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Oxidants/Antioxidants and COPD^*

^* From the Edinburgh Lung Environmental Group Initiative, Colt Research Laboratories, University of Edinburgh, Edinburgh, Scotland, UK.

Correspondence to: W. MacNee, MD, Respiratory Medicine, Edinburgh Lung Environmental Group Initiative, Colt Research Laboratories, Wilkie Building, Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland, UK; e-mail: w.macnee{at}ed.ac.uk

	Abstract

TOP Abstract Introduction Oxidants in Cigarette Smoke Cell-Derived Oxidants Oxidative Stress in the... Oxidative Stress and Neutrophil... Evidence of Oxidative Stress... Evidence of Systemic Oxidative... Other Mechanisms Related to... Evidence for a Relationship... Oxidative Stress and Gene... Oxidative Stress and... References

 
Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants. Oxidative stress is thought to play an important role in the pathogenesis of a number of lung diseases, not only through direct injurious effects, but by involvement in the molecular mechanisms that control lung inflammation. A number of studies have shown an increased oxidant burden and consequently increased markers of oxidative stress in the airspaces, breath, blood, and urine in smokers and in patients with COPD. The presence of oxidative stress has important consequences for the pathogenesis of COPD. These include oxidative inactivation of antiproteinases, airspace epithelial injury, increased sequestration of neutrophils in the pulmonary microvasculature, and gene expression of proinflammatory mediators. With regard to the latter, oxidative stress has a role in enhancing the inflammation that occurs in smokers and patients with COPD, through the activation of redox-sensitive transcriptions factors such as nuclear factor-B and activator protein-1, which regulate the genes for proinflammatory mediators and protective antioxidant gene expression. The sources of the increased oxidative stress in patients with COPD are derived from the increased burden of oxidants present in cigarette smoke, or from the increased amounts of reactive oxygen species released from leukocytes, both in the airspaces and in the blood. Antioxidant depletion or deficiency in antioxidants may contribute to oxidative stress. The development of airflow limitation is related to dietary deficiency of antioxidants, and hence dietary supplementation may be a beneficial therapeutic intervention in this condition. Antioxidants that have good bioavailability or molecules that have antioxidant enzyme activity may be therapies that not only protect against the direct injurious effects of oxidants, but may fundamentally alter the inflammatory events that play an important part in the pathogenesis of COPD.

Key Words: antioxidant • COPD • oxidants • reactive oxygen species

	Introduction

TOP Abstract Introduction Oxidants in Cigarette Smoke Cell-Derived Oxidants Oxidative Stress in the... Oxidative Stress and Neutrophil... Evidence of Oxidative Stress... Evidence of Systemic Oxidative... Other Mechanisms Related to... Evidence for a Relationship... Oxidative Stress and Gene... Oxidative Stress and... References

 

Abbreviations: ₁-AT = ₁-antitrypsin; AP = activator protein; BALF = BAL fluid; CSC = cigarette smoke condensate; ELF = epithelial lining fluid; -GCS = -glutamylcysteine synthetase; GP = glutathione peroxidase; GSH = glutathione; IL = interleukin; NAC = N-acetylcysteine; NAL = N-acystelyn; NF-B = nuclear factor-B; NO = nitric oxide; O₂^·- = superoxide anion; ROS = reactive oxygen species; RTLF = respiratory tract lining fluid; SOD = superoxide dismutase; TBARS = thiobarbituric acid reactive substances; TNF = tumor necrosis factor

COPD is a slowly progressive condition characterized by airflow limitation, which is largely irreversible.¹ A smoking history of at least 20 pack-years is usual, reflecting the fact that smoking is the main etiologic factor in this condition, which far outweighs any of the other risk factors. The pathogenesis of COPD is therefore strongly linked to the effects of cigarette smoke. Since cigarette smoke contains 10¹⁷ molecules per puff,² it has been proposed that an oxidant/antioxidant imbalance occurs in smokers, and an increased oxidant burden in smokers and patients with COPD.³

	Oxidants in Cigarette Smoke

TOP Abstract Introduction Oxidants in Cigarette Smoke Cell-Derived Oxidants Oxidative Stress in the... Oxidative Stress and Neutrophil... Evidence of Oxidative Stress... Evidence of Systemic Oxidative... Other Mechanisms Related to... Evidence for a Relationship... Oxidative Stress and Gene... Oxidative Stress and... References

 
Cigarette smoke is a complex mixture of > 4,700 chemical compounds of which free radicals and other oxidants are present in high concentrations.⁴ Free radicals are present in both the tar and the gas phases of cigarette smoke. The gas phase of cigarette smoke contains approximately 10¹⁵ radicals per puff, primarily of the alkyl and peroxyl types. Nitric oxide (NO) is another oxidant that is present in cigarette smoke in concentrations of 500 to 1,000 ppm.⁴ NO reacts quickly with the superoxide anion (O₂^·-) to form peroxynitrite, and with peroxyl radicals to give alkyl peroxynitrites.

The tar phase of cigarette contains more stable radicals, such as the semiquinone radical, which can react with oxygen to produce O₂^·-, the hydroxyl radical, and hydrogen peroxide.⁴ The tar phase is also an effective metal chelator and can bind iron to produce the tar-semiquinone + tar-Fe²⁺, which can generate hydrogen peroxide.^{5 6}

The epithelial lining fluid (ELF) and mucus are the first line of defense in the lungs against inhaled oxidants by quenching the short-lived radicals in the gas phase of cigarette smoke. However, cigarette smoke condensate (CSC), which forms in the ELF, may continue to produce reactive oxygen species (ROS) for a considerable in patients with COPD, as part of the pathogenesis of this condition.⁷

	Cell-Derived Oxidants

TOP Abstract Introduction Oxidants in Cigarette Smoke Cell-Derived Oxidants Oxidative Stress in the... Oxidative Stress and Neutrophil... Evidence of Oxidative Stress... Evidence of Systemic Oxidative... Other Mechanisms Related to... Evidence for a Relationship... Oxidative Stress and Gene... Oxidative Stress and... References

 
The direct increase in the oxidative burden produced by inhaling cigarette smoke can be further enhanced in smokers’ lungs by the release of oxygen radicals from inflammatory leukocytes, both neutrophils and macrophages, which are known to migrate into the lungs of cigarette smokers.⁸ Increased amounts of oxidants such as O₂^·- and hydrogen peroxide are released from the leukocytes of smokers, compared with those from nonsmokers.⁹

Iron is a critical element in many oxidative reactions. The generation of oxidants in ELF in smokers is further enhanced by the presence of increased amounts of free iron in the airspaces.¹⁰ The intracellular iron content of alveolar macrophages is increased in cigarette smokers and is increased further in those who develop chronic bronchitis, compared with nonsmokers.¹¹ Furthermore, macrophages from smokers release more free iron in vitro than those from nonsmokers.¹²

Free iron in the ferrous form can take part in the Fenton and Haber-Weiss reactions, which generate the hydroxyl radical, a free radical that is extremely damaging to all tissues, particularly to cell membranes, producing lipid peroxidation.

