Oxidative Stress Response of Honey Bee Colonies (Apis mellifera L.) during Long-Term Exposure at a Frequency of 900 MHz under Field Conditions.

In this study, oxidative stress and lipid peroxidation in honey bee larvae, pupae and the midguts of adult bees were investigated during a one-year exposure to radiofrequency electromagnetic fields (RF-EMFs) at a frequency of 900 MHz under field conditions. The experiment was carried out on honey bee colonies at three locations with electric field levels of 30 mV m-1, 70 mV m-1 and 1000 mV m-1. Antioxidant enzymes, glutathione-S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS) as indicators of lipid peroxidation were measured spectrophotometrically. The GST activity within the same developmental stage showed no significant differences regardless of electric field level or sampling time. The highest GST activity was found in the pupae, followed by activity in the larvae and midguts. Both CAT activity and TBARS concentration were the highest in the midguts, regardless of field level and sampling time. The larvae showed a significantly higher TBARS concentration at the location with an electric field level of 1000 mV m-1 compared to the locations with lower levels. Our results show that RF-EMFs at a frequency of 900 MHz can cause oxidative stress in honey bees, with the larval stage being more sensitive than the pupal stage, but there was no linear relationship between electric field level and effect in any of the developmental stages.

Phytotoxic Effects of Polystyrene and Polymethyl Methacrylate Microplastics on Allium cepa Roots.

Plastic contamination has become one of the most pressing environmental issues due to rapidly increasing production of disposable plastic products, their fragmentation into smaller pieces, and long persistence in the environment, which affects all living organisms, including plants. In this study, Allium cepa roots were exposed to 0.01, 0.1, and 1 g L-1 of commercial polystyrene (PS-MPs) and polymethyl methacrylate microparticles (PMMA-MPs) for 72 h. Dynamic light scattering (DLS) analyses showed high stability of both types of MPs in ultrapure water used for A. cepa treatment. Morphometric analysis revealed no significant change in root length compared to control. Pyrolysis hyphenated to gas chromatography and mass spectrometry (Py-GC-MS) has proven PS-MPs uptake by onion roots in all treatments, while PMMA-MPs were recorded only upon exposure to the highest concentration. Neither MPs induced any (cyto)toxic effect on root growth and PMMA-MPs even had a stimulating effect on root growth. ROS production as well as lipid and protein oxidation were somewhat higher in PS-MP treatments compared to the corresponding concentrations of PMMA-MP, while neither of the applied MPs induced significant damage to the DNA molecule assayed with a Comet test. Significantly elevated activity of H2O2 scavenging enzymes, catalase, and peroxidases was measured after exposure to both types of MPs. Obtained results suggest that onion roots take up PS-MPs more readily in comparison to PMMA-MPs, while both types of MPs induce a successful activation of antioxidant machinery in root cells that prevented the occurrence of toxic effects.

Effects of Silver Nanoparticles on Physiological and Proteomic Responses of Tobacco (Nicotiana tabacum) Seedlings Are Coating-Dependent.

