The impacts of mining on soil pollution with metal(loid)s in resource-rich Mongolia.

As Mongolia is considered one of the most resource extraction-dependent countries globally, significant mining-related environmental and human health risks are expected. The aim of this study was to (I) assess the impacts of mining on soil pollution with metals in Mongolia's key coal mining towns (Baganuur, Nalaikh and Sharyn Gol) and (II) review the current knowledge on soil pollution with metal(loid)s and related health risks in Mongolia. The results showed predominantly low soil contents of Cd, Cu, Pb and Zn and a related absence of severe pollution and potential health risk in the coal mining towns. Urban design, rather than the presence of mines, controlled the pollution distribution. Despite the methodological shortcomings of several studies on soil pollution in Mongolia, their results suggest a similarly low threat in the three largest cities (Ulaanbaatar, Darkhan, Erdenet) and several mining areas. While the generally highlighted risk of As seems like an artificially escalated issue, the content of Cr in urban soil may be a neglected threat. Further pollution research in Mongolia should focus on street dust and drinking water pollution.

Development and evaluation of the iron oxide-hydroxide based resin gel for the diffusive gradient in thin films technique.

An ion-exchange resin Lewatit FO 36 was used for the preparation of a new resin gel for the diffusive gradient in thin films technique (DGT). The DGT method was optimized for the accumulation of four bioavailable arsenic species (AsIII, AsV, monomethylarsonic acid, dimethylarsinic acid) in the aquatic environment. The total sorption capacity of Lewatit FO 36 resin gel was 535 µg As disc-1. The microwave-assisted extraction in the presence of NaCl (10 g L-1) and NaOH (10 g L-1) was used for the isolation of arsenic species from the Lewatit FO 36 resin gel. The elution efficiency of arsenic was 98.4 ± 2.0%. Arsenic was determined by the optimized electrothermal atomic absorption spectrometry (ET-AAS) method using palladium modifier, pre-atomization cool-down step and tungsten carbides coating of graphite tube. The Lewatit FO 36 resin gel provides accurate results (cDGT/cSOL ratio 0.86-1.00) in the pH range 4-8. No significant influence of experimental conditions was observed in the presence of chlorides (0-0.5 mol L-1) and humic acid (0-100 mg L-1). Only a very high concentration of phosphates (10 mg L-1) caused a slight decrease in the diffusion coefficients of MMA and AsV species (8.4% and 12.4%, respectively). The presence of iron (0-1 mg L-1) caused a decrease in the diffusion coefficients, but with regard to the common concentrations of iron (less than 0.3 mg L-1), the negative effect was considered not significant for AsIII and DMA in natural water. The DGT-ET-AAS method was applied for the determination of bioavailable arsenic species in the spiked river water samples and also in-situ in the water reservoir. The new resin gel was characterized by a homogeneous gel structure with excellent reproducibility (< 5% variation of results between batches) and high sorption capacity which suggests its possible long-term application (up to 286 days in the environment with the arsenic concentration of 100 µg L-1).

The strong reaction of simple phenolic acids during oxidative stress caused by nickel, cadmium and copper in the microalga Scenedesmus quadricauda.

This work has analysed the influence of CdCl2, NiCl2 and CuCl2 on simple phenolic acids, such as the 3PPP (phenylpyruvic, phenylacetic, and 4-hydroxyphenylpyruvic) and 2DR (3,4-dihydroxyphenylacetic and rosmarinic) phenolic acids for the first time and studied their interactions with antioxidant systems and the glutathione-ascorbate cycle in the freshwater green microalga Scenedesmus quadricauda. The compounds investigated are related to both the catabolic and anabolic pathways of l-phenylalanine and l-tyrosine, the main molecules in the biosynthesis of polyphenols. The concentrations of the simple phenolic acids responded significantly to NiCl2, CdCl2 and CuCl2 at 5, 20 and 40 µM concentrations. The 3PPP phenolic acid concentrations after 24 h were always higher in metal-treated cells than in controls, while the 2DR concentrations were significantly lower in the metal-treated cells than the controls. The GSH/GSSG ratio was lower in all experimental groups treated with the selected metals (especially so at 40 µM). Pearson correlation analysis indicated a strong negative correlation between ascorbate and rosmarinic acid content (-0.670; p < 0.05) in NiCl2-treated samples and reduced glutathione and 3,4-dihydroxyphenylacetic content (-0.700; p < 0.05) in CdCl2-treated samples. The GSSG content in samples exposed to CuCl2 was correlated with the concentrations of all of the investigated phenolic acids (4 negative, 1 positive).

