Oxidative Damage & Repair. Chemical, Biological and Medical Aspects

917 Pages·1991·33.947 MB·English

by  Kelvin J. A. Davies (Eds.)

Pergamon Title of Related Interest
Journal
Free Radical Biology & Medicine
Oxidative Damage & Repair
Chemical, Biological and Medical Aspects
Edited by
Kelvin J. A. Davies, PhD
Professor and Chairman
Department of Biochemistry & Molecular Biology
The Albany Medical College, Albany, New York
PERGAMON PRESS
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First edition 1991
Library of Congress Cataloging-in-Publication Data
Oxidative damage & repair : chemical, biological, and medical aspects/
[edited by] Kelvin J.A. Davies.
p. cm.
"This book was inspired by the oral presentations delivered at. . .the
5th Biennial Meeting of the International Society for Free Radical
Research. . .held from November 14-20, 1990 in Pasadena,
California, U.S.A."-Pref.
Includes index.
1. Active oxygen-Pathophysiology-Congresses. 2. Active oxygen in
the body-Congresses. 3. Free radicals (Chemistry)-Pathophysiology-
Congresses. 4. Lipids-Peroxidation-Congresses. 5. Antioxidants-
Therapeutic use-Congresses. I. Davies, Kelvin J. A. II. International
Society for Free Radical Research. Meeting (5th : 1990 : Pasadena,
Calif.)
[DNLM: 1. Antioxidants-congresses. 2. Free radicals-congresses.
3. Oxygen-adverse effects-congresses. 4. Oxygen-physiology-
congresses. QV 312 0945 1990]
RB170.095 1991 616.07-dc20 91-32039
ISBN 0-08-041749-3
Whilst every effort is made by the publishers and editorial board to see
that no inaccurate or misleading data, opinion or statement appears
in this book, they wish to make it clear that the data and opinions
appearing in the articles and advertisements herein are the sole
responsibility of the contributor or advertiser concerned. Accordingly,
the publishers, the editorial board and editors and their employers,
officers and agents accept no responsibility or liability whatsoever for
the consequences of any such inaccurate or misleading data, opinion or
statement.
All papers in Oxidative Damage & Repair: Chemical, Biological and
Medical Aspects have been selected for publication after refereeing
under the auspices of the Program/Organizing Committee(s) of the 5th
Biennial Meeting of the International Society for Free Radical Research.
Printed in Great Britain by BPCC Wheatons Ltd, Exeter
PREFACE
Free radicals, activated oxygen species, and photochemical excitation products have been
implicated in virtually every aspect of life. Oxidative Damage & Repair: Chemical, Biological,
and Medical Aspects seeks to provide a broad coverage of the field, while highlighting some
of the more novel or intriguing recent developments. To this end this volume includes
subjects such as DNA, protein, and lipid repair; expression of shock or stress genes and
proteins; and the use of antioxidants or oxidant repair strategies in cancer, heart attacks,
strokes, rheumatoid diseases, diseases of the eye, and atherosclerosis.
This book was inspired by the oral presentations delivered at Oxidative Damage &
Repair, the 5th Biennial Meeting of the International Society for Free Radical Research
(SFRR International). The meeting was held from November 14-20, 1990 in Pasadena,
California, U.S.A., and brought together over 1,000 of the most active investigators in the
field. SFRR International consists of four constituent member societies, SFRR Europe, SFRR
Japan, SFRR Australasia, and The Oxygen Society ("SFRR Americas"); The Oxygen Society
hosted the 5th Biennial Meeting. The 700 abstracts for Oxidative Damage & Repair, covering
both the oral presentations and the poster sessions, were published as Volume 9, Supplement
No. 1 of the journal, Free Radical Biology & Medicine (Pergamon Press).
Major sponsors for Oxidative Damage & Repair included the Institute for Toxicology of
the University of Southern California; the National Cancer Institute, the National Institute
of Environmental Health Sciences, and the National Heart, Lung, and Blood Institute (all of
the National Institutes of Health); the International Union of Biochemistry, and Society and
Association Services Corporation. Major corporate donors included Eli Lilly and Co., Gemini
Science Inc., Glaxo Research Laboratories, Henkel Corp., Johnson & Johnson, Marion
Merrell Dow Research Institute of Merrell Dow Inc., Monsanto Co., Parke Davis, Pergamon
Press Inc., Pfizer Inc., Roche Vitamins & Fine Chemicals, the Council for Tobacco Research-
USA Inc., the Upjohn Co., and Wyeth-Ayerst Laboratories.
