Free Radicals: From Health to Disease

Abstract

Over the past 40 years, there has been a tremendous amount of research on the dual role of free radicals as both toxic and beneficial species. Free radicals are produced as by-products of normal cellular metabolism or generated by chemicals in our external environment (e.g., cigarette smoke, air and water pollution, exposure to sunlight, gamma-irradiation, and certain chemotherapeutic drugs). At low to intermediate concentrations, free radicals exert their effects through regulation of cell signaling cascades. At high concentrations, they damage all macromolecules, inducing DNA damage, lipid peroxidation, protein modification, and eventually cell death. Free radicals have been implicated in the pathogenesis of a number of conditions, such as aging, atherosclerosis, ischemic heart disease, cancer, and Alzheimer’s disease. Aerobic organisms have evolved sophisticated antioxidant systems to protect themselves from cellular damage and death caused by free radicals. The ability to estimate chemical biomarkers of free radical damage in body fluids and tissues is an important step in understanding the mechanisms contributing to disease processes. This review of a large amount of research studies discusses formation of free radicals in normal cells, their basic properties, toxic effects on cellular processes, potential beneficial role in signaling and phagocytosis, and the part the oxidative stress plays in some major disease processes.

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Free Radicals: From Health to Disease
Arben Santo1, Hong Zhu2, and Y. Robert Li3‒5
1Department of Pathology, EVCOM, Virginia Tech CRC, Blacksburg, VA 24060, USA; 2Department of
Physiology and Pathophysiology and 3Department of Pharmacology, CUSOM, Campbell University, Buies
Creek, NC 27506, USA; 4Virginia Tech-Wake Forest University School of biomedical Engineering and
Sciences, Blacksburg, VA 24061, USA; 5Department of Biology, University of North Carolina, Greensboro,
NC 27412, USA
Correspondence: asanto@vcom.vt.edu
Santo A et al. Reactive Oxygen Species 2(4):245–263, 2016; ©2016 Cell Med Press
http://dx.doi.org/10.20455/ros.2016.847
(Received: November 28, 2015; Revised: January 22, 2016; Accepted: January 25, 2016)
ABSTRACT | Over the past 40 years, there has been a tremendous amount of research on the dual role of free
radicals as both toxic and beneficial species. Free radicals are produced as by-products of normal cellular
metabolism or generated by chemicals in our external environment (e.g., cigarette smoke, air and water
pollution, exposure to sunlight, gamma-irradiation, and certain chemotherapeutic drugs). At low to
intermediate concentrations, free radicals exert their effects through regulation of cell signaling cascades. At
high concentrations, they damage all macromolecules, inducing DNA damage, lipid peroxidation, protein
modification, and eventually cell death. Free radicals have been implicated in the pathogenesis of a number of
conditions, such as aging, atherosclerosis, ischemic heart disease, cancer, and Alzheimer’s disease. Aerobic
organisms have evolved sophisticated antioxidant systems to protect themselves from cellular damage and
death caused by free radicals. The ability to estimate chemical biomarkers of free radical damage in body
fluids and tissues is an important step in understanding the mechanisms contributing to disease processes. This
review of a large amount of research studies discusses formation of free radicals in normal cells, their basic
properties, toxic effects on cellular processes, potential beneficial role in signaling and phagocytosis, and the
part the oxidative stress plays in some major disease processes.
KEYWORDS | Antioxidants; Free radicals; Oxidative stress; Reactive oxygen species; Redox signaling
ABBREVIATIONS | CVD, cardiovascular disease; GPx, glutathione peroxidase; GR, glutathione reductase;
GSH, reduced form of glutathione; GSSG; glutathione disulfide; HNE, 4-hydroxy-2-nonenal; MDA,
malondialdehyde; MPO, myeloperoxidase; NO, nitric oxide; NOS, nitric oxide synthase; 8-OH-dG, 8-
hydroxy-2-deoxyguanosine; PUFA, polyunsaturated fatty acid; RNS, reactive nitrogen species; ROS, reactive
oxygen species; SOD, superoxide dismutase
CONTENTS
1. Definition of Free Radical
2. Oxygen: a Dangerous Friend

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3. Production of Free Radicals in Cells
3.1. Mitochondria
3.2. Endoplasmic Reticulum
3.3. Peroxisomes
3.4. Phagocytosis
3.5. Transition Metals
3.6. Nitric Oxide
4. Reactive Oxygen Species
4.1. Superoxide
4.2. Hydrogen Peroxide
4.3. Hydroxyl Radical
4.4. Hypochlorous Acid
5. Reactive Nitrogen Species
5.1. Nitric Oxide
5.2. Nitric Oxide Synthase
5.3. Peroxynitrite
6. Consequences of Oxidative Stress
7. Oxidative DNA Damage
8. Oxidative Stress and Lipid Peroxidation
8.1. Lipid Peroxidation by ROS
8.2. Lipid Peroxidation by RNS
9. Oxidative Protein Damage
9.1. Oxidative Cleavage of the Polypeptide Backbone
9.2. Oxidation of Amino Acid Residue Side Chains of Proteins
9.3. Generation of Protein–Protein Cross-Linkages
9.4. Accumulation of Oxidized Proteins
10. Antioxidant Defenses
10.1. Superoxide Dismutase
10.2. Catalase
10.3. Glutathione System
10.4. Nonenzymatic Antioxidants
11. ROS as Useful Molecules
11.1. ROS in Signaling Pathways
11.2. ROS in Phagocytosis
12. Measurement of Oxidative Tissue Injury
12.1. Biomarkers of Nucleic Acid Oxidation
12.2. Biomarkers of Lipid Peroxidation
12.3. Biomarkers of Oxidative Protein Damage
13. Free Radicals and Disease
13.1. Overview of the Relationship between Free Radicals and Disease
13.2. Examples of Free Radical Disease: Cardiovascular Disorders
14. Conclusion and Perspectives
1. DEFINITION OF FREE RADICAL
A free radical is any chemical species that contains a
single (unpaired) valence electron in the outermost
electron orbital. Free radicals are produced when the
covalent bond is broken and one electron from each
pair remains with each atom. A free radical is an un-
stable configuration and therefore highly reactive,
owing to the tendency of electrons to pair. Free radi-
cals can either donate an electron to or accept an
electron from other molecules, therefore behaving as
oxidants or reductants. A free radical lasts just for a

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few milliseconds, because it immediately reacts with
adjacent molecules, such as proteins, nucleic acids,
or lipids. When a free radical reacts with a non-
radical molecule, a chain reaction is initiated, where-
by the non-radical molecules are themselves con-
verted into free radicals. The chain reaction is
terminated when two free radicals react with each
other and cross-link two unpaired electrons forming
a covalent bond, with each radical contributing its
single unpaired electron [1, 2].
2. OXYGEN: A DANGEROUS FRIEND
Free radicals of most concern in biological systems
are derived from oxygen. Oxygen is an element in-
dispensable for aerobic life. Cells require oxygen to
generate energy in the mitochondrial electron
transport chain. This need for oxygen obscures the
fact that oxygen is a toxic gas that produces oxygen
radicals during the ATP synthesis in mitochondria.
About 90% of the oxygen taken up in the lungs is
utilized by the mitochondria to produce ATP. The
reminder (~10%) of oxygen is used in metabolism by
various oxidizing enzymes, which catalyze oxidation
of diverse chemical compounds, i.e., combination of
compounds with oxygen. It is worth mentioning that
some anaerobic bacteria survive by hiding in envi-
ronments into which oxygen does not penetrate (e.g.,
pockets of gums, in deeper layers of dental plaque, or
in colonic fecal content). These bacteria have insuf-
ficient antioxidant defenses to protect against oxygen
free radicals. They can often be killed by exposure to
21% oxygen, the atmospheric level [2, 3].
3. PRODUCTION OF FREE RADICALS IN
CELLS
Free radicals are generated from either endogenous
or exogenous sources. Exogenous free radicals result
from cigarette smoke, air and water pollution, pesti-
cides and herbicides, exposure to sunlight, alcohol,
heavy metals, certain chemotherapeutic drugs (e.g.,
doxorubicin, cyclosporine, tacrolimus), industrial
solvents, cooking, and gamma-irradiation (Figure 1).
But the biggest source of free radicals is our own
bodies. Free radical formation occurs continuously in
the cells as a consequence of both enzymatic and
nonenzymatic reactions. Enzymatic reactions, which
serve as sources of free radicals, include those in-
volved in the respiratory chain in mitochondria, cy-
tochrome P450 system in endoplasmic reticulum,
oxidative reactions in peroxisomes and during phag-
ocytosis, as well as transition metal ion-catalyzed re-
actions (Figure 2).
3.1. Mitochondria
The most important source of free radicals is the mi-
tochondrion. During the energy transduction in the
electron transport chain, a small number of electrons
(about 1‒3%) leak to oxygen prematurely forming
the radical superoxide. Superoxide is released into
mitochondrial matrix. The production of superoxide
by mitochondria may contribute to damage to mito-
chondrial proteins, lipids, and DNA. Mitochondrial
lipids damaged by free radical reactions form cross-
linked compounds that accumulate in the form of
yellow-brown lipofuscin pigment granules [4, 5].
3.2. Endoplasmic Reticulum
Cytochrome P450 mixed function oxidase enzymes
of endoplasmic reticulum are involved in the oxida-
tion of a wide range of substrates at the expense of
oxygen. Oxidation is any chemical reaction in which
a material gives up electrons, as when the material
combines with oxygen. The result is a compound
called oxide. Oxidases are capable of reducing oxy-
gen to superoxide before using it in oxidizing opera-
tions. Some superoxide always diffuses away from
FIGURE 1. Exogenous sources of free radi-
cals/ROS. As illustrated, a number of environmental
factors contribute to the production of free radicals
and ROS in biological systems.

