Influence of tobacco smoke on the appearance of oxidative stress in patients with lung cancer and chronic obstructive pulmonary diseases

Smoking is considered to be one of the primary etiological factors of many respiratory diseases, the most serious of which are chronic obstructive pulmonary diseases (COPD) and lung carcinoma (LC). Cigarette smokers have a higher mortality rate due to LC and COPD than non-smokers . Tobacco smoke is a complex mixture of over 4 700 chemical compounds, including high concentrations of oxidant agents, capable of interacting with cell constituents. Free radicals are atoms or molecules containg an impair number of electrons, which results in an unpaired electrons in the external orbit. In the human, oxygen, by means of its derivates referred to as reactive oxygen species acts as the fundamental oxidant. Once produced, these reactive metabolites can alter cellular and metabolic structures, interact with membrane unsaturated fatty acids, thus modifying the DNA and giving rise to mutations, with proteins and polysaccharides, creating more or less substantial alterations .


Introduction
Smoking is considered to be one of the primary etiological factors of many respiratory diseases, the most serious of which are chronic obstructive pulmonary diseases (COPD) and lung carcinoma (LC).Cigarette smokers have a higher mortality rate due to LC and COPD than non-smokers 1 .Tobacco smoke is a complex mixture of over 4 700 chemical compounds, including high concentrations of oxidant agents, capable of interacting with cell constituents.Free radicals are atoms or molecules containg an impair number of electrons, which results in an unpaired electrons in the external orbit.In the human, oxygen, by means of its derivates referred to as reactive oxygen species acts as the fun-damental oxidant.Once produced, these reactive metabolites can alter cellular and metabolic structures, interact with membrane unsaturated fatty acids, thus modifying the DNA and giving rise to mutations, with proteins and polysaccharides, creating more or less substantial alterations 2 .

Tobacco smoke and oxidative stress
All the individuals by the physiological breathing process produce free radicals.Oxidant -antioxidant balance in our organism is not stabile and many factors, such as tobacco smoke, infections, chemicals in environmental pollution can unbalance it, which result in the appearance of oxidative stress (Figure 1) 3 .Oxidative stress can be de- fined as an increased exposure to oxidants and/or a reduced defensive ability of the antioxidants 2 .The outcome of this condition at molecular level is an attack on various cell constituents ending in their irreversible damage and, consequently, damage to the cell structures and functions 1 .The human organism which is constantly attacked by damaging compounds in air, actuated mechanisms for defending and cleaning the lungs, which exert their action primarily against oxidant substances.Main natural antioxidants are: tracheobronchial mucus, alveolar lining, endocellular enzymatic substances (superoxide dismutase, catalase, gluthatione peroxidase, glutatione reductase), membrane scavengers (vitamine E, beta carotene), extracellular antioxidants (plasma proteins -albumin, ceruloplasmin, transferrin, alfa 1 -antitrypsin; low molecular weight scavengens -bilirubin, methionine, uric acid; glutathione, catalase, superoxide -dismutase, vitamin C, vitamin E) 1,3 .When natural antioxidants are ineffective, oxidative stress appears and it results in many respiratory disorders, including first and foremost COPD, LC and pulmonary fibrosis 4 .

REDUCED DEFENSIVE ABILITY OF THE ANTIOXIDANTS
Free radicals in smoke are present both in the gasphase and in the particulate phase (tar).The inhaled gas components contain approximately 10 15 radicals per puff (primarly alkyl and peroxyl types).Nitric oxide (NO), a free radical physiologically produced by the organism following antigenic stimulation, is also present in cigarette smoke.This compound reacts quickly with the superoxide anion (O 2 ) to form peroxynitrite and with peroxyl radicals to form alkyl peroxynitrites.
Whereas short lived radicals in the gas -phase of cigarette smoke can be inactivated immediately in the epithelial lining fluid (ELF), redox reactions in cigarette smoke condensate, which forms in the ELF, can produce reactive oxygen species for a considerable period 5 .
Tobacco smoke causes the burst of inflammatory cells expecially neutrophils, which in turn increase the quantity of oxidants in the airways.Moreover an alveolar macrophage and neutrophil accumulation has been observed in the lungs of smokers.

