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Study of antioxidant enzymes superoxide dismutase and glutathione peroxidase levels in tobacco chewers and smokers: A pilot study

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Context: Free radical associated damages play a major role in causation of cancer in tobacco habituates. The free radicals released by tobacco bring about alterations in antioxidant levels in humans and these free radical associated damages are reflected through antioxidant enzyme activities in blood. Aims: To evaluate the effects of tobacco consumption on the erythrocyte Antioxidant enzymes-Superoxide dismutase (SOD) and Glutathione Peroxidase (GPx) as they act as first line of defense antioxidants. Materials and Methods: A case control study comprising of 4 study groups of healthy controls (n = 27), smokers (n = 27), tobacco chewers (n = 30) and combination habit (n = 22) were included. Erythrocyte SOD and GPx enzyme activities were measured by spectrophotometry. The results were statistically analyzed using one way-Anova and Mann Whitney test. Results: The data analysis revealed an alteration in mean SOD levels as it was decreased in cases compared to control group where as mean GPx was seen to be increased in cases compared to controls. When SOD and GPx were compared for the frequency and duration of habit, GPx showed a significant decrease in chewers with increase in frequency and duration of habit. Conclusions: The present study gave us an insight about the relationship between antioxidant enzyme activity, oxidative stress and tobacco. The altered antioxidant enzyme levels observed in this study will act as a predictor for pre potentially malignant lesions. Therefore an early intervention of tobacco habit and its related oxidative stress would prevent the development of tobacco induced lesions.
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210 Journal of Cancer Research and Therapeutics - April-June 2013 - Volume 9 - Issue 2
Chundru
Venkata Naga
Sirisha, Ram
Manohar M1
Department of Oral
and Maxillofacial
Pathology,
C.K.S.Theja Institute
of Dental Sciences and
Research, Tirupathi,
Andhra Pradesh,
1Department of Oral
and Maxillofacial
Pathology, Educare
Institute of Dental
Sciences, Malappuram,
Kerala, India
For correspondence:
Dr. Chundru Venkata
Naga Sirisha,
Department of Oral
and Maxillofacial
Pathology, C.K.S Theja
institute of Dental
Sciences and Research,
Chadalawada nagar,
Renigunta Road,
Tirupathi, India.
E-mail:
sirisha@doctor.com
Access this article online
Website: www.cancerjournal.net
DOI: 10.4103/0973-1482.113352
PMID: ****
Quick Response Code:
Original Article
Study of antioxidant enzymes superoxide
dismutase and glutathione peroxidase levels in
tobacco chewers and smokers: A pilot study
ABSTRACT
Context: Free radical associated damages play a major role in causation of cancer in tobacco habituates. The free radicals released
by tobacco bring about alterations in antioxidant levels in humans and these free radical associated damages are reflected through
antioxidant enzyme activities in blood.
Aims: To evaluate the effects of tobacco consumption on the erythrocyte Antioxidant enzymes-Superoxide dismutase (SOD) and
Glutathione Peroxidase (GPx) as they act as first line of defense antioxidants.
Materials and Methods: A case control study comprising of 4 study groups of healthy controls (n = 27), smokers (n = 27), tobacco
chewers (n = 30) and combination habit (n = 22) were included. Erythrocyte SOD and GPx enzyme activities were measured by
spectrophotometry. The results were statistically analyzed using one way-Anova and Mann Whitney test.
Results: The data analysis revealed an alteration in mean SOD levels as it was decreased in cases compared to control group where
as mean GPx was seen to be increased in cases compared to controls. When SOD and GPx were compared for the frequency and
duration of habit, GPx showed a significant decrease in chewers with increase in frequency and duration of habit.
Conclusions: The present study gave us an insight about the relationship between antioxidant enzyme activity, oxidative stress and
tobacco. The altered antioxidant enzyme levels observed in this study will act as a predictor for pre potentially malignant lesions. Therefore
an early intervention of tobacco habit and its related oxidative stress would prevent the development of tobacco induced lesions.
KEY WORDS: Anti-oxidant enzymes, Glutathione Peroxidase (GPx), Oxidative stress, Superoxide Dismutase (SOD), tobacco.
