Department of Respiratory Medicine, Medical School, University
of Edinburgh, Edinburgh EH8 9AG, United Kingdom
Glutathione (GSH), a ubiquitous tripeptide
thiol, is a vital intra- and extracellular protective antioxidant in
the lungs. The rate-limiting enzyme in GSH synthesis is
-glutamylcysteine synthetase (-GCS). The promoter
(5'-flanking) region of the human -GCS heavy and light
subunits are regulated by activator protein-1 and antioxidant response
elements. Both GSH and -GCS expression are
modulated by oxidants, phenolic antioxidants, and inflammatory and
anti-inflammatory agents in lung cells. -GCS is regulated at both
the transcriptional and posttranscriptional levels. GSH plays a key
role in maintaining oxidant-induced lung epithelial cell function and
also in the control of proinflammatory processes. Alterations in
alveolar and lung GSH metabolism are widely recognized as a central
feature of many inflammatory lung diseases including chronic
obstructive pulmonary disease (COPD). Cigarette smoking, the major
factor in the pathogenesis of COPD, increases GSH in the lung
epithelial lining fluid of chronic smokers, whereas in acute smoking,
the levels are depleted. These changes in GSH may result from altered
gene expression of
-GCS in the lungs.
The mechanism of regulation of GSH in the epithelial lining fluid in
the lungs of smokers and patients with COPD is not known. Knowledge of
the mechanisms of GSH regulation in the lungs could lead to the
development of novel therapies based on the pharmacological or genetic
manipulation of the production of this important antioxidant in lung
inflammation and injury. This review outlines
1) the regulation of cellular GSH
levels and -GCS expression under oxidative
stress and 2) the evidence for lung
oxidant stress and the potential role of GSH in the pathogenesis of COPD.
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INTRODUCTION |
GLUTATHIONE (GSH) is a ubiquitous, essential tripeptide
(L--glutamyl-L-cysteinyl-glycine)
containing a sulfhydryl group that enables it to protect cells against
oxidants, electrophilic compounds, and xenobiotics. GSH, which accounts
for 90% of intracellular nonprotein thiols, is a key intracellular
reducing agent and is implicated in immune modulation and inflammatory
conditions (155, 157). GSH also serves as a storage and transport form
of cysteine and as a cofactor in several enzymatic reactions. Hence GSH
is emerging as one of the fundamental antioxidant defense mechanisms in
oxidant-induced lung injury and inflammation. Alterations in lung
lining fluid GSH levels have been shown in various inflammatory conditions. For example, it is decreased in the epithelial lining fluid
(ELF) of idiopathic pulmonary fibrosis (IPF) (40, 146), acute
respiratory distress syndrome (35), cystic fibrosis (217), and human
immunodeficiency virus-positive (235) patients.
Chronic obstructive pulmonary disease (COPD) is a condition
characterized by progressive and largely irreversible airway
obstruction and an influx of inflammatory cells into the lungs (201,
206, 232). Mortality and morbidity from the disease are
high in developed countries and are rising in developing countries
(232). The important events in the pathogenesis of COPD are considered
to be lung inflammation, an increased oxidant burden, and a
protease-antiprotease imbalance in the lungs (202, 205, 206, 232). The
increased oxidant burden derives from the fact that cigarette smoke
contains an estimated 1014 free
radicals/puff and that many of these, such as tar semiquinone, which
can generate
H2O2
by the Fenton reaction, are relatively long-lived (178, 194, 268). It
is reported that >90% of patients with COPD are smokers, but not all
smokers develop COPD (232). Fifteen to twenty percent of cigarette
smokers appear to be susceptible to its effects, show a rapid decline
in forced expiratory volume in 1 s
(FEV1), and develop the disease
(232). The reasons for this are not clear but may involve genetic
predisposition, dietary habits, differences in depth or pattern of
inhalation, variations in cellular and biochemical responses, and
differences in immune or regenerative capacity of lung cells.
Epidemiological evidence leaves no reasonable doubt that cigarette
smoke is the major causative agent of COPD, with atmospheric pollution
as an additional contributory factor. Studies carried out with an
animal model and an alveolar epithelial cell line (A549) in vitro
showed that the thiol antioxidant GSH is critical to lung cellular
antioxidant defenses, particularly in protection from oxidant injury
(129, 131). GSH is present in increased concentrations in the ELF of
chronic smokers, whereas this does not occur in the ELF of acute
smokers (42, 166). There is a large gap in our understanding of the
metabolism of GSH in both the various anatomic compartments and the
cell types within the lung. In addition, information is lacking on GSH
levels and GSH regulation in the lungs of smokers and patients with COPD.
