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R Kohen

Publications and source records attributed to R Kohen.

At least 73 records · Page 4Linked to original sources
PubMed1993

Chemiluminescence in activated human neutrophils: role of buffers and scavengers.

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the result of the interaction among H2O2, a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H2O2 was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn2+, Fe2+, Cu2+, and Co2+ very markedly enhanced CL induced by mixtures of H2O2 and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by superoxide dismutase (SOD) and by deferoxamine LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H2O2 probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.

Acridines↗
PubMed1993

Killing of endothelial cells and release of arachidonic acid. Synergistic effects among hydrogen peroxide, membrane-damaging agents, cationic substances, and proteinases and their modulation by inhibitors.

51Chromium-labeled rat pulmonary artery endothelial cells (EC) cultivated in MEM medium were killed, in a synergistic manner, by mixtures of subtoxic amounts of glucose oxidase-generated H2O2 and subtoxic amounts of the following agents: the cationic substances, nuclear histone, defensins, lysozyme, poly-L-arginine, spermine, pancreatic ribonuclease, polymyxin B, chlorhexidine, cetyltrimethyl ammonium bromide, as well as by the membrane-damaging agents phospholipases A2 (PLA2) and C (PLC), lysolecithin (LL), and by streptolysin S (SLS) of group A streptococci. Cytotoxicity induced by such mixtures was further enhanced by subtoxic amounts either of trypsin or of elastase. Glucose-oxidase cationized by complexing to poly-L-histidine proved an excellent deliverer of membrane-directed H2O2 capable of enhancing EC killing by other agonists. EC treated with rabbit anti-streptococcal IgG were also killed, in a synergistic manner, by H2O2, suggesting the presence in the IgG preparation of cross-reactive antibodies. Killing of EC by the various mixtures of agonists was strongly inhibited by scavengers of hydrogen peroxide (catalase, dimethylthiourea, MnCl2), by soybean trypsin inhibitor, by polyanions, as well as by putative inhibitors of phospholipases. Strong inhibition of cell killing was also observed with tannic acid and by extracts of tea, but less so by serum. On the other hand, neither deferoxamine, HClO, TNF, nor GTP gamma S had any modulating effects on the synergistic cell killing. EC exposed either to 6-deoxyglucose, puromycin, or triflupromazin became highly susceptible to killing by mixtures of hydrogen peroxide with several of the membrane-damaging agents. While maximal synergistic EC killing was achieved by mixtures of H2O2 with either PLA2, PLC, LL, or with SLS, a very substantial release of [3H]arachidonic acid (AA), PGE2, and 6-keto-PGF occurred only if a proteinase was also added to the mixture of agonists. The release of AA from EC was markedly inhibited either by scavengers of H2O2, by proteinase inhibitors, by cationic agents, by HClO, by tannic acid, and by quinacrin. We suggest that cellular injury induced in inflammatory and infectious sites might be the result of synergistic effects among leukocyte-derived oxidants, lysosomal hydrolases, cytotoxic cationic polypeptides, proteinases, and microbial toxins, which might be present in exudates. These "cocktails" not only kill cells, but also solubilize AA and several of its metabolites. However, AA release by the various agonists can be also achieved following attack by leukocyte-derived agonists on dead cells. It is proposed that treatment by "cocktails" of adequate antagonists might be beneficial to protect against cellular injury in vivo.

Animals↗
PubMed1993

The use of cyclic voltammetry for the evaluation of oxidative damage in biological samples.

