Particle Toxicology - Chapter 5 ppsx

If these reactions are numerous, they can causeextensive cellular damage, impaired cell functions, and in some cases, the influx of inflammatorycells to the sites of injury Freeman and Cra

5 Particle-Mediated Extracellular

Oxidative Stress in the Lung

Frank J Kelly and Ian S Mudway

Pharmaceutical Science Research Division, King’s College

CONTENTS

5.1 Health Effects of Particulate Matter and the Oxidative Stress Hypothesis 90

5.2 Antioxidant Defenses at the Air–Lung Interface 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

5.3 Induction of Oxidative Stress by Inhaled Particles 98

5.3.1 The Role 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 The Role 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 Oxidative Stress 103

5.3.5 Lipopolysacharide (LPS) Induced Oxidative Stress 104

5.3.6 Generation of ROS by Inflammatory Cells 104

5.4 The Role of Particle Size and Surface Area versus Composition as Determinants of PM Toxicity 104

89

5.5 Particle-Induced Toxicity: Lessons from Cigarette Smoke and Fiber Research 105

5.6 Evidence of Particle-Induced Oxidative Stress from Animal Studies 106

5.7 Concentrated Ambient and Diesel Exhaust 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–Lung Interface as a Predictor of Observed Health 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 political and scientific communities over the impact of airborne particulate matter (PM) on public health This concern has arisen primarily based on the findings of American epidemiological studies demonstrating an association between the mass concentration of PM (particularly particles with an aerodynamic diameter of less than 10 mm) 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 robust in numerous epidemiological studies worldwide (Brunekreef and Holgate 2002), with stronger associations usually associated with PM with an aerodynamic diameter of

!2.5 mm Moreover, the estimated decrease in life span associated with long-term residence in areas with high ambient PM is estimated to be between 1 and 2 years, which is large compared with other environmental risk factors Whilst the data demonstrating PM-induced health effects is irrefutable, major questions remain concerning the mechanisms by which these compositionally heterogeneous species elicit their toxic actions It has been argued that as exposure to a broad spectrum of particle types (vehicle emissions, cigarette and wood smoke PM, mineral dusts etc) elicits similar acute responses 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 a common mechanism Currently numerous groups are investigating the hypothesis that these common mechanisms may relate to the capacity of these particles to cause 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 lining fluid (RTLF) This compartment represents the first physical interface with which PM interacts, upon deposition in the airways A clear understanding of the initial reactions between

PM constituents and components of the RTLF is thus critical in understanding any observed toxicity

Oxidative stress is a relatively 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 a process which essentially involves the flow of electrons from one molecule to another within a biological setting The importance of this process lies in the reactivity of the molecules involved Under normal circumstances, electrons orbit around atoms in pairs, having opposite spins When an atom has a single unpaired electron, its reactivity increases markedly, and it is referred to as a free radical In a biological setting, free radicals are potentially very dangerous since they can react indiscriminately with neighboring molecules This process of “electron stealing” leads to oxidation of, for example, lipids, proteins, and nucleic acids, and as a consequence, altered

function or inactivation of these target molecules If these reactions are numerous, they can causeextensive cellular damage, impaired cell functions, and in some cases, the influx of inflammatorycells 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 thesespecialized molecules preferentially react with free radicals and give rise to products that oftenpossess low toxicity The damage arising from aberrant free radical activity is often loosely referred

to as oxidative stress which, in its simplest form is a potentially harmful process occurring whenthere is an excess of free radicals, a decrease in antioxidant defenses, or a combination of theseevents (Figure 5.1)

There is now a strong body of evidence demonstrating that disturbances in normal cellular andextracellular redox status in the lung, in response to PM exposure, can trigger inflammation via theactivation of redox-sensitive signaling pathways (Li et al 1996; Bonvallot et al 2001; Pourazar

et al 2005) The capacity of PM to elicit such a response has been explained by the delivery ofsurface 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 oxygenand nitrogen species (ROS and RNS) (Doelman and Bast 1990; Kelly 2003) The resultant airwayinflammation itself then results in an increased production of oxidants by activated phagocytes,recruited to the airways, perpetuating the cycle of oxidative injury In addition, ambient PM alsocontains appreciable concentrations of bacterial endotoxin that can also trigger inflammation

