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International Journal of Research in Pharmacy and Biosciences
Volume 3, Issue 6, 2016, PP 1-8
ISSN 2394-5885 (Print) & ISSN 2394-5893 (Online)
The Role of Reactive Oxygen Species and Antioxidants in
Oxidative Stress
Ozougwu, Jevas C. Ph.D
Physiology and Biomedical Research Unit, Department of Biological Sciences, College of Basic and Applied
Sciences, Rhema University Aba, Abia State, Nigeria
ABSTRACT
Under normal conditions, the physiologically important intracellular levels of reactive oxygen species (ROS) are
maintained at low levels by various antioxidant systems in the body but at high concentrations, they produce
adverse modifications to cell components such as lipids, proteins and DNA. In humans, oxidative stress is
involved in the development of cancer, parkinson's disease, alzheimer's disease, atherosclerosis, heart failure,
myocardial infarction, fragile X syndrome, sickle cell disease, lichen planus, vitiligo, autism, infection, chronic
fatigue syndrome and depression. However, reactive oxygen species can be beneficial, as they are used by the
immune system as a way to attack and kill pathogens, also short-term oxidative stress is important in the
prevention of aging by induction of mitohormesis. ROS can be from endogenous or exogenous sources and they
are produced from molecular oxygen as a result of normal cellular metabolism, they are divided into free radicals
and non-radicals. The major endogenous sources of ROS of physiological importance are superoxide anion,
hydroxyl radical, hydrogen peroxide, hypochlorous acid, peroxyl radical and hydroperoxyl radical while the
exogenous sources of ROS include cigarette smoke, ozone exposure, hyperoxia, ionizing radiation and heavy
metal ions. Aerobic organisms have integrated antioxidant systems, which include enzymatic and non-enzymatic
antioxidant that are usually effective in blocking harmful effects of reactive oxygen species. Enzymatic defense
is a system of enzymes which includes glutathione peroxidases, superoxide dismutases and catalase, which
decrease the concentration of the most harmful ROS whereas non-enzymatic antioxidants include low molecular
weight compounds such as vitamins C and E, β-carotene, uric acid and glutathione. Humans have a complex
antioxidant protection system, which functions interactively and synergistically to neutralize free radicals and
knowledge as presented in this review will be important in the management of oxidative stress and its associated
diseases.
Keywords: Role, Oxidative stress, Antioxidant, ROS
INTRODUCTION
Oxidative stress is an imbalance between the systemic manifestation of reactive oxygen species (ROS)
and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting
damage. Reactive oxygen species are produced by living organisms as a result of normal cellular
metabolism [1]. At low to moderate concentrations they function in physiological cell processes, but at
high concentrations, they produce adverse modifications to cell components such as lipids, proteins
and DNA [2, 3, 4]. Oxidative stress from oxidative metabolism causes base damage, as well as strand
breaks in DNA. Base damage is mostly indirect and caused by reactive oxygen species generated,
examples O2− (superoxide radical), OH (hydroxyl radical) and H2O2 (hydrogen peroxide). Some
reactive oxidative species act as cellular messengers in redox signaling. Thus, oxidative stress can
cause disruptions in normal mechanisms of cellular signaling. In humans, oxidative stress is thought to
be involved in the development of cancer, Parkinson's disease, Alzheimer's disease, [5, 6]
atherosclerosis, heart failure,[7] myocardial infarction, [8,9] fragile X syndrome, [10] Sickle Cell
Disease, [11] lichen planus, [12] vitiligo, [13] autism, [14] infection, [15] Chronic fatigue syndrome,
[16] and Depression. However, reactive oxygen species can be beneficial, as they are used by the
immune system as a way to attack and kill pathogens [17]. Short-term oxidative stress may also be
important in prevention of aging by induction of mitohormesis [18]. Aerobic organisms have
integrated antioxidant systems, which include enzymatic and non-enzymatic antioxidant that are
usually effective in blocking harmful effects of reactive oxygen species. However, in pathological
conditions, the antioxidant systems are overwhelmed by reactive oxygen species. In view of the central
role of oxidative stress in aging and disease progression, the critical role of antioxidant in maintaining
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Ozougwu, Jevas C. “The Role of Reactive Oxygen Species and Antioxidants in Oxidative Stress”
optimum health and well-being and the increased exposure to free radicals such as pollution, cigarette
smoke, drug, stress, exercise etc this review becomes necessary to x-ray the role of ROS and
antioxidant in oxidative stress.
