|Year : 2014 | Volume
| Issue : 1 | Page : 165-170
Assessment of cytogenic damage in the form of micronuclei in oral epithelial cells in patients using smokeless and smoked form of tobacco and non-tobacco users and its relevance for oral cancer
Anagha A Motgi1, Mahesh S Chavan1, Nikhil N Diwan1, Asha Chowdhery1, Pallavi P Channe1, Mrinal V Shete2
1 Department of Oral Medicine and Radiology, Dr. D. Y. Patil Dental College, Pimpri, Pune, Maharashtra, India
2 Department of Oral Pathology and Microbiology, Bharati Vidyapeeth University, Pune, Maharashtra, India
|Date of Web Publication||23-Apr-2014|
Anagha A Motgi
Department of Oral Medicine and Radiology, Dr. D. Y. Patil Dental College, Pimpri, Pune, Maharashtra
Source of Support: None, Conflict of Interest: None
Context: Early detection of cytological damages may help in reduction of morbidity and mortality in patients with oral cancer.
Aims: (1) The primary aim of this study is to assess the cytogenic damage in the form of micronuclei (MN) in patients with smokeless and smoked tobacco using habit. (2) The secondary aim of this study is to compare the MN score in patients using tobacco and patients with no tobacco habit. (3) To find out incidence of MN according to duration and frequency of tobacco usage.
Settings and Design: This is a clinical study.
Materials and Methods: A total of 100 patients each with the habit of smokeless tobacco (SLT) chewing, smoked tobacco usage and with no habit were included in the study. Epithelial cell smears were prepared and slides were stained with Papanicolaou stain. Scoring of at least 1000 cells was done and a MN frequency score was assigned for exfoliated oral mucosal cells.
Statistical Analysis: Analysis of variance and post hoc tests were used.
Results: The difference between the total number of cells with MN was not appreciable between the smokeless and smoked tobacco groups, though the total number of MN was higher in subjects using SLT. Total number of cells with MN and the total number of MN were significantly lower in non-tobacco users when compared with tobacco users. There was very weak positive correlation between the total number of MN as per the duration and frequency of the tobacco habit.
Conclusion: The use of smokeless and smoked tobacco are associated with cytotoxic and genotoxic effects. SLT seems to cause more damaging effects than the smoked form.
统计分析：方差分析和Post Hoc 检验。
Keywords: Micronuclei, oral cancer, smoked tobacco, smokeless tobacco
|How to cite this article:|
Motgi AA, Chavan MS, Diwan NN, Chowdhery A, Channe PP, Shete MV. Assessment of cytogenic damage in the form of micronuclei in oral epithelial cells in patients using smokeless and smoked form of tobacco and non-tobacco users and its relevance for oral cancer. J Can Res Ther 2014;10:165-70
|How to cite this URL:|
Motgi AA, Chavan MS, Diwan NN, Chowdhery A, Channe PP, Shete MV. Assessment of cytogenic damage in the form of micronuclei in oral epithelial cells in patients using smokeless and smoked form of tobacco and non-tobacco users and its relevance for oral cancer. J Can Res Ther [serial online] 2014 [cited 2020 Feb 26];10:165-70. Available from: http://www.cancerjournal.net/text.asp?2014/10/1/165/131454
| > Introduction|| |
Cancer is the second most common cause of mortality in developed countries and one of the 10 most common causes in developing countries like India. It is a complex disease with altered expression, abnormal growth and disruption of normal function of cells caused by genotoxic effects of chemical carcinogens.  Micronuclei (MN) are small chromatin bodies in the cytoplasm formed by condensation of acrocentric chromosomal fragments or by whole chromosomes, lagging behind the cell division and are sensitive indicators of genetic damage. It is the only biomarker that allows simultaneous evaluation of clastogenic and aneugenic effects in multiple cells, easily detected in interphase. According to previous studies significantly higher frequencies of MN exfoliated oral mucosal cells are seen in the tobacco habit groups irrespective of their type of chewing when compared with the healthy controls. 