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the enzyme before it is used in the chemical reaction
to oxidize the substrate [6, 7].
3.3. Peroxisomes
Peroxisomes are membrane-enclosed organelles that
contain enzymes involved in a variety of oxidative
reactions. A variety of organic molecules, such as
fatty acids, amino acids, and uric acid, are broken
down by a process of oxidation to produce hydrogen
peroxide. These organelles are called peroxisomes
because they produce large amounts of hydrogen
peroxide. To scavenge hydrogen peroxide produced
by these reactions, peroxisomes possess an apprecia-
ble amount of catalase [8] (also see Section 10.2).
3.4. Phagocytosis
Phagocytosis that takes place in neutrophils and mac-
rophages is associated with an oxidative burst that
releases large amounts of free radicals. These reac-
tive radical species serve as important components of
defense mechanism against invading pathogens.
Bursts of free radicals are produced through a con-
trolled reaction and are used to kill the phagocytized
bacteria [9, 10].
3.5. Transition Metals
Transition metals, such as iron and copper, are im-
portant in biology. Approximately 30% of enzymes
use metals because they facilitate enzyme catalysis.
Metalloproteins that contain a metal ion cofactor in-
clude hemoglobin, myoglobin, transferrin, ferritin,
hemosiderin, catalase, and cytochrome c, among
many others.
Transition metals, including iron and copper, can
undergo autoxidation and change valence. For exam-
ple iron exists in two oxidation states: ferrous state
(Fe2+) and ferric state (Fe3+). Copper also exists in
two oxidation states: cuprous (Cu1+) and cupric
(Cu2+) states. In normal cells, iron and copper ions
are typically not found in a free state, but are tightly
bound to proteins. The tightly bound iron is trans-
ported (as in transferrin) or stored (as in ferritin) to
discourage redox cycling. In this context, free iron is
considered a loose cannon, chemically. When ferrous
iron is converted into ferric iron, it donates one elec-
tron, and vice versa. However, because of its ability
to redox cycle between Fe2+ and Fe3+, iron may pro-
mote the formation of hydroxyl radical via the Fen-
ton reaction (H2O2 + Fe2+ → Fe3+ + OH˙ + OHˉ).
Transition metal ions in unbound forms are potential-
ly devastating since they catalyze unwanted free rad-
ical formation [11‒14].
3.6. Nitric Oxide
Nitric oxide (NO) is a free radical, i.e., its structure
includes an unpaired electron. Cells produce NO
from the amino acid L-arginine by the enzymatic ac-
tion of nitric oxide synthase (NOS). NO is known to
play important functional roles in a variety of phys-
iological systems. It is a vasodilator produced by en-
dothelial cells, signaling molecule generated by
neurons, and bactericidal agent produced by macro-
phages and other inflammatory cells during infec-
tion. NO reacts with superoxide to produce the
damaging oxidant peroxynitrite (ONOOˉ). Peroxyni-
trite is able to induce cell necrosis and apoptosis and
FIGURE 2. Endogenous sources of free radi-
cals/ROS. As depicted, the main endogenous
sources of free radicals and ROS include mitochon-
drial electron transport chain, cytochrome P450 en-
zyme system associated with endoplasmic reticulum,
peroxisomes, and oxidases in phagocytic cells.

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acts as a terminal mediator of cellular injury in vari-
ous pathophysiologic conditions [15‒20].
4. REACTIVE OXYGEN SPECIES
There are two major free radical groups: (1) oxygen
free radicals and (2) nitrogen free radicals. Reactive
oxygen species (ROS) is a collective term used to
denote oxygen-derived free radicals and related reac-
tive species. ROS represent the most important class
of free radicals and related reactive species generated
in the body. The most important ROS include super-
oxide radical (O2˙ˉ), hydroxyl radical (OH˙), and hy-
drogen peroxide (H2O2). Hypochlorous acid (HOCl)
is another important oxygen-derived reactive species
(also see Section 11.2).
4.1. Superoxide
Superoxide is the main precursor of most ROS in bi-
ology. It is formed when only a single electron is
added to the diatomic oxygen molecule. It is consid-
ered the primary ROS and can interact with other
molecules to generate secondary ROS. Superoxide is
produced mostly in mitochondria, caused by electron
leak from the respiratory chain. It is also a product of
oxidation reactions. Despite its ‘super’ name, super-
oxide seems relatively unreactive compared with
many other free radical species. But, irrespective of
its poor reactivity, superoxide is able to damage
some important enzymes of energy metabolism (i.e.,
Krebs cycle) and amino acid biosynthesis. The oxi-
dized enzymes have to be repaired to maintain their
activity. Superoxide does not attack DNA directly.
Superoxide generates other secondary radical species
by participating in the so-called iron-mediated Ha-
ber-Weiss reaction (H2O2 + O2˙ˉ → O2 + OH˙ +
OHˉ). In the Haber–Weiss reaction, superoxide re-
acts with hydrogen peroxide forming hydroxyl radi-
cal (OH˙) and hydroxide anion (OHˉ). Superoxide
dismutates spontaneously to hydrogen peroxide (2
O2˙ˉ + 2 H+ → H2O2 + O2) [1, 21‒23].
4.2. Hydrogen Peroxide
Hydrogen peroxide is a major biological ROS, ex-
cess of which can cause damage to cells and tissues.
It is a by-product of respiration and an end-product
of a number of metabolic reactions, particularly pe-
roxisomal oxidation pathways. Dismutation of su-
peroxide formed in the electron transport chain forms
hydrogen peroxide. Peroxisomes are another orga-
nelle known to produce hydrogen peroxide. Hydro-
gen peroxide is produced continually in all tissues
and can be detected in the exhaled breath as well as
the urine [1].
Hydrogen peroxide is a relatively weak ROS per
se, but gives rise to more damaging ROS. Production
of hydrogen peroxide is tightly regulated in the cells,
since a slight increase in its level may lead to cytox-
icity. Perhaps the most common reaction that hydro-
gen peroxide participates in is the Haber‒Weiss
reaction to produce hydroxyl radical (see Section
4.1). The O-O bond in hydrogen peroxide is relative-
ly weak and is susceptible to dissociation when hy-
drogen peroxide is subjected to heating, ionizing
radiation, ultraviolet radiation, or transition metals.
This gives rise to the hydroxyl radical, which is re-
sponsible for many of the strong oxidizing (or disin-
fecting) actions of hydrogen peroxide. In contrast to
superoxide and hydroxyl radical, the less reactive
hydrogen peroxide is involved in a wide variety of
biological processes, such as signal transduction, cell
differentiation and proliferation, and immune re-
sponses. Hydrogen peroxide is a major factor impli-
cated in the free radical theory of aging. According
to the theory, free radicals provoke oxidative dam-
age, and this is the cause of aging. There exists a
close association between ROS generation, ROS-
related damage, and aging. The classical physiologi-
cal role attributed to hydrogen peroxide is its capa-
bility to induce bacterial killing. Hydrogen peroxide
in conjunction with the amplification activity of
myeloperoxidase is responsible for bacterial killing
within the phagosome in the neutrophil cytoplasm
[24‒28] (also see Section 11.2).
4.3. Hydroxyl Radical
Hydroxyl radical is the most biologically active free
radical. This species is much more reactive than su-
peroxide, making it a very dangerous radical. Hy-
droxyl radical can damage virtually all types of
macromolecules, including nucleic acids, lipids, and
amino acids. Hydroxyl radical has a very short in vi-
vo half-life of approximately 10−9 seconds and a high
reactivity. This makes it a very dangerous species to
the organism [1]. It should be noted that hydroxyl
radical is distinct from hydroxyl or hydroxide ion

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(OHˉ), which is not a radical. Hydroxyl radical is
formed from the reaction between ferrous iron and
hydrogen peroxide (the Fenton reaction; see Section
3.5). Ultraviolet (UV) radiation of the skin generates
ROS such as superoxide, hydrogen peroxide, and the
hydroxyl radical. Hydroxyl radical is produced by
UV-induced splitting of hydrogen peroxide (gamma-
radiation + H2O2 → 2 OH˙). Gamma-radiation, the
most dangerous form of ionizing radiation, is high-
energy radiation. It passes through the human body,
like a microscopic bullet, until the radiation is
stopped by the tissues due to absorption. The first
consequence of ionizing radiation is ionization of
water. Since water represents 70% of the chemical
composition of the adult body, its chemical trans-
formation by ionizing radiation merits serious con-
sideration. Gamma-radiation splits water molecules
producing hydroxyl radical and hydrogen atom
(gamma-radiation + H2O → OH˙ + H˙). Of the ROS,
the highly reactive hydroxyl radical destroys biologi-
cally active molecules by either removing electrons
or removing hydrogen atoms. Cellular DNA, pro-
teins, and lipids can all be damaged by hydroxyl rad-
ical. This may lead to damage to cell membranes,
mitochondria, nucleus, and chromosomes, that either
inhibits cell division, results in cell death, or produc-
es a malignant cell [29, 30].
4.4. Hypochlorous Acid
Hypochlorous acid (HOCl) in biological systems is
produced by a heme protein, namely, myeloperoxi-
dase, which converts hydrogen peroxide to hypo-
chlorous acid in the presence of chloride ion (H2O2 +
Clˉ → HOCl + OHˉ) [1]. Myeloperoxidase is stored
in azurophilic granules of neutrophils. The powerful
bactericidal properties of hypochlorous acid have
been well documented. Hypochlorous acid is the ac-
tive ingredient in household bleach and the species
responsible for the antibacterial properties of chlo-
rinated water supplies [31‒33].
5. REACTIVE NITROGEN SPECIES
5.1. Nitric Oxide
Nitric oxide (also see Section 3.6) is a colorless gas.
As noted earlier, it possesses an unpaired electron
making it a reactive free radical. NO is generated in
cells by the action of the enzyme NOS which cata-
lyzes the production of NO from L-arginine [1]. NO
is an abundant chemical that acts as an important
biological signaling molecule in a variety of physio-
logical processes, such as smooth muscle relaxation,
neurotransmission in the enteric plexus, neurotrans-
mission in the central nervous system, defense
mechanisms, and immune regulation. NO relaxes
smooth muscle cells in arterioles during systole, with
resultant vasodilation and blood pressure regulation.
NO serves as an inhibitory neurotransmitter in the
gastrointestinal tract and mediates relaxation of both
circular and longitudinal muscle layers. NO serves as
a neurotransmitter in areas of the brain that are spe-
cialized in cognition. Last but not least, NO pro-
duced in macrophages during inflammation is central
in killing of engulfed microorganisms within phago-
lysosomes. Many of the reported toxic effects of NO
are more likely mediated by its oxidation products
rather than NO itself. NO may reversibly inhibit
some enzymes such as cytochrome P450 and ribonu-
cleotide reductase, but it generally does not directly
attack DNA. The cytotoxic action of NO results
largely from its oxidation product—the highly reac-
tive peroxynitrite (see Section 5.3).
5.2. Nitric Oxide Synthase
There are three types of NOS: endothelial (eNOS),
neuronal (nNOS), and inducible (iNOS). Endothelial
and neuronal NOS are constitutively expressed in
endothelial cells and neurons, respectively. Inducible
NOS is not expressed constitutively but is induced
by pro-inflammatory cytokines and lipopolysaccha-
ride (LPS) in macrophages in sites of inflammation.
iNOS and nNOS are soluble and found predominant-
ly in the cytosol, while eNOS is associated with cell
membranes. Both eNOS and nNOS synthesize NO, a
signaling molecule on demand, for short periods of
time (seconds to minutes) following enzyme activa-
tion. In contrast, iNOS is expressed after cell activa-
tion only and then produces NO for comparatively
long periods of time (hours to days). The long dura-
tion of NO production makes it a cytotoxic rather
than physiological molecule. iNOS activity may play
a detrimental role in experimental autoimmune or
chronic inflammatory processes. For example, over-
production of the vasodilator NO contributes to hy-
potension and vascular hyporeactivity to the
vasoconstrictor agents during septic shock.

