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.     

Inhibitory effect of eugenol on Cu2+-catalyzed lipid peroxidation in human erythrocyte membranes

1. The effects of eugenol on lipid peroxidation catalyzed by hydrogen peroxide (H2O2) or benzoyl peroxide (BPO) in the presence of copper ions were studied in human erythrocyte membranes. 2. The production of hydroxyl radicals was suggested in the peroxidation system catalyzed by H2O2/Cu2+. 3. H2O2/Cu2+-dependent peroxidation was inhibited by eugenol in a concentration-dependent manner; peroxidation was inhibited 62% by 200 microM eugenol. 4. In the presence of eugenol, the peroxidation catalyzed by BPO/Cu2+ was inhibited in a concentration-dependent manner, and more than 100 microM eugenol completely inhibited peroxidation. 5. The inhibitory effect of eugenol was non-competitive against Cu2+ in H2O2/Cu2+- and BPO/Cu2+-dependent peroxidation. 6. It is suggested that eugenol inhibits formation of hydroxyl radicals.     

In vitro effects of alloxan/copper combinations on lipid peroxidation, protein oxidation and antioxidant enzymes

The in vitro effects of alloxan and the product of its reduction dialuric acid (alone or in combination with copper ions) on lipid peroxidation, carbonyl content, GSH level and antioxidant enzyme activities in rat liver and kidney have been studied. The effects of Cu2+/alloxan and Cu2+/dialuric acid were compared with those of Fe3+/alloxan and Fe3+/dialuric acid. Unlike alloxan, dialuric acid increased liver and kidney lipid peroxidation; similar effects were registered in the presence of Fe3+. In the presence of Cu2+/dialuric acid, the lipid peroxidation was strongly inhibited and vice versa--the liver protein oxidation was increased. Alloxan and dialuric acid, as well as their combinations with Fe3+ had no effect on the total GSH level. Both substances did not affect the Cu2+-induced changes in GSH level, glucose-6-phosphate dehydrogenase and gluthatione reductase activities. In contrast, Cu2+ had no effect on dialuric-acid induced changes in gluthatione peroxidase and superoxide dismutase activities. The present in vitro results, concerning the metal dependence of the effects of alloxan and dialuric acid, are a premise for in vivo study of alloxan effects in metal-loaded animals.     

Effect of succinate on mitochondrial lipid peroxidation. 1. Comparative studies on ferrous ion and ADP · Fe/NADPH-induced peroxidation

Lipid peroxidation in isolated rat liver mitochondria, mitoplast, and mitochondrial inner membrane fragments was induced either by ferrous ions, or in an NADPH-dependent process by complexing with adenine nucleotides (ADP or ATP) iron. The Fe²⁺-induced lipid peroxidation is nonenzymic when inner membrane fragments are used, while the differences in the inhibitory effect of Mn²⁺ ions and the stimulatory effect of the ionophore A-23187 in mitochondria and inner membrane fragments suggest an enzymic mechanism for ferrous ion-induced lipid peroxidation in intact mitochondria. Contrary to this the ADP/Fe/NADPH-dependent lipid peroxidation is an enzymic process both in mitochondria and inner membrane preparations. We have shown that cytochrome P₄₅₀ is involved in the ADP/Fe/NADPH-induced lipid peroxidation. Succinate, a known inhibitor of NADPH-dependent lipid peroxidation, inhibited the Fe²⁺-induced process also, and there was no difference in this effect when inner membrane preparations, mitochondria, or mitoplasts were used.     

Origin of invaginations and vacuoles in liposomes under the effect of endocytosis inducers including oxidants

The action of various substances on the morphology of multilayer membranes made of lipids of egg yolk was studied. It has been shown that electroneutral and anionic compounds scaresly affected liposomes, whereas substances with pronounced basic properties, i.e. peroxidase, hemoglobin, cytochrome c, and RNAase, as well as lanthanium ions, induced the formation of invaginations, vesicles and aggregation of liposomes. Metals with variable valency: Cu2+, Cu4+, Ru6+, including lipid oxidants Fe3+ and UO+, produced similar morphological changes more intensely and moreover destroyed liposomal membranes. The activator of lipid peroxidation, namely ascorbic acid, intensified while antioxidizers such as alpha-tocopherylacetate and ionol removed the action of Fe3+ on liposomes. A protective effect was displayed by Ca2+ and Mg2+ ions and due to an increase in pH medium. Since many tested substances with the basic properties stimulate endocytosis of cells the processes of lipid peroxidation and electrostatic interactions are supposed to be part of endocytosis mechanism which does not involve the metabolic energy. It is also assumed that endocytosis may arise at the stage of protocells in terms of evolutions.     

