Anna Maria Santroni

Effect of three diaryl tellurides, and an organoselenium compound in trout erythrocytes exposed to oxidative stress in vitro

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.

Detection of DNA Damage in Stressed Trout Nucleated Erythrocytes Using the Comet Assay: Protection by Nitroxide Radicals

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.

Plasma Membrane Perturbation Induced by Organotins on Erythrocytes from Salmo irideus Trout

Tributyltin chloride and its degradation products monobutyltin and dibutyltin act as water pollutants, owing to the use of tributyltin chloride as a biocide in marine paint formulations. These compounds are lipid-soluble and undergo bioaccumulation and bioconcentration. Salmo irideus trout erythrocytes were studied to evaluate the possible effects of these compounds on freshwater fish, which could be exposed to long-term effects due to bioaccumulation of organotins. Data showed that tributyltin increases the haemolysis rate, starting at 10 μM, while dibutyltin has a scant protective effect at each concentration tested. Similar studies were performed in the presence of carbon monoxide (CO), which is protective against membrane oxidative stress due to haemoglobin (Hb) auto-oxidation. In these conditions all the organotins tested induced an increase in the haemolysis rate. These results suggest that the consequence of auto-oxidation of Hb could condition the effects of some organotin compounds. Steady-state fluorescence of probes embedded in the lipidic part of the membrane was used to evaluate the modifications induced by organotins to the physico-chemical state of phospholipids.

Effect of aromatic nitroxides on hemolysis of human erythrocytes entrapped with isolated hemoglobin chains.

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.

The Effect of Indolinic and Quinolinic Nitroxide Radicals on Trout Erythrocytes Exposed to Oxidative Stress

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.

Inactivation of glutathione peroxidase following entrapment of purified α or β hemoglobin chains in human erythrocytes

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 (α chains are more effective in the inactivation of the enzyme with respect to the β chains).

Plasma membrane perturbation induced by tributyltin chloride on density-separated trout erythrocytes

By using a discontinuous Percoll gradient in the 45–65% range it is possible to separate nucleated Salmo irideus erythrocytes in three different density fractions that are related to aging of the cell. The stability to lysis of the erythrocytes obtained from the three layers was examined in the presence and in the absence of Bu3SnCl4 (TBTC). The rate of hemolysis in the absence of the organotin was nearly the same for the three erythrocyte fractions. The addition of TBTC to the suspension increased the hemolysis but the effect depended on the density of the fraction. The change in the hemolysis rate was in the order bottom (B) > middle (M) > top (T). The effect of TBTC on 1,6-diphenyl-1,3,5-hexatriene (DPH) steady-state fluorescence anisotropy was measured in liposomes formed by the lipids extracted from the different fractions. Compared with the controls, the DPH anisotropy decrease in the presence of TBTC. Steady-state fluorescence of 2-dimethylamino-6-laurylnaphthalene (Laurdan) was measured to evaluate membrane polarity. The presence of TBTC on the same liposomes decreased the generalized polarization (GP340) in the top and middle fractions while it did not change in the bottom fraction. Copyright

Interaction of trout hemoglobin with H2O2: a chemiluminescence study

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.

Intraerythrocytic administration of a synthetic Plasmodium antigen elicits antibody response in mice, without carrier molecules or adjuvants.

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.

Superoxide anion handling by trout erythrocytes: A chemiluminescence study☆

Abstract Chemiluminescence is being increasingly employed for evaluating oxidative stress in biological systems. Using this technique, we examined the oxidative stress in trout erythrocytes. Hemolysates and erythrocyte ghosts were studied in whole and in density-separated trout erythrocytes, after exposure to Superoxide anion generated by the xanthine/xanthine oxidase system. The results show varying Chemiluminescence response in the studied fractions, possibly reflecting different degrees of oxidation.