ArticlePDF AvailableLiterature Review

Abstract and Figures

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.
Content may be subject to copyright.
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.
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:
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.
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.
) [18]. Moreover, as stated
above, vitamin B3 is able to modulate iNOS expression.
Riboflavin and Niacin are components of NADP
, 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
Taurine, a sulphur containing ß amino acid, is the most
abundant intracellular amino acid in humans, and is impli-
Vitamin B12
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).
Vitamin A
Vitamin E
Vitamin C
Vitamin K2
Vitamin D
. HCl
Vitamin B6
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
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
), 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
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
γ-Linoleic Acid
Arachidonic Acid
α-linolenic acid
Eicosapentenoic acid
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].
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).
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
, 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
[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
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).
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
+ 2GSH
R· + GSH
RH + GS·
GSSG·- + O
2 O
·- + 2H
+ O
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
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
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
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
) and with alkyl radicals (L
), derived from
oxidation. The reaction between vitamin E and lipid radical
in the membrane-water interphase where vitamin E
donates an hydrogen
ion to lipid radical with consequent
tocopheroxyl radical (TO
formation. Regeneration of the
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
radicals, especially singlet oxygen (via irreversible
of tocopherol), and as physical deactivators of singlet
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-
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
moieties found in several Lamiatae plants (rosmarinic
salvianolic acids, yunnaneic acids, etc.), the large group
of flavonoids, monomeric and oligomeric flavan-3-ols
of (+)-catechin or (-)-epicatechin, also known as
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
ions (termination of the Fenton reaction) [78]. Another
mechanism underlying the antioxidative properties
phenolics is the ability of flavonoids to alter peroxidation
kinetics by modification of the lipid packing order and to
fluidity of the membranes. These changes
1684 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
sterically hinder diffusion of free radicals and restrict
dative reactions. Moreover, it has been shown recently,
phenolic compounds can be involved in the hydrogen
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
, H
, OH
, and
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
. 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
in plants and bowel microflora) to form isothiocyanates.
vivo, isothiocyanates are conjugated with glutathione and
sequentially metabolized to mercapturic acids. These
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).
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
, NO
), 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
) 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).
Biochanin A O
Cinnamic Acid
Caffeic Acid
Gallic acid
1686 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
Fig. (9).
Fig. (10).
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
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].
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
is not water soluble,
and ii) Fe
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
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
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
ferric (Fe
), and ferryl (Fe
) 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
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
or H
, 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.
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-
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
, which is then rapidly reduced by catalase and/or
glutathione peroxidase to H
O and O
. The rapid methabol-
ism of O
and H
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
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
-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
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
Due to its lipophilicity ubiquinone interacts with dehydroge-
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-
in plasma. Urate (the soluble form of uric acid in
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
anion with nitric oxide that can injure cells by
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
the degradation of SOD3,
an enzyme critical in
maintaining endothelial and vascular function.
The removal
of O
by SOD3 prevents the reaction and
inactivation by
of the important endothelial vasodilator,
nitric oxide
(NO). SOD3, by removing O
, therefore
helps to maintain
NO levels and maintain endothelial function.
Normally, SOD3
is inactivated in the presence of H
, suggesting
a feedback
inactivation of the enzyme. However, uric acid blocks
inactivation by H
by regenerating SOD3 with the
of an urate radical. This latter radical, although
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
Fig. (11).
Uric acid
Inositol Esaphosphate
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
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.
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
NADPH = Nicotinamide adenine dinucleotide phosphate
NADH = Nicotinamide adenine dinucleotide
NO = Nitric oxide
NOS = NO synthases
= 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
SAMe = S-Adenosyl-L-methionine
Tf = Transferrin
References 151-153 are related articles recently published in
Current Pharmaceutical Design.
[1] Packer L, Valacchi G. Antioxidants and the response of skin to
oxidative stress: vitamin E as a key indicator. Skin Pharmacol Appl
Skin Physiol. 2002; 15: 282-90.
[2] Matsuo Y, Hirota K, Nakamura H, Yodoi J. Redox Regulation by
Thioredoxin and its Related Molecules. Drug News Perspect. 2002;
15: 575-80.
[3] Keys SA, Zimmerman WF. Antioxidant activity of retinol,
glutathione, and taurine in bovine photoreceptor cell membranes.
Exp Eye Res 1999; 68: 693-702.
[4] Hirokawa K, Oshaughnessy KM, Ramrankha P, Wilkins MR.
Inhibition of nitric oxide synthesis in vascular smooth muscle by
retinoids. Br J Pharmacol 1994; 113: 1448-54.
[5] Grosjean S, Devaux Y, Seguin C, et al. Retinoic acid attenuates
inducible nitric oxide synthase (NOS2) activation in cultured rat
cardiac myocites and microvascular endothelial cells. J Mol Cell
Cardiol 2001; 33: 933-45.
[6] Sano M, Fujita H, Morita I, Uematsu H, Murota S. Vitamin K-2
(menatetrenone) induces iNOS in bovine vascular smooth muscle
cells: no relationship between nitric oxide production and
carboxylation. J Nutr Sci Vitam 1999; 45: 711-23.
[7] Garcion E, Nataf S, Berod A, Darcy F, Brachet P. 1,25-
Dihydroxyvitamin D-3 inhibits the expression of inducible nitric
oxide synthase in rat central nervous system during experimental
allergic encephalomyelitis. Brain Res Mol Brain Res 1997; 45: 255-
[8] Masaki H, Okano Y, Ochiai Y, Obayashi K, Akamatsu H, Sakurai
H. Alpha-tocopherol increases the intracellular glutathione level in
HaCaT keratinocytes. Free Radic Res 2002; 36: 705-9.
[9] Naziroglu M, Kokcam I, Yilmaz S. Beneficial Effects of
Intraperitoneally Administered Alpha-Tocopheryl Acetate on the
Levels of Lipid Peroxide and Activity of Glutathione Peroxidase
and Superoxide Dismutase in Skin, Blood and Liver of Thermally
Injured Guinea Pigs. Skin Pharmacol Appl Skin Physiol 2003; 16:
[10] Calfee-Mason KG, Spear BT, Glauert HP. Vitamin E inhibits
hepatic NF-kappaB activation in rats administered the hepatic
tumor promoter, phenobarbital. J Nutr 2002; 132: 3178-85.
