Detection of Free Radicals Generated from the in vitro Metabolism of Carbon Tetrachloride Using Improved ESR Spin Trapping Techniques

The spin trapping chemistry of carbon tetrachloride has been previously investigated in rat liver, both in vitro and in vivo. In addition to the trichloromethyl radical, both a 'carbon-centred' and an 'oxygen-centred' radical have been detected in vitro. These spin adducts have been assigned to 'lipid' and 'lipid oxyl' radicals. However, no specific structural characterization has been provided to date. The spin trapping chemistry of this system was reinvestigated with the use of deuterated α-phenyl N-tert-butyl nitrones to obtain better spectral resolution. Results indicate that the PBN trapped carbon-centred lipid radical is of a primary alkyl type.     

The Role of Free Radicals in Paraquat-Induced Corneal Lesions

Paraquat is a synthetic bipyridylium salt widely used as herbicide and defoliant. Enzyme-catalyzed redox-cycling of paraquat generates oxygen radicals. The toxic, even lethal, effects of paraquat are due to free radical-mediated tissue injury. Ocular lesions, sometimes quite severe, have been observed following accidental splashing of paraquat solutions onto the eyes.

These studies were designed to document the generation of paraquat free radicals in corneal tissue, and to describe the histological nature of the corneal injuries in experimental animals (rabbits and monkeys). The EPR spectrum of rabbit corneas, 30 min. after intrastromal injection of paraquat, showed the signal of the free radical of paraquat. Ultrastructural studies of corneas 8 days after intrastromal injections (100μl) of paraquat solutions showed that the initial lesions occur at the epithelium/basement membrane interface. In rabbit cornea, dose dependent lesions were observed, i.e. whereas 50 mM paraquat caused only minimal damage to the epithelial basement membrane, 75 mM caused complete dissolution to the basement membrane with some damage to stromal collagen, and loss of epithelium with stromal ulceration and severe inflammatory response were observed with 150 mM paraquat. Monkey corneas were less susceptible than those of rabbits to the effects of paraquat. No lesions were observed following intrastromal injections of 50 mM or 75 mM paraquat. With higher concentrations of paraquat (100 mM and 150 mM) the primary injuries were to the proximal and lateral plasma membranes of basal epithelial cells; basement membrane alterations were detected only adjacent to areas of significant plasma membrane damage. The underlying Bowman's membrane and stroma were not affected. Anatomical differences between the corneas of rabbit and monkeys as well as possible biochemical differences may account for the species differences observed.     

Use of a Low-Frequency Esr Spectrometer: Implications for Spin-Trapping Free Radicals, In Situ

We have adapted the low-frequency ESR spectrometer, designed and built by H.J. Halpern, to the physiologic needs of organ preparations operating at 250 MHz. Initial studies have allowed us to detect nitroxides in an isolated perfused heart. These in siru measurements were made with nitroxides specifically designed to mimic the lipophilic nature of 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) and 2.2-dimethyl-S-hydroxy-l-pyrrolidinyloxyl (DMPO-OH). These spin labels provided information about the influence of dynamic factors of the heart, such as flow rate, different cell populations and unequal distribution between compartments on our ability to conduct and interpret spin trapping experiments. They also clarified the sacrifice in sensitivity involved in operating at the lower frequencies. To deal with this later problem. we have increased the sensitivity of the spin trapping method by synthesizing a family of ¹⁵N-and deuterium-containing DMPO analogs and by determining their ability to spin trap free radicals generated by the model superoxide system of xanthinelxanthinc oxidase. Finally, since activated neutrophils are one of the few cells known to generate free radicals as part of their physiologic function, we used these phagocytic cells, as a source of superoxide.     

Radical Intermediates and Antioxidants: An Esr Study of Radicals Formed on Carnosic Acid in the Presence of Oxidized Lipids

Carnosic acid, an antioxidant extracted from rosemary, is shown to produce radicals when in contact with oxidized methyl oleate in the absence of air above 50°C. Two radical species are formed: the first one, stable up to -110°C, is an hydroxy-phenoxy radical whose ESR spectrum was analyzed by studying its temperature dependence and its sensitivity to deuterium/proton exchange. The second species was observed above 110°C, its ESR spectrum was identical to the spectrum obtained when carnosol, another antioxidant extracted from rosemary, was heated at the same temperature in the presence of oxidized lipid. This observation is probably due to the transformation of carnosic acid into carnosol; the analysis of the corresponding ESR spectrum suggests the formation of a keto phenoxy radical exhibiting a great delocalization of the unpaired electron.     

