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Glutathione Homeostasis and Functions: Potential Targets for Medical

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Hindawi Publishing Corporation
Journal of Amino Acids
Volume 2012, Article ID 736837, 26 pages
doi:10.1155/2012/736837

Review Article
Glutathione Homeostasis and Functions: Potential Targets for
Medical Interventions

Volodymyr I. Lushchak
Department of Biochemistry and Biotechnology, Vassyl Stefanyk Precarpathian National University, 57 Shevchenko Street,
Ivano-Frankivsk 76025, Ukraine

Correspondence should be addressed to Volodymyr I. Lushchak, lushchak@pu.if.ua

Received 28 January 2011; Revised 30 August 2011; Accepted 24 October 2011

Academic Editor: Arthur J. L. Cooper

Copyright © 2012 Volodymyr I. Lushchak. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Glutathione (GSH) is a tripeptide, which has many biological roles including protection against reactive oxygen and nitrogen
species. The primary goal of this paper is to characterize the principal mechanisms of the protective role of GSH against reactive
species and electrophiles. The ancillary goals are to provide up-to-date knowledge of GSH biosynthesis, hydrolysis, and utilization;
intracellular compartmentalization and interorgan transfer; elimination of endogenously produced toxicants; involvement in
metal homeostasis; glutathione-related enzymes and their regulation; glutathionylation of sulfhydryls. Individual sections are
devoted to the relationships between GSH homeostasis and pathologies as well as to developed research tools and pharmacological
approaches to manipulating GSH levels. Special attention is paid to compounds mainly of a natural origin (phytochemicals)
which affect GSH-related processes. The paper provides starting points for development of novel tools and provides a hypothesis
for investigation of the physiology and biochemistry of glutathione with a focus on human and animal health.

1. Introduction called γ-glutamyl-L-cysteine ligase or γ-glutamylcysteine

synthase), and glutathione synthase (GLS, EC 6.3.2.3).
Glutathione (GSH) is a tripeptide (L-γ-glutamyl-L-cysteinyl- GSH is consumed in many ways, such as by oxidation,
glycine) with multiple functions in living organisms [1– conjugation, and hydrolysis [11]. GSH can be directly
4]. As a carrier of an active thiol group in the form of a oxidized by ROS and RNS or indirectly during GSH-
cysteine residue, it acts as an antioxidant either directly by dependent peroxidase-catalyzed reactions. Conjugation with
interacting with reactive oxygen/nitrogen species (ROS and
endogenous and exogenous electrophiles consumes a sub-
RNS, resp.) and electrophiles or by operating as a cofactor
stantial portion of cellular GSH. In addition, cells may lose
for various enzymes [5–8]. Glutathione is moderately stable
GSH due to export of its reduced, oxidized or conjugated
in the intracellular milieus because intracellular peptidases
can cleave peptide bonds formed by the α-carboxyl groups of forms. Extracellularly, GSH can be hydrolyzed by γ-L-
amino acids, but typically not the γ-carboxyl groups. glutamyl transpeptidase (GGT, EC 2.3.2.2) transferring the
The reduced and oxidized forms of glutathione (GSH γ-glutamyl functional group to water during hydrolysis to
and GSSG) act in concert with other redox-active com- form free glutamate [12]. The enzyme may also transfer the
pounds (e.g., NAD(P)H) to regulate and maintain cellular γ-glutamyl moiety of GSH to amino acids and peptides.
redox status [9]. The former is quantitatively described by the Frequently, products of GSH hydrolysis are taken up by
redox potential, calculated according to the Nernst equation. cells either as individual amino acids, or as dipeptides.
In most cells and tissues, the estimated redox potential for the The intra- and extracellular GSH levels are determined
GSH/GSSG couple ranges from −260 mV to −150 mV (cited by the balance between its production, consumption, and
after [10]). transportation. Due to important physiological functions of
GSH is synthesized in a two-step process catalyzed by GSH, these processes are tightly regulated. The activities of
L-glutamate: L-cysteine γ-ligase, (γGLCL, EC 6.3.2.2) (also the enzymes involved in GSH metabolism are controlled
2 Journal of Amino Acids

at transcriptional, translational, and posttranslational levels SH

[3, 11].
CH2 O
Since GSH participates not only in antioxidant defense O
γ
α
systems, but also in many metabolic processes, its role cannot C CH CH2 CH2 C NH CH C NH CH2 C
be overestimated. Therefore, it is not surprising that the GSH OH
HO NH2 O O
system has attracted the attention of pharmacologists as a
possible target for medical interventions. The main efforts Cysteine
Glutamate Glycine
in this field have been applied to decreasing or increasing
GSH levels in organisms. General strategies involve specific (a)
inhibition of γGLCL, a key enzyme of GSH biosynthesis, NH2 O O
and depletion of cellular reserves by externally added HO OH
electrophiles (usually for research purposes). The use of C CH CH2 CH2 C NH CH C NH CH2 C
α γ
buthionine sulfoximine (BSO) is probably the most popular O
CH2
O
approach to depleting GSH. BSO was first synthesised as the
D,L-form [13, 14] and later as the L-BSO enantiomer [15]. S
Usually a mixture of D- and L-BSO is used in experiments
[16–18]. GSH levels may be enhanced by supplementation S
with precursors, mainly cysteine in the form of different CH2
esters. However, during the the last decade a new approach O O
α γ
for the regulation of GSH-utilizing enzymes has emerged. It C CH CH2 CH2 C NH CH C NH CH2 C
is evident that many of these are induced at the transcrip- HO NH2 O O OH
tional level by mild oxidative stress, which involves binding
of the Nrf2 transcription factor to the antioxidant response (b)
element (ARE) (also called the electrophile response element; Figure 1: Glutathione is a tripeptide: L-γ-glutamyl-L-cysteinyl-
EpRE) in the promoter region of genes encoding certain glycine. In its reduced form (a) the N-terminal glutamate and
enzymes, particularly γGLCL and glutathione S-transferases cysteine are linked by the γ-carboxyl group of glutamate, preventing
[19–22]. cleavage by common cellular peptidases and restricting cleavage to
Glutathione has several additional functions in cells. For γ-glutamyltranspeptidase. The cysteine residue is the key functional
example, it is (i) a reserve form of cysteine, (ii) stores and component of glutathione, providing a reactive thiol group that
plays an essential role in its functions. Furthermore, cysteine
transports nitric oxide, (iii) participates in the metabolism
residues form the intermolecular dipeptide bond in the oxidized
of estrogens, leukotrienes, and prostaglandins, the reduction glutathione molecule (b).
of ribonucleotides to deoxyribonucleotides, the maturation
of iron-sulfur clusters of diverse proteins, (iv) involved in the
operation of certain transcription factors (particularly those Figure 1 shows the chemical structure of reduced and
involved in redox signalling), and (v) the detoxification of oxidised glutathione forms. GSH is formed from gluta-
many endogenous compounds and xenobiotics [11]. mate, cysteine, and glycine (Figure 1(a)), but it possesses
The present review will focus on the molecular mech- an unusual peptide bond. The N-terminal glutamate and
anisms of operation of the GSH system, with special cysteine residues are linked by the γ-carboxyl group of
attention to regulatory pathways controlling the expression glutamate, rather than the common linkage in proteins of an
of the enzymes involved. Information on GSH biosynthesis, α-carboxyl peptide bond. This specific peptide bond prevents
hydrolysis and utilization, intracellular compartmentaliza- GSH from being hydrolyzed by most peptidases that cleave
tion, and interorgan transfer will be highlighted. Special at the α-carboxyl peptide bond of N-terminal amino acids.
sections will deal with GSH functions, such as antioxidant This configuration also restricts the cleavage of GSH by GGT
properties and relationship to specific enzymes. On the basis localized on the external surface of certain cell types. As a
of these mechanisms, some potential approaches for medical result, GSH is relatively stable in the cell and is cleaved by
interventions will also be evaluated. GGT only at external sides on the membranes of certain cells.
In addition, the presence of the C-terminal glycine residue in
the GSH molecule protects it against cleavage by intracellular
2. Glutathione Biosynthesis, Hydrolysis, γ-glutamyl cyclotransferase. The major oxidized form of
Excretion, and Utilization glutathione (i.e., glutathione disulfide, GSSG) consists of two
residues of GSH that have been oxidized in such a fashion
Intracellular GSH concentrations usually range from 0.5 to as to be connected by an intermolecular disulfide bond
10 mM, whereas extracellular values in animals are one to (Figure 1(b)).
three orders of magnitude lower [2, 11]. GSH is commonly The steady-state level of cellular GSH is provided by the
the most abundant low molecular mass thiol in animal balance between production and consumption, as well as by
and plant cells. Most microorganisms also possess GSH in extrusion from the cell as reduced, oxidized, or bound forms
high concentrations, but there are some species and viable (summarized in Figure 2). GSH is produced in two steps.
mutants lacking GSH [23–25]. In the first step, the enzyme γGLCL forms a peptide bond
Journal of Amino Acids 3

