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Living beings have evolved over the past two billion years through adaptation, to an increasing atmospheric oxygen concentration, by both taking advantage of oxygen activating function and developing a complex control network. In these regards, potentially damaging species (reactive oxygen, nitrogen and chlorine species) arise as by-products of metabolism and also work as physiological mediators and signalling molecules. Oxidative stress may be an important factor in numerous pathological conditions, i.e. infection if micronutrients are deficient. Levels of these species are controlled by the antioxidant defence system, which is composed by antioxidants and pro-antioxidants. Several components of this system are micronutrients (e.g. vitamins C and E), are dependent upon dietary micronutrients (e.g. CuZn and Mn superoxide dismutase) or are produced by specific endogenous pathways. The antioxidant defences act, to control levels of these species, as a coordinated system where deficiencies in one component may affect the efficiency of the others. In this network some of the components act as direct antioxidants whereas others act indirectly (pro-antioxidants) either by modulation of direct agents or by regulation of the biosynthesis of antioxidant proteins. Thus, entities usually not considered as antioxidants, also act efficiently counteracting damaging effects of oxidative species. In this contest, the design of new molecules that take into account synergistic interactions among different antioxidants, could be useful both to address mechanistic studies and to develop possible therapeutic agents. In this review the principal categories of antioxidants and pro-antioxidants that goes from vitamins through phyto-derivatives to minerals, are critically reviewed, with particular emphasis on structure-function considerations, together with the perspective opened, in the design of possible therapeutic agents, by the antioxidants interplay.
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Current Pharmaceutical Design, 2004, 10, 1677-1694 1677
1381-6128/04 $45.00+.00 © 2004 Bentham Science Publishers Ltd.
The Antioxidants and Pro-Antioxidants Network: An Overview
Silvia Vertuani, Angela Angusti and Stefano Manfredini
*
Department of Pharmaceutical Science, University of Ferrara, Ferrara, Italy
Abstract: Living beings have evolved over the past two billon years through adaptation, to an increasing atmospheric
oxygen concentration, by both taking advantage of oxygen activating function and developing a complex control network.
In these regards, potentially damaging species (reactive oxygen, nitrogen and chlorine species) arise as by-products of
metabolism and also work as physiological mediators and signalling molecules. Oxidative stress may be an important
factor in numerous pathological conditions, i.e. infection if micronutrients are deficient. Levels of these species are
controlled by the antioxidant defence system, which is composed by antioxidants and pro-antioxidants. Several
components of this system are micronutrients (e.g. vitamins C and E), are dependent upon dietary micronutrients (e.g.
CuZn and Mn superoxide dismutase) or are produced by specific endogenous pathways. The antioxidant defences act, to
control levels of these species, as a coordinated system where deficiencies in one component may affect the efficiency of
the others. In this network some of the components act as direct antioxidants whereas others act indirectly (pro-
antioxidants) either by modulation of direct agents or by regulation of the biosynthesis of antioxidant proteins. Thus,
entities usually not considered as antioxidants, also act efficiently counteracting damaging effects of oxidative species. In
this contest, the design of new molecules that take into account synergistic interactions among different antioxidants,
could be useful both to address mechanistic studies and to develop possible therapeutic agents. In this review the principal
categories of antioxidants and pro-antioxidants that goes from vitamins through phyto-derivatives to minerals, are
critically reviewed, with particular emphasis on structure-function considerations, together with the perspective opened, in
the design of possible therapeutic agents, by the antioxidants interplay.
Key Words: Antioxidants, pro-antioxidants, structure-function relationships, drug design.
INTRODUCTION
ROS and RNS produced in vivo at levels that cannot be
adequately counteracted by endogenous antioxidant systems
can lead to the damage of lipids, proteins, carbohydrates and
nucleic acids. The oxidative modification of these molecules
by toxic levels of ROS and RNS represents an extreme event
that can lead to deleterious consequences such as loss of cell
function, this occurrence is known as “oxidative stress”.
More recently, however, interest has been focused on the
formation of these species at sub-toxic levels, for their
potential to act as biological signal molecules. Subtoxic ROS
and RNS production can lead to alterations in cellular and
extracellular redox state, and it is such alterations that signal
changes in cell functions. By the use of a variety of cell types
it has been shown that numerous cellular processes including
gene expression can be regulated by subtle changes in redox
balance [1]. Examples of this include the activation of
certain nuclear transcription factors, and the determination of
cellular fate by apoptosis or necrosis [2]. Cellular redox
balance is, under normal circumstances, probably under
genetic control and maintained by an array of enzymatic
systems that ensure that overall reducing conditions prevail.
Antioxidants are an heterogeneous family of molecules,
difficult to classify by common shared structural properties.
Moreover, other compounds should be also considered that
does not act directly as antioxidants, but just indirectly either
*Address correspondence to this author at the Department of Pharma-
ceutical Science, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy; Tel:
+39-0532-291292; Fax: +39-0532-291296; E-mail: s.manfredini@unife.it
by modulation of direct agents or by regulation of the bio-
synthesis of antioxidant proteins, promoting their synthesis
and/or availability. We may propose for these substances the
term of pro-antioxidants. Several classifications have been
attempted in the past taking into account the origin (natural
or synthetic), nature (enzymatic or non-enzymatic),
chemical-physical properties (hydrophilic or lipophilic),
structure (flavonoids, polyphenols, etc.), mechanism (preven-
tive, chain-breaking, etc.). Because antioxidants functions
are expressed through a complex network they would be
better characterized by function-structure considerations.
Taking this into account, several classes of antioxidant and
pro-antioxidants agents may be envisaged. The coverage of
all possible aspects and implications exceed the aim of this
work, in this review the principal classes of antioxidant and
pro-antioxidant molecules will be classified in view of
structure-function considerations.
Considering the wide number and different molecules
provided, directly and/or indirectly, of antioxidant effects we
may consider the following classes: vitamins; fats and lipids;
amino acids, peptides and proteins; plant derived antioxi-
dants; minerals; enzymes.
Vitamins
This class of molecules exerts its antioxidant activity
directly by an intrinsic free radical scavenging mechanism
(i.e. vitamin C) and/or indirectly participating to the
regulation and expression of enzymes (i.e. iNOS).
Retinol. Vitamin A presents both kind of antioxidant
activities: it is able to scavenge ROS [3] by a direct
1678 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
mechanism and also inhibits, by its oxidized metabolite
retinoic acid, NO production through inhibition of iNOS
gene transcription in different tissues as vascular, cardiac and
endothelial [4, 5]. 1,25-Dihydroxyvitamin D3 as well as
vitamin K2 and B3 (vitamin PP, Niacin) also inhibit iNOS
expression in smooth muscle and brain as well [6, 7].
Vitamin E is the most important antioxidant in lipids. It
also acts by at least two different mechanisms: directly
scavenges ROS and up-regulates antioxidant enzymes such
are GPX, CAT from liver, SOD, GST, GR and NAD(P)H:
quinone reductase
(DT-diaphorase)activities [8-11].
Vitamin C is the most important antioxidant present in
the hydrophilic compartments, its activity is expressed
through a variety of mechanisms, which still await to be
fully clarified. In principal it acts by scavenging ROS
directly, and among these species probably the most
important are superoxide and peroxynitrite [12]. Secondly, it
has been shown that ascorbate can recycle alpha-tocopherol,
which in turn helps to prevent lipids oxidation [13]. Without
ascorbate, the alpha-tocopheroxyl radical can assume a pro-
oxidant role and continue or even enhance the chain reaction
of lipid peroxidation [14]. The important role of ascorbic
acid within the network of recycling antioxidants has been
extensively investigated [15, 16] and although known since
long time, the studies on this vitamin are far to be complete.
For example, we have recently highlighted its possible role
in the transport of drugs, which do not cross itself the blood
brain barrier [17]. In these regards, it may act as a double
targeting molecule, working both as a carrier and as
antioxidant in neurodegenerative diseases.
Nicotinamide (Niacin, Vitamin B3) inhibits lipid peroxi-
dation induced by photosensitization with an activity com-
parable to that of glutathione and superior to that of vitamins
E and C. This activity is exerted through multiple mechan-
isms, which involves increase of GSH and GST levels and
direct quenching of ROS (i.e.
1
O
2
) [18]. Moreover, as stated
above, vitamin B3 is able to modulate iNOS expression.
Riboflavin and Niacin are components of NADP
+
/
NADPH, NAD
+
/NADH, and FAD/FADH
2
, that play a
fundamental role within the recycling antioxidant network,
restoring the reducing capabilities of antioxidant molecules
such is dihydrolipoate. The final acceptor of the oxidized
species, the CRS CAT activity is strictly related to NADPH
[19,20]. Moreover, both NADPH and FAD serves as
cofactors for glutathione reductase, which produces GSH
from GS-SG [21]. In addition, the production of NO, that
antagonizes superoxide production, requires NADPH [22].
