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Biochemistry of Oxidative Stress

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

2. Oxidative Damage to Nucleic Acids

This review article is concerned with oxidative damage


in biological systems. First of all, the nature of the damage
to organic compounds will be discussed. It will then be
shown that, despite being variable in terms of the different
kinds of compound afflicted, oxidative damage is ultimately exerted only by a small number of different reactive
oxygen species. Finally, some new aspects of defense and
repair in biological systems will be presented, i.e. antioxidants s e n m stricio as well as in a more general sense.
One-electron reduction of oxygen was widely studied
early this century (see Warburg and Battelli and
Mi~haelis~]
pointed out that one-electron steps of oxygen
reduction, leading to intermediate radical formation, may
be of general importance in biological chemistry. These radical forms of oxygen are Oa: and O e B and their protonated forms, HO: and HO. The perhydroxyl radical HO:
and the hydroxyl radical HOa were recognized by Haber
and we is^[^] in their study on the iron salt-catalyzed decomposition of H 2 0 2as being important in chemical, photochemical and electrochemical processes. Biological research on the superoxide anion radical OyB received exceptional stimulus after the discovery of superoxide dismutase by McCord and Frido~ich.[~
Other reactive species of oxygen are of a nonradical nature. Hydrogen peroxide (H,02) as the two-electron reduction state became important in the development of enzymology after the discovery of catalase Compound I by
Chance.16Further, the electronically excited states of oxygen such as singlet molecular oxygen or excited carbonyls
are also worthy of note. Kautskyf7realized quite early on
the potentially important role of a metastable active oxygen species (singlet oxygen), while Schenckf8Iand Gollnick made important fundamental contributions to the
photochemistry of photooxygenation reactions. F00te[~
identified the role of singlet oxygen in photooxygenation.
In recent years, much interest has been focused on the
biochemistry of oxygen activation and on the biological
significance of the reactive oxygen species.[-41More recent developments can be found in Refs. [15-201.

2.1. Causes and Effects

[I

Prof. Dr. H. Sies


lnstitut fur Physiologische Chernie I der Universitst
Moorenstrasse 5, D-4000 Dusseldorf (FRG)

1058

0 VCH Verlaq~qcwllwhafim h H . D-6940 Welnheim, 1986

Oxidative damage to D N A can be initiated by ionizing


radiation or photooxidation (UV light; visible light in the
presence of photosensitizers), hydroperoxides, oxygen
radicals or various other oxidizing agents. The nature of
the damage is often complex. For example, a variety of radicals can be formed upon y-irradiation of DNA, but only
the radical 1 with a free electron on C-4 leads to strand
breaks under anoxic conditions (Fig. 1). In the presence
of oxygen, the radical-mediated strand scission is more
complex and proceeds via peroxy radicals of the bases and
sugars. Base alteration has been studied most intensively
with thymine and guanine. Thymine glycol 2 (Fig. 2) or
5-(hydroxymethy1)uracil are formed from thymine (cf.
[231). The base most readily lost upon y-irradiation or photooxidation is guanine; some proposed reaction products
are shown in Figure 3. Adenine and cytosine, on the other
hand, appear to be more stable towards oxidation. Thymine dimers (Fig. 4) are also formed, and the chemical adducts upon reaction with epoxides, for example, usually
involve guanine as shown for the adduct with aflatoxin
epoxide (Fig. 5).
Strand scission by ionizing radiation is thought to be
due to the hydroxyl radical (see [21, 221). Likewise, the
strand scission observed with superoxide is due to the hydroxyl radical formed from it.[261However, in addition to
this low-molecular weight molecule, other reactive species
might be of interest; for example, the hydroperoxide of
linoleic acid, 13-hydroperoxylinoleic acid, was found to
cause the breakage of double-stranded DNA.f271The breakage site was specific for guanine. The ultimate chemical
agent interacting with the DNA, however, was not identified. Possibly a hydroxyl radical is generated from the hydroperoxide via homolytic scission, as has recently been
found in the case of hydrogen peroxide.[281
The damage to DNA (and possibly also to nucleoproteins) by ionizing radiation and by free radical oxidation
also leads to chromosome damage. This is manifested by
chromosome breaks and can be assayed by chromosome
and chromatid aberrations. Chromosome breaks are particularly prevalent in diseases such as Blooms syndr~me,~