Special thanks go to all the members of both the International Advisory Board and the
Local Organizing Committee; to Richard and Susan Burk of Society and Association Services
Corporation (SASC); to Linda Dorman and the staff of the Pasadena Convention and Visitors
Bureau; to Dr. Victor Vener of the Chamber Orchestra of Pasadena; and to Drs. Paul
Hochstein, Alex Sevanian, Enrique Cadenas, Joseph Landolph, Lynne Eddy, Anthony Verity,
and Joanna Davies of the University of Southern California.
The field of free radical research has grown exponentially during the past 10 years. Two
dedicated specialist journals (Free Radical Biology & Medicine, and Free Radical Research
Communications), special sections in several other journals, and a plethora of society and
independently organized meetings, attest to the strength and vitality of the field. Oxidative
Damage &. Repair was organized into 20 session topics representing many of the most active
research areas of free radical chemistry, biology, and medicine. Of the 140 oral presentations
delivered at the meeting, 60 speakers were invited by the organizers, and 80 speakers were
selected from the abstracts submitted. Each session topic had its own plenary speaker, session
chair, two invited afternoon speakers, and four additional afternoon speakers selected from
the submitted abstracts.
This book has been organized into 20 chapters which mirror the 20 session topics of
Oxidative Damage & Repair. I asked all speakers to contribute manuscripts describing their
presentations, and I asked all session chairs to write overview papers for their session topics.
It is my hope, and that of the publisher, that this book represents an up-to-date cross-section
of much of the most exciting research into oxidative damage and repair. I should like to
thank all the contributors for their efforts to make this wish a reality.
Kelvin J. A. Davies
XV
OXIDATIVE DAMAGE & REPAIR: INTRODUCTION AND OVERVIEW
Kelvin J. A. Davies^
Institute for Toxicology and Department of Biochemistry, The University of Southern
California, Health Sciences Campus, Los Angeles, California, U.S.A.
OXIDATIVE DAMAGE & THE OXYGEN PARADOX
Oxygen represents an unique problem for aerobic organisms. Oxygen plays a vital role
as the terminal electron acceptor during respiration, yet, due to its biradical nature, oxygen
and its metabolites are potentially cytotoxic and clastogenic.1""6 Univalent reduction of oxygen
produces the Superoxide radical (0 ~), whereas divalent reduction generates hydrogen
2
peroxide (H 0 ). Trivalent reduction of oxygen (or reaction of 0 ~ with H 0 ) generates the
2 2 2 2 2
highly reactive hydroxyl radical (ΌΗ).4 Reactions of 0 ~ and H 0 with metalloproteins can
2 2 2
produce other reactive species, such as ferryl, and perferryl states of heme groups (e.g., ferryl-
hemoglobin).
There are a large number of physiological and pathological sources of oxygen radicals
and related oxidants. Exogenous agents such as photochemical smog, ozone, pesticides,
xenobiotics, and ionizing radiation are all know to generate radicals/oxidants. Importantly, it
has also been well documented that a variety of endogenous processes (including cellular
metabolism in the form of mitochondrial respiration, cytochrome P-450 detoxification
reactions, phagocytic oxidative bursts, and peroxisomal leakage) are significant sources of
radicals/oxidants. Regardless of their origin, it is clear that oxygen radicals represent a real
threat to normal cellular function.
Virtually all cellular constituents appear to be sensitive to radical/oxidant damage.
Proteins,3'7-2* lipids,29""34 nucleic acids,4,35"40 and carbohydrates4,40'41 are all known to undergo
oxidative modification. The profile of damage products depends on both the quantity and
quality of radical/oxidant exposure.
ANTIOXIDANT DEFENSE SYSTEMS
In order to minimize the untoward effects of oxygen, aerobic organisms have developed
an extensive collection of antioxidant defenses (Fig. 1). Cellular antioxidants include multiple
compounds (e.g., vitamins E and C, uric acid, 0-carotene) which alleviate the oxidant load by
directly quenching reactive oxygen species before they damage vital cellular components.
