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 hydrogen peroxide(H₂O₂).

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:

1. The mitochondrial electronic transport chain, in which considerable quantities of radical superoxide (O₂^.) and hydrogen peroxide (H₂O2) (Cadenas, et al., 1977) are formed.

2. The enzymatic system of hypoxanthine/xanthine oxidase, especially in ischemia reperfusion (Radi, et al., 1992).

3. 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).

4. Activated fagocytes (Babior, 1978).

5. 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 organelles.

6. Many cytosolic enzymes are bound to membrane (aldehyde oxidase, nitric oxide synthase, cyclooxigenase, lipooxigenase).  They may produce free radicals and other reactive oxygen species.

7. 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 reaction.

8. 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:

1. 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.

2. Enzymes: These include catalase, superoxide dismutases and glutathione peroxidases.

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 synthesised.

The antioxidant action can be exercised in two ways:

1. 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 GSSG molecule.

2. Due to 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 oxidative stress.

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 peroxidation.

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.