Oxidative stress


1.1 Introduction

Oxidative stress is a state of the body in which there is an excess of free radicals present. A free radical is a chemical species in an unstable state because of the fact that it has an electron missing. To gain stability, this chemical species takes an electron from a nearby molecule. This can occur within any part of the cellular structure, ex. the cell membrane or the nucleus. Because the second molecule has been rendered unstable, it needs to regain its stability by taking electrons from another molecule. A domino effect of electron stealing is created. Without mediation this process may lead to the destruction of cellular structures . Free radicals are produced naturally throughout the body as by-products of cellular metabolic processes. Therefore our cells are able to protect themselves against damage by ROS and other radicals through repair processes, compartmentalization of free radical production, defence enzymes, and endogenous and exogenous antioxidants (free radical scavengers). However an issue arises when there is an excess of these free radical and the cells are not able to keep up with the removal of these radicals. This is called oxidative stress overload. Examples of free radicals include the Reactive Oxygen Species (ROS), such as Hydrogen Peroxide (H2O2), the Superoxide radical (O2-.), the hydroxyl radical (OH.) and Nitric Oxide (NO). (Lieberman et al, 2013)

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Factors which may cause the body to have an imbalanced load of oxidative stress include smoking, excessive exercise, a poor diet and radiation. If the continuous domino effect of stealing electrons were to continue with no intervention, cells would eventually die, leading to organ failure. Oxidative stress can also lead to chronic inflammation response and an inappropriate up regulation of the immune system both of which lead to chronic diseases such as coronary artery disease, allergies, cancer, diabetes and autoimmune disorders. Two behaviours that studies show are beneficial to reducing oxidative stress are a healthy diet and moderate exercise with the end result of both being an increase in antioxidants. Antioxidants are molecules which are able to donate electrons without themselves becoming unstable. Fruits and vegetables are a very rich source of antioxidants. (Sies, 1997)

1.2 The Radical Nature of Oxygen

The Oxygen molecule is both beneficial and harmful to the human body. It is needed for oxidation reactions in the pathways of adenosine triphosphate (ATP) and for detoxification. In some instances, O2 accepts single electrons to form reactive oxygen species which cause damage to cells. The generation of these ROS is a daily occurrence in our body. These ROS are formed as by-products of enzymatic or non-enzymatic reactions. (Lieberman et al, 2013)

A free radical may be defined as a species which contains a single unpaired electron in an orbital and is capable of independent existence. Radicals are highly reactive and are able to trigger chain reactions by extracting an electron from a neighbouring particle to complete their own orbital. (Halliwell and Gutteridge, 1989)

The O2 molecule is a biradical, i.e. it possesses two single electrons in different orbitals. Due to the fact that they have parallel spins, these electrons are not able to both travel in the same orbital. The single electrons of O2 cannot react rapidly with the electron pairs found in the covalent bonds of organic molecules and as a result O2 reacts slowly by accepting single electrons in reactions which require a catalyst to occur.

The two unpaired electrons of O2 which have the same parallel spin are called antibonding electrons. In contrast, C-H and C-C bonds both contain two electrons, each having antiparallel spins, and thus are able to form a thermodynamically stable pair. This results in O2 not being able to oxidize a covalent bond because one of its electrons would need to flip its spin around to make new pairs. This restriction in spin changing is called spin restriction. (Lieberman et al, 2013)

1.3 Characteristics of Reactive Oxygen Species

Reactive Oxygen Species (ROS) are O2 containing compounds which are highly reactive free radicals or compounds which are readily converted to these oxygen free radicals intracellularly. (Lieberman et al, 2013)

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These various radical species may be generated exogenously or formed within the cell from many different sources. Most of the intracellular ROS are formed within the mitochondria. The generation of these ROS occurs mainly at two points in the ETC, Complex I and Complex III. (Bovina et al, 1999)

Some of these species are extremely unstable with a very short half-lives whilst others are freely diffusable and have a relatively long half-life. It is this difference in half-lives which results in a great need of different types of defence mechanisms within the cells. (Sies, 1997)

1.4 Oxidants

The most important and biologically significant free radicals found in the body are the radical derivatives of the O2 molecule. A list of these free radicals together with a summary of their properties can be found in Table 1.

