Salivary SIRT3 Levels in Gutka and Alcohol Consumers: A Cross-Sectional Study on Habit-Associated Molecular Changes

Abstract

Background

Sirtuin-3 (SIRT3) regulates the production of cellular reactive oxygen species (ROS). This study aims to elucidate the molecular mechanisms involved in oral lesion development by exploring the relationship between SIRT3 expression, gutka consumption, and oral disease progression, with the objective of identifying potential biomarkers for early detection and risk assessment.

Methods

A cross-sectional study was conducted among individuals who regularly underwent oral health check-ups, excluding patients with premalignant and oral cancers from the study. Participants were categorized into three groups: Case 1 (gutka and alcohol consumers), Case 2 (gutka consumers without alcohol), and Control (non-consumers). Unstimulated saliva samples were collected and analysed using ELISA to measure SIRT3 levels. Statistical comparisons were performed using ANOVA followed by post hoc Tukey tests

Results

The mean SIRT3 levels were 7.57 ± 3.23 ng/mL (Case 1), 8.17 ± 2.87 ng/mL (Case 2), and 9.9 ± 2.78 ng/mL (control). The differences between the groups were statistically significant (p = .036), with the greatest difference observed between the case 1 group and the control group.

Conclusion

SIRT3 levels were lowest in individuals who consumed gutka and alcohol and were significantly lower than those in the control group. These findings suggest a potential negative impact of gutka and alcohol consumption on SIRT3 levels.

Clinical Implications:

This study highlights the significant impact of combined gutka and alcohol consumption on SIRT3 levels, which are associated with increased oxidative stress and cellular damage, potentially contributing to a greater risk of developing oral precancerous and cancerous lesions. Public health interventions aimed at reducing gutka and alcohol use are essential to mitigate these health risks. Further research is needed to establish the clinical significance of SIRT3 as a biomarker for the early detection and prevention of oral diseases. The use of saliva as a diagnostic fluid offers a non-invasive, easily accessible, and cost-effective approach, enhancing its potential for routine screening and early intervention.

Introduction

Tobacco use, whether through smoked or smokeless products, is the primary cause of oral cancer and has been recognized internationally as a significant public health concern. ¹ The prevalence of smokeless tobacco (SLT) is increasing globally, with its popularity rising rapidly. Gutka, a highly addictive form of smokeless tobacco (SLT), is widely consumed across various Indian communities and typically contains a mixture of crushed areca nut, slaked lime, catechu, sweet or flavourings, and tobacco.^2,3

Over 30 carcinogens have been recognized in SLT products, including tobacco-specific nitrosamines that are known to be potent carcinogens. SLT products also display signs of dependence, like in cigarette smokers, but pose a markedly different threat that may be due to the diversity in the product types and the variety in their composition.R4

According to the World Health Organization, approximately 21.4% of Indian adults use smokeless tobacco products, with gutka comprising a significant share. ⁴ States like Uttar Pradesh report a particularly high incidence of oral cancer, strongly associated with the prevalent use of SLT products such as gutka and paan. ⁵

A local survey in Wardha revealed that gutka consumption remains alarmingly high, with 46.4% of males and 20% of females reporting regular use, underscoring the urgent need for targeted public health interventions. ⁶ Gutka contains compounds that produce reactive oxygen species (ROS), which are known to disrupt DNA repair pathways. Over 30 carcinogens have been recognized in SLT products, including tobacco-specific nitrosamines that are known to be potent carcinogens. SLT products also display signs of dependence, like in cigarette smokers, but pose a markedly different threat that may be due to the diversity in the product types and the variety in their composition.4 The ingredients in commercially produced gutka include areca nut, slaked lime, catechu, and tobacco, which have been sun-dried, roasted, and finely chopped.^7–9

Proteins play a fundamental role in cellular metabolism, signalling, and structural integrity, acting as enzymes, transporters, and regulators of nearly every biological process. Among these, regulatory proteins are particularly crucial for maintaining cellular homeostasis and responding to environmental and metabolic cues.

