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   Table of Contents      
ORIGINAL ARTICLE
Year : 2018  |  Volume : 4  |  Issue : 1  |  Page : 12-17

Quantitative and Qualitative Analysis of Micronuclei in the Buccal Mucosal Cells of Individuals Associated with Tobacco


1 Department of Oral Pathology, Institute of Dental Studies and Technologies, Modinagar, Uttar Pradesh, India
2 Department of Oral Medicine & Radiology, Institute of Dental Studies and Technologies, Modinagar, Uttar Pradesh, India
3 Department of Pathology, UHSR, Rohtak, Haryana, India
4 AIMST University, Bedong, Malaysia

Date of Web Publication27-Mar-2018

Correspondence Address:
Shalu Rai
Department of Oral Medicine & Radiology, Institute of Dental Studies and Technologies, Kadrabad, Modinagar 201201, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mamcjms.mamcjms_64_17

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  Abstract 


Introduction: Micronucleus/micronuclei) have the potential to serve as an important biomarker in exfoliated cell. Exfoliated buccal mucosal cells can be evaluated using MN assay which can be applied to individuals practicing tobacco habits and are at risk of developing oral cancer. Material and Methods: 80 tobacco users and 20 non tobacco users as control were included in the study. Smears were taken and stained with H&E, feulgen, acridine orange. MN was counted using criteria given by Tolbert et al. Data was analyzed using SPSS software. Results: MN higher in tobacco users. The values obtained for the MN count in tobacco chewers using these stains were statistically significant (P<0.05). The values obtained for the MN count in tobacco smokers using these stains were statistically significant (P<0.05). Qualitatively a good score for staining intensity; MN outline and nuclear outline was obtained for acridine orange, followed by H&E and feulgen stain. Discussion: In our study quantitatively, MN was counted in maximum number using H&E stain, followed by acridine orange and feulgen. Qualitatively, MN count was best viewed using acridine orange stain under a fluorescence microscope, followed by H&E and feulgen under a light microscope. Conclusion: Assessment of MN in exfoliated cells is a promising tool to study epithelial carcinogens in the oral cavity.

Keywords: Buccal mucosa cells, cytogenic biomarker, micronuclei


How to cite this article:
Sharma S, Rai S, Misra A, Sharma A, Misra D, Dhanpal R. Quantitative and Qualitative Analysis of Micronuclei in the Buccal Mucosal Cells of Individuals Associated with Tobacco. MAMC J Med Sci 2018;4:12-7

How to cite this URL:
Sharma S, Rai S, Misra A, Sharma A, Misra D, Dhanpal R. Quantitative and Qualitative Analysis of Micronuclei in the Buccal Mucosal Cells of Individuals Associated with Tobacco. MAMC J Med Sci [serial online] 2018 [cited 2018 Aug 15];4:12-7. Available from: http://www.mamcjms.in/text.asp?2018/4/1/12/228657




  Introduction Top


Carcinogenesis is a multistep process characterized by genetic, epigenetic, and phenotypic changes. Many chemical, physical, and biological environmental agents are able to interact with deoxyribonucleic acid (DNA) to induce mutations. The normal function of DNA repair genes and/or cell proliferation and differentiation control genes, when lost as a consequence of mutations, increases the risk of cancer development. There are biomarkers that have been researched into, which can predict whether a premalignant lesion or condition is likely to develop into an aggressive metastasizing tumor.[1]

The lifestyle factors that are associated with genetic damage include smoking, alcohol consumption, and tobacco consumption. These are addictive and harmful to health in many ways.[2] Various forms of tobacco contain thousands of chemical compounds that are not only irritants and toxins, but also carcinogens. Among these, the most potent are tobacco-specific nitrosamines (TSNAs), polycyclic aromatic hydrocarbons, and reactive oxygen species.[3],[4],[5]

Currently, India has a large number of habitual chewers of betel quid (plain pan) and tobacco.[6] Betel quid contains tobacco in addition to areca nut, which contains polyphenols and several alkaloids that act as mutagens and clastogens.[7] Areca has been classified as carcinogenic to humans.[8],[9],[10] Tobacco used in any form can result in irreparable genetic injury.[5],[10] It is the most common chemical carcinogen causing oral cancer.[5] Tobacco is consumed in various forms. The two broad categories of tobacco consumption are smoked tobacco in the form of cigarettes, bidis, cigars, cheroots, reverse smoking, and hukkas and smokeless tobacco in the form of betel leaf, areca nut, slaked lime, catechu, pan masala, and gutka.[3]

