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The central role of glutathione in the pathophysiology of human disease

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The central role of glutathione in the pathophysiology of
human diseases
R. Franco a; O. J. Schoneveld a; A. Pappa b; M. I. Panayiotidis c
a
Laboratory of Signal Transduction, National Institute of Environmental Health
Sciences, National Institutes of Health, Research Triangle Park, NC
b
Department of Molecular Biology and Genetics, Democritus University of Thrace,
Alexandroupolis, Greece
c
Environmental & Occupational Health, School of Public Health, University of Nevada at Reno, Reno, NV

Online Publication Date: 01 October 2007

To cite this Article: Franco, R., Schoneveld, O. J., Pappa, A. and Panayiotidis, M. I. (2007) 'The central role of
glutathione in the pathophysiology of human diseases', Archives Of Physiology And Biochemistry, 113:4, 234 - 258
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REVIEW ARTICLE

The central role of glutathione in the pathophysiology of human diseases

R. FRANCO1*, O. J. SCHONEVELD1, A. PAPPA2, & M. I. PANAYIOTIDIS3

1
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, NC 27709, 2Department of Molecular Biology and Genetics, Democritus University of Thrace,
Dimitras 19, 68100 Alexandroupolis, Greece and 3Environmental & Occupational Health, School of Public Health, University
of Nevada at Reno, Reno, NV

Abstract
Reduced glutathione (L-g-glutamyl-L-cysteinyl-glycine, GSH) is the prevalent low-molecular-weight thiol in mammalian
cells. It is formed in a two-step enzymatic process including, first, the formation of g-glutamylcysteine from glutamate and
cysteine, by the activity of the g-glutamylcysteine synthetase; and second, the formation of GSH by the activity of GSH
sythetase which uses g-glutamylcysteine and glycine as substrates. While its synthesis and metabolism occur intracellularly, its
catabolism occurs extracellularly by a series of enzymatic and plasma membrane transport steps. Glutathione metabolism and
transport participates in many cellular reactions including: antioxidant defense of the cell, drug detoxification and cell
signaling (involved in the regulation of gene expression, apoptosis and cell proliferation). Alterations in its concentration have
also been demonstrated to be a common feature of many pathological conditions including diabetes, cancer, AIDS,
neurodegenerative and liver diseases. Additionally, GSH catabolism has been recently reported to modulate redox-sensitive
components of signal transduction cascades. In this manuscript, we review the current state of knowledge on the role of GSH
in the pathogenesis of human diseases with the aim to underscore its relevance in translational research for future therapeutic
treatment design.

Key words: GSH, apoptosis, human diseases, human disorders, ROS, free radicals, oxidative stress, thiols, glutathionylation,
glutathiolation, glutathione depletion, glutathione S-transferases, glutathione peroxidase, glutathione reductase, nitrosylation.
.
Abbreviations: GSH, reduced glutathione; GSSG, glutathione disulfide; GS , thyil radical; ROS, reactive oxygen
species; RNS, reactive nitrogen species; RS, reactive species; g-GCS, g-glutamylcysteine synthetase; GS, glutathione
synthetase (synthase); g-GT, g-glutamyl transpeptidase; GPX, glutathione peroxidase; GR, glutathione reductase;
GST, glutathione S-transferase; GSNO, nitrosoglutathione; OATP, organic anion transporting polypeptide; MRP,
multidrug resistance proteins; OA7, organic anions; NAC, N-acetyl-L-cysteine; NO, nitric oxide; OH, hydroxyl
.

radical; H2O2, hydrogen peroxide; O27, superoxide anion; ONOO7, peroxynitrite.

.

properties grow exponentially. Today it is widely

Introduction
Thiol containing compounds are transcendental in
many biochemical and pharmacological reactions
(Dickinson & Forman, 2002). In its reduced form,
glutathione (L-g-glutamyl-L-cysteinyl-glycine, GSH)
is the most abundant non-protein thiol in mamma-
lian cells and the prevalent low-molecular weight
peptide in eukaryotic cells (Figure 1). It was initially
described as a potent reducing agent; however, many
other cellular functions have been recently ascribed
to GSH, making the scientific interest in its biological
accepted that GSH not only acts as a reducing agent
and a major antioxidant within the cells maintaining
a tight control of the redox status, but it also acts as
mediator of many other physiological reactions
including cellular signaling (involved in cell cycle
regulation, proliferation and apoptosis), metabolism
of xenobiotics, thiol disulfide exchange reactions,
and also as an important reservoir of cysteine. It is
not surprising then that a large body of bibliography
is available on the role of GSH in several
human diseases including cancer, neurodegenerative

Correspondence: Dr. R. Franco, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences (NIEHS), National Institutes of
Health (NIH), PO Box 12233, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709. Tel: (919) 541-5387. Fax: (919) 541-1898. E-mail:
franco2@mail.nih.gov, *Unidad de Biomedicina, Facultad de Estudios Superiores-Istacala, Universidad Nacional Autónoma de México, Tlalnepantla, México,
México. E-mail: rfranco&campus.iztacala.unam.mx and Dr. M. I. Panayiotidis, Environmental & Occupational Health, School of Public Health, University of
Nevada at Reno, Reno, NV. Tel: 775-682-7082. Fax: 775-784-1340. E-mail: panayiotidism@unc.edu

Received for publication 16 March 2007. Accepted 20 July 2007.

ISSN 1381-3455 print/ISSN 1744-4160 online ª 2007 Informa UK Ltd.
DOI: 10.1080/13813450701661198

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Figure 1. Structure of reduced glutathione (GSH). Glutathione (L-g-
glutamyl-L-cysteinyl-glycine, GSH) is a linear tripeptide formed
from the amino acids glycine, cysteine and glutamate (MW
307.4 g mol71). The peptidic g-linkage between glutamate and
cysteine protects GSH from hydrolysis by intracellular peptidases,
while the presence of the C-terminal glycine protects it against
cleavage by intracellular g-glutamylcyclotransferases. The cysteinyl
moiety of GSH provides the reactive thiol group (-SH group) that
allows GSH to participate in a wide variety of metabolic processes
including oxidation-reduction (redox) reactions and nucleophilic
displacement or addition-type reactions.
diseases (Alzheimer, Parkinson), acquired immune
deficiency syndrome (AIDS), aging, cystic fibrosis,
liver and heart disease, ischemia, stroke, seizure,
sickle cell anemia and metabolic diseases (diabetes
and obesity) (Sies, 1999; Reid & Jahoor, 2001;
Jefferies et al., 2003; Pompella et al., 2003; Town-
send et al., 2003; Dalton et al., 2004; Wu et al.,
2004). The aim of this review is to summarize and
highlight the molecular events that regulate the
pathogenesis of distinct human diseases by altera-
tions in GSH metabolism, transport and catabolism.
Glutathione synthesis, transport and
catabolism
Glutathione is a ubiquitous molecule that is pro-
duced intracellularly in all organs and cell types, but
it is more abundant in liver and lung tissues. It is a
linear tripeptide formed from the amino acids
glycine, cysteine and glutamate (M.W. 307.4 g
mol71) (Figure 1), attaining concentrations of 1 to
10 mM in many different cell types. Plasma GSH
concentrations on the other hand are relatively low
*0.01 mM, mainly because of its rapid catabolism.
Intracellular GSH can exist as a monomer in its
reduced form, or as a disulfide dimmer formed due
to its oxidation (GSSG) which usually accounts for
less than 1% of the total intracellular glutathione
content. Additionally, an important fraction of total
intracellular GSH can be found as thioester, mercap-
tide or other thioester forms (GSH conjugates).
Glutathione is 85 to 90% freely distributed in the
cytosol, although it can be compartmentalized in
GSH in human health and disease 235
different organelles including mitochondria, peroxi-
somes, nuclear matrix and endoplasmic reticulum
after its cytosolic synthesis (Valencia et al., 2001b;
Valencia et al., 2001d; Jefferies et al., 2003; Wu et al.,
2004).
The intracellular concentration of GSH reflects a
dynamic balance between synthesis, consumption
rate (metabolism), and its transport. Glutathione
synthesis starts by the formation of g-glutamylcysteine
synthetase, a dipeptide form by the combination
of glutamate and cysteine catalyzed by the
g-glutamylcysteine synthetase (g-GCS), also known
as g-glutamylcysteine ligase (Figure 2). In this
reaction, the g-carboxyl group of glutamate reacts
with the amino group of cysteine to form a peptidic
g-linkage that protects GSH from hydrolysis by
intracellular peptidases. After this initial step, glycine
is added to g-glutamylcysteine by the activity of GSH
sythetase (GS). Both enzymatic steps consume one
molecule of ATP per catalytic cycle. The presence of
the C-terminal glycine protects it against cleavage by
intracellular g-glutamylcyclotransferases. Glutathione
acts as a feedback inhibitor of its synthesis since it can
competitively inhibit the g-GCS activity. On the other
hand, the availability of cysteine is the rate-limiting
factor in GSH formation (Sies, 1999; Dickinson &
Forman, 2002; Wu et al., 2004; Estrela et al., 2006).
Glutathione can also be synthesized through sal-
vage pathways that involve its catabolism (Figure 3),
or through its recycling after its oxidation (this is
discussed in the next section). Glutathione catabo-
lism occurs extracellularly by the activity of the
g-glutamyl transpeptidase (g-GT). The g-GT is
expressed mainly on the apical surface of cells and
initiates the catabolism of not only GSH, but of
glutathione S-conjugates and glutathione-complexes.
g-Glutamyl transpeptidase removes the g-glutamyl
moiety from GSH and GSH-conjugated compounds
by transferring it to other acceptors (amino acids or
other dipeptides), producing cysteinylglycine or
cysteinylglycine-conjugates. These products are
further hydrolyzed by ectoprotein dipeptidases
which remove the peptide bond between
cysteine and glycine. Cysteine, together with the
g-glutamyl-amino acids formed from the action of the
g-GT, are then uptaken by the activity of specific
transporters. The g-glutamyl-derivatives are further
substrates for the g-glutamyl cyclotransferase that
forms 5-oxoproline and the corresponding amino
acid. 5-Oxoproline is finally converted to glutamate
by the activity of the 5-oxoprolinase. On the other
hand, S-cysteine conjugates, as a result of GSH
S-conjugates breakdown by g-GT, are acetylated on
the amino group of the cysteinyl residue by intracel-
lular N-acetylfransferases to form mercapturic acids
(Paolicchi et al., 2002; Estrela et al., 2006).
Some studies have suggested the existence of a
Naþ-coupled GSH transport influx based on the idea
that rates of intracellular GSH synthesis and efflux
across the plasma membrane may not account by
 236 R. Franco et al.
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Figure 2. Overview of de novo glutathione synthesis. Reduced glutathione or GSH is a tripeptide formed by the amino acids glutamate, cysteine
and glycine, whose availability in the cell depends on cellular uptake by amino acid transporters or on other biochemical pathways.
Glutathione synthesis is mediated by the activity of the g-glutamylcysteine synthetase (g-GCT) and the glutathione synthetase (GS).
Glutathione is freely distributed in the cytosol, reaching a concentration of up to 10 mM in animal cells, and it can be compartmentalized (up
to 15%) to some degree in mitochondria, endoplasmic reticulum or nuclei matrix. Both cytosolic and compartmentalized GSH have been
reported to participate in distinct biological processes.

