Exp Toxic Pathol 1992; 44: 96-101 Gustav Fischer Verlag Jena
Polish Academy of Sciences, Medical Research Centre, Department of Neurochemistry and Laboratory of Ultrastructure of the Nervous System, Warsaw, Poland
Biochemical and morphological changes in rat brain synaptosomes after exposure to normobaric hyperoxia in vivo 1) By W. GORDON-MAJSZAK and B. GAJKOWSKA With 6 figures Received: October 25, 1989; Accepted: February 16, 1990
Address for correspondence: Dr. WANDA GORDON-MAJSZAK, Polish Academy of Sciences, Medical Research Centre, Department of Neurochemistry, Dworkowa 3,00-784 Warsaw, Poland Key words: synaptosomes; hyperoxia, normobaric; brain, hyperoxia; oxygen toxicity; thiobarbituric acid-reactive material; peroxide radicals
Summary Adult rats were submitted to normobaric hyperoxia for to 24 h, then the brain synaptosomes were isolated and their metabolic and morphologic properties were studied. Hyperoxia lasting 1-2 h .Bignificantly increased the content of thiobarbituric acid-reactive material (TBAR) and decreased the level of protein thid groups. During the next 5-8 h of hyperoxia SH groups as well as TBAR content became almost normal, reflecting adaptation of the animals to an elevated oxygen tension. After 24 h of hyperoxia a maximal increase in the TBAR content and parallel fall in protein thiol groups were noted. Simultaneously, significant morphological differences between control synaptosomes and synaptosomes isolated from rats exposed to 24 h oxygenation were observed in electron microscopy. The high-affinity dopamine uptake in hyperoxic synaptosomes was significantly increased in all experimental groups. A specific high sensitivity of the dopamine uptake system in synaptoplasmatic membranes to the free radical modification of the membrane structure is suggested.
Introduction Although oxygen is of vital importance to most higher organisms, it becomes toxic even at moderately elevated concentrations. The toxicity of oxygen is supposedly based on its tendency to form reactive intermediates, i.e. 0/, H2 0 2 • OH~ and 102 in the reduction to H2 0. Alternatively, more stable but more cytotoxic intermediates, e.g. aldehydes (4) may be formed by the reaction of free radicals with various cellular molecules. These activated oxygen species are believed to be detoxified by a cellular antioxygenic defence system which may be overwhelmed when the cells are exposed to conditions of increased "oxidative stress". Among the cellular 1) This work was supported by the Polish Academy of Sciences within project 06-02
96
Exp Toxic Pathol44 (1992) 2
defence systems against peroxidative damage, reduced glutathione (GSH) plays a major role (8, 15, 27). Prolonged exposure of man1mals to > 95 % O2 may lead to severe tissue damage. The LDso for adult rats is 72 h; however, rats generally survive exposure to 80-85 % O2 for 5 to 7 days and then they are resistant to subsequent exposure to > 95 % O 2 . This adaptation to hyperoxia is correlated with a increased activity of several antioxidant enzymes (3, 6, 7, 24). Prolonged hyperoxic exposure induces free radicals which participate in pathological mechanisms by causing direct pulmonary injury and/or by forming a secondary chemotactic factor for neutrophils (14). On the other hand, the effect of hyperbaric oxygen in producing seizures has been recognized for many years (1, 10, 25, 26, 29). Although descriptions of the clinical features of oxygen toxicity are numerous, the mechanisms involved have not been completely elucidated. Since the brain is a postmitotic tissue, oxidative events may be very destructive, and lead to irreversible neuronal injury. The brain is particularly prone to lipid peroxidation since it has a high content of fatty acids, such as 20: 4 and 22: 6 which are very susceptible to peroxidation. In certain parts of the brain, such as the extrapyramidal tract,especially in humans, there is also a large content of none-heme iron, which in very specific in vivo conditions may participate in the peroxidation processes (28). Our previous studies demonstrated a correlation between the time of oxygenation and peroxidation of lipid in the brain (9, 18). The relation between radical oxidation and in vivo yaminobutyric acid transport in isolated brain synaptosomes w,as also demonstrated (9). The aim of the present study was to examine, in an experimental model of in vivo normobaric hyperoxia, the interrelation between the development of morphological changes in synaptosomes and in dopamine transport in the isolated brain synaptosomes after induction of peroxidation processes in the brain by hyperoxia. Experiments were also conducted to determine the poss-
ible role of the protein thiol groups in the development of oxidative· damage.