One such group of regulatory proteins is the sirtuins, a family of NAD⁺-dependent histone deacetylases. These enzymes are evolutionarily conserved and are involved in a wide range of biological functions, including aging, inflammation, stress resistance, and energy metabolism. Sirtuins require nicotinamide adenine dinucleotide (NAD⁺) as a cofactor, linking their activity directly to the cell's metabolic state. In mammals, there are seven known sirtuins (SIRT1–SIRT7), each localized to specific cellular compartments. This compartmentalization allows sirtuins to regulate distinct cellular processes, from gene expression and DNA repair to mitochondrial function and oxidative stress response.

SIRT1 and SIRT2 are primarily found in the cytoplasm, SIRT3 and SIRT4 are localized in the mitochondria, and SIRT1, SIRT6, and SIRT7 are also present in the nucleus.^10–13

SIRT3 plays a crucial role in regulating mitochondrial protein deacetylation, a process essential for maintaining mitochondrial function and energy metabolism. Activation of SIRT3 has been reported to reduce reactive oxygen species (ROS) production and prevent cell death, while simultaneously enhancing ATP synthesis, promoting fatty acid β-oxidation, and stimulating the urea cycle, all of which are vital for cellular energy balance and detoxification.^14,15

SIRT3 is also critical for maintaining redox balance in normal human oral keratinocyte (HOK) cells and functions as an endogenous negative regulator under areca nut extract-induced oxidative stress. ¹⁶

SIRT3 is a critical modulator of oxidative stress and plays a crucial role in maintaining balance, functioning as a mitochondrial tumor suppressor by regulating reactive oxygen species (ROS) production. SIRT3 also acts as a Tumour Suppressor/Vulnerability Factor, as impairment of SIRT3 activity is identified as a factor increasing cancer susceptibility ¹⁷ :

Studies have also shown a specific non-synonymous point mutation (p.Val208Ile) in the SIRT3 gene, which is found in Oral Squamous Cell Carcinoma (OSCC) cell lines and is present as a germline alteration in nearly 24% of tested OSCC patients, severely reducing the catalytic efficiency of the SIRT3 protein. ¹⁸

Given the established role of SIRT3 in mitochondrial function, oxidative stress regulation, and cellular metabolism, investigating its expression in relation to gutka use is essential. Also, a clear articulation of the gap in non-invasive biomarker research for habit-associated oral disease risk is desirable. Also, a clearer articulation of the gap in non-invasive biomarker research for habit-associated oral disease risk is desirable. Hence, this study aimed to quantify and compare salivary SIRT3 levels among three groups: habitual gutka consumers, individuals who consume both gutka and alcohol, and non-consumers.

Materials and Methods

This cross-sectional study aimed to assess the differences in SIRT-3 levels between gutka consumers and non-consumers and was conducted among patients who visited the outpatient department of our institute for routine oral health check-ups.

Ethical Standards

The study received ethical approval from the Institutional Ethics Committee of Manipal College of Dental Sciences, Mangalore, with the IEC Protocol Reference Number 23084 on November 9, 2023. Saliva samples were collected at a single time point using a cross-sectional quantitative study design. The study's purpose and procedures were clearly explained to all participants, and informed written consent was obtained prior to participation. All methods were carried out in accordance with relevant guidelines and regulations. The reporting of this study conforms to STROBE guidelines. ¹⁹

Participants aged 21- 60 years who consumed gutka along with alcohol for more than a year were categorized into the Case 1 group. Those aged 21- 60 years who used gutka without alcohol consumption for more than a year were placed in the Case 2 group. Age- and sex-matched participants who were not consumers of either gutka or alcohol were included in the control group.

Inclusion criteria:

Participants aged 21- 60 years

Positive history of gutka consumption for more than a year

A positive history of alcohol consumption for more than a year

Participants willing to be part of the study by signing the informed consent form

Exclusion criteria:

Participants were outside the age range of 21- 60 years.

Individuals with systemic diseases that could influence salivary sirtuins.

Participants were on regular medications that could affect salivary flow and consistency.

Patients presenting with clinical features of malignant or premalignant lesions in the oral cavity.

Patients with a history of smoking, along with gutka and alcohol consumption

Patients with systemic illnesses on regular medications and salivary gland tumours were excluded from the study.