Figure 3. Glutathione salvage pathway. Glutathione can also be synthesized through salvage pathways that involve its catabolism. Glutathione
catabolism is mediated by the g-glutamyl transpeptidase (g-GT) expressed mainly on the apical surface of cells. It mediates the catabolism of
not only GSH, but of GSSG and GSH-conjugates. g-Glutamyl transpeptidase removes the g-glutamyl moiety from GSH and GSH-
conjugated compounds by its transfer to other acceptors (amino acids, aa, or other dipeptides), thus producing cysteinylglycine (Cys-Gly) or
cys-gly-conjugates, and g-glutamyl-amino acid (g-Glu-aa) products. These products are further hydrolyzed by ectoprotein dipeptidases
(DPT) which remove the peptide bond between cysteine and glycine. The g-glut-aa, glycine and cysteine (or cys-conjugates) formed from the
action of the g-GT are then uptaken by the activity of specific transporters. The g-glutamyl-derivatives are further substrates for the
g-glutamyl cyclotransferase (g-GCT) which forms 5-oxoproline and the corresponding amino acid. 5-Oxoproline is finally converted to
glutamate by the activity of the 5-oxoprolinase (OXP). On the other hand, S-cysteine conjugates, as a result of GSH S-conjugates breakdown
by g-GT, are acetylated on the amino group of the cysteinyl residue by intracellular N-acetylfransferases (NAT) to form mercapturic acids.
 GSH in human health and disease 237

themselves for the maintenance of relatively high protein (MRP, encoded by ABCC genes) that is a
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intracellular GSH concentrations. However, its ex-
istence is still controversial (Gukasyan et al., 2006).
In the basolateral membrane of renal proximal
tubular cells, the existence of two different GSH
influx transporters has been proposed. The first one
involves a Naþ-independent mechanism which has
been proposed to be mediated by the organic anion
transporters 1 and 3 (OAT1/3) that act as exchangers
of GSH for 2-oxoglutarate. On the other hand a
second Naþ-dependent influx of GSH has been attri-
buted to the sodium dicarboxylate cotransporter-2
(SDCT-2) (Lash, 2005). However, the biological
importance of these transporters in GSH uptake in
other tissues and cell lines remains to be explored.
Because GSH degradation (catabolism) occurs
extracellularly, the export of GSH, GSSG and
GSH-adducts is an important step in its turnover.
Relatively little is known at the molecular level about
GSH efflux transporters and pumps (Figure 4). To
date two GSH transporter families have been well
implicated in GSH efflux across the plasma mem-
brane. These include the multidrug resistance
subfamily of the ATP binding cassette transporters
(ABC transporters), and the organic anion transport
polypeptide proteins (OATP, encoded by SLCO
genes) (Hammond et al., 2001; Ballatori et al., 2005).
The MRP transporters have been demonstrated to
act as cotransporters of organic anions (OA7) and
GSH. In addition, they also act as transporters of
GSH-conjugated xenobiotics and GSH-conjugated
metabolites (e.g. the cysteinyl leukotriene C4) that
must be exported from the cells in which they are
formed to avoid deleterious effects. This efflux
confers drug resistance to tumor cells and can
protect normal cells from toxic insults. Multidrug
resistance proteins also export GSSG thus suggesting
a role of MRP proteins in cellular responses to
oxidative stress. In any case, MRP transport of
organic anions including drugs and conjugated OA7
requires the presence of GSH and the hydrolysis of
ATP (Ballatori et al., 2005; Cole & Deeley, 2006).
On the other hand the OATP transporters were
initially proposed to act as exchangers of GSH/OA7.
In this way, GSH efflux is stimulated by the presence

Figure 4. Glutathione transporters. Plasma membrane GSH efflux pumps. Two GSH transporter families have been implicated in GSH efflux
across the plasma membrane. The first group includes the multidrug resistant protein family (MRP) which acts as cotransporters of GSH
coupled to the extrusion of an OA7 (organic anion) and requires the use of energy. MRP proteins have also proposed to transport GSSG and
GSH-conjugates. The second group includes the organic anion transporting polipeptide proteins OATP which act as GSH/OA- exchanger.
This exchange is stimulated by the presence of high extracellular concentrations of OA7 and is driven by the electrochemical gradient of
GSH across the plasma membrane. Other proposed candidates for GSH efflux are hemichannels (connexins, CX), CFTR and RLIP76.
Mitochondrial GSH transporters. The charged nature of GSH (negative at physiological pH) suggests that GSH cannot passively diffuse into
the mitochondria, because the mitochondrial matrix has a negative potential relative to the cytoplasm. Some of the strongest candidates
involved in mitochondrial import of cytosolic GSH include the dicarboxylate transporter (DIC) and the 2-oxoglutarate transporter (OGC)
which act as exchangers of Pi2þ (inorganic phosphorous) and 2-oxoglutarate (2-OG27) for cytosolic GSH. This transport dissipates any
concentration gradient between cytoplasm and mitochondrial whose concentrations are both *5 mM. Additionally, mitochondrial
GSH transport allows this pool to be relatively independent from the cytosol, which when depleted does not affect the mitochondrial
GSH pool.
 238 R. Franco et al.
of a wide range of structurally unrelated OA7
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substrates (trans-stimulation) demonstrating the
wide inespecificity of the OA7 binding site in the
OATP proteins. Glutathione transport by OATP
proteins is driven by the outwardly directed electro-
chemical gradient across the plasma membrane, thus
it can be reversed by increases in the extracellular
GSH concentration demonstrating its potential
bidirectionality. As mentioned earlier, GSH is pre-
sent in high concentrations within the cells (10 mM),
whereas blood plasma concentrations are three
orders of magnitude lower (approximately
0.01 mM). Because GSH is negatively charged at
physiological pH, there is also a large negative
intracellular potential (730 to 760 mV) that facil-
itates its extrusion from the cell. In this way, the
combined chemical and electrical GSH gradients
serve as a powerful driving force for cellular uptake of
solutes by OATP (Hagenbuch & Meier, 2004;
Ballatori et al., 2005). Recently, however, some
studies have challenged the view of OATP transpor-
ters as GSH/OA7 exchangers. Bi-directional GSH/
OA7 has been reported in different cell types
including human cell lines (Garcia-Ruiz et al.,
1992; Sze et al., 1993; Iantomasi et al., 1997; Li
et al., 1998; Li et al., 2000; Lee et al., 2001; Ng et al.,
2003). Although OATP (human)/Oatp (rat, mouse)
proteins were initially reported to mediate this
exchange transport (Li et al., 1998; Li et al., 2000;
Hagenbuch & Meier, 2004), recent studies have
suggested that GSH/OA7 exchange is not mediated
by this family of transporters (Briz et al., 2006;
Mahagita et al., 2007). Thus there is a strong
possibility that GSH/OA7 is mediated by a different
and still uncharacterized entity.
Other less characterized pathways for GSH and
GSH-conjugates efflux transport are those involving
the cystic fibrosis transmembrane conductance
regulator (CFTR), the Ral-regulated effector pro-
tein (RLIP76), and hemichannels (connexins).
CFTR (ABCC7) is a member of the ABCC/MRP
family of transporters which in addition to its
function as a chloride channel, has also been
suggested to mediate either direct transport of
GSH or to modulate GSH transport by other
proteins (Hudson, 2001). RLIP76 (RALBP1) is a
76 kDa Ral-binding, Rho/Rac-GAP and Ral effector
protein which was recently proposed to be a novel
multispecific transporter of xenobiotics as well as
GSH-conjugates with inherent ATPase activity
(Awasthi et al., 2003). Finally, connexins are a
large family of transmembrane proteins that, when
apposed to other cells’ connexins, form functional
intercellular channels or gap junctions; and when
unapposed, form hemichannels. They control the
diffusion driven passage of ions and molecules of a
molecular mass of up to 1 kDa. Recently, hemi-
channels have been suggested to mediate the efflux
of glutathione in astrocytes (Contreras et al., 2004;
Rana & Dringen, 2006).
Distinct biological functions of GSH
metabolism
The cysteinyl moiety of GSH provides the reactive
thiol group (-SH group) that allows GSH to
participate in a wide variety of metabolic processes
including oxidation-reduction (redox) reactions and
nucleophilic displacement or addition-type reactions.
Glutathione reacts widely to form a number of
different metabolites. These reactions can be divided
into those involving principally the g-glutamyl por-
tion of the tripeptide such as in the case of the
reactions mediated by g-glutamyl transpeptidases,
and those involving the sulfhydryl moiety that
include oxidation-reduction and nucleophilic reac-
tions (Meister, 1995a; Meister, 1995b; Jefferies et al.,
2003; Wu et al., 2004).
Antioxidant and redox balance regulator
Glutathione is a strong reducing agent that con-
tributes to key antioxidant metabolic pathways by
acting either as a proton donor, or as a cofactor of
nucleophilic conjugates. Because GSH constitutes
the major intracellular antioxidant defense within the
cells, its depletion might be either a cause or a
prerequisite for the generation of ROS which could
potentially mediate a wide range of signaling events
(Figure 5, Table I). In this way, GSH has been
shown to scavenge a wide range of reactive species
(RS) including reactive oxygen species (ROS) and
reactive nitrogen species (RNS). In this way, GSH
can scavenge superoxide anion ( O .
2 ), hydroxyl
radical ( OH7), and singlet oxygen (1O2) directly,
.
by donating electrons and becoming oxidized to thyil
.
radical (GS ). It is important to mention that the
reductive ability of GSH, is associated with the
.
formation of thiyl radical (GS ) which if not efficiently
removed (by ascorbate for example), can lead to pro-
oxidant reactions and peroxidative injury (Wlodek,
.
2002). Two GS species can react further to form a
GSSG molecule. In general, RS are reduced or
inactivated through the generation of disulfide bonds
between two glutathione molecules to form GSSG.
Moreover, GSH can directly scavenge protein- and
DNA-radicals. Additionally, non-enzymatic reaction
of nitric oxide (NO) with GSH convey to the
formation of the relatively stable S-nitrosoglutathione
(GSNO), which can be inactivated by its conjugation
to proteins through S-nitrosylation reactions. Glu-
tathione can also catalytically detoxify cells from
hydroperoxides (H2O2), peroxynitrite (OONO7)
and lipid peroxides by the action of GSH peroxidases
(GPXs), peroxiredoxins (PXRs), and phospholipid
hydroperoxide GSH peroxidases (PHGPX) (Mates,
2000; Valko et al., 2007). Glutathione peroxidases
are selenoproteins that vary in their peroxide
substrate specificity. On the other hand, peroxire-
doxins catalyze the reduction of H2O2 by thiols
including GSH using cysteine in its thiolate (S7)
 GSH in human health and disease 239
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Figure 5. Metabolism of GSH. GSH metabolism can be arbitrarily classified in three different types of functions including antioxidant, as a
mediator of thiol/exchange reactions, and as a factor in nucleophile conjugation. As a strong reducing agent, GSH is involved in free radical
. .
or reactive species (RS) and metal oxido-reduction reactions that lead to the formation of the thyil radical GS . Two GS species can further
react and form a GSSG molecule (1). Additionally, GSH can catalytically detoxify cells from peroxides (PX) by the action of GSH
peroxidases (GPXs) peroxiredoxins (PXRs) and phospholipid hydroperoxide GSH peroxidases (PHGPX). Glutathione disulfide (GSSG) is
further reduced to GSH by NADPH through glutathione reductase (GR) (2). The antioxidant role of GSH is also mediated by its role in
other primary antioxidant systems such as that of the dehydroascorbate reductase (DHR) which regenerates oxidized ascorbate
(dehydroascorbate) (3). Glutathione also participates in thiol/exchange reactions (S-glutathionylation and S-nitrosylation) as a reversible
mechanism for post-translational modification of proteins. The main reactions involved in this phenomenon are protein-SH/GSSG exchange
reactions and NO mediated formation of S-nitrosoglutathione (GSNO) and which mediates S-nitrosylation of proteins. Disulfide linkages
are mainly removed (dethiolation or deglutathionylation) by the activity of enzymes such as glutaredoxin (GRX) or thioredoxin (TRX) (4).
Finally, as a nucleophile, GSH can react with a wide variety of electrophiles (including xenobiotics and endogenous metabolites) in a reaction
catalyzed by glutathione transferases (GST) thus leading to the formation of GSH-conjugates or adducts (5).

