Mechanisms of Ageing and Development, 51 (1990)283--297

283

ElsevierScientificPublishers Ireland Ltd.

GLUTATHIONE PEROXIDASE, SUPEROXIDE DISMUTASE, AND C A T A L A S E I N A C T I V A T I O N BY P E R O X I D E S AND OXYGEN DERIVED FREE RADICALS

E T I E N N E PIGEOLET*'**, P H I L I P P E CORBISIER**, A N D P ~ E HOUBION, DOMINIQUE LAMBERT, CARINE MICHIELS~', MARTINE RAES~, MARIE-DENISE ZACHARY** and JOSE R E M A C L E Laboratoire de Biochimie Ceilulaire, Facultes Universitaires N. D. de la Paix, 61 rue de Bruxelles, B-~O00 Namur (Belgium)

(Received March 6th, 1989) (Revision received May 23rd, 1989) SUMMARY Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase are the most important enzymes of the cell antioxidant defense system. However, these molecules are themselves susceptible to oxidation. The aim of this work was to estimate to what extent this system could be inactivated by its own substrates. We tested the effect of hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and hydroxyl and superoxide radicals on GPX, SOD and catalase. For GPX, a 50070 inactivation was observed at 10-~ M (30 min, 37 °C) for hydrogen peroxide, 3 x 10-4 M (15 rain, 37°C) for cumene hydroperoxide and 5 x 10-5 M (11 min, 37°C) for tbutyl hydroperoxide. Unlike the hydroxyl radicals, superoxide anions did not inactivate this enzyme. Cataiase was inactivated by hydroxyl radicals and by superoxide anions but organic peroxides had no effect. SOD was inactivated by 50070 by hydrogen peroxide at 4 x 10-4 M (20 min, 37°C), but organic peroxides and hydroxyl radicals were ineffective on this enzyme. Since the three enzymes of the antioxidant system are susceptible to at least one of the oxidative reactive molecules, in the case o f high oxidative stresses such an inhibition could take place, leading to an irreversible autocatalytical process in which the production rate of the oxidants will continuously increase, leading to cell death. K e y words: Antioxidant enzymes; Oxygen derived free radicals; Peroxides; Inactiva-

tion; Critical threshold *To whom reprint requestsshould be addressed. **Fellowof the I.R.S.I.A. t Researchassistant of the F.N.R.S. :[:Researchassociateof the F.N.R.S. 0047-6374/90/$03.50 Printed and Published in Ireland

© 1990ElsevierScientificPublishers Ireland Ltd.

284

INTRODUCTION

Living cells are in a steady state which is maintained by regulatory processes, mostly involving enzyme regulation. However, when submitted to excessive stresses, cells degenerate and die. This has been well exemplified in the case of excessive free radical production [ 1--4]. Free radicals are produced continuously, mainly by mitochondrial electron transport, autooxidation of molecules such as thiol molecules, quinones, etc., enzymes such as xanthine oxidase, aldehyde oxidase etc., and microsomal oxidations [5]. Oxidative stress can become harmful in some conditions such as ischaemia/reperfusion [2,6], inflammation [7], xenobiotic metabolism [8] and hyperoxia; in such conditions, excessive oxygen reactive species can be produced, damaging all biological molecules [5]. Cells are, however, well protected by antioxidant enzymes like glutathione peroxidase (GPX), superoxide dismutases (SOD) and catalase and by many antioxidants such as ascorbic acid and a-tocopherol. It has been proposed (Remacle, J., et al., unpublished) that during excessive production of free radicals the defence system of the cells could be overwhelmed so that a threshold of free radical production would exist above which an irreversible process of degradation would occur. This threshold of error accumulation could be decreased or increased by acting on the activity of the antioxidant enzymes. In this study, we wanted to perform a systematic investigation on the effect of three reactive oxygen species, the superoxide and hydroxyl radicals and some hydroperoxides, in order to have comparative values of their inactivation potency on the three antioxidant enzymes. In this way, a general view of the interrelationships between these three enzymes and their metabolites could be built up, leading to the idea that a threshold of free radical production can be reached, above which the antioxidant enzymes would become less active leading to an irreversible increase in free radical production. MATERIALS AND METHODS