Antioxidant enzymes such as catalases, glutathione peroxidases/reductases, and Superoxide
dismutases (SOD) function by catalyzing the decomposition of oxidants/radicals.4 It appears,
however, that these antioxidant defenses are not 100% effective, since signs of oxidative
damage to lipids, proteins, carbohydrates, and nucleic acids are apparent even in the
healthiest organisms.5,21'26,35,38,39'42,43 Clearly, it is to the cells' advantage to remove and/or
repair such damaged molecules.
*New Permanent Address: Prof. Kelvin J. A. Davies, Chairman, Department of Biochemistry
and Molecular Biology, The Albany Medical College, Albany, New York, U.S.A.
XVll
duction ate of that nthetic
efenses contribute to the proate of production, and the ration by catabolic enzymes ds) are conserved for biosy
ation by antioxidant ds is a function of the ratic repair, or degradatty acids, nucleic aci
ape detoxificd componentdirect enzymmino acids, f
air. Radicals that escstate level of damageecules may undergo Reusable blocks (acreted.
1: Pathways of Oxidative Damage and Repely damaged cell components. The steady nd/or repair. Oxidatively damaged biomolrecognize oxidatively damaged products. and irreversibly damaged molecules are ex
RE datival avely ses,
FIGUof oxiremovselectiproces
Introduction xix
OXIDANT REPAIR SYSTEMS
Defense Versus Damage Removal and Repair
The hypothesis that a group of "oxidant repair" enzymes may be involved in the
systematic recognition and removal of cellular components that have been damaged by radi-
cals/oxidants was proposed several years ago8 and has recently received support from many
experimental studies. Such repair systems include enzymes that attempt to directly restore
biomolecules to their native conformation, as well as catabolic enzymes that specifically
degrade non-functional proteins,3'7""28 lipids,19,30,32"34,44 and nucleic acids.36·45"51 Degrdation
of oxidatively modified macromolecules serves not only to remove them from the cytosol, but
also to replenish precursor pools for resynthesis (Fig. l).8·44»52
In Fig. 1, and the remainder of this review chapter, the term "repair" is often used in a
very broad sense. Direct molecular repair (i.e. the repair of individual damaged molecules)
certainly does occur, and examples are given in Fig. 1 and the text below. In the broader
sense, however, the processes of indirect repair may be more significant for overall
physiological homeostasis. Indirect repair mechanisms involve the degradation of oxidatively
damaged cell components (e.g. selective proteolytic cleavage of damaged proteins), with
conservation of undamaged constituents (e.g. undamaged amino acids), and excretion of
irreversibly modified constituents (e.g. excretion of covalently cross-linked or oxidized amino
acids). Conservation of the undamaged constituents of oxidatively damaged macromolecules
provides an immediate pool of building blocks for biosynthetic processes; including the
replacement of damaged proteins, lipids, and DNA (Fig. 1). Under normal circumstances the
production of oxidatively modified cellular components would have to be matched by a
comparable rate of removal/repair, thus maintaining a low steady state level of damage.
Evidence for a variety of oxidant removal/repair enzymes is presented below.
Protein Degradation and Repair
Proteins are certainly amoung the cellular components that are vulnerable to oxidant
damage.4'11"14'19,20'23,27'28,53. Our laboratory has investigated the fate of oxidatively modified
intracellular proteins.3,7"20'54 We have observed that cells which have been challenged with
radicals/oxidants undergo a proteolytic burst.9'17'18,20 This effect could be explained by an
activation of latent proteolytic enzymes, an increase in the proteolytic susceptibility of
oxidatively modified proteins, or both. To further investigate this phenomenon purified
proteins, oxidatively modified in vitro, and several cell-free systems were employed.
Purified proteins undergo a variety of changes when exposed to oxygen radicals or
H 0 .11"14,19,20^7,55 Such changes include decreases in native fluorescence, due to modification
2 2
of tryptophan and tyrosine; shifts in isoelectric points, due to charge modifications to amino
acid R-groups; increases in molecular weight, due to the formation of covalent and/or
hydrophobic intermolecular associations; and decreases in molecular weight, due to direct
scission of the peptide backbone to generate random peptide fragments (with production of
carbonyl and amide groups). Hunt et. al.41 have proposed a free radical mechanism by which
proteins undergo oxidative glycosylation. Stadtman, Levine, and co-workers have also
characterized mixed function oxidase catalyzed modifications to a variety of proteins;23'26'53
most notably glutamine synthetase.
