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.     

The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.     

Comparison of the ability of ferric complexes to catalyze microsomal chemiluminescence, lipid peroxidation, and hydroxyl radical generation

The interaction of microsomes with iron and NADPH to generate active oxygen radicals was determined by assaying for low level chemiluminescence. The ability of several ferric complexes to catalyze light emission was compared to their effect on microsomal lipid peroxidation or hydroxyl radical generation. In the absence of added iron, microsomal light emission was very low; chemiluminescence could be enhanced by several cycles of freeze-thawing of the microsomes. The addition of ferric ammonium sulfate, ferric-citrate, or ferric-ADP produced an increase in chemiluminescence, whereas ferric-EDTA or -diethylenetriaminepentaacetic acid (detapac) were inhibitory. The same response to these ferric complexes was found when assaying for malondialdehyde as an index of microsomal lipid peroxidation. In contrast, hydroxyl radical generation, assessed as oxidation of chemical scavengers, was significantly enhanced in the presence of ferric-EDTA and -detapac and only weakly elevated by the other ferric complexes. Ferric-desferrioxamine was essentially inert in catalyzing any of these reactions. Chemiluminescence and lipid peroxidation were not affected by superoxide dismutase, catalase, or competitive hydroxyl radical scavengers whereas hydroxyl radical production was decreased by the latter two but not by superoxide dismutase. Chemiluminescence was decreased by the antioxidants propylgallate or glutathione and by inhibiting NADPH-cytochrome P-450 reductase with copper, but was not inhibited by metyrapone or carbon monoxide. The similar pattern exhibited by ferric complexes on microsomal light emission and lipid peroxidation, and the same response of both processes to radical scavenging agents, suggests a close association between chemiluminescence and lipid peroxidation, whereas both processes can be readily dissociated from free hydroxyl radical generation by microsomes.     

Antioxidant protection by haemopexin of haem-stimulated lipid peroxidation.

Haem (ferrous protoporphyrin IX) is a reactive low-molecular-mass form of iron able to participate in oxygen-radical reactions that can lead to the degradation of proteins, lipids, carbohydrates and DNA. Oxygen-radical reactions are likely to occur upon tissue damage. Extracellular fluids rely on antioxidant mechanisms different from those found inside the cell, and circulating proteins limit radical reactions by converting pro-oxidant forms of iron into less-reactive forms. Of the compounds tested, only apohaemopexin and the chain-breaking antioxidant butylated hydroxytoluene inhibited (by more than 90%) haemin-stimulated peroxidation as measured by formation of conjugated dienes, thiobarbituric acid-reactive material from linolenic acid or peroxidation-induced phospholipid fluorescence. Haptoglobin, the haemoglobin-binding serum protein, was ineffective. Conversely, only haptoglobin significantly inhibited haemoglobin-stimulated lipid peroxidation. Iron-salt-induced lipid peroxidation was inhibited only by apotransferrin and the iron-chelator desferrioxamine. All lipid peroxidations were inhibited by the radical scavengers butylated hydroxytoluene and propyl gallate. These findings support the concept that transport and conservation of body iron stores are closely linked to antioxidant protection.     

Alloxan- and glutathione-dependent ferritin iron release and lipid peroxidation

The diabetogenic action of alloxan is believed to involve oxygen free radicals and iron. Incubation of glutathione (GSH) and alloxan with rat liver ferritin resulted in release of ferrous iron as assayed by spectrophotometric detection of ferrous-bathophenanthroline complex formation. Neither GSH nor alloxan alone mediated iron release from ferritin. Superoxide dismutase (SOD) and catalase did not affect initial rates of iron release whereas ceruloplasmin was an effective inhibitor of iron release. The reaction of GSH with alloxan resulted in the formation of the alloxan radical which was detected by ESR spectroscopy and by following the increase in absorbance at 310nm. In both instances, the addition of ferritin resulted in diminished alloxan radical detection. Incubation of GSH, alloxan, and ferritin with phospholipid liposomes also resulted in lipid peroxidation. Lipid peroxidation did not occur in the absence of ferritin. The rates of lipid peroxidation were not affected by the addition of SOD or catalase, but were inhibited by ceruloplasmin. These results suggest that the alloxan radical releases iron from ferritin and indicates that ferritin iron may be involved in alloxan-promoted lipid peroxidation.     

Selective in vitro antioxidant properties of bisphosphonates

The aim of this study was to investigate the in vitro antioxidant profile of different bisphosphonates. Bisphosphonates were tested for their xanthine oxidase and microsomal lipid peroxidation inhibiting capacity. Furthermore, the effect of these different compounds on DPPH, a stable radical, was investigated. Clodronate, risedronate, and pyrophosphate were further tested for their hydroxyl radical scavenging activity. None of the tested compounds showed xanthine oxidase inhibiting activity or DPPH scavenging activity. All the tested bisphosphonates exhibited inhibiting capacities on the microsomal lipid peroxidation. The hydroxyl radical scavenging activity was dependent on the order of adding the different reagents and was highest for risedronate. Bisphosphonates possess an inhibiting activity on the microsomal lipid peroxidation and the Fenton reaction. In these reactions iron plays an important role suggesting that the selective in vitro antioxidant properties of the bisphosphonates are due to their iron chelating characteristics.     

Role of hydroxyl radical in superoxide induced microsomal lipid peroxidation: Protective effect of anion channel blocker

Treatment of bovine pulmonary artery smooth muscle microsomes with the superoxide radical generating system hypoxanthine plus xanthine oxidase stimulated iron release, hydroxyl radical production and lipid peroxidation. Pretreatment of the microsomes with deferoxamine or dime thy lthiourea markedly inhibited lipid peroxidation, and prevented hydroxyl radical production without appreciably altering iron release. The superoxide radical generating system did not alter the ambient superoxide dismutase activity. However,addition of exogenous superoxide dismutase prevented superoxide radical induced iron release,hydroxyl radical production and lipid peroxidation. Simultaneous treatment of the microsomes with deferoxamine, dimethylthiourea or superoxide dismutase prevented hydroxyl radical production and liqid peroxidation. While deferoxamine or dimethylthiourea did not appreciably alter iron release, superoxide dismutase prevented iron release. However, addition of deferoxamine, dimethylthiourea or superoxide dismutase even 2 min after treatment did not significantly inhibit lipid peroxidation, hydroxyl radical production and iron release. Pretreatment of microsomes with the anion channel blocker 4,4’- dithiocyano 2,′- disulphonic acid stilbine did not cause any discernible change in chemiluminiscence induced by the superoxide radical generating system but markedly inhibited lipid peroxidation without appreciably altering iron release and hydroxial radical production.     

Ferritin, Lipid Peroxidation and Redox-Cycling Xenobiotics

A number of xenobiotics are toxic because they rcdox cycle and generate free radicals. Interaction with iron, either to produce reactive species such as the hydroxyl radical, or to promote lipid peroxidation, is an important factor in this toxicity. A potential biological source of iron is ferritin. The cytotoxic pyrimidines, dialuric acid, divicine and isouramil, readily release iron from ferritin and promote ferritin-dependent lipid peroxidation. Superoxide dismutase and GSH, which maintain the pyrimidines in their reduced form, enhance both iron release and lipid peroxidation. Microsomes plus NADPH can reduce a number of iron complexes, although not ferritin. Reduction of Adriamycin. paraquat or various quinones to their radicals by the microsomes enhances reduction of the iron complexes, and in some cases, enables iron release from ferritin. Adriamycin stimulates iron-dependent lipid peroxidation of the microsomes. Ferritin can provide the iron, and peroxidation is most pronounced at low PO₂. Compiexing agents that supress intraccllular iron reduction and lipid peroxidation may protect against the toxicity of Adriamycin.     

Noninvasive evaluation of in vivo free radical reactions catalyzed by iron using in vivo ESR spectroscopy.

The noninvasive, real time technique of in vivo electron spin resonance (ESR) spectroscopy was used to evaluate free radical reactions catalyzed by iron in living mice. The spectra and signal decay of a nitroxyl probe, carbamoyl-PROXYL, were observed in the upper abdomen of mice. The signal decay was significantly enhanced in mice subcutaneously loaded with ferric citrate (0.2 micromol/g body wt) and the enhancement was suppressed by pre-treatment with either desferrioxamine (DF) or the chain breaking antioxidant Trolox, but only slightly suppressed by the hydroxyl radical scavenger DMSO. To determine the catalytic form of iron, DF was administered at different times with respect to iron loading: before, simultaneously, and after 20 and 50 min. The effect of DF on signal decay, liver iron content, iron excretion, and lipid peroxidation (TBARs) depended on the time of the treatment. There was a good correlation between the signal decay, iron content, and lipid peroxidation, indicating that "chelatable iron" contributed to the enhanced signal decay. The nitroxyl probe also exhibited in vivo antioxidant activity, implying that the process responsible for the signal decay of the nitroxyl probe is involved in free radical oxidative stress reactions catalyzed by iron.     

Iron-catalyzed reactions may be responsible for the biochemical and biological effects of asbestos

The most carcinogenic forms of asbestos contain iron to levels as high as 36% by weight and catalyze many of the same biochemical reactions that freshly prepared solutions of iron do, i.e. oxygen consumption, generation of reactive oxygen species, lipid peroxidation and DNA damage. The participation of iron from asbestos in these reactions has been demonstrated using the iron chelator desferrioxamine B which inhibits iron-catalyzed reactions. Iron appears to be redox active on the asbestos fiber, but chelation and subsequent iron mobilization from asbestos by a variety of chelators, e.g. citrate, EDTA or nitrilotriacetate, makes the iron more redox active resulting in greater oxygen consumption and production of oxygen radicals in the presence of reducing agents. Iron also appears to be important for some of the asbestos-dependent biological effects on tissues or cells in culture, such as phagocytosis, cytotoxicity, lipid peroxidation and DNA damage. Therefore, redox cycling of iron to generate oxygen radicals at the surface of the fiber and/or in solution, as mobilized, low molecular weight chelates, may be very important in eliciting some of the biological effects of asbestos in vivo.     

The chelation of nonheme iron within sickle erythrocytes by the hydroxypyridinone chelator CP094.

Nonheme, nonferritin iron has been detected in membrane preparations from sickle erythrocytes and has been suggested to catalyze free radical reactions in these cells contributing to the development of membrane oxidation. In this study the hydroxypyridinone iron chelator, CP094, currently being evaluated as a potentially therapeutic chelator, and desferrioxamine have been studied for their abilities to chelate the nonheme iron within intact sickle erythrocytes under physiological conditions. The results suggest that CP094 can enter sickle erythrocytes, chelate nonheme iron and suppress membrane lipid peroxidation within a timescale in which desferrioxamine does not enter the cells. Suppression of lipid peroxidation showed no protective effect in an in vitro system inducing the formation of irreversibly sickled cells.     

Effect of pH and oxalate on hydroquinone-derived hydroxyl radical formation during brown rot wood degradation

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe2+ and H2O2 in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe3+ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe3+ at a rate constant of 4.5 x 10(3) M(-1)s(-1) at pH 4.0. Fe2+ is also very stable at a low pH. H2O2 generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe3+. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe3+. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 micro M), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.     

Induction of oxalate binding by lipid peroxidation in rat kidney mitochondria.

Enhanced oxalate binding (150-180% of control) was observed in kidney, liver, brain and heart, after subjecting them to lipid peroxidation in presence of iron. Kidney mitochondrial oxalate binding was stimulated by different promoters, and the order of stimulation was Fe2+ greater than t-BH greater than ascorbic acid greater than Fe3+ greater than H2O2. Oxalate binding was maximum when iron concentration was between 1-2 mM. The iron-induced oxalate binding was inhibited by reduced glutathione, beta-mercaptoethanol, alpha-tocopherol and hydroxyl ion scavengers, histidine and mannitol. Catalase inhibited both Fe(2+)-H2O2 induced oxalate binding and lipid peroxidation reactions, suggesting that the induced oxalate binding in mitochondria was mediated through the hydroxyl radical reaction mechanism.     

Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation

The generation of oxygen radicals and the process of lipid peroxidation have become a focus of attention for investigators in the fields of central nervous system (CNS) trauma and stroke (e.g., ischemia). Considering our level of understanding of free radical and lipid peroxidation chemistry, absolute proof for their involvement in the pathophysiology of traumatic and ischemic damage to the CNS has been meager. While direct, unequivocal evidence for the participation of free radicals and lipid peroxidation as primary contributors to the death of neuronal tissue waits to be established, numerous recent studies have provided considerable support for the occurrence of free radical and lipid peroxidation reactions in the injured or ischemic CNS. In addition, the pharmacological use of antioxidants and free radical scavengers in the treatment of experimental CNS trauma and ischemia has provided convincing, although indirect evidence, for the involvement of oxygen radicals and lipid peroxidation in these conditions. The intent of this and its companion paper is to review: 1) the biochemical processes which may give rise to free radical reactions in the CNS, 2) the environment of the ischemic cell as it may affect the generation of oxygen radicals and the catalysis of lipid peroxidation reactions, 3) the evidence for the involvement of free radical mechanisms in CNS trauma and ischemia, and 4) the pathophysiological consequences of these phenomena.     

Steady magnetic fields effect on lipid peroxidation kinetics

The effect of steady magnetic fields (ranging from 0 to 280 mT) has been investigated on the kinetics of non-enzymatic lipid peroxidation occurring in a model system consisting of liposomes obtained from 1, 2-dioleoylphosphatidylcholine by oxygen consumption. The process was found to be accelerated by weak steady magnetic fields. A computer simulation method was employed to detect the reactions that govern the process kinetics, to elucidate magneto-sensitive stages (initiation and reduction of iron(III), as well as lipid peroxide radical recombination) and to determine their rate constants at various external magnetic fields. The kinetics of peroxidation of lipid cell membranes have been modeled mathematically at oxygen and ‘free’ iron concentrations close to those in the cells and also at increased free iron concentrations at different external magnetic field values.     

An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation

A model lipid peroxidation system dependent upon the hydroxyl radical, generated by Fenton's reagent, was compared to another model system dependent upon the enzymatic generation of superoxide by xanthine oxidase. Peroxidation was studied in detergent-dispersed linoleic acid and in phospholipid liposomes. Hydroxyl radical generation by Fenton's reagent (FeCl₂ + H₂O₂) in the presence of phospholipid liposomes resulted in lipid peroxidation as evidenced by malondialdehyde and lipid hydroperoxide formation. Catalase, mannitol, and Tris-Cl were capable of inhibiting activity. The addition of EDTA resulted in complete inhibition of activity when the concentration of EDTA exceeded the concentration of Fe²⁺. The addition of ADP resulted in slight inhibition of activity, however, the activity was less sensitive to inhibition by mannitol. At an ADP to Fe²⁺ molar ratio of 10 to 1, 10 mm mannitol caused 25% inhibition of activity. Lipid peroxidation dependent on the enzymatic generation of superoxide by xanthine oxidase was studied in liposomes and in detergent-dispersed linoleate. No activity was observed in the absence of added iron. Activity and the apparent mechanism of initiation was dependent upon iron chelation. The addition of EDTA-chelated iron to the detergent-dispersed linoleate system resulted in lipid peroxidation as evidenced by diene conjugation. This activity was inhibited by catalase and hydroxyl radical trapping agents. In contrast, no activity was observed with phospholipid liposomes when iron was chelated with EDTA. The peroxidation of liposomes required ADP-chelated iron and activity was stimulated upon the addition of EDTA-chelated iron. The peroxidation of detergent-dispersed linoleate was also enhanced by ADP-chelated iron. Again, this peroxidation in the presence of ADP-chelated iron was not sensitive to catalase or hydroxyl radical trapping agents. It is proposed that initiation of superoxide-dependent lipid peroxidation in the presence of EDTA-chelated iron occurs via the hydroxyl radical. However, in the presence of ADP-chelated iron, the participation of the free hydroxyl radical is minimal.     

Acidic pH amplifies iron-mediated lipid peroxidation in cells

The goal of our study was to investigate the mechanism by which changes in extracellular pH influence lipid peroxidation processes. Ferrous iron can react with hydroperoxides, via a Fenton-type reaction, to initiate free radical chain processes. Iron is more soluble at lower pH values, therefore we hypothesized that decreasing the environmental pH would lead to increased iron-mediated lipid peroxidation. We used Photofrin, a photosensitizer that produces singlet oxygen, to introduce lipid hydroperoxides into leukemia cells (HL-60, K-562, and L1210). Singlet oxygen reacts with the PUFA of cells producing lipid hydroperoxides. Using EPR spin trapping with POBN, free radical formation from HL-60 cells was only detected when Photofrin, light, and ferrous iron were present. Free radical formation increased with increasing iron concentration; in the absence of extracellular iron, radical formation was below the limit of detection and lipid hydroperoxides accumulated in the membrane. In the presence of iron, lipid-derived radical formation in cells is pH dependent; the lower the extracellular pH (7.5-5.5), the higher the free radical flux; the lower the pH, the greater the membrane permeability induced in K-562 cells, as determined by trypan blue dye exclusion. These data demonstrate that lipid peroxidation processes, mediated by iron, are enhanced with decreasing extracellular pH. Thus, acidic pH not only releases iron from "safe" sites, but this iron will also be more damaging.     

Superoxide-dependent formation of hydroxyl radicals and lipid peroxidation in the presence of iron salts. Detection of `catalytic'' iron and anti-oxidant activity in extracellular fluids

Synovial fluid from rheumatoid patients and normal cerebrospinal fluid contains micromolar concentrations of non-protein-bound iron salts that can promote lipid peroxidation and also the superoxide-dependent formation of hydroxyl radicals from hydrogen peroxide. These iron catalysts of oxygen radical reactions cannot be detected by conventional assays unless interfering high-molecular-weight substances, probably proteins, are removed by ultrafiltration or inactivated by exposure to low pH values. The bleomycin assay for ;catalytic' iron [Gutteridge, Rowley & Halliwell (1981) Biochem. J.199, 263-265] does not suffer from these artifacts.     

Lipid peroxidation and tissue injury by ferric citrate in paraquat-intoxicated mice

To determine whether iron toxicity is caused by iron-catalyzed radical production, the in vivo effect of ferric citrate was studied in paraquat-intoxicated mice. Intraperitoneally injected Fe3+-citrate complex was distributed mainly in the liver and kidney, and promoted lipid peroxidation, as measured by expiratory ethane in both normal and paraquat-intoxicated mice. Plasma glutamic-oxaloacetic transaminase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) activity increased significantly only in paraquat and Fe3+-citrate-injected mice (PFe group). The rate of ethane production increased prior to the elevation of plasma glutamic-oxaloacetic transaminase levels, and was greater in the PFe group than in the mice, that were injected Fe3+-citrate alone. Pretreatment of animals with desferrioxamine mesylate inhibited both ethane production and elevation of plasma glutamic-oxaloacetic transaminase levels in the PFe group. Administration of 100% oxygen or glucose, which is expected to increase cellular NADPH, to the PFe group further elevated the plasma glutamic-oxaloacetic transaminase level, but had little effect on ethane production, indicating that tissue injury occurs independently of lipid peroxidation. These results suggest that iron toxicity is due to radical production and that, although iron stimulated lipid peroxidation, it might not be the only cause of tissue injury.     

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.