ZN-SOD for cell survival against oxidative stress

Free Radical Biology & Medicme,Vol. 17. No. 3. pp. 235-248. 1994 Copyright ,g, 1994 ElsevierScience Ltd Printed m the USA All rights reserved 0891-5849/94 $6.00 -¢- .00

Pergamon

0891-5849(94)E0056-O

Review Article IMPORTANCE OF SE-GLUTATHIONE PEROXIDASE, CATALASE, AND CU/ZN-SOD FOR CELL SURVIVAL AGAINST OXIDATIVE STRESS CARINE MICHIELS, 1VIARTINE RAES, OLIVIER TOUSSAINT. and Josl~ REMACLE Laboratoire de Biochimie Cellulaire, Facult~s Universitaires Notre Dame de la Paix, NAMUR Belgium

IReceived 3 August 1993; Revised 7 December 1993; Accepted 17 December 1993) Abstract--Eukao'otic cells have to constantly cope with highly reactive oxygen-derived free radicals. Their defense against these free radicals is achiexed by natural antioxidant molecules but also by antioxidant enzymes. In this paper, we review some of the data comparing the efficiency of three different antioxidant enzymes: Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase. and selenium-glutathione peroxidase. We perform our comparison on one experimental model (human fibroblasts) where the activities of these three antioxidant enzymes have been modulated inside the cells, and the repercusmon of these changes was investigated in different conditions. We also focus our attention on the protecting role of selenium-glutathione peroxidase. because this enzyme is veD' rarely studied due to the difficulties linked to its biochemical properties. These studies evidenced that all three anttoxtdant enzymes give protection for the cells. They show a high efficiency for selenium-glutathione peroxidase and emphasize the fact that each enzyme has a specific as well as an irreplaceable function. They are all necessary, for the surs ival of the cell even in normal conditions. In addition, these three enzymes act in a cooperative or synergistic way to ensure a global cell protection. Howexer, optimal protection is achieved only when an appropriate balance between the activities of these enzymes is maintained. Interpretation of the deleterious effects of free radicals has to be analyzed not only as a function of the amount of free radicals produced but also relative to the efficiency and to the activities of these enzymatic and chemical antioxidant systems. The threshold of protection can indeed vao' dramatically as a function of the level of activity of these enzymes. Keywords--Selenium-glutathione peroxidase. Cu/Zn-SOD. Catalase. Oxidative stress, Microinjection. Free radicals

INTRODUCTION

in r e s p i r a t i o n g i v e n its h i g h e l e c t r o c h e m i c a l p o t e n t i a l . H o w e v e r , b e c a u s e o f its e l e c t r o n i c s t r u c t u r e , t h e r e d u c -

O x y g e n s e r v e s as the o x i d a t i v e m o l e c u l e in all a e r o b i c o r g a n i s m s b e c a u s e it a l l o w s a h i g h e n e r g y p r o d u c t i o n

tion o f o x y g e n is a c h i e v e d t h r o u g h s i n g l e e l e c t r o n

Address correspondence to: Carine Michiels. Laboratoire de Biochlmie Cellulaire, Facultes Universitaires Notre Dame de la Palx. 61 rue de Bruxelles. 5000 NAMUR, Belgium. Carine Michiels got her PhD in 1989 at the University of Namur under the direction of Prof J. Remacle, with the highest honours. In 1990. she received a Young Investigator A~ard of the SFRR International and in 1993. a Fulbright grant to work in Dr Ltbby's laboratoD' at the Brigham and Women's Hospital. Boston. She is currently interested in the effect of hypoxia on the endothelial cells and their interactions with neutrophils and smooth muscle cells. Martine Raes got her PhD m 1983. at the University of Namur. in the laboratoD' of Cellular Biochemistry of Prof. J. Remacle, with the h~ghest honours. In 1986. she received a Distinguished Young Leadership Award (USA and UKL She is presently a permanent Research Associate of the National Fund for Scientific Research. invol~ed in cell aging and in cell activation in culture by cytokines. oxidative stresses, and biomaterials. Olivier Toussamt obtained his PhD in 1992 and the prize Rent de Cooman 1991-1993 for his research on free radical cellular toxicity and on accelerated cellular aging under different stresses, including oxldatixe stresses He is mostly interested in cellular aging

cals. It is t h u s h i g h l y toxic: e x p o s e d to a p u r e o x y g e n a t m o s p h e r e , all o r g a n i s m s d i e ) O n l y a f e w d e c a d e s

t r a n s p o r t s t e p s that l e a d to the p r o d u c t i o n o f free radi-

a g o , G e r s h m a n et al., 2 t h e n M c C o r d a n d F r i d o v i c h , -~ d i s c o v e r e d s u p e r o x i d e a n i o n a n d its i n v o l v e m e n t in o x y g e n toxicity. Now, different o x y g e n - d e r i v e d free

and the importance of the energetic factors for the resistance of cells to stresses like free radicals. Jos~ Remade got his PhD in Biochemistry at the University of Louvain under the direction of Profs. de Duve, Beaufays, and Trouet. He then did postdoctoral research at UCSD with Prof. J. Singer and worked recently at UMBC with Prof. G. Rao on the importance of free radicals as secondary messengers. He is now Professor at the Facult~s Universitaires of Namur. He is mostly concerned by the understanding of cellular aging and its relationship with stresses like free radicals. He developed a model of free radical production and elimination which shows interactions between these two opposite processes and the presence of stable and unstable domains. 235

236

C. MICHIELS e t al

radicals have been characterized as well as the mechanisms leading to cell death during oxidative stress.

OXYGEN-DERIVED FREE RADICALS

The formation and the sources of free radicals have been the subject of many reviews and will be just mentioned here. 4 The different oxygen-derived free radicals are formed by subsequent univalent reductions of molecular oxygen: superoxide anion (O-"'-~, hydrogen peroxide (H_~O_,, which is not a free radical1, hydroxyl radical CHO'I. and then water (H_,O). During respiration, oxygen is directly reduced into water by cytochrome oxidase: only a few percent ( 3 - 5 % ) of the oxygen used leads to superoxide anion formation due to leakage of electrons on the electron transport chain in mitochondria. ~ In addition. O_,'- and H_,O_, can be produced in the cells b~ various mechanisms: enzymatic activities (xanthine oxidase, cytochrome P450. NADPH oxidase, etc.), autooxidation of small molecules (catecholamines. flavines, etc.), or redox c~cle of xenobiotics (paraquat, nitrofurantoin, adriamycine. etc.1." HO" is formed b.~ the H a b e r - W e i s s reaction between H_,O_, and O-"'-. This reaction is catalyzed b 3 transition metals (mainl.~ iron)in the so-called Fenton reaction." Due to its reactivit}, this free radical reacts instantaneously with all cellular components and is therefore vers. toxic for the cell. All cellular components can react with ox}gen-derived free radicals at the level of unsaturated bonds and thiol groups (for a reviex~, see ref. 6). In proteins. some amino acids are ver}' sensitive to these attacks. inducing enzymatic activit} alterations or changes in conformation? HO" can also induce protein crosslinkings and cleave amino acid bonds, leading to fragmentation of the macromolecules." Nucleic acids are also a target for free radical attacks generating DNA strand breaks or base modifications, leading to point mutations, s However, the most important damage caused by these reactive species is lipid peroxidation. Polyunsaturated fatty acids are very sensitive to free radical attacks: when initiated, this reaction can be propagated by a chain reaction leading to the generation of lipid peroxides. " ~ Afterwards, these compounds can be cleaved into different molecules like aldehydes, with 4-hydroxynonenal being predominant. ~-" and they are all reD' toxic for the cells. ~3 These events induce profound changes in the structure of biological membranes, altering their different functions. ~ It is now thought that not only lipid peroxidation but also alterations of nucleic acids and proteins by free radicals are responsible for cell death during oxidative stress.'5

ANTIOXIDANT SYSTEMS

To defend themseh'es against these free radical attacks, cells have developed, during their evolution, different antioxidant systems: there are low molecular weight antioxidant molecules like ot-tocopherol, t6 ascorbic acid, ~ glutathione. ~s etc., and antioxidant enzymes: superoxide dismutases (SODs), catalase, and glutathione peroxidases) In physiological conditions, these defense mechanisms maintain a low stead} state concentration of free radicals in the cell and their activities are very precisely regulated. ~'~ SOD activity was discovered by McCord and Fridovich in 196¢: it dismutates two O2"- into H20_, and O~ (reaction 1 ) (for a review, see ref. 201. Afterwards. the}' showed that this enzyme is necessary to maintain life in aerobic conditions. 2j Three distinct enzymes have been described with the same kinetic properties: one containing iron in its active site found in prokaryotes, one with manganese in prokaryotes and eukats.'otic mitochondria, and one with copper and zinc (Cu/ Zn-SOD) in the cytoplasm of eukaryotic cells.:-" An extracellular Cu/Zn-SOD immunoiogically distinct from the classical cytosolic Cu/Zn-SOD has also been described. -"-~This enzyme seems to be the first line of defense against oxygen-derived free radicals and can be rapidly induced in some conditions when cells or organisms are exposed to an oxidative stress. -'~-'5 Catalase is a ubiquitous enzyme found in all known organisms. In eukaryotic cells, it is present in peroxisomes. -'~ This enzyme contains a heine in its active site responsible for its catalytic activity: t ~ o H_,O_, are transformed into two H20 and O_, Ireaction 21.-'" As for superoxide dismutase, this enzyme can be induced in some conditions by exposure of cells or organisms to oxidative stresses. -'~ Glutathione peroxidase is a selenoprotein (Se-Gpx) (for a review, see ref. 28). It is a tetrameric enzyme with four identical subunits of around 22 kD. each of them containing one atom of selenium as a selenocysteine invoh'ed in the catalytic activity. -'9 Sixty to seventy-five percent of this enzyme activity, is found in the cytoplasm of eukars'otic cells and 2 5 - 4 0 % in the mitochondria. *° Chambers et al. ~ have demonstrated that the selenocysteine was coded b~ a stop codon (UGAI in the mRNA of Se-Gpx. This Opal codon is recognized by a special tRNA. which is first charged with serine: serine is then converted to selenocysteine. This selenoamino acid is thus incorporated cotranslationally into the polypeptide Se-Gpx subunit chains.;" Selenium deficienc} in vivo or in vitro leads to dramatic decreases in Gpx activity. Gpx reduces lipidic or nonlipidic hydroperoxides as well as H202 while oxidizing two molecules of gluta-

Role of glutathione peroxidase, catalase, and SOD

thione (GSH) (reaction 3). It is very, specific for the H donor (GSH) but much less specific for the substrate. The catalytic mechanism is a ter-uni ping pong one, the reaction rate increases linearly with the substrate concentration and the enzyme is unsaturable by GSH. This kinetics is responsible for a very high rate of peroxide reduction, s3 s~ In this review, we will focus on the results obtained with the Se-Gpx. However, there exist other Gpx: some isoenzymes of GSH-S-transferase, ~` one selenium-containing Gpx in plasma which is glycosylated, s6 and one Gpx associated with membranes (PHGpx), which is able to reduce phospholipid hydroperoxides, unlike the soluble one. ~" Each of these enzymes has a particular function in the cell defense and their respective activities vary in the function of cell type. Gpx needs reduced GSH to detoxify peroxides. A high concentration of reduced GSH ( 0 . 5 - 1 0 x 10-' M) is maintained ,~sithin the cell. 3~ When oxidized. GSSG is reduced back to GSH by glutathione reductase (Gredl (reaction 4). Gred is a dimeric enzyme containing FAD in its active sites, which utilizes NADPH to reduce GSSG. ~ NADPH is regenerated from the reaction catalyzed b~ glucose-6-phosphate dehydrogenase in the hexose monophosphate shunt.

Reactions catalyzed by the d(~'erent anti¢z~idant enzymes SOD: O,'- + O_,'- + 2H + ---' H:O,_ + O.,

(1)

catalase: H_,Oz + H:O: ---, 2H,O + O:

(2)

Gpx: ROOH + 2GSH ---, ROH + H_,O + GSSG (3) Gred: GSSG + NADPH + H* --' 2GSH + NADP*

(4)

PROTECTING EFFECTS OF THE ANTIOXIDANT ENZYMES

Despite numerous reports on the activities and the properties of these different ant(oxidant enzymes, and on the fact that they play a very important role in the defense against oxygen-derived free radicals, their relative importance in the cell and their potential cooperation are still a subject of controversy. This is partly due to the heterogeneity of experimental models (in vitro or in vivo, cell types, animal species, etc.), and in the oxidative conditions used (with or without oxidative stress, the type of free radical-generating system, the location of their production, extra- or intracellular, the length of exposure, etc.). The interpretation of the results also varies according to the cellular response

237

selected to assess the protecting effects of the ant(oxidant enzymes: cell proliferation, cell survival. DNA strand breaks, tumorogenicity, etc. All these differences may lead to contradictoD' conclusions, the best example being the role of SOD. which is sometimes found protective and sometimes deleterious for the cells. ~t'

Comparison bent'een the d~fferent ant(oxidant etlT.,.Vtlles

The most conu'non experimental protocol to evaluate the protecting potential of the ant(oxidant enzymes is to expose animals or cells to a free radical generating system, then to add the different ant(oxidants and to test for their positive effect. The easiest way to perform such experiments is to add the enzymes extracellularl 3 to the cells exposed in vitro to a free radical-generating system. For example, Simon et al. ~1 have shown that catalase, but not Gpx or SOD, was able to protect human fibroblasts against free radicals generated by the acetaldehyde-xanthine oxidase system. Martin ~: also demonstrated that catalase but not SOD could inhibit nitrofurantoin-induced toxicity on lung parenchymal cells. These results are, however, diffcult to interprete because the enzyme is not in its natural location within the cells. The next step is to increase the intracellular amount of ant(oxidant enzymes using various systems, and to study the effects of this additionally supplied protection against oxidative stresses. Increased catalase activit3 by endocytosed PEG-enz.x me protected porcine endothelial cells against xanthine-xanthine oxidase-generated free radicals~: but SOD did not have any effect in this model. SOD brought into porcine endothelial cells by liposomes could protect these cells against hyperoxia. ~ By also using liposomes as carriers for the enzymes, Tanswell et al. as could protect rat fibroblasts against hyperoxia with SOD and catalase. CHO cells could be protected against paraquat by SOD, or, with less efficiency, by SOD and catalase but not by catalase alone, the enzymes being introduced into these cells by scrape-loading. ~6 Increasing Gpx activity by selenium supplementation led to an increased resistance of human endothelial cells against hyperoxia but not against paraquat. ~" Introduced by liposomes, Gpx, or catalase but not SOD could protect e%'throcytes against photohemolysis. In addition, combining SOD and catalase was not protective? ~ Because the experimental models aforementioned are vel3' different from each other, no strict comparison can be performed between the efficiency of the various ant(oxidant enzymes. In addition, due to the difficulties linked to the Gpx biochemical properties, the potential

C ]k|ICHIELS et al.

238

Table I. Comparison of the Efficienc.~ of the Three Antloxidant Enzymes When Injected Intracellularb for the Protectton of Human Fibroblasts Against Oxidative Stresses Concentrauon x~hich gave 20q, protection 2 arm 0.,

Enz3 mes Ctt/Zn-SOD Catalase Se-Gpx

1 arm Oz

5 ,¢ 10 ~ M Nttrofurantom

10 " Subunit mol/ml

Ratio to Native Concentration

10 + Subunit mol/ml

Ratio to Native Concentration

10 ~ Subumt mol/ml

Rauo to Native Concentration

6265 ° 20 3.1

035 66 fl 35

131 I e 10 08

133 33 I).09

12(~" 9.5 04

122 3.1 0 04

Soluuons containing different concentration~ of each enz.~me ~ere mlcromjected into human tibroblasts and their sur~i~al ~ a s followed ~hen e~posed to different o~idati~e stresses Gexpositlon under I or 2 atm of 95% O., or to 5 x 10 4 M mtrofurantom~. A protection percentage ~as calculated from these survival curves in comparison with the sur,m al of buffer-injected cells and the concentration of the injected solutions that gave 20% protection was calculated from the dose-response protectton curves. Native concentrations are 1.99 x 10-". 0 13 ,< 10- '~ and 0 32 x 10 ~- subumt tool/cell, respectivel3, for Cu/Zn-SOD. catalase, and Se-Gpx ° from ref. 49 from ref 50. • from ret. 51.

importance and the protecting role of this enzyme are very rarely studied. One possibility to tackle these problems is to take advantage of the microinjection technique I'~ hich allows the introduction of increasing concentrations of the antioxidant enzymes, alone or in combinations, into cells), and then to quantify the protective capacity of these enzs, mes. Increasing amounts of Cu/Zn-SOD, catalase, and Se-Gpx have been injected into human fibroblasts. The effect of such increased amounts of these enzymes in comparison to the corresponding heat-inactivated enzyme and to buffer alone, was then recorded as an increased cell resistance to oxidative stresses. Table 1 summarizes the results of three experiments obtained in different oxidative stresses: exposure to hyperoxia either under 1 or 2 arm of 95% O:. or incubation with 5 x 10 -~ M nitrofurantoin. 4~-5~ The results are expressed as the concentration of enzyme solutions that had to be injected to obtain 20% protection. These stresses induced cell death within, respectively. 6. 8. or 8 days. Each antioxidant enzyme, when injected into the cells before the oxidative stress exposure, was actually able to protect cells against this free radicalinduced toxicit.~ in a dose-dependent manner. However. their respectixe efficiency is very different: solutions containing 6250, 20. or 3.1 × 10 -9 subunit mol/ ml of, respectivel 3. Cu/Zn-SOD, catalase, or Se-Gpx had to be used to obtain the same protection against 2 arm O_,, which corresponds respectively to 635. 6.6. and 0.35 times the native activity of the cells. Lower amounts of these enzymes ~xere needed to afford 20% protection when fibroblasts were exposed to I atm O, or to nitrofurantoin, but the respective potency of the three enzymes was similar. As a mean for these three situations, Se-Gpx was 2200 times more potent on a

