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P7. Redox Regulation of Cell Proliferation and Apoptosis
P7. Redox Regulation of Cell Proliferation and Apoptosis P7-80 ZINC DEFICIENCY FACILITATES LEAD-INDUCED OXIDATIVE STRESS L. Aimo,1 G. G. Mackenzie,1 and P. I. Oteiza1 1
Departments of Nutrition and Environmental Toxicology, University of California, Davis, CA, USA, and Department of Biological Chemistry, IQUIFIB (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
Both zinc (Zn) deficiency and lead (Pb) intoxication induces oxidative damage to cell components and the triggering of oxidant-sensitive transcription factors. We investigated if Zn deficiency can increase the susceptibility of human neuroblastoma IMR-32 cells to Pbinduced oxidative stress. Cell oxidants concentration, determined with 5(and 6)-carboxy-2V7V-dichlorodihydrofluorescein diacetate (DCDCDHF), and the triggering of stress-activated MAPKs, p38 and JNK, were measured. Cells were cultured for 24 h in control (C) or chelated media containing 1.5 or 15 microM Zn (1.5 or 15Zn) without or with Pb acetate (5-100 microM). Pb exposure caused a dose-dependent decrease in cell viability in all groups, which was significantly lower in Zn deficient (1.5Zn) cells. Oxidants concentration increased with increasing Pb in all groups. At 50 microM Pb, significantly higher levels of oxidants were found in the 1.5Zn (3-fold increase) compared to C and 15Zn cells (2- and 1.8-fold increase, respectively). Pb caused a select phosphorylation of p-38 and JNK in the Zn deficient cells (32% and 20% increase, respectively). Results indicate that Zn deficiency could increase the susceptibility of neuronal cells to Pb toxicity, partially through an increased production of oxidants and the triggering of oxidantresponsive cell signals. Supported by grants from UBA (B054), CONICET (PIP 02120) and from the University of California, Davis.
Here, we report that the 50 kDa protein is a fragment of 110 kDa protein (p110) usually present in nuclei, and this protein functions on macrophage surface as a receptor for oxidized and apoptotic cells. Antibody against p110 (anti p110) bound on the surfaces of THP-1 cells and THP-1 macrophages, suggesting the presence of p110 on the cells. The antibody inhibited the binding of H2O2-oxidized and apoptotic Jurkat cells to THP-1 macrophages, suggesting involvement of the surface p110 in the binding. Transfection of p110 cDNA expression vector into HEK293 cells resulted in expression of p110 on cell surface, and the cells showed an ability to bind H2O2-oxidized and apoptotic Jurkat cells. The binding was inhibited by anti p110. We conclude that p110 on macrophages is a receptor for the carbohydrate-mediated recognition of oxidized and apoptotic cells.
P7-82 APOPTOSIS INDUCED BY HYPEROSMOTIC STRESS V. Eisner,1 C. Quiroga,1 A. Criollo,1 C. Olea,2 G. Dı´az-Araya,1 M. Chiong,1 and S. Lavandero1 1 FONDAP Center for Molecular Studies of the Cell, 2Department of Inorganic and Analytical Chemistry, Faculty Chemical and Pharmaceutical Sciences, University of Chile, Santiago, Chile
We have previously shown that hyperosmotic stress by sorbitol triggers cardiomyocyte apoptosis. However, in this cell death process, deleterious and protective signaling pathways activated by hyperosmotic stress remain unexplored. Our results showed that free radical production, evaluated by DCF fluorescence, was stimulated in cultured neonatal rat cardiomyocytes exposed to sorbitol (Sor, 600 mOsm), effect that was prevented by N-acetylcysteine (NAC, 5 mM). The OH radical generation was demonstrated by Electron Spin Resonance (ESR) using DMPO as spin trap. NAC, SN50 (a specific NFnB blocker) and AdInBa (adenovirus that overexpress an InBa mutated form) inhibited both the expression of a NFnB-LUX reporter gene and the nuclear accumulation of p65NFnB isoform induced by hyperosmotic stress. Sor (600 mOsm) also increased DNA fragmentation (assessed by agarose gel electrophoresis) and the number of anexin V positive cardiomyocytes (evaluated by flow cytometry); these effects were attenuated by NAC and increased by SN5O and AdInB. Lastly, cell viability was decreased by Sor and potentiated by NFnB inhibitors. We conclude that hyperosmotic stress stimulates free radical generation in cultured cardiomyocytes and also activates the protective NFnB pathway to attenuate apoptosis. FONDECYT 1010246, FONDAP 15010006, Chilean Society for Cardiology Grant, PG106 UCH. VE, AC and CQ are CONICYT fellows.
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P7-81 A MACROPHAGE RECEPTOR FOR THE CARBOHYDRATE-MEDIATED RECOGNITION OF OXIDIZED AND APOPTOTIC CELLS M. Beppu,1 K. Hirano,1 Y. Miki,1 Y. Hirai,1 R. Sato,1 A. Hayashi,1 and T. Itoh1 1 Tokyo University of Pharmacy and Life Science, Japan
We previously demonstrated that oxidatively stressed cells are susceptible to phagocytic recognition by macrophages and the recognition is mediated by sialylated-polylactosaminyl carbohydrate chains on oxidized cells. A lectin-like receptor for the recognition has been postulated to be present on macrophages, and a candidate protein of 50 kDa was isolated from the membrane of human monocytic THP-1 cells differentiated into macrophages (THP-1 macrophages).
