Free radical scavenging and antioxidant effects of lactate ion: an in vitro study.

Divergent literature data are found concerning the effect of lactate on free radical production during exercise. To clarify this point, we tested the pro- or antioxidant effect of lactate ion in vitro at different concentrations using three methods: 1) electron paramagnetic resonance (EPR) was used to study the scavenging ability of lactate toward the superoxide aion (O(2)(-).) and hydroxyl radical (.OH); 2) linoleic acid micelles were employed to investigate the lipid radical scavenging capacity of lactate; and 3) primary rat hepatocyte culture was used to study the inhibition of membrane lipid peroxidation by lactate. EPR experiments exhibited scavenging activities of lactate toward both O(2)(-). and.OH; lactate was also able to inhibit lipid peroxidation of hepatocyte culture. Both effects of lactate were concentration dependent. However, no inhibition of lipid peroxidation by lactate was observed in the micelle model. These results suggested that lactate ion may prevent lipid peroxidation by scavenging free radicals such as O(2)(-). and.OH but not lipid radicals. Thus lactate ion might be considered as a potential antioxidant agent.     

Damage to protein synthesis concurrent with lipid peroxidation in rat liver slices: effect of halogenated compounds, peroxides, and vitamin E1

Protein synthesis and lipid peroxidation were evaluated in rat liver slices incubated in the presence of oxidants and protein synthesis inhibitors. Protein synthesis by rat liver slices was evaluated by [3H]leucine incorporation into the trichloroacetic acid (TCA)-insoluble material, and lipid peroxidation was evaluated by thiobarbituric acid-reactive substances (TBARS) released into the incubation medium. Protein synthesis inhibition by bromotrichloromethane (BrCCl3) or t-butyl hydroperoxide (t-BOOH) depended on the incubation time and oxidant concentration. [3H]Leucine incorporation was decreased to 20 and 47% of control values and TBARS were enhanced from the control value of 16.9 to 45.3 and 62.5 nmol/g of liver by incubation for 1 h with 1 mM BrCCl3 and t-BOOH, respectively. Following incubation, both protein synthesis damage and lipid peroxidation were decreased in control and oxidant-treated slices prepared from rats injected with 200 mg of DL-alpha-tocopherol/kg of body wt. Release of lactate dehydrogenase was not enhanced by oxidant treatment. Protein synthesis inhibitors reversibly decreased [3H]leucine incorporation, but the effect of oxidants on protein synthesis was irreversible. Cumene hydroperoxide and methyl ethyl ketone peroxide, but not hydrogen peroxide, damaged protein synthesis and induced lipid peroxidation. The ability of carbon tetrabromide, benzyl chloride, bromoform, bromobenzene, carbon tetrachloride, chloroform, dichloromethane, and bromochloromethane to inhibit protein synthesis was correlated with their ability to induce lipid peroxidation, and with their LD50. The results suggest that oxidant-induced lipid peroxidation and protein synthesis damage occurred concurrently, and that protein synthesis inhibition may be involved in cell injury or death mediated by free radicals.     

Interactions of sulindac and its metabolites with phospholipid membranes: An explanation for the peroxidation protective effect of the bioactive metabolite

Non-steroidal anti-inflammatory drugs (NSAIDs) treat inflammatory processes by inhibition of cycloxygenase (COX). However, their action against lipid peroxidation can be an alternative pathway to COX inhibition. Since inflammation and lipid peroxidation are cell-surface phenomena, the effects of NSAIDs on membrane models were investigated. Peroxidation was induced by peroxyl radical (ROO?) derived from AAPH and assessed in aqueous or lipid media using fluorescence probes with distinct lipophilic properties: fluorescein; HDAF and DPH-PA. The antioxidant effect of Sulindac and its metabolites was tested and related with their membrane interactions. Drug–membrane interactions included the study of: drug location by fluorescence quenching; drug interaction with membrane surface by zeta-potential measurements; and membrane fluidity changes by steady-state anisotropy. Results revealed that the active NSAID (sulindac sulphide) penetrates into the lipid bilayer and protects the membrane against oxy-radicals. The inactive forms (sulindac and sulindac sulphone) present weaker interactions with the membrane and are better radical scavengers in aqueous media.     

