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Free Radicals as Mediators of Tissue Injury and Disease
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Critical Reviews in Toxicology
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Free radicals and related reactive species

as mediators of tissue injury and disease:
implications for Health
James P. Kehrer & Lars-Oliver Klotz
To cite this article: James P. Kehrer & Lars-Oliver Klotz (2015) Free radicals and related reactive
species as mediators of tissue injury and disease: implications for Health, Critical Reviews in
Toxicology, 45:9, 765-798, DOI: 10.3109/10408444.2015.1074159
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CRITICAL REVIEWS IN TOXICOLOGY, 2015
VOL. 45, NO. 9, 765–798
http://dx.doi.org/10.3109/10408444.2015.1074159
Free radicals and related reactive species as mediators of tissue injury and
disease: implications for Health
James P. Kehrer1 & Lars-Oliver Klotz2
1
Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada and 2Department of Nutrigenomics,
Institute of Nutrition, Friedrich Schiller University Jena, Jena, Germany
ABSTRACT KEYWORDS
A radical is any molecule that contains one or more unpaired electrons. Radicals are normal Antioxidants, autoxidation,
products of many metabolic pathways. Some exist in a controlled (caged) form as they perform cell signaling, free radicals,
essential functions. Others exist in a free form and interact with various tissue components. Such mitochondria, oxidative
interactions can cause both acute and chronic dysfunction, but can also provide essential control of stress, reactive oxygen
redox regulated signaling pathways. The potential roles of endogenous or xenobiotic-derived free species, redox cycling
Downloaded by [University of Alberta] at 08:10 30 November 2015
radicals in several human pathologies have stimulated extensive research linking the toxicity of
HISTORY
numerous xenobiotics and disease processes to a free radical mechanism. In recent years, Received 5 May 2015
improvements in analytical methodologies, as well as the realization that subtle effects induced by Revised 14 July 2015
free radicals and oxidants are important in modulating cellular signaling, have greatly improved our Accepted 15 July 2015
understanding of the roles of these reactive species in toxic mechanisms and disease processes. Published online 28 August
However, because free radical-mediated changes are pervasive, and a consequence as well as a 2015
cause of injury, whether such species are a major cause of tissue injury and human disease remains
unclear. This concern is supported by the fact that the bulk of antioxidant defenses are enzymatic
and the findings of numerous studies showing that exogenously administered small molecule
antioxidants are unable to affect the course of most toxicities and diseases purported to have a free
radical mechanism. This review discusses cellular sources of various radical species and their
reactions with vital cellular constituents, and provides examples of selected disease processes that
may have a free radical component.
Table of contents Transition metals ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 774
Introduction ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...766 Nanoparticles ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 775
Free radicals as causes and consequences of diseases Mechanisms of free radical toxicity ... ... ... ... ... ... ... ...776
and toxicities ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...766 Specific biological targets ... ... ... ... ... ... ... ... ... ... ... 776
Proving the involvement of free radicals in a biological Lipid oxidation ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 776
effect ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 766 Protein oxidation ... ... ... ... ... ... ... ... ... ... ... ... ... ... 778
General features of diseases and injuries DNA oxidation ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 778
associated with free radicals ... ... ... ... ... ... ... ... 766 RNA oxidation ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 779
Definitions ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...767 Carbohydrates ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 779
Chemistry and reactivity of free radicals in biological Cell signaling ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 779
systems ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...769 Apoptosis ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 781
Reactive oxygen species ... ... ... ... ... ... ... ... ... ... ... ... 769 Biological responses to free radicals ... ... ... ... ... ... ... ...781
Other radical species and their progeny ... ... ... ... ... 769 Cancer ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 781
Characteristic radical reactions ... ... ... ... ... ... ... ... ... ...770 Immune system and inflammatory processes ... ... 784
Cellular sources of free radicals ... ... ... ... ... ... ... ... ... ...771 Rheumatoid arthritis ... ... ... ... ... ... ... ... ... ... ... ... 785
NADPH oxidases ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 771 Chemoprevention ... ... ... ... ... ... ... ... ... ... ... ... ... 785
Mitochondrial electron transport ... ... ... ... ... ... ... ... 772 Cardiovascular disease ... ... ... ... ... ... ... ... ... ... ... ... 786
Microsomal electron transport ... ... ... ... ... ... ... ... ... 773 Chemoprevention ... ... ... ... ... ... ... ... ... ... ... ... ... 787
Soluble oxidase enzymes ... ... ... ... ... ... ... ... ... ... ... ... 774 The toxicity of oxygen ... ... ... ... ... ... ... ... ... ... ... ... ... 787
Auto-oxidation of endogenous or exogenous Phototoxicity ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 788
substrates ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 774 Diabetes and the metabolic syndrome ... ... ... ... ... 788
Correspondence: James P. Kehrer, PhD, Faculty of Pharmacy & Pharmaceutical Sciences, MS 2-35F, University of Alberta, 8613 - 114th St., Edmonton, Alberta
T6G 2H1, Canada. Tel: +1 780-492-1685. jkehrer@ualberta.ca
! 2015 Taylor & Francis
766 J. P. KEHRER & L.-O. KLOTZ
Tissue protection systems ... ... ... ... ... ... ... ... ... ... ... ... ...789 radical reactions. As some of these changes can also
Enzymes scavenging ROS ... ... ... ... ... ... ... ... ... ... ... 789 originate from non-radical reactions [e.g. the loss of
Enzymes catalyzing repair of ROS-induced damage 789 glutathione (GSH) can result from alkylation reactions as
Indirect antioxidant enzyme systems ... ... ... ... ... ... 790 well as oxidation], such measurements are rarely ideal.
Non-enzymatic antioxidants ... ... ... ... ... ... ... ... ... ... 790 Furthermore, many of the products measured are
Conclusions ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...791 produced at very low levels (e.g. isoprostanes) and are
Acknowledgements ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...791 unlikely to be directly linked to the observed toxicity.
Declaration of interest ... ... ... ... ... ... ... ... ... ... ... ... ... ...791 Nevertheless, various detectable products of free radical-
References ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...791 mediated reactions have been utilized as potential
biomarkers of the generation of reactive oxygen species
(ROS) (Table 1). Yet the detection of ROS, using probes or
Introduction biomarkers, is only one step in determining whether that
species is the cause of a biological effect elicited by
In 1993, one of us (J. P. K.) wrote a comprehensive review some stimulus. In order to link a specific reactive species
article for Critical Reviews in Toxicology (Kehrer 1993) to a biological effect, the same effect needs to be
covering the basics of free radical biochemistry, as well elicited upon exposure to a different source of the same
as our understanding at that time of the roles of free
reactive species. For example, if hydrogen peroxide

radicals in selected toxicities and diseases. In the (H2O2) is suspected to mediate the stimulation of
intervening years, there have been numerous advances signaling elicited by ultraviolet (UV) radiation, the same
in our understanding of these reactive species that signaling effect needs to be brought about by a different
should be considered by researchers in this field. Thus, source of H2O2. In other words, H2O2 needs to mimic UV
the current review was prepared to help those with an radiation with respect to the biological effect under
interest in free radicals to better understand their roles in investigation. Moreover, in a third step, interfering with
toxicities, health and diseases. In order to effectively the biological effect using a scavenger of the respective
cover the burgeoning literature in the very broad area of species, such as catalase or another peroxidase in the
free radical toxicology, many of the references cited case of H2O2, should be done (von Montfort et al. 2006).
herein are to other reviews that provide substantially
more depth on specific topics.
General features of diseases and injuries
associated with free radicals
Free radicals as causes and consequences of
A role for free radicals has been proposed in the toxicity
diseases and toxicities
of numerous chemicals and in the pathogenesis of many
Free radicals are generated in biological systems both as diseases (Table 2). The list of disorders in which free
a result of normal cellular aerobic metabolism as well as radicals are implicated is extensive because these
from abnormal reactions stimulated by some disease reactive molecules can produce most of the tissue
processes and xenobiotics (Sies 1986). Although free changes that have been identified during a variety of
radicals can explain the pathology of many toxicities and injurious processes. However, many of these changes
disease processes (Roberts et al. 2010), their causal role can be a consequence rather than a cause of damage
in specific disorders continues to be difficult to deter- and, with very few exceptions, the direct causal associ-
mine. For example, the same free radical-mediated ation of free radicals with any specific toxicity or disease
changes that may cause injury also occur secondary to has been difficult to establish. In this sense, more specific
that injury. This includes lipid peroxidation that clearly biological end points are essential. In the absence of
damages membranes but will also increase subsequent such endpoints, free radical mechanisms of injury will
to cell death. Thus, determining whether free radicals are continue to be invoked even though many of the
a cause, effect or both in toxicity and disease is parameters measured reflect tissue changes that are not
problematic. always specific for radical-mediated processes or even
linked solidly with cell damage.
The most important, and frequently overlooked,
Proving the involvement of free radicals in a
requirement for linking free radicals to a specific toxicity
biological effect
or disease is to be certain that the parameter(s) being
The strength of the links between free radicals and measured reflect injury. Many biological responses to
different diseases and toxicities is often dependent upon free radical-mediated perturbations are adaptive in
accurate measurements of changes related to free nature and may not be detrimental to the function of
CRITICAL REVIEWS IN TOXICOLOGY 767
cells or tissues; although this might change if the Table 1. Biomarkers of radical reactions.
changes become chronic. In addition, as already men- Lipids
tioned, some clearly deleterious phenomena, such as Lipid peroxides or products (lipid hydroxy acids or hydroperoxides)
Isoprostanes
lipid peroxidation, also occur subsequent to cell death Malondialdehyde (usually thiobarbituric acid reactive substances)
and membrane lysis, so that finding lipid peroxide Conjugated dienes
Ethane/pentane evolution
products in tissues in any disease or toxic process – Fatty acid analyses: loss of PUFA, formation of lipid hydroperoxides and/or
while suggesting the formation of ROS capable of lipid alcohols
Proteins
inducing lipid peroxidation – provides no evidence Protein oxidation
those free radicals were involved in causing that Sulfur oxidation (Cys disulfides, S-thiolation, Met sulfoxide,
mixed disulfides)
disorder. Rather, such products may represent secondary Protein carbonyls (side-chain aldehydes, ketones)
damage or they cause such secondary damage. Tyrosine cross-links, chlorination, nitrosation, hydroxylation
Tryptophanyl modifications
Hydro(pero)xy derivatives of aliphatic amino acids
Chloramines, deamination
Definitions Amino acid inter-conversions (e.g. His to Asn; Pro to OH-Pro)
Cross-links, aggregation, peptide bond cleavage
It is important to have a clear definition of injury so that DNA
homeostatic responses are not confused with damage. It Oxidized purines (8-hydroxydeoxyguanine) and pyrimidines
Adducts
should also be clear that, while free radicals may initiate Products of lipid peroxidation (e.g. malondialdehyde, HNE, acrolein)
a series of damaging biochemical events, they are not Products of amino acid oxidation (e.g. p-hydroxyphenylacetaldehyde);
Glycoxidation (e.g. carboxymethyllysine)
necessarily directly responsible for the final tissue Covalent binding to lipids
dysfunction. Conversely, free radicals may arise subse- Enzyme activities
Induction of antioxidant enzymes; both mRNA and protein levels
quent to some earlier events that are the proximate Loss or gain of activity of oxidant-sensitive enzymes
cause of tissue injury, but these radicals may have either Antioxidant contents
Loss of glutathione
no effect or ameliorate or aggravate the final damage. Formation of glutathione disulfide
This could result in there being no biomarker evidence Vitamin E
of free radical-mediated injury even though radicals did, Vitamin C
-carotene
in fact, contribute to the injury. Miscellaneous
In this review, injury is defined as any alteration that Myoglobin oxidation
detracts from the viability or essential functions of a cell,
tissue or organism. Of course, cell death is not always
undesirable to the organism as a whole (Bursch et al. scientific literature to refer to free radicals. These
1992). For example, radicals that induce apoptosis may include oxyradicals, oxygen free radicals and various
be necessary for the survival and optimal function of the combinations and permutations of these words. The
entire organism, but should not be considered to cause term ROS is generally preferred because singlet
injury (Ryter et al. 2007). oxygen, H2O2, hypochlorous acid and peroxide and
The sensitivity of the index used to assess cell and hydroperoxide and epoxide metabolites of endogen-
tissue damage will determine whether injury can be ous lipids and xenobiotics, contain chemically reactive
detected, but not always whether injury has actually oxygen-containing functional groups, but are not
occurred. For example, changes in glutathione (GSH)/ radicals and do not necessarily interact with tissues
glutathione disulfide (GSSG) ratios that are routinely through radical reactions. Furthermore, it must be
measured to assess oxidative stress have been observed remembered that, in biological systems, the super-
when damage to tissues or cell functions is not evident oxide radical (O 2 ) may even be a better reducing
(Mitchell et al. 1973; Robertson et al. 1998; Smith 1991). than oxidizing species. Thus, referring to ROS solely as
It also must be recognized that the ability to detect oxidizing agents is misleading. Carbon-, nitrogen- and
elevated levels of radicals directly using spin trapping sulfur-centered radicals also may occur in biological
agents prior to, or concomitant with, injury is consistent systems and are important in initiating and/or
with a role for these species in the damage, but does not propagating various types of injury.
establish a cause–effect relationship. Additional studies Radicals in the context of toxicology refer to those
are required to demonstrate that these radicals actually that exist in a free and uncombined state, and are able
interact with and damage tissue components (see to interact with various tissue components. Caged
above). radicals, that are retained within their sites of gener-
Free radicals are chemical species with one or more ation (e.g. those that transport electrons in the
unpaired electrons. A group of related (but not mitochondria or are involved in some enzymatic
necessarily synonymous) terms are used in the reactions), also exist in biological systems. Caged
Table 2. Diseases/tissue injuries that may have a free radical radicals are immobilized in a form that makes their
component. interaction with other molecules highly unlikely. In
Lung contrast, free radicals are diffusible, enhancing their
Normobaric hyperoxic injury
Bronchopulmonary dysplasia availability for interaction with other molecules.
Inhaled oxidants: SO2, NOx, O3 Moreover, the free (i.e. not spin-restricted; see
Asbestosis
Chemicals: paraquat, bleomycin
‘‘Reactive oxygen species’’ section) nature of the
Adult respiratory distress syndrome electrons in free radicals makes them able to combine
Emphysema readily with other unpaired and uncaged electrons
Cigarette smoke
Idiopathic pulmonary fibrosis within a tissue in order to achieve a more stable paired
Heart and cardiovascular system electron status.
Ischemia/reperfusion: after infarction or transplant
Chemicals: ethanol, doxorubicin Many changes can occur once a free radical interacts
Atherosclerosis with a tissue. These changes can be defined in terms of
Selenium deficiency (Keshan disease)
Hemochromatosis injury or other more general responses. The term
Kidney ‘‘oxidative stress’’ was originally defined to ‘‘denote a
Autoimmune nephrosis: inflammation
Chemicals: aminoglycosides, heavy metals disturbance in the prooxidant–antioxidant balance in
Liver favor of the former’’ (Sies 1985). However, this defin-
Ischemia/reperfusion
Chemicals: halogenated hydrocarbons, quinones, iron, acetaminophen,