Sample size: Ramesh et al ¹⁰ calculated the sample size for a 90% confidence interval, assuming a standard deviation of units per study and an acceptable margin of error of 0.5 in estimating the actual population mean. A total of 63 participants were selected for the present study. Since this was a triple-group study, the participants were divided into three groups of 21 each.

Procedure:

Data collection: A structured questionnaire form was used to gather the patients’ sociodemographic details, medical history, and habit history.

Salivary Examination: Unstimulated whole saliva samples from the subjects were collected between 8:00 AM and 10:30 AM. The participants were instructed not to consume food or water one hour before the appointment. Once the saliva accumulated, the participants were instructed to slightly open their mouths and spit the pooled saliva into a container. This process lasted for five minutes, and the flow rate was recorded in millilitres per minute. A salivary flow rate of less than 0.1 ml/min was classified as hyposalivation. Collected samples were promptly stored at −20 °C until further analysis

Sample collection and storage: Approximately 5 mL of saliva was collected in a sterile container within five minutes. Saliva samples were transferred to EDTA-coated centrifuge tubes via disposable plastic Pasteur pipettes and stored in a refrigerator until transportation. The samples were then transported in a sealed vaccine storage box with ice packs to maintain the cold chain to the Central Lab for total protein estimation on the same collection day. The saliva samples were centrifuged, and the clear supernatant was pipetted and transferred into sterile 2 mL Eppendorf tubes.

Assessment of Sirtuin 3 (SIRT3) in Saliva

The estimation of salivary Sirtuin 3 (SIRT3) in our study was carried out using advanced biochemical and proteomic approaches designed to detect and quantify low-abundance proteins or evaluate their enzymatic activity.

Quantification by Enzyme-Linked Immunosorbent Assay (ELISA)

The concentrations of SIRT3 were determined using a microplate reader (BioTek Instruments, Model ELx808) and a high-sensitivity EIA kit (DRG International Inc., USA), which provided the necessary reagents for the direct detection of sirtuins in human saliva.

Bio Plates in SIRT3 Estimation

Surface for Antibody Binding:

In ELISA-based assays, wells of the bioplate are coated with capture antibodies specific to SIRT3. These antibodies bind selectively to SIRT3 molecules present in the salivary sample

Quantitative Detection:

After antigen binding, enzyme-linked secondary antibodies and substrate reactions generate a colorimetric, fluorometric, or chemiluminescent signal. The intensity of the signal is proportional to the concentration of SIRT3 in the sample.

The suitable wells on the microplate received either standards or samples, along with a SIRT3-specific biotin-conjugated antibody. The TMB substrate solution was added, causing a color change only in the wells containing SIRT3, the biotin-conjugated antibody, and the enzyme-conjugated avidin. After incubation, the enzymatic reaction on the substrate was stopped with a sulfuric acid solution. The color change was measured via spectrophotometry within a wavelength range of 450 ± 10 nm. The obtained optical density (OD) values for the samples were compared against a standard curve to determine the SIRT3 concentration.

High-Throughput and Precision:

Bioplates allow for the simultaneous processing of multiple samples and standards, ensuring reproducibility, comparative analysis, and statistical accuracy. This makes them ideal for cross-sectional or comparative studies assessing SIRT3 levels in different subject groups (eg, Gutka users, alcohol consumers, and controls).

Integration with Analytical Instruments:

The signal produced in each well is read using a microplate reader (eg, BioTek ELx808), which quantifies absorbance or fluorescence, allowing for the accurate determination of SIRT3 concentration through standard curve calibration.

Statistical analysis was performed using SPSS software, version 29. The Shapiro‒Wilk test was performed to assess the normality of the data. Data that followed a normal distribution are presented as the means ± standard deviations. Comparisons between multiple groups were made via one-way ANOVA, followed by post hoc analysis with Tukey's test. A p-value of less than 0.05 was considered statistically significant.

Results

Comparison of SIRT-3 Levels in the Study Groups

The results revealed a statistically significant difference in the mean SIRT-3 levels among the three groups (p = .036).

Control Group:

This group exhibited the highest levels of SIRT-3 among all groups.