Antioxidant replenishment

Antioxidant losses Mucus

Organics Carbon core Transition metals

Inflammation ROS

by eliciting an inflammatory response resulting in a greater oxidative burden within the RTLF, whichultimately leads to cell death and tissue injury

Particle-Mediated Extracellular Oxidative Stress in the Lung 91

5.2 ANTIOXIDANT DEFENSES AT THE AIR–LUNG INTERFACE

Owing to its function, large surface area and exposure to high partial pressures of oxygen, the lung

is particularly susceptible to oxidative injury It is therefore logical that a robust extracellularantioxidant defense system exits to protect against undue oxidation of its delicate pulmonaryepithelial cells On entering the lung, ambient pollutants do not come into direct contact withthe respiratory epithelium, but rather they first interact with a liquid layer that covers the respiratoryepithelium from the nasal mucosa to the alveoli, the RT4 (Figure 5.2) Human RTLF is a complexand regionally heterogeneous compartment, ranging in depth from between 1 and 10 mm in theproximal airways to 0.2–0.5 mm in the distal airways and alveoli (Cross et al 1998) It consists of

Distal airways and alveoli

Transferrin Caeruloplasmin

Lactoferrrin Transferrin Caeruloplasmin

ecCuZn SOD ecGSHPx

CYS

UA

UA

AA A-Toc

secretions from underlying lung and resident immune cells, as well as plasma-derived exudates Inthe nasal and proximal airways, it exists as a two-phase structure consisting of a gel and sol phase,the former consisting of thiol-rich mucopolypeptide glycoprotein (mucins) This contrasts with thedistal airway and alveolar lining fluids, which are devoid of mucins, but contain surfactant lipidsand proteins Nasal, proximal, and distal lavage procedures have allowed a detailed examination ofthe RTLF in different lung compartments In addition to the mucin and surfactant components, theRTLF also contains a broad spectrum of low molecular weight antioxidants, as well as small

5.2.1 SMALLMOLECULARWEIGHTANTIOXIDANTS

An array of small molecular weight antioxidants has been measured in human RTLF, includingascorbate (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 (vitamin E) (Mudway et al 2001) As the lavageprocedure used to sample the airway introduces a large and variable dilution, the concentration ofthese antioxidants quoted in the literature are often difficult to interpret Whilst many groups simplyquote concentrations in the recovered lavage, others have made attempts to correct for the dilution,using a range of methods based on instilled dyes or endogenous dilution markers (Haslam andBaughma 1999) Of these, the most commonly adopted is the urea correction method that uses theratio of urea concentrations between the lavage sample and plasma as the basis for estimating thedilution This technique is often criticized because the instillation of a large volume of saline creates

a massive concentration gradient along which urea can move from cellular and interstitial sources.One way of limiting this potential problem is to reduce the lavage dwell time and not to performrepeat sampling of the same airway segment, as is often performed in lavage procedures The RTLFantioxidant concentration ranges quoted in this review will be based on values obtained using thesemethods, unless otherwise stated

5.2.1.1 Ascorbate

As a consequence of its high solubility, ascorbate is thought to diffuse freely between aqueous extraand intracellular compartments of the body including the RTLF, where concentrations(50–150 mM) comparable to those in plasma have been measured (Kelly, Buhl, and Sandstro¨m1999; van der Vliet 1999) In humans, this antioxidant vitamin originates solely from dietarysources in humans owing to the lack of the enzyme, gluconlactone oxidase, necessary for itsbiosynthesis (Halliwell and Gutteridge 1999) Differences in dietary intake may thereforeexplain the variability in ascorbate concentrations that have been measured in bronchoalveolarlavage (BAL) fluid from different individuals (Kelly, Buhl, and Sandstro¨m 1999) Ascorbate is anexcellent reducing agent and scavenges a variety of free radicals and oxidants in vitro, includingsuperoxide and peroxyl radicals, hydrogen peroxide, hypochlorous acid, and singlet oxygen.During its antioxidant activity, ascorbate readily undergoes two consecutive, but reversible, one-electron reductions The first one-electron reduction produces the semi-dehydroascorbate radical