Homocysteine, has a potent pro-oxidative activity and
might induce atherosclerosis by damaging the endothelium
either directly or by altering the oxidative status. For these
reasons this amino acid is considered a vascular disease risk
factor. Because vitamin B6 (pyridoxal-5'-phosphate) and
B12, along with folates, play a fundamental role in homocys-
teine metabolism, by serving as cofactors for methionine
synthase (B12), cystathionine synthase (B6), and cystathio-
nase (B6), they might be considered indirect antioxidants
because they are able to lower homocysteine levels and thus
the associated oxidative damages [23, 24]. Moreover,
vitamin B6 is a necessary co-factor for several TSS pathway
enzymes, this imply that a lack of B6 limits the availability
of cysteine, one of most important antioxidant amino acids,
involved in GSH biosynthesis [25].
Fig. (1).
Fats and Lipids
PUFAs (omega 3 and 6) and others. In general, this class
of molecules is readily oxidized by ROS; moreover, excess
intakes of lipids by foods enhances the amount of ROS
produced in the respiratory chain [26]. In these terms lipids
are pro-oxidant species and also induce iNOS expression in
many cell types. However, conflicting reports have appeared
in literature on this topic. Indeed, within the class of lipids,
PUFAs, and among them omega-3-PUFAs, contained in fish
oil (docosahexaenoic acid, eicosapentaenoic acid, and alpha-
linolenic acid), appear to behave differently, acting as inhi-
bitors of free radical generation. This mechanism is parti-
cular and involves the increase of expression of antioxidant
genes and the inhibition of iNOS expression and inducible
NO synthesis [27]. This in contrast to the parent omega-6-
PUFAs that appear to increase oxidative stress [28]. Very
recently this statement has been revised in a comparative
study on fish and olive oils. The results show that adminis-
tration of fish oil rich diets in rats, increased lipid peroxi-
dation and affected iron metabolism. On the other hand, the
olive oil rich diet did not increase oxidative stress and did
not alter iron metabolism. Based on these conflicting results,
we may conclude that the pattern is far to be completely
elucidated and that fish oil supplementation should be
advised carefully [29].
Aminoacids, Peptides and Proteins
Aminoacids
Taurine, a sulphur containing ß amino acid, is the most
abundant intracellular amino acid in humans, and is impli-
Vitamin B12
N
N
N
N
Co
H
3
C H
3
C
O
NH
2
O
NH
2
CH
3
CH
3
O
NH
2
CH
3
H
3
C
H
3
C
O
NH
2
H
2
N
O
H
2
N
O
CH
3
O
NH
CH
3
O
P
O
O
-
O
OH
O
HO
N
N
+
CH
3
CH
3
CN
The Antioxidants and Pro-Antioxidants Network Current Pharmaceutical Design, 2004, Vol. 10, No. 14 1679
cated in numerous biological and physiological functions and
is found in high concentrations in tissues containing
catecholamines [30]. Taurine has been suggested to have
cytoprotective actions via a number of different mechanisms.
In healthy individuals the diet is the usual source of taurine;
although in the presence of vitamin B6 it is also synthesised
from methionine and cysteine. Taurine has a unique chemical
structure that implies important physiological functions: bile
acid conjugation and cholestasis prevention, antiarrhythmic/
inotropic/chronotropic effects, central nervous system neuro-
modulation, retinal development and function, endocrine/
metabolic effects and antioxidant/anti-inflammatory proper-
ties. Taurine is a unique amino acid with antioxidant and
osmolytic properties, and its capability to inhibit oxidative
damage to DNA, through inhibition of quinone formation,
has been recently highlighted [31]. Sulfur containing amino
acids, such as taurine, could also serve to reduce cellular
damage associated with both NO and metal-stimulated
catecholamine oxidation [32]. Glutamine, the preferred fuel
for the gut, liver, and immune cells, also has an important
role because it serves as a precursor for antioxidants [33].
Glutamine and glutamate with proline, histidine, arginine
and ornithine, comprise 25% of the dietary amino acid intake
and constitute the "glutamate family" of amino acids, which
are disposed of through conversion to glutamate. It is a
component of the antioxidant glutathione and of the
polyglutamated folic acid [34].
L-Arginine is a semiessential amino acid with a terminal
guanidinium group, that serves as natural substrate for the
synthesis of NO by different NOS. The mechanism by which
L-arginine exerts its protective effects is still unclear. L-
arginine may act as direct antioxidant by scavenging oxygen-
derived free radicals [35]. The complex reaction involves the
transfer of electrons from NADPH, via the flavins FAD and
FMN in the carboxy-terminal reductase domain, to the haem
in the amino-terminal oxygenase domain, where the
Fig. (2).
CH
3
H
3
C
CH
3
CH
3
CH
3
OH
Vitamin A
O
CH
3
H
3
C
HO
CH
3
CH
3
CH
3
H
H
CH
3
H
3
CH
3
C
Vitamin E
O
O
HO
HO
OHHO
Vitamin C
O
O
CH
3
CH
3
Vitamin K2
CH
3
n
Vitamin D
N CH
3
OH
OH
HO
. HCl
Vitamin B6
N
COOH
Niacin
N
CNH
2
O
Nicotinamide
N
N
NH
N
H
3
C
H
3
C
O
O
CH
2
CH OH
CH
CH OH
OH
CH
2
OH
Riboflavin
CH
2
HO
H
3
C
H
H
3
C CH
3
CH
3
H
1680 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
substrate L-arginine is oxidised to L-citrulline and NO. The
haem is essential for dimerisation as well as NO production.
The pteridine/tetrahydrobiopterin is a key feature of NOS,
affecting dimerisation and electron transfer, although its full
role in catalysis remains to be determined. NOS can also
catalyse superoxide anion production, depending on subs-
trate and cofactor availability. There are three main isoforms
of the enzyme, named neuronal NOS, inducible NOS
(iNOS), and endothelial NOS (eNOS), which differ in their
dependence on Ca
++
, as well as in their expression and
activities. These unique features give rise to the distinct
subcellular localisations and mechanistic features, which are
responsible for the physiological and pathophysiological
roles of each isoform [36].
Histidine, can prevent LDL modification, both because it
may act as a singlet oxygen scavenger, but also because it
may complex Cu
++
ions and thus prevent lipid peroxidation
[37,38]. Based on this rational, zinc-histidine complex has
been recently proposed as synergistic approach to improve
zinc absorption, and contribute to the antioxidant status of
plasma, providing antioxidant properties against LDL
oxidation (as well as other patho-physiological processes)
through transition metal-chelating mechanisms [39]. Being
the component of the dipeptide carnosine, the behaviour of
these two antioxidants has been also compared. Histidine
was more effective at inhibiting copper-promoted formation
of carbonyls on bovine serum albumin than carnosine, but
carnosine was more effective in inhibiting copper-induced
ascorbic acid oxidation than histidine. However, neither
carnosine nor histidine resulted capable to inhibit 2,2'-azobis
(2-amidinopropane)dihydrochloride-promoted oxidation of
LDL; this result seems to indicate that their main antioxidant
mechanism is through copper chelation [40].
Glycine, as antioxidant, is involved in kidney protection
from massive injury induced by ischemia-reperfusion,
protects renal antioxidant enzymes and Na
+
-K
+
ATPase,
normalises malondialdehyde, and nitric oxide levels. Data
obtained on hypoxia/reperfusion injuries suggests a
protective role of glycine against ROS. The mechanism
proposed involves the regulation of antioxidant enzymes
such are SOD, GPX, CAT. As well as the NO production by
iNOS [41].
Thiols. Biothiols are quite efficient antioxidants able to
react with free radicals and thus protecting the cells against
their damages. In such reactions, thiols undergo one-electron
oxidation with the formation of thiyl radicals. Because of
these properties, thiols as attracted consistent attention. On
the contrary, the reactivity of the corresponding thiyl radicals
(RS
.
), formed simultaneously in these reactions, have been
often underestimated. Indeed, protective and repairing
efficacy of thiols depends not only on their capacity to
detoxify free radicals, but also on chemical character and
reactivity of the formed thiyl radical. Furthermore, quick and
efficient removal of RS radical leads to a disturbance in
balanced state of antioxidant reaction, which effectively
increases repairing capacity. Dangerous thiyl radicals, which
can cause peroxidative injury, should immediately undergo
regenerative reduction to thiols. Under physiological
conditions, thiyl radicals can react with thiolate anion
yielding disulfide radical anion (RSSR
•−
)
as an intermediate
and finally disulfides and superoxide radical anion (O
2
•−
),
which is next inactivated in the reaction catalysed by SOD.
Thiyl radicals can also be reduced to thiols by reacting with
ascorbate with the formation of low-activity ascorbyl radical
that subsequently enters disproportiation reaction [42].
Fig. (3).
Linoleic Acid
COOH
COOH
γ-Linoleic Acid
COOH
Arachidonic Acid
COOH
α-linolenic acid
COOH
Eicosapentenoic acid
COOH
Docahexenoic acid
The Antioxidants and Pro-Antioxidants Network Current Pharmaceutical Design, 2004, Vol. 10, No. 14 1681
Thiol groups are central to most, if not all, redox-
sensitive cell signalling mechanisms. Oxidation of thiol
groups is a reversible process that represents a sensitive
redox-regulated functional switch. Additionally, increasing
evidence suggests that thiol groups located on various
molecules act as redox sensitive switches thereby providing
a common trigger for a variety of ROS and RNS mediated
signalling events. Particular attention has been paid to the
importance of thiols and thiol-containing molecules in these
processes [43]. While reversible cysteine oxidation and
reduction is part of well-established signalling systems, the
oxidation and the enzymatically catalysed reduction of
methionine is just emerging as a novel molecular mechan-
ism for cellular regulation. Methionine sulfoxide reductase,
which reduces methionine sulfoxide to methionine in a thio-
redoxin-dependent manner, is therefore an enzyme important
for the repair of age- or degenerative disease-related protein
modifications. It is also a potential missing link, in the post-
translational modification cycle, involved in the specific
oxidation and reduction of methionine residues in cellular
signalling proteins, which may give rise to activity-
dependent plastic changes in cellular excitability [44].
N-Acetyl Cysteine (NAC) is another known precursor
for glutathione synthesis that has been shown to act on redox
balance and to be capable of significantly improving the
antioxidant potential by elevating reduced GSH levels.
Antioxidants such as NAC have been used as tools for
investigating the role of ROS in numerous biological and
pathological processes. NAC inhibits activation of c-Jun N-
terminal kinase, p38 MAP kinase; modulate redox-sensitive
activating protein-1 and nuclear factor kappa B transcription
factor activities, thus regulating expression of numerous
genes. NAC can also prevent apoptosis and promote cell
survival by activating extracellular signal-regulated kinase
pathway, a concept useful for treating certain degenerative
diseases [45]. NAC directly modifies the activity of several
proteins by its reducing activity [46]. A comprehensive
survey of the literature highlights the role of antioxidant
agents in counteracting the unfavorable effects of ROS and
oxidative stress in cancer progression and particularly in the
onset of tissue wasting and cancer-related anorexia/cachexia
syndrome (CACS) [47]. Recent results confirm a favorable
effect of antioxidant agents, such as ALA and NAC, on
several important T-cell functions in vitro in advanced-stage
cancer patients, which may potentially translate into a more
effective control of tumour cell growth and prevention/
reversal of CACS also in vivo [48]. Moreover, very recently
NAC was shown to inhibit TNF-alpha mediated phosphory-
lation of p65 (ser536) in vascular endothelial cells. This is an
interesting indication that natural antioxidants may serve as
potent Nuclear Factor kappa B (NF-kappaB) inhibitors in
vascular endothelial cells, yet acting through unique and
divergent pathways [49]. S-Adenosyl-L-methionine (SAMe)
is a precursor of cysteine, one of the 3 amino acids compo-
sing glutathione, and exerts many key functions in the liver.
SAMe is particularly important in opposing the toxicity of
free oxygen radicals generated by various pathogens, includ-
ing alcohol, which cause oxidative stress largely by the
induction of cytochrome P4502E1, and its metabolite acetal-
dehyde [50].
Peptides
Carnosine is a beta-alanyl-L-histidine dipeptide found in
skeletal muscle and nervous tissue, is a physiological
dipeptide, which can delay ageing and rejuvenate senescent
cultured human fibroblasts. The anti-oxidant, free radical-
and metal ion-scavenging activities of carnosine alone,
Fig. (4).
H
2
N N
H
OH
O O
L-Citrulline
H
2
N N
H
OH
NH O
L-Arginine
H
OH
H NH
2
O
Glycine
OH
H
O
H
2
N
SH
L-Cisteine
H
2
N
SO
3
H
Taurine
OH
H
2
N
O
H
HN+
NH
L-Histidine
H
N
N
H
O
SH
O
COOH
HOOC H
H
2
N
Gluthation
N
N
H
H
N
NH
2
OHO
O
Carnosine
H NH
2
H NH
2
OH
O
H NH
2
O
H
2
N
L-Glutamine
1682 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
cannot adequately explain these effects. Indeed, carnosine is
also able to reacts with small carbonyl compounds (aldehydes
and ketones) and protects macromolecules against their cross-
linking actions [51, 52]. This process is termed 'carnosinyla-
tion', and because ageing is associated with accumulation of
carbonyl groups on proteins, carnosine act by at least two
different mechanism (i.e. antioxidant and carnosinylation) as
an anti-ageing molecule. This reaction has been proposed to
occur in cultured fibroblasts and in vivo. In these studies
carnosine was able to suppress diabetes-associated increase
in blood pressure in fructose-fed rats, an observation
consistent with carnosine's anti-glycating actions [53].
Ascorbic acid (vitamin C) and the tripeptide thiol,
gamma-glutamyl cysteinyl glycine (GSH) are the major
low molecular weight soluble antioxidants in plant cells. The
pathway of glutathione biosynthesis is similar in animals and
plants while that of ascorbate biosynthesis differs consider-
ably between the two kingdoms. The mechanisms of thiol
metabolism and chemistry have particular relevance to both
cellular defences against toxicant exposure and to redox
signalling. The major pathways for GSH metabolism in
defence of the cell are reduction of hydroperoxides by GPX
and some peroxiredoxins, which yield GSSG, and conjuga-
tion reactions catalysed by glutathione-S-transferases. GSSG
can be reduced to GSH by glutathione reductase, but
glutathione conjugates are excreted from cells. The exoen-
zyme GGT removes the glutamate from extracellular GSH,
producing cysteinyl-glycine from which a dipeptidase then
generates cysteine, an amino acid often limiting for de novo
GSH synthesis. Synthesis of GSH from the constituent
amino acids occurs in two regulated, enzymatically catalysed
steps. The signalling pathways leading to activation of the
transcription factors that regulate these genes are a current
area of intense investigation [54]. GSH also participates in
redox signalling through the removal of H
2
O
2
, which has the
properties of a second messenger, and by reversing the
formation of sulfenic acid, a moiety formed by reaction of
critical cysteine residues in signalling proteins with H
2
O
2
[55]. Moreover, the concentrations of ascorbate and gluta-
thione are greatly modified in response to a variety of
environmental triggers, particularly those that cause increased
oxidative stress. It is essential that the signals and associated
signal transduction pathways that trigger enhanced antioxi-
dant accumulation are elucidated, as these offer an important
alternative means of achieving greater nutritional value in
edible plant organs [56]. GSH, which has one cysteine, and
the small protein thioredoxin, which has two cysteines in its
active site, often have complementary, if not overlapping
roles in cytoprotection. [57]. The role of GSH as a substrate
for GPX, to give GSSG, represents the predominant
mechanism for reduction of H
2
O
2
and lipid hydroperoxides,
[58] thus although it does not react directly with hydroper-
oxides, it works effectively as an antioxidant.
Fig. (5).
Another role for GSH in antioxidant defense depends
upon its ability to react with carbon radicals, acting in a
concerted manner with superoxide dismutase to prevent
oxidative damages [59].
Fig. (6).
Proteins
Generally speaking, insufficient protein intake may
induce indirect effects such as zinc deficiency with direct
effects on the bioavailability of Cu, Zn-SOD [60]. Proteins
such albumin and intracellular metallothionein, are zinc
carriers. Thus proteins paucity may induce oxidative stress
due to lack of antioxidant proteins.
Albumin. Albumin is the most abundant protein in the
circulation, and can function as an antioxidant. It has been
recently demonstrated that in vitro glycoxidation of human
serum albumin induced a marked loss of antioxidant activity
of this molecule in the regards of copper-mediated oxidation
of LDL, which may be caused by the generation of super-
oxide [61].
Thioredoxin. Almost all forms of ROS oxidize methio-
nine residues of proteins to a mixture of the R- and S-isomers
of methionine sulfoxide. Because organisms contain MSRs,
which can catalyse the thioredoxin-dependent reduction of
the sulfoxides back to methionine, it was proposed that the
cyclic oxidation/reduction of methionine residues might
serve as antioxidant system to scavenge ROS, and also to
facilitate the regulation of critical enzyme activities [62].
Lactoferrin. Lactoferrin is an iron binding protein
involved in a large spectrum of biological actions including
antimicrobial actions. Lactoferrin plays a central role in
ferrokinetics: it binds free iron with great affinity limiting the
amount of ions available for microorganism's metabolism.
Lactoferrin is also involved in the modulation of immune
system and recent studies indicate that lactoferrin directly
modulates both production and function of neutrophils and
monocytes. Lipid auto-oxidation in milk is affected by a
complex interplay of pro- and anti-oxidants. Several of these
compounds are also important nutrients in the human diet
and may have other physiological effects in the gastrointes-
tinal tract and other tissues. Lactoferrin has an important role
by binding pro-oxidative iron ions [63].
