Anna Bini

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.

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 μg 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 (α‐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.

Adrenocorticotropin normalizes the blood levels of nitric oxide in hemorrhage-shocked rats.

Anesthetized rats were subjected to volume-controlled hemorrhagic shock by stepwise bleeding. Besides cardiovascular and respiratory functions, nitric oxide (NO)-hemoglobin formation in arterial blood was directly evaluated by means of electron spin resonance spectroscopy. During hemorrhagic shock there was a massive increase in NO-hemoglobin, associated with a fall in mean arterial pressure, pulse pressure, respiratory rate and heart rate, and there was a further increase in NO-hemoglobin 15 min after intravenous (i.v.) treatment with saline. All rats died within 30 min. The reversal of the shock condition induced by the i.v. injection of the adrenocorticotropin (ACTH) fragment 1-24 (160 microg/kg, 5 min after bleeding termination) was associated with a prompt disappearance of NO-hemoglobin. Also S-methylisothiourea (3 mg/kg i.v.), a selective inhibitor of inducible NO synthase, provoked a disappearance of NO-hemoglobin and reversal of the shock condition. The present results provide a direct demonstration that volume-controlled hemorrhagic shock is associated with highly increased blood levels of NO, as indicated by increased NO-hemoglobin, and indicate that ACTH-induced reversal of the shock condition is associated with the normalization of NO blood levels, and a parallel improvement of cardiovascular and respiratory functions. This occurs probably through the inhibition of inducible NO synthase, as suggested by the fact that S-methylisothiourea, a selective inhibitor of this NO synthase isoform, produced the same results.

Red Wine Consumption Prevents Vascular Oxidative Stress Induced by a High-fat Meal in Healthy Volunteers

In order to investigate the effect of red wine on plasma lipid and oxidative stress parameters after a high-fat meal, fifteen healthy volunteers were studied: three days after a high-fat meal with 250 mL of water, they received the same meal with 250 mL of red wine. During both periods, serial blood samples were drawn before and 2, 4, and 8 hours after the meal to evaluate plasma lipids (cholesterol and triglycerides; retinyl palmitate), oxidative stress (D-ROM, and malondialdehyde) and antioxidant (total plasma antioxidant levels and uric acid) parameters. During the meal without wine, plasma lipid parameters increased significantly, whereas plasma total plasma antioxidant levels decreased, and a trend toward reduction of uric acid levels was seen). A similar trend in lipid parameters was observed after the meal with wine; no significant difference in individual lipid parameter trends after a meal with and without wine was observed. Wine ingestion induced higher total plasma antioxidant levels and uric acid; malondialdehyde levels remained constant after wine ingestion. Plasma D-ROM showed a significant postprandial increase in both experiments, but it was significantly lowered after wine ingestion. Our results give evidence of oxidative stress following a fat-rich meal in healthy subjects, suggesting that ingestion of red wine during a high-fat meal significantly reduces oxidative stress without inducing any significant modification in postprandial lipemia.

Age-related differences in the metabolism of sulphite to sulphate and in the identification of sulphur trioxide radical in human polymorphonuclear leukocytes

Sulphite oxidation and sulphur trioxide radical formation were studied in polymorphonuclear leukocytes (PMNs) isolated from healthy young, old and centenarian donors and from patients with Downs syndrome. The sulphur radical formation measured by electron spin resonance spectroscopy-spin trapping (EPR-ST) was correlated with the activity of sulphite oxidase and with the rate of sulphite oxidation to sulphate by PMNs. Sulphite metabolism was studied both in resting, and phorbol myristate acetate (PMA) stimulated freshly isolated cells. The rate of sulphur trioxide radical formation was demonstrated by use of the spin trapping agent 5,5-dimethyl-1-pyroline-1-oxide (DMPO) with subsequent formation of an adduct. The intensity of adduct formation was most intense in cells with low sulphite oxidase activity, while a mixture of the adduct and of DMPO hydroxyl radical was mainly observed in cells with high sulphite oxidase activity. Furthermore, experiments carried out on purified sulphite oxidase showed that in the presence of sulphite the enzyme could also give rise to a DMPO-OH adduct. Sulphite oxidase activity in cells isolated from healthy young and old donors was positive correlated with both rates of sulphur trioxide radical formation and sulphite oxidation to sulphate, respectively. However, sulphite oxidase activity in cells isolated from centenarians and patients with Downs syndrome seems to loose partly its rate of oxidising sulphite to sulphate. The intensity of the sulphur centred radical adduct increased in the two latter groups of population and the radical observed was predominantly sulphur trioxide radical.

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

Abstract‘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.

High blood levels of nitric oxide in rats subjected to prolonged respiratory arrest and their modulation during adrenocorticotropin-induced resuscitation

Anaesthetized rats, endotracheally intubated and mechanically ventilated with room air, were subjected to a 5-min period of asphyxia by turning off the ventilator. The ventilator was then turned back on and, simultaneously, the animals were treated with either the adrenocorticotropin fragment 1–24 [ACTH-(1–24), 160 µg/kg in a volume of 1 ml/kg i.v.] or an equivalent volume of saline. Nitric oxide (NO)-haemoglobin formation was detected ex vivo in arterial blood by electron spin resonance spectrometry; arterial blood pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for a 60-min observation period, or until prior death. During asphyxia, there was massive formation of NO (red cell concentrations 40–80 µM), associated with a dramatic fall in mean arterial pressure and pulse pressure, marked bradycardia and ECG signs of ischaemic damage, as well as an isoelectric EEG. Treatment with ACTH-(1–24) produced a prompt (within 15 min) and long-lasting drop in NO blood levels, associated with an almost immediate (within 1 min) restoration of cardiovascular function and with a more gradual recovery of EEG, which became normal after 30–40 min; all parameters remained stable throughout the 60-min observation period. In saline-treated rats, on the other hand, there was a further increase in NO blood levels, as detected 3 min after treatment, and all died within 5–8 min. Moreover, pretreatment and treatment with S-methylisothiourea sulphate (SMT, 3 mg/kg i.v.), a relatively specific inhibitor of inducible NO synthase, inhibited NO formation, but did not affect the mortality rate (100% within 5–8 min). The present results provide the first evidence that prolonged asphyxia is associated with high blood concentrations of NO, and that the life-saving effect of melanocortin peptides in severe hypoxic conditions is associated with a complete normalization of NO blood levels. However, the lack of SMT protection in this experimental model seems to rule out the possibility that the ACTH-(1–24)-induced resuscitation is due to an effect on NO overproduction.

Dietary fatty acids composition modulates oxidative stress and cellular injury in the liver of CBrCl3 intoxicated rats

Abstract Oxidative stress is a multifactorial event that has been proposed to be involved in the aethyology of a large number of human pathologies. The fatty acid composition of dietary lipid has been reported as one of major factors capable of modulating oxidative stress. In this paper we assess the effect of different types of dietary lipids on microsome fatty acids composition and on the oxidative stress induced by the administration of CBrCl 3 . Three groups of rats were fed different experimental diets providing 20% of energy from lipid derived by olive, coconut, and sunflower oils. Membrane fatty acid composition and antioxidant vitamins was determined at the end of six weeks diet. The dietary induced changes of membrane fatty acids composition were associated with different susceptibility to oxidative stress and cell damage, as assessed by conjugated dienes and cytosolic enzyme release respectively following CBrCl 3 intoxication. Feeding the CO diet leads to a low susceptibility to lipid peroxidation, but is associated with cell injury significantly higher than that observed in the other dietary treatments. No association was demonstrated between the extent of membrane lipid peroxidation and liver cell damage.

Adrenocorticotropin counteracts the increase in free radical blood levels, detected by electron spin resonance spectrometry, in rats subjected to prolonged asphyxia

We investigated the influence of the adrenocorticotropic fragment 1-24 [ACTH-(1-24)] on the blood levels of highly-reactive free radicals in a rat model of prolonged asphyxia. Anesthetized animals were endotracheally intubated and mechanically ventilated with room air; after a 10 min stabilization period, the ventilator was turned off to induce asphyxia for 5 min; then, the ventilator was turned back on, and, simultaneously, the rats were intravenously treated with either ACTH-(1-24) (160 microg/kg in a volume of 1 ml/kg) or equivolume saline. Free radicals were detected in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent employed (alpha-phenyl-N-tert-butylnitrone). Arterial pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for the 60 min observation period, or until prior death. At the end of the 5 min period of respiratory arrest, blood levels of free radicals were about four times higher than those of the basal, pre-asphyxia condition, arterial pressure had dramatically decreased, ECG showed marked bradycardia and signs of ischemic damage and the EEG had become isoelectric. Treatment with ACTH-(1-24) produced an immediate normalization of the blood levels of free radicals, associated with a restoration of cardiovascular function and full recovery of EEG within 30-45 min; all the saline-treated rats, on the other hand, died within 6.89 +/- 0.96 min. These results provide direct evidence that in a severe condition of prolonged asphyxia there is a rapid and massive production of highly-reactive free radicals and suggest that the resuscitating effect of adrenocorticotropin fragments in severe hypoxic conditions may be largely due to the inhibition of free radical overproduction during tissue reoxygenation.