Introduction

The habit of chewing betel nuts is prevalent in developing countries like India, Indonesia, and Bangladesh. In India, the use of chewable tobacco has increased in Gujarat. Tobacco smoking is a primary cause of oral diseases, including periodontal disease, significantly affecting patients' quality of life by impairing dental function and aesthetics (1). It weakens the immune system and slows wound healing, contributing to the degeneration of periodontal tissue (2). In rural areas of India and some third-world countries, cultural practices often lead to the combined use of chewable tobacco, such as pan masala/gutka, and betel nut, increasing the risk of oral submucous fibrosis (OSF), a premalignant condition (3). Early diagnosis of OSF is crucial; in later stages, it compromises the oral cavity, causing complications in speech, mastication, and swallowing (4).

Chewing betel nuts is as addictive as tea, coffee, and alcohol, containing psychoactive substances that provide partial recreational effects. Tobacco chewing can cause oral issues like tooth stains and thickened saliva, and may lead to symptoms in young adults, such as a dry face, constricted food pipe, and dizziness. Common complications among tobacco users include ulcers, pain while swallowing, and, in later stages, respiratory and cardiac symptoms like asthma, decreased blood pressure, and increased heart rate (5). Apart from that, the OSF increases the risk of acquiring infection with hepatitis (6). Tobacco consumption also leads to vitamin and nutritional deficiency and irritation while eating spicy food (7).

There may be compromised mouth opening due to various factors, including spasms, muscular dysfunction, and contraction of mucous membranes caused by OSF (8,9). Additionally, dysphagia is another standard clinical parameter in patients with reduced chewing and speaking abilities, adversely impacting their quality of life due to significant pain, malnutrition, and poor oral hygiene. These patients present challenges for intubation if surgery is required, potentially leading to life-threatening scenarios in a patient owing to immense pain, malnutrition and low oral hygiene. These patients are challenging to intubate if elected for any surgery, and it could create life-threatening scenarios (10,11).

Oral Squamous Cell Carcinoma (OSCC) is the most common type of oral cancer, affecting areas such as the lips, gums, tongue, and inner cheeks (12). In 2021, an estimated 54,010 new cases of oral cavity and oropharyngeal cancers were diagnosed in the U.S., with 10,850 deaths reported (13). The precise cause of OSCC is unknown, but key risk factors include tobacco and alcohol use, HPV infection, poor dental hygiene, and a weakened immune system. OSCC often begins as a white or red patch in the mouth, progressing to chronic sores or lumps. Diagnosis is confirmed via biopsy, with imaging (X-rays, CT, or MRI) used to assess malignancy. Treatment options such as surgery, radiation therapy, chemotherapy, or a combination of these are determined by the cancer’s stage and the patient’s overall health (14). Tobacco use remains a significant risk factor, with an estimated 1.3 billion global smokers, 80% residing in low- and middle-income nations, contributing to the burden of OSCC and related cancers (15,16)

Tobacco smoking is a major global health issue, causing over 6 million deaths annually, including many non-smokers exposed to second-hand smoke. It leads to chronic diseases such as cancer, heart disease, and lung diseases due to harmful components like nicotine and toxic chemicals. Smoking is especially prevalent in low- and middle-income countries, contributing to severe public health challenges.

Nicotine leads to both physical and psychological dependence, compounded by habitual smoking. Non-smokers, especially children exposed to second-hand smoke, also face serious health risks. Long-term smoking causes chronic respiratory issues, cardiovascular diseases, and cancers, weakening the respiratory system and increasing infection risk. The future impact of tobacco is alarming, particularly in developing nations where usage rates are rising. If this continues, tobacco-related deaths may escalate as younger smokers age. This situation underscores the urgent need for effective cessation programs and public health strategies. Additionally, research into biomarkers for diagnosing tobacco-related cancers, such as oral squamous cell carcinoma (OSCC) is progressing, which could enhance detection and treatment.