The harmful effects of silver nanoparticles (AgNPs) have been confirmed in many organisms, but the mechanism of their toxicity is not yet fully understood. In biological systems, AgNPs tend to aggregate and dissolve, so they are often stabilized by coatings that influence their physico-chemical properties. In this study, the effects of AgNPs with different coatings [polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB)] on oxidative stress appearance and proteome changes in tobacco (Nicotiana tabacum) seedlings have been examined. To discriminate between the nanoparticulate Ag form from the ionic one, the treatments with AgNO3, a source of Ag+ ions, were also included. Ag uptake and accumulation were found to be similarly effective upon exposure to all treatment types, although positively charged AgNP-CTAB showed less stability and a generally stronger impact on the investigated parameters in comparison with more stable and negatively charged AgNP-PVP and ionic silver (AgNO3). Both AgNP treatments induced reactive oxygen species (ROS) formation and increased the expression of proteins involved in antioxidant defense, confirming oxidative stress as an important mechanism of AgNP phytotoxicity. However, the mechanism of seedling responses differed depending on the type of AgNP used. The highest AgNP-CTAB concentration and CTAB coating resulted in increased H2O2 content and significant damage to lipids, proteins and DNA molecules, as well as a strong activation of antioxidant enzymes, especially CAT and APX. On the other hand, AgNP-PVP and AgNO3 treatments induced the nonenzymatic antioxidants by significantly increasing the proline and GSH content. Exposure to AgNP-CTAB also resulted in more noticeable changes in the expression of proteins belonging to the defense and stress response, carbohydrate and energy metabolism and storage protein categories in comparison to AgNP-PVP and AgNO3. Cysteine addition significantly reduced the effects of AgNP-PVP and AgNO3 for the majority of investigated parameters, indicating that AgNP-PVP toxicity mostly derives from released Ag+ ions. AgNP-CTAB effects, however, were not alleviated by cysteine addition, suggesting that their toxicity derives from the intrinsic properties of the nanoparticles and the coating itself.

Mechanisms of Kale (Brassica oleracea var. acephala) Tolerance to Individual and Combined Stresses of Drought and Elevated Temperature.

Rising temperatures and pronounced drought are significantly affecting biodiversity worldwide and reducing yields and quality of Brassica crops. To elucidate the mechanisms of tolerance, 33 kale accessions (B. oleracea var. acephala) were evaluated for individual (osmotic and elevated temperature stress) and combined stress (osmotic + temperature). Using root growth, biomass and proline content as reliable markers, accessions were evaluated for stress responses. Four representatives were selected for further investigation (photosynthetic performance, biochemical markers, sugar content, specialized metabolites, transcription level of transcription factors NAC, HSF, DREB and expression of heat shock proteins HSP70 and HSP90): very sensitive (392), moderately sensitive (395), tolerant (404) and most tolerant (411). Accessions more tolerant to stress conditions were characterized by higher basal content of proline, total sugars, glucosinolates and higher transcription of NAC and DREB. Under all stress conditions, 392 was characterized by a significant decrease in biomass, root growth, photosynthesis performance, fructan content, especially under osmotic and combined stress, a significant increase in HSF transcription and HSP accumulation under temperature stress and a significant decrease in NAC transcription under all stresses. The most tolerant accession under all applied stresses, 411 showed the least changes in all analyzed parameters compared with the other accessions.

Silver Nanoparticle Effects on Antioxidant Response in Tobacco Are Modulated by Surface Coating.

The antimicrobial properties of silver and enhanced reactivity when applied in a nanoparticle form (AgNPs) led to their growing utilization in industry and various consumer products, which raises concerns about their environmental impact. Since AgNPs are prone to transformation, surface coatings are added to enhance their stability. AgNP phytotoxicity has been mainly attributed to the excess generation of reactive oxygen species (ROS), leading to the induction of oxidative stress. Herein, in vitro-grown tobacco (Nicotiana tabacum) plants were exposed to AgNPs stabilized with either polyvinylpyrrolidone (PVP) or cetyltrimethylammonium bromide (CTAB) as well as to ionic silver (AgNO3), applied in the same concentrations, either alone or in combination with cysteine, a strong silver ligand. The results show a higher accumulation of Ag in roots and leaves after exposure to AgNPs compared to AgNO3. This was correlated with a predominantly higher impact of nanoparticle than ionic silver form on parameters of oxidative stress, although no severe damage to important biomolecules was observed. Nevertheless, all types of treatments caused mobilization of antioxidant machinery, especially in leaves, although surface coatings modulated the activation of its specific components. Most effects induced by AgNPs or AgNO3 were alleviated with addition of cysteine.

Laccase Activity in Fungus Cryphonectria parasitica Is Affected by Growth Conditions and Fungal-Viral Genotypic Interactions.

