Am J Physiol Lung Cell Mol Physiol 283: L246–L255, 2002;

10.1152/ajplung.00491.2001.

invited review
Antioxidant responses to oxidant-mediated lung diseases

SUZY A. A. COMHAIR AND SERPIL C. ERZURUM

Departments of Pulmonary and Critical Care Medicine and Cancer Biology,
Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Comhair, Suzy A. A., and Serpil C. Erzurum. Antioxidant re-

sponses to oxidant-mediated lung diseases. Am J Physiol Lung Cell Mol
Physiol 283: L246–L255, 2002; 10.1152/ajplung.00491.2001.—Reactive
oxygen species (ROS) and reactive nitrogen species (RNS) are generated
throughout the human body. Enzymatic and nonenzymatic antioxidants
detoxify ROS and RNS and minimize damage to biomolecules. An im-
balance between the production of ROS and RNS and antioxidant capac-
ity leads to a state of “oxidative stress” that contributes to the pathogen-
esis of a number of human diseases by damaging lipids, protein, and
DNA. In general, lung diseases are related to inflammatory processes
that generate increased ROS and RNS. The susceptibility of the lung to
oxidative injury depends largely on its ability to upregulate protective
ROS and RNS scavenging systems. Unfortunately, the primary intracel-
lular antioxidants are expressed at low levels in the human lung and are
not acutely induced when exposed to oxidative stresses such as cigarette
smoke and hyperoxia. However, the response of extracellular antioxidant
enzymes, the critical primary defense against exogenous oxidative
stress, increases rapidly and in proportion to oxidative stress. In this
paper, we review how antioxidants in the lung respond to oxidative
stress in several lung diseases and focus on the mechanisms that up-
regulate extracellular glutathione peroxidase.
redox; reactive oxygen species; reactive nitrogen species

OXYGEN IS ONE OF THE MOST abundant elements in our
world, constituting 21% of the air we breathe (20, 122).
It is essential for the oxidation of organic compounds,
which is the process by which mammalian cells gener-
ate the energy needed to sustain life. However, oxygen
may also damage the lung. Inhaled ozone and nitric
oxide may induce toxic processes that impair lung
function (20, 38, 82, 119, 122). Under normal condi-
tions, potentially toxic oxygen metabolites are gener-
ated at a low level in lung cells by the transfer of a
single electron during aerobic metabolism (25, 33, 46).
The resulting reactive oxygen species (ROS), which
include hydroxyl radicals, superoxide (O2⫺䡠), and hydro-
gen peroxide (H2O2), play an integral role in the mod-
ulation of several physiological functions but can also
be destructive if produced in excessive amounts (31, 38,
82, 99, 104, 107). Similarly, reactive nitrogen species
Address for reprint requests and other correspondence: S. A. A.
Comhair, Dept. of Pulmonary and Critical Care Medicine, Cleveland
Clinic Foundation, 9500 Euclid Ave./NB4-107, Cleveland, OH 44195
(E-mail: comhais@ccf.org).
L246 1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society
(RNS) such as nitric oxide, nitrite, and peroxynitrite
(ONOO⫺) are both physiologically necessary and po-
tentially destructive.
Another oxygen-mediated mechanism of damage is
inflammation, during which leukocytes, macrophages,
and mast cells release mediators that may cause bron-
choconstriction and edema as observed during an asth-
matic reaction (15, 38, 64). Lung tissue can also be
destroyed during reperfusion after an ischemic period
such as that produced by surgery (42, 94, 99, 104, 107,
134). All these mechanisms have one thing in common:
damage is at least partly mediated by oxidants and
nitrogen species.
To minimize oxidant damage to biological molecules,
the human lung is endowed with an integrated antiox-
idant system of enzymatic and expendable soluble an-
tioxidants. This system includes several antioxidant
defense mechanisms that detoxify reactive products or
convert them to products that are quenched by other
antioxidants (47, 58). If the oxidant burden is suffi-
ciently great, the reactive species may overwhelm or
inactivate the antioxidant system. The resulting excess
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 INVITED REVIEW
oxygen species can damage major cellular components,
including membrane lipids, protein, carbohydrates,
and DNA. The pathophysiological consequences of this
injury are inflammation and widespread tissue dam-
age (46).
OXYGEN AND REACTIVE OXYGEN SPECIES
More than 90% of all the oxygen we breathe under-
goes a concerted tetravalent reduction to produce wa-
ter in a reaction catalyzed by cytochrome oxidase in the
mitochondrial electron transport chain. Oxygen (O2)
can also be reduced via a nonenzymatic pathway
through four successive one-electron (e⫺) reductions (6,
34) (Eq. 1).
O 2 ⫹ 4H ⫹ ⫹ 4e ⫺ 3 2H 2O
Cytochrome oxidase is the terminal electron acceptor
in the respiratory chain and must donate its reducing
equivalents to oxygen to allow continued electron
transport. Otherwise, ATP production cannot continue.
Thus the major role for oxygen in all aerobic organisms
is simply to act as a sink or dumping ground for
electrons (34). The tetravalent reduction of oxygen by
the mitochondrial electron-transport chain is consid-
ered a relatively safe process. Nonetheless, the electron
carriers catalyze alternating one-electron oxidant-re-
duction reactions, and they can react with oxygen to
generate ROS such as O2⫺䡠 (47, 96, 97). Mitochondria
are the major intracellular sites of O2⫺䡠 generation
under physiological conditions (53). One other poten-
tially major source for the generation of O2⫺䡠 is the
NADPH oxidase enzymatic system, which is found in
neutrophils, monocytes, macrophages, cytochrome
P-450, monoamine oxidase, and lipooxygenase (4, 22,
31, 34). O2⫺䡠 is also generated by other mechanisms
such as molybdenum hydroxylase reactions (including
the xanthine, sulfite, and aldehyde oxidases) and ara-
chidonic acid metabolism.
O2⫺䡠 is relatively unstable, with a half-life of only
milliseconds. Because it is charged, it does not easily
cross cell membranes (6). O2⫺䡠 will react, however, with
proteins that contain transition metal prosthetic
groups, such as heme moieties or iron-sulfur clusters.
These reactions may damage amino acids or cause
protein/enzyme function to be lost (50, 138). Most of the
O2⫺䡠 generated in vivo undergoes a nonenzymatic or
superoxide dismutase (SOD)-catalyzed reaction, re-
sulting in the nonradical H2O2 (Eq. 2) (79). H2O2 can
also be directly produced by several oxidase enzymes,
including xanthine oxidase, monoamine, and amino
acid oxidase (24).
O 2⫺䡠 ⫹ O 2⫺䡠 ⫹ 2H ⫹ 3 H 2O 2 ⫹ O 2
H2O2 can be oxidized by eosinophil-specific peroxi-
dase (EPO) and neutrophil-specific peroxidase (MPO)
using halides (X⫺) as a cosubstrate to form the potent
oxidant hypohalous acids (HOX) and other reactive
halogenating species (Eq. 3) (44, 56, 70, 130).
H2O2 ⫹ X⫺ ⫹ H⫹ 3 HOX ⫹ H2O X ⫽ Br⫺, Cl
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Much of the damage done by O2⫺䡠 and H2O2 in vivo is
due to their production of hydroxyl radicals (䡠OH) in a
series of reactions catalyzed by traces of transition
ions. One such example is the iron-catalyzed Haber-
Weiss reaction in which Fe3⫹ is reduced to Fe2⫹, fol-
lowed by the Fenton reaction in which the Fe2⫹ cata-
lyzes the transformation of H2O2 into (䡠OH) (Eq. 4) (54).
O2⫺䡠 ⫹ Fe3⫹ 3 Fe2⫹ ⫹ O2 Haber-Weiss reaction
H2O2 ⫹ Fe 2⫹
3 Fe 3⫹ ⫺
⫹ OH ⫹ 䡠OH Fenton reaction
(4)
An alternative pathway for 䡠OH formation in vivo
may involve MPO and EPO. Under physiological con-
centrations of halides, MPO produces hypochlorous
(1) acid (HOCl), and EPO produces hypobromous acid
(HOBr). Studies of 䡠OH with spin-trapping agents (41,
124) and chemical traps (57, 95) have demonstrated
that hypohalous acids can generate 䡠OH after reacting
with O2⫺䡠 (Eq. 5). 䡠OH can react with different molecules
such as protein (16), DNA, and lipids (49).
O 2⫺䡠 ⫹ HOX 3 䡠OH ⫹ X ⫺ ⫹ O 2 (5)
RNS
The discovery that nitric oxide (NO) is endogenously
formed throughout the human body has led to intense
interest in the variety of roles this unique molecule
plays in vivo. NO is involved in a wide variety of
regulatory mechanisms. In addition, NO is also a cyto-
toxic agent present in environmental pollutants and
cigarette smoke (109). NO is formed from the semies-
sential amino acid L-arginine by the action of nitric
oxide synthase (NOS; Fig. 1) (5, 90). Several forms of
NOS have now been characterized, and several distinct
NOS genes have been identified (73). The NOS are
classified as either constitutive or inducible (76, 89,
(2)
Fig. 1. Reactive nitrogen species (RNS) synthesis. NO, nitric oxide;
NO3⫺, nitrate; NO2⫺ nitrite; HbO2, oxyhemoglobin; Hb3⫹, methemo-
globin; ONOOH, peroxynitrous acid; MPO, myeloperoxidase; HOCl,
hypochlorous acid; SNO: S-nitrosothiol; NOS II, nitric oxide syn-
(3) thase II.
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L248
131). The constitutive forms (NOS I and NOS III) are
cytosolic and originally described and cloned from neu-
ronal and endothelial cells, respectively. They are de-
pendent on Ca2⫹ and calmodulin and release low
amounts of NO for short periods in response to receptor
and physical stimulation (89). The inducible form
(NOS II) is independent of Ca2⫹. Once expressed, NOS
II generates NO in large amounts for long periods
(131). The biochemical effect of NO is largely defined by
the concentration of NO. The paramagnetic NO mole-
cule contains an odd number of electrons, which ex-
plains its highly reactive and radical nature (Fig. 1)
(65, 113). Autooxidation of NO with O2 results in the
formation of nitrite (NO2⫺). However, at physiological
concentrations of NO and O2, this reaction may be too
slow to be important in vivo (8, 66). NO2⫺ is also a
substrate for hemeperoxidases such as MPO and EPO,
which catalyze peroxidase-mediated oxidation and
chlorination of biological targets (40, 41, 66, 124, 130,
135, 136). Moreover, peroxidase-catalyzed oxidation of
NO2⫺ results in the formation of a nitrogen dioxide
radical (NO2 䡠) or related molecules. These substances
can contribute to the nitration of phenolic compounds
such as tyrosine to form dimerized (dityrosine) and
nitrated (3-nitrotyrosine) products, which are stable
(40, 41, 66, 124, 130, 135, 136).
Although NO2⫺ is a major end product of NO, it does
not accumulate in vivo but is rapidly oxidized to nitrate
(NO3⫺) (91, 131). NO is also rapidly oxidized by oxyhe-
moglobin (HbO2), which results in the formation of
methemoglobin (Hb3⫹) and NO3⫺ (1, 14, 51). The oxida-
tive metabolism of NO may also lead to the formation
of carcinogenic nitrosoamines (72, 75) and the rapid
loss of NO’s smooth muscle relaxant activity (59, 117).
The rapid reaction of NO with free radicals (radical-
radical reaction) has emerged as one of the major
routes to the formation of RNS. At present, the best
understood of these reactions is the reaction with O2⫺䡠
to form ONOO⫺ (91), a strong oxidant (113). Although
ONOO⫺ is relatively stable, it can be protonated to
yield peroxynitrous acid (ONOOH) (31), which then
rapidly decomposes to NO3⫺ via the intermediate for-
mation of 䡠OH and NO2-like species (31). ONOOH is
very unstable, highly reactive, and capable of both
oxidizing and nitrating reactions. For instance, irre-
versible ONOOH modifications include nitration of ar-
omatic amino acids, lipids, or DNA bases (127). The
amino acid tyrosine appears to be particularly suscep-
tible to nitration, and the formation of free or protein-
associated 3-nitrotyrosine has recently attracted inter-
est as a potential biomarker for the generation of RNS
in vivo (101, 125).
Reactions with thiol residues leading to the forma-
tion of S-nitrosothiols (SNO) have been proposed as a
mechanism whereby NO groups are transported and
targeted to specific effector sites, a potentially unique
signaling mechanism induced by nitrosative stress (77,
85, 92). The exact mechanism by which S-nitrosation
occurs in vivo is still unclear, but it involves the for-
mation of NO-derived intermediates with the redox
equivalence of NO⫹ (the primary candidates are N2O3
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and ONOOH) and (di)nitrosyl iron complex (52, 60, 66,
126, 132). Nitrosation of amines by these reactive ni-
trogen intermediates has been implicated in the muta-
genic properties of NO, presumably through nitrosa-
tive deamination of DNA bases (125). It is also of
interest that SNO such as S-nitroso-L-glutathione
(GSNO) may inhibit enzymes associated with the re-
sponse to oxidative stress in eukaryotic cells, including
glutathione peroxidase (GPx), glutathione reductase
(7), glutathione-S-transferase (24), and ␥-glutamyl cys-
teine synthase (55).
ANTIOXIDANTS
ROS and RNS play important physiological func-
tions and yet they can also cause extensive damage.
The balance between physiological functions and dam-
age is determined by the relative rates of formation
and the removal of ROS and RNS.
Normally, ROS and RNS are removed rapidly before
they cause cellular dysfunction and eventual cell
death. All aerobic organisms use a series of primary
antioxidant defenses to protect against oxidative dam-
age. Furthermore, numerous repair enzymes remove
and/or repair damaged molecules. However, an antiox-
idant cannot distinguish between radicals that play a
physiological role and those that cause damage (6). More-
over, some antioxidant compounds also have prooxidant
actions (6). This section will review the enzymatic and
nonenzymatic primary antioxidant defenses.
Enzymatic antioxidants. SOD (EC 1.15.1.11) is an
ubiquitous enzyme with an essential function in pro-
tecting aerobic cells against oxidative stress (79). It
catalyzes O2⫺䡠 radicals to H2O2. There are three forms
of SOD. The copper-zinc SOD is located in the cytosol,
the manganese SOD is primarily a mitochondrial en-
zyme, and extracellular SOD is usually found on the
outside of the plasma membrane (43).
Catalase (EC 1.11.1.6) is a tetrameric hemoprotein
that undergoes alternate divalent oxidation and reduc-
tion at its active site in the presence of H2O2 and
catalyzes the dismutation reaction (22, 36, 102). As a
result, catalase has appreciable reductive activity for
small molecules such as H2O2 and methyl or ethyl
hydroperoxide. It does not metabolize large molecular
peroxides such as lipid hydroperoxide products of lipid
peroxidation (129). Catalase is most effective in the
presence of high H2O2 concentrations. However, in the
presence of low concentrations of either H2O2 or other
peroxides, the glutathione system plays a critical role
(20).
The glutathione system is a central mechanism for
reducing H2O2. It complements catalase as a reducing
system for H2O2 but exceeds catalase in its capacity to
eliminate additional varieties of toxic peroxides (105).
Other metabolized substrate species include large mol-
ecule lipid peroxides, formed by free radical attack on
polyunsaturated lipid membranes and products of li-
pooxygenase-catalyzed reactions (58). The key enzyme
in the redox cycle responsible for the reduction of H2O2
is GPx. This reaction specifically requires reduced glu-
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tathione (GSH) to serve as the electron donor. The
glutathione disulfide (GSSG) formed in the course of
the reaction is subsequently reduced back to GSH by
glutathione reductase, which uses NADPH generated
from the hexose monophosphate shunt system as an
electron donor (37, 80). Healthy, nonstressed cells
maintain a high intracellular GSH:GSSG ratio to en-
sure the availability of GSH and thereby promote ac-
tive reduction of H2O2 through the glutathione system
(37, 80). In a role unrelated to its role in the GSH
system, free GSH can also function as a water-soluble
antioxidant by interacting directly with radical inter-
mediates in nonenzymatic catalyzed reactions. Scav-
enging of O2⫺䡠 by GSH leads to the formation of thiyl
radicals (GS 䡠 ) and H2O2 via several steps, which is a
radical propagation reaction. This reaction leads to the
formation of GS 䡠 and H2O2 and can occur in physiolog-
ically relevant concentrations. Hence, a substance that
is generally accepted to be an antioxidant may possess
prooxidant activity under certain conditions (6, 38).
Four GPx have been described, all selenium en-
zymes: 1) the classic cytosolic form, found in all cells
(9); 2) a membrane-associated GPx phospholipid H2O2
(39); 3) another cytoplasmic enzyme, gastrointestinal
GPx, which was first found in cells of the gastrointes-
tinal tract (23); and 4) an extracellular GPx (eGPx),
first identified as a distinct enzyme in human plasma
(137). All members of this family of enzymes can be
oxidized by organic hydroperoxides, hydroperoxide, or
both, and can subsequently be reduced by glutathione
(137). The existence of multiple forms of GPx is due to
the expression of different genes (103). All GPx contain
a selenium atom in the active site in the form of
selenocysteine.
Nonenzymatic antioxidants. Cells use nonenzymatic
antioxidant compounds to react directly with oxidizing
agents and disarm them. Such antioxidants are said to
be “scavengers”; their roles are unavoidably suicidal.
For example, vitamin E (␣-tocopherol) is a membrane-
bound antioxidant that terminates the chain reaction
of lipid peroxidase by scavenging lipid peroxyl radicals
(LOO 䡠 ) (6, 34, 123). In this reaction, vitamin E becomes
a radical, but it is much less reactive than LOO 䡠 (123).
However, at high concentrations, the radical form of
vitamin E may function as a prooxidant (6). Vitamin C
can also directly scavenge O2⫺䡠 and 䡠OH by forming the
semidehydroascorbate free radical that is subse-
quently reduced by GSH (78). Vitamin C, however, is
usually not considered a major antioxidant because it
also has prooxidant properties. It is probably the only
cellular reducing agent other than O2⫺䡠 capable of con-
verting Fe3⫹ to Fe2⫹, which then reacts with H2O2 to
form 䡠OH (106). Whether the prooxidant or antioxidant
properties of vitamin C prevail in any particular tissue
is determined by the extent of available iron stores;
iron overload favors excess oxidant generation (6, 106).
Other nonenzymatic antioxidants include ␤-carotene
(scavenger of O2⫺䡠 anions and peroxyl radicals), uric
acid (hydroxyl radical, O2⫺䡠, peroxyl radical scavenger),
glucose (hydroxyl radical scavenger), bilirubin (LOO䡠
scavenger), taurine (hypochlorous acid quencher), albu-
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min (transition metal binding), and cysteine and cys-
teamine (donators of sulfhydryl groups).
ANTIOXIDANTS IN THE LUNG
Lungs are unique because they have a large epithe-
lial surface area that is at risk for oxidant-mediated
attack. The tracheobronchial tree and the alveolar
space are exposed to reactive oxidizing species in the
form of inhaled airborne pollutants, tobacco smoke,
and products of inflammation. The lung, therefore,
requires additional antioxidant resources to prevent
airway-borne oxidant injury (58). The major airways
contain high-molecular-weight mucopolypeptide glyco-
proteins, which are synthesized by the epithelial cells
and glands that increase mucus production in the pres-
ence of inflammation (58). The lung contains intracel-
lular antioxidant enzymes to maintain a normal redox
state. The alveolar space can recruit additional antiox-
idant activity from the epithelial lining fluid (ELF).
This fluid contains large amounts of GSH (100-fold
higher than in plasma), 90% of which is in the reduced
form (19, 27, 35, 114, 115). The ELF also contains
catalase, SOD, and GPx (19, 27, 114, 115). Additional
antioxidants contained in ELF include ceruloplasmin,
transferrin, ascorbate, vitamin E, ferritin, other serum
proteins, and small molecules such as bilirubin (58).
The multiplicity of the antioxidant systems available to
the lung and their overlapping specific activities sug-
gest that to maintain normal pulmonary cellular func-
tion, it is critically important for the lung to adequately
control redox balance. Disequilibrium, either through
increased oxidant stress or decreased antioxidant re-
sources, can result in a series of pathophysiological
events in the lung that culminate in cellular death and
pulmonary dysfunction (58). A partial list of major
lung diseases associated with oxidants is presented in
Table 1.
EXTRACELLULAR ANTIOXIDANT RESPONSE IN LUNGS
EXPOSED TO OXIDATIVE STRESS
Normally, the homeostasis of cellular functions dur-
ing oxidative stress depends on the rapid induction of
protective antioxidant enzymes. Naturally occurring
antioxidants exist to protect cells and tissue against
the continuous production of ROS/RNS during normal
metabolism (58). Tissues and cells respond to mild
oxidative stress by increasing antioxidant defenses
(119). However, high levels of ROS/RNS may over-
whelm antioxidant defenses, resulting in oxidant-me-
diated injury or cell death (4, 20).
Numerous studies have revealed that oxidant stress
plays a crucial role in the initiation and progression of
a wide range of diseases and in the regulation of a
number of important biological processes. Pulmonary
diseases associated with oxidative stress include
asthma, hyperoxia, sarcoidosis, and chronic beryllium
disease (CBD). ROS play a key role in the initial lung
response to asbestos and silica that leads to interstitial
pulmonary fibrosis (87, 88). Interestingly, during the
development of pulmonary diseases, antioxidant re-
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Table 1. Lung diseases associated with oxygen radicals

Disease Mechanism References

Emphysema Tissue injury by oxidants in cigarette smoke (21, 63)

Adult respiratory distress syndrome
Hyperoxia
Idiopathic pulmonary fibrosis
Asthma
␣1PI, ␣-1-proteinase inhibitor.
Tissue injury by inflammatory cell oxidants ␣1PI inactivation by cigarette
smoke and inflammatory cells
Inflammatory cell release of oxidants ␣1PI oxidative inactivation by
inflammation cells (26)
Hyperoxia-mediated oxygen radical synthesis in cells (19)
Inflammatory cell oxidant release Glutathione deficiency (17, 18)
Inflammatory cell release of oxidants (35, 114)
Decrease in superoxide dismutase activity in bronchial epithelial cells

sponses are different. For example, asbestosis and sar- tive stress occurring in individuals with asthma or
coidosis lead to an increase of SOD, whereas there are CBD and in those who have been exposed to exogenous
no changes found in silicosis or hyperoxic lung injury oxidants such as cigarette smoke (Fig. 2) (2, 28–30).
(27, 62, 71). In contrast, SOD activity is significantly The upregulation of eGPx occurs rather late after ex-
lower in patients with asthma and decreases further posure (after 24 h) (28), which may explain why eGPx
during an asthmatic exacerbation. was not induced after 12 h of hyperoxia. In support of
The glutathione system is altered in lung inflamma- this, levels of eGPx mRNA and protein increase only
tory conditions. For instance, GSH levels are elevated after 72 h of hyperoxia in a mouse model (67). Induc-
in the ELF of chronic smokers and in chronic beryllium tion of eGPx mRNA in bronchial epithelial cells is
disease, an immune-specific granulomatous inflamma- associated with elevated protein levels in ELF, sug-
tion (29). Levels of GSH in ELF decrease rapidly in gesting that the increase of eGPx occurs, in part, by
patients with mild asthma during an asthma exacer- bronchial epithelial cell synthesis and secretion (28).
bation (27). Similarly, GSH levels are decreased in However, alveolar macrophages can also express eGPx
ELF in idiopathic pulmonary fibrosis (17, 74), asbesto- (2, 30). It is not known whether other lung cells or
sis (11), acute respiratory distress syndrome (13), and inflammatory cells upregulate eGPx gene in response
in human immunodeficiency virus-positive patients to oxidative stress.
(12). Levels of glutathione modulate the T helper type Bronchial epithelial cells significantly increase eGPx
1 (Th1) vs. the Th2 immune response pattern (93). For mRNA expression in response to increased intracellu-
example, high levels of glutathione in patients with lar or extracellular ROS in vitro. Supplementation of
CBD may contribute to the development and/or main- GSH in cell culture to physiological levels potentiates
tenance of a chronic Th1 cell-mediated immune re-
sponse to beryllium, whereas the low GSH levels may
contribute to the development or maintenance of Th2
cell immune response in asthma.
Other enzymes of the glutathione system are also
influenced by oxidant stress. Some studies have shown
increased GPx activity in ELF of smokers compared
with nonsmokers (29, 104), whereas others have shown
decreased GPx in smokers (81). The difference in GPx
activity may be due to the difference in smoking his-
tory (19, 86). GPx activity is not altered in asthma but
is increased in lungs of CBD patients. Overall, the
antioxidant response is inconsistent across different
oxidant-mediated lung diseases.
EGPX AND ITS ROLE IN OXIDATIVE STRESS

Expression of eGPx. eGPx transcripts have been

found in epithelial cells with well-developed brush bor-
ders that contain lipids and alkaline phosphatase ac-
tivity, e.g., the human airways, intestine, and renal
tubules (2, 3, 68, 120). Alveolar macrophages are also
able to synthesize and secrete eGPx (2, 28, 30). The
GPx family is an important enzymatic component of
the mechanisms for detoxifying ROS in the lung and
may play a significant role in preventing pulmonary
oxidant stress. eGPx gene expression is upregulated in
bronchial epithelial cells and ELF as a result of oxida-
Fig. 2. Expression of extracellular glutathione peroxidase (eGPx)
mRNA in human airway epithelial cells (means ⫾ SE). No significant
difference in the expression of eGPx mRNA was found in cells from
individuals with asthma, chronic beryllium disease (CBD), and
smoke exposure (P ⬎ 0.05), but in all 3 groups, levels were signifi-
cantly higher than in controls (P ⬍ 0.05). GAPDH, glyceraldehyde-
3-phosphate dehydrogenase.

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the induction of eGPx mRNA in response to ROS (28).

This effect is not reproduced by N-acetylcysteine, sug-
gesting that other effects of thiol groups are necessary
to achieve the synergistic effect on induction of the
eGPx gene (28). Although GSH is usually considered
an antioxidant, it can also act as an oxidant when
present at physiological levels (6). GSH may partici-
pate in oxidizing processes and/or accelerate the gen-
eration of ROS, specifically O2⫺䡠 (6, 84, 121, 133). Pre-
vious reports have shown that GPx can function as an
ONOO⫺ reductase and thereby prevent nitration reac-
tions caused by RNS (45). On the other hand, NO
donors (S-nitroso-N-acetyl-D,L, penicillamine/GSNO)
induce eGPx gene expression in a time- and dose-
dependent matter.
Transcriptional regulation. The regulation of genes
in response to oxidative stress occurs via transcription
and/or stabilization of mRNA. Studies support that the Fig. 4. Detoxification of ROS and RNS by eGPx in asthmatic indi-
ROS regulation of the eGPx gene expression occurs at viduals. a: Superoxide is detoxified through eGPx to H2O; b: in the
a transcriptional level (28). In general, ROS and RNS presence of low levels of superoxide dismutase, NO and superoxide
(O2⫺䡠) combine to form ONOOH; c: peroxynitrite is able to nitrate
regulate the expression of numerous genes via signal- tyrosine residues; d: nitrosation of reduced glutathione (GSH) by
ing mechanisms. Redox-sensitive transcription factors peroxynitrite leads to S-nitroso-L-glutathione (GSNO); e: detoxifica-
such as signal transducers and activators of transcrip- tion and liberation of NO through eGPx. GSSG, glutathione disul-
tion (STAT), nuclear factor-␬B, and transcription fac- fide.
tor activator protein-1 (AP-1) are activated in epi-
thelial cells and inflammatory cells during oxidative
STATs by oxidative stress is inhibited by antioxidants.
stress (98, 100). Although the STAT family of tran-
Several ROS-induced target genes have known STAT
scription factors is activated by many cytokines and
binding sites in their promoters. These include genes
growth factors, it can also be activated by oxidative
involved in antioxidant defense (111) such as SOD1
stress such as H2O2 (108, 112, 116). The activation of
(10) and genes involved in cell growth regulation such
as c-fos (128).
Studies from several laboratories have demonstrated
that oxidant stress such as cigarette smoke, treatment
with H2O2, depletion of intracellular GSH, or an in-
crease in the GSH:GSSG ratio stimulates AP-1 activa-
tion and binding (83, 99). AP-1 regulates many of the
inflammatory and immune genes in oxidant-mediated
diseases (69, 110, 118). AP-1 is a protein dimer com-
posed of the Jun and Fos gene products (100). These

Fig. 3. Proposed scheme for eGPx gene induction by oxidative stress.

Reactive oxygen species (ROS) and RNS cross the cell membrane,
which together with inflammatory mediators activate different tran-
scription factors, such as nuclear factor (NF)-␬B and activator pro-
tein-1 (AP-1). The 5⬘-flanking region of the eGPx gene necessary for
transcriptional activation shows that the inducible promoter ele- Fig. 5. Nitrotyrosine staining of asthmatic bronchial mucosa. Cyto-
ment contains the DNA binding element for AP-1. The activation and plasm of bronchial epithelial cells shows positive immunoreactivity
binding of AP-1 to the eGPx promoter is a possible mechanism for to nitrotyrosine (red; arrow); g, goblet cell; bm, basement membrane.
eGPx mRNA and protein induction. I␬B, inhibitor of NF-␬B. Magnification, ⫻40; hematoxylin counterstaining.

AJP-Lung Cell Mol Physiol • VOL 283 • AUGUST 2002 • www.ajplung.org

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L252 INVITED REVIEW
gene products can form homodimeric (Jun-Jun) or het-
erodimeric (Jun-Fos) complexes (100). The 5⬘-flanking
region of the eGPx gene contains the DNA-binding
element for AP-1 (137) (Fig. 3). Cigarette smoke in-
creases AP-1 DNA binding in human epithelial cells in
vivo (99). Based on our results and those of others,
we propose that increased formation of ROS and
RNS in inflammatory cells and epithelium leads to
alterations in the redox system, sensed by the epi-
thelial cells, and leads to the activation of transcrip-
tion factors such as STATs and AP-1 and down-
stream target gene expression.
Function. On the basis of our studies and the studies
of others, we have generated a model for the function of
eGPx in lung diseases associated with oxidative stress
(Fig. 3). NO is produced in mammalian airways, and
increased levels are found in many inflammatory lung
diseases such as asthma and hyperoxia (28, 30, 61).
Inflammation leads to increased levels of ROS (Fig. 4,
pathway a). NO reacts slowly with O2 to form the
cytotoxic compound NO2⫺ or very rapidly with O2⫺䡠 to
form ONOO⫺ (Fig. 4, pathway b) (51). This results in
tyrosine nitration in lung tissue (Fig. 5 and Fig. 4,
pathway c). Thus when O2⫺䡠 is produced at high rates
during lung inflammation, NO may accelerate the for-
mation of ONOO⫺, leading to tyrosine nitration and
aggravate lung damage (61). The NO/O2⫺䡠 pathway in
vitro appears to be significantly shifted toward the
formation of GSNO in the presence of physiological
levels of GSH (Fig. 4, pathway d) (30, 45, 77). Recent
studies have shown that GPx (Fig. 4, pathway e) can
protect against NO-mediated protein oxidation (48)
and can reduce GSNO. Thus the increased eGPx in
lung inflammation may have two functions: reduction
of ROS (e.g., H2O2 and O2⫺䡠) and possibly protection
against NO-mediated protein oxidation by regulation
of RSNO levels.
In conclusion, lung diseases occur in response to
oxidative and nitrosative stress. The lung’s ability to
respond to oxidative stress depends largely on its ca-
pacity to upregulate protective antioxidants. The anti-
oxidant responses to lung disease vary widely, but the
upregulation of eGPx in oxidant-mediated lung dis-
eases is likely important to defend airway surfaces
from ROS and RNS injury.
We thank J. H. J. M. van Krieken, F. B. J. M. Thunnissen, P. N. R.
Dekhuijzen, D. Roos, A. Bast, and P. Scheepers for helpful discussion
and comments.
This work was supported in part by National Heart, Lung, and
Blood Institute Grant HL-60917.
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