Oxidatively modified proteins are excellent substrates for degradation by proteolytic
systems in cell-free extracts, even though the extracts themselves have not been exposed to
any oxidant challenge.7'8,11"20,22"24,26"28 This suggests that modification of the protein substrate
is sufficient to generate a proteolytic response. Of all the primary, secondary, and tertiary
structural changes that we have measured in oxidatively modified proteins, the only one which
seems to correlate with the increased susceptibility to proteolysis is increased hydrophobicity
or denaturation.8'14'16'17 We have proposed the hypothesis that denaturation or unfolding may
expose previously buried hydrophobic residues that mark a protein for degradation8.'1,16,17
XX KELVIN J. A. DAVIES Ph.D.
As a natural next step, we sought to identify the proteolytic enzyme(s) responsible for
the recognition and selective degradation of oxidatively denatured proteins. We chose ΌΗ
treated hemoglobin, and H 0 treated Superoxide dismutase as physiological substrates for the
2 2
enzyme purification scheme. Chromatographie separation of red blood cell lysates revealed
that most of the proteolytic activity against our oxidatively modified protein substrates was
expressed by an unusually large proteolytic complex.17'20 This proteolytic complex has a native
molecular mass of 670 kDa, and appears to have at least three catalytically unique active sites.
The enzyme also appears to be ATP independent, with a pH optimum in the alkaline (7.8)
range.17'10
We have suggested the trivial name Macroxyproteinase (MOP) for this enzyme complex
due to its large size (macro-), its preference for oxidatively modified proteins (-oxy-), and its
catalytic activities (-proteinase).17,20 Rivett has proposed that a 650 kDa proteolytic complex
is responsible for the degradation of proteins oxidatively modified by mixed function
oxidases.24 Several other laboratories have described high molecular weight proteolytic
complexes which appear to be identical to MOP.25 These large complexes (which have been
called ingensin, macropain, macrosin, proteasome, and multicatalytic proteinase or MCP) have
been observed and purified from a large variety of eucaryotic cells. The ubiquitous
distribution of these proteolytic complexes, and their preference for oxidatively modified
protein substrates, suggests that MOP may play a major role in the proteolytic degradation
of aberrant proteins; thus preventing their accumulation and aggregation, and providing amino
acids for de novo synthesis of new proteins.
We have proposed that Superoxide (0"~) may be involved in the homeostasis of
2
intracellular SOD levels. SOD levels have been shown to increase as a result of oxidative
stress, yet SOD is irreversibly inactivated by its product (H0 ).19,20 We have demonstrated
2 2
that H 0 modified SOD is an excellent substrate for intracellular degradation by MOP.
2 2
Thus, Superoxide may be the signal for both SOD production and removal2.0
Some oxidatively modified proteins may be spared from proteolytic degradation by direct
repair mechanisms. Direct enzymatic re-reduction of sulfhydryl, and heme groups may restore
proteins to their native conformation. Enzymes which repair isoaspartyl peptides have been
described by Clarke et. A/.;56,57 the intriguing possibility that such enzymes may exist for
repairing oxidatively modified protein R-groups remains to be investigated.
Lipid Degradation and Repair
Lipid peroxidation was the first type of oxidative damage to be studied. Membrane
phospholipids are continually subjected to oxidant challenges. The process of lipid
peroxidation consists of a set of chain reactions which are initiated by the abstraction of a
hydrogen atom (from carbon) in an unsaturated fatty acyl chain3.1,32 In an aerobic
environment, oxygen will add to the fatty acid at the carbon centered lipid radical (L·) to give
rise to a lipid peroxyl radical (LOO·). Once initiated, LOO· can further propagate the
peroxidation chain reaction by abstracting a hydrogen atom from other vicinal unsaturated
fatty acids.31,32 The resulting lipid hydroperoxide (LOOH) can easily decompose into several
reactive species including: lipid alkoxyl radicals (LO·), aldehydes (e.g. malonyldialdehyde),
alkanes, lipid epoxides, and alcohols.31,32 Cholesterol has also been shown to undergo
oxidation, to give rise to a variety of epoxides and alcohols.
