5 Particle-Mediated Extracellular Oxidative Stress in the Lung Frank J. Kelly and Ian S. Mudway Pharmaceutical ScienceResearchDivision, King’sCollege CONTENTS 5.1Health Effects of Particulate Matter and the Oxidative Stress Hypothesis 90 5.2Antioxidant Defenses at the Air–LungInterface 92 5.2.1 Small Molecular Weight Antioxidants 93 5.2.1.1 Ascorbate 93 5.2.1.2 Urate 94 5.2.1.3 Reduced Glutathione 95 5.2.1.4 a -Tocopherol 96 5.2.2 Mucins 96 5.2.3 Enzymatic Antioxidant Defenses 96 5.2.3.1 Glutathione Peroxidase 96 5.2.3.2 EC-SOD 96 5.2.3.3 Catalase 96 5.2.4 Metal Chelation Proteins 97 5.2.4.1 Transferrin 97 5.2.4.2 Lacoferrin 97 5.2.4.3 Ferritin 97 5.2.4.4 Caeruloplasmin 97 5.3Induction of Oxidative Stress by Inhaled Particles 98 5.3.1 TheRole of Redox Active Metals 98 5.3.1.1 Iron (Fe) 98 5.3.1.2 Copper (Cu) 100 5.3.1.3 Manganese (Mn) 101 5.3.1.4 Vanadium (V) 101 5.3.1.5 Nickel (Ni) 101 5.3.1.6 Chromium (Cr) 102 5.3.2 TheRole of Nonredox Active Metals 102 5.3.2.1 Zinc (Zn) 102 5.3.2.2 Aluminium (Al) 102 5.3.2.3 Lead (Pb) 103 5.3.3 Particle-Associated Quinone Toxicity 103 5.3.4 Polycyclic Aromatic Hydrocarbons (PAHs) Induced OxidativeStress 103 5.3.5 Lipopolysacharide (LPS) Induced Oxidative Stress 104 5.3.6 Generation of ROS by Inflammatory Cells 104 5.4The Role of Particle Size and Surface Area versus Composition as Determinants of PM Toxicity 104 89 © 2007 by Taylor & Francis Group, LLC 5.5 Particle-Induced Toxicity: Lessonsfrom Cigarette Smoke and Fiber Research 105 5.6 Evidence of Particle-Induced Oxidative Stress from Animal Studies 106 5.7 Concentrated Ambient and DieselExhaust Particle Exposure in Human Subjects 107 5.8 Modeling the Interaction of Ambient PM with RTLF Antioxidants 109 5.9 Particle-Induced Oxidation Reactions at the Air–LungInterface as aPredictor of ObservedHealth Effects 109 5.10 Conclusions 110 Acknowledgments 110 References 110 5.1 HEALTH EFFECTS OF PARTICULATE MATTER AND THE OXIDATIVE STRESS HYPOTHESIS During the last decade, concerns have increased within both politicaland scientific communities over the impact of airborneparticulatematter (PM)onpublic health. This concern has arisen primarily based on the findings of American epidemiological studies demonstratinganassociation betweenthe massconcentration of PM (particularly particles with an aerodynamic diameter of less than 10 m m) in the air we breathe, and rates of respiratory and cardiac mortality and morbidity (Dockery et al. 1993; Pope et al. 1995;Samet et al. 2000). These associations have subsequently proven to be robustinnumerousepidemiological studies worldwide(Brunekreefand Holgate 2002), with stronger associations usually associated with PM with an aerodynamic diameter of ! 2.5 m m. Moreover,the estimated decrease in life span associated with long-term residence in areas with high ambient PM is estimated to be between1and 2years, which is large comparedwith otherenvironmental risk factors.Whilstthe data demonstratingPM-inducedhealtheffects is irrefutable, major questions remainconcerning the mechanisms by which thesecompositionally heterogeneous species elicittheir toxic actions.Ithas been argued that as exposure to abroad spectrumofparticle types (vehicleemissions,cigaretteand wood smoke PM, mineral dusts etc) elicits similar acuteresponses in humans, namely neutrophilic inflammation (Burns 1991;Salvi et al. 1999;Ghio 2000), reduced inspiratory capacity (Salvi et al. 1999;Stenfors et al. 2004)and heightened bronchial reactivity (Sherman et al. 1989; Menon et al. 1992; Nordenhall et al. 2000), they may act through acommon mechanism.Currently numerous groups are investigating the hypothesis that thesecommon mechanisms may relatetothe capacity of these particlestocause damaging oxidation reactions in the lung, as well as systemically. In this review we will consider this hypothesis in detail,with specific emphasis on initial interactions between inhaled PM and components of the thin liquid layer that overlies the respiratory epithelium, the respiratory tract liningfluid(RTLF). This compartment represents thefirst physicalinterface with which PM interacts, upon deposition in the airways. Aclear understanding of the initial reactions between PM constituents andcomponentsofthe RTLF is thus criticalinunderstanding any observed toxicity. Oxidative stress is arelatively new term in biology that was first introduced by Sies (1991),and defined as “a disturbance in the pro-oxidant-antioxidant balance in favor of the former,leading to potential damage.” Since then,many other definitions have been proposed, all of which attempt