TNT . J . RADIAT . BIOL .,
1979, VOL . 35,
NO .
4, 343-350
Protection of liposomal lipids against radiation induced oxidative damage A. W . T . KONINGS, J . DAMEN and W . B . TRIELING
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Laboratory of Radiopathology, Bloemsingel 1, University of Groningen, The Netherlands
(Received 3 May 1978; accepted 20 July 1978) Liposomes were prepared from phospholipids extracted from biological membranes . A comparison was made between the peroxidation rate in handshake liposomes and in sonicated liposomes . The smaller sonicated liposomes were more vulnerable to peroxidation, probably because of the smaller radius of curvature, which results in a less dense packing of lipid molecules in the bilayer and a facilitated action of water radicals produced by the X-irradiation . High oxygen enhancement ratios were obtained, especially at low dose rates, suggesting the operation of slowly progressing chain reactions initiated by ionizing radiation . Three compounds were tested for their ability to protect the liposomal membranes against lipid peroxidation . The naturally occurring compounds reduced glutathione (GSH) and vitamin E (a-T) and the powerful radiation protector cysteamine (MEA) . All three molecules could protect the liposomes against peroxidation . The membrane-soluble compound vitamin E was by far the most powerful . About 50 per cent protection was achieved by using 5 x 10 -6 M a-T, 10 -4 M GSH and 5 x 10 -4 M MEA . The fatty acid composition of the lipids altered drastically as a result of the irradiation . Arachidonic acid and docosahexanoic acid were the most vulnerable of the fatty acids . Very efficient protection of these polyunsaturated fatty acids could be obtained with relatively low concentrations of vitamin E built into the membranes .
1.
Introduction Although the cell membranes perform an important role in the functional organization of the cell, relatively little experimental work on radiation damage to membranes has been reported, as compared to radiation studies on nucleic acids . For a systematic approach to the problem of radiation-induced alterations in biomembranes, model systems are very suitable because different membrane components can be investigated separately . About half the mass of most cellular membranes consists of lipid molecules . The lipids are structured as bilayers, although the continuity is interrupted by nonbilayer regions (Singer and Nicolson 1972) . Liposomes are aqueous lipid dispersions, also organized as bilayers of lipid molecules (Bangham, Standish and Watkins 1965, Sessa and Weissmann 1968, Davison 1974) and thus provide an excellent model for radiation studies . The lipsomes used in the study to be reported here consisted of phospholipids extracted from biological membranes, and were prepared in the presence or absence of naturally occurring compounds with antioxidant capacity : glutathione and vitamin E . The action of radioprotective thiols such as gluthathione on living organisms has been well documented (Bacq 1{' :5, Bridges 1969) . A possible role of vitamin E as an in vivo radiation protector biological membranes has been indicated by recent work on the fragility of erythrocytes (Prince and Little 1973, Hoffer and Roy 1975) and by the observation of enhanced radiosensitivity of vitamin-E-deficient lymphosarcoma cells (Konings and Trieling 1977) . In this report the formation of conjugated diens in liposomes as a result of Xirradiation is described, and also the destruction of polyunsaturated fatty acids (PUFAs) in the phospholipids . It is well established that the methylene-interrupted PUFAs are vulnerable structures with respect to hydrogen abstraction, yielding
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conjugated dienes as the initial stage in lipid peroxidation, prior to chain scission (Milch and Klasse 1965) . Good correlation has been found to exist between the formation of conjugated dienes and the formation of products of lipid peroxidation such as malondialdehyde (Kenaston, Wilbur, Ottolenghi and Bernheim 1955, Dahle, Hill and Holman 1962, Pryor 1976) . Special attention will be paid to protective capacities against radiation-induced destruction of certain fatty acids as afforded by the naturally occurring compounds vitamin E and glutathione .
2. Materials and methods 2.1 . Subcellular fractionation C5,B1 mice, 3 to 4 months old, were killed by cervical dislocation and the livers were removed . A 10 per cent homogenate was made in the cold (4°C) in a Potter Elvehjem homogenizer with a motor-driven Teflon pestle (clearance 0 . 20 mm) in a medium consisting of 0 . 25 M sucrose in SO mM tris HCI, pH 7 .5, 2. 5 mM KC1 and 5 MM MgC1 2 . This solution (STKM) was used throughout the isolation procedure . The homogenate was filtered through two layers of cheesecloth and centrifuged for 10 min at 900g . The sediment contained the rough nuclear fraction and cell debris . The supernatant was centrifuged for 30 min at 15 OOOg to sediment the mitochondria and lysosomes (M + L fraction) . The microsomal fraction was obtained after centrifuging the 15 OOOg supernatant at 105 OOOg for 60min . All sediments were taken up in STKM, and the suspensions obtained were subjected to lipid extraction .
2 .2 . Isolation of phospholipids The lipid extraction and chromatographic separation procedures were generally performed as described previously (Konings 1970) . In most of the experiments described in this paper, phospholipids of the M + L fraction were used . To initiate the lipid extraction of the suspensions, two volumes of methanol and one volume of chloroform were added under thorough mixing in a glass-stoppered centrifuge tube . The extraction was completed according to Bligh and Dyer (1959) . Non-lipid contaminants were removed by Sephadex column chromatography (Siakatos and Rouser 1965) . The lipids were separated into neutral lipids and phospholipids by silicic acid column chromatography (Bergstrom 1952) . The total number of lipids and the proportions of neutral and phospholipids were quantitatively determined by evaporating aliquots of the extracts under a stream of nitrogen gas and weighing the lipid on a Cahn (Gram) electrobalance . To determine the phospholipid composition of the extract, thin layer chromatography on silica gel HF (Merck) was carried out, followed by destruction (Ames and Dubin 1960) of the separated fraction and phosphorus determination (Chen, Toribara and Marner 1956) . 2 .3 . Preparation of liposomes Solutions of phospholipids, isolated as described above were, if necessary, mixed with vitamin E in ethanol, added to round-bottomed flasks and the organic solvents evacuated at 30°C in rotatory evaporator . This resulted in a thin dry lipid coating on the flask wall . Small glass beads were added, and also double-distilled water in which, if necessary, glutathione and cysteamine were solubilized and brought to pH 7 . 4 . The fluid in the flask was purged with oxygen-free nitrogen and vigorously agitated on a Vortex mixer for 1 min . In order to obtain smaller liposomes the preparation received a sonication treatment with a Branson B12 Sonifier at 60-8 W .
