|Year : 2021 | Volume
| Issue : 2 | Page : 163-170
A study on micronuclei in tobacco and related habits
Twinkal S Patel, Anjani R Chaudhary, Bhavin B Dudhia, Parul V Bhatia, Purv S Patel, Yesha V Jani
Department of Oral Medicine and Radiology, Ahmedabad Dental College and Hospital, Ahmedabad, Gujarat, India
|Date of Submission||21-Aug-2020|
|Date of Decision||30-Jan-2021|
|Date of Acceptance||10-Apr-2021|
|Date of Web Publication||23-Jun-2021|
Dr. Twinkal S Patel
2, Sandhya society, Vijay Cross Road, Navrangpura, Ahmedabad, Gujarat
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Oral cancer is one of the ten most common causes of mortality in developing countries like India. A micronucleus (MN) is a small extra nucleus separated from the main one, generated during cellular division by late chromosomes or by chromosome fragments. Micronucleus is a potential biomarker for malignancy. Aim and Objectives: The study attempts to analyze micronuclei in buccal smear from patients with tobacco and related habits; and to determine its reliability as a biomarker for carcinogenesis. Study Design: A total of 400 consisting of normal subjects, patients with potentially malignant lesions and patients with malignant lesions were included in the study. Oral exfoliated cells were collected by scrapings of buccal mucosa of both control and study groups with a tooth brush. The scrapings collected from right and left buccal mucosa of each subject were studied under two separate slides. A total of 500 cells were examined from each slide. All the micronucleus cells located on each slide were examined again under the 100X magnification. Results: The P value for intergroup difference in average number of micronuclei in cells of buccal mucosa among all groups was <0.001 which was highly significant. The P value for micronucleated cells of buccal mucosa among all groups was <0.001 which was highly significant. Conclusion: A stepwise increase in MN counts from normal to potentially malignant to carcinoma suggested a link of this biomarker with neoplastic progression. Micronuclei can be used as a biomarker for early detection of premalignant and malignant lesions of the oral cavity.
Keywords: Biomarkers, exfoliated cells, micronucleated cells, micronuclei, premalignant lesions
|How to cite this article:|
Patel TS, Chaudhary AR, Dudhia BB, Bhatia PV, Patel PS, Jani YV. A study on micronuclei in tobacco and related habits. J Indian Acad Oral Med Radiol 2021;33:163-70
|How to cite this URL:|
Patel TS, Chaudhary AR, Dudhia BB, Bhatia PV, Patel PS, Jani YV. A study on micronuclei in tobacco and related habits. J Indian Acad Oral Med Radiol [serial online] 2021 [cited 2021 Dec 7];33:163-70. Available from: https://www.jiaomr.in/text.asp?2021/33/2/163/319062
| Introduction|| |
Cancer, a modern epidemic among the non-communicable diseases, is the second most common cause of mortality in developed countries. It remains one of the ten most common causes of mortality in developing countries like India.,,
Different diagnostic methods such as routine histopathology (H and E-stained sections), exfoliative cytology, and immunohistochemistry are available today. Out of these, oral exfoliative cytology is particularly valuable for mass screening purposes. It has been used in the detection of oral squamous cell carcinoma with high sensitivity (94%), specificity (100%) and accuracy (95%). It is the nucleus that expresses the genotypic alterations caused in the process of malignancy; and exfoliative cytology is a method that gives better insight of the nuclear changes in individual cells.
The genotoxic effects/risks in tobacco users on buccal mucosa can be evaluated by assessing the DNA damages such as chromosomal aberrations, sister chromatid exchanges and micronucleus test., Though the diagnosis of oral squamous cell carcinoma seldom presents difficulty, it is the cancer staging and histopathological grading that are more important for prognostication; and micronuclei are good prognostic indicators.
