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ORIGINAL ARTICLE
Year : 2021  |  Volume : 13  |  Issue : 6  |  Page : 1141-1148  

Genotoxic effect of various forms of tobacco on oral buccal mucosa and nuclear changes as a biomarker


Department of Oral and Maxillofacial Pathology, Best Dental Science College, Madurai, Tamil Nadu, India

Date of Submission16-Mar-2021
Date of Decision19-Aug-2021
Date of Acceptance24-Apr-2021
Date of Web Publication10-Nov-2021

Correspondence Address:
Murali Chinnakonda Raveendranath
Best Dental Science College, Madurai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpbs.jpbs_185_21

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   Abstract 


Aim: The present study aims to assess the genotoxic effect of various forms of tobacco users on buccal mucosa and nuclear changes as biomarkers. Materials and Methods: The study involves 150 cases, they were divided into three groups (two study groups and one control group). The buccal cytological smears were taken from three groups: Group I – 50 smokers, Group II – 50 nonsmokers (smokeless tobacco), and Group III – 50 control group. The buccal cells were transferred into a test tube containing Tris-ethylenediaminetetraacetic acid buffer (pH = 7) and was centrifuged (Remi® 1500 revolution/min [rpm]). Cell suspensions were transferred to the slides and fixed. The slides were stained using PAP and Feulgen stain. The MN and other nuclear abnormalities were studied and compared. Results: Nonsmokers (smokeless tobacco) had significantly increased frequency of all nuclear anomalies compared to smokers and healthy controls. Binucleation, karyorrhexis, micronuclei (MN), karyolysis, broken egg nuclei, and prominent nucleoli in nonsmokers (smokeless tobacco) and condensed chromatin in smokers were the most frequent anomalies. Binucleation and karyorrhexis were significantly more frequent in nonsmokers (smokeless tobacco) compared to smokers. The other nuclear abnormalities were not statistically significant in smokers and nonsmokers (smokeless tobacco). Conclusion: Numerous studies have stated that MN and other nuclear anomalies were present in higher frequency in smokers and nonsmokers. In our study, we found binucleation and karyorrhexis were statistically significant in nonsmokers (smokeless tobacco) compared to smokers. The other nuclear anomalies showed insignificant results. In order to further validate the significance of this study, a larger sample size has to be studied. On comparing the staining efficacy of smokers and nonsmokers using PAP and Feulgen stain, both the stains showed positive results. In the present study, DNA-specific Feulgen stain shows better staining of nuclear anomalies compared to DNA nonspecific PAP stain, which was found to be statistically significant.

Keywords: Micronuclei and other nuclear anomalies, nonsmokers (smokeless tobacco), smokers


How to cite this article:
Devadoss S, Raveendranath MC, Kathiresan T S, Ganesan K. Genotoxic effect of various forms of tobacco on oral buccal mucosa and nuclear changes as a biomarker. J Pharm Bioall Sci 2021;13, Suppl S2:1141-8

How to cite this URL:
Devadoss S, Raveendranath MC, Kathiresan T S, Ganesan K. Genotoxic effect of various forms of tobacco on oral buccal mucosa and nuclear changes as a biomarker. J Pharm Bioall Sci [serial online] 2021 [cited 2022 Oct 7];13, Suppl S2:1141-8. Available from: https://www.jpbsonline.org/text.asp?2021/13/6/1141/330001




   Introduction Top


Oral cancer is the sixth most common malignancy and one of the major causes of death worldwide.[1] Over 80% of oral cancer patients are tobacco users.[2] The habitual practice of tobacco products can be in two forms, namely smoking form as well as the smokeless tobacco chewing form.[3] Oral cancer may appear as an innocuous and asymptomatic lesion in the beginning stages and in the advanced stage of the tumor, symptomatic changes are seen and the patients report to the clinician in this stage; hence, the early diagnosis of the lesion is the key aspect in reducing the mortality associated with oral cancer.[4]

Surgical biopsy and histopathological examination is the gold standard for the definitive diagnosis of oral cancer. It is invasive and involves both psychological implications and technical difficulties for the patient and sample collection.[5],[6] Hence, the oral exfoliative cytology technique is one of the simple, nonaggressive, and relatively painless techniques which can be used for early detection of oral potentially malignant and malignant disorders in tobacco chewers.[7]