Transferrin. Iron transport occurs by the well-known
(Tf)-receptor system and by a second as yet uncharacterized
system [64]. The iron carrier protein Tf plays a prominent
antioxidant role in the lower respiratory tract and is present
H
2
O
2
+ 2GSH
GSHPx
2H
2
O + GSSG
ROOH + 2GSH ROH + GSSG
GSHPx
R· + GSH
RH + GS·
GS· + GS GSSG·-
GSSG·- + O
2
GSSG + O
-
2 O
2
·- + 2H
H
2
O
2
+ O
2
SOD
The Antioxidants and Pro-Antioxidants Network Current Pharmaceutical Design, 2004, Vol. 10, No. 14 1683
at elevated concentrations in lung epithelial lining fluid
relative to plasma [65].
Bilirubin. Bilirubin was long considered a useless
metabolite of heme catabolism, responsible for the clinical
manifestation of jaundice, and potentially toxic in high
doses, particularly in neonates. However, in the last 10 years,
in vitro and in vivo studies, have demonstrated that bilirubin
exhibits potent anti-oxidant properties preventing the oxida-
tive damage triggered by a wide range of oxidant-related
stimuli [66]. This suggests a beneficial and physiological
role for bilirubin in cytoprotection against short and long-
lasting oxidant-mediated cell injury [67]. Its role is probably
that of a physiological antioxidant present in human extra-
cellular fluids. However, other studies showed that bilirubin
in the presence of the transition metal ion Cu(II) causes
strand cleavage in DNA through generation of ROS, particu-
larly the hydroxyl radical [68]. Thus bilirubin possesses both
antioxidant and prooxidant properties. In order to understand
the chemical basis of the different biological properties of
bilirubin, structure-activity relationships of bilirubin and its
precursor biliverdin have been investigated. The bilirubin is
more active both as an antioxidant and oxidative DNA
cleaving agent as well, thus possessing antioxidant and toxic
properties at the same time. Further, it appear that the struc-
tural features of the bilirubin molecule which are important
for its prooxidant action, are also those that exert antioxidant
activity.
Ceruloplasmin. Ceruloplasmin is a ferroxidase that oxi-
dizes toxic ferrous iron to nontoxic ferric form. Recent
results indicate that ceruloplasmin plays an important role in
maintaining iron homeostasis in the CNS and in protecting
the CNS from iron-mediated free radical injury [69]. There-
fore, the antioxidant effects of ceruloplasmin could have
important implications for various neurodegenerative diseases
such as Parkinson's disease and Alzheimer's disease in which
iron deposition is known to occur.
Plants Derived Products
Phenols. Tocopherols and tocotrienols are essential
components of biological
membranes where they have both
antioxidant and non-antioxidant
functions. There are four
tocopherol and tocotrienol
isomers (α−, β−, γ−, δ−) which
structurally consist of
a chroman head group and a phytyl
side chain giving vitamin
E compounds amphipathic charac-
ter. Relative antioxidant activity of the tocopherol isomers
in
vivo is α>β>γ>δ, which is due to the
methylation pattern and
the amount of methyl groups present in
the phenolic ring of
the polar head structure. Hence, α−tocopherol,
with its three
methyl substituents, has the highest antioxidant
activity
among tocopherols. Vitamin E is a chain-breaking antioxi-
dant, i.e. it is able to
repair oxidizing radicals directly, preven-
ting the chain propagation
step during lipid autoxidation. It
reacts with alkoxy radicals (LO
), lipid peroxyl radicals
(LOO
) and with alkyl radicals (L
), derived from
PUFA
oxidation. The reaction between vitamin E and lipid radical
occurs
in the membrane-water interphase where vitamin E
donates an hydrogen
ion to lipid radical with consequent
tocopheroxyl radical (TO
)
formation. Regeneration of the
tocopherol
back to its reduced form can be achieved by
vitamin C (ascorbate),
reduced glutathione or coenzyme Q.
In addition, tocopherols act as chemical scavengers of
oxygen
radicals, especially singlet oxygen (via irreversible
oxidation
of tocopherol), and as physical deactivators of singlet
oxygen
by charge transfer mechanism.
TOH formation sustains pro-
oxidant action of tocopherol.
At high concentration toco-
pherols act as pro-oxidant synergist
with transition metal
ions, lipid peroxides or other oxidising
agents [70]. In
addition to antioxidant functions vitamin E has several non-
antioxidant
functions in membranes. Tocopherols have been
suggested to stabilize
membrane structures. Earlier studies
have shown that α-tocopherol
modulates membrane fluidity
in a similar manner to cholesterol,
and also membrane
permeability to small ions and molecules.
Indeed, the above discussed comparative scale of antioxi-
dant activity should be taken with caution, discrepancy in
relative antioxidant effectiveness are found in the respect of
the target of activity. Thus, γ-tocopherol was found to be
more potent than α-tocopherol in its interaction with reactive
nitrogen oxide species [71]. Also, attention should be paid,
when considering results obtained by tocopherols coming
from different origin (i.e. natural or synthetic, optically pure
or racemic). Moreover, antioxidant activity of tocotrienols
vs. tocopherols
is far less studied, α−tocotrienol is proven to
be a better antioxidant
than α−tocopherol in a membrane
environment [72]. Higher antioxidant activity was observed
with tocotrienol against lipid peroxidation in rat liver
microsomes than with α-tocopherol [73]. Similarly, palm
tocotrienol complex in rat brain mitochondria, showed a
stronger effect of γ-tocotrienol. Reasons for that may be
ascribed to different mechanisms including: i) a more
uniform distribution in the membrane lipid bilayer, ii) a more
efficient interaction of the chromanyl ring with lipid radicals,
and iii) a higher recycling efficiency from chromanoxyl
radicals [74, 75]. This data, taken together may suggest
important clinical implications for tocotrienols.
Polyphenols: Polyphenols comprise a wide number of
natural substances of plant
origin. Almost all of them exhibit
a marked antioxidant activity.
Typical examples in order of
increased complexity are hydroxy
stilbenes such as resvera-
trol, an antioxidant in grapes wine and Polygonum cuspida-
tum, [76] oligomeric catechol structures based on caffeic
acid
moieties found in several Lamiatae plants (rosmarinic
acid,
salvianolic acids, yunnaneic acids, etc.), the large group
of flavonoids, monomeric and oligomeric flavan-3-ols
[derivatives
of (+)-catechin or (-)-epicatechin, also known as
proanthocyanidins
or condensed tannins], or gallo- and
ellagitannins (hydrolyzable
tannins) [77].
Polyphenols possess the ideal
chemical structure for free
radical scavenging activity, and
they have been shown to be
more effective antioxidants in vitro
than tocopherols and
ascorbate. Antioxidative properties of
polyphenols arise from
i) their high reactivity as hydrogen or
electron donors, ii)
from the ability of the polyphenol-derived
radical to stabilize
and delocalize the unpaired electron (chain-breaking
function), and iii) from their ability to chelate transition
metal
ions (termination of the Fenton reaction) [78]. Another
mechanism underlying the antioxidative properties
of
phenolics is the ability of flavonoids to alter peroxidation
kinetics by modification of the lipid packing order and to
decrease
fluidity of the membranes. These changes
could
1684 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
sterically hinder diffusion of free radicals and restrict
peroxi-
dative reactions. Moreover, it has been shown recently,
that
phenolic compounds can be involved in the hydrogen
peroxide
scavenging cascade in plant cells [79]. Polyphenols
are reducing agents, and together with other dietary reducing
agents, such as vitamin C, vitamin E and carotenoids, protect
tissues against oxidative stress and associated pathologies
such as cancers, coronary heart disease and inflammation
[80]. These dietary phytophenolics have been recognized
largely as beneficial antioxidants that can scavenge harmful
active oxygen species including O
2
•−
, H
2
O
2
, OH
, and
1
O
2
,
but they can also act as pro-oxidant in some conditions. The
ESR signals of phenoxyl radicals are eliminated by mono-
dehydroascorbate radical (MDA) reductase, suggesting that
phenoxyl radicals, like the ascorbate radical, are enzyma-
tically recycled to parent phenolics [81]. Thus, phenolics in
plant cells can form an antioxidant system equivalent to that
of ascorbate. In contrast to their antioxidant activity, phyto-
phenolics also have the potential to act as pro-oxidants under
certain conditions. For example, flavonoids and dihydroxy-
cinnamic acids can impair DNA functions via the production
of radicals in the presence of Cu and O
2
. Phenoxyl radicals
can also initiate lipid peroxidation. Recently, Al, Zn, Ca, Mg
and Cd have been found to stimulate phenoxyl radical-
induced lipid peroxidation [82].
