Catalase enhances damage to DNA by bleomycin-iron(II): the role of hydroxyl radicals

Bleomycin degrades DNA under aerobic conditions when a ferrous salt is added. This reaction is enhanced by catalase and certain hydroxyl radical scavengers but inhibited by the addition of hydrogen peroxide. A ferricbleomycin complex is, however, stimulated by addition of hydrogen peroxide. These findings suggest that catalase removes hydrogen peroxide and in so doing prevents loss of ferrous ions and formation of hydroxyl radicals (OH.) by a Fenton-type reaction. It further suggests that OH. radicals, when formed, are more involved in the inactivation of bleomycin than in the release of thiobarbituric acid reactive material from DNA.     

The inhibition of oxygen radical release from human neutrophils by resting platelets is reversed by administration of acetylsalicylic acid or clopidogrel

Resting platelets inhibit oxygen radical release from neutrophils. Antiplatelet therapy may support this function by preventing platelet activation. Whether antiplatelet agents affect the antioxidative action of resting platelets in the absence of platelet activation is unknown. The effect of acetylsalicylic acid or clopidogrel administration on the antioxidative action of resting platelets was therefore studied in ten healthy volunteers. Preparations of resting platelets were obtained from 5 subjects each — before, during and after an eight-day course of daily treatment with 100 mg of acetylsalicylic acid or 75 mg of the thienopyridine clopidogrel. Human peripheral blood neutrophils were pretreated with the platelets at a ratio of 1/50 for 45 min; then formyl-Met-Leu-Phe-triggered oxygen radical release was measured fluorometrically. The inhibitory effect of platelets on oxygen radical release from neutrophils which was seen before treatment was abolished by antiplatelet therapy with either of the drugs, and inhibition was restored gradually after discontinuing acetlsalicylic acid/ clopidogrel intake. Results suggest that the protective role of resting platelets in controlling oxygen radical release from neutrophils in the absence of platelet activation may be impaired by antiplatelet therapy.     

Hydrogen peroxide causes the fatal injury to human fibroblasts exposed to oxygen radicals

Oxygen radicals are suspected as being a cause of the cellular damage that occurs at sites of inflammation. The phagocytic cells that accumulate in areas of inflammation produce superoxide, hydrogen peroxide, hydroxyl radical, and probably singlet oxygen in the extracellular fluid. The mechanism by which these oxygen molecules kill cells is unknown. To determine which of the oxygen species is responsible for the cellular killing, we exposed human fibroblasts in culture to oxygen radicals generated by the enzymatic action of xanthine oxidase upon acetaldehyde. Using the amount of chromium-51 released from labeled fibroblasts as an index of cellular death, we found that cells were protected only by interventions that reduce hydrogen peroxide concentration. Agents that inactivate superoxide, hydroxyl radical, and singlet oxygen were ineffective in limiting oxygen radical-induced cellular death.     

Superoxide-dependent oxidation of melatonin by myeloperoxidase

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.     

Chemical and biochemical aspects of superoxide radicals and related species of activated oxygen

The spectrum of biological processes in which oxygen is used by living systems is quite large, and the products include some damaging species of activated oxygen, particularly the superoxide radical (O-.2) and hydrogen peroxide (H2O2). Superoxide radicals and hydrogen peroxide, in turn, can lead to the formation of other damaging species: hydroxyl radicals (.OH) and singlet oxygen (1O2). Hydroxyl radicals react with organic compounds to give secondary free radicals that, in the presence of oxygen, yield peroxy radicals, peroxides, and hydroperoxides. Formation, interconversion, and reactivity of O-.2 and related activated oxygen species, methods available for their detection, and the basis of their biological toxicity are briefly reviewed.     

The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals

Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical "scavengers" compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method.     

Effects of various chemical compounds on spontaneous and hydrogen peroxide-induced reversion in strain TA104 of Salmonella typhimurium.

In experiments designed to determine which active oxygen species contribute to hydrogen peroxide (HP)-induced reversion in strain TA104 of Salmonella typhimurium, 1,10-phenanthroline (an iron chelator, which prevents the formation of hydroxyl radicals from HP and DNA-bound iron by the Fenton reaction), sodium azide (a singlet oxygen scavenger), and potassium iodide (an hydroxyl radical scavenger) inhibited HP-induced reversion. These results indicate that hydroxyl radicals generated from HP by the Fenton reaction, and perhaps singlet oxygen, contribute to HP-induced reversion in TA104. However, reduced glutathione (reduces Fe3+ to Fe2+ and/or HP to water), diethyldithiocarbamic acid (an inhibitor of superoxide dismutase), diethyl maleate (a glutathione scavenger), and 3-amino-1,2,4-triazole (an inhibitor of catalase) did not inhibit HP-induced reversion in TA104. Thus, superoxide radical anions and HP itself do not appear to be the cause of HP-induced reversion in this strain. In experiments on the effect of 5 common dietary compounds (beta-carotene, retinoic acid, and vitamins A, C and E), chlorophyllin (CHL), and ergothioneine, the frequency of revertants in TA104 increased above the spontaneous frequency in the presence of beta-carotene or vitamin C (about 2-fold) or vitamin A (about 3-fold). The 5 dietary antimutagens and CHL did not inhibit HP-induced reversion in TA104. However, L-ergothioneine inhibited HP-induced reversion in this strain. Therefore, it is likely that L-ergothioneine is a scavenger of hydroxyl radicals or an inhibitor of their formation, and perhaps of singlet oxygen, at the concentrations tested in TA104.     

Problems concerning the biochemical action of superoxide dismutase (erythrocuprein).

The decay of the tetraperoxochromate- (V) complex (CrO83theta) was examined to study the substrate specificity of erythrocuprein (super-oxide dismutase). The decay of CrO83theta proved rather complex in aqueous solutions. Apart from the two known oxygen species O2theta and singlet oxygen (1 deltagO2), H2O2 and probably OH radicals were formed. No unequivocal evidence for the appearance of superoxide was obtained. The possible electron transfer from Cr5 to Fe3 (cytochrome c) was also discussed. In Tris buffer, pH 7.8, there were absolutely no signs of superoxide or OH radical formation. In fact, pulse radiolysis measurements employing a homogeneous OH source demonstrated that the Tris and OH radicals react with each other. One mol of H2O2 was generated from 1 mol of CrO83theta in Tris buffer. By contrast, only 0.5 mol H2O2 could be determined when the CrO83theta decay was carried out in 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer, pH 7.8. The phenomenon of reducing oxidized cytochrome c could not fully be assigned to a superoxide-mediated reduction, since erythrocuprein was unable to inhibit this cytochrome c reduction efficiently. The energetic oxygen species (1deltag O2, OH etc.) appearing during the CrO83theta decay gave rise to a clearly detectable chemiluminescence. In this system, erythrocuprein was very active regardless of which buffer was used. Even in the absence of a chemiluminescent mediating agent, which might have interferred with the enzyme, erythrocuprein proved capable of inhibiting the CrO83theta-induced chemiluminescence in a rather specific way. No such specificity was seen in the presence of low molecular weight Cu-chelates including Cu(Tyr)2, Cu(Lys)2 and Cu(His)2. The ability to suppress chemiluminescence was approximately 3 orders of magnitude less pronounced than that of the native enzyme. It is presumed that erythrocuprein reacts with oxygen species other than the superoxide radical.     

Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide

Reactivities of chromium compounds with DNA were investigated by the DNA sequencing technique using 32P 5'-end-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Incubation of double-stranded DNA with sodium chromate(VI) plus hydrogen peroxide or potassium tetraperoxochromate(V) led to the cleavage at the position of every base, particularly of guanine. Even without piperidine, the formation of oligonucleotides was observed, suggesting the breakage of the deoxyribose-phosphate backbone. ESR studies using hydroxyl radical traps demonstrated that hydroxyl radical is generated both during the reaction of sodium chromate(VI) with hydrogen peroxide and the decomposition of potassium tetraperoxochromate(V), and that hydroxyl radical reacts significantly not only with mononucleotides but also with deoxyribose 5-phosphate. ESR studies using a singlet oxygen trap demonstrated that singlet oxygen is also generated both by the same reaction and decomposition, and reacts significantly with deoxyguanylate, but scarcely reacts with other mononucleotides. Furthermore, ESR studies suggested that tetraperoxochromate(V) is formed by the reaction of sodium chromate(VI) with hydrogen peroxide. These results indicate that sodium chromate(VI) reacts with hydrogen peroxide to form tetraperoxochromate(V), leading to the production of the hydroxyl radical, which causes every base alteration and deoxyribose-phosphate backbone breakage. In addition, sodium chromate(VI) plus hydrogen peroxide generates singlet oxygen, which subsequently oxidizes the guanine residue. The mechanism by which both hydroxyl radical and singlet oxygen are generated during the reaction of sodium chromate(VI) with hydrogen peroxide was presented. Finally, the possibility that this reaction may be one of the primary reactions of carcinogenesis induced by chromate(VI) is discussed.     

Generation of reactive oxygen species in water under exposure of visible or infrared irradiation at absorption band of molecular oxygen

It is found that in bidistilled water saturated with oxygen hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sourses, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.     

Generation of reactive oxygen species in water under exposure to visible or infrared irradiation at absorption bands of molecular oxygen

It is found that in bidistilled water saturated with oxygen, hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sources, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.     

The role of thrombocyte prostanoids and products secreted by dense granules in the platelet attachment, spreading and aggregation on collagen substrates

The role of platelet prostanoids and substances released from dense bodies (ADP and serotonin) in the initial attachment, spreading and aggregation of platelets on surfaces coated with I, III, IV and V genetic types of collagen was investigated. A positive linear correlation was found to exist between thrombi-like aggregate formation on collagen substrates and platelet prostanoid synthesis. No correlation was established between platelet aggregate formation and 14C-serotonin release. The cyclooxygenase inhibitor indomethacin and the antagonists of PG endoperoxides and TXA2 (13-APA and BM 13.177) completely block thrombi-like aggregate formation. Neither 13-APA nor BM 13.177 affect platelet spreading, while indomethacin inhibits this process by 25%. The ADP-scavenger CP/CPK inhibits platelet aggregation and spreading by 25-30%. The inhibitors of cyclooxygenase and CP/CPK do not influence the initial attachment of platelets. The data obtained suggest that thrombi-like aggregate formation on collagen substrates is mediated by the synthesis of PG endoperoxides and TXA2; however, in platelet spreading this synthesis plays a limited role. Spreading and aggregation of platelets on collagen substrates is only partly mediated by ADP and serotonin. Initial attachment of platelets does not depend on ADP and serotonin release and PG endoperoxide/TXA2 synthesis.     

Production of superoxide anions and hydrogen peroxide in Ehrlich ascites tumour cell nuclei.

Nuclei isolated from Ehrlich-Lettré ascites tumour cells catalyze the co-oxidation of epinephrine to adrenochrome in the presence of NADPH. Adrenochrome formation is sensitive to superoxide dismutase but not to scavengers of hydroxyl radicals or singlet oxygen. Addition of NADPH also initiates the production of hydrogen peroxide. Moreover measurements of superoxide dismutase activity indicate the presence of this enzyme in the ascites cell nuclei, although the sensitivity of adrenochrome formation to externally added superoxide dismutase indicates that the endogenous enzyme is not sufficient for a complete protection from superoxide radicals.     

Hydrogen peroxide release from human blood platelets

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10⁸ platelets showed a significant release of hydrogen peroxide (6.11 nmol/10⁹ platelets per 20 min, S.D., 0.26, n=9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10⁹ platelets per 20 min from resting platelets (S.D., 2, n=6) vs. 15 nmol/10⁹ platelets per 20 min from stimulated platelets (S.D., 2, n=6).     

The role of active forms of oxygen in platelet aggregation and deaggregation

The effect of hydrogen peroxide on ADP-induced platelet aggregation in the presence of active oxygen species scavengers was studied. It was shown that the superoxide radical and singlet oxygen, alongside with hydrogen peroxide, may play a role in platelet interactions.     

Singlet oxygen production from the peroxidase-catalyzed oxidation of indole-3-acetic acid

The aerobic oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase produces 1268 nm emission characteristic of singlet oxygen. Lactoperoxidase also oxidizes indole-3-acetic acid to produce singlet oxygen, but in contrast to horseradish peroxidase, this enzyme system requires hydrogen peroxide. In both of these systems, the intensity of the 1268 nm emission is small due to quenching of the singlet oxygen by indole-3-acetic acid and by reaction products derived from indole-3-acetic acid. The biomolecular reaction of peroxyl radicals via a Russell mechanism is a plausible mechanism for the singlet oxygen generation in these systems. Under typical conditions of p2H 4.0, 1 microM horseradish peroxidase, 1 mM indole-3-acetic acid, and 240 microM oxygen, the singlet oxygen yield was 15 +/- 1 microM or 13% of the amount predicted by the Russell mechanism.     

Actions of melatonin in the reduction of oxidative stress

Melatonin was discovered to be a direct free radical scavenger less than 10 years ago. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant. In terms of direct free radical scavenging, melatonin interacts with the highly toxic hydroxyl radical with a rate constant equivalent to that of other highly efficient hydroxyl radical scavengers. Additionally, melatonin reportedly neutralizes hydrogen peroxide, singlet oxygen, peroxynitrite anion, nitric oxide and hypochlorous acid. The following antioxidative enzymes are also stimulated by melatonin: superoxide dismutase, glutathione peroxidase and glutathione reductase. Melatonin has been widely used as a protective agent against a wide variety of processes and agents that damage tissues via free radical mechanisms.     

A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions

In human platelets stimulated by thrombin and collagen, diacylglycerol is rapidly produced from phosphatidylinositol. Concurrently, an endogenous protein having a molecular weight of about 40,000 (40K protein) is phosphorylated, and serotonin is released. These reactions are all inhibited by a prior treatment of platelets with prostaglandin E1, dibutyryl cyclic AMP, sodium nitroprusside, or with 8-bromo-cyclic GMP, which are known as potent inhibitors for platelet activation. Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) preferentially phosphorylates 40K protein. As judged by fingerprint analysis, the sites in 40K protein that are phosphorylated during the platelet activation appear to be identical with those phosphorylated by protein kinase C in a purified cell-free system. 12-O-Tetradecanoylphorbol-13-acetate, which directly activates protein kinase C by substituting for diacylglycerol, stimulates 40K protein phosphorylation and release reaction without inducing diacylglycerol formation. Tetracaine, which inhibits protein kinase C by competing with phospholipid, blocks 40K protein phosphorylation and serotonin release without inhibiting the receptor-linked diacylglycerol formation. The results indicate that thrombin and collagen activate platelets in almost similar mechanisms and that protein kinase C may lie on a common pathway which leads to the release of serotonin. However, analysis with indomethacin indicates that the role of thromboxane A2 appears to be more predominant for the action of collagen, and it is suggestive that this arachidonate metabolite activates platelets in an analogous mechanism to thrombin.     

Metabolic Transformation of Neuropeptide Carnosine Modifies Its Biological Activity

1. The ability of carnosine and carnosine-related compounds (CRCs) to interact with several free oxygen radicals is analyzed.2. Carnosine, the CRCs (imidazole, histidine, anserine), and ergothioneine were found to be equally efficient in singlet oxygen quenching. During generation of hydroxyl radicals from hydrogen peroxide in the Fenton reaction, carnosine was found to be more effective than the CRCs tested.3. By measuring the chemiluminescence produced by carnosine and CRCs in rabbit leukocytes in the presence of luminol or lucigenin, we conclude that carnosine and other CRCs play a stimulating role in superoxide oxygen production while suppressing the myeloperoxidase system.4. ADP-induced aggregation of human platelets is slightly stimulated by carnosine but is inhibited by acetylanserine.5. The following rank order of efficiency of CRCs was demonstrated while measuring the oxidation of human serum lipoproteins: acetylcarnosine < acetylanserine < homocarnosine = ophidine < carnosine < anserine.6. The results obtained demonstrate that metabolic transformation of carnosine into CRCs in tissues may play an important role in regulating the native antioxidant status of the organism.     

Participation of active oxygen species in the induction of chromosomal aberrations by cadmium chloride in cultured Chinese hamster cells

The effect of various scavengers of active oxygen species on the induction of chromosomal aberrations by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Incidences of chromosomal aberrations by CdCl2 were partially or fully reduced by the presence of catalase, mannitol (a scavenger of hydroxyl radicals) and butylated hydroxytoluene (BHT, an antioxidant). These findings may indicate participation of the active oxygen species such as hydrogen peroxide (H2O2) or hydroxyl radicals in the clastogenicity of cadmium. In contrast, superoxide dismutase (SOD) and dimethylfuran (a scavenger of singlet oxygen) did not influence incidences of chromosomal aberrations by CdCl2. These results suggest that superoxide anion and singlet oxygen are not directly involved in the clastogenicity of the metal. The presence of aminotriazole (an inhibitor of catalase) increased incidences of chromosomal aberrations by CdCl2. This emphasizes participation of H2O2 in the clastogenicity of cadmium.