Oxidative deamination by hydrogen peroxide in the presence of metals

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H 2 O 2 /Cu 2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O 2 on the oxidative deamination of benzylamine by H 2 O 2 . The formation of benzaldehyde was the greatest with Cu 2+ , and catalysis occurred with Co 2+ , VO 2+ , and Fe 3+ . The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O 2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O 2 /Cu 2+ and hemoglobin/H 2 O 2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H 2 O 2 /Cu 2+ system is discussed.     

Hydroxyl radical formation via iron-mediated Fenton chemistry is inhibited by methylated catechols

The differing effects of O-methylated catecholamines and their dihydroxyphenyl precursors on the production of ^?OH were quantified using a previously established specific salicylate hydroxylation assay in conjunction with a sensitive electrochemical detection system. The production of ^?OH by the Fenton reaction was diminished significantly by O-methylated catecholamines (O-methyldopa, O-methyldopamine, O-methyltyrosine, and N-acetyl-O-methyldopamine), whereas radical production was augmented by dihydroxyphenyls (DOPA, dopamine, and N-acetyldopamine), including those with methylated side chains (N-methyldopamine and α-methyldopa). Monohydroxyphenyls such as octopamine, tyramine, tyrosine, and α-methyltyrosine had little or no effect on radical production. These data show that a methyl group positioned on the side chain of a catecholamine does not alter its pro-oxidant behavior, while a methyl group positioned on the aromatic ring renders the catecholamine sterically or kinetically unfavorable for coordination with transition metals, thus preventing the promotion of Fenton chemistry. These results highlight the importance of O-methylation in forming catechols that are less reactive than their dihydroxyphenyl precursors. Thus, factors regulating the methylation of brain catecholamines may play a crucial role in mediating neuronal integrity during aging and in the pathogenesis of certain neurodegenerative disorders. Competitive side-chain methylation reactions may sustain or perpetuate some dihydroxyphenyls, creating an oxidatively less favorable environment for cells than would result from compounds formed by O-methylation.     

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.     

Degradation of Tribromophenol by Wood-Decaying Fungi and the 1,2-Dihydroxybenzene-Assisted Fenton Reaction

The degradation of 2,4,6-tribromophenol (TBP) by biological and chemical treatments was studied. Biological treatment involved the use of Laetoporeus sulfureus, Gloephyllum trabeum, and Ganoderma australe in liquid culture. Despite the inhibitory effects of TBP on the fungal growth, these fungi were able to degrade TBP after 15 days of biotreatment. At 66, 116, and 183 μ M TBP, the degradation by G. australe was the most efficient (71% to 77%), whereas G. trabeum and L. sulfureus degraded between 50% and 60% of three TBP concentrations. The removal of organic bromine reached values of 50% in all cases. The chemical treatment (1,2-dihydroxybenzene-assisted Fenton reaction) achieved up to 90% of TBP degradation. However, only 40% of TBP was mineralized and the toxicity level did not undergo changes during the chemical treatment. On the other hand, a 30% reduction in toxicity was obtained with a combined chemical-biological treatment.     

Enhanced hydroxyl radical production by dihydroxybenzene-driven Fenton reactions: implications for wood biodegradation

Brown rot fungi degrade wood, in initial stages, mainly through hydroxyl radicals (•OH) produced by Fenton reactions. These Fenton reactions can be promoted by dihydroxybenzenes (DHBs), which can chelate and reduce Fe(III), increasing the reactivity for different substrates. This mechanism allows the extensive degradation of carbohydrates and the oxidation of lignin during wood biodegradation by brown rot fungi. To understand the enhanced reactivity in these systems, kinetics experiments were carried out, measuring •OH formation by the spin-trapping technique of electron paramagnetic resonance spectroscopy. As models of the fungal DHBs, 1,2-dihydroxybenzene (catechol), 2,3-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid were utilized as well as 1,2-dihydroxy-3,5-benzenedisulfonate as a non-Fe(III)-reducing substance for comparison. Higher amounts and maintained concentrations of •OH were observed in the driven Fenton reactions versus the unmodified Fenton process. A linear correlation between the logarithms of complex stability constants and the •OH production was observed, suggesting participation of such complexes in the radical production.     

