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The influence of ozone on human red blood cells. Comparison with other mechanisms of oxidative stress
Biochimica et Biophysica Acta 924 (1987) 111-118 Elsevier
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BBA 22714
The influence of ozone on human red blood cells. Comparison with other mechanisms of oxidative stress
Jolanda Van der Zee, Karmi Tijssen-Christianse, Thomas M.A.R. Dubbelman and Johnny ~¢an Steveninck Department of Medical Biochemistry, Syloius Laboratories, Leiden (The Netherlands) (Received 1 September 1986) (Revised manuscript received 7 November 1986)
Key words: Ozone; Oxidative membrane damage; Lipid peroxidation; Potassium ion leakage; (Erythrocyte)
Exposure of red blood cells to ozone resulted in K + leakage, lipid peroxidation and inhibition of some membrane-associated enzymes. On the other hand, carrier-mediated transport of glucose, leucine, sulfate and glycerol and the nonspecific permeation of glycerol, L-glucose and erythritol were not affected by ozone. The cellular level of reduced glutathione declined, whereas the ATP content of the cells was quite insensitive to ozone exposure. It was shown that, most probably, lipid peroxidation and K ÷ leakage are not causally related. Further, K ÷ leakage did not reflect gradual, progressive loss of K + from all cells simultaneously, but occurred in an all-or-none fashion. Finally, ozone-induced damage was compared to damage induced by H202, t-butyl hydroperoxide and photosensitizers plus light. It appeared that the pathways leading to membrane deterioration are quite dissimilar in these various forms of oxidative stress.
Introduction Oxidative damage to biological systems is thought to form the basis of a number of physiological and pathological phenomena as diverse as inflammation, ageing and carcinogenesis [1,2]. In recent literature, many examples of the deleterious effects of reactive oxygen species on cells have been presented. In these studies erythrocytes have often been used as a convenient model cell. Membrane constituents proved to be very susceptible to oxidative agents and membrane deterioration frequently plays an important role in oxidative stress. For example, illumination of human erythrocytes with visible light in the presence of
Correspondence: J. van Steveninck, Department of Medical Biochemistry, Sylvius Laboratories, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
protoporphyrin as sensitizer leads to extensive membrane damage, reflected by K ÷ leakage and, ultimately, hemolysis. This is most likely caused by direct photooxidation of amino acids, such as histidine, tryptophan, tyrosine and cysteine, within membrane proteins. A limited lipid peroxidation was observed only after virtually all the K ÷ had leaked out of the cells [3,4]. H202-induced membrane damage in red blood cells is reflected by extensive lipid peroxidation, oxidation of membrane SH groups and increased passive cation permeability [5,6]. In this case, oxidation of SH groups accounted for the observed K ÷ leakage, whereas lipid peroxidation was not involved in this process. Utilizing t-butyl hydroperoxide to induce oxidative stress in erythrocytes, again lipid peroxidation and K+-leakage were observed. It could be shown, however, that the mechanisms of H202- and t-butyl hydroperoxide-induced lipid peroxidation were different. Also the pathway
0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
112 leading to K+-leakage appeared to differ with these two oxidants [6,7]. Although in all these cases of oxidative stress the ultimate result was complete deterioration of membrane structure and functions, the mechanisms of oxidative damage were apparently quite dissimilar. In this context the effects of ozone on red cell membranes were investigated in detail. It is known that ozone, an important component of photochemical air pollution and a very powerful oxidant, causes lipid peroxidation, crosslinking of membrane proteins and K + leakage in erythrocytes [8-10], but the mechanism by which ozone causes membrane damage is still unknown. The present investigations demonstrate that the mechanism of ozone-induced damage differs again from the pathways of H202-, t-butyl hydroperoxide and photodynamically induced damage. Methods Red blood cell suspensions, hemoglobin-free ghosts, linolenic acid and amino acid solutions were prepared as described before [6]. In experiments with intact cells the erythrocytes were suspended in phosphate-buffered saline, Ozone was generated by a Fisher-Ozone Generator Model 501 at a rate of 10 or 1 ~ m o l / m i n with an airflow of 0.4 m l / m i n . The 0 2 / 0 3 mixture was conducted over 10 ml of the stirred cell suspension or solution. Ozone production was measured by titration with KI-Na~S203-starch in 5 m M sodium phosphate (pH 7.4). K + efflux from red blood cells was determined with a flame photometer and expressed as percentage of total efflux, evoked by lysis of the cells in distilled water. Lipid peroxidation in cells was assayed by measuring thiobarbituric-acid-reactive products, as described by Stocks and D o r m a n d y [11]. Lipid peroxidation in ghosts or linolenic acid solutions was determined according to Asakawa and Matsushita [12]. Measurements of lipid peroxidation in the presence of paracetamol were complicated by the fact that paracetamol was oxidized by ozone, yielding a thiobarbituric-acidreactive product with an absorbance spectrum partially overlapping the spectrum of the thiobarbituric-acid-reactive lipid peroxidation products. The paracetamol-derived product, however,
exhibited an absorbance maximum at 548 nm, whereas the lipid peroxidation products had a maximal absorbance at 532 nm. Thus, lipid peroxidation could still be measured by absorbance readings at both wavelengths and appropriate correction of the absorbance at 532 nm. Chromolipids were measured as described by Jain and Hochstein [13]. The ( N A P + K~)-ATPase activity was determined by a modification of the method of Rega et al. [14]. Briefly, cells were exposed to ozone, centrifuged and resuspended at 5% hematocrit in a buffer containing 5 mM RbC1, 145 mM NaCI, 2 m M MgC12, 10 m M inosine and 10 m M Tris (pH 7.4) at 37°C. After 5 min incubation, S6Rb was added and the 86Rb influx was determined. Active Rb uptake was inhibited by the addition of 1 mM ouabain. The method of Ellman [15] was used for the assay of acetylcholinesterase activity and the method of Wu and Racker [16] for glyceraldehyde-3-phosphate dehydrogenase activity. The number of intact red blood cells was determined by a modification of the method of Devalia and McLean [17]. A Percoll solution was mixed with 10-times concentrated phosphatebuffered saline in the ratio of 9 : 1 (v/v). This was further diluted with normal-strength phosphatebuffered saline in the ratio of 42.5:57.5 (v/v). 2 ml of a 10% erythrocyte suspension were loaded on 8 ml of the Percoll solution and centrifuged for 10 min at 500 rpm. Intact cells were recovered in the bottom fraction and counted after delution in phosphate-buffered saline. This fraction of intact cells was also used in transport studies and for A T P and G S H determinations. The transport velocities of D-glucose, L-glucose, L-leucine, glycerol, erythritol and sulfate were assayed by measuring the efflux of radioactive substrate from preloaded cells. Cells were loaded with labeled substrate in the presence of 1 m M unlabeled substrate at 37°C in phosphate-buffered saline. After loading, the cells were exposed to ozone and subsequently centrifuged to remove extracellular label. Cells were resuspended in phosphate-buffered saline at 5% hematocrit. D-Ghicose, L-leucine and glycerol efflux was determined at 4°C. The other efflux experiments were performed at 20°C. Passive diffusion of glycerol was measured in the presence of 0.1 mM Cu 2+, completely inhibiting
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the facilitated diffusion of glycerol [18]. Cellular GSH content was determined according to Beutler [19]. GSH-depletion prior to exposure of the cells to ozone was brought about by treatment of the cells with diamide [20]. Hemoglobin release into the medium was measured according to Crosby et al. [21]. The ATP content of intact cells was measured with a BoehringerMannheim bioluminescence kit. Prior to this determination intact cells were centrifuged and lysed in distilled water. Histidine was determined according to Sokolovsky and Vallee [22], tryptophan as described by Spies and Chambers [23], tyrosine according to Uehara et al. [24], SH groups by the method of Sedlak and Lindsay [25], methionine according to McCarthy and Paille [26], phenylalanine as described by Ambrose [27]. Protein was determined by the method of Lowry et al. [28]. Results
TABLE I OXIDATION OF AMINO ACIDS AND LINOLENIC ACID IN S O L U T I O N BY O Z O N E 2 m M solutions of amino acids in 50 m M sodium phosphate buffer (pH 7.4) were exposed to ozone (flow rate, 10 /~mol O 3 / m i n ) at 0 ° C or at 2 0 ° C for 5 min. 3.5 m M linolenic acid solution was exposed to ozone (flow rate, 1 0 / t m o l O 3 / m i n ) at 0 ° C or at 2 0 ° C for 20 min. Initial A532 = 0. % decrease
Cysteine Histidine Tryptophan Tyrosine Methionine Phenylalanine
O°C
20oC
46 21 0 46 16 4
55 30 40 55 25 7
A532 urtits/0.1 ~mol Linolenic acid
0.325
0.655
ture to 0 ° C caused an increase of the velocity of lipid peroxidation (Table II).
Influence of ozone on solubilized substrates and
ghosts Exposure of amino acids in solution to ozone at 2 0 ° C caused oxidation of cysteine, histidine, tryptophan, methionine, tyrosine and phenylalanine. A similar treatment of linolenic acid resulted in a fast peroxidation. On lowering the temperature to 0 ° C the velocities of oxidation decreased (Table I). Addition of isopropanol (100 mM) to the amino acid or linolenic acid solutions inhibited the oxidation of phenylalanine completely and the oxidation of tyrosine for 20%. The oxidation of all other amino acids and linolenic acid was not influenced by isopropanol. Other scavengers, viz. butylated hydroxytoluene, vitamin E and thiourea, could not be used in these studies, as these compounds were rapidly degraded by ozone (unpublished results). Addition of hemoglobin (25 ttM) to the incubation mixture did not affect the oxidation of amino acids and linolenic acid peroxidation. With ghosts similar results were obtained. Again, at 20 o C, ozone caused oxidation of amino acid residues with a concomitant lipid peroxidation. In this case, however, lowering the tempera-
Influence of ozone on intact cells: membrane-associated enzymes, transport systems, A TP and G S H
Ouabain-sensitive Rb÷-transport, which reflects the ( N a ÷ + K÷)-ATPase activity, was very sensitive to ozone exposure, even at a flow rate of 1 #mol o z o n e / m in. Passive, ouabain-insensitive T A B L E II O X I D A T I O N OF A M I N O - A C I D RESIDUES A N D LIPID P E R O X I D A T I O N IN GHOSTS C A U S E D BY EXPOSURE TO O Z O N E Exposure of ghosts (1 mg protein/ml) in 10 m M sodium phosphate buffer (pH 7.4) to ozone (flow rate: 10 ~ m o l / m i n ) was carried out at 0 ° C or at 2 0 ° C for 60 min. Initia! A532 = 0. % decrease
Cysteine Histidine Tryptophan Tyrosine
O°C
20°C
55 26 56 12
60 55 56 40
A532 units/0.1 mg protein Lipid peroxidation
0.59
0.36
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Rb + permeability was concomitantly increased (Fig. 1). Glyceraldehyde-3-phosphate dehydrogenase and acetylcholinesterase activities also declined upon exposure of the erythrocyte suspension to ozone (Fig. 2). Glyceraldehyde-3-phosphate dehydrogenase activity was completely inhibited after 25 min at a flow rate of 10 ffmol o z o n e / m in, whereas acetylcholinesterase was less sensitive. Addition of isopropanol did not affect the ozone-induced inhibition of enzyme activities. Carrier-mediated transport of D-glucose, L-leucine, sulfate and glycerol, as well as passive permeability of L-glucose, glycerol and erythritol were unaffected by ozone until about 60% of the intracellular K + had leaked out of the cells. Beyond that point an increase of all transport velocities was observed, probably due to deterioration of the membrane structure. The intracellular ATP concentration was insensitive to ozone exposure, whereas complete GSH depletion occurred in about 40 min, at an ozone flow rate of 1 0 / ~ m o l / m i n (Fig. 3). Influence of ozone on intact cells: K + leakage and lipid peroxidation Exposure of erythrocytes to ozone led to K ÷ leakage and lipid peroxidation (Fig. 4). Lipid peroxidation was reflected by the formation of thiobarbituric-acid-reactive products and the formation of fluorescent chromolipids. These fluorescent chromolipids are Schiff's bases, formed by a slow reaction of malondialdehyde (a breakdown
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product of peroxidized fatty acids) with amino groups of phospholipids [13]. In all experiments a close parallel between the formation of thiobarbituric acid reactive products and fluorescent chromolipids was found. K + leakage and lipid peroxidation were not influenced by addition of 100 mM isopropanol. Conversion of cellular hemoglobin to carbonmonooxyhemoglobin (by treatment of the cells with CO prior to ozone exposure) did not affect K + efflux or lipid peroxidation. Even after very short ozone-exposure times K + leakage was
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Fig. 3. Effect of exposure of erythrocytes to ozone (10 p~mol/ min) on: O, GSH level (% of initial GSH content); ©, % K + leakage; ~q, % of non-intact cells.
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Fig. 4. K + leakage (© and * ) a n d formation of thiobarbituric-acid-reactive products (1~ and m), during different exposure times of erythrocytes to ozone at a flow rate of 10 ~ m o l / m i n . open symbols: continued ozone exposure; closed symbols: ozone exposure was stopped after 40 min.
attended with hemoglobin release into the medium. Initially the percentage of cell lysis, as judged from hemoglobin release, was only slightly lower than the percentage of K ÷ loss. After longer exposure times, however, cell lysis could not be assayed by this method, as a fast-increasing percentage of cellular hemoglobin was converted into
insoluble degradation products by ozone. Therefore, cell lysis was assayed by centrifugation of the cell suspension through a Percoll solution. It appeared that the percentage of lysed cells was only slightly lower than the percentage of K + loss during the entire ozone-exposure period (Fig. 3). When cells were exposed to ozone for 45 min and subsequently to air, both K ÷ loss and lipid peroxidation ceased (Fig. 4). GSH depletion prior to exposure to ozone had no influence on K ÷ loss and lipid peroxidation (not shown). Addition of 20 mM paracetamol to the incubation medium caused, initially, a virtual complete inhibition of K ÷ leakage, with no concomitant inhibition of lipid peroxidation. After 120 min, however, a very fast K ÷ loss with a concomitant increase of the rate of lipid peroxidation was observed (Fig. 5). With varying concentrations of paracetamol it appeared that the period of inhibition of K ÷ loss was roughly proportional to the paracetamol concentration, suggesting that the protective effect continued until all paracetamol had been oxidized by ozone (not shown). Finally, when the temperature during exposure to ozone was lowered from 2 0 ° C to 0 °C, the velocity of K ÷ leakage decreased about 10%, whereas lipid peroxidation increased considerably (Fig. 6).
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Fig. 5. (A) K + leakage after exposure of erythrocytes to ozone (10/~mol/min), without (©) and with (O) 20 m M paracetamol. (B) Formation of thiobarbituric acid reactive products by ozone (10 g m o l / m i n ) after exposure of erythrocytes (O), of 20 m M paracetamol (~,) and of erythrocytes in the presence of 20 m M paracetamol: C], paracetamol-derived products, and O, lipid peroxidation products.
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Fig. 6. K + leakage (open symbols) and thiobarbituric acid reactivity (A 532, closed symbols) after exposure of e~throcytes to ozone (10 ~ m o l / m i n ) at room temperature (© and O) or at 0 ° C ([] and m).
Discussion Due to the absence of DNA and subcellular structures, the red blood cell is a frequently used model to study the effects of oxidative stress on cellular membranes. The results presented in this paper indicate that exposure of human red blood cells to ozone leads to complete deterioration of the membrane structure, as was found before with other agents, causing oxidative stress, like H202 [5,6], t-butyl hydroperoxide [6,7] and photodynamically generated singlet oxygen [3,4]. Ozone may degrade under physiological conditions, yielding hydroxyl radicals (OH') [29]. As shown by Hoign6 and Bader [30], ozone-induced oxidations may occur by a direct reaction between a target molecule and ozone, or indirectly, with OH" as intermediates. Apparently, ozone-induced oxidation of phenylalanine proceeds via generation of OH', considering the complete inhibition of phenylalanine degradation by the OH'scavenger, isopropanol. However, in intact red blood cells lipid peroxidation, inhibition of enzyme activities and ozone-provoked K + loss were insensitive to isopropanol, indicating that most of the deleterious effects of ozone on membrane structure and function should be ascribed to a direct oxidation of cellular targets by ozone. According to the model experiments (Tables I and
II) possible targets include both oxidizable amino-acid residues and unsaturated fatty acids in membrane phospholipids. Some membrane-associated enzymes, viz. (Na ÷ + K +)-ATPase, glyceraldehyde-3-phosphate dehydrogenase and acetylcholinesterase were inactivated by ozone (Figs. 1 and 2). As these enzymes are also susceptible to other forms of oxidative stress [31,32], it seems, at first sight, conceivable that the mechanisms of enzyme inactivation will be similar, under various conditions of oxidative stress. In some cases this assumption may be valid. For instance, oxidative inactivation of glyceraldehyde-3-phosphate dehydrogenase has been ascribed to oxidation of an active site sulfhydryl group, both in photodynamic [32], H202induced [33] and in ozone-induced [34] enzyme inactivation. In other cases such a generalization is not appropriate, however, as can be shown in the case of ( N a + + K+)-ATPase inhibition. In a detailed study, Chan et al. [35] have shown that the ozone-induced inactivation of this enzyme in erythrocyte membranes is caused primarily by destruction of phospholipids necessary for the enzyme structural integrity. This mechanism can not be involved in photodynamic inactivation of this enzyme system [31], as photooxidative red cell membrane damage is not accompanied by phospholipid destruction [3,4]. More likely, photodynamic inactivation of the erythrocyte membrane (Na + + K+)-ATPase is caused by oxidation of an essential SH group [36]. The fact that different forms of oxidative stress may have different effects on membrane functions is also clearly demonstrated in the studies on transmembrane transport systems. Photodynamically induced oxidative stress leads to a rapid prelytic inhibition of cartier-mediated transport of glucose, leucine, sulfate and glycerol, and increased passive permeability of glycerol and other nonelectrolytes [37]. It could be shown that these effects were caused by photooxidation of membrane proteins. In contrast, ozone does not affect any of these transport systems in the prelytical phase, although in model systems the same amino acids, susceptible to photooxidation, are also degraded by ozone (Tables I and II). Presumably, the accessibility of the essential groups in intact membranes to ozone and photodynamically gener-
117 ated singlet oxygen is different. Also with respect to lipid peroxidation, different modalities of oxidative stress evoke different effects. Photodynamically generated singlet oxygen does not initiate appreciable lipid peroxidation in intact red blood cells, although with solubilized unsaturated fatty acids extensive photodynamic lipid peroxidation can be observed [4]. Apparently the short-living singlet oxygen can not reach the unsaturated fatty-acid chains in intact red cell membranes. H20~, t-butyl hydroperoxide [6] and ozone (Fig. 4), on the other hand, cause significant lipid peroxidation. Even so, the mechanisms involved are clearly different with each of these reactive species. With several oxidative species experimental evidence indicates a close interdependence between hemoglobin oxidation and lipid peroxidation [38]. With HzO ~ and t-butyl hydroperoxide, for instance, lipid peroxidation occurred in model systems only in the presence of hemoglobin [6]. In accordance, H202- and t-butyl hydroperoxide-induced lipid peroxidation in intact erythrocytes was significantly affected by conversion of cellular hemoglobin into carbonmonooxyhemoglobin. However, these experiments also indicated that the mechanisms of hemoglobin-dependent peroxidation were dissimilar with these two oxidative species: conversion of hemoglobin to carbonmonooxyhemoglobin strongly inhibited H~O2-induced lipid peroxidation, whereas it augmented lipid peroxidation, caused by t-butyl hydroperoxide [6]. Exposure of intact red blood cells to ozone caused a rapid oxidation of hemoglobin to insoluble degradation products. It seems unlikely, however, that ozone-induced oxidation of hemoglobin plays an intermediate role in the concomitantly occurring lipid peroxidation. Conversion of hemoglobin into carbonmonooxyhemoglobin prior to exposure to ozone had no effect on ozone-induced lipid peroxidation, contrary to the results with H zO2 and t-butyl hydroperoxide. Moreover, in model systems, ozone-induced lipid peroxidation appeared to be hemoglobin-independent (Table I), again in contrast to the results with H~Oz and t-butyl hydroperoxide [6]. Oxidative stress imposed on red blood cells causes K ÷ loss in all cases studied thus far. In this respect ozone is no exception, but the mechanism
of K + leakage is different from that in all other cases studied in sufficient detail. K + loss induced by photooxidation [3,4], H202 and t-butylhydroperoxide [6] is caused by an increased passive cation permeability of the membrane, leading to cell swelling and, ultimately, colloid-osmotic hemolysis. Ozone-induced K ÷ loss, on the other hand, exhibited the characteristics of an all-ornone response, viz. cessation of K + loss upon discontinuation of ozone exposure (Fig. 4) and a close parallel between the percentage of K ÷ loss and the percentage of disrupted cells (Fig. 3). The ozone-induced membrane disruption can not be attributed to ATP-depletion, as the cellular ATP concentration was not affected by ozone. GSH plays, in general, a key role in the protection of cells against oxidative stress [39]. Therefore, the ozone-induced GSH-depletion, as shown in Fig. 3, might be a major event in the context of ozone-induced K ÷ loss. However, further experiments showed that this is not the case. Pretreatment of erythrocytes with diamide leads to a virtually complete GSH-depletion [20]. Still, diamide pretreatment did not increase the sensitivity of red blood cells to ozone, despite the fact that diamide itself may cause some membrane damage by reaction with membrane sulfhydryl groups [6]. In previous papers it has been shown that there is no causal relationship between lipid peroxidation and K + leakage with photodynamically generated singlet oxygen, H202 and t-butyl hydroperoxide as oxidizing species [4,6]. Although the experimental results presented in this paper do not completely exclude a connection between ozoneinduced lipid peroxidation and K + loss, in this case, also several observations argue against a close causal relationship between these two phenomena. In the first place, both H202 and ozone cause K ÷ loss and lipid peroxidation. With both agents the extent of lipid peroxidation, as judged from the generation of thiobarbituric-acid-reactive products, is about the same when 50% of the K ÷ ions have been released to the medium. As it was shown previously that with H202 the observed K + loss was not caused by lipid peroxidation [6], it seems improbable that the same extent of lipid peroxidation during exposure to ozone would be causally related to the concomitant K ÷ loss. Further, when erythrocytes were exposed to ozone in
118
the presence of paracetamol, K + loss was almost completely inhibited, whereas lipid peroxidation was not affected (Fig. 5). Although the background of this paracetamol effect is not clear and is being studied in detail at present, this drug apparently dissociates ozone-induced K + loss and lipid peroxidation, rendering a causal relationship between these two phenomena unlikely. Finally, a similar dissociation was observed when the temperature during ozone exposure was lowered from 2 0 ° C to 0 ° C : K + loss was slightly reduced, whereas lipid peroxidation was significantly enhanced (Fig. 6). It is obvious that a generally applicable model to study the effects of oxidative stress in biological systems does not exist. For instance, whereas destruction of red blood cells by H202 is clearly caused by membrane desintegration [6], H202-induced killing of mouse 3T3 cells should presumably be attributed to DNA damage [40]. The results presented in this paper emphasize that similar restrictions exist when studying various forms of oxidative stress on the same biological system, e.g., the red blood cell. The ultimate effects may vary greatly, depending on the oxidative species. But even if the ultimate effects, like K + loss or lipid peroxidation, are identical, the underlying mechanisms may, apparently, be quite dissimilar with different modalities of oxidative stress.
Acknowledgement This work was supported by a grant from the Netherlands Organization for the Advancement of Pure Scientific Research, Z.W.O. (grant number 98-61).
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80oldstein, B.D. and Balchum, O.J. (1967) Proc. Sci. Exp. Biol. Med, 126, 356 358 9 Verweij, H. and Van Steveninck. J. (1979) Chemosphere 7. 443 -448 10 Verweij, H. and Van Steveninck, J. (1981) Biochem. Pharmacol. 30, 1033 1037 11 Stocks, J. and Dormandy, T.L. (1971) Br. J. Haematol. 20, 95 1ll 12 Asakawa, T. and Matsushita, S. (1980) Lipids 15, 137-140 13 Jain, S.K. and Hochstein, P. (1980) Biochem. Biophys. Res. Commun. 92, 247-254 14 Rega, A.F., Rothstein, A. and Weed, R.I. (1967) J. Cell. Physiol. 70~ 45-52 15 Ellman, G.L., Courtrey van Andres, K.D. and Featherstone, R.M. (1961) Biochem. Pharmacol. 7, 88-95 16 Wu, R. and Racker, E. (1959) J. Biol. Chem. 234, 1029-1035 17 Devalia, J.L. and McLean, A.E.M. (1983) Biochem. Pharmacol. 32, 2602-2603 18 Carlsen, A. and Wieth, J.O. (1976) Acta Physiol. Scand. 97, 501-513 19 Beut[er, E., Duron, O. and Kelly, B.H. (1963) J. Lab. Clin. Med. 61,882-890 20 Kosower, N.S., Song, K.R. and Kosower, E.M. (1969) Biochim. Biophys. Acta 192, 1-7 21 Crosby, W.H., Munn, J.I. and Furth, F.W. (1954) U.S. Armed Forces Med. J. 5, 693 22 Sokolovsky, M. and Vallee, B.L. (1966) Biochemistry 5, 3574-3581 23 Spies, J.R. and Chambers, D.C. (1949) Anal. Chem. 21, 1249-1266 24 Uehara, K., Mannen, S. and Kishida, K. (1970) J. Biochem. 68, 119-124 25 Sedlak, J. and Lindsay, R.H. (1968) Anal. Biochem. 25~ 192 - 205 26 McCarthy, T.C. and Paille, M.M. (1959) Biochem. Biophys. Res. Commun. 1, 29-33 27 Ambrose, J.A. (1969) Clin. Chem. 15, 15-23 28 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.I. (1951) J. Biol. Chem. 193, 265-275 29 Grimes, H.D., Perkins, K.K. and Boss, W.F. (1983) Plant Physiol. 72, 1016-1020 30 Hoign& J. and Bader, H. (1975) Science 190, 782 784 31 Schothorst, A.A., Van Steveninck, J., Went, L.N. and Suurmond, D. (197l) Clin. Chim. Acta 33, 207-213 32 Dubbelman, T.M.A.R., De Goeij, A.F.P.M. and Van Steveninck, J. (1978) Photochem. Photobiol. 28, 197-204 33 Little, C. and O'Brien, P.J. (1969) Eur. J. Biochem. 10, 533-538 34 Knight, K.L. and Mud& J.B. (1984) Arch. Biochem. Biophys. 229, 259-269 35 Chan, P.C., Kindya, R.J. and Kesner, L. (1977) J. Biol. Chem. 252, 8537-8541 36 Girotti, A.W. (1976) Photochem. Photobiol. 24, 525-532 37 Dubbelman, T.M.A.R., De Goeij, A.F.P.M. and Van Steveninck, J. (1980) Biochim. Biophys. Acta 595, 133-139 38 Szebeni, J., Winterbourn, C.C. and Carrell, R.W. (1984) Biochem. J. 220, 685-692 39 Meister, A. and Anderson, M.C. (1983) Annu. Rev. Biochem. 52, 711-760 40 Mello-Filho, A.C., Hoffmann, M.E. and Meneghini, R. (1984) Biochem. J. 218, 273 275
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BBA 22714
The influence of ozone on human red blood cells. Comparison with other mechanisms of oxidative stress
Jolanda Van der Zee, Karmi Tijssen-Christianse, Thomas M.A.R. Dubbelman and Johnny ~¢an Steveninck Department of Medical Biochemistry, Syloius Laboratories, Leiden (The Netherlands) (Received 1 September 1986) (Revised manuscript received 7 November 1986)
Key words: Ozone; Oxidative membrane damage; Lipid peroxidation; Potassium ion leakage; (Erythrocyte)
Exposure of red blood cells to ozone resulted in K + leakage, lipid peroxidation and inhibition of some membrane-associated enzymes. On the other hand, carrier-mediated transport of glucose, leucine, sulfate and glycerol and the nonspecific permeation of glycerol, L-glucose and erythritol were not affected by ozone. The cellular level of reduced glutathione declined, whereas the ATP content of the cells was quite insensitive to ozone exposure. It was shown that, most probably, lipid peroxidation and K ÷ leakage are not causally related. Further, K ÷ leakage did not reflect gradual, progressive loss of K + from all cells simultaneously, but occurred in an all-or-none fashion. Finally, ozone-induced damage was compared to damage induced by H202, t-butyl hydroperoxide and photosensitizers plus light. It appeared that the pathways leading to membrane deterioration are quite dissimilar in these various forms of oxidative stress.
Introduction Oxidative damage to biological systems is thought to form the basis of a number of physiological and pathological phenomena as diverse as inflammation, ageing and carcinogenesis [1,2]. In recent literature, many examples of the deleterious effects of reactive oxygen species on cells have been presented. In these studies erythrocytes have often been used as a convenient model cell. Membrane constituents proved to be very susceptible to oxidative agents and membrane deterioration frequently plays an important role in oxidative stress. For example, illumination of human erythrocytes with visible light in the presence of
Correspondence: J. van Steveninck, Department of Medical Biochemistry, Sylvius Laboratories, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
protoporphyrin as sensitizer leads to extensive membrane damage, reflected by K ÷ leakage and, ultimately, hemolysis. This is most likely caused by direct photooxidation of amino acids, such as histidine, tryptophan, tyrosine and cysteine, within membrane proteins. A limited lipid peroxidation was observed only after virtually all the K ÷ had leaked out of the cells [3,4]. H202-induced membrane damage in red blood cells is reflected by extensive lipid peroxidation, oxidation of membrane SH groups and increased passive cation permeability [5,6]. In this case, oxidation of SH groups accounted for the observed K ÷ leakage, whereas lipid peroxidation was not involved in this process. Utilizing t-butyl hydroperoxide to induce oxidative stress in erythrocytes, again lipid peroxidation and K+-leakage were observed. It could be shown, however, that the mechanisms of H202- and t-butyl hydroperoxide-induced lipid peroxidation were different. Also the pathway
0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
112 leading to K+-leakage appeared to differ with these two oxidants [6,7]. Although in all these cases of oxidative stress the ultimate result was complete deterioration of membrane structure and functions, the mechanisms of oxidative damage were apparently quite dissimilar. In this context the effects of ozone on red cell membranes were investigated in detail. It is known that ozone, an important component of photochemical air pollution and a very powerful oxidant, causes lipid peroxidation, crosslinking of membrane proteins and K + leakage in erythrocytes [8-10], but the mechanism by which ozone causes membrane damage is still unknown. The present investigations demonstrate that the mechanism of ozone-induced damage differs again from the pathways of H202-, t-butyl hydroperoxide and photodynamically induced damage. Methods Red blood cell suspensions, hemoglobin-free ghosts, linolenic acid and amino acid solutions were prepared as described before [6]. In experiments with intact cells the erythrocytes were suspended in phosphate-buffered saline, Ozone was generated by a Fisher-Ozone Generator Model 501 at a rate of 10 or 1 ~ m o l / m i n with an airflow of 0.4 m l / m i n . The 0 2 / 0 3 mixture was conducted over 10 ml of the stirred cell suspension or solution. Ozone production was measured by titration with KI-Na~S203-starch in 5 m M sodium phosphate (pH 7.4). K + efflux from red blood cells was determined with a flame photometer and expressed as percentage of total efflux, evoked by lysis of the cells in distilled water. Lipid peroxidation in cells was assayed by measuring thiobarbituric-acid-reactive products, as described by Stocks and D o r m a n d y [11]. Lipid peroxidation in ghosts or linolenic acid solutions was determined according to Asakawa and Matsushita [12]. Measurements of lipid peroxidation in the presence of paracetamol were complicated by the fact that paracetamol was oxidized by ozone, yielding a thiobarbituric-acidreactive product with an absorbance spectrum partially overlapping the spectrum of the thiobarbituric-acid-reactive lipid peroxidation products. The paracetamol-derived product, however,
exhibited an absorbance maximum at 548 nm, whereas the lipid peroxidation products had a maximal absorbance at 532 nm. Thus, lipid peroxidation could still be measured by absorbance readings at both wavelengths and appropriate correction of the absorbance at 532 nm. Chromolipids were measured as described by Jain and Hochstein [13]. The ( N A P + K~)-ATPase activity was determined by a modification of the method of Rega et al. [14]. Briefly, cells were exposed to ozone, centrifuged and resuspended at 5% hematocrit in a buffer containing 5 mM RbC1, 145 mM NaCI, 2 m M MgC12, 10 m M inosine and 10 m M Tris (pH 7.4) at 37°C. After 5 min incubation, S6Rb was added and the 86Rb influx was determined. Active Rb uptake was inhibited by the addition of 1 mM ouabain. The method of Ellman [15] was used for the assay of acetylcholinesterase activity and the method of Wu and Racker [16] for glyceraldehyde-3-phosphate dehydrogenase activity. The number of intact red blood cells was determined by a modification of the method of Devalia and McLean [17]. A Percoll solution was mixed with 10-times concentrated phosphatebuffered saline in the ratio of 9 : 1 (v/v). This was further diluted with normal-strength phosphatebuffered saline in the ratio of 42.5:57.5 (v/v). 2 ml of a 10% erythrocyte suspension were loaded on 8 ml of the Percoll solution and centrifuged for 10 min at 500 rpm. Intact cells were recovered in the bottom fraction and counted after delution in phosphate-buffered saline. This fraction of intact cells was also used in transport studies and for A T P and G S H determinations. The transport velocities of D-glucose, L-glucose, L-leucine, glycerol, erythritol and sulfate were assayed by measuring the efflux of radioactive substrate from preloaded cells. Cells were loaded with labeled substrate in the presence of 1 m M unlabeled substrate at 37°C in phosphate-buffered saline. After loading, the cells were exposed to ozone and subsequently centrifuged to remove extracellular label. Cells were resuspended in phosphate-buffered saline at 5% hematocrit. D-Ghicose, L-leucine and glycerol efflux was determined at 4°C. The other efflux experiments were performed at 20°C. Passive diffusion of glycerol was measured in the presence of 0.1 mM Cu 2+, completely inhibiting
113
the facilitated diffusion of glycerol [18]. Cellular GSH content was determined according to Beutler [19]. GSH-depletion prior to exposure of the cells to ozone was brought about by treatment of the cells with diamide [20]. Hemoglobin release into the medium was measured according to Crosby et al. [21]. The ATP content of intact cells was measured with a BoehringerMannheim bioluminescence kit. Prior to this determination intact cells were centrifuged and lysed in distilled water. Histidine was determined according to Sokolovsky and Vallee [22], tryptophan as described by Spies and Chambers [23], tyrosine according to Uehara et al. [24], SH groups by the method of Sedlak and Lindsay [25], methionine according to McCarthy and Paille [26], phenylalanine as described by Ambrose [27]. Protein was determined by the method of Lowry et al. [28]. Results
TABLE I OXIDATION OF AMINO ACIDS AND LINOLENIC ACID IN S O L U T I O N BY O Z O N E 2 m M solutions of amino acids in 50 m M sodium phosphate buffer (pH 7.4) were exposed to ozone (flow rate, 10 /~mol O 3 / m i n ) at 0 ° C or at 2 0 ° C for 5 min. 3.5 m M linolenic acid solution was exposed to ozone (flow rate, 1 0 / t m o l O 3 / m i n ) at 0 ° C or at 2 0 ° C for 20 min. Initial A532 = 0. % decrease
Cysteine Histidine Tryptophan Tyrosine Methionine Phenylalanine
O°C
20oC
46 21 0 46 16 4
55 30 40 55 25 7
A532 urtits/0.1 ~mol Linolenic acid
0.325
0.655
ture to 0 ° C caused an increase of the velocity of lipid peroxidation (Table II).
Influence of ozone on solubilized substrates and
ghosts Exposure of amino acids in solution to ozone at 2 0 ° C caused oxidation of cysteine, histidine, tryptophan, methionine, tyrosine and phenylalanine. A similar treatment of linolenic acid resulted in a fast peroxidation. On lowering the temperature to 0 ° C the velocities of oxidation decreased (Table I). Addition of isopropanol (100 mM) to the amino acid or linolenic acid solutions inhibited the oxidation of phenylalanine completely and the oxidation of tyrosine for 20%. The oxidation of all other amino acids and linolenic acid was not influenced by isopropanol. Other scavengers, viz. butylated hydroxytoluene, vitamin E and thiourea, could not be used in these studies, as these compounds were rapidly degraded by ozone (unpublished results). Addition of hemoglobin (25 ttM) to the incubation mixture did not affect the oxidation of amino acids and linolenic acid peroxidation. With ghosts similar results were obtained. Again, at 20 o C, ozone caused oxidation of amino acid residues with a concomitant lipid peroxidation. In this case, however, lowering the tempera-
Influence of ozone on intact cells: membrane-associated enzymes, transport systems, A TP and G S H
Ouabain-sensitive Rb÷-transport, which reflects the ( N a ÷ + K÷)-ATPase activity, was very sensitive to ozone exposure, even at a flow rate of 1 #mol o z o n e / m in. Passive, ouabain-insensitive T A B L E II O X I D A T I O N OF A M I N O - A C I D RESIDUES A N D LIPID P E R O X I D A T I O N IN GHOSTS C A U S E D BY EXPOSURE TO O Z O N E Exposure of ghosts (1 mg protein/ml) in 10 m M sodium phosphate buffer (pH 7.4) to ozone (flow rate: 10 ~ m o l / m i n ) was carried out at 0 ° C or at 2 0 ° C for 60 min. Initia! A532 = 0. % decrease
Cysteine Histidine Tryptophan Tyrosine
O°C
20°C
55 26 56 12
60 55 56 40
A532 units/0.1 mg protein Lipid peroxidation
0.59
0.36
114
Rb + permeability was concomitantly increased (Fig. 1). Glyceraldehyde-3-phosphate dehydrogenase and acetylcholinesterase activities also declined upon exposure of the erythrocyte suspension to ozone (Fig. 2). Glyceraldehyde-3-phosphate dehydrogenase activity was completely inhibited after 25 min at a flow rate of 10 ffmol o z o n e / m in, whereas acetylcholinesterase was less sensitive. Addition of isopropanol did not affect the ozone-induced inhibition of enzyme activities. Carrier-mediated transport of D-glucose, L-leucine, sulfate and glycerol, as well as passive permeability of L-glucose, glycerol and erythritol were unaffected by ozone until about 60% of the intracellular K + had leaked out of the cells. Beyond that point an increase of all transport velocities was observed, probably due to deterioration of the membrane structure. The intracellular ATP concentration was insensitive to ozone exposure, whereas complete GSH depletion occurred in about 40 min, at an ozone flow rate of 1 0 / ~ m o l / m i n (Fig. 3). Influence of ozone on intact cells: K + leakage and lipid peroxidation Exposure of erythrocytes to ozone led to K ÷ leakage and lipid peroxidation (Fig. 4). Lipid peroxidation was reflected by the formation of thiobarbituric-acid-reactive products and the formation of fluorescent chromolipids. These fluorescent chromolipids are Schiff's bases, formed by a slow reaction of malondialdehyde (a breakdown
300
100
80 7O 6O ~ 5O
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'°I , \
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60
80
100
120
time (rain)
Fig. 2. Ozone- (10 /~mol/min) induced inhibition of glyceraldehyde-3-phosphate dehydrogenase ([]) and acetylcholinesterase ( O ) activity in erythrocytes.
product of peroxidized fatty acids) with amino groups of phospholipids [13]. In all experiments a close parallel between the formation of thiobarbituric acid reactive products and fluorescent chromolipids was found. K + leakage and lipid peroxidation were not influenced by addition of 100 mM isopropanol. Conversion of cellular hemoglobin to carbonmonooxyhemoglobin (by treatment of the cells with CO prior to ozone exposure) did not affect K + efflux or lipid peroxidation. Even after very short ozone-exposure times K + leakage was
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~200
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Fig. t. Passive uptake (FI) and active transport ( O ) o f Rb ~ in
e~throcytes, both expressed as percentage of initial transport velocity, as a function of exposure time to ozone (1/~mol/min).
20
4.0 60 80 time (rain)
100
120
Fig. 3. Effect of exposure of erythrocytes to ozone (10 p~mol/ min) on: O, GSH level (% of initial GSH content); ©, % K + leakage; ~q, % of non-intact cells.
115 1.6 100 1.4
go
~ c~
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Fig. 4. K + leakage (© and * ) a n d formation of thiobarbituric-acid-reactive products (1~ and m), during different exposure times of erythrocytes to ozone at a flow rate of 10 ~ m o l / m i n . open symbols: continued ozone exposure; closed symbols: ozone exposure was stopped after 40 min.
attended with hemoglobin release into the medium. Initially the percentage of cell lysis, as judged from hemoglobin release, was only slightly lower than the percentage of K ÷ loss. After longer exposure times, however, cell lysis could not be assayed by this method, as a fast-increasing percentage of cellular hemoglobin was converted into
insoluble degradation products by ozone. Therefore, cell lysis was assayed by centrifugation of the cell suspension through a Percoll solution. It appeared that the percentage of lysed cells was only slightly lower than the percentage of K + loss during the entire ozone-exposure period (Fig. 3). When cells were exposed to ozone for 45 min and subsequently to air, both K ÷ loss and lipid peroxidation ceased (Fig. 4). GSH depletion prior to exposure to ozone had no influence on K ÷ loss and lipid peroxidation (not shown). Addition of 20 mM paracetamol to the incubation medium caused, initially, a virtual complete inhibition of K ÷ leakage, with no concomitant inhibition of lipid peroxidation. After 120 min, however, a very fast K ÷ loss with a concomitant increase of the rate of lipid peroxidation was observed (Fig. 5). With varying concentrations of paracetamol it appeared that the period of inhibition of K ÷ loss was roughly proportional to the paracetamol concentration, suggesting that the protective effect continued until all paracetamol had been oxidized by ozone (not shown). Finally, when the temperature during exposure to ozone was lowered from 2 0 ° C to 0 °C, the velocity of K ÷ leakage decreased about 10%, whereas lipid peroxidation increased considerably (Fig. 6).
16
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IO0 1.4 9O 12
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0.6
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120
14.0
20
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60 BO 100 time ( mini
120 140
Fig. 5. (A) K + leakage after exposure of erythrocytes to ozone (10/~mol/min), without (©) and with (O) 20 m M paracetamol. (B) Formation of thiobarbituric acid reactive products by ozone (10 g m o l / m i n ) after exposure of erythrocytes (O), of 20 m M paracetamol (~,) and of erythrocytes in the presence of 20 m M paracetamol: C], paracetamol-derived products, and O, lipid peroxidation products.
116
100 14. 90
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70
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04
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10 20
4.0 60 time (rain}
80
100
Fig. 6. K + leakage (open symbols) and thiobarbituric acid reactivity (A 532, closed symbols) after exposure of e~throcytes to ozone (10 ~ m o l / m i n ) at room temperature (© and O) or at 0 ° C ([] and m).
Discussion Due to the absence of DNA and subcellular structures, the red blood cell is a frequently used model to study the effects of oxidative stress on cellular membranes. The results presented in this paper indicate that exposure of human red blood cells to ozone leads to complete deterioration of the membrane structure, as was found before with other agents, causing oxidative stress, like H202 [5,6], t-butyl hydroperoxide [6,7] and photodynamically generated singlet oxygen [3,4]. Ozone may degrade under physiological conditions, yielding hydroxyl radicals (OH') [29]. As shown by Hoign6 and Bader [30], ozone-induced oxidations may occur by a direct reaction between a target molecule and ozone, or indirectly, with OH" as intermediates. Apparently, ozone-induced oxidation of phenylalanine proceeds via generation of OH', considering the complete inhibition of phenylalanine degradation by the OH'scavenger, isopropanol. However, in intact red blood cells lipid peroxidation, inhibition of enzyme activities and ozone-provoked K + loss were insensitive to isopropanol, indicating that most of the deleterious effects of ozone on membrane structure and function should be ascribed to a direct oxidation of cellular targets by ozone. According to the model experiments (Tables I and
II) possible targets include both oxidizable amino-acid residues and unsaturated fatty acids in membrane phospholipids. Some membrane-associated enzymes, viz. (Na ÷ + K +)-ATPase, glyceraldehyde-3-phosphate dehydrogenase and acetylcholinesterase were inactivated by ozone (Figs. 1 and 2). As these enzymes are also susceptible to other forms of oxidative stress [31,32], it seems, at first sight, conceivable that the mechanisms of enzyme inactivation will be similar, under various conditions of oxidative stress. In some cases this assumption may be valid. For instance, oxidative inactivation of glyceraldehyde-3-phosphate dehydrogenase has been ascribed to oxidation of an active site sulfhydryl group, both in photodynamic [32], H202induced [33] and in ozone-induced [34] enzyme inactivation. In other cases such a generalization is not appropriate, however, as can be shown in the case of ( N a + + K+)-ATPase inhibition. In a detailed study, Chan et al. [35] have shown that the ozone-induced inactivation of this enzyme in erythrocyte membranes is caused primarily by destruction of phospholipids necessary for the enzyme structural integrity. This mechanism can not be involved in photodynamic inactivation of this enzyme system [31], as photooxidative red cell membrane damage is not accompanied by phospholipid destruction [3,4]. More likely, photodynamic inactivation of the erythrocyte membrane (Na + + K+)-ATPase is caused by oxidation of an essential SH group [36]. The fact that different forms of oxidative stress may have different effects on membrane functions is also clearly demonstrated in the studies on transmembrane transport systems. Photodynamically induced oxidative stress leads to a rapid prelytic inhibition of cartier-mediated transport of glucose, leucine, sulfate and glycerol, and increased passive permeability of glycerol and other nonelectrolytes [37]. It could be shown that these effects were caused by photooxidation of membrane proteins. In contrast, ozone does not affect any of these transport systems in the prelytical phase, although in model systems the same amino acids, susceptible to photooxidation, are also degraded by ozone (Tables I and II). Presumably, the accessibility of the essential groups in intact membranes to ozone and photodynamically gener-
117 ated singlet oxygen is different. Also with respect to lipid peroxidation, different modalities of oxidative stress evoke different effects. Photodynamically generated singlet oxygen does not initiate appreciable lipid peroxidation in intact red blood cells, although with solubilized unsaturated fatty acids extensive photodynamic lipid peroxidation can be observed [4]. Apparently the short-living singlet oxygen can not reach the unsaturated fatty-acid chains in intact red cell membranes. H20~, t-butyl hydroperoxide [6] and ozone (Fig. 4), on the other hand, cause significant lipid peroxidation. Even so, the mechanisms involved are clearly different with each of these reactive species. With several oxidative species experimental evidence indicates a close interdependence between hemoglobin oxidation and lipid peroxidation [38]. With HzO ~ and t-butyl hydroperoxide, for instance, lipid peroxidation occurred in model systems only in the presence of hemoglobin [6]. In accordance, H202- and t-butyl hydroperoxide-induced lipid peroxidation in intact erythrocytes was significantly affected by conversion of cellular hemoglobin into carbonmonooxyhemoglobin. However, these experiments also indicated that the mechanisms of hemoglobin-dependent peroxidation were dissimilar with these two oxidative species: conversion of hemoglobin to carbonmonooxyhemoglobin strongly inhibited H~O2-induced lipid peroxidation, whereas it augmented lipid peroxidation, caused by t-butyl hydroperoxide [6]. Exposure of intact red blood cells to ozone caused a rapid oxidation of hemoglobin to insoluble degradation products. It seems unlikely, however, that ozone-induced oxidation of hemoglobin plays an intermediate role in the concomitantly occurring lipid peroxidation. Conversion of hemoglobin into carbonmonooxyhemoglobin prior to exposure to ozone had no effect on ozone-induced lipid peroxidation, contrary to the results with H zO2 and t-butyl hydroperoxide. Moreover, in model systems, ozone-induced lipid peroxidation appeared to be hemoglobin-independent (Table I), again in contrast to the results with H~Oz and t-butyl hydroperoxide [6]. Oxidative stress imposed on red blood cells causes K ÷ loss in all cases studied thus far. In this respect ozone is no exception, but the mechanism
of K + leakage is different from that in all other cases studied in sufficient detail. K + loss induced by photooxidation [3,4], H202 and t-butylhydroperoxide [6] is caused by an increased passive cation permeability of the membrane, leading to cell swelling and, ultimately, colloid-osmotic hemolysis. Ozone-induced K ÷ loss, on the other hand, exhibited the characteristics of an all-ornone response, viz. cessation of K + loss upon discontinuation of ozone exposure (Fig. 4) and a close parallel between the percentage of K ÷ loss and the percentage of disrupted cells (Fig. 3). The ozone-induced membrane disruption can not be attributed to ATP-depletion, as the cellular ATP concentration was not affected by ozone. GSH plays, in general, a key role in the protection of cells against oxidative stress [39]. Therefore, the ozone-induced GSH-depletion, as shown in Fig. 3, might be a major event in the context of ozone-induced K ÷ loss. However, further experiments showed that this is not the case. Pretreatment of erythrocytes with diamide leads to a virtually complete GSH-depletion [20]. Still, diamide pretreatment did not increase the sensitivity of red blood cells to ozone, despite the fact that diamide itself may cause some membrane damage by reaction with membrane sulfhydryl groups [6]. In previous papers it has been shown that there is no causal relationship between lipid peroxidation and K + leakage with photodynamically generated singlet oxygen, H202 and t-butyl hydroperoxide as oxidizing species [4,6]. Although the experimental results presented in this paper do not completely exclude a connection between ozoneinduced lipid peroxidation and K + loss, in this case, also several observations argue against a close causal relationship between these two phenomena. In the first place, both H202 and ozone cause K ÷ loss and lipid peroxidation. With both agents the extent of lipid peroxidation, as judged from the generation of thiobarbituric-acid-reactive products, is about the same when 50% of the K ÷ ions have been released to the medium. As it was shown previously that with H202 the observed K + loss was not caused by lipid peroxidation [6], it seems improbable that the same extent of lipid peroxidation during exposure to ozone would be causally related to the concomitant K ÷ loss. Further, when erythrocytes were exposed to ozone in
118
the presence of paracetamol, K + loss was almost completely inhibited, whereas lipid peroxidation was not affected (Fig. 5). Although the background of this paracetamol effect is not clear and is being studied in detail at present, this drug apparently dissociates ozone-induced K + loss and lipid peroxidation, rendering a causal relationship between these two phenomena unlikely. Finally, a similar dissociation was observed when the temperature during ozone exposure was lowered from 2 0 ° C to 0 ° C : K + loss was slightly reduced, whereas lipid peroxidation was significantly enhanced (Fig. 6). It is obvious that a generally applicable model to study the effects of oxidative stress in biological systems does not exist. For instance, whereas destruction of red blood cells by H202 is clearly caused by membrane desintegration [6], H202-induced killing of mouse 3T3 cells should presumably be attributed to DNA damage [40]. The results presented in this paper emphasize that similar restrictions exist when studying various forms of oxidative stress on the same biological system, e.g., the red blood cell. The ultimate effects may vary greatly, depending on the oxidative species. But even if the ultimate effects, like K + loss or lipid peroxidation, are identical, the underlying mechanisms may, apparently, be quite dissimilar with different modalities of oxidative stress.
Acknowledgement This work was supported by a grant from the Netherlands Organization for the Advancement of Pure Scientific Research, Z.W.O. (grant number 98-61).
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