucleus of buccal mucosa cells in nonsmokers, first group (smoking history less than 10 years) and second group (smoking history more than 10 years) was 0.94 ± 0.94, 1.89 ± 0.62 and 2.01 ± 0.93 respectively. The difference was statistically significant (P < 0.002). Considering the number of micronuclei of the buccal mucosa cells, the difference between groups 1 and 2 was not significant (P < 0.6). The mean percentage of micronucleated cells in nonsmokers, group 1 and group 2 was 2.26 ± 2.17%, 13.9 ± 5.90 and14.3 ± 7.97, respectively. The difference was statistically significant (P < 0.001).The difference between the percentage of the cells with micronucleus in smokers with a smoking history of less or more than 10 years was not significant (P < 0.6). Conclusion: The mean number of micronuclei in buccal mucosa cells of the nonsmokers was significantly lower than that of the smokers. However, the mean number of micronucleus of buccal mucosa cells in smokers who smoked more than 10 years was higher than smokers who smoked less than 10 years. Increasing the smoking duration could heighten the frequency of micronucleus; however, the difference was not significant.



How to cite this article:
Naderi NJ, Farhadi S, Sarshar S. Micronucleus assay of buccal mucosa cells in smokers with the history of smoking less and more than 10 years.Indian J Pathol Microbiol 2012;55:433-438


How to cite this URL:
Naderi NJ, Farhadi S, Sarshar S. Micronucleus assay of buccal mucosa cells in smokers with the history of smoking less and more than 10 years. Indian J Pathol Microbiol [serial online] 2012 [cited 2020 Oct 25 ];55:433-438
Available from: https://www.ijpmonline.org/text.asp?2012/55/4/433/107774


Full Text

 Introduction



Nowadays cigarette smoking has been recognized as an important risk factor for several types of cancer, mainly oral cancer. [1] It has been shown that there is a dose-response relation between smoking and development of oral cancer. [2],[3],[4],[5] Cigarette smoking, chewing of tobacco and their derivations are the major risk factors of oral cavity cancers. [6] Cigarette contains several carcinogens. These materials activate in different tissues, which cause the DNA adducts products. The time influence on DNA adducts has controversial results. [7]

About 25 years ago, Stich et al. [8] introduced a method for micronucleus assay in exfoliated buccal mucosa cells. Micronucleus is very similar to the cell's nucleus. These structures are the result of chromosomal alterations. [8],[9],[10]

Micronucleus assay is performed in cytopathologic samples. It has been believed that the number of micronucleus is related to increasing the effects of carcinogens. The important point is that, this event has happened before clinical symptoms of cancer appear. [11]

Kamboj and Mahajan have indicated that evaluation of buccal mucosa epithelial cells is a reliable biomarker for early detection of premalignant and malignant lesions. [12]

The changes in buccal mucosa of tobacco chewers and cigarette smokers were demonstrated by micronucleus assay. [13],[14]

Cigarette smoking is one of the most important causes of oral cancer. [15] However, the question is whether the duration of smoking has an effect on chromosomal changes.The number of micronuclei is a good indicator of chromosomal alterations in cytological samples. With increasing micronuclei number, the risk of chromosomal alterations will become higher.

The aim of this study is to assay the frequency of micronucleus in buccal mucosa cells of smokers with the history of smoking less and more than 10 years and its comparison with nonsmokers.

 Materials and Methods



The present study is a historical cohort study. The statistical population comprises smokers and nonsmokers who were referred to dental school of Shahed University in 2011. The inclusion criteria were the usage of mix diet and nonexposure to dental radiography beam in recent 6 months. The exclusion criteria included the following: suffering from systemic disease, consuming any type of drugs and disagreement of subjects to join the study. The 10 year cutoff point was determined for evaluating the duration of smoking. In the first step, the pilot study was performed to determine the number of samples in the nonsmokers group and smokers group based on the quantity of the years of smoking. The number of micronuclei in nonsmokers group (n = 11) and smokers (n = 14) were 1.36 ± 1.12 and 2.46 ± 0.49, respectively. According to these findings and with the 95% confidence level and 90% strength of a test, the number of samples in each group was determined as 15.

The smokers were categorized into 2 groups: 1) subjects with the history of smokingless than 10 years (14 samples) and 2) subjects with the history of smoking more than 10 years (26 samples). The control group was selected from non smokers (23 samples).Control group was matched with smokers about age and sex. All participants were males. After taking the informed consent of subjects to participate in the study, the information was entered in to the registration form and was coded. All subjects in three groups were matched from a social and economic point of view.

