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The Impact of Smoking Status
on Antioxidant Enzyme Activity
and Malondialdehyde Levels
in Chronic Periodontitis
Mine O
¨ztu
¨rk Tongucx,* O
¨nder O
¨ztu
¨rk,
†
Recep Su
¨tcxu
¨,
‡
Betu
¨l Mermi Ceyhan,
‡
Gizem Kılıncx,*
Yonca So
¨nmez,
§
Zuhal Yetkin Ay,* U
¨nal Sxahin,
†
Esra Baltacıog
˘lu,
i
and F.Yesxim Kırzıog
˘lu*
Background: The aim of this study is to investigate the im-
pact of smoking status on the systemic and local superoxide
dismutase (SOD), glutathione peroxidase (GSH-Px), and cat-
alase (CAT) activities and malondialdehyde (MDA) levels in
subjects with chronic periodontitis (CP).
Methods: Sixty-five CP patients (23 smokers [CP-S], 23 for-
mer smokers [CP-FS], and 19 non-smokers [CP-NS]) and 20
periodontally healthy non-smoker controls (PH-NS) were in-
cluded in the study. After the clinical measurements, serum
and gingival tissue samples were collected. SOD, GSH-Px,
and CAT activities and MDA levels in hemolysates and gingi-
val tissue samples were spectrophotometrically assayed.
Results: Blood MDA levels in all the periodontitis groups
were higher than in the PH-NS group but only the difference
between CP-FS and PH-NS groups was significant (P<0.01).
Gingival tissue MDA levels in the periodontitis groups were
significantly higher than that in the control group (P<0.01).
However, the control group had the highest gingival SOD,
GSH-Px, and CAT activities compared with all the peri-
odontitis groups (P<0.01). The CP-S group had the highest
gingival MDA levels and SOD, GSH-Px, and CAT activities
among the periodontitis groups, whereas the lowest values
were observed in the CP-NS group (P<0.01). The blood and
gingival MDA levels in the CP-FS group were similar in the
CP-NS group, whereas they were lower than in the CP-S group.
Conclusions: Systemic and local MDA levels are increased
by smoking in addition to the impact of periodontitis. The de-
creased local SOD, GSH-Px, and CAT activities observed in
periodontitis patients may increase with smoking. J Periodon-
tol 2011;82:1320-1328.
KEY WORDS
Antioxidants; oxidative stress; periodontitis; smoking.
Smoking causes the development
of various chronic diseases.
1
In
addition, cigarette smoking is
a well-established risk factor for peri-
odontitis and is associated with an in-
creased risk for periodontal attachment
loss (AL) and bone loss.
2
Smoking has
several detrimental effects on periodon-
tal tissues. These effects include chronic
reduction of blood flow, altered neutro-
phil function and cytokine and growth
factor production, inhibition of fibroblast
growth and attachment, and decreased
collagen production and vascularity.
2
Furthermore, smoking stimulates the
oxidative burst of neutrophils; increases
reactive oxygen species (ROS) produc-
tion; and leads to lipid peroxidation
(LPO), oxidation of protein thiols, and
alterations in protein carbonyls in
plasma.
3-5
The compulsory use of the
body’s reserves of antioxidants (AO) to
detoxify the excess free radicals in
smokers results in the alteration of the
level of the AO.
6
ROS are toxic substances that attack
and damage biologic molecules. ROS
are also involved in the pathogenesis of
several inflammatory disorders, such as
type 2 diabetes
7
and vascular diseases.
8
The human body has an array of non-
enzymatic and enzymatic AO defense
mechanisms to remove harmful ROS
to prevent their deleterious effects. The
* Department of Periodontology, Faculty of Dentistry, Suleyman Demirel University,
Isparta, Turkey.
† Department of Chest Diseases, Faculty of Medicine, Suleyman Demirel University.
‡ Department of Biochemistry, Faculty of Medicine, Suleyman Demirel University.
§ Department of Public Health, Faculty of Medicine, Suleyman Demirel University.
iDepartment of Periodontology, Faculty of Dentistry, Karadeniz Technical University,
Trabzon, Turkey.
doi: 10.1902/jop.2011.100618
Volume 82 • Number 9
1320
non-enzymatic AOs include vitamins E, A, and C; uric
acid; bilirubin; reduced glutathione; albumin; trans-
ferrin; lactoferrin; ceruloplasmin; and haptoglobin.