Esr and Spin-Trapping Study of Free Radicals in γ-Irradiated Solid Lysozyme

Free radicals produced by γ-irradiation of solid lysozyme were investigated by a technique combining ESR, spin-trapping and enzymatic digestion. MNP and DMPO were used as spin-trapping reagents. The solid lysozyme was first γ-irradiated and then dissolved in an aqueous solution containing the spin-trapping reagent to stabilize free radicals. The spin adducts of lysozyme were digested to oligopeptides to get ESR spectra having a well-resolved hyperfine structure. The ESR spectra obtained showed that carbon-centered radicals, CH-, at the side chains of amino acids, and thiyl radicals, -CH₂-_-S^-, at disulfide bridges were produced in γ-irradiated solid lysozyme.     

Electron Paramagnetic Resonance and Spin Trapping Study of Radicals Formed During Reaction of Aromatic Amines with isoAmyl Nitrite Under Aprotic Conditions

Diazotization of primary aromatic amines with isoamyl nitrite in benzene at room temperature was studied employing EPR and spin trapping techniques. Nitrosodurene (ND). 2-methyl-2-nitrosopropane (MNP). and 5,5-dimethyl-pyrroline N-oxide (DMPO) were used as spin trapping agents. Aryl radicals were detected employing ND and MNP. Using DMPO as a spin trap most of the amines produced EPR spectra ascribed to adducts with aniline-type radicals (N-centred radicals). The assignments were verified using ¹⁵JN-labeled anilines. Similar spectra of DMPO adducts were recorded from amines treated with benzoyl peroxide or benzophenone plus UV. Possible mechanisms of formation of these adducts (radical trapping versus nucleophilic addition to DMPO followed by oxidation) during treatment of the amines with isoamyl nitrite are discussed.     

Electron Paramagnetic Resonance and Spin Trapping Study of Radicals Formed During Reaction of Aromatic Amines with isoAmyl Nitrite Under Aprotic Conditions

Diazotization of primary aromatic amines with isoamyl nitrite in benzene at room temperature was studied employing EPR and spin trapping techniques. Nitrosodurene (ND). 2-methyl-2-nitrosopropane (MNP). and 5,5-dimethyl-pyrroline N-oxide (DMPO) were used as spin trapping agents. Aryl radicals were detected employing ND and MNP. Using DMPO as a spin trap most of the amines produced EPR spectra ascribed to adducts with aniline-type radicals (N-centred radicals). The assignments were verified using ¹⁵JN-labeled anilines. Similar spectra of DMPO adducts were recorded from amines treated with benzoyl peroxide or benzophenone plus UV. Possible mechanisms of formation of these adducts (radical trapping versus nucleophilic addition to DMPO followed by oxidation) during treatment of the amines with isoamyl nitrite are discussed.     

Hypoxia-Induced Generation of Nitric Oxide Free Radicals in Cerebral Cortex of Newborn Guinea Pigs

Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate (NMDA) receptor activation and receptor-mediated increase in intracellular calcium which may activate Ca⁺⁺-dependent nitric oxide synthase (NOS). The present study tested the hypothesis that tissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of newborn guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance (ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO₂ = 21%, n = 5) or hypoxic (FiO₂ = 7%, n = 5) groups. Prior to exposure, animals were injected subcutaneously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO₄.7H₂O (40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or 7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted. The difference of spectra between 2.047 and 2.027 gauss represents production of NO free radical. In hypoxic animals, there was a difference (16.75 ± 1.70 mm/g dry brain tissue) between the spectra of NO spin adducts identifying a significant increase in NO free radical production. In the normoxic animals, however, there was no difference between the two spectra. We conclude that hypoxia results in Ca²⁺- dependent NOS mediated increase in NO free radical production in the cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in presence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteins including the NMDA receptor, a potential mechanism of hypoxia-induced modification of the NMDA receptor resulting in neuronal injury.     