GSH de novo synthesis

5-oxoprolinase
COOH
Cell membrane H2 N CH COOH
H2 N C H γ-glutamylcysteine ligase
CH2 GSH
CH2 salvage
R H OH synthesis
O N CH2 H2 N CH COOH
Transported CH2 COOH
H C NH2 O
amino acid C CH2
C NH C H O
COOH O 5-oxoproline OH
R
Glutamate
ATP SH GSH
Cysteine
γ-glutamyl amino ADP
acid GSH
feedback COOH γ-glutamyl-
Glutathione inhibition
H2 N H
transpeptidase
C
COOH O H O
ADP CH2
H C CH2 CH2 C NH C C NH CH2
ATP O
CH2 CH2 COOH H
NH2 COOH
H2 N CH C N CH COOH
SH C NH C H
GSSG O CH2 H
Glutathione CH2
Glutathione SH Cysteinyl glycine
reductase synthetase SH
γ-glutamylcysteine
GSH Leucyl aminopeptidase
Salvage H2 N CH COOH

cycle H
Glyoxalase I Glycine
GSH O O GSH consumptive
O
H3 C C CH Spontaneous thiolation
H3 C CSG Methylglyoxal Glutathione-S-transferases
COOH C Prostaglandin-H-synthetase
+ H3 C
GSH C Leukotriene synthetase
H H OH
OH Formaldehyde dehydrogenase
D-lactate S-D-lactoyl Maleylacetoacetate isomerase
GSH DDT-dehydrochlorinase
Glyoxalase II

Figure 2: Glutathione homeostasis involves both intra- and extracellular mechanisms. Glutathione is synthesized in both de novo and
salvage synthesis pathways. De novo synthesis requires the three amino acids and energy in the form of ATP. Glutamate may be provided in
part from the conversion of a γ-glutamyl amino acid to 5-oxoproline, which is then converted to glutamate. Two ATP molecules are used for
the biosynthesis of one GSH molecule. Salvage synthesis involves either reduction of GSSG or uses precursors formed from the hydrolysis
of GSH or its conjugates by γ-L-glutamyl transpeptidase at the external surface of the plasma membrane that are transported back into the
cell as amino acids or dipeptides. GSH is consumed in various processes. In addition to detoxification of reactive species and electrophiles
such as methylglyoxal, GSH is involved in protein glutathionylation and several other processes, such as the biosynthesis of leukotrienes and
prostaglandins, and reduction of ribonucleotides. Modified from [27].

between the γ-carboxyl of glutamate and the amino group of [30, 31]. Direct oxidation leads to the production of thiyl
cysteine using energy provided by the hydrolysis of ATP: radicals [32], the fusion of which results in GSSG formation
(Figure 2). GSH is extensively used as a cosubstrate by glu-
γ-L-Glutamate + L-cysteine + ATP
(1) tathione peroxidases (GPx, EC 1.11.1.9) reducing hydrogen
−→ γ-L-glutamyl-L-cysteine + ADP + Pi peroxide (H2 O2 ) or organic peroxides (generally abbreviated
as ROOH or LOOH in the case of lipid peroxides) with the
In the next step, the dipeptide is combined with glycine production of GSSG, water, or alcohols. Figure 3 shows the
by glutathione synthetase (GLS), again driven by the hydrol- dismutation of H2 O2 by catalase.
ysis of ATP: How do catalases and GPxs cooperate in H2 O2
γ-L-Glutamyl-L-cysteine + glycine + ATP catabolism? Firstly, they are mainly localized in different cel-
(2) lular compartments—GPxs are cytosolic residents, whereas
−→ GSH + ADP + Pi catalases are found mainly in peroxisomes. Secondly, the
affinity of GPx for H2 O2 is one to two orders of magnitude
It should be noted that, in some cases, the provision of higher than that of catalase. So, one may conclude that the
ATP for GSH synthesis can be a limiting factor for GSH two enzymes operate in concert, complementing each other.
metabolism [26]. The first step, catalyzed by γGLCL, is the GSSG produced from the consumption of GSH can be either
rate-limiting step for overall GSH biosynthesis process. The restored again by the action of glutathione reductase (GR, EC
enzyme is inhibited by GSH, the end product of the pathway, 1.6.4.2) (reaction (3)), or excreted from the cell.
indicating that its biosynthesis is regulated via a negative
feedback control mechanism.
GSH may be oxidized directly by oxidants such as
hydroxyl radical (HO• ) [28, 29] or peroxynitrite (ONOO− ) GSSG + NADPH + H+ −→ 2GSH + NADP+ (3)
4 Journal of Amino Acids

Methionine
GS•
GSH
GS• GSSG
O2

ē • NO ONOO−
NADP+ G6P
O•2 −
GR G6PDH

ē 2H+
NADPH 6PGL
2GSH GSSG
Catalase

x
O2 + H 2 O H2 O2

Gp
H2 O

ē
Peroxisome GS•
GSH
HO•
+ GS• + H2 O
OH− +
ē H
H2 O
Cell membrane

Figure 3: Involvement of glutathione in elimination of reactive oxygen and nitrogen species. Hydroxyl radical and nitric oxide (after
oxidation to the NO+ form) or peroxynitrite may interact directly with GSH leading to GSSG formation. Hydrogen peroxide may be removed
by catalase or by glutathione peroxidase (GPx). The latter requires GSH to reduce peroxide.

Glutathione excretion from cells is inhibited by methion- by a dipeptidase to cysteine and glycine. The products,
ine [33]. Three forms of glutathione, namely, GSH, GSSG, namely, amino acids and γ-glutamyl amino acids, may be
and GSH-conjugates, can be excreted into extracellular transported back into cells and used for GSH resynthesis or
spaces. There the conjugates are mainly hydrolysed to differ- other needs. This provides the basis for recycling of excreted
ent components and reabsorbed. However, cysteine residues GSH and GSSG (salvage cycle) by the cell of origin or by
usually remain conjugated to xenobiotics and are released by other cells [34]. Upregulation of this process provides an
organisms in feces. Most glutathione S-conjugates are metab- additional mechanism for GSH maintenance in the cell.
olized to the corresponding N-acetyl cysteine S-conjugates
(mercapturic acids) and released in the urine and bile [34].
Glutamate and glycine residues are usually recovered, but the
3. Intracellular Compartmentalization and
cysteine residues remain conjugated and are lost. Both GSH Interorgan Transfer
and GSSG are substrates for the extracellular membrane-
Although GSH is synthesized in the cytosol, it is distributed
bound enzyme GGT:
to different intracellular organelles where it is used in
organelle-specific functions related to its role in the regu-
GSH + amino acid −→ γ-glutamyl-amino acid
(4) lation of cellular redox status. In addition to the cytosolic
+ L-cysteinyl-glycine pool, GSH functions in somewhat independent pools in the
endoplasmic reticulum (ER), nucleus, and mitochondria. In
most of these compartments GSH is typically found in a
GSH + H2 O −→ L-glutamate + L-cysteinyl-glycine highly reduced state, but in the ER a substantial portion
(5) is oxidised and the ratio [GSH]/[GSSG] may be as high as
3 : 1, whereas in the cytoplasm the oxidized form is usually
γ-L-Glutamyl transpeptidase cleaves only the γ- on the order of about 1% of the total or less [35, 36].
peptide linkage. The enzyme can transfer the γ-glutam- In the ER, GSSG is the main source of oxidizing power
yl group of GSH, GSSG, or GSH-conjugates onto amino that supports the efficient production of the functional
acid acceptors to form γ-glutamyl peptides and cysteinyl- conformation of nascent polypeptides by the formation of
glycine (reaction (4)), or to water thereby hydrolyzing GSH the required intramolecular disulfide bonds between cysteine
and related compounds to glutamate and cysteinylglycine residues. In the nucleus, GSH maintains the appropriate
(reaction (5)). Cysteinylglycine can be further hydrolyzed redox status of the sulfhydryl groups in proteins involved
Journal of Amino Acids 5