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5.3. Peroxynitrite
Exposure of cells to excess NO can cause toxic ef-
fects, which are due to production of secondary reac-
tive nitrogen species (RNS). NO reacts with
superoxide producing peroxynitrite (also see Section
3.6). Peroxynitrite is synthesized when cells produce
large amounts of both NO and superoxide (NO +
O2˙ˉ → ONOOˉ). iNOS can make substantial con-
centrations of NO when expressed. Peroxynitrite is
also a strong oxidant but reacts relatively slowly with
most biological molecules, which makes it unusually
selective as an oxidant. Peroxynitrite is able to cross
biological membranes and diffuse one to two cell di-
ameters. This allows it to interact with most critical
biomolecules, such as DNA, proteins, and lipids.
Peroxynitrite reacts with lipids causing lipid peroxi-
dation and with DNA causing DNA fragmentation.
Peroxynitrite reacts with the tyrosine residues in pro-
teins converting them to 3-nitrotyrosine. Nitration of
proteins leads to enzyme inactivation. Tissue levels
of 3-nitrotyrosine are widely used as a biomarker of
peroxynitrite generation. Elevated levels of 3-
nitrotyrosine have been observed in a vast range of
diseases [15, 34‒37].
6. CONSEQUENCES OF OXIDATIVE STRESS
In healthy organisms, production of free radicals is
approximately balanced by antioxidant defense sys-
tem. This balance is not perfect. Cells cannot elimi-
nate reactive oxygen and nitrogen species
(ROS/RNS) completely since free radicals are useful
in controlled amounts. Oxidative stress refers to an
imbalance between oxidants and antioxidants due to
either increased oxidants or decreased antioxidants,
or both. Cells exposed to oxidative stress are rela-
tively unable to detoxify the reactive intermediates or
to repair the resulting damage. Oxidative stress can
result in adaptation or oxidative damage. Mild oxida-
tive stress can result in adaptation. In this case, cells
are able to cope with elevated concentrations of free
radicals because of increased synthesis of endoge-
nous antioxidants.
Oxidative stress adaptation is an important mecha-
nism by which cells are able to accommodate with
shifts in the level of oxidative stress. Body cells are
exposed to a degree of oxidative stress that is not
static but shifts with changes in environment, metab-
olism, diet, and age. Cells adapt to temporary chang-
es in stress levels, through changing enzyme activity
and overexpressing of a large number of protective
genes. The adapted cell is then significantly more
resistant to oxidative stress. If the stress is reduced to
a lower level, cells gradually lose their previous ad-
aptation [38].
Experimental work has demonstrated that adapted
cells respond differently to single acute stress expo-
sures. Initially, cells are exposed to a single, mild,
non-toxic, dose of an oxidant such as hydrogen per-
oxide, then allowed to adapt for a period of time. The
cells are then re-exposed to a much higher dose of
the oxidant, which would normally be toxic without
the pretreatment and adaptive period. Pretreated cells
become considerably more resilient to the toxic chal-
lenge level of oxidative stress compared to non-
pretreated cells [38].
Oxidative stress leads to potential cell damage.
Such damage is called oxidative damage. It is note-
worthy that the terms ‘‘oxidative stress’’ and ‘‘oxi-
dative damage’’ are not synonymous. Oxidative
damage usually occurs when important biological
molecules, such as DNA, proteins, or cell membrane
lipids, are robbed of their electrons and suffer chemi-
cal modification that can lead to cellular dysfunction.
Oxidative damage occurs continuously in every cell.
Examples of oxidative damage include DNA strand
breakage, lipid peroxidation, and protein oxidation.
Oxidatively damaged molecules can be repaired or
replaced. Cell death (apoptosis, necrosis) occurs
when oxidative stress is severe [39‒41].
7. OXIDATIVE DNA DAMAGE
Of the ROS, hydroxyl radical, the most potent one,
reacts with DNA by addition to double bonds of
DNA bases. Oxidation damage of DNA matters
more than that of other macromolecules because it
can lead to genetic mutations associated with can-
cers. The guanine base is highly susceptible to oxida-
tive stress. The remaining nucleotides are much
harder to oxidize [40]. The most extensively studied
DNA lesion is the formation of 8-hydroxy-2'-
deoxyguanosine (8-OH-dG). 8-OH-dG is formed af-
ter hydroxyl radical addition to C8 of guanosine. 8-
OH-dG is the most extensively studied DNA lesion.
The levels of this guanine analogue increase with ox-
idative stress. Once formed, 8-OH-dG is a major pre-

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mutagenic lesion. Further oxidation of 8-OH-dG
opens the imidazole ring of the guanine (guanine is a
purine) to give rise to a pyrimidine such as thymine.
During the cell division and DNA replication, thy-
mine pairs with adenine. As a consequence of this
miscoding, the pair GC (guanine-cytosine) is re-
placed by the pair TA (thymine-adenine). If it is not
corrected via base excision repair, GC-TA transver-
sion mutations would lead to potential deleterious
consequences. Many oxidative base lesions are mu-
tagenic. For this reason, they have been considered
as intermediate markers of cancer. GC→TA trans-
versions, potentially derived from 8-OH-dG, have
been observed in vivo in the RAS oncogene and the
p53 tumor suppressor gene in lung and liver cancer.
Also, tissue concentration of 8-OH-dG has been
found increased 10‒20 fold in breast carcinoma.
Augmented urinary levels of 8-OH-dG have been
found in many cancers [41‒43].
8. OXIDATIVE STRESS AND LIPID
PEROXIDATION
Lipid peroxidation refers to a process under which
free radicals attack lipids containing carbon-carbon
double bond(s), especially polyunsaturated fatty ac-
ids (PUFAs). The greater the number of double
bonds in a fatty acid, the more readily it undergoes
peroxidation. Saturated fatty acids such as palmitic
and stearic acid, and monounsaturated fatty acids
such as oleic acid do not undergo peroxidation. The
highly unsaturated fatty acids are very susceptible to
peroxidation.
PUFAs are structural components of lipid bilayer
cellular membranes. Many of the permeability char-
acteristics of cellular membranes depend on lipid bi-
layer integrity. The permeability characteristics of
the bilayer allow gradients of metabolite and electro-
lyte concentrations to exist between the intra‒ and
extracellular spaces. Damage of PUFAs would lead
to rapid loss in membrane integrity, dissipation of
these gradients, and compromise of organelle or cel-
lular function. The destruction of membrane lipids
by a free radical attack and the end‒products of lipid
peroxidation reactions are especially dangerous for
the viability of cells.
The two most prevalent reactive species that can
affect profoundly the lipids are hydroxyl radical and
peroxynitrite. The overall process of lipid peroxida-
tion consists of three steps: initiation, propagation,
and termination.
8.1. Lipid Peroxidation by ROS
The first step in lipid peroxidation by oxygen free
radicals is abstraction of a hydrogen atom from the
acyl chain to form a lipid radical. The next step is the
addition of molecular oxygen to the lipid radical
yielding lipid peroxyl radical. When lipid peroxyl
radical abstracts a hydrogen atom from one of the
adjacent PUFAs, a hydroxyperoxy fatty acid (also
called lipid hydroperoxide) is formed. Transition
metals reduce hydroxyperoxy fatty acids yielding
alkoxyl radicals and hydroxy fatty acids. Another
possible direction of peroxidation reactions is the
generation of complex cyclic compounds via intra-
molecular reactions yielding endoperoxides. The
most studied endoperoxide products of peroxidation
are isoprostanes. These are prostaglandin‒like com-
pounds formed via peroxidation of arachidonic acid
without the direct action of cyclooxygenase (COX)
enzymes. Isoprostanes are biologically active; they
are potent vasoconstrictors acting via the thrombox-
ane receptor.
The ultimate direction of peroxidation reactions is
the fragmentation of fatty acid carbon chain. Frag-
mentation products include some of the most exten-
sively studied products of lipid peroxidation, such as
malondialdehyde (MDA), acrolein, and 4‒hydroxy‒
2‒nonenal (HNE). MDA is a naturally occurring
end‒product of lipid peroxidation that is mutagenic
and carcinogenic. It is able to induce large insertions
and deletions in the DNA, but base pair substitutions
have also been detected. MDA causes up to a 15‒
fold increase in mutation frequency compared to
background levels [44]. This level of increase is
comparable with the mutation frequencies induced
by ultraviolet light. Acrolein formed in vivo during
lipid peroxidation exhibits facile reactivity with vari-
ous key macromolecules, including proteins and
phospholipids, has the potential to inhibit many en-
zymes, quickly deplete cellular glutathione levels,
and potentially induce cell death. HNE is a highly
abundant fragmentation product that results from li-
pid peroxidation of arachidonic acid. Elevated HNE
concentrations have been found in several pathologi-
cal conditions, such as atherosclerosis and cardiovas-
cular diseases. HNE depletes cellular glutathione
levels, easily forms adducts to cellular proteins lead-

