Table Of ContentSuperoxide Ion:
Chemistry and
Biological
Implications
Volume II
Oxygen Radicals in Biology
Author
Igor B. Afanas’ev, D.Sc.
Chief
Laboratory of Chemical Kinetics
All-Union Vitamin Research Institute
Moscow, Soviet Union
First published 1991 by CRC Press
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PREFACE TO VOLUME II
This book is a continuation of the first volume of Superoxide Ion: Chemistry and
Biological Implications, and it is concerned with the problems of production and reactions
of the superoxide ion and other oxygen radicals in biological processes. By contrast with
chemical experiments, it is virtually impossible to produce the superoxide ion alone in
biological systems; moreover, since the in vitro studies are always carried out to model real
in vivo processes, the attempt to study the role of the superoxide ion in some biological
systems without other oxygen species is not only unrealistic but is actually not very fruitful.
Therefore, in this book we consider all the processes in which the participation of oxygen
radicals, i.e., superoxide ion, perhydroxyl radical, hydroxyl radicals, and active iron-oxygen
intermediates formed in the Fenton reaction during the oxygenation of iron ions (“crypto-
hydroxyl” radicals, perferryl and ferryl ions), is suggested. There is now an essential change
in understanding of the role of oxygen radicals in biological processes: if earlier they were
considered only as some damaging species, at present, their important regulatory role is
becoming evident. Both damaging and regulatory functions of oxygen radicals will be
discussed here. Since superoxide ion is a precursor of all other oxygen species, their reactions
should be considered as a consequence of the formation of superoxide ion.
This book consists of five chapters. The questions of classification and reactivity of
oxygen radicals and other oxygen metabolites are briefly discussed in Chapter 1. The three
subsequent chapters are concerned with the modes of production of oxygen radicals in
biological systems. In Chapter 5, the interaction of oxygen radicals with naturally and
biologically active compounds, cellular components and cells, and the reactions of oxygen
radicals in biological systems are discussed. There is a big difference in conducting oxygen
radical studies in chemistry and in biology. As we have seen (Volume I), chemists actually
use only a few methods for superoxide production (radiolysis, photolysis, electrochemical
reduction of dioxygen, and alkali metal superoxides) while in biological systems, superoxide
ion is formed as a result of numerous redox processes in which natural substrates, enzymes,
xenobiotics, cellular components, and whole cells take part. Therefore, much attention is
given to studying the mechanisms of superoxide production in cells and cellular components.
The difference in the experimental techniques used in biological and chemical investi
gations should also be stressed. Again, in chemical studies, workers do not apply many
specific methods for detection and identification of oxygen radicals: they are ESR and optical
spectroscopy, pulse radiolysis and flash photolysis, electrochemical technique, and the usual
chemical methods based on the identification of reaction products. All these methods are
also applied in biological systems for the identification of oxygen radicals and the investi
gation of their reactions. However, in addition to these methods, numerous biochemical,
physiological, immunological, and pathological experiments are carried out in which im
portant information concerning the role of oxygen radicals in physiological and pathological
processes is obtained.
In this book an attempt was made to describe if not all, at least most biological processes
in which oxygen radicals are produced. Due to the great number of such works, it is
impossible (and even pointless, because it can be correctly done only by scientists working
in specific experimental fields) to consider the experimental details of these works. However,
I tried to compile a special index of experimental techniques applied in cited works which
I hope will be useful to readers. Furthermore, since there is great growth in the oxygen
radical studies in biology, about 400 papers were published during the compilation of Volume
II. Therefore, I included short discussions on these works in Additions to all chapters.
As the reader can see, Volume II does not contain all data concerning oxygen radicals
in biology. Thus many questions such as the role of oxygen radicals in pathological processes
which we wanted to discuss in this Volume (see Preface to Volume I) are not considered.
The reason is obvious: too many works in this field, especially concerned with pathobiological
aspects of this problem, appear in the current literature. I hope that these questions, including
the studies of oxygen radicals in organs and a whole organism, the role of oxygen radicals
in free radical pathologies (ischemia, cancer, inflammation, environmental-associated dis
eases, etc.), and the role of antiradical, antioxidative, and chelating drugs in the treatment
of these diseases, will be discussed in subsequent work.