OS70-0833/86/12~2-1OS8$ 02.50/0

Angew. Chem. Int. Ed. Engl. 25 (1986) 1058-1071

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.[~~]

3. Oxidative Damage to Amino Acids and Proteins


The oxidation of amino acid side-chains in proteins has
recently been recognized as being a potentially important
signal in biological systems. The reversible oxidation-reduction of thiol groups is intimately linked to oxidative
stress in a number of respects (see Sections 3.1, 3.4, and
3.5). Other types of oxidation of reactive groups are also
reversible; for example, the oxidation of methionine to
methionine sulfoxide and its enzymatic reduction back to
m e t h i ~ n i n e . ~However,
~'~
irreversible oxidative damage to
amino acids may also occur; for instance, the ring cleavage
in histidine or in tryptophan (see Fig. 7).

Fig. 7. End products or intermediates of the oxidatwe breakdown of some


amino acids of biological interest.

dized. Oxidation to the sulfoxide can be associated with


loss of function. A particularly striking example is that of
a,-antitrypsin. Oxidation of methionine-358 at the active
site results in a dramatic decrease in inhibitory activity toward e l a ~ t a s e . [This
~ ~ ] loss of control of elastase is thought
to be responsible for the development of pulmonary emp h y ~ e m a . ' ~Indeed,
~]
the development of emphysema can
be limited by the intravenous injection of a , - a n t i t r y p ~ i n . [ ~ ~ ~
It is of interest to note that recently the problem of oxidative inactivation has been circumvented by producing an
a,-antitrypsin with valine at position 358 instead of methionine. This was achieved by site-directed mutagenesis
and recombinant DNA techniques,[461thus providing further evidence that critically targeted methionine oxidation

Table I. Some Proteins and peptides affected by oxidation


Oxidized
amino acid

Protein or peptide

Ref.

Methionine

Ribosomal protein L 12 ( E . coli)


Lysozyme
Pepsin
Ribonuclease

[421

3.1. Methionine

Methionine can be oxidized by HOO, '02or H 2 0 zto the


sulfoxide and then further to the sulfone. This reaction sequence has now been established as an interesting metabolic signal.[421Table l contains a list of enzymes in which
a specific methionine residue has been shown to be oxi1060

Tryptophon endoperoxide

Histidine

Phosphoglucomutase
a,-Proteinase inhibitor
Calmodulin
ACTH (adrenocorticotropic hormone)
Chemotactic factors
Met-Leu-Phe
Complement C5A
a ,-Antitrypsin

[431

Glutamine synthetase ( E . coli)

[48, 491

Angew. Chem. lnt.

Ed. Engl. 28 (1986) 1088-1071

is biologically significant. The mechanism of inactivation


of the human a,-proteinase inhibitor by gas-phase cigarette smoke has been investigated (for mechanism, see
Ref. [47]).

3.2. Histidine

Histidyl residues in proteins can play a role similar to


that described for methionine. The selective loss of one
histidine residue from sixteen per subunit leads to the
inactivation of glutamine synthetase from E. coZi.i48,491
Several other enzymes of interest in cellular metabolism were
also found to be inactivated upon exposure to oxidizing
conditions, as established, for example, in a mixed-functional system consisting of NADPH, cytochrome P-450 reductase, and cytochrome P-450.[49' It has been discussed
that this "marking" of the protein by histidine oxidation
may serve for its recognition by proteases, thus serving a
function in protein turnover.
The products of histidine oxidation have not yet been
completely identified. Loss of histidine is accompanied by
the introduction of a carbonyl group, detectable as a stable
d i n i t r o p h e n y l h y d r a z ~ n e . [Singlet
~ ~ ~ oxygen can lead to the
products shown in Figure 7.['01