Antioxidant status of rats' blood and liver affected by sodium selenite and selenium nanoparticles.

BACKGROUND: Selenium is an essential element; however, at higher doses, it can be toxic. Therefore, alternative nanotechnological solutions are required to overcome toxicological issues, rather than conventional alternatives. Nanoparticles show new and promising properties that may be able to suppress toxicity while maintaining the positive effects of selenium on an organism. The aim of the experiment was to determine the influence of sodium selenite and selenium nanoparticles (SeNPs) on the antioxidant status of rats. METHODS: The males of the outbreed rat strain Wistar albino were selected as a model organism. Animals were fed different forms of selenium. The control group was given a mixture without selenium addition, whereas other groups were fed a mixture containing sodium selenite, Se-49, and Se-100 SeNPs respectively. The duration of the trial was 30 days. RESULTS: Analysis of blood and liver was performed where the concentration of reduced (GSH) and oxidised (GSSG) glutathione, and total selenium content were measured. In the liver, a significant reduction in GSSG was found for all experiment groups. Blood samples showed a significant reduction in GSH and an increase in GSSG. DISCUSSION: These results show that SeNPs may be an alternative to dietary selenium for animal organisms.

Separation, identification and quantification of carotenoids and chlorophylls in dietary supplements containing Chlorella vulgaris and Spirulina platensis using High Performance Thin Layer Chromatography.

In this study, 14 commercial products (dietary supplements) containing alga Chlorella vulgaris and cyanobacteria Spirulina platensis, originated from China and Japan, were analysed. UV-vis spectrophotometric method was applied for rapid determination of chlorophylls, carotenoids and pheophytins; as degradation products of chlorophylls. High Performance Thin-Layer Chromatography (HPTLC) was used for effective separation of these compounds, and also Atomic Absorption Spectrometry for determination of heavy metals as indicator of environmental pollution. Based on the results obtained from UV-vis spectrophotometric determination of photosynthetic pigments (chlorophylls and carotenoids), it was confirmed that Chlorella vulgaris contains more of all these pigments compared to the cyanobacteria Spirulina platensis. The fastest mobility compound identified in Chlorella vulgaris and Spirulina platensis using HPTLC method was ß-carotene. Spectral analysis and standard calibration curve method were used for identification and quantification of separated substances on Thin-Layer Chromatographic plate. Quantification of copper (Cu2+, at 324.7 nm) and zinc (Zn2+, at 213.9nm) was performed using Flame Atomic Absorption Spectrometry with air-acetylene flame atomization. Quantification of cadmium (Cd2+, at 228.8 nm), nickel (Ni2+, at 232.0nm) and lead (Pb2+, at 283.3nm) by Electrothermal Graphite Furnace Atomic Absorption Spectrometry; and quantification of mercury (Hg2+, at 254nm) by Cold Vapour Atomic Absorption Spectrometry.

Reactions of Melolontha hippocastani adults to high manganese content in food.

Rapid adaptation of insect species to environmental changes can be advantageous for their development. Forest cockchafer (Melolontha hippocastani) is herbivorous insect species with high food intake that has been widely studied within European conditions. It can cause huge economic and ecological losses especially in oak-dominated forest stands. Food preference of this species is mainly comprised of trees with a potential to accumulate manganese in their assimilation apparatus. In order to determine cockchafer's reactions to increased Mn concentration in its food, the adults were reared in laboratory conditions controlled for temperature, humidity, and light on a natural diet of oak (Quercus petraea) leaves. Food was contaminated by soaking in solutions of MnCl2·4H2O in concentrations from 0.5 to 10mgml-1. Food consumption, activity period of adults, quantity of eggs laid, body weights of individuals, as well as Mn concentrations in unconsumed food, excrement, and the bodies of cockchafers were determined to indicate how these change in reaction to food quality altered in terms of Mn content. The results showed that very high Mn content in the diet significantly influenced food consumption by M. hippocastani adults and thereby their development. This was in spite of the tendency of individuals to expel Mn through the faeces and incorporate it into undetermined parts of their bodies. Negative reactions of the experimental population were determined in treatments fed leaves with Mn concentration of 1800ppm and more. Our experiment proved that Mn can greatly influence the phytophagous cockchafer (decreased feeding time, food consumption, and fertility).

Comparison of vascular and non-vascular aquatic plant as indicators of cadmium toxicity.

Antioxidative and microscopic responses in non-vascular (moss Taxiphyllum barbieri) and vascular (Ceratophyllum demersum) aquatic plants exposed to short-term (24 h) cadmium (Cd) excess (10 and 100 µM) were compared. Ceratophyllum considerably accumulated Cd but less pronounced symptoms of oxidative stress were detected by confocal microscopy (at the level of general ROS, hydrogen peroxide, hydroxyl radical/peroxynitrite and superoxide) that could be related to enhanced activities of antioxidative enzymes (SOD, CAT, APX). Amounts of ascorbic acid and non-protein thiols were higher in Ceratophyllum than in Taxiphyllum and increased with increasing Cd dose, which may help to better regulate circulation of free metal ions in Ceratophyllum mainly. Besides, it was observed that citric acid increased in Ceratophyllum while malic acid in Taxiphyllum in response to Cd which may also contribute to Cd chelation. Our data indicate that Ceratophyllum is a suitable species for Cd bioaccumulation while Taxiphyllum is more sensitive to Cd excess and thus suitable as indicator species. It was also proven that sensitive microscopic techniques allow the visualization of Cd-induced changes in aquatic plants even after short-term exposure when no morphological signs of damage are visible.

Dandelion Taraxacum linearisquameum does not reflect soil metal content in urban localities.

Accumulation of selected heavy metals (Cd, Pb, Ni, Cr, Fe, and Zn) and phenolic metabolites (total soluble phenols, cichoric and caftaric acid) in dandelion organs (leaves, roots, inflorescences/anthodia) collected from six localities within the industrial town Kosice (eastern Slovakia) were studied. Localities from the vicinity of a steel factory (Cd, Fe) and heavy traffic (Pb, Ni, Cr, Zn) contained the highest amount of individual metals in the soil but a significant correlation between soil and organ metal content was found only for Cr in the leaves (r2 = 0.7679). The amount of Cd and partially Pb differed among localities in all organs and especially in the leaves and anthodia, indicating probably the impact of atmospheric pollution. The bioaccumulation factor was <1 for almost all metals, suggesting that given dandelion species is not metal accumulator. Translocation factor did not reach values close to or over 1 only for Cd, indicating a root-to-shoot movement of Pb, Ni and Zn though the impact of air pollution on leaves cannot be excluded. A strong correlation between leaf Cd and leaf total phenols, cichoric and caftaric acids was observed (r2 = 0.7926, 0.8682 and 0.8830, respectively), indicating that phenolic metabolites act in the protection of dandelion against Cd excess. Overall, our data indicate low pollution of urban soil by Cd (5.53-113.8 ng g-1) and partially by Cr and the suitability of above-ground organs of dandelion species for the monitoring of air pollution mainly by Cd.

Content of metals and metabolites in honey originated from the vicinity of industrial town Kosice (eastern Slovakia).

Composition of three types of honey (mixed forest honey and monofloral-black locust and rapeseed honeys) originated from the vicinity of an industrial town (Kosice, Slovak Republic) was compared. Higher content of minerals including toxic metals in forest honey (1358.6 ng Ni/g, 85.6 ng Pb/g, and 52.4 ng Cd/g) than in rapeseed and black locust honeys confirmed that botanical origin rather than the distance for eventual source of pollution (steel factory) affects metal deposition. Benzoic acid derivatives were typically more accumulated in forest but cinnamic acid derivatives and some flavonoids in rapeseed honey (in free and/or glycoside-bound fraction). In terms of quantity, p-hydroxybenzoic and p-coumaric acids were mainly abundant. Total phenols, thiols, and proteins were abundant in forest honey. Some metals and phenols contributed to separation of honeys based on principal component analysis (PCA). Native amount of 5-(hydroxymethyl)furfural was not related to honey type (~11 µg/g) and was elevated after strong acid hydrolysis (200-350 µg/g) but it did not interfere with the assay of phenols by Folin-Ciocalteu reagent. This is the first report of metals and metabolites in the same study, and data are discussed with available literature. We conclude that black locust (acacia) honey is the most suitable for daily use and that central European monofloral honeys contain lower amounts of toxic metals in comparison with other geographical regions.

Age affects not only metabolome but also metal toxicity in Scenedesmus quadricauda cultures.

Responses of Scenedesmus quadricauda grown in vitro and differing in age (old culture-13 months, young culture-1 month) to short-term cadmium (Cd) or nickel (Ni) excess (24h) were compared. Higher age of the culture led to lower amount of chlorophylls, ascorbic acid and glutathione but higher signal of ROS. Surprisingly, sucrose was detected using DART-Orbitrap MS in both old and young culture and subsequent quantification confirmed its higher amount (ca. 3-times) in the old culture. Cd affected viability and ROS amount more negatively than Ni that could arise from excessive Cd uptake which was also higher in all treatments than in respective Ni counterparts. Surprisingly, nitric oxide was not extensively different in response to age or metals. Strong induction of phytochelatin 2 is certainly Cd-specific response while Ni also elevated ascorbate content. Krebs cycle acids were more accumulated in the young culture but they were rather elevated in the old culture (citric acid under Ni excess). We conclude that organic solid 'Milieu Bristol' medium we tested is suitable for long-term storage of unicellular green algae (also successfully tested for Coccomyxa sp. and Parachlorella sp.) and the impact of age on metal uptake may be useful for bioremediation purposes.

Chromium speciation and biochemical changes vary in relation to plant ploidy.

Uptake of trivalent chromium (Cr(III)-chloride), Cr speciation and consequences for the metabolism in chamomile plants with two ploidy levels have been studied. Depletion of fresh biomass, tissue water content and soluble proteins in response to high (120 µM) Cr(III) was ploidy-independent. Cr mainly accumulated in the roots (only negligibly in the shoots) and total root Cr amount was higher in tetraploid ones including the proof with specific fluorescent indicator (naphthalimide-rhodamine) of Cr(III). Quantification of Cr(VI) detected its higher content in tetraploid roots (up to 4.2% from total Cr), indicating partial oxidation of applied Cr(III). Higher H2O2 presence but lower activities of peroxidases were observed in tetraploid roots while nitric oxide, superoxide dismutase and glutathione reductase activities did not differ extensively. Soluble phenols, lignin, non-protein thiols, individual thiols (glutathione and phytochelatin 2) and ascorbic acid responded to high Cr(III) similarly in both cultivars while decrease of minerals was more pronounced in tetraploid ones. It seems that Cr(III)-induced oxidative stress arises from high root Cr uptake and Cr(VI) presence and is related to depletion of thiols. Assay of Krebs cycle acids confirmed rather depletion under 120 µM Cr(III) in both cultivars but increase in citric acid may indicate its involvement in root Cr chelation. Subsequent comparison of Cr(III)-chloride and Cr(III)-nitrate showed similar influence on Cr accumulation and majority of biochemical responses while different impact on phytochelatin 2 amount was the most distinct feature.

Lanthanum rather than cadmium induces oxidative stress and metabolite changes in Hypericum perforatum.

Physiology, oxidative stress and production of metabolites in Hypericum perforatum exposed to moderate Cd and/or La concentration (10 µM) were studied. La evoked increase in reactive oxygen species, malondialdehyde and proline but suppressed growth, tissue water content, glutathione, ascorbic acid and affected mineral nutrient contents more than Cd while the impact of Cd+La was not synergistic. Similar trend was observed at the level of superoxide dismutase gene expression. Shoot Cd amount increased in Cd+La while only root La increased in the same treatment. Extensive quantification of secondary metabolites revealed that La affected phenolic acids more pronouncedly than Cd in shoots and roots. Flavonols were suppressed by La that could contribute to the appearance of oxidative damage. Procyanidins increased in response to La in the shoots but decreased in the roots. Metabolic responses in Cd+La treatment resembled those of La treatment (almost identically in the roots). Phenylalanine ammonia-lyase activity was mainly suppressed by La. The presence of La also depleted amount of hypericin and expression of its putative gene (hyp-1) showed similar trend but accumulation of hyperforin increased under Cd or La excess. Clear differences in the stem and root anatomy in response to Cd or La were also found. Overall, H. perforatum is La-sensitive species and rather Cd ameliorated negative impact of La.

Physiology and methodology of chromium toxicity using alga Scenedesmus quadricauda as model object.

Physiological responses of Scenedesmus quadricauda to Cr(VI) and Cr(III) excess were studied in buffer with circumneutral pH (6.5). Total Cr content was similar in low (1 µM of both oxidation states) but higher in 10 µM Cr(VI) treatment and high accumulation potential was detected (80-82% and 41-65% in 1 and 10 µM treatments, respectively). Specific fluorescence indicator (6-((anthracen-9-yl) methyleneamino)-2H-chromen-2-one) confirmed partial reduction of Cr(VI) to Cr(III) under exposure conditions. Viability and chlorophyll autofluorescence were more depleted by Cr(VI) while Cr(III) stimulated increase in ROS and lipid peroxidation. Antioxidative enzyme activities showed significantly higher values in 10 µM treatments of both Cr oxidation states. Depletion of mitochondrial proteins was not reflected in alteration of total soluble proteins indicating sensitivity of this organelle to Cr and TTC test showed no clear oxidation state-related effect. In this view, "Cr(VI) is not more toxic than Cr(III)" at least for some parameters. Subsequent study with the application of 10 µM Cr(VI) confirmed that HEPES buffer is more suitable exposure solution for toxicological studied than water or inorganic salts (higher chlorophyll autofluorescence was observed) and pH 6.5 is more suitable than low or high pH (4.5 or 8.5) in terms of Cr uptake. Another known Cr(III) fluorescence indicator (naphthalimide-rhodamine) also confirmed partial reduction of Cr(VI) to Cr(III) at acidic pH but only traces were seen at alkaline pH.

Development of Lymantria dispar affected by manganese in food.

We studied the response of gypsy moth (Lymantria dispar (Linnaeus) (Lepidoptera: Lymantriidae)) to the content of manganese in food in the laboratory breeding of caterpillars. The food of the caterpillars {Betula pendula Roth (Fagales: Betulaceae) leaves} was contaminated by dipping in the solution of MnCl2 · 4H2O with manganese concentrations of 0, 0.5, 5 and 10 mg ml(-1), by which differentiated manganese contents (307; 632; 4,087 and 8,124 mg kg(-1)) were reached. Parameters recorded during the rearing were as follows: effect of manganese on food consumption, mortality and length of the development of caterpillars, pupation and hatching of imagoes. At the same time, manganese concentrations were determined in the offered and unconsumed food, excrements, and exuviae of the caterpillars, pupal cases and imagoes by using the AAS method. As compared with the control, high manganese contents in the food of gypsy moth caterpillars affected the process of development particularly by increased mortality of the first instar caterpillars (8 % mortality for caterpillars with no Mn contamination (T0) and 62 % mortality for subjects with the highest contamination by manganese (T3)), by prolonged development of the first-third instar (18.7 days (T0) and 27.8 days (T3)) and by increased food consumption of the first-third instar {0.185 g of leaf dry matter (T0) and 0.483 g of leaf dry matter (T3)}. The main defence strategy of the caterpillars to prevent contamination by the increased manganese content in food is the translocation of manganese into frass and exuviae castoff in the process of ecdysis. In the process of development, the content of manganese was reduced by excretion in imagoes to 0.5 % of the intake level even at its maximum inputs in food.

Toxicity of naturally-contaminated manganese soil to selected crops.

The impact of manganese excess using naturally contaminated soil (Mn-soil, pseudototal Mn 6494 vs 675 µg g(-1) DW in control soil) in the shoots of four crops was studied. Mn content decreased in the order Brassica napus > Hordeum vulgare > Zea mays > Triticum aestivum. Growth was strongly depressed just in Brassica (containing 13696 µg Mn g(-1) DW). Some essential metals (Zn, Fe) increased in Mn-cultured Brassica and Zea, while macronutrients (K, Ca, Mg) decreased in almost all species. Toxic metals (Ni and Cd) were rather elevated in Mn-soil. Microscopy of ROS, NO, lipid peroxidation, and thiols revealed stimulation in all Mn-cultured crops, but changes were less visible in Triticum, a species with low shoot Mn (2363 µg g(-1) DW). Antioxidative enzyme activities were typically enhanced in Mn-cultured plants. Soluble phenols increased in Brassica only while proteins rather decreased in response to Mn excess. Inorganic anions (chloride, sulfate, and phosphate) were less accumulated in almost all Mn-cultured crops, while the nitrate level rather increased. Organic anions (malate, citrate, oxalate, acetate, and formate) decreased or remained unaffected in response to Mn-soil culture in Brassica, Hordeum, and Triticum but not in Zea. However, the role of organic acids in Mn uptake in these species is not assumed. Because control and Mn-soil differed in pH (6.5 and 3.7), we further studied its impact on Mn uptake in solution culture (using Mn concentration ∼5 mM deducted from water-soluble fraction of Mn-soil). Shoot Mn contents in Mn-treated plants were similar to those observed in soil culture (high in Brassica and low in Triticum) and pH had negligible impact. Fluorescence indicator of "general ROS" revealed no extensive or pH-dependent impact either in control or Mn-cultured roots. Observed toxicity of Mn excess to common crops urges for selection of cultivars with higher tolerance.

Comparison of oxidative stress in four Tillandsia species exposed to cadmium.

This is first study comparing four morphologically variable species of the genus Tillandsia and therefore various responses to the cadmium (Cd) action were expected. In accordance, Cd accumulation increased in order Tillandsia fasciculata < Tillandsia brachycaulos < Tillandsia pruinosa < Tillandsia capillaris, reaching 29.6 and 197.4 µg g(-1) DW in first and last species after watering with 2 µM Cd(2+) solution over 30 days. Fluorescence visualization of oxidative stress confirmed increase in ROS and especially elevation in hydroperoxides though no visible symptoms appeared on the plants. At the same time, nitric oxide generation and nitroso-glutathione depletion by Cd treatment were typically observed. Fluorescence staining of Cd using two dyes (PhenGreen and Leadmium) showed that Leadmium fits better with AAS quantification. Macro- and micro-nutrients were not considerably affected except for zinc. Reduced glutathione content was the highest in control T. fasciculata while oxidized glutathione in T. capillaris. Ascorbic acid amount revealed extreme quantitative differences among species and decreased in T. fasciculata only. Free amino acids accumulation was similar among species except for T. capillaris and Cd caused both depletion and increase but without high quantitative differences. Data are explanatively discussed in the context of limited literature related to oxidative stress in epiphytic plants and with general responses of plants to cadmium/heavy metals.

Hexavalent chromium damages chamomile plants by alteration of antioxidants and its uptake is prevented by calcium.

Toxicity of low (3µM) and high (60 and 120µM) concentrations of hexavalent chromium/Cr(VI) in chamomile plants was studied. Fluorescence staining confirmed reduction of Cr(VI) to Cr(III). Cr was mainly accumulated in the roots with translocation factor <0.007. Notwithstanding this, both shoots and roots revealed increase in oxidative stress and depletion of glutathione, total thiols, ascorbic acid and activities of glutathione reductase and partially ascorbate peroxidase mainly at 120µM Cr. Though some protective mechanisms were detected (elevation of nitric oxide, enhancement of GPX activity and increase in phenols and lignin), this was not sufficient to counteract the oxidative damage. Consequently, soluble proteins, tissue water content and biomass production were considerably depleted. Surprising increase in some mineral nutrients in roots (Ca, Fe, Zn and Cu) was also detected. Subsequent experiment confirmed that exogenous calcium suppressed oxidative symptoms and Cr uptake but growth of chamomile seedlings was not improved. Alteration of naturally present reductants could be a reason for Cr(III) signal detected using specific fluorescence reagent: in vitro assay confirmed disappearance of ascorbic acid in equimolar mixture with dichromate (>96% at pH 4 and 7) while such response of glutathione was substantially less visible.

Unexpected behavior of some nitric oxide modulators under cadmium excess in plant tissue.

Various nitric oxide modulators (NO donors--SNP, GSNO, DEA NONOate and scavengers--PTIO, cPTIO) were tested to highlight the role of NO under Cd excess in various ontogenetic stages of chamomile (Matricaria chamomilla). Surprisingly, compared to Cd alone, SNP and PTIO elevated Cd uptake (confirmed also by PhenGreen staining) but depleted glutathione (partially ascorbic acid) and phytochelatins PC2 and PC3 in both older plants (cultured hydroponically) and seedlings (cultured in deionised water). Despite these anomalous impacts, fluorescence staining of NO and ROS confirmed predictable assumptions and revealed reciprocal changes (decrease in NO but increase in ROS after PTIO addition and the opposite after SNP application). Subsequent tests using alternative modulators and seedlings confirmed changes to NO and ROS after application of GSNO and DEA NONOate as mentioned above for SNP while cPTIO altered only NO level (depletion). On the contrary to SNP and PTIO, GSNO, DEA NONOate and cPTIO did not elevate Cd content and phytochelatins (PC2, PC3) were rather elevated. These data provide evidence that various NO modulators are useful in terms of NO and ROS manipulation but interactions with intact plants affect metal uptake and must therefore be used with caution. In this view, cPTIO and DEA NONOate revealed the less pronounced side impacts and are recommended as suitable NO scavenger/donor in plant physiological studies under Cd excess.

Manganese-induced oxidative stress in two ontogenetic stages of chamomile and amelioration by nitric oxide.

Impact of manganese (Mn(2+)) excess (100, 500 and 1000 µM over 7 days) on two ontogenetic stages (7-week-old plants and 7-day-old seedlings) of Matricaria chamomilla was compared. Mn excess depressed growth of seedlings (but not germination) and stimulated oxidative stress (ROS and lipid peroxidation) in both plants and seedlings. Growth inhibition could be evoked by higher Mn uptake and higher translocation factor in seedlings than in plants. Total thiols staining revealed elevation in almost all treatments. In 7-week-old plants, activity of peroxidases increased slightly and rather decreased under high Mn doses. Superoxide rather than hydrogen peroxide contributed to visualized ROS presence. Fluorescence of nitric oxide (NO) showed stimulation in plants but decrease in seedlings. Impact of exogenous nitric oxide donor (sodium nitroprusside/SNP) was therefore tested and results showed amelioration of 1000 µM Mn-induced oxidative stress in seedlings (decrease in H2O2 and increase in NO content while antioxidative enzyme activities were variably affected) concomitantly with depleted Mn accumulation. It is concluded that NO participates in tolerance to Mn excess but negative effects of the highest SNP dose were also observed. Extensive fluorescence microscopy is also explanatively discussed.

Chromium uptake and consequences for metabolism and oxidative stress in chamomile plants.

Chromium Cr(III) toxicity toward chamomile metabolism and oxidative stress-related parameters after 7 days of exposure was studied. Cr preferentially accumulated in the roots and evoked extensive both dose-dependent and dose-independent increase in fluorescence signals of ROS, NO and thiols. Superoxide increased mainly at the highest Cr dose, whereas H2O2 accumulation revealed a discontinuous trend in relation to external Cr supply, and this could be owing to variation in activities of peroxidases. Glutathione and ascorbate quantification, using LC-MS/MS equipment, revealed strong stimulation despite low shoot Cr amounts. Phenolic enzyme activities, except for PAL, were depressed by Cr presence, whereas phenolic metabolites were stimulated, indicating various time dynamics. Among free amino acids, their sum and even proline decreased in the roots, whereas soluble proteins increased. Mineral nutrients showed negligible responses with only Zn and Cu being depleted in both shoots and roots. Cr staining using Cr(III)-specific (naphthalimide-rhodamine) and metal nonspecific (Phen Green) dyes indicated that the former correlated well with AAS quantification of Cr amount. Use of Phen Green is also discussed. These data indicate that Cr-induced oxidative stress is not simply a function of exposure time and applied concentration. Microscopic observations in terms of oxidative stress and chromium uptake are presented here for the first time.