Aerobic oxidation of aminoacetone, a threonine catabolite: iron catalysis and coupled iron release from ferritin

Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cri-du-chat and threoninemia syndromes and, more recently, implicated as a contributing source of methylglyoxal (MG) in diabetes mellitus. Oxidation of AA to MG, NH(4)(+), and H(2)O(2) has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by Cu(II) ions. We here study the mechanism of AA aerobic oxidation, in the presence and absence of iron ions, and coupled to iron release from ferritin. Aminoacetone (1-7 mM) autoxidizes in Chelex-treated phosphate buffer (pH 7.4) to yield stoichiometric amounts of MG and NH(4)(+). Superoxide radical was shown to propagate this reaction as indicated by strong inhibition of oxygen uptake by superoxide dismutase (SOD) (1-50 units/mL; up to 90%) or semicarbazide (0.5-5 mM; up to 80%) and by EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which detected the formation of the DMPO-(*)OH adduct as a decomposition product from the DMPO-O(2)(*)(-) adduct. Accordingly, oxygen uptake by AA is accelerated upon addition of xanthine/xanthine oxidase, a well-known enzymatic source of O(2)(*)(-) radicals. Under Fe(II)EDTA catalysis, SOD (<50 units/mL) had little effect on the oxygen uptake curve or on the EPR spectrum of AA/DMPO, which shows intense signals of the DMPO-(*)OH adduct and of a secondary carbon-centered DMPO adduct, attributable to the AA(*) enoyl radical. In the presence of iron, simultaneous (two) electron transfer from both Fe(II) and AA to O(2), leading directly to H(2)O(2) generation followed by the Fenton reaction is thought to take place. Aminoacetone was also found to induce dose-dependent Fe(II) release from horse spleen ferritin, putatively mediated by both O(2)(*)(-) and AA(*) enoyl radicals, and the co-oxidation of added hemoglobin and myoglobin, which may be viewed as the initial step for potential further iron release. It is thus tempting to propose that AA, accumulated in the blood and other tissues of diabetics, besides being metabolized by SSAO, may release iron and undergo spontaneous and iron-catalyzed oxidation with production of reactive H(2)O(2) and O(2)(*)(-), triggering pathological responses. It is noteworthy that noninsulin-dependent diabetes has been frequently associated with iron overload and oxidative stress.     

Site-specific DNA damage induced by cobalt(II) ion and hydrogen peroxide: role of singlet oxygen

The effect of Co(II) ion on the reaction of hydrogen peroxide with DNA was investigated by a DNA sequencing technique using 32P-5'-end-labeled DNA fragments obtained from human c-Ha-ras-1 protooncogene. Co(II) induced strong DNA cleavage in the presence of hydrogen peroxide even without alkali treatment. Guanine residues were the most alkali-labile site, and the extent of cleavages at the positions of thymine and cytosine was dependent on the sequence. Adenine residues were relatively resistive. Diethylenetriaminepentaacetic acid, present in excess over Co(II), inhibited DNA cleavage. Singlet oxygen scavengers (dimethylfuran, sodium azide, 1,4-diazabicyclo[2.2.2]octane, dGMP), sulfur compounds (methional, methionine), and superoxide dismutase inhibited DNA cleavage completely. Hydroxyl radical scavengers were not so effective as singlet oxygen scavengers. ESR studies using 2,2,6,6-tetramethyl-4-piperidone as a singlet oxygen trap suggest that Co(II) reacts with hydrogen peroxide to produce singlet oxygen or its equivalent. ESR studies using 5,5-dimethylpyrroline N-oxide (DMPO) showed that the hydroxyl radical adduct of DMPO was also formed. The results suggest that Co(II) ion binds to DNA and subsequently reacts with hydrogen peroxide to produce singlet oxygen and hydroxyl radicals and that singlet oxygen plays a more important role in the DNA damage than hydroxyl free radicals.     

Free radical production and site-specific DNA damage induced by hydralazine in the presence of metal ions or peroxidase/hydrogen peroxide.

