Low Molecular Mass Antioxidants Research Articles

Glutathione: Role in Oxidative/Nitrosative Stress, Antioxidant Defense, and Treatments

AbstractGlutathione (GSH) is the most abundant cellular tripeptide (L‐γ‐glutamate‐L‐cysteinyl‐glycine) which is as critical as oxygen and water. This low molecular mass antioxidant has a very high intracellular concentration that ranges from 1–10 mM and reaches extreme concentration points in malignant cell types. This defender of the cell is compartmentalized in mitochondria, nucleus, peroxisomes, endoplasmic reticulum (ER), and cytosol where it is synthesized. The enzymes involved in GSH redox cycling are important for both cellular free radical and non‐radical detoxification. The present review article is covering the crucial roles of GSH to combat oxidative/nitrosative stress related to different diseases/disorders and possible drug designing for new therapies.

Influence of Lichenin and Pectin on Mice During Chronic Intoxication with Cadmium(II) Chloride

Lichenin from thalli of Flavocetraria cucullata and pectin from vegetative mass of Amaranthus retroflexus were isolated. Their monomeric compositions were established. The polysaccharides lichenin and pectin were shown to promote removal of cadmium ions from mice upon chronic intoxication. Increased levels of lipid peroxidation, elevated contents of total low-molecular-mass antioxidants (LMAO), and activation of superoxide dismutase in red blood cells were not observed in experimental groups of mice that received aqueous solutions (1 and 2%) of pectin and lichenin relative to a control. Administration of 1% solutions of lichenin and pectin was shown to be most effective upon chronic intoxication with cadmium(II) chloride. The malondialdehyde content and total LMAO remained at the level of the intact group. This may be due to the fact that 2% solutions of pectin and lichenin had higher viscosities that reduced their in vivo bioavailability in mice.

Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.

'Reactive oxygen species' (ROS) is an umbrella term for an array of derivatives of molecular oxygen that occur as a normal attribute of aerobic life. Elevated formation of the different ROS leads to molecular damage, denoted as 'oxidative distress'. Here we focus on ROS at physiological levels and their central role in redox signalling via different post-translational modifications, denoted as 'oxidative eustress'. Two species, hydrogen peroxide (H2O2) and the superoxide anion radical (O2·-), are key redox signalling agents generated under the control of growth factors and cytokines by more than 40 enzymes, prominently including NADPH oxidases and the mitochondrial electron transport chain. At the low physiological levels in the nanomolar range, H2O2 is the major agent signalling through specific protein targets, which engage in metabolic regulation and stress responses to support cellular adaptation to a changing environment and stress. In addition, several other reactive species are involved in redox signalling, for instance nitric oxide, hydrogen sulfide and oxidized lipids. Recent methodological advances permit the assessment of molecular interactions of specific ROS molecules with specific targets in redox signalling pathways. Accordingly, major advances have occurred in understanding the role of these oxidants in physiology and disease, including the nervous, cardiovascular and immune systems, skeletal muscle and metabolic regulation as well as ageing and cancer. In the past, unspecific elimination of ROS by use of low molecular mass antioxidant compounds was not successful in counteracting disease initiation and progression in clinical trials. However, controlling specific ROS-mediated signalling pathways by selective targeting offers a perspective for a future of more refined redox medicine. This includes enzymatic defence systems such as those controlled by the stress-response transcription factors NRF2 and nuclear factor-κB, the role of trace elements such as selenium, the use of redox drugs and the modulation of environmental factors collectively known as the exposome (for example, nutrition, lifestyle and irradiation).

Effects of Ascorbic Acid and Reduced Glutathione on the Alleviation of Salinity Stress in Olive Plants

ABSTRACTThe aim of this study was to evaluate the effects of low molecular mass antioxidants and NaCl salinity on growth, ionic balance, proline, and water contents of ‘Zard’ olive trees under controlled greenhouse conditions. The experiment was carried out by spraying 2 mM of ascorbic acid (Asc) and 3 mM of reduced glutathione (GSH) on the plants that were treated with two salinity levels (0 and 100 mM NaCl) on their root medium. Plant growth parameters (leaf fresh weight, leaf dry weight, leaf number, total fresh weight, and total dry weight) were significantly improved by Asc compared with growth parameters in GSH and control plants. Higher concentrations of Na+ and Cl– were observed in salt-stressed plants, while Na+ and Cl– concentrations were decreased in the olive leaves that were sprayed with Asc. Salinity in the root zone caused a considerable decline in both K+ concentration and K/Na ratio. K+ concentration and K/Na ratio were significantly increased by application of Asc on plant leaves. Salinity caused an increase in electrolyte leakage (EL) compared with the control plants. Lowest EL and tissue water content (TWC) was obtained in Asc-sprayed plants, whereas TWC was increased in salt-stressed plants. Plants were subjected to salt stress and showed a higher relative water content (RWC) than the control plants. Salt stress induced proline accumulation in olive leaves. In conclusion, exogenous application of Asc is recommended to improve tolerance of olive plants under saline conditions.