This study aims to evaluate the severity of oral squamous fibrosis (OSF) using clinical parameters such as MPC, MMO, Dysphagia, and Thyromental distance. A survey of biomarker detection at the early stage may create a bridge between the Mallampati classification and the differences between tobacco and non-tobacco chewers. Hence, we hypothesised to determine the difference in the Mouth opening grading, Mallampati classification, Dysphagia, and Thyromental distance between tobacco chewers and non-tobacco chewers, as well as biomarker detection.

Material and Methods

Study Design

The prospective comparative study was meticulously conducted over a span of two years, from June 2023 to August 2025. This research focused on gathering vital data from hospitals located in the Anand region of Gujarat, a vibrant area known for its diverse population and healthcare challenges. Before the commencement of the study, an advisory scientific research committee rigorously reviewed and approved the research protocol. In alignment with ethical guidelines, informed consent was obtained from all participants, ensuring their understanding and willingness to partake in the study, as outlined by IEC-9/ARIBAS/2022-23/E-7. This comprehensive approach underscores the commitment to ethical standards and the integrity of the research process.

Study Sample

In the present study, 200 patients were included, out of which 131 were male, and 69 were female, in the age range between 18 and 90 years. Out of 200 patients, 100 were tobacco chewers, and 100 were non-tobacco chewers. Patients visiting the hospital for general surgeries, such as cholecystectomy, hernioplasty, appendectomy, and Circumcision, were included in the study. Patients should be between 18 and 90 years old based on the inclusion criteria. Patients below the age of 18, facial traumas, emergency patients, unwillingness to give consent, cigarette smokers and “bidi” smokers were excluded from the study. We classified the patients into two groups: tobacco-consuming patients as “group T” and non-tobacco consumers as “group NT”.

Oral Assessment

During the study, patients underwent a thorough oral examination in a sterile environment to assess the clinical status of the internal structures of the mouth. A skilled anaesthesia technologist took the measurements under the supervision of an anaesthesiologist. All the gathered data was meticulously documented using a specialised form explicitly designed for this research study. Modified Mallampati Classification (MMC), Maximal mouth opening (MMO) and Thyromental Distance (TD) were used to evaluate the internal structures of the oral cavity. Patients were asked if they had difficulties swallowing food (Dysphagia). Mallampati classification was used to assess the internal structures of the oral cavity, such as the uvula, soft palate, faucets, and pillars. MMO is a technique used to check the adequacy of the mouth opening of a patient by measuring the distance between the upper and lower incisors. Mouth opening was assessed with the help of a Vernier calliper. TD was used to evaluate if there is any decrease in the distance between the thyroid cartilage and the tip of the chin with the neck fully extended. Further, the MMC, MMO and TD were classified as.

Statistical Analysis

Statistical analysis was done with IBM SPSS Statistics for Windows, version 26 (IBM Corp., Armonk, N.Y., USA). Differences in demographic features such as age and gender were measured using the Student's t-test and the chi-square test. The clinical parameters, including Mallampati classification, maximal mouth opening, dysphagia, and Thyromental distance, were compared between the T and NT-consuming groups using a chi-square test. The statistical analysis by SPSS was considered significant when the P value was < 0.05.

Biomarker detection study

From the data procured from the previous objectives, we recruited 5 females (35-45 years) and 35 males (30-75 years) who expressed their willingness to participate in this study. Participants had to be free of fever or cold, non-smokers, and have good oral hygiene. Participants were asked to refrain from eating and drinking for two hours before saliva collection to obtain a relatively constant baseline.

Sample collection: Saliva samples were collected from individuals who regularly chew tobacco, following ethical guidelines and informed consent procedures. To minimise contamination, the participants were asked to refrain from eating, drinking, or chewing tobacco for at least 30 minutes before collection. Using sterile plastic saliva collection tubes, approximately 2–3 mL of unstimulated saliva was collected by having participants naturally enter the tubes. The samples were immediately transported on ice to the laboratory to prevent degradation, where they were stored at -40°C until further analysis. The collection procedure ensured minimal contamination and maintained the integrity of the samples for downstream molecular analyses such as DNA extraction and biomarker detection (17).

