Biochemistry of Oxidative Stress

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

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 ulti- mately 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 senm 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~hael is‘~] 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 O:a and OeB 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 de- composition of H 2 0 2 as being important in chemical, pho- tochemical and electrochemical processes. Biological research on the superoxide anion radical OyB received ex- ceptional 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 enzy- mology after the discovery of catalase Compound I by Chance.16’ Further, the electronically excited states of oxygen such as singlet molecular oxygen or excited carbonyls are also worthy of note. Kautskyf7’ realized quite early on the potentially important role of a “metastable active oxygen species” (singlet oxygen), while Schenckf8”I and Goll- nick‘”’ 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.[’”-’41 More recent developments can be found in Refs. [15-201.

[‘I Prof. Dr. H. Sies lnstitut fur Physiologische Chernie I der Universitst Moorenstrasse 5, D-4000 Dusseldorf (FRG)

2. Oxidative Damage to Nucleic Acids

2.1. Causes and Effects

Oxidative damage to DNA 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. Thy- mine dimers (Fig. 4) are also formed, and the chemical ad- ducts 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.[261 However, 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.f271 The 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 nucleopro- teins) 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 Bloom’s syndr~me,~”’

1058 0 VCH Verlaq~qcwllwhafi mhH. D-6940 Welnheim, 1986 OS70-0833/86/12~2-1OS8 $ 02.50/0 Angew. Chem. Int. Ed. Engl. 25 (1986) 1058-1071

I 0-@-

I I 0-@- 0-@--

I 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])

Fig. 2 Oxidative degradation of thymine residues in DNA. In addition to thymine glycol 2, 5-hydroxymethyl uracil may also be formed (after [24]).

R = Ribosyl

- ROH NH NH2 I R

Fig. 3. Scheme for the oxidative degradation of guanine by photooxygenation. Sens. = sensitizer (after [25]).

&NH N>

. . . __

\ ( 3 3

. . .

. . . NK 0

N -0

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).

[Tg o o ‘ OCH,

4

0 I

DNA

Fig. 5. Formation of the N-7 guanyl adduct 01 aflatoxin B, 4 from the 2,3- epoxide.

lupus e r y t h e m a t o s ~ s [ ~ ~ ] or Fanconi’s Superox- ide dismutase has an anticlastogenic (anti-chromosome- break) 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.

2.2. Repair

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 op- erate 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 Angew. Chem. In[ . Ed. Engl. 25 (1986) 1058-1071

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-(hydroxy- methy1)uracil and 5-(hydroxymethyl)-2'-deoxyuridine; a 5- hydroxymethyluracil glycosylase has recently been described.[3x1

Incision endonuclease 1 DNA- glycosylase I 1

i h h Damaged

deoxynucleoside

t

+. Damaqed

Apurin iclApyrimidinic base

endonuclease

Exctsion exonuclease 1

+ Polymerase. Llgase

888888 Fig. 6 . Two 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 pre- dominant lesion in UV-irradiated DNA (Fig. 4) cannot only be repaired by the dimer-specific endonuclease, fol- lowed by DNA polymerase,[391 but also by a light-dependent repair reaction. This photoreactivation is catalyzed by a f lavoen~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).

3.1. Methionine

Methionine can be oxidized by HOO, '02 or H 2 0 z to the sulfoxide and then further to the sulfone. This reaction sequence has now been established as an interesting metabolic signal.[421 Table l contains a list of enzymes in which a specific methionine residue has been shown to be oxi-

N H ? N H ? C H 2 - L - C O O ~ ( 3 ; C H 1 -COO@

0-0 H

Histidine endoperoxides

F2 N H ?

Tryptophon hydroperoxide Tryptophon endoperoxide

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 em- p 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,[461 thus providing further evidence that critically targeted methionine oxidation

Table I. Some Proteins and peptides affected by oxidation

Oxidized Protein or peptide amino acid

Ref.

