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Comparative study on the antioxidants levels in smokers and non-smokers with chronic periodontitis
i n d i a n j o u r n a l o f d e n t i s t r y 4 ( 2 0 1 3 ) 6 7 e7 1
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ijd
Original Article
Comparative study on the antioxidants levels in smokers and non-smokers with chronic periodontitis M.K. Reejamol a,*, Mythilli Swaminathan b a
Senior Lecturer, Department of Periodontics, PMS College of Dental Science and Research, Vattappara, Trivandrum, Kerala, India Prof & Head, Department of Periodontics, Rajah Muthiah Dental College & Hospital, Annamalai University, Chidambaram 608002, Tamil Nadu, India b
article info
abstract
Article history:
Aim: The aim of the study is to evaluate the effect of cigarette smoking on periodontal
Received 21 October 2012
damage in terms of free radicals and antioxidants.
Accepted 31 December 2012
Materials and methods: Fifteen male patients (non-smokers) with chronic periodontitis (age 25e55) were selected as control & 15 male patients (smokers) with chronic periodontitis
Keywords:
(age 25e55) were selected as experimental group; which is further subdivided as three
Antioxidants
groups based on the number of cigarettes smoked per day (group I: <10 cigarettes/day,
Free radicals
group II: 10e30 cigarettes/day, group III: >30 cigarettes/day). Gingival tissue samples were
Periodontitis
collected from test and control subjects during surgery and were subjected for the esti-
Smoking
mation of lipid peroxide, reduced Glutathione, Superoxide dismutase, Catalase and total Thiol. Results: The levels of lipid peroxide (TBARS), Catalase and total Thiol in gingival tissues showed maximum levels in smokers than non-smokers whereas Superoxide dismutase and reduced Glutathione (GSH) showed elevated levels in non-smokers than smokers. Among the smokers group, group III exhibited significantly higher values of lipid peroxide (TBARS), Catalase and total Thiol followed by group II & I; whereas, the levels of Superoxide dismutase and reduced Glutathione (GSH) showed reduction in such a way that group I exhibited higher levels followed by group II and III. Conclusion: Based on the results of present investigation it is concluded that smoking increases the level of free radicals in periodontal tissues, which in turn may augment the level of periodontal destruction. ª 2013 Indian Journal of Dentistry. All rights reserved.
1.
Introduction
Periodontal diseases are inflammatory disorders that give rise to tissue damage and loss, as a result of complex interactions
between pathogenic bacteria and host immune response.1 The tissue destructive mechanism involves the release of proteolytic enzymes and free oxygen radicals from activated neutrophil.2 The most important reactive oxygen species
* Corresponding author. E-mail address: [email protected] (M.K. Reejamol). 0975-962X/$ e see front matter ª 2013 Indian Journal of Dentistry. All rights reserved. http://dx.doi.org/10.1016/j.ijd.2012.12.009
68
i n d i a n j o u r n a l o f d e n t i s t r y 4 ( 2 0 1 3 ) 6 7 e7 1
implicated in inflammatory injuries to tissues are the hydroxyl radical (OH), the superoxide anion (O 2 ), the nitrous oxide radical (NO), hypochlorous acid, hydrogen peroxide and singlet oxygen.3 Antioxidants are substances which when present at low concentration when compared to an oxidizable substrate will significantly delay or inhibit the oxidation of that substrate.4In normal physiology, there is a dynamic equilibrium between reactive oxygen species (ROS) activity and antioxidant defence capacity. Once that equilibrium shifts in favour of ROS, either by a reduction in antioxidant defences or an increase in ROS production or activity, it results in oxidative stress leading to potential damage.4 Unbalanced radical and non-radical reactive oxygen species will damage cells by variant mechanisms including peroxidation of lipid membranes, protein inactivation, introduction of DNA damage and cytokine induced tissue damage.5 Smoking exerts a major effect on protective elements of immune response, resulting in an increase in the extent and severity of periodontal destruction.6 Smoking may have an adverse effect on fibroblast function, chemotaxis and phagocytosis of neutrophils, immunoglobulin, production and induction of peripheral vasoconstriction.7 Exposure to nicotine results in increased secretion of Prostaglandin E2 (PGE2) by monocytes, elevated levels of tumour necrosis factor (TNF) and gingival crevicular fluid (GCF), elevated levels of neutrophil elastase and matrix metalloproteinase-8.6 A major exogenous source of free radicals is cigarette smoke which contains two phases of free radicals; one in gas phase and other in tar.8 The obligatory use of body reserve of antioxidants to detoxify the excess of free radicals in smokers results in alteration in level of different antioxidants. The antioxidant disturbance in smokers may be further enhanced by their lower intake of both supplemental and dietary antioxidants.8 Cigarette smoke contains free radicals and other oxidants in abundance. In one puff of a cigarette, the gas phase of smoke exposes 1015 free radicals and oxidants than in tar phase. The direct exposure to cigarette smoke represent only a portion of total oxidative stress and contributes to additional endogenous oxidant formation through effects on inflammatory immune response.9 In this backdrop, an attempt has been made in the present study to evaluate the relationship between smoking and the levels of antioxidants and free radicals in chronic periodontitis patients.
