Membrane damage caused by free radicals--radiation perspective

Irradiation experiments were performed with Ehrlich ascites tumour cells grown in the peritoneal cavity of mice. A 20 MeV proton beam was used as the source of ionizing radiation. The generation of free radicals was investigated with EPR spectrometry. Lipid peroxidation products were determined by the thiobarbiturate reaction, UV-absorption, and fluorometric measurements. Membrane injuries were visualized by electron microscopy. Free radicals (like H and OH) were formed in the cells during the irradiation. Radical reactions were transferred to membrane lipids producing secondary radicals, diene conjugation, malondialdehyde formation and fluorescent end products. By using electron microscopy severe damage could be detected in cell membranes.     

Differential cytotoxicity of daunomycin in tumour cells is related to glutathione-dependent hydrogen peroxide metabolism

Addition of 0.5mm-daunomycin, a quinone anti-cancer drug, causes severe inhibition of respiration in Ehrlich ascites cells, whereas Yoshida ascites cells were almost as resistant as rat hepatocytes. An inverse relationship appears to exist in the two types of tumour cells (which are both catalase-deficient) between the extent of cellular damage brought about by intracellular formation of superoxide anion occurring on reaction with O₂ of the drug free radical and the efficiency of the glutathione-mediated H₂O₂-detoxifying system.     

A study of hyperlipidaemia induced by ascites tumours.

In mice four stages of hyperlipidaemia induced by Ehrlich ascites tumour could be distinguished. Hyperlipidaemia is characterized mainly by increased serum VLDL content accompanied by high triglyceride concentration. The only exception was the regressive stage III where the serum lipid level (VLDL) has temporarily decreased. From the results obtained with the simultaneous examination of the liver, mesenteric fat tissue, tumour cell and ascites plasma lipids, it may be concluded that the endogeneous fat mobilization induced by tumour cells via increased VLDL synthesis and secretion of liver will lead to hyperlipidaemia and to the total depletion of the lipid stores. Rapid and reversible fall in lipid level following the withdrawal of ascites fluid in tumorous animals demonstrated clearly the direct effect of the tumour cells on lipid-lipoprotein metabolism of the host organism.     

PAF-like lipids- and PAF-induced gallbladder muscle contraction is mediated by different pathways in guinea pigs

H2O2 stimulates gallbladder muscle contraction and scavengers of free radicals through the generation of PGE2. Oxidative stress causes lipid peroxidation and generation of platelet-activating factor (PAF) or PAF-like lipids. The present studies therefore were aimed at determining whether either one induced by H2O2 mediates the increased generation of PGE2. Dissociated muscle cells of guinea pig gallbladder were obtained by enzymatic digestion. Both PAF-like lipids and PAF-induced muscle contraction was blocked by the PAF receptor antagonist CV-3988. This antagonist also blocked the increased PGE2 production caused by PAF-like lipids or PAF. Actions of PAF-like lipids were completely inhibited by indomethacin, but those of PAF were only partially reduced by indomethacin or by nordihydroguaiaretic acid and completely blocked by their combination. PAF-like lipids-induced contraction was inhibited by AACOCF3 (cystolic phospholipase A2 inhibitor), whereas the actions of PAF were blocked by MJ33 (secretory phospholipase A2 inhibitor). Receptor protection studies showed that pretreatment with PAF-like lipids before N-ethylmaleimide protected the contraction induced by a second dose of PAF-like lipids or PGE2 but not by PAF. In contrast, pretreatment with PAF protected the actions of PAF and PGE2 but not that of PAF-like lipids. Both PAF-like lipids and PAF-induced contractions were inhibited by anti-Galphaq/11 antibody and by inhibitors of MAPK and PKC. In conclusion, PAF-like lipids seem to activate a pathway different from that of PAF probably by stimulating a different PAF receptor subtype.     