Subjects were asked to rinse their mouth twice with normal saline, before obtaining the buccal mucosa cells. The exfoliated buccal mucosa cells were scrapped using spatula and they were spread over the glass slides. Feulgen reaction following the modified method of Thomas et al.[16] was used for determination of micronuclei. The prepared smears were fixed in Carnoy's fixative (methanol and glacial acetic acid in a ratio of 3:1) for 30-35 min. Then the slides were dried at room temperature. Thereafter, slides were dipped for 10 min in 1 N HCL at 60 and were rinsed in distilled water for 3 min. The smears were placed in Schiff's reagent for about 90 min and were transferred to normal saline for 10 min. Then the slides were placed 3 times in 0.5% sodium metabisulfite solution and were rinsed with tap water. The slides were stained with 1% light green about 15 min and finally were rinsed with tap water. Finally, they were dried and mounted for microscopic evaluation.

Cellular evaluation was performed using optic microscope (ZEISS, Germany) with ×400 and ×1000 magnification by two pathologists in this double blind study.

To create unanimity between two pathologists, several samples were selected randomly and were projected for observers to make settlement between them based on the aspect of selected region, shape and number of the micronuclei. In that primary examination, final results between two pathologists were made equal.

For counting the micronuclei, the cells with distinct margin and nuclei were counted. The overlapped cells were not considered for counting.

500 cells per samples were counted. The presence of micronucleus in all subjects and the mean number of micronuclei in nuclei were determined. To determine the mean number of the micronuclei in nuclei, the following formula was applied:

[INLINE:1]

The quantum of smoking exposure was also calculated by the number of packs × years of exposure.

Statistical analysis was performed using T-test and Pearson correlation coefficient test. P < 0.05 was considered as significant.

 Results



The average age for nonsmokers group was 37.75 ± 17.25, for group 1 (smoking history less than 10 years) was 34.78 ± 11.13 and for group 2 (smoking history more than 10 years) was 40.26 ± 12.43. The range of the smoking duration in group 1 was from 3 to 8 years with the mean duration 5 years and it was from 11 to 52 years with the mean duration 20 years in group 2.

In microscopic evaluation, the micronuclei were one third of the main nucleus. The micronuclei were round or ovoid with the same color as main nucleus [Figure 1], [Figure 2].{Figure 1}{Figure 2}

The least percentage of the cells with micronucleus was observed in nonsmokers.The mean percentage of the micronucleated cells in group 1 and group 2 was 13.9 ± 5.90 and 14.3 ± 7.97, respectively.The difference was statistically significant (P < 0.001).The difference in percentage of the cells that have micronucleus between two smoker groups was not significant (P < 0.6). The percentage of the cells with micronucleus in buccal mucosa cells of case and control groups is shown in [Table 1].{Table 1}

The mean number of micronucleus in buccal mucosa cells was 0.94 ± 0.94 for nonsmokers. The mean number of micronucleus in buccal mucosa cells was 1.89 ± 0.62 in group 1. The difference between group 1 and control group was statistically significant (P < 0.002). The mean number of micronucleus in buccal mucosa cells of group 2 was 2.01 ± 0.93.The difference between group 2 and control group was significant (P < 0.001).On the other hand, there was no significant statistical difference between both smokers groups (P < 0.6). [Table 2] shows the number of micronucleus in buccal mucosa cells of case and control groups. [Table 3] has summarized the clinical features and micronucleus assay of all subjects.{Table 2}{Table 3}

The minimum and maximum number of pack years was 3 and 13140, respectively. The pack-years smoking quantum showed degrees of correlation with percentage of cells containing micronucleus (r = 0.27), and also with average of micronucleus number (r = 0.29). However, the correlations were not significant.

The pack-years data are summarized in [Table 4].{Table 4}

 Discussion



The present study shows that the mean number of micronuclei in buccal mucosa cells of smokers was higher than that of nonsmokers. The prevalence of micronucleus in individuals with the smoking history of more than 10 years was very higher than the persons with the history of smoking less than 10 years.

The mean prevalence of cells with micronucleus in the general populations is 0.0 to 0.9%. [11] Any different range of micronucleus number can be the result of chromosomal alternations.