Hydrogen peroxide (H202) , c u m e n e hydroperoxide (CUOOH), t-butyl hydroperoxide (TBHP), ascorbate, EDTA, FeSO4, phosphate, triethanolamine hydrochloride (TRAP), cyanide and iodoacetate were purchased from Merck AG (Darmstadt, F.R.G.). Catalase (bovine liver), Cu/Zn superoxide dismutase (bovine erythrocytes), Mn superoxide dismutase (E. coh), xanthine oxidase (grades I and III from butter milk), GSH, NADPH, glutathione reductase (type IV from yeast), epinephrine, xanthine and 2-thiobarbituric acid were from Sigma Chemicals (St Louis, MO., USA). Before use, catalase was desalted in order to remove thymol which is present in commercial preparations. For this purpose 25/~l of catalase was diluted into 0.15 ml of 50 mM phosphate buffer (pH 7.4) containing 0.1 mM EDTA. The solution was fractionated on a Sephadex G25 column (25 X 0.6 cm) equilibrated in the same buffer. The protein fraction of the void volume was used. Acetaldehyde was from Fluka Chemie (Buchs, Switzerland), desferal from Ciba Geigy (Groot

285 Bijgaarden, Belgium), mannitol from UCB (Brussels, Belgium) and trichloroacetic acid from Farmitalia, Carlo Erba (Milan, Italy). GPX was purified from bovine erythrocytes following the method of Grossmann and Wendel [9].

Enzyme assays GPX activity was measured by using the consecutive glutathione reductase reaction. This reaction was monitored by oxidation of NADPH followed at 366 nm [10]. The assay mixture contained 70/~M NADPH, 0.9 mM GSH, 0.4 mM TBHP, 2 /~1 GSH reductase (500 U (Sigma)/2.3 ml) in a final volume of 0.6 ml of phosphate buffer (60 mM KH2PO4 (pH 7.9), 1 mM EDTA) at 20°C. Spontaneous oxidation of GSH in this mixture was used for correction of the enzymatic reaction. SOD activity was measured by its ability to inhibit oxidation of epinephrine by the superoxide anions. To 1 ml of TRAP buffer (20 raM, pH 9.0), containing 0.25 M sucrose and 2 mM EDTA, were added successively 10/~1 of 0.1 M epinephrine, 20 ~1 of 0.1 M xanthine and 2 ~1 of xanthine oxidase (25 U (Sigma)/0.6 ml, grade I); the formation of adrenochrome was measured by the absorption difference at 480 and 575 nm, in a double wavelength spectrophotometer (Perkin Elmer, Hitachi Ltd, Tokyo, Japan). SOD solution (100/~1) was added and the decrease of the adrenochrome formation was observed. SOD activity is expressed as inhibition percentage of the adrenochrome formation. Catalase activity was measured by the disappearance rate of H202 monitored at 240 nm in a spectrophotometer Zeiss PM6 (F.R.G.) following the method of Aebi [ll]. Incubation o f GPX, SOD and catalase with H20 z, CUOOH and TBHP Purified enzymes were diluted in 50 mM phosphate buffer and 1 mM EDTA (pH 7.4) in the presence or absence of these molecules at 10-3 M. This solution was incubated at 37°C and residual activity was measured as a function of incubation time. When inactivation occurred, different concentrations of peroxides were tested for a fixed period of incubation time. The incubated solutions were diluted before the enzyme assays in order to avoid interference of incubated peroxides with assay procedures. Incubation o f GPX, SOD and catalase with hydroxyl radicals The system used for producing hydroxyl radicals was ascorbate/iron (Fe2÷) Ascorbate (10-2 M) and iron (5 × 10-5 M) were dissolved in 50 mM phosphate buffer (pH 7.4) and 1 mM EDTA. The production of hydroxyl radicals was assayed by the deoxyribose oxidation method described by Halliwell and Gutteridge [12]. For the incubation with hydroperoxides, enzymes were incubated in a small volume at 37 °C with ascorbate and iron and then diluted in phosphate, EDTA buffer before assay. Incubation o f GPX, SOD and catalase with superoxide anions Superoxide anions were produced by xanthine oxidase using acetaldehyde as a

286 substrate. Acetaldehyde was freshly distilled before use. The production of superoxide anions was checked by following the oxidation of epinephrine at 480 nm: in 1 ml o f 50 m M phosphate buffer (pH 7.4), 1 m M (EDTA), we added 2/~l of xanthine oxidase (grade III, 5 U (Sigma)/0.7 ml), 20/~l of 100 m M or 250 m M acetaldehyde (final concentration o f 2 m M or 5 mM) for different quantitative production of superoxide radicals and 10/al o f 0.1 M epinephrine. Enzymes solutions were incubated at 37°C (SOD, G P X ) or at 23 °C (catalase) in small volumes (in order to dilute before assay) with the same xanthine oxidase and acetaldehyde final concentrations. Inhibition o f G P X by iodoacetate and cyanide: in order to test the redox state of G P X , we incubated G P X with 1 m M iodoacetate or with 10 m M K C N following the method of Kraus et al. [13]. RESULTS

Production o f hydroxyl radicals and superoxide anions Halliwell and Gutteridge [12] described a simple colorimetric assay for the detection of hydroxyl radicals which react with deoxyribose to form a thiobarhituric acid reactive substance. This method was used to check the production of hydroxyl radicals in the ascorbate/iron system. A time-dependent accumulation of chromogen formed between thiobarhituric acid and deoxyribose degradation products in the presence o f iron and ascorbate is shown in Fig. 1. The production is stable for at least 90 min. Evidence of the involvement of hydroxyl radicals is given by the lowering of this accumulation by desferal and mannitol. The first is a strong 0,4

0,3

~O 0,2

0,1

0,0 . ~ o

|

5o

1 oo

15o

Time (min) Fig. 1. Production of hydroxyl radicals. Deoxyriboseis incubated for increasing periods of time with 5 X 10-5 M iron and 10-2 M ascorbate. Hydroxyl radicals produced react with deoxyribose which can in turn react with 2-thiobarhituric acid forming a chromogen measured at 532 nm. Ascorbate + iron ( • ), ascorbate + iron + 10-3M desferioxamine([]), ascorbate + iron + mannitol 0.1 M (In).

287 iron chelator, preventing the participation o f this metal ion in the Fenton reaction; the second is a potent scavenger o f hydroxyl radicals. The time dependent oxidation o f epinephrine by superoxide anions produced by xanthine oxidase and acetaldehyde is shown in Fig. 2. Acetaldehyde was used as a substrate rather than xanthine because o f its slow utilization by xanthine oxidase compared to xanthine, allowing a slower production of superoxide radicals. Addition o f 25 U of Mn SOD (Sigma) abolished adrenochrome formation while inactive SOD did not.

Glutathione peroxidase G P X was incubated for 30 min with increasing concentrations of H202. The residual activity o f the enzyme was then measured. The residual activity of the control (56 _+ 5.8~/0 o f initial activity) was considered as 100%. Residual activities o f the tests were calculated with regard to the control (Fig. 3). By extrapolation o f the curve, the concentration o f H20 2 inducing a 50~0 inactivation of the enzyme was roughly estimated at 10-1 M, under these conditions. Since the inactivation of G P X in the presence o f 10-3 M T B H P or C U O O H was much faster, we used a shorter time of incubation for these two compounds. The results show a 50% inactivation o f the enzyme with T B H P at 5 × 10-5 M and C U O O H at 3 × 10 -4 M. Residual activities o f the controls were o f 67.3 ± 8.7~70 o f the initial activity for the T B H P experiment and of 72.1 _+ 1.1 ~70o f the initial activity for the C U O O H experiment. Figure 4 shows the time-dependent inactivation rate of G P X in the presence of the ascorbate/iron system or with iron alone (control). There is a clear inactivation

0.20I 0.15 i 0,10 0,05 0,00

0

10

20

30

40

50

Time (rain) Fig. 2. Production of sdperoxide anions. Xanthine oxidase/acetaldehyde reaction produces superoxide anions which oxidize epinephrine forming adrenochrome absorbing at 480 nm. Xanthine oxidase 4- 2 mM acetaldehyde (e), xanthine oxidase 4- 5 mM acetaldehyde (A), 5 mM acetaldehyde without xanthine oxidase (D), 5 mM acetaldehyde + xanthine oxidase + Mn SOD ( , ) .

288

100"

80 v

60

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20 O

l

-9

-7

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I

-5

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Log. [ hydroperoxides] Fig. 3. Effect o f peroxides on G P X activity. G P X was incubated at 37°C with increasing concentrations o f H~O 2 (Tq), C U O O H ( m ) a n d T B H P ( e ) . After the incubation period o f 30 min, 15 min, and 11 min for H , O 2, C U O O H a n d T B H P , respectively, the residual activities o f G P X were measured. The results are expressed as % o f the G P X incubated without peroxides.

100

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60

40 n20

0 0

i

I

I

l

30

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120

Time (rain) Fig. 4. Activity in the course o f time of G P X incubated at 37°C with 5 x 10-5 M iron alone ( e ) , with 5 x 10-s M iron and 10-2 ascorbate ( I ) ; with 5 x 10-5 M iron, 10-2 m ascorbate and 100 m M mannitol

(c]).

