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1.
Mutat Res ; 464(2): 269-77, 2000 Jan 24.
Article in English | MEDLINE | ID: mdl-10648914

ABSTRACT

Previous literature reports have demonstrated that nucleated trout erythrocytes in conditions of oxidative stress are subjected to DNA and membrane damage, and inactivation of glutathione peroxidase. The present study was undertaken to evaluate the ability of three diaryl tellurides and the organoselenium compound ebselen to protect trout (Salmo irideus) erythrocytes against oxidative stress, induced thermally and by a variation of pH. The antioxidant ability of these molecules was evaluated through chemiluminescence. Impairment of DNA was assessed using the comet assay, a rapid and sensitive single cell gel electrophoresis technique, used to detect primary DNA damage in individual cells. At low concentrations (<10 microM), all the compounds used presented a protective effect on DNA damage without altering the hemolysis rate. In higher concentrations, they accelerated the hemolysis rate and two of the diaryl tellurides were strongly genotoxic.


Subject(s)
Antioxidants/toxicity , Azoles/toxicity , Erythrocytes/drug effects , Erythrocytes/metabolism , Organometallic Compounds/toxicity , Organoselenium Compounds/toxicity , Oxidative Stress/drug effects , Animals , Comet Assay , DNA Damage/drug effects , Dose-Response Relationship, Drug , Erythrocytes/cytology , Hemolysis/drug effects , Hot Temperature/adverse effects , Hydrogen-Ion Concentration , Isoindoles , Luminescent Measurements , Mutagenicity Tests , Oncorhynchus mykiss , Organometallic Compounds/chemistry , Tellurium/chemistry
2.
Free Radic Res ; 28(5): 507-16, 1998 May.
Article in English | MEDLINE | ID: mdl-9702531

ABSTRACT

The purpose of this study was to evaluate the ability of indolinic and quinolinic nitroxide radicals to protect trout (Salmo irideus) erythrocytes against oxidative stress. By using laurdan as a fluorescence probe, it was observed that the nitroxides inhibited the shift towards a gel phase of liposomes prepared with phospholipids extracted from trout erythrocyte membranes prior to the hemolytic event. In addition, the presence of 100 microM nitroxides in these liposomes protected the latter against lipid peroxidation determined by monitoring conjugated diene formation. However, the short chain analogue of the indolinic nitroxide and the quinolinic nitroxide had a negative effect on trout hemolysis, contrary to what has already been observed in previous studies on human RBCs (red blood cells). The half-time (t1/2) of the hemolytic process was 174 +/- 4.02 min for the former and 184 +/- 4.30 min for the latter compared to the control, 283 +/- 5.05 min. Furthermore, the nitroxides remarkably increased the autoxidation rate of both trout and human hemoglobin to met-Hb. Even though protection at the membrane level is conferred by the nitroxides during the early stages of lipid peroxidation, their antioxidative ability might be overwhelmed at a later stage by other mechanisms such as the increased autoxidation of hemoglobin in the presence of the nitroxides, thus giving a possible explanation for the early induction of hemolysis induced by the nitroxides. The superoxide scavenging ability of all the nitroxides used was also evaluated through chemiluminescence.


Subject(s)
Cyclic N-Oxides/pharmacology , Erythrocytes/drug effects , Indoles/pharmacology , Oxidative Stress , Protective Agents/pharmacology , Quinolines/pharmacology , Animals , Cyclic N-Oxides/chemistry , Erythrocytes/metabolism , Free Radical Scavengers/metabolism , Free Radicals/metabolism , Free Radicals/pharmacology , Indoles/chemistry , Luminescent Measurements , Oxidation-Reduction , Protective Agents/chemistry , Quinolines/chemistry , Trout
3.
Free Radic Biol Med ; 24(7-8): 1310-5, 1998 May.
Article in English | MEDLINE | ID: mdl-9626588

ABSTRACT

Because previous literature reports have demonstrated that nucleated trout erythrocytes in conditions of oxidative stress are subjected to both membrane damage and a decrease in the enzymatic defense systems (glutathione peroxidase), which in turn lead to hemolysis, the present study was undertaken to determine whether DNA may be affected too, prior to the hemolytic event. Impairment of DNA in stressed trout erythrocytes was assessed using the comet assay--a rapid and sensitive, single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. In addition, indolinic and quinolinic nitroxide radicals were included in the study to determine their efficacy as antioxidants against free-radical-induced DNA damage. The parameters, tail length, tail intensity, and tail moment, used as an index of DNA damage, have shown that trout erythrocytes exposed to oxidative stress experience DNA damage prior to hemolysis and that the nitroxides significantly prevent this damage. This result provides further information about the potential use of these compounds as antioxidants in biological systems.