Radiation effects on liposomes
345
This was done after the Vortex mixing, during four periods of 30s separated by pauses of 15 s at room temperature under a stream of nitrogen .
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2 .4 . Irradiation
X-rays from a Philips-Muller MG 300 X-ray machine were used, which operated at 200 kV and 15 mA . The beam was filtered with 0. 5 mm Cu and 0. 5 mm Al; the h .v .l . was 1 . 1 mm Cu . Different dose rates were obtained by changing the focus-object distance . The samples were continuously gassed with either nitrogen or air before and during the irradiation . All irradiations were performed at room temperature . 2 .5 . Lipid peroxidation
The formation of conjugated dienes was determined after adding 10 volumes of ethanol to one volume of the liposomes and measuring U .V . absorption in quartx cuvettes at 233 and 215 rim. The absorbancy ratio was taken as a measure of lipid peroxidation (Klein 1970) . 2 .6 . Determination of fatty acid composition
Aliquots of liposomal suspensions were subjected to lipid extraction as described above . For transesterification purposes about 1 mg of dry phospholipid (under N 2) was solubilized in 10 ml methanol/sulphuric acid (95 : 5, v/v) in glass-stoppered tubes. The solution was purged with N 2 for 2 min, the tubes closed and placed in a water bath at 70°C for 2 hours . After cooling, 5 ml of water was mixed into the solution . The solution was then extracted twice with 10 ml of hexane, and the gathered hexane phases were extracted twice with 10 ml of water and subsequently dried over anhydrous sodium sulphate . The solution was filtered and evaporated under N 2 . The resulting methylated fatty acids were taken up in 50 or 100 µl of hexane . Mostly 1 µl of hexane sample was applied to a Becker Packard Gaschromatograph Model 419, equippped with a flame ionization detector and integrator system . As a stationary phase a cyanosilicone column was used that consisted of 10 per cent SP-2330 on 100/120 Chromosorb W AW (Supelco Inc ., Bellefonte, Pennsylvania) . The heating was carried out at an initial temperature of 165 ° C for 2 min, followed by an increase of 3 ° C per minute up to 200 ° C . The methylesters were identified by comparison with retention times of authentic standards and controlled on a different GLC column (diethyleneglycolsuccinate) . 3. Results Liposomes of different sizes and at different concentrations were prepared in order to establish the effect of X-irradiation on lipid peroxidation . The approximate phospholipid composition of the liposomes was 65 per cent phosphatidylcholine (PC), 20 per cent phosphatidylethanolamine (PE), 5 per cent phosphatidylinositol (PI) plus phosphatidylserine (PS), and 10 per cent diphosphatidylglycerol (DPG) . The data in table 1 are expressed as the ratio between the absorptions at 233 and 215 nm, which is a quantitative measure of the formation of conjugated dienes (Klein 1970) . The highest yields were obtained at concentrations of about 0 . 3 to 0. 5 mg of phospholipid per ml . The sonicated liposomes are smaller and were more vulnerable than the unsonicated larger liposomes . Sonicated liposomes were used throughout the rest of the investigation . Table 2 gives the results of an experiment in which an X-ray dose of 200 Gy was given at both (0 . 8 Gy/min) and (8 . 0 Gy/min)
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Lipid concentration (mg/ml)
Unsonicated
Sonicated
Control
Irradiated
Difference
Control
Irradiated
Difference
0-3 0.5 0.7
0-26 0 . 28 028
0-72 0 . 81 0 . 56
0-45 0 . 53 0 . 28
0-45 0. 44 0. 46
1 . 09 1 . 13 0 . 99
0-64 0. 69 0-53
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Table 1 . Lipid peroxidation in X-irradiated liposomes . Each datum is expressed as the ratio of absorptions at 233 and 215 nm . The irradiation was performed in air with a dose of 100 Gy at a dose rate of 0. 8 Gy/min . Radiation conditions Dose rate (Gy/min)
Air
O .e .r .
Control
Irradiated
Difference
Control
Irradiated
Difference
0. 48 0-36
1 . 28 0-62
0-80 0-26
0-28 0-25
0-34 0-30
0 .06 0 .05
0. 8 8-0 Table 2 .
Hypoxia
13 5
Effects of dose rate on o .e .r . Sonicated liposomes were X-irradiated with a dose of 200 Gy ; the phospholipid concentration was 0-5 mg/ml .
dose rates under oxic and hypoxic conditions . While almost no dose-rate effects were observed under hypoxic conditions, enhanced peroxidation was found under aerobic conditions when a low dose rate was applied . As a consequence, the oxygen enhancement ratio (o .e .r .) clearly increased . The protective ability of reduced glutathione, cysteamine and vitamin E were then compared with respect to lipid peroxidation induced by X-irradiation . The results of a representative experiment are given in table 3 . From the first line of the table it can be seen that the three compounds protected the lipids against peroxidation, during both the process of preparing the liposomes and the time that the irradiated liposomes were exposed to the X-rays . Protection against radiation-induced peroxidation was observed for all three compounds, as shown in the last line of table 3 . Vitamin E was by far the most Formation of conjugated dienes None
Additions GSH (M)
2VIEA (A'I)
5 x 10 -5
10 -4
5 x 10 -5
10 -4
3 x 10 -e
10 -5
Control
0 . 50
0-46
0-44
0-45
0 . 40
0-35
0-30
100 Gv X-rays
0-98
0-84
0-70
0 . 90
0-77
0-68
0-30
Percentage protection
(0)
21
46
6
23
31
100
Table 3 . Protection against lipid peroxidation by glutathione (GSH), cysteamine (MEA) and vitamin E (a-T) . The irradiation data are expressed as ratios of the absorptions at 233 and 215 nm . The dose rate was 0-8 Gy/min and the liposome concentration 0-5 mg/ml .
effective protector in the system . The concentrations yielding 50 per cent protection were roughly estimated to be 10 - 'M GSH, 5 x 10 - ' M MEA and 5 x 10 -6 M a-T . The liposomes consist of phospholipids containing PUFAs . Irradiation results in a change of the fatty acid pattern, as shown in table 4 . The compositions are all taken relative to the saturated fatty acid palmitic acid (16 : 0), which is set at 100 per cent . In particular, arachidonic acid (20 :4) and docosahexaenoic acid (22 :6) disappear, a small decrease in linoleic acid (18 : 2) is also observed . The irradiation
Radiation effects on liposomes
Fatty acids-
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16 18 18 18 20 22
:0 :0 :1 :2 :4 :6
Control
347
Irradiated
No addition
No addition
10 -4M GSH
10 -3 M GSH
10 -4M MEA
3 x 10 -6 1b 1 1 x-T
10 -5 M x-T
(100) 51 53 67 53 45
(100) 51 52 59 32 23
(100) 52 52 64 43 35
(100) 53 52 68 51 44
(100) 52 50 60 38 31
(100) 50 52 62 43 32
(100) 51 52 66 55 47
-1 Fatty acids are denoted by (number of carbon atoms : number of double bonds) . The compositions are all taken relative to palmitic acid (16 :0) . Table 4 . Protection of PUFAs during X-irradiation of liposomes . X-irradiation was performed with a dose of 100 Gy and a dose rate of 0 . 8 Gy/min under aerobic conditions . The liposome concentration was 0 . 5 mg/ml .
in this experiment was performed with a dose of 100 Gy ; higher doses of X-rays may destroy almost all PUFAs (data not shown) . The different protective substances used in the experiments mentioned before were also tested for their effect on the fatty acid composition . A good correlation appears to exist between the capacity of the compounds to protect against the formation of lipid peroxides (see table 3) and the ability to maintain the original fatty acid composition (see table 4) . Almost full protection was given by 10 -4 M GSH, but this was not the case with 10 -4 M MEA . A concentration of 10 -5 M x-T fully protected the PUFAs in the phospholipids of the liposomes . 4.
Discussion The possible importance of biological membranes as critical targets in irradiated cells has often been stressed, especially by Alper (1971, 1976) . Irradiation of cells with electron beams of selective penetration (Zermeno and Cole 1969) and with alpha-particles (Datta, Cole and Robinson 1976) have shown that the peripheral region of the cell nucleus is the most radiosensitive part of the cell, suggesting that the nuclear membrane is the main radiation target . This finding corresponds with the idea of the cell membrane being the site for DNA replication in prokaryotic systems (Kornberg, Lockwood and Worcel 1974) and of nuclear membranes having a similar function in eukaryotes (Dye and Tollivier 1975, Hobart, Duncan and Infante 1977) . The effect of irradiation on membrane-dependent functions is illustrated by activation of the plasma membrane enzyme alkaline phosphatase (Konings and Drijver 1975) and the nuclear membrane enzyme NAD glycohydrolase (Konings, Brauer and Streffer 1975) . The enhanced activity caused by relatively low doses of radiation has been explained as the result of radiation damage to the micro-(lipid)-environment of the enzyme in the membrane . The constraint normally imposed by the lipid environment is often essential for efficient functioning of membrane-dependent enzymes under physiological conditions . The radiation effect on the plasma membrane-bound enzyme alkaline phosphatase could be mimicked by mild detergent treatment of isolated plasma membranes (Konings and Drijver 1975) . The degree of saturation of phospholipids is known to influence the conformation of membrane proteins . As shown in this paper, the PUFAs arachidonic acid (20 : 4) and docosahexaenoic acid (22 : 6) are easily destroyed by ionizing irradiation of liposomes . This is an oxygen- and dose-rate-dependent reaction . High o .e .r .s were obtained when
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A . W. T . Konings et al.
irradiation was performed under hypoxic and oxic conditions at low dose rates . As early as 1952 Mead found an oxygen dependence of linoleate oxidation during irradiation (Mead 1952). High oxygen effects have been obtained in radiation experiments on isolated lysosomes in which the release of enzymes through the membrane was taken as the measure of radiation damage (Watkins 1970). The observation that more PUFAs are destroyed at a low dose rate is understandable in terms of slowly progressing chain reactions initiated by ionizing irradiation (Pryor 1976, Petkau and Chelak 1976, Raleigh, Kremers and Gaboury 1977) . More radiation damage occurred in sonicated liposomes with an optimal concentration of about 0 .5 mg PL per ml . The concentration dependence is an indication of the optimal formation of water radicals in relation to the amount of vulnerable lipid molecules . The dependence on radius of curvature, as enhanced by sonication, may have implications for biological membrane systems because there are many examples of very small radius of curvature such as the highly convoluted cristae of the mitochondrial inner membrane, the brush borders of intestinal epithelial cells and the nuclear membrane at the site of the pores . To obtain more knowledge of processes controlling radiation -induced lipid oxidation, two naturally occurring compounds, vitamin E and gluthathione, have been used and compared with cysteamine MEA, a very effective radiation protector in-vivo . These compounds have been tested for their capacity to protect the PUFAs present in biological membranes . The results of the experiments in this report show that all three compounds possess protective properties against radiation damage to arachidonic acid (20 : 4) and docosahexaenoic acid (22 : 6) . From the data presented it is also clear, however, that the lipid-soluble compound vitamin E is a much more effective protector than the water-soluble compounds glutathione and cysteamine . These thiol compounds are thought to react effectively with the radicals formed in the radiolysis of water (Jayson, Own and Wilbraham 1967), and it may be that the concentration close to the site of the vulnerable fatty acids in the bilayer is not as high as with vitamin E . The protective capacity of vitamin E against radiation damage to PUFAs probably must also be ascribed to a high degree of physical interaction of this compound with polyunsaturated phospholipids (Diplock and Lucy 1973, Maggio, Diplock and Lucy 1977) . It is very possible that membranes of living cells are protected against all kinds of oxidative stress by a complicated integrated defence mechanism . Vitamin E may be an antioxidant of primary importance to the unsaturated fatty acids because of the structural properties mentioned, while thiol compounds may take part in a secondary systems of defence with respect to this fatty acid oxidation . Acknowledgments The authors are very grateful to Mr . A . van Doorn for his advice on the use of the gas chromatograph . Financial support for these studies was obtained from the IRS, Interuniversity Institute for Radiopathology and Radiation Protection in The Netherlands . Des liposomes ont ete prepares a partir des phospholipides extraits des membranes biologiques . Une comparaison est faite entre les taux de peroxidation de lipides dans le systeme de liposomes ]'handshake' et les liposomes sonices . Les liposomes qui sont les plus petits etaient tres vulnerables, probablement parce que ]'existence d'une curvature plus petite, resultant cans un emballage de molecules lipidiques moins clos en structure de `bilayer' et one action plus facile des radicaux oxydants formes dans la phase aqueuse induite par l'irradiation de rontgen . Des rations d'augmentation d'oxygene tres elevees etaient obtenues, specialement aux velocities des doses tres bases, sugerant ('operation des chaInes de reactions progressant
Radiation effects on liposomes
349
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tres lentement, initiees par le rayonnement ionise . Trois combinaisons chimiques ont ete examinees pour tester leur qualite a proteger les membranes liposomales contre faction de peroxidation des lipides . Les combinaisons chimiques qui se trouvent clans la nature, la glutathione reduite (GSH) et la vitamine E (aT) et aussi le radioprotecteur tres fort, la cysteamine (MEA) . Toutes les trois molecules etaient capables de proteger les liposomes contre 1'action peroxidative . La combinaison vitamine E qui est soluble aux membranes etait la plus efficace. Les concentrations qui donnent un pourcentage de protection de 50 pour cent etaient 5 x 10 -6 M vitamine E, 10 -4 M GSH et 5 x 10-4M MEA . La composition des acides gras des lipides changeait radicalement comme resultat du rayonnement . L'acide arachidonique et l'acide docosahexaenoique etaient les acides gras les plus vulnerables . Une protection tres efficace de ces acides gras nonsatures etait obtenue avec la vitamine E aux concentrations tres basses qui sont incorporees aux membranes .
Aus Phospholipiden, die aus biologischen Membranen extrahiert worden waren, wurden Liposome prapariert . Die Peroxidationsrate wurde zwischen hand geschuttelten and mit Ultraschall behandelten Liposomen verglichen . Die kleineren, beschallten Liposome zeigten sich anfalliger gegenuber der Peroxidation, wahrscheinlich wegei i der kleinern Krummung welche, zu einer weniger dichten Packung der Lipidmolekule in der Doppelschicht fuhrt and somit die Einwirkung von Wasseradikalen nach Rontgen Bestrahlung erleichtert . Es wurden hohe Sauerstoffverstarkungsfaktoren erhalten, besonders bei niedrigen Dosisleistungen, was auf langsam ablaufende Kettenreaktionen hinweist, welche durch die Bestrahlung ausgelost werden . In bezug auf eine mogliche Schutzwirkung gegen Lipidperoxidation wurden drei Substanzen getestet, die naturlich vorkommen reduziertes Glutathion (GSH) and Vitamin E (a-T) sowie die wirksame Strahlenschu .substanz Cysteamin (MEA) . Alle drei Molek6le zeigten eine Schutzwirkung in Bezug auf die Lip idper~xidierung . Dabei war das membranlosliche Vitamin E das mit Abstand wirkungsvollste . Die Konzentrationen fur eine 50"0ige Schutzwirkung waren 5 x 10 -6 M bei aT, 10 - 'M bei GSH un 5 x 10 -4 M bei MEA . Die Fettsaurenzusammensetzung war als Folge der Bestrahlung drastisch verandert, dabei erwiesen sich Arachiodonsaure and Dokosahexamiensaure als die anfalligste . Dutch Einbau relativ geringer Mengen von Vitamin E in die Membranen konnte ein sehr wirksamer Schutz dieser mehrfach ungesattigen Fettsauren erreicht werden .
References ALPER, T ., 1971, Biophysical Aspects of Radiation Quality (London : H .M .S .O .), p . 171 ; 1976, Radiation and Cellular Control Processes, edited by J . Kiefer (New York : Springer-Verlag), p . 307 . AMES, B . N ., and DUBLIN, D . T ., 1960, J. biol. Chem ., 235, 769 . BACQ, Z. M ., 1965, Chemical Protection Against Ionizing Radiation (Springfield, Illinois : Charles . C . Thomas) . BANGHAM, A . D ., STANDISH, M ., and WATKINS, J . C ., 1965 . Y . molec . Biol., 13, 238 . BERGSTROM, B ., 1952, Acta physiol . scand ., 25, 101 . BLIGH, E . C ., and DYER, W . J ., 1959, Can . J. Biochem Physiol ., 37, 911 . BRIDGES, B . A ., 1969, Adv . Radiat . Biol ., 19, 651 . CHEN, P . S ., TORIBARA, T . Y ., and WARNER, H ., 1956, Analyt . Chem ., 28, 1756 . DAHLE, L . K ., HILL, E . G ., and HOLMAN, R . T ., 1962, Arehs Biochem . Biophys ., 98, 253 . DATTA, R ., COLE, A ., and ROBINSON, S ., 1976, Radiat. Res ., 34, 300 . DAVISON, S . G ., 1974, Progress in Surface Science, Vol . 4, Part 2 . DIPLOCK, A . T ., and LUCY, J . A ., 1973, FEBS Lett ., 29, 205 . DYE, D . M ., and ToLIVER, A . P ., 1975, Biochim . biophys . Acta, 414, 173 . HOBART, P ., DUNCAN, R ., and INFANTE, A . A ., 1977, Nature, Land ., 267, 544 . HOFFER, A ., and Roy, R . M ., 1975, Radiat . Res ., 61, 439 . JAN- SON, G . G ., OWEN, T . C ., and WILBRAHAM, A . C ., 1967, Y . chem . Soc . B, p . 944 . KENASTON, C . B ., WILBUR, K . M ., OTTOLENGHI, A ., and BERNHEIM, F ., 1955, Y. Am . Oil Chem . Soc ., 32, 33 . KLEIN, R . A ., 1970, Biochim . biophys . Acta, 210, 486 . KONINGS, A . W . T ., 1970, Biochim . biophys . Acta, 223, 398 ; 1977, Int . ,Y . Radiat . Biol ., 31, 381 . KONINGS, A . W . T ., BRAUER, W ., and STREFFER, C ., 1975, Int . Y . Radiat . Biol., 28, 589 . KONINGS, A . W . T ., and DRIJVER, E . B ., 1975, Br . Y. Cancer, 32, 755 . KONINGS, A . W . T ., and TRIELING, W . B ., 1977, Int . Y. Radiat . Biol ., 31, 397 . KORNBERG, T ., LOCKWOOD, A ., and WORCEL, A ., 1974, Proc . natn . Acad. Sci . U.S . A ., 71, 3189 . MAGGIO, B ., DIPLOCK, A . T ., and LUcY, J . A ., 1977, Biochem . Y ., 161, 111 . MEAD, J . F ., 1952, Science, N .Y ., 115, 470 . MILCH, R . A ., and KLASSE, G . A ., 1965, Nature, Lond ., 205, 1106 . PETKAU, A ., and CHELAK, W . S ., 1976, Biochim . biophys . Acta, 433, 445 . PRINCE, E . W ., and LITTLE, J . B ., 1973, Radiat . Res ., 53, 49 . PRYOR, W . A ., 1976, Free Radicals in Biology (New York : Academic Press) . R .B .
2B
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and GABOURY, B ., 1977, Int . J . Radiat . Biol ., 31, 20 G ., and WEISSMANN, G ., 1968, Y. Lipid Res ., 9, 310 . SIAKATOS, A . N ., and RAUSER, G ., 1965, Y . Am ., Oil Chem . Soc ., 42, 913 . SINGER, S . J ., and NICOLSON, G . L ., 1972, Science, N .Y ., 175, 720 . RALEIGH, J . A ., KREMERS, W ., SESSA,
WATKINS, D . K .,
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ZERMENO, A .,
and
1970, Adv . biol . med . Phys ., 13, 289 . COLE, A ., 1969, Radiat . Res ., 39, 669 .
1979, VOL . 35,
NO .
4, 343-350
Protection of liposomal lipids against radiation induced oxidative damage A. W . T . KONINGS, J . DAMEN and W . B . TRIELING
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Laboratory of Radiopathology, Bloemsingel 1, University of Groningen, The Netherlands
(Received 3 May 1978; accepted 20 July 1978) Liposomes were prepared from phospholipids extracted from biological membranes . A comparison was made between the peroxidation rate in handshake liposomes and in sonicated liposomes . The smaller sonicated liposomes were more vulnerable to peroxidation, probably because of the smaller radius of curvature, which results in a less dense packing of lipid molecules in the bilayer and a facilitated action of water radicals produced by the X-irradiation . High oxygen enhancement ratios were obtained, especially at low dose rates, suggesting the operation of slowly progressing chain reactions initiated by ionizing radiation . Three compounds were tested for their ability to protect the liposomal membranes against lipid peroxidation . The naturally occurring compounds reduced glutathione (GSH) and vitamin E (a-T) and the powerful radiation protector cysteamine (MEA) . All three molecules could protect the liposomes against peroxidation . The membrane-soluble compound vitamin E was by far the most powerful . About 50 per cent protection was achieved by using 5 x 10 -6 M a-T, 10 -4 M GSH and 5 x 10 -4 M MEA . The fatty acid composition of the lipids altered drastically as a result of the irradiation . Arachidonic acid and docosahexanoic acid were the most vulnerable of the fatty acids . Very efficient protection of these polyunsaturated fatty acids could be obtained with relatively low concentrations of vitamin E built into the membranes .
1.
Introduction Although the cell membranes perform an important role in the functional organization of the cell, relatively little experimental work on radiation damage to membranes has been reported, as compared to radiation studies on nucleic acids . For a systematic approach to the problem of radiation-induced alterations in biomembranes, model systems are very suitable because different membrane components can be investigated separately . About half the mass of most cellular membranes consists of lipid molecules . The lipids are structured as bilayers, although the continuity is interrupted by nonbilayer regions (Singer and Nicolson 1972) . Liposomes are aqueous lipid dispersions, also organized as bilayers of lipid molecules (Bangham, Standish and Watkins 1965, Sessa and Weissmann 1968, Davison 1974) and thus provide an excellent model for radiation studies . The lipsomes used in the study to be reported here consisted of phospholipids extracted from biological membranes, and were prepared in the presence or absence of naturally occurring compounds with antioxidant capacity : glutathione and vitamin E . The action of radioprotective thiols such as gluthathione on living organisms has been well documented (Bacq 1{' :5, Bridges 1969) . A possible role of vitamin E as an in vivo radiation protector biological membranes has been indicated by recent work on the fragility of erythrocytes (Prince and Little 1973, Hoffer and Roy 1975) and by the observation of enhanced radiosensitivity of vitamin-E-deficient lymphosarcoma cells (Konings and Trieling 1977) . In this report the formation of conjugated diens in liposomes as a result of Xirradiation is described, and also the destruction of polyunsaturated fatty acids (PUFAs) in the phospholipids . It is well established that the methylene-interrupted PUFAs are vulnerable structures with respect to hydrogen abstraction, yielding
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conjugated dienes as the initial stage in lipid peroxidation, prior to chain scission (Milch and Klasse 1965) . Good correlation has been found to exist between the formation of conjugated dienes and the formation of products of lipid peroxidation such as malondialdehyde (Kenaston, Wilbur, Ottolenghi and Bernheim 1955, Dahle, Hill and Holman 1962, Pryor 1976) . Special attention will be paid to protective capacities against radiation-induced destruction of certain fatty acids as afforded by the naturally occurring compounds vitamin E and glutathione .
2. Materials and methods 2.1 . Subcellular fractionation C5,B1 mice, 3 to 4 months old, were killed by cervical dislocation and the livers were removed . A 10 per cent homogenate was made in the cold (4°C) in a Potter Elvehjem homogenizer with a motor-driven Teflon pestle (clearance 0 . 20 mm) in a medium consisting of 0 . 25 M sucrose in SO mM tris HCI, pH 7 .5, 2. 5 mM KC1 and 5 MM MgC1 2 . This solution (STKM) was used throughout the isolation procedure . The homogenate was filtered through two layers of cheesecloth and centrifuged for 10 min at 900g . The sediment contained the rough nuclear fraction and cell debris . The supernatant was centrifuged for 30 min at 15 OOOg to sediment the mitochondria and lysosomes (M + L fraction) . The microsomal fraction was obtained after centrifuging the 15 OOOg supernatant at 105 OOOg for 60min . All sediments were taken up in STKM, and the suspensions obtained were subjected to lipid extraction .
2 .2 . Isolation of phospholipids The lipid extraction and chromatographic separation procedures were generally performed as described previously (Konings 1970) . In most of the experiments described in this paper, phospholipids of the M + L fraction were used . To initiate the lipid extraction of the suspensions, two volumes of methanol and one volume of chloroform were added under thorough mixing in a glass-stoppered centrifuge tube . The extraction was completed according to Bligh and Dyer (1959) . Non-lipid contaminants were removed by Sephadex column chromatography (Siakatos and Rouser 1965) . The lipids were separated into neutral lipids and phospholipids by silicic acid column chromatography (Bergstrom 1952) . The total number of lipids and the proportions of neutral and phospholipids were quantitatively determined by evaporating aliquots of the extracts under a stream of nitrogen gas and weighing the lipid on a Cahn (Gram) electrobalance . To determine the phospholipid composition of the extract, thin layer chromatography on silica gel HF (Merck) was carried out, followed by destruction (Ames and Dubin 1960) of the separated fraction and phosphorus determination (Chen, Toribara and Marner 1956) . 2 .3 . Preparation of liposomes Solutions of phospholipids, isolated as described above were, if necessary, mixed with vitamin E in ethanol, added to round-bottomed flasks and the organic solvents evacuated at 30°C in rotatory evaporator . This resulted in a thin dry lipid coating on the flask wall . Small glass beads were added, and also double-distilled water in which, if necessary, glutathione and cysteamine were solubilized and brought to pH 7 . 4 . The fluid in the flask was purged with oxygen-free nitrogen and vigorously agitated on a Vortex mixer for 1 min . In order to obtain smaller liposomes the preparation received a sonication treatment with a Branson B12 Sonifier at 60-8 W .
Radiation effects on liposomes
345
This was done after the Vortex mixing, during four periods of 30s separated by pauses of 15 s at room temperature under a stream of nitrogen .
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2 .4 . Irradiation
X-rays from a Philips-Muller MG 300 X-ray machine were used, which operated at 200 kV and 15 mA . The beam was filtered with 0. 5 mm Cu and 0. 5 mm Al; the h .v .l . was 1 . 1 mm Cu . Different dose rates were obtained by changing the focus-object distance . The samples were continuously gassed with either nitrogen or air before and during the irradiation . All irradiations were performed at room temperature . 2 .5 . Lipid peroxidation
The formation of conjugated dienes was determined after adding 10 volumes of ethanol to one volume of the liposomes and measuring U .V . absorption in quartx cuvettes at 233 and 215 rim. The absorbancy ratio was taken as a measure of lipid peroxidation (Klein 1970) . 2 .6 . Determination of fatty acid composition
Aliquots of liposomal suspensions were subjected to lipid extraction as described above . For transesterification purposes about 1 mg of dry phospholipid (under N 2) was solubilized in 10 ml methanol/sulphuric acid (95 : 5, v/v) in glass-stoppered tubes. The solution was purged with N 2 for 2 min, the tubes closed and placed in a water bath at 70°C for 2 hours . After cooling, 5 ml of water was mixed into the solution . The solution was then extracted twice with 10 ml of hexane, and the gathered hexane phases were extracted twice with 10 ml of water and subsequently dried over anhydrous sodium sulphate . The solution was filtered and evaporated under N 2 . The resulting methylated fatty acids were taken up in 50 or 100 µl of hexane . Mostly 1 µl of hexane sample was applied to a Becker Packard Gaschromatograph Model 419, equippped with a flame ionization detector and integrator system . As a stationary phase a cyanosilicone column was used that consisted of 10 per cent SP-2330 on 100/120 Chromosorb W AW (Supelco Inc ., Bellefonte, Pennsylvania) . The heating was carried out at an initial temperature of 165 ° C for 2 min, followed by an increase of 3 ° C per minute up to 200 ° C . The methylesters were identified by comparison with retention times of authentic standards and controlled on a different GLC column (diethyleneglycolsuccinate) . 3. Results Liposomes of different sizes and at different concentrations were prepared in order to establish the effect of X-irradiation on lipid peroxidation . The approximate phospholipid composition of the liposomes was 65 per cent phosphatidylcholine (PC), 20 per cent phosphatidylethanolamine (PE), 5 per cent phosphatidylinositol (PI) plus phosphatidylserine (PS), and 10 per cent diphosphatidylglycerol (DPG) . The data in table 1 are expressed as the ratio between the absorptions at 233 and 215 nm, which is a quantitative measure of the formation of conjugated dienes (Klein 1970) . The highest yields were obtained at concentrations of about 0 . 3 to 0. 5 mg of phospholipid per ml . The sonicated liposomes are smaller and were more vulnerable than the unsonicated larger liposomes . Sonicated liposomes were used throughout the rest of the investigation . Table 2 gives the results of an experiment in which an X-ray dose of 200 Gy was given at both (0 . 8 Gy/min) and (8 . 0 Gy/min)
346
A . W. T . Konings et al.
Lipid concentration (mg/ml)
Unsonicated
Sonicated
Control
Irradiated
Difference
Control
Irradiated
Difference
0-3 0.5 0.7
0-26 0 . 28 028
0-72 0 . 81 0 . 56
0-45 0 . 53 0 . 28
0-45 0. 44 0. 46
1 . 09 1 . 13 0 . 99
0-64 0. 69 0-53
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Table 1 . Lipid peroxidation in X-irradiated liposomes . Each datum is expressed as the ratio of absorptions at 233 and 215 nm . The irradiation was performed in air with a dose of 100 Gy at a dose rate of 0. 8 Gy/min . Radiation conditions Dose rate (Gy/min)
Air
O .e .r .
Control
Irradiated
Difference
Control
Irradiated
Difference
0. 48 0-36
1 . 28 0-62
0-80 0-26
0-28 0-25
0-34 0-30
0 .06 0 .05
0. 8 8-0 Table 2 .
Hypoxia
13 5
Effects of dose rate on o .e .r . Sonicated liposomes were X-irradiated with a dose of 200 Gy ; the phospholipid concentration was 0-5 mg/ml .
dose rates under oxic and hypoxic conditions . While almost no dose-rate effects were observed under hypoxic conditions, enhanced peroxidation was found under aerobic conditions when a low dose rate was applied . As a consequence, the oxygen enhancement ratio (o .e .r .) clearly increased . The protective ability of reduced glutathione, cysteamine and vitamin E were then compared with respect to lipid peroxidation induced by X-irradiation . The results of a representative experiment are given in table 3 . From the first line of the table it can be seen that the three compounds protected the lipids against peroxidation, during both the process of preparing the liposomes and the time that the irradiated liposomes were exposed to the X-rays . Protection against radiation-induced peroxidation was observed for all three compounds, as shown in the last line of table 3 . Vitamin E was by far the most Formation of conjugated dienes None
Additions GSH (M)
2VIEA (A'I)
5 x 10 -5
10 -4
5 x 10 -5
10 -4
3 x 10 -e
10 -5
Control
0 . 50
0-46
0-44
0-45
0 . 40
0-35
0-30
100 Gv X-rays
0-98
0-84
0-70
0 . 90
0-77
0-68
0-30
Percentage protection
(0)
21
46
6
23
31
100
Table 3 . Protection against lipid peroxidation by glutathione (GSH), cysteamine (MEA) and vitamin E (a-T) . The irradiation data are expressed as ratios of the absorptions at 233 and 215 nm . The dose rate was 0-8 Gy/min and the liposome concentration 0-5 mg/ml .
effective protector in the system . The concentrations yielding 50 per cent protection were roughly estimated to be 10 - 'M GSH, 5 x 10 - ' M MEA and 5 x 10 -6 M a-T . The liposomes consist of phospholipids containing PUFAs . Irradiation results in a change of the fatty acid pattern, as shown in table 4 . The compositions are all taken relative to the saturated fatty acid palmitic acid (16 : 0), which is set at 100 per cent . In particular, arachidonic acid (20 :4) and docosahexaenoic acid (22 :6) disappear, a small decrease in linoleic acid (18 : 2) is also observed . The irradiation
Radiation effects on liposomes
Fatty acids-
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16 18 18 18 20 22
:0 :0 :1 :2 :4 :6
Control
347
Irradiated
No addition
No addition
10 -4M GSH
10 -3 M GSH
10 -4M MEA
3 x 10 -6 1b 1 1 x-T
10 -5 M x-T
(100) 51 53 67 53 45
(100) 51 52 59 32 23
(100) 52 52 64 43 35
(100) 53 52 68 51 44
(100) 52 50 60 38 31
(100) 50 52 62 43 32
(100) 51 52 66 55 47
-1 Fatty acids are denoted by (number of carbon atoms : number of double bonds) . The compositions are all taken relative to palmitic acid (16 :0) . Table 4 . Protection of PUFAs during X-irradiation of liposomes . X-irradiation was performed with a dose of 100 Gy and a dose rate of 0 . 8 Gy/min under aerobic conditions . The liposome concentration was 0 . 5 mg/ml .
in this experiment was performed with a dose of 100 Gy ; higher doses of X-rays may destroy almost all PUFAs (data not shown) . The different protective substances used in the experiments mentioned before were also tested for their effect on the fatty acid composition . A good correlation appears to exist between the capacity of the compounds to protect against the formation of lipid peroxides (see table 3) and the ability to maintain the original fatty acid composition (see table 4) . Almost full protection was given by 10 -4 M GSH, but this was not the case with 10 -4 M MEA . A concentration of 10 -5 M x-T fully protected the PUFAs in the phospholipids of the liposomes . 4.
Discussion The possible importance of biological membranes as critical targets in irradiated cells has often been stressed, especially by Alper (1971, 1976) . Irradiation of cells with electron beams of selective penetration (Zermeno and Cole 1969) and with alpha-particles (Datta, Cole and Robinson 1976) have shown that the peripheral region of the cell nucleus is the most radiosensitive part of the cell, suggesting that the nuclear membrane is the main radiation target . This finding corresponds with the idea of the cell membrane being the site for DNA replication in prokaryotic systems (Kornberg, Lockwood and Worcel 1974) and of nuclear membranes having a similar function in eukaryotes (Dye and Tollivier 1975, Hobart, Duncan and Infante 1977) . The effect of irradiation on membrane-dependent functions is illustrated by activation of the plasma membrane enzyme alkaline phosphatase (Konings and Drijver 1975) and the nuclear membrane enzyme NAD glycohydrolase (Konings, Brauer and Streffer 1975) . The enhanced activity caused by relatively low doses of radiation has been explained as the result of radiation damage to the micro-(lipid)-environment of the enzyme in the membrane . The constraint normally imposed by the lipid environment is often essential for efficient functioning of membrane-dependent enzymes under physiological conditions . The radiation effect on the plasma membrane-bound enzyme alkaline phosphatase could be mimicked by mild detergent treatment of isolated plasma membranes (Konings and Drijver 1975) . The degree of saturation of phospholipids is known to influence the conformation of membrane proteins . As shown in this paper, the PUFAs arachidonic acid (20 : 4) and docosahexaenoic acid (22 : 6) are easily destroyed by ionizing irradiation of liposomes . This is an oxygen- and dose-rate-dependent reaction . High o .e .r .s were obtained when
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A . W. T . Konings et al.
irradiation was performed under hypoxic and oxic conditions at low dose rates . As early as 1952 Mead found an oxygen dependence of linoleate oxidation during irradiation (Mead 1952). High oxygen effects have been obtained in radiation experiments on isolated lysosomes in which the release of enzymes through the membrane was taken as the measure of radiation damage (Watkins 1970). The observation that more PUFAs are destroyed at a low dose rate is understandable in terms of slowly progressing chain reactions initiated by ionizing irradiation (Pryor 1976, Petkau and Chelak 1976, Raleigh, Kremers and Gaboury 1977) . More radiation damage occurred in sonicated liposomes with an optimal concentration of about 0 .5 mg PL per ml . The concentration dependence is an indication of the optimal formation of water radicals in relation to the amount of vulnerable lipid molecules . The dependence on radius of curvature, as enhanced by sonication, may have implications for biological membrane systems because there are many examples of very small radius of curvature such as the highly convoluted cristae of the mitochondrial inner membrane, the brush borders of intestinal epithelial cells and the nuclear membrane at the site of the pores . To obtain more knowledge of processes controlling radiation -induced lipid oxidation, two naturally occurring compounds, vitamin E and gluthathione, have been used and compared with cysteamine MEA, a very effective radiation protector in-vivo . These compounds have been tested for their capacity to protect the PUFAs present in biological membranes . The results of the experiments in this report show that all three compounds possess protective properties against radiation damage to arachidonic acid (20 : 4) and docosahexaenoic acid (22 : 6) . From the data presented it is also clear, however, that the lipid-soluble compound vitamin E is a much more effective protector than the water-soluble compounds glutathione and cysteamine . These thiol compounds are thought to react effectively with the radicals formed in the radiolysis of water (Jayson, Own and Wilbraham 1967), and it may be that the concentration close to the site of the vulnerable fatty acids in the bilayer is not as high as with vitamin E . The protective capacity of vitamin E against radiation damage to PUFAs probably must also be ascribed to a high degree of physical interaction of this compound with polyunsaturated phospholipids (Diplock and Lucy 1973, Maggio, Diplock and Lucy 1977) . It is very possible that membranes of living cells are protected against all kinds of oxidative stress by a complicated integrated defence mechanism . Vitamin E may be an antioxidant of primary importance to the unsaturated fatty acids because of the structural properties mentioned, while thiol compounds may take part in a secondary systems of defence with respect to this fatty acid oxidation . Acknowledgments The authors are very grateful to Mr . A . van Doorn for his advice on the use of the gas chromatograph . Financial support for these studies was obtained from the IRS, Interuniversity Institute for Radiopathology and Radiation Protection in The Netherlands . Des liposomes ont ete prepares a partir des phospholipides extraits des membranes biologiques . Une comparaison est faite entre les taux de peroxidation de lipides dans le systeme de liposomes ]'handshake' et les liposomes sonices . Les liposomes qui sont les plus petits etaient tres vulnerables, probablement parce que ]'existence d'une curvature plus petite, resultant cans un emballage de molecules lipidiques moins clos en structure de `bilayer' et one action plus facile des radicaux oxydants formes dans la phase aqueuse induite par l'irradiation de rontgen . Des rations d'augmentation d'oxygene tres elevees etaient obtenues, specialement aux velocities des doses tres bases, sugerant ('operation des chaInes de reactions progressant
Radiation effects on liposomes
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tres lentement, initiees par le rayonnement ionise . Trois combinaisons chimiques ont ete examinees pour tester leur qualite a proteger les membranes liposomales contre faction de peroxidation des lipides . Les combinaisons chimiques qui se trouvent clans la nature, la glutathione reduite (GSH) et la vitamine E (aT) et aussi le radioprotecteur tres fort, la cysteamine (MEA) . Toutes les trois molecules etaient capables de proteger les liposomes contre 1'action peroxidative . La combinaison vitamine E qui est soluble aux membranes etait la plus efficace. Les concentrations qui donnent un pourcentage de protection de 50 pour cent etaient 5 x 10 -6 M vitamine E, 10 -4 M GSH et 5 x 10-4M MEA . La composition des acides gras des lipides changeait radicalement comme resultat du rayonnement . L'acide arachidonique et l'acide docosahexaenoique etaient les acides gras les plus vulnerables . Une protection tres efficace de ces acides gras nonsatures etait obtenue avec la vitamine E aux concentrations tres basses qui sont incorporees aux membranes .
Aus Phospholipiden, die aus biologischen Membranen extrahiert worden waren, wurden Liposome prapariert . Die Peroxidationsrate wurde zwischen hand geschuttelten and mit Ultraschall behandelten Liposomen verglichen . Die kleineren, beschallten Liposome zeigten sich anfalliger gegenuber der Peroxidation, wahrscheinlich wegei i der kleinern Krummung welche, zu einer weniger dichten Packung der Lipidmolekule in der Doppelschicht fuhrt and somit die Einwirkung von Wasseradikalen nach Rontgen Bestrahlung erleichtert . Es wurden hohe Sauerstoffverstarkungsfaktoren erhalten, besonders bei niedrigen Dosisleistungen, was auf langsam ablaufende Kettenreaktionen hinweist, welche durch die Bestrahlung ausgelost werden . In bezug auf eine mogliche Schutzwirkung gegen Lipidperoxidation wurden drei Substanzen getestet, die naturlich vorkommen reduziertes Glutathion (GSH) and Vitamin E (a-T) sowie die wirksame Strahlenschu .substanz Cysteamin (MEA) . Alle drei Molek6le zeigten eine Schutzwirkung in Bezug auf die Lip idper~xidierung . Dabei war das membranlosliche Vitamin E das mit Abstand wirkungsvollste . Die Konzentrationen fur eine 50"0ige Schutzwirkung waren 5 x 10 -6 M bei aT, 10 - 'M bei GSH un 5 x 10 -4 M bei MEA . Die Fettsaurenzusammensetzung war als Folge der Bestrahlung drastisch verandert, dabei erwiesen sich Arachiodonsaure and Dokosahexamiensaure als die anfalligste . Dutch Einbau relativ geringer Mengen von Vitamin E in die Membranen konnte ein sehr wirksamer Schutz dieser mehrfach ungesattigen Fettsauren erreicht werden .
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