Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

Selective promotion of ferrous ion-dependent lipid peroxidation in Ehrlich ascites tumor cells by histidine as compared with other amino acids

Among tumors in general, Ehrlich ascites tumor cells are particularly resistant to lipid peroxidation. In this study lipid peroxidation was measured in terms of the formation of malondialdehyde-equivalent material in Ehrlich tumor cells during incubation in vitro. It was shown that the high antioxidant potential of these cells could be overcome by a strong radical-promoting agent like ferrous ion. Various amino acids were tested for their capability to augment the effect of Fe(II). Histidine and its 3-methyl-derivative turned out to be the most effective pro-oxidants, whose action could be ascribed to the presence of the imidazole group. From studies with homogenized and denatured cells it was concluded that lipid peroxidation stimulated by Fe(II)-histidinate is an autoxidation process and that no carrier effect of iron by histidine is predominating. The stimulatory action of Fe(II)-histidinate could be completely suppressed by vitamin C, which was shown to be a potent anti-oxidant under the conditions used. The combined application of Fe(II)-histidinate and vitamin C may offer a means to study lipid peroxidation of Ehrlich tumor cells in a controlled manner.     

Pantothenic acid and its derivatives protect ehrlich ascites tumor cells against lipid peroxidation

Preincubation of Ehrlich ascites tumor cells at 22 or 32°C, but not at 0°C, with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine reduced lipid peroxidation (measured by production of thiobarbituric acid-reactive compounds) induced by the Fenton reaction (Fe²⁺ + H₂O₂) and partly protected the plasma membrane against the leakiness to cytoplasmic proteins produced by the same reagent. Pantothenic acid and its derivatives did not inhibit (Fe²⁺ + H₂O₂)-induced peroxidation of phospholipid multilamellar vesicles, thus indicating that their effect on the cells was not due to the scavenging mechanism. Homopantothenic acid and its 4′-phosphate ester (which are not precursors of CoA) neither protected Ehrlich ascites tumor cells against lipid peroxidation nor prevented plasma membrane leakiness under the same conditions. Incubation of the cells with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine significantly increased the amount of cellular CoA and potentiated incorporation of added palmitate into phospholipids and cholesterol esters. It is concluded that pantothenic acid and its related compounds protect the plasma membrane of Ehrlich ascites tumor cells against the damage by oxygen free radicals due to increasing cellular level of CoA. The latter compound may act by diminishing propagation of lipid peroxidation and promoting repair mechanisms, mainly the synthesis of phospholipids.     

Membrane fluidity in Ehrlich ascites tumor cells treated with adriamycin

The incubation of Ehrlich ascites tumor cells with adriamycin resulted in an increase in lipid peroxide content and a decrease in membrane fluidity as measured by electron spin resonance using the paramagnetic probe 5-doxylstearic acid. Coincidently, the incorporation of [3H]thymidine into tumor cells was progressively inhibited as the concentration of adriamycin was increased. The results indicate that adriamycin induces changes in the plasma membrane of Ehrlich ascites tumor cells after exposure to a low, but cytotoxic, level of this agent.     

Detection of 4-hydroxynonenal as a product of lipid peroxidation in native Ehrlich ascites tumor cells

4-Hydroxynonenal, which is a major product of lipid peroxidation in rat liver microsomes, was detected in native Ehrlich ascites tumor cells. Its formation was stimulated either by ferrous ions or by Fe(II)-histidinate. The identification was based on chromatographic (TLC/HPLC) and ultraviolet-spectroscopic evidence using synthetic 4-hydroxynonenal as reference. Highest values of 4-hydroxynonenal concentration (about 0.1 microM in the cell suspension) after 30 min of incubation were observed with Fe(II)-histidinate as stimulant. Saturation was already reached after an incubation period of 10 min. The results confirm the expectation by Schauenstein and Esterbauer (in Submolecular Biology and Cancer, Ciba Foundation Series 67 (1979) pp. 225-244, Excerpta Medica, Amsterdam) that endogenous lipid peroxidation gives rise to a distinct intracellular level of alpha, beta-unsaturated aldehydes. A simple hypothetical mechanism for the formation of 4-hydroxynonenal from n-6-polyunsaturated fatty acids is presented.     

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.     

Tumor growth, angiogenesis and inflammation in mice lacking receptors for platelet activating factor (PAF)

Tumor growth is associated with angiogenesis and inflammation and the endogenous lipid, platelet activating factor (PAF), is a pro-inflammatory and pro-angiogenic mediator. We therefore measured tumor growth, angiogenesis and inflammation in normal (WT) mice and those lacking the receptor for PAF, through gene deletion (PAFR-KO). Growth of solid tumors derived from colon 26 cells was not altered but that from Ehrlich cells was markedly (5-fold) increased in the PAFR-KO mice, relative to the WT strain. Angiogenesis, as tumor content of VEGF or hemoglobin, was increased in both tumors from the mutant strain. Inflammation, as neutrophil and macrophage accumulation and chemokine (CXCL2 and CCL2) content of tumors, was decreased or unchanged in the tumors implying an overall decrease in the inflammatory response in the PAFR-KO strain. We also assessed growth of the Ehrlich tumor in its ascites form, after i.p. injection. Here growth (ascites volume) was inhibited by about 30%, but neutrophil and macrophage numbers were increased in the ascites fluid from the PAFR-KO mice. Angiogenesis in the peritoneal wall, which is not invaded by the tumor cells, was increased but leukocyte infiltration decreased in the mutant strain. Our results show, unexpectedly, that tumor-induced angiogenesis was increased in mice lacking response to PAF, from which we infer that in normal (WT) mice, PAF is anti-angiogenic. Further, although growth was still associated with angiogenesis in PAFR-KO mice, growth was not correlated with inflammation (leukocyte accumulation).     

Lipidg Peroxidation in Liver and Ehrlich Ascites Cell Mitochondria

Ehrlich ascites cell mitochondria are highly resistant to lipid peroxidation as compared to liver mitochondria from host animals. Succinate protects mitochondria from peroxidative damage, proteins from crosslinks, enzymes from inactivation of the enzymes and membranes from permeability changes. The sensitivity of Ehrlich ascites cell mitochondrial membranes to lipid peroxidation is significantly increased in sub-mitochondrial particles. Lipid peroxidation in tumour mitochondrial membranes can not be diminished by succinate as effectively as in liver mitochondria. Ascites cell mitochondria seems to be protected very efficiently from peroxidative damage by a glutathione-dependent mechanism.     

Thyroid hormones and the level of divalent iron ions in the rabbit liver

Experimental hypothyreosis in animals was obtained by means of extirpation of thyroid gland; hyperthyreosis was obtained by thyroxine injections intraperitoneally for 10 days. Content of Fe2+ ions of the free pool (not bound with ferritine) was estimated with the ESR method, after treatment with sodium nitrite. A decrease in Fe2+ ions content is observed in liver homogenate of hypothyroid rabbits, and an increase is observed in that of the hyperthyroid animals. Content of Fe2+ ions in cells of liver tissues correlates with the oxidation capacity of the mitochondrial lipids. The changes in the rate of lipid peroxidation under thyroid pathology is supposed to result from changes in free ferrum content.     

Uptake of ferrous iron histidinate, a promoter of lipid peroxidation, by Ehrlich ascites tumor cells

The kinetics of the uptake of Fe(II)-histidinate, a known promoter of lipid peroxidation, into Ehrlich ascites tumor (EAT) cells and the intracellular binding of iron were studied in vitro. EAT cells (27.10(6)/ml) were incubated in Hanks' balanced salts solution at 37 degrees C for various time intervals in the presence of FeSO4 (1 mM) and L-histidine (10 mM). Total iron was determined by the 1,10-phenanthroline/ascorbate method and ferric iron by reaction with 5-sulfosalicylic acid; the difference was ascribed to ferrous iron. Total iron decreased rapidly in the medium (242 nmol within the first 10 min), and a corresponding increase of total iron (saturation value 376 nmol after 60 min) was determined within the cells, after the cellular proteins had been solubilized with 6 M urea. In the absence of EAT cells, Fe(II)-histidinate was readily oxidized to Fe(III)-histidinate by oxygen, but this reaction was strongly retarded by the tumor cells. The uptake of iron histidinate occurred in the oxidized state, while an uptake of ferrous iron could not be proven unambiguously. When EAT cells were saturated with iron, it was found that 93% of intracellular iron was bound to water-insoluble proteins and 7% was associated with soluble proteins, while no unbound iron was detectable by the method used. It was concluded that, despite the high uptake of total iron, only a very small portion of the intracellular iron was available as a redox catalyst for lipid peroxidation.     

Lipid peroxidation in Ehrlich ascites cell mitochondria is not determined by the polyunsaturated fatty acid content of the membrane

Lipid peroxidation intensity is compared in Ehrlich Ascites Cell and in liver mitochondria, prepared from tumor bearing mice. Malondialdehyde formation is negligible in intact ascites tumour mitochondria, but it is significantly increased in permeabilised mitochondria and in isolated mitochondrial membranes. We suggest that the resistance against oxidative stress is a consequence of efficient protective mechanisms operating in the intact tumour mitochondria and the low level of polyunsaturated fatty acids under these circumstances cannot be the rate limiting factor in lipid peroxidation. Succinate, an effective inhibitor of mitochondrial lipid peroxidation in liver, cannot determine malondialdehyde formation in ascites tumour mitochondria.     

Correlation of the membrane and genetic effects of lipid peroxidation

Linoleic acid hydroperoxides delivered to Ehrlich ascites tumor cells in vitro in a concentration reaching a threshold one (approximately 10(-5) M) sharply decrease their ionic permeability and the value of membrane capacity. With suprathreshold hydroperoxide concentrations (greater than 10(-5) M), the specific yield of chromosome aberrations and the share of aberrant cells increase while the mitotic index decreases. The correlation between general regularities and the equality of the threshold concentrations, with a reference to membrane and genetic effects of hydroperoxides, is thought to be an indication of a close relationship between membrane lesions caused by the development of induced lipid peroxidation and injury to genetic apparatus of the cell.     

Lipid peroxidation in rat liver microsomes. II. Response of hydroperoxide formation to iron concentration

When rat liver microsomes were incubated with NADPH, the major products were hydroperoxides which increased with time indicating that endogenous iron content is able to promote lipid peroxidation. The addition of either 5 microM Fe2+ or Fe3+ ions strongly enhanced the hydroperoxide formation rate. However, due to the hydroperoxide breakdown, hydroperoxide concentration decreased with time in this case. Higher ferrous or ferric iron concentration did not change the situation much, in that both hydroperoxide breakdown and formation were similar to those when NADPH only was present in the incubation medium. After lipid peroxidation, analysis of fatty acids indicated that the highest amount of peroxidized PUFA occurred in the presence of 5 microM of either Fe2+ or Fe3+. This analysis also showed that after 8 min incubation with low iron concentration, PUFA depletion was about 77% of that observed after 20 min, whereas without any iron addition or in the presence of 30 microM of either Fe3+, PUFA decrease was only about 37% of that observed after 20 min. As far as the optimum Fe2+/Fe3+ ratio required to promote the initiation of microsomal lipid peroxidation in rat liver is concerned, the highest hydroperoxide formation was observed with a ratio ranging from 0.5 to 2. These results indicate that microsomal lipid peroxidation induced by endogenous iron is speeded up by the addition of low concentrations of either Fe2+ or Fe3+ ions, probably because free radicals generated by hydroperoxide breakdown catalyze the propagation process. In experimental conditions unfavourable to hydroperoxide breakdown the principal process is that of the initiation of lipid peroxidation.     

Measurement of 'in situ' mitochondrial membrane potential in Ehrlich ascites tumor cells during aerobic glycolysis

(1) A method is presented for continuous and simultaneous monitoring of the 'in situ' mitochondrial membrane potential (delta psi m) and respiration rate of Ehrlich ascites tumor cells. The method involves permeabilization of the plasma membrane, achieved by treatment with low digitonin concentration, and the use of a TPP+ selective electrode attached to an oxygraph vessel. Binding of the probe inside the cells was analyzed assuming a proportional relationship between the amount of bound TPP+ and the free concentration of the lipophilic cation. (2) Evidence is reported that the addition of glucose to digitonin-permeabilized Ehrlich ascites tumor cells causes a decrease of mitochondrial membrane potential that coincided with a transient enhancement of the respiration rate and remained unchanged during the subsequent Crabtree effect. We have characterized the effect of glucose on delta psi m by determining its dependent on the glycolytic pathway and its sensitivity towards oligomycin. The mutual relationships between glucose and ADP effects on the mitochondrial membrane potential were also studied. A plausible mechanism underlying the depolarization of mitochondrial membrane induced by glucose is presented.     

Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+

Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.     

The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation

Intense lipid peroxidation of brain synaptosomes initiated with Fenton's reagent (H2O2 + Fe2+) began instantly upon addition of Fe2+ and preceded detectable OH. formation. Although mannitol or Tris partially blocked peroxidation, concentrations required were 10(3)-fold in excess of OH. actually formed, and inhibition by Tris was pH dependent. Lipid peroxidation also was initiated by either Fe2+ or Fe3+ alone, although significant lag phases (minutes) and slowed reaction rates were observed. Lag phases were dramatically reduced or nearly eliminated, and reaction rates were increased by a combination of Fe3+ and Fe2+. In this instance, lipid peroxidation initiated by optimal concentrations of H2O2 and Fe2+ could be mimicked or even surpassed by providing optimal ratios of Fe3+ to Fe2+. Peroxidation observed with Fe3+ alone was dependent upon trace amounts of contaminating Fe2+ in Fe3+ preparations. Optimal ratios of Fe3+:Fe2+ for the rapid initiation of lipid peroxidation were on order of 1:1 to 7:1. No OH. formation could be detected with this system. Although low concentrations of H2O2 or ascorbate increased lipid peroxidation by Fe2+ or Fe3+, respectively, high concentrations of H2O2 or ascorbate (in excess of iron) inhibited lipid peroxidation due to oxidative or reductive maintenance of iron exclusively in Fe2+ or Fe3+ form. Stimulation of lipid peroxidation by low concentrations of H2O2 or ascorbate was due to the oxidative or reductive creation of Fe3+:Fe2+ ratios. The data suggest that the absolute ratio of Fe3+ to Fe2+ was the primary determining factor for the initiation of lipid peroxidation reactions.     

Cytochrome P450 inactivation in microsomal membranes damaged by Fe2+-ascorbate-dependent lipid peroxidation

It has been established that Fe2+-ascorbate-dependent lipid peroxidation in rat liver microsome membranes is followed by the decrease of microsome cytochrome P450 content and the increase of the reduced haemoprotein inactivation rate. These changes are proportional to the amount of lipid peroxidation products (malonic dialdehyde) accumulating in the membranes.     

On the mechanism of glycolysis stimulation by neutral detergents in 3T3 and Ehrlich ascites tumor cells

Glycolysis of 3T3 and Ehrlich ascites tumor cells was greatly enhanced by Nonidet P-40 or Triton X-100 at about 100 micrograms/mg cell protein. This enhanced glycolysis was partly sensitive to rutamycin and partly sensitive to ouabain, suggesting that the detergent released the control of the ATPase of the mitochondria and of the plasma membrane Na+K+-ATPase. Nonidet P-40 had no effect on glycolysis in cell-free extracts from Ehrlich ascites tumor cells to which soluble mitochondrial ATPase was added. Measuring ouabain-sensitive 22Na efflux and using ouabain-sensitive lactate production as a measure of ATP hydrolysis by the Na+K+ pump, it was shown that Nonidet P-40 greatly decreased the efficiency of the Na+K+ pump. Quercetin increased the efficiency of pumping in EAT cells both in the absence and presence of the detergent.     

Activation of lipid peroxidation in the adrenal cortex by metal ions

The processes of lipid peroxidation have been studied in bovine adrenal cortex in vitro. The lipid peroxidation rate in this tissue is shown to be dependent on the content of metal ions. EDTA, deferroxamine and penicyllamine inhibit spontaneous lipid peroxidation by 25, 50 and 42%, respectively. The ability to activate the process permits arranging metal ions in the following sequence: Fe2+ greater than Fe3+ greater than Cu2+ greater than Mg2+ greater than Mn2+. The maximum activation of lipid peroxidation is observed at Fe2+ and Fe3+ concentrations within the range of 5 x 10(-6) x 10(-4) M.