Reactions of Adriamycin with Microsomal Iron and Lipids

Iron plays a central role in oxidative injury, reportedly because it catalyzes superoxide- and hydrogen peroxide-dependent reactions yielding a powerful oxidant such as the hydroxyl radical. Iron is also thought to mediate the cardiotoxic and antitumour effects of adriamycin and related compounds. NADPH-supplemented microsomes reduce adriamycin to a semiquinone radical, which in turn re-oxidizes in the presence of oxygen to form superoxide and hence hydrogen peroxide. During this redox cycling membrane-bound nonheme iron undergoes superoxide dismutase- and catalase-insensitive reductive release. Membrane iron mobilization triggers lipid peroxidation, which is markedly enhanced by simultaneous addition of superoxide dismutase and catalase. The results indicate that : i) lipid peroxidation is mediated by the release of iron, yet the two reactions are governed by different mechanisms; and ii) oxygen radicals are not involved in or may actually inhibit adriamycin-induced lipid peroxidation. Microsomal iron delocalization and lipid peroxidation might represent oxyradical-independent mechanisms of adriamycin toxicity.     

Peroxidation of Phospholipids Promoted by Myeloperoxidase

Myeloperoxidase was found to promote peroxidation of phospholipids under acidic conditions in the presence of hydrogen peroxide and iodide ions The peroxidation was markedly enhanced by pyrophosphate-chelated ferric iron and was inhbited by desferrioxamine and superoxide dismutase. This observation adds lipid peroxidation to the oxidative damage caused by myeloperoxidase, which is a phagocytic cell enzyme involved in phapocyte-mediated cell destruction.     

Lipid peroxidation in mitochondrial inner membranes. I. An integrative kinetic model

An integrative mathematical model was developed to obtain an overall picture of lipid hydroperoxide metabolism in the mitochondrial inner membrane and surrounding matrix environment. The model explicitly considers an aqueous and a membrane phase, integrates a wide set of experimental data, and unsupported assumptions were minimized. The following biochemical processes were considered: the classic reactional scheme of lipid peroxidation; antioxidant and pro-oxidant effects of vitamin E; pro-oxidant effects of iron; action of phospholipase A₂, glutathione-dependent peroxidases, glutathione reductase and superoxide dismutase; production of superoxide radicals by the mitochondrial respiratory chain; oxidative damage to proteins and DNA. Steady-state fluxes and concentrations as well as half-lives and mean displacements for the main metabolites were calculated. A picture of lipid hydroperoxide physiological metabolism in mitochondria in vivo showing the main pathways is presented. The main results are:(a) perhydroxyl radical is the main initiation agent of lipid peroxidation (with a flux of 10⁻⁷Ms⁻¹); (b) vitamin E efficiently inhibits lipid peroxidation keeping the amplification (kinetic chain length) of lipid peroxidation at low values (10); (c) only a very minor fraction of lipid hydroperoxides escapes reduction via glutathione-dependent peroxidases; (d) oxidized glutathione is produced mainly from the reduction of hydrogen peroxide and not from the reduction of lipid hydroperoxides.     

Comparison of iron-catalyzed DNA and lipid oxidation

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.     

Effect of flooding on the activities of some enzymes of activated oxygen metabolism,the levels of antioxidants,and lipid peroxidation in senescing tobacco leaves

Effects of flooding on the activities of some enzymes of activated oxygen metabolism, the levels of antioxidants, and lipid peroxidation in senescing leaves of tobacco were investigated. As judged by the decrease in chlorophyll and protein levels, flooding accelerated the senescence of tobacco leaves. Total peroxide and the lipid peroxidation product, malondialdehyde, increased in both control and flooding-treated leaves with increasing duration of the experiment. Throughout the duration of the experiment, flooded leaves had higher levels of total peroxide and malondialdehyde than did control leaves. Flooding resulted in an increase in peroxidase and ascorbate peroxidase activities and a reduction of superoxide dismutase activity in the senescing leaves. Glycolate oxidase, catalase, and glutathione reductase activities were not affected by flooding. Flooding increased the levels of total ascorbate and dehydroascorbate. Total glutathione, reduced form glutathione, or oxidized glutathione levels in flooded leaves were lower than in control leaves during the first two days of the experiment, but were higher than in control leaves at the later stage of the experiment. Our work suggests that senescence of tobacco induced by flooding may be a consequence of lipid peroxidation possibly controlled by superoxide dismutase activity. Our results also suggest that increased rates of hydrogen peroxide in leaves of flooded plants could lead to increased capacities of the scavenging system of hydrogen peroxide.Abbreviations GSH reduced form glutathione - GSSG oxidized form glutathione - GSSG reductase glutathione reductase - MDA malondialdehyde - SOD superoxide dismutase     