Professor I. B. Afanas’ev
THE AUTHOR
Igor B. Afanas’ev, D.Sc., Professor, is Chief of the Laboratory of Chemical Kinetics,
All-Union Vitamin Research Institute, Moscow.
Dr. Afanas’ev graduated in 1958 from Moscow Chemico-Technological Institute, re
ceived his Candidate Sc. and D.Sc. degrees (Chemistry) in 1963 and 1972, respectively.
He became a Professor of Chemical Kinetics and Catalysis in 1982.
Dr. Afanas’ev has presented lectures and reports at many national and international
conferences on chemistry, biology, and medicine of free radicals, including “Oxygen Rad
icals in Chemistry and Biology’’, Munich, 1983; “Superoxide and Superoxide Dismutase
in Chemistry, Biology, and Medicine’’, Rome, 1985; and International Conference on
Medical, Biochemical, and Chemical Aspects of Free Radicals, Kyoto, 1988; etc.
Dr. Afanas’ev is a Chairman of the U.S.S.R. annual seminar on oxygen radicals in
chemistry and biology. He has published more than 130 research papers, among them about
30 papers in English. His current research interests include studying the reactions of oxygen
radicals in chemical and biological systems and developing new antioxidative drugs for
combating free radical pathologies.
TABLE OF CONTENTS
Chapter 1
Who Is the Villain? (Classification and Reactivities of Oxygen Species in
Biological Systems)..................................................................................................................... 1
Chapter 2
Nonenzymatic and Enzymatic Production of Oxygen Radicals in
Biological Systems.........................................................................................................................5
I. Introduction.........................................................................................................................5
II. Nonenzymatic Production of the Superoxide Ion..........................................................5
III. Enzymatic Production of the Superoxide Ion ...............................................................9
A. Xanthine Oxidase .................................................................................................9
B. Peroxidases, Catalase, PGH Synthase, Lipoxygenase, and
Other Enzymes....................................................................................................10
C. Production of the Superoxide Ion by Mitochondria........................................11
D. Production of the Superoxide Ion by Microsomes..........................................13
E. Production of the Superoxide Ion by Cell Nuclei............................................15
IV. Production of Hydroxyl, Crypto-Hydroxyl, and Site-Specific Radicals.................15
A. Enzymatic and Nonenzymatic Reduction of Ferric Ions and
Complexes...........................................................................................................16
B. Physiological Metal Ion Catalysts of the Fenton Reaction...........................17
V. Additions...........................................................................................................................18
References.......................................................................................................................................19
Chapter 3
Production of Oxygen Radicals by Xenobiotics..................................................................27
I. Introduction.......................................................................................................................27
II. Production of Oxygen Radicals by Reducible Compounds.......................................27
A. Anthracyclines.......................................................................................................27
1. The Interaction of Anthracycline Antibiotics with
Dioxygen and the Superoxide Ion........................................................28
2. Enzymatic and Nonenzymatic Reduction of
Anthracyclines........................................................................................32
3. “Oxidation” of Anthracyclines..........................................................34
4. Metal Complexes of Anthracyclines and their
Participation in Redox Processes..........................................................35
5. Generation of Hydroxyl Radicals by Anthracyclines......................38
6. Free Radical Mechanisms of Cytotoxicity and
Anticancer Activity of Anthracyclines...............................................40
a. Stimulation of Lipid Peroxidation..........................................40
b. Deoxyribose Degradation.........................................................42
c. Free Radical Degradation of DNA........................................42
d. Other Possible Modes of Free Radical Damage
Induced by Anthracyclines......................................................43
e. Anthracycline-Dependent Free Radical Damage
to Whole Cells..........................................................................44
f. In Vivo Investigations of Free Radical Activity of
Anthracyclines............................................................................45
g. Evolution of Free Radical Hypothesis of Toxicity
and Anticancer Activity of Anthracycline
Antibiotics...................................................................................45
B. Synthetic Analogs of Anthracyclines..............................................................47
C. Other Quinone Anticancer Drugs: Mitomycins, Actinomycins,
Streptonigrin, and Aziridinylquinones.............................................................49
D. Benzoquinones and Naphthoquinones.............................................................52
E. Pyridinium Compounds......................................................................................54
F. Nitroaromatic and Other Reducible Compounds............................................55
III. Production of Oxygen Radicals by Reductants...........................................................58
IV. Other Producers of Oxygen Radicals............................................................................61
V. Photogeneration of Oxygen Radicals in the Presence of Xenobiotics....................63
VI. Additions..........................................................................................................................65
References....................................................................................................................................68
Chapter 4
Production of Oxygen Radicals by Cells............................................................................87
I. Introduction.......................................................................................................................87
II. Neutrophils.......................................................................................................................88
A. Detection of the Superoxide Ion Produced by Stimulated
Neutrophils...........................................................................................................88
B. The Mechanism of Superoxide Production by Neutrophils...........................90
1. NADPH Oxidase Is a Main Generator of Superoxide
Production by Neutrophils....................................................................90
2. Stimulation of NADPH Oxidase by Phagocytosable
Particles....................................................................................................92