Subject(s)
Antioxidants/pharmacology , DNA Damage , Erythrocytes/drug effects , Erythrocytes/metabolism , Nitrogen Oxides/pharmacology , Oncorhynchus mykiss/blood , Animals , Electrophoresis, Agar Gel/methods , Free Radicals/blood , Hemolysis , In Vitro Techniques , Oxidative Stress
4.
Mutat Res ; 397(2): 353-60, 1998 Feb 02.
Article in English | MEDLINE | ID: mdl-9541660

ABSTRACT

The 'comet' assay is being increasingly employed for evaluating DNA damage in biological systems. Using this technique, we examined DNA damage in whole in density-separated trout erythrocytes. Results clearly show that all the three considered parameters (tail length, tail intensity and tail moment) increased with the density of the fractions, possibly reflecting different degrees of DNA damage. Probably, this behaviour is due to different periods of exposure of the density fractions to the hazard of active oxygen radicals; older cells have been exposed to oxidative stress for a longer time.


Subject(s)
DNA Damage , Erythrocytes/ultrastructure , Animals , DNA Fragmentation , Mutagenicity Tests , Trout
5.
Biochem Biophys Res Commun ; 242(3): 560-4, 1998 Jan 26.
Article in English | MEDLINE | ID: mdl-9464255

ABSTRACT

The antioxidant activity of hemoglobin was examined by studying both its peroxidase activity and its interaction with the superoxide anion. The peroxidase activity of both the subunits (alpha and beta) was reduced with respect to the alpha 2 beta 2 tetramer and heme-oxidation was found to be associated with a decrease in this activity. Lucigenin-amplified chemiluminescence experiments have shown that at low pH, the presence of hemoglobin reduces the level of superoxide anion generated by the xanthine/xanthine oxidase system (met-Hb is more efficient in reducing the level of O2- than oxy-hemoglobin). These results confirm that hemoglobin may be of importance in providing protection against oxidative damage to erythrocytes.


Subject(s)
Antioxidants/metabolism , Hemoglobins/metabolism , Acridines/metabolism , Dopamine/metabolism , Erythrocytes/metabolism , Humans , Hydrogen-Ion Concentration , Kinetics , Luminescent Measurements , Oxidative Stress , Peroxidases/metabolism , Spectrophotometry , Superoxides/metabolism , Xanthine/metabolism , Xanthine Oxidase/metabolism
6.
Biochem Biophys Res Commun ; 238(2): 301-4, 1997 Sep 18.
Article in English | MEDLINE | ID: mdl-9299499

ABSTRACT

The stability of trout hemoglobin was examined in the presence of some organotin compounds. Tributyltin chloride (TBTC) and triphenyltin chloride (TPTC) protect HbI most efficently from the oxidation. On the other hand, the same compounds accelerate the precipitation process in HbIV to a great extent. Parahydroxymercuribenzoate (PMB), an agent blocking free SH-groups of the protein, abolished the ability of TPTC to decrease the oxidation rate of HbI.


Subject(s)
Hemoglobins/drug effects , Organotin Compounds/pharmacology , Trialkyltin Compounds/pharmacology , Trout/blood , Animals , Hemoglobins/chemistry , Hemoglobins/metabolism , Organotin Compounds/chemistry , Oxidative Stress , Trialkyltin Compounds/chemistry
7.
J Biolumin Chemilumin ; 12(2): 79-85, 1997.
Article in English | MEDLINE | ID: mdl-9336009

ABSTRACT

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H2O2 using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H2O2 produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H2O2.


Subject(s)
Hemoglobins/metabolism , Luminescent Measurements , Trout/blood , Animals , Evaluation Studies as Topic , Half-Life , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , In Vitro Techniques , Methemoglobin/metabolism
8.
Free Radic Biol Med ; 23(2): 278-84, 1997.
Article in English | MEDLINE | ID: mdl-9199890

ABSTRACT

An in vitro model of thalassemia was produced by entrapment of isolated hemoglobin chains in human erythrocytes, thus subjecting the loaded cells to oxidative stress. The presence of these unpaired chains induced physico-chemical modifications at the membrane level as studied by laurdan fluorescence. The polarity of the lipid bilayer was shown to decrease with a concomitant shift towards a gel phase in alpha-loaded erythrocytes. The determination of conjugated dienes before the hemolytic event was used as an oxidation index; the results obtained demonstrate that beta thalassemia is associated with oxidative stress. Furthermore, the presence of indolinic and quinolinic nitroxide radicals, a new class of antioxidants, in suspensions of alpha-loaded erythrocytes protected the erythrocytes from the hemolytic event. However, the protective effect exerted by the nitroxide radicals is not related to effects on membrane polarity and lipid peroxidation, even though a chemiluminescence study has demonstrated the superoxide scavenging activity of these nitroxide radicals.


