Teresa Carbonell

Simultaneous generation of nitric oxide and superoxide by inflammatory cells in rats

It has recently been shown that peroxynitrite anion is a powerful oxidant than can initiate lipid peroxidation. As this oxidant is the product of the reaction between nitric oxide and superoxide, we have studied whether cells isolated from an inflammatory exudate can release both radicals simultaneously under physiological conditions. The carrageenin-induced granuloma model in rats was used. Cells from the inflammatory pouch were stimulated with opsonized zymosan in the absence or in the presence of exogenous L-arginine. Nitric oxide production without exogenous L-arginine was detectable after 15 min (0.29 nmol NO2-) and increased with time (1.65 nmol NO2- at 4 h). When nitrite released from cells was expressed as a rate a burst was shown in the first few minutes. Between 0 and 15 min, cells produced NO2- at the following rates: 20 pmol NO2-/1 x 10(6) cells/min without exogenous L-arginine and 83 pmol NO2-/1 x 10(6) cells/min with exogenous L-arginine. Production was further stimulated with opsonized zymosan (92 pmol NO2-/1 x 10(6) cells/min), and inhibited by L-NMMA and L-NIO. The production of superoxide increased for up to 2 h and then stabilized. A significant increase in nitrite was observed in the presence of SOD, whereas L-NIO increased superoxide generation. These results suggest that peroxynitrite anion may be formed by inflammatory cells.

Nitric oxide inhibits superoxide production by inflammatory polymorphonuclear leukocytes.

Nitric oxide (NO ⋅) has a complex role in the inflammatory response. In this study, we modified the levels of endogenous NO ⋅ in vivo in an acute model of inflammation and evaluated the interactions between NO ⋅ and superoxide anion ([Formula: see text]) produced by polymorphonuclear leukocytes (PMNs) accumulated in the inflamed area. We injected phosphate-buffered saline (control group), 6 μmol ofl- N 5-(1-iminoethyl)ornithine (l-NIO group), or 6 μmol ofl-arginine (l-arginine group) into the granuloma pouch induced by carrageenan in rats.[Formula: see text] plus[Formula: see text] (indicative of NO ⋅ generation) was 188 nmol in the exudate of the control group, but it decreased in the l-NIO group ( P < 0.05) and increased in thel-arginine group ( P < 0.05). When PMNs from treated rats were incubated in vitro, the production of superoxide anion ([Formula: see text]) decreased by ∼46% in thel-arginine group. Furthermore,[Formula: see text] was inhibited in PMNs whenl-arginine was added to the incubation medium before phorbol 12-myristate 13-acetate stimulation but not when added simultaneously. Our results suggest a protective role for NO ⋅ in inflammation, through the inactivation of NADPH oxidase and the consequent impairment of[Formula: see text] production for cell-mediated injury.Nitric oxide (NO.) has a complex role in the inflammatory response. In this study, we modified the levels of endogenous NO. in vivo in an acute model of inflammation and evaluated the interactions between NO. and superoxide anion (O2-.) produced by polymorphonuclear leukocytes (PMNs) accumulated in the inflamed area. We injected phosphate-buffered saline (control group), 6 mumol of L-N5-(1-iminoethyl)ornithine (L-NIO group), or 6 mumol of L-arginine (L-arginine group) into the granuloma pouch induced by carrageenan in rats. NO2- plus NO3- (indicative of NO. generation) was 188 nmol in the exudate of the control group, but it decreased in the L-NIO group (P < 0.05) and increased in the L-arginine group (P < 0.05). When PMNs from treated rats were incubated in vitro, the production of superoxide anion (O2-.) decreased by approximately 46% in the L-arginine group. Furthermore, O2-. was inhibited in PMNs when L-arginine was added to the incubation medium before phorbol 12-myristate 13-acetate stimulation but not when added simultaneously. Our results suggest a protective role for NO. in inflammation, through the inactivation of NADPH oxidase and the consequent impairment of O2-. production for cell-mediated injury.

Iron intake increases infarct volume after permanent middle cerebral artery occlusion in rats

Experimental and clinical data suggest an important role of iron in cerebral ischaemia. We measured infarct volume and analysed the oxidative stress, and also the excitatory and inflammatory responses to brain injury in a rat stroke model after an increased oral iron intake. Permanent middle cerebral artery occlusion (MCAO) was performed in ten male Wistar rats fed with a diet containing 2.5% carbonyl iron for 9 weeks, and in ten control animals. Glutamate, interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) were determined in blood samples before and at 2, 4, 6, 8, 24 and 48 h after MCAO, and thiobarbituric acid reaction substances (TBARS) were analysed at 48 h. Infarct volume was measured at 48 h by image analysis on brain slices stained with 1% TTC. Tissue iron was measured by atomic absorption spectrophotometry. Infarct volume was 66% greater in the iron fed rats than in the control group (178+/-49 mm(3) versus 107+/-53 mm(3), P<0.01). Significant higher levels of glutamate, IL-6 and TNF-alpha were observed in the group with iron intake (peak values were obtained at 6, 8 and 4 h, respectively). Iron-fed animals also showed significantly higher levels of TBARS than those receiving a normal diet (6.52+/-0.59 vs. 5.62+/-0.86 micro mol/l, P=0.033) Liver iron stores (3500+/-199 vs. 352+/-28 micro g Fe/g, P<0.0001), but not brain iron stores (131 vs. 139 micro g Fe/g, P=0.617), were significantly higher in the iron fed rats group. These results suggest that iron intake is associated with larger infarct volumes after MCAO in the rat. This effect seems to be associated with higher oxidative stress, excitotoxicity and inflammatory responses.

Different roles for nitrogen monoxide and peroxynitrite in lipid peroxidation induced by activated neutrophils.

We studied the roles of nitrogen monoxide (NO&z.rad;) and peroxynitrite produced by the polymorphonuclear leukocytes (PMNs) isolated from an inflammatory exudate. PMNs were incubated either in a medium with a submicromolar concentration of iron or in a diethylenetriaminepenta-acetic acid (DTPA)-containing medium, and stimulated with phorbol 12-myristate 13-acetate (PMA) to generate free radicals. In both conditions superoxide anion (O(2)(*)(-)), NO&z.rad; and peroxynitrite were produced. In the presence of arachidonic acid, malondialdehyde (MDA) was generated. This MDA was generated in one of two way; the peroxynitrite iron-independent mechanism (40%) and the Fenton reaction, caused by free iron (60%). We also observed that the addition of L-arginine was followed by a 42% reduction in MDA, which can be explained by the antioxidant effect of NO&z.rad;. These results indicate that lipid peroxidation can occur in the absence of iron, through a peroxynitrite-mediated mechanism, and that NO&z.rad; may act as an antioxidant when it is produced in large amounts.

Oxidative Stress and Antioxidant Activity in Hypothermia and Rewarming: Can RONS Modulate the Beneficial Effects of Therapeutic Hypothermia?

Hypothermia is a condition in which core temperature drops below the level necessary to maintain bodily functions. The decrease in temperature may disrupt some physiological systems of the body, including alterations in microcirculation and reduction of oxygen supply to tissues. The lack of oxygen can induce the generation of reactive oxygen and nitrogen free radicals (RONS), followed by oxidative stress, and finally, apoptosis and/or necrosis. Furthermore, since the hypothermia is inevitably followed by a rewarming process, we should also consider its effects. Despite hypothermia and rewarming inducing injury, many benefits of hypothermia have been demonstrated when used to preserve brain, cardiac, hepatic, and intestinal function against ischemic injury. This review gives an overview of the effects of hypothermia and rewarming on the oxidant/antioxidant balance and provides hypothesis for the role of reactive oxygen species in therapeutic hypothermia.

Proteasome inhibitors protect the steatotic and non-steatotic liver graft against cold ischemia reperfusion injury.

BACKGROUND The dramatic shortage of organs leads to consider the steatotic livers for transplantation although their poor tolerance against ischemia reperfusion injury (IRI). Ubiquitin proteasome system (UPS) inhibition during hypothermia prolongs myocardial graft preservation. The role of UPS in the liver IRI is not fully understood. Bortezomib (BRZ) treatment at non-toxic doses of rats fed alcohol chronically has shown protective effects by increasing liver antioxidant enzymes. We evaluated and compared both proteasome inhibitors BRZ and MG132 in addition to University of Wisconsin preservation solution (UW) at low and non-toxic dose for fatty liver graft protection against cold IRI. EXPERIMENTAL Steatotic and non-steatotic livers have been stored in UW enriched with BRZ (100 nM) or MG132 (25 μM), for 24h at 4°C and then subjected to 2-h normothermic reperfusion (37 °C). Liver injury (AST/ALT), hepatic function (bile output; vascular resistance), mitochondrial damage (GLDH), oxidative stress (MDA), nitric oxide (NO) (e-NOS activity; nitrates/nitrites), proteasome chymotrypsin-like activity (ChT), and UPS (19S and 20S5 beta) protein levels have been measured. RESULTS ChT was inhibited when BRZ and MG132 were added to UW. Both inhibitors prevented liver injury (AST/ALT), when compared to UW alone. BRZ increased bile production more efficiently than MG132. Only BRZ decreased vascular resistance in fatty livers, which correlated with an increase in NO generation (through e-NOS activation) and AMPK phosphorylation. GLDH and MDA were also prevented by BRZ. In addition, BRZ inhibited adiponectin, IL-1, and TNF alpha, only in steatotic livers. CONCLUSION MG132 and BRZ, administrated at low and non toxic doses, are very efficient to protect fatty liver grafts against cold IRI. The benefits of BRZ are more effective than those of MG132. This evidenced for the first time the potential use of UPS inhibitors for the preservation of marginal liver grafts and for future applications in the prevention of IRI.