Flavonoids. Flavonoids are constituents of fruits, vege-
tables, and plant-derived beverages, as well as components
in herbal-containing dietary supplements, with established in
vitro antioxidant properties and potential cardioprotective
effects. Several compounds belong to flavonoid family, such
are flavones (apigenin, luteolin, kaempferol, quercetin, myri-
cetin and rutin), isoflavonoids (genistein, daidzein, biochanin
A, and genistin), flavanones (taxifolin, naringenin and
naringin) and a flavanol (catechin). The antioxidant and pro-
oxidant activities of flavonoids, belonging to several classes,
have been studied in detail to establish their structure-
activity relationships against different oxidants [83]. Activity
behaviour is particularly related to hydroxyl groups present
on the molecule scaffold. Special attention has been paid to
the flavonoids quercetin (flavone), taxifolin (flavanone) and
catechin (flavanol), which possess different basic structures
but the same hydroxylation pattern (3,5,7,3'4'-OH). It was
found that these three flavonoids exhibited comparable
antioxidant activities against different oxidants leading to the
conclusion that the presence of ortho-catechol group (3',4'-
OH) in the B-ring is determinant for a high antioxidant
activity. The flavone kaempferol (3,5,7,4'-OH), however, in
spite of bearing no catechol group, also presents a high
antioxidant activity against some oxidants. This fact can be
attributed to the presence of both 2,3-double bond and the 3-
hydroxyl group, meaning that the basic structure of
flavonoids becomes important when the antioxidant activity
of B-ring is small [84].
Biochanin A. Biochanin A belongs to the isoflavone
class of flavonoids. It is also classified as a phytoestrogen
since it is a plant-derived nonsteroidal compound that posse-
ses estrogen-like biological activity. Biochanin A has been
found to have weak estrogenic activity. The polyphenolic
structures of flavonoids and isoflavonoids give them the
ability to either scavenge free radicals and chelate transition
metals, a basis for their potent antioxidant abilities. Another
possible contribution to their antioxidant activities derives
from their ability to stabilize membranes by decreasing
membrane fluidity. As stated above, localization of
flavonoids and isoflavonoids into the membrane and the
resulting restrictions on fluidity of membrane components
could sterically hinder diffusion of free radicals and thereby
decrease the kinetics of free radical reactions [85].
Glucosinolates. This class of molecules is a large group
of sulfur-containing glucosides (β-thioglucoside N-hydroxy-
sulfates) and isothiocyanates, widely distributed in Cruci-
ferous vegetable (syn. Brassicaceae). This latter is com-
prised of familiar
foods of the species Brassica oleracea (e.g.
cabbage, broccoli,
cauliflower, Brussels sprouts, kohlrabi and
kale) as well as
>350 other genera that include a variety of
food plants (e.g.
arugula, radish, daikon, watercress, horsera-
dish and wasabi). A growing number of studies confirms the
chemopreventive activity of cruciferous vegetables, due to
the high glucosinolates content [86]. They induce phase-2
detoxication enzymes, increase antioxidant
status, and pro-
tect against chemically induced cancer, at least in animals.
Glucosinolates are hydrolyzed by myrosinase (an enzyme
found
in plants and bowel microflora) to form isothiocyanates.
In
vivo, isothiocyanates are conjugated with glutathione and
then
sequentially metabolized to mercapturic acids. These
metabolites
are collectively designated dithiocarbamate [87,
88]. The cancer preventive effects of cruciferous vegetables
could be related to protection from mutagenic oxidative
DNA damage [89].
Carotenoids. Dietary carotenoids are thought to provide
health benefits, particularly in decreasing the risk of certain
Fig. (7).
O
CH
3
HO
H
3
C
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
(R)-α-Tocotrienol
O
CH
3
HO
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
β-tocotrienol
O
CH
3
HO
CH
3
CH
3
CH
3
CH
3
CH
3
H
3
C
γ-tocotrienol
O
CH
3
HO
CH
3
CH
3
CH
3
CH
3
CH
3
δ-tocotrienol
The Antioxidants and Pro-Antioxidants Network Current Pharmaceutical Design, 2004, Vol. 10, No. 14 1685
cancers and eye diseases. The carotenoids that have been
most studied in this regards are β-carotene, lycopene, lutein,
and zeaxanthin. The earliest role established for β-carotene
in animals was as a vitamin A precursor, a role it shares with
other pro-vitamin A carotenoids. In part, the beneficial
effects of carotenoids are thought to be due to their role as
antioxidants, because carotenoids are excellent scavengers of
singlet oxygen and respectable scavengers for other reactive
oxygen species [90]. The ability of dietary carotenoids such
as β-carotene and lycopene to act as antioxidants in biolo-
gical systems is dependent upon a number of factors. While
the structure of carotenoids, especially the conjugated double
bond system, gives rise to many of the fundamental proper-
ties of these molecules, it also affects how these molecules
are incorporated into biological membranes. This implies
that in vivo behaviour may differ significantly from the
results observed in vitro. Moreover, interaction with other
antioxidants, particularly vitamins E and C, greatly improve
the effectiveness of carotenoids as antioxidants [91]. On the
other hand, carotenoids, may lose their antioxidant activity at
high concentrations or at high partial pressures of oxygen
and this behaviour has been related with the low probability
of pro-oxidants effects in vivo [92]. Increasing data supports
pro-oxidants properties of carotenoids as well as possible
implications in human health [93, 94]. The antioxidant
activity of carotenoids is exerted through the reaction with
different ROS (CCl
3
O
2
, RSO
2
, NO
2
), which produce via
electron transfer, the radical cation of the carotenoid. Reac-
tion with arylperoxyl radicals, instead produce, by hydrogen
atom transfer, the neutral carotene radical. The interaction of
carotenoids and carotenoid radicals with other antioxidants is
of importance with respect to anti- and possibly pro-oxidative
reactions of carotenoids. Indeed, in polar environments the
vitamin E radical cation is deprotonated (TOH
+
--> TO
+
H
+
) and TO
does not react with carotenoids, whereas in
nonpolar environments such as hexane, TOH
+
is converted
to TOH by hydrocarbon carotenoids. However, the nature of
the reaction between the tocopherol and various carotenoids
shows a marked variation depending on the specific toco-
pherol homologue. Similarly, the radical cations of the
carotenoids all react with vitamin C restoring the parent
carotenoids [95]. Increasing studies, are continuing to deepen
the knowledge on carotenoids interactions. Other aspects
regards the less investigated polar carotenoids, lutein and
zeaxanthin, that constitute the macular pigment were they
exert a recently disclosed role in protecting the eye from the
blue light [96]. Moreover, antineoplastic activity of carote-
noids have been also related to their antioxidant properties,
[97] although the formation of retinoids from diverse
carotenoids may also account for their action. Dietary intakes
of tomatoes and tomato products containing lycopene have
been shown to be associated with decreased risk of chronic
diseases such as cancer and cardiovascular diseases in
numerous studies [98]. Serum and tissue lycopene levels
have also been inversely related to the risk of lung and
prostate cancers, in this regards, lycopene can trap singlet
oxygen and reduce mutagenesis in the Ames test [99]. It has
to be considered that, most of the studies have been
conducted with either tomato products or lycopene extracted
from tomatoes that also contain other carotenoids in various
proportions. Therefore, the results cannot be attributed to the
solely effects of lycopene [100]. Food sources of these
compounds include a variety of fruits and vegetables,
although the primary sources of lycopene are tomato and
tomato products, but these carotenoids have now become
very popular and also available in supplement form [101].
Fig. (8).
O
OOH
HO
OCH
3
Biochanin A O
OOH
O
OH
O
HO
HO
O
HO
O
CH
3
HO
HO
H
HO
Naringin
O
OOH
HO
OH
OH
OH
Quercetin
O
OH
HO
OH
OH
OH
Catechin
OH
HO
OH
Resveratrol
COOH
Cinnamic Acid
OH
COOH
HO
Caffeic Acid
OH
OHHO
COOH
Gallic acid
1686 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
Fig. (9).
Fig. (10).
H
3
C
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
H
3
C CH
3
Licopene
CH
3
CH
3
CH
3
CH
3
CH
3
H
3
C
CH
3
H
3
C
CH
3
H
3
C
α-carotene
CH
3
CH
3
CH
3
CH
3
CH
3
H
3
C
CH
3
H
3
C
CH
3
H
3
C
β-carotene
CH
3
CH
3
CH
3
CH
3
CH
3
H
3
C
CH
3
H
3
C
CH
3
H
3
C
γ-carotene
CH
3
CH
3
CH
3
CH
3
CH
3
H
3
C
CH
3
H
3
C
CH
3
H
3
C
δ-carotene
H
3
C CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
H
3
C
H
3
C CH
3
OH
HO
Luthein
H
3
C CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
H
3
C
H
3
C CH
3
OH
HO
Zeaxanthin
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
Canthaxantin
O
CH
3
O
The Antioxidants and Pro-Antioxidants Network Current Pharmaceutical Design, 2004, Vol. 10, No. 14 1687
Phytic acid. Phytic acid (myo-inositol hexaphosphate)
has been extensively studied in animals and is being
promoted as an anti-cancer agent in health food stores. It is
naturally found in legumes, wheat bran, and soy foods. It is
believed to be the active ingredient that gives these
substances their cancer fighting abilities [102]. Its cancer
chemopreventive activity is thought to be related to its ability
in inhibiting the generation of reactive oxygen species from
H
2
O
2
by chelating metals [103].
