Inactivation of Heart Dihydrolipoamide Dehydrogenase by Copper Fenton Systems. Effect of Thiol Compounds and Metal Chelators

Copper Fenton systems (Cu(II)/H₂O₂ and Cu(II)/Asc) inactivated the lipoamide reductase and enhanced the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). Cupric ions alone were less effective. As a result of Cu(II)/H₂O₂ treatment, the number of titrated thiols in LADH decreased from 6 to 1 per subunit. NADH and ADP (not NAD⁺ or ATP) enhanced LADH inactivation by Cu(II). NADH also enhanced the effect of Cu(II)/H₂O₂. Dihydrolipoamide, dihydrolipoic acid, Captopril, acetylcysteine, EDTA, DETAPAC, histidine, bathocuproine, GSSG and trypanothione prevented LADH inactivation. 100 μM GSH, DL-dithiothreitol, N-(2-mercaptopropionylglicine) and penicillamine protected LADH against Cu(II)/Asc and Cu(II), whereas 1.0 mm GSH and DL-dithiothreitol also protected LADH against Cu(II)/H₂O₂. Allopurinol provided partial protection against Cu(II)/H₂O₂. EthanoI, mannitol, Na benzoate and superoxide dismutase failed to prevent LADH inactivation by Cu(II)/H₂O₂ or Cu(II). Catalase (native or denaturated) and bovine serum albumin protected LADH but that protection should be due to Cu binding. LADH inhibited deoxyribose oxidation and benzoate hydroxylation by Cu(II)/H₂O₂. It is concluded that site-specifically generated HO, radicals were responsible for LADH inactivation by Cu(II) Fenton systems. The latter effect is discussed in the context of ischemia-reoxygenation myocardial injury.     

Bactericidal Activity of Catecholamine Copper Complexes

Washed or growing E. coli cells are killed by epinephrine, norepinephrine or dopamine in the presence of non lethal concentrations of Cu(II). Killing is enhanced by anoxia and by sublethal Concentrations of H₂O₁. The rate of killing is proportional to the rate of catecholamine oxidation. The copper epinephrine complex binds to E. coli cells, induces membrane damage and depletion of the cellular ATP pool. The cells may be partially protected by SOD or catalase but not by OH radical scavengers. Addition of H²O₂ to cells which were sensitized by preincubation with the epinephrine-copper complex, causes rapid killing and DNA degradation. Sensitized cells are not protected by BSA.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

Trypanosoma cruzi dihydrolipoamide dehydrogenase as target of reactive metabolites generated by cytochrome c/hydrogen peroxide (or linoleic acid hydroperoxide)/phenol systems

Abstract

This study determines that cytochrome c (cyt c) catalyses the oxidation of phenol compounds (Phen) in the presence of H₂O₂ or linoleic acid hydroperoxide (LOOH), generating Phen-derived free radicals or other reactive metabolites. These products irreversibly inactivated the dihydrolipoamide dehydrogenase from Trypanosoma cruzi (T cruzi LADH), depending on: the Phen structure, peroxide type, activated cyt c, incubation time and presence of an antioxidant. Nordihydroguaiaretic acid (NDGA) and caffeic acid (CAFF) with cyt c/H₂O₂ or cyt c/LOOH were the most effective inhibitors of T cruzi LADH. The comparison of inactivation values for T cruzi and mammalian heart enzymes demonstrated a greater sensitivity of T cruzi LADH to Phen. GSH, N-acetylcysteine, NAD(P)H, ascorbate and trolox, prevented T cruzi LADH inactivation by acetaminophen. The role of the Phen as potential trypanocidal systems is discussed.     

Inactivation of Copper,Zinc superoxide dismutase by H2O2 : Mechanism of protection

Cu,Zn SOD is known to be inactivated by HO₂⁻ and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO₂⁻. We now report that protective actions of both formate and NADH increase as the pH was raised in the range 8.0–9.5. This is taken to indicate increased accessibility of the Cu site with rising pH and/or increased reactivity of the bound oxidant toward exogeneous substrates at high pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate. The same is likely also true of NADH.     

