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Biomedical subjects

A Tomasi

Publications and source records attributed to A Tomasi.

At least 37 records · Page 2Linked to original sources
PubMed1996

Role of cytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol.

We have previously shown that the treatment with diallyl sulfide (DAS) and phenylethyl isothiocyanate (PIC) of rats receiving ethanol in the alcohol tube-feeding model effectively suppressed the induction of cytochrome P4502E1 (CYP2E1) by ethanol. Here we report that rat treatment with DAS and PIC significantly decreased the trapping of hydroxyethyl free radicals in liver microsomes incubated in vitro with ethanol. Furthermore, these inhibitors also greatly reduced the production of hydroxyethyl radical-derived epitopes detectable in vivo in the liver of ethanol-fed rats. The action of DAS and PIC on the formation of hydroxyethyl radicals paralleled their inhibitory effect on lipid peroxidation as monitored using, respectively, liver malonildialdehyde (MDA) and plasma lipid hydroperoxide levels as well as by the titers of antibodies versus MDA adducts to proteins. Thus, these results indicated a link between the induction of CYP2E1 by ethanol, the formation of hydroxyethyl radicals and the stimulation of lipid peroxidation. The pathological scores in the livers of rats fed with ethanol plus or minus DAS and PIC also correlated with levels of hydroxyethyl radical-derived epitopes. Rats fed intragastrically with ethanol developed antibodies and the formation of these antibodies was greatly reduced by DAS and PIC. Taken together these results suggest that CYP2E1 plays an important role in the generation of hydroxyethyl radicals during chronic alcohol feeding and that ethanol-derived free radicals might play a role in the onset of liver injury in this model of alcohol administration.

Allyl Compounds↗
PubMed1996

'Free' iron, as detected by electron paramagnetic resonance spectroscopy, increases unequally in different tissues during dietary iron overload in the rat.

'Free' iron concentration, as determined by electron paramagnetic resonance (EPR) spectroscopy, and lipid peroxidation (LPO), as determined by thiobarbituric acid test, were assessed in the lung, heart, liver, spleen, brain and kidney of rats subjected to experimental iron overload. Two tests, Desferal- and NO-available iron, were used to measure 'free' iron and gave comparable results. The most pronounced accumulation of 'free' iron was observed in liver, kidney and spleen. Differences between control and iron loaded animals increased during the initial 90 days of treatment. Between 90 and 180 days 'free' iron concentration reached a steady state level, or even decreased, as in the case of liver. Lipid peroxidation level, measured in the organs of both treated and matched controls, did not give any significant difference during the initial 90 days of treatment. A significant augmentation was observed in liver, kidney, spleen and heart at 180 days. The results of the present research show that, under conditions of moderate siderosis, the occurrence of LPO is partially related to the level of 'free' iron.

Animals↗
PubMed1996

Effectiveness of pentoxifylline in semen preparation for intrauterine insemination.

Pentoxifylline, an inhibitor of cAMP phosphodiesterase activity, favours intracellular cAMP concentration increase. In-vitro treatment of semen with pentoxifylline leads to marked augmentation of sperm motility, enhancement of acrosome reaction, increase of sperm penetration into zona-free hamster oocytes, and protection of the sperm plasma membrane. Such properties indicate that the drug may be a useful tool for semen preparation in assisted reproduction, but its real effectiveness in improving fertilization rates is still uncertain, mainly in association with intrauterine insemination (IUI). Theoretically sperm motility should play an extremely important role for positive results in IUI. Therefore, a retrospective clinical trial was planned in order to evaluate whether addition of pentoxifylline to the previously standardized in-vitro treatment of semen had improved the percentage of pregnancies after homologous IUI. The study involved 55 sterile couples (33 classified infertile for male factor and 22 for other factors) who underwent a total of 150 cycles of homologous IUI: 101 for male factor infertility and 49 for other factors (anovulation n = 26, endometriosis n = 2, idiopathic n = 21). Out of the 101 cycles performed for male factor infertility, 61 underwent the standard preparation of semen and were followed by seven pregnancies (pregnancy rate = 11.5%) while 40 had a semen preparation with pentoxifylline addition and were followed by 11 pregnancies (pregnancy rate = 27.5%) with a significant difference between the two procedures (P < 0.05). Out of the 49 cycles carried out for factors different from male infertility, 10 underwent the standard preparation of semen and were followed by two pregnancies (pregnancy rate = 20.0%), while 39 had pentoxifylline addition and were followed by nine pregnancies (pregnancy rate = 23.1%). The difference between the two groups was not significant. Abortions and malformations were equally distributed in the standard treatment and in the pentoxifylline group.