Identification of platelet-activating factor as the inflammatory lipid mediator in CCl4-metabolizing rat liver

Unmitigated oxidative stress is deleterious, as epitomized by CCl4 intoxication. In this well-characterized model of free radical-initiated damage, liver metabolism of CCl4 to CCl3. causes lipid peroxidation, F-ring isoprostane formation, and pathologic leukocyte activation. The nature of the mediator that couples oxidation to the hepatotoxic inflammatory response is uncharacterized. We found that oxidatively modified phosphatidylcholines were present in the livers of CCl4-exposed rats and not in livers from control animals, that CCl4 metabolism generated lipids that activated 293 cells stably transfected with the human platelet-activating factor (PAF) receptor, and that this PAF-like activity was formed as rapidly as isoprostane-containing phosphatidylcholine (iPC) during oxidation. iPC and the PAF-like activity also had similar chromatographic properties. The potential for iPC activation of the PAF receptor has been unexplored, but we conclude that iPC themselves did not activate the PAF receptor, as phospholipase A1 hydrolysis completely destroyed iPC, but none of the PAF-like bioactivity. Oxidatively fragmented phospholipids are potent agonists of the PAF receptor, but mass spectrometry characterized PAF as the major inflammatory component coeluting with iPC. Oxidatively fragmented phospholipids and iPC are markers of free radical generation in CCl4-intoxicated liver, but PAF generation by activated hepatic cells generated the inflammatory agent.     

Free Radicals Scavengers Attenuate Platelet-Activating Factor (PAF)-And Endotoxin-Induced Intestinal Myoelectric Disturbances in Rats

Pretreatment with radical scavengers significantly reduced the intestinal myoelectric disturbances following either E. coli endotoxin or platelet-activating factor (PAF) injection in the rat indicating that free radicals might be involved in the intestinal motor alterations observed in endotoxin shock and that PAF acts partially via free radical production. Moreover, dimethylsulfoxide (DMSO) was found to be more effective in inhibiting the endoxotin-induced intestinal motor alterations, than superoxide dismutase (SOD) and allopurinol. BN 52021, a specific PAF antagonist, was able to reduce the effects of endotoxin on intestinal motility, However, when BN 52021 was combined with free radical scavengers, no additive effect was observed. It is concluded that free radicals involved in endotoxin-induced intestinal motility alterations are at least in part produced in response to PAF.     

Utilization of ascites plasma very low density lipoprotein triglycerides by Ehrlich cells

Much of the lipid present in the ascites plasma in which Ehrlich cells grow is contained in very low density lipoproteins (VLDL). Chemical measurements indicated that triglycerides were taken up by the cells during in vitro incubation with ascites VLDL. When tracer amounts of radioactive triolein were incorporated into the ascites VLDL, the percentage uptakes of glyceryl tri[1-(14)C]oleate and triglycerides measured chemically were similar. The cells also took up [2-(3)H]glyceryl trioleate that was added to VLDL, but the percentage of available (3)H recovered in the cell lipids was 30-40% less than that of (1 4)C from glyceryl tri[1-(1 4)C]oleate. This difference was accounted for by water-soluble (3)H that accumulated in the incubation medium, suggesting that extensive hydrolysis accompanied the uptake of VLDL triglycerides. Radioactive fatty acids derived from the VLDL triglycerides were incorporated into cell phospholipids, glycerides, and free fatty acids, and they also were oxidized to CO(2). Triglyceride utilization increased as the VLDL concentration was raised. These results suggest that one function of the ascites plasma VLDL may be to supply fatty acid to the Ehrlich cells and that the availability of fatty acid to this tumor is determined in part by the ascites plasma VLDL concentration. Although Ehrlich cells incorporate almost no free glycerol into triglycerides, considerable amounts of [2-(3)H]glyceryl trioleate radioactivity were recovered in cell triglycerides. This indicates that at least some VLDL triglycerides were taken up intact. The net uptake of VLDL protein and cholesterol was very small relative to the triglyceride uptake, suggesting that intact triglycerides are transferred from the ascites VLDL to the Ehrlich cells and that hydrolysis occurs after the triglyceride is associated with the cells.     

Synthesis and In Vitro Antitumor Activity of New Substituted Thiopyrimidine Acyclic Nucleosides and Their Thioglycoside Analogs

Some new thiopyrimidine acyclic nucleosides and thioglycoside derivatives 3a-c, 4a-c, 6a,b, and 7a,b were synthesized. The cytotoxicity and antitumor evaluation of all prepared compounds have been tested in vitro against Ehrlich's ascites carcinoma cell line and their activity against glutathione peroxidase and catalase were reported. The role of the prepared compounds as free radical regulators and the therapeutic antitumor effect of a balanced generation of free radicals are discussed. Compounds 2, 3b, 3c, 4a, and 4c inhibited significantly in a dose dependent manner the growth of Ehrlich ascites carcinoma cells while the other compounds did not show any antitumor activity even at higher concentrations.     

The inhibitory effect of l-cysteine and its derivatives on glycolysis in Ehrlich ascites tumour cells

(1) l-Cysteine inhibits aerobic glycolysis and restores the Pasteur effect in Ehrlich ascites tumour cells or in their supernatants, while d-cysteine has no effect on this process. (2) Other compounds which have configuration l at the α-carbon and a thiol group in the β-position (penicillamine) or restore them in vivo (3-mercaptopyruvate, cystine or l-serine together with l-homocysteine) also show inhibitory properties. (3) dl-Homocysteine with a free thiol group in the γ-position, reduced glutathione, methionine and products of cysteine oxidation (cysteic acid, taurine) do not inhibit tumour aerobic glycolysis. (4) Glycolysis of normal tissue supernatants (mouse liver and muscle) is not sensitive to the inhibitory effect of cysteine. (5) Metabolic studies showing a cysteine-induced decrease in ATP content, coupled with cross-over of the pyruvate and 2-phosphoenolpyruvate concentrations in Ehrlich ascites tumour cells, indicate that tumour pyruvate kinase is an enzyme sensitive to cysteine inhibition. (6) Enzymatic studies carried out both after preincubation of Ehrlich ascites tumour cells with cysteine or during direct action of this substance on tumour and normal tissue supernatants indicate the presence of a cysteine-sensitive isoenzyme besides the normal cysteine-insensitive pyruvate kinase in tumour material.     

The inhibitory effect of various fatty acids on aerobic glycolysis in Ehrlich ascites tumour cells

Inhibition of glycolysis in Ehrlich ascites tumour cells by saturated fatty acids, added either in form of potassium salts or incorporated into phosphatidylcholine liposomes, increases with the increasing carbon atom chain length and is independent of the concentration within the range of 0.1 to 1.0 mM. In contrast, the inhibition of glycolysis in the cytosolic fraction from Ehrlich ascites cells depends on the concentration of fatty acids. The content of ATP in Ehrlich ascites cells incubated with fatty acids increases with increasing carbon atom chain length, which leads to a crossing-over in the concentrations of pyruvate and 2-phosphoenolpyruvate. Lowering of the sum of both these metabolites by palmitate and stearate points to the inhibition not only of pyruvate kinase but also of other enzymes of early steps of glycolysis. Fatty acids in intact Ehrlich ascites cells inhibit all three key glycolytic enzymes but added to the cytosolic fraction affect mainly the activity of phosphofructokinase. The inhibition of pyruvate kinase by fatty acids is smaller in the cytosolic fraction from tumour cells than from liver and muscles.     

Effect of the diabetic environment on the lipid metabolism of Ehrlich ascites cells

One strain of Ehrlich ascites cells lacking of insulin receptors, was grown into control and diabetic mice and cells harvested from diabetic mice reimplanted into control mice. The fatty acid composition of neutral and polar lipids was analyzed and several parameters calculated. Results showed that it is possible to produce similar changes in the lipid fatty acid unsaturation of Ehrlich cells to those observed in the liver of the diabetic bearing mice. These changes may be reverted by growing these cells into control mice. The diabetic environment also promoted a relative increase in the radioactivity from incorporated in vitro into neutral lipids of Ehrlich cells. This metabolic adjustment, probably due to an induction of the enzyme diglyceride acyltransferase, was completely reverted by transplanting these cells in control mice. The metabolic adaptation of Ehrlich ascites cells to the diabetic environment did not modify their biological behaviour as pointed out by their mean generation time. The evidence presented here, showing relatively normal growth of Ehrlich cells in association with changes in the lipid fatty acid pattern and in lipid metabolism, indicates the adaptation of these cells, lacking of insulin receptors, to the environment provided by the diabetic mice.     

Free radicals and their interpretations

Oxygen free radicals can be blamed for evoking gastric mucosal damage, because of the protective effect of some lipid soluble free radical scavengers (vitamin A related compounds, Vitamin E). Direct determination of free oxygen radicals related chemical entities in the gastric tissue during ulcerogenesis yielded controversial results. Aluminum antacid compound together with acid binding property exhibited cytoprotection too, elevating the tissue PGE2 level substantially. Magnesium containing antacid according to our model experiments on red blood cells damage by free radicals, is capable to bind free radicals as well as to counteract with the dangerous intracellular calcium accumulation. It has been concluded that aluminum-magnesium antacid has a cytoprotective effect via: 1. acid binding; 2. prostaglandin generation; 3. free radical scavenging; 4. calcium antagonist activity.     

Biphasic kinetics of metal ion reactivation of trypsin-thiol complexes

This report describes biphasic kinetic data obtained when trypsin was inhibited by a thiol-containing inhibitor present in Ehrlich ascites tumour cells and then subjected to addition of Hg2+, Cu2+ or Ag+. This resulted in an initial re-activation of the trypsin, followed by inhibition of the enzyme with the addition of higher concentrations of these ions. The significance of these observations is 2-fold: (i) help to elucidate the mechanism of metal ion activation of latent enzymes, and (ii) also indicate that, in certain circumstances, the concentration of added metal ion determines whether the metal acts as an activator or an inhibitor of enzyme activity.     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.     

Ribonucleotide reductase in ascites tumour cells detected by electron paramagnetic resonance spectroscopy

Tyrosine radicals localized in the M2 subunits of ribonucleotide reductase have been detected by electron paramagnetic resonance (EPR) in ordinary ascites tumour cells. The intensity of its doublet EPR spectrum is higher in rapidly proliferating cells. Hydroxyurea, a specific inhibitor of this enzyme, decreases the concentration of the tyrosine radical. Whereas in different ascites tumours the doublet EPR spectrum dominates at g = 2.004, in solid tumours another more intense EPR spectrum from nitrosyl-hemoproteins appears. In conclusion, EPR spectroscopy can be used to monitor the content and variations of active M2 subunits of ribonucleotide reductase in intact ascites tumour cells.     

Kinetic analysis of the free-radical-induced lipid peroxidation in human erythrocyte membranes: evaluation of potential antioxidants using cis-parinaric acid to monitor peroxidation.

cis-Parinaric acid (PnA), cis-trans-trans-cis-9, 11, 13, 15-octadecatetraenoic acid, is fluorescent (epsilon = 74,000 at 324 nm) when partitioned into a lipid environment and the fluorescence is destroyed upon reaction with free radicals. It has been used to monitor semiquantitatively free-radical-induced lipid peroxidation in human erythrocyte membranes. We have applied this assay to the quantitative evaluation of potential antioxidants. The kinetics of the reaction of PnA with free radicals were measured in erythrocyte ghosts. After initiation of free radical generation by cumene hydroperoxide and cupric ion, a steady-state rate of fluorescence decay is rapidly established. In the steady state the oxidation of PnA and, hence, the loss of fluorescence is a first-order process. In the presence of antioxidants, such as vitamin E, the rate constant of fluorescence loss decreases, thereby indicating that the antioxidant decreases the steady-state concentration of free radicals. By adding various concentrations of potential antioxidants, pseudo-first-order rate constants [k1] which measure the reactivity of antioxidants with free radicals were determined. Results show that, when incorporated into erythrocyte membranes, U-78, 517f, a vitamin E analog, is a potent free radical scavenger, being approximately 50% as effective as vitamin E and 10-15 times more potent than the aminosteroids evaluated (see Table 1).     

Increased ultra weak chemiluminescence emission from rat heart at postischemic reoxygenation: protective role of vitamin E

Aim of this study was to confirm an increased free radical generation rate during ischemia-reoxygenation, by ultra-weak chemiluminescence detection at the surface of perfused rat heart. We observed that reoxygenation following 30 min global ischemia, induces an increase of ultraweak chemiluminescence emission in isolated perfused heart only if partial depletion of vitamin E is induced by dietary manipulation. Moreover, in normal diet fed rats, vitamin E is partially consumed during global ischemia, but not during reoxygenation. Since chemiluminescence increases during post-ischemic reperfusion, when vitamin E myocardial content is lowered, the most probable free radicals involved are the hydroperoxyl radical derivatives of lipids. These radicals, indeed, are known both to produce photoemission by disproportion and to react with vitamin E. On the other hand, the nature of the reaction that consumes vitamin E during ischemia is still obscure. Accordingly, the basal level of vitamin E myocardial content seems to be a key factor for protecting the heart against reoxygenation injury and its consumption during ischemia could be a determinant of myocardial sensitivity to oxidative stress during reperfusion.     

Transport and metabolism of thiamine in Ehrlich ascites-carcinoma cells

1. Aerobic or anaerobic incubation at 37 degrees of Ehrlich ascites-carcinoma cells in Krebs-Ringer bicarbonate medium containing glucose and labelled thiamine results in accumulation in the cell of labelled thiamine, so that the concentration of total labelled thiamine in the cells greatly exceeds (by a factor 7) that in the medium. This concentration ratio is approximately constant for small initial external concentrations of labelled thiamine but diminishes when the latter exceed 0.4mum. 2. All the labelled thiamine in the tumour cells is present as thiamine phosphates. 3. The uptake of labelled thiamine is markedly diminished by decrease of temperature. At 9 degrees concentration ratio (cells/medium) 0.5 is observed whereas at 37 degrees the concentration ratio is 8.6. 4. The extent of phosphorylation of labelled thiamine depends on the period of incubation. 5. The influx of labelled thiamine is diminished by the presence of its analogues, pyrithiamine and Amprol, and also by the presence of thiamine monophosphate and thiamine diphosphate, which are potent inhibitors of thiamine phosphorylation in Ehrlich ascites cells. 6. Labelled thiamine phosphates leak from the cell into the medium, so that eventually all the labelled thiamine, both in the cell and medium, is converted into thiamine phosphates. However, in the presence of 2,4-dinitrophenol (0.1mm) and iodoacetate (1mm) thiamine phosphorylation is diminished, the concentration ratio for labelled thiamine (cells/medium) falls to half its normal value and little or no labelled thiamine phosphates leaks into the medium. 7. In the presence of thiamine phosphates, free labelled thiamine accumulates in Ehrlich ascites cells against a concentration gradient, concentration ratios (cells/medium) greater than unity being evident. 8. The evidence supports the conclusion that thiamine is transferred into the Ehrlich ascites cell by a carrier-mediated energy-assisted process.     

Glutamine and glucose as energy substrates for Ehrlich ascites tumour cells

Energy metabolism of freshly harvested Ehrlich ascites tumour cells in the presence of 5 mM glucose and/or 0.5 mM glutamine was studied. The rate of oxygen utilization was not altered by the addition of 0.5 mM glutamine; 5 mM glucose induced an inhibition of respiration. In the presence of both glucose and glutamine, the Crabtree effect decreased. In these conditions, the rates of oxygen uptake, the CO2 evolution and the changes in the redox states of cytochromes indicate that glucose is preferred by Ehrlich ascites tumour cells as energy substrate. Glucose decreased the rate of glutamine utilization by 34%. On the other hand, glutaminolysis did not inhibit glycolysis.     

Radiation inactivation of ion channels formed by gramicidin A. Protection by lipid double bonds and by alpha-tocopherol.

The conductance induced by the channel-forming peptide gramicidin A in lipid membranes is reduced by many orders of magnitude on exposure of the membrane and its aqueous environment to ionizing radiation. This results from an interaction of free radicals of water radiolysis with the tryptophan residues of gramicidin A. The sensitivity of the ion channels towards irradiation is strongly reduced in the presence of either vitamin E or of highly unsaturated lipids. An increase of the D37 dose up to a factor of 50 was found. The phenomena are interpreted via a reduction of the effective concentration of free radicals (such as OH.) in the membrane by reaction with unsaturated fatty acid residues or with vitamin E.