Biomedical subjects

V Vannini

Publications and source records attributed to V Vannini.

At least 19 recordsLinked to original sources

PubMed1992

Detection of free radicals during brain ischemia and reperfusion by spin trapping and microdialysis.

Extracellular free radicals were detected in rat striatal perfusate samples by intracerebral microdialysis coupled to the spin trapping technique. Five Sprague-Dawley rats were subjected to 30 min of global ischemia followed by reperfusion; throughout the experimental period the intrastriatal dialysing probe was perfused with Ringer's solution containing the spin trap agent pyridyl-N-oxide-t-butylnitrone (100 mM) together with the iron chelating agent diethylentriaminepentacetic acid (100 microM). A radical adduct occurred during ischemia and early reperfusion, but not in basal conditions; the spin adduct was characterized as a carbon centered radical, consistent with the presence of an oxidative attack on membrane lipids. The direct evidence of the formation of free radicals supports the hypothesis that free radicals play a role in the pathogenesis of the histological damage during brain ischemia.

Animals↗

PubMed1992

Detection and quantitation in rat tissues of the superparamagnetic magnetic resonance contrast agent dextran magnetite as demonstrated by electron spin resonance spectroscopy.

RATIONALE AND OBJECTIVES: The compound studies in this article is a superparamagnetic macromolecular complex of magnetite cores coated with hydrophilic dextran, which is under active investigation as a contrast agent for magnetic resonance imaging (MRI) in liver and spleen. The biodistribution of paramagnetic compounds is problematic and is usually studied by histochemical reactions or by radiolabeling the compound under study. The purpose of this article is to show how electron spin resonance (ESR) spectroscopy detects dextran magnetite (DM) particles in tissues. METHODS: DM injected intravenously in the experimental animal was detected in some reticulo-endothelial organs by ESR. The spectroscopic study was validated using electron microscopy and electron-probe microanalysis. RESULTS: DM exhibits an ESR spectrum; ESR delineated the distribution of DM distribution in liver, spleen, bone marrow, and blood as a function of time. The blood clearance was biphasic, dependent on the size of particles. CONCLUSIONS: ESR spectroscopy is a highly sensitive and reproducible method of studying DM distribution.

Animals↗

PubMed1991

Enzymatic recycling of oxidized ascorbate in pig heart: one-electron vs two-electron pathway.

Enzymatic systems able to reduce either dehydroascorbate or ascorbyl radical back to ascorbate by "recycling" vitamin C may contribute to lowering the nutritional requirement of it and to increase tissue antioxidant capacity. The activities of two enzymatic activities, GSH-dehydroascorbate reductase (two-electron reduction pathway) and NADH-semidehydroascorbate reductase (one-electron reduction pathway) in pig tissues, have been investigated. The activity of glutathione-dependent reduction of dehydroascorbate, although measurable, appeared negligible taking into consideration the low physiological substrate concentration. On the other hand, the one-electron reduction of ascorbyl radical resulted fast enough to slow down the consumption of the antioxidant vitamin.

Animals↗

PubMed1991

Blood clearance of dextran magnetite particles determined by a noninvasive in vivo ESR method.

Dextran magnetite (DM) is a potential MR contrast agent with superparamagnetic properties. Its fast clearance from the blood and selective uptake by tissue macrophages provide advantages for imaging tumors in the liver and spleen. DM consists of a suspension of solid particles with a wide distribution of sizes. In this study we have used ESR spectroscopy to determine the blood clearance of DM injected iv in mice. The spectra are obtained on living animals by inserting the tail of a mice into the waveguide cavity of the ESR spectrometer and recording the ESR spectrum continuously. This procedure allows the direct measurement of the plasma clearance of DM from individual animals, without blood sampling. We applied this method to study the clearance of suspensions of DM particles with different average sizes.

Animals↗

PubMed1990

Metabolism of nitroxide spin labels in subcellular fraction of rat liver. I. Reduction by microsomes.

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.

Animals↗

PubMed1990

Metabolism of nitroxide spin labels in subcellular fractions of rat liver. II. Reduction in the cytosol.

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of five nitroxides in cytosol derived from rat hepatocytes. The nitroxides were chosen to provide information on the effects of the type of charge and the ring on which the nitroxyl group is located. The rates of reduction were fastest for a six-membered positively charged nitroxide ('CAT-1') and slowest for an anionic five-membered ring nitroxide ('PCA'). Changing levels of glutathione, sulphydryl groups in general, NADPH or NADH had little or no effect on the rates of reduction, while the addition of ascorbate oxidase essentially abolished reduction of the nitroxides. The products of reduction by the cytosol were the corresponding hydroxylamines. The overall rates of reduction of neutral or anionic nitroxides were much slower than those observed with intact cells. We conclude that the primary source of metabolism of nitroxides by cytosol is reduction by ascorbate and that under most conditions reduction of nitroxides in the cytosol is not a major factor in the metabolism of nitroxides by cells.

