PubMed Health⌕ Search

Biomedical subjects

A Samuni

Publications and source records attributed to A Samuni.

At least 37 records · Page 2Linked to original sources
PubMed1990

Superoxide reaction with nitroxides.

Stable, free radical nitroxides are commonly used ESR spectroscopy tools. However, it has recently been found that ESR observable signal from 5-membered ring spin-adducts or stable label nitroxides is lost or diminished by reaction with superoxide. A similar radical-radical annihilation was not found for six membered ring nitroxide radicals. To discern why six-membered ring nitroxides are not reduced under superoxide flux generated by hypoxanthine/xanthine oxidase, spectrophoptmetric (Cyt CIII) and chemiluminescence (lucigenin) and ESR assays were used to follow the reactions. Spectrophotometry and chemiluminescence clearly demonstrated that the six-membered piperidine-1-oxyl compounds (TEMPO, TEMPOL, and TEMPAMIN) rapidly react with superoxide: rate constants at pH 7.8 ranging from 7 x 10(4) to 1.2 x 10(5) M-1 s-1. The absence of detectable ESR signal loss results from facile re-oxidation of the corresponding hydroxylamine by superoxide. To fully corroborate the efficiency of the 6-membered nitroxide superoxide dismutase activity, they were shown to protect fully mammalian cells from oxidative damage resulting from exposure to the superoxide and hydrogen peroxide generating system hypoxanthine/xanthine oxidase. Since six-membered cyclic nitroxides react with superoxide about 2 orders of magnitude faster than the corresponding 5-membered ring nitroxides, they may ultimately be more useful as superoxide oxide dismutase mimetic agents.

Animals↗
PubMed1989

Mechanistic aspects of paraquat toxicity in E. coli. A spin trapping study.

Mechanistic aspects of paraquat monocation radical (PQ.+) and copper involvement in paraquat toxicity have been examined using E. coli B cells. Electron spin resonance (ESR) spectrometry combined with cell survival studies were used to explore the correlation between radical production and biological damage. The line broadening agent oxalato-chromiate (CrOx) was used to characterize the anoxic partition of PQ.+ inside and outside the cell. In the presence of CrOx the ESR signal was totally eliminated, indicating that intracellular species were undetectable and that, contrary to previous reports, PQ.+ exclusively accumulates outside the cell. The PQ.+ radical does not react with H2O2 but disappears in the presence of H2O2 when catalytic traces of Cu(II) are present. Spin-trapping studies using DMPO showed that in aerobic environment paraquat-induced O2 radicals are detectable exclusively in the extracellular compartment. The correlation between PQ.+ appearance and the biological damage is not simple. PQ.+ non-toxically accumulates, in the absence of oxygen and either Cu(II) or H2O2. By contrast, with both H2O2 and Cu(II) the cells are rapidly killed but PQ.+ was undetectable. These results reconfirm the key catalytic mediatory function of transition metals in paraquat toxicity.

Aerobiosis↗
PubMed1989

Free radicals induced by adriamycin-sensitive and adriamycin-resistant cells: a spin-trapping study.

The radicals generated by adriamycin-sensitive (CHO-AB) and adriamycin-resistant (CHO-C5) Chinese hamster ovary cells as well as by adriamycin-sensitive and -resistant human breast cancer cells (MCF7-WT and MCF7-ADR) have been studied with spin-trapping and ESR spectroscopy. During anoxic exposure to adriamycin (ADR) both pairs of cell lines produced the broad ESR singlet characteristic of ADR semiquinone (AQ.). By use of tris(oxalato)chromate (CrOx) as an extracellular line-broadening agent, the distribution of AQ. between the intra- and extracellular compartments was studied. For cell densities of (1-3) X 10(7) cells/mL, CrOx eliminated most, though not all, of the ESR signal, indicating that the AQ. radicals freely diffuse and partition between the intra- and extracellular compartments proportionally to their respective volumes. Similar behavior was exhibited by all four cell lines studied. Upon introduction of oxygen to anoxic cells in the presence of the spin trap 5,5-dimethylpyrroline N-oxide (DMPO), the AQ. signal was replaced by that of the DMPO-OH spin adduct. Metal chelators such as desferrioxamine had no effect on DMPO-OH or AQ. formation. Superoxide dismutase, not catalase, totally eliminated the ESR signal, indicating that DMPO-OH produced by ADR-treated cells originates from superoxide rather than from .OH produced from H2O2. In the presence of CrOx, the DMPO-OH signal was not distinguishable from the background noise, thus excluding any contribution to the signal by intracellular spin adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Evaluation of dibromonitrosobenzene sulfonate as a spin trap in biological systems.