to explain aprocess which essentially involves the flow of electrons from one molecule to another within abiological setting. Theimportanceofthis process lies in the reactivity of the molecules involved.Under normal circumstances,electrons orbit around atomsinpairs, having opposite spins. When an atom has asingle unpaired electron, its reactivity increases markedly, and it is referred to as afree radical. In abiological setting, free radicals are potentially very dangerous since they can react indiscriminately with neighboringmolecules. This process of “electron stealing” leads to oxidation of, for example, lipids, proteins, and nucleic acids, and as aconsequence, altered Particle Toxicology90 © 2007 by Taylor & Francis Group, LLC function or inactivation of these target molecules. If these reactions are numerous,they can cause extensive cellular damage, impaired cellfunctions, and in some cases, the influx of inflammatory cells to the sites of injury (Freeman and Crapo 1982; Halliwell and Gutteridge 1999; Droge 2002). The extent of damage is related to the availability of neutralizing antioxidant defenses,since these specialized molecules preferentially react with free radicals and give rise to products that often possess low toxicity. The damage arising from aberrant free radical activityisoften loosely referred to as oxidative stress which, in its simplest form is apotentially harmfulprocess occurring when there is an excessoffree radicals, adecrease in antioxidant defenses,oracombination of these events (Figure 5.1). There is now astrong body of evidence demonstrating that disturbances in normal cellular and extracellular redox status in the lung, in response to PM exposure, can triggerinflammation via the activation of redox-sensitive signaling pathways (Lietal. 1996; Bonvallot et al. 2001; Pourazar et al. 2005). The capacity of PM to elicit such aresponse has been explained by the deliveryof surface adsorbed (redox active) metals (Aust et al. 2002)and organic contaminants (Squadrito et al. 2001)into the lung that drive oxidation reactions with the generation of cytotoxic reactive oxygen and nitrogenspecies (ROS and RNS) (Doelman and Bast 1990; Kelly 2003). The resultant airway inflammation itself then results in an increased productionofoxidants by activated phagocytes, recruited to the airways, perpetuating the cycleofoxidative injury. In addition, ambient PM also containsappreciable concentrations of bacterial endotoxinthat can also trigger inflammation. Antioxidant replenishment Antioxidant losses Mucus Organics Carbon core Transition metals Inflammation ROS Oxidativedamage Blood -Ve A-O X OX OX A-O X +Ve RTLF FIGURE 5.1 The imposition of oxidative stress at the air–gas interface. Under physiological conditions, the production of oxidant species (OX) in the aqueous respiratory tract lining fluid (RTLF) compartment is kept in check by the endogenous antioxidant defenses(A-OX). Whenthe production of reactive oxygen species increases, either throughthe inhalation of pro-oxidants or thedevelopmentofairwayinflammation,the RTLF antioxidant network may become overwhelmed, permitting oxidative damage to occur to protein and lipid components of this compartment. The underlying epithelium can respond to this oxidative stress in a graded fashion, either up-regulating the expression of antioxidant defenses or mobilizing them to the RTLF, or by elicitinganinflammatoryresponseresulting in agreateroxidative burdenwithinthe RTLF,which ultimately leads to cell death and tissue injury. Particle-Mediated Extracellular OxidativeStress in the Lung 91 © 2007 by Taylor & Francis Group, LLC 5.2 ANTIOXIDANT DEFENSES AT THE AIR–LUNG INTERFACE Owingtoits function, large surface area and exposure to high partial pressuresofoxygen, the lung is particularly susceptibletooxidative injury. It is thereforelogicalthat arobustextracellular antioxidantdefense systemexits to protectagainst undue oxidation of itsdelicate pulmonary epithelial cells. On entering the lung, ambient pollutantsdonot comeinto direct contact with the respiratory epithelium, but rather they first interact with aliquid layer that covers the respiratory epithelium from the nasal mucosa to the alveoli, the RT4 (Figure5.2). Human RTLF is acomplex and regionally heterogeneous compartment, ranging in depth from between 1and 10 m minthe proximal airways to 0.2–0.5 m minthe distal airwaysand alveoli (Cross et al. 1998). It consists of Atmosphere Nasal airways Proximal airways Distal airwaysand alveoli Mucus Mucins Surfactant AA GSH GSH ecCuZn SOD ecGSHPx Lactoferrrin