The Evolution of Free Radicals and

Oxidative Stress
Joe M. McCord, PhD
The superoxide free radical has come to occupy an amazingly
central role in a wide variety of diseases. Our metabolic focus on
aerobic energy metabolism in all cell types, coupled with some
chemical peculiarities of the oxygen molecule itself, contribute
to the phenomenon. Superoxide is not, as we once thought, just
a toxic but unavoidable byproduct of oxygen metabolism.

Rather it appears to be a carefully regulated metabolite capable

of signaling and communicating important information to the
cells genetic machinery. Redox regulation of gene expression
by superoxide and other related oxidants and antioxidants is
beginning to unfold as a vital mechanism in health and disease.
Am J Med. 2000;108:652 659. 2000 by Excerpta Medica, Inc.

tion. Reduction-oxidation (or redox) reactions are at the

core of our metabolic machinery. Redox reactions involve the transfer of electrons or hydrogen atoms from
one reactant to another. (The process of taking away electrons is called oxidation, because oxygen does it so well.
The substance receiving electrons becomes reduced.)
Life on our planet has evolved into two main categories of
lifeforms: 1) photosynthesizing plants that capture solar
energy and use it to drive thermodynamically unfavorable reactions producing reduced carbonaceous compounds, and 2) the rest of us, who eat these reduced carbonaceous compounds and burn them in thermodynamically favorable reactions. The latter reactions allow
the electrons (or hydrogen atoms) to return to the molecule that wants them most, molecular oxygen, with the
release of large amounts of energy that we can cleverly
trap and use to power our lives. This process might be
straightforward but for the quirky electronic structure of
the oxygen molecule. Oxygen itself is a diradical. (Chemically, a free radical is any molecule containing a single,
unpaired electron. Usually, paramagnetic transition
metal ions are not considered to be free radicals, although
by technical definition they are.) Most molecules contain
only pairs of electrons with opposite spins that reside in
discrete molecular orbitals and that may or may not participate in bond formation. Oxygen contains two electrons that are not spin paired, and each resides in an orbital of its own. Thermodynamically, oxygen wants to
take on additional electrons (two per atom, or four per
molecule) to produce water molecules, which have much
lower free energy. The unconventional distribution of
electrons in the oxygen molecule, however, makes it impossible for oxygen to accept a spin-matched pair of electrons, as badly as it may want them, until one of its unpaired electrons undergoes a spontaneous spin reversal to
make pairing possible. At ordinary collisional frequency,
the period of contact is too brief for this spin reversal to
occur, imposing a kinetic barrier (ie, a large energy of
activation) to most oxidative reactions. It is this kinetic
barrier that saves us from reacting explosively with an
atmosphere of huge thermodynamic oxidizing potential.

ree radicals, oxidative stress, and antioxidants have

become commonly used terms in modern discussions of disease mechanisms. The purpose of this
article is to provide some understanding as to why free
radical metabolism seems to occupy a central and remarkably common position in the mechanisms of so
many seemingly unrelated types of human disease. In articles to follow in this series, experts in a broad spectrum
of clinical specialties will examine more specifically the
free radical mechanisms known to be operative in the
inflammatory process and in ischemia/reperfusion injury, as they affect a variety of organ systems. In addition,
there are some free radical mechanisms that are unique to
certain tissue locations, especially those affecting the central nervous system.
In a general sense, substantial insight regarding free
radical production can come from the recognition of a
few basic facts about the nature of the chemical reactions
that support life as we know it, and from an understanding of the quirky structure of molecular oxygen, upon
which our metabolism is based. We can gain further insight as we consider the course of our evolution in a redox
active milieu. As evolutionary pressures tend to make the
best of any bad situation, we will see examples of evolutionary steps to contain, control, or avoid unpleasant
toxic species, and other examples of how these reactive
species can be turned to constructive purposes contributing to our defense, or even to our internal regulation and
communication processes.

A REDOX PRIMER
At the risk of inducing unpleasant flashbacks to college
chemistry courses, let us consider the class of chemical
reactions upon which our bodies rely for energy producFrom Webb-Waring Institute, University of Colorado Health Sciences
Center, Denver, Colorado.
Correspondence should be addressed to Joe M. McCord, PhD, Box
C-321, University of Colorado, 4200 East Ninth Avenue, Denver, Colorado 80262.
652

2000 by Excerpta Medica, Inc.

