Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: implication in hemodialysis mediated atherogenic LDL modification

Objective: Al³⁺ stimulates Fe²⁺ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al³⁺ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al³⁺ on LDL oxidation.

Materials and methods: Using different LDL modifying systems (Fe²⁺, Cu²⁺, free radical generating compounds, human endothelial cells, hemin/H₂O₂ and HOCl), the influence of Al³⁺ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.

Results: Al³⁺ could stimulate the oxidizability of LDL by Fe²⁺, but not in the other systems tested. Al³⁺ and Fe²⁺ were found to bind to LDL and Al³⁺could compete with Fe²⁺ binding to the lipoprotein. Fluorescence polarization data indicated that Al³⁺ does not affect the phospholipid compartment of LDL.

Conclusions:The results indicate that increased LDL oxidation by Fe²⁺ in presence of Al³⁺ might be due to blockage of Fe²⁺ binding sites on LDL making more free Fe²⁺ available for lipid oxidation.     

Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Radical-Induced Damage to Proteins: E.S.R. Spin-Trapping Studies

The reactions of hydroxyl radicals generated from Fe¹¹/H₂O₂ and Cu¹¹/H₂O₂ redox couples with a variety of proteins (BSA, histones, cytochrome c, lysozyme and protamine) have been investigated by e.s.r. spin trapping. The signals obtained, which are generally anisotropic in nature, characterize the formation of partially-immobilized spin-adducts resulting from attack of the HO- radicals on the protein and subsequent reaction of the protein-derived radicals with the spin trap. Similar spin adducts are observed on incubation of two haem-proteins (haemoglobin and myoglobin) with H₂O₂ in the absence of added metal ions implying a reaction at the haem centre followed by internal electron transfer reactions.

Two strategies have been employed to obtain information about the site(s) of radical damage in these proteins. The first involves the use of a variety of spin traps and in particular DMPO: with this particular trap the broad spectra from largely immobilized radicals show characteristic a(β-H) values which enable carbon-, oxygen- and sulphur-centred radicals to be distinguished. The second involves the use of enzymatic cleavage of first-formed adducts to release smaller nitroxides, with isotropic spectra, which allow the recognition of β-proton splittings and hence information about the sites of radical damage to be obtained. These results, which allows backbone and side-chain attack to be distinguished, are in agreement with random attack of the HO^. radical on the protein and are in accord with studies carried out on model peptides. In contrast the use of less reactive attacking radicals [N₃·, ·CH(CH₃)OH] and oxidising agents (Ce⁴⁺) provides evidence for selective attack on these proteins at particular residues.     

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.     

The Effect of Vitamin E-Deficiency in Isolated Rat Heart on the Cellular Defence System Against Free Radicals During Normal Reperfusion After Hypoxic, Ischemic and Ca2+-Free Perfusion

We investigated whether vitamin E plays a role in the protection against potential free radical formation and related biochemical changes in hypoxic, ischemic and Ca²⁺-depleted rat heart upon normal reperfusion.

In the heart of normally fed rats a decrease in the activity of superoxide dismutase and the capacity of the glutathione system, factors of the cellular protective mechanisms against free radicals, occurred upon exposure to the above mentioned treatments. This decrease was not further enhanced if vitamin E-deficient rat hearts were treated. Vitamin E-deficiency, however, led to detectable peroxidation of lipids if Ca²⁺-depleted or hypoxic hearts were reperfused. Lipid peroxidation was measured as the formation of thiobarbituric acid reactive material, which is readily formed during this process. Reflow after ischemia did not induce lipid peroxidation either in normal or in vitamin E-deficient rat heart.