Oxidant-sensitive transcription factors such as activator protein-1
(AP-1), which consists mainly of c-Fos and c-Jun homo- or heterodimers
are known to play a key role in proinflammatory processes such as the
transcription of cytokine genes and also in upregulating protective
antioxidant genes (196). Recent evidence (204) suggested that oxidants,
phenolic antioxidants, and inflammatory and anti-inflammatory agents
modulate the activities of AP-1. AP-1 has also been reported to
modulate the expression of -glutamylcysteine synthetase (-GCS),
the rate-limiting enzyme in de novo GSH synthesis. -GCS consists of
a catalytic heavy subunit (-GCS-HS) and a regulatory light subunit
(-GCS-LS). It has recently been shown that the promoter
(5'-flanking) regions of the human catalytic
-GCS-HS and regulatory subunit
-GCS-LS genes contain a putative
AP-1 and an antioxidant response element (ARE) that are necessary for -GCS expression in response to diverse
stimuli (81, 199, 203). It is possible that differences in ELF GSH in
various inflammatory lung diseases are due to changes in the molecular
regulation of GSH synthesis in lung cells by AP-1 and ARE. There are
excellent reviews available describing aspects of the antioxidant GSH
(58, 83), oxidant-induced lung injury (26, 202, 212) and toxicity (208,
230), and the protective role of antioxidants (86, 252). The primary
objective of this review is to present a detailed account of the
current knowledge of the regulation of lung GSH and -GCS in
conditions of oxidative stress in smokers and patients with airway
diseases such as COPD. Second, this review explores the molecular
mechanisms by which this antioxidant molecule is modulated in
oxidant-mediated lung injury and inflammation.
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BIOSYNTHESIS OF GSH |
The synthesis of GSH requires the presence of two enzymes; ATP;
Mg2+; and the amino acids glycine,
cysteine, and glutamate, with cysteine being the rate-limiting
substrate. The tripeptide GSH is formed by the consecutive actions of
-GCS and GSH synthetase (Fig. 1) (157).
Both enzymes are exclusively cytosolic, and the rate of GSH synthesis
is controlled by the amount of -GCS present, the availability of
L-cysteine, and feedback
inhibition excerted by GSH on -GCS (214).
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Fig. 1.
Steps in de novo glutathione (GSH) biosynthesis and degradation of
extracellular GSH in lung cells.
DL-Buthionine-(SR)-sulfoximine
(BSO) is an inhibitor of -glutamylcysteine synthetase (-GCS)
enzyme. -Glutamyl transpeptidase, -GCS, and glutathione
synthetase enzymes are discussed in text. SH, sulfhydryl group.
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GSH synthetase apparently has no regulatory role; once synthesized,
-glutamylcysteine is rapidly converted to GSH. The activity of
-GCS determines the rate of GSH synthesis. -GCS-HS contains binding sites for all three substrates and all essential catalytic residues. The mammalian -GCS holoenzyme is a heterodimer consisting of a 73-kDa -GCS-HS and a 30-kDa -GCS-LS (223). Although the HS
contains all of the catalytic activity, HS activity can be modulated by
the association with the regulatory -GCS-LS (100). It has been
calculated that 80% of the cytosolic -GCS protein is inactive under
physiological conditions due to binding with GSH (100). Thus a decrease
in GSH triggers the release of the GSH bound to -GCS, which, in
turn, results in increased levels of active -GCS and hence enhanced
synthesis of GSH. This process does not require de novo synthesis of
-GCS protein and is one way by which cells control their GSH levels
when challenged by agents that lead to an initial depletion of
intracellular GSH.
The regulatory properties of -GCS-LS are proposed to be mediated by
a disulfide bridge between the subunits that would allow conformational
changes in the active site depending on the oxidative state of the cell
(100). An important cysteine residue has been identified in the active
site of -GCS-HS by site-directed mutagenesis, which is involved in
heterodimer formation between -GCS-HS and -GCS-LS (247). This
suggests that the potential for increasing the rate of GSH synthesis
exists under conditions of GSH depletion.
