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Table of Contents
ORIGINAL ARTICLE
Year : 2018  |  Volume : 6  |  Issue : 4  |  Page : 69-72

Buccal mucosal alterations: A mirror of diabetes mellitus


Department of Oral Pathology and Microbiology, M. A. Rangoonwala College of Dental Sciences and Research Centre, Pune, Maharashtra, India

Date of Web Publication14-May-2019

Correspondence Address:
Dr. Sanskriti Gandhi
Department of Oral Pathology and Microbiology, M. A. Rangoonwala College of Dental Sciences and Research Centre, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/INJO.INJO_13_18

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  Abstract 

Background: Oral exfoliative cytology can be used as a simple chairside investigation for microscopic study of Diabetes Mellitus. Exfoliative cytology gives an insight to the cellular changes occurring in the buccal mucosa of diabetic individuals, serving as a mirror for diabetes mellitus. Aim: To evaluate the qualitative changes in oral epithelial cells using exfoliative cytology and its correlation with glycosylated hemoglobin in patients with Type 2 Diabetes Mellitus. Materials and Methods: Study group consisted of 100 patients with Type 2 Diabetes. Approval from the ethics committee was taken. HbA1c was measured. Buccal smears were collected using exfoliative cytology and were subjected to Rapid Papanicolaou (PAP) stain. Smears were scrutinized under Leica Research Microscope. Individuals who smoke, are dependent on alcohol, or have malignancy were excluded to eliminate its effect on cellular shape and morphology. Statistical Analysis: Spearman Rank Correlation. Results: A statistically significant increase in cytological changes are seen in the buccal mucosa as the levels of glycosylated hemoglobin increase. Exfoliated buccal cells revealed micronuclei, enlarged nuclear area, and increased inflammatory cells. Conclusion: On the basis of the results obtained, a strong correlation exists between the levels of glycosylated hemoglobin and buccal mucosal alterations. We are encouraged to venture with a suggestion that exfoliative cytology can be significant as a chairside adjunctive diagnostic tool in the detection of Diabetes Mellitus, apart from regular standard tests.

Keywords: Diabetes mellitus, exfoliative cytology, glycosylated hemoglobin, micronuclei


How to cite this article:
Dhebri M, Gandhi S, Kazi RB, Gore V, Baldawa P, Koshy A. Buccal mucosal alterations: A mirror of diabetes mellitus. Int J Oral Care Res 2018;6:69-72

How to cite this URL:
Dhebri M, Gandhi S, Kazi RB, Gore V, Baldawa P, Koshy A. Buccal mucosal alterations: A mirror of diabetes mellitus. Int J Oral Care Res [serial online] 2018 [cited 2019 May 21];6:69-72. Available from: http://www.ijocr.org/text.asp?2018/6/4/69/253704




  Introduction Top


Diabetes Mellitus is a metabolic disorder characterized by chronic hyperglycemia due to absolute or relative deficiency of insulin associated with long-term complications, which include dysfunction and failure of various organs.[1] In 2000, 31.7 million people were diagnosed with Diabetes Mellitus in India, which topped the world’s highest number of people with Diabetes Mellitus among other countries. It is predicted that by 2030, diabetes may afflict up to 79.4 million individuals in India.[2] It is also the fifth most common chronic condition and sixth most frequent cause of death among the elderly.[3] Diabetes produces morphological and functional alterations in the oral mucosa. The oral complications include xerostomia, candidiasis, increased incidence of caries, gingivitis, periodontitis, traumatic ulcers, glossodynia, taste impairment and abscess formation.[3],[4]

Several clinical and paraclinical techniques are available for studying oral mucosal changes. Biopsy is the most reliable technique to study these changes. However, in Diabetes Mellitus, changes in blood glucose reduce the viability of a biopsy.[4] Hence, exfoliative cytology can be used as it is a painless and noninvasive technique that will help to investigate the cytological alterations of the cells. Thus, the goal of this study was to evaluate the levels of glycosylated hemoglobin in patients with Type 2 Diabetes Mellitus and its correlation with mucosal changes using exfoliative cytology, and to establish its role as a diagnostic criterion and introduce it as a chairside investigation during routine dental examination.