Table 1: Reactive Oxygen Species and Reactive Nitrogen Oxygen Species

Reactive Species


Superoxide anion (O2-.)

Produced by the electron transport chain and various other sites. Cannot diffuse far from the site of origin. Generates other Reactive Oxygen Species.

Hydrogen Peroxide (H2O2)

Not a free radical but is able to generate free radicals by reaction with a transition metal. Can diffuse through the cell membrane.

Hydroxyl Radical (OH.)

The most reactive species in attacking biologic molecules. Produced from (H2O2) in the Fenton reaction in the presence of Cu+ or Fe2+.

Hypochlorous Acid (HOCl)

Produced in neutrophils during the respiratory burst to destroy pathogens. Causes halogenation and oxidation reactions. Attacking species is OCl-.

Nitric Oxide (NO)

A free radical produced endogenously by nitric oxide synthase. Binds to metal ions. Combines with O2 or other oxygen containing radicals to produce additional Reactive Nitrogen-Oxygen Species.

Lieberman et al., 2013

1.4.1 Superoxide Radical

The first molecular species formed in the univalent pathway of oxygen reduction is the superoxide radical and it is most commonly generated in the mitochondrial ETC by the autoxidation of a semiquinone species. (Sun and Trumpower, 2003)

It is produced by all aerobic cells in the body, predominantly the macrophages, monocytes, neutrophils and eosinophils. The superoxide produced is utilised to eradicate any invading pathogens in a process called oxygen dependant killing.

The superoxide radical is formed when there is reduction of O2 with an electron being transferred to its outer shell.

O2 + e- "" O2-

It is thought to play a very vital role in the formation of other reactive intermediates. This is due to the fact that it is a main source for the generation of H2O2 and acts as a reducant of transition metals which play a role in the formation of the hydroxyl radical through the Fenton reaction. (Azzi et al, 1974)

1.4.2 Hydrogen peroxide

Hydrogen peroxide is a weak oxidizing agent which has all its electrons paired and is thus not considered a free radical. However it still falls under the category of a free radical since it is the main source of hydroxyl radicals in the presence of transition metal ions like Fe3+ . It is also responsible for the production of HOCl by neutrophils. The formation of H2O2 occurs by the two electron reduction of oxygen. In the human body, two superoxide molecule contribute to the generation of H2O2 and H2O. (Augusto and Miyamoto, 2011)

2 O2- + 2 H+ "" H2O2 + O2

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This reaction is termed a dismutation reaction because of the fact that the radicals reacting together give rise to non-radical products.

1.4.3 Hydroxyl Radical

The hydroxyl radical is a highly reactive oxidising radical and is the primary cause of free radical induced cellular damage. Hydroxyl radical formation mainly occurs by decomposition of superoxide and hydrogen peroxide, catalysed by transition metals such as copper and iron. (Woodside and Young, 2001)

The Fenton reaction devised by Henry Fenton shows how a transition metal such as iron, in the free state, can act as a catalyst to generate the hydroxyl radical.

Under normal conditions, the amount of free iron available in the body is very limited in order to avoid this reaction. (Vercellotti, 1996)

O2-. + Fe3+ "" Fe2+ + O2

H2O2 + Fe2+ "" Fe3+ + OH- + O.

Nitric Oxide

Nitric Oxide is a gaseous free radical which plays a vital part in vascular physiology and is referred to as endothelium derived relaxing factor. In the body, NO plays the role of a vasodilator, a neurotransmitter and an antithrombic and anti-inflammatory agent.

It is generated by neutrophils, macrophages and the vascular endothelium in an reaction catalysed by the enzyme nitric oxide synthetase. Its generation may be further stimulated by interleukins, cytokines and tumour necrosis factor. (Darley-Usmar et al, 1995).

Weakening of the tumouricidal and microbicidial actions of macrophages may result from the inhibiton of the generation of nitric oxide.

