Comparison of various iron chelators and prochelators as protective agents against cardiomyocyte oxidative injury

Oxidative stress is a common denominator of numerous cardiovascular disorders. Free cellular iron catalyzes the formation of highly toxic hydroxyl radicals, and iron chelation may thus be an effective therapeutic approach. However, using classical iron chelators in diseases without iron overload poses risks that necessitate more advanced approaches, such as prochelators that are activated to chelate iron only under disease-specific oxidative stress conditions. In this study, three cell-membrane-permeable iron chelators (clinically used deferasirox and experimental SIH and HAPI) and five boronate-masked prochelator analogs were evaluated for their ability to protect cardiac cells against oxidative injury induced by hydrogen peroxide. Whereas the deferasirox-derived agents TIP and TRA-IMM displayed negligible protection and even considerable toxicity, the aroylhydrazone prochelators BHAPI and BSIH-PD provided significant cytoprotection and displayed lower toxicity after prolonged cellular exposure compared to their parent chelators HAPI and SIH, respectively. Overall, the most favorable properties in terms of protective efficiency and low inherent cytotoxicity were observed with the aroylhydrazone prochelator BSIH. BSIH efficiently protected both H9c2 rat cardiomyoblast-derived cells and isolated primary rat cardiomyocytes against hydrogen peroxide-induced mitochondrial and lysosomal dysregulation and cell death. At the same time, BSIH was nontoxic at concentrations up to its solubility limit (600 μM) and in 72-h incubation. Hence, BSIH merits further investigation for prevention and/or treatment of cardiovascular disorders associated with a known (or presumed) component of oxidative stress.     

The hydrolytic susceptibility of prochelator BSIH in aqueous solutions

The prochelator BSIH ((E)-N′-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isonicotinohydrazide) contains a boronate group that prevents metal coordination until reaction with peroxide releases the iron chelator SIH ((E)-N′-(2-hydroxybenzylidene)isonicotinohydrazide). BSIH exists in aqueous buffer and cell culture media in equilibrium with its hydrolysis products isoniazid and (2-formylphenyl)boronic acid (FBA). The relative concentrations of these species limit the yield of intact SIH available for targeted iron chelation. While the hydrolysis fragments are nontoxic to retinal pigment epithelial cells, these results suggest that modifications to BSIH that improve its hydrolytic stability yet maintain its low inherent cytotoxicity are desirable for creating more efficient prochelators for protection against cellular oxidative damage.     

Prochelators triggered by hydrogen peroxide provide hexadentate iron coordination to impede oxidative stress

Prochelators are agents that have little affinity for metal ions until they undergo a chemical conversion. Three new aryl boronate prochelators are presented that are responsive to hydrogen peroxide to provide hexadentate ligands for chelating metal ions. TRENBSIM (tris[(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)-2-aminoethyl]amine), TRENBSAM (tris[(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-2-aminoethyl]amine), and TB (tris[(2-boronic acid-benzyl)2-aminoethyl]amine) convert to TRENSIM (tris[(salicylideneamino)ethyl]amine), TRENSAM (tris[(2-hydroxybenzoyl)-2-aminoethyl]amine), and TS (tris[2-hydroxybenzyl)2-aminoethyl]amine), respectively. The prochelators were characterized by ¹¹B NMR, and the structures of TRENBSAM, TRENBSIM, and the Fe(III) complex of TS were determined by X-ray crystallography. Of the three prochelator/chelator pairs, TB/TS was identified as the most promising for biological applications, as they prevent iron and copper-induced hydroxyl radical generation in an in vitro assay. TB has negligible interactions with metal ions, whereas TS has apparent binding constants (log K′) at pH 7.4 of 15.87 for Cu(II), 9.67 Zn(II) and 14.42 for Fe(III). Up to 1 mM TB was nontoxic to retinal pigment epithelial cells, whereas 10 μM TS induced cell death. TS protected cells against H₂O₂-induced death, but only within a 1-10 μM range. TB, on the other hand, had a much broader window of protection, suggesting that it may be a useful agent for preventing metal-promoted oxidative damage.     

Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage

The role of intracellular iron, copper, and calcium in hydrogen peroxide-induced DNA damage was investigated using cultured Jurkat cells. The cells were exposed to low rates of continuously generated hydrogen peroxide by the glucose/glucose oxidase system, and the formation of single strand breaks in cellular DNA was evaluated by the sensitive method, single cell gel electrophoresis or "comet" assay. Pre-incubation with the specific ferric ion chelator desferrioxamine (0.1-5.0 mM) inhibited DNA damage in a time- and dose-dependent manner. On the other hand, diethylenetriaminepentaacetic acid (DTPA), a membrane impermeable iron chelator, was ineffective. The lipophilic ferrous ion chelator 1,10-phenanthroline also protected against DNA damage, while its nonchelating isomer 1,7-phenanthroline provided no protection. None of the above iron chelators produced DNA damage by themselves. In contrast, the specific cuprous ion chelator neocuproine (2,9-dimethyl-1,10-phenanthroline), as well as other copper-chelating agents, did not protect against H(2)O(2)-induced cellular DNA damage. In fact, membrane permeable copper-chelating agents induced DNA damage in the absence of H(2)O(2). These results indicate that, under normal conditions, intracellular redox-active iron, but not copper, participates in H(2)O(2)-induced single strand break formation in cellular DNA. Since BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), an intracellular Ca(2+)-chelator, also protected against H(2)O(2)-induced DNA damage, it is likely that intracellular Ca(2+) changes are involved in this process as well. The exact role of Ca(2+) and its relation to intracellular transition metal ions, in particular iron, needs to be further investigated.     

Protection of mammalian cells by o-phenanthroline from lethal and DNA-damaging effects produced by active oxygen species

Active oxygen species are suspected as being a cause of the cellular damage that occurs at the site of inflammation. Phagocytic cells accumulate at these sites and produce superoxide ion, hydrogen peroxide and hydroxyl radical. The ultimate killing species, the cellular target and the mechanism whereby the lethal injury is produced are unknown. We exposed mouse fibroblasts to xanthine oxidase and acetaldehyde, a system which mimics the membrane of phagocytic cells in terms of production of oxygen species. We observed that the generation of these species produced DNA strand breaks and cellular death. The metal chelator o-phenanthroline completely abolished the former effect, and at the same time it effectively protected the cells from lethal injuries. Because complexing iron o-phenanthroline prevents the formation of hydroxyl radical by the Fendon reaction (Fe(II) + H2O2----Fe(III) + OH- + OH.), it is proposed that most of the cell death and DNA damage are brought about by OH radical, produced from other species by iron-mediated reactions.     

Reactive oxygen metabolite-induced toxicity to cultured bovine endothelial cells: Status of cellular iron in mediating injury

We aimed to determine the status of iron in mediating oxidant-induced damage to cultured bovine aortic endothelial cells. Chromium-51-labeled cells were exposed to reaction mixtures of xanthine oxidase/hypoxanthine and glucose oxidase/glucose; these produce superoxide and hydrogen peroxide, or hydrogen peroxide, respectively. Xanthine oxidase caused a dose dependent increase of ⁵¹Cr release. Damage was prevented by allopurinol, oxypurinol, and extracellular catalase, but not by superoxide dismutase. Prevention of xanthine oxidase-in-duced damage by catalase was blocked by an inhibitor of catalase, aminotriazole. Glucose oxidase also caused a dose-dependent increase of ⁵¹Ci release. Glucose oxidase-induced injury, which was catalase-inhibitable, was not prevented by extracellular superoxide dismutase. Both addition of and pretreatment with deferoxamine (a chelator of Fe³⁺) prevented glucose oxidase-induced injury. The presence of phenanthroline (a chelator of divalent Fe²⁺) prevented glucose oxidase-induced ⁵¹Cr release, whereas pretreatment with the agent did not. Apotransferrin (a membrane impermeable iron binding protein) failed to influence damage. Neither deferoxamine nor phenanthroline influenced cellular antioxidant defenses, or inhibited lysis by non-oxidant toxic agents. Treatment with allopurinol and oxypurinol, which inhibited cellular xanthine oxidase, failed to prevent glucose oxidase injury. We conclude that (1) among the oxygen species extracellularly generated by xanthine oxidase/hypoxanthine, hydrogen peroxide induces damage via a reaction on cellular iron; (2) deferoxamine and phenanthroline protect cells by chelating Fe³⁺ and Fe²⁺, respectively; and (3) reduction of cellular stored iron (Fe³⁺) to Fe²⁺ may be a prerequisite for mediation of oxidantinduced injury, but this occurs independently of extracellular superoxide or cellular xanthine oxidase-derived superoxide. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Role of cellular superoxide dismutase against reactive oxygen metabolite injury in cultured bovine aortic endothelial cells.

