The Role of Reactive Oxygen Species
and Oxidative Stress in Mediating
Particulate Matter Injury
Tian Xia, MD, PhDa,b, Michael Kovochich, BSa,b,
Andre Nel, MD, PhDa,b,*
aDivision of Clinical Immunology and Allergy, Department of Medicine,
University of California Los Angeles, 52-175 CHS, 10833 Le Conte Avenue,
Los Angeles, CA 90095-1680, USA
bSouthern California Particle Center, 650 Charles E. Young Drive South,
Box 951772, Los Angeles, CA 90095-1772, USA
Several plausible mechanisms have been proposed to explain the adverse
health effects of particulate matter (PM) in polluted ambient air . To date,
experimental support has been provided for the role of local and systemic
inflammation, cytokine and chemokine production, increased bone marrow
production of myeloid lineage cells, free oxygen radical production in the
chest, endotoxin-mediated cellular and tissue responses, stimulation of irri-
tant receptors, and covalent modification of key cellular enzymes [1,2]. Best
characterized in humans are the effects of PM on airway inflammation .
Several human and animal studies have shown that inhalation of diesel
exhaust particles (DEP), a model particulate pollutant, and concentrated
ambient particles elicit proinflammatory effects, cytokine production, and
enhancement of allergic responses in the upper and lower airways [2–4].
The mechanistic link between the PM exposure and inflammation depends
Support for this article provided by US Public Health Service grants U19AI 070453
(UCLA Asthma and Allergic Disease Clinical Research Center), RO1 ES10553 (National
Institute of Environmental and Health Science), RO1 ES15498, RO1 ES13432, and RO1
ES10253 and the US Environmental Protection Agency STAR award (RD-83241301) to the
Southern California Particle Center and Supersite. This work has not been subjected to the
Environmental Protection Agency for peer and policy review and does not necessarily reflect
the views of the agency. No official endorsement should be inferred.
* Corresponding author. Division of Clinical Immunology and Allergy, Department of
Medicine, University of California Los Angeles, 52-175 CHS, 10833 Le Conte Avenue, Los
Angeles, CA 90095-1680.
E-mail address: firstname.lastname@example.org (A. Nel).
1526-0046/06/$ - see front matter ? 2006 Elsevier Inc. All rights reserved.
Clin Occup Environ Med
5 (4) 817–836
on generation of reactive oxygen species (ROS) and oxidative stress [2,5–7].
chemical compounds and transition metals play a role in ROS production
[8,9]. The large reactive surface area of ambient ultrafine particles (UFP)
lial cells and macrophages also generate ROS in response to particle compo-
nents by involving biologic processes [2,6,8].
ROS are capable of damaging key cellular components. To defend against
this damage, cellsuse glutathione (GSH)andotherhigh molecular weight an-
tioxidants to inactivate oxygen radicals and protect cellular components
against damage. In the setting of overwhelming ROS production, GSH de-
pletion can induce a state of redox stress (ie, oxidative stress in the cell)
[2,11]. Oxidative stress acts as a trigger that initiates a series of cellular re-
sponses, which can either be protective (eg, induction of antioxidant en-
zymes) or injurious in nature . In this article we discuss the mechanisms
and particle characteristics that contribute to ROS production and oxidative
stress, including the experimental and clinical evidence that oxidative stress
leads to clinically relevant disease. Finally, we review the biology of oxidative
stress to explain the pathogenesis of PM-induced adverse health effects.
Evidence that reactive oxygen species and oxidative stress are involved
in particular matter–induced injury
diovascular system. Pulmonary effects include small airway inflammation,
which can lead to the exacerbation of asthma and chronic bronchitis, airway
obstruction, and decreased gas exchange [2,13,14]. PM also can interfere with
the clearance and inactivation of bacteria in the lung, which can lead to respi-
diovascular effects can be explained by enhanced atherogenesis and by sys-
temic proinflammatory effects that impact the endothelium and blood
cular and pulmonary adverse health effects is PM-induced inflammation. We
discuss the possibility that these proinflammatory effects constitute an oxida-
tive stress response that is related to the ability of PM to induce ROS genera-
tion in specific airway and vascular target tissue.
In vitro evidence for the ability of PM to induce oxidative stress has
emerged from several studies using DEP as a model air pollutant. Exposure
of macrophages and bronchial epithelial cells, two of the principal targets
for PM in the lung, to DEP leads to increased H2O2and superoxide (O2?? )
production in a time- and dose-dependant manner [16–18]. The kinetics of
ROS generation in macrophages exhibit two phases, namely an early phase
XIA et al
of mostly H2O2production, followed by a later phase of O2??production
[16,17]. These phases could reflect different biologic events, as is discussed
later. Intact particles and their methanol extracts can induce ROS produc-
tion, which is accompanied by GSH depletion and induction of oxidative
stress [17–19]. N-acetylcysteine (NAC), a thiol antioxidant, interferes in
these cellular responses [7,20], further substantiating the role of oxidative
stress in PM injury. Studies involving ambient concentrated particles, in-
cluding ambient UFP, show similar effects on ROS production, GSH deple-
tion, and induction of oxidative stress responses [8,21,22].
In vivo evidence for the ability of PM to induce ROS production and
oxidative stress has been provided by using chemiluminescence sensors
[21,22]. Using photomultiplier techniques, researchers demonstrated that
H2O2production increases in a time- and dose-dependent manner over
the mediastinum of rats receiving concentrated ambient particles [21,22].
This effect was accompanied by decreased heart rate variability and the gen-
eration of thiobarbituric reactive substances in that organ [21,22]. These
effects were inhibited by NAC, which further suggested the importance of ox-
idative stress after PM exposure [21,22]. Another approach involves using
electron spin resonance to detect ROS generation on particles and in biologic
tissue that come into contact with particles [19,23]. This approach includes
the detection of oxygen radicals by spin trap markers in the bronchoalveolar
lavage fluid of rats receiving intratracheal DEP . Indirect evidence for the
role of oxidative stress in vivo comes from studies that examine the adjuvant
effects of DEP in a murine model of allergic inflammation and asthma .