289

effect of hydroxyl radicals, which is largely counteracted by 100 mM mannitol. The slight difference from the control curve could be explained by the fact that even with 100 mM mannitol, some hydroxyl radicals escape scavenging (Fig. 1). G P X was also exposed to the xanthine oxidase/acetaldehyde reaction in order to test the effect of the superoxide anions, and the time-dependent inactivation o f the enzyme was measured. Figure 5 shows that incubation with xanthine oxidase and 5 mM acetaldehyde did not inactivate the enzyme compared with the control. In the same way, when using 2 mM and 10 mM acetaldehyde with xanthine oxidase, we could not demonstrate any inactivating effect of the superoxide anions (not shown). In order to test the redox state of GPX, we measured the inhibitory effect of iodoacetate and cyanide following the method of Kraus et al. [13]. We found with both molecules the same inhibition reaching 75070 in regard with the control incubated without the two inhibitors. Superoxide dismutase Figure 6a shows the effect of H 2 0 2 o n C u / Z n SOD. The enzyme was incubated at 37°C for 20 min with increasing amounts of H 2 0 2 . I n these conditions, 50°70 inactivation was reached at 4 X 10-4 M H 2 0 2 . Organic peroxides like T B H P and C U O O H did not seem to affect the enzyme, since no inactivation was observed in the same conditions, even at 10-3 M. Longer incubation periods up to 2 h did not give any further inactivation (not shown). In the same way the ascorbate/iron system producing hydroxyl radicals did not inactivate the enzyme (Fig. 7). Superoxide anions were not tested as they are the substrate o f SOD.

100

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Time (rain) Fig. 5. Effect of superoxide anions on GPX activity. GPX was incubated with acetaldehyde and xanthine oxidas¢ at 37°C for increasing times. The residual activity was then measured. Xanthine oxidase alone ( • ) ; xanthin¢ oxidas¢ and 5 mM acetaldehyde ([]).

290 120 100 80

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Log. [ hydroperoxides] Fig. 6. Residual activity of SOD incubated at 37°C for 20 rain with increasing concentrations of H202 ([~), relative to the enzyme incubated without H202 ( . . . . ). Two other hydroperoxides, TBHP ( • ) and CUOOH (O) were also tested at 10-3 M.

Catalase C a t a l a s e was e x p o s e d t o o r g a n i c peroxides: Figures 8a an d 8b show t h a t they h ad no i n a c t i v a t i n g effect o n catalase. H o w e v e r , w h e n catalase was i n c u b a t e d with a s c o r b a t e a n d i r o n (Fig. 9) a r a p i d i n a c t i v a t i o n was o b s e r v e d , with loss o f 50°70 o f the initial activity. It m u s t be n o t e d , h o w e v e r , t h a t m a n n i t o l at 100 m M h ad no p r o t e c t i n g e f f e c t (not s h o w n ) . 120 100 80 .> 60 40 Q: 20 0 0

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Time (rain) Fig. 7. Activity in the course of time of SOD incubated at 37 °C with 5 X 10-5 M iron alone ( • ) or with 5 X 10-5 M iron and 10-2 M ascorbate (El).

291

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Time (min) Fig. 8. (A) Activity in the course of time of catalase incubated at 37°C alone (I]]) or with 10-~ M TBHP (O). (B) Activity in the course of time of catalase incubated at 37°C alone (--[]--) or with 10-3 M CUOOH (- - • - -).

I n c u b a t i o n o f c a t a l a s e with the x a n t h i n e o x i d a s e / 2 m M a c e t a l d e h y d e system is p r e s e n t e d in Fig. 10. A r a p i d b u t slight i n a c t i v a t i o n is observed. This i n a c t i v a t i o n is i n d u c e d b y the s u p e r o x i d e a n i o n s since the a d d i t i o n o f active M n S O D (25 U Sigma) to the system, prevents the e n z y m e i n a c t i v a t i o n . T h e curve o b t a i n e d with M n S O D i n a c t i v a t e d b y h e a t at 95 °C f o r 60 m i n is the s a m e as the curve o b t a i n e d w i t h o u t M n S O D . Surprisingly, t h e C u / Z n S O D c o u l d n o t p r o t e c t catalase. A s c h e m a t i c s u m m a r y o f all these results is p r e s e n t e d in T a b l e I.

292

120 100 A

80" o:~

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40 20 0

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Time (min) Fig. 9. Activity in the course of time of catalase incubated at 37°C with 5 × 10-~ M iron alone ( e ) or with 5 x 10-5 M iron a n d 10-2 M ascorbate ([7).

.>

I/)

4O

er 2O

0 0

10

20

30

40

50

60

70

Time (rain) Fig. 10. Effect o f superoxide anions on catalase activity. Catalase was incubated with 5 m M acetaldehyde and xanthine oxidase at 23 °C for increasing times. The residual activity was then measured. Catalase with 5 m M acetaldehyde alone ( 1 ) ; with 5 m M acetaldehyde, xanthine oxidase and inactive Mn SOD ( e ) ; with 5 m M acetaldehyde, xanthine oxidase a n d active Mn SOD ([]).

293

TABLE I SUMMARY OF T H E RESULTS OF A N T I O X I D A N T ENZYME I N A C T I V A T I O N BY THE T H R E E PEROXIDES, SUPEROXIDE ANIONS A N D HYDROXYL RADICALS The figures, where given, are the concentrations of oxidants and incubation times at 37 °C, which result in 50% inactivation of the enzymes with regard to the controls. ( + ) Inactivation without precise quantification, ( - ), no inactivation,(n.t.) not tested.

TBHP

CUOOH

Hydrogen peroxide

Superoxide anion

Hydroxyl radical

15 rain 3 × 10-4M -

+

n.t.

-

CAT

-

-

30 min 10-~M 20 min 4 × 10-4M n.t.

-

SOD

11 min 5 × 10-SM -

+

+

GPX

DISCUSSION

The susceptibility of the main cellular antioxidant enzymes, GPX, SOD and catalase to different oxidant metabolites was tested. Inactivation of GPX in the presence of hydroperoxides revealed the sensitivity of this enzyme to its own substrates. Indeed, we can see that organic peroxides are very potent inactivators, since 11 min at 5 × 10-5 M of TBHP and 15 min at 3 x 10.4 M of CUOOH are sufficient to inactivate 50% of the enzyme. Results show that TBHP is the most effective molecule, followed by CUOOH and H202. There is no strict correlation between inactivation ability of these molecules and their size (H202 < TBHP < CUOOH), neither is there a strict correlation between this effectiveness and the hydrophobicity of the molecules (H202 < TBHP < CUOOH). But there could be a balance between these two characteristics leading to their inactivating ability. The more hydrophobic and the smaller, the more potent inactivator would be the molecule. Condell and Tappel [14] also reported an inactivating effect of H202, though slightly weaker, and of linoleic hydroperoxide. Blum and Fridovich [15] also report that the inactivation of GPX by TBHP is an irreversible process. The effect of peroxides on GPX by the way of free radical production cannot be ruled out since traces of iron are present in the buffer, even if at very low concentrations. However, this effect must be very small since at neutral pH the reaction of peroxides with metals is very slow [16] and we observed effects of hydroxyl radicals only at high concentrations. It could appear at first that incubation of GPX with hydroperoxides alone is far from in vivo conditions, where the enzyme is surrounded by glutathione molecules and consequently destroys these peroxides. In conditions of oxidative stress, however, when the concentration of antioxidant molecules such as glutathione is decreased, the inactivating effect of these peroxides could become crucial and explain the irreversible damage caused by the free radical derivatives in such pathological situations.