  Materials and Methods Top


Source and method of collection of data

We chose the sample by convenient sampling technique, and the sample size was determined depending on the prevalence of patients with diabetes in the area of research. All patients with Diabetes Mellitus under treatment and regular follow-up were included in the study (N = 100). Ethical clearance for the study was obtained from the institutional ethical committee and review board. Written informed consent was also obtained.

Exclusion criteria

The exclusion criteria included the patients with

  • Habitual alcohol intake.
  • Potentially malignant conditions.
  • Smoking and tobacco chewing habits.
  • Clinically evident nutritional deficiencies (especially anemia).


Method of measuring glycosylated hemoglobin

A blood sample of 4 µL was obtained at the collection leg of the reagent pack. The reagent pack was inserted into the cartridge. The cartridge was inserted into the Clover A1c Analyzer. Glycosylated hemoglobin was photometrically measured.

Method of obtaining exfoliated cells

Participants were asked to rinse their mouth gently with water, and buccal mucosal surface scrapings were obtained using a wooden spatula. The spatula was used only in one direction over the buccal mucosa, with moderate pressure, thus obtaining clear epithelial cells. Smears from left and right sides of the buccal mucosa were taken to reduce the effect of localized inflammation on the results. The cells were immediately smeared on the center of clean, dry microscopic slides and spread over a large area, preventing the clumping of cells. The smears were fixed with absolute alcohol. Air drying of smears was strictly avoided as it leads to alterations in the cellular morphology.

Procedure for staining exfoliated cells

All cytological smears were stained by Rapid Papinicolaou (PAP) stain and observed under Leica Research Microscope at low magnification (x100) and high magnification (x400) for observing inflammatory cells, altered nuclear cytoplasmic ratio and micronuclei. Pap-stained smears were observed in a stepwise manner, moving from left to right and then down and across, in order to avoid studying the same cells again.

Heddle initially described the well-established basic criteria for micronuclei (MN).[5] However, the criteria for identifying cells for inclusion into MN frequency count were not provided. Later, Tolbert et al. developed the criteria for choosing the cells and this being most widely applied.[6],[7] It consists of the following parameters:

  • Cytoplasm intact and lying relatively flat.
  • Little or no overlap with adjacent cells.
  • Little or no debris.
  • Nucleus normal and intact, nuclear perimeter smooth and distinct.
  • Less than 1/3rd of the diameter of the associated nucleus but large enough to discern shape and color.
  • PAP positive
  • Staining intensity is similar to that of nucleus.
  • Texture similar to that of nucleus.
  • Same focal plane as nucleus.
  • Absence of overlap or bridge to nucleus.



  Results Top


The following parameters were studied:

  • The levels of glycosylated hemoglobin (GHb) were recorded, which indicate the degree of glycemic control achieved:
    • Well-controlled diabetics (WCD): GHb ≤ 8%
    • Moderately controlled diabetics (MCD): GHb > 8% and GHb ≤ 10%
    • Poorly controlled diabetics (PCD): GHb > 10% and GHb ≤ 12%
    • Uncontrolled diabetics (UCD): GHb > 12%
  • The alterations in the buccal mucosa were noted. Cytological changes prevalent were increased nuclear area, occurrence of micronuclei, and the presence of inflammatory cells. The smears were scored based on the presence of aforementioned parameters [Figure 1], [Figure 2], [Figure 3]:
    • Score 1: one of the above parameters present
    • Score 2: two of the above parameters present
    • Score 3: three of the above parameters present
      Figure 1: Increased inflammatory cells observed. (x 40)

      Click here to view
      ,
      Figure 2: Altered nuclear cytoplasmic ratio observed. (x 100)

      Click here to view
      ,
      Figure 3: Presence of micronuclei observed. (x 100)

      Click here to view


    Using the aforementioned criteria, the following results were obtained:

    • Of 100 patients, 79 showed the presence of inflammatory cells, 52 increased nuclear area, and 53 the presence of micronuclei.
    • Of 100 patients, 33 showed a smear score of 3, which stated the presence of inflammatory cells, increased nuclear area, and the presence of micronuclei; 25 showed a smear score of 2, which stated the presence of either of the two aforementioned changes; 35 showed a smear score of 1, which stated the presence of either of the one aforementioned change; and 7 showed no cytological changes in smears.