ROLE OF ROS IN OXIDATIVE STRESS
Pro-oxidants are chemicals that induce oxidative stress, either by generating reactive oxygen species or
by inhibiting antioxidant systems [19]. Oxidative stress produced by these chemicals can damage cells
and tissues, for example an overdose of paracetamol (acetaminophen) damaged the liver of
experimental rats, partly through its production of reactive oxygen species [20, 21]. Some of the
conditions that make a substance serve as pro-oxidants are the concentration of the chemical and if
oxygen or transition metals are present. Reactive oxygen species are produced from molecular oxygen
as a result of normal cellular metabolism. They are divided into free radicals and non-radicals (Table
1). Molecules containing one or more unpaired electrons and thus giving reactivity to the molecule are
called free radicals. When two free radicals share their unpaired electrons, non-radical forms are
created. ROS can be from endogenous or exogenous sources. The major endogenous sources of ROS
of physiological importance are superoxide anion, hydroxyl radical, hydrogen peroxide, hypochlorous
acid, peroxyl radical and hydroperoxyl radical (Table 1). Superoxide anion is formed by the addition
of one electron to the molecular oxygen [22]. Hydrogen peroxide is produced by xanthine oxidase,
amino acid oxidase and NAD(P)H oxidase [23, 24] and in peroxisomes by consumption of molecular
oxygen in metabolic reactions. Hydroxyl radical is the most reactive of reactive oxygen species and
can damage proteins, lipids, carbohydrate and DNA. It can also start lipid peroxidation by taking an
electron from polyunsaturated fatty acids [1]. Hypochlorous acid (HOCl) is highly oxidative and plays
an important role in killing of the pathogens in the airways [25]. HOCl can also react with DNA and
induce DNA protein interactions and produce pyrimidine oxidation products and add chloride to DNA
bases [26, 27]. Other oxygen-derived free radicals are the peroxyl radicals (ROO-), the simplest form
of these radicals is hydroperoxyl radical (HOO-) and has a role in fatty acid peroxidation. Free radicals
can trigger lipid peroxidation chain reactions by abstracting a hydrogen atom from a side-chain
methylene carbon. The lipid radical then reacts with oxygen to produce peroxyl radical. Peroxyl
radical initiates a chain reaction and transforms polyunsaturated fatty acids into lipid hydroperoxides.
Lipid hydroperoxide are very unstable and easily decompose to secondary products such as aldehydes
and malondialdehydes. Peroxidation of lipids disturbs the integrity of cell membranes and lead to
rearrangement of membrane structure. Exogenous sources of ROS includes cigarette smoke, ozone
exposure, hyperoxia, ionizing radiation and heavy metal ions. Free radicals are electrically charged
molecules that are unpaired electron, which causes them to seek out and capture electrons from other
substances in order to neutralize themselves. The initial attack of free radicals cause the free radical to
become neutralized, another free radical is formed in the process causing a chain reaction to occur
[28], which continues until subsequent free radicals are deactivated, thousands of free radical reactions
can occur within seconds of the initial reaction.
Table1. Some Important Reactive Oxygen Species in Living Organisms
FREE RADICALS
Hydroxyl radical
Superoxide radical
Nitric oxide radical
Lipid peroxyl radical
Hydroperoxyl radical
NON RADICALS
Hydrogen peroxide
Singlet oxygen
Hypochlorous acid
ozone
OH+
O2+
NO+
LOO+
HOO
H2O2
1O2
HOCl
O3
All ROS are capable of reacting with membrane lipids, nucleic acids, proteins and enzymes and other
small molecules resulting in cellular damage [28]. Reactive oxygen species are produced continuously
in the human body as a consequence of normal metabolic processes. If free radicals are not inactivated,
their chemical level can damage all types of cellular macromolecules, including proteins,
carbohydrates, lipids and nucleic acids [29]. Free radicals and other reactive oxygen species in the
human body are derived either from normal internal metabolic processes or from external sources as
shown in Table 3.