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GSH AND ITS REDOX SYSTEM |
The GSH redox system is crucial in maintaining intracellular GSH
homeostasis, which is critical to normal cellular physiological processes and represents one of the most important antioxidant defense
systems in the lung (39). This system uses GSH as a substrate in the
detoxification of peroxides such as hydrogen peroxide
(H2O2) and lipid peroxides, a reaction that
involves glutathione peroxidase (GPx). This reaction generates oxidized GSH (GSSG), which is subsequently reduced by glutathione reductase in a
reaction that requires the hexose monophosphate shunt pathway utilizing NADPH (Fig. 2). Physiologically,
the glutathione reductase reaction is driven strongly in favor of GSH,
with the GSH-to-GSSG ratio normally >90%. Maintenance of the high
GSH-to-GSSG ratio minimizes intracellular accumulation of disulfides.
However, if oxidant stress or other stress alters this ratio, the
consequent shift in the GSH-to-GSSG redox buffer influences a variety
of cellular processes such as activation of the transcription factors AP-1 and nuclear factor-
B (NF-B). The protective functions of GSH
involve enzymatic as well as nonenzymatic processes. GSH is a strong
nucleophile and often inactivates electrophilic reactive compounds
either by nonenzymatic direct conjugation or by an enzyme-catalyzed reaction involving glutathione
S-transferase (GST).
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Fig. 2.
GSH redox cycle. GSH converts hydrogen and lipid peroxides to nontoxic
hydroxy fatty acids and/or water. Glutathione disulfide (GSSG) is
subsequently reduced to GSH in presence of NADPH and glutathione
reductase, which are linked with hexose monophosphate (HMP) shunt.
G-6-PD, glucose-6-phosphate dehydrogenase.
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COMPOSITION OF GSH IN LUNGS |
The lung is constantly exposed to many atmospheric pollutants such as
cigarette smoke, ozone, and nitrogen dioxide and is also at risk from
oxidant injury by inhalation of high concentrations of oxygen. It
contains the largest endothelial surface area of any organ, which makes
the lung a major target site for circulating oxidants and xenobiotics.
It is therefore no surprise that the human lung is one of the important
storage areas for GSH (6.1-17.5 nmol/mg lung) (19, 49). Lung
extracellular ELF is rich in the antioxidant GSH, which detoxifies
oxidants, free radicals, organic polyaromatic hydrocarbons, and
electrophilic compounds (200, 208). Thus extracellular GSH in the lungs
can protect alveolar macrophages, pulmonary epithelial cells, and
pulmonary endothelial cells from oxidative stresses and helps to
maintain functional surfactant (52, 93, 226, 245). GSH concentrations vary throughout the respiratory tract, being lower in nasal lining fluid than in alveolar lining fluid (52). GSH levels in the ELF
(200-400 µM) of the lungs are ~100 times higher
than those in plasma (2-4 µM) (52). The half-life of cytosolic
GSH in the lungs is not known, but its half-life is 0.5 and 3 h
in kidney and liver cells, respectively, compared with that in human
plasma where its half-life is <2 min (259).
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ROLE OF  -GLUTAMYL TRANSPEPTIDASE IN THE REGULATION OF GSH
LEVELS IN LUNGS |
Intact GSH is not taken up at a significant rate by the lungs.
Extracellular GSH is broken down into its constituent amino acids
by -glutamyl transpeptidase (-GT) and is resynthesized intracellularly rather than by direct cellular uptake (1, 89). The
enzyme -GT is a plasma membrane enzyme, with its active site directed toward the outside of the cell, present in lung epithelial cells. This enzyme breaks the -glutamyl bond of
-glutamyl-cysteinyl-glycine (Fig. 1) (218). The glutamyl moiety is
then transferred to an amino acid, a dipeptide, or GSH itself,
producing its -glutamyl derivative. Thus -GT acts as a salvage
enzyme for cellular GSH synthesis (72). The lung epithelium has been
shown to have high levels of -GT activity and utilizes extracellular
GSH from the alveolar lining fluid (17). Hence most of the plasma GSH
is catabolized by the enzyme -GT in lungs (39, 90). As a result, -GT may be important in determining the levels of GSH in lung ELF. Endothelial cells, alveolar macrophages, and fibroblasts have
lower -GT levels and therefore less easily use extracellular GSH for
intracellular GSH synthesis (17, 218).