ition does neither address the potential for detrimental
ethanol effects of such a change on the function of a tissue, nor
Endotoxin
GI tract
it indicate whether this disturbance results from
Ischemia/reperfusion increased ROS production or decreased tissue homeo-
Chemicals: non-steroidal anti-inflammatory agents static responses. Importantly, this definition also does
Blood
Chemicals: phenylhydrazine, primaquine and related drugs, sulfonamides, not consider deviations from steady-state in that the
lead prooxidant–antioxidant balance cannot be changed
Protoporphyrin photoxidation
Malaria system-wide, only in ‘‘compartments’’. As a result,
Various anemias: sickle cell, favism other definitions and caveats have been proposed,
Eye
Retinopathy of prematurity (oxygen) such as the more recent definition of oxidative stress as
Photic retinopathy ‘‘an imbalance between oxidants and antioxidants in
Cataracts
Skin favor of the oxidants, leading to a disruption of redox
Radiation: solar or ionizing signaling and control and/or molecular damage’’ (Sies &
Thermal injury
Chemicals: photosensitizers (i.e. tetracyclines)
Jones 2007). The merits and pitfalls of the term
Contact dermatitis oxidative stress have been recently considered by Sies
Porphyria (2015).
Muscle
Muscular dystrophy Despite these caveats, many investigators have
Multiple sclerosis equated oxidative stress with the increased generation
Exercise
Miscellaneous/general of ROS, and have implied that this term is synonymous
AIDS with damage. Associating oxidative stress with injury
Aging
Radiation injury might be expected inasmuch as this is the logical
Trauma consequence of a disturbance in the redox status of a
Chemicals
Alloxan (diabetes); cell by reactive oxygen or other free radical species.
Iron overload However, tissue changes that meet the definition of
Radiosensitizers
Immune disorders
oxidative stress also could arise from changes in
Rheumatoid arthritis antioxidant defenses or repair systems and would not
Autoimmune diseases such as Lupus necessarily be detrimental to the tissue or cell. In fact,
Inflammation
Nervous system some redox reactions are critical for normal cell func-
Aluminum-induced neuropathy tions including cell signaling (Barthel & Klotz 2005;
Alzheimer’s disease
Amyotrophic lateral sclerosis Burhans & Heintz 2009; Go & Jones 2010; Jones 2008; Ray
Down’s syndrome et al. 2012; Sies 2014; Watson et al. 2004) and apoptosis
Hyperbaric hyperoxic injury
Myasthenia gravis (Ray et al. 2012).
Neuronal ceroid-lipofuscinosis (Batten’s disease) As noted above, oxidation must be accompanied by
Neurotoxins (e.g. 6-hydroxydopamine, MPTP)
Parkinson’s disease reduction. Thus, a focus on oxidation may miss other
Ischemia/reperfusion compartmentalized changes that are critical for dysfunc-
Werdnig–Hoffman disease
tion. For example, concomitant ‘‘reductive stress’’ might
affect radical production and cell functions including
redox-controlled enzymes and transcription factors molecules resulting in the difficulties in conclusively
(Finkel 2003; Forman et al. 2010). Overall, although linking free radicals with injury and disease.
research has focused on oxidation, the obligatory Superoxide is relatively unreactive toward most bio-
reductive reactions may also contribute to cell dysfunc- logical molecules – except for its known interaction with
tion (Kehrer 2007; Levonen et al. 2014). transition metals and transition metal complexes,
including iron/sulfur clusters (Bielski & Cabelli 1991),
and with other radicals, particularly nitrogen monoxide
Chemistry and reactivity of free radicals in (NO, see below) and O 2 itself (in terms of a dispro-
biological systems portionation reaction). Interestingly, O 2 may act both
Reactive oxygen species as a moderately active reductant and oxidant and forms
the uncharged hydroperoxyl radical (HOO) in aqueous
Although technically a diradical (having two unpaired
media (pK &4.8). At physiological pH (7), the predom-
electrons), ground state or triplet oxygen is poorly
inant species is the anion radical, but a local more acidic
reactive due to spin restriction. The addition of sufficient
environment (including in the vicinity of biological
energy to change the spin of one of these two unpaired
membranes) will shift the equilibrium towards the
electrons removes this spin restriction and produces two
formation of HOO, which is more reactive than the
reactive forms at different energy levels called singlet
superoxide anion radical, i.e. is both a better reductant

oxygen (1O2). One of these forms – ‘‘singlet oxygen
and oxidant (Buettner 1993).
sigma’’ – has such a short lifetime (Weldor et al. 1999)
Although the direct biological reactivity of H2O2 is
that it is unlikely to be of relevance in a biological
relatively modest, concentrations in vivo are kept very
setting. In contrast, the formation of the more stable
low. In contrast to the charged superoxide anion, H2O2
‘‘singlet delta’’ form, with a half-life of microseconds in
can penetrate membranes to some extent. This pene-
an aqueous milieu, is detectable in biological systems.
tration is augmented by cellular water channels, the
As 1O2 may be generated during lipid peroxidation
aquaporins (AQPs): because of its close relationship to
through reactions involving the decomposition of lipid
water, H2O2 readily enters cells via AQPs such as AQP3
hydroperoxides (Miyamoto et al. 2014) and following the
and AQP8, crossing cell membranes more rapidly than
stimulation of neutrophils (Briviba et al. 1997; Klotz
by diffusion (Henzler & Steudle 2000; Vieceli Dalla Sega
2002), and because it readily oxidizes numerous bio- et al. 2014). Importantly, H2O2 can accept an electron
logical molecules (Klotz et al. 2003), its role in toxicology from transition metals, principally Cu+ and Fe2+, to
was once considered significant. However, this needs to generate the hydroxyl radical, a far more reactive
be more critically evaluated as the debate on whether species.
1
O2 is of significance in the modulation of (patho-) The hydroxyl radical is the most reactive species of
physiological processes is ongoing (Pryor et al. 2006). oxygen. It has a half-life in biological systems of about
Nevertheless, there is significant evidence that 1O2 is one nanosecond and reacts indiscriminately with
formed in vivo, particularly photochemically in areas organic molecules at diffusion-limited rates (Buxton
exposed to UV radiation, and that it may mediate et al. 1988). Based on this lack of specificity, the
adverse effects of UV radiation (Klotz 2002). Moreover, biological effectiveness of purported hydroxyl radical
1
O2 plays a key role in the toxicity of photosensitizers scavengers is questionable. Specifically, in biological
(Briviba et al. 1997, see ‘‘Phototoxicity’’ section) and may systems, the concentrations of such scavengers would
be involved in the toxicity of TiO2 nanoparticles in need to be equal to the concentrations of all present
human keratinocytes (Fenoglio et al. 2013; see biological molecules (i.e. in the order of 50 molar) in
‘‘Transition metals’’ section). order to inhibit the initial reactions by half. Overall, spin-
In addition to adding energy, the spin restriction on trapping studies have demonstrated that hydroxyl
the reactivity of oxygen also can be removed by forming radicals are formed in biological systems but their
partially reduced species. The one-, two- and three- significance in specific types of injury remains unclear.
electron reduction products of oxygen, O 2 , H2O2 and
the hydroxyl radical, respectively, are clearly produced in
biological systems. These ROS participate in numerous Other radical species and their progeny
reactions essential to aerobic organisms. However, their The prevalence of oxygen in biological systems means
reactive nature can lead to injury if not controlled by that oxygen-centered radicals are the most common
endogenous antioxidant systems. The chemistry of free type found. However, organic molecules contain other
radical reactions is complex and this complexity is atoms that can exist as free radicals, and these can also
further heightened by interactions with biological play a role in tissue injury. For example, sulfur-centered
thiyl radicals (RS) can be formed from endogenous thiol precursors metabolized to quinones (e.g. vicine, as found
compounds such as GSH, and subsequent to homolytic in fava beans), and certain drugs or metal ions (Figure 1).
cleavage of disulfide bonds in proteins. These radicals The roles of nitrogen monoxide (nitric oxide, NO) as a
are quite reactive and form peroxysulfenyl radicals neurotransmitter and vasodilator are now well-estab-
(RSOO) upon interacting with molecular oxygen. lished. However, it is important to remember that NO is
Although a thiyl radical may combine with another a free radical and a key component of the immune
thiyl to generate a disulfide, hydrogen abstraction by a response (Bogdan 2001). Although only moderately
thiyl radical is a more likely consequence unless two thiyl reactive itself, allowing for diffusion distances required
radicals are generated in close proximity. Disulfide for NO to perform the above tasks, NO will react readily
formation is more likely to occur through non-radical with other radicals and can contribute to tissue injury
reactions such as the nucleophilic attack of a thiolate (Pacher et al. 2006). For example, NO reacts with O2 in
(RS) at a sulfenic acid moiety (RS-OH) (Jacob et al. a diffusion-limited reaction to form peroxynitrite, a
2003). Similarly, although GSSG may in theory be strong oxidant and nitrating species. It is short-lived as
generated from two GS radicals (which is unlikely in compared to NO, but has a sufficient lifetime to be of
vivo based on the low steady-state concentrations of significance in vivo (Radi 2013). In addition to its direct
GS), it is more likely that GS react with GSH/GS to toxicity, peroxynitrite decomposes to other damaging
form the glutathione disulfide anion radical (GSSG),
radicals including the hydroxyl radical and, in the