Alternative pathways as mechanism for the negative effects associated with overexpression of superoxide dismutase

One of the most important antioxidant enzymes is superoxide dismutase (SOD), which catalyses the dismutation of superoxide radicals to hydrogen peroxide. The enzyme plays an important role in diseases like trisomy 21 and also in theories of the mechanisms of aging. But instead of being beneficial, intensified oxidative stress is associated with the increased expression of SOD and also studies on bacteria and transgenic animals show that high levels of SOD actually lead to increased lipid peroxidation and hypersensitivity to oxidative stress. Using mathematical models we investigate the question how overexpression of SOD can lead to increased oxidative stress, although it is an antioxidant enzyme. We consider the following possibilities that have been proposed in the literature: (i) Reaction of H(2)O(2) with CuZnSOD leading to hydroxyl radical formation. (ii) Superoxide radicals might reduce membrane damage by acting as radical chain breaker. (iii) While detoxifying superoxide radicals SOD cycles between a reduced and oxidized state. At low superoxide levels the intermediates might interact with other redox partners and increase the superoxide reductase (SOR) activity of SOD. This short-circuiting of the SOD cycle could lead to an increased hydrogen peroxide production. We find that only one of the proposed mechanisms is under certain circumstances able to explain the increased oxidative stress caused by SOD. But furthermore we identified an additional mechanism that is of more general nature and might be a common basis for the experimental findings. We call it the alternative pathway mechanism.     

The antioxidant action of ergothioneine.

Ergothioneine is a product of plant origin that accumulates in animal tissues. Its suggested ability to act as an antioxidant has been evaluated. Ergothioneine is a powerful scavenger of hydroxyl radicals (.OH) and an inhibitor of iron or copper ion-dependent generation of .OH from hydrogen peroxide (H2O2). It is also an inhibitor of copper ion-dependent oxidation of oxyhaemoglobin, and of arachidonic acid peroxidation promoted by mixtures of myoglobin (or haemoglobin) and H2O2. Ergothioneine is a powerful scavenger of hypochlorous acid, being able to protect alpha 1-antiproteinase against inactivation by this molecule. By contrast, it does not react rapidly with superoxide (O2-) or hydrogen peroxide (H2O2) and it does not inhibit microsomal lipid peroxidation in the presence of iron ions. Overall, our results show that ergothioneine at the concentrations present in vivo could act as an antioxidant.     

Distribution of oxidized and HNE-modified proteins in U87 cells

Protein modification is one of the important processes during oxidative stress. This modification of proteins is either due to direct oxidation of proteins by various oxidants or due to secondary modification by lipid peroxidation products, e.g. 4-hydroxynonenal. In the here presented work we compare the intracellular distribution of protein modification products after treatment of human U87 astrocytoma cells with hydrogen peroxide or HNE. The treatment with hydrogen peroxide leads mainly to a cytosolic formation of oxidized proteins whereas HNE treatment is forming HNE-adducts throughout the cell. Therefore, we concluded that HNE diffusion distance in cells enables this lipid peroxidation product to act as a second messenger within the cell and on the other hand is the reason for the genotoxic properties of this compound.     

β-Amyloid Neurotoxicity In Vitro: Evidence of Oxidative Stress but Not Protection by Antioxidants

Abstract: Recent data from several groups suggest that the primary mechanism of β-amyloid neurotoxicity may be mediated by reactive oxygen species. To evaluate this hypothesis, we first compared the efficacy of antioxidant agents in preventing toxicity caused by oxidative insults (iron, hydrogen peroxide, and tert -butyl hydroperoxide) and β-amyloid peptides in cultured rat hippocampal neurons. Tested antioxidants (propyl gallate, Trolox, probucol, and promethazine) generally provided significant protection against oxidative insults but not β-amyloid peptides. Next, we examined whether β-amyloid causes oxidative stress, by comparing levels of lipid peroxidation after exposure to either iron or β-amyloid. In a cell-free system, iron but not β-amyloid generated lipid peroxidation. In culture, both insults caused rapid increases in lipid peroxidation, with iron inducing higher levels at later time points. Pretreatment with the antioxidant probucol significantly reduced lipid peroxidation caused by both insults but only attenuated iron toxicity, suggesting that lipid peroxidation does not contribute directly to cell death induced by β-amyloid. Finally, we observed that increasing basal levels of oxidative stress by pretreating cultures with subtoxic doses of iron significantly increased neuronal vulnerability to β-amyloid. The ability of β-amyloid to induce oxidative stress and the demonstration that oxidative stress potentiates β-amyloid toxicity support the clinical use of antioxidants for AD. However, these data do not support the theory that the primary mechanism of β-amyloid toxicity involves oxidative pathways, indicating a continued need to identify additional cellular responses to β-amyloid that underlie its neurodegenerative actions.     

Ferritin and superoxide-dependent lipid peroxidation

Ferritin was found to promote the peroxidation of phospholipid liposomes, as evidenced by malondialdehyde formation, when incubated with xanthine oxidase, xanthine, and ADP. Activity was inhibited by superoxide dismutase but markedly stimulated by the addition of catalase. Xanthine oxidase-dependent iron release from ferritin, measured spectrophotometrically using the ferrous iron chelator 2,2'-dipyridyl, was also inhibited by superoxide dismutase, suggesting that superoxide can mediate the reductive release of iron from ferritin. Potassium superoxide in crown ether also promoted superoxide dismutase-inhibitable release of iron from ferritin. Catalase had little effect on the rate of iron release from ferritin; thus hydrogen peroxide appears to inhibit lipid peroxidation by preventing the formation of an initiating species rather than by inhibiting iron release from ferritin. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide was used to observe free radical production in this system. Addition of ferritin to the xanthine oxidase system resulted in loss of the superoxide spin trap adduct suggesting an interaction between superoxide and ferritin. The resultant spectrum was that of a hydroxyl radical spin trap adduct which was abolished by the addition of catalase. These data suggest that ferritin may function in vivo as a source of iron for promotion of superoxide-dependent lipid peroxidation. Stimulation of lipid peroxidation but inhibition of hydroxyl radical formation by catalase suggests that, in this system, initiation is not via an iron-catalyzed Haber-Weiss reaction.     

Effect of oxidation on Ca2+ -ATPase activity and membrane lipids in lens epithelial microsomes.

Membrane oxidation may contribute to cataractogenesis. In our pursuit to understand the etiology of cataracts, we assessed the effect of membrane oxidation products on the activity of the lens epithelium calcium pump. Microsome preparations from bovine lens epithelium were oxidized to varying degrees with a ferrous and ferric ascorbate system to generate hydrogen peroxide and superoxide. Ca2+ -ATPase activity was measured using a colorometric assay. Lipid oxidation was quantified by infrared spectroscopy. Ca2+ -ATPase activity decreased as a function of ascorbate concentration between 0 and 200 microM. The level of Ca2+ -ATPase inhibition was correlated to both the level of lipid oxidation and the degree of lipid hydrocarbon chain order. At 25 degrees C when lipids are more ordered, the Ca2+ -ATPase activity was similar to that observed in the oxidized system measured at 37 degrees C. Glutathione, mercaptoethanol, and iodoacetate were able to reverse the oxidative inhibition of the calcium pump, suggesting that the ascorbate/iron oxidant directly oxidized the protein sulfhydryl moieties. To further probe the mechanism of Ca2+ ATPase inhibition, hydrogen peroxide was used to oxidize muscle sarcoplasmic reticulum Ca2+ -ATPase reconstituted in its native lipid vesicles, egg phosphatidylcholine, and dihydrosphingomyelin, with saturated hydrocarbon chains. In these systems, oxidation inhibited the Ca2+ -ATPase pump by 60-80%. There was no statistical difference between the level of oxidative inhibition and the percentage of dihydrosphingomyelin. Because dihydrosphingomyelin cannot be oxidized, whereas egg phosphatidylcholine (PC) can, and because the percentage of inhibition was the same for reconstituted systems using either lipid, the mechanism of inhibition is likely not via a secondary process involving oxidation-induced lipid structural changes or products of lipid oxidation.     

Cytochrome c-catalyzed membrane lipid peroxidation by hydrogen peroxide.

Cytochrome c(3+)-catalyzed peroxidation of phosphatidylcholine liposomes by hydrogen peroxide (H2O2) was indicated by the production of thiobarbituric acid reactive substances, oxygen consumption, and emission of spontaneous chemiluminescence. The iron chelator diethylenetriaminepentaacetic acid (DTPA) only partially inhibited peroxidation when H2O2 concentrations were 200 microM or greater. In contrast, iron compounds such as ferric chloride, potassium ferricyanide, and hemin induced H2O2-dependent lipid peroxidation which was totally inhibitable by DTPA. Cyanide and urate, which react at or near the cytochrome-heme, completely prevented lipid peroxidation, while hydroxyl radical scavengers and superoxide dismutase had very little or no inhibitory effect. Changes in liposome surface charge did not influence cytochrome c3+ plus H2O2-dependent peroxidation, but a net negative charge was critical in favoring cytochrome c(3+)-dependent, H2O2-independent lipid auto-oxidative processes. These results show that reaction of cytochrome c with H2O2 promotes membrane oxidation by more than one chemical mechanism, including formation of high oxidation states of iron at the cytochrome-heme and also by heme iron release at higher H2O2 concentrations. Cytochrome c3+ could react with mitochondrial H2O2 to yield "site-specific" mitochondrial membrane lipid peroxidation during tissue oxidant stress.     

Supression of hemin-mediated oxidation of low-density lipoprotein and subsequent endothelial reactions by hydrogen sulfide (H2S)

Heme-mediated oxidative modification of low-density lipoprotein (LDL) plays a crucial role in early atherogenesis. It has been shown that hydrogen sulfide (H₂S) produced by vascular smooth muscle cells is present in plasma at a concentration of about 50 µmol/L. H₂S is a strong reductant which can react with reactive oxygen species like superoxide anion and hydrogen peroxide. The current study investigated the effect of H₂S on hemin-mediated oxidation of LDL and oxidized LDL (oxLDL)-induced endothelial reactions. H₂S dose dependently delayed the accumulation of lipid peroxidation products—conjugated dienes, lipid hydroperoxides (LOOH), and thiobarbituric acid reactive substances—during hemin-mediated oxidation. Moreover, H₂S decreased the LOOH content of both oxidized LDL and lipid extracts derived from soft atherosclerotic plaque, which was accompanied by reduced cytotoxicity. OxLDL-mediated induction of the oxidative stress responsive gene, heme oxygenase-1, was also abolished by H₂S. Finally we have shown that H₂S can directly protect endothelium against hydrogen peroxide and oxLDL-mediated endothelial cytotoxicity. These results demonstrate novel functions of H₂S in preventing hemin-mediated oxidative modification of LDL, and consequent deleterious effects, suggesting a possible antiatherogenic action of H₂S.     

Redox-dependent trafficking of 2,3,4,5, 6-pentafluorodihydrotetramethylrosamine, a novel fluorogenic indicator of cellular oxidative activity

The trafficking of 2,3,4,5,6-pentafluorodihydrotetramethylrosamine (PF-H(2)TMRos, also known as RedoxSensor Red), a new fluorogenic indicator for oxidative activity, was evaluated in a contact-inhibited cell line, normal rat kidney fibroblast (NRK-49F), using quantitative fluorescence microscopy. After cells were incubated with 1-5 microM dye at 37 degrees C for 10 to 30 min, fluorescent staining of its oxidized product (PF-TMRos) distributed in mitochondria and/or lysosomes. This distribution pattern varied depending on the proliferation state of cells. In proliferating cells, PF-H(2)TMRos was internalized through a nonendocytic pathway, then oxidized in the cytosol, followed by immediate targeting to active mitochondria, resulting in fluorescent staining in this organelle. Photo-oxidation experiments demonstrated that PF-H(2)TMRos is not directly transported to mitochondria. On the contrary, in contact-inhibited cells whose proliferation is inhibited, PF-H(2)TMRos enters cells and is transported to lysosomes before it is oxidized. This results in lysosomal rather than mitochondrial staining. In both proliferating and quiescent cell states, subcellular distribution of the oxidized dye PF-TMRos can be altered by treatment with an oxidant (hydrogen peroxide) or an antioxidant (N-acetyl-L-cysteine), indicating a regulatory relationship between cell proliferation and oxidative activity. In solution assay, this probe can be oxidized by a broad spectrum of oxidizing species including horseradish peroxidase, hydrogen peroxide and horseradish peroxidase, cytochrome c, cytochrome c and hydrogen peroxide, superoxide and hydrogen peroxide, nitric oxide (or nitrite), peroxynitrite, and lipid hydroperoxide. Based on its subcellular distribution and its oxidation by a broad range of oxidizing species, PF-H(2)TMRos is demonstrated to be a novel indicator for cellular oxidative stresses.     

Protective effects of curcumin on biochemical and molecular changes in sodium arsenite‐induced oxidative damage in embryonic fibroblast cells

The present study was aimed at determining the oxidative damage caused by sodium arsenite in 3T3 fibroblast cells and the possible protective role of curcumin (Cur) against sodium arsenite toxicity. Embryonic fibroblast cells were exposed to sodium arsenite (0.01, 0.1, 1, and 10 μM) in the presence and absence of Cur (2.5 μM) for 24 hours. Cell viability, cytotoxicity, lipid peroxidation, hydroxyl radical, hydrogen peroxide, antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione‐S‐transferase) and expression levels of antioxidant genes (superoxide dismutase, catalase, and glutathione peroxidase) were measured in embryonic fibroblast cells. Results demonstrated that sodium arsenite directly affects antioxidant enzymes and genes in 3T3 embryonic fibroblast cells and induces oxidative damage by increasing the amount of hydrogen peroxide, hydroxyl radical, and lipid peroxidation in the cell. Furthermore, the study indicated that Cur might be a potential ameliorative antioxidant to protect the fibroblast cell toxicity induced by sodium arsenite.     

Role of Oxidative Damage in Friedreich's Ataxia

Plasma malondialdehyde (MDA) levels were raised in Friedreich's ataxia (FRDA) patients. These levels correlated with increasing age and disease duration, suggesting lipid peroxidation increased with disease progression. Using fibroblasts from FRDA patients we observed that GSH levels and aconitase activities were normal, suggesting their antioxidant status was unchanged. When exposed to various agents to increase free radical generation we observed that intracellular superoxide generation induced by paraquat caused enhanced oxidative damage. This correlated with the size of the GAA1 expansion, suggesting decreased frataxin levels may render the cells more vulnerable to mild oxidative stress. More severe oxidative stress induced by hydrogen peroxide caused increased cell death in FRDA fibroblasts but was not significantly different from control cells. We propose that abnormal respiratory chain function and iron accumulation may lead to a progressive increase in oxidative damage, but increased sensitivity to free radicals may not require detectable respiratory chain dysfunction.     

The flavonoids, quercetin and isorhamnetin 3-O-acylglucosides diminish neutrophil oxidative metabolism and lipid peroxidation.

Two natural flavonoids, quercetin and isorhamnetin 3-O-acylglucosides, were examined for their inhibitory influence on the in vitro production and release of reactive oxygen species in polymorphonuclear neutrophils (PMNs). The generation of superoxide radical, hydrogen peroxide and hypochlorous acid were measured by, respectively, cytochrome c reduction, dichlorofluorescin oxidation and taurine chlorination. Membrane lipid oxidation was studied by the thiobarbituric acid method in mouse spleen microsomes. The addition of flavonoids at the concentration range 1-100 microM inhibited PMNs oxidative metabolism and lipid peroxidation in a dose-dependent manner. The results suggest that these flavonoids suppress the oxidative burst of PMNs and protect membranes against lipid peroxidation.     

Role of calcium in AMF-mediated alleviation of the adverse impacts of cadmium stress in Bassia indica [Wight] A.J. Scott

The aim of this study was to evaluate cadmium stress induced changes in the growth, lipid peroxidation and antioxidant activity of Bassia indica associated with arbuscular mycorrhizal fungi (AMF) and their amelioration by calcium application. Cadmium stress can cause alterations in the physiological and biochemical processes in plants. A calcium application combined with an AMF treatment resulted in the reduction of lipid peroxidation and the production of hydrogen peroxide, thereby mediating the mitigation of cadmium induced oxidative stress. The activity of antioxidant enzymes increased with cadmium application, whereas AMF inoculation combined with a calcium application further enhanced their activity. An increase in the content of non-enzymatic antioxidants such as ascorbate, reduced glutathione (GSH), oxidized glutathione (GSSG) and S-nitrosoglutathione (GSNO) in AMF-inoculated and calcium-treated plants further suggests their role in strengthening the antioxidant defense system that results in maintained growth. The application of calcium combined with the AMF treatment caused a significant reduction in lipid peroxidation and in the production of hydrogen peroxide, thereby mediating the mitigation of the cadmium induced oxidative stress. Increased proline accumulation was clearly evident in stressed plants, and the calcium application as well as the AMF inoculation further induced proline synthesis, thereby providing efficient protection against cadmium stress by increasing the maintenance of the systemic resistance criteria.     

Inhibition of the Fenton reaction by the protein caeruloplasmin and other copper complexes. Assessment of ferroxidase and radical scavenging activities

The copper-containing protein caeruloplasmin is an important biological extracellular protein. By catalysing the oxidation of ferrous ions to the ferric state (ferroxidase activity) it can inhibit lipid peroxidation and the Fenton reaction. This activity is readily destroyed by heat-denaturation. When a ferric-EDTA complex is added to hydrogen peroxide, OH X radicals are formed in a reaction inhibitable by superoxide dismutase (SOD). This reaction is also inhibited by caeruloplasmin both before and after heat-denaturation, suggesting a non-catalytic scavenging role for the protein. A combination of ferroxidase and radical scavenging activities in fluids containing iron complexes and hydrogen peroxide, but no SOD or catalase, would make caeruloplasmin an important extracellular antioxidant.     

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.