Direct oxidative damage to components of the lung matrix (such as elastin and collagen) can results from oxidants in cigarette smoke.¹³ Elastin synthesis and repair can also be impaired by cigarette smoke,¹⁴ which can augment proteolytic damage to matrix components and thus enhance the development of emphysema.

Increased oxidative stress in the airspaces can initiate a number of early inflammatory events in the lungs.

	Oxidative Stress in the Airspaces

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By virtue of its direct contact with the environment, the airspace epithelial surface of the lung is particularly vulnerable to the effects of oxidative stress. The respiratory tract lining fluid (RTLF) forms an interface between the epithelial cells and the external environment, and thus constitutes a first line of defense against inhaled oxidants. At least three processes may be responsible for oxidant injury to the respiratory tract epithelial cells from cigarette smoke: (1) a direct toxic interaction of constituents of cigarette smoke (including free radicals) that have penetrated the protective antioxidant shield of the RTLF; (2) damage to the cells by toxic reactive products generated by interaction between cigarette smoke and RTLFs; and (3) reactions occurring subsequent to activation of inflammatory-immune processes initiated by (1) and/or (2), above.^{15 16 17}

Injury to the epithelium may be an important early event following exposure to cigarette smoke, and is shown by an increase in airspace epithelial permeability.¹⁸ Lannan and colleagues¹⁹ demonstrated the injurious effect of both whole and vapor phases of cigarette smoke on human alveolar epithelial cell monolayers, as shown by increased epithelial cell detachment, decreased cell adherence, and increased cell lysis.

These effects were in part oxidant mediated, since they were partially prevented by the antioxidant glutathione (GSH) in concentrations (500 µM) that are present in the ELF. Extra- and intracellular GSH appears to be critical to the maintenance of epithelial integrity following exposure to cigarette smoke. This was shown in studies by Li et al^{20 21} and Rahman et al,²² which demonstrate that the increased epithelial permeability of epithelial cell monolayers in vitro and in rat lungs in vivo following exposure to CSC was associated with profound changes in the homeostasis of the antioxidant GSH. Concentrations of GSH were considerably decreased, concomitant with a decrease in the activities of the enzymes involved in the GSH redox cycle such as GSH peroxidase (GP) and glucose-6-phosphate dehydrogenase. In addition, depletion of lung GSH alone, by treatment with the GSH-synthesis inhibitor buthionine sulfoxamine, can induce increased airspace epithelial permeability both in vitro and in vivo.^{21 22 23}

Similar results to these in vitro and animal studies were shown in human studies demonstrating increased epithelial permeability in chronic smokers compared with nonsmokers, as measured by increased ^99mtechnicium-diethylenetriaminepentacetate lung clearance, with a further increase in ^99mtechnicium-diethylenetriaminepentacetate clearance following acute smoking.²⁴ Thus, cigarette smoke has a detrimental effect on alveolar epithelial cell function that is, in part, oxidant mediated, since antioxidants provide protection against this injurious event.

The oxidant burden in lungs may be further enhanced in smokers by the increased numbers of neutrophils (by 10-fold) and macrophages (by two- to fourfold) in the alveolar space.^{25 26} In vitro studies have shown that the spontaneous release of ROS from alveolar leukocytes in cigarette smokers is increased, compared to those from nonsmokers.^{27 28 29 30 31} Recent evidence from bronchial biopsy and lung resection studies indicates that increased numbers of neutrophils are present in both bronchial and alveolar walls in smokers with moderately severe COPD.³²

Xanthine/xanthine oxidase, which generates O₂^·-, has also been shown to be increased in the BAL fluid (BALF) from patients with COPD.³³

	Oxidative Stress and Neutrophil Sequestration and Migration in the Lungs

TOP Abstract Introduction Oxidants in Cigarette Smoke Cell-Derived Oxidants Oxidative Stress in the... Oxidative Stress and Neutrophil... Evidence of Oxidative Stress... Evidence of Systemic Oxidative... Other Mechanisms Related to... Evidence for a Relationship... Oxidative Stress and Gene... Oxidative Stress and... References

 
The first step in the recruitment of neutrophils to the airspaces is the sequestration of these cells in the lung microcirculation.³⁴ This occurs under normal circumstances in the pulmonary capillary bed, as a result of the size differential between neutrophils (average diameter, 7 µm) and pulmonary capillary segments (average diameter, 5 µm). Thus a proportion of the circulating neutrophils have to deform in order to negotiate the smaller capillary segments. Studies using a variety of techniques, including radiolabeled or fluorescent-labeled neutrophils, have supported the idea that the lungs contain a large pool of noncirculating neutrophils, which are either retained or slowly moving within the pulmonary microcirculation. In healthy subjects, radiolabeled neutrophil studies indicate that a proportion of neutrophils are normally delayed in the pulmonary circulation, compared to radiolabeled erythrocytes.³⁵ In normal subjects, studies have shown a correlation between neutrophil deformability measured in vitro and the subsequent sequestration of these cells in the pulmonary microcirculation following their reinjection—the less deformable the cells, the increased sequestration of these cells occurs in the pulmonary circulation.³⁵ This provides a mechanism for the creation of a pool of sequestered or noncirculating cells in the pulmonary microcirculation, without the need to invoke margination of neutrophils in the postcapillary venules, which is the mechanism for the noncirculating pool of cells in the systemic circulation.³⁶ The sequestration of neutrophils in the pulmonary capillaries allows time for the neutrophils to interact with the pulmonary capillary endothelium, resulting in their adherence to the endothelium and thereafter their transmigration across the alveolar capillary membrane to the interstitium and airspaces of the lungs in response to inflammation or infection.