Animals↗

PubMed1990

[Morphological research on the dental pulp of the calf].

Morphological researches have been carried out through histological studies with fine and semifine sections at the electronic scan microscope on the dental pulp of calves. The general aim was to identify the antioxidant properties of pulp and study cellular density and microfibrillar architecture. The dental pulps of calves of 5-6 months taken immediately after slaughtering were used with immersions into the fixing liquid. The scan microscope showed fibroblasts in the deepest regions of the pulp in a stroma of collagenic fibres, not organised but scattered among the cells of the connective tissue. The odontoblasts presented well ordered one beside the others with well visible details such as the swollen basal portion, the nucleus and a very tight villosity. No clear interodontoblastic connections were evident. The electron transmission microscope revealed typical cells with histiocyte appearance with microfilaments evident in the cytoplasma (myofibroblasts) presenting a wide variety of cytoplasmatic interconnections with interdigitations.

Animals↗

PubMed1989

Biochemical mechanism of GSH depletion induced by 1,2-dibromoethane in isolated rat liver mitochondria. Evidence of a GSH conjugation process.

HPLC measurements of GSH and GSSG levels in isolated rat liver mitochondria, on addition of 1,2-dibromoethane (DBE), revealed the presence of a glutathione (GSH)-conjugating pathway of DBE. This process required the structural integrity of the mitochondrial matrix and inner membrane complex and was inhibited by the uncouplers of oxidative phosphorylation, particularly 2,4-dinitrophenol. On the other hand it was not affected by the energetic state of the mitochondria, since other mitochondrial inhibitors like KCN and oligomycin did not have any effect on it. This process also did not require the involvement of mitochondrial inner membrane transport systems, based on the measurement of the mitochondrial transmembrane potential. The involvement of mitochondrial GSH-S-transferases, located either in the matrix or in the intermembrane space, is discussed.

Animals↗

PubMed1989

Metabolism in rat liver microsomes of the nitroxide spin probe tempol.

Paramagnetic nitroxide spin labels have been extensively used to probe various biophysical and biochemical properties of the cellular environment. Recently nitroxides have been proposed as contrast enhancing agents in proton magnetic resonance imaging and contrast enhancement has been demonstrated in animal studies. Nitroxides, possessing a stable unpaired electron, increases the relaxation rates of protons, providing an enhancement of contrast. Nitroxides are metabolized intracellularly principally via reversible reduction to hydroxylamines. Rates of reduction depend on the physical characteristics of the nitroxides, in general 5-membered pyrrolidine ring are reduced more slowly than those with a 6-membered piperidine ring. Oxidation back to the nitroxide is relevant for lipid soluble hydroxylamines, while is low for water soluble ones. It is known that nitroxides are metabolized by subcellular fractions (cytosol, mitochondria, microsomes), though the enzymatic and non-enzymatic systems involved are poorly characterized. In the present study, the first of the necessary steps toward a systematic study of the metabolism of nitroxides by subcellular organelles, we have chosen to study the metabolism of 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl in isolated rat liver microsomes. Microsomes were able to reduce Tempol slowly without any substrate addition; when NADPH was added, the reduction rate substantially increased. In phenobarbitone induced rats the reduction rate was significantly higher than in not-induced microsomes. NADPH-dependent reduction rate was inhibited by thallium chloride (an inhibitor of the flavin-centered cytochrome P-450 reductase), superoxide dismutase, and by N-ethylmaleimide; menadione increased it. The Tempol reduction rate was not significantly affected by various cytochrome P-450 inhibitors with the sole exception of metyrapone. A solution containing purified cytochrome P-450 reductase and NADPH readily reduced Tempol. Microsomes fortified with NADPH were able to reduce Tempol at an appreciable rate. In order to distinguish between reduction of nitroxides to hydroxylamine or destruction of nitroxides following nitroxide reduction, microsomal suspensions were treated with a mild oxidant (ferricyanide 0.5-10 mM). The recovery varied from 40 to 60%, indicating a process of probe destruction leading to as yet unknown metabolites. The present study clearly indicates that, in this model system, cytochrome c (P-450) reductase and not cytochrome P-450 is responsible for the observed Tempol metabolism; along with hydroxylamine formation, other Tempol derived metabolites are formed during the process.

Animals↗

PubMed1989

Metabolism of aqueous soluble nitroxides in hepatocytes: effects of cell integrity, oxygen, and structure of nitroxides.