Laccase activity reduction in the chestnut blight fungus Cryphonectria parasitica usually accompanies the hypovirulence caused by the infection of fungus with Cryphonectria hypovirus 1 (CHV1). However, the different methods utilized for assessing this phenomenon has produced varied and often conflicting results. Furthermore, the majority of experimental setups included only one prototypic system, further confounding the results. Considering the diversity of fungal isolates, viral strains, and variability of their effects on the phytopathogenic process observed in nature, our goal was to ascertain if laccase activity variability is affected by (1) different C. parasitica isolates infected with several CHV1 strains, and (2) growth conditions. We have demonstrated that some CHV1 strains, contrary to previous assumptions, increase the activity of C. parasitica laccases. The specific fungal isolates used in the experiments and culture conditions also affected the results. Furthermore, we showed that two commonly used laccase substrates, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 2,4-dimethoxyphenol, cannot be used interchangeably in C. parasitica laccase activity measurements. Our results illustrate the importance of conducting this type of study in experimental systems and culture conditions that resemble natural conditions as much as possible to be able to infer the most relevant conclusions applicable to natural populations.

Phytotoxicity of Silver Nanoparticles on Tobacco Plants: Evaluation of Coating Effects on Photosynthetic Performance and Chloroplast Ultrastructure.

Silver nanoparticles (AgNPs) are the most exploited nanomaterial in agriculture and food production, and their release into the environment raises concern about their impact on plants. Since AgNPs are prone to biotransformation, various surface coatings are used to enhance their stability, which may modulate AgNP-imposed toxic effects. In this study, the impact of AgNPs stabilized with different coatings (citrate, polyvinylpyrrolidone (PVP), and cetyltrimethylammonium bromide (CTAB)) and AgNO3 on photosynthesis of tobacco plants as well as AgNP stability in exposure medium have been investigated. Obtained results revealed that AgNP-citrate induced the least effects on chlorophyll a fluorescence parameters and pigment content, which could be ascribed to their fast agglomeration in the exposure medium and consequently weak uptake. The impact of AgNP-PVP and AgNP-CTAB was more severe, inducing a deterioration of photosynthetic activity along with reduced pigment content and alterations in chloroplast ultrastructure, which could be correlated to their higher stability, elevated Ag accumulation, and surface charge. In conclusion, intrinsic properties of AgNP coatings affect their stability and bioavailability in the biological medium, thereby indirectly contributing changes in the photosynthetic apparatus. Moreover, AgNP treatments exhibited more severe inhibitory effects compared to AgNO3, which indicates that the impact on photosynthesis is dependent on the form of Ag.

Surface Coating-Modulated Phytotoxic Responses of Silver Nanoparticles in Plants and Freshwater Green Algae.

Silver nanoparticles (AgNPs) have been implemented in a wide range of commercial products, resulting in their unregulated release into aquatic as well as terrestrial systems. This raises concerns over their impending environmental effects. Once released into the environment, they are prone to various transformation processes that modify their reactivity. In order to increase AgNP stability, different stabilizing coatings are applied during their synthesis. However, coating agents determine particle size and shape and influence their solubility, reactivity, and overall stability as well as their behavior and transformations in the biological medium. In this review, we attempt to give an overview on how the employment of different stabilizing coatings can modulate AgNP-induced phytotoxicity with respect to growth, physiology, and gene and protein expression in terrestrial and aquatic plants and freshwater algae.

Coating-Dependent Effects of Silver Nanoparticles on Tobacco Seed Germination and Early Growth.

Silver nanoparticles (AgNPs) are used in a wide range of consumer products because of their excellent antimicrobial properties. AgNPs released into the environment are prone to transformations such as aggregation, oxidation, or dissolution so they are often stabilised by coatings that affect their physico-chemical properties and change their effect on living organisms. In this study we investigated the stability of polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB) coated AgNPs in an exposure medium, as well as their effect on tobacco germination and early growth. AgNP-CTAB was found to be more stable in the solid Murashige and Skoog (MS) medium compared to AgNP-PVP. The uptake and accumulation of silver in seedlings was equally efficient after exposure to both types of AgNPs. However, AgNP-PVP induced only mild toxicity on seedlings growth, while AgNP-CTAB caused severe negative effects on all parameters, even compared to AgNO3. Moreover, CTAB coating itself exerted negative effects on growth. Cysteine addition generally alleviated AgNP-PVP-induced negative effects, while it failed to improve germination and growth parameters after exposure to AgNP-CTAB. These results suggest that the toxic effects of AgNP-PVP are mainly a consequence of release of Ag+ ions, while phytotoxicity of AgNP-CTAB can rather be ascribed to surface coating itself.

Comparative proteomic study of phytotoxic effects of silver nanoparticles and silver ions on tobacco plants.

Widespread application of silver nanoparticles (AgNPs), due to their antibacterial and antifungal properties, increases their release into the environment and potential detrimental impact on living organisms. Plants may serve as a potential pathway for AgNPs bioaccumulation and a route into the food chain, hence investigation of AgNP phytotoxic effects are of particular importance. Since proteins are directly involved in stress response, studies of their abundance changes can help elucidate the mechanism of the AgNP-mediated phytotoxicity. In this study, we investigated proteomic changes in tobacco (Nicotiana tabacum) exposed to AgNPs and ionic silver (AgNO3). A high overlap of differently abundant proteins was found in root after exposure to both treatments, while in leaf, almost a half of the proteins exhibited different abundance level between treatments, indicating tissue-specific responses. Majority of the identified proteins were down-regulated in both tissues after exposure to either AgNPs or AgNO3; in roots, the most affected proteins were those involved in response to abiotic and biotic stimuli and oxidative stress, while in leaf, both treatments had the most prominent effect on photosynthesis-related proteins. However, since AgNPs induced higher suppression of protein abundance than AgNO3, we conclude that AgNP effects can, at least partially, be attributed to nanoparticle form.

Coexisting lacertid lizard species Podarcis siculus and Podarcis melisellensis differ in dopamine brain concentrations.

In the eastern Adriatic, Podarcis siculus, an invasive species, competitively excludes the native Podarcis melisellensis. Monoamine neurotransmitters-serotonin (5HT), dopamine (DA), and noradrenaline (NA)-are implicated in social behavior, and could lie at the basis of the direct behavioral interference of P. siculus with P. melisellensis. To understand the relationship between social behavior and monoamines, as well as the differences in behavior between P. siculus and P. melisellensis, we developed a high-performance liquid chromatography (UV/VIS detection) method with which we were able to reliably measure concentrations of 5HT, DA, and NA in 32 brains of the two lizard species. We observed no statistically significant influence of species, sex, or their interaction on brain NA and 5HT concentrations. Statistically significant influence of species on dopamine levels were recorded, with P. siculus having twice as much dopamine in their brains. Taking into account that a significant aggressive relationship, with P. siculus dominating over P. melisellensis, has been previously observed, and that dopamine directly influences this behavior, the observed differences in dopamine levels could represent a trait in these species and may contribute to the competitive exclusion of P. melisellensis by P. siculus in the eastern Adriatic.

Physiological, ultrastructural and proteomic responses of tobacco seedlings exposed to silver nanoparticles and silver nitrate.