form in their active site rather than selenium.
Furthermore, H2O2 can spontaneously react with
S7 to form sulfenic acid. Since these S7 molecules
are often part of active sites of a variety of proteins
including protein tyrosine phoshatases, alteration of
protein function by H2O2 can be modulated by GSH.
Other enzymes including GSH-transferases (GST)
have been shown to help in the metabolism of RS like
NO and peroxides. Finally, GSH also participates in
the antioxidant defense against carbon-centered
radicals. (Mates, 2000; Valko et al., 2007).
Glutathione disulfide formed in the process of RS
scavenging, also known as oxidized GSH, is reduced
to glutathione by the action of glutathione reductase
(GR) (Wu et al., 2004) (Figure 5). Glutathione
reductase is a flavoenzyme represented as a single-
copy gene in humans. It requires reduced nicotina-
mide adenine dinucleotide phosphate (NADPH) as
an electron donor reductant, which is maintained
predominantly by the pentose phosphate shunt.
Under physiological conditions GSSG is maintained
at *1% of total glutathione concentration and
increases in GSSG are counteracted by its rapid
reduction by GR (Forman et al., 2002). However,
severe oxidative stress impairs GR activity leading to
increased GSSG accumulation in the cell which in
most cases can be actively extruded through specific
transporters. The reducing power of ascorbate also
helps to maintain systemic GSH homeostasis
(Gutierrez-Correa & Stoppani, 1997; Vander Jagt
et al., 1997). Glutathione reductase has been
reported regulated at the expression level by several
oxidative stress conditions (Masella et al., 2005).
Recently the activity of both GR and GPX have been
shown to be important predictors of the general
tissue redox state (Yang et al., 2006a).
The antioxidant function of GSH is also mediated
by its role in other primary antioxidant systems.
Several GSH-dependent enzymes have been de-
scribed to have dehydroascorbate reductase activity.
In addition, novel dehydroascorbate reductases
(DHR) have been reported to act as GSH-dependent
enzymes. In this case, DHR activity is provided by
the electron donor capacity of GSH that allows the
 240 R. Franco et al.
regeneration of oxidized ascorbate (dehydroascor-
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bate) (Meister, 1994; Maellaro et al., 1997; Masella
et al., 2005). Glutathione may also aid in the storage
and transfer of cysteine because of its reducing
properties. Cysteine’s rapid autoxidation to cystine
produces potentially toxic oxygen radicals and so
most of the non-protein cysteine is stored as GSH.
Thus, the ability of many cells to transport GSH into
the extracellular compartment may help in the
transport of cysteine and might also protect the cell
membrane by reducing harmful reactive compounds
such as RS (Meister, 1995a; Meister, 1995b; Jefferies
et al., 2003; Wu et al., 2004).
Changes in the intracellular thiol-disulfide (GSH/
GSSG) balance within the cell can be used as an
indicator of the redox status of the cell. As mentioned
above, under conditions of oxidative stress, GSH is
consumed by scavenging free radicals while GSSG
levels increase. The ratio of [GSH/GSSG] and the
reduction potential (mV) are two ways to express
the intracellular redox environment of the cell. The
GSH/GSSG couple is considered to be a cellular
redox buffer because it provides a very large pool of
reducing equivalents. Whereas many proteins are
active when the key sulfhydryls are in the thiol form,
others require them to be in the oxidized disulfide
Table I. Examples of GSH-mediated oxido-reduction reactions. At
physiological pH (7.4) and in solution, GSH becomes dissociated
to GS7 (thiolate anion) þ Hþ. Thus, GSH in its GS7 form
mediates most of the reactions described below.
Precursors Products
GSH þ OH7
. .
GS þ H2O
GSH þ O
.
þ GS þ H2O
2 þH
GSH þ NO þ O2 GSNO þ O2
. .
GSH þ R GS þ RH
GSH þ DNA
. .
GS þ DNA
2GSH þ H2O2 GSSG þ 2H2O
2GSH þ LOOH GSSG þ LOH þ H2O
2GSH GSSG þ Ascorbic acid
þ Dihydroascorbate
2GSH þ Protein-SSX GSSG þ Protein-(SH2)X
GSH þ Protein-SSG GSSG þ Protein-SH
GSH þ Mn Mn71 þ GS
.
GSH þ PGG2 GSSG þ PGH2
.
GS þ GS
.
GSSG
GSSG þ NADPH þ Hþ 2GSH þ NADPþ
Abbreviations: GSH, reduced glutathione; GSSG, glutathione
.
disulfide; GS , thyil radical; GPX, glutathione peroxidase; GR,
glutathione reductase; GSNO, nitrosoglutathione; NO, nitric
oxide; OH, hydroxyl radical; H2O2, hydrogen peroxide; O27,
. .
superoxide anion; NADPH, b-nicotinamide adenine dinucleotide
phosphate; Protein-SSG; protein mixed disulfide; Protein-SSX,
protein disulfide bond; LOOH, lipid hydroperoxide; LOH,
. .
alcohol; R , secondary radical; DNA , DNA radical; M, metals;
Protein-SH, protein sulhydril or protein thiol; PGG2, prostaglan-
din G2; PGH2, prostaglandin H2; PES; Prostaglandin endoper-
oxide synthetase; GRX, glutaredoxin.
form. Thus, changes in the GSH/GSSG equilibrium
within the cell may regulate certain metabolic path-
ways by activating or inactivating key enzymes.
Without a process to reduce protein disulfides,
vulnerable cysteinyl residues of essential enzymes
might remain oxidized, leading to changes in their
catalytic activity (see section below). Higher-order
thiol cell systems like metallothioneins and thior-
edoxins are regulated by the GSH/GSSG balance
(Schafer & Buettner, 2001; Jones, 2006).
Thiol/exchange reactions. Glutathionylation
(glutathyolation) of proteins
Several proteins involved in cellular signaling have
critical thiols whose function can be altered by redox
modifications, including receptors, kinases, tran-
scription factors and proteins involved in ubiquitina-
tion. Proteins with sulhydryls 7SH (thiol groups)
can respond to oxidative stress by forming disulfides.
The formation of disulfide bonds within a protein
(intra) or between two proteins (inter) are reversed
by protein disulfide isomerases. On the other hand,
the formation of mixed disulfides with low-molecular
mass thiols such as cysteine, homocysteine and GSH
(s-thiolation) generates mixed protein/non-protein
disulfides (s-thiolated proteins). Because GSH is the
most available substrate in these reactions most of the
S-thiolation processes involve S-glutathionylation of
proteins. The importance of GSH in mixed disulfide
formation is demonstrated by the observation that
nearly 85% of mixed protein disulfides contain GSH.
Potential modes for S-glutathionylation modification
of protein thiols are exemplified in Table II. The
Table II. Potential modes for GSH mediated S-glutathionylation
and S-nitrosylation.
S-Glutathionylation
Precursors Products
GSSG þ Protein-SH Protein-SSG þ GSH
GSH þ Protein-SOH Protein-SSG þ H2O
. .
GS þ Protein-S Protein-SSG
GS-OH þ Protein-SH Protein-SSG þ H2O
.
GSNO þ Protein-S Protein-SSG þ NO
GSNO þ Protein-S7 Protein-SSG þ NO7
GSH þ Protein-SNO Protein-SSG þ NO
Protein  SSG þ O2
.
GSH þ Protein-S þ O2
GS þ Protein-S7 þ O2 Protein  SSG þ O2
.
S-Nitrosylation
GSNO þ Protein-SH Protein-SNO þ GSH
Abbreviations: GSH, reduced glutathione; GSSG, glutathione
.
disulfide; GS , thyil radical; GSNO, nitrosoglutathione; NO, nitric
oxide; O27, superoxide anion; Protein-SSG; protein mixed
.
disulfide; Protein-SSX, protein disulfide bond; Protein-SH,
protein sulhydryl or protein thiol; Protein-SOH, Protein sulfenic
acid; Protein-SNO, protein nitrosothiol; Protein-S7, protein
.
thiolate anion; Protein S , protein thyil radical.

