Comparative analysis of geotypic variations in the proteome of Nostoc commune.

Cyanobacterium Nostoc commune is a filamentous terrestrial prokaryotic organism widely distributed, which suggest its high adaptive potential to environmental or abiotic stress. Physiological parameters and proteomic analysis were performed in two accession of N. commune with the aim to elucidate the differences of physiological trails between distant geotypes, namely Antarctic (AN) and central European (CE). The result obtained clearly showed that the AN geotype demonstrates elevated levels of total phenols, flavonoids, carotenoids, and phycobiliproteins, indicative of its adaptation to environmental stress as referred by comparison to CE sample. Additionally, we employed LC-MS analysis to investigate the proteomes of N. commune from AN and CE geotypes. In total, 1147 proteins were identified, among which 646 proteins expressed significant (up-regulation) changes in both accessions. In the AN geotype, 83 exclusive proteins were identified compared to 25 in the CE geotype. Functional classification of the significant proteins showed a large fraction involved in photosynthesis, amino acid metabolism, carbohydrate metabolism and protein biosynthesis. Further analysis revealed some defense-related proteins such as, superoxide dismutase (SOD) and glutathione reductase, which are rather explicitly expressed in the AN N. commune. The last two proteins suggest a more stressful condition in AN N. commune. In summary, our findings highlight biochemical processes that safeguard the AN geotype of N. commune from extreme environmental challenges, not recorded in CE accession, probably due to less stressful environment in Europe. This study brings the first ever proteomic analysis of N. commune, emphasizing the need for additional investigations into the climate adaptation of this species with rather plastic genome.

Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix.

(1) Background: Lichens, as an important part of the terrestrial ecosystem, attract the attention of various research disciplines. To elucidate their ultrastructure, transmission electron microscopy of resin-embedded samples is indispensable. Since most observations of lichen samples are generated via chemical fixation and processing at room temperature, they lack the rapid immobilization of live processes and are prone to preparation artefacts. To improve their preservation, cryoprocessing was tested in the past, but never widely implemented, not least because of an extremely lengthy protocol. (2) Methods: Here, we introduce an accelerated automated freeze substitution protocol with continuous agitation. Using the example of three lichen species, we demonstrate the preservation of the native state of algal photobionts and mycobionts in association with their extracellular matrix. (3) Results: We bring to attention the extent and the structural variability of the hyphae, the extracellular matrix and numerous crystallized metabolites. Our findings will encourage studies on transformation processes related to the compartmentation of lichen thalli. They include cryopreserved aspects of algal photobionts and observations of putative physiological relevance, such as the arrangement of numerous mitochondria within chloroplast pockets. (4) Conclusions: In summary, we present accelerated freeze substitution as a very useful tool for systematic studies of lichen ultrastructures.

Mechanisms of Copper Toxicity and Tolerance in the Aquatic Moss Taxiphyllum barbieri.

Aquatic habitats are very frequently polluted with different kinds of xenobiotics, including heavy metals. For biomonitoring studies of aquatic pollution, algae are frequently used, as they do not contain protective cuticle on the surface of their thalli and can accumulate pollutants over the whole surface of thalli. However, this is a feature of most cryptogams. For this reason, we assessed the sensitivity of the aquatic moss Taxiphyllum barbieri (Java moss) to copper excess in a short-term study. Moss T. barbieri belongs to the common aquatic plants originating from Southeast Asia. To test the sensitivity (or tolerance) of the moss to excess Cu, selected concentrations (50, 250 and 500 µM) were employed in our 24 h studies. Total and intracellular Cu accumulation positively correlated with Cu availability in the water. This total and intracellular Cu accumulation was negatively correlated with decreased intracellular K content. Excess Cu negatively affected the composition of assimilation pigments and soluble proteins. Cu caused increased peroxidation of membrane lipids assessed using TBARS assay. Excess Cu decreased GSH to GSSG ratio and ascorbic acid content. We did not observe phytochelatin synthesis in this moss. The roles of selected amino acids, their intermediates and derivatives, as well as S-containing nucleosides and phenolic acids in Cu homeostasis and toxicity or tolerance were evaluated. We assume that this moss has potential for future employment in water quality evaluation.

Atranorin, a Secondary Metabolite of Lichens, Exhibited Anxiolytic/Antidepressant Activity in Wistar Rats.