MN assay in exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage in humans in comparison to obtaining blood samples for lymphocyte and erythrocyte assays or tissue biopsies. Oral mucosal cells are the first barrier for inhalation or ingestion route and are capable of metabolizing proximate carcinogens to reactive products. ,,, Approximately, 90% of human cancers originate from oral epithelial cells.  They represent a preferred target site for early genotoxic events induced by carcinogenic agents entering the body through inhalation and ingestion. MN frequency in buccal cells has not been adequately addressed and quantified. Its predictive value for cancer risk has also not been studied comprehensively.  Buccal vestibule is the easier, readily available site for collection of shed cells. Hence, it is preferred over other oral epithelial sites. Furthermore, samples from the tongue may include more materia alba. Our study separately assesses and compares the effects of smokeless and smoked tobacco on the oral mucosa and compares with habit-free controls. We have also evaluated whether the frequency and duration of tobacco habit are proportional to the damage caused.
| > Materials and Methods|| |
A total of 100 patients each with the habit of smokeless tobacco (SLT), smoked tobacco and without any tobacco habit were selected.
Patients with extremely poor oral hygiene, patients under long-term orthodontic therapy, patients with other chronic source of irritation in the oral cavity, patients with oral precancerous or cancerous lesions, patients with alcohol usage habit and patients with occupational exposure to various chemicals were excluded from the study.
Collection of oral epithelial cells
Oral mucosal cells were collected from each subject using a metal spatula. The site of exfoliative cytology was labial or buccal vestibule depending upon the site of placement of tobacco product.
Slide preparation and staining
Epithelial cell smears were prepared by spreading the cells on a clean slide. The slides were stained with Papanicolaou stain.
Criteria developed by Tolbert et al. (Tolbert's criteria) , for choosing the cells are the most widely used. They consist of following parameters for cell inclusion in the cells to be scored: (a) Intact cytoplasm and relatively flat cell position on the slide, (b) little or no overlap with adjacent cells, (c) little or no debris; and (d) normal nucleus and intact, nuclear perimeter smooth and distinct.
Suggested criteria for identifying MN
Suggested criteria for identifying MN are: (a) Rounded smooth perimeter suggestive of a membrane, (b) less than a third the diameter of associated nucleus, but large enough to discern shape and color, (c) Feulgen positive, i.e. pink in bright field illumination, (d) staining intensity similar to that of nucleus, (e) texture similar to that of nucleus, (f) same focal plane as nucleus; and (g) absence of overlap with or bridge to the nucleus.
Scoring of at least 1000 cells was done under high power compound binocular microscope with Battlefield zigzag method and a MN frequency score was assigned for exfoliated oral mucosal cells as number of MN/1000 cells ×40 magnification was used. It was done by three subject specialist observers to avoid bias. Kappa test was later done for the same.
Master charts were prepared for MN frequency and distribution per 1000 cells separately for patients with smokeless, smoked tobacco habit and individuals with no form of tobacco habit. The mean and standard deviation were calculated. Analysis of variance followed by Bonferroni's post hoc test was then applied to compare the groups between themselves. For MN frequency according to the duration and frequency of tobacco usage, information was obtained regarding negative, positive or no correlation between the said parameters.
| > Results|| |
As said earlier, Kappa test was done to rule out inter-observer discrepancy. Cohen's kappa coefficient value was 0.92, which showed good inter-observer agreement.
[Table 1] gives us the total number of cells with MN and the total number of MN in three study groups. [Table 2] shows intergroup comparison of the total number of cells with MN and the total number of MN. [Table 3] provides the mean difference, value of significance and confidence interval by between group comparison of the total number of cells with MN and the total number of MN (Bonferroni's post hoc test). [Table 4] provides a number of MN in patients according to their duration and frequency of tobacco usage habit. [Table 5] gives intergroup comparison of the total number of MN according to their duration and frequency of tobacco usage habit. [Table 6] gives between group comparison of the total number of MN according to their duration and frequency of tobacco usage habit (Bonferroni's post hoc test).
