key: cord-0006479-5dlbf1oe
authors: Kelly, F. J.; Mudway, I. S.
title: Protein oxidation at the air-lung interface
date: 2003-07-31
journal: Amino Acids
DOI: 10.1007/s00726-003-0024-x
sha: a92ffafc26e9a0ca675c0715336c042542b2c544
doc_id: 6479
cord_uid: 5dlbf1oe

Whilst performing its normal functions the lung is required to deal with a range of toxic insults. Whether these are infectious agents, allergens or air pollutants they subject the lung to a range of direct and indirect oxidative stresses. In many instances these challenges lead to oxidative alterations of peptides and proteins within the lung. Measurement of protein oxidation products permits the degree of oxidative stress to be assessed and indicates that endogenous antioxidant defences are overwhelmed. The range of protein oxidation products observed is diverse and the nature and extent of specific oxidation products may inform us about the nature of the damaging ROS and NOS. Recently, there has been a significant shift away from the measurement of these oxidation products simply to establish the presence of oxidative stress, to a focus on identifying specific proteins sensitive to oxidation and establishing the functional consequences of these modifications. In addition the identification of specific enzyme systems to repair these oxidative modifications has lead to the belief that protein function may be regulated through these oxidation reactions. In this review we focus primarily on the soluble protein components of within the surface liquid layer in the lung and the consequence of their undue oxidation.

The lung, like the skin (Thiele, 2001) and the surface of the eye (Rose et al., 1998) , presents the first physical interface between the outside environment and the body. As such it is susceptible to a range of environmental toxicants, both viable, such as bacteria, viruses, allergen and fungi, and non-viable, pollutant oxidant gases (ozone, nitrogen dioxide) and airborne particulate matter. Whilst these toxicants are diverse, in all cases their pathophysiological action on the lung seems to involve the imposition of an oxidative stress (the excess generation of reactive oxygen species (ROS) in relation to available antioxidant defences). This may occur in the case of oxidant gases through the direct oxidation of biomolecules at the surface of the lung (Pryor, 1994) , or in the case of micro-organisms because they engender airway inflammation (Morcillo et al., 1999; Babior, 2000) . All pulmonary conditions where inflammation has been implemented (asthma, COPD, cystic fibrosis) appear to involve a component of oxidative stress. In this review we examine the impact of these challenges on the lung, focusing specifically on protein oxidation events within the thin amphipathic layer overlaying the respiratory epithelium: the respiratory tract lining fluid (Bastacky et al., 1995; van der Vliet et al., 1999) . In this context the endogenous antioxidant defences within this compartment will be outlined, as are the adaptive antioxidant pathways which function to mitigate against oxidative injury. Central to our review of the current literature will be the issue of whether protein oxidation is simply a biomarker of oxidative stress (allowing some discrimination of the radical species involved) or actually a mechanistically significant event, with major impacts on protein function and lung physiology. Whilst a considerable body of work has been performed on pulmonary tissues, examining protein oxidation in a variety of disease settings, comparatively little work has focused on protein modifications within the RTLF. This, allied to the critical position of this compartment suggests that identification of oxidatively modified proteins within this compartment is likely to represent a fertile area of future research. Whilst much of the focus of this review will be on the soluble protein components of RTLF we will also deal with the oxidation of proteins within lung tissue itself.

This aliphatic compartment consists of a two-layer structure, comprising a lower aqueous sol phase and an upper mucus, gel phase. The gel phase is largely composed of high molecular weight mucopolypeptide glycoproteins derived from sub-mucosal glands (Quinton, 1979) . Whilst these compounds may act as sacrificial antioxidants in vivo due to their thiol content (van der Vliet and Cross, 2001) their main function is to trap microorganisms and large particles from the air-stream. These particles are then transported by mucociliary action to the posterior pharynx and swallowed; therefore acting as a mechanical clearance mechanism to limit the penetration of inhaled toxicants into the lung. As a consequence of this action the gel phase of the RTLF is continually turning over. The lower, sol phase which has been shown to have a wide range of antioxidant defences (van der Vliet et al., 1999; Cantin et al., 1987; Mudway et al., 2001) in contrast turns over relatively slowly, permitting secreted antioxidants to concentrate within this compartment (Cantin et al., 1987) . In the distal airways, where mucus-secreting cells are absent, this layer is the first physical interface inhaled materials come into contact with. Furthermore, in airway inflammation the majority neutrophil, eosinophil, and macrophage actions occur within this compartment. Therefore much of the discussion in this review focusing on the antioxidant defences within the RTLF relates to events occurring with the sol compartment. Not all aspects of lung lining fluid are currently understood. For example, although it is appreciated that the depth of the fluid layer varies along the respiratory tree, its precise thickness is still under debate (Duneclift et al., 1997; Widdicombe et al., 1997) . In the upper airways, lung-lining fluid may be 1-10 "m thick compared with 0.2-0.5 "m in the distal bronchoalveolar regions. In addition whether the RTLF comprises a continuous sheet covering the pulmonary epithelium throughout its length, as supported by high resolution EM-studies (Bastacky et al., 1995) , or is patchy toward the more distal structures (Pryor et al., 1995) remains a point of contention. Both of these issues are critical in understanding the nature of oxidation reactions occurring at the lung surface as the RTLF may prevent or limit the extent of the reaction of inhaled toxicants with the lung epithelium.

RTLF can be obtained from human volunteers and patients using a variety of bronchoscopy based techniques, bronchial wash and bronchoalveolar lavage (BAL). More recently the use of less invasive techniques have also become widely employed: nasal lavage, (Noah et al., 1995; Housley et al., 1995) , sputum induction with hypertonic saline (Nordenhall et al., 2001; Jones et al., 2001) , and the condensation of exhaled breath (Antczak et al., 2002) . There is currently little data to demonstrate the quantitative relationship between indirect proximal airway measures of pulmonary inflammation with the more traditional gold standard bronchoscopy based techniques. Despite this important proviso, these less invasive techniques have permitted more detailed time course studies in human investigations, and investigation of inflammatory responses in patients with severe airways disease such as asthma and COPD. These procedures, with the exception of the collection of breath condensate yield both cell pellets and dilute samples of the RTLF after separation of these components by centrifugation. In addition bronchoscopy based techniques often incorporate the collection of bronchial biopsies. These samples can therefore all be analysed for antioxidant concentrations and a range of oxidation markers. Collection and analysis of the RTLF in lavage fluid supernatants however presents a number of problems: First, the RTLF proteome may be contaminated with intra-cellular and plasma proteins if the lavage, or post aspiration processing is too robust. Second, prolonged saline, or PBS dwell times in the lung can result in erroneously high antioxidant concentrations, as these molecules move down a concentration gradient from cellular stores. Finally, unless lavage is performed rapidly, pH and contaminating transition metal sources are tightly controlled and samples are processed and stored appropriately, RTLF components may become oxidised due to the lavage procedure.

The protein constituents of the RTLF have been reviewed in detail previously (Hatch, 1992) , however recent developments in proteomics using 2D-gel maps of lavage fluid samples have produced a fuller picture (Noel-Georis et al., 2002; Lenz et al., 1993; Sabounchi-Schutt et al., 2001; Lindahl et al., 1995; Wattiez et al., 1999) . These techniques have allowed the identification of protein profiles characteristic in a range of pulmonary diseases (sarcidosis, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis) and following acute airway insults (cigarette smoke) (Wattiez et al., 1999; Lindahl et al., 1996; Lenz et al., 1993; Lindahl et al., 1998; Lindahl et al., 1999a, b) . BAL protein components are diverse and derived from a variety of sources: local synthesis and export by epithelial and resident inflammatory cell populations and via transduction of plasma proteins across the blood-air barrier. Proteins constitute approximately 0.5-1% of the dry weight of bronchial secretions (Creeth, 1978) , and total protein concentrations in the RTLF have been reported to be in the order of 3-5 mg=mL in healthy human subjects Thompson et al., 1990) . These values are derived from BAL-fluid concentrations corrected for lavage dilution using the urea correction. Whilst there are acknowledged problems with this dilutional control (Haslam et al., 1999) , this is the most widely used approach in the literature and does give a indication of overall dilution, as long as the lavage dwell time is short (van der Vliet et al., 1999) .

The major protein constituents of RTLF are albumin (50-60% of the total protein pool), transferrin (5-6%), 1-antitrypsin (3-5%) and the immunolglobulins A and G (30%) (Hatch, 1992; Bell et al., 1981; Blomberg et al., 1999) . The ratio of these major protein species varies depending on the compartment sampled, with albumin concentrations lower (12-20%), and lysozyme (15-35%) and lactoferrin (2-4%) concentrations higher in the nasal and bronchial RTLFs. These regional differences are also apparent in the expression of specific proteins, such as surfactant proteins in the distal lung, and mucins in the upper airways. Proteomic analysis by the group of Bernard has identified over 1200 silver stained spots, corresponding to approximately 78 protein species on 2D-gels of BALfluid proteins (Wattiez et al., 1999) . Classification of this large number of proteins has been made on the basis of their origin: proteins derived from the plasma pool via transduction onto the surface of the lung, proteins synthesised in the lung, but also present in the plasma, and proteins unique to the surface of the lung. Using this approach it has been shown that the vast majority of the proteins found within the nasal RTLFs are derived from the plasma, whilst the distal lung has a significant complement of lung specific proteins (Noel-Georis et al., 2002) . BAL proteins can also be classified according to their function: proteases=anti-proteases (-1-trypsin, -1-Antitrypsin, -1 antichymotrypsin, -1-antiplasmin, -1-antiprotease, 2-Macroglobulin, secretory leukoprotease inhibitor); antioxidant enzymes (AOEB166, superoxide dismutase, glutathione S transferase, thioredoxin, plasma glutathione peroxidase), metal-chelating proteins (transferrin, lactoferrin, ceruloplasmin); surfactant proteins (SP-A=B=C and D), proteins involved in tissue repair and cellular proliferation (fibronectin, calcyclin, calgizzarin, cathepsin D, zinc--2 glycoprotein, transthyretin, clusterin, intestinal trefoil factor, 2-HS-glycoprotein); regulating airway immune responses (IgG, IgA, Ig-binding protein, complement fragments 3 and 4, lipocortin-1, Clara cell protein 16, lipocalin, 1-acid glycoprotein); anti-microbial proteins (defensins, cystatin S); lipid metabolism (acyl-CoA-binding protein, FABP-E, FABP-A, saposin D); and calcium binding (calvasculin, calreticulin, calgranulin A). Detection of certain proteins by this method is problematic due to issues of hydrophobicity, low molecular mass of certain proteins and also low expression levels. In addition some of the proteins detected are clearly intra-cellular (-and -actin, tubulin 2, tropomyosin, haemoglobin) suggesting cellular lysis. This list is far from exhaustive and does not include the wide range of cytokines=chemokines (IL-1=4=5=6=8= 10=18, TNF-Gro-, Rantes) and inflammatory cell activation markers (myeloperoxidase, eosinophil peroxidase, eosinophil cationic protein, and s-Selectin) that have been detected in BAL-fluid, both basally and during inflammatory episodes.

