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The Effect Of Envıronmental Tobacco Smoke On The Quantıty Of Oxıdant/Antıoxıdant Markers In Exhaled Breath Condensate 1 Doruk S, 2 Ozyurt H, 1 Inonu H, 3 Erkorkmaz U, 2 Saylan O, 1 Seyfikli Z Gaziosmanpasa University Departmant of 1 Pulmonary Medicine, - PowerPoint PPT Presentation
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The Effect Of Envıronmental Tobacco Smoke
On The Quantıty Of Oxıdant/Antıoxıdant Markers
In Exhaled Breath Condensate
1Doruk S, 2Ozyurt H, 1Inonu H, 3Erkorkmaz U, 2Saylan O, 1Seyfikli Z
Gaziosmanpasa University Departmant of 1Pulmonary Medicine, 2Biochemistry, 3Bioistatistics ,Tokat, Turkey
*Supported by the ‘Committe of Scientific Investigation of Gaziosmanpasa University’
Cigarette smoking most common type of tobacco use
In 2020; expected to nine million deaths annually
World Health 2002, 2003 Report
Macnee and Rahman 1999, Reddy 2002, Puri 2008, Mak 2008, Mates 1999
Cigarette smoke (CS) is a complex mixture of chemical
compounds, in which high concentrations of free radicals and
other oxidants contain more than 1016-1017 oxidant molecules per
puff and affects antioxidant defense of the lungs
Inbalance between oxidant and antioxidant activities; increased
oxidative stress in the lungs
Enzymatic antioxidants;
Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px)
Nonenzymatic antioxidants;
Vitamin C (Vit C), Vitamin E (Vit E)
Toxic products associated with lipid peroxidation;
Malondialdehyde (MDA) and nitric oxid (NO)
(They react with protein and DNA. NO is also one of the major
reactive oxygen species in CS)
Mak 2008, Mccall and Frei 1999, Abuja and Albertini 2001, Kanabrocki 2006, Rahman 2005
8-OHdG; An alternative biomarker of oxidative DNA
damage associated with chemical exposure demonstrates
the balance between DNA formation and repair
Kanabrocki 2006, Pilger and Rüdiger 2006, Halliwell 1999
Oxidative stress in smokers may be critical for the
inflammatory response to CS
The detection of oxidant/antioxidant markers may reflect
the affect of CS on the oxidative-antioxidative balance in lungs
Macnee and Rahman 1999, Morrow 1995
Oxidative stress markers; in the epithelial lining
fluid (ELF), breath, urine and blood
Several antioxidants (SOD, GSH-Px, vitamins) are
present in the lungs. These antioxidants provide a first
line defense against inhaled, also endogenously
produced oxidants
Macnee and Rahman 1999, Mak and Chan-Yeung 2006, Cross 1994, van der Viliet 1999, Abuja and Albertini 2001
AIM
To investigate the oxidative and antioxidative
activities in lungs associated with TS exposure via
measurement of the levels of MDA, NO, 8-OHdG,
SOD, GSH-Px, Vit E and Vit C in EBC samples
Group I: Current smokers who were defined by a history of
cigarette smoking ≥ 7 pack/years (n=26)
Group II: Nonsmokers, but had ETS more than 3 hours in a day in
his/her house/workplace/Turkish coffeehouse (n=21)
Group III: Nonsmokers, without ETS exposure in any where
(n=22)
Fidan 2005
EXCLUSION CRITERIA
♦ Atopy
♦ Chronic pulmonary diseases (asthma, COPD, bronchiectasis)
♦ Systemic diseases
(diabetes mellitus regulated with insulin, chronic renal failure,
collagen vasculary disases, inflamatory bowel diseases
♦ Gastroeosephagial reflux
♦ Upper respiratory tract infection within in the last 2 weeks
♦ Previous or concurrent use of vitamins
♦ Cancer
Exhaled Breath Condansate (EBC)
To breath at a normal frequency and tidal
volume, wearing a noseclip, for a period of 15
minutes through a mouthpiece and a two way
non-rebreathing valve
The collected condensate samples was at least
2 ml and stored at –70°CBreath condensate collector
Measurements of Oxidative and Antioxidative Markers
Total SOD (U/mL); Sun et al.
GSH-Px (U/L); Paglia and Valentine
MDA (µmol/L); Esterbaur and Cheeseman
NO (µmol/L); Cortas and Wakid
8-OHdG (ng/ml); ELISA
Vit C (µgram/dl); Chromsystems Diagnostic Test,
HPLC (High Performance Liquid Chromatography)
Vit E (µmol/L); HPLC
Sun 1988, Paglia and Valentine 1967, Esterbaur and Cheeseman 1990, Cortas and Wakid 1990
Pulmonary Functıon Tests (PFTs): to exclude
obstructive pulmonary diseases, to determine
differences between the groups
Forced vital capacity (FVC), forced expiratory volume
in one second (FEV1), FEV1/FVC ratio, maxium mean
expiratory flow (MMEF) rates were expressed
Quanjer 1993
Group I
(n=26)
Group II
(n=21 )
Group III
(n=22)
All Cases
(n=69 )
*Gender Male 3 (11.5) 6 (28.6) 12 (54.5) 21 (30.4)
Female 23 (88.5) 15 (71.4) 11 (45.5) 48 (69.6)
**Mean age ± Std 33.9±6.1 38.8±11.0 39.4±13.2 37.1±10.5
*p=0.005 **p=0.126
FEV1 and FVC were lower in smokers than nonsmokers,
FEV1/FVC and MMEF similar
Group I (n=26)
Group II (n=21 )
Group III(n=22) p
MDA (µmol/L) 1.06±0.47 0.87±0.35 0.52±0.25 <0.001
8-OHdG (ng/ml) 0.52±0.15 0.31±0.10 0.36±0.09 <0.001
NO (µmol/L) 52.28±12.18 64.43±15.65 95.17±25.05 <0.001
SOD (U/ml) 1.92±0.60 1.11±0.71 0.81±0.58 <0.001
GSH-Px (U/L) 456.18±163.76 317.82±69.98 259.07±46.15 <0.001
Vit C (µg/dl) 0.13±0.02 0.13±0.02 0.13±0.02 0.925
0102030405060708090
NO (µmol/L)0
0,2
0,4
0,6
0,8
1
1,2
MDA (µmol/L)
smoker
nonsmoker
p<0,001p<0,001
050
100150200250300350400450500
GSH-Px (U/L)0
0,020,040,060,08
0,10,120,14
Vit C (µg/dl)
smokernonsmoker
p<0,001p=0,760
00,20,40,60,8
11,21,41,61,8
2
SOD (U/ml)
smokernonsmoker
0
0,1
0,2
0,3
0,4
0,5
0,6
8-OHdG (ng/ml)
p<0,001 p<0,001
p<0,05p<0,05
A positive correlation (0.241); SOD and FEV1(L)
A negative correlation (-0.305); 8-OHdG and FEV1 (L)
A negative correlation (-0.298); 8-OHdG and MMEF (%)
(p<0.05)
Induced sputum, BAL fluid or bronchial biopsy are used for direct
assesments of airway inflammation
EBC is the liquid phase of the exhaled air sampled by cooling.