INTRODUCTION
Smoking and chewing of tobacco is a vice that
has been practiced by millions of people all over
the world. It is estimated that among 400 million
individuals aged 15 years and above in India, 47%
use tobacco in one or the other form. Of the 250
million kilograms of tobacco cleared for domestic
consumption in India, 86% is used for smoking and
14% is used in the smokeless form (1% as snuff).[1]
Tobacco use in various forms is considered
as the major etiological factor for oral cancer
development, accounting for 30-40% of all cancer
cases in India. Tobacco consumption generates
free radicals and results in increased oxidative
stress and lipid peroxidation.[2] Lipid peroxidation
is a chain reaction providing a continuous supply
of free radicals that initiate further peroxidation
unless checked by antioxidants.
In normal cells there is an intricate balance between
pro-oxidant and antioxidant states but in oxidative
stress this balance shifts towards pro-oxidants.[3] The
imbalance between pro-oxidants and antioxidants
linked to decreased smoke related antioxidant
capacity and increased free radical generation
especially in arterial tissue might render smokers
more prone to peroxidative stress.[4]
The heat (generated during smoking) as well as
pH (change during chewing) of body fluids due to
tobacco consumption affects the formation and
stabilization of free radicals. The alkaline conditions
observed in betel nut chewing are reported to favor
the formation of free radicals.[2] Reactive oxygen
metabolites (ROMs) such as superoxide anion (O2
),
hydrogen peroxide (H2O2) and hydroxyl radical
(OH), malondialdehyde (MDA) and nitric oxide
(NO) are directly involved in multi stage process
of carcinogenesis by bringing out a continuous
endogenous damage to cellular DNA.[2]
Inactivation and removal of these reactive
oxygen species depend on reactions involving the
antioxidative defense system. Antioxidants have
a shielding role by scavenging the free radicals.
The free radicals released by tobacco are known to
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Journal of Cancer Research and Therapeutics - April-June 2013 - Volume 9 - Issue 2
Sirisha, et al.: Antioxidant enzymes and tobacco
bring about alterations in antioxidant levels in humans, and
these free radical-associated damages are reflected through
antioxidant enzyme activities in blood. Thus, it seems that
studying biological parameters like antioxidant enzyme system
would be of fundamental importance in evaluating the role of
tobacco on antioxidant status in tobacco users.
Since the first line defense antioxidants are SOD, GPx and
Catalase (CAT), the present study was undertaken to establish
the effects of tobacco chewing and smoking on the antioxidant
enzymes SOD and GPx levels.
The objectives of this study were as follows:
1. To study the levels of antioxidant enzymes SOD and GPx
in erythrocytes of healthy individuals with the habit of
tobacco usage either in the form of smoking, chewing or
both (combination habit)
2. To compare these levels with healthy controls without any
tobacco habit
MATERIALS AND METHODS
The study was carried out in 106 age matched healthy male
subjects enrolled from College of Dental Sciences, Bapuji Dental
College and Hospital, Davangere.
The study included 30 healthy tobacco chewers, 27 smokers,
and 22 subjects with combination habit. For comparison
purpose we selected 27 age matched healthy males without
any tobacco habits. The age group of patients ranged from 19
years to 68 years. They were divided into age matched groups
for age wise comparison.
Ethical committee clearance and prior informed consent of all
the subjects was obtained before conducting the study.
Cases and controls were grouped into 4 groups as follows:
GROUP I - Chewers
GROUP II - Smokers
GROUP III - Combination habit
GROUP IV - Controls
All subjects were personally interviewed through a
questionnaire. It included details of tobacco consumption
(duration, type & frequency), dietary supplements such as
vitamins, drug intake, alcohol, medical history etc. Though the
Inclusion criteria was duration of habit not less than 1 year,
only one out of the 79 cases enrolled had one year of habit
duration and rest had minimum of 3 years and maximum of 45
years of habit duration. Group III subjects interestingly had one
of the habit for longer duration compared to the other habit.
Exclusion criteria were Alcohol, infectious diseases, systemic
diseases such as Diabetes mellitus, Hypertension which may
also influence the antioxidant status. Only healthy subjects
with strong history of tobacco habit and devoid of any tobacco
related lesions were included to see if habit alone could have
caused an alteration of antioxidant enzyme levels long before
the onset of any tobacco related lesions as these findings can
have different impact on the outcome of the study.
