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By Helmut Sies
As a normal attribute of aerobic life, structural damage to organic compounds of a wide
variety (DNA, proteins, carbohydrates and lipids) may occur as a consequence of oxidative
reactions. Oxidative damage inflicted by reactive oxygen species has been called oxidative
stress. Biological systems contain powerful enzymatic and nonenzymatic antioxidant systems, and oxidative stress denotes a shift in the prooxidant/antioxidant balance in favor of
the former. Diverse biological processes such as inflammation, carcinogenesis, ageing, radiation damage and photobiological effects appear to involve reactive oxygen species. This
field of research provides new perspectives in biochemical pharmacology, toxicology, radiation biochemistry as well as pathophysiology.
1. Introduction
[I
1058
OS70-0833/86/12~2-1OS8$ 02.50/0
I
0-@-
0-@-
0-@--
HO
Fig. I . Hydrolysis of the C-4-radical 1 in DNA to yield strand breaks (anaerobic). The situation is
more complex under aerobic conditions (modified after [21])
[Tg
o
OCH,
DNA
Fig. 5. Formation of the N-7 guanyl adduct 01 aflatoxin B, 4 from the 2,3epoxide.
Fig. 2 Oxidative degradation of thymine residues in DNA. I n addition to
thymine glycol 2, 5-hydroxymethyl uracil may also be formed (after [24]).
lupus e r y t h e m a t o s ~ s [ or
~ ~ Fanconis
]
Superoxide dismutase has an anticlastogenic (anti-chromosomebreak)
It is assumed that double-strand breaks of
DNA are the initial lesion leading to chromosome breaks.
The exact mechanism of chromosome breakage is not yet
known.
R
= Ribosyl
2.2. Repair
- ROH
NH
I
NH2
R
Fig. 3. Scheme for the oxidative degradation of guanine by photooxygenation. Sens. = sensitizer (after [25]).
...
__
&NH
>N
...
NK
0
...
-0
\
(33
Fig. 4 Adjacent thymine r w d u e s in D N A can join upon irradiation to form
a cyclobutylthymine dimer (sugar and phosphate residues omitted).
Angew. Chem. In[. Ed. Engl. 25 (1986) 1058-1071
Oxidized bases and strand breaks can be repaired enzymatically. It should be noted that oxidative damage is just
one way of altering DNA; further routes of damage include errors in replication by recombination to produce
mismatches, or by other ways of chemical alteration such
as deamination. It has been estimated that the DNA of a
mammalian cell probably loses 5000- 10 000 purine and
200-500 pyrimidine residues per 20 h generation
Nucleases involved in DNA repair have been shown to operate in several ways :[35.361 examples include nucleotide excision repair, base excision repair, recombinational repair,
mismatch repair and error-prone repair. A given thymine
glycol residue in DNA (cf. 2 in Fig. 2) may either be released as thymine glycol by a specific glycosylase, or it
1059
may be released as thymidine glycol by an excision exonuclease (Fig. 6). These bases can be traced in the urine and
have potential value for the screening of oxidative damage in man.["1 A similar pair of metabolites are 5-(hydroxymethy1)uracil and 5-(hydroxymethyl)-2'-deoxyuridine;a 5hydroxymethyluracil glycosylase has recently been described.[3x1
NH?
( 3 ; C H
CH2-L-COO~
Incision endonuclease
F2
0-0
DNA- glycosylase
NH?
1 -COO@
Histidine endoperoxides
t
NH?
+.
Damaqed
Apurin iclApyrimidinic
base
endonuclease
ihh
Damaged
deoxynucleoside
Tryptophon hydroperoxide
Exctsion e x o n u c l e a s e
Polymerase. Llgase
888888
Fig. 6 . T w o pathways of excision repair. The left sequence is known as nucleotide excision repair, whereas the right sequence is base excision-repair
(modified after [35]).