Isolation of DNA from a saliva sample:

DNA isolation from saliva begins with collecting a saliva sample, typically by having the donor spit into a paper cup. Once the sample is collected, the cells are lysed by adding two drops of dishwashing liquid, which breaks down the cell membranes and releases the DNA. Next, DNA purification is carried out using the precipitation method. This involves adding ½ cup of isopropyl alcohol to ¼ cup of the saliva sample, which causes the DNA to precipitate out of the solution. The quality and quantity of the purified DNA are then assessed using gel electrophoresis. Finally, the DNA is stored in a TE buffer to maintain its integrity for future applications.

PCR Amplification and Sequencing

Primers for cathepsin V, ADAM9, and kallikrein 5 were designed to target specific regions of interest, typically ranging from 18–25 base pairs in length, with melting temperatures (Tm) between 55–62°C. The detailed primer sequences and their corresponding Tm values are presented in Table 2. PCR amplification was carried out in a final reaction volume of 25 µl, containing 15 µl of Hi-Chrom PCR Master Mix (2X), 1 µl of forward primer (10 µM), 1 µl of reverse primer (10 µM), and 2 µl of template DNA (minimum 100 ng), with the volume adjusted using molecular biology-grade water. Amplification was performed in a thermal cycler with an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94 °C for 45 seconds, annealing at gene-specific temperatures (62°C for cathepsin V, 60°C for kallikrein 5, and 59°C for ADAM9) for 30 seconds, and extension at 72°C for 30 seconds. A final extension was conducted at 72°C for 5 minutes (cathepsin V) or 7 minutes (kallikrein 5 and ADAM9), after which reactions were held at 4 °C until further analysis. All positively amplified products, including controls, were purified and subjected to sequencing for validation. Sequencing data were analyzed to determine similarities, differences, and evolutionary relationships. Throughout DNA isolation and PCR processing, products were quantitatively assessed using Nanodrop (ND-1000) and qualitatively evaluated by gel electrophoresis.

Results and Discussion

The habit of tobacco chewing was significantly higher in males than in females. (P 0.0018). Comparing the clinical parameters for the oral assessment revealed that the number of individuals in the later stages of MPC was significantly higher. (P= 0.000) in the tobacco chewing group as compared to non-tobacco chewers. Restricted mouth opening was observed in almost 80% of the tobacco chewers, while in non-tobacco chewers, the numbers were meagre (P= 0.000). An increased number of patients among tobacco-chewing groups complained about difficulty swallowing, as there were no such complications in non-tobacco chewers. (P= 0.000) There was no difference in the Thyromental distance between the groups, and we didn’t find any significance either.

Figure 1: Gender-wise distribution of tobacco and non-tobacco consumers

Figure 2 (A): Severity of Mallampati classification in both groups. (B): The assessment has outlined the variations in mouth-opening capability observed between the two groups."(T and NT).
Figure 3 (A): Graphical distribution of patients having difficulty in swallowing for both groups. (B): Graphical distribution of thyromental distances among both groups.

The analysis revealed the successful amplification of specific genetic products, confirming the presence of a 218 base pair (bp) fragment corresponding to the Kallikrein gene in 4 samples having tobacco chewing habits for 10 years or more. Additionally, a larger amplified product, the Cathepsin V gene and the adam9 gene, was not observed in any of the samples. These results not only validate the targeted amplification but also provide critical insights into the genetic characteristics of the samples analysed.

Figure 4: A Phylogenetic analysis showing the distribution of amplified product with the reference gene specifically focused on the kallikrein gene family, which encodes serine proteases involved in various physiological processes. The tree helps visualise which kallikrein variants are more closely related (connected by shorter branches) versus those that diverged earlier in evolutionary history (connected by longer branches or more nodes).