Bortezomib enhances fatty liver preservation in Institut George Lopez-1 solution through adenosine monophosphate activated protein kinase and Akt/mTOR pathways

The aim of this study is to investigate the protective mechanisms induced by bortezomib added to Institut George Lopez (IGL)‐1 preservation solution to protect steatotic livers against cold ischaemia reperfusion injury and to examine whether these mechanisms occur through the activation of adenosine monophosphate activated protein kinase (AMPK), Akt/mTOR pathways.

Hypothermic protection in an acute hypoxia model in rats: Acid-base and oxidant/antioxidant profiles

AIM OF THE STUDY Recent works demonstrate the benefits of hypothermia when used to preserve brain, cardiac, hepatic, and intestinal function against hypoxic-ischemic injury. However, it is also known that hypothermia affects systemic parameters and also induces the generation of reactive oxygen species in cells and tissues. Here we studied the acid-base related parameters and the antioxidant-oxidant effects of deep hypothermia induction before an acute hypoxic insult in rats. METHODS Acid-base indicators and parameters related to oxidative stress were analyzed in hypothermic rats (21-22 degrees C) breathing room air during 2h (control hypothermia), and hypothermic animals switched to hypoxic air (10% O(2)) during the second hour (hypothermia hypoxia group), and they were compared with corresponding normothermia groups maintained at 37 degrees C (control normothermia and normothermia hypoxia groups). RESULTS Mild metabolic acidosis appeared early in arterial blood during hypothermia. After exposure to hypoxia, evidence of tissue injury (plasma transaminases and blood lactate) and oxidative stress (increase in lipid peroxidation, decrease in glutathione levels and in the glutathione reduction potential in liver) was found. In contrast, in the hypothermia hypoxia group, plasmatic parameters remained as the control values, and the hepatic glutathione reduction potential were significantly more negative when compared with the normothermia hypoxia group. CONCLUSIONS We propose that acidosis induced by hypothermia contributes to the maintenance of intracellular reduction potential in liver, regarding the GSSG/2GSH couple and may help to increase plasmatic antioxidant pool. Our findings provide new insights into the protective effects of hypothermia in vivo.

Fish Oil and Oxidative Stress by Inflammatory Leukocytes

We investigated the effects of diets with different fatty acid composition upon the oxidative stress of inflammatory leukocytes of rats. After weaning, two groups of rats were fed isoenergetic semipurified diets for five weeks containing 5% of corn oil or menhaden oil. Polymorphonuclear leukocytes from rats fed menhaden oil diet incorporated n-3 polyunsaturated fatty acids into phospholipid membranes at the expense of arachidonic acid. These cells showed diminished superoxide production and, as a consequence, the total antioxidant status in the inflammatory exudate was increased. However, nitric oxide production was not affected by diet. Free malondialdeyde concentration increased in the exudate because of lower mitochondrial activity. These results add new aspects that help clarifying the anti-inflammatory mechanisms of n-3 polyunsaturated fatty acids.

The use of a reversible proteasome inhibitor in a model of Reduced-Size Orthotopic Liver transplantation in rats☆

Ischemia/reperfusion injury (IRI), inherent in liver transplantation (LT), is the main cause of initial deficiencies and primary non-function of liver allografts. Living-related LT was developed to alleviate the mortality resulting from the scarcity of suitable deceased grafts. The main problem in using living-related LT for adults is graft size disparity. In this study we propose for the first time that the use of a proteasome inhibitor (Bortezomib) treatment could improve liver regeneration and reduce IRI after Reduced-Size Orthotopic Liver transplantation (ROLT). Rat liver grafts were reduced by removing the left lateral lobe and the two caudate lobes and preserved in UW or IGL-1 preservation solution for 1h liver and then subjected to ROLT with or without Bortezomib treatment. Our results show that Bortezomib reduces IRI after LT and is correlated with a reduction in mitochondrial damage, oxidative stress and endoplasmic reticulum stress. Furthermore, Bortezomib also increased liver regeneration after reduced-size LT and increased the expression of well-known ischemia/reperfusion protective proteins such as nitric oxide synthase, heme oxigenase 1 (HO-1) and Heat Shock Protein 70. Our results open new possibilities for the study of alternative therapeutic strategies aimed at reducing IRI and increasing liver regeneration after LT. It is hoped that the results of our study will contribute towards improving the understanding of the molecular processes involved in IRI and liver regeneration, and therefore help to improve the outcome of this type of LT in the future.