[11] Tauler P, Aguilo A, Fuentespina E, Tur JA, Pons A. Diet
supplementation with vitamin E, vitamin C and beta-carotene
cocktail enhances basal neutrophil antioxidant enzymes in athletes.
Pflugers Arch 2002; 443: 791-7.
[12] May JM. How does ascorbic acid prevent endothelial dysfunction?
Free Radic Biol Med 2000; 28: 1421-9.
[13] Diaz MN, Frei B, Vita JA, Keaney JF. Antioxidants and
atherosclerotic heart disease. N Engl J Med 1997; 337: 408-416.
[14] Doba T, Burton GW, Ingold KU. Antioxidant and co-antioxidant
activity of vitamin C. The effect of vitamin C, either alone or in the
presence of vitamin E or a water-soluble vitamin E analogue, upon
peroxidation of aqueous multilamellar phospholipid liposomes.
Biochim Biophys Acta 1985; 835: 298-303.
[15] Guo Q, Packer L. Ascorbate-dependent recycling of the vitamin E
homologue Trolox by dihydrolipoate and glutathione in murine skin
homogenates. Free Radic Biol Med 2000; 29: 368-74.
[16] Thiele JJ, Schroeter C, Hsieh SN, Podda M, Packer L. The
antioxidant network of the stratum corneum. Curr Probl Dermatol
2001; 29: 26-42.
[17] Manfredini S, Pavan B, Vertuani S, Scaglianti M, Compagnone D,
Biondi C, et al. Design, synthesis and activity of ascorbic acid
prodrugs of nipecotic, kynurenic and diclophenamic acids, liable to
increase neurotropic activity. J Med Chem 2002; 45: 559-62.
[18] Kamat JP, Devasagayam TP. Methylene blue plus light-induced
lipid peroxidation in rat liver microsomes: inhibition by
nicotinamide (vitamin B3) and other antioxidants. Chem Biol
Interact 1996; 99: 1-16.
[19] Cossins E, Lee R, Packer L. ESR studies of vitamin C regeneration,
order of reactivity of natural source phytochemical preparations.
Biochem Mol Biol Int 1998; 45: 583-97.
[20] Zamocky M, Koller F. Understanding the structure and function of
catalases: clues from molecular evolution and in vitro mutagenesis.
Prog Biophys Mol Biol 1999; 72: 19-66.
[21] Holmgren A. Antioxidant function of thioredoxin and glutaredoxin
systems. Antioxid Redox Signal 2000; 2: 811-20.
[22] Wu G, Haynes TE, Li H, Yan W, Meininger CJ. Glutamine
metabolism to glucosamine is necessary for glutamine inhibition of
endothelial nitric oxide synthesis. Biochem J 2001; 353: 245-52.
[23] Mennen LI, de Courcy GP, Guilland JC, Ducros V, Bertrais S,
Nicolas JP, et al. Homocysteine, cardiovascular disease risk factors,
and habitual diet in the French Supplementation with Antioxidant
Vitamins and Minerals Study. Am J Clin Nutr 2002; 76: 1279-89.
[24] Earnest C, Cooper KH, Marks A, Mitchell TL. Efficacy of a
complex multivitamin supplement. Nutrition 2002; 18: 738-42.
[25] Hsu PC, Guo YL. Antioxidant nutrients and lead toxicity.
Toxicology 2002; 180: 33-44.
[26] Dobarganes C, Marquez-Ruiz G. Oxidized fats in foods. Curr Opin
Clin Nutr Metab Care 2003; 6: 157-63.
[27] Komatsu W, Ishihara K, Murata M, Saito H, Shinohara K.
Docosahexaenoic acid suppresses nitric oxide production and
inducible nitric oxide synthase expression in interferon-gamma plus
lipopolysaccharide-stimulated murine macrophages by inhibiting
the oxidative stress. Free Radic Biol Med. 2003; 34: 1006-16.
[28] Yun-Zhong Fang, Sheng Yang, Guoyao Wu. Free Radicals,
Antioxidants and Nutrition. Nutrition 2002; 18: 872-9.
[29] Miret S, Saiz MP, Mitjavila MT. Effects of fish oil- and olive oil-
rich diets on iron metabolism and oxidative stress in the rat. Br J
Nutr 2003; 89: 11-8.
[30] Lourenco R, Camilo ME. Taurine: a conditionally essential amino
acid in humans? An overview in health and disease. Nutr Hosp
2002; 17: 62-70.
[31] Messina SA, Dawson R Jr. Attenuation of oxidative damage to
DNA by taurine and taurine analogs. Adv Exp Med Biol 2000; 483:
[32] Biasetti M, Dawson R Jr. Effects of sulfur containing amino acids
on iron and nitric oxide stimulated catecholamine oxidation. Amino
Acids 2002; 22: 351-68.
[33] Boelens PG, Houdijk AP, de Thouars HN, Teerlink T, van
Engeland MI, Haarman HJ, et al. Plasma taurine concentrations
increase after enteral glutamine supplementation in trauma patients
and stressed rats. Am J Clin Nutr 2003; 77: 250-6.
[34] Tapiero H, Mathe G, Couvreur P, Tew KD. II. Glutamine and
glutamate. Biomed Pharmacother 2002; 56: 446-57.
[35] Lass A, Suessenbacher A, Wolkart G, Mayer B, Brunner F.
Functional and analytical evidence for scavenging of oxygen
radicals by L-arginine. Mol Pharmacol 2002; 61: 1081-8.
[36] Andrew PJ, Mayer B. Enzymatic function of nitric oxide synthases.
Cardiovasc Res 1999; 43: 521-31.
[37] Patterson RA, Leake DS. Human serum, cysteine and histidine
inhibit the oxidation of low density lipoprotein less acidic
pH. FEBS Lett 1998; 434: 317-21.
[38] van Hinsbergh WM, Havekes L, Kempen HJM. Role of endothelial
cells and their products in the modification of low-density
lipoproteins. Biochim Biophys Acta 1986; 878: 49-64.
[39] Yeomans VC, Rechner AR, Rice-Evans CA. The mechanism of
action of zinc-histidine complex (Curazink) as an antioxidant. Free
Radic Res 2002; 36: 717-8.
[40] Decker EA, Ivanov V, Zhu BZ, Frei B. Inhibition of low-density
lipoprotein oxidation by carnosine histidine. J Agric Food Chem
2001; 4: 511-6.
[41] Matilla B, Mauriz JL, Culebras JM, Gonzalez-Gallego J, Gonzalez
P. Glycine: a cell-protecting anti-oxidant nutrient. Nutr Hosp 2002;
17: 2-9.
