Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Protein Radical Formation Resulting from Eosinophil Peroxidase-catalyzed Oxidation of Sulfite

Eosinophil peroxidase (EPO) is an abundant heme protein in eosinophils that catalyzes the formation of cytotoxic oxidants implicated in asthma, allergic inflammatory disorders, and cancer. It is known that some proteins with peroxidase activity (horseradish peroxidase and prostaglandin hydroperoxidase) can catalyze oxidation of bisulfite (hydrated sulfur dioxide), leading to the formation of sulfur trioxide anion radical (^·SO₃⁻). This free radical further reacts with oxygen to form peroxymonosulfate anion radical (⁻O₃SOO^·) and the very reactive sulfate anion radical (SO₄^˙̄), which is nearly as strong an oxidant as the hydroxyl radical. However, the ability of EPO to generate reactive sulfur radicals has not yet been reported. Here we demonstrate that eosinophil peroxidase/H₂O₂ is able to oxidize bisulfite, ultimately forming the sulfate anion radical (SO₄^˙̄), and that these reactive intermediates can oxidize target proteins to protein radicals, thereby initiating protein oxidation. We used immuno-spin trapping and confocal microscopy to study protein oxidation by EPO/H₂O₂ in the presence of bisulfite in a pure enzymatic system and in human promyelocytic leukemia HL-60 clone 15 cells, maturated to eosinophils. Polyclonal antiserum raised against the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) detected the presence of DMPO covalently attached to the proteins resulting from the DMPO trapping of protein free radicals. We found that sulfite oxidation mediated by EPO/H₂O₂ induced the formation of radical-derived DMPO spin-trapped human serum albumin and, to a lesser extent, of DMPO-EPO. These studies suggest that EPO-dependent oxidative damage may play a role in tissue injury in bisulfite-exacerbated eosinophilic inflammatory disorders.     

Basic Principles of Reactivity in Free Radical Chemistry

This review is concerned with an overall survey of reactivity in free radical chemistry. A concise classification is given of elementary reaction steps which can be combined in different ways to account for overall chemical transformations: radical forming reactions, radical transformations, and radical destroying reactions. From this is derived the concept of the chain reaction which leads on to an up-to-date theory for understanding reactivity in free radical processes. Finally, a few aspects of autoxidation are discussed.     

Basic Principles of Reactivity in Free Radical Chemistry

This review is concerned with an overall survey of reactivity in free radical chemistry. A concise classification is given of elementary reaction steps which can be combined in different ways to account for overall chemical transformations: radical forming reactions, radical transformations, and radical destroying reactions. From this is derived the concept of the chain reaction which leads on to an up-to-date theory for understanding reactivity in free radical processes. Finally, a few aspects of autoxidation are discussed.     

Free Radicals in Wheat Flour Change During Storage in Air and are Influenced by the Presence of Ozone During the Growing Season

Electron paramagnetic resonance (EPR) spectra of wheat flour show components from Fe(III), Mn(II) and free radicals (FR). The metal signals were higher in the samples from the stressed plants, and reflected the higher total levels of these elements determined analytically. They remained essentially constant throughout the experiment, but the FR signal increased progressively with time over a period of 4-6 months after milling, after which it reached a maximum. The rate of increase in the FR signal during this period was considerably higher in the flour from plants that had been exposed to elevated ozone levels.     

Free radicals in wheat flour change during storage in air and are influenced by the presence of ozone during the growing season

Electron paramagnetic resonance (EPR) spectra of wheat flour show components from Fe(III), Mn(II) and free radicals (FR). The metal signals were higher in the samples from the stressed plants, and reflected the higher total levels of these elements determined analytically. They remained essentially constant throughout the experiment, but the FR signal increased progressively with time over a period of 4-6 months after milling, after which it reached a maximum. The rate of increase in the FR signal during this period was considerably higher in the flour from plants that had been exposed to elevated ozone levels.     

A Novel Interleukin Stimulating Free Radical Production by Granulocytes

Polymorphonuclear granulocytes (PMN) are potent producers of free oxygen-derived radicals. Since other granulocyte functions are affected by interleukins, we investigated whether free-radical production can be initiated by a similar mediator. For estimation of free radical production, SOD-inhibitable lucigenin-dependent chemiluminescence and SOD-inhibitable cytochrome C reduction were used. As a source of interleukins, serum-free 24 h culture supernatants of human mononuclear cells (MNC) stimulated with bacterial lipopolysaccharide were prepared. Addition of such supernatants to PMN caused stimulation of sod-inhibitable chemiluminescence and superoxide production. Studies with separated MNC showed that monocytes were the cellular source of the activity. Biochemically, this activity of the supernatants was due to a heat-labile glycoprotein with a MW of approx. 60KDa. This mediator, termed granulocyte chemiluminescence inducer (GCI), appears to be distinct from interleukin 1 (a and j?) and interferon (a and y). In conclusion we describe a novel monokine, granulocyte chemiluminescence inducer (GCI), which initiates granulocyte free radical production. This interaction of monocytes and granulocytes may also in vivo constitute a new and potent pathway leading to stimulation of free oxygen production by granulocytes.     

A Novel Interleukin Stimulating Free Radical Production by Granulocytes

Polymorphonuclear granulocytes (PMN) are potent producers of free oxygen-derived radicals. Since other granulocyte functions are affected by interleukins, we investigated whether free-radical production can be initiated by a similar mediator. For estimation of free radical production, SOD-inhibitable lucigenin-dependent chemiluminescence and SOD-inhibitable cytochrome C reduction were used. As a source of interleukins, serum-free 24 h culture supernatants of human mononuclear cells (MNC) stimulated with bacterial lipopolysaccharide were prepared. Addition of such supernatants to PMN caused stimulation of sod-inhibitable chemiluminescence and superoxide production. Studies with separated MNC showed that monocytes were the cellular source of the activity. Biochemically, this activity of the supernatants was due to a heat-labile glycoprotein with a MW of approx. 60KDa. This mediator, termed granulocyte chemiluminescence inducer (GCI), appears to be distinct from interleukin 1 (a and j?) and interferon (a and y). In conclusion we describe a novel monokine, granulocyte chemiluminescence inducer (GCI), which initiates granulocyte free radical production. This interaction of monocytes and granulocytes may also in vivo constitute a new and potent pathway leading to stimulation of free oxygen production by granulocytes.     

Free Radical Scavenging Properties of Sulfinpyrazone

Sulfinpyrazone, a potent uricosuric drug, was tested in vitro for its scavenging action against oxygen free radicals. In this study, sulfinpyrazone was able to scavenge 1,1-diphenyl-2-picrylhydrazyl radical with IC 50 value of 29.82 &#119 g/ml compared to butylated hydroxytoluene (BHT, IC 50 value=20.15 &#119 g/ml) and Trolox (IC 50 value=16.01 &#119 g/ml). It was able to scavenge superoxide anion with IC 50 value of 27.72 &#119 g/ml compared to Trolox (IC 50 value=22.08 &#119 g/ml) and ascorbic acid (IC 50 value=14.65 &#119 g/ml). The hydroxyl radical scavenging activity of sulfinpyrazone is in a concentration-dependent fashion. In the range of concentrations used, sulfinpyrazone was not a scavenger toward H 2 O 2 . However, the intracellular H 2 O 2 -induced 2',7'-dichlorofluorescin diacetate (DCF-DA) fluorescence in HL-60 cells was significantly reduced by sulfinpyrazone during 30-60 min of incubation. Finally, phorbol-12-myristate-13-acetate induced-lucigenin chemiluminescence in whole blood was markedly inhibited by sulfinpyrazone. Our results suggest a new direction for the pharmacological actions of sulfinpyrazone in free radical scavenging properties.     

Detection of Hydroxyl free Radicals in the Reperfused Primate Heart

Early reperfusion of an ischemic region can result in significant salvage of the area at risk. We show the presence of hydroxyl free radicals at the time of post ischemia reperfusion using electron paramagnetic resonance (EPR) spectroscopy in a macaque model. These free radicals may be formed as a result of reperfusion or may be an un-involved bystander. It is possible that they may be involved in reperfusion injury.     

The Chemical Origin of Free Radicals in Coffee and Other Beverages

Sugars or carbohydrates are identified as the source of free radicals in coffees, ersatz coffees, a number of other food flavouring and colouring agents formed by processes involving heating, and in beers and stouts. The radicals are not derived from phenolic constituents, in contrast to those in wine, and are unlikely to be due solely to the occurrence of Maillard reactions.     

The prosthetic group of methanol dehydrogenase from Hyphomicrobium X: electron spin resonance evidence for a quinone structure.

Partial reduction of the isolated prosthetic group of methanol dehydrogenase yields a free radical with the same characteristics as the one contained in the enzyme. The Electron Spin Resonance spectrum in alkaline aqueous solution displays hyperfine structure and is interpreted in terms of an isotropic g-value, hyperfine coupling constants and nuclear spins. The magnitudes of these parameters indicate that the prosthetic group is a quinone containing two nitrogen atoms.     

Photogeneration of free radicals (*OH and HB*-) and singlet oxygen (1O2) by hypocrellin B in TX-100 micelles microsurroundings

To solve the problems faced in clinical use of hypocrellins, a water-soluble preparation of Hypocrellin B (HB), HB-Triton X-100 (TX-100) micelles, was prepared. To evaluate the photodynamic activity, the free radicals (^•OH and HB^•¯) and singlet oxygen (

1

O

2

) generated via photosensitization of the preparation in aqueous solution were detected by using electron paramagnetic resonance (EPR) and spectrophotometric methods. It was observed that

1

O

2

was formed with a quantum yield of 0.72, similar to that for HB in organic solvents, further, hydroxyl radicals (

•

OH) could also be efficiently produced by the new preparation, which have never before been detected following HB photoactivities. In addition, the semiquinone anion radicals (HB^•-) could also be generated via the self-electron transfer between an excited triplet state and a ground state molecule. The accumulation of HB^•- would replace that of

•

OH or

1

O

2

with the depletion of oxygen in the system. All these findings suggested that the HB-TX-100 micelles could play the photodynamic action through not only the type I mechanism by free radicals (^•OH, O₂^•- and HB^•-) but also the type II mechanism by singlet oxygen (

1

O

2

). It can be concluded further that the new preparation basically maintains the inherent photodynamic activity of HB, or even higher.     

Characterization of Free Radical Defense System in High Glucose Cultured HeLa-tat Cells: Consequences for Glucose-induced Cytotoxicity

HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1 has a decreased antioxidant potential. In this work, we used this model to investigate the effect of a high glucose level (20 mM) on the glucose induced cytotoxicity and on the antioxidant system. In comparison to cell culture under control medium, HeLa-wild cell cultured under 20 mM glucose did not exhibit necrosis or apoptosis, contrary to HeLa-tat cell presenting a significant increase in necrotic or apoptotic state. Moreover after 48 h culture under high glucose level the HeLa-tat proliferation rate was not higher than the one of HeLa-wild cells. In HeLa-wild cell high glucose level resulted in an induction of glutathione reductase activity in opposition to HeLa-tat cells where no change was observed. High glucose level resulted in 20% increase in GSSG/GSH ratio in HeLa-wild cells and 38% increase in HeLa-tat cells. Moreover, high glucose level resulted in a dramatic cytosolic thiol decrease and an important lipid peroxidation in HeLa-tat cells. No significant change of these two parameters was observed in HeLa-wild cells. In both cell lines, high glucose resulted in an increase of total SOD activity, as a consequence of the increase in Cu,Zn-SOD activity. High glucose did not result in an increase of Mn-SOD activity in both cell lines. As a consequence of tat tranfection Mn-SOD activity was 50% lower in HeLa-tat cells in comparison to HeLa-wild cells. This work emphasizes the importance of the antioxidant system in the glucose induced cytotoxicity.     

抗氧化剂与自由基清除剂对硒性白内障形成的影响

本文观察了8种化合物(抗氧化剂及自由基清除剂)对大鼠亚硒酸钠性白内障的影响。实验分为正常对照组,亚硒酸钠组及药物对抗组。亚硒酸钠组系给13日龄大鼠皮下注射亚硒酸钠(6μmoles/kg体重),间日一次,逐次递增1μmole/kg体重,连续5次,药物对抗组则同时腹腔注射抗氧化剂或自由基清除剂,每日观察并记录白内障的发生频率及程度,实验表明,一些抗氧化剂及自由基清除剂能够有效的对抗亚硒酸钠性白内障的发生发展,其中AC1、AC3及AC3的效果尤为明显。本文的结果为探讨白内障形成机理及防治提供了实验依据。     