These adjuvant effects include increased ovalbumin-specific IgE production
in mice exposed to DEP plus ovalbumin . The thiol antioxidants, NAC
and bucillamine, were found to be effective inhibitors of these pro-oxidative
and adjuvant effects of DEP . Research also has shown that treatment
of mice with DEP increased nitric oxide (NO) production and NO syn-
thase (iNOS) expression [6,25]. NO can combine with O2?? to generate
the peroxynitrite (ONOO?) radical, which can further damage the airway
epithelium . Pretreatment with N-G-monomethyl L-arginine, an iNOS
inhibitor, significantly reduced the airway hyperresponsiveness induced by
There is limited evidence for the production of oxygen radicals and oxi-
dative stress after PM exposure in humans. One study showed increased CO
levels in the exhaled air of human subjects after exposure to DEP, likely
reflecting the induction of heme oxygenase (HO-1) expression as a sensitive
oxidative stress response protein . HO-1 is an enzyme that is responsible
for the catabolism of heme, leading to the generation of CO and biliverdin
as catalytic products of that reaction . NO in the expired air also has
been shown to be a sensitive marker for assessment of inflammatory lung
diseases . Several human studies have shown that various components
of air pollution are associated with increased levels of NO in exhaled air
ROS AND OXIDATIVE STRESS IN MEDIATING PM INJURY
Particle components, including absorbed particulate matter chemicals,
are responsible for spontaneous particulate matter–induced reactive
oxygen species production
The mechanisms by which ambient PM generates ROS include biologic
events and particle-induced redox chemistry. These events are key to under-
standing disease pathogenesis and PM toxicity. Although there has been
much debate about whether the particles or their absorbed chemicals are re-
sponsible for PM injury, it is our view that the particles and their absorbed
chemicals play important roles in ROS production. This notion is derived
from the observations that PM (1) contains organic and inorganic chemical
compounds that are capable of ROS generation [8,32], (2) can spontane-
ously generate O2??and hydroxyl radicals (?OH) in the absence of a biologic
catalyst [8,33], and (3) size and reactive surface area are independent phys-
ical variables that influence PM ability to generate oxidative stress and
inflammation in experimental animals (Table 1) [8,10].
Emerging evidence indicates that particle size, surface area, and chemical
composition are the chief characteristics that determine PM health risks .
Based on size, PM can be classified into coarse, fine, or ultrafine particles
(Table 1) . Coarse particles, which have an aerodynamic diameter of
2.5 to 10 mm, are mostly derived from soil and sea salts. Fine particles,
which range in diameter from 0.1 to 2.5 mm, and UFP, with diameters
!0.1 mm, are predominantly derived from fossil fuel combustion processes.
Combustion particles are comprised of a carbonaceous core that consists of
elemental carbon, which is coated with a layer of chemicals, including or-
ganic hydrocarbons, metals, nitrates, and sulfates [11,34]. Although several
chemical components could contribute to particle toxicity, the principal
components that are responsible for ROS production are transition metals
and redox cycling organic chemical compounds [12,18,35].
Characterization of different sized particles
Coarse (PM10)Fine (PM2.5)Ultrafine
Carbon content (elemental)
Carbon content (organic)
Redox activity (DTT assay)
Abbreviations: DTT, dithiothreitol; PAH, polycyclic aromatic hydrocarbons.
Data from Li N, Sioutas C, Cho A, et al. Ultrafine particulate pollutants induce oxidative
stress and mitochondrial damage. Environ Health Perspect 2003;111(4):455–60; Donaldson K,
Tran CL. Inflammation caused by particles and fibers. Inhal Toxicol 2002;14(1):5–27.
XIA et al
Transition metals, such as iron, copper, vanadium, chromium, nickel,
and cobalt are responsible for the conversion of H2O2into?OH by the Fen-
Fe2þþ H2O2/Fe3þþ?OH þ OH?
tive stress in vitro and in vivo [36,37]. Residual oil fly ash contains high con-
centrations of transition metals and is often used as a model for the
induction of ROS production and inflammation by transition metals. For
instance, it has been shown that residual oil fly ash can activate mitogen-
activated protein kinases and induce the expression of interleukin-8, interleu-
kin-6, and tumor necrosis factor-a by a chelator-sensitive pathway . The
induction of inflammation in the rat lung and proinflammatory cytokines in
human bronchial epithelial cells by ambient PM also can be reduced by treat-
ment with metal chelators . It is also interesting that the metal content of
ity to induce oxidative stress .
outgrowth of the observation that methanol extracts made from DEP are
capable of mimickingthe pro-oxidative andproinflammatory effects of intact
particles [18,38]. Chemical fractionation of organic DEP extracts by silica gel
chromatography, using the elution principle of increasing polar solvents, has
defined two major chemical groups that contribute to ROS production and
oxidative stress [38,39]. The first is aromatic compounds, including polycyclic
aromatic hydrocarbons (PAHs). PAHs are cyclical compounds that contain
two to seven phenyl rings (Fig. 1). In the presence of cellular targets, such as
bronchial epithelial cells, PAHs are capable of inducing oxidative stress after
diol dehydrogenase . These enzymes convert PAHs to redox cycling qui-
nones (see later discussion). Organic DEP extracts have been shown to
induce the expression of cytochrome P450 1A1 in bronchial epithelial cells
and rat lungs . The specific PAH profile of ambient PM varies with com-
bustion source (eg, heavy duty and light duty diesel engines preferentially
emit low and high molecular weight PAHs, respectively) . This profile is
of environmental importance because PAHs are semivolatile substances
that can be released and repartition to the particle surfaces, depending on
the ring size and environmental temperature . Ambient UFPs, which to
a large extent are derived from diesel exhaust emissions, exhibit a higher
PAH content in the winter versus the summer months, which correlates
with the increased propensity of ambient PM collected in winter months to
generate oxidative stress . Among the ambient particles collected in the
Los Angeles basin, UFPs have a higher PAH content than coarse and fine
erate oxidative stress in epithelial cells and macrophages . Molecular
ROS AND OXIDATIVE STRESS IN MEDIATING PM INJURY
epidemiologic studies in environmentally exposed populations also have
tions, formation of DNA adducts, and intrauterine growth retardation
The second major class of organic chemical compounds that could con-
tribute to PM-induced ROS production is polar substances, including oxi-
dized forms of PAH (eg, ketones and quinones) [11,38]. The biologic
mechanism by which quinones contribute to ROS generation is described
in the section on biologic mechanisms.
In addition to providing a backbone for the adsorption of chemicals, the
particle surface area and size are independent variables that determine ROS
production and excitation of airway inflammation [47,48]. The same particle
characteristics are also important in determining the pro-oxidative and
proinflammatory effects of mineral dust particles and the generation of air-
way inflammation by experimental metal oxide particles [47,48]. There are
several possible ways to explain these findings, including the fact that parti-
cle size is intimately linked to surface area. Table 2 shows the physical char-
acteristics of a cloud of airborne particles of varying size but with a fixed
mass of 10 mg/m3. These calculations show that as the particle size
Fig. 1. Representative pro-oxidative organic chemical compounds, PAHs, and quinones. (Mod-
ified from Li N, Hao M, Phalen RF, et al. Particulate air pollutants and asthma: a paradigm for
the role of oxidative stress in PM-induced adverse health effects. Clin Immunol 2003;109(3):254;
XIA et al
decreases, the number of particles per unit air volume (1 cc) increases in ex-
ponential fashion, along with an exponential increase in the surface area.
The increase in surface area places a larger number of potentially reactive
chemical groups on the particle surface. This finding is of particular rele-
vance to particles in the nano-size range (ie, UFPs), because the number
of surface molecules is inversely related to the particle size. For instance,
in a particle of 30-nm size, approximately 10% of the molecules are ex-
pressed on the surface, whereas at 10 and 3 nm, the ratios increase to
20% and 50%, respectively. The number of atoms or molecules on the par-
ticle surface determines characteristics such as electron storage and transfer
to molecular dioxygen (O2). At this stage it is unknown which chemicals or
molecules are responsible for the formation of particle-associated reactive
surface groups. What is known, however, is that even after organic extrac-
tion, the particulate core is still able to generate ROS, although in decreased
amounts . One possibility is that transition metals cooperate with elec-
trophilic organic chemicals, such as quinone, to generate ROS on the parti-
cle surface , as demonstrated in Fig. 2.
Particle number and surface area varies with particle size as demonstrated for airborne particles
with a fixed mass concentration of 10 mg/m3
Particle diameter (mm) Particles/cc in the airParticle surface area (mm2/cc)
Data from Donaldson K, Tran CL. Inflammation caused by particles and fibers. Inhal
Fig. 2. Hypothetical model for particle-induced ROS production wherein organic chemicals co-
operate with transition metals in the generation of the?OH radical. Quinones can redox cycle
on the particle surface. This reaction involves electron capture by the quinone (Q) to form
a semiquinone. The semiquinone, in turn, transfer the electrons to O2molecule to generate
O2?? . Spontaneous dismutation of O2??can lead to H2O2production, which in the presence of
ferrous ion on the particle surface can generate?OH through the Fenton reaction.
ROS AND OXIDATIVE STRESS IN MEDIATING PM INJURY
Biologic mechanisms of particulate matter–induced reactive oxygen
The origins of PM-induced ROS in biologic systems and target cells are
from mixed subcellular sources (Fig. 3), including (1) catalytic conversion of
PAHs to quinones by cytochrome P450 1A1 in the endoplasmic reticulum
(Fig. 4), (2) quinone redox cycling by NADPH-dependent P450 reductase
in microsomes (Fig. 4) , (3) mitochondrial perturbation leading to elec-
tron leakage in the inner membrane, and (4) NADPH oxidase activation on
the cell membrane or the phagosome of macrophages.
Redox cycling organic chemicals are a major source of ROS generation in
target tissue, such as bronchial epithelial cells, macrophages, and endothelial
cells [17,51]. Quinones can act as catalysts to produce ROS during cellular
responses to DEP . Quinones are byproducts of diesel combustion and
the enzymatic conversion of PAH in lung tissue . Redox cycling
Fig. 3. Sources of ROS production and their effects on cells. Quinones can redox cycle to pro-
duce ROS in the endoplasmic reticulum under the catalytic influence of NADPH-cytochrome
P450 reductase. Phagocytosis can induce the assembly and activation of NADPH oxidase to
produce superoxide. PM can interfere in electron transduction in the mitochondrial inner mem-
brane and in the perturbation of the PT pore to generate ROS. ROS leads to lipid peroxidation
in the cell membrane and can crosslink protein SH groups and induce redox equilibrium
through a depletion of GSH. Depending on the level of oxidative stress this could induce
Nrf2 release to the nucleus, activation of mitogen-activated protein kinases and NF-kB signal-
ing cascades, or cytotoxicity. Nrf2 interaction with the antioxidant response element leads to the
expression of HO-1 and other phase II enzymes at low level of oxidative stress, whereas mito-
gen-activated protein kinases and NF-kB signaling cascades lead to proinflammatory responses
(eg, cytokine production) at higher levels of oxidative stress. At the highest oxidative stress
level, ROS can lead to opening of mitochondrial PT pore, followed by cytochrome c release,
caspase-3 activation, and apoptosis or necrosis.
XIA et al
quinones undergo one-electron reductions by NADPH cytochrome P450 re-
ductase to form semiquinones (Fig. 4). The semiquinones are metastable and
donateelectronsto O2,leadingto theformation ofO2??asabyproduct . In
theprocess,the original quinones arebeing regeneratedandcancontributeto
multiple rounds of O2??generation (ie, redox cycle). Because of their high con-
tent of organic chemicals, ambient UFPs contribute proportionally more
ability of UFPs to generate O2??in the dithiothreitol assay [8,33]. The dithio-
threitol assay has been developed to measure the content of redox cycling
chemicals present in fresh ambient PM samples collected by particle concen-
trators from ambient air [8,33].
der physiologic conditions [52–54]. Mitochondria catalyze ATP production,
which is linked to the activity of an electron transduction chain that operates
NADH and FADH2, consists of three multiprotein complexes in the inner
membrane: the NADH dehydrogenase complex (I), the cytochrome c reduc-
tase complex (III), and the cytochrome c oxidase complex (IV). The chain
also includes two diffusible molecules, ubiquinone and cytochrome c, which
function as electron transporters between complexes I and III and between
complexes III and IV, respectively. Electrons are transferred in a stepwise
fashion along this chain, moving from a high to a low redox potential, which
ultimately leads to the formation of H2O [52–54]. The dissipation in electron
against a concentration gradient. This event leads to the formation of the
Fig. 4. PAHs can be converted to quinones by cytochrome P450 1A1. Reduction of quinones
by NADPH P450 reductase leads to the formation of semiquinones, which are metastable and
donate their electron to O2to form O2? . In the process, the original quinone is regenerated and
can participate in several additional rounds of redox cycling. (Modified from Li N, Hao M,
Phalen RF, et al. Particulate air pollutants and asthma: a paradigm for the role of oxidative
stress in PM-induced adverse health effects. Clin Immunol 2003;109(3):254; with permission.)
ROS AND OXIDATIVE STRESS IN MEDIATING PM INJURY
mitochondrial membrane potential (DJm) [52–54]. Although efficient, this
electron transfer process is not perfect. For instance, during the Q cycle that
operates between complexes I and III, a two-step oxidation occurs in which
ubiquinol is transformed into ubiquinone via an intermediary ubisemiqui-
none. The ubisemiquinone is capable of transferring electrons to molecular
O2, leading to the formation of O2? [52–54].
Organic DEP chemicals are able to interfere in the electron transfer chain
in mitochondria to generate ROS [16,17,39], which includes interference by
polar DEP chemicals, including redox cycling quinones such as 9,10-
phenanathraquinone (see Fig. 1). We have demonstrated that this interfer-
ence takes place between complexes I and III, which suggests that PM
redox cycling chemicals may disrupt the Q cycle at this juncture of the
electron transfer chain . This disruption could favor the accumulation
of ubisemiquinones, thereby contributing to O2??production in the mito-
In addition to the depolarizing effect on the inner membrane, organic
DEP chemicals such as quinones and PAHs can perturb the mitochondrial
permeability transition (PT) pore . The PT pore is a redox-, pH-, cal-
cium-, and DJm-dependent protein complex that plays a pivotal role in
regulating mitochondrial function and controlling apoptosis in cells
[57–59]. DEP chemicals lead to oxidation of vicinal thiol groups that reg-
ulate the open/closed state of the mitochondrial PT pore. This oxidation,
together with PM-induced increases in intracellular free calcium, results
in PT pore opening . The Ca2þdependence is demonstrated by the
ability of cyclosporin A, an inhibitor of the Ca2þ-dependant chaperone,
cyclophilin D, to interfere in PM-induced PT pore opening . Redox
cycling quinones, such as phenanathraquinone and naphthoquinone
induce Ca2þ-dependent, cyclosporin A sensitive PT pore opening in iso-
lated mitochondria, whereas a non–redox-cycling quinone, 9,10-anthra-
quinone, was inactive . Large-scale PT pore opening decreases
DJm, increases O2??generation, releases apoptotic factors to the cytosol,
disrupts ATP synthesis, and ultimately could lead to cell death [16,39,53].
Mitochondria have been shown to be a direct subcellular target for
ambient UFP [8,16,39]. Ambient UFP lodge in the mitochondria of tar-
get cells such as macrophages and epithelial cells after their exposure to
an aqueous suspension of UFP . Morphologically, it manifests as dis-
ruption of the mitochondrial integrity, with disappearance of their cristae.
Functionally, these changes are accompanied by a loss of mitochondrial
membrane potential, decrease in mitochondrial mass, opening of the PT
pore, ROS production, and cell death [8,39]. In contrast, fine and coarse
particles donot lodge in mitochondria but can indirectlyaffectmitochondrial
function through ROS generation and intracellular calcium flux elsewhere in
the cell [8,12,34]. Why UFPs target the mitochondria is unknown.
in phagocytic cells . The holoenzyme consists of two membrane-bound
XIA et al
p47PHOX, and p67PHOX) and a small GTPase Rac1/2 [60,61]. In its unassem-
bled form under basal cellular conditions, this enzyme is inactive. In the pres-
ence of agonists such as fMLP and opsonized zymosan, however, which
interact with membrane receptors, and stimuli generated in the course of
phagocytosis, NADPH oxidase is assembled and activated in the membrane
cytoplasmic subunitstothemembrane,where theyinteractwithgp91phoxand
p22phox. The catalytically active holoenzyme generates large amounts of O2?
[60,61]. NanosizedDEP have been found to selectively damage dopaminergic
initiation of an oxidative insult . This effect is decreased in cells collected
from NADPH oxidase deficient (PHOX?/?) mice and by a phagocytic inhib-
itor, cytochalasin D .
andp22phox) andthree cytoplasmicsubunits(p40PHOX,
The biology of oxidative stress explains several particulate matter–induced
adverse health effects
ROS are oxygen molecules that contain unpaired electrons. Fig. 5 depicts
the four-electron transfer that can transform O2to H2O. In the process, the
addition of one, two, or three electrons can generate O2? , H2O2, and?OH re-
active species. ROS readily react with cellular components, such as proteins,
lipids, membranes, and DNA, leading to structural damage [53,54]. ROS
also can deplete cellular GSH, which leads to the accumulation of glutathi-
one disulfide [7,17]. The accompanying decrease in the cellular GSH/gluta-
thione disulfide ratio indicates a state of the redox disequilibrium (ie,
oxidative stress) [8,17]. Oxidative stress acts as a stimulus that can initiate
further cellular responses, thereby contributing to the pathogenesis of
Fig. 5. ROS generation by a chain of electron acquisitions that involve the formation of super-
oxide, H2O2, the hydroxyl radical, and finally H2O. (Modified from Li N, Hao M, Phalen RF,
et al. Particulate air pollutants and asthma: a paradigm for the role of oxidative stress in PM-
induced adverse health effects. Clin Immunol 2003;109(3):251; with permission.)
ROS AND OXIDATIVE STRESS IN MEDIATING PM INJURY
Cellular responses to oxidative stress include protective and injurious
events. To explain this apparent paradox, we used biologic and proteome
analyses to explore the evolving series of events in target cells, such as epi-
thelial cells and macrophages during exposure to pro-oxidative PM chemi-
cals [5,7,20,21,38]. This experiment led to the characterization of
a hierarchical oxidative stress model (Table 3), which posits that at a lower
level of oxidative stress (Tier 1), cells generate protective antioxidant and de-
toxification enzymes by acting on a genetic response element that requires
the bZIP transcription factor, Nrf2 [7,43]. Nrf2 drives the antioxidant re-
sponse element in the promoter of phase II genes, leading to the expression
of antioxidant and cytoprotective enzymes . Several of these phase II en-
zymes that have been shown to be responsive to DEP, ambient UFP, and
organic DEP extracts in PM target tissue in the lung, including HO-1, glu-
tathione-S-transferase, NADPH quinone oxidoreductase, catalase, superox-
ide dismutase, glutathione peroxidase, and UDP-glucoronosyltransferase
[38,63]. These phase II enzymes protect against oxidative stress injury (Tiers
2 and 3), such that a reduced or compromised Tier 1 response may promote
oxidant PM injury. Clinically, a compromise in Tier 1 responses can occur
because of phase II enzyme polymorphisms in phase II genes or null geno-
types. For instance, the glutathione-S-transferase M1 null genotype predis-
poses atopic people to asthma and to an enhanced allergic inflammatory
response by DEP challenge in the nose . Conversely, the induction of
a phase II response may help people to adapt to a polluted environment
and may explain why only a relatively small number of people in a popula-
tion get sick when confronted with a sudden surge in ambient PM levels .
Adaptation can explain why repeated low-dose concentrated ambient
particle exposures fail to elicit persistent lung inflammation .
If Tier 1 protection fails, a further increase in oxidative stress could lead
to the generation of proinflammatory (Tier 2) or cytotoxic (Tier 3) effects
Biology of oxidative stress
Oxidative stress level
Tier 1Tier 2 Tier 3
Antioxidant defenseInflammation Toxicity
Nrf2 NF-kB & MAPK
perturbation (PT pore)
Apoptosis, necrosisBiologicPhase II antioxidant
? Increased ROS
generation? ? epithelial
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(Table 3) at the cellular level. Tier 2 responses are linked to the activation of
intracellular signaling pathways that impact cytokine and chemokine gene
promoters [11,32,66]. An example is activation of the mitogen-activated pro-
tein kinase (MAPK) cascades . These cascades are responsible for the ex-
pression and activation of AP-1 transcription factors (eg, c-Jun and c-Fos),
which play a role in the transcriptional activation of proinflammatory genes,
such as the genes that encod for cytokines, chemokines, and adhesion mol-
ecules. Tier 3 responses involve mitochondrial perturbation by pro-oxidative
chemicals [16,18,32,43]. Although the in vivo significance of the mitochon-
drial pathway is uncertain, it has been demonstrated in tissue culture cells
that PM interference in mitochondrial electron transfer can contribute to
ROS production and the induction of apoptosis [18,32,43]. These effects
can be mimicked by organic DEP extracts and redox cycling quinones
and functionalized aromatic hydrocarbons present in these extracts .
Using the strengths of proteomics to find DEP-induced oxidative stress
proteins, the principles of a hierarchical oxidative stress response could be
confirmed in macrophages and epithelial cell cultures [7,32]. Two-dimen-
sional gel electrophoresis demonstrated the appearance of O50 newly
expressed proteins, which could be subtracted from the expression profile
by pretreatment of the cells with NAC . At the lowest tier of oxidative
stress (Tier 1), the expression of catalase, superoxide dismutase, and HO-1
confirm the involvement of Nrf2-regulated enzymes that play a role in the
suppression of inflammation through their antioxidant activities [63,66].
This finding was confirmed by showing that DEPs and UFPs increase the
accumulation of Nrf2 in the nucleus, including their ability to activate the
antioxidant response element . The Nrf2 translocation to the nucleus de-
pends on a prolongation of the half-life of this transcription factor by inter-
ference in its proteasomal degradation . Under basal conditions, Nrf2
exhibits a short (approximately 15-minute) half-life because it is continu-
ously being shuttled to the 20S proteosome by a chaperone, Keap-1 .
In the presence of electrophilic chemicals, Nrf2 uncouples from Keap-1,
which leads to its withdrawal from the proteasomal degradation pathway
and the accumulation in the cell and the nucleus [63,67]. Phosphoproteome
analysis performed in parallel with cytokine array has confirmed that acti-
vation of the ERK, p38, and Jun kinase cascades are linked to the induction
of proinflammatory responses .
Each tier of oxidative stress is sensitive to the effects of the thiol antiox-
idant, NAC [7,16,18]. NAC is capable of quenching oxygen radicals and act-
ing as a precursor to GSH synthesis [7,16]. NAC is capable of covalent
modification of electrophilic chemicals and cellular proteins . These inter-
actions prevent redox cycling of organic PM chemicals and can protect key
cellular cysteine residues against oxidative cross-linking. It is possible, there-
fore, that low levels of oxidative stress may target NAC-protected cysteines
that are involved in the binding of Nrf2 to Keap-1 . Similarly, at Tiers 2
and 3 of the hierarchical oxidative stress response, it is possible that NAC
ROS AND OXIDATIVE STRESS IN MEDIATING PM INJURY
may protect SH groups that are involved in the inactivation of mitogen-
activated protein kinase phosphatases or in the opening of the mitochon-
drial PT pore . A possible unifying hypothesis to explain the hierarchical
response is that the cysteines that regulate each of these tiers are differen-
tially sensitive to oxidative modification by ROS or electrophilic chemicals.
A key question is whether the hierarchical oxidative stress model is rele-
vant to in vivo disease outcomes in PM-exposed people. Although it is dif-
ficult to envisage a large-scale oxidative stress response that targets an entire
organ or the intact body, it is probably more practical to think of the tiered
response as a dynamic equilibrium between pro-oxidant and antioxidant
forces at the level of specific tissue targets. For instance, in an individual
who has asthma and suffers from low-grade airway inflammation, a sudden
surge in ambient PM levels could increase particle deposition at airway
bifurcation points. These so-called ‘‘hot spots’’ of deposition are richly
endowed with bronchial epithelial cells, macrophages, and immune cells,
such as antigen-presenting cells and lymphocytes . The ability of the
epithelium, macrophages, and antigen-presenting cells to mount a phase
II response may prevent or suppress a potential chain of immune activating
events that can culminate in IgE switching in B cells because of functional
changes in helper T lymphocytes . The converse also may be true, how-
ever; namely, that abundant ROS generation that pushes the local tissue re-
sponse to Tier 2 may perturb antigen-presenting cell activity and immune
activation to complete the IgE switch. At this point, the committed response
of memory T cells and B cells could transfer the immune response to the
entire respiratory mucosa. A focal area in which proinflammatory dominate
anti-inflammatory responses may lead to more generalized effects. Similar
scenarios can be envisaged in the pathophysiology of an atherosclerotic pla-
que, in which pro- and antioxidative influences may determine whether
lipid-laden macrophages undergo apoptosis or whether the overlying endo-
thelium may express adhesion molecules that favor platelet adhesion and
thrombus formation. Both events contribute to fatal heart attacks, which
are epidemiologically associated with a sudden rise in ambient PM levels .
The importance of oxidative stress in future research and therapy
for particulate matter–induced adverse health effects
The recognition of the role of oxidative stress in PM-induced adverse
health effects is an important cornerstone for future research. First, it is im-
portant to characterize all the chemical components that play a role in ROS
generation for toxicologic and regulatory purposes. If PAH and quinone
analyses prove to be accurate predictors of adverse health effects, it may
be important to monitor these substances in ambient air. In this regard,
the high PAH content of UFPs collected in the Los Angeles basin has
been shown to be a good predictor of the ability of these particles to gener-
ate oxidative stress in macrophages and epithelial cells . Even if PAHs are
XIA et al
not directly responsible for ROS generation, their assessment could serve as
a proxy for redox cycling chemicals that are responsible for adverse health
effects. Second, the recognition that particle size and reactive surface area
play an important role in predicting pro-oxidative particle effects could
mean that in addition to a mass standard, particle number and surface
area may need to be incorporated into the regulatory assessments. This
addition could be of considerable importance in following PM effects in
areas in which vehicular traffic is a major contributor to ambient PM levels,
especially as it pertains to UFP levels. A third consideration is the develop-
ment of in vivo markers for oxidative stress that can be used to look for sub-
clinical PM responses. This consideration is particularly important for
dissecting the apparent disconnect between macroscale epidemiology, which
shows considerable morbidity and mortality caused by ambient PM expo-
sure, and smaller scale exposure studies, which often fail to show significant
health effects. A possible explanation is that only a small percentage (eg,
1%–2%) of individuals is truly sensitive to PM and more likely to be ex-
cluded on a chance basis when selections are made for human exposure
studies. To identify susceptible individuals, it is important to develop in
vivo oxidative stress markers that can be used to identify susceptible people
for study purposes.
Understanding of the role of oxidative stress in disease pathogenesis is
also important from a therapy perspective. Based on our principal hypoth-
eses, namely that (1) oxidative stress is a key mechanism by which PM
impacts allergic inflammation and asthma exacerbation and (2) Nrf2-
mediated antioxidant defense protects against the proinflammatory effects
of PM, we predict that therapeutic interventions that lead to phase II
enzyme expression could provide a novel treatment approach for asthma.
Although several types of antioxidants have been tried in patients who
have asthma [69–71], there is no clear consensus that antioxidant therapy
is useful in this disease. In our opinion, previous attempts have been ham-
pered by the lack of appreciation that certain asthmatic subsets (eg, atopic
subjects with a glutathione-S-transferase M1 null genotype ) may be
more prone to the adverse biologic effects of oxidative stress. There may
be a lack of appreciation of the redundancy of oxidative stress pathways
once ROS production is already in progress. Initial sources of ROS produc-
tion could be redox cycling and electrophilic chemicals, which generate ROS
through spontaneous particle-mediated and enzymatic reactions. This could
yield to or be overtaken by additional sources of ROS once tissue inflamma-
tion is involved. We propose that interference in oxidative stress must com-
mence before the initial ROS production by pro-oxidative chemicals to be
effective and prevent the redundant mechanisms that operate once radical
production is in place. Increased expression of phase II enzymes is an attrac-
tive candidate to accomplish the goal of interfering in lung and heart
diseases. Based on the detoxification and antioxidant effects of phase II
enzymes, they could be effective in neutralizing the adverse biologic effects
ROS AND OXIDATIVE STRESS IN MEDIATING PM INJURY
of redox cycling organic chemicals by scavenging a wide range of ROS and
detoxifying the inducing chemicals . Phase II enzymes also enhance
GSH synthesis and specialize in the removal of a wide range of ROS .
Phase II enzyme expression can be achieved by oral administration of
two chemical compounds, a-lipoic acid (aLA) [72–74] and sulforaphane
. Both compounds function through Nrf2 release and antioxidant re-
sponse element activation. aLA is a disulfide derivative of octanoic acid
that forms an intracellular disulfide bond in its oxidized form. High electron
density that results from special positioning of the two sulfur atoms in the
1,2-dithiolane ring allows aLA to reduce redox-sensitive molecules. aLA
in its reduced form (DHLA) is a strong reductant that is more easily oxi-
dized than monothiols. Exogenously supplied aLA is absorbed, transported
to tissues, and reduced to DHLA. aLA and DHLA are highly reactive
against various ROS in vitro. aLA and DHLA redox also are capable
of regenerating GSH. This action includes Nrf2-mediated expression of
g-glutamylcysteine ligase, the rate-limiting enzyme in GSH synthesis .
In a murine asthma model, aLA treatment significantly reduced airway
hyperreactivity, BAL eosinophilia, and airway inflammation . Sulfora-
phane is a chemical found in foods such as broccoli and is capable of induc-
ing various phase II enzymes, which results in the enhancement of cellular
antioxidant capacity . Sulforaphane induces phase II enzyme expression
by uncoupling Nrf2 from its chaperone, Keap-I, and allowing the transcrip-
tion factor to accumulate in the nucleus [63,75].
Numerous reports have linked oxidative stress to PM-induced adverse
health effects. ROS production is related to redox cycling organic chemicals
and transition metals bound to the PM surface and may originate from the
particle surface and enzymatically catalyzed reactions in target cells. UFPs
are more toxic than larger ambient particles in the Los Angeles basin, based
on their ability to generate ROS. The PM-induced oxidative stress response
at cellular level is a hierarchical event. Oxidative stress responses range from
protective phase II enzyme expression, at low levels of oxidative stress, to
activation of proinflammatory signaling pathways at higher levels of oxida-
tive stress. This dynamic equilibrium may determine who responds to PM,
what type of response is generated, and who could benefit from rational
antioxidant therapy that leads to increased phase II enzyme expression.
 National Academy of Science National Research Council. Research priorities for airborne
DC: National Academy of Science National Research Council; 1998.
XIA et al
 Nel AE, Diaz-Sanchez D, Ng D, et al. Enhancement of allergic inflammation by the interac-
tion between diesel exhaust particles and the immune system. J Allergy Clin Immunol 1998;
102(4 Pt 1):539–54.
 Ghio AJ, Devlin RB. Inflammatory lung injury after bronchial instillation of air pollution
particles. Am J Respir Crit Care Med 2001;164(4):704–8.
 Muranaka M, Suzuki S, Koizumi K, et al. Adjuvant activity of diesel-exhaust particulates
for the production of IgE antibody in mice. J Allergy Clin Immunol 1986;77(4):616–23.
 Gurgueira SA, Lawrence J, Coull B, et al. Rapid increases in the steady-state concentration
of reactive oxygen species in the lungs and heart after particulate air pollution inhalation.
Environ Health Perspect 2002;110(8):749–55.
 Lim HB, Ichinose T, Miyabara Y, et al. Involvement of superoxide and nitric oxide on air-
way inflammation and hyperresponsivenessinducedby diesel exhaust particles in mice. Free
Radic Biol Med 1998;25(6):635–44.
 Xiao GG, Wang M, Li N, Loo JA, et al. Use of proteomics to demonstrate a hierarchical
oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line.
J Biol Chem 2003;278(50):50781–90.
 Li N, Sioutas C, Cho A, et al. Ultrafine particulate pollutants induce oxidative stress and
mitochondrial damage. Environ Health Perspect 2003;111(4):455–60.
 Silbajoris R, Ghio AJ, Samet JM, et al. In vivo and in vitro correlation of pulmonary MAP
kinase activation following metallic exposure. Inhal Toxicol 2000;12(6):453–68.
 Donaldson K, Tran CL. Inflammation caused by particles and fibers. Inhal Toxicol 2002;
 Nel AE, az-Sanchez D, Li N. The role of particulate pollutants in pulmonary inflammation
and asthma: evidence for the involvement of organic chemicals and oxidative stress. Curr
Opin Pulm Med 2001;7(1):20–6.
 Nel A. Atmosphere: air pollution-related illness: effects of particles. Science 2005;308(5723):
 Samet JM, Dominici F, Curriero FC, et al. Fine particulate air pollution and mortality in 20
US cities, 1987–1994. N Engl J Med 2000;343(24):1742–9.
 Dockery DW, Pope CA III, Xu X, et al. An association between air pollution and mortality
in six US cities. N Engl J Med 1993;329(24):1753–9.
for healthcare professionals from the Expert Panel on Population and Prevention Science of
the American Heart Association. Circulation 2004;109(21):2655–71.
 Hiura TS, Li N, Kaplan R, et al. The role of a mitochondrial pathway in the induction of
apoptosis by chemicals extracted from diesel exhaust particles. J Immunol 2000;165(5):
 Li N, Wang M, Oberley TD, et al. Comparison of the pro-oxidative and proinflammatory
effects of organic diesel exhaust particle chemicals in bronchial epithelial cells and macro-
phages. J Immunol 2002;169(8):4531–41.
 Hiura TS, Kaszubowski MP, Li N, et al. Chemicals in diesel exhaust particles generate reac-
tive oxygen radicals and induce apoptosis in macrophages. J Immunol 1999;163(10):
 Kumagai Y, Arimoto T, Shinyashiki M, et al. Generation of reactive oxygen species during
interaction of diesel exhaust particle components with NADPH-cytochromeP450 reductase
and involvement of the bioactivation in the DNA damage. Free Radic Biol Med 1997;22(3):
 Whitekus MJ, Li N, Zhang M, et al. Thiol antioxidants inhibit the adjuvant effects of
aerosolized diesel exhaust particles in a murine model for ovalbumin sensitization. J Immu-
 Rhoden CR, Lawrence J, Godleski JJ, et al. N-acetylcysteine prevents lung inflammation
after short-term inhalation exposure to concentrated ambient particles. Toxicol Sci 2004;
ROS AND OXIDATIVE STRESS IN MEDIATING PM INJURY
 Rhoden CR, Wellenius GA, Ghelfi E, et al. PM-induced cardiac oxidative stress and
dysfunction are mediated by autonomic stimulation. Biochim Biophys Acta 2005;1725(3):
 Knaapen AM, Shi T, Borm PJ, et al. Soluble metals as well as the insoluble particle fraction
exhaust particles and endotoxin. Am J Respir Crit Care Med 2005;171(4):379–87.
 Sanbongi C, Takano H, Osakabe N, et al. Rosmarinic acid inhibits lung injury induced by
diesel exhaust particles. Free Radic Biol Med 2003;34(8):1060–9.
 Nabeyrat E, Jones GE, Fenwick PS, et al. Mitogen-activated protein kinases mediate
peroxynitrite-induced cell death in human bronchial epithelial cells. Am J Physiol Lung
Cell Mol Physiol 2003;284(6):L1112–20.
 Nightingale JA, Maggs R, Cullinan P, et al. Airway inflammation after controlled exposure
to diesel exhaust particulates. Am J Respir Crit Care Med 2000;162(1):161–6.
Biol Med 2005;39(1):1–25.
 Silkoff PE. Noninvasive measurement of airway inflammation using exhaled nitric ox-
ide and induced sputum: current status and future use. Clin Chest Med 2000;21(2):
 Steerenberg PA, Nierkens S, Fischer PH, et al. Traffic-related air pollution affects peak ex-
piratory flow, exhaled nitric oxide, and inflammatory nasal markers. Arch Environ Health
 van Amsterdam JG, Verlaan BP, van Loveren H, et al. Air pollution is associated with in-
creased level of exhaled nitric oxide in nonsmoking healthy subjects. Arch Environ Health
 Wang M, Xiao GG, Li N, et al. Use of a fluorescent phosphoprotein dye to characterize
oxidative stress-induced signaling pathway components in macrophage and epithelial
cultures exposed to diesel exhaust particle chemicals. Electrophoresis 2005;26(11):
 Cho AK, Sioutas C, Miguel AH, et al. Redox activity of airborne particulate matter at dif-
ferent sites in the Los Angeles Basin. Environ Res 2005;99(1):40–7.
 Donaldson K, Stone V, Borm PJ, et al. Oxidative stress and calcium signaling in the
adverse effects of environmental particles (PM10). Free Radic Biol Med 2003;34(11):
 Carter JD, Ghio AJ, Samet JM, et al. Cytokine production by human airway epithelial cells
 LayJC,BennettWD,KimCS,etal.Retentionandintracellulardistributionof instillediron
oxide particles in human alveolar macrophages. Am J Respir Cell Mol Biol 1998;18(5):
 Ghio AJ, Hall A, Bassett MA, et al. Exposure to concentrated ambient air particles alters
hematologic indices in humans. Inhal Toxicol 2003;15(14):1465–78.
 Li N, Venkatesan MI, Miguel A, et al. Induction of heme oxygenase-1 expression in macro-
phages by diesel exhaust particle chemicals and quinones via the antioxidant-responsive
element. J Immunol 2000;165(6):3393–401.
 Xia T, Korge P, Weiss JN, et al. Quinones and aromatic chemical compounds in particulate
matter induce mitochondrial dysfunction: implications for ultrafine particle toxicity. Envi-
ron Health Perspect 2004;112(14):1347–58.
 Penning TM, Burczynski ME, Hung CF, et al. Dihydrodiol dehydrogenases and polycyclic
Res Toxicol 1999;12(1):1–18.
XIA et al
 Baulig A, Garlatti M, Bonvallot V, et al. Involvement of reactive oxygen species in the met-
abolic pathways triggered by diesel exhaust particles in human airway epithelial cells. Am
J Physiol Lung Cell Mol Physiol 2003;285(3):L671–9.