IAI 05
TBAR
Materials and Methods Experiments were carried out on male Wistar rats (180-220 g). The animals were exposed to 100% oxygen in normobaric conditions for I, 3, 5, 8 and 24 h. The oxygen was passed at a rate of 3l!min through the 8.4-1-chamber. After hyperoxia the animals were decapitated and the synaptosomes were isolated from the cerebral cortex as described by BOOTH and CLARK (2). The content of thiobarbituric acid-reactive material (TBAR) in the homogenate from the cerebral cortex was measured by the technique of SLATER and SAWYER (20). Protein thiol groups were detemlined by the method described by SEDLAK and LINDSAY (19). For measurements of dopamine (DA) uptake, synaptosomes (2 mg protein per ml) were suspended in Krebs-Hanseleit medium supplemented with 2.5 mM CaCh, 10 mM glucose and 10 ~lM pargiline. After preincubation for 5 min at 30°C, the uptake was initiated by addition of eHlDA (final concentration 0.4 ~M; 34.2 Ci/mmol). Aliquots of 200 ~l were withdrawn at time intervals from 20 to 300 s and rapidly centrifuged (Beckman Microfuge) through a layer of silicon oil (specific gravity 1.03, General Electric Waterford N.Y.). The pellets were solubilized with an NCS tissue solubilizer and radioactivity was measured in a Beckman LS 9000 liquid scintillation counter with use of the Bray scintillation fluid. Electron microscopy
Synaptosomes from the cerebral cortex of control animals and animals exposed to 100 % O2 for 5 and 24 h were placed in 2.5 % glutaraldehyde solution in 0.1 M cacodylate buffer for I h and then fixed in 2 % solution osmium tetroxide in 0.1 M cacodylate buffer. The slide was then dehydrated in successive alcohols, carried through propylene oxide mixtures and embedded in Epon 812 resin according to the standard procedure. Sections of approximately 90-60 m~ were cut on a Reichert OmU microtome and stained with Reynold's solution. The slides were examined and pictures made with a JEM 7 A electron microscope. Total protein was measured by the method of LOWRY et al. (12) with bovine serum albumin as standard. The significance of differences between the treated and the control groups was determined by Student's t-test.
Results The exposure of rats to hyperoxia lasting 1-2 h significantly elevated the level of TBAR and decreased the content of the protein thiol groups in the isolated fraction of rat brain synaptosomes. The level of the SH group as well as TBAR production almost returned to the control value during the next 8 h. Prolonged hyperoxia up to 24 h again increased TBAR in the cerebral cortex. This coincides with the considerable parallel fall in protein thiol groups (fig. 1). eHlDA uptake by synaptosomes isolated after 1, 3, 5, 8 and 24 h of hyperoxygenation was still higher than the control value with maximal accumulation after 24 h of oxygenation (fig. 2). Morphological observations under the electron microscope revealed changes in the synaptosomes. After 5 h we noted that pre- and postsynaptic parts mostly did not form contacts. In presynaptic endings numerous synaptic 4
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~ 10
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Fig.l. Changes in the level of TBAR (A) and protein thiol groups (B) in the rat cerebral cortex during in vivo hyperoxygenation. Results are means ± SEM from 8 experiments. *Values significantly differing from control; p < 0.01 by Student's t-test.
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Fig.2. Effect of in vivo hyperoxia on eHlDA uptake to rat brain synaptosomes. Results are means ± SEM of 6 experiments performed in duplicates. *Values significantly differing from control; p < 0.05 by Student's t-test. Exp Toxic Pathol 44 (1992) 2
97
Fig. 3. Synaptosomes received from cerebral cortex of animals exposed to 100 % O2 for 5 h . Pre- and postsynaptic parts did not form contacts . Note numerous and regularly dispersed synaptic vesicles (SV) and fragments of cytoskeleton elements (asterisk) in presynaptic parts , and mitochondria (M) in condensed form in postsynaptic parts. x 48 ,000 .
Fig. 4. Synaptosomes received from cerebral cortex of animals exposed to 100 % O2 for 5 h. Pre- and postsynaptic parts form close contacts. Note concentration of synaptic vesicles (SV) and very electron-dense synaptic cleft (asterisk) . In postsynaptic part swollen mitochondria (M) and electron-lucent vacuoles (V) are present. x48 ,000. 98
Exp Toxic Pathol 44 (1992) 2
Fig. 5. Control synaptosomal fraction. Ultrastructurally unchanged pre- and postsynaptic parts form contacts (asterisk). x48,000.
vesicles were present, of normal ultrastructure and regularly dispersed through the synaptosome, and mitochondria usually in condensed form. In postsynaptic parts swollen mitochondria could be seen exhibiting'ftn electron-lucent matrix and shortened cristae between unchanged organelles (fig. 3). Occasionally synaptosomes were observed in which pre- and postsynaptic parts formed close contacts (fig. 4). In these synaptosomes we observed a concentration of synaptic vesicles in presynaptic
parts, and the synaptic cleft was electron-dense. This picture was similar to the control (fig. 5). After 24 h of hyperoxia the membranes limiting the pre- and postsynaptic endings exhibited abnormalities in their ultrastructure. These membranes did not show the typical ultrastructural picture in some places. Swelling of synaptoplasm and a decreased number of synaptic vesicles were observed in some presynaptic endings.
Fig. 6. Synaptosomes received from cerebral cortex of animals exposed to 100 % O 2 for 24 h. Pre- and postsynaptic parts did not form contacts. The limiting membranes exhibited abnormalities in their ultrastructure (asterisk). Note greatly changed mitochondria (M) in pre- and postsynaptic parts. x48,000. Exp Toxic Pathol 44 (1992) 2
99
Swollen mitochondria, fragments of the cytoskeleton element, numerous electron-lucent vacuoles of various size were noted in swollen postsynaptic parts (fig. 6). Pre- and postsynaptic parts did not form any contacts.
Discussion Our results suggest that damage to brain neurons during hyperoxia may be related to the direct peroxidation of the cell membrane lipid. This may lead to rupture of double bonds and to the formation of free peroxide radicals (9, 16, 21). By an autocatalytic mechanism these in tum could further peroxidize other membrane lipids. A rapid increase in the level of TBAR products coincides with a fall in protein thiol groups. A simple model of experimental peroxidation conditions - normobaric hyperoxia in the rat - used in this study allows to follow dynamic metabolic changes in the brain. The evolution of TBAR varied significantly, since after a very rapid initial increase the level of TBAR dropped quickly to only slightly elevated values during oxygenation prolonged to 8 h. Simultaneously the content of protein thiol groups in the brain changed in the reverse direction. Adaptation of animals submitted to elevated oxygen pressure during prolonged normobaric hyperoxia may be postulated. This adaptation is correlated with an increase in activities of several antioxidant enzymes - including the seleniumcontaining enzyme glutathione peroxidase (3, 6, 23, 24). This agrees with the here observed elevated level of protein thiol groups. Glutathione peroxidase, )Vhich utilizes the reducing equivalent of GSH to catalyze the decomposition of toxic hydroperoxides, and metabolically related enzymes, GSH reductase and glucose6-phosphate (G-6-P) dehydrogenase, have been suggested to be the key mechanism for cellular protection against the deleterious effects of hydroperoxides. However, prolonged exposure of mammals to oxygen for up to 24 h may damage this antioxygenic defence system. Morphological electron microscopic observation of control synaptosomes and synaptosomes isolated from rats exposed to oxygen for 5 h does not show dramatic differences, whereas after 24-h-hyperoxia these changes are more pronounced, i.e. swelling of synaptoplasm, vacuolization, swollen mitochondria, a great number of lysosomes and loss of connections between pre- and post-synaptic endings. It is, of course, possible that the membrane changes are so subtle that they may be overlooked, and certainly the biochemical evidence of lipid peroxidation is clear. Our previous studies indicated that in vitro lipoperoxidation changes the properties of biomembranes and modifies in different ways the accumulation in synaptosomes of dopamine and other neurotransmitters such as yaminobutyric acid and choline (5, 17). We can postulate that in vivo hyperoxia changes the biomembranes and interferes with dopamine uptake and level in a similar manner to that observed in "in vitro" studies (11, 13, 17). In the present study we observed increased dopamine uptake during the whole period of hyperoxia with a maximum after 24 h. The relationship observed between peroxidation and dopamine uptake is consistent with the view that lipid peroxidation increases the activity of the dopamine translocase system (22). The increase in the rate of dopamine accumulation probably reflects the conformational changes in the carrier and/or its environment in the membrane. The results obtained strongly suggest that presynaptic nerve 100
Exp Toxic Pathol 44 (1992) 2
terminals are particularly sensitive to free radical attack, which is manifested in altered morphological and functional properties of the isolated synaptosomal fracion. Disturbances in synaptosomal transport and metabolism of neurotransmitters in hyperoxia and other pathological conditions inducing peroxidation indicate that processes of synaptic transmission are likely targets for free radical modification. Therefore peroxidation may play an important role in the pathogenesis of several neuropsychiatric disorders.