3.3. Tryptophan, Lysine and Tyrosine


These amino acids are also subject to oxidation at appreciable rates, but no detailed study of their oxidation in
proteins seems to have been carried out as yet. Dityrosine
and isodityrosine are oxidation products of tyrosine.

drastically alter biological functions. Alteration in the


thiol/disulfide status, for example, has been found to lead
to biological consequence^,[^^-^'^ including changes in enzyme properties ( K , or urn;,, effects) (see Table 2). Thus,
the thiol redox status seems to serve as a metabolic signal.
Intracellular proteins in general are predominantly present
in the thiol form and have a low cysteine content (1.6%),
whereas extracellular proteins (e.g. in blood plasma) are
primarily disulfide proteins and have a high half-cystine
content (4. 1%).'~~1

Table 2. Enzyme activities modified by thiol/disulfide exchange [ 5 5 ] .


GSSG =disulfide of glutathione (GSH): cystamhe= bis(2-aminoethyl) disulfide; DTNB = bis(3-carboxy-4-nitrophenyl)disulfide (dithionitrobenzoate).
Enzyme

Activation by

Glucose 6-phosphate dehydrogenase


Collagenase (Leukocytes)
Acid phosphatase (Spinach)
Fructose 1,6-diphosphatase
6-Aminolaevulinate synthetase

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

Proline in proteins may constitute a preferential target


for the hydrolysis of peptide bonds, yielding new N-terminal glutamate residues in the fragments["] (Fig. 8). Collagen was found to be particularly susceptible to oxidative
attack.i521
R-OC,

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

+ GSSG +CoASSG + GSH.

In isolated mitochondria, respiration and ATP synthesis


with CoA-dependent substrates such as pyruvate or 2-0x0glutarate are abolished, whereas there is little effect, for
example, with 0-hydroxybutyrate or succinate as substrate.@']
1061

Thiol groups may be further oxidized to alkylthio radicals and subsequently add oxygen:
RS'

+ Oz -+

oxidation before release from the phospholipid may serve


as a marker for the phospholipase.

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

In radiation biochemistry, interest has recently focused


on reactions of the hydroxyl radical (HOO) and the peroxyl radical (ROO0) with proteins (see [70b, 711). Inactivation of yeast alcohol dehydrogenase and the protection
of the enzyme by antioxidant~["~
or the fragmentation of
bovine serum albumin into pep tide^"^.^^] are examples of
studies in this area. As mentioned above, fragmentation
via hydroxyl radicals is thought to occur preferentially at
proline residues; reaction with a peroxyl radical leads to a
2-pyrrolidone intermediate, hydrolysis of which yields a
new N-terminal glutamate r e ~ i d u e l(cf.
~ ' ~Fig. 8).

The specific enzymatic oxidation of polyunsaturated


fatty acids should not be considered as a damaging event,
since it leads to a realm of extremely potent and biologically important signal compounds. The products of this
specific oxidation are prostaglandins, thromboxane A2,
prostacyclin, and the leukotrienes (see [791). Unspecific oxidation of polyunsaturated fatty acids is known as lipid
peroxidation, a radical-mediated pathway (Fig. 10) leading
to a number of stable degradation products (Table 3). The
product pattern depends essentially on the nature of the
initial fatty acid involved; for example, pentane is gener-

4. Oxidative Damage to Carbohydrates


R@

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]).

Table 3. Products of lipid peroxidation [Sl].


~

Chain cleavage and recurrent oxidarion products:

5. Oxidative Damage to Lipids


5.1. Causes and Effects

Polyunsaturated fatty acids have become a central area


of interest in the chemistry and biochemistry of oxidative
reactions. These fatty acids, largely present in phospholipids of biological membranes, can be released into the
pool of free fatty acids via the action of phospholipase Az
before or after oxidative attack (Fig. 9). It is possible that
1062

Alkanes, alkenes, n-alkanals, '-alkenals, 2,4-alkadienals, alkatrienals,


hydroxyaldehydes, hydroperoxyaldehydes, 4-hydroxyalkenals, 4-hydroperoxyalkenals, rnaionaldehyde, dicarbonyl compounds, saturated and
unsaturated ketones

Rearrangement and consecuriue products:


Hydroxy acids, keto acids, ketohydroxy acids, epoxyhydroxy acids, dihydroxy acids, ketodihydroxy acids, trihydroxy acids

Further peroxidation products:


Cycloendoperoxides (prostaglandin GJ and analogous compounds
Dimers and polymers
Dimers and polymers linked by -0-, -0-0-, -C-C-

bridges

Angew. Chem. Int. Ed. Eiigl. 25 (1986) 1058-1071

ated from an w-6-polyunsaturated fatty acid, and ethane


from an w-3-polyunsaturated fatty acid. The chemistry of
lipid peroxidation is complex, and will not be discussed
here in detail (see Ref. [SZ]).

Table 4. Reactive oxygen species of interest in oxidative stress.


Species
000

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.["']

6. Reactive Oxygen Species


The reactive oxygen species involved in biological systems are listed in Table 4. Most of them are free radicals;
the term oxygen-free radicals is used more or less synonymously with reactive or aggressive oxygen species. However, it should be noted that ground state (triplet) molecular
oxygen as a diradical is much less reactive than molecular
oxygen in the excited state ('Agoz abbrev. lo2).
This singlet oxygen is diamagnetic and not of a radical nature.["I
Thus, non-radical excited states of molecular oxygen and
of oxygen in organic compounds, e.g. in excited carbonyl
compounds and dioxetanes,["] as well as ozone fall into
the category of reactive oxygen species of biological interest. The kinetic constants of the reactions of HO:/O:"
with more than 300 organic and inorganic compounds in
aqueous solution have recently been compiled.[901
These reactive oxygen species are formed via several
pathways, enzymatic and nonenzymatic. One-electron reduction, initially leading to the formation of the superoxide anion radical O:", is a major source (Table 5). Direct
two-electron reduction generates hydrogen peroxide; an
example for this is given by the fatty acyl-CoA oxidase in
Anyeit, Chem. I n t .

Ed. Engl. 25 11986) 1058-1071

Remarks

Superoxide

One-electron reduction state, formed


in many autoxidation reactions (e.g.
flavoproteins; redox cycling)
Protonated form of O?", more lipidsoluble
Two-electron reduction state, formed
from Ofo (HO?) by dismutation, or
directly from O2
Three-electron reduction state,
formed by Fenton reaction, metal(iron)-catalyzed Haber-Werss reaction; highly reactive
Oxygen-centered organic (e. g. lipid)
radical
Formally formed from organic (e.g.
lipid) hydroperoxide, ROOH, by hydrogen abstraction
Organic hydroperoxide (e. g. lipid-,
thymine-OOH)
First excited state, 22 kcal/mol above
ground state (triplet) ' 0 2 red
; (dimol)
or infrared (monornol) photoemission
Excited carbonyl compound, bluegreen photoemission (e. g., formed
via dioxetane as intermediate)

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 peroxisome. The formation of the hydroxyl radical


may occur directly through radiolysis of water, and also
via the Fenton reaction:
FeZB+ HzOz+Fe3@i OH' + O H Q

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

Table 5. Formation of the 0yoradical in biological systems [176].


I . Of" formation by autoxidation (inclusive of "redox
cycling")

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

2. Ofo formation by enzymes or enzyme complexes

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

3. Increased (enzymatic) O?O formation by xenobiotics


Antimycin
Adriamycin
Paraquat

[I891
[194, 1951
[196, 1971

4. O?" formation by physical factors

Ultraviolet light, Ultrasound,


X-rays, y-rays

1063

sources is difficult to assess in a general fashion; they


could contribute to a large extent in maintaining the cellular steady state concentration of the 0:" radical. Autoxidation reactions such as redox cycling (Fig. 12) are certainly also growing in importance for explaining the oxidative stress caused by several xenobiotics; these include
compounds such as quinones, which are used in cancer
~hemotherapy.["~
The steady-state oxygen concentration is of particular importance. At low 0, partial pressure, in the area of hypoxia, there is an optimum for damage by lipid peroxidation,
since reductive steps such as the transfer of electrons in
redox cycling (Fig. 12) or the reduction of haloalkanes by
cytochrome P-450occur simultaneously with 02-dependent steps of lipid peroxidation (Figs. 10 and 13).1911
For example, the optimum 0, partial pressure was found to be
2 mm Hg for liver microsomal lipid peroxidation by CCI,
or halothane (CF3CHBrCI) using an oxystat
e0

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

tion, but also two-electron reduction products. Whereas


the dismutation of 0:' and of H 2 0 2by superoxide dismutase and catalase, respectively, yield ground-state triplet
oxygen, the dismutation of prostaglandin G, or of organic
hydroperoxides as products of lipid peroxidation affords
singlet molecular oxygen.
Singlet oxygen is formed in biological systems (a) via
photosensitization reactions, an appropriate sensitizer being electronically excited and then transferring energy to
oxygen, and (b) via chemical excitation reactions. These
latter pathways of singlet oxygen formation occur without
activation by light, and are therefore also referred to as
"photochemistry in the dark."1891
In principle there are two pathways for the chemical excitation of oxygen. One proceeds via a radical-radical int e r a ~ t i o n l ' ~ (Russell's
.~~~
mechanism) (see Fig. 13), the
other via oxene transfer using heme iron (Fe3+):1941

+ ROOH
(Fe=O) + ROOH
(Fe3')

--f

+ ROH
(Fe3@) + ROH + '02

(Fe=O)

---t

2ROH

+ '02

Singlet oxygen may be of particular interest in biological


systems, because it is capable of diffusing an appreciable
distance in membranes. In stearate monolayers, the diffusion path for singlet oxygen for half-deactivation was estimated to be 115 (Fig. 14).1951The formation of singlet
oxygen in cellular systems has so far been demonstrated
mainly by the detection of photoemission via the dimol
reaction:

'0,

I064

ROOH

2ROOH

2PG G2

ROOQ

Enzyme

Reaction

2HZOZ

R@

R'R''Cff

Table 6. "Dismutation reactions" of oxygen metabolites; PG = prostaglandin.

20y0

a -T-0

Fig. 13. Generation o f excited oxygen (singlet oxygen; left-hand branch) or


excited carbonyls R'R"CO* (triplet carbonyls; right-hand branch) from lipid
peroxyl radicals, according to Russell's mechanism 192, 931. a-l-OHsignifies
a-tocopherol (see Fig. 19).

02

H2O2. OH @ , ROO

a -T-OH
02

+ '0,

2'02

+ hv

(A=634 nm, 703 nm).

Angew. Chem. Inf. Ed. Engl. 25 (1986) 1058-1071

-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

7. Defense Against Oxidative Stress:


Biochemical Antioxidants

: 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

The detoxication of reactive oxygen species is one of the


prerequisites of aerobic life; the multiple lines of defense
which have evolved form a veritable antioxidant defense
system (Table 7). The repertoire to counteract the potentially hazardous reactions initiated by oxygen metabolites
includes all levels of protection: prevention, interception,
and repair. It comprises nonenzymatic scavengers and
quenchers referred to under the term antioxidants in the
more narrow sense, and also enzymatic systems.

Table 7. Anitoxidant defense in biological systems


System
Non-enzymatic
a-Tocopherol (vitamin E)
Ascorbic acid (vitamin C )
Flavonoids
Chemical antioxidants

B-Carotene (vitamin A)
Uric acid
Plasma proteins

Superoxide dismutases
GSH peroxidases

Catalase

CuZn enzyme, Mn enzyme


Selenoenzyme: non-Se enzyme; some
GSH transferases. e.g., isoenzymes B
and AA; cytosol and mitochondria1
matrix
Heme enzyme, predominantly in
peroxisomal matrix

Ancillary enzymes

3'
GSSG-reductase
GSH synthesizing enzymes
NADPH supply

Membrane-bound; receptors'? Regeneration from chromanoxyl radical?