which may further react to reduce oxygen to generate presence of carbon dioxide and via peroxynitrosocarbo-
superoxide and GSSG (Winterbourn & Hampton 2008). nate formation and decomposition, the carbonate and
However, the vast majority of GSSG produced endogen- nitrogen dioxide radicals. In mammalian cells, peroxyni-
ously is from enzymatic pathways involving the GSH trite has numerous deleterious effects, ranging from
peroxidases (Ross 1988). oxidation and nitration of biomolecules to the disturb-
Carbon-centered radicals may arise in biological ance of signaling processes and the induction of cell
systems following hydrogen abstraction from unsatur- death (Klotz 2002; Radi 2013; Radi et al. 2001; Szabo et al.
ated bonds in fatty acids, or the metabolism of xeno- 2007).
biotics such as carbon tetrachloride. They are usually
short-lived because of very rapid reactions with molecu-
Characteristic radical reactions
lar oxygen to generate peroxyl radicals (ROO). For
example, the carbon-centered radicals produced by There are five basic reactions characteristic of radicals
abstraction of bis-allylic hydrogen atoms from polyun- (Table 3). These fall into three categories: (1) initiation
saturated fatty acids react with oxygen leading to lipid reactions where the net numbers of radical species
peroxidation. increase. These reactions can involve oxidation, reduc-
Redox cycling can be a source of ROS in biological tion or homolytic cleavage of a covalent bond; (2)
systems and lead to toxicity (Kappus & Sies 1981). For propagation reactions where the numbers of radicals do
example, semiquinone radicals can have extremely long not change; and (3) termination reactions where the
half-lives (up to days at 37 C) and tend to be neither products contain no radical species. Free radical reac-
reactive nor toxic. However, semiquinone radicals can tions constantly affect biological molecules such as DNA,
donate their excess electron to molecular oxygen, protein and lipids as a consequence of the aerobic
thereby generating O 2 . If the parent quinone is again environment in which we live. Fortunately, cells have
reduced by an endogenous enzyme, a redox-cycling developed a battery of defenses to prevent and/or
process is established that generates substantial quan- repair the injury associated with oxidative changes
tities of superoxide and H2O2 and depletes reduced to DNA, protein and lipids (see ‘‘Tissue protection
pyridine nucleotides (Figure 1) leading to toxicity. systems’’ section). It is only when the homeostatic
This cycling reaction is normally prevented in vivo by mechanisms fail to keep pace with these reactions that
NAD(P)H:quinone oxidoreductase-1 (NQO-1). This detrimental effect become evident.
enzyme catalyzes a two-electron reduction of quinones Products arising from each of the characteristic radical
thereby bypassing the radical intermediates and allow- reactions have been identified in biological systems and
ing for coupling of the resulting hydroquinone form to studied in relation to tissue injury. In addition, radicals
hydrophilic moieties such as sulfate or glucuronic acid in can function as signaling species, both directly and
phase II metabolism (Nishiyama et al. 2010). Redox through numerous redox-regulated transcription factors
cycling is the basis of ROS generation in cells exposed to (Ray et al. 2012). This suggests that free radical-mediated
various xenobiotics, including natural quinones (e.g. effects in biological systems can be both overt (i.e. gross
naphthoquinones; Klaus et al. 2010; Klotz et al. 2015), damage), and subtle (i.e. changes in signaling).
Figure 1. Redox cycling of quinones and transition metal ions. Quinone moieties of many xenobiotics can accept a single electron
(denoted as [H]) from endogenous flavin enzyme systems, producing a semiquinone. The semiquinone can either be reduced further
(dashed arrows) to generate a hydroquinone – in part through disproportionation –, or be re-oxidized by molecular oxygen, which is
thereby reduced to superoxide. NADPH:quinone oxidoreductase-1 (NQO-1) can perform a two-electron reduction of quinones to the
hydroquinone, thereby diminishing the possibility of redox cycling.
Most free radical products formed in biological Table 3. Free radical reactions.
systems are the result of propagation reactions; hydro- Initiation RH + energy ! R
gen abstraction, electron transfer and addition. Hydrogen abstraction A + RH ! AH + R
Propagation reactions are eventually halted through Electron transfer X + Y ! X + Y-
Addition X + RCH ¼ CHR !
termination or disproportionation reactions. True ter- Termination A + A ! A2
mination reactions result from the interaction of two Disproportionation CH3CH2 + CH3CH2 ! CH3CH3 + CH2¼CH2
radicals. In contrast, non-radical antioxidants, such as
a-tocopherol, only retard the propagation of radical
reactions by the generation of radical species of much monocytes). When activated to begin phagocytosis
lower reactivities. (although phagocytosis itself does not have to occur),
these cells exhibit a marked increase in oxygen con-
sumption. This ‘‘oxidative burst’’ (or respiratory burst) of
Cellular sources of free radicals
activated phagocytes was shown by Babior et al. (1973)
There are a number of intracellular sources for free to involve the rapid reduction of oxygen to O 2 .
radicals and ROS that have been identified (Figure 2). Subsequent work demonstrated that this reaction is
The importance of each source in any specific cell and catalyzed by a plasma membrane-bound NADPH oxi-
disorder is often unknown, and the relative role each dase with the extracellular production of large amounts
plays in tissue injury seems certain to vary with the of ROS (Figure 3). Phagocytes also generate hypohalous
specific experimental conditions used. As noted previ- acids and NO (Moncada & Higgs 1993) that contribute
ously, it also appears likely that radical formation is often to signaling and microbicidal activities, and tissue injury
secondary to the primary disease process. For example, (Calcerrada et al. 2011).
following many types of tissue injury, including trauma, The importance of ROS in the normal functioning of
cells will rupture and release their contents. These the immune system is exemplified by the enhanced
contents will include transition metal ions, such as iron, susceptibility to infections of patients suffering from
that can rapidly catalyze additional radical-mediated chronic granulomatous disease. This disorder is caused
tissue injury. Phagocyte activation and the disruption of by a defect in the NADPH oxidase activity in phagocytes,
mitochondrial function also may result in the formation which impairs the killing of invading organisms (Kuhns
of excess ROS. As these processes routinely accompany et al. 2010). However, the role for free radicals in the
and aggravate tissue injury, separating cause from effect immune system extends well beyond this effect. For
is difficult. example, free radicals may play a role in generating
chemotactic factors that recruit additional phagocytic
cells (Petrone et al. 1980). The damage produced by
NADPH oxidases
radicals liberated from phagocytic cells can spill beyond
A well-recognized biological source of free radicals is the intended target and produce injury to surrounding
activated phagocytic cells (e.g. neutrophils and tissues leading to a continuation of the damaging
Xanthine oxidase
Electron transport /
Autoxidation (e.g., flavins; catechols)
respiratory chain
Transition metal ions (Fe, Cu)
Cytoplasm
Oxidases & flavoproteins
Myeloperoxidase
(phagocytes)
Lipoxygenases
Prostaglandin synthase
NADPH oxidases
Mixed-function oxidase electron

transport
Cytochromes P450, etc.
Figure 2. Cellular sources of free radicals. Free radicals and other reactive species are produced by cells through the action of various
soluble and membrane-bound enzymes. The capacity of specific pathways to produce free radicals varies with the cell type, but all
aerobic cells are capable of some production. The cell diagram is reproduced courtesy of Stefano Tartarotti.
process (Figure 3) (Babior 2000). Additional roles for publications as demonstrating that mitochondria are a
radicals in modulating the immune system have been major source of ROS. What has been ignored is that
identified in recent years and are discussed in more this 2% figure was calculated from data obtained in a
detail in the ‘‘Immune system and inflammatory highly artificial system, using isolated mitochondria
processes’’ section. Taken together, the available data with succinate as the sole reducing substrate, and that
clearly identify free radicals as a critical component in it only applies to state 4 respiration. Under conditions
the functioning of the immune system. of state 3 respiration, production of ROS decreases
Although originally identified as membrane-bound (Cortassa et al. 2014). Although it has been demon-
flavoenzymes responsible for the generation of O 2 in strated in the perfused rat liver that mitochondria
phagocytes upon their stimulation, NADPH oxidase approach state 4, and it is possible that state 4
complexes (with five different isoforms of the catalytic conditions are obtained under hypoxic, ischemic and
subunit, NOX 1 through 5, identified) are present in some toxic conditions, the normal in vivo conditions of
many non-phagocytes, activated by numerous stimuli respiring mitochondria are unknown. Reports suggest
including proinflammatory factors, and are crucial medi- the actual production of radicals by intact mitochondria
ators in cellular signaling processes (Katsuyama et al. in healthy tissues may be as low as 0.15% of total
2012; Schulz et al. 2014). oxygen consumption (Nohl et al. 2003; St-Pierre et al.
2002). On the other hand, the steady-state production
of H2O2 in perfused rat liver is about 2% of total
Mitochondrial electron transport
oxygen consumption (Oshino et al. 1973). While this
The mitochondrial electron transport system has been higher level likely involves other cellular sources
long known to generate ROS, although the extent of besides mitochondria, it is apparent that tissues
this production in vivo is unclear. It has been calculated produce significant quantities of ROS.
that up to 2% of total mitochondrial oxygen consump- The specific ROS generated by mitochondria appear
tion goes toward the production of ROS (Boveris et al. to be O
2 and, following its dismutation, H2O2. The rate
1972). This figure has been cited in numerous of ROS production is directly proportional to the rate of
ubiquinone/coenzyme Q itself (James et al. 2004).

However, coenzyme Q is unlikely to be a major
source of radicals and, in fact, functions as an antioxi-
dant, protecting cells from free radical damage
(Bentinger et al. 2007).
Interestingly, ROS release from mitochondria may be
stimulated by NOX-derived O 2 and its derivatives, H2O2
and peroxynitrite, by initiating a loss of mitochondrial
inner membrane potential and resulting ROS production
(Schulz et al. 2014). Similarly, these NOX-derived ROS can
contribute to oxidation and uncoupling of NO synthase,
rendering this enzyme (e.g. eNOS) a O 2 source, rather
than a producer of NO (Schulz et al. 2014).
Figure 3. The production of reactive species by phagocytes.
Phagocytic cells are capable of generating large amounts of ROS Microsomal electron transport
as part of the oxidative burst, mediated by NADPH oxidase
(NOX). Hydrogen peroxide produced during this reaction can

undergo Fenton-type reduction to form the hydroxyl radical, or The endoplasmic reticulum contains a family of mixed-
be metabolized by myeloperoxidase (MPO) with the resultant function oxidase (or monooxygenase) enzymes (cyto-
formation of hypohalous acids that are reactive. Nitric oxide chromes P450, CYPs) that oxidize a wide range of
synthase (NOS) will produce NO, another radical that can yield endogenous substances and xenobiotics. These oxida-
further reactive species, such as peroxynitrite. Cytokines are also
produced which modulate the entire process. tions involve the transfer of electrons from NADPH via
cytochrome P450 reductase to heme at the CYP active
site. Once reduced, the heme binds and splits oxygen,
mitochondrial oxygen utilization in vitro (Boveris & releasing one atom of oxygen as water and leaving the
Chance 1973). Alterations in this rate may be induced other bound to heme. The oxygen atom bound to
in response to various toxic insults, including changes in heme is a powerful oxidant that reacts with the bound
the pH or ionic environment of the mitochondrion. xenobiotic substrate. Although not a major source of
Mitochondria isolated from ischemic or hypoxic heart ROS under basal conditions because these reactions
tissue exhibit a decreased formation of ROS are tightly coupled, the disruption of normal mixed-
(Paraidathathu & Kehrer 1992; Shlafer et al. 1987). function oxidase metabolism through injury or the
However, once again, such analyses have been per- presence of a xenobiotic, may cause leakage and
formed in vitro or ex vivo under strictly controlled greatly enhance this source of free radicals (Puntarulo &
conditions, and are not clear that decreased radical Cederbaum 1998).
production would be found in vivo where intracellular Like CYPs (Mishin et al. 2010), a second group of
calcium and sodium may be increased as a result of microsomal monooxygenases, the flavin monooxy-
diminished ATP availability. Indeed, one study used genases (FMOs), are also linked to the generation of
cytochemical techniques to show an increased mito- ROS by leakage. FMOs are distinct from CYP in that
chondrial production of H2O2 in intact ischemic heart they contain flavin (as FAD) but no heme, and in that
tissue (Vandeplassche et al. 1989), while a second study they take NADPH as a direct cosubstrate (rather than
showed that calcium enhanced ROS production by receive its electrons via a reductase, as in the case of
mitochondria from hypoxic heart tissue (Paraidathathu CYPs). Following reduction by NADPH and interaction
& Kehrer 1992). with molecular oxygen, FAD is present as a FAD-
Mitochondrial electron transport is normally a tightly hydroperoxide (FAD-OOH) at the active site of FMO,
coupled process. The production of ROS by mitochon- oxygenating substrates that usually harbor soft oxygen
dria can involve NADH:ubiquinone (complex I), succin- acceptors, such as nitrogen or sulfur (Jakoby & Ziegler
ate:ubiquinone (complex II) and ubiquinol:cytochrome c 1990; Krueger & Williams 2005). As with all mono-
oxidoreductases (complex III). A non-heme iron–sulfur oxygenases, one of the oxygen atoms ends up in the
cluster appears to be involved in the transfer of substrate, whereas the other is released as water. FAD-
electrons to oxygen at each site. Most of this transfer OOH may decompose to release (i.e. ‘‘leak’’) super-
is tightly coupled, but a small amount of leakage oxide/hydrogen peroxide that, interestingly, appears to
occurs, primarily from the NADH:ubiquinone oxidore- be more pronounced in the presence (rather than
ductase complex, and from the autooxidation of absence) of substrate (Siddens et al. 2014).
In addition to leakage of ROS, the metabolism of Parkinson’s disease received extensive attention a few
xenobiotics by microsomal enzyme systems can directly decades ago because of the discovery that 1-methyl-4-
from free radicals, such as the CCl3 radical generated by phenyl-1,2,3,6-tetrahydropyridine (MPTP) can produce
metabolism of carbon tetrachloride by CYPs (Weber damage virtually identical to this disease, and is redox-
et al. 2003), or other reactive metabolites, such as FMO cycled with the generation of ROS. Although most data
reaction products that can undergo redox cycling now suggest that this pathway is not responsible for
(Henderson et al. 2004). These free radicals may, in iatrogenic Parkinson’s disease, the enzymatic reduction
turn, mediate tissue injury. of certain chemicals can yield unstable species able
to reduce molecular oxygen and regenerate the par-
ent compound. The most commonly studied chemicals
Soluble oxidase enzymes
of this sort are the quinones (Bolton et al. 2000) (Figure
There are numerous soluble enzymes that can oxidize 1), but other substances also can undergo this redox
endogenous and exogenous substrates. The most well- cycling process.
known of these enzymes is xanthine oxidase, a molyb-
denum and FAD-dependent oxidoreductase that can
directly reduce molecular oxygen to O
Transition metals
2 and H2O2 while
oxidizing purines. Although this reaction is widely used The ability of transition metal ions to move electrons is
to generate ROS in vitro, its importance in vivo, where the basis for their importance as enzyme cofactors but
the dehydrogenase form that uses NAD+ as an electron also, unfortunately, for the propagation of many of the
acceptor predominates, is questionable. Under some most toxic radical reactions. For example, O 2 is
pathological conditions, conversion to the oxidase form, relatively non-reactive in aqueous solution. However, it
that uses oxygen as the electron acceptor (Stirpe & Della may reduce transition metal ions like Fe3+ (or Cu2+) that,
Corte 1969), can occur in vivo. Although this has in their reduced form, interact with H2O2 to generate the
implicated xanthine oxidase as a potential source of extremely reactive hydroxyl radical. This pathway, known
ROS, most studies indicate that it is not an important as the iron- (or metal-)catalyzed Haber–Weiss reaction
source in vivo (Coudray et al. 1994; Nishino 1994; Werns (Reaction I), is a type of Fenton chemistry and has been
et al. 1991). studied extensively (Kehrer 2000). In addition, free
Other oxidases include FAD- or Cu-dependent amine radicals can themselves cause the release of metals
oxidases that catalyze the oxidation of amines to their from storage sites that can then catalyze the decom-
respective aldehydes, generating H2O2. These aldehydes position of existing organic peroxides. The end result of
may, in turn, be further oxidized by aldehyde oxidase or these reactions is tissue damage (Jomova & Valko 2011).
xanthine oxidase – again producing O 2 =H2 O2 .
Peroxisomal enzymes like D-amino acid oxidase and O
2 þ Fe
3þ
! O2 þ Fe2þ ðIaÞ
fatty acyl CoA oxidase also can generate H2O2. With the
exceptions of aldehyde oxidase (Kundu et al. 2012) and
Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH ðIbÞ
xanthine oxidase (Nishino 1994), these enzymes have
not been well-studied in terms of free radical toxicology,
and little is known of their contribution to tissue injury. Net : O
2 þ H2 O2 ! OH þ OH þ O2 ðIÞ
The same is true of the peroxidases, although radical
species formed by some of these, such as the The intracellular environment is highly reducing, and
lipoxygenases and prostaglandin H synthase, may be transition metal ions probably exist in their reduced
involved in xenobiotic metabolism via co-oxidation forms in vivo. Although the role of the hydroxyl radical in
mechanisms (O’Brien 2000). pathology is not well established, the extensive meas-
ures taken by cells to minimize the presence of free
metal ions such as iron and copper indirectly indicates
Auto-oxidation of endogenous or exogenous
that such reactions are detrimental to biological systems.
substrates
In the case of transition metal ions, the primary strategy
The autooxidation of epinephrine has been used to that has evolved to prevent free radical-mediated injury
generate free radicals in a variety of experimental is to produce chelating proteins. A number of these
situations. This process can lead to tissue injury in exist, including the relatively non-specific metallothio-
isolated organ or cell systems, and has been implicated neins (zinc, copper, but also toxic metal ions such as
in neurotoxicity in vivo (Napolitano et al. 2011). The cadmium) and the highly specific ferritin (iron). These
possibility that the oxidation of dopamine is involved in permit almost no unchelated iron or copper to exist in
body fluids. The importance of maintaining very low generate ROS. Furthermore, oxidant gases such as ozone
levels of metal ions in plasma is evident in the case of and nitrogen oxides can be adsorbed onto nanoparticles
iron where there is a threefold excess of transferrin- and enhances radical reactions. In addition, nanoparti-
binding capacity relative to the amount of iron normally cles can interact with cells affecting mitochondrial
transported. respiration and activate immune cells thereby leading
Despite the normally low levels of free metal ions, to the production of free radicals and subsequent injury
some tissues appear to have an enhanced susceptibility through mechanisms described elsewhere in this review.
to ROS-mediated damage through metal-mediated The potential for nanoparticles to damage cells has
reactions. For example, there is a high content of been assessed mostly in vitro. While toxicity can clearly
bound iron in brain that, if released in response to some be induced, the clinical relevance of these studies is not
insult, cannot readily be re-sequestered due to the always evident. This is, in part, because the toxic
absence of significant iron-binding capacity in the concentrations of nanoparticles are not always known.
cerebrospinal fluid. The release of this iron might In addition, the likelihood of exposure to toxic levels of
damage the polyunsaturated fatty acids found in brain nanoparticles needs to be considered, as does whether a
tissue. This may explain the ease with which certain threshold for toxicity exists and whether the toxicity
neurotoxic drugs are able to damage nerve terminals, differs from that induced by larger particles of the same
the suggested role for ROS in the pathogenesis of chemical composition.