The intrastrand thymidine dimers that form as the predominant lesion in UV-irradiated DNA (Fig. 4) cannot
only be repaired by the dimer-specific endonuclease, followed by DNA polymerase,[391but also by a light-dependent repair reaction. This photoreactivation is catalyzed by
a flavoen~yme.[~~]
Protein or peptide
Ref.
Methionine
[421
3.1. Methionine
Tryptophon endoperoxide
Histidine
Phosphoglucomutase
a,-Proteinase inhibitor
Calmodulin
ACTH (adrenocorticotropic hormone)
Chemotactic factors
Met-Leu-Phe
Complement C5A
a ,-Antitrypsin
[431
[48, 491
3.2. Histidine
Activation by
GSSG
GSSG
GSSG
Cystamine
GSSG
Enzyme
Inhibition by
Pyruvate kinase
Phosphorylase phosphatase
Phosphofructokinase
Glycogen synthase D
HMG-CoA-reductase
Adenylate cyclase (brain)
Ribonucleotide reductase ( E . coli)
Hexokinase
Tyrosine aminotransferase
PDH kinase
Fatty acid synthetase
y-Glutamylcysteine synthetase
Papain, trypsin
GSSG
GSSG
GSSG
GSSG
GSSG
GSSG
GSSG
Tetraethylcystamine
Cystine
DTNB
DTN B
Cystamine
Dimethyl disulfide
3.4. Proline
CO-NH-R'
HO @/O,
R-OC,
CO-NH-R'
0
- RCOOH
CO-NH-R'
HOOC
H2
HZO
spontaneous hydrolysis
Also of interest is the reversible formation of mixed disulfides between proteins and low-molecular weight thiols,
in particular glutathione (GSH). ProtSSG have been observed in the soluble cytoplasm as well as in memb r a n e ~ . ~ ' ~The
- ~ ' ~S-thiolation of proteins in heart cells
treated with diamide and tert-butyl hydroperoxide showed
distinct patterns, notably proteins with molecular masses
of 23, 42 and 97 kD.ls9] The levels of protein-glutathione
mixed disulfides are low, for example, about 20-30 nmol/g
of liver.i60,6'1When the GSSG levels were increased, the
concentration of mixed disulfides also increased,i60-621
possibly catalyzed by thiol transferases.'"]
There are also mixed disulfides of glutathione and coenzyme A (CoASSG). The fluctuations in their concentration
can be metabolically significant; during oxidative challenge with tert-butyl hydroperoxide, the cellular free
CoASH pool can be decreased to such an extent that coenzyme A-dependent processes are blocked :[64,651
23'"-",""'
Fig. 8 Reaction scheme for cleavage of proteins at proline residues as proposed in [Sl]. In a first complex step initiated by hydroxyl radicals, hydrogen
abstraction occurs and, in presence of OZrleads to a 2-pyrrolidone intermediate 5 via an organic peroxy radical. This then undergoes hydrolysis at the
peptide bond. The resulting pyroglutamate 6 may hydrolyze to a new Nterminal glutamate.
3.5. Cysteine
Although the formation of disulfide bonds as such cannot be considered as damage, because it is a reversible
process, disulfide bridges in peptides and proteins may
Angew Chem. Int. Ed. Engl. 25 (1986) 1058-1071
CoASH
Thiol groups may be further oxidized to alkylthio radicals and subsequently add oxygen:
RS'
+ Oz -+
RSOY
Further rearrangements and oxidation steps lead to sulfenic, sulfinic, and, finally, sulfonic acids (cf. Fig. 7). The latter are stable enough to be detectable in assays of enzymatic oxidation; for example, glutathione sulfonate was
detected in the enzymatic oxidation of glutathione by xanthine oxidasei"61and horseradish p e r ~ x i d a s e . ~ ~ ~ ]
Alkylthio radicals have been detected by ESR methods
in horseradish peroxidase-catalyzed reactions in
but no information is as yet available on the metabolic
generation of these radicals in cells. Oxidation products of
disulfides such as cystine S-monoxide or cystine SS-dioxide can also be formed, and they possibly occur in keratin
Cyclooxygenose
Lipoxygenose
Phospholipose A2
Lipocortin( -)
Phosphohpids
Prostaglandins
Thrornboxones
Leukotrienes
+Arochidonote
02
Fig. 9. Scheme of release of arachidonate lrorn phospholipids and of eicosanoid formation. The inhibition of phospholipase AZ by lipocortin, the inhibitory peptide inducible by glucocorticoids (see 1771) and the positive effect of
organic hydroperoxides on cyclooxygenase 1781 and lipoxygenase ("peroxide
tone") are indicated.