The enzymatic AOs include superoxide dismutase
(SOD), glutathione peroxidase (GSH-Px), and cata-
lase (CAT).
9
There is a balance between the produc-
tion of ROS and tissue concentration of AOs in the
body.
Periodontal disease is associated with reduced total
AO capacity and increased oxidative damage within
the oral cavity.
10-14
Furthermore, it has been shown
that patients with periodontitis have elevated levels
of local and systemic LPO.
15-21
Recently, it was dem-
onstrated that smoking increases the levels of free
radicals
22
and LPO
23
in periodontal tissues. In addi-
tion, decreased AO levels in blood, gingival tissue,
saliva, and gingival crevicular fluid (GCF) have been
shown in patients with periodontitis and gingivitis who
smoke.
22,24,25
Quitting smoking is essential for improving the
chances of a favorable outcome before beginning
periodontal treatments.
26
It was suggested that the
effects of smoking are reversible, because the risk
of developing periodontitis was clearly reduced on
smoking cessation.
27
In light of these findings, a de-
crease in LPO and oxidative damage in periodontal
tissues can be expected after smoking cessation.
There are no data in the literature regarding the sys-
temic and local periodontal SOD, GSH-Px, and CAT
activities and LPO levels in former smoker patients
with periodontitis. In addition, the mechanism for
the negative effects of smoking on the periodontium
is still unclear.
The aims of this study are to investigate the impact
of smoking status on the blood and gingival tis-
sue SOD, GSH-Px, and CAT activities and malondial-
dehyde (MDA) levels in patients with periodontitis
who are current, former, or non-smokers and to ex-
plore the relationships between the periodontal pa-
rameters and SOD, GSH-Px, and CAT activities and
MDA levels.
MATERIALS AND METHODS
A total of 65 otherwise healthy patients with chronic
periodontitis (CP) (32 males and 33 females; age
range: 20 to 50 years) and 20 periodontally healthy
non-smoker (PH-NS) volunteers (11 males and 9
females; age range: 25 to 49 years) were recruited
for the study. The patients with chronic periodontitis
were chosen from patients admitted to the Suleyman
Demirel University, Faculty of Dentistry, Department
of Periodontology, Isparta, Turkey, from October
2006 to July 2008. None of the patients had received
periodontal therapy or used antibiotics, non-steroidal
analgesics, sympathomimetics, or immunosuppres-
sive agents in the last 6 months. Patients with sys-
temic disease, those using regular supplementary
vitamins, and pregnant or lactating females were ex-
cluded from the study. Patients with moderate gener-
alized chronic periodontitis (i.e., £3mmAL,<5mm
throughout £30% of the mouth)
28
were recruited for
the study to make a clear distinction among the par-
ticipants in the groups of smokers, non-smokers, and
former smokers.
Participants with chronic periodontitis were classi-
fied into one of three groups as follows: 1) the current
smoker chronic periodontitis (CP-S) group consisted
of 23 smoker patients with chronic periodontitis
who have smoked £10 cigarettes per day for >5
years;
29
2) the former smoker chronic periodontitis
(CP-FS) group consisted of 23 former smoker sub-
jects with chronic periodontitis who had quit smoking
for >6 months; and 3) the non-smoker chronic peri-
odontitis (CP-NS) group consisted of 19 non-smoker
patients with chronic periodontitis who never smoked.
The control group (PH-NS) consisted of 20 non-
smoker volunteers who were admitted to Suleyman
Demirel University, Faculty of Dentistry, and Depart-
ment of Oral Surgery for tooth extraction, during the
same time period as the periodontitis patients, who
had neither history of periodontal disease nor tooth
loss caused by periodontitis and had no clinical signs
of periodontitis (clinical attachment level [CAL] <1
mm, probing depth [PD] £3 mm, gingival index [GI]
<1).
Written informed consent was obtained from all
patients. Ethical approval for the study was obtained
fromtheethicscommitteeofSuleymanDemirel
University, Faculty of Medicine (decree number:
02.06.2006 to 01/11).
All periodontal patients underwent supragingival
scaling to prepare tissues for biopsy. One week after
periodontal clinical measurements, gingival tissue,
and blood samplings were performed in the morning
hours after overnight fasting.