several neurological diseases, and why vitamin E defi- Overall, nanoparticles do not appear to be inherently
ciency or other free radical-based insults lead to a variety hazardous. Nevertheless, various organs can exhibit
of neurological disorders (Kovacic & Somanathan 2014). nanoparticle-induced free radical toxicity. The lung has
been the most frequently studied target because of
inhalation exposures. However, damage to most internal
Nanoparticles
organs has been reported. In addition, genotoxicity,
Considering the aforementioned endogenous mechan- possibly involving free radicals, has been reported in
isms of ROS formation, it comes as no surprise that response to nanoparticles (Kermanizadeh et al. 2015).
exogenous stimuli that trigger these processes will cause Despite significant research over the past decade, the
oxidative damage in exposed cells. For example, expos- risk of experiencing toxicity of nanoparticles, and the
ure of tissues to redox-active metal ions may cause ROS role of free radicals in this toxicity, remains poorly
generation as described in the ‘‘Transition metals’’ understood.
section. Similarly, xenobiotic substrates of enzyme sys- Figure 4 shows the mechanisms contributing to the
tems described above, or adverse agents causing generation of ROS in cells exposed to nanoparticles.
inflammatory reactions, will contribute to oxidative Some types of nanoparticles act as photosensitizers,
damage in tissues. facilitating the generation of 1O2 and O2 from ground
In lieu of a detailed treatise on the mechanistic state molecular oxygen under the influence of light (Shi
aspects of oxidative damage elicited by exogenous et al. 2013). In tissues that are not exposed to daylight,
stimuli, we will briefly focus on the role of ROS in the several other types of reactions may account for ROS
biological effects of nanoparticles, thereby providing an generation, such as the release of organic matter from
example that combines several of the above aspects of a combustion-derived nanoparticles, including quinones
disturbance of endogenous circuits. prone to redox cycling, or precursors thereof.
Numerous nanoparticles are cytotoxic (Lewinski et al. Furthermore, transition metal ions may be released
2008) and some of this toxicity has been linked to free from nanomaterial preparations, partly derived from
radicals (Manke et al. 2013). Nanoparticles have been particle impurities, catalyzing Fenton-type reactions.
also demonstrated to modulate intra- and inter-cellular In addition to such chemical reactivities, the mere
signaling, and these effects appear to have an oxidative interaction of particles with, or proximity to, subcellular
component (Ale-Agha et al. 2009, 2010; Unfried et al. structures involved in the catalysis of redox reactions
2007). may modulate the generation of ROS. Hypothetical
Toxic nanoparticles include those composed of targets include the plasma membrane with its enzyme
metals, metal oxides, fullerenes and carbon. Free radicals complexes, including NADPH oxidases, whose activity
and oxidative stress generated by nanoparticles is due to and regulation may be affected by interaction with
physical factors such as particle surface, composition, nanoparticles (a in Figure 4B). Moreover, nanoparticle
size and the presence or absence of metals, particularly interactions with mitochondria or the endoplasmic
transition metals. For example, silica oxide radicals reticulum (b and c in Figure 4B, respectively) are
present on the surface of crystalline silica particles will anticipated to contribute to ROS formation. For example,
the physical interaction between nanoparticles and cell clearly indicate the ability of these species to have far
structures might directly affect electron flow and leak- more subtle effects on cell and tissue functions.
age from the inner mitochondrial membrane. In fact, the Free radicals may not only oxidize diverse substrates
enhanced generation of ROS, and damage to mitochon- but also initiate a number of addition reactions that can
dria, as well as the presence of ultrafine particulate result in the covalent binding of xenobiotics, or oxida-
matter inside mitochondria of exposed cells, was tion products of endogenous molecules, to various
demonstrated (Li et al. 2003; Xia et al. 2006). The biomolecules, including lipids (Smith et al. 1984). The
interaction of nanomaterials with mitochondria and functional consequences of covalent binding to lipids
endoplasmic reticulum, two major cellular Ca2+ stores, are not known, but are considered minimal and thus of
may cause the dysregulation of calcium ion levels. This, little toxicologic importance. Covalent binding of xeno-
in turn, might activate calcium-dependent enzymes biotic species to critical macromolecules such as DNA or
involved in the generation of ROS/RNS, such as NO proteins, however, may be directly related to cell injury.
synthases (Christen et al. 2014; Huang et al. 2009; Yet the relationship between covalent binding and
Unfried et al. 2007). toxicity is not clear inasmuch as there are compounds
that bind extensively but apparently do not damage
cells (Roberts et al. 1990). Nevertheless, covalent binding
Mechanisms of free radical toxicity
is indicative of the formation of reactive species.

Numerous experimental systems, most in vitro, have Depending on the specific molecules attacked, the
been used to show that radicals and other reactive irreversible binding of free radical metabolites to tissue
species can cause cellular damage. The general targets macromolecules seems capable of disrupting function
of these species include cellular macromolecules, and a and is a plausible mechanism leading to cell death.
vast range of molecular changes have been investigated However, the critical determinants of the toxicity of
to explain the underlying mechanism(s) of toxicity. reactive species include the specific molecules affected,
However, acute experimental systems may miss import- the specific molecular natures of the modifications, and
ant cumulative effects induced by chronic or repetitive the cellular disposition of the altered molecules, not the
exposures to much lower levels of free radicals. In simple fact of binding (Zhou et al. 2005).
addition, the reactive nature of free radicals led originally
to the assumption that the damage produced was non-
Lipid oxidation
specific. However, more recent data have shown that
highly reactive species can exhibit specificity in terms of Lipids have been studied extensively in terms of free
the molecules that are modified and the nature of the radical-mediated injury due to their critical structural
modifications effected. Furthermore, these alterations and functional role in membranes, and due to the
may not always be undesirable, as shown by the ability ease with which some lipid peroxidation products
of radicals and oxidants to modulate signal transduction can be measured. Unfortunately, the simplicity of some
mechanisms and immune system functions (Dröge lipid peroxidation assays (particularly the thiobarbituric
2002), and of radicals and alkylating agents to induce acid reaction) has led to many published reports that
apoptosis (Circu & Aw 2010). have not considered the numerous limitations and
problems with this assay (Janero 1990). Furthermore,
cause versus effect is often ignored. As membrane lipids
Specific biological targets
become much more susceptible to peroxidation after
Lipids, proteins (and small peptides), carbohydrates, cell death, this is an important consideration when
DNA and RNA are all targets for free radical reactions. A assessing peroxidation as a mechanism rather than a
vast array of changes have been identified that could marker of cell death. Nevertheless, fatty acid hydroper-
provide a mechanistic explanation for the damage oxides are detrimental to cells, and mechanisms exist to
observed (Figure 5). Which change(s) are the most remove them from membrane phospholipids (van Kuijk
important in response to any specific insult appears to et al. 1987), including membrane-bound glutathione
some extent to depend on the model used. In general, it peroxidase (GPx-4). When such processes are over-
would appear that the cumulative effect of multiple whelmed, it seems clear that cell injury will ensue.
changes induced by acute exposure to high levels of free The homoconjugated double bonds found in poly-
radicals is the proximate cause of cell death, much as is unsaturated fatty acids are particularly susceptible to
seen with other non-specific toxins such as alkylating free radical-mediated reactions. The abstraction of
agents. However, more recent data showing the ability bisallylic hydrogen atoms from these double bonds is
of free radicals to affect molecular signaling pathways favored by the low homolytic dissociation energies.
Figure 4. The production of reactive species by nanoparticles (NP). (A) NP-induced generation of ROS as a result of the chemical
composition of NPs. Transition metal ions (Men+) or organic compounds, released or accessible at the particle surface, may serve as
initiators of metabolic reactions that generate ROS. (B) NPs may physically interact with subcellular compartments (a – NADPH oxidase
(NOX) complexes; b – mitochondria; c – endoplasmic reticulum), affecting their integrity and activity, and resulting in the diversion of
electrons or release of calcium from intracellular stores. Elevation of calcium concentrations may result in the stimulation of Ca2+/
Reaction of the resultant radicals with molecular oxygen chemically modify target molecules, usually at cysteines
is rapid and these products can then propagate the and amino moieties, and generate Michael-type adducts
reaction (i.e. lipid peroxidation) by abstraction of a or Schiff bases to both alkylate and cross-link proteins,
hydrogen atom from another polyunsaturated fatty acid resulting in signaling. In a way, therefore, HNE would
molecule. As these reactions progress and products display second messenger effects (Chapple et al. 2013;
accumulate, ion channels may be affected, membrane Forman et al. 2014). These effects are primarily seen on
transport proteins or enzymes may be inactivated, or the vascular endothelial and smooth muscle cells, and
lipid bilayer itself may become more rigid and/or include the activation of kinases and effects on prolif-
permeable. eration. HNE can also induce apoptosis at high concen-
In recent years, the signaling effects of products of trations and has been implicated as a factor in
lipid peroxidation have become evident. Of particular atherosclerosis, diabetes and some neurodegenerative
interest has been 4-hydroxy-2-nonenal (HNE), a major diseases (Chapple et al. 2013). Similarly, the isoprostanes
electrophilic product of lipid peroxidation that can that were originally identified as markers of oxidative
Free Radicals/ROS/RNS
(Transition metals)
Lipids Protein DNA
Anoxidant & Repair Systems
Lipid peroxidaon Amino acid oxidaon Oxidized DNA bases

