Journal of Cytology
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 Table of Contents    
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 30  |  Issue : 3  |  Page : 174-178
Alteration in buccal mucosal cells due to the effect of tobacco and alcohol by assessing the silver-stained nucleolar organiser regions and micronuclei


Department of Oral and Maxillofacial Pathology, M.M College of Dental Sciences and Research, Mullana, Haryana, India

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Date of Web Publication5-Sep-2013
 

   Abstract 

Background: Oral habits such as alcohol consumption and tobacco chewing are considered to be initiators of dysplastic changes in the oral mucosa.
Aim: The aim of this study was to determine and compare the alteration in apparently normal buccal mucosal cells due to effect of alcohol and tobacco by assessing silver-stained nucleolar organiser regions (AgNORs) and micronuclei.
Materials and Methods: The study comprised a total of 100 subjects which were divided into four groups with 25 subjects having alcohol consuming habit, 25 subjects were tobacco consumer, 25 were both alcohol and tobacco consumer and 25 formed control group who neither consumed alcohol nor tobacco. Two cytological smears were taken from each subject with the help of cytological brush. The smear was then wet fixed and stained with AgNOR and acridine orange staining technique and assessed for nucleolar organiser region and micronuclei count respectively. 500 cells per slide were counted to note the changes.
Results: Mann-Whitney test was applied to assess the variation in the number of AgNORs and micronuclei count between different groups. Cytological changes in each group revealed the increase in mean AgNORs and micronuclei count in subjects with combined alcohol and tobacco consumption when compared with individual groups.
Conclusions: Tobacco and alcohol consumption produce alteration in apparently normal buccal mucosal cells, which may cumulatively lead to carcinomatous changes. Result of these changes may be used as educational tool in cessation of habits.

Keywords: Acridine orange; micronuclei; silver-stained nucleolar organiser regions

How to cite this article:
Jindal S, Chauhan I, Grewal HK. Alteration in buccal mucosal cells due to the effect of tobacco and alcohol by assessing the silver-stained nucleolar organiser regions and micronuclei. J Cytol 2013;30:174-8

How to cite this URL:
Jindal S, Chauhan I, Grewal HK. Alteration in buccal mucosal cells due to the effect of tobacco and alcohol by assessing the silver-stained nucleolar organiser regions and micronuclei. J Cytol [serial online] 2013 [cited 2019 Dec 5];30:174-8. Available from: http://www.jcytol.org/text.asp?2013/30/3/174/117667



   Introduction Top


Cytodiagnosis is one of the most readily available means of cancer diagnosis. The clinician's use of exfoliative cytology as a method of diagnosis was first introduced by Papanicolaou in 1943. [1] It is considered to be an easy, non-invasive procedure carried out even on the slightest suspicion regarding the nature of the lesion. [2]

Oral habits such as alcohol consumption and tobacco chewing are considered to be initiators of dysplastic changes in the oral mucosa. World-wide, 48% of the total adult population (approximately 2 billion people), consume alcohol and 33% (approximately 1.3 billion people) consume tobacco. [3] Around two-third of oral squamous cell carcinoma can be attributed to the use of tobacco and alcohol consumption. The aim of this study was to determine and compare the alteration in apparently normal buccal mucosal cells due to effect of alcohol and tobacco, by assessing silver-stained nucleolar organiser regions (AgNORs) and micronuclei and to detect the earliest possible changes due to these genotoxic agents.


   Materials and Methods Top


A total of 100 subjects were selected in the age group of 20-30 years and were divided into 4 groups of 25 subjects each. Group I - Control group (individuals without any tobacco or alcohol habits); Group II - Tobacco consumers; Group III - Alcohol consumers; and Group IV - Alcohol and tobacco consumers.

The Group II comprised of individuals who consumed tobacco in the form of bidi/cigarette with total quantity in the range of 5-20 bidis/cigarettes a day. Group III comprised of individuals who consumed only alcohol (country liquor) without any tobacco habit with the total quantity in the range of 50-500 mL/day. Duration of above habits ranges from 5 years to 10 years.

Individuals who had undergone any oral radiograph in past 6 months, were suffering from malignancy in any part of the body, or those with premalignant lesion/condition in the oral cavity, suffering from any systemic condition, or those on medical treatment of any kind were not included in the study. All subjects were well informed about the study. A detail case history and informed consent were taken before participating in the study. After a thorough examination of the oral cavity, two smears were made from the same right buccal mucosa of all subjects using cytological brush. Each smear was then immediately fixed in 95% alcohol for 20 min. One smear thus fixed was stained with AgNOR staining process for nucleolar organiser regions (NORs) analysis. The second was stained with acridine orange staining process for micronuclei count. 500 cells were counted in zigzag method in each stained smear for AgNORs and micronuclei. The slides were evaluated by two observers, both for AgNOR and micronuclei count and the inter observer variability came out to be nil in the present study.