[42] Wlodek L. Beneficial and harmful effects of thiols. Pol J Pharmacol
2002; 54: 215-23.
1692 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
[43] Moran LK, Gutteridge JM, Quinlan GJ. Thiols in cellular redox
signalling and control. Curr Med Chem 2001; 8: 763-72.
[44] Hoshi T, Heinemann S. Regulation of cell function by methionine
oxidation and reduction. J Physiol 2001; 53: 1-11.
[45] Zafarullah M, Li WQ, Sylvester J, Ahmad M. Molecular
mechanisms of N-acetylcysteine actions. Cell Mol Life Sci 2003;
60: 6-20.
[46] Behr J, Maier K, Degenkolb B, Krombach F, Vogelmeier C.
Antioxidative and clinical effects of high-dose N-acetylcysteine in
fibrosing alveolitis. Adjunctive therapy to maintenance
immunosuppression. Amer. J. Respir. Crit. Care Med 1997; 156:
[47] Mantovani G, Maccio A, Lai P, Massa E, Ghiani M, Santona MC.
Cytokine activity in cancer-related anorexia/cachexia: role of
megestrol acetate and medroxyprogesterone acetate. Semin Oncol.
1998; 25: 45-52.
[48] Mantovani G, Maccio A, Melis G, Mura L, Massa E, Mudu MC.
Restoration of functional defects in peripheral blood mononuclear
cells isolated from cancer patients by thiol antioxidants alpha-lipoic
acid and N-acetyl cysteine. Int J Cancer 2000; 86: 842-7.
[49] Schubert SY, Neeman I, Resnick N. A novel mechanism for the
inhibition of NF-kappaB activation in vascular endothelial cells by
natural antioxidants. FASEB J 2002; 16: 1931-3.
[50] Lieber CS. S-adenosyl-L-methionine: its role in the treatment of
liver disorders. Am J Clin Nutr 2002; 76: 1183S-7S.
[51] Hipkiss AR, Brownson C. A possible new role for the anti-ageing
peptide carnosine. Cell Mol Life Sci. 2000; 57: 747-53.
[52] Aldini G, Carini M, Beretta G, Bradamante S, Facino RM.
Carnosine is a quencher of 4-hydroxy-nonenal: through what
mechanism of reaction? Biochem Biophys Res Commun. 2002;
298: 699-706.
[53] Hipkiss AR, Brownson C, Carrier MJ. Carnosine, the anti-ageing,
anti-oxidant dipeptide, may react with protein carbonyl groups.
Mech Ageing Dev 2001; 122: 431-45.
[54] Dickinson DA, Iles KE, Watanabe N, Iwamoto T, Zhang H,
Krzywanski DM, et al. 4-hydroxynonenal induces glutamate
cysteine ligase through JNK in HBE1 cells. Free Radic Biol Med.
2002; 33: 974-87.
[55] Dickinson DA, Forman HJ. Glutathione in defense and signaling:
lessons from a small thiol. Ann N Y Acad Sci 2002; 973: 488-504.
[56] Foyer CH. Prospects for enhancement of the soluble antioxidants,
ascorbate and glutathione. Biofactors 2001; 15: 75-8.
[57] Dale A. Dickinson and Henry Jay Forman Cellular glutathione and
thiols metabolism. Biochem Pharmacol 2002; 64: 1019-26.
[58] Cohen G, Hochstein P. Glutathione peroxidase: the primary agent
for the elimination of hydrogen peroxide in erythrocytes.
Biochemistry 1963; 2: 1420-28.
[59] Winterbourn CC. Superoxide as an intracellular radical sink. Free
Radic Biol Med 1993; 14: 85-90.
[60] Machilin LJ, Bandito A. Free radical tissue damage: protective role
of antioxidant nutrients. FASEB J 1987; 1: 441-52.
[61] Sakata N, Moh A, Takebayashi S. Contribution of superoxide to
reduced antioxidant activity of glycoxidative serum albumin. Heart
Vessels 2002; 17: 22-9.
[62] Stadtman ER, Moskovitz J, Berlett BS, Levine RL.Cyclic oxidation
and reduction of protein methionine residues is an important
antioxidant mechanism. Mol Cell Biochem 2002; 234-235: 3-9.
[63] Lindmark-Mansson H, Akesson B. Antioxidative factors in milk. Br
J Nutr 2000; 84: 103-10.
[64] Kaplan J. Mechanisms of cellular iron acquisition: another iron in
the fire. Cell 2002; 11: 603-6.
[65] Widera A, Beloussow K, Kim KJ, Crandall ED, Shen WC.
Phenotype-dependent synthesis of transferrin receptor in rat
alveolar epithelial cell monolayers. Cell Tissue Res. 2003; 312:
[66] Tomaro ML, Batlle AM. Bilirubin: its role in cytoprotection against
oxidative stress. Int J Biochem Cell Biol 2002 ; 34: 216-20.
[67] McGeary RP, Szyczew AJ, Toth I. Biological properties and
therapeutic potential of bilirubin. Mini Rev Med Chem 2003; 3:
[68] Asad SF, Singh S, Ahmad A, Khan NU, Hadi SM. Prooxidant and
antioxidant activities of bilirubin and its metabolic precursor
biliverdin: a structure-activity study. Chem Biol Interact 2001; 137:
[69] Patel BN, Dunn RJ, Jeong SY, Zhu Q, Julien JP, David S.
Ceruloplasmin regulates iron levels in the CNS and prevents free
radical injury. J Neurosci 2002; 22: 6578-86.
[70] Yoshida Y, Niki E, Noguchi N. Comparative study on the action of
tocopherols and tocotrienols as antioxidant: chemical and physical
effects. Chem Phys Lipids 2003 Mar; 123: 63-75.
[71] Cooney RV, Franke AA, Harwood PJ, Hatch-Pigott V, Custer LJ,
Mordan LJ. Gamma-tocopherol detoxification of nitrogen dioxide:
Superiority to alpha-tocopherol. Proc Natl Acad Sci USA 1993; 90:
[72] Packer L, Weber SU, Rimbach G. Molecular aspects of alpha-
tocotrienol antioxidant action and cell signalling. Journal of
Nutrition 2001: 131; 369S-373S.
