Gamal Kenawy*, Abdul Fattah Amer**, Akram El awady**, Hisham Mahdy***, and Radi Massoud**

1. Antioxidant Enzymes Activity in Gingiva and Gingival Crevicular Fluid in Chronic Periodontitis Patients: Correlation with Some Potent Periodontopathogens Gamal Kenawy*, Abdul Fattah Amer**, Akram El awady**, Hisham Mahdy***, and Radi Massoud** * Medical Biochemistry, Riyadh Colleges of Dentistry and Pharmacy** Oral Medicine and Periodontology, College of Dentistry, Al-Azhar University*** Microbiology, College of Medicine, Al-Azhar University

2. Introduction

3. Periodontal Disease • Periodontal disease is a common chronic adult condition that, left untreated, can lead to tooth loss. • Chronic periodontitis results in inflammation within the supporting tissues of the teeth, progressive attachment and bone loss. This is the most frequently occurring form of periodontal disease.

4. Chronic Periodontitis • Chronic periodontitis has now been linked to heart disease, stroke, lung infections, pre-term and low birth weight babies, oral cancer, osteoporosis, and other chronic diseases.

5. Chronic periodontitis • Causes • Chronic gingivitis • Occlusal trauma • Improper application of orthodontic appliance (excess force) Pathology • Destruction of periodontal ligament • Formation of periodontal pocket • Resorption of alveolar bone • Loosening of teeth

6. Bacteria Colonisation Invasion Destruction Environmental Smoking HostSusceptibility Genetic Acquired Periodontal Diseases

7. Chronic Periodontitis • Chronic periodontitis results from an exuberant inflammation induced by pathogenic oral microorganisms that stimulate host cells to release pro-inflammatory cytokines and exhibit increased production of Reactive Oxygen Species (ROS) as part of the host response to infection.

8. What are Reactive Oxygen Species (ROS)? • ROS are highly reactive oxidizing agents include: • Oxygen derived free radicals (e.g. Superoxide anion O2.-) • Oxygen-derived non radical species (e.g. H2O2) • ROS are potentially harmful to cells, causing oxidation of lipids, proteins and DNA.

9. ROS and Chronic Periodontitis • It has been suggested that ROS are capable of inducing periodontal tissue destruction and are associated with osteoclastic bone resorption, commonly associated with periodontitis.

10. Smoking 10 Quad Trillion free radicals per cigarette! External Sources of ROS • Cigarette smoke • Environmental pollutants • Radiations • Ultraviolet radiations • Ozone • Certain drugs • Pesticides • Anesthetics

11. Internal Sources of ROS • Mitochondria • Inflammation • Phagocytes • Xanthine oxidase • Arachidonate pathways • Ischemia/Reperfusion

12. Antioxidants • Antioxidants neutralize ROS in body tissues. • Antioxidant compounds include: • Ascorbic acid (vitamin C) • α-Tocopherol (vitamin E) • Glutathione • Lipoic acid • Uric acid • Carotenes

13. Antioxidant Sources A diet rich in FRUITS and VEGETABLES And Nutritional Supplements

14. Antioxidant Enzymes • Antioxidant enzymes, including Superoxide Dismutase (SOD), Catalase (CAT), and Glutathione Peroxidase (GPx) are naturally produced enzymes that have evolved for cellular protection against oxidative stress and ROS. • They detoxify ROS to harmless substances such as water and ordinary oxygen.

15. The Constant Battle ROS are toxins that cause cell and DNA damage Antioxidants combat ROS to prevent cell damage and maintain health

17. ROSvs. Antioxidants • In health, the balance is maintained among ROS and antioxidants while under pathological conditions, the balance may be tilted towards the oxidative stress with increase in ROS levels.

18. Periodontopathogens • The bacteria Porphyromonas gingivalis (P. gingivalis)and Fuesbacterium nucleatum (F. nucleatum) have been implicated in the etiology of chronic periodontitis. P. gingivalis

19. Periopathogenic plaque (mostly gram negative anaerobes) Healthy plaque (mostly gram positive bacteria) There is a strong clinical correlation between the bacterial plaque composition and the innate host defense status

20. Aim of Work

21. Aim of Work • In this study, the activity of antioxidant enzymes in gingival crevicular fluid and gingival tissue from patients with chronic periodontitis and periodontally healthy controls were compared. • In addition, correlation of antioxidant enzyme activities with the total viable count of P. gingivalis and F. nucleatum were studied.