In an animal model, rats exposed to hyperoxia exhibited low -GT
activity in ELF, which was associated with low ELF GSH levels (252,
254). -GT expression is increased in rat lung
epithelial cells by oxidants such as menadione and
t-butylhydroquinone (122), suggesting
that -GT might play a role in the protection against oxidative
stress. However, cigarette smoke condensate and oxidative stress had no
effect on -GT activity in human type II alveolar epithelial cells
(A549 cells) (197). The possible explanation for the differential
regulation of -GT activity in response to oxidants may be due to
differential expression of the
-GT gene in different
cell lines and organs and in different species. Furthermore, the direct
involvement of -GT in the regulation of GSH levels in the lungs of
smokers remains unproven.
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REGULATION OF ELF GSH |
Lung ELF GSH may come from a variety of sources. Simple diffusion from
the plasma is unlikely because blood levels of GSH are 100 times lower
than those in ELF, with values on the order of 0.5-5 µM (52,
259). It is likely that GSH is transported out of cells; intracellular
GSH levels are regulated in part by the rate of such a membrane
bidirectional transport system as in lung and liver cells (142, 253).
The function of such a GSH transport system is influenced by the redox
or thiol status of the cell, the membrane potential, and the presence
of cations in the extracellular environment (102, 141). GSH-related
structural compounds, such as glutathione S-conjugates and GSH ethyl
ester (GEE), inhibit cellular GSH uptake or influx (102, 253).
Furthermore, a more oxidized extracellular environment stimulates cells
to retain GSH, whereas a more reduced extracellular state facilitates GSH efflux (138, 253). However, these effects are in direct contrast
with the situation in vivo in the lungs because the increased oxidant
burden imposed by smoking and endogenous oxidative stress should cause
lung cells to retain rather than release GSH into the ELF. This
mechanism is difficult to explain by the presence of such a
bidirectional GSH transporter in the lung. Thus the mechanisms that
determine the levels of GSH in lung ELF are not fully understood.
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REGULATION OF CYSTINE TRANSPORT AND GSH LEVELS IN LUNG
CELLS: EFFECTS OF OXIDATIVE STRESS |
The rate-limiting step in the biosynthesis of GSH is the availability
of cysteine as a substrate within the cell (157). Cystine, an oxidized
form of cysteine, is efficiently transported into cells by the specific
inducible Na+-independent anionic
amino acid transport x
c mechanism
and subsequently reduced for use in various metabolic processes
including GSH synthesis in lungs (13, 58, 63). Intracellular transport
of cystine is accompanied by the extracellular release of glutamate.
Cysteine is also transported into cells by
Na+-dependent pathways (A or ASC)
shared with glutamine and serine (11). It has been reported that
isolated rat alveolar type II cells have a constitutive noninducible
Na+-dependent active uptake system
that transports exogenous GSH and its -glutamyl analogs into the
cells against a concentration gradient (10, 34, 93). These transport
systems may act to increase intracellular GSH in lung cells.
Various forms of oxidant stress and nitric oxide (NO) also increase the
activity of membrane cystine and glutamate transport, leading to
increased GSH synthesis in lung cells (57, 63, 127). It has been
clearly shown that cystine uptake is the rate-limiting step for GSH
synthesis in cultured lung cells, especially under conditions of
oxidative stress (12, 62). Glutamate or glycine is rarely rate
limiting. Oxidants (hyperoxia and
H2O2)
and agents such as sodium arsenite, cadmium, electrophilic compounds,
and diethyl maleate also induce cystine transport in various lung cells, macrophages, and erythrocytes that is analogous to the xc transport system, a
Na+-independent inducible system
specific for intracellular transport of cystine and glutamate (56, 57,
188, 237). Deneke and colleagues (59, 61) have shown that exposure of
rats to hyperoxia resulted in increases in total lung GSH within 24 h.
It is therefore possible that the induction of cystine or cysteine
transport could contribute to the increased GSH levels in the lungs
after exposure to hyperoxia (59, 61).
The regulation of cystine-glutamate transport is governed by the
availability of extracellular cysteine or cystine as well as by the
extracellular redox state (which is, in part, determined by
extracellular GSH levels) (13, 189). Treatment with reducing agents
such as
N-acetyl-L-cysteine
(NAC) or GSH increases intracellular GSH levels by reducing cystine to
cysteine in bovine pulmonary artery endothelial cells (58).
Furthermore, NAC increases intracellular GSH levels in bovine pulmonary
artery endothelial cells even in the absence of cystine in the medium,
possibly not mediated by mixed disulfide formation (189). This suggests
that a different transport mechanism independent of the
xc system may be involved in type
II epithelial cells to increase GSH levels in response to various
stresses (34). This is one of the mechanisms whereby lung cells
increase intracellular GSH levels under various stresses (either
oxidant stress or GSH depletion).
