Journal of Dr. NTR University of Health Sciences

: 2021  |  Volume : 10  |  Issue : 1  |  Page : 27--32

Estimation of salivary superoxide dismutase, glutathione peroxidase, catalase individuals with and without tobacco habits

Afroz K Syed, Divya Sri Godavarthy, Kattappagari K Kumar, Chandra Sekhar Poosarla, Gontu S Reddy, Baddam V R. Reddy 
 Department of Oral Pathology and Microbiology, SIBAR Institute of Dental Sciences, Takkellapadu, Guntur District, Andhra Pradesh, India

Correspondence Address:
Dr. Afroz K Syed
Door No. 22-8-18, Kollavari Street, Kothapet, Tenali, Andhra Pradesh - 522 201


Context: Tobacco usage is associated with the derailment of antioxidant status. Salivary studies for diagnostic potential as a biofluid. Saliva may provide acumen into disease pathogenesis. Aims: To determine the influence of tobacco on salivary superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) enzyme levels in individuals with and without tobacco habits. Settings and Design: One mL of unstimulated whole saliva was collected, centrifuged immediately at 2000 RPM, and stored at 4°C for analysis. The supernatant was aspirated and assayed. Methods and Material: Eighty males subjects aged 25–40 years were selected and included 20 controls, 20 smokers, 20 chewers, and 20 with both habits. The saliva samples were collected and SOD, GPX, CAT levels were analyzed using UV spectrophotometric assay. Statistical Analysis Used: Mann-Whitney U test was used for comparison taking the probability value of P ≤ 0.05 as statistically significant using SPSS 20.0 version. Results: SOD, CAT, GPX enzyme levels in the saliva were significantly lower in those with tobacco habit than in the controls (P < 0.05). A significant correlation existed between SOD, CAT, GPX levels, and the type of the habit. The antioxidant status is affected by the impact of tobacco. Conclusions: This study emphasizes the importance of saliva as an easy noninvasive tool in diagnosing patients who are more prone to precancerous lesions and conditions, and its importance in patient education and motivation programs for tobacco habit cessation.

How to cite this article:
Syed AK, Godavarthy DS, Kumar KK, Poosarla CS, Reddy GS, Reddy BV. Estimation of salivary superoxide dismutase, glutathione peroxidase, catalase individuals with and without tobacco habits.J NTR Univ Health Sci 2021;10:27-32

How to cite this URL:
Syed AK, Godavarthy DS, Kumar KK, Poosarla CS, Reddy GS, Reddy BV. Estimation of salivary superoxide dismutase, glutathione peroxidase, catalase individuals with and without tobacco habits. J NTR Univ Health Sci [serial online] 2021 [cited 2021 Jun 23 ];10:27-32
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Salivary studies are being done extensively to elucidate its diagnostic potential as a biofluid for local and systemic diseases. Saliva offers a preferable substitute to blood because its collection is safe for both the donor and collector, noninvasive, and low in cost.[1] Molecules from serum enter saliva through capillaries by passive diffusion, the secretory cells by active transport, and between acinar and ductal cells by ultrafiltration through spaces.[2],[3] Therefore, within the present study, the impact of tobacco on the secretion of salivary superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) levels were measured and compared thereupon of the nonusers of tobacco.

 Materials and Methods

The present study was carried out in the Department of Oral Pathology and Microbiology, SIBAR Institute of Dental Sciences, Guntur, Andhra Pradesh. Ethical clearance for the study was obtained. Eighty Male participants of the age range of twenty-five to forty years. After obtaining informed consent, unstimulated saliva was collected. Among these participants, twenty each were nonsmokers- group I, smokers- group II, tobacco chewers- group III, both tobacco chewers and smokers- group IV.

Collection of saliva

The selected participants were asked not to eat or drink 2 h before saliva collection. The smokers and tobacco chewers were also prohibited from smoking for 1 h before sample collection. The sampling was performed in a quiet room between 9 am and 2 pm to prevent any variations which may be attributable to the circadian rhythm. The participants were instructed to rinse the mouth using distilled water. The unstimulated whole saliva was collected for at least 5 min in a container and kept at low temperature. Following saliva collection, the samples were centrifuged to remove cell debris for 5 min at 2000 to 2500 RPM. The supernatant was stored at 4°C until tests were performed.

Determination of antioxidant enzymes

The levels of Salivary SOD, catalase, and GPX were measured using standard procedures. SOD activity in saliva was measured by using nitro blue tetrazolium as a substrate after suitable dilution as per the method suggested by Menami and Yoshikawa (1979).[4] Salivary catalase activity was determined according to the method of Gregory EM, Fridovich (1974).[5] GPX activity was ascertained by the method of Paglia and Valentine (1967). The activity was determined spectrophotometrically. Data entry, database management, and all statistical analysis were performed using the SPSS 20.0 version. The mean values were analyzed using the Mann-Whitney U test. The probability value of P ≤ 0.05 was set as statistically significant.


The mean age of Group I, II, III, IV were 20.60, 19.45, 28.45, 29.25 years, respectively. In Group II, 95% number of subjects had a habit of smoking cigarettes. In Group III, 75% number of subjects had a habit of chewing khaini. Group IV had a habit of cigarette and Khaini in 60% of subjects. In group II, the enzyme catalase was highest for subjects with 4 years of habit [Graph 1], GPX for 2 years of habit [Graph 2], SOD for 4 years of habit [Graph 3]. In group III, the enzyme catalase was highest for subjects with 1 year of habit, GPX for 3 years of habit, SOD for 4 years of habit. In group IV, the enzyme catalase was highest for subjects with 2 years of habit, GPX for 2 years of habit, SOD for 1 year of habit. Regarding the duration of the habit, there was no significant change in the enzyme levels of SOD, catalase, and GPX in all the habit groups. The SOD, GPX, catalase levels were lower in the groups with tobacco habit than without tobacco habit. The SOD, GPX, catalase were least in group IV than groups II and III indicating a synergistic effect [Table 1], [Table 2], [Table 3].[INLINE:1][INLINE:2][INLINE:3]{Table 1}{Table 2}{Table 3}


Tobacco use is known as one of the most important risk factors for oral diseases.[6],[7],[8] When a cell with DNA damage divides, metabolism and duplication of cells become deranged and mutations can arise, which is an important factor in carcinogenesis.[9],[10] It is noted that cigarette smokers have risks of oral cancer 2 to 5 times that of nonsmokers.[11],[12] Cigarette smoke is an important exogenous source of free radicals as well as nonradical oxidants. Oral squamous cell carcinoma is 4–7 times more prevalent in cigarette smokers.[13] The human body has a variety of antioxidant defense mechanisms (nonenzymatic and enzymatic antioxidants) to eliminate reactive oxygen species and prevent their harmful consequences on the host.[14],[15] Peroxide–antioxidant imbalance leads to oxidative stress, potentially damaging DNA, proteins, lipids, and cells. Smoking also correlates with oxidative stress and DNA damage, causing cancers and other diseases. All the body fluids and tissues have antioxidants that stabilize or deactivate and protect against free radicals.[16] Saliva is the first biological fluid to come into contact with external materials, foods, drinks, and inhaled cigarette smoke. Two types of antioxidants exist in the body and saliva: enzymatic antioxidants such as catalase, SOD, peroxidase, and GPX, and nonenzymatic antioxidants or diet supplements and small molecules such as vitamin C, vitamin E, and uric acid.[17],[18] Therefore, its antioxidative system has an essential role in the anticancerous capacity of the saliva. This antioxidative system consists of different molecules and enzymes such as uric acid, SOD, catalase, and peroxidase system.[19],[20] However, very limited studies have evaluated saliva. Recently, it has been shown that the imbalances in free radical levels and reactive oxygen species with antioxidants may play a key role in the onset and development of several inflammatory oral pathologies.[15],[21] This evidence emphasizes the role of cigarette smoking on salivary antioxidants in the pathogenesis of oral cancers. The most critical intracellular enzymes which protect cells and tissues from the oxygen-derived free radicals are SOD, which rapidly causes dismutation of the superoxide anion to hydrogen peroxide, GPX, which reduces hydrogen peroxide (a precursor of more potent radical species) and/or lipid hydrogen peroxides by the oxidation of reduced glutathione or S-nitrosoglutathione, and catalase (CAT), which scavenges hydrogen peroxide by converting it to oxygen and water.

