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.

A circular dichroism study of the proton-linked transition in the carbomonoxy derivative of the hemoglobin component IV from trout.

Near ultraviolet and visible circular dichroism spectra of the carbomonoxy derivative of the hemoglobin component IV from trout Salmo irideus are pH-dependent in the range 6.2-7.8, and are affected by the presence of inositol hexaphosphate at pH 6.2. On the basis of previous studies, the spectral changes observed can be associated to the pH-dependent R4 leads to T4 transition occurring in the liganded protein. The CD spectra above 500 nm at low pH can be interpreted as due to release of the heme asymmetry in the T liganded conformation, in agreement with the movement of the iron toward the proximal histidine.

Encapsulation of proteins into human erythrocytes: a kinetic investigation.

Moderate osmotic shocks of human erythrocytes by hypotonic dialysis (0.06 mosmol/kg) induce cell swelling and formation of pores, without causing apparent lysis. Using 125I-labeled macromolecules of different molecular weight and net charge, we followed the kinetics and efficiency of their encapsulation into erythrocytes. After a 20-30 min period of cell dialysis, macromolecules of up to 50 kDa begin diffusing into the swollen cells by a process which can be described by a first-order two-compartment kinetics. Adsorption to the external cell surface was insignificant, while adsorption to the inner membrane surface was substantial (15-20%) only for positively charged proteins, at physiological pH. After resealing, pores of a 12-14 kDa cut-off might remain open allowing some release of entrapped material (20-30%), depending on the final cytocrit, while the remaining might be associated with inner membrane or cytosolic components. Although the method of hypotonic dialysis is known to affect minimally the biophysical and immunological properties of red blood cell membranes, the interaction of encapsulated material with cell constituents would need to be further assessed when considering red cells as macromolecular carriers.

Cyanide dissociation from the hemoglobin of Parascaris equorum.

The reduction of cyanomethemoglobin by dithionite leads to the appearance of an intermediate, the complex of cyanide with ferrous hemoglobin, whose dissociation is easily followed in a stopped flow apparatus. This reaction was studied in the hemoglobin from the parasitic nematode Parascaris equorum, whose extremely high oxygen affinity is due to a very low dissociation rate. The rate of cyanide dissociation from ferrous Parascaris hemoglobin is not so dramatically different from that of other hemoglobins and myoglobins. Other features of the reaction are: (i) the rate constant of cyanide release is pH independent, an observation which is agreement with the possible absence of the distal histidine, given the mechanism suggested in a previous study (Bellelli, A., Antonini, G., Brunori, M, Springer, B.A. and Sligar, S.G. (1990) J. Biol. Chem. 265, 18898-18901), and (ii) the time-course shows no kinetic cooperativity. The structural basis of the extremely high oxygen affinity of Parascaris hemoglobin cannot be explained on the basis of the results here reported. This study also confirms that, even though cyanide binding to ferrous hemoglobins is controlled by distal interactions, the functional behaviour of this ligand is characteristic and differs from the behaviour of oxygen.

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.

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.

Properties of trout hemoglobin covalently bound to a solid matrix.

This paper reports the ligand binding properties of the major hemoglobin component from trout (Salmo irideus) covalently bound to a solid matrix (Sepharose or Sephadex). A comparison between the functional properties of this protein in solution and of the protein-matrix complex shows significant changes although the basic properties of the molecule are maintained on covalent binding to Sepharose (or Sephadex). Thus the Root effect, characteristic of Hb trout IV, is still present while the heme-heme interactions are, on the average, smaller in the matrix bound protein as compared to the soluble form. No differences in the O2 binding properties were observed when the protein was coupled to the resin, as the ligand bound or as the ligand free derivative. Although an unequivocal interpretation of the data is made difficult by the lack of information on the number and identity of the groups involved in the coupling, the main changes in the protein functional properties may be related to the chemical modifications "per se" more than to the immobilization imposed to the macromolecule by coupling to the matrix. Structural changes which mainly involve perturbation of the tertiary structure of the molecule may qualitatively rationalize the data.

Functional properties of partially oxidized trout hemoglobins.

This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The O2 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However, the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the pH dependence of the apparent "on" constant in the oxidation mixtures is similar to that characteristic of the native molecule. The results of the O2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is an increase in the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.

Red cell lysis induced by microorganisms as a case of superoxide- and hydrogen peroxide-dependent hemolysis mediated by oxyhemoglobin.

Some bacteria, isolated from the blood of hospitalized patients, have been shown to hemolyze red blood cells through a mechanism which was dependent on the oxygenated state of intracellular hemoglobin, since transformation of hemoglobin into the CO-derivative inhibited the lysis. Hemolysis was also inhibited by superoxide dismutase and catalase, while only catalase prevented the formation of methemoglobin in experiments where isolated oxyhemoglobin was exposed to metabolizing bacteria. Production by bacteria of extracellular superoxide was demonstrated. It is suggested that hemolysis is due to interaction of O-2 and/or H2O2 with intracellular hemoglobin and that some product of such interaction is the lytic agent.

Mini-myoglobin: native-like folding of the NO-derivative.

