the inflammatory and immune reactions induced by the bacterial plaque represent the main characteristics of periodontitis , and this disease represents a particularly cogent example of 
 problem arising from the phenomenon .
studies have demonstrated that periodontal disease affects between 10% and 15% of the 
 world 's population , representing the greatest cause of tooth loss  
 .
there is strong evidence that this disease affects a specific , predisposed group of the 
 population that presents an exacerbated inflammatory / immune response to the 
 periodontopathogenic bacteria that accumulate on the teeth and around the 
 gingival tissue , which in turn may lead to tissue damage [ 1 , 3 ] .
the exact mechanism of periodontitis development , including the prior agents or 
 mediators involved , is not clear .
periodontitis manifests itself as a 
 multifactor phenomenon , including the generation of reactive oxygen species 
 ( ros ) . the strong evidence linking ros to the pathological destruction of the 
 connective tissue during periodontal disease rests on the presence of 
 neutrophils infiltration as the main event in the host 's response to bacterial 
 invasion [ 1 , 5 , 
 6 ] .
furthermore , hydroxyl radical 
 ( oh ) is 
 most active in damaging important molecules such as dna proteins and lipids , 
 while hydrogen peroxide ( h2o2 ) , even not being considered a potent ros , is capable of crossing the nuclear membrane and also damaging the 
 dna .
quantitatively , the main source of superoxide anion 
 ( o2 ) and other ros responsible for initiation reactions is the respiratory chain . 

however , its presence in the periodontal tissue results first and foremost from 
 the activation of phagocytes ( neutrophils and macrophages ) , such as 
 antibacterial agents [ 1 , 5 , 8 ] . it has been suggested that superoxide anion is 
 involved in bone reabsorption which has been corroborated by studies that have 
 demonstrated the presence of this anion in reabsorption zones adjacent to the osteoclasts 
 .
the hydroxyl radical is able to initiate a classical chain reaction 
 known as lipid peroxidation leading to the vasodilation production and rat bone 
 reabsorption .
an example of the damage caused by hydrogen peroxide is that it can stimulate the phosphorylation of the nf - kb - ikb 
 complex activating the nk - kb and facilitating nuclear 
 translocation and downstream of proinflammatory cytokines , including 
 interleukin-2 ( il-2 ) , interleukin-6 ( il-6 ) , interleukin-8 ( il-8 ) , 
 -interferon , 
 and tumor necrosis factor- ( tnf- ) that are 
 very important in the pathogenesis of periodontal disease [ 1012 ] .
ros production is inevitable in all aerobic organisms including humans , who necessarily posses a complex system of antioxidant defense [ 8 , 13 ] .
if the homeostasis is interrupted in favor of ros , an oxidative stress situation is 
 created .
the aim of this study was to evaluate the involvement of proinflammatory and oxidative stress markers in individuals with chronic periodontal disease .
september , 2003 in a single centre ( peridontologic centre , florianopolis , santa catarina , brazil ) .
eighteen subjects were divided into 2 
 groups : experimental group ( e ) , comprising individuals with chronic periodontitis , age 52.95.0 
  ( 4 men and 5 women ) , with the following inclusion criteria : 
 presence of chronic inflammation ( pain , redness , heat , swelling ) , diagnosed 
 according to bleeding on probing , at least 5 or 6 sites with probing depth 
 5  mm , attachment loss 3  mm , and extensive radiographic bone loss 
 .
control group ( c ) , was composed of healthy individuals , age 51.19.6 ( 4 men and 5 women ) with no prior history of periodontal disease .
for both groups , the following items were considered as exclusion 
 criteria : infection , cardiovascular and/or neurological illness , renal 
 insufficiency and/or diabetes ; pregnancy ; smoking ; use of antibiotics and/or 
 hormonal or nonhormonal anti - inflammatory drugs 6 months prior to tissue 
 collection . in the day of the surgical procedures ,
this study was in agreement with the ethical principles of the world 
 medical association declaration of helsinki ( 1964 ) .
permission for this study 
 was obtained from the ethical committee for human research of the federal 
 university of santa catarina ( project no .
210/2002 ) and the study included 
 only individuals that agreed to participate after reading and signing a free 
 and informed consent form , except those with difficulty in understanding and 
 communicating , with physical handicap , or both , which could have compromised 
 the sample collection .
all of the surgical procedures were assessed and performed by a periodontist , 
 according to the necessity for each treatment .
the patients with chronic 
 periodontitis ( experimental group ) were submitted a pocket depth reduction technique 
 from palatal / lingual , buccal , and interproximal sites .
the biopsies were 
 obtained from inflammatory granulation tissues , connective and epithelium 
 tissues .
the samples collected from the control group were obtained from quarantined 
 mucosa during the surgical procedures of impacted third molars removed 
 following orthodontic recommendation or after the reopening of dental implants .
all samples from the experimental and control groups were removed 
 during the surgical and were immediately frozen in liquid nitrogen ( 170c ) subsequently latter laboratory analysis .
on the day of 
 the experiments , the samples were deathward at room temperature to determine 
 the different parameters : cat , gpx , gst , gr , mpo activities , and the contents 
 of tg , gsh , gssg , and tbars .
the method described by aebi was employed to measure the 
 catalase activity ( cat ) by measuring the decay of a freshly prepared 10  mm 
 hydrogen peroxide solution at 240  nm .
glutathione peroxidase ( gpx ) was measured at 340  nm through the 
 glutathione / nadph / glutathione reductase system by the dismutation of tert - butyl 
 hydroperoxide .
glutathione s - transferase ( gst ) activity was determined at 340  nm using cdnb ( 1-chloro-2 , 4-dinitrobenzene ) as substrate and a 0.15  m gsh concentration .
finally , glutathione reductase ( gr ) activity was assayed at 340  nm by measuring the 
 rate of nadph oxidation .
results were expressed as mmolg(cat ) and mol ming ( gpx , gst , and gr ) .
nonprotein thiols , mostly present as the reduced form of glutathione ( gsh ) , were measured at 412  nm according to beutler using elmann 's reagent ( dtnb : 2-dithionitrobenzoic acid ) . 
 immediately after thawing ,
acid extracts were obtained by adding tissue 
 portions to 12% trichloroacetic acid ( 1 : 4 w / v ) , which were then centrifuged at 
 15  000  g for 5 minutes at 5c .
supernatants from the acid extracts were added to a buffer 
 containing 0.25  mm dtnb in 0.1  m na2po4 , ph 8.0 , and the 
 formation of the thiolate anion was immediately determined .
total glutathione 
 ( tg ) was also measured at 412  nm in acid extracts according to the enzymatic 
 method of tietze .
oxidized 
 glutathione ( gssg ) was also determined by calculating the difference ( in 
 equivalents of gsh ) between total glutathione and reduced glutathione contents . 

