Antioxidant Enzymes Activity in Gingiva and Gingival Crevicular Fluid in Chronic Periodontitis Patients: Correlation with Some Potent Periodontopathogens.

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

Introduction

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.

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.

Chronic periodontitisChronic periodontitis CausesCauses

Chronic gingivitisChronic gingivitis Occlusal traumaOcclusal trauma Improper application of orthodontic appliance (excess force)Improper application of orthodontic appliance (excess force)

PathologyPathology Destruction of periodontal ligamentDestruction of periodontal ligament Formation of periodontal pocketFormation of periodontal pocket Resorption of alveolar boneResorption of alveolar bone Loosening of teethLoosening of teeth

Environmental Smoking

Host

Susceptibility Genetic Acquired

Periodontal Diseases

BacteriaColonisation

InvasionDestruction

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.

What are Reactive Oxygen Species (ROS)?

• ROS are highly reactive oxidizing agents include:

a. Oxygen derived free radicals (e.g. Superoxide anion O2.-)

b. Oxygen-derived non radical species (e.g. H2O2)

• ROS are potentially harmful to cells, causing oxidation of lipids, proteins and DNA.

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.

•Cigarette smoke•Environmental pollutants•Radiations•Ultraviolet radiations•Ozone•Certain drugs•Pesticides•Anesthetics

External Sources of ROS

SmokingSmoking 1010 Quad Trillion free Quad Trillion free

radicals per cigaretteradicals per cigarette!!

Internal Sources of ROS

• Mitochondria

• Inflammation

• Phagocytes

• Xanthine oxidase

• Arachidonate pathways

• Ischemia/Reperfusion

Antioxidants

Antioxidants neutralize ROS in body tissues.Antioxidant compounds include:Ascorbic acid (vitamin C)α-Tocopherol (vitamin E)GlutathioneLipoic acidUric acidCarotenes

Antioxidant SourcesA diet rich in FRUITS and

VEGETABLES

And Nutritional

Supplements

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.

The Constant Battle

ROS are toxins that cause cell and DNA damage

Antioxidants combat ROS to prevent cell damage and maintain health

ROS vs. 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.

Periodontopathogens

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

P. gingivalis

Healthy plaque(mostly gram positive

bacteria)

Periopathogenic plaque (mostly gram

negative anaerobes)

There is a strong clinical correlation between the bacterial plaquecomposition and the innate host defense status

Aim of Work

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.

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.

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/l) and GT (U/mg tissue homogenate) samples were determined.

1 Sun et al. Clin Chem 34: 497-500, 19882 Aebi, H. Methods Enzymol 105: 121 – 126, 19843 Paglia and Valentine. J. Lab. Clin. Med. 70: 158 – 169, 1967

Methods

The total viable count of P. gingivalis and F. 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. gingivalis and F. nucleatum was calculated.

Identification criteria of P. gingivalis

Test

Result

Color of colony Black Gram reaction -ve Cell shape Short rod Spore stain -ve Relation to O2 Obligate anaerobe Catalase -ve Growth in bile: 10% 20%

+ve -ve

Hydrolysis of: Starch Gelatin Lipid Casein Licithin Hippurate Esculin

-ve +ve -ve +ve -ve -ve -ve

Nitrate reduction -ve Arginine dihydrolase -ve Voges proskauer test +ve Indole formation +ve Methyl red +ve

Holdeman et al.Int. J. Syst. Bacteriol., 32: 125-131, 1982.

P. gingivalis Agar (P.GING)

Identification criteria of F. nucleatum Test Result

Gram reaction -ve Shape of cells Spindle or rods Spore stain -ve Reaction to oxygen Anaerobic Blood hemolysis -ve Starch hydrolysis -ve Gelatin hydrolysis +ve Lipid hydrolysis -ve Esculin hydrolysis -ve Hippurate hydrolysis -ve Motility -ve Milk: Coagulation Peptonization

-ve -ve

Indole production +ve Acetation -ve Nitrate reduction -ve Lecithinase -ve Growth in 20% bile salts -ve Acid from: Trehalose -ve D-Xylose -ve L-Arabinose -ve Mannose -ve Glucose -ve Fructose -ve Galactose -ve Lactose -ve Maltose -ve Sucrose -ve Melibiose -ve Mannitol -ve Sorbitol -ve Cellobiose -ve Starch -ve Inulin & Salicin -ve

Holdeman et al.Int. J. Syst. Bacteriol., 32: 125-131, 1982.

