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