Case 1 Group:

This group showed the lowest SIRT-3 levels in comparison to the others.

Case 2 Group:

SIRT-3 levels in this group were lower than those in the control group but higher than those in the Case 1 group.

The control group, which did not consume gutka or alcohol, presented the highest SIRT-3 levels, suggesting that the absence of these addictive substances might be associated with increased SIRT-3 levels. The case 2 group, which consumed gutka without alcohol, had intermediate SIRT-3 levels, whereas the case 1 group, which consumed both gutka and alcohol, had the lowest levels. This pattern suggests a potential negative impact of combined gutka and alcohol consumption on SIRT-3 levels, as shown in Table 1.

Table 1.

Differences in the Mean SIRT-3 Levels Between the Study Groups.

Group	Mean SIRT-3 Level ng/mL (mean ± SD)	p Value
Case 1 group (n = 21)	7.57 ± 3.23	.036*
Case 2 group (n = 21)	8.17 ± 2.87
Control group (n = 21)	9.9 ± 2.78

The results from Table 2 indicate the following:

Table 2.

Post hoc Comparison of the Mean SIRT-3 Levels among the Three Groups.

Comparison Groups	Mean Difference	p Value
Case 1 group versus Case 2 group	0.6	.788
Case 1 group versus control group	2.34	.035*
Case 2 group versus control group	1.73	.149

Case 1 Group versus Control Group:

A statistically significant difference in SIRT-3 levels was observed between the Case 1 group and the control group (p = .035). The control group exhibited notably higher levels of SIRT-3 compared to the Case 1 group.

Case 2 Group versus Control Group:

Although the control group showed higher SIRT-3 levels than the Case 2 group, the difference was not statistically significant (p > .05).

Case 1 Group versus Case 2 Group:

The comparison between Case 1 and Case 2 groups revealed a slight variation in SIRT-3 levels, but this difference did not reach statistical significance (p > .05).

Discussion

The primary objective of this study was to evaluate the impact of gutkha and alcohol consumption, both independently and in combination, on the levels of Sirtuin 3 (SIRT3) detectable in saliva, as a potential non-invasive indicator of cellular stress and precancerous risk. The analysis yielded a distinct pattern across the three cohorts: the Control Group (no addictive substance use) exhibited the highest mean salivary SIRT3 level (9.9 ± 2.78 ng/mL), followed by the Case 2 Group (gutkha only) (8.17 ± 2.87 ng/mL), and finally, the Case 1 Group (gutkha and alcohol combined) showed the lowest levels (7.57 ± 3.23 ng/mL) as shown in Table 1. This hierarchy suggests a significant negative correlation between the consumption of these substances and salivary SIRT3 levels, with combined use demonstrating the most profound reduction, implying a potential synergistic or additive detrimental effect.

SIRT3, a primary mitochondrial deacetylase, is a crucial regulator of cellular redox balance, metabolism, and genomic stability.^20–21 Its presence is detectable in human saliva, suggesting that saliva's utility lies in its use as a surrogate for monitoring systemic and oral cellular health. ²² The decrease in salivary SIRT3 concentration observed in the consuming groups may reflect widespread disruption of the cellular antioxidant defence system triggered by chronic exposure to carcinogens found in smokeless tobacco (SLT) products, such as gutkha. SLT use induces a shift toward an oncogenic state, strongly characterized by indicators of oxidative stress.

The significantly lower salivary SIRT3 concentration observed in the Case 1 and Case 2 Groups likely correlates with this dysfunctional state in mitochondrial homeostasis, reflecting a compromised ability to suppress oxidative stress induced by gutkha chewing.

Impact of Combined Gutkha and Alcohol Consumption- The study's finding that the Case 1 Group (gutkha + alcohol) exhibited the lowest SIRT3 levels supports the hypothesis of an enhanced negative impact resulting from the co-consumption of these two risk factors.

Alcohol consumption is well-documented to induce systemic and local stress, often compounding the effects of tobacco use. Specifically, alcohol metabolism can lead to mitochondrial dysfunction and protein hyperacetylation, ultimately resulting in reduced SIRT3 activity and impaired antioxidant defences. ²² Furthermore, chronic alcohol consumption induces oxidative stress and generates reactive nitrogen species (RNS).