results were expressed as mol g. myeloperoxidase activity was measured 
 according to the method developed by rao et al . , and was estimated by 
 colorimetric measurement at 450  nm on an elisa plate reader 
 .
thiobarbituric acid - reactive substance ( tbars ) contents were determined to assess endogenous 
 lipid oxidation in gingival tissue according to ohkawa   et   al .   and bird and draper .
after thawing , 
 gingival portions were immediately added to 12% trichloroacetic acid ( 1 : 4 v / v ) 
 and were then centrifuged at 15  000  g for 5 minutes at 5c .
supernatants were added to 50  mm tris - hcl ph 7.0 , vortexed for 20 seconds , added to 0.67% ( w / v ) 2-thiobarbituric 
 acid , maintained in boiling water for 60 minutes , cooled at 5c for 30 minutes , and then analyzed 
 spectrophotometrically at 535  nm .
concentrations were expressed as nmol tbars / g 
 wet tissue using 535=153  mmcm .
all the biochemical parameters described 
 above were measured in duplicate , except for the tbars determinations , which 
 were measured in triplicate .
the results were expressed as mean  sem . statistical differences 
 between groups were determined by independent student   t test 
 analysis . for all analyses , p<.05 was used to assess overall differences .
these results show that there were no significant differences in 
 catalase activity in the experimental group ( e ) compared to the control group ( p=.523 ) .
however , a significant increase in gpx activities in the experimental 
 group when compare to the control group ( p=.006 ) was detected ( see table 1 ) .
furthermore , in the experimental group , a significant increase of 
 glutathione s - transferase ( gst ) values compared to the control group ( p=.001 ) was also observed ( see table 1 ) .
the analysis of glutathione reductase 
 ( gr ) revealed no differences between the studied groups ( p=.481 ) ( see table 1 ) .
the myeloperoxidase activity 
 revealed a significant increase of this inflammatory biomarker in the 
 experimental group ( p=.003 ) ( see table 1 ) . in relation to the total 
 glutathione ( tg ) and the reduced glutathione ( gsh ) contents , no differences in the 
 values of the experimental group compared to the control group were found ( p>.05 ) ( table 1 ) .
however , the values obtained for oxidized glutathione 
 ( gssg ) showed a significant increase in the experimental group when compared to 
 the control group ( p=.019 ) ( see table 1 ) .
lipoperoxidation was 
 measured through tbars contents , which were significantly a higher increase in 
 the experimental group ( p=.015 ) ( see table 1 ) .
few studies have considered 
 the effect of the imbalance between oxidants and antioxidants in patients with 
 periodontitis , which in turn predisposes such individuals to the damaging 
 effects of ros in the periodontium .
ellis and collaborators analyzed gingival tissues from patients with 
 severe periodontal disease and showed that the activity of catalase was 
 decreased . in the present study , the activity of catalase was 
 not different when the experimental and control groups were compared .
one 
 possible explanation for these different responses is that the patients with 
 periodontal disease were in distinct stages of the disease . in this regard
, it 
 is well known that the antioxidant responses found in different pathologies 
 depend on the severity or extension suffered by the patients , and long - term 
 chronic conditions may have jeopardized the antioxidant defenses .
the analysis of the enzyme glutathione peroxidase 
 revealed a significant increase in the experimental group . a gpx   increase in 
 gingival samples from dogs and humans with periodontal disease
the gpx increase may represent possible 
 antioxidant compensation in detoxification reactions of organic peroxides 
 produced during oxidative stress in gingival tissue .  
furthermore , glutathione s - transferase ( gst ) also 
 revealed a significant increase in its activities in the experimental group . 