In the present study dibromonitrosobenzene sulfonate (DBNBS) was examined for its suitability for spin trapping for ESR detection of superoxide radicals in biological systems. This nitroso spin trap recently has been reported to yield very persistent spin adducts with O2. as well as with various carbon-centered radicals. In the present work the possible toxicity of DBNBS, the partitioning of its spin adducts into cells, and the stability of the adducts and the parent compound inside cells were studied. No significant toxicity was found. In cellular systems, however, DBNBS did not produce detectable adducts with O2.; it also did not detectably trap superoxide generated in the xanthine/xanthine oxidase system. Both DBNBS and a DBNBS adduct performed extracellularly and then added to cell suspensions were rapidly metabolized by cells. Intracellular spin adducts were not detected under any condition. Evidently, in spite of its promising features, DBNBS will not be useful for spin trapping radicals in cellular systems or for detecting superoxide radicals in any biological system.

Benzenesulfonates↗
PubMed1989

Superoxide reaction with nitroxide spin-adducts.

The reactions of superoxide radical with persistent nitroxide spin-adducts or with stable spin-labels were studied using ESR spectrometry. Superoxide radicals were produced enzymatically using xanthine - xanthine oxidase or chemically by dissolving potassium superoxide in DMSO. Hydroxyl and methyl spin-adducts of the spin-trap DMPO were performed by sonolysis and subsequently reacted with superoxide radical. Superoxide-induced depletion of DMPO--OH obeyed second order kinetics. Contrary to previously published mechanisms, the reaction requires neither transition metal ions nor thiols. The depleted spin-adducts could not be restored by reoxidation with ferricyanide or copper +H2O2; thus, the superoxide-mediated destruction does not result in a mere one-electron reduction product. Superoxide also depletes other DMPO spin-adducts including DMPO--CH3 and DMPO--H, but not PBN--CH3. In addition, some 5-membered ring stable nitroxides are depleted by superoxide in a pseudo-zero order reaction. In studying systems which generate O2- and OH, the superoxide-induced destruction of DMPO--OH may well lead to erroneous conclusions regarding the primary radicals produced. In particular this reaction might be operative under circumstances where elevated rates of superoxide production take place, such as during oxygen consumption "burst" in phagocytosis, degranulation, or paraquat intoxication.

Cyclic N-Oxides↗
PubMed1989

The cellular-induced decay of DMPO spin adducts of .OH and .O2.

In a recent report, it was concluded that DMPO, often considered the spin trap of choice for detection of superoxide and hydroxyl radical adducts in biological systems, may be unsuitable for many biological uses because of its instability in cellular systems. It was demonstrated in red blood cells and in hamster V79 cells that the DMPO spin adducts of .O2- and .OH are metabolized very rapidly so that even if formed, they may not be detected in many experiments with cells. Because of the potential importance of these findings to experiments already reported on the occurrence of oxygen radicals in cellular systems, and the implications of these findings for future experiments, we have extended the studies on DMPO to other cellular, systems. We have also investigated the role of oxygen in this system because it has been shown recently that very hypoxic cells reduce some nitroxides much more rapidly than oxic cells and therefore it seemed possible that the rapid loss of radical adducts of DMPO was due to the hypoxic conditions under which the previous experiments were carried out. The results of the present experiments indicate that the loss of the DMPO spin adducts occurs in other cell systems as well, that the decomposition rate is independent of the concentration of oxygen, and that the final products of cellular metabolism of DMPO adducts are different from those of most nitroxides. There is no evidence that intracellular DMPO-spin adducts of oxygen radicals can be observed under conditions similar to those used in this study. We conclude that DMPO is not likely to be a suitable agent for studying intracellular oxygen radicals.

Animals↗
PubMed1988

A novel metal-free low molecular weight superoxide dismutase mimic.

2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH), the one-electron reduction product of the stable nitroxide radical, 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO), is reportedly oxidized by superoxide, and its oxidation has been proposed as a method for assaying superoxide. We find that superoxide can both reduce OXANO and oxidize OXANOH. The respective rate constants, k1 and k2, were determined using two superoxide-generating systems (xanthine oxidase/xanthine as well as ionizing radiation). OXANOH oxidation and OXANO reduction are both inhibitable by superoxide dismutase, pH-dependent (4.5-9.3), and result in a steady state distribution of [OXANO] and [OXANOH], independent of their initial concentrations, i.e. the OXANO/OXANOH couple exhibits a metal-independent superoxide dismutase-like function. Thus it provides a prototype for future development of improved low molecular weight superoxide dismutase mimics which will also function in cellular hydrophobic (aprotic) compartments such as membranes.