main non-enzymatic mechanisms for S-glutathiony-
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lation of proteins involve: (1) reaction of GSH with
partially oxidized protein-(SH) groups (sulfenic
acids); (2) protein-SH/GSSG exchange reactions;
.
(3) protein-SH/GSH-GS radical reactions; and (4)
NO-mediated formation of S-nitrosoglutathione
.
(GSNO) and GS radicals (S-nitrosation or
S-nitrosylation). It has been demonstrated that a
significant basal level of protein mixed disulfides
(protein-SSG) of around 1% is found within cells,
and where there is a significant increase in GSSG
content due to oxidative stress, protein-SSG will
concomitantly increase. S-Glutathionylation has also
been observed under basal conditions in the absence
of oxidative insults. S-Glutathionylation of proteins
(considered a reversible post-translational modifica-
tion of proteins) has a dual role in protecting against
irreversible protein thiol oxidation and in regulating
protein function. Once formed, mixed disulfide
linkages are removed by changes in the intracellular
GSH/GSSG balance and/or the activities of protein
disulfide reductase, thioredoxin, glutaredoxin (thiol-
transferase) enzymes and other reducing agents
(dethiolation or deglutathionylation). The enzymatic
specificity and efficiency of dethiolating reactions has
been observed to depend on the structural con-
formation of the disulfide (Pompella et al., 2003;
Giustarini et al., 2004; Di Simplicio et al., 2005;
Shackelford et al., 2005; Biswas et al., 2006; Cross &
Templeton, 2006).
Glutathionylation of electrophiles
As a nucleophile, GSH can react with electrophiles
that are generated as a consequence of metabolic
processes involving both endogenous compounds
and xenobiotics (Figure 5, Table III). Conjugation of
GSH is an essential aspect of both xenobiotic and
normal physiological metabolism. In general, the
Table III. GSH-mediated conjugation reactions.
Precursors Products
2GSH þ M2þ GS-M-SG
GSH þ PGA1 S-(PGA1)-GSH
GSH þ Hepoxilin A3 Hepoxilin A3-C
GSH þ 4-HNE 4-HNE-GSH
GSH þ Dopamine þ 2O2 SS-gluathionyl-
dopamine þ 2HO2
GSH þ Methylgyoxal D-lactate þ GSH
2GSH þ 2ATP þ Trypanothione þ
Spermine 2ADP þ 2Pi
Abbreviations: GSH, reduced glutathione; M, metal; PGA1,
prostaglandin A1; 4-HNE, 4-hydroxynonenal; GXI, glyoxylase I;
GXII, glyoxylase II; GSS; glutathionylspermidine synthetase; TS,
trypanothione synthetase.
GSH in human health and disease 241
binding of GSH to endogenous compounds serves
to: (a) limit and regulate the activity of chemicals; (b)
facilitate their membrane transport and elimination
from the cell and organism; and (c) result in the
formation of important intermediates (van Bladeren,
2000; Townsend et al., 2005). Glutathione forms
conjugates with a great variety of highly reactive
electrophiles by either direct interaction (i.e. non-
catalyzed); or by a reaction catalyzed by GST
(S-glutathionylation). Glutathione reacts with var-
ious electrophiles (e.g. arene oxides, unsaturated
carbonyls, organic halides), physiological metabolites
(e.g. estrogen, melanins, prostaglandins and leuko-
trienes), and xenobiotics (e.g. bromobenzene and
acetaminophen). In this way, GSH forms thioether
conjugates with compounds like leukotrienes, pros-
taglandins, hepoxilins, nitric oxide hydroxyalkenals,
ascorbic acid, dopa, dopamine and maleic acid, while
it forms thioesthers with cysteine, coenzyme A,
proteins and other cellular thiols (Eaton & Bammler,
1999; van Bladeren, 2000). Via non-catalyzed reac-
tions GSH can also form thermodynamically stable
but kinetically labile mercaptides with a high affinity
for metals including mercury, silver, cadmium,
arsenic, lead, gold, zinc, and copper. In addition,
GSH also functions in the mobilization and delivery
of metals between ligands, in the transport of metal
across cell membranes, as a source of cysteine for
metal binding, and as a reductant or cofactor in
redox reactions involving metals (Wang & Ballatori,
1998).
The group of GSTs forms a class of enzymes with
overlapping substrate specificity, that generate a large
set of thioesthers, called glutathione S-conjugates.
Most of the GST isoforms are present in the
cytoplasm, although some are also present in the
endoplasmic reticulum and mitochondrial matrix.
This allows them to participate in the detoxification
of a chemically diverse group of compounds includ-
ing chemical carcinogens, environmental pollutants
and anti-tumor agents. The most common reactions
by GST involve nucleophilic attack by GSH to an
electrophilic carbon. These include those of satu-
rated (e.g. alkyl halides, lactones and epoxides) and
unsaturated carbon atoms (e.g. a, b-unsaturated
compounds, quinones, quinonimines and esters) as
well as aromatic carbon atoms (e.g. aryl halides and
aryl nitro compounds) (van Bladeren, 2000). The
electrophilic centers of these compounds undergo
nucleophilic substitution, nucleophilic addition to a
a, b-unsaturated ketones or epoxides, and nucleo-
philic attack on electrophilic oxygen (Townsend
et al., 2005). Glutathione-S-transferase also mediates
formation of GSNO from alkyl nitrite and GSH.
Moreover, GST can also serve as peroxidases,
isomerases and thiol transferases. In addition,
protein-protein interactions between GSTs and
signaling proteins such as JNK have been shown to
regulate signal transduction (Pompella et al., 2003;
Hayes et al., 2005). Finally, GSH conjugation has
 242 R. Franco et al.
been demonstrated to have a pro-oxidant activity
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by itself that can give rise to the formation of
compounds that are at least as reactive as the parent
electrophiles. In this way, GSH-conjugates extrusion
is an important mechanism of electrophile detoxifi-
cation although this may result in severe GSH
depletion. In addition, GSH-conjugation of metal
ions has been reported to generate ROS including
.
O27 and H2O2 (Pompella et al., 2003).
Glutathione compartmentalization
Glutathione compartmentalization is also an impor-
tant process which participates in many other
biological processes. Significant pools are found
within mitochondria, endoplasmic reticulum and
nuclear matrix. Mitochondria are highly dependent
on GSH for the prevention of oxidative damage due
to aerobic respiration and they rely on the salvage of
GSSG by GR and on the uptake of cytosolic GSH
across the outer membrane since they are incapable
of de novo synthesis. It has been proposed that GSH
must be actively transported into the mitochondria or
exchanged for another anion not only because the
matrix space of the mitochondria possesses a negative
membrane potential relative to the cytoplasm, but
also because GSH is a negatively charged molecule at
physiological pH. Some of the possible candidates
involved in mitochondrial import of cytosolic GSH
include the monocarboxylate, dicarboxylate, 2-ox-
oglutarate, tricarboxylate, and glutamate-hydroxide
and glutamate-aspartate transporters (Figure 4)
(Lash, 2006).
In contrast to the cytosol and mitochondria, the
lumen of the endoplasmic reticulum (ER) contains a
relatively higher concentration of GSSG which
allows the formation of disulfide bonds and their
isomerization through the activity of protein disulfide
isomerases and oxidoreductases. The high concen-
tration of GSSG in the ER is maintained by the influx
of reducing equivalents by either protein transloca-
tion, which provides with available cysteine residues,
and/or selective transport of GSH from the cytosol to
the ER. Protein disulfide isomerase (PDI) catalyzes
the formation of disulfide bonds, then, the resulted
reduced PDI is further oxidized by the flavoprotein
Ero1. The later generates ROS as byproducts that are
detoxified by GSH thereby generating high levels of
GSSG. Protein disulfide isomerase can be either
reduced by GSH or oxidized by GSSG. High luminal
GSSG concentrations are then secreted from the ER
(Chakravarthi et al., 2006).
Nuclear matrix is another important pool for
GSH. No active transport mechanisms for GSH
have been described so far for its nuclear import;
however, it is assumed that GSH is capable of
diffusing into the nucleus across nuclear pores.
Recent reports have suggested the critical role of
nuclear GSH pools in protecting DNA from oxida-
tive modifications that might, in turn, affect the
efficiency of the DNA repair machinery (Smith et al.,
1996; Green, 2003; Green et al., 2006).
Other biological functions of GSH
Glutathione has been reported to participate in
several other important biological processes. It also
serves as a substrate for the formaldehyde dehydro-
genase that converts formaldehyde and GSH to
S-formyl-glutathione. This is of physiological im-
portance because formaldehyde is a potent carcino-
gen produced from the metabolism of methionine,
choline, methanol sarcosine, and xenobiotics.
Glutathione is required as well for the conversion
of the arachidonic acid metabolite, prostaglandin H2,
into prostaglandins D2 and E2 by endoperoxide
isomerase. Furthermore, GSH is involved in the
glyoxylase system, which converts methylgyoxal to
D-lactate in microorganisms. Finally, GSH is a
cofactor for transhydrogenases in the reduction of
oxidized centers on DNA, proteins, and other
biomolecules (Table III) (Meister, 1995b; Sies,
1999; Jefferies et al., 2003; Wu et al., 2004).
Glutathione as a central player in human
diseases
Alterations in GSH homeostasis and metabolism
have been widely reported to be correlated with
several human diseases. Most of them have been
associated with depletion of cellular GSH levels.
Glutathione depletion can be induced by three
different mechanisms: (1) its oxidation by RS, (2)
its conjugation to proteins or electrophiles, and (3)
its extrusion across the plasma membrane. Under
these conditions, the cell demands GSH replenish-
ment through de novo GSH synthesis or salvage
pathways (catabolism). For example, GSH depletion
has been associated with the progression of cellular
death and cytotoxicity. On the other hand, high
intracellular GSH concentrations are responsible for
multidrug resistance observed in transformed (carci-
nogenous) cells. Finally, alterations in GSH-depen-
dent enzymes have been described in different
disease conditions. In the next section, we briefly
summarize the role of alterations in intracellular
GSH content and/or GSH-dependent enzymes or
processes in the progression of several pathological
conditions.
Apoptosis
Apoptosis or programmed cell death is a ubiquitous,
evolutionary, highly conserved process involved in
several biological phenomena. Under physiological
conditions, it is important not only in the constant
turnover of cells in all tissues but also during the
normal development and senescence of the organism.
Moreover, its deregulation has been observed to
occur as either a cause or consequence of distinct
 GSH in human health and disease 243

pathologies (Fadeel & Orrenius, 2005). Apoptosis is a changes involved in the generation of a permissive
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highly organized process characterized by the pro-
gressive activation of precise pathways leading to
specific biochemical and morphological alterations.
Initial stages of apoptosis are characterized by RS
formation, changes in intracellular ionic homeostasis,
cell shrinkage, loss of membrane lipid asymmetry and
chromatin condensation. Later stages associated with
the execution phase of apoptosis are characterized by
activation of execution caspases and endonucleases,
apoptotic body formation and cell fragmentation
(Hengartner, 2000; Franco et al., 2006).
Despite the large efforts on the characterization of
the molecular events leading to apoptosis, recent
reports have underlined the role of changes in the
intracellular milieu of the cells that are necessary for
the progression of the cell death program. Intracel-
lular GSH loss is an early hallmark in the progression
of cell death in response to different apoptotic stimuli
(Beaver & Waring, 1995; Ghibelli et al., 1998;
Obrosova et al., 1999; Hammond et al., 2004; Kern &
Kehrer, 2005), and it is thought to be part of the
apoptotic environment necessary for the activation
of apoptotic enzymes. Glutathione is essential for
survival as g-GCS knock-out mouse, which induces
GSH depletion, die from apoptotic cell death
(Dalton et al., 2004). Several studies have shown a
correlation between GSH depletion and the progres-
sion of apoptosis (Figure 6), for example, inhibition
of GSH release is able to rescue cells from apoptosis
(Ghibelli et al., 1998; Franco & Cidlowski, 2006;
Hammond et al., 2007). Moreover high [GSH]i
levels have been associated with an apoptotic
resistant phenotype (Chiba et al., 1996; Kohno
et al., 1996; Mehlen et al., 1996; Watson et al.,
1997; Meredith et al., 1998; Xu et al., 1999; Friesen
et al., 2004; Cazanave et al., 2006). Glutathione
depletion during apoptosis has been associated with
the activation of a plasma membrane transport
mechanism during apoptosis that is induced by both
extrinsic and intrinsic apoptotic pathway activators
(Ghibelli et al., 1995; van den Dobbelsteen et al.,
1996; Bojes et al., 1997; Ghibelli et al., 1998;

Figure 6. Role of GSH in apoptosis and cytotoxicity. Glutathione depletion is an important hallmark for apoptosis and cytotoxicity, and can be
depleted by different mechanisms. Apoptosis induced by physiological stimuli such as death receptor activation or cytokine withdrawal has
been shown to induce GSH loss by the activation of a plasma membrane efflux transport for GSH and not by its oxidation to GSSG (1).
Distinct apoptotic stimuli (death receptors), stress conditions (osmotic, hypoxia), and cytotoxic agents (such as xenobiotics and pollutants)
can generate or increase reactive species (RS) formation either by organelle-mediated damage, or by simple chemical reactions, which can
induce GSH depletion through its oxidation (GSSG formation) and/or conjugation (2). GSH depletion and RS formation can trigger an
amplification cascade which utilizes both increased RS formation and GSH depletion (3). Mitochondrial GSH pool has also been shown to
act as an important regulator of cellular death. The resulted GSSG and/or GSH-conjugates generated are toxic to the cell and must be
extruded by efflux pumps. This further depletes intracellular GSH pools by the impairment of GSSG recycling to GSH through the action of
the GR and NADPH. Finally, GSH depletion and RS formation might regulate the induction of apoptosis or cytotoxicity by RS-mediated
signaling (through ROS or RNS), thiol-exchange reactions or protein oxidation-modifications (S-glutathionylation, S-nitrosylation), or GSH
conjugation (by the action of GST) (4). GSH-T, GSH transporter involved in GSH depletion during apoptosis.
 244 R. Franco et al.
He et al., 2003; Hammond et al., 2004; Franco &
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Cidlowski, 2006; Hammond et al., 2007). On the
other hand, oxidation of GSH by RS has been
demonstrated to mediate its depletion during
apoptosis induced by cytotoxic agents that by them-
selves induce oxidative stress (see next section)
(Hengartner, 2000; Fu et al., 2001; Hiroi et al.,
2005; Cai et al., 2007). Recent studies have suggested
that both MRP (He et al., 2003; Benlloch et al., 2005;
Hammond et al., 2007) and GSH/OA7 exchange
transporters might be involved in GSH efflux during
apoptosis (Franco & Cidlowski, 2006). Additionally,
a recent study has shown that glutamate-L-cysteine
ligase also known as g-glutamyl cysteine synthetase,
the rate limiting enzyme in GSH biosynthesis, is a
direct target of caspase 3 (Franklin et al., 2002). This
may represent another mechanism for diminishing
GSH levels during apoptosis.
The mechanisms involved in GSH-induced mod-
ulation of apoptosis remain elusive. Due to its action
as one of the main antioxidant defenses within the
cell, depletion of intracellular GSH content has
primarily been thought to reflect the generation of
RS (Wu et al., 2004). The role of RS in apoptosis has
been extensively studied (Chandra et al., 2000;
Carmody & Cotter, 2001). However, such role has
been suggested mainly through the use of RS
generation systems that induce cell death. On the
other hand, the role of RS in apoptosis induced by
physiological stimuli, like that of death receptor
activation, remains largely controversial primarily
due to reports showing contradictory roles including
both inhibitory and stimulatory effects of RS on
apoptosis. For example, apoptosis induced by
activation of the death receptor Fas/CD95/Apo-1 by
its ligand (FasL) has been suggested to depend on
the generation of H2O2 formation (Devadas et al.,
2003; Perez-Cruz et al., 2003; Sato et al., 2004;
Reinehr et al., 2005), while other studies report an
inhibitory role of H2O2 on Fas-mediated cell death
(Borutaite & Brown, 2001; Aronis et al., 2003).
Many reports have correlated ROS and Fas-induced
apoptosis by the protective effect of extracellular
thiols such as GSH and N-acetyl-L-cysteine (NAC)
in preventing cell death, effects ascribed to their
known capacities as antioxidants (Gulbins et al.,
1996; Um et al., 1996; Deas et al., 1997; Devadas
et al., 2003; Sato et al., 2004; Franco & Cidlowski,
2006). Interestingly, protective effects of thiol-com-
pounds in the absence of any ROS formation are also
observed (Deas et al., 1997). Additionally, apoptosis
has been reported to occur under anaerobic
conditions i.e. in the absence of ROS formation
(Jacobson & Raff, 1995; Shimizu et al., 1995), or in
cells lacking mitochondrial DNA, i.e. deficient in
respiration and mitochondrial oxidative phosphory-
lation (Jacobson et al., 1993; Jiang et al., 1999).
Thus, inhibition of apoptosis by using any
experimental intervention that prevents GSH
depletion or replenishes it might be an indicative of
a role of GSH content in the regulation of apoptotic
enzymes, rather than the involvement of RS.
Glutathione depletion has been shown to either
predispose cells to apoptosis or directly trigger cell
death by modulation of both the permeability
transition pore formation, and the activation of
execution caspases (Ueda et al., 1998; Obrosova
et al., 1999; Coppola & Ghibelli, 2000; Haouzi et al.,
2001; Armstrong & Jones, 2002; Nagai et al., 2002;
Varghese et al., 2003; Valverde et al., 2006). In vitro
studies have shown that a reduction in the GSH
content is necessary for the formation of the
apoptosome (Sato et al., 2004). Additionally, GSH
depletion activates the intrinsic apoptotic pathway
initiator Bax, by its oxidation-dependent dimeriza-
tion (D’Alessio et al., 2005). It has also been
proposed recently that released cytochrome C from
the mitochondria needs to become oxidized for its
pro-apoptotic activation, which will require cytosolic
GSH levels to be depleted (Coppola & Ghibelli,
2000; Hancock et al., 2003).
Several GSH-dependent enzymes have been re-
ported to protect cells from undergoing apoptosis.
For example, overexpression of GPX has been
reported to protect against Fas-induced apoptosis
(Gouaze et al., 2001). However, in vivo studies have
shown that there are no difference in animals
deficient of GPX compared to WT in their response
to death receptor-(Fas and TNF) mediated cell death
(Bajt et al., 2002). Thiol exchange reactions have also
been reported to directly modulate apoptotic en-
zymes (England & Cotter, 2005). For example,
caspases have been reported to be glutathionylated
and nitrosylated under basal conditions (Zech et al.,
1999; Klatt et al., 2000). Activation and/or transloca-
tion of these enzymes during apoptosis has been
reported to require deglutathionylation (Pan & Berk,
2007) and denitrosylation (Dimmeler et al., 1997;
Haendeler et al., 1997; Kim et al., 1997; Li et al.,
1997; Melino et al., 1997; Mohr et al., 1997; Kim
et al., 2000; Melino et al., 2000; Bernassola et al.,
2001; Mannick et al., 2001; Kim & Tannenbaum,
2004). Additionally, anti-apoptotic functions of Bcl-
2, FLICE inhibitory protein (FLIP) and dynamin
also depend on their degree of nitrosylation (Chan-
vorachote et al., 2005; Moungjaroen et al., 2006;
Kang-Decker et al., 2007). Interestingly, inhibition of
GRX which regulates protein glutathionylation by
cadmium triggers apoptosis in the absence of any
other apoptotic stimuli (Chrestensen et al., 2000).
Stress response and cytotoxicity
The intracellular content of GSH is highly responsive
to environmental factors. Many different conditions
of environmental stress have been demonstrated to
alter GSH content (Figure 6). These include heavy
metals, high glucose concentrations, heat shock, RS
or compounds that can generate RS. Modulation of
GSH content by xenobiotics has been shown to be