Carbonyl formaon
• Membrane damage (De-) Acvaon of enzmes or • Mutaon

• LPO products as signaling molecules • DNA damage
signaling molecules
(HNE, MDA) Inability to maintain ion Altered gene expression
gradients
Cell & Tissue Injury
Figure 5. Mechanisms by which free radicals damage cells. When free radicals interact with lipids, proteins, and DNA there is the
potential for numerous changes that could affect cell function and lead to injury. Antioxidant and repair systems will prevent, or
minimize these changes.
stress can exert potent biological effects. These include products (see ‘‘Enzymes catalyzing repair of ROS-induced
vasoconstriction, effects on platelets, and hypertrophy of damage’’ section), damaged proteins are not repaired
cardiac tissue (Milne et al. 2011). and are removed primarily by proteolytic systems. For
example, oxidized proteins are much more susceptible
to proteolysis by the 20S proteasome (Davies 2001; Jung
Protein oxidation
& Grune 2008). Interestingly, if protein oxidation is too
Proteins, including specific amino acid residues (Yang extensive, oxidized proteins may overwhelm and clog
et al. 2001), have received significant attention as targets the proteasome, impairing further degradation and
for free radical damage (Davies 2005). Oxidation of these resulting in the accumulation of protein oxidation
residues can lead to a loss of critical sulfhydryl groups products (Grune et al. 2001; Höhn et al. 2014).
through simple thiol oxidation, the formation of mixed Lysosomal exocytosis, resulting in excretion of oxida-
disulfides or the oxidation of methionine residues to the tively modified proteins, offers another mechanism for
corresponding sulfoxides. In addition, modifications of removal of damaged proteins (Dunlop et al. 2009).
amino acids leading to the formation of aldehydes or However, no system is perfect and damaged proteins
ketones (i.e. carbonyls), hydroperoxides and their can accumulate with time and impair proteasome and
reduced hydroxy species, or ring cleavage in histidine mitochondrial function. Overall, increases in oxidized
or tryptophan residues, can occur. These modifications proteins may be associated with age-related losses of
can impair signaling (Spickett & Pitt 2012), and both the selected biochemical and physiological functions, and
functional and structural activities of proteins (Dean may reflect unrepaired damage to other cellular macro-
et al. 1997), thereby having significant impacts on a molecules such as DNA (Baraibar et al. 2012).
myriad of disorders.
As with lipids, the removal of oxidized proteins from
DNA oxidation
biological systems is an ongoing process, and it is only
when the rate at which they are produced exceeds their The interaction of free radicals with DNA can affect both
removal and/or replacement with fresh fully functional the integrity and the regulation of certain genes, which
molecules that cell injury will become evident. With the may result in changes both detrimental and beneficial to
exception of cysteine and methionine oxidation cells. These interactions may involve direct modifications
of DNA by oxidation (e.g. resulting in the generation of 8- on DNA, RNA and proteins, as well as mono- and
oxo-deoxyguanosine moieties) or adduct formation (e.g. polysaccharides. Damage to sugars in DNA nucleotides
lipid peroxidation product-derived adducts to bases) appears to be repaired by fairly typical nucleases
(Winczura et al. 2012), or they may be mediated through (Demple & Levin 1991) and such damage is not generally
changes in transcription factors or enzymes involved in considered a critical factor in toxicity. However, free
regulating gene expression or repair. Much of the work glucose can undergo several oxidation reactions result-
on gene expression effects has been conducted by ing in the formation of advanced glycation end products
researchers interested in the role of ROS in carcinogenesis (AGEs) (Ott et al. 2014). Glucose can either slowly modify
and is discussed further in the ‘‘Cancer’’ section. proteins to form Schiff bases that may undergo further
Reactive oxygen species (ROS) can directly modify rearrangements, or be reduced to sorbitol, followed by
both DNA bases and sugars (Cooke et al. 2003; Regulus further oxidation. Some of the resultant carbohydrate
et al. 2007). If DNA repair processes are inadequate, this oxidation products can enolize in the presence of
can lead to permanent DNA damage (which, in turn, may transition metals to become ketoaldehydes that interact
entail cell death), permanently altered DNA sequences with proteins to generate AGEs. These products may
(mutation), and errors in transcription. Peroxide- further stimulate the generation of ROS in cells by
mediated DNA damage can activate poly(ADP-ribose) activating an NADPH-oxidase-coupled receptor of AGEs
polymerase (Luo & Kraus 2012). Once activated, this (RAGE) (Ott et al. 2014). AGEs may be involved in
enzyme will utilize large amounts of NAD to repair diabetic complications (Jomova & Valko 2011; Ott et al.
damaged DNA (Bürkle & Virag 2013). This may impair the 2014; Rains & Jain 2011).
ability of a cell to produce ATP, thereby further stressing
cells. Although this alone is insufficient to cause cell
Cell signaling
death (Andreoli 1989), it seems clear that ROS can
disrupt pathways critical for the maintenance of normal The same mechanisms described above that cause
adenine and pyridine nucleotide status. damage to biological molecules through oxidation or
alkylation reactions are frequently recognized by the cell
as a stimulus to initiate cellular signaling processes that
RNA oxidation
may ultimately lead to adaptation or cell death (see
In recent years, the importance of RNA, and in particular ‘‘Apoptosis’’ section). Hence, these signaling processes
small RNA molecules such as microRNAs, in regulating may both contribute to remediation of the cellular
genes and other cell functions has become recognized. landscape following oxidative damage and be respon-
As discussed in the previous section, the oxidation of sible for the toxic effects of xenobiotics. This is because
DNA can affect gene regulation. The same is true for such signaling, in the absence of the natural triggers,
RNA, and oxidized RNA is found in many diseases, may be regarded as an undue perturbation of the
especially Alzheimer’s disease (Nunomura et al. 2012). usually tightly controlled cellular regulatory circuits
Oxidation of RNA bases leads primarily to strand breaks (Figure 6).
and base modifications. In the case of RNA involved in Calcium (as Ca2+), one of the classical second
protein synthesis, this can result in decreased as well as messengers, is often invoked as a mediator of cell
abnormal protein production (e.g. misfolding). Cells can injury. The intracellular content of calcium is normally
recognize this problem and degrade the affected pro- 10 000-fold less than that in the extracellular fluid.
teins, but if extensive or prolonged, apoptosis may be Maintenance of this concentration gradient requires
triggered. Overall, evidence suggests that RNA oxidation voltage-dependent and -independent channels, plus
is involved in several diseases including diabetes and energy-dependent pumping systems in cell membranes.
cardiovascular disease (Poulsen et al. 2012). However, Any perturbation that affects calcium transport, or
considering the transiency of RNA molecules such as impairs mitochondrial and endoplasmic reticular calcium
mRNAs or microRNAs, substantial additional research is storage, is capable of seriously affecting cell function. As
needed to fully understand how the oxidation of RNA a consequence of improper calcium release from intra-
can affect the development and progression of diseases cellular stores, Ca2+/calmodulin-dependent enzymes,
and toxicities. such as endothelial or neuronal nitrogen monoxide
synthases (eNOS or nNOS), may be activated resulting in
the production of NO and peroxynitrite.
Carbohydrates
The calcium ATPase enzymes have critical thiol groups
Cells contain numerous sugars that are susceptible to and can be inactivated by ROS (Ariki & Shamoo 1983;
attack by free radicals. These include the sugar moieties Schöneich & Sharov 2006). ROS-mediated damage can
Figure 6. Reactive oxygen species (ROS)/reactive nitrogen species (RNS) and signal transduction. There is substantial evidence that
ROS/RNS can affect the genome. Prolonged exposure to ROS/RNS can, in some instances, induce the activities of antioxidant enzymes
and repair systems. ROS/RNS can alter kinase activities that, in turn, can modulate the activities of various genes. Changes in gene
activities may also be mediated by cytokines released in response to ROS/RNS, or from phagocytes. ROS are also able to affect the
activity of transcription factors and oncogenes, potentially leading to tumorigenesis.
also decrease cellular energy, further compromising the Redox cycling of quinones (as depicted in Figure 1) is
ability of cells to maintain calcium (and other ion) attenuated in vivo by NQO1 (also known as DT diaph-
gradients. Thus, it is clear that free radicals have the orase), whose expression is regulated at the transcrip-
ability to alter the homeostasis of calcium and other tional level by antioxidant response elements (AREs) in
ions. Although the relationship between such changes the gene’s promoter region (Li & Jaiswal 1992). AREs are
and cell injury is far from clear, it is unlikely that calcium recognized by the transcription factor nuclear factor
functions as a simple final common pathway to injury. erythroid 2-related factor 2 (Nrf2) (Venugopal & Jaiswal
There have been some excellent reviews discussing the 1996). Nrf2 is stimulated by alkylating agents/electro-
roles of calcium in tissue injury (Harman & Maxwell 1995; philes and ROS. The expression of numerous other
Orrenius et al. 2011). proteins considered protective and important in cellular
Reactive oxygen species (ROS)-mediated alterations in stress responses is regulated by Nrf2, including phase II
gene regulation can induce changes that are either enzymes, heme oxygenase or enzymes involved in GSH
beneficial (e.g. adaptive) or detrimental to cells (Allen & biosynthesis, as well as antioxidant enzymes (Kansanen
Tresini 2000). Research in prokaryotes was the first to et al. 2013). In cells, most of the synthesized Nrf2 is kept
identify regulons, such as oxyR and soxR, that when in check in the cytoplasm by a cysteine-rich protein,
oxidized activate the expression of genes involved in Keap-1, that mediates proteasomal degradation of Nrf2
protecting prokaryotes from stress (Fleischhacker & Kiley by forming a complex consisting of two Keap-1 mol-
2011). Adaptive responses to ROS are more complex in ecules, Nrf2 and the Cul3 component of a ubiquitin
eukaryotes, and several stress-responsive signaling cas- ligase. Keap1 may be oxidized by ROS (forming disul-
cades and transcription factors exist to mediate adaptive fides) and alkylated by certain electrophiles, both
responses acutely and at the level of gene expression. leading to the release of Nrf2. Free Nrf2 will then
Thiols are involved in the activation of these factors and translocate into the nucleus to form heterodimers with
thus ROS influence this activation (Covas et al. 2013). another transcription factor molecule, such as a Maf
monomer, to stimulate ARE-dependent transcription (Ma redox sensors to transmit H2O2 signals (Sobotta et al.
2013). 2015). While an attractive hypothesis, additional work is
Forkhead box, class O (FoxO) transcription factors are needed to determine whether this mechanism has a
another redox-regulated family controlling the expres- major role in redox signaling.
sion of antioxidant enzymes (Klotz et al. 2015). These In addition to the mounting evidence implicating
factors regulate the expression of genes involved in fuel H2O2 in cell signaling, free radicals may play a role,
metabolism, and of genes coding for antioxidants particularly in terms of how they may affect thiols
such as manganese superoxide dismutase (MnSOD) (Winterbourn 2015). The evidence for radical mechan-
(Kops et al. 2002), selenoprotein P (Speckmann et al. isms remains limited, but it is clear that radicals are
2008; Walter et al. 2008) and ceruloplasmin (Leyendecker generated when signaling pathways are activated. Such
et al. 2011). Activity of the FoxO factors is modulated by correlations are only suggestive, but do support the
ROS, such as H2O2, but also by potentially toxic metal need for additional research in the area of thiol-based
ions such as copper (Hamann et al. 2014a; Walter et al. redox signaling.
2006) and metalloid compounds such as arsenite
(Hamann & Klotz 2013; Hamann et al. 2014b).
Apoptosis
In recent years, H2O2 was recognized as an important
messenger molecule (Sies 2014). As it is non-charged, Damage induced by any toxicant, including free radicals,
and molecularly similar to water, it is readily diffusible can result in cell death by apoptosis or necrosis.
and thus acts in ways analogous to signaling gases such Apoptosis is a structured form of cell death characterized
as NO, carbon monoxide or hydrogen sulfide. H2O2- by cell rounding, a decrease in cell volume, nuclear
mediated signaling involves relatively specific targeting condensation and plasma membrane blebbing. In con-
of cysteinyl residues. As noted by Sies (2014), this can trast, necrosis is characterized by cellular swelling and
occur via direct oxidation, oxidation via a highly reactive rupture of the plasma membrane. The initiation and
sensor protein, activation of a target protein subsequent execution of apoptosis are mediated by a family of
to the oxidation and dissociation of an inhibitor, cysteine-aspartate proteases (caspases). These enzymes
secondary oxidation of a target protein (i.e. the signaling are activated by intrinsic (mitochondria-mediated) and
oxidation occurs via a product first oxidized by H2O2), extrinsic (receptor-mediated) signaling pathways
inactivation of a scavenging protein and association of a (Figure 7). ROS alone can induce apoptosis through
target protein with a hydrogen-peroxide-generated ROS-mediated damage to mitochondria and the release
protein leading to site-directed oxidation. H2O2 can of cytochrome c that initiates caspase activation
also indirectly affect signaling by oxidizing GSH leading (Orrenius et al. 2007). In addition, as noted previously,
to protein glutathionylation. low levels of ROS can induce or facilitate apoptosis in
There are numerous signaling targets of H2O2. Among response to death receptor ligands by modulating the
the first to be identified were factors associated with activities of several transcription factors and numerous
insulin (May & de Haen 1979). Since that time, numerous protein kinase cascades (Zhivotovsky & Kaminskyy 2014).
additional targets have been identified that are either Overall, the effects of free radicals on apoptosis are
directly or indirectly affected by H2O2 including platelet- integral to their effects on other pathways and generally
derived-, epidermal-, fibroblast- and vascular endothe- are not a common mediator of this form of cell death.
lial-growth factor receptors, as well as many serine/
threonine kinases including Akt and the MAP kinases
Biological responses to free radicals
(Barthel & Klotz 2005; Breton-Romero & Lamas, 2014;
Klotz 2002; Sies 2014; Truong & Carroll 2013). H2O2 is As noted previously, there are extremely large number
also involved in regulating many transcription factors; of xenobiotics and pathologies associated with free
e.g. through modulation of upstream signaling cascades, radicals. No single review article can cover all of these.
such as the FoxOs that are phosphorylated by H2O2- Those covered here were selected based on the strength
responsive kinases such as Akt or JNKMAPK, resulting in of evidence supporting a role for free radicals, their
FoxO inactivation (Akt) or activation (JNKMAPK). H2O2 may prevalence and the authors’ interests.
also directly interfere with transcription factor structure
or interact with co-regulators (Marinho et al. 2014).
Cancer
Importantly, recent data indicate that signaling by H2O2
may not be the result of direct oxidation. Rather, the The ability of ionizing radiation to stimulate the devel-
peroxiredoxins are far more efficient at interacting with opment of cancer has been long recognized. The direct
H2O2 than putative thiol targets and may function as absorption of energy by DNA resulting in damage to this
Figure 7. Major apoptotic pathways in mammalian cells. In the extrinsic pathway, the DISC complex is formed upon ligand binding to
specific receptors and caspase-8 is activated leading to the activation of caspase-3 and apoptosis. In the intrinsic pathway,
cytochrome c is released from the mitochondria resulting in formation of the apoptosome and activation of caspase-9, then caspase-3
and apoptosis.
molecule may be part of the basis for radiation-induced bases can cause miscoding (Lord & Ashworth 2012), and
carcinogenesis. However, in biological systems, most of that exogenously generated ROS induce expression of
the energy from ionizing radiation is absorbed by water some oncogenes (Ziech et al. 2011). Moreover, mice
(because water represents 70% of an organism, it also deficient in Ogg1, the 8-oxo-guanine-specific DNA
represents the major target for radiation) leading to the glycosylase involved in base excision repair of the
formation of ROS. The attack of these ROS on DNA to corresponding oxidized DNA regions, accumulate spon-
produce double strand breaks, as well as on RNA and taneous mutations more readily than control mice,
other portions of a cell, is generally believed to provide although no progression to cancer was observed (Epe
the basis for the formation of radiation-induced cancer 2002; Klungland et al. 1999). Thus, although a direct
(Barcellos-Hoff & Nguyen 2009; Chadwick & Leenhouts connection between oxidatively modified DNA and
2011). There also is substantial evidence that free cancer is not available, there is substantial support of a
radicals (including ROS) are involved in initiation and role for free radicals in carcinogenesis. In addition,
progression of cancers upon exposure to non-ionizing products of lipid peroxidation, such as HNE, have been
radiation (such as UV; see also ‘‘Phototoxicity’’ section) implicated in signaling pathways associated with cancer.
(Scharffetter-Kochanek et al. 1997) and in chemical Mitochondria appear to be particularly important in this
carcinogenesis via various metabolic pathways (Poirier process as HNE generated from the oxidation of
2004). Thus, it is logical to assume that free radicals cardiolipin in this organelle can modify mitochondrial
formed by other means would be carcinogenic. proteins, lipids and DNA (Zhong & Yin 2015).
Surprisingly, most of the data have not supported this A better understanding of the molecular events
conclusion. leading to cancer was greatly facilitated by the two-
Free radicals, generated chemically or enzymatically, stage theory of carcinogenesis. Subsequent research
are capable of oxidatively modifying DNA both in vivo refined this concept to include several other stages, and
and in vitro. Of interest are the correlations between 7,8- organs other than the skin where it was first described
dihydro-8-oxo-2’-deoxyguanosine (8-oxo-dG) formation and has been studied most extensively. Although the
and carcinogenesis (Floyd 1990), the increased content transformation of a normal cell into one that proliferates
of oxidized DNA bases in skin tumors, that oxidized in an uncontrolled fashion is a highly complex multistep
process (Slaga et al. 1980), a consideration of the basic antioxidant defense systems. For example, superoxide
two-stage theory remains useful for discussing the dismutase (SOD) and catalase activities are decreased in
potential role of free radicals. mouse epidermal cells following treatments with several
The first stage of carcinogenesis involves a permanent different promoters (Solanki et al. 1981). However, in
genetic change called initiation. This change may be contrast to this finding, SOD activity was increased in
latent, without any overt cancer development during the response to TPA in tumor necrosis factor-resistant but
lifetime of an animal. However, at any time, a promoter not sensitive cell lines (Fujii & Taniguchi 1991), and
can act to stimulate the initiated tissue to transform. The glutathione peroxidase activity shows a variable
effect of promoters appears to be reversible because it response to TPA (Perchellet et al. 1985). Thus, changes
requires repeated administration to reveal the cancer, in the activities of antioxidant enzymes appear unlikely
and this administration must be done at frequent time to play a consistent role in the carcinogenic response of
intervals (1–2 weeks in the case of skin) or the incidence of tissues to tumor promoters.
cancer is decreased. The administration of a promoter As the production of ROS generally correlates with
without prior initiation will infrequently induce cancer. In tumor promoting activity, scavenging these radicals with
contrast, the only way to verify the presence of initiated antioxidants would be expected to antagonize tumor
cells is to apply the promoter. Some carcinogens are promoters. Protection has been seen in vitro or in
labeled as complete because they promote as well as specialized cancer systems with most antioxidants tested
initiate, while others require a second chemical promoter. (Slaga 1995). There is evidence that an increased cellular
There is extensive evidence supporting a role for free GSH content is protective but that GSH may also
radicals in the promotion process (Kensler & Taffe 1986). promote cancer by affecting resistance to drug treat-
Specifically, tumor promoters can activate phagocytic ments (Singh et al. 2012). Importantly, studies with
cells (which then produce ROS), antioxidants have anti- transgenic mice have clearly shown the importance of
promoting activity, and many free radical-generating MnSOD in regulating the development of cancer (Dhar &
compounds are tumor promoters. Tumor promoters, St. Clair 2012).
particularly the phorbol esters such as 2-O-tetradeca- In all cases, the antioxidant species must be present at
noylphorbol-13-acetate (TPA), are highly potent activa- the time of exposure to the promoter in order to provide
tors of the oxygen burst in phagocytic cells. This process protection. As this is the time that free radical produc-
appears to be mediated through protein kinase C and tion is enhanced, it suggests that the antioxidant activity
protein phosphatases, as well as by inflammation and of these materials, and not some cell repair effect, is the
tumor necrosis factor a (Fujiki et al. 2013). However, critical determinant of their anti-cancer action. In con-
because some incomplete tumor promoters (those that trast to these protective effects, antioxidants can have
require a second chemical to function as a promoter) can no effect on the development of cancer, or may even
produce as much or more ROS as complete promoters have a promoting effect (Bauer et al. 2001). This
(Marks & Fürstenberger 1985), it is obvious that additional promotion may, of course, be unrelated to the antioxi-
factors are involved. Various signal transduction path- dant activity and instead be due to inflammation (Bauer
ways and phospholipase A2 are examples of such factors et al. 2001) or tissue damage (e.g. lung damage
that can be activated by oxidants (among other things), produced only in mice by agents such as butylated
and may be important in tumor promotion (Reuter et al. hydroxytoluene) (Witschi 1990).
2010). Indirect evidence for a role of ROS in carcinogenesis is
Various chemical and enzymatic free radical-generat- available from the long-recognized association between
ing systems can mimic the biochemical actions of tumor cancer and several genetic and chronic inflammatory
promoters, further suggesting a role of ROS in their disorders (Kamp et al. 2011). For example, patients with
activity. For example, transformation of cultured cells, inflammatory bowel diseases have an increased risk of
otherwise initiated with benzo[a]pyrene or ionizing developing adenocarcinoma of the colon; patients with
radiation, can be achieved by addition of activated hepatic cirrhosis run an increased risk of developing
neutrophils or xanthine/xanthine oxidase to these cells hepatocellular carcinoma; and patients with ataxia
(Weitzman et al. 1985; Zimmerman & Cerutti 1984). telangiectasia, Fanconi anemia and Bloom’s syndrome
Organic peroxides also can function as tumor promoters, (all of which exhibit abnormal oxygen metabolism) have
although they are largely inactive as initiators. These an increased incidence of cancer. However, it is not clear
peroxides require cell-mediated activation to free radical whether inflammation alone (e.g. through the formation
products. of ROS and oxidation products) is sufficient to initiate
In addition to increasing the generation of ROS, and promote cancer formation (i.e. similar to a complete
several tumor promoters modulate endogenous carcinogen), or whether another carcinogenic stimulus
must also be involved. Nevertheless, it is commonly showed that b-carotene actually increased the risk of
accepted that UV radiation (both UVB and UVA) is a cancer (Potter 2014). Results with folate, vitamin C,
complete carcinogen, and that photochemical selenium and calcium were also negative (Potter 2014).
ROS formation is a major contributor to cancer Positive results have been seen with low-dose aspirin in
(Scharffetter-Kochanek et al. 1997). terms of colon cancer (Chan et al. 2011), but it seems