References 1. BEHNKE, A. R., JOHNSON, F. S., POPPEN, J. R., MOTLEY, E. P.: The effect of oxygen on man at pressures from 1 to 4 atmospheres. Am. J. Physio!. 1935; 110: 565-569. 2. BOOTH, R. F., CLARK, 1. B.: A rapid method for the preparation of relatively pure metabolically competent, synaptosomes from rat brain. Biochem. J. 1978; 176: 365-370. 3. BROUSSOLE, B., BURNET, H., BARET, A., FOLIGUET, B., MICHEL, P., MARCHAL, L.: Adaptation to pulmonary chronic hyperoxia in rats: Demonstration by a study of pulmonary surfactant, tissue superoxide dismutase and histology (eds. QUANJER, PH. H., WIDDICOMBE, J. G.). Bull. Eur. Physiopatho!. Respir. 1978, pp. 133-135. 4. CHIO, K. S., TAPPEL , A. L.: Inactivation of ribonuclease and other enzymes by peroxidizing lipids and by malonaldehyde. Biochemistry 1969; 8: 2827-2832. 5. D~BROWIECKI, Z., GORDON-MAJSZAK, W., LAZAREWICZ, J.: Effect of lipid peroxidation on neurotransmitters uptake by rat synaptosomes. Pol. J. Pharmacol. Pharm. 1985; 37: 321-327. 6. FORMAN, H. J., FISHER, A. B.: Role of glutathione peroxidase in tolerance and adaptation of rats to hyperoxia. In: Oxygen Radicals in Chemistry and Biology (eds. BORs, W., SARAN, M., TAIT, D.). Walter de Gruyter, Berlin, New York 1984, pp. 699-704. 7. FRIDOVICH, 1., FREEMAN, B.: Antioxidant defenses in the lung. Ann. Rev. Physiol. 1986; 48: 693-792. 8. GERSCHMAN, R., GILBERT, D. L., CACCAMISE, D.: Effects of various substances on survival times of mice exposed to different high oxygen tensions. Am. J. Physiol. 1958; 192: 563-571. 9. GORDON-MAJSZAK, W., RAFAtowSKA, U., LAZAREWICZ, J.: Metabolic changes in rat brain synaptosomes after exposure to normobaric hyperoxia in vivo. Bull. Acad. Pol. Sci. 1987; 35: 97-104. 10. JOANNY, P., BRUE, F., CORRIOL, J.: Effect of hyperbaric oxygen (OHP) on lipid peroxidation in the rat brain: Some relationships with the occurrence of OHP-induced convulsions. Bull. Eur. Physiopathol. Res. 1981; 17: 165P-166P. 11. LEUNG, T. K. c., LAI, J. C. K., TRJCKLEBANK, M., DAVISON, A. N., LIM, L.: Chronic manganese treatment of rats alters synaptosomal uptake of dopamine and the behavioural response to amphetamine administration. J. Neurochem. 1982; 39: 1496-1499. 12. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., RANDALL, R. J.: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 1951; 193: 265-275. 13. MESSRIPOUR, M., HADDADY, H.: Effect of ascorbic acid administration on copper-induced changes of rat brain hypothalamic catecholamine contents. Acta Neurol. Scand. 1988; 77: 481-485. 14. MURRAY, H., NATHAN, C. F., COHN, Z. A.: Macrophage oxygendependent antimicrobial activity. IV. Role of endogeneous scavengers of oxygen intermediates. J. Exp. Med. 1980; 152: 1610-1624. 15. NISHIKI, K., JAMIESON, D., OSHINO, N., CHANCE, B.: Oxygen toxicity in the perfused rat liver and lung under hyperbaric conditions. Biochem. J. 1976; 160: 343-355. 16. NODA, Y., MCGEER, P. L., MCGEER, E. G.: Lipid peroxide distribution in brain and the effect of hyperbaric oxygen. J. Neurochem. 1983; 40: 1329-1332. 17. PASTUSZKO, A., GORDON-MAJSZAK, W., D~BROWIECKJ, Z.: Dopamine uptake in striatal synaptosomes exposed to peroxidation in vitro. Biochem. Pharmacology 1983;32: 141-146.
D~BROWIECKJ, Z.: Consequences of exposure of rats to hyperoxygenation for actomyosin-like protein from synaptosomes of brain. J. Neurochem. Pathol. 1986; 5:
24. TIERNEY, D. F., AYERS, L., HERZOG, S., YANG, J.: Pentose pathway
107-115. 19. SEDLAK, J., LINDSAY, R. H.: Estimation of total, proteinbound and
25. VION-DuRY, J., LEGAL LA SALLE, G., ROUGIER, I., PAPY, J. J.:
18. RAFALOWSKA, U., GORDON-MAJSZAK, W.,
nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. 1968; 25: 192-205. 20. SLATER, T. F.: Free-radical mechanisms in tissue injury. Biochem. J.
1984;222: 1-15. 21. - SAWYER, B. C.: The stimulatory effects of carbon tetrachloride and other halogenoalkanes on peroxidative reactions in rat liver fraction in vitro. Biochem. J. 1971; 123: 805-814. 22. SOKOLOV, V. S., CHURAKOVA, T. D., BULGAKOV, V., KAGAN, E., BILENKO, M. V., BOGUSLAVSKY, L. I.: Study of action mechnisms of the products of lipid peroxide oxidation on permeability of bilayer lipid membranes. Biophysica 1981; 26: 147-149 (in Russian). 23. STEVENS, J. B., Am'oR, A. P.: Proposed mechanism for neonatal rat tolerance to normobaric hyperoxia. Fed. Proc. 1980; 39: 3138-3143.
and production of reduced nicotinamide adenine dinucleotide phosphate. Am. Rev. Respir. Dis. 1973; 108: 1348-1351. Effects of hyperbaric and hyperoxic conditions on amygdala-kindled seizures in rat. Exp. Neurol. 1986; 92: 513-521.
26. WOOD, J. D.: Seizures induced by hyperbaric oxygen and cerebral yaminobutyric acid in chicks during development. J. Neurochem. 1970; 17: 573-579. 27. YOUNGMAN, R. J.: Oxygen activation: Is the hydroxyl radical always biologically relevant? TIBS 1984; 9: 280-283.
28. ZALESKA, M. M., FLOYD, R. A.: Regional lipid peroxidation in rat brain in vitro: possible role of endogenous iron. Neurochem. Res. 1985; 10: 397-410. 29. ZIRKLE, L. G., MENGEL, C. E., HORTON, B. D., DUFFY, E.: Studies of oxygen toxicity in the central nervous system. Aerospace Med.
1965; 36: 1027-1032.
Exp Toxic Pathol 44 (1992) 2
101
Polish Academy of Sciences, Medical Research Centre, Department of Neurochemistry and Laboratory of Ultrastructure of the Nervous System, Warsaw, Poland
Biochemical and morphological changes in rat brain synaptosomes after exposure to normobaric hyperoxia in vivo 1) By W. GORDON-MAJSZAK and B. GAJKOWSKA With 6 figures Received: October 25, 1989; Accepted: February 16, 1990
Address for correspondence: Dr. WANDA GORDON-MAJSZAK, Polish Academy of Sciences, Medical Research Centre, Department of Neurochemistry, Dworkowa 3,00-784 Warsaw, Poland Key words: synaptosomes; hyperoxia, normobaric; brain, hyperoxia; oxygen toxicity; thiobarbituric acid-reactive material; peroxide radicals
Summary Adult rats were submitted to normobaric hyperoxia for to 24 h, then the brain synaptosomes were isolated and their metabolic and morphologic properties were studied. Hyperoxia lasting 1-2 h .Bignificantly increased the content of thiobarbituric acid-reactive material (TBAR) and decreased the level of protein thid groups. During the next 5-8 h of hyperoxia SH groups as well as TBAR content became almost normal, reflecting adaptation of the animals to an elevated oxygen tension. After 24 h of hyperoxia a maximal increase in the TBAR content and parallel fall in protein thiol groups were noted. Simultaneously, significant morphological differences between control synaptosomes and synaptosomes isolated from rats exposed to 24 h oxygenation were observed in electron microscopy. The high-affinity dopamine uptake in hyperoxic synaptosomes was significantly increased in all experimental groups. A specific high sensitivity of the dopamine uptake system in synaptoplasmatic membranes to the free radical modification of the membrane structure is suggested.
Introduction Although oxygen is of vital importance to most higher organisms, it becomes toxic even at moderately elevated concentrations. The toxicity of oxygen is supposedly based on its tendency to form reactive intermediates, i.e. 0/, H2 0 2 • OH~ and 102 in the reduction to H2 0. Alternatively, more stable but more cytotoxic intermediates, e.g. aldehydes (4) may be formed by the reaction of free radicals with various cellular molecules. These activated oxygen species are believed to be detoxified by a cellular antioxygenic defence system which may be overwhelmed when the cells are exposed to conditions of increased "oxidative stress". Among the cellular 1) This work was supported by the Polish Academy of Sciences within project 06-02
96
Exp Toxic Pathol44 (1992) 2
defence systems against peroxidative damage, reduced glutathione (GSH) plays a major role (8, 15, 27). Prolonged exposure of man1mals to > 95 % O2 may lead to severe tissue damage. The LDso for adult rats is 72 h; however, rats generally survive exposure to 80-85 % O2 for 5 to 7 days and then they are resistant to subsequent exposure to > 95 % O 2 . This adaptation to hyperoxia is correlated with a increased activity of several antioxidant enzymes (3, 6, 7, 24). Prolonged hyperoxic exposure induces free radicals which participate in pathological mechanisms by causing direct pulmonary injury and/or by forming a secondary chemotactic factor for neutrophils (14). On the other hand, the effect of hyperbaric oxygen in producing seizures has been recognized for many years (1, 10, 25, 26, 29). Although descriptions of the clinical features of oxygen toxicity are numerous, the mechanisms involved have not been completely elucidated. Since the brain is a postmitotic tissue, oxidative events may be very destructive, and lead to irreversible neuronal injury. The brain is particularly prone to lipid peroxidation since it has a high content of fatty acids, such as 20: 4 and 22: 6 which are very susceptible to peroxidation. In certain parts of the brain, such as the extrapyramidal tract,especially in humans, there is also a large content of none-heme iron, which in very specific in vivo conditions may participate in the peroxidation processes (28). Our previous studies demonstrated a correlation between the time of oxygenation and peroxidation of lipid in the brain (9, 18). The relation between radical oxidation and in vivo yaminobutyric acid transport in isolated brain synaptosomes w,as also demonstrated (9). The aim of the present study was to examine, in an experimental model of in vivo normobaric hyperoxia, the interrelation between the development of morphological changes in synaptosomes and in dopamine transport in the isolated brain synaptosomes after induction of peroxidation processes in the brain by hyperoxia. Experiments were also conducted to determine the poss-
ible role of the protein thiol groups in the development of oxidative· damage.
IAI 05
TBAR
Materials and Methods Experiments were carried out on male Wistar rats (180-220 g). The animals were exposed to 100% oxygen in normobaric conditions for I, 3, 5, 8 and 24 h. The oxygen was passed at a rate of 3l!min through the 8.4-1-chamber. After hyperoxia the animals were decapitated and the synaptosomes were isolated from the cerebral cortex as described by BOOTH and CLARK (2). The content of thiobarbituric acid-reactive material (TBAR) in the homogenate from the cerebral cortex was measured by the technique of SLATER and SAWYER (20). Protein thiol groups were detemlined by the method described by SEDLAK and LINDSAY (19). For measurements of dopamine (DA) uptake, synaptosomes (2 mg protein per ml) were suspended in Krebs-Hanseleit medium supplemented with 2.5 mM CaCh, 10 mM glucose and 10 ~lM pargiline. After preincubation for 5 min at 30°C, the uptake was initiated by addition of eHlDA (final concentration 0.4 ~M; 34.2 Ci/mmol). Aliquots of 200 ~l were withdrawn at time intervals from 20 to 300 s and rapidly centrifuged (Beckman Microfuge) through a layer of silicon oil (specific gravity 1.03, General Electric Waterford N.Y.). The pellets were solubilized with an NCS tissue solubilizer and radioactivity was measured in a Beckman LS 9000 liquid scintillation counter with use of the Bray scintillation fluid. Electron microscopy
Synaptosomes from the cerebral cortex of control animals and animals exposed to 100 % O2 for 5 and 24 h were placed in 2.5 % glutaraldehyde solution in 0.1 M cacodylate buffer for I h and then fixed in 2 % solution osmium tetroxide in 0.1 M cacodylate buffer. The slide was then dehydrated in successive alcohols, carried through propylene oxide mixtures and embedded in Epon 812 resin according to the standard procedure. Sections of approximately 90-60 m~ were cut on a Reichert OmU microtome and stained with Reynold's solution. The slides were examined and pictures made with a JEM 7 A electron microscope. Total protein was measured by the method of LOWRY et al. (12) with bovine serum albumin as standard. The significance of differences between the treated and the control groups was determined by Student's t-test.
Results The exposure of rats to hyperoxia lasting 1-2 h significantly elevated the level of TBAR and decreased the content of the protein thiol groups in the isolated fraction of rat brain synaptosomes. The level of the SH group as well as TBAR production almost returned to the control value during the next 8 h. Prolonged hyperoxia up to 24 h again increased TBAR in the cerebral cortex. This coincides with the considerable parallel fall in protein thiol groups (fig. 1). eHlDA uptake by synaptosomes isolated after 1, 3, 5, 8 and 24 h of hyperoxygenation was still higher than the control value with maximal accumulation after 24 h of oxygenation (fig. 2). Morphological observations under the electron microscope revealed changes in the synaptosomes. After 5 h we noted that pre- and postsynaptic parts mostly did not form contacts. In presynaptic endings numerous synaptic 4
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0.3
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8 oxygenation time
5
24h
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60
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~ 10
1 2 3
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8 oxygenation time
24h
Fig.l. Changes in the level of TBAR (A) and protein thiol groups (B) in the rat cerebral cortex during in vivo hyperoxygenation. Results are means ± SEM from 8 experiments. *Values significantly differing from control; p < 0.01 by Student's t-test.
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Fig.2. Effect of in vivo hyperoxia on eHlDA uptake to rat brain synaptosomes. Results are means ± SEM of 6 experiments performed in duplicates. *Values significantly differing from control; p < 0.05 by Student's t-test. Exp Toxic Pathol 44 (1992) 2
97
Fig. 3. Synaptosomes received from cerebral cortex of animals exposed to 100 % O2 for 5 h . Pre- and postsynaptic parts did not form contacts . Note numerous and regularly dispersed synaptic vesicles (SV) and fragments of cytoskeleton elements (asterisk) in presynaptic parts , and mitochondria (M) in condensed form in postsynaptic parts. x 48 ,000 .
Fig. 4. Synaptosomes received from cerebral cortex of animals exposed to 100 % O2 for 5 h. Pre- and postsynaptic parts form close contacts. Note concentration of synaptic vesicles (SV) and very electron-dense synaptic cleft (asterisk) . In postsynaptic part swollen mitochondria (M) and electron-lucent vacuoles (V) are present. x48 ,000. 98
Exp Toxic Pathol 44 (1992) 2
Fig. 5. Control synaptosomal fraction. Ultrastructurally unchanged pre- and postsynaptic parts form contacts (asterisk). x48,000.
vesicles were present, of normal ultrastructure and regularly dispersed through the synaptosome, and mitochondria usually in condensed form. In postsynaptic parts swollen mitochondria could be seen exhibiting'ftn electron-lucent matrix and shortened cristae between unchanged organelles (fig. 3). Occasionally synaptosomes were observed in which pre- and postsynaptic parts formed close contacts (fig. 4). In these synaptosomes we observed a concentration of synaptic vesicles in presynaptic
parts, and the synaptic cleft was electron-dense. This picture was similar to the control (fig. 5). After 24 h of hyperoxia the membranes limiting the pre- and postsynaptic endings exhibited abnormalities in their ultrastructure. These membranes did not show the typical ultrastructural picture in some places. Swelling of synaptoplasm and a decreased number of synaptic vesicles were observed in some presynaptic endings.
Fig. 6. Synaptosomes received from cerebral cortex of animals exposed to 100 % O 2 for 24 h. Pre- and postsynaptic parts did not form contacts. The limiting membranes exhibited abnormalities in their ultrastructure (asterisk). Note greatly changed mitochondria (M) in pre- and postsynaptic parts. x48,000. Exp Toxic Pathol 44 (1992) 2
99
Swollen mitochondria, fragments of the cytoskeleton element, numerous electron-lucent vacuoles of various size were noted in swollen postsynaptic parts (fig. 6). Pre- and postsynaptic parts did not form any contacts.
Discussion Our results suggest that damage to brain neurons during hyperoxia may be related to the direct peroxidation of the cell membrane lipid. This may lead to rupture of double bonds and to the formation of free peroxide radicals (9, 16, 21). By an autocatalytic mechanism these in tum could further peroxidize other membrane lipids. A rapid increase in the level of TBAR products coincides with a fall in protein thiol groups. A simple model of experimental peroxidation conditions - normobaric hyperoxia in the rat - used in this study allows to follow dynamic metabolic changes in the brain. The evolution of TBAR varied significantly, since after a very rapid initial increase the level of TBAR dropped quickly to only slightly elevated values during oxygenation prolonged to 8 h. Simultaneously the content of protein thiol groups in the brain changed in the reverse direction. Adaptation of animals submitted to elevated oxygen pressure during prolonged normobaric hyperoxia may be postulated. This adaptation is correlated with an increase in activities of several antioxidant enzymes - including the seleniumcontaining enzyme glutathione peroxidase (3, 6, 23, 24). This agrees with the here observed elevated level of protein thiol groups. Glutathione peroxidase, )Vhich utilizes the reducing equivalent of GSH to catalyze the decomposition of toxic hydroperoxides, and metabolically related enzymes, GSH reductase and glucose6-phosphate (G-6-P) dehydrogenase, have been suggested to be the key mechanism for cellular protection against the deleterious effects of hydroperoxides. However, prolonged exposure of mammals to oxygen for up to 24 h may damage this antioxygenic defence system. Morphological electron microscopic observation of control synaptosomes and synaptosomes isolated from rats exposed to oxygen for 5 h does not show dramatic differences, whereas after 24-h-hyperoxia these changes are more pronounced, i.e. swelling of synaptoplasm, vacuolization, swollen mitochondria, a great number of lysosomes and loss of connections between pre- and post-synaptic endings. It is, of course, possible that the membrane changes are so subtle that they may be overlooked, and certainly the biochemical evidence of lipid peroxidation is clear. Our previous studies indicated that in vitro lipoperoxidation changes the properties of biomembranes and modifies in different ways the accumulation in synaptosomes of dopamine and other neurotransmitters such as yaminobutyric acid and choline (5, 17). We can postulate that in vivo hyperoxia changes the biomembranes and interferes with dopamine uptake and level in a similar manner to that observed in "in vitro" studies (11, 13, 17). In the present study we observed increased dopamine uptake during the whole period of hyperoxia with a maximum after 24 h. The relationship observed between peroxidation and dopamine uptake is consistent with the view that lipid peroxidation increases the activity of the dopamine translocase system (22). The increase in the rate of dopamine accumulation probably reflects the conformational changes in the carrier and/or its environment in the membrane. The results obtained strongly suggest that presynaptic nerve 100
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terminals are particularly sensitive to free radical attack, which is manifested in altered morphological and functional properties of the isolated synaptosomal fracion. Disturbances in synaptosomal transport and metabolism of neurotransmitters in hyperoxia and other pathological conditions inducing peroxidation indicate that processes of synaptic transmission are likely targets for free radical modification. Therefore peroxidation may play an important role in the pathogenesis of several neuropsychiatric disorders.
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