in nucleic acid biosynthesis and DNA repair in addition to and the dissociation of cytochrome c. ROS also induce
standard antioxidant functions. In this compartment, it is an increase in permeability of the internal mitochondrial
also used in the reduction of ribonucleotides to produce membrane for calcium. Enhanced ROS and calcium levels,
deoxyribonucleotides by ribonucleotide reductase [37]. acting in concert, may trigger the cell death machinery via
About 10–15% of cellular GSH is located in mitochon- apoptosis or necrosis. Hence, mitochondrial GSH clearly
dria. Since mitochondria have a very small volume, the local has an important role in preventing apoptosis triggered by
GSH concentration in these organelles is usually higher than cytochrome c release from the inner membrane.
that in the cytosol. Of the various subcellular compartments, Not surprisingly, therefore, a decrease in mGSH levels is
most attention has been paid to the mitochondrial GSH closely associated with certain pathologies in both humans
pool (mGSH) because of the close relationship between and animals. This relationship has been described for
mGSH and cell survival that has been demonstrated in hypoxia/reperfusion injury [47, 48], certain liver diseases
many cases. This topic is covered in an excellent recent such as alcoholic steatohepatitis [49, 50], nonalcoholic
review of Mari et al. [38] and readers are directed to this steatohepatitis [51, 52], and liver cirrhosis [53, 54], neuro-
review for extensive details. Here, I will mention just a logical diseases such as Alzheimer and Parkinson diseases,
few important aspects of the mGSH system. As mentioned diabetes mellitus and associated complications [55–57].
above, GSH is synthesized only in the cytosol and is Many of the abovementioned pathologies are included in
transported into intracellular organelles. It easily crosses the the group of so-called age-related diseases and, therefore,
outer mitochondrial membrane through porin channels but, it is not easy to differentiate aging as a normal physio-
being an anion, cannot diffuse across inner mitochondrial logical process and age-related or age-induced pathologies.
membrane into the matrix. At least two systems are believed Harman [58] proposed the oxidative stress theory of aging,
to be involved in GSH import into the mitochondria across which he later modified to the mitochondrial theory of
the inner membrane. GSH transport into the matrix must aging [59]. This theory suggested that oxidative damage to
overcome an unfavourable electrochemical gradient [39– organisms is connected with the progressive accumulation
44]. This is provided by two mitochondrial membrane of oxidized/modified products of ROS attack that ultimately
carriers [45, 46] that exchange GSH for dicarboxylates determine the lifespan of organisms. Insofar as they are
and 2-oxoglutarate (α-ketoglutarate). These two antiport cornerstones of the oxidative stress and/or mitochondrial
carriers provide electroneutral exchange of selected anions theories of aging, ROS and mitochondrial function are
across the inner mitochondrial membrane with no charge intimately regulated by GSH and the [GSSG]/[GSH] ratio,
transfer. The role of these two mitochondrial GSH carriers thereby linking these theories of aging to mitochondrial
was also evidenced by a reconstitution of recombinant GSH levels. Other pathologies, such as several diseases of
mitochondrial dicarboxylate carriers into proteoliposomes the lungs (e.g., chronic pulmonary disease, acute respiratory
[45]. However, it should be noted that during GSH import distress syndrome, neonatal lung damage, and asthma)
the mitochondria lose important intermediates of the Krebs and of the immune system are also associated with a
cycle so that anaplerotic mechanisms may be needed to compromised mitochondrial GSH system [60–62]. Finally,
replenish these. It should also be noted that GSSG cannot mGSH involvement in combating the toxicity of different
leave the mitochondria and therefore needs to be regenerated xenobiotics, particularly drugs such as cisplatin, is clearly
in the matrix by GR using NADPH (reaction (3)). evident [63–65].
In addition to its “classic” functions, GSH plays One more important point related to mGSH should also
organelle-specific roles in the mitochondria and a few of be mentioned here. The correct analysis of the mitochon-
them will be mentioned here. Due to the pivotal role of drial GSH pool is an experimentally complicated issue. To
mitochondria in programmed cell death (apoptosis) as well study this, cells are typically disrupted in order to isolate
as extensive ROS involvement in this process, and adding the mitochondria and this can substantially affect not only
fact that mitochondria produce over 90% of cellular ROS, redox status, but also total GSH content. Hence, there is a
the role of GSH in cell protection cannot be overestimated.
need to introduce new techniques for the proper evaluation
GSH may either directly bind some ROS species or serve as
of the operation of the mitochondrial GSH system. Some
a source of reductive power for certain antioxidant systems.
interesting ideas on this topic can be found in recent studies
The inner mitochondrial membrane is particularly rich
by Winther and colleagues [66, 67].
in cardiolipin, whereas it is virtually absent from other
membranes and only the outer mitochondrial membrane Another important topic is GSH distribution between
contains minor amounts of this phospholipid. When mGSH different organs of animals. Glutathione can be transported
levels are compromised, cardiolipin is one of the important across the plasma membrane, which is the first step of a
targets of oxidative damage. Due to its unique chemical complicated interorgan transfer network [4, 13]. Liver is the
structure among phospholipids, cardiolipin confers stability main source of GSH exported into the blood [68–71]. The
and fluidity to the mitochondrial membrane. In addition, export of GSH and its conjugates from liver cells occurs
cytochrome c is normally bound to the inner mitochondrial via transporters referred to as organic anion-transporting
membrane via its association with cardiolipin. By protecting polypeptides (OATPs), which are generally believed to carry
cardiolipin from oxidative damage, GSH prevents changes out electroneutral exchange, in which the cellular uptake
in the physicochemical properties of the mitochondrial of organic anions is coupled to the efflux of anions such
inner membrane that lead to membrane destabilization as HCO3 − , GSH, GSSG, and/or glutathione S-conjugates
6 Journal of Amino Acids

[72, 73]. Both GSH and GSSG are circulated and are used products from ROS-promoted oxidation of lipids such as
to supply other organs, particularly kidney. The production malonic dialdehyde and 4-hydroxy-2-nonenal [79, 80], and
in liver and export from it are related to GSH functions, probably many other products of ROS interaction with
and at least two principles may be implicated. The first cellular components [11, 19, 81, 82]. The thiyl radicals
one involves epithelial cells that contact with the exterior, formed from these reactions can also combine with different
such as intestine and lungs. The primary GSH function molecules, as well as with other thiyl radicals leading to
here is directed to detoxification of injurious external agents the formation of oxidized glutathione (glutathione disulfide,
to prevent damage to the organism. There is a large body GSSG) in the latter instance. GSSG is also produced in
of data indicating that this is an important role of GSH reactions catalyzed by GPx (reaction (6)) and glutaredoxins
in normal intestinal function. The lungs are exposed to (reaction (7)):
high oxygen levels and also to inhaled toxins. Alveolar
macrophages provide an additional ROS source in this tissue. ROOH + 2GSH −→ ROH + GSSG + H2 O (6)
Hence, there are multiple reasons for maintaining adequate
GSH levels in lungs. The second principle is related to
high intensity oxygen-based metabolism and detoxification Oxidized glutaredoxin + 2GSH
of certain compounds by internal organs. Liver and kidney (7)
are probably the best representatives of this group. The portal −→ reduced glutaredoxin + GSSG
vein brings blood from the intestine to the liver and, if not
GSSG may be either excreted from the cell, or reduced
detoxified in the intestine, xenobiotics must be neutralized
by GR at the expense of NADPH (reaction (3)). Most of the
by hepatocytes [52, 74–76]. In addition, the liver is an
reductive power for this reaction is provided by the pentose
important biosynthetic organ where ROS are produced in
phosphate shunt-two molecules of NADPH are produced per
substantial amounts as side products of energy production
molecule of glucose-6-P that cycle through the pathway. The
in the mitochondrial electron transport chain or as the
first and limiting step is catalyzed by glucose-6-phosphate
result of biosyntheses involving diverse oxygenases. Kidney
dehydrogenase (G6PDH, EC 1.1.1.49):
also requires a highly efficient GSH system to perform its
functions [13, 77, 78]. The problems with extracellular GSH Glucose-6-phosphate + NADP+
investigation and intertissue transfer are to a large extent (8)
based on inadequate methodology. Since the concentrations −→ 6-phosphoglucolactone + NADPH + H+
of extracellular GSH are more than an order of magnitude
lower than intracellular levels, correct redox ratios are often The second molecule of NADPH is provided by the
difficult to determine. next pentose phosphate shunt reaction, catalyzed by 6-
phosphogluconate dehydrogenase (6-PGDH). These two
enzymes are not the only cellular NADPH producers.
4. Glutathione Functions NADPH is also formed by NADP-dependent isocitrate
dehydrogenase, malic enzyme, and some others, but it is
The chemical structure of GSH determines its potential func- widely believed that most cellular NADPH is generated by
tions and its broad distribution among all living organisms the pentose phosphate pathway.
reflects its important biological role. GSH has been found As mentioned above, the glutathione couple GSH/GSSG
in all mammalian cells. Probably most importantly, GSH is a critically important redox player and together with
is responsible for protection against ROS and RNS, and other redox active couples, including NAD(P)/NAD(P)H,
detoxification of endogenous and exogenous toxins of an FAD/FADH2 , regulates and maintains cellular redox status.
electrophilic nature. Other functions include (i) maintaining The estimated in vivo redox potential for the GSH/GSSG
the essential thiol status of proteins and other molecules; (ii) couple ranges from −260 mV to −150 mV depending on the
storage of cysteine reserves both in the cell and for interorgan conditions (cited after [10]).
transfer; (iii) involvement in the metabolism of estrogens,
Under normal conditions, when a cell is not stressed,
leukotrienes, and prostaglandins; (iv) participation in the
the processes that generate ROS are well counterbalanced by
reduction of ribonucleotides to deoxyribonucleotides; (v)
antioxidant systems. In this respect, GSH is often considered
participation in the maturation of iron-sulfur clusters in pro-
to be a key player of the defense system. However, under
teins; (vi) copper and iron transfer; (vii) signal transduction
various circumstances the steady-state ROS level increases
from the environment to cellular transcription machinery.
leading to oxidative damage to the cell, called “oxidative
The above-listed GSH functions and a few others will be
stress,” the term first defined by Sies [83] “Oxidative stress”
covered in this section.
“came to denote a disturbance in the prooxidant-antioxidant
balance in favor of the former.” The definition was later
4.1. Elimination of Reactive Oxygen and Nitrogen Species. expanded to “An imbalance between oxidants and antioxi-
GSH is an important antioxidant, directly reacting with dants in favour of the oxidants, potentially leading to damage,
ROS, RNS, and other reactive species, particularly HO• , is termed “oxidative stress”” to emphasize the damage to
HOCl, RO• , RO2 • , 1 O2 , and ONOO− , often resulting in certain cellular components [84]. Owing to extensive studies
the formation of thiyl radicals (GS• ) (Figure 3). GSH is on oxidative stress and the discovery of many intricacies
also involved as an antioxidant in the detoxification of related to this phenomenon over the two last decades, the
Journal of Amino Acids 7

definition could be modified to “Oxidative stress is a situation Oxidative stress

where the steady-state ROS concentration is transiently or
chronically enhanced, disturbing cellular metabolism and its Quasistationary ROS level
regulation and damaging cellular constituents” [81]. This Acute
definition underlines the dynamic nature of the processes of Oxidant oxidative
stress