|Table 1: Total number of cells with MN and total number of MN in the three study groups|
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|Table 2: Intergroup comparison of the total number of cells with MN and total number of MN|
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|Table 3: Between group comparison of the total number of cells with MN and total number of MN (Bonferroni's post hoc test)|
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|Table 4: Number of micronuclei in patients according to their duration and frequency of tobacco usage habit|
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|Table 5: Intergroup comparison of total number of MN according to their duration and frequency of tobacco usage habit|
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|Table 6: Between group comparison of total number of MN according to their duration and frequency of tobacco usage habit (Bonferroni's post hoc test)|
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The difference between total number of cells with MN was not appreciable between smokeless and smoked tobacco groups, though the total number of MN was higher in subjects using SLT. The total number of cells with MN and the total number of MN were significantly lower in non-tobacco users as compared with tobacco users. P <0.05 is significant. After the application of Bonferroni's post hoc test, it was observed that the difference in relation with the total number of cells with MN was not significant between the two groups i.e. subjects using SLT and subjects using smoked tobacco. All other values were significant between three groups in relation with the total number of cells with MN and total number of MN. Mean difference is taken as significant at 0.05 level. As observed by further statistical analysis, there was minimal significance or very weak positive correlation between the total number of MN as per the duration and frequency of the tobacco usage habit.
| > Discussion|| |
Deoxyribonucleic acid (DNA) damage caused due to the use of tobacco and related products can be assessed by MN test. It is found to be most sensitive when compared with other tests as it neither requires tedious procedures like cell culture and metaphase preparation, nor it requires any specific DNA stains. MN is a microscopically visible round or oval cytoplasmic chromatin mass in the extra nuclear vicinity. They originate from mitosis and consist of eccentric chromosomes, chromatid fragments or whole chromosomes, which failed to reach spindle poles during mitosis. They are used as biomarkers for assessment of DNA damages.  Aqueous extract of N-nitroso compound related to areca nut is highly cytotoxic and genotoxic to human buccal epithelial cells and potentially import in the induction of tumors in betel quid chewers. Such assays play a significant role in predicting pre-cancerous stage. MN test is a better indicator for genotoxicity damage than chromosomal aberrations or sister chromatid exchange.  Increased MN frequency has a higher risk for the development of oral cancer.  The MN were regarded as potential intermediate marker for cancer chemoprevention. They may be used as a quantifiable estimate of the extent of recent DNA injury.  Their frequency is increased in the oral mucosa of individuals with known carcinogenic exposure, such as the site of tobacco quid placement as compared with grossly normal-appearing mucosa.
In 2008, a study was done to assess the association between water pipe smoking and a cytogenetic measure of tobacco harm. There was a definite rise in the number of MN in smokers. The total number of MN/1000 cells per subject and the number of MN - containing cells per individual were compared.  Higher MN frequencies were noted within the oral cavity where the quid or tobacco mixture was kept as compared to the opposite, control site. A reliable and minimally invasive biomarker is a necessity to improve the implementation of biomonitoring, diagnostics and treatment of diseases caused by or associated with genetic damage. The MN assay in exfoliated buccal cells is potentially an excellent candidate to serve as such a biomarker.  Buccal cells are the first barrier for the inhalation or ingestion route and are capable of metabolizing proximate carcinogens to reactive products. Around 90% of human cancers originate from epithelial cells.  Hence, oral epithelial cells are a preferred target site for early genotoxic events induced by carcinogenic agents entering the body through inhalation or ingestion. Few studies reported an elevation of MN count in exposed individuals as compared with control groups, but the observed effects were relatively small, ranging between 1.1 and 4-fold.  MN in the buccal mucosa are also said to predict cancer risk for the upper aerodigestive tract, including premalignant stages such as oral preleukoplakia.  According to another study conducted in 2011, the mean MN frequency in oral exfoliated cells was also significantly raised in malignant and potentially malignant groups as compared to the control group. For oral cancer, it can be used as a screening prognostic and educational tool in community centers of oral pre-cancer and cancer.  Fareed et al. also conducted a study in 2011 on MN investigation in buccal mucosal cells among pan masala/gutkha chewers. MN frequency was very high in such patients when compared to control; indicating that they may be at high risk for developing oral cancer.  A number of other variables may affect MN frequency in buccal cells such as age, gender, genotype, season, diet, oral hygiene, dental health, life-style, recreational drugs etc., A study was conducted in 1997 to evaluate the MN in buccal mucosal cells of habitual Maras powder users. The subjects were divided into three groups - SLT users, smokers and non-smokers/non-users. The mean percentage of MN cells was significantly higher in SLT users and smokers than in non-smokers/non-users (P < 0.01). The mean percentage of MN cells was 1.86 ± 0.26 in users and 1.99 ± 0.30 in smokers. There was no difference between the mean percentages of MN cells in these two groups.  According to a study conducted by Bansal et al.,  MN cells were found to be significantly higher in SLT users than in smokers and controls. A positive correlation is found between increased MN frequency and tobacco-using habits. Hence, MN assay can be used as a biomarker of genotoxicity. This study was also in accordance with the previous studies conducted by Patel et al.  and Palaskar and Jindal,  who observed that the score of the MN frequency decreased as we moved from the SLT chewers to the smokers and then to the non-users and the non-smokers. A study carried out by Sardas et al.  states that the oral use of SLT represents a genotoxic hazard, which is even higher than the DNA damage observed in cigarette smokers.