Hydralazine caused site-specific DNA damage in the presence of Cu(II), Co(II), Fe(III), or peroxidase/H2O2. The order of inducing effect of metal ions on hydralazine-dependent DNA damage [Cu(II) greater than Co(II) greater than Fe(III)] was related to that of accelerating effect on the O2 consumption rate of hydralazine autoxidation. Catalase completely inhibited DNA damage by hydralazine plus Cu(II), but hydroxyl radical (.OH) scavengers and superoxide dismutase did not. On the other hand, DNA damage by hydralazine plus Fe(III) was inhibited by catalase and .OH scavengers. Hydralazine plus Cu(II) induced piperidine-labile sites predominantly at guanine and some adenine residues, whereas hydralazine plus Fe(III) caused cleavages at every nucleotide. Activation of hydralazine by peroxidase/H2O2 caused guanine-specific modification in DNA. ESR-spin trapping experiment showed that .OH and superoxide are generated during the Fe(III)- or Cu(II)-catalysed autoxidation of hydralazine, respectively, and that nitrogen-centered radical is generated during the Cu(II)- or peroxidase-catalysed oxidation. The generation of nitrogen-centered radical was also supported by HPLC-mass spectrometry. The results suggest that the guanine-specific modification by the enzymatic activation of hydralazine is due to the nitrogen-centered hydralazyl radical or derived active species, whereas .OH participates in DNA damage by hydralazine plus Fe(III). The mechanism of hydralazine plus Cu(II)-induced DNA damage is complex. The possible role of the DNA damage induced by hydralazine in the presence of Cu(II) or peroxidase/H2O2 is discussed in relation to hydralazine-induced lupus, mutation, and cancer.     

Aniline-, phenylhydroxylamine-, nitrosobenzene-, and nitrobenzene-induced hemoglobin thiyl free radical formation in vivo and in vitro

We have employed the ESR spin trapping technique in vivo to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats following administration of either aniline, phenylhydroxylamine, nitrosobenzene, or nitrobenzene. This DMPO adduct was a six-line, strongly immobilized, radical adduct. Using rat red blood cells, both phenylhydroxylamine and nitrosobenzene were able to induce the formation of the DMPO/glutathiyl free radical adduct and the same DMPO/hemoglobin thiyl free radical adduct was detected in in vivo samples. In experiments using purified rat oxyhemoglobin, a four-line, weakly immobilized, DMPO/hemoglobin thiyl free radical adduct was detected, in addition to the six-line strongly immobilized adduct. When this study was repeated using human red blood cells, we detected only the DMPO/glutathiyl free radical adduct and, when purified human oxyhemoglobin was employed, only the four-line, weakly immobilized, DMPO/hemoglobin thiyl radical adduct could be detected. In a study using reduced glutathione, we found that phenylhydronitroxide free radicals were reduced by glutathione and that glutathione was concomitantly oxidized to its thiyl free radical. We propose that the species responsible for the oxidation of the thiols to yield the thiyl free radicals in vivo and in vitro was the phenylhydronitroxide radical produced from the reaction of phenylhydroxylamine with oxyhemoglobin.     

Site-specific DNA damage by phenylhydrazine and phenelzine in the presence of Cu(II) ion or Fe(III) complexes: roles of active oxygen species and carbon radicals.

Phenylhydrazine cleaved isolated DNA in the presence of Cu(II), Mn(III), hemin, Fe(III)-EDTA, or peroxidase/H2O2, while phenelzine cleaved in the presence of Cu(II). DNA cleavage by phenylhydrazine in the presence of Mn(III), hemin, or Fe(III)-EDTA occurred without marked site specificity. Inhibitory effects of scavengers of hydroxyl free radical (.OH) on the DNA damage suggest the involvement of .OH. On the other hand, Cu(II)-mediated DNA cleavage by phenylhydrazine or phenelzine was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, but not by .OH scavengers. The predominant cleavage site was the thymine residue of 5'-GTC-3' sequence. Since the cleavage pattern was similar to that induced by Cu(I) plus H2O2 but not to that induced by Cu(II) plus H2O2, it is speculated that the copper-oxygen complex derived from the reaction of H2O2 with Cu(I) participates in DNA damage by phenylhydrazine or phenelzine in the presence of Cu(II). A comparison between scavenger effects on the DNA damage and those on radical production detected with ESR suggests that carbon-centered radicals (phenyl radical, 2-phenylethyl radical) do not play an important role in Cu(II)-, hemin-, or Fe(III)-EDTA-mediated DNA damage by phenylhydrazine or phenelzine of relatively low concentrations (less than 0.5 mM). However, during the oxidation of a high concentration (10 mM) of phenylhydrazine by ferricyanide, phenyl radical seemed to cause DNA damage, especially the breakage of the deoxyribose phosphate backbone. The possibility that active oxygen species (copper-oxygen complex, .OH) are more important in DNA damage induced by hydrazines in vivo than carbon-centered radicals is discussed.     