Allicin. Various preparations of garlic, mainly aged
garlic extract (AGE), have been shown to have promising
antioxidant potential. However, the presence of more than
one compound in garlic, with apparently opposite biological
effects, has added to the complexity of the subject. The
organosulfur compounds, responsible, at least in part for the
antioxidant activity of AGE are S-allyl-L-cysteine and S-
allylmercapto-L-cysteine [104]. Raw garlic homogenate has
been reported to exert antioxidant potential but higher doses
have been shown to be toxic to the heart, liver and kidney
[105]. In view of its strong antioxidant properties, garlic, has
been suggested, as a “panacea”, for the reduction of risks of
several pathological events such are: cardiovascular disease,
increased platelet aggregation, thrombus formation, cancer
and diseases associated with cerebral aging, arthritis, cataract
formation. Moreover it has been reported to rejuvenate skin,
improve blood circulation and energy levels [106].
Minerals
The role of minerals in enzyme functions has been
studied extensively in therapy, nutrition and biochemistry
[107]. For example, magnesium is a cofactor for glucose-6-
phosphate dehydrogenase and 6-phosphogluconate dehydro-
genase, two pentose-cycle enzymes catalyzing the produc-
tion of NADPH from NADP
+
. Thus, a deficiency of dietary
magnesium reduces glutathione reductase activity and results
in radical-induced protein oxidation (indicated by the
generation of protein carbonyls) and marked lesions in
tissues (e.g. skeletal muscle, brain, and kidney). Iron is the
most abundant trace element in the body, and almost all iron
occurs bound to proteins. Free iron concentrations are
particularly low for two reasons: i) Fe
3+
is not water soluble,
and ii) Fe
2+
participates in the generation of free radicals.
Thus, an increase in extracellular or intracellular iron concen-
trations, which can result from dietary protein deficiency,
dietary iron loading, low concentrations of iron-binding pro-
teins, or cell injury, promotes ROS production, lipid peroxi-
dation, and oxidative stress. Increasing the extracellular
concentration of non-heme iron also enhances iNOS protein
expression and inducible NO synthesis in many cell types,
including cultured proximal tubule cells and macrophages,
further exacerbating oxidative damage via peroxynitrite
generation.
Zinc. Zinc is present in all organs, tissues, and fluids of
the body. Zinc binds to a number of biologic molecules and
influences their conformation, stability and activity. Zinc
serves as a catalyst for enzymes responsible for DNA repli-
cation, gene transcription, and RNA and protein synthesis.
Therefore, the ability of zinc to retard oxidative processes
has been recognized since many years. Although the
evidence for the antioxidant properties of zinc is compelling,
the mechanisms are still unclear. In general, the mechanism
of antioxidation can be divided into acute and chronic effects
[108]. Chronic effects involve exposure of an organism to
zinc on a long-term basis, resulting in the production of other
antioxidant molecules, such as the metallothioneins. In these
terms zinc act as a precursor (pro-antioxidant). Thus, zinc
paucity results in lack of defences against some kind of
oxidative stress. The acute effects regards either i) protection
of protein sulfhydryls or ii) reduction of OH
formation from
H
2
O
2
through the antagonism of redox-active transition
metals, such as iron and copper. Zn
++
may induce metallo-
thionein synthesis, forming a zinc-thiolate moiety that
functions as a preferred sacrificial site for oxidant attacks,
preserving skin and its components [109, 110]. Different
mechanisms have been proposed for protein sulfhydryl
groups protection, among them: i) binding of zinc to the
sulfhydryl, ii) steric hindrance by in close proximity to the
sulfhydryl group on the protein or iii) a conformational
change from binding to some other site on the protein. From
these data it appears that much needs to be done to fully
elucidate antioxidant properties of this mineral. It is likely
that from these researches new antioxidant functions and
possibly new applications for zinc will rise up.
Iron. It is essential for aerobic life and is required for the
biosynthesis of a variety of iron-containing proteins and for
DNA synthesis. Iron is utilized as a catalyst at the active site
of numerous enzymes involved in oxygen metabolism and
also within proteins involved in oxygen and electron trans-
port, and storage. It can exist in a variety of redox states, but
in biological systems is mainly restricted to the ferrous (Fe
II
),
ferric (Fe
III
), and ferryl (Fe
IV
) states. The living beings has
developed strategies to control, by specific enzymes,
negative iron effects and to transport it in a non reactive form
binded to specific proteins. On the other hand, low molecular
mass (LMrFe) iron form, behave differently. Indeed, LMrFe
may act both as a pro-oxidant, leading to the formation of
ROS or act as a redox signal molecule [111]. In view of its
capability to drive one-electron reactions, iron is the
principal responsible in the production and metabolism of
free radicals in biological systems. In these regards, ferrous
iron (Fe
++
) can catalyse the decomposition of peroxides to
hydroxyl radical from hydrogen peroxide or alkoxyl radicals.
Moreover, oxygen is reduced to superoxide radical by Fe
++
,
in turn this latter is promptly restored to the Fe
+++
form by
different reducing agents (i.e. vitamin C) resulting in the
production of superoxide anion from molecular oxygen,
which can finally dismutate to give oxygen and hydrogen
peroxide. Thus, the participation of iron is essential in the
production of hydroxyl radical and in the propagation of free
radical reactions by decomposing peroxides [112].
Copper. It is a transition metal, essential for life, capable
of undergoing one-electron oxidation-reduction conversions.
It has several important roles in the body, apparently related
to maintenance of immune function, bone health, arterial
compliance, haemostasis and protection against oxidative
and inflammatory damage [113]. Copper attends to important
catalytic functions in a number of enzymes such as Cu, Zn-
superoxide dismutase, cytochrome oxidase, and ceruloplas-
min. In these enzymatic reactions, copper tightly binds to
proteins such that redox activity of the resulting chelate
formed is strictly regulated. Copper and zinc, and manganese
1688 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
are indispensable metals for the activities of Cu, Zn-SOD
and Mn-SOD, respectively. Therefore, dietary deficiencies of
these minerals markedly decrease tissue Cu,Zn-SOD and
Mn-SOD activities and result in peroxidative damage and
mitochondrial dysfunction. A deficiency of copper or zinc in
rats also enhances cytochrome P-450 activity in microsomes
of liver and lung, stimulates ROS generation, and increases
intestinal iNOS expression. Such effects render the animal
more susceptible to lipid peroxidation and gastrointestinal
infection. However, the beneficial redox properties of copper
may also result in a pro-oxidant activity: it can catalyse
production of free radical intermediates from molecular
oxygen, particularly when is released from proteins [114].
Indeed, when the copper-binding domains are altered the
redox activity of copper is enhanced and cell damage and
death are observed. Thus it is not surprisingly that the delivery
of copper is rigorously controlled in cells and biological
fluids. Similarly to iron, which is bound to ferritin or
transferring, copper is transported and inactivated by specific
binding proteins. In plasma, ceruloplasmin and albumin are
the two major proteins (along with transcuprein) responsible
for binding and transport of copper and prevention of its
detrimental redox activity [115]. Taken together these data
suggest copper as an essential element, with a complex
pattern of utilization, to be taken in high consideration in any
issue related to nutrition and supplementation as well.
Selenium. It is essential for the functionality of the
immune system in both animals and humans [116]. The
antioxidant effects of selenium appear to be
mediated
through the GPX [117] that removes
potentially damaging
lipid hydroperoxides and hydrogen peroxide.
Since the
discovery of glutathione peroxidase as a selenium-dependent
enzyme, selenium has been identified as an essential cofactor
for selenoprotein P and other selenoproteins. At least five of
these peroxidases have now been identified
as operating in
different cell and tissue compartments.
Thus, selenium can
act as an antioxidant in the extracellular
space, the cell
cytosol, in association with cell membranes preserving
structure integrity [118]. The Keshen disease may be taken
as a paradigm of the essential role of selenium in promoting
and preserving human health. The name derives from the
village in China were the effects of selenium deficiency were
firstly discovered [119]. However, excess of selenium intake
(selenosis), for example with diet or supplementation, may
be also very harmful, leading to loss of hair and nails, skin
and nervous system lesions and death [120].
Toxic metals (lead, cadmium, mercury and arsenic) are
widely found in our environment. Humans are exposed to
these metals from numerous sources, including contaminated
air, water, soil and food. A growing number of studies
indicate that transition metals act as catalysts in the oxidative
reactions of biological macromolecules, therefore the
toxicities associated with these metals might be due to
oxidative tissue damage [121]. Redox-active metals, such as
iron, copper and chromium, undergo redox cycling whereas
redox-inactive metals, such as lead, cadmium, mercury and
others deplete cells' major antioxidants, particularly thiol-
containing antioxidants and enzymes. Either redox-active or
redox-inactive metals may cause an increase in production of
ROS such as HO
, O
2
•−
or H
2
O
2
, thus leading to oxidative
stress that can be partially responsible for the toxic effects of
heavy metals. Although these data suggest a possible role for
antioxidants in counteracting and preventing some of the
dangerous aspect of heavy metals, the biochemical basis for
metal-induced oxidative stress needs to be further investi-
gated prior to suggest a general use of antioxidants in heavy
metals related diseases [122]. Several studies are currently
underway to determine the effect of antioxidant supplemen-
tation following heavy metal exposure.