AgNOR staining procedure

The fixed smears were subjected to the silver-staining method for AgNOR according to Ploton's method. The final working solution was freshly prepared by mixing one volume of 2% gelatin in 1% formic acid solution and two volumes of 50% aqueous silver nitrate solution. Slides were incubated in darkness with this silver solution for 40 min at 45°C.

AgNOR stained smears were evaluated under light microscope at (×1000) magnification under immersion oil (Nikon Eclipse 80i). AgNORs appeared as discrete black brown dots present within the nucleus [Figure 1].
Figure 1: Silver-stained nucleolar organiser regions (AgNOR) stained smear showing AgNORs in exfoliated buccal mucosal cells

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Acridine orange staining procedure

The staining solution is prepared from 0.10% (10 mg) aqueous stock solution by diluting with 5 mL of distilled water and 5 mL of phosphate buffer. The phosphate buffer is a combination of 1/15 M KH 2 PO (0.90 g/100 mL of distilled water) and 1/15 M Na 2 HPO (0.94 g/100 mL of distilled water) and mixed in the right proportion to attain the pH 6.

The fixed smears prepared passed through regressive changes of alcohol 80%, 70%, 50% and distilled water for 2 min each. These were then dipped in 1% acetic acid for 5 dip to prevent rapid fading of fluorescence. The slides were then washed with distilled water for 2 min and stained in acridine orange staining solution for 5 min, followed by phosphate buffer for 1 min to remove excess stain and eventually 0.10 M CaCl 2 for 2 min to bring about differentiation between ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The sections were then rinsed with phosphate buffer and mounted.

Acridine orange stained smear were evaluated under a fluorescent microscope at (×400) magnification (Nikon Eclipse 80i). In the present study, criteria used for the identification of micronuclei were suggested by Sarto et al. [4] Micronuclei were scored only when chromatin structure and color intensity were similar to or weaker than those of the main nucleus, borders were distinctly recognizable, they were round and were included within the same cell cytoplasm. Dead or degenerating cells (karyolysis, karyorrhexis, pyknosis and nuclear fragmentation) were excluded from evaluation. Micronuclei appeared as discrete green dots present in orange cytoplasm of exfoliated cells [Figure 2].
Figure 2: Smear showing micronuclei in exfoliated buccal mucosal cells (Acridine Orange stain, x400)

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Statistical analysis

Mann-Whitney test was applied to assess the variation in the number of AgNORs and micronuclei count between different groups. Multiple comparisons were carried out to determine the significance of the mean difference.


   Results Top


[Figure 3] depict the result with statistical observation. AgNOR count showed highly statistically significant increase in various study groups when compared with the control group (P < 0.05). Micronuclei count showed statistically significant increase in tobacco and combined habit group when compared with the control group (P < 0.05), but insignificant increase was seen in the alcohol group when compared with the control group (P > 0.05). The mean AgNOR count was higher in the tobacco group (4.162 ± 0.5338) than the alcohol group (3.980 ± 0.7582), but this difference was statistically insignificant. Similarly, insignificant difference was seen in micronuclei count between these two groups. We observed that mean AgNORs and micronuclei count were found to be statistically higher in combined habit group as compared to individual alcohol and tobacco habit group.
Figure 3: Frequency of distribution of silver-stained nucleolar organiser regions and micronuclei in buccal mucosal cells in various groups

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   Discussion Top


Exfoliative cells from oral epithelium have been widely used in cytology to detect abnormal nuclear and cellular morphology depicting precancerous and cancerous changes. Genetic changes in these cells are of particular interest. [5] Buccal mucosal cells are seen to be widely affected as more surface area of the buccal mucosa is exposed to the insult in the oral cavity and the fact that these epithelial cells are non-keratinized, makes them more vulnerable to change. [6]

Biomarker is a measurable DNA and RNA characteristic that is used as an indicator of biologic and pathogenic process. The biomarkers can be translated into the relationship between exposure and disease and thus act as an indicator of the disease process. [7] In the present study, two biomarkers used to assess the proliferation potential of cells and DNA damage were AgNORs and micronuclei assay respectively.

NORs are intimately related to cell cycle and thus may be related to proliferation. In rapidly, proliferating cell nuclear disaggregation may take place resulting in dispersion of individual AgNORs, which appear as black brown dots of varying size in the nucleus. Because of its simple technique and high reliability for cellular proliferation AgNOR staining was used. However, there are certain limitations to this such as risk of obscuring some AgNORs by superimposition and fusion of small AgNOR dots due to continuous deposition of silver for a long time. [8]

Micronucleus assay can be used to measure DNA damage in the proliferative cell as these arise from chromosomal fragment lagging behind during cell division, which appear as green dots in the yellow orange cytoplasm of an exfoliated cell stained with acridine orange. [6] In the present study, acridine orange stain was used over other DNA specific stains such as Feulgen stains as it consumed less time and micronuclei could easily be assessed because of fluorescence. There are certain limitations regarding the identification of micronuclei such as binuclei, lobed nuclei, blebed nuclei and notched nuclei, may be misdiagnosed as micronuclei.

