Biomedical subjects

Rui Curi

Publications and source records attributed to Rui Curi.

At least 37 records · Page 2Linked to original sources

PubMed2006

Comparative toxicity of fatty acids on a macrophage cell line (J774).

In the present study, the cytotoxicity of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on a macrophage cell line (J774) was investigated. The induction of toxicity was investigated by changes in cell size, granularity, membrane integrity, DNA fragmentation and phosphatidylserine externalization by using flow cytometry. Fluorescence microscopy was used to determine the type of cell death (Acridine Orange/ethidium bromide assay). The possible mechanisms involved were examined by measuring mitochondrial depolarization, lipid accumulation and PPARgamma (peroxisome-proliferator-activated receptor gamma) activation. The results demonstrate that fatty acids induce apoptosis and necrosis of J774 cells. At high concentrations, fatty acids cause macrophage death mainly by necrosis. The cytotoxicity of the fatty acids was not strictly related to the number of double bonds in the molecules: palmitic acid>docosahexaenoic acid>stearic acid=eicosapentaenoic acid=arachidonic acid>oleic acid>linoleic acid. The induction of cell death did not involve PPARgamma activation. The mechanisms of fatty acids to induce cell death involved changes in mitochondrial transmembrane potential and intracellular neutral lipid accumulation. Fatty acids poorly incorporated into triacylglycerol had the highest toxicity.

Animals↗

PubMed2006

Modulation of lipopolysaccharide-induced acute lung inflammation: Role of insulin.

The present study was undertaken to investigate the influence of insulin on lipopolysaccharide (LPS)-induced acute lung injury. Diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 30 days) and controls were instilled with saline containing LPS (750 microg/0.4 mL) or saline alone. The following analyses were performed 6 h there after: (a) total and differential cell counts in bronchoalveolar lavage (BAL) fluid, (b) quantification of tumor necrosis factor alpha, interleukin (IL) 1beta, IL-10, and cytokine-induced neutrophil chemoattractant 1 in the BAL (enzyme-linked immunosorbent assay), (c)immunohistochemistry for intercellular adhesion molecule 1 and E-selectin on lung vessels, and (d) quantification of metalloproteinases (MMP) 2 and 9 in the BAL (zymography). Relative to controls, diabetic rats exhibited a reduction in the number of neutrophils (80%) and reduced concentrations of tumor necrosis factor alpha (56%), IL-1beta (66%), and IL-10 (35%) after LPS instillation. Cytokine-induced neutrophil chemoattractant 1 levels did not differ between groups. Increased levels of MMP-2 (90%) and MMP-9 (500%) were observed in diabetic rats compared with controls. Treatment of diabetic rats with neutral protamine Hagedorn insulin (4 IU, s.c.), 2 h before LPS instillation, completely restored the number of neutrophils and concentrations of cytokines in the BAL fluid. Despite no significant differences between diabetic and control groups, there was a remarkable increase in intercellular adhesion molecule 1 and E-selectin expression on lung vessels after insulin treatment. Levels of MMP-2 and MMP-9 did not change after treatment with insulin. Levels of corticosterone were equivalent among groups. Data presented suggest that insulin modulates the production/release of cytokines and the expression of adhesion molecules controlling, therefore, neutrophil migration during the course of LPS-induced acute lung inflammation.

Animals↗

PubMed2005

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle.

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O(2) consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.

Animals↗

PubMed2005

Induction of apoptosis by pterocarpans from Platymiscium floribundum in HL-60 human leukemia cells.

(+)-2,3,9-Trimethoxy-pterocarpan (1) (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4) are cytotoxic pterocarpans isolated from the native Brazilian plant Platymiscium floribundum. The purpose of the present study was to examine whether induction of apoptosis and/or inhibition of DNA synthesis is involved in the cytotoxicity of these pterocarpans in human leukemia cells. The effect on cell viability determined using the trypan exclusion assay revealed that all compounds tested reduced the number of viable cells, while only in the presence of 3 and 4, there was an increase of nonviable cells. The analysis of membrane integrity and morphological modifications by flow cytometry in the presence of these two compounds indicated that treated cells undergo necrosis, while 1 and 2 trigger apoptosis. DNA synthesis seemed to be affected since BrdU incorporation was inhibited in a dose-dependent manner in the presence of all tested compounds. Pterocarpan treatment also induced an increase in the amount of subdiploid DNA, indicating internucleosomal DNA breakdown, mitochondrial depolarization and caspase-3 activation, which indicate apoptosis induction.

