A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo

Free radical formation in heme proteins is recognised as a factor in mediating the toxicity of peroxides in oxidative stress. As well as initiating free radical damage, heme proteins damage themselves. Under extreme conditions, where oxidative stress and low pH coincide (e.g., myoglobin in the kidney following rhabdomyolysis and hemoglobin in the CSF subsequent to subarachnoid hemorrhage), peroxide can induce covalent heme to protein cross-linking. In this paper we show that, even at neutral pH, the heme in hemoglobin is covalently modified by oxidation. The product, which we term OxHm, is a "green heme" iron chlorin with a distinct optical spectrum. OxHm formation can be quantitatively prevented by reductants of ferryl iron, e.g., ascorbate. We have developed a simple, robust, and reproducible HPLC assay to study the extent of OxHm formation in the red cell in vivo. We show that hemoglobin is oxidatively damaged even in normal blood; approximately 1 in 2,000 heme groups exist as OxHm in the steady state. We used a simple model (physical exercise) to demonstrate that OxHm increases significantly during acute oxidative stress. The exercise-induced increase is short-lived, suggesting the existence of an active mechanism for repairing or removing the damaged heme proteins.     

On the Cytoprotective Role of Ferritin in Macrophages and its Ability to Enhance Lysosomal Stability

Macrophages have a great capacity to take up (eg. by endocytosis and phagocytosis) exogenous sources of iron which could potentially become cytotoxic, particularly following the intralysosomal formation of low-molecular weight, redox active iron, and under conditions of oxidative stress. Following autophago-cytosis of endogenous ferritin/apoferritin, these compounds may serve as chelators of such lysosomal iron and counteract the occurrence of iron-mediated intralysosomal oxidative reactions. Such redox-reactions have been shown to lead to destabilisation of lysosomal membranes and result in leakage of damaging lysosomal contents to the cytosol. In this study we have shown: (i) human monocyte-derived macrophages to accumulate ferritin in response to iron exposure; (ii) iron to destabilise macrophage secondary lysosomes when the cells are exposed to H₂O₂; and (iii) endocytosed apoferritin to act as a stabiliser of the acidic vacuolar compartment of iron-loaded macrophages. While the endogenous ferritin accumulation which was induced by iron exposure was not sufficient to protect cells from the damaging effects of H₂O₂, exogenously added apoferritin, as well as the potent iron chelator desferrioxamine, afforded significant protection. It is suggested that intralysosomal formation of haemosiderin, from partially degraded ferritin, is a protective strategy to suppress intralysosomal iron-catalysed redox reactions. However, under conditions of severe macrophage lysosomal iron-overload, induction of ferritin synthesis is not enough to completely prevent the enhanced cytotoxic effects of H₂O₂.     

Tyrosine residues as redox cofactors in human hemoglobin: implications for engineering nontoxic blood substitutes

Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and protein-based free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves "through-protein" electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the alpha and beta chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the alpha chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the beta chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.     

Characteristics and mechanism of formation of peroxide-induced heme to protein cross-linking in myoglobin.

At acidic pH values heme-protein cross-linked myoglobin (Mb-H) forms as a product of a peroxide-induced ferric-ferryl redox cycle. There is evidence that this molecule acts as a marker for heme-protein-induced oxidative stress in vivo and may exacerbate the severity of oxidative damage due to its enhanced prooxidant and pseudoperoxidatic activities. Therefore, an understanding of its properties and mechanism of formation may be important in understanding the association between heme-proteins and oxidative stress. Although the mechanism of formation of heme-protein cross-linked myoglobin is thought to involve a protein radical (possibly a tyrosine) and the ferryl heme, we show that this hypothesis needs revising. We provide evidence that in addition to a protein-based radical the protonated form of the oxoferryl heme, known to be highly reactive and radical-like in nature, is required to initiate cross-linking. This revised mechanism involves radical/radical termination rather than attack of a single radical onto the porphyrin ring. This proposal better explains the pH dependence of cross-linking and may, in part, explain the therapeutic effectiveness of increasing the pH on myoglobin-induced oxidative stress, e.g., therapy for rhabdomyolysis-associated renal dysfunction.     