fiber^.""^
3.6. Damage to Proteins by y-Irradiation or Free Radical
Attack
As mentioned in Section 2, deoxyribose is prone to oxidative degradation. This property has even been exploited
for the development of an assay of the formation of freeradicals. In the thiobarbiturate assay, deoxyribose yields a
degradation product that is almost identical to the product
obtained with m a l ~ n d i a l d e h y d e . ' ~ ~ ]
Polysaccharides such as hyaluronic acid can be degraded by oxidative attack; superoxide dismutase was
found to be capable of protecting hyaluronic acid against
depolymerization in synovial f l ~ i d . ~Proteoglycans
' ~ ~ ~ ~ ~
may be subject to oxidative breakdown in a similar manne~.i'~]
Fig. 10. initial steps of' lipid peroxidation, schernatlc lor d i i unsalurated nalkane (RH): H abstraction, diene conjugation, oxidation and reaction with
RH (after [SO]).
bridges
5.2. Atherogenesis
It has been suggested that lipid peroxidation is involved
in the development of atherosclerotic lesions. It was found
that modified forms of human low-density lipoprotein
(LDL) cause a n accumulation of large amounts of cholesterol esters in macrophages, e.g. after treatment of LDL
with malonaldehyde.[8s14-Hydroxynonenal is also capable
of modifying LDL, and the concentrations required are
one hundred to one thousand fold less than with malonaldehyde.[*'] In addition to the enhanced accumulation of
cholesterol esters, the binding of modified LDL to macrophages also leads to an increased release of a number of
The molecular basis of modificalysosomal
tion of LDL by endothelial cells that leads to its enhanced
uptake by macrophages remains to be defined.["']
Remarks
Superoxide
or hyperoxide
HO?
Perhydroxyl
H202
Hydrogen peroxide
HO"
Hydroxyl
ROO
R-oxyl, e.g.
alkoxyl
R-dioxyl, e.g.
alkoxydioxyl
ROO"
The oxidation of cholesterol is of special biological interest, yielding a 5,6-epoxide and, in addition, the 5a-hydroperoxide (Fig. 11). This cholesterol epoxide occurs in
high concentrations in human breast fluid[s31and has been
identified as a directly acting mutagen.[841
Name
ROOH
R-hydroperoxide
' 4 0 2
Singlet molecular
oxygen
(also Of
or l o 2 )
'R'R"C0
(also
R'R"CO*)
Triplet carbonyl
The sou-rces of the radical 0:" produced via one-electron reduction of molecular dioxygen in the cell include
the mitochondria1 and microsomal respiratory chains as
well as the bactericidal activity of leukocytes and macrophages. The physiological importance of the cellular
Hydroquinones (semiquinones)
Flavins
Hemoglobin(s)
Glutathione and other Thiols
Catecholamines
Transition metal ions
Ref.
I I771
[177, 1781
1179, 1801
[ I S l , 1821
11831
[184, 1851
Flavin-dependent oxidations
Photosynthetic oxygen reduction
Mitochondria1 respiratory chain
Microsomal oxygenation
NADPH-dependent oxygen reduction by granulocytes
and macrophages
[186, 1871
[ I881
11891
11901
[ 19 I - 1931
[I891
[194, 1951
[196, 1971
1063
R e o c t i v e oxygen species
@,
RO
@,
'0,
Fig. 12. Scheme of redox cycling. The superoxide anion radical, O:", is generated under aerobic conditions, driven by electrons coming from NADPH
as catalyzed by various reductases.