Animals↗
PubMed1996

Influence of ACTH-(1-24) on free radical levels in the blood of haemorrhage-shocked rats: direct ex vivo detection by electron spin resonance spectrometry.

1. The influence of ACTH-(1-24) on the blood levels of highly reactive free radicals in haemorrhagic shock was studied in rats. 2. Volume-controlled haemorrhagic shock was produced in adult rats under general anaesthesia (urethane, 1.25 g kg-1 intraperitoneally) by stepwise bleeding until mean arterial pressure stabilized at 20-23 mmHg. Rats were intravenously (i.v.) treated with either ACTH-(1-24) (160 micrograms kg-1 in a volume of 1 ml kg-1) or equivolume saline. Free radicals were measured in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent (alpha-phenyl-N-tert-butylnitrone). 3. Blood levels of free radicals were 6490 +/- 273 [arbitrary units (a.u.) ml-1 whole blood, before starting bleeding, and 30762 +/- 2650 after bleeding termination (means +/- s.e. mean of the values obtained in all experimental groups). All rats treated with saline died within 30 min, their blood levels of free radicals being 35450 +/- 5450 a.u. ml-1 blood, 15 min after treatment. Treatment with ACTH-(1-24) produced a rapid and sustained restoration of arterial pressure, pulse pressure, heart rate and respiratory function, with 100% survival at the end of the observation period (2 h); this was associated with an impressive reduction in the blood levels of free radicals, that were 12807 +/- 2995, 10462 +/- 2850, 12294 +/- 4120, and 10360 +/- 2080 a.u. ml-1 blood, 15, 30, 60 and 120 min after ACTH-(1-24) administration, respectively. 4. These results provide a direct demonstration that (i) in haemorrhagic shock there is a rapid and massive production of highly reactive free radicals, and that (ii) the sustained restoration of cardiovascular and respiratory functions induced by the i.v. injection of ACTH-(1-24) is associated with a substantial reduction of free radical blood levels. It is suggested that ACTH-(1-24) prevents the burst of free radical generation during blood mobilisation and subsequent tissue reperfusion, and this may be an important component of its mechanism of action in effectively preventing death for haemorrhagic shock.

Animals↗
PubMed1996

No direct evidence of increased lipid peroxidation in hemodialysis patients.

Lipid peroxidation, as measured by the thiobarbituric acid test, has been reported to have increased in hemodialysis (HD) patients, even though the test has low specificity in vivo. Conjugated diene fatty acid (CDFA) hydroperoxides are formed during lipid peroxidation, but not all conjugated dienes (CD) detected in humans originate from lipid peroxidation: octadeca-9,11-dienoic acid, a nonhydroperoxide CD derivative of linoleic acid (CDLA), has a dietary origin. We evaluated CDFA hydroperoxides, CDLA and linoleic acid, using high-performance liquid chromatography, in lipids extracted from plasma, adipose tissue and RBC membranes obtained from 25 patients treated with HD, 16 patients treated with hemodiafiltration (HDF) and 29 controls. No differences in the levels of CDFA hydroperoxides and linoleic acid were seen in any of the groups. Concentrations of CDLA were found to be significantly high in the adipose tissue and low in the RBC membranes of HD patients. HDF-treated patients showed the same results as HD patients. No direct evidence of increased lipid peroxidation was found in HD patients. This does not exclude the possibility that lipid peroxidation is increased and escapes direct detection due to the body's homeostatic control eliminating the increased production of hydroperoxides. Both HD- and HDF-treated patients showed a significant change in CDLA concentrations, either in the adipose tissue, or in the RBC membranes. These dietary CD may be mistaken for markers of lipid peroxidation by conventional methodologies.

Adipose Tissue↗
PubMed1995

Scavenging effect of silipide, a new silybin-phospholipid complex, on ethanol-derived free radicals.