Clinical Measurements
Plaque index (PI),
30
GI,
31
PD, CAL, and presence of
bleeding on probing (BOP)
32
were measured at six
sites and recorded on each tooth, except third molars.
All clinical periodontal measurements were per-
formed by the same examiner (MO
¨T).
Blood Sampling
Venous blood samples were collected from the
antecubital vein in tubes with K
3
EDTA. The anticoa-
gulated blood was separated into plasma and erythro-
cytes by centrifugation at 1,500 ·g for 10 minutes at
4C. The erythrocyte samples were washed three
times in cold isotonic saline (0.9%, vol/wt) and then
hemolyzed with 2 mL of bidistilled water. All samples
were kept in -80C conditions until the date of analy-
sis. The SOD, GSH-Px, and CAT activities and MDA
J Periodontol • September 2011 O
¨ztu
¨rk Tongucx,O
¨ztu
¨rk, Su
¨tcxu
¨
1321
levels of the blood were measured in the hemolysates.
Butylated hydroxytoluene and EDTA were added
to the sample and reaction mixture to minimize the
oxidation of lipids that contributes artifactually dur-
ing sample processing and the thiobarbituric acid
reaction.
Gingival Tissue Sampling
Tissues were collected from patients before any peri-
odontal surgical procedure including scaling and root
planing under asepsis and local anesthesia, avoiding
local anesthetic infiltration into the biopsy site.
33
Gin-
gival tissue samples were harvested from the sulcular
margins of the gingival papillae between premolar or
molar teeth with moderate periodontal destruction
(2 mm <PD <4mmor2mm<CAL <5 mm) to stan-
dardize the gingival sampling.
The gingival tissue samples of the control group
were harvested from the gingival margins of the pre-
molar or molar teeth undergoing extraction because
of profound occlusal caries or orthodontic reasons.
All gingival samples were washed in saline solution
immediately after excision and dried with gauze at
4C. Tissue samples were stored in firmly wrapped
sterile Eppendorf tubes at -80C until analysis.
Laboratory Analyses
Preparation of the tissue homogenates. The gingival
tissue samples were weighed by an ocular sensitive
scale and homogenized
¶
(1:5, wt/vol) in 100 mmol/
L phosphate buffer (pH 7.4) in an ice bath. The ho-
mogenate was sonicated
#
for 30 seconds and centri-
fuged at 10,000 ·g for 10 minutes at 4C to remove
debris. The clear upper supernatant was taken and as-
says were carried out on this part. The preparation
procedures of tissue homogenates were performed
in an anaerobic environment. Protein concentrations
of homogenates were determined by the method of
Lowry et al.
34
Measurements of MDA levels in hemolysates
and gingival tissue homogenates. To evaluate the
LPO levels of hemolysates and gingival tissues,
levels of MDA were determined by the double heating
method of Draper and Hadley.
35
The principle of this
method is spectrophotometric measurement of the
color developed during reaction to thiobarbituric
acid with MDA. The concentration of MDA is ex-
pressed as nanomole per milligram hemoglobin
and nanomole per milligram protein in hemolysates
and tissues.
Measurements of SOD, GSH-Px, and CAT
activities in hemolysates and gingival tissue
homogenates. SOD, GSH-Px, and CAT activities in
the hemolysates and the gingival tissues were deter-
mined. The measurement of SOD was based on the
principle that xanthine reacts with xanthine oxidase
to generate superoxide radicals, which react with
2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazo-
lium chloride to form a red formazan dye. SOD activity
is then measured by the degree of inhibition of this re-
action.
36
The determination of GSH-Px activity was
based on the method of Paglia and Valentine.
37
The
activities of SOD and GSH-Px are expressed as unit
per milligram hemoglobin and unit per milligram pro-
tein in hemolysates and tissues. CAT activity was
measured according to the method of Aebi.
38
The
principle of the assay is based on the determination
of the rate constant (k, s-1) of decomposition of hy-
drogen peroxide by the enzyme CAT. The activity of
CAT is expressed as k/mg hemoglobin and milligram
protein in hemolysates and tissues.
Hemoglobin concentration was determined by the
cyanomethemoglobin method from the hemolyzed
erythrocytes.
39
An autoanalyzer** was used to de-
termine the activities of SOD and GSH-Px, and a
spectrophotometer
††
was used to estimate the activ-
ities of the enzyme CAT.
