|Year : 2013 | Volume
| Issue : 2 | Page : 210-214
Study of antioxidant enzymes superoxide dismutase and glutathione peroxidase levels in tobacco chewers and smokers: A pilot study
Chundru Venkata Naga Sirisha1, Ram M Manohar2
1 Department of Oral and Maxillofacial Pathology, C.K.S.Theja Institute of Dental Sciences and Research, Tirupathi, Andhra Pradesh, India
2 Department of Oral and Maxillofacial Pathology, Educare Institute of Dental Sciences, Malappuram, Kerala, India
|Date of Web Publication||13-Jun-2013|
Chundru Venkata Naga Sirisha
Department of Oral and Maxillofacial Pathology, C.K.S Theja institute of Dental Sciences and Research, Chadalawada nagar, Renigunta Road, Tirupathi
Source of Support: Partly funded by Colgate –Palmolive, (INDIA) Ltd.,
Mumbai,, Conflict of Interest: None
Context: Free radical associated damages play a major role in causation of cancer in tobacco habituates. The free radicals released by tobacco bring about alterations in antioxidant levels in humans and these free radical associated damages are reflected through antioxidant enzyme activities in blood.
Aims: To evaluate the effects of tobacco consumption on the erythrocyte Antioxidant enzymes-Superoxide dismutase (SOD) and Glutathione Peroxidase (GPx) as they act as first line of defense antioxidants.
Materials and Methods: A case control study comprising of 4 study groups of healthy controls (n = 27), smokers (n = 27), tobacco chewers (n = 30) and combination habit (n = 22) were included. Erythrocyte SOD and GPx enzyme activities were measured by spectrophotometry. The results were statistically analyzed using one way-Anova and Mann Whitney test.
Results: The data analysis revealed an alteration in mean SOD levels as it was decreased in cases compared to control group where as mean GPx was seen to be increased in cases compared to controls. When SOD and GPx were compared for the frequency and duration of habit, GPx showed a significant decrease in chewers with increase in frequency and duration of habit.
Conclusions: The present study gave us an insight about the relationship between antioxidant enzyme activity, oxidative stress and tobacco. The altered antioxidant enzyme levels observed in this study will act as a predictor for pre potentially malignant lesions. Therefore an early intervention of tobacco habit and its related oxidative stress would prevent the development of tobacco induced lesions.
Keywords: Anti-oxidant enzymes, Glutathione Peroxidase (GPx), Oxidative stress, Superoxide Dismutase (SOD), tobacco.
|How to cite this article:|
Naga Sirisha CV, Manohar RM. Study of antioxidant enzymes superoxide dismutase and glutathione peroxidase levels in tobacco chewers and smokers: A pilot study. J Can Res Ther 2013;9:210-4
|How to cite this URL:|
Naga Sirisha CV, Manohar RM. Study of antioxidant enzymes superoxide dismutase and glutathione peroxidase levels in tobacco chewers and smokers: A pilot study. J Can Res Ther [serial online] 2013 [cited 2020 Apr 9];9:210-4. Available from: http://www.cancerjournal.net/text.asp?2013/9/2/210/113352
| > Introduction|| |
Smoking and chewing of tobacco is a vice that has been practiced by millions of people all over the world. It is estimated that among 400 million individuals aged 15 years and above in India, 47% use tobacco in one or the other form. Of the 250 million kilograms of tobacco cleared for domestic consumption in India, 86% is used for smoking and 14% is used in the smokeless form (1% as snuff). 
Tobacco use in various forms is considered as the major etiological factor for oral cancer development, accounting for 30-40% of all cancer cases in India. Tobacco consumption generates free radicals and results in increased oxidative stress and lipid peroxidation.  Lipid peroxidation is a chain reaction providing a continuous supply of free radicals that initiate further peroxidation unless checked by antioxidants.
In normal cells there is an intricate balance between pro-oxidant and antioxidant states but in oxidative stress this balance shifts towards pro-oxidants.  The imbalance between pro-oxidants and antioxidants linked to decreased smoke related antioxidant capacity and increased free radical generation especially in arterial tissue might render smokers more prone to peroxidative stress. 
The heat (generated during smoking) as well as pH (change during chewing) of body fluids due to tobacco consumption affects the formation and stabilization of free radicals. The alkaline conditions observed in betel nut chewing are reported to favor the formation of free radicals.  Reactive oxygen metabolites (ROMs) such as superoxide anion (O 2• ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH• ), malondialdehyde (MDA) and nitric oxide (NO) are directly involved in multi stage process of carcinogenesis by bringing out a continuous endogenous damage to cellular DNA. 
