Linking molecular and population stress responses in Daphnia magna exposed to cadmium

DNA microarrays can be used to measure environmental stress responses. If they are to be predictive of environmental impact, we need to determine if altered gene expression translates into negative impacts on individuals and populations. A large cDNA microarray (14000 spots) was created to measure molecular stress responses to cadmium in Daphnia magna,the most widely used aquatic indicator species, and relate responses to population growth rate (pgr). We used the array to detect differences in the transcription of genes in juvenile D. magna (24 h old) after 24 h exposure to a control and three cadmium concentrations (6, 20, and 37 mu g Cd2+ L-1). Stress responses at the population level were estimated following a further 8 days exposure. Pgr was approximately linear negative with increasing cadmium concentration over this range. The microarray profile of gene expression in response to acute cadmium exposure begins to provide an overview of the molecular responses of D. magna, especially in relation to growth and development. Of the responding genes, 29% were involved with metabolism including carbohydrate, fat and peptide metabolism, and energy production, 31% were involved with transcription/translation, while 40% of responding genes were associated with cellular processes like growth and moulting, ion transport, and general stress responses (which included oxidative stress). Our production and application of a large Daphnia magna microarray has shown that measured gene responses can be logically linked to the impact of a toxicant such as cadmium on somatic growth and development, and consequently pgr.

Common variants of apolipoprotein A-IV differ in their ability to inhibit low density lipoprotein oxidation

Apolipoprotein A-IV (apoA-IV) inhibits lipid peroxidation, thus demonstrating potential anti-atherogenic properties. The aim of this study was to investigate how the inhibition of low density lipoprotein (LDL) oxidation was influenced by common apoA-IV isoforms. Recombinant wild type apoA-IV (100 mu g/ml) significantly inhibited the oxidation of LDL (50 mu g protein/ml) by 5 mu M CuSO4 (P < 0.005), but not by 100 mu M CuSO4, suggesting that it may act by binding copper ions. ApoA-IV also inhibited the oxidation of LDL by the water-soluble free-radical generator 2,2'-azobis(amidinopropane) dihydrochloride (AAPH; I mM), as shown by the two-fold increase in the time for half maximal conjugated diene formation (T-1/2; P < 0.05) suggesting it can also scavenge free radicals in the aqueous phase. Compared to wild type apoA-IV, apoA-IV-S347 decreased T-1/2 by 15% (P = 0.036) and apoA-IV-H360 increased T-1/2 by 18% (P = 0.046). All apoA-IV isoforms increased the relative electrophoretic mobility of native LDL, suggesting apoA-IV can bind to LDL and acts as a site-specific antioxidant. The reduced inhibition of LDL oxidation by apoA-IV-S347 compared to wild type apoA-IV may account for the previous association of the APOA4 S347 variant with increased CHD risk and oxidative stress. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Ascorbate does not protect macrophages against apoptosis induced by oxidised low density lipoprotein

Apoptosis of macrophages and smooth muscle cells is observed in atherosclerotic lesions and may play an important role in the disease progression. Oxidised low density lipoprotein (LDL) is cytotoxic and induces apoptosis in a variety of cell types. We reported previously that ascorbate protects arterial smooth muscle cells from apoptosis induced by oxidised LDL containing the peak levels of lipid hydroperoxides. We now demonstrate that macrophages undergo apoptosis when treated with this species of oxidised LDL, as detected by increased annexin V binding and DNA fragmentation. Ascorbate treatment of macrophages did not protect against the cytotoxicity of oxidised LDL, and modestly increased the levels of annexin V binding and DNA fragmentation. Oxidised LDL treatment also increased the expression of the antioxidant stress protein heme oxygenase-1 in macrophages; however, this increase was markedly attenuated by ascorbate pretreatment. Although apoptosis induced by oxidised LDL was modestly promoted by ascorbate, ascorbate apparently decreased the levels of oxidative stress in macrophages, suggesting that this pro-apoptotic effect was not mediated by a pro-oxidant mechanism, but may instead have been due to intracellular protection of the apoptotic machinery by ascorbate. (c) 2006 Elsevier Inc. All rights reserved.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes, Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye (TM) and analysed by two-dimensional difference gel. electrophoresis. Gel images analysed off-line, using the DeCyder (TM) image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Role of oxygen radicals in DNA damage and cancer incidence

