Free Radicals in the Physiological Control
of Cell Function
WULF DRO
¨
GE
Division of Immunochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany
I. Introduction 48
A. From oxidative damage to redox regulation: historic background 48
B. About this review 49
II. Major Types of Free Radicals and Their Derivatives in Living Organisms 49
A. Reactive oxygen species 49
B. Reactive nitrogen species 49
C. Key message from section
II 50
III. Oxidative Stress Response as a Model of Redox Signaling 50
A. Maintenance of “redox homeostasis” 50
B. Examples of redox signaling in the maintenance of redox homeostasis 53
C. Key message from section
III 55
IV. Nitric Oxide and Reactive Oxygen Species as Regulatory Mediators of Physiological Responses 55
A. Regulated production of free radicals in higher organisms 55
B. Regulation of vascular tone and other regulatory functions of NO 57
C. ROS formation as a sensor for changes in oxygen concentration: control of ventilation 57
D. The oxygen sensor in the regulation of erythropoietin production: redox regulation through the
transcription factor hypoxia-inducible factor 1 58
E. Redox regulation of cell adhesion 58
F. Redox-mediated amplification of immune responses 59
G. Role of ROS in programmed cell death 59
H. Regulatory role of ROS in plants 60
I. Key messages from section
IV 61
V. Redox-Sensitive Targets in Signaling Cascades 61
A. Role of ROS in receptor-mediated signaling pathways: the EGF receptor as a case in point 61
B. Enhancement of signaling cascades by oxidative inhibition of protein tyrosine phosphatases 62
C. Role of ROS in the regulation of insulin receptor kinase activity 63
D. Activation of cytoplasmic protein kinases by ROS 64
E. Oxidative activation of MAPK cascades 65
F. Oxidative activation of protein kinase C isoforms 65
G. ROS-induced changes in cytosolic Ca
2⫹
concentrations
66
H. Activation of the transcription factor AP-1 66
I. Activation of the transcription factor NF-
B66
J. Importance of the intracellular glutathione level 67
K. Differential redox requirements in the induction and execution of signal cascades 67
L. Key messages from section
V 69
VI. Role of Reactive Oxygen Species in Senescence, Stress Conditions, and Disease: Pathophysiological
Implications of Redox Regulation 69
A. Mediators of excessive ROS production 69
B. The free radical theory of aging 70
C. Indications for an age-related increase in ROS levels 70
D. Replicative senescence as a putative consequence of redox-mediated dysregulation 71
E. Oxidative induction of telomere shortening 72
F. Factors contributing to changes in ROS production 72
G. ROS production in skeletal muscle tissue during immobilization and intensive physical exercise 73
H. Oxidative stress as a frequent complication in disease conditions 74
I. Malignant diseases 74
J. Diabetes mellitus 74
K. Atherosclerosis 75
L. Neurodegenerative diseases 75
Physiol Rev
82: 47–95, 2002; 10.1152/physrev.00018.2001.
www.prv.org 470031-9333/02 $15.00 Copyright © 2002 the American Physiological Society
M. Rheumatoid arthritis 76
N. HIV infection 76
O. Ischemia and reperfusion injury 77
P. Obstructive sleep apnea 77
Q. Key messages from section
VI 77
VII. Conclusions 78
A. Physiological aspects of redox regulation 78
B. Molecular aspects of redox regulation: gain of function, loss of function, or outright destruction 79
C. Regulated versus uncontrolled free radical production: increased ROS levels in old age
and disease 79
D. Chances for therapeutic intervention and perspectives 80
Dro¨ ge, Wulf. Free Radicals in the Physiological Control of Cell Function. Physiol Rev 82: 47–95, 2002; 10.1152/
physrev.00018.2001.—At high concentrations, free radicals and radical-derived, nonradical reactive species are
hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, however,
nitric oxide (NO), superoxide anion, and related reactive oxygen species (ROS) play an important role as regulatory
mediators in signaling processes. Many of the ROS-mediated responses actually protect the cells against oxidative
stress and reestablish “redox homeostasis.” Higher organisms, however, have evolved the use of NO and ROS also
as signaling molecules for other physiological functions. These include regulation of vascular tone, monitoring of
oxygen tension in the control of ventilation and erythropoietin production, and signal transduction from membrane
receptors in various physiological processes. NO and ROS are typically generated in these cases by tightly regulated
enzymes such as NO synthase (NOS) and NAD(P)H oxidase isoforms, respectively. In a given signaling protein,
oxidative attack induces either a loss of function, a gain of function, or a switch to a different function. Excessive
amounts of ROS may arise either from excessive stimulation of NAD(P)H oxidases or from less well-regulated
sources such as the mitochondrial electron-transport chain. In mitochondria, ROS are generated as undesirable side
products of the oxidative energy metabolism. An excessive and/or sustained increase in ROS production has been
implicated in the pathogenesis of cancer, diabetes mellitus, atherosclerosis, neurodegenerative diseases, rheumatoid
arthritis, ischemia/reperfusion injury, obstructive sleep apnea, and other diseases. In addition, free radicals have
been implicated in the mechanism of senescence. That the process of aging may result, at least in part, from
radical-mediated oxidative damage was proposed more than 40 years ago by Harman (J Gerontol 11: 298–300, 1956).
There is growing evidence that aging involves, in addition, progressive changes in free radical-mediated regulatory
processes that result in altered gene expression.
I. INTRODUCTION
A. From Oxidative Damage to Redox Regulation:
Historic Background
The presence of free radicals in biological materials
was discovered less than 50 years ago (114). Soon ther-
after, Denham Harman hypothesized that oxygen radicals
may be formed as by-products of enzymic reactions in
vivo. In 1956, he described free radicals as a Pandora’s
box of evils that may account for gross cellular damage,
mutagenesis, cancer, and, last but not least, the degener-
ative process of biological aging (234, 235).
The science of free radicals in living organisms en-
tered a second era after McCord and Fridovich (386)
discovered the enzyme superoxide dismutase (SOD) and,
finally, convinced most colleagues that free radicals are
important in biology. Numerous researchers were now
inspired to investigate oxidative damage inflicted by rad-
icals upon DNA, proteins, lipids, and other components of
the cell (reviewed in Ref. 49).
A third era began with the first reports describing
advantageous biological effects of free radicals. Mittal
and Murard (394) provided suggestive evidence that the
superoxide anion (O
2
⫺
䡠), through its derivative, the hy
-
droxyl radical, stimulates the activation of guanylate cy-
clase and formation of the “second messenger” cGMP.
Similar effects were reported for the superoxide deriva-
tive hydrogen peroxide (615). Ignarro and Kadowitz (272)
and Moncada and colleagues (456) discovered indepen-
dently the role of nitric oxide (NO) as a regulatory mole-
cule in the control of smooth muscle relaxation and in the
inhibition of platelet adhesion. Roth and Dro¨ge (472)
found that in activated T cells the superoxide anion or low
micromolar concentrations of hydrogen peroxide in-
crease the production of the T-cell growth factor interleu-
kin-2, an immunologically important T-cell protein. Keyse
and Tyrrell (300) showed that hydrogen peroxide induces
the expression of the heme oxygenase (HO-1) gene. Storz
and colleagues (551) reported the induction of various
genes in bacteria by hydrogen peroxide, and Schreck and
Baeuerle (501) reported the activation of the transcription
factor nuclear factor
B (NF-
B) by hydrogen peroxide in
mammalian cells.
At the beginning of the 21st century, there is now a
large body of evidence showing that living organisms
48 WULF DRO
¨
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have not only adapted to an unfriendly coexistence with
free radicals but have, in fact, developed mechanisms for
the advantageous use of free radicals. Important physio-
logical functions that involve free radicals or their deriv-
atives include the following: regulation of vascular tone,
sensing of oxygen tension and regulation of functions that
are controlled by oxygen concentration, enhancement of
signal transduction from various membrane receptors in-
cluding the antigen receptor of lymphocytes, and oxida-
tive stress responses that ensure the maintenance of re-
dox homeostasis (see Table 1). The field of redox
regulation is also receiving growing attention from clini-
cal colleagues in view of the role that oxidative stress has
been found to play in numerous disease conditions. These
pathological conditions demonstrate the biological rele-
vance of redox regulation. The delicate balance between
the advantageous and detrimental effects of free radicals
is clearly an important aspect of life. The science of
biological “redox regulation” is a rapidly growing field of
research that has impact on diverse disciplines including
physiology, cell biology, and clinical medicine.
B. About This Review
We are now living in a particularly exciting time of
redox research where information from different fields
and independent approaches is falling into place and be-
ginning to reveal a meaningful picture. This is a good time
for a broad overview that summarizes the main principles
of redox regulation. In textbook style, this review de-
scribes the current knowledge and paradigms but does
not discuss future research directions, historical contro-
versies, or experimental models. Moreover, it was not
within the scope of this review to deal with all the details.
Even the more than 600 references cited here do not
cover all relevant publications in the field. For the inter-
ested reader, a number of more detailed and specific
reviews on this topic are recommended (see Refs. 13, 20,
45, 66, 122, 125, 183, 211, 212, 251, 294, 333, 337, 397, 446,
506, 510, 512, 632, 659).
II. MAJOR TYPES OF FREE RADICALS
AND THEIR DERIVATIVES
IN LIVING ORGANISMS
A. Reactive Oxygen Species
The superoxide anion is formed by the univalent
reduction of triplet-state molecular oxygen (
3
O
2
). This
process is mediated by enzymes such as NAD(P)H oxi-
dases and xanthine oxidase or nonenzymically by redox-
reactive compounds such as the semi-ubiquinone com-
pound of the mitochondrial electron transport chain (see
Fig. 1). SODs convert superoxide enzymically into hydro-
gen peroxide (130, 187). In biological tissues superoxide
can also be converted nonenzymically into the nonradical
species hydrogen peroxide and singlet oxygen (
1
O
2
)
(549). In the presence of reduced transition metals (e.g.,
ferrous or cuprous ions), hydrogen peroxide can be con-
verted into the highly reactive hydroxyl radical (䡠OH)
(101). Alternatively, hydrogen peroxide may be converted
into water by the enzymes catalase or glutathione perox-
idase (Fig. 1). In the glutathione peroxidase reaction glu-
tathione is oxidized to glutathione disulfide, which can be
converted back to glutathione by glutathione reductase in
an NADPH-consuming process (Fig. 1).
Because superoxide and NO are readily converted by
enzymes or nonenzymic chemical reactions into reactive
nonradical species such as singlet oxygen (
1
O
2
), hydrogen
peroxide, or peroxynitrite (ONOO
⫺
), i.e., species which
can in turn give rise to new radicals, the regulatory effects
of these nonradical species have also been included in
this review. Most of the regulatory effects are indeed not
directly mediated by superoxide but rather by its reactive
oxygen species (ROS) derivatives. Frequently, different
reactive species coexist in the reactive environment and
make it difficult to identify unequivocally which agent is
responsible for a given biological effect.
B. Reactive Nitrogen Species
The NO radical (NO䡠) is produced in higher organ-
isms by the oxidation of one of the terminal guanido-
TABLE
1. Important physiological functions that involve free radicals or their derivatives
Type of Radical Source of Radical Physiological Process
Nitric oxide (NO䡠) Nitric oxide synthase Smooth muscle relaxation (control of vascular tone) and various other
cGMP-dependent functions
Superoxide (O
2
⫺
䡠 ) and related ROS
NAD(P)H oxidase Control of ventilation
Control of erythropoietin production and other hypoxia-inducible functions
Smooth muscle relaxation
Signal transduction from various membrane receptors/enhancement of
immunological functions
Superoxide (O
2
⫺
䡠 ) and related ROS
Any source Oxidative stress responses and the maintenance of redox homeostasis
ROS, reactive oxygen species.
FREE RADICALS IN REGULATION OF PHYSIOLOGICAL FUNCTIONS
49
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nitrogen atoms of L-arginine (437). This process is
catalyzed by the enzyme NOS. Depending on the micro-
environment, NO can be converted to various other reac-
tive nitrogen species (RNS) such as nitrosonium cation
(NO
⫹
), nitroxyl anion (NO
⫺
) or peroxynitrite (ONOO
⫺
)
(546). Some of the physiological effects may be mediated
through the intermediate formation of S-nitroso-cysteine
or S-nitroso-glutathione (207).
C. Key Message From Section
II
The most relevant radicals in biological regulation
are superoxide and NO (see Table 1). These radicals are
formed by two groups of enzymes, i.e., the NAD(P)H
oxidase and NOS isoforms, respectively. Many regulatory
effects are mediated by hydrogen peroxide and other ROS
that are chemically derived from superoxide.
III. OXIDATIVE STRESS RESPONSE AS A
MODEL OF REDOX SIGNALING
A. Maintenance of “Redox Homeostasis”
The term redox signaling is widely used to describe
a regulatory process in which the signal is delivered
through redox chemistry. Redox signaling is used by a
wide range of organisms, including bacteria, to induce
protective responses against oxidative damage and to
reset the original state of “redox homeostasis” after tem-
porary exposure to ROS.
1. Oxidant-antioxidant balance
Free radicals and reactive nonradical species derived
from radicals exist in biological cells and tissues at low
but measurable concentrations (228, 527). Their concen-
trations are determined by the balance between their
rates of production and their rates of clearance by various
antioxidant compounds and enzymes, as illustrated sche-
matically in Figure 2. Halliwell and Gutteridge (228) have
defined antioxidants as substances that are able, at rela-
tively low concentrations, to compete with other oxidiz-
able substrates and, thus, to significantly delay or inhibit
the oxidation of these substrates. This definition includes
the enzymes SOD, glutathione peroxidase (GPx), and
catalase, as well as nonenzymic compounds such as
␣
-to-
copherol (vitamin E),

-carotene, ascorbate (vitamin C),
and glutathione.
In addition, there are compounds that have a rela-
tively low specific antioxidative activity, i.e., on a molar
basis, but, when present at high concentrations, can con-
tribute significantly to the overall ROS scavenging activ-
ity. The most prominent examples of such high-level,
low-efficiency antioxidants are free amino acids, pep-
tides, and proteins. Practically all amino acids can serve
as targets for oxidative attack by ROS, although some
amino acids such as tryptophan, tyrosine, histidine, and
cysteine are particularly sensitive to ROS (126, 128, 545).
Because the cumulative intracellular concentration of
free amino acids is on the order of 10
⫺1
M, free amino
acids are quantitatively important ROS scavengers (see
sect. IIIB5).
2. Oxidized proteins as substrates for proteolytic
digestion and their contribution to
redox homeostasis
Oxygen radicals and other ROS cause modifications
of proteins (reviewed in Ref. 220). These oxidative mod-
ifications may lead to changes in protein function, chem-
ical fragmentation, or increased susceptibility to proteo-
lytic attack (124, 543, 631). Proteolytic degradation is
FIG.
1. Pathways of reactive oxygen species (ROS)
production and clearance. GSH, glutathione; GSSG,
glutathione disulfide.
50 WULF DRO
¨
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executed mainly by proteasomes (219). In one of the
studies, proteolysis was estimated to increase more than
11-fold after exposure to superoxide or hydrogen perox-
ide (127). Proteolysis is enhanced by 20–400
M hydro-
gen peroxide, whereas millimolar concentrations inhibit
proteolysis and may lead to the intracellular accumula-
tion of oxidized proteins (220, 544).