A micronucleus (MN) is a small extra nucleus separated from the main one, generated during cellular division by late chromosomes or by chromosome fragments. It is a microscopically visible round to oval cytoplasmic chromatin mass in the extra nuclear vicinity. They are induced in cells by numerous genotoxic agents that damage the chromosomes. The damaged chromosomes, in the form of acentric chromatids or chromosome fragments, lag behind in anaphase when centric elements move towards the spindle poles.,
MN assays provide information on the cytogenetic damage in the tissues, that are targets of human carcinogens and from which carcinomas can develop. MN is induced in oral exfoliated cells by a variety of substances, including genotoxic agents and carcinogenic compounds in tobacco, betel nut, and alcohol, UV radiation, infrared rays, X-radiations, and chemicals.,, Tobacco-specific nitrosamines have been reported to be potent clastogenic and mutagenic agents which are thought to be responsible for the induction of chromatid/chromosomal aberrations resulting in production of MN. It is a known fact that habits such as use of tobacco in various forms, alcohol consumption, pan chewing and use of commercial pan products are associated with increased risk for development of oral cancer.,, Oral squamous cell carcinomas are characterized by complex karyotypes that involve many chromosomal deletions, translocations and structural abnormalities.
Recent studies are attempting to show a correlation between the frequency of MN in oral exfoliated cells and histopathological grading of oral squamous cell carcinoma. There are very few studies analyzing and comparing micronuclei and micronucleated cells from buccal smear between subjects who have tobacco and related habits with oral lesions and without lesions with control groups of habit with oral cancer and without habit without lesions. Hence, this study was undertaken to analyze micronuclei in buccal smear from subjects with tobacco and related habits as a biomarker for carcinogenesis.
| Materials and Methods|| |
A total of 400 subjects attending the outpatient Department of Oral Medicine and Radiology of a Dental College within a period of 2 years, were included in the study irrespective of their age and gender. The study has been conducted as per the IRB guidelines and Helsinki Declaration and after approval from the ethical committee of Scientific Research Committee of a dental college, Gandhinagar (letter Reference ECR/1101/Inst/GJ/2018). Data sampling was done by convenience sampling method, using the sample size formula (n) = 4pq/L2 wherein p is anticipated proportion of samples showing micronuclei in percentage, q is (1-p) and L is permissible error in estimation of p. According to this formula, the estimated sample size was calculated to be 277.77.
The study sample of 400 subjects was divided into following four groups:
- Group 1: 150 subjects with tobacco and related habits and having habit related oral lesions (but not oral cancer).
- Group 2: 150 subjects with tobacco and related habits but no habit related oral lesion.
- Group 3 (Control group): 50 subjects with tobacco and related habits and having oral cancer.
- Group 4 (Control group): 50 healthy subjects with no habit and no habit related oral lesion.
- The subjects of Groups 1, 2 and 3 having oral lesions on clinical examination were included in the study as per the following inclusion and exclusion criteria:
- Subjects having tobacco and related habits atleast since last 5 years.
- Subjects with tobacco related oral lesions or oral cancer.
- Subjects having oral lesions other than tobacco related oral lesions.
- Subjects already treated for tobacco related oral lesions.
- Subjects having any systemic disease.
- Subjects who have underwent any radiographic procedure before 1 month.
- Subjects exposed to known DNA-damaging agent or ionizing radiation.
- Subjects were informed about the procedure of the study and written informed consent was obtained from them.
- Each subject was made to rinse his/her mouth thoroughly with 0.12% chlorhexidine mouthwash (undiluted) and tap water.
- Cytosmear was taken from right and left buccal mucosa with a baby toothbrush (toothbrush head length: 1 inch, width: 5/16 inch, number of rows: 4 rows of bristles, number of tufts: 8 tufts per row, number of bristles: 75 bristles per tuft) with mild pressure and spread onto pre-cleaned and marked slides. [Figure 1]
- Two slides were made (one each from the right and left buccal mucosa) for each subject. [Figure 2]
- The smears were fixed with 95% alcohol spray and stained with Papanicolaou (PAP) stain commercially available as a Rapid pap staining kit [Figure 3].
|Figure 1: Scraping of oral exfoliated cells from buccal mucosa with a brush|
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- The Tolbert et al. criteria was used for counting the micronuclei and micronucleated cells.
- From each slide, approximately 500 cells were examined under the 40X magnification [Figure 4].
|Figure 4: Exfoliated cells seen under 40X magnification showing micronucleated cells (arrow) with micronuclei|
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Parameters for micronucleated cell inclusions (Tolbert's criteria)
- Intact cytoplasm and relatively flat cell position on the slide.
- Little or no overlap with adjacent cells.
- Little or no debris.
- Nucleus normal and intact, nuclear perimeter smooth and distinct.
- Wherever micro-nucleated cells were located, they were examined under the 100X magnification for counting of micronuclei [Figure 5].
|Figure 5: Micronucleated cell under 100X showing micronuclei (arrow). (<1/3rd diameter of nucleus)|
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Criteria for identifying micronuclei (Tolbert's criteria)
- Rounded smooth perimeter suggestive of a membrane.
- Less than a third of the diameter of the associated nucleus, but large enough to discern shape and color.
- Feulgen-positive, i.e., pink in bright field illumination.
- Staining intensity similar to that of the nucleus.
- Texture similar to that of the nucleus.
- Same focal plane as nucleus.
- Absence of nucleus overlap with or bridge to the nucleus.
Observation of data
- Micronuclei and micronucleated cells were observed independently by three observers.
- The data that was common in at-least two out of three observations was considered final.
- The observation for all the patients were recorded in Windows 7 Microsoft Excel worksheet.
The data were analyzed using the discriminate procedure of the Statistical package for the Social Sciences (SPSS 17.0). SPSS 17.0. ANOVA test was applied for age, gender, oral lesions and habit distribution. Paired t-test was applied for average number of micronuclei in cells and micronucleated cells of right and left buccal mucosa; intergroup difference in average number of miccronucleated cells and micronuclei in cytosmear of buccal mucosa. Discriminate function analysis was used to determine variables that discriminate between habit with oral lesions and habit without oral lesions. P < 0.05 was considered as statistically significant in all statistical analysis.
| Results|| |
The study was conducted on 400 subjects (287 males and 113 females) with age ranging from 18 to 75 years. Statistically significant differences were not observed for gender or age (p > 0.05). The interobserver agreement kappa values for the micronuclei were 0.91 (observer one vs observer two), 0.88 (observer two vs. observer three) and 0.85 (observer one vs. observer three). For the micronucleated cells, the kappa values were 0.81 (observer one vs. observer two), 0.83 (observer two vs. observer three) and 0.90 (observer one vs. observer three). For micronuclei as well as micronucleated cells; the agreement was excellent.
[Table 1] depicts the distribution of subjects of study groups according to different age groups and gender. Out of 150 subjects of Group I; age group 15-25 had 14 patients of OSMF and 6 patients of tobacco quid lesion, age group 26-35 had 42 patients of OSMF and 10 patients of tobacco quid lesion, age group 36-45 had 27 patients of OSMF, 6 patients of leukoplakia and 8 patients of tobacco quid lesion, age group 46-55 had 8 patients of OSMF, 7 patients of leukoplakia and 6 patients of tobacco quid lesion, age group 56-65 had 4 patients of OSMF, 8 patients of leukoplakia and 2 patients of tobacco quid lesion, age group 66-75 had 1 patient of OSMF and 1 patient of leukoplakia.
|Table 1: Distribution of subjects of study groups according to age and gender and oral lesions|
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The age wise distribution of subjects of Group II are shown in [Table 1]. Out of 50 subjects of Group III; age group 15-25 had no subjects, age group 26-35 had 8 subjects (8 males and 0 females), age group 36-45 had 12 subjects (11 males and 1 female), age group 46-55 had 15 subjects (13 males and 2 females), age group 56-65 had 11 subjects (8 males and 3 females) and age group 66-75 had 4 subjects (all males).
Out of 50 subjects of Group IV; age group 15-25 had 2 subjects (both males), age group 26-35 had 19 subjects (all males), age group 36-45 had 21 subjects (all males), age group 46-55 had 3 subjects (1 male and 2 females), age group 56-65 had 5 subjects (3 males and 2 females) and age group 66-75 had no subjects.
The distribution of subjects of different study groups according to different types of habits are shown in [Graph 1]. Group IV was without habit and lesion, which is excluded from this graph. In 150 subjects of group I, 16.3% subjects had smoking, 57.1% chewing and 26.6% had placement habit. In 150 subjects of Group II, 8.2% subjects had smoking, 69.4% chewing and 22.3% had placement habit. In 50 subjects of Group III, 14% had smoking, 35.3% chewing, 42.3% placement, 7.1% rubbing tobacco and 1.2% had alcohol habits.
[Table 2] depicts comparison of average number of micronuclei and micronucleated cells of right and left buccal mucosa in study groups. In Group I subjects, the average number of micronuclei in cells of right buccal mucosa was 27.98 and that of left buccal mucosa was 27.15 (p value 0.193) while average number of micronucleated cells of right buccal mucosa was 21.73 and that of left buccal mucosa was 20.91 (p value 0.254). In Group II subjects, the average number of micronuclei in cells of right buccal mucosa was 18.50 and that of left buccal mucosa was 18.19 (p value 0.891) while average number of micronucleated cells of right buccal mucosa was 14.52 and that of left buccal mucosa was 14.98 (p value 0.953). In Group III subjects, the average number of micronuclei in cells of right buccal mucosa was 44.76 and that of left buccal mucosa was 46.14 (p value 0.066) while average number of micronucleated cells of right buccal mucosa was 22.38 and that of left buccal mucosa was 22.40 (p value 0.965). In Group IV subjects, the average number of micronuclei in cells of right buccal mucosa was 7.30 and that of left buccal mucosa was 7.22 (p value 1.00) while average number of micronucleated cells of right buccal mucosa was 6.82 and that of left buccal mucosa was 6.68 (p value 0.350). P value for all comparisons was non-significant.
|Table 2: Comparison of average number of micronuclei in cells and micronucleated cells of right and left buccal mucosa in study groups of right and left buccal mucosa in study groups|
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[Table 3] shows discriminant functional analysis to assess presence and absence and number of micronuclei and micronucleated cells of buccal mucosa that can be used to determine the habit status and presence of lesions and its malignant potentials.
|Table 3: Discriminant functional analysis for number of micronuclei and micronucleated cells|
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It depicts the percentage of subjects diagnosed correctly predicted as having habit with lesions and habit without lesion by counting micronuclei and micronucleated cells. It was observed that out of 150 subjects with oral habit and lesions; 135 (90%) were predicted correctly having micronuclei and 138 (92%) were predicted correctly having micronucleated cells. It was observed that out of 150 subjects with oral habit without lesions; 141 (94%) were predicted correctly having micronuclei and 132 (88%) were predicted correctly having micronucleated cells. Hence, out of total 300 subjects; 276 (92%) were predicted correctly having micronuclei and 270 (90%) were predicted correctly having micronucleated cells. As per [Table 3] number of micronuclei in cells of buccal mucosa are 92% accurate and number of micronucleated cells of buccal mucosa which are 90% accurate.
The intergroup difference in average number of micronucleated cells in cytosmear of buccal mucosa is seen in [Graph 2]. In group I average number of micronucleated cells was 21.30, in Group II it was 13.41, in Group III it was 22.39 and in group IV the average number of micronucleated cells was 6.75. P value for intergroup difference in average number of micronucleated cells in cytosmear of buccal mucosa among all groups was <0.001 except that between Group I and Group III which was 0.006, all of which were significant.
The intergroup difference in average number of micronuclei in cells of buccal mucosa is depicted by [Graph 3]. In Group I average number of micronuclei was 28.20, in Group II it was 16.85, in Group III it was 45.45 and in Group IV the average number of micronuclei was 7.26. P value for intergroup difference in average number of micronuclei in cells of buccal mucosa among all groups was <0.001 which were all highly significant.
| Discussion|| |
In humans, MN can be easily assessed in erythrocytes, lymphocytes, and exfoliated epithelial cells (e.g., oral, urothelial, nasal) to obtain a measure of genome damage induced in vivo. Buccal cells are the first barrier for the inhalation or ingestion route and are capable of metabolizing proximate carcinogens to reactive products. Approximately, 90% of human cancers originate from epithelial cells. Therefore, oral epithelial cells represent a preferred target site for early genotoxic events induced by carcinogenic agents entering the body via inhalation and ingestion. Furthermore, MN assay can be performed in buccal cells without the need for ex vivo nuclear division., The present study was conducted on cytosmear taken from right and left buccal mucosa in all subjects.
In this study, oral mucosal cells were scraped by baby toothbrush which reaches up to lamina propria, thus obtaining epithelial cells through the full thickness of the epithelium., Similar studies have been performed on micronucleus frequency in cytosmear by using baby toothbrush Smita Jyoti et al., (2013) and Swati Gupta et al. (2014).
The micronuclei assay in exfoliated oral mucosal cells depends strongly on the staining method. Papanicolaou stain (PAP), which is the most commonly used cytological stain, was found to show better staining results as compared to the May-Grünwald Giemsa (MGG) stain and Romanowsky's stain.,,,,,, Hence, commercially available RAPIDPAP kit was used for this study. Also, the current study examined 500 cells from each slide which is in accordance with previous studies conducted by Varsha Ajit Sangle et al. (2016) and Dahal S et al., (2013),,
In this study, tobacco and betel quid placement and chewing habits were found to be common habits in Gandhinagar district. This is in accordance with the Indian literature which states that the prevalence of chewing and placement habit is more common than smoking habit.,,,,, Previous studies conducted by Kawatra Abhishek et al. (2012), Farhadi S et al. (2016), Vandana G et al. (2016), Gopal KS et al. (2018) and Benkassar A et al. (2019) also concluded that tobacco placement and chewing habits are more common habits.
In this study, criteria of Tolbert et al., was used for consideration of micronuclei and micronucleated cells which was in accordance with other studies.,,,,, Similar studies of counting micronuclei and micronucleated cells by using Tolbert's criteria was conducted by Varsha Ajit Sangle et al., (2016) and for counting micronuclei alteration by Shally Khanna et al. (2016), Vandana A et al. (2016), Aroumougam A et al. (2018), Somya S et al. (2018) and Kamini K et al. (2018).
The present study revealed that the number of micronuclei and number of micronucleated cells were less in number in cytosmear from buccal mucosa of healthy subjects, increased in subjects with tobacco and related habits without oral lesions, further increased in subjects with tobacco and related habits with oral lesions and even more increased in subjects with tobacco and related habits with oral cancer. (p < 0.05) This stepwise increase in MN counts from normal to potentially malignant lesions to carcinoma suggested a link of this biomarker with neoplastic progression. These findings are in accordance with the studies conducted by Gupta S et al., (2014) and Gupta S et al., (2014) and Gupta J et al. (2019). These observations indicate cytogenic damage of the epithelial cells from tobacco. The various possible explanations for MN formation in potentially malignant conditions include chromosome loss/breakage, chromosomal aberrations, mitotic apparatus dysfunctions, aneuploidy, and genetic instability., Hence, as a biomarker of genomic damage, MN has been proved to be an important upcoming marker of tumorogenesis.
Limitations and future prospects
This study was performed on the subjects before treatment; if the same study can be extended on the subjects after treatment it would be better. We included subjects with different habits and various lesions in same study; studies could be conducted on subjects with different habits and various lesions separately. The study was conducted on 500 cells for micronuclei and micronucleated cells in each subject but it might give better result if study can include more cells.
| Conclusion|| |
Number of micronuclei and number of micronucleated cells were minimum in cytosmear of buccal mucosa of healthy subjects, increased in subjects with tobacco and related habits without oral lesions, further increased in subjects with tobacco and related habits with oral lesions and were maximum in subjects with tobacco and related habits with oral cancer.
This stepwise increase in micronuclei and micronucleated cells assay can be useful as a biomarker for genotoxicity and epithelial carcinogenic progression. This assay is sensitive, minimally invasive, simple, economical and quick.
Hence, micronuclei can be used as a biomarker for early detection of premalignant and malignant lesions of the oral cavity; making it an essential tool for the oral physician to convince the patient for further investigations (e.g., biopsy) as well as to motivate the patient towards the cessation of the adverse oral habit(s).
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]