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Table3. Some Sources of Free Radicals
Internally Generated Sources
Mitochondria
Phagocytes
Xanthine oxidase
Reactions involving iron and other transition metals
Arachidonate pathways
Peroxisomes
Exercise
Inflammation
Ischemia/reperfusion
Externally Generated Sources
Cigarette smoke
Environmental pollutants
Ionizing radiation
Ultraviolet light
Certain drugs, pesticides, anaesthetics and industrial solvent
Ozone exposure
Heavy metal ions
Source: Langseth (1995).
Free radicals are not always harmful, they serve useful purposes in the human body. For example, free
radicals play a role in the destruction of disease-causing micro-organisms by specialized blood cells
called phagocytes [29]. Reactive oxygen species are generated by a number of pathways. [28]
reported that most of the ROS produced by cells occur as:
i A consequence of normal aerobic metabolism and approximately 90% of the oxygen utilized by the
cell is consumed by the mitochondrial electron transport system.
ii Oxidative burst from phagocytes (white blood cells) as part of the mechanism by which bacteria
and viruses are killed and by which foreign proteins (antigens) are denatured.
iii Xenobiotic metabolism, that is, detoxification of toxic substances. Consequently, vigorous exercise,
which accelerates cellular metabolism, chronic inflammation, infections and other illnesses;
exposure to allergens and the presence of “leaky gut” syndrome and exposure to drugs or toxins
such as cigarette smoke, pollution, pesticides and insecticides may all contribute to an increase in
the body’s oxidant load.
ROLE OF ANTIOXIDANTS IN OXIDATIVE STRESS
Antioxidants are substances which can delay or inhibit oxidation. Cell damage caused by free radicals
is the major contributor to aging and to degenerative diseases of aging such as cancer, cardiovascular
disease, cataracts, immune system decline and brain dysfunction [30]. Free radicals have been
implicated in the pathogenesis of at least 50 diseases [31]. Fortunately, free radical formation is
controlled naturally by various beneficial compounds known as antioxidant. It is when the availability
of antioxidant becomes limited that damage caused by ROS can become cumulative and debilitating.
Antioxidant are capable of stabilizing or deactivating free radicals before they attack cells which make
them absolutely critical for maintaining optimal cellular and systemic health and well-being [28]. The
human body has several mechanisms for defense against free radicals and other reactive oxygen
species. The various defenses are complementary to one another because they act on different ROS or
in different cellular compartments [29]. These defenses can be grouped into enzymatic and nonenzymatic antioxidant. Enzymatic defense is a system of enzymes which includes glutathione
peroxidases, superoxide dismutases and catalase, which decrease the concentration of the most
harmful ROS. Superoxide dismutases are a family of antioxidant enzymes which are important in the
catalytic decomposition of the superoxide radical to hydrogen peroxide and oxygen [29]. Catalase
specifically catalyzes the decomposition of hydrogen peroxide and glutathione peroxidases are a
family of antioxidant enzymes containing selenium which are important in the reduction of hydro
peroxides. Non-enzymatic antioxidants include low molecular weight compounds such as vitamin C
and E, β- carotene, uric acid and glutathione. To protect the cells and organ systems of the body
against reactive oxygen species, humans have evolved a highly sophisticated and complex antioxidant
protection system which functions interactively and synergistically to neutralize free radicals [32]. [28]
reported that these components include:
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Ozougwu, Jevas C. “The Role of Reactive Oxygen Species and Antioxidants in Oxidative Stress”
i Nutrient-derived antioxidant like ascorbic acid (vitamin C), tocopherols and tocotrienols (Vitamin
E), carotenoids, and other low molecular weight compounds such as glutathione and lipoic acid.
ii Antioxidant enzymes for example superoxide dismutase, glutathione peroxidase and glutathione
reductase, which catalyze free radical quenching reactions.
iii Metal binding proteins, such as ferritin, lactoferrin, albumin, and ceruloplasmin that sequester free
iron and copper ions that are capable of catalyzing oxidative reactions.
iv Numerous other antioxidant phytonutrients present in a wide variety of plant foods.
Table4. Various ROS and Corresponding Neutralizing Antioxidant
Reactive oxygen species (ROS)
Hydroxyl radical
Superoxide radical
Hydrogen peroxide
Lipid peroxides
Neutralizing antioxidant
Vitamin C, glutathione, flavonoids, lipoic acid
Vitamin C, glutathione, flavonoids, SOD
Vitamin C, glutathione, beta carotene, vitamin E, CoQ10 Flavonoids,
lipoic acid
Beta carotene, vitamin E, ubiquinone, flavonoids, glutathione peroxidase
Source: Percival (1998).