The collection of buccal cells is a simple, painless, and least invasive method available for measuring DNA damage in humans which may be produced by environmental exposure to genotoxins such as radiation, chemicals, micronutrient deficiency, and lifestyle factors (e.g. alcohol, smoking, and stress) which damage either directly or indirectly the integrity of the macromolecules.[8] Microscopic changes that occur earlier in the buccal mucosa include MN and other nuclear anomalies such as karyorrhexis, karyolysis, pyknosis, binucleation, condensed nuclei, hyperchromatism, prominent nucleoli, broken egg nuclei, nuclear–cytoplasmic ratio, and irregular nuclear borders.[9] Micronuclei (MN) tests in exfoliated buccal cells are popular biomarkers of genetic damage.[8]

This study was carried out to assess the effects of tobacco in smoking (bidi and cigarette) and smokeless forms (chewing tobacco, snuff, and betel quid) on buccal cells of tobacco users at the chromosomal level, through assessment of different nuclear anomalies as biomarkers.

In this study, Pap-Papanicolaou Stain and Feulgen stains were used to identify the nuclear anomalies in both smoking and smokeless form of tobacco users.


   Methodology Top


Sample preparation

Prior to the sample collection, informed consent was obtained from the participant, and relevant history of frequency, number, duration of smoking, and smokeless form of tobacco was recorded. The subjects were asked to rinse their mouth thoroughly before taking the smear to remove food particles, debris, and bacteria from the oral cavity. Using a Cytobrush® (Medscand, US), the two smears were taken from the buccal mucosa.[10],[11] The buccal cells were transferred into a test tube containing Tris-ethylenediaminetetraacetic acid buffer (pH = 7) and was centrifuged (Remi® 1500 revolution/min [rpm]).[12],[13],[14],[15]

Slide preparation

Cell suspensions were transferred to the slides and fixed with Zenker fluid and then air-dried at room temperature. The slides were then stained with PAP and Feulgen.

Staining procedure

Feulgen staining

The slides were immersed in 1Mol hydrochloric acid (HCl) at 60°C for 5–6 min, followed by placement in 1Mol HCl at room temperature for 1 min and were then immersed in Schiff reagent for 30 min and washed under running tap water for 10 min.[16],[17],[18],[19],[20],[21],[22],[23]

PAP staining

The slides were dip fixed for 3 min in tap water and the excess water was blown out. They were then dipped for 60 s in PAP stain. Three drops of Scotte's tap water buffer were added and the smear was washed after 10 s, followed by dipping with two changes in PAP dehydrant for 30 s, each then dipped for 45 s in the working cytoplasmic stain, washed in tap water, dehydration was repeated in a second bath of PAP dehydrant for 30 s and dried in the air, dipped in xylene, dried, and mounted with a cover glass using a drop of distyrene plasticizer xylene.[11],[24],[25]

Scoring

The observation was carried out using OLYMPUS BX41© research microscope and the MN analysis was done at ×400 magnification as per the criteria given by Tolbert et al., 1992, and 1000 cells/slide were examined. The frequency of MN was evaluated along with other metanucleated anomalies such as binucleated cells, karyorrhexis, and karyolysis.

The morphological criteria for MN given by Tolbert et al. are as follows:

  1. Rounded smooth perimeter suggestive of a membrane
  2. Less than a third of the diameter of the associated nucleus but large enough to discern shape and color
  3. Staining intensity similar to that of the nucleus
  4. Texture similar to that of the nucleus
  5. Same focal plane as nucleus
  6. Absence of overlap with the bridge to the nucleus.[26]



   Results Top


Overall, 150 cases were studied and out of 150 cases, 50 cases were smokers, 50 cases were nonsmokers (smokeless tobacco), and 50 cases were control, which is shown in [Graph 1]. Among 100 cases, 50 cases were males in smokers. In nonsmokers (smokeless tobacco), 31 cases were males and 19 cases were females, as shown in [Table 1]. In the present study, we have analyzed 11 nuclear abnormalities in all patients. The nuclear anomalies are MN, karyolysis, binucleation, hyperchromatism, broken egg nuclei, irregular nuclear borders, karyorrhexis, pyknosis, condensed chromatin, prominent nucleoli, and nuclear–cytoplasmic ratio. These nuclear abnormalities were compared using two different stains, i.e., PAP stain and Feulgen stain to assess their staining efficacy.

Table 1: Sex and group distribution

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In our study, on comparing the nuclear abnormalities with smokers and nonsmokers (smokeless tobacco), we found binucleation, karyorrhexis, MN, karyolysis, broken egg nuclei, and prominent nucleoli to be predominantly positive in nonsmokers (smokeless tobacco) when compared to smokers, whereas condensed chromatin was predominant in smokers compared to nonsmokers [Graph 2].