Action of lead(II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions

Lead (Pb2+) ions accelerate the lipid peroxidation observed when Fe2+ ions are added to phospholipid liposomes at pH 5.5 or pH 7.4, although Pb2+ ions alone do not induce any peroxidation. Similarly, aluminium (Al3+) ions increase Fe2+-dependent liposomal peroxidation at pH 5.5. Both Pb2+ and Al3+ accelerate the peroxidation of erythrocytes induced by high concentrations of H2O2 in the presence of azide, and they also increase the peroxidation that occurs when Fe2+ or Fe2+-ADP is added to rat liver microsomes at pH 7.4. It is proposed that increased lipid peroxidation may contribute to the toxic actions of Pb2+ in humans.     

Ferric(III) ions inhibits copper(II)/hydrogen peroxide-catalyzing lipid peroxidation in human erythrocyte membranes.

1. Effect of ferric ions (Fe3+) on the lipid peroxidation catalyzed by copper ions (Cu2+) and hydrogen peroxide (H2O2) was studied in human erythrocyte membranes. 2. The formation of thiobarbituric acid-reactive products elicited by CuCl2/H2O2 was inhibited by FeCl3 in a concentration-dependent manner; 0.25 mM FeCl3 were enough to cause 50% inhibition of the formation of peroxides. 3. The inhibitory effect of FeCl3 is not due to competition against Cu2+. 4. FeCl3 inhibited the initiation, but did not inhibit the propagation of Cu2+/H2O2-catalyzing lipid peroxidation. 5. In the heat- or trypsin-treated erythrocyte membranes, FeCl3 had no inhibitory effect on Cu2+/H2O2-catalyzing lipid peroxidation. 6. Sodium azide, an inhibitor of catalase, had no effect on the inhibitory effect of FeCl3. 7. These results suggest that a protein factor(s), which is not catalase, is involved in the inhibition of Cu2+/H2O2-catalyzing lipid peroxidation by Fe3+.     

Binding of divalent cations with low density lipoproteins. A study by ESR

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.     

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.     

Importance of Fe2+-ADP and the relative unimportance of OH in the mechanism of mitomycin C-induced lipid peroxidation

The mechanism of mitomycin C-induced lipid peroxidation has been studied at pH 7.5, using systems containing phospholipid membranes (liposomes) and an Fe3+-ADP complex with purified NADPH-cytochrome P-450 reductase. Both O2- and H2O2 are generated during the aerobic enzyme-catalyzed reaction in the presence of mitomycin C. Hydroxyl radical is formed in the reaction by the reduction of H2O2. This is catalyzed by the Fe2+-ADP complex in a phosphate buffer or to a lesser extent when in a Tris-HCl buffer. The reduction of Fe3+-ADP to Fe2+-ADP is mainly achieved by O2-. The resulting Fe2+-ADP in the presence of O2 forms a perferryl ion complex which is a powerful stimulator of lipid peroxidation. However, the formation of such an iron-oxygen complex is strongly inhibited by phosphate ions, which do not interfere with the generation of OH radicals. These findings suggest that, since lipid peroxidation occurs in a Tris-HCl buffer (but not in a phosphate buffer), the OH radical is unlikely to be involved in the observed lipid peroxidation process.     

Lipid peroxidation and 4-hydroxy-2-nonenal formation by copper ion bound to amyloid-beta peptide

The lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is proposed to be a toxic factor in the pathogenesis of Alzheimer disease. The primary products of lipid peroxidation are phospholipid hydroperoxides, and degraded reactive aldehydes, such as HNE, are considered secondary peroxidation products. In this study, we investigated the role of amyloid-beta peptide (A beta) in the formation of phospholipid hydroperoxides and HNE by copper ion bound to A beta. The A beta1-42-Cu2+ (1:1 molar ratio) complex showed an activity to form phospholipid hydroperoxides from a phospholipid, 1-palmitoyl-2-linoleoyl phosphatidylcholine, through Cu2+ reduction in the presence of ascorbic acid. The phospholipid hydroperoxides were considered to be a racemic mixture of 9-hydroperoxide and 13-hydroperoxide of the linoleoyl residue. When Cu2+ was bound to 2 molar equivalents of A beta(1-42) (2 A beta1-42-Cu2+), lipid peroxidation was inhibited. HNE was generated from one of the phospholipid hydroperoxides, 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylcholine (PLPC-OOH), by free Cu2+ in the presence of ascorbic acid through Cu2+ reduction and degradation of PLPC-OOH. HNE generation was markedly inhibited by equimolar concentrations of A beta(1-40) (92%) and A beta(1-42) (92%). However, A beta(1-42) binding 2 or 3 molar equivalents of Cu2+ (A beta1-42-2Cu2+, A beta1-42-3Cu2+) acted as a pro-oxidant to form HNE from PLPC-OOH. These findings suggest that, at moderate concentrations of copper, A beta acts primarily as an antioxidant to prevent Cu2+-catalyzed oxidation of biomolecules, but that, in the presence of excess copper, pro-oxidant complexes of A beta with Cu2+ are formed.     

Copper-mediated Lipid Peroxidation Processes in Photosynthetic Membranes

The phytotoxic effect of Cu via the photosynthetic electron transport system was studied with isolated spinach chloroplasts. Cu(II) ions induce a light-driven peroxidation of membrane lipids leading to ethylene formation, the latter dominating over a concurrent ethane production. Seemingly, the hydroxyl radical originating from superoxide anion is the starting reactive O₂ species. Cu ions inhibit photosynthetic electron transport and apparently catalyze the formation of hydroxyl radical and Fenton-type reactions that result in destruction of unsaturated membrane fatty acids. The concept on the mode of action of Cu(II) and Cu(I) ions in lipid peroxidation as presented here suggests the influence of Cu on different reactions. Two sites are in the photosynthetic redox system; Cu participates in two Fenton-type reactions and in the conversion of ethyl radical to ethylene and ethane.     

Antioxidant/prooxidant effects of dietary non-flavonoid phenols on the Cu2+-induced oxidation of human low-density lipoprotein (LDL)

A central role in the oxidative development of atherosclerotic lesions has been ascribed to the peroxidation of plasma low-density lipoprotein (LDL). Dietary supplementation with virgin olive oils increases the total plasma antioxidant status and the resistance of low-density lipoprotein to ex vivo oxidation. We have studied the effects of some dietary non-flavonoid phenols from Olea europaea L., both in purified form or in complex mixtures obtained by biotransformation of olive leaf extracts, on the LDL oxidation induced by Cu2+ ions. Cu2+-Induced LDL oxidation is inhibited by oleuropein and hydroxytyrosol in the initiation phase of the reaction at concentrations of phenols higher than that of Cu2+ ions. Interestingly, at lower concentration, both phenols anticipated the initiation process of LDL oxidation, thus exerting prooxidant capacities. Although similar effects are already described for flavonoids, such as quercetin, rutin, and apigenin, it is the first time that a prooxidant effect of dietary non-flavonoid phenols, such as oleuropein and hydroxytyrosol, on the LDL oxidation is reported. Our results show that a net effect of oleuropein and hydroxytyrosol on Cu2+-induced LDL peroxidation is determined by a balance of their pro- and antioxidant capacities. It is worth to underline that, during Cu2+-induced LDL oxidation in the presence of bioreactor eluates, we have evidence of a synergistic effect among phenolic compounds that enhance their antioxidant capacities so avoiding the prooxidant effects.     

UVA-induced oxidative damage to rat liver nuclei: reduction of iron ions and the relationship between lipid peroxidation and DNA damage

Lipid peroxidation and DNA damage and the relationship between the two events were studied in rat liver nuclei irradiated with low dose UVA. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by spectrophotometric method and as malondialdehyde-TBA adduct by HPLC, and DNA damage was measured as 8-hydroxy-deoxyguanosine (8-OH-dGu) and strand breakage (or loss of double-stranded DNA) by a fluorometric analysis of alkaline DNA unwinding method. The results show that UVA irradiation by itself increased nuclear lipid peroxidation but caused little or no DNA strand breakage or 8-OH-dGu. When 0.5 mM ferric (Fe+3) or ferrous (Fe+2) ions were added to the nuclei during UVA irradiation, lipid peroxidation and DNA damage, measured both as 8-OH-dGu and loss of double-stranded DNA, were strongly enhanced. Lipid peroxidation occurred concurrently with the appearance of 8-OH-dGu. Fe3+ ions were reduced to Fe2+ in this UVA/Fe2+/nuclei system. Lipid peroxidation and DNA damage were neither inhibited by scavengers of hydroxyl radical and singlet oxygen nor inhibited by superoxide dismutase and catalase. Inclusion of EDTA or chain-breaking antioxidants, butylated hydroxytoluene (BHT) and diphenylamine (an alkoxy radical scavenger), inhibited lipid peroxidation but not the level of 8-OH-dGu. BHT also did not inhibit the loss of double-stranded DNA in this system. This study demonstrates the reduction of exogenous Fe+3 by UVA when added to rat liver nuclei, and, as a result, oxidative damage is strongly enhanced. In addition, the results show that DNA damage is not a result of lipid peroxidation in this UVA/Fe2+/nuclei system.     

The effect of ionic strength on the lipid peroxidation of porcine intestinal brush-border membrane vesicles

The effects of salt concentration gradient (inside to outside) on the lipid peroxidation of porcine intestinal brush-border membrane vesicles have been studied and several interesting features of the peroxidation have been elucidated. The addition of dithiothreitol and Fe2+ is far more effective in induction of the lipid peroxidation than any of the other metal ion species tested (Fe3+, Cu2+, Ni2+, Zn2+ and Cr3+). The peroxidation rate of the membrane vesicles induced by dithiothreitol plus Fe2+ was sensitive for the incubation temperature and was increased with increase of the temperature. Imposition of an inward salt concentration gradient on the membrane vesicles preloaded with 300 mM mannitol by addition of 100 mM chloride of K+, Na+, Li+, Rb+, NH4+ or choline to medium produces a very large reduction of the lipid peroxidation induced by dithiothreitol plus Fe2+. The membrane peroxidation is depressed more with the mannitol (300 mM)-preloaded vesicles than with the K2SO4 (100 mM)-preloaded vesicles when they are incubated in medium containing 20-100 mM of K2SO4. Addition of membrane-permeant anions such as SCN- and I-, but not addition of NO3-, to incubation medium has been found to decrease markedly the lipid peroxidation of the mannitol-preloaded vesicles. From these results it is suggested that the lipid peroxidation of the brush-border membranes by addition of dithiothreitol plus Fe2+ is sensitively changed with change in ionic strength.     

Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA.

Membrane lipid peroxidation processes yield products that may react with DNA to cause oxidative modifications. We have investigated this possibility and have found that calf thymus DNA exposed to autooxidized lipids causes the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG). 8-OH-dG formation in DNA was measured using high-pressure liquid chromatography with electrochemical detection. Methyl linolenate oxidized for different lengths of time was exposed to DNA. The amount of 8-OH-dG formed in DNA was proportional to the amount of lipid peroxidation as measured by the thiobarbituric reactive substances present. The formation of 8-OH-dG in DNA by autooxidized methyl linolenate was dependent on the presence of the transition metal ions Cu or Fe and was inhibited by various scavengers, including superoxide dismutase and catalase. This implicates the involvement of oxygen free radicals in the process. Liposomes formed from phosphatidylcholine (82%) and methyl arachidonate (18%) were peroxidized for different lengths of time and then exposed to DNA. 8-OH-dG was formed in DNA by exposure to Cu(II) and peroxidized liposomes. Under these conditions, Fe(III) was slightly less effective than Cu(II) in mediating 8-OH-dG formation. These observations clearly show that 8-OH-dG formation in DNA may result from processes that may occur during intracellular lipid peroxidation.     

Antioxidative activity of copper in root nodules of yellow lupin plants

Copper nutrition inhibited lipid peroxidation in root nodules of yellow lupin plants at the early growth stages by about 50 %. The antioxidative activity of copper in the process of lipid peroxidation could be associated with Cu taking part in oxidative reaction of nodule catechol-like siderophores and its effect on iron accumulation and reactivity. The obtained results, for the first time, suggest that the ability of copper to inhibit lipid peroxidation in nodules could be considered as a major function for Cu requirements by symbiotic N₂ fixation in grain legume nodules.     

Non-linear pattern of radiation induced lipid peroxidation is not affected by vitamin E, Fe2+ ions and molecular oxygen.

Results of the present study on liposomes have clearly shown that non-linear pattern of radiation-induced lipid peroxidation was not changed even in the presence of vitamin E, Fe2+ ions or molecular oxygen. These results are important from biological point of view as lipid peroxidation is used as a measure of membrane damage.     

Lipid peroxidation induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system.

Cu,Zn-superoxide dismutase (SOD) can catalyze hydroxyl radical generation using H2O2 as a substrate. Lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system was investigated. When linoleic acids micelles or phosphatidylcholine liposomes were incubated with Cu,Zn-SOD and H2O2, lipid peroxidation was gradually increased in a time-dependent manner. The extent of lipid peroxidation was proportional to Cu,Zn-SOD and H2O2 concentrations. Hydroxyl radical scavengers and copper chelator inhibited lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system. These results suggest that lipid peroxidation is mediated by the Cu,Zn-SOD and H2O2 system via the generation of hydroxyl radicals by a combination of the peroxidative reaction of Cu,Zn-SOD and the Fenton-like reaction of free copper released from oxidatively damaged SOD.     

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with alpha-linolenic acid

Lipid hydroperoxide (LOOH)-dependent lipid peroxidation was induced in alpha-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 microM. The effects of structurally related flavonoids at concentrations from 10 to 500 microM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid-metal complexes.