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Year : 2016  |  Volume : 12  |  Issue : 1  |  Page : 228-231

Quantitative analysis of agnor counts of buccal mucosal cells of chewers and non chewers of gutkha: A comparative cytologic study

1 Department of Oral Pathology, Yogita Dental College and Hospital, Khed, Maharashtra, India
2 Department of Oral Pathology, K. M. Shah Dental College, Sumandeep Vidyapeeth University Campus, Vadodara, Gujarat, India
3 Department of Oral Pathology and Microbiology, P M Nadagowda Memorial Dental College and Hospital, Bagalkot, Karnataka, India
4 Department of Basic Dental Sciences, College of Dentistry, Al-Hawaiya, Taif University, Taif, Kingdom of Saudi Arabia
5 Department of Oral and Maxillofacial Sciences, Alfarabi College of Dentistry, Riyadh, Kingdom of Saudi Arabia

Date of Web Publication13-Apr-2016

Correspondence Address:
Nikhil I Malgaonkar
Block num. 18, 3rd Floor, ‘Laxmi Prasad’, Khandkar Lane, Tilak Nagar, Dombivli (East), Thane - 421 201, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.148713

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 > Abstract 

Aims and Objectives: The present study was taken up to evaluate the AgNOR counts in the buccal mucosa cells of gutkha chewers and compare that with the sex-matched controls.
Materials and Methods: In all, 100 gutkha chewers and 50 sex-matched non-chewers (controls) were chosen. None of the patients in both groups had any clinical oral lesions or systemic diseases. After rinsing with 0.9% sodium chloride, cytologic smears were prepared and stained using the AgNOR method and observed in immersion oil at 1000 × magnification. Finally, 50 cells were selected at random; AgNOR dots were counted and their mean was recorded. The student t-test was used for analysis of data.
Results: Comparison between mean AgNOR counts of gutkha chewers (2.68 ± 0.23) and non-chewers (2.01 ± 0.14) was found to be statistically significant.
Conclusion: Cytology associated with AgNOR staining can effectively detect the early molecular changes within buccal mucosa cells of oral mucosa.

Keywords: AgNOR, cytology, cytobrush, Gutkha, smear

How to cite this article:
Malgaonkar NI, Dagrus K, Vanaki SS, Puranik RS, Sharanesha MB, Tarakji B. Quantitative analysis of agnor counts of buccal mucosal cells of chewers and non chewers of gutkha: A comparative cytologic study. J Can Res Ther 2016;12:228-31

How to cite this URL:
Malgaonkar NI, Dagrus K, Vanaki SS, Puranik RS, Sharanesha MB, Tarakji B. Quantitative analysis of agnor counts of buccal mucosal cells of chewers and non chewers of gutkha: A comparative cytologic study. J Can Res Ther [serial online] 2016 [cited 2021 Apr 15];12:228-31. Available from: https://www.cancerjournal.net/text.asp?2016/12/1/228/148713

 > Introduction Top

Squamous cell carcinoma is one of the commonly occurring malignancies in oral cavity. The early diagnosis and consequent treatment of oral cancer could prevent a large number of deaths due to this disease.[1] It is an established fact that oral squamous cell carcinomas are preceded by oral precancerous lesions (potentially malignant disorders) in most of the cases. There exists a strong relationship between gutkha chewing and squamous cell carcinoma.[2] Molecular changes appear much prior to any lesion being evident clinically. These ultra-structural changes can be detected by special techniques like flow cytometry, AgNOR or micronuclei count technique.[3],[4] AgNOR in cytology has not been used as frequently and efficiently as in paraffin embedded sections. No particular technique or diagnostic modality is designed to predict the accurate malignancy conversion rate of potentially malignant disorders.[5] Also, there is no specific technique that would accurately predict whether the potentially malignant disorder would transform into a malignancy. It is important that techniques are developed to aid in the diagnosis of early oral cancer especially in predicting the behaviour of those lesions which display epithelial dysplasia but no overt malignancy.[6] Proliferation markers like proliferating cell nuclear antigen (PCNA), ki-67 and economical substitutes like AgNOR and micronuclei evaluation techniques can give an arbitrary estimate of the changes the cells undergo in potentially malignant disorders or even the changes that cells undergo before the lesions appear.[7],[8] If not precise, but an arbitrary prediction about the molecular changes that take place in the buccal mucosa cells of gutkha chewers in individuals without any lesion, can hint towards occurrence of any lesion in those individuals in future. Hence, this study was conducted with an intention of detecting changes in buccal mucosa cells of gutkha chewers by comparing their AgNOR counts with the counts of buccal mucosalcells of non-gutkha chewers (normal).