Aminoglutethimide-induced protein free radical formation on myeloperoxidase: a potential mechanism of agranulocytosis

Aminoglutethimide (AG) is a first-generation aromatase inhibitor used for estrogen-dependent breast cancer. Unfortunately, its use has also been associated with agranulocytosis. We have investigated the metabolism of AG by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that AG oxidation by MPO/H2O2 would produce an AG cation radical that, in the absence of a biochemical reductant, would lead to the oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms only by the reaction of DMPO with a protein free radical. We found that AG metabolism by MPO/H2O2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. Glutethimide, a congener of AG that lacks the aromatic amine, did not cause DMPO-MPO formation, indicating the necessity of oxidation of the aniline moiety in AG. When analyzed by electron spin resonance spectroscopy, we detected a phenyl radical adduct, derived from AG, which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with AG/H2O2 and was found to contain DMPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by one of two free radical drug metabolites of AG, one of which was characterized by spin trapping with 2-methyl-2-nitrosopropane. These results are the first demonstration of MPO free-radical detection by the anti-DMPO antibody that results from drug oxidation. We propose that drug-dependent free radical formation on MPO may play a role in the origin of agranulocytosis.     

Requirement of glutathione and cysteine in guanine-specific oxidation of DNA by carcinogenic potassium bromate.

Potassium bromate (KBrO3), a food additive, induces renal-cell tumors in rats. KBrO3 induced 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human leukemia cell line HL-60 as well as in its H2O2-resistant clone, HP100, suggesting no involvement of H2O2. Depletion of GSH by buthionine sulfoximine (BSO) had a little inhibitory effect on KBrO3-induced 8-oxodG formation. However, the amount of 8-oxodG was still significantly higher than that in control, suggesting that intracellular Cys can affect KBrO3 to oxidize DNA, when GSH decreased. KBrO3 caused 8-oxodG in isolated DNA in the presence of GSH (tripeptide; gamma-GluCysGly), gamma-GluCys, CysGly, or Cys. Methional completely inhibited 8-oxodG formation induced by KBrO3 plus GSH, but typical hydroxyl radical scavengers, SOD and catalase, had little or no inhibitory effects. When bromine solution (BrO(-)) was used instead of BrO3(-), similar scavenger effects were observed. Experiments with 32P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene suggested that KBrO3 induced 8-oxodG formation at 5'-site guanine of GG and GGG sequences of double-stranded DNA in the presence of GSH and that treatment of formamidopyrimidine-DNA glycosylase led to chain cleavages at the guanine residues. ESR spin-trapping studies showed that 1:2:2:1 quartet DMPO (5,5-dimethyl-1-pyrroline N-oxide) spectrum similar to DMPO/hydroxy radical (*OH) adduct, but the signals were not inhibited by ethanol. Therefore, the signal seemed not to be due to *OH but byproduct due to oxidation of DMPO by the reactive species. The signals were suppressed by the addition of dGMP, but not by other mononucleotides, suggesting the specific reactivity with guanine. On the basis of our results and previous literature, it is speculated that reduction of KBrO3 by SH compounds in renal proximal tubular cells yields bromine oxides and bromine radicals, which are the reactive species that cause guanine oxidation, leading to renal carcinogenesis of KBrO3.     