Since changes in Ca²⁺ -homeostasis are thought to be primarily responsible for the Ca²⁺-reperfusion injury, a role for Ca²⁺-ions in lipid peroxidative processes, either directly or indirectly, seems indicated. Furthermore the results imply that even a sharp and extensive decrease of reduced glutathione, as seen upon Ca²⁺ -repletion after a period of Ca²⁺ -depletion, does not necessarily induce peroxidation of lipids in heart tissue. Obviously, vitamin E is very important in the protection of cardiac membranes. Replenishment of the water-soluble protective factors in the heart seems, however, more important during above mentioned treatments, especially since repair of the vitamin E-free radical is dependent on water-soluble factors.     

Spin Trapping by Use of N-Tert-Butylhydroxylamine. Involvement of Fenton Reactions

Spin trapping of short-lived R. radicals is done by use of N-tert-butylhydroxylamine (1) and H²O². The hydroxylamine is oxidized to the radical t-BuN(O)H (2) which is converted into the spin trap 2-methyl-2-nitrosopropane (3). Simultaneously, hydroxyl radicals. OH are formed from H²O². The latter radical species abstracts hydrogen atoms from suitable molecules HR to give R. radicals, which are trapped with the formation of aminooxyl radicals, i. e., t-BuN(O)R (4) detectable by EPR spectroscopy. The reaction is enhanced by the presence of iron ions. The cleavage of H²O² into. OH radicals is considered to involve both a radical-driven (t-BuN(O)H 2) and an iron-driven Fenton reaction.     

The Effect of the Phenolic Antioxidant Ferulic Acid on the Oxidation of Low Density Lipoprotein Depends on the Pro-oxidant Used

The action of ferulic acid during the oxidation of LDL has been investigated using both copper ions and the haem protein metmyoglobin as pro-oxidants. The results demonstrate the ability of ferulic acid to act as a pro-oxidant when LDL oxidation is induced by copper at concentrations of the phenolic acid which are protective when the LDL oxidation is mediated by metmyoglobin. The suggested mechanism involves the reduction of Cu²⁺ to Cu⁺ by ferulic acid resulting in the production of the ferulic phenoxyl radical.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

Cu2+-catalyzed oxidative degradation of thyroglobulin

Thyroglobulin (Tg) was subjected to metal-catalyzed oxidation, and the oxidative degradation was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. In contrast to no effect of hydrogen peroxide (H₂O₂) alone on the Tg degradation, the inclusion of Cu²⁺ (30 μM), in combination with 2 mM H₂O₂, caused a remarkable degradation of Tg, time- and concentration-dependent. The action of Cu²⁺ was not mimicked by Fe²⁺, suggesting that Tg may interact selectively with Cu²⁺. A similar degradation of Tg was also observed with Cu²⁺corbate system, and the concentration of Cu²⁺ (5-10 μM), in combination with ascorbate, required for the effective degradation was smaller than that of Cu²⁺ (10-30 μM) in combination with H₂O₂. In support of involvement of H₂O₂ in the Cu²⁺ corbate action, catalase expressed a complete protection. However, hydroxyl radical scavengers such as dimethylsulfoxide or mannitol failed to prevent the oxidation of Tg whereas phenolic compounds, which can interact with Cu²⁺, diminished the oxidative degradation, presumably consistent with the mechanism for Cu²⁺-catalyzed oxidation of protein. Moreover, the amount of carbonyl groups in Tg was increased as the concentration (3-100 μM) of Cu²⁺ was enhanced, while the formation of acid-soluble peptides was not remarkable in the presence of Cu²⁺ up to 200 μM. In further studies, Tg pretreated with heat or trichloroacetic acid seemed to be somewhat resistant to Cu²⁺-catalyzed oxidation, implying a possible involvement of protein conformation in the susceptibility to the oxidation. Based on these observations, it is proposed that Tg could be degraded non-enzymatically by Cu²⁺-catalyzed oxidation.     