The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species

Myoglobin catalyses the breakdown of lipid hydroperoxides (e.g., HPODE) during which the absorption band of the lipid conjugated diene (234 nm) is partially bleached. The constant for this process is strongly pH-dependent (k = 9.5 x 10(-3)s(-1), pH 7: k = 2.3 x 10(-1)s(-1), pH 5). This rate enhancement is not due to acid-induced changes in protein conformation or the involvement of protein-based radical species, as demonstrated by an almost identical pH dependence of the same reaction catalyzed by ferric haemin. The rate constants for ferryl formation and auto-reduction show different pH dependencies, with a pK of 8.3 for ferryl formation and a projected pK of 3.5 for ferryl auto-reduction. The pH dependence for the auto-reduction of the ferryl species is the same as that of the myoblobin catalyzed breakdown of HPODE. We propose that the protonated form of ferryl myoglobin (Fe(4+) - OH(-)) is the reactive species regulating the peroxidatic activity of myoglobin. The protonated ferryl species abstracts an electron from either the protein or porphyrin, allowing fast regeneration of the ferric species. Alkaline conditions stabilize the ferryl species, making myoglobin considerably less reactive towards lipids and lipid hydroperoxides. These findings are significant for understanding myoglobin-induced oxidative stress in vivo and the development of therapies.     

The role of iron in ferritin- and haemosiderin-mediated lipid peroxidation in liposomes.

Ferritin and haemosiderin were shown, by the measurement of malondialdehyde production and loss of polyunsaturated fatty acids, to stimulate lipid peroxidation in liposomes. At pH 7.4 ascorbate was additionally required to achieve peroxidation; however, peroxidation occurred at pH 4.5 in the presence of iron-proteins alone. The damage was completely inhibited by the incorporation of chain-breaking antioxidants (alpha-tocopherol and butylated hydroxytoluene) into the liposomes. Metal chelators (desferrioxamine and EDTA) also completely inhibited lipid peroxidation. These and further results indicate that, at pH 4.5, even in the absence of a reducing agent, iron is released from haemosiderin and can mediate oxidative damage to a lipid membrane.     

Biologically relevant metal ion-dependent hydroxyl radical generation. An update.

Transition metal ions, especially iron, appear to be important mediators of oxidative damage in vivo. Iron(II) reacts with H2O2 to give more-reactive radicals. On the basis of ESR spin-trapping data with DMPO, supported by aromatic hydroxylation studies and patterns of DNA base modification, it is concluded that hydroxyl radical (OH.) is likely to be the major damaging species formed in Fenton Systems under biologically-relevant conditions (which include iron concentrations no higher than the micromolar range). Although reactive oxo-iron species (such as ferryl and perferryl) may also be important, direct chemical evidence for their formation and identity in biologically relevant Fenton systems is currently lacking. Studies at alkaline pH values show that iron(IV) and iron(V) species are highly oxidizing under those reaction conditions, with a pattern of reactivity different from that of OH..     

Nanoparticle and other metal chelation therapeutics in Alzheimer disease

Current therapies for Alzheimer disease (AD) such as the anticholinesterase inhibitors and the latest NMDA receptor inhibitor, Namenda, provide moderate symptomatic delay at various stages of disease, but do not arrest disease progression or supply meaningful remission. As such, new approaches to disease management are urgently needed. Although the etiology of AD is largely unknown, oxidative damage mediated by metals is likely a significant contributor since metals such as iron, aluminum, zinc, and copper are dysregulated and/or increased in AD brain tissue and create a pro-oxidative environment. This role of metal ion-induced free radical formation in AD makes chelation therapy an attractive means of dampening the oxidative stress burden in neurons. The chelator desferioxamine, FDA approved for iron overload, has shown some benefit in AD, but like many chelators, it has a host of adverse effects and substantial obstacles for tissue-specific targeting. Other chelators are under development and have shown various strengths and weaknesses. In this review, we propose a novel system of chelation therapy through the use of nanoparticles. Nanoparticles conjugated to chelators show a unique ability to cross the blood-brain barrier (BBB), chelate metals, and exit through the BBB with their corresponding complexed metal ions. This method may prove to be a safe and effective means of reducing the metal load in neural tissue thus staving off the harmful effects of oxidative damage and its sequelae.     