undergo a further one-electron reduction to dehydroascorbate (DHA) The ascorbyl radical isrelatively unreactive, owing to its unpaired electron being in the delocalized p-system, and inthe absence of a further oxidation, two ascorbyl radicals will undergo a disproportionate reaction

to regenerate one molecule of ascorbate and one molecule of DHA The DHA, once formed, rapidly

poten-tially cytotoxic in significant concentrations, and therefore cells possess enzymes that convert eitherSDA or DHA back to ascorbate at the expense of GSH or NADPH (Diliberto et al 1982; Park andParticle-Mediated Extracellular Oxidative Stress in the Lung 93

Levine 1996) In addition, glutathione has been shown to recycle DHA nonenyzmatically (Winkler,Orselli, and Rex 1994) in vitro, although at a rate insufficient to prevent substantial losses of DHA.Little is currently understood about the turnover of ascorbate within the RTLF as to date, onlyascorbate and dehydroascorbate have been routinely measured At present, it is not clear whethersuch regenerative mechanisms exist in the RTLF, but certainly the 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 ascorbate within the RTLF would constitute asignificant drain on bodily stores of this antioxidant Many cell types, including neutrophils, areable to take up DHA rapidly via the facilitative glucose transporters GLUT1 and GLUT3 (Rumsey

et al 1997) Once internalized, the DHA is rapidly reduced back to ascorbic acid by glutaredoxin atthe expense of glutathione (Park and Levine 1996) Thus, it may be possible that DHA formed inthe RTLF under normal or pathological conditions may be reduced back to ascorbate by cellularuptake by the epithelium (Nualart et al 2003)

In addition to having a direct scavenging action, ascorbate also acts indirectly to prevent lipidperoxidation through its reaction with the membrane bound a-tocopherol radical In vitro studieshave demonstrated that in this way, ascorbate is able to reduce the a-tocopherol radical, therebyregenerating the vitamin E molecule (Packer, Slater, and Wilson 1979; Niki, Yamamoto, andKamiya 1985; Doba, Burton, and Ingold 1985) This synergistic action has not yet however beenreported in vivo This potential interaction, together with the recycling of DHA by glutathione,

Whilst ascorbate represents a critical protective component of the RTLF, it also has the

formation of the damaging hydroxyl radical in the presence of hydrogen peroxide

This situation occurs rarely under normal physiological conditions, as both Fe and Cu aresequestered into transport and storage proteins to limit these potentially damaging reactions.5.2.1.2 Urate

Urate is an oxidized purine base present in all RTLF compartments of the lung, but at particularlyhigh concentrations in the nasal and proximal airways (200–500 mM) where it appears to be thepredominant antioxidant defense (Cross et al 1994; Blomburg et al 1998) In the nasal airways itappears to be secreted from gland cells along with lactoferrin (Peden et al 1990, 1993), but little isknown about its transport into the lower airways This antioxidant can directly scavenge hydroxylradicals, oxyhaem oxidants formed between the reactions of hemoglobin and peroxy radicals,peroxyl radicals themselves, and singlet oxygen (Becker 1993) In these reactions it acts in asacrificial mode, in that it is irreversibly damaged to produce a range of oxidation products,including allantoin (Ames et al 1981) As with ascorbate, relatively little is known about theturnover of urate in the RTLF In addition to its role as a free radical scavenger, urate has alsobeen reported to chelate iron and protect against iron-mediated free radical damage of ascorbate andlipids (Davies et al 1986; Ghio et al 1994) Increased urate concentrations have been reported inrats challenged with iron salts and silica-iron in concert with increased lung xanthine oxido-reductase activities This would imply that elevated RTLF urate might reflect a regulatedadaptive response to limit Fe-induced oxidative injury (Ghio et al 2002)