[73] Serbinova E, Kagan V, Han D, Packer L. Free radical recycling and
intramembrane mobility in the antioxidant properties of α-
tocopherol and α-tocotrienol. Free Radic Biol Med 1991; 10: 263-
[74] Fuchs J, Weber S, Podda M, Groth N, Herrling T, Packer L, et al.
HPLC analysis of vitamin E isoforms in human epidermis:
correlation with minimal erythema dose and free radical scavenging
activity. Free Radic Biol Med. 2003; 34: 330-6.
[75] Weber SU, Thiele JJ, Han N, Luu C, Valacchi G, Weber S, et al.
Topical alpha-tocotrienol supplementation inhibits lipid
peroxidation but fails to mitigate increased transepidermal water
loss after benzoyl peroxide treatment of human skin. Free Radic
Biol Med 2003; 34: 170-6.
[76] Brandolini V, Maietti A, Tedeschi P, Durini E, Vertuani S,
Manfredini S. Capillary Electrophoresis Determination, Synthesis
and Stability of Resveratrol and related 3-O-beta-D-
Glucopyranosides J Agr Food Chem 2002; 50: 7407-11.
[77] Bors W, Michel C. Chemistry of the antioxidant effect of
polyphenols. Ann N Y Acad Sci 2002; 957: 57-69.
[78] Shafiee M, Carbonneau MA, d'Huart JB, Descomps B, Leger CL.
Synergistic antioxidative properties of phenolics from natural origin
toward low-density lipoproteins depend on the oxidation system. J
Med Food. 2002; 5: 69-78.
[79] Blokhina O, Virolainen E, Fagerstedt KV. Antioxidants, oxidative
damage and oxygen deprivation stress: a review. Ann Bot (Lond)
2003; 91: 179-94.
[80] Tapiero H, Tew KD, Ba GN, Mathe G. Polyphenols: do they play a
role in the prevention of human pathologies? Biomed Pharmacother
2002; 56: 200-7.
[81] Sakihama Y, Cohen MF, Grace SC, Yamasaki H. Plant phenolic
antioxidant and prooxidant activities: phenolics-induced oxidative
damage mediated by metals in plants. Toxicology 2002; 177: 67-80.
[82] Sakihama Y, Cohen MF, Grace SC, Yamasaki H. Plant phenolic
antioxidant and prooxidant activities: phenolics-induced oxidative
damage mediated by metals in plants. Toxicology 2002; 177: 67-80.
[83] Mira L, Fernandez MT, Santos M, Rocha R, Florencio MH,
Jennings KR. Interactions of flavonoids with iron and copper ions:
a mechanism for their antioxidant activity. Free Radic Res 2002;
36: 1199-208.
[84] Silva MM, Santos MR, Caroco G, Rocha R, Justino G, Mira L.
Structure-antioxidant activity relationships of flavonoids: a re-
examination. Free Radic Res 2002; 36: 1219-27.
[85] Arora A, Byrem TM, Nair MG, Strasburg GM. Modulation of
liposomal membrane fluidity by flavonoids and isoflavonoids. Arch
Biochem Biophys 2000; 373: 102-9.
[86] Fahey JW, Zalcmann AT, Talalay P. The chemical diversity and
distribution of glucosinolates and isothiocyanates among plants.
Phytochemistry 2001; 56: 5-51.
[87] Shapiro TA, Fahey JW, Wade KL, Stephenson KK, Talalay P.
Chemoprotective glucosinolates and isothiocyanates of broccoli
sprouts: metabolism and excretion in humans. Cancer Epidemiol
Biomarkers Prev 2001; 10: 501-8.
[88] Johnson IT. Glucosinolates: bioavailability and importance to
health. Int J Vitam Nutr Res 2002; 72: 26-31.
[89] Deng XS, Tuo J, Poulsen HE, Loft S. Prevention of oxidative DNA
damage in rats by brussels sprouts. Free Radic Res 1998; 28: 323-
[90] Handelman GJ. The evolving role of carotenoids in human
biochemistry. Nutrition 2001; 17: 818-22.
[91] Palozza P, Piccioni E, Avanzi L, Vertuani S, Calviello G,
Manfredini S. Design, synthesis, and antioxidant activity of
FeAOX-6, a novel agent deriving from a molecular combination of
The Antioxidants and Pro-Antioxidants Network Current Pharmaceutical Design, 2004, Vol. 10, No. 14 1693
the chromanyl and polyisoprenyl moieties. Free Radic Biol Med
2002; 33: 1724-35.
[92] Young AJ, Lowe GM. Antioxidant and prooxidant properties of
carotenoids. Arch Biochem Biophys 2001; 385: 20-7.
[93] Palozza P. Prooxidant actions of carotenoids in biologic systems.
Nutr Rev 1998; 56: 257-65.
[94] Palozza P. Evidences of prooxidant effects of carotenoids in vitro
and in vivo: implications in health and disease. In: Kinsky N,
Mayne S, Sies H Eds. Carotenoids in health and disease. N.Y.
Marcell Dekker Inc. 2003, in press.
[95] Mortensen A, Skibsted LH, Truscott TG. The interaction of dietary
carotenoids with radical species. Arch Biochem Biophys 2001;
[96] Gale CR, Hall NF, Phillips DI, Martyn CN. Lutein and zeaxanthin
status and risk of age-related macular degeneration. Invest
Ophthalmol Vis Sci 2003; 44: 2461-5.
[97] Epstein KR. The role of carotenoids on the risk of lung cancer.
Semin Oncol 2003; 30: 86-93.
[98] Oh WK, Small EJ. Complementary and alternative therapies in
prostate cancer. Semin Oncol 2002; 29: 575-84.
[99] Heber D, Lu QY. Overview of mechanisms of action of lycopene.
Exp Biol Med 2002; 227: 920-3.
[100] Rao AV. Lycopene, tomatoes, and the prevention of coronary heart
disease. Exp Biol Med 2002; 227: 908-13.
[101] Johnson EJ. The role of carotenoids in human health. Nutr Clin
Care 2002; 5: 56-65.
[102] Fox CH, Eberl M. Phytic acid (IP6), novel broad spectrum anti-
neoplastic agent: a systematic review. Complement Ther Med 2002;
10: 229-34.
[103] Midorikawa K, Murata M, Oikawa S, Hiraku Y, Kawanishi S.
Protective effect of phytic acid on oxidative DNA damage with
reference to cancer chemoprevention. Biochem Biophys Res
Commun. 2001-2; 288: 552-7.