Atranorin (ATR) is one of lichens' many known secondary metabolites. Most current studies have investigated the various effects of ATR in vitro and only sporadically in vivo. The latest data indicate that ATR may have anxiolytic/antidepressive effects. This study aimed to analyze the potential of ATR in a depression-like state in male Wistar rats. Pregnant females were stressed by restricting their mobility in the final week of pregnancy three times a day for 45 min each, for three following days. After birth, progeny aged 60 days was stressed repeatedly. The male progeny was divided into three groups as follows: CTR group as a healthy control (n = 10), DEP group as a progeny of restricted mothers (n = 10), and ATR group as a progeny of restricted mothers, treated daily for one month with ATR (n = 10; 10 mg/kg of body weight, p.o.). Our results show that ATR acts as an antioxidant and markedly changes animal behavior. Concomitantly, hippocampal neurogenesis increases in the hilus and subgranular zone, together with the number of NeuN mature neurons in the hilus and CA1 regions. Our results indicate a potential antidepressant/anxiolytic effect of ATR. However, further studies in this area are needed.

Usnic Acid Isolated from Usnea antarctica (Du Rietz) Reduced In Vitro Angiogenesis in VEGF- and bFGF-Stimulated HUVECs and Ex Ovo in Quail Chorioallantoic Membrane (CAM) Assay.

Natural products include a diverse set of compounds of drug discovery that are currently being actively used to target tumor angiogenesis. In the present study, we evaluated the anti-angiogenic activities of secondary metabolite usnic acid isolated from Usena antarctica. We investigated the in vitro effects on proliferation, migration, and tube formation of VEGF- and bFGF-stimulated HUVECs. Ex ovo anti-angiogenic activity was evaluated using the CAM assay. Our findings demonstrated that usnic acid in the concentration of 33.57 µM inhibited VEGF (25 ng/mL) and bFGF (30 ng/mL)-induced HUVECs proliferation, migration, and tube formation. The ex ovo CAM model was used to confirm the results obtained from in vitro studies. VEGF- and bFGF-induced vessel formation was inhibited by usnic acid after 72 h in over 2-fold higher concentrations compared to in vitro. Subsequently, histological sections of affected chorioallantoic membranes were stained with hematoxylin-eosin and alcian blue to determine the number and diameter of vessels as well as the thickness of the individual CAM layers (ectoderm, mesoderm, endoderm). Usnic acid was able to suppress the formation of VEGF- and bFGF-induced vessels with a diameter of less than 100 µm, which was demonstrated by the reduction of mesoderm thickness as well.

A Comparative Study of Isolated Secondary Metabolites from Lichens and Their Antioxidative Properties.

Free radicals play a critical role in the chemical processes that occur in all cells. Pharmaceutical companies manufacture a variety of synthetically prepared antioxidants, but it is known that many of these can be carcinogenic. As a result, efforts are being made to find natural antioxidants that do not have these side effects. Lichens may be suitable candidates because they contain secondary metabolites with proven antioxidant properties. This could be explained by the presence of compounds with phenolic groups in lichens. The radical scavenging reaction is a chemical reaction governed by stoichiometry, and our aim is to determine the efficacy of these reactions. The aim of this study is to compare metabolite activity based on the same amount of substance involved in radical scavenging, calculated in micromoles rather than weight concentration. This provides an accurate way of comparing radical scavenging activity. We tested superoxide anion scavenging activity and free radical scavenging activity of isolated lichen secondary metabolites and their mixtures in different ratios. The following compounds were isolated and tested for antioxidant activity: gyrophoric acid (Umbilicaria hirsuta), evernic acid (Evernia prunastri), physodic acid, 3-hydroxyphysodic acid, physodalic acid and atranorin (Hypogymnia physodes), and usnic acid (as a synthetic compound). Of all the tested compounds, 3-hydroxyphysodic acid, as well as mixtures containing this metabolite, showed the strongest scavenging activity. The results also demonstrated that calculation by amount of substance leads to a new consideration of antioxidant activity.

The Absence of the AtSYT1 Function Elevates the Adverse Effect of Salt Stress on Photosynthesis in Arabidopsis.