Isolated Cerebral and Cerebellar Mitochondria Produce Free Radicals when Exposed to Elevated Ca2+ and Na+: Implications for Neurodegeneration

Abstract: The evidence is compelling that free radicals, plus increases in free cytosolic Ca²⁺ and Na⁺, figure prominently in neuronal death after exposure to glutamate and dicarboxylic excitotoxins such as NMDA and kainate. However, neither the source of these radicals nor the direct connection between Ca²⁺ mobilization and radical production has been well defined. Electron paramagnetic resonance studies reported here indicate that intact mitochondria isolated from adult rat cerebral cortex and cerebellum generate extremely reactive hydroxyl (•OH) radicals, plus ascorbyl and other carbon-centered radicals when exposed to 2.5 µ M Ca²⁺, 14 m M Na⁺, plus elevated ADP under normoxic conditions, circumstances that prevail in the cytoplasm of neurons during excitotoxin-induced neurodegeneration. In a feed-forward cycle, exposure of isolated mitochondria to •OH significantly increases subsequent radical production five- to 16-fold (average = 8.8 ± 1.6 SE, n = 6, p > 0.01) with succinate as substrate, and also selectively impairs function of NADH-CoQ dehydrogenase activity (electron transport complex 1). These effects are also reflected by respiration rates that are reduced 48% with complex 1 substrates, but increased 27% with complex 2 substrate, after •OH exposure. Comparable complex 1 dysfunction is observed in mitochondria isolated from the substantia nigra of Parkinson's disease patients, from platelets of Huntington's disease patients, and from neocortex of Alzheimer's disease patients. Mitochondrial radical production provides a testable model, based on oxyradical toxicity, oxidative enzyme inactivation, and mitochondrial dysfunction, for the final common pathway of neuronal necrosis during excitotoxicity, and in a host of neurodegenerative disorders.     

Differences in the reactivity of phthalic hydrazide and luminol with hydroxyl radicals

The reactivity of 5-amino-2,3-dihydro-phthalazine-1,4-dione (luminol) and phthalic hydrazide with hydroxyl radicals was studied. HO^·-radicals were generated by the Fenton reaction as well as by water radiolysis. Both luminol and phthalic hydrazide react with hydroxyl radicals under intense chemiluminescence (CL) emission. However, exclusively the CL arising from phthalic hydrazide oxidation can be quenched by competition (e.g. by the addition of carbohydrates), whereas luminol CL is enhanced.

The reactivities of both compounds with HO^·-radicals were further studied by time-resolved spectroscopy (pulse radiolysis), competition methods, NMR spectroscopy and mass spectrometry. Whereas only slight differences were detectable by pulse radiolysis, the analysis of competition kinetics in the presence of p-nitroso-dimethylaniline (NDMA) gave a two-fold-enhanced reactivity for luminol (4.8 × 10⁹l mol^-1 s^-1) in comparison to phthalic hydrazide (2.0 × 10⁹l mol^-1s^-1).

NMR and mass spectrometric analyses revealed significant differences in the reactivity of HO^·-radicals: whereas in luminol solutions hydroxylation of the aromatic ring system predominated, hydroxylated products were not detectable upon irradiation of phthalic hydrazide. A hypothetical mechanism is proposed which may explain the observed differences.     

Cu ions interact with cell membranes

The influence of Cu²⁺ ions on the physical properties of resealed human erythrocyte membranes was studied by fluorescence spectroscopy. A net ordering effect was observed at the hydrophobic–hydrophilic interface both in the bulk as well as in the lipid–protein boundary. The explanation for this result was found by X-ray diffraction performed in multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Cu²⁺ did not significatively affect the structure of DMPE; however, DMPC polar head and hydrocarbon chain arrangements were perturbed at low but reordered at high Cu²⁺ concentrations. These effects were respectively explained in terms of a limited and extended interaction between Cu²⁺ ions and DMPC PO₄⁻ groups. Thus, the ordering effect in the erythrocyte membrane could be based on the interaction of this cation with phosphatidylcholine phosphate groups located in its outer leaflet. This binding, besides producing a decrease of membrane fluidity, might also induce a change in its electric field. These two effects should affect the activity of membrane proteins, particularly of ion channels. In fact, it was found that increasing concentrations of Cu²⁺ ions applied to either the mucosal or serosal surface of the isolated toad skin elicited a dose-dependent decrease of the short-circuit current (SCC) and of the potential difference (PD). These results lead to the conclusion that Cu²⁺ ions inhibited Na⁺ transport across the epithelial cell membranes.     