[104] Ryu K, Ide N, Matsuura H, Itakura Y. N alpha-(1-deoxy-D-fructos-
1-yl)-L-arginine, an antioxidant compound identified in aged garlic
extract. J Nutr 2001; 131(3s): 972S-6S.
[105] Banerjee SK, Mukherjee PK, Maulik SK. Garlic as an antioxidant:
the good, the bad and the ugly. Phytother Res 2003; 17: 97-106.
[106] Rahman K. Garlic and aging: new insights into an old remedy.
Ageing Res Rev 2003; 2: 39-56.
[107] Ames BN, Wakimoto P. Are vitamin and mineral deficiencies a
major cancer risk? Nat Rev Cancer 2002; 2: 694-704.
[108] Powell SR. The antioxidant properties of zinc. J Nutr 2000; 130 (5S
Suppl): 1447S-54S.
[109] Rostan EF, DeBuys HV, Madey DL, Pinnell SR. Evidence
supporting zinc as an important antioxidant for skin. Int J Dermatol
2002; 41: 606-11.
[110] Onderci M, Sahin N, Sahin K, Kilic N. Antioxidant properties of
chromium and zinc: in vivo effects on digestibility, lipid
peroxidation, antioxidant vitamins, and some minerals under a low
ambient temperature. Biol Trace Elem Res. 2003; 92: 139-50.
[111] Quinlan GJ, Evans TW, Gutteridge JM. Iron and the redox status of
the lungs. Free Radic Biol Med 2002; 33: 1306-13.
[112] Fraga CG, Oteiza PI. Iron toxicity and antioxidant nutrients.
Toxicology 2002; 180: 23-32.
[113] O'Connor JM, Bonham MP, Turley E, McKeown A, McKelvey-
Martin VJ, Gilmore WS, et al. Copper supplementation has no
effect on markers of DNA damage and liver function in healthy
adults (FOODCUE Project). Ann Nutr Metab 2003; 47: 201-6.
[114] Videla LA, Fernandez V, Tapia G, Varela P. Oxidative stress-
mediated hepatotoxicity of iron and copper: role of Kupffer cells.
Biometals 2003; 16: 103-11.
[115] Gryzunov YA, Arroyo A, Vigne JL, Zhao Q, Tyurin VA, Hubel
CA. Binding of fatty acids facilitates oxidation of cysteine-34 and
converts copper-albumin complexes from antioxidants to
prooxidants. Arch Biochem Biophys 2003; 413: 53-66.
[116] Arthur JR, McKenzie RC, Beckett GJ. Selenium in the immune
system. J Nutr 2003; 133: 1457S-9S.
[117] Arthur JR. The glutathione peroxidases. Cell Mol Life Sci 2000; 57:
[118] May SW. Selenium-based pharmacological agents: an update.
Expert Opin Investig Drugs 2002; 11: 1261-9.
[119] Yang GQ, Wang SZ, Zhou RH, Sun SZ. Endemic selenium
intoxication of humans in China. Am J Clin Nutr 1983; 37: 872.
[120] Tinggi U. Essentiality and toxicity of selenium and its status in
Australia: a review. Toxicol Lett 2003; 137: 103-10.
[121] Casanueva E, Viteri FE. Iron and oxidative stress in pregnancy. J
Nutr 2003; 133: 1700S-08S.
[122] Ercal N, Gurer-Orhan H, Aykin-Burns N. Toxic metals and
oxidative stress part I: mechanisms involved in metal-induced
oxidative damage. Curr Top Med Chem. 2001; 1: 529-39.
[123] Maier CM, Chan PH. Role of superoxide dismutases in oxidative
damage and neurodegenerative disorders. Neuroscientist 2002; 8:
[124] Bowler RP, Crapo JD. Oxidative stress in airways: is there a role
for extracellular superoxide dismutase? Am J Respir Crit Care Med
2002; 166: S38-43.
[125] Pong K. Oxidative stress in neurodegenerative diseases: therapeutic
implications for superoxide dismutase mimetics. Expert Opin Biol
Ther 2003; 3: 127-39.
[126] Wood ZA, Schroder E, Robin Harris J, Poole LB. Structure,
mechanism and regulation of peroxiredoxins. Trends Biochem Sci
2003; 28: 32-40.
[127] Georgiou G, Masip L. Biochemistry. An overoxidation journey
with a return ticket. Science 2003; 300: 592-4.
[128] Kitani K, Minami C, Yamamoto T, Maruyama W, Kanai S, Ivy
GO, et al. Do antioxidant strategies work against aging and age-
associated disorders? Propargylamines: a possible antioxidant
strategy. Ann NY Acad Sci 2001; 928: 248-60.
[129] Ozen T, Korkmaz H. Modulatory effect of Urtica dioica L.
(Urticaceae) leaf extract on biotransformation enzyme systems,
antioxidant enzymes, lactate dehydrogenase and lipid peroxidation
in mice. Phytomedicine 2003; 10: 405-15.
[130] Nohl H, Gille L, Staniek K. The biochemical, pathophysiological,
and medical aspects of ubiquinone function. Ann NY Acad Sci
1998; 854: 394-409.
[131] Niki E. Mechanisms and dynamics of antioxidant action of
ubiquinol. Mol Aspects Med 1997; 18 Suppl: S63-70.
[132] Johnson RJ, Kang DH, Feig D, Kivlighn S, Kanellis J, Watanabe S,
et al. Is there a pathogenetic role for uric Acid in hypertension and
cardiovascular and renal disease? Hypertension. Epub 2003; 41:
[133] Hink HU, Santanam N, Dikalov S, McCann L, Nguyen AD,
Parthasarathy S, et al. Peroxidase properties of extracellular
superoxide dismutase: role of uric acid in modulating in vivo
activity. Arterioscler Thromb Vasc Biol 2002; 22: 1402–8.
[134] Ostdal H, Davies MJ, Andersen HJ. Reaction between protein
radicals and other biomolecules. Free Radic Biol Med 2002; 33:
[135] Rustin P. The use of antioxidants in Friederich’s ataxia treatment.
Expert Opin Investig Drugs 2003; 12: 569-75.
[136] Maxwell SRJ. Prospects for the use of antioxidant therapies. Drugs
1995; 49: 345-61.
[137] Elsayed NM. Antioxidant mobilization in response to oxidative
stress: a dynamic environmental-nutritional interaction. Nutrition
2001; 17: 828-34.
[138] O'Donnell VB. Free radicals and lipid signaling in endothelial cells.