Mini-myoglobin is a polypeptide fragment (residues 32-139) obtained by limited proteolysis of horse heart apomyoglobin and reconstituted with the natural heme. Its functional properties are very similar to those of native myoglobin and therefore it may represent a model for testing the functional role of the protein fragment encoded by the central exon of myoglobin gene (residues 31-105). Here we have investigated some properties of the nitric oxide derivative of mini-myoglobin in comparison with those of NO-myoglobin, to provide more structural information on the heme pocket residues in addition to that already acquired by electron paramagnetic resonance of the cobalt-substituted mini-myoglobin. At pH 7.0, optical and circular dichroism Soret spectra, as well as electron paramagnetic resonance spectra reveal that the heme orientation in the pocket and the coordination state of the ferrous iron in NO-mini-myoglobin are similar to those of the whole protein. The spectra of the NO-mini-myoglobin complex are very sensitive to pH changes at variance to what is observed for the NO-myoglobin derivative in the same pH range (5.5-9.5). In particular, increasing or decreasing pH from 7.0, results in a decrease of the extinction coefficient and of the ellipticity in the Soret region and in a change of the shape of the electron paramagnetic resonance signal. The spectral changes are diagnostic for a transition from a hexa-coordinated (at pH 7.0) to a penta-coordinated heme (at pH 5.5 or 9.5), with the proximal histidine-iron bond either broken or stretched dramatically. Thus, although mini-myoglobin is able to bind NO in a geometry similar to that of the native protein, the resulting NO derivative shows a much higher pH dependence, suggesting that the two lacking side domains are mainly involved in enhancing the stability of the hemoprotein core.

Mini-myoglobin. The structural significance of haem-ligand interactions.

The properties of purified mini-myoglobin, the fragment 32-139 of horse heart myoglobin reconstituted with protohaem, have been investigated from a structural and functional view point. The recovery of secondary structure observed in the carbon monoxide derivative of mini-myoglobin, as shown by circular dichroism, and the overall similarity of the haem pocket to that of myoglobin, as deduced from the fluorescence properties of the complex with 1-anilino-8-naphthalene sulphonate, indicate that, in the presence of the constraints imposed by the haem and its ligands, the miniprotein reacquires a conformation close to that of native myoglobin. These spectroscopic data parallel the conclusions drawn from the results of ligand combination and dissociation kinetics; stopped-flow experiments indicate that carbon monoxide and oxygen bind to mini-myoglobin with rates almost identical with those of myoglobin itself. The significance of mini-myoglobin as a model of an oxygen-carrying protein, with some of the expected functional characteristics of an ancestor haemoprotein, is discussed, with reference to the mosaic structure of the myoglobin gene and the role of different exons in the evolution of proteins.

Mini-myoglobin: preparation and reaction with oxygen and carbon monoxide.

A domain of 108 amino acid residues (32 to 139), obtained by digestion of horse heart apomyoglobin with clostripain, was found to bind protoheme in a 1 to 1 molar ratio. This domain is 33 amino acid residues larger than the protein segment encoded by the central exon in seal myoglobin. Flash photolysis experiments have shown that reconstituted "mini-myoglobin" is very similar to myoglobin in the combination reaction with carbon monoxide and with oxygen, and in the oxygen replacement reaction by carbon monoxide. These experiments provide for the first time direct evidence for the presence of a structural and functional domain, closely corresponding to the segment encoded by the central exon of the myoglobin gene, which contains the information for binding the natural heme and for maintaining the native folding typical of a respiratory protein.

Mini-myoglobin. Electron paramagnetic resonance and reversible oxygenation of the cobalt derivative.

Mini-myoglobin, obtained by limited proteolysis of horse heart myoglobin (residues 32 to 139), represents a good model for testing the correlation between an exon and a protein domain. We have shown that ligand binding kinetics, spectral and folding features of mini-myoglobin are very similar to those of native myoglobin. In order to develop further the analysis of the structure-function relationship in this mini-protein, mini-globin was reconstituted with the heme moiety in which iron is replaced by cobalt. The Soret absorption spectra of oxy and deoxy cobaltous mini-myoglobin are very similar to those of cobaltous myoglobin derivatives; in addition. Co-mini-myoglobin binds oxygen reversibly with an n value approximately 1 and a p50 value of 45 to 50 mm Hg (the same as Co-myoglobin). Oxy Co-mini-myoglobin shows a well-resolved electron paramagnetic resonance (e.p.r.) spectrum typical of an oxygenated hemoprotein, while the spectrum of the deoxy derivative, although similar to that of deoxy Co-myoglobin, displays a lower resolution of the complex hyperfine structure. Moreover, photodissociation experiments on oxy Co-mini-myoglobin allow e.p.r. detection of an intermediate state, already observed in most hemoproteins and diagnostic for the interaction of bound oxygen with the distal histidine residue. Thus, reconstitution of mini-globin with cobalt protoprophyrin IX has provided, for the first time, a stable oxygenated complex that reflects a correct folding of the protein surrounding the heme pocket and possesses the functional behaviour typical of a hemoprotein.

Stereochemistry of ATP and GTP bound to fish haemoglobins. A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study.

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3'-endo; the P-O5'-C5'-C4' torsion angle lies in the trans domain (180 degrees +/- 20 degrees); the P alpha-O-P beta and P beta-O-P gamma angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO4) gamma can accept hydrogen bonds from Val NA1 beta 2 and from Arg H21 beta 1, while (PO4) beta and (PO4) alpha can accept hydrogen bonds from Lys EF6 beta 1 and beta 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 beta 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 beta 1; in addition, Val Na1 beta 1 can donate a hydrogen bond to O2' of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2 beta in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2 beta and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1 beta and O2' of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.

Glutathione peroxidase and oxidative hemolysis in trout red blood cells.

Red blood cells from the trout Salmo irideus contain several hemoglobin components that are prone to oxidation with production of oxygen radicals. The rate of hemolysis has been correlated to the extent of methemoglobin formation. A difference in the rate of hemolysis between red blood cells saturated with either CO or O2 was evident only when diminished glutathione peroxidase activity was observed. These results confirm the important role of this enzyme in providing protection against or repair of oxidative damage to the red cell membrane.

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.

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.

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.

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.