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MOLECULAR REGULATION OF GSH SYNTHESIS IN LUNG CELLS |
Transcriptional Level
The liver and lungs are the major sources of GSH metabolism and have
higher levels of -GCS than other tissues (39, 87, 157). Studies have
been performed on alveolar epithelial type II cells to elucidate the
potential role of these cells in the regulation of cellular GSH
turnover in the lung (252). Alveolar epithelial type II cells are more
metabolically active than other lung cells (51, 64) and represent a
relatively small proportion (4-5%) of the total air space cell
population (79). The molecular mechanisms of GSH synthesis and
regulation in type II alveolar epithelial cells in response to various
environmental, oxidant, and inflammatory stimuli have been studied. We
(203) and other investigators (238, 243) have recently reported that
the promoter (5'-flanking) region of the human
-GCS-HS gene is
regulated by a putative c-Jun homodimeric complex-AP-1 sequence. This
sequence is located at the proximal region of the -GCS-HS TATA box
in various cell lines including human alveolar epithelial cells (203, 238, 243). Monova and Mulcahy (161) and Mulcahy et al.
(170), however, have reported an ARE containing an
embedded phorbol 12-myristate 13-acetate response element (TRE/AP-1)
and an electrophile response element (EpRE; or its functional
equivalent, ARE), which play a key role in the regulation of
-GCS-HS and -GCS-LS,
respectively, in response to a planar aromatic xenobiotic compound, the
phenolic antioxidant 
-naphthoflavonone, specifically in a hepatoma
cell line (HepG2 cells). They also showed that the internal AP-1 site is important for the constitutive expression of the
-GCS-LS gene (161).
However, recently, Galloway et al. (82) were unable to show a role for
ARE in the induction of -GCS-LS by oxidants such as t-butylhydroquinone in
HepG2 cells. They suggested that an AP-1 site was the critical element
for the constitutive regulation of this subunit.
A role for NF-B in the modulation of
-GCS-HS gene
expression has also been suggested (103, 250). It has been shown that blocking the activation of NF-B that is present at the
transcriptional site of the -GCS-HS promoter by various strategies
prevented the oxidant- or cytokine-induced increase in
-GCS-HS transcription in mouse endothelial
cells and hepatocytes (36, 250). However, mutation and deletion
strategies in the -GCS-HS promoter region have ruled out the
possible involvement of NF-B in the transcriptional upregulation of
the -GCS-HS gene in
alveolar epithelial cells and other cell lines in response to tumor
necrosis factor (TNF)-
and oxidative stress (164, 196, 198, 203,
224). In addition, the role of the metal response element-binding
transcription factor-1 (MTF-1), which is present in the promoter
region of -GCS-HS, has been suggested in the
transcriptional control of
-GCS-HS gene
expression in response to heavy metals (92). The transcription of
-GCS-HS mRNA is largely diminished in the
livers of MTF-1-null mice, establishing a potential link between the
MTF-1 in the regulation of GSH biosynthesis and protection from
metal-induced oxidative stress. Therefore, it is likely that the
expression of the -GCS genes is regulated distinctly in a variety of cells at the
transcriptional level by different regulatory signals in response to
diverse stimuli.
Translational Level
Modulation of GSH synthesis has also been described at the
posttranslational levels in the rat liver in vivo (15). Various inflammatory agents such as cAMP and intracellular calcium that are
released during inflammation may inhibit GSH synthesis at the
translational level (140). It has been shown that -GCS activity is
inhibited by agonists of various signal transduction pathways in rat
hepatocytes (140), suggesting a role for signaling mechanisms in the
regulation of GSH levels. Lu et al. (140) reported that hepatic GSH
synthesis is downregulated in response to hormones known to mediate
their effects through the activation of distinct signal transduction
pathways. Using various specific inhibitors of signaling pathways,
these investigators determined that the hormone-specific inhibition of
GSH synthesis was mediated by the activation of protein kinase A,
protein kinase C, and
Ca2+/calmodulin-dependent kinase
II. This inhibition of GSH synthesis was correlated with the direct
phosphorylation of -GCS-HS on serine and threonine residues in a
Mg2+ concentration-dependent
fashion. Phosphorylation of -GCS-HS was also detected in rat
hepatocytes treated with dibutyryl cAMP, resulting in the inhibition of
-GCS activity in vivo (236). Thus phosphorylation-dephosphorylation
may regulate -GCS activity (236) and may provide a mechanism for
altering GSH levels in lung cells during oxidative stress.