Antioxid Redox Signal 2003; 5: 195-203.
[139] Maulik N, Das DK. Redox signaling in vascular angiogenesis. Free
Radic Biol Med 2002; 33(8): 1047-60.
[140] Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as
intracellular messengers during cell growth and differentiation. Cell
Physiol Biochem 2001; 11: 173-86.
[141] Palozza P, Serini S, Di Nicuolo F, Calviello G. Mitogenic and
apoptotic signaling by carotenoids: involvement of a redox
mechanism. IUBMB Life 2001; 52: 77-81.
[142] Guo Q, Packer L. Ascorbate-dependent recycling of the vitamin E
homologue Trolox by dihydrolipoate and glutathione in murine skin
homogenates. Free Radic Biol Med 2000; 29: 368-74.
[143] Chan AC, Chow CK, Chiu D. Interaction of antioxidants and their
implication in genetic anemia. Proc Soc Exp Biol Med 1999; 222:
[144] Bohm F, Edge R, Mcgarvey DJ, Truscott TG. ß-carotene with
vitamins E and C offers synergistic cell protection against NOx.
FEBS Lett 1998; 436: 387-89.
[145] Stahl W, Heinrich U, Jungmann H, Sies H, Tronnier H. Carotenoids
and carotenoids plus vitamin E protect against ultraviolet light-
induced erythema in humans. Am J Clin Nutr 2000; 71: 795-8.
[146] Manfredini S, Vertuani S, Manfredi B, Rossoni G, Calviello G,
Palaozza P. Novel antioxidant agents deriving from molecular
combinations of vitamins A and E analogues: 3,4-dihydroxy-
5(R,S)-(6-hydroxy-2,5,7,8 tetramethylchroman-2(R,S)-yl-methyl)-
1694 Current Pharmaceutical Design, 2004, Vol. 10, No. 14 Vertuani et al.
[1,3]dioxolan-4(S)-yl]-5H-furan-2-one and 3-O-octadecyl
derivatives. Bioorg Med Chem 2000; 8: 2791-801.
[147] Ninfali P, Biagiotti E, Bacchiocca M, Avanzi L, Vertuani S,
Manfredini S. Capacità antiossidante di prodotti agroalimentari e di
antiossidanti naturali o di sintesi. Industrie Alimentari 2002; 412,
[148] Geromel V, Darin N, Chretien D, Benit P, DeLonlay P, Rotig A,
Munnich A, Rustin P. Coenzyme Q(10) and idebenone in the
therapy of respiratory chain diseases: rationale and comparative
benefits. Mol Genet Metab 2002; 77: 21-30.
[149] MJ. Antioxidants and Friedreich's ataxia. Lancet 1999; 354: 1300-1.
[150] Manfredini S, Vertuani S, Avanzi L, Durini E, Palozza P, Piccioni
E, et al. Design, synthesis and activity of idebenone analogues. 12th
European Meeting on Fat Solubile Vitamins 2003, Rieti, Italy
Manuscript submitted.
[151] Abraham NG. Therapeutic applications of human heme oxygenase
gene transfer and gene therapy. Curr Pharm Design 2003; 9(30):
[152] Pag U. and Sahl HG. Multiple activities in lantibiotics--models for
the design of novel antibiotics? Curr Pharm Design 2002; 8(9):
[153] Ploug M. Structure-function relationships in the interaction
between the urokinase-type plasminogen activator and its receptor.
Curr Pharm Design 2003; 9(19): 1499-528.
... 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]. ...
Full-text available
Oxygen metabolism in the mitochondria is essential for biological activity, and reactive oxygen species (ROS) are produced simultaneously in the cell. Once an imbalance between ROS production and degradation (oxidative stress) occurs, cells are damaged. Sensory organs, especially those for hearing, are constantly exposed during daily life. Therefore, almost all mammalian species are liable to hearing loss depending on their environment. In the auditory pathway, hair cells, spiral ganglion cells, and the stria vascularis, where mitochondria are abundant, are the main targets of ROS. Excessive generation of ROS in auditory sensory organs is widely known to cause sensorineural hearing loss, and mitochondria-targeted antioxidants are candidates for treatment. This review focuses on the relationship between acquired hearing loss and antioxidant use to provide an overview of novel antioxidants, namely medicines, supplemental nutrients, and natural foods, based on clinical, animal, and cultured-cell studies.
... In addition, oxidative stress transforms 3CLpro (a key proteinase for SARS-CoV-2 replication and a significant target for antiviral drug development) into an insoluble and enzymatically active form, leading to increased viral replication/transcription, suggesting the therapeutic potential of antioxidants in the clinical treatment of COVID-19 patients [5]. Antioxidants are compounds used by the body to eliminate free radicals, which are highly reactive chemicals that introduce oxygen into the cells and cause oxidation in different cell parts, changes in the DNA, and various changes that accelerate the body's aging [6]. However, the body's antioxidant defenses are sometimes not entirely efficient, and there is an increase in the formation of free radicals, i.e., oxidative stress. ...
Full-text available
In the present research, an orange extract (OE) was obtained and encapsulated in a zein matrix for its subsequent physicochemical characterization and evaluation of its antioxidant capacity. The OE consists of phenolic compounds and flavonoids extracted from orange peel (Citrus sinensis) by ultrasound-assisted extraction (UAE). The results obtained by dynamic light scattering (DLS) and scanning electron microscopy (SEM) indicated that zein nanoparticles with orange extract (NpZOE) presented a nanometric size and spherical shape, presenting a hydrodynamic diameter of 159.26 ± 5.96 nm. Furthermore, ζ-potential evolution and Fourier transform infrared spectroscopy (FTIR) techniques were used to evaluate the interaction between zein and OE. Regarding antioxidant activity, ABTS and DPPH assays indicated no significant differences at high concentrations of orange peel extract and NpZOE; however, NpZOE was more effective at low concentrations. Although this indicates that ultrasonication as an extraction method effectively obtains the phenolic compounds present in orange peels, the nanoprecipitation method under the conditions used allowed us to obtain particles in the nanometric range with positive ζ-potential. On the other hand, the antioxidant capacity analysis indicated a high antioxidant capacity of both OE and the NpZOE. This study presents the possibility of obtaining orange extracts by ultrasound and coupling them to zein-based nanoparticulate systems to be applied as biomedical materials functionalized with antioxidant substances of pharmaceutical utility.
