P8. Oxygen Reactive Species in Inflammation and Disease

P8. Oxygen Reactive Species in Inflammation and Disease

P8-92 OXIDATIVE DAMAGE OF GABAa RECEPTORS BY 5-AMINOLEVULINATE A. Adhikari,1 C. A. A. Penatti,1 E. J. H. Bechara,1 and L. R. G. Britto1 1 Instituto de Quı´mica and Instituto de Ciencias Biome´dicas, Universidade de Sao Paulo, Brazil

Porphyrias are diseases associated with enzymatic deficiencies in heme biosynthesis as in the case of intermittent acute porphyria (IAP) and lead poisoning, where 5aminolevulinate (ALA) accumulates. Effects of ALA on the central nervous system during both acute phases and chronic evolution of IAP have been explained by strong binding of ALA to g-aminobutyric acid (GABA) receptors, followed by receptor lesions from oxyradicals and 4,5-dioxovalerate (DOVA) generated in iron-catalyzed ALA oxidation. Previous experiments revealed that GABAa receptors of cortical synaptosomes obtained from ALA-treated mice have decreased affinity for the radiolabeled agonist [3H]-muscimol. Furthermore, human neuroblastoma cells lost total gabaergic binding capacity when exposed to DOVA. We now present an in vivo study of ALA effects on the GABAa receptors density in brains of rats treated with either ALA or succinylacetone (SA), an inhibitor of ALA dehydratase and hence able to induce ALA accumulation in the brain and other tissues. Immunohistochemical assays indicate that ALA and SA given to rats for 15 days diminish the density of gabaergic receptors in rat diencephalon (habenular complex and hypothalamic arcuate nucleus), whose disruption can be correlated with the neuropsychiatric and autonomic symptoms in IAP. In conclusion, these findings support the hypothesis that ALA-promoted damage to GABAa receptors may indeed be implicated in the neurological manifestations of IAP and lead poisoning. Support: FAPESP, PRONEX, CNPq.

P8-93 BENEFICIAL EFFECT OF OXYKINE IN DIABETIC NEPHROPATHY IN DB/DB MICE S. Akagiri,1 Y. Naito,1 K. Uchiyama,1 H. Ichikawa,1 N. Yoshida, and T. Yoshikawa1 1

Inflammation and Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Japan

Oxidative stress is implicated as an important mechanism by which diabetes causes nephropathy. In this study, we examined whether chronic administration of oxykine, which is the first orally effective, vegetarian form of superoxide dismutase (SOD) from cantaloupe melon and gliadin, could prevent the progression of diabetic nephropathy induced by oxidative stress in mice. We used female C57BL/KsJ-db/db mice, a rodent model of type 2 diabetes, and their non-diabetic db/m littermates.

The mice were divided into three groups as follows: nondiabetic db/m, diabetic db/db, and diabetic db/db treated with oxykine. Blood glucose level, body weight, urinary albumin, and urinary 8-hydroxydeoxyguanosine (8-OHdG) were measured during the experiments. After 12 weeks of treatment, the oxykine-treated group showed almost the same high level of blood glucose as that of the non-treated db/db group. The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were significantly inhibited by chronic treatment with oxykine. In this study, treatment with oxykine ameliorated the progression and acceleration of diabetic nephropathy in the rodent model of type 2 diabetes. The results suggested that the antioxidative activity of oxykine reduced the oxidative stress on the kidneys and prevented renal cell damage. In conclusion, administration of oxykine might be a novel approach for the prevention of diabetes nephropathy.

P8-94 MODULATION OF PRO-INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES, AND ANTIOXIDATIVE PROFILE IN EXPERIMENTAL ARTHRITIS BY AN INDIAN HERBAL PLANT, Nyctanthes arbor tristis L. A. A. Mahdi,1 B. Rathore, and B. N. Paul 2 1 Department of Biochemistry, King George’s Medical University, Lucknow, India, 2Immunobiology Division, Industrial Toxicology Research Centre, Lucknow, India

Rheumatoid arthritis is an autoimmune disease that causes inflammation in joint tissue, and is characterized by concomitant destruction of bone and cartilage. Cytokines play a major role in pathogenesis of rheumatoid arthritis. Dysregulated expression of TNF-a in experimental animals has been shown to cause destructive arthritis; also development of arthritis is markedly suppressed in interleukin-1h deficient collagen induced arthritis (CIA). Interleukin-6 gene disrupted mice were found resistant to antigen induced arthritis. These studies indicate the role of inflammatory cytokines like TNF-a, IL-1h and IL-6 in arthritis and are therefore therapeutically potential targets. Recently, evidences prove a possible role of highly reactive products of oxygen termed as free radicals in the pathogenesis of rheumatoid arthritis because oxidative stress exacerbates inflammation and worsens joint tissue. In view of the above considerations we investigated the pro-inflammatory and anti-inflammatory cytokine profiles and the levels of lipid peroxides and anti-oxidants enzymes superoxide dismutase, catalase and glutathione peroxidase in aged arthritic Swiss albino mice joint. Moreover, we also studied the effect of extracts of Nyctanthes arbor tristis L. (Oleaceae), a plant widely used in the traditional medicinal systems of India and reported to possess hepatoprotective, antileishmanial, antiviral and antifungal

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activities. Pro-inflammatory cytokines like TNF-a, IL-1h and IL-6 and lipid peroxide levels were found elevated while the activity of anti-oxidant enzymes was found depleted in arthritic mice. Furthermore, in Nyctanthes treated arthritic mice we observed significant protective changes in the cytokine and lipid peroxide profiles and anti-oxidant enzyme activities. On the basis of the results of our study we conclude that the modulation of cytokines by plant extract along with protecting the oxidantanti-oxidant balance will find general acceptance worldwide and will open up a new vistas in devising better treatment modalities for rheumatoid arthritis.

P8-95 NITRIC OXIDE METABOLISM IN MUSCLE MITOCHONDRIA DURING ENDOTOXEMIA S. Alvarez1 and A. Boveris1 1 Laboratory of Free Radical Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

Endotoxemia and septic shock occur with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle appears as one of the main target organs in septic shock, showing an increased nitric oxide (NO) production, an early oxidative stress, and contractile failure. Kinetic aspects of mtNOS were studied. The KM for O2 and KM for L-arg were calculated for diaphragm (4.6 and 37 AM) and heart (3.3 and 36 AM). Optimum pH for assessing its activity was 6.5 for diaphragm and 7.0 for heart. Diaphragm and heart submitochondrial membranes isolated from endotoxemic rats showed an increased mtNOS activity in nmol NO/min. mg prot.; diaphragm: control 0.77 F 0.08 and treated 1.71 F 0.15; heart: control 0.69 F 0.05 and treated 0.92 F 0.09. H2O2 and O2 production showed 1.5 – 1.8 increased rates, respectively. Catalase activity did not show significant changes, although Mn – SOD activity showed a 2 fold increase in mitochondria from treated animals. One of the current hypothesis for the molecular mechanisms of septic shock is that the enhanced NO production by mitochondrial nitric oxide synthase (mtNOS) leads to excessive peroxynitrite (ONOO ) production and protein nitration in the mitochondrial matrix, to mitochondrial dysfunction and to contractile failure.