regulated by modulation of the g-GCS subunit gene
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expression by EpRE (electrophile response element,
known as well as antioxidant response element,
ARE), TRE (activatior protein-1) and TRE-like
transcription enhancer elements, which are modu-
lated by oxidants, phenolic antioxidants, growth
factors, inflammatory and anti-inflammatory agents
in various cells (Dickinson & Forman, 2002).
Exposure to agents such as 4-hydroxy-2-nonenal
(4-HNE) increase the content of GSH by increasing
the rate of GSH synthesis, which facilitates 4-HNE
detoxification from GST. The GSH-conjugate or
adduct of 4-HNE is by itself an inhibitor of GST, and
has been suggested to be detoxified from the cell by
RLIP76. 4-Hydroxy-2-nonenal has also been de-
monstrated to activate the JNK pathways, which in
turn activate the activator protein-1 (AP-1) transcrip-
tion complex that can further regulate GSH bio-
synthesis (Sharma et al., 2004; Yadav et al., 2007).
Oxidant-mediated damage has been widely re-
ported as the main mechanism of metal-induced
cytotoxicity. Iron (Fe)-, copper (Cu)-, chromium
(Cr)-, cobalt (Co)-, vanadium (Va)-, cadmium
(Cd)-, arsenic (As)- and nickel (Ni)-mediated
toxicity have been widely reported induced by free
radicals-mediated DNA damage, lipid peroxidation
and changes in calcium and sulphydril homeostasis
(Valko et al., 2005; Valko et al., 2006). Arsenic-
induced GSH depletion is one of the most studied
models for metal-induced toxicity. Studies so far
have suggested three distinct mechanisms by which
arsenic depletes cellular GSH pools: (1) via GSH-
mediated reduction of pentavalent to trivalent
arsenicals where GSH acts as an electron donor;
(2) via direct interaction of GSH with arsenic; and
(3) by its oxidation through arsenic-induced genera-
tion of free radicals (Kumagai & Sumi, 2007). On the
other hand, both beneficial and harmful effects of
GSH metabolism have been reported during metal-
induced toxicity. For example, Chromium (VI) does
not react with DNA in vitro or in isolated nuclei, until
it reacts with cellular reductants, causing a wide
variety of DNA lesions including Cr – DNA adducts,
DNA – protein crosslinks, DNA – DNA crosslinks
and oxidative damage. Glutathione has been shown
to rapidly form a complex with Cr(VI), followed by a
slow reduction of Cr(VI) to yield Cr(V) leading to
.
the formation of GS . In addition to the cellular
. .
damaging effect of the GS radical, GS can react
further with other thiol molecules in oxygenated
conditions to give O27. Furthermore, Cr(V) can be
.
.
reduced by GSH to Cr(IV) generating OH . It has
also been demonstrated that in the presence of Co
(II), Fe(III), Ni(II) and Cu(II), endogenous reduc-
tants such as GSH cause DNA damage by exacer-
bation of oxidative stress (Valko et al., 2005;
Valko et al., 2006). Similarly, the reduction of Fe(III)
to Fe(II) by GSH in particular has been suggested
to be mediated through the g-GT-mediated GSH
cleavage that triggers an iron-redox cycling process
GSH in human health and disease 245
that results in the production of free radicals and
ROS (GS ,  O27, H2O2) and oxidative stress
. .
(Paolicchi et al., 2002). Glutathione reductase
activity has also been suggested to play an important
role in the enzymatic reduction of heavy metals
including Cr (VI) and vanadate (Ning & Grant,
2000; Gunaratnam & Grant, 2001). In contrast,
GSH supplementation (via NAC, GSH or GSH-
monoester), alone or in combination with other
antioxidants, has been shown to have protective
effects against experimental models of iron-induced
neurodegeneration, chromium picolate-induced
toxicity, cobalt particle-induced inflammation, ar-
senic-induced lipid peroxidation, and nickel-induced
oxidative stress (Valko et al., 2005; Valko et al.,
2006). Cell death mediated by oxidative stress from
arsenic exposure has been demonstrated to be largely
prevented by GSH or NAC (Choi et al., 2002; Hu
et al., 2003; Kang et al., 2003; McCafferty-Grad
et al., 2003; Baysan et al., 2007; Habib et al., 2007;
Piga et al., 2007; Santra et al., 2007). In addition,
overexpression of GST has also been shown to be
protective by decreasing arsenic trioxide-induced
H2O2 formation (Lu et al., 2004; Zhou et al., 2005;
Habib et al., 2007). Finally, GSH supplementation of
cells with NAC prevents apoptosis induced by heavy
metals including cadmium (Oh & Lim, 2006),
chromium (Hayashi et al., 2004) and copper (Zhai
et al., 2000).
Cellular stress has also been widely reported to
induce apoptosis primarily through oxidant toxicity.
In general, GSH depletion has been shown to
sensitize cells to compounds that produce oxidative
cytolysis (Meister, 1991). Overexpression of
PHGPX and GSTs has been reported to be
protective against apoptosis induced by ultraviolet
radiation (UV), cytokine withdrawal, stress and
xenobiotics which is associated with lipid peroxida-
tion and RS formation (Nomura et al., 2001;
Sharma et al., 2004; Briz et al., 2006). Additionally,
overexpression of the g-GCS protects HEK293 cells
against UV irradiation-induced cell death by inhibi-
tion of the JNK1 and caspase-3 activation (Fan
et al., 2005). Accordingly, GSH supplementation
with NAC also prevents apoptosis induced by UV
radiation (Bush et al., 1999), chemotherapeutics
(Park et al., 2000; Custodio et al., 2002; Kurosu
et al., 2003; Jiang et al., 2007), drugs (Rosati et al.,
2004) and cytokine withdrawal (Kirkland &
Franklin, 2001).
Cancer
Numerous factors contribute to the initiation, devel-
opment and progression of cancer including those of
genetic, environmental and dietary influences.
Glutathione and GSH-metabolism participate in
different ways in cancer prevention and progression
(Figure 7). Both clinical and epidemiological studies
have shown the role of RS produced by UV radiation
 246 R. Franco et al.
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Figure 7. Role of GSH in carcinogenesis. Glutathione has been shown to regulate the progression of carcinogenesis in different ways. Its
protective role in cellular transformation involves the detoxification of carcinogens (RS or xenobiotics) produced by UV exposure, chemical
agents, environment, inflammation and diet. This is mediated either by its antioxidant role or by GST-mediated phase II detoxification
reactions (1). Transformed cells have shown to have increased levels of expression for several GSH enzymes whose function could be also
altered by polymorphisms. Glutathione inhibition of apoptosis and thiol/exchange reactions also contribute to cellular transformation and
immortalization. In addition, in tumoral cells, abnormal levels of several ROS and RNS regulate post-translational modifications through
S-glutathionylation or S-nitrosylation of redox-sensitive transcription factors and proteins that have been shown to be involved in
tumorigenesis (2). Finally, GSH also contributes to cell’s resistance to antineoplastic treatments by either inhibition of apoptosis, or
impairment of chemotherapeutic drug detoxification by GSH-conjugation (3). In this way, therapeutic strategies based on GSH depletion
(by its extrusion or metabolism) have been shown to sensitize cells to anti-cancer therapy.

exposure, chemical carcinogens, environmental modifications through S-glutathionylation and/or

agents, inflammation and diet, in cancer etiology.
By its antioxidant properties, GSH participates in the
defense against carcinogens by protecting cells from
ROS-mediated DNA-damage and ROS-induced
regulation of gene expression (Valko et al., 2006).
Reduced GR and GPX activity has been reported in
tumor tissues (Tanriverdi et al., 2007). In addition,
GPX polymorphisms have been associated with
cancer (Moscow et al., 1994). Glutathione can also
protects cells from potential carcinogens by GST-
mediated phase II detoxification reactions that
mediate the conjugation of GSH with potential
carcinogenic compounds (Rosado et al., 2006).
Recent studies have as well linked GST polymorph-
isms that are associated with reduced enzymatic
activity and increased risk of blood, bladder, gastric,
colorectal, skin, breast, kidney and liver cancers
(McIlwain et al., 2006). Moreover, redox regulation
of thiol groups has also been involved in carcino-
genesis. For instance, many malignant cell types
posses abnormal levels of several ROS and RNS
(Giles, 2006) that when combined with GSH result
in increases in GSSG and GSNO concentrations.
This might further mediate post-translational
S-nitrosylation of redox-sensitive transcription fac-
tors (e.g. NF-kB, Oxy R and AP-1) and enzymes
(e.g. PKC and Ras) that have been reported to be
involved in tumorigenesis (England & Cotter, 2005;
Biswas et al., 2006; Fukumura et al., 2006).
Glutathione metabolism has also been reported to
participate in chemotherapy resistance. Increased
expression of GSTs and GSH-transporters as well
as high GSH concentration are common features of
transformed cells that, in turn, are associated with
high resistance to chemotherapeutic agents (Yang
et al., 2006b). When antineoplastic or chemother-
apeutic drugs enter cancer cells, they are conjugated
with glutathione and are excreted through GSH-
pumps of the MRP family of transporters. It is known
that MRP transporters are highly expressed in
malignant cells. Thus, inhibitors of MRP-mediated
transport of GSH-conjugates have been largely used
as a common adjuvant in anti-cancer therapy (Cole &
Deeley, 2006). High intracellular GSH content in
tumor cells is a consequence of high g-GCS expres-
sion and can promote tumor cell survival by inhibi-
tion of apoptotic cell death activation. In accordance,
modulation of intracellular GSH content has been