 Seagrave J, Gigliotti A, McDonald JD, et al. Composition, toxicity, and mutagenicity of
particulate and semivolatile emissions from heavy-duty compressed natural gas-powered
vehicles. Toxicol Sci 2005;87(1):232–41.
 Li N, Kim S, Wang M, et al. Use of a stratified oxidative stress model to study the biological
effects of ambient concentrated and diesel exhaust particulate matter. Inhal Toxicol 2002;
mental pollutants: effects of polycyclic aromatic hydrocarbons (PAHs) in environmental
pollution on exogenous and oxidative DNA damage. Mutat Res 2003;544(2–3):397–402.
environmental pollutants on the fetus. Environ Health Perspect 1999;107(Suppl 3):451–60.
 Dejmek J, Solansky I, Benes I, et al. The impact of polycyclic aromatic hydrocarbons and
fine particles on pregnancy outcome. Environ Health Perspect 2000;108(12):1159–64.
 Donaldson K, Stone V. Current hypotheses on the mechanisms of toxicity of ultrafine par-
ticles. Ann Ist Super Sanita 2003;39(3):405–10.
 Oberdo ¨ rster G, Oberdo ¨ rster E, Oberdo ¨ rster J. Nanotoxicology: an emerging discipline
evolving from studies of ultrafine particles. Environ Health Perspect 2005;113(7):823–39.
 Pan CJ, Schmitz DA, Cho AK, et al. Inherent redox properties of diesel exhaust particles:
catalysis of the generation of reactive oxygen species by biological reductants. Toxicol Sci
 Valavanidis A, Fiotakis K, Bakeas E, et al. Electron paramagnetic resonance study of the
generation of reactive oxygen species catalysed by transition metals and quinoid redox
cycling by inhalable ambient particulate matter. Redox Rep 2005;10(1):37–51.
 Sagai M, Saito H, Ichinose T, et al. Biological effects of diesel exhaust particles. I. In vitro
production of superoxide and in vivo toxicity in mouse. Free Radic Biol Med 1993;14(1):
 Andreyev AY, Kushnareva YE, Starkov AA. Mitochondrial metabolism of reactive oxygen
species. Biochemistry (Mosc) 2005;70(2):200–14.
 Papa S, Skulachev VP. Reactive oxygen species, mitochondria, apoptosis and aging. Mol
Cell Biochem 1997;174(1–2):305–19.
 Raha S, Robinson BH. Mitochondria, oxygen free radicals, disease and ageing. Trends Bio-
chem Sci 2000;25(10):502–8.
 Walter L, NogueiraV,LeverveX, etal. Threeclassesof ubiquinoneanalogsregulatethe mi-
tochondrial permeability transition pore through a common site. J Biol Chem 2000;275(38):
 FontaineE,Ichas F, Bernardi P. A ubiquinone-bindingsite regulatesthe mitochondrialper-
meability transition pore. J Biol Chem 1998;273(40):25734–40.
 Zamzami N, Kroemer G. The mitochondrion in apoptosis: how Pandora’s Box opens. Nat
Rev Mol Cell Biol 2001;2:67–71.
 Halestrap AP, McStay GP, Clarke SJ. The permeability transition pore complex: another
view. Biochimie 2002;84(2–3):153–66.
 Bernardi P, Petronilli V, Di Lisa F, et al. A mitochondrial perspective on cell death. Trends
Biochem Sci 2001;26(2):112–7.
 Babior BM. NADPH oxidase. Curr Opin Immunol 2004;16(1):42–7.
 Brandes RP, Kreuzer J. Vascular NADPH oxidases: molecular mechanisms of activation.
Cardiovasc Res 2005;65(1):16–27.
 Block ML, Wu X, Pei Z, et al. Nanometer size diesel exhaustparticles are selectively toxic to
dopaminergic neurons: the role of microglia, phagocytosis, and NADPH oxidase. FASEB J
ROS AND OXIDATIVE STRESS IN MEDIATING PM INJURY
 Li N, Alam J, Venkatesan MI, et al. Nrf2 is a key transcription factor that regulates antiox- Download full-text
idant defense in macrophages and epithelial cells: protecting against the proinflammatory
and oxidizing effects of diesel exhaust chemicals. J Immunol 2004;173(5):3467–81.
 Fryer A, Bianco A, Hepple M, et al. Polymorphism at the glutathione S-transferase GSTP1
locus: a new marker for bronchial hyperresponsiveness and asthma. Am J Respir Crit Care
particles (CAPs) in mice. I. Introduction, objectives, and experimental plan. Inhal Toxicol
of oxidative stress in PM-induced adverse health effects. Clin Immunol 2003;109(3):250–65.
 Zhang DD, Hannink M. Distinct cysteine residues in Keap1 are required for Keap1-
dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents
and oxidative stress. Mol Cell Biol 2003;23(22):8137–51.
 Hao M, Comier S, Wang M, et al. Diesel exhaust particles exert acute effects on airway in-
flammation and function in murine allergen provocation models. J Allergy Clin Immunol
 Bylin G, Hedenstierna G, Lagerstrand L, et al. No influence of acetylcysteine on gas
exchange and spirometry in chronic asthma. Eur J Respir Dis 1987;71(2):102–7.
 Pearson PJK, Lewis SA, Britton J, et al. Vitamin E supplements in asthma: a parallel group
randomised placebo controlled trial. Thorax 2004;59(8):652–6.
 Ram FS, Rowe BH, Kaur B. Vitamin C supplementation for asthma. Cochrane Database
Syst Rev 2004;3:CD000993.
 Flier J, Van Muiswinkel FL, Jongenelen CA, et al. The neuroprotective antioxidant alpha-
lipoic acid induces detoxication enzymes in cultured astroglial cells. Free Radic Res 2002;
 Suh JH, Shenvi SV, Dixon BM, et al. Decline in transcriptional activity of Nrf2 causes age-
related loss of glutathione synthesis, which is reversible with lipoic acid. Proc Natl Acad Sci
U S A 2004;101(10):3381–6.
 Sook Cho Y, Lee J, Lee TH, et al. a-Lipoic acid inhibits airway inflammation and hyperres-
ponsiveness in a mouse model of asthma. J Allergy Clin Immunol 2004;114(2):429–35.
 Lee JS, Surh YJ. Nrf2 as a novel molecular target for chemoprevention. Cancer Lett 2005;
XIA et al