Dietary Antioxidant
Vitamin C, vitamin E and Beta carotene are among the most widely studied dietary antioxidant. The
food sources of antioxidant vitamins are shown in Table 5.
Table5. Food Sources of Antioxidant Vitamins.
Vitamin E
Best sources: vegetable oils, cold pressed seed oils, wheat germ
Other significant sources: vegetables, fruits, meat/poultry/ fish
Vitamin C
Fruits: especially citrus fruits, strawberries, cantaloupe melon
Vegetables: especially tomatoes, leafy greens, cabbage family vegetables such as broccoli and cauliflower.
Carotenoids
β- carotene: yellow-orange vegetables and fruits, dark green vegetables
α- carotene: carrots
Lycopene: tomatoes
Lutein and zeaxanthin: dark green leaf vegetables, broccoli
Β- cryptoxanthin: citrus fruits
Source: Langseth (1995).
Vitamin C is considered the most important water-soluble antioxidant in extracellular fluids [28]. It is
capable of neutralizing ROS in the aqueous phase before lipid peroxidation is initiated. Vitamin E, a
major lipid-soluble antioxidant, is the most effective chain-breaking antioxidant within the cell
membrane where it protects membrane fatty acids from lipid peroxidation. Vitamin C has been
reported as being capable of regenerating vitamin E [30]. Beta carotene and other carotenoids are also
believed to provide antioxidant protection to lipid-rich tissues. Research suggests that beta carotene
may work synergistically with vitamin E [32, 33]. A diet that is excessively low in fat may negatively
affect beta carotene and vitamin E absorption, as well as other fat-soluble nutrients. Fruits and
vegetables are major sources of vitamin C and carotenoids, while whole grains and high quality,
properly extracted and protected vegetable oils are major sources of vitamin E [28]. A number of other
dietary antioxidant substances exist beyond the traditional vitamins. Many plant-derived
phytochemicals are becoming increasingly known for their antioxidant level. Phenolic compounds
such as flavonoids are ubiquitous within the plant kingdom, approximately 3,000 flavonoid substances
have been described [34]. In plants, flavonoids serve as protectors against a wide variety of
environmental stresses while, in humans, flavonoids appear to function as biological response
modifiers. Flavonoids have been demonstrated to have anti-inflammatory, antiallergenic, anti-viral,
anti-aging and anti-carcinogenic level [35]. The broad therapeutic effects of flavonoids can be largely
attributed to their antioxidant properties [28]. In addition to an antioxidant effect, flavonoid
compounds may exert protection against heart disease through the inhibition of cyclooxygenase and
lipoxygenase level in platelets and macrophages [31]. The best way to ensure an adequate intake of
phytonutrients is to eat a diet rich in a wide variety of fresh fruits and vegetables.
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Endogenous Antioxidant
The body relies on several endogenous defense mechanisms to help protect against free radicalinduced cell damage. The antioxidant enzymes – glutathione peroxidase, catalase and superoxide
dismutase (SOD) - metabolize oxidative toxic intermediates and require micronutrient cofactors such
as selenium, iron, copper, zinc and manganese for optimum catalytic level [28]. It has been suggested
that an inadequate dietary intake of these trace minerals may compromise the effectiveness of these
antioxidant defense mechanisms [37]. Research indicates that consumption and absorption of these
important trace minerals may decrease with aging [37]. Glutathione, an important water-soluble
antioxidant, is synthesized from the amino acids glycine, glutamate, and cysteine. Glutathione directly
quenches ROS such as lipid peroxides and also plays a major role in xenobiotic metabolism [28].
