A clinical comparative study was conducted at Narayana Dental College and Hospital for a period of 1 month (September to October 2016) to assess the effect of tobacco chewing and smoking on the salivary total antioxidant power among the male subjects (35-50 years old), who satisfied the inclusion criteria and who gave a written consent. Study subjects were selected from the people visiting Narayana Medical and Dental Hospitals conveniently, who were not patients (people accompanying the patients). The ethical clearance for the study was obtained from the Institutional Review Board of the Narayana Dental College and Hospital, Nellore.

To estimate the sample size, a power analysis was performed based on the data obtained from a previous study conducted by Jenifer HD et al., with standard deviations of 8.11, least detectable difference of 9.74 [7]. The sample size arrived was 11.64, which was rounded off to 15. Therefore, sample size for each group was 15. As we had three groups, a total of 45 subjects were included in this study.

Tobacco chewers: Subjects with a history of daily consumption of chewable tobacco for at least 10 years, with no habit of smoking.

Smokers: Subjects with a habit of smoking for at least 10 years and with no habit of tobacco chewing.

Controls: Subjects with no habit of tobacco chewing and smoking in their life time.

Subjects with both smoking and chewing habit, subjects with oral mucosal lesions and systemic diseases, subjects with a history of chronic alcohol consumption and dental caries and subjects with clinical attachment loss of less than 4 mm were excluded from the study.

Saliva collection: Unstimulated saliva sample was collected by spitting method [12]. Saliva was collected while the subject was sitting upright with the head slightly tilted forward and the eyes open. The patient was advised to refrain from intake of any food or beverage (water exempted) one hour before the test session. Smoking, chewing gum and intake of coffee were prohibited during this hour. The subject was advised to rinse his mouth several times with deionized (distilled) water and then to relax for five minutes. Saliva was allowed to accumulate in the mouth and subject spitted into the graduated test tube every 60 seconds for five minutes. So that salivary flow rate can be measured.

Salivary pH assessment: The pH of the saliva was assessed using digital pH meter (Ri, model 152-R). In between each reading the electrode was cleaned with a stream of distilled water and placed in a standard solution of pH 7.0. This ensured stable readings and a constant check on drift.

Storage of saliva: Then saliva samples were immediately centrifuged at 800 g for 10 minutes at 4°C to remove cell debris. The resulting supernatants were immediately deep-frozen at -80°C and stored for later analysis.

Total antioxidant power of saliva: Total antioxidant power of saliva was measured by FRAP assay. FRAP was used to determine the antioxidant potential in a given sample. This assay is inexpensive, reagents are simple to prepare, results are highly reproducible, and the procedure is straightforward and speedy. It gives putative index of antioxidant, or reducing potential of biological fluids within the technological reach of laboratory and researcher interested in oxidative stress and its effects. FRAP utilises the reducing potential of the antioxidant to react with a Ferric Tripyridyltriazine (TPTZ) complex. This produces a coloured ferrous tripyridyltriazine form. The change in absorbance at 593 nm can then be compared with a standard to determine the antioxidant potential in a given sample [8].

The collected data were analysed using Statistical Package for the Social Sciences (SPSS) version 20.0. Analysis of Variance (ANOVA) was used for comparison of three groups with respect to salivary total antioxidant power, flow rate and pH. Tukeys post-hoc analysis was used for pairwise comparison of study groups with respect to salivary total antioxidant power, flow rate, pH. A p-value less than 0.05 was considered as statistically significant.

A total of 45 subjects participated in the study with 15 each in smoker group, tobacco chewer group and control group with a mean age of 44.23±4.001. All the participants were males and there was no significant difference in the mean age between the three study subjects so they were comparable [Table/Fig-1]. The mean duration of smoking was 16.73±3.411 years and mean duration of tobacco chewing was 14.53±4.138 years among the smokers and chewers respectively.

Salivary total antioxidant power was lowest in tobacco chewers (407±48), compared to smokers (573±60). Controls (800±67) had the highest total antioxidant power. This difference was statistically significant. Post-hoc assessment for one to one comparison confirmed a statistically significant difference between smokers vs. chewers, smokers vs. controls and chewers vs. controls in terms of total antioxidant power, indicating that tobacco chewers had significant lower total antioxidant power compared to other groups [Table/Fig-2].