Reactive oxygen (hydroxyl radicals, superoxide, and hydrogen peroxide) and nitrogen species (nitric oxide, nitrite, and peroxynitrite) are produced in the body as a part of normal metabolism: through leakage from electron transport chains, as by-products of a number of enzyme reactions (cytochrome-P450, liposoxygenase, cycloxygenases, peroxidases), and through secretion from activated leukocytes. Whilst these species may play a modulating role in a range of physiological functions (Conner and Grisham, 1996) , over production in a range of conditions has been shown to cause tissue injury (Repine et al., 1997) . Endogenous antioxidant defences offset the potential damaging action of these reactive oxygen and nitrogen species. These may either act to directly detoxify these species or by converting them to less damaging species that can be removed by other components of the antioxidant network. However, if these defences are overwhelmed damage to cellular components will occur. By virtue of its large surface area and daily exposure to inhaled toxins the lung is particularly susceptible to oxidative and nitrosative injury. A number of other unique features of this compartment may also predispose it to oxidative injury. First, there is evidence, that the RTLF, unlike virtually every compartment in the body has a significant pool of non-transferrin bound iron (Gutteridge et al., 1996) . Second, this extracellular compartment is poorly invested with antioxidant enzyme defences compared with intra-cellular levels. Although both extra-cellular SOD (Mudway et al., 2001) and plasma glutathione peroxidase (Avissar et al., 1996) have been reported in the RTLF of healthy subjects, catalase and glutathione reductase are absent. Compounding this is the high concentration of peroxidase-like enzymes within this compartment: lactoperoxidase, myeloperoxidase and eosinophil peroxidase.

To offset this considerable burden the lung has evolved a range of specialized antioxidant defences within the RTLF (Heffner and Repine, 1989) . These not only include the enzymatic antioxidants described in the previous section but also a range of small molecular weight antioxidants that act as direct free radical scavengers. It is important at this point, and in the context of expanding this discussion into the oxidation of proteins to state that the RTLF antioxidant network should not however be thought of as a static pool opposing any imposed oxidative insult. Instead the defences within this compartment should be thought of as a dynamic equilibrium with the antioxidant defences within the epithelium and the more remote plasma pool. The most characteristic features of all oxidative challenges are the range of adaptive antioxidant responses observed; either through increased synthesis (Rahman and MacNee, 2000) or the enhanced movement of reducing equivalents to the RTLF (Freed et al., 1999) . A fine balance therefore exists between basal antioxidant levels, oxidant fluxes and adaptive modifications to antioxidant reserves all of which modulate the capacity of ROS and RNS to oxidise protein amino acids.

As a consequence of its high water solubility, L-ascorbate (vitamin C) is widely distributed throughout the body, including the RTLF. Ascorbate is an excellent reducing agent and scavenges a variety of free radicals and oxidants in vitro, including superoxide, peroxyl radicals, hydrogen peroxide, hypochlorous acid, singlet oxygen, ozone (Mudway and Kelly, 1998; Frei et al., 1989; Langford et al., 1995) and nitrogen dioxide (Kelly and Tetley, 1997; Postlethwait et al., 1995) . During this scavenging activity, ascorbate loses one electron resulting in the formation of the semi-dehydroascorbate radical (Buettner and Jurkiewicz, 1996) , which rapidly decomposes to dehydroascorbate and a range of further oxidation products, terminating in oxalate. As oxalate is cytotoxic, most cells contain a variety of dehy-droascorbic reductase enzymes that catalyse the regeneration of ascorbate from dehydroascorbate at the expense of GSH and NADPH (Wang et al., 1997; Washburn and Wells, 1999; May et al., 1997) . Notably, whilst a range of enzymes has been reported to have DHA-reductase activity intracellularly (glutaredoxin, NADPH-dependent-3-hydroxysteriod DHA reductase, erythrocyte glutathione peroxidase) only glutathione peroxidase has been reported in the RTLF. GSH can regenerate ascorbate from DHA non-enzymatically (Winkler et al., 1994) but again this is unlikely at RTLF concentrations. These two observations suggest that the ascorbate pool on the surface of the lung is likely to be highly liable, a contention further supported by the presence of non-transferrin bound iron in this compartment (Gutteridge et al., 1996) , and basal hydrogen peroxide concentrations . Thus maintenance of adequate ascorbate concentrations at the lung surface require it to be rapidly transported from cellular sources or from the plasma pool. Which of these pathways occurs is currently not understood but absence of a clear association between plasma and lavage fluid ascorbate concentrations (over a wide range of plasma concentrations, 20-100 "M) suggests that simple diffusion from the plasma pool does not explain RTLF levels. Further, undiluted RTLF concentrations have been reported at 100-400 "M (Cantin et al., 1987; van der Vliet et al., 1999) above plasma saturation levels, suggesting that a simple diffusion gradient does not exist. In addition to the direct scavenging action of ascorbate it also acts indirectly to prevent lipid peroxidation through its reaction with membrane-bound tocopherol. In in vitro studies, it has been demonstrated that ascorbate is able to reduce the tocopherol radical back to tocopherol, thereby restoring its scavenging activity (Halpner et al., 1998) . This synergistic action of ascorbate though clearly demonstrated in vitro, has importantly, not yet been conclusively demonstrated in vivo. Whilst ascorbate has many antioxidant actions, it also has the capacity to act as a pro-oxidant in the presence of transition metals. There is now a considerable body of evidence indicating that much of the toxicity attributed to inhaled airborne particles may be due to the presence of transition metals absorbed onto their surface (Ghio and Devlin, 2001) . One might therefore speculate that these particles may actually subvert much of the protective character of the RTLF, with ascorbate acting as a pro-oxidant, resulting in tissue injury, instead of protecting against it.

Glutathione and glutathione enzymes RTLF contains high concentrations of glutathione (L--glutamyl-L-cysteinylglycine) at concentrations approximately 100 times greater than those in plasma, and in a predominately (>90% GSH) reduced form (Cantin et al., 1987) . Initial estimates of the concentration of GSH in undiluted RTLF were approximately 400"M (Cantin et al., 1987 #869) , but concentrations are now considered to range between 100-200"M (van der Vliet et al., 1999) , with the concentration of GSSG being critically dependent on the magnitude of airway inflammation, but typically < 90% of the total pool (Cantin et al., 1987) . A number of cell types, alveolar macrophages, Clara cells and alveolar Type II cells have been reported to contain high levels of glutathione (4.8, 3.2 and 0.54 mM, respectively (Horton et al., 1987) . Poor reabsorption of glutathione from the respiratory tract is also important. The cell surface enzyme responsible for the uptake of glutathione from the extra cellular space, -glutamyl transpeptidase is present in much lower concentrations in the lung compared with other organs but may be located in specific epithelial cells such as Clara and Type II cells (Dinsdale et al., 1992) . RTLF is also replenished relatively slowly therefore any glutathione exported into this compartment is likely to remain there for prolonged periods. Glutathione is particularly good at defending against inflammatory cell derived oxidants such as superoxide, hypochlorous acid and hypobromous acid (Winterbourn, 1985) . Though GSH concentrations are high in the RTLF, the glutathione redox cycle is not operative in this compartment due to the absence of glutathione reductase. Thus any GSSG formed either directly through the reaction of GSH with ROS=RNS, or through the detoxifying actions of glutathione peroxidase will tend to accumulate. Whilst glutathione reductase has been reported in lavage in some studies its presence has generally been taken as evidence of cell lysis. Therefore glutathione probably functions more in a sacrificial mode in the RTLF, reacting directly with ROS with the production of thiyl radicals (GSÁ) which can subsequently be converted to glutathione disulphide (GSSG) through a radical transfer process. Thiyl radicals should not however be considered wholly harmless as they have been shown to react with other targets propagation free radical induced injury (Pryor, 1994; Kalyanaraman, 1995) .

The concentration GSH has been shown to be altered in a range of pulmonary conditions, either decreased, in asthma during exacerbations , idiopathic pulmonary fibrosis (Cantin et al., 1989) , asbestosis (Brown et al., 2000) , acute respiratory distress syndrome (Bunnell and Pacht, 1993) and in HIV positive patients (Pacht et al., 1997) ; or increased, in chronic smoking (Cantin et al., 1987) , chronic beryllium disease (Comhair et al., 1999) , or following exposure to ozone and nitrogen dioxide .

In addition, the concentration and activity of plasma glutathione peroxidase is also influenced by oxidative stress. Its activity in the RTLF of smokers is increased (Melloni et al., 1996) , while decreased activities are seen after ozone exposure (Avissar et al., 2000) , and unaltered activities following exposure to nitrogen dioxide (Avissar et al., 2000) . Recent studies have also provided evidence for GSH-dependent enzymes glutathione S transferase and thioredoxin within the RTLF (Noel-Georis et al., 2002) . The role of these two enzymes within this compartment has not currently been investigated in human subjects. Glutathione S transferases represent a family of enzymes responsible for the detoxification of endobiotic and xenobiotic compounds by covalently linking them to glutathione. It is therefore possible that they may play a significant role in the detoxification of organic radicals inhaled in cigarette smoke and introduced into the lung on the surface of inhaled particulates . This function if operative in the RTLF could explain the reported loss of GSH in certain acute lung insults where there is no evidence of an increase in GSSG. Thioredoxin catalyses thiol disulphide exchange reactions and is therefore critical in the regulation of protein function by thiol redox control. Given the propensity for numerous RTLF proteins to undergo thiol modification under oxidative stress, investigations into the regulation of this enzyme in a number of pulmonary conditions is clearly warranted.

Uric acid is also an important antioxidant in RTLF (Ames et al., 1981) especially in the upper airways where its concentration is high (Peden et al., 1990) . Uric acid is an oxidised purine base which is an effective scavenger of hydroxyl radicals, ONOO -, and oxyhaem oxidants formed between the reactions of haemoglobin and peroxy radicals, peroxyl radicals themselves and singlet oxygen (Becker, 1993) . It acts in these reactions in a sacrificial mode, in that it is irreversibly damaged through the interaction producing a range of oxidation products including allantoin (Ames et al., 1981) . It is also a highly effective scavenger of both inhaled ozone (Mudway et al., 1996; Mudway et al., 1999) and nitrogen dioxide (Kelly and Tetley, 1997) . From our observations to date, we have noted that RTLF uric acid concentration seems to be associated closely with protein concentration. This seems to be the case at all levels of the respiratory tract. This finding infers that RTLF uric acid comes from either a direct flux from the plasma pool (Becker, 1993; Housley et al., 1995) , or alternatively some UA may be stored and then actively secreted from sub-mucosal glands (Peden et al., 1993) .

