Long-lived radicals of amino acids induced by X-ray radiation are the source of hydrogen peroxide in aqueous medium

Formation of long-lived radicals in solutions of casein and its hydrolysate with an equimolar mixture of amino acids was compared by measuring the X-ray-induced chemiluminescence. It was shown that free amino acids constituting the protein produce long-lived radicals. It was demonstrated that some amino acids (Leu, Ile, Val, Ser, Trp, Met, Pro, Arg, Gly, Phe) emit light of visible spectrum over a long period of time after irradiation, which indicates generation of long-lived radicals of these amino acids. The half-life times of these radicals are several hours. Dissolution of irradiated dry amino acids capable of luminescing over a long time causes formation of hydrogen peroxide in the aqueous medium.     

Formation of hydrogen peroxide and hydroxyl radicals in aqueous solutions of L-amino acids by the action of X-rays and heat

The action of 1 mM solutions of L-amino acids in 5 mM phosphate buffer, pH 7.4, on the production of hydrogen peroxide and hydroxyl radicals under the action of X-rays and heating has been studied. Hydrogen peroxide was estimated by the method of enhanced luminescence in a system luminol-paraiodophenol-peroxidase and hydroxyl radicals were determined by using the fluorescence probe coumarin-3-carboxylic acid. It was shown that amino acids can be divided by their influence on H202 formation into three groups: those that reduce the yield of H202, that do not influence it, and that increase it. A similar action of amino acids was observed upon heating, but the composition of the groups was different. All amino acids lowered the formation of hydroxyl radicals under the action of X-rays, and the most effective among them were Cys > His > Phe = Met = Trp > Tyr. Met, His and Phe lowered the amount of hydroxyl radicals by heating, Ser raised it, whereas Tyr and Pro did not change it. Thus, amino acids differently influence the formation of reactive oxygen species by the action of X-rays and heat, and some of amino acids reveal themselves as effective natural antioxidants.     

Long-lived mutagenic radicals induced in mammalian cells by ionizing radiation are mainly localized to proteins

We have provided evidence that long-lived radicals, produced by ionizing radiation, are highly mutagenic and transforming in mammalian cells. Long-lived radicals are scavenged effectively by vitamin C or by epigallocatechin-3-O-gallate (EGCG). Long-lived radicals are not involved in lethality or in the induction of chromosome aberrations. We now report the results of experiments that define the relative amounts of long-lived radicals in DNA and proteins and identify the major protein radicals as sulfinyl radicals (R-CH2-S-O*). To make these assignments, yields of long-lived radicals in gamma-irradiated salmon sperm DNA and albumin were compared by ESR. ESR spectra of long-lived radicals produced in irradiated Syrian hamster embryo (SHE) cells were analyzed precisely and compared with ESR parameters obtained by density functional theory calculations. Long-lived radicals yields of 99.8% were produced in proteins. We also identified a new type of long-lived radical as H-added phenylalanine radicals. While our evidence does not rule out the possibility of important biological consequences of the low-level long-lived radicals created by radiation, it implicates radicals in proteins as playing a key role in genetic effects of ionizing radiation. We suggest that these novel radicals, wherever they reside, need to be considered in explanations of biological sequela of radiation.     

Epoxidation of cottonseed oil by aqueous hydrogen peroxide catalysed by liquid inorganic acids

The kinetics of epoxidation of cottonseed oil by peroxyacetic acid generated in situ from hydrogen peroxide and glacial acetic acid in the presence of liquid inorganic acid catalysts were studied. It was possible to obtain up to 78% relative conversion to oxirane with very less oxirane cleavage by in situ technique. The rate constants for sulphuric acid catalysed epoxidation of cottonseed oil were in the range 0.39-5.4 x 10(-6)L mol(-1)s(-1) and the activation energy was found to be 11.7 kcal mol(-1). Some thermodynamic parameters such as enthalpy, entropy, and free energy of activation were determined to be of 11.0 kcal mol(-1), -51.4 cal mol(-1)K(-1) and 28.1 kcal mol(-1), respectively. The order of effectiveness of catalysts was found to be sulphuric acid>phosphoric acid>nitric acid>hydrochloric acid. Acetic acid was found to be superior to formic acid for the in situ cottonseed oil epoxidation.     

An EPR study of the peroxyl radicals induced by hydrogen peroxide in the haem proteins