of 5 mm in at least 4 sites per quadrant) were selected and were categorized as smokers and non-smokers. The control group were non-smokers comprised of 15 patients with chronic periodontitis. Experimental group were smokers with chronic periodontitis and were subdivided into three sub groups based on the number of cigarette consumption per day. They were, Group-I: 5 subjects smoking less than 10 cigarettes/day. Group-II: 5 subjects smoking 10-30 cigarettes/day. Group-III: 5 subjects smoking more than 30 cigarettes/day.
2.2.
Gingival tissue was obtained during modified Widman Flap Surgery. The excised tissue was thoroughly washed with normal saline so to remove blood and then stored in vials containing 0.15 M potassium chloride.
2.3.
Materials and method
2.1.
Patient selection criteria
The study was conducted at Rajah Muthiah Dental College and Hospital (RMDCH), Annamalai University. Patients devoid of any systemic disease and had not received any periodontal treatment or medication for last 6 months were selected. Written informed consent was obtained from each included subject prior to commencement of the study. Thirty male patients of age group between 25 and 55 years with chronic periodontitis (with probing pocket depth and attachment loss
Biochemical analysis
The gingival tissues thus obtained were subjected for biochemical tests and the levels of lipid peroxidize, Superoxide dismutase (SOD), Catalase, total Thiol group and reduced Glutathione were assessed.
2.4.
Preparation of tissue homogenate
The gingival sample was weighed and a minimum of 400 mg of tissue sample was obtained. Tissue sample was then homogenized in cold 0.15 m KCl using a tissue homogenizer.
2.5.
Estimation of lipid peroxidation in tissue
Lipid peroxidation in tissue was estimated by the method of Ohkawa et al.10 A 0.2 ml sample of tissue homogenate was mixed with 0.2 ml of 8% aqueous sodium dodecyl sulphate, 1.0 ml of 20% acetic acid adjusted to pH of 4.0 using concentrated sodium hydroxide and 1.5 ml of 0.5% aqueous solution of Thio-barbituric acid (TBA). The mixture was incubated in a boiling water bath for 1 h and then centrifuged at 10,062 g for 15 min. The clear supernatant obtained after centrifugation was used for measuring the absorbance at 532 nm using a spectrophotometer.
2.6.
2.
Experimental design
Estimation of Superoxide dismutase (SOD)
Superoxide dismutase (SOD) activity was assayed by the method of Kakkar et al. (1984).11 0.5 ml of tissue homogenate was added with 0.5 ml of water, 2.5 ml of ethanol and 1.5 mol of chloroform. This mixture (5 ml) is shaked at 4 C and the centrifuge supernatant was taken for analysis. The SOD activity is expressed as nmol/mg protein for tissues.
2.7.
Estimation of Catalase
Catalase activity was estimated calorimetrically by the method of Sinha (1972).12 0.1 ml of tissue homogenate was added with 1 ml of phosphate buffer and 0.4 ml of hydrogen
69
i n d i a n j o u r n a l o f d e n t i s t r y 4 ( 2 0 1 3 ) 6 7 e7 1
peroxide. Incubated for 90 s and added 2 ml of dichromate acetic acid. The mixture was incubated in a boiling water bath for 10 min and then centrifuged at 10,062 g for 15 min. The clear supernatant obtained after centrifugation was used for measuring the absorbance at 620 nm using a spectrophotometer.
2.8.
Estimation of total thiol group
The total Thiol level was estimated in tissue by the method of Hu (1986).13 A 0.2 ml sample of 10% plasma tissue homogenate was mixed with water to make the volume upto 0.5 ml. Two millilitres of 0.3 M disodium hydrogen phosphate was added to each sample and 0.25 ml of 5, 50 -dithio-bis-2-nitrobenzoic acid (DTNB) reagent was added just before measuring the absorbance at 412 nm. Total thiol groups were calculated using an absorptivity of 13,600/cm/M.
