Oxidative Stress and Neurotoxicity of Cadmium and Zinc on Artemia franciscana.

Despite not being redox-active metals, Cd and Zn can disrupt cellular redox homeostasis by acting pro-oxidatively. The aim of this study was to examine the effects of exposure to Zn (14 and 72 mg/L) and Cd (7.7 and 77 mg/L) for 24 and 48 h on oxidative and antioxidative parameters and the activity of glutathione-S-transferase in Artemia franciscana tissue. In addition, the neurotoxicity of the metals was examined by determining the activity of acetylcholinesterase (AChE). In A. franciscana tissue, Cd (0.0026 ± 0.0001 mg/L) was detected only after 48 h of exposure to 77 mg/L Cd. After 24 h, the 14- and 72-mg/L Zn treatments resulted in significant increases in the Zn concentration (0.54 ± 0.026 mg/L (p < 0.01) and 0.68 ± 0.035 (p < 0.0001), respectively) in A. franciscana tissue compared with the control level, and significant increases were also detected after 48 h (0.59 ± 0.02 (p < 0.0001) and 0.79 ± 0.015 (p < 0.0001), respectively). The malondialdehyde (MDA) concentration in the metal-treated samples was increased after 24 h of exposure, whereas after 48 h, an increase in the MDA concentration was detected only with 7.7. mg/L Cd. A significant increase in the H2O2 concentration after 24 h was measured only after treatment with 72 mg/L Zn. The treatment with 7.7 mg/L Cd for 24 h induced a significant increase in the AChE activity, whereas 48 h of treatment with 77 mg/L Cd and 14 mg/L Zn significantly inhibited AChE. The results indicate that lipid peroxidation resulting from metal toxicity may constitute the basis of neurotoxicity.

Protective effects of extracts of lichen Dirinaria consimilis (Stirton) D.D. Awasthi in bifenthrin- and diazinon-induced oxidative stress in rat erythrocytes in vitro.

The intoxication of insecticides such as bifenthrin and diazinon has been reported to generate free radicals, and thereby alter the antioxidant defense system in erythrocytes. The present study is aimed to investigate the protective effects of acetone (DA) and methanolic (DM) extracts of lichen Dirinaria consimilis against bifenthrin and diazinon toxicity in rats' erythrocytes in vitro. Rats' erythrocytes were exposed to bifenthrin and diazinon, individually and also in combination with DA or DM at 1 ppm for 3 h at 37 ˚C. By using spectrophotometric methods, all the samples were estimated for changes in hemoglobin (Hb) concentration, malondialdehyde (MDA) levels, and enzyme [Superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferases (GST)] activities. The outcomes showed that both the insecticides were highly toxic to rats' erythrocytes. Among all groups, both the insecticides and DA exposed groups showed very low levels of MDA content, and GST activity in rats' erythrocytes, when compared to the control. Besides, DA groups pretreated with both insecticides showed significant improvement of total Hb concentration, SOD, and CAT activities, with respect to the control. Hence, the present results indicate that the extracts of D. consimilis act as an antioxidant agent that reduces oxidative stress burden in insecticides toxicity.

Beneficial Effects of Liposomal Formulations of Lichen Substances: A Review.

Lichens are commonly used as essential traditional medicines to treat various conditions, including skin disorders, wounds, digestive, respiratory, obstetric, and gynecological problems in many cultures in Africa, Asia, Europe, Haitian, Oceania, and North and South America. Lichens have been deeply investigated for their phytochemical properties and, to date, numerous compounds (also known as substances) have been successfully isolated from the extracts. However, the low solubility and bioavailability of pure lichen substances have been widely recognized as significant issues hindering their biological applications. Recently, several groups have investigated the properties and the potential applications of lichen metabolites-based liposomal formulations and revealed a substantial improvement in their solubility, bioactivity, and toxicity in the animal. Thus, in this topical review, we aimed to provide an overview of liposomal structures, the efficacy of liposomal formulations, as well as their beneficial effects as compared to the free compounds themselves.

Phenolic compounds as antidiabetic, anti-inflammatory, and anticancer agents and improvement of their bioavailability by liposomes.

Phenolic compounds, widespread in plants, are a necessary part of the human regimen due to their antioxidant and pro-oxidative properties. Naturally, phenolics structurally range from a very simple phenolic molecule moiety to an intricate polymer. For decades, phenolic compounds have gained pronounced attention because of their protective effects against degenerative disorders such as inflammation, diabetes and cancer. Physico-chemical properties (eg, solubility) restricted their bioactivity and also limited their usage as nutraceutical ingredients. However, encapsulation technology like liposomal formulations has been developed for the delivery of phenolic compounds without affecting their original aesthetic and organoleptic property. Hence, this review outlines the antioxidant and pro-oxidative properties of phenolic compounds and focuses on biological activity reports of flavonoids and phenolic acids as antidiabetic, anti-inflammatory and anticancer agents. Also, the delivery applications of phenolic compounds as liposomes are discussed with few examples.

The protective role of melatonin under heavy metal-induced stress in Melissa Officinalis L.

Heavy metals, due to their inability to degrade, pose a serious environmental and nutritional problem. The accumulation of essential and non-essential heavy metals in living organisms reduces normal growth and development, resulting in acute poisoning, disease and even death of organisms. Melatonin is a very important multifunctional molecule in protecting plants from oxidative stress due to its ability to directly neutralize reactive oxygen species (ROS). Also, melatonin has a chelating property, which may contribute in reducing metal-induced toxicity. In this paper, the protective role of melatonin in counteracting metal-induced free radical generation was highlighted. Using the HPLC-FLD technique melatonin was identified and quantified in the roots and leaves of lemon balm ( Melissa officinalis L.), grown under photoperiod conditions. Furthermore, the response of plants pre-treated with exogenous 0.1 mM melatonin to the increased zinc (Zn) and cadmium (Cd) concentrations was observed, with changes in mineral (Ca, Mg), physiological and antioxidant status of the plant during heavy metals stress. The obtained melatonin concentrations were the highest published for dry plants so far. Elevated Cd and Zn levels in soil caused alternation in biochemical and physiological parameters of lemon balm leaves and roots. However, melatonin pre-treatment increased plant tolerance to heavy metals stress. Increased Cd and Zn uptake and their translocation into the leaves were also improved, indicating the possible use of melatonin in phytoremediation.

Wild garlic extract reduces lipid peroxidation in terbuthylazine-treated human erythrocytes.

Background: Among other negative effects, herbicides induce oxidative stress, leading to lipid peroxidation and protein oxidation. Therefore, there is a growing need to identify natural compounds with sufficient antioxidant capacity and mitigate the negative effects of herbicides without side effects.Objective: Our study aimed to examine the protective effect of the phenolic extract of wild garlic (WG) leaves on terbuthylazine-treated erythrocytes.Material and methods: In human erythrocytes treated with the herbicide terbuthylazine (4.5 mg/L) alone and a combination of terbuthylazine and WG extract, we measured malondialdehyde (MDA) and haemoglobin (Hb) concentrations and the antioxidant activities of CuZn superoxide dismutase (SOD1; EC 1.15.1.1) and catalase (CAT; EC 1.11.1.6) in vitro.Results: In comparison with terbuthylazine, WG extract reduced the concentrations of MDA and Hb from 59.69 to 43.45 nmol/gHb (27%, p < 0.001) and 165.08 to 128.64 g/L (22%, p < 0.05), respectively. Catalase activity was induced for samples treated with both WG extract and terbuthylazine compared with terbuthylazine alone (p < 0.05).Conclusions: The results demonstrated that WG may reduce the toxicity of terbuthylazine, and the erythrocyte membrane may be the primary site of phenolic action. Therefore, the lipid peroxidation intensity could be a biomarker of oxidative damage caused by terbuthylazine and the protective effect of WG.

Modulations of the antioxidants defence system in two maize hybrids during flooding stress.

Flooding stress nowadays is one of the major stressors for plants under climate change. This kind of stress may cause severe depression of the plant's growth through inhibition of photosynthesis and oxidative cell damage as well as changes in cell respiration. The present work aimed to study the effect of flooding stress on oxidative and antioxidative parameters in leaves of two maize hybrids (ZP 555 and ZP 606). Leaves of maize plants at the stage of three fully developed leaves were harvested after 6, 24, 72, and 144 h of applied flooding stress. Leaves were used for determination of physiological (the content of photosynthetic pigments and soluble proteins), oxidative stress parameters (the content of malondialdehyde (MDA) and H2O2) as well as antioxidants (the total polyphenols content, and activity of antioxidative enzymes [catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), and Class III peroxidases (POX, EC, 1.11.1.7)]). Results indicated that flooding stress-induced time-dependent changes of measured parameters and those hybrids differ in response to stress. The noticeable difference between hybrids was detected in the H2O2 and MDA content. An increase in the activity of SOD, POX and polyphenols content, with the most pronounced changes in POX activity and polyphenols concentration, could minimize the cellular damage caused by flooding. The results of the present study suggest that a more robust antioxidative metabolism is essential under flooding stress and could be a protective strategy against oxidative damage induced by flooding in ZP 606 maize plants compared to ZP 555 plants.

Tyrosinase-crosslinked pea protein emulsions: Impact of zein incorporation.

The effect of tyrosinase-crosslinking of pea protein and pea-zein complexes on the properties of concentrated o/w emulsions was studied in the present work. Emulsions comprising 2% pea protein (w/w) solubilized in the aqueous phase (60% w/w) with or without zein solubilized in the oil phase (40% w/v), were fabricated by using high pressure homogenization. Tyrosinase treated emulsions (TyrBm-crosslinked) and non-crosslinked emulsions were evaluated after 2 h of incubation. Crosslinked pea protein stabilized emulsions led to better stability, larger particle size, increased viscosity and a paste-like structure, compared to non-crosslinked pea protein stabilized emulsions. Zein incorporation in the crosslinked pea-zein stabilized emulsions, contributed to significant improvement of stability and an increase in G' concurrently with a gel-like structure formation (G' > G″), compared to the non-crosslinked pea-zein and crosslinked pea protein stabilized emulsions. In general, crosslinked emulsions showed higher protein adsorption percentage compared to non-crosslinked emulsions, while the fraction adsorbed at the oil/water interface contained crosslinked convicilin/vicilin and zein fractions. Altogether, results demonstrate that enzymatic covalent bond formation in pea protein or zein-pea protein complexes is a useful approach to design and formulate sauces, cheese and meat replacements, and other vegetarian or vegan emulsion based foods. In addition, this work represents a step forward in application of functionalized zein in concentrated oil-in-water-emulsions.

Antioxidative and antifungal response of woody species to environmental conditions in the urban area.

The complexity of ecological conditions in urban areas imposes the plant species need for the development of various biochemical and physiological adaptive strategies. The aim of our research was to examine the antioxidative and antifungal metabolism of species Pinus nigra, Picea omorika, Tilia cordata and Betula pendula from the area of Banja Luka City (urban area) during two vegetation seasons (spring and autumn) and compared with the same species from forest habitats. Changes in the protein concentration, activity and isoenzyme profiles of peroxidases (POD, EC 1.11.1.7), content and antioxidative activity of total phenols and antifungal activity in leaves and needles of the plants from the urban area and forest habitats were monitored. The obtained results indicate that urban areas induce changes in antioxidative metabolism in all examined species, but that the response is species specific. The most sensitive parameter that indicates different adaptation strategy of Pinus nigra, Picea omorika, Tilia cordata and Betula pendula to environment conditions in the urban area were peroxidase isoenzyme patterns. Less specific parameter was phenol content even though there are some indications for role of their antioxidative capacity in the adjustment to specific habitat. In addition, each species had different metabolic strategy to cope with the changes caused by the urban environment.

Oil-in-water emulsions stabilized by tyrosinase-crosslinked potato protein.

Potato protein (PP) holds great promise as a non-allergenic food ingredient with high nutritional value. Attempts to modulate its functional properties by crosslinking have not been reported to date. The effect of tyrosinase-mediated crosslinking of PP on the properties of o/w emulsions was studied in the present work. Among the various PPs, protease inhibitors were efficiently crosslinked by the enzyme as determined by SDS-PAGE analysis. Concentrated emulsions comprising 40% olive oil and 6.1% PP (w/w) were fabricated by a shear stress homogenizer. The PP-stabilized emulsions were evaluated after one and 4h of incubation with tyrosinase. Emulsions were characterized by their droplet size distribution, rheological behavior, creaming resistance and microstructure. The crosslinked emulsion had a self-standing elastic gel-like structure after 1h of incubation. Unlike the Newtonian non-crosslinked emulsion, the crosslinked emulsion exhibited a shear-thinning behavior with a 20-fold increase in viscosity. The longer incubation time coupled with shaking at 250rpm for up to 4h resulted in the disruption of the droplets structure and led to a 2-fold decrease in viscosity, compared to the 1-hour crosslinked emulsion. Droplet size distribution showed formation of large particles in the crosslinked emulsion. Microscopy imaging demonstrated formation of aggregated and dense emulsion droplets network, which also contributed to the emulsion gel-like behavior. In the case of the non-crosslinked emulsion, severe flocculation and coalescence was observed, regardless of incubation time. The results suggest that tyrosinase crosslinking is a useful method to modulate the properties of PP-based food formulations.

Pre-fractionation strategies to resolve pea (Pisum sativum) sub-proteomes.

Legumes are important crop plants and pea (Pisum sativum L.) has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula Gaertner) allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins). Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed.

Cell wall-bound cationic and anionic class III isoperoxidases of pea root: biochemical characterization and function in root growth.

Cell wall isolated from pea roots was used to separate and characterize two fractions possessing class III peroxidase activity: (i) ionically bound proteins and (ii) covalently bound proteins. Modified SDS-PAGE separated peroxidase isoforms by their apparent molecular weights: four bands of 56, 46, 44, and 41kDa were found in the ionically bound fraction (iPOD) and one band (70kDa) was resolved after treatment of the cell wall with cellulase and pectinase (cPOD). Isoelectric focusing (IEF) patterns for iPODs and cPODs were significantly different: five iPODs with highly cationic pI (9.5-9.2) were detected, whereas the nine cPODs were anionic with pI values between pH 3.7 and 5. iPODs and cPODs showed rather specific substrate affinity and different sensitivity to inhibitors, heat, and deglycosylation treatments. Peroxidase and oxidase activities and their IEF patterns for both fractions were determined in different zones along the root and in roots of different ages. New iPODs with pI 9.34 and 9.5 were induced with root growth, while the activity of cPODs was more related to the formation of the cell wall in non-elongating tissue. Treatment with auxin that inhibits root growth led to suppression of iPOD and induction of cPOD. A similar effect was obtained with the widely used elicitor, chitosan, which also induced cPODs with pI 5.3 and 5.7, which may be specifically related to pathogen defence. The differences reported here between biochemical properties of cPOD and iPOD and their differential induction during development and under specific treatments implicate that they are involved in specific and different physiological processes.

Generation of hydroxyl radical in isolated pea root cell wall, and the role of cell wall-bound peroxidase, Mn-SOD and phenolics in their production.

The hydroxyl radical produced in the apoplast has been demonstrated to facilitate cell wall loosening during cell elongation. Cell wall-bound peroxidases (PODs) have been implicated in hydroxyl radical formation. For this mechanism, the apoplast or cell walls should contain the electron donors for (i) H(2)O(2) formation from dioxygen; and (ii) the POD-catalyzed reduction of H(2)O(2) to the hydroxyl radical. The aim of the work was to identify the electron donors in these reactions. In this report, hydroxyl radical (.OH) generation in the cell wall isolated from pea roots was detected in the absence of any exogenous reductants, suggesting that the plant cell wall possesses the capacity to generate .OH in situ. Distinct POD and Mn-superoxide dismutase (Mn-SOD) isoforms different from other cellular isoforms were shown by native gel electropho-resis to be preferably bound to the cell walls. Electron paramagnetic resonance (EPR) spectroscopy of cell wall isolates containing the spin-trapping reagent, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used for detection of and differentiation between .OH and the superoxide radical (O(2)(-).). The data obtained using POD inhibitors confirmed that tightly bound cell wall PODs are involved in DEPMPO/OH adduct formation. A decrease in DEPMPO/OH adduct formation in the presence of H(2)O(2) scavengers demonstrated that this hydroxyl radical was derived from H(2)O(2). During the generation of .OH, the concentration of quinhydrone structures (as detected by EPR spectroscopy) increased, suggesting that the H(2)O(2) required for the formation of .OH in isolated cell walls is produced during the reduction of O(2) by hydroxycinnamic acids. Cell wall isolates in which the proteins have been denaturated (including the endogenous POD and SOD) did not produce .OH. Addition of exogenous H(2)O(2) again induced the production of .OH, and these were shown to originate from the Fenton reaction with tightly bound metal ions. However, the appearance of the DEPMPO/OOH adduct could also be observed, due to the production of O(2)(-). when endogenous SOD has been inactivated. Also, O(2)(-). was converted to .OH in an in vitro horseradish peroxidase (HRP)/H(2)O(2) system to which exogenous SOD has been added. Taken together with the discovery of the cell wall-bound Mn-SOD isoform, these results support the role of such a cell wall-bound SOD in the formation of .OH jointly with the cell wall-bound POD. According to the above findings, it seems that the hydroxycinnamic acids from the cell wall, acting as reductants, contribute to the formation of H(2)O(2) in the presence of O(2) in an autocatalytic manner, and that POD and Mn-SOD coupled together generate .OH from such H(2)O(2).

Characterization of polyphenol oxidase changes induced by desiccation of Ramonda serbica leaves.

Resurrection plants are able to dehydrate/rehydrate rapidly without cell damage by a mechanism, the understanding of which may be of ecological importance in the adaptation of crop plants to dry conditions. The o-diphenol oxidase in Ramonda serbica Pan. & Petrov, a rare resurrection plant of the Balkan Peninsula, was characterized in respect to different isoforms, preferable substrates and specific inhibitors. Two anionic isoforms with pI 4.6 and 4.7 were separated from turgid leaves. Three additional anionic isoforms (pI 5.1, 5.3 and 5.6) and three neutral isoforms (pI from 6.8 to 7.4) were induced in desiccated leaves. Based on apparent K(m) values, the affinity for reducing substrates decreased as follows: methyl catechol > chlorogenic acid > 3,4-dihydroxyphenylalanine > caffeic acid > pyrogallol. Polyphenol oxidase (PPO) activity was specifically sensitive to diethyldithiocarbamate and also inhibited by KCN, DTT and salicylic hydroxamic acid but with no inhibitory effect of Na3N. Plants were subjected to drought-to-near complete water loss (approximately 2% relative water content, RWC) and several fold higher PPO activity was detected in desiccated leaves. Ramonda leaves contain high levels of phenolics, which decreased during drought. Rehydration of dry leaves from 2% RWC to 95% RWC led to transient inhibition of PPO in the first few hours. Within a day, the levels completely recovered to those determined in desiccated leaves. The finding of desiccation-induced high activity of PPO and new isoforms, which were also present in rehydrated turgid leaves, indicates a substantial role for PPO in the adaptation mechanism of resurrection plants to desiccation and also to the oxidative stress during rehydration.

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.).

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms. Three POD isoforms with pI values of approximately 4.5, 4.8, and 8.4, and one additional pair of isoforms with a pI value of approximately 8.8 were separated from tissue extracts of all transgenic lines. Isoelectrofocusing patterns revealed the induction of one isoform of SOD with a pI of about 5.6 in all transgenic lines compared with non-transformed embryogenic tissue. These results indicate that the process of transformation may disrupt redox homeostasis in alfalfa tissues.

Senescence- and drought-related changes in peroxidase and superoxide dismutase isoforms in leaves of Ramonda serbica.

Ramonda sp. (Gesneriaceae) is an endemic and relic plant in a very small group of poikilohydric angiosperms that are able to survive in an almost completely dehydrated state. Senescence- and drought-related changes in the activity of peroxidase (POD; EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11), and superoxide dismutase (SOD; EC 1.15.1.1) were determined in leaves of different age and relative water content. The results indicate that different POD isoforms were stimulated during senescence and dehydration. Two of the soluble POD isoforms were anionic with pI 4.5, and two were cationic with pI 9.3 and 9.0. The activity of ascorbate peroxidase remained unchanged either by drought or senescence. For the first time, SOD isoforms have now been determined in this resurrection plant. Several SOD isoforms, all of the Mn type, were found to be anionic with pI 4 and a few others had pI from 5 to 6, while one band of FeSOD with a lower molecular weight was neutral. Rehydration brought about a remarkable decrease over the first hour in the activity of all the antioxidant enzymes examined but activity recovered 1 d after rehydration. The results confirmed that dehydration and senescence caused disturbance in the redox homeostasis of Ramonda leaves, while inducing different POD isoforms. A physiological role of peroxidase reaction with hydroxycinnamic acids in conservation and protection of cellular constituents of desiccated Ramonda leaves is suggested.

Ascorbic acid and the oxidative processes in pea root cell wall isolates: characterization by fluorescence and EPR spectroscopy.

A comparative fluorescence and oxygen radical-sensitive spin trap EPR spectroscopic study of isolated cell walls (with proteins or deproteinated), in the presence and absence of ascorbate and H(2)O(2) is presented. Fluorescence spectra indicate the presence of at least two fluorophores, one degraded and the other synthesized after reduction or oxidation, indicating phenol di/polymerization. DEPMPO spin trap measurements show that isolated cell walls are capable of oxygen-dependent hydroxyl radical generation in the absence of NADH or other reductants, ascorbate addition, or deproteination of the cell wall abolishing the signal due to hydroxyl radicals.