5.2.1.3 Reduced Glutathione

High levels of glutathione (100–200 mM) have been reported in proximal airway and alveolarRTLFs from healthy individuals, with some reports suggesting concentrations as high as 400 mM(Cantin et al 1987) This is around 100 times higher than normal plasma concentrations (Kelly and

The source of GSH has not been established, although in the light of low concentrations in plasmaand poor re-adsorption from the respiratory tract, it is likely that this antioxidant is produced via asecretary mechanism from the cells in the lung (Smith, Anderson, and Shamsuddin 1992) As well

as acting as a substrate for the glutathione redox cycle, glutathione is known to react with a widerange of compounds in vitro including hydroxyl radicals, hypochlorous acid, peroxynitrite,peroxyl radicals, carbon centred radicals, and singlet oxygen In its reactions with free radicals,thiyl radicals are produced, which can subsequently be converted to oxidized glutathione through aradical transfer process (Halliwell and Gutteridge 1999) Glutathione is particularly good atdefending against oxidants such as hypochlorous acid and hypobromous acid (Winterbourn1985), which are released from neutrophils and eosinophils, respectively The presence of thisantioxidant in lung lining fluid may therefore be particularly important in defending the extra-cellular surface of the lung against activated inflammatory cells

NADP

GSH

GSH GSSG

GSSG GSSG reductase

Glutaredoxin

GLUT1/3 ROOHROH

ROOHROH

UA

UA SDA

SDA

ROO-

RO-FIGURE 5.3 Cooperativity of RTLF antioxidants Peroxyl (ROO%) and alkoxyl (RO%) lipid radicals formedthough the oxidation of polyunsaturated fatty acids (PUFA) can be reduced to harmless alcohols (ROH,ROOH) through the scavenging action of membrane-bound a-tocopherol The tocopherol radical can bere-reduced at the expense of ascorbate (AA), which is converted to the ascorbyl radical (SDA) and hence todehydroascorbic 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 knownwhether a similar DHA reductase activity exists within the RTLF, but GSH can recycle DHA to a limitedextent in an non-enzyme catalyzed mechanism Urate (UA) can also scavenge ROO%and RO%Radicals, andthe redox potential of the urate radical suggests that UA may also be recycled by ascorbate

Particle-Mediated Extracellular Oxidative Stress in the Lung 95

5.2.1.4 a-Tocopherol

RTLF a-Tocopherol is believed to be derived from type II cells (Rustow et al 1993) and is present

at relatively low concentrations within lung lining fluid It is, however, a powerful antioxidant, both

in terms of scavenging free radicals such as singlet oxygen, alkoxy radicals, peroxynitrite, nitrogendioxide, ozone, and superoxide (Wang and Quinn 1999) and its ability to terminate lipidperoxidation (Witting 1980; Burton and Ingold 1981; Niki, Takahashi, and Komuro 1986;Nakamura et al 1987) It is thought that the reactivity of a-tocopherol with organic peroxyl radicalsaccounts for the majority of its biological activity (Witting 1980; Burton and Ingold 1981), areaction that yields a relatively stable lipid hydroperoxide and a vitamin E radical, thereby effec-tively interrupting the lipid peroxidation chain reaction (McCay 1985)

5.2.2 MUCINS

In addition to providing a physical barrier and clearance mechanism to remove inhaled toxins,mucin components of the gel phase of the upper airway RTLF also have significant antioxidantproperties, by virtue of their cysteine-rich domains and carbohydrate moieties (Cross, Halliwell,and Allen 1984; Hiraishi et al 1993) Mucins have been shown to have metal binding properties(Cooper, Creeth, and Donald 1985), as well as the capacity to scavenge hydroxyl radicals (Cross,Halliwell, and Allen 1984) In addition, the thiol moieties imply that mucins should also provide

5.2.3 ENZYMATICANTIOXIDANTDEFENSES

RTLF also contains antioxidant enzymes, whose role it is to scavenge oxidants or to repair damagecaused by ROS These enzymatic antioxidants are a complex set 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 a selenoprotein that catalyzes the reduction of hydrogen peroxideand lipid peroxides, at the expense of glutathione (Brigelius-Flohe´ and Traber 1999) Of the fourtypes of GPx, namely cellular or cytosolic or classical GPx, gastrointestinal GPx, extracellular orplasma GPx, and phospholipid hydroperoxide GPx (Takebe et al 2002), Avissar et al (1996) hasshown that RTLFs contains selenium-dependent cellular GPx and extracellular GPX, each contri-buting approximately 50% 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 mocytes (Oury et al 1996), and can be found in the RTLF (Mudway and Kelly 2000) and airwayepithelial cell junctions EC-SOD catalyzes the dismutation of the superoxide free radical tohydrogen peroxide thus in concert with glutathione peroxidase, protects lung interstitium againstfree radical generated during inflammation (Oury et al 1996)

5.2.4 METALCHELATIONPROTEINS

Metal-chelating proteins present in the RTLF also perform an important antioxidant function bybinding free transition metals, thus preventing them from participating in potentially damagingredox 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 shown to be a potent inhibitor of lipid peroxidation in vivo (Pacht andDavis 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 occurs at pH!4.5 This is pertinent toparticle-association metal chemistry in the RTLF, in that the pH of RTLF from healthy individualshas been reported to be between 7 and 7.5, but in certain disease states, such as asthma, the pH isapproximately pH 5 (Hunt et al 2000) One might speculate, therefore the chelation of iron bytransferrin to be sub-optimal in asthmatics, this would increase the presence of unbound iron andmay, in turn, contribute to the low levels of RTLF antioxidants seen in asthmatics (Kelly, Buhl, andSandstro¨m 1999; Mudway and Kelly 2000)

5.2.4.2 Lacoferrin

Lacoferrin is able to bind and transport iron and release it again at specific receptor cells in thehuman intestine High concentrations have been reported in the upper airways, whilst studies haveshown that concentrations are increased in the lower respiratory tract of chronic bronchitics(Thompson et al 1990) Like transferrin, lactoferrin is a potent inhibitor of lipid peroxidation

in vivo (Pacht and Davis 1988) Lactoferrin can bind free iron with high affinity and thus function

as a local antioxidant, protecting the immune cells against the free radicals produced by them(Britigan, Serody, and Cohen 1994) Furthermore, reports suggest that lactoferrin can bind free ironreleased from dying cells, thereby protecting the phagocytic cells, as well as adjacent tissues, fromROS produced from the Haber–Weiss reaction (Britigan, Serody, and Cohen 1994)

5.2.4.3 Ferritin

Ferritin controls the storage of iron within the body, and thus can also provide protection againstiron-generated ROS Concentrations of ferritin in alveolar cells and on the alveolar surface areincreased in patients with a variety of respiratory disorders (Stites et al 1999) and it is likely that thepresence of ferritin in the RTLF reflects the release of cell contents from dying cells A number ofinflammatory mediators can also induce intracellular up-regulation, as well as the cleavage of ironfrom 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 cysticfibrosis as a consequence of mobilizing iron

5.2.4.4 Caeruloplasmin

Caeruloplasmin is a glycoprotein involved in serum copper transport It represents the main oxidant defense against copper in human plasma, but also promotes the incorporation of iron intotransferrin without the formation of toxic iron products (Koc et al 2003) It is primarily synthesized

anti-in the liver and secreted to the blood; however, recent studies have identified the lung as anothermajor site of synthesis In particular, Yang et al (1996) have suggested that the airway epithelialcells are the major source of ceruloplasmin identified in RTLF

Particle-Mediated Extracellular Oxidative Stress in the Lung 97

5.3 INDUCTION OF OXIDATIVE STRESS BY INHALED PARTICLES

Particulate matter is a complex mixture of chemical components in terms of their chemical tion, dependant on the emission source and in combustion scenarios, the type of fuel being burnt.For example, ultrafine particles from sources of combustion generally comprise a carbonaceouscore with absorbed substances condensed onto the surface during combustion and atmosphericprocesses 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

composi-as bacterial endotoxin, composi-as well composi-as sulphate, nitrate, chloride, and ammonium The compositiondictates the surface reactivity of the particles, an important factor in determining particle toxicity(Fubini 1997) and its adverse health effects Whether these PM components ultimately result insubstantial oxidative damage, inflammation, and injury ultimately depends on their initialinteractions with the antioxidant defenses within the RTLF These potential interactions are

stress, with low-level oxidative stress resulting in an up-regulation of endogenous extra- andintracellular responses, prior to the induction of substantial toxicity (Li et al 2002) The various