Statistical Analyses
The sex differences of the groups were investigated by
x
2
test. The normality of the data distribution was ex-
amined by Kolmogorov-Smirnov test. The only nor-
mally distributed data were age. Age was expressed
as mean –SD and the group comparisons regarding
age were made by using an independent samples t
test. The other parameters were presented as median
values (min-max). The Kruskal-Wallis test followed
by the Mann-Whitney Utest with Bonferroni correc-
tion was used for the group comparisons for the
non-normally distributed data. Pearson correlations
were used to look at the relationships among smoking
status, blood and tissue AO enzyme, and MDA levels
and clinical parameters. A significance level of P
<0.05 was used for the Kruskal-Wallis test and P
<0.01 was accepted as the significance level for the
Mann-Whitney Utest with Bonferroni correction.
The statistical analyses were performed by a package
program.
‡‡
RESULTS
Clinical Findings
Demographic variables and median (min-max)
values of clinical periodontal measurements are given
in Table 1. There were no statistically significant dif-
ferences among the study groups regarding age and
sex (matching variables).
There were no statistically significant differences
among the periodontitis groups regarding periodontal
clinical parameters except PI. The PI value of the CP-S
¶ Ultra Turrax T25, Janke &Kunkel, Staufen, Germany.
# Bandelin Sonoplus UW 2070, Berlin, Germany.
** Aeroset, Abbott, Abbott Park, IL.
†† UV-1601, Shimadzu, Kyoto, Japan.
‡‡ SPSS 15, IBM, Chicago, IL.
Smoking and Antioxidant Enzyme Activity in Periodontitis Volume 82 • Number 9
1322
group was significantly higher than that of the CP-FS
group (P<0.01). There was no significant difference
between the periodontitis groups regarding the mean
PD of the gingival tissue sampling site (P>0.05). How-
ever, there were significant differences between the
PH-NS group and all of the periodontitis groups re-
garding the PD of the sampling region (P<0.01).
Laboratory Findings
The median (min-max) MDA levels and SOD, GSH-
Px, and CAT activities in hemolysates and gingival
tissues of the groups are given in Table 2. Blood
MDA levels in all the periodontitis groups were higher
than in the PH-NS group but only the difference be-
tween CP-FS and PH-NS groups was significant (P
<0.01). Although blood MDA levels and SOD and
CAT activities were close to each other in all peri-
odontitis groups (P>0.05), the GSH-Px activity in
the CP-S group was significantly lower compared to
that of the CP-NS group (P<0.01). The lowest blood
MDA levels and SOD and GSH-Px activities were ob-
served in the PH-NS group, whereas the highest blood
CAT activity was detected in this group, compared to
the periodontitis groups.
Gingival tissue MDA levels in the periodontitis
groups were significantly higher than that in the
PH-NS group (P<0.01). There was a progressive re-
duction in the gingival tissue MDA levels and SOD,
GSH-Px, and CAT activities from the CP-S to the
CP-FS to the CP-NS patients. The highest AO enzyme
activity and the lowest MDA levels were observed in
the gingival tissues of the PH-NS compared to the
other groups (P<0.01). The gingival MDA levels de-
tected between the groups were statistically signifi-
cantly different (P<0.01), except for the difference
between the CP-NS and the CP-FS groups.
Gingival tissue SOD activity in the CP-NS group
was significantly lower than in the other groups (P
<0.01). However, there was no significant difference
in tissue SOD levels between the CP-S and CP-FS
groups.
Tissue GSH-Px activity levels of the CP-S and CP-
FS groups were close, but that of the CP-NS group
was significantly lower than those of the other groups
(P<0.01). The tissue CAT activities were significantly
different between the groups (P<0.01). The lowest
tissue CAT activity was observed in the CP-NS group,
whereas the same and the highest CAT activities were
observed in the CP-S and PH-NS groups.
Correlations
There were significant correlations between periodon-
tal parameters, smoking-related parameters, and AO
enzyme activity and MDA levels in the blood and gin-
giva. The correlations between the parameters are
presented in Table 3.
Ta b l e 1 .