ROS concentration
ROS generation and elimination, damage to cellular core and
regulatory pathways, and potential negative consequences of Chronic oxidative stress
enhanced ROS levels either acutely or chronically. If cells are Steady-state (stationary) ROS level
not capable of coping with the intensity of oxidative stress,
this can culminate in their death via necrosis or apoptosis. Time
The dynamics of ROS-related processes are shown in Reductive stress
Figure 4. Under control conditions, steady-state ROS levels
fluctuate over a certain range [81, 82, 85]. However, ROS
Figure 4: The dynamics of reactive oxygen species in biological
levels can exceed this range due to an increase in ROS
systems. Steady-state levels of reactive oxygen species fluctuate over
production either as a result of internal physiological changes a certain range under normal conditions. However, under stress
or external induction. If the cellular antioxidant potential is ROS levels may increase or decrease beyond the normal range
high enough, acutely increased ROS levels can be quickly resulting in acute or chronic oxidative or reductive stress. Under
reduced again back to the initial (control) range. But if the some conditions, ROS levels may not return to their initial range
existing antioxidant potential is not capable of eliminating and stabilize at a new quasistationary level.
extra ROS, the cell can increase its antioxidant defenses,
but it will require some time to respond, and this will also 4.2. Elimination of Endogenously Produced Toxicants. The
consume energy and important biomolecules (e.g., amino role of GSH in detoxification of the end products of lipid
acids). Upregulation of the antioxidant potential may result peroxidation such as malonedialdehyde and 4-hydroxy-2-
in the restoration of ROS levels back into the initial range, nonenal was mentioned above. Many other toxic metabo-
or due to a prolonged increase in ROS levels the cell may lites are produced as side-products of the normal cellular
enter a state of “chronic oxidative stress” (Figure 4). In many metabolism. For example, methylglyoxal (2-oxopropanal) is
cases, acute oxidative stress has no serious consequences for one of these and it can be generated both enzymatically and
organisms, but the chronic state may lead to or accompany nonenzymatically [92, 93]. Glycolysis appears to be the main
certain pathologies. Oxidative stress is well-documented to source of methylglyoxal where it is produced from triose
occur, for example, in cardiovascular and neurodegenerative phosphates, particularly due to spontaneous decomposition
diseases, diabetes mellitus, cancer, and aging [9, 12, 47, 51, of glyceraldehyde-3-phosphate [94, 95]. Methylglyoxal toxic-
86–89]. Under some circumstances, ROS levels do not return ity is based on its capacity to interact with any molecule con-
to the initial range and the system may be stabilized at new, taining free amino groups such as amino acids, nucleotide
higher ROS level referred to as “quasistationary” that occurs bases of nucleic acids, and cysteine residues in proteins [96–
in various pathological states [81]. Interestingly, the opposite 99]. Methylglyoxal and other α-dicarbonyls, in turn, may be
situation of decreased ROS levels can occur in some instances involved in ROS generation. Glutathione acts as a cofactor
and is sometimes called “reductive stress.” However, there has in the system of methylglyoxal elimination which consists
been very little investigation of this situation and, therefore, of two enzymes called glyoxalases [92, 100, 101]. The first
it will not be further discussed here. enzyme in this pathway, glyoxalase I (Glo I, EC 4.4.1.5),
The above short excursion into oxidative stress theory catalyses the isomerization of hemiacetal adducts, which are
underscores not only the importance of GSH for ROS formed in a spontaneous reaction between a glutathione and
combating in unstressed conditions, but also the augmented aldehydes such as methylglyoxal:
role that GSH must play during oxidative stress. Enhanced
ROS levels may require not only enhanced GSH action to glutathione + methylglyoxal ←→ hemithioacetal adduct
maintain redox status, but also enhanced energy and material ←→ (R)-S-lactoylglutathione
consumption to replace consumed GSH and/or transport it
(9)
to the places where it is needed.
As mentioned above, GSH may be involved in detoxi- The second enzyme, glyoxalase II (Glo II, EC 3.1.2.6),
fication of RNS [6]. For example, nitric oxide (• NO) was catalyzes the hydrolysis of the product of the above reaction:
initially thought to interact directly with GSH to produce
S-nitrosoglutathione (GSNO). However, further investiga- (R)-S-Lactoyl-GSH + H2 O −→ D(-)lactic acid + GSH
tion demonstrated • NO must first be converted to NO+ (10)
(nitrosonium ion) in an iron- or copper-catalyzed reaction
before reacting with GSH to form GSNO [90, 91]. It should This pathway is the main route for methylglyoxal
be noted that GSNO and other nitrosothiols can be used catabolism in yeasts [3, 95, 102, 103] and mammals [104–
for storage and transportation of • NO because as unstable 107].
compounds they can be decomposed easily to generate • NO GSH also may be involved in the detoxification of
and GSSG. endogenously produced formaldehyde. For example, some
8 Journal of Amino Acids

yeasts produce formaldehyde as part of methanol catabolism the effects of insulin [128, 129]. It is worth noting that
[108–111]. The reaction is catalyzed by formaldehyde although Cr3+ is thought to be a regulator of carbohydrate
dehydrogenase (FaDH, EC 1.1.1.1) which uses GSH as a metabolism, the capacity of biological systems to reduce Cr6+
cosubstrate: with the participation of the GSH system may be used to
Formaldehyde + GSH + NAD+ −→ S-formylglutathione deliver chromium into biological systems.
GSH plays a more specific and well-documented role in
+ NADH + H+ the metabolism of copper and iron. GSH is believed to be
(11) responsible for the mobilization and delivery of copper ions
for the biosynthesis of copper-containing proteins [118]. In
Formaldehyde also may be produced from the catabolism this case, GSH is involved in (i) reduction of Cu2+ to Cu+ ,
of certain amino acids and, therefore, reaction (11) may (ii) mobilization of copper ions from stores, and (iii) delivery
be important for its detoxification in animals and plants of copper ions during the formation of “mature” proteins.
[112, 113]. Interestingly, formaldehyde dehydrogenase also For the last function, Cu2+ must be reduced to Cu+ before
catalyzes the decomposition of S-nitroso-glutathione and it it can be incorporated into apoproteins, and GSH provides
is not limited to yeasts [24], but also found in plants and the reducing power [130]. Interestingly, GSH is not only the
animals [114–117]. carrier for Cu+ , but is also involved in copper mobilization
from metallothioneins in a reversible manner. The Cu(I)-
4.3. Metal Homeostasis. GSH can interact with certain metal GSH complex is used for copper incorporation into Cu,Zn-
ions. It contains six potential coordination sites for metal superoxide dismutase (Cu,Zn-SOD) from bovine erythro-
ion binding such as cysteinyl sulfhydryl, glutamyl amino, cytes [131] lobster apohemocyanin [132], and blood plasma
glycyl, and glutamyl carboxyl groups, and two peptide bonds. albumin [133].
Among these, the sulfhydryl group possesses the highest The role of GSH in iron metabolism is not as well
affinity for metal cations, particularly cadmium, copper, zinc, studied. However, by analogy with copper, GSH may be
silver, mercury, arsenic, and lead [118]. The interaction of a involved in iron reduction, transportation, mobilization
metal ion with the GSH sulfhydryl group can be stabilized from different stores, and incorporation into certain target
by coordination with other potential binding sites. The most molecules. GSH involvement in iron metabolism in the yeast
stable complexes are formed by divalent cations in a 1 : 2 S. cerevisiae, has been investigated in details [134]. GSH was
ratio with GSH. The complexes form spontaneously because not required for iron adsorption, delivery to mitochondria,
they are thermodynamically favored and the resulting mer- maintenance of mitochondrial Fe,S-proteins, or for their
captides are relatively stable. Several metabolic functions for maturation. However, the maturation of extramitochondrial
these metal-GSH complexes have been proposed: (i) they Fe,S-proteins required GSH. Although the precise role of
can help in the mobilization and transfer of cations between GSH in this process is not clear, GSH involvement in
ligands; (ii) they can serve to transport metal ions across facilitated transport of components of Fe,S-clusters was
membranes; (iii) they serve as a source of cysteine, playing a suggested [134].
central role in metal homeostasis; (iv) they serve as a cofactor
for redox reactions yielding metal compounds with different
speciation or biochemical forms [118]. The remainder of this 5. Glutathione Peroxidases and Transferases
section focuses only on GSH involvement in the metabolism and Their Regulation
of chromium, copper, and iron ions.
GSH is involved in Cr6+ reduction in many organisms These enzymes play very specific roles in cellular metabolism
(reviewed in [82, 119]). GSH-dependent reduction of Cr6+ that should be specially highlighted. As mentioned above,
results in the formation of Cr3+ , effectively converting the GPx catalyzes the GSH-dependent reduction of many per-
ion from an anionic form (Cr2 O7 2− or CrO4 2− ) to a cationic oxides (reaction (6)). GPx enzymes are particularly involved
form [82, 120]. Cr6+ in its anion form (associated with in the removal of LOOH, thereby terminating lipid peroxi-
oxygen) is readily transported into cells via nonspecific anion dation chain reactions and protecting biological membranes.
carriers, but Cr3+ as a cation is not so bioavailable and is Four isoenzymes of GPx have been identified in mammalian
believed to be less toxic due to its interaction with many tissues [135, 136]. The active site of these enzymes contains
cellular ligands [121]. Therefore, Cr6+ reduction to Cr3+ can a selenocysteine residue which is responsible for the catalytic
be characterized as a way to decrease chromium toxicity activity. Mammalian isoenzymes GPx-1, GPx-2, and GPx-3
[122]. Although Cr6+ can be reduced nonenzymatically, reduce H2 O2 and peroxides of free fatty acids, whereas GPx-
studies suggest that in cells GSH and GSH-dependent 4 reduces peroxides of phospholipids and cholesterol [137].
enzymes, either alone or in concert with ascorbic acid and Certain glutathione S-transferases (GST, EC 2.5.1.18)
cysteine, play an important role in these processes [119, 123– catalyze GSH conjugation with electrophiles, but some
126]. For example, the inhibition of GR by carmustine also catalyze the reduction of lipid peroxides and as a
prevented Cr6+ reduction in isolated rat hepatocytes [127]. consequence they are also called selenium-independent per-
Nontoxic biological effects of chromium are also associated oxidases [138]. These GSTs do not possess a selenocysteine
with GSH-related transformation of Cr6+ . Although it is not residue in their active site. GSTs are an enzyme super-
clear how this occurs, these effects are related to the ability family responsible for biotransformation of electrophilic
of chromium to affect carbohydrate metabolism potentiating compounds. In this way GSTs protect organisms against
Journal of Amino Acids 9