The cellular changes associated with SLT users are more than that in smokers.  It indicates more carcinogenic potential of SLT. Carcinogenic and mutagenic compounds, including tobacco-specific nitrosamines, which are present in SLT forms,  are believed to be responsible for the induction of MN. These compounds are produced from nicotine by bacterial or enzymatic activity. The same formation occurs in the mouth under the influence of saliva.  The risk of cancer in SLT users has been attributed to the presence of tobacco specific nitrosamines (TSNAs).  In India, SLT processing is performed by individual farmers and small companies with little control over fermentation and curing, which increases the production of TSNAs. Furthermore, SLT is not homogeneous in India, since the tobacco is often combined with betel leaf (Piper betle) and sliced areca nut (Areca catechu) and/or powdered slaked lime, which are additives that enhance the toxicity as well as the psychotropic effect of tobacco.  Furthermore, studies have proven that the nicotine content of the Indian brands of smoking tobacco is slightly high as compared to that of the international brands. The nicotine content in commercially available chewing tobacco products was found to be much lower than that in the smoking form of tobacco, but the average daily consumption has made it comparable to the smoking form.  According to the center for disease control, chewing tobacco which was used 7-8 times a day could be equivalent to smoking 30-40 cigarettes/day. Other factors such as use of slaked lime and continuous contact with the oral mucosa, led to more absorption of nicotine through SLT use. Additionally, in contrast to the smokers who absorbed nicotine primarily through the pulmonary vasculature, chewing tobacco users were found to absorb nicotine through the buccal mucosa and the gastrointestinal tract mucosa. Chewing tobacco is said to have increased the carcinogenic and genotoxic potential. The odds for oral cancer are estimated to be 7.3 in smokers, 1.3 in alcoholics and 11.4 in those who are habituated to chewing tobacco.  Genotoxic effects are caused by tobacco and areca-nut specific nitrosamines, which leach out in the saliva of chewers. The aqueous extract of N-nitroso compound related to areca nut i.e. 3-(N-nitrosomethylamino) propionaldehyde is highly cytotoxic and genotoxic to human buccal epithelial cells and potentially import in the induction of tumors in betel quid chewers.  According to a study conducted by Saran et al., a step-wise increase in the DNA damage was observed in buccal epithelial cells from control to precancer patients and from pre-cancer to cancer patients. 
Besides the use of smokeless or smoked forms of tobacco, there are multiple other reasons for occurrence of MN cells. It has been established that genomic damage is produced by exposure to genotoxic substances, medical procedures (radiation and chemicals),micronutrient deficiency (folic acid), life-style (alcohol, smoking, drugs, stress), urban pollution, chronic contact with arsenic and chromium and genetic factors, such as defects in metabolism and/or in the repair of DNA.  Bloching et al. said that the subgingival plaque and synthetic dental materials in addition to chronic alcohol and tobacco consumption might cause genotoxicity in the oral cavity.  Mahimkar et al. also stated that the degree of overlap in MNC frequency across the study groups is due to the influence of environmental and host factors. Hence, this assay serves as an indicator of DNA damage in a group and fails to predict risk at an individual level. 
Severity of buccal changes associated with the use of SLT was more as compared with the smoking habits. Though weak, there is a positive correlation between duration and frequency of habit and occurrence of MN.
For conclusive and reliable results, still larger samples should be studied for longer duration. Few patients are usually unwilling to reveal the duration, frequency and other associated habits during case history recording. The buccal exfoliated cells should also be collected after stoppage of habit to check for the decrease in the number of MN and level of genotoxicity. We should also check other causes of formation of MN besides tobacco and related products, difference in the degree and mechanism of action of smokeless and smoked variants of tobacco and also the effects of various habit patterns in relation to occurrence of MN.
| > Acknowledgement|| |
This study has been approved by ICMR in its entirety.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]