Inactivation and removal of these reactive oxygen species depend on reactions involving the antioxidative defense system. Antioxidants have a shielding role by scavenging the free radicals. The free radicals released by tobacco are known to bring about alterations in antioxidant levels in humans, and these free radical-associated damages are reflected through antioxidant enzyme activities in blood. Thus, it seems that studying biological parameters like antioxidant enzyme system would be of fundamental importance in evaluating the role of tobacco on antioxidant status in tobacco users.
Since the first line defense antioxidants are SOD, GPx and Catalase (CAT), the present study was undertaken to establish the effects of tobacco chewing and smoking on the antioxidant enzymes SOD and GPx levels.
The objectives of this study were as follows:
- To study the levels of antioxidant enzymes SOD and GPx in erythrocytes of healthy individuals with the habit of tobacco usage either in the form of smoking, chewing or both (combination habit)
- To compare these levels with healthy controls without any tobacco habit
| > Materials and Methods|| |
The study was carried out in 106 age matched healthy male subjects enrolled from College of Dental Sciences, Bapuji Dental College and Hospital, Davangere.
The study included 30 healthy tobacco chewers, 27 smokers, and 22 subjects with combination habit. For comparison purpose we selected 27 age matched healthy males without any tobacco habits. The age group of patients ranged from 19 years to 68 years. They were divided into age matched groups for age wise comparison.
Ethical committee clearance and prior informed consent of all the subjects was obtained before conducting the study.
Cases and controls were grouped into 4 groups as follows:
GROUP I - Chewers
GROUP II - Smokers
GROUP III - Combination habit
GROUP IV - Controls
All subjects were personally interviewed through a questionnaire. It included details of tobacco consumption (duration, type & frequency), dietary supplements such as vitamins, drug intake, alcohol, medical history etc. Though the Inclusion criteria was duration of habit not less than 1 year, only one out of the 79 cases enrolled had one year of habit duration and rest had minimum of 3 years and maximum of 45 years of habit duration. Group III subjects interestingly had one of the habit for longer duration compared to the other habit. Exclusion criteria were Alcohol, infectious diseases, systemic diseases such as Diabetes mellitus, Hypertension which may also influence the antioxidant status. Only healthy subjects with strong history of tobacco habit and devoid of any tobacco related lesions were included to see if habit alone could have caused an alteration of antioxidant enzyme levels long before the onset of any tobacco related lesions as these findings can have different impact on the outcome of the study.
| > Blood Samples|| |
About 2 ml of venous blood was collected from all study subjects during mornings between 9-11 am so as to see that the time of blood collection was similar for all the subjects using heparin as anticoagulant. 0.5 ml of whole blood was centrifuged for 10 minutes at 3000 rpm and plasma was separated. Erythrocytes were then washed 4 times with 3 ml of 0.9% NaCl solution centrifuging for 10 minutes at 3000 rpm after each wash. The washed and centrifuged erythrocytes were then made up to 2 ml with cold re-distilled water, mixed and left to stand at +4 0 c for 15 minutes. The lysate was diluted with 0.01mol/L Phosphate buffer pH 7.0, So that percentage inhibition falls between 30% and 60%.
SOD estimation was based on the method of McCord and Fridovich  where as GPx was estimated by the method of Paglia and Valentine.  Enzymes SOD and GPx were determined using Randox kits - RANSOD and RANSEL respectively.
SOD was analyzed on XL-600 Auto analyzer and GPx on Erba-Chem-Pro semi Auto analyzer for spectrophotometry.
Finally, the results obtained were carefully recorded and statistically analyzed by using one way ANOVA and Mann-Whitney test.
| > Results|| |
Sample size and age wise distribution of cases and controls is illustrated in [Table 1].
When one way Anova test was used, no statistically significant difference for age was found between all the 4 groups. The values of mean with standard deviation, median and range of SOD and GPx were calculated for all the 4 groups and are given in [Table 2].
When SOD activity was compared between cases and controls, mean SOD value showed a trend of gradual depletion in cases compared to control group, though the depletion was statistically non-significant [Figure 1]. GPx showed an inverse pattern where mean GPx was lower in controls compared to cases. The increase in GPx in cases was statistically insignificant [Figure 2].
Mean SOD and GPx were compared among cases for frequency of the habit [Table 3]. On application of Mann-Whitney test for pair wise comparison between the groups, a significant difference (P < 0.05) was found for GPx with increase in frequency of habit for chewers and smokers.
Mean SOD and GPx were compared among cases for the duration of tobacco habit [Table 4]. Mean GPx has significantly decreased (P < 0.05) with increase in duration of habit for chewers while it has slightly decreased in smokers and combination users.
Relationship between age and SOD, GPx was shown in [Table 5].
Pearson's correlation coefficient showed positive correlation for age for SOD among all the 4 groups.
