Oxidative stress biomarkers of exposure in the blood of cichlid species from a metal-contaminated river

The oxidative stress biomarkers of exposure, such as reduced glutathione (GSH), activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and the levels of lipid peroxidation (LPO), were measured in the blood of three cichlid fish (Oreochromis niloticus, Tilapia rendalli, and Geophagus brasiliensis) taken during two seasons from two sites, unpolluted and polluted by industrial effluents, to evaluate the effectiveness of these biomarkers in assessing the impact of water contamination. The LPO levels in the blood were higher in fish from the metal-contaminated site and the chronic exposure led to significant changes in GPx, CAT, and SOD activities in all three cichlid species. The considerable variation of responses in these cichlids to water contamination evidenced differences in sensitivity to the metal contamination and/or in the potential to respond to it highlighting the importance of using a set of related biomarkers to assess the impact of water contamination. (C) 2007 Elsevier Inc. All rights reserved.

Zn uptake, physiological response and stress attenuation in mycorrhizal jack bean growing in soil with increasing Zn concentrations

The influence of arbuscular mycorrhizal fungi (AMF) inoculation on Canavalia ensiformis growth. nutrient and Zn uptake, and on some physiological parameters in response to increasing soil Zn concentrations was studied. Treatments were applied in seven replicates in a 2 x 4 factorial design, consisting of the inoculation or not with the AMF Glomus etunicatum, and the addition of Zn to soil at the concentrations of 0, 100, 300 and 900 mg kg(-1). AMF inoculation enhanced the accumulation of Zn in tissues and promoted biomass yields and root nodulation. Mycorrhizal plants exhibited relative tolerance to Zn up to 300 mg kg(-1) without exhibiting visual symptoms of toxicity, in contrast to non-mycorrhizal plants which exhibited a significant growth reduction at the same soil Zn concentration. The highest concentration of Zn added to soil was highly toxic to the plants. Leaves of plants grown in high Zn concentration exhibited a Zn-induced proline accumulation and also an increase in soluble amino acid contents; however proline contents were lower in mycorrhizal jack beans. Plants in association or not with the AMF exhibited marked differences in the foliar soluble amino acid profile and composition in response to Zn addition to soil. In general, Zn induced oxidative stress which could be verified by increased lipid peroxidation rates and changes in catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase activities. In summary, G. etunicatum was able to maintain an efficient symbiosis with jack bean plants in moderately contaminated Zn-soils, improving plant performance under those conditions, which is likely to be due to a combination of physiological and nutritional changes caused by the intimate relation between fungus and plant. The enhanced Zn uptake by AMF inoculated jack bean plants might be of interest for phytoremediation purposes. (C) 2009 Elsevier Ltd. All rights reserved.

Biochemical responses of the ethylene-insensitive Never ripe tomato mutant subjected to cadmium and sodium stresses

In order to further address the known interaction between ethylene and components of the oxidative system, we have used the ethylene-insensitive Never ripe (Nr) tomato (Solanum lycopersicum L) mutant, which blocks ethylene responses. The mutant was compared to the control Micro-Tom (MT) cultivar subjected to two stressful situations: 100 mM NaCl and 0.5 mM CdCl(2). Leaf chlorophyll, lipid peroxidation and antioxidant enzyme activities in roots, leaves and fruits, and Na and Cd accumulation in tissues were determined. Although we verified a similar growth pattern and Na and Cd accumulation for MT and Nr, the mutant exhibited reduced leaf chlorophyll degradation following stress. In roots and leaves, the patterns of catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX), superoxide dismutase (SOD) enzyme activity as well as malondialdehyde (MDA) and hydrogen peroxide (H(2)O(2)) production under the stressful conditions tested were very similar between MT and Nr mutant. However, Nr fruits showed increased H(2)O(2) production, reduced and enhanced APX activity in NaCl and CdCl(2), respectively, and enhanced GPOX in NaCl. Moreover, through non-denaturing PAGE, a similar reduction of SOD I band intensity in both, control MT and Nr mutant, treated with NaCl was observed. In leaves and fruits, a similar SOD activity pattern was observed for all periods, genotypes and treatments. Overall the results indicate that the ethylene signaling associated with NR receptor can modulate the biochemical pathways of oxidative stress in a tissue dependent manner, and that this signaling may be different following Na and Cd exposure. (C) 2011 Elsevier B.V. All rights reserved.