Water-soluble
Plant antioxidants (rutin, quercetin,
etc.)
Food additives, e.g. BHA (butylated
hydroxyanisole), BHT (butylated hydroxytoluene) [a]
Singlet oxygen quencher
Singlet oxygen quencher, radical scavenger'?
e.g., Coeruloplasmin

Enzymatic

NADPH-quinone oxidoreductase
(DT-diaphorase)
Epoxide hydrolase
Conjugation enzymes

f2
.-

Remarks

Transport systems

Two-electron reduction, dicoumarolsensitive


UDP-glucuronyl transferase
Sulfotransferase
GSH transferases

Glucose 6-phosphate dehydrogenase


6-Phosphogluconate dehydrogenase
Isocitrate dehydrogenases
Malate enzyme
Energy-linked transhydrogense
GSSG export
Conjugate export

Semidehydroascorbate reductase
Methionine sulfoxide reductase
DNA repair enzymes
-

0
0

[a] BHA = 2-tert-butyl-4-methoxyphenol; BTA = 2,6-di-tert-butyl-4-methylphenol (see 8 and 9 in Fig. 19)

650 690
h Inrnl-

7.1. Nonenzymatic Antioxidants


Fig. 15. I)imol eminion spectrum of low-level chemiluminescence of singlet
oxygen. Generation of ' 0 2 :a) in the hypochlorite-H202 reaction (after
[loll): h) enzyme-catalyzed in the prostaglandin cyclooxygenase reaction
(after [Y 41).
Angeu Cheni. I n ( . Ed. Engl. 28 (1986) 1058-1071

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.

7.2. Enzymatic Antioxidants


The essential enzymes are the intensively studied superoxide dismutases and various hydroperoxidases such as
gfutathione peroxiduse, caralase, and other hemoprotein
peroxidases. They are characterized, in general, by high
specific cellular content, by specific organ and subcellular
localizations which often overlap in a complementary way,
and by a specific form of metal involvement, especially of
copper, manganese, iron (heme) and selenium, in the catalysis. These antioxidant systems have a wide distribution in
nature, underscoring their importance for coping with the
damaging effects of reactive oxygen metabolites in biological systems. Their distribution is crucial in target organ
toxicity.['111A comprehensive discussion of these central
enzymes in antioxidant defense is beyond the scope of this
article (see Refs. [13-201). In the following, some more recent aspects of reactions involved more indirectly in antioxidant defense will be presented.
A novel protein which protects membranes from peroxidation and exhibits GSH peroxidase activity toward phosphatidylcholine hydroperoxides has been identified and
characterized as a selenoenzyme."lzl Also new is a GSHdependent heat-labile factor which inhibits lipid peroxidation in biological membranes.["31 Most recent findings indicate that this cytosolic protein is not one of the known
GSH-dependent
Some specific cell types may use extracellular GSH. The
basolateral membrane of intestinal epithelial cells contains
an Na@-dependentGSH uptake system, so that exogenous
GSH protects these cells from oxidative injury." 14'] Most
other cell types cannot utilize GSH as such, but have to
synthesize their GSH intracellularly from the constituent
amino acids.
It should be noted that a number of additional or ancillary systems are of crucial importance. For example, many
of the radical or nonradical reactions in cells may lead to
the oxidation of thiols to disulfides, i.e., the oxidation of
glutathione (GSH) to form GSSG. Thus, the regenerative
reaction of reduction to GSH as catalyzed by GSSG reductase can become pivotal in antioxidant defense. Naturally,
the provision of reducing equivalents for this enzyme is
also of importance. Thus, the NADPH regenerating systems (Table 7) are also of interest.
Diminution of the steady-state levels of reactive compounds capable of generating reactive oxygen species also
1066

results in a decreased expression of oxidative stress; in this


respect, the two-electron reduction of quinones by
NADPH :quinone oxidoreductase (DT diaphorase) and
the subsequent conjugation reaction of the hydroquinone
are part of the antioxidant defense.[97,I "I
Obviously, the export of reactive species in free or conjugated form also serves as a detoxication function, so that
transport of conjugates as well as of GSSG from cells is of
interest here. The binding of conjugates of glutathione to
GSSG binding sites may have metabolic significance. It
has been shown in kinetic and X-ray crystallographic studies that glutathione conjugates bind to the GSSG-binding
site in the active center of GSSG reductase, causing inhibition of enzymatic
(Fig. 16). An increase in
GSSG levels causes metabolic perturbations, including an
inhibition of protein synthesis (see Ref. [53]).
a)