Methionine Ribosomal protein L 12 ( E . coli) [421 Lysozyme Pepsin Ribonuclease Phosphoglucomutase a,-Proteinase inhibitor Calmodulin ACTH (adrenocorticotropic hormone) Chemotactic factors

Met-Leu-Phe Complement C5A a , -Antitrypsin [431

Histidine Glutamine synthetase ( E . coli) [48, 491

1060 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 cigar- ette 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 Sev- eral 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-func- tional 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 in i t rophenylhydraz~ne . [~~~ 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.

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). Col- lagen was found to be particularly susceptible to oxidative attack.i521

R-OC, CO-NH-R' R-OC, CO-NH-R' HO @/O, -

0

- RCOOH spontaneous hydrolysis i 23'"-",""' CO-NH-R'

HZO

HOOC H2 7 0

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 OZr leads 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 N- terminal 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

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 GSSG Phosphorylase phosphatase GSSG Phosphofructokinase GSSG Glycogen synthase D GSSG HMG-CoA-reductase GSSG Adenylate cyclase (brain) GSSG Ribonucleotide reductase ( E . coli) GSSG Hexokinase Tetraethylcystamine Tyrosine aminotransferase Cystine PDH kinase DTNB Fatty acid synthetase DTN B y-Glutamylcysteine synthetase Cystamine Papain, trypsin Dimethyl disulfide

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 mem- b 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.i60,6'1 When 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 coen- zyme 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 coen- zyme A-dependent processes are blocked :[64,651

CoASH + GSSG + CoASSG + GSH.

In isolated mitochondria, respiration and ATP synthesis with CoA-dependent substrates such as pyruvate or 2-0x0- glutarate are abolished, whereas there is little effect, for example, with 0-hydroxybutyrate or succinate as substrate.@']

Angew Chem. Int. Ed. Engl. 25 (1986) 1058-1071 1061

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

RS' + Oz -+ RSOY

Further rearrangements and oxidation steps lead to sulfen- ic, 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 xan- thine oxidasei"61 and 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-diox- ide can also be formed, and they possibly occur in keratin 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). Inacti- vation of yeast alcohol dehydrogenase and the protection of the enzyme by ant ioxidant~["~ 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).

4. Oxidative Damage to Carbohydrates

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 free- radicals. In the thiobarbiturate assay, deoxyribose yields a degradation product that is almost identical to the product obtained with mal~ndia ldehyde . '~~]

Polysaccharides such as hyaluronic acid can be de- graded 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 man- ne~. i '~]

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

oxidation before release from the phospholipid may serve as a marker for the phospholipase.

Phospholipose A2 Cyclooxygenose Lipoxygenose

Prostaglandins Phosphohpids + Arochidonote Thrornboxones

Leukotrienes

Lipocortin( -)

0 2

Fig. 9. Scheme of release of arachidonate lrorn phospholipids and of eicosa- noid 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.

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-

R @

Fig. 10. initial steps of' lipid peroxidation, schernatlc lor d i i unsalurated n- alkane (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:

Alkanes, alkenes, n-alkanals, '-alkenals, 2,4-alkadienals, alkatrienals, hydroxyaldehydes, hydroperoxyaldehydes, 4-hydroxyalkenals, 4-hydro- peroxyalkenals, rnaionaldehyde, dicarbonyl compounds, saturated and unsaturated ketones

Rearrangement and consecuriue products: Hydroxy acids, keto acids, ketohydroxy acids, epoxyhydroxy acids, di- hydroxy 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

1062 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]).

