Free radical research communications最新文献

The reaction of superoxide with the spin trap DMPO (5,5-dimethylpyrroline-N-oxide) is widely used to study superoxide production as well as issues associated with the superoxide-related formation of hydroxyl radical. However, the interpretation of observed intensities of DMPO/.OOH and DMPO/.OH signals in electron paramagnetic resonance spin trapping experiments is not without its difficulties. In this paper, I report experiments that demonstrate: 1. That the flux of superoxide formation in a DMPO spin trapping experiment can alter the apparent importance of weak DMPO/.OH signals; 2. That iron can influence the DMPO/.OOH spin trapping results; 3. That there is very little spontaneous breakdown of DMPO/.OOH to form DMPO/.OH.

Porcine stress syndrome (PSS) which is an example of malignant hyperthermia (MH) in swine has previously been attributed to oxidative stress primarily due to an inherited antioxidant abnormality in MH susceptible (MHS) animals. C-phenyl-N-tert-butyl nitrone (PBN), a free radical spin trap, was selected to investigate whether free radicals are involved in MH. If free radicals cause the MH stress attack, then PBN should alter the time required for the onset of the stress attack, or perhaps protect the animal from experiencing the stress attack. In vivo phosphorus-31 (31P) magnetic resonance spectroscopy (MRS) was used to monitor metabolism in three to four week old normal and MHS piglets administered halothane as the stress challenge. Malignant hyperthermia was not reproducibly induced by halothane anesthesia. For those animals which did develop MH a dramatic fall in the level of PCr and a rise in the level of Pi was detected by 31P MRS. Intravenous administration of PBN prior to halothane exposure had no effect on the number of animals experiencing the stress attack. PBN does not appear to prevent, delay or reverse the onset of halothane-induced MH in three to four week old MHS piglets. The primary events leading to the MH syndrome do not appear to be influenced by the intervention of the type of free radicals normally trapped by PBN.

We describe expression of alpha, mu and pi class glutathione S-transferase (GST) and, CuZn- and Mn superoxide dismutase (SOD) in human synovium and cultured synovial fibroblasts. Immunohistochemical and immunoblotting studies showed synovium and cultured cells expressed pi GST and both isoforms of SOD. Cellular localisation was largely perinuclear. No expression of alpha or mu GST was detected even though polymerase chain reaction analysis showed 4/6 subjects had positive genotypes at the polymorphic, mu class GSTM1 locus. Incubation of cultured synovial fibroblasts with H2O2, IL-1 alpha and the cyclooxygenase and lipoxygenase inhibitor, Tenidap, did not induce expression of alpha, mu or pi GST though treatment with IL-1 alpha caused a marked increase in the expression of Mn SOD.

The production of formaldehyde from tris(hydroxymethyl) aminomethane(Tris) by interaction with hydroxyl radicals(.OH) was studied, since the reaction mixture from the Fenton reaction performed in Tris/HCl buffer was found to be color-developed by colorimetric determination of formaldehyde. The absorption spectrum of chromogens was identical to that of authentic formaldehyde. Color development, which required the presence of Tris, hydrogen peroxide and cupric ions in the Fenton reaction mixture, was inhibited by the addition of hydroxyl radical scavengers such as glucose or hyaluronic acid. These results indicated that formaldehyde was produced when Tris interacted with .OH. With structures similar to Tris, Good's buffers were also found to produce formaldehyde by interaction with .OH. Analysis of formaldehyde derived from these buffers may provide a simple and convenient assay for detecting .OH generation. In evaluating effects of .OH on the biological system in Tris/HCl buffer or certain Good's Buffers, .OH loss may be due to interactions of .OH with these buffers. The formaldehyde produced as a result of such interactions may affect biological systems.

The ability of synaptosomes, prepared from striata, to take up 3H-dopamine declined rapidly during incubation at 37 degrees C, in an oxygenated Krebs-Ringer medium with 0.1 mM ascorbic acid. Ascorbic acid was responsible for this decrease. Its effectiveness after a 60 min incubation was concentration dependent from 1 microM and virtually complete for 0.1 mM. Furthermore, a decrease of synaptosomal membrane fluidity was revealed by measurements of fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene. This decrease was potentiated by Fe2+ ions (1 microM). In contrast, it was prevented by the Fe2+ ion chelator, desferrioxamine (0.1 mM), by the Ginkgo biloba extract EGb 761 [2-16 micrograms/ml], as well as by the flavonoid quercetin (0.1 microM). This preventive effect was shared by trolox C (from 0.1 mM). It is concluded that peroxidation of neuronal membrane lipids induced by ascorbic acid/Fe2+ is associated with a decrease in membrane fluidity which, in turn, reduces the ability of the dopamine transporter to take up dopamine.