-Tocopherol -Tocopherol (vitamin E) is present within RTLF, albeit at relatively low concentrations: 50-200 nM. It is thought that this -tocopherol is secreted by type II cells in to RTLF along with surfactant (Rustow et al., 1993) . -Tocopherol is a powerful antioxidant, both in terms of its direct free radical scavenging activity, and through its ability to terminate lipid peroxidation (Burton et al., 1981; Burton et al., 1983) . Vitamin E functions as a chain-breaking antioxidant in the lipid phase. Evidence of this function is however indirect, focusing almost entirely on observations made using in vitro systems (Burton et al., 1983) . It is thought that reactivity with organic peroxyl radicals accounts for the majority of the biological activity of -tocopherol (Burton et al., 1981) . This reaction is of considerable importance because tocopherols reacting with lipid peroxyl radicals yield a relatively stable lipid hydroperoxide and a vitamin E radical, which effectively interrupts the lipid peroxidation chain reaction (McCay, 1985) .

Whilst 2D-Gel maps have reported the presence of Mn-SOD (Noel-Georis et al., 2002) in RTLF it is likely that this antioxidant enzyme is present as a contaminant due to cell lysis during lavage and lavage fluid processing. RTLF has also been shown to contain appreciable concentrations of extracellular SOD, both in nasal and BALfluid samples (Mudway et al., 2001) presumably closely associated with proteoglycan at the epithelial surface. The association of this antioxidant enzyme with epithelial and endothelial surfaces may be significant as it may function to limit the production of peroxynitrite in local environments in which superoxide and nitric oxide are produced.

The RTLF has also been shown to contain a range of metal binding proteins: caeruloplasm, lactoferrin, transferrin, (Pacht and Davis, 1988; Noel-Georis et al., 2002) . These chelators perform an important antioxidant function in vivo by regulating free iron concentrations and hence limiting the potential for Fenton-like chemistry, with the formation of the damaging hydroxyl radical. Despite detectable concentrations of these proteins in lavage fluid from healthy control subjects (Gutteridge et al., 1996) , and evidence of their upregulation during a variety of pulmonary conditions and acute oxidant insults (Gutteridge et al., 1996; Nelson et al., 1996; Ghio et al., 1998) , there is evidence that RTLF contains a significant 'free' non-transferrin bound iron pool (Gutteridge et al., 1996; Nelson et al., 1996) , which is available for potential redox reactions.

Although it is clear that there are differences in antioxidants between different regions of the respiratory tract, the extent of these differences has not yet been clarified. In the nasal cavity, uric acid is by far the most prevalent antioxidant (Peden et al., 1990; Housley et al., 1995) . It should be noted however that there are gender differences with women having significantly lower levels of uric acid than men . A similar gender difference exists for plasma concentrations of uric acid, indicating that the major source of this antioxidant in NL is from plasma. However, Peden et al. demonstrated that in the nasal cavity, RTLF Sol phase uric acid concentration is increased following cholinergic stimulation of the airways (Peden et al., 1991) . Furthermore, they found that the increase in uric acid correlated positively with lactoferrin concentration, but not with albumin. As lactoferrin is predominately derived from mucosal gland secretions (Gel phase) this supports the contention that RTLF uric acid in the upper airways is derived, in part, from glandular secretions. Control of uric acid secretion in this compartment may therefore be closely related to mucus secretion. Glutathione and ascorbic acid are also present in nasal cavity RTLF however at significantly lower concentrations. Furthermore, although not always the case, low concentrations of -tocopherol are sometimes observed. In the peripheral airways (BL fluid) and bronchoalveolar region (BAL fluid) uric acid, ascorbic acid, GSH and -tocopherol are usually present.

The respiratory tract is exposed to a variety of ROS and RNS, predominately through exposure to exogenous oxidants (ozone, nitrogen dioxide) and inflammation. During inflammatory processes a number of cell types can release ROS and RNS. Neutrophils, eosinophils, B-lymphocytes and monocytes (but not macrophages, except under special circumstance) all contain the NADPH oxidase enzyme system which catalyses the production of superoxide from oxygen and NADPH (Babior, 2000) . In phagocytic cell populations the superoxide, released into the extra-cellular environment by this enzyme, is involved in the destruction of microorganisms. Superoxide is relatively unstable, though unreactive (redox potential À330 mV) radical, which will dismutate to hydrogen peroxide, in SOD catalysed and un-catalysed reactions. Whilst superoxide is relatively unreactive it will participate in the liberation of Fe 2 þ from ferritin (Harris et al., 1994) , and aconitase (Gardner et al., 1995) with a loss of protein function and release potential dangerous 'free'-iron. Ultimately therefore hydrogen peroxide is the predominate oxidant formed by phagocytes. Hydrogen peroxide is also a relatively weak oxidising species, which allied to cellular glutathione peroxidase and catalase defences means its toxicity in vivo is not thought to be significant. Hydrogen peroxide can also be produced directly by a variety of oxidase enzymes including xanthine oxidase, monoamine, and amino acid oxidase. The damaging actions of hydrogen peroxide are therefore predominately related to its reduction to a variety of more damaging oxidant species. Hydrogen peroxide is a key substrate for the neutrophil derived myeloperoxidase, and the eosinophil derived, eosinophil peroxidase enzymes which catalyses the oxidation of halide anions (MPO, Cl -, Brand I -; EPO, Brand Ionly) to form hypochlorous (HOCl) and hypobromous acid (HOBr) (Foote et al., 1983; Thomas et al., 1995) . These highly reactive oxidants have been shown to react with thiols, and thiolethers (Vissers and Winterbourn, 1995) , halogenate nucleotides, and with amine groups to form halamines which may have even greater reactivity than the parent radical (Thomas et al., 1983) .

Hydrogen peroxide can also undergo a transition metal catalysed reduction to the highly damaging hydroxyl radical. A range of biological reductants including ascorbate can then reduce the metal oxidised in this reaction. This reaction is critically dependent on the presence of 'free' Fe 2 þ and Cu þ . In vivo, these metals are typically sequestered into a range of proteins: ferritin, transferrin, lactoferrin, ceruloplasmin etc. Therefore whether this is a significant pathway in vivo is questionable. Hydroxyl radicals can also be formed in the reaction between HOCl and superoxide but again the significance of this pathway in vivo has not been fully established (Kettle and Winterbourn, 1994) . Whilst the hydroxyl radical is highly reactive, not only does its formation require the presence of 'free' or unchealated iron, but such is its rate of reaction with a broad spectrum of organic molecules that its toxic action will tend to be diluted out by its very promiscuity. Therefore, whilst undoubtedly highly reactive, the hydroxyl radical is more likely to interact with non-vital cellular components than critical targets.

The hydrophobic gas nitric oxide (NO) is produced endogenously in vivo where it plays a role in a range of important functions in the maintenance of normal airway physiology and host defense: modulating the expression of inflammatory cytokines, adhesion molecules and enzymes of the eicsanoid biosynthetic, as well as having a modulating influence on a range of antioxidant systems Brennan and Moncada, 2002) . Nitric oxide is produced from L-arginine by a variety of cells within the airway, both epithelial (type II pneumocytes) and inflammatory (macrophages, neutrophils, mast cells) through the action of constitutive (Ca 2 þ dependent, short term, low capacity) and inducible (Ca 2 þ independent, long term, high capacity) forms of nitric oxide synthase. The expressions of these enzymes are increased during inflammation with concomitant increases in exhaled NO, as observed in asthmatics (Kharitonov et al., 1997) , in patients with bronchiectasis, during airway infections and following inhalation of oxidant gases (Balint et al., 2001; Kharitonov and Barnes, 2002; Kharitonov and Barnes, 2000; Nightingale et al., 1999; Kharitonov and Barnes, 2001) . Similarly the end products of NO metabolism, nitrite (NO 2 À ) and nitrate (NO 3 À ) are increased in RTLF in these conditions (Balint et al., 2001) . Whilst most of the NO detected in exhaled breath is derived from the nasal airways and sinuses it is clear that NO can be produced throughout the length of the airway. Although a free radical itself, NO is not particularly reactive allowing it to diffuse readily in vivo from its site of production to its site of action. It can however react with a range of other radical species (superoxide, thiol, lipid and protein radicals) to generate a variety of highly damaging radical intermediates of considerably greater toxicity than itself. Nitric oxide can react with oxygen to form nitrogen oxides: the powerful oxidant nitrogen dioxide (NO 2 ), as well as nitrite (NO 2 À ) and nitrate (NO 3 À ) but these reactions are slow and not favoured vivo (Beckman and Koppenhol, 1996) . Instead much interest has focused on the rapid reaction between NO and superoxide (out competing its dismutation by SOD) with the formation of the highly damaging oxidant species: peroxynitrate (ONOO -) . It is also clear that both the inflammatory cells of the airways and the epithelium itself are capable of producing both NO and superoxide (Munozfernandez et al., 1992) . It is now believed that much of the toxicity previously ascribed to the hydroxyl, superoxide and nitric oxide in vivo is now often attributed to peroxynitrite and its derivatives (Tien et al., 1999) . The ability of ONOOto nitrite proteins as well as induce lipid peroxidation and DNA damage has been demonstrated in vitro. In contrast to the hydroxyl radical the rate with which peroxynitrite will react with biomolecules is rather slow, favouring selective reactions with its major targets: sulphydryls (Stamler et al., 1992) , iron=sulphur centres (Hausladen and Fridovich, 1994) and zinc-fingers. Peroxynitrate is surprisingly uncreative toward reduced glutathione and is therefore not an effective scavenger for this radical. Indeed unlike most oxidants, peroxynitrite will oxidize protein targets without having to first deplete endogenous antioxidant defences . This potent species will participate in a wide range of oxidation reactions including the oxidation of protein and non-protein sulphydryls, deoxyribonucleic acid and lipids, as well as nitration of tyrosine residues by catalysed (low molecular weight transition metals and superoxide dismutase) and un-catalysed reactions.