The synergistic effect suggested by the lowest SIRT3 levels in the Case 1 Group (gutkha + alcohol) indicates that alcohol consumption exacerbates the pre-existing mitochondrial and oxidative damage initiated by tobacco chewing. This combined stress may lead to a more severe depletion of SIRT3 protein or activity than either substance alone, accelerating cellular impairment relevant to oral cancer initiation.

The results of our study are in strong concordance with the findings reported by Ramesh et al ¹⁰ who investigated SIRT3 expression across a spectrum of oral tissue types, ranging from normal oral mucosa and smokers without lesions (SWLs) to oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC). Their analysis revealed a statistically significant variation in SIRT3 expression among these groups (p < .001), with a marked increase in SIRT3 levels observed in OSCC patients compared to OL, as determined by a two-tailed unpaired Student's t-test.

Our study similarly demonstrated a significant difference in SIRT3 expression between the Case 1 group and the control group, reinforcing the hypothesis that elevated SIRT3 expression may be associated with the progression from regular usage of addictive tobacco-containing products leading to potentially malignant disorders and eventual malignancy. These findings not only validate our results but also strengthen the evidence supporting SIRT3 as a potential biomarker for malignant transformation in oral tissues. ²³

The observed post-hoc analysis of salivary Sirtuin 3 (SIRT3) levels across the study groups provides compelling insights into the potential synergistic detrimental effects of combined gutkha and alcohol consumption on this critical mitochondrial regulator. The data delineate a clear hierarchy of SIRT3 levels, with the Control Group exhibiting the highest levels, followed by the Case 2 Group (Gutkha only), and the Case 1 Group (Gutkha + Alcohol) showing the lowest mean concentration.

The exposure associated with smokeless tobacco (SLT) products, such as gutkha, is well-established as a potent inducer of chronic oxidative stress in oral tissues. SLT use causes a shift toward an oncogenic state, strongly characterized by indicators of oxidative stress. Consistent with severe cellular compromise, studies show that dysregulated mitochondrial proteins are decreased in SLT users relative to controls, providing evidence of the generalized impact of tobacco chewing-induced oxidative stress. ²⁴

The observation that both Case groups showed lower mean salivary SIRT3 levels compared to the Control Group aligns with the understanding that chronic exposure to tobacco-specific carcinogens and ROS induces systemic stress and likely compromises mitochondrial integrity.

Significance of Combined Exposure (Case 1 vs Control)

The post-hoc analysis highlighted that the decrease in SIRT3 levels was statistically significant only when comparing the Case 1 Group (Gutkha + Alcohol) to the Control Group This finding strongly suggests that alcohol acts synergistically with gutkha to accelerate the disruption of cellular homeostasis, resulting in a quantifiable collapse of SIRT3 levels that overcomes biological variability.

This result is biologically plausible given the known impact of alcohol:

Exacerbated Oxidative Stress: Chronic alcohol consumption induces stress and generates reactive nitrogen species (RNS), further compounding the effects of tobacco carcinogens, which are known to increase ROS production. Both tobacco and alcohol exposures increase oxidative stress and reduce antioxidant enzymes. ²⁵

SIRT3 Activity Impairment: Alcohol consumption is specifically linked to mitochondrial dysfunction and protein hyperacetylation, leading to reduced SIRT3 activity and impaired antioxidant defences. Chronic alcohol ingestion also enhances tumor necrosis factor-alpha (TNF-α) expression and salivary gland apoptosis. ²⁶

Therefore, the profound, statistically verifiable reduction in SIRT3 observed only in the Case 1 Group suggests that the combined insults of oxidative stress from gutkha components (like betel quid carcinogens) and the direct mitochondrial damage mediated by alcohol consumption push the defence mechanisms beyond a critical threshold, resulting in a significant loss of the key mitochondrial regulatory enzyme.

Although SIRT3 has a complex role that sometimes positions it as a tumor promoter in advanced oral squamous cell carcinoma (OSCC) by assisting highly stressed cancer cells to survive, the finding of significantly depressed SIRT3 levels in the high-risk group (Case 1) is relevant when viewed through the lens of impaired protective function and increased susceptibility.