In the present study, binucleation and karyorrhexis were statistically significant in nonsmokers (smokeless tobacco) when compared to smokers.

On comparing the nuclear abnormalities with smokers and control group, the following nuclear abnormalities MN, karyolysis, broken egg nuclei, karyorhexis, condensed chromatin, prominent nucleoli, and binucleation were predominant in smokers, whereas pyknosis was less predominant in smokers [Graph 3].



In this study, MN, binucleation, broken egg nuclei, condensed chromatin, and prominent nucleoli were statistically significant in smokers when compared to the control group.

[Graph 4] shows the prevalence of nuclear abnormalities in nonsmokers (smokeless tobacco) and control group.



On comparing the nuclear abnormalities in nonsmokers (smokeless tobacco) and control group, we found MN, binucleation, broken egg nuclei, karyorrhexis, pyknosis, condensed chromatin, and prominent nucleoli to be the predominant in nonsmokers (smokeless tobacco) compared to control group and karyolysis was predominant in the control group compared to nonsmokers (smokeless tobacco).

In the present study, MN, binucleation, broken egg nuclei, karyorrhexis and prominent nucleoli were statistically significant in nonsmokers (smokeless tobacco) compared to the control group.

In the present study, the staining efficacy of PAP and Feulgen stain in smokers and nonsmokers (smokeless tobacco) are as follows:

[Graph 5] shows the staining efficacy of smokers using PAP and Feulgen stain.



In the present study, both the DNA-specific and nonspecific stains showed positive results and Feulgen stain was found to be greater, followed by PAP stain. The staining efficacy of Feulgen was found to be the highest when compared to PAP stain and it was statistically significant (P = 0.001).

[Graph 6] shows the staining efficacy of nonsmokers (smokeless tobacco).



In nonsmokers (smokeless tobacco), both the stains show positive results and Feulgen stain was found to be greater when compared to PAP stain which was statistically significant [P = 0.001].

[Graph 7] shows comparison of staining efficacy of smokers and nonsmokers (smokeless tobacco) using PAP stain.



Both the smokers and nonsmokers (smokeless tobacco) using PAP stain showed positive results, but it was not significant.

[Graph 8] shows comparison of staining efficacy of smokers and nonsmokers (smokeless tobacco) using Feulgen stain.



Both the smokers and nonsmokers (smokeless tobacco) using Feulgen stain showed positive results, but it was not significant.


   Discussion Top


Tobacco use (either by smoking or chewing) has adverse effects on buccal tissue. The major content of tobacco is nicotine, tar, and polycyclic hydrocarbons. One of the major causes of tobacco toxicity is a reactive oxygen species. Assessment of tobacco toxicity with various forms of tobacco showed that tar and nicotine content has genotoxic effects on buccal cells manifesting with different nuclear anomalies which can be used as biomarkers for potentially malignant disorders.

In the present study, we found that tobacco in both smoking and smokeless forms had significant effects on the buccal exfoliating cells causing several nuclear anomalies. We also found that chewers of tobacco were more affected than smokers and controls.

In our study, on comparing nuclear abnormalities between smokers and nonsmokers (smokeless tobacco), we found that binucleation, karyorrhexis, MN, karyolysis, broken egg nuclei, and prominent nucleoli were predominantly positive in nonsmokers (smokeless tobacco) when compared to smokers, whereas condensed chromatin was found to be predominant in smokers when compared to nonsmokers [Table 2].
Table 2: Total level of awareness of the interns regarding jaw relation procedures

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In the present study, binucleation and karyorrhexis were statistically significant in nonsmokers (smokeless tobacco) compared to smokers.

There is a corresponding increase in the nuclear anomalies, with an increase in frequency and duration of the tobacco usage in both smoking and smokeless forms. Further on comparing smoking and smokeless form of tobacco users, the nuclear anomalies were found to be substantially more in the smokeless form of tobacco users. This can be attributed to the longer duration of mucosal contact in smokeless form of tobacco users and consuming raw form of tobacco (chewing tobacco, snuff, and betel quid) which might have a more toxic impact than individuals who are taking a refined form of tobacco (cigarettes).

At the same time, more nuclear anomalies were seen in older age people compared to younger age group due to decreased thickness, integrity, and regenerative capacity of the buccal epithelium in older people.

These nuclear anomalies can be used as biomarkers to assess the genotoxic effect of tobacco on buccal exfoliated cells.