genotoxic and carcinogenic compounds of both exogenous II detoxification enzymes via interaction with antioxidant
(xenobiotics) and endogenous origin. Mammalian GSTs are response elements (ARE) in regulatory regions of many of
organized in multiple classes designed by Greek letters. the genes that encode antioxidant enzymes [21, 159–168]
Major classes include Alpha, Mu, Pi, abbreviated in Roman (the same gene region is also known as the electrophile
capitals as A, M, P. [139]. Traditionally, GST activity response element (EpRE) to designate its involvement in the
is measured with 1-chloro-2, 4-dinitrobenzene (CDNB), cellular response to electrophiles). In animals, the activities
cumene hydroperoxide, or tert-butyl hydroperoxide as the of many phase II detoxifying enzymes, including GSTs
substrates. Due to selenium-independent GPx activity, α- and GPxs, are also upregulated via the Nrf2/Keap1 system.
class GSTs can efficiently reduce peroxides of free fatty acids The dilemma of the simultaneous regulation of GSTs and
and phospholipids, as well as cholesterol hydroperoxides antioxidant enzymes was solved when the mechanism by
[140]. It is worth noting that α-class GSTs can reduce which the Nrf2/Keap1 system operation was uncovered
peroxides of membrane phospholipids without requiring (Figure 5). Under normal (nonstressed) conditions Nrf2
phospholipase A2 -mediated release of the peroxidized fatty protein interacts with Keap1 in the cytosol and is quickly
acids from the membrane phospholipids [141, 142]. The role ubiquitinated followed by the proteasomal degradation.
of α-GST in peroxide metabolism is highlighted in excellent However, when ROS levels rise, Keap1 is oxidized and
reviews of Awasthi and colleagues [140, 143]. becomes incapable of binding Nrf2. This results in its
By regulating the level of certain electrophiles, GSTs and migration (possibly related to phosphorylation by certain
GSH may indirectly affect regulatory pathways controlled protein kinases) into the nucleus. In the nucleus, Nrf2 binds
by these compounds. For example, 4-hydroxynonenal (4- to the ARE (EpRE) DNA element of target genes together
HNE) is a well-known product of lipid peroxidation, which with a small Maf protein and perhaps with other proteins.
has a key role in stress-mediated signalling. Its steady-state The complex stimulates the expression of target genes,
intracellular level is determined by the balance between including those encoding GSTs and antioxidant enzymes.
production due to lipid peroxidation and elimination via Clearly, enhanced expression of antioxidants and phase II
various pathways. One of the subgroups of the anionic α- detoxification enzymes is an important factor in increasing
class of GSTs can utilize 4-HNE as a preferred substrate, cellular resistance to xenobiotics. In addition to GSTs, a
conjugating it to GSH with high efficiency [140]. The enzyme key enzyme of GSH-biosynthesis, γGLCL, is also among the
shows a much higher affinity toward 4-HNE than to most targets of the Nrf2/Keap1 regulatory pathway. Because of its
xenobiotics suggesting its critical role in the regulation of involvement in the regulation of diverse physiological pro-
cellular 4-HNE levels. The adduct formed, GS-HNE, is cesses, and especially those related to GSH, the Nrf2/Keap1
exported from cells in an ATP-dependent manner by a system has gained attention not only at the basic biological
primary transport system similar to the system that extrudes level, but also from a pharmacological viewpoint.
other GSH conjugates [144, 145]. Detoxification of xenobiotics in animals is usually,
However, GSTs may not only play positive roles in but not always, provided by a specific system consisting
cell protection against xenobiotics. In certain cases, they of so-called phase I, phase II, and phase III enzymes.
can be responsible for the need to increase the doses of Phase I enzymes are represented by hydroxylases such as
specific drugs. For example, in many solid tumors enhanced endoplasmic reticulum members of the cytochrome P450
resistance to drugs is associated with the increased activity of family, which introduce oxygen onto molecules of hydropho-
GSTs that detoxify xenobiotics [27, 146]. GST was identified bic xenobiotics and endogenous compounds, transform-
as a prominent protein in many cases and is overexpressed ing them in more hydrophilic forms. Phase II detoxi-
in many cancers resistant to several drugs. These GSTs have fication enzymes catalyze conjugation reactions that add
been proven to be a viable target for prodrug activation glutathione, amino acids, sulphate, glucuronic, acetyl, or
with at least one candidate in late-stage clinical development methyl residues to activated xenobiotics. Plasma membrane
[146]. antiporters represent phase III detoxification; these energy-
The activities of GPxs and GSTs, like other antioxidant dependent pumps export conjugates from the cell, thereby
enzymes, are regulated in many ways. Most attention has decreasing their intracellular concentration. Although this
been paid to their upregulation via specific regulatory path- system of nomenclature for the detoxification of xenobiotics
ways involving ROS or electrophiles at certain stages. Many
can be useful, the classification may not always hold for
reviews extensively describe these pathways [147–152], and
detoxification reactions involving GSH. For example, many
here we will describe just a few of them where GSH is known
electrophilic xenobiotics can react directly with GSH without
to be an active participant. OxyR-related regulatory protein
was described in bacteria about 20 years ago (reviewed in the prior need for activation by phase I enzymes [34].
[153–158]). Subsequently, the YAP1/GPx3-regulated system
was found to be responsible for augmentation of antioxidant 6. Glutathionylation of Cellular Sulfhydryls
potential in yeast [85, 154, 157, 158]. Finally, in animals
the operation of ROS-based regulatory cascades, involving An increase in cellular levels of mixed disulfides formed
GSH and GSH-related enzymes, has been identified. In between GSH and protein thiols, a process called glutathi-
this context, the Nrf2/Keap1 system of animals is often onylation, was demonstrated to be caused by oxidative stress
considered to be the most important and finely controlled about three decades ago [169–171]. Since that time many
pathway that regulates the activities of antioxidant and phase studies of the role of glutathionylation have been carried out.
10 Journal of Amino Acids

Cul3
Keap1 Ub
Nrf2 Ub
SHSH
Nrf2
SH SH Ub
Ub
Keap1 Ub
Keap1 Proteasomal
Modification
SH SH
of
Nrf2/Keap1

Degradation

Proteine kinases: ERK,

Prooxidants Nucleophiles ZNK, P38, MAP kinase,
PKC, CK-2, etc.

Keap1 Keap1 Keap1

l3 l3 l3
Cu S S Cu SHSH Cu SH SH
S S SHSH SHSH
Keap1 Keap1 Keap1

Nrf2 Nrf2 Nrf2 P

Stabilization Phosphorylation

Cytosol
Nucleus

Nrf2 Map Antioxidant/detoxifying enzymes

ARE/EpRA Gene

Figure 5: Operation of the Nrf2/Keap1 system during response to oxidative stress in animals. Under nonstressed conditions the transcription
factor Nrf2 binds to the Keap1 homodimer. The resulting protein complex can then further complex with Cullin 3 leading to ubiquitination
of Nrf2 followed by proteasomal degradation. Following an oxidative insult or electrophilic attack, Keap1 cannot bind Nrf2 which allows
Nrf2 to diffuse into the nucleus and, in concert with small Maf proteins (sMaf), Map and others, Nrf2 binds to the ARE/EpRE elements of
regulatory regions in genes encoding antioxidant or phase 2 detoxification enzymes. Nrf2 migration into the nucleus is promoted by at least
three different mechanisms: oxidation of Keap thiol groups to form disulfides, modification of Keap1 cysteine residues by electrophiles, or
phosphorylation of Nrf2 by protein kinases that, in turn, may be activated by oxidants.

Work from the laboratory of Sies and others implicated the residues are important for protein function. Therefore, in
process in the regulation of the activity of specific enzymes addition to direct reduction of sulfenic acid to cysteine,
and certain regulatory pathways [6, 172–176]. From this, glu- living organisms possess other ways of dealing with this
tathionylation was recognised as one of the physiologically moiety (Figure 6). Sulfenic acid residues may interact with
relevant mechanisms of posttranslational modification of reduced glutathione forming mixed disulfides [182, 183].
certain proteins. Exposure of cysteine residues of proteins to This issue is not so straightforward, because formation
ROS leads to their oxidation with the consequent formation of this dithiol can be implicated in the regulation of
of stable sulfenic, sulfinic, or sulfonic acid derivatives and some metabolic pathways. Many proteins are subject to
unstable transient forms (Figure 6). Sulfenic acid may be glutathionylation and some of them lose biological activity
returned to the original cysteine form by several reductases as the result, whereas others may be activated [182]. In
([6, 177] and cited therein) whereas sulfinic acid can be human T lymphocytes, Fratelli and colleagues [184] found
reduced only by the specific action of sulfiredoxin [178– that cell exposure to oxidants (diamide and H2 O2 ) enhanced
181]. It is believed that sulfonic acid cannot be reduced glutathionylation of certain proteins. These included cyto-
in living organisms. Cysteine oxidation to sulfenic acid skeletal proteins (vimentin, myosin, tropomyosin, cofilin,
may be used for ROS sensing and in this case it plays a profilin, and actin), metabolic enzymes (enolase, aldolase, 6-
positive role in cell adaptation. However, more frequently the phosphoglucolactonase, adenylate kinase, ubiquitin-conju-
oxidation may inhibit certain proteins if the oxidized cysteine gating enzyme, phosphoglycerate kinase, triose phosphate
Journal of Amino Acids 11