Autoxidation of the antitumor drug 9-hydroxyellipticine and its derivatives

In aqueous solution and using molecular oxygen as electron acceptor, the antitumor drug 9-hydroxyellipticine (9-OH-E) undergoes a spontaneous oxidation to give hydrogen peroxide (H2O2), the quinone imine 9-oxoellipticine (9-oxo-E), and a dimer of 9-OH-E(9-OH-E2). Electron paramagnetic resonance (EPR) experiments performed either in alkaline Me2SO or in phosphate buffer in the presence of the spin trap 5,5-dimethylpyrroline 1-oxide (DMPO) suggest that the oxidation process involves the initial formation of superoxide anion (O2- .) and the free radical of the drug. In aqueous medium, this step is followed by the dismutation of both O2- . and free radicals of the drug generating, respectively, H2O2 and 9-oxo-E. 9-Oxo-E further reacts with the 9-OH-E remaining in the solution to form the dimer 9-OH-E2 as the terminal product. The autoxidation process is strongly enhanced by superoxide dismutase and manganese ions. In the ellipticine series, all drugs that have an OH group in position 9 exhibit the ability to transfer one electron on molecular oxygen to generate O2- .. This property may be involved in the cytotoxic activities of these drugs.     

ESR evidence for the hydroxyl radical formation in aqueous suspension of quartz particles and its possible significance to lipid peroxidation in silicosis

Electron spin resonance (ESR) spectrum of the hydroxyl (.OH) radical spin adduct with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide has been obtained in suspensions of freshly ground quartz particles. The concentration of the spin adduct (and hence of the .OH radicals) increases with the amount of grinding. The dust's potential for the generation of the .OH radicals is maximum when fresh (i.e., immediately after grinding) and decreases to 50% in about a day on storage in air. Studies involving metal chelates indicate that the .OH radical formation involves mainly the silica surface and H2O rather than the Fenton reaction. The results suggest that hydroxyl radical reaction(s) could be important in the lipid peroxidation and fibrogenicity by quartz dust, particularly in acute silicosis.     

Hydroxyl radical-induced oxidation of a phenolic C-linked 2'-deoxyguanosine adduct yields a reactive catechol

Phenolic toxins stimulate oxidative stress and generate C-linked adducts at the C8-site of 2'-deoxyguanosine (dG). We previously reported that the C-linked adduct 8-(4″-hydroxyphenyl)-dG (p-PhOH-dG) undergoes oxidation in the presence of Na(2)IrCl(6) or horseradish peroxidase (HRP)/H(2)O(2) to generate polymeric adducts through phenoxyl radical production [ Weishar ( 2008 ) Org. Lett. 10 , 1839 - 1842 ]. We now report on reaction of p-PhOH-dG with two radical-generating systems, Cu(II)/H(2)O(2) or Fe(II)-EDTA/H(2)O(2), which were utilized to study the fate of the C-linked adduct in the presence of hydroxyl radical (HO(?)). The radical-generating systems facilitate (i) hydroxylation of the phenolic ring to afford the catechol adduct 8-(3″,4″-dihydroxyphenyl)-dG (3″,4″-DHPh-dG) and (ii) H-atom abstraction from the sugar moiety to generate the deglycosylated base p-PhOH-G. The ratios of 3″,4″-DHPh-dG to p-PhOH-G were ～1 for Cu(II)/H(2)O(2) and ～0.13 for Fe(II)-EDTA/H(2)O(2). The formation of 3″,4″-DHPh-dG was found to have important consequences in terms of reactivity. The catechol adduct has a lower oxidation potential than p-PhOH-dG and is sensitive to aqueous basic media, undergoing decomposition to generate a dicarboxylic acid derivative. In the presence of excess N-acetylcysteine (NAC), oxidation of 3″,4″-DHPh-dG produced mono-NAC and di-NAC conjugates. Our results imply that secondary oxidative pathways of phenolic-dG lesions are likely to contribute to toxicity.     