22. Subjects • Forty subjects were included in this study; divided into two groups: • 1- Chronic periodontitis (CP) group: 20 patients with chronic periodontitis. • 2- Control group: 20 periodontal healthy subjects.

23. Methods • The activities of Superoxide Dismutase1 (SOD, EC 1.15.1.1), Catalase2 (CAT, EC 1.11.1.6), and Glutathione Peroxidase3 (GPx, EC 1.11.1.9) enzymes in GCF (U/ml) and GT (U/mg tissue homogenate) samples were determined. 1 Sun et al. Clin Chem 34: 497-500, 1988 2 Aebi, H. Methods Enzymol 105: 121 – 126, 1984 3 Paglia and Valentine. J. Lab. Clin. Med. 70: 158 – 169, 1967

24. Methods • The total viable count of P. gingivalisandF. nucleatum (cfu/ml) recovered from subgingival plaque samples, cultured anaerobically and were estimated. • The correlation between the enzyme activities and the total viable count of P. gingivalisandF. nucleatum was calculated.

25. Identification criteria of P. gingivalis Holdeman et al.Int. J. Syst. Bacteriol., 32: 125-131, 1982.

26. P. gingivalis Agar (P.GING)

27. Identification criteria of F. nucleatum Holdeman et al.Int. J. Syst. Bacteriol., 32: 125-131, 1982.

28. Gram-negative stained culture ofF. nucleatum

29. RESULTS

30. Mean clinical parameters in CP and control groups Control CP GI = Gingival index; PI = Plaque index; PD = Probing depth; CAL = Clinical attachment loss p <0.01

31. P. gingivalis and F. nucleatum total viable counts (cfu/ml) in CP and control groups CP Control p <0.001

32. Superoxide dismutase (SOD) activity in GCF and GT in CP and control groups

33. Superoxide dismutase (SOD) activity in GCF and GT in CP and control groups Control CP p <0.001

34. Catalase (CAT) activity in GCF and GT in CP and control groups

35. Catalase (CAT) activity in in GCF and GT in CP and control groups Control CP p <0.01

36. Glutathione peroxidase (GPx) activity in GCF and GT in CP and control groups

37. Glutathione peroxidase (GPx) activity in GCF and GT in CP and control groups Control CP p <0.05

38. Pearson’s correlation coefficient between clinical parameters and periodontal pathogens ** significant Correlation at p< 0.01 * p <0.01

39. Pearson’s correlation coefficient between P. gingivalis, F. nucleatum total viable count and SOD, CAT and GPx in chronic periodontitis patients * p< 0.05 , ** p< 0.01 * p<0.05, ** p<0.01

40. Correlation of GT SOD with P. gingivalis total viable count in chronic periodontitis patients • r = -0.761, p<0.01

41. Correlation of GT SOD with F. nucleatum total viable count in chronic periodontitis patients • r = -0.752 , p<0.01

42. Correlation of GT CAT with P. gingivalis total viable count in chronic periodontitis patients • r = -0.537, p<0.05

43. Correlation of GT CAT with F. nucleatum total viable count in chronic periodontitis patients • r = -0.631, p<0.01

44. Conclusion

45. Conclusion • The lower activities of antioxidant enzymes in the GCF and GT in chronic periodontitis patients can participate directly and indirectly in tissue destruction that coincident to periodontal disease and, probably may have an inference in treatment modalities of periodontal disease.

46. Conclusion • P. gingivalisandF. nucleatum could be implicated as true pathogens in predisposition of chronic periodontitis. • These peridontopathogens may have a role in suppression of antioxidant enzymes synthesis or decreasing their activities.

47. Conclusion • The negative correlation between total viable count of P. gingivalis and F. nucleatum with the antioxidant enzyme activities in chronic periodontitis patients may be applied as diagnostic and/or prognostic periodontal tool.

48. Conclusion • The findings of the present study may provide opportunities to develop a novel antioxidant therapy that function not only as antioxidants in the traditional sense but, also, act as anti-inflammatory agent to overcome the unwanted effects of the inflammatory process upon periodontal tissues.

49. Thank you for your attention