3. Stimulation of NADPH Oxidase by Calcium-Dependent
Soluble Stimuli......................................................................................92
4. Stimulation of NADPH Oxidase by Activators of
Protein Kinase C.....................................................................................93
5. Stimulation of NADPH Oxidase by Other Soluble
Stimuli......................................................................................................94
6. Kinetics, Duration, and Other Features of Superoxide
Production by Stimulated Neutrophils.................................................96
7. Inhibition of Neutrophil Superoxide Production...............................98
C. Production of Hydroxyl Radicals by Neutrophils..........................................100
III. Eosinophils and Basophils...........................................................................................102
IV. Monocytes.......................................................................................................................103
V. Macrophages....................................................................................................................104
VI. Generators of Oxygen Radicals in Phagocytes, Other than NADPH
Oxidase............................................................................................................................108
VII. The Production of Oxygen Radicals by Other Cells.................................................109
VIII. Plant Cells.......................................................................................................................112
IX. Additions........................................................................................................................113
References...................................................................................................................................116
Chapter 5
The Interaction of Oxygen Radicals with Natural and Biologically
Active Compounds, Cellular Components, and Cells......................................................135
I. Introduction.....................................................................................................................135
II. Reactions of Oxygen Radicals with Natural Substrates and Biologically
Active Compounds.........................................................................................................136
A. NADH................................................................................................................136
B. Catechols and Melanins...................................................................................137
C. Thiols..................................................................................................................138
D. Sugars, Nucleotides, and Nucleosides...........................................................139
E. Other Examples of the Interaction of Oxygen Radicals with
Biologically Active Compounds....................................................................141
III. The Interaction of Oxygen Radicals with Lipids.....................................................144
A. Experimental Methods of Studying Lipid Peroxidation..............................144
B. Superoxide-Mediated Initiation of Lipid Peroxidation................................146
C. Oxygen Radicals in NADPH-Dependent Lipid Peroxidation......................147
D. Participation of Oxygen Radicals in Other Peroxidation Systems.............148
E. The Role of Oxygen Radicals and Iron in Lipid Peroxidation..................149
F. Natural Iron-Containing Catalysts of Lipid Peroxidation ...........................151
IV. Interaction of Oxygen Radicals with Proteins..........................................................152
A. Heme-Containing Proteins................................................................................152
1. Cytochromes..........................................................................................152
2. Hemoglobins...........................................................................................154
3. Catalase and Peroxidases ....................................................................154
4. Indoleamine 2,3-Dioxygenase.............................................................157
B. Superoxide Dismutase (SOD).........................................................................157
C. Other Enzymes Possessing Dismutase Activity............................................161
D. Oxygen Radicals in Activation and Inactivation of Enzymes....................161
E. Proteins Without Enzymatic Activity.............................................................165
V. Interaction of Oxygen Radicals with DNA and Oxygen Radical-
Mediated Mutagenesis..................................................................................................168
A. DNA Damage by Enzymatic and Nonenzymatic Generators of
the Superoxide Ion............................................................................................168
B. DNA Damage by Oxygen Radicals Formed in the Fenton
Reaction..............................................................................................................170
C. DNA Damage by Oxygen Radicals Released by Phagocytes....................171
D. Oxygen Radicals as Mediators of the Mutagenic Effect of
Xenobiotics.........................................................................................................172
E. Formation of 8-Hydroxydeoxyguanosine from DNA Mediated
by Oxygen Radicals..........................................................................................173
VI. Action of Oxygen Radicals on Cellular Components...............................................174
VII. Action of Oxygen Radicals on Cells...........................................................................176
A. Stimulatory and Regulatory Effects of Oxygen Radicals on
Cells.....................................................................................................................176
B. Damaging Effects of Oxygen Radicals on Cells..........................................178
1. Erythrocytes..........................................................................................178
2. Endothelial Cells...................................................................................180
3. Other Animal Cells..............................................................................181
4. Bactericidal Activity of Oxygen Radicals.........................................182
VIII. Some Extracellular Functions of Oxygen Radicals...................................................184
IX. Additions........................................................................................................................184
References....................................................................................................................................187
Appendix I
Experimental Techniques Used in Oxygen Radical Studies.................................................207
Appendix II
Abbreviations...............................................................................................................................209
Index............................................................................................................................................211
Volume II 1
Chapter 1
WHO IS THE VILLAIN?