The reaction of hydrogen peroxide H(2)O(2) with horse heart metmyoglobin (HH metMb), sperm whale metmyoglobin (SW metMb) and human metHb (metHbA) was studied at pH 6-8 by low temperature (10 K) EPR spectroscopy with the emphasis on the peroxyl radicals formed during the reaction. The same type of peroxyl radical was found in both myoglobin systems, as was concluded from close similarities in the spectroscopic properties of the radicals and in their kinetic dependences. This is consistent with previous reports of the peroxyl radical being localised on the Trp14 of SW and HH myoglobins. There are two types of peroxyl radical found in the metHbA/H(2)O(2) system, one (ROO-I) having spectral parameters, kinetic and pH dependences similar to those of the peroxyl radical found in both myoglobin systems. The other peroxyl radical (ROO-II) found in metHbA treated with H(2)O(2) has slightly different, though distinguishable, spectral parameters and a significantly different kinetic dependence as compared to those of the peroxyl radical common for all three proteins studied (ROO-I). The concentration of ROO-I radical formed in the three proteins on addition of H(2)O(2) correlates with the effectiveness of incorporating molecular oxygen into styrene oxide reported before for these three proteins. It is shown that a different distance from Trp14 to haem iron in the three proteins might be the structural basis for the different yield of the peroxyl radical and the different efficiency of incorporation of molecular oxygen into styrene. The site of the peroxyl radical found only in metHbA (ROO-II) is speculated to be the Trp37 residue of the beta-subunit of HbA.     

Glutathione peroxidase in lens and a source of hydrogen peroxide in aqueous humour

1. Glutathione peroxidase has been demonstrated in cattle, rabbit and guineapig lenses. 2. The enzyme will oxidize GSH either with hydrogen peroxide added at the start of the reaction or with hydrogen peroxide generated enzymically with glucose oxidase. 3. No product other than GSSG was detected. 4. Oxidation of GSH can be coupled with oxidation of malate through the intermediate reaction of glutathione reductase and NADPH₂. 5. Traces of hydrogen peroxide are present in aqueous humour: it is formed when the ascorbic acid of aqueous humour is oxidized. 6. Hydrogen peroxide will diffuse into the explanted intact lens and oxidize the contained GSH. The addition of glucose to the medium together with hydrogen peroxide maintains the concentration of lens GSH. 7. Glutathione peroxidase in lens extracts will couple with the oxidation of ascorbic acid. 8. It is suggested that, as there is only weak catalase activity in lens, glutathione peroxidase may act as one link between the oxygen of the aqueous humour and NADPH₂.     

Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide

Cell killing by ionizing radiation has been shown to be caused by hydroxyl free radicals formed by water radiolysis. We have previously suggested that the killing is not caused by individual OH free radicals but by the interaction of volumes of high radical density with DNA to cause locally multiply damaged sites (LMDS) (J. F. Ward, Radiat. Res. 86, 185-195, 1985). Here we test this hypothesis using hydrogen peroxide as an alternate source of OH radicals. The route to OH production from H2O2 is expected to cause singly damaged sites rather than LMDS. Chinese hamster V79-171 cells were treated with H2O2 at varying concentrations for varying times at 0 degree C. DNA damage produced intracellularly was measured by alkaline elution and quantitated in terms of Gray-equivalent damage by comparing the rate of its elution with that of DNA from gamma-irradiated cells. The yield of DNA damage produced increases with increasing concentration of H2O2 and with time of exposure. H2O2 is efficient in producing single-strand breaks; treatment with 50 microM for 30 min produces damage equivalent to that formed by 10 Gy of gamma irradiation. In the presence of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), the yield of damage decreases with increasing DMSO concentration consistent with the scavenging of hydroxyl radicals traveling an average of 15 A prior to reacting with the DNA. In contrast to DNA damage production, cell killing by H2O2 treatment at 0 degree C is inefficient. Concentrations of 5 X 10(-2) M H2O2 for 10 min are required to produce significant cell killing; the DNA damage yield from this treatment can be calculated to be equivalent to 6000 Gy of gamma irradiation. The conclusion drawn is that individual DNA damage sites are ineffectual in killing cells. Mechanisms are suggested for killing at 0 degree C at high concentrations and for the efficient cell killing by H2O2 at 37 degrees C at much lower concentrations.     

Caspases are reversibly inactivated by hydrogen peroxide

Hydrogen peroxide (H₂O₂) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H₂O₂ inhibited the activity of recombinant caspase-3 and caspase-8, half-inhibition occurring at about 17 μM H₂O₂. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H₂O₂ added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H₂O₂-producing NADPH oxidase in macrophages also caused catalase-sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H₂O₂.     

Oxidation of nitroxide radicals by the reaction of hemoglobin with hydrogen peroxide

The ESR signal of 4-hydroxy-1-oxyl-2,2,6,6-tetramethylpiperidine in hemoglobin solution decreased drastically by the addition of hydrogen peroxide. The results of ion-exchange chromatography and sodium tetraphenylborate on the reaction solution showed an oxidation of the nitroxide radical to cation form. On the basis of the comparison of thin layer-chromatogram with the reaction products of the nitroxide radicals with HCl or Br2, the formation of 4-hydroxy-1-oxo-2,2,6,6- tetramethylpiperidinium cation was demonstrated. This result was supported by the 13C NMR measurement.     