P8-96 EXTRACELLULAR ATP METABOLISM AND OXIDATIVE STRESS IN STIMULATED PERITONEAL MACROPHAGES L. F. De Souza,1 D. P. Gelain,1 F. Dal Pizzol,1and E. A. Bernard1 1 Departamento de Bioquı´mica/ICBS/Universidad Federal de Rio Grande do Sul e Departamento de Medicina Universidad do Extremo Sul Catarinense, Criciuma, Brasil

Macrophage generated reactive oxygen species are implicated in both, tissue destruction, via lipid peroxidation, and the activation of these cells. Many works have shown

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that purinergic receptors are involved in the cellular response to oxidative stress, and our previous work showed that extracellular inosine is involved on Sertoli cells (’unprofessional’ macrophage) response to oxidant treatment. In the current study we examined the extracellular ATP metabolism and some parameters involved in oxidative response of peritoneal macrophages (PM), submitted to both in vivo and ex vivo inflammation models. Rat PM were treated 24 hours with lipopolyssacharide (LPS: 10Ag/ml), with or without inosine/adenosine. In vivo model: sepsis was induced by cecal ligadure and puncture, and rats treated with either basic support (volume restoration and antibiotics: BS) or BS/antioxidants (trolox and N-acetylcysteine). Extracellular ATP degradation was increased in LPS-treated PM, as well as lipoperoxidation, measured by thiobarbituric acid reactive substances (TBARS). Extracellular inosine inhibited LPS-stimulated TBARS. In PM of septic animals, no alterations in extracellular ATP degradation and TBARS were observed, however, carbonylation of proteins, catalase and SOD activity were increased, being prevented by BS/antioxidant treatment. The ex vivo response to LPS by PM is different from that of septic animals. The reversion by BS/antioxidant treatment on the oxidative parameters modified by sepsis, could be involved in the improvement of the response in PM.

P8-97 EFFECT OF TNF-A ON EXTRACELLULAR PURINES AND THEIR RELATION WITH LIPOPEROXIDATION AND NO PRODUCTION ON A REPRESENTATIVE CELL LINE OF HEPATIC STELLATE CELLS. F. R. Jardim,1 E. L. M. Guimaraes,1 L. F. de Souza,1 R. Borojevic,2 F. C. R. Guma,1 and E. A. Bernard1 1 Depto de Bioquı´mica, ICBS, UFRGS, 2Depto Histologia e Embriologia, ICB, UFRJ, Brazil

Hepatic stellate cells (HSC) are major source of extracellular matrix, which during fibrosis, undergo a process of activation, characterized by increased proliferation and collagen synthesis. TNF-a plays an important role during HSC activation, including production of nitric oxide (NO). Extracellular nucleotides participate in many processes on HSC, and adenosine has been reported to act as a hepatic protector to injuries known to generate oxidative stress, as CCl4. The aim of this study is to analyze the effect of TNF-a on extracellular purines, NO production and lipoperoxidation on the HSC representative cell line GRX. These cell line express the myofibroblast phenotype under standard culture conditions, and can be induced in vitro to display a more activated state when treated wit TNF-a. The results show an increase on extracellular ATP and adenosine in cells treated with TNF-a for 5 min. Treatment of GRX with TNF-alpha, during 24 hs, increased NO production and lipoperoxidation. Addition of ATP augments NO production, but diminishes lipoperoxidation, and also lower TNF-a effect on NO. The present data

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indicate that both TNF-a and ATP increases the production of NO and that this cytokine modulates the metabolism of extracellular nucleotides, suggesting a possible role for TNF-a as inductor of oxidative stress and ATP as a protector.

P8-98 IS SOD/CAT RELATION ASSOCIATED WITH OXIDATIVE DAMAGE DURING SEPSIS DEVELOPMENT? M. E. Andrades, C. Ritter, F. Bonatto, J. C. F. Moreira, and F. Dal-Pizzol 1 Centro de Estudos em Estresse Oxidativo, Depto. Bioquı´mica, UFRGS, 2 Laborato´rio de Fisiopatologia Experimental – Dpto. Medicina – UNESC, Brazil

The imbalance between free radicals generation and its degradation is thought to participate in sepsis generation and progression. To analyze the importance of the relationship between superoxide dismutase (SOD) and catalase (CAT) in oxidative damage in sepsis, we performed a cecal ligation and puncture (CLP) in male Wistar rats with two needle size—18 gauge to mild and 14 gauge to severe sepsis. Analysis of SOD and CAT activities were performed in lung, diaphragm, kidney, liver, and heart by measuring inhibition of epinephrine oxidation (480nm) and H 2 O 2 decay (240nm), respectively. As an index of oxidative damage, we measured TBARS (532nm). In mild sepsis the increase in SOD/CAT relation did not cause increase in oxidative damage. In contrast, severe sepsis present a strong correlation between SOD/CAT increase and oxidative damage in some organs (lung r2 = 0,91 > diaphragm r2 = 0,82 > kidney r2 = 0,78). Basic support plus antioxidants (N-acetylcysteine plus deferoxamine) were effective in reverse this correlation. Our results suggest that in severe sepsis, the increase in SOD activity without a compensatory increase in CAT activity lead a overload of H2O2 and hydroxyl generation by Fenton reaction. Furthermore, SOD/CAT analysis is more accurate to indicate the sepsis severity than each enzyme alone.

P8-99 PROTECTIVE EFFECTS OF N-ACETYLCYSTEINE PLUS DEFEROXAMINE FROM SEPSIS-INDUCED MITOCHONDRIAL DAMAGE IN RAT LIVER C. Ritter,1 M. E. Andrades,1 F. Bonatto,1 L. F. Souza,1 D. Gelain,1 A. A. Cunha,1 J. C. F. Moreira,1 and F. Dal-Pizzol1 1 Centro de Estudos em Estresse Oxidativo, Departamento de Bioquı´mica, ICBS, UFRGS, Laborato´rio de Fisiopatologia Experimental, Departamento de Medicina, UNESC, Brazil

It is well defined the occurrence of mitochondrial damage during sepsis development. This seems to be of major importance during organ damage and progression to death. We had previously demonstrated

that administration of N-acetylcysteine plus deferoxamine improves survival in a rat model of severe sepsis. Thus the aim of the present study is determine the effects of N-acetylcysteine plus deferoxamine on mitochondrial function during sepsis development. Sepsis was induced by cecal ligature and puncture (CLP) in male Wistar rats. Rats subjected to CLP were treated with either N-acetylcysteine plus deferoxamine or vehicle with ‘‘basic support’’ (fluid resuscitation plus antibiotics). After 24 hours of induction liver mitochondria was isolated for analyses of mitochondrial superoxide production, mitochondrial lipid peroxidation, mitochondrial swelling and mitochondrial cytochrome c content. Sepsis increases mitochondrial superoxide production and lipid peroxidation. In addition, induces mitochondrial swelling and reduction of mitochondrial cytochrome c content, which is reversed by the addition of cyclosporin A, suggesting the formation of mitochondria permeability transition pore. N-acetylcysteine plus deferoxamine reduces superoxide production, lipid peroxidation, mitochondrial swelling and cytochrome c release. These results suggest that the efficacy of Nacetylcysteine plus deferoxamine in the treatment of sepsis is, in part, secondary to the reduction of oxidative effects on mitochondria.