    Using Spearman rank correlation between cytological changes observed and the levels of HbA1c P = 0.000 which is a statistically significant value, signifying that as the levels of HbA1c increase there is a concomitant increase in the number of buccal alterations [Graph 1].
    Graph 1: Comparison of buccal smear scores with HbA1c values in patients with type 2 diabetes

    Click here to view



      Discussion Top


    Diabetes Mellitus is one of the most common endocrine metabolic disorders and its prevalence has been increasing worldwide because of population growth, aging, increase in prevalence of obesity, and urbanization.[8]

    Diabetes frequently predisposes an individual to oral health complications. Patients with diabetes have abnormality in the formation of final glycosylated products. This impairs cross-linking of collagen, leading to delayed wound healing. These necrosed cells on the surface of buccal mucosa exfoliate. In compensation, basal cells proliferate with a defective nucleus, which come to the surface and shed.[9] These cells, indicative of the underlying defects, can be studied by exfoliative cytology.

    Buccal smears studied showed a significant increase in inflammatory cells; increase in nuclear area, resulting in the decreased cytoplasmic nuclear ratio; and the presence of micronuclei. The studies by Chavez et al. have shown that subjects with poorly controlled Diabetes Mellitus had significantly lower stimulated parotid salivary flow rates, and alterations in the salivary composition with increased concentration of mucin result in a dry, atrophic mucosa with accompanying mucositis.[10] This state of poor lubrication in the buccal mucosa results in increased exfoliation of cells on contact. Studies have shown that there are modifications in the contents of salivary antimicrobial proteins such as lactoferrin, lysozyme, and lactoperoxidase. This gives way to increased proliferation of pathogenic microorganisms, thereby increasing the susceptibility to buccal infections such as candidiasis. Hence, polymorphonuclear leucocytes are produced in response to the microbial colonization.

    In this study, the possible hypothesis in explaining the increase in mean nuclear area is that the nucleus contains the genomic DNA, histones and several proteins. The nuclear size can, therefore, be altered by change in the contents of DNA or proteins. There is usually twice as much of protein as DNA in the nucleus.[11] Hyperglycemia induces a compensatory increase in insulin secretion which in turn causes increase in protein formation. Insulin has several mitogenic functions, including initiation of DNA synthesis in certain cells. These may account for the increase in nuclear area seen in diabetic patients.[12]

    The reduction in cytoplasmic area could be due to the dehydrated condition of the diabetics. Increase blood glucose causes dehydration, polyuria, polydipsia, intra cellular, extra cellular dehydration. Glucose does not defuse easily through the pores of the cell membrane causing as increase in osmotic pressure. This increase in osmotic pressure in extra cellar fluid causes osmotic transfer of water out of the cells explaining the reduction in cytoplasmic area.[13]

    Diabetes Mellitus has been associated with elevated levels of DNA damage and decreased efficacy of DNA repair. Micronuclei are chromatin masses with appearance of small nuclei that arise from chromosomal fragments or whole chromosomes that lack behind at the anaphase stage of cell division. Their presence in cells means the number of chromosomal aberrations arising during mitosis developing a risk of cancer. Also, these structures can be used for individual’s biomonitoring by acting as biomarkers.[12] Through our study, we used the exfoliated buccal mucosal cells as sources for this biomonitoring. Oxidative stress plays a vital role in Type 2 Diabetes Mellitus due to hyperglycemia by intensifying DNA synthesis and leads to increase in free radical release that destroys the DNA and enzymes that take part in repair process. Hyperglycemia also aggravates an increase in the levels of reactive species by suppression of glutathione synthesis. These reactive species cause oxidative damage to DNA and thus, lead to breakage of DNA strands forming micronuclei.[13] Collective evidences indicate that diabetes is a pathophysiological state of oxidative stress and DNA damage that can lead to various types of mutations to cause aberrations in cells forming micronuclei. Hence, increased micronuclei formation can be associated with early events in carcinogenesis.