Oxidative modification of ferritin induced by hydrogen peroxide

Excess free iron generates oxidative stress that may contribute to the pathogenesis of various causes of neurodegenerative diseases. In this study, we assessed the modification of ferritin induced by H(2)O(2). When ferritin was incubated with H(2)O(2), the degradation of ferritin L-chain increased with the H(2)O(2) concentration whereas ferritin H-chain was remained. Free radical scavengers, azide, thiourea, and N-acetyl-(L)-cysteine suppressed the H(2)O(2)-mediated ferritin modification. The iron specific chelator, deferoxamine, effectively prevented H(2)O(2)-mediated ferritin degradation in modified ferritin. The release of iron ions from ferritin was increased in H(2)O(2) concentration-dependent manner. The present results suggest that free radicals may play a role in the modification and iron releasing of ferritin by H(2)O(2). It is assumed that oxidative damage of ferritin by H(2)O(2) may induce the increase of iron content in cells and subsequently lead to the deleterious condition.     

Fragmentation of human ceruloplasmin induced by hydrogen peroxide

We investigated the fragmentation of human ceruloplasmin induced by H2O2 to study its oxidative damage. When ceruloplasmin was incubated with H2O2, the frequency of the protein fragmentation increased in a proportion to the concentration of H2O2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H2O2. Incubation of ceruloplasmin with H2O2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H2O2 is due to hydroxyl radicals formed by a copper-dependent Fenton-like reaction.     

Extraction of amino acids by aqueous two-phase partition

Summary Amino acids, including lysine, glutamic acid, and phenylalanine, in pure solution or in fermentation broth, were extracted with the aqueous two-phase system consisting of polyethylene glycol and salts, giving a very sharp separation. The partition is influenced by the type and the amount of salts used, pH and components of the broth.     

Superoxide anions are required for the inactivation ofEscherichia coli induced by high concentrations of hydrogen peroxide

The wild-type strain and mutants ofEscherichia coli lacking Mn-superoxide dismutase (Sod A) or Fe-superoxide dismutase (Sod B) are compared for their sensitivity to the H₂O₂ insult (exposure for 15 min at 37°C, in M9 salts). Whereas mode one killing is similar in superoxide dismutase mutants and wild-type cells, the latter strain appears to be more resistant than the former ones to mode two lethality. Furthermore, Sod B cells, as well as wild-type cells but unlike Sod A cells, are capable of reversing the toxicity of the oxidant (even in the presence of chloramphenicol), this effect being observed by gradually increasing the H₂O₂ concentration from 2.5 to 10 mM. It is concluded that (a) superoxide ions may not be involved in the production of mode one killing by H₂O₂, although further experiments are needed to validate or modify this hypothesis; (b) superoxide ions mediate mode two killing by H₂O₂, possibly by reducing trivalent iron to the divalent form; and (c) the intervening zone of partial resistance observed in wild-type and Sod B cells exposed to intermediate H₂O₂ concentrations is not a consequence of Mn-superoxide dismutase induction; it would appear, however, that cells lacking this superoxide dismutase isoenzyme are not proficient in this acquired response.     

Oxidation of amino acids and peptides in reaction with myeloperoxidase, chloride and hydrogen peroxide

Oxidation was studied of N-acetyl derivatives of cystine, cysteine, methionine and glycyltryptophan employing the myeloperoxidase-Cl--H2O2 system at pH 4.5, 6.0 and 7.0. Moreover, oxidation of pentapeptide composed of Leu-Trp-Met-Arg-Phe-COOH with myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and hypochlorite was also studied. It was found that amino-acid derivatives having an amino group bound to an acetyl residue react with functional groups of the side-chain. The -SH groups of N-acetylcysteine and the -SS- group of cystine oxidize to cysteic acid. Methionine residues oxidize to methionine sulphoxide, and tryptophan residues to a derivative of 2-oxoindolone. The same reaction products were obtained when respective amounts of hypochlorous acid were used instead of myeloperoxidase, Cl- and H2O2. Differences in the stoichiometry of reactions of myeloperoxidase-mediated oxidation and hypochlorite oxidation suggest differences in the reaction mechanisms of both studied systems. Interaction of the studied pentapeptide with myeloperoxidase-Cl(-)-H2O2 system as well as with hypochlorite showed that in the peptide molecule individual amino acids oxidize consecutively according to their susceptibility to oxidation. No splitting of peptide bonds was observed. Therefore, a modified peptide with methionine sulphoxide and and oxidized tryptophan incorporated into the molecule was obtained.     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.     

Mechanism of oxyhemoglobin oxidation induced by hydrogen peroxide]

The process of oxyhemoglobin oxidation initiated by hydrogen peroxide in low (10(-7) M) concentrations was investigated. It was found, that H2O2 in this concentration is able to induce the process of chain oxidation of oxyhemoglobin to methemoglobin. The following observations indicate that the process is essentially the chain reaction: 1) The amount of the methemoglobin in haem groups, produced in the reaction, exceed by 20 times the quantity of hydrogen, added initially, to induce the oxidation. 2) Catalase stopped this process at any stage of the reaction. This fact implies that the chain process involves generation of new molecules of H2O2 in the course of oxidation of oxyhemoglobin. The chain reaction proceeded only in the presence of oxygen. But if oxygen was introduced into hemoglobin solution, preincubated with H2O2 in vacuum, than again the oxidation of hemoglobin developed. Apparently, H2O2 in low concentrations appears, mainly, as an inductor of the oxyhemoglobin autooxidation.