Redox Parameters Research Articles

Genetically encoded molecular tools for H 2 O 2 imaging and manipulation

Synthetic biology approaches in modern biosciences allow obtaining living systems with new properties by trans-species and even trans-kingdom transfer of molecular machines. Examples are optogenetics and thermogenetics, genome editing by GRISPR-Cas systems, fluorescent proteins and biosensors. After 200 years from discovery of hydrogen peroxide by Thenard, we now use synthetic biology principles to study functions of H2O2 in biological systems. Tools that we have include genetically encoded fluorescent biosensors for H2O2 based on fluorescent proteins from hydrozoan and anthozoan species and bacterial sensing domains, and metabolic engineering tools that are yeast and prokaryotic enzymes with unique capabilities placed in a context of an animal cell. This allow us to precisely monitor and modulate H2O2 levels inside compartments and sub-compartments of the living cells, in a cell- and tissue-specific way. In my talk I will show how antioxidant systems of the cell maintain engineered intracellular H2O2 gradients and define intercompartment H2O2 traffic. I will also give an overview of several new metabolic engineering tools that allow control over key redox parameters in the living cells.

Redox zonation for different groundwater flow paths during bank filtration: a case study at Liao River, Shenyang, northeastern China

The spatial and temporal distribution of redox zones in an aquifer is important when designing groundwater supply systems. Redox zonation can have direct or indirect control of the biological and chemical reactions and mobility of pollutants. In this study, redox conditions are characterized by interpreting the hydrogeological conditions and water chemistry in groundwater during bank infiltration at a site in Shenyang, northeast China. The relevant redox processes and zonal differences in a shallow flow path and deeper flow path at the field scale were revealed by monitoring the redox parameters and chemistry of groundwater near the Liao River. The results show obvious horizontal and vertical components of redox zones during bank filtration. Variations in the horizontal extent of the redox zone were controlled by the different permeabilities of the riverbed sediments and aquifer with depth. Horizontally, the redox zone was situated within 17 m of the riverbank for the shallow flow path and within 200 m for the deep flow path. The vertical extent of the redox zone was affected by precipitation and seasonal river floods and extended to 10 m below the surface. During bank filtration, iron and manganese oxides or hydroxides were reductively dissolved, and arsenic that was adsorbed onto the medium surface or coprecipitated is released into the groundwater. This leads to increased arsenic content in groundwater, which poses a serious threat to water supply security.

Oxidative effects of the acute exposure to a pesticide mixture of cypermethrin and chlorpyrifos on carp and zebrafish – A comparative study

The use of commercial pesticides combinations increases the risk of intoxication in non-target aquatic organisms. Here, we investigate the potential of a commercial pesticide formulation containing (CYP) plus chlorpyrifos (CPF) to induce oxidative damage on two fish species (common carp and zebrafish). Carp and zebrafish were exposed for 96 h under laboratory conditions. Fish were divided in three different groups: CTL, 0.3 μg L−1 or 0.6 μg L−1 of CYP and 0.5 or 1 μg L−1 of CPF in commercial formulation. Both carp and zebrafish showed an increase in lipid peroxidation (LPO) and glutathione-S-transferase (GST) activity when compared to control group. Other oxidative parameters responded differently to exposure in carp and zebrafish. There were an increase in ascorbic acid (ASA) levels and decrease in catalase (CAT) activity and non-protein thiols (NPSH) levels in treated groups of carps. In the other hand, zebrafish showed significant decrease in ASA and increase in CAT activity and NPSH levels. Overall, we demonstrate noxious effects on redox parameters in two fish experimental models and different effects were observe in each fish species exposed to commercial pesticide formulation. This difference responses observed can be related with specific mechanisms of detoxification and antioxidant defense system of each species.