Kauser et al. studied micronucleus and other nuclear abnormalities among betel quid chewers with or without sadagura, a unique smokeless tobacco preparation, in a population from North East India. In their study, they found a higher frequency of MN among betel quid chewers with or without sadagura as compared to the unexposed control group. Betel quid chewers showed a higher frequency of binucleation as compared to controls. They concluded that sadagura was a harmful tobacco form in causing genotoxicity when consumed with betel quid.

Similar to this study, we also found binucleation to be significantly higher in tobacco chewers. This indicates a cytokinesis defect in the exposed cells. Areca nut ingredients may cause cytotoxic effect by deregulation of cell cycle control, mitochondrial function, or reactive oxygen species and this may further produce a cytotoxic effect on buccal cells.

Sharma et al. did a comparative study on the oral epithelium in tobacco chewers and alcohol consumers. They observed a higher frequency of karyolysis and condensed chromatin in both the groups. Similarly in the present study, we found condensed chromatin higher in smokers. This indicates cell death because of chronic exposure to betel leaf ingredients.

Saranya et al. studied cytomorphological changes in buccal epithelial cells of khaini chewers in different age groups. Their study showed a significant increase in nuclear changes in older people along with extended usage of khaini. They concluded that the prolonged use of khaini causes morphological changes in exfoliated buccal cells. In older people, there was an increase in the cytomorphological changes with an increase in tobacco usage.

In our study, we observed more nuclear anomalies in the older age group of smokers and nonsmokers (smokeless tobacco) compared to the younger age group. With an increase in duration of the exposure, there is a corresponding increase in nuclear anomalies, as well as aging can produce mutagenic lesions, due to decreased thickness, integrity, and regenerative capacity of the buccal epithelium.

U Nair et al., MS Joshi et al., H Bansal et al., and S. Baxi et al. stated a higher frequency of MN in tobacco chewers in comparison with smokers and controls.

Apart from tobacco, there are other confounding factors which are responsible for increased MN. They are anemia, dietary vitamin deficiencies, environmental pollution exposure to organic solvents, diesel derivatives, polycyclic aromatic hydrocarbons, lead-containing paints, remnants of pesticides in agricultural products and solvents, and arsenic-contaminated drinking water.

Sohini et al. studied the genotoxic effect of tobacco on buccal cells. They stated that the chewers had a significantly highest frequency of all nuclear anomalies compared to smokers and healthy controls. The presence of each nuclear anomaly was significantly greater in older ages in all the study groups; hence, they concluded that tobacco can cause and increase the rate of nuclear anomalies in both smoking and smokeless forms compared to healthy controls. The genotoxic effect of tobacco on buccal cells was partly age related.

In the present study, we found that MN, broken egg nuclei, prominent nucleoli, binucleation, karyorrhexis, karyolysis, and pyknosis were higher in nonsmokers (smokeless form of tobacco) compared to smokers and condensed chromatin was higher in smokers compared to nonsmokers (smokeless form of tobacco).

S Baxi et al. did a study to assess the nuclear anomalies and cytological features in buccal mucosa of tobacco users. They stated that there was no statistical difference for mean MN and other nuclear anomalies between tobacco smokers and tobacco chewers. They concluded that the nuclear anomalies and MN frequency were increased in any form of tobacco and it can be used as a screening tool for genotoxicity.

Similar to this study, we also found an increased frequency of MN in nonsmokers (smokeless tobacco) compared to smokers and control group, but there was no statistical difference.

Rajkokila et al. studied the nuclear anomalies in exfoliated buccal epithelial cells of petrol station attendants in Tamil Nadu, South India. They observed increased frequency of MN, binucleation, karyorrhexis, and karyolysis in the study group compared to the control group. They concluded that it was necessary to educate the working population about the genotoxic effect of petroleum derivatives.

Similar to this study, we showed an increased frequency of karyorrhexis and binucleation in nonsmokers (smokeless tobacco) compared to smoker's group.

In our study, we have evaluated the staining efficacy of nuclear anomalies in smokers and nonsmokers (smokeless tobacco) using DNA-specific Feulgen stain over the routinely used nonspecific DNA stain PAP.

Feulgen stain showed better staining efficacy compared to PAP stain. The staining efficacy of MN using Feulgen stain was found to be better than the PAP stain.

Other nuclear abnormalities such as broken egg nuclei, karyolysis, karyorrhexis, prominent nucleoli, condensed chromatin, and binucleation were found to be better when compared to routine nonspecific DNA stain PAP.