A study conducted in Gujarat indicates an increase in tobacco consumption, resulting in various related complications. Approximately 85% of the tobacco-chewing demographic are in their early 30s (18). Historically, males have dominated tobacco use, particularly in the age range of 40 to 60 years (19). The current research shows a higher prevalence of tobacco consumption among males compared to females. The average age, along with standard deviation, for tobacco users is 48.02 ± 16.45 years, while for non-tobacco users it is 46.52 ± 13.39 years. The traumatic effects of chewing tobacco can severely damage the oral mucosa, leading to peeling and exfoliation of surrounding tissue (20). Wollina et al. note that chewing betel quid causes oral cavity fibrosis, which can mutilate the soft palate, uvula, and surrounding areas. The buccal mucosa and soft palate are notably affected by excessive consumption (21).

The Mallampati classification is commonly employed to predict difficult intubation in pre-operative assessments. Previous studies have shown that an MPC of III or higher correlates with difficulty in intubation within the tobacco-chewing population. In our research, we applied the MPC to assess the oral cavity structures of both groups and identified a significant difference. The tobacco-consuming group had a higher incidence of patients with MPC III or greater compared to non-tobacco consumers: only three patients out of 100 in Group NT versus 58 in Group T, showing MPC III or above. Evidence points to significant difficulties with mouth opening in patients who chew tobacco, attributed to the formation of fibrous bands (21).

During our study, mouth opening was measured using a Vernier calliper, revealing that tobacco chewers had restricted mouth openings (interincisor gap less than 4 cm) in comparison to non-tobacco users. This restriction likely stems from prolonged tobacco use. Previous studies have linked dysphagia, or difficulty in swallowing, to restricted mouth openings (22). This condition can deteriorate a person’s nutritional status and quality of life (23).

In our current study, we inquired about patients' swallowing difficulties, finding a greater prevalence of dysphagia among the tobacco-consuming group compared to non-users. Mittal et al. concluded that caries rates were higher among smokers when compared to non-smokers, whereas tobacco chewers exhibited lower rates; however, any form of tobacco use is tied to multiple health issues, including cancer, low birth weight, and heart and lung disorders. The adverse effects of tobacco on oral health are evident (24). Many patients reported pain while swallowing food.

Due to limited previous studies, we undertook another assessment to examine the thyromental distance in patients, finding no clinically significant differences between the groups. No link was established between reduced thyromental distance and tobacco use. Variations in space sizes among individuals made it challenging for experts to define limitations for specific patients. Nevertheless, body metrics such as height, weight, and age remain crucial in diagnosis (25).

Most patients in our study were from rural areas with low literacy rates. The reluctance of patients to participate posed a significant challenge, affecting both the sample size and the overall validity of the research.

A pioneering primary study investigating biomarkers showed that a collaborative approach could effectively merge clinical diagnostic methods with molecular techniques. Notably, 10% of the 40 patients enrolled in this study were quickly identified as having elevated Kallikrein levels. This underscores the potential of rapid diagnostic methods, which could be developed to support early cancer predictions, particularly among both tobacco chewers and non-chewers. Such innovations might lead to improved screening and intervention strategies in the future.

Conclusion

This investigation examines the oral health differences between tobacco users and non-users. We found significant differences in key health measures, such as the Modified Mallampati Classification, mouth opening, and swallowing difficulties, all with low p-values (p=0.000). This shows these measures can identify the risk of oral submucous fibrosis (OSF) early in tobacco users. Our analysis shows a male predominance among tobacco users (p=0.0018), but no significant age difference (p=0.4805). This highlights the need for targeted prevention efforts. By combining clinical assessments with genetic analysis, our study offers a comprehensive method for detecting OSF risk in tobacco chewers. Additionally, our finding of no significant difference in thyromental distance (p=0.761) suggests it may not be helpful for OSF assessment. Overall, this research emphasises the importance of combining clinical and molecular factors for better OSF risk management. We also discovered Kallikrein gene amplification in 10% of participants, suggesting it could be a marker for tobacco-related oral changes. While no amplification was found in adam and cathepsin genes, the Kallikrein expression warrants further study to understand its role in oral tissue inflammation and fibrosis.

Funding

No Funding Source.

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors thank the respective institutions for providing work facilities and the hospitals for their permission. Special thanks go to Dr. Harshil Chavda for their permission during data collection.

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