The development of cancer in humans and animals is a multistep process. The complex series of cellular and molecular changes participating in cancer development are mediated by a diversity of endogenous and exogenous stimuli. One type of endogenous damage is that arising from intermediates of oxygen (dioxygen) reduction - oxygen-free radicals (OFR), which attacks not only the bases but also the deoxyribosyl backbone of DNA. Thanks to improvements in analytical techniques, a major achievement in the understanding of carcinogenesis in the past two decades has been the identification and quantification of various adducts of OFR with DNA. OFR are also known to attack other cellular components such as lipids, leaving behind reactive species that in turn can couple to DNA bases. Endogenous DNA lesions are genotoxic and induce mutations. The most extensively studied lesion is the formation of 8-OH-dG. This lesion is important because it is relatively easily formed and is mutagenic and therefore is a potential biomarker of carcinogenesis. Mutations that may arise from formation of 8-OH-dG involve GC. TA transversions. In view of these findings, OFR are considered as an important class of carcinogens. The effect of OFR is balanced by the antioxidant action of non-enzymatic antioxidants as well as antioxidant enzymes. Non-enzymatic antioxidants involve vitamin C, vitamin E, carotenoids (CAR), selenium and others. However, under certain conditions, some antioxidants can also exhibit a pro-oxidant mechanism of action. For example, beta-carotene at high concentration and with increased partial pressure of dioxygen is known to behave as a pro-oxidant. Some concerns have also been raised over the potentially deleterious transition metal ion-mediated (iron, copper) pro-oxidant effect of vitamin C. Clinical studies mapping the effect of preventive antioxidants have shown surprisingly little or no effect on cancer incidence. The epidemiological trials together with in vitro experiments suggest that the optimal approach is to reduce endogenous and exogenous sources of oxidative stress, rather than increase intake of anti-oxidants. In this review, we highlight some major achievements in the study of DNA damage caused by OFR and the role in carcinogenesis played by oxidatively damaged DNA. The protective effect of antioxidants against free radicals is also discussed.

A randomised clinical trial to assess the effect of total enteral and total parenteral nutritional support on metabolic, inflammatory and oxidative markers in patients with predicted severe acute pancreatitis (APACHE II >= 66)

Background: Total enteral nutrition (TEN) within 48 h of admission has recently been shown to be safe and efficacious as part of the management of severe acute pancreatitis. Our aim was to ascertain the safety of immediate TEN in these patients and the effect of TEN on systemic inflammation, psychological state, oxidative stress, plasma glutamine levels and endotoxaemia. Methods: Patients admitted with predicted severe acute pancreatitis (APACHE II score 15) were randomised to total enteral (TEN; n = 8) or total parenteral nutrition (TPN; n = 9). Measurements of systemic inflammation (C-reactive protein), fatigue ( visual analogue scale), oxidative stress ( plasma thiobarbituric acid- reactive substances), plasma glutamine and anti-endotoxin IgG and IgM antibody concentrations were made on admission and repeated on days 3 and 7 thereafter. Clinical progress was monitored using APACHE II score. Organ failure and complications were recorded. Results: All patients tolerated the feeding regime well with few nutrition-related complications. Fatigue improved in both groups but more rapidly in the TEN group. Oxidative stress was high on admission and rose by similar amounts in both groups. Plasma glutamine concentrations did not change significantly in either group. In the TPN group, 3 patients developed respiratory failure and 3 developed non-respiratory single organ failure. There were no such complications in the TEN group. Hospital stay was shorter in the TEN group [ 7 (4-14) vs. 10 (7-26) days; p = 0.05] as was time to passing flatus and time to opening bowels [1 (0-2) vs. 2 (1-5) days; p = 0.01]. The cost of TEN was considerably less than of TPN. Conclusion: Immediate institution of nutritional support in the form of TEN is safe in predicted severe acute pancreatitis. It is as safe and as efficacious as TPN and may be beneficial in the clinical course of this disease. Copyright (C) 2003 S. Karger AG, Basel and IAP.

Effects of chronic and acute fruit and vegetable juice consumption on cardiovascular disease risk factors

Diets low in fruit and vegetables are reportedly responsible for 2.7 million deaths annually from cardiovascular diseases (CVD) and certain cancers. A daily fruit and vegetable intake of five 80 g portions is recommended for chronic disease prevention. However, in the UK, average adult consumption is less than three portions. It is suggested that fruit juice should only count as one portion. However, fruit juices are a beneficial source of phytochemicals. The preliminary results of two randomized, controlled, crossover, dietary intervention studies investigating the effects of chronic and acute consumption of fruit and vegetable puree and juice based drinks (FVPJ) on bioavailability, antioxidant status, vascular reactivity, and risk factors for CVD are reported. In the first study, 39 volunteers consumed 200 ml FVPJ, or fruit-flavoured control, daily for six weeks. In the second study, 24 volunteers consumed 400 mL FVPJ, or sugar-matched control, on the morning of the study day. Blood and urine samples were collected throughout both studies and real-time measurements of vascular tone were performed using laser Doppler imaging with iontophoresis. Overall, the studies showed that the fruit and vegetable puree and juice based drink increased dietary phytochemicals. There was a trend towards increased vasodilation following both acute and chronic fruit juice consumption. Measurements of antioxidant status, oxidative stress and other cardiovascular disease risk factors are currently being determined.

Effects of chronic and acute consumption of fruit- and vegetable-puree-based drinks on vasodilation, risk factors for CVD and the response as a result of the eNOS G298T polymorphism

The average UK adult consumes less than three portions of fruit and vegetables daily, despite evidence to suggest that consuming five portions daily could help prevent chronic diseases. It is recommended that fruit juice should only count as one of these portions, as juicing removes fibre and releases sugars. However, fruit juices contain beneficial compounds such as vitamin C and flavonoids and could be a useful source of dietary phytochemicals. Two randomised controlled cross-over intervention studies investigating the effects of chronic and acute consumption of commercially-available fruit- and vegetable-puree-based drinks (FVPD) on bioavailability, antioxidant status and CVD risk factors are described. Blood and urine samples were collected during both studies and vascular tone was measured using laser Doppler imaging. In the chronic intervention study FVPD consumption was found to significantly increase dietary carotenoids (P = 0.001) and vitamin C (P = 0.003). Plasma carotenoids were increased (P = 0.001), but the increase in plasma vitamin C was not significant. There were no significant effects on oxidative stress, antioxidant status and other CVD risk factors. In the acute intervention study FVPD were found to increase total plasma nitrate and nitrite (P = 0.001) and plasma vitamin C (P = 0.002). There was no effect on plasma lipids or uric acid, but there was a lower glucose and insulin peak concentration after consumption of the FVPD compared with the sugar-matched control. There was a trend towards increased vasodilation following both chronic and acute FVPD consumption. All volunteers were retrospectively genotyped for the eNOS G298T polymorphism and the effect of genotype on the measurements is discussed. Overall, there was a non-significant trend towards increased endothelium-dependent vasodilation following both acute and chronic FVPD consumption. However, there was a significant time x treatment effect (P < 0.05) of acute FVPD consumption in individuals with the GG variant of the eNOS gene.

Age-associated changes in protein oxidation and proteasome activities in rat brain: modulation by antioxidants

The free radical theory of ageing postulates that age-associated neurodegeneration is caused by an imbalance between pro-oxidants and antioxidants resulting in oxidative stress. The current study showed regional variation in brain susceptibility to age-associated oxidative stress as shown by increased lipofuscin deposition and protein carbonyl levels in male rats of age 15-16 months compared to control ones (3-5 months). The hippocampus is the area most vulnerable to change compared to the cortex and cerebellum. However, proteasomal enzyme activity was not affected by age in any of the brain regions studied. Treatment with melatonin or coenzyme Q10 for 4 weeks reduced the lipofuscin content of the hippocampus and carbonyl level. However, both melatonin and coenzyme Q10 treatments inhibited beta-glutamyl peptide hydrolase activity. This suggests that these molecules can alter proteasome function independently of their antioxidant actions. (c) 2005 Elsevier Inc. All rights reserved.

Role of rpoS in the Development of Cell Envelope Resilience and Pressure Resistance in Stationary-Phase Escherichia coli

This work investigated the role of rpoS in the development of increased cell envelope resilience and enhanced pressure resistance in stationary phase cells of Escherichia coli. Loss of both colony-forming ability and membrane integrity, measured as uptake of propidium iodide (PI), occurred at lower pressures in E. coli BW3709 (rpoS) than in the parental strain (BW2952). The rpoS mutant also released much higher concentrations of protein under pressure than the parent. We propose that RpoS-regulated functions are responsible for the increase in membrane resilience as cells enter stationary phase and that this plays a major role in the development of pressure resistance. Strains from the Keio collection with mutations in two RpoS-regulated genes, cfa (cyclopropane fatty acyl phospholipid synthase) and osmB (outer membrane lipoprotein), were significantly more pressure-sensitive and took up more PI than the parent strains with cfa having the greatest effect. Mutations in the bolA morphogene and other RpoS-regulated lipoprotein genes (osmC, osmE, osmY and ybaY) had no effect on pressure resistance. The cytoplasmic membranes of the rpoS mutant failed to reseal after pressure treatment and strains with mutations in osmB and nlpI (new lipoprotein) were also somewhat impaired in the ability to reseal their membranes. The cfa mutant, though pressure-sensitive, was unaffected in membrane resealing implying that the initial transient permeabilization event is critical for loss of viability rather than the failure to reseal. The enhanced pressure sensitivity of polA, recA and xthA mutants suggested that DNA may be a target of oxidative stress in pressure-treated cells.

Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Hydrogen sulfide (H(2)S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H(2)S in response to pro-inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H(2)S. HACs and MPCs were exposed to IL-1β, IL-6, TNF-α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H(2)S synthesizing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) were determined by Western blot and zinc-trap spectrophotometry, respectively. Cellular oxidative stress was induced by H(2)O(2), the peroxynitrite donor SIN-1 and 4-hydroxynonenal (4-HNE). Cell death was assessed by 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (DCm) was determined in situ by flow cytometry. Endogenous H(2) S synthesis was inhibited by siRNA-mediated knockdown of CSE and CBS and pharmacological inhibitors D,L-propargylglycine and aminoxyacetate, respectively. Exogenous H(2)S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H(2)S in a CBS-dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL-1β, TNF-α, IL-6 or LPS. Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment. These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs. H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

Neuroprotective effects of phenolic antioxidant tBHQ associate with inhibition of FoxO3a nuclear translocation and activity

The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K) – Akt signalling pathways retains FoxO3a in the cytoplasm thereby inhibiting the transcriptional activation of death promoting genes. We hypothesised that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K-Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localisation of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and upregulated Fas ligand expression. In contrast the phenolic antioxidant tBHQ caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a prevented NMDA-induced upregulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration.