Nitrogen fixation in several grain legume species with contrasting sensitivities to copper nutrition

The nitrogen fixation response to copper nutrition in faba bean, yellow lupin and soybean was studied. Copper nutrition significantly increased the pod yields of all tested grain legumes but faba bean gave the greatest Cu-use efficiency for pod and grain production. The accumulation of dry matter in vegetative parts, nodules, N and leghemoglobin concentration in nodules and nitrogen accumulation in the whole plants were increased by copper supply in faba bean and yellow lupin in contrast with soybean. Cu nutrition significantly increased the Cu concentrations in nodules of all cultivated plants. The differential sensitivity of N₂ fixation in tested grain legume species to copper nutrition could be connected with the level of phenols in nodules and depended on both the host plants and strains of rhizobia, which differ in their ability to produce catechol-like siderophores. Copper requirements by symbiotic N₂ fixation could also depend on the nature of phenols in nodules (presence of o-dihydroxyphenols or number of hydroxyls in molecule).     

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.     

NADPH-dependent electron transport chain in microsomes and lipid peroxidation catalyzed by metal ions.

Like iron ions copper ions are also able to stimulate the NADPH-dependent lipid peroxidation in rat liver microsomes. This effect is strongly dependent on the concentration of Cu2+ added. Initial concentrations of Cu2+ above 50 microM completely inhibit the formation of malonaldehyde. The activator and inhibitor functions may be interpreted by a simultaneous participation of Cu+ ions formed in the chain branching and termination reaction of the free radical lipid peroxidation process. Inhibition studies with pCMB and the His-reagent diethyl pyrocarbonate indicate an essential role of cysteine and histidine residues in the Cu+-NADPH-dependent lipid peroxidation process.     

Ameliorative Role of Castasterone on Copper Metal Toxicity by Improving Redox Homeostasis in <Emphasis Type="Italic">Brassica juncea</Emphasis> L.

The transition metal elements like copper act as double-edged sword for living cells. Cu, a redox active metal, is essential for various biological processes, but at higher concentrations it leads to toxicity by inducing production of reactive oxygen species (ROS). Thus, the objective of the present study was to investigate the effects of exogenously applied castasterone on oxidative stress markers and redox homeostasis managers in Brassica juncea plants subject to copper stress for 30 days. Copper-exposed plants showed accumulation of free radicals (H₂O₂ and superoxide anion) and lipid peroxidation. However, the exogenous treatment of seeds via the seed soaking method with different concentrations of castasterone reduced H₂O₂ production, superoxide anion radical content, and lipid peroxidation, thus indicating improved detoxification of ROS. Enzyme activity was increased by 19.19% for guaiacol peroxidase, 16.20% for superoxide dismutase, 35.74% for glutathione peroxidase, 27.58% for dehydroascorbate reductase, and 42.75% for ascorbate peroxidase, with castasterone pre-soaking under copper stress. The levels of non-enzymatic antioxidants were also increased with castasterone pre-treatment under copper stress. It may be concluded that castasterone treatment enhanced redox homeostasis managers in addition to increased levels of osmoprotectants.     

Involvement of zinc in intracellular oxidant/antioxidant balance

The effect of zinc (Zn) on cellular oxidative metabolism is complex and could be explained by multiple complementary interactions. In this study, we evaluated the impact of Zn on the pro-oxidant/ antioxidant balance of HaCaT keratinocytes. Cells were submitted to a diffusible metal chelator able to induce intracellular Zn deprivation, TPEN, in combination or not with Zn chloride (ZnCl₂), in the culture medium. The intracellular amount of Zn, copper (Cu), and iron (Fe) was determined, as well as CuZnSOD and MnSOD activities and glutathione reserves. The consequence of the modulation of Zn concentration on lipid peroxidation was also evaluated. TPEN induced a significant dose-dependent decrease in intracellular Zn and Cu (from 394–181 and 43–21 Μg/g protein, respectively, after 6 h of TPEN 50 ΜM). No significant change in intracellular Fe concentration was found following TPEN exposure. The SOD activities were unchanged after 6 h of TPEN 50 ΜM application, either CuZnSOD or MnSOD. Cells exposure to TPEN induced a deep time- and dosedependent decrease in their glutathione content (from 65–8 ΜM/g protein after 6 h of TPEN 50 ΜM), and a concomittant increase in glutathione in the cell-culture supernatants. No significant change in lipid peroxidation products was detected.     

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.     

Cytoprotective effect of copper(II) complexes against ethanol-induced damage to rat gastric mucosa.

The cytoprotective effect of various copper(II) complexes on the gastric mucosa damage induced by acute intragastric administration of ethanol was investigated. For in vitro experiments, the following copper(II) complexes were tested: Cu(II)(L-Trp)(L-Phe), Cu(II)(L-Leu)Cu(II)(L-Leu-Leu)(L-Leu), Cu(II)(L-Phe-L-Leu), Cu(II)(Gly-His-Lys), and Cu(II)(cyHis)2(ClO4)2. Inorganic copper such as CuSO4 was also tested. The free radical generating system, acting for 2 hr on cardial and fundic mucosa scrapings or mucosal microsomes, was Fe++ (20 microM)/ascorbate (0.25 mM). We found a marked inhibition to 75% of lipid peroxidation in the range 10-100 mM, regardless of whether copper was given in complexed or inorganic form. The results suggest that nontoxic copper(II)-amino acid complexes are able to neutralize oxygen-derived free radicals. In addition, copper(II) complexes suppressed membrane lipid peroxidation when mucosa homogenates were exposed to t-butyl hydroperoxide (1-20 microM) plus Fe++ (50 microM). In vivo experiments on rat stomachs, pretreated p.o. by gavage either with Cu(II)(L-Trp)(L-Phe) as paradigmatic agent or with copper sulphate at equivalent doses in the range 3-30 mg/kg body weight showed a significant decrease (30 min after 95% ethanol administration) in the number and severity of mucosal hemorrhagic lesions. In the gastric mucosa scrapings of copper-treated rats after ethanol exposure, we found that malondialdehyde and conjugated diene levels were unchanged compared to those of untreated controls; five enzyme activities released from lysosomes were near control values. In isolated mucosal cells, whether or not pretreated with 200 microM solution of either Cu(II)(L-Trp)(L-Phe) or CuSO4, the release of cathepsin D activity was also unmodified. The results suggest that the cytoprotective effect of Cu(II) complexes against ethanol-induced mucosal lesions was not associated in vivo to lipid peroxidation.     

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+.     

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.     

Reactivity of Cu2(lonazolac)4, a lipophilic copper acetate derivative

An acetate-like copper complex of lonazolac (3-(p-chlorophenyl)-1-phenyl-pyrazole-4-acetate) was prepared and characterized. The copper of Cu₂-(lonazolac)₄ was spin-coupled and remained EPR-silent. Water and organic solvents did not affect this magnetic interaction. Superoxide dismutase activity of the Cu complex in micromolar concentrations was detectable in the presence of up to 900 μg per ml of serum albumin or whole serum protein. At 700 μM albumin concentration, a ternary complex between Cu₂(lonazolac)₄ and the protein was formed. The original acetate-copper coordination changed to a biuret-type copper bonding as seen from EPR and electron absorption spectrometry. Lonazolac did not induce a detectable conformational change of the protein near or at the copper binding site. Equilibrium dialysis and optical titration experiments revealed that essentially all copper of the Cu₂-(lonazolac)₄ complex was bound in the specific binding site of serum albumin. The copper complex proved to be an effective inhibitor of lipid peroxidation.     

Antioxidant capacity of <Emphasis Type="Italic">Brassica juncea</Emphasis> plants exposed to elevated levels of copper

Brassica juncea L. eight-day-old seedlings treated with various concentrations (50–200 µM) of copper for 48 h accumulated Cu more in the roots than in leaves. Accumulation of copper resulted in more active lipid peroxidation and depletion of glutathione (GSH) pools in both roots and shoots, which was attributed to copper-induced additional oxidative stress. Activities of ascorbate peroxidase and superoxide dismutase were higher in both roots and shoots while catalase activity increased in leaves but remained unchanged in roots in response to copper accumulation. Changes in lipid peroxidation, GSH content, and antioxidant enzyme activities suggest that oxidative damage may be involved in copper toxicity.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 233–237.Original English Text Copyright © 2005 by Devi, Prasad.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.     

同型半胱氨酸诱导血管内皮细胞凋亡的研究

观察不同浓度同型半胱氨酸(homocysteine,HCY)在Cu2 介导下,能否诱导培养的人脐静脉内皮细胞凋亡,以揭示HCY致血管内皮损伤的机制。采用细胞计数板检测脱落细胞量;比色法分别测定乳酸脱氢酶释放率、细胞内丙二醛含量和超氧化物歧化酶、谷胱甘肽过氧化物酶活力的改变;Hoechst 33258染色观察凋亡细胞核形态变化及流式细胞术定量测定细胞凋亡。结果表明HCY在生理浓度Cu2 的介导下,可能通过氧化应激损伤的机制而导致血管内皮细胞凋亡,这提示在体内可能通过此途径诱发动脉粥样硬化。     

Regulation of exogenous spermidine on the reactive oxygen species level and polyamine metabolism in <Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> (Mart.) Griseb under copper stress

Effects of exogenous spermidine (Spd) on the reactive oxygen species level and polyamine metabolism against copper (Cu) stress in Alternanthera philoxeroides (Mart.) Griseb leaves were investigated. Cu treatment induced a marked accumulation of Cu and enhanced contents of malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and the generation rate of O₂ ^·−. It also significantly increased putrescine (Put) levels but lowered spermidine (Spd) and spermine (Spm) levels. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and polyamine oxidase (PAO) were all elevated with the increase of Cu concentration. However, application of exogenous Spd effectively decreased H₂O₂ content and the generation rate of O₂ ^·−, prevented Cu-induced lipid peroxidation and reduced Cu accumulation. Moreover, it declined level of endogenous Put and increased levels of Spd and Spm. Activities of ADC, ODC and PAO were all inhibited by exogenous Spd. The results indicated that application of exogenous Spd could enhance the tolerance of A. philoxeroides to Cu stress by reducing the reactive oxygen level and balancing polyamine metabolism.     

Lipid peroxidation in peribacteroid membranes from French-bean nodules

Enriched peribacteroid membranes were prepared from Phaseolus vulgaris nodules and, in the presence of metleghemoglobin and H₂O₂, membranal lipid peroxidation was observed. The initial rate of the reaction was low and increased with time. Ferrous leghemoglobin was unable to induce this peroxidation with H₂O₂. Thus, it appears that leghemoglobin (IV) is not the activated species involved in this process. Heme plays a role in this peroxidation and the hydroxyl radical is not an intermediate of the reaction. Lipid peroxidation in peribacteroid membranes was also observed in the presence of iron ions. A mixture of iron (III) and iron (II) produced a maximal peroxidation. Senescing nodule extracts were able to provoke membranal lipid peroxidation; they contained nonprotein-bound iron. Peribacteroid membranes were more sensitive than microsomes to peroxidation, as measured by malonaldehyde formation.     

Calcium-mediated enhancement of copper tolerance in Elodea canadensis

The alleviative effects of exogenous calcium on copper phytotoxicity were investigated in Elodea canadensis plants. There was a significant accumulation of Cu in the plants after their exposure to 0.01 mM Cu accompanied by many symptoms of toxicity. Increased uptake of Cu severely reduced content of photosynthetic pigments, soluble proteins, and free proline. The total antioxidant capacity (T-AOC), reduced glutathione (GSH), and non-protein thiol (NP-SH) were severely suppressed in Cu-stressed plants resulting in a rapid increase in content of superoxide anion (O₂ ^·?), hydrogen peroxide, lipid peroxidation, and cell death. Simultaneous application of Ca markedly increased the content of photosynthetic pigments, soluble proteins, free proline, T-AOC, GSH, and NP-SH, and reduced oxidative damage as indicated by lowered content of MDA, O₂ ^·?, and H₂O₂; and decreased cell death. Furthermore, application of Ca reduced Cu uptake and effectively reversed the Cu-induced nutrient imbalance.     

Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation.

The degree of plasma membrane fatty acid unsaturation and the copper sensitivity of Saccharomyces cerevisiae are closely correlated. Our objective was to determine whether these effects could be accounted for by differential metal induction of lipid peroxidation. S. cerevisiae S150-2B was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Potassium efflux and colony count data indicated that sensitivity to both copper (redox active) and cadmium (redox inactive) was increased in 18:2-supplemented cells and particularly in 18:3-supplemented cells. Copper- and cadmium-induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements with the membrane probe trimethylammonium diphenylhexatriene. Levels of thiobarbituric acid-reactive substances (lipid peroxidation products) were up to twofold higher in 18:2-supplemented cells than in unsupplemented cells following metal addition, although this difference was reduced with prolonged incubation up to 3 h. Conjugated-diene levels in metal-exposed cells also increased with both the concentration of copper or cadmium and the degree of cellular fatty acid unsaturation; maximal levels were evident in 18:3-supplemented cells. The results demonstrate heavy metal-induced lipid peroxidation in a microorganism for the first time and indicate that the metal sensitivity of PUFA-enriched S. cerevisiae may be attributable to elevated levels of lipid peroxidation in these cells.     

The effect of vitamin E on lipid peroxidation in the copper-deficient rat

The present study was designed to determine whether the supplementation of vitamin E in the copper-deficient diet would ameliorate the severity of copper deficiency in fructose-fed rats. Lipid peroxidation was measured in the livers and hearts of rats fed a copper-deficient diet (0.6 microg Cu/g) containing 62% fructose with adequate vitamin E (0.1 g/kg diet) or supplemented with vitamin E (1.0 g/kg diet). Hepatic lipid peroxidation was significantly reduced by vitamin E supplementation compared with the unsupplemented adequate rats. In contrast, myocardial lipid peroxidation was unaffected by the level of vitamin E. Regardless of vitamin E supplementation, all copper-deficient rats exhibited severe signs of copper deficiency, and some of the vitamin E-supplemented rats died of this deficiency. These findings suggest that although vitamin E provided protection against peroxidation in the liver, it did not protect the animals against the severity of copper deficiency induced by fructose consumption.     

Mechanisms of Hemolysis Induced by Copper

An excess of copper is the cause of hemolysis in a number of clinical conditions. Incubation of human erythrocyte (RBC) suspensions with copper (II) causes the formation of methemoglobin, lipid peroxidation and hemolysis.

A new variant of the thiobarbituric acid (TBA) method, which minimizes the formation of interfering chromophores, was used to detect lipid peroxidation. Lipid peroxidation precedes hemolysis and the antioxidant vitamins C and E, which inhibit lipid peroxidation, also inhibit hemolysis. Consequently lipid peroxidation appears to be the cause of RBC destruction. Lipid peroxidation arises mostly from the oxidation of oxyhemoglobin by copper as it is inhibited in RBCs with carbon monoxyhemoglobin or methemoglobin. A direct interaction of copper with the red cell membrane seems to play only a minor role. Copper effects depend on the presence of free SH groups. Lipid peroxidation is probably initiated by activated forms of oxygen as it is increased by an inhibitor of catalase and reduced by hydroxyl radical scavengers. With higher copper concentrations hemolysis is greater: its mechanism appears different as lipid peroxidation is smaller but hemoglobin alterations, namely precipitation, are more pronounced.     

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.