Binding of the intramitochondrial ADP and its relationship to adenine nucleotide translocation

Adriamycin under partially anaerobic conditions degrades deoxyribose with the release of thiobarbituric acid-reactive products. This reaction is seen when electrons are transferred to adriamycin by xanthine oxidase or ferredoxin reductase to form the semiquinone free radical. Under the conditions described, damage to deoxyribose was inhibited by hydroxyl radicals scavengers, catalase and iron chelators. When the ratio of iron chelator to iron salt is varied both EDTA and diethylenetriamino penta-acetic acid (DETAPAC) show stimulatory properties whereas desferrioxamine remains a potent inhibitor of all reaction.     

Iron mobilisation from lactoferrin by chelators at physiological pH

Several alpha-ketohydroxypyridine, 2-hydroxypyridine N-oxide and 8-hydroxyquinoline chelators were shown to mobilise iron from diferric 59Fe-labelled human lactoferrin at physiological pH without the use of mediators or reducing agents. 1,2-Dimethyl-3-hydroxypyrid-4-one was found to be the most effective chelator, removing 90% of 59Fe from [59Fe]lactoferrin, in contrast to desferrioxamine, which was ineffective under the same conditions.     

Effect of iron chelators on the cytotoxic and genotoxic action of hyperoxia in Chinese hamster ovary cells.

The iron chelators o-phenanthroline and desferrioxamine were tested for their ability to protect Chinese hamster ovary cells against the cytotoxic and genotoxic effects of normobaric hyperoxia. Desferrioxamine added at sub-toxic concentrations (up to 2.5 microM) over a period of several days had no protective effect on hyperoxia-induced clonogenic cell killing and growth inhibition. The clastogenic effect of hyperoxia was strongly potentiated by desferrioxamine, while the induction of sister-chromatid exchanges (SCEs) by hyperoxia was unaffected. Similarly, o-phenanthroline (up to 0.25 microM) had no protective effect on hyperoxia-induced cell killing, growth inhibition, and SCE induction, while also this compound potentiated the clastogenic effect of hyperoxia. These results do not support a critical role for cellular iron in the mechanism of toxicity by normobaric hyperoxia in CHO cells. However, the results may still be consistent with a critical involvement of particular iron fraction(s) not susceptible to the chelators used. Furthermore, our results show that concentrations of iron chelators known to protect against short-term (up to 1 h) toxic exposure to oxidative stress become toxic themselves when applied chronically, i.e., in the order of days.     

Effects of (-)-epigallocatechin gallate on the redox reactions of human hemoglobin

The toxicity of acellular hemoglobin (Hb)-based therapeutics has been attributed in part to the uncontrolled oxidative reactions. A variety of antioxidant strategies to ameliorate potential oxidative damage in vivo have been suggested. We have examined the effects of (-)-epigallocatechin gallate (EGCG), a green tea polyphenol compound widely regarded as a chain-breaking antioxidant, on the oxidative stability of diaspirin crosslinked Hb (DBBF) and its cytotoxic ferryl intermediate. DBBF (ferrous) was rapidly oxidized to the ferric form in the presence of EGCG relative to the normal spontaneous oxidation of this Hb. The fast elimination of ferrous Hb is probably due to the ability of EGCG to produce hydrogen peroxide (H2O2) as these reactions were almost completely reversed by the addition of catalase and superoxide dismutase to the reaction medium. EGCG, however, effectively reduced ferryl back to ferric Hb in a biphasic kinetic reaction at physiological pH. At acidic pH where the autoreduction of protonated ferryl Hb is enhanced, a monophasic reduction process of the ferryl heme is achieved. A balance between pro and antioxidant properties of EGCG should be taken into account if EGCG is used in combination therapy with redox active acellular Hbs.     

