INTRODUCTION

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An increasing amount of research has focused on the proposal that an oxidant/antioxidant imbalance occurs in smokers and in patients with chronic obstructive pulmonary disease (COPD) as part of the pathogenesis of this condition (1). The reason for this is obvious since cigarette smoke, which is the major etiological factor in COPD, has been estimated to contain between 10¹⁶ and 10¹⁷ oxidant molecules per puff (2). The traditional role for oxidants in the pathogenesis of COPD, whether inhaled in the form of cigarette smoke or released from activated neutrophils, is the inactivation of ₁-proteinase inhibitor (₁-PI), producing a functional deficiency of ₁-PI in the airspaces, an event that is thought to be critical to the proteinase/ antiproteinase imbalance that occurs as part of the pathogenesis of emphysema. However, it has been difficult to prove this theory in vivo since it is complicated by the presence of other proteinases and antiproteinases and by the fact that few studies in this field have controlled for the acute effects of cigarette smoking. One study that did assess the acute effect of smoking on the elastase inhibitory capacity of bronchoalveolar lavage found a small but significant decrease in elastase inhibitory capacity 1 h after smoking a cigarette (3). However, other studies of chronic cigarette smokers, where the smoking history has not been controlled, have been inconclusive. Several other targets, both in the lungs and in the blood, for the oxidative stress that occurs in smokers and in patients with COPD are discussed in this article. Particular emphasis is given to newer data that demonstrate the importance of oxidative stress as a trigger for the upregulation of proinflammatory genes and protective mechanisms such as the upregulation of genes for antioxidant mechanisms. The development of antioxidant therapy must take account of these fundamental molecular mechanisms in the inflammatory response to cigarette smoke, in order to protect effectively against both the injurious and proinflammatory effects of oxidative stress.

	THE OXIDANT BURDEN IN SMOKERS AND PATIENTS WITH COPD

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Oxidants in Cigarette Smoke

Cigarette smoke is a complex mixture of more than 4,700 chemical compounds, including high concentrations of free radicals and other oxidants (2). Free radicals in cigarette smoke are derived from both the gas and the tar phase. Pryor and Stone have reported that the gas-phase cigarette smoke contains approximately 10¹⁵ radicals per puff, primarily of the alkyl and peroxyl types. In addition, nitric oxide is present in cigarette smoke in concentrations of 500-1,000 ppm (4). Nitric oxide (NO·) reacts quickly with the superoxide anion (O₂^·) to form peroxynitrite (ONOO), and with peroxyl radicals to give alkyl peroxynitrites (ROONO).

The radicals in the tar phase of cigarette are more stable and are predominantly organic, such as the semiquinone radical, which can react with oxygen to produce O₂^·. The tar phase also contains the hydroxyl radical (·OH) and hydrogen peroxide (H₂O₂) (4). Cigarette smoke tar contains more than 10¹⁸ free radicals per gram, and is also an effective metal chelator and can bind iron to produce the tar-semiquinone + tar-Fe²⁺, which can generate H₂O₂ (5). Whereas short-lived radicals in the gas phase of cigarette smoke may be quenched immediately in the epithelial lining fluid (ELF), redox reactions in cigarette smoke condensate, which forms in the epithelial lining fluid, may produce reactive oxygen species (ROSs) for a considerable time.

Cell-derived Oxidants

The oxidative burden produced by inhaling cigarette smoke can be further enhanced in the lungs of cigarette smokers by the release of oxygen radicals from the influx and activation of inflammatory leukocytes, both neutrophils and macrophages, which occurs in the lungs of cigarette smokers. Leukocytes from smokers have been shown to release increased amounts of oxidants such as O₂^· and H₂O₂, compared with those from nonsmokers (6).

The generation of oxidants in epithelial lining fluid in smokers is further enhanced by the presence of increased amounts of free iron in the airspaces (7). 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. Furthermore, macrophages from smokers release more free iron in vitro than those from nonsmokers (7). 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.

	OXIDANT DAMAGE

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Components of the lung matrix (such as elastin and collagen) can be directly damaged by oxidants in cigarette smoke (8). Cigarette smoke can also interfere with elastin synthesis and repair (9), which can augment proteolytic damage to matrix components and thus enhance the development of emphysema.