Chronic irritation at any tissue site increases the risk of unlikely that this effectiveness is related to any antioxi-
cancer at that site. The underlying mechanisms are, as dant activity. Rather the inhibition of cyclooxygenase-2
usual, complex and involve both inflammatory cells and or the modulation of transcription factors and the
inflammatory mediators. As noted previously, inflamma- induction of apoptosis may be involved (Chan et al.
tory cells release ROS and RNS as well as numerous 2011).
signaling molecules such as NF-kB, STAT3, PI3K/Akt and The reasons why chemoprevention with antioxidants
ERKMAPK that affect signaling pathways involved in has been a failure are unclear. In general, a single
inflammatory responses, and in the control of prolifer- treatment for patients of different ages, lifestyle, health,
ation and apoptosis. etc. is always unlikely to be effective. It is also obvious
Thorough consideration of the evidence outlined that trials involving only a single agent do not mimic the
above provides a strong case that ROS species are complexity of dietary chemoprevention that has shown
important in tumor promotion, and may be initiation some benefits. There may be selection for resistant cells
factors in combination with ionizing radiation or meta- when using single agents and chemoprevention with
bolic activation of various xenobiotics. The mechanisms antioxidants may have both positive and negative
by which ROS affect tumorigenesis seem likely to be effects on signaling pathways that are used by free
multifactorial and may involve changes in both cytokine radicals. Despite the clear failures of most chemopreven-
production and gene expression. Ongoing work con- tion trials, this approach continues to have supporters, at
tinues to improve our understanding of the molecular least for high-risk individuals (Adhami et al. 2014).
details of these effects. Interestingly, the generation of ROS is frequently seen
In summary, ROS are mutagenic to human cells (Lost in cancer therapy; e.g. during exposure to ionizing
& Poulsen 1996) and may function as tumor initiators radiation, in photodynamic therapy or during chemo-
(Sun 1990). ROS can alter the activities of kinases (Barthel therapy using redox cyclers such as doxorubicin or
& Klotz 2005), and oxidative DNA modifications may mitomycin (Begleiter 2000). A therapeutic approach
activate oncogenes or inactivate tumor suppresser successfully tested in cultured breast cancer cells and
genes (Fujiki et al. 2013). ROS can both inhibit and in mice was based upon overexpression of MnSOD to
stimulate cell proliferation, depending upon the cell catalyze the dismutation of O 2 to H2O2, combined with
type and condition, but a direct interaction of ROS with an impairment of peroxide removal through drugs
DNA is not required to affect cell proliferation. Thus, in interfering with glutathione metabolism (Sun et al.
addition to genotoxic effects, free radicals can affect the 2009, 2010). This approach is based on the assumption
development of cancer through non-genotoxic mech- that their already elevated levels of ROS (Aykin-
anisms (Benigni et al. 2013), such as through modulation Burns et al. 2009) would make cancer cells more
of signal transduction pathways (Ray et al. 2012). susceptible to ‘‘death by ROS’’, such as by ROS
Chemoprevention of cancer (i.e. using pharmaco- generated during exposure to redox cycling quinones
logical agents) is an important therapeutic goal due to (Klaus et al. 2010).
the difficulties associated with treating existing cancer. Overall, despite extensive advances in our under-
However, despite the apparent role for free radicals, and standing of carcinogenic mechanisms, it is clear that no
some promising preliminary results with antioxidants, single mechanism can explain the entire process.
the identification of specific agent(s) that could be used Although free radicals may be a major cause of some
widely has not been realized. For example, preliminary cancers, and although there clearly are regulatory roles
research indicated that vitamin E was likely to be for ROS at all levels of carcinogenesis (Poillet-Perez et al.
effective in preventing prostate cancer. Unfortunately, 2015), it is highly unlikely that they are necessary in all
an extensive clinical trial ultimately showed that vitamin cases because alternate pathways of initiation and
E did not decrease prostate cancer and, in fact, slightly promotion clearly exist.
increased the incidence of this disease (Klein et al. 2011).
Similarly, although the consumption of vegetables and
Immune system and inflammatory processes
fruits high in antioxidants is inversely correlated with the
risk of some cancers, and low serum retinol is associated The importance of free radicals in immune responses to
with an increased risk of lung cancer, several trials invading organisms is well-established (Reuter et al.
2010). This role includes the creation of chemotactic of time, inflammation may become the disease process
factors when superoxide interacts with albumin-bound (Figure 8). This appears to be the underlying basis of
lipids, recruiting additional phagocytic cells to the site of rheumatoid arthritis (McInnes & Schett 2011). The trigger
injury (Petrone et al. 1980). Importantly, over the last for producing the antibodies that attack joint tissue in
decade, extensive research has shown that free radicals this autoimmune disease is not known. There is no
and oxidants play critical roles in cell communication for doubt, however, that by binding to various joint proteins
the immune system. For example, radicals and oxidants these antibodies stimulate the accumulation of activated
activate some immunologically relevant transcription neutrophils. The production of ROS and other inflam-
factors and are involved in the modulation of several matory mediators by these activated cells is believed to
signal transduction pathways (Brigelius-Flohe & Flohe contribute to the damage that occurs in the inflamed
2011; Ray et al. 2012). rheumatoid joint. The subsequent recruitment of more
Cell-to-cell signaling is critical for the normal func- activated neutrophils leads to the chronic inflammation
tioning of the immune system. This communication and gradual degeneration of the joints characteristic of
involves the release of cytokines from tissues in response this disease.
to insults (i.e. disease, drugs, antigens and radicals). Evidence suggesting a role for ROS in rheumatoid
Optimal activation of some genes, as well as the function arthritis has been increasing (Datta et al. 2014).
of lymphocytes, depends to some extent on cellular Research using the Nrf2-knockout mouse has indicated
thiols (Dröge et al. 1994). In general, exposure to high that ROS are important factors in the breakdown of
levels of free radicals has a negative impact on the collagen in experimental arthritis (Wruck et al. 2011).
immune system. Conversely, antioxidants appear to Other work has supported a role for myeloperoxidase
enhance some aspects of immune function (Knight (Stamp et al. 2012). Recognizing that correlations do
2000). The molecular bases for these effects remain not reflect causation, it is still apparent that ROS are a
unclear, but are likely related to a balance between the likely culprit in at least some of the pathology of this
roles of radicals as immune toxicants and their roles in disease.
cellular communication. Although it has not been proven that ROS are
Although integral to the function of the immune pathologic in various forms of arthritis, the preponder-
system at both intra- and extracellular levels, radicals ance of evidence supports the contention that they are
can escape and initiate injury or aggravate existing involved at some level. In terms of rheumatoid arthritis,
tissue damage. This injury can be significant, particu- the extent of this involvement must not be overstated
larly in chronic inflammatory disorders such as inasmuch as the underlying cause of this disease is
rheumatoid arthritis (see below), but varies from case certainly autoimmune in nature, and thus antioxidant
to case, or even time to time, as the inflammatory therapy will be treating only a symptom. Furthermore,
process evolves. Despite the potential for inducing the effects of the numerous other inflammatory medi-
injury, inflammation is a normal response of injured ator substances and proteases released by phagocytic
tissue and is generally not pathologic. As in other cells will be significant, and perhaps major, contributors
situations in which radicals are normal metabolic to the disease process.
products, their production during immune reactions
(excluding autoimmune reactions) is controlled and
Chemoprevention
targeted as well as possible. This control is, of course,
not absolute and thus radicals can aggravate existing The available evidence implicating free radicals in
damage. Importantly, the concept that inflammation inflammation has led to a number of trials for antioxi-
recedes simply as a consequence of proinflammatory dant therapies in several inflammatory disorders includ-
molecules dissipating as the injury resolves is being ing rheumatoid arthritis. Although there is some
replaced by the knowledge that tissues can and do evidence of success in osteoarthritis (Mobasheri et al.
actively secrete molecules that shut down this process 2014) with high doses of natural antioxidants, including
(Serhan & Petasis 2011). Whether or not this involves vitamins and phytochemicals, these agents have largely
free radicals has not been determined. failed to yield benefits in rheumatoid arthritis (Canter
et al. 2007). SOD has shown some benefit in rat models
of arthritis as well as in some human patients (Carillon
Rheumatoid arthritis
et al. 2013).
Although inflammation represents a normal response of The success of therapies with enzymatic antioxidants
injured tissue, when uncontrolled, initiated by an has been limited, possibly due to the numerous barriers
abnormal stimulus, or occurring for prolonged periods associated with delivering them to the necessary
Figure 8. The role of free radicals in inflammation. The inflammatory process involves a complex series of events with a large number
of chemical mediators. Free radicals are involved in the overall response including the generation of chemotactic factors. Tissue
damage associated with chronic inflammation is mediated in part by the endogenous production of reactive species.
anatomical sites. Simple chemical antioxidants such as system began with the discovery that one of the earliest
vitamins E and C also appear to have only modest anti- changes evident in pre-atherosclerotic arterial walls is
inflammatory effects (Bruunsgaard et al. 2003; Tahan the accumulation of lipids by macrophages, resulting in
et al. 2011). The development of advanced delivery the formation of foam cells. This process is believed to
systems for antioxidant proteins with limited cell pene- be critical to the pathogenesis of atherosclerosis, and
tration and short half-lives, such as liposomally encap- extensive studies have shown that oxidized lipids may
sulated or polyethylene glycol conjugated-SOD, may play an important role (Stocker & Keaney 2004).
yield better results. However, the safety and practical use However, definitive evidence that lipid oxidation is
of such therapies remain to be determined. In general, essential for human atherosclerosis is lacking (Libby
the concept that exogenous antioxidants will be bene- et al. 2011).
ficial in inflammation may be flawed because radicals, Increases in plasma LDL content are clearly linked to
oxidants and antioxidants can have competing effects foam cell formation in vivo, and these cells are central in
on normal signaling pathways. the development of atherosclerosis (Howell et al. 2011).
An alternative approach to treating disorders caused However, macrophages will not accumulate normal LDL,
by abnormal phagocytic ROS production may be to even when incubated with high levels in vitro (Goldstein
inhibit the NADPH oxidase enzyme responsible for this et al. 1979). Thus, the exact mechanism of foam cell
process. Several small molecule inhibitors of this enzyme formation is unclear. Phagocytic cells are capable of
complex that is composed of six proteins have been generating ROS and fatty acids found in LDL are
developed, but lack specificity. The most well-known of susceptible to peroxidation. It is also known that
these is diphenylene iodonium. Small peptide inhibitors, oxidized, and other modified forms of LDL, are taken
with greater specificity, have also been developed (El- up by macrophages (Aviram 1993). Recent data indicate
Benna et al. 2012), but their use depends on the ability that toll-like receptor 4 is necessary for oxidized LDLs to
to deliver such peptides at effective concentrations to stimulate the differentiation of macrophages into foam
appropriate in vivo sites of interest. cells (Goyal et al. 2012; Howell et al. 2011). Cultured
endothelial cells, smooth muscle cells and fibroblasts, in
addition to activated phagocytes, can promote the
Cardiovascular disease
oxidation of LDL lipids (Heinecke et al. 1986; Henrickson
A role for free radicals in cardiovascular diseases, et al. 1981; Steinbrecher et al. 1990). There is a lag time
including atherosclerosis, hypertension and congestive in vitro from the onset of oxidizing conditions until
heart failure, has been supported by numerous studies modified LDLs are evident. This lag is believed to be
(Sugamura & Keaney 2011). Interest in the potential determined by the time it takes to lose endogenous
pathologic effects of free radicals in the cardiovascular antioxidants from LDL particles (Esterbauer et al. 1987).
Oxidized LDL particles are rapidly cleared from the numerous experiments confirmed his belief and estab-
circulation by sinusoidal cells (Pitas et al. 1985) as well as lished that the toxicity of molecular oxygen is based on
by endothelial cells through lectin-type LDL receptors. the ability of this molecule to undergo a four-electron
Once oxidized, macrophages and monocytes can phago- reduction to water and the inevitable leakage of some of
cytize modified LDLs resulting in the formation of the partially reduced intermediates that are reactive. In
foam cells. order to survive this constant bombardment by ROS, all
Foam cells appear to be required for fatty streak aerobic organisms have developed a number of
formation, and this precursor stage to atherosclerosis defenses (see ‘‘Tissue protection systems’’ section).
recruits additional macrophages. The generation of ROS However, these antioxidant defenses are not perfect,
and RNS, and the release of degradative enzymes, and the free radical theory of aging (Harman 1988) is
cytokines and growth factors by these cells, as well as based on the premise that a small amount of injury
the direct cytotoxicity of oxidized LDL to endothelial and occurs daily, culminating in the changes characteristic of
fibroblast cells (Cathcart et al. 1985; Hessler et al. 1983), aging. Although circumstantial evidence suggests this
can then damage endothelial cells leading, under theory has some validity, as noted previously, many of
chronic conditions, to the development of an athero- the effects of ROS are adaptive in nature and thus the
sclerotic lesion. free radical theory of aging may need to be reconsidered
(Liochev 2013).
The toxicity of oxygen to cells is directly related to the
Chemoprevention
partial pressure of oxygen (pO2) these cells are exposed
As antioxidants slow the oxidation of LDLs (Jialal & Fuller to. Breathing 100% oxygen at 0.21 atmospheres of
1995), and based on other data supporting a role for pressure is, in principle, non-toxic because the partial
oxidants in cardiovascular diseases, there have been a pressure is the same as that in ambient air. Under
large number of clinical studies conducted to assess the conditions of hyperoxia, i.e. a fraction of inspired oxygen
ability of antioxidant therapy to modulate the progress (FIO2) exceeding 0.21 (achieved either with a pO2 4 0.21
of these diseases (Sugamura & Keaney 2011). atmosphere at one atmosphere of barometric/ambient
Unfortunately, most of these studies have failed to pressure, or a pO2 of 0.21 atmosphere with a barometric
demonstrate any clinical efficacy of antioxidant treat- pressure 4 one atmosphere), the lung is the organ most
ments (Myung et al. 2013). The reasons for these failures severely damaged because that is the only site where
are similar to those discussed previously regarding the the pO2 is greatly increased above normal. In contrast,
chemoprevention of cancer. They include the complex exposure to 100% oxygen at an ambient pressure above
effects of ROS on both damaging and protective 1 atmosphere (i.e. hyperbaric oxygen: two or more
pathways, and a lack of knowledge of optimal dosing atmospheres that dissolves substantially more oxygen in
protocols. There is also the possibility of genetic factors the blood and tissues) damages not only the lung, but
influencing the outcome meaning that effects on patient also the central nervous system, resulting in convulsions
subpopulations may have been overwhelmed by a lack due to increased brain pO2.
of response in the general population. As a change in the pO2 is the only element involved in
Overall, although the preponderance of evidence pulmonary oxygen toxicity, this disorder is one of the
indicates that LDL oxidation occurs in vivo and may play few in which ROS can be firmly linked to tissue injury.
a role in atherosclerotic disease (Goyal et al. 2012), there Exposure of all mammalian species to 100% oxygen for
is no firm link between oxidative damage and this 24–72 h is sufficient to induce measurable tissue injury,
disease. Thus, the premise of using antioxidants to and in some species (e.g. rat), it is sufficient to cause
prevent atherosclerotic disease may be flawed. Even if death (Kalleet & Matthay 2013). The mechanism of this
correct, blanket strategies to ameliorate any disease by damage appears to be related to the increased intracel-
antioxidants have generally failed. Additional research lular utilization of oxygen and the subsequent increased
will eventually clarify the mechanism of atherosclerosis production of ROS. Nevertheless, similar to other
and determine whether antioxidant therapies can pro- diseases discussed previously, treatments with exogen-
vide benefits. ous antioxidants have been largely ineffectual in pre-
venting this damage. Animals are consistently protected
when their endogenous antioxidant defense systems
The toxicity of oxygen
can be induced. Specifically, neonates of species that are
The possibility that oxygen might be toxic was resistant to hyperoxia are able to increase their lung
recognized by Joseph Priestley in 1775 when he first content of antioxidant enzymes, while species that were
discovered this molecule. In the intervening years, susceptible were unable to do so (Frank et al. 1978).
Subsequent studies demonstrated that treatments of assume an excited state, allowing for further reactions
adult rats that resulted in resistance to oxygen toxicity with suitable reaction partners (including biological
also were associated with increases in pulmonary molecules or oxygen), i.e. energy transfer to O2 or an
antioxidant defenses (Frank et al. 1980). Conversely, initiation of electron transfer/hydrogen abstraction.
manipulations designed to decrease antioxidant These photosensitized electron transfer (with the
defenses enhanced oxygen toxicity. Transgenic mice generation of radicals, such as O2 ) and energy transfer
expressing elevated levels of copper/zinc SOD are more (with non-radical intermediates such as 1O2) reactions
resistant to oxygen toxicity (White et al. 1991) and other are referred to as type I and type II photosensitized
studies have shown that all three forms of SOD are reactions, respectively (Cadet et al. 2012). Alternatively,
important in this regard (Tsan 1997). However, the failure these reactions can be distinguished not with respect to
to achieve more complete protection was somewhat radical/non-radical intermediates, but with respect to
surprising and indicated that antioxidant enzymes were the dependence or independence of the reaction on
not acting alone in protecting cells and tissues from free oxygen. Type I reactions would be those where an
radical-mediated damage. excited photosensitizer reacts with anything but oxygen
As with other disorders where free radicals are (such as solvent), whereas in type II reactions an excited
implicated in the etiology, antioxidant therapy was photosensitizer would react with oxygen irrespective of
whether O 1
expected to be beneficial, and some positive effects 2 or O2 was generated (Foote 1991).
have been seen with antioxidant enzymes such as SOD In skin, endogenous photosensitizers include porphy-
and catalase delivered in forms to increase their plasma rins, flavins and others (for a comprehensive list, see
half-life and tissue penetration (Tanswell & Freeman Wondrak et al. 2006), and their presence contributes to
1987; White et al. 1989). In addition, various sulfur- the formation of ROS in tissues upon exposure to light,
containing reagents are protective against oxidant lung including UV radiation (UVB or UVA). The severity of
injury. For example, N-acetyl cysteine (NAC) has been diseases resulting from uncontrolled accumulation of
tested as a protective agent in various lung pathologies endogenous photosensitizers, such as porphyrias, is a
related to the production of ROS. NAC is frequently used testament to the biological significance of these photo-
as a mucolytic – in some countries available even oxidation processes. Accordingly, xenobiotic photosen-
without a prescription. NAC is a membrane-permeant sitizing compounds may trigger the photogeneration of
version of cysteine and therefore serves as a precursor ROS, resulting in various phototoxic effects, including
for the intracellular synthesis of GSH. In fact, the the death of exposed cells. In fact, this phenomenon is
application of NAC increases GSH concentrations in made use of in photodynamic therapy, with a photo-
bronchoalveolar lavage fluid of idiopathic pulmonary sensitizer applied to the area/tissue of interest, followed
fibrosis patients (Meyer et al. 1994) and ameliorates the by exposure to light of an appropriate wavelength
generation of oxidative stress biomarkers in several (Buchczyk et al. 2001).
animal models of lung injury (Ji et al. 2010; Sciuto et al.
1995). However, translating this work to the clinic has
Diabetes and the metabolic syndrome
not been successful at all levels. Recent reports even link
NAC consumption to the enhanced progression of The role of ROS, and more recently RNS, in the
certain types of lung cancer in mice (Sayin et al. 2014). pathogenesis of the metabolic syndrome, as well as
In part, this may be because ROS have indirect effects on both Type 1 and Type 2 diabetes, has been studied
cytokines that, in turn, activate defense systems. Thus, as extensively (Jialal et al. 2012; Rochette et al. 2014; Son
noted previously, antioxidants may have direct protect- 2012). Both an enhanced generation of ROS and a
ive effects that are counterbalanced by their indirect dysfunctional cellular antioxidant response appear to be
anti-protective effects, i.e. their blocking ROS-induced factors. For example, elevated blood glucose and
adaptation. saturated fatty acid levels are linked to an enhanced
production of superoxide/hydrogen peroxide through
stimulated mitochondrial metabolism, as well as through
Phototoxicity
activation of NADPH oxidases (Rochette et al. 2014). In
In cells exposed to light, activation of molecular oxygen addition, the hyperglycemia associated with poorly
by electron and energy transfer reactions (to yield O2 controlled diabetes can induce oxidative stress and
and 1O2, respectively) may occur in the presence of may play an important role in diabetic complications,
photosensitizers, i.e. molecules that facilitate these especially b-cell damage. As in other diseases associated
processes upon absorption of light of an appropriate with oxidative or nitrative stress, antioxidants have been
wavelength. Following irradiation, photosensitizers studied as potential treatments. Unfortunately, such
treatments are not effective (Golbidi et al. 2011) very rapidly with a second molecule of H2O2. Other
although research continues (Karunakaren & Park 2013; peroxidases, such as the five selenium-dependent gluta-
Rochette et al. 2014). For a recent review on the role of thione peroxidases also reduce H2O2, as well as organic
ROS and FoxO transcription factors in diabetes, refer hydroperoxides, by transferring reducing equivalents
Klotz et al. (2015). from GSH, producing water and GSSG (Brigelius-Flohe &
Maiorino 2013). Finally, in addition to heme-based and
selenocysteine-based peroxidases, cysteine (i.e. thiol-
Tissue protection systems
based) peroxidases, the peroxiredoxins, are major con-
The evolutionary steps that led to oxidative phosphor- tributors to H2O2 reduction – and act as peroxide sensors
ylation resulted in a far more efficient method of by relaying H2O2 signals (Rhee et al. 2012).
generating energy from sugars than glycolysis, but
came at the price of existing in an aerobic environment
Enzymes catalyzing repair of ROS-induced
with the resultant presence of ROS. This necessitated the
damage
evolution of efficient enzymatic and non-enzymatic
antioxidant systems. Although these provide sufficient
protection under normal circumstances, high levels of Despite the presence of a range of enzymatic and non-
enzymatic antioxidant systems, damage to cellular
oxidants and free radicals can overwhelm their protect-