Since silver nanoparticles (AgNPs) are a dominant nanomaterial in consumer products, there is growing concern about their impact on the environment. Although numerous studies on the effects of AgNPs on living organisms have been conducted, the interaction of AgNPs with plants has not been fully clarified. To reveal the plant mechanisms activated after exposure to AgNPs and to differentiate between effects specific to nanoparticles and ionic silver, we investigated the physiological, ultrastructural and proteomic changes in seedlings of tobacco (Nicotiana tabacum) exposed to commercial AgNPs and ionic silver (AgNO3) from the seed stage. A higher Ag content was measured in seedlings exposed to AgNPs than in those exposed to the same concentration of AgNO3. However, the results on oxidative stress parameters obtained revealed that, in general, higher toxicity was recorded in AgNO3-treated seedlings than in those exposed to nanosilver. Ultrastructural analysis of root cells confirmed the presence of silver in the form of nanoparticles, which may explain the lower toxicity of AgNPs. However, the ultrastructural changes of chloroplasts as well as proteomic study showed that both AgNPs and AgNO3 can affect photosynthesis. Moreover, the majority of the proteins involved in the primary metabolism were up-regulated after both types of treatments, indicating that enhanced energy production, which can be used to reinforce defensive mechanisms, enables plants to cope with silver-induced toxicity.

Changes in Cryphonectria parasitica Populations Affect Natural Biological Control of Chestnut Blight.

Invasive species, especially plant pathogens, have a potential to completely eradicate native plant species and remodel landscapes. Tripartite interactions among sweet chestnut (Castanea sativa), chestnut blight-causing invasive fungus Cryphonectria parasitica, and hyperparasitic virus Cryphonectria hypovirus 1 (CHV1) were studied in two populations. The number of different vegetative compatibility (vc) types of C. parasitica more than doubled over the 10 years, while the hypovirulence incidence dropped in one population and slightly increased in the other one. Over the course of our 3-year monitoring experiment, the prevalence of hypovirulent isolates obtained from monitored cankers increased slowly (i.e., more hypovirulent isolates were being obtained from the same cankers over time). Within studied cankers, considerable changes in vc type and CHV1 presence were observed, indicating a highly dynamic system in which virulent and hypovirulent mycelia, sometimes of discordant vc types, often appeared together. The increase in hypovirulence prevalence did not have any observable curative effect on the cankers and, occasionally, reactivation of healed cankers by new, virulent C. parasitica isolates was observed. Both short- and long-term observations and revalidation of the infected plant populations are necessary to accurately estimate disease progress and formulate an adequate disease management strategy.

Phytotoxic effects of silver nanoparticles in tobacco plants.

The small size of nanoparticles (NPs), with dimensions between 1 and 100 nm, results in unique chemical and physical characteristics, which is why they are implemented in various consumer products. Therefore, an important concern is the potential detrimental impact of NPs on the environment. As plants are a vital part of ecosystem, investigation of the phytotoxic effects of NPs is particularly interesting. This study investigated the potential phytotoxicity of silver nanoparticles (AgNPs) on tobacco (Nicotiana tabacum) plants and compared it with the effects of the same AgNO3 concentrations. Accumulation of silver in roots and leaves was equally efficient after both AgNP and AgNO3 treatment, with predominant Ag levels found in the roots. Exposure to AgNPs did not result in elevated values of oxidative stress parameters either in roots or in leaves, while AgNO3 induced oxidative stress in both plant tissues. In the presence of both AgNPs and AgNO3, root meristem cells became highly vacuolated, which indicates that vacuoles might be the primary storage target for accumulated Ag. Direct AgNP uptake by root cells was confirmed. Leaf ultrastructural studies revealed changes mainly in the size of chloroplasts of AgNP-treated and AgNO3-treated plants. All of these findings indicate that nano form of silver is less toxic to tobacco plants than silver ions.

Electromagnetic fields at a mobile phone frequency (900 MHz) trigger the onset of general stress response along with DNA modifications in Eisenia fetida earthworms.