Spin Trapping of Free Radicals Produced in vivo in Heart and Liver During Endotoxemia

Studies using free radical scavengers and measurements of lipid peroxidation have suggested that free radicals are generated during endotoxemia. Conclusions from these studies have implied that free radicals may participate in the sequence of pathologic events following endotoxin challenge in the experimental animal. Current inferences of free radical generation and involvement have been derived from indirect evidence and are therefore inconclusive. To quantitate the generation of free radicals in vivo during endotoxemia this study employed the use of electron paramagnetic resonance spectroscopy (EPR) combined with spin trapping techniques. Five minutes before intraperitoneal endotoxin administration, trimethoxy-a-phenyl-t-butyl-nitrone [(MeO), PBN] was administered intraperitoneally. Experimental animals were always matched with control animals receiving no endotoxin. At either five minutes or twenty-five minutes following endotoxin administration animals were decapitated and hearts and livers were rapidly taken for lipid extraction and EPR evaluation. Analysis of the EPR spectra revealed hyperfine splitting constants that indicated the presence of carbon-centered radical spin adducts in both organ tissues from animals exposed to endotoxin for twenty-five minutes. No signals were present in hearts and livers taken five minutes after endotoxin administration. EPR evaluation did not indicate spin adduct formation in control tissue. These data directly demonstrate that activation of processes in vivo involving free radical generation occur early during endotoxemia, but are not detectable immediately after the endotoxin challenge.     

Free Radicals and Sod Activity of Jaw Cyst. Direct Measurement and Spin Trapping Studies by Esr

Free radicals produced in the fluid of jaw cysts were directly measured at room temperature using ESR. With these samples, SOD activity of the cyst fluid was measured by the ESR spin trapping method with DMPO as a trapping agent. Freeze-dried samples of cyst fluid showed a broad ESR signal at g = 2.005. Relative signal intensity of samples from jaw cysts with inflammation was higher than jaw cysts without inflammation. SOD activity of cyst fluid with high viscosity showed higher values than that of cyst fluid with low viscosity. We suggest that free radicals produced in jaw cyst damage tissues while higher SOD activity of cyst fluid play a role in a self-defense mechanism against free radicals.     

Free Radicals and Sod Activity of Jaw Cyst. Direct Measurement and Spin Trapping Studies by Esr

Free radicals produced in the fluid of jaw cysts were directly measured at room temperature using ESR. With these samples, SOD activity of the cyst fluid was measured by the ESR spin trapping method with DMPO as a trapping agent. Freeze-dried samples of cyst fluid showed a broad ESR signal at g = 2.005. Relative signal intensity of samples from jaw cysts with inflammation was higher than jaw cysts without inflammation. SOD activity of cyst fluid with high viscosity showed higher values than that of cyst fluid with low viscosity. We suggest that free radicals produced in jaw cyst damage tissues while higher SOD activity of cyst fluid play a role in a self-defense mechanism against free radicals.     

Direct Observation of Spin-Trapped Carbon Dioxide Radicals in Hepatocytes Exposed to Carbon Tetrachloride

Carbon dioxide radical adducts of the spin trapping agent, α-phenyl N-t-butyl nitrone (PBN), have been observed to occur in the urine and bile of rats exposed to carbon tetrachloride as well as in perfusates of liver in which the perfusion medium contained carbon tetrachloride (Connor er al., J. Biol. Chem., 261, 4542, (1986)). The carbon dioxide adduct was proven to be derived from CCI, by use of 13-C-labelled compound. These adducts were not observed in the liver itself suggesting that they might be rapidly secreted from the liver. However, using isolated hepatocytes, we have demonstrated that the carbon dioxide radical adduct can be observed directly in the liver cells as it is formed. Since this water-soluble adduct cannot be extracted by non-aqueous solvents such as chloroform or toluene, its formation in liver in vivo or in perfused livers was not detected. Lowering the oxygen tension in the system diminished the intensity of production of the carbon dioxide adduct, consistent with the adduct being produced as a result of ·OOCCl₃ generation. It is not clear the extent to which this adduct is formed as a result of the ·CO₂ radical or is produced by metabolic oxidation of the trichloromethyl radical adduct of PBN per se to the carbon dioxide radical adduct. The intensity of the signal of the carbon dioxide radical adduct suggests that adduct conversion may be the route of formation since it seems unlikely that a sufficient amount of the halocarbon could be metabolized to ·COCl or ·CO₂ radicals to generate a signal of the magnitude involved. The ·CO₂ adduct is readily observed in intact hepatocytes, but the ·CCl₃ adduct is not (although we know the ·CCl₃ adduct has been produced in these cells), indicating that the ·CO₂ adduct is present in considerable abundance compared to the ·CCl₃ adduct.     