NADPHbinding
site

GIy- Cys-~Glu

His-L67'

Solvent

Fig. 16. a) GSSG binding site of glutathione reduciabe, b) the blnding of


S-( 1,2-dinitrophenyl)glutathione (thick lines) at this site (after [ 1161).

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).

cardiac plasma membrane was half-maximal at (ATPI


ADP),,,, ratios of = 10 in the intact perfused rat heart preparation."'"
Interestingly, a prominent GSH transferase activity in
the heart is isozyme 4-4, accepting 4-hydroxynonenal as a
substrate"221 and, therefore, capable of detoxifying this
biologically active product of lipid p e r ~ x i d a t i o n . " ' ~Re~
cently, another high-activity isozyme (8-8) was described.'"']
It should also be pointed out that some GSH transferase
isozymes have the capacity to catalyze the GSH peroxidase
reaction with organic hydroperoxides as substrate."24i
These non-selenium-dependent activities may become essential in states of selenium deficiency when the Se enzyme, GSH peroxidase, is very low in its cellular activity.
However, since the GSH transferases d o not accept H 2 0 2
as substrate, there is no complete substitute for the selenoenzyme. This may explain why in Se-deficiency there
can be overt clinical symptoms such as the cardiomyopathy known as Keshan disease.['''l This disease, which led
to death in about one percent of the male population of
school-age Chinese in the Keshan province, was found to
be completely treatable by the oral application of 1 mg of
sodium selenite per week."2s1
7.3. DNA Repair

Possibly the biologically most important line of defense


for the organism consists in preserving the identity of the
genetic material by repair after oxidative damage (cf. Fig.
6).13@In addition, an equally important process for microorganisms is enzymatically controlled mutagenesis in order
to ensure adaption to changing environmental conditions.
The repair system seems to be less sensitive to ionizing
radiation than DNA. In yeast cells, the repair of singlestrand breaks is dependent on the irradiation dose (up to
high doses of 2400 G Y ) . " ~The
~ ] repair enzymes are thus to
be considered in a broad sense as a central component of
the antioxidant enzymes for the cell.
Angew Chem In1

Ed. Engl 25 (1986) 1058-1071

7.4. Control of the Antioxidant Capacity

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-

Table 8. Spontaneous mutagenesis in relation to the presence of the OxyR


regulon in S . typhimurium tester strains. Strains with His G428 containing
plasmid pKMlOl and the oxyR regulon (oxyR+) or overexpressed (oxyR1)
or deleted (oxyA2) i n oxyR were assayed for spontaneous mutations in the
His reversion assay (Ames test). For description of phenotype, zones of inhibition by HZOZon the agar plates are also shown 11341.
Strain

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

7.6. Ebselen, a Novel Organoselenium Compound

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

5-Azacytidine(rng/kg body wt)

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

7.5. Synthetic Antioxidants

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

ebselen. The role of selenium as an antioxidant"511and as


an anticarcinogen, as well as its potential as a carcinogen
and as a cytotoxic agent, has been recently r e ~ i e w e d . " ~ ~ " ~

lntracellular
Reversible l o s s ]

7.7. The Selenoenzyrne GSH Peroxidase


This enzyme has recently been sequenced;"52b' its strucThe
ture has been studied by X-ray
gene from the mouse was cloned and it was found that the
selenocysteine in the active site is encoded by the termination codon TGA.1'5Zd1
Likewise, this codon was found in
formate dehydrogenase of E. ~ ~ l i . ~ ~ ~
7.8. Superoxide Dismutase as a Drug