It is of interest to note that reactive oxygen intermediates often seem to be metabolized according to the principle of dismutation (Table 6). Not only the one-electron
reduction state is eliminated in a disproportionation reac-
RH
-57
+ 2He
--*
+2H20
2 ROOH
--t
H 2 0 2 O2
Superoxide dismutase
Catalase
0 2
2PG H2
+ '02
PG hydroperoxidase
2 ROH
+ '02
Cytochrome P-450
' 0 2
Cytochrome P-450
-t
2PhI=Oh2PhI+
hu
703
hv
(340-460 nrn)
Dioxetone
+ ROOH
(Fe=O) + ROOH
(Fe3')
--f
+ ROH
(Fe3@) + ROH + '02
(Fe=O)
---t
2ROH
+ '02
'0,
I064
ROOH
2ROOH
2PG G2
ROOQ
Enzyme
Reaction
2HZOZ
R@
R'R''Cff
20y0
a -T-0
02
H2O2. OH @ , ROO
a -T-OH
02
+ '0,
2'02
+ hv
-1 50
B
blue)
3
U
P-450 and iodosobenzene as substrate."021The monomolphotoemission of singlet oxygen occurs at A= 1270 nm (in
the IR). Recently, the lactoperoxidase reaction was shown
to photoemit at A= 1270 nm."031
10
.-P
: 5
X
.0
3
az
Diphenylanthracene (eostne)
Fig. 14. Yield of photooxidation products in the reaction of rubrene and diphenylanthracene, respectively, in the presence of the sensitizers methylene
blue and eosine. The photosensitizer was spaced by stearate monolayers of
thicknesa d (after [95]).
Sensitive photon-counting equipment has made it possible to use this "low-level" chemiluminescence as an indicator of '0, in intact cells and organs. The spectra as well
as the enhancement of photoemission by diazabicyclooctane (DABCO) and the decrease of photoemission by
azide indicate the occurrence of singlet oxygen in intact
liver during the redox cycling of 2-methyl-l,4-naphthoquinone ( r n e n a d i ~ n e ) ~ ~or
' ] 1,l '-dimethyL4,4'-bipyridinium
(paraquat).[981Thus, singlet oxygen may be responsible for
the toxic effects observed with these compounds. Menadione is mutagenic,1991and singlet oxygen generated by microwave discharge has been shown to affect biologically
active DNA, causing a loss of transforming activity in an
assay system using the plasmid pBR 322.['Ooa1Similar results were obtained when ' 0 , was generated from a thermodissociable naphthalene endoperoxide.['OOhl
In isolated enzymatic systems, the photoemission specTwo
tra resemble that of the H,O,/NaOCI
peaks near the 634-nm and 703-nm regions were observed
with isolated ram seminal vesicle prostaglandin hydroperoxidase ( c y c l o ~ x y g e n a s e ) (Fig.
~ ~ ~ ' 15), o r with cytochrome
B-Carotene (vitamin A)
Uric acid
Plasma proteins
Superoxide dismutases
GSH peroxidases
Catalase
Ancillary enzymes
3'
GSSG-reductase
GSH synthesizing enzymes
NADPH supply
Enzymatic
NADPH-quinone oxidoreductase
(DT-diaphorase)
Epoxide hydrolase
Conjugation enzymes
f2
.-
Remarks
Transport systems
Semidehydroascorbate reductase
Methionine sulfoxide reductase
DNA repair enzymes
-
0
0
650 690
h Inrnl-
a-Tocopherol (vitamin E, cf. Fig. 19)['04] is the most important lipid-soluble antioxidant;"051its unique function in
1065
the membrane may be aided by the specific physicochemical interaction between the phytyl residue and the fatty
acid residues of the polyunsaturated phospholipids in the
membrane.'"'61 The proper insertion of the vitamin into the
membrane increases its effectiveness by about 5 0 - f 0 l d . [ ' ~ ~ ~
Ascorbic acid (vitamin C), together with glutathione, is
the important antioxidant in the aqueous phase. It can
react with the vitamin E radical (chromanoxyl) and thus
can regenerate tocopherol in the membrane. Further aspects of interest, e.g. in nutrition[lo91(normal requirement:
12 mg of vitamin E and 75 mg of vitamin C per day["01)
cannot be discussed in detail here.