Nerseseyan et al. studied the MN frequencies in oral mucosa cells of heavy smokers and nonsmokers which were evaluated with nonspecific (Giemsa, May-Grunwald Giemsa Stain) and DNA-specific (4, 6diamidino-2-phenylindole, Feulgen, acridine orange) stains. They showed that the MN frequencies scored with Giemsa and May–Grunwald–Giemsa were significantly higher in smokers than in nonsmokers. These findings indicate that keratin bodies may be misinterpreted as MN with nonspecific DNA stains and lead to false-positive results in studies with cells of epithelial origin. They found out that exposure to tobacco smoke does not lead to the induction of MN in these cells.

On contrary to this study, we found an increased frequency of MN in nonsmokers compared to smokers in our study.

Similarly, Grover et al. did a comparative study for selectivity of MN in oral exfoliated epithelial cells. They showed an increased frequency of MN in PAP and H and E stains compared to Feulgen stain. The reasons for misinterpretation of MN in PAP stain were due to the formation of keratin granules that are found in degenerated cells with nuclear anomalies. These granules do not contain DNA and might be misinterpreted as MN.

Similar to this study, we also found an increased frequency of MN in PAP stain compared to Feulgen stain.

Manish Kumar et al. conducted a study to determine the genotoxic effect of smoking and chewing tobacco using Mn assay and other nuclear anomalies associated with it. They found that the individuals having tobacco habits (smoking and chewing) with lesions had a significant number of Mn cells and hence could be used as a reliable biomarker for assessing the genotoxic effect of tobacco in the oral mucosa.

Similar to this study, we also found an increased frequency of MN in nonsmokers (smokeless tobacco) compared to smokers and control group.

In the present study, we found an increase in MN and other nuclear anomalies in nonsmokers except condensed chromatin which was higher in smokers. However, binucleation and karyorrhexis were found to be statistically significant in nonsmokers compared to smokers in our study.

On comparison of staining efficacy of smokers and nonsmokers (smokeless tobacco) using PAP and Feulgen stain, Feulgen stain shows better staining efficacy compared to DNA nonspecific PAP stain.

Using Feulgen stain, we avoided chances of misinterpretation of MN.

The possible explanation for better staining efficacy in Feulgen stain could be because of high DNA specificity and a clear transparent appearance of the cytoplasm, which could be helpful for easy identification of MN and other nuclear anomalies.

Groover et al. observed misinterpretation of MN in PAP-stained smears, due to the following reasons:

  1. Misinterpretation of nuclear anomalies such as karyorrhexis, karyolysis, condensed chromatin, and binucleates as MN was observed using DNA nonspecific stains, which was similar to a study carried out by Nersesyan et al.
  2. Formation of keratin granules that are found in the cytoplasm of degenerated epithelial cells showed resemblance to nuclear anomalies. These round cytoplasmic bodies, which are formed as a consequence of cell injury, do not contain DNA, and may be classified as MN with nonspecific stains
  3. Contamination by bacteria that were commonly found in the mouth and can interfere with MN scoring. Bacteria can be differentiated from MN by their characteristic shape, smaller size, color, staining intensity, and their presence upon and between buccal cells on the slide
  4. Another common confounding issue is the small dye granules that may sometimes resemble MN but usually have a slightly different refractivity and color intensity.


The present study has revealed that nuclear anomalies in buccal epithelial cells can be used as a biomarker for smokers and nonsmokers in identifying potentially malignant disorders of the oral cavity.


   Conclusion Top


Smoking and smokeless form of tobacco can produce significant genotoxic damage on buccal exfoliating epithelial cells. These toxic effects of tobacco can produce various nuclear anomalies which can easily be studied through cytological smears as it very simple, noninvasive, and a cost-effective procedure compared to tissue biopsy. The present study has revealed that many nuclear anomalies such as MN, karyorrhexis, karyolysis, broken egg nuclei, binucleation, and prominent nucleoli were seen with increased frequency in nonsmokers compared to smokers in buccal epithelial cells. Only condensed chromatin was seen in smokers with increased frequency compared to nonsmokers (smokeless tobacco) in the present study.

The present study has shown an increase in the frequency of nuclear anomalies in nonsmokers when compared to smokers which was similar to the previous studies. These nuclear anomalies showed better staining with Feulgen stain when compared to PAP stain which was also similar to previous studies.

In the present study, confounding factors such as anemia, dietary vitamin deficiencies, environmental pollution (exposure to organic solvents), diesel derivatives, polycyclic aromatic hydrocarbons, lead-containing paints, remnants of pesticides in agricultural products and solvents, and arsenic-contaminated drinking water were not excluded.

To further validate the genotoxic effects on buccal epithelial cells, these confounding factors have to be excluded from the study design.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2]



 

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