Cytological Alterations in Buccal Mucosa and Molecular Interaction of Nicotine with XRCC1 In Young Male Gutkha Chewers: A Cross-Sectional and in Silico Study

Abstract

Background: Smokeless tobacco products such as gutkha are widely consumed in South Asia and are strongly linked to oral potentially malignant disorders and oral cancer. Nicotine, the principal addictive alkaloid in tobacco, is absorbed through the oral mucosa and may compromise DNA repair capacity, yet the combined cytological and molecular evidence linking gutkha use to early genotoxic change in young users remains limited.
Objective: To assess awareness of the harmful effects of tobacco among young male gutkha users, to characterise cytological alterations in exfoliated buccal epithelial cells, and to evaluate the molecular interaction between nicotine and the DNA-repair protein X-ray repair cross-complementing protein 1 (XRCC1).
Methods: A cross-sectional study was conducted among 60 male gutkha users aged 18–25 years and 60 age-matched non-tobacco-using controls. A structured, pre-validated questionnaire captured knowledge, attitude and practice (KAP) related to tobacco use. Buccal smears were stained by the Papanicolaou (PAP) and May–Grünwald Giemsa (MGG) methods and screened for nuclear and cytoplasmic aberrations. Molecular docking of nicotine with XRCC1 was performed in AutoDock 4.2 following Lamarckian genetic algorithm optimisation; ligand toxicity was profiled with ProTox-3.0.
Results: All participants (100%) recognised chewing tobacco as harmful, yet 81.67% believed their preferred brand was comparatively safe, and none cited social media as a source of anti-tobacco information. Cytological screening revealed a marked excess of broken egg nuclei, micronuclei, pyknosis, karyorrhexis, karyolysis and enucleated cells in tobacco chewers relative to controls across both staining methods (e.g micronuclei present in 100% of chewer smears vs 30–37% of control smears). Molecular docking demonstrated a thermodynamically favourable interaction between nicotine and XRCC1, with a binding energy of −6.1 kcal/mol, mediated by an alkyl interaction with the Pro135 residue.
Conclusion: Habitual gutkha chewing is associated with early, detectable cytological damage in the buccal mucosa of young men, and nicotine forms a stable interaction with XRCC1 that may impair base excision repair. Buccal cytology, combined with molecular insight into nicotine–DNA-repair-protein interactions, supports a non-invasive, low-cost strategy for early risk stratification and reinforces the urgency of youth-targeted cessation programmes.
Novelty Statement
This study is among the first to combine field-based knowledge–attitude–practice assessment, dual-stain (PAP and MGG) exfoliative cytology, and structure-based molecular docking in a single investigative framework focused specifically on adolescent and young adult male gutkha users. By anchoring the observed cytological damage to a specific, residue-level molecular interaction between nicotine and XRCC1, the study moves beyond descriptive cytopathology toward a mechanistic hypothesis for tobacco-induced impairment of the base excision repair pathway in a high-risk, under-studied population.