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ing to cellular dysfunction, disrupts cellular cyto-
skeleton, and induces apoptotic cell death [45].
8.2. Lipid Peroxidation by RNS
PUFAs can undergo modification by RNS as well.
Attack to fatty acid double bonds by RNS gives rise
to lipid nitrosylation. The initial steps in lipid nitro-
sylation are the formation of nitrosyl lipid radical
and dinitro-fatty acids. Further steps in the formation
of nitrated lipids give rise to a multiplicity of com-
pounds, such as nitroalkene and alkyl nitrate. Bioac-
tivities of nitrated lipids have been well characterized
in the literature [44, 46‒51].
9. OXIDATIVE PROTEIN DAMAGE
Protein modifications that occur due to reactive spe-
cies generated during oxidative stress include frag-
mentation of the polypeptide chain, oxidation of
amino acid side chains, and generation of protein–
protein cross-linkages.
9.1. Oxidative Cleavage of the Polypeptide
Backbone
Oxidation of a protein molecule is initiated by ab-
straction of a hydrogen atom from the polypeptide
chain to form water and a carbon-centered radical.
The next step is formation of a peroxyl radical as a
consequence of the addition of molecular oxygen.
Peroxyl radical is readily converted to protein perox-
ide and alkoxyl radical by reactions with transition
metals. The protein alkoxyl radical can undergo pep-
tide bond cleavage and fragmentation of the polypep-
tide chain. It has been demonstrated that it is the
oxidation of proline residues that is most susceptible
to peptide bond cleavage [52, 53].
9.2. Oxidation of Amino Acid Residue Side
Chains of Proteins
Amino acid residues of protein side chains that are
most susceptible to oxidation by free radicals include
cysteine and methionine. Cysteine and methionine
residues of proteins are by far the most sensitive to
oxidation by almost all kinds of free radicals. The
primary oxidation products of cysteine residues are
protein disulfides. Other resides such as tyrosine can
also be modified by free radicals. Abstraction of a
hydrogen atom from the tyrosine residue forms the
tyrosine radical. A tyrosine radical can react with
RNS to form 3-nitrotyrosine. This modification is
biologically irreversible. Tyrosine oxidation prod-
ucts, especially 3-nitrotyrosine, are considered as bi-
omarkers for oxidative stress [54].
Protein carbonylation refers to a process that forms
reactive ketones or aldehydes under oxidative stress
conditions. Lysine, arginine, proline, and threonine
residues of proteins are particularly susceptible to
this process. Protein carbonylation reactions are con-
sidered biologically irreversible. Protein carbonyla-
tion is a commonly used marker for oxidative stress.
The carbonyl content of proteins has become the
most generally used marker for estimation of oxida-
tive stress-mediated protein oxidation [55].
9.3. Generation of Protein–Protein Cross-
Linkages
Free radical-mediated oxidation of proteins can lead
to the formation of protein–protein cross-linked de-
rivatives. Examples of oxidative cross-linking in-
clude reaction of a carbonyl group in one protein
with the amino group of a lysine residue of another
protein. Cysteine or methionine oxidation leads to
intramolecular disulfide formation with another cys-
teine or methionine within the oxidized protein or
intermolecular disulfide formation with a cyste-
ine/methionine from another protein. These oxida-
tions lead to alterations in protein conformation and
loss of biological activity of the oxidized proteins.
Protein–protein cross-links can be formed also by the
interaction of lysine residues of two different pro-
teins with the aldehyde groups of MDA produced
during lipid peroxidation of PUFAs. Cross-linkages
are also produced by a combination of tyrosine radi-
cals in two different proteins [53].
9.4. Accumulation of Oxidized Proteins
Proteins which have been damaged through oxida-
tive imbalances are degraded by the proteolytic ma-
chinery to their constitutive amino acids. There is
evidence that mildly oxidized proteins are removed
in a proteasome-dependent fashion. Heavily oxidized
proteins and protein–protein cross-linked derivatives
are misfolded and do not undergo ubiquitin-
dependent degradation. Heavily modified substrates

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are incompletely degraded, and they tend to aggre-
gate and accumulate within the lysosomal compart-
ments resulting in the formation of lipofuscin-like
aggregates. Accumulation of misfolded proteins
eventually results in impaired cell function, cell
death, and tissue injury. Aggregation of oxidized
proteins has been implicated in the development of
cardiomyopathies, Alzheimer's disease, and protein
folding diseases such as amyloidosis [53, 56‒60].
10. ANTIOXIDANT DEFENSES
One of the biological mechanisms to counteract oxi-
dative stress is by producing antioxidants. An antiox-
idant is a substance capable of preventing, reducing,
or repairing the oxidative damage of a targeted mac-
romolecule [61]. The human body is equipped with a
wide variety of antioxidants, which are either endog-
enous or exogenously supplied through foods and/or
dietary supplements. A narrow definition of antioxi-
dants would include molecules that are capable of
removing, neutralizing, or scavenging ROS/RNS and
their intermediates. But antioxidant defense mecha-
nisms also include the inhibition of free radical gen-
eration, binding of redox-active metal ions needed
for catalysis of oxidative species production, and up-
regulation of antioxidant defense activity. In fact, the
elimination of free radicals by cells is not accom-
plished by a single biochemical pathway but is car-
ried out by several cascades of intricately related
events or processes.
Endogenous antioxidants can be classified into two
categories: enzymatic and non-enzymatic. The major
enzymatic antioxidants directly involved in the neu-
tralization of ROS include superoxide dismutase,
catalase, glutathione peroxidase, and glutathione re-
ductase, among many others [62‒64].
10.1. Superoxide Dismutase
Superoxide dismutases (SODs) are metalloproteins
that catalyze dismutation of superoxide into hydro-
gen peroxide and molecular oxygen (2 O2˙ˉ + 2 H+
→ H2O2 + O2). Depending on the transition metal
ion found at their active site, SODs can be catego-
rized into three types: copper, zinc superoxide dis-
mutase (Cu,ZnSOD or SOD1), manganese
superoxide dismutase (MnSOD or SOD2), and extra-
cellular superoxide dismutase (ECSOD or SOD3).
SOD1 is primarily a cytoplasmic enzyme, SOD2 re-
sides in mitochondria, and SOD3, which contains
copper and zinc in its active sites, is the major extra-
cellular antioxidant enzyme. The role of SODs in
modulating the pathophysiology of diverse disease
processes has been demonstrated in genetically engi-
neered mice [65, 66].
10.2. Catalase
Catalase is an enzyme found predominantly in perox-
isomes. The main function of catalase is decomposi-
tion of hydrogen peroxide to water and molecular
oxygen (2 H2O2 → 2 H2O + O2). Catalase thus limits
the accumulation of hydrogen peroxide, which is
generated by various oxidases in cells and serves as a
substrate for the Fenton reaction to generate the
highly reactive hydroxyl radical (see Section 3.5 for
the Fenton reaction). Catalase plays an important
role in removing intracellular H2O2 and is the key
enzyme for disposal of hydrogen peroxide in peroxi-
somes. It is a heme-containing homotetrameric pro-
tein, ubiquitously present in all types of mammalian
cells. Catalase is a significant defense against oxida-
tive tissue injury in various disease conditions. For
example, mice deficient in catalase undergoing ne-
phrectomy are more susceptible to oxidant tissue in-
jury and renal fibrosis [8, 67, 68].
10.3. Glutathione System
The glutathione system is a major cellular antioxi-
dant defense and consists of the reduced form of glu-
tathione (GSH) and the enzymes that are involved in
either its synthesis/regeneration or using it as an
electron donor to detoxify reactive species. GSH is a
tripeptide; its synthesis from glutamate, cysteine, and
glycine is catalyzed sequentially by two cytosolic
enzymes, γ-glutamylcysteine synthetase (GCS),
which is more formally known as γ-glutamylcysteine
ligase (GCL), and GSH synthetase.
Most of the cellular GSH is present in the cytosol.
GSH scavenges free radicals and ROS (e.g., hydro-
gen peroxide, hydroxyl radical, peroxynitrite, and
lipid peroxyl radical) directly and indirectly through
enzymatic reactions. In such reactions, GSH is oxi-
dized to form glutathione disulfide (GSSG). GSH is
then regenerated from GSSG by the enzyme gluta-
thione reductase (GR). In addition, glutathione pe-
roxidase (GPx) catalyzes the GSH-dependent

