Enhanced sensitivity to oxidative stress in Cu,ZnSOD depleted rat erythrocytes

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~~chimica et Biophysjca Acta, 1123 (1992) 29 l-295

0 1992 Elsevier Science Publishers B.V. All rights reserved ~5-2760/92/$05.~

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Enhanced sensitivity to oxidative stress in Cu,ZnSOD depleted rat erythrocytes Gianna Maria Bartoli ‘, Paola Palozza 2 and Elisabetta Piccioni 2 ’ ~epffrtment of Biology, ~n~~ersi~ of ‘Tor Vergata ; Rome ~Ita~y~and ’ Institute of General Path~iu~, Catholic ~ni~ers~~, Rome (Italyl

(Received 17 December 1990) (Revised manuscript received 10 September 1991)

Key words: Lipid peroxidation; Superoxide dismutase; Copper; Oxidative stress; (Rat erythrocyte)

The effects on red blood cells of superoxide dismu~se (Cu,ZnSOD) depletion, induced by feeding Wistar rats with a copper deficient diet, were investigated. SOD depleted red blood cells were more sensitive to pe~xidation and to hemolysis than normal cells when exposed to bed-bu~lhyd~peroxide (t-BOOH). Membranes isolated from SOD depleted cells showed a Iower content of vitamin E and higher (Na+, K ‘1 and Mg’+ ATPase activities. These results support the view that superoxide dismutase plays an important role in cellular oxidative metabolism.

Introduction Many reports emphasize the primary role of oxidative stress in the development of different pathological processes [l-3]. Experimental evidence demonstrates that oxidative stress can be generated either by increasing endogenous production of reactive oxygen species or by decreasing intracellular antioxidant defenses. The modulation of antioxidant enzymes such as glutathione peroxidase or superoxide dismutase (Cu,ZnSODl can affect the rate of intracellular radical production inducing modifications in cellular structure and functions [4-61. Previous reports showed that feeding animals with a copper deficient diet results in a Cu,ZnSOD depletion in many tissues. This depletion is specific enough to study the correlation between the loss of this enzymatic activity and a possible consequent oxidative damage [7,81. The connection between the loss of Cu,ZnSOD, induced in liver cells by this treatment, with modifications of lipid composition and an impairment of cal-

Correspondence: G.M. Bartoli, Institute of General Catholic Unive~ity, L.go F. Vito 1, 00168 Rome, Italy.

Pathology,

cium metabolism in microsomal membranes has already been established [9,10]. Among other cells, erythrocytes are also affected, in terms of SOD depletion, by a copper deficient diet. These cells have been extensively used as a model to investigate oxidative membrane damage, since they constitute a large reservoir of oxygen from which the potentially dangerous radicals may be derived. The presence of hemoglobin with its potentiality to catalyze formation and degradation of oxyradicals may increase the danger [11,12]. The oxidative membrane damage, evidenced by the increased cation permeability and hemolysis, has been ascribed to protein modifications [13-15j or lipid peroxidation [11,16-191. The relative involvement of these two processes is a matter of debate [ZO]. ~e$~-ButyIhydroperoxide (t-BOOH) reacting with hemoglobin generates oxidative damage in red blood cells and produces ion leakage and hemolysis. These phenomena seem more related to oxidative damage of proteins, than to lipid peroxidation as indicated by several reports [21-231. Many papers suggest that ion leakage and lipid peroxidation are expressions of parallel events [24-271. This study was set up to investigate whether Cu,ZnSOD depletion modifies etythrocyte structure and functions, making them more sensitive to oxidative insults.

292 Wistar rats were fed a copper deficient diet for ten weeks to deplete Cu,ZnSOD. Red blood cells and erythrocyte membranes were isolated from both normal and copper deficient rats and exposed to oxidative stress by r-BOOH. Lipid peroxidation, hemoglobin status, membrane fatty acid composition and vitamin E content were compared. The membrane ATP-ase activity was also measured to verify the damaging effect induced by Cu,ZnSOD depletion. Materials and methods Weaning male rats (21 days) of the Wistar strain were used. The animals were kept for ten weeks on a copper deficient diet. Control rats were fed the same synthetic diet containing copper. Both control and copper-deficient diets had the following composition: casein 15%, corn oil lO%, acetyl-cellulose powder 2%, vitamin mix in glucose 5%, salt mix 3%, oil with vitamin A + D OS%, sucrose 64.5%. The copper-deficient diet contained 0.8 mg &/Kg. Food and deionized water were supplied ad libitum. The diets were purchased from Piccioni, Brescia, Italy. Cu,ZnSOD was extracted from red blood cells following Winterbourn et al. 1281. The enzyme content was measured following the autoxidation of epinephrine to adrenochrome as described by Misra and Fridovich

Lw. The red blood cells were washed three times in buffered isotonic NaCI solution and resuspended at 5% hematocrit in Krebs Ringer buffer. Erythrocyte membranes were prepared by the method of Hanahan et al. 1301. Pelieted ghosts were washed and resuspended in 10 mM phosphate buffer (pH 7.4) at a concentration of 1 mg/ml. AI1 incubations were carried out at 37”C, under 0,. t-BOOH was 0.5 mM in the experiments with cells and I mM with membranes.

