Release of iron from haemoglobin--a possible source of free radicals in diabetes mellitus

Increased blood glucose in diabetes mellitus stimulates nonenzymatic glycosylation of several proteins, including haemoglobin. Although iron is tightly bound to haemoglobin, it is liberated under specific circumstances yielding free reactive iron. Studies with purified haemoglobin from normal individuals and diabetic patients revealed that concentration of free iron was significantly higher in the latter cases and increased progressively with extent of the disease. In vitro glycosylation of haemoglobin also led to increase in release of iron from protein. This increase in free iron, acting as a Fenton reagent, might produce free radicals, which, in turn might be causing oxidative stress in diabetes.     

Lytic polysaccharide monooxygenases promote oxidative cleavage of lignin and lignin–carbohydrate complexes during fungal degradation of lignocellulose

Overcoming lignocellulosic biomass recalcitrance, especially the cleavage of cross-linkages in lignin–carbohydrate complexes (LCCs) and lignin, is essential for both the carbon cycle and industrial biorefinery. Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in fungal polysaccharide oxidative degradation. Nevertheless, comprehensive analysis showed that LPMOs from a white-rot fungus, Pleurotus ostreatus, correlated well with the Fenton reaction and were involved in the degradation of recalcitrant nonpolysaccharide fractions in this research. Thus, LPMOs participated in the extracellular Fenton reaction by enhancing iron reduction in quinone redox cycling. A Fenton reaction system consisting of LPMOs, hydroquinone, and ferric iron can efficiently produce hydroxy radicals and then cleave LCCs or lignin linkages. This finding indicates that LPMOs are underestimated auxiliary enzymes in eliminating biomass recalcitrance.     

Copper catechol-driven Fenton reactions and their potential role in wood degradation

Metals can potentially play a role in the non-enzymatic processes involved in wood biodegradation. Dihydroxybenzenes reduce Cu(II)–Cu(I), which then react with H₂O₂ driving a Fenton reaction. In this work the degradation of veratryl alcohol (VA), the simplest non-phenolic lignin model compound, via a cuprous Fenton reaction mediated by 1,2-dihydroxybenzene (catechol, CAT) was studied. A factorial experimental design was performed to assess the impact of several experimental variables including, pH, and CAT, CuCl₂ and H₂O₂ concentrations on VA degradation. Optimized conditions were determined using a response surface modeling methodology (RSM). The greatest amount of VA degradation occurred at a CAT:CuCl₂:H₂O₂ ratio of 0.287:0.313:4.062, a pH of 3.6. A time-course measurement for VA degradation was performed under these experimental conditions and after an 8 h reaction period, 31% of the VA was degraded. Under the same experimental conditions, VA degradation by an iron CAT-driven Fenton reaction was more effective than the copper CAT-driven Fenton reaction. In a similar experiment, carboxymethyl cellulose (CMC) depolymerization was also determined. Only the iron CAT-driven Fenton reaction was found to depolymerize CMC. We suggest that the greater redox potential of the Fe(III)CAT complex compared to the Cu(II)CAT complex would dictate that under most environmental conditions, degradation of VA would occur by the iron complex only. This research has important implications for the mechanisms of brown rot fungal degradation in wood because it eliminates a pathway that had previously been proposed as a mechanism explaining free radical generation in the oxidative depolymerization of cellulose in the cell wall.     

Activated bleomycin. A transient complex of drug, iron, and oxygen that degrades DNA

"Activated bleomycin" is an oxygenated iron drug complex which embodies the drug's DNA-cleaving activity. This activity is exercised on DNA, if present, but if DNA is absent, the drug itself is inactivated. Hyperfine interactions in the EPR spectra of activated bleomycin prepared with 57Fe(II) and 17O2 demonstrate the presence of iron as Fe(III) and of bound oxygen originating in dioxygen. Bleomycin can also be activated with Fe(III) and either H2O2 or ethyl hydroperoxide. These latter reactions do not produce a ferrous intermediate nor do they require O2. But O2 is required for the reaction of activated bleomycin with DNA to yield the malondialdehyde-like chromogens used to monitor DNA degradation. The attack on DNA is quantitatively concurrent with the decay of activated bleomycin, however generated.     