A method using cyclic voltammetry to evaluate oxidative damage in biological systems is presented. Three biological systems were tested: Escherichia coli cells, the rat jejunal mucosa, and the enzyme, lactate dehydrogenase. Exposure of E. coli cells to oxidative stress resulted in a rapid decrease in their survival and a decrease in their ability to accumulate 14C-leucine. This was accompanied by a significant increase in the oxidation potential of the cells. Similar results were obtained when the rat jejunal mucosa was exposed in a perfusion system to oxidative stress induced by the hydroxyl radical produced by either hydrogen peroxide and ferrous ions or the combination of ascorbic acid and copper ions. Loss of cellular potassium was taken as an indication of damage to the rat jejunum. Exposure of lactate dehydrogenase to oxidative stress induced by hydroxyl and peroxyl radicals also resulted in a significant loss of enzyme activity along with a pronounced change in the cyclic voltammogram of the enzyme. It was concluded that measurement of the oxidation potentials of these biological systems can give an indication of the occurrence of oxidative damage.

Animals↗
PubMed1993

Prevention of oxidative damage in the rat jejunal mucosa by pectin.

The role of the soluble non-starch polysaccharide pectin in the prevention of oxidative damage induced by peroxy, superoxide and hydroxyl radicals to the rat jejunal mucosa was studied. The oxidative stress was introduced to the rat jejunal mucosa by means of a closed-loop perfusion system and was characterized biochemically by monitoring the enterocyte activity of the enzyme lactate dehydrogenase (EC 1.1.1.27) and the K+ level. Aqueous solutions of pectin were perfused into the rat jejunum before the oxidative stress inducers. The possible protection effect was evaluated by comparing the mucosal integrity (as measured by biochemical variables) to the values obtained after perfusion with the oxidative stress inducers only. We found that: (a) mucosal damage was detected following the perfusion of peroxy and hydroxyl radicals in the rat jejunum, but not following perfusion of the superoxide radical; (b) a significant reduction in the mucosal damage was noted when pectin was perfused before the perfusion with the peroxy radical induction; (c) full protection against the mucosal damage induced by hydroxyl radicals was achieved when pectin was perfused before the damage induction.

Animals↗
PubMed1993

Lung responses to ultrasound-guided fetal treatments with corticosteroids in preterm lambs.

Maternal corticosteroid treatments augment lung function in the human preterm infant. However, not all fetuses respond, the response requires > or = 48 h of exposure, and multiple maternal doses expose the mother to potential risks. To evaluate the potential of direct fetal therapy, we used ultrasound to direct fetal intramuscular or intravascular injections of corticosteroids or saline in sheep and subsequently delivered the preterm lambs at 128 days gestational age to assess postnatal lung function. Relative to saline-injected controls, 0.5 or 2 mg/kg betamethasone given as a single intramuscular dose 48 h before delivery increased compliance and the efficiency of ventilation (as measured by an indicator that included ventilatory pressures and CO2 values) nearly twofold (P < 0.05). Lung volumes, measured from deflation pressure-volume curves, also increased (P < 0.05). However, the 2 mg/kg dose caused severe pulmonary interstitial emphysema in 5 of 13 lambs, suggesting adverse effects. An intravascular fetal dose of 12.5 mg/kg hydrocortisone or an intramuscular dose of 0.1 mg/kg betamethasone had no effect on postnatal lung function. In separate studies, the 2 mg/kg dose improved all indicators of lung function almost twofold after only 24 h of fetal exposure and delivery at 128 days gestational age (P < 0.01). There was a dose-dependent suppression of the postnatal cortisol surge in treated animals, although fetal treatment did not alter cord cortisol levels. Single-dose fetal hormone treatments can cause large and rapid improvements in postnatal lung function in preterm lambs.

Adrenal Cortex Hormones↗
PubMed1992

The role of cationized catalase and cationized glucose oxidase in mucosal oxidative damage induced in the rat jejunum.

