Radiation-induced changes in permeability in unilamellar phospholipid liposomes

Gamma-radiation-induced oxidative damage in unilamellar dipalmitoylphosphatidylcholine liposomes was investigated using a fluorescence technique. Liposomal changes in permeability induced by gamma radiation were monitored by measuring the leakage of pre-encapsulated 6-carboxyfluorescein, and alterations in lipid bilayer fluidity were determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization. The changes in permeability and fluidity in the bilayer were found to be dependent on the radiation dose in a biphasic fashion. The results are interpreted in terms of lipid bilayer fluidization after exposure to doses up to 1 kGy, but rigidization of the bilayer at higher doses. These results indicate a relationship between alterations in permeability and fluidity in the lipid bilayer after irradiation. The vesicles were protected significantly against radiation-induced oxidative damage in the presence of alpha-tocopherol and ascorbic acid. Radiation-induced changes in the permeability of the liposomes after exposure to gamma radiation and their modification by antioxidants indicate the involvement of a free radical mechanism in the production of damage, which may offer new insights in to the modification of cellular radiosensitivity by modulation of membrane damage.     

Radioprotective effects of lipid A, liposomes, and liposomes containing lipid A in mice

Lipid A from Gram-negative bacterial lipopolysaccharide (endotoxin) was incorporated into liposomal membranes and examined as a prophylactic radioprotectant compound in lethally irradiated mice. Splenic hematopoietic activity, resulting in increased numbers of spleen cell colonies, was induced both by lipid A alone or more strongly by liposomal lipid A. Increased survival of lethally irradiated animals was induced to a slight extent by liposomes alone, to a greater extent by lipid A, and at the highest level by liposomes containing lipid A. Under conditions where 100% of untreated or saline-treated animals died of acute radiation syndrome after 20 days, more than 90% of the animals pretreated with liposomal lipid A were still alive 30 days after irradiation. We conclude that lipid A had substantial radioprotectant activity by itself, and the activity was enhanced by incorporation into liposomes. Liposomes alone also exhibited mild radioprotectant effects.     

Antioxidant properties of resveratrol and piceid on lipid peroxidation in micelles and monolamellar liposomes

The antioxidant activities of trans-resveratrol (trans-3,5,4′-trihydroxystilbene) and trans-piceid (trans-5,4′-dihydroxystilbene-3-O-β-d-glucopyranoside), its more widespread glycosilate derivative, have been compared measuring their inhibitory action on peroxidation of linoleic acid (LA) and the radical scavenging ability towards different free radicals (such as DPPH) and radical initiators. It has been found that the two stilbenes have similar antioxidant capacity, while the comparison with BHT (2,6-di-tert-butyl-4-methylphenol) and -tocopherol (vitamin E, vit. E), taken as reference, points out a slower but prolonged protective action against lipid peroxidation. Furthermore, piceid appears more efficacious than resveratrol as a consequence of the reaction of the latter with its radical form.

The DSC profiles of phosphatidylcholine liposomes of various chain lengths, and EPR measurements of spin labelled liposomes demonstrated that the susceptible hydroxyl group of these compounds are located in the lipid region of the bilayer close to the double bonds of polyunsatured fatty acids, making these stilbenes particularly suitable for the prevention and control of the lipid peroxidation of the membranes.     

Radiation-induced lipid peroxidation: influence of oxygen concentration and membrane lipid composition

Radiation-induced lipid peroxidation in phospholipid liposomes was investigated in terms of its dependence on lipid composition and oxygen concentration. Non-peroxidizable lipid incorporated in the liposomes reduced the rate of peroxidation of the peroxidizable phospholipid acyl chains, possibly by restricting the length of chain reactions. The latter effect is believed to be caused by interference of the non-peroxidizable lipids in the bilayer. At low oxygen concentration lipid peroxidation was reduced. The cause of this limited peroxidation may be a reduced number of radical initiation reactions possibly involving oxygen-derived superoxide radicals. Killing of proliferating mammalian cells, irradiated at oxygen concentrations ranging from 0 to 100 per cent, appeared to be independent of the concentration of peroxidizable phospholipids in the cell membranes. This indicates that lipid peroxidation is not the determining process in radiation-induced reproductive cell death.     

The thermal properties of dilauryl phosphatidylethanolamine liposomes are affected by lipid source and preparation

Dilauryl phosphatidylethanolamine (DLPE) dispersions in water ('liposomes') display phase metastability. We find, by differential scanning calorimetry (d.s.c.), that the new phases are dependent upon lipid source and temperature of initial hydration. These observations may explain inconsistencies in the reported metastability behaviour of saturated PEs.     

Effect of lipid hydroperoxide on lipoxygenase kinetics.