Iron Complexes and Their Reactivity in the Bleomycin Assay for Radical-Promoting Loosely-Bound Iron

The sensitivity of the bleomycin assay for loosely-bound iron depends on the concentration of bleomycin and ascorbic acid and the pH of the reaction. The non-haem-iron proteins transferrin, conalbumin and ferritin release iron at an acid pH value, whereas the haem-iron proteins release iron more readily at an alkaline pH. In addition, haem proteins are liable to release iron when peroxides are present. Organic peroxides and hydrogen peroxide can be produced during the bleomycin reaction leading to iron release from haem proteins. However, this can be prevented from reacting with bleomycin by adding zinc ions to the reaction following addition of the sample. Iron already bound to bleomycin is not displaced by zinc whereas zinc bound to bleomycin is not displaced by iron allowing 'free' and 'released' iron to be discriminated.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

Effect of chelating agents and metal ions on the degradation of DNA by bleomycin.

The degradation of DNA by bleomycin was studied in the absence and in the presence of added reducing agents, including 2-mercaptoethanol, dithiothreitol, reduced nicotinamide adenine dinucleotide phosphate, H2O2, and ascorbate, and in the presence of a superoxide anion generating system consisting of xanthine oxidase and hypoxanthine. In all cases, breakage of DNA was inhibited by low concentrations of chelators; where examined in detail, deferoxamine mesylate was considerably more potent than (ethylenedinitrilo)tetraacetic acid. Iron was found to be present in significant quantities in all reaction mixtures. Thus, the pattern of inhibition observed is attributed to the involvement of contaminating iron in the degradation of DNA by bleomycin. Cu(II), Zn(II), and Co(II) inhibit degradation of DNA by bleomycin and Fe(II) in the absence of added reducing agents. A model is proposed in which the degradation of DNA in these systems is dependent on the oxidation of an Fe(II)-bleomycin-DNA complex.     

Influence of tropolone on Poria placenta wood degradation

Fenton reactions are believed to play important roles in wood degradation by brown rot fungi. In this context, the effect of tropolone (2-hydroxycyclohepta-2,4,6-trienone), a metal chelator, on wood degradation by Poria placenta was investigated. Tropolone (50 micro M) strongly inhibits fungal growth on malt agar, but this inhibition could be relieved by adding iron salts. With an experimental system containing two separate parts, one supplemented with tropolone (100 micro M) and the other not, it was shown that the fungus is able to reallocate essential minerals from the area where they are available and also to grow in these conditions on malt-agar in the presence of tropolone. Nevertheless, even in the presence of an external source of metals, P. placenta is not able to attack pine blocks impregnated with tropolone (5 mM). This wood degradation inhibition is related to the presence of the tropolone hydroxyl group, as shown by the use of analogs (cyclohepta-2,4,6-trienone and 2-methoxycyclohepta-2,4,6-trienone). Furthermore, tropolone possesses both weak antioxidative and weak radical-scavenging properties and a strong affinity for ferric ion and is able to inhibit ferric iron reduction by catecholates, lowering the redox potential of the iron couple. These data are consistent with the hypothesis that tropolone inhibits wood degradation by P. placenta by chelating iron present in wood, thus avoiding initiation of the Fenton reaction. This study demonstrates that iron chelators such as tropolone could be also involved in novel and more environmentally benign preservative systems.     

Bleomycin-metal interaction: ferrous iron-initiated chemiluminescence

Chemiluminescence often accompanies the spontaneous degradation of intermediates in an electronically excited state. The interaction of iron with bleomycin results in the activation of bleomycin to a reactive intermediate which can alter DNA or undergo self-inactivation. This report demonstrates that the interaction of ferrous iron with bleomycin results in chemiluminescence, that this response is iron-specific and that the presence of DNA prevents the generation of chemiluminescence. These observations suggest that the activated bleomycin intermediate may be in an electronically excited state.     