Formation of Hydroxyl Radicals on Reaction of Hypochlorous Acid with Ferrocyanide, a Model IRON(II) Complex

Hypochlorous acid reacts with the model iron(II) complex, ferrocyanide (Fe(CN)₆^4-) in aqueous solution with the rate constant 220 ± 15 dm³ mol^-1 s^-1. Free hydroxyl radicals are formed in this reaction in 27% yield as shown by the hydroxylation of benzoate to give a product distribution identical to that of free (radiolytically generated) hydroxyl radicals. This reaction is three orders of magnitude faster than the analogous reaction involving hydrogen peroxide (the Fenton reaction), suggesting that the hypochlorous acid generated by activated neutrophils may be a source of hydroxyl radicals.     

Role of Oxygen-Derived Free Radicals in Gastric Mucosal Injury Induced by Ischemia or Ischemia-Reperfusion in Rats

Oxygen-derived free radicals have been implicated as possible mediators in the development of tissue injury induced by ischemia and reperfusion. Clamping of the celiac artery in rats reduced the gastric mucosal blood flow to 10% of that measured before the clamping. The area of gastric erosions and thiobarbituric acid (TBA) reactants in gastric mucosa were significantly increased 60 and 90 min after clamping. These changes were inhibited by treatment with SOD and catalase. Thirty and 60 min after reoxyganation, produced by removal of the clamps following 30 min of ischemia, gastric mucosal injury and the increase in TBA reactants were markedly aggravated compared with those induced by ischemia alone. SOD and catalase significantly inhibited these changes. The serum a-tocopherol/cholesterol ratio, an index of in vivo lipid peroxidation, was significantly decreased after long periods of ischemia (60 and 90 min), or after 30 and 60 min of reperfusion following 30 min of ischemia. These results indicated that active oxygen species and lipid peroxidation may play a role in the pathogenesis of gastric mucosal injury induced by both ischemia alone and ischemia-reperfusion. Although, allopurinol inhibited the formation of gastric mucosal injury and the increase in TBA reactants in gastric mucosa, the depletion of polymorphonuclear leukocytes (PMN) counts induced by an injection of anti-rat PMN antibody did not inhibit these changes. As compared with the hypoxanthine-xanthine oxidase system, PMN seem to play a relatively small part in the formation of gastric mucosal injury induced by ischemia-reperfusion.     

Regional Distributions of Thiobarbituric Acid-Reactive Products, Activities of Enzymes Regulating the Metabolism of Oxygen Free Radicals, and Some of the Related Enzymes in Adult and Aged Rat Brains

Regional distributions of thiobarbituric acid-reactive products, activities of enzymes regulating metabolism of oxygen free radicals, and some of the related enzymes were studied in 10 areas of adult and aged rat brains. Thiobarbituric acid-reactive products were lower in cerebral cortex, septal area, hippocampus, caudate-putamen, and substantia nigra compared with other areas studied in adult rats; however, they increased significantly in the former areas with aging. A slight but significant reduction in superoxide dismutase activity was noted in frontal cortex, septal area, caudate-putamen, and substantia nigra with aging. Glutathione peroxidase and reductase activities were highest in caudate-putamen and in substantia nigra. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were lowest in cortical areas. Phosphofructokinase activity was lowest in septal area and hippocampus in aged rats. Glyceraldehyde-3-phosphate dehydrogenase activity showed only small regional and evolutional changes. Lactate dehydrogenase activity declined with age in most of the areas studied. sn-Glycerol-3-phosphate dehydrogenase activity showed small changes with aging except in hippocampus, where 40% reduction was noted. Generally, cerebral cortical areas, hippocampus, and septal areas were not particularly enriched in enzymes regulating the metabolism of oxygen free radicals. The results were discussed in relation to the role of free radicals in aging.     

Role of Oxygen Free Radicals in Respiratory Distress Induced by Arachidonic Acid In the Rat

The purpose of our present study is the possible implication of oxygen free radicals in the respiratory distress induced in rats by intravenous administration of arachidonic acid (20mg/kg). The respiratory frequency was measured and plasma TXB2 concentration was assayed by RIA from blood withdrawn I min after arachidonic acid administration. The substances studied were: SOD, catalase, manifold, DMSO, BHT, imidazole. All the drugs, except imidazole, significantly protect the rats from the respiratory distress induced by arachidonic acid. SOD, catalase, BHT and imidatole inhibit whereas mannitol and DMSO increase the plasma levels of TXB2. We suggest that oxygen free radicals generated in the respiratory burst induced by arachidonic acid are mainly responsible for the consequent respiratory distress.     