Any circumstances that lead to a decrease in neutrophil deformability will potentially increase neutrophil sequestration in the lungs.

Decreased neutrophil deformability occurs in cell activation due to the assembly of the cytoskeleton, in particular the polymerization of microfilaments (F actin), resulting in cell stiffening. Neutrophils can be activated while in transit in the pulmonary microcirculation by a number of mediators, including cytokines released from resident lung cells, alveolar macrophages, and epithelial and endothelial cells. Noxious inhaled agents, such as cigarette smoke, could influence the transit of cells in the pulmonary capillary bed. Studies in man using radiolabeled neutrophils and RBCs show a transient increase in neutrophil sequestration in the lungs during smoking,³⁷ which returns to normal after cessation of smoking. Using an in vitro positive pressure cell filtration technique, it has been shown that cells exposed to cigarette smoke in vitro decrease their deformability.³⁸ A similar decrease in deformability can be demonstrated in vivo for neutrophils in blood obtained from subjects who are actively smoking (Fig 1 ).³⁹ Since each puff of cigarette smoke contains 10¹⁶ oxidant molecules, it has been suggested that the effect of cigarette smoke on neutrophil deformability is oxidant mediated. Support for this hypothesis comes from in vitro studies that show that the decrease neutrophil deformability induced by cigarette smoke exposure is abolished by antioxidants, such as GSH (Fig 2 ).³⁸ There is also evidence that oxidative stress may reach the circulation during cigarette smoking, which could decrease the deformability of neutrophils, so increasing their sequestration in the pulmonary microcirculation.⁴⁰ Oxidants appear to affect neutrophil deformability by altering the cytoskeleton by polymerizing actin.³⁸

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 1.. Effects of smoking in eight healthy smokers on 6-min filtration pressure of neutrophils harvested from arterial blood as a measure of deformability. Smoking two cigarettes increased the ex vivo filtration pressure significantly (p < 0.05), which represents a decrease in deformability of peripheral blood neutrophils (modified from Drost et al39 ).

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 2.. Deformability measured as in vitro 6-min filtration pressure of neutrophils harvested from peripheral blood of healthy subjects. As filtration pressure increases, deformability of the cells decreases. Exposure of these cells to smoke in vitro in phosphate-buffered saline/bovine serum albumin (PBS/BSA) produced an increase in filtration pressure, a decrease in deformability. The addition of RBCs, GSH, or plasma decreased the increase in filtration pressure produced by cigarette smoke. (**p = 0.01, *p < 0.05 compared with PBS/BSA smoked exposed; modified from Drost et al38 ).

Once sequestered, components of cigarette smoke can alter neutrophil adhesion to endothelium by upregulating CD18 integrins,^{39 40 41} which is known to upregulate the nicotinamide–adenine dinucleotide phosphate oxidase-hydrogen peroxide (NADPH) generating system.⁴² Cigarette smoke has also been shown to alter neutrophil adhesion.^{40 41} Inhalation of cigarette smoke by hamsters increases neutrophil adhesion to the endothelium of both arterioles and venules.⁴⁰ This increased neutrophil adhesion is thought to be mediated by (O₂^·-) derived from cigarette smoke, since it was inhibited by pretreatment with CuZnSOD.⁴¹ Neutrophils sequestered in the pulmonary circulation of the rabbit following cigarette smoke inhalation also show increased expression of CD18 integrins,⁴¹ which is known to upregulate the NADPH oxidase-O₂·^- generating system.⁴³

Increased expression of adhesion molecules in smoke-exposed animals may result from the secondary inflammatory effects of smoking, through the release of cytokines, since direct smoke exposure in vitro does not produce increased expression of neutrophil adhesion molecules, nor does it enhanced functional adherence.⁴⁴ Thus, several mechanisms involving oxidants cause neutrophil sequestration in the pulmonary microcirculation in smokers. Oxidant-mediated mechanisms may also result in the increased sequestration of neutrophils, which occurs in the microcirculation during exacerbations of COPD.^{45 46}

These sequestered neutrophils may subsequently respond to chemotactic components in cigarette smoke and become more adhesive to pulmonary vascular endothelial cells, in preparation for migration into the airspaces. Studies in animal models of smoke exposure⁴⁷ have demonstrated increased neutrophil sequestration in the pulmonary microcirculation in situ, associated with upregulation of adhesion molecules on the surface of these cells.⁴² Activation of neutrophils sequestered in the pulmonary microvasculature⁴⁸ could also induce the release of reactive oxygen intermediates and proteases within a microenvironment, with limited access for free radical scavengers and antiproteases. Thus, destruction of the alveolar wall, as occurs in emphysema, could result from a proteolytic insult derived from the intravascular space, without the need for the neutrophils to migrate into the airspaces.

As indicated above, several studies have shown that there are increased numbers of neutrophils in the BAL in chronic cigarette smokers.^{24 26} Neutrophil sequestration in the microcirculation allows chemotaxis to occur. Smoke exposure in humans results in increased chemotactic activity or levels of chemotactic factors in the airspaces.⁴⁹

	Evidence of Oxidative Stress in Smokers and Patients With COPD

TOP Abstract Introduction Oxidants in Cigarette Smoke Cell-Derived Oxidants Oxidative Stress in the... Oxidative Stress and Neutrophil... Evidence of Oxidative Stress... Evidence of Systemic Oxidative... Other Mechanisms Related to... Evidence for a Relationship... Oxidative Stress and Gene... Oxidative Stress and... References

 
There is now overwhelming evidence for the presence of increased oxidative stress in smokers and patients with COPD.^{50 51 52} Direct measurements of specific markers of oxidative injury resulting from excessive free radical activity can be made by electron spin resonance, which cannot be applied to the study of tissues at present. Most studies have therefore relied on indirect measurements of free radical activity in biological fluids. Although these markers suggest that oxidative stress has occurred, they do not indicate that this event is necessarily involved in the pathogenesis of the condition that is being studied. Markers of oxidative stress have been shown to occur in the ELF, in the breath, and in the urine in cigarette smokers and patients with COPD.