Hydrogen-Ion Concentration↗
PubMed1988

Hydroxyl radical production by stimulated neutrophils reappraised.

Release of active oxygen species during the human neutrophil respiratory burst is thought to be mandatory for effective defense against bacterial infections and may play an important role in damage to host tissues. Part of the critical bacterial and host tissue damage has been attributed to hydroxyl radicals produced from superoxide and hydrogen peroxide. Because of the short life time of the very reactive hydroxyl radical, direct study of hydroxyl radical production is not possible; therefore, indirect detection methods such as electron spin resonance (ESR) coupled with appropriate spin-trapping agents such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) have been used. Superoxide production during the oxidative burst has been unambiguously demonstrated. Recent reports claim that hydroxyl radicals are not made during neutrophil stimulation and offer as an explanation the presence of granular components that interfere with hydroxyl radical production. When using the spin-trap agent DMPO, absence of the relatively long-lived adducts DMPO-OH and DMPO-CH3 has been assumed to be prima facie evidence for lack of hydroxyl radical participation. We show that high superoxide flux produced during stimulation of human neutrophils rapidly destroys both DMPO-OH and DMPO-CH3. In accord with previous implications, our results provide an alternative explanation for the absence of .OH adduct in spin-trapping studies and corroborate results obtained using other methods that implicate hydroxyl radical production during neutrophil stimulation.

Cyclic N-Oxides↗
PubMed1987

Stimulation of murine cultured mast cells under anaerobic conditions: inhibition of arachidonic acid release.

The exocytosis of beta-hexosaminidase from either IgE-antigen- or calcium ionophore A23187-stimulated murine bone-marrow-derived mast cells was not affected by oxygen-depleted conditions regardless of the absence of glucose from the medium. No detectable changes in the content of ATP were observed when the cells were triggered immunologically under anaerobic conditions in the absence of glucose in the medium. Depletion of oxygen from mast cells activated by both stimuli almost completely inhibited the specific release of arachidonic acid, which indicates that arachidonate does not play a significant role in the secretion of preformed mediators.

Adenosine Triphosphate↗
PubMed1987

Ascorbic acid oxidation and DNA scission catalyzed by iron and copper chelates.

The asorbic acid (AH-) auto-oxidation rates catalyzed by copper chelates of 1,10-phenanthroline (OP) or by iron chelates of bleomycin (BLM) are only slightly higher than the oxidation rates catalyzed by the metal ions. AH- oxidation in the presence of DNA is accompanied by degradation of the DNA. The rates of DNA scission by the metal chelates are markedly higher than the rates induced by the free metal ions. AH- oxidation is slowed down in the presence of DNA which forms ternary complexes with the chelates. The ternary complexes react slowly with AH- but induce DNA double strand breaks more efficiently than the free metal chelates. With OP, DNA is degraded by the reaction of the ternary complex, DNA-(OP)2Cu(I), with H2O2. AH- oxidation in the presence of DNA was biphasic, showing a marked rate increase after DNA was cleaved. We suggest that this sigmoidal pattern of the oxidation curves reflects the low initial oxidative activity of the ternary complexes, accelerating as DNA is degraded. Using O2- produced by pulse radiolysis as a reductant, we found that AH- oxidation with (OP)2Cu(II) induced more DNA double strand breaks per single strand break than bipyridine-copper. The site specific DNA damaging reactions indicated by these results are relevant to the mechanism of cytotoxic activities of bleomycin and similar antibiotics or cytotoxic agents.

Ascorbic Acid↗
PubMed1986

Cellular targets of adriamycin-induced damage in Escherichia coli.

The cellular targets of adriamycin (ADR) activity were studied in Escherichia coli by following colony forming ability and various cellular functions. The parameter exhibiting the best correlation with mortality was inhibition of RNA synthesis. Total DNA synthesis was inhibited to a lesser extent, but may reflect a concurrent inhibition of replication and stimulation of DNA repair activity. Protein synthesis, membrane function and rate of oxygen consumption were affected later. No extensive DNA fragmentation was observed. The inhibition of RNA synthesis was independent of the stringent response and of inhibition of DNA synthesis induced by nalidixic acid. ADR activated the SOS repair system, and the lesions induced by the drug could be repaired by recA dependent functions. These results indicate that the primary activity of ADR was directed against the DNA and interfered with the DNA template function.

Adenosine Triphosphate↗
PubMed1986

On the spin trapping and ESR detection of oxygen-derived radicals generated inside cells.

