INFLUENCE OF DIABETES-MELLITUS ON THE GLUTATHIONE REDOX SYSTEM OF HUMAN RED-BLOOD-CELLS

Several components of the erythrocyte-dependent glutathione redox system (reduced glutathione, GSH; oxidized glutathione, GSSG; glutathione peroxidase, GSH-Px; glutathione reductase, GSH-Red) were determined in patients with types I and II diabetes mellitus (DM). All groups studied were male subjects: G1, 20 young healthy individuals (aged 23.7 +/- 4.2 years); G2, 15 young insulin-treated type I DM patients; G3, 20 older insulin-treated type II DM patients; 04, 21 older oral hypoglycemic agent-treated type II DM patients; G5, 28 aged healthy individuals (aged 68.9 +/- 11.5 years). There were no differences between G1 and G2, G3 or G4 regarding erythrocyte GSH, GSSG, and GSH-Red (without FAD) levels. GSH-Px activity was significantly lower in G2 when compared to G1 (15.2 +/- 4.9 vs 20.6 +/- 6.6 IU/g Hb). The GSH-Red and GSH-Px activities and GSH levels were significantly higher in 03 (4.6 +/- 1.7 IU/g Hb, 20.2 +/- 8.7 IU/g Hb and 3.5 +/- 1.3-mu-M/g Hb) and G4 (5.0 +/- 2.2 IU/g Hb, 16.9 +/- 6.1 IU/g Hb and 5.0 +/- 2.3-mu-M/g Hb) when compared to G5 (3.4 +/- 0.9 IU/g Hb, 12.0 +/- 3.6 IU/g Hb and 2.3 +/- 0.9-mu-M/g Hb). The findings suggest that treatment of DM can stimulate the redox activity of red blood cells in aged subjects.

Associação entre a concentração de selênio, o polimorfismo Pro198Leu da glutationa peroxidase 1 e a mortalidade em pacientes com choque séptico

Pós-graduação em Fisiopatologia em Clínica Médica - FMB

Effect of mixture of diazinon and benzo[a]pyrene in Glutathione S-transferase of Nile tilapia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene expression and physiological changes of the Antarctic krill Euphausia superba under different hypoxia intensities

Antarctic krill (Euphausia superba) from South Georgia comprise one of the most northern and abundant krill stocks. South Georgia waters are undergoing rapid warming, as a result of climate change, which in turn could alter the oxygen concentration of the water. We investigated gene expression in Antarctic krill related to aerobic metabolism, antioxidant defence, and heat-shock response under severe (2.5% O2 saturation or 0.6 kPa) and threshold (20% O2 saturation or 4 kPa) hypoxia exposure compared to in situ levels (normoxic; 100% O2 saturation or 21 kPa). Biochemical metabolic and oxidative stress indicators complemented the genic expression analysis to detect in vivo signs of stress during the hypoxia treatments. Expression levels of the genes citrate synthase (CS), mitochondrial manganese superoxide dismutase (SODMn-m) and one heat-shock protein isoform (E) were higher in euphausiids incubated 6 h at 20% O2 saturation than in animals exposed to control (normoxic) conditions. All biochemical antioxidant defence parameters remained unchanged among treatments. Levels of lipid peroxidation were raised after 6 h of severe hypoxia. Overall, short-term exposure to hypoxia altered mitochondrial metabolic and antioxidant capacity, but did not induce anaerobic metabolism. Antarctic krill are swarming organisms and may experience short periods of hypoxia when present in dense swarms. A future, warmer Southern ocean, where oxygen saturation levels are decreased, may result in smaller, less dense swarms as they act to avoid greater levels of hypoxia.