ive capacity allowing injury to develop. In addition, molecules is inevitable. At times, this damage, if
chronic exposure to free radicals resulting from the confined and regulated, is desired and a requirement
inherent inefficiencies of biological protective systems for cellular adaptation processes as it serves as a
may contribute to a number of disorders. molecular signal that can be sensed by appropriate
Numerous studies have demonstrated that the tox- signaling molecules. Cells have also developed means to
icity of agents that generate reactive species is increased repair or remove oxidized molecules – both to prevent
when endogenous antioxidant systems are impaired. As further damage and to render an oxidative signal
discussed previously in the sections relating to specific reversible. As discussed in the ‘‘Protein oxidation’’
disorders, the reverse effect, achieving protection by section, the oxidation of amino acids causes physical
enhancing antioxidant activities, has proven much less changes in proteins, including fragmentation and aggre-
effective in most instances (Omenn 2007). gation. This increases the susceptibility of proteins to
proteolysis, thereby removing them before cellular
functions are impaired (Höhn et al. 2013). Similarly,
Enzymes scavenging ROS phospholipases will remove damaged fatty acid moieties
Mammalian enzymatic antioxidant defense systems in phospholipids (Sevanian & Kim 1987) thereby pre-
include the SODs, a family of metalloenzymes that venting adverse effects such damage could cause.
convert two superoxide anions to triplet oxygen and Cysteine residues are a major target of oxidants in
H2O2. The enzymatic rate of dismutation is approxi- mammalian cells, and several mechanisms exist to
mately 10 000 times greater than the spontaneous reduce oxidized cysteines, including thioredoxin (Trx)/
disproportionation (Fridovich 1983). The major mamma- Trx reductase-based systems (Lu & Holmgren 2014), or
lian forms of SOD are the cytosolic Cu/Zn enzyme, the the glutaredoxins that catalyze either the reduction of
mitochondrial Mn enzyme and the extracellular Cu/Zn- protein disulfides or the reversible S-glutathionylation of
containing SOD. Although molecularly different, all proteins (Lillig et al. 2008). This may serve to protect
catalyze the same reaction. One or more of the SOD cysteines from further (irreversible) oxidation.
enzymes are found in all aerobic organisms and it would Trx is present in all life-forms and knockout studies
appear that this enzyme is a critical antioxidant. have demonstrated its importance for life (Matsui et al.
Interestingly, however, only knocking out MnSOD 1996; Nonn et al. 2003). Trx contains a redox-active
(Lebovitz et al. 1996; Li et al. 1995), but neither cytosolic dithiol that can be oxidized to the corresponding
(Reaume et al. 1996) nor extracellular (Carlsson et al. disulfide (Figure 9). Trx influences cellular responses to
1995) SOD, is lethal. toxicants, especially oxidants and electrophiles (Watson
Multiple enzyme systems exist to deal with H2O2. et al. 2004). The direct scavenging of reactive species by
Notably, these include heme peroxidases, such as Trx is chemically feasible, but because Trx is present at
catalase, that catalyzes the dismutation of H2O2 to much lower concentrations (mM) than other endogenous
water and molecular oxygen. Catalase requires the antioxidants and nucleophiles such as GSH (mM), it is
presence of a small amount of H2O2 bound at the unlikely that Trx plays a major role as a direct scavenger.
active heme site (catalase compound I) in order to react However, Trx and related redoxins have specific
regulatory functions that affect apoptosis, cell prolifer- Non-enzymatic antioxidants