Oxidative Stress and Proteinase/Antiproteinase Imbalance
The concept that a proteinase/antiproteinase imbalance occurs in the lungs as part of the pathogenesis of emphysema in smokers developed from studies of ₁–antitrypsin (₁-AT)-deficient patients. In the case of smokers with normal levels of ₁-AT, the elastase burden may be increased as a result of increased recruitment of leukocytes to the lungs, and there may be a functional deficiency of ₁-AT due to inactivation in the lungs by oxidation, which is considered to contribute to the pathogenesis of this condition.

A large body of literature has been published in an attempt to prove the protease/antiprotease theory of the pathogenesis of emphysema. It is clear that an imbalance between an increased elastase burden in the lungs and a functional deficiency of ₁-AT due to its inactivation by oxidants is an oversimplification, not least because other proteinases and other antiproteinases are likely to have a role. Early studies showed that the function of ₁-AT in BAL was reduced by around 40% in smokers, compared with nonsmokers.⁵³ This functional ₁-AT deficiency is thought to be due to inactivation of the ₁-AT by oxidation of the methionine residue at its active site by oxidants in cigarette smoke, as part of the pathogenesis of emphysema.⁵⁴ Another major inhibitor of neutrophil elastase is secretary leukoprotease, which can also be inactivated by oxidants.^{55 56}

This theory was supported by in vitro studies showing loss of ₁-AT inhibitory function when treated with oxidants,⁵⁷ including cigarette smoke.⁵⁸ In addition, oxidation of the methionine residue in ₁-AT was confirmed in the lungs of healthy smokers.^{59 60} These studies supported the concept of inactivation of ₁-AT by oxidation of the active site of the protein. Other studies showed that macrophages from the lungs of smokers release increased amounts of ROS, which could also inactivate ₁-AT in vitro.⁵⁴ However, most of the ₁-AT in cigarette smokers remains active, and is therefore still capable of protecting against the increased protease burden. Later studies have provided conflicting data on whether ₁-AT function lavage is altered in cigarette smokers,⁶⁰ which may be due to technical differences between the studies that may have affected ₁-AT function.

The acute effects of cigarette smoking on the functional activity of ₁-AT in BALF have shown a transient, but nonsignificant fall in the antiprotease activity of BALF 1 h after smoking.^{61 62} Thus studies assessing the function of ₁-AT in either chronic or acute cigarette smoking have failed to produce a clear picture.

Antioxidants in BALF
The major antioxidants in RTLF include mucin, reduced GSH, uric acid, protein (largely albumin), and ascorbic acid.^{15 63} Mucin is a glycoprotein rich in cysteine residues (sulfydryls), and hence is an important antioxidant of the RTLF. Mucins have metal binding properties⁶⁴ that effectively scavenge hydroxyl radicals⁶⁵ and would be expected to scavenge OCL^-/HOCL because of the abundance of sulfydryl and disulphide moieties in their structure. Toxic inhalants increase the secretion of mucins, which therefore represent a major antioxidant in the upper RTLFs. However, oxygen radicals are known to degrade mucus glycoproteins.¹⁶ It is therefore likely that cigarette smoke oxidants also react with this respiratory tract secretory glycoprotein.

There is limited information on the respiratory epithelial antioxidant defenses in smokers, and less in COPD. Several studies have shown that GSH is elevated in BALF in the airways of chronic smokers.^{24 66 67}

Despite the twofold increase in BALF GSH in chronic smokers, GSH may not be present in sufficient quantities to deal with the excessive oxidant burden during acute smoking when acute depletion of GSH may occur.²⁰ Rahman and colleagues^{22 68} studied the acute effects of CSC on GSH metabolism in a human alveolar epithelial cell line in vitro, and in vivo in rat lungs after intra-tracheal CSC instillation. They found a dose and time-dependent depletion of intracellular GSH, concomitant with the formation of GSH conjugates, which is supported by similar results in studies in animal lungs in vivo.^{22 68} Furthermore, the activities of GSH redox system enzymes, such as GP and glucose 6-phosphate dehydrogenase, were transiently decreased in alveolar epithelial cells and in rat lungs after CSC exposure, possibly as a result of the action of highly electrophilic free radicals on the active site of the enzymes. GSH homeostasis may also play a central role in the maintenance of lung airspace epithelial barrier integrity. In particular, lowering the levels of GSH in epithelial cells leads to loss of barrier function and increased permeability.^{21 22}

Pacht and coworkers⁶⁹ showed reduced levels of vitamin E in the BALF of smokers compared with nonsmokers. By contrast, Bui and colleagues⁷⁰ found a marginal increase in vitamin C in BALF of smokers, compared to nonsmokers. Similarly, alveolar macrophages from smokers have both increased levels of ascorbic acid and augmented uptake of ascorbate.⁷¹ Enhanced activity of antioxidant enzymes (superoxide dismutase [SOD], and catalase) in alveolar macrophages from young smokers has also been reported.⁷² However, Kondo and coworkers⁷³ found that increased superoxide generation by alveolar macrophages in elderly smokers was associated with decreased antioxidant enzyme activities, when compared with nonsmokers. The activities of CuZnSOD, GSH-S-transferase, and GP were found to be decreased in alveolar macrophages from elderly smokers. This reduced activity was not associated with decreased gene expression, but was due to modification at the post-translational level.⁷⁴

The apparent discrepancies between these studies of the levels of the different antioxidants in BALF and alveolar macrophages may be due to different smoking histories in chronic smokers, particularly the time of the last cigarette in relation to the sampling of BALF.

McCusker and Hoidal⁷² demonstrated enhanced alveolar macrophage antioxidant enzyme activities following cigarette smoke exposure, which resulted in reduced mortality when the hamsters were subsequently exposed to > 95% oxygen. They speculated that mammalian alveolar macrophages undergo an adaptive response to chronic oxidant exposure that may ameliorate potential damage to lung cells from further oxidant stress. The mechanisms for the induction of antioxidants enzymes in erythrocytes,⁷⁵ alveolar macrophages,⁷² and lungs⁷⁴ by cigarette smoke exposure are currently unknown. However, it is likely to be due to the induction of antioxidant genes (see below).