Salivary flow rate was lowest in tobacco chewers (1.43±0.70) compared to smokers (2.31±0.65). Controls had (3.09±0.48) the highest salivary flow rate. This difference was statistically significant. Post-hoc assessment for one to one comparison confirmed a statistically significant difference between smokers vs. chewers, smokers vs. controls and chewers vs. controls in terms of salivary flow rate [Table/Fig-3]. Salivary pH was lowest in tobacco chewers (6.34±0.25) compared to smokers (6.73±0.17). Controls had the highest pH (7.05±0.20). The difference was statistically significant. Post-hoc assessment for one to one comparison confirmed a statistically significant difference between smokers vs. chewers, smokers vs. controls and chewers vs. controls in terms of pH [Table/Fig-4]. This Indicates a significant lower salivary flow rate and pH among tobacco chewers compared to others.

Initially clinical examinations and health questionnaires were used to identify patients at risk of developing diseases. Haematologic, serologic and imaging diagnostic methods were used to assess these patients further. In recent years, saliva-based diagnostic tests have been increased because of their non-invasive nature. Technologies are available that use saliva to diagnose, follow and assess the risk and severity of diseases [13]. Given the importance of saliva and its defence system, and due to the limited studies in this field, the present study was aimed to assess the effect of tobacco chewing and smoking on the salivary total antioxidant power.

In the present study, total antioxidant power of the saliva was measured by using FRAP assay. FRAP reagent was prepared by mixing 25 mL acetate buffer, 2.5 mL Ferric TPTZ solution, and 2.5 mL FeCl₃.6H₂O solution. It is a novel method for assessing “antioxidant power.” Ferric to ferrous ion reduction at low pH causes a coloured ferrous-tripyridyltriazine complex. FRAP values are obtained by comparing the absorbance change at 593 nm in test reaction mixtures with those containing ferrous ions in known concentration [8].

Age influences the total antioxidants status of saliva. As elderly people have significantly reduced total salivary antioxidant capacity [14]. We included this particular age group in the current study. Subjects with dental caries were excluded in this study, as the total antioxidant capacity in saliva of children with early childhood caries will be significantly greater than in the group without caries [15]. Subjects with extensive periodontitis and chronic alcoholics were also excluded in this study, as periodontal disease is associated with reduced salivary antioxidant status [16] and the alcohol-dependent subjects showed significantly lower total antioxidant capacity in saliva compared to the controls [17].

The most important and interesting finding of the present study was that salivary total antioxidant power of smokers and tobacco chewers was decreased compared to healthy controls. The findings of the current study were similar to the findings of the other studies. Study conducted by Falsafi P et al., showed that the levels of antioxidants in smokers were lower than non-smokers [18]. Study conducted by Greabu M et al., showed that total antioxidant capacity of saliva was lower in smokers than non-smokers [19]. The study conducted by Shetty AV et al., showed that smokers with and without caries showed decrease in total antioxidant level compared to the non smokers with and without caries [20]. Patel T et al., observed that oral submucous fibrosis patients consuming tobacco quid showed low levels of antioxidant enzymes than the healthy controls [1].

Free radicals are very unstable and react quickly with other compounds. Antioxidants can neutralize free radicals by donating their electrons [9]. A comprehensive mechanism is suggested for the initiation of oral cancer by cigarette smoking, saliva loses its antioxidant capacity and becomes a potent pro-oxidant in the presence of cigarette smoke. This mechanism is based on the well-known observation that oral cancer mostly occurs in oral epithelial cells exposed to tobacco products [3].

In present study, salivary flow rate and pH were lowest in the tobacco chewers and smokers compared to controls, which was similar to study conducted by Kanwar A et al. They showed that salivary flow rate decreases appreciably among tobacco users especially more among smokeless form. A lower salivary pH was observed in tobacco users than in controls. Decrease in salivary flow rate in smokers and chewers are probably due to the effect of nicotine on the taste nerve apparatus. Decrease in pH may be due to the alteration in electrolytes and ions as they interact with the buffering systems of saliva [21].

Saliva is the first body fluid which encounters the cigarette smoking and tobacco chewing. Antioxidant system of saliva plays a significant role in the anticariogenic and antibacterial effect of saliva. Results of the present study indicated that oxidant-antioxidant balance of saliva is degraded in favour of free radicals. Degradation of this oxidant-antioxidant balance possibly will contribute to worsening of oral hygiene and oral cancer development in smokers and tobacco chewers.

Main limitation of present study was that smoking status was recorded by the self-reporting of subjects. However, estimation of nicotine intake by serum cotinine assay would give more reliable information regarding tobacco consumption. As salivary antioxidants in smokers and chewers were significantly decreased, it is recommended to incorporate antioxidants in food supplements, tooth pastes and mouth rinses in order to prevent the harmful effects of tobacco.

Tobacco chewers and smokers had a lower salivary total antioxidant power compared to controls. This evidence of the decreased salivary antioxidants in the tobacco chewers and smokers emphasises the role of smoking and tobacco chewing in the pathogenesis of oral potentially malignant disorders and cancers.