TR

OH O O
S• OH
SH S S O S OH
ROS OH− ROS ROS
Pr Pr Pr Pr Pr
Cysteine Cysteinyl Sulfenic Sulfinic Sulfonic
residue radical acid Srx acid acid

GSSG
GSH
GSSG
GSH
in
ox ase
red sfer
a
ut an GSH
Gl oltr S SG
i
th
Pr SH
SH
Pr
Pr

GSH [GS–OH] GS–O–SG

Glutathione Glutathione
sulfenate disulfide
S-oxide

Figure 6: Oxidation of protein cysteine residues to sulfenic, sulfinic, or sulfonic derivatives and formation of glutathionylated proteins. In
biological systems, sulfenic and sulfinic derivatives may be reduced by thioredoxin (TR) and sulfiredoxin (Srx), respectively, whereas sulfonic
moieties cannot be reduced. Glutathionylated proteins are formed by direct interaction of GSH with sulfenic acid derivatives, exchange
between cysteine residues and GSSG, or interaction with oxidized glutathione forms.

isomerase and pyrophosphatase), redox enzymes (perox- fructose-1,6-bisphosphatase was activated by CoASSG [188].
iredoxin 1, protein disulfide isomerase, and cytochrome c A very potent vasoconstrictory effect of CoASSG has also
oxidase), cyclophilin, stress proteins (HSP70 and HSP60), been described [189].
nucleophosmin, transgelin, galectin, and fatty acid binding GSTs also play regulatory roles in many cellular processes
protein. S-Glutathionylation is thought to be one of the in ways that are not usually directly related to their catalytic
mechanisms preventing ROS-induced irreversible protein activity. Frequently their direct interaction with certain
inactivation under oxidative stress insults. During recovery, regulatory enzymes/proteins has been shown to be involved
GSH residues can be removed from the glutathionylated in cellular responses to oxidative stress, regulation of pro-
proteins resulting in restoration of their functional activity. liferation, differentiation, and apoptosis. Most information
Figure 6 shows the known pathways of glutathionyla- on these mechanisms is associated with the pi-type GSTs
tion/deglutathionylation and oxidation of cysteine residues (GSTπ). For example, GSTπ inhibits c-Jun aminoterminal
in cellular processes. The routes leading to the formation of kinase (JNK) [183]. JNK phosphorylation activates c-Jun
mixed disulfides are interactions between: (i) sulfenic acid and triggers activation of multiple downstream effectors
derivatives and GSH, (ii) GSSG and protein cysteine residues, related to proapoptotic signalling and certain cytotoxicities
(iii) protein cysteine residues and glutathione sulfenate, and, but its sequestration in a complex with GSTπ blocks these
finally (iv) protein cysteine residues and glutathione disulfide events. Under oxidative or nitrosative stresses the above
S-oxide. Connected to the protein via a disulfide bond, complex dissociates, and GSTπ undergoes glutathionylation
GSH can be removed via a thiol-disulfide exchange reaction. with subsequent oligomerization. The GSTπ isoenzyme is
GSH is used by glutaredoxin releasing GSSG. It is now clear believed to be the main isoenzyme involved in this effect,
that glutathionylation as a posttranslational modification of although other soluble isoforms of GST may also be involved
proteins can be involved in the regulation of the activity of in this type of regulation [183].
diverse proteins. The glutathionylation process is thought to be
In addition to formation of mixed thiols with proteins, responsible for the anticancer effect of PABA/NO [O2 -{2,4-
GSH may also form mixed disulfides with low molecular dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl}1-(N,N-
mass thiols. In many cases, the biological relevance is dimethylamino)diazen-1-ium-1,2-diolate] [190]. Overex-
uncertain, but in the case of coenzyme A the formation pression of GSTπ in solid tumors is linked to the
of the mixed disulfide may be biologically important. For development of resistance to a number of anticancer
example, CoASSG was found to inhibit GR [185], phospho- agents. PABA/NO is catalytically activated by GSTπ releasing
•
fructokinase [186], and fatty acid synthase [187], whereas NO that elicits antitumor activity both in vitro and in vivo
12 Journal of Amino Acids

[191]. Locally produced • NO extensively modifies specific (iii) phosphorylation of Nrf2 by different protein kinases,
target proteins, particularly protein disulfide isomerase and (iv) ubiquitination of Keap1 followed by proteasomal
(PDI). Nitrosylation or glutathionylation of PDI leads to hydrolysis (Figure 5).
enzyme inactivation, activation of the unfolded protein Deciphering the mechanisms of operation of the
response (UPR), and cancer cell death. It has been suggested Nrf2/Keap1 system helped to explain various previously
that • NO itself may not be directly responsible for the puzzling data on chemoprevention in several disease states.
toxicity of PABA/NO, but rather that peroxynitrite, which Chemoprevention has attracted much attention as one of
is much more reactive, provides the effect. Peroxynitrite the most practical and realistic strategies for decreasing
is a product of the interaction between nitric oxide and the global burden of diseases related to xenobiotics and
superoxide anion radical and is known to be a powerful certain oxidants. A mechanistic approach has gained accep-
nitrosating agent [190]. tance recently because it not only provides the rationale
to reveal potential mechanisms, but it also predicts and
identifies potentially effective chemicals. A broad spectrum
7. Regulation of Transcription of of substances have been reported that exhibit chemopre-
GSH-Related Genes ventive potential, and it is noticeable that many of these
substances were identified in plants, particularly those that
Being an important antioxidant either directly, or via GSH- are medicinal and/or edible. Numerous phytochemicals
related enzymes, GSH is a key component in the regulation derived from fruits, vegetables, grains, spices, and herbs
of redox homeostasis. It is well known that changes in GSH are capable of affecting certain diseases related to disrupted
levels or deregulation of the redox status are caused or at GSH homeostasis. Extensive reviews on chemopreventive
least are associated with diverse pathologies and aging. The phytochemicals have been published. Thus, there is no need
most thoroughly investigated cases include cardiovascular for in depth coverage of this field, and interested readers are
and neurodegenerative diseases, cancer, AIDS, cystic fibrosis, directed instead to several excellent recent reviews [21, 22, 86,
liver disorders, diabetes mellitus, and associated complica- 166, 195–198]. In the present review, discussion will be lim-
tions. Regulation of the activities of GSH-related enzymes ited to well-studied phytochemicals that operate by affecting
is often considered as a way to prevent or ameliorate the the Nrf2/Keap1 system. These have been exceptionally well
disease. Several cellular signalling systems are known to discussed by Surh and colleagues [21] and are summarized
be involved. However, the mostly efficient approaches are in Table 1.
related to the possibility of manipulating GSH biosynthesis Sulforaphane [1-isothiocyanato-(4R,S)(methylsulfinyl)
and phase II detoxification enzymes. In the former case, butane] is an isothiocyanate found in broccoli and other
attention is focused on the first key enzyme of GSH cruciferous plants. It is a known inducer of genes encoding
synthesis, γGLCL, and in the latter case on GSTs. These phase II defense and antioxidant enzymes including GPx,
enzymes are mainly regulated at the expression level and GST, and γGLCL [196, 211]. Sulforaphane appears to
some of the mechanisms involved have been deciphered. modulate upstream MAP kinases, but reliably demonstrated
Although it is known that the promoter regions of the genes effects are associated with Nrf2 activation via the direct
encoding γGLCL and GSTs possess binding sites for such modification of Keap1 cysteine residue(s) [199]. As an ele-
transcriptional regulators as NF-κB, AP-1, AP-2, SP-1, and ctrophile, sulforaphane directly interacts with protein thiols
others [192–194], most attention has been concentrated on forming thionoacyl adducts. In addition, sulforaphane
the Nrf2/Keap1 system [160, 195]. This is connected, at induces structural changes in Keap1 leading to its polyu-
least partially, to its high sensitivity to effectors relative to biquitination and proteasomal degradation [200].
other regulatory systems [81]. The Nrf2/Keap1 system is
Curcumin (diferuloylmethane) is derived from the
responsive to many challenges, particularly to oxidants and
rhizomes of turmeric (Curcuma longo). It stimulates the
electrophiles. As mentioned above, Nrf2 operates in concert
expression of antioxidant and phase II detoxification enzyme
with an adaptor protein, Keap1, a cytoplasmic resident. In
genes in several experimental models [212–214]. Curcumin-
nonstressed cells the binding of Nrf2 to Keap1 promotes
induced expression is also mediated via Nrf2 activation in a
ubiquitination of Nrf2 followed by proteasomal degrada-
ROS-related manner. ROS activate PKC and P38 MAP kinase
tion. This system is tightly regulated in cells (Figure 5).
which then have downstream effects by phosphorylation of
Enhanced levels of oxidants or electrophiles, as well as
Nrf2 [201, 215].
activation of various protein kinases disrupt the Nrf2/Keap1
association resulting in Nrf2 stabilization and migration into Epigallocatechin gallate (EGCG) is a major active cate-
the nucleus. Therein Nrf2 binds to the ARE/EpRE in the chin of green tea that exerts antioxidant, anti-inflammatory
promoter region of target genes and in concert with small and chemopreventive properties [86, 216, 217]. It stimulates
proteins of the Maf family stimulates their transcription. In Akt, ERK1/2 and P38 MAP kinase leading to Nrf2 phospho-
a series of elegant studies several mechanisms that direct rylation and its import into the nucleus [202, 218].
Nrf2 into the nucleus have been described (reviewed in [81]): Several allyl sulfides, namely, diallyl sulfide (DAS), diallyl
(i) oxidation of specific cysteine residues of Keap1 resulting disulfide (DADS), and diallyl trisulfide (DATS) are major
in its inability to bind Nrf2, (ii) interaction of nucleophilic components of garlic that are capable of inducing phase II
molecules with cysteine residue(s) of Keap1 leading to detoxification enzymes in a Nrf2-dependent manner [203,
the formation of adducts that prevent binding to Nrf2, 204, 219]. DAS transiently increases ROS concentrations
Journal of Amino Acids 13

Table 1: Phytochemicals that are known to activate the Nrf2/Keap1 signalling pathway in human and animal systems with identified
mechanisms.

