Cadmium accumulation and biochemical responses in Sparus aurata following sub-lethal Cd exposure.

Cadmium (Cd), a heavy metal with limited biological function, is widely distributed in the aquatic environment as a result of natural and anthropogenic activities. The effect of 4 and 11 days exposure of gilthead sea bream Sparus aurata to sub-lethal concentrations of Cd was evaluated as levels of Cd content and Cd-metallothionein (MT) presence in different organs. The possible genotoxic effect was also evaluated in erythrocytes by using the "comet assay", a promising tool for estimating DNA damage at the single-cell level. The results obtained show that in the controls, Cd content was significantly higher in gills compared to in liver, but the treatment of fish with 0.1mg/l Cd induced a stronger accumulation of metal in liver depending on the length of the exposure period. Cd traces were found in plasma, muscle and kidney. Cd forms complexes in the cytosol with MT only in the liver but Cd-MT content significantly increased after 11 days of exposure to the metal, while after 4 days of treatment the protein level was similar to the control. The "comet assay" performed on S. aurata eryhtrocytes isolated from fish treated for 4 and 11 days with 0.1mg/l Cd, showed that there was no DNA damage at both exposure periods.

Effect of different organotins on DNA of mollusk (Scapharca inaequivalvis) erythrocytes assessed by the comet assay.

The alkaline comet assay, employing a single-cell gel-electrophoresis, is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual cells. In this study, we report data about the effect of different organotin compounds (MBTC, DBTC and TBTC) on DNA from erythrocytes of the Scapharca inaequivalvis bivalve mollusc. Our results show significant DNA damage after 30 min in vitro incubation with 10microM of organotins. Since TBTC turned out to be the most genotoxic compound, followed by MBTC and DBTC, we exposed the molluscs to 50ppb of TBTC for 11 days. A significant increase of comet parameters was measured in our experimental conditions. The use of the comet test as a high-throughput screening assay to monitor the effect of environmental pollutants on marine organisms has been proposed.

Lymphocyte DNA damage in rats challenged with a single bout of strenuous exercise.

Exercise induces extensive generation of reactive oxygen species, which are responsible for tissue damage: enzymes inactivation, lipid peroxidation and single strand breaks in DNA. Defense system against free radicals is consisting of enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and numerous non-enzymatic antioxidants. The study was performed to evaluate the effect of a single bout of submaximal running exercise, on the lymphocyte DNA strand breaks and also to test how supplementation with tempol - a membrane-permeable SOD-mimetic (0.2 mmol/kg/day) influences the eventually evoked damage. Male, Wistar rats were challenged with graded 50 min. running on treadmill at intensity up to 75-85% of predicted VO(2)max. The DNA strand breaks in individual lymphocytes were determined by using a gel electrophoretic technique - "comet" assay. We found substantial lymphocyte DNA damage 60 min. after the exercise. Tempol failed to prevent from oxidative damage in rats challenged with exercise. Moreover tempol by itself induced higher DNA damage than the exercise bout.

Lead-induced changes in human erythrocytes and lymphocytes.

In the present work we studied, by chemiluminescence measurements, the influence of lead on the production of reactive oxygen species (ROS) in haemolysates obtained from human erythrocytes incubated in the presence of different concentrations of lead acetate. Moreover, we evaluated the modification of proteins and lipids in human erythrocyte and lymphocyte membranes by using the fluorescence probes N-(1-pyrene)maleimide (PM), laurdan and pyrene. No significant changes in chemiluminescence were detected for erythrocytes incubated with 1-10 microM lead acetate for 3 h at 37 degrees C. By increasing the lead acetate concentration in cell suspensions up to 50 microM for the same incubation time, the percentage of chemiluminescence inhibition was ca. 20%. It was shown that, after incorporating fluorescence probes in the membrane lipid bilayer of erythrocytes and lymphocytes treated with 10 and/or 50 microM lead acetate, the total fluorescence intensity and the excimer to monomer intensity ratio of PM decreased and the generalized fluorescence polarization of laurdan decreased by 10-15%. The pyrene excimerization coefficient (kappa(ex)) increased by 20% (in comparison with a magnitude of kappa(ex) for white membranes isolated from intact erythrocytes) with 6-10 microM lead acetate for 3 h at 37 degrees C. The data obtained suggest that the effect of low concentrations of lead acetate does not cause production of ROS in erythrocytes in vitro, but can change the physicochemical state of proteins and lipids in erythrocyte and lymphocyte membranes. This effect is important because it influences the enzymatic activity and the functionality of receptors and channels present at the plasma membrane level, thus modulating the molecular composition of the intracellular space and cell functions.