2.9.
Estimation of reduced Glutathione
Reduced Glutathione was measured in tissue according to the method of Beutler and Kelley (1963).14 This method was based on development of yellow colour when DTNB reagent was added to compounds containing sulfhydryl groups.
2.10.
Statistical analysis
Levels of lipid peroxide (Thio-barbituric acid and reactive substances -TBARS), reduced Glutathione, Superoxide dismutase, Catalase and total Thiol in tissues of control and experimental groups were statistically treated with nonparametric regression analysis. The P values were crosschecked for unravelling the level of statistical significance in such a way that if p < 0.01 indicates a significant difference.
3.
Results
Mean and standard deviation of tissue lipid peroxide (TBARS) (nmol/mg protein), tissue Catalase (nmol/mg protein), tissue Superoxide dismutase (U/mg protein), tissue Glutathione (nmol/mg protein), tissue total Thiol (nmol/mg protein) in non-smokers and smokers are presented in Table 1. The results indicate that the tissue lipid peroxide level revealed an increase in smokers compared to non-smokers where maximum value is recorded in group III. When control is compared
with group I and II, P value is 0.000 (P < 0.01) which reveals a significant difference. The results revealed that the tissue Superoxide dismutase level exhibited a decreasing trend from control to experimental groups in such a way that control group recorded the maximum value. When comparing control with experimental groups I, II and III, P < 0.01 which proves that there is a significant difference. The results showed that the tissue catalase level exhibited an increasing trend from control to experimental groups in such a way that group III recorded the maximum value. When comparing control with experimental groups I, II and III, P < 0.01 which proved that there is a significant difference. The results revealed that the tissue Glutathione level proved a decreasing trend from control group to experimental group IeIII in such a way that group I recorded the maximum value. When control was compared with experimental groups I, II and III, P < 0.01 that underlines the fact that there exist a significant difference. The results revealed that the tissue total Thiol showed an increasing trend from control to experimental groups in such a way that group III recorded the maximum value. When control are compared with group I, P value is 0.098 (P < 0.01) which reveals no significant difference. In case of group II and III when compared with control, P value is 0.017 and 0.003 respectively which proved to be statistically significant.
4.
Discussion
The strongest evidence implicating ROS in periodontal destruction of the connective tissues during periodontal diseases arises in considering PMN infiltration as a key event of host response against bacterial invasion.15 The tissue concentrations of antioxidants or free radical scavengers may be either in the form of enzymes (eg. Superoxide dismutase (SOD), Catalase and Glutathione peroxide) or as low molecular weight free radical scavengers (eg. vitamin E, total thiol, Glutathione etc). When there is an excess of free radicals from exogenous or endogenous sources, the available tissue antioxidant system may become overwhelmed leading to oxidative damage to tissues.8 In smokers, the obligatory use of reserve antioxidants to detoxify the excess free radicals, results in alteration of the level of different antioxidants. The antioxidant disturbance in smokers may be further enhanced by lower intake of both
Table 1 e Mean and standard deviation of biochemical parameters between smokers (groups IeIII) and non-smokers. Parameters
Control
Experimental groups Group I
Lipid peroxide Superoxide dismutase (SOD) Catalase Total thiol group Reduced Glutathione
Group II
Group III
Mean
SD
Mean
SD
P value
Mean
SD
P value
Mean
SD
P value
1.520 99.6 1.4630 0.1790 70.160
0.457 1.198 0.373 0.052 2.046
4.140 79.840 2.4280 0.2500 87.920
0.462 4.731 0.2986 0.025 1.594
0.000 0.001 0.013 0.098 0.000
5.160 71.680 3.4420 0.2860 61.700
0.365 2.893 0.2099 0.019 1.654
0.000 0.000 0.002 0.017 0.002
6.360 63.360 5.1520 0.4420 56.400
0.467 5.264 0.1806 0.043 5.171
0.000 0.000 0.000 0.003 0.006
70
i n d i a n j o u r n a l o f d e n t i s t r y 4 ( 2 0 1 3 ) 6 7 e7 1
supplemental and dietary antioxidants.8 Studies emphasized elevated levels of lipid hydroperoxides in gingival tissues of patients with periodontitis.15,16 The present study exhibited an increase in lipid peroxide level, which is more significant in group III than group I and II, when compared to non-smokers because of increased number of cigarettes per day will influence an increase in the production of free radicals.17 In the present investigation, SOD level were found to be decreased in smokers than non-smokers which might be due to the inactivation by hydrogen peroxide. Among smokers, group III exhibited more reduction in SOD level when compared to group I and II which might be due to increased production of hydrogen peroxide from smoking. Similar results were obtained by Bray et al suggested that free radicals, particularly hydrogen peroxide are generated by the direct interaction between smoke and periodontal tissues that will lead to a significant