Collection of EBC is noninvasive method and can reflect
acid/oxidative stress and inflammation
ATS/ERS 2003, Hoffmeyer 2009
Acute smoke exposure; increases the products of lipid peroxidation
MDA is the most mutagenic product of lipid peroxidation and can be determined in
serum, BALF, and tissues
Increased levels of MDA may be associated with current smoking status
Bloomer ; higher MDA levels in smokers than nonsmokers
MDA levels were higher in smokers than nonsmokers.
The levels of MDA in ETS exposed group were higher than that ETS nonexposed group
ETS exposure may cause the initiation of oxidant damage in lungs
van der Vaart 2004, Kanabrocki 2006, Petruska 1990, Kelly 1996, Rumley 2004, Bloomer 2007
NO levels decrease in smokers. This reduction is reversible,
NO levels increase after smoking cessation
In our study the highest NO levels were determined in Group
III, the lowest NO levels were detected in smokers
We did not detect any correlation between NO levels and
PFT’s. This result could be associated with reverseble decrease
in NO levels
Kharitonov 1995, Robbins 1997
Smoking; increased level of 8-OHdG in lung tissue
Howard; 8-OHdG increases in blood in subjects with ETS exposure
8-OHdG levels in smokers were significiantly higher than nonsmokers,
ETS exposure did not affect 8-OHdG levels. A negative correlation was
revealed between FEV1, MMEF and 8-OHdG levels
This outcome may be indicated the development of DNA damage in
healthy individuals with normal PFTs
Asami 1997, Chen 2007, Howard 1998
Patel; GSH-Px levels were higher in smokers than nonsmokers
Hilbert; smoking is associated with increased activities of GSH-Px in
BALF cells
Valenca; GSH-Px activity in BAL fluid/blood increases with TS exposure
Yildiz; GSH-Px activity is higher in healty control than active/passive
smokers
GSH-Px activity were higher than nonsmokers. We did not dedect difference
between groups with or without ETS exposure
Patel 2005, Hilbert and Mohsenin 1996, Valenca 2008, Yildiz 2002
▪Vit C; the most abundant antioxidant substance in the extracellular
lining fluid of the lung and inhibits lipid peroxidation
▪ Repine, Lykkesfeldt; smokers have lower serum levels of Vit C and Vit E
▪ Hilbert, Pacth; smokers have higher Vit E levels in BALF
▪ Rahman; reduced levels of Vit E in the BALF of smokers
▪Pacht; Vit E levels was undetectable (<2 ng/ml) in the BALF of smokers
Martin and Frei 1997, Slade 1993, Eiserich 1995, Repine 1997, Lykkesfeldt 1997, Hilbert and Mohsenin 1996, Pacth 1986, Rahman and Macnee 1996
The cut-off value was foe Vit E was 0.58 µmol/L in this study, we
could not determine Vit E levels
Schock; the cut-off value for Vit E in BALF was 0.0028 µmol/L
Inability of measurement Vit E levels might be due to high cut-off value
Hilbert, Rahman; increase in Vit C levels in the BALF of smokers
We did not find difference between the groups
Schock 2003, Hilbert and Mohsenin 1996, Rahman and Macnee 1996
Valenca; SOD activity is higher in BALF and lung homogenates of
the mice exposed to CS
Melloni; ≤ normal SOD in BALF from smokers
DiSilvistro; SOD activity is lower in smokers
Valence 2008, DiSilvistro 1998, Melloni 1996, Yildiz 2002
In present study; SOD activity was highest in smokers
SOD activity was higher in cases with ETS exposure than
nonexposed group but this difference is not significiant
A positive correlation between SOD levels and FEV1 were detected
This finding might demonstrate the contribution of decrease in the activity
of the antioxidant marker SOD on decrease in FEV1
CONCLUSION
▪ SOD, MDA and GSH-Px may be useful markers of
oxidative stress in the lung inflammation induced by exposure
to CS
▪ ETS exposure alters the levels of NO and MDA which
reflect oxidative state
▪ Increased levels of 8-OHdG which reflect DNA damage
developed as a result of CS exposure in cases with normal PFTs. These
higher levels of 8-OHdG may be an early of respiratory tract diseases
in the future
BALLICA CAVE, TOKAT, TURKIYE
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