... A study by Aydın et al. [30] revealed that iron overload leads to a decrease in the tGSH and glutathione/oxidized glutathione ratio. Oxidative stress was suggested to first induce GSH depletion and then increase ROS in tissues [88], which is considered the result of the actions of increased antioxidant products to prevent ROS generation or to remove them [89]. We also found that HFe-induced elevation of antioxidant gene products, including HO-1, GPX4, and NQO1, participates in iron redox conversion [90,91]. ...
Full-text available
Iron supplementation is recommended during pregnancy and fetal growth. However, excess iron exposure may increase the risk of abnormal fetal development. We investigated the potential side effects of high iron levels in fetuses and through their adult life. C57BL/6J pregnant mice from 2 weeks of gestation and their offspring until 30 weeks were fed a control (CTRL, FeSO4 0 g/1 kg) or high iron (HFe, FeSO4 9.9 g/1 kg) diets. HFe group showed higher iron accumulation in the liver with increased hepcidin, reduced TfR1/2 mRNAs, and lowered ferritin heavy chain (FTH) proteins in both liver and adipose tissues despite iron loading. HFe decreased body weight, fat weight, adipocyte size, and triglyceride levels in the blood and fat, along with downregulation of lipogenesis genes, including PPARγ, C/EBPα, SREBP1c, FASN, and SCD1, and fatty acid uptake and oxidation genes, such as CD36 and PPARα. UCP2, adiponectin, and mRNA levels of antioxidant genes such as GPX4, HO-1, and NQO1 were increased in the HFe group, while total glutathione was reduced. We conclude that prolonged exposure to high iron from the fetal stage to adulthood may decrease fat accumulation by altering ferritin expression, adipocyte differentiation, and triglyceride metabolism, resulting in an alteration in normal growth.
... Damage to DNA can cause mutations and possibly cancer, if not reversed by DNA repair mechanisms (Nakabeppu, et al. 2006 andValko, et al. 2004), while damage to proteins causes enzyme inhibition, denaturation and protein degradation (Standtman, 1992). Supplementation of antioxidants in diets, drinks, drugs, etc has helped somewhat in limiting the activities of these ROS that are harmful (Vertuani, et al. 2004). The relative importance and interactions between these different antioxidants is a very complex question, with the various metabolites and enzyme systems having Because of vitamin C (L-ascorbic acid) nutritional importance, the distribution of ascorbate has been extensively quantified in plants; it has been shown to have essential role in several physiological processes in plants and animals including growth, differentiation and metabolism (Foyer, 1993). ...
Full-text available
vitamin c effect on gamma glutamyl transferase enzyme activity in paraquat intoxicated rats.
... The main function of antioxidant protein is to prevent cellular components from oxidative damages (Sies 1997;Vertuani 2004). There are many research reports available as to the significances of antioxidant proteins in protecting cellular components from oxidative damages in honeybee embryo in the younger queen with Corona et al (2005). ...
Full-text available
A honeybee (Apis mellifera L.) colony is a highly organized insect society consisting of two castes; a single queen, thousands of workers. In spite of same genetic makeup, the queen and the workers show alternative morphologies, behavior and physiology. The female queen is large in size and specializes in reproduction, while workers are small and engage in colony maintaining activities. Their life spans also vary that the queen lives for 1-2 years, while the workers only live 6-7 weeks. Existing information indicate that this alternative morphology, behavior and physiology are driven by nutritional difference at their 3.5 days old larval stage. Despite the successive investigations on the underlying causes of this honeybee caste polymorphism, information at proteome levels that considers early developmental stages (less than 3.5 days old larvae) is limited. In this study, we analyzed the caste determination mechanisms of the queen and the worker destined larvae using mitochondrial and nuclear proteomes at 72, 96 and 120 hours and through their total proteome at 48 hours developmental stages. Combinations of differential centrifugation, two dimensional electrophoresis, mass spectrometry, bioinformatics and quantitative real time PCR were applied. There were significant qualitative and quantitative protein expression differences between the two castes at three developmental stages both at mitochondria and nuclear levels. Interestingly, the queen-destined larvae upregulated large proportions of proteins at all the developmental stages from both sub-cells. In particular, the queen larvae upregulated 95% of the mitochondrial and 69% of nuclear located proteins at 72 hours. Although wide-ranging mitochondrial proteomes participate to shape the larvae metabolic, physiologic and anatomic differences between the two castes at 72 hours, physiometabolic-enriched proteins (metabolism of carbohydrate and energy, amino acid and fatty acid, protein biosynthesis) as well as protein folding were found as the major modulators of the profoundly marking of this caste differentiation. As well, the prospective queen larvae exclusively up regulated most of the nuclear enriched proteins (cytoskeleton, development and nucleic acids) that have nuclear functions to regulate DNA and RNA activities during the process of caste formations. The proteins differential expressions from both subcellular enriched were further verified by functional enrichment and biological interaction network analyses as a direct link with metabolic rates and cellular responses to hormones and DNA/RNA functions. In general, the changing mitochondrial and nuclear proteome of the two castes intended larvae indicate that the two larvae are on different trajectories as early as before 72 hours and further recommended a research works that considers the larvae age less than 72 hours old. Further research attempt of comparing the two caste intended larvae with differential protein expression at 48 hours indicate the queen intended larvae upregulated 60% of the total 47 identified proteins. This suggests that the two larvae have already on different trajectories at 48 hours. To our knowledge, these first subcellular and this early stage global proteomic data explore the innermost biological makings of honeybee society’s polymorphism and pave way to other eusocial insect caste pathway decision mechanisms. In particular, the global proteome results significantly advance the time of caste decision to 48 hours. This is a major step forward in the analysis of the fundamental causes and mechanisms of honeybee caste pathway decision and greatly contributes to the knowledge of honeybee biology. In particular, the consistency between the proteins and mRNA expressions from the subcellular proteomes provides us important target genes for the reverse genetic analysis of caste pathway modulation through RNA interference.