The proteins may differ strongly in their susceptibil-
ity to oxidative damage. The redox-sensitive amino acids
of bovine serum albumin, for example, were shown to be
oxidized about twice as fast as those of glutamine syn-
thase (54), and intact proteins are less sensitive to oxida-
tion than misfolded proteins (161). These findings impli-
cate that 1) phylogenetic evolution has selected for
protein structures that are relatively well-protected
against oxidation and 2) ROS scavenging activities of
intact proteins are weaker than those of misfolded pro-
teins or equivalent concentrations of their constituent
amino acids. Protein oxidation and enhanced proteolytic
degradation cause, therefore, a net increase in ROS scav-
enging capacity as schematically illustrated in Figure 2.
Preliminary experiments showed that treatment of human
skeletal muscle cells with proteasome inhibitors causes a
substantial increase in intracellular ROS levels and that
this increase is reversed by the addition of free amino
acids (R. Breitkreutz and W. Dro¨ ge, unpublished observa-
tions). More systematic studies are needed to determine
the relative contribution of proteins, free amino acids, and
classical antioxidant compounds and enzymes to the total
ROS scavenging capacity of different cells and tissues.
3. Changes in oxidant-antioxidant balance as a
trigger for redox regulation: the theory of redox
homeostasis and the existence of different
quasi-stable states
Living cells and tissues have several mechanisms for
reestablishing the original redox state after a temporary
exposure to increased ROS or RNS concentrations. The
production of NO (NO䡠), for example, is subject to direct
feedback inhibition of NOS by NO (see sect.
IIIB1). Ele-
vated ROS concentrations induce in many cells the ex-
pression of genes whose products exhibit antioxidative
activity (Fig. 2). A major mechanism of redox homeosta-
sis is based on the ROS-mediated induction of redox-
sensitive signal cascades that lead to increased expres-
sion of antioxidative enzymes or an increase in the cystine
transport system, which, in turn, facilitates in certain cell
types the increase in intracellular glutathione (see Fig. 2;
for details see sect.
III, B2–B4). Moreover, because pro-
teins generally provide less ROS scavenging activity than
an equivalent amount of the free amino acids contained in
them, it is reasonable to assume that oxidative enhance-
ment of proteolysis also contributes, at least to some
extent, to the maintenance of redox homeostasis (see Fig.
2 and sect.
IIIB5).
Cells or tissues are in a stable state if the rates of ROS
production and scavenging capacity are essentially con-
stant and in balance (see Fig. 2 and baseline level in Fig.
3). Redox signaling requires that this balance is disturbed,
either by an increase in ROS concentrations or a decrease
in the activity of one or more antioxidant systems (Fig. 3).
In higher organisms, such an oxidative event may be
induced in a regulated fashion by the activation of endog-
enous RNS- or ROS-generating systems (see sect.
IVA).
However, similar responses may be induced by oxidative
stress conditions generated by environmental factors (see
sect.
IIIA4). If the initial increase in ROS is relatively small,
the antioxidative response may be sufficient to compen-
sate for the increase in ROS and to reset the original
balance between ROS production and ROS scavenging
capacity. Thus physiological manifestations of redox reg-
ulation involve typically a temporary increase and/or a
temporary shift of the intracellular thiol/disulfide redox
state toward more oxidative conditions, as illustrated in
FIG.
2. Mechanisms of redox homeostasis.
Balance between ROS production and various
types of scavengers. The steady-state levels of
ROS are determined by the rate of ROS produc-
tion and their clearance by scavenging mecha-
nisms. Certain antioxidative enzymes including
superoxide dismutase (SOD), glutathione per-
oxidase, catalase, and thioredoxin are potent
ROS scavengers but occur in cells only at rela-
tively low concentrations. The same is true for
nonenzymic antioxidants. Amino acids and pro-
teins are also ROS scavengers. Amino acids are
less effective than the classical antioxidants on
a molar basis, but their cumulative intracellular
concentration is ⬎0.1 M.
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