Delayed alteration of membrane fluidity in intact cultured B-16 melanoma cells affected by ultraviolet irradiation

Lipid peroxidation in the plasma membrane has been reported to decrease membrane fluidity. We examined membrane fluidity in relation to lipid peroxidation processes after UV-B exposure of cultured B-16 melanoma cells. UV exposure promptly increased TBA-positive material(s), but alteration of membrane fluidity was delayed. Plasma membrane fluidity increased significantly 6 hours after exposure when the TBA-value(s) had become under the control level. To examine the direct effect of lipid peroxides on the fluidity, tert-butyl hydroperoxide was added to B-16 melanoma cells. Similar results were obtained with respect to membrane fluidity. These results suggest that lipid peroxidation at UV doses maintaining cell viability does not directly induce a significant alteration of membrane fluidity, but may influence the fluidity either during metabolizing processes of UV-induced lipid peroxides or during repair processes following oxidative cell membrane damage.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

Alpha-adrenergically mediated changes in membrane lipid fluidity and Ca2/ binding in isolated rat liver plasma membranes

Noradrenaline (0.1-5 microM, in the presence of 5 microM propranolol to block beta-receptors), ATP (100 microM) and angiotensin II (0.1 microM), which are thought to increase cytosolic Ca2+ concentration by mobilizing Ca2+ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the alpha-antagonists phenoxybenzamine (50 microM) and phentolamine (1 microM). The response to a maximal dose of noradrenaline (5 microM) and that to ATP (100 microM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 microM) along with the foreign amphiphile Na+-oleate (1-30 microM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg2+, reducing free Ca2+ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca2+ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 microM) and angiotensin II (0.1 microM) also promoted a small release of 45Ca2+ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the alpha-antagonist phentolamine (5 microM). It is proposed that noradrenaline, via alpha-adrenergic receptors and other Ca2+ -mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca2+ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.     

Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles.

P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues. P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport [3H]daunomycin. In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of [3H]daunomycin and [3H]vinblastine. Accumulation of both cytotoxic agents was stimulated by ATP, exhibited temperature dependence and osmotic sensitivity, and followed Michaelis-Menten kinetics. Alterations in CMV lipid fluidity were induced by the known fluidizers, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) and benzyl alcohol, and were assessed by fluorescence polarization techniques using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). Both A2C (2.5-5.0 microM) and benzyl alcohol (10-20 mM) produced a dose-dependent increase in CMV lipid fluidity. Moreover, both fluidizers, at the above doses, significantly inhibited (p < 0.05) the ATP-dependent accumulation of [3H]daunomycin. [3H]Vinblastine accumulation was also inhibited by A2C (p < 0.05). Lower doses of A2C (0.6 microM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation. Kinetic analysis revealed that A2C (5.0 microM) noncompetitively inhibited [3H]daunomycin accumulation and uncompetitively inhibited [3H]vinblastine accumulation with apparent Ki values of approximately 1.5 and approximately 1.2 microM, respectively. Verapamil competitively inhibited P-gp-mediated accumulation of [3H]daunomycin but failed to alter the fluidity of CMV. Taken together, the present results demonstrate that while increases in membrane fluidity of CMV are not necessarily required to inhibit P-gp-mediated drug accumulation, they can inhibit these processes, at least in CMV. Alterations in the physical state of CMV, therefore, appear to be at least one important modulator of P-gp function.     

Amyloid β-Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes

Abstract: Amyloid β-peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ_1–40 and Aβ_25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer-eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly ( p ≤ 0.02) by Aβ_1–40. Similar effects on fluidity were observed for Aβ_25–35 ( p ≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased ( p ≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water-soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ-induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.     

Effects of tannins on Chinese hamster cell line B14

Tannins, naturally occurring plant phenols, have been recognized as antioxidants, but toxic effects have also been observed. In the current investigation, the interaction of this type of compounds with Chinese hamster cells (cell line B14) has been examined. This study reports on the results of experiments in which B14 cells were exposed to tannins: tannic, ellagic and gallic acids in the concentration range 15-240 microM. The cytotoxic and genotoxic effects of these compounds were studied. The colorimetric MTT assay to assess cytotoxicity and the Comet assay for detection of DNA damage were used. In this paper, we also demonstrated the influence of tannins on the fluidity of the plasma membrane. This experiment was carried out by a spectrofluorometric method using two fluorescent probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 12-(9-anthroyloxy)stearic acid (12-AS). The tannins increased the fluidity in the internal region of the lipid bilayer, but no changes at the surface of the plasma membrane were observed. The results of the MTT assay showed that tannins could decrease the viability of cells and that their cytotoxicity was highest at the concentration of 60 microM. The degree of toxicity of these compounds was not correlated with the concentration used. The data obtained from the Comet assay showed that the tannins could also contribute to formation of DNA single-strand breaks.     

Phencyclidine actions measured intracellularly in hippocampal CA1 neurons

The electrophysiological effects of phencyclidine (PCP) were measured intracellularly in guinea pig hippocampal CA1 neurons in vitro. At all doses tested (0.2 microM - 10 mM), PCP increased the width of action potentials (APs). Doses of 10 microM and higher were associated with decreased action potential amplitude. PCP decreased inhibitory postsynaptic potentials and excitatory postsynaptic potentials but did not alter responses to focally applied GABA. At the lowest dose (0.2 microM), PCP decreased the input resistance (Rin), while at all other doses Rin was increased. PCP decreased post-spike train afterhyperpolarizations at low and medium doses. PCP effects persisted in low calcium medium and also in medium containing 10(-6) M tetrodotoxin. It is concluded that in these central neurons, PCP primarily blocks potassium conductances at all doses and, at anesthetic doses, depresses sodium-dependent spikes.     

Changes in plasma membrane fluidity of immortal rodent cells induced by anticancer drugs doxorubicin, aclarubicin and mitoxantrone

The aim of this study was to examine the effect of three structurally different anticancer drugs-the pro-oxidative anthracyclines doxorubicin (DOX) and aclarubicin (ACL), and antioxidative anthraquinone mitoxantrone (MTX) on the fluidity of plasma membrane of immortalized rodent fibroblasts using fluorescence spectroscopy and electron spin resonance (ESR) techniques. Two kinds of fluorescent probes (TMA-DPH and 12-AS) and spin labels (5-DS and methyl-12-DS) were used to monitor fluidity in the hydrophobic core and in the polar headgroup region of the lipid bilayer. Immortalized hamster B14 and NIH 3T3 mouse fibroblasts were exposed to DOX, ACL and MTX. We demonstrate that these drugs influence predominantly the hydrophobic core of the lipid bilayer, inducing significant decrease in its fluidity at low concentrations (2-5 microM). A decreased membrane fluidity at the surface of the lipid bilayer was observed only at a higher concentration (20 microM) of the drugs, which indicates that DOX, ACL and MTX intercalate mainly into the hydrophobic core of the membrane, thereby perturbing its structure.     

Pentachlorophenol inhibition of succinate oxidation by the respiratory chain in submitochondrial particles from the bovine heart

Study on the effect of pentachlorophenol on the succinate oxidase activity of submitochondrial particles and on the reduction level of cytochromes b revealed that the Ki value for PCP is equal to 2-4 microM. The succinate-DCPIP-reductase activity is noncompetitively inhibited with PCP (by 75-85%) (Ki = 3.6 microM). In the case of the succinate-PMS-reductase activity PCP at micromolar concentrations decreases the value of V only by 40% (C50 = 2 microM) with a simultaneous increase of the Km value for PMS. The identity of Ki values for PCP under these conditions suggests that the effect of PCP is due to the inhibitor interaction with the same component of the succinate dehydrogenase complex. The type of action of PCP on the succinate-acceptor-reductase activities indicates that the inhibiting effect of PCP on succinate oxidations is similar to that exerted by traditional inhibitors of succinate dehydrogenase--tenoyltrifluoroacetone and carboxins. Since PCP inhibits succinate dehydrogenase at low concentrations, it seems likely that the biological (pesticidal) effect of PCP is provided for not only by its uncoupling action but also by the inhibition of succinate oxidation in the respiratory chain.     

Indole-3-propionic acid, a melatonin-related molecule, protects hepatic microsomal membranes from iron-induced oxidative damage: relevance to cancer reduction

Excessive free iron and the associated oxidative damage are commonly related to carcinogenesis. Among the antioxidants known to protect against iron-induced oxidative abuse and carcinogenesis, melatonin and other indole compounds recently have received considerable attention. Indole-3-propionic acid (IPA), a deamination product of tryptophan, with a structure similar to that of melatonin, is present in biological fluids and is an effective free radical scavenger. The aim of the study was to examine the effect of IPA on experimentally induced oxidative changes in rat hepatic microsomal membranes. Microsomes were preincubated in presence of IPA (10, 3, 2, 1, 0.3, 0.1, 0.01 or 0.001 mM) and, then, incubated with FeCl(3) (0.2 mM), ADP (1.7 mM) and NADPH (0.2 mM) to induce oxidative damage. Alterations in membrane fluidity (the inverse of membrane rigidity) were estimated by fluorescence spectroscopy and lipid peroxidation by measuring concentrations of malondialdehyde+4-hydroxyalkenals (MDA+4-HDA). IPA, when used in concentrations of 10, 3 or 2 mM, increased membrane fluidity, although at these concentrations it did not influence lipid peroxidation significantly. The decrease in membrane fluidity due to Fe(3+) was completely prevented by preincubation in the presence of IPA at concentrations of 10, 3, 2 or 1 mM. The enhanced lipid peroxidation due to Fe(3+) was prevented by IPA only at the highest concentration (10 mM). It is concluded that Fe(3+)-induced rigidity and, to a lesser extent, lipid peroxidation in microsomal membranes may be reduced by IPA. However, IPA in high concentrations increase membrane fluidity. Besides melatonin, IPA may be used as a pharmacological agent to protect against iron-induced oxidative damage to membranes and, potentially, against carcinogenesis.     

5-methoxytryptophol preserves hepatic microsomal membrane fluidity during oxidative stress

Lipid peroxidation is a degenerative chain reaction in biological membranes that may be initiated by exposure to free radicals. This process is associated with changes in the membrane fluidity and loss of several cell membrane-dependent functions. 5-methoxytryptophol (ML) is an indole isolated from the mammalian pineal gland. The purpose of this study was to investigate the effects of ML (0. 01mM-10mM) on membrane fluidity modulated by lipid peroxidation. Hepatic microsomes obtained from rats were incubated with or without ML (0.01-10 mM). Then lipid peroxidation was induced by FeCl(3), ADP, and NADPH. Membrane fluidity was determined using fluorescence spectroscopy. Malonaldehyde (MDA) +4-hydroxyalkenals (4-HDA) concentrations were estimated as an indicator of the degree of lipid peroxidation. With oxidative stress, membrane fluidity decreased and MDA+4-HDA levels increased. ML (0.01-3 mM) reduced membrane rigidity and the rise in MDA+4-HDA formation in a concentration-dependent manner. 10 mM ML protected against lipid peroxidation but failed to prevent the membrane rigidity. In the absence of oxidative reagents, ML (0.3-10 mM) decreased membrane fluidity whereas MDA+4-HDA levels remained unchanged. This indicates that ML may interact with membrane lipids. The results presented here suggest that ML may be another pineal indoleamine (in addition to melatonin) that resists membrane rigidity due to lipid peroxidation.     

Effect of quercetin on nicotine-induced biochemical changes and DNA damage in rat peripheral blood lymphocytes

We elucidated the protective effect of quercetin, a polyphenolic flavonoid, on lipid peroxidation, endogenous antioxidant status and DNA damage during nicotine-induced toxicity in cultured rat peripheral blood lymphocytes as compared to N-acetylcysteine (NAC), a well-known antioxidant. Lymphocytes were exposed to nicotine (3 mM) with and without quercetin and NAC (1 mM) in RPMI-1640 medium for 1 h. In preliminary experiments to fix the effective dose of quercetin, different doses of quercetin (25, 50, 75, 100 and 200 microM) were administered to lymphocytes with nicotine, and lipid peroxidation markers (thiobarbituric acid reactive substances and hydroperoxides) were analysed. A 75 microM dose of quercetin was found to be effective as evidenced by decreased lipid peroxidation. To evaluate the protective potential of quercetin against genotoxic effects of nicotine we used comet and micronucleus assays, which are valid parameters to assess genetic damage. In addition, biochemical changes including lipid peroxidation and antioxidant status were assessed. There were significant increases in the levels of lipid peroxidation, comet parameters and micronuclei frequencies, followed by decrease in the endogenous antioxidant status, in nicotine-treated lymphocytes, which were brought back to near normal by quercetin or NAC treatment. The protective effect of quercetin against nicotine toxicity was comparable to that of NAC. These findings suggest that quercetin can be as effective as NAC in protecting rat peripheral lymphocytes against nicotine-induced cellular and DNA damage.     

Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4 degrees C.

Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.     

The effect of 2,4-dichlorophenol and pentachlorophenol on antioxidant system in the leaves of Phalaris arudinacea

The purpose of this work was to evaluate the effect of 2,4-dichlorophenol (2,4-DCP) and pentachlorophenol (PCP) on the activity of antioxidative system and lipid peroxidation in the leaves of reed canary grass (Phalaris arudinacea). The activity of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferase (GST) as well as the content of glutathione, ascorbate and phenolic compounds were determined. An induced-increase in the APX, CAT, GPX and GR activities was stronger for PCP, while a significant increase in the GST activity was noted only for 2,4-DCP. Both compounds increased the content of phenolic compounds, oxidized and reduced glutathione as well as the content of ascorbic acid. PCP induced stronger increase in lipid peroxidation than 2,4-DCP. The observed changes revealed that chlorophenols induce oxidative stress and oxidative damage in the leaves of reed canary grass.     

克山病病区粮喂养大鼠红细胞膜脂流动性的变化

本文观察了低硒的克山病病区粮和克山病病区粮补硒后喂养大鼠对其红细胞膜脂流动性的影响。实验结果表明克山病病区粮喂养的大鼠红细胞膜脂流动性较正常对照降低,其原因可能与机体处于低硒状态下红细胞膜结合硒含量降低、红细胞膜胆固醇含量及脂质过氧化产物升高有关,克山病病区粮补硒后喂养大鼠,其红细胞膜脂流动性恢复至正常对照。     

Alterations in lipid composition and fluidity of liver plasma membranes in copper-deficient rats

In view of the importance of membrane fluidity on cell functions, the influence of phospholipid acyl groups on membrane fluidity, and the changes in lipid metabolism induced by copper (Cu) deficiency, this study was designed to examine the influence of dietary Cu on the lipid composition and fluidity of liver plasma membranes. Male Sprague-Dawley rats were divided into two dietary treatments, namely Cu deficient and Cu adequate. After 8 weeks of treatment, liver plasma membranes were isolated by sucrose density gradient centrifugation. The lipid fluidity of plasma membranes, as assessed by the intramolecular eximer fluorescence of 1,3-di(1-pyrenyl) propane, was significantly depressed by Cu deficiency. In addition, Cu deficiency significantly reduced the content of arachidonic and palmitoleic acids but increased the docosatetraenoic and docosahexaenoic acids of membrane phospholipids. This alteration in unsaturated phospholipid fatty acid composition, especially the large reduction in arachidonic acid, may have contributed to the depressed membrane fluidity. Furthermore, Cu deficiency also markedly altered the fatty acid composition of the triacylglycerols associated with the plasma membranes. Thus, the lipid composition and fluidity of liver plasma membranes are responsive to the animal's Cu status.     

Relationships among lipid peroxidation, glutathione peroxidase, superoxide dismutase, sperm parameters, and competitive index in dairy bulls

Sperm membranes contain high concentrations of polyunsaturated fatty acids that are highly susceptible to oxidative damage that interferes with fertilization ability. The objective of this study was to determine associations among lipid peroxidation (thiobarbituric-acid-reactive substance concentration), antioxidant enzymatic activities in frozen spermatozoa, and competitive indices. Semen from multiple ejaculates collected in succession from each bull (four Holstein and four Jersey) was pooled. Heterospermic doses (20x10(6)sperm/0.5mL straw) were made to obtain 16 Holstein/Jersey combinations (equal number of sperm from each bull). Cows were inseminated on observed or synchronized estrus. The sire of calves (N=460) was determined; based on the number of calves sired, a competitive index was obtained for each bull. Prior to preparation of the heterospermic doses, a sub-sample of semen from each bull was taken, processed, frozen, and stored concurrent with heterospermic samples. After thawing, these homospermic samples were assessed for lipid peroxidation, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, DNA fragmentation index (DFI), plasma membrane integrity (PMI), and total progressive motility (assessed by CASA). Sperm lipid peroxidation and the competitive index were negatively correlated (r=-0.78; P<0.05), the DFI and sperm lipid peroxidation were positively correlated (r=0.86; P<0.001), and there were negative correlations (P<0.05) for sperm lipid peroxidation and both PMI and total progressive motility (r=-0.78 and -0.83, respectively). There was neither significant association between SOD activity and competitive index, nor between GPx activity and competitive index. In conclusion, bulls with lower sperm lipid peroxidation had higher chances of siring calves; this was attributed to the deleterious effects of lipid peroxidation on sperm plasma membrane integrity and sperm DNA, which may reduce sperm fertilizing potential.     

Comparison of the relative activities of alpha-tocopherol and PMC on platelet aggregation and antioxidative activity.

In this study, PMC (2,2,5,7,8-pentamethyl-6-hydroxychromane), a potent antioxidant derived from alpha-tocopherol, dose-dependently inhibited agonist-induced platelet aggregation in human platelet-rich plasma. PMC is over 5-10 times more potent than alpha-tocopherol in inhibiting human platelet aggregation. Moreover, PMC (25-350 microM) dose-dependently reduced the relative fluorescence intensity of platelet membrane tagged with diphenylhexatriene (DPH). PMC is about 6-times more potent than alpha-tocopherol on this effect. Furthermore, antioxidative activity of PMC was investigated using two in vitro models. PMC inhibited non-enzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC50 value of 0.21+/-0.05 microM. It was more potent than alpha-tocopherol or other classical antioxidants. PMC also scavenged the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). The concentration of PMC resulting in a decrease of 0.20 in the absorbance of DPPH was about 12.1+/-3.6 microM, was comparable in potency to alpha-tocopherol, butylated hydroxytoluence and Trolox. The antiplatelet activity of PMC may possibly be due initially to an increase in fluidity of the platelet membrane followed by inhibition of platelet aggregation. Our results indicate that PMC is a potentially effective antioxidant and antiaggregating agent, and could be helpful the design of compounds with more clinical effectiveness.     

UVB-induced hemolysis of rat erythrocytes: protective effect of procyanidins from grape seeds

Besides erythema and sunburn reactions, UVB stress can promote erythrocyte extravasation from skin capillaries and hemolysis, and photosensitized hemoglobin can in turn lead to an overload of free radicals in dermis which exacerbates photodamage. The objective of this study was to investigate in rat erythrocytes (RBC) the pattern of events leading to membrane peroxidation and hemolysis following UVB insult (1.5-8.5 J/cm2), and the protective action of grape seed procyanidins. UVB causes a dramatic dose-dependent decrease of intracellular glutathione (paralleled by the formation of pro-oxidant ferryl-hemoglobin), of intramembrane vitamin E and of membrane fluidity, then a rise of conjugated dienes (CD), and thiobarbituric acid-reactive substances (TBARS) and finally a strong hemolytic effect. Procyanidins prevent membrane peroxidation (but not intracellular GSH depletion nor ferryl-hemoglobin formation), with a minimal effective concentration of 0.1 microM (IC50 for TBARS and CD after 120 min UVB exposure: 0.71 microM and 0.56 microM) and dose-dependently delay the onset of hemolysis, by 30 min at 0.1 mciroM, by 90 and 120 min at 0.5 and 1.0 microM. Epigallocatechin-3-O-gallate (EGCG) and catechin, typical constituents of the fraction, were significantly less potent. This since procyanidins (1 microM) inhibit the formation of phospholipid hydroperoxides of the inner (phosphatidylserine, phosphatidylethanolamine) and outer (phosphatidylcholine) layers of the RBC membrane (HPLC analysis), suppress the decrease in membrane fluidity due to lipid and protein thiol oxidation and spare vitamin E from consumption in a dose-dependent manner (0.1-1 microM). Hence procyanidins, preserving membrane phospholipids, since their strong antilipoperoxidant activity, may maintain in vivo the integrity of RBC in sub-epidermal capillaries and effectively counteract in dermis the onset/exacerbation of the UVB-induced skin photodamage.     

Effects of simvastatin and pravastatin on peroxidation of erythrocyte plasma membrane lipids in patients with type 2 hypercholesterolemia

Since hypercholesterolemia directly modifies the composition of erythrocytes plasma membrane, the influence of statins on erythrocytes has been researched. The beneficial effects of statins on clinical events may involve mechanisms that modify endothelial dysfunction, plaque stability, thrombus formation and inflammatory responses. The aim of the study was to evaluate the hypolipemic efficacy and effects of pravastatin and simvastatin on erythrocyte membrane fluidity and damage of erythrocytes in patients with type 2 hypercholesterolemia in comparison with a control group of healthy subjects. The study involved 53 patients affected by type 2 hypercholesterolemia (mean age, 53.3 +/- 10.3) with initial total serum cholesterol (TC) levels > 250 mg/dL, LDL-cholesterol (LDL-C) levels > 170 mg/dL, and triglycerides (TG) levels < 400 mg/dL. The control group consisted of 30 healthy individuals (mean age 56.9 +/- 6.3). Statins were given for 12 weeks. The dosages for oral administration of simvastatin and pravastatin were 20 mg/day. Laboratory tests were carried out before and after 4 and 12 weeks of the pharmacological treatment. The damage to plasma membrane of erythrocytes was measured on the basis of lipid peroxidation. The fluidity of plasma membrane of erythrocytes was determined by electron paramagnetic resonance (EPR) spectroscopy, using two spin labels: 5-DSA and 16-DSA. The cholesterol level in the membrane of red blood cells was estimated. Simvastatin and pravastatin reduced the total cholesterol concentration and LDL-cholesterol in plasma, as well as the cholesterol concentration in erythrocytes membranes. Hypercholesterolemia induced changes in the basic properties of human erythrocyte plasma membrane, including its fluidity and the intensity of lipid peroxidation. These results indicate that the simvastatin and pravastatin therapy reverses the alteration in the erythrocyte plasma membrane properties.