Further important antioxidants are listed in Table 7.
NADPHbinding
site
GIy- Cys-~Glu
His-L67'
Solvent
The transport systems for GSSG and glutathione conjugates (thioethers) have been studied in some detail recently""l (see Fig. 17). In liver, there is mutual competition of
biliary export between these two types of glutathione derivatives,"181 indicating that the canalicular carrier system
may accept both these substrates for transport. There appears to be a GSSG activatable ATPase in the hepatic
plasma membrane.'' 19] Mutual competition for export of
GSSG and Cis-conjugates was also detected in the
heart.['"] Using the creatine-kinase reaction as an indicator system, it was found that GSSG transport across the
Angew. Chem. In[. Ed. Engl. 25 11986) 1058-1071
dtracellular space
Extracellular
Subce
I.
rner
X-SG
GSSG
Fig. 17. Relations between GSH peroxidase and GSH transferase reactions.
Glutathione disulfide (GSSG) and glutathione thioethers (X-SG) are exported from the cells, competing for transport. X-SGs inhibit GSSG reductase
(after [ 1201).
The level of antioxidant defense is regulated. For example, the induction of catalase and superoxide dismutase
(SOD) in microorganisms such as E . coli or S . typhirnuriurn
during anaerobic shifts['271or by treatment with H2021'281
has been observed; there are also adaptation phenomena.112u-1311
A d ouble mutant of E. coli devoid of the two
S O D activities was unable to grow on minimal glucose medi~m.~'~
During
''
adaptation of S . typhirnurium to H202,30
proteins are induced, and it was shown that nine of them
are under positive control of a regulon for defense against
oxidative stress, called ~ x y R . [ ' ~ The
' l oxyR regulon controls a global response to a D N A damaging agent, in addition to three other global responses (the SOS response, adaptation to alkylating agents, and heat-shock). In cells in
which the oxyR regulon is deleted, the rate of spontaneous
mutagenesis is dramatically increased, and the level of mutagenesis is less than in the controls if the oxyR gene is
o v e r e ~ p r e s s e d " ~(Table
~]
8). Similarly, the E. coli mutants
lacking S O D exhibit oxygen-dependent mutager~esis."~~'
While control of the patterns of antioxidant enzymes,
and also the control of the levels of antioxidants such as
vitamin E, are not well-characterized in mammalian cells,
it appears that adaptation phenomena of this nature may
also be important in eukaryotes. I n this regard, the changes
in the biochemical pattern exhibited in cells in hepatic nodules may be considered as adaptive. These nodules contain clones of hepatocytes in which a new state of liver differentiation is acquired, and this is considered as a physiological response to environmental perturbation^,"^"] like
the oxyR response mentioned above. The changes observed in the nodules refer to some of the ancillary antioxidant enzymes mentioned in Table 7; these consist of increases in the cellular activities of some isozymes of glucuronyl transferases, glutathione transferases, y-glutamyl
transferase, epoxide hydrolase, and NADPH :quinone oxidoreductase. These enzymes are classified as belonging to
the Phase I1 group of enzymes involved in xenobiotic
transformation. Interestingly, the enzymes of Phase I,
namely cytochromes P-450 and b5, have drastically decreased cellular activities. It appears that these changes in
gene expression are related to D N A methylation (cf. I137,
1381). In experiments to decrease D N A methylation at the
cytosine residues by treatment of animals with the drug
analog, 5-azacytidine, the content of cytochromes P-450
whereas the content of sevand b5 was found to
eral isozymes of the glutathione transferases as well as of
NADPH :quinone oxidoreductase in mouse liver was in-
Description
Colonies
per plate
Zone of inhibition
[mml
TA 41 I7
TA 4118
TA 41 I9
oxyR
oxyRl
oxyA2
33 ? 6
12+4
1408f L60
18.0
12.5
33 5
1067
This synthetic organoselenium compound, 2-phenyl- 1,2benzoselenazol-3(2H)-one (ebselen), has been found to exhibit antioxidant capacity.['431In an assay of lipid peroxidation using rat liver microsomes, the lag phase preceding
the onset of ascorbate/ADP-Fe-induced lipid peroxidation
is increased by the addition of ebselen, whereas the sulfur
analog is inactive (Fig. 20); this pertains not only to the
low-level chemiluminescence, but also to other parameters
of lipid peroxidation like the evolution of ethane and npentane or the production of thiobarbiturate-reactive material.'941 In addition to this antioxidant activity, the compound acts catalytically in the GSH peroxidase reacI
50
25
2GSH
Fig. 18. Increase in NADPH :quirtone oxidoreductase (DT-diaphorase) and
decrease in cytochrome P-450 in mouse liver after injection of azacytidine for
inhibition of D N A (cytosine-5) methyl transferase (after [ 1401).
(Fig. 18). Recently, NADPH :quinone oxidoreductase was cloned and was described to be hypomethylated in persisting liver n ~ d u l i . [ ' ~ ' ]
Thus, there are interesting relationships between the status of DNA methylation and the expression pattern for
some enzymes of importance in defense against oxidative
challenge (see also [ 1421).
+ ROOH
GSSG i- ROH
Ebselen
+ H20
Sulfur onologue
Numerous drugs serving as antioxidants have been synthesized and tested in biological environments, ranging
from phenolic antioxidants added to foodstuffs to drugs
used in medicine (Fig. 19). This subject cannot be treated
here in detail. However, two selective aspects from our
own research interest will be briefly mentioned.
R1
R2
a-T
CH3
CH3
8-T
CH3
7-T
CH3
R2
R'
OH
11
OH
Fig. 19. Some antioxidant inhibitors of peroxidation reactions. '7: a- bis Stocopherol; 8 : BHA=2-1er/-butyl-4-methoxyphenol; 9 : BHT=2.6-di-tertbutyl-4~methylphenol: 10: propyl gallate; I 1 : N D G A = nordihydroguaiaretic acid.
I068
I[rninl
Fig. 20. Ebselen and its sulfur analog. The organoselenium compound exhibits antioxidant activity, shown by prolongation of lag phase in microsomal
lipid peroxidation assay (after [143]).
This activity is thought to be responsible for the protection of isolated hepatocytes against oxidative challenge.
Significant protection was afforded against ADP-Feinduced cell damage in control cells, whereas cells previously rendered deficient in GSH were not protected by
e b ~ e l e n . " ~The
~ " ~ cytotoxicity of anticancer quinones in
Ehrlich ascites cells was significantly decreased by ebselen."4sh1Recently, it was found that ebselen also exhibits an
inhibiting activity in the lipoxygenase pathway."461
Whether this can be explained by the removal of activatory
hydroperoxide through the GSH peroxidase reaction (cf.
Fig. 9), or by yet another site of action, is not clear.
Selenium displays a variety of biological effects (see
[147]), a prominent one being its role as selenocysteine in
the active center of GSH p e r o x i d a ~ e . " ~ It
* ~might
' ~ ~ ~be of
interest to compare the mechanism of catalysis of the GSH
peroxidase reaction identified for the enzyme with that of
Angew. Chem. In,. Ed. Engl. 25 (1986) 1058-1071
lntracellular
Reversible l o s s ]
GSSG
Protein-SSG
Acy I- S G
"Bound" GS H
Amino a c i d s -GSH'
Excretion {kidney) ]
Amino acids
\Hydrolysis]
GSH-Efflux(Sinusoida1 s p a c e + bile)
GSSG-Efflux (Bile)
Complexes with heavy m e t a l s (bile)
(Kidney and
epithelial organs1
Fig. 21. Processes (chemical and translocation) that influence the intrahepatic and extrahepatic glutathione status (after 1551).
1069
1070
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