Transferrin Caeruloplasmin Lactoferrrin Transferrin Caeruloplasmin ecCuZn SOD ecGSHPx CYS UA UA AA A-Toc Epithelium RTLF thickness Interstitium Endothelium A-Toc CYS Organics Carbon core Tr ansition metals FIGURE 5.2 RTLF antioxidant network in the nasal/proximal airways and alveolar regions of the lung. Both the upper- and lower-airways RTLFs contain cysteine (CYS) and ascorbate (AA), but there are especially high concentrations of urate (UA) in the upper airways, especially the nasal mucosa. In contrast, glutathione (GSH) concentrations appear to be greatest in the terminal airways and alveoli. a -Tocopherol is present throughout theairwayRTLFs,but at lowconcentrations.Extracellular superoxide dismutase(ecCuZnSOD) and glutathione peroxidase (ecGSH-Px) are also present in RTLFs. Catalase and glutathione reductase have also been reported in human RTLF, but it is not clear whether these are derived from cellular lysis and so are not included. Metal-ion chelator proteins within the RTLF are also illustrated, with lactoferrin emphasized in the upper airway where its concentration is highest. Particle Toxicology92 © 2007 by Taylor & Francis Group, LLC secretions from underlying lung and residentimmune cells, as well as plasma-derived exudates.In the nasal and proximal airways, it exists as atwo-phase structure consisting of agel and sol phase, the former consisting of thiol-rich mucopolypeptide glycoprotein(mucins). This contrasts with the distal airwayand alveolarlining fluids, which are devoid of mucins, but contain surfactant lipids and proteins. Nasal, proximal, and distal lavageprocedureshave allowed adetailed examination of the RTLF in different lung compartments. In addition to the mucin and surfactant components, the RTLF also contains abroad spectrum of lowmolecular weight antioxidants,aswellassmall concentrations of antioxidant enzymes (Kelly et al. 1995; 1999)(Figure5.2). 5.2.1 S MALL M OLECULAR W EIGHT A NTIOXIDANTS An array of small molecular weight antioxidants has been measured in human RTLF, including ascorbate(vitamin C) (Willis and Kratzing 1974; Skoza, Snyder,and Kikkawa 1983; van der Vliet et al. 1999), urate (Peden et al. 1990), reduced glutathione (GSH) (Cantin et al. 1987; Jenkinson, Black,and Lawrence 1988), and a -tocopherol (vitaminE)(Mudway et al. 2001). As the lavage procedure used to sample the airway introduces alarge and variable dilution, the concentration of these antioxidants quoted in the literature are often difficult to interpret. Whilst manygroups simply quote concentrations in the recovered lavage, others have made attemptstocorrect for the dilution, using arange of methods based on instilled dyes or endogenousdilution markers (Haslamand Baughma1999). Of these, the mostcommonly adopted is the urea correctionmethod that uses the ratio of urea concentrations between the lavage sample and plasma as the basis for estimating the dilution. This technique is often criticized because the instillation of alarge volume of saline creates amassive concentration gradient along which urea can move from cellular and interstitial sources. One way of limiting this potential problem is to reducethe lavage dwell time and not to perform repeat sampling of the same airway segment,asisoften performed in lavage procedures. The RTLF antioxidant concentration rangesquoted in this review will be based on values obtained usingthese methods,unless otherwise stated. 5.2.1.1 Ascorbate As aconsequence of its high solubility, ascorbateisthought to diffuse freely between aqueousextra andintracellular compartmentsofthe body including theRTLF, where concentrations (50–150 m M) comparable to those in plasma have been measured (Kelly, Buhl, and Sandstro ¨ m 1999; vander Vliet1999).Inhumans, this antioxidantvitamin originates solely from dietary sourcesinhumansowing to thelackofthe enzyme,gluconlactone oxidase, necessaryfor its biosynthesis (Halliwell andGutteridge 1999).Differences in dietary intakemay therefore explain the variabilityinascorbateconcentrations that have been measured in bronchoalveolar lavage (BAL) fluid from different individuals (Kelly, Buhl, and Sandstro ¨ m1999). Ascorbate is an excellent reducing agent and scavenges avariety of free radicals and oxidants in vitro,including superoxide andperoxyl radicals, hydrogenperoxide,hypochlorous acid, andsinglet oxygen. During its antioxidant activity, ascorbate readily undergoes twoconsecutive, but reversible, one- electron reductions. The first one-electron reduction produces the semi-dehydroascorbate radical (SDA) also known as the ascorbyl radical (Asc % )(Buettner and Jurkiewicz 1996). Asc % can also undergoafurther one-electronreduction to dehydroascorbate(DHA).The ascorbyl radical is relatively unreactive, owing to its unpaired electron being in the delocalized p -system, and in the absence of afurther oxidation, two ascorbyl radicalswill undergo adisproportionate reaction to regenerate one molecule of ascorbate and one molecule of DHA.The DHA,once formed,rapidly undergoeshydrolysis to 2,3-diketo- L -gulonic acid,ultimatelydegradingtoadiverse range of products, including oxalic and L -threonic acids. Dehydroascorbateand its metabolitesare poten- tially cytotoxic in significant concentrations,and therefore cells possess enzymes that convert either SDA or DHA back to ascorbate at the expenseofGSH or NADPH. (Diliberto et al. 1982; Park and Particle-Mediated Extracellular OxidativeStress in the Lung 93 © 2007 by Taylor & Francis Group, LLC Levine1996). In addition, glutathione has been shown to recycle DHA nonenyzmatically (Winkler, Orselli, and Rex1994) in vitro,although at arate insufficient to prevent substantiallosses of DHA. Little is currently understoodabout the turnover of ascorbate within the RTLF as to date, only ascorbateand dehydroascorbatehave been routinely measured. At present, it is not clear whether such regenerative mechanismsexist in the RTLF, but certainlythe high concentrations of gluta- thione in this compartment may act to limit the oxidative losses of ascorbate. Clearly in the absence of some recycling mechanism,the oxidative losses of ascorbatewithin the RTLF would constitute a significant drain on bodily stores of this antioxidant. Many celltypes, including neutrophils, are able to take up DHA rapidly via the facilitative glucose transporters GLUT1and GLUT3 (Rumsey et al. 1997). Once internalized, the DHA is rapidlyreduced back to ascorbic acid by glutaredoxin at the expenseofglutathione (Park and Levine1996). Thus, it may be possible that DHA formed in the RTLF under normal or pathologicalconditions may be reduced back to ascorbatebycellular uptake by the epithelium (Nualart et al. 2003). In addition to having adirect scavenging action, ascorbatealso acts indirectly to prevent lipid peroxidation through its reaction with the membrane bound a -tocopherol radical. In vitro studies have demonstrated that in this way, ascorbate is able to reduce the a -tocopherol radical, thereby regenerating thevitamin Emolecule(Packer,Slater,and Wilson 1979; Niki,Yamamoto, and Kamiya 1985; Doba, Burton, and Ingold1985). This synergistic action has not yet however been reported in vivo.Thispotential interaction, togetherwith the recycling of DHA by glutathione, illustrates that the antioxidant networkwithin the RTLF is highlysynergistic (Figure5.3). Whilst ascorbaterepresents acriticalprotective component of theRTLF,italsohas the potential to act as apro-oxidant. In the presence of ferric iron(Fe 3 C )orcupric copper (Cu 2 C ) ions,ascorbate can promote reduction to ferrous (Fe 2 C )and cuprous forms (Cu 2 C ), permitting the formation of the damaging hydroxyl radical in the presence of hydrogen peroxide. Fe 3 C C ascorbateð reducedÞ / Fe 2 C C ascorbateð oxidized Þ Fe 2 C C H 2 O 2 / Fe 3 C C OH $ C OH K This situation occurs rarely under normal physiological conditions, as both Fe andCuare sequestered into transportand storage proteins to limit these potentially damaging reactions. 5.2.1.2 Urate Urate is an oxidizedpurine base present in all RTLF compartments of the lung, but at particularly high concentrations in the nasal and proximal airways (200–500 m M) where it appears to be the predominant antioxidant defense (Cross et al. 1994; Blomburg et al. 1998). In the nasal airways it appears to be secreted from glandcells alongwith lactoferrin (Pedenetal. 1990, 1993), but little is knownabout its transportinto the lower airways. This antioxidant can directly scavenge hydroxyl radicals,oxyhaem oxidantsformedbetweenthe reactionsofhemoglobinand peroxy radicals, peroxyl radicals themselves,and singletoxygen(Becker 1993).Inthese reactionsitactsina sacrificialmode, in that it is irreversiblydamaged to producearange of oxidationproducts, including allantoin (Amesetal. 