In an attempt to explain the role of antioxidants in various pathologies, Panjamurthy et al. reported that the enzymatic antioxidant activity (SOD, CAT, and GPX) in plasma, erythrocytes, erythrocyte membrane, and gingival tissue of periodontitis patients was significantly higher in the chronic periodontitis group relative to parameters found in healthy patients. A positive correlation between serum and salivary SOD levels in the ischemic heart disease group and the healthy group is in accordance with the study by Al-Rawietal where the salivary SOD followed serum SOD. In the present study, we attempted to estimate and compare the levels of SOD, GPX, CAT enzyme levels among individuals with a tobacco habit. In the present study, the mean levels of all the three enzymes SOD, GPX, and CAT were significantly lower in the saliva of smokers than controls. The present study is consistent with the results of Agnihotri et al. showed that smoking decreases the activity of salivary SOD than nonsmokers.[22] Kelin et al. reported that the activity of salivary SOD decreases up to 70% in smokers. Abdolsamadi et al. showed that the activities of salivary SOD, GPX, and peroxidase enzymes in smokers were significantly compared to those in nonsmokers.[23],[24] In addition, Reznick et al. reported that even after smoking one cigarette there is a rapid decrease in the activity of salivary peroxidase in both smokers and nonsmokers.[25] In the study of Huseyin Kurku et al., they assessed the changes in the total antioxidant capacity of saliva among both acute and chronic smokers and concluded that both acute and chronic habit of smoking increased oxidative conditions may be a significant sign of the destructive effects of smoking.[26] However, in the study by Kanehira et al., the salivary levels of superoxide dismutase were higher in smokers than nonsmokers. Despite increased H2O2 level as a defense system induced by SOD, detoxification of H2O2 might be deteriorated in the oral cavity of smokers.[27] The present observation is in contradiction to the study of Baharvand et al. who demonstrated that the activity of superoxidase as a component of the antioxidative system of the body was higher in smokers compared to nonsmokers. It appears that such an increase can decrease the production of free radicals in smokers.[28] However, there was no significant change in the level of the SOD, GPX, and CAT with the duration of the habits. This observation is in accordance with the study of Masoomeh Shirzaiy. In their study, they showed that there was no statistically significant association between salivary Total antioxidant capacity (TAoC) and the duration of the smoking habit.[29] Hammo Mahmoud et al. measured the plasma level of TAoC in smokers' antecubital venous blood using a similar test to that of the present study and showed a significant reduction in total antioxidant status in smokers.[30] GPX is a selenium-containing peroxidase and shares substrate with catalase. It alone can react effectively with lipid and other organic hydroperoxides. Glutathione peroxidase provides protection against low levels of oxidant stress, whereas catalase becomes more significant in defending against severe oxidant stress.[19],[31],[32],[33] The present study is consistent with the study of, Guica et al., which showed a significant decrease in glutathione peroxide levels in smokers; while SOD levels did not significantly increase in smokers and nonsmokers. In another study by Greabu et al., they revealed a significant decrease in salivary levels of GPX in smokers.[34],[35] Saggu et al. analyzed unstimulated saliva of 100 smokers by measuring the activity of salivary SOD and GPX and showed them a significantly higher mean value of SOD activity in the smokers' group, while the levels of GPX activity were significantly higher in the nonsmoking group. Many studies suggested these measurements could be helpful for determining the level of oxidative stress caused by cigarette smoke and may help in patient's education regarding the harmful effects of smoking and estimating the evolution of various oral diseases.