largely studied for potential anti-cancer therapies.
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Such therapeutic interventions have been shown to
increase cellular sensitivity to treatment with ionizing
radiation and also decrease tumor resistance to
chemotherapeutic agents (Balendiran et al., 2004).
Furthermore, GSH depletion sensitizes tumor cells to
undergo apoptosis overriding Bcl-2 protection and
thus suggesting a link between GSH content and the
anti-apoptotic function of the Bcl-2 family of anti-
apoptotic proteins (Kern & Kehrer, 2005). Another
potential role for anti-neoplastic therapy is the
modulation of GSH efflux by acting on its transpor-
ters or pumps. Recently, it has been shown that
transformed cells are sensitized to cell death when
intracellular GSH is depleted through stimulation of
GSH efflux pumps (MRP, CFTR and OATP
proteins) (Estrela et al., 2006; Franco & Cidlowski,
2006), whose expression varies in different cancer cell
lines (Cui et al., 2003; Perez-Tomas, 2006).
Diabetes
Metabolic diseases arise either when a critical
enzyme is disabled, or if a control mechanism for a
metabolic pathway is affected. Diabetes mellitus is a
metabolic disease characterized by hyperglycemia
and oxidative stress (Maritim et al., 2003; Green
et al., 2004; Opara, 2004; Pennathur & Heinecke,
2004; Robertson, 2004; Robertson et al., 2004).
Enhanced production of free radicals and oxidative
stress play a central role in the pathogenesis of
diabetes and its complications, including obesity,
which is the most critical factor in the emergence of
metabolic diseases. However, controversy still exists
about the nature, magnitude, and localization of
oxidative stress in diabetes. Moreover, it is still
unclear whether oxidative stress is a primary event
that occurs early in the progression of diabetes or if it
is a secondary phenomenon as part of the end-stage
tissue damage (Pennathur & Heinecke, 2004).
Several studies have demonstrated that diabetes
elicits dynamic and profound alterations in GSH
metabolism and transport, that mediate alterations in
the cell’s redox state (Figure 8) (Gandhi & Roy
Chowdhury, 1979; Murakami et al., 1989; Collier
et al., 1990; Paolisso et al., 1992; Yoshida et al., 1995;
Lu et al., 1997; Giron et al., 1999; Obrosova et al.,
2000; Sharma et al., 2000; Yang et al., 2000; Abou-
Seif & Youssef, 2001; Yue et al., 2003; Raza & John,
2004; Winiarska et al., 2004; Darmaun et al., 2005;
Sampathkumar et al., 2005). Several pathological
complications of diabetes including peripheral nerve
lesions, embryonic malformations, angiopathies- and
artherosclerosis-mediated tissue hypoxia, diabetic
encephalopathy, and gastric damage have been
proposed to be associated with GSH depletion
(Goldin et al., 1997; Sakamaki et al., 1999; Gumie-
niczek et al., 2001; Donma et al., 2002; Mastrocola
et al., 2005; Bonfigli et al., 2006; Ho et al., 2006;
Mehta et al., 2006). For example, GSH depletion
GSH in human health and disease 247
mediated by oxidative stress has been shown to play
an important role in regulating transient outward Kþ
currents in rat ventricular myocytes that might
mediate impaired contractility and abnormal elec-
trical properties during diabetes (Xu et al., 2002).
Despite that GSH depletion has been suggested to be
a cause of free radicals formation, such depletion has
also been associated to its metabolism independent
from its oxidation (Beard et al., 2003; Januel et al.,
2003).
The mitochondrial GSH pool also seems to be an
important player in diabetes. Hyperglycemia has
been shown to increase the enzymatic conversion of
glucose to sorbitol by the sorbitol dehydrogenase,
with concomitant decreases in NADPH and glu-
tathione. This depletion in reducing equivalents
results in augmented sensitivity to oxidative stress
(Brownlee, 2003; Rolo & Palmeira, 2006). Diabetic
streptozotocin rats have been reported to have
reduced GPX activity, and increased levels of GSSG
and ROS in the mitochondria (Awasthi et al., 2003;
Green et al., 2004; Ghosh et al., 2005; Mastrocola
et al., 2005). Hyperglycemia-induced oxidative stress
and GSH depletion have been suggested to be
regulated by the inner membrane potential of the
mitochondria (Gustafsson et al., 2004), and oxidative
stress has been reported to impair insulin secretion
(Krippeit-Drews et al., 1999; Maechler et al., 1999).
Nicotinamide nucleotide transhydrogenase (NNT)
has been demonstrated to protect against mitochon-
drial oxidative stress by mediating NADPþ and
NADH conversion to NADPH that is necessary for
GSSG recycling to GSH (Arkblad et al., 2005).
Recently, it has been proposed that alterations in
NNT activity in pancreatic b cells mediate impaired
insulin secretion during type 2 diabetes. The
proposed mechanism involves the impairment of
GR-mediated GSH recycling after its oxidation that
is required for ROS-detoxification by GPX. This is
thought to lead to enhanced ROS accumulation
decreased ATP production and enhanced KATP
channel activity (ATP-inhibited Kþ channels) there-
by producing hyperpolarization of the plasma mem-
brane and preventing Ca2þ-dependent insulin
secretion (Freeman et al., 2006a; Freeman et al.,
2006b).
Although GST polymorphisms together with
altered GST distribution and activity have been
reported to occur in distinct models of diabetes, their
physiological significance is still elusive (Fujita et al.,
2001; Traverso et al., 2002; Raza et al., 2004; Wang
et al., 2006; Yalin et al., 2006). In this respect, insulin
treatment has been shown to up-regulate the anti-
oxidant defenses of the cells by increasing GST
expression through the PI3K/Akt/p70S6K pathway
(Kim et al., 2003; Kim et al., 2006), while glucagon
on the other hand, a physiological antagonist of
insulin, decreases GST expression (Kim et al., 2003).
Additionally, insulin treatment in patients with type 2
diabetes mellitus reduces intracellular oxidative
 248 R. Franco et al.
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Figure 8. Mechanisms for ROS formation and GSH depletion during hyperglycemia. During diabetes, elevated plasma glucose and free fatty acids
(FFA) lead to a rise in its cytoplasmic concentration by its uptake and diffusion respectively. Increase glucose and FFA oxidation lead to
increased production of reducing equivalents that mediate ROS generation through the respiratory electron transport chain (1). This is
followed by a concomitant decrease in mitochondrial NADPH and GSH. NNT mutation or deficiency has been proposed as a possible
mechanism involved in the impairment of NADPH formation and by consequence GR-mediated GSH recycling after its oxidation, which is
necessary for ROS-detoxification by GPX (2). This leads to increased ROS accumulation that have been shown to decrease ATP and
enhance KATP channels producing hyperpolarization of the plasma membrane and preventing Ca2þ-dependent insulin secretion (3).
Reactive oxygen species have been proposed to act as the major mechanism mediating cytosolic GSH depletion, although other mechanisms
including GSH efflux might also contribute to its depletion (4). Oxidative stress and GSH depletion might further alter insulin signaling
through thiol/exchange reactions. Diabetes has also been shown to decrease the expression of GSH-dependent antioxidant enzymes such as
GST and GPX (5). Finally, GSH depletion might also contribute to FFA and ROS-mediated apoptosis in b-cells (6). GLUT4, glucose
transporter; ETC, electron transport chain; NNT, Nicotinamide nucleotide transhydrogenase; ATP-S, ATP synthase.

stress through an increased GSH/GSSG ratio (Bravi
et al., 2006), while GSH infusion in type 2 diabetes
mellitus increases insulin sensitivity (De Mattia et al.,
1998). Glutathione reductase activity has also been
reported to be protective against streptozotocin-
induced diabetes (Nagaoka et al., 2004). In addition,
diabetes has been reported to either impair GPX
activity (Bhor et al., 2004; Manea et al., 2004; Sindhu
et al., 2004), which will decrease the antioxidant
defenses; or to increase GPX activity as a compensa-
tory response to ROS formation (Ulusu et al., 2003).
Glutathione peroxidase activity has been observed to
have both beneficial and deleterious effects on
diabetes. For example GPX overexpression has been
shown to protect pancreatic beta cells from oxidative
stress induced by glucose toxicity (Tanaka et al.,
2002; Robertson et al., 2003). However, a recent
report demonstrates a development of insulin resis-
tance in mammals with elevated expression of the
antioxidant enzyme GPX1 whose activity may inter-
fere with insulin function by over quenching in-
tracellular ROS that are required for insulin
sensitization (McClung et al., 2004). Thiol-exchange
reactions might also play an important role in
diabetes. Cellular levels of protein S-nitrosylation
and S-glutathionylation have been demonstrated to
be elevated in type 2 diabetic patients (Niwa et al.,
2000; Sampathkumar et al., 2005; Kaneki et al.,
2007). Additionally, S-nitrosylation of the insulin
receptor, insulin receptor substrate-1, and Akt/PKB,
have been reported in skeletal muscle of diabetic
mice where the Akt/PKB pathway was shown to be
reversibly inactivated (Yasukawa et al., 2005).
A reduction in the number of insulin producing
b-cells through apoptosis also contributes to the
progression of diabetes. Hyperglycemia has been
shown to induce apoptosis via activation of NFkB,
mitochondrial cytochrome C, formation of ROS,
and overexpression of the apoptotic genes Bad, Bik
and Bid (Federici et al., 2001; Mandrup-Poulsen,
2003). Additionally, free fatty acid-induced b-cell
apoptosis is suggested to involve the formation of
ceramide and NO. Finally, high glucose has been
shown to mediate upregulation of Fas in human