All rights reserved.

0002-9343/00/$see front matter

PII S0002-9343(00)00412-5

The Evolution of Free Radicals and Oxidative Stress/McCord

It is this same kinetic restriction that makes oxygen an

ideal terminal electron acceptor for biological systems.
Enzymes have binding sites that can hold oxygen in contact with an oxidizable substrate for a much longer time
than would occur by simple collision, overcoming the
kinetic barrier to reaction. At the same time, enzymes
may be designed to trap much of the energy released as
the oxidation occurs in a useful high-energy compound,
such as ATP.

OXYGEN-DERIVED FREE RADICALS AND

OXIDANTS
Our relationship with oxygen is, at best, a difficult one to
manage. Occasionally, under normal biological conditions, oxygen does manage to steal away electrons from
other molecules by nonenzymatic autoxidations. Because
it cannot accommodate a spin-matched pair, it must settle for stealing electrons one at a time. This breaking up of
electron pairs results in free radical formation. The oneelectron reduction product of oxygen is the superoxide
radical, O
2 . If two electrons are transferred, the product
is hydrogen peroxide, H2O2, which is not a radical. It is
nonetheless still eagerly receptive of two more electrons,
causing hydrogen peroxide to be a cytotoxic oxidant.
Certain chelates of ferrous iron and cuprous copper are
capable of transferring a third electron to hydrogen peroxide, causing lysis of the O-O bond. One fragment is
reduced to the state of water; the other fragment is the
hydroxyl free radical, HO, one of the most potent oxidants known. It can initiate lipid peroxidation, cause
DNA strand breaks, and indiscriminately oxidize virtually any organic molecule. The fact that it is so indiscriminate actually works in our favor, as most of its targets are
expendable. Reactivity and toxicity are not synonymous
(1). Despite the much lesser reactivity of superoxide, its
toxicity appears to be substantial precisely because its targets are focused. Cyanide, for example, is not particularly
reactive but is extremely toxic, because it effectively
strikes one crucial target, cytochrome oxidase.
The family of reactive intermediates resulting from the
incomplete reduction of oxygen therefore includes superoxide radical, hydrogen peroxide, and hydroxyl radical. It is not correct to refer to this group as oxygen-derived
free radicals, because one member, hydrogen peroxide, is
not a radical. Accordingly, several terms are now in use to
refer to this family. Reactive oxygen metabolites (ROM)
or active oxygen (AO) or variations thereon are the most
common. Occasionally, the terms are expanded to include electronically excited oxygen (singlet oxygen), hypochlorous acid (produced from oxygen by the neutrophil enzymes NADPH oxidase and myeloperoxidase),
peroxynitrite, and even nitric oxide, as all of these oxidants are derived from molecular oxygen.