Peroxynitrite will also undergo protonation in acid environments to produce the strongly oxidizing peroxynitrous acid radical, which decomposes to nitrate via intermediate formation of the hydroxl radical and a nitrogen dioxide-like species (Beckman et al., 1990) . This may represent a significant nitrating species on the surface of the lung of asthmatic subjects where RTLF has been demonstrated to be acidic (Hunt et al., 2000) . Peroxynitrite will also react rapidly with CO 2 to form nitrogen dioxide and the CO 2 adduct nitrosperoxocarbonate (ONOOCO 2 À ), which has been proposed to represent the major damaging peroxynitrate derived intermediate formed under physiological conditions (Tien et al., 1999) . The ability of ONOOto nitrite proteins as well as induce lipid peroxidation and DNA damage has been demonstrated both in vitro and in vivo. It therefore appears that peroxynitrite, and its derivatives, is one of the major damaging oxidants formed in vivo, and certainly accounts for much of the toxicity associated with the increased production of nitric oxide. Notably, in the absence of these physiological catalysts the rates of nitration seen in model systems are low and indeed diminish significantly as addition components are added to the models (Ischiropoulos et al., 1992) . Protein nitration does not always imply the local production of ONOO -. Nitration by ONOOcatalysed by MnSOD occurs through a mechanism thought to involve the cleavage of the peroxide bond to produce a nitryl cation like intermediate. Due to the short half-life of this species it does not exist free. NO can also be converted to the highly reactive NO 2 radical in vivo through various biological pathways: The auto-oxidation of NO, via one electron reactions with antioxidants, thiols, aromatic amino acids, unsaturated lipids (Pryor and Squadrito, 1995) and via the one electron reduction of nitrite. In addition under acidic conditions (gastric compartment, phagosomes, tissue acidosis) nitrite can be protonated to nitrous acid (HNO 2 ), which can decompose to NO and NO 2 (Ohshima et al., 1990) . Nitrite also acts as a substrate for MPO and EPO in a reaction producing the highly reactive NO 2 radical. High concentrations of these peroxidases can be found at sites of leukocyte activation, where they have been shown to be associated with evidence of protein nitration (Heinecke, 1997) . This pathway is dependent on the concentrations of nitrite (10-140 uM in nasal and 10-15 uM in alveolar RTLF). Significantly nitrite does not accumulate in vivo being further metabolised to nitrate (Parks et al., 1981) but levels do increase during inflammatory episodes.

The lung is also susceptible to oxidative damage from exogenous oxidant species. The secondary pollutant gas, ozone, represents an excellent example in that through its powerful oxidising ability it will react with numerous biomolecules. Despite the broad target specificity of ozone, the rate at which reactions occur with different molecules can vary over several orders of magnitude (Pryor, 1993) . Indeed when ozone encounters an array of potential substrates it reacts initially with those with the highest intrinsic reactivities toward it. Uric acid, ascorbic acid and glutathione have all been shown (in defined settings) to display high intrinsic reactivates toward ozone (Pryor et al., 1995; Cross et al., 1992; Mudway and Kelly, 1998; Postlethwait et al., 1994) . It has therefore been proposed that RTLF antioxidants function as sacrificial substrates for ozone (Pryor et al., 1985) . By reacting with ozone they effectively remove it with only the production of ostensibly harmless products. It should however be noted that thiyl and ascorbyl radicals are not without some biological significance. When these defences are overwhelmed oxidation to lipid and protein moieties can occur. As ozone is relatively insoluble it is unlikely that unreacted ozone can penetrate the RTLF to directly oxidise epithelial cell membranes. Instead its toxicity is transmitted to the underlying cells through the generation of a range of lipid oxidation products (LOPs) (Pryor et al., 1995) , which have been reported to have pro-inflammatory properties (Kafoury et al., 1999) .

The primary gaseous pollutant nitrogen dioxide is also highly oxidizing and its uptake into the RTLF is also coupled to its reaction with RTLF substrates, predominately reduced glutathione and ascorbate (Postlethwait et al., 1991; Connor et al., 2001) . Because absorption is directly coupled to these reactions the products clearly must drive the events leading to tissue injury. The initial reaction products of these reactions are the thiyl and ascorbyl radicals and nitrite. Thiyl and ascorbyl radicals are able to generate ROS through a variety of mechanisms (Buettner and Jurkiewicz, 1993; Miller and Aust, 1989; Scarpa et al., 1983) . Thiyl radicals may react with thiolated (GS -) to yield superoxide and GSSG, or molecular oxygen to produce GSOO . and GSO2OO . (Buettner and Jurkiewicz, 1993) . The reaction between NO 2 and ascorbate can also result in the generation of oxidants: the ascorbyl radical and nitrite. Whilst the latter comprises a substrate for heme peroxidase catalysed formation of RNS, the ascorbyl radical can react with free iron, ultimately resulting in the formation of hydrogen peroxide (Miller and Aust, 1989; Scarpa et al., 1983) . Therefore the reaction of both of these pollutant gases with the RTLF can give rise to a variety of radical species.

Cigarette smoke also contains an extensive variety of free radicals and oxidant species (including NO 2 ), both in the gas and tar phase (Kim et al., 2002) and therefore will both introduce directly radicals to the lung surface, as well as generate them as a result of oxidation reactions at the RTLF -air interface. Much recent work in the air pollution field has focused on the damaging role of inhaled particulate matter of an aerodynamic diameter of <10 "m. These particles induce oxidative stress within the lung via the introduction of transition metal (Ghio et al., 1998) and stable organic radicals into the lung. All of these exogenous agents are also capable of eliciting acute inflammatory responses in the human lung and hence a secondary source of ROS and RNS (Committee of the Environmental and Occupational Health Assembly of the American Thoracic Society, 1996).

Reactive oxygen and nitrogen species can react directly or indirectly with proteins to cause a range of oxidative modifications. The key question when addressing the significance of these modifications is whether they are simply markers for the presence of oxidative stress, or have some substantive consequence on protein function that impacts on tissue injury or disease progression. To achieve this it is important to show that the amino acid modification is related to altered function, and not simply a parallel event. Here we will describe the major oxidative modifications, which have been reported, specifically focusing on those studies where oxidation has been related to altered function. By necessity this will require a consideration of findings derived from other organ systems, as to date only protein nitrosylation, methionine oxidation and protein carbonyl formation have been dealt with in any detail in the lung, and then mostly focusing on BAL cells or tissue biopsies. In the previous section considering the RTLF proteome it was clear that the major proteins could be classified into four groups: those related to (a) protease=anti-protease balance, (b) antioxidant defence and metal ion chelation, (c) regulation of inflammatory response, and (d) tissue repair and remodelling. It is therefore reasonable to assume that if oxidation of these proteins in other body compartment alters function (under an appropriate stress), these findings will be of relevance to the RTLF compartment. These types of detailed analysis in lavage fluid are still hampered by the low total protein concentrations in this compartment, though the use of proteomic technologies should help to resolve these issues.

Reactive oxygen and nitrogen species and their secondary lipid and sugar oxidation products may interact with proteins to cause oxidation of the polypeptide backbone of the protein, peptide bond cleavage, protein-protein crosslinking, and a range of amino-acid side chain modifications. All amino acids are potential targets for oxidation by reactive oxygen and nitrogen species but the derived products have only been fully characterised in a few cases. In the physiological context the major aromatic amino acids tyrosine, tryptophan, phenylalanine, the sulphur containing amino acids, cysteine, methionine, as well as the aliphatic amino acids arginine, lysine, proline, and histidine are the major targets of ROS=RNS attack. Cysteine and methionine, the two sulphur-containing amino acids appear especially sensitive to oxidation.

Methionine is easily oxidized to methionine sulfoxide derivatives in a mechanism mediated by a wide range of ROS (chloroamines, peroxynitrite, superoxide, hydrogen peroxide, hypochlorous acid) at physiological pHs. This oxidative modification can be reversed through the action of methionine sulfoxide reductase . Further oxidation products of methionine sulphoxide such as the sulphone cannot be reduced to methionine. The reversible oxidation-reduction of this residue has led to suggestions that this might act as a means of regulating enzyme activity, as well as protecting vital MET residue from irreversible oxidation with a corresponding loss of protein function . The oxidation of methionine residues has also been shown to result in an increase in protein surface hydrophobicity (Chao et al., 1997) . This is related to the exposure of hydrophobic residues usually buried within the protein, as structural integrity is lost due to amino-acid oxidations. This increased hydrophobicity has been shown to increase the susceptibility of proteins to ligation with ubiquitin (Hershko and Ciechanover, 1986) , targeting the proteins for proteolytic degradation by the proteosome.

A wide range of proteins has been shown to display altered function following methionine oxidation. Focusing on those proteins likely to be of significance on the surface of the lung, exposure of -1-anti-trypsin to a variety of ROS in vitro has been shown to cause oxidation of surface exposed methionine residues at its reactive centre (met-351, met-358) associated with a loss of activity (Johnson and Travis, 1979; Taggart et al., 2000) . Treatment with methionine reductase restored anti-elastase activity suggesting that the functional impairment was directly related to the methionine oxidation (Carp et al., 1983) . Similarly, the proteinase inhibitor -2 macroglobulin has been shown to possess oxidisable methionine residues (Reddy et al., 1994) though their oxidation in this case did not appear to result in a loss of antiprotease function. Instead their oxidation appeared to spare the oxidation of a critical tryptophan residue related to the protein function suggesting that in certain proteins methionine may have protective antioxidant functions. Other antiproteases have also been reported to have their function modified through oxidation of their methionine residues: -2-antiplasmin (Geary and Gonias, 1989) , mucus proteinase inhibitor (Boudier and Bieth, 1994) and secretory leukocyte proteinase inhibitor (Tomova, 1994) . Methionine oxidation has also been implemented in altered function in a variety of proteins regulating the inflammatory response, present within the RTLF: Interferon (Keck, 1996) , IL-6 (Nishimura et al., 1991) , and lysozyme (Jori et al., 1968) . In these studies the involvement of methionine oxidation in the modulation of protein function was generally examined in simple in-vitro models; whether these specific oxidations occur in vivo remains an open question. There is however evidence for methionine oxidation of RTLF proteins in vivo. Elevated concentrations of oxidised methionine residues have been detected in BAL proteins obtained from lung transplantation patients suffering from bronchiolitis obliterans syndrome (Behr et al., 2000; Hirsch et al., 1999) . Increased concentrations of methionine sulphoxide have also been detected in BAL fluid obtained from patients with acute and chronic bronchitis (Maier et al., 1992) , chronic obstructive pulmonary disease (Maier et al., 1992) , and lung cancer patients under-going radio chemotherapy (Beinert et al., 2000) . In each of these cases, the protein species that has been modified remains unknown. The measurement of methionine sulfoxide concentrations is simply used as an index of oxidative stress. Whilst it might be possible to relate a loss of a candidate proteins function, one which is known to have altered function related to specific methionine oxidation, with generic total protein levels of methionine sulphoxide, there is still a requirement to identified the modified protein if a direct causation is to made.