Keap1 Nrf2
Phytochemical References
Oxidation Alkylation Ubiquitination Phosphorylation
Sulforaphane − + ? ? [199, 200]
Curcumin + − − ? [201]
Epigallocatechin gallate + [202]
Allyl sulfides ? + [203, 204]
Resveratrol + [205]
Capsaicin + [206]
(10)-Shogaol + [207]
Lycopene [208]
Carnosol + [209]
Xanthohumol + [210]

stimulating, ERK and P38 MAP kinase which phosphorylate pathway, but it is likely that many more remain to be
Nrf2 [203, 220]. discovered.
Resveratrol (trans-3,5,4 -trihydroxystilbene) is a poly- Many diverse studies on the involvement of Nrf2 and
phenol found in grapes, bilberry, blueberry, other berries, associated components were discussed above. However, in
and other plant species. It exerts antioxidant, anti-inflam- our opinion, the authors have not always provided clear
matory, antiaging, and chemopreventive activities affecting evidence of direct or mediated Nrf2 involvement in the
cellular signalling [205, 221, 222]. These activities are upregulation in certain systems. Although Nrf2 involvement
mediated, at least partially, by Nrf2 phosphorylation. could be expected logically, other signalling pathways should
Pungent vanilloids such as capsaicin (trans-8-methyl- also be investigated. This is especially true when dealing with
N-vanillyl-6-nonenamide), a major pungent of hot chili natural extracts instead of pure compounds because even a
pepper (Capsicum annuum) [206, 223], and (10)-shogaol minor component in the extract may affect the system via
from ginger (Zingiber officinale) also activate phase II detox- an unidentified pathway(s) and imitate Nrf2 involvement.
ification enzyme expression in a Nrf2-dependent manner Unfortunately, in many cases the data presented do not
[207]. The former acts in a ROS-dependent manner via provide definitive evidence to support the involvement of
PI3/Akt mediated Nrf2 phosphorylation, whereas the latter Nrf2.
acts via electrophilic alkylation of Keap1. The chemopreventive efficacy of various phytochemicals
that has been demonstrated in cell models frequently
Lycopene, a natural carotenoid found in tomato and
cannot be extrapolated to whole organisms due to low
tomato products also exerts chemopreventive activity in an
bioavailability. Only a very small portion of consumed
Nrf2-dependent manner [208, 224]. However, there is no
phytochemicals is absorbed in the gastrointestinal tract,
available information on the mechanisms involved. It should
usually much less than 1% [231, 232]. In addition, there
be noted, that absorption of lycopene by the intestine is much
are often potentially negative effects on organisms due
more efficient from processed tomatoes than from fresh
to supposedly useful phytochemicals. They often activate
tomatoes due to a higher bioavailability in the processed
the expression of genes encoding phase I detoxification
products [225–227].
enzymes such as cytochrome P450. This can create problems
Carnosol, an orthophenolic diterpene found in rosemary because many xenobiotics may be activated by oxidation
(Rosmarinus officinalis), also enhances the expression of mediated by these oxygenases and thereby express their toxic
phase II detoxification enzyme genes in an Nrf2-related
potential. In this case, the transcriptional activation of genes
manner [228]. Upregulation of ERK, P38 MAP kinase,
encoding these oxygenases would be considered a negative
and JNK pathways was found to be responsible for the side effect of phytochemical treatment. In some cases, these
effects, which potentially show the involvement of Nrf2 compounds may simultaneously activate the expression of
phosphorylation [228]. Cinnamaldehyde from dried bark of phase I and phase II enzyme genes, in which case the
Cinnamomum cassia also induced phase II enzyme expres- final result would be unpredictable in many circumstances.
sion via Nrf2 translocation into the nucleus [209, 229, 230]. Simultaneous induction of the expression of genes encoding
Xanthohumol, a sesquiterpene from hop (Humulus lupulus) phase II detoxification and antioxidant enzymes may take
also shows chemopreventive activity, inducing antioxidant place with so-called phase III detoxification enzymes which
and phase II detoxification enzymes [210]. Its action was are membrane pumps providing active extrusion of GSH
linked with Nrf2 activation resulting from the alkylation of conjugates of electrophiles that are formed either sponta-
Keap1. Hence, a great variety of diverse agents of natural neously or enzymatically in GST-catalyzed reactions. A final
origin have been found that activate the Nrf2 signalling important issue must be emphasized when analyzing effects
14 Journal of Amino Acids

due to phytochemicals. Phase II and phase III detoxification via supplementation of substrates and energy, (2) increased
enzymes may be responsible for catabolizing certain drugs enzymatic potential to produce GSH and reduce GSSG,
(such as drugs used to treat cancer) via conjugation with (3) increased activities of detoxification enzymes that use
GSH and extrusion from cells. This could lead to the need GSH, and (4) activation of routes for extrusion of GSSG
to increase doses of some drugs to make them effective or and glutathione S-conjugates from cells. It is clear from this
could even result in resistance to the drugs. list that there are several good targets for pharmacological
The mechanism of induction of phase II enzyme expres- interventions in pathologies in which oxidative stress may be
sion by plant polyphenols has been elucidated by Zoete a contributing factor.
and colleagues [233]. They investigated the ability of these The uptake of xenobiotics and their interaction with
compounds and their synthetic analogs to induce the activity biomolecules in living organisms depend on various fac-
of NADP(H) quinone reductase (NQ01), a prototypic phase tors such as their chemical and physical properties, type
II detoxification enzyme. By using quantum-mechanical of organism, and its physiological state. Here, we will
methods the authors calculated the tendency of these not focus on specific aspects, but rather will provide
compounds to release electrons by the energy of the highest the general principles of xenobiotic metabolism leading
occupied molecular orbital (EHOMO ). They found that the to pathologies, GSH involvement and potential protective
smaller the absolute EHOMO of an agent (i.e., the lower effects of certain phytochemicals. Some xenobiotics can be
its reduction potential), the stronger its electron donor directly autoxidized leading to ROS production and the
property was and the greater its inducer potency. That potential pathological consequences were described above.
allowed inducers to be ranked and led to predictions of However, most xenobiotics are not autoxidized directly
the efficiency of inducers based on their reduction potential and contribute to pathology only after transformation via
[233]. However, it should be noted that the experiments different mechanisms. Many xenobiotics are oxidized by
were carried out in cell culture, which does not take into various endogenous oxygenases with the production of ROS
account factors such as the absorption and transportation at this stage. The biotransformed xenobiotics that result
of polyphenols when they are administered to the whole may also have enhanced potential to induce pathology via
organism. However, the approach may give some clues for direct interaction with cellular constituents due to their
the prediction of the biological effects of polyphenols in electrophilic nature. Biotransformed xenobiotics may also
regulating the activity of antioxidant and phase II and III undergo autoxidation with concomitant ROS generation.
detoxification enzymes. In order to prevent this scenario, cells utilize phase II
detoxification enzymes. GSH plays a prominent role in
this process, either directly conjugating with xenobiotics
8. Relationship between GSH Homeostasis
or participating as a substrate in enzymatically catalyzed
and Pathologies conjugation reactions. Finally, conjugates are excreted from
Elevated ROS levels as well as the presence of different the cell by the phase III detoxification system of plasma
xenobiotics are well-known factors in various pathologies membrane active transporters. However, cellular GSH is
not lost to a great extent; most is reclaimed via GSH
and aging, but in some cases these relationships are not
salvage processes (Figure 2). This means that extracellular
straightforward. Many details of GSH involvement in these
transpeptidases cleave the conjugates releasing different GSH
processes including regulation of GSH-related enzymes were
components which may be reabsorbed by cells and reused
discussed above. Therefore, the current section will provide a
for tripeptide resynthesis. Overall, then, GSH may prevent
general summary as well as highlight some potentially useful
the development of pathology related to electrophiles either
therapeutic avenues.
by directly interacting with them or in an enzyme-catalyzed
Figure 7 shows general routes of enhanced ROS levels manner. Some phytochemicals also directly interact with
and/or the presence of xenobiotics associated with various
electrophiles, but their action may also be realized through
pathologies. Elevated ROS levels are a key finding in many
activation of GSH synthesis/resynthesis and reduction. Acti-
diseases [234] including cardiovascular and neurodegen-
vation of phase II and III detoxification enzymes is thought
erative diseases, cancer, diabetes mellitus, and aging [88,
89, 197, 235–237]. ROS concentration may be enhanced to be the main route for xenobiotic detoxification and
for many reasons of both an internal or external nature, excretion from the organism. Activation of the transcription
such as inflammation or exposure to xenobiotics. GSH can of genes encoding enzymes that combat xenobiotics is one of
interact directly with ROS to reduce their levels and in the main pharmacological strategies for treating xenobiotic-
this manner delay the development of pathologies. The induced diseases. As described above, the Nrf2/Keap1 sys-
potential of various phytochemicals to disrupt this link tem, in concert with other signal transduction systems,
between ROS elevation and increased pathology may be regulates the expression of genes encoding many of the
related to the inherent antioxidant activity possessed by enzymes involved in phase I, II, and phase III xenobiotic
various plant components. However, potentially more potent detoxification. Some phytochemicals may stimulate phase I
protective effects of phytochemicals may arise from indirect detoxification enzymes and also increase cellular potential
effects. Since this review is focused on GSH, the ways for detoxification of drugs, which may cause either a decrease
in which GSH participates in these processes must be in sensitivity to the drug or even complete resistance.
highlighted. They include (1) activation of GSH biosynthesis This emphasizes the need for a clear understanding of the
Journal of Amino Acids 15