The successful prevention of hydrogen peroxide-induced damage to the rat jejunal mucosa by cationized catalase is described in this study. Biological damage was induced in a closed circulating intestinal loop of the rat by hydrogen peroxide and by hydroxyl radicals induced in situ via the metal-mediated Haber-Wiess reaction. The mucosal activity of lactate dehydrogenase and the amount of potassium ions were used to quantitatively characterize the tissue damage. Catalase was cationized by reacting it with N,N'-dimethyl-1,3-propanediamine to give a soluble product or with polyhistidine to give an insoluble product. The activity of the modified enzymes was assessed, and their ability to protect the rat jejunal mucosa against oxidative stress was studied. It was found that in all cases the cationized enzymes were superior to the native catalase in their shield capability. A significant protection against Fe(II)/H2O2 and ascorbic acid/copper ion-mediated damage was obtained when the cationized enzymes were used. In the presence of glucose, native glucose oxidase failed to cause damage in the rat jejunal mucosa; however, the cationized enzyme caused profound tissue injury. These findings indicate the potential therapeutic merit of cationized enzymes for the treatment of pathological processes in the intestine, whenever oxidative stress is involved.

Animals↗
PubMed1992

Synergism among oxidants, proteinases, phospholipases, microbial hemolysins, cationic proteins, and cytokines.

A striking similarity exists between the pathogenetic properties of group A streptococci and those of activated mammalian professional phagocytes (neutrophils, macrophages). Both types of cells are endowed by the ability to adhere to target cells; to elaborate oxidants, hydrolases, and membrane-active agents (hemolysins, phospholipases); and to freely invade tissues and destroy cells. From the evolutionary point of view, streptococci might justifiably be considered the forefathers of "modern" leukocytes. Our earlier findings that synergy between a streptococcal hemolysin (streptolysin S, SLS) and a streptococcal thiol-dependent proteinase and between cytotoxic antibodies+complement and streptokinase-activated plasmin readily killed tumor cells, led us to hypothesize that by analogy to the pathogenetic mechanisms of streptococci, the mechanisms of tissue destruction initiated by activated leukocytes in inflammatory sites, as well as in tissues undergoing episodes of ischemia and reperfusion, might also be the result of the synergistic effects among leukocyte-derived oxidants, phospholipases, proteinases, cytokines, and cationic proteins. The current report extends our previous synergy studies with endothelial cells to two additional cell types--monkey kidney epithelial cells and rat beating heart cells. Monolayers of 51Cr-labeled cells that had been treated by combinations of sublytic amounts of hydrogen peroxide (generated either by glucose oxidase, xanthine-xanthine oxidase, or by paraquat) and with sublytic amounts of a variety of membrane-active agents (streptolysin S, phospholipases A2 and C, lysophosphatides, histone, chlorhexidine) were killed in a synergistic manner (double synergy). Crystalline trypsin markedly enhanced cell killing by combinations of oxidant and the membrane-active agents (triple synergy). Injury to the cells was characterized by the appearance of large membrane blebs that detached from the cells and floated freely in the media, looking like lipid droplets. Cytotoxicity induced by the various combinations of agonists was depressed, to a large extent, by scavengers of hydrogen peroxide (catalase, dimethyl thiourea, and by Mn2+) but not by SOD or by deferoxamine. When cationic agents were employed together with hydrogen peroxide, polyanions (heparin, polyanethole sulfonate) were also found to inhibit cell killing. It is proposed that in order to effectively combat the deleterious toxic effects of leukocyte-derived agonists on cells and tissues, antagonistic "cocktails" comprised of cationized catalase, cationized SOD, dimethylthiourea, Mn(2+)+glycine, proteinase inhibitors, putative inhibitors of phospholipases, and polyanions might be concocted. The current literature on synergistic phenomena pertaining to mechanisms of cell and tissue injury in inflammation is selectively reviewed.

Animals↗
PubMed1992

The reductive capacity index of saliva obtained from donors of various ages.