Peroxidized membranes become rigid, lose selective permeability and, under extreme
conditions, can lose their integrity. Water-soluble lipid peroxidation products (most notably
the aldehydes) have been shown to diffuse from membranes into other sub-cellular
compartments.29,32'34,58 Dialdehydes can act as crosslinking reagents, and are thought to play
a role in the protein aggregation which forms the age pigment lipofuscin3. Several
laboratories are investigating the possibility that lipid peroxidation products may form DNA
adducts, thus giving rise to mutations and altered patterns of gene expression.
Introduction xxi
Others have noted inhibition of enzyme function by lipid peroxidation products. It is
very clear that the process of lipid peroxidation, and its products, can be detrimental to cell
viability. Cumulative effects of lipid peroxidation have been implicated as underlying
mechanisms in numerous pathological conditions including: atherosclerosis, hemolytic anemias,
and ischemia/reperfusion injuries.32
Lipid bilayers which have been oxidized become better substrates for phospholipase
enzymes. Phospholipase A acts at the sn-2 position of the phospholipid glycerol backbone
2
to generate a free fatty acid and a lysophospholipid. Phospholipase A has been shown to
2
preferentially hydrolyze fatty acids from oxidized liposomes.33 Structural perturbations due
to changes in membrane microviscosity, and the increased hydrophilic nature of oxidized lipids
may be responsible for the increased susceptibility to phospholipase A action.34,44 Removing
2
fatty acid hydroperoxides from the membrane compartment can help prevent further
propagation reactions. Additionally, it has been demonstrated that fatty acid hydroperoxides
released into the cytosol are substrates for glutathione peroxidase. Glutathione peroxidase
detoxifies fatty acid hydroperoxides by reducing them to the corresponding hydroxy-fatty
acids.44 Lysophospholipids left in the membrane posses potential detergent properties which
have been shown to disrupt membrane structure and function. Lysophospholipids can serve
as substrates for reacylation reactions (readdition of fatty acids to the sn-2 position) to
regenerate intact phospholipids.30'52
Recent work suggests that it is possible to reduce fatty acid hydroperoxides (to their
corresponding alcohols) without hydrolysis and release from the membrane compartment.59"62
A glutathione peroxidase which acts preferentially on phospholipid hydroperoxides has been
characterized by Ursini, Maiorino and their co-workers.60"62 A glutathione transferase with
activity towards lipid hydroperoxides has also been extracted from nuclei.59
Peroxidized membranes and lipid oxidation products represent a constant threat to
aerobic cells. It is now widely held that in addition to preventing initiation of peroxidation
(with compounds like vitamin E), cells have also developed a variety of mechanisms for
maintaining membrane integrity and homeostasis by repairing oxidatively damaged
components.
DNA Degradation and Repair
Ribo- and deoxyribo- nucleic acids are also vulnerable to oxidative damage. mRNA,
tRNA, rRNA and, perhaps most importantly, DNA have all been shown to suffer oxidative
damage in v/vo.4,35"40 Although DNA is a relatively simple biopolymer, made up of only four
different nucleic acids, its integrity is vital to cell division and survival. Oxidative alterations
to nucleic acid polymers has been shown to disrupt transcription, translation, and DNA
replication, and to give rise to mutations and (ultimately) cell senescence or death.5,6,40"43,63'64
Despite the precious nature of the genetic code, it too appears to be a target for oxidative
damage. The amount of oxidative damage, even under normal physiological conditions, may
be quite extensive with estimates as high as 1 base modification per 130,000 bases in nuclear
DNA.39 Damage to mitochondrial DNA is estimated to be even higher at 1 per 8,000 bases.