Enzymes
Antioxidant defence mechanisms are present to counteract
both metabolic and environmental sources of ROS. Antioxi-
dant defence mechanisms include small non-enzymatic
molecules and enzymes. Antioxidant enzymes have been well
conserved from bacteria to humans. Antioxidant enzymes
include the families of SOD, CAT, GPX, glutathione S-
transferase,
and thioredoxin. Each family has isoenzymes
that are distinguished
primarily by their distribution. For
instance, the three mammalian
SODs are cytosolic (SOD1),
mitochondrial (SOD2) [123], extracellular (SOD3),
and the
two thioredoxins are cytosolic
(Trx1) and mitochondrial
(Trx2) [124]. Superoxide dismutase (SOD) converts O
2
•−
into
H
2
O
2
, which is then rapidly reduced by catalase and/or
glutathione peroxidase to H
2
O and O
2
. The rapid methabol-
ism of O
2
•−
and H
2
O
2
is critical because competing mechan-
ism may lead to the generation of more active and toxic
radical species [125]. Peroxiredoxins (Prxs) are enzymes
lacking prosthetic groups that catalyse the reduction of H
2
O
2
and organic hydroperoxides [126]. They are ubiquitous pro-
teins found in organisms ranging from bacteria to humans. In
mammalian cells, they typically constitute 0.1 to 0.8% of the
total soluble protein of the cell. In particular, PrxII is the
second most abundant protein in mouse red blood cells. The
abundance of Prxs partly compensates for their moderate
catalytic proficiency. For example, other antioxidant
enzymes such as catalase and glutathione peroxidase reduce
peroxide at rates that are one to three orders of magnitude
greater than the peroxide reduction rate of the peroxi-
redoxins. In eukaryotic cells, the Prxs are both antioxidants
and regulators of H
2
O
2
-mediated signaling. Importantly,
human PrxII has been implicated in several disease states
including cancer and neurodegenerative disorders [127].
Antioxidant enzyme may be induced by different stimulus
[128]. For example, very recently, extracts of Urtica dioica
L. have been found able to induce GST, DTD, SOD and
CAT activity in the forestomach and SOD and CAT activity
in the lung [129].
Coenzyme Q. Ubiquinone is a biological compound that
is widely
distributed in plants, animals, and in most
microorganisms.
It is present in all tissues associated with
biomembranes. However,
its biological function is not clear
so far. In accordance with
the chemistry of redox-cycling
ubiquinone, one may assume that
this compound acts both as
an electron carrier and proton translocator.
In mitochondria,
coenzyme Q is involved in energy-linked redox
processes.
Due to its lipophilicity ubiquinone interacts with dehydroge-
nases
and shuttles a pair of two single electrons to cyto-
chromes by
diffusion [130]. Coenzyme Q is known to play
an important role as a mobile redox proton carrier in the
energy-transducing membranes of mitochondria and
chloroplasts. The reduced form of Coenzyme Q, ubiquinol,
The Antioxidants and Pro-Antioxidants Network Current Pharmaceutical Design, 2004, Vol. 10, No. 14 1689
has been shown to act as an antioxidant against free radical-
mediated oxidations in membranes and lipoproteins [131].
Uric acid. Uric acid is one of the most important antioxi-
dants
in plasma. Urate (the soluble form of uric acid in
the
blood) can scavenge superoxide, hydroxyl radical, and singlet
oxygen and can chelate transition metals. Peroxynitrite
is a
particularly toxic product formed by the reaction of
superoxide
anion with nitric oxide that can injure cells by
nitrosylating
the tyrosine residues (nitrotyrosine formation)
of proteins.
Uric acid can also block this reaction [132].
Recently, Hink et al. [133] reported that uric acid may also
prevent
the degradation of SOD3,
an enzyme critical in
maintaining endothelial and vascular function.
The removal
of O
2
•−
by SOD3 prevents the reaction and
inactivation by
O
2
•−
of the important endothelial vasodilator,
nitric oxide
(NO). SOD3, by removing O
2
•−
, therefore
helps to maintain
NO levels and maintain endothelial function.
Normally, SOD3
is inactivated in the presence of H
2
O
2
, suggesting
a feedback
inactivation of the enzyme. However, uric acid blocks
SOD
inactivation by H
2
O
2
by regenerating SOD3 with the
production
of an urate radical. This latter radical, although
potentially
pro-oxidant, has been found to be markedly less
reactive [134] than
classic oxidants and can be rapidly
regenerated back to urate
in the presence of ascorbate.
What is Behind the Corner?
Several diseases have been recognized as directly related
to oxidative damages. In few cases the oxidative activity is
as central pathophysiological feature (i.e. amyotrophic lateral
sclerosis, ALS), in many other cases the oxidative damage is
part or determining factor in the cellular disfunctions and
thus symptoms (i.e. neurodegeneration, cataract, diabetes)
[135]. Thus, in contrast to the current receptor-based phar-
macological approaches, the idea of an antioxidant therapy
that could work as a general protection against damages
induced by oxidized species is particularly attractive [136].
Moreover, during the last decade, the concept of health
promotion has become a legitimate part of health care. In the
attempt to counteract the oxidative stress damages, the stra-
tegy of implementing the diet with antioxidants, especially
deriving from natural sources, is becoming more and more
convincing [137]. Therefore, antioxidants have become very
well recognized nutraceutical ingredients. Although the great
number of positive reports, several problems still remain to
be solved before such interventions may become standard
tools in the treatment of major diseases and health promotion.
1) First of all free radical pathways appear as very complex
to be regulated and much need to be done in terms of
basic studies in order to highlight the mechanisms at the
base of their action. This also involves possible toxic
effects, related to impairment of such pathways. As an
example, it has been very recently reported that lipid
mediators generated by oxidative pathways play essential
roles in vascular disease but also in homeostasis,
activating signal transduction pathways that control a
variety of cellular functions [138, 139]. Several other
evidences are also reported in literature [140].
2) The problem of stability: as higher the activity as higher
will be instability and reactivity with oxidized and free
radical species. This aspect is also related to possible
toxic effects due to antioxidants over-reactivity: some of
them may become pro-oxidant in certain circumstances
[141].
Fig. (11).
Ubiquinone
N
H
NH
O
O
H
N
N
H
O
Uric acid
O
O
O
O
OO
P
P
O
-
O OH
O
-
O
OH
P
O
-
OH
O
P
O
-
O
OH
P
-
O
O
OH
P
O
-
HO
O
Inositol Esaphosphate
CH
3
O
O
O
O
CH
3
CH
3
CH
3
CH
3
H
3
C
H
3
C
1690 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
3) Free radical species are highly diffusible entities,
involved in radical-chain reactions and with variable
half lives which ranges from nanoseconds to minutes
and hours. Thus, it is difficult to imagine that the
antioxidant will be present at the exact moment and
place where the oxidative damage will occur.
4) Antioxidant effects may be acute or chronic, resulting
either from antioxidant or pro-antioxidant activities.
Moreover, most antioxidants works in a synergistic
manner, with a recycling kind of mechanism: thus α-
tocopherol works because restored, after quenching of
peroxyl radicals, by ascorbate, which is itself
regenerated by the dihydrolipoate-lipoate couple that is
finally restored by the NAD-NADH system [142, 143].
5) These latter issues are also complicated by distribution
problems: it is difficult to imagine that supplementation
by ascorbate and α-tocopherol, one hydrophilic and the
other lipophilic, will reach the same biological
compartment at the same time, to quench the free-
radicals to work synergistically.
These considerations point to the fact that more efforts
should be directed toward the development of synthetic
compounds, able to overcome the above stated problems but
endowed with the versatility of their natural counterparts.
Newer molecules could be useful both to address
mechanistic studies and to develop possible therapeutic
agents.
The Centaur Tactic
In Greek mythology, the centaurs were the half-man,
half-horse creature, descendants of Centaurus, a son of the
music god Apollo. Much like the centaur, the satyr combined
the qualities of the hoofed with the human. Our recently
developed strategy for the discovery of novel antioxidants,
can be illustrated on the base of these mythological
considerations. Indeed, we have obtained molecular
combinations of antioxidants, designed in the aim to improve
the pharmacology, bioavailability and stability of the parent
compounds. Increasing evidences support the idea that a
combination of antioxidants may offer a better overall
protection against oxidative stress than that exerted by
individual antioxidants. In these regards, the existence of
cooperative interactions between carotenoids and
tocopherols, tocopherol and ascorbic acid, in biological
systems, has been reported [144, 145]. In view of such
potential interactions, we have recently designed novel
entities, deriving from the conjugation of antioxidant
cooperative moieties, through stable chemical bonds. In
particular, we have recently proposed molecular
combinations of the pharmacophores of synergistic
antioxidants, i.e vitamin E with vitamin C or carotenoids, as
a tool to improve the antioxidant activity by concomitant
scavenging of lipoperoxylradicals (preferred by α-
tocopherol) and ROS (preferred by vitamin C) [146, 91].