molar basis than Cu/Zn-SOD. and 14 times more than catalase. We have to stress that in no case could the increase in one antioxidant enzyme give a protection higher than 4 0 ~ , even with Se-Gpx. There exists a limit in the possible protection of these cells. Both hyperoxia and nitrofurantoin treatments seem to induce similar effects on the fibroblasts, because a similar pattern of protection by the different antioxidant enzymes is obtained. Both stresses induced generation of free radicals within the cell. Youngman et al. ~" evidenced the production of H_,O: and O:'- by cells exposed to nitrofurantoin, and Frank and Massaro ~ and Housset et al. ~ described that H_,O: x~as an important factor of the toxicity under h) peroxia. The fact that catalase and Se-Gpx are far more efficient than Cu/ Zn-SOD in protecting fibroblasts against the oxidative stresses confirms that HzO_, and its derivatixes are the most deleterious species inxoh'ed in this toxicit.~. White et al. 5~ also drew the same conclusion, showing a preponderant protective role of the glutathione-cycle enzymes for protection of rat lungs from hyperoxia, From a kinetic point of xiew. catalase and Gpx are both able to destroy H,O_,, but Gpx has a much higher affinity for HzO., than catalase. 5~' suggesting that H_,O: is principally degraded b 3 Gpx in normal conditions. Hiraishi et al. ~" have indeed shown that rat gastric cells in culture depend mainl) on their GSH-cycle enzymes but not on their catalase activity for their protection against HzO:. Dobrina and Patriarca 5~ have also demonstrated that in endothelial cells, 70% of the H_,O, generated by activated polymorphonuclear neutrophils t PMNI or by glucose-glucose oxidase were destroyed by Gpx. However, when the H,O: concentration increases, as in severe oxidative conditions, the catalase contribution for its degradation concomitantl) in-

Role of glutathione peroxidase, catalase, and SOD Table 2. Protection Afforded by Combinations of Injected Antioxidant Enzymes Protection When Injected With Protection When Injected Alone

Se-Gpx 3 U/ml

Se-Gpx 3 Cu/Zn-SOD 0.4.5 x 10~ Cu/Zn-SOD 0.6 × 10~

19% 2~c 21c:.c

22% 32%

Se-Gpx 3 Catalase 28 Catalase 55

19c~ 4% 20%

36~ 48c2

Catalase 28 Cu/Zn-SOD 0.01 x 10" Cu/Zn-SOD 0.6 x 10~

20% 0e~ 16%

Enzyme tUIml~

Catalase 28 U/ml

-25% 30%

Human fibroblasts were microinjected with solutions containing different concentrauons of Cu/Zn-SOD. catalase, or Se-Gpx alone or in combination and their survival was followed when exposed to 2 arm O.,. A protection percentage ~ a s calculated from these survival cur~es m comparison with the survival of buffer-injected cells t from ref. 65 L

creases. 59 Moreover, the development of resistance to high H~O., concentrations in chinese hamster fibroblasts was concomitant with a 20-fold increase in catalase activity. 6° Those cells were also more resistant than the parental line to hyperoxia, and this resistance was due to the increased catalase activity, but also to a 2-fold increase in Gpx activity. However, it must also be stressed that a HeLa line resistant to hyperoxia has been selected that does not demonstrate higher levels of antioxidant enzymes. 6~6: Gpx also catalyzes the reduction of lipidic peroxides in addition to H20:, thus acting on more sensitive cellular targets. McCay et al. 63 showed that Gpx protects biological membranes by preventing lipid peroxidation propagation. This specific action, in addition to the destruction of H202, explains the very high efficiency of exogenous Gpx added intracellularly for the protection of cells against oxidative stresses. Effect of enzyme combinations Despite the fact that the three main antioxidant enzymes are present in various amounts according to the cell type, and are localized in specific subcellular compartments, it was hypothesized that they could influence each other to give a resulting global protection. We could thus a priori postulate that additive or even synergistic cooperations between these enzymes could exist. This hypothesis has been tested by the simultaneous injection in cells of different concentrations of each enzyme alone or in combinations, and by estimating the protection obtained under hyperoxic oxidative stress. Table 2 presents a summary of experimental results

239

showing that the protections afforded by Cu/Zn-SOD and Se-Gpx are additive, while the ones given by catalase and Se-Gpx are synergistic. However, a combination of catalase and Cu/Zn-SOD leads to either positive (additive) or negative (toxic) effects according to the Cu/Zn-SOD concentration tested. In other experimental systems, either cooperation 64 or negative results ~6~s have been reported when catalase was combined with SOD. These three reports seem contradictor),, but the dose response curve obtained by injecting increasing amounts of the enzymes can explain this discrepancy. 6-~ The effect was very much SOD concentration-dependent: when increasing SOD concentrations are added to a constant concentration of catalase, a negative or even toxic effect is observed at low SOD concentrations, while a protection, even additive, is achieved at higher SOD concentrations. Changing the SOD activity can have opposite effects depending on the magnitude of the change and on the ratio of the resulting SOD activity to the native catalase activity. For example, Omar et al. 4° found that a bell-shaped curve is obtained for the protection of SOD in the reoxygenated heart. Explanations for such a complex behaviour are multiple. Most obviously, according to the respective amounts of SOD and catalase present, both O:'- and H20_, are present at the same time, so that the HaberWeiss reaction will occur giving rise to the highly reactive hydroxyl radical I reaction 5). When either SOD and/or catalase concentrations are high enough, the Haber-Weiss reaction is inhibited, because at least one of the reagents is missing. Another negative effect of SOD is that it inhibits the termination reaction involving the superoxide anion and the lipid hydroperoxide radicals. In the presence of SOD, O:'- concentration x~ill decrease so that the termination reaction of the hydroperoxide radical cycle will be lowered, and the damages to the membrane lipids could increase Ireaction 61. Finally', SOD can indirectly' stimulate the autooxidation of hydroquinones through the presence of higher concentrations of H:O_~ Ireaction 71, and so it lowers the termination reaction involving 02"- and the semiquinone radical ~6~" (reaction 8). All these observations could explain why overexpression of SOD in cells or organisms did not produce overresistant cells, as observed by Scott et al. 6~ or Elroy-Stein et al. 69 It is the level of SOD activity relative to catalase activity that is the key factor for estimating the efficiency' of SOD. Three possible reactions explaining the enhanced toxicity observed in some experimental conditions with increased SOD concentration Increase of the Haber-Weiss reaction: O:'- + H_,O: --" HO" + OH- + O_~