Fragments of oxidatively modified mitochondrial DNA have been implicated in cancer and
aging.38
Oxidants can elicit a wide variety of DNA damage products several of which have been
carefully characterized.40,65 The types of DNA damage can be grouped into: strand breaks
(single and double), sister chromatid exchange, DNA-DNA and DNA-protein cross links, and
base modifications. All four DNA bases can be oxidatively modified, however, the pyrimidines
(cytosine and, especially, thymidine) appear to be most susceptible.64 Bases undergo ring
saturation, ring opening, ring contraction, and hydroxylation. These types of alteration usually
result in a loss of aromaticity and planarity, which can cause local distortions in the double
helix.64 Depending on the type and extent of damage, the altered bases can be found either
XX11 KELVIN J. A. DAVIES Ph.D.
attached to, or dissociated from the DNA molecule to generate apurinic/apyrimidinic (AP)
sites.37·49'64
Radical interactions with DNA appear to be fairly nonspecific; hence the phosphodiester
backbone may also be oxidatively damaged. Damage to the sugar and phosphate moieties,
which form the backbone, may result in strand breaks4.0'65 Depending on the site of radical
attack, unusual 3' and 5' ends (Le. non 3'-OH, non 5'-P0) can be generated. These
f 4
abnormal ends are not substrates for DNA polymerases, and must be removed before any
repair can occur.49
There is evidence that several procaryotic and eucaryotic enzymes repair oxidatively
damaged DNA by both direct repair processes59,64'65 and by (indirect) excision-repair
mechanisms.36'37,45"47'49'50'51'63-66 The actual repair steps in the excission-repair process are
carried out by DNA polymerases which replace the excised (damaged) bases, and DNA ligases
which seal the new 3' and 5' ends. Pilot studies using cell-free extracts in vitro indicated that
some endo- and exo- nucleases preferentially cleave oxidized DNA. Bacterial mutants
deficient in these putative DNA oxy-repair enzymes: nth' (endonuclease III), xth' (exonuclease
III), and nfo' (endonuclease IV) were used to continue these studies in vivo.67 It was found
that strains deficient in exonuclease III (xth') are hypersensitive to H 0 .67 Endonuclease IV
2 2
deficiency (nfo') also produces a hypersensitivity to organic hydroperoxides, and to oxidants
generated by bleomycin.67 When the nfo and xth mutations are combined, oxidant damage
and cell death are drastically increased.
The activity of these procaryotic enzymes, in the repair of oxidatively damaged DNA,
has been extensively studied.47'66"69 Endonuclease III has been shown to cleave at the 3* side
of AP sites.68 Endonuclease III also appears to posses an N-glycosylase activity for thymine
glycol and urea residues; two common products of oxidative damage to DNA. Exonuclease
III posses a 3' to 5' nucleolytic activity which may be responsible for removing the sugar
fragments generated during oxidative strand breakage.65,68 Exonuclease III is really a poor
name for this enzyme which actually expresses 85% of the 5' AP endonucleolytic activity in
E. coli. Endonuclease IV is also a 5' AP endonuclease.
Eucaryotic studies are still in their initial phases, however, several parallels to the results
of procaryotic studies have already been found.45'46·48,49 Several DNA glycosylases which act
on DNA oxidation products have been characterized. A 3'-repair diesterase in yeast is
apparently responsible for removing damaged 3' termini left by free radical reactions4.9 A
mammalian endonuclease has been isolated based on its specificity for oxidatively modified
DNA.45,46 It bears remarkable similarities to the bacterial endonuclease III including:
molecular weight (~30 kDa), lack of divalent cation requirement, and substrate specificity.
The term "redoxyendonucleases" has been proposed for all nucleases which participate in
repairing oxidatively damaged DNA.45
There is mounting evidence that redoxyendonucleases function in higher eucaryotic cells.
DNA damage which appears in cells as a result of oxidant challenge (including base damage
and single strand breaks) has been shown to diminish as a function of time3.6 These results
suggest that a removal of lesions is being carried out by intracellular systems. Oxidatively
damaged bases, such as 8-hydroxy-deoxyguanosine and thymine glycol, have been measured
in animal urine.39 Again this suggests that there is systematic excision and excretion of
oxidized DNA bases in vivo.
In addition to excision repair mechanisms, there may be direct repair of oxidatively
altered DNA bases. Reports of glutathione transferases49 and peroxidases using thymidine
hydroperoxide as a substrate have been published. DNA may also undergo oxidative
demethylation.64'65 DNA methylases may play an important role in restoring methylation
patterns and maintaining epigenetic phenomena. Inhibition of poly (ADP-ribose) polymerase
has been shown to exacerbate H 0 genotoxicity, although the mechanism for this is not yet
2 2
clear.63