Ethanol metabolism by cytochrome P4502E1 (CYP2E1) produces free radical intermediates, identified as hydroxyethyl radicals. We have observed that in vitro addition or in vivo pretreatment of rats with Silipide, a new 1:1 complex of silybin with phosphatidyl-choline, is able to decrease the spin trapping of hydroxyethyl radicals in microsomes from chronic alcohol-fed rats. This effect is not due to an interference with the metabolism of ethanol by CYP2E1, but is rather related to the capacity of the silybin molecule to scavenge hydroxyethyl radicals. However, such an effect is lost when pure silybin in amounts comparable to those present in Silipide is administered instead, due to the low bioavailability of uncomplexed flavonoid. Further experiments in vivo have shown that Silipide administration also decreases hydroxyethyl radical signals detectable in the bile of rats acutely treated with ethanol. The ability of Silipide to scavenge ethanol-derived radicals along with its antioxidant activity suggests that this drug might be potentially useful in counteracting free radical-mediated injuries involved in the development of liver damage caused by alcohol abuse.

Animals↗
PubMed1995

Ex vivo demonstration of nitric oxide in the rat brain: effects of intrastriatal endothelin-1 injection.

Nitric oxide (NO) is a novel transmitter with multiple functions in endothelium and neuronal tissue. In particular, it has been implicated in the pathogenesis of neurodegenerative diseases. The aim of the present study was to demonstrate the ex vivo detection of NO in basal conditions and after ET-1 intrastriatal injection by means of electron paramagnetic resonance (EPR) spectroscopy using locally injected hemoglobin (Hb) as a NO trapping agent. The extent of neostriatal damage after Hb and ET-1 injections was assessed by means of immunocytochemistry with a monoclonal antibody against dopamine and cAMP-phosphoprotein M(r) 32 (DARPP-32), which is considered a marker of striatal intrinsic neurons. In the absence of local Hb injection, no signal related to endogenous NO was detected in the neostriatum, suggesting that endogenous NO trapping agents are not sufficiently concentrated to allow NO detection with the present technique. Instead, 1 h after Hb injection, a clear nitrosyl-Hb signal can be detected in neostriatal homogenates. ET-1, a powerful vasoconstrictor agent, was used to cause neuronal loss in the neostriatum. No change in nitrosyl-Hb signal was observed in neostriatal 1 h after ET-1 injection, whereas an almost 3-fold increase in the signal intensity was present 24 h after ET-1 injection. The analysis of neostriatal damage showed that Hb injection did not cause either significant damage of striatal tissue or potentiation of ET-1-induced lesions. In conclusion, the present technique allows ex vivo detection of NO in the brain. The delayed increase in NO observed after ET-1 injection indicates that this molecule may participate in the development of slowly progressive neuronal damage occurring at late post-ischemic times.

Animals↗
PubMed1995

Activation of alkylhydrazines to free radical intermediates by ethanol-inducible cytochrome P-4502E1 (CYP2E1).

Electron spin resonance (EPR) spectroscopy analysis using the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) was used to measure the formation of free radical intermediates during NADPH-dependent oxidation of 1-methyl-, 1-ethyl-, and 1-isopropylhydrazine in rat liver microsomes and in reconstituted enzyme systems. The experiments in microsomes revealed that the specific activation of the hydrazines, as measured by the EPR signal intensities, was about two-fold higher, when expressed per nmol of P-450, in microsomes from rats treated with ethanol (EtOH) as compared to membranes isolated from either phenobarbital (PB)-, beta-naphthoflavone (beta-NF)-treated or control rats. Furthermore, kinetic experiments revealed that EtOH-microsomes had an apparent affinity for 1-ethylhydrazine about one order of magnitude higher than PB-microsomes. In reconstituted vesicular systems composed of phospholipids, NADPH cytochrome P-450 reductase and P-450, the intensities of EPR signals produced by the formation of the methyl-, ethyl- and isopropyl-free radicals, were 3- to 5-fold more intense in membrane vesicles containing ethanol-inducible CYP2E1 than phenobarbital-inducible CYP2B1. By contrast, CYP1A2, CYP2B4 and CYP2C4 were inefficient catalysts of radical formation. Desferrioxamine, catalase and superoxide dismutase did not influence the extent of ethyl radicals formed in EtOH-microsomes, indicating that hydroxyl radicals are not involved in the CYP2E1-dependent activation of 1-ethylhydrazine. Addition of cytochrome b5, an efficient donor of the second electron to P-450 and hence an inhibitor of the formation of the oxy-cytochrome P-450 complex, increased to be consistent with the results, did not influence the amount of ethyl radicals trapped. In liver microsomes from untreated rats selective substrates of CYP2E1, such as diethyl-dithiocarbamate and p-nitrophenol, as well as anti-CYP2E1-IgG, inhibited the free radical formation from 1-ethylhydrazine by about 60%. The anti-CYP2E1 IgG used significantly inhibited ethyl radical production also in human liver microsomes incubated with 1-ethylhydrazine and 4-POBN. Taken together, these results indicate that CYP2E1, as compared to other rat liver cytochromes P-450, is an efficient catalyst of transformation of alkylhydrazines to free radical intermediates, a finding that might be of importance in the development of the toxicity of these compounds.

Animals↗
PubMed1995

Induction of ferritin synthesis by oxidative stress. Transcriptional and post-transcriptional regulation by expansion of the "free" iron pool.

Ferritin, by regulating the "free" intracellular iron pool, controls iron-catalyzed generation of reactive oxygen species, but its role in oxidative damage is still unclear. We show that ferritin synthesis is significantly stimulated in the liver of rats subjected to oxidative stress by treatment with phorone, a glutathione-depleting drug. RNA-bandshift assays document reduced activity of iron regulatory factor, in particular of IRFB, the cytoplasmic protein that post-transcriptionally controls ferritin mRNA translation. Furthermore, Northern blot analysis shows increased accumulation of H and L subunit mRNAs, and nuclear run-on experiments provide evidence of transcriptional activation. Direct measurements of intracellular free iron levels by EPR indicate that the increased ferritin synthesis can be mediated by an expansion of the free iron pool. An early drop of ferritin content after phorone treatment indicates that part of the iron that fuels the free pool might derive from ferritin degradation. Present data seem to suggest that, under conditions of oxidative stress, liver ferritin can represent either a pro- or an anti-oxidant in a time-dependent manner. In fact, its early degradation contributes to expand the intracellular free iron pool that, later on, activates multiple molecular mechanisms to reconstitute ferritin content, thus limiting the pro-oxidant challenge of iron.

Animals↗
PubMed1995

Prenatal detection of short arm deletion and isochromosome 18 formation investigated by molecular techniques.

A patient was referred for amniocentesis because of advanced maternal age and polyhydramnios. The fetal karyotype was a mosaic 46,XX,del(18)(p11.1)/46,XX,-18,+i(18q)de novo. The deletion appeared to encompass the whole short arm as evidenced by G banding and in situ hybridisation. However, telomere sequences were found on both ends of the deleted chromosome as well as the isochromosome. The normal 18 and the isochromosome showed more alphoid sequences than the del(18). Subsequent passages of the cell lines showed an increase in the frequency of the isochromosome from 20% to about 30%. Possible mechanisms are discussed.

Adult↗
PubMed1994

Free radical activation of acetaldehyde and its role in protein alkylation.

The formation of carbon centered free radicals, identified as methylcarbonyl species, was observed using ESR spectroscopy and the spin trapping agent 4-pyridyl-1-oxide-N-t-butyl nitrone (4-POBN) during the oxidation of acetaldehyde by xanthine oxidase. The reaction was dependent upon the presence of OH. radicals and was inhibited by the addition of superoxide dismutase, catalase or OH. radical scavengers. The generation of methylcarbonyl radicals was associated with a doubling of stable acetaldehyde adducts with serum albumin, and 4-POBN or superoxide dismutase and catalase, completely blocked this effect. Thus, methylcarbonyl radicals contributed to acetaldehyde-mediated protein alkylation which is involved in causing toxic as well as immunological reactions ascribed to acetaldehyde.

Acetaldehyde↗
PubMed1994

Alternative pathways of sulfite oxidation in human polymorphonuclear leukocytes.

Sodium sulfite is metabolized by human polymorphonuclear leukocytes by two alternative pathways, one enzymatic route dependent on sulfite oxidase and one non-enzymatic which involves intermediate formation of sulfur trioxide anion radicals. Initiation of the oxidative burst by phorbol myristate acetate significantly stimulates sulfate formation through the second pathway. The activity of sulfite oxidase in polymorphonuclear leukocytes varies greatly among individuals, a variation consistent with the suggested polymorphic distribution of sulfite oxidase in the human population.

Chromatography, High Pressure Liquid↗
PubMed1993

Free radical production during metabolism of organic hydroperoxides by normal human keratinocytes.