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reduction of hydrogen peroxide to water.
GSH/GSSG is the major redox couple that deter-
mines the antioxidative capacity of cells. The ratio of
intracellular GSH to GSSG is often used as an indi-
cator of the cellular redox state. This ratio is 10:1 to
100:1 under normal conditions.
GPx is a family of enzymes with peroxidase activi-
ty. There are eight isozymes encoded by different
genes, which vary in cellular location and substrate
specificity. In mammalian tissues, GPx1, 2, 3, and 4
are selenoproteins with a selenocysteine in the cata-
lytic center. All GPx enzymes are able to catalyze
the reduction of hydrogen peroxide to water using
GSH as a reductant. During the reaction GSH is oxi-
dized to GSSG which can then be reduced back to
GSH by GR [69, 70].
10.4. Nonenzymatic Antioxidants
Nonenzymatic antioxidants include low-molecular-
weight compounds, such as vitamins C and E. Vita-
min C (also known as ascorbic acid) is a water solu-
ble antioxidant. Vitamin C is a cofactor for at least
eight enzymes. Vitamin C acts as a reducing agent,
donating electrons to various enzymatic reactions.
Most mammalian organisms actually synthesize vit-
amin C, and the synthesis involves a series of en-
zymes. Humans must ingest vitamin C because we
lack the last enzyme in the series of steps needed to
make this molecule. Deficiency of vitamin C causes
scurvy. Sources of vitamin C are vegetables and
fruits (e.g., lemons, lime). Vitamin C is regarded as
the first line natural antioxidant defense in plasma
and a powerful scavenger of superoxide, hydroxyl
radical, and peroxynitrite.
Vitamin E (α-tocopherol) is a generic term for a
group of fat-soluble organic compounds known as
tocopherols and tocotrienols. Vitamin E exists in
eight different forms: 4 tocopherols (alpha, beta,
gamma, and delta) and 4 tocotrienols (alpha, beta,
gamma, and delta). The most biologically active
form of vitamin E is α-tocopherol.
α-Tocopherol is a potent peroxyl radical scaven-
ger, which protects cell membranes from lipid perox-
idation. This would prevent the oxidation reaction
from continuing. The oxidized α-tocopherol radical
produced in this process may be reduced by a hydro-
gen donor (such as vitamin C). Compared with to-
copherols, tocotrienols occur at very low levels in
cells and tissues [71, 72].
11. ROS AS USEFUL MOLECULES
Although historically viewed as associated with tis-
sue injury, ROS also play a major physiological role
in several aspects of intracellular signaling and de-
fense mechanisms against infection. Summarized be-
low are representative scenarios where ROS play a
part in cell signal transduction and innate immunity.
11.1. ROS in Signaling Pathways
The epidermal growth factor receptor (EGF-R) is a
cell surface receptor that has an intrinsic ligand-
activated protein tyrosine kinase activity. Ligand
binding to the EGF-R leads to receptor auto-
phosphorylation on specific tyrosine residues, which
mediates cell response through a signal transduction
pathway which, in turn, activates mitogen-activated
protein (MAP) kinase. Studies have demonstrated
that binding of ligands to EGF-R cannot produce
enough phosphorylation to allow a full response.
Transient inhibition of phosphatases by ROS is also
required (Figure 3). The prevention of ROS accumu-
lation by antioxidants blocks MAP kinase activation
after EGF-R ligand binding. Moreover, direct expo-
sure of cells to exogenous hydrogen peroxide, to
mimic oxidative stress, leads to activation of EGF-R
and MAP kinase pathways. The mechanism of EGF-
R activation by ROS is not known. A plausible hy-
pothesis is that oxidative modification of critical
amino acid residues of the proteins participating in
this signal transduction pathway is necessary for full
activation of MAP kinase pathways [9, 73, 74].
11.2. ROS in Phagocytosis
It has been well documented that neutrophils and
macrophages are able to generate ROS and use them
to kill phagocytized bacteria (Figure 4). After phag-
ocytizing a microorganism, activation of the phago-
cyte NADPH oxidase results in a rapid increase in
oxygen uptake which is known as the respiratory
burst. The increase in oxygen uptake during the res-
piratory burst can be 50 times the resting O2 con-
sumption of neutrophils. A key enzyme in this
process is the oxygen radical-generating NADPH ox-
idase. It consists of six subunits, which are separated
under physiological conditions to prevent spontane-
ous generation of free radicals with consequent cell
damage [75]. In order to become activated, the subu

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nits of NADPH oxidase have to assemble to form the
multi-subunit enzyme. Two of these subunits,
gp91phox and p22phox (phox refers to phagocyte oxi-
dase according to the currently accepted nomencla-
ture) are located within the phagolysosome
membrane. These two subunits form cytochrome b
which is the catalytic core of the enzyme, essential
for the generation ROS in phagocytes. After neutro-
phil activation, three additional subunits (p67phox,
p47phox, and p40phox) along with GTPase binding pro-
tein Rac1 are dislocated from cytosol to phagolyso-
some membrane [9].
Activation of NADPH oxidase results in the gen-
eration of significant amounts of superoxide which
accumulates in phagolysosomes. Subsequently, su-
peroxide is converted into much more potent bacteri-
cidal oxidants species. Superoxide is rapidly
converted to hydrogen peroxide by spontaneous dis-
mutation. On the other hand, activated neutrophils
undergo fusion of the cytoplasmic azurophilic gran-
ules with the phagolysosome with release of the
granule content. Azurophilic granules are loaded
with the enzyme myeloperoxidase (MPO). MPO is
an abundant heme protein that accounts for 5% of the
total neutrophil protein content. MPO produces hy-
pochlorous acid from hydrogen peroxide and chlo-
ride anion (also see Section 4.4). Last but not least,
hydroxyl radical is formed in phagolysosome by
transition metal-catalyzed Haber‒Weiss reaction or
Fenton reaction (also see Sections 3.5 and 4.1). This
reaction requires the presence of iron or copper,
which is believed to be provided by the cell itself.
Hydroxyl radical is a very powerful bactericidal
agent [32, 76, 77].
12. MEASUREMENT OF OXIDATIVE TISSUE
INJURY
Oxygen free radicals have been discovered more
than fifty years ago. However, only within the last
two decades, has there been an explosive research
about their roles in the development of diseases. Free
radicals are widely believed to contribute to the de-
velopment of a large number of diseases and per-
haps, even to the aging process itself. For example,
diseases in which oxidative damage has been impli-
cated include cancer, Alzheimer’s disease, other neu-
rodegenerative diseases, atherosclerosis, diabetes,
hypertension, and among many others. But the causal
role of oxidative damage in the onset and progres-
sion of many specific diseases remains to be eluci-
dated. One way to study the participation of free rad-
radicals in pathology is to be able to accurately
measure chemical biomarkers of free radical damage
in body fluids and tissues.
12.1. Biomarkers of Nucleic Acid Oxidation
The spectrum of oxidation products involving nucle-
ic acids includes more than 20 different types of base
damage. The most studied oxidation product is 8-
OH-dG (also see Section 7). 8-OH-dG is used as a
marker of DNA oxidation to evaluate carcinogenicity
of ROS-forming chemicals. Cancer tissues contain
abundant amounts of 8-OH-dG [78]. Tumor tissue
from lung cancer patients contains 2‒3 times higher
amounts of 8-OH-dG than the apparently normal
lung tissue. Smokers excrete 50% more 8-OH-dG in
urine than nonsmokers. Also, there is a considerable
amount of evidence supporting the involvement of
DNA oxidation in the pathogenesis of neurodegener-
FIGURE 3. ROS in MAP kinase signaling path-
way. See text (Section 11.1) for detailed description.

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ative diseases, especially Alzheimer’s disease. In-
creased levels of 8-OH-dG have been found in the
brains of patients with Alzheimer’s disease [79].
12.2. Biomarkers of Lipid Peroxidation
There is evidence that lipid peroxidation of PUFAs is
involved in the onset and progression of many spe-
cific disorders, such as diabetes, atherosclerosis, can-
cer, and neurodegenerative diseases (e.g.,
Parkinson’s disease). Studies have demonstrated in-
creased levels of products of lipid peroxidation, such
as HNE, MDA, acrolein, and isoprostanes, in the
brains of patients with Parkinson’s disease. Immuno-
histochemistry studies have demonstrated increased
HNE levels in dopaminergic cells in the substantia
nigra and cerebrospinal fluid in patients with Parkin-
son’s disease [80].
12.3. Biomarkers of Oxidative Protein Damage
Common markers of protein oxidation include pro-
tein carbonylation and protein nitration, which are
used to study the possible involvement of oxidative
stress in a variety of disease processes. For instance,
increased amounts of carbonylated proteins have
been found in the synovial fluid in patients with
rheumatoid arthritis. Biomarkers of oxidative protein
damage have been demonstrated in postmortem brain
tissue of patients with amyotrophic lateral sclerosis
(ALS). Protein carbonyls and nitrotyrosine have been
shown to be elevated in the spinal cord of patients
with familial and sporadic ALS [81‒84].
13. FREE RADICALS AND DISEASE
13.1. An Overview of the Relationship between
Free Radicals and Disease
The significance of oxidative stress has become in-
creasingly recognized to the point that it is now con-
sidered to be a component of almost every disease.
Researchers in the field of oxidative stress can be
classified into two large groups: enthusiasts and crit-
ics. Enthusiasts believe that increased free radical
formation accompanies tissue injury in most if not all
diseases, and oxidative stress may play an important
casual role in many disease conditions. Critics, how-
ever, believe that in most disorders, oxidative stress
is a mere consequence and not a cause of the primary
disease process [83, 85].
In many diseases, oxidative stress may be just an
unimportant consequence of and not a cause of the
disease pathophysiology. Muscular dystrophy is a
good example of this category: affected muscles
demonstrate increased lipid peroxidation, which is a
consequence of tissue damage, but makes no contri-
bution to the damage. Attempts to slow down the
course of muscular dystrophy with antioxidants have
so far been unsuccessful [86]. On the other hand,
some diseases are caused directly by oxidative stress
(Figure 5). Examples include acute radiation syn-
drome, carbon tetrachloride intoxication, acetamino-
phen intoxication, and ischemia/reperfusion injury,
among others [87‒89]. In the third group of diseases,
FIGURE 4. ROS in bacterial killing process in-
side the phagosomes of phagocytic cells. See text
(Section 11.2) for detailed description.
FIGURE 5. Diseases directly induced by oxida-
tive stress. Illustrated in the scheme are some repre-
sentative pathophysiological conditions in which
oxidative stress may play a causal role.