Absence of Synproportionation Between Oxy and Ferryl Leghemoglobin

The synproportionation reaction between ferryl leghemoglobin and oxyleghemoglobin does not occur, at least under conditions where this process could be clearly demonstrated with myoglobin and hemoglobin. In contrast, a cross synproportionation can occur between oxyleghemoglobin and ferryl myoglobin or between ferryl leghemoglobin and oxymyoglobin. The non-exposure, at the surface of the leghemoglobin molecule, of the nearest tyrosine residue to the heme group could explain this behaviour. Thus leghemoglobin per se does not appear to be able to act as an antioxidant in removing H₂O₂ by synproportionation. However, in the presence of ascorbate and/or glutathione which can reduce ferryl leghemoglobin, this hemoprotein could act as an H₂O₂-removing antioxidant, in a process similar to that described for myoglobin. This could also explain why, despite the absence of synproportionation, ferryl leghemoglobin is not detected in nodule extracts.     

Vitamin B12b enhances the cytotoxicity of dithiothreitol

It has been found previously that vitamin B12b amplifies significantly the cytotoxic effects of ascorbic acid by catalyzing the formation of reactive oxygen species, and the antioxidant dithiothreitol (DTT), in contrast to catalase, does not prevent the cytotoxicity. Therefore, in this study we examined whether B12b is able to enhance the cytotoxicity of DTT. It was revealed that B12b strongly increases the cytotoxic effect of DTT. Vitamin B12b added to DTT catalyzed the generation and drastic accumulation of hydrogen peroxide in culture medium to a concentration of 260 microM within 7 min. The extracellular oxidative burst induced by the combination of B12b and DTT (DTT + B12b) was accompanied by intracellular oxidative stress, the destabilization of lysosomes, and damage to DNA. The accumulation of DNA lesions led to the initiation of apoptotic cell death, including the activation of caspase-3 and the release of cytochrome c. The antioxidants pyruvate and catalase completely prevented the DTT + B12b-induced oxidative stress and cell death. The iron chelators desferrioxamine and phenanthroline prevented the geno- and cytotoxic action of the combination although they did not reduce the exogenous oxidative burst, indicating a key role for intracellular iron in the cytotoxicity of the combination. Thus, vitamin B12b dramatically enhances the cytotoxicity of DTT, catalyzing the generation of hydrogen peroxide and inducing extra- and intracellular oxidative stress, early destabilization of lysosomes, and iron-dependent DNA damage.     

DNA damage induced by catechol derivatives

We investigated the effect of catechol derivatives, including dopa, dopamine, adrenaline and noradrenaline, on DNA damage and the mechanisms of DNA strand breakage and formation of 8-hydroxyguanine (8HOG). The catechol derivatives caused strand breakage of plasmid DNA in the presence of ADP-Fe(3+). The DNA damage was prevented by catalase, mannitol and dimethylsulfoxide, suggesting hydroxyl radical (HO..)-like species are involved in the strand breakage of DNA. Iron chelators, such as desferrioxamine and bathophenanthroline, and reduced glutathione also inhibited the DNA damage. Deoxyribose, a molecule that is used to detect HO,, was not degraded by dopa in the presence of ADP-Fe(3+). By adding EDTA, however, dopa induced the marked deoxyribose degradation in the presence of ADP-Fe(3+), indicating that EDTA may extract iron from ADP-Fe(3+) to catalyze HO. formation by dopa. Thus, EDTA was a good catalyst for HO.-generation, whereas it did not promote the strand breakage of DNA. However, calf thymus DNA base damage, which was detected as 8-HOG formation, was caused by dopa in the presence of EDTA-Fe(3+), but not in the presence of ADP-Fe(3+). The 8HOG formation was also inhibited by catalase and HO. scavengers, indicating that HO&z.rad; was involved in the base damage. These results suggest that DNA strand breakage is due to ferryl species rather than HO., and that 8HOG formation is due to HO. rather than ferryl species.     

Dengue haemorrhagic fever and dengue shock syndrome: are they tumour necrosis factor-mediated disorders?

Growth of Bacteroides fragilis under anaerobic conditions in the presence of either haemin or protoporphyrin IX was inhibited by the ferrous iron chelator bipyridyl. The ferric-iron chelator desferrioxamine inhibited growth in the presence of protoporphyrin but not haemin, suggesting that even under anaerobic conditions Fe3+ is involved in uptake of non-haem iron, which is required in the absence of haemin. However, the ferric iron chelators 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and pyridoxal isonicotinoyl hydrazone (PIH) were only weakly inhibitory. Apotransferrin, which also binds Fe3+, inhibited growth, but this was not simply due to binding of iron in the medium, as under the reducing conditions present, transferrin was unable to bind iron. This study suggests that even under anaerobic conditions, uptake of non-haem iron by B. fragilis may involve conversion of Fe2+ to Fe3+.     