Evidence of a relationship between tumor production induced by various organic (hydro)peroxides and free radical formation has been shown in cultured murine keratinocytes and human skin-tumor cell line. In the present study the bioactivation of cumene hydroperoxide, t-butyl-hydroperoxide, and benzoyl peroxide via one-electron oxidation or reduction was compared in freshly isolated and in cultured normal human keratinocytes. The formation of methyl free radicals during the metabolism of cumene and t-butyl-hydroperoxide was shown by the electron spin resonance-spin trapping technique. Radical formation increased under hypoxic conditions. An intracellular activation site was demonstrated by the use of two spin-trapping agents, the hydrophilic, membrane-impermeable, 3,5-dibromo-4-nitrosobenzenesulfonic acid and the lipophilic, membrane-permeable alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone. At 30 min incubation and 25 mM concentration, hydroperoxides exhibited cytotoxicity, as indicated by trypan blue exclusion and lactate dehydrogenase release assay; free radicals were concurrently trapped. Hydroperoxides at a lower concentration (1 mM) did not significantly affect cell viability. However, free radical production was still detected using a membrane-permeable spin trap. The incubation of keratinocytes with benzoyl peroxide did not show any peroxide-dependent radical adduct. No significant differences in bioactivation capability were demonstrated between freshly isolated and cultured human keratinocytes. The results indicate that cultured human keratinocytes can be used as a model system for the study of the metabolic activation to free radical intermediates of toxic and carcinogenic compounds in the epidermis.

Benzene Derivatives↗
PubMed1993

Conjugated diene fatty acids in patients with chronic renal failure: evidence of increased lipid peroxidation?

Conjugated diene fatty acids (CDFA) were evaluated by second derivative spectrophotometry in the plasma and adipose tissue of 42 chronic renal failure (CFR) patients in conservative treatment, 40 patients treated by hemodialysis (HD) with cuprophane, cellulose acetate or hemophan, 29 treated by hemodiafiltration (HDF) with polysulfone, polyacrylonitrile or polyamide, and 28 healthy controls. Plasma CDFA were also evaluated at the beginning, at 30 min and at the end of the dialytic session. CDFA were unchanged in CRF patients with creatinine clearance (Ccr) > 10 ml/min respect to the controls, CRF patients with Ccr < 10 ml/min showed a higher level of CDFA both in plasma and adipose tissue (p < 0.02). HD patients showed values similar to those of the control group. The lowest level of CDFA was found in HDF patients (p < 0.01 for plasma, p < 0.05 for adipose tissue versus both control and any other group). A significant relationship between plasma and adipose tissue CDFA was found in all groups. In the group of CRF patients with Ccr < 10 ml/min, females exhibited a higher level of CDFA both in plasma and adipose tissue. No significant change was found during dialytic session, independently from the membrane used. CDFA are not only primary products of lipid peroxidation, but also have a dietary origin, primarily from dairy products. Taking into account the reduced dietary intake, the increase in end-stage CRF may be due to an enhanced oxidative stress and/or to abnormalities in CDFA metabolism. Uremic patients, particularly in the predialytic stage, should be considered at risk for increased oxidative stress. HDF treatment better corrects the abnormality compared to conventional HD.

Adipose Tissue↗
PubMed1993

Tert-butylhydroperoxide bioactivation to methyl radical in rat liver mitochondria and submitochondrial particles.

Electron paramagnetic resonance spectroscopy (EPR) coupled to the spin trapping technique was used to detect carbon-centered radicals in rat liver mitochondria and submitochondrial particles exposed to t-butyl-hydroperoxide (TBH), using the spin trapping agent 3,5-dibromo-4-nitroso-benzenesulfonic acid (DBNBS). The signal recorded was unambiguously assigned to the methyl radical adduct. DBNBS was added to isolated rat liver mitochondria energized with succinate, and the methyl radical adduct was observed. The addition of NADH, NADPH, inhibitors of the respiratory chain, and of monoaminoxidase (MAO) inhibitors did not cause any relevant modification in the yield of radical adduct formation. Boiling and the addition of a non-ionic detergent inhibited the formation of the radical adduct, while experiments carried out under hypoxic conditions generated a significant increase in methyl radical formation. Further experiments were carried out on sub-mitochondrial particles (SMP) giving rise to, basically, the same results. From the above results, we are proposing that haem prosthetic groups are the likely source of TBH bioactivation in mitochondria.

Animals↗