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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 16  |  Issue : 1  |  Page : 63-67

Cytomorphometric evaluation of the epithelial cells of buccal mucosa in smokeless tobacco users: An In vivo study


Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences (Deemed to be University), Wardha, Maharashtra, India

Date of Submission13-Oct-2020
Date of Decision08-Feb-2021
Date of Acceptance14-Feb-2021
Date of Web Publication29-Jul-2021

Correspondence Address:
Dr. Alka Harish Hande
Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences (Deemed to be University), Sawangi (Meghe), Wardha - 442 001, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_361_20

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  Abstract 


Background: Smokeless tobacco (SLT) used in various formulations induces certain oral mucosal lesions commonly known as oral potentially malignant disorders (OPMDs). The prevalence of transformation of OPMD to oral squamous cell carcinoma is high. Thus, early diagnosis and prompt intervention may prevent this progression and further malignant transformation. Aim and Objectives: We aimed to assess the effect of tobacco chewing on epithelial cells of buccal mucosa in SLT users using cytomorphometry. Materials and Methods: The study group comprised 90 patients divided into three groups (A, B, and C). The comparison of the cellular diameter, nuclear diameter, and the nuclear–cellular diameter ratio of epithelial cells of buccal mucosa of control group (A) with tobacco chewing habit but no oral mucosal lesion (B) and tobacco chewing habits and oral mucosal premalignant lesions (C) was carried out. Results: Univariate analysis of variance showed a significant group effect for cellular diameter, nuclear diameter, and ratio of nuclear–cellular diameter. Multiple comparison tests by Tukey's honestly significant difference procedure revealed a significant decrease in the mean cellular diameter, increase in the nuclear diameter, and ratio of nuclear–cellular diameter. Conclusion: Cytomorphometric changes could be the earliest indicators of cellular alterations. There is a progressive decrease in cellular diameter, increase in nuclear diameter, and ratio of nuclear–cellular diameter in epithelial cells of buccal mucosa from all smokeless tobacco users, as compared to normal controls. This indicates that there could be cause–effect relationship between tobacco and quantitative alterations.

Keywords: Cytomorphometry, exfoliative cytology, oral squamous cell carcinoma, smokeless tobacco


How to cite this article:
Batra M, Hande AH, Gawande MN, Patil SK, Sonone A, Sharma PN. Cytomorphometric evaluation of the epithelial cells of buccal mucosa in smokeless tobacco users: An In vivo study. J Datta Meghe Inst Med Sci Univ 2021;16:63-7

How to cite this URL:
Batra M, Hande AH, Gawande MN, Patil SK, Sonone A, Sharma PN. Cytomorphometric evaluation of the epithelial cells of buccal mucosa in smokeless tobacco users: An In vivo study. J Datta Meghe Inst Med Sci Univ [serial online] 2021 [cited 2021 Sep 16];16:63-7. Available from: http://www.journaldmims.com/text.asp?2021/16/1/63/322632




  Introduction Top


Environmental factors have proven to be contributing to etiology of cancers of the orofacial region. This can be attributed to the fact that the prevalence of orofacial malignancies is seen more in specific areas as compared to other areas of the same country or other countries of the world. Tobacco which is used by means other than smoking is termed as smokeless tobacco (SLT). It can be used as a snuff, either moist or dry which is usually inhaled through the nose or can be chewed. SLT is known to have harmful chemicals like nicotine which are suspected to have carcinogenic potential. Various studies have shown that in the Asian population, the consumption of tobacco in chewable form is far more common than any other form, whereas snuff is more commonly used in the US.[1] Tobacco usage shows variations in context to age, gender, and socioeconomic status. In the Southeast Asian population, a smokeless form of tobacco is used on a large scale.[2]

SLT is used in various formulations such as gutkha, paan, khaini, zarda, and naswar, which induces certain oral mucosal lesions which clinically represent as leukoplakia, erythroplakia, tobacco lime lesions, and lichen planus, which are commonly known as oral potentially malignant disorders (OPMDs). The prevalence of transformation of OPMD to oral squamous cell carcinoma (OSCC) is high. Thus, early diagnosis and prompt intervention may prevent this progression and further malignant transformation.

Oral exfoliative cytology is a simple diagnostic technique for the early revelation of premalignant and malignant lesions.[3] It has been demonstrated that exfoliative cytology can also be used for the assessment of clinically suspicious lesions and carcinomatous lesions after definitive treatment.[3] Awareness regarding the clinical signs and symptoms of OPMDs is very important among the general population and physicians to prevent its progression into OSCC.

Formulation of diagnosis of oral malignancies can be made using morphometry, which is widely used for the determination of quantitative parameters such as nuclear size, cellular size, optical density, nuclear shape, nuclear texture, and nuclear–cytoplasmic ratio. The significant parameters among all above-stated ones are nuclear size, cellular size, and ratio of a nuclear–cellular diameter which occur in actively proliferating cells.[4],[5] Papanicolaou staining technique is used in exfoliative cytology as it confers varied color to the cytoplasm of epithelial cells, which is based on the degree of differentiation of epithelial cells. The fundamental of the adaptations of any cell initiates at the molecular level which activate a series of reactions and thereby affecting the whole cell system and accordingly its morphology. The common biological activities are reflected greatest in the nucleus whereas functional in cytoplasm.[6]

The present study was designed to evaluate the alterations due to SLT consumption on buccal mucosal cells and assess them by their cytomorphometric parameters in SLT users.


  Materials and Methods Top


The present study was conducted on patients from the department of oral pathology and microbiology. The study protocol was approved by the institutional ethical committee (DMIMS(DU)/IEC/2019-20/126). The study population consisted of 90 individuals whose demographic data pertaining to age, gender, detailed history of relevant habit with its dose and duration, and site of the lesion were recorded. The selection criteria of individuals for the study were as follows: tobacco usage 4–5 times daily for a minimum period of 8 years. Group A consisted of 30 individuals with no tobacco consumption history and no oral mucosal lesion which was taken as the control group. Group B included 30 individuals having tobacco chewing habit but no oral mucosal lesion. Group C included 30 individuals having tobacco chewing habits and oral mucosal premalignant lesions. The premalignant lesions included in the study were leukoplakia, erythroplakia, leukoerythroplakia, lichen planus, and tobacco pouch keratosis. After taking the written informed consent, a blood sample and cytological smear were taken from the participants. Venipuncture of all the participants was done for the determination of hemoglobin levels. The exclusion criteria were as follows: for female population – hemoglobin level: 11 g/dl and for male population – 12 g/dl. Moistened cytobrush with normal saline was used for scraping of the buccal mucosa. Scrapings were taken from clinically normal-appearing buccal mucosa in Groups A and B. In Group C, a representative area of the lesion was scraped. A smear of area 2.5 cm × 2.5 cm was prepared on a glass side from scrapings. Spray fixative was used to fix the smear. All cytological smears were stained using Papanicolaou stain using a commercially available staining kit – RapidPap™ (Biolab Diagnostics, Tarapur, Maharashtra). Cellular diameter, nuclear diameter, and nuclear–cellular diameter ratio were calculated using an automated image analysis from 100 cells from each smear. To compare the mean of cellular diameter, nuclear diameter, and ratio of nuclear diameter to cellular diameter, one-way analysis of variance (ANOVA) was carried out for the three groups.

The comparison of the mean values between the groups was made using multiple comparison tests by Tukey's honestly significant difference (HSD) procedure, using the statistics package SPSS 10.0 for Windows (SPSS Inc., Chicago, IL).

Ethical clearance

The Institutional Ethics Committee of DMIMSDU has approved the Research work proposed to be carried out at Sharad Pawar Dental College & Hospital, Sawangi(M), Wardha. Date : 11th April 2019 with Reference no DMIMS(DU)/IEC/2019-20/126.

[TAG:2]Results and Observations [/TAG:2]

A total of 90 participants were selected for the study, which was further categorized into three groups. ANOVA was used to compare between Groups A, B, and C with respect to various cytomorphometric parameters, and multiple comparisons were done using post hoc Tukey's HSD test [Table 1]. The age range of participants of the study was 21–62 years, with a mean age of 34.6 ± 2.8 years. On comparison of the cellular diameter, we observed that the mean cellular diameter was progressively reduced from normal (Group A: 1017.97 ± 177.96), through history of tobacco chewing but without any mucosal lesion (Group B: 784.65 ± 79.06), to oral mucosal premalignant lesions (Group C: 560.35 ± 52.13). Through Tukey's HSD test, the multiple comparisons of cellular diameter among the different groups demonstrated statistically significant variation (P ≤ 0.001) [Table 2] and [Graph 1]. On evaluation of the nuclear diameter, there was a progressive increase from normal (Group A: 66.26 ± 9.14), through history of tobacco chewing but without any mucosal lesion (Group B: 90.94 ± 9.06), to oral mucosal premalignant lesions (Group C: 113.06 ± 26.06). In comparison of the mean nuclear diameter between Groups A, B, and C, the mean difference was statistically significant (P ≤ 0.001) [Table 3] and [Graph 2]. Further, on comparison of the nuclear–cellular diameter ratio, we observed a progressive increase from normal (Group A: 0.06 ± 0.01), through history of tobacco chewing but without any mucosal lesion (Group B: 0.11 ± 0.01), to oral mucosal premalignant lesions (Group C: 0.19 ± 0.04). A statistically significant difference was observed in mean nuclear–cellular diameter ratio among Groups A, B, and C (P ≤ 0.001) [Table 4] and [Graph 3].
Table 1: Mean of cellular diameter, nuclear diameter, and nuclear-cellular diameter ratio

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Table 2: Comparison between groups with respect to cellular diameter by post hoc Tukey's test

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Table 3: Comparison between groups with respect to nuclear diameter by post hoc Tukey's test

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Table 4: Comparison between groups with respect to nuclear-cellular diameter ratio by post hoc Tukey's test

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  Discussion Top


Tobacco consumption in any form can cause cancers of the orofacial region. Oral cancer in SLT users is often seen in alveolar or buccal surfaces, the area where the quid is kept. In the Central Maharashtra rural population, tobacco mixed with lime, commonly referred to as kharra/khaini, is placed commonly in the mandibular canine premolar area.[7] Intraorally, a yellowish-white, thick lesion is appreciable occasionally associated with loose tissue tags. It has been suggested that scraping off the surface layers of buccal mucosal cells may be possible because of the caustic action of the mixture used which has an approximate pH 8.3. They concluded that tobacco pouch keratosis was four times more common than leukoplakia in the Central Maharashtra rural population. During the transformation of a normal mucosal lesion into potentially malignant disorder or cancer, the molecular changes or the microscopical changes appear earlier than the appreciable clinical changes. Early diagnosis and prompt management at premalignant stages could help us limit the debilitating status of the individual, morbidity, and mortality rates associated with OSCC. Screening of high-risk patients, by clinical examination, can be done at early-stage disease, as it is curable and thereby improves the prognosis of the patient.[8]

The smear procured by exfoliative cytology can be qualitatively and quantitatively analyzed.[7] An accurate diagnosis can be made on the basis of different cytological parameters such as nuclear diameter, cellular diameter, nuclear-to-cellular diameter ratio, nuclear shape, nuclear continuity variations, and the texture of the nucleus.[9] Nuclear diameter, cellular diameter, and their ratio prove to be the most accurate parameters for assessment of oral epithelial lesions.[4],[5] Exfoliative cytology can also be used for the assessment of posttreatment malignant lesions and clinically suspicious lesions.[1],[3],[7]

In our study, we observed increased nuclear diameter, decreased cellular diameter, and also a decrease in the ratio of nuclear to cellular diameter in Groups B and C as compared to Group A.

Our study results are in accordance with Ramaesh et al.,[10] Einstein and Sivapathasundharam,[11] Khot et al.,[12] Babuta et al.,[13] Syamala et al.,[14] Nivia et al.,[15] and Udayashankar et al.[16] The various researchers have evaluated the nuclear and cellular diameters of exfoliated mucosal cells in tobacco chewers as well as tobacco smokers. The observations are further compared with dysplastic and malignant epithelial cells. Thus, the absolute spectrum of disease that begins from cellular and nuclear alterations secondary to tobacco exposure which further leads to dysplastic changes in tissue toward invasive malignancy has been enlightened. The tobacco products consist of a variety of nitrosamine and nitrosonornicotine, which are released and infiltrate into mucosa when in close contact during their uses. The cellular alterations in the form of decreased cellular and increased nuclear diameter as well as their ratio observed in the present study can be to some extent accredited to this fact which further may influence cellular morphometry. In additional, oral buccal mucosal cells have a reduced turnover rate, so cells remain in the cell cycle for extended periods which results in a delayed cell division, ultimately increases the nuclear size.[12] Thus, we may postulate that these cellular alterations could be adaptation in response to exposure of tobacco and its byproducts. It was also suggested that alterations seen in cellular and nuclear dimensions such as decreased cellular dimension and increased nuclear dimension are indicative of early malignant changes. Nuclear diameter increased as the DNA content of the nucleus and the nuclear diameter exhibit a direct relationship with each other. Our observations suggested that SLT may be the factor that produced significant nuclear and cellular alterations in the B and C groups. In Group B, that is tobacco users with no buccal mucosal lesion, the oral mucosa seemed to be normal on clinical examination, however, the mean difference of cellular size showed statistically significant variations in comparison with Group C. Thus, the alterations may be at molecular level secondary to exposure of tobacco, which are not discernible clinically. This study confirmed only the etiology–effect relation between SLT consumption and quantitative nuclear and cellular alterations.[17],[18],[19],[20],[21]


  Conclusion Top


It was found that SLT in any form proves to be one of the important etiologies in causing precancer and cancer as it produces significant changes on the oral mucosa, which can be evaluated by studying the cytomorphometric parameters of the oral epithelial cells. These parameters show notable variations with respect to nuclear size, cellular size, and their ratios when compared with normal mucosa. Molecular changes start to occur comparatively earlier than clinical changes. These molecular changes can be detected accurately using cytomorphometry aiding in the correct and early diagnosis. Cytomorphometry is comparatively easy, less time consuming, and cost-effective. Thus, cytomorphometric measurements provide an objective method for the evaluation of epithelial dysplasia and to predict their potential of malignant transformation. If oral mucosal premalignant lesions are detected at earlier stages, their potential of malignant transformation may be prevented. Early intervention of precancerous lesions improves the prognosis of the condition limiting the morbidity and mortality rates. Thus, the quantitative analysis of cellular and nuclear diameter in SLT lesions may be used as an effective diagnostic tool.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Lewin F, Norell SE, Johansson H, Gustavsson P, Wennerberg J, Biörklund A, et al. Smoking tobacco, oral snuff, and alcohol in the etiology of squamous cell carcinoma of the head and neck: A population-based case-referent study in Sweden. Cancer 1998;82:1367-75.  Back to cited text no. 1
    
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