[36],[37],[38] On the other hand, Shetty et al., reported a higher activity of salivary SOD in patients with leukoplakia and oral cancer compared to the controls that can be due to the fact that the majority of participants with leukoplakia and oral cancers were smokers suggesting SOD level as a biomarker for oral cancers indicating that subjects with tobacco smoking habit are more prone to oxidative stress due to increasing load of free radicals in the body due to chemical carcinogens present in tobacco.[39] Many other studies also liked the use of saliva in the development of cancer.[33],[40],[41],[42] In the present study, salivary GPX, SOD, and catalase levels were decreased significantly in the saliva of chewers than controls. In the present study, 75% had a habit of chewing khaini, 15% with paan masala, and 10% with gutka. Maximum subjects in the present study had the habit for a duration of 1 year. However, when the duration of the habit was compared for the enzymes CAT, GPX, and SOD there was no significant change obtained in the present study. The present study is consistent with the study of Shwetha et al. In their study, they evaluated the effect of tobacco chewing and smoking on salivary flow rate, pH, and salivary total antioxidant power. They concluded that the total antioxidant levels were lower in tobacco chewers and that the decreased salivary antioxidants in the tobacco chewers emphasize the role of smoking and tobacco chewing in the pathogenesis of oral cancers.[43] In another study by Tarboush et al., they concluded that chronic tobacco chewing is associated with reduced levels of total antioxidant capacity (TAC) and CAT among a population of adult men in comparison with nontobacco -chewing controls. These findings suggest that the pro-oxidative effect of tobacco chewing may be a contributing mechanism for various oral diseases including cancer.[44] In a study by Begum et al. the levels of malondialdehyde (MDA) and nitric oxide (NO2 and NO3) and the antioxidant enzyme activities were assessed. They found out that the activity levels of antioxidant enzymes were decreased in the saliva of the smokeless tobacco users in comparison with normal controls. In the present study, 60% had a habit of cigarette + khaini, 10% with cigarette + paan masala, and 30% with cigarette + ghutka. A maximum number of subjects had the habit for 4 years. In our study, SOD, GPX, CAT was significantly lower in the saliva of chewers than controls. This is in accordance with studies of Arbabi-Kalati who concluded from his study that the use of tobacco products decreases the antioxidative activity of the saliva.[45] The levels of SOD, GPX, CAT, when compared between the groups, were least among group IV, than other groups signifying a synergistic effect. This observation was consistent with the study of C Wen et al. who proposed that to a large extent, the serious health consequences suffered by betel quid chewers were the result of the combined effects of smoking and chewing.[46] Also in the study by Tsai KY et al., they proposed that betel quid chewing and cigarette smoking patients are more likely to be diagnosed with oral cavity cancer at a younger age than those who have just one habit or none.[47] The GPX, CAT levels (though not significantly) were lesser in smokers than chewers. But the SOD levels were lesser in chewers than smokers. However, when the duration of the habit was compared for the enzymes CAT, GPX, SOD, there was no significant change obtained in the present study. Saliva is abundant and sampling is much easier, less costly, and better tolerated by a patient.


The present study enlightens the possible relationship between antioxidant enzyme levels, oxidative stress, and tobacco habit. The decrease in the antioxidant levels of salivary SOD, catalase, GPX depicts the oxidant burden faced by the system due to the usage of tobacco. Also, the synergistic effect of smoking and chewing was observed in the individuals with both habits as noted by the lower enzyme levels. These biochemical changes occurring in blood during the disease process occur simultaneously in saliva. Human saliva is an increasingly attractive medium for biomarker discovery on account of its amenability to noninvasive and repeated sampling, ease of collection, and processing. Hence, as a future perspective, saliva can be considered as a noninvasive diagnostic fluid over blood analysis in disease diagnosis.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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