Defense Responses to Tetrapyrrole-Induced Oxidative Stress in Transgenic Plants with Reduced Uroporphyrinogen Decarboxylase or Coproporphyrinogen Oxidase Activity1

We analyzed the antioxidative defense responses of transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase. These plants are characterized by necrotic leaf lesions resulting from the accumulation of potentially photosensitizing tetrapyrroles. Compared with control plants, the transformants had increased levels of antioxidant mRNAs, particularly those encoding superoxide dismutase (SOD), catalase, and glutathione peroxidase. These elevated transcript levels correlated with increased activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD. Total catalase activity decreased in the older leaves of the transformants to levels lower than in the wild-type plants, reflecting an enhanced turnover of this photosensitive enzyme. Most of the enzymes of the Halliwell-Asada pathway displayed increased activities in transgenic plants. Despite the elevated enzyme activities, the limited capacity of the antioxidative system was apparent from decreased levels of ascorbate and glutathione, as well as from necrotic leaf lesions and growth retardation. Our data demonstrate the induction of the enzymatic detoxifying defense system in several compartments, suggesting a photosensitization of the entire cell. It is proposed that the tetrapyrroles that initially accumulate in the plastids leak out into other cellular compartments, thereby necessitating the local detoxification of reactive oxygen species.

Thermosensitive phenotype of transgenic mice overproducing human glutathione peroxidases.

Exposure of humans and other mammals to hyperthermic conditions elicits many physiological responses to stress in various tissues leading to profound injuries, which eventually result in death. It has been suggested that hyperthermia may increase oxidative stress in tissues to form reactive oxygen species harmful to cellular functions. By using transgenic mice with human antioxidant genes, we demonstrate that the overproduction of glutathione peroxidase (GP, both extracellular and intracellular) leads to a thermosensitive phenotype, whereas the overproduction of Cu,Zn-superoxide dismutase has no effect on the thermosensitivity of transgenic mice. Induction of HSP70 in brain, lung, and muscle in GP transgenic mice at elevated temperature was significantly inhibited in comparison to normal animals. Measurement of peroxide production in regions normally displaying induction of HSP70 under hyperthermia revealed high levels of peroxides in normal mice and low levels in GP transgenic mice. There was also a significant difference between normal and intracellular GP transgenic mice in level of prostaglandin E2 in hypothalamus and cerebellum. These data suggest direct participation of peroxides in induction of cytoprotective proteins (HSP70) and cellular mechanisms regulating body temperature. GP transgenic mice provide a model for studying thermoregulation and processes involving actions of hydroxy and lipid peroxides in mammals.

Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

Although cytosolic glutathione S-transterase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes. GST1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GsTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals. e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence Suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestivc tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

Diet Carotenoid Lutein Modulates The Expression Of Genes Related To Oxygen Transporters And Decreases Dna Damage And Oxidative Stress In Mice.

Lutein (LT) is a carotenoid obtained by diet and despite its antioxidant activity had been biochemically reported, few studies are available concerning its influence on the expression of antioxidant genes. The expression of 84 genes implicated in antioxidant defense was quantified using quantitative reverse transcription polymerase chain reaction array. DNA damage was measured by comet assay and glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were quantified as biochemical parameters of oxidative stress in mouse kidney and liver. cDDP treatment reduced concentration of GSH and increased TBARS, parameters that were ameliorated in treatment associated with LT. cDDP altered the expression of 32 genes, increasing the expression of GPx2, APC, Nqo1 and CCs. LT changed the expression of 37 genes with an induction of 13 mainly oxygen transporters. In treatments associating cDDP and LT, 30 genes had their expression changed with a increase of the same genes of the cDDP treatment alone. These results suggest that LT might act scavenging reactive species and also inducing the expression of genes related to a better antioxidant response, highlighting the improvement of oxygen transport. This improved redox state of the cell through LT treatment could be related to the antigenotoxic and antioxidant effects observed.

Overexpression Of Ucp1 In Tobacco Induces Mitochondrial Biogenesis And Amplifies A Broad Stress Response.

Uncoupling protein one (UCP1) is a mitochondrial inner membrane protein capable of uncoupling the electrochemical gradient from adenosine-5'-triphosphate (ATP) synthesis, dissipating energy as heat. UCP1 plays a central role in nonshivering thermogenesis in the brown adipose tissue (BAT) of hibernating animals and small rodents. A UCP1 ortholog also occurs in plants, and aside from its role in uncoupling respiration from ATP synthesis, thereby wasting energy, it plays a beneficial role in the plant response to several abiotic stresses, possibly by decreasing the production of reactive oxygen species (ROS) and regulating cellular redox homeostasis. However, the molecular mechanisms by which UCP1 is associated with stress tolerance remain unknown. Here, we report that the overexpression of UCP1 increases mitochondrial biogenesis, increases the uncoupled respiration of isolated mitochondria, and decreases cellular ATP concentration. We observed that the overexpression of UCP1 alters mitochondrial bioenergetics and modulates mitochondrial-nuclear communication, inducing the upregulation of hundreds of nuclear- and mitochondrial-encoded mitochondrial proteins. Electron microscopy analysis showed that these metabolic changes were associated with alterations in mitochondrial number, area and morphology. Surprisingly, UCP1 overexpression also induces the upregulation of hundreds of stress-responsive genes, including some involved in the antioxidant defense system, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione-S-transferase (GST). As a consequence of the increased UCP1 activity and increased expression of oxidative stress-responsive genes, the UCP1-overexpressing plants showed reduced ROS accumulation. These beneficial metabolic effects may be responsible for the better performance of UCP1-overexpressing lines in low pH, high salt, high osmolarity, low temperature, and oxidative stress conditions. Overexpression of UCP1 in the mitochondrial inner membrane induced increased uncoupling respiration, decreased ROS accumulation under abiotic stresses, and diminished cellular ATP content. These events may have triggered the expression of mitochondrial and stress-responsive genes in a coordinated manner. Because these metabolic alterations did not impair plant growth and development, UCP1 overexpression can potentially be used to create crops better adapted to abiotic stress conditions.

Water Stress Reveals Differential Antioxidant Responses Of Tolerant And Non-tolerant Sugarcane Genotypes.

The biochemical responses of the enzymatic antioxidant system of a drought-tolerant cultivar (IACSP 94-2094) and a commercial cultivar in Brazil (IACSP 95-5000) grown under two levels of soil water restriction (70% and 30% Soil Available Water Content) were investigated. IACSP 94-2094 exhibited one additional active superoxide dismutase (Cu/Zn-SOD VI) isoenzyme in comparison to IACSP 95-5000, possibly contributing to the heightened response of IACSP 94-2094 to the induced stress. The total glutathione reductase (GR) activity increased substantially in IACSP 94-2094 under conditions of severe water stress; however, the appearance of a new GR isoenzyme and the disappearance of another isoenzyme were found not to be related to the stress response because the cultivars from both treatment groups (control and water restrictions) exhibited identical changes. Catalase (CAT) activity seems to have a more direct role in H2O2 detoxification under water stress condition and the shift in isoenzymes in the tolerant cultivar might have contributed to this response, which may be dependent upon the location where the excessive H2O2 is being produced under stress. The improved performance of IACSP 94-2094 under drought stress was associated with a more efficient antioxidant system response, particularly under conditions of mild stress.

Activation Of The Low Molecular Weight Protein Tyrosine Phosphatase In Keratinocytes Exposed To Hyperosmotic Stress.

Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.

Perfil proteico e metabolismo oxidativo de cordeiros experimentalmente infectados pelo Haemonchus contortus e suplementados com selênio e vitamina E

Para avaliar a influência da suplementação com selênio e vitamina E sobre o perfil proteico e metabolismo oxidativo de cordeiros infectados experimentalmente pelo Haemonchus contortus, trinta cordeiros fêmeas foram distribuídos em quatro grupos: G1 (n=10): animais infectados; G2 (n=10): infectados e suplementados; G3 (n=5): controle; e G4 (n=5): não infectados e suplementados. Os grupos 1 e 2 receberam 500 larvas de H. contortus (L3), via oral, por um período de 20 dias, com intervalo de dois dias entre as doses. A suplementação nos grupos 2 e 4 foi realizada no dia zero, com 0,1mg kg-1 de Selenito de sódio (1,67%) e com 2.000UI de vitamina E por via intramuscular (IM). Somente a vitamina E foi reaplicada no dia 30. As coletas de sangue para determinação do perfil proteico (proteína total, albumina, alfa, beta e gamaglobulina) e metabolismo oxidativo (espécies reativas ao ácido tiobarbitúrico-TBARS e a enzima glutationa peroxidase (GSPX) foram realizadas nos dias zero, 20, 30, 45, 60 e 80. OPG foi quantificado nos dias 0, 20 ,45 e 80. Em relação aos valores de proteínas totais, albumina, betaglobulina e gamaglobulina, as principais diferenças foram observadas quando os grupos parasitados foram comparados com o grupo somente suplementado; e este manteve valores mais elevados. Conclui-se que não há influência da suplementação com selênio e vitamina E no perfil proteico e metabolismo oxidativo quando os cordeiros se encontram severamente parasitados por H.contortus.

O papel das selenoproteínas no câncer

Evidências têm demonstrado que distúrbios do metabolismo são comuns em células tumorais, levando ao aumento do estresse oxidativo. A elevação na produção de espécies reativas de oxigênio (EROs) associada à baixa atividade antioxidante tem sido relacionada a vários tipos de câncer. O selênio, micronutriente antioxidante, pode funcionar como um agente antimutagênico, prevenindo transformações malignas de células normais. Realizou-se um levantamento bibliográfico no período 2000 a 2009 mediante consulta à base de dados PubMed (National Library of Medicine´s Medline Biomedical Literature, USA), selecionando-se 39 artigos que avaliaram a relação entre câncer, estresse oxidativo e suplementação com selênio. O efeito protetor desse mineral é especialmente associado à sua presença na glutationa peroxidase e na tioredoxina redutase, enzimas protetoras do DNA e outros componentes celulares contra o dano oxidativo causado pelas EROs. Vários estudos têm demonstrado a expressão reduzida destas enzimas em diversos tipos de câncer, principalmente quando associados a uma baixa ingestão de selênio, que pode acentuar os danos causados. A suplementação de selênio parece ocasionar redução do risco de alguns tipos de câncer diminuindo o estresse oxidativo e o dano ao DNA. No entanto, mais estudos são necessários para esclarecer as doses de selênio adequadas para cada situação (sexo, localização geográfica e tipo de câncer)

Hypervalent organochalcogenanes as inhibitors of protein tyrosine phosphatases

A series of organochalcogenanes was synthesized and evaluated as protein tyrosine phosphatases (PTPs) inhibitors. The results indicate that organochalcogenanes inactivate the PTPs in a time- and concentration-dependent fashion, most likely through covalent modification of the active site sulfur-moiety by the chalcogen atom. Consequently, organochalcogenanes represent a new class of mechanism-based probes to modulate the PTP-mediated cellular processes.

Fatty acids decrease catalase activity in human leukaemia cell lines

Fatty acid (FA) may disturb the redox state of the cells not only by an increase in reactive oxygen species (ROS) generation but also due to a reduction in antioxidant enzyme activities. The effect of various FAs (palmitic, stearic, oleic, linoleic, gamma-linolenic and eicosapentaenoic acids (EPAs)) on Jurkat and Raji cells, (human T and B leukaemic cell lines was investigated). The following measurements were carried out: FA composition of the cells, cell proliferation and activities of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). The protective effect of alpha-tocopherol on cell death was also investigated. Each cell line presented a specific FA composition. All the tested ENS reduced catalase activity. The toxic effect of FA was abolished by the pre-incubation with physiological concentrations of alpha-tocopherol. The findings support the proposition that the increase in oxidative stress induced by FA partially occurs due to a reduction in catalase activity. In spite of the decrease in the enzyme activity, catalase protein and mRNA levels were not changed, suggesting a post-translational regulation. Copyright (C) 2007 John Wiley & Sons, Ltd.