Eisenia fetida earthworms were exposed to electromagnetic field (EMF) at a mobile phone frequency (900 MHz) and at field levels ranging from 10 to 120 V m-1 for a period of two hours (corresponding to specific absorption rates ranging from 0.13 to 9.33 mW kg-1). Potential effects of longer exposure (four hours), field modulation, and a recovery period of 24 h after two hours of exposure were addressed at the field level of 23 V m-1. All exposure treatments induced significant DNA modifications as assessed by a quantitative random amplified polymorphic DNA-PCR. Even after 24 h of recovery following a two hour-exposure, the number of probe hybridisation sites displayed a significant two-fold decrease as compared to untreated control earthworms, implying a loss of hybridisation sites and a persistent genotoxic effect of EMF. Expression of genes involved in the response to general stress (HSP70 encoding the 70 kDa heat shock protein, and MEKK1 involved in signal transduction), oxidative stress (CAT, encoding catalase), and chemical and immune defence (LYS, encoding lysenin, and MYD, encoding a myeloid differentiation factor) were up-regulated after exposure to 10 and modulated 23 V m-1 field levels. Western blots showing an increased quantity of HSP70 and MTCO1 proteins confirmed this stress response. HSP70 and LYS genes were up-regulated after 24 h of recovery following a two hour-exposure, meaning that the effect of EMF exposure lasted for hours.

Cryphonectria hypovirus 1-Induced Changes of Stress Enzyme Activity in Transfected Phytopathogenic Fungus Cryphonectria parasitica.

Cryphonectria parasitica is a phytopathogenic fungus introduced from Eastern Asia to North America and to Europe, where it causes chestnut blight, a devastating disease of chestnut trees. The disease can be biologically controlled utilising the mycovirus Cryphonectria hypovirus 1 (CHV1), which changes the physiology of the host, reducing its virulence towards chestnut. We measured fungal growth in vitro and activities of glutathione S-transferase, catalase and superoxide dismutase, enzymes involved in oxidative stress response, to elucidate the effects of CHV1 infection on the host. Six CHV1 strains of different subtypes and three fungal isolates were used in different combinations to better represent natural conditions, where higher genetic diversity of both fungus and virus is expected. The infection with different CHV1 strains decreased in vitro growth rate of infected fungal isolates and increased activity of their stress enzymes in most of the studied fungus/virus combinations, indicating increased oxidative stress following CHV1 infection. All our field CHV1 strains belong to the Italian subtype, but while strain M56-1 had equal or even stronger effect on its fungal host than prototypic strain EP713 of French subtype F1, strain B11 had no effect. Thus, the severity of the observed effects depended on a particular virus strain, fungal isolate, and the combination of the two, rather than solely on the virus subtype. Since previous research showed discordance between accumulation of mRNA and stress-related proteins in CHV1 infected C. parasitica, our results emphasise the importance of enzymes' activity measurements as an invaluable extension of transcriptomic and proteomic analyses.

Toxicity of silver ions and differently coated silver nanoparticles in Allium cepa roots.

Silver nanoparticles (AgNPs) are the dominating nanomaterial in consumer products due to their well-known antibacterial and antifungal properties. To enhance their properties, different surface coatings may be used, which affect physico-chemical properties of AgNPs. Due to their wide application, there has been concern about possible environmental and health consequences. Since plants play a significant role in accumulation and biodistribution of many environmentally released substances, they are also very likely to be influenced by AgNPs. In this study we investigated the toxicity of AgNO3 and three types of laboratory-synthesized AgNPs with different surface coatings [citrate, polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB)] on Allium cepa roots. Ionic form of Ag was confirmed to be more toxic than any of the AgNPs applied. All tested AgNPs caused oxidative stress and exhibited toxicity only when applied in higher concentrations. The highest toxicity was recorded for AgNPs-CTAB, which resulted with increased Ag uptake in the roots, consequently leading to strong reduction of the root growth and oxidative damage. The weakest impact was found for AgNPs-citrate, much bigger, negatively charged NPs, which also aggregated to larger particles. Therefore, we can conclude that the toxicity of AgNPs is directly correlated with their size, overall surface charge and/or surface coating.

Growth and photosynthetic responses of Lemna minor L. exposed to cadmium in combination with zinc or copper.

Metals have a variety of negative outcomes on plants, essential components of any ecosystem. The effects of CdCl2 (5 µmol L-1), ZnCl2 (25 or 50 µmol L-1), and CuCl2 (2.5 or 5 µmol L-1) and combinations of CdCl2 with either ZnCl2 or CuCl2 on the growth, photosynthetic pigments, and photosystem II (PSII) efficiency of duckweed (Lemna minor L.) were investigated. All of the treatments caused growth inhibition and remarkable metal accumulation in plant tissue after 4 and 7 days. In the combined treatments, the accumulation of each metal applied was lesser in comparison to treatments with single metals. After 4 days, all of the treatments generally diminished chlorophyll a content and decreased the maximum quantum yield (Fv/Fm) and effective quantum yield (ΔF/F'm) of PSII. However, after 7 days of exposure to a combination of Cd and Zn, pigment content and PSII activity recovered to control levels. A higher concentration of Cu (5 µmol L-1) as well as Cd in combination with Cu had a prolonged inhibitory effect on photosynthetic features. Our results suggest that growth inhibition was due to the toxic effect of absolute metal quantity in plant tissue. Zn counteracted Cd uptake, as seen from the recovery of pigment content and PSII efficiency in plants exposed for 7 days to the Cd and Zn combination. Cu-induced oxidative stress led to a prolonged inhibitory effect in plants treated both with a higher concentration of Cu (5 µmol L-1) and simultaneously with Cd and Cu. Our findings could contribute to general knowledge on anthropogenic and environmental contaminants that endanger plant communities and significantly disrupt the sensitive balance of an ecosystem by influencing photosynthetic mechanisms.

The effects of cadmium-zinc interactions on biochemical responses in tobacco seedlings and adult plants.

The objective of the present study was to investigate the effects of cadmium-zinc (Cd-Zn) interactions on their uptake, oxidative damage of cell macromolecules (lipids, proteins, DNA) and activities of antioxidative enzymes in tobacco seedlings as well as roots and leaves of adult plants. Seedlings and plants were exposed to Cd (10 µM and 15 µM) and Zn (25 µM and 50 µM) as well as their combinations (10 µM or 15 µM Cd with either 25 µM or 50 µM Zn). Measurement of metal accumulation exhibited that Zn had mostly positive effect on Cd uptake in roots and seedlings, while Cd had antagonistic effect on Zn uptake in leaves and roots. According to examined oxidative stress parameters, in seedlings and roots individual Cd treatments induced oxidative damage, which was less prominent in combined treatments, indicating that the presence of Zn alleviates oxidative stress. However, DNA damage found in seedlings, and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity recorded in both seedlings and roots, after individual Zn treatments, indicate that Zn accumulation could impose toxic effects. In leaves, oxidative stress was found after exposure to Cd either alone or in combination with Zn, thus implying that in this tissue Zn did not have alleviating effects. In conclusion, results obtained in different tobacco tissues suggest tissue-dependent Cd-Zn interactions, which resulted in activation of different mechanisms involved in the protection against metal stress.

Gene flow vs. pollution pressure: genetic diversity of Mytilus galloprovincialis in eastern Adriatic.

Environmental pollution may modify all the evolutionary processes involved in shaping the genetic patterns of exposed populations. In order to evaluate the pollution impact on the genetic diversity of Mediterranean mussel Mytilus galloprovincialis ten populations inhabiting differently polluted sites along the eastern Adriatic coast, from pristine bays to heavily trafficked harbours, were studied. Pollution pressure was assessed through an integrated study of biological effects and responses across different levels of biological organization. Eight microsatellite markers were analysed to assess genetic diversity of investigated populations. Both the principal component analysis (PCA) of the biomarker data set as well as the biomarker response index (BRI) confirmed substantial pollution pressure at the highly polluted sites, and very low pollution exposure at the three reference sites. Very shallow genetic differentiation was found in respect to maritime distances or pollution status, and this was attributed to a high gene flow among the populations. However, populations inhabiting polluted sites exhibited higher levels of genetic diversity and evolutionary mechanisms underlying this phenomenon are discussed.