The reducing ability of saliva is decreased with donor age as indicated by cyclic voltammetry measurements of saliva. It has been shown that saliva possesses a strong reducing power that is influenced by the induction of oxidative stress. The exact capacity of saliva to cope with oxidative stress has never been studied in detail. We used a new approach to detect the role of oxidative stress on saliva. This approach included the detection of the total reducing capacity of the saliva without determination of the exact concentration of a specific reducing compound present in the saliva. Induction of oxidative stress resulted in a significant change in the reductive capacity detected. Measurements of the reducing potential of saliva revealed that there is a correlation between the donor age and the reductive capacity observed. Also, saliva obtained from donors in various disease states have shown a significant change in the potential recorded, as compared to controls. The possible use of this method as a diagnostic tool in various diseases is discussed.

Adolescent↗
PubMed1992

The biological reductive capacity of tissues is decreased following exposure to oxidative stress: a cyclic voltammetry study of irradiated rats.

The reductive capacity of rat tissue homogenates and body fluids was determined by cyclic voltammetric measurements. The reductive capacity of rat lung, liver and kidney homogenates was significantly reduced four days after total body gamma-ray irradiation with 5.5 Gy as compared to controls. In parallel, reduced ability of the irradiated organ homogenates to scavenge hydroxyl radicals and to destroy hydrogen peroxide was recorded. However, no difference in their superoxide dismutase activity was found. The possible use of cyclic voltammetry as a method for qualitative evaluation of the ability of biological tissues to cope with oxidative stress is discussed.

Animals↗
PubMed1991

The SOD like activity of copper:carnosine, copper:anserine and copper:homocarnosine complexes.

Carnosine, anserine and homocarnosine are natural compounds which are present in high concentrations (2-20 mM) in skeletal muscles and brain of many vertebrates. We have demonstrated in a previous work that these compounds can act as antioxidants, a result of their ability to scavenge peroxyl radicals, singlet oxygen and hydroxyl radicals. Carnosine and its analogues have been shown to be efficient chelating agents for copper and other transition metals. Since human skeletal muscle contains one-third of the total copper in the body (20-47 mmol/kg) and the concentration of carnosine in this tissue is relatively high, the complex of carnosine:copper may be of biological importance. We have studied the ability of the copper:carnosine (and other carnosine derivatives) complexes to act as superoxide dismutase. The results indicate that the complex of copper:carnosine can dismute superoxide radicals released by neutrophils treated with PMA in an analogous mechanism to other amino acids and copper complexes. Copper:anserine failed to dismute superoxide radicals and copper:homocarnosine complex was efficient when the cells were treated with PMA or with histone-opsonized streptococci and cytochalasine B. The possible role of these compounds to act as physiological antioxidants that possess superoxide dismutase activity is discussed.

Anserine↗
PubMed1988

Cytoplasmic membrane is the target organelle for transition metal mediated damage induced by paraquat in Escherichia coli.

Bacterial survival indicates that copper or iron is an essential mediator in paraquat toxicity in Escherichia coli [Kohen, R., & Chevion, M. (1985) Free Radical Res. Commun. 1, 79-88; Korbashi, P., Kohen, R., Katzhendler, J., & Chevion, M. (1986) J. Biol. Chem. 261, 12472-12476]. In this study we have identified the cytoplasmic membrane as a target organelle in metal-mediated paraquat toxicity and have demonstrated the complete correlation of the membrane damage with the levels of adventitious copper (or iron). The extent of membrane damage was related by use of four parameters: (a) the level of cellular ATP, (b) the level of cellular potassium, (c) the cellular capacity to accumulate and retain radiolabeled leucine, and (d) the cellular integrity as reflected by transmission electron microscopy (TEM). Exposure of bacterial cells to a combination of paraquat and copper caused a marked decline in parameters a, b, and c. This decline was found to occur in parallel with, or even to precede, the sharp loss of survival of E. coli under the same conditions. Likewise, TEM micrographs clearly indicated alterations in cellular structure that possibly reflect sites of detachment of the cytoplasmic membrane from the bacterial capsule. In contradistinction, copper alone or paraquat alone could not bring about similar changes in cellular structure. These findings are in accord with the suggested site-specific metal-mediated Haber-Weiss mechanism for paraquat toxicity and support our notion that specific chelators of transition metals could reduce or prevent the biological deleterious effects of this herbicide.