In order to investigate the activation of lipoxygenase and to clarify the role of the oxygenation product hydroperoxide in this process, the effect of 13-hydroperoxylinoleic acid (P, 0-35 microM) on linoleic acid (S, 1-80 microM) oxygenation catalysis by 12 nM lipoxygenase-1 from soybean was studied at pH 10, 25 degrees C, and 240 microM O2 with rapid kinetic techniques. The following observations were made: (1) Iron(II) and iron(III) lipoxygenases are kinetically different: reactions started with the Fe(II) enzyme form show a lag phase, whereas iron(III) lipoxygenase induces an initial burst. (2) Oxidation of the enzyme alone is not sufficient to abolish the lag phase: at [S] greater than 50 microM, the initial burst in the iron(III) lipoxygenase curves is still followed by a lag. The lag phase disappears completely only in the presence of micromolar quantities of P. (3) The approximate dissociation constants for S and P are 15 and 24 microM, respectively, 1 order of magnitude smaller than the corresponding values in the absence of oxygen. The observed kinetics are predicted by numerical integration of the rate equations of a model based on the single lipid binding site mechanism for the anaerobic lipoxygenase reaction [Ludwig et al. (1987) Eur. J. Biochem. 168, 325-337; Verhagen et al. (1978) Biochim. Biophys. Acta 529, 369-379]. A quasi-steady-state approximation of the model suggests that a high [S]/[P] the fraction of active iron(III) lipoxygenase is small and that, therefore, a lag phase is intrinsic to the mechanism.     

Linoleic acid hydroperoxide favours hypochlorite- and myeloperoxidase-induced lipid peroxidation

Liposomes composed of soybean phosphatidylcholine were peroxidized using the reagent sodium hypochlorite or the myeloperoxidase-hydrogen peroxide-Cl^- system. Linoleic acid hydroperoxide previously prepared from linoleic acid by means of lipoxidase was incorporated into liposomes. The yield of thiobarbituric acid reactive substances (TBARS) continuously increased with higher amounts of hydroperoxide groups after the initiation of lipid peroxidation by hypochlorous acid producing systems. The accumulation of TBARS was inhibited by scavengers of free radicals such as butylated hydroxytoluene and by the scavengers of hypochlorous acid, taurine and methionine. Lipid peroxidation was also prevented by sodium azide or chloride free medium in the myeloperoxidase-hydrogen peroxide-Cl^- system. Here we show for the first time that the reaction of hypochlorous acid with a biologically relevant hydroperoxide yields free radicals able to cause further oxidation of lipid molecules.     

Activity of soybean lipoxygenase in the absence of lipid hydroperoxide

Soybean lipoxygenase was assayed under conditions such that the concentration of the enzyme was in excess of the concentration of the substrate, arachidonic acid. Under these conditions, the concentration of lipid hydroperoxides present as contaminants in the substrate was negligible relative to the enzyme concentration, and the concentration of lipid hydroperoxide product could be determined accurately. The ferric form of the enzyme was observed to be fully active and to catalyze the oxidation of arachidonic acid at a near-diffusion-controlled rate, 1.4 X 10(7) M-1 s-1 at 0 degree C, at concentrations of lipid hydroperoxides as low as 5% of the enzyme concentration. From this, it can be concluded that the higher oxidation states that would be accessible by oxidation of Fe(III) by hydroperoxide are not required for catalysis by soybean lipoxygenase. Surprisingly, the activation of the ferrous form of the enzyme was also observed at insignificantly low lipid hydroperoxide concentrations. This activation presumably involves oxidation of the ferrous to the ferric form of the enzyme and must be more facile than has hitherto been reported. This result may rationalize previous reports that the ferrous and the ferric forms of the enzyme are both active.     

On the specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase from Arabidopsis thaliana

Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well.     

Carboxymethylated glucan inhibits lipid peroxidation in liposomes

Protective capabilities were studied of carboxymethylated (1-->3)-beta-D-glucan from Saccharomyces cerevisiae cell wall against lipid peroxidation in phosphatidylcholine liposomes induced by OH radicals produced with Fenton's reagent (H2O2/Fe2+) and also by microwave radiation using absorption UV-VIS spectrophotometry. A significant decrease in the conjugated diene production, quantified as Klein oxidation index, was observed in the presence of a moderate amount of added glucan. Increase of the oxidation index was accompanied with enhanced carboxyfluorescein leakage as a result of liposome membrane destabilization. This process was markedly suppressed with glucan present in the liposome suspension. Therefore, glucan may be considered as a potent protector against microwave radiation-induced cell damage.     

Radiation-induced damage to mitochondrial D-beta-hydroxybutyrate dehydrogenase and lipid peroxidation

Radiation-induced damage to the reconstituted system of membrane-bound enzyme, D-beta-hydroxybutyrate dehydrogenase obtained from rat liver mitochondria, was investigated in relation to the lipid peroxidation of membranes. The activity of D-beta-hydroxybutyrate dehydrogenase in fresh mitochondria was very low in general and was not affected by irradiation because of little incorporation of substrates into mitochondria. However, the enzyme activity in one-day-aged mitochondria or submitochondrial particles was five times higher than that of fresh mitochondria and decreased with increasing radiation dose accompanying the increase in peroxidation of membrane lipids. The activity of D-beta-hydroxybutyrate dehydrogenase in the reconstituted system of the purified enzyme with irradiated liver microsomes or irradiated liposomes was decreased considerably in comparison with either unirradiated control or irradiated enzyme. Therefore, the radiation-induced decrease in the enzyme activity was thought to be caused mainly by peroxidation of membrane lipids and not to be due to direct damage by radiation to the enzyme molecule itself. Irradiation of microsomes, a component of the reconstituted system, caused decreases in phosphatidylcholine and phosphatidylethanolamine content and an increase in lysophosphatidylcholine content. In addition, arachidonic acid contents in phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were also markedly decreased with increasing radiation dose. These results are discussed in terms of a mechanism involving radiation-induced damage to membrane function and structures.     

Fluorescent probes for asymmetric lipid bilayers: Synthesis and properties in phosphatidyl choline liposomes and erythrocyte membranes

Summary We have synthesized three sets of fluorescent probes which we believe will be useful in studies of asymmetric membranes and have studied their interactions with model lipid bilayers and erythrocyte membranes. The probes were designed to partition preferentially into one face of a lipid bilayer with asymmetrically disposed phospholipids and to report lipid transitions in that monolayer.We synthesized more than twenty probes containing anthroyl-, dansyl-, or pyrene rings with acidic, basic, and neutral functional groups and alkyl spacers of various lengths. The interactions of these probes with liposomes of phosphatidyl choline and with erythrocyte membranes were characterized to determine whether probe insertion was asymmetric, how deeply the probe penetrated the bilayer, and whether the probe reflected thermotropic phase transitions in model membranes. The set of variously charged anthroyl esters, analogs of local anaesthetics, appears to be promising for studies of asymmetric membranes.Fluorescent probes have been used extensively to provide information on the lipid regions of biological membranes. Membrane fluidity, a composite of molecular packing and motion of acyl chains in lipid bilayers, has been assessed with a variety of fluorescent probes, the fluorescence of which undergoes some measurable change at the temperature of the membrane's thermotropic phase transition. A large number of fluorescent probes have been used for this purpose. Bashford, Morgan and Radda (Bashford, C.L., Morgan, C.G., Radda, G.K. 1976;Biochim. Biophys. Acta 426: 157) and Thulborn and Sawyer (Thulborn, K.R., Sawyer, W.H. 1978;Biochim. Biophys. Acta 511: 125) synthesized several fatty acid derivatives in which an anthracene group is attached (in ester linkage) along the acyl chain at various positions, and have shown that this set of probes may be useful in probing membrane fluidity at differentdepths within the bilayer.This report describes the synthesis and properties of several sets of amphipathic fluorescent probes, which may partition unequally into the two faces of an asymmetric lipid bilayer, and may therefore provide information about membranes complementary to that obtainable with existing probes.     

The effect of dietary lipid hydroperoxide on lymphoid tissues in mice

Effects of dietary lipid hydroperoxides on lymphoid tissue were studied in mice. When graded amounts (190, 270 and 310 mg) of methyl linoleate hydroperoxide (MLHPO) were orally administered to male C57BL/6 mice (6 weeks old), necrosis was observed in lymphocytes located among the reticular network in the thymus, and thymus weight was significantly decreased 24 h after the treatment. The spleen weight of mice given MLHPO tended to decrease. Spontaneous chemiluminescence of the thymus was remarkably increased after the dose. Thiobarbituric acid reactants in the liver, thymus and blood were also increased after the dose of MLHPO. At intervals of 3, 6, 12 and 24 h after a dose of 14C-labeled MLHPO, 14C was detected in the blood and liver. Fatty infiltration of the liver was found after the treatment with MLHPO. These findings indicate that oral intake of lipid hydroperoxides causes significant damage to lymphoid tissues of mice.     

Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide.

The increase in light emission of hydroperoxide-supplemented cytochrome c observed on addition of lipid vesicles was related to the degree of unsaturation of the fatty acids of the phospholipids: dipalmitoyl phosphatidylcholine was without effect, whereas dioleoyl phosphatidylcholine and soya-bean phosphatidylcholine enhanced chemiluminescence 2- and 3-fold respectively. Effects on light-emission were similar to those on O2 uptake. The chemiluminescence of the present system was sensitive to cyanide and to the radical trap 2,5-di-t-butylquinol, indicating a catlytic activity of cytochrome c and the presence of free-radical species respectively. Lipid-vesicle enhanced chemiluminescence showed different kinetic behaviours, apparently depending on unsaturation: three phases are described for soya-bean phosphatidylcholine, whereas only one phase was present in mixtures containing dipalmitoyl and dioleoyl phospholipids. Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide showed similar kinetic patterns with H2O2 and primary (ethyl) and tertiary (t-butyl and cumene) hydroperoxides. Participation of singlet molecular oxygen, mainly on the phase III of chemiluminescence, is suggested by the increase of light-emission by 1,4-diazabicyclo[2.2.2]-octane as well as by data from spectral analysis.     

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

Singlet oxygen generation from phosphatidylcholine hydroperoxide in the presence of copper

This study pursued whether singlet oxygen ((1)O2) is generated from phosphatidylcholine hydroperoxide (PCOOH), the oxidized modification product of a major constituent of biomembranes and serum lipoproteins. The (1)O2 formation was detected, by utilizing the oxidation of 2,2,6,6-tetramethyl-4-piperidone (TMPD) by (1)O2 to yield 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (TEMPONE), which generates electron spin resonance (ESR) signals. The TEMPONE signal was detected in human plasma with addition of PCOOH by ESR determination after introducing copper(II). The TEMPONE formation was proportional to the amounts of PCOOH added according to moles of active oxygen. The TEMPONE signal intensity was weakened significantly in the presence of beta-carotene and histidine in a concentration-dependent manner, but was not at all decreased by mannitol, Mn-superoxide dismutase and catalase. In addition, HPLC-chemiluminescence analysis demonstrated that incubation with the PCOOH/Cu(II) combination oxidized cholesterol, a relatively oxidation-resistant component, to the cholesterol hydroperoxide. These results reveal that (1)O2 is generated from PCOOH in contact with copper(II). In conclusion, this in-vitro study provides directly the (1)O2 formation in living organisms following the advancement of peroxidation of constitutive lipids.     

Studies on cytochrome P-450-dependent lipid hydroperoxide reduction

A reconstituted mixed-function oxidase system containing cytochrome P-450, cytochrome P-450 reductase, phosphatidylcholine, and NADPH catalyzed the reduction of 13-hydroperoxy-9,11-octadecadienoic acid to 13-hydroxy-9,ll-octadecadienoic acid. Activity was stimulated by the addition of type I substrates, while carbon monoxide and oxygen inhibited the reaction. Perfluoro-n-hexane stimulated the reduction of lipid hydroperoxide to lipid alcohol in the reconstituted system but not by cytochrome P-450 alone. Incubation of cytochrome P-450 with only lipid hydroperoxide resulted in destruction of the hemoprotein. Addition of substrates such as aminopyrine decreased cytochrome P-450 destruction. Addition of reducing equivalents from a reconstituted electron transport system also decreased cytochrome P-450 destruction.     

Fusion of liposomes with planar lipid bilayers

The fusion of liposomes with planar lipid bilayers was monitored by two different methods. (a) Liposomes consisting of phospholipids and cholesterol were added to the aqueous phase bathing the cholesterol-deficient planar lipid bilayers in the presence of nystatin. The resulting increase in the planar lipid bilayer's electrical conductance was considered indicative of fusion. (b) Transplanar lipid bilayer injection of ³⁵SO₄^2? trapped inside the liposomes.It is shown by both methods that fusion is specifically dependent on the presence of negatively charged phospholipids both in the liposomes and the planar lipid bilayers and on Ca²⁺ in the aqueous phase of the fusion system.     

Studies on lipid oxidation in fish phospholipid liposomes

Fish phospholipid liposomes were prepared and used as an artificial membrane system to study factors influencing-lipid oxidation. The extent of lipid oxidation was indexed by measuring the amount of thiobarbituric acid reactive substances (TBARS) produced. Fe²⁺, Fe³⁺, and Cu²⁺ were potent prooxidants in catalysing lipid oxidation. These metal ions induced lipid oxidation in a dose dependent manner. However, Zn²⁺, Ni²⁺, and Mn²⁺ did not significantly (p>0.05) affect lipid oxidation at all the concentrations (1, 10, or 100 μM) studied. Morin, luteolin (flavonoids), butein (chalcone), tannic acid, ellagic acid (polyphenols), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) (synthetic antioxidants) were potent antioxidants (producing <50% TBARS compared to control) of Fe²⁺-catalyzed lipid oxidation. Morin, luteolin, and butein possess two hydroxyl substituents, a C₄ ketone structure and a 2–3 double bond, all of which contributed to their antioxidative potential. Fe²⁺ caused some losses of polyunsaturated fatty acids (PUFA), whereas tannic acid protected the oxidation of several of the PUFA including C 16∶1 (Palmitoleic acid), C 18∶3 (Linolenic acid), C 20∶4 (Arachidonic acid), C 20∶5 (Eicosapentaenoic acid), and C 22∶6 (Docosahexaenoic acid).