Procainamide, but not N-acetylprocainamide, induces protein free radical formation on myeloperoxidase: a potential mechanism of agranulocytosis

Procainamide (PA) is a drug that is used to treat tachycardia in postoperative patients or for long-term maintenance of cardiac arrythmias. Unfortunately, its use has also been associated with agranulocytosis. Here, we have investigated the metabolism of PA by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that PA oxidation by MPO/H 2O 2 would produce a PA cation radical that, in the absence of a biochemical reductant, would lead to the free radical oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms by the reaction of DMPO with a protein free radical. We found that PA metabolism by MPO/H 2O 2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. N-acetyl-PA did not cause DMPO-MPO formation, indicating that the unsubstituted aromatic amine was more oxidizable. PA had a lower calculated ionization potential than N-acetyl-PA. The DMPO adducts of MPO metabolism, as analyzed by electron spin resonance spectroscopy, included a nitrogen-centered radical and a phenyl radical derived from PA, either of which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with PA/H 2O 2 and was found to contain DMPO using the anti-DMPO antibody. Mass spectrometry analysis confirmed the identity of the protein as human MPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by free radical metabolites of PA, which we characterized by spin trapping. We propose that drug-induced free radical formation on MPO may play a role in the origin of agranulocytosis.     

Electron paramagnetic resonance spectrometry evidence for bioreduction of tirapazamine to oxidising free radicals under anaerobic conditions

Tirapazamine (SR 4233) is a bioreductive antitumour drug in Phase III clinical trial which is activated in hypoxic tumour regions to generate a cytotoxic species. Electron paramagnetic resonance (EPR) spectrometry was used to investigate directly the formation of free radicals as the result of tirapazamine reduction by NADPH-supplemented liver microsomes. Under anaerobic conditions, the tirapazamine nitroxide free radical EPR signal was not evident over a range of rat or human liver microsomal protein (1–5 mg) concentrations. However, in combination with 1,1′,5,5′-dimethylpyrolline-1-N-oxide (DMPO), a spin trap for short-lived free radicals, tirapazamine resulted in formation of a 1:1:1:1:1:1 spectrum with hyperfine splitting A_N = 15.8 G A_H = 22.3 G consistent with generation of DMPO-R, a carbon-centered radical adduct. Addition of DMSO increased the signal intensity of the carbon-centred radical by at least twofold. The hyperfine splitting constants associated with DMPO-R could be indicative of a tirapazamine carbon-centred radical per se or, more likely, carbon radicals from endogenous materials (or DMSO) in the biological matrix as a result of oxidative attack by the tirapazamine primary radical. Formation of DMPO-OH, the hydroxyl radical spin adduct, by tirapazamine in the absence of air indicates that liberation of a hydroxyl radical may be a consequence of tirapazamine bioreduction under anaerobic conditions. The reactivity of tirapazamine free radicals with endogenous microsomal substances to generate reactive carbon-centred radicals indicates that tirapazamine may disrupt a wide range of cellular activities.     

Evidence for intracellular superoxide formation following the exposure of guinea pig enterocytes to bleomycin

Spin trapping of free radicals during the exposure of guinea pig enterocytes to bleomycin (BLM) was investigated using an in vitro cell suspension. The spin traps employed in this study were 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3-diethyl-5,5-dimethyl-1-pyrroline-1-oxide (DEDMPO). The hydroxyl radical spin-trapped adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed with DMPO. In the presence of dimethyl sulfoxide (DMSO), the only 2,2,5-trimethyl-1-pyrrolidinyloxyl (DMPO-CH3) observed was that expected from hydroxyl radical formation by the decomposition of the superoxide spin-trapped adduct 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH). Production of hydroxyl radical was not detected in the presence of DEDMPO, which is a nitrone that will spin trap hydroxyl radical, but not superoxide, at cellular concentrations. Thus, these data indicate that superoxide was produced during the exposure of guinea pig enterocytes to BLM and that DMPO-OH resulted from the cellular bioreduction of DMPO-OOH by glutathione peroxidase. Addition of superoxide dismutase to the in vitro reaction mixture indicated that superoxide production was intracellular.     