Smoking, oxidative stress and chronic obstructive pulmonary diseases
Oxidative stress plays a pathogenic role of primary importance in COPD 6 .
The clinical manifestations of COPD are influenced by a number of risk factors including alpha-1-antitrypsin deficiency, low birth weight, air pollution, socio-economic status, recurrent respiratory infections and tobacco smoke, which plays a leading role (Figure 2) 1,7 .About 90% of all COPD patients are smokers or ex-smokers.They are chronically exposed to exogenous oxidant overloads and have chronic bronchial inflammation with an increase and activation of macrophages and neutrophil granulocytes.These inflammatory cells, by damaging oxidant compounds formation, inhibit the antiprotease defence systems and alter mucociliary clearance, leading to the known patho-anatomical and functional transformations 1 .
It is possible to identify several oxidative stress indicators in smokers with COPD 1 .Since oxygen radicals direct measurment is difficult in lung tissue or in exhaled air, an alternative is to measure the biomolecules involved in a free radical damage such as lipids and hydrogen peroxide (H 2 O 2 ).Patients with COPD have a chronic formation of oxygen radicals in the lung, which can be measured by means of the H 2 O 2 in exhaled air.Alveolar macrophages from smokers release significantly more superoxide anion when compared to healthy controls.
Free radicals also trigger a lipid peroxidation chain reaction.The resulting lipid radical reacts with oxygen to make a peroxyl radical, which then transforms polyunsaturated fatty acid into lipid hydroperoxides, capable of producing other radical compounds.Lipid peroxidation can impair membrane function, increase membrane permeability and inactivate membrane -bound receptors and enzymes 8 .

Oxidant radicals in lung cancer
Lung cancer arises as a focal transformation of chronically injured epithelium by different factors, the most wellknown of which is tobacco smoke.One of the most significant effects of oxygen radicals is DNA damage.Different compounds in tobacco smoke can react directly to form radicals, while other substances are pre-carcinogens and need to be activated by one or more of the P450 cytochromes 9 .
Gackowski, et al. 10 , confirmed significantly higher values of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in leukocyte DNA in smokers with LC and ex-smokers with LC than in healthy smokers and healthy nonsmokers.The mean levels of vitamin C in the plasma is significantly higher in healthy nonsmokers than in smokers with LC.Vitamin E, as measured antioxidant in plasma, was reduced in patients with LC (smokers or ex-smokers) compared to healthy smokers.Their results suggest that a high level of 8-oxodGuo in leukocyte DNA and low concentration of vitamin E in blood may predict lung cancer risk.
In another study, authors measured oxidative DNA damage biomarkers (urinary excretion of 8-hydroxy-2'deoxyguanosine = 8-OH-dGuo and 8-hydroxyguanine = 8-OH-Gua) in LC patients who were smokers, healthy smokers and healthy nonsmokers.The mean level of biomarkers in urine was significantly lower in nonsmokers than in smoker groups (cancer patients and healthy smokers) 11 .
Paz-Elizur, et al. 12 investigated the activity of DNA repair enzyme 8-oxoguanine DNA N-glucosylase (OGG) which repairs the oxidative DNA lesion 8-oxoguanine in patients with LC.They measured low OGG activity in patients with LC.They concluded that low OGG activity is associated with an increased risk of LC.Authors suggested that smoking cessation in individuals with reduced OGG activity might be an effective strategy in LC prevention.

Oxidative stress in interstitial lung disease
There were an oxidant-antioxidant inbalance also in the pathogenesis of interstitial lung diseases 13 .Idiopathic pulmonary fibrosis is characterised by alveolitis with accumulation of macrophages and neutrophils, which preceedes injury to the lung parenchima.In pulmonary fibrosis glutathion deficiency occurs in the alveolar lavage fluid, which further contributes to determine and explain oxidative cell damage 14 .There are increases in the number of inflammatory cells and they release large amounts of highly reactive oxygen free radicals.
The study confirmed that there was a reduced glutathione concentration in the ELF of patients with idiopathic pulmonary fibrosis 14 .Levels were increased following the treatment with oral N-acetylcystein.The significance of this study is based on increasing glutathion levels in order to enhance the antioxidant protection of the lung and the control of the proliferation of fibroblasts.

Conclusion
Tobacco smoke is one of the well-known risk factors for oxidative stress which plays an important pathogenic role in the appearance of various pulmonary diseases including first and foremost COPD, LC and pulmonary fibrosis.

Fig. 2 −
Fig. 2 − Schematization of the processes causing pulmonary oxidative damage that contributes in different inflammatory mechanisms in COPD