The micronuclei are the result of genomic damages to the cells. These structures can be used for individual's biomonitoring. Exfoliated buccal mucosa cells are good sources for this biomonitoring. The micronuclei assay is a non-invasive and simple technique for evaluating the DNA damages. [17]

The average rate of the cells with micronucleus is related to premalignant lesions and leukoplakia. [18]

Different forms of smoking and related agents such as cigarette, Betel nut and Quid and reverse smoking are associated with increase in the number of micronucleus in exfoliated buccal mucosa cells. [8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19]

According to this study, the mean number of micronuclei in buccal mucosa cells of smokers was very higher than nonsmokers; the result was compatible with the previous studies that have showed smoking causes the increase in number of micronucleus. [20],[21],[22],[23]

In the present study, several different points in design and performance of research method have been considered in comparison with other previous studies. In other investigations, cigarette and other shape of tobacco were compared with each other, the subjects were consumers of several different agents. In this study, only the effect of smoking cigarettes on the mean of micronucleus has been evaluated. When the subjects have used some agents such as cigarette and smokeless tobacco, the contemporary effect of these agents has caused nuclear degeneration and some structures which were very similar to micronucleus were appered; this event has impressed the count of micronuclei and its calculation. [17]

None of the subjects were alcohol consumers, so the role of confounding factors especially consumption of alcohol has omitted completely. This affair has caused the omission of synergic effect of these two agents on the number of micronuclei.The significant increase in number of micronuclei in smokers definitely has been attributed to smoking cigarette. It has been shown that the synergic effect of cigarette smoking and alcohol consumption is more than 5.5 fold. [24]

In this study, Feulgen staining has been used to represent the micronucleus. Nersesyan et al. [14] have shown that this staining method has higher accuracy in examining the micronucleus as compared with nonspecific DNA staining such as Giemsa.

Our findings have demonstrated that the mean micronuclei frequency in smokers is higher than that of nonsmokers.

Konopacka et al. [25] have reported that the micronuclei frequency was 1.50% ± 0.47% in smokers and 0.55% ± 0.32% for nonsmokers. Ozkul et al.[21] also have reported the mean micronuclei frequency in smokers was 1.99 ± 0.3.These findings are in agreement with the present study.

The results about the mean number of micronuclei in men and women were controversial; some researches have shown higher micronuclei values in men and the others have reported higher values in women. [26],[27],[28] Contradiction also exists about the age of the subjects. [21],[26],[29],[30] To omit the effect of age and sex on the obtained results, in the present study all the samples were men and were age matched.

The correlation between pack-years smoking quantum with the percentage of cells containing micronucleus and average of micronucleus's number were not significant.

Wu et al.[22] have reported the positive relation between micronuclei frequency and smoking intensity. The micronuclei frequency in buccal cells was higher in heavy smokers.

We have contributed these differences to different sample size and method of smoking quantum calculation.

All the studies have shown that smoking increases the mean number of micronuclei in buccal mucosa cells. In this study, the role of time in increasing the mean number of the cell's micronucleus has been evaluated for the first time. The mean number of micronucleus of buccal mucosa cells in smokers with the smoking history of more than 10 years was higher in comparison with smokers with a smoking history of less than 10 years. The percentage of the cells with micronucleus in smokers who smoked either less or more than 10 years was not significant. Smoking of cigarettes cause chromosomal alternations in smokers and these alternations are probably not related to the duration of smoking. It seems that cigarette has the ability to manifest chromosomal alternations from the early time of smoking.

Several reports have indicated that cigarette and other forms of tobacco increase the micronucleus in oral mucosa. However, Stich and Rosin have obtained different results. [24]

This discrepancy and other differences that have mentioned before can be the result of using different staining procedures, distinction in number of samples and evaluation of different smoking and smokeless agents. It should be noted that, chemical compound in cigarettes from different factories and smokeless agents are different. Thus, obtaining more decisive results requires standardization of the applied techniques.

 Conclusion



The mean number of micronucleus in buccal mucosa cells of nonsmokers was significantly lower than smokers. However, the mean number of micronucleus of buccal mucosa cells in smokers who smoked more than 10 years was higher than smokers who smoked less than 10 years. Increasing the smoking duration could heighten the frequency of micronucleus; however, the difference was not significant.

 Acknowledgments



We thank Mr. N. Valayi for his contribution as research advisor.

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