Arabidopsis thaliana SYNAPTOTAGMIN 1 (AtSYT1) was shown to be involved in responses to different environmental and biotic stresses. We investigated gas exchange and chlorophyll a fluorescence in Arabidopsis wild-type (WT, ecotype Col-0) and atsyt1 mutant plants irrigated for 48 h with 150 mM NaCl. We found that salt stress significantly decreases net photosynthetic assimilation, effective photochemical quantum yield of photosystem II (ΦPSII), stomatal conductance and transpiration rate in both genotypes. Salt stress has a more severe impact on atsyt1 plants with increasing effect at higher illumination. Dark respiration, photochemical quenching (qP), non-photochemical quenching and ΦPSII measured at 750 µmol m-2 s-1 photosynthetic photon flux density were significantly affected by salt in both genotypes. However, differences between mutant and WT plants were recorded only for qP and ΦPSII. Decreased photosynthetic efficiency in atsyt1 under salt stress was accompanied by reduced chlorophyll and carotenoid and increased flavonol content in atsyt1 leaves. No differences in the abundance of key proteins participating in photosynthesis (except PsaC and PsbQ) and chlorophyll biosynthesis were found regardless of genotype or salt treatment. Microscopic analysis showed that irrigating plants with salt caused a partial closure of the stomata, and this effect was more pronounced in the mutant than in WT plants. The localization pattern of AtSYT1 was also altered by salt stress.

Potential Effect of Pseudevernia furfuracea (L.) Zopf Extract and Metabolite Physodic Acid on Tumour Microenvironment Modulation in MCF-10A Cells.

Lichens comprise a number of unique secondary metabolites with remarkable biological activities and have become an interesting research topic for cancer therapy. However, only a few of these metabolites have been assessed for their effectiveness against various in vitro models. Therefore, the aim of the present study was to assess the effect of extract Pseudevernia furfuracea (L.) Zopf (PSE) and its metabolite physodic acid (Phy) on tumour microenvironment (TME) modulation, focusing on epithelial-mesenchymal transition (EMT), cancer-associated fibroblasts (CAFs) transformation and angiogenesis. Here, we demonstrate, by using flow cytometry, Western blot and immunofluorescence microscopy, that tested compounds inhibited the EMT process in MCF-10A breast cells through decreasing the level of different mesenchymal markers in a time- and dose-dependent manner. By the same mechanisms, PSE and Phy suppressed the function of Transforming growth factor beta (TGF-ß)-stimulated fibroblasts. Moreover, PSE and Phy resulted in a decreasing level of the TGF-ß canonical pathway Smad2/3, which is essential for tumour growth. Furthermore, PSE and Phy inhibited angiogenesis ex ovo in a quail embryo chorioallantoic model, which indicates their potential anti-angiogenic activity. These results also provided the first evidence of the modulation of TME by these substances.

Biological activity of selected lichens and lichen-based Ag nanoparticles prepared by a green solid-state mechanochemical approach.

Lichens dispose a wide spectrum of bioactive compounds known as secondary metabolites. Their biological effects like antioxidant and antibacterial activities are widely studied. Green synthesis of silver nanoparticles (AgNPs) is a method where the compounds/substances present in plants are used for reduction of AgNO3instead of toxic chemicals. However, this methodology is usually a two-step process (extract preparation step and the synthesis step) performed under the elevated temperatures nad in the case of lichens, the redicing compounds are insoluble in water. These disadvantages can be overcome by a solid-state mechanochemical synthesis applied in the present study. As microorganisms are becoming more resistant to commercial antibiotics, AgNPs prepared in an environmentally friendly way represent an interesting alternative. In the present study, we compared the processing of lichen material of Pseudevernia furfuracea and Lobaria pulmonaria for extraction as well as for synthesis of AgNPs, and tested the antibacterial and antioxidant activity of the extracts. Both selected lichen species could be successfully used as reducing agents to produce AgNPs. Six different bacterial strains were tested for antibacterial activity of AgNPs-containing products and it was highly effective on all strains. However, the antioxidant activity of lichen extracts showed the lowest effect even if AgNPs are present which positively correlated with the content of total phenols and flavonoids. Both phenols and flavonoids are natural antioxidants and react with silver nitrate. Due to this fact, we observed a decrease of total phenols, total flavonoids as well as antioxidant activity when processing of lichen extracts with silver nitrate was used. We demonstrated that the formation of AgNPs increased the antibacterial activity but on the other hand reduced the antioxidant activity. Thus, antibacterial and antioxidant effects have to be treated differentially.

Oxidative stress mediated by gyrophoric acid from the lichen Umbilicaria hirsuta affected apoptosis and stress/survival pathways in HeLa cells.