Chemiluminescence of the Fenton reaction and a dihydroxybenzene-driven Fenton reaction

A dihydroxybenzenes(DHB)-driven Fenton reaction was found to be more efficient than a simple Fenton reaction based on OH radical and activated species production. The reason for this enhanced reactivity by [Fe DHB] complexes is not well understood, but results suggest that it may be explained by the formation of oxidation species different from those formed during the classic Fenton reactions. In previous work, greater concentrations, and more sustained production of OH over time were observed in DHB driven Fenton reactions versus neat Fenton and Fenton-like reactions. In this work, chemiluminescence (CL) was monitored, and compared to OH production kinetics. The CL of the DHB-driven Fenton reaction was shorter than that for sustained production of OH. CL appears to have been caused by excited Fe(IV) species stabilized by the DHB ligands initially formed in the reaction. Formation of this species would have to have occurred by the reaction between OH and Fe(III) in a DHB complex.     

Egg Yolk Phosvitin Inhibits Hydroxyl Radical Formation from the Fenton Reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical (^?OH) formation from H₂O₂ in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited ^?OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the ^?OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H₂O₂. These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

Radical oxidation of the adenine moiety of nucleoside and DNA: 2-hydroxy-2'-deoxyadenosine is a minor decomposition product

A method involving high performance liquid chromatography (HPLC) separation associated with tandem mass spectrometry (MS/MS) detection in the multiple reaction monitoring mode was set-up for the measurement of 2-hydroxy-2'-deoxyadenosine (2-OHdAdo). This modified nucleoside, arising from the radical oxidation of 2'-deoxyadenosine (dAdo), has been described in the literature as a potential biological marker of the Fenton reaction. Using the specific and sensitive HPLC-MS/MS assay, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 4,6-diamino-5-formamidopyrimidine and 2-hydroxy-2'-deoxyadenosine (2-OHdAdo) were measured within 2'-deoxyadenosine and DNA solutions either exposed to γ-rays or treated under Fenton reaction conditions. It was found that the yield of 2-OHdAdo was low compared to that of 8-oxodAdo under most of the oxidative conditions studied. In particular and in contrast to previous works, the formation of 2-OHdAdo was shown to be a minor process both upon gamma irradiation and under Fenton reaction conditions. However, a significant yield of formation of 2-OHdAdo was observed either upon incubation with high concentrations of Fe 2+ ions in the absence of hydrogen peroxide or upon γ-radiolysis of a nucleoside solution in the presence of the copper/ ( o )-phenanthroline complex.     

Inhibition of the Fenton reaction by nitrogen monoxide

The toxicity of iron is believed to originate from the Fenton reaction which produces the hydroxyl radical and/or oxoiron(2+). The effect of nitrogen monoxide on the kinetics of the reaction of iron(II) bound to citrate, ethylenediamine-N,N′-diacetate (edda), ethylenediamine-N,N,N′,N′-tetraacetate (edta), (N-hydroxyethyl)amine-N,N′,N′-triacetate (hedta), and nitrilotriacetate (nta) with hydrogen peroxide was studied by stopped-flow spectrophotometry. Nitrogen monoxide inhibits the Fenton reaction to a large extent. For instance, hydrogen peroxide oxidizes iron(II) citrate with a rate constant of 5.8×10³ M⁻¹ s⁻¹, but in the presence of nitrogen monoxide, the rate constant is 2.9×10² M⁻¹ s⁻¹ . Similar to hydrogen peroxide, the reaction of tert-butyl hydroperoxide with iron(II) complexes is also efficiently inhibited by nitrogen monoxide. Generally, nitrogen monoxide binds rapidly to a coordination site of iron(II) occupied by water. The rate of oxidation is influenced by the rate of dissociation of the nitrogen monoxide from iron(II). Electronic Supplementary Material Supplementary material is available for this article at     

Chlorella vulgaris Aldehyde Reductase Is Capable of Functioning as Ferric Reductase and of Driving the Fenton Reaction in the Presence of Free Flavin

The free flavin-dependent Fenton reaction was detected in cell-free extracts of Chlorella. The corresponding enzyme was purified to homogeneity, and its N-terminal sequence was highly homologous to those of aldo-keto reductase family enzymes. The purified enzyme displayed aldehyde reductase activity in the presence of NADPH. Additionally, it showed ferric reductase activity and drove the Fenton reaction in the presence of free FAD and NADH.     

Quantitation of Hydroxyl Radicals Produced by Radiation and Copper-Linked Oxidation of Ascorbate by 2-Deoxy-d-Ribose Method

We have established controlled conditions for studying the reaction of chemically and radiolytically produced hydroxyl radical (OH) with 2-deoxy-D-ribose (2-DR). Ascorbate (ASC) or dithiothreitol (DTT) and cuprous or cupric ions were used to generate the OH-radical. The OH-radical was detected using the classical method of measuring the amount of thiobarbituric acid reactive products (TBARP) formed by OH-mediated 2-DR degradation, but using sensitive fluorescent detection of the TBARP production to quantify the OH-radical. All experiments were performed with adequate O₂ concentrations. The copper reaction with ASC consumes O₂ in a manner that is strongly dependent on copper concentration, and less dependent on ascorbate concentration. For an independent check of the Cu²⁺ catalyzed ASC oxidation kinetics, the decay of ASC absorbency at 265 nm, as well as the increase of H₂O₂ absorbency at 240 nm, were also monitored. These spectral changes agree well with the O₂ consumption data. TBARP production from 2-DR incubated with a Cu²⁺–ASC mixture or γ-irradiated were also compared. γ-Irradiation of 2-DR solutions shows a dose and 2-DR concentration dependent increase of TBARP generation. Other electron donors, such as DTT, are more complicated in their mechanism of OH-radical production. Incubation of 2-DR with Cu²⁺-DTT mixtures shows a delay (50 min) before OH-radical generation is detected. Our results suggest that the Cu²⁺-ASC reaction can be used to mimic the effects of ionizing radiation with respect to OH-radical generation. The good reproducibility and relative simplicity of the 2-DR method with fluorescence detection indicates its usefulness for the quantitation of the OH-radical generated radiolytically or chemically in carefully controlled model systems. © 1997 Elsevier Science Inc.     

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with ^•NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged ^•NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief ^•NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief ^•NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of ^•NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of ^•NO.     

The effect of pyrophosphate, tripolyphosphate and ATP on the rate of the Fenton reaction

It has been recently reported that pyrophosphate, tri-polyphosphate, ATP and analogous ligands considerably decrease the yield of hydroxyl radicals by the Fenton reaction under conditions where [H₂O₂] > > [Fe(II)L_n]. It was suggested that this effect is due to the slowing down of the Fenton reaction by these ligands. This suggestion seemed surprising as polyphosphate ligands stabilize Fe(III). Indeed, a kinetic study points out that these ligands accelerate the rate of the Fenton reaction by several orders of magnitude. Thus it is suggested that the effect of the ligands on the yield of the hydroxyl radicals is due to the stabilization of the Fe(III) complexes which slows down, or inhibits, their reduction by the radicals formed in the system and thus decreases the overall yield of hydroxyl radicals.     

Modification of a Reaction Vessel for the Determination of Total Mercury in Hair by Cold Vapor Atomic Absorption Spectrometry

A classical cold vapor atomic absorption spectrometric (CVAAS) method for the determination of total mercury in blood has been previously adapted for application to samples of human hair. The reaction vessel specified in the original adaptation was, however, large and difficult to use with small hair samples. In the present study, the reaction vessel has been modified and reduced in size and the protocols have been optimized in order to provide an analytical method that is more efficient, less time-consuming, and gives lower blank values than the original adaptation. The optimized method was validated by multiple, independent, replicated assays of certified reference hair samples, and the mean recovery obtained (98.7%) indicated an efficient performance of the digestion and detection processes. The method was applied to the assay of 144 hair samples derived from populations that had or had not been exposed to mercury from environmental sources. The results from all of the samples analyzed were consistent with those published previously for similar samples.     

Monosaccharide-H2O2 reactions as a source of glycolate and their stimulation by hydroxyl radicals

An analysis of the H(2)O(2)-induced breakdown and transformation of different keto-monosaccharides at physiological concentrations reveals that glycolate and other short-chained carbohydrates and organic acids are produced. Depletion of monosaccharides and glycolate synthesis occurs at increased rates as the length of the carbohydrate chain is decreased, and is significantly increased in the presence of trace amounts of Fe(2+) ions (10 microM). Rates of monosaccharide depletion (initial concentration of 3 mM) observed were up to 1.55 mmol h(-1) in the case of fructose, and 2.59 mmol h(-1) in the case of dihydroxyacetone, depending upon pH, H(2)O(2) concentration, temperature and the presence or absence of catalytic amounts of Fe(2+). Glycolate was produced by dihydroxyacetone cleavage at rates up to 0.45 mmol h(-1) in the absence, and up to 1.88 mmol h(-1) in the presence of Fe(2+) ions (pH 8). Besides glycolate, other sugars (ribose, glyceraldehyde, glucose), glucitol (sorbitol) and organic acids (formic and 2-oxogluconic acid) were produced in such H(2)O(2)-induced reactions with fructose or dihydroxyacetone. EPR measurements demonstrated the participation of the OH radical, especially at higher pH. Presence of metal ions at higher pH values, resulting in increased glycolate synthesis, was accompanied by enhanced hydroxyl radical generation. Observed changes in intensity of DEPMPO-OH signals recorded from dihydroxyacetone and fructose reactions demonstrate a strong correlation with changes in glycolate yield, suggesting that OH radical formation enhances glycolate synthesis. The results presented suggest that different mechanisms are responsible for the cleavage or other reactions (isomerisation, auto- or free-radical-mediated oxidation) of keto-monosaccharides depending of experimental conditions.     

Lysozyme conjugate immune complex formation and the effects on substrate hydrolysis

The defined estrone glucuronide-lysozyme conjugate E3, that is acylated solely at K33, was used as a probe for the steric requirements of the active site cleft of chicken type lysozymes. When the immune complex was formed with an anti-estrone glucuronide antiserum, the rate of lysis of the E3 conjugate with the large bacterial substrate Micrococcus lysodeikticus was inhibited by over 90%. However, when the small hexamer of N-acetyl glucosamine was used as the substrate, the rate of hydrolysis by the immune complex was accelerated by 350% compared with the control rate. Thus, inhibition by the anti-estrone glucuronide cannot be caused simply by steric occlusion of the active site. Other factor(s) in the immune complex activate the hydrolysis reaction, most likely by favouring the conformations that lead to the transition state.     

Oxidation of bovine serum albumin initiated by the Fenton reaction--effect of EDTA, tert-butylhydroperoxide and tetrahydrofuran

Oxidation of bovine serum albumin (BSA) was investigated using different oxidants: The water-soluble azo-initiator 2,2'azo-bis-(2-amidinopropane) hydrochloride (AAPH), a combination of FeCl₃ and ascorbate or the Fenton oxidant consisting of FeCl₂, H₂O₂ and EDTA. In addition, the effects of exogenous compounds such as tert-butyl hydroperoxide (tBuOOH) or solvents such as tetrahydrofuran (THF), often used in model systems, was evaluated. The extent of protein damage was studied by measuring protein carbonyl groups and protein hydroperoxides. The interaction between Fenton oxidant and EDTA, THF or tBuOOH was further characterized using spin trapping electron spin resonance (ESR) spectroscopy. The results showed that the extent of protein oxidation depended on the oxidant used. The Fenton oxidant was the most reactive of the initiators tested. However, in the absence of EDTA, the Fenton system produced protein carbonyl groups on BSA equivalent to that obtained with the other oxidants, however, significantly more protein hydroperoxide was produced. Surprisingly, it was also found that addition of tBuOOH or THF to BSA reduced protein damage when the oxidation was initiated with the Fenton oxidant. ESR investigation showed that EDTA played a key role in the generation of free radicals. It was also revealed that in an EDTA containing system both tBuOOH and THF were able to react with radicals without inducing protein damage in effect protecting BSA from oxidative damage.     

Dna Radiation Damage and its Modification by Metallothionein

Thiol compounds have long been known to protect living cells against the harmful effects of ionizing radiation. Maetallothionein is a naturally occurring low molecular weight polypeptide rich in cysteine residues and may be useful in protection against low-level radiation effects.

Radiation damage to DNA and its nucleotide components and the radioprotective effect of metallothionein have been studied in model chemical systems and compared to its effect on cells. Metallothionein acts both as a free radical scavenger and a reductant, and its radioprotective effectiveness has been studied as a function of dose, drug concentration, and in the presence and absence of oxygen. It is more effective in protecting against sugar-phosphate damage under hypoxic conditions. The chemical modification is greater than that of cell killing as measured by the loss of colony-forming ability. Dose reduction factors greater than two are observed for DNA radioprotection, but the values in cells are much lower. These findings will be discussed in terms of the molecular mechanisms and their implications.