O,OY....,.“““‘.‘.“‘l. 0

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Percent hemolysis was calculated as described by Goldberg and Stern [31]. Lipid peroxidation was assayed by measuring thiobarbituric acid reactive substances (TBARS), as described by Stocks and Dormandy [32]. Oxyhemoglobin, methemoglobin and intact hemoglobin (intact hemoglobin is defined as the sum of oxyand methemoglobin) were measured as described in Ref. 17. Fatty acids from erythrocyte ghosts were extracted by saponification and differential extraction in petroleum ether according to Entenman [33]. For analysis of fatty acids by gas-liquid chromatography, methyl esters were prepared as described by Borrello et al. [34] and examined with a Carlo Erba HRGC 5300 chromatograph. Vitamin E was extracted in acetone and analysed by high performance liquid chromatography with fluorescence detection, using a reversed-phase coloumn [35]. Na+, K+-ATPase activity in erythrocyte membranes was measured spectrophotometrically as described by Scharschmidt et al. [Xl. Fatty acid and vitamin E standards, I-butylhydroperoxide, ADP, ATP, NADH, phosphoenolpyruvate, Iactate deydhrogenase and pyruvate kinase were purchased from Sigma Chemical Co. (St. Louis). Thiobarbituric acid and all other chemicals were of analytical grade and were purchased from Merck (Darmstadt). Organic solvents of chromatographic grade were products of Fisher. Results As we have demonstrated by studying liver [9], ten weeks feeding Wistar rats with a copper deficient diet induces in the erythrocytes a Cu,ZnSOD depletion of 72% the enzyme content being 1.2 5 0.08 (7) pg/mg Hb in control cells and 0.34 i 0.02 (8) pg/mg Hb in

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0) and copper-deficient Fig. 1. TBARS release (A) and hemolysis (B) from erythrocytes of control f*production and hemolysis were triggered by f-BOOH 0.5 mM. The incubation was performed at 37 ‘C under 0, Ringer buffer. Values are means + SE. of Z-22 experiments.

n ) rats. TBARS fm with 5% hematocrit in Krebs

293 Cu-deficient cells. The possibility that loss of Cu, ZnSOD increases the reactive oxygen species production rate, thus inducing modifications in cell structure and functions, was studied. Normal and SOD depleted red blood cells were exposed to oxidative stress, induced ‘in vitro’ by t-BOOH. Fig. 1A shows lipid peroxidation measured as TBARS production in both kinds of cells. After 15 min incubation, SOD depleted cells begin to be markedly sensitive to peroxidation since they release higher amounts of TBARS than normal cells. This suggests an important role of SOD in counteracting the damaging effects of t-BOOH. We have compared lipid peroxidation to hemolysis induced by r-BOOH, as shown in Fig. 1B. SOD depleted cells undergo hemolysis at a higher rate than normal cells, although hemolysis starts 60 min later than TBARS release in both kinds of cells. As demonstrated by Trotta et al. 1171 the degree of lipid peroxidation is greatly dependent on the hemoglobin status; HbO, is an excellent initiator of lipid peroxidation as are low concentrations of MetHb, while high levels of MetHb are inhibitors. The hemoglobin oxidation and the concomitant lipid peroxidation is dependent on the reactivity of hemoglobin towards t-BOOH, which is higher for MetHb than HbO,. After 60 min of exposure to t-BOOH Hb02 is 61%, metHb 25% and non intact Hb is 12% in both kinds of cells; further incubation of erythrocytes for 2 h does not increase hemoglobin degradation products in both kinds of cells, indicating that SOD depletion does not modify hemoglobin reactivity to t-BOOH. Nevertheless, SOD depleted cells show a very high sensitivity to an oxidative stress probably due to the impairment of antioxidant status. To determine whether the high sensitivity to peroxidation elicited by erythrocytes could be related to modifications of membrane composition and structure we studied red cell membranes isolated from normal and copper deficient animals. The increased susceptibility to peroxidation observed in whole cells is present, although at a lower extent, in isolated membranes, TBARS production induced by t-BOOH being significantly higher in ghosts isolated from SOD depleted erythrocytes than in normal membranes (Fig. 2). The sensitivity to peroxidation shown by these membranes can be correlated to either a different degree of fatty acid unsaturation or a modified antioxidant membrane status. As we have previously shown, copper deficiency induces a fatty acid pattern characterized by a higher degree of saturation in liver microsomal membranes. In such membranes we have observed a decreased sensitivity to peroxidation. In this case we suggested a close relationship between the degree of fatty acid unsaturation and the susceptibility to peroxidation 191.Under the same conditions membranes isolated from SOD-depleted red blood cells do not modify the fatty acid composition and show the same

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minutes Fig. 2. TBARS production of erythrocyte membranes isolated from normal (o0) and SOD-depleted (m n) rat red blood cells exposed to 1 mM t-BOOH. The incubation was performed at 37 o C under O2 with 1.0 mg protein/ml in 10 mM phosphate buffer (pH 7.4). Values are means tr SE. of 3-6 experiments.

degree of unsaturation as normal membranes (not shown). To determine the relationship between the membrane antioxidant status and its susceptibility towards lipid peroxidation we measured the content of vitamin E, which is 0.623 f 0.006 pg/mg prot. (4) and 0.233 * 0.023 pg/mg prot (3) in normal and SOD depleted membranes, respectively. Thus membranes isolated from SOD depleted erythrocytes show a loss of more than 60% of their vitamin E content, which explains their higher sensitivity to peroxidation ‘in vitro’. This

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minutes Fig. 3. Vitamin E consumption in erythrocyte membranes isolated from normal (0 -01 and SOD-depleted (m -R) rat red blood cells, during lipid peroxidation initiated by 1 mM t-BOOH. Incubation conditions were the same as in Fig. 2. Vitamin E was extracted and measured as described in Material and Methods.

294

Erythrocyte membranes were added to I ml of reaction buffer pH 7.4 containing 0.5 mM NADH, 2.5 mM phosphoenolpyruvate, 5 mM ATP and 10 U each of LDH and PK (with or without I mM ouahain: ouabain-suppressible portion of total ATPase is NaK-AT&e activity). The activity is measured by NADH oxidation. Values are means + S.E. (number of experiments) Control nmoi/min/mg Total ATPase Mg-ATPase NaK-ATPase

3.7 + 0.3 (61 I.9+_O.f (61 1.7+0.2 16)

Cu-deficient protein 5.3 & 0.3 (9) 3.1 +O.t (91 2.2 + 0.1 (9)

enhanced susceptibility to peroxidation is confirmed by the observation that these membranes almost completely lack vitamin E after only 5 min of incubation with t-BOOH (Fig. 3). Since lipid peroxidation afso damages membrane proteins, we hypothesized that an increase of susceptibility to peroxidatio~ could be the cause of membrane functions alteration. To explore this possibility NaC, K+- and Mg2*-ATPase activities were measured (Table I). As expected, we found that SOD depleted membranes show modified activities in both enzymes, expecially Mg2 ‘-ATPase activity, which appears higher than in normal membranes. Discussion

Erythrocytes depleted of Cu,ZnSOD by a long term copper deficient diet show a marked sensitivity to lipid peroxidation and hemolysis when exposed to an ‘in vitro’ oxidative stress, which is the indication of the important role played by superoxide dismutase in the reguIation of the antioxidant defenses of these cells, It has been established that the primary oxidative damage exerted by t-BOOH on red blood cells is due to its reaction with hemoglobin that generates the initiators of lipid peroxidation [ 17-191. Hemoglobin degradation regulates lipid peroxidation because HBO, is an excellent initiator, while high levels of MetHb inhibit it. Although phospholipid peroxidation is regarded by many investigators as the major event underlying oxidative membrane damage [1,2,17,18], recent data indicate that oxidative hemofysis is mainly due to membrane protein modifications [24-271. Our studies show that the loss of superoxide dismutase makes red cells more sensitive to peroxidation as well as to hemolysis, but does not modify the hemoglobin oxidation rate. Moreover, the hypothesis that a higher degree of hemolysis, shown by SOD depleted cells, is connected with an increased susceptibility to peroxidation is confirmed by the observation that these cells have a lower

content of vitamin E. Converseiy, as shown by fchikawa et al. changes in the level of a-tocopherof in erythrocyte membranes do not influence the hemofysis rate, when hemofysis is not related to lipid peroxidation [37]. The loss of antioxidant defense determined by SOD depletion can induce oxidative stress and produce membrane modifications. SOD depleted erythrocytes could undergo lipid peroxidation and produce malondialdheyde, which forms adducts with membrane phosphohpids, as shown by Jain in aged human erythrocytcs (381. The occurrence of these events may influence membrane fluidity and fipid-protean interactions, with consequent enzyme modifications. The current idea that oxidative damage is most probably due to membrane protein modifications 124-273 is confirmed by the higher ATP-ase activity observed in SOD depleted cells. Alterations at the level of such an important membrane function in cellular homeostasis can deeply influence cellular integrity. From the data presented above we can conclude that SOD plays an important role in maintaining erythrocyte oxidative equilibrium and that its loss alters cellular metabolism, producing damaging effects mainly at the level of membrane functions. Acknowledgements

G.M.B. gratefully acknowfedges Prof. TI Galeotti (Institute of General Pathology, Catholic University, Rome) for helpful criticism and revision of the manuscript. This work has been partiafly supported by a grant MPI 60%. Accounts of this work have been given at the 5th Conference on Superoxide and Superoxide Dismutase held in Jerusalem, Israel, September 1989. P.P was recipient of a fellowship from Associazione Italiana per la Ricerca sul Cancro. References I Halliwell. l-14. 2 3 4 5 6 7 8 9 IO 11

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