Transcript P. gingivalis

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 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
Bacteria
Colonisation
Invasion
Destruction
Environmental
Smoking
Host
Susceptibility
Genetic
Acquired
Periodontal Diseases
Chronic Periodontitis
Chronic periodontitis results from an
exuberant inflammation induced by
pathogenic oral microorganisms that
stimulate host cells to release proinflammatory
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.
External Sources of ROS
•Cigarette smoke
•Environmental
pollutants
•Radiations
•Ultraviolet radiations
•Ozone
•Certain drugs
•Pesticides
•Anesthetics
Smoking
10 Quad Trillion free
radicals 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)
 Glutathione
 Lipoic acid
 Uric acid
 Carotenes
Antioxidant Sources
A 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
There is a strong clinical correlation between the bacterial plaque
composition and the innate host defense status
Healthy plaque
(mostly gram positive
bacteria)
Periopathogenic
plaque (mostly gram
negative anaerobes)
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/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
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
Result
Test
Color of colony
Gram reaction
Cell shape
Spore stain
Relation to O2
Catalase
Growth in bile:
10%
20%
Hydrolysis of:
Starch
Gelatin
Lipid
Casein
Licithin
Hippurate
Esculin
Nitrate reduction
Arginine dihydrolase
Voges proskauer test
Indole formation
Methyl red
Black
-ve
Short rod
-ve
Obligate anaerobe
-ve
+ve
-ve
-ve
+ve
-ve
+ve
-ve
-ve
-ve
-ve
-ve
+ve
+ve
+ve
Holdeman et al.Int. J. Syst. Bacteriol., 32: 125-131, 1982.
P. gingivalis Agar (P.GING)
Identification criteria of F. nucleatum
Test
Gram reaction
Shape of cells
Spore stain
Reaction to oxygen
Blood hemolysis
Starch hydrolysis
Gelatin hydrolysis
Lipid hydrolysis
Esculin hydrolysis
Hippurate hydrolysis
Motility
Milk:
Coagulation
Peptonization
Indole production
Acetation
Nitrate reduction
Lecithinase
Growth in 20% bile salts
Acid from: Trehalose
D-Xylose
L-Arabinose
Mannose
Glucose
Fructose
Galactose
Lactose
Maltose
Sucrose
Melibiose
Mannitol
Sorbitol
Cellobiose
Starch
Inulin & Salicin
Holdeman et al.Int. J. Syst. Bacteriol., 32: 125-131, 1982.
Result
-ve
Spindle or rods
-ve
Anaerobic
-ve
-ve
+ve
-ve
-ve
-ve
-ve
-ve
-ve
+ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
 Gram-negative stained culture of F. nucleatum
RESULTS
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
P. gingivalis and F. nucleatum total viable
counts (cfu/ml) in CP and control groups
p < 0.001
Control
CP
Superoxide dismutase (SOD) activity in
GCF and GT in CP and control groups
Group
GCF SOD
GT SOD
Range
Mean ± SD
P
<0.001
Min
Max
Control (n=20)
8.19
16.70
11.83 ± 2.46
CP (n=20)
2.73
9.90
3.56 ± 1.63
Control (n=20)
11.30
23.00
16.09 ± 3.39
CP (n=20)
3.00
8.60
5.86 ± 1.50
<0.001
Superoxide dismutase (SOD) activity in
GCF and GT in CP and control groups
Control
p < 0.001
CP
Catalase (CAT) activity in GCF and GT in CP
and control groups
Group
GCF CAT
GT CAT
Range
Mean ± SD
P
<0.01
Min
Max
Control (n=20)
6.00
19.00
10.59 ± 3.95
CP (n=20)
3.30
6.40
4.46 ± 0.97
Control (n=20)
7.00
22.20
14.06 ± 4.04
CP (n=20)
3.00
6.90
4.77 ± 1.18
<0.01
Catalase (CAT) activity in in GCF and GT in CP
and control groups
Control
p < 0.01
CP
Glutathione peroxidase (GPx) activity in
GCF and GT in CP and control groups
Group
GCF GPx
GT GPx
Range
Mean ± SD
P
<0.05
Min
Max
Control (n=20)
0.88
1.84
1.15 ± 0.23
CP (n=20)
0.53
1.66
0.87 ± 0.19
Control (n=20)
0.88
1.88
1.41 ± 0.31
CP (n=20)
0.53
1.60
1.00 ± 0.39
<0.05
Glutathione peroxidase (GPx) activity in
GCF and GT in CP and control groups
Control
p < 0.05
CP
Pearson’s correlation coefficient between
clinical parameters and periodontal pathogens
P. gingivalis
F. 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
* p < 0.01 Correlation at p< 0.01
Pearson’s correlation coefficient between P.
gingivalis, F. nucleatum total viable count and SOD,
CAT and GPx in chronic periodontitis patients
SOD
GCF
GT
CAT
GPx
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<
* p<0.05,
** 0.01
p<0.01
Correlation of GT SOD with P. gingivalis total
viable count in chronic periodontitis patients
 r = -0.761, p<0.01
Correlation of GT SOD with F. nucleatum total
viable count in chronic periodontitis patients
 r = -0.752 , p<0.01
Correlation of GT CAT with P. gingivalis total
viable count in chronic periodontitis patients
 r = -0.537, p<0.05
Correlation of GT CAT with F. nucleatum total
viable count in chronic periodontitis patients
 r = -0.631, p<0.01
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 antiinflammatory
agent
to
overcome the unwanted
effects of the inflammatory
process upon periodontal
tissues.
Thank you
for your attention