Interactions of sulindac and its metabolites with phospholipid membranes: an explanation for the peroxidation protective effect of the bioactive metabolite

Non-steroidal anti-inflammatory drugs (NSAIDs) treat inflammatory processes by inhibition of cycloxygenase (COX). However, their action against lipid peroxidation can be an alternative pathway to COX inhibition. Since inflammation and lipid peroxidation are cell-surface phenomena, the effects of NSAIDs on membrane models were investigated. Peroxidation was induced by peroxyl radical (ROO*) derived from AAPH and assessed in aqueous or lipid media using fluorescence probes with distinct lipophilic properties: fluorescein; HDAF and DPH-PA. The antioxidant effect of sulindac and its metabolites was tested and related with their membrane interactions. Drug-membrane interactions included the study of: drug location by fluorescence quenching; drug interaction with membrane surface by zeta-potential measurements; and membrane fluidity changes by steady-state anisotropy. Results revealed that the active NSAID (sulindac sulphide) penetrates into the lipid bilayer and protects the membrane against oxy-radicals. The inactive forms (sulindac and sulindac sulphone) present weaker interactions with the membrane and are better radical scavengers in aqueous media.     

The origin of red cell fluorescence caused by hydrogen peroxide treatment

Fluorescence in red cells following hydrogen peroxide treatment has been attributed to lipid peroxidation of the membrane. The putative relationship between lipid peroxidation and fluorescence was questioned by the finding that BHT and alpha-tocopherol, which are thought to inhibit lipid peroxidation, do not inhibit the fluorescence detected by flow cytometry. Furthermore, lipid peroxidation induced in red cells by the Fe(III)-ADP-ascorbate system did not produce fluorescence. These results require an alternative explanation for the hydrogen peroxide-induced fluorescence. A role for reduced hemoglobin is indicated by the inhibition of fluorescence by pretreatment of cells with CO that binds strongly to ferrohemoglobin and nitrite that oxidizes ferrohemoglobin. Our earlier studies have shown the formation of fluorescent heme degradation products during the reaction of purified hemoglobin with hydrogen peroxide, which was also inhibited by CO and nitrite pretreatment. The fluorescence produced in red cells after the addition of hydrogen peroxide can, therefore, be attributed to fluorescent heme degradation products.     

Dynamics of antioxidant action of ubiquinol: a reappraisal.

The dynamics of action of ubiquinol as an antioxidant against lipid peroxidation was reinvestigated and compared with that of alpha-tocopherol. It was found that ubiquinol was 2.5 and 1.9 times more reactive than alpha-tocopherol toward phenoxyl and peroxyl radicals, respectively, at 25 degrees C in ethanol and that it was capable of donating two hydrogen atoms toward oxygen radicals but that the apparent stoichiometric number decreased in the inhibition of lipid peroxidation, to even smaller than 1, due to its autoxidation. The autoxidation of ubiquinol proceeded even in the micelles and liposomal membranes in aqueous dispersions as well as in organic homogeneous solution. The apparent antioxidant activity of ubiquinol was smaller than that of alpha-tocopherol against lipid peroxidation in organic solution as judged from either rate of oxidation or duration of inhibition period. They exerted similar antioxidant potency against lipid peroxidation in the membranes and micelles in aqueous dispersions. The combination of ubiquinol and alpha-tocopherol was suggested to be effective.     

Cytotoxic effects of paraquat and inhibition of them by vitamin E

Paraquat causes failure of multiple organs including the liver in humans. The kinetics and mechanism of paraquat intoxication were studied using cultured rat hepatocytes. Paraquat induced time- and dose-dependent lactate dehydrogenase release, lipid peroxidation, and cell death, estimated as decrease in protein in cells attached to culture dishes. However, the increase in lipid peroxidation occurred after lactate dehydrogenase release had reached a plateau. Vitamin E inhibited the inductions of all these cytotoxic effects of paraquat. Kinetic studies showed that lipid peroxidation was a better indicator of cell death than lactate dehydrogenase release, because vitamin E inhibited the induction of cell death even when added 6 h after paraquat, when lactate dehydrogenase release had reached a plateau but lipid peroxidation had not. The present results strongly suggest that paraquat exerts its cytotoxicity by a mechanism involving oxidation reactions.     

Corroding iron as a hydrogen source for sulphate reduction in growing cultures of sulphate-reducing bacteria

Summary In anaerobic corrosion experiments, hydrogenase-positiveDesulfovibrio strains, grown with limiting lactate concentrations in the presence of steel wool, formed more sulphide than expected or observed with lactate alone. The additional sulphide obviously originated from sulphate reduction with cathodically formed hydrogen from the steel surface. The hydrogenasenegativeD. sapovorans did not produce additional sulphide. The observations agree with the theory of von Wolzogen Kühr and van der Vlugt (1934) that explains anaerobic corrosion as a cathodic depolarization of iron surfaces by hydrogen-consuming sulphate-reducing bacteria. The influence of the iron surface area, the salt concentration and the pH-value on the utilization of cathodically formed hydrogen was investigated. The significance of an additional organic electron donor for the corrosion of iron in aqueous environments is discussed.     

Effect of Ascorbate on Red Blood Cell Lipid Peroxidation

The present study investigates the effect of ascorbate on red cell lipid peroxidation. At a concentration between 0.2 mmol-20 mmol/l ascorbic acid reduces hydrogen peroxide-induced red blood cell lipid peroxidation resulting in a marked decrease in ethane and pentane production as well as in haemolysis. Ascorbic acid also shows an antioxidant effect on chelated iron-catalyzed hydrogen peroxide-induced peroxidation of erythrocyte membranes. At a concentration of 10 mmol/l ascorbic acid totally inhibits oxidative break-down of polyunsaturated fatty acids by radicals originating from hydrogen peroxide.

Our results indicate that ascorbate at the chosen concentration has an antioxidant effect on red blood cell lipid peroxidation.     

The influence of NADPH-dependent lipid peroxidation on the progesterone biosynthesis in human placental mitochondria.

In an in vitro system consisting of human term placental mitochondria and an NADPH-generating system plus Fe2+, significant lipid peroxidation was observed along with a concomitant inhibition of progesterone biosynthesis. This inhibition could be markedly blocked by Mn2+, superoxide dismutase and dimethylfuran, inhibitors of NADPH-dependent lipid peroxidation. In addition, it has been found that malondialdehyde formation is accompanied by a corresponding decrease in placental mitochondrial cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in cell-free systems. These measurements provide the first evidence that the inhibition of progesterone biosynthesis by a NADPH-dependent lipid peroxidation in placental mitochondria is a consequence of cytochrome P-450 degradation due 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+.     

Probucol as a potent inhibitor of oxygen radical-induced lipid peroxidation and DNA damage: in vitro studies

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H2O2). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H2O2-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H2O2 (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10-34% inhibition; P < 0.05) of Fe-NTA plus H2O2-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36-71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H2O2. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.     

Inhibition of adriamycin-stimulated microsomal lipid peroxidation by mitoxantrone and ametantrone,two new anthracenedione antineoplastic agents

Ametantrone and mitoxantrone, two new anthracenedione antineoplastic agents, produced a concentration-dependent inhibition of hepatic microsomal lipid peroxidation. Malondialdehyde production was diminished from 10.6 nmoles/mg/60 min to 3.3 and 5.4 nmoles/mg/60 min, in the presence of 100 μM mitoxantrone and ametantrone, respectively. Under similar conditions, Adriamycin stimulated lipid peroxidation over twofold. In addition, both mitoxantrone and ametantrone inhibited Adriamycin-stimulated lipid peroxidation, with 50% inhibition occurring at concentrations of 4 and 6 μM, respectively. Microsomal superoxide production was not significantly inhibited at anthracenedione concentrations which markedly decreased lipid peroxidation, suggesting that inhibition of lipid peroxidation was not the result of inhibition of superoxide generation. These results correlate with the lack of anthracenedione cardiotoxicity and also demonstrate anthracenedione inhibition of lipid peroxidation at micromolar concentrations; an observation with potential therapeutic significance.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.     

Oxygen-dependent reperfusion injury in the isolated rat lung.

To further define the relationship between oxygen dependence of lung injury during ischemia and ischemia-reperfusion, we used the isolated, perfused, and ventilated rat lung model, so that oxygenation and perfusion could be separated. During ischemia, lungs were ventilated with various oxygen concentrations and then ventilated with 95% oxygen during the 60-min reperfusion period. Other lungs were ventilated with 0% oxygen (nitrogen) during ischemia, and the reperfusion phase oxygen concentration was varied. Tissue and perfusate lipid peroxidation products (thiobarbituric acid-reactive substances and conjugated dienes), dry-to-wet weight ratio, and lactate dehydrogenase were measured as indexes of lung damage. In addition, electron microscopy of some lungs was performed. Results demonstrate an oxygen dependence of lipid peroxidation in both the ischemic and reperfusion phases, but lipid peroxidation is severalfold greater in the reperfusion than in the ischemic phase. Products of lipid peroxidation closely correlate with indexes of lung injury (dry-to-wet weight ratio, lactate dehydrogenase, and electron microscopy).     

Changes in oxidative stress parameters and acid phosphatase activity in the pre-regressing and regressing tail of Indian jumping frog Polypedates maculatus (Anura,Rhacophoridae)

Activities of acid phosphatase (normal and Co²⁺-sensitive), superoxide dismutase and catalase and levels of lipid peroxidation, hydrogen peroxide were compared in the tails of tadpoles of stage III, XVIII, XXI and XXIII, respectively, of the Indian Jumping frog Polypedates maculatus. It is noticed that acid phosphatase activity (normal and Co²⁺-sensitive), and levels of lipid peroxidation and hydrogen peroxide increased during tail regression. There is also an increase in the level of superoxide dismutase and catalase in the regressing tail. A positive correlation between activity of acid phosphatase and lipid peroxidation, hydrogen peroxide and lipid peroxidation, acid phosphatase and hydrogen peroxide was noticed in the tail of tadpoles during different developmental stages, suggesting a critical interaction between reactive oxygen species and lysosomal activity during metamorphosis.     

Lipid peroxidation initiated by superoxide-dependent hydroxyl radicals using complexed iron and hydrogen peroxide

Iron salts stimulate lipid peroxidation by decomposing lipid peroxides to produce alkoxyl and peroxyl radicals which initiate further oxidation. In aqueous solution ferrous salts produce OH. radicals, a reactive species able to abstract hydrogen atoms from unsaturated fatty acids, and so can initiate lipid peroxidation. When iron salts are added to lipids, containing variable amounts of lipid peroxide, the former reaction is favoured and OH. radicals contribute little to the observed rate of peroxidation. When iron is complexed with EDTA, however, lipid peroxide decomposition is prevented, but the complex reacts with hydrogen peroxide to form OH. radicals which are seen to initiate lipid peroxidation. Superoxide radicals appear to play an important part in reducing the iron complex.     

Effects of anthrapyrazole antineoplastic agents on lipid peroxidation

The effects of three anthrapyrazoles and an aminoacridine derivative on doxorubicin- and iron-stimulated lipid peroxidation in rabbit hepatic microsomes have been characterized. Two anthrapyrazoles, CI-937 and CI-942, were potent inhibitors of lipid peroxidation with 15 microM drug inhibiting the rate of peroxidation 70 to 90%. In contrast CI-941 was relatively ineffective in inhibiting lipid peroxidation with only 35% inhibition occurring at 100 microM drug. CI-921, an aminoacridine derivative, diminished lipid peroxidation by 65% at 15 microM. All four drugs failed to decrease the rate of doxorubicin-stimulated NADPH oxidation at concentrations less than 50 microM, suggesting that inhibition of lipid peroxidation was not the result of diminished enzyme activity. CI-937 formed a 2:1 complex with ferric ion, KD = 47 microM, which was reversible with EDTA.     

Assessment of cell damage caused by spontaneous lipid peroxidation in rabbit spermatozoa

Damage to the plasma membrane of rabbit epididymal spermatozoa during spontaneous lipid peroxidation was examined by means of trypan blue uptake and expression of activity of the intracellular enzymes, lactate dehydrogenase and pyruvate kinase. Both the dye uptake and the expression of enzyme activity probe cell damage from lipid peroxidation as loss of integrity of the plasma membrane. A linear correlation was obtained between trypan blue staining of the cells and malondialdehyde production, a quantifiable measure of the extent of lipid peroxidation. At the point of trypan blue staining of all cells, 0.5 nmol malondialdehyde/10(8) cells was produced. This is the same amount produced at the point of complete loss of motility and superoxide dismutase activity. We have defined this as the "lipoperoxidative lethal end point." Expression of lactate dehydrogenase and pyruvate kinase activities increased with time of aerobic incubation. In the high Na+ medium, NTP, in which lipid peroxidation is slow, there is a linear correlation between increase in expressed enzyme activities and malondialdehyde production. But in the high K+ medium, KTP, in which lipid peroxidation is rapid, there is an initial rapid rise in expressed enzyme activity over 3 h, followed by a slower increase. Activities of rabbit sperm lactate dehydrogenase, pyruvate kinase, and flagellar ATPase were unaffected by aerobic incubations for up to 48 h, double the incubation period used for the assay of enzymatic activities for the first two. The activity of glyceraldehyde-3-phosphate dehydrogenase decreased during aerobic incubation, the time course matching the loss of motility. The subcellular distribution of lactate dehydrogenase in rabbit spermatozoa was determined: 4% in the mitochondrial matrix, 10% in the plasma membrane and 85% in the cytosolic compartment.     

Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

Carbon tetrachloride (CCl(4)) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl(4)-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation, and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl(4) with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl(4) exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl(4) and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl)ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl(4) is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl(4).