Oxidative stress may be defined as an
imbalance between pro-oxidant and antioxidant agents, in
favour of the former (Sies, 1986); this imbalance may be due
to an excess of pro-oxidant agents, a deficiency of
antioxidant agents or both factors simultaneously. The
origin of oxidative stress is an alteration of the redox
status in cells, leading to a cellular response to
counteract the oxidising action (Sies, 1986).
Pro-oxidant agents are all those that can
directly or indirectly oxidise molecules.
The most important pro-oxidant agents in
biological systems are those derived from oxygen, more
commonly known as reactive oxygen species (ROS), although
there are also reactive species derived from nitrogen (RNS)
or sulphur (RSS) (Halliwell, 1994). Some of these molecules
exhibit great reactivity, such as hydroxyl radicals (HO.),
and others present mild reactivity per se. The
biological importance of the latter relies on their capacity
to be easily transformed into the hydroxyl radical,
especially in the presence of iron (Fenton, 1894), as in the
case of superoxide radicals (O2.) or
There are free radicals within these groups
of reactive species. A free radical is a chemical species
with an unpaired electron and it is symbolised by a dot (.).
The presence of an unpaired electron in an atom or molecule
provides great reactivity, thus shortening its half life (Simic
and Taylor, 1988).
The production of these reactive species
occurs continuously in the organism; this production may be
endogenous or exogenous (Freeman and Crapo, 1982; Frei,
1994). Some of these reactive species are generated as
“chemical accidents”, i.e. undesired secondary reactions
between biomolecules or alternatively in the detoxification
of xenobiotics. Other reactive species, however, are
generated in vivo for a specific aim such as in the
case of activated phagocytes which produce O2.
and H2O2 (Halliwell, 1996).
The production of these reactive species via
exogenous sources is due to xenobiotics or the ionizing
action of radiation or situations such as hyperoxia.
Amongst the very varied endogenous sources, it is worth
mentioning the following main production points of free
radicals and reactive species:
mitochondrial electronic transport chain, in which
considerable quantities of
radical superoxide (O2.) and hydrogen
peroxide (H2O2) (Cadenas, et al., 1977) are
The enzymatic system of hypoxanthine/xanthine
oxidase, especially in ischemia reperfusion (Radi, et al.,
The electronic transport systems of the
endoplasmic reticulum, which contain cytochromes P450 and b5
that can oxidise xenobiotics and unsaturated fatty acids
(Dolphin, 1988; Foster and Estabrook, 1993).
Activated fagocytes (Babior, 1978).
Peroxisomes or microsomes. These organules
participate in fatty acid oxidation and contain
peroxide-producing enzymes. Moreover, they also contain
cytochrome P450. Catalase is also a peroxisomal enzyme
which metabolises the hydrogen peroxide formed in these
Many cytosolic enzymes are bound to membrane
(aldehyde oxidase, nitric oxide synthase, cyclooxigenase,
lipooxigenase). They may produce free radicals and other
reactive oxygen species.
Iron, which in the oxidation state (II)
promotes that both superoxide and hydrogen peroxide give
rise to the hydroxyl radical by the Fenton and Haber Weiss
Direct oxidation of molecules by oxygen. In
the organism, many molecules react directly with oxygen
mainly giving rise to the superoxide radical.
These free radicals and other activated
oxygen species are continuously formed in our body and on
top of their physiological function they may also be
damaging to the cellular integrity due to its high
reactivity. They react with all the present biomolecules
and they affect their normal function. Thus, living
organisms have developed a number of defence mechanisms
known as the “antioxidant defence system”. The action of
these systems is multifactorial. In the first instance,
they try to prevent the production of reactive oxygen
species. On a second level, they try to reduce these
molecules, and on a third level the repair the damage caused
by such molecules. These defence mechanisms may be
organised in the following way:
Non-enzymic system: Molecules that can react
directly with reactive oxygen species and other free
radicals, or with the products of these reactions without
the involvement of any special enzyme. These antioxidants
include glutathione, vitamin C, vitamin E, betacarotenes,
uric acid and the flavonoids.
These include catalase, superoxide dismutases and
Moreover, in the antioxidant defence,
compartmentalisation is specially important, particularly
that of glutathione.
Glutathione is the most abundant non-protein
thiol in mammalian cells. It is present mainly in the
reduced form (GSH). The oxidised form (GSSG) is less than
10% of the reduced one. GSSG is a dymer of two reduced
glutathione molecules bound by a disulphide bond.
Glutathione carries out an important number of metabolic
functions and one of the most important is protection of
cells against oxidants and other xenobiotics. Glutathione
is synthesised and degraded by the gamma glutamyl cycle and
the liver is the major organ where this peptide is
The antioxidant action can be exercised in
Due to its thiol group. Reduced glutathione
may take one electron from a free radical and be converted
into the glutathionil radical. Then two radicals of the
glutathonil radical may be bound together giving rise to a
the glutathione redox cycle. In this case, glutathione
reacts with free radicals and reactive oxygen species
through a reaction catalysed by glutathione peroxidase and
thus glutathione is oxidised (GSSG). In this manner,
through glutathione peroxidase glutathione is oxidised and
it will eventually be reduced by glutathione reductase using
NADPH as reducing equivalents. This is one of the most
effective mechanisms against oxidative stress.
The degree of oxidative damage suffered by an
organism, tissue or organ may be evaluated by the
measurement of a number of molecules which are indexes of
The attack of radicals on membrane lipids
gives rise to lipid peroxidation. This lipid damage leads
to a loss in membrane fluidity. Malondialdehyde,
4-hydroxynonenal, pentane or ethane may be indexes of lipid
The attack of these reactive oxygen species
on DNA causes oxidation of DNA bases. This induces
mutagenic phenomena and thus carcinogenesis. The most
frequent indicator of oxidative damage to DNA is
8-hydroxy-2’-deoxyguanosine (oxo-dG) which rises from the
oxidative attack to deoxyguanosine.
Carbohydrates and proteins also suffer an
oxidative attack. Carbonyl groups in proteins are excellent
indicators of oxidative damage to these molecules.
One of the most interesting parameters to
determine oxidative stress is the glutathione redox status.
Indeed, a glutathione redox ratio (GSH-GSSG) gives us an
indication of the redox state of the cells and thus
indicates a global level of oxidation of the whole organism.