The goal of my lecture is to provide a discussion of recent advances regarding a group of oxidized lipids termed the isoprostanes (IsoPs). The IsoPs are a series of PG-like compounds formed in vivo from the free radical-catalyzed peroxidation of arachidonate. Recent discoveries related to the chemistry of IsoP formation will be detailed. The first class of IsoPs discovered were the F2-IsoPs that possess an F-type prostane ring, analogous to PGF2a. Since then, other IsoPs have been characterized that contain E/D-, A/J-, and thromboxane ring structures. A number of these IsoPs possess potent pro-inflammatory biological activity, and their potential role in mediating certain aspects of the effects of oxidant stress will be examined. A portion of my talk will deal with the utility of measuring IsoPs as markers of oxidant injury. In 2007, IsoPs are recognized as the most accurate index of oxidant stress in humans. Two diseases that have been studied with regards to the role IsoPs play in their pathogenesis are atherosclerosis and neurodegenerative disease and these will be discussed. Over the past several years, we have undertaken a number of studies to examine the impact of various dietary, lifestyle, and therapeutic interventions on IsoP formation in vivo. I will summarize these findings. In addition to the formation of IsoPs from arachidonic acid, we have also determined that other polyunsaturated fatty acids can undergo peroxidation to form IsoP-like compounds. One that we have extensively studied is eicosanpentaenoic acid (EPA), an abundant polyunsaturated fatty acids in fish oil. Consumption of EPA exerts anti-inflammatory effects and prevents diseases such as atherosclerosis. The mechanism by which this occurs is unknown but we have recently determined that consumption of EPA reduces the formation of pro-inflammatory 2-series IsoPs derived from arachidonate. In addition we have identified a class of reactive EPA-derived IsoPs termed cyclopentenone IsoPs (A3/J3-IsoPs) that possess potent antioxidant propterties by stabilizing the transcription factor Nrf2, increasing Nrf2 gene expression and lowering oxidant stress. This may account, in addition to other mechanisms, for the anti-inflammatory properties of fish oil.
Peroxynitrite (PN ) is formed in vivo by a fast combination of NO and superoxide and is able to oxidatively modify amino acids in proteins as a basis for redox signalling. We here show that the biosynthesis of prostanoids is regulated by PN at several steps. Likewise cyclooxygenase ( COX )-1 and –2 are activated by 20 and 2 nM PN, respectively. At levels of 100 nM prostacyclin synthase is nitrated and becomes inactivated whereas the closely related thromboxane synthase remains unaffected. However, thromboxane synthesis in stimulated macrophages can be inhibited by the PN degradation product nitrite through nitration of the essential Tyr at COX-2. This requires NO-synthase –2 induction. In stimulated smooth muscle cells this induction is suppressed allowing prostacyclin synthesis via COX-2 without COX or prostacyclin synthase nitration. Control of PN formation can occur by several mechanisms from which the sources of NO as well as superoxide are the main determinants together with the pathways of PN degradation. Conflicting data exist on the reactivity of PN with NO and superoxide. Our results as well as others support a network of redox reactions that contribute to the regulation of important physiological and pathophysiological events.
Redox processes involving enzymatic ROS generation via NADPH oxidase play a prominent role in vascular pathophysiology, but mechanisms that account for their regulation and integration to other cellular stress responses are insufficiently understood. Protein disulfide isomerase (PDI) is a thioredoxin superfamily dithiol disulfide oxidoreductase chaperone from the endoplasmic reticulum (ER) that exerts key role in redox protein folding and traffic. We provide evidence that PDI closely regulates NADPH oxidase activation secondary to angiotensin II in vascular smooth muscle cells (VSMC). Moreover, PDI physically interacts at least with subunits p22phox and with Nox isoforms 1and 4. We further postulated that PDI/NADPH oxidase interaction is a pathway bridging the unfolded protein response/ER stress signaling cascade to oxidative stress. Incubation of VSMC with ER stressors upregulate ROS generation and NADPH oxidase activity in VSMC and enhance Nox4 expression. UPR disruption induced through eIF-2-alfa dephosphorylation or caspase-12 siRNA decreases superoxide production. Conversely, ROS contribute to sustain UPR, since catalase overexpression decreases expression of chaperone markers. Also, Nox4 siRNA promoted reduced expression of proapoptotic CHOP-GADD153. Loss or gain-of-function experiments further support the involvement of PDI as a mediator of the convergence between oxidative stress and ER stress. In addition, PDI expression increases following sustained ER stress. PDI as well as ER stress markers are markedly upregulated during vascular repair after injury, mainly in neointima. Thus, neointimal oxidative stress occurs in the context of an integrated stress response involving ER stress signaling, PDI and NADPH oxidase. In addition, we provide evidence that a similar interaction between PDI and NADPH oxidase occurs in endothelial cells. Moreover, PDI is a central regulator of NO bioavailability during sustained laminar shear stress in endothelial cells. Such effect appears to result from a balance between PDI assistance of NADPH oxidase-mediated superoxide production and PDI-mediated NO internalization via trans-nitrosation reactions. Finally, recent observations indicate that PDI also displays functional interaction with phagocyte NADPH oxidase. The combination of PDI redox sensitivity and its multiple cellular effects enable PDI to act as a central regulator of redox signaling processes (Supported by FAPESP, CNPq Milênio Redoxoma).
Palabras clave: NADPH oxidase, Protein disulfide isomerase, Endoplasmic reticulum stress
| 10:55 - 11:20 | Invited Lecture | 05.Nitric Oxide, Peroxynitrite and Oxidants in Redox Vascular Biology
Endothelial damage mediated by Cyclosporine A: a model for nitroxidative damage in search for targets
Lamas, S 1(*); Redondo, M 2; Martinez-Ruiz, A 3; Vazquez, J 4; Martinez, P 4
1 - Centro de Investigaciones Biologicas | (*) Spain
Cyclosporine A (CsA) is an immunosuppresant which has been used in the clinical practice for about 25 years. Even when it represents a significant advance for the prevention of transplant rejection its use is associated with serious side effects, among which hypertension and vascular damage is one of the most prominent. For the past 15 years our group has been interested in clarifying the mechanisms of endothelial cell damage, as a means to understand and prevent vascular injury. We have shown that upon exposure to CsA, peroxynitrite is formed, this leading to increased nitration of endothelial cells and of the vascular wall. This is associated with significant cellular toxicity and prevented by the use of antioxidants, such as N-acetylcysteine, and of the permeable analog of MnSOD, MnTMPyP. In an effort to identify specific targets for this damage we focused on the mitochondrial SOD isoform and detected increased nitration of this protein in the presence of CsA, both in vitro and in endothelial cells. Studies using MS-ESI approaches have assigned nitration to Tyr 34 as one of the target residues. The functional inactivation of MnSOD by this postranslational modification has been well established by several groups. As a result of this inactivation, cellular detoxification mechanisms triggered by increased nitroxidative stress may be severely compromised. We propose that nitration of MnSOD by CsA provides a biochemical mechanism to explain the cellular toxicity of this immunosuppresant, as well as a potential therapeutic target for specific pharmacological or biochemical interventions.
Endothelial responses in atherosclerosis are linked to various factors, including ROS, lipid peroxidation and defective NO production. 4-Hydroxy-2-nonenal (4-HNE) is an abundant lipoperoxidation product that mediates oxidative stress by mechanisms that remain controversial. Here, the effect of 4-HNE in endothelial NO/O2·- fluxes was examined as a potential mechanism by which lipid peroxidation products mediate oxidative vascular dysfunction. To better understand the amount of 4-HNE involved in the cellular responses, the uptake of 4-HNE was examined by HPLC with UV detection. It was shown that BAECs accumulate 0.46±0.06 nmol/mg protein of free 4-HNE after exposure to 25µM 4-HNE for 4h with a considerable decrease thereafter. No significant changes in GSH levels were detected under these experimental conditions. The 4-HNE build up was accompanied by a dose-dependent inhibition of NO that correlated with losses of hsp90 and phosphorylated eNOS-serine1179, but not eNOS protein levels. 4-HNE failed to inhibit NO production in sepiapterin- and ascorbate-supplemented cells, suggesting that tetrahydrobiopterin (BH4) is a limiting factor in non-supplemented cells. This was verified in the analysis of BH4 levels which were depleted. It was also shown decreased GTP cyclohydrolase I (GCH) protein levels and activity after 4-HNE treatment. Lactacystin pre-treatment inhibited the decline in both GTPCH and hsp90 indicating that depletion of BH4 by 4-HNE is due to specific mechanisms involving proteasome activation. Using fluorogenic substrates, it was shown that 4-HNE increases the ATP-dependent 26S proteasome activity in a dose-dependent fashion. The decline in GCH activity and intracellular BH4 causes eNOS uncoupling stimulating O2·- production, as indicated by the dose-dependent increase in 2-hydroxyethidium levels. The unbalanced production of NO/O2·- in 4-HNE-treated cells showed a good correlation with increases in caspase-3 activity. This activation, however, was counteracted by either sepiapterin or DETANONoate supplementation, indicating that loss of NO is a significant source of cellular stress. In summary, 4-HNE by altering BH4 and eNOS homeostasis specifically regulates NO/ROS fluxes in the endothelium with important consequences to cell survival mechanisms.
In recent years, the gaseous biological mediator hydrogen sulfide (H2S) emerged as a modulator of vascular function and as a mediator or modulator of cell death. The pharmacological and biological actions of H2S include vasodilatation, inhibition of mitochondrial respiration as well as induction of suspended animation. The cellular actions of H2S include cytoprotection (occurring at lower concentrations, and including a protection against the cytotoxic effects of oxyradicals and peroxynitrite) and (at higher concentrations) cytotoxicity. Emerging data in the literature indicate that H2S can exert cytoprotective effects in vitro, it can induce myocardial preconditioning in cardiac myocytes and isolated perfused hearts, it can reduce myocardial infarct size in coronary ischemia-reperfusion models in rodents and large animals, and it can improve myocardial contractility in cardiopulmonary bypass models. The mechanism of cardiac protection elicited by H2S is probably multiple and may include antioxidant mechanisms, activation of KATP channels, as well as inhibition of inflammatory cell activation. While exogenously administered H2S is cardioprotective, pharmacological inhibition of endogenously produced H2S exacerbates myocardial ischemic-reperfusion injury. There are some clinical data indicating that plasma levels of H2S are decreased in patients with cardiac disease. In many ways, the cardiac characteristics of H2S resemble those of nitric oxide (NO), another endogenous gaseous mediator with vasodilatory, cytoprotective and cardioprotective actions. The current presentation will overview the available data on the cardioprotective effects of hydrogen sulfide, and will outline potential avenues for the therapeutic exploitation of this effect.
In this presentation, mass spectrometric strategies to detect and characterize nitrated lipids will be discussed. Nitro fatty acid derivatives (NO2-FA) have been reported to mediate anti-inflammatory responses. Furthermore, NO2-FA stimulate peroxisome proliferator activated receptor gamma (PPARg) activity to extents that rival the thiazolidinedione class of PPARg agonists. This observation has generated interest into defining the endogenous concentrations and structural diversity of this class of lipid mediators. Due to its sensitivity and qualitative analytical abilities, mass spectrometry is an essential tool in the discovery and structural analysis of NO2-FA. Triple quadrupole-, GC- and ion trap-based mass spectrometric analysis revealed that the key identifying fragment of this class of lipid mediators is the loss of the nitro anion. This diagnostic fragment has been used to detect other endogenous NO2-FA species. These strategies are also applicable to complex lipids containing nitrated fatty acids. These include nitrated phospholipids and tri- and diglycerides. Recently, nitrocholesterol linoleate has been identified, indicating lipid nitration to be a ubiquitous covalent modification of endogenous lipids. The structural identification of low abundance free and esterified NO2-FA in vivo is a major analytical challenge. Helpful strategies include alternative mass spectrometric scan modes and the use of mono- and divalent cations to form adducts whose charge is locked at the carboxylic acid functional group, which greatly facilitates structural elucidation. Recently, NO2-FA derivatives containing a nitroalkene functional group were identified as electrophiles that react with protein thiols and histidines to form adducts; these post-translational modifications are identified using proteomic approaches. Capitalizing on the electrophilicity of nitroalkenes, small molecular weight thiols (e.g., b-mercaptoethanol; BME) can be used to chemically “freeze” nitroalkenes to prevent side reactions with biomolecules during isolation. This strategy has been extended to ‘strip’ protein-NO2-FA adducts of the lipid moiety with BME in a trans-nitroalkylation reaction to form BME adducts that can then be quantitated.
Palabras clave: Nitroalkenes, Mass spectrometry, electrophiles
15:30 a 17:30 - Poster Session
| Poster Presentation | 10.Neurodegeneration and Neuroprotection
Evidence that cis-4-decenoic acid induces oxidative stress in brain of young rats
Schuck, PF 1(*); Ceolato, PC 1; Ferreira, GC 1; Tonin, A 1; Zanatta, A 1; Latini, A 2; Wajner, M 3
1 - Universidade Federal do Rio Grande do Sul | (*) Brazil
Patients affected by medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most prevalent inborn error of fatty acid oxidation, suffer from acute episodes of encephalopathy whose underlying mechanisms are poorly known. The present study investigated the in vitro effect of cis-4-decenoic acid (cDA), the pathognomonic metabolite that accumulates in MCADD, on various parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBA-RS), chemiluminescence, carbonyl content, sulfhydryl oxidation, 2’,7’-dihydrodichlorofluorescein (DCFH) oxidation, total-radical trapping antioxidant potential (TRAP), reduced glutathione (GSH) levels, and the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in homogenates from brain of young rats. It was observed that cDA significantly induced lipid peroxidation, by eliciting increased levels of chemiluminescence and TBA-RS, and protein oxidation, reflected by increased carbonyl formation. cDA also increased DCFH oxidation, suggesting an increased production of reactive species. On the other hand, cDA diminished the cortical non-enzymatic antioxidant defenses evaluated by TRAP, TAR and GSH values. In contrast, the sulfhydryl content and the activities of the antioxidant enzymes CAT, SOD and GPx were not altered by cDA exposition. Our present results showing a cDA-induced increase of lipid and protein oxidative damage and a decrease of tissue non-enzymatic antioxidant defences strongly indicate that oxidative stress might be involved in the pathophysiology of the brain injury in MCADD. Financial support: CNPq, FAPERGS, PRONEX and the FINEP research grant Rede Instituto Brasileiro de Neurociência (IBN-Net) # 01.06.0842-00.
The brain is dependent on the maintenance of the levels of vitamin A to its well function. However, vitamin A in excess might be toxic to the central nervous system (CNS). This vitamin is a redox-active molecule and our group has studied its prooxidant potential. Oxidative stress is a condition characterized by an overload in prooxidant molecules. Here, we investigated the effects of an acute (3 or 7 days) or chronic (28 days) vitamin A supplementation at therapeutic (1,000 or 2,500 IU/kg/day) or excessive (4,500 or 9,000 IU/kg/day) doses on the striatal redox environment of adult rats. We gavaged the animals once a day during each period of investigation. Lipoperoxidation (Draper and Hadley, 1990), protein carbonylation (Levine et al., 1990), and the protein and non-protein thiol content (Ellman, 1959) in the striatum were investigated as previously described. The activities of the antioxidant enzymes superoxide dismutase (SOD) (Misra and Fridovich, 1972), catalase (CAT) (Aebi, 1984), and glutathione peroxidase (GPx) (Flohé and Günzler, 1984) were also analyzed. Additionally, the non-enzymatic antioxidant defense (TRAP) was quantified (Wayner et al., 1985). We found that vitamin A at therapeutic and excessive doses increased lipoperoxidation, protein carbonylation, and decreased the protein thiol content acutely and chronically. Furthermore, vitamin A supplementation increased SOD activity and decreased CAT activity, inducing an imbalance in the SOD/CAT ratio. GPx activity did not change after vitamin A supplementation. Moreover, acute or chronic vitamin A supplementation induced a decrease in the striatal non-enzymatic antioxidant defense. Therefore, we conclude that oral vitamin A at therapeutic doses impairs the striatal redox environment after acute or chronic vitamin A supplementation at therapeutic doses.Financial Support: CNPq, FAPERGS, PROPESQ/UFRGS
Palabras clave: vitamin A, oxidative stress, adult striatum
| Poster Presentation | 09.Mitochondria and Apoptosis
DIETARY THIOPROLINE AND LIFE CONDITIONS THAT IMPROVE MITOCHONDRIAL AND BRAIN FUNCTION AND SURVIVAL IN MICE
Sanchez-Pino, MJ 1(*); Gomez, C 2; Bandez, MJ 3; Cadenas, E 4; Boveris, A 5; Navarro, A 3
1 - Depatment of Biochemistry and Molecular Biology, School of Medicine, University of Cadiz | (*) Spain
Dysfunctional mitochondria in aged rodents are characterized, besides decreased electron transfer and O2 uptake, by an increased content of oxidation products, a decreased membrane potential, and increased size and fragility. Inner membrane enzyme activities, such as those of complexes I and IV and mtNOS, decrease upon aging and afford aging markers. The activities of these three enzymes in mice brain are linearly correlated with neurological performance, as determined by the tightrope and the T-maze tests. The same enzymatic activities correlated positively with mice survival and negatively with the mitochondrial content of lipid and protein oxidation products. Male mice on a diet supplemented with thioproline (l-thiazolidine-4-carboxylic acid), a physiological metabolite of 5-hydroxytryptamine, at 2.0 g/kg of food from 28 wk of age and for mice entire life, showed a 29-23 % increased median and maximal life span. These survival increases were associated with improved neurological functions. Compared to control mice, thioproline-supplemented mice had a 20 % lower integral spontaneous food intake, and 10 % lower body weight at 100 wk of age. Body weight showed a statistically significant inverse relationship with survival and neurological performances. Thioproline-supplemented mice exhibited a 58-70 % decrease of the age-dependent oxidative damage in brain and liver mitochondria at 52 wk (old mice) and 78 wk (senescent mice) of age, respectively. The age-associated decrease of brain mitochondrial enzyme activities, NADH-dehydrogenase, cytochrome c oxidase, and mitochondrial nitric oxide synthase (mtNOS), in old and senescent mice were markedly prevented (51-74 %) by thioproline. It is surmised that thioproline induces an anorexic effect associated with improved survival and neurological function through a decreased oxidative damage and regulation that may involve hypothalamic appetite centers. Conditions that increase survival, as dietary thioproline, moderate physical exercise, vitamin E dietary supplementation, and high spontaneous neurological activity, ameliorate mitochondrial dysfunction in aged brain and liver. The prevention of the mitochondrial dysfunction is likely to improve mitochondrial biogenesis and turnover mediated with the pleiotropic signaling of H2O2 and NO diffusion to the cytosol.
Palabras clave: anorexia, aging, mtNOS
| Poster Presentation | 01.Oxygen Radical Chemistry and Detection
Characterization of Adduct Derived from the Reaction of Glutathione with trans,trans-2,4-Decadienal
Monteiro, RVM 1(*); Medeiros, MHG 2; Di Masccio, P 1
Trans,trans-2,4-decadienal (DDE) is an important breakdown-product of lipid peroxidation. This aldehyde is cytotoxic to mammalian cells and is known to be implicated in DNA and protein damage. Therefore, attempts were made in the present work to assess the reactivity of DDE with glutathione (GSH), one of the mostly likely candidates for the consumption of DDE in cytosol. An HPLC system was developed for the assay of DDE-glutathione conjugates with a UV/Vis diode array detector operating at 274 nm. To optimize the synthesis condition, DDE (1 mM in 10 mM phosphate buffer, pH 7,4) was incubated with GSH at different molar ratios (1:1; 1:2; 1:5;1:10) and different time periods (2, 4, 6 and 24 h) at 37ºC. The results indicate that GSH is able to quench DDE. The best condition for GSH-DDE conjugation was obtained with molar ratio 1:1 and 24 h incubation time. The ESI-MS (direct infusion) of the 24 h incubation mixture (positive-ion mode) shows a peak at m/z 460 indicating a Michael adduct formation. MS/MS analysis of the ion at m/z 460 gave as the main fragments ions at m/z 442 [M+H-H2O]+ and m/z 308 due to the cleavage of Michael adduct at C-5 of DDE via retro-Michael reaction. Conjugation of GSH with aldehydes to give Michael adducts, is a well recognized pathway of detoxification of reactive aldehydes in cells. Acknowledgements: FAPESP, CNPq, Instituto do Milênio Redoxoma.
Shear stress is a major determinant of endothelial function. Generally, sustained laminar shear (LS) leads to eNOS-derived NO production and is vasodilator, anti-inflammatory and anti-atherogenic. However, increases in LS trigger transient superoxide production via NADPH oxidase complex. Recently, we showed that NADPH oxidase undergoes thiol-dependent regulation by thioredoxin superfamily chaperone Protein Disulfide Isomerase (PDI). PDI is known to promote NO internalization via trans-nitrosation reactions. We hypothesized that PDI-dependent support of NADPH oxidase activity affects NO output during sustained LS. Rabbit aortic endothelial cells (RAEC) submitted to LS (15 dynes/cm2) in a cone-plate system for 18h exhibited (vs. static controls): a) Decreased (~50%) superoxide production (HPLC analysis of DHE oxidation); b) Decreased (~20%) NADPH-triggered hydrogen peroxide production in membrane fraction (Amplex Red assay); c) Decreased mRNA expression of Nox1 (67%) and Nox4 (45%) (real-time QPCR); d) Increased eNOS expression (~50%, western blot) and nitrite levels in culture medium (Δ = 7.1±2.5 μM, Griess reaction); e) Decrease in total and membrane fraction PDI protein expression (~20%) without changes in membrane fraction/total ratio of PDI. RAEC were transfected with c-myc-tagged plasmid coding for wild-type PDI (WT) or PDI mutated in 4 thioredoxin-motif cysteine residues. Forced expression (2-fold) of mutated but not WT PDI led to increase in nitrite output after LS (Δmutated = 17.2±2.9 μM vs. ΔWT = 7.0±1.5 μM, p<0.05). Confocal microscopy indicated similar subcellular localization between WT and mutated PDI. PDI co-imunoprecipitated with p22phox NADPH oxidase subunit, but not with eNOS or caveolin-1, either in static condition or after LS. Fractionation studies in sucrose gradients showed that PDI is distributed throughout several fractions in static condition, including caveolin-1-enriched fractions, but migrates to higher-density fractions, not containing caveolin-1, during sustained LS. These results suggest that PDI is involved in regulation of NO output during LS via its effects on NADPH oxidase activity (Supported by FAPESP and CNPq Milênio Redoxoma)
Palabras clave: Protein Disulfide Isomerase, Nitric oxide , NADPH oxidase
| Poster Presentation | 01.Oxygen Radical Chemistry and Detection
Enhanced oxidative stress in the digestive gland of the clam Anamolocarida brasiliana exposed to domestic sewage
Faria, P, E. P. 1(*); Parisotto, B, E. 1; Pilatti, K, F. 1; Backes, P 1; Budni, P 1; Possamai, P, F 1; Avila, Jr. S 2; Cesa, M 1; Wilhelm, F, D. 1
Bivalve mollusks are filter-feeding animals that are therefore able to bioacculmulate environmental contaminants. The digestive gland of the Brazilian clam Anomalocardia brasiliana (berbigão) is an excellent biomarker of aquatic contamination. Several biomarkers of oxidative stress (activity of catalase and glutalthione S-transferase, GSH and TBARS contents were evaluated in clams collected at a site contaminated by domestic sewage and compared to clams collected at a pristine site. The clams sampled at the contaminated site showed enhanced ipoperoxidation (TBARS) levels and higher GST activity compared to the pristine site, whilst no differençes were found for catalase activity and GSH contents in the digestive gland of clams sampled at both sites. We concluded that mollusks chronically exposed to aquatic contaminants might not adequatelly compensate their antioxidant system.
Nitric oxide (NO) has emerged as an important mediator of Ca2+ homeostasis and myocardial contractile function and would be involved in the mechanism of protection against ischemia/reperfusion. The effects of endogenous NO on papillary muscle mechanical activity during the response to increasing Ca2+ concentration and to hypoxia/reoxygenation were evaluated in 12 months old rats. Both papillary muscles of the left ventricle of each rat were simultaneously studied, one supplemented with the substrate L-arginine (L-arg) 2 mM to obtain the maximal NO level and the other with the NOS inhibitor L-NNA 2 mM to block NO generation. Developed tension (DT), maximal rate of rise in DT (+T), and maximal rate of relaxation (-T) were determined under isometric conditions at 0.6, 0.8, 1.3, 1.8, 2.3, and 2.8 mM Ca2+, and at the highest Ca2+concentration, during a period of 60 min of hypoxia and 30 min of reoxygenation. Significant results (p < 0.05), L-arg versus L-NNA, are expressed as mean ± SE. At maximal Ca2+, DT (g/mm2): 1.16 ± 0.08 vs 0.85 ± 0.07, +T (g/mm2.s): 22.6 ± 1.5 vs 15.6 ± 1.6, -T (g/mm2.s): 15.9 ± 0.5 vs 11.4 ± 1.5. After hypoxic recovery, +T: 16.2 ± 1.5 vs 12.6 ± 0.6, -T: 11.3 ± 1.0 vs 8.0 ± 0.7. Results showed that, under these conditions, endogenous NO would positively modulate mechanical activity of the heart, increase the maximal Ca2+ response (27-31% for the different parameters) and contribute to the protection against contractile dysfunction caused by hypoxia/reoxygenation (22-29%). These effects are qualitatively in agreement with the correlation of mtNOS activity and contractile parameters found in our previous work (J Appl Physiol, 2005) and quanitatively in the range of mtNOS functional activity in the regulation of mitochondrial respiration (Boveris et al, 2003).
Superoxide (O2•) and nitric oxide (•NO) are generated by blood vessels and can rapidly react to produce peroxynitrite (ONOO), a powerful oxidant that modifies lipoproteins making them more atherogenic. This study was designed to determine the effect of diphenyl diselenide ((PhSe)2, a new synthetic organoselenium compound under investigation) in comparison with the well known ebselen, on peroxynitrite-mediated endothelial damage. Bovine aortic endothelial cells (BAEC) in primary cultures were treated with authentic peroxynitrite and cell viability, intracellular gluthatione (GSH) content and gluthatione peroxidase (GPx) activity were assessed. Experimental results showed that a long pre-incubation (24 h) with (PhSe)2 (0.5 and 1M) protected endothelial cells from the damage promoted by peroxynitrite exposure, in a more effective way than ebselen. The intracellular levels of GSH were almost completely consumed by peroxynitrite and although the compounds did not restore the normal levels, (PhSe)2 per se increases significantly GSH in a concentration-dependent manner. This effect may be related with the significant increase in cellular GPx activity promoted by this compound, which revealed to be even more active as a glutathione peroxidase mimic, than ebselen. In conclusion, our data suggest a new role for (PhSe)2 as a potential anti-atherogenic agent.
Objective: To investigate the effects of acute exercise on the vasomotor response mediated by nitric oxide (NO) in rat aorta, and to analyze some mechanisms underlying NO bioavailability. Methods: Forty-five rats (male, Wistar, 332±14.6g) were distributed in non-exercise control (CTR, n=21) and exercise (EX, n=24) groups. Immediately after a single acute exercise bout (treadmill, 60min, 60-70% maximal capacity), the rats were sacrificed and their thoracic aorta was removed and cut into rings (5mm). Two rings were suspended in an organ bath connected to an isometric force transducer to evaluate the vasodilatation (Acetylcholine, ACh 10-10 to 10-4 M) and vasoconstriction response (Norepinephrine, NE 10--7 M), after a 30-min period of incubation with L-NAME (10-3M) or without incubation. The remaining rings were used to evaluate the nitrate and nitrite concentration and superoxide levels by chemiluminescence, and SOD activity by spectrophotometry. Results: Acute exercise significantly increased aortic relaxation (101.8±1.6 vs. 91.8±1.8%; p<0.05) and decreased constriction responses compared to CTR group (1.17±0.07 vs. 1.75±0.16g; p<0.05). However, after incubation with L-NAME, the vasomotor response was similar between CTR and EX groups (ACh: EX: 47.1±10.5 vs. CTR: 63.23±8.05%; NE: EX: 2.73±0.22 vs. CTR: 2.73±0.27g). Acute exercise significantly increased nitrate levels (15.1±4.2 vs. 6.5±1.2 nmol/mg prot; p<0.05), nitrite levels (0.13±0.04 vs. 0.07±0.02 nmol/mg prot; p<0.05), superoxide levels (1555±60 vs. 1212±94 cpm/mg dry weight; p<0.05), and SOD activity (32±1.3 vs. 25±2.0 U/mg prot, p<0.05) compared to CTR. Conclusion: These data suggest that despite of exercise-induced increase in superoxide levels, it does not compromise exercise-induced increase in vascular NO bioavailability, which might be one of the causes of the lower aortic vasoconstriction and higher vasodilation response in exercised rats.
Purpose: Heart Failure (HF) is a clinical syndrome characterized by skeletal muscle abnormalities, such as early fatigue and exercise intolerance (EI). Although previous studies suggested that oxidative stress plays a pathological role in worsening skeletal myopathy, little is known about its involvement in muscle dysfunction and morphological changes associated with HF. As aerobic exercise training (ET) decreases oxidative stress, we tested whether ET would reverse muscle atrophy and improve redox status in mice lacking both α2A/ α2C AR subtypes (KO), which develop cardiomyopathy induced by sympathetic hyperactivity.
Methods: It was studied a cohort of wild type (WT, n=23) and KO (n=27) mice in a C57BL/6J background, randomly assigned to resting or ET (8-wk treadmill running sessions of 60 min, 5 days/wk). Exercise capacity was estimated by using a graded treadmill protocol. Skeletal fiber cross-sectional area (CSA) was evaluated by myosin-ATPase histochemistry, lipid hydroperoxides were evaluated by the ferrous oxidation-xylenol orange technique (FOX2) and reduced glutathione (GSH) measurement was performed by HPLC system with electrochemical detection. Total GSH was measured after GSSG was regenerate to GSH by NADP and glutathione reductase at pH 7.5. The concentration of GSSG was estimated by subtracting the measured GSH from the measured total GSH.Results: KO mice presented significantly higher lipid hydroperoxides levels (54±3.8 vs. 43±1.4 mM.mg-1 protein, p< 0.05) while decreased GSH:GSSG ratio (3.9±0.4 vs. 11.4±0.8, p< 0.05), paralleled by fiber type shift (I→IIa), decreased CSA mainly in IIa (27%), and lower exercise capacity (259±18 vs. 351±19 m, p< 0.05) compared to WT mice. In KO mice, ET restored lipid hydroperoxides levels and GSH/GSSG ratio to WT levels, which was associated with increased CSA and improved exercise capacity. Conclusions: collectively, these results provide evidence that ET improves exercise tolerance in KO mice and this response is associated, at least in part, to restored redox status and skeletal muscle throphism.
Caffeine is consumed worldwide in the form of beverages such as coffee and tea. In vitro, antioxidant properties have been described for it. In modern societies, caffeine consumption may frequently be associated to stress. It has been reported that stress may induce oxidative damage and change the balance between pro-oxidant and antioxidant factors in the brain. As the hippocampus is the most stress susceptible brain structure, the aim of this study is to verify the effect of repeated restraint stress and chronic consumption of caffeine on oxidative stress and DNA damage in the hippocampus of rats. 24 male, adult, Wistar rats were divided into two groups: (1) control and (2) submited to repeated stress, 1h/day, 5 days a week, during 40 days. These 2 groups were subdivided into receiving or not caffeine, which was offered in the drinking water (0.3g/L or 1g/L caffeine). After 40 days, the animals were sacrificed. The hippocampi were taken and immediately used (the right hippocampus) in single-cell gel electrophoresis (Comet) assay in order to detect DNA damage. The other hemi-hippocampus (the left side) was kept at -70ْC for further investigation of lipid peroxide levels (by TBARS levels) and total radical-trapping potential (TRAP). Results were analysed by two-way ANOVA. Caffeine (0.3g/L and 1g/L) decreased TBARS levels and protected against the effects induced by stress (marginally significant interaction, P = 0.06). Both stress and either doses of caffeine (0.3g/L and 1g/L) increased DNA damage (P<0.05); in addition, there was a significant interaction between these two factors. No effect was observed on TRAP. These results suggest that chronic stress induce oxidative stress on rat hippocampus, which may be involved in the DNA damage observed. Caffeine consumption protects against these effects, but has effects per se on DNA damage.
5-Oxoproline (pyroglutamic acid) accumulates in glutathione synthetase (GS) deficiency, an autossomic recessive inherited disorder clinically characterized by hemolytic anemia, metabolic acidosis and severe neurological symptoms. The mechanisms of brain damage in this disease are poorly known. However, 5-oxoproline was already shown to exhibit neurotoxic actions. We have recently reported that 5-oxoproline increases reactive species production and decreases non-enzymatic antioxidant defenses in vitro in rat brain, thus promoting oxidative stress. In the present study we investigated the possible role of oxidative stress in 5-oxoproline neurotoxicity in vivo in order to further clarify its participation in the brain damage mechanisms responsible for the neurological impairment observed in GS-deficient patients. To accomplish that, rats received a single dose of 5-oxoproline subcutaneously (1 g/Kg body weight). This dose produced plasma and brain levels of 5-oxoproline (2.82 mM and 0.80 mM, respectively, determined by GC-MS) similar to those in GS-deficient patients. Total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), reduced glutathione (GSH) content, chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), carbonyl content, and the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in cerebral cortex and cerebellum of 14-day-old rats. Chemiluminescence, TBA-RS and carbonyl content were significantly enhanced while TRAP was reduced both in cerebral cortex and cerebellum. Catalase and glutathione peroxidase activities were significantly reduced only in the cerebral cortex. In contrast, TAR, GSH content and the activity of superoxide dismutase were not altered. These results indicate that the acute administration of 5-oxoproline causes lipid peroxidation and protein oxidation, and also impairs brain antioxidant defenses. Taken together, it may be presumed that the acute administration of 5-oxoproline elicits oxidative stress in cerebral cortex and cerebellum of young rats. If these effects also occur in the brain of patients affected by GS deficiency, it is possible that they may contribute, at least in part, to the neurological dysfunction characteristic of this disease. Financial support: CNPq, CAPES, Propesq/UFRGS, Pronex and Finep/IBN-Net (01.06.0842-00).
Upon UV-irradiation, the skin is exposed to photooxidative damage, which is induced by the formation of reactive oxygen species. Photooxidative damage affects cellular lipids, proteins, DNA, and is considered to be involved in the pathobiochemistry of erythema, premature aging, development of photodermatoses, and skin cancer. Photoprotection by topically or orally administered compounds such as carotenoids or flavonoids becomes a focus of life-long major interest. The aim of this research is to provide basic information on the photoprotective activities of natural and new synthetic carotenoids and flavonoids. Based on such information, the development of different photoprotectants can be initiated. New compounds, named “carotenylflavons” and differing in the polyene chain length, were synthesized. These compounds have a portion in their structure similar to carotenoid bounded to a flavonoid-like structure. Liposomes containing or not carotenylflavons were constructed and exposed to different UVB or UVA radiation doses. Astaxanthin and epicatechin-loaded liposomes were also constructed and tested for comparisons. Lipid peroxidation was evaluated through malonaldehyde (MDA) analysis, and phospholipids were measured. The unique structure of carotenoids with a system of conjugated double bonds determines their photoprotective activity, and is also responsible for their antioxidant properties. In contrast, these polyenes can act as a prooxidant, depending on the conditions and on their structure, mainly the end groups, chain length and position of methyl groups. The results demonstrated that the new synthetic compound with the longest conjugated system decreased the relative MDA formation when the liposomes were exposed to UVB, in a concentration-dependent manner, presenting a higher protection than astaxanthin. In the other hand, when the liposomes were exposed to UVA, lipid peroxidation was enhanced, demonstrating a high prooxidative activity. This effect was even higher than the prooxidative effect presented by astaxanthin-loaded liposomes in the same conditions. Epicatechin significantly reduces lipid peroxidation and did not show any prooxidant activity. Another compound similar to carotenylflavon, but with a shorter polyene chain, was also tested and it was verified an enhancement in the prooxidative activity. These results suggest that different mechanisms are involved in the UVA and UVB-induced lipid peroxidation in the liposomes containing these new synthetic polyenes.
Pathways linking oxidant to overall vascular cell stress are unclear. Recently we described close spatial/functional interdependence of NADPH oxidase and endoplasmic reticulum thiol oxireductase PDI in VSMC. Here we hypothesized that such pathway connects oxidative stress to UPR. UPR inducer Tunicamycin (Tn) (2mg/ml) led to eIF2α phosphorylation (Western) and, after 12 h, higher expression of Bip, GRP94 and calreticulin plus decreased levels of procaspase12. Cell loss occurred after 48 hs. HPLC analysis of dihydroethidium products showed Tn-induced increase (by 35%) in VSMC O2·- levels. Significant parallel decreases in SOD activity and GSH/GSSG ratio indicate oxidative stress at levels similar to known oxidase agonist angiotensin-II (100nM). Also, UPR induction by a novel TAP-direct peptide bearing N-glycosylation motifs, confirmed by increased Bip/GRP94 expression, strongly increased O2·- level (by 60%) after 2hs. Tn-mediated O2·- production was markedly decreased after UPR disruption promoted either by caspase12 siRNA or forced expression of GADD34 cDNA, which promotes eIF2α dephosphorylation. Conversely, induced catalase overexpression in VSMC decreased UPR markers after Tn exposure, indicating bidirectional UPR convergence to oxidative stress. Tn induced concentration and time-dependent increase in VSMC membrane NADPH oxidase activity, which correlated with increased Nox4 mRNA expression and PDI translocation to membrane fraction. VSMC membrane incubation with PDI inhibitors (DTNB, bacitracin, PDI antibody) led to decreased Tn-induced NADPH oxidase activity. Moreover, the role of PDI in O2·- production was further supported by gain and loss-of-function experiments with transfection of sense or antisense PDI cDNA. Redox western analysis suggested increased PDI oxidation with Tn vs. baseline or angiotensin-II. Nox4 siRNA decreased GRP94 protein expression, in parallel with decreased nuclear expression of CHOP(GADD153) monomer and induction of CHOP-C/EBP heterodimer transcriptional complex, thus suggesting a role for Nox 4 in VSMC terminal differentiation and/or apoptosis during UPR. In vivo pathophysiological relevance of such integrated stress response was assessed after vascular balloon injury, known to promote NAD(P)H oxidase upregulation. The neointima also co-overexpressed PDI and UPR markers and showed strongly increased level of procaspase12 cleavage. Thus, PDI/NADPH oxidase interaction couples UPR and oxidative stress in VSMC and may underlie cell differentiation/apoptosis in vascular disease. (Supported by FAPESP, CNPq-Milênio-Redoxoma).
Palabras clave: NADPH oxidase, Protein disulfide isomerase, Endoplasmic reticulum stress
| Poster Presentation | 06.Inflammation, Oxidants and Myeloperoxidase in Disease
Lipoteichoic acid stimulated nitric oxide production is regulated by A2A and A2B adenosine receptors in RAW 264.7 macrophages.
Souza, LF 1(*); Franke, C 1; Nunes, CC 1; Jardim, FR 1; Margis, R 1; Bernard, EA 1
1 - Departamento de Bioquímica - ICBS/UFRGS | (*) Brazil
Several studies have proposed the therapeutic role of agonists and antagonists of adenosine receptors in inflammatory conditions, mainly on account of their effect in the modulation of inflammatory mediators, like nitric oxide and tumor necrosis factor alpha, when macrophages were exposed to inflammatory stimulus, such as lipopolisacharide and interferon gamma. In the last years, the importance of gram-positive bacterial infections has increased, and are predominant in intensive care units. However, no studies have investigated the role of adenosine receptors in gram-positive sepsis. Lipoteichoic acid (LTA) is the main antigen of gram-positive bacterias and its ligation to Toll-Like receptor 2 stimulates inflammatory response in macrophages, including nitric oxide production. In this work was investigated the role of A2A and A2B adenosine receptors in lipoteichoic acid stimulated nitric oxide production in RAW 264.7 macrophages. LTA increased A2A and A2B adenosine receptors mRNA levels in 12 hours of treatment, with no effect at 24 hours. Extracellular ATP degradation was increased in 24 hours LTA stimulated macrophages, resulting in increased extracellular AMP and adenosine accumulation. No effect of LTA was observed in extracellular adenosine degradation. A2A and A2B adenosine receptors antagonists increased basal and LTA stimulated nitric oxide production. Additionally, an adenosine receptor unspecific agonist inhibited LTA stimulated nitric oxide production. This effect was blocked in the presence of A2A antagonist. These results suggest that A2A and A2B adenosine receptors are differentially involved in LTA stimulated nitric oxide production. The A2A adenosine receptor seems to be more important in adenosine down regulation of LTA stimulated nitric oxide production, becoming the main target for future pharmacological and therapeutic studies. FinancialSupport: CNPq
Mammalian mitochondria can degrade nitric oxide (NO) through a non-enzymatic reaction with superoxide anion (O2-) produced mainly by electron leakage from complex III of the respiratory chain. Plant mitochondria have unique components, such as external NAD(P)H dehydrogenases, the physiological importance of which is still incompletely understood. In this work, we analyzed O2- production and NO consumption activities of mitochondria isolated from potato tubers. Superoxide generation was monitored as the formation of peroxide detected with an amplex red fluorescent probe in the presence of superoxide dismutase (SOD). NO and oxygen concentrations in the reaction medium were measured simultaneously using electrochemical sensors connected to a free radical analyzer. When energized with NAD(P)H, potato mitochondria produced larger amounts of O2- than in the presence of succinate or malate. Higher SOD-sensitive NO degradation rates were also observed in NAD(P)H-energized mitochondria, resulting in a smaller, transient inhibitory effect of NO on oxygen consumption and on the safranine-estimated inner membrane electrical potential. In the presence of succinate or malate, mitochondrial O2- production and NO degradation were stimulated by antimycin-A, suggesting a role for complex III in this process. Although favored by antimycin-A, NAD(P)H-mediated O2- generation and NO consumption were not abolished by myxothiazol, indicating the existence of an additional mechanism for O2- production, independent of complex III. This NAD(P)H-dependent activity was unaffected by rotenone or capsaicin, but was attenuated by EGTA. These results exclude a contribution by complex I and suggest the participation of Ca2+-dependent external NAD(P)H dehydrogenases in this process. Overall, our results indicate that, in addition to electron leakage from complex III, NAD(P)H oxidation by alternative external proteins contributes to O2- generation that favors NO degradation by potato tuber mitochondria. These findings indicate the existence of an additional mechanism for preventing the deleterious effects of NO on respiratory activity in plant mitochondria. (Supported by FAPESP).
Mitochondrial ATP-sensitive K+ channels (mitoKATP) are regulators of mitochondrial reactive oxygen species (ROS) release. These channels mediate cardiac protection promoted by ischemic preconditioning. We have previously shown  that these channels are activated by mitochondrial ROS released by preconditioned hearts and cardiomyocytes. We now study the specificity of mitoKATP toward activation by ROS by following K+ uptake into isolated heart mitochondria using the swelling technique . We found that ATP-sensitive, K+-specific swelling attributable to mitoKATP was not enhanced by t-butil hydroperoxide, but was sensitive to nanomolar concentrations of H2O2 and low levels of superoxide radicals generated by xanthine/xanthine oxidase. ATP-sensitive K+ transport in heart mitochondria was also augmented by SNAP, suggesting mitoKATP are regulated by nitric oxide. Our data indicate that mitoKATP are activated by specific reactive oxygen and nitrogen species and support the idea that these channels act as redox sensors that regulate mitochondrial ROS release. Supported by FAPESP, CNPq, Redoxoma and the Guggenheim Foundation.  Free Radic Biol Med 2007 42:1039-48;  Am J Physiol 2001 280:H649-57
Palabras clave: mitochondrial, potassium channel, reactive oxigen species
| Poster Presentation | 10.Neurodegeneration and Neuroprotection
Evidence that 3-hydroxy-3-methylglutaric acid induces oxidative stress in cerebral cortex of young rats
Leipnitz, G. 1(*); Seminotti, B. 1; Haubrich, J. 1; Solano, A. 1; Bortoli, G. de 1; Amaral, A.U. 1; Dutra Filho, C.S. 1; Latini, A. 2; Wajner, M. 3
3-Hydroxy-3-methylglutaric aciduria (HMGA) is a disorder of leucine catabolism and ketone body synthesis, biochemically characterized by predominant tissue accumulation of 3-hydroxy-3-methylglutarate (HMG). The objective of the present investigation was to investigate the in vitro effect of HMG on various parameters of oxidative stress in rat cerebral cortex supernatants in order to clarify the pathogenesis of the brain injury in HMGA. It was observed that HMG induced lipid peroxidation by significantly increasing chemiluminescence and thiobarbituric acid-reactive substances (TBA-RS) levels. Moreover, pre-incubation of cortical supernatants with the antioxidant scavenger melatonin prevented the HMG-increased TBA-RS levels, suggesting that free radicals are involved in the lipid oxidative damage. HMG also induced protein oxidation in cortical supernatants. On the other hand, HMG significantly reduced total-radical trapping antioxidant potential, total antioxidant reactivity and reduced glutathione (GSH) levels, indicating a decrease of the non-enzymatic antioxidant defenses in rat cerebral cortex. Finally, HMG did not alter the activity of the main antioxidant enzymes. Our data is strongly indicative that oxidative stress is involved in the pathophysiology of the brain injury in HMGA Financial support: CNPq, FAPERGS, PRONEX and the FINEP research grant Rede Instituto Brasileiro de Neurociência (IBN-Net) # 01.06.0842-00.
Air pollution is a major environmental risk for human health. The mutagenicity of polar organic extracts collected from São Paulo city was recently investigated. The most mutagenic fractions contained ketones, aldehydes and quinolines. Higher levels of acetaldehyde and formaldehyde were found in the atmosphere of São Paulo. It is know that these aldehydes induce oxidative stress and cellular death. We have investigated cell death (apoptosis), lipid peroxidation and DNA damage in IMR-90 cells pre incubated with 20 μM lycopene for 2 h before a 3 h treatment with acetaldehyde (0.5 mM to 10 mM). Acetaldehyde caused lipid peroxidation in a dose dependent manner as monitored by the quantification of malondialdehyde (MDA) using HPLC coupled to fluorescence detection. A significant amount of DNA strand breaks, measured by the Comet assay, and apoptosis monitored by FACS were observed in cells treated with the aldehyde compared to control. Preincubation with lycopene protect against cell death and prevented biomolecule oxidative damage. Our results confirm that acetaldehyde is highly cytotoxic and that DNA oxidative damage can be an important causative agent of the mutagenicity of São Paulo’s atmosphere extracts. Our results indicate that lycopene reduce biomolecule damage induced by aldehyde exposition and possibly play a protective role against cancer risk associated with air pollution. Acknowledgements: FAPESP, CNPq, Instituto do Milênio Redoxoma.
Scientific interest in compounds from plant secondary metabolism has increased, since epidemiological studies suggest that diets with a high intake of vegetables, herbal extract and fruits may reduce the incidence of some degenerative diseases. Several classes of phytochemicals are examined in food the most notable are the polyphenolics. The reputed human health-promoting properties of polyphenolics lie in their antioxidant activities and anti-inflammatory activities. Small fruits like "guabiju" are rich in flavonoid poliphenolics including anthocyanins and flavonol glycosides. In this work, different lyophilized fruit of Myrcianthes pungens (Berg.) Lerg. from Embrapa- Clima Temperado- RS, were analyzed for anthocyanins (European Pharmacopeia, 2002), flavonoids, and total polyphenols (Brazilian Pharmacopeia, 2004), In addition, the radical scavenging activity of the compounds against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was studied by the method described by CAVIN et al. 2002. The total antioxidant capacity of different cultivars studied ranged from a low of 79.6% to 88.8% Trolox equivalents /mg of anthocyanins. These results indicate that extract of Myrcianthes pungens (Berg.) Lerg., can protect of reactive oxigen species. In conclusion there is a positive correlation between antioxidant activity potential and amount of phenolic compounds of the ethanolic extracts of guabiju. (CNPQ/BR, UFRGS/PROPESQ)
Palabras clave: guabijú, DPPH, anthocyanins
| Poster Presentation | 12.Antioxidant Interventions and Repair Systems
TOXICITY AND ANTIOXIDANT CAPACITY OF EUGENOL DERIVATIVES
Hidalgo, M.E. 1(*); De la Rosa, C. 1; Carrasco, H. 2; Cardona, W. 2
Several studies demonstrated the antioxidant capacity of the Eugenol and related compounds (like isoeugenol), to inhibit the lipid peroxidation induced by reactive oxygen species. Also inhibits the superoxide radical formation in the system xanthine-xanthine oxidase, as well as the generation of the hydroxyl radical, preventing therefore the Fe+2 oxidation in the Fenton reaction. The toxicity and antioxidant capacity of eugenol derivatives (E1= 4-Alyl-2-methoxyphenol, E2= 2-methoxy-4-[1-propenylphenyl]acetate, E3= 4-Alyl-2-methoxyphenylacetate, E4=4-Alyl-2-methoxy-4-nitrophenol, E5= 5-Alyl-3-nitrobencene-1,2-diol, E6= 4-Alyl-2-methoxy-5-nitrophenyl acetate) were evaluated in order to determine the influence of the sustituents. These derivatives (E1 to E6) were synthesized from eugenol (4-alil-2-metoxifenol). The acethylate compounds were obtained by the treatment with acetic anhydride and dimethylamine piridine (DMPA). The non acethylate nitrated derivative, was obtained by treatment with a mixture of sodium nitrate, hydrogen potassium sulphate and humid silice with dichloromethane as solvent at room temperature. Other nitrated compounds were obtained using a sulphonitric mixture at 0ºC. Antioxidant capacity evaluated by DPPH (1,1-Diphenyl-2-picrylhidrazile) and ORAC fluorescein demonstrated that E1 and E5 have a higher capacity and the minor toxicity evaluated by red blood cells haemolysis and the Artemia saline test. These compounds (E1-E5) could be used in the pharmaceutical, cosmetic and or food industries.
Palabras clave: eugenol, antioxidant, toxicity
| Poster Presentation | 09.Mitochondria and Apoptosis
SNP: Sodium NitroPrusside or Sham NO-dependent death Process?
Cardaci, S 1(*); Filomeni, G 1; Ciriolo, MR 2
1 - Dept. of Biology, “Tor Vergata” University, Via della Ricerca Scientifica, 1, I-00133 Rome, Italy | (*) Italy
In the nervous system, nitric oxide (NO) is involved in a huge number of physio-pathological processes, ranging from neurotransmission to neurotoxicity. The mechanisms underlying NO effects are investigated using NO donors with different half-life, chemical properties and kinetics of release. One of the most used NO-releasing drugs is the disodium nitroprusside (SNP), an iron nitrosyl complex, Na2[NO-Fe(CN)5], often reported to reproduce the deleterious effect of NO overproduction. Here we demonstrated that SNP-induced apoptosis in neuroblastoma cells is independent on NO production, since one-week light exhausted SNP triggers apoptosis at the same extent. Atomic absorption analyses of increased intracellular iron content, together with the rescue of cell viability by incubations with the iron chelator deferoxamine mesylate, indicate that the iron moiety of SNP is the principal mediator of apoptosis in neuroblastoma cells. Alterations in intracellular glutathione (GSH) levels, and increases in reactive oxygen species (ROS) and ROS-derived damages to proteins and DNA demonstrate that iron uptake is tightly associated with oxidative stress. Moreover, incubation with the hydroxyl radical (OH.) scavenger, dimethylthiourea suggests that OH., reasonably produced by an iron-mediated Fenton reaction, could be the genuine inducer of cell damages and death. Gastric adenocarcinoma cells, which are particularly enriched in GSH and GSH-related antioxidants, although sensitive to NO-mediated cell death, are completely resistant to SNP-induced injuries and apoptosis. Overall, our results are of particular importance in order to discern between toxicity mediated by chemical NO-donors and NO itself.
Palabras clave: nitric oxide, apoptosis, iron
| Poster Presentation | 09.Mitochondria and Apoptosis
Effects of two novel isatin-Schiff base copper(II) complexes on mitochondria and nuclei.
Filomeni, G 1(*); Cerchiaro, G 2; Da Costa Ferreira, AM 2; Rotilio, G 1; Ciriolo, MR 1
1 - Dept. of Biology, “Tor Vergata” University, Via della Ricerca Scientifica, 1, I-00133, Rome, Italy | (*) Italy
We previously demonstrated that newly synthesized isatin-Schiff bases copper(II) complexes are able to induce apoptosis owing to their characteristic chemical properties. In this study we selected the [Bis(2-oxindol-3-ylimino)-1,3-diaminopropane N,N’,O,O’] copper(II), and the Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N’]copper(II), named compound 1 and 3, respectively, and we further investigated their cytotoxic effects. Compounds 1 and 3 cross cell membranes and accumulate within organelles, as evidenced by atomic absorption analysis of mitochondrial and nuclear fractions. Fluorescence microscopy analyses of mitochondrial transmembrane potential show that both compounds 1 and 3 affect mitochondrial integrity, phenomenon which is associated with a time-dependent decrease in ATP levels. Nevertheless, polarographic assays of purified mitochondria evidence that both compounds increase O2 consumption, suggesting for them a role as uncoupler-like molecules. For what compound 3 concerns, a decrease in the 39-kDa subunit of Complex I and subunit II of cytochrome c oxidase is also observed in mitochondrial fraction, confirming a more toxic effect for this compound. Compounds 1 and 3 are also able to induce nuclear damage, as shown by DNA ladder appearance and the increase of the phospho-active form of the histone H2A.X, which is phosphorylated after DNA double strand break. Taken together, these results emphasize the selectivity of compound 1 and 3 in targeting cellular organelles, behaving as delocalized lipophilic cations (DLC)-like. molecules
Palabras clave: copper, mitocondria, DNA
| Poster Presentation | 06.Inflammation, Oxidants and Myeloperoxidase in Disease
(–)-Epicatechin elevates nitric oxide in HUVEC via O-methylation leading to inhibition of NADPH oxidase
Schewe, T. 1(*); Steffen, Y. 1; Sies, H 1
1 - Heinrich Heine University Duesseldorf, Institute of Biochemistry and Molecular Biology I | (*) Germany
Dietary (-)-epicatechin is known to improve bioactivity of ·NO in arterial endothelium of humans, but the mode of action is unclear. We used the fluorophore 4,5-diaminofluorescein diacetate to visualize the ·NO level in living human umbilical vein endothelial cells (HUVEC). Untreated cells showed only a weak signal, whereas pretreatment with (-)-epicatechin (10 µM) or apocynin (100 µM) elevated the ·NO level. The effects were more pronounced when the cells were treated with angiotensin II with or without preloading of the cells with ·NO via PAPA-NONOate. The increase in ·NO was paralleled by a corresponding increase in the level of cGMP, indicating that the elevated ·NO is bioavailable.
While (-)-epicatechin scavenged O2•-, its O-methylated metabolites prevented O2•- generation through inhibition of endothelial NADPH oxidase activity, even more strongly than apocynin. From the effect of 3,5-dinitrocatechol, an inhibitor of catechol-O-methyltransferase (COMT), on HUVEC as well as Western blotting with antibody against human COMT, it is concluded that (-)-epicatechin serves as "prodrug" for COMT-dependent conversion to apocynin-like NADPH oxidase inhibitors.
These data indicate an ·NO-preserving effect of (-)-epicatechin via suppression of O2•--mediated loss of ·NO, whereas expression and activity of endothelial ·NO synthase were not modulated by (-)-epicatechin or its metabolites under these conditions.
Palabras clave: Epicatechin, NADPH oxidase, NO imaging
| Poster Presentation | 10.Neurodegeneration and Neuroprotection
Effect of intracerebroventricular administration of 3-hydroxy-3-methylglutaric acid on lipid peroxidation and reduced glutathione levels in cerebral structures of young rats
Amaral, A.U. 1(*); Leipnitz, G. 1; Seminotti, B. 1; Beskow, A.P. 1; Ribeiro, C.A.J. 1; Haubrich, J. 1; Bortoli, G. de 1; Solano, A. 1; Dutra Filho, C.S. 1; Latini, A. 2; Wajner, M. 3
3-Hydroxy-3-methylglutaric aciduria is a neurometabolic disorder caused by deficiency of 3-hydroxy-3-methylglutaril CoA lyase. The disease is biochemically characterized by predominant accumulation and high urinary excretion of 3-hydroxy-3-methylglutaric acid (HMG). Considering that the pathophysiology of the neurologic damage of this disorder is not yet established, in the present study we investigated the effect of intracerebroventricular administration of HMG (5 µmol) to 30-day-old rats on parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBA-RS) and glutathione levels. The parameters were evaluated in cerebral cortex, hippocampus and striatum at 2 and 24 h after HMG injection. We observed that HMG significantly induced lipid peroxidation (increased TBA-RS levels) in hippocampus and striatum. Furthermore, HMG diminished GSH values in hippocampus. Our present preliminary results suggest that HMG induces lipid oxidative damage and a decrease of tissue non-enzymatic antioxidant defences in vivo. Taken together, the data indicate that oxidative stress might be involved in the pathophysiology of the brain injury occurring in 3-hydroxy-3-methylglutaric aciduria. Financial support: CNPq, FAPERGS, PRONEX and the FINEP research grant Rede Instituto Brasileiro de Neurociência (IBN-Net) # 01.06.0842-00.
Ligaria cuneifolia (Lc) is a hemiparasite species with anti-inflammatory and vasopressor activities, employed in Argentine folk medicine. Its pharmacological actions may be explained by the probable antioxidant activity of the phytochemical content. The aim of this work was to evaluate the in vitro and in vivo antioxidant activity of Lc extracts. We first assayed the crude, methanol, aqueous and ethyl acetate (EAF) Lc fractions with the ABTS and DPPH methods to estimate the in vitro hydrosoluble and liposoluble antioxidant capacity, respectively. All extracts tested showed significant antioxidant activities with both methods, being the EAF the one with the higher response. We then tested the EAF in in vitro assays with biological substrates and in vivo. The inhibition of the spontaneous chemiluminescence (CL) of rat brain homogenates and TBARS production in rat liver homogenates by the EAF showed, in both cases, a dose-dependent response. The EC50 values obtained from CL and TBARS were 26 mg/mL and 0.27 mg/mL of dried EAF, respectively. It is known that exposure of mouse skin to low dose UVA leads to oxidative stress, which is directly indicated by in vivo organ CL. Topical application with the EAF produced a 38% decrease in mouse skin CL after 30 min of irradiation (UVA irradiated: 42 ± 3 cps/cm2). The EAF of Lc proved to be an efficient in vitro and in vivo antioxidant. These properties may be one of the mechanisms by which Lc exerts its pharmacological actions.
The ORAC methodology (Oxygen Radical Absorbance Capacity) is widely employed for the evaluation of antioxidant capacity of complex mixtures present in human diet.1 This methodology is based on the competitive reactions of a target molecule (fluorescein) and antioxidants towards peroxyl radicals.2 However, the low reactivity of fluorescein in comparison with high reactive flavonoids, leads to ORAC indexes largely affected by stoichiometric factors. Recently, we have proposed the use of pyrogallol red (PGR) as target molecule to evaluate the reactivity of polyphenols (ORAC-PGR).3,4 In the present work, we have extended the use of ORAC-PGR to evaluate antioxidant capability of fruit extracts. Kinetics profiles, followed by UV-vis spectroscopy, correspond to two different pathways: - An induction time (raspberry), related to ascorbic acid concentration (evaluated using ascorbate oxidase), and - A reduced rate of PGR consumption (blackberry and blueberry), related to competitive reactions between PGR and phenols. ORAC-PGR is considered to be an appropriated method to estimate both, the reactivity of polyphenols and the presence of ascorbic acid in fruit extracts. References: 1. Perez D, Leighton F, Aspée A, Aliaga C, Lissi E. Biol. Res. 2000;33:71-77. 2. Ou B, Hampsch-Woodill M, Prior R. J. Agric. Food Chem. 2001;49:4619-4626. 3. López-Alarcón C, Lissi E. Free Radic. Res. 2005;39:729-736. 4. López-Alarcón C, Lissi E. Free Radic. Res. 2006;40:979-985. Acknowledgments: This work was supported by Vicerrectoría Adjunta de Investigación y Doctorado (VRAID), Pontificia Universidad Católica de Chile (DIPUC nº2006/28) and by FONDECYT (n°11060323).
Palabras clave: ORAC, Pyrogallol red, Fruit extracts
NADPH oxidase is a multicomponent enzyme which catalyses the generation of superoxide in phagocytes. Upon activation, a cytosolic complex consisting of the oxidase subunits p47phox, p67phox and p40phox together with Rac2 associates with the membrane-bound cytochrome b588 comprised of p22phox and gp91phox. NADPH oxidase activation is inhibitable by thiol oxidants, not affected by the global redox cell state. Thiol oxidoreductases are effective modulators of the thiol redox state on cell membranes, endoplasmic reticulum (ER) and cytosol. Since spontaneous thiol-disulfide exchange reactions tend to be sufficiently slow and nonspecific to account for efficient target protein regulation, we hypothesized that thiol oxidoreductases could regulate NAD(P)H oxidase activity. The goal of the present study was to investigate the role of protein disulfide isomerase (PDI), a ubiquitous multifunctional protein of the thioredoxin family, in the activation of neutrophil NAD(P)H oxidase. Western blotting confirmed robust PDI expression in neutrophils. PDI antagonism with bacitracin or scrambled RNase led to 60-100 % inhibition (p<0.05) of NADPH oxidase activity in a recombinant cell free system assessed with SOD inhibitable cytochrome c reduction. Neutrophil activation with phorbol myristate acetate (PMA) led to a significant increase in oxidase activity, which was followed by the translocation of PDI, p47phox and p67phox, from the cytosol to the membrane. Co-immunoprecipitation experiments using anti-PDI and anti-oxidase subunit antibodies indicated close spatial interaction between PDI and the oxidase subunits p67phox and p22phox. PDI is normally an ER protein that can migrate to plasma membrane. Immunofluorescence labeling showed that in neutrophils PDI is co-localized with p47phox, p67phox, p22phox and gp91phox. This co-localization is increased in PMA stimulated neutrophils. ER stress is a situation where PDI expression and/or membrane traffic is increased. Incubation of neutrophils with ER stressor tunicamycin (5mg/ml) led to marked time-dependent (0-120 min) increase in NAD(P)H oxidase activity (p< 0.05). Therefore, PDI displays spatial and functional interaction with the oxidase. In particular, PDI can modulate neutrophil NAD(P)H oxidase activation. Altogether, these results suggest that PDI closely associates with neutrophil NADPH oxidase and could act as a novel redox sensitive regulatory protein potentially affecting subunit translocation and superoxide generation.
Palabras clave: PDI, NADPH oxidase, neutrophil
| Poster Presentation | 06.Inflammation, Oxidants and Myeloperoxidase in Disease
Time course of nitric oxide synthases and oxidative stress in ovine sepsis
Lange, M 1(*); Enkhbaatar, P 1; Connelly, R 1; Traber, LD 1; Traber, DL 1
1 - University of Texas Medical Branch | (*) United States
Background: Previous studies revealed the important roles of different isoforms of nitric oxide synthases (NOS) and determinants of oxidative stress in the pathophysiology of cardiopulmonary derangements in sepsis, thereby offering potentially new treatment options such as inhibition of NOS or inhibition of reactive nitrogen species and poly(ADP-ribose) polymerase. When considering possible treatment strategies, however, it is crucial to identify the time changes of the expression of the pathogenic factors that are involved in the pathophysiology of sepsis. The present study was conducted to determine the time course of endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), 3-nitrotyrosine (3-NT), and poly(ADP-ribose) (PAR) in lung tissue using an established model of ovine sepsis. Methods: Twenty-four sheep were instrumented for chronic study. After inhalation of 48 breaths of cotton smoke, live Ps. aeruginosa was instilled into both lungs. Sheep were sacrificed at 4, 8, 12, 18, and 24 h after induction of sepsis (n=4 per group). Additional four sheep received sham injury and were sacrificed after 24 h. Results: In all sheep, PaO2/FiO2 ratio was below 200 at 18 h, indicating acute respiratory distress syndrome (ARDS). Lung wet-to-dry weight ratio, an indicator of lung water content, started to increase 4 h after injury and reached a peak at 24 h. Expression of eNOS was increased between 4 and 18 h after injury (p<0.05), reaching a peak at 12 h. Expression of iNOS was increased between 8 and 18 h (p<0.05), peaking also at 12 h. These changes were associated with a rise in plasma nitrite/nitrate levels (p<0.05). Expression of nNOS did not increase throughout the study. Expression of 3-NT, a stable marker of peroxynitrite, was increased between 4 and 12 h (p<0.05), peaking at 8 h. Expression of PAR started to increase 4 h after injury (p<0.05), peaking also at 8 h. Conclusion: The increased expression of eNOS and iNOS was associated with increases in both 3-nitrotyrosine and PAR expression. The time changes of oxidative stress correlated with the early development of ARDS and pulmonary edema in this model.
Mitochondria play an important role in the regulation of the apoptosis. Selenium compounds are known to act as quimiopreventive agents due its ability to oxidize thiol groups and generate reactive oxygen species (ROS) leading to a mitochondrial dysfunction, and consequently apoptotic cell death. Based on the ability of the diphenyl ditelluride, ebselen and diphenyl diselenide oxidize thiol groups, our objective was to study the effect of these organochalchogens on mitochondrial function, in order to establish a link between these organochalchogens and the apoptotic cell death via mitochondrial dysfunction. Ebselen (5 ìM), diphenyl diselenide (20 ìM) and diphenyl ditelluride (20 ìM) caused a rapid and almost complete mitochondrial depolarization measured by the Rhodamine 123 fluorescence. Associated with the depolarization, both ebselen (5 ìM), diphenyl ditelluride (20 ìM) and diphenyl diselenide (20 ìM) induced a mitochondrial swelling, and oxidation of the mitochondrial nucleotides (NAD(P)H). The effect of these organochalchogens was partially prevented by the reducing agent DTT. However, the organochalchogens-induced mitochondrial dysfunction was not prevented by ruthenium red (RR; 4 ìM), vitamin E (1 mM), or cyclosporin A (CsA; 1 ìM). All the studied compounds were able to induce the formation of protein aggregates, which were prevented by DTT. These results suggest that the studied compounds are able to induce mitochondrial dysfunction, as evidenced by the mitochondrial depolarization, swelling, NAD(P)H oxidation, and formation of protein aggregates. The effect of these compounds seems to be due to their ability to oxidize mitochondrial thiols groups, because the reducing agent DTT afforded some protection. Furthermore, their effects are not Ca2+ or ROS dependent as evidenced by the lack of effect of RR or vitamin E, respectively. The inability of CsA protect against the effect of this compounds suggest that the MPT pore opening induced by these organochalchogens are CsA insensitive. Supported by CAPES, FAPERGS, and CNPq
Palabras clave: Selenium, Tellurium, Mitochondria
| Poster Presentation | 09.Mitochondria and Apoptosis
MARINE SPONGE EXTRACTS INDUCES ROS PRODUCTION AND APOPTOSIS IN HUMAN U138MG GLIOMA CELL LINE
Frota Jr, MLC 1(*); Braganhol, E 2; Hoff, MLM 1; Delgado-Cañedo, A 3; Apel, MA 4; Mothes, B 5; Lerner, C 5; Battastini, AMO 2; Henriques, AT 4; Moreira, JCF 1
1 - Centro de Estudos em Estresse Oxidativo (CEEO), Depto. de Bioquímica, ICBS - UFRGS | (*) Brazil
Marine organisms have been prominently featured in the area of cancer research. In this work, we examine the effects of crude extracts (aqueous and organic) of marine sponge Polymastia janeirensis in the U138MG human glioma cell line. Briefly, when cells were exposed to sponge extracts ranging from 10 to 100 µg/ml for 24 hours, nuclear changes characteristic for apoptosis was observed at 10 µg/ml, as demonstrated by fluorescence microscopy; the higher doses were cytotoxic and necrosis cell death was observed. Furthermore, a dose-dependent increase in reactive oxygen species (ROS) production, a decrease in cell proliferation and cell viability, and an increase in annexin V-FITC binding were observed, as assayed by DCF, cell counting, MTT and flow cytometry, respectively. These results suggest that this marine sponge has metabolites which are capable of interfering with the survival of human glioma cells in culture, indicating that, at least in part, ROS production is required for herein presented data. Additional studies are required to understand the exact mechanism by which these extracts works to regulate ROS production and cell death in U138MG human glioma cell line, as well as its significance in a normal neuronal tissue. This study is part of a collaborative work among several Brazilian institutions (Centro de Estudos em Estresse Oxidativo, Depto. Bioquímica - UFRGS; Fundação Zoobotânica do Rio Grande do Sul - MCN; and Faculdade de Farmácia, UFRGS) for the collection and screening of Brazilian marine sponges for pharmacological activities, with the aim of identifying new sponge species and novel molecules with promising and potentially useful therapeutic activities. This work was supported by CNPq, CAPES, Fundação Zoobotânica do Rio Grande do Sul – Museu de Ciências Naturais, and PROPESQ – UFRGS. All experiments carried out comply with current Brazilizn laws.
Palabras clave: Glioma, Apoptosis, Marine sponges
| Poster Presentation | 12.Antioxidant Interventions and Repair Systems
Antioxidant defenses during the desiccation and reoxygenation cycles in the gastropod Nacella (P.)deaurata
Malanga, G 1(*); Calvo, J 2; Puntarulo, S 3
1 - 1Centro Austral de Investigaciones Científicas (CADIC-CONICET), Bernardo Houssay 200, 9410, Ushuaia, Tierra del Fuego, Argentina | (*) Argentina
Nacella (P.) deaurata is a limpet that inhabits the intermareal and the submareal area of the Beagle Channel. In the natural environment the limpets, experiment physiological alterations between water submersion and exposure to air. This study investigated the effects of desiccation and re-oxygenation on the generation of reactive oxygen species and the activity of antioxidant enzymes in digestive glands of N. (P.) deaurata. Sampling was carried out in September 2004 in Punta Occidental (54°50´S, 68°20´W) at the Beagle Channel. The limpets were exposed to air during 5 h, then they were submerged in seawater for periods of 15 to 180 min under the same salinity and temperature conditions that in their natural environment. Immediately after each treatment the digestive glands were separated and conserved at -30ºC. The control limpets were placed in oxygen-saturated seawater over the same time period. Oxygen radical generation in limpets, evaluated as DCF-DA oxidation, showed an increase over the control values of 138%, and 48% after 15 and 30 min of re-oxygenation, respectively. DCF-DA oxidation returned approximately to control levels after 180 min of re-oxygenation. The activities of the antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) were spectrophotometrically determined. After 5 h of drying, the activity of superoxide dismutase in digestive glands decreased by 28% as compared to control values. After 30 min of rehydration superoxide dismutase activity increased by 39%, and returned to control values after 3 h of rehydration. Both catalase and glutathione peroxidase activities showed a similar profile. The results presented here indicate that the enzymatic antioxidant defenses of these marine organisms are a complex adaptive system to preserve cellular functioning that respond to the oxygenation cycles and dependent on the tides. This study was supported by grants from the University of Buenos Aires, ANPCYT and CONICET
Palabras clave: , ,
| Poster Presentation | 06.Inflammation, Oxidants and Myeloperoxidase in Disease
OXIDATIVE STRESS IN ELDERLY RATS SUBMITTED TO A VENTILATORY STRATEGY
Cavassani, SS 1(*); Junqueira, VBC 2; Oliveira-Júnior, IS 1
1 - Universidade Federal de São Paulo | (*) Brazil
Mechanical ventilation (MV) is critical for life support in patients who cannot maintain adequate alveolar ventilation. We propose in this paper that increases in lung oxygen concentration, due to MV, may result in increasing oxygen reactive species production leading to alveolar damage, mainly in the elderly. For this purpose we have studied in elderly and adult rats the effects of short-duration MV on lung inflammatory neutrophils and lipid-derived oxidation products. Adult (3-5 mo) and elderly (18-20 mo) Wistar rats were divided into 4 groups: non-ventilated adults (NVA), ventilated adults (VA), non-ventilated elderly (NVE) and ventilated elderly (VE). The ventilatory strategy was: 3 hs, VT:6 mL/Kg bw, PEEP: 5cm H2O, RR:50-60 breaths/min, respiratory flow:10mL/s and FiO2:1.0. Elderly rats showed a marked neutrophil infiltration in the lung (expressed as % of neutrophils in lugn-washing medium) (NVE: 19±2.5, NVA: 11±4.5; p<0.05) which markedly increase after MV (VE: 38±3, VA: 26±3; p<0.05). Lipid-derived oxidation products, as measured by thiobarbituric acid reactants (LPO), was found increased in NVE animals (268±4 ìM) as compared to VA (111±3 ìM) (p<0.05). MV elicits an LPO significant increase in the lung of elderly animals (VE:715±8 ìM) as compared to adult animals (VA: 354± 15 ìM) (p<0.05). This ventilatory strategy leads to a more marked increase in inflammatory cells in the lung of elderly animals that may be responsible for the increased lipid-derived oxidation products. Moreover, this increase was found to be related to a decrease in the corticosterone levels in elderly rats (NVA:107±12 ng/mL; NVE:84±9 ng/mL; p<0.05). Financial support: FAPESP (grant 06/60834-9). I.S. Oliveira-Jr is a CAPES pos doctoral fellowship recipient.
In the southern South America the subantarctic stone crab Paralomis granulosa constitutes the most important crustacean in the region. After their capture, animals spend several hours alive exposed to air on deck until they arrive to the fishery company where they are processed. As a direct consequence of interrupted water flow through the gills, the reactive oxygen species start to increase triggering oxidative stress. Hence, the goal of this work was to determine the levels of antioxidant enzyme activities due to air exposure in different tissues of P. granulosa. During winter 2004, 50 male crabs of legal size (carapace length >82 mm) were captured in Beagle Channel (54º 50´S, 68º 20´W). Experiments of air exposure were done using five groups of 10 crabs each, where each one was exposed to dryness at 8 ºC for 0, 3, 6, 12 or 24 h, respectively. Samples of gills, muscle, hepatopancreas and haemolymph were analized to determine enzymatic activity of superoxide dismutase (SOD), catalase (CAT) and glutathione-S transferase (GST). Furthermore, protein oxidation (PO) and lipid peroxidation (LPO) levels were measured in the same tissues.
The activity of the enzymes involved in the antioxidant defense of P. granulosa varied with the time of air exposure. The maximum enzyme activity was obtained between 3 and 6 h of air exposure. After that, values decreased to those observed at time 0. CAT and SOD showed coordinated activity in muscle, hepatopancreas and haemolymph. Gills were the only tissue where SOD activity decreased with time of air exposure. PO levels varied significantly in gills. LPO levels increased significantly in muscle and hepatopancreas. The high activity of antioxidant enzymes registered during anoxic condition was named preparation for oxidative stress. This process seems to be the key to minimize tissue damage during reoxygenation, since tissues are prepared to deal with oxyradicals. The preparation for oxidative stress in a crab that does not inhabit intertidal coastal as P. granulosa is still intriguing, and could be a feature of the intertidal lineage of this group that has been retained by the current species. Supported by CONICET, ANPCyT, and DNA.
Palabras clave: oxidative stress, antioxidant enzymes, air exposure
| Poster Presentation | 10.Neurodegeneration and Neuroprotection
L-Tyrosine decreases antioxidant defenses in rat brain in vitro
Sgaravatti, A. M. 1(*); Vargas, B. A. 1; Zandoná, B. R. 1; Deckmann, K. B. 1; Rockenbach, F. J. 1; Zanin, F. R. 1; Meska, C. P. 1; Scapin, F. 1; Vaz, B. V. 1; Streck, E. S. 1; Sgarbi, M. B. 1; Pederzolli, C. D. 1; Wajner, M. 1; Dutra-Filho, C. S. 1
Tyrosinemia type II is an inherited metabolic disease caused by a deficiency of hepatic cytosolic tyrosine aminotransferase (TAT) that leads to increased plasma and tissue tyrosine levels, which in turn are associated with eye, skin, and neurological alterations. However, the mechanisms underlying the brain damage are poorly known. Thus, in the present study, the in vitro effect of 0.1-4.0 mM L-tyrosine was investigated on the following oxidative stress parameters in cerebral cortex homogenates of 14-day-old Wistar rats: total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), thiol content, carbonyl groups, as well as the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). TRAP and TAR measurements were markedly diminished by L-tyrosine, as well as total thiol content and CAT activity. In contrast, chemiluminescence, TBA-RS levels, carbonyl groups, mitochondrial membrane protein thiol content, and SOD and GPX activities were not affected by L-tyrosine. These results indicate that L-tyrosine in vitro decreases enzymatic and non-enzymatic antioxidant defenses in cerebral cortex of young rats which became the CNS prone to reactive species. This suggests that oxidative stress may represent a pathophysiological mechanism in TAT-deficient patients, in which this amino acid accumulates. Financial support: CNPq, CAPES, Propesq/UFRGS, Pronex and Finep/IBN-Net (01.06.0842-00).
Palabras clave: L-tyrosine, antioxidant defenses, rat brain
| Poster Presentation | 12.Antioxidant Interventions and Repair Systems
Antioxidant activity of new pyrazoline derivatives
Torres, J V 1(*); Martins, D M 2; Spohr, P R 1; Machado, P 3; Emanuelli, T 2; Martins, M A P 3; Zanatta, N 3; Bonacorso, H G. 3
1 - Department of Alimentary Technology and Science - Federal University of Santa Maria | (*) Brazil
Reactive oxygen species can induce some neuronal damage and cause disorders as Parkinson’s and Alzheimer’s disease. In the current study, we extended our investigations on the antioxidant action of pyrazole derivative compounds (5-trifluoromethyl-4,5-dihydro-1H-pyrazoles). Thus, we investigated the antioxidant effect of six newly synthesized compounds: pz1=3-metil-5-triflorometil-4,5-diidro-1H-formil-pirazol, pz2=3-metil-5-hidroxi-5-triflorometil-4,5-diidro-1H-1-carboximetil-pirazol, pz3=3-metil-5-triflorometil-4,5-diidro-1-H-1 carboxiamidepyrazole, pz4=3-metil-5-hidroxi-5-triflorometil-4,5-diidro-1H-1- benzoil-pirazol, pz5=3-metil-5-hidroxi-5-triflorometil-4,5-diidro-1H-1-(2-hidroxibenzoil) and pz6=3-metil-5-hidroxi-5-triflorometil-4,5-diidro-1H-1-carboxi(4-metoxifenil)-pirazol (0, 15, 30 or 150 µM); against lipid peroxidation (thiobarbituric acid reactive substances-TBARS) and against glutathione (GSH) oxidation in the absence or presence of H2O2. For TBARS assay, cerebral tissue was homogenized and incubated at 37°C for 1h in the absence or presence of 50µM FeCl2, 1mM H2O2 or 5 µM sodium nitroprusside (SNP) and pyrazole compounds. All compounds evaluated were effective to inhibit TBARS formation with the exception the compound pz1. Pz5 was the most effective compound to inhibit TBARS. Only pz5 (150µM) prevented GSH oxidation in the presence of H2O2 (3.96 vs. 41.16% GSH degradation in the absence of the compound), while the others compounds did not prevent GSH oxidation in the presence or absence of H2O2. These results indicate the antioxidant power of pz5. Further investigations are required to elucidate its mechanism of action. Keywords: TBARS, Ferro, Glutathione
Palabras clave: TBARS, Ferro, Glutathione
| Poster Presentation | 12.Antioxidant Interventions and Repair Systems
PENTOXIFYLLiNE REDUCE OXIDATIVE STRESS IN HYDROCHLORIC ACID-INDUCED LUNG INJURY
Oliveira-Júnior, IS 1(*); Oliveira, WRS 2; Maganhin, CC 2; Carbonel, AAF 2; Simões, MJ 2; Monteiro, CMR 3; Barreiro, E 4; Junqueira, VBC 5
1 - Univerisdade Federal de São Paulo | (*) Brazil
Gastric aspiration is a frequent occurrence in unconscious patients and in the development of ARDS (mortality rate up to 30%). Pentoxifylline (PTX), a phosphodiesterase inhibitor, has been shown to decrease inflammatory mediators concentration in the lung. This study was performed to analyze the effects of pre- and/or post-PTX treatment and mechanical ventilation (MV) with low tidal volume (VT) in a hydrochloric acid-induced lung injury model. Adult male Wistar rats were anesthetized and received bilateral instillation of hydrochloric acid (HCl) and pre- or post-treatment with PTX followed by 3 hrs of MV. The ventilatory strategy was: VT 6 mL/kg, PEEP 5 cmH2O, RR at 50-60/min, FiO2 at 1. Rats were divided into 4 groups (n=7 each): Control: only MV; HCl: HCl-instillation and MV; HCl+PTX: PTX infusion 10 min after HCl instillation and MV; and PTX+HCl: PTX infusion performed 10 min before HCl instillation and MV. Measurements were done in bronchoalveolar lavage (BAL) or lungs. PTX elicited a decrease in total protein, as well as in wet-dry ratio, and in TNF-α in the HCl+PTX group (36±3 mg/mL, 11.2±1.8 g, 329±44 pg/mL, respectively) being more evident in PTX+HCl group (28±3 mg/mL, 8.6±0.5 g, 249±59 pg/mL, respectively). Corticosterone level was increased in the HCl+PTX (16±2 ng/mL) and PTX+HCl (27±2 ng/mL) as compared to HCl group (7.6±1.3 ng/mL). PTX treatment of animals either before or after, elicited a decrease in lipid-derived oxidation products (LPO) (HCl; 869±7 ng/mL; HCl+PTX: 688±26 ng/mL; PTX+HCl: 481±21; p<0.01). In conclusion, PTX pre-treatment decreased the levels of an inflammatory mediator in BAL, as well as in LPO levels, by ameliorating the corticosterone level. Financial support: Fapesp (grant: 03/14098-0), CAPES and CNPq.
Changes in the production of reactive oxygen species on aquatic organisms had been related to environmental or physiological variations. Carotenoids are often found among sea invertebrates protecting their lipid constituents against oxidation.and influencing on their coloration. Loxechinus albus, is a edible sea urchin inhabiting the Beagle Channel. In this study, dietary carotenoids were fed to L. albus as prepared diets to test their effects on the sea urchin´s gonads oxidative metabolism. The animals were fed with one of the following diets: (A) enriched with all-trans β-carotene 0.02%; (B) enriched with all-trans β-carotene 0.045%, or (C) control diet (macroalgae) for 16 weeks. The artificial diets had the same basic content of protein, lipid, carbohydrate, ash and energy. Total carotenoid concentration in the gonad were measured spectrophotometrically. The content of lipid antioxidant (a-tocopherol and b-carotene) was quantified by reverse-phase HPLC. At the end of the tested period (16 weeks) the gonads did not showed any differences in either total carotenoid concentration or b-carotene content from animals feed with diet A, B or C. However, both parameters showed two fold increase in the gonads isolated from animals feed with diet B as compared to either animals control or animals feed diet A, after the initial 8 weeks of treatment. Over the feeding period (16 weeks) the a-tocopherol content increased in gonads from an value of 0.22±0.05 nmol/mgFW in animals control to 0.95±0.33 and 1.1±0.5 nmol/mgFW for diet A and B, respectively. The content of TBARS, as an index of lipid peroxidation, showed an initial value of 19±2 nmol/mgFW, and was decreased after 16 weeks of treatment to 7.6±0.9, 15 ±4 and 17±2 nmol/mgFW for the animals feed with diet A, B and C, respectively). Overall, the results presented here suggest that the exogenous supplementation with all-trans β-carotene 0.045% over an 8 weeks period afforded the maximum level of protection against lipid peroxidation since endogenous cellular protection mechanisms could be triggered under those conditions, and further increases in either antioxidant content or time of supplementation did not lead to increase in protection. Supported by University of Buenos Aires, ANPCYT, CONICET and PNUD ARG/02/018
In a previous work, in a rat liver preneoplastic model, we have demonstrated that IFN-α2b reduces the number and volume of altered hepatic foci (AHF) inducing apoptosis through a mechanism mediated by TGF-β1. In this study, it was analyzed the involvement of reactive oxygen species (ROS) on the apoptotic mechanism induced by IFN-α2b in rat preneoplastic liver. For in vitro studies, male Wistar rats were subjected to a 2-phase model of hepatic preneoplasia. Hepatocytes were obtained and cultured at different times: a) without any treatment, b) with IFN-α2b, c) with IFN-α2b plus anti-TGF-β1, d) with IFN-α2b plus ascorbic acid (ASC). IFN-α2b induced increase of TGF-β1 mRNA levels at 4 h of culture and a two-fold increase of TGF-b1 secretion at 7h of culture. ASC totally abolished the increase of TGF-β1 mRNA levels, and TGF-β1 levels in the culture media remained unchanged. IFN-α2b-treatment increased hepatocytes apoptosis since 20h of culture onwards. ASC or anti-TGF-b1 completely abolished it. After IFN-α2b treatment there were 2 peaks of ROS at 1h and 9h of culture. Anti-TGF-β1 did not block the production of the first peak of ROS whereas totally blocked the appearance of the second one. ASC abolished the production of both peaks. IFN-α2b induced NADPH oxidase activity increase at 1h of culture, reaching a maximum at 9h. Anti-TGF-β1 did not block activation at 1h, but did at 9h. Always, ASC totally blocked NADPH oxidase activation. Then, we determined the relevance of ROS on the onset of the apoptotic process in vivo in the whole preneoplastic liver. Treatment of preneoplastic rats with IFN-α2b + ASC abolished the IFN-α2b apoptotic effects observed in IFN-α2b-treated rats. Results indicate that IFN-α2b induces hepatocytic TGF-β1 production and secretion by induction of ROS formation through the activation of a NADPH oxidase complex. TGF-β1, induces programmed cell death. It was also demonstrated that treatment of rats with IFN-α2b plus ascorbic acid, abolishes the apoptotic effect of IFN-α2b in rat preneoplastic livers, increasing the foci volume. In conclusion, these findings strongly suggest that ROS have a fundamental role as signaling molecules in the IFN-α2b-induced apoptosis in hepatic preneoplastic cells.
Thioredoxin-1 (TRX-1) is a redox active protein that mediates reduction or oxidation of disulfide bonds. TRX-1 exists in the extracellular, cytoplasmic and nuclear compartments. In cells exposed to oxidants TRX-1 migrates to the nuclear compartment. In addition, cellular localization of TRX-1 was found to be associated with the expression of the Thioredoxin Interacting Protein (TXNIP). Our group has observed that nuclear translocation of TRX-1 is induced by SNAP (NO-donor) and involves the p21Ras-ERK1/2 survival pathway. The present study investigated TRX-1 localization in HeLa cells exposed to SNAP and treated with PD98059, a MEK inhibitor. Confocal microscopy showed that in cells exposed to PD98059, SNAP-induced TRX-1 nuclear migration was inhibited. In order to evaluate TXNIP expression and its effects on TRX-1 localization, real time RT-PCR experiments were performed. After stimulation with SNAP and PD98059, we observed that TXNIP levels were up-regulated, suggesting that ERK1/2 participates in the regulation of TXNIP expression. We have also developed a TXNIP promoter-fusion-GFP construction to analyze the TXNIP regulation. A fragment ~2000bp of the 5´ region of TXNIP was subcloned in a pEGFP vector and transfected into HeLa cells. Further studies using the TXNIP-based construction will be presented. In conclusion, our results suggest that ERK1/2 and TXNIP are involved in TRX-1 nuclear translocation process. Financial support was provided by FAPESP and CNPq/Milênio Redoxoma.
Recently, we demonstrated that the hydro-ethanolic extract of Bauhinia microstachya leaves (HEBM) is a source of phenolic compounds and the properties to inhibit lipid peroxidation, scavenge hydroxyl and superoxide radicals observed in vitro are linked to phenolic content and phytochemical profile. In this study, we have investigated the effects of a single intragastric dose per day of HEBM in a short-term treatment and during 4 weeks in alloxan-induced diabetic rats. After diabetic model confirmation by immunohistochemistry (insulin antibody) some rats received different doses of HEBM (0.01, 0.1 and 1 mg/Kg) or saline. Blood glucose was determined before and intervals following the treatment. Doses of 0.1 and 1 mg/Kg reduce the glycemic levels around of 23% 120 min after oral treatment, however, only 1 mg/Kg increase the plasma antioxidant potential quantified by TRAP assay. In other hand, long-term treatment (1 mg/Kg) not showed significant effects in blood glucose levels, in plasma antioxidant potential, in addition, diabetic animals treated with HEBM lost weight in manner similar to diabetic-saline group. Catalase (CAT) and superoxide dismutase (SOD) activity were determined in liver, kidney and gastrocnemius muscle and when an imbalance in the SOD/CAT ratio was observed an increase in lipid peroxidation was detected by TBARS assay. The treatment reduced tissue lipid damage and HbA1c; however, the HEBM increased plasma lipids, ALT and AST activity and decreased liver glutathione s-transferase activity. Hyperglycemic altered the accumulation of glycogen in tissues and HEBM modulated it in target organs. Our results suggest that HEBM treatment in alloxan-induced diabetic rats posses hypoglycemic properties and in vivo antioxidant effect in an acute investigation. However, in long-term study, only tissues damage was avoided by HEBM related to SOD/CAT modulation. Reduction in HbA1c and blood glucose associated to glycogen content variation by HEBM treatment may be related to popular use of B. microstachya leaves against diabetes as well as another species from Bauhinia genus, however, the increase in plasma lipids, ALT and AST activity and the decrease in liver glutathione s-transferase activity may be an indicative of liver damage. Supported by: CNPq, FAPERGS and PROPESQ-UFRGS.
A great number of experimental evidences have indicated a key role for reactive species (RS) as cytotoxic agents. An imbalance between the antioxidant defenses and RS generation can cause cellular damage. Oximes are a class of compounds normally used to reverse the acetylcholinesterase inhibition caused by organophosphates. More recently, they are being considered as a second generation antidepressant. Conversely, researches focusing on the possible antioxidant properties of these compounds are lacking in the literature. The aim of this study was to investigate the capacity of 3-(phenylhydrazono)butan-2-one oxime to prevent the damage induced by RS and its potential toxicity to mice. Our findings indicate that the oxime did not modify d-ALA-D activity in vitro and ex vivo. The in vitro experiments depicted that peroxidation of lipids induced by H2O2 presented a significant reduction by the oxime at 0.1mM (p < 0.05) and the lipid peroxidation induced by malonate and iron was significantly decreased at 0.4mM (p< 0.05), indicating a antioxidant action of the oxime. Ex vivo results depicted that oxime pre-treatment was unable to decrease the basal peroxidation level or prevent the induced lipid peroxidation. The treatment with oxime did not change non-protein thiol levels ex vivo. The data obtained in vitro suggest that oximes could be a good antioxidant compound, once that presents an effective antioxidant activity in vitro. Further studies are needed to elucidate the exact antioxidant mechanism of action of oxime as well as to investigate its pharmacological proprieties. In conclusion, our data show that some metabolic processes are required to produce molecules derived from the oxime with antioxidant properties and the metabolite is probably not toxic at all.
Palabras clave: oximes, antioxidant, oxygen reactive species
| Poster Presentation | 03.Redox Biology: Metals and Thiols
Influence of the coordination on the redox activiy of Cu(dipeptide) complexes.
Copper metalloproteins are involved in various biological functions such as electron transfer and substrate oxidation. These functions depend on the redox properties of the copper species, modulated by the coordination around the copper (coordinating index, type of ligands and geometry of the environment). In proteins this is achieved by the variation of the amino acid ligands and their arrangement at the active site. As a part of our research devoted to the study of the structure, reactivity and biological activity of copper complexes as models, the redox behaviour of a series of Cu(L-dipeptide) complexes, determined by cyclic voltammetry, is presented and related with their structure in solution. Besides, superoxide scavenger activity was evaluated as the SOD mimetic activity. As shown in the following table, the studied complexes presented two different voltammetric behaviours.
From these data, it can be observed that the coordination of the deprotonated amidic nitrogens (which occurs in the complexes of the first group leads to the decrease of the Cu(II)/Cu(III) potential (usually higher), thus rendering the Cu(III) state more easily obtainable. Besides his-gly and his-ala complexes stabilized the Cu(II) species. All the studied complexes are able to scavenge superoxide radicals, as shown by their SOD mimetic activity.
Diethyl-2-phenyl-2-tellurophenyl vinylphosphonate (DPTVP) is an organotellurium compound which is very low toxic to mice, even when administered for 12 days by the subcutaneous or intraperitoneal routes. Also, its antioxidant activity was already demonstrated in vitro and ex vivo, at low concentrations/doses. Neverteheless, the mechanisms which underlies these effects are not well-understood. In view of this, we aimed to investigate the ability of this compound to scavenge some reactive oxygen/nitrogen species, as hydrogen peroxide (H2O2), hydroxyl radical (OH.) and radical anion nitric oxide (NO.). For this purpose, we performed, in vitro, the dichlorofluoresceine diacetate (DCF-DA) oxidation assay (for H2O2 and OH. scavenger activity) DPPH assay (specific for OH. scavenging) chemiluminescence assay (specific for H2O2 scavenging) and NO. scavenger activity assay. Liver, kidney and brain homogenates of mice were used for the DCF-DA oxidation experiments. DPTVP was prepared in DMSO, ethanol or methanol, according with the protocol of the assays in the literature, and incubated at concentrations ranging 1-100µM. We observed that DPTVP showed H2O2 scavenger activity, since it was able to decrease the oxidation of DCF-DA in all tissues, as well as to decrease the chemiluminescence of luminol in the presence of H2O2(p<0.001). Besides, the decrease in the DCF-DA oxidation demonstrates that the organotellurium compound also possesses OH. scavenger activity, associated with the fact that the DPPH generation of OH. was also decreased(p<0.001). On the other hand, DPTVP did not demonstrated ability to scavenge the NO. in any of the tested concentrations. Interestingly, the scavenger activities in the absence of tissue were significantly observed only from 50 µM, which is 25 fold higher than obtained previously on TBARS and protein carbonilation assays and for DCF-DA oxidation, in which tissue is incubated with compound and with prooxidant agents. This lead us to believe that the compound is probably metabolized in tissues, generating the telluroxide form of the compound, which is described in the literature as the antioxidant form of organotellurium compounds.
Skeletal muscle tissue injuries are commonly characterized by an increase in reactive species (RS) generation. The mechanism of muscular lesions frequently involves a strain injury to muscle cells structure. In this study we investigated the oxidative damage induced by a partial muscular strain injury model in rats, and the potential antioxidant profile of heat and cold therapeutics modalities. This study was performed with 81 Wistar rats divided into 6 groups. 1: basal group not submitted to the standard muscular strain injury (n=15); 2: sham group submitted to standard chirurgic procedure but without lesion (n=15); 3: control group submitted to muscular lesion without any treatment (n=15); 4: therapeutic cold (n=12); 5: therapeutic heat (n=12); and 6: therapeutic heat + therapeutic cold (n=12) groups submitted to the standard lesion followed by the indicated treatments. The biochemical analyses were performed immediately after the standard lesion, and after one, five, ten, or fifteen days of treatment. The standard strain injury elicited a significant increase in plasma creatine kinase (CK) levels (p≤0.05), and the therapeutic cold treatment was able to maintain the basal values (p≤0.05). Our data reveal a significant increase in dichlorofluoreceine diacetate (DCFA-DA) oxidation both in muscle, plasma and erythrocytes preparations under control conditions in comparison to basal or sham procedure (p≤0.05). The malondialdehyde (MDA) levels were also significantly increased both in muscle and total blood preparations in animals submitted to standard strain injury muscle (p≤0.05). In addition, the standard lesion elicited a significantly increase in muscle catalase activity (p≤0.05). The therapeutic cold was able to effectively counteract the increase in DCFA and MDA levels both in muscle and blood preparations (p≤0.05). Moreover the muscle catalase activity in cold treated group was significantly lesser than in control group. The obtained results point to a possible antioxidant effect of therapeutic cold in counteracting the oxidative damage resulting from RS generation in a standard strain injury model induced in rats.
Palabras clave: antioxidant, cold, heat
| Poster Presentation | 12.Antioxidant Interventions and Repair Systems
Exploratory study of Vitamin C, E and beta-carotene consumption in an aleatory population from Valparaiso, V region, Chile.
Exploratory study of Vitamin C, E and beta-carotene consumption in an aleatory population from Valparaiso, V region, Chile. María José Muñoz & María Eliana Hidalgo. Faculty of Science, University of Valparaíso, Chile. email@example.com, firstname.lastname@example.org This work tries to show a quantitative vision on the food consumption with greater antioxidant amounts. The data were obtained from a nutritional survey, validated by the WHO, that was applied to 53 patients who are made the analyses of Oxidative Stress and Antioxidant defences in the Free Radicals Laboratory of the University of Valparaiso. Vitamin E consumption from the main food sources was : tinned tuna (0,4 mg*day), olive oil (0,4 mg*day) and sunflower oil (0.6 mg*day), considering for each case, as recommended doses: 1,1, 0,6 and 1,2 mg*day, respectively. Vitamin C consumption from the foods that represented mayor contribution were: cereals (1,6 mg*day, white cabbage (7,2 mg/day), red ground pepper (31,1 mg*day) and oranges (30,5 mg/day), being calculated optimal dose: 53, 25,7, 61,2 and 73 mg/day, respectively. Finally the beta-carotene sources were concentrated in red beets (322 mcg/day), tomatoes (268,3 mcg/day),ground peppers (201,1 mcg/day) and carrots (2333,4 mcg/day), recommended doses are 1360, 499, 395,6 and 6107,1 mcg/day respectively. The shown results demonstrated the low level of antioxidant consumption from their natural source, although the people who included themselves in this survey are worried about their oxidative level, since voluntarily they asked for the mentioned analysis , demonstrating the higher risk to which they can be exposed to suffer diseases related to oxidative stress and the undesirable diet common in our country.
Palabras clave: Vitamin C, E, betacarotene, consumption
| Poster Presentation | 05.Nitric Oxide, Peroxynitrite and Oxidants in Redox Vascular Biology
Glycolaldehyde impairs coagulation in a free radicals independent way.
Andrades, Michael Éverton 1(*); Lorenzi, Rodrigo 2; Fonseca Moreira, José Cláudio 3; Dal Pizzol, Felipe 4
Glycolaldehyde (GA) is generated physiologically and pathologically by several pathways, such as glucose oxidation, glycated proteins, lipid peroxidation, and oxidative attack on amino acids. This aldehyde is highly reactive and is known to produce superoxide. Since the sources of GA are highly activated in pathologies that present coagulation dysfunction, we aim to investigate what effects glycolaldehyde has on the intrinsic, extrinsic and fibrinolysis pathway. Further, we investigate the role of free radicals in this process. Human plasma (n=6) incubated in presence of GA (1mM) for 8 hours had coagulatory parameters - Activated Partial Thromboplastin Time (aPTT), Prothrombin Time (PT) and Thrombin Time (TT) - delayed when compared with plasma incubated in absence of GA (control). Moreover, incubation of samples with aminoguanidine (AMG) - an anti-glycating agent - reverted the effects of GA. Since oxidative damage impairs coagulatory pathway, control and GA plasma were incubated in presence of antioxidants (2mM glutathione, 1mM vitamin C and 0.1mM deferoxiamine) and challenged with GA but we found no inhibitory effect, suggesting no involvement of ROS. To access the stability of the fibrin clot, we triggered the coagulation using 10mM CaCl2 and after the clot formation we added trypsin (1.7-17ug/mL). Absorbance was followed up to 4 hours in a microplate reader (405nm). Fibrin clot formed in GA treated plasma was more resistant to degradation than control plasma. Despite the absence of evidences for free radicals participation in our study, we found - 8-fold increase in carbonyl in GA treated plasma when compared with control, which was reversed by AMG. Taken together, our results indicate that despite delaying coagulation, GA stabilizes clot in a ROS independent way. The role of GA in inflammatory pathologies is not well understood, but it seems that it may participate on thrombus generation, contributing to hematological dysfunctions seen in diabetes and sepsis. Supported by: CNPq,
Taurine, a sulfur-containing amino acid that is not incorporated into proteins, is presented at high concentrations – reaching 50 mM – within mammalian cells. It is the most abundant free amino acid in the body and plays an important role in several essential biological processes. Taurine is believed to be a weaker scavenger of reactive oxygen/nitrogen species but the concentrations used to test its antioxidant properties were way lower than physiological. In this study we found that taurine, at physiological concentrations (milimolar range), is an efficient scavenger of peroxyl radical (ROO∙), measured by TRAP assay, as compared to Trolox®. No reactivity against H2O2 was obtained. We also observed that taurine reacts in vitro and ex vivo with nitric oxide. Using cortex brain slices, taurine prevented the inhibitory effect of sodium nitroprusside (a NO∙ donor) on Na+/K+ ATPase activity – a well-known target of NO∙ oxidation. To support a physiological role of the observed antioxidant capacity, taurine concentrations were determined by HPLC in subcellular fractions (cytosolic, mitochondrial and nuclear) from different tissues. Moreover, we demonstrated that the mitochondrial fraction is the most enriched subcellular fraction with taurine. These results are in agreement with previous studies of our group, which demonstrated that taurine is taken up preferentially by mitochondria (Klamt & Shacter, J Biol Chem 280:21346-52, 2005). Our data suggest that physiological taurine concentrations have antioxidant capacity against NO∙ and peroxyl radical, playing a pivotal role in the mitochondrial oxidative defense. (Supported by: CNPq, PROPESQ/UFRGS)
Euchariotic cytochrome c (cytc) is a hemeprotein involved in the respiratory chain electron transport and in the caspase activation at the onset of the apoptosis. Cytc properties can be modulated by changes in the structure and spin state promoted by membranes. In this work, it was studied the catalytic activity of Fe3+ alternative low spin state cytc (ALSScytc) induced by SDS micelles and AOT/hexane reverse micelles. ALSScytc exhibits heme iron in a less rhombic symmetry and Met80 probably replaced by Lys79 at the sixth coordination position. The peroxidase and oxidase activities of ALSScytc respectively on tert-butylhydroperoxide (t-BuOOH), diphenylacetaldehyde (DPAA) and dithiothreitol (DTT) were compared with that exhibited by native cytc. The Fe3+ ALSScytc retained the capacity to cleave t-BuOOH and to be reduced by DPAA and DTT. During the reaction with t-BuOOH, ALSScytc was more susceptible to the attack of free radicals than the native form, probably due to a more open heme crevice. Contrariwise to that exhibited by the native form, the oxidase activity of ALSScytc on DPAA resulted in heme iron reduction concomitant with the conversion to the high spin form. The reduction of native cytc by DPAA probably involves electron transfer via heme edge without displacement of the heme iron ligands maintaining the reduced protein in the low spin state. However, ALSScytc, probably due to a more opened heme crevice, permitted DPAA free radical to attack and displace Lys79 postulated as the heme iron sixth ligand. By using DTT as the reducing agent, Fe2+ALSScytc exhibits spectral changes suggestive of the heme iron in the low spin state, however the spectral differences relative to the corresponding native protein pointed out the possibility of a non-oxidized DTT -SH group at Fe2+ heme iron sixth coordination position. The oxidase activity of SDS and AOT-induced ALSScytc on DPAA produced the aldehyde-derived peroxyl radical detected by EPR in the presence of DMPO. This species should be preceded by the carbon-centered radical that was not detected in these conditions. These results contribute for the comprehension of the membrane effects on the cytc function. Supported by FAPESP, CNPq, CAPES and FAEP-UMC
The peroxidase activity of Manganese-Microperoxidase-11 and -meso-tetrakis(2,6-diclore-sulfonatophenilporphyrin) - MnTDC(SO3Na)PP was investigated at pH 7.4 and 9.1, in homogeneous and heterogeneous media (CTAB micelles and DODAB liposomes) and in the presence and absence of histidine. Both for the hemepeptide and MnTDC(SO3Na)PP, the electronic absorption and electron paramagnetic resonance (EPR) spectra changed with the pH increase and the presence of micelles and liposomes. These changes are respectively compatible with deprotonation of the water molecule coordinated with the porphyrin metal and the partition of the catalysts in the hydrophobic environment of micelles and liposomes. On the other hand, the presence of histidine did not induce changes in the catalyst UV-visible and EPR spectra. Similarly to that described for iron-microperoxidases, the increase of pH accelerated the reaction rate of the catalysts with hydrogen peroxide. This effect was due to the deprotonation of the coordinating water that favored the formation of Compound 0, the first intermediate of the reaction. CTAB micelles and DODAB liposomes decreased the reaction rate of the catalysts with hydrogen peroxide. This effect could be attributed to the low partition of hydrogen peroxide in micelles and liposomes and the absence of a proton donor for the completion of the catalytic process. At pH 7.4 but not at pH 9.1, in all investigated media, the presence of histidine decreased the reaction rate to a half. This effect was not expected since the amino acid did not change the catalyst EPR and UV-visible spectra and deserves future investigations. The comprehension of the mechanisms able to modulate porphyrin and microperoxidase activities is important because these compounds could be used in the development of nanotechnological devices and drugs. Supported by CAPES, CNPq, FAPESP and FAEP-UMC
Palabras clave: Manganese, Porphyrin, EPR
| Poster Presentation | 07.Peroxynitrite Chemistry and Biochemistry
Positive correlation between the nitric oxide-derived reactive nitrogen species and thiocyanate levels in tobacco-smoking human whole saliva
Human whole saliva is a mixed fluid comprising secretions from major and minor salivary glands, a serum-derived transudation from gingival crevices as well as components from oral microorganisms, leukocytes, and epithelial cells. In recent years, estimation of some of the organic components of saliva has become of considerable diagnostic interest. (Pseudo)halides iodide (I-), bromide (Br-), and thiocyanate (SCN-) are oxidized to respective hypo(pseudo)halides by human salivary peroxidases in the presence of hydrogen peroxide (H2O2), resulting in end products of wide antimicrobial potential. Thus, the role of SCN- as an oxidizable cofactor in the lactoperoxidase/SCN-/ H2O2 antibacterial system is the only known function of this ion in human saliva. After absorption via the pulmonary, nasal, and oral mucosa, cyanide compounds in tobacco smoke are transformed into thiocyanates (SCN) and excreted into urine, saliva, and sweat. Thiocyanate has thus been proposed as a biochemical marker of smoking exposure (Lahti et al., 1999). Saliva tests, generally considered the most sensitive, have the additional advantage of easy, non-invasive sampling. In this work, a total of 32 smokers and 28 nonsmokers were analyzed for salivary SCN- concentration. Ages of the 60 male/female volunteers ranged from 18 to 60 years. After the use of a systematized sialometry method, SCN- ions were measured by using a Köning colorimetric assay. Smoking statistically increased the amount of SCN-, comparing to nonsmokers. Comparing to previous analysis of increased nitrite (NO2-) concentration in smokers (Souza & Genestra, 2006), where positive correlations were statistically significant, these data suggests that NO2- could be a potential substrate for peroxidases even in the presence of Cl- and/or SCN- in smokers (Ihalin et al., 2006). Nitrate (NO3-) excreted by salivary glands is reduced to NO2- by bacteria presence in the surface of tongue, and in the present of peroxidases and H2O2, NO2- is further oxidized to NO2 radical, which participates in nitration reactions. The increase in the SCN- concentration in smokers led to an increased ability of the lactoperoxidase/SCN-/H2O2 antibacterial system to inhibit the growth of Lactobacillus acidophilus.
Palabras clave: Saliva, Nitrite, Thiocyanate
| Poster Presentation | 06.Inflammation, Oxidants and Myeloperoxidase in Disease
Aspirin-triggered lipoxin A4 differentially modulates HO-1 expression and NADPH oxidase activity in endothelial cells: Implications in pro-oxidative, proinflammatory processes.
Arruda, MA 1(*); Nascimento-Silva, V 1; Barja-Fidalgo, C 1; Fierro, IM 1
1 - Departamento de Farmacologia, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro | (*) Brazil
Lipoxins (LX) and aspirin-triggered LX (ATL) are eicosanoids generated during inflammation via transcellular biosynthetic routes, which have emerged as mediators of key events in endogenous anti-inflammation and resolution. We have reported that an aspirin-triggered lipoxin A4 analog (ATL-1) induces the expression heme oxygenase 1 (HO-1) in endothelial cells (EC), an inducible enzyme closely related with the resolution phase of inflammation. This process requires de novo protein synthesis via LXA4 receptor. Moreover, ATL-1-triggered HO-1 expression inhibited VCAM and E-selectin expression induced by proinflammatory cytokines, what may lead to an efficient impairment of leukocyte-endothelium interactions during the onset of inflammation. These data showed, for the first time, the convergence of these two pillars of resolution of inflammation. More recently, we observed that ATL-1 also impairs ROS generation by EC, a phenomenon closely related to pro-oxidant, proinflammatory and pro-thrombotic cardiovascular conditions, such as hypertension, atherosclerosis, and heart failure. Pre-treatment of EC with ATL-1 (1 - 100 nM) completely blocked ROS production triggered by different agents, as assessed by dihydrorhodamine 123 and hydroethidine. Furthermore, ATL-1 inhibited the phosphorylation and translocation of the cytosplamic NAD(P)H oxidase subunit p47phox to the cell membrane as well as NAD(P)H oxidase activity. ATL-1 treatment also impaired the redox-sensitive activation of the transcription factor NF- kappaB, a critical step in events associated to inflammatory and vascular pathologies. The involvement of distinct protein kinases/phosphatases in this phenomenon are under investigation. We believe that a better understanding of ATL ability to modulate ROS-sensitive processes, interfering with two pathways closely related to the maintenance of cellular redox homeostasis, may lead to the development of new strategies in order to control the redox imbalance associated with several pathological situations associated with a proinflammatory, pro-oxidative component.
Chronological aging in yeast represents an interesting model because of its resemblance with aging in mammalian, post-mitotic tissues, that show low proliferation but conserved metabolic activity. The survival in the stationary phase in yeast is strongly associated with oxidative-stress resistance. In the present study hypo- and hyper-glycogenic phenotypes of S. cerevisiae strains with deletions (Euroscarf, Yeast Deletion Project) of glucose-metabolism enzymes were selected, a comparison of their chronological lifespans was achieved, and the following assays were performed in the emerged candidates: stress sensitivity, ROS levels, and apoptosis markers during aging. Among the strains that accumulated greater amounts of glycogen, the deletion of glycogen phosphorylase, gph1Δ (59 vs. 29 μg glycogen/108 cel. in wt. p<.05), was the unique in showing a shortened lifespan, stress intolerance, and higher levels of ROS during its survival. The transcription of SOD1 and 2 were analyzed in gph1Δ, the transcript levels being 4- and 3-fold lower than in wt at the end of the stationary phase (8 and 5 vs. 31 and 16 AU in wt, respectively. p<.05), and during aging. Hypo-glycogenic deletions such as the one of glycogen synthase, gsy2Δ (8 μg/108 cel. p<.05), demonstrated a little longevity advantage but similar stress tolerance, ROS and RNA levels of SOD1/2 compared with the wt. Low-copy-plasmid-mediated overexpression of SOD1 and SOD2 together rescued gph1Δ from its accelerated aging and stressed phenotype. The incapability to degrade glycogen (deletion of GPH1), produced a rapid-aging strain, what would be attributed, at least in part, to the impoverished stress resistance associated to the decreased transcript levels of both SOD in this mutant. It remains to further clarify the putative dialogue between glycogen availability and the negative regulation of these genes in the aging process.
The transition metals ions nickel (Ni2+), cobalt (Co2+)and copper (Cu2+)are present in very low concentration in human body, but the acute or chronic exposition to them produces citotoxicity and oxidative damage to lipids by a mechanism similar to the Fenton/Haber-Weiss (catalized by Fe2+). The aim of this work is to study the participation of these ion metals in the initiation step of lipid peroxidation mediated by reactive oxygen species. Liposomes composed of 60:40 mol/mol of 1-palmytoil-2-oleoil-sn-glycero-3-phosphocholine and 1-palmytoil-2-oleoil-sn-glycero-3-phospho-L-serine, were incubated (0.25 mg of lipid /0.5 mL) for 90 min at 37°C for promoting hydroperoxide generation, and incubated in the presence of Fe2+, Cu2+,Co2+,and Ni2+ (5-200 mM) solutions, with and without the addition of H2O2 (5-50 mM). Lipid peroxidation was evaluated at the end of the incubation as 2-thiobarbituric acid-reactive substances (TBARS) formation. Stimulation of lipid peroxidation was calculated as: (TBARS of the corresponding ion plus Fe2+) / (TBARS plus Fe2+). Fe2+and Cu2+ producelipid peroxidation by both mechanisms, the direct homolysis of lipid-hydroperoxides (343 % for Fe2+ and 210 % for Cu2+), and the H2O2 decomposition by Fenton/Haber-Weiss reaction with the HO. generation. The Fe2+-promoted lipid peroxidation takes place by a second order kinetic reaction, but the reaction catalized by Cu2+ corresponds to a first order kinetic (50% of the Fe2+ effect on Fenton reaction). The Co2+and Ni2+ ions do not induce either lipid peroxidation by themselves, or H2O2 decomposition. Nevertheless, Ni2+ and Cu2+ mixed with Fe2+, stimulate the Fe2+-initiated lipid peroxidation by a positive competitive mechanism (t1/2: 45 and 40 min respectively) and Co2+inhibits Fe2+-initiated lipid peroxidation (t1/2: 16 min). The presence of Cu2+and Ni2+ stimulate the initial phase of lipid peroxidation catalyzed by Fe2+,by a Fenton/Haber-Weiss mechanism, and Co2+ inhibit the Fe2+-promoted lipid peroxidation. The control of the initial phase of lipid peroxidation would prevent the oxidative damage that causes cellular death.
Oxidative damage is the pathogenic mechanism involved in the development of the lesions induced by choline-deficiency (CD). It produces oxidative damage in liver, heart, kidney and brain by lipid peroxidation, decrease of antioxidants, and consumption of tissue alpha-tocopherol. Weanling Wistar male rats were divided into two groups: one group fed a choline-deficient diet (CD); and the other fed a choline-supplemented diet as control (CS). Liver, heart, brain and both kidneys were removed for oxidative damage determinations: thiobarbituric reactive substances (TBARS), and tert-butyl hydroperoxide initiated chemiluminescence (CL-BOOH), and histopathological analysis. The histopathological study showed: hepatic esteatosis (first day), renal necrosis (fifth day), and heart necrosis (seventh day). In tissues homogenates, TBARS increased by 35 % in liver (third day, p<0.05); 80 % in kidney (third day, p<0.05); more than 100 % in heart (fifth day, p<0.01), and 70% in brain (sixth day, p<0.05). Oxidative stress reaches systemic significance in CD since 4-times increased plasma levels of TBARS with a t1/2 (the time for half maximal effect) of 2.8 days were observed. The normal rat plasma level (1.5 microM) was increased by 200% at the third day (p<0.02), 400% at the fifth day (p<0.04) and 300% at the sixth day (p<0.05). CL-BOOH was increased at the sixth day by 45% in liver (p<0.05), 83% in kidney (p< 0.01), 50% in heart and 114% in brain (p<0.05 respectively). TBARS is the earliest sign of the pathogenic effect of CD, followed by the increase in CL-BOOH, meaning decreased tissue levels of lipid soluble antioxidants, i.e. endogenous alpha-tocopherol, and by the histopathological damage. In the liver, the morphological damage is previous to the lipid peroxidation and the consumption of endogenous antioxidants. In kidney and heart, indeed, lipid peroxidation and oxidative damage are preceding the necrosis. These results indicate that lipid peroxidation plays a role in the pathogenesis of CD. Oxidative damage in liver is associated with hepatic lipid metabolism, and may be affecting the absorption and transport mechanisms of alpha-tocopherol in this organ. TBARS is an early marker of lipid peroxidation in plasma and tissues.
Copper is an essential but also potentially toxic trace element. Upon entering the cells, Cu2+ ions rapidly react with reduced glutathione (GSH) to form Cu+1 ions. In a subsequent reaction, Cu1+ ions are sequestered by other GSH molecules to form a Cu(I)-[GSH]2 complex.. The oxygen concentration [O2] of a solution containing Cu2+ plus GSH (1:3) mixtures was found to remain constant in time (0-2 hrs). Yet, upon addition of SOD, O2 underwent a rapid and sustained decrease. Such result was interpreted as evidence that the Cu(I)-[GSH]2 complex is continuously generating O2-· anions, and that the drop in O2 occurs onlyupon removal of this radical species by SOD. In solutions containing the complex and O2,EOH levels markedly increased along time. The latter was not affected by catalase addition (thus, H2O2-independent), but totally blocked by SOD (O2-·-dependent). The possibility that in addition to O2-· anions, HO· radicals are also generated by the Cu(I)-[GSH]2 complex was ruled out since no decrease in fluorescence was seen when the complex was incubated with fluorescein, a O2-·-independent probe. To investigate whether the O2-·-generating capacity of the complex is conc.-dependent, increasing conc. of the 1:3 mixture were added to a solution containing a fixed DHE concentration. Contrarily to what was expected, the rate of DHE oxidation, instead of increasing, decreased linearly as the conc. of the Cu2+/GSH mixtures was increased. The latter can be explained if the expected higher rates of O2-·-generation translate immediately into higher rates of O2-·-autodismutation. In fact, our results show that a concomitant increase in the rate of H2O2 formation occurs in such mixtures. In conclusion, the Cu(I)-[GSH]2 complex is redox-active towards oxygen. The present data indicate that, in the absence of O2-· interceptors (such as SOD or DHE), the complex would function as a continuous source of O2-· radicals (and/or H2O2). We suggest that in a scenario in which other interceptors are present, such as various biological targets, the complex could well play a pro-oxidant role (Fondecyt # 1070613).
Palabras clave: GLUTATHIONE, SUPEROXIDE, COPPER
| Poster Presentation | 01.Oxygen Radical Chemistry and Detection
STUDY BY ELECTRON SPIN RESONANCE (ESR) OF IRRADIATED BEEHIVES STRUCTURES IN WOOD
Huarte, M.G. 1(*); Rubín de Celis, E. 1; Magnavaca, C. 2
1 - CÁTEDRA DE FÍSICA. FACULTAD DE FARMACIA Y BIOQUIMICA. UBA. | (*) Argentina
Irradiation treatments have been widely used for sterilization, disinfection, preservation (mainly for food), or improvement of commercial and valuable products. For example, beehives structures in wood are irradiated to avoid acaricide use between beekeeping cycles. For Argentina, the first world-wide exporter of honey, the availability of methods to know if these materials were or not irradiated could become important. Among the effects of ionizing radiation that might lead to identify irradiated materials, the irradiation by-products free radicals signals have conclusive value. As far as every cellulose material, in wood cases, free radicals are trapped enough to be detected for quite a long time. The aim of this work consisted in evaluating ESR spectroscopy as a methodology to detect the presence or absence of irradiation in wood of beehives in different conditions along time. Beehives structures in wood, new (NW) and recycled (RW), were irradiated with 0-10-15 and 20 kGy (kGy: unit of absorbed irradiation dose) in the presence of chemical dosimeters for dose control. Its splinters were loaded into quartz tubes and measured using an X-band ESR spectrometer Bruker ECS-106. Samples of non-irradiated wood have shown a native signal. Results of irradiated showed: (i) an increase of the height of this signal (h0), proportional to the irradiation dose, (ii) the appearance of two “satellite” peaks on both side of the signal in the irradiated samples exclusively, which remained for a long time (more than 250 days), (iii) differences in the kinetic behavior in the decay of h0 for RW (half-time: 5-20 days) and NW (half-time: 1-5 days), (iv) the relation h0/dose was higher for RW than NW, and (v) the saturation power behavior indicated a fast saturation for NW in comparison with RW. These results suggest that ESR spectroscopy could be useful to evaluate different types of irradiation wood along time and might be repeated in other wood goods.
Palabras clave: wood, irradiation, ESR
| Poster Presentation | 01.Oxygen Radical Chemistry and Detection
Detection of ONOO– using dihydroethidium
Pignitter, M 1(*); Rechberger, G 2; Gorren, AC 1; Schmidt, K 1; Mayer, B 1
1 - Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, Karl-Franzens University Graz, Austria | (*) Austria
Dihydroethidium (HE) is widely used as fluorescence dye for the detection of reactive oxygen species. In contrast to the well established and specific reaction of superoxide (O2•–) with HE to yield 2-hydroxy-ethidium (2-OH-ethidium), little is known about the product arising from the interaction of peroxynitrite (ONOO–) with HE. The present study was designed to identify this product and to clarify whether it may serve as a specific marker for cellular ONOO– formation.
Our data clearly demonstrate that incubation of HE with authentic peroxynitrite does not induce the generation of the O2•–-specific product, 2-OH-ethidium, but yields a distinct product that can be detected by HPLC. Attempts to identify this product by mass spectrometry (MS) revealed the formation of a dimeric HE derivative (dimer 1) with a distinctive mass to charge ratio of 627. Dimer 1 exhibited fluorescence characteristics similar to that of the parent compound HE. When increasing the ONOO–-to-HE ratio from 1:1 to 4:1, formation of dimer 1 was markedly reduced and a distinct dimeric compound with a mass to charge ratio of 313 (dimer 2) appeared.
Formation of dimer 1 was also observed upon incubation of HE with SIN-1, which decomposes nonenzymatically to yield equimolar fluxes of NO/O2•–. Interestingly, incubation of HE with NO alone (released from the NO donors DEA/NO or SPER/NO) also triggered the generation of dimer 1, whereas H2O2 and O2•– (generated with the hypoxanthine-xanthine oxidase system), as well as the NO oxidation products NO2– and NO3– were ineffective. Summarized, our results demonstrate that HE reacts with ONOO–, NO/O2•– and NO to a dimeric HE derivative. These findings provide additional insights into the molecular mechanisms underlying HE chemistry.
Cryopreservation of human spermatozoa is a useful therapeutic option in the treatment of infertility, but this procedure may cause structural and functional oxidative damage to the male gamete. The purpose of this study was to analyze seminal and oxidative stress parameters, before and after using resveratrol, a major antioxidant, for the cryopreservation of semen of fertile and infertile men. The study group consisted of 20 patients who were infertile or had oligozoospermia (spermatozoa concentration below 20 millions/mL, or less than 40 million total spermatozoa), and a control group of 10 fertile patients who donated a semen sample before vasectomy for sterilization purposes. The results in this study show that the spermatozoa of infertile men are less resistant to the oxidative damage caused by its freezing and later thawing, compared to those obtained from fertile patients. A negative correlation (r=-0.623, p¡Ü0.05) was observed between spermatic motility of fresh semen and the lipoperoxidation levels measured in the cryopreserved samples. Both groups of patients sampled presented diminished levels of lipoperoxidation in the added aliquots of resveratrol, compared to the respective post-freezing controls. The data shown in this study may lead to a better understanding of the cryobiology of human spermatozoa and an increased understanding of the role of resveratrol in the redox balance involved in the human spermatozoa cryopreservation procedures.
Peroxynitrite is a strong oxidant and nitrating agent. Many decades ago, the reduction of violet MnO4- to green MnO42- was used to determine the concentration of peroxynitrite [1,2]. We investigated the kinetics of this reaction and those of nitrogen monoxide, and superoxide with by high-pressure stopped-flow spectrophotometry, and pulse radiolysis. The reaction of MnO4- with peroxynitrite is zero-order in MnO4-; the rate constant is (0.020 ± 0.002) s−1 at pH 11.7. The reactions of MnO4- with nitrogen monoxide and superoxide proceed with rate constants of (3.5 ± 0.7) × 106 M−1s−1 and (6.1 ± 0.8) × 105 M−1s−1, respectively, at pH 11.7. ONOO- = NO● + O2●- (1) We assume that Reaction 1 is the rate determining step. The activation volume of 12.6 ± 0.8 cm3mol−1 supports our hypothesis of a homolytic N-O scission. The temperature dependent studies yield an activation energy of 102 ± 2 kJ mol-1. Since both NO● and O2●− are consumed rapidly by MnO4−, we are able to use the rate constant k1 of 0.020 s−1 and the backward rate constant k-1 of 1.6 ´ 1010 M−1s−1  to calculate ΔfG°(ONOO−) = 68 kJ mol−1. Furthermore, it is feasible to derive a ΔfG•(ONOOH) of 31 kJ mol-1, with help of pKa(ONOOH) = 6.5. MnO4− can be used conveniently to determine the concentrations of H2O2-free solutions of ONOO−.  K. Gleu and E. Roell, Zeitschr. anorg. allgem. Chemie 1929, 179, 233-266.  H. M. Papée and G.L. Petriconi, Nature 1964, 204, 142-144.  T. Nauser and W.H. Koppenol, J. Phys. Chem. A., 2002, 106, 4084-4086.
Nanotechnology is a fast growing field with emerging development of modern materials for electronics and information technology industries, cosmetics, health and medicine and in a variety of manufactured goods. Single-Walled Carbon Nanotubes (SWCNT) with unique physico-chemical, electronic and mechanical properties are novel materials of technological importance. Pulmonary exposure to SWCNT revealed toxicity within the range of doses relevant to exposure limits established by regulatory agencies for fine size carbon particles. In C57BL/6 mice exposed to SWCNT, pulmonary toxicity was associated with a dose-dependent augmentation of biomarkers of cell injury and oxidative stress quantified by cell counts, total protein, lactate dehydrogenase and g-glutamyltranspeptidase activities, reduced level of GSH, total antioxidant reserve along with the accumulation of lipid peroxidation products found in bronchoalveolar lavage (BAL) fluid and in the lung. Markers of pulmonary cytotoxicity corresponded to early development of acute inflammation, collagen accumulation, and progressive fibrosis. In mice maintained on vitamin E deficient diet, SWCNT significantly enhanced collagen deposition as compared to the vitamin E sufficient group. Overall, our data suggest that pharyngeal aspiration of SWCNT elicited a robust acute inflammatory response with early onset of progressive pulmonary fibrosis whose expression and severity was associated with the intensity of oxidative stress in the lung of the exposed C57BL/6 mice. Acknowledgements: supported by NIOSH OH008282, NORA 92700Y.
The red blood cell (RBC) is the major scavengers of peroxynitrite in blood. About half of peroxynitrite crosses the cell membrane and then reacts with hemoglobin (Hb) producing metHb. MetHb can be back reduced through the activation of glucose metabolism. Our study is aimed at deciphering the signaling pathways induced in RBC by peroxynitrite. RBCs were exposed to peroxynitrite and then incubated in the presence of 10 mM [2-13C] glucose and analyzed by 1H and 13C NMR spectroscopy to determine the fraction of glucose metabolized by both the glycolytic and the pentose phosphate pathways (PPP). The PPP flux, estimated from the ratio of 13C-enrichment in lactate carbon 3 relative to carbon 2, was calculated under baseline conditions or after peroxynitrite treatment. Moreover, RBCs incubated in the presence of deoxy-D-glucose, an inhibitor of the glycolytic pathway, and treated with peroxynitrite (0-1 mM) revealed a fast consumption of deoxy-D-glucose 6 phosphate, suggesting PPP activation. In peroxynitrite-treated RBCs, but not in the presence of deoxy-D-glucose, tyrosine phosphorylation of Band 3 was upregulated. In RBCs this signal activates the translocation and activation of some glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, aldolase, pyruvate kinase phosphofructokinase, and lactate dehydrogenase). Band 3 phosphorylation was induced through inhibition of phosphotyrosine phosphatases and consequent activation of Lyn, a tyrosine kinase of the Src family.
Palabras clave: peroxynitrite, red blood cell, signaling
| Poster Presentation | 01.Oxygen Radical Chemistry and Detection
Analysis of the chronological life span associated with advanced glication end-products (AGEs) in Saccharomyces cerevisiae.
The reactive carbon species (RCS) are formed from the oxidation of carboydrates, lipids, amino acids, and DNA.The glycolytic metabolism is an important mechanism of RCS induction associated with protein modification by advanced glication end-products (AGEs). In this sense, the formation of AGEs is a natural phenomenon of biological systems elevated rates of AGEs has been evidenced in several diseases and aging mechanism.Thus, the objective of this work was to evaluate how different carbon sources (e.g. glucose, frutose and glicerol) affects the metabolism and the chronological life span of the yeast Saccharomyces cerevisiae. For this purpose, the analysis of chronological life span (CLS) of a wild-type yeast strain was carried in synthetic complete (SynCo) medium containing glucose and fructose at high (5% w/v) and low (3% w/v) concentration at 30 ° C. At each 2 days, the cells were collected, washed, and plated in rich medium (YEPD) followed by plate incubation for 2 days at 30 ° C and colony counting. The number of colonies was used to determine the survival number (%) of cells. In addition, the CLS of a non-fermenting yeast culture was determined in SynCo medium containing glycerol 3% (v/v) at 30 ° C. The results indicated a reduction of the survival of yeast cells in SynCo media containing glucose 5% (w/v) and frutose 5%(w/v) after 6 days of culture (38% and 35%, respectively). For yeast cells growth after 6 days in SynCo media containing glucose 0.5% (w/v) and frutose 0.5% (w/v), it was observed a survival level of 58% and 37%, respectively. Interestingly, yeast cultured in SynCo medium containing glycerol 3% (v/v) remained viable at elevated levels after 6 days of growth. These data indicate that high concentrations of (glucose and frutose) induce a reduction of cells survival when compared to yeast cells growth in low carbohydrate concentration or in glycerol, which seems to be correlated with the formation of AGEs and the reduction of CLS in yeast cells.
Reactive oxygen species (ROS) are involved in alteration of excitation-contraction coupling and muscle fatigue. However, results concerning their effect on maximum tetanic force, sensibility of myofibrils to calcium and fatigability are contradictory. Difficulties arise due to the use of slow, high-affinity calcium dyes and different fatiguing protocols and experimental conditions. We reinvestigated this theme using hand-dissected bundles of less than 20 fibers from Flexor Digitorum Brevis muscles of adult mice. The bundles were mounted in a chamber with two platinum plate electrodes for field stimulation, attached to a force transducer for tension measurements and bathed in Tyrode solution at 24-24,5°C. Fibers were fatigued by repetitive tetanic stimulation (350 ms at 100 Hz), with progressively increasing frequency, from 0,25 to 0,5 tetani/s. With this protocol 3 fatigue phases were observed. During the 1st phase tension fell by 22±5% (n=13) in the first minute, during the 2nd phase fell 30±15% (n=9) within the following 3-4 min and during the 3rd phase tension diminished rapidly to very low values in less than 90 s, when stimulation was stopped (before eliciting 150 tetani in 8 out of 9 cases). The use of the ROS scavenger Tiron (4 mM) caused no change in 1st phase but produced a huge prolongation of the 2nd phase and a great delay (>10 min) in the onset of phase 3 (n=8), with tension remaining as big as 0,4P0 after more than 210 tetani in 3 experiments. When Tiron was applied at the beginning of 3rd phase, tetanic tension was partially recovered (40±1%, n=2) and the fibers were brought back into phase 2 and never presented phase 3, in one case despite more than 350 tetani. In one experiment, with Tiron applied at the end of phase 1, almost original tension values were reestablished with fatigue following its normal time course. Tiron also reduced the fatigue-induced change of slope in the linear phase of relaxation (Westerblad & Lännergren. 1991). The results indicate that ROS play an important role in the genesis of the 3rd phase of fatigue and in the alteration of relaxation, even at room temperature. FONACIT G-2001000637.
The ionic homeostase is fundamental for the maintenance of the major cellular physiologic processes. Considering all metallic ions that are essential for cellular viability, the Fe2+/Fe3+ occupies a central role on metabolism. In addition, Fe2+/Fe3+ are also necessary for the mechanisms associated to tolerance against reactivate oxygen species (ROS). In this sense, the absorption of Fe2+/Fe3+ from the environment depends on an iron high affinity system formed by a metal reductase, iron-dependent oxidase and an iron permease. In Saccharomyces cerevisiae, two proteins Fet3 and Fet5 acts as iron-dependent oxidase, converting Fe2+ to Fe3+. Once converted, Fe3+ is internalized through Ftr1p (an iron permease). Inside the cell, the homeostasis of Fe3+ is maintained by specialized proteins, e.g. Fsf1p (a sideroflexin-homologous sequence. Interestingly, little is known about effects of the effects of iron high-affinity transport system on the generation and/or tolerance to ROS. Thus, the objective of this work was to evaluate the sensitivity of proficient and deficient yeast strains for the iron high-affinity transport system of S. cerevisiae in the presence of different concentrations of H2O2. The yeast strains sensitivity analysis was performed by drop test in synthetic complete medium (SynCo) containing H2O2 at 0.5 mM, 1.0 mM, 1.25 mM, 1.5 mM, and 2.5 mm. The results demonstrated that the strains defectives for Fet3p and Ftr1p were highly sensitive to H2O2 when compared to wild-type, fet5D and fsf1D strains, indicating the importance of. the iron high-affinity transport system for the tolerance of ROS generated by H2O2 in the presence of Fe2+/Fe3+.
Insulin is an important protein hormone that is secreted by pancreatic islet β-cells primarily in response to increasing levels of blood concentrations of glucose and it may be a potential target when accumulating peroxynitrite (ONOO‾)is formed in β-cells that are exposed to a relatively large amount of NO∙ and O2∙‾. The inactivation of insulin by ONOO‾ is accompanied by oxidative and nitrative modifications of critical tyrosine which can generate a change in the charge distribution and promote the conversion of the soluble protein into amyloid fibrils. It may alter insulin function and conformation leading to significant functional consequences. It has been earlier reported that mono-nitro-insulin reduced about 31% of insulin receptor binding potency and the hypoglycemic capacity of mono-nitro-insulin was about 75% of that of native insulin. In order to find out specific tyrosine targets for the inactivation of insulin by ONOO‾, to reveal the relation between changes of insulin secondary structure induced by ONOO‾ and insulin nitration, we analyzed the dose-dependent insulin tyrosine modification indicated by spectral changes. Also, the nitrated sites of insulin were identified by HPLC-ESI-MS/MS analysis. The results show that an infusion of ONOO‾ caused a maximum observed yield of 2.7 3-nitrotyrosine per insulin subunit and led to different nitrated species. Moreover, low concentration ONOO‾ can cause a maximum 2.5-fold more dityrosine in ONOO‾-modified insulin to form insoluble amyloid fibrils. The results also demonstrated that all tyrosine residues are nitrated by ONOO‾ except tyr-A14, in which tyr-B26 appeared to be the most susceptible residue concerning with ONOO‾ mediated nitration and the β-sheet structure are increased in the nitrated protein, which may be result in the inactivation of insulin.
Palabras clave: insulin, peroxynitrite, nitration
| Poster Presentation | 06.Inflammation, Oxidants and Myeloperoxidase in Disease
OXIDATIVE METABOLISM DURING PREADIPOCYTES DIFFERENTIATION
Sapochnik, D 1(*); Julianelli, V 2; Garate, H 1; Calvo, J.C. 2; Guerra, L.N. 1
1 - Dep de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires | (*) Argentina
Oxidative stress plays critical roles in the pathogenesis of diabetes, hypertension and atherosclerosis. Some authors showed that fat accumulation correlates with systemic oxidative stress in human and mice, but relationship of lipid production and oxidative metabolism is still under study. We performed assays with preadipocytes 3T3-L1 (undifferentiated cells: UC), which under defined conditions are capable of differentiating in mature adipocytes (differentiated cells: DC) and accumulate lipids. DC present higher content of triglycerides than UC (DC: 0.45 + 0.03 ug Tg / ug protein vs UC: 0.23 + 0.02 ugTg /ug protein, p < 0.05). Treatment with N-acetylcistein (NAC) inhibited lipid accumulation: DC vs DC + 10uM NAC: 0.21 + 0.03 ug Tg / ug protein, p < 0.05; DC vs DC + 5uM NAC: 0.31 + 0.02 ugTg /ug protein, no significantly different). Intracellular ROS generation in adipocytes was measured with 2,7dichlorofluorescein (DCHF) and ROS production was determined from an H2O2 standard curve: in DC we determined 7.3 x 103 nmol ROS/ml, meanwhile preadipocytes (UC) showed 3. 4 x 103 nmol ROS/ml. We performed a microscopy assay and observed that 53% of differentiated cells and 24% of undifferentiated cells were positive to oxidized DCHF, a control was performed with H2O2 (30mM). As antioxidant enzymes we determined Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, both were higher in DC than in UC. SOD (DC: 41.5 ± 2.4 U/g protein vs UC: 26.0 ± 0.4 U / g protein, p< 0.01) and GPx (DC: 225 ± 12 U/g protein vs UC: 35 ± 13 U / g protein, p< 0.01). We demonstrated that ROS production was increased in 3T3-L1 adipocytes in parallel with fat accumulation, NAC inhibited accumulation in a inhibitor concentration dependent manner. Antioxidant enzymes might be activated in response to oxidative stress.
| Poster Presentation | 03.Redox Biology: Metals and Thiols
Dioxygen Reactivity and Heme Redox Potential of Truncated Human Cystathionine β-Synthase
Carballal, S. 1(*); Madzelan, P. 2; Zinola, C. F. 3; Graña, M. 4; Radi, R 5; Banerjee, R. 2; Alvarez, B. 6
1 - Laboratorio de Enzimología, Facultad de Ciencias; Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay | (*) Uruguay
Cystathionine β-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to possess a regulatory role. The heme in CBS is unusual, it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations of CBS showed a reversible response with a reduction potential of -287 ± 2 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.03 ± 0.01) X 105 M-1 s-1 at pH 7.4 and 25 °C for the direct reaction of Fe(II)CBS with dioxygen. Heme oxidation led to superoxide radical (O2.-) generation, which was detected by the SOD-inhibitable oxidation of epinephrine. Our results show that Fe(II)CBS could represent a previously unrecognized source of cytosolic superoxide radical.
During stimulation, macrophages release superoxide anion (O2•-) through NADPH oxidase complex activation. Thiols are required to maintain cellular functions. For instance, glutathione (GSH) is an important reductant to repair oxidized biomolecules, to cycle proteins involved in cell signaling and to scavenge bioradicals, although the relatively reactive glutathionyl radical (GS•) is produced in the latter case. To investigate GS• participation in macrophage respiratory burst, we employed the nitroxide 4-9((-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl (Ac-Tempo) whose interaction with GS• and carbon-centered radicals switches off Ac-Tempo EPR signal while switching on acridine moiety fluorescence (lexc 361 nm, lemi 440 nm) (Borisenko et al JACS 126, 9221, 2004). Thus, inflammatory macrophages (106 cells/ml) were incubated with Ac-Tempo (50 µM, 5 min), and PMA (100 ng). Ac-Tempo fluorescence response and EPR signal were determined in parallel to O2•- release through cytochrome c reduction. Unstimulated cells displayed low fluorescence accompanied by maintenance of Ac-Tempo EPR signal. Cells stimulated with PMA in the presence of Ac-Tempo elicited a dose-dependent fluorescence in synchrony with EPR signal decay and O2•- release inhibition. Changes in fluorescence and EPR signal intensity were dependent on GSH levels as demonstrated by pre-treatment of cells with buthionine sulfoximine and N-ethylmaleimide. Fluorescence decrease matched the inhibition of O2•- release. These results suggest a role of thiyl radicals, mainly GS•, in the maintenance of the respiratory burst of macrophages and may provide a therapeutic target for inflammation management. Supported by CNPq, Instituto do Milênio Redoxoma.
Biacetyl, like methylglyoxal and other α-dicarbonyl compounds, is reported as cytotoxic and genotoxic. It is a food and cigarette contaminant and related with alcohol hepatotoxicity. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the radical anion superoxide with nitric oxide [k2 = 2 x 1010 M-1 s-1; pKa(ONOOH) = 6.8]. We have found that peroxynitrite reacts with biacetyl predominantely to acetate in normally equilibrated phosphate buffer (k = 1.5x 104 M-1s-1; optimum pH 7.2) at rates near those with carbon dioxide and dependent on both reagent and phosphate concentrations. EPR spin-trapping studies with MNP were conducted in normally aerated biacetyl- and peroxynitrite-containing phosphate buffer to detect and possibly identify carbon-centered radical intermediates. The intensity of an EPR signal, assignable to a MNP-acetyl radical adduct (aN = 8.3 mT; lit. NIEHS 7.8-8.3 mT), enhances at increasing concentrations of either biacetyl or peroxynitrite. Upon treatment of Arg, His, Lys, Phe, Thr, Trp, and Tyr (1 mg/mL in all cases) with 2.0 mM biacetyl and 2.0 mM peroxynitrite in 200 mM phosphate buffer, pH 7.2, at room temperature, the corresponding monoacetyl-products were found in the spent reaction mixtures by CE-MS-ESI: m/z = 217, 198, 189, 208, 162, 247 and 224, respectively. Acetylation/deacetylation of histones, transcription factors, nuclear import factors, α-tubulin, lenticular crystallin, adenylate kinase, cytochrome c, etc, has been reported to regulate diverse cellular functions, including DNA repair, hormone signaling and cell cycle (Backs & Olson, 2006; Ito et al., 2007). Our results raise the possibility that, at conditions of nitrosative and carbonyl stress, free-radical acetylation of proteins may take place in cells together with the well documented acetylase-catalyzed reactions, leading to protein gain or loss of function. Support: FAPESP, CNPq, Milênio Redoxoma.
Hypoxic tumor cells are resistant to radiation and chemotherapy and may lead to increased metastasis and accelerated malignant progression. However, the hypoxic environment of the tumor offers an attractive difference between normal and tumor cells that may be exploited with the use of bioreducible cytotoxins. These compounds are prodrugs that undergo bioreductive activation to yield cytotoxic metabolites. The reduction is facilitated by appropriate reductases under the environment of low oxygen tension. It was hypothesized that hybrid compounds that possess an N-oxide and a ð-DNA-stacking moiety would be a new generation of bioreductive compounds. These kind of hybrid compounds could damage hypoxic cells by generating ·OH and after bioreduction, damage the hypoxic cells by direct DNA interaction or DNA-related biomolecules This idea together with our interest in the development of new hypoxic selective cytotoxic agents encourages us to synthesize phenazine 5,10-dioxide derivatives, structurally related to other bioreductive compounds and possessing potential DNA-stacking moieties. Some derivatives showed excellent behavior in hypoxic conditions. In order to determine the possible mechanism of action of the compounds we have studied the bioreductive profile of some phenazine-5,10-dioxide derivatives using cytosolic and microsomal liver fractions from Wistar rats. The bioreductive profile of the compounds was studied under hypoxic and normoxic conditions. We found that compounds with cytotoxic selective profile were selective bioreduced under hypoxic conditions. Compounds that showed low cytotoxicity under both conditions were not bioreduced and compounds with high cytotoxicity but no selectivity were bioreduced in both conditions. Also it appears that several different enzymes participate in the bioreduction of the various selective hypoxic cytotoxic agents. So, we studied the relative participation of reductive enzymes as DT-diaphorase, cytochrome P450, xantine oxidase and aldehyde oxidase. We found that DT-diaphorase and cytochrome P450 were involved in the bioreductive process of our selective hypoxic cytotoxins. These results confirm that the designed prodrugs are excellent bioreductive cytotoxins.
Oxidative stress and inflammation have been implicated in the pathogenesis of atherosclerosis. We recently found that the iron chelator, desferrioxamine (DFO), suppresses NADPH oxidase-mediated oxidative stress and expression of vascular cell adhesion molecule-1 (VCAM-1) in mice treated with lipopolysaccharide (LPS). The objective of the present study was to investigate the mechanism by which iron enhances, and DFO inhibits, NADPH oxidase activity in human aortic endothelial cells (HAEC). Incubation of HAEC with LPS (5 ìg/ml) for 24 hrs increased NADPH oxidase activity in a time-dependent manner, starting at 12 hrs of incubation. This effect was inhibited by DFO, but not DFO pre-loaded with an equimolar amount of iron. Treatment with LPS also enhanced the intracellular concentration of iron, measured by inductively coupled plasma optical emission spectroscopy, and cellular levels of heme and p22phox protein, a catalytic, heme-containing subunit of NADPH oxidase. Interestingly, incubating HAEC with iron (100 ìmol/L) for 48 hrs exerted similar effects as LPS. However, LPS did not change protein levels of heme oxygenase-1 and p47phox, a non-heme containing subunit of NADPH oxidase, or mRNA levels of p47phox and NOX4, assessed by quantitative real-time RT-PCR. In contrast, mRNA levels of p22phox and VCAM-1 were increased after 3-6 hrs of incubation with LPS, and these effects were blocked by DFO. Our data suggest that LPS activates NADPH oxidase in HAEC by increasing cellular iron uptake, which subsequently increases heme synthesis and p22phox protein levels. Iron chelation by DFO effectively inhibits NADPH oxidase activity and endothelial adhesion molecule expression, which may be beneficial in inhibiting atherosclerosis.
The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown to negatively regulate LPS-induced acute inflammatory responses. We previously observed that the metabolic thiol antioxidant, á-lipoic acid (LA), inhibits LPS-induced expression of cellular adhesion molecules and adherence of monocytes to human aortic endothelial cells. Here, we investigated the mechanism by which LA attenuates LPS-induced monocyte activation in vitro and acute inflammatory responses in vivo. Incubation of human monocytic THP-1 cells with LA induced phosphorylation of Akt in a time- and dose-dependent manner. In cells pretreated with LA followed by LPS, Akt phosphorylation was elevated initially and further increased during incubation with LPS. This LA-dependent increase in Akt phosphorylation was accompanied by inhibition of LPS-induced NFêB DNA binding activity and upregulation of TNFá and monocyte chemoattractant protein-1 (MCP-1). Lipoic acid-dependent Akt phosphorylation and inhibition of NFêB activity were abolished by the PI3K inhibitors, LY294002 and wortmannin. Furthermore, LA treatment of LPS-exposed C57BL/6N mice strongly enhanced phosphorylation of Akt and GSK3â in blood cells; inhibited the LPS-induced increase in serum concentrations and/or tissue expression of adhesion molecules, MCP-1, and TNFá; and attenuated NFêB activation in lung, heart, and aorta. Lipoic acid also improved survival of endotoxemic mice. All of these anti-inflammatory effects of LA were abolished by treatment of the animals with wortmannin. We conclude that LA inhibits LPS-induced monocyte activation and acute inflammatory responses in vitro and in vivo by activating the PI3K/Akt pathway. Lipoic acid may be useful in the prevention of sepsis and inflammatory vascular diseases.
Lipid hydroperoxides are the primary products of lipid peroxidation. They can react with reactive nitrogen species yielding nitrated/nitrosylated products, which can decompose to generate NO and other reactive species. It is well known that direct nitration of hydrogen peroxide and organic hydroperoxides yields pernitric acid (HOONO2) and peroxynitrates (ROONO2), respectively. The purpose of this work was to investigate the generation of singlet oxygen (1O2) in the reaction of oleic acid hydroperoxides and the nitronium ion. Oleic acid hydroperoxides (OLOOH) were synthesized by photooxidation and purified by column chromatography. The formation of 1O2 was monitored by measuring the 1O2 monomol light emission in the near-infrared region at 1270 nm. The reaction of OLOOH with the nitrating agent, NO2BF4, yielded an intense light emission, indicating the formation of 1O2. Moreover, the presence of 1O2 was unequivocally demonstrated by the direct spectral characterization of the near-infrared light emission. For comparison, we also tested the reaction of hydrogen peroxide, cumene and tert-butylhydroperoxide with NO2BF4. Interestingly, all these hydroperoxides also led to the formation of 1O2. These data indicates that hydrogen peroxide and organic hydroperoxides, including lipid hydroperoxides, can react with nitronium ion to generate 1O2. The mechanism of 1O2 formation is under investigation. Supported by: FAPESP, CNPq, CNPq/Instituto do Milênio: Redoxoma, L´OREAL/UNESCO.
Palabras clave: lipid hydroperoxides , Singlet molecular oxygen , nitronium ion
| Poster Presentation | 01.Oxygen Radical Chemistry and Detection
Novel Liposoluble Spin Traps derived from Amidinoquinoxaline N-oxides: Characterization by ESR.
Facorro, G. 1(*); García, B. 2; Piehl, L 1; Orelli, L 2; Rubin de Celis, E 1
1 - Cátedra de Física, Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires. Argentina | (*) Argentina
Spin trapping is a unique technique that allows the detection of too short lived free radicals for direct electron spin resonance (ESR) spectroscopy observation. The spin adducts have a longer half-life than the trapped free radicals. Nevertheless, spin adducts are frequently labile. In the present work, a novel series of nitrones was synthesized and their methyl spin adducts were characterized. Eight compounds derived from the 2,3-dihydro-1H-pyrimido[1,2a]quinoxaline 6-oxide core with C5-substituents: A: 4-methoxyphenyl, B: phenyl, C: 4-clorophenyl, D: 4-nitrophenyl, E: 2-clorophenyl, F: 2-nitrophenyl, G: methyl and H: benzyl, were studied. Methyl radical was generated using the Fenton reaction in PBS pH 7.6 in the presence of 33% DMSO. The spin trapped adduct signals were followed by recording their ESR spectra at different times between 0 and 480 minutes. The novel spin traps were compared with the widely used 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Negative controls were also carried out. All compounds, except for F, showed spin trap properties. Spin adducts were characterized spectrally by simulation of the experimental ESR spectra, showing a characteristic ESR spectrum with 9 peaks. Interestingly,all of them were more persistent than the DMPO adduct, presenting different stabilities. The decay of the spin trap adducts followed a pseudo-first order kinetics. Their half-life times were: A=97 min, B=143 min, C=23 min, D=133 min, E=46 min, G=61 min, and H=20 min. DMPO in similar experimental conditions, showed a half-life of 9 min. Spin adducts formation kinetics were observed for A, B and D compounds during the first minutes of the Fenton reaction (up to 30-40 minutes). Competition studies with DMPO revealed the persistence of the signal of the novel adducts after DMPO adduct signal disappearance. The trapping efficiencies of the new nitrones, evaluated by normalized double integration, were equivalent or higher than that of DMPO. The liposolubility and the high stability of some of the novel spin traps might be an advantage for the detection of free radicals in biological systems.
During the infection, Trypanosoma cruzi invades different cell types, and parasites are exposed to reactive oxygen and nitrogen species inside the phagolysosome and even in the cytosol. Trypanosomatids lack catalase and selenium-dependent glutathione peroxidase. Tryparedoxin peroxidases belong to the widespread peroxiredoxin family of peroxidases that use redox-active cysteine residues to reduce hydrogen peroxide (H2O2), organic peroxides and peroxynitrite. T. cruzi has a cytosolic 2-Cys Prx (TccTXNPx), and a mitochondrial 2-Cys Prx (TcmTXNPx). They both use tryparedoxins as reducing substrates, which in turn are reduced by trypanothione. In this work, we report the kinetic characterization of these two peroxiredoxins reaction with peroxynitrite and H2O2. Pre-steady state kinetic analysis using cysteine mutants of bothTXNPx isoforms identified one conserved cysteine residue (Cys52 in TccTXNPx and Cys81 in TcmTXNPx) as the primary target of H2O2 and peroxynitrite oxidation. These peroxidatic cysteines are oxidized to their sulfenic acid derivative before they react with the resolving cysteine, to form a disulfide bond. We studied the rate of peroxynitrite decomposition in the presence of cytosolic TcTXNPx and its two mutants in the peroxidatic and resolving cysteine residues (C52S and C173S respectively). While the C52S mutant lacked activity, the second order rate constant of the reaction between peroxynitrite and the wt enzyme was 1 x 106 M-1s-1 at pH 7.4 and 25 ºC. TccTXNPx C173S mutant was also capable of decomposing peroxynitrite but the rate of the reaction was somewhat slower than that observed with the wt enzyme. Preliminary studies of peroxynitrite reduction by TcmTXNPx showed that the reaction was faster than that of TccTXNPx. Moreover, using a competitive pre-steady state approach, we determined the rate constant of H2O2-mediated TccTXNPx oxidation as 1.3 x 107 M-1s-1 at pH 7.4 and 25ºC, which was faster than H2O2-mediated TcmTXNPx oxidation.
Background: Fatty acids bind to and regulate the activity of peroxisome proliferator-activated (PPAR) and liver X receptors (LXR).However, the role of nitrolinoleate (LNO2) on signalling pathways regulating these nuclear receptors is poorly understd. Thus, we investigated the effects of LNO2 on signal transduction pathway important for activation of PPAR and LXR. Methods: LNO2 was synthesized from linoleic acid and NO2BF4. LNO2 was purified and characterized using HPLC and LC-ESI/MS/MS-based techniques. HEK 293 cells were tansfected with plasmids containing the luciferase gene and LXR response elements using JetPi. After overnight transfection, cells were treated for 24h LNO2 (1-100μM), T1317 (1μM) and DMSO (vehicle) for 24h.The luciferase expression was analyzed by luciferase assay. The eletrophoretic mobility shift assay was used to evaluate the LNO2-mediated activation of PPAR g in THP-1 cells. Western blot analysis was used to evaluate the participation of the MAP kinases ERK 1/2, p38 and p21Ras in the PPAR g activation by LNO2. Results: LNO2 atphysiologically relevant concentrations (10 nM) activates PPAR. By contrast, linoleic acid a natural ligand for PPAR only activated the receptor at much higher concentrations (10 mM). The LXR α and β were insensitive to LNO2 regulation when compared with T1317, an LXR specific agonist. LNO2 activated p21 Ras more intensivelty than linoleic acid in the same conditions (10 nM). LNO2 activated the ERK1/2 MAP kinases but had no effects on p38 MAP kinase. In addition, the MEK inhibitor PD98059 blocked PPAR g activation by LNO2, suggesting a connection between ERK1/2 MAP kinases and the activation of this transcription factor. Concluding remarks: LNO2 has distinct effects on the activation of the nuclear receptors LXR and PPAR. While LNO2 is is a potent activator of PPAR g in THP-1 cells, it did not affect LXR activation. This LNO2 differencial effect on activation of nuclear receptors is helpful to explain the mechanisms involved in the biological actions of this nitrated fatty acid. Financial Support: FAPESP,CAPES/CNPQ - Instituto do Milênio Redoxoma.
Palabras clave: nitrolinoleate, PPAR, LXR
| Poster Presentation | 02.Cyclooxygenase, Lipid Peroxidation and Nitrolipids
Nitrolinoleate increases zymosan phagocytosis by THP-1 macrophages
Ferderbar, S 1(*); Faine, L 1; Rudnicki, M 1; De las Heras, B 2; Abdalla, DSP 3; Bosca, L 4
1 - Faculdade de Ciencias Farmaceuticas/USP | (*) Brazil
Background: Phagocytosis plays an essential role in host defense mechanisms through the uptake and destruction of infectious pathogens and contributes to inflammation and the immune response. Macrophage phagocytosis is mediated by distinct phagocytic receptors, such as mannose, complement and Fc receptors. These receptors induce activation of complex signalling cascades that lead to rearrangement of actin, cytoskeleton, and extension of the membrane and particle engulfment. Considering that LNO2 is a potent signalling molecule, the present study was designed to investigate the effect of LNO2 on the signalling pathways that govern the macrophage phagocytosis process in THP-1 cells differentiated to macrophage. Methods: THP-1 cells were differentiated to macrophages with 30nM PMA for 48h. LNO2 was synthesized from linoleic acid and NO2BF4 and characterized using HPLC and LC-ESI/MS/MS-based techniques. For evaluation of the phagocytic activity, cells were stimulated for 24h with LNO2 (10 uM), subsequently incubated for 2h with FITC-zymosan particles, and then analysed by flow cytometry.
Results: Incubation with LNO2 increased phagocytic activity of THP-1 cells differentiated to macrophages (48%) when compared to control cells. Pre-treatment of cells with PD98059 (MEK inhibitor) and LY294002 (PI3K inhibitor) decreased the phagocytic activity by 47% and 43%, respectively. On the other hand, the inhibitors SB203580 (p38 inhibitor) and GO6983 (PKC inhibitor) had no effect on LNO2-stimulated phagocytosis. Concluding remarks: Our results indicated that LNO2 increased THP-1 phagocytic activity by acting on ERK and PI3K that are kinases involved in the signalling pathway of phagocytosis. Financial Support: FAPESP, CAPES,CNPQ - Instituto do Milênio Redoxoma.
Sulfenic acid is a central intermediate in the redox modulation by reactive species of an increasing number of proteins. However, techniques for quantifying or even detecting it are scarce. Previous work from our group has shown that the single thiol in human serum albumin can be oxidized to a relatively stable sulfenic acid (HSA-SOH). We have developed a technique for quantifying HSA-SOH based on its reaction with the yellow thiol thionitrobenzoate (TNB). The reaction of HSA-SOH with TNB was biphasic. The first phase, which was ~20 times faster than the second phase and first order in oxidized HSA and TNB (105 ± 11 M-1 s-1), allowed us to quantify HSA-SOH. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37 °C) led to the formation of 0.20 ± 0.04 moles of HSA-SOH per HSA. Using this technique, we determined that HSA-SOH reacts with H2O2 at 0.4 ± 0.2 M-1 s-1 and confirmed that HSA-SH reacts with H2O2 at 2.7 ± 0.7 M-1 s-1 (37 °C, pH 7.4). Dimedone and sodium arsenite, two reagents used as RSOH probes, reacted with second order rate constants of 0.027 ± 0.009 and 0.036 ± 0.009 M-1 s-1 (37 °C, pH 7.4), respectively. Remarkably, ascorbic acid and uric acid were unable to reduce HSA-SOH back to thiol. In contrast, a competition approach with TNB allowed us to determine the rate constants of the reaction of HSA-SOH with cysteine, glutathione and homocysteine as 21.6 ± 0.2, 2.9 ± 0.5 and 9.3 ± 0.9 M-1 s-1 (25 °C, pH 7.4), respectively. This systematic study will aid in the design of suitable probes besides illustrating on the possible biological fates of this key intermediate.
Palabras clave: sulfenic acid, human serum albumin, hydrogen peroxide
| Poster Presentation | 01.Oxygen Radical Chemistry and Detection
Genotoxicity and oxidative stress in freshwater planarians (Dugesia schubarti) exposed to different formulations of herbicide glyphosate
Bordin, D.L. 1(*); dos Santos, D.R. 1; Salvador, M. 1; Erdtmann, B. 1
Glyphosate is a post-emergent and systemic herbicide with a broad spectrum used for the control of crop weeds. Glyphosate has been reported to have a low acute toxicity to different animal species. In spite of the extensive use of glyphosate in terrestrial and aquatic environments, there are relatively few studies about the impact of this product on the environment. In this study it were evaluated the toxicity of three formulations of glyphosate (Roundup Original® , Roundup Transorb® and Roundup Ready® ) on freshwater planarians (Dugesia schubarti). The parameters analyzed included oxidative stress by determination of thiobarbituric acid reactive substances (TBARS), superoxide dismutase (Sod) and catalase (Cat) activities and DNA damages by the Comet assay. The planarians were exposed during 4 hours to zero (control), 0.5 and 1.0 mg/l concentrations of glyphosate. The results indicated an increase in TBARS levels and DNA damage at 0.5mg/l, and a decrease in Sod and Cat activities for all three formulations. The planarians tested with 1.0 mg/l of glyphosate presented more DNA damages than controls, while the oxidative stress was not so evident showing a slightly increasing of both enzymes (Sod and Cat). The Roundup Original formulation induced a low level of oxidative stress than the others formulations. No differences were found in the Comet assay among the three formulations. Considering the results of oxidative stress and DNA damage, the three formulations of glyphosate are not risk-free for the environment. It must be noted that the concentrations of glyphosate used in these tests, for limited time, are lower than those detected in the environment.
Sticholysin II (StII) is a cytolysin from the sea anemone Stichodactyla helianthus. The toxin is highly haemolytic at the nanomolar range. Its haemolytic activity can be modified by oxidative modification of the toxin and/or the target red blood cell. In the present communication we briefly discuss the mechanism of the toxin action and how its activity is modified by pretretment of the toxin or the target cell with peroxynitrite or peroxyl radicals. StII haemolytic activity was enhanced in peroxyl-damaged red blood cells and senescent erythrocytes, a process closely related to the intracellular lost of K. On the other hand, toxin pre-treatment with micromolar peroxynitrite concentrations reduces its activity in a concentration-dependent way. In fact, even a few number of added peroxynitrite molecules per toxin molecule strongly reduces the haemolytic activity elicited by toxin addition to a red blood cell ensemble; these data are compared to those obtained when the toxin is pre-exposed to a source of peroxyl radicals (AAPH). This work has been supported by Fondecyt (Grant) 1070285
Palabras clave: Toxin, peroxynitrite, ROS
| Poster Presentation | 01.Oxygen Radical Chemistry and Detection
Oxidative stress in the blood of coal mining workers from South Brazil
Avila, Jr. S 1(*); Possamai, P, F 1; Backes, P 1; Parisotto, B, E. 1; Faria, E, P 1; Rizielio, M, V 1; Bortolotto, T 2; Ficagna, L 2; Pilatti, K, F 1; Wilhelm, F, D. 1
Mineral coal is responsible for 66% of the non-renewed energetic resources in Brazil, and the exploration of coal in the state of Santa Catarina uses surface and underground techniques. The air contamination generated from this activity increases the exposition risk to several environmental pollutants. The aim of this study was to evaluate the antioxidant profile and the possible cell damage in the blood of directly exposed mining workers and indirectly exposed subjects (residents) to the coal mining. Biomarkers of oxidative stress (TBARS, GSH and GST) and genotoxicity (Comet assay) were evaluated in blood samples of mining workers. Four groups (n=15 each group) were studied: surface miners, underground miners, residents near the mining area, and a control group. Plasma TBARS levels were significantly increased and GSH levels were decreased in residents, surface and underground workers when compared with control group. However, no significant differences were obtained through the Comet assay. The results indicate that the pollutants from the coal mining are probably acting as stress factors in all groups directly and directly exposed to the mining activity. Further studies are been carried out to better evaluate the associated oxidative stress, including the evaluation of heavy metal content in the blood of the same subjects.
Palabras clave: oxidative stress, coal miners, DNA damage
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 after benznidazol treatment (5mg/kg/day during 2 months). Antioxidant enzymes in erythrocytes and contents of reduced glutathione were analyzed before and after BZN treatment 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 gama-glutamil transferase as well as reduced glutathione contents in red cells were unchanged but lipoperoxidation levels in plasma, measured as TBARS contents, were significantly increased after BZN treatment. It seems that the antioxidant status is jeopardized after BZN treatment.
| Poster Presentation | 03.Redox Biology: Metals and Thiols
Tissue distribution of the thiol oxidase quiescin Q6/sulfhydryl oxidase (QSOX) during mouse development
Portes, KF 1(*); Ikegami, CM 2; Getz, J 1; Martins, AP 1; Noronha, L 1; Zischler, LF 1; Klassen, G 2; Camargo, AA 3; Bevilacqua, E 4; Zanata, SM 2; Nakao, LS 1
1 - Núcleo de Investigação Molecular Avançada -PUCPR | (*) Brazil
Thiol-oxidoreductase enzymes have been demonstrated to actively participate in several developmental redox processes. For instance, thioredoxin-1 and -2 knockout mice are not viable. Quiescin/sulfhydryl oxidases (QSOX) are novel thiol oxidases, that contain a thioredoxin domain and introduce disulfide bonds into proteins and peptides, with the concomitant hydrogen peroxide formation. It has been described in several tissues, such as male reproductive tract, nervous system, secreting tissues, epidermis, egg white and fetal lung fibroblasts. We have recently demonstrated its presence and activity in fetal serum. Then, to assess if QSOX could have a role during fetal development, here we analyzed the expression profile of QSOX in mouse E13.5, E16.5 fetuses and in P1 newborn by immunohistochemistry. For this purpose, recombinant mouse QSOX was first expressed as a fusion protein (His-tagged) in E. coli, purified by affinity chromatography and used to generate polyclonal antibodies in rabbits. The anti-QSOX produced was shown to be specific, since western blotting revealed a specific band in the expected molecular weight (65 kDa) in seminal vesicle extract from both mouse and rat. Moreover, in agreement with the literature, immunohistochemical staining of such adult tissue demonstrated a specific QSOX localization in epithelial cells. In the fetal and neonatal tissues, QSOX distribution was analyzed by immunohistochemistry in sagittal and transversal sections of tissues embedded in paraffin. Counterstaining was performed with hematoxylin. Results showed that QSOX staining profile is similar in all stages analyzed, and was preferentially observed in mesenchyme-derived tissues, such as glial cells, cardiac and skeletal muscles, vessels and connective tissues from lung, kidney, liver, esophagus, and intestine. Exception for the similarities was fetal epithelium and hepatocytes in which QSOX was absent, in contrast to the newborn tissues. Additionally, QSOX was observed both intra and extracellularly, in agreement with its possible role in extracellular matrix remodeling. In conclusion, QSOX is expressed in several tissues during mouse development, but preferentially in those derived from the mesenchyme, suggesting a putative role in redox modulation of developmental programs.Supported by Fundação Araucária, Instituto do Milênio Redoxoma (CNPq) and PIBIC
Cationic Mn(III) porphyrins are among the best functional SOD-mimics and the most potent catalytic redox modulators. We show here that the design of effective SOD mimics based on anionic porphyrins is hindered by inappropriate electrostatics. However, if the Mn porphyrin stability and uptake is suitably tuned, in vivo redox modulation may be achieved via an alternative mechanism, in which Mn porphyrin acts as an Mn carrier; such mode of action is unprecedented within Mn-porphyrins and was observed here for an anionic derivative. Structure activity of Mn-based SOD mimics indicates that optimal in vitro activity (on thermodynamic grounds) is expected for compounds possessing a redox couple at approximately +300 mV vs NHE, which is the midway potential between the oxidation and reduction of superoxide. The â-octabromination of the prototypical anionic Mn(III) porphyrins meso-tetrakis(p-carboxylatophenyl)porphinatomanganese(III) (MnTCPP3- or MnTBAP3-; E½ = ‑194 mV) and meso-tetrakis(p-sulfonatophenyl)porphinatomanganese(III) (MnTSPP3-; E½ = ‑160 mV), which have unsuitable Mn(III)/Mn(II) potentials and are not SOD mimics, resulted in the anionic analogues MnBr8TCPP3- (E½ = +213 mV) and MnBr8TSPP3- (E½ = +209 mV), respectively. Despite the Mn(III)/Mn(II) reduction potential of these â-octabromo derivatives being close to the optimum and nearly identical to that of the cationic meso-tetrakis(N-methylpyridinium-2-yl)porphinatomanganese(III) (MnTM-2-PyP5+; E½ = +220 mV), the SOD activity of both anionic â-octabromo porphyrins (MnBr8TCPP3-, log kcat = 5.07; MnBr8TSPP3-, log kcat = 5.56) are considerably lower than that of a potent, cationic derivative (MnTM-2-PyP5+, log kcat = 7.79); this is consistent with an unfavorable electrostatic facilitation in the anionic porphyrins, whose negative charges hamper the approach of the superoxide anion. MnTCPP3-, MnTSPP3-, and MnBr8TCPP3- do not protect SOD-deficient E. coli grown aerobically in restricted, 5 amino acid medium. Yet, MnBr8TSPP3-, with surprisingly high SOD-deficient E. coli protectiveness, operates not as a redox modulator itself, but presumably as an Mn-carrier; accumulation of the free-ligand was verified in vivo. Control experiments with corresponding levels of MnCl2 show no significant protection. The molecular basis of this process was also explored chemically and is consistent with kinetics and thermodynamics of metal-porphyrin interactions within this class of compounds. [NIH-IR21-ESO/3682, NIH-U19A167798-01, NIH/NCI DCCC 5-P30-CA14236-29, CNPq, FAPEMIG]
Two simple cobalt salts, CoCl2·6H2O and Co(Ac) 2·4H2O, have the abilities to catalyze the nitration of free tyrosine and BSA tryrosine residues to produce 3-nitrotyrosine (3-NY). The percentage of added peroxynitrite recovered as nitrated tyrosine was enhanced from 11% to 24% for free tyrosine, 8% to 13% for BSA by CoCl2 and from 11% to 25% for free tyrosine, 8% to 15% for BSA by Co(Ac)2 respectively when the concentration of cobalt salts increased from 0 to 0.1 mmol·L-1. The reaction rate of tyrosine nitration by peroxynitrite was strictly linear with cobalt salt concentration at 4, 15, 25 and 37 ℃. The apparent activation energy for CoCl2 and Co(Ac)2 - catalyzed nitration of tyrosine were 15.7 and 15.5 kcal·mol-1 respectively which compared favorably with the energy required for heterolytic cleavage of peroxynitrite into nutronium ion (NO2+) and hydroxyl ion (OH-) (13 ± 2 kcal·mol-1 ) in water at pH 7.0. The yield of another product, dityrosine or dimmer BSA which came form the combination of two Tyr· decreased sharply in the presence cobalt salts. Our former studies showed that the hydroxyl radical scavengers can inhibit the spontaneous nitration of tyrosine but have little effect on the yield of 3-NY in the presence of cobalt salts. Taken together, cobalt salts changed the free radical nitration mechanism which occurred spontaneously and catalyze the nitration of tyrosine through the formation of nitronium ion-like species (NO2δ+―δ-O―CoⅡ) but not through the homolysis of O-O bond in Co(ONOO-) to form Co-O· and NO2· which was reported previously.
DMPOX (5,5-dimethyl-1-pyrrolidone-2-oxyl) is an oxidation product of the spin probe DMPO (5,5-dimethyl-1-pyrroline N-oxide). We characterized DMPOX formation in the reaction between DMPO (10 mM) and thallium (III) (Tl(III): 5-100 mM). DMPOX was determined by ESR at room temperature using a Bruker ECS 106 with an ER 4102ST cavity. The obtained spectrum in water solution was a primary nitrogen triplet with each line split into triplets with a 1:2:1 pattern with aN(1) = 7.3 ± 0.1 G and aH(2) = 3.9 ± 0.1 G. Superoxide dismutase (SOD, 100 mU), catalase (CAT, 100 mU), mannitol (50 mM), ethanol (50 %) or methanol (50%) did not affect the intensity of DMPOX signal, suggesting that oxygen free radicals are not involved in this process. However, DMPOX signal intensity decreased 82 % (p < 0.01) in 90 % (v/v) methanol, and no signal was detected in 100 % (v/v) methanol, indicating that water is necessary for this reaction. Tl(III) and Tl(I) concentrations were determined by the reaction with trifluoperazine dihydrochloride, and by capillary electrophoresis with UV detection at 214 nm, respectively. In these conditions, Tl(III) disappearance rate was 1.6- times higher than DMPOX formation rate, and equal to Tl (I) formation rate, suggesting a stoichiometry 1.5:1 for Tl(III):DMPOX and 1:1 for Tl(III):Tl(I). Additionally, Tl(III) did not oxidize the aliphatic nitrones PBN and POBN. In conclusion, we propose that the reaction between Tl(III) and DMPO to produce DMPOX is a non-radical mediated process, and that it could proceed through a Forrester-Hepburn mechanism where Tl (III) induces a nucleophilic attack from water to the 2-position of DMPO ring, forming the intermediate DMPO-OH. Next, Tl(III) mediates the two-electron oxidation of DMPO-OH to a keto group, rendering DMPOX and Tl(I). Supported by UBA (B802, B128, B072) and CONICET.
Palabras clave: nitrones, thallium, esr
| Poster Presentation | 11.Nitration, Nitrosation and Oxidation in Protein Function
The influence of the time of haemodialysis treatment under delta aminolevulinate dehydratase activity
Valentini, J. 1(*); Piva, S.J. 1; Brucker, N. 1; Tonello, R. 1; Grotto, D. 1; Barbosa Jr, F. 2; Vicentini, J.T. 1; Garcia, S.C. 1
Delta aminolevulinate dehydratase (d-ALA-D) is an enzyme involved in the heme biosynthesis that requires thiol groups for their activity. ALA-D has been suggested as a marker for oxidative stress, it is involved in many pathophysiological processes, with higher prevalence in patients with chronic renal failure (CRF), and this situation is amplified by haemodialysis (HD) treatment. In addition, long time of HD treatment may intensify the oxidative stress. Thus, the aim of this study was to evaluate the effect of prolonged HD treatment under activity and reactivation index of the ALA-D. Three groups were evaluated: healthy subjects (control, n=40), HD patients with recent (n=36; HD duration: 17.7 ± 1.71 months), and long (n=26; HD duration: 82.2 ± 6.32 months) time of treatment. ALA-D activity and reactivation index was determined in the blood by method of Sassa (1982). Results were expressed as means ± standard error (S.E.M). Dates were analyzed using the Statistica® 6.0 software. Blood ALA-D activity was significantly lower in HD patients when compared to the control group (p<0.0001), however it didn’t is different between patients with recent and long time of treatment, RT and LT, respectively. The involvement of SH-groups was measurement by reactivation index of the enzyme ALA-D, and it was significantly higher in HD patients in comparison with controls (p<0.0001). Additionally, the reactivation index was significantly different between patients with RT and LT of HD treatment (p<0.05), and it was positively correlated with the time of treatment (r=0.30; p<0.05). In conclusion, this work demonstrated that situations associated with overproduction of free radicals result in ALA-D oxidation, and the measurement of ALA-D activity can be used as biomarker of oxidative stress. The increase in the reactivation index demonstrated that free radicals can interact directly with thiol groups of proteins, oxidizing them to disulfides. Additionally, the reactivation index correlated positively with the time of HD treatment, while the ALA-D activity didn’t suffer influence. Thus, we could suggest that in chronic processes, as HD treatment with long time, the assessed of ALA-D reactivation index can be important such as biomarker of oxidative damage in thiol enzymes.
The present report attempts to address the involvement of reactive oxygen species (ROS) in L. major. Macrophages derived from NOS2-deficient mice (nos2 KO) produce increased amounts of ROS in response to L. major infection or other stimuli, when compared to WT macrophages. Such result suggests a compensatory effect in the absence of NO•. However, despite preserved and even augmented production of ROS, nos2 KO macrophages and NOs2-/- mice display a partial control of Leishmania replication. In order to further evaluate the involvement of ROS in resistance to L. major, NADPH deficient (gp91phox knockout or phox KO) mice were infected. Phox KO animals exhibited similar lesion and tissue parasitism in footpads, draining lymph nodes, spleens and livers when compared to wild-type controls. These results point to a minor role of ROS in this model. However, when NO• was inhibited in vitro, the parasite proliferation was higher in phox KO macropahges than in WT controls. Therefore, aminoguanidine treatment during in vivo infection showed that ROS play a role in controlling parasite visceralization to the liver in absence of NO•. Supported by FAPEMIG and CAPES
The main mechanism involved in control of Leishmania in mice is the production of NO by activated machrophages. However, mice that do not express the nitric oxide synthase (iNOS-/-) present some resistance to infection when compared to mice that do not express interferon-gamma (IFN-g-/-), the main macrophage-activating cytokine. Hence, we raised the hypothesis that reactive oxygen species (ROS) might be responsible for the partial resistance to infection in iNOS-/- mice. To test this hypothesis, iNOS-/-, phox-/- and wild type (wt) mice were used for in vivo and in vitro assays to establish what phenotypes were connected with susceptibility to Leishmania amazonensis. Elicited peritoneal machophages from iNOS-/- were more permissive to infection in vitro, and macrophages from phox-/- mice did not differ from macrophages from wt animals. On the other hand, in vivo infection revealed an important role of ROS in the inhibition of parasite growth, since phox-/- mice had larger footpad lesions than wt. When treated with an inhitor of iNOS, aminoguanidine, these mice showed augmented parasite load and lesion size in the footpad. Our data suggest the presence of an important mechanism independent of nitric oxide, and introduce another model of study involving reactive oxygen species and the immunology of parasitic disease. Support: CNPq, CAPES and FAPEMIG.
Tyrosine nitration under conditions of oxidative stress is well established. One of the pathways proposed for nitration in vivo involves heme peroxidases in the presence of hydrogen peroxide and nitrite. In this work, horseradish peroxidase (HRP), a heme–containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of organic and inorganic compounds, was used as a model of heme peroxidase. The reaction was performed in the presence of hydrogen peroxide, nitrite and tyrosine, and the products of oxidation 3,3’–dityrosine and 3–nitrotyrosine were quantified by HPLC. Computer–assisted simulations with the software Gepasi, using reported rate constants, explained the relatively low yields of 3–nitrotyrosine and 3,3’–dityrosine, supporting a free radical mechanism. Physiological concentrations of glutathione (5 mM) completely inhibited nitration, due to the fast recombination reaction between nitrogen dioxide (•NO2) and tyrosyl radical, regenerating nitrite and tyrosine, respectively. Paradoxically, with increasing concentrations of glutathione, we observed a bell–shaped behaviour for 3,3’–dityrosine. The suicide inactivation of heme peroxidases by hydrogen peroxide is well–documented. In our study, hydrogen peroxide caused a mild inactivation of HRP, with 800 turnovers before achieving complete inactivation. Contrary to the protective effect of substrates, the inactivation was significantly enhanced in the presence of nitrite (500 μM) and HRP itself appeared nitrated. Both tyrosine (500 μM) and glutathione (2 mM) prevented the inactivation of HRP. Our results indicate that •NO2 is not only responsible for tyrosine nitration, but also for enzyme inactivation.
Background: Nitrolinoleate (LNO2), a nitroalkene derivative of linoleic acid (LA) that belongs to a new class of lipid mediators, has been demonstrated to donate nitric oxide (·NO) and act as a mediator in cell signaling. As ·NO donors are involved in the regulation of angiogenesis and compelling evidence suggests that the process of neovascularization might be associated with both atherosclerotic plaque growth and destabilization, our aim was to investigate the effects of LNO2 on the angiogenesis process. Methods: The effects of LNO2 on angiogenesis was examined using in vitro and in vivo model systems. Proliferation and cell migration were evaluated by human umbilical endothelial cells (HUVEC) and endothelial cell sprouting by the rat aortic ring assay. In addition, the effects of LNO2 on the neovascularization process was also investigated through the chick chorioallantoic membrane (CAM) model. Results: No effect of the LNO2 on the endothelial cell proliferation (1-10 uM) was observed. However, LNO2 (1–10 uM) significantly induced not only HUVEC migration but also endothelial cell sprouting from rat aortic rings. Interestingly, equimolar doses of its precursor, the linoleic acid, showed no effect on endothelial cell migration and sprouting. Furthermore, the LNO2 effects on endothelial cell migration and sprouting were significantly prevented by a ·NO scavenger pretreatment [2-(4-carboxyphenyl)-4,4,5,5 -tetramethylimidazoline -1-oxyl-3-oxide (carboxy PTIO, 100 uM)]. Moreover, although the pretreatment with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 500 uM) has not shown any significant effect on LNO2-induced cell migration, LNO2-induced endothelial cell sprouting was significantly prevented by a L-NMMA pretreatment. Finally, LNO2 at 10 uM also significantly augmented neovascularization in the CAM model. Conclusions: Our findings suggest that LNO2 may be a mediator of the neoangiogenesis process through modulation of endothelial cell migration and sprouting by a ·NO-dependent mechanism. Financial Support: FAPESP,CNPQ/INSTITUTO do MILÊNIO REDOXOMA.
Trypanossoma cruzi epimastigotes (EPI) proliferate inside of the hematophagous insects that usually ingest in a single meal about 10mM heme bound to hemoglobin. Recently we showed thatEPI acquires extracellular heme from their invertebrate vectors and the addition of heme increases drastically the parasite proliferation (Lara et al., 2007, BBRC). Although heme induces the growth in these cells this molecule is a tetrapyrrole containg a central ion and a powerful generator of reactive oxygen species (ROS) that promotes an imbalance redox. In order to investigate the oxidative stress induced by heme in these cells, T. cruzi epimastigotes Dm28c, were maintained in BHI supplemented with 10% FCS at 28ºC without addition of heme for two passages. Afterwards, cells were incubated in the absence or in the presence of different concentrations of heme. Thus, lipid peroxidation using TBARS assay and 4-HNE immunoblotting was examined. We observed that the lipoperoxide formation increased according to heme addition. This result is supported by ROS formation analysis through flow cytometry and epifluorescense microscopy of CM-H2DCFDA. Interestingly, the fluorescent signal is proportional to heme concentration and decreases in a short period of time, indicating that T. cruzi might have selected effective mechanisms against high heme concentration found in its environment. Supported by FAPERJ, CNPq and PIBIC/UERJ.
Selenium is a constituent of several selenoproteins, including glutathione peroxidase, thioredoxin reductase, selenoprotein P and selenoprotein R, which are primarily involved in oxidant function and redox state. Organoselenium (OS) compounds have been described as antioxidant, enzyme inhibitor, neuroprotective, anti-tumor and anti-infectious agents. Diphenyl diselenide (DPDS) is an OS compound that has presented interesting pharmacological effects such as hepatoprotective, neuroprotective, anti-inflamatory and antinociceptive. Besides of these pharmacological properties, DPDS presents some toxic effects. The chronic exposure to high doses of DPDS causes central effects in mouse brain and there is also evidence for teratogenic effects. Moreover, it has been shown that DPDS acts as pro-oxidant by depleting free glutathione (GSH) and it was able to induce frameshift mutations in Salmonella typhimurium and in Saccharomyces cerevisiae and increased crossing over and gene conversion frequencies in diploid strains of S. cerevisiae. 3’3-ditrifluoromethyldiphenyl diselenide (DFDD) is a novel OS and it is a structural analog of DPDS. In contrast of DPDS, it has been poorly studied. Furthermore, we have previously described that DFDD prevented against the apomorphine-induced stereotypy in mice. Therefore, the aim of this work was to investigate the possible protective effect of DFDD on the mutagenicity induced by hydrogen peroxide in TA102 Salmonella typhimurium and XV185-14c Saccharomyces cerevisiae strains. DFDD was able to protect against the hydrogen peroxide – induced mutagenicity in both strains and concentrations used. In addition, as also cited by previous works, DFDD showed opposite features to those observed for DPDS in the same systems and the CF3 functional groups presents in DFDD might be involved in this antioxidant action. Indeed, these results suggest that the DFDD is a promising molecule with possible antioxidant actions. Nevertheless, more studies are needed to elucidate the mechanisms of action of this OS compound.
The aim of this study is to reveal the current situation of the Chilean population (Valparaiso region), exposed to oxidative risk’s factors. A group of 100 people, who answered part of the selected survey (Festy 2003), including factors that induce oxidative damage as: fruits and vegetables daily intake (1), Smoking habits (2), alcohol consumption (3), sedentariness (4), environment pollution (5), medicine drug consumption (6), direct solar light exposition (7), age over 50 years old (8), excessive work (9), overweight (10) and familiar background of pathologies bounded to oxidative stress (11) The results were based on the amount of affirmative answers. An arrangement of the factors (included in the alluded survey) that would induce oxidative damage was made, resulting that 3% of the population would be in high risk, 61% would present medium risk and 36% low risk to suffer oxidative damage. In addition 5 relations between the measured factors were made, in order to evaluate a possible synergism between the different factors. The relationship between factors Nº 1, 4 and 10, demonstrated that overweight is not necessarily a consequence to sedentariness, and would be due to a low vegetable intake. The overweight increases when a low antioxidant intake and sedentariness are present. Relationship between factors Nº 6 and Nº 8 demonstrated that the higher medicine drug consumption (76.6%) was in people over 50 years old. This investigation reveals that the mayor difference related with age was drug consumption (over 50 years old) and smoking habits (under 50 years old). Antioxidants guide practique. Danièle Festy, 2003 LEDUC. S Èditions
Palabras clave: oxidative , risk, habits
| Poster Presentation | 03.Redox Biology: Metals and Thiols
MOLECULAR ALTERATIONS ASSOCIATED WITH OXIDATIVE STRESS INDUCED BY HEAVY METALS IN THE MACROALGA GRACILARIA TENUISTIPITATA
Tonon, AP 1(*); Zaini, P 1; Cabral, MC 1; Colepicolo, P 1
1 - Instituto de Química, Universidade de São Paulo | (*) Brazil
Gracilaria tenuistipitata is a red algae economically important for production of the agar, utilized by pharmaceutical, nutritional and biotechnological industries. Moreover, due to its tolerance to salinity, temperature and pH, this species is widely studied as a model for many biochemical analyses. One of the problems that affect the development of this alga is the contamination by chemical agents such as heavy metals that induce severe stress reactions in organisms. Previous studies showed variations in concentration of some enzymes important for the metabolism. Such variations were caused by stress induced by metals. Using DNA microarray techniques for Gracilaria tenuistipitata, it is possible to analyse differential expression of several enzymes when the algae is submitted to 50% of a sublethal concentration of Cu2+ and Cd2+ metals. It was used in the microarray construction a sequenced cDNA library, representing normal culture conditions. These library has in total 3631 ESTs, 2387 unique sequences and 53,4% redundancy. Preliminary analysis of the microarrays results from our laboratory have shown that Cu2+ and Cd2+ cause increased oxidative stress. The additions of heavy metals induce increase content of catalase, superoxide dismutase, glutathione peroxidase, ascorbate peroxidase and others. Our results are being processed and analysed. Preliminary data revealed differential expression in these enzymes when the G. tenuistipitata is submitted to stress by heavy metals. They, present in the cDNA library, are up-regulated in response to stress as a defense mechanism.
Reactive oxygen species (ROS) generated during ischemia (I)/reperfusion (R) enhance myocardial injury, but brief periods of myocardial ischemia followed by reperfusion (ischemic preconditioning, IP) induce cardioprotection. Ischemia is reported to stimulate glucose uptake through the translocation of Glut-4 from the intracellular vesicles to sarcolemma. In the present study we demonstrated involvement of ROS in IP mediated Glut-4 translocation along with increased expression of caveolin-3, p-eNOS, p-Akt and decreased expression of caveolin-1. The rats were divided into: Control Sham, NAC Sham (NS) [N=n-acetyl cysteine (NAC), free radical scavenger], IR, IP + I/R (IP), IPN. IP was performed by 4 cycles of 4 min I and 4 min R followed by 30 min of ischemia and 3, 24, 48 hrs of R depending on protocol. Increased mRNA expression of Glut-4 and caveolin-3 was observed after 3h of R in IP compared to other groups. IP increased expression of Glut-4, caveolin-3, p- fractions as compared to IR. Co-immunoprecipitation demonstrated decreased association of cav-1 / eNOS in IP group as compared to IR. Significant Glut-4 & caveolin-3 association was also observed in IP group. This association was disrupted when NAC was used in conjunction with IP. It clearly documents significant role of ROS signaling in Akt/eNOS/caveolin-3 mediated Glut-4 translocation and association in IP myocardium. In conclusion, we demonstrated a novel redox mechanism in IP induced eNOS and Glut-4 translocation and the role of caveolar paradox in making the heart euglycemic during the process of ischemia leading to myocardial protection in a clinically relevant rat ischemic model.
Palabras clave: Redox, Diabetes, Heart
| Poster Presentation | 12.Antioxidant Interventions and Repair Systems
Heart antioxidant enzyme activities during ischemia-reperfusion injury are modulated by exercise and anabolic steroid treatment
Chaves, EA 1(*); Hamilton, JHM 2; Oliveira, MF
1 - Federal University of Rio de Janeiro - Medical Biochemistry Institute | (*) Brazil
The beneficial effects of exercise against ischemia/reperfusion (I/R) injury are well known. Previous studies showed that exercise cardioprotection would be mediated by increasing the antioxidant enzyme activities that were impaired by the anabolic steroid nandrolone decanoate (DECA) treatment. Here, we evaluated the effects of exercise and DECA treatment on the activities of the four antioxidant enzymes superoxide dismutase (SOD), catalase (cat), glutathione peroxidase (GPx) and glutathione reductase (GR) in three periods: pre-ischemia (PI); after 30 minutes of ischemia (I); after 30 minutes of ischemia and 60 minutes of reperfusion (I/R). The activity of all antioxidant enzymes were increased during I period in all groups. Control trained rats (CT) showed higher activities of SOD and GPx at PI, I and I/R periods when compared to control sedentary (CS), DECA sedentary (DS) and DECA trained (DT) rats. GR activity was increased in CT rats at PI and I/R periods compared to DS and DT. Cat activity was higher in CT group at PI period compared to DS. Together, these results indicate that the exercise-induced heart antioxidant enzyme activities was impaired by DECA treatment.
Flavonoids are phenolic substances present in substantial amounts in vascular plants. Among the many significant medicinal and nutritional values, antioxidant properties of flavonoids have been by far the most studied. These molecules interact strongly with cations, and experimental evidence also show that the antioxidant activity is modified in the presence of metal ions. The research on the structure-activity relationship has been hampered by the lack of a full understanding of the flavonoid-cation interactions with biologically relevant cations. In this work we report a detailed potentiometric study of the complexation of flavonoids (quercetin, rutin, rhamnetin and isorhamnetin) with Mg2+, Ca2+, Cu2+, Zn2+, Mn2+, Fe2+, Fe3+ and Al3+ under experimental conditions resembling the physiological ones (0.15 M NaClO4 either at 37.0 or 25.0 °C). The UV-vis spectra studies of some selected systems in methanolic solution and the characterizaton of solid complexes are also included. Besides, we report the study of the free radical scavenging activity of the ligands and their corresponding metal complexes. The results show the formation of stable species of flavonoids with these metal ions, most of them having a 1:1 metal to ligand ratio. The flavonoids are present in these species in deprotonated forms. The observed free radical scavenging activity of flavonoid:metal ion mixtures (equimolar amounts) was similar to that observed for the flavonoids alone in the same conditions. The unique case in which the activity is markedly increased upon addition of the metal ion is Cu-qrc. But, when one equivalent of the base triethylamine was additionaly added to the 1:1 flavonoid-metal ion mixture, an increase in the antioxidant activity was verified for systems containing qrc or rha, due to the higher percentage of metal complexes upon deprotonation of the ligands. The observed activity was always higher than that showed by the corresponding flavonoid with the addition of one equivalent of the base. The higher variation in this sense was observed for qrc and rha complexes of Zn and Mn. Other assayed ligand to metal molar ratios yielded similar or lower antioxidant activity.
Palabras clave: flavonoids, antioxidant activity, chemical speciation
| Poster Presentation | 12.Antioxidant Interventions and Repair Systems
In vitro evaluation of antioxidant activity and potential toxicity of phenyl(phenylselanyl)sulfane and (2-chlorophenyl)(phenylselanyl)sulfane
Pinton, S. 1(*); Borges, V. C. 1; Dadalt, G. 1; Savegnago, L 1; Schneider, C. C. 1; Nogueira, C. W. 1
1 - Laboratório de Síntese, Reatividade e Avaliação Farmacológica e Toxicológica de Organocalcogênios. Departamento de Química. Centro de Ciências Naturais Exatas. Universidade Federal de Santa Maria | (*) Brazil
The rapid growth, in recent years, of the role of reactive oxygen species in pathology has brought with new ideas for the therapy of variety of diseases. Consequently, the search for new antioxidants as potential drugs is an active field of medicinal chemistry.In this way, several reports have appeared describing the antioxidant activity of ebselen and other organoselenium compoundsin different experimental models. The present study investigated the antioxidant potential, thiol peroxidase activity, and rate of reduced glutathione (GSH) oxidation of phenyl(phenylselanyl)sulfane (Compound 1) and (2-chlorophenyl)(phenylselanyl)sulfane (Compound 2). Compound 1 protected against lipid peroxidation up to 200 ìM, around 30.6%. Compound 2 protected, at 100 ìM, around 22.5% against lipid peroxidation induced by Fe/EDTA. Compounds 1 and 2 significantly decreased reactive species (RS) in the fluorimetric assay from 50 and 100µM, respectively. Compound 1 showed thiol-peroxidase activity in lower concentrations (5-40 ìM) at 30, 60 and 120 min. In contrast, compound 2 did not present a catalytic effect in this reaction.Compounds 1 and 2 (up to 50 ìM) significantly increased the rate of GSH oxidation at 30, 60 and 120 minutes. In conclusion, the present results demonstrate that phenyl(phenylselanyl)sulfane and (2-chlorophenyl)(phenylselanyl)sulfane can be considered potential antioxidant compounds. However, more detailed pharmacological studies involving organoselenium compounds are needed. Acknowledgements The financial support by UFSM, FAPERGS, CAPES and CNPq is gratefully acknowledged.
In previous work, upregulation of heart mitochondrial nitric oxide synthase (mtNOS) during acclimatization of young rats to hypobaric hypoxia up to maturity was associated to preservation of contractile function and increased tolerance to O2 deprivation (J Appl Physiol 98:2363-75, 2005). In the present study, the persistence of these and other effects of chronic hypoxia during deacclimatization were analyzed. Seven-week-old rats were submitted to 53.8 kPa in a hypopressure chamber, whereas a same number of controls (C) remained at 101.3 kPa. After 5 mo, hypopressure was interrupted and hypoxic rats returned to sea-level atmospheric pressure. At 0.4, 2, and 5 mo, hematocrit was determined, heart was removed, and both papillary muscles and mitochondria were isolated from the left ventricle of 5 rats of each group. Hypoxia-induced increased hematocrit and decreased body weight were normalized at 2 mo; right ventricle weight, 100% increased in acclimatized rats, was still 30% higher than in C at 5 mo. Papillary muscle mechanical activity, measured under isometric conditions at increasing Ca2+ concentrations, was significantly higher in all deacclimatized groups than in C at 0.8 to 2.8 mM Ca2+. At maximal Ca2+, developed tension (DT) was 65, 58 and 40% higher under basal conditions and 129, 107 and 71% higher after a period of 60 min hypoxia and 30 min reoxygenation, at 0.4, 2, and 5 mo, respectively. Maximal rates of contraction and relaxation behave in a similar way as DT. Production of NO by submitochondrial membranes (SMM), determined by the oxyhemoglobin assay, was 40, 27 and 21% increased at 0.4, 2, and 5 mo, respectively. Western blot analysis performed in SMM revealed a higher expression of iNOS and nNOS in previously hypoxic rats, which were normalized at 5 mo. The correlation of left ventricle mtNOS activity with papillary muscle parameters of contractility, before as well as after recovery from in vitro hypoxia, sustains the hypothesis of an involvement of mtNOS in cardioprotection afforded by acclimatization. This model would provide the most persistent effect known at present on preservation of myocardial mechanical activity and tolerance to O2 deprivation.
We recently reported that hypovolemic state induced by acute hemorrhage triggered a heterogeneous and dynamic nitric oxide synthases (NOS) activation modulating the cardiovascular response in anesthetized rats. The involvement of nitric oxide (NO) pathway occurs in an isoform-specific and time dependent manner. We demonstrated, for the first time, that increased cardiac endothelial NOS expression is an early molecular response to regulate heart rate after blood loss. The inducible NOS become a major source of cardiac NO production in the later stages, which could be determinant of the heart dysfunction after 120 min of sustained hemorrhagic shock. Our aim was to evaluate the involvement of mitochondrial NOS (mtNOS) activity in the cardiovascular adaptation to hemorrhagic shockc after 120 min of bleeding in conscious and anesthetized rats. The study comprised 3 groups of animals (n=5 per group): C: normotensive conscious and anesthetized rats; CH: conscious hemorrhaged rats (20% blood loss); AH: anesthetized hemorrhage rats. Heart mitochondria isolated from control, conscious and anesthetized hemorrhaged rats showed high respiratory control, indicating that the organelles were well coupled and able to effectively carry out oxidative phosphorylation (C=1.8; CH=1.8 and AH=1.5). No differences in the succinate-supported state 4 and 3 respiration (ng-at O/min.mg protein) were observed among the experimental groups (Cst4 = 130±9.8, Cst3 =234±16; CHst4 = 112±10, CHst3 = 204±15; AHst4 = 158±11, AHst3 = 240±11). Blood loss did not modify heart mitochondrial hydrogen peroxide production (nmol H2O2/min.mg protein) compared with C (C=0.577±0.05; CH=0.569±0.1; AH=0.524±0.06). However, the hypovolemic state modified heart mtNOS functional activity determined by enhancing H2O2 production (C=59%; CH=85%; AH=67%). Taking into account that changes in mtNOS functional activity reflect variations in mitochondrial NO production and steady state concentration, higher mitochondrial NO levels would be involved in the cardiovascular adaptation to volume depletion observed in conscious animals.
Cytotoxic effects of the cationic (Fe(III)TMPyP) or anionic (Fe(III)TPPS4) mesoporphyrins, in the dark, on LNCaP tumor cells was dose-dependent. The IC50 was about 15 µM, after 48 h of incubation, for either porphyrin. After 1 hour incubation in the presence of any of these compounds, at 10 µM, the number of dead cells were negligible. However, a further period of 3 min under irradiation (radiation wavelength of 660 nm using a diode laser) induced cell death either by apoptosis or necrosis, both preceded by a significant increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) and Reactive Oxygen Species (ROS) production. Chelation of the [Ca2+]cyt with BAPTA/AM protected LNCaP from porphyrins-induced cell death. The presence of either DPI, a NADPH oxidase inhibitor, or FCCP, a mitochondrial uncoupler, decreased ROS production. This suggests the participation of both NADPH oxidase and mitochondria in this process of oxidative stress. Indeed, irradiation of LNCaP in the presence of each porphyrin resulted in cyclosporin-A but not in FK 506 sensitive decrease in apoptotic and necrotic cell death. It is proposed that irradiation of these porphyrins stimuled ROS generation followed by mitochondrial permeability transition and cell death mediated by increased [Ca2+]cyt. Supported by FAPESP, CNPq and FAEPEX/UNICAMP.
Palabras clave: Reactive oxygen species, Irradiated porphyrins, Tumor cell death
| Poster Presentation | 07.Peroxynitrite Chemistry and Biochemistry
Peroxynitrite, carbon dioxide and iron(II) cytochrome /c/.
Domazou, A. 1(*); van der Meijden, B. 1; Koppenol, W.H. 1
The oxidation of iron(II) cytochrome c by peroxynitrite in the presence of various concentrations of CO2 was studied at pH 7.1 and 25 °C, with iron(II) cytochrome c and CO2 always in excess. Peroxynitrite is known to react with CO2 to form the intermediate adduct, ONOOCO2-;, that decomposes to CO3•− and NO2 with yields of 30% or less than 5%. Fe(II)cytochrome c is oxidized to Fe(III)cytochrome c. The formation of Fe(III)cytochrome c follows first-order kinetics and the observed first-order rate constants are linearly dependent on the CO2 concentration. The derived apparent second-order rate constant is quite comparable with that reported for the reaction between peroxynitrite and CO2 at the same pH. The yield of the Fe(III)cytochrome c formed relative to peroxynitrite increased slightly with increasing CO2 concentration and reached a plateau value of 65 ± 5% at around 0.6 mM of CO2. Pulse radiolysis experiments showed that NO2· does not oxidize iron(II) cytochrome c to iron(III) cytochrome c. We already showed that the trioxidocarbonate radical(•1−), CO3•−, oxidizes Fe(II)cytochrome c with formation of Fe(III)cytochrome c with 100% yield. Assuming that these radicals are the only reactive species formed, the maximum yield of Fe(III)cytochrome c formed should be 30%. The higher yield of the Fe(III)cytochrome c formed reported here implies that, apart from the CO3•− / NO2 radicals, another reactive intermediate is formed, capable to oxidize Fe(II)cytochrome c.
Flavonoids have recently attracted a great interest as potential therapeutic agents againts a large variety of diseases, most of which involve radical damage. Morin (2´, 3, 4´, 5, 7-pentahydroxyflavone) is one kind of flavonoid widely distributed in tea, coffee, cereal grains and a variety of fruits and vegetables. In pharmaceutical product development, b-cyclodextrins, a category of pharmaceutical excipients, have been widely used to improve solubility, chemical stability and bioavailability of a number of poorly soluble compounds. The present work was designated to study the complexation of morin utilizing three different cyclodextrins, (HP-bCD, DM-bCD and bCD) to improve the solubility and to determine the effect of the complexation process on their antioxidant capacity using ORACFL. Also we obtained the thermodynamic properties for all complexes. Through complexation with the three cyclodextrins, bCD, DM-bCD and HP-bCD, the aqueous solubility of morin has been improved in neutral aqueous solutions forming 1:1 inclusion complexes. The inclusion ability of âCD and its derivatives was the order: HP-bCD > DM-bCD > bCD. ORACFL assay indicated that antioxidant activities of the all complexes were better than morin free. Finally, the DM-bCD complex is primarily enthalpy driven process meanwhile HP-bCD and bCD entropy driven process.
Palabras clave: MORIN, ANTIOXIDANT, CYCLODEXTRIN
| Poster Presentation | 09.Mitochondria and Apoptosis
COMPLEX I INHIBITORS AND MITOCHONDRIAL NITRIC OXIDE SYNTHASE FUNCTIONAL ACTIVITY
Gomez, C 1(*); Sanchez-Pino, MJ 2; Bandez, MJ 3; Boveris, A 4; Navarro, A 2
1 - Depatment of Biochemistry and Molecular Biology, School of Medicne, University of Cádiz | (*) Spain
Rotenone and pyridaben have been recognized as selective inhibitors of mitochondrial complex I activity. The selective inhibition of mitochondrial NADH-dehydrogensae activity by pesticides as rotenone and pyridaben may constitute part of the pathogenic mechanism of Parkinson´s disease. The Parkinsonian syndrome induced by pesticides is associated with the impairment of mitochondrial function. Rotenone and pyridaben show a selective inhibition of O2 uptake and respiratory control in rat brain mitochondria in the presence of NAD-dependent substrates. The IC50 of rotenone and pyridaben for complex I inhibition were in the range 1.7-2.2 microM. The determination of NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome oxidase activities in rat brain mitochondrial membranes showed again the selective inhibition of complex I by rotenone and pyridaben. The effect of rotenone and pyridaben in rat brain mitochondria showed an exponential dependence on membrane potential, as determined by Rh-123 fluorescence.The mitochondrial respiration that is affected by supplementation with mtNOS substrates and inhibitors, the mtNOS functional activity, provides an experimental approach to the regulation of mtNOS activity by complex I activity. In rat brain mitochondria, rotenone and pyridaben markedly decreased mtNOS functional activity with NAD-dependent substrates but not when the substrate was succinate. This suggests than the activity of the mtNOS inserted in the inner mitochondrial membrane depends on complex I activity. This regulation and the role of mitochondrial NO diffusion as a signal for mitochondrial biogenesis could have a role in the etiopathology of Parkinson´s disease.
Physical exercise is known to induce oxidative stress leading to the generation of free radicals. The objective of present study was understand the combined effects of intense physical exercise and the possible antioxidant effect of diphenyl diselenide (PhSe)2, pretreatment in prevent this oxidative damages in lung of mice. Male Swiss mice (25-35 g) were divided into ten groups. The groups that received (PhSe)2 for seven day per oral route 5 mg/kg/day was (PhSe)2 = (PhSe)2, (PhSe)2 + exercise killed immediately after = (Ex. Im. + (PhSe)2), (PhSe)2 + exercise killed after 1 hour = (Ex. 1h + (PhSe)2), (PhSe)2 killed after 24 hours = ((PhSe)2 24h), (PhSe)2 + exercise killed after 24 hour = (Ex. 24h + (PhSe)2). The other groups were control group killed immediately = (Control), exercise killed immediately after = (Ex. Im), exercise killed after 1 hour = (Ex. 1h), canola oil killed after 24 hours = (Control 24h), exercise killed after 24 hours = (Ex. 24h). The physical activity was forced swim with 20 minutes of duration. The lung of all animals was removed and thiobarbituric acid reactive species (TBARS), catalase activity, non-protein thiols (NPSH) and ascorbic acid was mensured in this tissue. TBARS levels increased on groups (Ex. Im) and (Ex. 1h) and the (PhSe)2 was significantly efficient in restore the lipidic peroxidation to the (Control) levels. Did not have significant difference in 24h between the groups. In fact catalase activity was increased in groups (Ex. 1h), (Ex. 1h + (PhSe)2)and(Ex. 24h + (PhSe)2), however, in the groups that received (PhSe)2 the activity of the enzyme was significantly lower that groups (Ex. 1h) and (Ex. 24h). NPSH levels were increased in the groups that received (PhSe)2 in all the times and the intense physical exercise group did not change the NPSH levels. Ascorbic acid levels were reduced in groups (Ex. Im) and (Ex. Im. + (PhSe)2). (PhSe)2 treatment partially retored the ascorbic acid levels. In conclusion datas of this study indicate that the pretreatment with (PhSe)2 minimizes oxidative stress caused for the practical of intense physical exercise.
| Poster Presentation | 07.Peroxynitrite Chemistry and Biochemistry
Inactivaction of cystathionine β-Synthase by peroxynitrite
Celano, L. 1(*); Denicola, A 2; Alvarez, B. 3
1 - Laboratorio de Enzimología, Facultad de Ciencias; Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay | (*) Uruguay
Cystathionine β-synthase (CBS) is a key enzyme of homocysteine metabolism. It catalyzes the condensation of homocysteine and serine to form cystathionine in the transsulfuration pathway that yields cysteine. Increases in plasma homocysteine constitute an independent risk factor for cardiovascular disease. Human CBS is a pyridoxal 5´-phosphate (PLP) dependent enzyme that contains an heme-thiolate of unknown function. In this study, human dimeric CBS was purified from a recombinant expression system. Exposure of Fe(III)CBS (5 µM) to peroxynitrite led to inactivation with an IC50 of ~100 µM at pH 7.4. Inactivation was higher at alkaline pH. The rate of peroxynitrite decay increased in the presence of Fe(III)CBS according to a second order rate constant of 2.4 x 104 M-1 s-1 at pH 7.4 and 37 ºC. Although free PLP reacted with peroxynitrite, no changes in the PLP cofactor of CBS were observed, while tryptophan fluorescence decreased. Peroxynitrite exposure resulted in tyrosine nitration that was inhibited by 25 mM bicarbonate. The absorbance peak of Fe(III)CBS at 429 nm decreased in intensity and blue-shifted without dectectable formation of intermediates. In addition, the ligand to metal charge transfer band at 650 nm was lost. These changes suggest that peroxynitrite leads to loss of heme-thiolate coordination, which could be related to inactivation.
Palabras clave: CBS, peroxynitrite, heme thiolate
17:00 a 17:40 - Café y alfajores
17:40 a 19:45 - Colloquium 07
Peroxynitrite Chemistry and Biochemistry
Chairpersons: Dr. Gerardo Ferrer Sueta - FQB Facultad de Ciencias (Uruguay)
Phagocytes are remarkable engines of oxidative stress that, depending upon their metabolic capabilities, generate diverse types of oxidative toxins. Research in our laboratory has focused upon two cell types, namely the neutrophil and RAW 264.7 macrophage-like transformed murine cells, which represent extreme opposites with respect to the identities of chemical oxidants produced. For neutrophils, the prevailing viewpoint is that respiratory stimulation following agonist binding leads to intraphagosomal generation of lethal amounts of hypochlorous acid (HOCl), whereas, for RAW cells, production of chlorinating agents is negligible, but reactive nitrogen species are thought to play a major role in microbicidal function. Our recent research with neutrophils has been directed at resolving a vexing controversy concerning whether the physiological role of myeloperoxidase (MPO) in neutrophils is really that of a chlorine peroxidase; if not, then the long-standing notion that HOCl is an important neutrophil-generated microbicide would have to be abandoned. We have addressed this issue by developing GFP-IgG antibody conjugates for which GFP fluorescence quenching is HOCl-specific. This conjugate bound strongly to the bacterial cell envelope of Staphylococcus aureus. Subsequent exposure to neutrophils led to MPO-dependent fluorescence quenching of phagocytosed cells on the same timescale as phagocytosis and respiratory stimulation. We have additionally shown that DNA isolated from Escherichia coli exposed to neutrophils contained elevated levels of 5-chlorouracil, another HOCl-specific marker. Thus, we have demonstrated that copious amounts of HOCl are generated in the neutrophil phagosome under physiological conditions, i.e., when bacteria are phagocytosed. Our recent studies with RAW cells have involved determination of the onset, localization and extent of formation of superoxide ion, hydrogen peroxide and nitrite ion, as well as respiratory rates and specific enzyme activities, following activation. The observed pattern of reactivity has led us to propose that cyclooxygenase (COX-2) functions to generate bactericidal levels of NO2• from accumulated nitrite ion and hydrogen peroxide; the microbicidal capabilites of COX-2 are currently under investigation.
Decomposition of peroxynitrite to nitrite and dioxygen at neutral pH may involve formation of an adduct of ONOOH and ONOO-, followed by disproportionation to NO2- and O2NOO-. In order to test this hypothesis, we initiated the decomposition by mixing alkaline 0.5 - 2.5 mM peroxynitrite with buffers (final pH near 7), followed by excess acetate buffer (final pH near 4.5) after 0.1 - 3.2 s. At pH 4.5, the decomposition is quenched and any remaining peroxynitrite is converted to nitrate. Solutions were examined for oxidative activity and UV/VIS absorption after the pH was returned to neutrality. During decomposition of 0.1 -2.5 mM tetramethylammonium peroxynitrite solutions 1ΔgO2 was observed between 1180-1350 nm and by trapping, with a yield of ca 1% relative to peroxynitrite. When the decomposition of peroxynitrite in amine-type buffers (TRIS, HEPES) is stopped by acidification, then the resulting solution should not be oxidizing. However, for 1-2 minutes after quenching, the solutions oxidized iodide rapidly to iodine, but solutions with equivalent concentrations of nitrite did not. The oxidant has a half-life of about 1 s at neutral pH, but lives much longer at lower pH. This observation suggests that the oxidizing compound is peroxynitrate, which is supported by the detection of 1O2. The yields of oxidant, or peroxynitrate, and 1O2 were distinctly lower in phosphate than in amine buffers. However, the ability to oxidize iodide lasted longer in phosphate, and was detectable even after 10 minutes. As peroxyphosphate could be this oxidant, we precipitated the phosphate after quenching with an excess of aluminium sulfate, isolated and washed the precipitate with water and dissolved it at pH 3. The resulting solution oxidized iodide. The addition of a bolus of peroxynitrite to a solution buffered by phosphate may result in, aside from oxidation and nitration by peroxynitrite, oxidations by 1O2, peroxynitrate and peroxyphosphate. Supported by: FAPESP, CNPq/Instituto do Milênio: Redoxoma and ETH Zürich.
Palabras clave: peroxynitrite, singlet oxygen, peroxynitrate
The title reactions have been investigated for NONOates of the general structure X[N(O)NO]-, where X is R2N (amino NONOates; R = C2H5, H2NC2H4, or H2NC3H6) or O- (Angeli´s anion, N2O32-). Although these compounds are extensively used in biochemical, physiological, and pharmacological studies due to their ability to slowly release NO and/or its congeneric nitroxyl (HNO), the mechanisms of these processes remain obscure, even controversial. In variances with the previous reports, it is found that all NONOates studied exhibit a single acid-base equilibration in the pH range 10-2 and the attendant pKa values are substantially revised. In all cases the protonation occurs on the [N(O)NO]- group and only the monoprotonated forms undergo decomposition with rates that are exceptionally sensitive to the nature of X group; the deprotonated forms are stable. Kinetic and product analyses suggest that none of the NONOates is a "clean" donor of NO or HNO, instead these species are released concurrently, albeit in different proportions for the amino NONOates and Angeli´s anion. The NO/HNO ratios are also pH-dependent. At lower pH, the decompositions are subject to additional acid catalysis and, in the case of Angeli´s anion, to catalysis by nitrous acid. The mechanisms of these reactions have been unraveled.
Similarly, photolysis of NONOates generates a unique spectrum of primary products including the NO- and HNO nitroxyl species, and NO. The photoinduced decomposition of NONOates, especially if it can be photosensitized, holds the potential for highly-controlled photoactivated delivery of NO and/or HNO to particular physiological targets. In neutral solutions, the distributions of the NO-, HNO, and NO photolysis products show that NONOates are not highly selective photochemical generator of NO-/HNO or NO, although the selectivity of amino NONOates for NO release is considerably greater than Angeli´s anion selectivity for producing HNO.
These findings are significant because NO and HNO have been reported to produce distinctly different physiological effects. In addition, production of HNO from NONOates opens up previously unrecognized reaction pathways. Specifically, NO addition to HNO generates a highly oxidizing N2O2·- radical and O2 addition to HNO can generate peroxynitrite.
N-t-BOC L-tyrosine tert-butyl ester (BTBE) is a hydrophobic tyrosine analog which was incorporated into phosphatidyl choline (PC) liposomes to study tyrosine nitration mechanisms in hydrophobic environments. We have previously shown (Bartesaghi et al. Biochemistry 2006, 45, 6813-6825) that tyrosine nitration by peroxynitrite in saturated phospholipids-containing liposomes (1,2-dilauroyl- and 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DLPC and DMPC) is a free radical-dependent process. Herein, we report that at pH 7.4, 3-NO2-BTBE yields (respect to added peroxynitrite) were similar in DLPC and DMPC liposomes (3 %); interestingly, nitration yields were higher in egg PC (4 %) and even considerable in soybean PC (0.96 %) in spite of their substantial content of polyunsaturated fatty acids (PUFA) (~ 24% and 57 %, respectively) which out-competed BTBE in the reaction with .OH and initiated lipid peroxidation. Further experiments performed in liposomes with variable content of DLPC/PLPC (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine) resulting on different degree of unsaturation, revealed significant nitration yields throughout the range of 0-100 % of PLPC. The data suggest that BTBE nitration in PUFA-containing liposomes must require the participation of lipid-derived radicals for the one-electron oxidation of BTBE (Eo´~ +0.88 V), the essential first-step in the radical mechanism of tyrosine nitration. Thermodynamically, lipid alkoxyl (Eo´ = +1.76 V) and peroxyl radicals (Eo´=+1.02 V) are able to perform the postulated reaction. Further experimental support was provided by 1) a profound inhibition of BTBE nitration under low oxygen tensions, concomitantly with a drastic drop on lipid oxidation yields, 2) generation of higher yields of 3,3´-di-BTBE from peroxynitrite in unsaturated PC liposomes and 3) the formation of 3,3´-di-BTBE during hemin-induced peroxidation in unsaturated PC liposomes. Once formed, BTBE phenoxyl radical would react at diffusion-controlled rates with .NO2 to yield 3-NO2-BTBE.The relationship between tyrosine nitration in hydrophobic environments and lipid radical-dependent processes was further substantiated by computer-assisted kinetic simulations which recapitulated the experimental data.
Glycosaminoglycans, large linear polysaccharides consisting of repeating disaccharide units, are important components of the extracellular matrix. Damage to the extracellular matrix – which plays a key role in providing mechanical strength and elasticity to tissues (e.g. the artery wall) and modulates cellular behaviour – is believed to be a factor in the development of a number of pathologies, including atherosclerosis. Hyaluronan and other glycosaminoglycans have previously been shown to be susceptible to fragmentation. The processes that result in fragmentation in vivo are incompletely understood, though evidence has been presented for a role for both oxidants (radical or molecular) and enzymatic reactions. Peroxynitrite/peroxynitrous acid (ONOO-/ONOOH), formed in vivo by the reaction of superoxide and nitric oxide radicals, has been implicated in the fragmentation of glycosaminoglycans. We have previously shown that ONOO-/ONOOH induces the fragmentation of isolated glycosaminoglycans via a •OH-like mechanism, resulting in the formation of specific polymer fragments in a concentration-dependent manner (Free Radic. Biol. Med. (2007) 42, 1278-1289). Here we extend this work to investigate the contributions of •OH, CO3-•, NO3•, NO2•, products of the further reactions and decomposition of ONOO-/ONOOH, to glycosaminoglycan fragmentation. The small molecule radicals of interest were generated in a specific manner by 60Co-irradiation of aqueous solutions containing HCO3-, NO3-, or NO2-, and hyaluronan or chondroitin sulfate. Each radical, •OH, CO3-•, NO3•, NO2•, caused the formation of specific disaccharide fragments of the glycosaminoglycans in a dose-dependent manner, though with markedly different efficiencies. At each of the radiation doses examined (in the range 100 – 1000 Gray), the efficacy of polymer fragmentation was •OH > CO3-• >NO3• > NO2•. These results are consistent with •OH being the predominant intermediate involved in ONOO-/ONOOH-mediated damage to glycosaminoglycans at sites of inflammation.