Hemoglobin components from trout (Salmo irideus): determination of their peroxidative activity.

The peroxidative activity of trout hemoglobins, HbI and HbIV, which differ in their conformation, was compared with that of HbA. Artificial substrates (guaiacol and dopamine) and more physiological substrates such as model lipid membranes containing unsaturated fatty acids were used. The results indicate that all the hemoglobin molecules assayed show different levels of peroxidative activity. The capability to act as peroxidases is greater in HbIV than in HbI and HbA. In contrast, native globins did not show peroxidase activity. The different peroxidative activity of the Hbs is discussed in relation to stability both vs. protein oxidation and protein dissociation. The results confirm the view that hemoglobin may be of importance in establishing the life span of the erythrocyte itself.

Mitochondrial membrane potential in density-separated trout erythrocytes exposed to oxidative stress in vitro.

Previous literature reports have demonstrated that nucleated trout erythrocytes in condition of oxidative stress are subjected to DNA and membrane damage, and inactivation of glutathione peroxidase. The present study was undertaken to investigate if mitochondrial membrane potential in stressed conditions was also influenced. Density-separated trout erythrocyte fractions, obtained using a discontinuous Percoll gradient, were submitted to stress conditions and the mitochondrial membrane potential was determined by means of cytofluorimetric analysis after incubation of each subfraction with JC-1, a mitochondrial specific fluorescent probe. The results clearly show that the mitochondrial membrane potential decreased significantly in all erythrocyte fractions, also if the oxidative effect on mitochondria is more severe with increased density (age) of the cell. Ebselen was very effective in preventing mitochondrial depolarization in young as well as in old erythrocytes.

Oxidative response and membrane modification of diabetic platelets challenged with PAF.

Alterations in the functional activities of platelets (PLT) in type I diabetes have been widely observed. These changes play a key role in the development of cardiovascular complications in diabetes. Various functional activities of PLT are the result of the interaction of numerous stimuli with PLT plasma membrane. This study was designed to evaluate the oxidative response and membrane modifications of diabetic PLT stimulated by platelet activating factor (PAF). The oxidative response was assessed by employing luminol- and lucigenin-amplified chemiluminescence. Luminol-amplified chemiluminescence is sensitive to the release of hydrogen peroxide whereas lucigenin-amplified chemiluminescence is sensitive to the production of superoxide anion. Membrane fluidity and polarity were studied using fluorescence spectroscopy. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). The diabetic group consisted of 20 type I diabetic children with good metabolic control. Our results show a significant decrease in the luminol- and lucigenin-amplified chemiluminescence of PAF stimulated PLT in the diabetic group with respect to controls. These data indicate a decrement in the release of reactive oxygen species by diabetic PLT. We observed a significant increase in steady-state fluorescence anisotropy of diabetic PLT membrane that reflects a decrease in membrane fluidity. Laurdan showed a blue shift of the fluorescence emission and excitation spectra in diabetic PLT with respect to the control group, indicating a decrease in membrane polarity. The addition of PAF to PLT induced a red shift of Laurdan spectra in both groups, indicating an increase in membrane polarity. Our study [table: see text] demonstrates an altered oxidative response to PAF stimulation of diabetic PLT, probably due to altered generation or handling of reactive oxygen species, and alterations in the physico-chemical properties of the plasma membrane which could influence various functional activities of PLT.

Steady-state and time resolved fluorescence of albumins interacting with N-oleylethanolamine, a component of the endogenous N-acylethanolamines.