Cysteine residues are easily oxidised resulting in enzyme inactivation, alterations in protein tertiary structure and protein cross-linking (Thomas et al., 1995) . Like methionine, cysteine residues can be oxidised to their corresponding sulfoxides: sulfenic (RSOH), sulfinic (RO 2 H), and sulfonic acid (RSO 3 H) by a range of reactive oxygen and nitrogen species (Claiborne et al., 1999) . Of these, only the formation of sulfenic acid is reversible under physiological conditions, though this species readily oxidises to irreversible oxidation products referred to above (Prutz, 1992) , or reacts with GSH to form a disulphide bond (Claiborne et al., 1993) . The conversion of critical cysteine residues to sulfenic acids by S-nitrosoglutathione, and other reactive nitrogen species has been implemented in the inactivation of glutathione reductase (Becker et al., 1998) , and Cathepsin K (Percival et al., 1999) in in vitro models.

The most studied oxidative modifications of cysteine are the formation of disulphide bonds both within and between proteins and the formation of mixed disulfides with small molecular weight thiols: cysteine, GSH, homocysteine (Klatt and Lamas, 2000) . Under conditions of oxidative stress these thiolation reactions occur as a result of either partially oxidised protein sulfhydryl (thiyl radicals and sulfenic acid intermediates) with thiols such as cysteine and glutathione, or by thiol=disulfide exchange reactions with GSSG (Thomas et al., 1995) . Further the formation of these mixed disulphides has been shown to alter the function of a diverse range of proteins under conditions of oxidative stress: ion translocators (Shattock and Matsuura, 1993) , protein kinases (Ward et al., 1998) , phosphatases (Mahadev et al., 2001) , antioxidant enzymes (Schinina et al., 1996) , G-proteins (Mallis et al., 2001) , metabolic (Cappiello et al., 1996) proteosome complexes (Demasi et al., 2001) and structural proteins (Eaton et al., 2002; Hanson et al., 1999) , as well as DNA isomerases (Wang et al., 2001) . These modifications are easily reversed, either through changes in intra-cellular redox, or via enzymatic reduction by thioredoxin, glutaredoxin, or disulfide isomerase at the expense of NADPH (Grant, 2001) . Many studies have shown that S-thiolated proteins formed during oxidative stress are readily de-thiolated once the stress is removed (Seres et al., 1996) associated with complete restoration of function (Ravichandran et al., 1994) . It has therefore been proposed that thiolation=de-thiolation may regulate protein function in much the same way as other reversible posttranslational modifications, such phosphorylation and adenylation. Regulatory cysteine residues are generally located in protein regions characterised by high pKa's, as these areas favour the formation of reactive thiolated ions which are sensitive to oxidative modification by a number of oxidants: GSSG, nitric oxide, nitrosothiols, peroxynitrite, hypochlorous acid, and reactive lipids (Padgett and Whorton, 1998; Mohr et al., 1999; Pullar et al., 2001; Stamler et al., 2001) .

The formation of mixed disulfides has been observed in a wide range of cells and tissues exposed to oxidative stress, both in vitro and in vivo. Mixed disulphides have been reported in rat lung tissue after exposure to cigarette smoke (Park et al., 1998) and hyperoxia (Awasthi et al., 1998) , in the human lens during cataract formation , in models cadmium-induced oxidative stress (Figueiredo-Pereira et al., 1998) , and during ishemia=reperfussion in isolated rat hearts (Eaton et al., 2002) . Further, stimulation of respiratory burst in isolated human neutrophils (Seres et al., 1996; Chai et al., 1994) and monocytes (Ravichandran et al., 1994) has been shown to result in the rapid formation of disulfide bonds between intra-cellular low molecular weight thiols (GSH, -glutamylcysteine, homocysteine and cysteine) and a range of proteins (glygeraldehyde-3phosphate dehydrogenase, actin) in a hydrogen peroxide dependent mechanism. These disulphides were subsequently reduced back to sulphydryls in a thioredoxin reductase dependent manner as the respiratory burst attenuated (Seres et al., 1996) . As well as a potential regulatory role on protein function, S-thiolation has been proposed to play a protective role by preventing the oxidation of protein cysteine residues to irreversible oxidation products. Supportive evidence for this has been observed in the glutathiolation of the -glutamyl transpeptidase which appears to protect this membrane-bound enzyme from the hydrogen peroxide produced during its catabolism of GSH in the presence of metal cations (Dominici et al., 1999) .

Whilst nitric oxide and many of its reactive derivatives have been implemented in the formation of mixed disulphide, probably through the oxidation of free thiols and subsequent disulphide exchange reactions, NO can also react directly with free and protein bound thiols with the formation of S-nitrosothiols (SNO). The products of these S-nitrosation reactions have been proposed to simultaneously facilitate the transport of NO in vivo (Kharitonov et al., 1995) , conferring NO properties on the carrier, whilst mitigating against its toxic effects (Stamler et al., 1992) . A number of proteins have been identified as physiological NO reservoirs permitting the transport of this bioactive gas to effector sites in vivo: haemoglobin (Bonaventura et al., 2002) , albumin (Tsikas et al., 2001) and calbindin D 28K (Tao et al., 2002) . In addition to this beneficial role nitrosylation of cysteine residues has also been shown to inactivate, or indeed activate, a broad spectrum of proteins: cathepsin B (Stamler et al., 1992) , ion channels (Bolotina et al., 1994; Sun et al., 2001) , receptors (Lipton et al., 1993) , G-proteins (Lander et al., 1995) , nuclear proteins and transcription factors (Marshall and Stamler, 2002; Laval and Wink, 1994) , metabolic proteins (Mohr et al., 1996) . S-nitroso compounds such as S-nitroso-L-glutathione (GSNO) may inhibit enzymes responsible for the modulation of the response to oxidative stress: glutathione peroxidase (Becker et al., 1995) , glutathione reductase (Becker et al., 1995) and glutathione S transferase (Clark and Sinclair, 1988) . NO can be liberated from these S-nitrosoative cysteine residues through a variety of mechanisms including metal catalysed homolytic cleavage, non-enzymatic and enzymatically catalysed transnitrosation reactions and metabolism of S-nitrosothiols by various enzymes (Hogg, 2002) .

Increased concentrations of S-nitrosothiols have been reported in BAL fluid recovered from patients following lung transplantation (Gaston et al., 1993) and associated with infective pneumonia (Gaston et al., 1994) . Increased concentrations have also been reported in exhaled breath condensate of patients with severe asthma, COPD, CF and in current smokers (Corradi et al., 2001) . The formation of nitrosothiols depends on the availability of GSH within the RTLF, and there is some evidence that concentrations of this antioxidant are in a range of chronic pulmonary conditions (Smith et al., 1993; Cross et al., 1994) , and following acute airway insults Cantin et al., 1987) . At question is whether the formation of these nitrosothiols represents an adaptive response of the airway to nitrosative stress and related to this, what the functional consequences of these elevated concentrations are.

During the oxidation of the aromatic amino acid tyrosine, tyrosine radicals are formed, which in the absence of repair by cellular reductants may combine to form di-tyrosines and 3,4-dihydroxyphenylalanine, generally referred to as DOPA (Davies et al., 1999) . As di-tyrosine derivatives are fluorescent this property has been made use of to assess oxidative damage to proteins in a range of model systems. Under physiological conditions the formation of these species are relatively minor but their detection has proven a reliable method of assessing protein oxidation (Huggins et al., 1993; Giulivi and Davies, 1994) . Hypochlorous acid released from activated neutrophils also reacts with tyrosine residues in protein to produce 3-chlorotyrosine and 3,5dichloro tyrosine (Kettle, 1996) . Although this is a relatively minor reaction, it is the only known physiological source of chlorotyrosine. As such, chlorotyrosine may be a specific marker for oxidant activity of cells that contain MPO such as neutrophils and monocytes. Elevated chlorotyrosine concentrations have been reported in atherosclerotic lesions (Podrez et al., 2000) and lungs of patients with adult respiratory distress syndrome (ARDS), cystic fibrosis and following lung transplantation (Lamb et al., 1999; De Andrade et al., 2000; van der Vliet et al., 2000) . Similarly, hypobromous acid released from eosinophils can lead to the bromination of tyrosine residues. The generation of bromotyrosine is thought to be particularly important as this product has been shown to increase 10-fold in BAL fluid obtained from asthmatic subjects following allergen challenge (Wu et al., 2000) and 100-fold in severe asthmatics admitted to emergency departments (MacPherson et al., 2001) .

The most studied oxidative modification of tyrosine residues is their nitration to 3-nitrotyrosine (3-NT). This may occur through a variety of mechanisms: via the reaction of superoxide with nitric oxide to form peroxynitrate and its derivative species, by peroxidase dependent nitrite oxidations, catalysed by MPO and EPO (Eiserich et al., 1998) , and through nitric oxide interactions with tyrosyl radicals (Gunther et al., 1997) , and nitrous acid (Knowles et al., 1974) . These mechanisms have all been demonstrated in simple in vitro models, however questions remain about which mechanisms operate in vivo, where competing reactions (including those with antioxidants) will limit the degree of protein nitration. For example the toxic action of peroxynitrite can be seriously impaired in the presence of urate (Reiter et al., 2000) . Despite this proviso, there is clear evidence that nitration of proteins does occur in vivo (MacMillan-Crow et al., 1996; Gole et al., 2000; Viner et al., 1996; Strong et al., 1998) . Elevated concentrations have been detected in animal models of hyperoxia (Haddad et al., 1994) , endotoxemia (Wizemann et al., 1994) , lung ischaemia=reperfusion (Ischiropoulos et al., 1995) , influenza induced pneumonia (Akaike et al., 1996) and asbestosis (Tanaka et al., 1998) . Nitrotyrosine concentrations have been shown to be increased in a range of pulmonary diseases: asthma (Saleh et al., 1998 ; Kaminsky et al., 1999) idiopathic pulmonary fibrosis (Walker et al., 2001) ; Cystic fibrosis (van der Vliet et al., 1997) ; lung transplantation (McDermott et al., 1997) ; as well as in acute lung injury (Kooy et al., 1995; Haddad et al., 1994) . A global increase in nitrotyrosine has been demonstrated in lung autopsy tissues from paediatric patients who had died of acute pulmonary conditions (Kooy et al., 1995) , as well as in the RTLF of intensive care patients at risk of developing pneumonia and ARDS (Lamb et al., 1999; Mathy-Hartert et al., 2000) . Concentrations of nitrite, nitrate and 3-NT have been shown to be elevated in BAL-fluid, cells and lung biopsy samples obtained from patients with ARDS (Gole et al., 2000; Lamb et al., 1999) . Increased concentrations of 3-NT have been reported in asthma, with evidence that these levels were reduced following inhaled corticosteriod treatment (Saleh et al., 1998) . Elevated concentrations of 3chlorotyrosine, 3,3 0 di-tyrosines, nitrite, nitrate and 3-NT have been reported in induced sputum samples obtained from CF patients (van der Vliet et al., 2000) .