Subject(s)
Antioxidants/pharmacology , Cyclic N-Oxides/pharmacology , Hemoglobins/metabolism , Hemolysis/drug effects , Indoles/pharmacology , Quinolines/pharmacology , Erythrocytes/drug effects , Erythrocytes/metabolism , Free Radical Scavengers/pharmacology , Hemoglobins/chemistry , Humans , In Vitro Techniques , Luminescent Measurements , Models, Biological , Oxidative Stress , Superoxides/metabolism , beta-Thalassemia/blood , beta-Thalassemia/etiology
9.
Arch Biochem Biophys ; 336(1): 157-62, 1996 Dec 01.
Article in English | MEDLINE | ID: mdl-8951047

ABSTRACT

Erythrocytes of Salmo irideus trout were separated in the range from 45 to 65% Percoll, yielding three well-separated different fractions. Steady-state fluorescence of probes embedded in erythrocyte membranes and/or in liposomes from extracted lipids was used to characterize their physicochemical properties. Furthermore, the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH), embedded in the same liposomes, was measured by a frequency decay fluorometer. DPH decay was analyzed on the assumption of continuous distribution of lifetimes, for evaluating modifications of membrane microheterogeneity. Significant differences were observed in the parameters measured for the three erythrocyte fractions, possibly connected with the specific lipid composition of the samples.


Subject(s)
Erythrocyte Membrane/chemistry , Animals , Cell Fractionation , Chemical Phenomena , Chemistry, Physical , Cholesterol/blood , Erythrocyte Aging , Fluorescence Polarization , Lipid Peroxides/blood , Liposomes/chemistry , Membrane Fluidity , Membrane Lipids/blood , Phosphatidylcholines/chemistry , Temperature , Trout/blood
10.
Clin Chim Acta ; 217(2): 187-92, 1993 Aug 31.
Article in English | MEDLINE | ID: mdl-8261627

ABSTRACT

Inactivation of glutathione peroxidase correlates with the rate of hemoglobin chain oxidation. The enzyme inactivation is mainly present in those conditions where the autoxidation of the oxygenated chains is followed by transformation of the oxidized molecule into a hemichrome. Free hemoglobin chains have been encapsulated in human red blood cells by a dialysis technique that involves transient hypotonic hemolysis followed by isotonic resealing. Chain-loaded erythrocytes represent a good in vitro model of thalassemia. The presence of free human chains in the cell alters the intraerythrocytic glutathione peroxidase activity (alpha chains are more effective in the inactivation of the enzyme with respect to the beta chains).


Subject(s)
Erythrocytes/metabolism , Glutathione Peroxidase/blood , Hemoglobins/metabolism , Humans , In Vitro Techniques , Reactive Oxygen Species/metabolism
11.
Biotechnol Appl Biochem ; 16(3): 269-74, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1282323

ABSTRACT

Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.


Subject(s)
Aprotinin , Drug Carriers , Erythrocytes , alpha 1-Antitrypsin , Aprotinin/metabolism , Dialysis , Drug Compounding , Erythrocytes/chemistry , Hemolysis , Humans , Protein Binding , alpha 1-Antitrypsin/metabolism
12.
Int J Parasitol ; 20(8): 1109-11, 1990 Dec.
Article in English | MEDLINE | ID: mdl-2074141

ABSTRACT

The synthetic peptide (NANP)40, reproducing the tandem-repeated epitope of the circumsporozoite protein of Plasmodium (Laverania) falciparum, was entrapped into murine, autologous erythrocytes by a hypotonic dialysis method. Mice immunized intravenously with minute amounts of encapsulated peptide produced considerable antibody titres. This result indicates that intraerythrocytic antigen administration may have a potential as an immunization system for humans, since it dispenses with adjuvants and carrier molecules.


Subject(s)
Antibodies, Protozoan/biosynthesis , Antigens, Protozoan/immunology , Erythrocytes/immunology , Immunization/methods , Plasmodium falciparum/immunology , Protozoan Proteins , Animals , Antigens, Protozoan/chemical synthesis , Mice , Mice, Inbred C57BL
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