Recently several attempts to identify oxygen-derived radicals in whole cells by spin trapping and electron spin resonance have been reported by using 5,5-dimethyl-1-pyrroline-N-oxide as the spin trap. In the present study, the feasibility of this method is examined. Chinese hamster V79 cells and human erythrocytes served as the test systems, while OH radicals were generated by gamma radiolysis. Several spin traps were used to scavange the radicals and a distinction between exo- and endocellular ESR observable species was achieved using tri(oxalato) chromiate(III) as a line broadening agent. To distinguish between exo- and endocellular sites of radical formation, we studied the effects of high molecular weight scavengers (polyethylene glycols), which do not enter the cell. Various possible obstacles associated with trapping and detecting the radicals inside the cells were examined. The results indicate that the primary radicals react with the spin traps. However, these spin adducts decayed within the cells. Cellularly induced decay of 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl radical presented the major difficulty in detecting the endogenous radicals, and potential experimental approaches to overcome this difficulty are discussed.

Animals↗
PubMed1986

Enhancement of misonidazole cytotoxicity by iron.

The toxicity of misonidazole (MISO) to hypoxic Chinese hamster ovary (CHO) cells in serum-free medium is enhanced by Fe(III)-EDTA. Enhancement of MISO cytotoxicity by a factor of 1.6 was seen with 2 microM Fe(III)-EDTA, while 200 microM Fe(III)-EDTA results in sensitization by a factor of 2.0. Treatment of CHO cells with the iron chelator desferal resulted in protection against the hypoxic cytotoxicity in MISO (approximate protection factor of 2.5 with 100 microM desferal). Similar results were obtained with Chinese hamster V79 cells. Fe(III)-EDTA also enhanced binding of [2-14C] MISO to cellular macromolecules while desferal decreased binding of MISO to cellular macromolecules. These results suggest that iron plays an important role in the reductive metabolism of MISO and that modification of the intracellular metal ion status may be a useful approach to modulating the biological effect of nitro compounds.

Animals↗
PubMed1979

Quaternary structure of methemoglobin. Pulse radiolysis study of the binding of oxygen to the valence hybrid.

The pulse radiolysis of solutions of adult human methemogolbin was used in order to reduce a single heme iron within the protein tetramers. The valence hybrids thus formed were reacted with oxygen. Kinetics of the reactions were studied. The effects of pH and inositol hexaphosphate were examined. The kinetics of the ligation of oxygen to stripped valence hybrids showed a single phase behavior at the pH range 6.5 to 9. As the pH was lowered below 6.5, a second, slower phase became apparent. In the presence of inositol hexaphosphate, above pH 8, the kinetics of oxygen binding was of a single phase. As the pH was lowered, a transition to a second, slower phase was noticed. Below pH 7, the slower phase was the only detectable one. The analysis of the relative contribution of the faster phase to the total reaction as a function of the pH showed a typical transition curve characterized by a pK = 7.5 and a Hill parameter n = 2.9. On this basis, it is concluded that human adult stripped methemoglobin resides in an R quarternary structure, while the presence of IHP stabilizes the T structure at pH below 7.5. This transition between the quaternary structures of methemoglobin cannot be accounted for by the switch between the high spin and the low spin states of the ferric iron. This switch of spin state takes place at pH greater than 8.2.

Heme↗
PubMed1979

Dynamics of pH-induced spectral changes in bacteriorhodopsin.

The kinetics of the spectral shift induced in bacteriorhodopsin by low pH are investigated by using the rapid-mixing, stopped-flow technique. The generation of the acid form of the chromophore (A605) occurs in two distinct steps: a fast process (t1/2I = 21 +/- 4 ms) is followed by a much slower reaction (t1/2II = 6 +/- 2 s). The observations are interpreted in terms of neutralization of an acid group in the neighborhood of the retinyl chromophore, the double-staged kinetics being attributed to cooperative effects between chromophores. The method may serve as a tool for studying the kinetics of proton migration across the purple membrane.

Bacteriorhodopsins↗
PubMed1978

Quaternary states of methemoglobin and its valence-hybrid. A pulse radiolysis study.

Using the pulse radiolysis technique on solutions of stripped adult human methemoglobin, we found that the heme-iron within a single subunit in the tetramer was reduced to iron(II). The valence-hybrid thus formed was reacted with oxygen and with carbon monoxide. Kinetics of the reactions were studied. The effects of pH, inositol hexaphosphate, and temperature on these reactions were examined. The kinetics of the ligation of O2 and CO were used to characterize the affinity states of the valence-hybrid and its parent methemoglobin. Our results support the description of stripped methemoglobin A as residing in an R state. In the presence of inositol hexaphosphate methemoglobin is stabilized in a T state, but it switches into a high affinity state when the pH is raised a0ove 8.0. This structural transition was not found to coincide with the switch of spin state of the heme-iron that accompanies the ionization of water in aquomethemoglobin A.

Adult↗