电子顺磁共振技术研究一枝蒿总黄酮抗氧化作用

目的　探讨一枝蒿总黄酮清除自由基的作用。方法　采用电子顺磁共振技术 (EPR)分别检测一枝蒿总黄酮对超氧阴离子自由基 (O·2 ) ,羟自由基 (·OH)的清除作用。结果　一枝蒿总黄酮对超氧阴离子自由基、羟自由基具有不同程度的清除作用 ,且存在明显的药物浓度—清除效用关系。结论　一枝蒿总黄酮有明显的抗氧化作用     

Studies on the activation of molecular oxygen and the biological defence mechanism against active oxygen species

One of the active oxygen species, superoxide (O2-), was generated by the electrolytic reduction of molecular oxygen in acetonitrile. O2- was determined by the ultraviolet (UV) (lambda max/nm = 255, epsilon = 1.48 x 10(3) M-1 cm-3) and the electron spin resonance (ESR) (g parallel = 2.083, g perpendicular = 2.008) spectrum. O2- could easily react with tocopherols (vitamin E and its derivatives) to give the corresponding chromanoxyl radicals of which structures were determined by ESR. ESR studies of the reactions of O2- with tocopherols or their model compounds indicate that the radical concentrations from tocopherol models correlate with the physiological activities of the tocopherols. O2- could also react with some biologically active quinones such as vitamin K3 and vitamin E quinone to give the corresponding semiquinone radicals. The fact that vitamin E quinone, an irreversible metabolite of vitamin E, was reduced by O2- to the semiquinone radical suggests that, like vitamin E, vitamin E quinone may also scavenge O2- and protect living cells from the effects of O2- in a hydrophobic environment. Further, O2- could react with some metalloporphyrins. In this case, non-redox metalloporphyrins such as Zn(II)TPP (TPP: tetraphenylporphine), Cd(II)TPP, Mg(II)TPP generated the superoxide adduct by the reaction with O2-. On the other hand, redox-active metalloporphyrins such as Cr(III)TPP.Cl, Mn(III)TPP.Cl, Co(II)TTP TTP: tetra-p-tolylporphine) and Co(III)TTP.Cl underwent the addition and/or redox reactions with O2-. Another active oxygen species, hydroxyl radical (OH.), was first detected from some copper (II) coplexes such as Cu(en)2 (en: ethylenediamine) with hydrogen peroxide (H2O2) by ESR spin trapping and thiobarbituric acid (TBA) methods. Further, by using Cu(en)2-H2O2 system the most active OH. scavenger was determined. This Cu(en)2-H2O2 system will be useful for determing the antioxidant ability against OH..     

EPR studies on the oxidation of hydroxyurea to paramagnetic compounds by oxyhemoglobin

N. Hydroxyurea forms methemoglobin from oxyhemoglobin with concomitant formation of the aminocarbonylaminooxyl radical H2N-CO-NHO., as detected with electron paramagnetic resonance spectroscopy (EPR). This radical could be detected for several hours in a low steady-state concentration. Approximately 1 hr after the reaction had been started, the EPR spectra of two additional paramagnetic intermediates could be detected at low temperature (77 degrees K), a low-spin ferric methemoglobin complex with hydroxyurea (MetHb-NHOH-CO-NH2) and the hemoglobin-nitric oxide adduct (Hb2(+)-NO). The intensities of their EPR spectra increased steadily over the range of more than 64 hr. The low-spin ferric methemoglobin complex was immediately formed when hydroxyurea was dissolved in a methemoglobin whereas the nitric oxide complex was possibly an oxidation product of the MetHb-hydroxyurea adduct. Its oxidative degradation is known to lead to the very toxic compounds nitric oxide and nitrogen dioxide which can therefore contribute to the toxic action of hydroxyurea.     