Urine isoprostane F₂-III, which is an isomer of prostaglandin, formed by free radical peroxidation of arachidonic acid, has recently been shown to be elevated in patients with COPD, compared with healthy control subjects, and to be even more elevated in exacerbations of the condition.⁷⁶ This is one of a number of surrogate markers of oxidative stress that have been shown to be elevated in patients with COPD.^{77 78 79 80}

	Evidence of Systemic Oxidative Stress

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There has recently been considerable interest in the systemic effects of COPD. One manifestation of a systemic effect is the presence of markers of oxidative stress in the blood in patients with COPD. This is reflected in the increased sequestration of neutrophils in the pulmonary microcirculation during smoking and during exacerbations of COPD which, as described above, is an oxidant-mediated event.^{37 38 39 40 46}

Rahman and colleagues⁴⁰ demonstrated increased production of superoxide anion from peripheral blood neutrophils obtained from patients with acute exacerbations of COPD, which returned to normal when the patients were restudied when clinically stable. Other studies have shown that circulating neutrophils from patients with COPD have upregulated surface adhesion molecules, which may also be an oxidant-mediated effect.^{40 81} Neutrophil activation may be even more pronounced in neutrophils that are sequestered in the pulmonary microcirculation in smokers and in patients with COPD, since animal models of lung inflammation have shown that neutrophils which are sequestered in the pulmonary microcirculation release more ROS than circulating neutrophils in the same animal.⁴⁸ Thus, neutrophils which are sequestered in the pulmonary microcirculation may be a source of oxidative stress, which may have a role of inducing airway injury in COPD, particularly during exacerbations.

Polyunsaturated fats and fatty acids in cell membranes are a major target for free radical attack, resulting in lipid peroxidation, a process that may continue as a chain reaction to generate peroxides and aldehydes. Products of lipid peroxidation reactions can be measured in body fluids as thiobarbituric acid reactive substances (TBARS). The levels of TBARS in plasma or in BALF, are significantly increased in healthy smokers and patients with acute exacerbations of COPD, compared with healthy nonsmokers.^{9 40} There is, however, a problem with the specificity of thiobarbituric acid-malondialehyde assays as a measure of lipid peroxidation, since this assay does not directly measure the lipid peroxidation reaction. Other studies have measured conjugated levels of dienes of linoleic acid, a secondary product of lipid peroxidation, and shown the levels in plasma were elevated in chronic smokers.⁸² In addition, circulating levels of F₂-isoprostane, which is a more direct measurement of lipid peroxidation, have been found in smokers.⁸³ Similarly Lapenna and colleagues⁸⁴ demonstrated increased levels of fluorescent products of lipid peroxidation in smokers.

A recent study directly examined the balance between oxidants/antioxidants in smokers and patients with acute exacerbations of COPD by measuring changes in the antioxidant capacity in the blood. Rahman and coworkers⁴⁰ found that the plasma antioxidant capacity was significantly decreased in smokers 1 h after smoking and in patients with acute exacerbations of COPD, when compared with plasma from age- matched nonsmoking control subjects (Fig 3 ). The decrease in plasma antioxidant capacity in smokers may be due to a profound depletion of plasma protein sulfydryls as demonstrated following cigarette smoke exposure in vitro.^{85 86 87 88 89} Thus, there is clear evidence that oxidants in cigarette smoke, either in vitro or in vivo, markedly decrease low molecular plasma antioxidants both in vitro and in vivo. Depletion of plasma antioxidants reduces the protection against cigarette smoke-induced plasma membrane peroxidation.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 3.. The effect of cigarette smoking on the antioxidant capacity (trolox equivalent antioxidant capacity [TEAC]) of plasma in both chronic and acute cigarette smokers. Chronic cigarette smokers have a lower antioxidant capacity than nonsmokers, and the antioxidant capacity falls further immediately after smoking (modified from Rahman et al40 ).

Likewise, investigators have measured the major plasma antioxidants in smokers.^{90 91 92 93 94 95 96 97 98} These studies show a depletion of ascorbic acid vitamin E, beta-carotene, and selenium in the serum of chronic smokers.^{92 93 94 95 96 97} Moreover, decreased vitamin E and vitamin C levels were measured in leukocytes from smokers.^{96 97 98} However, circulating RBCs from cigarette smokers contain increased levels of SOD and catalase, despite similar activity of GP, and are better able to protect endothelial cells from the effects of hydrogen peroxide, when compared with cells from nonsmokers.⁷⁵

Plasma ascorbate may be a particularly important antioxidant in the plasma because the gas phase of cigarette smoke induces lipid peroxidation in plasma in vitro that is decreased by ascorbate.⁸⁷ Inhalation of NO from cigarette smoke, as well as NO and O₂^·- released by activated phagocytes react to form peroxynitrite, which is cytotoxic. Peroxynitrite has recently been shown to decrease plasma antioxidant capacity by rapid oxidation of ascorbic acid, uric acid, and plasma sulfydryls.⁹⁹ Evidence of NO/peroxynitrite activity in plasma has been demonstrated in cigarette smokers. Nitration of tyrosine residues or proteins in plasma leads to the production of 3-nitrotyrosine.⁹⁹ Petruzzelli and colleagues¹⁰⁰ demonstrated the presence of 3-nitrotyrosine in plasma in smokers, which were possibly in higher levels than in a small group of nonsmokers. They also confirmed low levels of antioxidant capacity in smokers, which were negatively correlated with the levels of 3-nitrotyrosine.¹⁰⁰ The levels of antioxidant capacity in the plasma have a negative correlation with the increased release of oxygen radicals from circulating neutrophils in patients with exacerbations of COPD.⁴⁰

	Other Mechanisms Related to the Pathogenesis of COPD Involving Oxidants

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The majority of the information that is available on the pathogenesis of COPD relates to the development of emphysema. COPD also includes the other conditions of chronic bronchitis and small airways disease. It is presumed that the factors that initiate inflammation and the effects of proteolytic and oxidant-induced damage are also relevant to these conditions, although much less information is available.