Manganese and copper promote the binding of dopamine to “serotonin binding proteins” in bovine frontal cortex

The authors previously reported that Fe²⁺ is capable of increasing the binding of dopamine and of serotonin to “serotonin binding proteins” which are present in soluble extracts from calf brain. In this study, it is shown that Mn²⁺ and Cu²⁺ are also capable of increasing the binding, but for dopamine only. As for Fe²⁺, Mn²⁺ and Cu²⁺ are likely to promote the binding by virtue of their ability to enhance the oxidation of dopamine into dopamine-O-quinone, a derivative which is known to undergo covalent association with sulfhydryl groups of proteins. Data such as the irreversible nature of the majority of the binding, the inhibitory action of reducing agents (sodium ascorbate) and of reagents which contain, or modify sulfhydryl groups (reduced glutathione) are compatible with such a mechanism. The three metal ions are also capable of inactivating part of the binding sites on SBP directly; this effect is more pronounced for Cu²⁺ than for Fe²⁺ and it is only weak for Mn²⁺. The Fe²⁺-mediated binding of dopamine is inhibited by the superoxide dismutase enzyme, and it was therefore suggested that Fe²⁺ enhances the oxidation of dopamine by virtue of its ability to produce superoxide radicals out of dissolved molecular oxygen. Such a mechanism does not appear to take place in the case of Mn²⁺ and Cu²⁺. Instead, it is likely that Cu²⁺ and dopamine form a complex which is highly susceptible towards oxidation by dissolved molecular oxygen. Mn²⁺, on the other hand, can easily be oxidized into Mn³⁺, which is capable to oxidize dopamine by itself. Chronic manganese intoxication (from exposure to manganese) and Wilson's disease (related to inadequate elimination of copper) go along with neurological symptoms which are very similar to those encountered in Parkinson's disease. Our data indicate that manganese and copper ions accelerate the oxidation of catecholamines to produce toxic quinones. These quinones could, at least in part, account for the degeneration of dopamininergic neurons in such pathologies.     

Kinetics of the Competitive Degradation of Deoxyribose and Other Molecules by Hydroxyl Radicals Produced by the Fenton Reaction in the Presence of Ascorbic Acid

The competition method in which the Fenton reaction is employed as an OH radical generator and deoxyribose as a detecting molecule, has been used to determine the rate constants for reactions of the OH radical with its scavengers. Nonlinear competition plots were obtained for those scavengers which reacted with the Fenton reagents (Fe²⁺ or H₂O₂). Ascorbic acid is believed to overcome this problem. We have investigated the kinetics of deoxyribose degradation by -OH radicals generated by the Fenton reaction in the presence of ascorbic acid, and observed that the inclusion of ascorbic acid in the Fenton system greatly increased the rate of OH radical generation. As a result, the interaction between some scavengers and the Fenton reagents became negligeable and linear competition plots of A7A vs scavenger concentrations were obtained. The effects of experimental conditions such as, the concentrations of ascorbic acid, deoxyribose, H₂O₂ and Fe²⁺-EDTA, the EDTA/Fe²⁺ ratio as well as the incubation time, on the deoxyribose degradation and the determination of the rate constant for mercaptoethanol chosen as a reference compound were studied. The small standard error, (6.76± 0.21) ±' 10⁹M^-1s^-1 observed for the rate constant values for mercaptoethanol determined under 13 different experimental conditions, indicates the latter did not influence the rate constant determination. This is in fact assured by introducing a term, k_x, into the kinetic equation. This term represents the rate of-OH reactions with other reagents such as ascorbic acid, Fe²⁺-EDTA, H₂O₂ etc. The agreement of the rate constants obtained in this work with that determined by pulse radiolysis techniques for cysteine, thiourea and many other scavengers, suggests that this simple competition method is applicable to a wide range of compounds, including those which react with the Fenton reagents and those whose solubility in water is low.     