 > Materials and Methods Top

This study comprised of cytological examination of buccal mucosa cells in 100 gutkha chewers as study sample group and 50 sex-matched non-chewers as controls using AgNOR staining.

Criteria for inclusion of subjects

The study group consisted of gutkha chewers with the following criteria:

  • Consumption of minimum of five commercially available packs or sachets of gutkha per day
  • Duration of consumption of gutkha for more than three years
  • Clinical absence of any apparent lesion in the oral cavity
  • No history of any systemic disease/malignancy/recurrence.

The control group comprised of age- and sex-matched subjects without any lesion, systemic illness or any other tobacco habits resulting in alteration of buccal mucosa cells.

Materials used

  • Cytobrush for brush biopsy
  • AgNOR stain
  • Binocular light microscope
  • Microscopy slides
  • Fixative (90% ethyl alcohol).

Procedure employed

The subjects from the study group and control group were well informed about the study and their consent was obtained prior to the commencement of the study. The details of the subjects and controls were noted on the case history proforma and examined for presence of any lesion. After rinsing with 0.9% sodium chloride, scrapings from buccal mucosae of the individuals were taken and fixed on microscopy slides using 90% ethyl alcohol. The slides were stained with AgNOR stain using Ploton's method. The number of AgNOR has been thought to be related to cellular activity. After horizontally screening of the sections from left to right, AgNORs were counted in the nuclei of the first 50 non-overlapped nucleated cells. Cells with pyknotic nuclei were not counted. The AgNOR count was made adopting the method described by Ahmed and Babiker [9] by using a binocular light microscope at 1000 × magnification (oil immersion power) by three different observers. The average AgNOR count per smear was calculated by dividing the sum of nucleolar organizing regions counted in the cells by the total number of cells counted.

 > Results Top

AgNOR counts of fifty cells per smear of subjects and control were counted by three observers and the mean of the three was taken. An overall mean of AgNOR count per smear was counted in subjects and controls and a correlation was evaluated using student t- test and the difference was found to be statistically significant (P value = 0.0000). The average AgNOR count was found to be 2.6811 ± 0.2306 in gutkha chewers whereas the average AgNOR count in control group was found to be 2.0114 ± 0.1478 [Table 1] and [Figure 1].
Table 1: Comparison of chewers and non-chewers with respect to average counts by t-test

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Figure 1: Graphical comparison of chewers and non-chewers with respect to average counts

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A comparison of AgNOR counts was done between two groups of gutkha chewers; for more than 10 years (2.6943 ± 0.2683) and less than 10 years (2.6735 ± 0.2061) and the difference was not statistically significant.

 > Discussion Top

It is not correct to perform an incisional biopsy on a clinically normal appearing mucosa and in such a case, exfoliative cytology can be a better substitute for biopsy procedures. Exfoliative cytology has proved to be a fair adjunct for routine histopathological techniques. Newer techniques like flow cytometry, or the economical substitutes like AgNOR staining procedures can be combined with exfoliative cytology for effective evaluation of cells for molecular changes.[10] AgNOR stains have been used successfully on paraffin-embedded sections previously but use of AgNOR stain in cytology has not been explored.

Since prevalence of potentially malignant disorders and malignancies is more associated with habits like smoking, gutkha chewing and tobacco chewing,[11] detection of molecular changes in individuals with habits becomes more relevant and rational.[12] A study done by Illana Kaplan et al., suggested that AgNOR method seems to be sensitive and enables earlier identification of nuclear changes.[13] Mean AgNOR counts increased gradually from normal epithelium to non-dysplastic to dysplastic leukoplakia to squamous cell carcinoma.[14] In spite of the rampant and fast increasing prevalence of gutkha chewing habit in India, no study using AgNOR in cytology of gutkha chewers has been documented in the literature.