Antineoplastic Agents, Phytogenic↗

PubMed2005

Comparative toxicity of oleic and linoleic acid on human lymphocytes.

Commercially available lipid emulsions for parenteral nutrition are mainly composed by long chain triacylglycerol containing a high proportion of linoleic acid (LA) or oleic acid (OA). The immunological impact of such therapy is particularly important because parenteral diets are often administered to critically ill patients as a mechanism to supply adequate nutrition during catabolic stress conditions. The comparative toxicity of OA and LA on human lymphocytes and the type of cell death induced by these fatty acids were determined in vitro. Parameters of cell death were investigated by flow cytometry-cell viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, neutral lipid accumulation and production of reactive oxygen species-and by fluorescence microscopy-chromatin condensation. Additionally a spectrofluorometric assay was employed to determine the activities of caspase--3, 6 and 8. Evidence is presented herein that OA is less toxic to human lymphocytes than LA. However, both fatty acids promoted apoptosis and necrosis of these cells. The mechanism of cell death induced by OA involved activation of caspase 3 while the mechanism of death induced by LA involved mitochondrial depolarization and ROS production. Importantly, neutral lipid accumulation may be a mechanism to protect lymphocytes against the toxicity induced by OA. OA may offer an immunological less problematic alternative to LA with respect to fatty acid composition of parenteral nutritional emulsions.

Cell Survival↗

PubMed2005

Lifelong exposure to dietary fish oil alters macrophage responses in Walker 256 tumor-bearing rats.

Supplementation of the diet with fish oil (FO) decreases growth of the Walker 256 tumor and decreases the cachexia associated with tumor-bearing. The mechanisms by which FO inhibits tumor growth and cachexia are unknown. Macrophages are very important in host defence against tumors since they produce several anti-tumor agents which in turn have been shown to be modified by dietary FO, but rarely in the setting of tumor bearing and never in relation to lifelong exposure. In this study, we compared the effects of supplementation of the diet of pregnant and lactating rats and subsequent supplementation of the offspring with coconut fat or FO on macrophage activities involved in anti-tumor defence. FO supplementation was able to induce an increase in phagocytosis, in O2-, H2O2, nitric oxide, and TNF-alpha production by macrophages and in lysosomal volume in non-tumor-bearing rats. However, phagocytosis, production of O2- and H2O2 and lysosomal volume were not affected by the FO diet when rats were bearing tumors, although nitric oxide production was higher in these animals. It appears that tumor bearing activates the innate immune system and that dietary FO has little effect on innate immunity in the presence of Walker 256 tumors. Thus, it is still unclear how FO decreases the growth of Walker 256 tumors and the associated cachexia.

Aging↗

PubMed2005

Oxidative state in platelets and erythrocytes in aging and Alzheimer's disease.

Several studies have shown involvement of peroxynitrite anion, a potent oxidative agent, in Alzheimer's disease (AD) neuropathology. Herein, we assessed in platelets and erythrocytes of AD patients, age-matched and young adults controls: thiobarbituric acid-reactive substances (TBARS) production; superoxide dismutase (SOD), nitric oxide synthase (NOS) and Na,K-ATPase activities; cyclic GMP (cGMP) content, both basal and after sodium nitroprusside (SNP) stimulation. Aging was associated with an increase in TBARS production and NOS activity, a decrease in basal cGMP content and no change in SOD and Na,K-ATPase activities. AD patients, compared to aged controls, have: increase in TBARS production and in NOS, SOD and Na,K-ATPase activities but no alteration in basal cGMP content. SNP increased cGMP platelets production in all groups. In conclusion, we demonstrated in platelets and erythrocytes a disruption in systemic modulation of oxidative stress in aging and with more intensity in AD.

Aged↗

PubMed2005

Comparative toxicity of oleic acid and linoleic acid on Raji cells.

OBJECTIVE: Parenteral diets are often administered to critically ill patients. To study one of the effects of commercially available parenteral lipid diets, rich in triacylglycerol esters of omega-6 polyunsaturated fatty acids or omega-9 monounsaturated fatty acids, on the immune system of such patients, we evaluated the cytotoxicity of oleic and linoleic acids on Raji cells that had been derived from human B-lymphocytes. METHODS: Cell death intensity and type were investigated by flow cytometry by quantitation of cell volume, granularity, DNA fragmentation, mitochondrial depolarization, and lipid accumulation. Fluorescence microscopy was used to determine chromatin condensation and type of cell death (acridine orange/ethidium bromide assay). Gene expression of BCL-XL, BCL-XS, C-MYC, and P53 was studied by reverse transcriptase polymerase chain reaction. RESULTS: Oleic acid was less toxic than linoleic acid to Raji cells. Both fatty acids promote apoptosis and necrosis of these cells. The mechanism of cell death induced by these fatty acids seemed to involve mitochondrial depolarization, lipid accumulation, and overexpression of C-MYC and P53. CONCLUSION: Oleic acid may offer a less harmful alternative to linoleic acid in parenteral diets with respect to patient B-lymphocyte-mediated immunologic activity.

Apoptosis↗

PubMed2005

Toxicity of fatty acids on murine and human melanoma cell lines.

High concentrations of certain fatty acids can cause cell death via apoptosis or necrosis. The aim of this study was to investigate the toxicity of saturated and unsaturated fatty acids on melanoma cell lines, which was evaluated by either loss of membrane integrity and/or DNA fragmentation using flow cytometric analysis. Evidence is presented that saturated and unsaturated fatty acids exert toxic effects on melanoma cells through loss of membrane integrity and/or DNA fragmentation. Arachidonic and linoleic acids were the most effective in decreasing the number of viable S91 murine melanoma cells, causing loss of membrane integrity and DNA fragmentation at 100 microM concentration already after 24 h in culture. In B16F10 murine melanoma cells, palmitic acid was the most toxic, inducing cell death by both apoptosis and necrosis. The human melanoma cell lines were more resistant to the toxic effect of fatty acids. In SK-Mel 23 cells, indications of cytotoxicity were detected only after 48 h treatment with arachidonic, linoleic, palmitic and palmitoleic acids at 200 microM concentration. Linoleic acid was the most toxic for this cell line. In SK-Mel 28 human cells, only palmitic acid caused a significant decrease of the number of viable cells, inducing DNA fragmentation after 24 and 48 h treatments at 200 microM concentration.

Animals↗

PubMed2005

Toxicity of a mixture of fatty acids on human blood lymphocytes and leukaemia cell lines.

The effect of a mixture of fatty acids upon lymphocyte and leukaemia cell death was examined. Peripheral lymphocytes from healthy subjects and two human leukaemia cell lines-Jurkat (T lymphocyte) and Raji (B lymphocyte) cells-were treated with increasing concentrations (0.1-0.4 mM) of a fatty acid mixture in a proportion mimicking that of the free fatty acids in plasma. Features of cell death were then evaluated. Phosphatidylserine externalization, and DNA fragmentation (apoptosis), and loss of cell membrane integrity (necrosis) and mitochondrial depolarization (common feature of cell death) were observed in leukaemia cells after the fatty acid treatment for up to 48 h. Human lymphocytes, however, when submitted to the same treatment presented apoptotic feature only. These findings indicate that a free fatty acid mixture (mimicking the proportion found in plasma) triggers apoptosis of leukaemia cell lines followed by loss of cell membrane integrity, whereas in human circulating lymphocytes the same treatment causes apoptosis only. Evidence is presented herein that mitochondria from leukaemic cells are more susceptible to the toxicity of the fatty acids than mitochondria from human circulating lymphocytes.

B-Lymphocytes↗

PubMed2005

Regulation of reactive oxygen species (ROS) production by C18 fatty acids in Jurkat and Raji cells.

In the present study, the effects of C18 fatty acids with different numbers of double bonds, SA (stearic acid; C18:0), OA (oleic acid; C18:1), LA (linoleic acid; C18:2) and gamma-LNA (gamma-linolenic acid; C18:3), on ROS (reactive oxygen species) production by Jurkat (a human T-lymphocyte-derived cell line) and Raji (a human B-lymphocyte-derived cell line) cells were investigated. ROS production was determined by NBT (Nitro Blue Tetrazolium) reduction (intracellular and extracellular ROS production) and by dihydroethidium oxidation using flow cytometry (intracellular ROS production). The effectiveness on ROS production was gamma-LNA<SA<OA<LA in Jurkat cells and SA<gamma-LNA<OA<LA in Raji cells. LA (found in corn, soya bean and sunflower oils) was more potent than OA (found in olive oil) in stimulating ROS production in both Raji and Jurkat cells. The lower ROS production by OA compared with LA may be one of the benefits of olive oil consumption. As SA and gamma-LNA acids had little or no effect, further studies on the site of ROS production in these cells were carried out with OA and LA only. Activation of NADPH oxidase via PKC (protein kinase C) was found to be the major mechanism of ROS production induced by OA and LA in Jurkat and Raji cells.

Analysis of Variance↗

PubMed2005

Ratio of n6 to n-3 fatty acids in the diet affects tumor growth and cachexia in Walker 256 tumor-bearing rats.

In this study we investigate the impact of the dietary ratio of n-6 to n-3 fatty acids (FAs) from postweaning until adult age upon tumor growth, lipid peroxidation in tumor tissue, and metabolic indicators of cancer cachexia in Walker 256 tumor-bearing rats. Weanling male Wistar rats received a normal low-fat (40 g/kg diet) chow diet or high-fat diets (300 g/kg) that included fish oil (FO) or sunflower oil or blends of FO and sunflower oil to yield n-6 to n-3 FA ratios of approximately 6:1, 30:1, and 60:1 ad libitum. After 8 wk, half of each group was inoculated with 1 ml of 2 x 10(7) Walker 256 cells. At the 14th day after tumor inoculation, the animals were killed, and tumors and blood were removed. The different diets did not modify the blood parameters in the absence of tumor bearing, except the high-FO diet, which decreased serum cholesterol and triacylglycerol concentrations. Tumor weight in chow-fed rats was 19 g, and these rats displayed cancer cachexia, characterized by hypoglycemia, hyperlacticidemia, hypertriacylglycerolemia, loss of body weight, and food intake reduction. Tumor weight in FO-fed rats was 7.7 g, and these animals gained body weight (14.6 g) and maintained blood metabolic parameters similar to non-tumor-bearing animals. Tumor weight in rats fed the diet with an n-6 to n-3 FA ratio of 6:1 was similar to tumor-bearing, chow-fed rats, but they gained 2 g in the body weight and blood metabolic parameters were similar to those in non-tumor-bearing rats. However, a further increase in the n-6 FA content of the diet did not change the cachectic state associated with tumor bearing. In this experimental model, a dietary n-6 to n-3 FA ratio of 6:1 was able to increase food intake and body weight, restore the biochemical blood parameters of cachexia, and prevent the development of cancer cachexia.

Animals↗

PubMed2005

S-nitrosation of the insulin receptor, insulin receptor substrate 1, and protein kinase B/Akt: a novel mechanism of insulin resistance.

Evidence demonstrates that exogenous nitric oxide (NO) and the NO produced by inducible nitric oxide synthase (iNOS) can induce insulin resistance in muscle. Here, we investigated whether this insulin resistance could be mediated by S-nitrosation of proteins involved in early steps of the insulin signal transduction pathway. Exogenous NO donated by S-nitrosoglutathione (GSNO) induced in vitro and in vivo S-nitrosation of the insulin receptor beta subunit (IRbeta) and protein kinase B/Akt (Akt) and reduced their kinase activity in muscle. Insulin receptor substrate (IRS)-1 was also rapidly S-nitrosated, and its expression was reduced after chronic GSNO treatment. In two distinct models of insulin resistance associated with enhanced iNOS expression-diet-induced obesity and the ob/ob diabetic mice-we observed enhanced S-nitrosation of IRbeta/IRS-1 and Akt in muscle. Reversal of S-nitrosation of these proteins by reducing iNOS expression yielded an improvement in insulin action in both animal models. Thus, S-nitrosation of proteins involved in insulin signal transduction is a novel molecular mechanism of iNOS-induced insulin resistance.

Animals↗

PubMed2004

Metabolism of the microregions of human breast cancer.

Glucose and glutamine metabolism of two microregions of human infiltrating ductile breast cancer, the center and the periphery, was studied and the results were compared with those of healthy mammary glands. In general, the activities of glycolytic enzymes and of phosphate-dependent glutaminase were as follows: center>periphery>mammary gland. Insulin caused a marked increase of glucose consumption and lactate production by incubated slices of mammary gland but had no effect on both microregions of the tumor. Therefore, human breast cancer presents metabolic microregions.

Adolescent↗

PubMed2004

Fatty acid flip-flop and proton transport determined by short-circuit current in planar bilayers.

The effect of palmitic acid (PA) and oleic acid (OA) on electrical parameters of planar membranes was studied. We found a substantial difference between the effects of PA and OA on proton transfer. PA induced a small increase in conductance, requiring a new technique for estimating proton-mediated currents across low-conductance planar bilayers in which an electrometer is used to measure the transmembrane current under virtual short circuit (SCC). Open-circuit voltage and SCC were used to determine proton and leak conductances. OA caused a marked increase in membrane conductance, allowing the use of a voltage-clamp technique. From SCC data, we were able to estimate the flip-flop rate constants for palmitate (1 x 10(-6) s(-1)) and oleate (49 x 10(-6) s(-1)) anions. Cholesterol, included in the membrane-forming solution, decreased importantly the leak conductance both in membranes unmodified by FA and in membranes modified by PA added to the bath.

Biochemical Phenomena↗

PubMed2004

Identification of genes regulated by oleic acid in Jurkat cells by suppressive subtractive hybridization analysis.

In this study, the effect of oleic acid (50 microM) on gene expression of Jurkat cells (human T lymphocytes cell line) was examined using the suppressive subtractive hybridization approach. This technique allowed us to identify genes with higher or lower expression after cell treatment with oleic acid as compared to untreated cells. Oleic acid upregulated the expression of the translation elongation factor alpha 1 and ATP synthase 8 and downregulated gp96 (human tumor rejection antigen gp96), heat-shock protein 60 and subtilisin-like protein 4. These results suggest that oleic acid, at plasma physiological concentration, can regulate the expression of important genes to maintain the machinery that ensures cell functioning.

Blotting, Northern↗

PubMed2004

Cachexia induced by Walker 256 tumor growth causes rat lymphocyte death.

Death induction by Walker 256 tumor cachexia in non-tumor-infiltrating lymphocytes was investigated. Lymphocytes from cachectic tumor-bearing rats presented a higher proportion of cells with ruptured membranes, indicating necrotic cell death. The cachexia induced by Walker 256 tumor also increased by 3.6-fold the percentage of cells with fragmented DNA, suggestive of apoptotic cell death. The mitochondria involvement was examined by analysis of mitochondria transmembrane potential using rhodamine 123. Lymphocytes from cachectic tumor-bearing rats presented a more pronounced depolarization of mitochondrial transmembrane potential in comparison with cells from the control group. The expression of important proapoptotic (Bcl-xs, Bax, p53, caspase-3) and antiapoptotic genes (Bcl-2 and Bcl-xL) was also altered by tumor cachexia. These results suggest that the immunosuppression induced by Walker 256 tumor cachexia is at least in part a result of lymphocyte death. Evidence was found for the involvement of mitochondria and important proapoptotic genes in the process of lymphocyte death by Walker 256 tumor cachexia.

Animals↗

PubMed2004

Indole-3-acetic acid increases glutamine utilization by high peroxidase activity-presenting leukocytes.

Indole-3-acetic acid (IAA) is toxic for human tumor cells and in association with horseradish peroxidase (HRP) can be used as a new prodrug/enzyme combination for targeted cancer therapy. The toxic effect of IAA on neutrophils, macrophages and lymphocytes is associated with cell peroxidase activity, which is high in neutrophils and low in lymphocytes. The effect of IAA on glucose and glutamine metabolism in leukocytes presenting different peroxidase activities: neutrophils, thioglycollate-elicited macrophages and lymphocytes was investigated. A time-course effect (from 6 to 48 h in culture) of IAA on glucose and glutamine metabolism of neutrophils, thioglycollate-elicited macrophages, and lymphocytes was then carried out. Addition of IAA (0.25 mM) did not have a marked effect on glucose utilization and lactate formation by the three cell types but it raised glutamine consumption and glutamate production by neutrophils and macrophages. IAA had no effect on glutamine consumption and glutamate production by lymphocytes. A strong relationship was found between glutamine utilization (0.999) and glutamate production (0.999) and peroxidase activity. IAA did not change the activities of hexokinase, glucose-6-phosphate dehydrogenase, citrate synthase, lactate dehydrogenase, and phosphate-dependent glutaminase of 24 h cultured neutrophils and lymphocytes. The effect of IAA (1 mM) on glucose and glutamine metabolism was also investigated by 1 h incubated leukocytes in PBS. IAA did not affect glucose and glutamine metabolism of lymphocytes but enhanced glucose and glutamine metabolism by 1 h incubated neutrophils and thioglycollate-elicited macrophages. IAA caused a marked increase on oxygen consumption by neutrophils, which was more pronounced in the presence of the glutamine as compared to glucose. The stimulation of oxygen consumption leads to a reduction in NADH/NAD+ ratio that activates the flux of substrates through the Krebs cycle. Since glutamine is mainly metabolized through the left hand side of the Krebs cycle, a reduction in the redox state of the cells may accelerate the flux of substrates through glutaminolysis. The toxic results presented here show that the affect of IAA in association with peroxidase involves activation of glutamine metabolism.

Analysis of Variance↗