Salivary Oxidative Stress Assessment in Tobacco Users with and without Potentially Malignant Disorders and Micronuclei Estimation using Fluorescent Microscopy

Abstract

Abstract Background: Consumption of tobacco in either smoking or nonsmoking form produces free radicals, contributing to an increased risk of oral cancer as a result of oxidative stress. Biochemical changes in antioxidant enzymes are responsible for tobacco-associated oral carcinogenesis. Due to the production of reactive oxygen species and oxidative stress, the promotion of carcinogenesis occurs in the cells. Oral potentially malignant disorders (OPMDs) is an assembly of benign lesions with morphologically altered clinical or histopathological tissue that has a greater than normal risk of transforming into malignant lesions after diagnosis at a later date. Micronuclei are the goal site for early genotoxic events and are involved in carcinogenic events. Micronuclei in exfoliated cells are useful biomarkers in assessing cytogenetic damage. Early detection will help to devise appropriate treatment plans, thereby improving patient survival rates. Aim and Objectives: To estimate the salivary superoxide dismutase (SOD) levels and the expression of micronuclei in exfoliated buccal cells of tobacco users before the onset of tobacco-induced oral lesions and correlating the same with patients with OPMDs to determine if this can be useful in predicting early malignant potential in subjects with habitual tobacco usage. Materials and Methods: The study included three groups. Group I – Tobacco users with OPMD (n = 50), Group II – Tobacco users without OPMD (n = 50), and Group III – Healthy controls (n = 50). Sandwich enzyme-linked immunosorbent assay was done for saliva samples for quantification of salivary SOD. Exfoliated buccal cells were smeared and stained with acridine orange stain and viewed under a fluorescent microscope for micronuclei estimation. Results: The study showed a significant difference in salivary SOD levels and increased micronuclei expression among tobacco users than healthy controls. Conclusion: Our study results revealed a gradual and steady decrease of SOD levels and increased micronuclei expression from healthy control to tobacco users without lesions and tobacco users with lesion, and this can be used as an effective noninvasive early diagnostic biomarker in assessing malignant progression in tobacco users. Furthermore, the evaluation of micronuclei estimation can serve as a simple, quick, and noninvasive chair-side procedure for regular oral cancer screening in patients with tobacco usage.