islets which might induce apoptosis in neighboring
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b-cells constitutively expressing FasL (Mandrup-
Poulsen, 2003). Altered GSH homeostasis and
oxidative stress in diabetes are also important
factors modulating cell death in different tissues.
As explained above, GSH is a central regulator of
apoptosis. MRP-mediated GSH efflux and mito-
chondrial GSH depletion are required for caspase 9-
and 3-dependent cardiomyocyte apoptosis in short
term diabetic rats (Ghosh et al., 2004; Ghosh et al.,
2005). Furthermore, NAC supplementation pro-
tects these cells from oxidative stress-mediated
injury (Fiordaliso et al., 2004; Patriarca et al.,
2005). Finally, it has been demonstrated that skin
cells derived from obese mouse are more sensitive
to oxidative stress induced by UV radiation
(Katiyar & Meeran, 2007).
Glutathione synthetase deficiency
Glutathione synthetase deficiency is a rare autosomal
recessive disorder which prevents the production of
GSH due to a mutation and malfunctioning of the
GS as the result of a mutation in the corresponding
gene (Njalsson & Norgren, 2005). Glutathione
synthetase deficiency can be classified into three
types based on the severity of the disease. Mild GS
deficiency results in the destruction of red blood cells
and concomitant hemolytic anemia. These patients
also excrete large amounts of 5-oxoproline in their
urine (5-oxoprolinuria), which builds up when GSH
is not processed correctly in cells (Al-Jishi et al.,
1999), while individuals with moderate GSH defi-
ciency also present elevated acidity in the blood
and tissues (metabolic acidosis). Patients suffering
from the severe form of this disorder experience
recurrent bacterial infections, and neurological
symptoms including seizures, generalized slowing
down of physical reactions, movements, and
speech (psychomotor retardation), mental retarda-
tion, and a loss of coordination (ataxia) (Ristoff &
Larsson, 1998; Njalsson, 2005). More than 20
mutations in the GS gene have been described in
patients suffering from GS deficiency. These muta-
tions change single amino acids in the GS or disrupt
how genetic information from the GS gene is
spliced to make a blueprint for producing this
enzyme. The mutated GS enzyme is either unstable,
shorter than usual, or in the wrong conformation,
and in all cases shows reduced activity that disrupts
the g-glutamyl cycle and production of GSH
(Njalsson et al., 2005).
Lung diseases
Lungs are amongst the major sources of GSH
storage (6.1 – 17.5 nmol/mg lung) and have higher
levels of g-GCS than other tissues. Alterations in the
levels of reduced GSH in the lung lining fluid have
been shown in various inflammatory conditions.
GSH in human health and disease 249
Glutathione deficiency has been largely correlated
with pulmonary diseases including chronic pulmon-
ary disease, acute respiratory distress syndrome,
neonatal lung damage and asthma. Glutathione
levels in the epithelial lining fluid (ELF), the first
line of defense against exposure to oxygen, airborne
toxins and oxygen radicals released from lung
phagocytes, were observed to be depleted in idio-
pathic pulmonary fibrosis and acute distress syn-
drome. Moreover, GSH depletion in the ELF
contributes to the imbalance between oxidants and
antioxidants in the lungs amplifying inflammatory
responses and potentiating lung damage. It is
possible that GSH differences in ELF in various
inflammatory lung diseases are due to changes in the
molecular regulation of GSH synthesis and the
production of lipid peroxidation products (Rahman
et al., 2005). Glutathione content in lung cells has
also been shown to be critical to the lung antioxidant
defenses against oxidant/free radical (cigarette
smoke/air particulate)-mediated injury and inflam-
mation. Air pollution gases such as sulfur dioxide
and nitrogen dioxide generate an inflammatory
response and increase alveolar permeability by
depleting GSH levels in the lung. Additionally, it
has been suggested that extracellular GPX secreted
into ELF by alveolar epithelial cells and macro-
phages, along with the GSH redox system, provide a
strong defense against oxidants (Rahman, 2005).
It is known that supplemental oxygen can cause
significant lung damage in prematurely born infants
which puts them at risk for developing pulmonary
oxygen toxicity. This is mainly ascribed to cellular
damage which is probably mediated by increased
production of ROS in the mitochondria. Protection
against these ROS is provided mainly by GSH, and
depletion of mitochondrial GSH has been asso-
ciated with an increased susceptibility to oxidant
injury (O’Donovan & Fernandes, 2000). Cystic
fibrosis is a genetic disorder ascribed to a recessive
mutation of the organic anion efflux channel
CFTR. CFTR contributes to the cellular homeo-
static balance of GSH and in this way cystic
fibrosis is associated with systemic and cellular
GSH deficiency, by altered GSH efflux. GSH
deficiency in these conditions has been observed
to regulate inflammatory cytokines through NFkB
activation and to promote high levels of oxidative
stress and lipid peroxidation byproducts (Hudson,
2001).
Other human diseases and pathological conditions
Alterations in GSH homeostasis have also been
described in several other pathological conditions;
however, its implication in the pathogenesis of these
conditions is still unclear. For example, GSH has
been found to be consistently lower in children with
oedematous protein energy malnutrition (PEM)
(such as in kwashiorkor and marasmic-kwashiorkor)
 250 R. Franco et al.
compared with non-oedematous PEM, suggesting a
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direct relation with oedematous malnutrition. It has
been suggested that the limited cysteine content in
the diet is the underlying cause for the reduced rate
of GSH synthesis in these patients (Reid & Jahoor,
2001).
Glutathione levels are also depleted in plasma,
epithelial lining fluid, peripheral blood mononuclear
lymphocytes and monocytes of symptom-free HIV
seropositive individuals, asymptomatic HIV-infected
individuals and AIDS patients. However, the me-
chanisms by which alterations in GSH metabolism
regulate HIV progression remain unclear. Glu-
tathione deficiency has been associated with HIV
infection. HIV-viral maturation depends on protease
activity regulated by glutathionylation. Additionally,
depleted GSH levels are suggested to activate NFkB
which allows HIV expression. Finally, GSH deple-
tion has also been suggested to be a modulator of
apoptosis in CD4þ T-cell lymphocytes which pre-
sent low GSH content during HIV progression. In
this way, NAC-based treatment for replenishing
GSH stores has been demonstrated to have beneficial
effects (Droge & Breitkreutz, 2000; Shackelford
et al., 2005).
Liver is one of the major pools of GSH, and
alterations in its content are either the cause or
consequence of several pathologies. Alcoholism-
mediated depletion of mitochondrial GSH mediates
ROS-dependent cell death (triggered by cytokines),
that in turn contribute to the progression of
cirrhosis. This has been reported to be associated
to the inactivation of the mitochondrial import
transport for GSH (Fernandez-Checa & Kaplowitz,
2005). Additionally, chronic hepatitis C infection is
associated with depleted levels of GSH in hepatic
and plasma fractions, as well as in peripheral blood
mononuclear cells. Acetaminophen in addition to
other pharmaceutical and/or environmental xeno-
biotics can deplete liver GSH. Finally, GSH levels
after partial hepatectomy have been reported to be
doubled due to increased g-GCS expression and
activity. In this conditions, it has been proposed that
an increased reducing environment caused by
increased GSH may be generated in order to ensure
proper NFkB activity and hepatocyte survival
(Han et al., 2006).
Like GS deficiency, g-GCS deficiency is also
associated with haemolytic anemia. On the other
hand g-GT deficiency exhibits marked glutathionuria
and glutathionaemia (excess glutathione in the
blood), which can lead to an imbalance in glutamic
acid homeostasis (Schulman et al., 1975). Patients
with Crohn’s disease and ulcerative colitis present
low level activities of g-GCT, g-GT and GST
(Jefferies et al., 2003). On the other hand, perturba-
tions in S-glutathionylated protein homeostasis is
associated with hyperlipidemia, chronic renal failure,
uremia, as well as with Friederich’s ataxia and
Down’s syndrome (Giustarini et al., 2004).
The aging process has also been associated with
deterioration of GSH homeostasis by increased
levels of GSSG with a concomitant reduction of
intracellular GSH. Additionally, disruption of the
microsomal GST gene significantly reduces life-
span in Drosophila suggesting a role of lipid
peroxidation in cell aging and senescence. A down-
regulation of the g-GCS has been reported to occur
in the rat brain during aging. In the brain, a
complex interaction between astrocytes and neurons
exists, where astrocytes supply neurons with cy-
steine for GSH synthesis that is necessary for the
defense against oxidative-stress based pathologies
including those of Parkinson’s, Alzheimer’s and
Huntington’s disease, as well as in ischemia,
schizophrenia and epilepsy. Parkinson’s disease
progression is associated with a depletion of GSH
levels and an increase in ROS formation in the
substantia nigra. Finally, some reports have also
attributed a signaling role to GSH as a hormone
(Dringen & Hirrlinger, 2003).
Modulation of GSH content as a means of
therapeutic approach
Because of the relevance of GSH as a central element
in the regulation of several human diseases, there is
an increasing interest in the design of new strategies
to modulate the GSH content. As indicated earlier,
several pathological conditions are associated with
GSH depletion either by its increased metabolism,
transport or impaired synthesis. In this way ther-
apeutic treatments have been focused on the
supplementation of cells and tissues with GSH.
Dietary amino acid balance has an important effect
on GSH homeostasis. In particular, the adequate
provision of sulfur-containing amino acids as well as
glutamate (glutamine) and glycine (serine) is critical
for optimal GSH synthesis (Valencia et al., 2001a;
Valencia et al., 2001e; Valencia et al., 2001c; Parcell,
2002; Wu et al., 2004). Glutamine is an efficient
precursor for glutamate involved in GSH synthesis.
Cysteine is not only the rate limiting precursor to
GSH but it is by itself an important reducing agent.
Cysteine can be given orally to increase GSH;
however, cysteine has been shown to have toxic
effects due to its instability and pro-oxidant reac-
tions. Because cysteine can be generated via the
trans-sulfuration pathway, dietary methionine
(through the synthesis of S-adenosylmethionine)
can replace cysteine to support GSH synthesis
in vivo (Wu et al., 2004). Administration of oral
GSH increases hepatic GSH levels; however, the
mechanisms involved in its absorption are still
unclear. Glutathione is thought to be taken up poorly
by cells and rapidly degraded in the gut and
circulation. Moreover, GSH does not readily cross
the blood – brain barrier. In this way, intravenous or
oral administration of GSH serves only to provide
brain cells with constituent amino acids. Indeed,

GSH has to be broken down into its amino acids,
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transported into the cell and then resynthesized.
Thus, the use of GSH precursors is a more efficient
way to increase GSH levels (Valencia & Hardy, 2002;
Njalsson & Norgren, 2005).
Precursors of GSH include NAC, GSH
monoethyl or diethyl esters, and 2-oxothiazolidine-
4-carboxylate (OTC or procysteine). N-acetyl-
cysteine is a derivative of the sulfur-containing
amino acid cysteine and an intermediary (along
with glutamic acid and glycine) in the conversion of
cysteine to GSH. It is intracellularly hydrolyzed to
cysteine before it serves as a precursor for GSH
formation and has limited toxicity compared to
other thiols. Oral NAC administration leads to an
increase in intracellular cysteine and GSH levels,
however, intravenous administration is preferably
recommended since nausea and vomiting limit its
effectiveness in oral therapy. Glutathione ethyl
esters are lipid soluble compounds that are readily
transported into cells where they are de-esterified
by esterases and increase the levels of GSH.
Glutathione diethyl ester is more rapidly trans-
ported into cells than the monoethyl ester. 2-
Oxothiazolidine-4-carboxylate is transported into
the cell and metabolized forming cysteine; however,
OTC and other thiazolidines are of limited value
because of the by-products formed such as for-
maldehyde. It is important to mention that pre-
cursors of cysteine or its dipeptides have been
shown to have limited usefulness in raising GSH
levels in patients with GS deficiency or altered acti-
vity (Wernerman & Hammarqvist, 1999; Schafer &
Buettner, 2001; Valencia et al., 2002).
High intracellular GSH levels are also important
contributors to pathologies such as cellular transfor-
mation and resistance to antineoplastic treatments in
cancer cells. In this way, other tools are available
in order to decrease intracellular GSH levels.
L-buthionine-SR-sulfoximide (BSO) has been widely
used as a specific inhibitor of g-GCS thereby
inhibiting the synthesis of g-glutamylcysteine and
the formation of GSH. This experimental approach
has been shown to efficiently deplete cells from
cytosolic GSH, although mitochondrial GSH has
been observed to remain largely intact. Thus, a
combination of BSO and thiol oxidants such as
diethylmalate (DEM) or diamide, have proven
efficient in depleting cells from intracellular GSH,
although coupled to a high degree of cytotoxicity.
Ethacrynic acid is another compound reported to
deplete cells from GSH via GST. It has been
reported to deplete both cytosolic and mitochon-
drial GSH. Moreover, 1, 3-Bis(2-chloroethyl)-N-
nitrosourea (BCNU, or carmustine) is another
compound that carbonylates the lysine residue in
the active site of GR. This NADPH-dependent
reaction is fast and inhibits GR, thereby preventing
the recycling of GSSG to GSH (Schafer & Buettner,
2001).
GSH in human health and disease 251
Conclusions and perspectives
Glutathione is a central component of many meta-
bolic pathways. Studies so far have uncovered that
GSH is not only one of the most important
antioxidant defenses and a major regulator of the
redox balance in the cell, but it is also involved in a
myriad of other cellular processes. It is a key
mediator of many physiological events including
cellular signaling, detoxification, and thiol disulfide
exchange reactions. Moreover, its deregulation has
been associated with a wide range of pathological
conditions such as AIDS, ischemia, neurodegenera-
tive, metabolic, and inherited diseases. Throughout
this review article, we have highlighted the role of
GSH in the pathophysiology of several human
diseases where glutathione metabolism has shown
to play a transcendental role in disease progression.
Clearly, it becomes apparent the importance in
designing pharmacologically-based strategies aimed
for specific targets in the metabolism, transport and
catabolism of GSH, as potential therapeutic treat-
ments against human diseases.
Acknowledgements
This work was supported, in part, by the Intramural
Research Program of the NIH/National Institute of
Environmental Health Sciences (Franco and Scho-
neveld); by the European Community through a
Marie Curie International Reintegration Grant with-
in the 6th European Community Framework Pro-
gram (Pappa); and the American Institute for Cancer
Research (AICR) through a Marilyn Gentry Fellow-
ship (Panayiotidis).
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