(Bi)sulfite metabolism in human granulocytes: age-dependent formation of free radical intermediates.
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Adult↗
Biomedical subjects
A Tomasi
Publications and source records attributed to A Tomasi.
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↗
In vitro and in vivo evidence for the formation of methyl radical from procarbazine: a spin-trapping study.
Electron spin resonance (ESR) analysis combined with the use of 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) and dibromonitroso benzenesulfonic acid (DBNBS) as spin-trapping agents was used to characterize free radical generation during the metabolism of the anticancer agent procarbazine [N-isopropyl-a-(2-methylhydrazino)-p-toluamide hydrochloride]. The formation of free radical species, identified as methyl radicals, was observed during oxidation of procarbazine in rat liver microsomes and isolated hepatocytes in vitro, as well as in several organs following administration of the drug in vivo. A cytochrome P450-mediated reaction, involving P450IA and IIB isoenzymes, was responsible for the activation process. The metabolic pathway leading to free radical formation was characterized using various procarbazine metabolites and revealed strict analogies with previously published data on methane production from procarbazine. These results supported the identification of the trapped species as methyl free radical and suggested that C-oxidation of azoprocarbazine is the main source of radical intermediates derived from this anticancer drug.
Animals↗
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↗
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↗
Role of ethanol-inducible cytochrome P450 (P450IIE1) in catalysing the free radical activation of aliphatic alcohols.
Incubation of rat liver microsomes with 1-propanol and 1-butanol in the presence of NADPH and of the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) allowed the detection of free radical intermediates tentatively identified as 1-hydroxypropyl and 1-hydroxybutyl radical, respectively. Microsomes isolated from rats treated chronically with ethanol (EtOH) or with the combination of starvation and acetone treatment (SA), exhibited a two-fold increase in the ESR signal intensity as compared to untreated controls, whereas no increase was observed in phenobarbital-induced (PB) microsomes. Consistently, in reconstituted membrane vesicles, ethanol-inducible cytochrome P450IIE1 was twice as active as phenobarbital-inducible P450IIB1 in producing 1-butanol free radicals. In the microsomal preparations from EtOH and SA pretreated rats the addition of antibodies against cytochrome P450IIE1, but not of preimmune IgGs, lowered the ESR signal of 1-butanol radicals by more than 50%. The same antibodies decreased the free radical production by untreated microsomes by 35-40%, but were ineffective on microsomes from PB-treated animals. This indicated that cytochrome P450IIE1 is the major enzyme responsible for the free radical activation of alcohols in control and ethanol-fed rats. The generation of 1-hydroxybutyl radicals by EtOH microsomes was inhibited by 40, 48 and 68%, respectively, by the addition of isoniazid, tryptamine and octylamine, compounds known to specifically affect the NADPH oxidase activity of this isoenzyme. This effect was not due to the scavenging of the alcohol radical since none of these compounds affected the ESR signals originated from 1-butanol in a xanthine-xanthine oxidase system. When added to reconstituted membrane vesicles isoniazid, tryptamine and octylamine also decreased 1-butanol radical formation by P450IIE1 by 54, 38 and 66%, respectively. Such an inhibition corresponded to the effect exerted by the same compounds on O2- release from P450IIE1 containing vesicles. These results indicate that the capacity of cytochrome P450IIE1 to reduce oxygen is related to its ability to generate alcohol free radicals and suggest that ferric cytochrome P450-oxygen complex might act as oxidizing species toward alcohols.
1-Butanol↗
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↗
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↗
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↗
Lipid composition and fluidity of liver mitochondria, microsomes and plasma membrane of rats with chronic dietary iron overload.
The effect of chronic dietary iron overload on the lipid composition and physical state of rat liver mitochondria, microsomes and plasma membranes was investigated. After 9 weeks of iron treatment, a significant decrease of polyunsaturated and a parallel increase of saturated fatty acids was observed in mitochondrial and plasma membrane phospholipids. By contrast, no appreciable modification of the fatty acid composition of microsomal membranes was detected. The cholesterol/phospholipid molar ratio as well as the lipid/protein ratio, did not reveal any significant difference in any of the fractions studies. Finally, no change in the molecular order of the various membranes, as assessed by electron spin resonance spectrometry, was observed following iron intoxication. These data indicate that, although in vivo chronic hepatic iron overload induces a modification of fatty acid profile in cellular structures consistent with the in vivo occurrence of lipid peroxidation, these changes do not bring about appreciable modifications of other physico-chemical parameters relevant to membrane integrity and cell viability.
Animals↗
Oxidative stress, poly(ADP)ribosylation and aging: in vitro studies on lymphocytes from normal and Down's syndrome subjects of different age and from patients with Alzheimer's dementia.
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Adolescent↗
Studies on the antioxidant and free radical scavenging properties of IdB 1016 a new flavanolignan complex.
Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has been evaluated. One hour after the intragastric administration to rats of IdB 1016 (1.5 g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5 micrograms/mg protein corresponding to a final concentration in the microsomal suspension used of about 10 microM. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CCl4 and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine. These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.
Administration, Oral↗
[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↗
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↗
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↗
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↗
Lipid composition and fluidity of liver plasma membranes from rats with chronic dietary iron overload.
Liver plasma membranes isolated from rats with chronic dietary iron overload showed a large modification of their phospholipid fatty acid composition. Specifically, a significant decrease in polyunsaturated fatty acids and a parallel increase in saturated fatty acids was observed. This pattern was consistent with the in vivo occurrence of lipoperoxidative reactions in the liver plasma membranes. However, neither change in the cholesterol/phospholipid molar ratio nor in the lipid/protein ratio was detected. Direct measurement of the plasma membrane fluidity state by electron spin resonance spectrometry did not reveal any difference between control and iron-treated rats. These findings indicate that chronic dietary iron overload can induce lipid peroxidation of rat liver plasma membranes, but this event does not bring about modification in the physical state of the membrane.
Animals↗
Free radical activation of monomethyl and dimethyl hydrazines in isolated hepatocytes and liver microsomes.
Isolated hepatocytes and liver microsomes incubated with monomethyl-1,1 dimethyl- and 1,2 dimethyl-hydrazines produced free radical intermediates which were detected by ESR spectroscopy by using 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) as spin trapping agent. The spectral features of the spin adducts derived from all three hydrazine derivatives corresponded to the values reported for the methyl free radical adduct of 4-POBN. In the microsomal preparations inhibitors of the mixed function oxidase system and the destruction of cytochrome P450 by pretreating the rats with CoCl2 all decreased the free radical formation. Methimazole, an inhibitor of FAD-containing monoxygenase system, similarly decreased the activation of 1,1 dimethyl-hydrazine, but not that of monomethyl- and 1,2 dimethyl-hydrazines. The addition to liver microsomes of physiological concentrations of glutathione (GSH) lowered by approx. 80% the intensities of the ESR signals. Consistently, incubation of isolated hepatocytes with methyl-hydrazines decreased the intracellular GSH content, suggesting that GSH can effectively scavenge the methyl free radicals. The results obtained suggest that methyl free radicals could be the alkylating species responsible for the toxic and/or carcinogenic effect of methyl-hydrazines.
Animals↗