O2
Biotransformation Detoxification
Xenobiotics Electrophiles Conjugates
Phase I Cyt P450 Phase II T
GS
Phase III
GSH
Other ROS O•2 − H2 O2 HO• Export
sources
Phytochemicals
ROS disbalance
GSH
Phytochemicals

Pathology

Figure 7: Involvement of glutathione in the detoxification of xenobiotics and reactive oxygen species, its relationship with pathological
development and the potential role of different phytochemicals. Glutathione is responsible for helping to maintain redox balance by directly
or indirectly interacting with ROS, and is also involved in detoxification of electrophiles either via direct interactions or via enzyme-
catalysed conjugation. Certain phytochemicals may affect GSH action on ROS and electrophiles either by directly interacting with ROS
and electrophiles, or by upregulating defensive enzymes.

molecular mechanisms of both drug and phytochemical On the other hand, cysteine in protein-bound form, particu-
action for the development of new medical strategies. larly as a component of whey, has some potential to increase
GSH levels [255–258]. The above compounds are used as
9. Research Tools and Pharmacological precursors for GSH biosynthesis, both experimentally and
in some therapies; for example, NAC is broadly used in
Approaches to Manipulate Glutathione Levels therapies that combat HIV [259–261] and other infections
The role of GSH in the function of living organisms is clearly [262–264]. Although used less frequently than NAC, cysteine
reflected by a phrase coined by Sies [4]—the term “inevitable precursor in the form of prodrug, 2-oxothiazolidine-4-
GSH.” The great importance of GSH has been revealed in carboxylate (OTC), is also used to enhance cellular GSH level
multiple experiments either by depletion or repletion of [247]. Experimentally, the overexpression of certain genes
cellular GSH reserves. involved in GSH production also may enhance its level.
At least one important factor needs to be taken into
Cellular GSH reserves can be depleted in at least three account when treatments are used to elevate GSH. It is
different ways—by increasing GSH oxidation, by inhibition well known that many “classic” antioxidants can, under
of biosynthesis, or by inactivation of the genes encoding the certain conditions, become prooxidants. These include low
enzymes of GSH synthesis. Experimentally, the cellular GSH molecular mass antioxidants such as ascorbic acid [265,
pool can be reduced by treatment with different oxidants 266], epigallocatechin-3-gallate [267], α-tocopherol [268],
such as hydrogen peroxide (H2 O2 ), tert-butyl hydroperoxide and retinol [265], as well as antioxidant enzymes such as
[238, 239], or diamide [240, 241]. In 1979, a specific inhibitor superoxide dismutase [269, 270]. Although information on
of γGLCL was synthesized—buthionine sulfoximine (BSO) possible prooxidant properties of GSH is very limited [271–
[13, 14], that when introduced into cells depletes GSH 273], its potential prooxidant effects cannot be ignored.
reserves [16, 17, 242]. These approaches have helped Virtually all compounds known as antioxidants possess
researchers investigate the function of cellular GSH. Since prooxidant properties [274]; these are two sides of the
the use of oxidants to deplete GSH pools in the treatment of same coin. The relationship between pro- and antioxidant
different pathologies usually causes many side effects, BSO properties depends on the nature of the compound and
was soon tested not only for basic research purposes, but also specific conditions.
for clinical investigations in cancer research. For example,
local BSO application to certain skin cancers may sensitize 10. Conclusions and Perspectives:
them to irradiation [243], drug [244], and photodynamic Glutathione—Two Faced Janus
[245] treatments.
Pharmacological Target
Frequently used tools in GSH research and therapy are
interventions that increase GSH levels [246]. This is usually GSH has a very complicated pattern of involvement in
achieved by supplementation with GSH monoesters and diverse biological processes. Consequently, any experimental
diesters [247–249], GSH precursors such N-acetyl cysteine and clinical intervention should be undertaken with pre-
(NAC); [250–252] or α-mercaptopropionylglycine [87, 253]. caution due to the complicated, interrelated, and tightly
Importantly, cysteine is not usually utilized as a precursor regulated networking of living processes. In many cases, any
presumably due to its toxicity at high concentrations [254]. modification of one parameter may result in unpredictable
16 Journal of Amino Acids

responses from diverse processes. For example, at first glance, of potential drugs. They allow rapid testing of diverse
an increased GSH level through supplementation of its esters potential compounds at low cost. This approach is especially
may augment defense mechanisms of not only normal cells, helpful for revealing molecular mechanisms of investigated
but also of cancer cells, especially considering that cancer processes. In some cases, simpler cellular models such as
cells may be rather aggressive in sequestering resources. This bacteria and unicellular yeasts can also be used as models,
can result in a need to enhance the doses of anticancer drugs. but in many cases their pathways of xenobiotic catabolism
The same ideology can be applied to upregulation of are very different from those of mammals thereby limiting
detoxification and antioxidant enzymes. They are frequently their use. However, all isolated cell systems have at least
regulated at the transcriptional level via enhanced Nrf2 two serious limitations. The first is that isolated cells are
binding to ARE/EpRE DNA elements. However, in many not under systemic control by the whole organism, lacking
cases, phase II and III detoxification enzymes are also factors such as the regulatory effects of endocrine and
responsible for the detoxification of anticancer drugs and nervous systems, which may substantially modify cellular
their extrusion from the cell. In addition, some inducers responses. The second is that chemicals or mixtures for
of these enzymes affect phase I detoxification enzymes, testing are applied directly to cells, which avoids complicated
which frequently may transform procarcinogens to actual whole organism processes such as absorpton, transportation,
carcinogens via metabolic activation by hydroxylases such as transformation, and excretion. These processes can lead to
cytochrome P450. large differences in the responses of isolated cells versus cells
However, taking into account the potential undesirable in intact organisms, emphasizing the fact that both basic
effects of pharmacological interventions, there is a need to and applied studies must ultimately rely on the use of whole
investigate them carefully and many different models may animal models.
be used for that purpose. Based on available information, Animal models also have some limitations, both tech-
some specific molecules with expected properties can be nical and ethical. The second is beyond the scope of this
synthesized and tested. Several important notes should be review, and, therefore, we will focus only on the first
provided in this case. Many potential effectors can exist item. First, animal experimental models are much more
in several forms and chemical synthesis may lead to the expensive and require many more resources than cellular
production of, for example, mixtures of different racemates models. Certainly, mammalian models are the most valuable
or diastereoisomers, some of which may be pharmaceutically because these animals are closest to the human condition.
effective, but others of which may cause deleterious effects However, much information may be gained from simple
such as what occurred with thalidomide. One of its racemates animal models that may be ultimately applied to mammals.
was teratogenic [275]. The second important consideration The fruit fly, Drosophila melanogaster, is one of the most
in the chemical synthesis of putative drugs is related to the popular and tractable animal models. Although it is an
production of intermediates and side products, which needs invertebrate, it is easy to care for, thousands of different
special attention and investigation. strains exist, and it is possible to manipulate its genome. As
Another important factor should be reiterated here. a result several experimental models of human pathologies
Innumerable studies have shown that GSH is an antioxidant. have been developed in D. melanogaster, making it a very
However, virtually any antioxidant can, under certain condi- useful biomedical tool. Many biological processes and their
tions, act as a prooxidant [274]. For example, in studies with regulation are highly conserved in eukaryotes, particularly
yeast we found that superoxide dismutase may act either as from yeasts through insects and to vertebrates. For example,
an anti- or prooxidant depending on its expressed activity the Nrf2/Keap1 system has recently been described in D.
[269, 270]. Under certain conditions GSH also can be a melanogaster [277] and fish [278]. Warm-blooded mammals,
prooxidant [276]. Therefore, precaution should be paid to such as rats, mice, and primates are also extremely useful
interventions that enhance GSH levels. subjects, but ethical issues often substantially limit the use
Because of the above caveats, modern pharmacology of mammalian models. As a result, cellular models are
research has refocused on natural products, mainly of plant often preferred to animal models. Certainly, clinical trials in
origin, although bacteria, fungi, and animal sources cannot human populations are the final step before introduction of
be ignored. The ideal situation is when these components certain drugs.
are possessed by edible vegetables, fruits, herbs, and spices or One more aspect which is frequently ignored should be
products formed during their processing. Excellent examples highlighted here. This is the problem of accurate measure-
of these include sulforaphane from cruciferous plants [196, ment of the levels of different glutathione forms, particularly
211], epigallocatechin gallate from green tea [86, 216, 217], reduced (GSH) and oxidized (GSSG) forms, their ratio (an
curcumin from turmeric [201, 212–215], allyl sulfides from index of redox potential), and mixed thioethers need further
garlic [203, 204, 219, 220], anthocyanins and resveratrol experimental development. This is very important because
from different berries and grapes [196, 205, 221, 222], and these parameters are used to characterize the development of
carnosol from rosemary [228]. These and other examples oxidative or nitrosative stresses under some circumstances,
demonstrate the great potential for discovery of natural particularly in certain pathologies [9, 10, 27, 146]. When
compounds that can be used as pharmaceuticals that may dealing with cell cultures or unicellular organisms it is
affect GSH homeostasis. practically impossible to isolate cells from the cultivation
Careful selection of experimental models is very impor- media and fix GSH level quickly. Other problems exist
tant. Cell cultures are extremely useful for the identification when studying multicellular organisms. One is the need
Journal of Amino Acids 17

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