~5)

240

C. MICHIELS et al.

Decrease of the termination reaction of hydroperoxides: ROO" + O_,'- --' ROOH + O2

~61

Increase of the autooxidation of hydroquinones: QH_, + H_,O: --" Q ' - + HO" + O H - + 2H + (71 instead of the termination reaction: Q ' - + o2"- + 2H + --* QH_, + 02

(81

in addition to these considerations, we must also stress the importance of Se-Gpx: combinations of SeGpx with either catalase or Cu/Zn-SOD were very beneficial. As Gpx detoxifies H_,O2 with higher efficiency than catalase, it is not surprising to observe a good complementarity with SOD, better than the combination of catalase and SOD. Interestingly, in some cases. cells having higher SOD activity than normal also induce their Gpx activity. "°'~ This is also observed in cells from trisomy 21. but the presence of one extra cop> of chromosone 21 could also influence the level of Gpx activity. "2 Gpx also reduces lipid peroxides, thus indirectly' protecting the h.~drophobic membrane compartment of the cell. On the other hand, catalase and SOD principally act in hydrophilic regions. Consequently, when one or the other is combined with Gpx. the protection to the hydrophobic and hydrophilic compartments are complementao', and such a combination better maintains the cellular integrity against the free radical attacks.

THRESHOLD OF FREE RADICAL ATTACKS AND MODULATION BY ANTIOXIDANT ENZYMES

Several studies have shown that the recovery capacity of cells exposed to an oxidative stress depends on the intensity of the stress: the higher the stress, the lower the recovery. 4"~3''t These results suggest the presence of a threshold of oxidative damages that cannot be totally repaired and that impairs cell division. It was previously shown that antioxidant enzymes could protect cells exposed to a continuous oxidative stress. We can thus hypothesize that this threshold of oxidative damage, belo~ which recove O is still possible, can also be modulated by the antioxidant enzyme activities of the cells; the idea is that if the antioxidant enzyme activities are increased, the cell could undergo a more severe oxidative stress and still be able to recover.

This hypothesis was tested in the above-described

experimental model: human fibroblasts x~ere submitted to h)peroxia for increasing times and their recox er~ in normal atmosphere was foilo~ed, as well as their ability to divide. "s Hyperoxia incubation strongl3 affects the dividing capacity of these cells: after 15 h of h~ peroxia the proliferation ceased, and after 20 h the cells began to die. even when allowed to recover in nonnal conditions. "~ Therefore, a threshold of oxidatixe damage exists that limits the dividing capacity of the cells. Different concentrations of Cu/Zn-SOD, catalase, or Se-Gpx were then injected in the cells before exposition under hyperoxia for 5 h, l0 h, 15 h, or 2{) h, and the dividing capacit3 of the injected cells xxas follox~ed during the recover3 period. The results showed that cells incubated for a short time and injected with a high concentration of one of the three enz~ rues divided like nonoxygen-incubated cells: the longer the incubation, the higher the enzyme concentration needed for complete recovery. Se-Gpx was also. in these conditions, the most potent enzyme: from the concentrations of the injected enzyme solutions, x~hich gax e 20q- protection for the recoveo of the cells after short periods of hyperoxia, we can calculate that Se-Gpx is 1354 times more efficient than Cu/Zn-SOD and 4 times more efficient than catalase. However. when cells were incubated for too long, cells were either less protected or not protected any more. and could no longer divide, Figure 1 gives a schematic representation of the threshold level that is obtained by injecting increasing amounts of Se-Gpx. and which allows cells to resist longer exposures under hyperoxia. -5 Opposite results ~ere obtained when the activites were lox~ered b3 incubating cells with enzyme inhibitors, and the3 are also schematically presented in Fig. 1.'" A similar picture can be drawn from the experimental results lor catalase and Cu/Zn-SOD. The experiments were repeated in another experimental model where human fibroblasts were exposed to tert-bub'lhydroperoxide (TBHPI. The concentration of TBHP needed to inhibit cell division, without inducing cell death, x~as determined in normal condition~ or in cells where Se-Gpx had been inhibited by mercaptosuccinate to different levels. The results showed a high correlation between the Se-Gpx acti~it3 of the cells and the concentration of TBHP needed to inhibit cell proliferation. "~ One conclusion of such obserxations is that the threshold of oxidative attacks that cells can sustain can be modulated by their content of antioxidant enzymes: either this threshold is decreased when the enz 3mes are partially inhibited or it is increased by supplementation of antioxidant enzy'mes. The presence of a critical threshold is intrinsic to the system of production and elimination of free radi-

Role of glutathione peroxlda~,e,catalase, and SOD

241

+ 12 U/ml ~

Q,.(n

U,'ml

tO

~natwe

conlent

~mh~bmon of Gpx 0

5'

10 '

15 '

2o

2

30

Hyperoxia mcubatton limes (h) F~g I Exoluuon of the prohferauon capacity of human fibroblasts exposed to increasing h.,,peroxm incubation times. This capaot.~ ~as alread~ depressed after 5 h incubation, but cells injected with increasing concentrations of Se-Gpx can sustain increaqng h.~peroxia incubauons before losing their proliferauon capacity. When native Se-Gpx is inhibited, cells can no longer d~ ~de exen under normoxia.

cals when the inhibition of the antioxidant enzymes by peroxides and free fradicals is taken into account. "~ A mathematical model of free radical production and elimination was constructed, taking into consideration the main reactions of the free radical system, and it ~ a s then analyzed for its stability. TM It clearly shows the presence of stable and unstable domains according to the free radical production and to the antioxidant enz>me activities, mainly Se-Gpx and catalase, but not Cu/Zn-SOD. The limit between the stable and the unstable domains represents the critical threshold, which is experimentally illustrated in Fig. i. Another important conclusion can be drawn when the experimental data are expressed as ratios between the concentrations of the injected catalase or Cu/ZnSOD solutions giving 20% protection, and the concentration of Se-Gpx achieving the same protection, in these various oxidative conditions. These ratios are presented in Table 3. They are smaller for short periods of oxidative stress, which induced inhibition of cell division but not cell death. On the other hand, these ratios are more than twice higher for more severe oxidative stresses inducing cell degeneration and death: either after 20 h exposure to hyperoxia or during continuous oxidative stresses (more than 72 h) induced by high ox)gen pressure, or in the presence of nitrofurantoin. The difference in these protective enzyme concentration ratios means that mitosis and cell survival are differently sensitive to the different molecular species of ox~.gen-derived free radicals. While remaining the most efficient enzyme, in comparison with catalase and Cu/Zn-SOD, Se-Gpx would be relatively less involved

in the protection of the mitotic capacity than in the protection of the whole cell su~'ival capacity. So. O.,'and H_,O: would be relatively more responsible for the mitotic arrest, which occurs first, while hydroperoxides would affect the general long-term cell survival through progressive membrane damages. Housset et al. ~4 has already postulated that Gpx can protect endothelial cells against some aspects of oxygen toxicity. but not in the division process. These data led to similar. but not so categorical conclusions.

EFFECTS OF INHIBITION OF THE DIFFERENT

ANTIOXIDANT ENZYMES In all the experiments described above, the concentrations of the different antioxidant enzymes were increased and their protective effects reported. These results were obtained under oxidative stress so that the)' give no clues for the respective role of these enzymes in physiological conditions. One way to approach this question is to keep cells in normal conditions and to look for the consequences of the inhibition of each of these antioxidants. Different means have been used to achieve this purpose: deficiency in one metabolite necessary for the synthesis of the defense systems, for instance lowered cysteine to decrease intracellular glutathione s° or reduced selenium for decreasing Se-Gpx activity, -~4 or direct inhibition by specific antibodies st or by specific chemical inhibitors. 76~-" Kaczmarek et al. s3 first showed that antibodies raised against an enzyme, in this case DNA polymerase

242

C. MICHIELSel al.

Table 3 Evolution of the Ratios of Protective Concentrations of Catalase/Se-Gpx and Cu/Zn-SOD/SeGpx as a Function of Increasing Oxidative Stress Intensity Oxidatixe Stress 5h 2 arm O_, 1Oh 2 atm O: 15h 2 atm O: 20h 72h 72h 72h

2 5 I 2

Mnotic .~'rest

Cell Death

+ + +

arm O. x 11) i3,I NF arm 02 arm O.-

+ + + +

Catalase/Se-Gpx

Cu/Zn-SOD/Se-Gp~

-I 17 3.06 3 37

321" 346" 246 ~

9.16 23.75 12.59 6.45

720 ~ 1500 b 809' 1008 J

Different concentrations of each enz~,me were rejected into human fibroblasts and their survival was followed when exposed to different oxtdative stresses (NF = nitrofurantom). A protection percentage was calculated from the survtval cur,,es in comparison with the survival of buffer-injected ceils, and the concentration of the injected solutions that gave 20% protection was calculated from the dose-response protection curves. Ratios between these concentrations obtained for catalase and Se-Gpx or Cu/Zn-SOD and Se-Gpx ~ere then calculated. from ref. 75. from ref 51 from ref. 50 from ref. 49.

a. can be microinjected into cells and are able to inhibit intracellularly the corresponding enzyme. The same approach was then used by Michiels et al. 8~ to inhibit the two antioxidant enzymes that are in the cytosol and can be reached by the injected antibodies: Cu/ZnS O D and Se-Gpx. The results showed that both antiS O D or anti-Gpx antibodies increased the mortality of fibroblasts exposed to hyperoxia. Such an increased sensitivity indicates that both native enzymes are actually necessary for cell protection against free radical attacks when exposed to an oxidative stress. In normoxia, the injection of anti-Se-Gpx antibodies into fibroblasts was also deleterious: a decrease in the proliferation rate of these cells was observed. Se-Gpx is thus also absoluteb necessary for the survival of the cells under normoxia. Chance et al. '7 have demonstrated that a steady state level of H.,O_, and 02"- is always present within cells due to normal metabolism. The fact that a decrease in Se-Gpx even in normoxia has a toxic effect, indicates that this basal production can be damaging for the cell and must be kept under control. However. the inhibition of Cu/Zn-SOD in normoxia was not detrimental but rather increased the division rate of the cells. As already mentioned, changing the Cu/Zn-SOD activity alone may have unexpected results because Cu/Zn-SOD increases the formation of H_,O,. which if not destroyed could have more detrimental effects than 02"- alone, a°6869 We can conclude from these observations that decreasing H,,O2 production by decreasing Cu/Zn-SOD can be a favourable event as long as the cell does not have to cope with an oxidative stress, in which case the amount of 0_,'would be excessive and should be eliminated by Cu/ Zn-SOD. Ceils may have evolved to optimize their

S O D content to fulfill these two conditions. These data stress again, as in the studies where combined exogenous enzymes were injected, the relative efficiency of Cu/Zn-SOD according to the balance between the radical production and the activity of the other antioxidant systems, as well as the high efficiency of Se-Gpx. Another approach to study the influence of decreased antioxidant activites is to use specific chemical inhibitors. Puglia and Powell s-" have already used this approach, and demonstrated an increased toxicity on rats under hyperoxia when Gred was inhibited by bischloroethylnitrosourea (BCNU). 84 Different works have also compared the role of the glutathione-cycle enzymes vs. catalase to protect cells against oxidative stress. Andreoli et ai. 8-~ exposed endothelial cells to activated PMN or to xanthine-xanthine oxidase and demonstrated an increased sensitivity to these free radical-generating systems when either catalase or GSH synthesis was inhibited by aminotriazole and buthionine suifoximine (BSO). respectively. They also showed by a similar approach that the G S H redox cycle is more important than catalase for protection against xanthine oxidase-derived free radicals in different cell types. 86 Suttorp et al. 8~ also showed that inhibition of the GSH-cycle enzymes by BSO or BCNU, but not the inhibition of catalase by aminotriazole, decreased the resistance of endothelial cells to free radicals generated by activated PMN or the g l u c o s e - g l u c o s e oxidase system. They also have drawn similar conclusions for endothelial cells exposed to hyperoxia, and, moreover. stressed the important role of Gpx in this resistance. 88 Aerts et al., 89 using BSO, showed that GSH synthesis is essential for pneumocyte survival under hyperoxia. Also, in vivo and in normal conditions, the decrease

