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Biomedical subjects

A Samuni

Publications and source records attributed to A Samuni.

At least 55 records · Page 3Linked to original sources
PubMed1991

Growth inhibition of Plasmodium falciparum involving carbon centered iron-chelate radical (L., X-)-Fe(III) based on pyridoxal-betaine. A novel type of antimalarials active against chloroquine-resistant parasites.

Malaria parasites have been shown to be more susceptible to oxidative stress than their host erythrocytes. In the present work, a chloroquine resistant malaria parasite, Plasmodium falciparum (FCR-3) was found to be susceptible in vitro to a pyridoxal based iron chelator--(1-[N-ethoxycarbonylmethylpyridoxlidenium]-2-[2'-pyridyl ] hydrazine bromide--(code named L2-9). 2h exposure to 20 microM L2-9 was sufficient to irreversibly inhibit parasite growth. Desferrioxamine blocked the drug effect, indicating the requirement for iron. Oxygen however, was not essential. Spectrophotometric analysis showed that under anoxic conditions, L2-9-Fe(II) chelate undergoes an intramolecular redox reaction which presumably involves a one electron transfer and is expected to result in the formation of free radical. Spin trapping coupled to electron spin resonance (ESR) studies of L2-9-iron chelate showed that L2-9-Fe(II) produced free radicals both in the presence and absence of cells, while L2-9-Fe(III) produced free radicals only in the presence of actively metabolising cells.

Animals↗
PubMed1991

Fe (II)-chelates based on redox-active pyridoxal-betaines as C-centered radicals causing single- and double-strand scissions to DNA.

The ability of 1-[N-Ethoxycarbonylmethylpridoxylidenium]-2-[2'- pyridyl]hydrazine bromide code name - [L2.9 = L+,X-]-FE(II) chelate [L2-9-Fe(II)] to induce breaks both in the 43kb linear double-strand lambda phage DNA, and in the 4363 base pair supercoiled pBR322 plasmid DNA is herein described. Neither the free ligand nor FE(II) alone demonstrated any effect on the DNA. The cleaving ability is shown to occur instantaneously under strictly anaerobic conditions, either in the presence or absence of the enzyme catalase. It is also shown to be dose dependent. Thus, at lambda DNA:L2-9-Fe(II) molar ratio of 3.7:1.0, the linear DNA is randomly cleaved into fragments ranging from 23.1kb to 4.3kb, whereas at approximately 1:1 molar ratio, the range extends down to 2.5kb fragments. By contrast, at 1:2.7 [plasmid DNA]: chelate-Fe(II) molar ratio, a single-strand nick was observed, and a double strand break was noted at a 1:50 ratio [( plasmid DNA]: chelate-Fe(II). A multi-stage redox cycling involving a carbon-centered (L,X-)-Fe(III) radical capable of transferring an electron to the DNA to form high unstable [DNA].- anion-radical is invoked to explain the degradation of the chain macromolecule. Possible modes for regeneration of the chelate-Fe(III) radical both at the cell-free and at the cell levels are proposed.

DNA Damage↗
PubMed1991

Superoxide production by stimulated neutrophils: temperature effect.

Activation of neutrophils results in a one-electron reduction of oxygen to produce the superoxide anion and other oxygen-derived, microbicidal species. Evidence from many kinetic studies of oxygen-derived radicals generated by stimulated neutrophils in vitro shows that radical production is optimal at 37 degrees C but only lasts several minutes and then rapidly subsides. These findings support the widely held perception that the neutrophil's "oxidative burst" is a transitory event that peaks within minutes of stimulation and ends shortly thereafter. However, while some studies have shown that under controlled conditions stimulated neutrophils can generate superoxide continuously for several hours, others have observed that the superoxide formation by neutrophils stimulated in buffer at 37 degrees C does not persist. To reconcile the conflicting findings and to better understand neutrophil function, we have reinvestigated the effect of temperature on the kinetics of radical generation by PMA-stimulated cells. Electron paramagnetic resonance spectroscopy coupled with spin-trapping and SOD-inhibitable ferricytochrome c reduction were used to monitor superoxide production by neutrophils stimulated at either 25 degrees C or 37 degrees C in RPMI 1640 medium or in Hank's balanced salt solution. When oxygen was supplied continuously, neutrophils stimulated at 25 degrees C in buffer or in medium generated superoxide for several hours but at 37 degrees C, particularly in HBSS, O2- formation strikingly and rapidly decreased. This cessation of superoxide generation was reversible by lowering the temperature back to 25 degrees C. These data imply that in vivo neutrophils may be capable of generating oxy-radicals for prolonged periods. In part, our results may also explain the often observed termination of neutrophil-derived radical formation in vitro and help to dispel the perception that neutrophil-derived oxy-radical production is an ephemeral phenomenon.