change in the level of antioxidant enzyme SOD.18 The present study observed an increase in catalytic activity in smokers than non-smokers which might be caused by higher levels of hydrogen peroxide formation, which is evidenced in group III than other two groups. Similar trend was observed in the findings of Halliwell et al in which they observed that an initial rate of hydrogen peroxide removal is directly proportional to its concentration. They emphasized that the enzyme Catalase come into action only after an optimum concentration of hydrogen peroxide has been obtained; below which Catalase has no role to play.19 Works done on the status of antioxidants, mainly Glutathione peroxidase (GPx) and reduced Glutathione in saliva, GCF and serum of chronic periodontitis patients proved that GSH is a scavenger of hydroxyl radicals and singlet oxygen.20,15,21 It functions as a substrate for the hydrogen peroxide removing enzyme, Glutathione peroxidase (GPx). The present study revealed a reduction of GSH levels in smokers, among which group III exhibited least level. This might be due to more flux of hydrogen peroxide and hydroxyl radicals influenced by smoking. This is in accordance with the study done by Tribble et al in which they reported that exposure of tissues to a large flux of hydrogen peroxide and hydroxyl radicals might result in an imbalance of GSH and oxidized Glutathione (GSSG) ratio. They stated that GSSG accumulates in tissues and it paves way to inactivation of various enzymes which lead to lower levels of GSH in smokers.22 The observation done by Wayner et al postulated that among smokers, there might be some structural modifications in proteins because of oxidative activity, that results in the exposure of protein linked thiol groups so to react freely and thus depicts a significantly higher total thiol level.23 The present study is in favour of the above findings with elevated level of total thiol in smokers compared to non-smokers. An insignificant increase in total thiol in tissues of group I smokers (smoking < 10 cigarettes/day) might be due to increased free radical production but in amount that are scavenged by antioxidants, such that the damage is limited to few protein molecules. The present study suggests that free radical production induced by cigarette smoke is highly toxic and will impair the oxidant-antioxidant balance, which is a major risk factor in periodontal disease.
Studies pertain to this line can be undertaken by including factors such as past history and duration of smoking in smokers, which will help in unravelling the possible cumulative effects of those parameters on the production of free radicals leading to periodontal disease.
Conflicts of interest All authors have none to declare.
references
1. Battino M, Ferrero MS, Gallardo, Newman HN, Bullon P. The antioxidant capacity of saliva. J Clin Periodontol. 2002;29:189e194. 2. Gustafsson A, Asman B. Increased release of free oxygen radicals from peripheral neutrophils in adult periodontitis after FCR receptor stimulation. J Clin Periodontol. 1996;23:38e44. 3. Chappel ILC. Reactive oxygen species and antioxidants in inflammatory diseases. J Clin Periodontol. 1997;24:287e296. 4. Iain L, Chapple C, Mathews JB. The role of relative oxygen and antioxidant species in periodontal tissue destruction. Periodontol 2000. 2007;43:160e232. 5. Budueli N, Kardester, Wills CS, Scott PA. Effects of smoking and gingival inflammation on salivary antioxidant capacity. J Clin Periodontol. 2006;33:159e164. 6. Carranza FA, Newman G, Takei H, Khokkevold PR. Carranza’s Clin Periodont. 2007. 10th ed. 251e256. 7. Kinane DF, Radavar M. Smoking on mechanical and antimicrobial periodontal therapy. J Periodontol. 1997;68:467e472. 8. Garg N, Sing R, Dixit J, Jain A, Tewari V. Levels of lipid peroxide and antioxidants in smokers and non-smokers. J Periodont Res. 2006;41:405e410. 9. Alberg Anthony J. The influence of cigarette smoking on circulatory concentrations of anti-oxidant micronutrients. Toxicology. 2002;180:121e137. 10. Ohkawa H, Ohisi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Annal Biochem. 1979;95:315e357. 11. Kakkar P, Das B, Viswanathan P. A modified spectrophotometric assay of super oxide dismutase. Indian J Biochem Biophys. 1984;21:130e132. 12. Sinha KA. Calorimetric assay of catalase. Anal Biochem. 1972;13:114e117. 13. Hu M. Measurement of protein thiol groups and glutathione plasma. Meth Enzymol. 1986;233:380e385. 14. Beutler E, Kelley BM. The effect of sodium nitrate on RBC glutathione. Experientia. 1963;19:96e97. 15. Battino M, Ferreiro MS, Bompadre S, et al. Elevated hydroperoxide levels and antioxidants patterns in PapilloneLefevre syndrome. J Periodontol. 2001;72:1760e1766. 16. Tuter G, Kurtis B, Serdar M. Interleukin-1B and thiobarbituric acid reactive substance (TBARS) levels after phase I periodontal therapy in patients with chronic periodontitis. J Periodontol. 2001;72:883e888. 17. Bluhm AC, Western J, Sonsa. Free radical in tobacco smoke. Nature; 1971:229e240. 18. Bray RC, Cockle SA. Reduction and inactivation of super oxide dismutase by hydrogen peroxide. Biochem J. 1974;139:43e48. 19. Halliwell B, Gutterdge JMC. Free Radical in Biology and Medicine. Oxford: Claredon Press; 1989. 86e92.
i n d i a n j o u r n a l o f d e n t i s t r y 4 ( 2 0 1 3 ) 6 7 e7 1
20. Moore S, Calder KAC, Miller NJ, et al. Antioxidant activity of saliva and periodontal disease. Free Radic Res. 1994;21(6):417e425. 21. Chappel ILC, Mason GI, Garner I. Enhanced chemiluminescent assay for measuring the total antioxidant capacity of serum, saliva and crevicular fluid. Ann Clin Biochem. 1997;34(4):412e421.
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22. Tribble DL, Jones DP. Oxygen dependence of oxidative stress. Rate of NaDPH supply for maintaining the GSH pool during hypoxia. Biochem Pharmacol. 1990;39:729e736. 23. Wayner DDM, Buston GW, Infol KU. The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical trapping antioxidant capacity of human blood plasma. Biochim Biophys Acta. 1987;924:408e419.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ijd
Original Article
Comparative study on the antioxidants levels in smokers and non-smokers with chronic periodontitis M.K. Reejamol a,*, Mythilli Swaminathan b a
Senior Lecturer, Department of Periodontics, PMS College of Dental Science and Research, Vattappara, Trivandrum, Kerala, India Prof & Head, Department of Periodontics, Rajah Muthiah Dental College & Hospital, Annamalai University, Chidambaram 608002, Tamil Nadu, India b
article info
abstract
Article history:
Aim: The aim of the study is to evaluate the effect of cigarette smoking on periodontal
Received 21 October 2012
damage in terms of free radicals and antioxidants.
Accepted 31 December 2012
Materials and methods: Fifteen male patients (non-smokers) with chronic periodontitis (age 25e55) were selected as control & 15 male patients (smokers) with chronic periodontitis
Keywords:
(age 25e55) were selected as experimental group; which is further subdivided as three
Antioxidants
groups based on the number of cigarettes smoked per day (group I: <10 cigarettes/day,
Free radicals
group II: 10e30 cigarettes/day, group III: >30 cigarettes/day). Gingival tissue samples were
Periodontitis
collected from test and control subjects during surgery and were subjected for the esti-
Smoking
mation of lipid peroxide, reduced Glutathione, Superoxide dismutase, Catalase and total Thiol. Results: The levels of lipid peroxide (TBARS), Catalase and total Thiol in gingival tissues showed maximum levels in smokers than non-smokers whereas Superoxide dismutase and reduced Glutathione (GSH) showed elevated levels in non-smokers than smokers. Among the smokers group, group III exhibited significantly higher values of lipid peroxide (TBARS), Catalase and total Thiol followed by group II & I; whereas, the levels of Superoxide dismutase and reduced Glutathione (GSH) showed reduction in such a way that group I exhibited higher levels followed by group II and III. Conclusion: Based on the results of present investigation it is concluded that smoking increases the level of free radicals in periodontal tissues, which in turn may augment the level of periodontal destruction. ª 2013 Indian Journal of Dentistry. All rights reserved.
1.