The presence of oxidative stress in the airspaces and in the blood, manifest by the measurements described above, initiates a number of early events in the inflammation in the lungs. All tissues are vulnerable to oxidant damage but, by virtue of its direct contact with the environment, the airspace epithelial surface of the lung is particularly vulnerable. The respiratory tract lining fluid (RTLF) forms the interface between the epithelial cells and the external environment and thus constitutes the first line of defense against inhaled oxidants. Alveolar epithelial cells are important in maintaining the integrity and fluid balance of the lungs and in the control of inflammation. Increased epithelial permeability is one of the earliest effects of cigarette smoking and may enhance the inflammatory process by allowing easier access for inflammatory and injurious mediators between the blood, interstitium, and alveolar space. In a number of studies both in vitro and in vivo, we have shown that increased epithelial permeability, produced by cigarette smoking, is likely to be oxidant mediated, through depletion of the major lung antioxidant glutathione (GSH) (1).

Among the earliest events in the inflammatory response in the lungs to cigarette smoking is an increase in the normal sequestration of neutrophils within the pulmonary microcirculation (10). During cigarette smoking there is an acute increase in the number of neutrophils sequestered in the pulmonary microcirculation (11). In a further series of experiments we have shown that this increased sequestration of neutrophils in the lungs is due to the effects of systemic oxidative stress reaching the circulation, and either directly or indirectly through release of cytokines, decreasing the deformability of neutrophils, and thus increasing their sequestration (12). Once sequestered, components of cigarette smoke can alter neutrophil adhesion to endothelium by upregulating CD18 integrins (13). 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 enhance functional adherence (14). 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 (15, 16).

	EVIDENCE OF OXIDATIVE STRESS IN SMOKERS AND PATIENTS WITH COPD

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There is now overwhelming evidence of the presence of increased oxidative stress in smokers and patients with COPD (17). 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. However, these markers indicate that oxidative stress has occurred, but not that this event is necessarily involved in the pathogenesis of the condition that is being studied. Markers of oxidative stress have been demonstrated in the epithelial lining fluid, in the breath, and in the urine in cigarette smokers and patients with COPD, and there has been interest in evidence of systemic oxidative stress, measured in the blood. In the breath hydrogen peroxide and nitric oxide, direct measurements of oxidants generated by cigarette smoking, or released from inflammatory leukocytes and epithelial cells, have been shown to be increased in patients with both stable and acute COPD in the case of hydrogen peroxide (18) and in exacerbations of the condition in the case of nitric oxide (19).

Alveolar leukocytes from smokers and patients with chronic bronchitis have increased ability to release oxygen radicals, compared with those from healthy controls (20). Xanthine/ xanthine oxidase, which generates superoxide anion, has also been shown to be increased in the bronchoalveolar lavage fluid from patients with COPD (21). Antioxidant levels vary in epithelial lining fluid; for example, vitamins E and C are depleted in chronic cigarette smokers, but glutathione is elevated (1, 6, 22). These variable results of measurements of the levels of antioxidants in epithelial lining fluid probably reflect differences in the acute on chronic effects of cigarette smoking (see below). Urine isoprostane F₂-III, which is an isomer of prostaglandin, formed by free radical peroxidation of arachidonic acid, has been shown to be elevated in patients with COPD, compared with healthy controls, and to be even more elevated in exacerbations of the condition (23).

There has been considerable interest in the systemic effects of COPD (24). There is accumulating evidence of a systemic oxidative oxidant burden leading to systemic oxidative stress in patients with COPD. This is reflected in the increased sequestration of neutrophils in the pulmonary microcirculation during smoking and during exacerbations of COPD and, as described above, this is an oxidant-mediated event (11, 15).