Positive correlation with age was noted for GPx among controls and negative correlation among chewers, smokers and combination habit and was statistically significant among chewers and smokers (P < 0.05).
| > Discussion|| |
Free radical associated damages leads to an imbalance between pro-oxidant and anti-oxidant states. This imbalance plays an important causative role in carcinogenesis.  Anti-oxidants have a shielding role by scavenging the free radicals and SOD, GPx form the first line defense anti-oxidants. 
As free radical associated damages are reflected through anti-oxidant enzyme activities in blood, the present study was undertaken to establish the effects of tobacco on the above anti-oxidant enzymes.
Mean SOD was found to be decreased in cases compared to controls. Cases are more prone to oxidative stress due to increasing load of free radicals in body due to chemical carcinogens present in tobacco.
The slight decrease in SOD observed in cases compared to controls in our study probably relates to its utilization in scavenging the free radicals as SOD catalyses the dismutation of Superoxide O 2• to Hydrogen peroxide (H 2 O 2 ), which then must be removed by GPx or CAT
Our study was similar to that of Zhou J  who found significantly low levels of erythrocyte SOD in smokers compared to non-smokers.
Mean GPx was found to be slightly increased in cases compared to controls as it acts after SOD and this increase could be co-related to an adaptive phenomenon when free radical generation exceeds the quenching capacity of SOD.
Our study was similar to that of Beena P Patil  and Yildiz  who showed that erythrocyte SOD was lowered and GPx was elevated in tobacco users compared to non tobacco users.
Our study showed that an increase in duration and frequency of tobacco habit significantly reduced GPx levels in chewers emphasizing that frequency and duration of tobacco habit has a definitive impact on antioxidant enzyme status.
Though the increase in frequency appeared to have caused increase in GPx levels among smokers, the same showed a decrease with increased duration of habit.
The present study results were comparable to that of Anderson HR et al. where in GPx negatively co-relates with tobacco consumption for increase in frequency and duration of habit probably indicating a decrease in GPx levels on prolonged duration of tobacco usage.
| > Conclusion|| |
The present study enlightens the possible relationship between anti-oxidant enzyme levels, oxidative stress and tobacco habit. The results of this pilot study can be validated with a larger sample size, there by opening up avenues for using these altered antioxidant enzyme levels as a predictor for pre potentially malignant lesions. Tobacco habit coupled with the lesion would probably have synergistic effect in lowering the bodies antioxidant status paving way for an enhanced oxidative stress. But further studies are required in this direction before we can come to a conclusion.
An early intervention of tobacco habit and its related oxidative stress is recommended.
| > References|| |
|1.||Mehta FS, Hamner JE. Tobacco-related oral mucosal lesions and conditions in India - A guide for dental students, dentists and physicians. Bombay: Basic dental research institute, Tata Institute of fundamental research; 1993. |
|2.||Patel BP, Rawal UM, Shah PM, Prajapati JA, Rawal RM, Dave TK, et al. Study of tobacco habits and alterations in enzymatic antioxidant system in oral cancer. Oncology 2005;68:511-9. |
|3.||Uikey AK, Hazarey VK, Vaidhya SM. Estimation of serum antioxidant enzymes Superoxide dismutase and Glutathione peroxidase in Oral submucous fibrosis: A biochemical study. J Oral Maxillofac Pathol 2003;7:44-5. |
|4.||Marangon K, Herbeth B, Lecomte E, Paul-Dauphin A, Grolier P, Chancerelle Y, et al. Diet, antioxidant status, and smoking habits in French men. Am J Clin Nutr 1998;67:231-9. |
|5.||McCord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (Hemocuprein). J Biol Chem 1969;244:6049-55 |
|6.||Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158-69. |
|7.||Beevi SS, Rasheed AM, Geetha A. Evaluation of oxidative stress and nitric oxide levels in patients with oral cavity cancer. Jpn J Clin Oncol 2004;34:379-85. |
|8.||Ray G, Husain SA. Oxidants, antioxidants and carcinogenesis. Indian J Exp Biol 2002;40:1213-32. |
|9.||Zhou J, Guo F, Qian Z. Effects of cigarette smoking on antioxidant vitamin and activities of antioxidases. Zhonghua Yu Fang Yi Xue Za Zhi 1997;31:67-70. |
|10.||Yildiz L, Kayaoglu N, Aksoy H. The changes of Superoxide dismutase, Catalase and Glutathione peroxidase activities in erythrocytes of active and passive smokers. Clin Chem Lab Med 2002;40:612-5. |
|11.||Andersen HR, Jesper BN, Flemming N, Philippe G. Antioxidative enzyme activities in human erythrocytes. Clin Chem 1997;43:562-8. |
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]