294 It is well known that hydroxyl radicals are very active molecules reacting with all types of biological molecules. Epp e t al. [17] have shown that G P X has a tryptophan residue in its active site. This tryptophan is probably involved in the binding o f the hydrophobic peroxides and is particularly sensitive to hydroxyl radicals. However, due to the high reactivity of hydroxyl radicals with many other amino acids, other mechanisms o f G P X inactivation could be involved. In the conditions where superoxide anions were produced, our results showed that this oxygen reactive species does not inactivate GPX. This is in accordance with Searle et al. [18] but in contradiction with the results obtained by Michelson and Maral [19] and Blum and Fridovich [15]. In their work, Blum and Fridovich [15] found that the enzyme was susceptible to superoxide anions when in the reduced form but insensitive when in the oxidized form. We therefore wanted to demonstrate the redox state o f the G P X in the conditions o f work. The discrimination between the oxidized or the reduced form is usually done based on the inhibition by CN- or iodoacetate [ 13,20]. Unfortunately, there was no clear difference in the inhibition of these two molecules on the GPX. Such a situation is typical of a special oxidized form of G P X which has been stored for long periods o f time [13]. This is the case in these experiments and we can assume that also in this case this oxidized form of G P X is not susceptible to superoxide anions. The results obtained for SOD are in accordance with other workers [21--25]. H202 inactivates SOD. Blech and Borders [22] investigated the mechanism of H202 inactivation o f C u / Z n SOD and proposed that in fact the reactive species is the H O E anion leading to one histidine oxidation. SOD is, however, insensitive to organic hydroperoxides and hydroxyl radicals. One could suspect SOD to be susceptible to these radicals as it contains Cu which can replace iron in the Fenton reaction [4]. These authors also report the involvement of Cu in the oxidation o f ascorbate producing hydrogen peroxide and hydroxyl radicals. Ascorbate/Cu mixtures inactivate many proteins, probably by formation o f hydroxyl radicals. It is possible that SOD could be slightly damaged in the ascorbate/iron system without loss of activity. Indeed, SOD submitted to hydroxyl radicals is more susceptible to proteolysis than native SOD [26]. The active site of catalase is not easily accessible to solvents. It is located at the end of a narrow channel and although this channel is lined with hydrophobic amino acids, the narrowness probably does not allow organic peroxides to reach the catalytic site [27, 28]. It is therefore not surprising to observe no effect of these peroxides on the enzyme. Inactivation of catalase by ascorbate alone or in the presence of a metal like Cu has already been reported by several workers [29--33]. Davison et al. [34] studied the mechanism of this inhibition. They concluded that semidehydroascorbate is the irreversible inhibitor acting probably on a histidine residue. They do not exclude, however, the possibility of a site specific hydroxyl radical production with a possible role for semidehydroascorbate as a Fenton electron donor. They do not consider the

295

possibility of the formation of hydroxyl radicals resulting from the Fenton reaction with iron directly present in the catalytic site. This possibility was raised by the results of Kono and Fridovich [35] since the ferric center of the catalytic site can be reduced to a ferro oxy compound by the superoxide radicals. In this situation the absence of protection by mannitol would be understandable since the deleterious effect of the radical would be worked out immediately at the site of production in the active site, well isolated from the surrounding medium. The same mechanism would also explain the inactivation obtained with the superoxide anions [35]. This inactivation, already mentioned by other workers [35,36], was slight, probably because of the low accessibility of the active site. The fact that only the Mn SOD could be effective for preventing this inactivation, contrary to the Cu/Zn SOD, was a very surprising observation which can perhaps be explained by the peculiar mechanism of inactivation of the active site. Kono and Fridovich [35] also reported the same observation; they suggested that Mn SOD could be active because of its resistance to H202. The results summarized in Table I clearly show that the three most important enzymes of the cellular antioxidant defense system are inactivated by at least one of the oxygen reactive species. These three enzymes protect each other from inactivation either directly or indirectly. Indeed, SOD by destroying the superoxide anions lowers the reduction of oxidized iron and so reduces the possibility of production of hydroxyl radicals, which in turn are important triggers of the lipid peroxidation. The products of this lipid peroxidation, the lipid peroxides, are also potent inactivators of GPX. These conclusions stress the importance of iron in oxidative damages. Iron availability is very low in normal conditions, but it can be enhanced drastically from its storage pools, such as ferritin and transferrin, during oxidative stress. [26,37--40]. SOD is itself protected by GPX and eatalase, which remove H202. The latter enzyme, sensitive to hydroxyl and superoxide radicals, could also be protected by SOD. The relevance of these data to physiological conditions is difficult to assess because of the differences in the medium composition in which the enzymes are located, the source of oxidant production and the distance to the active site of the enzymes. It is clear, however, that these inhibitions do not take place in normal conditions since the amounts of these reactive molecules are very low. It has been reported that the concentrations of O 3 and H202 could be, respectively, in the order of 10-H to 10-t2 M and 10-9 M [41] in the cells and are thus far below the concentrations used in the in vitro experiments described herein our experimental conditions, the O 3 production is about 2.1 x 10-5 M with 2 mM acetaldehyde after 30 min and about 3.2 x 10-s M with 5 mM acetaldehyde after 30 min). However, in pathological situations, as for example in the case of reperfusion after ischaemia, the amounts of these free radicals can increase drastically and what we postulate here is that above a critical level of production, these free radicals will inactivate the antioxidant enzymes, leading to an autocatalytical irreversible process where production rates of the oxidants will increase exponentialy.

296 REFERENCES l

2 3 4 5 6 7 8 9

10 11 12

13 14

15 16 17 18 19

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