5.3.1 THEROLE OF REDOXACTIVEMETALS

Redox-active metals are defined as those containing unpaired electrons in their d-orbital, and arecapable of generating free radical species via redox cycling mechanisms with biological reductants.Using this definition, the entire first row elements in the d-block of the periodic table, with theexception of zinc, qualify: Sc, Ti, V, Cr, Mn, Fe, Co, Ni, and Cu Of these, the most commoncomponents of PM are listed below

5.3.1.1 Iron (Fe)

form in the storage protein ferritin, or is associated with transport or receptor proteins, therebyfacilitating the removal of reactive iron from areas including the respiratory tract In the presence ofexcess iron (for example, when associated with PM), these proteins become overwhelmed, andsince the body has no way of actively excreting iron, it is possible for a free pool to accumulate inthe body Such a pool is an excellent target for Fenton chemistry (Halliwell and Gutteridge 1999),and the subsequent production of superoxide, hydrogen peroxide, and hydroxyl radicals, all ofwhich have been associated with oxidative stress in the human body (see below) The Fentonreaction, in particular the Haber–Weiss (superoxide-driven) form, has been implicated in thepulmonary toxicity of iron (Winterbourn 1995; Turi et al 2003)

2K

2O$

In vivo, the rate of Fe-catalyzed radical production is critically dependent on the concentration

some transitional metal-dependent radical production will occur prior to the sequestering of theexogenous Fe in transferrin and lactoferrin It should be noted, however, that in the presence ofbiological reductants such as ascorbate, these reactions are cyclic with the ferric iron being reduced

to its ferrous form, promoting further ROS production

Mucus

Transition metals Carbon core Organics

Transition metals Carbon core Organics

Nitration Oxiadtion

Oxidised products LOPs

Altered extrzcellur redox

P

P P P

P

P LLL L P P

P

L

L A A

L A A

P P

P L L P

A

P

P P P P P P P

P P P

P

P P

P

P P P

P

P

AP P P

FIGURE 5.4 (Seecolor insert) Particle-RTLF interactions Inhaled particles depositing in the airways may become trapped by the layer of mucus and transported fromthe airways by the mucocillary elevator Those particles that are retained in the airways can either leach soluble components into the RTLF that oxidize antioxidants, lipid,and protein components of the RTLF, or absorb the oxidized or native RTLF components onto their surface These interactions therefore alter both the redox state of theRTLF, which will impact upon the underlying cells, as well as modifying the particle surface that ultimately reaches the underlying epithelium, or that is intercepted byalveolar macrophages within the extracellular compartment

H2O2ROS

2 Þ, hydrogen peroxide (H2O2), and finally the damaging hydroxyl radical (%OH).(II) Quinones on the particle surface can also redox cycle in the presence of biological reductants to form thesemi-quinone radical (SQ%K) that will also yield superoxide and hydrogen peroxide Both of these pathwayswill result in the loss of extracellular antioxidants and hence, an altered, at least transiently, extracellular redox.(III) Bacterial endotoxin associated with the particle surface has been shown to trigger inflammation throughits interaction with the TLr4/CD14/MD2 receptor, which further adds to the oxidative burden in the airways.(IV) Polyaromatic hydrocarbons (PAHs) have no intrinsic oxidative activity, but they can undergo biotrans-formations intracellularly through the action of P450cyp1A1 to form reactive electrophiles and reactiveoxygen species (ROS) (V) The particle surface itself has been shown to cause oxidative stress in vivo,though the mechanism by which this occurs is not well defined Overall then exposure to inhaled particlesresults in losses of extra- and intra-cellular antioxidants, especially ascorbate and glutathione This alteredredox state results in the upregulation of redox sensitive signalling partway and transcription factors (NFkBand AP-1) leading to the increased production of cytokines and the development of airway inflammation

much more likely to drive damaging oxidations reactions within the RTLF Furthermore, Cu hasbeen shown to be a much more effective catalyst of radical production than any of the other major

participate in a redox cycling reaction that produces hydrogen peroxide (and benzoquinone) viaoxidation of hydroquinone Reactive oxygen species produced in this manner have been linked toplasma DNA damage and cleavage (Li et al 1995)