Our study findings are in alignment with those reported by Shajedul Islam et al, ¹¹ who investigated the molecular effects of betel quid (BQ) chewing. Their research demonstrated that reduced levels of SIRT1 in BQ chewers were associated with DNA hypermethylation, suggesting a mechanistic link between SIRT1 downregulation and the development of oral cancer. They proposed that this epigenetic alteration in the oral mucosa could serve as a predictive marker for malignant transformation.

Similarly, our study observed a reduction in SIRT3 expression among Gutkha consumers(similar to BQ in composition), mirroring the trend seen with Sirtuin levels in the above study. This suggests that sirtuin family members, including SIRT3, may play a broader role in the epigenetic regulation and carcinogenic processes associated with Gutkha and BQ usage. The consistency between our findings and those of Shajedul Islam et al reinforces the potential of sirtuins as biomarkers for early detection and risk assessment in oral carcinogenesis among Gutkha consumers.

Noor et al investigated the role of SIRT3 in deacetylating mitochondrial proteins, regulating reactive oxygen species (ROS), and maintaining mitochondrial quality control, which are essential processes for preventing mitochondrial dysfunction and ensuring cellular health. Their study revealed that smokers presented significantly lower SIRT3 levels than nonsmokers did, suggesting potential mitochondrial dysfunction. ¹² Similarly, in our study, we observed that the SIRT3 levels were relatively low in the group of individuals who consumed gutka with alcohol compared with those in the non-habit control group.

Our study findings are in strong agreement with those reported by Sripodok et al, ²⁰ who examined the expression of SIRT1, SIRT6, and SIRT7 in patients diagnosed with oral squamous cell carcinoma (OSCC). Their research highlighted elevated levels of SIRT1 and SIRT6 in OSCC tissues. Notably, they also identified an inverse relationship between BQ consumption and SIRT3 expression, suggesting that lifestyle habits may negatively influence mitochondrial function and contribute to oral carcinogenesis.

Consistent with these observations, our study found that individuals with habits such as gutka consumption and alcohol intake exhibited significantly lower levels of SIRT3 compared to the non-habit control group. This reinforces the hypothesis that lifestyle-related exposures can downregulate SIRT3, potentially impairing mitochondrial regulation and promoting malignant transformation. Together, these findings underscore the importance of Sirtuins as a potential biomarker for habit-associated oral cancer risk and progression.

Our study's findings are consistent with those of Jasim et al, ²⁶ who explored the impact of BQ chewing on oral health. Their research highlighted that arecoline, a major alkaloid in areca nuts (a key ingredient in gutka), contributes to the development of oral submucous fibrosis and its progression to oral cancer. This occurs through the stimulation of transforming growth factor-beta (TGF-β), which in turn may epigenetically silence SIRT1 by upregulating MeCP2, a methyl-CpG-binding protein involved in gene repression. Similarly, our study observed reduced SIRT3 levels in individuals who consumed gutka, which also contains areca . This suggests that arecoline may influence oral cancer progression through epigenetic mechanisms affecting multiple sirtuin family members, including SIRT3. ²⁵

L. Naia, et al found that SIRT1 methylation levels were significantly higher in smear samples taken from the clinically healthy buccal mucosa of BQ chewers compared to non-chewers. Their study demonstrated that even in the absence of visible lesions, the oral epithelium of BQ users exhibited elevated methylation of SIRT1, suggesting early epigenetic changes linked to carcinogenesis. In line with these findings, our study observed reduced SIRT3 expression specifically among gutka chewers, indicating that similar epigenetic mechanisms may be affecting other sirtuin family members and contributing to oral cancer risk in individuals with such habits. ²⁷

The difference in SIRT3 levels between the Case 2 Group (Gutkha only) and the Control Group was notable (1.733 ng/mL difference) but not statistically significant (p > .05). This indicates that while gutkha chewing likely initiates oxidative stress and compromises SIRT3 levels, the damage may be less severe or more variable in the absence of alcohol's potentiating effect, preventing the result from reaching statistical significance in this study design.