Effects of hypoxia and glutathione depletion on hemoglobin- and myoglobin-mediated oxidative stress toward endothelium

We investigated the toxicity of hemoglobin/myoglobin on endothelial cells under oxidative stress conditions that include cellular hypoxia and reduced antioxidant capacity. Bovine aorta endothelial cells (BAECs), grown on microcarrier beads, were subjected to cycles of hypoxia and reoxygenation in a small volume of medium, and endothelial cell monolayers were depleted of their intracellular glutathione (GSH) by treatment with buthionine sulfoximine. Incubation of diaspirin cross-linked hemoglobin (DBBF-Hb) or horse skeletal myoglobin (Mb) with BAECs subjected to 3 h of hypoxia caused transient oxidation of the hemoproteins to the ferryl form (Fe(4+)). Formation of the ferryl intermediate was decreased in a concentration-dependent manner by the addition of L-arginine, a substrate of NO synthase, after 3 h of hypoxia. Optimal inhibition of ferryl formation, possibly due to the antioxidant action of NO, was achieved with 900 microM L-arginine. Addition of hydrogen peroxide to GSH-depleted cells in the presence of DBBF-Hb or Mb significantly decreased cell viability. Ferryl Mb, but not ferryl DBBF-Hb, was observed in samples analyzed at the end of treatment, which may explain the greater toxicity observed with Mb as opposed to DBBF-Hb. This model may be utilized to identify causative agent(s) associated with hemoprotein cytotoxicity and in designing strategies to suppress or control heme-mediated injury under physiologically relevant conditions.     

Copper ions mediate the lethality induced by hydrogen peroxide in low iron conditions in Escherichia coli

Iron ions mediate the formation of lethal DNA damage by hydrogen peroxide. However, when cells are depleted of iron ions by the treatment with iron chelators, DNA damage can still be detected. Here we show that the formation of such damage in low iron conditions is due to the participation of copper ions. Copper chelators can inhibit cell inactivation, DNA strand breakage and mutagenesis induced by hydrogen peroxide in cells pre-treated with iron chelators. The Fpg and UvrA proteins play an important role in the repair of DNA lesions formed in these conditions, as suggested by the great sensitivity of the uvrA and fpg mutant strains to the treatment when compared to the wild type strain.     

Glucose-6-phosphate dehydrogenase deficiency: a new hypothesis for an old disease

Summary

We have previously shown insulinoma (HIT-T15 and RINm5F) cells in culture to be very sensitive, in comparison with a reference cell line (J-774), to the oxidative stress that is created when alloxan reacts extracellularly with reducing agents, forming superoxide and hydrogen peroxide. The toxic effects are prevented by catalase added to the medium, suggesting that alloxan does not need to be taken up in order to affect cells. Rather, alloxan seems to exert its action through extracellular formation of hydrogen peroxide that influences the stability of the cells' lysosomes following diffusion into them. To further analyse the mechanisms in operation, we studied the influence of induced autophagocytosis on the sensitivity to ensuing oxidative stress. Starvation for 60–120 min in PBS at 37°C markedly enhanced autophagocytosis and, in parallel, increased the cytotoxic effect and lysosomal vulnerability of ensuing exposure to hydrogen peroxide, while not significantly changing the antioxidative status or the energy balance. Autophagocytosis increased the size of the intralysosomal pool of reactive, low-molecular-weight, iron, probably by degradation of metallo-proteins, as shown by autometallography and HPLC demonstration of desferrioxamine-reactive intracellular iron. Moreover, exposure to the iron-chelator desferrioxamine before treatment with hydrogen peroxide prevented lysosomal destabilization and cellular death of both starved and control cells, further proving the importance of intralysosomal iron for the response to oxidative stress. We hypothesize that β-cells which, like insulinoma cells, have a weak antioxidative defence system under conditions of enhanced general autophagocytosis, or crinophagy, might become vulnerable to even low, or moderate, oxidative stress.