(Classification and Reactivities of Oxygen Species in Biological Systems)
There is now abundant literature concerning the role of reactive oxygen species in
biological processes. However, authors do not always clearly determine even hypothetical
structures of these compounds. In part, it is due to the confusing terminology when such
terms as “free oxygen radicals”, “oxygen-centered free radicals”, “reactive oxygen spe
cies” (ROS), “active oxygen”, “oxygen metabolites”, and “partly reduced oxygen” are
applied indiscriminately. We have already discussed the structures of various oxygen radicals
in connection with the mechanism of the superoxide-driven Fenton reaction (Volume I,
Chapter 9) and will again consider the mechanisms of their formation in subsequent chapters.
However, it seems necessary to discuss the questions of classification and some disputable
questions of the structures of oxygen radicals at the beginning of this volume.
It is certain that all reduced oxygen species, excluding H20, are reactive intermediates
which may mediate important physiological processes and simultaneously may induce the
cellular damage leading to various pathologies. Hence, the most general terms for them are
“reactive oxygen species” or “oxygen metabolites”, which include free radicals (superoxide
ion 0¿, perhydroxyl H02*, and hydroxyl HO*), hydrogen peroxide, singlet oxygen, and
oxygenated transition metal complexes (crypto-hydroxyl radicals, ferry 1 Fe02+, and perferryl
Fe02+ ions). Thus oxygen radicals, which are the principal subject of this book, are only
a part of a whole body of ROS. However, there are important reasons to consider biological
processes mediated by oxygen radicals and oxygenated transition metal complexes separately
from those promoted by other ROS.
As is known, free radicals are species possessing an unpaired electron. The occurrence
of an unpaired electron, as a rule, but not always, sharply increases the reactivity of a
compound especially in hydrogen atom abstraction reactions, in the addition to double bonds
of organic compounds, and in other reactions. The enhanced reactivity of free radicals is a
basis of free radical hypothesis in biology, explaining the development of many pathological
processes by the destructive action of free radicals on cellular components (mitochondria,
microsomes, nuclei, lipid membranes, etc.), and on the cells themselves. Thus, in accord
with this hypothesis, free radicals are our major villains.
However, here some difficulties begin in both the classification and the understanding
of the role of oxygen radicals, other free radicals, and other oxygen species in biological
processes. Oxygen radicals are not the only free radicals forming in cells. For example, it
is well known that such a destructive process as lipid peroxidation is mediated by lipid alkyl
and peroxy radicals. Superoxide ion, hydroxyl radicals, and oxygenated transition metal
complexes are important initiators of lipid peroxidation (see Chapter 5). However, their role
is insignificant in the subsequent stages (propagation and termination) of this process. Within
the limits of free radical hypothesis, all free radicals should be considered. This point of
view is shown in the term “oxygen-centered” free radicals, which includes both oxygen
and peroxy free radicals. Nonetheless, the distinctions in the modes of formation and chemical
properties of peroxy and oxygen radicals which make their roles different in physiological
and pathological processes justify separate consideration of both types of free radicals.
Singlet oxygen and hydrogen peroxide are frequently united with oxygen radicals in
studying the effects of ROS on cells and cellular components. There have been many
discussions about the possibility of singlet oxygen forming in biological systems. After the
original enthusiasm in 1970 to 1980, no further light was shed on its role in biological
processes until about 1985. At present, the formation of singlet oxygen in cells cannot be
completely excluded. However, the main question remains to be decided: is the formation