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ROS play a role in furthering tissue injury even when
they are not the cause of the disease. Examples of
this group include hereditary hemochromatosis and
Wilson disease [90, 91]. A detailed discussion of the
role of oxidative stress in specific diseases is beyond
the scope of this article. Nevertheless, the section be-
low uses cardiovascular disease as an example to il-
lustrate the complexity of the oxidative stress
mechanism of disease pathophysiology and antioxi-
dant-based intervention.
13.2. Examples of Free Radical Disease:
Cardiovascular Disorders
Cardiovascular disease (CVD) is the leading cause of
death in the United States. Approximately 1.5 mil-
lion cases of myocardial infarction are diagnosed an-
nually and approximately 800,000 deaths related to
the coronary artery disease and other CVDs occur
each year [92]. In most cases, myocardial infarction
occurs because of rupture or fissuring of a coronary
artery atherosclerotic plaque containing a lipid-rich
necrotic core with superimposed thrombosis causing
reduced blood flow and consequent ischemic myo-
cardial cell death [93].
Studies have shown that ischemic death of myo-
cardial cells increases as a function of the duration of
ischemia [94]. Fundamental experimental studies of
Jennings et al. in the 1960s demonstrated that a 15-
minute temporary occlusion of the left circumflex
coronary artery in dogs caused no irreversible injury
and was not associated with histopathologic changes
of necrosis in the ischemic region. On the other hand,
temporary ischemic episode lasting 20 minutes or
longer was associated with ischemic necrosis of my-
ocardial cells in the ischemic region [95]. Timely
reperfusion of acute ischemic myocardium is essen-
tial for myocardial salvage. The case fatality rate of
acute myocardial infarction has fallen dramatically in
the past 3 decades, in part because of the widespread
use of reperfusion therapy. Reperfusion is achieved
through interventional procedures such as balloon
angioplasty or the use of thrombolytic agents. Reper-
fusion of the ischemic tissue is beneficial because it
supplies oxygen. However, there is evidence that
reperfusion itself contributes to oxidative damage to
myocardium. Hence, reperfusion is like a double-
edged sword as ischemic tissues release myocardi-
um-damaging ROS when reperfused with fresh oxy-
genated blood [93].
In classical experiments using open-chest, anesthe-
tized dogs, a coronary occlusion lasting 15 minutes
and followed by reperfusion caused no irreversible
injury, but this episode of transient ischemia led to a
period of hypokinesis of the previously ischemic
wall after the restoration of flow. This mechanical
dysfunction was not accounted for by myocardial ne-
crosis as no histological signs of irreversible injury
to cardiomyocytes existed. The contractile function
usually restored several days or weeks later. This
phenomenon of reversible reduction of function of
heart contraction not accounted for by tissue damage
or reduced blood flow after reperfusion has been re-
ferred to as myocardial stunning.
The degree of myocardial stunning and the level of
the functional depression are proportional to dura-
tion, severity, and extent of the myocardial ischemia.
The coronary occlusion-reperfusion cycles in open-
chest dogs were shown to be associated with recur-
rent bursts of free radical formation. It was demon-
strated that these bursts were markedly inhibited by
mercaptopropionyl glycine, which is a hydroxyl rad-
ical scavenger [94]. Inhibition of free radical genera-
tion by mercaptopropionyl glycine resulted in
attenuation of the ensuing postischemic contractile
depression, indicating that free radical formation is
necessary for the development of severe myocardial
stunning in this setting. Taken together, the results of
these experimental studies support an important
pathogenic role of hydroxyl radical in myocardial
stunning [94].
Recently, multiple antioxidant compounds with
distinct structure have been shown to be protective
against myocardial stunning in animal models [96].
However, it remains unknown if any of the antioxi-
dant modalities would be effective in the clinical set-
ting. In fact, for the entire spectrum of cardiovascular
diseases, ranging from hypertension to ischemic
heart disease and to heart failure, diverse antioxidant
therapies have been shown to be effective in animal
models, but none have been conclusively demon-
strated to be effective in human patients [97‒100].
This, on the one hand, highlights the complexity of
the free radical nature of cardiovascular injury, and
on the other hand, points to the need for further
mechanistic studies of the oxidative stress mecha-
nism of tissue injury and development of innovative
mechanistically based strategies for the intervention
of oxidative tissue injury in human patients with car-
diovascular diseases as well as other disorders.

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14. CONCLUSION AND PERSPECTIVES
The generation of oxygen and nitrogen free radicals
is an unavoidable consequence of aerobic life.
Sources of free radicals are numerous, and the major
ones include electron leak from the mitochondrial
electron transport chain, xenobiotics metabolism by
the cytochrome P450 enzyme system in the endo-
plasmic reticulum, and activation of NADPH oxidase
in inflammatory cells. The unpaired electron is re-
sponsible for the extremely high biological reactivity
of free radicals. Oxygen and nitrogen free radicals
represent a constant source of assaults upon cellular
lipids, proteins, nucleic acids, and other biomole-
cules. Yet, it would be inaccurate to consider solely
the damaging biological effects of ROS. At low or
moderate concentrations, free radicals are vital to
body health. They are important for host defense
mechanisms, and play a key role in the regulation of
intracellular signaling cascades in various types of
nonphagocytic cells. Exposure of cells to elevated
concentrations of reactive species through either en-
dogenous or exogenous insults gives rise to oxidative
stress. The harmful effect of oxidative stress is coun-
teracted by the action of both antioxidant enzymes
and non-enzymatic antioxidants. Oxidative stress has
been recognized as a contributor to a number of
pathophysiological conditions, especially cardiovas-
cular injury. In this context, antioxidant compounds
of diverse structures have been demonstrated to be
protective in many disease processes in diverse ani-
mal models. However, for most, if not all, of the an-
tioxidant-based modalities shown to be effective in
animal models, their clinical efficacy in human pa-
tients remains to be established. As new knowledge
on free radicals in health and disease increases, new
opportunities would become available to help devel-
op innovative mechanistically based modalities for
the intervention of free radical disease without com-
promising the physiological function of these reac-
tive species.
REFERENCES
1. Cheeseman KH, Slater TF. An introduction to free
radical biochemistry. Br Med Bull 1993;
49(3):481‒93.
2. Halliwell B, Gutteridge JMC. Oxygen: boon yet
bane—introducing oxygen toxicity and reactive
species. In: Free Radicals in Biology and Medicine
(B. Halliwell, JMC Gutteridge), 5th ed. Oxford
University Press, Oxford, UK. 2015, p. 1‒29.
3. Pierre JL. Chemistry of dioxygen and its activated
species. In: Analysis of Free Radicals in Biological
Systems (A Favier, J Cadet, B. Kalyanaraman, M
Fontecave, JL Pierre). Birkhäuser, Basel,
Switherland. 1995, p. 1‒11.
4. Cadenas E, Davies KJ. Mitochondrial free radical
generation, oxidative stress, and aging. Free Radic
Biol Med 2000; 29(3‒4):222‒30.
5. Figueira TR, Barros MH, Camargo AA, Castilho
RF, Ferreira JC, Kowaltowski AJ, et al.
Mitochondria as a source of reactive oxygen and
nitrogen species: from molecular mechanisms to
human health. Antioxid Redox Signal 2013;
18(16):2029‒74. doi: 10.1089/ars.2012.4729.
6. Cederbaum AI. Molecular mechanisms of the
microsomal mixed function oxidases and biological
and pathological implications. Redox Biol 2015;
4:60‒73. doi: 10.1016/j.redox.2014.11.008.
7. Gorlach A, Klappa P, Kietzmann T. The
endoplasmic reticulum: folding, calcium
homeostasis, signaling, and redox control. Antioxid
Redox Signal 2006; 8(9‒10):1391‒418. doi:
10.1089/ars.2006.8.1391.
8. Kodydkova J, Vavrova L, Kocik M, Zak A. Human
catalase: its polymorphisms, regulation and
changes of its activity in different diseases. Folia
Biol (Praha) 2014; 60(4):153‒67.
9. Manda-Handzlik A, Demkow U. Neutrophils: the
role of oxidative and nitrosative stress in health and
disease. Adv Exp Med Biol 2015; 857:51‒60. doi:
10.1007/5584_2015_117.
10. Hager M, Cowland JB, Borregaard N. Neutrophil
granules in health and disease. J Intern Med 2010;
268(1):25‒34. doi: 10.1111/j.1365-
2796.2010.02237.x.
11. McCord JM. Iron, free radicals, and oxidative
injury. Semin Hematol 1998; 35(1):5‒12.
12. Jomova K, Valko M. Advances in metal-induced
oxidative stress and human disease. Toxicology
2011; 283(2‒3):65‒87. doi:
10.1016/j.tox.2011.03.001.
13. Valko M, Jomova K, Rhodes CJ, Kuca K, Musilek
K. Redox- and non-redox-metal-induced formation
of free radicals and their role in human disease.
Arch Toxicol 2016; 90(1):1‒37. doi:
10.1007/s00204-015-1579-5.
14. Halliwell B, Gutteridge JM. Oxygen toxicity,
oxygen radicals, transition metals and disease.
Biochem J 1984; 219(1):1‒14.

REVIEW ARTICLES
REACTIVE OXYGEN SPECIES ǀ aimsci.com/ros 260 VOLUME 2 ǀ ISSUE 4 ǀ JULY 2016
ROS
15. Pacher P, Beckman JS, Liaudet L. Nitric oxide and
peroxynitrite in health and disease. Physiol Rev
2007; 87(1):315‒424. doi:
10.1152/physrev.00029.2006.
16. Calcerrada P, Peluffo G, Radi R. Nitric oxide-
derived oxidants with a focus on peroxynitrite:
molecular targets, cellular responses and
therapeutic implications. Curr Pharm Des 2011;
17(35):3905‒32.
17. Thomas DD. Breathing new life into nitric oxide
signaling: a brief overview of the interplay between
oxygen and nitric oxide. Redox Biol 2015; 5:225‒
33. doi: 10.1016/j.redox.2015.05.002.
18. Esquivel-Solis H, Vallecillo AJ, Benitez-Guzman
A, Adams LG, Lopez-Vidal Y, Gutierrez-Pabello
JA. Nitric oxide not apoptosis mediates differential
killing of Mycobacterium bovis in bovine
macrophages. PLoS One 2013; 8(5):e63464. doi:
10.1371/journal.pone.0063464.
19. Vazquez-Torres A, Jones-Carson J, Mastroeni P,
Ischiropoulos H, Fang FC. Antimicrobial actions of
the NADPH phagocyte oxidase and inducible nitric
oxide synthase in experimental salmonellosis. I.
Effects on microbial killing by activated peritoneal
macrophages in vitro. J Exp Med 2000;
192(2):227‒36.
20. Szabo C, Ischiropoulos H, Radi R. Peroxynitrite:
biochemistry, pathophysiology and development of
therapeutics. Nat Rev Drug Discov 2007; 6(8):662‒
80. doi: 10.1038/nrd2222.
21. Mani S. Production of reactive oxygen species and
its implication in human diseases. In: Free
Radicals in Human Health and Disease (V Rani,
UCS Yadav). Springer, Berlin, Germany. 2015, p.
3‒15.
22. Benov L. How superoxide radical damages the cell.
Protoplasma 2001; 217(1‒3):33‒6.
23. Kehrer JP. The Haber‒Weiss reaction and
mechanisms of toxicity. Toxicology 2000;
149(1):43‒50.
24. Rani V MS, Yadav T, Yadav UCH, and Kohli S.
Hydrogen peroxide sensing and signaling. In: Free
Radicals in Human Health and Disease (V Rani,
UCS Yadav). Springer, Berlin, Germany. 2015, p.
105‒16.
25. Winterbourn CC. The biological chemistry of
hydrogen peroxide. Methods Enzymol 2013;
528:3‒25. doi: 10.1016/B978-0-12-405881-
1.00001-X.
26. Pandey KB, Rizvi SI. Redox biology of aging:
focus on novel biomarkers. In: Free Radicals in
Human Health and Disease (V Rani, UCS Yadav).
Springer, Berlin, Germany. 2015, p. 279‒90.
27. Wittmann C, Chockley P, Singh SK, Pase L,
Lieschke GJ, Grabher C. Hydrogen peroxide in
inflammation: messenger, guide, and assassin. Adv
Hematol 2012; 2012:541471. doi:
10.1155/2012/541471.
28. Liochev SI. Reactive oxygen species and the free
radical theory of aging. Free Radic Biol Med 2013;
60:1‒4. doi: 10.1016/j.freeradbiomed.2013.02.011.
29. Lipinski B. Hydroxyl radical and its scavengers in
health and disease. Oxid Med Cell Longev 2011;
2011:809696. doi: 10.1155/2011/809696.
30. Sies H. Strategies of antioxidant defense. Eur J
Biochem 1993; 215(2):213‒9.
31. Tyrrell RM. Ultraviolet radiation and free radical
damage to skin. Biochem Soc Symp 1995; 61:47‒
53.
32. Pullar JM, Vissers MC, Winterbourn CC. Living
with a killer: the effects of hypochlorous acid on
mammalian cells. IUBMB Life 2000; 50(4‒5):259‒
66. doi: 10.1080/713803731.
33. Winterbourn CC. Biological reactivity and
biomarkers of the neutrophil oxidant, hypochlorous
acid. Toxicology 2002; 181‒182:223‒7.
34. Modun D, Giustarini D, Tsikas D. Nitric oxide-
related oxidative stress and redox status in health
and disease. Oxid Med Cell Longev 2014;
2014:129651. doi: 10.1155/2014/129651.
35. Kroncke KD, Fehsel K, Kolb-Bachofen V. Nitric
oxide: cytotoxicity versus cytoprotection–how,
why, when, and where? Nitric Oxide 1997;
1(2):107‒20. doi: 10.1006/niox.1997.0118.
36. Kroncke KD, Fehsel K, Kolb-Bachofen V.
Inducible nitric oxide synthase in human diseases.
Clin Exp Immunol 1998; 113(2):147‒56.
37. Beckman JS, Koppenol WH. Nitric oxide,
superoxide, and peroxynitrite: the good, the bad,
and ugly. Am J Physiol 1996; 271(5 Pt 1):C1424‒
37.
38. Pickering AM, Vojtovich L, Tower J, KJ AD.
Oxidative stress adaptation with acute, chronic, and
repeated stress. Free Radic Biol Med 2013;
55:109‒18. doi:
10.1016/j.freeradbiomed.2012.11.001.
39. Hybertson BM, Gao B, Bose SK, McCord JM.
Oxidative stress in health and disease: the
therapeutic potential of Nrf2 activation. Mol
Aspects Med 2011; 32(4‒6):234‒46. doi:
10.1016/j.mam.2011.10.006.
40. Haliwell B, Gutteridge JMC. Oxidative stress and
redox regulation: adaptation, damage, repair,

REVIEW ARTICLES
REACTIVE OXYGEN SPECIES ǀ aimsci.com/ros 261 VOLUME 2 ǀ ISSUE 4 ǀ JULY 2016
ROS
senescence and death. In: Free Radicals in Biology
and Medicine (B. Halliwell, JMC Gutteridge), 5th
ed. Oxford University Press, Oxford, UK. 2015, p.
199‒284.
41. Betteridge DJ. What is oxidative stress?
Metabolism 2000; 49(2 Suppl 1):3‒8.
42. Cooke MS, Evans MD, Dizdaroglu M, Lunec J.
Oxidative DNA damage: mechanisms, mutation,
and disease. FASEB J 2003; 17(10):1195‒214. doi:
10.1096/fj.02-0752rev.
43. Nemera DB, Jones AR, Merino EJ. DNA
oxidation. In: Molecular Basis of Oxidative Stress:
Chemistry, Mechanisms and Disease Pathogenesis
(FA Villamena). Willey, Hoboken, NJ, USA. 2013,
p. 93‒112.
44. Niedernhofer LJ, Daniels JS, Rouzer CA, Greene
RE, Marnett LJ. Malondialdehyde, a product of
lipid peroxidation, is mutagenic in human cells. J
Biol Chem 2003; 278(33):31426‒33. doi:
10.1074/jbc.M212549200.
45. Poli G, Schaur RJ. 4-Hydroxynonenal in the
pathomechanisms of oxidative stress. IUBMB Life
2000; 50(4‒5):315‒21. doi: 10.1080/713803726.
46. Ayala A, Munoz MF, Arguelles S. Lipid
peroxidation: production, metabolism, and
signaling mechanisms of malondialdehyde and 4-
hydroxy-2-nonenal. Oxid Med Cell Longev 2014;
2014:360438. doi: 10.1155/2014/360438.
47. Davies S, Guo L. Lipid peroxidation and nitration.
In: Molecular Basis of Oxidative Stress: Chemistry,
Mechanisms and Disease Pathogenesis (FA
Villamena). Willey, Hoboken, NJ, USA. 2013, p.
49‒70.
48. Hogg N, Kalyanaraman B. Nitric oxide and lipid
peroxidation. Biochim Biophys Acta 1999; 1411(2‒
3):378‒84.
49. Violi F, Marino R, Milite MT, Loffredo L. Nitric
oxide and its role in lipid peroxidation. Diabetes
Metab Res Rev 1999; 15(4):283‒8.
50. Lovell MA, Xie C, Markesbery WR. Acrolein, a
product of lipid peroxidation, inhibits glucose and
glutamate uptake in primary neuronal cultures.
Free Radic Biol Med 2000; 29(8):714‒20.
51. Spickett CM. The lipid peroxidation product 4-
hydroxy-2-nonenal: advances in chemistry and
analysis. Redox Biol 2013; 1:145‒52. doi:
10.1016/j.redox.2013.01.007.
52. Hawkins CL, Davies MJ. Generation and
propagation of radical reactions on proteins.
Biochim Biophys Acta 2001; 1504(2‒3):196‒219.
53. Berlett BS, Stadtman ER. Protein oxidation in
aging, disease, and oxidative stress. J Biol Chem
1997; 272(33):20313‒6.
54. Giulivi C, Traaseth NJ, Davies KJ. Tyrosine
oxidation products: analysis and biological
relevance. Amino Acids 2003; 25(3‒4):227‒32. doi:
10.1007/s00726-003-0013-0.
55. Dalle-Donne I, Giustarini D, Colombo R, Rossi R,
Milzani A. Protein carbonylation in human
diseases. Trends Mol Med 2003; 9(4):169‒76.
56. Hougland J, Darling J, Flynn S. Protein
posttranslational modification. In: Molecular Basis
of Oxidative Stress: Chemistry, Mechanisms and
Disease Pathogenesis (FA Villamena). Willey,
Hoboken, NJ, USA. 2013, p. 71‒92.
57. Stadtman ER. Protein oxidation and aging. Free
Radic Res 2006; 40(12):1250‒8. doi:
10.1080/10715760600918142.
58. Stadtman ER, Moskovitz J, Levine RL. Oxidation
of methionine residues of proteins: biological
consequences. Antioxid Redox Signal 2003;
5(5):577‒82. doi: 10.1089/152308603770310239.
59. Suzuki YJ, Carini M, Butterfield DA. Protein
carbonylation. Antioxid Redox Signal 2010;
12(3):323‒5. doi: 10.1089/ars.2009.2887.
60. Dunlop RA, Brunk UT, Rodgers KJ. Oxidized
proteins: mechanisms of removal and
consequences of accumulation. IUBMB Life 2009;
61(5):522‒7. doi: 10.1002/iub.189.
61. Zhu H, Wang J, Santo A, Li Y. Downregulation of
antioxidants and phase 2 proteins. In: Molecular
Basis of Oxidative Stress: Chemistry, Mechanisms
and Disease Pathogenesis (FA Villamena). Willey,
Hoboken, NJ, USA. 2013, p. 113‒22.
62. Birben E, Sahiner UM, Sackesen C, Erzurum S,
Kalayci O. Oxidative stress and antioxidant
defense. World Allergy Organ J 2012; 5(1):9‒19.
doi: 10.1097/WOX.0b013e3182439613.
63. Gupta RK, Patel AK, Shah N, Chaudhary AK, Jha
UK, Yadav UC, et al. Oxidative stress and
antioxidants in disease and cancer: a review. Asian
Pac J Cancer Prev 2014; 15(11):4405‒9.
64. Valko M, Leibfritz D, Moncol J, Cronin MT,
Mazur M, Telser J. Free radicals and antioxidants
in normal physiological functions and human
disease. Int J Biochem Cell Biol 2007; 39(1):44‒
84. doi: 10.1016/j.biocel.2006.07.001.
65. Mruk DD, Silvestrini B, Mo MY, Cheng CY.
Antioxidant superoxide dismutase–a review: its
function, regulation in the testis, and role in male
fertility. Contraception 2002; 65(4):305‒11.