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OXIDATIVE STRESS: INTRACELLULAR GSH AND -GCS REGULATION IN
LUNG CELLS AND CELLULAR TOLERANCE |
As a result of various oxidative stresses, GSH may form a protein-mixed
disulfide with sulfhydryl (-SH) group-rich protein moieties such as
albumin (52, 157). GSH also undergoes oxidation to form GSSG and a
thiyl group, which are toxic to the cells (83, 157, 208). The
relationship between decreased GSH content, increased formation of GSSG
or protein-mixed disulfide, and increased cellular sensitivity to a
variety of agents that impose oxidative stress is well established
(83).
Oxidative stress may initially deplete GSH, followed by an increase in
intracellular GSH levels, as a result of induction of -GCS-HS
(197-199). Rapid depletion of intracellular GSH has been shown to
occur with exposure to cigarette smoke or its condensate in epithelial
cells in vitro and in rat lungs in vivo (129, 200). This is followed by
a later rebound increase in GSH in epithelial cells as an adaptive
response to oxidative stress, which occurs as a result of upregulation
of -GCS-HS and activation of AP-1 (199). This
adaptive response may explain the increase in GSH in ELF in chronic
smokers (42, 166). In addition, after the initial depletion of GSH by
oxidants such as
H2O2,
redox recycling, menadione, and hyperoxia, there is also a later
increase in GSH at 12-24 h in lungs in vivo and in human alveolar
and bronchial epithelial and endothelial cells in vitro (96, 108, 191). This is associated with an increased expression of mRNA for the -GCS subunit genes.
Thus oxidants appear to upregulate the gene for GSH synthesis (Table
1). This presumably acts as a protective mechanism against oxidative stress. Table 1 categorizes the main inducers of -GCS-HS and
-GCS-LS in lung cells. However, there are other
conditions that induce GSH synthesis in other cells, and they may be of
relevance to lung cells.
A potential role for GSH has been shown in the modulation of
c-fos and
c-jun gene expression by cigarette
smoke condensate in Swiss 3T3 fibroblasts and conducting airway
epithelium (144, 172). The c-fos and
c-jun genes belong to a family of
stress- and differentiation-related immediate-early response genes, the expression of which generally represents the first measurable response
to a variety of chemical and physical stimuli (174). Cigarette smoke
condensate exposure led to the induction of the c-fos gene, and this effect was
mimicked by peroxynitrite and smoke-related aldehydes in concentrations
that are present in cigarette smoke condensate (174, 175). The effects
of cigarette smoke condensate can be enhanced by pretreatment of the
cells with
DL-buthionine-(SR)-sulfoximine
(BSO) to decrease intracellular GSH and can be prevented by treatment
with NAC (173). Thus depletion of GSH by cigarette smoke condensate
leads to induction of c-fos and
c-jun, components of AP-1, which may
then act to induce
-GCS-HS gene
expression as a feedback mechanism.
Oxidative stress produced by hyperoxia, ozone, xanthine/xanthine
oxidase,
H2O2,
menadione, lipid peroxidation products (4-hydroxy-2-nonenal), oxidized
low-density lipoprotein, ionizing radiation, BSO, and heat shock leads
to sustained increases in GSH levels by upregulation of
-GCS-HS mRNA in alveolar epithelial cells in
vitro and in vivo in lungs (45, 118, 136, 165, 242, 257). NO and its donors such as
S-nitroso-N-penicillamine
or DETA NONOate cause a transient depletion of GSH followed by
induction of GSH synthesis by enhanced expression of
-GCS-HS in rat aortic vascular smooth muscle
cells (158), pulmonary fibroblasts (261), and bovine aortic
endothelial cells (159). The increase in GSH caused by NO donors is a
further potential mechanism to protect cells against oxidative stress.
-GCS-LS is also concomitantly induced in
response to oxidants and phenolic antioxidants in rat lung epithelial
L2 cells and liver HepG2 cells, suggesting that concomitant induction of both subunits may provide a potential mechanism to enhance cellular
GSH synthesis and so develop cellular tolerance to oxidative stress
(81, 168, 242). Support for this comes from studies (136, 171) of rat
epithelial L2 cells exposed to sublethal oxidative stress that showed
increased GSH content associated with the development of tolerance to
further oxidant assault in these cells. Furthermore, ozone exposure in
rats and monkeys was associated with an initial decrease in GSH
followed by a significant increase in GSH levels in airway epithelial
cells (66). The increase in GSH levels was associated with tolerance of
the airway cells to further oxidative stress (66).