ation and iron metabolism (Hanschmann et al. 2013).
Non-enzymatic antioxidants that are important in bio-
Another amino acyl residue prone to oxidation is
logical systems include the lipid soluble tocopherols/
methionine, and enzymes exist (e.g. the methionine
tocotrienols (vitamin E) and carotenoids, as well as the
sulfoxide reductases) to catalyze the reduction of
water-soluble ascorbic acid (vitamin C), urate and GSH.
methionine sulfoxides back to methionine at the
The phenolic hydroxyl of vitamin E is the basis for the
expense of NADPH (Drazic & Winter, 2014).
antioxidant activity of this molecule and can donate an
There are numerous mechanisms by which cells are
electron forming the tocopheroxyl radical. This radical is
able to repair oxidative damage to nucleic acids (Svilar
minimally reactive and effectively ends radical reactions.
et al. 2011). These are essentially the same excision/
b-Carotene is a vitamin A precursor and can scavenge
repair pathways that have been studied extensively in 1
O2 in vitro with very high efficiency. However, recent
the context of genetics and cancer. Although nuclear
data indicate that 1O2 is not efficiently inactivated in
DNA is quantitatively the most important target of free
mammalian cells (Bosio et al. 2013). Nevertheless, the
radical injury, damage to mitochondrial DNA that codes
carotenoids have significant biologic antioxidant effects,
for only 13 proteins, may be functionally critical. In
particular when consumed in the diet, and are believed
recent years, it has become clear that there are highly
to be an important part of the overall antioxidant system
efficient mitochondrial DNA repair pathways to minimize

(Stahl & Sies 2005).
this potential adverse effect (Muftuoglu et al. 2014). Ascorbic acid is a vitamin by virtue of its role as
cofactor for enzymes such as prolyl hydroxylase. Its
Indirect antioxidant enzyme systems cofactor activity is based on its ability to undergo redox
reactions. This, along with its ability to chemically react
In addition to directly acting antioxidant enzymes, there with reactive species, has resulted in vitamin C being
are other enzymatic systems such as the glutathione S- considered an important water-soluble antioxidant.
transferases and the UDP-glucuronyl transferases that However, vitamin C can also function as a prooxidant
can remove biologically reactive electrophilic intermedi- (particularly in the presence of transition metal ions), and
ates through conjugation reactions. The export of these its overall importance to cellular antioxidant defenses
conjugates, as well as of other species formed during when present at physiologic levels remains unclear.
oxidative stress (including GSSG), can also protect cells Uric acid, the end product of purine metabolism in
from adverse effects. For example, the removal of humans, is a potent antioxidant (Ames et al. 1981) and
disulfides will limit S-thiolation reactions that can affect can complex transition metal ions (Yu 1994). Despite
cell signaling and will also help tissues maintain normal problems associated with its poor aqueous solubility (i.e.
thiol redox ratios. gout), there is evidence that it may play an important
Another indirect antioxidant strategy involves bypass- role in vivo as an antioxidant (Glantzounis et al. 2005).
ing potential radical production reactions. For example, Glutathione (GSH) and other cellular thiols are import-
NQO-1 catalyzes the two-electron reduction of quinones ant antioxidants, both as cofactors and as direct-acting
to the corresponding non-toxic hydroquinone forms scavengers. The free thiol group on these molecules is
(Vasiliou et al. 2006). By avoiding a one-electron reduc- nucleophilic, and will react rapidly with many electro-
tion to the semiquinone form, redox cycling with the philes. The glutathione S-transferases significantly speed
concomitant formation of free radicals is prevented this reaction in some cases, but these reactions can occur
(Figure 1). without enzymatic intervention. Relatively low reaction
Thioredoxin reductase
HSe SH
NADPH FAD reduced Trx-(S)2 Substrate
ox (SH)
NADP+ FADH2 oxidized Trx-(SH)2 Substrate

red (S-S)
Se S
Figure 9. Oxidation and reduction of thioredoxin. Thioredoxin (Trx) contains a redox-active dithiol that can be oxidized to the
corresponding disulfide. The disulfide is reduced by thioredoxin reductase.
rate constants for direct interactions between GSH and beneficial effects seen with antioxidant therapies. Future
various ROS are compensated by the high intracellular research should focus upon understanding additional
GSH concentrations (mM). Furthermore, metal chelating details regarding the roles of free radicals in cellular
molecules such as ferritin, metallothionein or albumin in signaling, particularly within the immune system, so that
plasma, protect against Fenton-like chemistry by neu- agents may be identified, or designed, that can more
tralizing redox-active metal ions. specifically affect cells and lead to effective treatments for
Various other compounds have been frequently the wide range of disorders associated with free radicals.
brought forth as potential endogenous antioxidants,
including the heme degradation product, bilirubin Acknowledgements
(Stocker 2011) and melatonin (N-acetyl-5-methoxytryp-
The authors thank Helmut Sies for helpful comments. We also
tamine; Galano et al. 2011; Hardeland 2014). While
extend our thanks to the three reviewers who provided
indeed interacting with certain ROS with high reaction exceptionally helpful comments that were used to improve the
rate constants, and therefore somewhat justifiably manuscript.
categorized as efficient ROS scavengers, these com-
pounds do not necessarily undergo recycling (like GSH, Declaration of interest
ascorbate and the tocopherols, all of which can be
The affiliation of the authors is as shown on the cover page. Both
recycled to their reduced forms at the direct or indirect

authors are employed by academic institutions and have been
expense of pyridine nucleotides). It also needs to be kept
throughout their careers. The manuscript was prepared during
in mind that in vivo concentrations of melatonin are very the normal course of their employment without outside
low, thus rendering it unlikely that any antioxidant effect financial support for this particular work. Over 95% of the
by melatonin is elicited by direct ROS scavenging. authors’ research on free radicals has been supported by public
Specifically, physiological plasma concentrations of funds. They have not received any personal compensation from
any private entities with a commercial interest in free radicals.
melatonin are a maximum of &0.5 nM (120 pg/ml).
Over the past 10 years, neither author has been engaged in any
Although transient plasma concentrations of up to legal or regulatory proceedings concerning free radicals other
&100 nM were reported after consumption of mela- than as an independent expert for federal agencies. This work
tonin supplements (Sanders et al. 1999), even these high product was conceptualized by the authors and represents an
concentrations are negligible compared to the levels of independent professional evaluation of the literature. The
conclusions drawn are exclusively those of the authors.
ascorbate, urate and albumin found in plasma.
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Free Radicals as Mediators of Tissue Injury and Disease

Article in Critical Reviews in Toxicology · February 1993

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ISSN: 1040-8444 (Print) 1547-6898 (Online) Journal homepage: http://www.tandfonline.com/loi/itxc20

Free radicals and related reactive species

James P. Kehrer & Lars-Oliver Klotz

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reactive species. For example, if hydrogen peroxide

Chemicals: halogenated hydrocarbons, quinones, iron, acetaminophen,

superoxide anion radical, i.e. is both a better reductant

radicals including the hydroxyl radical and, in the

Oxidases & flavoproteins

Mixed-function oxidase electron

ubiquinone/coenzyme Q itself (James et al. 2004).

(NOX). Hydrogen peroxide produced during this reaction can

the suggested role for ROS in the pathogenesis of chemical composition.

is indicative of the formation of reactive species.

Lipids Protein DNA

Anoxidant & Repair Systems

Lipid peroxidaon Amino acid oxidaon Oxidized DNA bases

• Membrane damage (De-) Acvaon of enzmes or • Mutaon

Cell & Tissue Injury

oxidants and free radicals can overwhelm their protect-

regulatory functions that affect apoptosis, cell prolifer- Non-enzymatic antioxidants

efficient mitochondrial DNA repair pathways to minimize

NADP+ FADH2 oxidized Trx-(SH)2 Substrate

recycled to their reduced forms at the direct or indirect

Toxicology 2001;169:1–15. with a focus on peroxynitrite: molecular targets, cellular

Drazic A, Winter J. The physiological role of reversible

JC, Toll-like receptor 4 mediates oxidized LDL-induced

inflammation: superoxide-dependent activation of a neutro- Methods 1998;8:233–244.

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