Microplastics (MPs), an emerging pollutant, are of global concern due to their wide distribution and large quantities. In addition to MPs themselves, various additives within MPs (such as plasticizers, flame retardants, antioxidants and heavy metals) may also have harmful effects on the environment. Most of these additives are physically bound to plastics and can therefore be leached from the plastic and released into the environment. Aging of MPs in the actual environment can affect the migration and release of additives, further increasing the ecotoxicological risk of additives to organisms. This work reviews the functions of several commonly used additives in MPs, and summarizes the representative characterization methods. Furthermore, the migration and leaching of additives in the human environment and marine environment are outlined. As aging promotes the internal chain breaking of MPs and the increase of specific surface area, it in turn stimulates the release of additives. The hazards of additive exposure have been elucidated, and various studies from the laboratory have shown that more toxic additives such as phthalates and brominated flame retardants can disrupt a variety of biological processes in organisms, including metabolism, skeletal development and so on. Increase of MPs ecological risk caused by the leaching of toxic additives is discussed, especially under the effect of aging. This study presents a systematic summary of various functional and environmental behaviors of additives in plastics, using weathering forces as the main factor, which helps to better assess the environmental impact and potential risks of MPs.
In present study, various extracts using different solvents such as petroleum ether, ethyl acetate and ethanol were subjected to in vitro antioxidant and antidiabetic activities. Ethanolic extract showed the highest activities in all antioxidant studies such as DPPH scavenging, ABTS scavenging and nitric oxide methods but it is comparatively weaker than standard drug ascorbic acid. In vitro antidiabetic activities were performed by using α-amylase and α-glucosidase inhibition in both method ethanolic extract showed highest activity, but the standard drug acarbose showed more potent than all other extracts. The highest antioxidant and antidiabetic activity of ethanolic extract may be due to the highest content phenols and flavanoids. Therefore, the ethanolic extracts contains potent antioxidant and antidiabetic compounds their isolation will help the invention of a new potent drug for the treatment diabetes and oxidative stress.
Diabetes mellitus is a metabolic disorder which is characterized based on the blood glucose level. This can be due to the lack of efficiency of utilizing insulin or lack of production of insulin. There are numerous therapies and medications which are available for the treatment of this disease which can reduce the risk of diabetes. But there is no permanent cure found. Nutritional antioxidants show a foremost role in sustaining the homeostasis of the oxidative equilibrium. They have imparted their electron donor efficacy in preventing aging and in cancer. Vitamin C, E, β-carotene, carotenoids, polyphenols and selenium have been appraised as antioxidant constituents in the human diet nourishment. This paper emphasizes on the role of antioxidants which help in reducing or maintaining the level of glucose in the body. Antioxidants are substances that reduces the damages to the cells caused by free radicals. The available treatment and medications and how the supplementation of antioxidants is different from them is also discussed. Different type of antioxidants and their treatment in curing the disease is further focused in this paper. Graphical abstract:
Full-text available
Ceruloplasmin is a ferroxidase that oxidizes toxic ferrous iron to its nontoxic ferric form. We have previously reported that a glycosylphosphatidylinositol-anchored form of ceruloplasmin is expressed in the mammalian CNS. To better understand the role of ceruloplasmin in iron homeostasis in the CNS, we generated a ceruloplasmin gene-deficient (Cp(-/-)) mouse. Adult Cp(-/-) mice showed increased iron deposition in several regions of the CNS such as the cerebellum and brainstem. Increased lipid peroxidation was also seen in some CNS regions. Cerebellar cells from neonatal Cp(-/-) mice were also more susceptible to oxidative stress in vitro. Cp(-/-) mice showed deficits in motor coordination that were associated with a loss of brainstem dopaminergic neurons. These 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. Therefore, 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.
An endemic disease was discovered in 1961 in parts of the population of Enshi County, Hubei Province of the People's Republic of China. During the years of the highest prevalence, from 1961 to 1964, the morbidity was almost 50% in the 248 inhabitants of the five most heavily affected villages; its cause was determined to be selenium intoxication. The most common sign of the poisoning was loss of hair and nails. In areas of high incidence, lesions of the skin, nervous system, and possibly teeth may have been involved. A case is reported of a middle-aged, female hemiplegic, whose illness and death apparently were related to selenosis. Daily dietary intakes of selenium, estimated after the peak prevalence had subsided, averaged 4.99 (range 3.20 to 6.69) mg and hair and blood selenium levels averaged 32.2 and 3.2 µg/ml, respectively. Up to l000× differences occurred when selenium contents of vegetables, cereals, scalp hair, blood, and urine from the selenosis areas were compared with those from Keshan disease (selenium deficiency) areas. The ultimate environmental source of selenium was a stony coal of very high selenium content (average more than 300 µg/g; one sample exceeded 80,000 µg/g). Selenium from the coal entered the soil by weathering and was available for uptake by crops because of the traditional use of lime as fertilizer in that region. This particular outbreak of human selenosis was due to a drought that caused failure of the rice crop, forcing the villagers to eat more high-selenium vegetables and maize and fewer protein foods.
Conference Paper
Selenium as an essential component of selenocysteine-containing protein is involved in most aspects of cell biochemistry and function. As such, there is much potential for selenium to influence the immune system. For example, the antioxidant glutathione peroxidases are likely to protect neutrophils from oxygen-derived radicals that are produced to kill ingested foreign organisms. When the functions of all selenoproteins are described, only then will it be possible to fully understand their role in maintaining optimal immune function.
In the redox antioxidant network, dihydrolipoate can synergistically enhance the ascorbate-dependent recycling of vitamin E. Since the major endogenous thiol antioxidant in biological systems is glutathione (GSH) it was of interest to compare the effects of dihydrolipoate with GSH on ascorbate-dependent recycling of the water-soluble homologue of vitamin E, Trolox, by electron spin resonance (ESR). Trolox phenoxyl radicals were generated by a horseradish peroxidase (HRP)–hydrogen peroxide (H2O2) oxidation system. In the presence of dihydrolipoate, Trolox radicals were suppressed until both dihydrolipoate and endogenous levels of ascorbate in skin homogenates were consumed. Similar experiments made in the presence of GSH revealed that Trolox radicals reappeared immediately after ascorbate was depleted and that GSH was not able to drive the ascorbate-dependent Trolox recycling reaction. However, at higher concentrations GSH was able to increase ascorbate-mediated Trolox regeneration from the Trolox radical. ESR and spectrophotometric measurements demonstrated the ability of dihydrolipoate or GSH to react with dehydroascorbate, the two-electron oxidation product of ascorbate in this system. Dihydrolipoate regenerated greater amounts of ascorbate at a much faster rate than equivalent concentrations of GSH. Thus the marked difference between the rate and efficiency of ascorbate generation by dihydrolipoate as compared with GSH appears to account for the different kinetics by which these thiol antioxidants influence ascorbate-dependent Trolox recycling.
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.
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.
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.