In rabbits, exposure to hypoxia-reoxygenation decreases lung GSH
content associated with an increase in GSSG levels (104). Oxidative
stress imposed by heavy metals such as selenium (46), iron (183),
methylmercury (265), sodium arsenite (12), and cadmium (12, 96) also
induces GSH synthesis in various organs in both rats and mice. These
metals may activate AP-1, induce protein phosphorylation (236), and
activate c-Jun NH2-terminal kinase
(249). All these phenomena may be linked to the induction of
-GCS expression. Other cytotoxic agents such as
radiation (165) and chemotherapeutic agents such as cisplatin (88, 224, 266) and melphalan (168) that act through the generation of reactive
oxygen species (ROS) also increase GSH synthesis in cancer cell
lines. However, it is possible that GSH synthesis and a
tolerance mechanism in response to various stimuli described in various cell lines may differ in lung epithelial cells.
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ROLE OF PHENOLIC ANTIOXIDANTS IN THE REGULATION OF GSH SYNTHESIS |
Gene regulation by phenolic antioxidants has been demonstrated to be
the result of enhanced transcription factor binding to a
cis-acting element known as the ARE or
electrophile response element. The sequences for
cis-acting ARE regions contain AP-1 or
AP-1-like elements in the consensus region (106). It has been demonstrated that the AP-1 or ARE sites are critical in the regulation of -GCS subunit genes
(82, 161, 170, 196, 203, 224, 243). Exposure to phenolic antioxidants
such as dietary
3-t-butyl-4-hydroxyanisole and
butylated hydroxytoluene as well as the synthetic indolic antioxidant
5,10-dihydroindeno(1,2-b)indole
leads to induction of -GCS in mouse liver and
kidney cell lines (69, 137, 248). The plant-derived phenolic
antioxidant apocynin (4-hydroxy-3-methoxyacetophenone) also induces GSH
synthesis in human alveolar epithelial cells (125). These effects of
phenolic antioxidants are associated with the activation of
mitogen-activated protein kinases, AP-1, and ARE (106, 179). Therefore,
in addition to their scavenging abilities, phenolic antioxidants may
provide additional protection from oxidant-induced injury by
upregulating the expression of -GCS and
increasing GSH. More recently, pyrrolidine dithiocarbamate, a
sulfhydryl-modifying antioxidant compound possessing both antioxidant and prooxidant properties, has been shown to enhance DNA binding and
transactivation of AP-1 and induce
-GCS-HS and
-GCS-LS gene expression, resulting in de novo GSH synthesis in liver HepG2 cells
(262). Hence many direct or indirect oxidant stresses lead to an
increase in GSH synthesis and, consequently, tolerance of further
oxidative stress. Further identification and characterization of the
types of naturally occurring and synthetic phenolic antioxidant compounds, which could act as potent inducers of the
-GCS subunits, should aid in the development of
effective pharmacological strategies for antioxidant treatment
involving GSH regulation in airway disease.
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ROLE OF DIETARY AMINO ACIDS IN THE REGULATION OF LUNG GSH |
Dietary GSH and cysteine are absorbed intact in the small intestine and
increase GSH levels in plasma and various tissues (94, 95, 239). Oral
administration of GSH (100 mg/kg) in mice detected higher levels of GSH
in the plasma within 30 min of administration (9). This was associated
with substantially increased GSH concentrations in various organs
including the lungs. The regulation of tissue GSH concentration by diet
and nutritional status and the potential to restore GSH in humans have
been reviewed in detail (23).
Deneke et al. (61) and other investigators (67) have reported that
total lung GSH is dependent on the amount of sulfur-containing amino
acids, particularly the level of cysteine in the diet. This observation
is supported by nutritional experiments showing that the availability
of cysteine is a limiting factor for GSH synthesis in cases where the
diet is deficient in sulfur-containing amino acids (60). GSH levels in
the lungs from rats on a protein-deficient diet supplemented with
cysteine were lower than those in control rats but increased more
rapidly than those in control rats after exposure to hyperoxia (59).