Exposure of the liver to xenobiotic substances induces oxidative reactions through the up regulation of
detoxification enzymes that is cytochrome P -450 mixed-function oxidase. When an individual is
exposed to high levels of xenobiotics, more glutathione is utilized for conjugation making it less
available to serve as an antioxidant. Research suggests that glutathione and vitamin C work
interactively to quench free radicals and that they have a sparing effect upon each other. Lipoic acid,
another important endogenous antioxidant, categorized as a thiol or biothiol, is a sulfur-containing
molecule that is known for its involvement in the reaction that catalyzes the oxidative decarboxylation
of alpha-keto acids, such as pyruvate and alphaketoglutarate, in the Krebs cycle. Lipoic acid and its
reduced form, dihydrolipoic acid, are capable of quenching free radicals in both lipid and aqueous
domains and as such has been called a universal antioxidant. Lipoic acid may also exert its antioxidant
effect by chelating with pro-oxidant metals. Research reports indicate that lipoic acid has a sparing
effect on other antioxidant [38]. Animal studies have demonstrated supplemental lipoic acid protect
against the symptoms of vitamin E or vitamin C deficiency. Other physiological antioxidant are shown
in Table 6.
Table6. Antioxidant Protection System
Endogenous antioxidant
Bilirubin
Thiols, eg glutathione,lipoic acid, N-acetyl cysteine
NADPH and NADH
Ubiquinone (coenzyme Q10)
Uric acid
Enzymes: copper/zinc and manganese-dependent superoxide dismutase (SOD), Iron –Dependent Catalase,
selenium-dependent glutathione peroxidase.
Dietary antioxidant
Vitamin c
Vitamin E
Beta carotene and other carotenoids and oxycarotenoids, e.g lycopene and lutein.
Polyphenols e.g flavonoids, flavones, flavonols and proanthocyanidins.
Metal binding proteins
Albumins (coopper)
Ceruloplasmin (copper)
Metallothionein (copper)
Ferritin (iron)
Myoglobin(iron)
Transferring(iron)
Source: Percival (1998).
EFFECTS OF OXIDATIVE STRESS ON DNA, LIPIDS AND PROTEINS
DNA modifications as a result of ROS occurs in several ways, which involves degradation of bases,
single or double stranded DNA breaks, purine, pyrimidine or sugar bound modifications, mutations,
deletions or translocations and cross-linking with proteins. Most of these DNA modifications are
highly relevant to carcinogenesis, aging, neurodegenerative, cardiovascular and autoimmune diseases.
Lipid peroxidation can be induced by ROS which disrupts the membrane lipid bilayer arrangement
that may inactivate membrane bound receptors and enzymes and increase tissue permeability [39].
Products of lipid peroxidation, such as MDA and unsaturated aldehydes, are capable of inactivating
many cellular proteins forming protein cross-linkage [40, 41, 42]. 4- Hydroxy-2-nonenal causes
depletion of intracellular GSH and induces peroxide production [43, 44], activates epidermal growth
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Ozougwu, Jevas C. “The Role of Reactive Oxygen Species and Antioxidants in Oxidative Stress”
factor receptor [45] and induces fibronectin production [46]. Lipid peroxidation products, such as
isoprostanes and thiobarbituric acid reactive substances, have been used as indirect biomarkers of
oxidative stress and increased levels were shown in the exhaled breath condensate or bronchoalveolar
lavage fluid or lung of chronic obstructive pulmonary disease patients or smokers [47, 48, 49].
ROS can cause fragmentation of the
peptide chain, alteration of electrical charge of proteins,
cross-linking of proteins and oxidation of specific amino acids and therefore lead to increased
susceptibility to proteolysis by degradation by specific proteases [50]. Cysteine and methionine
residues in proteins are particularly more susceptible to oxidation [51]. Oxidation of sulfhydryl groups
or methionine residues of proteins cause conformational changes, protein unfolding and degradation
[51, 52]. Enzymes that have metals on or close to their active sites are especially more sensitive to
metal catalyzed oxidation. Oxidative modification of enzymes has been shown to inhibit their
activities [53, 54].
CONCLUSIONS
Damage to the body cells caused by ROS play central role in the aging process and in disease
progression. Under normal conditions, the physiologically important intracellular levels of reactive
oxygen species (ROS) are maintained at low levels by various antioxidant systems in the body but at
high concentrations, they produce adverse modifications to cell components such as lipids, proteins
and DNA. Reactive oxygen species can be beneficial, as they are used by the immune system as a way
to attack and kill pathogens and short-term oxidative stress is important in prevention of aging by
induction of mitohormesis. Humans have evolved a complex antioxidant protection system, which
functions interactively and synergistically to neutralize free radicals and knowledge as presented in
this review will be beneficial in the management of oxidative stress and its associated diseases.
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International Journal of Research in Pharmacy and Biosciences V3● I6● 2016