Following the same approach, we have more recently
explored molecular combinations between vitamin C and
polyphenols, also obtaining a consistent improvement in the
antioxidant activity [147]. Other very recent results of this
strategy also indicate potential usefulness against
degenerative diseases based on oxidative damages [148,
149]. We have very recently reported on molecular
combination between idebenone and other cooperative
antioxidant entities, obtaining an increase of activity in the
respect of the parent compounds [150].
Finally, the molecular combination strategy was
afterwards positively considered by us, as a new approach
for CNS drug targeting. We have recently investigated the
conjugation of ascorbic acid with neurotropic drugs, as a
possible means to improve the entry of such CNS drugs, that
difficulty cross the blood brain barrier. [17] Further studies
and results are currently being submitted for publication.
Taking this into account, modification of antioxidants can
represent a possibility for exponentially increase their
antioxidant activity, and in view of our results, we think the
centaur tactic as an important tool in the i) study the
mechanism of interaction of cooperative antioxidants; ii)
evaluation of new approaches to potential therapeutic agents
in chronic diseases in which a free radical damage is
involved; iii) investigation of new endogenous carriers to
improve the drug bioavailabity of the transported drugs; iv)
improvement of stability of antioxidant compounds.
ABBREVIATIONS
AGE = Aged garlic extract
ALA = Alpha-lipoic acid
ALS = Amyotrophic lateral sclerosis
CAT = Catalase
CNS = Central nervous system
GGT = Gamma-glutamyltranspeptidase
ESR = Electron spin resonance
4-HNE = 4-Hydroxynonenal
MSRs = Methionine sulfoxide reductases
ROS = Reactive oxygen species
RNS = Reactive nitrogen species
CRS = Cellular reduction systems
GSH = Glutathione
GPX = Glutathione peroxidase
GS-SG = Glutathione disulfide
GST = Glutathione S-transferase,
GR = Glutathione reductase
NADP
+
/
NADPH = Nicotinamide adenine dinucleotide phosphate
NAD
+
/
NADH = Nicotinamide adenine dinucleotide
NO = Nitric oxide
NOS = NO synthases
FAD/
FADH
2
= Flavin adenine dinucleotide
FMN = Flavin mononucleotide
MAP = Mitogen activated protein
The Antioxidants and Pro-Antioxidants Network Current Pharmaceutical Design, 2004, Vol. 10, No. 14 1691
Prxs = Peroxiredoxins
PUFAs = Poly unsaturated fatty acids
TNF = Tumour necrosis factor
TSS = Trans-sulfuration
NF- = Nuclear Factor kappa B
kappaB
SAMe = S-Adenosyl-L-methionine
Tf = Transferrin
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... The hydrophilic antioxidant reacts with the oxidants present in blood plasma along with cell cytosol, whereas the cell membrane is prevented by hydrophobic antioxidants [3]. All these compounds can be produced in a body as well as could be found from food and other food products [4]. There are present different oxidants in the body fluids and tissues, example glutathione or ubiquinone which is mostly present in the cell, whereas few other like uric acid is uniformly distributed. ...
... Interaction among the antioxidants is such a complicated issue, having most of an antioxidant compounds as well as antioxidant enzyme system with interdependent along with synergistic effect on each other [6,7]. The mode of action of the antioxidant depends on the functioning of the all members of an antioxidant system [4]. The amount of protection delivered through any antioxidant can also be governed by its reactivity to the ROS being deliberated, the antioxidant as well as concentration status having its interacting [4]. ...
... The mode of action of the antioxidant depends on the functioning of the all members of an antioxidant system [4]. The amount of protection delivered through any antioxidant can also be governed by its reactivity to the ROS being deliberated, the antioxidant as well as concentration status having its interacting [4]. ...
... Prevalence of the oxidative reaction causes the development of processes described as oxidative stress [2]. The result of the reaction above is a change in the protein structure and function, oxidation of cell membranes and DNA damage [3]. The result of oxidative stress is lipid peroxidation [4], a process of oxidation of the unsaturated fatty acids, constituting elements of phospholipids. ...
... The number of DNA lesions is estimated to be ten thousand per day; however, DNA damage is quickly repaired by intrinsic ROS scavengers [99][100][101]. To remove ROS, antioxidant enzymes such as catalase, SOD, and peroxidase detoxify ROS [102][103][104]. Excess ROS that cannot be degraded by antioxidant enzymes are known to cause various diseases, including neurodegenerative diseases such as Parkinson's disease [105] and Alzheimer's disease [106,107], carcinogenesis [108,109], lifestyle-related diseases [110,111], and aging [20,112]. ...
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Molecular combinations of two antioxidants (i.e., ascorbic acid and the pharmacophore of α-tocopherol), namely the 2,3-dihydroxy-2,3-enono-1,4-lactone and the chromane residues, have been designed and tested for their radical scavenging activities. When evaluated for their capability to inhibit malondialdehyde (MDA) production in rat liver microsomal membranes, the 3,4-dihydroxy-5R-2(R,S)-(6-hydroxy-2,5,7,8-tetramethylchroman-2(R,S)yl-methyl)-1,3]dioxolan-4S-yl]-5H-furan-2-one (11a–d), exhibited an interesting activity. In particular the 5R,2R,2R,4S and 5R,2R,2S,4S isomers (11c,d) displayed a potent antioxidant effect compared to the respective synthetic α-tocopherol analogue (5) and natural α-tocopherol or ascorbic acid, used alone or in combination. Moreover, the mixture of stereoisomers 11a–d also proved to be effective in preventing damage induced by reperfusion on isolated rabbit heart, in particular at the higher concentration of 300 μM. In view of these results our study represents a new approach to potential therapeutic agents for applications in pathological events in which a free radical damage is involved. Design, synthesis and preliminary biological activity are discussed.
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The present study investigates the reactivity of bovine serum albumin (BSA) radicals towards different biomolecules (urate, linoleic acid, and a polypeptide, poly(Glu-Ala-Tyr)). The BSA radical was formed at room temperature through a direct protein-to-protein radical transfer from H(2)O(2)-activated immobilized horseradish peroxidase (im-HRP). Subsequently, each of the three different biomolecules was separately added to the BSA radicals, after removal of im-HRP by centrifugation. Electron spin resonance (ESR) spectroscopy showed that all three biomolecules quenched the BSA radicals. Subsequent analysis showed a decrease in the concentration of urate upon reaction with the BSA radical, while the BSA radical in the presence of poly(Glu-Ala-Tyr) resulted in increased formation of the characteristic protein oxidation product, dityrosine. Reaction between the BSA radical and a linoleic acid oil-in-water emulsion resulted in additional formation of lipid hydroperoxides and conjugated dienes. The results clearly show that protein radicals have to be considered as dynamic species during oxidative processes in biological systems and that protein radicals should not be considered as end-products, but rather as reactive intermediates during oxidative processes in biological systems hereby supporting recent data.
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A majority of the LDL preparations from various donors could be modified by incubation with endothelial cells from human arteries, veins and microvessels. These alterations comprise changes in electrophoretic mobility, buoyant density and lipid composition of LDL, the generation of thiobarbituric acid reactive substances in the medium, and a decrease in primary amino groups of LDL. Furthermore, the association of endothelial cell proteins with LDL was demonstrated by [35S]methionine incorporation and trichloroacetic acid precipitation of reisolated endothelial cell-modified LDL. After SDS-polyacrylamide gel electrophoresis of the reisolated modified LDL particles, radioactivity was mainly found at a molecular mass of 48 kDa and at one or two bands with a molecular mass of more than 100 kDa. The 48 kDa protein was identified as a latent plasminogen activator inhibitor. Cell viability was necessary for the cell-mediated LDL modification, which indicates that endothelial cells are actively involved in this process. The Ca2+ ionophore A23187 and monensin did not influence LDL modification. LDL modification was markedly inhibited by antioxidants. It was not prevented by cyclooxygenase and lipoxygenase inhibitors, which indicates that non-enzymatic lipid peroxidation is involved. Transition metal- (copper-) induced lipid peroxidation results in similar physicochemical alterations of the LDL particle as found with endothelial cells; it is prevented by the presence of superoxide dismutase. In contrast, endothelial cell LDL modification was not influenced by superoxide dismutase. Catalase or singlet oxygen and hydroxyl radical scavengers also did not affect it. We suggest that yet unidentified radicals or lipid peroxides are generated in the cells or on the cell membrane and that these reactive molecule(s) will react with LDL after leaving the cell. HDL and lipoprotein-depleted serum prevented LDL modification markedly, and to a larger extent than that by copper ions. We speculate that LDL modification by endothelial cells will only occur under those conditions in which the balance between the generation of reactive oxygen molecules and the cellular protection against these reactive species is disturbed.