Inactivation of the plasma membrane ATPase ofSchizosaccharomyces pombe by hydrogen peroxide and by the fenton reagent (Fe2+/H2O2): Nonradicalvs. Radical-induced oxidation

In the absence of added Fe²⁺, the ATPase activity of isolatedSchizosaccharomyces pombe plasma membranes (5–7 μmolP _i per mg protein per min) is moderately inhibited by H₂O₂ in a concentration-dependent manner. Sizable inactivation occurs only at 50–80 mmol/L H₂O₂. The process, probably a direct oxidative action of H₂O₂ on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the radical scavengers mannitol and Tris, and involves a decrease of both theK _m of the enzyme for ATP and theV of ATP splitting. On exposing the membranes to the Fenton reagent (50 μmol/L Fe²⁺ +20 mmol/L H₂O₂), which causes a fast production of HO⁻ radicals, the ATPase is 50–60% inactivated and 90% of added Fe²⁺ is oxidized to Fe³⁺ within 1 min. The inactivation occurs only when Fe²⁺ is added before H₂O₂ and can thus bind to the membranes. The lack of effect of radical scavengers (mannitol, Tris) indicates that HO⁻ radicals produced in the bulk phase play no role in inactivation. Blockage of the inactivation by the iron chelator deferrioxamine implies that the process requires the presence of Fe²⁺ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe²⁺ for H₂O₂, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide radical that gives rise to delayed HO⁻ production.     

Trypanosoma cruzi dihydrolipoamide dehydrogenase is inactivated by myeloperoxidase-generated “reactive species”

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. With MPO/H₂O₂/NaCl, LADH lipoamide reductase and diaphorase activities significantly decreased as a function of incubation time. Iodide, bromide, thiocyanide and chloride effectively supplemented the MPO/H₂O₂ system, KI and NaCl being the most and the least effective supplements, respectively. LADH inactivation by MPO/H₂O₂/NaCl and by NaOCl was similarly prevented by thiol compounds such as GSH, L-cysteine, N-acetylcysteine, penicillamine and N-(2-mercaptopropionyl-glycine) in agreement with the role of HOCl in LADH inactivation by MPO/H₂O₂/NaCl. LADH was also inactivated by MPO/NADH/halide, MPO/H₂O₂/NaNO₂ and MPO/NADH/NaNO₂ systems. Catalase prevented the action of the NADH-dependent systems, thus supporting H₂O₂ production by NADH-supplemented LADH. MPO inhibitors (4-aminobenzoic acid hydrazide, and isoniazid), GSH, L-cysteine, L-methionine and L-tryptophan prevented LADH inactivation by MPO/H₂O₂/NaNO₂. Other MPO systems inactivating LADH were (a) MPO/H₂O₂/chlorpromazine; (b) MPO/H₂O₂/monophenolic systems, including L-tyrosine, serotonin and acetaminophen and (c) MPO/H₂O₂/di- and polyphenolic systems, including norepinephrine, catechol, nordihydroguaiaretic acid, caffeic acid, quercetin and catechin. Comparison of the above effects and those previously reported with pig myocardial LADH indicates that both enzymes were similarly affected by the MPO-dependent systems, allowance being made for T. cruzi LADH diaphorase inactivation and the greater sensitivity of its LADH lipoamide reductase activity towards the MPO/H₂O₂/NaCl system and NaOCl.     