      Conclusion Top


    Oral exfoliative cytology can be used as a simple chairside investigation that is quick, simple, less technically demanding, painless and noninvasive for microscopic study of the mucosa as compared to the conventional invasive procedures such as biopsy. Exfoliative cytology demonstrates its importance in the field of diagnosis, based on the principle that any change in the superficial cells is the reflection of the change in the immediate underlying tissue. Through this study, exfoliative cytology gives an insight into the cellular changes occurring in the buccal mucosa of patients with diabetes even in the absence of clinical manifestations serving as a mirror for Diabetes Mellitus. It can also be used as an educative tool in a dental office for raising awareness of mucosal alterations occurring in patients with Diabetes Mellitus.

    Hence, the dentist plays a key role in diagnosis and therapeutics of Diabetes Mellitus. Responsibility in the detection of Diabetes Mellitus and in referral of uncontrolled diabetics to proper medical care is important.

    Financial support and sponsorship

    Nil.

    Conflicts of interest

    There are no conflicts of interest.



     
      References Top

    1.
    Haslett C, Chilvers ER, Hunter JA, Boon NA. Davidson’s Principles and Practice of Medicine. 18th ed. Great Britain: ELBS with Churchill Livingstone; 2000. p. 472.  Back to cited text no. 1
        
    2.
    Kaveeshwar SA, Cornwall J. The current state of diabetes mellitus in India. Australas Med 2014;7:45-8.  Back to cited text no. 2
        
    3.
    Jajarm HH, Mohtasham N, Rangiani A. Evaluation of oral mucosa epithelium in type II diabetic patients by an exfoliative cytology method. J Oral Sci 2008; 50:335-40.  Back to cited text no. 3
        
    4.
    Survana M, Anuradha C, Kiran Kumar K, Chandra Sekhar P, Lalith Prakash Chandra K, Ramana Reddy BV. Cytomorphometric analysis of exfoliative buccal cells in type II diabetic patients. J Dr NTR Univ Health Sci 2012;1:33-7.  Back to cited text no. 4
        
    5.
    Heddle SM. Short term tests for chemical carcinogens. In: StichSR, editor. The Micronucleus Assay. I. In Vivo. Berlin/New York: Springer Verlag; 1981. pp. 243-9.  Back to cited text no. 5
        
    6.
    Tolbert PE, Shy SM, Allen JW. Micronuclei and other nuclear anomalies in buccal smears: Methods development. Mutat Res 1992;271:69-77.  Back to cited text no. 6
        
    7.
    Tolbert PE, Shy SM, Allen JW. Micronuclei and other nuclear anomalies in buccal smears: A field test in snuff users. Am J Epidemiol 1991;134:840-50.  Back to cited text no. 7
        
    8.
    Hu FB. Globalization of diabetes: The role of diet, lifestyle and genes. Diabetes Care 2011;34:1249-57.  Back to cited text no. 8
        
    9.
    Kumaresan GD, Jagannathan N. Exfoliative cytology: A predictive diagnostic tool. Int J Pharm Pharm Sci. 2014;6:1-3.  Back to cited text no. 9
        
    10.
    Chavez EM, Borrell LN, Taylor GW, Ship JA. A longitudinal analysis of salivary flow in control subjects and older adults with Type 2 Diabetes Mellitus. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:166-173.  Back to cited text no. 10
        
    11.
    Bibbo M, David W. Comprehensive Cytopathology. 3rd ed. Philadelphia, PA: Saunders; 2008.  Back to cited text no. 11
        
    12.
    Salamone MF, Heddle JA, Hite M, Kirkhart B, Mavournin K, MacGregor JT, et al. The induction of micronuclei as a measure of genotoxicity: A report of the U.S. environmental protection agency Gene-Tox program. Mutat Res 1983;123:61-118.  Back to cited text no. 12
        
    13.
    Rachana TS, Basu S. Oxidative stress and diabetes. In: RaniV, YadavU, editors. Free Radicals in Human Health and Disease. New Delhi: Springer; 2015. pp. 241-57.  Back to cited text no. 13
        


        Figures

      [Figure 1], [Figure 2], [Figure 3], [Graph 1]



     

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