Mechanism of the Interaction of Superoxide Ion and Ascorbate with Anthracycline Antibiotics

The interaction of superoxide ion and ascorbate anion with anthracycline antibiotics (adriamycin and aclacinimycin A) as well as with their Fe³⁺ complexes has been studied in aprotic and protic media. It was found that both superoxide and ascorbate reduce anthracyclines to deoxyaglycons via a one-electron transfer mechanism under all conditions studied. The reaction of ascorbate anion with adriamycin and aclacinomycin A in aqueous solution proceeded only in the presence of Fe³⁺ ions; it is supposed that an active catalytic species was Fe³⁺ adriamycin. It is also supposed that the reduction of anthracycline antibiotics by O,⁷ and ascorbate in cells may increase their anticancer effect.     

Investigation of the interaction of superoxide ion with adriamycin and the possible origin of cardiotoxicity of the anthracycline anticancer antibiotics

Interaction of superoxide ion with adriamycin in an aprotic medium has been studied. It was shown that superoxide ion reacts irreversibly with adriamycin, giving a diamagnetic product (the dimer or oligomer of semiquinone) which can be reoxidized to adriamycin. This product was also obtained when adriamycin reacted with benzosemiquinone, ubisemiquinone, and the semiquinones of tocopherylquinone and vitamin K₁. It is suggested that the cardiotoxicity of adriamycin and other anthracycline anticancer antibiotics is caused by the high elcctron-attracting properties of these antibiotics, while the ability of natural quinones to reduce cardiotoxicity and to induce recovery of respiration in mitochondria is due to their interaction with the semiquinone states of the antibiotics.     

Purification of Oat Debranching Enzyme and Occurrence of Inactive Debranching Enzyme in Cereals

The interaction of some anthracycline antibiotics (adriamycin, daunomycin, aclacinomycin-A) with bacteriophage ?X174 was investigated. Adriamycin and daunomycin inactivated the infectivity of both free ?X174 phage and naked single-stranded ?X174 DNA without DNA strand scission, but aclacinomycin-A did not show this action. The phage inactivation reaction was reversibly inhibited by Superoxide dismutase, catalase or other oxygen radical scavengers. The inactivation of ?X174 by adriamycin and aclacinomycin-A was stimulated by the addition of Cu²⁺, while the ?X174 inactivation by daunomycin was inhibited by the addition of Cu²⁺. The ?X174 inactivation by adriamycin and aclacinomycin-A in the presence of Cu²⁺ was caused by degradation of DNA, and this inactivation reaction was inhibited irreversibly by oxygen radical scavengers. These results indicate that anthracycline antibiotics bind to ?X174 DNA in the form of free radicals and that during the auto-oxidation of these antibiotics in the presence of Cu²⁺, oxygen radicals were generated to cause the degradation of ?X174 DNA.     

Interaction of anthracycline antibiotics with actin and heavy meromyosin.

For the purpose of elucidating the biochemical mechanism of anthracycline cardiomyopathy, the interaction with actin and heavy meromyosin (HMM) was studied. HMM and acto-HMM Mg²⁺-ATPase reactions were inhibited by daunorubicin and adriamycin; but not significantly by aclacinomycin A. The three antibiotics induced G-actin polymerization. Difference absorption spectra showed a direct interaction of adriamycin or aclacinomycin A with actin or HMM. Equilibrium dialysis and spectrofluorometric studies indicated that actin monomer possesses one binding site for adriamycin or aclacinomycin A with the same order of association constants (1.4 – 7.2 × 10⁴ M^?1). Adriamycin exhibited significantly higher affinity for HMM than aclacinomycin A.     

An Unusual Nad(P)H-Dependent Oi-Generating Redox System in Hepatoma 22a Nuclei

Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes⁷ which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.     

In vitro antioxidant activity of silymarin

Silymarin, a known standardized extract obtained from seeds of Silybum marianum is widely used in treatment of several diseases of varying origin. In the present paper, we clarified the antioxidant activity of silymarin by employing various in vitro antioxidant assay such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH·) scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by Fe³⁺ ? Fe²⁺ transformation method and Cuprac assay, superoxide anion radical scavenging by riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe²⁺) chelating activities. Silymarin inhibited 82.7% lipid peroxidation of linoleic acid emulsion at 30 μg/mL concentration; butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox indicated inhibition of 83.3, 82.1, 68.1 and 81.3% on peroxidation of linoleic acid emulsion at the same concentration, respectively. In addition, silymarin had an effective DPPH· scavenging, ABTS^√+ scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe³⁺) reducing power by Fe³⁺ ? Fe²⁺ transformation, cupric ions (Cu²⁺) reducing ability by Cuprac method, and ferrous ions (Fe²⁺) chelating activities. Also, BHA, BHT, α-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. Moreover, this study, which clarifies antioxidant mechanism of silymarin, brings new information on the antioxidant properties of silymarin. According to the present study, silymarin had effective in vitro antioxidant and radical scavenging activity. It could be used in the pharmacological and food industry because of its antioxidant properties.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects

Iron and copper ions, in their unbound form, may lead to the generation of reactive oxygen species via Haber–Weiss and/or Fenton reactions. In addition, it has been shown that copper ions can irreversibly and non-specifically bind to thiol groups in proteins. This non-specific binding property has not been fully addressed for iron ions. Thus, the present study compares both the pro-oxidant and the non-specific binding properties of Fe³⁺ and Cu²⁺, using rat liver cytosol and microsomes as biological systems. Our data show that, in the absence of proteins, Cu²⁺/ascorbate elicited more oxygen consumption than Fe³⁺/ascorbate under identical conditions. Presence of cytosolic and microsomal protein, however, differentially altered oxygen consumption patterns. In addition, Cu²⁺/ascorbate increased microsomal lipid peroxidation and decreased cytosolic and microsomal content of thiol groups more efficiently than Fe³⁺/ascorbate. Finally, Fe³⁺/ascorbate and Cu²⁺/ascorbate inhibited in different ways cytosolic and microsomal glutathione S-transferase (GST) activities, which are differentially sensitive to oxidants. Moreover, in the absence of ascorbate, only Cu²⁺ decreased the content of cytosolic and microsomal thiol groups and inhibited cytosolic and microsomal GST activities. Catechin partially prevented the damage to thiol groups elicited by Fe³⁺/ascorbate and Cu²⁺/ascorbate but not by Cu²⁺ alone. N-Acetylcysteine completely prevented the damage elicited by Cu²⁺/ascorbate, Fe³⁺/ascorbate and Cu²⁺ alone. N-Acetylcysteine also completely reversed the damage to thiol groups elicited by Fe³⁺/ascorbate, partially reversed that of Cu²⁺/ascorbate but failed to reverse the damage promoted by Cu²⁺ alone. Our data are discussed in terms to the potential damage that the accumulation of iron and copper ions can promote in biological systems.     

A peptide-mediated and hydroxyl radical HO*-involved oxidative degradation of cellulose by brown-rot fungi

A special low-molecular-weight peptide named Gt factor, was isolated and purified from the extracellular culture of brown-rot fungi Gloeophyllum trabeum via gel filtration chromatography and HPLC. It has been shown to reduce Fe³⁺ to Fe²⁺. Electron paramagnetic resonance (EPR) spectroscopy revealed Gt factor was able to drive H₂O₂ generation via a superoxide anion O₂ ^.- intermediate and mediate the formation of hydroxyl radical HO^. in the presence of O₂. All the results indicated that Gt factor could oxidize the cellulose, disrupt the inter- and intrahydrogen bonds in cellulose chains by a HO^. -involved mechanism. This resulted in depolymerization of the cellulose, which made it accessible for further enzymatic hydrolysis.     

Effects of Melanins on Lipid Peroxidation

Effects of melanins obtained from cultured Cladosporium cladosporidae fungi and Alpha grape on Fe²⁺-induced, Fe²⁺–ascorbate-induced, and NADPH-induced lipid peroxidation in rat liver, brain, and eye were studied. Melanins were shown to inhibit the accumulation of lipid peroxidation products in vitro. The inhibitory effects of melanins were not due to direct interactions of these pigments with superoxide anion (O ₂ ). However, melanins may interact with other free radicals. Melanins were demonstrated to have the ability to oxidize NADPH, which is probably one of the mechanisms of their antioxidant effects.     

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase requires both Fe²⁺ ion and ascorbate and the level of activation is dependent on the concentrations of Fe²⁺ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe²⁺ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3mM, respectively. Several common divalent metal ions, including Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe²⁺ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe²⁺/ascorbate system is a specific process. Moreover, H₂O₂, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe²⁺/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe²⁺/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.     

Study of the Kinetics of Oxidation of Monophenols by Tyrosinase. The Effect of Reducers

Kinetics of oxidation of monophenols by tyrosinase from the fungus Aspergillus flavipes 56003 and the effect of Fe²⁺, serine, and ascorbic acid on this reaction were studied. The effectors were shown to accelerate the oxidation of monophenols, decreasing the lag-time of the reaction. It is assumed that the activation of the tyrosinase in the presence of Fe²⁺ is due to a direct reduction of the active site copper ions. Serine and ascorbic acid are supposed to affect the reaction of quinone transformation. The activation of the enzyme in the presence of Fe²⁺ suggests that the oxidation of monophenols is an autocatalytic process.     

Effect of prooxidants on yeast mitochondria

Tightly coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts were used in this study. The two yeasts are aerobes containing the fully competent respiratory chain with three energy conservation sites. Interaction of the yeast mitochondria with prooxidants (diamide, menadione, oxaloacetate, phenylarsine oxide, hydrogen peroxide, t-butyl peroxide, and ascorbate plus Fe²⁺) was studied. The prooxidants, depending on their chemical nature, either caused uncoupling (e.g., activated state 4 respiration) or inhibited oxidation of respiratory substrates. All of the agents dissipated the membrane potential without megachannel formation (no large-scale swelling of mitochondria was observed). Except for combined application of ascorbate and Fe²⁺, the prooxidant-induced decrease in the membrane potential was specifically prevented by ATP, even in the cases when classic antioxidants, e.g., N-acetylcysteine, were ineffective. No permeabilization of yeast mitochondria was observed under concerted action of prooxidants and Ca²⁺, suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with Ca²⁺ uptake.     

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase requires both Fe²⁺ ion and ascorbate and the level of activation is dependent on the concentrations of Fe²⁺ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe²⁺ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3mM, respectively. Several common divalent metal ions, including Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe²⁺ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe²⁺/ascorbate system is a specific process. Moreover, H₂O₂, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe²⁺/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe²⁺/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.Abbreviations MAPK mitogen-activated protein kinase - MCO metal ion-catalyzed oxidation - kinase F_A the activating factor of ATP.Mg-dependent protein phosphatase - I₂ inhibitor-2 - EDTA ethylenediaminetetraacetic acid - MBP myelin basic protein     

Effects of iron chelates on the transferrin-free culture of rat dermal fibroblasts through active oxygen generation

Summary Effects of nonchelating and chelating agents at 10 mM on the serum-free culture of rat dermal fibroblasts were investigated. A strong iron-chelating agent, iminodiacetic acid (IDA), and a weak one, dihydroxyethylglycine (DHEG), decreased iron permeation into preconfluent fibroblasts. A weak iron-chelating agent, glycylglycine (GG), a nonchelating agent, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and human apotransferrin (10 μg/ml) increased the permeation with time. Iron may be essential for survival of fibroblasts because subconfluent fibroblasts exposed to 100 μM FeSO₄ in combination with transferrin, HEPES, or GG significantly decreased to release lactate dehydrogenase into the medium. Superoxide dismutase and dimethyl sulfoxide blocked the enzyme release, suggesting that superoxide and hydroxyl radical induce cellular damage but hydrogen peroxide (H₂O₂) generated by superoxide dismutation does not. GG significantly reduced H₂O₂ cytotoxicity. DHEG acted as a potent promoter of the iron-stimulated cellular damage if ascorbate or H₂O₂ was added to the medium. FeSO₄ and FeCl₃ (50 to 100 μM) individually combined with IDA maximally promoted fibroblast proliferation. Ascorbate increased formation of thiobarbituric acid-reactive substances from deoxyribose in the medium supplemented with FeSO₄ and either IDA or DHEG. Conversely, ascorbate decreased the formation in the medium with FeSO₄ and with or without other agents. Fibroblast proliferation may thus be stimulated through the active oxygen generation mediated by a redox-cycling between Fe³⁺ and Fe²⁺, which are dissolved in the medium at a high concentration, rather than through delivery of iron into the cells.     

A role for oxalic acid generation in ozone‐induced signallization in Arabidopis cells

Ozone (O₃) is an air pollutant with an impact increasingly important in our industrialized world. It affects human health and productivity in various crops. We provide the evidences that treatment of Arabidopsis thaliana with O₃ results in ascorbate‐derived oxalic acid production. Using cultured cells of A. thaliana as a model, here we further showed that oxalic acid induces activation of anion channels that trigger depolarization of the cell, increase in cytosolic Ca²⁺ concentration, generation of reactive oxygen species and cell death. We confirmed that O₃ reacts with ascorbate in the culture, thus resulting in production of oxalic acid and this could be part of the O₃‐induced signalling pathways that trigger programmed cell death.     

Clofibric acid or diethylmaleate supplemented diet decrease blood pressure in DOCA-salt treated male Sprague-Dawley rats - relation with liver antioxidant status

The effects of ascorbate and a-tocopherol as antioxidants and as co-operative factors against NADPH-dependent lipid peroxidation in human placental mitochondria have been studied. The addition of ascorbate at low concentration (up to 50 M) to the NADPH-generating system resulted in increasing lipid peroxidation and Fe³⁺ to Fe²⁺ reduction. High concentration of ascorbate (150 M), which produced maximal rate of ascorbate-dependent lipid peroxidation, was found to inhibit almost completely NADPH-dependent lipid peroxidation by maintaining too much iron in its reduced form. Either stimulatory or inhibitory effect of ascorbate on NADPH-dependent lipid peroxidation depends on the appropriate Fe³⁺/Fe²⁺ ratio. -Tocopherol caused a decrease of NADPH-dependent lipid peroxidation, inhibiting completely this process at 150 M concentration. The inhibitory effect of -tocopherol increased rapidly with the increasing ascorbate concentration, almost complete inhibition of NADPH-dependent lipid peroxidation being obtained at 25 M -tocopherol and 50 M ascorbate. This strong inhibitory combined effect of -tocopherol and ascorbate was independent of the Fe³⁺/Fe²⁺ ratio, as a-tocopherol is not able to reduce Fe³⁺ to Fe²⁺ under the conditions employed. These findings suggest that antioxidant effects of ascorbate in placental mitochondria are mediated by recycling of a-tocopherol rather than by strong reduction of Fe³⁺ to Fe²⁺. On the basis of the results obtained, we assume that adequate concentrations of a-tocopherol and ascorbate in placental tissue may prevent the release of lipid peroxide from placental mitochondria and therefore could be protective against the development of preeclampsia.     

How superoxide radical damages the cell

Summary Superoxide is considered to be poorly reactive, and cell damage has been attributed to HO· generated via the Haber-Weiss reaction. The function of O₂ ^– in this reaction is only to reduce Fe³⁺ to Fe²⁺. In vivo, however, superoxide could not out-compete cellular reductants such as glutathione, NADPH, and ascorbate, which makes the observed O₂ ^– toxicity rather puzzling. Little attention has been paid to the idea that, irrespective of its poor chemical reactivity, superoxide might be capable of interacting directly with specific intracellular targets; and that even the Haber-Weiss reaction might be a consequence of such direct interactions. This paper summarizes latest data that support the concept of such a mechanism.Abbreviation SOD Superoxide dismutase     

Biotransformation of D‐panthenol and its action in protection of activation of oxidizing processes in brain

It is a well known fact that 3H‐panthenol (PL) has a high bioavailability, so we studied its biotransformation and its protective action against lipoperoxide activation in homogenates and mitochondrial‐synaptosomal fraction (11 000 g) of rat brain. The lipoperoxidation was initialized by Fe²⁺‐ascorbate complex (Fe²⁺‐Asc). In experiments in vivo, after 30 min, we demonstrated accumulation of intermediate products of CoA biosynthesis – pantothenic acid (PA), phospho‐PA, and phosphopantetheine – in postmitochondrial fraction of brain, by using a HPLC technique. Addition of the PL (10 mm ) to brain hemispheres homogenates or mitochondrial‐synaptosomal fraction caused a remarkable reduction of malondialdehyde production. However, 30 min preincubation with the PL, but not with PA, was ineffective. The data obtained may be a reason for a high neuroprotective activity of PL in curing brain diseases with vessel or alcohol‐induced damages.