The functions of N-acylethanolamines, minor constituents of mammalian cells, are poorly understood. It was suggested that NAEs might have some pharmacological actions and might serve as a cytoprotective response, whether mediated by physical interactions with membranes or enzymes or mediated by activation of cannabinoid receptors. Albumins are identified as the major transport proteins in blood plasma for many compounds including fatty acids, hormones, bilirubin, ions, and many drugs. Moreover, albumin has been used as a model protein in many areas, because of its multifunctional binding properties. Bovine (BSA) and human (HSA) serum albumin are similar in sequence and conformation, but differ for the number of tryptophan residues. This difference can be used to monitor unlike protein domains. Our data suggest that NOEA binds with high affinity to both albumins, modifying their conformational features. In both proteins, NOEA molecules are linked with higher affinity to hydrophobic sites near Trp-214 in HSA or Trp-212 in BSA. Moreover, fluorescence data support the hypothesis of the presence of other NOEA binding sites on BSA, likely affecting Trp-134 environment. The presence of similar binding sites is not measurable on HSA, because it lacks of the second Trp residue.

Morphological and functional changes of mitochondria from density separated trout erythrocytes.

Density separated trout erythrocytes, using a discontinuous Percoll gradient, yielded three distinct subfractions (top, middle and bottom) since older cells are characterized by increasing density. Cells from each subfraction were incubated with mitochondria-specific fluorescent probe Mitotracker and JC-1 in order to assess mitochondrial mass and membrane potential by means of cytofluorimetric analysis, confocal microscopy and subsequent computer-aided image analysis allowing a detailed investigation at single cell level. Both cytofluorimetric data and image analysis revealed changes in size and redistribution of mitochondria starting from the light fraction to the bottom. In particular in young erythrocytes small mitochondria were detected localized exclusively around the nucleus in a crown-like shape, the middle fraction revealed enlarged mitochondria partially scattered throughout the cytosol, whereas the last fraction represented again mitochondria with reduced size being distinctly dispersed throughout the cytosol in the cells. Concerning membrane potential considerations, our study revealed a dramatic decrease of DeltaPsi(m) in the bottom layer cell mitochondria compared to the top and unusual membrane potential increase of a subpopulation of enlarged mitochondria. DeltapH was also investigated in the three fractions by pretreating the cells with nigericin, allowing to confirm a mitochondrial energetic impairment in older cells.

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.

Steady-state fluorescence and circular dichroism of trout hemoglobins I and IV interacting with tributyltin.

The effect of tributyltin chloride (TBTC) on rainbow trout (Salmo irideus) hemoglobin I (HbI) and hemoglobin IV (HbIV) was characterized by the steady-state fluorescence of intrinsic and extrinsic fluorescent probes. The fluorescence emission spectrum (lambdaex 280 nm) is greatly increased in intensity by the presence of the organotin in both proteins. Circular dichroism spectra in the same samples show a small decrease in theta222, a measure correlated with the percentage of the alpha-helical content. Morever, important changes in near-UV, Soret, and visible regions of CD were induced by TBTC. The correlation of data obtained with trout hemoglobins (HbI and HbIV) with similar measurements on globins suggests that the presence of heme is necessary for the interaction of the organotin compound with the proteins.

Plasma membrane fluidity and polarity of polymorphonuclear leukocytes from children with type I diabetes mellitus.

Polymorphonuclear leukocytes (PMN) from diabetic subjects have been found to be abnormal in various functional activities. These activities are mediated by the plasma membrane. This study was designed to evaluate plasma membrane fluidity and polarity in children with type I diabetes mellitus using fluorescence spectroscopy. PMN membrane fluidity and polarity were assessed in a group of 32 diabetic children. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy and fluorescence decay of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH), whereas membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). TMA-DPH and Laurdan are known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer. Our data show a significant increase in steady-state fluorescence anisotropy in diabetic PMN that reflects a decrease in membrane fluidity, and a decrease in TMA-DPH lifetime distribution indicating a decrease in membrane heterogeneity. Laurdan shows a blue shift of the fluorescence emission and a red shift of the excitation spectra in diabetic PMN with respect to the control group, indicating a decrease in membrane polarity. The results demonstrate a decrease in the phospholipid order at the membrane surface and a decrease in membrane polarity in diabetic PMN. These alterations in the physico-chemical properties of the plasma membrane could be the basis of the modifications in functional activities of PMN. The changes in the plasma membrane of PMN could be the result of metabolic and chemical modification associated with type I diabetes.

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.

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.

Extent of DNA damage in density-separated trout erythrocytes assessed by the 'comet' assay.

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.

Antioxidant activities of different hemoglobin derivatives.

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.

Effect of organotin compounds on trout hemoglobins.

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.

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.

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.

Physicochemical characterization of plasma membranes from density-separated trout erythrocytes.

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.