Cyclic N-Oxides↗
PubMed1991

Nitroxide SOD-mimics: modes of action.

Low molecular weight superoxide dismutase mimics have been shown to afford protection from oxidative damage. Such SOD-mimics can readily permeate cell membrane achieving sufficiently high levels both inside and outside the cell to effectively detoxify intracellular O2-.. Preliminary findings also indicated that metal-based and metal-free SOD-mimics can protect hypoxic cells from H2O2-induced damage. The present study explored the possibility that SOD-mimics such as desferrioxamine-Mn(III) chelate [DF-Mn] or cyclic nitroxide stable free radicals could protect from O2-.-independent damage. Killing of monolayered V79 Chinese hamster cells was induced by H2O2 or by t-butyl hydroperoxide (t-BHP) and assayed clonogenically. Neither catalase nor native SOD protected the cells from t-BHP. In contrast, both DF-Mn and cyclic nitroxides protected suggesting cytotoxic processes independent of O2-. or of O2-.-derived active species. The inhibition of the damage by both metal-free and metal-based SOD mimics is attributable to either SOD-mimic reacting with reduced transition metal to block the Fenton reaction and/or intercepting and detoxifying intracellular organic free radicals.

Animals↗
PubMed1990

Time-dependent modifications of ferric-adriamycin.

The biological and chemical properties of the ferric-Adriamycin complex changed with time after its preparation. Our experiments demonstrated that the toxicity of the iron-chelate in mice decreased as a function of its age. The reduced toxicity can be correlated with changes in the difference spectrum of ferric-Adriamycin vs Adriamycin (ADR), where a peak around 610 nm shifted to the 570 nm region. When ferric-Adriamycin "aged", the affinity of the drug for iron increased. Concurrently, the redox properties of the chelate changed, such that the bound iron was no longer reduced by glutathione or cysteine. The time-dependent changes observed did not involve the formation of polynuclear iron, as shown by electron spin resonance. Thin-layer chromatography showed that ADR undergoes accelerated degradation in the presence of iron. The iron-catalyzed degradation was oxygen independent. The changes evolving in the spectral and chemical properties of the chelate were shown to stem from transfer of the iron from ADR to one of the degradation products.

Anaerobiosis↗
PubMed1990

Biologically active metal-independent superoxide dismutase mimics.

Superoxide dismutase (SOD) is an enzyme that detoxifies superoxide (O2.-), a potentially toxic oxygen-derived species. Attempts to increase intracellular concentrations of SOD by direct application are complicated because SOD, being a relatively large molecule, does not readily cross cell membranes. We have identified a set of stable nitroxides that possess SOD-like activity, have the advantage of being low molecular weight, membrane permeable, and metal independent, and at pH 7.0 have reaction rate constants with O2.- ranging from 1.1 x 10(3) to 1.3 x 10(6) M-1 s-1. These SOD mimics protect mammalian cells from damage induced by hypoxanthine/xanthine oxidase and H2O2, although they exhibit no catalase-like activity. In addition, the nitroxide SOD mimics rapidly oxidize DNA-FeII and thus may interrupt the Fenton reaction and prevent formation of deleterious OH radicals and/or higher oxidation states of metal ions. Whether by SOD-like activity and/or interception of an electron from redox-active metal ions they protect cells from oxidative stress and may have use in basic and applied biological studies.

Animals↗
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

Reappraisal of the association between adriamycin and iron.

The nature of the association between Adriamycin (ADR) and iron was reinvestigated spectroscopically. It is shown that ADR and Fe3+ do not necessarily form a colloidal aggregate, but rather form a true chelate, Fe3+ ADR3, having a 602-nm molar extinction coefficient of 16.4 mM-1.cm-1. In contrast to the high nominal binding constant for ferric-ADR, beta = 10(33.4), it is shown that under actual conditions of metal hydrolysis and ADR protonation, the effective binding constant, Keff, is strongly pH dependent and is only 10(16.2) M-3 at pH 7.4. These properties are reflected in a progressive dissociation of Fe3+ ADR3 upon dilution and at decreasing pH. Maximal iron chelation by ADR is not achieved at [ADR]:[iron] ratios lower than 10:1, and at [ADR] below the 0.1 mM range. These observations necessitate a reevaluation of previous conclusions regarding the involvement of iron in ADR activity. The clinical implications are important, because at ADR concentrations obtained in vivo, and contrary to common assumptions, ADR will not bind adventitious iron to form a binary chelate. Furthermore, a preformed Fe3+ ADR3 chelate will dissociate when injected. This precludes the involvement of a binary ferric-ADR chelate in the mechanism of action of ADR in vivo.

Algorithms↗
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↗