THE EVOLUTION OF THE AEROBIC

LIFESTYLE
Life on this planet first evolved in a reducing atmosphere.
It was not until photosynthetic algae appeared that oxygen began to be introduced into the atmosphere in ever
increasing quantities. This shift from a reducing environment to an oxidizing one undoubtedly resulted in some
serious evolutionary pressures. One might be surprised
when examining modern metabolic pathways to find that
very few enzymes actually deal with molecular oxygen,
despite the fact that our bioenergetics scheme is completely dependent on the transfer of electrons to this acceptor. In fact, approximately 98% of the oxygen we metabolize is handled by a single enzyme, the cytochrome
oxidase in our mitochondria, which transfers four electrons to oxygen in a concerted reaction to produce two
molecules of water as the product. The enzyme is structurally quite complex, containing four redox centers (two
hemes and two copper ions), each of which can store a
single electron. When all centers are reduced, the simultaneous transfer of four electrons to an oxygen molecule
occurs with no detectable intermediate steps. One probable reason for this dominance by cytochrome oxidase in
the reduction of oxygen is the chemical difficulty of carrying out the reaction in a safe and controlled manner. As
discussed above, the reduction of oxygen by anything less
than the full complement of four electrons results in the
production of active oxygen species. Surely, as primitive
metabolic systems were struggling with the shift toward
energy-rich oxidative pathways, there must have been
many contenders that lost out in the end, because unacceptable levels of active oxygen products were produced.
Hence, one evolutionary strategy for survival in an oxidative environment is to restrict opportunities for poorly
controlled transfer of electrons to oxygen. Although
many enzymes involved in redox pathways exist as reduced intermediates with great thermodynamic potential
for the transfer of their electrons to oxygen, the evolutionary pressure has led to kinetic barriers against such
reactivity. Instead, most of these high-energy electrons
are transferred to NADP to produce NADPH, which is
itself kinetically resistant to reaction with oxygen. Thus,
our electron-conducting metabolic circuits are insulated
by evolutionary design to prevent the inadvertent development of short-circuits, much as the electron-conducting circuits in a modern electronic circuit board are insulated to keep electrons flowing in the proper channels.
Our insulation is not perfect. At least two sites have been
identified in the electron-transport chain (Complex I and
ubisemiquinone) where electrons may leak out through
breaks in the insulation to waiting oxygen molecules, resulting in the formation of superoxide (2). These sites are
diagrammatically represented in Figure 1 as the primary
sources of intracellular superoxide generation. It has been
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Figure 1. A schematic representation of signal transduction pathways for superoxide radical. Mitochondrial respiration accounts for
most of the superoxide generated in a cell with leakage sites at Complex I and at ubisemiquinone. The steady-state concentration of
superoxide is kept low in all compartments by SODs, not shown. The low levels of the radical remaining may modulate various
kinases, or may activate transcription factors directly to effect gene regulation in the nucleus. It is interesting to speculate on the
existence of a cell surface receptor for superoxide (R), which might transduce various responses within the cell by means of kinase
activation, for example. There is presently no direct evidence for such a receptor.

estimated that this leakage amounts to 1% to 2% of total

electron flux through the mitochondria. Because working
myocardium consumes oxygen at approximately 8 mM
per minute, rates of superoxide production could exceed
0.1 mM per minute. So, as well designed as we may be,
mitochondria are still the major source of accidental free
radical production. During exposure to hyperoxia, the
rates of leakage from these sites in lung mitochondria are
believed to increase in direct proportion to the increased
oxygen tension. Healthy adult rats will die within 72
hours if placed in an atmosphere of 100% oxygen, only
five times the normal concentration at sea level (3).

THE EVOLUTION OF ANTIOXIDANTS

AND ANTIOXIDANT ENZYMES
In addition to evolutionary attempts to avoid the production of reactive byproducts of oxidative metabolism, another very important direction was the ability to synthesize or accumulate antioxidantsmolecules that would
avidly react with and annihilate active oxygen species before they could inflict oxidative damage to vital components, such as DNA or cell membranes. The result was
hundreds of kinds of such antioxidant molecules, especially in plants. Among the most successful of these molecules are the water-soluble antioxidant ascorbic acid (vitamin C) and the lipid-soluble antioxidant -tocopherol
(vitamin E) (4).
The most efficient way to eliminate undesirable toxic
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species, of course, is by means of catalysis. Families of

antioxidant enzymes have evolved for this purpose, including superoxide dismutases for the elimination of the
superoxide radical, and catalases and glutathione peroxidases for the elimination of hydrogen peroxide and organic peroxides. Humans have three genes encoding superoxide dismutases (SOD), which localize in the mitochondria, the cytosol, or the extracellular spaces (5).
These genes are derived from two ancestral genes. One
gene gave rise to the copper-and-zinc containing enzymes; the other gave rise to the manganese- or ironcontaining enzymes. The genes can be traced back to the
most primitive organisms with high degrees of homology
around the active sites, indicating that the genes that
evolved early as life forms were figuring out how to survive and thrive in the presence of oxygen. The SODs (6)
catalyze the reaction:
O2 O2 2H 3 H2O2 O2

This dismutation or disproportionation reaction makes

use of the fact that superoxide is both an oxidant and a
reductant, eager to get rid of its extra electron or to take
on another. The enzyme uses one superoxide radical to
oxidize another. Catalases work in much the same way,
because hydrogen peroxide can be a weak reductant as
well as a fairly strong oxidant:
H2O2 H2O2 3 2H2O O2

In higher organisms, glutathione peroxidases appear to

have largely supplanted the need for catalase. These en-

The Evolution of Free Radicals and Oxidative Stress/McCord

zymes use NADPH as the reducing species for hydrogen

peroxide:
NADPH H H2O2 3 2H2O NADP

They can reduce lipid peroxides as well as hydrogen peroxide and are very important enzymes in the prevention
of lipid peroxidation to maintain the structure and function of biologic membranes.