Whilst the presence of 3-NT residues in protein serves as a useful marker of nitrosative stress, whether these modifications have any impact on protein function, and allied to this whether they may play an active role in tissue pathology is a critical issue. In simple model systems in which target proteins are exposed to peroxynitrite there is considerable evidence that this results in a loss of function in range of proteins: nuclear proteins (Chazotte-Aubert et al., 2000; Haqqani et al., 2002) ; metabolic enzymes (Souza and Radi, 1998) ; calcium binding proteins (Rowan et al., 2002) ; surfactant proteins Haddad et al., 1996; Haddad et al., 1993) ; tyrosine kinases (Knapp et al., 2001; MacMillan-Crow et al., 1996) ; phosphatases (Takakura et al., 1999) , ion channels (Viner et al., 1996; Hu et al., 1994) , inflammatory proteins (Sato et al., 1999; Zou et al., 1997) and antioxidant enzymes (MacMillan-Crow et al., 1996) . Further, recent studies have been able to show that this loss of function is due to nitrosylation of critical tyrosine residues and not simply parallel oxidations within the protein. Of specific interest to the lung, nitration of tyrosine residues 161, 164 and 166 in SP-A have been shown to be related to its loss lipid aggregation capacity . Nitration of Tyr67 on cytochrome C has been shown to have profound effects on its redox-regulating properties (Cassina et al., 2000) . In studies with purified enzymes peroxynitrite has been shown to be a potent inhibitor of prostaglandin I2 synthase, related to the nitration of a critical tyrosine residue within the active site (De Andrade et al., 2000) . Cytoskeletal proteins are also thought to be significant targets for nitrosylation due to their intra-cellular abundance and high tyrosine content (Eiserich et al., 1999) . Further nitration of Tyr34 allied to the formation of 3,3 0 -dityrosine accounts for much of the inactivation of MnSOD seen in vivo (MacMillan-Crow et al., 1996; Yamakura et al., 1998) . This list of specific modifications is increasing, but there is still a requirement for their identification in vivo. Circulating levels of free 3-NT has been reported in a range of pathological conditions (Fukuyama et al., 1997 and has been shown to be taken up into A549 cells where it is irreversibly incorporated into -tubulin through the action of tyrosine ligase, disrupting binding of microtubule-associated proteins with potential impacts on cell growth, differentiation and motility (Eiserich et al., 1999; Chang et al., 2002) . Whilst these studies can demonstrate altered protein function in specific proteins in simple cell and cell free models exposed to considerable nitrosative stresses, only a limited number of studies have identified the specific proteins nitrated in vivo (Gole et al., 2000; MacMillan-Crow et al., 1996) . In an important study by Gole et al plasma ceruloplasmin, transferrin, 1-protease inhibitor, 1-antichymotrypsin and fibrinogen were all shown to be nitrated with an associative loss of function in ARDs patients (Gole et al., 2000) . Whilst this approach partially bridges the conceptual gap between the formation of 3-NT and altered protein function, there still remains the possibility that the parallel oxidation of other amino acids within the protein drives the effect. Therefore at present it is probably best to view these identified protein as having altered function due to general oxidation as illustrated by the presence of 3-NT. Significantly, the recent identification of a de-nitrase activity isolated from rat spleen and lung (Kamisaki et al., 1998) has raised the possibility that many of these tyrosine nitrations reactions may be reversible and hence provide a mechanism for regulating protein activity.

As mentioned previously, recent studies have demonstrated that the heme peroxidases, myeloperoxidase and eosinophil peroxidase can also catalyse tyrosine nitration from nitrite in the presence of hydrogen peroxide, providing a significant alternative pathway for their formation in vivo during inflammation (Eiserich et al., 1998) . Increased 3-NT staining in tissue sections are often colocalised with neutrophils, and associated with elevated concentrations of MPO. Clearly the operation of this pathway is critically dependent on the concentrations of nitrite within the local environment and notably significant concentrations have been reported in the RTLF: 10-140 "M in nasal and 10-15 "M in alveolar compartment, increasing markedly during inflammation. The involvement of MPO in the formation of 3-NT in vivo has been illustrated by the observation of diminished 3-NT concentrations in the livers of MPO deficient mice compared with wild type (Baldus et al., 2001) . Recent studies have revealed that MPO released from inflammatory cells at the endothelium may undergo cellular transcytosis to the basolateral cell surface where they associate with endothelial cell glycoasinoglycans, especially fibronectin (Baldus et al., 2001) . This mechanism is significant as it implies that the site of action of MPO may both be remote from its site of production and extended.

There is now good evidence that eosinophils can use nitrite as a substrate for the nitration of tyrosine residues (Wu et al., 1999) . Pulmonary inflammation in asthma is characterized by the differential recruitment of eosinophils to the airway mucosa and lumen. Activated eosinophils release large amounts of cationic proteins from morphologically distinct cytoplasmic granules (Erjefalt and Persson, 2000) . The most abundant of these granule proteins is a cationic heme-containing protein, eosinophil peroxidase (EPO). EPO appears to be specific for the eosinophil cell type and represents nearly 25% of the total protein mass of the secondary granule (Carlson et al., 1985) . The release of EPO, together with the generation of superoxide and hydrogen peroxide from the respiratory burst (Elsner et al., 1996) , leads to the generation of hypobromous acid (Slungaard and Mahoney, 1991) . This potent defense mechanism can kill invading pathogens such as bacteria (Jong et al., 1980) . Indeed in model systems EPO is a more potent catalyst of protein nitration than MPO under physiological conditions (Wu et al., 1999) raising the possibility that much of the nitration seen in asthma in vivo is related to its action. A recent study has shown a significantly higher concentration of 3-bromotyrosine in asthmatics compared with control subjects (Aldridge et al., 2002) . Notably, levels of 3-chlorotyrosine were not elevated in this group emphasising the importance of EPO derived oxidants in asthma.

There is a considerable body of evidence that the reaction of reactive oxygen and nitrogen species with lys, arg, cys, his, pro, glu, asp and thr residues in proteins leads to an increase in carbonyl groups . The introduction of these groups into proteins can arise through five distinct oxidative mechanisms: (1) through oxidative cleavage of polypeptide bonds by -amidation or oxidation of glu and asp amino acid side chains; (2) by direct oxidation of lys, arg, pro and thr residues; (3) Michael addition of unsaturated aldehydes such as 4-hydroxy-2-nonenal to lys amino groups, cys sulfhydryl groups, and histidine imidazole groups; (4) by the reaction of malondialdehyde with lys to form Schiff base derivatives; and finally (5) through the reaction of reducing sugars and their oxidation products with lys in glycation reactions. These mechanisms have been reviewed in detail by a number of authors previously . In contrast to many of the specific amino acid modifications already considered, where the absolute numbers of modifications are low, carbonyl modification may occur in numerous residues and may therefore have profound structural changes to a broad spectrum of proteins. A variety of highly sensitive HPLC and antibody-based techniques are available to measure protein carbonyls and thus their determination has been widely used to assess oxidative stress in a number in vitro and in vivo models. Increased concentrations of protein carbonyls have been measured following exposure of model protein solutions to ozone (Mudway et al., 1998; Berlett et al., 1996) , transitional metal based free radical generating systems (Requena and Stadtman, 1999) , cigarette smoke (Reznick et al., 1992) and activated neutrophils (Oliver, 1987) . Increased concentrations have also been detected in the lungs of rats exposed to hyperoxia (Winter and Liehr, 1991) and paraquat (Winter and Liehr, 1991) . Carbonyls concentrations are also increased in a murine model of emphysema (Yoshida et al., 2001 ) associated with increased NF-B expression and ROS formation by alveolar macrophages. Elevated protein carbonyls concentrations have also been reported in plasma from ARDS patients (Quinlan et al., 1994) and in plasma BAL fluid obtained from in critically ill patients following major trauma or sepsis. Here the increased concentration of carbonyls correlated well with lipid oxidation product measurements and indices of neutrophilia and neutrophil activation . Increased levels of protein oxidation have also been observed in tracheal aspirates from premature infants undergoing ventilation therapy, similarly related to pulmonary inflammation . Similar results have been shown by other groups where the increase in protein carbonyl concentrations has been shown to be positively associated with increased concentrations of matrix-metalloproteinase-9, and its inhibitor, TIMP-1 (Schock et al., 2001) suggesting that the processing of epithelial remodelling may be regulated through oxidative stress. There appears to be little evidence at present that protein carbonyl levels are elevated in asthma (Aldridge et al., 2002) , although some evidence of a small increase has been reported in subjects following allergen challenge (Foreman et al., 1999) related to eosinophilia migration. Notably, in the later study, as with many of the protein oxidation markers described previously, the major protein oxidised was 1antitrypsin.

Histidine residues are converted to 2-oxohistidine (Uchida and Kawakishi, 1993) or to asparagine or aspartic acid (Berlett et al., 1996) by a variety of reactive oxygen species and oxidants. The rate of these reactions and the ultimate product formed is a function both of the oxidising species and the local amino acid environment. Whilst ozone and hydroxyl radicals randomly attack histidine residues, metal catalysed oxidation is relatively site-specific targeting residue near the metal binding site (Stadtman and Oliver, 1991) . Phenylalanine can also be oxidised to a range of products: ortho-and metaptyrosine (Huggins et al., 1996) , 2-, 3-, 4-hydroxy and 2,3-dihydroxy derivatives Dean et al., 1993) . Proteincross-linking in response to oxidation results in the formation of larger molecular weight species. These aggregates are formed through a variety of mechanisms: the formation of disulphide bridges, through covalent C-C bond formation arising from the interaction between two carbon-centred radicals, by interactions between lysine residues on separate proteins with lipid peroxidation products, or finally, via formation of a Schiff base cross link between a carbonyl group on one protein and a N-amino group of a lysine residue on a second. None of these modifications have been addressed in detail in models of pulmonary injury to date.

Recently considerable attention has focused on the formation of protein lipid oxidation product adducts, specifically with the lipid peroxide degradation products 4-hydroxynonenal and malondialdehyde in a number of diseases characterised by oxidative stress (Eaton et al., 2001; Walker et al., 2001; Houglum et al., 1990) . The focus on this protein oxidative modification has been driven by the availability of commercially available antibodies against 4-HNE-protein adducts, permitting identification and quantification of modified proteins by Western blotting and immunohistochemistry (Eaton et al., 2001) . In addition, elevated concentrations of these lipid oxidation species have been identified in almost all of the major pulmonary diseases characterised by acute and chronic inflammation making the formation of these adducts highly feasible in vivo. Increased concentrations of these adducts have been reported in the lungs of smokers with and without COPD . Notably, 4-HNE concentrations in the pulmonary epithelium, airway endothelium and particularly in neutrophils of the COPD patients were found to be inversely associated with lung function. Significantly, the formation of these protein-lipid adducts have been shown to have profound effects on protein function. For example the binding of lipid oxidation products to the epidermal growth factors receptor has been shown to result in the phosphorylation of the receptors, with subsequent stimulation of the ERK pathway (Liu et al., 1999) .