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[s31 and has been identified as a directly acting mutagen.[841

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.[8s1 4-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 lysosomal The molecular basis of modifica- 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 synony- mously with reactive or aggressive oxygen species. Howev- er, 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

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

Species Name Remarks

000 Superoxide One-electron reduction state, formed in many autoxidation reactions (e.g. flavoproteins; redox cycling)

HO? Perhydroxyl Protonated form of O?", more lipid- soluble

H202 Hydrogen peroxide Two-electron reduction state, formed from Ofo (HO?) by dismutation, or directly from O2

formed by Fenton reaction, metal(iron)-catalyzed Haber- Werss reaction; highly reactive

or hyperoxide

HO" Hydroxyl Three-electron reduction state,

ROO R-oxyl, e.g. Oxygen-centered organic (e. g. lipid)

ROO" R-dioxyl, e.g. Formally formed from organic (e.g. alkoxyl radical

alkoxydioxyl lipid) hydroperoxide, ROOH, by hydrogen abstraction

thymine-OOH) ROOH R-hydroperoxide Organic hydroperoxide (e. g. lipid-,

' 4 0 2 Singlet molecular First excited state, 22 kcal/mol above (also Of oxygen ground state (triplet) '02; red (dimol) or l o 2 ) or infrared (monornol) photoemission 'R'R"C0 Triplet carbonyl Excited carbonyl compound, blue- (also green photoemission (e. g., formed R'R"CO*) via dioxetane as intermediate)

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 0yo radical in biological systems [176].

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

Hydroquinones (semiquinones) Flavins Hemoglobin(s) Glutathione and other Thiols Catecholamines Transition metal ions

I I771 [177, 1781 1179, 1801 [ I S l , 1821 11831 [184, 1851

2. Ofo formation by enzymes or enzyme complexes

Flavin-dependent oxidations [186, 1871 Photosynthetic oxygen reduction [ I881 Mitochondria1 respiratory chain 11 891 Microsomal oxygenation 11901

and macrophages NADPH-dependent oxygen reduction by granulocytes

[ 19 I - 1931

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

Antimycin [I891 Adriamycin [194, 1951 Paraquat [196, 1971

4. O?" formation by physical factors

Ultraviolet light, Ultrasound, X-rays, y-rays

Anyeit, Chem. Int . Ed. Engl. 25 11986) 1058-1071 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. Autoxi- dation reactions such as redox cycling (Fig. 12) are cer- tainly 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 hyp- oxia, 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-450 occur 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

0 2

R e o c t i v e oxygen species

H2O2. OH @ , ROO @, 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-

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

Reaction Enzyme

2 0 y 0 + 2He --* H202 + O2

2HZOZ + 2H20 + 0 2

2PG G2 --t 2PG H2 + ' 0 2

2 ROOH -t 2 ROH + ' 0 2

Superoxide dismutase

Catalase

PG hydroperoxidase

Cytochrome P-450

2 P h I = O h 2 P h I + ' 0 2 Cytochrome P-450

a -T-OH a - T - 0 02

R @ ROOQ ROOH R H -57

R'R' 'Cff

i hu

703

Dioxetone

hv (340-460 nrn)

Fig. 13. Generation of 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-OH signifies a-tocopherol (see Fig. 19).

tion, but also two-electron reduction products. Whereas the dismutation of 0:' and of H 2 0 2 by 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 in- t e r a ~ t i o n l ' ~ . ~ ~ ~ (Russell's mechanism) (see Fig. 13), the other via oxene transfer using heme iron (Fe3+):1941

(Fe3') + ROOH ---t (Fe=O) + ROH

(Fe=O) + ROOH - (Fe3@) + ROH + '02

2ROOH --f 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 diffu- sion path for singlet oxygen for half-deactivation was estimated to be 115 A (Fig. 14).1951 The formation of singlet oxygen in cellular systems has so far been demonstrated mainly by the detection of photoemission via the dimol reaction:

'0, + '0, - 2'02 + hv (A=634 nm, 703 nm).