We examined the protective effect of cellular superoxide dismutase against extracellular hydrogen peroxide in cultured bovine aortic endothelial cells. 51Cr-labeled cells were exposed to hydrogen peroxide generated by glucose oxidase/glucose. Glucose oxidase caused a dose-dependent increase of 51Cr release. Pretreatment with diethyldithiocarbamate enhanced injury induced by glucose oxidase, corresponding with the degree of inhibition of endogenous superoxide dismutase activity. Inhibition of cellular superoxide dismutase by diethyldithiocarbamate was not associated either with alteration of other antioxidant defenses or with potentiation of nonoxidant injury. Enhanced glucose oxidase damage by diethyldithiocarbamate was prevented by chelating cellular iron. Inhibition of cellular xanthine oxidase neither prevented lysis by hydrogen peroxide nor diminished enhanced susceptibility by diethyldithiocarbamate. These results suggest that, in cultured endothelial cells: 1) cellular superoxide is involved in mediating hydrogen peroxide-induced damage; 2) superoxide, which would be generated upon exposure to excess hydrogen peroxide independently of cellular xanthine oxidase, promotes the Haber-Weiss reaction by initiating reduction of stored iron (Fe3+) to Fe2+; 3) cellular iron catalyzes the production of a more toxic species from these two oxygen metabolites; 4) cellular superoxide dismutase plays a critical role in preventing hydrogen peroxide damage by scavenging superoxide and consequently by inhibiting the generation of the toxic species.     

Elucidation of endemic neurodegenerative diseases--a commentary

Recent investigations of scrapie, Creutzfeldt-Jakob disease (CJD), and chronic wasting disease (CWD) clusters in Iceland, Slovakia and Colorado, respectively, have indicated that the soil in these regions is low in copper and higher in manganese, and it has been well-known that patients of ALS or Parkinson's disease were collectively found in the New Guinea and Papua islands, where the subterranean water (drinking water) contains much Al3+ and Mn2+ ions. Above facts suggest that these neurodegenerative diseases are closely related with the function of a metal ion. We have investigated the chemical functions of the metal ions in detail and established the unique mechanism of the oxygen activation by the transition metal ions such as iron and copper, and pointed out the notable difference in the mechanism among iron, aluminum and manganese ions. Based on these results, it has become apparent that the incorporation of Al(III) or Mn(II) in the cells induces the "iron-overload syndrome", which is mainly due to the difference in an oxygen activation mechanism between the iron ion and Al(III) or the Mn(II) ion. This syndrome highly promotes formation of hydrogen peroxide, and hydrogen peroxide thus produced can be a main factor to cause serious damages to DNA and proteins (oxidative stress), yielding a copper(II)- or manganese(II)-peptide complex and its peroxide adduct, which are the serious agents to induce the structural changes from the normal prion protein (PrP(c)) to abnormal disease-causing isoforms, PrP(Sc), or the formation of PrP 27-30 (abnormal cleavage at site 90 of the prion protein). It seems reasonable to consider that the essential origin for the transmissible spongiform encephalopathies (TSEs) should be the incorporation and accumulation of Al(III) and Mn(II) ions in the cells, and the sudden and explosive increase of scrapie and bovine spongiform encephalopathy (BSE) in the last decade may be partially due to "acid rain", because the acid rain makes Al(III) and Mn(II) ions soluble in the subterranean aquifers.     

Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide. Evidence for the participation of hydroxyl radicals formed by an iron-catalyzed Haber-Weiss reaction