Animal models of elastase-induced emphysema also show features of airways diseases with goblet cell hyperplasia.¹⁰¹ Neutrophil elastase is known to be a potent secretagogue for mucous glands, and therefore may contribute to the hyper-mucous secretion in chronic bronchitis.¹⁰² Oxidant-generated systems, such as xanthine/xanthine oxidase have also been shown to cause the release of mucous.¹⁰³

	Evidence for a Relationship Between Oxidant/Antioxidant Balance and the Development of Airways Obstruction

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The neutrophil appears to be a critical cell in the pathogenesis of COPD. Previous epidemiologic studies have shown a relationship between circulating neutrophil numbers and the FEV₁,^{104 105} and indeed a relationship has been shown between the change in peripheral blood neutrophil count and the change in airflow limitation over time.¹⁰⁵ Other studies have provided supportive evidence of a role for ROS released from circulating neutrophils and the development of airflow limitation. Richards and colleagues²⁸ have shown a relationship between peripheral blood neutrophil chemiluminescence and measures of airflow limitation in young cigarette smokers. Even passive cigarette smoking has been associated with increased peripheral blood leukocyte counts and enhanced release of oxygen radicals.¹⁰⁶ Oxidative stress, measured as TBARS in plasma, has also been shown to correlate inversely with the percent predicted FEV₁ in a population study, indicating that lipid peroxidation is associated with airflow limitation in the general population.¹⁰⁷

An association between dietary intake of antioxidant vitamins and lung function has been demonstrated in the general population. Britton and coworkers¹⁰⁸ showed in a population of 2,633 subjects an association between dietary intake of the antioxidant vitamin E and lung function, supporting the hypothesis that this antioxidant may have a role in protecting against the development of COPD; hence, vitamin supplementation may be a possible preventive therapy against the development of COPD. Such intervention studies have been difficult to carry out,¹⁰⁹ but there is at least some evidence to suggest that antioxidant vitamin supplementation reduces oxidant stress, measured as a decrease in pentane levels in breath as an assessment of lipid peroxides.¹¹⁰

	Oxidative Stress and Gene Expression

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Proinflammatory Genes
There is overwhelming evidence that COPD is associated with airway and airspace inflammation, as shown for example by recent biopsy studies.³² Numerous markers of inflammation have been shown to be elevated in the sputum of patients with COPD, such as interleukin (IL)-8 and tumor necrosis factor (TNF)-.¹¹¹

Genes for many inflammatory mediators, such as the cytokines IL-8, TNF-, and nitric oxide (NO) are regulated by transcription factors such as TNF-B). NF-B is present in the cytosol in an inactive form linked to its inhibitory protein IB. Many stimuli, including cytokines and oxidants, activate NF-B, resulting in ubiquination cleaving of IB from NF-B and the destruction of IB in the proteozome.¹¹² This critical event in the inflammatory response is redox sensitive. We have shown in preliminary studies in vitro, using both macrophage cell lines and alveolar and bronchial epithelial cells, that oxidants cause the release of inflammatory mediators such as IL-8, IL-1, and NO, and that these events are associated with increased expression of the genes for these inflammatory mediators and increased nuclear binding or activation of NF-B.^{113 114}

Thiol antioxidants such as N-acetylcysteine (NAC) and nacystelin, which have potential as therapies in COPD, have been shown in in vitro experiments to block the release of these inflammatory mediators from epithelial cells and macrophages, by a mechanism involving increasing intracellular GSH and decreasing NF-B activation.^{113 114}

Antioxidant Genes
As described above, there is considerable evidence for an increased oxidant burden in the lungs of smokers and patients with COPD. An important effect of oxidative stress is the upregulation of protective antioxidant genes. The antioxidant GSH is concentrated in ELF compared with plasma,¹¹⁵ and appears to have an important protective role, together with its redox enzymes in the airspaces and intracellularly in epithelial cells. To illustrate the protective role of GSH against the effects of cigarette smoke, we have developed models in vivo in the rat and in vitro using monolayer cultures of alveolar epithelial cells, to assess the injurious effects of cigarette smoke. Human studies have shown that GSH is elevated in ELF in chronic cigarette smokers, compared with nonsmokers,⁶³ an increase that does not occur during acute cigarette smoking.⁹ The effects of acute and chronic cigarette smoking can be mimicked following intratracheal instillation of CSC in the rat and exposure of epithelial cell monolayers to cigarette smoke in vitro.^{20 21 68} Following exposure to cigarette smoke, there is a profound decrease in GSH in BAL in the rat that is mirrored by a fall in total lung GSH 6 h after exposure.^{21 68} Similarly, there is a fall in intracellular GSH in epithelial cells following exposure to CSC.^{20 68} There is an association between the fall in lung and intracellular GSH both in vivo and in vitro and the increase in epithelial permeability, as described above.

We have used a rat model of intratracheal instillation of CSC in vivo and exposure of epithelial cell monolayers in vitro to study the regulation of GSH and its redox system in response to CSC and other oxidants, and in particular to investigate the discrepancy between GSH levels in chronic and acute cigarette smoking. After exposure of airspace epithelial to CSC in vitro, there is an initial decrease in intracellular GSH with a rebound increase when the cells are washed and culture is continued for 24 h.¹¹⁶ This effect in vitro was mimicked by a similar change in GSH in rat lungs in vivo following intratracheal instillation of CSC,⁶⁸ associated with an increase in oxidized GSH. We also examined the activity of the major enzymes involved in GSH synthesis and in the GSH redox system in response to CSC both in vivo and in vitro. The initial fall in lung and intracellular GSH after treatment with CSC was associated with a decrease in the activity of -glutamylcysteine synthetase (-GCS), the rate-limiting enzyme of GSH synthesis, with recovery of the activity by 24 h.^{68 115} We hypothesize that the increased levels of GSH following CSC exposure may be due to induction of the -GCS gene by components within cigarette smoke. Using reverse transcriptase-polymerase chain reaction, we showed an increase in -GCS messenger RNA expression 12 to 24 h after airspace epithelial cells were exposed to CSC in vitro (Fig 4 ).^{116 117} We also demonstrated that the upregulation of -GCS gene expression occurred at the transcriptional level (Fig 4) . We suggested that this might be due to activation of redox-sensitive transcription factors involved in the regulation of -GCS expression. In a series of experiments using both the gel mobility shift assay and reporter system in which the promoter region of -GCS gene was transfected into airway epithelial cells, we showed that CSC activated the transcription factor activator protein (AP)-1.^{118 119} In deletion experiments and using site-directed mutagenesis in a reporter system, we demonstrated that a proximal AP-1 is critical for the regulation of -GCS gene expression in response to various oxidants including cigarette smoke (Fig 5 ),¹¹⁹ and hence GSH synthesis in lung epithelial cells. Thus oxidative stress, including that produced by cigarette smoking, causes upregulation of an important gene involved in the synthesis of GSH as an adaptive or protective effect against oxidative stress. These events are likely to account for the increased GSH levels seen in the ELF in chronic cigarette smokers, which acts as a protective mechanism, whereas the more injurious effects of cigarette smoke may occur repeatedly during and immediately after cigarette smoking when the lung is depleted of antioxidants, including GSH. The cytokine TNF, which is thought to have a role in the lung inflammation in COPD, also decreases intracellular GSH levels initially in epithelial cells by a mechanism involving intracellular oxidative stress, which is followed 12 to 24 h thereafter by a rebound increase in intracellular GSH as a result of AP-1 activation and an increased -GCS expression.¹¹⁹ Corticosteroids have been used as anti-inflammatory agents in COPD, but there is still doubt over their effectiveness in reducing airway inflammation in COPD. Interestingly dexamethasone also causes a decrease in intracellular GSH in airspace epithelial cells, but no rebound increase compared with the effects of TNF.¹¹⁹ Moreover, the rebound increase in GSH produced by TNF in epithelial cells is prevented by cotreatment with dexamethasone.¹¹⁹ These effects may have relevance for the treatment of COPD patients with corticosteroids.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 4.. Effect of cigarette smoke on -GCS messenger RNA expression in alveolar epithelial cells (A549) exposed to CSC. -GCS gene expression by reverse transcriptase-polymerase chain reaction increase following exposure to CSC is shown (upper panel). This effect was blocked by actinomycin D (AD) but not by cycloheximide (CX). The densitometry of the -GCS band relative to ß-actin is shown (lower panel). These results show that cigarette smoke increases the expression of -GCS messenger RNA in alveolar epithelial cells, an effect which is at the transcriptional level (modified from Rahman et al116 ).

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 5.. Constituitive and inducible regulation of the -GCS-HS 5'flanking region in a chloramphenicol acetyl transferase (CAT) reporter system. The 5'-flanking successive deletion sequence of the -GCS-HS gene from oligonucleotides - 1050, - 818, - 511, - 305, - 201 to + 82 base pair were cloned into pCAT-Basic and co-transfected into A549 alveolar epithelial cells along with the control plasmid pressure support ventilation (PSV)-ß-Gal. Left: the relative positions of the cis-acting DNA elements and a restriction map of the promoter region cloned in pCRII vector. The numbers in the figure represent the nucleotide positions from the transcriptional start site of the -GCS-HS gene, which is indicated by the bent arrow on the right. The dotted line on the left indicates an additional 50 base pair from multiple cloning sites of the pCRII vector. The structure of the -GCS-HS CAT plasmids are shown below on the right. Deletion mutants were ligated to upstream of the CAT gene in pCAT Basic vector (pCB), and its constitutive transcriptional CAT activity was measured 36 to 48 h after transfection. Various deleted constructs were transfected into A549 cells and exposed to hydrogen peroxide (100 µM) and menadione (100 µM). After 24-h incubation, the cells were harvested and assayed for CAT activity. Right: Transcriptional activity among different constructs was standardized by the amount of CAT activity relative to ß-Gal activity. The results are shown as percentages of the CAT concentration compared to that of pCBGCS. Each histogram represents the mean and the bars the SEM of four independent transfections, each performed in duplicate with the activity pCBGCS set at 100%. **p < 0.01, ***p < 0.001, compared to pCBGCS (modified from Rahman and MacNee118 ). Abbreviations: GAL = galactosidase activity; Kpn = restriction enzyme that cuts at 5'-GGTACC-3'; BAL = restriction enzyme that cuts at TGGCCA at 3 CGATGG-5'; TATA = repeating units of T and A oligonucleotides from where transcription starts; DRA = restriction enzyme that cuts at 5'-TTTAAA-3'; NSI = restriction enzyme that cuts at 5'-ATGCAT-3'; pCBGCS = PCAT basic linked to the GCS-HS promoter; CAT = chloramphenicol acetyltransferase.

The cfos gene belongs to a family of growth- and differentiation-related immediate early genes, the expression of which generally represents the first measurable response to a variety of chemical and physical stimuli.¹¹² Studies in various cell lines have shown enhanced gene expression of cfos in response to CSC.¹²¹ These effects of CSC can be mimicked by peroxynitrite and smoke-related aldehydes in concentrations that are present in CSC.¹²¹ The effects of CSC can be enhanced by pretreatment of the cells with buthionine sulfoxamine to decrease intracellular GSH and can be prevented by treatment with NAC, a thiol antioxidant.¹²¹ These studies emphasize the importance of the intracellular levels of the antioxidant GSH in gene expression.

Thus oxidative stress, including that produced by cigarette smoke, causes increased gene expression of both proinflammatory genes by oxidant-mediated activation of transcription factors such as NF-B, and activation of protective genes such as -GCS through other transcription factors, which in the case of -GCS, is the transcription factor AP-1. A balance may therefore exist between pro- and anti-inflammatory gene expression in response to cigarette smoke, which may be critical to whether cell injury is induced by cigarette smoking (Fig 6 ). Knowledge of the molecular mechanisms that regulate these events may open new therapeutic avenues in the treatment of COPD.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 6.. The effect of oxidants on intracellular GSH redox status and NF-B and AP-1 nuclear translocation. Oxidants cause nuclear translocation of NF-B, which upregulates genes for cytokines such as TNF and IL-1, which themselves can produce oxidative stress through the release of oxidants from mitocondrial electron transfer chain that may result in decreased intracellular GSH. In addition, oxidants can upregulate AP-1, which can cause nuclear translocation of AP-1, which has been shown to increase messenger RNA for -GCS, which will increase intracellular GSH. An imbalance may occur between these pro- and anti-inflammatory effects of oxidant-mediated gene regulation.

	Oxidative Stress and Susceptibility to COPD

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Polymorphisms of various genes have been shown to be more prevalent in smokers who develop COPD than in nonsmokers.¹²³ A number of these polymorphisms may have functional significance, such as the association between the TNF- gene polymorphism (TNF-2) which may be associated with increased TNF levels in response to inflammation, and the development of chronic bronchitis.¹²⁴ Relevant to the effects of cigarette smoke is a polymorphism in the gene for microsomal epoxide hydrolase, which is an enzyme involved in the metabolism of highly reactive epoxide intermediates that are present in cigarette smoke.¹²⁵ The proportion of individuals with a slow microsomal epoxide hydrolase activity (homozygoes) was significantly higher in patients with COPD and a subgroup of patients shown pathologically to have emphysema (COPD, 22%; emphysema, 19%) compared with control subjects (6%). It may be that a panel of the susceptibility polymorphisms of functional significance in enzymes involved in xenobiotic metabolism or antioxidant enzyme genes may allow individuals to be identified as being susceptible to the effects of cigarette smoke.

Therapeutic Options to Redress the Oxidant/Antioxidant Imbalance in COPD
Having demonstrated evidence for an oxidant/antioxidant imbalance in smokers and its probable role in the pathogenesis of COPD, do we have any therapeutic options?

Various approaches have been tried to redress this imbalance. One approach would be to target the inflammatory response by reducing the sequestration or migration of leukocytes from the pulmonary circulation into the airspaces. Possible therapeutic options for this are drugs that alter cell deformability, so preventing neutrophil sequestration or the migration of neutrophils, either by interfering with adhesion molecules necessary for migration, or preventing the release of inflammatory cytokines such as IL-8 or leukotriene-B4, which result in neutrophil migration. It should also be possible to use anti-inflammatory agents to prevent the release of oxygen radicals from activated leukocytes or to quench those oxidants once they are formed, by enhancing the antioxidant screen in the lungs.

There are various options to enhance the lung antioxidant screen. One approach would be the molecular manipulation of antioxidant genes, such as GP or genes involved in the synthesis of GSH, such as -GCS or by developing molecules with activity similar to those of antioxidants enzymes such as catalase and SOD.

Another approach would simply be to administer antioxidant therapy. This has been attempted in cigarette smokers using various antioxidants such as vitamin C and vitamin E.^{124 125 126 127 128 129} Attempts to supplement lung GSH have been tried using GSH or its precursors.¹³⁰ GSH itself is not efficiently transported into most animal cells, and an excess of GSH may be a source of the thyl radical under conditions of oxidative stress.¹³¹ Nebulized GSH has also been used therapeutically, but this has been shown to induce bronchial hyperreactivity.¹³² Cysteine is a thiol that is the rate-limiting amino acid in GSH synthesis.¹³³ Cysteine administration is not possible since it is oxidized to cysteine that is neurotoxic.¹³⁴ The cysteine-donating compound NAC acts as a cellular precursor of GSH and becomes de-acetylated in the gut to cysteine following oral administration. It reduces disulphide bonds and has the potential to interact directly with oxidants. The use of NAC in an attempt to enhance GSH in patients with COPD has met with varying success.^{135 136} NAC given orally in low dosages, 600 mg/d, to normal subjects results in very low levels of NAC in the plasma for up to 2 h after administration.¹³⁵ Bridgeman and colleagues¹³⁶ showed after 5 days of NAC, 600 mg tid, that there was a significant increase in plasma GSH levels. However, there was no associated rise in BAL GSH or in lung tissue. These data seem to imply that producing a sustained increase in lung GSH is difficult using NAC in subjects who are not already depleted of GSH. In spite of this, continental European studies have shown that NAC reduces the number of exacerbation days in patients with COPD.^{137 138} This was not confirmed in a British Thoracic Society study of NAC.¹³⁹ The contradictory results of these studies may result from several reasons. Firstly, the positive studies of NAC were in patients who had relatively mild COPD, whereas in the British study the patients had more severe COPD. Secondly, a relatively small dose of NAC was given in both studies.

N-acystelyn (NAL) is a lysine salt of N-acetylinecysteine. It is also a mucolytic and oxidant thiol compound that, in contrast to NAC, which is acid, has a neutral pH. NAL can be aerosolized into the lung without causing significant side effects.¹⁴⁰ Studies comparing the effects of NAL and NAC found that both drugs enhanced intracellular GSH in alveolar epithelial cells¹⁴⁰ and inhibited hydrogen peroxide and superoxide anion release from neutrophils harvested from peripheral blood from smokers and patients with COPD.¹⁴¹

Most animal cells normally export GSH, and do not take up intact GSH. GSH ethyl ester contains an ethyl group that is esterified to the glycine of GSH. GSH ethyl ester is more lipophylic and thus passes more readily into cells than GSH. The monoester is then hydrolyzed to GSH by cytosolic nonspecific esterase.¹⁴² GSH monoethyl ester is resistant to the cleavage by the enzyme -glutamylcysteine transpeptidase and has been used to increase GSH in vitro.¹⁴³ Thiazolidine is a potentially useful compound for cysteine delivery and can be shown to protect against oxidative injury.¹⁴⁴ However, there are no studies in humans that validate these compounds for clinical trials.

Molecular regulation of GSH synthesis by targeting -GCS has great promise in as a means of treating oxidant medicated injury in the lungs. Cellular GSH may be increased by increasing -GCS activity. This may be possible by gene transfer techniques, although this would be an expensive treatment that may not be considered for a condition such as COPD. However, knowledge of how -GCS is regulated may allow the development of other compounds that may act to enhance GSH.

In summary, there is now very good evidence for an oxidant/antioxidant imbalance in COPD and increasing evidence that this imbalance is important in the pathogenesis of this condition. There are a number of important effects of oxidative stress in smokers that are relevant to the development of COPD (Fig 7 ). Oxidative stress may also be critical to the inflammatory response to cigarette smoke, through the upregulation of redox-sensitive transcription factors and hence proinflammatory gene expression; but it is also involved in the protective mechanisms against the effects of cigarette smoke by the induction of antioxidant genes. Inflammation itself induces oxidative stress in the lungs, and polymorphisms on genes for inflammatory mediators or antioxidant genes may have a role in the susceptibility to the effects of cigarette smoke. Knowledge of the mechanisms of the effects of oxidative stress should in the future allow the development of potent antioxidant therapies that test the hypothesis that oxidative stress is involved in the pathogenesis of COPD, not only by direct injury to c