This cytology based study to evaluate the AgNOR counts in normal appearing mucosa of gutkha chewers shows increased number of AgNOR dots in gutkha chewers [Figure 2] as compare to non-gutkha chewers [Figure 3]. The increased AgNOR count in gutkha chewers is due to the ingredients of gutkha. The study done by Jeng JH et al., concluded that areca nut induces unscheduled DNA synthesis in keratinocytes.[15] The non-significant difference between the AgNOR counts of gutkha chewers that were categorized depending upon gutkha chewing period could be attributed to the variable amount of gutkha being chewed per day. Similar studies were done on smokers that have shown that cellular proliferation is significantly higher in smokers and this causes an increase in the nuclear dimensions of oral mucosal cells.[16]
Figure 2: Photomicrograph showing four AgNOR dots in epithelial cells of gutkha chewer (AgNOR stain, ×40 magnification)

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Figure 3: Photomicrograph showing one AgNOR dot in epithelial cells of Non-gutkha chewer (AgNOR stain, ×40 magnification)

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Various follow-up studies have been done that provide a significant correlation between the AgNOR count and prognosis was in pre-malignant and malignant lesions of the cervix [17], colorectal cancer [18], benign and malignant effusions [19], adenoid cystic carcinoma [20] and breast carcinoma.[21] However, it is proven to be of prognostic value in ovarian cancer,[22] transitional cell carcinoma of the bladder [23] and glottic cancer.[24]

 > Conclusion Top

Occurrence of keratotic and white lesions has consistently been seen in association with gutkha consumption habit. Timely detection of molecular changes prior to occurrence of clinically evident lesions can be helpful in creating awareness in the individuals with habits. Thus, cytological screening of the patients, with high risk of oral neoplastic lesions and without any macroscopically apparent oral lesion, can be of extreme importance.[9]

Exfoliative cytology combined with AgNOR is an economical noninvasive procedure that can be helpful in evaluating high-risk group individuals. Follow-up of the individuals associated with the habit, is advised in situ ations where they present with increased AgNOR counts.