The isolation of antioxidant enzymes from mature tomato (cv. Micro-Tom) plants

The activity of catalase (CAT), guaiacol peroxidase (GPOX), ascorbate peroxidase (APX), glutathione reductase (GR), and the isoenzymes of superoxide dismutase (SOD) were determined in the organs of tomato (Lycopersicon esculentum) cultivar Micro-Tom after 104 days of development. The total activities of CAT, GPOX, and GR were higher in the stem than in others tissues, whereas the stem exhibited the lowest APX activity. Activity staining analysis following gel electrophoresis revealed the existence of four SOD isoenzymes in leaves, three in fruits, but only two in the roots and stems. This characterization is essential for an investigation into the effect of abiotic and biotic stresses on the oxidative stress responses by this plant model system.

Effects of the herbicides acetochlor and metolachlor on antioxidant enzymes in soil bacteria

The aim of this study was to investigate the antioxidant responses of three bacteria (SD1. KD and K9) isolated from soil previously treated with the herbicides metolachlor and acetochlor. By 165 rRNA gene sequencing, we determined that SD1 is phylogenetically related to Enterobacter asburiae, while KD and K9 have divergent genomes that more closely resemble that of Enterobacter amnigenus. Decreased levels of lipid peroxidation were observed in SD1 and KD following treatment with 34 mM metolachlor or 62 mM acetochlor, respectively, indicating that both bacteria were able to adapt to an increase in ROS production. In the presence of 34 mM metolachlor or 62 mM acetochlor, all bacterial isolates exhibited increases in total catalase (CAT) activity (81% for SDI, 53% for KD and 59% for K9), whereas total SOD activity (assessed based on the profile and intensity of the bands) was slightly reduced when the bacteria were exposed to high concentrations of the herbicides (340 mM metolachlor or 620 mM acetochlor). This effect was due to a specific reduction in SOD IV (K9 and KD isolates) by 45% and 90%, respectively, and SOD V (SD1 isolate) isoenzymes by 60%. The most striking result was obtained in the SD1 isolate, where two novel isoenzymes of glutathione reductase (GR) that responded specifically to metolachlor were identified. In addition, acetochlor was shown to induce the expression of a new 57 kDa protein band in the K9 and KD isolates. The bacteria isolated from the herbicide-contaminated soil exhibited an efficient antioxidant system response at herbicide concentrations of up to 34 mM metolachlor or 62 mM acetochlor. These data suggest a mechanism for tolerance that may include the control of an imbalance in ROS production versus scavenging. The data suggest that specific isoenzymes of CAT and GR could be involved in this herbicide tolerance mechanism. (C) 2011 Elsevier Ltd. All rights reserved.

Antioxidant response of Nicotiana tabacum cv. Bright Yellow 2 cells to cadmium and nickel stress

Plant cell cultures are a suitable model system for investigation of the physiological mechanisms of tolerance to environmental stress. We have determined the effects of Cd (0.1 and 0.2 mM CdCl(2)) and Ni (0.075 and 0.75 mM NiCl(2)) on Nicotiana tabacum L. cv. Bright Yellow (TBY-2) cell suspension cultures over a 72-h period. Inhibition of growth, loss of cell viability and lipid peroxidation occurred, in general, only when the TBY-2 cells were grown at 0.2 mM CdCl(2) and at 0.75 mM NiCl(2). At 0.1 mM CdCl(2), a significant increase in growth was determined at the end of the experiment. Increases in the activities of all of the four enzymatic antioxidant defence systems tested, were induced by the two concentrations of Cd and Ni, but at different times during the period of metal exposure. Overall, the cellular antioxidant responses to Cd and Ni were similar and were apparently sufficient to avoid oxidative stress at the lower concentrations of Cd and Ni. The activities of glutathione reductase and glutathione S-transferase increased early but transiently, whereas the activities of catalase and guaiacol peroxidase increased in the latter half of the experimental period. Therefore it is likely that the metabolism of reduced glutathione was enhanced during the initial onset of the stress, while catalase and guaiacol-type peroxidase appeared to play a more important role in the antioxidant response once the stress became severe.

Biochemical responses of glyphosate resistant and susceptible soybean plants exposed to glyphosate

Glyphosate is a wide spectrum, non-selective, post-emergence herbicide. It acts on the shikimic acid pathway inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), thus obstructing the synthesis of tryptophan, phenylalanine, tyrosine and other secondary products, leading to plant death. Transgenic glyphosate-resistant (GR) soybean [Glycine max (L.)] expressing an glyphosate-insensitive EPSPS enzyme has provided new opportunities for weed control in soybean production. The effect of glyphosate application on chlorophyll level, lipid peroxidation, catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GOPX) and superoxide dismutase (SOD) activities, soluble amino acid levels and protein profile, in leaves and roots, was examined in two conventional (non-GR) and two transgenic (GR) soybean. Glyphosate treatment had no significant impact on lipid peroxidation, whilst the chlorophyll content decreased in only one non-GR cultivar. However, there was a significant increase in the levels of soluble amino acid in roots and leaves, more so in non-GR than in GR soybean cultivars. Root CAT activity increased in non-GR cultivars and was not altered in GR cultivars. In leaves, CAT activity was inhibited in one non-GR and one GR cultivar. GOPX activity increased in one GR cultivar and in both non-GR cultivars. Root APX activity increased in one GR cultivar. The soluble protein profiles as assessed by 1-D gel electrophoresis of selected non-GR and GR soybean lines were unaffected by glyphosate treatment. Neither was formation of new isoenzymes of SOD and CAT observed when these lines were treated by glyphosate. The slight oxidative stress generated by glyphosate has no relevance to plant mortality. The potential antioxidant action of soluble amino acids may be responsible for the lack of lipid peroxidation observed. CAT activity in the roots and soluble amino acids in the leaves can be used as indicators of glyphosate resistance.

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro-Tom) plants to cadmium-induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro-Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl(2), respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl(2), whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl(2). Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl(2) after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl(2), but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl(2) when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl(2), whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl(2). The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl(2). However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl(2) from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.

A role for ferritin in the antioxidant system in coffee cell cultures

Iron (Fe) is an essential nutrient for plants, but it can generate oxidative stress at high concentrations. In this study, Coffea arabica L. cell suspension cultures were exposed to excess Fe (60 and 240 mu M) to investigate changes in the gene expression of ferritin and antioxidant enzymes. Iron content accumulated during cell growth, and Western blot analysis showed an increase of ferritin in cells treated with Fe. The expression of two ferritin genes retrieved from the Brazilian coffee EST database was studied. CaFER1, but not CaFER2, transcripts were induced by Fe exposure. Phylogenetic analysis revealed that CaFER1 is not similar to CaFER2 or to any ferritin that has been characterised in detail. The increase in ferritin gene expression was accompanied by an increase in the activity of antioxidant enzymes. Superoxide dismutase, guaiacol peroxidase, catalase, and glutathione reductase activities increased in cells grown in the presence of excess Fe, especially at 60 mu M, while the activity of glutathione S-transferase decreased. These data suggest that Fe induces oxidative stress in coffee cell suspension cultures and that ferritin participates in the antioxidant system to protect cells against oxidative damage. Thus, cellular Fe concentrations must be finely regulated to avoid cellular damage most likely caused by increased oxidative stress induced by Fe. However, transcriptional analyses indicate that ferritin genes are differentially controlled, as only CaFER1 expression was responsive to Fe treatment.

Cultivation of hitherto-uncultured bacteria belonging to the Verrucomicrobia subdivision 1 from the potato (Solanum tuberosum L.) rhizosphere

The role of dominant bacterial groups in the plant rhizosphere, e.g., those belonging to the phyla Acidobacteria and Verrucomicrobia, has, so far, not been elucidated, and this is mainly due to the lack of culturable representatives. This study aimed to isolate hitherto-uncultured bacteria from the potato rhizosphere by a combination of cultivation approaches. An agar medium low in carbon availability (oligotrophic agar medium) and either amended with potato root exudates or catalase or left unamended was used with the aim to improve the culturability of bacteria from the potato rhizosphere. The colony forming unit numbers based on colonies and microcolonies were compared with microscopically determined fluorescence-stained cell numbers. Taxonomical diversity of the colonies was compared with that of library clones made from rhizosphere DNA, on the basis of 16S rRNA gene comparisons. The oligotrophic media amended or not with catalase or rhizosphere extract recovered up to 33.6% of the total bacterial numbers, at least seven times more than the recovery observed on R2A. Four hitherto-uncultured Verrucomicrobia subdivision 1 representatives were recovered on agar, but representatives of this group were not found in the clone library. The use of oligotrophic medium and its modifications enabled the growth of colony numbers, exceeding those on classical agar media. Also, it led to the isolation of hitherto-uncultured bacteria from the potato rhizosphere. Further improvement in cultivation will certainly result in the recovery of other as-yet-unexplored bacteria from the rhizosphere, making these groups accessible for further investigation, e.g., with respect to their possible interactions with plants.

Effect of free or protein-associated soy isoflavones on the antioxidant status in rats

BACKGROUND: The objective of this study was to investigate the effect of chronic ingestion of free and protein-associated soy isoflavones on the antioxidant status in male Wistar rats. Free isoflavone (iso), protein-associated soy isoflavone (iso + prot) and soy protein (prot) extracts were administered for 30 days by gavage to the rats at a dosage of 1 mg aglycone isoflavones per 200 g body weight, adjusted daily, and the prot group was given the same concentration of soy protein received by the iso + prot group. Antioxidant capacity of plasma, thiobarbituric acid-reactive substance (TBARS) and glutathione (GSH) levels and catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities in plasma, erythrocytes and tissues and gene expression levels in liver and kidney were evaluated. RESULTS: Chronic ingestion of free but not of protein-associated soy isoflavones nor of solely soy protein increased plasma antioxidant capacity and GPx activity in erythrocytes. Soy protein increased CAT activity and gene expression in liver. SOD activity in erythrocytes was increased by all treatments. CONCLUSION: The overall results confirm that dietary soy isoflavones have a positive effect on antioxidant status, enhancing antioxidant capacity of plasma and antioxidant enzymes in various tissues, but the effects are dependent on the form of administration and on a complex mechanism of antioxidant status balance on the organism. (C) 2010 Society of Chemical Industry

Antioxidant status in rats after long-term intake of anthocyanins and ellagitannins from blackberries

BACKGROUND: This study reported the effects of the daily intake of anthocyanins and ellagitannins (ET) extracted from blackberries on the markers for oxidative status in healthy rats. RESULTS: The phenolic compounds were administered from three different extracts: an aqueous extract of blackberry (BJ) and its two derived fractions: anthocyanin-enriched (AF) and ET-enriched (EF) fractions. After 35 days` administration, the AF and EF extracts significantly reduced thiobarbituric acid reactive substance levels and increased glutathione levels in the liver, kidney and brain. Plasma antioxidant capacity increased only in the group that received AF. Antioxidant enzyme activity and expression did not follow a pattern of response varying according to the tissues and extracts. A significant increase in the catalase activity was observed only in the plasma of the groups administered anthocyanin-containing extracts, which were the BJ and AF groups. Glutathione peroxidase activity was significantly increased in the liver and brain after EF treatment, and the highest increase in its expression was observed in the livers and brains of rats that received AF and EF, respectively. CONCLUSION: The results demonstrate that long-term intake of anthocyanin and ET through diet affects antioxidant enzyme activity and expression, and enhances oxidative markers in healthy rats. (C) 2010 Society of Chemical Industry

Antioxidant Status in Humans after Consumption of Blackberry (Rubus fruticosus L.) Juices With and Without Defatted Milk

The present study was designed to evaluate the possible effect of the consumption of blackberry juices (BJ) prepared with water (BJW) and defatted milk (BJM) on the plasma antioxidant capacity and the. enzymatic and nonenzymatic antioxidants. A significant (p < 0.05) increase in the ascorbic acid content in the plasma was observed after intake of both BJs. However, no changes were observed in the plasma urate and a-tocopherol levels. An increase on the plasma antioxidant capacity, by ORAC assay, was observed only after consumption of BJW but not statistically significant. Plasma antioxidant capacity had a good positive correlation with ascorbic acid (r = 0.93) and a negative correlation with urate level (r = -0.79). No correlation was observed between antioxidant capacity and total cyanidin or total ellagic acid contents. Further, it was observed that plasma catalase increased following intake of BJ`s. No change was observed on the plasma and erythrocyte CAT and glutathione peroxidase activities. A significant decrease (p < 0.05) in the urinary antioxidant capacity between 1 and 4 h after intake of both BJs was observed. A good correlation was observed between total antioxidant capacity and urate and total cyanidin levels. These results suggested association between anthocyanin levels and CAT and a good correlation between antioxidant capacity and ascorbic acid in the human plasma after intake of BJs. Follow-up studies investigating the antioxidant properties and health benefits are necessary to demonstrate the health benefits of polyphenols.

Effect of n-3 fatty acids and statins on oxidative stress in statin-treated hypercholestorelemic and normocholesterolemic women

Introduction: Although the combination of statins with n-3 fatty acids seems to be beneficial under the lipid profile aspect, there is little information about the interaction of these two compounds on oxidative stress. Objective: Evaluate the interaction between statins and n-3 fatty acids on oxidative stress in women, using a 2(2) factorial design. Methods: Forty-three women participated in this crossover design. They were separated into two groups in which 20 were under statin treatment for more than 6 months, and 23 were normolipidemic. Within each group, half of the patients received capsules containing 2.4 g/day of a mixture of EPA and DHA for 6 weeks, while the other half received a mixture of soya and corn oil. After a period of 90 days of washout, the groups were switched, and received the supplementation for 6 weeks more. Results: Statins reduced serum LDL and increased SOD expression. n-3 fatty acids increased the plasma malondialdehyde and SOD activity but reduced catalase expression (p < 0.05). The interaction involving statins and n-3 fatty acids was nearly significant to the serum triacylglycerol reduction (p = 0.054). Conclusion: Combining statins and n-3 fatty acids is an excellent strategy to reduce plasma cholesterol and triacylglycerol concentration in women. However, n-3 fatty acids increased the oxidative stress and the pleiotropic effect of statins seemed to be not enough to counterbalance this result. Our data also suggested that the mechanism by which n-3 fatty acids interfere in oxidative stress can be associated with antioxidant enzymes expression and activity. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

On the mechanisms of phenothiazine-induced mitochondrial permeability transition: Thiol oxidation, strict Ca(2+) dependence, and cyt c release

Phenothiazines (PTZ) are drugs widely used in the treatment of schizophrenia. Trifluoperazine, a piperazinic PTZ derivative, has been described as inhibitor of the mitochondrial permeability transition (MPT). We reported previously the antioxidant activity of thioridazine at relatively low concentrations associated to the inhibition of the MPT (Brit. J. Pharmacol., 2002;136:136-142). In this study, it was investigated the induction of MPT by PTZ derivatives at concentrations higher than 10 mu M focusing on the molecular mechanism involved. PTZ promoted a dose-response mitochondrial swelling accompanied by mitochondrial transmembrane potential dissipation and calcium release, being thioridazine the most potent derivative. PTZ-induced MPT was partially inhibited by CsA or Mg(2+) and completely abolished by the abstraction of calcium. The oxidation of reduced thiol group of mitochondrial membrane proteins by PTZ was upstream the VIP opening and it was not sufficient to promote the opening of PTP that only occurred when calcium was present in the mitochondrial matrix. EPR experiments using DMPO as spin trapping excluded the participation of reactive oxygen species on the PTZ-induced MPT. Since 117 give rise to cation radicals chemically by the action of peroxidases and cyanide inhibited the PTZ-induced swelling, we propose that VIZ bury in the inner mitochondrial membrane and the chemically generated 117 cation radicals modify specific thiol groups that in the presence of Ca(2+) result in MPT associated to cytochrome c release. These findings contribute for the understanding of mechanisms of MET induction and may have implications for the cell death induced by PTZ. (C) 2010 Elsevier Inc. All rights reserved.