Role of glutath~one peroxidase, catalase, and SOD

243

Table 4. Comparison Between the Percentage of Inhibition of the Different Anuoxidant Enzymes Leading to a Loss of 50°~ in Cell Number Concentrations of the inhibitor Leading to 50% Cell Loss cpb, l) Enz~ mes/Inhlbitors Catalase/AT Se-Gpx/M S Gred/BCNLI GSH/BSO

Inhibition of the Corresponding Enzyme fc'c

Normoxia

H> peroxia

Normoxia

H} peroxm

2500 1 83 0.068

76000 0.058 35 0.34

55 21 bI 27

92 17 82 34

Correlations ,.,,ere made bet~een the concentrations of aminotriazole IATL mercaptosuccinate tMSI. bischloroethylnitrosourea IBCNUp. and buthionine sulfoximine IBSO~ that led to a decrease of 50c~ in the cell number enther under normoxm or h~peroxia t I atm O:p. and the inhibition of the corresponding enz.~ me that these precise concentrations ga',e ~ hen added to intact cells ifrom ref. 76~

in Se-Gpx activity induced by selenium deficiency seems to be more detrimental than catalase inhibition by aminotriazolefl ~'~ In an attempt to quantify the consequences of enzyme inhibitions, a systematic analysis was performed, which correlates the level of the enzyme inhibitions with their detrimental effect on cell survival. "6 Catalase was inhibited by aminotriazole, "n Cu/Zn-SOD by diethydithiocarbamate, ~n Se-Gpx by mercaptosuccinate. 3~ Gred by BCNU? '4 and GSH synthesis by BSO? '-~ Dose-response curves for each of these inhibitors on the intracellular activity of the corresponding enzymes were performed. Viability of cells incubated with increasing concentrations of these inhibitors was then studied under normoxia and under hyperoxia. The decrease in cell division rate 1under normoxia~ or the acceleration of cell death lunder hyperoxia) was then correlated with the corresponding levels of enzyme inhibition for each inhibitor. The results of this anal}sis have been summarized in Table 4. We can first observe that the inhibition of each enzyme leads to a decrease of the cell number, and that the inhibition levels are rather similar under both normoxia and hyperoxia, which means that each of these enzymes is equally necessary to protect cells against oxidative stress and to ensure the cell-dividing capacity in normal conditions. A similar conclusion was already suggested by the results of the experiments of inhibition of Se-Gpx by specific antibodies. There are, however, differences between the different enzymes. In normal conditions, 21% of the Se-Gpx activity has to be inhibited to decrease the cell number by 50%. while 55% inhibition of the catalase activity is needed to achieve the same effect. In these experiments. Se-Gpx was also found to be a very important enzyme to protect cells in normal conditions and under oxidative stress. It is particularly interesting to note

that in these inhibitor3' experinaents, and considering the residual activities of these enzymes, Se-Gpx seemed to be 10.4 times more potent than catalase when compared on a residual activity basis: this is very similar to the value 17-20) obtained on a molar basis, when the enzymes ~,ere injected in the same cell type exposed to oxidative stresses (Table 11. A second important observation is the differences between the inhibitor}' values of Se-Gpx compared to Gred or GSH synthesis: as a mean, 71%, inhibition of Gred but only 19% for Se-Gpx was needed to reduce the cell number to 50%. These data also reinforce the idea of the importance of Se-Gpx. Gred is a GSHrecycling enzyme; with physiological GSH concentrations, a low Gred activity could still be sufficient to reduce enough GSSG for the Gpx activity. On the other hand. Gpx reduces hydroperoxides, which, if no longer detoxified, will accumulate and amplify lipid peroxidation leading rapidly to cell death. This is also confirmed by the fact that a mild decrease in GSH concentration (30%1 induced a comparable toxicity. All the antioxidant systems are thus necessary for the survival of the cell even in normoxia, but they do not seem to have the same importance. A more refined relationship has been obtained between the Se-Gpx activity and the steady state level of hydroperoxides, using a mathematical model describing the free radical production and elimination reactions within a cell. TM This relationship is presented in Fig. 2. The lower curve represents the steady state level of hydroperoxides as a function of Se-Gpx activity. It increases when the Se-Gpx activity decreases. The upper curve represents the maximum hydroperoxide concentration that the system can sustain without being destabilized: it decreases when Se-Gpx activity decreases. Such a relationship has been validated experimentally and the critical value, below which the

C. MICHIELS e t al

244 b

8

unstable

I

stable

t

Gpx activity Fig. 2 Relauonship between [he Se-Gpx activit} and the stead x state level of hydroperoxides (ROOHP x~h]ch is represented b} the lower curxe. The upper cur~e represents the maximum level of ROOH that the 'L~stem can sustain before reaching an unstable domain 4redra~ n from Remacle et al. "~ ~ ~th permission,

cell can no longer sustain the stress and dies, has been found to be 56% of the native Se-Gpx activity for human fibroblasts.'" T R A N S F E C T I O N OF A N T I O X I D A N T ENZYME CDNA

Recently. new technical developments have been used to confer a long term resistance to oxidative stresses: it is to transfer the cDNA for SOD. catalase, or Gpx into cells (transfection) or into organisms ~transgenic mice). This cDNA will then incorporate in the genome of the recipient cells, leading to overexpression of the corresponding enzyme and to increased resistance to oxidative stresses. However. the physiological repercussion of such an increase was not always the one expected. MnSOD transfected mouse cells overexpressing SOD activity were indeed more resistant to paraquat ~' or to hyperoxia."" and Huang et al/"- demonstrated a clear correlation between the magnitude of the increase in SOD activity and the resistance to paraquat in different trisomic, transfected, or transgenic mice derivedcell lines. However, in many reports, an increase in SOD activity was not alwa}s associated with an increased resistance to oxidative stress. SOD-enriched bacteria showed an increased sensitivity to hyperoxia) ~ to ionizing radiations, ' ' or to paraquat'r"): mouse and human SOD transfected fibroblasts seemed more resistant to paraquat but exhibited increased levels of lipid peroxidation/'~ Cells derived from trisomy 21 patients also contain an increased Cu/Zn-SOD activity due to gene dosage and are also more sensitive to oxidative stress, u~ SOD transgenic mice have also been generated, n'-" which demonstrated similar patterns of pathologic changes as those observed in trisomic patients.L°'