All parts of the oral cavity are susceptible to cancer from tobacco smoking or chewing, including the lip, tongue, palate, gingiva and cheek.[11],[12] The buccal mucosa (BM) provides a barrier to potential carcinogens that can be metabolized to generate potential reactive products. As up to 90% of all cancers appear to be epithelial in origin, the BM could be used to monitor early genotoxic events that result due to potential carcinogens entering the body through ingestion or inhalation. The exfoliated buccal cells have been used noninvasively to successfully show the genotoxic effects of lifestyle factors such as tobacco smoking and/or chewing.[13]

Micronucleus (MN) assay is a simple epidemiologic tool that can be applied to individuals having the habit of tobacco consumption in various forms and are at a risk of developing oral carcinomas.[14],[15] The MN test on the exfoliated buccal mucosal cells can be helpful in quantitating a synergistic effect between these habits.[5],[14],[16],[17],[18] They can be detected under light microscopy using hematoxylin and eosin (H&E) staining or using fluorescent microscopy with DNA-specific stains such as acridine orange and feulgen.[14],[19],[20] This method can be used on a regular basis in general dental practice on a day-to-day basis and helps to screen suspected patients and large populations. This is an inexpensive procedure that uses easily available equipments and stains that can be employed easily in clinical setups.

The aim of the study was to quantify the MN in chewers and smokers with a habit of tobacco consumption for more than 10 years and compare their data with that obtained from healthy individuals. The study also assessed the variation of MN count and quality using three different stains (H&E, acridine orange, and feulgen).


  Materials and Methods Top


This cross-sectional prospective study was conducted at the Department of Oral and Maxillofacial Pathology and Microbiology in a Dental College in Ghaziabad, UP. The smears were collected from the patients with a habit of chewing and smoking tobacco for a minimum of 10 years reporting to the Outpatient Department. Because the formation of MN is time bound and a habit-based phenomenon, this action will vary from person to person. Our study emphasized on the MN count in participants with prolonged habits; hence, we kept 10 years as the minimum baseline for the same.

Participants for the study were selected after a detailed history regarding the tobacco form used, as well as the duration and frequency of habit, was obtained. Smears were taken from the BM of the patients associated with tobacco. Inclusion criteria were as follows: (i) participants with only one kind of habit for a minimum of 10 years and (ii) smears that were positive for MN. Exclusion criteria were as follows: (i) participants with mixed habits, (ii) participants with systemic disease, and (iii) participants undergoing radiotherapy. The study was approved by the institutional ethical committee of our institution. Consent forms were signed, and permission was obtained before collecting the smears.

The study was conducted with 100 patients, among which 40 patients were chewers and 40 were smokers. A total of 20 control participants (without any habits) were also used in the study to see the difference in the MN counts from those observed in the patients with a habit of tobacco consumption. All the smears collected from the participants were stained using H&E, feulgen, and acridine orange [[Figure 1][Figure 2][Figure 3]].
Figure 1: A buccal smear of a smoker showing micronuclei (H&E: 40×)

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Figure 2: A buccal smear of a smoker showing micronuclei (feulgen: 40×)

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Figure 3: A buccal smear of a chewer showing micronuclei (acridine orange: 40×)

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The participants were asked to rinse their mouth thoroughly with water before the smear was collected. A wet wooden spatula was used to scrape the BM vigorously. The scraping obtained was cytocentrifuged in neutral Tris hydrochloric acid buffer. The pellet obtained was smeared on three clean microscopic slides. The slides were fixed with Biofix and air dried. H&E, acridine orange, and feulgen were used to stain the prepared slides. The H&E and feulgen stained slides were viewed under a light microscope, and acridine orange stained slides were viewed under a fluorescent microscope. A total of 500 cells with intact nuclei and cell boundaries were counted using a zigzag method. The MN was counted as per the criteria given by Tolbert et al.[12]

The resulting data were analyzed using the Statistical Package for the Social Sciences software (SPSS Inc., Chicago, IL, United States). Data were expressed as mean and standard deviation. Differences among the different variables were analyzed using Student’s t-test, chi-square test, interclass correlation, one-way analysis of variances (ANOVAs), post hoc test followed by Bonferroni test, and Friedman test (F value). A P value <0.05 was considered to be significant.