Glutathione-dependent enzyme activities and concentrations of glutathione, vitamin E and sulfhydryl groups in barbel (Barbus barbus) and its intestinal parasite Pomphorhynchus laevis (Acanthocephala)

Barbel (Barbus barbus) is the final host of the adult worm Pomphorhynchus laevis (Acanthocephala), one of the most abundant and widespread intestinal parasites of European freshwater fish. During the course of the present study, we analyzed the activities of the glutathione-dependent enzymes glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione-S-transferase (GST) as well as the concentrations of total glutathione (GSH), sulfhydryl (SH) groups and vitamin E (Vit E) in the liver and intestine of B. barbus and in its intestinal parasite P. laevis. The fish were caught from the Danube River (Serbia) in spring and summer. We detected that GSH-Px activity in fish liver was higher in spring. GR activity was significantly higher in spring in all investigated samples, while GST activity was significantly higher in spring in fish liver and in the parasite. At that time, GST activity was ten times higher in the parasite than in fish tissues. The concentration of GSH was increased in barbel liver in spring. In summer, the concentration of SH groups was significantly increased, while the concentration of Vit E was significantly decreased in fish tissues and in the parasite. We performed Canonical Discriminant Analysis, which revealed differentiation among the examined tissues during both seasons based on the all measured antioxidant components. We found that the seasonal patterns of antioxidant defense in the parasite are closely correlated with seasonal variation and physiological change in the host and represent the parasite's adaptation to changes in the host's antioxidant system. The present investigation contributes to general knowledge and provides a basis for future studies of glutathione-dependent enzymes and non-enzymatic low molecular mass antioxidants as potential biomarkers for monitoring the influence of the environment on fishes and their parasites.

Обмеження вмісту вуглеводів у дієті личинок спричинює оксидативний стрес у дорослих комах Drosophila melanogaster

The influence of 20 and 1% glucose and fructose, which were components of larval diet, on the level of oxidized proteins and lipids, low molecular mass antioxidant content as well as activities of antioxidant and associated enzymes in adult fruit fly Drosophila melanogaster were investigated. The restriction of carbohydrates in larval diet leads to oxidative stress in adult insects. It is supported by 40-50% increased content of protein carbonyl groups and by 60-70% decreased level of protein thiol groups as well as by a 4-fold increase of lipid peroxide content in 2-day-old flies of both sexes, developed on the diet with 1% carbohydrates. Oxidative stress, induced by carbohydrate restriction of the larval diet, caused the activation of antioxidant defence, differently exhibited in male and female fruit flies. Caloric restriction increased activity of superoxide dismutase and thioredoxin reductase associating only in males with 2-fold higher activity of NADPH-producing enzymes--glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase. Carbohydrate restriction in the larval diet caused the increase of uric acid content, but the decrease in catalase activity in males. In females the values of these parameters were changed in opposite direction compared with males. The obtained results let us conclude the different involvement of low molecular mass antioxidants, glutathione and uric acid, and antioxidant enzyme catalase in the protection of male and female fruit fly macromolecules against oxidative damages, caused by calorie restriction of larval diet.

Evaluation of zinc accumulation potential of Hydrilla verticillata

Biofortification of foods with essential micronutrients and phytoremediation of the contaminated sites are the two sides of the same coin for metals like zinc. In the present study, Zn accumulation potential, growth and antioxidant status of Hydrilla verticillata (L.f.) Royle plants were studied upon supplementation of Zn (0–5 000 µM) for 2 and 7 d. At 5000 µM Zn, plants accumulated about 7.60 and 18.07 mg(Zn) g−1(d.m.) after 2 and 7 d, respectively. Plants exposed to Zn concentrations up to 500 µM showed significantly increased contents of low molecular mass antioxidants and activities of antioxidant enzymes in comparison with controls. Only upon exposure of plants to 5 000 µM Zn, toxicity was observed after 7 d. Therefore, owing to their high Zn accumulation capacity, Hydrilla plants may be used both as a Zn source (via culturing in ca. 100 µM Zn supplemented nutrient medium) or as a phytoremediator.

Production of reactive oxygen species and development of antioxidative systems during in vitro growth and ex vitro transfer

Ex vitro transfer is often stressful for in vitro grown plantlets. Water stress and photoinhibition, often accompanying the acclimatization of in vitro grown plantlets to ex vitro conditions, are probably the main factors promoting production of reactive oxygen species (ROS) and in consequence oxidative stress. The extent of the damaging effects of ROS depends on the effectiveness of the antioxidative systems which include low molecular mass antioxidants (ascorbate, glutathione, tocopherols, carotenoids, phenols) and antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase). This review is focused on ROS production and development of antioxidative system during in vitro growth and their further changes during ex vitro transfer.

Direct and simultaneous determination of reduced and oxidized glutathione and homoglutathione by liquid chromatography–electrospray/mass spectrometry in plant tissue extracts

A simple, highly selective, sensitive, and reproducible liquid chromatography–electrospray ionization/mass spectrometry (time of flight) method has been developed for the direct and simultaneous determination of glutathione and related compounds such as homoglutathione in different plant tissues. These compounds are low-molecular mass antioxidants involved in cellular redox homeostasis in plants, and efforts are being made to develop methods to determine the concentrations of oxidized and reduced forms of these compounds and their ratio. Many of the methodologies developed so far, however, are time-consuming and complex; therefore, analytes can decompose and their redox status can change during the analysis process. The method we have developed allows the simultaneous determination of reduced forms (glutathione [GSH] and homoglutathione [hGSH]) and oxidized forms (glutathione disulfide [GSSG]) of these compounds and is also suitable for the determination of ascorbic acid (ASA) and S-nitrosoglutathione (GSNO). Quantification was done using isotopically labeled GSH and ASA as internal standards. All compounds were base peak resolved in less than 6 min, and limits of detection were 60 pmol for GSH, 30 pmol for hGSH, 20 pmol for GSSG, 100 pmol for ASA, and 30 pmol for GSNO. The intraday repeatability values were approximately 0.4 and 7% for retention time and peak area, respectively, whereas the interday repeatability values were approximately 0.6 and 9% for retention time and peak area, respectively. Analyte recoveries found were between 92 and 105%. The method was used to determine the concentrations of GSH, GSSG, hGSH, and ASA in extracts from several plant tissues.

Molecular identification and characterisation of the glycine transporter (GLYT1) and the glutamine/glutamate transporter (ASCT2) in the rat lens

Glutathione (GSH) is an essential antioxidant required for the maintenance of lens transparency. In the lens, GSH is maintained at unusually high concentrations as a result of direct GSH uptake and/or intracellular de novo synthesis from its precursor amino acids; cysteine, glycine and glutamine/glutamate. With increasing age, the levels of GSH, particularly in the core of the lens, are significantly reduced. It has been proposed that alterations in the transport of GSH and/or its precursor amino acids may contribute to the changes in GSH levels in older lenses. As considerable uncertainty exists about the molecular identity of GSH transporters in the lens, we have focused on identifying transporters involved in the uptake of the precursor amino acids required for GSH synthesis. Previously, we identified an uptake system for cyst(e)ine mediated by the Xc − exchanger and the Excitatory Amino Acid Transporters (EAATs) in the rat lens. In this current study, we have identified and localised additional uptake systems that contribute to GSH synthesis. Transcripts for GLYT1 (glycine transporter) and ASCT2 (glutamine/glutamate transporter) were detected in rat lens fiber cells using the reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of both GLYT1 and ASCT2 at the protein level. Immunocytochemistry revealed GLYT1 expression to be restricted to cortical regions of the lens. Labelling was predominantly cytoplasmic with some labelling of the membrane. In contrast, ASCT2 was expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, ASCT2 expression was predominantly cytoplasmic. However, with deeper distance into the lens, labelling became more membraneous indicating insertion of ASCT2 into the membranes of mature fiber cells of the lens core. The molecular identification and localisation of GLYT1 and ASCT2 in the lens suggests that these transporters may be responsible for the uptake of the precursor amino acids, glycine and glutamine, which are involved in GSH synthesis. Moreover, the presence of ASCT2 in the centre of the lens raises the possibility that ASCT2 may work with the Xc − exchanger to accumulate cysteine where it can potentially act as a low molecular mass antioxidant.

Effect of thyroxine on antioxidant defense system in the liver of rats of different age

The effects of altered thyroid state on the antioxidant defense system in the liver of differently aged rats were examined. Male rats aged 15, 45 and 75 days were treated with L-thyroxine, T(4) (40 microg/100 g body mass, s.c., one dose per day) for 14 days (finally aged 30, 60 and 90 days, respectively). The following antioxidant defense enzymes were measured: superoxide dismutases (both copper zinc, CuZn-SOD and manganese containing, Mn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), glutathione reductase (GR), as well as the content of low molecular mass antioxidant glutathione (GSH). The effect of T(4) on antioxidant defense system in the liver differs with respect to age. T(4) treatment decreased CAT and GST activities, as well as the content of GSH in animals aged 60 and 90 days. The same treatment elevated GR activity in rats at 30 days of age, this phenomenon was not observed in older animals. The different response of immature rats to thyroxine compared to older animals could be attributed to the differences in thyroxine metabolism and the developmental pattern. Direct effect of T(4) on mature rats can be considered as a part of its overall catabolic action.

Antioxidants, oxidative damage and oxygen deprivation stress: a review.

Oxidative stress is induced by a wide range of environmental factors including UV stress, pathogen invasion (hypersensitive reaction), herbicide action and oxygen shortage. Oxygen deprivation stress in plant cells is distinguished by three physiologically different states: transient hypoxia, anoxia and reoxygenation. Generation of reactive oxygen species (ROS) is characteristic for hypoxia and especially for reoxygenation. Of the ROS, hydrogen peroxide (H(2)O(2)) and superoxide (O(2)(.-)) are both produced in a number of cellular reactions, including the iron-catalysed Fenton reaction, and by various enzymes such as lipoxygenases, peroxidases, NADPH oxidase and xanthine oxidase. The main cellular components susceptible to damage by free radicals are lipids (peroxidation of unsaturated fatty acids in membranes), proteins (denaturation), carbohydrates and nucleic acids. Consequences of hypoxia-induced oxidative stress depend on tissue and/or species (i.e. their tolerance to anoxia), on membrane properties, on endogenous antioxidant content and on the ability to induce the response in the antioxidant system. Effective utilization of energy resources (starch, sugars) and the switch to anaerobic metabolism and the preservation of the redox status of the cell are vital for survival. The formation of ROS is prevented by an antioxidant system: low molecular mass antioxidants (ascorbic acid, glutathione, tocopherols), enzymes regenerating the reduced forms of antioxidants, and ROS-interacting enzymes such as SOD, peroxidases and catalases. In plant tissues many phenolic compounds (in addition to tocopherols) are potential antioxidants: flavonoids, tannins and lignin precursors may work as ROS-scavenging compounds. Antioxidants act as a cooperative network, employing a series of redox reactions. Interactions between ascorbic acid and glutathione, and ascorbic acid and phenolic compounds are well known. Under oxygen deprivation stress some contradictory results on the antioxidant status have been obtained. Experiments on overexpression of antioxidant production do not always result in the enhancement of the antioxidative defence, and hence increased antioxidative capacity does not always correlate positively with the degree of protection. Here we present a consideration of factors which possibly affect the effectiveness of antioxidant protection under oxygen deprivation as well as under other environmental stresses. Such aspects as compartmentalization of ROS formation and antioxidant localization, synthesis and transport of antioxidants, the ability to induce the antioxidant defense and cooperation (and/or compensation) between different antioxidant systems are the determinants of the competence of the antioxidant system.

Kinetics of antioxidative defence responses to photosensitisation in porphyrin-accumulating tobacco plants

Tobacco ( Nicotiana tabacum L.) plants expressing antisense genes for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase suffered from photodynamic damage due to their accumulating respective tetrapyrrolic substrates. In the light, unbound pigments caused massive formation of reactive molecules that triggered a cascade of deleterious reaction in these photosensitised plants. Photosensitisation strongly depended on environmental conditions. The formation of necrotic leaf lesions was suppressed by low light dose or decreased oxygen concentration, triggered by extended light periods and enhanced by elevated temperature. An experimental system was established in which the responses to tetrapyrrole-induced oxidative stress were resolved in time during extended photoperiods. Initial differences in antioxidant responses between transgenic and WT plants were observed on enzyme activity level, followed by changes in antioxidant transcript and protein accumulation. Induction of the defence system was accompanied by changes in the redox state of low molecular mass antioxidants (ascorbate, glutathione). In response to spreading porphyrins and derived reactive oxygen species, the enhanced antioxidant capacity in different cellular compartments did not prevent cellular damage. The results are discussed particularly in view of the known antioxidant responses to photosensitisation by porphyrins in plants.

Oxidative stress: oxidants and antioxidants.

An imbalance between oxidants and antioxidants in favour of the oxidants, potentially leading to damage, is termed 'oxidative stress'. Oxidants are formed as a normal product of aerobic metabolism but can be produced at elevated rates under pathophysiological conditions. Antioxidant defense involves several strategies, both enzymatic and non-enzymatic. In the lipid phase, tocopherols and carotenes as well as oxy-carotenoids are of interest, as are vitamin A and ubiquinols. In the aqueous phase, there are ascorbate, glutathione and other compounds. In addition to the cytosol, the nuclear and mitochondrial matrices and extracellular fluids are protected. Overall, these low molecular mass antioxidant molecules add significantly to the defense provided by the enzymes superoxide dismutase, catalase and glutathione peroxidases.