Adenosine Triphosphate↗
PubMed1988

Antioxidant activity of carnosine, homocarnosine, and anserine present in muscle and brain.

Carnosine, homocarnosine, and anserine are present in high concentrations in the muscle and brain of many animals and humans. However, their exact function is not clear. The antioxidant activity of these compounds has been examined by testing their peroxyl radical-trapping ability at physiological concentrations. Carnosine, homocarnosine, anserine, and other histidine derivatives all showed antioxidant activity. All of these compounds showing peroxyl radical-trapping activity were also electrochemically active as reducing agents in cyclic voltammetric measurements. Furthermore, carnosine inhibited the oxidative hydroxylation of deoxyguanosine induced by ascorbic acid and copper ions. Other roles of carnosine, such as chelation of metal ions, quenching of singlet oxygen, and binding of hydroperoxides, are also discussed. The data suggest a role for these histidine-related compounds as endogenous antioxidants in brain and muscle.

Amidines↗
PubMed1988

Iron enhancement of ascorbate toxicity.

Iron has been shown to enhance ascorbate-induced damage to both acetylcholine esterase and E. coli B in a manner analogous to previous studies with ascorbate and copper ions. It is suggested that the mechanism of damage entails interaction of iron with biological macromolecules, followed by its reduction by ascorbate. Subsequently, the iron (II) could participate in generating hydroxyl radicals from hydrogen peroxide via the Fenton reaction, which in turn, could damage biomolecules in a site-specific and multiple hit fashion. The high abundance of iron in biological systems, especially in certain storage disorders, may indicate an important toxicological role of the combination of iron and ascorbate.

Acetylcholinesterase↗
PubMed1986

Iron mediates paraquat toxicity in Escherichia coli.

The role of iron ions in paraquat toxicity was studied in bacterial system. We show that addition of ferrous iron led to an enhancement of the bacterial killing, whereas addition of chelating agents, such as nitrilotriacetate and desferrioxamine, markedly reduced, up to a total abolishment, the toxic effects. The calculated rates of bacterial killing are proportional to both paraquat and iron concentrations, and conform to the rate equation: dN/dt = -k[paraquat] [Fe2+]. The killing constant for iron, k, is 24-fold smaller than the corresponding value for copper. Mannitol, an OH. scavenger, has a partial protective effect: 15-35% at concentrations range of 1-50 mM, respectively. Histidine, on the other hand, provided a more efficient protection that may be due to a combination of various effects. Induction of endogenous superoxide dismutase and catalase provided partial protection (about 25%). These findings, together with an earlier study on the role of copper in paraquat toxicity (Kohen, R., and Chevion, M. (1985) Free Rad. Res. Commun. 1, 79-88) indicate that transition metals play a central catalytic role in the production of the deleterious effects of paraquat, probably by redox cycling and producing OH. via the site-specific Fenton reaction.

Catalase↗
PubMed1986

Quantitation of single- and double-strand DNA breaks in vitro and in vivo.

This communication describes a rapid and convenient procedure for quantitation of strand breaks in bacterial DNA, both in vitro and in vivo, using agarose gel electrophoresis. The electrophoretic determination of single strand breaks is carried out in alkaline medium, followed by renaturation of the gel and intercalation of the fluorescent dye, ethidium bromide. Double-strand breaks are determined by electrophoresis in neutral medium containing the dye. The distribution of DNA fragment sizes, the determination of the number-average molecular weight, the quantitation of the average number of DNA breaks per molecule, and the ratio between the single- and double-strand breaks are evaluated from microdensitometric scanning of the gels. The application of this analysis to damage caused by a combination of ascorbate and copper is demonstrated.

Ascorbic Acid↗