Radical production by hydrogen peroxide/bicarbonate and copper uptake in mammalian cells: modulation by Cu(II) complexes

The presence of the bicarbonate/carbon dioxide pair is known to accelerate the transition metal ion-catalysed oxidation of various biotargets. It has been shown that stable Cu(II) complexes formed with imine ligands that allow redox cycling between Cu(I) and Cu(II) display diverse apoptotic effects on cell cultures. It is also reported that Cu(II)-tetraglycine can form a stable Cu(III) complex. In the present study, radical generation from H₂O₂ and H₂O₂/HCO₃⁻ in the presence of these two different classes of Cu(II) complexes was evaluated by monitoring the oxidation of dihydrorhodamine 123 and NADH and by the quantitative determination of thiobarbituric acid reactive substances (TBARs method). Cu(II)-imine complexes produced low levels of reactive species whereas Cu(II)-Gly-derived complexes, as well as the free Cu(II) ion, produced oxygen-derived radicals in significantly larger amounts. The effects of these two classes of complexes on mammalian tumour cell viability were equally distinct, in that Cu(II)-imine complexes caused apoptosis, entered in cell and remained almost unaffected in high levels whilst, at the same concentrations, Cu(II)-Gly peptide complexes and Cu(II) sulphate stimulated cell proliferation, with the cell managing copper efficiently. Taken together, these results highlight the different biological effects of Cu(II) complexes, some of which have been recently studied as anti-tumour drugs and radical system generators, and also update the effects of reactive oxygen species generation on cell cycle control.     

A kinetic study on the copper-albumin catalyzed oxidation of ascorbate

Serum albumin can specifically bind one Cu(II)-ion, and is proposed to function as a copper transport protein in vivo. Cu(II)-albumin is rapidly reduced by ascorbate. A second order rate constant of 0.54 mM^–1 min^–1 was estimated for the reaction. The oxidation process is catalytic, the Cu(I)-albumin molecule being reoxidized by molecular oxygen. The reaction was found to follow Michaelis-Menten kinetics, characterized by an apparent K_m-value of 0.89 mM, and a catalytic constant of 0.066 M O₂/min. An apparent inhibition of oxygen uptake was obtained with catalase (but not with superoxide dismutase), suggesting the formation of H₂O₂ in the system. Wilson's disease patients usually have increased amounts of non-ceruloplasmin copper in plasma. The low level of plasma ascorbate observed in such patients could possibly be due, at least in part, to an oxidation by Cu(II)-albumin.     

Ascorbic Acid Oxidation and DNA Scission Catalyzed by Iron and Copper Chelates

The asorbic acid (AH^?) auto-oxidation rates catalyzed by copper chelates of 1,10-phenanthroline (OP) or by iron chelates of bleomycin (BLM) are only slightly higher than the oxidation rates catalyzed by the metal ions. AH^? oxidation in the presence of DNA is accompanied by degradation of the DNA. The rates of DNA scission by the metal chelates are markedly higher than the rates induced by the free metal ions. AH^? oxidation is slowed down in the presence of DNA which forms ternary complexes with the chelates. The ternary complexes react slowly with AH^? but induce DNA double strand breaks more efficiently than the free metal chelates. With OP, DNA is degraded by the reaction of the ternary complex, DNA-(OP)₂Cu(I), withH₂O₂

AH^? oxidation in the presence of DNA was biphasic, showing a marked rate increase after DNA was cleaved. We suggest that this sigmoidal pattern of the oxidation curves reflects the low initial oxidative activity of the ternary complexes, accelerating as DNA is degraded.

Using O₂^?produced by pulse radiolysis as a reductant, we found that AH^? oxidation with (OP)₂Cu(II) induced more DNA double strand breaks per single strand break than bipyridine-copper.

The site specific DNA damaging reactions indicated by these results are relevant to the mechanism of cytotoxic activities of bleomycin and similar antibiotics or cytotoxic agents.     