Pro-oxidative interactions of cobalt with human neutrophils

The primary objectives of this study were to investigate the effects of cobalt(II) chloride (Co, 1.5-25 microM) on the reactivity of hydrogen peroxide (H2O2, 100 microM) or oxidants generated by activated human neutrophils. The prooxidative interactions of Co with H2O2 or cells were measured by luminol-enhanced chemiluminescence (LECL), and according to the extent of oxidative inactivation of added alpha-1-proteinase inhibitor (API). Cobalt dramatically potentiated the oxidation of luminol and API by both H2O2 and neutrophils activated with phorbol 12-myristate 13-acetate (5 ng/ml), without affecting the assembly of NADPH oxidase or the magnitude of oxygen consumption by the cells. Using 5,5-dimethyl-pyrolline 1-oxide-based electron spin resonance spectroscopy we were unable to detect hydroxyl radical formation by Co in the presence of either H2O2 or activated neutrophils, while the corresponding LECL responses were unaffected by the hydroxyl radical scavengers benzoate and mannitol (50 mM). These observations indicate that Co potentiates the reactivity of neutrophil-derived oxidants, primarily H2O2, which if operative in vivo during exposure to the heavy metal may pose the risk of oxidant- and protease-mediated tissue injury.     

Antioxidant activity of nasunin, an anthocyanin in eggplant peels

The free radical scavenging activities and inhibitory effect of lipid peroxidation of a delphinidin derivative in eggplant were investigated. Delphinidin-3-(p-coumaroylrutinoside)-5-glucoside (nasunin), an anthocyanin, was isolated as purple colored crystals from eggplant peels. Using electron spin resonance spectrometry and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), hydroxyl radicals (OH) or superoxide anion radicals (O(2)(-)) generated by the Fenton reaction or the hypoxanthine-xanthine oxidase system were measured as DMPO-OH or DMPO-OOH spin adducts. L-Ascorbic acid 2-[3, 4-dihydro-2,5,7,8-tetramethyl-2-(4,8, 12-trimethyltridecyl)-2H-1-benzopyran-6yl-hydrogen phosphate] potassium salt (EPC-K1) and bovine erythrocyte superoxide dismutase (SOD) were used as standards for OH and O(2)(-) scavengers, respectively. Nasunin showed potent O(2)(-) scavenging (143+/-8 SOD-equivalent U/mg) and OH scavenging (0. 65+/-0.07 EPC-K1-equivalent micromol/mg) activities. Then, by changing the concentration of DMPO to vary the trapping rate of OH, the presence of a competitive reaction between nasunin and OH was studied. The 50% inhibition dose (ID(50)) obtained from the inhibition curve did not change, indicating OH scavenging of nasunin is not due to direct scavenging but inhibition of OH generating system by chelating ferrous ion. Nasunin protection against H(2)O(2)-induced lipid peroxidation in rat brain homogenate was measured at 586 nm using the indicator of malonaldehyde and 4-hydroxyalkenals. Nasunin (<50 microM) protected against lipid peroxidation of brain homogenates. The findings suggest that nasunin is a potent O(2)(-) scavenger and has protective activity against lipid peroxidation.     

Traditional Chinese medicine formula Qing Huo Yi Hao as superoxide anion scavenger in high glucose-treated endothelial cells

Aim:

To investigate the effects of a traditional Chinese medicine formula Qing Huo Yi Hao (QHYH) and its components on hydroxyl radical (HO^•) production in vitro and the activity of QHYH against free radicals in cultured endothelial cells induced by high glucose.

Methods:

Hydroxyl radicals (HO^•) were generated through Fenton reactions in vitro, and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a spin trap to form DMPO/HO^• adducts detected using electron paramagnetic resonance (EPR). Immortalized mouse cerebral microvascular endothelial (bEnd.3) cells were treated with high glucose (35 mmol/L). The free radical scavenging ability of QHYH in the cells was evaluated using EPR. Superoxide dismutase (SOD) was used to identify the free radicals scavenged by QHYH in the cells.

Results:

QHYH and its 8 components concentration-dependently reduced DMPO/HO^• signaling. The DMPO/HO^• adduct scavenging ability of QHYH was 82.2%, which was higher than each individual component. The free radical scavenging ability of 1% QHYH in high glucose-treated bEnd.3 cells was approximately 70%. In these cells, the free radicals were also specifically reduced by SOD (400 U/mL), implying that the free radicals were primarily superoxide anions.

Conclusion:

The results demonstrate that the QHYH formula is potent antioxidant acting as scavenge of superoxide anions in high glucose-treated endothelial cells.