5.3.1.4 Vanadium (V)

Vanadium represents one of the key metal components of PM, and is derived from the burning offuel oil, along with Fe and Ni PM pollution generated from oil powered power stations has been amajor environmental issue in the United States, and as such, a considerable body of work has beenperformed addressing the toxicity of residual oil fly ash (ROFA) and its V component V has severaloxidation states, V(V), V(IV), V(III), and V(II), of which the 5C form is the most common Theproduction of hydroxyl radicals by vanadate has been demonstrated by ESR during co-incubationswith liver microsomes (Shi and Dalal 1992) which appear to occur through Fenton-like chemistryrather than the Haber–Weiss reaction (Shi and Dalal 1993)

V(IV) has also been shown to promote the decomposition of lipid peroxides The production ofROS by V(V) can also be promoted by its reduction to V(IV) by cellular reductants, such asascorbate and glutathione, thus raising the possibility that the reaction between PM V and anti-oxidants within the RTLF may actually sponsor, rather than inhibit, radical production inthis compartment

5.3.1.5 Nickel (Ni)

Excess nickel is a well-documented carcinogen in humans, especially when inhaled as insolubleparticulates such as nickel subsulphide (Kasprzak 1991) Despite evidence that nickel compoundscause the oxidation of lipids (Athar, Hasan, and Srivastava 1987; Stinson et al 1992), proteins(Zhuang, Huang, and Costa 1994) and nucleic acids (Stinson et al 1992; Lynn et al 1997),intracellular radical production (Huang, Klein, and Costa 1994), and intracellular glutathionedepletion (Rodriguez et al 1991; Herrero et al 1993; Li, Zhao, and Chou 1993), there is little

Particle-Mediated Extracellular Oxidative Stress in the Lung 101

This ligand-dependent Ni2C/Ni3C redox cycling of nickel results in the formation of oxygenradicals via Fenton-type reactions (Klein, Frenkel, and Costa 1991; Lin, Zhuang, and Costa 1992).

induce the release of reactive oxygen species from phagocytic cells (Costa and Mollenhauer 1980).These insoluble forms of Ni have been shown to be more carcinogenic than soluble nickel and

in vitro studies have demonstrated that they induce greater intracellular free radical production thansoluble nickel salts (Huang et al 1994a, 1994b)

5.3.1.6 Chromium (Cr)

yield the hydroxyl radical (Galaris and Evangelou 2002)

responsible for eliciting DNA strand breaks (Stohs and Bagchi 1995)

5.3.2 THEROLE OF NONREDOXACTIVEMETALS

In addition to transition metals, particulate air pollution can contain nonredox active metals, whichcan influence the toxic effects of transition metals, either exacerbating or lessening the production

of free radicals

5.3.2.1 Zinc (Zn)

Although the literature regarding the oxidative potential of zinc is rather ambiguous, the generalconsensus from in vitro studies is that zinc acts in an antioxidant capacity For example, zinc

oxidation product (Zago and Oteiza 2001)—whilst Chevion et al 1990, showed that excess zincinhibits the toxicity of paraquat through a mechanism involving the displacement of copper from itsbinding sites, inhibiting free radical production at that site Zinc has also been seen to interactfavorably with negatively charged phospholipids, occupying potential binding sites, preventing thebinding of redox-active metals, and thereby blocking the initiation or propagation of lipidoxidation Evidence does exist, however, that zinc can catalyze iron- and copper-induced oxidation(Zago and Oteiza 2001), presumably by displacing iron and copper atoms from chelators andmaking them more bioavailable to catalyze free radical generation

avai-lability and concentration of iron, and thus potential free radical formation (Zatta et al 2002)