活性氧自由基诱发的胆红素聚合、聚集及稳定自由基的形成

 为了深入了解胆红色素类结石形成的触发机制,我们研究了活性氧自由基与胆红素的作用。结果表明:·O_2和·OH均可诱发胆红素聚合和聚集,井使胆红素转变成稳定的自由基,从而引起脂质过氧化和透明质酸分子降解。经自由基处理的胆红素溶解度减小,粒度分布趋向颗粒变大.根据以上事实,重点讨论了自由基触发的胆色素类结石的致病过程。     

Free radical generation during chemical depolymerization of heparin

Low-molecular weight heparins (LMWHs), as compared with unfractionated heparin (UFH), present superior bioavailability, much longer plasma half-life, and lower incidence of side effects. For these reasons, over the past two decades LMWHs have become the drugs of choice for the treatment of deep venous thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina. Furthermore, their use in acute ischemic stroke is currently under study. LMWHs are obtained by UFH depolymerization, which can be performed using various methods, including nitrous acid depolymerization, cleavage by beta-elimination of benzyl ester, enzymatic depolymerization, and peroxyl radical-dependent depolymerization. This article addresses the chemical depolymerization, obtained by free radical attack (mainly hydroxyl radical), of heparin. The electron spin resonance (ESR) spectroscopy, coupled to the spin trapping technique, was employed to study this reaction. Free radical-mediated heparin depolymerization was performed under different chemical conditions. The final products of the reactions were purified and classified on the basis of their molecular weight and other characteristics. The level of heparin fragmentation was different depending on the type of depolymerization reaction used. Moreover, the level of reproducibility and the resulting radical species were different for every type of reaction performed.     

Tyrosinase-Induced Free Radical Formation From VP-16,213: Relationship To Cytotoxicity

Tyrosinase-dependent activation of hydroxybenzenes forms reactive compounds, including catechols and o-quinones, and some of which show antitumor activity against pigmented melanomas. Since VP-16 is a phenoxy-containing antitumor drug, forms free radicals and reactive o-quinones during peroxidative activation, wc evaluated the cytotoxicity of VP-16 to both tyrosinase-containing and non-tyrosinasecontaining tumor cells. Our results show that VP-16 is significantly more cytotoxic to B-16/F-10 melanoma cells than human MCF-7 breast tumor cells. Phenylthiocarbamide, an inhibitor of tyrosinase activity, selectively decreased VP-16 toxicity only in melanoma cells. Furthermore, VP-16 was readily activated to its phenoxy free radical intermediate by purified tyrosinase, indicating tyrosinase may play a role in VP-16 toxicity in pigminted melanomas.     

The role of tetrahydrobiopterin in superoxide generation from eNOS: enzymology and physiological implications

Tetrahydrobiopterin (BH₄) is a ubiquitous pteridine metabolite that serves as a NOS cofactor. Recently, we showed that BH₄ efficiently inhibits superoxide generation from the heme group at the oxygenase domain of eNOS. This role indicates that BH₄ acts as a redox switch in the catalytic mechanism of the enzyme, which may have important consequences in the physiology of the endothelium. Here the mechanism by which BH₄ inhibits superoxide release from eNOS and the "uncoupling" effects of oxidized BH₄ metabolites are presented. The implications of the disparate actions of fully reduced and oxidized BH 4 metabolites in the control of eNOS biochemistry are discussed in the light of clinical data indicating that BH 4 levels are important in the regulation of superoxide levels and of endothelial reactivity.     

The Role of Tetrahydrobiopterin in Superoxide Generation from eNOS: Enzymology and Physiological Implications

Tetrahydrobiopterin (BH₄) is a ubiquitous pteridine metabolite that serves as a NOS cofactor. Recently, we showed that BH₄ efficiently inhibits superoxide generation from the heme group at the oxygenase domain of eNOS. This role indicates that BH₄ acts as a redox switch in the catalytic mechanism of the enzyme, which may have important consequences in the physiology of the endothelium. Here the mechanism by which BH₄ inhibits superoxide release from eNOS and the "uncoupling" effects of oxidized BH₄ metabolites are presented. The implications of the disparate actions of fully reduced and oxidized BH 4 metabolites in the control of eNOS biochemistry are discussed in the light of clinical data indicating that BH 4 levels are important in the regulation of superoxide levels and of endothelial reactivity.