Oral habits such as tobacco and alcohol consumption are said to be important etiologic factors for carcinogenic cytological change. [5] Around two-third of squamous cell carcinoma and 75% of head and neck cancer have been attributed to tobacco and tobacco consumption. The analysis of cell proliferation and DNA damaged has gained popularity as an in vitro genotoxicity test. In the present study, results showed that in case of tobacco consumer's proliferation potential in the form of 4.162 mean AgNOR count and DNA damaged in the form of 5.360 mean micronuclei count were significantly higher when compared with the control group. These finding are consistent with previous studies. Sampaio et al. [9] used AgNORs silver soaking technique in exfoliative cytology smears collected from the clinically healthy oral mucosal cells, comparing smokers and non-smokers. They showed that the number of AgNORs was higher within the smokers group, indicating an increase in the proliferative activity of healthy oral mucosal cells in smokers.

Genotoxic carcinogens, mainly N-nitrosonornicotine, polycyclic aromatic hydrocarbons present in the tobacco act on the keratinocytes and then enter the nucleus, where they are metabolized by cytochrome P450 and glutathione S transferase. The electrophilic intermediate thus produced, binds with DNA by direct stimulation of heat of cigarette and chemical action of volatile products of tobacco causing altered cell proliferation and DNA damage. These events lead to unrepaired alterations or mutations in the DNA that may further progress to carcinogenesis. [10] This was in accordance with the present study as the cell proliferation and DNA damage was increased in the tobacco consumers, which might cumulatively lead to carcinomatous changes.

While in alcohol consumers, cell proliferation potential in the form of 3.98 mean AgNOR count was significantly higher, when compared with control, but in DNA damaged in the form of 4.320 mean micronuclei count was insignificantly higher, when compared with the control group.

Bohrer et al. [11] in 2005 assessed the presence of micronuclei in exfoliated oral mucosal cells collected from three anatomic sites in patients exposed to tobacco and alcohol and found a trend toward an increased number of micronucleated cells in tobacco and alcohol users at all anatomic sites.

Reis et al. [12] in 2002 did a study to assess the frequency of micronuclei in exfoliated cells from the tongue and buccal mucosa of alcoholic individuals and found that frequency of micronuclei in buccal mucosa cells was higher in the group of alcoholic individuals, when compared to the control group, although the difference was not statistically significant (P > 0.05). The results of these studies are consistent with the present study.

The synergistic effect of alcohol and tobacco showed a significantly higher proliferation index and DNA damage, when compared to either of the two substances alone. This was depicted by significantly higher AgNOR and micronuclei count in this group. Alcoholic beverages exert synergistic effect with tobacco. [13] In a similar study, Stich and Rosin [14] observed the effect of tobacco and alcohol on the exfoliated cell of buccal mucosa by using the micronucleus assay. They found a strong synergistic effect between smoking and alcohol consumption as seen by the elevated frequency of micronucleated buccal mucosa cells Alcoholic beverages contain alcohol, acetaldehyde and nitrosamine that alter the rate of penetration of substances from the oral environment across the mucosa that have a role in carcinogenesis. Acetaldehyde produced by microflora by the oxidation of ethanol is predominantly responsible for alcohol associated carcinogenesis. It binds with the DNA and protein result in hyperproliferation. [13] It is clear that oral cancer risk is related to both intensity and duration of alcohol and tobacco consumption. According to World Health Organisation over 1 billion people are currently associated with tobacco smoking and nearly 2 billion adults worldwide are estimated to consume alcoholic beverages regularly, with average daily consumption of 13 g of ethanol (about one drink). [15] Alcohol use (>5 drinks/day) along with tobacco use (>20 cigarettes/day) increase the risk of oral cancer than expected based upon the independent effects of the same amount of alcohol or tobacco alone. [16] In addition, tobacco smoking is estimated to account for approximately 4-5 million deaths a year worldwide. This number is projected to increase to approximately 10 million a year by 2030. [17]


   Conclusions Top


This study, thus, shows that tobacco and/or alcohol may cause alterations in the oral mucosal cells with alcohol causing less severe effects than tobacco and together, their synergistic effect cause even more severe changes at cellular levels, which may increase the chances of progression to oral cancer. This study supports and extends the view that cytological changes in oral mucosa due to tobacco and alcohol can serve as a useful diagnostic adjunct for the early detection of oral premalignant changes and cancer. Furthermore, these may also be used as an educational tool for population awareness programs to help in cessation of these oral habits.

 
   References Top

1.Bertalanffy FD. Cytodiagnosis of cancer using acridine orange with fluorescence microscopy. Triangle 1961;5:152-6.  Back to cited text no. 1
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Correspondence Address:
Harshaminder Kaur Grewal
Department of Oral and Maxillofacial Pathology, M.M College of Dental Sciences and Research, Mullana, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9371.117667

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