P8-100 ROLE OF OXIDATIVE STRESS IN EPILEPSY J. Bozorgmehr,1 Y. Saffari,1 and S. M. Hossein Sadrzadeh1 1

University of Washington, USA

Epilepsy is a disorder with still unknown biochemical etiology. Iron and iron compounds such as hemoglobin (Hb), via oxidative damage may be involved in etiology of epilepsy. Haptoglobin (Hp) is a plasma glycoprotein that strongly binds Hb. Hp has three phenotypes (Hp 11, Hp 2-2 and Hp 2-1). Recently, we showed Hp 2-2 was overrepresented in epilepsy. This study investigates the association of Hp phenotypes and oxidative stress in epilepsy. Blood was collected from 12 idiopathic epileptic patients with seizure and 26 healthy controls. Oxidative status was assessed by measuring plasma thiobarbituric acid reactive substances, TBARS, beta-carotene and total antioxidant capacity (TAOC). Results show that mean TAOC was significantly lower in our patients than controls (1.303 F 0.04 vs. 1.508 F 0.05, mmol/l, mean F SEM, patients vs. controls, respectively) (p = 0.0019). Patients with Hp 2-2 had the lowest (1.234 F 0.04 mmol/l) and those with Hp 1-1 had the highest TAOC (1.44 F 0.01 mmol/l). Also, mean TBARS levels were higher in patients than in controls (0.84 F 0.07 vs. 0.64 F 0.07, Amol TBARS/mg protein, mean F SEM, patients vs controls, respectively) (p < 0.05). Mean TBARS levels were highest in our patients with Hp 2-2 (0.96 F 0.08 Amol TBARS/mg protein) and lowest in Hp 1-1 (0.8 F 0.03 Amol TBARS/mg protein). Beta-carotene levels in patients were significantly lower than in controls (0.36 F 0.03 vs. 0.53 F 0.05 Ag/dl, mean F SEM, patients vs. controls, respectively (p < 0.05).

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Mean beta-carotene levels were lowest in our patients with Hp 2-2 (0.29 F 0.04 Ag/dl) and highest in Hp 1-1(0.47 F 0.05 Ag/dl). In conclusion, epileptic patients with Hp 2-2 have elevated oxidative stress and low antioxidant defense.

P8-101 PROLIFERATION AND WOUND HEALING OF VASCULAR CELLS TRIGGER THE GENERATION OF EXTRACELLULAR REACTIVE OXYGEN SPECIES AND LDL OXIDATION C. Duval,1 A. V. Cantero,1 N. Auge,1 L. Mabile,1 J. C. Thiers,1 A. Negre-Salvayre,1 and R. Salvayre1 1

Inserm U-466 and Department of Biochemistry, CHU Rangueil, Toulouse, France

Cell proliferation of vascular cells is a key feature in vascular biology, wound healing, and pathophysiological processes such as atherosclerosis and restenosis. In atherosclerotic intima, cell proliferation colocalizes with oxidized LDL that indicate a local oxidative stress. This study aims to investigate whether cell proliferation is causally related with extracellular ROS generation and subsequent LDL oxidation. Sparse proliferating endothelial and smooth muscle cells generate higher levels of extracellular ROS (superoxide and H2O2) and LDL oxidation than confluent contact-inhibited cells. During wound healing of confluent cell layer, cell proliferation associated with healing also induced enhanced extracellular ROS generation and LDL oxidation. Proliferation-associated extracellular ROS generation is mediated through mitogenic signaling pathways, involving ERK1/2 and PKC, but is independent of de novo DNA synthesis, gene expression and protein synthesis. Data obtained with inhibitors of oxidases suggest that proliferation-associated extracellular ROS are not generated by a single ROS-generating system and are not essential for cell proliferation. In conclusion, our data show that proliferating vascular cells (in sparse culture or during wound healing) generate high levels of extracellular ROS and LDL oxidation through regulation of ROS-generating systems by mitogenic signaling. This constitutes a link between proliferative events and oxidative stress/LDL oxidation in atherosclerotic lesions and restenosis.

P8-102 LUNG OXIDATIVE STRESS BY EXPOSURE TO AIR POLLUTION PARTICLES P. Evelson,1 S. Martin,2 P. Mandalunis,2 L. Dawidowski,3 and D. R. Tasat 2,3 1 Facultad de Farmacia y Bioquı´mica, UBA, 2Escuela de Ciencia y Te´cnica. UNSAM, 3Unidad de Actividad Quı´mica, CNEA. Buenos Aires, Argentina

There is an increasing interest in the study of the health effects of particulate matter (PM), a constituent of urban air pollution. Several studies suggest that oxidative stress may be involved in the mechanism of

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disease initiation and progression following exposure to these particles. In the present study we compared the occurrence of oxidative stress in the lung by exposure to Residual oil fly ash (ROFA) and Buenos Aires (BA) air pollution particles. Their composition and morphology was also evaluated. The occurrence of oxidative stress was assayed using tert-butyl hydroperoxide-initiated chemiluminescence (CL), total antioxidant potential (TRAP) and thiobarbituric reactive substances (TBARS) as end points. PM analysis showed that no transition metals were present in BA whereas in ROFA a peak for vanadium (V) was detected. BA were spherical and smaller than ROFA. Regarding the oxidative stress markers, ROFA and BA increased CL by 60 % (control value: 3500 F 300 pmol/mg prot) and TBARS by 55% (control value: 12.5 F 1.3 pmol/ mg prot). TRAP values showed no changes in both groups when compared to the control animals (2.46 F 0.46 nmol Trolox /mg prot). In conclusion, the present study showed that despite their different composition, ROFA and BA induced the generation of oxidant species, suggesting the occurrence of oxidative stress.

P8-103 OXIDATIVE STRESS IN ENDOTOXEMIC RATS ASSESSED BY THE RATIO ASCORBYL RADICAL/ ASCORBATE CONTENT IN PLASMA M. Galleano,1 N. Cardoso,1 L. Aimo,1 and S. Puntarulo1 1

Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

The aim of this work was to evaluate oxidative stress in a model of endotoxemia employing the ratio ascorbyl S radical/ascorbate content (A /AH ) in rat plasma. Female Sprague-Dawley rats were administered with lipopolysaccharide from Escherichia coli (serotype 0127:B8) (ip 4 mg/kg) (LPS) and samples were obtained after 0 h, 1.5 h, 3 h and 6 h of the treatment. Nitric oxide production was followed by measuring NO-Hb in blood by EPR and showed a significant increase starting 3 h after LPS administration. Carbonyl groups content in plasma proteins were increased significantly over the same period (+28%), while plasma TBARS and the content of thiol groups were unaffected by the treatment. Total iron content in plasma decreased significantly after 6 h of LPS administration (30 F 5 AM and 13 F 3 AM for controls and S 6 h-treated rats, respectively). Plasma A was measured by EPR at room temperature and plasma AH was S detected by reverse phase HPLC. The ratio A /AH was significantly higher in endotoxemic rats ([6.0 F 0.5]10 4 and [10.4 F 0.9]10 4 for control and 6 h-treated rats S respectively). These data suggest that the ratio A /AH is an early and sensitive indicator of oxidative stress in this experimental model, however modifications in the content of catalytically active iron in plasma should be further study to asses iron role under these conditions. Supported by University of Buenos Aires, ANPYCT and CONICET.

