Manganese protects wheat from the mycotoxin zearalenone and its derivatives.

Searching for factors that reduce zearalenone (ZEN) toxicity is an important challenge in wheat production, considering that this crop is a basic dietary ingredient. ZEN, absorbed by cells, is metabolized into α-zearalenol and α-zearalanol, and this study focused on the function of manganese ions as potential protectants against the mycotoxins. Stress effects were invoked by an application of 30 µM ZEN and its derivatives. Manganese ions were applied at 100 µM, not stress-inducing concentration. Importance of the biomembrane structures in the absorption of the mycotoxins was demonstrated in in vitro wheat calli and on model membranes. ZEN showed the greatest and α-zearalanol the smallest stressogenic effect manifested as a decrease in the calli growth. This was confirmed by variable increase in antioxidant enzyme activity. Mn ions added to the toxin mixture diminished stressogenic properties of the toxins. Variable decrease in total lipid content and the percentage of phospholipid fraction detected in calli cells exposed to ZEN and its metabolites indicated significance of the membrane structure. An analysis of physicochemical parameters of model membranes build from phosphatidylcholine, a basic lipid in native membranes, and its mixture with the tested toxins made by Langmuir technique and verified by Brewster angle microscopy, confirmed variable contribution of ZEN and its derivatives to the modification of membrane properties. The order of toxicity was as follows: ZEN ≥ α-zearalenol > α-zearalanol. Manganese ions present in the hydrophilic phase interacted with polar lipid groups and reduced the extent of membrane modification caused by the mycotoxins.

Changes in content of steroid regulators during cold hardening of winter wheat - Steroid physiological/biochemical activity and impact on frost tolerance.

The purpose of experiments was to describe the alterations of content of steroid regulators (brassinosteroids, progesterone) during cold hardening of winter wheat. Further we studied physiological and biochemical changes induced by these steroids in cold hardened winter wheat together with estimation of plant frost tolerance. The endogenous brassinosteroid content was elevated in winter wheat during cold hardening while level of progesterone was lowered. A higher content of brassinosteroids (but not progesterone) was connected to better frost tolerance of winter wheat cultivars. Plant supplementation with brassinosteroid (24-epibrassinolide) and progesterone before cold hardening reduced frost damage. Tests with the inhibitors of the biosynthesis of brassinosteroids and progesterone suggested that these steroids are one of players in regulating the antioxidant system in winter wheat during cold hardening. Their role in regulating the expression of Rubisco or the Rubisco activase gene was less clear. Steroid regulators did not affect the content of the stress hormone ABA. Model studies of the membranes, made on a Langmuir bath, showed an increase in the value of the parameter describing differences in membrane compressibility (resulting from stronger interactions among the molecules in the monolayers). This suggests that 24-epibrassinolide and progesterone enter into the lipid layer and - in a similar way to sterols - stabilise the interaction among lipids. It may be significant step for better frost tolerance. The use of steroid regulators (especially brassinosteroids) as agrochemicals improving frost tolerance of winter cereals will be discussed.

Exposure of human lymphoma cells (U-937) to the action of a single mycotoxin as well as in mixtures with the potential protectors 24-epibrassinolide and selenium ions.

The progressive contamination of food products by mycotoxins such as zearalenone (ZEN) has prompted the search for specific substances that can act as protectors against an accumulation of these toxins. This paper discusses the effect of selenium ions and 24-epibrassinolide (EBR) as non-organic and organic compounds that preserve human lymphoblastic cells U-937 under ZEN stressogenic conditions. Based on measurements of cell viability and a DAPI test, concentrations of ZEN at 30 µmol/l, Se at 2.5 µmol/l and EBR at 0.005 µmol/l were selected. The addition of both protectors resulted in an increase in the viability of ZEN-treated cells by about 16%. This effect was connected with a decrease in lipid peroxidation (a decrease in the malonyldialdehyde content) and the generation of reactive oxygen species, which were determined by a cellular ROS/superoxide detection assay and the SOD activity. The Se protection was observed as the blocking of the all excess ROS, while the EBR action was mainly concentrated on something other than the superoxide radical itself. The experiments on the model lipid membranes that mimic the environment of U-937 cells confirmed the affect of ZEN on the structure and physicochemical properties of human membranes. Although the presence of both Se and EBR reduced the effect of ZEN by blocking its interaction with a membrane, the action of Se was more evident.

Impact of polyphenol-rich green tea extracts on the protection of DOPC monolayer against damage caused by ozone induced lipid oxidation.

Effectiveness of green tea (compared to two single polyphenols) in removing ozone derived reactive oxygen species acting on dioleoylphosphatidylcholine monolayers was determined. Lipid oxidation was followed by changes in mechanical properties of the layer spread on the aqueous subphase containing various amounts of ozone in the presence and absence of polyphenolics. It was shown that the tea extract (containing 8.5 x 10-4 mg/cm3 polyphenols) is capable of inactivating 0.4 ppm ozone. The DPPH radical scavenging test set polyphenols in the order of increasing activity, consistent with their protective effect in relation to lipid oxidation, showing the highest efficacy of EGCG.

Regulation of the membrane structure by brassinosteroids and progesterone in winter wheat seedlings exposed to low temperature.

Steroids constitute one of the most important groups of compounds of regulatory properties both in the animal and plant kingdom. In plants, steroids such as brassinosteroids or progesterone, by binding to protein receptors in cell membranes, regulate growth and initiate processes leading to increased tolerance to stress conditions. Due to their structural similarities to sterols, these steroids may also directly interact with cellular membranes. Our aim was to determine the changes of the structural parameters of lipid membranes under the influence of hydrophobic steroid compounds, i.e., 24-epibrassinolide (EBR) and its precursor-24-epicastasterone (ECS) and progesterone (PRO). Lipids were isolated from wheat seedlings with different tolerances to frost, grown at low temperatures (5 °C) for 1.5 and 3 weeks (acclimation process). Control plants were cultured continuously at 20 °C. From galactolipids and phospholipids, the main polar lipid fractions, the monolayers were formed, using a technique of Langmuir trough. EBR and ECS were introduced into monolayers, together with lipids, whereas the PRO was dissolved in the aqueous sub-phase upon which the monolayers were spread. Measurements performed at 25 °C and 10 °C showed a significant action of the tested compounds on the physicochemical properties of the monolayers. EBR and PRO increased the area per lipid molecule in monolayers, resulting in formation of more flexible surface structures while the presence of the ECS induced the opposite effect. The influence of the polarity of lipids and steroids on the interactions in the monolayer was discussed. Lipids extracted from the membranes of wheat with the most tolerance to frost were characterized by the highest fatty acid unsaturation and steroids had a relatively weak effect on the parameters of the structure of their monolayers.

The Effects of the Structure and Composition of the Hydrophobic Parts of Phosphatidylcholine-Containing Systems on Phosphatidylcholine Oxidation by Ozone.

The degree of lipid unsaturation is a parameter used to describe membrane susceptibility to oxidation. This paper highlights the importance of double bond distribution in the hydrophobic parts of lipid layers. The problem was studied by determining the effects induced by ozone dissolved in an aqueous phase acting on layers of unsaturated cholines of various molecular structures, including bi-unsaturated (DOPC), mono-unsaturated (POPC) and natural origin (soy PC). The destructive effects of ozone were quantified as the ratio of areas per molecule, which corresponded to a 1 mN/m rise in the layer surface pressure for oxidized to non-oxidized lipids (A lift/A lift0 ). The experimental results showed different behaviours among the studied lipids. Layers of DOPC with both unsaturated fatty acyl chains exhibited the greatest disruption compared with that of PC extracted from soy, which maintained stability despite high degree of unsaturation. Mono-unsaturated ozonized layers of POPC did not exhibit any disruption, but their modified properties indicated structural changes caused by the appearance of oxidation products. The stability of mixed layers (of the same unsaturation degree as the soy PC) composed of DOPC and fully saturated lipid increased, however, not reaching the soy PC level. Comparisons of the behaviour of tested systems indicated that the fraction of lipids containing one saturated acyl chain is the parameter most important for stability of the oxidized layer. The stabilizing effects of the cholesterol admixture were also quantified. Results obtained for lipid layers were supported by measurements of liposome size, zeta potential and surface tension of liposome suspension.

Studies of Lipid Monolayers Prepared from Native and Model Plant Membranes in Their Interaction with Zearalenone and Its Mixture with Selenium Ions.

The impact of zearalenone and selenate ions on the monolayers of 1,2-dipalmitoyl-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and the lipid mixtures (phospholipids and galactolipids) extracted from wheat plasmalemma has been studied using Langmuir trough technique and Brewster angle microscopy (BAM). The zearalenone is a mycotoxin that exerts toxic effects on the cells of plants and animals. Monolayers' properties were characterized by surface pressure (π)-molecular area (A) isotherms. It was found that zearalenone interacts with lipid monolayers causing their expansion. The selenate ions, added to the subphase together with zearalenone, reduce the effect of this mycotoxin on the surface properties of lipid films.

α-Tocopherol/Gallic Acid Cooperation in the Protection of Galactolipids Against Ozone-Induced Oxidation.

The protective ability of α-tocopherol (TOH) and gallic acid (GA) acting simultaneously at the moment of oxidizer application was evaluated by determination of galactolipid layers' oxidation degree. Addition of GA resulted in a significant decrease of ozone-derived radicals shifting the threshold of lipid sensitivity by an amount approximately corresponding to the GA intake in bulk reaction with ozone. TOH presence in lipid layers results in a change of the role of GA which additionally may be involved in the reduction of tocopheroxyl radical formed during oxidation. This leads to a decrease in effectiveness of GA in diminishing the amount of ozone radicals. Such an effect was not observed for mixed layers containing galactolipid and pre-oxidized tocopherol where the ozone threshold level was associated with a stoichiometry of GA + O3 reaction. It was concluded that probably subsequent transformations of tocopheroxyl radical to less reactive forms prevent its reaction with GA the entire quantity of which is used for radicals scavenging. This result shows the role of time parameter in systems where substrates are engaged in various reactions taking place simultaneously. The inactivation of 1,1-diphenyl-2-picrylhydrazyl radical by studied antioxidants in homogeneous system confirmed observations made on the basis of lipid layer properties indicating their antagonistic action (at least at studied conditions). Formation of layers in post-oxidation situation did not depend whether tocopherol was oxidized during oxidation of lipid/tocopherol mixture or was introduced as pre-oxidized. This may be interpreted as indication that products of tocopherol oxidation may stabilize lipid layers.

Physicochemical aspects of reaction of ozone with galactolipid and galactolipid-tocopherol layers.

The impact of reaction of galactolipids with ozone on the physicochemical properties of their monolayers was examined. In Megli and Russo (Biochim Biophys Acta, 1778:143-152, 2008), Cwiklik and Jungwirth (Chem Phys Lett, 486:99-103, 2010), Jurkiewicz et al. (Biochim Biophys Acta, 1818:2388-2402, 2012), Khabiri et al. (Chem Phys Lett, 519:93-99, 2012), and Conte et al. (Biochim Biophys Acta, 1828:510-517, 2013), the properties of layers formed from model mixtures composed of chosen lipids and selected oxidation products were studied, whereas in this work, question was raised as to how the oxidation reactions taking place in situ affect the physical properties of the galactolipid layers. So, set experiment should take into account the effect of all reaction products. The mechanical characteristics of monolayers of monogalactosyldiacyl-glycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were determined by Langmuir trough technique, and the electrical properties of liposomes formed from these lipids by measuring their electrophoretic mobility. Considerable loss of galactolipid molecules forming monolayers was found at ozone concentrations (in aqueous medium) higher than 0.1 ppm with a stronger effect measured for MGDG. That goes along with the greater amounts of MDA found in the extracts of oxidized MGDG films compared with DGDG. Based on this, it was concluded that an additional galactose group present in DGDG molecules acts protectively under oxidative conditions. The surface tension of the solutions (of small volume) contacting the oxidized galactolipids films was significantly reduced, indicating the presence of soluble in polar media, surface active reaction products. The presence of α-tocopherol in mixtures with tested galactolipids at a molar ratio of lipid to tocopherol equal to 1.7:1 caused some inhibition of lipid oxidation, reducing the decrease of amount of lipid particles forming the monolayer. Here, also protective effect of α-tocopherol was greater for the MGDG compared to DGDG.

Mechanical and electrokinetic effects of polyamines/phospholipid interactions in model membranes.

The mechanical and electrical properties of phospholipids layers influenced by interaction with polyamines were determined by measuring surface pressure and compression modulus of monolayers and zeta potential of liposomes. The saturated derivative of phosphatidic acid (DPPA) formed layers of the organization varying with compression degree. Contact of DPPA layers with polyamines present in the subphase resulted in changing their mechanical properties and the conditions in which the layer reorganization appears. The parameters corresponding to the layer reorganization depended on the size and charge of polyamines' molecules. The values of: area per DPPA molecule, surface pressure at the point of layer structure reorganization, and surface pressure at the point of collapse characterizing of DPPA layers in the studied systems were determined. It was found that polyamines influenced to a much lesser extent the mechanical properties of monolayers formed from unsaturated derivative of phosphatidic acid slightly increasing its mechanical resistance in the range of higher molecular packing. The results of electrokinetic measurements revealed that surface charge of phosphatidic acid liposomes was effectively neutralized in the presence of polyamines. A similar effect was observed for phosphatidyl glycerol and for negatively charged polystyrene latex particles used as a reference. The influence of polyamines on the mechanical properties of DPPA layers was interpreted assuming a possibility of penetration of the lipid layer by polyamines' molecules. Comparison of action of putrescine and calcium ions and effects of polyamines on phosphatidyl glycerol provided additional justification for the proposed interpretation of the observed effects.

Protective effect of ascorbic acid after single and repetitive administration of cadmium in Swiss mice.

The aim of the study was to delineate the protective effect of ascorbic acid with plausible mechanism after single and repetitive cadmium administration to Swiss mice. The effects of single high dose administration of CdCl(2) (6 mg/kg) or ascorbic acid (AsA) (50 mg/kg) and chronic (three times) administration of Cd at low dose (2 mg/kg) or AsA at same dose (50 mg/kg) were compared in Swiss albino mice. Changes of lipid peroxidation [determined by the malonyldialdehyde (MDA) concentration] were taken as a measure of the oxidative stress intensity. Lipid fatty acid's unsaturation related to the permeability of cell membranes was also examined. Mobilization of the immune system was determined by analyzing changes in antioxidant concentrations of AsA and glutathione (GSH), and by measuring the activation of antioxidant enzymes SOD, GPx and CAT. In addition, the level of free polyamines and variation in their proportions were examined. In conclusion, exposure to higher levels of cadmium will have more deleterious effects on the body rather than chronic exposure at lower levels with this toxic metal, while this study clearly demonstrated the protective effects of AsA in a mouse model.