since gst has a direct role in the neutralization of hydroperoxides derived 
 from the lipoperoxidation processes , increases in gst activities are probably 
 related to the oxidative stress caused by the periodontal inflammatory process 
 [ 8 , 29 ] .
gst comprises a group of enzymes that are also able to detoxify a 
 variety of compounds including xenobiotics derived from pathogenic 
 microorganisms , catalyzing their conjugation with gsh .
hence , increases in gst activities are excellent indicators of endogenous detoxification from 
 exogenous sources
.   the enzyme 
 glutathione reductase ( gr ) has an important accessory antioxidant function 
 related to glutathione peroxidase and glutathione s - transferase .
gr 
 intervention continuously regenerates gsh from gssg in the presence of nadph , 
 therefore preventing cellular loss of gsh .
however , in the current study , 
 no differences in gr activities were detected in gingival tissue between the 
 two groups .  
the ubiquitous tripeptide glutathione ( gsh ) acts 
 directly as a generic ros scavenger or as a cofactor of gpx and gst , either by 
 catalyzing the reduction of hydrogen peroxide and lipid hydroperoxides or by 
 the conjugation / excretion processes of the so - called phase ii reactions . 

total and reduced glutathione revealed a tendency to increase , but the values 
 were not significantly different in patients with periodontitis compared to the 
 controls .   despite gsh , these results suggest a de novo synthesis of glutathione , which is 
 extremely necessary for the homeostasis of cells . some periodontopathogenic 
 bacteria deplete gsh , and
this may explain the amount of this antioxidant was 
 not elevated in the gingival tissue of patients with periodontitis combined with an increase of the gpx activity in the affected tissue [ 33 , 34 ] .
a similar result was obtained in gingival 
 tissue and blood , but lower levels of gsh were detected in the crevicular 
 gingival fluid of patients with chronic periodontitis , when compared to normal 
 subjects [ 27 , 35 ] .  
on the other hand , a significant increase in gssg 
 concentrations was detected in the experimental group , which is a clear 
 biomarker of oxidative stress detected in inflammatory processes linked to 
 periodontitis .
2002 ) found less gssg in gingival 
 cervical fluid of patients with chronic periodontitis .
consistent with the 
 results for gssg , tissue lipoperoxidation , measured as tbars contents in the 
 gingival tissue , also displayed a significant increase ( p=.015 ) in 
 individuals affected by periodontitis , and oxidative stress , in the gingival 
 tissue associated with periodontal disease .  
the systemic depletion of antioxidants clearly 
 indicates that in chronic periodontitis the antioxidant system is affected by a 
 relatively strong oxidation insult , which can also deplete nutritional 
 antioxidants such as vitamin e and c in plasma and also vitamin e in red cell 
 membrane .
moreover , myeloperoxidase activity in 
 gingival tissue showed a significant increase in patients with periodontal 
 disease when compared to the control group , an indicative of a chronic 
 inflammatory process also reflected at a systemic level .
these results were 
 similar to the measurements obtained from the analysis of crevicular gingival 
 fluid in humans with periodontal disease .
oxidative stress processes and alterations in the 
 immune system are closely related and have been described in different 
 diseases , thus both the aspects also seem to be linked to the 
 pathogenesis of periodontal disease , and can also be detected in the 
 plasma of patients with periodontitis [ 8 , 27 , 
 35 ] .
however , the extent to which 
 ros overgeneration influences the initiation and progression of periodontal 
 diseases is still unknown . in conclusion , in spite of the 
 limited number of samples examined in the present study , the 
 results indicate a relationship between proinflammatory and oxidative stress 
 biomarkers and periodontal disease .
 objective . to evaluate the involvement of proinflammatory and oxidative stress markers in gingival tissue in individuals with chronic periodontitis . 
 subject and methods . 
 eighteen subjects were divided in two groups : experimental ( age 52.95.0 ) and control ( age 51.19.6 ) . 
 the activities of enzymatic antioxidants such as catalase , glutathione peroxidase ( gpx ) , glutathione s - transferase ( gst ) , glutathione reductase , nonenzymatic antioxidants : total glutathione and reduced glutathione , oxidized glutathione ( gssg ) , thiobarbituric acid reactive substances ( tbars ) , and myeloperoxidase activity ( mpo ) were evaluated in gingival tissues from interproximal sites . 
 statistical differences between groups were determined by independent student t test and p<.05 . 
 results . 
 individuals with periodontal disease exhibited a significant increase in the activities of mpo , gpx , gst , and also in tbars and gssg levels in gingival tissue compared to the control group ( p<.05 ) . 
 conclusion . 
 the results of the present work showed an important correlation between oxidative stress biomarkers and periodontal disease . 
 
1. INTRODUCTION
2. SUBJECTS AND METHODS
3. STATISTICAL ANALYSIS
4. RESULTS
5. DISCUSSION