The lung is assaulted on a daily basis by a range of toxic insults. Whether these are infectious agents, allergens, or air pollutants they act upon the lung by eliciting oxidation reactions, either directly, or through inflammatory processes. Measurement of protein oxidation products therefore permits the degree of oxidative stress to be assessed and indicates that endogenous antioxidant defences are overwhelmed. The range of protein oxidation products observed is diverse and the nature and extent of specific oxidation products may inform us about the nature of the damaging ROS and NOS. Recently, there has been a significant shift away from the measurement of these oxidation products simply to establish the presence of oxidative stress, to a focus on identifying specific proteins sensitive to oxidation and establishing the functional consequences of these modifications. In addition the identification of specific enzyme systems to repair these oxidative modifications has led to the belief that protein function may be regulated through these oxidation reactions.

A major challenge for researchers working in this area will be to try and establish which specific amino acid oxidations are related to altered protein function. Oxidised proteins are likely to carry a range of oxidative modifications and thus determining which are the crucial changes will require careful work. In this respect it is clear that some protein modifications occur at a low frequency, i.e., try and met oxidations, while others have a much more global effect, e.g. carbonyl formation. Determining which, if any, of these modifications are most important in vivo is a clear challenge. Much more work is required to clarify specific oxidation products that arise due to the wide range of ROS and RNS that are present under defined conditions. Likewise, the interaction between endogenous antioxidant defences and protein oxidation requires more study. It is likely that certain amino acids are oxidised on proteins to spare other critical residues from oxidation, i.e. they are performing an antioxidant role. Much of this work will most easily be approached with in vitro studies. However, clearly it will be very important to test the relevance of these data within the in vivo situation.

Pathogenesis of influenza virusinduced pneumonia: involvement of both nitric oxide and oxygen radicals

Eosinophil peroxidase produces hypobromous acid in the airways of stable asthmatics

Uric acid provides an antioxidant defense in humans against oxidant-and radicalcaused aging and cancer: a hypothesis

Increased exhaled cysteinyl-leukotrienes and 8-isoprostane in aspirininduced asthma

Extracellular glutathione peroxidase in human lung epithelial lining fluid and in lung cells

Ozone, but not nitrogen dioxide, exposure decreases glutathione peroxidases in epithelial lining fluid of human lung

Protein oxidation biomarkers in hyperoxic lung injury in rats: effects of U-74389

Phagocytes and oxidative stress

Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration

Increased nitric oxide metabolites in exhaled breath condensate after exposure to tobacco smoke

Alveolar lining layer is thin and continuous: low-temperature scanning electron microscopy of rat lung

Towards the physiological function of uric acid

Inhibition of human glutathione reductase by S-nitrosoglutathione

Enzyme inactivation through sulfhydryl oxidation by physiologic NOcarriers

Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly

Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide

Oxidative chemistry of peroxynitrite

Evidence for oxidative stress in bronchiolitis obliterans syndrome after lung and heart-lung transplantation. The Munich Lung Transplant Group

Further evidence for oxidant-induced vascular endothelial growth factor up-regulation in the bronchoalveolar lavage fluid of lung cancer patients undergoing radio-chemotherapy

Plasma proteins of the bronchoalveolar surface of the lungs of smokers and nonsmokers

Comparison of the effects of ozone on the modification of amino acid residues in glutamine synthetase and bovine serum albumin

Ozone-induced lung function decrements do not correlate with early airway inflammatory or antioxidant responses

Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle

Responses of normal and sickle cell hemoglobin to S-nitroscysteine: implications for therapeutic applications of NO in treatment of sickle cell disease

Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor

From pollutant gas to biological messenger: the diverse actions of nitric oxide in cancer

Biochemistry and physiological role of methionine sulfoxide residues in proteins

Depletion of glutathione and ascorbate in lung lining fluid by respirable fibres

Ascorbate free radical as a marker of oxidative stress: an EPR study

Catalytic metals, ascorbate and free radicals: combinations to avoid

Oxidized glutathione is increased in the alveolar fluid of patients with the adult respiratory distress syndrome

An improved procedure for the isolation of ghost membranes from human red blood cells

Vitamin E as an antioxidant in vitro and in vivo

Elevated protein carbonyls and lipid peroxidation products correlating with myeloperoxidase in tracheal aspirates from premature infants

Normal alveolar epithelial lining fluid contains high levels of glutathione

Glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis

Specifically targeted modification of human aldose reductase by physiological disulfides

Human eosinophil peroxidase: purification and characterization

Human methionine sulfoxide-peptide reductase, an enzyme capable of reactivating oxidized alpha-1-proteinase inhibitor in vitro

Cytochrome c nitration by peroxynitrite

Protein S-thiolation in hepatocytes stimulated by t-butyl hydroperoxide, menadione, and neutrophils

Alteration of the C-terminal amino acid of tubulin specifically inhibits myogenic differentiation

Modification of protein surface hydrophobicity and methionine oxidation by oxidative systems

Nitric oxide nitrates tyrosine residues of tumor-suppressor p53 protein in MCF-7 cells

Protein-sulfenic acid stabilization and function in enzyme catalysis and gene regulation

Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation

The Meisenheimer complex of glutathione and trinitrobenzene. A potent inhibitor of the glutathione S-transferase from Galleria mellonella

Increased glutathione and glutathione peroxidase in lungs of individuals with chronic beryllium disease

Rapid loss of superoxide dismutase activity during antigen-induced asthmatic response

Committee of the Environmental and Occupational Health Assembly of the

Inflammation, free radicals, and antioxidants

NO2 interfacial transfer is reduced by phospholipid monolayers

Increased nitrosothiols in exhaled breath condensate in inflammatory airway diseases

Constituents of mucus and their separation

Oxidative damage to plasma constituents by ozone

Oxidants, antioxidants, and respiratory tract lining fluids

Stable markers of oxidant damage to proteins and their application in the study of human disease

Protein nitration, metabolites of reactive nitrogen species, and inflammation in lung allografts

Reactive species and their accumulation on radical-damaged proteins

Role of free radicals in the toxicity of airborne fine particulate matter

Glutathiolation of the proteasome is enhanced by proteolytic inhibitors

The ultrastructural immunolocalization of gamma-glutamyltranspeptidase in rat lung: correlation with the histochemical demonstration of enzyme activity

Redox modulation of cell surface protein thiols in U937 lymphoma cells: the role of gammaglutamyl transpeptidase-dependent H2O2 production and S-thiolation

Estimation of thickness of airway surface liquid in ferret trachea in vitro

Lipid hydroperoxide modification of proteins during myocardial ischaemia

Detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion

Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils

Microtubule dysfunction by posttranslational nitrotyrosination of alpha-tubulin: a nitric oxide-dependent mechanism of cellular injury

Human eotaxin represents a potent activator of the respiratory burst of human eosinophils

New aspects of degranulation and fates of airway mucosal eosinophils

Disruption of the intracellular sulfhydryl homeostasis by cadmium-induced oxidative stress leads to protein thiolation and ubiquitination in neuronal cells

Assessment of chlorination by human neutrophils

Role of the eosinophil in protein oxidation in asthma: possible effects on protei-nase=antiproteinase balance

Antioxidant transport modulates peripheral airway reactivity and inflammation during ozone exposure

Ascorbate is an outstanding antioxidant in human blood plasma

Clinical evidence of peroxynitrite formation in chronic renal failure patients with septic shock

Superoxide radical and iron modulate aconitase activity in mammalian cells

Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways

Inactivation of alpha 2-antiplasmin by limited reaction with cis-dichlorodiammineplatinum (II)

Inflammatory lung injury after bronchial instillation of air pollution particles

Disruption of normal iron homeostasis after bronchial instillation of an iron-containing particle

Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins

Dityrosine: a marker for oxidatively modified proteins and selective proteolysis

Plasma proteins modified by tyrosine nitration in acute respiratory distress syndrome

Role of the glutathione=glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions

Nitric oxide trapping of the tyrosyl radical of prostaglandin H synthase-2 leads to tyrosine iminoxyl radical and nitrotyrosine formation

Prooxidant iron is present in human pulmonary epithelial lining fluid: implications for oxidative stress in the lung

Mechanisms of peroxynitrite-induced injury to pulmonary surfactants

Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury

Nitration of surfactant protein A results in decreased ability to aggregate lipids

Nitric oxide and peroxynitrite. The ugly, the uglier and the not so good: a personal view of recent controversies

Protection by vitamin C of loss of vitamin E in cultured rat hepatocytes

Thiolation of the gammaB-crystallins in intact bovine lens exposed to hydrogen peroxide

Selective nitration of histone tyrosine residues in vivo in mutatect tumors

Iron release from ferritin and its sensitivity to superoxide ions differs among vertebrates

Report of ERS Task Force: guidelines for measurement of acellular components and standardization of BAL

Comparative biochemistry of airway lining fluid. In: Parent R (ed) Treatise on pulmonary toxicology

Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not

Pulmonary strategies of antioxidant defense

Pathways for oxidation of low density lipoprotein by myeloperoxidase: tyrosyl radical, reactive aldehydes, hypochlorous acid and molecular chlorine

The ubiquitin pathway for the degradation of intracellular proteins

Bronchiolitis obliterans syndrome after (heart-)lung transplantation. Impaired antiprotease defense and increased oxidant activity

The biochemistry and physiology of S-nitrosothiols

Glutathione biosynthesis from sulfur-containing amino acids in enriched populations of Clara and type II cells and macrophages freshly isolated from rabbit lung

Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload

Depletion of urate in human nasal lavage following in vitro ozone exposure

Gender difference in the concentration of the antioxidant uric acid in human nasal lavage

Peroxynitrite inhibition of oxygen consumption and sodium transport in alveolar type II cells

Formation of o-tyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation

Endogenous airway acidification. Implications for asthma pathophysiology

Peroxynitrite formation from macrophage-derived nitric oxide

Reactive species in ischemic rat lung injury: contribution of peroxynitrite

The oxidative inactivation of human alpha-1-proteinase inhibitor. Further evidence for methionine at the reactive center

The tolerability, safety, and success of sputum induction and combined hypertonic saline challenge in children

Bactericidal activity of eosinophil peroxidase

Selective and reversible photo-oxidation of the methionyl residues in lysozyme

Induction of inflammatory mediators in human airway epithelial cells by lipid ozonation products

Thiyl radicals in biological systems: significant or trivial?

Nitrotyrosine formation in the airways and lung parenchyma of patients with asthma

An activity in rat tissues that modifies nitrotyrosinecontaining proteins

The use of t-butyl hydroperoxide as a probe for methionine oxidation in proteins

Antioxidant kinetics in lung lavage fluid following exposure of humans to nitrogen dioxide

Nitrogen dioxide depletes uric acid and ascorbic acid but not glutathione from lung lining fluid

Neutrophils convert tyrosyl residues in albumin to chlorotyrosine

Superoxide-dependent hydroxylation by myeloperoxidase

Clinical aspects of exhaled nitric oxide

Exhaled markers of pulmonary disease

Biomarkers of some pulmonary diseases in exhaled breath

Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen

Nasal nitric oxide is increased in patients with asthma and allergic rhinitis and may be modulated by nasal glucocorticoids

Glutathione prevents inhibition of fibroblast-mediated collagen gel contraction by cigarette smoke

Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress

Peroxynitriteinduced tyrosine nitration and inhibition of protein kinase C

Interaction of nitrite with proteins at gastric pH

Evidence for in vivo peroxynitrite production in human acute lung injury

Oxidative damage to proteins of bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome: evidence for neutrophil-mediated hydroxylation, nitration, and chlorination

p21ras as a common signaling target of reactive free radicals and cellular redox stress

Ozone-reactive absorption by pulmonary epithelial lining fluid constituents

Inhibition by nitric oxide of the repair protein, O6-methylguanine-DNA-methyltransferase

Bronchoalveolar lavage fluid proteins in human lung disease: analysis by two-dimensional electrophoresis

Oxidation of methionine in proteins: roles in antioxidant defense and cellular regulation

Two-dimensional gel electrophoresis of nasal and bronchoalveolar lavage fluids after occupational exposure

Two dimensional protein patterns of bronchoalveolar lavage fluid from non-smokers, smokers, and subjects exposed to asbestos

Protein patterns of human nasal and bronchoalveolar lavage fluids analyzed with twodimensional gel electrophoresis

Newly identified proteins in human nasal and bronchoalveolar lavage fluids: potential biomedical and clinical applications

Demonstration of different forms of the anti-inflammatory proteins lipocortin-1 and Clara cell protein-16 in human nasal and bronchoalveolar lavage fluids

A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds

4-hydroxynonenal triggers an epidermal growth factor receptor-linked signal pathway for growth inhibition

Correlation of nuclear color and opalescence with protein S-thiolation in human lenses

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts

Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species

Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade

Increased oxidized methionine residues in BAL fluid proteins in acute or chronic bronchitis

Oxidative modification of H-ras: S-thiolation and S-nitrosylation of reactive cysteines

Nitrosative Stress-induced Apoptosis through Inhibition of NF-kappa B

Nitrated proteins in bronchoalveolar lavage fluid of patients at risk of ventilator-associated bronchopneumonia

Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reductase

Vitamin E: interactions with free radicals and ascorbate

Immunohistochemical localization of nitric oxide synthase and the oxidant peroxynitrite in lung transplant recipients with obliterative bronchiolitis

Antioxidant activity in bronchoalveolar lavage fluid from patientswith lung cancer

Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation

Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase by S-nitrosylation and subsequent NADH attachment

Nitric oxideinduced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase

Oxidative stress and pulmonary inflammation: pharmacological intervention with antioxidants

Antioxidant consumption and repletion kinetics in nasal lavage fluid following exposure of healthy human volunteers to ozone

Modeling the interactions of ozone with pulmonary epithelial lining fluid antioxidants

Differential depletion of human respiratory tract antioxidants in response to ozone challenge

Differences in basal airway antioxidant concentrations are not predictive of individual responsiveness to ozone: a comparison of healthy and mild asthmatic subjects

Activation of human macrophages for the killing of intracellular Trypanosoma cruzi by TNF-alpha and IFN-gamma through a nitric oxide-dependent mechanism

Regional variation in iron and iron-binding proteins within the lungs of smokers

Effect of inhaled ozone on exhaled nitric oxide, pulmonary function, and induced sputum in normal and asthmatic subjects

Chemical modification and 1H-NMR studies on the receptor-binding region of human interleukin 6

Nasal lavage cytokines in normal, allergic, and asthmatic school-age children

Database of bronchoalveolar lavage fluid proteins

Diesel exhaust enhances airway responsiveness in asthmatic subjects

Nitrotyrosine as a new marker for endogenous nitrosation and nitration of proteins

Inactivation of enzymes and oxidative modification of proteins by stimulated neutrophils

Role of transferrin and ceruloplasmin in antioxidant activity of lung epithelial lining fluid

Alveolar fluid glutathione decreases in asymptomatic HIV-seropositive subjects over time

Cellular responses to nitric oxide: role of protein S-thiolation=dethiolation

Oxidative damage in tissues of rats exposed to cigarette smoke

Nitrogen-13-labeled nitrite and nitrate: distribution and metabolism after intratracheal administration

Uric acid is a major antioxidant in human nasal airway secretions

Human nasal glandular secretion of novel antioxidant activity: cholinergic control

Nasal secretion of the ozone scavenger uric acid

Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid

Myeloperoxidase-generated oxidants and atherosclerosis

Interfacial transfer kinetics of NO2 into pulmonary epithelial lining fluid

Determinants of inhaled ozone absorption in isolated rat lungs

NO2 reactive absorption substrates in rat pulmonary surface lining fluids

Catalytic reduction of Fe(III)-cytochrome-c involving stable radiolysis products derived from disulphides, proteins and thiols

Ozone in all its reactive splendor

Mechanisms of radical formation from reactions of ozone with target molecules in the lung

The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide

The cascade mechanism to explain ozone toxicity: the role of lipid ozonation products

A new mechanism for the toxicity of ozone

Glutathione oxidation by hypochlorous acid in endothelial cells produces glutathione sulfonamide as a major product but not glutathione disulfide

Oxidative damage to plasma proteins in adult respiratory distress syndrome

Composition and control of secretions from tracheal bronchial submucosal glands

Oxidative stress and regulation of glutathione in lung inflammation

4-Hydroxy-2-nonenal, a specific lipid peroxidation product, iselevatedin lungsofpatientswith chronic obstructive pulmonary disease

S-thiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the phagocytosis-associated respiratory burst in blood monocytes

Oxidative dissociation of human alpha 2-macroglobulin tetramers into dysfunctional dimers

Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite

Fractional processing of sequential bronchoalveolar lavage to separate bronchial and alveolar samples

Oxidative stress in chronic obstructive pulmonary disease. Oxidative Stress Study Group

Conversion of lysine to N(epsilon)-(carboxymethyl)lysine increases susceptibility of proteins to metalcatalyzed oxidation

Modification of plasma proteins by cigarette smoke as measured by protein carbonyl formation

Ocular oxidants and antioxidant protection

Nitration of annexin II tetramer

Type II pneumocytes secrete vitamin E together with surfactant lipids

Detection and identification of human bronchoalveolar lavage proteins using narrow-range immobilized pH gradient DryStrip and the paper bridge sample application method

Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis

Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid

Effects of reactive oxygen and nitrogen metabolites on RANTES-and IL-5-induced eosinophil chemotactic activity in vitro

Superoxide ion as active intermediate in the autoxidation of ascorbate by molecular oxygen. Effect of superoxide dismutase

Amino acid sequence of chicken Cu, Zn-containing superoxide dismutase and identification of glutathionyl adducts at exposed cysteine residues

Oxidative stress in lavage fluid of preterm infants at risk of chronic lung disease

Protein S-thiolation and dethiolation during the respiratory burst in human monocytes. A reversible post-translational modification with potential for buffering the effects of oxidant stress

Measurement of Na( þ )-Kþ pump current in isolated rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Inhibition of the pump by oxidant stress

Bromide-dependent toxicity of eosinophil peroxidase for endothelium and isolated working rat hearts: a model for eosinophilic endocarditis

Increased levels of glutathione in bronchoalveolar lavage fluid from patients with asthma

Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite

Quinoid redox cycling as a mechanism for sustained free radical generation by inhaled airborne particulate matter

Protein oxidation

Metal-catalyzed oxidation of proteins. Physiological consequences

S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds

Nitrosylation. the prototypic redoxbased signaling mechanism

Nitration of the low molecular weight neurofilament is equivalent in sporadic amyotrophic lateral sclerosis and control cervical spinal cord

Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO

Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity

Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite

Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura of the rat

S-nitrosation of Ca(2 þ )-loaded and Ca(2 þ )-free recombinant calbindin D(28K) from human brain

Oxidative targets in the stratum corneum. A new basis for antioxidative strategies

Myeloperoxidasedependent effect of amines on functions of isolated neutrophils

Oxidation of bromide by the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase. Formation of bromamines

Protein sulfhydryls and their role in the antioxidant function of protein S-thiolation

Lower respiratory tract lactoferrin and lysozyme arise primarily in the airways and are elevated in association with chronic bronchitis

Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation

Selective oxidation of methionyl residues in the human recombinant secretory leukocyte proteinase inhibitor. Effect on the inhibitor binding properties

S-Transnitrosylation of albumin in human plasma and blood in vitro and in vivo in the rat

van der Vliet A, Cross CE (2001) Innate antioxidant defense systems in the respiratory tract

Interactions of peroxynitrite with human plasma and its constituents: oxidative damage and antioxidant depletion

Oxidative stress in cystic fibrosis: does it occur and does it matter?

Myeloperoxidase and protein oxidation in cystic fibrosis

Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining fluids

The oxidative inactivation of sarcoplasmic reticulum Ca(2 þ )-ATPase by peroxynitrite

Oxidation of intracellular glutathione after exposure of human red blood cells to hypochlorous acid

Oxidative stress and reactive nitrogen species generation during renal ischemia

Stimulation of topoisomerase II-mediated DNA damage via a mechanism involving protein thiolation

Ascorbate recycling in human neutrophils: induction by bacteria

Irreversible inactivation of protein kinase C by glutathione

Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase

Human bronchoalveolar lavage fluid: two-dimensional gel electrophoresis, amino acid microsequencing and identification of major proteins

Regulation of depth and composition of airway surface liquid

The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective

Free radical-induced carbonyl content in protein of estrogen-treated hamsters assayed by sodium boro[3H]hydride reduction

Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite

Protein carbonyl measurements show evidence of early oxidative stress in critically ill patients

Effects of acute endotoxemia on production of cytokines and nitric oxide by pulmonary alveolar and interstitial macrophages

Eosinophil peroxidase nitrates protein tyrosyl residues. Implications for oxidative damage by nitrating intermediates in eosinophilic inflammatory disorders

Eosinophils generate brominating oxidants in allergen-induced asthma

Inactivation of human manganese-superoxide dismutase by peroxynitrite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosine

Surfactant protein D regulates NF-kappa B and matrix metalloproteinase production in alveolar macrophages via oxidant-sensitive pathways

Nitration of surfactant protein A (SP-A) tyrosine residues results in decreased mannose binding ability

Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages

Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite