Protection of human erythrocytes against oxidative stress by berries

Berries contain several bioactive compounds that can protect against oxidative stress. In this study we evaluated the protective effect of different sequential extracts (ethyl acetate, ethanol and water) of seven berry species: bilberry (Vaccinium myrtillus), blackcurrant (Ribes nigrum), elderberry (Sambucus nigra), lingonberry (Vaccinium vitis-idaea), rose hips (Rosa sp.), sea buckthorn (Hippohae rhamnoides) and strawberry (Fragaria × ananassa). The protective effect was tested on human erythrocytes and the antioxidant capacity was also evaluated in vitro by the FRAP assay. In the erythrocyte assay all sea buckthorn extracts were superior in antioxidant effect to other berry extracts. The ethyl acetate extract of bilberries, and the ethanol and water extracts of blackcurrants, also protected the erythrocytes from oxidation. In contrast, water extracts of rose hips, bilberries and strawberries had a pro-oxidant effect on erythrocytes. The water extract of rose hips was superior to the other berry extracts in the FRAP assay. Thus, the results of the erythrocyte assay did not correlate with the results of the FRAP assay, but provided additional insights into the potential protective effects of berry extracts against oxidative stress. © 2012 - IOS Press and the authors. All rights reserved.

Revealing 4-hydroxynonenal-histidine adducts as qualitative and quantitative biomarker of oxidative stress, lipid peroxidation and oxidative homeostasis

Findings on growth regulating activities of the end-product of lipid peroxidation 4-hydroxy-2-nonenal (HNE), which acts as a “second messenger of free radicals”, overlapped with the development of antibodies specific for the aldehyde-protein adducts. These led to qualitative immunochemical determinations of the HNE presence in various pathophysiological processes and to the change of consideration of the aldehyde’s bioactivities from toxicity into cell signalling. Moreover, findings of the HNE-protein adduct in various organs under physiological circumstances support the concept of “oxidative homeostasis”, which implies that oxidative stress and lipid peroxidation are not only pathological but also physiological processes. Reactive aldehydes, at least HNE, could play important role in oxidative homeostasis, while complementary research approaches might reveal the relevance of the aldehydic-protein adducts as major biomarkers of oxidative stress, lipid peroxidation and oxidative homeostasis. Aiming to join efforts in such research activities researchers interacting through the International 4-Hydroxynonenal Club acting within the SFRR-International and through networking projects of the system of the European Cooperation in Science and Technology (COST) carried validation of the methods for lipid peroxidation and further developed the genuine 4-HNE-His ELISA founding quantitative and qualitative methods for detection of 4-HNE-His adducts as valuable tool to study oxidative stress and lipid peroxidation in cell cultures, various organs and tissues and eventually for human plasma and serum analyses [1]. Reference: 1. Weber, Daniela. Lidija, Milkovic. Measurement of HNE-protein adducts in human plasma and serum by ELISA—Comparison of two primary antibodies. Redox Biol. 2013. 226-233.

The Nrf2-ARE activation protect neurones against oxidative stress

Oxidative stress has been implicated in the pathogenesis of many neurodegenerative diseases including Alzheimer’s disease. The transcription factor, Nrf2 (nuclear factor E2-related factor 2) that binds to the antioxidant responsive element (ARE) activates a battery of genes encoding enzymes and factors essential for neuronal survival. We have investigated the hypothesis that a downstream product of cyclooxygenase(COX-2), 15-deoxy-delta (12, 14)-prostagland in J2 (15d-PGJ2) has protective effects by activating the Nrf2 pathway during oxidative stress.Human neuroblastoma cells (SHSY5Y) were differentiated intoneuronal-like cells as described previously (Gimenez-Cassina et al.,2006). SHSY5Y cells were co-treated with 10 mM buthionine sulfoximine (BSO) 7 10 mM 15d-PGJ2. Cell viability was measured by MTT assay and cellular glutathione (GSH) levels were measured after treating cells for0.5-24 hours by GSH recycling assay. Cellular Nrf2 levels were determined by immunoblotting. IL-6 levels were measured by ELISA.15d-PGJ2 alone lowered GSH levels 30min after the treatment(12.870.64 nmol/mg protein) and returned to untreated control levels at 16hours (28.173.6 nmol/mg protein; Po0.01). Compared to intracellular GSH levels in untreated cells (27.871.8 nmol/mg protein) BSO treatment alone significantly decreased GSH (9.672.1 nmol/mg protein;Po0.001) but co-incubation with 15d-PGJ2 for 24 hours prevented the depletion elicited by BSO(21.372.7 nmol/mg protein). Compared to untreated cells BSO treatment decrease dIL-6 secretion (from 0.941.6ng/ml to 0.6971.3ng/ml) and total Nrf2 protein levels (by21%). Co-incubation with15d-PGJ2 for 24 hours with BSO did not change IL-6(0.6771.4ng/ml) or total Nrf2 level at any time point. This study suggests that neuronal toxicity resulting from glutathione depletion canbere stored by the induction of Nrf2-ARE pathway and the role of the Nrf2 signalling merits further investigation in neurodegenerative diseases.

Mechanism of action of a tumour derived lipid mobilising factor

Cancer cachexia comprises unintentional and debilitating weight loss associated with certain tumour types. Fat loss in cachexia is mediated by a 43kDa Lipid Mobilising Factor (LMF) sharing homology with endogenous Zinc-α2-Glycoprotein (ZAG). LMF and ZAG induced significant lipolysis in isolated epidydimal adipose tissue. This is attenuated by co-incubation with 10μM of antagonist SR59230A and partially attenuated by 25μM PD098059 (indicating β3-AR and MAPK involvement respectively). LMF/ZAG induced in vitro lipid depletion in differentiated 3T3-L1 adipocytes that seen to comprise a significant increase in lipolysis (p<0.01), with only a modest decrease in lipid synthesis (p=0.09). ZAG significantly increased in vitro protein synthesis (p<0.01) in C2C12 myotubes (without an effect on protein degradation). This increase was activated at transcription and attenuated by co-incubation with 10μM SR59230A. Proteolytic digestion of ZAG and LMF followed by sephadex G50 chromatography yielded active fragments of 6-15kDa, indication the entire molecule was not required for bioactivity. Cachexigenic MAC16 cells demonstrated significant in vitro ZAG expression over non-cachexigenic MAC13 cells (p<0.001). WAT and BAT excised from MAC16 mice of varying weight loss demonstrated increased ZAG expression compared to controls. Dosing of NMRI mice with s/c ZAG failed to reproduce this up-regulation, thus another cachectic factor is responsible. 0.58nM LMF conferred significant protection against hydrogen peroxide, paraquat and bleomycin-induced oxidative stress in the non-cachexigenic MAC13 cell line. This protection was attenuated by 10μM SR59230A indicating a β3-AR mediated effect. In addition, 0.58nM LMF significantly up regulated UCP2 expression (p<0.001), (a mitochondrial protein implicated in the detoxification of ROS) implying this to be the mechanism by which survival was achieved. In vitro, LMF caused significant up-regulation of UCP1 in BAT and UCP2 and 3 in C2C12 myotubes. This increase in uncoupling protein expression further potentiates the negative energy balance and wasting observed in cachexia.

Evidence for sensitisation of DNA to oxidative damage during isolation

The oxidative base lesion 8-oxo-deoxyguanosine (8-oxo-dG) has been identified in DNA isolated from normal tissue and may occur at elevated levels during disease. However, the use of phenol during DNA extraction may artificially elevate the detected levels of this lesion. Herein, we have performed a comparative methodological study using both pronase E and phenol extraction techniques; native or oxidatively stressed DNA was isolated to determine the validity of each extraction technique for the subsequent determination of 8-oxo-dG. Whilst the yields of DNA were comparable, after pronase E extraction there was no detectable induction of 8-oxo-dG in reextracted naked DNA or peripheral blood mononuclear cell DNA that had been oxidatively stressed. However, phenol extraction enhanced the basal levels of 8-oxo-dG detected, and also induced a significant increase in levels of the modified base after exposure to oxidative stress. The latter was dependent on the presence of foetal calf serum in the extracellular medium. We have confirmed that phenol extraction sensitises native DNA to subsequent oxidative damage. In addition, this work shows that the extent of sensitisation occurring during phenol extraction varies with the degree of oxidative damage already incurred and infers that labile guanine sites generated during oxidative stress may be detected as 8-oxo-dG residues after phenol extraction.

Tissue transglutaminase and the stress response

The expression of the protein crosslinking enzyme tissue transglutaminase (TG2, tTG), the ubiquitous member of transglutaminase family, can be regulated by multiple factors. Although it has been suggested that TG2 can be involved in apoptotic cell death, high levels of enzyme have also been associated with cell survival in response to different stimuli. Furthermore, evidence indicates that increases in TG2 production cause enzyme translocation to cell membrane. Cell stress can also lead to TG2 accumulation on the cell surface and in the extracellular matrix resulting in changes in cell-matrix interactions. Here, we discuss the underlying mechanisms of TG2 up-regulation induced by various stimuli including glutamate exposure, calcium influx, oxidative stress, UV, and inflammatory cytokines. These findings agree with a postulated role for transglutaminases in molecular mechanisms involved in several diseases suggesting that cross-linking reactions could be a relevant part of the biochemical changes observed in pathological conditions. © 2007 Springer-Verlag.

Aβ(1-42) induced metabolic effects in a stem cell derived neuron and astrocyte network.

Alzheimer’s Disease (AD) is the most common form of dementia currently affecting more than 35 million people worldwide. Hypometabolism is a major feature of AD and appears decades before cognitive decline and pathological lesions. This has a detrimental impact on the brain which has a high energy demand. Current models of AD fail to mimic all the features of the disease, which has an impact on the development of new therapies. Human stem cell derived models of the brain have attracted a lot of attention in recent years as a tool to study neurodegenerative diseases. In this thesis, neurons and astrocytes derived from the human embryonal carcinoma cell line (NT2/D1) were utilised to determine the metabolic coupling between neurons and astrocytes with regards to responses to hypoglycaemia, neuromodulators and increase in neuronal activity. This model was then used to investigate the effects of Aß(1-42) on the metabolism of these NT2-derived co-cultures as well as pure astrocytes. Additionally primary cortical mixed neuronal and glial cultures were utilised to compare this model to a widely accepted in vitro model used in Alzheimer’s disease research. Co-cultures were found to respond to Aß(1-42) in similar way to human and in vivo models. Hypometabolism was characterised by changes in glucose metabolism, as well as lactate, pyruvate and glycogen. This led to a significant decrease in ATP and the ratio of NAD+/NADH. These results together with an increase in calcium oscillations and a decrease in GSH/GSSG ratio, suggests Aß-induces metabolic and oxidative stress. This situation could have detrimental effects in the brain which has a high energy demand, especially in terms of memory formation and antioxidant capacity.

Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells:role of ERK1/2 in H2O2-induced cell death

Reactive oxygen species including H2O2 activate an array of intracellular signalling cascades that are closely associated with cell death and cell survival pathways. The human neuroblastoma SH-SY5Y cell line is widely used as model cell system for studying neuronal cell death induced by oxidative stress. However, at present very little is known about the signalling pathways activated by H2O2 in SH-SY5Y cells. Therefore, in this study we have investigated the effect of H2(O2 on extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (PKB) activation in undifferentiated and differentiated SH-SY5Y cells. H2O2 stimulated time and concentration increases in ERK1/2, JNK and PKB phosphorylation in undifferentiated and differentiated SH-SY5Y cells. No increases in p38 MAPK phosphorylation were observed following H2O2 treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 ((2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited H2O2-induced increases in ERK1/2 and PKB phosphorylation. Furthermore, H2O2-mediated increases in ERK1/2 activation were sensitive to the MAPK kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK responses were blocked by the JNK inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Treatment of SH-SY5Y cells with H2O2 (1 mM; 16 h) significantly increased the release of lactate dehydrogenase (LDH) into the culture medium indicative of a decrease in cell viability. Pre-treatment with wortmannin, SP 600125 or SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; p38 MAPK inhibitor) had no effect on H2O2-induced LDH release from undifferentiated or differentiated SH-SY5Y cells. In contrast, PD 98059 and LY 294002 significantly decreased H2O2-induced cell death in both undifferentiated and differentiated SH-SY5Y cells. In conclusion, we have shown that H2O2 stimulates robust increases in ERK1/2, JNK and PKB in undifferentiated and differentiated SH-SY5Y cells. Furthermore, the data presented clearly suggest that inhibition of the ERK1/2 pathway protects SH-SY5Y cells from H2O2-induced cell death.

In vitro protective effect and antioxidant mechanism of Resveratrol induced by Dapsone Hydroxylamine in human cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDSNHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10-1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.