Hypochlorous Acid

HClO is another non-radical species which


Certain drugs play a role in the generation of ROS in the presence of high oxygen tensions. Examples of these types of drugs include non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, cancer drugs and anti-viral drugs as can be seen in Table 2. (Deaval et al, 2012)

Free radicals generated from such drugs may promote ROS generation and also interfere with the body's natural defence mechanisms against such compounds. In addition, ROS generated from these drugs may reduce ascorbic acid levels in the body leading to an increase in lipid peroxidation and cause the inactivation of the protease enzymes. (Bondy, 1992)


Exposure of the cells to radiation may result in oxidizing events and the formation of radicals which is caused by the transfer of energy to cellular components such as H2O resulting in bond breakage. Furthermore, the oxidative damage induced may also spread from the affected cells to neighbouring cells via intercellular communication mechanisms. (Azzam et al, 2012)

Examples of ionising radiation include cosmic rays, X-rays and radioactive chemicals.

Precautions must be taken in the practice of radiotherapy since this may lead to cellular injury which is caused by the formation of free radicals. (Beena et al, 2009)


The process of autoixdation is a result of the aerobic internal milieu. Examples of molecules

that are capable of autoxidation include thiol, Hb, reduced cytochrome C, catecholamines

and myoglobin, resulting in the formation of the oxygen diradical and the formation of ROS,

the most common being the superoxide radical. In certain cases, there is oxidation of ferrous

iron (Fe2+), resulting in the formation of superoxide and ferric iron (Fe3+).

Enzymatic Oxidation

A variety of enzyme systems is capable of generating significant amounts of free radicals, including xanthine oxidase (activated in ischemia-reperfusion), prostaglandin synthase, lipoxygenase, aldehyde oxidase, and amino acid oxidase. The enzyme myeloperoxidase produced in activated neutrophils, utilizes hydrogen peroxide to oxidize chloride ions into the powerful oxidant hypochlorous acid (HOCl) (Halliwell et al. 1995)

Respiratory Burst

Is a term used to describe the process by which phagocytic cells consume large amounts of oxygen during phagocytosis. Between 70 and 90% of this oxygen consumption can be accounted for in terms of superoxide production (Baboir BM; 1984). These phagocytic cells possess a membrane bound flavoprotein cytochrome-b-245 NADPH oxidase system. Cell membrane enzymes such as the NADPH-oxidase exist in an inactive form. It is the exposures to immunoglobulin-coated bacteria, immune complexes, complement 5a, or leukotriene, however, which activate the enzyme NADPH-oxidase. This activation initiates a respiratory burst at the cell membrane to produce superoxide (Baboir BM, 1978). H2O2is then formed from superoxide by dismutation with subsequent generation of OH and HOCl by bacteria (Rosen H, Rikata R, Waltersdorph AM, Klebanoff S; 1987).

Transition metal ions

Iron and copper play a major role in the generation of free radicals injury and the facilitation of lipid peroxidation. Transition metal ions participate in the Haber-Weiss reaction that generates ?OH from O2?⁻and H2O2.

H2O2+ Fe2⁺?OH + OH⁻+ Fe3⁺

The Haber-Weiss reaction accelerates the nonenzymatic oxidation of molecules such as epinephrine and glutathione that generates O2?⁻and H2O2and subsequently ?OH.

Subcellular organelles

Organelles such as mitochondria, chloroplasts, microsomes, peroxisomes and nuclei have been shown to generate O2?⁻and this is easily demonstrated after the endogenous superoxide dismutase has been washed away (Asada and Kiso, 1973). Mitochondria are the main cellular organelle for cellular oxidation reactions and the main source of reduced oxygen species in the cell. The leaks in mitochondrial electron transport system allow O2to accept a single electron forming O2?⁻(Kalra et al. 1994; Haliwell, 1995). It has been shown that superoxide production by the mitochondria increases in two conditions; either when the oxygen concentration is greatly increased or when the respiratory chain becomes fully reduced (as happens during ischemia).

Microsomes are responsible for 80% of the H2O2produced in vivo at 100% hyperoxia sites (Jamieson et al. 1986). Peroxisomes are known to produce H2O2, but not O2?⁻, under physiologic conditions (Chance et al. 1979). Although the liver is the primary organ where peroxisomal contribution to the overall H2O2production is significant, other organs that contain peroxisomes are also exposed to these H2O2-generating mechanisms. Peroxisomal oxidation of fatty acids has recently been recognized as a potentially important source of H2O2production with prolonged starvation.