I064 Angew. Chem. Inf. Ed. Engl. 25 (1986) 1058-1071

1 50 - B I

c

3 U e P

10

.- : 5 X 0

0 3z a .-

0

blue)

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 indi- cator of '0, in intact cells and organs. The spectra as well as the enhancement of photoemission by diazabicyclooc- tane (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-naphthoqui- none ( rnenadi~ne)~~ ' ] or 1,l '-dimethyL4,4'-bipyridinium (paraquat).[981 Thus, singlet oxygen may be responsible for the toxic effects observed with these compounds. Mena- dione is mutagenic,1991 and singlet oxygen generated by mi- crowave discharge has been shown to affect biologically active DNA, causing a loss of transforming activity in an assay system using the plasmid pBR 322.['Ooa1 Similar results were obtained when '0, was generated from a ther- modissociable naphthalene endoperoxide.['OOhl

In isolated enzymatic systems, the photoemission spectra resemble that of the H,O,/NaOCI Two 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), or with cytochrome

3'

f 2 .- C -

1

0 0 650 690

h Inrn l -

Fig. 15. I)imol eminion spectrum of low-level chemiluminescence of singlet oxygen. Generation of '02: a) in the hypochlorite-H202 reaction (after [loll): h) enzyme-catalyzed in the prostaglandin cyclooxygenase reaction (after [Y 41).

Angeu Cheni. In ( . Ed. Engl. 28 (1986) 1058-1071

P-450 and iodosobenzene as substrate."021 The monomol- photoemission of singlet oxygen occurs a t A= 1270 nm (in the IR). Recently, the lactoperoxidase reaction was shown to photoemit a t A= 1270 nm."031

7. Defense Against Oxidative Stress: Biochemical Antioxidants

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 Remarks

Non-enzymatic a-Tocopherol (vitamin E)

Ascorbic acid (vitamin C) Flavonoids

Chemical antioxidants

B-Carotene (vitamin A) Uric acid

Plasma proteins

Enzymatic Superoxide dismutases GSH peroxidases

Catalase

Ancillary enzymes

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

GSSG-reductase GSH synthesizing enzymes NADPH supply

Transport systems

Semidehydroascorbate reductase Methionine sulfoxide reductase DNA repair enzymes

Membrane-bound; receptors'? Regen- eration from chromanoxyl radical? Water-soluble Plant antioxidants (rutin, quercetin, etc.) Food additives, e.g. BHA (butylated hydroxyanisole), BHT (butylated hy- droxytoluene) [a] Singlet oxygen quencher Singlet oxygen quencher, radical scav- enger'? e.g., Coeruloplasmin

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

Two-electron reduction, dicoumarol- sensitive

UDP-glucuronyl transferase Sulfotransferase GSH transferases

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

-

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

7.1. Nonenzymatic Antioxidants

a-Tocopherol (vitamin E, cf. Fig. 19)['04] is the most important lipid-soluble antioxidant;"051 its unique function in

1065

the membrane may be aided by the specific physicochemi- cal 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.['111 A 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 phos- phatidylcholine hydroperoxides has been identified and characterized as a selenoenzyme."lzl Also new is a GSH- dependent 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@-dependent GSH 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.

I t 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

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 con- jugated 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)

/ NADPH- binding site

GI y- Cys-~Glu His-L67'

1 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 de- rivatives,"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 indica- tor system, it was found that GSSG transport across the

1066 Angew. Chem. In[ . Ed. Engl. 25 11986) 1058-1071

Subce

I. Extracellular dtracellular

rner - X-SG GSSG

space

Fig. 17. Relations between GSH peroxidase and GSH transferase reactions. Glutathione disulfide (GSSG) and glutathione thioethers (X-SG) are ex- ported from the cells, competing for transport. X-SGs inhibit GSSG reductase (after [ 1201).

cardiac plasma membrane was half-maximal a t (ATPI ADP),,,, ratios of = 10 in the intact perfused rat heart prep- aration."'"

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 per~xida t ion ." '~~ 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 cardiomyopa- thy 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 single- strand 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.