1981). As with ascorbate, relatively little is knownabout the turnover of urate in the RTLF. In addition to its role as afreeradical scavenger, urate has also been reported to chelate ironand protect against iron-mediated free radical damage of ascorbateand lipids (Davies et al. 1986; Ghio et al. 1994). Increased urate concentrations have been reported in rats challengedwith iron saltsand silica-iron in concert with increased lung xanthine oxido- reductaseactivities.Thiswouldimply that elevated RTLF uratemight reflect aregulated adaptiveresponse to limit Fe-induced oxidative injury (Ghio et al. 2002). Particle Toxicology94 © 2007 by Taylor & Francis Group, LLC 5.2.1.3 Reduced Glutathione High levels of glutathione (100–200 m M) have been reportedinproximal airway andalveolar RTLFs from healthy individuals, with some reports suggesting concentrations as high as 400 m M (Cantin et al. 1987). This is around100 timeshigher than normal plasma concentrations (Kelly and Richards 1999)with over 90%present in its reduced form (GSH; L -glutamyl- L cysteinylglycine). The sourceofGSH has not been established, although in the light of low concentrations in plasma and poor re-adsorption from the respiratory tract,itislikely that this antioxidant is produced via a secretary mechanism from the cells in the lung (Smith, Anderson, and Shamsuddin1992). As well as acting as asubstrate for the glutathione redox cycle, glutathione is known to reactwith awide range of compounds in vitro including hydroxyl radicals, hypochlorous acid, peroxynitrite, peroxyl radicals, carbon centred radicals, and singlet oxygen. In its reactions with freeradicals, thiyl radicals are produced, which can subsequently be converted to oxidizedglutathione through a radical transfer process(Halliwell andGutteridge 1999). Glutathione is particularlygood at defending againstoxidantssuchashypochlorous acid andhypobromous acid(Winterbourn 1985), which are released from neutrophils and eosinophils, respectively. The presence of this antioxidant in lung lining fluid may therefore be particularly important in defendingthe extra- cellular surface of the lung against activated inflammatory cells. Airway lumen AA AA UA ROO - PUFA ROS ROOH DHA DHA AA RTLF AA α -tocopherol α -tocopherol α -tocopherol radial Epithelial cell NADPH NADPH NADP NADP GSH GSH GSSG GSSG GSSG reductase GSSG reductase Glutaredoxin GLUT1/3 ROOH ROH ROOH ROH UA UA SDA SDA ROO - RO - FIGURE 5.3 Cooperativity of RTLF antioxidants. Peroxyl (ROO % )and alkoxyl (RO % )lipid radicals formed thoughthe oxidation of polyunsaturated fattyacids(PUFA)can be reduced to harmlessalcohols(ROH, ROOH) throughthe scavenging action of membrane-bound a -tocopherol.The tocopherol radicalcan be re-reduced at the expense of ascorbate (AA), which is converted to the ascorbyl radical (SDA) and hence to dehydroascorbic acid (DHA). As DHA rapidly, it is normally rapidly reduced back to ascorbate intracellularly by glutaredoxin at the expense of the oxidation of glutathione (GSH) to glutathione disulphide. It is not known whether asimilar DHA reductase activity exists within the RTLF, but GSH can recycle DHA to alimited extent in an non-enzyme catalyzed mechanism. Urate (UA) can also scavenge ROO % and RO % Radicals, and the redox potential of the urate radical suggests that UA may also be recycled by ascorbate. Particle-Mediated Extracellular OxidativeStress in the Lung 95 © 2007 by Taylor & Francis Group, LLC 5.2.1.4 a -Tocopherol RTLF a -Tocopherolisbelievedtobederived from type II cells (Rustow et al. 1993)and is present at relatively low concentrations within lung lining fluid. It is, however,apowerful antioxidant, both in terms of scavenging free radicals such as singlet oxygen, alkoxyradicals, peroxynitrite, nitrogen dioxide, ozone, andsuperoxide(Wang andQuinn 1999)and its ability to terminate lipid peroxidation(Witting1980; Burtonand Ingold1981; Niki,Takahashi,and Komuro 1986; Nakamura et al. 1987). It is thought that the reactivity of a -tocopherol with organic peroxyl radicals accounts forthe majorityofits biologicalactivity(Witting 1980;Burtonand Ingold 1981), a reaction that yields arelatively stable lipid hydroperoxide and avitamin Eradical, thereby effec- tivelyinterrupting the lipid peroxidation chain reaction (McCay 1985). 5.2.2 M UCINS In addition to providing aphysical barrier and clearance mechanism to remove inhaled toxins, mucin components of the gel phase of the upper airway RTLF also have significant antioxidant properties, by virtue of their cysteine-rich domains and carbohydrate moieties (Cross, Halliwell, and Allen 1984; Hiraishi et al. 1993). Mucinshave been showntohave metal binding properties (Cooper, Creeth, and Donald 1985), as well as the capacity to scavenge hydroxyl radicals(Cross, Halliwell, and Allen1984). In addition, the thiol moieties imply that mucins shouldalso provide considerable protections against hypochlorous acid (HOCl K ). 