Rahman and colleagues (16) demonstrated, in peripheral blood neutrophils obtained from patients with acute exacerbations of COPD, increased production of superoxide anion, which returned to normal when the patients were restudied when clinically stable. Circulating neutrophils in patients with COPD show upregulation of their surface adhesion molecules, which may be an oxidant-mediated effect (25). Activation of neutrophils in COPD may be even more pronounced in neutrophils that are sequestered in the pulmonary microcirculation in smokers and in COPD, since animal models have shown that neutrophils that are sequestered in the pulmonary microcirculation in lung inflammation release more reactive oxygen species than do circulating neutrophils in the same animal (26). These studies suggest that the neutrophils sequestered in the pulmonary microcirculation induce oxidative stress, which may have a role in inducing airway injury in COPD, particularly during exacerbations.

A major site of free radical attack is on polyunsaturated fats and fatty acids in cell membranes, producing lipid peroxidation, a process that may continue as a chain reaction to generate peroxides and aldehydes. The levels of products of lipid peroxidation in plasma or in bronchoalveolar lavage fluid, measured as thiobarbituric acid-reactive substances (TBARS), have been shown to be significantly increased in healthy smokers and patients with acute exacerbations of COPD, compared with healthy nonsmokers (6, 24). However, there is a problem with the specificity of thiobarbituric acid-malondialdehyde assays measuring lipid peroxidation as TBARS, since this does not directly measure the lipid peroxidation reaction. Duthie and coworkers (27) found that plasma levels of conjugated levels of dienes of linoleic acid, a secondary product of lipid peroxidation, were increased in chronic smokers. Furthermore, Morrow and coworkers (28) showed increased levels of circulating products of lipid peroxidation (F₂-isoprostane), which is a more direct measurement of lipid peroxidation in smokers.

We have also shown good evidence that oxidative stress reaches the circulation as shown by a fall in the antioxidant capacity of blood in chronic smokers, with a further decrease in acute smokers (24). A similar fall in plasma antioxidant capacity occurs in exacerbations of COPD (16, 24). The decrease in antioxidant capacity in smokers could be due to depletion of a number of factors in the plasma, including protein sulfydryls, which are depleted after exposure of plasma to cigarette smoke in vitro (29). Other investigators have shown that other major plasma antioxidants such as ascorbic acid, vitamin E, -carotene, and selenium are depleted in the serum of chronic cigarette smokers (reviewed in [1]). 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 (29). Increased inhalation of NO^· in cigarette smokers, as well as NO^· and superoxide anion released by activated phagocytes, react to form peroxynitrite. Peroxynitrite is cytotoxic, and has been shown to decrease plasma antioxidant capacity by rapid oxidation of ascorbic acid, uric acid, and plasma sulfhydryls (30). Evidence of NO^·/peroxynitrite activity in plasma has been demonstrated in cigarette smokers (31). Nitration of tyrosine residues on proteins in plasma leads to the production of 3-nitrotyrosine. Petruzzelli and colleagues (31) demonstrated the presence of 3-nitrotyrosine in plasma in smokers, possibly at higher levels than in a small group of nonsmokers. They also confirmed low levels of antioxidant capacity, as we have previously shown in smokers (24), which were negatively correlated with the levels of 3-nitrotyrosine (31). The fall in antioxidant capacity also correlates with the increased release of oxygen radicals from circulating neutrophils in patients with exacerbations of COPD (24).

	EVIDENCE OF A RELATIONSHIP BETWEEN OXIDANT/ANTIOXIDANT BALANCE AND THE DEVELOPMENT OF AIRWAY OBSTRUCTION

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The neutrophil appears to be a critical cell in the pathogenesis of COPD. Previous epidemiological studies have shown a relationship between circulating neutrophil numbers and the FEV₁ (32) and, indeed, the change in peripheral blood neutrophil count and the change in airflow limitation over time (33). Richards and coworkers have shown a relationship between peripheral blood neutrophil chemiluminescence and measures of airflow limitation in young cigarette smokers (34). Even passive cigarette smoking has been associated with increased peripheral blood leukocyte counts and enhanced release of oxygen radicals (35). 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 (36).

An association between dietary intake of antioxidant vitamins and lung function has been demonstrated in the general population. Britton and coworkers (37), in a population of 2,633 subjects, showed 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. This study supports the concept that vitamin supplementation may be a possible preventive therapy against the development of COPD (38). Such intervention studies have been difficult to carry out, 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.