Cultured hepatocytes pretreated with the ferric iron chelator deferoxamine were resistant to the toxicity of H2O2 generated by either glucose oxidase or by the metabolism of menadione (2-methyl-1,4-naphthoquinone). Ferric, ferrous, or cupric ions restored the sensitivity of the cells to H2O2. Deferoxamine added to hepatocytes previously treated with this chelator prevented the restoration of cell killing by only ferric iron. The free radical scavengers mannitol, thiourea, benzoate, and 4-methylmercapto-2-oxobutyrate protected either native cells exposed to H2O2 or pretreated hepatocytes exposed to H2O2 and given ferric or ferrous iron. Superoxide dismutase prevented the killing of native hepatocytes by either glucose oxidase or menadione. With deferoxamine-pretreated hepatocytes, superoxide dismutase prevented the cell killing dependent upon the addition of ferric but not ferrous iron. Catalase prevented the killing by menadione of deferoxamine-pretreated hepatocytes given either ferric or ferrous iron. Deferoxamine pretreatment did not prevent the toxicity of t-butyl hydroperoxide but did, however, prevent that of cumene hydroperoxide. It is concluded that both ferric iron and superoxide ions are required for the killing of cultured hepatocytes by H2O2. The toxicity of H2O2 is also dependent upon its reaction with ferrous iron to form hydroxyl radicals by the Fenton reaction. The ferrous iron needed for this reaction is formed by the reduction of cellular ferric iron by superoxide ions. Such a sequence corresponds to the so-called iron-catalyzed Haber-Weiss reaction, and the present report documents its participation in the killing of intact hepatocytes by H2O2. Cumene hydroperoxide but not t-butyl hydroperoxide closely models the toxicity of hydrogen peroxide.     

Labile iron pool correlates with iron content in the nucleus and the formation of oxidative DNA damage in mouse lymphoma L5178Y cell lines

Labile iron pool (LIP) constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need for iron, its uptake and storage. In this study we investigated oxidative DNA damage in relation to the labile iron pool in a pair of mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S) differing in sensitivity to hydrogen peroxide. The LY-R cells, which are hydrogen peroxide-sensitive, contain 3 times more labile iron than the hydrogen peroxide-resistant LY-S cells. Using the comet assay, we compared total DNA breakage in the studied cell lines treated with hydrogen peroxide (25 microM for 30 min at 4 degrees C). More DNA damage was found in LY-R cells than in LY-S cells. We also compared the levels of DNA lesions sensitive to specific DNA repair enzymes in both cell lines treated with H(2)O(2). The levels of endonuclease III-sensitive sites and Fapy-DNA glycosylase-sensitive sites were found to be higher in LY-R cells than in LY-S cells. Our data suggest that the sensitivity of LY-R cells to H(2)O(2) is partially caused by the higher yield of oxidative DNA damage, as compared to that in LY-S cells. The critical factor appears to be the availability of transition metal ions that take part in the OH radical-generating Fenton reaction (very likely in the form of LIP).     

Evidence against transition metal-independent hydroxyl radical generation by xanthine oxidase

It is shown by the use of EPR spectroscopy that formation of the hydroxyl radical adduct with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the xanthine-xanthine oxidase system is hydrogen peroxide-independent. Production of the DMPO-hydroxyl radical adduct is inhibited by superoxide dismutase but is unaffected by purified grades of catalase. Hydroxyl radicals are a secondary product of the decomposition of the DMPO-superoxide radical adduct and are also formed as a result of trace metals such as iron present in the buffer. These results are in contrast with a recent report (Kuppusamy, P., and Zweier, J. W. (1989) J. Biol. Chem. 264, 9880-9884) in which the assertion is made that the hydroxyl radical adduct arises from the trapping of hydroxyl radicals generated via the direct reduction of hydrogen peroxide by xanthine oxidase. It is demonstrated here that treatment of phosphate buffer with the chelator deferoxamine mesylate is not in itself sufficient to suppress the effect of contaminating adventitious metal ions in xanthine-xanthine oxidase incubations.     

Protection Against Free Radical-Induced and Transition Metal-Mediated Damage: The Use of “Pull” and “Push” Mechanisms

Free radicals have been incriminated in a variety of injurious processes including the toxicity of the herbicide paraquat and the damage following ischemia and reperfusion of different organs.

Based on the assumption that iron and copper could serve as mediators for the transformation of relatively low reactive species (such as superoxide radicals, hydrogen peroxide, axorbate, and others) to the highly reactive species, in the site-specific metal-mediated mechanism, two new modes for intervention have been tried out. The first is the introduction of specific chelators that “pull” out redox-active and available metals, and by this reduce the apparent damage. Desferrioxamine was shown to protect bacterial cells and mammals against the poisonous effects of paraquat. Using the retrogradly perfused isolated rat heart, we have demonstrated that the chelator neocuproine, which effectively binds both iron and copper provides a major protection against hydrogen peroxide-induced cardiac damage and against ischemia/ reperfusion-induced arrhythmias. Likewise, TPEN a heavy metal chelator. provides almost total (> 90%) protection against ischemia/reperfusion-induced arrhythmias.