As explained earlier, the sensitivity to higher oxidative stress of cells overexpressing SOD may be due to an enhanced generation of H,O:. Cells indeed t~' to compensate for that negative effect by increasing their Gpx activity while their catalase activity remains unchanged.":'-'-" or by inducing the catalase activity. ~'" A correlation can even be drawn bet~een the increased Gpx activit} and the paraquat resistance in SODtransfected routine fibroblasts. "° In addition. Amstad et al. ~''' have demonstrated that, in mouse epidermic cells, an increased SOD activity by transfection increased the sensitivity of these cells to oxidative stress. but this sensitivity can be largely diminished even below that of the parental line if catalase activity is concomitantl} increased. Ceballos-Picot et al. K'6 also evidenced that SOD transgenic mice showed an enhanced rate of basal lipid peroxidation in the brain. The}' h}pothesized that the balance bet~ een SOD and catalase plus Gpx is more important for the sensitivity to free radicals than the absolute amount of a single antioxidant enzyme. A transgenic Drosophila melanogaster overexpressing catalase has been obtained by Orr and Sohal. u'' but enhanced levels of catalase did not prolong the lifespan nor did the}, provide enhanced protection against oxidative stresses induced by paraquat or hyperoxia, However, high resistance to H_,O, was observed. Some transfectants with human Se-Gpx cDNA have been recently obtained in human breast cancer cells. E.... and in this case, a clear enhanced resistance to different oxidant molecules like H,O,, cumene hydroperoxide, or menadione could be obtained. ~°° Transfection of COS cells with the human Se-Gpx gene was also achieved, ~° but their resistance to oxidative stresses was not tested. CONCLUSIONS In normal conditions, there is a stead}' state balance between the production of oxygen-derived free radicals and their destruction by the cellular antioxidant systems. However. this balance can be broken naturally or experimentally either by increasing the free radical production or by decreasing the defense systems. These two approaches have been used to understand the respective role and the efficiency of the different antioxidant enzymes for protection against the free radical-induced decrease in cell proliferation and cell death. The results clearly demonstrate the protective role of each antioxidant enzyme in different conditions, stressing the importance of the balance between Cu/ Zn-SOD and catalase + Se-Gpx activities, evidencing the high potency of selenium-glutathione peroxidase,

Role of glutathione peroxidase, catalase, and SOD

245

Gpx concentration offreeradicals

~

Catalase SOD I I I I N

enzyme activity

Fig. 3. E'.olution of the concentration of free radicals that the cell can sustain ~xlthout ds ,ng. Each curve represents the e~olution of such critical concentration in function of the actw]t.s, respectivel), of Se-Gpx. catalase, and Cu/Zn-SOD. This critical concentration in normoxia and at native enzyme actixities ,s represented by the arrow Iredra~n from Remacle et al.TM with permissions. N: natixe enzyme act,~,ties: P: free radical concentrations under phssiological conditions.

and clearly supporting the existence o f a threshold level o f free radical production, which is determined by the c o m b i n a t i o n of these three ant,oxidant enzymes. A s u m m a r y o f the efficiency o f Se-Gpx. catalase, and C u / Z n - S O D is illustrated in Fig. 3. It shows that a great protection is produced with increasing concentration o f S e - G p x , while a very small protective effect is produced when C u / Z n - S O D activity is increased. On the other hand. a high toxic effect is achieved when S e - G p x is inhibited under physiological conditions. while inhibition of C u / Z n - S O D does not lead to toxicity. Catalase has a b e h a v i o u r in between, being relatively efficient to protect cells against free radicals and cells being relatively sensitive to its inhibition. We also focussed our attention on some more specific roles of these enzymes. If we e m p h a s i z e d many times the high efficiency o f S e - G p x in protecting cells against oxidative stress, we also stressed its crucial importance for cell su~'ival, as well as for the maintenance of cell-dividing capacity in normal conditions. These two aspects seem to be protected differently by the different ant,oxidant e n z y m e s , which means that they seem to be differentially affected by the various radical species. A second important observation is that even in normal conditions, and all other protecting systems being present, the inhibition o f only one of the ant,oxidant e n z y m e s leads to deleterious effects. Each o f these systems thus has a specific irreplaceable role in cell defense, and all o f them have to be present to ensure total protection. These ant,oxidant e n z y m e s indeed seem to act c o o p e r a t i v e l y and even in synergy. The optimal protection in the cell is then the result of equilibrated activities between each of these three e n z y m e s , not to mention the other chemical and e n z s matic ant,oxidants. W e have to stress that the study of the effects o f the variation of these three ant,oxidant

e n z y m e activities that have been obtained in these studies has also to take into account the basal protection given by the other ant,oxidant enzymes, such as the P H G p x or the M n - S O D . The repercussion o f changing the level of one of these e n z y m e s by tranfection, for e x a m p l e , is not yet fully understood and can bring unexpected results, as in the case o f cells o v e r e x p r e s s i n g SOD. The c o m p e n sation for S O D overexpression by increasing G p x activity illustrates v e ~ ' well that cells adapt themselves to ensure an optimal efficiency o f their ant,oxidant defense systems. This work e m p h a s i z e s that. despite the difficulties linked to the G p x b i o c h e m i c a l properties, its characterization in biological systems in relation to S O D and catalase is necessary to obtain a precise picture of the ant,oxidant defenses within the cell and to d e v e l o p a strategy for designing new well-balanced ant,oxidant therapies based on this general concept. A c k m , w l e d g e m e m s - - T h i s work ~as performed with the support of

the lnstitut pour l'Encouragement de la Recherche Scientifique dans I'lndustrie et I"Agriculture. Bruxelles IIRSIA ). of the Fonds National de la Recherche Scientifique. Bruxelles (FNRSp. and of the Fonds pour la Recherche Scientlfique Mrdicale. Bruxelles I FRSM t. C. M. ,s a Senior Research Assistant. M.R.i.~ a Research Associate. and O.T. is a Research Assistant at FNRS

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