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P7-83 HYDROGEN PEROXIDE REGULATES CELL CYCLE PROGRESSION IN TUMORAL CELLS S. Galli,1 M. C. Carreras,1 and J. J. Poderoso1 1 Laboratory of Oxygen Metabolism, University Hospital, University of Buenos Aires, Buenos Aires, Argentina
Mammalian cells express mitogen activated protein kinases (MAPKs) that integrate extracellular signals and transduction pathways, ending in specific responses (proliferation, cell cycle arrest, apoptosis). Mitochondria generates reactive oxygen species, implicated in physiological cell signaling. Hydrogen peroxide (H2O2) promotes tyrosine-phosphorylation of proteins, included ERK1/2 and p38. Steady-state H2O2 concentration ([H2O2]ss) depends on rates of mitochondrial production and degradation by cytosolic enzymes; proliferating tissues (fetal liver and brain) and tumors exhibit diminished [H2O2]ss, while quiescent tissues (adult liver) display two-fold higher [H2O2]ss. In this work we studied H2O2 modulation on cell cycle progression of tumoral cell line LP07. In these cells, 1 mM H2O2 promotes proliferation and higher doses (>10 mM) cell cycle arrest or apoptosis, with differential MAPKs phosphorylation. At 1 mM H2O2, activation of ERK1/2 is persistent while 50 mM H 2O 2 only induces transient ERK1/2 activation, with high and low of cyclin D1 expression, respectively. Inhibition of MEK1/2 limited cyclin D1 expression by 1 mM H2O2, indicating that the proliferative signal is driven by ERK1/2. Additionally, redox effects associated to ERK1/2 specific mitochondrial translocation and activation. These results suggest that persistent tumoral proliferation depends on differential activation and cellular traffic of MAPKs in connection with low [H2O2]ss in transformed cells.
P7-84 MECHANISM OF APOPTOSIS IN THE MAMMARY GLAND OF WEANED RATS; IMPLICATION OF XANTHINE OXIDASE A. Diana Rus,1 M. D. Royo,1 M. C. Gome´z-Cabrera,1 R. Zaragoza,1 C. Garcı´a,1 J. Sastre,1 J. Vin˜a,1 J. R. Vin˜a,1 and F. V. Pallardo´1 1 Departments of Physiology and Biochemistry, Faculty of Medicine, University of Valencia, Spain
We previously demonstrated the role of mitochondrial oxidative stress in the induction of apoptosis and the importance of decreased GSH in the involution of the mammary gland during weaning. Here, we show the importance of xanthine oxidase (XO) activity in apoptosis using lactating mammary gland of Wistar rats at 6, 12, 24, 48 hour after weaning. XO is one of the major sources of reactive oxygen and nitrogen species (ROS, RNS). During apoptosis in the mammary gland XO activity rose and the GSSG/GSH in blood and mammary gland increased. The maximum of the protein nitration and percentage of apoptosis were found
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when XO activity reached a pick. Using Allopurinol, specific inhibitor of XO activity, we obtained a reduction of the apoptotic process, evidenced by TUNEL and DNA fragmentation. JNK and p38, stress activated protein kinases involved in apoptosis, were phosphorylated during weaning. The expression of p53, a proapoptotic gene, was induced too. In conclusion, high XO activity may be one of the triggers in the apoptosis process by increasing ROS production and controlling the p53 expression. XO maintains apoptosis by inducing RNS production and controlling p38 phosphorylation.
P7-85 CYTOTOXIC EFFECT AND CELL DEATH INDUCED BY NITROIMIDAZOLE IN HUMAN MONONUCLEAR CELLS M. M. Lo´pez Nigro,1 M. A. Carballo,1 and J. Bustamante2 1 CIGETOX-Human Citogenetic and Genetic Toxicology, Department of Clinic Biochemistry, 2Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
5-Nitroimidazoles are a well-established group of antiprotozoal and antibacterial agents. These chemotherapeutic agents inhibit the growth of both anaerobic bacteria and certain anaerobic protozoa, such as Trichomonas vaginalis, Entamoeba histolytica and Giardia lamblia. The aim of this study was to characterize their cytotoxic and genotoxic activity and to evaluate the cell death mechanism associated. Peripheral blood lymphocyte cultures were treated with Metronidazole (MTZ) and Tinidazole (TNZ) at concentrations of 0.1, 1, 10 and 50 Ag/ml. A significant decrease in Mitotic Index (p < 0.0001) as well as an increase in Chromosomal Aberrations (p < 0.0001) frequencies for the two drugs were observed. No modifications in Replication Index were found. The evaluation of the cell death by hipodiploid DNA level showed 17% and 18% at 10 Ag/ml, and 30% and 34.5% at 50 Ag/ml for MTZ and TNZ respectively. Acridine orange/ethidium bromide staining showed typical characteristic nuclei shrinkage at the higher MTZ and TNZ doses; cell necrosis was also evaluated showing 32% and 23% for 50 Ag/ml after 4 hours of culture with MTZ and TNZ respectively. Flow cytometry of DCFH-DA loaded cells showed a 10 % fluorescence increase after 30 min of MTZ and TNZ exposure. The results suggest that MTZ and TNZ are able to induce a genotoxic and cytotoxic effect related with apoptotic and necrotic process, associated with an increased level of oxidant status at early stages.
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P7-86 A DECREASE IN NEURONAL ZINC TRIGGERS THE OXIDANT-MEDIATED ACTIVATION OF NF-KB AND AP-1 IN NEUROBLASTOMA CELLS G. G. Mackenzie,1,2,3 M. P. Zago,1 A. G. Erlejman,1 L. Aimo,2,3 C. L. Keen,2,4 and P. I. Oteiza1,2,3 1 Department of Biological Chemistry, IQUIFIB (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina, 2Departments of Nutrition, 3Environmental Toxicology and 4Internal Medicine, University of California, Davis, CA, USA
The role of oxidants on NF-nB- and AP-1-activation associated with a decrease in intracellular zinc levels was investigated. IMR-32 cells were incubated in control media or chelated media containing 1.5, 5 or 15 AM zinc, without or with 0.5 mM a-lipoic acid (LA) or 1 mM N-acetyl-L-cysteine (NAC) for 24 h. H2O2 and total oxidant cell concentrations were higher and total glutathione concentrations were lower in the low zinc groups (1.5 and 5 Zn) compared to control and 15 Zn groups. LA or NAC prevented the increase in H2O2 and total oxidants levels, and restored glutathione concentration in the low zinc cells. Zinc deficiency induced the activation of early steps of NF-nB (InB phosphorylation) and AP-1 (JNK and p38 phophorylation) cascades and a high NF-nB- and AP-1-DNA binding activity in total cell extracts. LA and NAC prevented InB, JNK and p38 phosphorylation, and inhibited NF-nB- and AP-1-DNA binding activity in the low zinc cells. Results demonstrate that alterations in the cell concentrations of oxidants and/or thiols, associated with a decrease in intracellular zinc, trigger NF-nB- and AP-1-activation in neuronal cells. Supported by grants from UBA (B054), CONICET (PIP 02120) and NIH (HD 01743)
P7-87 EVIDENCE FOR OXIDATIVE DAMAGE DURING HUMAN SERUM INDUCED APOPTOSIS IN THE UNICELLULAR PARASITE Trypanosoma cruzi L. Piacenza,1 F. Irigoı´n,1 G. Peluffo,1 and R. Radi1 1 Departamento de Bioquı´mica, Facultad De Medicina, Uruguay
Trypanosoma cruzi is the etiological agent of Chagas disease that afflicts 18 million people in South and Central America. We have shown that fresh human serum (FHS) induces apoptosis in T. cruzi in a Larginine inhibitable manner. Mitochondrial function was evaluated by determination of membrane potential using JC-1 and oxygen consumption. FHS induced a initial (+40%, <1 hr.) dose-dependent hyperpolarization response with a final depolarization ( 25%, 18 hs). Oxidants production increased in the presence of the death stimuli with concomitant oxidation of the major antioxidant non-protein thiol trypanothione [T(SH)2] and inhibition of trypanothione reductase. Levels of T(SH2) oxidation similar to that observed in the presence of FHS, were
achieved using 2,3-Dimethoxy-1-naphtoquinone (DMNQ) as an intracellular superoxide source (30 AM, =0.7 AM superoxide/min/parasite). In addition, protein oxidative damage was detected as 5,5V-dimethyl-pyrolline-N-oxide/protein adducts. Apoptosis was inhibited by L-arginine-derived nitric oxide and nitric oxide donors, which counteract the effects induced by FHS. Hyperpolarization, inhibition of cell respiration and oxygen radical formation were prevented by L-arginine in a NOS-dependent process. We hypothesize that mitochondria is a target organelle involved in FHS-induced apoptosis with the generation of reactive oxygen species and that nitric oxide plays a modulatory role on mitochondrial functions in T. cruzi.
P7-88 ROLE OF OXIDATIVE STRESS IN THE OVULATION E. F. Sato,1 E. Nakagawa-Okamoto,1 A. Nishida,1 and M. Inoue1 1 Department of Biochemistry and Molecular Pathology, Osaka City University Medical School, Osaka, Japan
Ovulation occurs using an inflammation-like reaction in the ovary. We previously showed that oxidative stress underlies the ovulatory process by some SODinhibitable mechanism. We also found that L-carnitine effectively inhibits oxidative injury of mitochondria thereby suppressing apoptosis of various cell types. The present work shows that L-carnitine significantly promotes the ovulation in the rats. Experiments were carried out using 22-day-old Wistar female rats. Lcarnitine was subcutaneously administrated to ras at indicated time. Animals were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin (PMSG). After 48 h, ovulation was triggered by injecting 10 IU of human chronic gonadotropin (hCG). At 16 h after hCG injection, the number of ova in the ampullary site of both tubers was counted. At the indicated times, plasma 17beta-estradiol and ovarian levels of L-carnitine, organic cation transporters (OCTNs) and apoptotic cells were analyzed. After treating animals with PMSG, ovarian levels of OCTN2 markedly increased. Ovarian levels of carnitine significantly increased after PMSG treatment. The number of ova and plasma level of 17beta-estradiol significantly increased in L-carnitine-treated animals. Carnitine suppressed the apoptosis of granulosa cells in the ovarium. These results indicate that mitochondriadependent and L-carnitine-inhibitable apoptosis of granulosa cells underlies the mechanism of PMSG/ hCG-induced ovulation.
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P7-89 NEUROPROTECTIVE EFFECTS OF ;-TOCOTRIENOL AND A-TOCOPHEROL AGAINST H2O2-INDUCED OXIDATIVE STRESS IN ASTROCYTES CULTURE S. M. Then,1 M. Musalmah,1 M. T. Gapor,2 and W. Z. Wan Ngah1 1
Department of Biochemistry, Medical Faculty, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur and 2Department of Chemistry, Malaysian Palm Oil Board, 43500 Bangi, Selangor, Malaysia
There is accumulative evidence that oxidative stress is one of the factors that lead to decreased neuronal cell viability and this could be inhibited by antioxidant agents. a-Tocopherol (ATF) has been widely studied for its antioxidant properties as well as neuroprotective effects. But recent studies have shown that tocotrienols also have similar reactivity towards radicals and is more readily absorbed into the membranes than tocopherols. We compared the effects of GTT and ATF on oxidative stress-induced cytotoxicity using primary culture of astrocytes. Results showed that both ATF and GTT have different range of cytotoxicity. ATF is toxic at concentrations greater than 750 mM whereas GTT is toxic from 100mM. ATF and GTT protected cells against cytotoxicity of hydrogen peroxide (H2O2) at different concentrations: 50 mM-75 0mM and at 40 mM-60 mM respectively. Therefore, GTT is more effective in protecting cells against superoxide radicals (O2 ) at lower concentration compare to ATF. This might be due to the fact that GTT is more readily transferred between membranes and incorporated into membranes. In conclusion, these results show that both ATF and GTT protects astrocytes against H2O2 induced-oxidative stress, albeit at different concentrations, presumably reflecting the different absorption rates and antioxidant mechanisms of actions of the two vitamin E subfamilies.
P7-90 APOPTOSIS OF NEURAL PRECURSOR CELLS FOLLOWING GAMMA IRRADIATION IS MODULATED BY REACTIVE OXYGEN AND NITROGEN SPECIES (ROS/RNS) E. Robello,1 D. Dubner,1 S. Michelin,1 M. R. Perez,1 S. Puntarulo,2 and P. Gisone1 Autoridad Regulatoria Nuclear (ARN), Gerencia de Apoyo Cientı´fico, Laboratorio de Radiopatologı´a, Avenida del Libertador 8250, (C1429BNP) Buenos Aires, Argentina, 2Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
Enhanced induced generation of reactive oxygen (ROS) and nitrogen species (RNS) has been observed after exposures to low radiation doses. This study was aimed to address the participation of radiation-induced ROS,
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and endogenous NO generation in the radiation-induced apoptosis of neural cortical precursors. Cortical cells obtained from rats at 17 gestational day (gd) were irradiated with doses from 0.2 Gy to 2 Gy at a dose-rate of 0.3 Gy/m. A significant decrease of the luminol-dependent chemiluminescence was observed in cells after 30 min of irradiation, and basal levels were reached after 120 min. Chemiluminescence in irradiated cells significantly decreased by incubation with superoxide dismutase. Cellular NO content, estimated as NO2 + NO3 released to the culture medium, showed a significant increase in irradiated cells as compared to control values, up to 60 min post-irradiation. Apoptosis was evaluated by flow cytometry, morphology and DNA fragmentation after 24 h of irradiation. A significant increase was detected with 0.4 Gy doses. Pre-treatment of the cells with 10 mM N-acetylcyteine (NAC), a precursor for GSH sunthesis, lead to a significant decrease in cellular apoptosis, and inhibition of nitric oxide synthase (NOS) by L-NAME significantly increased apoptosis in irradiated cells. We conclude that a complex cross-talk between ROS and RNS play a pivotal role in the early signaling pathway leading to radiation-induced cell death.
P7-91 GLYCOXIDATIVE STRESS INDUCES NEURONAL APOPTOSIS TROUGH PKC-DELTA ACTIVATION M. Nitti,1 D. Verzola,1 S. Rossi,1 P. Odetti,1 D. Cottalasso,1 M. A. Pronzato,1 U. M. Marinari,1 and C. Domenicotti1 1
DiMeS University of Genoa, Italy
Accumulation of AGEs (Advanced Glycation End products), found in almost all body tissues including brain during ageing, leads to cell functional injury inducing alterations of intracellular redox balance. In this work, post-mitotic neuron-like cells were incubated for 48 h with increasing amounts of glycated foetal serum (G-FBS) containing 750 – 1500 pmol/ml of pentosidine, a well known glycoxidative marker. This treatment was able to induce an increase in apoptosis linear with AGE concentration up to necrotic death with highest AGE amount (3000pmol/pentosidine). Analysing the effect of 10% GFBS exposure (corresponding to 40% of apoptotic cell number), we found an early generation of intracellular reactive oxygen species (ROS): +26 – 28 % at 1.5 – 3 h incubation respectively and +40% at 6 h. In these conditions, the PKC-delta activity was increased approximately two fold and DNA binding activity of redox transcription factor AP-1, strictly linked to this isoform, was enhanced 2.5 fold. A relationship between oxidative stress, PKC delta activity, AP-1 activation and apoptosis, was demonstrated pre-treating neurons with 500 AM Vitamin E, a well-known antioxidant, with 20 Ag/ml of Gingko Biloba extract, and with 3 AM Rottlerin, inhibitor of PKC delta, that were able to completely prevent all effects dependent on glycoxidative stress. (Grants from COFIN 2002 Prof. Marinari and FIRB 2001 Prof. Poli).
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Departments of Nutrition and Environmental Toxicology, University of California, Davis, CA, USA, and Department of Biological Chemistry, IQUIFIB (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
Both zinc (Zn) deficiency and lead (Pb) intoxication induces oxidative damage to cell components and the triggering of oxidant-sensitive transcription factors. We investigated if Zn deficiency can increase the susceptibility of human neuroblastoma IMR-32 cells to Pbinduced oxidative stress. Cell oxidants concentration, determined with 5(and 6)-carboxy-2V7V-dichlorodihydrofluorescein diacetate (DCDCDHF), and the triggering of stress-activated MAPKs, p38 and JNK, were measured. Cells were cultured for 24 h in control (C) or chelated media containing 1.5 or 15 microM Zn (1.5 or 15Zn) without or with Pb acetate (5-100 microM). Pb exposure caused a dose-dependent decrease in cell viability in all groups, which was significantly lower in Zn deficient (1.5Zn) cells. Oxidants concentration increased with increasing Pb in all groups. At 50 microM Pb, significantly higher levels of oxidants were found in the 1.5Zn (3-fold increase) compared to C and 15Zn cells (2- and 1.8-fold increase, respectively). Pb caused a select phosphorylation of p-38 and JNK in the Zn deficient cells (32% and 20% increase, respectively). Results indicate that Zn deficiency could increase the susceptibility of neuronal cells to Pb toxicity, partially through an increased production of oxidants and the triggering of oxidantresponsive cell signals. Supported by grants from UBA (B054), CONICET (PIP 02120) and from the University of California, Davis.
Here, we report that the 50 kDa protein is a fragment of 110 kDa protein (p110) usually present in nuclei, and this protein functions on macrophage surface as a receptor for oxidized and apoptotic cells. Antibody against p110 (anti p110) bound on the surfaces of THP-1 cells and THP-1 macrophages, suggesting the presence of p110 on the cells. The antibody inhibited the binding of H2O2-oxidized and apoptotic Jurkat cells to THP-1 macrophages, suggesting involvement of the surface p110 in the binding. Transfection of p110 cDNA expression vector into HEK293 cells resulted in expression of p110 on cell surface, and the cells showed an ability to bind H2O2-oxidized and apoptotic Jurkat cells. The binding was inhibited by anti p110. We conclude that p110 on macrophages is a receptor for the carbohydrate-mediated recognition of oxidized and apoptotic cells.
P7-82 APOPTOSIS INDUCED BY HYPEROSMOTIC STRESS V. Eisner,1 C. Quiroga,1 A. Criollo,1 C. Olea,2 G. Dı´az-Araya,1 M. Chiong,1 and S. Lavandero1 1 FONDAP Center for Molecular Studies of the Cell, 2Department of Inorganic and Analytical Chemistry, Faculty Chemical and Pharmaceutical Sciences, University of Chile, Santiago, Chile
We have previously shown that hyperosmotic stress by sorbitol triggers cardiomyocyte apoptosis. However, in this cell death process, deleterious and protective signaling pathways activated by hyperosmotic stress remain unexplored. Our results showed that free radical production, evaluated by DCF fluorescence, was stimulated in cultured neonatal rat cardiomyocytes exposed to sorbitol (Sor, 600 mOsm), effect that was prevented by N-acetylcysteine (NAC, 5 mM). The OH radical generation was demonstrated by Electron Spin Resonance (ESR) using DMPO as spin trap. NAC, SN50 (a specific NFnB blocker) and AdInBa (adenovirus that overexpress an InBa mutated form) inhibited both the expression of a NFnB-LUX reporter gene and the nuclear accumulation of p65NFnB isoform induced by hyperosmotic stress. Sor (600 mOsm) also increased DNA fragmentation (assessed by agarose gel electrophoresis) and the number of anexin V positive cardiomyocytes (evaluated by flow cytometry); these effects were attenuated by NAC and increased by SN5O and AdInB. Lastly, cell viability was decreased by Sor and potentiated by NFnB inhibitors. We conclude that hyperosmotic stress stimulates free radical generation in cultured cardiomyocytes and also activates the protective NFnB pathway to attenuate apoptosis. FONDECYT 1010246, FONDAP 15010006, Chilean Society for Cardiology Grant, PG106 UCH. VE, AC and CQ are CONICYT fellows.
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P7-81 A MACROPHAGE RECEPTOR FOR THE CARBOHYDRATE-MEDIATED RECOGNITION OF OXIDIZED AND APOPTOTIC CELLS M. Beppu,1 K. Hirano,1 Y. Miki,1 Y. Hirai,1 R. Sato,1 A. Hayashi,1 and T. Itoh1 1 Tokyo University of Pharmacy and Life Science, Japan
We previously demonstrated that oxidatively stressed cells are susceptible to phagocytic recognition by macrophages and the recognition is mediated by sialylated-polylactosaminyl carbohydrate chains on oxidized cells. A lectin-like receptor for the recognition has been postulated to be present on macrophages, and a candidate protein of 50 kDa was isolated from the membrane of human monocytic THP-1 cells differentiated into macrophages (THP-1 macrophages).
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P7-83 HYDROGEN PEROXIDE REGULATES CELL CYCLE PROGRESSION IN TUMORAL CELLS S. Galli,1 M. C. Carreras,1 and J. J. Poderoso1 1 Laboratory of Oxygen Metabolism, University Hospital, University of Buenos Aires, Buenos Aires, Argentina
Mammalian cells express mitogen activated protein kinases (MAPKs) that integrate extracellular signals and transduction pathways, ending in specific responses (proliferation, cell cycle arrest, apoptosis). Mitochondria generates reactive oxygen species, implicated in physiological cell signaling. Hydrogen peroxide (H2O2) promotes tyrosine-phosphorylation of proteins, included ERK1/2 and p38. Steady-state H2O2 concentration ([H2O2]ss) depends on rates of mitochondrial production and degradation by cytosolic enzymes; proliferating tissues (fetal liver and brain) and tumors exhibit diminished [H2O2]ss, while quiescent tissues (adult liver) display two-fold higher [H2O2]ss. In this work we studied H2O2 modulation on cell cycle progression of tumoral cell line LP07. In these cells, 1 mM H2O2 promotes proliferation and higher doses (>10 mM) cell cycle arrest or apoptosis, with differential MAPKs phosphorylation. At 1 mM H2O2, activation of ERK1/2 is persistent while 50 mM H 2O 2 only induces transient ERK1/2 activation, with high and low of cyclin D1 expression, respectively. Inhibition of MEK1/2 limited cyclin D1 expression by 1 mM H2O2, indicating that the proliferative signal is driven by ERK1/2. Additionally, redox effects associated to ERK1/2 specific mitochondrial translocation and activation. These results suggest that persistent tumoral proliferation depends on differential activation and cellular traffic of MAPKs in connection with low [H2O2]ss in transformed cells.
P7-84 MECHANISM OF APOPTOSIS IN THE MAMMARY GLAND OF WEANED RATS; IMPLICATION OF XANTHINE OXIDASE A. Diana Rus,1 M. D. Royo,1 M. C. Gome´z-Cabrera,1 R. Zaragoza,1 C. Garcı´a,1 J. Sastre,1 J. Vin˜a,1 J. R. Vin˜a,1 and F. V. Pallardo´1 1 Departments of Physiology and Biochemistry, Faculty of Medicine, University of Valencia, Spain
We previously demonstrated the role of mitochondrial oxidative stress in the induction of apoptosis and the importance of decreased GSH in the involution of the mammary gland during weaning. Here, we show the importance of xanthine oxidase (XO) activity in apoptosis using lactating mammary gland of Wistar rats at 6, 12, 24, 48 hour after weaning. XO is one of the major sources of reactive oxygen and nitrogen species (ROS, RNS). During apoptosis in the mammary gland XO activity rose and the GSSG/GSH in blood and mammary gland increased. The maximum of the protein nitration and percentage of apoptosis were found
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when XO activity reached a pick. Using Allopurinol, specific inhibitor of XO activity, we obtained a reduction of the apoptotic process, evidenced by TUNEL and DNA fragmentation. JNK and p38, stress activated protein kinases involved in apoptosis, were phosphorylated during weaning. The expression of p53, a proapoptotic gene, was induced too. In conclusion, high XO activity may be one of the triggers in the apoptosis process by increasing ROS production and controlling the p53 expression. XO maintains apoptosis by inducing RNS production and controlling p38 phosphorylation.
P7-85 CYTOTOXIC EFFECT AND CELL DEATH INDUCED BY NITROIMIDAZOLE IN HUMAN MONONUCLEAR CELLS M. M. Lo´pez Nigro,1 M. A. Carballo,1 and J. Bustamante2 1 CIGETOX-Human Citogenetic and Genetic Toxicology, Department of Clinic Biochemistry, 2Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
5-Nitroimidazoles are a well-established group of antiprotozoal and antibacterial agents. These chemotherapeutic agents inhibit the growth of both anaerobic bacteria and certain anaerobic protozoa, such as Trichomonas vaginalis, Entamoeba histolytica and Giardia lamblia. The aim of this study was to characterize their cytotoxic and genotoxic activity and to evaluate the cell death mechanism associated. Peripheral blood lymphocyte cultures were treated with Metronidazole (MTZ) and Tinidazole (TNZ) at concentrations of 0.1, 1, 10 and 50 Ag/ml. A significant decrease in Mitotic Index (p < 0.0001) as well as an increase in Chromosomal Aberrations (p < 0.0001) frequencies for the two drugs were observed. No modifications in Replication Index were found. The evaluation of the cell death by hipodiploid DNA level showed 17% and 18% at 10 Ag/ml, and 30% and 34.5% at 50 Ag/ml for MTZ and TNZ respectively. Acridine orange/ethidium bromide staining showed typical characteristic nuclei shrinkage at the higher MTZ and TNZ doses; cell necrosis was also evaluated showing 32% and 23% for 50 Ag/ml after 4 hours of culture with MTZ and TNZ respectively. Flow cytometry of DCFH-DA loaded cells showed a 10 % fluorescence increase after 30 min of MTZ and TNZ exposure. The results suggest that MTZ and TNZ are able to induce a genotoxic and cytotoxic effect related with apoptotic and necrotic process, associated with an increased level of oxidant status at early stages.
SFRR 2004
P7-86 A DECREASE IN NEURONAL ZINC TRIGGERS THE OXIDANT-MEDIATED ACTIVATION OF NF-KB AND AP-1 IN NEUROBLASTOMA CELLS G. G. Mackenzie,1,2,3 M. P. Zago,1 A. G. Erlejman,1 L. Aimo,2,3 C. L. Keen,2,4 and P. I. Oteiza1,2,3 1 Department of Biological Chemistry, IQUIFIB (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina, 2Departments of Nutrition, 3Environmental Toxicology and 4Internal Medicine, University of California, Davis, CA, USA
The role of oxidants on NF-nB- and AP-1-activation associated with a decrease in intracellular zinc levels was investigated. IMR-32 cells were incubated in control media or chelated media containing 1.5, 5 or 15 AM zinc, without or with 0.5 mM a-lipoic acid (LA) or 1 mM N-acetyl-L-cysteine (NAC) for 24 h. H2O2 and total oxidant cell concentrations were higher and total glutathione concentrations were lower in the low zinc groups (1.5 and 5 Zn) compared to control and 15 Zn groups. LA or NAC prevented the increase in H2O2 and total oxidants levels, and restored glutathione concentration in the low zinc cells. Zinc deficiency induced the activation of early steps of NF-nB (InB phosphorylation) and AP-1 (JNK and p38 phophorylation) cascades and a high NF-nB- and AP-1-DNA binding activity in total cell extracts. LA and NAC prevented InB, JNK and p38 phosphorylation, and inhibited NF-nB- and AP-1-DNA binding activity in the low zinc cells. Results demonstrate that alterations in the cell concentrations of oxidants and/or thiols, associated with a decrease in intracellular zinc, trigger NF-nB- and AP-1-activation in neuronal cells. Supported by grants from UBA (B054), CONICET (PIP 02120) and NIH (HD 01743)
P7-87 EVIDENCE FOR OXIDATIVE DAMAGE DURING HUMAN SERUM INDUCED APOPTOSIS IN THE UNICELLULAR PARASITE Trypanosoma cruzi L. Piacenza,1 F. Irigoı´n,1 G. Peluffo,1 and R. Radi1 1 Departamento de Bioquı´mica, Facultad De Medicina, Uruguay
Trypanosoma cruzi is the etiological agent of Chagas disease that afflicts 18 million people in South and Central America. We have shown that fresh human serum (FHS) induces apoptosis in T. cruzi in a Larginine inhibitable manner. Mitochondrial function was evaluated by determination of membrane potential using JC-1 and oxygen consumption. FHS induced a initial (+40%, <1 hr.) dose-dependent hyperpolarization response with a final depolarization ( 25%, 18 hs). Oxidants production increased in the presence of the death stimuli with concomitant oxidation of the major antioxidant non-protein thiol trypanothione [T(SH)2] and inhibition of trypanothione reductase. Levels of T(SH2) oxidation similar to that observed in the presence of FHS, were
achieved using 2,3-Dimethoxy-1-naphtoquinone (DMNQ) as an intracellular superoxide source (30 AM, =0.7 AM superoxide/min/parasite). In addition, protein oxidative damage was detected as 5,5V-dimethyl-pyrolline-N-oxide/protein adducts. Apoptosis was inhibited by L-arginine-derived nitric oxide and nitric oxide donors, which counteract the effects induced by FHS. Hyperpolarization, inhibition of cell respiration and oxygen radical formation were prevented by L-arginine in a NOS-dependent process. We hypothesize that mitochondria is a target organelle involved in FHS-induced apoptosis with the generation of reactive oxygen species and that nitric oxide plays a modulatory role on mitochondrial functions in T. cruzi.
P7-88 ROLE OF OXIDATIVE STRESS IN THE OVULATION E. F. Sato,1 E. Nakagawa-Okamoto,1 A. Nishida,1 and M. Inoue1 1 Department of Biochemistry and Molecular Pathology, Osaka City University Medical School, Osaka, Japan
Ovulation occurs using an inflammation-like reaction in the ovary. We previously showed that oxidative stress underlies the ovulatory process by some SODinhibitable mechanism. We also found that L-carnitine effectively inhibits oxidative injury of mitochondria thereby suppressing apoptosis of various cell types. The present work shows that L-carnitine significantly promotes the ovulation in the rats. Experiments were carried out using 22-day-old Wistar female rats. Lcarnitine was subcutaneously administrated to ras at indicated time. Animals were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin (PMSG). After 48 h, ovulation was triggered by injecting 10 IU of human chronic gonadotropin (hCG). At 16 h after hCG injection, the number of ova in the ampullary site of both tubers was counted. At the indicated times, plasma 17beta-estradiol and ovarian levels of L-carnitine, organic cation transporters (OCTNs) and apoptotic cells were analyzed. After treating animals with PMSG, ovarian levels of OCTN2 markedly increased. Ovarian levels of carnitine significantly increased after PMSG treatment. The number of ova and plasma level of 17beta-estradiol significantly increased in L-carnitine-treated animals. Carnitine suppressed the apoptosis of granulosa cells in the ovarium. These results indicate that mitochondriadependent and L-carnitine-inhibitable apoptosis of granulosa cells underlies the mechanism of PMSG/ hCG-induced ovulation.
SFRR 2004
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P7-89 NEUROPROTECTIVE EFFECTS OF ;-TOCOTRIENOL AND A-TOCOPHEROL AGAINST H2O2-INDUCED OXIDATIVE STRESS IN ASTROCYTES CULTURE S. M. Then,1 M. Musalmah,1 M. T. Gapor,2 and W. Z. Wan Ngah1 1
Department of Biochemistry, Medical Faculty, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur and 2Department of Chemistry, Malaysian Palm Oil Board, 43500 Bangi, Selangor, Malaysia
There is accumulative evidence that oxidative stress is one of the factors that lead to decreased neuronal cell viability and this could be inhibited by antioxidant agents. a-Tocopherol (ATF) has been widely studied for its antioxidant properties as well as neuroprotective effects. But recent studies have shown that tocotrienols also have similar reactivity towards radicals and is more readily absorbed into the membranes than tocopherols. We compared the effects of GTT and ATF on oxidative stress-induced cytotoxicity using primary culture of astrocytes. Results showed that both ATF and GTT have different range of cytotoxicity. ATF is toxic at concentrations greater than 750 mM whereas GTT is toxic from 100mM. ATF and GTT protected cells against cytotoxicity of hydrogen peroxide (H2O2) at different concentrations: 50 mM-75 0mM and at 40 mM-60 mM respectively. Therefore, GTT is more effective in protecting cells against superoxide radicals (O2 ) at lower concentration compare to ATF. This might be due to the fact that GTT is more readily transferred between membranes and incorporated into membranes. In conclusion, these results show that both ATF and GTT protects astrocytes against H2O2 induced-oxidative stress, albeit at different concentrations, presumably reflecting the different absorption rates and antioxidant mechanisms of actions of the two vitamin E subfamilies.
P7-90 APOPTOSIS OF NEURAL PRECURSOR CELLS FOLLOWING GAMMA IRRADIATION IS MODULATED BY REACTIVE OXYGEN AND NITROGEN SPECIES (ROS/RNS) E. Robello,1 D. Dubner,1 S. Michelin,1 M. R. Perez,1 S. Puntarulo,2 and P. Gisone1 Autoridad Regulatoria Nuclear (ARN), Gerencia de Apoyo Cientı´fico, Laboratorio de Radiopatologı´a, Avenida del Libertador 8250, (C1429BNP) Buenos Aires, Argentina, 2Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
Enhanced induced generation of reactive oxygen (ROS) and nitrogen species (RNS) has been observed after exposures to low radiation doses. This study was aimed to address the participation of radiation-induced ROS,
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and endogenous NO generation in the radiation-induced apoptosis of neural cortical precursors. Cortical cells obtained from rats at 17 gestational day (gd) were irradiated with doses from 0.2 Gy to 2 Gy at a dose-rate of 0.3 Gy/m. A significant decrease of the luminol-dependent chemiluminescence was observed in cells after 30 min of irradiation, and basal levels were reached after 120 min. Chemiluminescence in irradiated cells significantly decreased by incubation with superoxide dismutase. Cellular NO content, estimated as NO2 + NO3 released to the culture medium, showed a significant increase in irradiated cells as compared to control values, up to 60 min post-irradiation. Apoptosis was evaluated by flow cytometry, morphology and DNA fragmentation after 24 h of irradiation. A significant increase was detected with 0.4 Gy doses. Pre-treatment of the cells with 10 mM N-acetylcyteine (NAC), a precursor for GSH sunthesis, lead to a significant decrease in cellular apoptosis, and inhibition of nitric oxide synthase (NOS) by L-NAME significantly increased apoptosis in irradiated cells. We conclude that a complex cross-talk between ROS and RNS play a pivotal role in the early signaling pathway leading to radiation-induced cell death.
P7-91 GLYCOXIDATIVE STRESS INDUCES NEURONAL APOPTOSIS TROUGH PKC-DELTA ACTIVATION M. Nitti,1 D. Verzola,1 S. Rossi,1 P. Odetti,1 D. Cottalasso,1 M. A. Pronzato,1 U. M. Marinari,1 and C. Domenicotti1 1
DiMeS University of Genoa, Italy
Accumulation of AGEs (Advanced Glycation End products), found in almost all body tissues including brain during ageing, leads to cell functional injury inducing alterations of intracellular redox balance. In this work, post-mitotic neuron-like cells were incubated for 48 h with increasing amounts of glycated foetal serum (G-FBS) containing 750 – 1500 pmol/ml of pentosidine, a well known glycoxidative marker. This treatment was able to induce an increase in apoptosis linear with AGE concentration up to necrotic death with highest AGE amount (3000pmol/pentosidine). Analysing the effect of 10% GFBS exposure (corresponding to 40% of apoptotic cell number), we found an early generation of intracellular reactive oxygen species (ROS): +26 – 28 % at 1.5 – 3 h incubation respectively and +40% at 6 h. In these conditions, the PKC-delta activity was increased approximately two fold and DNA binding activity of redox transcription factor AP-1, strictly linked to this isoform, was enhanced 2.5 fold. A relationship between oxidative stress, PKC delta activity, AP-1 activation and apoptosis, was demonstrated pre-treating neurons with 500 AM Vitamin E, a well-known antioxidant, with 20 Ag/ml of Gingko Biloba extract, and with 3 AM Rottlerin, inhibitor of PKC delta, that were able to completely prevent all effects dependent on glycoxidative stress. (Grants from COFIN 2002 Prof. Marinari and FIRB 2001 Prof. Poli).
SFRR 2004