Free Radicals Developed in the Amino-Garbonyl Reaction of Sugars with Amino Acids

GA_l5, GA₃, GA₅, GA₁₉, GA₂₀ and GA₂₃ were identified by GC-MS in the acidic ethyl acetate-soluble fraction from the seeds of sweet potato (Ipomoea batatas Lam.). GA₁₉ and GA₂₃ were major GAs in the mature seeds, their contents being about 200 and 160 μg/kg fresh weight, respectively, while those of GA₁₉ and GA₂₃ in immature seeds were below 100 μg/kg fr. wt. The occurrence of glycosyl conjugates of GA₃, GA₅, GA₈, GA₁₇, GA₁₉, GA₂₀, GA₂₃ and GA₄₄ in the butanol fraction from mature seeds was shown by GC/MS analysis after enzymatic hydrolysis.

Besides the endogenous GAs in sweet potato, those in immature seeds of several other Convolvulaceae plants were investigated. The species of endogenous GAs were discussed in terms of chemotaxonomy.     

康复新液联合表皮生长因子预防急性放射性肠炎的疗效研究

目的:探讨康复新液联合表皮生长因子保留灌肠对盆腔肿瘤放疗后并发急性放射性肠炎的预防作用及其机制。方法:86例盆腔恶性肿瘤行放射性治疗的患者随机分为观察组和对照组,2组患者均行外照射治疗,观察组康复新液与表皮因子联合保留灌肠,每次50 mL,每日一次,对照组不加任何预防用药。比较2组急性放射性肠炎的发生率以及发生时间,并于放射治疗10次、20次后静脉采血,酶联免疫吸附法检测血清TNF-α、IL-6、IL-8,黄嘌呤氧化酶法检测MDA、SOD的含量。结果:观察组、对照组的急性放射性肠炎发生率分别为9.31%、53.49%,组间差异有统计学意义(P0.05),且观察组出现急性放射性肠炎的时间迟于对照组。观察组放射治疗10次及20次后,MDA含量明显低于对照组,SOD含量高于对照组(P0.05)。TNF-α、IL-6、IL-8含量明显低于对照组(P0.05)。结论:康复新液联合表皮生长因子可预防急性放射性肠炎的发生,其机制可能与抗细胞炎性因子与氧自由基释放有关。     

Generation of Free Radicals during Decomposition of Hydroperoxide in the Presence of Myeloperoxidase or Activated Neutrophils

It was shown with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone that myeloperoxidase (MPO) in the presence of its substrates H2O2 and Cl- as well as activated neutrophils destroy tert-butyl hydroperoxide producing two adducts of O-centered radicals which were identified as peroxyl and alcoxyl radicals. Inhibitory analysis performed with traps of hypochlorite (taurine and methionine), free radical scavengers (2,6-di-tret-butyl-4-methylphenol and mannitol), and MPO inhibitors (salicylhydroxamic acid and 4-aminobenzoic acid hydrazide) revealed that the destruction of the hydroperoxide group in the presence of isolated MPO or activated neutrophils was directly caused by the activity of MPO: some radical intermediates appeared as a result of the chlorination cycle of MPO at the stage of hypochlorite generation, whereas the other radicals were produced independently of hypochlorite, presumably with involvement of the peroxidase cycle of MPO. The data suggest that the activated neutrophils located in the inflammatory foci and secreting MPO into the extracellular space can convert hydroperoxides into free radicals initiating lipid peroxidation and other free radical reactions and, thus, promoting destruction of protein-lipid complexes (biological membranes, blood lipoproteins, etc.).     

Effect of Free Radicals and Temperature on Sister Chromatid Exchanges in Hordeum vulgare L.

The clastogenic (chromosome-damaging) effect of many chemical and physical agents is believed to be mediated by reactive oxygen-detived radicals. The interaction of these free radicals with DNA and the significance of the radical-induced DNA lesions in mutagenesis and carcinogenesis have been the subjects of increasing interest during recent years. Sister chromatid exchange (SCE) reflects an interchange between DNA molecules at homologous loci within a replicating chromosome. SCE analysis was found to have increased use for monitoring the exposure of cell to mutagenic carcinogens. The authors found that the induction of SCEs in cells of Hordeum vulgare L. by ascorbic acid, mitomycin C, adriamycin and maleic hydrazid was through the action of free radicals. They also studied the influence of growth temperature on average generation time(AGT) and SCEs. and disclosed a close correlation between AGT and SCEs. The Brdu-Giemsa techniques were used for the detection of SCEs and AGT in cytological preparations of metaphase chromosomes.