Similarly, GSH supplementation to preterm rabbits attenuated the
changes in lung mechanics and injury caused by hyperoxia (30). The
requirement of dietary cysteine in GSH synthesis was confirmed in rats
fed protein-deficient diets, which produced enhanced toxicity, with a
failure of elevation in lung GSH levels on exposure to hyperoxia. This
observation may have implications in smokers where less dietary intake
of sulfur-containing amino acids is associated with abnormal cellular function and possibly low lung function (31). Replenishment of
sulfur-containing amino acids in the protein-deficient diets elevated
lung GSH and prevented enhanced oxygen-mediated toxicity or
inflammation (77, 101). In vivo studies of GSH levels in the lung and
other organs are complicated by the fact that there is considerable
diurnal fluctuation of GSH levels in the various organs and that the
fluctuations are not synchronized (14). For example, lung GSH levels
fluctuate by 200% in rats. Thus, in addition to cystine transport, the
nutritional requirement of cysteine, particularly in smokers, is an
important step in the regulation of GSH in lungs in vivo.
Dietary regulation of the key enzymes involved in the synthesis of GSH
has been demonstrated in the rat liver (15). Rats fed a basal
low-protein diet for 2 wk had lower activity of -GCS. This suggests
that diet plays an important role in the regulation of GSH
biosynthesis. The lower enzyme activity was associated with lower
expression of -GCS-HS and
-GCS-LS in the rat liver, implicating the
potential role of diet (protein or sulfur-containing amino acids) in
the regulation of -GCS expression.
The biological levels of GSH may also depend on the quality of the food
and its processing and preservation. Jones et al. (107) measured the
concentration of total GSH in various food samples. They found that
dairy products, cereals, and breads are generally low in GSH; fruits
and vegetables have moderate to high amounts of GSH; and freshly
prepared meats are relatively high in GSH. Generally, frozen foods are
thought to contain similar levels of GSH as fresh foods, whereas other
forms of processing and preservation may result in an extensive loss of
GSH (107).
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MITOCHONDRIAL GSH AND OXIDATIVE STRESS |
Ten to twenty percent of the intracellular GSH is in the mitochondria
and a small percentage is in the endoplasmic reticulum (78). The
mitochondrial GSH pool is solely derived from the activity of a
mitochondrial transporter that translocates GSH from the cytosol to the
mitochondrial matrix (156). Mitochondria do not possess the enzymes
-GCS or -GT (156). Mitochondria normally produce a substantial
quantity of ROS (e.g.,
H2O2
and O2·), which are normally
broken down by GSH-dependent peroxidase-catalyzed reactions. Hence it
is possible that the generation of ROS either endogenously or under
oxidative stress may partly be regulated by mitochondrial GSH.
Mitochondrial GSH deficiency leads to injury to lung cells and lamellar
body formation (150). Animals treated with BSO, an inhibitor of
-GCS, show a low cytosolic GSH level and a 40% decrease in
mitochondrial GSH levels in the cells (150). However, Smith and
Anderson (231) have reported that there is no relationship between
mitochondrial GSH levels and the susceptibility to oxygen-induced
lung damage in mice. This study, however, was performed in whole lung
tissue, and it may be that individual cells such as alveolar epithelial and capillary endothelial cells are susceptible to oxidant-induced damage.
Mitochondrial GSH may also be susceptible to the oxidative stress
imposed by TNF- and by products of chemotherapeutic drug metabolism
in various cell lines and in human lungs (213, 222). TNF- is known
to deplete cytosolic GSH levels transiently in lung epithelial cells
(196). This depletion by TNF- is thought to be due to oxidative
stress from mitochondrial generation of O2· in the electron transport
chain (190). Cigarette smoke, which contains many electrophilic
compounds and ROS, also depletes cytosolic GSH levels in alveolar
epithelial cells in vitro and in lungs in vivo (129, 200) and
mitochondrial DNA mutation in human lungs (76). It is likely that
mitochondrial GSH plays a key role in maintaining cellular antioxidant
defense system and thus cell integrity under conditions of various
oxidative stress. Recent studies (6, 182) have shown that mitochondrial gene transfer of glutathione reductase and overexpression of
GPx in various cell lines provided protection against oxidative
stress. This suggests that the GSH redox system and its enzymes such as glutathione reductase and GPx may be important in the protection of
mitochondrial and cellular functions under oxidative stress such as
cigarette smoke in the lungs.
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OXIDANT-ANTIOXIDANT IMBALANCE IN SMOKERS AND PATIENTS WITH COPD:
ROLE OF LUNG GSH |
TOP
ABSTRACT
INTRODUCTION
-GLUTAMYL...