	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 biopsy studies. Numerous markers of inflammation have been shown to be elevated in the sputum of patients with COPD. Prominent among these are interleukin (IL)-8 and tumor necrosis factor  (TNF-) (39).

Genes for many inflammatory mediators, such as the cytokines IL-8, TNF-, and nitric oxide, are regulated by transcription factors such as nuclear factor-B (NF-B). NF-B is present in the cytosol in an inactive form linked to its inhibitory protein I-B. Many stimuli, including cytokines and oxidants, activate NF-B, resulting in ubiquination cleaving of I-B from NF-B and the degradation of I-B by the proteosome (40). 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 nitric oxide 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 (41).

Thiol antioxidants such as N-acetylcysteine 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 glutathione and decreasing NF-B activation (41).

Antioxidant Genes

An important effect of oxidative stress is the upregulation of protective antioxidant genes. We have studied the antioxidant glutathione and its redox enzymes, since glutathione, which is concentrated in epithelial lining fluid compared with plasma (1), appears to have an important protective role in the airspaces and intracellularly in epithelial cells. To illustrate the protective role of glutathione 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. Studies in humans have shown that glutathione is elevated in epithelial lining fluid in chronic cigarette smokers, compared with nonsmokers (22). This increase does not occur during acute cigarette smoking (6). The differential effects of acute and chronic cigarette smoking can be mimicked after intratracheal instillation of cigarette smoke condensate in the rat and exposure of epithelial cell monolayers to cigarette smoke in vitro (42, 43). After exposure to cigarette smoke there is a profound decrease in GSH in bronchoalveolar lavage (BAL) in the rat that is mirrored by a fall in total lung GSH 6 h after exposure (42, 43). Similarly there is a profound fall in intracellular GSH in epithelial cells after exposure to cigarette smoke condensate (44). The presence of extracellular glutathione in concentrations of 500 µM, which are similar to concentrations present in lung epithelial lining fluid, are totally protective against the increased epithelial permeability that is induced by cigarette smoke condensate in in vitro experiments (44).

There is an association between the fall in lung and intracellular glutathione both in vivo and in vitro and the increase in epithelial permeability. Support for this relationship being causal is shown by studies in which lung and intracellular glutathione was depleted with buthionine sulfoxamine, an inhibitor of -glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione synthesis, which produced increased permeability of airspace epithelial cell monolayers in vitro and rat lungs in vivo (43, 44).

To investigate the discrepancy between glutathione levels in chronic and acute cigarette smoking, we have used a rat model of intratracheal instillation of cigarette smoke condensate in vivo and exposure of epithelial cell monolayers in vitro to study the regulation of glutathione and its redox system in response to cigarette smoke condensate and other oxidants. After exposure of airspace epithelial cells to cigarette smoke condensate 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 (45). This effect in vitro was mimicked by a similar change in glutathione in rat lungs in vivo after intratracheal instillation of cigarette smoke condensate (42, 44), associated with an increase in the oxidized form (GSSG). We also examined the activity of the major enzymes involved in glutathione synthesis and in the glutathione redox system in response to cigarette smoke condensate both in vivo and in vitro. The initial fall in lung and intracellular glutathione after treatment with cigarette smoke condensate was associated with a decrease in the activity of GCS, the rate-limiting enzyme of glutathione synthesis, with recovery of the activity by 24 h (42, 45). We hypothesize that the increased levels of glutathione after cigarette smoke condensate exposure may be due to induction of the GCS gene by components within cigarette smoke. By using the reverse transcriptase polymerase chain reaction we showed an increase in GCS mRNA expression 12-24 h after airspace epithelial cells were exposed to cigarette smoke condensate in vitro (45, 46) (Figure 1). We also demonstrated that the upregulation of GCS gene expression occurred at the transcriptional level (Figure 1). 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 a reporter system in which the promoter region of the GCS gene was transfected into airway epithelial cells, we showed that cigarette smoke condensate activated the transcription factor activator protein 1 (AP-1) (45, 47). In deletion experiments and by using site- directed mutagenesis in a promoter system we demonstrated that a proximal AP-1 is critical for the regulation of -glutamylcysteine synthetase gene expression in response to various oxidants including cigarette smoke (Figure 2) (47, 48), and hence glutathione 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 glutathione as an adaptive or protective effect against oxidative stress. These events are likely to account for the increased glutathione levels seen in the epithelial lining fluid 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 glutathione. The cytokine TNF-, which is thought to have a role in the lung inflammation in COPD, also decreases intracellular glutathione levels initially in epithelial cells by a mechanism involving intracellular oxidative stress, which is followed 12-24 h thereafter by a rebound increase in intracellular glutathione as a result of AP-1 activation and increased GCS expression (48). Corticosteroids have been used as antiinflammatory agents in COPD, but there is still doubt concerning their effectiveness in reducing airway inflammation in COPD. It is interesting that dexamethasone also causes a decrease in intracellular glutathione in airspace epithelial cells, but no rebound increase compared with the effects of TNF- (46). Moreover, the rebound increase in glutathione produced by TNF in epithelial cells is prevented by cotreatment with dexamethasone (48). These effects may have relevance for the treatment with corticosteroids of patients with COPD.