5.2.3 E NZYMATIC A NTIOXIDANT D EFENSES RTLF also containsantioxidant enzymes,whose role it is to scavenge oxidants or to repair damage causedbyROS. These enzymatic antioxidantsare acomplexset of proteins and include extra- cellular CuZn superoxide dismutase (EC-SOD), catalase, and glutathione peroxidase (GPx). 5.2.3.1 Glutathione Peroxidase Glutathione peroxidase (GPx) is aselenoprotein that catalyzes the reduction of hydrogen peroxide and lipid peroxides, at the expenseofglutathione (Brigelius-Flohe ´ and Traber1999). Of the four typesofGPx, namely cellular or cytosolic or classical GPx, gastrointestinal GPx, extracellularor plasma GPx, and phospholipid hydroperoxide GPx (Takebe et al. 2002), Avissaretal. (1996) has shownthat RTLFs contains selenium-dependent cellular GPx and extracellular GPX, each contri- buting approximately50% to the total GPx activity. 5.2.3.2 EC-SOD EC-SOD is expressed in especially high levels in mammalian lungs by the alveolar type II pneu- mocytes (Oury et al. 1996), and can be found in the RTLF (Mudway and Kelly 2000)and airway epithelialcell junctions.EC-SOD catalyzes thedismutationofthe superoxide freeradicalto hydrogen peroxide thus in concert with glutathione peroxidase, protects lung interstitium against free radical generated during inflammation (Oury et al. 1996). 5.2.3.3 Catalase Catalaseisaheme-containing tetramerous protein, primarily present in peroxisomes, but also found cytoplasm, mitochondria, and BALF (Fridovich 1998; Halliwelland Gutteridge 1999; Vallyathan et al. 2000). Catalaseisimportant in the dismutation of hydrogen peroxide to water and oxygen (Quinlan, Evans, and Gutteridge 2002). As with many of the enzymatic antioxidants reportedin RTLF, there is someuncertainty regarding whether they are authentic extracellular proteins, or are derived from cellular disruption during the lavage procedure. Particle Toxicology96 © 2007 by Taylor & Francis Group, LLC 5.2.4 M ETAL C HELATION P ROTEINS Metal-chelatingproteins present in the RTLF also perform an important antioxidant function by binding free transition metals, thus preventing them from participating in potentially damaging redox reactions (Halliwell and Gutteridge 1999). 5.2.4.1 Transferrin Transferrin controls the transport of iron in the body. It is the predominant metal-chelating protein in the RTLF and has been showntobeapotent inhibitor of lipid peroxidation in vivo (Pacht and Davis 1988). Of interest, the affinity of iron for transferrin is pH-dependent,such that in plasma (pH 7.4), binding is very strong, whereas virtually no binding occursatpH ! 4.5. This is pertinent to particle-association metal chemistry in the RTLF, in that the pH of RTLF from healthy individuals has been reportedtobebetween 7and 7.5, but in certain disease states, such as asthma, the pH is approximately pH 5(Hunt et al. 2000). One might speculate, therefore the chelation of iron by transferrin to be sub-optimal in asthmatics, this wouldincreasethe presence of unboundiron and may, in turn, contribute to the low levels of RTLF antioxidantsseen in asthmatics (Kelly, Buhl, and Sandstro ¨ m1999; Mudway and Kelly 2000). 5.2.4.2 Lacoferrin Lacoferrin is able to bind and transportiron and release it again at specific receptor cells in the human intestine. High concentrations have been reported in the upper airways, whilststudies have shownthatconcentrations are increased in thelower respiratorytract of chronicbronchitics (Thompson et al. 1990). Liketransferrin, lactoferrin is apotent inhibitor of lipid peroxidation in vivo (Pacht and Davis 1988). Lactoferrin can bind free iron with high affinityand thus function as alocal antioxidant,protectingthe immune cells against the freeradicals produced by them (Britigan, Serody, and Cohen 1994). Furthermore, reports suggestthat lactoferrin can bind free iron released from dying cells, thereby protectingthe phagocytic cells, as well as adjacent tissues, from ROS produced from the Haber–Weiss reaction (Britigan, Serody,and Cohen 1994). 5.2.4.3 Ferritin Ferritin controls the storage of ironwithin the body, and thus can also provide protection against iron-generated ROS.Concentrations of ferritin in alveolar cells and on the alveolar surface are increased in patients with avarietyofrespiratory disorders (Stites et al. 1999)and it is likely that the presence of ferritin in the RTLF reflects the release of cell contents from dying cells. Anumber of inflammatory mediators can also induce intracellular up-regulation, as well as the cleavage of iron from extracellular ferritin, thereby allowing the free iron to be redox active (Reif 1992). Indeed, Stites et al. (1999) reported that oxidant injury could be promoted in lungs of patients with cystic fibrosis as aconsequence of mobilizing iron. 5.2.4.4 Caeruloplasmin Caeruloplasmin is aglycoprotein involved in serum copper transport. It represents the main anti- oxidant defense against copper in human plasma, but also promotes the incorporation of iron into transferrin without the formation of toxic iron products (Koc et al. 2003). It is primarily synthesized in the liver and secreted to the blood; however,recent studies have identified the lung as another major site of synthesis. In particular, Yangetal. (1996) have suggestedthat the airwayepithelial cells are the major source of ceruloplasminidentified in RTLF. Particle-Mediated Extracellular OxidativeStress in the Lung 97 © 2007 by Taylor & Francis Group, LLC 5.3 INDUCTION OF OXIDATIVE STRESS BY INHALED PARTICLES Particulate matter is acomplex mixture of chemical components in terms of their chemical composi- tion, dependant on the emission sourceand in combustion scenarios, the type of fuel being burnt. For example, ultrafineparticles from sources of combustion generally comprise acarbonaceous core with absorbed substances condensed onto the surface during combustion and atmospheric processes. These substances may include organic and elemental carbon; polycyclic aromatic hydro- carbons (PAHs); metals(both redox active and non-redox active); and biological compounds such as bacterial endotoxin, as well as sulphate, nitrate, chloride, and ammonium. The composition dictates the surface reactivity of the particles, an important factor in determining particle toxicity (Fubini 1997) and its adverse health effects. Whether thesePMcomponents ultimatelyresult in substantial oxidativedamage, inflammation, andinjuryultimately depends on their initial interactionswith theantioxidant defenses within theRTLF. These potential interactions are illustrated in Figure 5.4.Itisuseful to grade the response of the lung to particle-induced oxidative stress, with low-leveloxidativestress resultinginanup-regulation of endogenous extra- and intracellular responses,prior to the induction of substantial toxicity (Lietal. 2002). The various pathways by which inhaled PM can elicit injury to the airway are illustratedinFigure 5.5. 5.3.1 T HE R OLE OF R EDOX A CTIVE M ETALS Redox-active metalsare defined as thosecontaining unpaired electrons in their d -orbital, and are capable of generating free radical species via redox cycling mechanismswith biological reductants. Using this definition, the entire first row elements in the d-block of the periodic table, with the exception of zinc, qualify: Sc, Ti, V, Cr, Mn, Fe, Co, Ni, and Cu. Of these, the most common components of PM are listed below. 5.3.1.1 Iron (Fe) As outlined earlier, under normal conditions iron is sequestered in the chemically less active Fe 3 C form in the storage protein ferritin, or is associated with transportorreceptorproteins, thereby facilitating the removal of reactive iron from areas including the respiratory tract.Inthe presence of excessiron (for example, when associated with PM), these proteins become overwhelmed, and since the body has no way of actively excreting iron, it is possible for afree pool to accumulate in the body. Such apool is an excellent target for Fenton chemistry (Halliwell and Gutteridge 1999), and the subsequent production of superoxide, hydrogen peroxide, and hydroxyl radicals, all of which have been associated with oxidativestress in the humanbody (see below).The Fenton reaction,inparticular the Haber–Weiss (superoxide-driven) form,has been implicated in the pulmonary toxicity of iron (Winterbourn1995; Turi et al. 2003). Fe 2 C O 2 / Fe 3 C O $ 2 K 2O $ 2 K C 2H/ H 2 O 2 C O 2 H 2 O 2 C Fe 2 C / Fe 3 C HO $ C OH In vivo,the rate of Fe-catalyzed radical production is critically dependent on the concentration of endogenous chealators, reducing agents, and O 2 concentration. Thus, in the lung, it is likelythat sometransitional metal-dependentradical production will occur prior to the sequestering of the exogenous Fe in transferrin and lactoferrin. It should be noted, however, that in the presence of biological reductants such as ascorbate, these reactions are cyclic with the ferriciron being reduced to its ferrous form, promoting further ROS production. Particle Toxicology98 © 2007 by Taylor & Francis Group, LLC [...]