BLOOD SAMPLES
About 2 ml of venous blood was collected from all study
subjects during mornings between 9-11 am so as to see that
the time of blood collection was similar for all the subjects
using heparin as anticoagulant. 0.5 ml of whole blood was
centrifuged for 10 minutes at 3000 rpm and plasma was
separated. Erythrocytes were then washed 4 times with 3 ml
of 0.9% NaCl solution centrifuging for 10 minutes at 3000 rpm
after each wash. The washed and centrifuged erythrocytes
were then made up to 2 ml with cold re-distilled water, mixed
and left to stand at +40c for 15 minutes. The lysate was diluted
with 0.01mol/L Phosphate buffer pH 7.0, So that percentage
inhibition falls between 30% and 60%.
SOD estimation was based on the method of McCord and
Fridovich[5] where as GPx was estimated by the method of Paglia
and Valentine.[6] Enzymes SOD and GPx were determined using
Randox kits - RANSOD and RANSEL respectively.
SOD was analyzed on XL-600 Auto analyzer and GPx on Erba-
Chem-Pro semi Auto analyzer for spectrophotometry.
Finally, the results obtained were carefully recorded and
statistically analyzed by using one way ANOVA and Mann-
Whitney test.
RESULTS
Sample size and age wise distribution of cases and controls is
illustrated in Table 1.
When one way Anova test was used, no statistically significant
difference for age was found between all the 4 groups. The values
of mean with standard deviation, median and range of SOD and
GPx were calculated for all the 4 groups and are given in Table 2.
When SOD activity was compared between cases and controls,
mean SOD value showed a trend of gradual depletion in
cases compared to control group, though the depletion was
Table 1: Sample size and age wise distribution of cases and
controls
Age group
(years)
Chewers
n
Smokers
n
Smoking +
chewing n
Controls
n
16-30 11 7 6 9
31-45 13 9 9 9
46-70 6 11 7 9
Total 30 27 22 27
Mean age ± SD 36.2 ± 11.5 41.9 ± 13.2 38.7 ± 11.6 37.6 ± 12.3
Range 19-60 19-68 19-58 19-64
ANOVA, F = 1.08, P = 0.36 NS
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212 Journal of Cancer Research and Therapeutics - April-June 2013 - Volume 9 - Issue 2
Sirisha, et al.: Antioxidant enzymes and tobacco
statistically non-significant [Figure 1]. GPx showed an inverse
pattern where mean GPx was lower in controls compared
to cases. The increase in GPx in cases was statistically
insignificant [Figure 2].
Mean SOD and GPx were compared among cases for frequency
of the habit [Table 3]. On application of Mann-Whitney test
for pair wise comparison between the groups, a significant
difference (P < 0.05) was found for GPx with increase in
frequency of habit for chewers and smokers.
Mean SOD and GPx were compared among cases for the
duration of tobacco habit [Table 4]. Mean GPx has significantly
decreased (P < 0.05) with increase in duration of habit for
chewers while it has slightly decreased in smokers and
combination users.
Relationship between age and SOD, GPx was shown in Table 5.
Pearson’s correlation coefficient showed positive correlation
for age for SOD among all the 4 groups.
Positive correlation with age was noted for GPx among
controls and negative correlation among chewers, smokers
and combination habit and was statistically significant among
chewers and smokers (P < 0.05).
DISCUSSION
Free radical associated damages leads to an imbalance between
pro-oxidant and anti-oxidant states. This imbalance plays an
important causative role in carcinogenesis. [7] Anti-oxidants
have a shielding role by scavenging the free radicals and SOD,
GPx form the first line defense anti-oxidants.[8]
As free radical associated damages are reflected through anti-
oxidant enzyme activities in blood, the present study was
undertaken to establish the effects of tobacco on the above
anti-oxidant enzymes.
Table 3: Relationship between frequency of habit and SOD, GPx
Chewers Smokers Chewing and smoking
Type No. SOD GPx Freq/day No. SOD GPx Freq/day No. SOD GPX
Gutkha 10 289.1 ±
205.0
17189 ±
5786
Cig < 10 14 290.6 ±
180.8
10149 ±
5359
< 20 13 276.6 ±
151.3
10996 ±
5313
Plain tobacco 20 248.7±
154.3
10463±
6771
Cig ≥ 10 13 321.7 ±
142.5
17860 ±
8946
≥ 20 9 255.2 ±
135.6
13223 ±
7969
Signicance* P = 0.88
NS
P < 0.05
S
P = 0.34
NS
P <0.05
S
P = 0.74
NS
P = 0.64
NS
* Mann-Whitney test, P < 0.05 Signicant, P > 0.05 Not signicant
Table 2: SOD and GPx levels in various groups
Groups No. Particulars SOD (U/ml) GPx (U/l)
I. Chewers 30 Mean ± SD 262.2 ± 170.3 12705 ± 7129
Median 218 11902
Range 63 - 741 1173 - 29712
II. Smokers 27 Mean ± SD 305.5 ± 161.1 13861 ± 8168
Median 253 10689
Range 76 - 697 3108 - 34116
III. Smokers
& chewers
22 Mean ± SD 267.9 ± 142.1 11907 ± 6448
Median 273 9455
Range 69 - 564 4485 - 31517
IV. Controls 27 Mean ± SD 322.4 ± 191.4 11759 ± 6266
Median 269 10074
Range 88 - 694 4141 - 30324
ANOVA, F F = 0.81, F = 0.48,
P = 0.49, NS P = 0.69, NS
Mean SOD: I < III < II < IV, Mean GPx: IV < III < I < II
Figure 1: SOD levels in control and study groups
Figure 2: GPx levels in control and study groups
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213
Journal of Cancer Research and Therapeutics - April-June 2013 - Volume 9 - Issue 2
Mean SOD was found to be decreased in cases compared
to controls. Cases are more prone to oxidative stress due
to increasing load of free radicals in body due to chemical
carcinogens present in tobacco.
The slight decrease in SOD observed in cases compared to
controls in our study probably relates to its utilization in
scavenging the free radicals as SOD catalyses the dismutation
of Superoxide O2
to Hydrogen peroxide (H2O2), which then
must be removed by GPx or CAT
O2
+ O2
+ 2 H + SOD H2O2 + O2
H2O2 + 2GSH GPx GSSG + 2 H2O
Our study was similar to that of Zhou J[9] who found
significantly low levels of erythrocyte SOD in smokers
compared to non-smokers.
Mean GPx was found to be slightly increased in cases compared
to controls as it acts after SOD and this increase could be
co-related to an adaptive phenomenon when free radical
generation exceeds the quenching capacity of SOD.
Our study was similar to that of Beena P Patil[2] and Yildiz[10]
who showed that erythrocyte SOD was lowered and GPx was
elevated in tobacco users compared to non tobacco users.
Our study showed that an increase in duration and frequency
of tobacco habit significantly reduced GPx levels in chewers
emphasizing that frequency and duration of tobacco habit has
a definitive impact on antioxidant enzyme status.
Though the increase in frequency appeared to have caused
increase in GPx levels among smokers, the same showed a
decrease with increased duration of habit.
The present study results were comparable to that of Anderson
HR et al.[11] where in GPx negatively co-relates with tobacco
consumption for increase in frequency and duration of habit
probably indicating a decrease in GPx levels on prolonged
duration of tobacco usage.
CONCLUSION
The present study enlightens the possible relationship
between anti-oxidant enzyme levels, oxidative stress and
tobacco habit. The results of this pilot study can be validated
with a larger sample size, there by opening up avenues for
using these altered antioxidant enzyme levels as a predictor
for pre potentially malignant lesions. Tobacco habit coupled
with the lesion would probably have synergistic effect in
lowering the bodies antioxidant status paving way for an
enhanced oxidative stress. But further studies are required in
this direction before we can come to a conclusion.
An early intervention of tobacco habit and its related oxidative
stress is recommended.
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Table 5: Relationship between age and SOD, GPx
Correlation
between
SOD GPx
Gr-I
Chewers
Gr-II
Smokers
Gr-III
Smoking +
Chewing
Gr-IV
Controls
Gr-I
Chewers
Gr-II
Smokers
Gr-III
Smoking +
Chewing
Gr-IV
Controls
Age and SOD r-value 0.03 0.26 0.12 0.16 –0.36 –0.41 –0.28 0.23
Age and GPx P-value 0.87 NS 0.20 NS 0.59 NS 0.44 NS <0.05 S <0.05 S 0.21 NS 0.25 NS
* Pearson’s correlation coefcient (-ve sign indicates inverse correlation with age), P <0.05 signicant, P> 0.05 not signicant
Table 4: Relationship between duration of habit and SOD, GPx
Duration of
habits (yrs)
Chewers Smokers Chewing + smoking
nSOD (U\ml) GPx (U\l) nSOD (U\ml) GPx (U\l) nSOD (U\ml) GPx (U\l)
≤ 10 16 250.6 ± 117.4 15617 ± 6588 11 279.4 ± 206.2 13897 ± 9169 11 288.5 ± 171.6 13937 ± 8144
11-20 6 232.3 117.0 11392 ± 8383 9 308.2 ± 145.3 14790 ± 7886 4 240.3 ± 147.0 10119 ± 4656
≥ 21 8 307.7 ± 198.8 7867 ± 4446 7 343.2 ± 103.0 12612 ± 7933 7 251.2 ± 96.1 9738 ± 2997
ANOVA F = 0.40
P = 0.68
NS
F = 3.94
P <0.05
S
F = 0.32
P = 0.73
NS
F = 0.13
P = 0.88
NS
F = 0.22
P = 0.80
NS
F = 1.11
P = 0.35
NS
One – way Anova, Mann-Whitney test, P < 0.05 Signicant, P > 0.05 Not signicant
Sirisha, et al.: Antioxidant enzymes and tobacco
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214 Journal of Cancer Research and Therapeutics - April-June 2013 - Volume 9 - Issue 2
Cite this article as: Naga Sirisha CV, Manohar RM. Study of antioxidant
enzymes superoxide dismutase and glutathione peroxidase levels in
tobacco chewers and smokers: A pilot study. J Can Res Ther 2013;9:210-4.
Source of Support: Partly funded by Colgate –Palmolive, (INDIA) Ltd.,
Mumbai, Conict of Interest: Nil.
5. McCord JM, Fridovich I. Superoxide dismutase. An enzymic function
for erythrocuprein (Hemocuprein). J Biol Chem 1969;244:6049-55
6. Paglia DE, Valentine WN. Studies on the quantitative and qualitative
characterization of erythrocyte glutathione peroxidase. J Lab Clin
Med 1967;70:158-69.
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nitric oxide levels in patients with oral cavity cancer. Jpn J Clin Oncol
2004;34:379-85.
8. Ray G, Husain SA. Oxidants, antioxidants and carcinogenesis. Indian
J Exp Biol 2002;40:1213-32.
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vitamin and activities of antioxidases. Zhonghua Yu Fang Yi Xue Za
Zhi 1997;31:67-70.
10. Yildiz L, Kayaoglu N, Aksoy H. The changes of Superoxide dismutase,
Catalase and Glutathione peroxidase activities in erythrocytes of
active and passive smokers. Clin Chem Lab Med 2002;40:612-5.
11. Andersen HR, Jesper BN, Flemming N, Philippe G. Antioxidative
enzyme activities in human erythrocytes. Clin Chem 1997;43:562-8.
Sirisha, et al.: Antioxidant enzymes and tobacco
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... The decrease in the SOD activity is thought to be due to the excessive production of H2O2 by free radicals, especially those originating from the tar phase, such as quinone/semiquinone. 28,33 The excessive amount of free radicals also makes other antioxidant enzymes, such as glutathione peroxidase and catalase, inactive so that H2O2 is not degraded, eventually inactivating SOD. 33,34 The decrease in NO bioavailability is a central mechanism in the pathophysiology of endothelial dysfunction. ...
... 28,33 The excessive amount of free radicals also makes other antioxidant enzymes, such as glutathione peroxidase and catalase, inactive so that H2O2 is not degraded, eventually inactivating SOD. 33,34 The decrease in NO bioavailability is a central mechanism in the pathophysiology of endothelial dysfunction. eNOS is an enzyme that produces NO in endothelial cells; therefore, the level of eNOS can represent the NO bioavailability in endothelial cells. ...
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... The present study observed a decrease in the enzymatic activity in smokers than nonsmokers which might have been caused by higher levels of hydrogen peroxide formation. Similar trend was also observed in the findings of Naga-Sirisha and Manohar (2013) [23] , in which they observed that an initial rate of hydrogen peroxide removal is directly proportional to its concentration. ...
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... The primary products of free radical oxidation-lipoperoxides, have been discovered in atherosclerotic plaques in the early studies. The lack of equilibrium between the oxidative and antioxidant systems together with hyperlipidemia creates favorable conditions for the initiation of lipid peroxidation processes [26]. ...
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