REVIEW ARTICLES
REACTIVE OXYGEN SPECIES ǀ aimsci.com/ros 262 VOLUME 2 ǀ ISSUE 4 ǀ JULY 2016
ROS
66. Buettner GR. Superoxide dismutase in redox
biology: the roles of superoxide and hydrogen
peroxide. Anticancer Agents Med Chem 2011;
11(4):341‒6.
67. Ho YS, Xiong Y, Ma W, Spector A, Ho DS. Mice
lacking catalase develop normally but show
differential sensitivity to oxidant tissue injury. J
Biol Chem 2004; 279(31):32804‒12. doi:
10.1074/jbc.M404800200.
68. Kobayashi M, Sugiyama H, Wang DH, Toda N,
Maeshima Y, Yamasaki Y, et al. Catalase
deficiency renders remnant kidneys more
susceptible to oxidant tissue injury and renal
fibrosis in mice. Kidney Int 2005; 68(3):1018‒31.
doi: 10.1111/j.1523-1755.2005.00494.x.
69. Wu G, Fang YZ, Yang S, Lupton JR, Turner ND.
Glutathione metabolism and its implications for
health. J Nutr 2004; 134(3):489‒92.
70. Brigelius-Flohe R, Maiorino M. Glutathione
peroxidases. Biochim Biophys Acta 2013;
1830(5):3289‒303. doi:
10.1016/j.bbagen.2012.11.020.
71. Chawla J, Kvarnberg D. Hydrosoluble vitamins.
Handb Clin Neurol 2014; 120:891‒914. doi:
10.1016/B978-0-7020-4087-0.00059-0.
72. Sen CK, Khanna S, Roy S. Tocotrienols in health
and disease: the other half of the natural vitamin E
family. Mol Aspects Med 2007; 28(5‒6):692‒728.
doi: 10.1016/j.mam.2007.03.001.
73. Son Y, Cheong YK, Kim NH, Chung HT, Kang
DG, Pae HO. Mitogen-activated protein kinases
and reactive oxygen species: how can ros activate
mapk pathways? J Signal Transduct 2011;
2011:792639. doi: 10.1155/2011/792639.
74. Finkel T. Signal transduction by reactive oxygen
species. J Cell Biol 2011; 194(1):7‒15. doi:
10.1083/jcb.201102095.
75. Quinn M. NADPH oxidases: structure and
function. In: Molecular Basis of Oxidative Stress:
Chemistry, Mechanisms and Disease Pathogenesis
(FA Villamena). Willey, Hoboken, NJ, USA. 2013,
p. 137‒78.
76. Magder S. Reactive oxygen species: toxic
molecules or spark of life? Crit Care 2006;
10(1):208. doi: 10.1186/cc3992.
77. Halliwell B, Gutteridge JMC. Reactive species can
be useful: some more examples. In: Free Radicals
in Biology and Medicine (B. Halliwell, JMC
Gutteridge), 5th ed. Oxford University Press,
Oxford, UK. 2015, p. 411‒62.
78. Shen J, Deininger P, Hunt JD, Zhao H. 8-Hydroxy-
2'-deoxyguanosine (8-OH-dG) as a potential
survival biomarker in patients with nonsmall-cell
lung cancer. Cancer 2007; 109(3):574‒80. doi:
10.1002/cncr.22417.
79. Gabbita SP, Lovell MA, Markesbery WR.
Increased nuclear DNA oxidation in the brain in
Alzheimer's disease. J Neurochem 1998;
71(5):2034‒40.
80. Barnham KJ, Masters CL, Bush AI.
Neurodegenerative diseases and oxidative stress.
Nat Rev Drug Discov 2004; 3(3):205‒14. doi:
10.1038/nrd1330.
81. Sultana R, Cenini G, Butterfield DA. Biomarkers
of oxidative stress in neurodegenerative diseases.
In: Molecular Basis of Oxidative Stress: Chemistry,
Mechanisms and Disease Pathogenesis (FA
Villamena). Willey, Hoboken, NJ, USA. 2013, p.
359‒76.
82. Halliwell B, Whiteman M. Measuring reactive
species and oxidative damage in vivo and in cell
culture: how should you do it and what do the
results mean? Br J Pharmacol 2004; 142(2):231‒
55. doi: 10.1038/sj.bjp.0705776.
83. Dalle-Donne I, Rossi R, Giustarini D, Milzani A,
Colombo R. Protein carbonyl groups as biomarkers
of oxidative stress. Clin Chim Acta 2003; 329(1‒
2):23‒38.
84. Datta S, Kundu S, Ghosh P, De S, Ghosh A,
Chatterjee M. Correlation of oxidant status with
oxidative tissue damage in patients with
rheumatoid arthritis. Clin Rheumatol 2014;
33(11):1557‒64. doi: 10.1007/s10067-014-2597-z.
85. Pala FS, Gürkan H. The role of free radicals in
ethiopathogenesis of diseases. Advances in
Molecular Biology 2008; (1):1‒9.
86. Kohlstadt I. Scientific Evidence for
Musculoskeletal, Bariatric, and Sports Nutrition.
CRC Press, Boca Raton, FL, USA. 2006.
87. Azzam EI, Jay-Gerin JP, Pain D. Ionizing
radiation-induced metabolic oxidative stress and
prolonged cell injury. Cancer Lett 2012; 327(1‒
2):48‒60. doi: 10.1016/j.canlet.2011.12.012.
88. Hinson JA, Roberts DW, James LP. Mechanisms
of acetaminophen-induced liver necrosis. Handb
Exp Pharmacol 2010; (196):369‒405. doi:
10.1007/978-3-642-00663-0_12.
89. Knockaert L, Berson A, Ribault C, Prost PE,
Fautrel A, Pajaud J, et al. Carbon tetrachloride-
mediated lipid peroxidation induces early
mitochondrial alterations in mouse liver. Lab Invest
2012; 92(3):396‒410. doi:
10.1038/labinvest.2011.193.

REVIEW ARTICLES
REACTIVE OXYGEN SPECIES ǀ aimsci.com/ros 263 VOLUME 2 ǀ ISSUE 4 ǀ JULY 2016
ROS
90. Shizukuda Y, Bolan CD, Nguyen TT, Botello G,
Tripodi DJ, Yau YY, et al. Oxidative stress in
asymptomatic subjects with hereditary
hemochromatosis. Am J Hematol 2007; 82(3):249‒
50. doi: 10.1002/ajh.20743.
91. Kalita J, Kumar V, Misra UK, Ranjan A, Khan H,
Konwar R. A study of oxidative stress, cytokines
and glutamate in Wilson disease and their
asymptomatic siblings. J Neuroimmunol 2014;
274(1‒2):141‒8. doi:
10.1016/j.jneuroim.2014.06.013.
92. Chou R, High Value Care Task Force of the
American College of P. Cardiac screening with
electrocardiography, stress echocardiography, or
myocardial perfusion imaging: advice for high-
value care from the American College of
Physicians. Ann Intern Med 2015; 162(6):438‒47.
doi: 10.7326/M14-1225.
93. Bagai A, Dangas GD, Stone GW, Granger CB.
Reperfusion strategies in acute coronary
syndromes. Circ Res 2014; 114(12):1918‒28. doi:
10.1161/CIRCRESAHA.114.302744.
94. Bolli R, Zughaib M, Li XY, Tang XL, Sun JZ,
Triana JF, et al. Recurrent ischemia in the canine
heart causes recurrent bursts of free radical
production that have a cumulative effect on
contractile function: a pathophysiological basis for
chronic myocardial "stunning". J Clin Invest 1995;
96(2):1066‒84. doi: 10.1172/JCI118093.
95. Reimer KA, Lowe JE, Rasmussen MM, Jennings
RB. The wavefront phenomenon of ischemic cell
death. 1. Myocardial infarct size vs duration of
coronary occlusion in dogs. Circulation 1977;
56(5):786‒94.
96. Chin KY, Qin C, May L, Ritchie RH, Woodman
OL. New pharmacological approaches to the
prevention of myocardial ischemia-reperfusion
injury. Curr Drug Targets 2015 [Epub ahead of
print].
97. Bielli A, Scioli MG, Mazzaglia D, Doldo E,
Orlandi A. Antioxidants and vascular health. Life
Sci 2015; 143:209‒16. doi:
10.1016/j.lfs.2015.11.012.
98. He F, Zuo L. Redox roles of reactive oxygen
species in cardiovascular diseases. Int J Mol Sci
2015; 16(11):27770‒80. doi:
10.3390/ijms161126059.
99. Madmani ME, Yusuf Solaiman A, Tamr Agha K,
Madmani Y, Shahrour Y, Essali A, et al.
Coenzyme Q10 for heart failure. Cochrane
Database Syst Rev 2014; 6:CD008684. doi:
10.1002/14651858.CD008684.pub2.
100. Vardi M, Levy NS, Levy AP. Vitamin E in the
prevention of cardiovascular disease: the
importance of proper patient selection. J Lipid Res
2013; 54(9):2307‒14. doi: 10.1194/jlr.R026641.