  Results Top


In our study, the results showed that the number of the micronucleated cells was higher among tobacco chewers and smokers in comparison to the control. The mean percentage of the MN cell count in chewers was 7.63 ± 3.62, which in number showed a mean of 10.63 ± 3.21. Similarly, the MN cell count percentage in smokers was 5.23 ± 3.15, showing a mean of 7.34 ± 2.14. The MN cell count percentage in the control group was 1.87 ± 2.75, showing a mean of 2.41 ± 2.02. The mean difference between the two groups was statistically significant [[Table 1], [Figure 4]].
Table 1: Comparison of micronucleated cells and the micronuclei count among the chewers, smokers, and the control group (under H&E)

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Figure 4: Quantitative analysis of micronucleated cell (MNC) count and micronuclei (MN) count among the chewers, smokers, and the control group

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On quantitatively comparing the results from chewers using the three stains, the mean count of the MN was 6.98 ± 1.07 using acridine orange, 5.30 ± 0.93 using feulgen, and 10.63 ± 3.21 using H&E. The values obtained for the MN count using these stains were statistically significant (P < 0.05) [[Table 2], [Figure 5]].
Table 2: Comparison of the micronuclei count among the participants in the chewer group using different stains (H&E, feulgen, and acridine orange)

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Figure 5: Comparison of micronuclei count between chewers and smokers under H&E, acridine orange, and feulgen stains

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Similarly, for smokers, the mean of the MN count was 6.25 ± 3.41 using acridine orange, 5.43 ± 1.01 using feulgen, and 7.34 ± 2.14 using H&E. The values obtained for the MN cell count and the MN count using the stains were statistically significant (P < 0.05) [[Table 3], [Figure 5]].
Table 3: Comparison of the micronuclei count among the participants in the smoker group using different stains (H&E, feulgen, and acridine orange)

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The qualitative analysis for this study was performed by two observers, and the interobserver comparison taking three parameters was used to analyse MN by Kappa test [Table 4]. The results showed a good score for all the three parameters (1–staining intensity; 2–MN outline; 3–nuclear outline) on using acridine orange, followed by H&E; least score was observed with feulgen stain. Sensitivity and specificity were calculated as 90 and 100%, respectively.
Table 4: Qualitative analysis showing the comparison of readings between Observer 1 and Observer 2 under the parameters used with the Kappa values

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  Discussion Top


Exfoliative cytology forms the basis of many epidemiological studies that help to assess the level of damage in individuals who may be prone to life-threatening diseases such as oral cancer. The cytological diagnosis of exfoliative epithelium has been increasingly used during the last few decades for biomonitoring human populations exposed to genotoxic agents.[21]

The use of cells from the BM provides a unique opportunity to study the regenerative capacity of the epithelial tissue, which is of ectodermal origin in humans. The assessment of the micronuclei in the exfoliated cells is a promising tool for the study of epithelial carcinogens in the oral cavity.[22] The epithelial tissues are in immediate contact with inhaled and ingested genotoxic agents, because it lines all the mucosal surfaces. Most cancers arise in the epithelial tissues. In many cases, these tissues are the actual targets of carcinogens, as indicated by the sites of cancers being related to the exposures. The epithelial cells can be easily collected from the mouth, nose, and bladder by noninvasive procedures.[23]

The human buccal cells display diverse changes that are associated with smoked and smokeless tobacco. In addition, clinicopathologic studies have correlated the changes in the human buccal cell with oral cancer. The buccal cell changes that have been reported include micronuclei formation, bacterial adherence, genetic mutations, DNA polymorphisms, carcinogen–DNA adducts, and chromosome abnormalities.[24],[25]

The standard laboratory procedure for a MN assay is cheap and accurate. This assay technique involves the examination of epithelial smears to determine the prevalence of the cells containing micronuclei, which are extranuclear bodies composed of chromosomal fragments or entire chromosomes that failed to be incorporated into daughter nuclei at mitosis. The assay can be used to detect chromosome breakage or mitotic interference events thought to be relevant to carcinogenesis.[22]

MN mainly originates from acentric chromosome fragments, acentric chromatid fragments, or whole chromosomes that fail to be included in the daughter nuclei at the completion of telophase during mitosis. These displaced chromosomes or chromosome fragments are eventually enclosed by a nuclear membrane and, except for their smaller size, are morphologically similar to nuclei after conventional nuclear staining. These micronuclei can be induced by carcinogenic agents.[26]

Tobacco chewing and smoking are popular oral habits with potential links to the occurrence of oral cancer. Many studies reveal that areca nut extract may demonstrate mutagenic and genotoxic effects, in addition to inducing preneoplastic as well as neoplastic lesions of the oral cavity.[4],[6],[7] More than 60 known carcinogens have been detected in the smoked forms of tobacco, which include polycyclic aromatic hydrocarbons, nitrosamines, and aromatic amines with genotoxic potential that are capable of initiating and promoting events in carcinogenesis at the cellular level.[6],[8]

In chewers, reactive oxygen species produced during the autoxidation of areca nut polyphenols are crucial in the initiation and promotion of oral cancer. The risk of cancer in smokeless tobacco users has been attributed to the presence of TSNAs.