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P8-104 SYSTEMIC AND PULMONARY OXIDATIVE STRESS BIOMARKERS IN SEPSIS AND IN MECHANICAL-VENTILATION INDUCED LUNG INJURY J. Gorrasi,1,2 C. Batthyany,2 H. Botti,2 H. Correa,1 and R. Radi 2 1 Centro de Tratamiento Intensivo. Hospital de Clı´nicas, 2Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Repu´blica, Montevideo, Uruguay

Systemic and pulmonary oxidative stress in patients was assessed during sepsis and during mechanical ventilation (MV) without severe lung injury. We studied plasma and broncho alveolar lavage fluid (BALF) samples from 15 septic and 10 MV patients. Ten healthy control subjects were included. Severity of illness was categorized by APACHE II score and lung dysfunction by lung injury score (LIS). The biomarkers studied were a-tocopherol (a-TH) and malondialdehyde (MDA) by RP-HPLC and 3-nitrotyrosine by ELISA. At study admission, significant differences in a-TH concentrations between MV and septic groups with HCS was observed (7.5 F 6.5 and 2.7 F 3.3 versus 40 F 8, respectively; p < 0.05). There was no significant difference between sepsis and MV group. At seven days of ICU evolution a-TH decreased further to 0.2 F 0.5 and 4.6 F 4.8 in the MV and septic groups respectively (p < 0.05). In the septic group at day one plasma a-TH concentrations were inversely correlated with APACHE II (r = 0.82, p = 0.001, n = 12) and LIS (r = 0.34, p = 0.018, n = 16) respectively. Thus, sepsis and MV patients without severe lung injury have plasma and pulmonary oxidative stress showed by low a-TH concentrations, in addition to changes observed in MDA and 3nitrotyrosine as well. We suggest that a-TH could be a relevant biomarker to follow up critically ill patients.

P8-105 CERAMIDE-MEDIATED OXIDATIVE STRESS ON A REPRESENTATIVE CELL LINE OF HEPATIC STELLATE CELL E. L. M. Guimaraes,1 R. O. Birnfeld,1 R. Borojevic,2 and F. C. R. Guma1 Universidade Federal do Rio Grande do Sul, (1Depto de Bioquı´mica, ICBS, UFRGS, 2Depto Histologia e Embriologia, ICB, UFRJ), Brazil

Increased production of free radicals has been implicated in the pathogenesis of a number of conditions that lead to hepatic damage. Activation of hepatic stellate cells results in the differentiation of the quiescent phenotype to one characterized by increased collagen production known, as myofibroblast. Reactive oxygen species have been shown to increase collagen production and initiate the activation of hepatic stellate cell. Ceramide has been proposed as a coordinator of stress response. The role of this sphingolipid in the induction of oxidative stress has been elucidated in many cell types, however little is known about the effects of ceramide on the hepatic stellate cells. The aim of this study is to determine the effects of ceramide on the generation of oxidative stress

in the experimental model of mouse hepatic stellate cells, the lineage GRX. We used as parameter of oxidative stress the generation of lipid peroxidation measured by the method of Thiobarbituric acid reactive species and the activity of catalase. The treatment with ceramide-C6 during 24 hours increased lipid peroxidation and diminished the activity of catalase. This results suggest a possible involvement of ceramide during induction of oxidative stress on the hepatic stellate cells.(CAPES, PIBIC-CNPq/UFRGS, PROPESQ/UFRGS)

P8-106 LANSOPRAZOLE, A PROTON PUMP INHIBITOR, REDUCES THE SEVERITY OF INDOMETHACIN-INDUCED RAT ENTERITIS H. Ichikawa,1 T. Takagi,1 Y. Isozaki,1 K. Katada,1 M. Kuroda,1 M. Shimozawa,1 N. Nakabe,1 Y. Naito,1 N. Yoshida,1 and T. Yoshikawa1 1 Kyoto Prefectural University of Medicine, Japan

Lansoprazole is an antiulcer benzimidazole proton pump inhibitor that acts on gastric (H+/K+) ATPase of parietal cells. Recently, the anti-inflammatory action of lansoprazole has been noted, and research has proceeded from a variety of angles. We have evaluated the biological effects of lansoprazole in the setting of experimental rat nonsteroidal antiinflammatory drug-induced enteritis. The animals were given indomethacin subcutaneously and the intestinal mucosa was examined 24 h later. Lansoprazole was given subcutaneously just after following indomethacin injection. Single administration of indomethacin at 10 mg/kg provoked severe hemorrhagic lesions in the small intestine, mostly the jejunum and ileum. The levels of thiobarbituric acid-reactive substances (TBA-RS), the myeloperoxidase activity (MPO) and the content of cytokine-induced neutrophil chemoattractant-1 (CINC-1) in the intestinal mucosa significantly increased in indomethacin-treated groups compared with the sham-operated groups. The development of intestinal lesions in response to indomethacin was dosedependently prevented by lansoprazole at a dose of 5mg/kg s.c. together with significant suppression of the level of TBARS, MPO activities and the content of CINC-1 in the small bowel. These results suggest that lansoprazole administered exogenously prevented the small intestine against indomethacin-induced damage, the action being dependent on its anti-inflammatory and anti-oxidative responses.

P8-107 REBAMIPIDE PROTECTS AGAINST ACUTE REFLUX ESOPHAGITIS IN RATS Y. Isozaki,1 N. Yoshida,1 K. Katada,1 M. Kuroda,1 T. Takagi,1 S. Kokura,1 H. Ichikawa,1 Y. Naito,1 and T. Yoshikawa1 1

Kyoto Prefectural University of Medicine, Japan

Rebamipide has been widely used as an anti-ulcer agent, which has anti-neutrophil, anti-cytokine and anti-oxidative effects. The aim of the present study

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was to investigate the effect of rebamipide on acute reflux esophagitis model in rats. Esophagitis was induced in male Wistar rats weighed 200g by the ligation both at limiting ridge and at lower portion of duodenum (below papilla duodeni major) with silk thread. Vehicle or rebamipide (30 mg/kg) were given intraduodenally from the anal side of duodenal ligation. Esophagus was taken out 12 hours after the induction of esophagitis and lesion index (LI) was estimated macroscopically. Thiobarbituric acid-reactive substances (TBA-RS) and tissue-associated myeloperoxidase (MPO) activity were measured in esophageal mucosa as indices of lipid peroxidation and neutrophil infiltration. The protein concentration and mRNA expression of TNF-a and CINC-1 were determined by ELISA and RT-PCR. The LI was significantly decreased by the pretreatment with rebamipide. TBA-RS, MPO activity and the contents of both mucosal TNF-a and CINC-1 were also significantly increased in esophagitis group. These increases were significantly reduced in the pretreatment with rebamipide. These results indicate that rebamipide protects against the occurrence of esophagitis induced by reflux of gastro-duodenal contents via inhibiting mucosal inflammatory mediators.

P8-108 INHIBITION OF HUMAN NEUTROPHIL RESPIRATORY BURST BY NIMESULIDE METABOLITES IS HIGHER THAN THAT OF THE PARENT DRUG A. Kanashiro,1 F. E. Mingatto,2 F. M. Kabeya,1 D. J. Dorta,1 A. C. M. Polizello,1 A. C. Santos,1 C. Curti,1 and Y. Lucisano-Valim1 1 Faculdade de Ciencias Farmaceuticas de Ribeirao Preto – USP. Av. Cafe´ s/n., Ribeirao Preto-SP, Brasil, 14040-903, 2Instituto de Ciencias da Saude – UNIP, Ribeirao Preto-SP, Brasil

Nimesulide (Nim; N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide) is a nonsteroidal anti-inflammatory drug widely used in clinical practice. Some of its adverse effects have been attributed to the hepatotoxicity of Nim itself and its metabolites. However, the nitro-reduced Nim metabolite (Nim-H) was recently found to be non-toxic to hepatocytes (Mingatto et al., 2002). One of the mechanisms underlying the anti-inflammatory activity of Nim is the inhibition of neutrophil-related functions, including suppression of reactive oxygen species generation. In this study, we found that Nim and its Nim-H and 4-OH (Nim-OH) metabolites inhibited the human neutrophil oxidative burst, triggered by opsonized zymosan and measured by lucigenin- and luminol-enhanced chemiluminescence assays, in a concentration-dependent manner, at the tested range of concentrations (10 to 250 Amol/L). Interestingly, Nim-H and Nim-OH were more potent than the parent compound Nim. Moreover, at the assessed conditions, all tested compounds had no toxic effect on neutrophils (evaluated by lactate dehydrogenase release) and had no scavenger activity (as evaluated by DPPH test and H2O2-horseradish peroxidase-luminol system). Taken together, these results suggest that clinical use of the Nim metabolites should be considered as an

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alternative to the parent compound for treatment of inflammatory diseases.

P8-109 THE ROLE OF FREE RADICALS IN THE DEVELOPMENT OF ACUTE PANCREATITIS T. Manabe,1 U. Okada,1 A. Nonaka,1 and K. Tamura1 1

Dept of Gastroenterological Surgery, Nagoya City University, Japan

The role of free radicals in the development of acute pancreatitis was evaluated by measuring the activity of the endogenous scavengers, superoxide dismutase (SOD), catalase(CAT) and glutathione peroxide(GSHpx) as indicators of the defense system, and the level of lipid peroxide(LPO) in the pancreas as indicator of the offense system using caerulein-induced pancreatitis in mice. The direct effect of active oxygen species on the pancreas was also studied in rat pancreas. Acute pancreatitis was induced by 5 hourly intraperitoneal administration of cerulean (50 Ag/kg) and confirmed by the serum amylase levels and microscopical features as edematous acute pancreatitis in mice. The SOD level was decreased significantly to 60% of the basal level at 12 h (p < 0.01). The CAT level also decreased significantly to 58% of the basal level (p < 0.01). GSHpx increased to 150% of the basal level (p < 0.01). The LPO level increased to 286% of the basal level (p < 0.01). After a continuous infusion of xanthine / xanthine oxidase into the celiac artery supplying the pancreas in rats, the amylase and lipase in the peritoneal fluid rose significantly and extensive edema with hemorrhage developed in the pancreas. The subsequent infusion of SOD completely suppressed edema and hemorrhage and the amylase and lipase levels. After injection of hydrogen peroxide marked hemorrhage and edema appeared in the pancreas with the higher levels of amylase and lipase. These abnormalities were significantly suppressed by the infusion of the catalase. These results indicate that active oxygen species have a direct effect on the pancreas and tissue imbalances of the offense system and of the defense system may play an important role in the development of acute pancreatitis induced by oxygen derived free radicals.

P8-110 INCREASED NITROTYROSINE AND iNOS EXPRESSION DURING FETAL HYPOXIA A. Martı´nez,1 R. Castellanos,1 L. Briozzo,1 M. Gonza´lez,1 C. Batthya´ny,1 P. Cassina,1 J. Alonso,1 and H. Rubbo1 1 Department of Obstetrics and Gynecology, Pereira Rossell Hospital, Departments of Biochemistry and Histology and Center for Free Radical and Biomedical Research, School of Medicine, University of the Republic, Montevideo, Uruguay

Intrapartum asphyxia is one of the main causes of perinatal morbimortality. Fetal hypoxia is related with oxidative stress, which may determine severe tissue

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damage to fetuses and newborns. In addition, fetal hypoxia can determine umbilical cord acidosis at birth (pH < 7.2 in umbilical artery). It has been shown that hypoxia increases expression of all types of nitric oxide synthases (NOS) in different tissues suggesting that nitric oxide (NO) could be involved in perinatal hypoxiaassociated damage. To further understand the involvement of NO and NO-derived oxidants in tissue damage during fetal hypoxia, we compared the expression of inducible nitric oxide synthase (iNOS) and nitrotyrosine in the umbilical cord of newborns with and without acidosis at birth. An increase in iNOS immunoreactivity was detected at the muscular level both in umbilical arteries and veins in cases which pH was < 7.0 (severe acidosis). This immunoreactivity was present at significantly lower extents at the endothelial level and in control umbilical samples nitrotyrosine immunoreactivity, a footprint of nitration processes, exhibited a similar pattern to that described for iNOS. Our results suggest that NO is over produced in the umbilical cord vessels during severe cord acidosis, and that NO-derived oxidants such as peroxynitrite may play a critical role in this process.

P8-111 CARDIAC AND HEPATIC CYTOCHROME P450 DECLINE FOLLOWING CONTINUOUS TREATMENT OF ORGANIC NITRATE—IMPACT OF CLINICAL PHARMACOTHERAPY Y. Minamiyama, S. Takemura, K. Yamasaki, S. Hai, S. Kodai, K. Hirohashi, S. Suehiro, and S. Okada Department of Food and Health Science, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan, Departments of Hepato-Biliary-Pancreastic Surgery and Cardiovascular Surgery, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan

We have reported P450-catalyzed conversion of organic nitrate to nitric oxide (NO) by purified P450 isoforms, and had some insight into the relationship between P450 expression and nitrate tolerance following continuous infusion of organic nitrates in rats. The hypotensive effect of a nitroglycerin (NTG) bolus injection was abolished in rats that had been provided a continuous 48 hr infusion of NTG or 96 hr infusion of isosorbide dinitrate (ISDN). A gradual but marked decrease in plasma and urinary nitrate levels were observed by continuous infusion of either NTG or ISDN. Nitrate tolerance was reversible; the decline in the hypotensive effect and P450 levels of the heart observed after continuous infusion was followed by restoration to control levels two days after cessation of the infusion. Similarly, the hypotensive action disappeared in P450-depleted, and -inhibited rats. Hepatic P450 was also drastically decreased after 48 hr or 72 hr of continuous NTG or ISDN infusion, but it recovered 48 hr after cessation of the drug administration. HO-1 was induced within 24 hr of continuous NTG infusion, but it returned to normal levels 48 hr after cessation of the NTG. The administration

of sodium nitroprusside, an agent to which the animals showed no tolerance, did not induce HO-1 or P450 depletion. These results suggest that P450-dependent bioactivation in the heart and drug metabolism in the liver may be drastically affected after continuous organic nitrate administration. Interventions that maintain or increase P450 activity may be a strategy to provide relief from patients with nitrate tolerance.

P8-112 SINGLET OXYGEN GENERATION IN THE REACTION OF HYPOCLORITE WITH LIPID HYDROPEROXIDES S. Miyamoto,1 G. R. Martinez,1 M. H. G. Medeiros,1 and P. Di Mascio1 1 Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sao Paulo, CP 26077 CEP 05513-970, Sao Paulo, SP, Brazil

Hypochlorite (HOCl) is known to react with hydrogen peroxide generating singlet oxygen (1O2). In this study we report the generation of 1O2 in the reaction of HOCl with lipid hydroperoxides at physiological pH. Characteristic chemiluminescence emission at 1270 nm is observed upon injection of HOCl into linoleic acid hydroperoxide or egg yolk phosphatidylcholine hydroperoxide in solution or in liposomal membranes. The generation of 1O2 was confirmed by: (i) direct measurement of the monomol light emission of 1O2 at 1270nm; (ii) spectral characterization of the light emitted in the near-infrared region using a photomultiplier coupled to a monochromator; and (ii) the enhancing effect of deuterium oxide and the quenching effect of azide. A mechanistic aspect of the reaction was also studied using 18 O-labeled linoleic acid hydroperoxide (LA18O18OH) and HPLC coupled to mass spectrometry. Using the water-soluble anthracene-9,10-diyldiethyl disulfate as a specific 1O2 trapping agent, we have detected the formation of 18O-labeled 1O2. These novel observations open new insights into the mechanism of 1O2 production in biological system where HOCl and lipid hydroperoxides can be generated. Financial support: FAPESP, CNPq and PRONEX.

P8-113 ANTIOXIDANTS PREVENT RENAL GLYCOXIDATIVE DAMAGE IN DIABETIC RATS S. Patriarca,1 S. Menini,2 A. L. Furfaro,1 E. Balbis,1 P. Odetti,3 C. Pesce,2 D. Cottalasso,1 U. M. Marinari,1 M. A. Pronzato,1 and N. Traverso1 1 DIMES (Section of General Pathology), 3DIMI, 2DISTBIMO (Centre of Anatomical Pathology), University of Genova, Italy

Glycoxidation is believed the keystone in the development of long-term diabetic complications (1). In this study the effect of some antioxidants (oxerutin, N-acetylcysteine, taurine, and N-acetylcysteine+taurine)

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on kidney glycoxidative damage has been evaluated by immuno-histochemistry in streptozotocined rats after 6 months of diabetes. Diabetic rats showed marked positivity in the glomeruli for carboxymethyllysine and protein adducts with malondialdehyde and 4-hydroxynonenal, while the samples from Control rats were negative. All antioxidants except taurine were able to protect the glomeruli from the accumulation of carboxymethyl-lysine and aldehydic adducts; the failure of taurine may be due to residual oxidative properties of its derivatives. These data are consistent with our previous morphometric study (2), showing that all antioxidants except taurine provided protection from diabetes-induced glomerular enlargement, increased apoptotic rate and decreased cell density. This attests to a role of glycoxidative damage in the diabetes-dependent morphological damage of the kidney, and indicates that specific antioxidants can prevent or attenuate the diabetic nephropathy. Grants from Ateneo ex60%, Cofin 2001#2001064293_005 (Coord. Naz. Prof E. Bergamini) and FIRB 2001 (Coord. Naz. Prof G. Poli). (1) Horie K et al. J Clin Invest. 1997; 100: 2995 – 3004. (2) Odetti P et al. Diabetes 2003; 52: 499 – 505.

P8-114 ESR AND SPIN TRAPPING STUDIES OF NITROFURAN DERIVATIVES AS POTENTIAL ANTITRYPANOSOMA DRUGS C. Rigol,1 C. Olea-Azar,1 F. Mendiza´bal,2 M. Gonza´lez,3 H. Cerecetto,3 A. Morello,4 and J. D. Maya4 1

Department of Inorganic and Analytical Chemistry. Faculty of Chemical and Pharmaceutical Sciences, University of Chile, P.O. Box 233, Santiago 1, Chile, 2Department of Chemistry, Faculty of Sciences, University of Chile, P.O. Box 653, Santiago, Chile, 3 Department of Organic Chemistry, Faculty of Chemistry, University of the Republic, Montevideo, Uruguay, 4Department of Molecular and Clinical Pharmacology. Faculty of Medicine. University of Chile, P.O. Box 70000, Santiago 7, Chile

The Electron Spin Resonance (ESR) spectra of radicals generated by electrolytic and Trypanosoma cruzi reduction from eight tiosemicarbazone nitrofurans analogues of the antiprotozoal drug nifurtimox were analyzed. The electrochemistry of these compounds was first studied using Cyclic Voltammetry. DFT calculations using 6-31G* and IGLO-III basis sets were performed to obtain the optimized geometries and spin distribution respectively in order to assign the experimental coupling constants obtained with a simulation program. Chemically generated free radical spectra were also recorded in order to calculate the decay rate constants and half lives of these radicals. DMPO Spin Trapping was used to investigate free radicals generation in the trypanosome microsomal system (T. Cruzi). The ESR spectra and signal intensities were consistent with the trapping of either OH or ArNO2 , showing the generation of Reactive Oxygen Species (ROS) by the interaction between the free radical species with the O2. The antiparasitic activities of the nitrofurans studied were better than nifurtimox (drugs

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used clinically). These values were also compared with the radical half lives and the Spin Trapping results. Finally, biological studies and the ESR experiment with the T. cruzi system indicate that nitrofuranes family. produced the radical species derivative and the antiparasitic activities could be explain by production of oxidative stress by the redox recycling of the nitrofurane family.

P8-115 OXIDATIVE STRESS AND CARDIAC DYSFUNCTION IN RATS EXPOSED TO PARTICULATE MATTER FOR SHORT-TERM. C. R. Rhoden,1 G. Wellenius,1 E. Ghelfi,1 and B. Gonzalez-Flecha1 1 Physiology Program, Department of Environmental Health, School of Public Health, Harvard University, Boston, MA, USA

Epidemiological studies show an association between increases in the levels of ambient particulate matter (PM) and increased mortality and hospital admissions. Increases in the autonomic influence on the heart as well as increased sympathetic tone have been shown in animals exposed to concentrated ambient particles (CAPs). The aim of this study was to determine the role of reactive oxygen species (ROS) in the development of cardiac malfunction and to identify neural mediators of cardiac oxidative stress and cardiac dysfunction. Adult Sprague – Dawley rats received 5mg/Kg of atenolol (AT, i.v.), 0.30 mg/kg of glycopyrrolate (GLY, i.v.) or saline immediately before intra-tracheal instillation of 750 Ag urban ambient particles (UAP-CSRM 1649). To determine the role of ROS in the development of cardiac malfunction after CAPs exposure we used pre-administration of N-acetylcysteine (NAC). Thirty min after UAP instillation the animals were assayed for cardiac level of oxidants by in situ heart chemiluminescence(H-CL). Intratracheal instillation of UAP led to a 3.8 fold increase in H-CL (sham = 10+/ 1 cps/cm2; UAP = 38 cps/ cm2; p < 0.01). Atenolol, glycopyrrolate and NAC pretreatment effectively prevented cardiac oxidative stress (AT = 11+/ 1cps/cm2, GLYC = 10+/ 0.5 cps/cm2, NAC = 17+/ 1 cps/cm2, p < 0.01). Similar results were found with CAPs in aerosol (700+/ 200 mg/m3, 5 h): CAPs = 20+/ 4 cps/cm2, sham = 12+/ 1cps/cm2. Pre-treatment with AT, GLY or NAC effectively prevented the increases of CAPs-induced CL. To investigate cardiac function, heart rate (HR) and heart rate variability (SDNN) was monitored after UAP instillation. UAP instillation increased HR values immediately after exposure (HRUAP = 372+/ 13 bpm, HR sham = 350+/ 5 bpm) and over the next 30 min. SDNN values were increase after UAP instillation. These data suggest increased sympathetic influence immediately after exposure, with a sympathetic withdrawal and/or a compensatory increase in parasympathetic tone in the period following exposure. Changes in cardiac function occurred simultaneously with increases in oxidant levels and were prevented by pre-treatment with generic antioxidants. In conclusion, PM exposure increases cardiac oxidants via autonomic signals and the resulting oxidative stress is associated with significant

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functional alterations in the heart. Funding: Health Effects Institute NIH (R01 HL68073) U.S. Environmental Protection Agency (R827353), and FFFCMPA-Brazil.

P8-116 URSODEOXYCHOLIC ACID PROTECTS AGAINST BILIARY CIRRHOSIS VIA UP-REGULATION OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE AND PREVENTING MITOCHONDRIAL OXIDATIVE STRESS G. Serviddio,1 F. Pallardo,1 G. Vendemiale,1 G. Poli,1 J. Vin˜a,1 and J. Sastre1 1

University of Foggia and University of Torino, Italy; University of Valencia, Spain

Background/Aim: Cholestatic liver disorders represent a significant clinical problem in children and adults. The underlying cellular mechanisms relating cholestasis to liver injury and fibrosis are not completely understood. Hydrophobic bile acids increase hydroperoxide production in hepatocytes and mitochondria, and hepatic lipid peroxidation during chronic cholestasis occurs. Ursodeoxycholic acid (UDCA) improves liver function and histology, but the mechanism for this beneficial effect is not completely known. Aim of this study was to investigate the in vivo effect of UDCA on mitochondrial GSH status and hepatic GSH metabolism during secondary biliary cirrhosis in rats. Methods: Bile duct ligation was performed in male Wistar rats fed a normal or 0.25% supplemented UDCA diet. Animals were sacrificed after 21 or 28 weeks of duct ligation. GSH, GSSG and ratio and the rate of peroxide production were determined in liver mitochondria. The expression of gamma-GCS and gamma-cystathionase was assessed by real time PCR. Results: Chronic cholestasis altered mitochondria morphology, increased mitochondria peroxide production, and diminished mitochondrial GSH levels via down-regulation of the expression of gamma-GCS and gamma-cystathionase. UDCA reduced peroxide production and increased the rate og GSH synthesis and mitochondrial GSH levels via up-regulation of gamma-GCS expression. Conclusions: This effect may explain the hepatoprotective action of UDCA in vivo.

P8-117 AZELNIDIPINE, A NOVEL Ca2+-CHANNEL BLOCKER, REDUCES ENDOTHELIAL INFLAMMATORY RESPONSE THROUGH THE INHIBITION OF INTRACELLULAR REACTIVE OXYGEN SPECIES PRODUCTION M. Shimozawa,1 Y. Naito,1 H. Manabe,1 K. Uchiyama,1 N. Nakabe,1 M. Kuroda,1 K. Katada,1 N. Yoshida,1 and T. Yoshikawa1 1

Kyoto Prefectural University of Medicine, Japan

Introduction: The endothelial cell surface adhesion molecules is regulated, at least in part, by reactive

oxygen species (ROS) generated locally at sites of tissue injury or inflammation. The objectives of the present study were to determine in vitro effects of azelnidipine on expression of these adhesion molecules mediated by intracellular ROS. Materials and Methods: We used human aortic endothelial cell (HAEC) and monocytes cell-line (U937). The surface expression and mRNA level of VCAM-1 on HAEC stimulated by 7-ketocholesterol (7KC) and TNF-a were determined by EIA and RT-PCR. Intracellular ROS productions and the amount of adherent monocytes on HAEC were determined fluorometrically. Results: Azelnidipine significantly reduced the expression of protein and mRNA levels of VCAM-1, whereas niphedipine did not. Less monocytes adhered to HAEC pretreated by azelnidipine. Also, azelnidipine significantly reduced the intracellular ROS production induced by 7KC and TNF-a. Conclusion: These results suggest that azelnidipine works as anti-atherogenic agent through inhibiting ROS-dependent expression of endothelial cell adhesion molecules induced by 7KC and TNF-a.

P8-118 DAPSONE INDUCES OXIDATIVE STRESS AND IMPAIRS ANTIOXIDANT ENZYME FUNCTION IN RAT LIVER L. M. Veggi,1 F. A. Crocenzi,1 R. M. Aguero,1 A. Nocito,1 M. G. Roma,1 and A. D. Mottino1 1 Instituto de Fisiologı´a Experimental (CONICET) Facultad de Ciencias Me´dicas (UNR), Rosario, Santa Fe´. Argentina

Dapsone, a drug used in the treatment of leprosy, induces hepatic injury by unknown mechanisms. We evaluated whether dapsone induces oxidative stress in rat liver through determination of biliary output of glutathione species, lipid peroxidation and activity of antioxidant enzymes. Dapsone was administered i.p. to male Wistar rats for 4 days (60 mg/Kg/day, N = 4). Biliary output of total glutathione (reduced + oxidized, GSH + GSSG) was significantly increased by the drug (Dapsone 6.89 F 0.74 vs. Control 3.06 F 0.21, means F SD, p < 0.05), as the result of an increase in GSSG output (Dapsone 2.05 F 0.29 vs. Control 0.22 F 0.06 nmol/min/g liver, p < 0.05). The hepatic content of GSH and GSSG were not modified by Dapsone. The content of thiobarbituric acid-reactive substances in liver homogenates was neither affected. Dapsone significantly decreased the activity of catalase (Dapsone 712 F 200 vs. Control 1043 F 166 U/mg protein, p < 0.05) and glutathione peroxidase (Dapsone 376 F 39 vs. Control 440 F 30 mU/mg protein, p < 0.05) determined in liver homogenates, whereas the activity of superoxide dismutase was not modified. The increase in GSSG biliary output suggests that dapsone induces oxidative stress as a potential mechanism to explain liver injury. The decreased activity of antioxidant enzymes and in consequence, increased susceptibility to oxidative damage, may represent an aggravating factor.

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P8-119 CHANGE IN MARKERS OF ENDOTHELIAL FUNCTION PRODUCED BY OZONE INHALATION IN RATS R. Mendoza,1,2 M. A. Moro,3 J. Sanchez-Gonzalez,1,4 C. CastilloHenkel,1 J. A. Blanco-Tufino,5 R. Cobilt-Catana,5 and C. Villanueva1 1

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Escuela Superior de Medicina del IPN, Mexico, AstraZeneca, Universidad Complutense de Madrid, Facultad de Medicina, Espan˜a, 4Escuela Medico Militar, UDEFA, Mexico, 5Hospital Central Militar, Mexico 3

Ozone exposure produces systemic oxidative stress. Some endothelial diseases have been related to oxidative stress. The aim of this study was to investigate if ozone exposure could change some markers of endothelial function in rats. Male Wistar rats were exposed to ozone (0.25ppm, 4 h a day) or filtered air (<0.05 ppm of ozone). After ozone exposure, blood samples were taken to measure NO2 / NO3 (NOx , stable product of nitric oxide), 6-Keto-Prostaglandin F1alpha (6-Keto-PGF1a, stable product of prostacyclin), dehydro-thromboxane B2 (DHTxB2, stable product of thromboxane A2) and endothelin-1. After 14 days of ozone exposure, NOx- increased (p < 0.05) while the other markers fell (p < 0.05). 14 days later 6-ketoPGF1a remained low (p < 0.05) and DHTXB2 increased (p < 0.05). It is concluded that ozone exposure produced endothelial dysfunction evidenced by changes in endothelial function markers.

P8-120 EVALUATION OF OXIDATIVE STRESS IN HUMAN PERIODONTAL DISEASE I. Borges, Jr.,1 E. A. M. Moreira,1 D. Wilhelm Filho,2 T. B. Oliveira,2 M. S. Lunardi,2 and M. C. Wiese 1 Programa de Po´s-Graduacß a˜o em Nutricßa˜o, CCS, 2Depto. de Ecologia e Zoologia, CCB, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil

Reactive oxygen species (ROS) are enhanced in inflammatory process, among them periodontitis. Oxidative stress was evaluated in gingival tissue in healthy subjects (controls, probing depth until 3mm, n = 9 each group) and pre-obese subjects having chronic periodontal (probing depth between 6 to 9mm). Nutritional evaluation was also carried out through food consumption and anthropometric measurements. Control group showed body mass indices compared to the pre-obese-periodontitis group, and food consumption containing antioxidants was higher in the control group compared to the experimental group. Subjects with periodontal disease showed enhanced GSSG and TBARS concentrations in gingival tissue compared to healthy subjects. Moreover, they also showed enhanced GPx, GST, MPO activities, and GT concentrations in gingival tissue, compared to controls, while the CAT and GR activities showed no significant differences. Summing up the results, TBARS and GSSG contents, activities of antioxidant enzymes such as GPx and GST, together with MPO activity and evaluation of

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the nutritional status, indicate a strong correlation between biomarkers of oxidative stress, periodontitis and the nutritional status of the subjects. Support: DWF is a recipient of a research fellowship from CNPq (Brazil).

OXIDATIVE STRESS IN CHRONIC CARDIOPATHY ASSOCIATED WITH CHAGAS’ DISEASE T. Bittencourt de Oliveira,1 A. d’Agostini Zottis,1 D. Wilhelm Filho,1 and R. Couri Pedrosa 2 1

Depto. de Ecologia e Zoologia, CCB, Universidade Federal de Santa Catarina, Floriano´polis, 2Depto. de Cardiologia do Hospital Universita´rio Clementino Fraga, UFRJ, Rio de Janeiro, Brazil

The unbalance between prooxidant and antioxidant defenses is known as oxidative stress, and is a common phenomenon associated with inflammatory processes of many diseases. The Chagas’ disease is a chronic pathology characterized by chronic inflammation similar to autoimmune pathologies which ultimate mechanisms are unknown. The present work measured some components of antioxidant system present in the blood of chagasic cardiopathy patients. Antioxidant enzymes in erythrocytes and contents of reduced glutathione were analyzed in four groups of patients (n = 10 each group) with different degrees of severity of the disease, according to the classification of Los Andes. Catalase and superoxide dismutase activities showed decreased values parallel to the severity degree, while GST activity showed an inverse profile. The intraerythrocytic contents of GSH showed also lowered values according to the progression of the disease. It seems that the antioxidant status is jeopardized with the extension of the disease, being exposed to an progressive oxidative stress condition. New perspectives for the treatment of Chagas’ disease may include an antioxidant therapy in order to attenuate consequences related to oxidative insult. Support: DWF is a recipient of a research fellowship from CNPq (Brazil).

P8-122 NON-STEROIDAL ANTI-INFLAMMATORY DRUGS AS EFFECTIVE SCAVENGERS FOR REACTIVE OXYGEN AND NITROGEN SPECIES D. Costa,1 E. Fernandes,1 S. A. Toste,1 J. L. F. C. Lima,1 and S. Reis1 1 REQUIMTE, Departamento de Quı´mica-Fı´sica, Faculdade de Farma´cia, Universidade do Porto, Rua Anı´bal Cunha, 164, 4099-030 Porto, Portugal

The present study was undertaken to evaluate the scavenging activity for reactive oxygen species (ROS) and reactive nitrogen species (RNS) by several structurallyrelated nonsteroidal anti-inflammatory drugs (NSAIDs), namely indoleacetic derivatives (indomethacin, acemetacin, etodolac), and pyrroleacetic derivatives (tolmetin and ketorolac). The scavenging activity of these NSAIDs was evaluated against an array of ROS (ROO , HO , O2 , H2O2, and HOCl) and RNS ( NO and ONOO ) using

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non-cellular in vitro systems. The results obtained showed that ROO is effectively scavenged by etodolac, being the other compounds much less active. The same applies to HO , in which etodolac was also the most active scavenger for HO , although the other tested compounds were also shown to be active. Tolmetin and ketorolac were not active against O2 , while acemetacin, indomethacin and etodolac exhibited a concentration dependent effect. In what concerns H2O2 scavenging activity, acemetacin, indomethacin and etodolac showed to be the most potent, while tolmetin and ketorolac only had a small effect. None of the studied compounds had any scavenging effect for HOCl. Indomethacin and etodolac were the most effective scavengers for NO, while indomethacin and etodolac and ketorolac showed the most potent scavenging activities for ONOO . Taking into account that some of the observed effects were obtained within therapeutical concentrations, these effects may strongly contribute for the anti-inflammatory therapy to be attained with the studied NSAIDs. Acknowledgements The authors greatly acknowledge FCT financial support POCTI / FCB / 47186 / 2002. David Costa acknowledges FCT and FSE for his PhD grant (SFRH/BD/10483/2002).

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37jC. In vivo, positive H2O2 producing cells were analyzed by DHR oxidation after injection of GOLs in mice (C57BL/6J). After 120 minutes 40 % of granulocytes were fluorescent and the result correlated with the half life of GOLs of 80 minutes in blood. The bactericidal activity of granulocytes with NADPH oxidase defect towards Staph. aureus was reconstituted after treatment of cells with GOL (Gerber et al., Blood, 2001). In vivo the bacterial load of Staph. aureus infected mice (NADPH oxidase deficient) could be reduced after i.v. injection of GOLs. GO activity could be detected in organs (liver, spleen), indicating the high stability and functionality of the incorporated enzyme. Further experiments will refine the analyses of GOL effects in the infection model. The work was supported by the DFG (GE 1241/1, SCHU 800/ 4-1) and the SSAI Switzerland.

P8-124 CHOLESTEROL OZONATION PRODUCTS IN INFLAMMATION AND DISEASE C. Takeuchi,1 J. Nieva,1 R. A. Lerner,1 and P. Wentworth, Jr.1 1

P8-123 GLUCOSE OXIDASE CONTAINING LIPOSOMES FOR TREATMENT OF CGD: COMPARISON OF IN VITRO AND IN VIVO REACTIONS C. Gerber,1 G. Bruchelt,1 D. Niethammer,2 and R. Schubert 2 1 University Children’s Hospital, Dept. of Hematology/Oncology, 2 University of Freiburg, Dept. of Pharmaceutical Technology, Germany

Liposomes containing GO (GOL) are phagocytosed by granulocytes and monocytes, producing then H2O2 and HOCl intracellularly. The lacking oxidative burst of cells with NADPH oxidase deficiency can therefore be reconstituted. In vitro, H2O2 and methemoglobin were only measurable in very high concentrations [GO > 2 U/mL] after incubation of GO in whole blood. The steady state concentration of H2O2 was at 20 nM over 4 – 8 hours at

The Scripps Research Institute, USA

Recent studies have suggested that trioxygen species are formed in biological systems during the antibodycatalyzed water oxidation pathway, and that excised human atherosclerotic tissue can generate trioxygen species in vitro. Investigations into the downstream effects of these oxidants demonstrate that ozonolysis of cholesterol generates a number of compounds that are cytotoxic to circulating leukocytes, vascular smooth muscle and endothelial cells. These ozone-derived oxysterols, the so-called atheronals, induce the formation of foamy macrophages and lead to apoliproprotein-B misfolding. Atheronal-B has been detected in the plasma of patients about to undergo endarterectomy. Our observations offer a link between the production of trioxygen species, elevated cholesterol levels and the pathogenesis of atherosclerosis. Such effects may well be applicable to the development of other inflammatory diseases.

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