Introduction
Periodontal diseases are inflammatory disorders that give rise to tissue damage and loss, as a result of complex interactions
between pathogenic bacteria and host immune response.1 The tissue destructive mechanism involves the release of proteolytic enzymes and free oxygen radicals from activated neutrophil.2 The most important reactive oxygen species
* Corresponding author. E-mail address: [email protected] (M.K. Reejamol). 0975-962X/$ e see front matter ª 2013 Indian Journal of Dentistry. All rights reserved. http://dx.doi.org/10.1016/j.ijd.2012.12.009
68
i n d i a n j o u r n a l o f d e n t i s t r y 4 ( 2 0 1 3 ) 6 7 e7 1
implicated in inflammatory injuries to tissues are the hydroxyl radical (OH), the superoxide anion (O 2 ), the nitrous oxide radical (NO), hypochlorous acid, hydrogen peroxide and singlet oxygen.3 Antioxidants are substances which when present at low concentration when compared to an oxidizable substrate will significantly delay or inhibit the oxidation of that substrate.4In normal physiology, there is a dynamic equilibrium between reactive oxygen species (ROS) activity and antioxidant defence capacity. Once that equilibrium shifts in favour of ROS, either by a reduction in antioxidant defences or an increase in ROS production or activity, it results in oxidative stress leading to potential damage.4 Unbalanced radical and non-radical reactive oxygen species will damage cells by variant mechanisms including peroxidation of lipid membranes, protein inactivation, introduction of DNA damage and cytokine induced tissue damage.5 Smoking exerts a major effect on protective elements of immune response, resulting in an increase in the extent and severity of periodontal destruction.6 Smoking may have an adverse effect on fibroblast function, chemotaxis and phagocytosis of neutrophils, immunoglobulin, production and induction of peripheral vasoconstriction.7 Exposure to nicotine results in increased secretion of Prostaglandin E2 (PGE2) by monocytes, elevated levels of tumour necrosis factor (TNF) and gingival crevicular fluid (GCF), elevated levels of neutrophil elastase and matrix metalloproteinase-8.6 A major exogenous source of free radicals is cigarette smoke which contains two phases of free radicals; one in gas phase and other in tar.8 The obligatory use of body reserve of antioxidants to detoxify the excess of free radicals in smokers results in alteration in level of different antioxidants. The antioxidant disturbance in smokers may be further enhanced by their lower intake of both supplemental and dietary antioxidants.8 Cigarette smoke contains free radicals and other oxidants in abundance. In one puff of a cigarette, the gas phase of smoke exposes 1015 free radicals and oxidants than in tar phase. The direct exposure to cigarette smoke represent only a portion of total oxidative stress and contributes to additional endogenous oxidant formation through effects on inflammatory immune response.9 In this backdrop, an attempt has been made in the present study to evaluate the relationship between smoking and the levels of antioxidants and free radicals in chronic periodontitis patients.
of 5 mm in at least 4 sites per quadrant) were selected and were categorized as smokers and non-smokers. The control group were non-smokers comprised of 15 patients with chronic periodontitis. Experimental group were smokers with chronic periodontitis and were subdivided into three sub groups based on the number of cigarette consumption per day. They were, Group-I: 5 subjects smoking less than 10 cigarettes/day. Group-II: 5 subjects smoking 10-30 cigarettes/day. Group-III: 5 subjects smoking more than 30 cigarettes/day.
2.2.
Gingival tissue was obtained during modified Widman Flap Surgery. The excised tissue was thoroughly washed with normal saline so to remove blood and then stored in vials containing 0.15 M potassium chloride.
2.3.
Materials and method
2.1.
Patient selection criteria
The study was conducted at Rajah Muthiah Dental College and Hospital (RMDCH), Annamalai University. Patients devoid of any systemic disease and had not received any periodontal treatment or medication for last 6 months were selected. Written informed consent was obtained from each included subject prior to commencement of the study. Thirty male patients of age group between 25 and 55 years with chronic periodontitis (with probing pocket depth and attachment loss
Biochemical analysis
The gingival tissues thus obtained were subjected for biochemical tests and the levels of lipid peroxidize, Superoxide dismutase (SOD), Catalase, total Thiol group and reduced Glutathione were assessed.
2.4.
Preparation of tissue homogenate
The gingival sample was weighed and a minimum of 400 mg of tissue sample was obtained. Tissue sample was then homogenized in cold 0.15 m KCl using a tissue homogenizer.
2.5.
Estimation of lipid peroxidation in tissue
Lipid peroxidation in tissue was estimated by the method of Ohkawa et al.10 A 0.2 ml sample of tissue homogenate was mixed with 0.2 ml of 8% aqueous sodium dodecyl sulphate, 1.0 ml of 20% acetic acid adjusted to pH of 4.0 using concentrated sodium hydroxide and 1.5 ml of 0.5% aqueous solution of Thio-barbituric acid (TBA). The mixture was incubated in a boiling water bath for 1 h and then centrifuged at 10,062 g for 15 min. The clear supernatant obtained after centrifugation was used for measuring the absorbance at 532 nm using a spectrophotometer.
2.6.
2.
Experimental design
Estimation of Superoxide dismutase (SOD)
Superoxide dismutase (SOD) activity was assayed by the method of Kakkar et al. (1984).11 0.5 ml of tissue homogenate was added with 0.5 ml of water, 2.5 ml of ethanol and 1.5 mol of chloroform. This mixture (5 ml) is shaked at 4 C and the centrifuge supernatant was taken for analysis. The SOD activity is expressed as nmol/mg protein for tissues.
2.7.
Estimation of Catalase
Catalase activity was estimated calorimetrically by the method of Sinha (1972).12 0.1 ml of tissue homogenate was added with 1 ml of phosphate buffer and 0.4 ml of hydrogen
69
i n d i a n j o u r n a l o f d e n t i s t r y 4 ( 2 0 1 3 ) 6 7 e7 1
peroxide. Incubated for 90 s and added 2 ml of dichromate acetic acid. The mixture was incubated in a boiling water bath for 10 min and then centrifuged at 10,062 g for 15 min. The clear supernatant obtained after centrifugation was used for measuring the absorbance at 620 nm using a spectrophotometer.
2.8.
Estimation of total thiol group
The total Thiol level was estimated in tissue by the method of Hu (1986).13 A 0.2 ml sample of 10% plasma tissue homogenate was mixed with water to make the volume upto 0.5 ml. Two millilitres of 0.3 M disodium hydrogen phosphate was added to each sample and 0.25 ml of 5, 50 -dithio-bis-2-nitrobenzoic acid (DTNB) reagent was added just before measuring the absorbance at 412 nm. Total thiol groups were calculated using an absorptivity of 13,600/cm/M.
2.9.
Estimation of reduced Glutathione
Reduced Glutathione was measured in tissue according to the method of Beutler and Kelley (1963).14 This method was based on development of yellow colour when DTNB reagent was added to compounds containing sulfhydryl groups.
2.10.
Statistical analysis
Levels of lipid peroxide (Thio-barbituric acid and reactive substances -TBARS), reduced Glutathione, Superoxide dismutase, Catalase and total Thiol in tissues of control and experimental groups were statistically treated with nonparametric regression analysis. The P values were crosschecked for unravelling the level of statistical significance in such a way that if p < 0.01 indicates a significant difference.
3.
Results
Mean and standard deviation of tissue lipid peroxide (TBARS) (nmol/mg protein), tissue Catalase (nmol/mg protein), tissue Superoxide dismutase (U/mg protein), tissue Glutathione (nmol/mg protein), tissue total Thiol (nmol/mg protein) in non-smokers and smokers are presented in Table 1. The results indicate that the tissue lipid peroxide level revealed an increase in smokers compared to non-smokers where maximum value is recorded in group III. When control is compared
with group I and II, P value is 0.000 (P < 0.01) which reveals a significant difference. The results revealed that the tissue Superoxide dismutase level exhibited a decreasing trend from control to experimental groups in such a way that control group recorded the maximum value. When comparing control with experimental groups I, II and III, P < 0.01 which proves that there is a significant difference. The results showed that the tissue catalase level exhibited an increasing trend from control to experimental groups in such a way that group III recorded the maximum value. When comparing control with experimental groups I, II and III, P < 0.01 which proved that there is a significant difference. The results revealed that the tissue Glutathione level proved a decreasing trend from control group to experimental group IeIII in such a way that group I recorded the maximum value. When control was compared with experimental groups I, II and III, P < 0.01 that underlines the fact that there exist a significant difference. The results revealed that the tissue total Thiol showed an increasing trend from control to experimental groups in such a way that group III recorded the maximum value. When control are compared with group I, P value is 0.098 (P < 0.01) which reveals no significant difference. In case of group II and III when compared with control, P value is 0.017 and 0.003 respectively which proved to be statistically significant.
4.
Discussion
The strongest evidence implicating ROS in periodontal destruction of the connective tissues during periodontal diseases arises in considering PMN infiltration as a key event of host response against bacterial invasion.15 The tissue concentrations of antioxidants or free radical scavengers may be either in the form of enzymes (eg. Superoxide dismutase (SOD), Catalase and Glutathione peroxide) or as low molecular weight free radical scavengers (eg. vitamin E, total thiol, Glutathione etc). When there is an excess of free radicals from exogenous or endogenous sources, the available tissue antioxidant system may become overwhelmed leading to oxidative damage to tissues.8 In smokers, the obligatory use of reserve antioxidants to detoxify the excess free radicals, results in alteration of the level of different antioxidants. The antioxidant disturbance in smokers may be further enhanced by lower intake of both
Table 1 e Mean and standard deviation of biochemical parameters between smokers (groups IeIII) and non-smokers. Parameters
Control
Experimental groups Group I
Lipid peroxide Superoxide dismutase (SOD) Catalase Total thiol group Reduced Glutathione
Group II
Group III
Mean
SD
Mean
SD
P value
Mean
SD
P value
Mean
SD
P value
1.520 99.6 1.4630 0.1790 70.160
0.457 1.198 0.373 0.052 2.046
4.140 79.840 2.4280 0.2500 87.920
0.462 4.731 0.2986 0.025 1.594
0.000 0.001 0.013 0.098 0.000
5.160 71.680 3.4420 0.2860 61.700
0.365 2.893 0.2099 0.019 1.654
0.000 0.000 0.002 0.017 0.002
6.360 63.360 5.1520 0.4420 56.400
0.467 5.264 0.1806 0.043 5.171
0.000 0.000 0.000 0.003 0.006
70
i n d i a n j o u r n a l o f d e n t i s t r y 4 ( 2 0 1 3 ) 6 7 e7 1
supplemental and dietary antioxidants.8 Studies emphasized elevated levels of lipid hydroperoxides in gingival tissues of patients with periodontitis.15,16 The present study exhibited an increase in lipid peroxide level, which is more significant in group III than group I and II, when compared to non-smokers because of increased number of cigarettes per day will influence an increase in the production of free radicals.17 In the present investigation, SOD level were found to be decreased in smokers than non-smokers which might be due to the inactivation by hydrogen peroxide. Among smokers, group III exhibited more reduction in SOD level when compared to group I and II which might be due to increased production of hydrogen peroxide from smoking. Similar results were obtained by Bray et al suggested that free radicals, particularly hydrogen peroxide are generated by the direct interaction between smoke and periodontal tissues that will lead to a significant change in the level of antioxidant enzyme SOD.18 The present study observed an increase in catalytic activity in smokers than non-smokers which might be caused by higher levels of hydrogen peroxide formation, which is evidenced in group III than other two groups. Similar trend was observed in the findings of Halliwell et al in which they observed that an initial rate of hydrogen peroxide removal is directly proportional to its concentration. They emphasized that the enzyme Catalase come into action only after an optimum concentration of hydrogen peroxide has been obtained; below which Catalase has no role to play.19 Works done on the status of antioxidants, mainly Glutathione peroxidase (GPx) and reduced Glutathione in saliva, GCF and serum of chronic periodontitis patients proved that GSH is a scavenger of hydroxyl radicals and singlet oxygen.20,15,21 It functions as a substrate for the hydrogen peroxide removing enzyme, Glutathione peroxidase (GPx). The present study revealed a reduction of GSH levels in smokers, among which group III exhibited least level. This might be due to more flux of hydrogen peroxide and hydroxyl radicals influenced by smoking. This is in accordance with the study done by Tribble et al in which they reported that exposure of tissues to a large flux of hydrogen peroxide and hydroxyl radicals might result in an imbalance of GSH and oxidized Glutathione (GSSG) ratio. They stated that GSSG accumulates in tissues and it paves way to inactivation of various enzymes which lead to lower levels of GSH in smokers.22 The observation done by Wayner et al postulated that among smokers, there might be some structural modifications in proteins because of oxidative activity, that results in the exposure of protein linked thiol groups so to react freely and thus depicts a significantly higher total thiol level.23 The present study is in favour of the above findings with elevated level of total thiol in smokers compared to non-smokers. An insignificant increase in total thiol in tissues of group I smokers (smoking < 10 cigarettes/day) might be due to increased free radical production but in amount that are scavenged by antioxidants, such that the damage is limited to few protein molecules. The present study suggests that free radical production induced by cigarette smoke is highly toxic and will impair the oxidant-antioxidant balance, which is a major risk factor in periodontal disease.
Studies pertain to this line can be undertaken by including factors such as past history and duration of smoking in smokers, which will help in unravelling the possible cumulative effects of those parameters on the production of free radicals leading to periodontal disease.
Conflicts of interest All authors have none to declare.
references
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