There are four principal compounds:
  1. N-nitrosonornicotine (NNN);
  2. 4-methyl-Nnitrosamino-1-(3-pyridyl)-1-butanone (NNK);
  3. N-nitrosoanatabine;
  4. N-nitrosoanabasine.


Only two TSNAs, NNN and NNK, are considered to be potential carcinogens (International Agency for Research on Cancer, IARC Monographs 1985).[4],[5],[6]

Carcinogenic and mutagenic compounds, including TSNAs, which are present in the smoked and smokeless tobacco, are believed to be responsible for the induction of micronuclei.[5],[20],[27],[28]

Majority of the studies from 1985 till date have shown significant increase in the number of MN in the buccal exfoliated cells among chewers and smokers compared to healthy individuals. In our study, we assessed the MN count in these habit groups. Our study reported the MN count using the different grades of smear and the interobserver variability of the quantitative and qualitative assessments of the micronuclei under three different stains and parameters.

In our study, the MN count was higher among chewers than smokers. These findings are consistent with the studies of Sellappa et al.[28] and Patel et al.,[29] wherein the MN count in smokeless tobacco users was higher than that in the control group. Similarly, an increase in the frequencies of MN among pan masala and gutkha consumers has also been reported.[30],[31] MN formation has been observed in the precancerous lesions of the oral cavity of chewers.[32] Similar results were obtained by Fareed et al.,[5] wherein they compared the MN count between gutkha chewers and a control group and concluded that the MN frequency obtained in the case of tobacco users was much higher, which clearly indicated that chewers are at high risk for developing oral cancer.

MN count was more in chewers compared to smokers using H&E and acridine orange stains, but was slightly more in smokers when detected using feulgen stain. Comparison between these three stains has not been done in any study previously. A comparative study was conducted by Grover et al.,[33] wherein they used H&E, feulgen, and Papanicolaou (PAP) stains. They also obtained the least MN score using feulgen stain and maximum MN count using H&E stain. The possible explanation for the lowest count of MN with the feulgen stain could be because of its high DNA specificity and a clear transparent appearance of the cytoplasm. Apart from being highly technique sensitive, it is relatively lengthy method and may lead to the underscoring of MN. Scoring of MN does not require any special training. However, certain features within the cytological smear may be confused with MN. These include interference with bacterial colonies, small dye granules, or cellular structures such as keratohyaline granules that may lead to false negative results.[34]

In the second part of our study we qualitatively compared the three stains under the parameters of (i) staining intensity, (ii) MN outline, and (iii) nuclear outline. Our results showed that staining intensity was best for acridine orange stain, followed by H&E. A lower score for staining intensity was reported using feulgen. MN outline was best seen using acridine orange stain, followed by H&E; a poor score was given to MN outline with feulgen stain. Similarly, nuclear outline was best seen using acridine orange, followed by H&E. A poor scoring of nuclear outline was reported with feulgen stain. Thus, most cases scored a good staining result for all the three parameters using acridine orange s under fluorescence microscope, followed by H&E under a light microscope. Feulgen got the least score in most cases on considering all three parameters. As mentioned by Torres et al.[35] acridine orange is a specific stain for DNA and, therefore, allows differentiation with ribonucleic acid. Other stains such as feulgen, Schiff, PAP, Orcein, and H&E are acidic–basic stains and may produce contrast between cytoplasm and nucleus. This study is unique because we did not come across any study in literature in which qualitative analysis using such parameters has been done before.

The results of this study suggest that H&E stain under light microscope and acridine orange stain under fluorescent microscope are best to view the MN in cytological smears. The drawback of feulgen stain is that it is a sensitive technique and a time-consuming procedure as compared to the other two staining procedures.


  Conclusion Top


The following points can be summarized from this study:

MN count is increased in the participants belonging to the chewer and smoker groups in comparison to those of the normal control group. Quantitatively, MN was counted in maximum number using H&E stain, followed by acridine orange and feulgen. Qualitatively, MN count was best viewed using acridine orange stain under a fluorescence microscope, followed by H&E and feulgen under a light microscope.

Hence, the biological significance of the micronuclei in the buccal cells of the oral mucosa is that the micronuclei are a manifestation of a readily identifiable clastogenic event as studied herein and have been associated with tobacco chewing and smoking. Keeping this aspect in view, MN assay might bridge the gap between clinical assessment and outright invasive procedure. The classical methodology of viewing smears for cytological changes can be used along with an investigation of MN as a routine procedure to assess early genotoxic damage in suspected patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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