The cell's defence system against ROS falls under the category of antioxidant enzymes,

The antioxidant enzymes which confer protection against ROS include:

  • Superoxide Dismutase (SOD): converts O2-. to H2O2 and O2
  • Catalase: converts H2O2 to H2O and O2
  • Glutathione Peroxidase : reduces H2O2 to H2O

Superoxide Dismutase is an endogenous enzyme present within all cells in the body. It is a metalloenzyme with a very important anti-oxidant role in human health, conferred by its ability to scavenge the superoxide anion. SODs are the primary defence in the detoxification of products resulting from oxidative stress. The reaction which it catalyses may be represented as follows:

2 O2 -. + 2 H+ + SOD "" H2O2 + O2

Three isoenzyme forms of SOD exist in nature. SOD1 Cu-Zn-SOD is a copper and zinc containing homodimer and is found in intracellular cytoplasmic spaces. SOD2/Mn-SOD exists as a tetramer and is found exclusively in the mitochondrial spaces. SOD3/EC-SOD exists as a copper and zinc containing tetramer and is found exclusively in the extracellular fluid.

An enzyme which is capable of reducing hydrogen peroxide is catalase. Once hydrogen peroxide is formed it must be immediately reduced to water to prevent it from forming the hydroxyl radical in the Fenton or the Haber-Weiss reaction.

H2O2 "" 2 H2O + O2

This enzyme is found mainly in peroxisomes and to a lesser extent in microsomes and the cytoplasm of a cell. The kidney, liver and erythrocytes are examples of tissues in which there is a high concentration of this enzyme present while in tissues such as the brain, heart and skeletal muscle, it is found in lower concentrations.


Vitamin E (α-tocopherol) is the most widely distributed antioxidant in nature which mainly acts to protects against lipid peroxidation in membranes. Vitamin E consists of several tocopherols which differ in their pattern of methylation. Among these, α-tocopherol is present in the largest amounts in our diets and is the most potent antioxidant.

Vitamin E is an efficient antioxidant and nonenzymatic terminator of free radical

chain reactions, and it has little pro-oxidant activity. When vitamin E donates

an electron to a lipid peroxy radical, it is converted to a free radical form that is

stabilized by resonance. If this free radical form were to act as a pro-oxidant and

abstract an electron from a polyunsaturated lipid, it would be oxidizing that lipid

and actually propagate the free radical chain reaction. The chemistry of vitamin E

is such that it has a much greater tendency to donate a second electron and go to

the fully oxidized form

Glutathione (_-glutamylcysteinylglycine) is one of the body’s principal means of

protecting against oxidative damage (see also Chapter 29). Glutathione is a tripeptide

composed of glutamate, cysteine, and glycine, with the amino group of cysteine

joined in peptide linkage to the _-carboxyl group of glutamate (Fig. 24.15).

In reactions that are catalyzed by glutathione peroxidases, the reactive sulfhydryl

groups reduce hydrogen peroxide to water and lipid peroxides to nontoxic Glutathione (_-glutamylcysteinylglycine) is one of the body’s principal means of

protecting against oxidative damage (see also Chapter 29). Glutathione is a tripeptide

composed of glutamate, cysteine, and glycine, with the amino group of cysteine

joined in peptide linkage to the _-carboxyl group of glutamate (Fig. 24.15).

In reactions that are catalyzed by glutathione peroxidases, the reactive sulfhydryl

groups reduce hydrogen peroxide to water and lipid peroxides to nontoxic

alcohols. In these reactions, two glutathione molecules are oxidized to form a

single molecule, glutathione disulfide. The sulfhydryl groups are also oxidized in

nonenzymatic chain-terminating reactions with organic radicals.

Glutathione peroxidases exist as a family of selenium enzymes with somewhat

different properties and tissue locations. Within cells, they are found principally in

the cytosol and mitochondria and are the major means for removing H2O2 produced

outside of peroxisomes. They contribute to our dietary requirement for selenium and

account for the protective effect of selenium in the prevention of free radical injury.

Once oxidized glutathione (GSSG) is formed, it must be reduced back to the

sulfhydryl form by glutathione reductase in a redox cycle (see Fig. 24.15C).

Glutathione reductase contains a flavin adenine dinucleotide (FAD) and catalyzes

transfer of electrons from NADPH to the disulfide bond of GSSG. NADPH is thus

essential for protection against free radical injury.