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['271 or 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 SOD activities was unable to grow on minimal glucose me- d i ~ 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 . [ ' ~ ' l The oxyR regulon controls a global response to a DNA 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 SOD 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. In 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 dif- ferentiation 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 DNA methylation (cf. I137, 1381). In experiments to decrease DNA methylation at the cytosine residues by treatment of animals with the drug analog, 5-azacytidine, the content of cytochromes P-450 and b5 was found to whereas the content of several 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) in oxyR were assayed for spontaneous mutations in the His reversion assay (Ames test). For description of phenotype, zones of inhibition by HZOZ on the agar plates are also shown 11341.

Strain Description Colonies Zone of inhibition per plate [mml

TA 41 I7 oxyR + 33 ?6 18.0 TA 4118 oxyRl 12+4 12.5 TA 41 I9 oxyA2 1408f L60 33 5

Angew Chem In1 Ed. Engl 25 (1986) 1058-1071 1067

I I 1 0 25 50

5-Azacytidine(rng/kg body wt) - 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 DNA (cytosine-5) methyl transferase (after [ 1401).

(Fig. 18). Recently, NADPH : quinone oxidoreductase was cloned and was described to be hypometh- ylated 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).

7.5. Synthetic Antioxidants

Numerous drugs serving as antioxidants have been syn- thesized 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.

1 R1 R2 R2

a - T CH3 CH3

8-T H CH3

R' 7 - T CH3 H

OH

OH 11

Fig. 19. Some antioxidant inhibitors of peroxidation reactions. '7: a- bis S- tocopherol; 8: BHA=2-1er/-butyl-4-methoxyphenol; 9 : BHT=2.6-di-tert- butyl-4~methylphenol: 10: propyl gallate; I 1 : NDGA= nordihydroguaiar- etic acid.

7.6. Ebselen, a Novel Organoselenium Compound

This synthetic organoselenium compound, 2-phenyl- 1,2- benzoselenazol-3(2H)-one (ebselen), has been found to exhibit antioxidant capacity.['431 In 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 n- pentane or the production of thiobarbiturate-reactive material.'941 In addition to this antioxidant activity, the compound acts catalytically in the GSH peroxidase reac-

2GSH + ROOH - GSSG i- ROH + H 2 0

Ebselen Sulfur onologue

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-Fe- induced cell damage in control cells, whereas cells pre- viously 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."4sh1 Recently, 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

I068 Angew. Chem. In,. Ed. Engl. 25 (1986) 1058-1071

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

7.7. The Selenoenzyrne GSH Peroxidase

This enzyme has recently been sequenced;"52b' its structure has been studied by X-ray The gene from the mouse was cloned and it was found that the selenocysteine in the active site is encoded by the termina- tion 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 ism~tase[ '~ 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 free- radical involvement in ischemic myocardial1i561 or 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 endothelium- derived 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 al- ready 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['641 re- quire 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 trap- ping.""" Selenite and Vitamin E were found to inhibit ra- diogenic and chemically induced cell transformation in vi- troilhhl but, 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

lntracellular

Reversible loss] GSSG Protein-SSG Acy I- S G "Bound" GS H

I Amino a c i d s -GSH' I

Excretion {kidney) ]

GSH-Efflux(Sinusoida1 space + bile) GSSG-Efflux (Bile) Amino acids - Complexes with heavy meta ls (bile)

I \Hydrolysis] (Kidney and epithelial organs1

Fig. 21. Processes (chemical and translocation) that influence the intra- hepatic 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 availa- b1e1169,1701 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 nox- ious challenge against which the organism must defend it- self. As was recently pointed numerous physiologi- cally important chemical reactions occur in cells via reactive oxygen species and free radical reactions. These include the vital functions of macro phage^"'^^ and neutro- p 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 far- reaching pathophysiological consequences, for example clinical states such as ischemia and

The work carried out in the author's laboratory was sup- ported by the Deutsche Forschungsgerneinschaft (Schwer- punktprogramm "Mechanismen toxischer Wirkungen uon Fremdstoffen '7 , by the National Foundation for Cancer Re- search, 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- work- ers in the laboratory whose contributions are cited in the text.

Received. April 7. 1986; [A 599 IE]

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Biochemistry of Oxidative Stress

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