Effects of hypochlorite on cultured respiratory epithelial cells

Neutrophils and eosinophils are involved in the pathogenesis of many respiratory diseases. The enzymes myeloperoxidase and eosinophil peroxidase catalyze the reaction of H₂O₂ with Cl to produce the reactive oxygen species HOCl.

Normal human bronchial epithelial (NHBE) cells were exposed to 0.18-0.90 mM HOCl for 48 h, and studied with immunohistochemical, metabolic and morphological studies.

The ability of the cells to attach to each other and/or to the matrix was altered. Immunohistochemical studies showed a decreased amount of desmosomes and focal adhesion sites, although the morphology of the cells was not affected. The ability of the mitochondria to oxidize glucose was reduced. HOCl-exposed cells had an increased production of NO, probably by an increased activity of cNOS, due to increased intracellular Ca²⁺. The antioxidant N-acetylcysteine inhibited both the NO production and the effects of HOCl on glucose oxidation. The cNOS-inhibitor N-propyl-L-arginine inhibited HOCl-induced NO production. X-ray microanalysis showed an increase in the intracellular Na⁺/K⁺ ratio, which indicates cell damage.

In conclusion, exposure to HOCl results in cell detachment and metabolic alterations in normal human bronchial epithelial cells. Oxygen radicals could in part mediate the effects. Oxygen radicals could hence contribute to the observed epithelial damage in respiratory diseases.     

Hydroxyl Radical-Induced Reactions in Polyadenylic Acid as Studied by Pulse Radiolysis: Part I. Transformation Reactions of Two Isomeric OH-Adducts

The absorption spectra of polyadenylic acid (polyA) radicals in N₂0 saturated aqueous solution have been measured as a function of time (up to 15 s) following an 0.4μS electron pulse. The spectra and their changes were analysed by comparison with those from monomeric adenine derivatives (nucleosides and nucleotides) which had been studied by Steenken.¹

The reaction of OH^· radicals with the adenine moiety in poly A results in the formation of two hvdroxvl adducts at the positions C-4 [polyA40H^·] and C-8 [polyA80H^·]. Each OH-adduct undergoes a unimol-ecular transformation reaction before any bimolecular or other unimolecular decay occurs. These reactions are characterized by different rate constants and _pH dependencies. The polyA40H^· adduct undergoes a dehydration reaction to yield a neutral N⁶ centered radical (rate constant K_deh= 1.4 × 10⁴s^-1 at ^pH7.3). This reaction is strongly inhibited by H⁺. In comparison with the analogous reactions in adenosine phosphates, the kinetic _pK value for its inhibition is two ^pH units higher. This shift is the result of the counter ion condensation or double-strand formation. The polyA80H^· adduct undergoes an imidazole ring opening reaction to yield an enol type of formamidopyrimidine radical with the resulting base damage (k^r.o. = 3.5 × 10⁴ s ^-1 at pH7.3). This reaction in contrast is strongly catalysed by H⁺and OH^-, similar as for adenosine but different compared to the nucleotides.     

Transition Metals Potentiate Paraquat Toxicity

The involvement of transition metal ions in paraquat toxicity was studied in bacterial model system. We show that the addition of micromolar, or lower, concentrations of copper dramatically enhanced the rate of bacterial inactivation. In contrast, the addition of chelating agents totally eliminated the killing of E. coli. No inactivation was observed under anaerobic exposure to paraquat, both in the absence and presence of copper. However, in the presence of copper, the anaerobic addition of hydrogen peroxide resulted in complete restoration of inactivation as under aerobiosis.

Paraquat either produces superoxide ions or directly reduces bound copper ions in a catalytic mode. The reduced cuprous complexes react with hydrogen peroxide to locally form hydroxyl radicals (OH) which are probably responsible for the deleterious effects.

This study indicates the involvement of a site-specific metal-mediated Haber-Weiss mechanism in paraquat toxicity. It is in agreement with earlier observations that copper unusually enhance biological damage induced by either superoxide or ascorbate.     

Iron (II) Ions Induced Oxidation of Ascorbic Acid and Glucose

Lipid peroxidation (LPO) of polyunsaturated fatty acids (PUFAs) is suspected to be involved in the generation of chronic diseases. A model reaction for LPO is the air oxidation of PUFAs initiated by Fe²⁺ and ascorbic acid. In the course of such model reactions glycolaldehyde (GLA) was detected as main aldehydic product. Since it is difficult to explain the generation of GLA by oxidation of PUFAs, it was suspected that GLA might be derived by oxidation of ascorbic acid. This assumption was verified by treatment of ascorbic acid with Fe²⁺.

Produced aldehydic compounds were trapped by addition of pentafluorobenzylhydroxylamine hydrochloride (PFBHA-HCl), trimethylsilylated and finally identified by gas chromatography/mass spectrometry (GC/MS). Oxidation of ascorbic acid with O₂ in presence of iron ions produced not only glycolaldehyde (GLA), but also glyceraldehyde (GA), dihydroxyacetone (DA) and formaldehyde. Glyoxal (GO) and malondialdehyde (MDA) were detected as trace compounds.

The yield of the aldehydic compounds was increased by addition of lipid hydroperoxides (LOOH) or H₂O₂. The buffer influenced the reaction considerably: Iron ions react with Tris buffer by producing dihydroxyace-tone (DA). Since ascorbic acid is present in biological systems and Fe²⁺ ions are obviously generated by cell damaging processes, the production of GLA and other aldehydic components might add to the damaging effects of LPO.

Glucose suffers also oxidation to short-chain aldehydic compounds in aqueous solution, but this reaction requires addition of equimolar amounts of Fe²⁺ together with equimolar amounts of H₂O₂ or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HPODE). Therefore this reaction, also influenced by the buffer system, seems to be not of biological relevance.     

Reactions of captopril and epicaptopril with transition metal ions and hydroxyl radicals: An EPR spectroscopy study

In the present study, using the technique of EPR spin trapping with DMPO a spin trap, we demonstrated formation of thiyl radicals from thiol-containing angiotensin converting enzyme (ACE) inhibitor captopril (CAP) and from its stereoisomer epicaptopril (EPICAP), a non-ACE inhibitor, in the process of ^.OH radical scavenging. Splitting constants of DMPO/thiyl radical adducts were identical for both thiols and were a_N = 15.3 G, and a_H = 16.2 G. Bimolecular rate constants for the reaction of CAP and EPICAP with ^.OH radicals were close to a diffusion-controlled rate (≈ 2 × 10¹⁰ M⁻¹s⁻¹). Our data also show that both CAP and EPICAP reduce Fe(III) ions and that their respective thiyl radicals are formed in this reaction. In the presence of Fe(III), H₂O₂, and CAP, or EPICAP, ^.OH radicals were produced by a thiol-driven Fenton mechanism. Copper(II) ions were also reduced by these thiols, but no thiyl radicals could be detected in these reactions, and no ^.OH or other Fenton oxidants were observed in the presence of H₂O₂. Our data show direct evidence that thiol groups of CAP and EPICAP are involved in scavenging of ^.OH radicals. The direct ^.OH radical scavenging, together with the reductive “repair” of other sites of ^.OH radical attack, may contribute to the known protective effect of CAP against ischemia/reperfusion-induced arrhythmias. The formation of reactive thiyl radicals in the reactions of the studied compounds with ^.OH radicals and with Fe(III) ions may play a role in some of the known adverse effects of CAP.