The Cu,Zn-enzyme superoxide d i s m ~ t a s e [ 'has


~ found a
variety of applications as a n antioxidant. In clinical medicine, the enzyme has been employed mainly in topical, e.g.
intraarticular, applications, in the knee joint["31 and it was
found to be active as an antiinflammatory agent. The enzyme has been cloned;Li541
the recombinant human superoxide dismutase has been expressed in
The use
of the human enzyme as obtained by gene-technological
procedures may permit the systemic application and treatment of a variety of clinical conditions associated with
oxidative stress. For example, there is evidence of freeradical involvement in ischemic myocardial1i561
o r intestinal
injury or p a n ~ r e a t i t i s . ~Recently,
'~~'
it was suggested that
superoxide is involved in the breakdown of endotheliumderived vascular relaxing factor (EDRF).1'58'However, this
area of clinical research awaits further rigorous testing of
the therapeutic benefits of oxygen radical scavengers.

8. Biology of Oxidative Stress: Some Aspects


The biological implications of oxidative stress have already been discussed at length (see, e.g., [13-20, 1591). It
seems that almost all complex biological processes can be
implicated with respect to reactive oxygen species and free
radical involvement. Therefore, it seems that an in-depth
treatment of such topics as the role of prooxidant states in
tumor promorion"601 or in spontaneous mutagenesisl'611 can
only be referred to here. Similarly, the ageing process[i62.
1631
and the complex relations in inflammatory states['641require a critical treatment from the biochemical standpoint.
There is an intricate interorgan relationship in physiological and pathophysiological states; Figure 21 shows some
of these for glutathione.
The biochemical events associated with radiation effects
occur through free radicals (see Section 3.6). Recently, free
radicals generated in brain, spleen and liver during y-irradiation of mice ( 5 Gy) were detected by in vivo spin trapping.""" Selenite and Vitamin E were found to inhibit radiogenic and chemically induced cell transformation in vitroilhhlbut, conversely, depletion of these antioxidants for
six weeks had no effect on the radiation response in mice
in v ~ v o . ~ ' "Radioprotectors
~
and anticarcinogens should be
viewed
Biochemical-biological studies on oxidative stress have
now reached a state where very careful and cautious use of
Anqen. C'hrm. Inr. Ed Engl. 25 llY86) 1058-1071

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).

methods and concepts is called for. In this respect it


should be mentioned that two volumes dealing with methods in this area of research have recently become availab1e1169,1701
and should be consulted by workers interested in
the biological effects of reactive oxygen species.
Finally, it should be pointed out that the term oxidative
stress should not be interpreted solely in terms of a noxious challenge against which the organism must defend itself. As was recently pointed
numerous physiologically important chemical reactions occur in cells via reactive oxygen species and free radical reactions. These include the vital functions of macro phage^"'^^ and neutrop h i l ~ , ~formate-pyruvate
'~~]
l ~ a s e ,ribonucleotide
~'~~~
reductase necessary for deoxyribonucleotide p r o d ~ c t i o n , ~ ' ~ ~ ' ' ~
and the vast area of selective oxidation of polyunsaturated
fatty acids to produce the eicosanoids (see [791) with farreaching pathophysiological consequences, for example
clinical states such as ischemia and
The work carried out in the author's laboratory was supported by the Deutsche Forschungsgerneinschaft (Schwerpunktprogramm "Mechanismen toxischer Wirkungen uon
Fremdstoffen '7, by the National Foundation for Cancer Research, Washington, by the Ministerium fur Wissenschaji
und Forschung des Landes Nordrhein- Wesrfalen, and by the
Fonds der Chemischen Industrie. Thanks are due to numerous colleagues for valuable discussions, and to my co- workers in the laboratory whose contributions are cited in the
text.
Received. April 7. 1986;
supplemented. August 25, 1986 [A 599 IE]
German version. Angew Chem 98 (1986) 1061
[ 1J 0. Warburg: Uber die katalvtrschen Wirkungen der Iebendigm Substan;,

Springer, Berlin 1928.


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