 > References Top

Cowpe JG, Longmore RB, Green MW. Quantitative exfoliative cytology of abnormal oral mucosal smears. J R Soc Med 1988;81:509-13.  Back to cited text no. 1
Nair U, Bartsch H, Nair J. Alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha and pan masala: A review of agents and causative mechanisms. Mutagenesis 2004;19:251-62.  Back to cited text no. 2
Epstein JB, Zhang L, Rosin M. Advances in the diagnosis of oral premalignant and malignant lesions. J Can Dent Assoc 2002;68:617-21.  Back to cited text no. 3
Sirri V, Roussel P, Hernandez-Verdun D. The AgNOR proteins: Qualitative and quantitative changes during the cell cycle. Micron 2000;31:121-6.  Back to cited text no. 4
Reibel J. Prognosis of oral pre-malignant lesions: Significance of clinical, histopathological, and molecular biological characteristics. Crit Rev Oral Biol Med 2003;14:47-62.  Back to cited text no. 5
Sciubba JJ. Improving detection of precancerous and cancerous oral lesions. Computer-assisted analysis of the oral brush biopsy. U.S. Collaborative OralCDx Study Group. J Am Dent Assoc 1999;130:1445-57.  Back to cited text no. 6
Huang WY, Coltrera M, Schubert M, Morton T, Truelove E. Histopathologic evaluation of proliferating cell nuclear antigen (PC10) in oral epithelial hyperplasias and premalignant lesions. Oral Surg Oral Med Oral Pathol 1994;78:748-54.  Back to cited text no. 7
Piattelli A, Rubini C, Fioroni M, Iezzi G, Santinelli A. Prevalence of p53, bcl-2, and Ki-67 immunoreactivity and of apoptosis in normal oral epithelium and in premalignant and malignant lesions of the oral cavity. J Oral Maxillofac Surg 2002;60:532-40.  Back to cited text no. 8
Ahmed HG, Babiker AE. Assessment of cytological atypia, AgNOR and nuclear area in epithelial cells of normal oral mucosa exposed to toombak and smoking. Rare Tumors 2009;1:e18.  Back to cited text no. 9
Rajput DV, Tupkari JV. Early detection of oral cancer: PAP and AgNOR staining in brush biopsies. J Oral Maxillofac Pathol 2010;14:52-8.  Back to cited text no. 10
[PUBMED]  Medknow Journal  
Sujatha D, Hebbar PB, Pai A. Prevalence and correlation of oral lesions among tobacco smokers, tobacco chewers, areca nut and alcohol users. Asian Pac J Cancer Prev 2012;13:1633-7.  Back to cited text no. 11
Sampaio Hde C, Loyola AM, Gomez RS, Mesquita RA. AgNOR count in exfoliative cytology of normal buccal mucosa. Effect of smoking. Acta Cytol 1999;43:117-20.  Back to cited text no. 12
Kaplan I, Engelberg J, Dayan D. The effect of desalivation on 4-nitroquinoline-1-oxide-induced tongue carcinogenesis: A morphometric study of nucleolar organizer regions. J Oral Pathol Med 2001;30:48-52.  Back to cited text no. 13
Chattopadhyay A, Ray JG, Caplan DJ. AgNOR count as objective marker for dysplastic features in oral leukoplakia. J Oral Pathol Med 2002;31:512-7.  Back to cited text no. 14
Jeng JH, Hahn LJ, Lin BR, Hsieh CC, Chan CP, Chang MC. Effects of areca nut, inflorescence piper betle extracts and arecoline on cytotoxicity, total and unscheduled DNA synthesis in cultured gingival keratinocytes. J Oral Pathol Med 1999;28:64-71.  Back to cited text no. 15
Orellana-Bustos AI, Espinoza-Santander IL, Franco-Martínez ME, Lobos-James-Freyre N, Ortega-Pinto AV. Evaluation of keratinization and AgNORs count in exfoliative cytology of normal oral mucosa from smokers and non-smokers. Med Oral 2004;9:197-203.  Back to cited text no. 16
Pratibha D, Kuruvilla S. Value of AgNORs in pre-malignant and malignant lesions of the cervix. Indian J Pathol Microbiol 1995;38:11-6.  Back to cited text no. 17
Jin W, Gao MQ, Lin ZW, Yang DX. Multiple biomarkers of colorectal tumor in a differential diagnosis model: A quantitative study. World J Gastroenterol 2004;10:439-42.  Back to cited text no. 18
Akhtar G, Chaudhary NA, Tayyab M, Khan SA. AgNOR staining in malignant and benign effusions. Pak J Med Sci 2004;20:29-32.  Back to cited text no. 19
Fonseca I, Soares J. Adenoid cystic carcinoma: A study of the nucleolar organizer regions (AgNORs) counts and their relation to prognosis. J Pathol 1993;169:255-8.  Back to cited text no. 20
Sivridis E, Sims B. Nucleolar organizer regions: New prognostic variable in breast carcinomas. J Clin Pathol 1990;43:390-2.  Back to cited text no. 21
Ghazizadeh M, Sasaki Y, Araki T, Konishi H, Aihara K. Prognostic value of the proliferative activity of ovarian carcinoma as revealed by PCNA and AgNOR analyses. Am J Clin Pathol 1997;107:451-8.  Back to cited text no. 22
Lipponen P. Image analysis of the AgNOR proteins in transitional cell bladder cancer. J Pathol 1993;171:279-83.  Back to cited text no. 23
Yamamoto Y, Itoh T, Saka T, Sakakura A, Takahashi H. Nucleolar organizer regions in glottic carcinoma: Comparison of DNA cytoflurometry and clinicopathological analysis. Eur Arch Otorhinolaryngol 1995;252:499-503.  Back to cited text no. 24


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]


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