The other mode of intervention is the use of redox-inactive metal ions that could compete for the binding sites of iron and copper, and by this “push” these metal ions out, lead to their displacement, and divert the site of free radical attack. Applying Zn(II) complexes provided a marked protection against metal mediated free radical-induced damage in the copper-mediated paraquat toxicity to E. coli, and in the arrhythmias induced by ischemia and reperfusion.

It is proposed that the complex zinc-desferrioxamine would be the ultimate protector being effective by both the “pull” and “push” mechanisms.     

Calcium chelator Quin 2 prevents hydrogen-peroxide-induced DNA breakage and cytotoxicity

Exposure of cultured Chinese hamster ovary (CHO) cells to hydrogen peroxide results in the production of extensive DNA breakage which can be prevented by the intracellular calcium chelator Quin 2. This effect occurs at Quin 2 AM concentrations as low as 0.1 microM and is maximal at 1 microM. Addition of the extracellular calcium chelator, EGTA, does not affect the level of DNA breakage generated by H2O2. Quin 2 also significantly reduces cellular toxicity caused by the oxidant. Experiments with spin-trapping techniques demonstrate that Quin 2 does not affect the formation of hydroxyl radicals generated by the action of Fe2+ on H2O2. Quin 2 at high concentrations, similar to those reached within the cell, actually enhanced generation of hydroxyl radical in the absence of other iron chelators under our experimental conditions. These results suggest that H2O2 or H2O2-derived radicals do not directly induce DNA strand breakage in intact mammalian cells; rather, these radicals may disturb intracellular Ca2+ homeostasis which results in secondary reactions ultimately leading to DNA strand breakage. In addition to strand breakage, membrane and protein oxidation probably contribute to the cytotoxic effect of H2O2.     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.     

Hypothermia injury/cold-induced apoptosis--evidence of an increase in chelatable iron causing oxidative injury in spite of low O2-/H2O2 formation.

When incubated at 4 degrees C, cultured rat hepatocytes or liver endothelial cells exhibit pronounced injury and, during earlier rewarming, marked apoptosis. Both processes are mediated by reactive oxygen species, and marked protective effects of iron chelators as well as the protection provided by various other antioxidants suggest that hydroxyl radicals, formed by classical Fenton chemistry, are involved. However, when we measured the Fenton chemistry educt hydrogen peroxide and its precursor, the superoxide anion radical, formation of both had markedly decreased and steady-state levels of hydrogen peroxide did not alter during cold incubation of either liver endothelial cells or hepatocytes. Similarly, there was no evidence of an increase in O2-/H2O2 release contributing to cold-induced apoptosis occurring on rewarming. In contrast to the release/level of O2- and H2O2, cellular homeostasis of the transition metal iron is likely to play a key role during cold incubation of cultured hepatocytes: the hepatocellular pool of chelatable iron, measured on a single-cell level using laser scanning microscopy and the fluorescent indicator phen green, increased from 3.1 +/- 2.3 microM (before cold incubation) to 7.7 +/- 2.4 microM within 90 min after initiation of cold incubation. This increase in the cellular chelatable iron pool was reversible on rewarming after short periods of cold incubation. The cold-induced increase in the hepatocellular chelatable iron pool was confirmed using the calcein method. These data suggest that free radical-mediated hypothermia injury/cold-induced apoptosis is primarily evoked by alterations in the cellular iron homeostasis/a rapid increase in the cellular chelatable iron pool and not by increased formation of O2-/H2O2.     

Mechanisms of wood degradation by brown-rot fungi: chelator-mediated cellulose degradation and binding of iron by cellulose

Iron, hydrogen peroxide, biochelators and oxalate are believed to play important roles in cellulose degradation by brown-rot fungi. The effect of these compounds in an 'enhanced' Fenton system on alpha-cellulose degradation was investigated specifically in regard to molecular weight distribution and cellulose-iron affinity. This study shows that the degradative ability of an ultrafiltered low molecular weight preparation of chelating compounds isolated from the brown-rot fungus Gloeophyllum trabeum (termed 'Gt chelator') increased with increasing Gt chelator concentration when the FeIII to Gt chelator ratio was greater than about 30:1. When this ratio was less than 30:1, increasing Gt chelator concentration did not accelerate cellulose degradation. In excess hydrogen peroxide, cellulose degradation increased and then decreased with increasing iron concentration when FeIII was present in excess of the Gt chelator. The critical ratio of FeIII to Gt chelator varied depending on the concentration of hydrogen peroxide in the system. Increasing iron concentration above a critical iron:chelator ratio inhibited cellulose degradation. The optimum pH for cellulose degradation mediated by Gt chelator was around 4.0. A comparison of the effects of 2,3-DHBA (a chelator that reduces iron similarly to Gt chelator) and Gt chelator with respect to cellulose degradation demonstrated the same pattern of cellulose degradation. Cellulose-iron affinity studies were conducted at three pH levels (3.6, 3.8, 4.1), and the binding constants for cellulose-FeIII, cellulose-FeII and cellulose-FeIII in the presence of Gt chelator were calculated. The binding constants for cellulose-FeIII at all three pH levels were much higher than those for cellulose-FeII, and the binding constants for cellulose-FeIII in the presence of Gt chelator were very close to those for cellulose-FeII. This is probably the result of FeIII reduction to FeII by Gt chelator and suggests that chelators from the fungus may be able to sequester iron from cellulose and reduce it in near proximity to the cellulose and thereby better promote depolymerization. The free radical generating system described has potential for use in a variety of industrial processing and pollution control applications.     

The effect of melanin on iron associated decomposition of hydrogen peroxide

The effects of melanin on the iron-catalyzed decomposition of hydrogen peroxide to hydroxyl radicals and hydroxyl ions have been studied using electron spin resonance, spin trapping and visible light spectrophotometry. Melanin altered these reactions by several different mechanisms and consequently, depending on conditions, can significantly increase or decrease the yield of reactive products, including hydroxyl radicals. For low concentrations of ferrous ions, melanin decreased the yield of hydroxyl radicals due to binding of ferrous ions by melanin; ferrous ions bound to melanin did not decompose H2O2 efficiently. Melanins increased the rate of hydroxyl radical production if the predominant form of iron was ferric, due to the ability of melanin to reduce ferric to ferrous iron. Hydroxyl radical production in the presence of a strong chelator (e.g. EDTA) and melanin was greater than in the presence of a weak chelator (e.g. ADP) and melanin. Melanin also increased the rate of destruction of the DMPO-OH adduct.     

Dark production of reactive oxygen species in photosystem II membrane particles at elevated temperature: EPR spin-trapping study

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH.) and singlet oxygen ((1)O2), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 degrees C). Production of OH., evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH. production. These observations reveal that OH. is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH., whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH. is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH. scavengers. This finding indicates that the production of these two species was independent and that the production of (1)O2 is not closely linked to PSII donor side.     

Metal ions as potential regulatory factors in the biosynthesis of red hair pigments: a new benzothiazole intermediate in the iron or copper assisted oxidation of 5-S-cysteinyldopa

In the presence of iron or copper ions, the course of the oxidation in air of 5-S-cysteinyldopa (1), the main biosynthetic precursor of pheomelanins and trichochromes, was markedly changed affording two main products. One of these was identified as the oxobenzothiazine 8, previously obtained under nonphysiologically relevant conditions, while the other was characterized as the novel hydroxybenzothiazole 9. Besides 8 and 9, carboxylated and noncarboxylated benzothiazine products were obtained by persulfate oxidation of 1 in the presence of iron or copper ions. The ratio of formation yields of carboxylated/noncarboxylated benzothiazines, determined after reduction of the mixture, was lower than that of the control reaction run in the absence of metal ions, and much lower than that of the oxidation carried out in the presence of zinc ions, in agreement with a recent report. Notably, 8 and 9 were formed in variable yields under different oxidation conditions including tyrosinase/O(2), peroxidase/hydrogen peroxide, and the hydrogen peroxide/or (9E,11Z,13S)-13-hydroperoxyoctadeca-9,11-dienoic acid/Fe(III) systems. Mechanistic routes to 8 and 9 were proposed based on the results of experiments involving in situ generation of labile benzothiazine intermediates. Overall, these results allow to formulate an improved biosynthetic scheme in which metal ions act as critical regulatory factors determining pheomelanin vs. trichochromes formation.