Green synthesis of silver nanoparticles using Stevia leaves extracts

Three extracts of Stevia rebaudiana (Bertoni) were prepared using different types of raw materials: leaves of plants grown ex situ, leaves of plants grown in vitro, callus culture formed on damaged leaves. Composition of the extracts was studied by means of high-performance liquid chromatography and laser desorption/ionization mass spectrometry; total phenol content was estimated using Folin–Ciocalteau method. Flavonoids and hydroxycinnamic acids were found to be the main groups of phenol antioxidants available in the Stevia leaves, with the amount of these compounds in the extract being dependent on the type of raw material. The reducing properties of phenol compounds identified in the extracts were characterized using quantum chemical method; flavonoids and hydroxycinnamic acids were found to have similar redox parameters. Silver nanoparticles (AgNPs) colloids were synthesized using three Stevia extracts; AgNPs size distribution were characterized by means of scanning electron microscopy. All the extracts revealed significant activity in AgNPs synthesis; the nanoparticles of predominantly spherical shape with the average sizes of 16–25 nm were formed. The reducing properties of the extracts were found to correlate with total phenol content; the activity of extracts from the leaves of plants grown ex situ and from callus culture in Ag+ ions reduction was similar to each other and exceeded the activity of extract from the leaves of plants grown in vitro.

A Biochemical Approach to Detect Oxidative Stress in Infertile Women Undergoing Assisted Reproductive Technology Procedures.

Oxidative stress plays a major role in critical biological processes in human reproduction. However, a reliable and biologically accurate indicator of this condition does not yet exist. On these bases, the aim of this study was to assess and compare the blood and follicular fluid (FF) redox status of 45 infertile subjects (and 45 age-matched controls) undergoing in vitro fertilization (IVF), and explore possible relationships between the assessed redox parameters and IVF outcomes. Reactive Oxygen Species (ROS) production, assessed by flow cytometry analysis in blood leukocytes and granulosa cells, significantly increased (p < 0.05) in infertile patients. Also, oxidative stress markers—ThioBarbituric Acid-Reactive Substances (TBARS) as an index of lipid peroxidation, and Oxygen Radical Absorbance Capacity (ORAC) to account for total antioxidant capacity, both assayed by fluorometric procedures—in blood and FF were significantly (p < 0.001) modified in infertile patients compared to the control group. Moreover, a significant correlation between blood redox markers and FF redox markers was evident. An ORAC/TBARS ratio, defined as the redox index (RI), was obtained in the plasma and FF of the patients and controls. In the patients, the plasma RI was about 3.4-fold (p < 0.0001) lower than the control, and the FF RI was about six-fold (p < 0.0001) lower than the control. Interestingly, both the plasma RI and FF RI results were significantly correlated (p < 0.05) to the considered outcome parameters (metaphase II, fertilization rate, and ongoing pregnancies). Given the reported findings, a strict monitoring of redox parameters in assisted reproductive techniques and infertility management is recommended.

Antioxidant defense capacity of ovarian tissue after vitrification in a metal closed system.

ObjectiveThe present study analyzed the quality of bovine ovarian tissue after vitrification in a metal closed chamber, in terms of putative changes in tissue viability (lactate dehydrogenase -LDH- release), anti-oxidant defenses, and redox parameters caused by cryopreservation.MethodsSmall and large fragmented bovine ovarian tissue specimens were vitrified in a metal chamber. After rewarming, tissue samples were fixed or cultured for 48 hours. Glutathione (GSH), protein sulfhydryl content, Total Radical Trapping Antioxidant Potential (TRAP), and lactate dehydrogenase were analyzed immediately after rewarming and after tissue culture.ResultsNo changes in antioxidant parameters or viability of rewarmed tissue samples were found immediately or 48h after vitrification. The method of vitrification in a metal closed chamber used in this study preserved the quality of bovine ovarian tissue. Furthermore, our data showed that the size of the tissue specimens did not affect post-vitrification biochemical viability parameters.ConclusionsWe believe that the vitrification methodology employed in the present study is safe and effective, and should be evaluated for use in humans.

Proapoptotic and Antimigratory Effects of Pseudevernia furfuracea and Platismatia glauca on Colon Cancer Cell Lines.

SUMMARYThe aim of this study is to investigate cytotoxic, proapoptotic, antimigratory and pro-antioxidant effects of methanol, acetone and ethyl acetate extracts of lichens Pseudevernia furfuracea and Platismatia glauca on colorectal cancer (HCT-116 and SW-480) cell lines. We compared the cytotoxic effects on colorectal cancer cells with the effects obtained from normal human fibroblast (MRC-5) cell line. Tetrazolium (MTT) test evaluated the cytotoxic effects, Transwell assay evaluated cell migration, acridine orange/ethidium bromide (AO/EB) fluorescent method followed the apoptosis, while prooxidant/antioxidant effects were determined spectrophotometrically through concentration of redox parameters. The tested extracts showed considerable cytotoxic effect on cancer cells with no observable cytotoxic effect on normal cells. Ethyl acetate and acetone extract of P. furfuracea induced the highest cytotoxicity (IC50=(21.2±1.3) µg/mL on HCT-116, and IC50=(51.3±0.8) µg/mL on SW-480 cells, respectively, after 72 h), with noteworthy apoptotic and prooxidant effects, and antimigratory potential of methanol extract. P. glauca extracts induced cytotoxic effects on HCT-116 cells after 72 h (IC50<40 μg/mL), while only methanol and acetone extracts had cytotoxic effects on SW-480 cells after 24 h, with proapoptotic/necrotic activity, as a consequence of induced oxidative stress. In conclusion, lichen extracts changed to a great extent cell viability and migratory potential of colorectal cancer cell lines. HCT-116 cells were more sensitive to treatments, P. furfuracea had better proapoptotic and antimigratory effects, and both investigated lichen species might be a source of substances with anticancer activity.

Redox status of patients before cardiac surgery

ABSTRACTObjectives: Redox regulation plays a crucial role in balancing the cardiovascular system. In this prospective study we aimed to identify currently unknown correlations valuable to cardiovascular research and patient management.Methods: Blood samples from 500 patients were collected directly before cardiosurgical interventions (Ethics Committee reference number 85/11). Four central redox parameters were determined together with about 30 clinical, anthropometric, and metabolic parameters.Results: Creatinine levels and pulmonary hypertension were significant predictors of the total antioxidant status (TAOS) in the patients; total glutathione levels were linked to C-peptide, and creatinine, gender, and ventricular arrhythmia influenced nitrate/nitrite levels. Notably, significant interactions were found between medication and redox parameters. Calcium channel blockers (CCBs) were positive predictors of total glutathione levels, whereas angiotensin-converting enzyme inhibitors and CCBs were negative predictors of NOx levels. Age showed the highest correlation with the duration of the intensive care stay, followed by NOx levels, creatinine, TAOS, and C-reactive protein.Discussion: In this prospective study we determined multiple correlations between redox markers and parameters linked to cardiovascular diseases. The data point towards so far unknown interdependencies, particularly between antihypertensive drugs and redox metabolism. A thorough follow-up to these data has the potential to improve patient management.Abbreviations: A: absorption; ΔA: absorption difference; ABTS: 2,2′-azino-di(3-ethylbenzothiazoline sulfonate); ACE: angiotensin-converting enzyme; AO: antioxidant; ARB: angiotensin receptor blocker; BMI: body mass index; CAD: coronary artery disease; CCB: calcium channel blocker; CDC: coronary heart diseases; COPD: chronic obstructive pulmonary disease; CRP: C-reactive protein; CVD: cardiovascular diseases; Cu-OOH: cumene hydroperoxide; D: dilution factor; DAN: 2,3-diaminonaphtalene; DMSO: dimethylsulfoxide; DNA: deoxyribonucleic acid; DTNB: 5,5-dithiobis(2-nitrobenzoate); ε: extinction coefficient; EDRF: endothelium-derived relaxing factor; fc: final concentration; GPx: glutathione peroxidases; (h)GR: (human) glutathione reductase; GSH: (reduced) glutathione; GSSG: glutathione disulfide; GST: glutathione-S-transferase; Hb: hemoglobin; HDL: high-density lipoprotein; Hk: hematocrit; H2O2: hydrogen peroxide; ICS: intensive care stay; LDH: lactate dehydrogenase; LDL: low-density lipoprotein; MI: myocardial infarction; NED: N-(1-naphthyl)-ethylendiamine-dihydrochloride; NOS: nitric oxide synthase; NOx: nitrate/nitrite; NR: nitrate reductase; PBS: phosphate buffered saline; PCA: principle component analysis; PH: pulmonary hypertension; ROS: reactive oxygen species; RNS: reactive nitrogen species; RT: room temperature (25°C); SA: sulfanilamide; SOD: superoxide dismutase; SSA: sulfosalicylic acid; TAC: total antioxidant capacity; TAOS: total antioxidant status; TEAC: trolox equivalent antioxidative capacity; TG: triglycerides; tGSH: total glutathione; TNB-: 2-nitro-5-thiobenzoate; U: unit; UV: ultraviolet; VA: volume activity; Wc: working concentration; WHR: waist-hip ratio.

Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD+ ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD+ ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD+ ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD+ ratio, and this increase is mitigated by the presence of NAD+-generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD+ ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD+ ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real-time measurements of the ratio in living, functioning liver cells, overcoming many limitations of previous methods for measuring this important redox parameter. The feasibility of using Peredox in liver slices is particularly attractive because slices allow preservation of hepatic microanatomy and metabolic zonation of hepatocytes.

Coordination, redox properties and SOD activity of Cu(II) complexes of multihistidine peptides

The results of electrochemical and SOD activity measurements of such copper(II) complexes of terminally protected multihistidine peptides that may mimic the active site of CuZnSOD enzyme are submitted and completed with solution equilibrium studies of some copper(II)-ligand systems. The equilibrium data confirm that the thermodynamic stabilities increase with the increasing number of histidyl residues in the amino acid sequence, the stability order, however, can be finely tuned by the number and quality of amino acids between histidine residues. Based on the cyclic voltammetric studies we concluded that the formal reduction potential values of imidazole nitrogen coordinated complexes decrease with the increasing number of imidazole donor atoms in the coordination sphere. However, the redox parameters of [CuH−1L]+ and [CuH−2L] complexes containing amide nitrogen coordination can be determined as well. All formal potential values of [CuL]2+, [CuH−1L]+ and [CuH−2L] complexes fall in the middle potential range of SOD activity. Finally, after the detailed analysis of species distribution curves based upon the equilibrium data SOD activity of copper(II) containing systems at two pH (pH=6.8 and 7.4) were determined. The imidazole coordinated [CuL]2+ complexes of the multihistidine peptide containing the HXH sequence exhibit the most significant activity, but the presence of amide nitrogen coordinated species with slightly distorted geometry could considerably contribute to the SOD activity.

Peripheral Oxidative Stress Biomarkers in Spinocerebellar Ataxia Type 3/Machado-Joseph Disease.

Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a polyglutamine disorder with no current disease-modifying treatment. Conformational changes in mutant ataxin-3 trigger different pathogenic cascades, including reactive oxygen species (ROS) generation; however, the clinical relevance of oxidative stress elements as peripheral biomarkers of SCA3/MJD remains unknown. We aimed to evaluate ROS production and antioxidant defense capacity in symptomatic and presymptomatic SCA3/MJD individuals and correlate these markers with clinical and molecular data with the goal of assessing their properties as disease biomarkers. Molecularly confirmed SCA3/MJD carriers and controls were included in an exploratory case-control study. Serum ROS, measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) as well as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) antioxidant enzyme activities, levels were assessed. Fifty-eight early/moderate stage symptomatic SCA3/MJD, 12 presymptomatic SCA3/MJD, and 47 control individuals were assessed. The DCFH-DA levels in the symptomatic group were 152.82 nmol/mg of protein [95% confidence interval (CI), 82.57-223.08, p < 0.001] higher than in the control and 243.80 nmol/mg of protein (95% CI, 130.64-356.96, p < 0.001) higher than in the presymptomatic group. The SOD activity in the symptomatic group was 3 U/mg of protein (95% CI, 0.015-6.00, p = 0.048) lower than in the presymptomatic group. The GSH-Px activity in the symptomatic group was 13.96 U/mg of protein (95% CI, 5.90-22.03, p < 0.001) lower than in the control group and 20.52 U/mg of protein (95% CI, 6.79-34.24, p < 0.001) lower than in the presymptomatic group and was inversely correlated with the neurological examination score for spinocerebellar ataxias (R = -0.309, p = 0.049). Early/moderate stage SCA3/MJD patients presented a decreased antioxidant capacity and increased ROS generation. GSH-Px activity was the most promising oxidative stress disease biomarker in SCA3/MJD. Further longitudinal studies are necessary to identify both the roles of redox parameters in SCA3/MJD pathophysiology and as surrogate outcomes for clinical trials.

Evaluation of distribution, redox parameters, and genotoxicity in Wistar rats co-exposed to silver and titanium dioxide nanoparticles

ABSTRACTThe increasing production of silver nanoparticles (AgNPs) and titanium dioxide nanoparticles (TiO2NPs) has resulted in their elevated concentrations in the environment. This study was, therefore, aimed at determining the distribution, redox parameters, and genotoxic effects in male Wistar rats that were treated with either AgNP or TiO2NP individually, as well as under a co-exposure scenario. Animals were exposed via oral gavage to either sodium citrate buffer (vehicle), 0.5 mg/kg/day TiO2NP, 0.5 mg/kg/day AgNP or a mixture of TiO2NPs and AgNPs. Exposure lasted 45 days after which rats were sacrificed, and tissue biodistribution of Ag and Ti measured. The blood concentration of glutathione (GSH) and activities of glutathione peroxidase (GPx) and catalase (CAT) were determined while the genotoxicity was analyzed using the comet assay in peripheral blood and liver cells. The tissue concentrations of Ag followed the order; blood > liver > kidneys while for Ti the order was kidneys > liver > blood. There was no significant change in the measured redox parameters in animals that were exposed to TiO2NPs. However, there was a significant increase in GSH levels accompanied by a reduction in the GPx activity in AgNP-treated and co-exposed groups. The individual or co-exposure to TiO2NP and AgNP did not markedly induce genotoxicity in blood or liver cells. Data showed that TiO2NP did not produce significant oxidative stress or genotoxicity in rats at the dose used in this study while the same dose level of AgNPs resulted in oxidative stress, but no noticeable adverse genotoxic effects.

Correlation of Low Birth Weight of Neonates to Placental Levels of Zinc, Copper, Iron, Lipid Peroxidation and Glutathione

This study was conducted to seek association between placental redox (antioxidant/oxidant) parameters and essential metals status (zinc, copper, iron) with birth weight. Placental tissue of pregnant women was collected and levels of zinc, copper and iron were analyzed by Inductively Coupled Plasma-Mass spectrometry. The level of glutathione (GSH) along with the metals was found to markedly decrease in study group, while MDA concentration was significantly higher as compared to control group. Glutathione was found to be positively associated with the levels of zinc and birth weight inspite of negative correlation being established between birth weight and MDA. Placental iron level was indicated to be constructive association with birth weight and was unlikely related with level of MDA. Our findings from the study group supports that damaged antioxidant defence mechanism status is well linked with elevated level of MDA and decreased concentrations of essential metals rendering lower birth weight in neonates.

Glioma sensitive or chemoresistant to temozolomide differentially modulate macrophage protumor activities

BackgroundGlioblastomas are the most devastating brain tumor characterized by chemoresistance development and poor prognosis. Macrophages are a component of tumor microenvironment related to glioma malignancy. The relation among inflammation, innate immunity and cancer is accepted; however, molecular and cellular mechanisms mediating this relation and chemoresistance remain unresolved. ObjectiveHere we evaluated whether glioma sensitive or resistant to temozolomide (TMZ) modulate macrophage polarization and inflammatory pathways associated. The impact of glioma-macrophage crosstalk on glioma proliferation was also investigated. MethodsGL261 glioma chemoresistance was developed by exposing cells to increasing TMZ concentrations over a period of 6months. Mouse peritoneal macrophages were exposed to glioma-conditioned medium or co-cultured directly with glioma sensitive (GL) or chemoresistant (GLTMZ). Macrophage polarization, in vitro and in vivo glioma proliferation, redox parameters, ectonucleotidase activity and ATP cytotoxicity were performed. ResultsGLTMZ cells were more effective than GL in induce M2-like macrophage polarization and in promote a strong immunosuppressive environment characterized by high IL-10 release and increased antioxidant potential, which may contribute to glioma chemoresistance and proliferation. Interestingly, macrophage-GLTMZ crosstalk enhanced in vitro and in vivo proliferation of chemoresistant cells, decreased ectonucleotidase activities, which was followed by increased macrophage sensitivity to ATP induced death. ConclusionsResults suggest a differential macrophage modulation by GLTMZ cells, which may favor the maintenance of immunosuppressive tumor microenvironment and glioma proliferation. General significanceThe induction of immunosuppressive environment and macrophage education by chemoresistant gliomas may be important for tumor recovery after chemotherapy and could be considered to overcome chemoresistance development.

P 193 - Redox control of the 20 S proteasome gating: Implications on the chronological life span of yeast cells

Two Cys residues located in the α5-subunit of the yeast 20 S proteasome (20SPT) are post-translationally modified by S-glutathionylation. Such modification implies on the opening of the 20SPT gate when compared to the DTT-reduced purified preparations. The site-specific mutation of the highly conserved (yeast to human) α5-Cys76 residue induced either decreased cell viability or life span when compared to the wild type strain. The α5-C76S-mutated strain presented decreased resistance to oxidative stress without any alteration on major redox parameters. The closed gate conformation of the 20SPT pool in that short-lived strain represents 70% of total 20SPT pool against 35% in the wild type counterpart. Mass spectrometry analysis of the α5-subunit in the α5-C76S-mutated strain showed S-glutathionylation of the remaining α5-C221 residue suggesting that redox modification of the conserved α5-C76, but not of the α5-C221, is the one involved in the gate opening. Circular dichroism analyses of recombinant α5-WT and α5-C76S subunits showed similar pattern between them regarding their secondary and tertiary structures. The differences between the wild type and short-lived strains regarding prevalence of 20SPT gate conformation, clearly demonstrate that the 20SPT opening is relevant to maintain cellular homeostasis and life span.

P 065 - Effect of GCEE or GCEE-loaded microspheres Administration on the Levels of Glutathione and Thiol Redox Molecules in Rat Brain

It is known that activity of thiol redox molecules such as Trx, TrxR and Ref-1 is regulated by glutathione (GSH). γ-glutamylcysteine ethyl ester (GCEE) is a precursor of GSH biosynthesis and neuroprotective agent. Little is known about boosting or regulating capacity of GCEE or its pharmaceutical preparations for GSH levels or thiol redox molecules in rat brain under normal physiological conditions. Experimentally GCEE (10 mg/kg/daily) was administered to the rats as acute or chronic (five consecutive days). GSH levels determined in brain tissues by DTNB-GSH reductase assay. Trx, TrxR and Ref-1 mRNAs were quantified by qPCR. Lipid/water/lipid technique was used for the preparation of GCEE- loaded microspheres and administered to the rats with the dose of 2 mg/ml. Following the administration of GCEE- loaded microspheres, the levels of GSH in hippocampus and cortex regions were determined at 12 different time periods ranging 0–96 h by HPLC pharmacokinetically. Acute or chronic treatments with GCEE significantly increased GSH levels, and Trx mRNA expression was the most regulated parameter. Pharmacokinetically the most remarkable change in GSH levels in brain tissues was observed at 4–6 h. As a conclusion, GCEE is able to regulate the levels of GSH and thiol redox parameters, and GCEE-loaded microspheres can be used to deliver this agent to the brain successfully.

Long term biochemical changes in offspring of rats fed diet containing alpha-cypermethrin

To investigate the possible developmental programming, we analyzed the effects of maternal and postnatal low dose alpha-cypermethrin exposure on metabolic and redox parameters in the offspring. Postnatal changes in plasma biochemical parameters and plasma and tissue oxidative stress markers were determined in offspring of dams fed standard chow or diet containing alpha cypermethrin at 1.50mg/kg/day during gestation and lactation, weaned on to standard chow or on treated diet until adulthood (5months).Our results showed that exposure to alpha cypermethrin induced a significant reduction in body weight, food intake and metabolic alterations such as an increase in plasma glucose, triglyceride, urea, creatinine and AST levels in both postnatal and prenatal/postnatal treated female and male rats. This increase was more pronounced in prenatal/postnatal exposed rats. Alpha-cypermethrin exposure resulted in an imbalance of oxidant/antioxidant status, marked by high levels of carbonyl proteins and MDA, and low levels of antioxidants in erythrocytes, liver and kidney of both male and female offspring. Offspring of exposed dams have pre-existing oxidative stress that was accentuated with postnatal pesticide exposure.In conclusion, maternal alpha-cypermethrin exposure affected metabolism leading to permanent changes in biochemical parameters, enzyme activities and redox markers in the offspring. These abnormalities in offspring were worsened under postnatal pesticide exposure from weaning to adulthood.

Antioxidant mechanism of mitochondria-targeted plastoquinone SkQ1 is suppressed in aglycemic HepG2 cells dependent on oxidative phosphorylation

Previously suggested antioxidant mechanisms for mitochondria-targeted plastoquinone SkQ1 included: i) ion-pairing of cationic SkQ1+ with free fatty acid anions resulting in uncoupling; ii) SkQ1H2 ability to interact with lipoperoxyl radical; iii) interference with electron flow at the inner ubiquinone (Q) binding site of Complex III (Qi), involving the reduction of SkQ1 to SkQ1H2 by ubiquinol. We elucidated SkQ1 antioxidant properties by confocal fluorescence semi-quantification of mitochondrial superoxide (Jm) and cytosolic H2O2 (Jc) release rates in HepG2 cells. Only in glycolytic cells, SkQ1 prevented the rotenone-induced enhancement of Jm and Jc but not basal releases without rotenone. The effect ceased in glutaminolytic aglycemic cells, in which the redox parameter NAD(P)H/FAD increased after rotenone in contrast to its decrease in glycolytic cells. Autofluorescence decay indicated decreased NADPH/NADH ratios with rotenone in both metabolic modes. SkQ1 did not increase cell respiration and diminished Jm established high by antimycin or myxothiazol but not by stigmatellin. The revealed SkQ1 antioxidant modes reflect its reduction to SkQ1H2 at Complex I IQ or Complex III Qi site. Both reductions diminish electron diversions to oxygen thus attenuating superoxide formation. Resulting SkQ1H2 oxidizes back to SkQ1at the second (flavin) Complex I site, previously indicated for MitoQ10. Regeneration proceeds only at lower NAD(P)H/FAD in glycolytic cells. In contrast, cyclic SkQ1 reduction/SkQ1H2 oxidation does not substantiate antioxidant activity in intact cells in the absence of oxidative stress (neither pro-oxidant activity, representing a great advantage). A targeted delivery to oxidative-stressed tissues is suggested for the effective antioxidant therapy based on SkQ1.

Effect of different dietary methionine levels on the growth performance and tissue redox parameters of turkeys

A total of 630 8-week-old female Hybrid Converter turkeys were divided (based on their body weights) into 6 groups, with 7 replicates per group and 15 birds per replicate. All birds were fed identical isocaloric and isonitrogenous wheat-soybean meal-based diets without (group 1) or with (groups 2 to 6) increasing levels of supplemental methionine (Met). The total content of Met in diets 1 to 6 was as follows (%): 0.29, 0.32, 0.40, 0.47, 0.56, and 0.61 at 9 to 12 wk of age and 0.24, 0.28, 0.34, 0.42, 0.47, and 0.55 at 13 to 16 wk of age. In both feeding phases, dietary Met levels in group 3 corresponded to those recommended by the National Research Council (NRC) (1994). Different dietary Met concentrations had no influence on feed intake, the final body weights of turkeys or carcass dressing percentage. Only in the first experimental feeding period (9 to 12 wk), the lowest dietary Met content significantly deteriorated the feed conversion ratio (FCR), whereas the highest Met content led to a significant improvement in FCR. After 8 wk of experimental feeding, dietary treatment 1 contributed to a significant increase in the activity of catalase (CAT) (blood and breast muscles) and superoxide dismutase (liver), an increase in lipid peroxides concentrations (blood, breast muscle) and a decrease in total glutathione (GSH+GSSG) content (breast muscles), in comparison to treatment 3 which is comparable to NRC recommendations. The highest level of dietary Met significantly increased blood total antioxidant potential (FRAP) values and glutathione content in the liver. To sum up, in the final feeding period between 9 and 16 wk of age, the growth performance of female turkeys was not deteriorated by dietary Met deficiency or excess (–30% and up to +50% relative to NRC recommendations, respectively). The total antioxidant potential can be effectively increased by dietary Met supplementation, but the highest Met level may lead to unbalanced oxidative changes in the body as indicated by lower FRAP values and a lower GSH/GSSG ratio in the liver.

Artificial rearing influences the morphology, permeability and redox state of the gastrointestinal tract of low and normal birth weight piglets

BackgroundIn this study the physiological implications of artificial rearing were investigated. Low (LBW) and normal birth weight (NBW) piglets were compared as they might react differently to stressors caused by artificial rearing. In total, 42 pairs of LBW and NBW piglets from 16 litters suckled the sow until d19 of age or were artificially reared starting at d3 until d19 of age. Blood and tissue samples that were collected after euthanasia at 0, 3, 5, 8 and 19 d of age. Histology, ELISA, and Ussing chamber analysis were used to study proximal and distal small intestine histo-morphology, proliferation, apoptosis, tight junction protein expression, and permeability. Furthermore, small intestine, liver and systemic redox parameters (GSH, GSSG, GSH-Px and MDA) were investigated using HPLC.ResultsLBW and NBW artificially reared piglets weighed respectively 40 and 33% more than LBW and NBW sow-reared piglets at d19 (P < 0.01). Transferring piglets to a nursery at d3 resulted in villus atrophy, increased intestinal FD-4 and HRP permeability and elevated GSSG/GSH ratio in the distal small intestine at d5 (P < 0.05). GSH concentrations in the proximal small intestine remained stable, while they decreased in the liver (P < 0.05). From d5 until d19, villus width and crypt depth increased, whereas PCNA, caspase-3, occludin and claudin-3 protein expressions were reduced. GSH, GSSG and permeability recovered in artificially reared piglets (P < 0.05).ConclusionThe results suggest that artificial rearing altered the morphology, permeability and redox state without compromising piglet performance. The observed effects were not depending on birth weight.