... Doelman, C J and Bast, A., Pro- and anti-oxidant factors in rat lung cytosol, Adv Exp Med Biol., 264, 455 , 1990 Donaldson, K and Tran, C L., Inflammation caused by particles and fibers, Inhal Toxicol., 14, 5, 2002 Donaldson, K and Tran, C L., An introduction to the short-term toxicology of respirable industrial fibers, Mutat Res., 55 3, 5, 2004 Donaldson, K et al., Asbestos-stimulated tumour necrosis factor... P Modified P Alveolar macrophage Anti-proteases anti-oxidant enzymes P Particle- Mediated Extracellular Oxidative Stress in the Lung Transition metals Blood FIGURE 5. 4 (See color insert) Particle- RTLF interactions Inhaled particles depositing in the airways may become trapped by the layer of mucus and transported from the airways by the mucocillary elevator Those particles that are retained in the airways... randomised, placebo-controlled crossover study, Lancet, 363, 119, 2004 Gilmour, P S et al., Detection of surface free-radical activity of respirable industrial fibers using supercoiled phi-x174 rf1 plasmid DNA, Carcinogenesis, 16, 2973, 19 95 Gilmour, P S et al., Pulmonary and systemic effects of short-term inhalation exposure to ultrafine carbon black particles, Toxicol Appl Pharmacol., 1 95, 35, 2004 Gong,... free radicals and ascorbate, Ann Rev Nutr., 5, 323, 19 85 Manoli, E et al., Profile analysis of ambient and source emitted particle- bound polycyclic aromatic hydrocarbons from three sites in northern Greece, Chemosphere, 56 , 867, 2004 Menon, P et al., Passive cigarette smoke-challenge studies: increase in bronchial hyper-reactivity, J Allergy Clin Immunol., 89, 56 0, 1992 Monn, C et al., Ambient PM(10) extracts... pulmonary disease, Thorax, 51 , 348, 1996 Rahman, I., Skwarska, E., and MacNee, W., Attenuation of oxidant/antioxidant imbalance during treatment of exacerbations of chronic obstructive pulmonary disease, Thorax, 52 , 56 5, 1997 Rahman, I et al., Systemic oxidative stress in asthma, COPD, and smokers, Am J Respir Crit Care Med., 154 , 1 055 , 1996a Rahman, I et al., Induction of gamma-glutamylcysteine synthetase... cells via ironmediated redox chemistry (Gilmour et al 19 95) 5. 6 EVIDENCE OF PARTICLE- INDUCED OXIDATIVE STRESS FROM ANIMAL STUDIES Convincing evidence to support a role for PM in eliciting adverse health effects comes from animal toxicology studies In vivo animal exposures to diesel exhaust particles (DEPs) (Sagai et al 1993; Ichinose et al 19 95) and a range of surrogate PM, such as carbon black (Li... Mol Biol., 25, 51 5, 2001 Borm, P J et al., Chronic inflammation and tumour formation in rats after intratrachial instillation of high doses of coal dusts, titanium dioxide and quartz, Inhal Toxicol., 12, 2 25, 2000 Brigelius-Flohe, R and Traber, M G., Vitamin E: function and metabolism, Faseb J., 13, 11 45, 1999 Britigan, B E., Serody, J S., and Cohen, M S., The role of lactoferrin as an anti-inflammatory... Taylor & Francis Group, LLC Particle- Mediated Extracellular Oxidative Stress in the Lung 1 05 particles interacting with both inflammatory and epithelial cells, or a function of the particle surface area, ultrafine particles may be highly effective for inducing adverse effects, irrespective perhaps of surface oxidants Indeed, Brown and colleagues (2000) have shown that ultrafine particles without transition... Tlr4 CD14 Fe(II) GSH AA GSH AA O2 H2O2 Epithelial cell R(n+1) O 2- SQ- R(n) RTLF ROS (IV) PAH CYP1A1 ROS electrophiles (V) Particle surface ROS NFκB Ap1 FIGURE 5. 5 Pathways of particle induced toxicity at the air–lung interface Particles elicit oxidative stress through five inter-related mechanisms: (I) Through the introduction of redox active metals such as iron, into the lung, which redox cycle in... this response is attenuated in transgenic mice over-expressing EC-SOD (Ghio et al 2002a) Furthermore, previous work has shown that elevated lung extracellular GSH concentrations in PM-challenged is due to an increased activity of c-glutamylcysteine synthase (Rahman 1996a, 1996b) Urate concentrations are also © 2007 by Taylor & Francis Group, LLC Particle- Mediated Extracellular Oxidative Stress in the . 96 5. 2.2 Mucins 96 5. 2.3 Enzymatic Antioxidant Defenses 96 5. 2.3.1 Glutathione Peroxidase 96 5. 2.3.2 EC-SOD 96 5. 2.3.3 Catalase 96 5. 2.4 Metal Chelation Proteins 97 5. 2.4.1 Transferrin 97 5. 2.4.2. 101 5. 3.1.4 Vanadium (V) 101 5. 3.1 .5 Nickel (Ni) 101 5. 3.1.6 Chromium (Cr) 102 5. 3.2 TheRole of Nonredox Active Metals 102 5. 3.2.1 Zinc (Zn) 102 5. 3.2.2 Aluminium (Al) 102 5. 3.2.3 Lead (Pb) 103 5. 3.3. 97 5. 2.4.3 Ferritin 97 5. 2.4.4 Caeruloplasmin 97 5. 3Induction of Oxidative Stress by Inhaled Particles 98 5. 3.1 TheRole of Redox Active Metals 98 5. 3.1.1 Iron (Fe) 98 5. 3.1.2 Copper (Cu) 100 5. 3.1.3