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

An EPR Investigation of Human Methaemoglobin Oxidation by Hydrogen Peroxide: Methods to Quantify all Paramagnetic Species Observed in the Reaction

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglabin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H₂O₂ was added at 3- and 10-fold molar excess to 100 μM metHb in 50 mM phosphate buffer, pH 7.4, 37°C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H₂O₂ the EPR signals (- 196°C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (- 196°C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g° = 2.035 and g° = 2.0053), and the other an isotropic singlet (g = 2.0042, AH = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic Pehaviour of both peroxyl (ROO^*) and nonperoxyl (P^*) free radicals were similar, we concluded that ROO^* radicals are not formed from P^* radicals by addition of O₂. The time courses for both radicals showed a steady state during the time required for H₂O₂ to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 ± 10^-3 s^-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H₂O₂. The formation of the rhombic Fe(III) non-haemcentres with g = 4.35 was found. Their yield was proportional to the H₂O₂ concentration used and the centers were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at —196°C were used.     

The enhancement of oxidative DNA damage by anti-diabetic metformin,buformin, and phenformin,via nitrogen-centered radicals

Metformin (N,N-dimethylbiguanide), buformin (1-butylbiguanide), and phenformin (1-phenethylbiguanide) are anti-diabetic biguanide drugs, expected to having anti-cancer effect. The mechanism of anti-cancer effect by these drugs is not completely understood. In this study, we demonstrated that these drugs dramatically enhanced oxidative DNA damage under oxidative condition. Metformin, buformin, and phenformin enhanced generation of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) in isolated DNA reacted with hydrogen peroxide (H₂O₂) and Cu(II), although these drugs did not form 8-oxodG in the absence of H₂O₂ or Cu(II). An electron paramagnetic resonance (EPR) study, utilizing alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide as spin trapping agents, showed that nitrogen-centered radicals were generated from biguanides in the presence of Cu(II) and H₂O₂, and that these radicals were decreased by the addition of DNA. These results suggest that biguanides enhance Cu(II)/H₂O₂-mediated 8-oxodG generation via nitrogen-centered radical formation. The enhancing effect on oxidative DNA damage may play a role on anti-cancer activity.     

Specific features of the kinetics of H2-O2-O2(a 1Δ g ) mixtures: I. formation and quenching of electronically and vibrationally excited (A′) molecules in H2-O2-O2(A 1Δ g ) mixtures at a temperature of 300 K

Available data on the kinetic processes in H₂-O₂-O₂(a ¹Δ _g ) mixtures are analyzed theoretically, and the ranges in which the rate constants of these processes can vary are determined. The processes of energy transformation in an O₂(a ¹Δ _g )-H₂-H-HO₂ system in the approximations of the fast and slow (in comparison with the vibrational relaxation time of the HO₂ radical) quenching of the electronically excited state are considered. The experiments on the quenching of singlet delta oxygen (SDO) molecules O₂(a ¹Δ _g ) excited in a microwave discharge at a temperature of 300 K and pressure of 6 Torr in a lean hydrogen-oxygen mixture are simulated (by a lean fuel mixture is meant a mixture in which the ratio of the fuel to the oxidizer mass fraction is less than the stoichiometric ratio, which is 2: 1 for a hydrogen-oxygen mixture). It is shown that, over the experimental observation times, the SDO quenching frequency depends on the concentration of molecular hydrogen, the residual odd oxygen fraction in the gas flow, and the ratio between the rate constants of kinetic processes involving HO₂ and HO₂* radicals. Simulations of the microwave discharge and the transport of excited oxygen along the drift tube make it possible to determine the residual odd oxygen concentration in the gas flow. Recommendations on the choice of the rate constants for the O₂(a ¹Δ _g ) + HO₂)A″, v₃″ = 0) ? O₂ + HO₂*(A′, v₃′ = 1), HO₂*(A′v₃′ ≤ 1) + O₂(a ¹Δ _g ) → HO₂*(A′,v₃′ ≥ 6) + O₂, and HO₂*(A′,v₃′ ≤ 1) + O₂(a ¹Δ _g ) → H + O₂ + O₂ processes are given. It is shown that, in the case of slow quenching in a H₂-O₂-O₂(a ¹Δ _g ) mixture at a low temperature, the intensity of hydrogen oxidation is enhanced due to the reaction + HO₂*(A′,v₃′ ≤ 1) + O₂(¹Δ) → H + O₂ + O₂.     

Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H₂O₂) prevents polyspermy in Arbacia punctulata. We found that it is not H₂O₂ but probably hypochlorous acid/hypochlorite (HOCl/OCl^?) derived from H₂O₂ that is toxic to the supernumerary sperm. The spermicidal activity of H₂O₂ is potentiated by at least one order of magnitude by cupric ions (Cu²⁺). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu²⁺. More-over, substitution of Cu²⁺ by ferrous ions (Fe²⁺), which are known to cause formation of ·OH from H₂O₂, had no effect on fertilization even at 10²?10³ times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), an HOCl/OCl^? scavenger, totally reversed the toxic effects of Cu²⁺. Furthermore, we found that HOCl/OCl^? is generated in solutions of H₂O₂ and Cu²⁺ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl^?. HOCl/OCl^? generated by Cu²⁺ from H₂O₂ and Cl^?, a low concentration of exogenously added HOCl/OCl^?, or increased concentrations of H₂O₂ has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl^? from the Cl^? present in sea water through reaction with H₂O₂ generated by the newly fertilized egg.     

Characterisation of the major autoxidation products of 3-hydroxykynurenine under physiological conditions

3-Hydroxykynurenine (3-OHKyn) is a tryptophan metabolite that is readily autoxidised to products that may be involved in protein modification and cytotoxicity. The oxidation of 3-OHKyn has been studied here with a view to characterising the major products as well as determining their relative rates of formation and the role that H₂O₂ and hydroxyl radical (HO^·) may play in modifying the autoxidation process. Oxidation of 3-OHKyn generated several compounds. Xanthommatin (Xan), formed by the oxidative dimerisation of 3-OHKyn, was the major product formed initially. It was, however, found to be unstable, particularly in the presence of H₂O₂, and degraded to other products including the p-quinone, 4,6-dihydroxyquinolinequinonecarboxylic acid (DHQCA). A compound that has a structure consistent with that of hydroxy-xanthommatin (OHXan) was also formed in addition to at least two minor species that we were unable to identify. Hydrogen peroxide was formed rapidly upon oxidation of 3-OHKyn, and significantly influenced the relative abundance of the different autoxidation species. Increasing either pH (from pH 6 to 8) or temperature (from 25°C to 35°C) accelerated the rate of autoxidation but had little impact on the relative abundance of the autoxidation species. Using electron paramagnetic resonance (EPR) spectroscopy, a clear phenoxyl radical signal was observed during 3-OHKyn autoxidation and this was attributed to xanthommatin radical (Xan^·). Hydroxyl radicals were also produced during 3-OHKyn autoxidation. The HO^· EPR signal disappeared and the Xan^· EPR signal increased when catalase was added to the autoxidation mixture. The HO^· did not appear to play a role in the formation of the autoxidation products as evidenced using HO^· traps/scavengers. We propose that the cytotoxicity of 3-OHKyn may be explained by both the generation of H₂O₂ and by the formation of reactive 3-OHKyn autoxidation products such as the Xan^· and DHQCA.     

The amylin peptide implicated in type 2 diabetes stimulates copper-mediated carbonyl group and ascorbate radical formation

Human amylin (hA), which is toxic to islet β-cells, can self-generate H₂O₂, and this process is greatly enhanced in the presence of Cu(II) ions. Here we show that carbonyl groups, a marker of oxidative modification, were formed in hA incubated in the presence of Cu(II) ions or Cu(II) ions plus H₂O₂, but not in the presence of H₂O₂ alone. Furthermore, under similar conditions (i.e., in the presence of both Cu(II) ions and H₂O₂), hA also stimulated ascorbate radical formation. The same observations concerning carbonyl group formation were made when the histidine residue (at position 18) in hA was replaced by alanine, indicating that this residue does not play a key role. In complete contrast to hA, rodent amylin, which is nontoxic, does not generate H₂O₂, and binds Cu(II) ions only weakly, showed none of these properties. We conclude that the hA-Cu(II)/Cu(I) complex is redox active, with electron donation from the peptide reducing the oxidation state of the copper ions. The complex is capable of forming H₂O₂ from O₂ and can also generate ^•OH via Fenton chemistry. These redox properties of hA can explain its ability to stimulate copper-mediated carbonyl group and ascorbate radical formation. The formation of reactive oxygen species from hA in this way could hold the key to a better understanding of the damaging consequences of amyloid formation within the pancreatic islets of patients with type 2 diabetes mellitus.     

Oxidation of ethanol by hepatic microsomes of acatalasemic mice

Hepatic microsomes of acatalasemic Cs^b mice subjected to heat inactivation displayed decreased catalatic activity but NADPH dependent microsomal ethanol oxidation (MEOS) remained active and unaffected. Even without heat inactivation, in the Cs^b strain, the NADPH dependent metabolism of ethanol was much more active than the H₂O₂ mediated one whereas microsomes of Cs^a control mice displayed equal rates of H₂O₂ and NADPH dependent ethanol oxidation. Addition of catalase to liver microsomes in vitro abolished this difference whereas the catalase inhibitor azide established in the Cs^a mice a pattern similar to that of the Cs^b, namely a much more active NADPH dependent than a H₂O₂ mediated ethanol oxidation. The selective persistence in the Cs^b mice of NADPH dependent ethanol oxidation contrasting with the reduction in the H₂O₂ mediated metabolism of ethanol supports the existence of a microsomal ethanol oxidizing system independent of catalase.     

Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence

The ability of paraquat radicals (PQ^+.) generated by xanthine oxidase and glutathione reductase to give H₂O₂-dependent hydroxyl radical production was investigated. Under anaerobic conditions, paraquat radicals from each source caused chain oxidation of formate to CO₂, and oxidation of deoxyribose to thiobarbituric acid-reactive products that was inhibited by hydroxyl radical scavengers. This is in accordance with the following mechanism derived for radicals generated by γ-irradiation [H. C. Sutton and C. C. Winterbourn (1984) Arch. Biochem. Biophys.235, 106–115] PQ^+. + Fe³⁺ (chelate) → Fe²⁺ (chelate) + PQ⁺⁺ H₂O₂ + Fe²⁺ (chelate) → Fe³⁺ (chelate) + OH^? + OH.. Iron-(EDTA) and iron-(diethylenetriaminepentaacetic acid) (DTPA) were good catalysts of the reaction; iron complexed with desferrioxamine or transferrin was not. Extremely low concentrations of iron (0.03 μm) gave near-maximum yields of hydroxyl radicals. In the absence of added chelator, no formate oxidation occurred. Paraquat radicals generated from xanthine oxidase (but not by the other methods) caused H₂O₂-dependent deoxyribose oxidation. However, inhibition by scavengers was much less than expected for a reaction of hydroxyl radicals, and this deoxyribose oxidation with xanthine oxidase does not appear to be mediated by free hydroxyl radicals. With O₂ present, no hydroxyl radical production from H₂O₂ and paraquat radicals generated by radiation was detected. However, with paraquat radicals continuously generated by either enzyme, oxidation of both formate and deoxyribose was measured. Product yields decreased with increasing O₂ concentration and increased with increasing iron(DTPA). These results imply a major difference in reactivity between free and enzymatically generated paraquat radicals, and suggest that the latter could react as an enzyme-paraquat radical complex, for which the relative rate of reaction with Fe³⁺ (chelate) compared with O₂ is greater than is the case with free paraquat radicals.