THE REACTIVITY AND TOXICITY OF

SUPEROXIDE RADICAL
Although the chemical reactivity of the superoxide radical is modest, its toxicity is quite easily demonstrated.
Escherichia coli contains three genes for SODs: one enzyme uses manganese as its cofactor, one uses iron, one
uses copper and zinc. Disruption of the two major genes
encoding the manganese and iron enzymes results in a
bacterium unable to grow aerobically in minimal medium but still able to grow anaerobically (7). Aerobically,
it displays multiple auxotrophies and can grow if all
amino acids are added to the minimal medium, indicating that several biosynthetic pathways for amino acids are
sensitive to inactivation by the radical. Indeed, certain
dehydratases in E. coli have subsequently been shown to
be sensitive to inactivation by superoxide radical: the ,
-dihydroxyisovalerate dehydratase (8) and 6-phosphogluconate dehydratase (9). More than a dozen other important enzymes have similarly been shown to be inactivated by superoxide, including the following mammalian
enzymes: catalase (10), glyceraldehyde-3-phosphate dehydrogenase (11), ornithine decarboxylase (12), glutathione peroxidase (13), myofibrillar ATPase (14), adenylate
cyclase (15), creatine phosphokinase (16), and glutamine
synthase (17).
The toxicity of superoxide is seen not only in its ability
to inhibit certain enzymes and thereby attenuate vital
metabolic pathways, but also in its effects on other major
classes of biological molecules. E. coli deficient in SOD
activity show increased rates of mutagenesis (18), illustrating the role of the radical, directly or indirectly, in
DNA damage. In conditions of ischemia and reperfusion,
the most acute problem resulting from the overproduction of superoxide appears to be greatly increased rates of
lipid peroxidation. Here, superoxide radical plays paradoxical roles, in that it can both initiate and terminate
lipid peroxidation chains (19). This dichotomy of good
and bad will be explored further below. The practical result, however, is that a proper balance between oxidants
and antioxidants is required. Superoxide radical is not all
bad, as we shall see in other ways.
Knockout mice have now been produced for each of
the three mammalian SOD genes separately, but not yet
in combination. Surprisingly, SOD1 knockouts, missing

the cytosolic copper-zinc SOD, get along quite well until

they are stressed. They do show increased neurologic and
histologic damage after focal cerebral ischema and reperfusion (20) and increased motor neuron death after axonal injury (21). Similarly, SOD3 knockouts missing the
extracellular SOD do well until stressed. They show increased pulmonary damage after exposure to hyperoxia
(22). It is the homozygous SOD2 knockouts, however,
that dramatically illustrate how toxic superoxide can be.
SOD2 encodes the manganese-containing SOD that localizes to the mitochondrion, the site that is responsible
by far for most of the cellular production of superoxide.
SOD2 knockouts are born small, but alive, but they die
within days of birth with a dilated cardiomyopathy (23).

IF LIFE GIVES YOU LEMONS, MAKE

LEMONADE
One of the most fascinating aspects of evolution is the
ability to make the best of a bad situation, to make a silk
purse from the proverbial sows ear. There are clear examples of how active oxygen products, which we generally try to avoid producing at all costs, can actually be put
to constructive uses. The best example is the evolution of
our phagocytic NADPH oxidase. When first discovered
as a biologic metabolite, it appeared that the superoxide
radical was simply a noxious cytotoxic byproduct that
served no good purpose. That view changed when Babior
et al (24) realized that the radical is an important player in
our defense against invading microbes. It is now universally accepted that the production of superoxide radical
by activated polymorphonuclear leukocytes and other
phagocytes is an essential component of their bactericidal
armamentarium (25). Precisely because superoxide is cytotoxic, this NADPH oxidase has evolved to circumvent
the kinetic stability of NADPH and specifically to allow its
oxidation by molecular oxygen with the production of
superoxide radical. It equips phagocytes (which have
evolved mechanisms to detect and engulf invading microorganisms) with a way to destroy chemically the ingested microbes. In effect, superoxide serves as an extremely broad spectrum antibiotic. The neutrophil is also
destroyed in the process, a Kamikaze mission, by its own
artillery. In addition, surrounding healthy host cells may
be injured or even killed in the crossfire (26 28). In effect, superoxide is a mediator of inflammation, and SODs
display anti-inflammatory activity. It should be noted
that our immune defense system tends to overreact to any
challenge, as too timid a response may be fatal. The damage associated with the inflammatory process is the price
we pay for a vigilant defense system. From a medical therapeutic point of view, our own ingenuity may now have
evolved to a position of greater intelligence than our immune system, enabling us to treat ourselves with more
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targeted synthetic antibiotics (that our bodies have not

yet learned to produce) and allowing us to attenuate selectively our inflammatory response to spare ourselves
the damage associated with it.
We now believe that the superoxide radical plays additional constructive roles that may be more subtle in nature. When organisms evolved from single-celled creatures to complex, multicellular, multiorgan creatures, a
huge paradigm shift took place. For a single-celled organism, the biological imperative is simply to grow and divide without restraint when times are good and food is
plentiful. For higher organisms, only epithelial cells
(which are continuously being sloughed) are in this
grow-and-divide mode. Thus, nearly all the cells in our
bodies are under tight constraints that override the biologic imperative that tells our individual DNA molecules
to replicate themselves. Certain types of cells are able to
escape from the restraints under certain circumstances.
For example, fibroblasts are able to proliferate to form
scar tissue that is necessary for wound closure and healing. Lymphocytes capable of producing needed antibodies are able to proliferate to create a clone of such cells
when appropriately stimulated. In both cases, it appears
that superoxide may serve as the signal to override the
postmitotic constraints, and both cases may have evolved
as secondary responses to the oxidative nature of the
primitive immune systems superoxide-generating machinery.
The boundaries of an open wound become a battlefield
for phagocytes versus microbes. The objectives are twofold: to sterilize the wound, and to close the wound. The
NADPH oxidase of neutrophils becomes activated, and
superoxide and other oxidants derived therefrom, including hydrogen peroxide and hypochlorous acid, accomplish the first objective. It then appears that the
phagocyte-generated superoxide serves to stimulate fibroblasts to enter a proliferative mode (29), laying down
collagen fibrils and forming scar tissue to close and seal
the wound against further infection. The proliferative response of fibroblasts to exposure to superoxide is easily
demonstrated in the laboratory (30,31). Similarly, the
clonal expansion of stimulated lymphocytes may be
driven largely by superoxide production. B lymphocytes,
in particular, contain an NADPH oxidase closely related
to the one found in neutrophils and macrophages. When
the oxidase is stimulated to produce superoxide by mitogens, the result is cellular proliferation giving rise to a
clone of antibody-producing cells.

REDOX REGULATION OF GENE

EXPRESSION
If oxidative status can signal cells to respond in various
ways, we must ask how these signals are transduced, car656

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ried, and interpreted, especially by the cells genetic machinery. The study of redox regulation of gene expression
has exploded in recent years and clearly suggests that oxidants are major determinants of gene expression. Reactive oxygen intermediates have been implicated in the
activation of a variety of kinases [such as the Src kinase
family (32); protein kinase C (33); mitogen-activated
protein kinase, MAPK (34); and receptor tyrosine kinases
(35)] and transcriptional factors, such as AP-1 and
NF-B (34,36). An additional layer of complexity is offered by oxidant modification of redox-sensitive proteins, such as thioredoxin (37,38), which can regulate the
activity of certain stress kinases (39). Figure 1 depicts a
schematic representation of how reactive oxygen species
may regulate gene expression. This extensive interface between oxidants and reductants and the cells genetic machinery results in responsiveness to exogenous oxidant
exposure and to remarkably effective mechanisms governing redox homeostasis under normal conditions. The
unfolding complexity of the system further suggests just
how badly things can go wrong when a cells redox status
is upset.

OXIDATIVE STRESS AND MALIGNANCY

Reining in a cells biological imperative to proliferate and
placing constraints on the natural inclination to replicate
DNA and divide is no small feat. Indeed, it may require
more sophisticated cellular engineering to squelch the desire to proliferate than to promote it. The connection
between mild oxidative stress and cellular growth may
date back to the primordial soup. When food is plentiful,
metabolism is running at full speed, and there is sufficient
energy to support cell division, the rate of superoxide
production will also be high (at least in aerobic organisms), producing a state of mild oxidative stress. Conversely, when food supply nears exhaustion, the rate of
oxidant production within the cell would drop, possibly
signaling insufficient energy production to support the
cells entry into a vulnerable period of replication.
For a normal postmitotic cell to become malignantly
transformed, several conditions may have to be met. It
may be necessary to relieve certain evolutionary constraints that tell the cell not to enter the cell cycle leading
to mitosis. It may be necessary to provide mild oxidative
stress to serve as the driving force for proliferation. It may
even be necessary to disable yet another set of evolutionary constraints designed to prevent cells from running
amok by triggering apoptosis (40). This latter set of constraints can, in fact, be triggered by oxidative stress per se
(41). Although wild proliferation may be a mark of success for a bacterium, it is a very dangerous situation in a
cell that is part of a human being. The entire organism can
be brought down by what begins as a single errant cell that

The Evolution of Free Radicals and Oxidative Stress/McCord

has broken free of its evolutionary constraints. Thus, we

have evolved a failsafe system that can detect out-of-control proliferation and can cause programmed self destruction in any cell showing this behavior.
How might a wannabe cancer cell achieve and maintain the condition of mild oxidative stress necessary to
drive its proliferation? It has been shown that many types
of human cancer cells have reduced manganese superoxide dismutase (MnSOD) (42). In most cases, the reduced
activity has been assumed to be the result of defective
expression of the gene (ie, changes in the promotor region of the gene) (43). Oberley, St. Clair, and others have
observed in numerous studies that transfection with the
gene for human MnSOD can reverse the malignant phenotype of tumor cells, suggesting that MnSOD functions
as a tumor suppressor (44 48). Very recently, Xu et al
(49) reported finding a variant sequence containing a
cluster of three mutations in the promoter regions of the
MnSOD genes from 5 of 14 human cancer cell lines examined. All 5 cell lines were heterozygous for the variant
sequence. The mutations change the binding pattern of
transcription factor AP-2 and cause a marked diminution
in the efficiency of the promoter using a luciferase reporter assay system. Alternatively, mutations in the coding region of MnSOD may adversely affect catalytic efficiency or the stability of the protein.

OXIDATIVE STRESS AND HUMAN

DISEASE
Perhaps the most noteworthy observation concerning the
role of oxidative stress in human disease is the commonality of it. Oxidative stress is now thought to make a significant contribution to all inflammatory diseases [arthritis (26,50), vasculitis (51), glomerulonephritis (52),
lupus erythematosus (53), adult respiratory distress syndrome (54)], ischemic diseases [heart disease (55), stroke
(56), intestinal ischemia (57)], hemochromatosis (58),
acquired immunodeficiency syndrome (AIDS) (59), emphysema (60), organ transplantation (61,62), gastric ulcers (63), hypertension (64) and preeclampsia (65), neurologic diseases [multiple sclerosis (66), Alzheimers disease (67), Parkinson disease (68), amyotrophic lateral
sclerosis (69), muscular dystrophy (70)], alcoholism
(71), smoking-related diseases (72), and many others.
The reason that overproduction of free radicals is a feature of such a broad spectrum of diseases derives from the
fact that oxidative metabolism is a necessary part of every
cells metabolism. If that cell is sick or injured in any way
that results in mitochondrial injury (calcium influx, leaky
membranes, and so forth), then increased production of
superoxide is likely to result. In the series of articles to
follow, experts in many of these areas will delineate spe-

cific roles for free radicals and oxidative stress in a number of the diseases mentioned above.

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