In a survey of a number of heavy metal ions for effects on the oxidative metabolism (respiratory burst) of human polymorphonuclear leukocytes (neutrophils) we have found that mercury(II) and silver ions in micromolar concentration significantly increase the production of superoxide anions in cells, initiated by formyl-methionyl-leucylphenylalanine (fMLP). The stimulation of radical formation induced by a certain ion concentration varied considerably in cells isolated from different blood donors, from a moderate increase to a very large (up to 400% of control values). When the soluble stimulator phorbol myristate acetate (PMA) or the particulate stimulator Zymosan were used to initiate the cell respiratory burst, no additional stimulating effects by the metal ions on superoxide anion formation were observed. This fact might indicate that the effect of the metal ions on the fMLP-dependent initiation of cell activity is a mechanism coupled to the interaction between the chemotactic peptide and its corresponding receptor molecules on the cell surface. By increasing the concentration of silver ions during pre-incubation of resting neutrophils, a spontaneous activation of the cells could be recorded at a concentration exceeding 5 microM. However, the silver ion concentration at which such spontaneous initiation of the respiratory burst occurred varied significantly between blood samples from different donors with a concentration range of 5 to 15 microM. This effect could not be shown for mercuric ions due to the toxicity of the metal above 5 microM. Blood samples from some donors contained neutrophils that could be activated by either mercuric- or silver ions at concentration as low at 1 microM. The spontaneous activation of neutrophils with elevated concentrations of silver ions is kinetically similar to the PMA-induced. The onset of superoxide anion formation is preceded by a lag period whose length varies in time with the concentration of agent applied to the cells. It is a known fact that once the neutrophils have been activated with fMLP it is not possible to reactivate the cells by a second supplementation of fMLP. However, after cessation of the fMLP-induced activation, addition of PMA or silver ions gives rise to renewed production of superoxide anions. We propose two different mechanisms of action of silver ions on oxidative metabolism of neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)

The one-electron oxidation of a reduced nitroxide (2,2,6,6-tetramethyl-1,4-dihydroxypiperidine, TOLH), detected by ESR, was used to resolve and quantify oxidants arising from the reaction of heme proteins with hydroperoxides, including chelatable iron released subsequent to oxidative cleavage of the porphyrin ring. Released iron was distinguished from protein radicals and ferryl heme by analyzing TOLH oxidation in the presence of different chelating agents. Metmyoglobin (metMb) treatment with one mole of H2O2 per mole of heme produced protein-bound oxidants that oxidized about two molecules of TOLH per heme. Some of the oxidizing species responsible for TOLH oxidation were highly persistent (t1/2 for the decay was 3 hrs at 25 degrees C). Iron release, metMb bleaching and the catalysis of Fenton-type chemistry were compared in metMb solutions treated with tert-butyl hydroperoxide (tBH). Iron release required about five-fold higher hydroperoxide concentrations than did metMb bleaching. Alkoxyl and methyl radical production was catalyzed by iron released from metMb but not by protein-bound iron in oxidized metMb solutions treated with tBH and ascorbic acid. The results suggest that ascorbate-mediated hydroxyl and alkoxyl radical production by hydroperoxide-treated metMb is due to released iron and that the protein-bound non-heme iron that arises during bleaching is at most a weak Fenton reagent.

Hamamelitannin, which is a component of bark extract of hamamelis (Hamamelis virginior L.), was found to be a potent scavenger of superoxide anion radicals. Superoxide anion scavenging activity of the compound was evaluated by ESR-spin trap method using DMPO (5,5'-dimethyl-1-pyrroline-N-oxide) as a spin trapping agent. The IC50 value (the concentration producing 50% inhibition of superoxide anion radicals) of hamamelitannin was found to be 1.38 +/- 0.06 microM much lower than that of ascorbic acid (23.31 +/- 2.23 microM). Supporting the superoxide scavenging activity of hamamelitannin, the compound showed both suppressive ability against depolymelization of hyaluronic acid and protective ability against cytotoxicity induced by superoxide anion radicals. Hamamelitannin increased the survival rate of fibroblast to 85.5 +/- 3.3%, compared with that of control (27.2 +/- 4.3%).