Gram-negative stained culture of F. nucleatum

RESULTS

Mean clinical parameters in CP and control groups

0.03 0.04

1.66

0

2.392.62

6.63

3.75

0

1

2

3

4

5

6

7

mean s

core

1 2

GI

PlI

PD

CAL

GI = Gingival index; PI = Plaque index; PD = Probing depth; GI = Gingival index; PI = Plaque index; PD = Probing depth; CAL = Clinical attachment lossCAL = Clinical attachment loss

p <p < 0.010.01

CPControl

P. gingivalis and F. nucleatum total viable counts (cfu/ml) in CP and control groups

54.73 37.51

619.55

403.63

0

100

200

300

400

500

600

700

tota

l via

ble

count

1 2

Pg

Fn

p <p < 0.0010.001CPControl

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

Group Range Mean ± SD P

MinMax

GCF SODControl (n=20) 8.19 16.70 11.83 ± 2.46 <0.001

CP (n=20) 2.73 9.90 3.56 ± 1.63

GT SODControl (n=20)11.30 23.00 16.09 ± 3.39 <0.001

CP (n=20) 3.00 8.60 5.86 ± 1.50

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

11.83

16.09

3.56

5.86

0

2

4

6

8

10

12

14

16

18

mean a

ctiv

ity le

vel

1 2

GCF SOD

GT SOD

p <p < 0.0010.001

CPControl

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

Group Range Mean ± SD P

MinMax

GCF CATControl (n=20) 6.00 19.00 10.59 ± 3.95 <0.01

CP (n=20) 3.30 6.40 4.46 ± 0.97

GT CATControl (n=20)7.00 22.20 14.06 ± 4.04 <0.01

CP (n=20) 3.00 6.90 4.77 ± 1.18

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

10.59

14.06

4.46 4.77

0

2

4

6

8

10

12

14

16

mean a

ctiv

ity le

vel

1 2

GCF CAT

GT CAT

p <p < 0.010.01CPControl

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

Group Range Mean ± SD P

MinMax

GCF GPxControl (n=20) 0.88 1.84 1.15 ± 0.23 <0.05

CP (n=20) 0.53 1.66 0.87 ± 0.19

GT GPxControl (n=20)0.88 1.88 1.41 ± 0.31 <0.05

CP (n=20) 0.53 1.60 1.00 ± 0.39

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

1.15

1.41

0.871

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

mean a

ctiv

ity le

vel

1 2

GCF GPx

GT GPx

p <p < 0.050.05CPControl

Pearson’s correlation coefficient between clinical parameters and periodontal pathogens

P. gingivalisF. nucleatum

Gingival Index (GI)0.738*0.757*

Plaque Index (PI)0.703*0.779*

Probing Depth (PD)0.751*0.818*

Clinical Attachment Loss (CAL)

0.638*0.795*

** significant Correlation at p< 0.01* p <* p < 0.010.01

Pearson’s correlation coefficient between P. gingivalis, F. nucleatum total viable count and SOD, CAT and GPx in chronic periodontitis patients

SODCATGPx

GCF GT GCF GT GCF GT

P. gingivalis-0.393-0.761**-0.153-0.537*-0441-0.417

F. nucleatum-0.315-0.752**-0.354-0.631**-0.174-0.241

* p< 0.05 , ** p< 0.01* p<0.05, ** p<0.01

Correlation of GT SOD with P. gingivalis total viable count in chronic periodontitis patients

r = -0.761, p<0.01

0

1

2

3

4

5

6

7

8

9

10

0 200 400 600 800 1000

GT

SO

D

Correlation of GT SOD with F. nucleatum total viable count in chronic periodontitis patients

r = -0.752 , p<0.01

0

1

2

3

4

5

6

7

8

9

10

0 100 200 300 400 500 600

GT

SO

D

Correlation of GT CAT with P. gingivalis total viable count in chronic periodontitis patients

r = -0.537, p<0.05

0

1

2

3

4

5

6

7

8

0 200 400 600 800 1000

GT

CA

T

Correlation of GT CAT with F. nucleatum total viable count in chronic periodontitis patients

r = -0.631, p<0.01

0

1

2

3

4

5

6

7

8

0 100 200 300 400 500 600

GT

CA

T

Conclusion

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.

Conclusion

P. gingivalis and F. 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.

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.

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.

Thank you

for your attention