The optimum use of nitroxides in viable biological systems, including live animals, requires knowledge of the metabolism of nitroxides by major organ systems, especially the liver. We report here details of the metabolism of several prototypic aqueous soluble nitroxides in suspensions of freshly isolated hepatocytes. The general patterns of metabolism were similar to those observed in other types of cells (previous studies have been done principally in cells from tissue culture, such as CHO cells) including the primary initial reaction being reduction to the hydroxylamine, an increased rate of metabolism of some nitroxides in hypoxic cells, faster rates of reduction of nitroxides on six-membered piperidine rings compared to five-membered pyrrolidine rings, and most metabolism being intracellular. Metabolism in hepatocytes differed from other cell lines in having (1) significant reduction in the extracellular medium due to ascorbate that was released from damaged hepatocytes; (2) decreased rates of metabolism in freeze-thawed cells due to damage to subcellular organelles. These results provide much of the data needed to understand the role of the liver in the metabolism of nitroxides by intact animals and explain some previously puzzling results which indicated an apparent unusually high rate of metabolism of a charged nitroxide (Cat1) by hepatocytes. Our results also indicate that the use of freshly isolated cells or tissue homogenates may introduce experimental artifacts in the study of the metabolism of nitroxides.

Air↗

PubMed1989

Free radicals and lipid peroxidation in liver of rats kept on a diet devoid of choline.

Rodents kept on a choline devoid (CD) diet up to 14 months develop hepatic lesions progressing through two broad stages. The first is characterized by severe steatosis and increase in cell turnover, the second by a gradual clearance of the deposited fat and fibrosis. Hepatocellular carcinomas eventually arise in rats fed for over 12 months, even though the animals aer not exposed to chemical carcinogens. It has been suggested that the diet may trigger generated thereby may be responsible for initiation of liver cancer and promotion. The radicals would lead to DNA damage, and the altered DNA in a proliferating liver would result in initiation of the carcinogenic process. In this communication we present evidence that the diet used in the above studies contained stable fatty acid isomers with conjugated dienes, which are absorbed and deposited in rat liver. This finding cast doubts on whether a CD diet does indeed cause a peroxidation of cellular membrane lipids. Electron spin resonance (ESR) spectroscopy was also used to investigate whether any abnormal pattern of free radicals exists in the liver of rats fed a CD diet. No significant differences were noted in ESR spectra of either transition metal-centered signals, or organic free radicals.

Animals↗

PubMed1987

Free-radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation.

Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.

Animals↗

PubMed1987

Detection of free radical intermediates in the oxidative metabolism of carcinogenic hydrazine derivatives.

Hydrazine derivatives are widely used in agriculture, in industry, as rocket propellants, and in medicine. Hydrazines also occur naturally in tobacco and mushrooms. Many hydrazines tested in animal studies appear to be carcinogenic and induce tumors in various target tissues in mice, hamsters, and rats. The use of hydrazine derivatives in humans is often complicated by adverse side-effects such as liver injury and rheumatoid arthritis. A number of studies have demonstrated that hydrazine derivatives are activated to reactive intermediates, such as free radicals, through a variety of cellular oxidative metabolic pathways. The aim of this work is to demonstrate the occurrence of free radical intermediates during the metabolic activation of various hydrazine derivatives and to characterize the enzymatic system(s) responsible for the activation to free radical species. The hydrazines studied are acetylhydrazine, isoniazid, isopropylhydrazine, iproniazid, methylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine. The model systems chosen are those of rat liver microsomes and isolated hepatocytes. Free radical intermediates have been demonstrated by the electron spin resonance spectroscopy coupled to spin trapping technique. The activation mechanism has been characterized using inhibitors of the mixed function oxidase system and of the FAD-dependent oxygenase system. Glutathione was able to scavenge, with high efficiency, the free radicals produced.

Animals↗

PubMed1987

Free radical metabolism of alcohols by rat liver microsomes.

By using e.s.r. spectroscopy coupled with the spin trapping technique we have detected the formation of free radical intermediates by rat liver microsomes incubated with either ethanol, 2-propanol or 2-butanol in the presence of a NADPH regenerating system and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) as spin trap. The e.s.r. spectra have been identified as due to the hydroxyalkyl free radical adducts of 4-POBN. The free radical formation depends upon the activity of the microsomal monoxygenase system and is blocked by omitting NADP+ from the incubation mixture, by anaerobic incubation or by enzyme denaturation. The involvement of hydroxyl radicals (OH.) produced through a Fenton-type reaction from endogenously formed hydrogen peroxide is suggested by the opposite effects exerted on the e.s.r. signal intensity by azide and catalase. Consistently, iron chelation by desferrioxamine inhibits the free radical formation, while the supplementation of EDTA-iron increases it by several fold. Inhibitors of cytochrome P450-dependent monoxygenase system reduce to various extents the production of free radical intermediates suggesting that reactive oxygen species might be formed at the active site of cytochrome P450 where they react with alkyl alcohol molecules. The data presented support the hypothesis that free radical species are generated during the microsomal metabolism of alcohols and suggest the possibility that ethanol-derived radicals might play a role in the pathogenesis of the liver lesions consequent upon alcoholic abuse.

1-Propanol↗

PubMed1986

Induction of calcium efflux from isolated rat-liver mitochondria by 1,2-dibromoethane.

Addition of 1,2-dibromoethane to rat-liver mitochondria induces a concentration-dependent depletion of mitochondrial glutathione. This event seems to be associated with the induction of Ca2+ release from mitochondria pre-loaded with a low pulse of Ca2+. The enhancement of the energy-dissipating process to reaccumulate the released Ca2+ ('Ca2+ cycling') results in a progressive drop of membrane potential. Addition of EGTA (ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid), when the membrane potential has reached the lowest level, restitutes it to a normal value. All these findings and the observation that Ca2+ release also occurs under non cycling conditions (e.g., in the presence of ruthenium red) suggest that 1,2-dibromoethane induces a Ca2+ efflux by activating a selective pathway which is sensitive to critical sulfhydryl groups.

Animals↗

PubMed1986

Decrease of hepatic mitochondrial glutathione and mitochondrial injury induced by 1,2-dibromoethane in the rat in vivo: effect of diethylmaleate pretreatment.

Diethylmaleate (DEM) potentiated the 1,2-dibromoethane (DBE)-induced hepatic morphological lesion in fasted male Wistar rats, as revealed by light and electron microscopy examination. The subcellular structures involved in such lesions were the mitochondria. The potentiating effect of DEM appeared to be due to enhancement of the depletion of hepatic mitochondrial glutathione (GSH) caused by DBE. DEM, however, failed to potentiate the DBE-induced release in the plasma of hepatic enzymes. The relationship between loss of mitochondrial GSH, mitochondrial injury, and the importance of the mitochondrial lesion in DBE-induced hepatotoxicity is discussed.

Animals↗

PubMed1985

Activation of chloroform and related trihalomethanes to free radical intermediates in isolated hepatocytes and in the rat in vivo as detected by the ESR-spin trapping technique.

When hepatocytes isolated from phenobarbital-induced rats were incubated with chloroform and the spin trap phenyl-t-butyl nitrone (PBN) under anaerobic conditions, a free radical-spin trap adduct was detectable by ESR spectroscopy. A similar incubation of hepatocytes in the presence of air resulted in an ESR signal that was eight times less intense than that seen under anaerobic conditions; incubation mixtures exposed to pure oxygen had no detectable adduct signal. A significant reduction in the signal intensity was also produced by the addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone and carbon monoxide, indicating that free radical formation depended upon the reductive metabolism of chloroform mediated by the mixed oxidase system. The origin of the CHCl3-derived free radical has been confirmed by using [13C]CHCl3, while the comparison between the ESR spectra obtained in the presence of deuterated chloroform (CDCl3) and bromodichloro-methane (CHBrCl2) suggests that the free radical derived from CHCl3 may be CHCl2. Free radical intermediates were also detected during the aerobic and anaerobic incubation of isolated hepatocytes with bromoform (CHBr3), and iodoform (CHI3). The intensity of the ESR signal obtained with the various trihalomethanes increases in the order CHCl3 less than CHBrCl2 less than CHBr3 less than CHI3. The formation of PBN-free radical adducts has also been observed in phenobarbital-induced rats in vivo when intoxicated with chloroform, bromoform or iodoform, suggesting that the reductive metabolism of trihalomethanes might be of relevance to their established toxicity in the whole animal.

Animals↗

PubMed1984

Toxicity of 1,2-dibromoethane in isolated hepatocytes: role of lipid peroxidation.

Treatment of isolated hepatocytes with 1,2-dibromoethane (DBE) caused a concentration dependent depletion of cellular glutathione (GSH) content and a parallel increase in the covalent binding of reactive intermediates to cell proteins, as a consequence of the haloalkane activation. The reduction of the hepatocyte GSH content, induced by DBE, stimulated the onset of lipid peroxidation, as measured by malondialdehyde (MDA) accumulation. N-Acetylcysteine (1 mM) was found to partially prevent GSH loss and to inhibit MDA formation, whereas equal concentrations of cysteine and methionine were ineffective on these respects. The stimulation of the peroxidative reactions appeared to be also associated with an increase in the leakage of lactate dehydrogenase (LDH) from the cells, indicative of a severe hepatocyte injury. Antioxidants such as alpha-tocopherol, N,N'-phenyl-phenylenediamine (DPPD) and promethazine, as well as N-acetylcysteine reduced MDA formation to various extents and also protect against LDH release, yet without interfering with the covalent binding of DBE reactive intermediates to hepatocyte proteins. These results suggest the involvement of lipid peroxidation, consequent to GSH depletion, in the pathogenesis of liver cell necrosis due to DBE.

Animals↗