Intergroup Comparisons of Demographic Variables and Clinical Periodontal Parameters
Parameters CP-S (n =23) CP-FS (n =23) CP-NS (n =19) PH-NS (n =20)
Sex (male/female)
a
11/12 12/11 9/10 11/9
Age median (min-max) 20 to 47 22 to 49 20 to 50 25 to 49
Age (mean –SD)
b
(36.08 –8.33) (36.65 –8.14) (36.00 –9.32) (35.21 –9.07)
Packs/year (SD)
c
15.82 –6.16 15.38 –12.25
†
00
Smoking duration (year) (SD)
c
16.95 –8.26 17.47 –7.58
†
00
Time since smoking cessation (year) (SD)
c
0 3.43 –4.04 0 0
PI median (min-max)
c
1.28 (0.47 to 2.79) 0.87 (0.38 to 2.29)*0.98 (0.47 to 2.56) 0.44 (0.29 to 0.68)
‡§i
GI median (min-max)
c
0.96 (0.05 to 1.29) 0.64 (0.03 to 1.93) 0.76 (0.27 to 1.57) 0.27 (0.07 to 0.65)
‡§i
PD (mm) median (min-max)
c
2.78 (1.18 to 4.24) 2.56 (1.06 to 4.32) 2.41 (1.57 to 4.59) 2.05 (1.67 to 2.32)
‡§i
PD gingival tissue sampling site (mm)
median (min-max)
c
3 (2 to 4) 3 (2 to 4) 3 (2 to 3) 1 (1 to 2)
‡§i
CAL (mm) median (min-max)
c
3.24 (1.96 to 5.08) 3.24 (1.06 to 5.04) 2.55 (1.57 to 5.06) 2.05 (1.67 to 2.32)
‡§i
BOP (%) median (min-max)
c
62 (0 to 100) 64 (0 to 100) 78 (0 to 100) 28 (10 to 31)
‡§i
* Significant difference between CP-S and CP-FS groups (P<0.01).
† Significant difference between CP-FS and CP-NS groups (P<0.01).
‡ Significant difference between CP-S and PH-NS groups (P<0.01).
§ Significant difference between CP-FS and PH-NS groups (P<0.01).
iSignificant difference between CP-NS and PH-NS groups (P<0.01).
a=x
2
test; b =Independent samples ttest; c =Mann-Whitney Utest with Bonferroni correction.
J Periodontol • September 2011 O
¨ztu
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¨tcxu
¨
1323
DISCUSSION
The findings of this study indicate that blood and gin-
gival tissue MDA levels and SOD and CAT activities
and gingival tissue GSH-Px activity are increased
in smoker patients with periodontitis compared with
former smoker and non-smoker patients with peri-
odontitis. There are studies conducted on smoker
and non-smoker patients with periodontitis regarding
the oxidative capacity of blood, saliva, gingiva, or
GCF.
22-25,40
However, we did not come across any
analogous studies conducted on current, former,
and non-smoker patients with periodontitis. To our
knowledge, this is the first study investigating the
blood and gingival tissue MDA levels and SOD,
GSH-Px, and CAT activities in current, former, and
non-smokers who have the same level of periodontal
AL and comparing them to those of periodontally
healthy patients who do not smoke.
The studies investigating local AO capacity and
oxidative stress related to periodontal disease
Table 2.
Median (min-max) Values and Intergroup Comparisons of Malondialdehyde and
Antioxidant Enzyme Levels in Blood and Gingival Tissue
Biochemical Parameters CP-S (n =23) CP-FS (n =23) CP-NS (n =19) PH-NS (n =20)
Blood MDA (nmol/mg hemoglobin) 45.9 (27 to 138.2) 44.8 (26.5 to 161.6) 42.5 (27.8 to 180.5) 36.3 (30 to 54.3)*
SOD (U/mg hemoglobin) 1,500 (991.2 to 2,639.8) 1,393.4 (425 to 3,786.6) 1,426.9 (344.6 to 2,184.5) 1,023.8 (343.8 to 1,596.8)
†‡
GSH-Px (U/mg hemoglobin) 10.1 (2.6 to 29.2) 16.5 (1.1 to 75.7) 17.5 (6.6 to 26.7)
§
7.4 (3.4 to 18.6)*
‡
CAT (CAT/mg hemoglobin) 19.7 (5.4 to 46.4) 18.3 (7.8 to 95.8) 19.1 (8 to 39.9) 51 (3.8 to 539.8)*
†‡
Gingival Tissue MDA (nmol/mg protein) 1.21 (1.12 to 2.54) 1.05 (0.86 to 1.31)
i
1.00 (0.84 to 1.40)
§
0.63 (0.34 to 1.62)*
†‡
SOD (U/mg protein) 1.37 (1.08 to 1.95) 1.30 (1.25 to 1.67) 1.12 (1.08 to 1.50)
§¶
2.42 (1.58 to 2.80)*
†‡
GSH-Px (U/mg protein) 173.7 (156.8 to 257.3) 155.8 (102.3 to 215.4) 135.6 (73.4 to 189.27)
§¶
284.4 (237.3 to 421.4)*
†‡
CAT (CAT/mg protein) 2.63 (0.87 to 6.53) 1.60 (0.62 to 2.36)
i
0.88 (0.33 to 1.19)
§¶
2.63 (0.85 to 2.89)*
‡
* Significant difference between CP-FS and PH-NS groups (P<0.01).
† Significant difference between CP-S and PH-NS groups (P<0.01).
‡ Significant difference between CP-NS and PH-NS groups (P<0.01).
§ Significant difference between CP-S and CP-NS groups (P<0.01).
iSignificant difference between CP-S and CP-FS groups (P<0.01).
¶ Significant difference between CP-FS and CP-NS groups (P<0.01).
Table 3.
Correlations Among Periodontal Parameters, Smoking-Related Parameters, Blood and
Gingival Tissue, Malondialdehyde, and Antioxidant Enzyme Levels in all Participants
Correlations rP Correlations rP
Age-PD 0.331 0.002 CAL-GT SOD -0.425 0.000
Age-CAL 0.384 0.000 CAL-GT GSH-Px -0.348 0.000
PI-packs/year 0.407 0.000 BOP-GT SOD -0.493 0.000
PI-smoking duration 0.362 0.001 BOP-GT GSH-Px -0.445 0.000
PI-GT-SOD -0.467 0.000 B GSH-Px-B MDA 0.337 0.002
PI-GT GSH-Px -0.423 0.000 GT SOD-packs/year -0.383 0.000
GI-GT SOD -0.494 0.000 GT SOD-smoking duration -0.357 0.001
GI-GT GSH-Px -0.513 0.000 GT GSH-Px-time since smoking cessation -0.340 0.000
GI-packs/year 0.344 0.001 GT GSH-Px-GT SOD 0.777 0.000
PD-GT SOD -0.345 0.001 GT GSH-Px-GT CAT 0.449 0.001
CAL-packs/year 0.461 0.000 GT CAT-GT MDA 0.442 0.000
CAL-smoking duration 0.465 0.000 GT CAT-GT SOD 0.344 0.001
B=blood; GT =gingival tissue; r=Pearson correlation coefficient.
Smoking and Antioxidant Enzyme Activity in Periodontitis Volume 82 • Number 9
1324
were mostly conducted with saliva or GCF
samples.
14,17,20,21,23-25,41-43
The number of studies
investigating LPO and AO levels in gingival tissue
was quite small and these gingival samples were ob-
tained during flap surgery followed by initial periodontal
treatment.
19,22,44,45
The reduction of oxidative stress
and increase in AO capacity after the non-surgical
periodontal therapy were reported in the litera-
ture.
23,42,46
In contrast D’Aiuto et al.
47
demonstrated
that acute increases in reactive oxygen metabolites
and systemic inflammation occurred after periodontal
therapy. In addition, it was suggested that periodontal
therapy could not only alter the local ROS production
and AO state, but also the host systemic oxidative
state.
47
In the present study, to eliminate the effects
of periodontal therapy on the local and systemic
SOD, GSH-Px, and CAT activities and MDA levels,
and to evaluate the additional effects of smoking on
the tissues having moderate periodontitis accurately,
the blood and tissue samples were obtained before
non-surgical periodontal treatment.
An increase inLPO in the blood and periodontium of
patients with periodontitis has been reported.
16,20,21
In agreement with those results, blood and gingival
tissue MDA levels in all the periodontitis groups were
found to be elevated compared to patients who were
periodontally healthy. However, only the gingival tis-
sue MDA levels in the periodontitis groups and the
blood MDA level of former smoker patients with peri-
odontitis were significantly different than those of the
control group.
Smoking increases the level of free radicals and
causes oxidative damage in the tissues. A study inves-
tigating the effects of smoking on blood oxidative
parameters irrespective of periodontal status demon-
strated that blood MDA levels of active smokers were
higher than those of non-smokers.
41
In addition, it was
reported that MDA levels increased in the gingival bi-
opsy specimen and saliva of the smoker patients with
periodontitis.
22,23
The results of our study supported
the theory that the MDA levels in smokers were ele-
vated and demonstrated that the MDA levels in the
gingival tissues of the former smokers were signifi-
cantly lower than in current smokers.
It has been reported that total AO capacity and non-
enzymatic AO levels are decreased in patients with
periodontitis,
13,14,19,23,42,43
but the results related to
enzymatic AO activity in patients with periodontitis
were conflicting. Some studies investigating the effect
of periodontitis on the SOD activity in the literature
suggested that the SOD activity in plasma,
19,44
eryth-
rocytes,
19
gingival tissue,
19,44
saliva,
46
and GCF
44
of
the patients with periodontitis were higher than those
of periodontally healthy patients, whereas others re-
ported a decrease in the SOD activity levels in the
gingival tissue,
45
serum,
43,48,49
GCF,
43,48,49
and
saliva
49
of the patients with periodontitis. Similar to
these results, we observed higher blood and lower gin-
gival SOD activity in the subjects with periodontitis
compared to the non-smoker healthy controls. It
was reported that the SOD activity in the blood and
periodontium of the smoker patients with peri-
odontitis was lower than in the non-smoker patients
with periodontitis.
22,23,25
In contrast, we observed
an insignificant increase in the CP-S group and an
insignificant decrease in the CP-FS group compared
with the CP-NS group as regards blood SOD activ-
ity, whereas the gingival SOD activities of the CP-S
and CP-FS groups were significantly higher than
that of the CP-NS group. This difference may be
caused by the low levels of periodontal destruction
of the sampling region in our study compared to the
other studies.
22,23,25
The studies evaluating the ef-
fects of smoking on SOD activity regardless of peri-
odontal status suggested that smoking increases the
SOD activity in the blood and saliva.
40,41,50-52
More-
over, it was reported that after the smoking cessation,
the increased SOD activity had decreased to those
found in non-smoker subjects.
51
The results of our
study support these findings.
The increased GSH-Px activity in the plasma,
19
sa-
liva,
20,23
GCF,
17,20,53
erythrocytes,
19
and gingival tis-
sues
19
of the patients with periodontitis were reported.
Similar with these results, the GSH-Px activity in the
blood of the non-smoker patients with periodontitis
was significantly higher than that of non-smoker con-
trols, whereas GSH-Px activity in gingival tissue was
significantly lower. Guentsch et al.
23
reported that
GSH-Px activity in the saliva of the smoker patients
with or without periodontitis was higher than the
matched non-smoker controls. In the present study, a
slight and insignificant increase in blood GSH-Px ac-
tivity was observed in the CP-S group compared to the
PH-NS group. However, the blood GSH-Px activity of
CP-S group was lower than that of CP-FS and CP-NS
groups. These results were supported by the results of
other studies that reported decreased plasma and sa-
liva GSH-Px activity of smoker subjects.
50,52
As differ-
ent from the other studies, the gingival tissue GSH-Px
activity in the CP-S, CP-FS, and CP-NS groups was
significantly lower than in the PH-NS groups, and
the CP-S group had the highest gingival GSH-Px
activity among the periodontitis groups.
There was only one study in the literature regarding
the effects of periodontitis on CAT activity of plasma,
erythrocytes, and gingival tissues of the non-smoker
subjects.
19
The increased CAT activity in non-smoker
patients with periodontitis was reported.
19
In contrast,
we found decreased blood and gingival tissue CAT
activity of the non-smoker patients with periodon-
titis compared to healthy controls. McCusker and
Hoidal
51
suggested that the CAT activity in alveolar
J Periodontol • September 2011 O
¨ztu
¨rk Tongucx,O
¨ztu
¨rk, Su
¨tcxu
¨
1325
macrophages of patients who smoke were twice that
found in non-smoker patients. The increased CAT ac-
tivity in blood and gingival tissues of the smoker pa-
tients with periodontitis compared to non-smoker
patients with periodontitis were also reported.
22
The
findings of our study support these results.
The principal radical in the tar phase of cigarette
smoke is a quinine-hydroquinone complex that can
reduce molecular oxygen to superoxide radicals.
6
Polymorphonuclear leukocytes and macrophages
produce superoxide as an antibacterial agent in case
of bacterial challenge to the periodontium.
10
Because
periodontal tissues are directly exposed to ciga-
rette smoke, the increase of superoxide level in the
periodontal tissues of the smoker patients with peri-
odontitis is an expected result. Superoxide is removed
from tissues by dismutation to hydrogen peroxide
spontaneously or catalyzed by SOD. The hydrogen
peroxide formed is removed by CAT in the intracellu-
lar environments or by GSH-Px in the extracellular
environments.
10
It was suggested that although
smoking induces selective increase of AO enzyme ac-
tivity in the tissues as self-defense mechanism,
51
this
increase is not sufficient to protect the tissues from the
harmful effects of smoking.
54
In the present study, in-
creased gingival SOD, GSH-Px, and CAT activities in
smokers compared to periodontally matched non-
smoker and former smoker patients may be the result
of a protective and adaptive mechanism developing
in the tissue.
In accordance with the fact that cigarette smoking
is one of the important risk factors for periodontal AL,
strong positive correlations between CAL and smok-
ing duration and yearly cigarette consumption were
observed in this study. In addition, it was determined
that there were strong negative correlations between
gingival tissue GSH-Px levels and smoking duration
and yearly cigarette consumption.
The studies in the literature regarding the effects of
quitting smoking on periodontal health reported that
preferable results
24
and greater attachment gain
25
were obtained with periodontal treatment and that
there was an increase in blood flow to the gingival tis-
sues
27
in patients who quit smoking. Bergstro
¨m
et al.
55
found that former smokers have better peri-
odontal health than current smokers, although worse
than that of non-smokers.
The other interesting finding of our study was the re-
duction in the blood and gingival MDA levels and AO
enzyme activity except the blood GSH-Px activity in
former smoker patients with periodontitis compared
to smoker patients with periodontitis. Moreover, the
MDA levels and enzymatic AO activities of former
smokers were close to those of the non-smoker pa-
tients with periodontitis. It was observed that there
was negative correlation between time since smoking
cessation and the gingival tissue GSH-Px activity in
the former smoker patients with periodontitis. Our
findings are consistent with the study suggesting that
the filtration of smoke within the tissues induced the
increase in AO activities and that after smoking ces-
sation, the increased activities had returned to those
found in non-smoker patients.
50
There were a number of limitations to the study.
The non-longitudinal design of the study made it dif-
ficult to evaluate the effects of smoking cessation on
the MDA levels and AO enzyme activity of the peri-
odontium. The lack of current and former smoker
periodontally healthy groups limited the evaluation
of the effects of periodontitis and smoking on the
MDA levels and AO enzyme activity separately.
CONCLUSIONS
Within the limitation of the study, we conclude that
both periodontitis and smoking lead to significant
changes in MDA production and AO enzyme activity
in blood and gingival tissues. The combination of
smoking and periodontitis resulted in significant in-
creases in MDA levels and alterations in SOD, GSH-
Px, and CAT activities in periodontium. Quitting
smoking can reverse the negative effects of smoking
on the AO balance of the periodontium. Smoking ces-
sation programs should be a standard component of
periodontal therapy. Further longitudinal and cross-
sectional studies are needed for the determination
of when the decrease in the oxidative stress begins
after smoking cessation and to clarify the role of
smoking on the oxidative and AO status in healthy
periodontal tissues.
ACKNOWLEDGMENTS
This study was supported by a grant from the Scien-
tific Research Commission of Suleyman Demirel
University (grant number: 1300-M 06). The authors
report no conflicts of interest related to this study.
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Correspondence: Dr. Mine O
¨ztu
¨rk Tongucx, Department of
Periodontology, Faculty of Dentistry, Suleyman Demirel
University, Isparta, Turkey. Fax: 90-246-2370607; e-mail:
mineperio@gmail.com.
Submitted October 12, 2010; accepted for publication
December 24, 2010.
Smoking and Antioxidant Enzyme Activity in Periodontitis Volume 82 • Number 9
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