Furthermore, the difference between the Case 1 (G + A) and Case 2 (G only) groups was small (0.603 ng/mL) and not statistically significant (p > .05). While Case 1 exhibited the lowest overall mean, suggesting combined factors are indeed most detrimental, the lack of statistical significance between the two consuming groups reflects the challenge of disentangling independent and synergistic effects in biological samples and points toward the need for larger studies to confirm the relative contribution of alcohol versus isolated gutkha exposure on SIRT3 levels.

SIRT3 plays a critical role in maintaining mitochondrial health by regulating oxidative stress and energy metabolism. Reduced SIRT3 levels among gutka chewers lead to an accumulation of reactive oxygen species (ROS) within cells. Elevated ROS levels can cause oxidative damage and stabilize hypoxia-inducible factor 1-alpha (HIF-1α), a transcription factor that promotes metabolic reprogramming in cancer cells^28–29

The present study is the first to report a reduction in SIRT3 levels in saliva samples collected from individuals who consume gutkha in combination with alcohol. This is a significant finding, as both substances are known to induce epigenetic alterations, particularly DNA methylation changes. The observed downregulation of SIRT3 may be attributed to hypermethylation of its promoter region, a mechanism that silences gene expression and has been implicated in various cancers. ³⁰

This potential hypermethylation of SIRT3 may serve as an early predictive biomarker for oral carcinogenesis in individuals with these high-risk habits. By identifying such epigenetic changes in non-invasive samples like saliva, our study opens new avenues for early detection and risk assessment of oral cancer, especially in populations with prevalent gutkha and alcohol use. ³¹

The data demonstrate that chronic exposure to gutkha, and particularly the combination of gutkha and alcohol, significantly depletes salivary SIRT3 levels compared to controls. This reduction is physiologically relevant, as it links established risk behaviors to the suppression of a key mitochondrial regulator that protects against oxidative stress and maintains genomic stability. These results support the utility of salivary SIRT3 quantification as a non-invasive biomarker for assessing heightened risk for oral potentially malignant disorders and oral cancer among tobacco chewers, pending further validation in larger, longitudinal studies. While SIRT3 reduction is noted, the study does not establish its diagnostic accuracy (eg, sensitivity/specificity, ROC curves) or link to clinical outcomes (eg, progression to oral cancer).

Limitations- This study has several limitations that need to be acknowledged. First, the sample size was relatively small, which may limit the generalizability of the findings. In addition, the study did not include a detailed assessment of the participants’ habit history, such as duration of use, frequency, or quantity of smokeless tobacco or alcohol consumption. We also did not record confounders such as diet, oral hygiene practices, and betel nut use. We also recommended that future work include all these data on duration and frequency of habit use, diet, as a control to account for the variables. Additionally, the study focused exclusively on individuals with lifestyle habits such as gutkha chewing and alcohol consumption, while excluding participants with clinically diagnosed potentially malignant disorders (PMDs) or oral cancer. The study population did not include individuals with premalignant or malignant oral lesions, and the findings of our study suggest a potential molecular link but do not establish a direct clinical correlation with oral cancer. As a result, the findings may not fully capture the spectrum of SIRT3 alterations across different stages of oral disease progression.

Conclusion

This study underscores the potential role of SIRT3 as a non-invasive salivary biomarker for evaluating oral cancer risk in individuals with high-risk habits such as gutkha chewing and alcohol consumption. The observed reduction in SIRT3 levels suggests a link between these lifestyle factors and early epigenetic changes that may contribute to mitochondrial dysfunction and tumour development. While the findings are novel, particularly in identifying reduced SIRT3 in gutkha and alcohol users, limitations such as a small sample size, short study duration, and exclusion of patients with PMDs or oral cancer highlight the need for further research to validate these results and explore the mechanistic role of SIRT3 in oral carcinogenesis. Suggest that future studies should incorporate detailed habit history (eg, years of use, frequency per day) to explore dose–response relationships. Such investigations will be essential to determine whether SIRT3 can serve as a reliable non-invasive biomarker for early detection and risk assessment in oral disease progression.