View larger version (57K): [in this window] [in a new window]   View larger version (26K): [in this window] [in a new window]   Figure 1.   Transcriptional regulation of GCS-HS mRNA in A549 cells. (A) Effects of actinomycin D and cycloheximide on cigarette smoke condensate (CSC)-induced GCS-HS mRNA expression. (B) Densitometric quantitation of GCS-HS mRNA compared with -actin mRNA. ***p < 0.001 compared with control. (Used with permission from Reference 45.)

View larger version (17K): [in this window] [in a new window]   View larger version (35K): [in this window] [in a new window]   Figure 2.   Constitutive and inducible regulation of the GCS-HS 5' flanking region in a chloramphenicol acetyltransferase (CAT) reporter system. (A) The 5' successive deletion sequences with the restriction enzymes used are shown by the arrows, which were cloned into pCAT-basic and cotransfected into A549 alveolar epithelial cells. (B) Transcriptional activity among different constructs measured as CAT activity in response to the oxidants H2O2 and menadione (MQ). **p < 0.01; ***p < 0.001 compared with pCBGCS. (Reprinted from Reference 47.)

Gilks and coworkers (49) have shown, in rats exposed to whole cigarette smoke for up to 14 d, increased expression of a number of antioxidant genes in the bronchial epithelial cells in these animals. Whereas mRNA of manganese superoxide dismutase (MnSOD) and metallothionein (MT) was increased at 1-2 d and returned to normal by 7 d, mRNA for glutathione peroxidase did not increase until after 7 d of exposure, suggesting the importance of the glutathione redox system as a mechanism for chronic protection against the effects of cigarette smoke (49).

The c-fos 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 (40). Studies in various cell lines have shown enhanced gene expression of the c-fos in response to cigarette smoke condensate (50). These effects of cigarette smoke condensate can be mimicked by peroxynitrite and smoke-related aldehydes in concentrations that are present in cigarette smoke condensate (50). The effects of cigarette smoke condensate can be enhanced by pretreatment of the cells with buthionine sulfoximine to decrease intracellular glutathione and can be prevented by treatment with N-acetylcysteine, a thiol antioxidant (50). These studies emphasize the importance of intracellular levels of the antioxidant glutathione in gene expression.

Thus oxidative stress, including that produced by cigarette smoke, causes increased gene expression of both injurious proinflammatory genes by oxidant-mediated activation of transcription factors such as NF-B, but also activation of protective genes such as -glutamylcysteine synthetase through other transcription factors, which in the case of GCS is the transcription factor AP-1. A balance may therefore exist between pro- and "antiinflammatory" gene expression in response to cigarette smoke, which may be critical to whether cell injury is induced by cigarette smoking (Figure 3). Knowledge of the molecular mechanisms that regulate these events may open new therapeutic avenues in the treatment of COPD.

View larger version (37K): [in this window] [in a new window]   Figure 3.   Schematic diagram of the role of oxidants and the antioxidant glutathione (GSH) in proinflammatory and -glutamyl cysteine synthetase (-GCS) gene expression.