Influence of Tropolone on Poria placenta Wood Degradation

Fenton reactions are believed to play important roles in wood degradation by brown rot fungi. In this context, the effect of tropolone (2-hydroxycyclohepta-2,4,6-trienone), a metal chelator, on wood degradation by Poria placenta was investigated. Tropolone (50 μM) strongly inhibits fungal growth on malt agar, but this inhibition could be relieved by adding iron salts. With an experimental system containing two separate parts, one supplemented with tropolone (100 μM) and the other not, it was shown that the fungus is able to reallocate essential minerals from the area where they are available and also to grow in these conditions on malt-agar in the presence of tropolone. Nevertheless, even in the presence of an external source of metals, P. placenta is not able to attack pine blocks impregnated with tropolone (5 mM). This wood degradation inhibition is related to the presence of the tropolone hydroxyl group, as shown by the use of analogs (cyclohepta-2,4,6-trienone and 2-methoxycyclohepta-2,4,6-trienone). Furthermore, tropolone possesses both weak antioxidative and weak radical-scavenging properties and a strong affinity for ferric ion and is able to inhibit ferric iron reduction by catecholates, lowering the redox potential of the iron couple. These data are consistent with the hypothesis that tropolone inhibits wood degradation by P. placenta by chelating iron present in wood, thus avoiding initiation of the Fenton reaction. This study demonstrates that iron chelators such as tropolone could be also involved in novel and more environmentally benign preservative systems.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on ⋅OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as β-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against ^⋅OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for ^⋅OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of ^⋅OH from H₂O₂. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H₂O₂. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on ^⋅OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

The antioxidant activity of haptoglobin towards haemoglobin-stimulated lipid peroxidation

Haemoglobin stimulates the peroxidation of lipids in two discernable phases. The first phase is inhibited by binding haemoglobin to the protein haptoglobin. The second phase is stimulated by complexable iron released from the haemoglobin molecule during the process of lipid peroxidation. This latter peroxidation is inhibitable by transferrin and the iron chelator desferrioxamine. Heat-denatured haemoglobin and haemin both stimulated lipid peroxidation but this is not inhibitable by haptoglobin. It is suggested that the haptoglobins play an important antioxidant role in vivo by preventing iron-stimulated formation of oxygen radicals.     

Heme and non-heme iron transporters in non-polarized and polarized cells

Background  

Heme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs), and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs.     

<Emphasis Type="Italic">Synechocystis</Emphasis> ferredoxin-NADP<Superscript>+</Superscript> oxidoreductase is capable of functioning as ferric reductase and of driving the Fenton reaction in the absence or presence of free flavin

We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP⁺ oxidoreductase (FNR _S ). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme, indicating that Synechocystis FNR _S may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR _S in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic chelate iron compounds. The Synechocystis FNR _S is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR _S was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic chelate iron compounds.     

The influence of pH on OH scavenger inhibition of damage to deoxyribose by Fenton reaction

Summary Hydroxyl radicals (OH') can be formed in aqueous solution by direct reaction of hydrogen peroxide (H₂O₂) with ferrous salt (Fenton reaction). OH' damage to deoxyribose, measured as formation of thiobarbituric acid-reactive material, was evaluated at different pHs to study the mechanism of action of classical OH' scavengers. OH' scavenger effect on Fe²⁺ oxidation was also evaluated in the same experimental conditions. In the absence of OH' scavengers, OH' damage to deoxyribose is higher at acidic compared to neutral and moderately basic pH. At acidic pH deoxiribose is per se able to inhibit Fe²⁺ oxidation by H₂0₂. Most of OH' scavengers tested inhibit deoxyribose damage and Fe²⁺ oxidation in a similar manner: both inhibitions are most relevant at acidic pH and decrease by increasing the pH. These results are not due to OH' scavenger inhibition of Fenton reaction. The influence of pH on the parameters studied appears to be due to the competition of deoxyribose and OH' scavengers for iron. These results suggest the prominent role of iron binding in the degradation of deoxyribose and in the OH' scavenging ability of different compounds. Results obtained with triethylenetetramine, a iron chelator with a low rate constant with OH', confirm that both deoxyribose and the OH' scavengers interact with iron bringing about a site specific Fenton reaction; that the OH' formed at these sites oxidize these molecules to their radical forms which in turn reduce the Fe^3– produced by Fenton reaction. The results presented indicate that most of classical OH' scavengers exert their effect predominantly by preventing the site specific reaction between Fe²⁺ and H₂0₂ on the deoxyribose molecule.     

Egg yolk phosvitin inhibits hydroxyl radical formation from the fenton reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical ((.-)OH) formation from H(2)O(2) in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited (.-)OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the (.-)OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H(2)O(2). These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> FtnA acts as an iron buffer for re-assembly of iron–sulfur clusters in response to hydrogen peroxide stress

Iron–sulfur clusters are one of the most ubiquitous redox centers in biology. Ironically, iron-sulfur clusters are highly sensitive to reactive oxygen species. Disruption of iron-sulfur clusters will not only change the activity of proteins that host iron–sulfur clusters, the iron released from the disrupted iron–sulfur clusters will further promote the production of deleterious hydroxyl free radicals via the Fenton reaction. Here, we report that ferritin A (FtnA), a major iron-storage protein in Escherichia coli, is able to scavenge the iron released from the disrupted iron–sulfur clusters and alleviates the production of hydroxyl free radicals. Furthermore, we find that the iron stored in FtnA can be retrieved by an iron chaperon IscA for the re-assembly of the iron–sulfur cluster in a proposed scaffold IscU in the presence of the thioredoxin reductase system which emulates normal intracellular redox potential. The results suggest that E. coli FtnA may act as an iron buffer to sequester the iron released from the disrupted iron–sulfur clusters under oxidative stress conditions and to facilitate the re-assembly of the disrupted iron–sulfur clusters under normal physiological conditions.     

Study of a Fenton type reaction: effect of captopril and chelating reagents.

The purpose of this study was to determine if captopril, an angiotensin-converting enzyme inhibitor, could interact with iron ions and so modify a Fenton type reaction. Results indicate that different degrees of thiobarbituric acid-reactive substance from deoxyribose are obtained in an ascorbate-driven Fenton system depending on the order of addition of captopril and iron to the incubation medium. Similar results were obtained with the chelating reagents ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, indicating that the buffer solution plays a relevant role when a particular iron complex is formed with a chelating agent. These metal complexes produce oxidizing species in a Fenton type system whose nature is discussed.     

Spin-trapping studies of the oxidation-reduction reactions of iron bleomycin in the presence of thiols and buffer.

The reaction of ferrous bleomycin with dioxygen is reexamined to clarify whether radical species derived from molecular oxygen are generated. Detection of low levels of spin-trapped oxyradicals confirm the production of OH during this reaction when bleomycin is present in excess, but not when iron and drug concentrations are equal. In phosphate buffer, hydroxyl radicals continue to be spin trapped for at least 15 min after Fe(II)bleomycin has been oxidized to Fe(III)bleomycin. In HEPES buffer, detection of a HEPES radical in the absence of spin trap over the same period independently supports the conclusion that reactive radicals are present after the initial oxidation of Fe(II)bleomycin is complete. When glutathione is included in the aerobic reaction mixture, thiyl radical species are spin trapped. The reaction of Fe(III)bleomycin with cysteine produces thiyl radical without spin-trapped hydroxyl radical.     

Model Studies of the Iron-Catalysed Haber-Weiss Cycle and the Ascorbate-Driven Fenton Reaction

Complementary hydroxylation assays and stopped-flow e.s.r. techniques have been employed in the investigation of the effect of various iron chelators (of chemical, biological and clinical importance) on hydroxyl-radical generation via the Haber-Weiss cycle and the ascorbate-driven Fenton reaction.

Chelators have been identified which selectively promote or inhibit various reactions involved in hydroxyl-radical generation (for example, NTA and EDTA promote all the reactions of both the Haber-Weiss cycle and the ascorbate-driven Fenton reaction, whereas DTPA and phytate inhibit the recycling of iron in these reactions). The biological chelators succinate and citrate are shown to be relatively poor catalysts of the Haber-Weiss cycle, whereas they are found to be effective catalysts of ·OH generation in the ascorbate-driven Fenton reaction.

It is also suggested that continuous redox-cycling reactions between iron, oxygen and ascorbate may represent an important mechanism of cell death in biological systems.