BACKGROUND: Lichens produce a huge diversity of bioactive compounds with several biological effects. Gyrophoric acid (GA) is found in high concentrations in the common lichen Umbilicaria hirsuta, however evidence for biological activity was limited to anti-proliferative activity described on several cancer cell lines. METHODS: We developed and validated a new protocol for GA isolation, resulting in a high yield of highly pure GA (validated by HPLC and NMR) in an easy and time saving manner. Anti-proliferative and pro-apoptotic activity, oxygen radicals formation and stress/survival proteins activity changes was study by flow cytometry. RESULTS: The highly purified GA showed anti-proliferative activity against HeLa (human cervix carcinoma) and other tumor cells. Moreover, GA threated cells showed a significant increase in caspase-3 activation followed by PARP cleavage, PS externalization and cell cycle changes mediated by oxidative stress. Production of oxygen radicals led to DNA damage and changes in stress/survival pathways activation. CONCLUSIONS: GA treatment on HeLa cells clearly indicates ROS production and apoptosis as form of occurred cell death. Moreover, DNA damage and changing activity of stress/survival proteins as p38MAPK, Erk1/2 and Akt mediated by GA treatment confirm pro-apoptotic potential. The pharmacological potential of U. hirsuta derived GA is discussed.

Insights into physiological responses of mosses Physcomitrella patens and Pohlia drummondii to lichen secondary metabolites.

It is widely accepted that allelopathy among mosses and lichens do exist due to its similar ecological needs, though it is rarely documented. With an aim to test whether there is an effect of allelochemicals to mosses, we grow axenically two moss species (namely Physcomitrella patens and Pohlia drummondii) in controlled conditions and use them to test the effect of lichen Pseudevernia furfuracea acetone extracts containing active compounds: atranorin, chloratranorin, and physodic acid. The photosynthesis value and the biochemical parameters were measured to detect changes in moss organisms upon application of different concentration of lichen extract. The results obtained clearly showed that both moss species reacted to allelochemicals applied in test but to different extent. This suggests that tested moss species have various patterns on reaction to allelochemicals, and that the process of allelopathy is rather a recently coevolving one, than pre-defined. The lichen secondary metabolites are allelochemicals effective also to moss species that are not selected lichen cohabitants.

Usnic acid, as a biotic factor, changes the ploidy level in mosses.

Lichens and mosses often share the same environmental conditions where they compete for substrate and other essential factors. Lichens use secondary metabolites as allelochemicals to repel surrounding plants and potential rivals. In mosses, endoreduplication leads to the occurrence of various ploidy levels in the same individual and has been suggested as an adaptation to abiotic stresses. Here, we show that also biotic factors such as usnic acid, an allelochemical produced by lichens, directly influenced the level of ploidy in mosses. Application of usnic acid changed the nuclei proportion and significantly enhanced the endoreduplication index in two moss species, Physcomitrella patens and Pohlia drummondii. These investigations add a new aspect on secondary metabolites of lichens which count as biotic factors and affect ploidy levels in mosses.

Toxicity of Diclofenac in the Fern Azolla filiculoides and the Lichen Xanthoria parietina.

This study investigated the occurrence of toxicity, expressed as damage to the photosynthetic apparatus, in the aquatic fern Azolla filiculoides and the lichen Xanthoria parietina following treatments with diclofenac at different concentrations (0.1, 1, 10 and 100 mg/L) and different exposure times (24, 48, 72 and 240 h). Measurements of photosynthetic efficiency, chlorophyll content and chlorophyll degradation indicated dose- and time-dependent toxicity, since significant differences with control samples as well as among treatments, emerged mainly for the highest concentration (100 mg/L) and the longest time (240 h). In addition, also the mycobiont of the lichen X. parietina showed similar toxic effects, expressed as ergosterol content. The absence of relevant alterations at the lowest concentration (0.1 mg/L) suggested a very limited susceptibility of these species to environmentally relevant levels of this pharmaceutical.

A drug delivery system based on switchable photo-controlled p-coumaric acid derivatives anchored on mesoporous silica.

A stimuli-responsive drug delivery system consisting of mesoporous silica with its surface modified by p-coumaric acid derivatives (CA) as photo-switchable ligands was studied for the delivery of the non-steroidal anti-inflammatory drug (NSAID) naproxen. For this purpose, MCM-41 mesoporous silica material was prepared and post-synthetically modified by the grafting of a triethoxysilyl derivative of p-coumaric acid, which undergoes a reversible photo-dimerization under UV irradiation and creates the "valves" on the surface of silica allowing targeted opening/closing of the pores. Naproxen was encapsulated into grafted MCM-41 and drug release studies were performed in two different media, in a simulated gastric fluid (pH = 2) and in a simulated body fluid (pH = 7.4). Differences in drug release were observed after irradiation of the material using UV light at λ = 365 nm (closed pore configuration) and UV light at λ = 254 nm (opened pore configuration).

Lichen secondary metabolites affect growth of Physcomitrella patens by allelopathy.

Lichen secondary metabolites can function as allelochemicals and affect the development and growth of neighboring bryophytes, fungi, vascular plants, microorganisms, and even other lichens. Lichen overgrowth on bryophytes is frequently observed in nature even though mosses grow faster than lichens, but there is still little information on the interactions between lichens and bryophytes.In the present study, we used extracts from six lichen thalli containing secondary metabolites like usnic acid, protocetraric acid, atranorin, lecanoric acid, nortistic acid, and thamnolic acid. To observe the influence of these metabolites on bryophytes, the moss Physcomitrella patens was cultivated for 5 weeks under laboratory conditions and treated with lichen extracts. Toxicity of natural mixtures of secondary metabolites was tested at three selected doses (0.001, 0.01, and 0.1 %). When the mixture contained substantial amounts of usnic acid, we observed growth inhibition of protonemata and reduced development of gametophores. Significant differences in cell lengths and widths were also noticed. Furthermore, usnic acid had a strong effect on cell division in protonemata suggesting a strong impact on the early stages of bryophyte development by allelochemicals contained in the lichen secondary metabolites.Biological activities of lichen secondary metabolites were confirmed in several studies such as antiviral, antibacterial, antitumor, antiherbivore, antioxidant, antipyretic, and analgetic action or photoprotection. This work aimed to expand the knowledge on allelopathic effects on bryophyte growth.

Murine breast carcinoma 4T1 cells are more sensitive to atranorin than normal epithelial NMuMG cells in vitro: Anticancer and hepatoprotective effects of atranorin in vivo.

The aim of this study was to evaluate the anticancer effect of atranorin (ATR) on murine 4T1 breast carcinoma cells and compare its sensitivity with normal mammary epithelial NMuMG cells in vitro. Anti-tumor and hepatoprotective activity of ATR-therapy was examined on mouse model of 4T1-induced cancer disease. ATR significantly reduced clonogenic ability of carcinoma 4T1 cells at the concentration of 75 µM, but clonogenicity of normal NMuMG cells was not affected by any of ATR concentrations tested. Moreover, flow cytometric and BrdU incorporation analysis did not confirm the inhibited entry into S-phase of the cell cyle after ATR incubation, and on the contrary, it induced apoptosis associated with the activation of caspase-3 and PARP cleavage in 4T1 cells. Although ATR did not cause any significant changes in Bcl-xL protein expression in NMuMG cells, an apparent depletion of Bcl-xL protein in 4T1 cells after 48 h ATR therapy was confirmed. Based on this result as well as the result of the total cell number decline, we can conclude that 4T1 cells are more sensitive to ATR therapy than NMuMG cells. ATR administration resulted in significantly longer survival time of BALB/c mice inoculated with 4T1 cells, what was associated with reduced tumor size and the higher numbers of apoptotic 4T1 cells. No differences were recorded in the number of BrdU-positive tumor cells between ATR-treated group and controls. Results indicate that ATR has rather proapoptotic than antiproliferative effect on 4T1 cells in vitro and in vivo and normal NMuMG cells are less sensitive to ATR. Furthermore, our studies revealed protective effect of ATR against oxidative stress in the livers of the tumor-bearing mice.

Effects of acute NH3 air pollution on N-sensitive and N-tolerant lichen species.

Lichens are sensitive to the presence of ammonia (NH3) in the environment. However, in order to use them as reliable indicators in biomonitoring studies, it is necessary to establish unequivocally the occurrence of certain symptoms following the exposure to NH3 in the environment. In this paper, we simulated an episode of acute air pollution due to the release of NH3. The biological effects of acute air pollution by atmospheric NH3 have been investigated using N-sensitive (Flavoparmelia caperata) and N-tolerant (Xanthoria parietina) species. Lichen samples were exposed to ecologically relevant NH3 concentrations for 8 weeks, simulating three areas of impact: a control area (2 µg/m(3)), an area of intermediate impact (2-35 µg/m(3)) and an area of high impact (10-315 µg/m(3)), with a peak of pollution reached between the fourth and fifth week. Ammonia affected both the photobiont and the mycobiont in F. caperata, while in X. parietina only the photosynthetic performance of the photobiont was altered after exposure to the highest concentration. In the photobiont of F. caperata we recorded chlorophyll degradation as indicated by OD435/415 ratio, decrease of the photosynthetic performance (as reflected by the maximum quantum yield of primary photochemistry FV/FM and the performance index PIABS); in the mycobiont, ergosterol reduction, membrane lipid peroxidation (as reflected by the increase of thiobarbituric acid reactive substances), alteration (decrease) of the secondary metabolite usnic acid. No effects were detected on caperatic acid and dehydrogenase activity. In X. parietina, the only signal determined by NH3 was the alteration of FV/FM and the performance index PIABS. The results suggest that physiological parameters in N-sensitive lichens well reflect the effects of NH3 exposure and can be applied as early indicators in monitoring studies.

Uptake and toxicity of glyphosate in the lichen Xanthoria parietina (L.) Th. Fr.

This study investigated if treatment of the lichen Xanthoria parietina (L.) Th. Fr. with glyphosate caused uptake of this herbicide as well as physiological alterations. Samples were treated with Glifene SL®, a common commercial glyphosate-based herbicide, at the lowest recommended doses (3.6g/L) as well as with doses slightly higher than the highest suggested (36 g/L). The results clearly showed glyphosate uptake in X. parietina proportionally to the dose provided. Adverse physiological effects were evident on the photosynthetic apparatus (photosynthetic efficiency, chlorophyll a content, chlorophyll degradation) as well as on the fungal respiration rates and cell membrane integrity (ergosterol content, dehydrogenase activity) already after 24h from treatment, also at the low application dose. It is concluded that lichens are suitable organisms for monitoring unwanted biological effects from the application of glyphosate-based herbicides, as well as for detecting the accumulation of this compound in the biota, thus screening for its environmental fate.

Epiphytic lichens as indicators of environmental quality around a municipal solid waste landfill (C Italy).

Epiphytic lichens have been used as indicators of environmental quality around a municipal solid waste landfill in C Italy. An integrated approach, using the diversity of epiphytic lichens, as well as element bioaccumulation and physiological parameters in the lichen Flavoparmelia caperata (L.) Hale was applied along a transect from the facility. The results highlighted the biological effects of air pollution around the landfill. The Index of Lichen Diversity (ILD) increased and the content of heavy metals (Cr, Cd, Cu, Fe, Ni and Zn) decreased with distance from the landfill. Clear stress signals were observed in lichens growing in front of the facility, i.e. discoloration, necrosis, membrane lipid peroxidation, lower ergosterol content, higher dehydrogenase activity. Decreased photosynthetic efficiency, altered chlorophyll integrity and production of secondary metabolites were also found. The results suggested that lichens can be profitably used as bioindicators of environmental quality around landfills.

Changes of metal-induced toxicity by H2O 2/NO modulators in Scenedesmus quadricauda (Chlorophyceae).

Effect of nitric oxide donor (sodium nitroprusside, SNP, 500 µM) or hydrogen peroxide scavenger (dithiothreitol, DTT, 500 µM) on cadmium (Cd) or copper (Cu) uptake (150 µM solutions) and toxicity using Scenedesmus quadricauda was studied. Combined treatments (Cd or Cu + DTT or SNP) usually ameliorated metal-induced toxicity at the level of pigments, proteins, and mineral nutrients in comparison with respective metal alone. Viability tests (MTT and TTC) showed the lowest values preferentially in Cu treatments, indicating higher toxicity in comparison with Cd. Cd showed low impact on amino acids while strong Cu-induced depletion was mitigated by DTT and SNP. Amount of ROS and NO showed the most pronounced responses in SNP variants being rather reciprocal than parallel and regulated ascorbate peroxidase activity. Blot gel analyses of hsp70 protein did not reveal extensive changes after given exposure period. Phenols were elevated by DTT alone while all Cu treatments revealed depletion. Total Cu content decreased while total Cd content increased in metal + SNP or metal + DTT. Subsequent experiment using lower Cd, SNP or DTT doses (10 and 100 µM) revealed concentration-dependent impact on Cd uptake. Overall, DTT was found to be more suitable for the amelioration of metal-induced toxicity.