	OXIDATIVE STRESS AND SUSCEPTIBILITY TO COPD

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It is well recognized that only a proportion15 to 20% of cigarette smokersappears to be susceptible to the effects of smoking, shows a rapid decline in FEV₁, and develops COPD. One reason for the susceptibility of some smokers to the development of COPD could be a genetic factor in which there has been interest (51). Polymorphisms of various genes have been shown to be more prevalent in smokers who develop COPD than in nonsmokers (51). A number of these polymorphisms may have functional significance, such as the association between the TNF- gene polymorphism (TNF2), which may be associated with increased TNF- levels in response to inflammation, and the development of chronic bronchitis (reviewed in [51]). 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 (52). The proportion of individuals with low microsomal epoxide hydrolase activity (homozygotes) 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 FOR REDRESSING THE OXIDANT/ANTIOXIDANT IMBALANCE IN COPD

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Having demonstrated evidence of 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 by 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 antiinflammatory 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 glutathione peroxidase or genes involved in the synthesis of glutathione, such as GCS, or by developing molecules with activity similar to those of antioxidant enzymes such as catalase and superoxide dismutase.

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 (reviewed in [1]). The results have been rather disappointing, although, as already described, the antioxidant vitamin E has been shown to reduce oxidative stress in patients with COPD (38). Attempts to supplement lung glutathione have been tried, using glutathione or its precursors (53). Glutathione itself is not efficiently transported into most animal cells and an excess of glutathione may be a source of the thiyl radical under conditions of oxidative stress. Nebulized glutathione has also been used therapeutically but this has been shown to induce bronchial hyperreactivity (54). Cysteine is a thiol that is the rate-limiting amino acid in GSH synthesis. Cysteine administration is not possible since it is oxidized to cystine, which is neurotoxic. The cysteine-donating compound N-acetylcysteine (NAC) acts as a cellular precursor of GSH and becomes deacetylated in the gut to cysteine after oral administration. It reduces disulfide bonds and has the potential to interact directly with oxidants. The use of N-acetylcysteine in an attempt to enhance GSH in patients with COPD has met with varying success (reviewed in [53]). NAC given orally in low doses of 600 mg/d to normal subjects results in low levels of NAC in the plasma for up to 2 h after administration. Bridgeman and colleagues (55) showed after 5 d of NAC (600 mg three times daily) 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 glutathione. In spite of this, continental European studies have shown that NAC reduces the number of exacerbation days in patients with COPD (56). This was not confirmed in a British Thoracic Society study of NAC (57). The contradictory results of these studies may result from several reasons; first, the positive studies of NAC were in patients who had relatively mild COPD, whereas in the British study the patients had more severe COPD. Second, a relatively small dose of N-acetylcysteine was given in both studies.

Nacystelyn (NAL) is a lysine salt of N-acetylcysteine. It is also a mucolytic and oxidant thiol compound; in contrast to NAC, which is acid, it has a neutral pH. NAL can be aerosolized into the lung without causing significant side effects (58). Studies comparing the effects of NAL and NAC found that both drugs enhanced intracellular glutathione in alveolar epithelial cells (58) and inhibited hydrogen peroxide and superoxide anion release from neutrophils harvested from the peripheral blood of smokers and patients with COPD (59).

Most animal cells normally export glutathione, and do not take up intact glutathione. Glutathione ethyl ester contains an ethyl group that is esterified to glutathione. Glutathione ethyl ester is more lipophilic and thus passes more readily into cells than glutathione. The monoester is then hydrolyzed to glutathione by cytosolic nonspecific esterase. Glutathione monoethyl ester is resistant to cleavage by the enzyme -glutamylcysteine transpeptidase and has been used to increase glutathione in vitro (60). Thiazolidine is a potentially useful compound for cysteine delivery and can be shown to protect against oxidative injury (61). However, there are no studies in humans that validate these compounds for clinical trials.

Molecular regulation of glutathione synthesis, by targeting GCS, has great promise as a means of treating oxidant-mediated 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 good evidence of 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 (Figure 4). 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 of 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 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 cells, but also as a fundamental factor in inflammation in smoking- related lung disease.

View larger version (30K): [in this window] [in a new window]   Figure 4.   Summary diagram of oxidant-mediated lung injury in smokers.