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The 2021 EU Action Plan 'Towards Zero Pollution for Air, Water and Soil' highlighted that climate change, environmental pollution, biodiversity loss and unsustainable use of natural resources pose multiple risks to human, animal and ecosystem health and are the four main environmental challenges to be addressed. Toxicologists and ecotoxicologists with solid skills are needed to fulfil the ambitious objectives of this Action Plan as it aims, in particular, to improve the assessment of the toxicity of pollutants (especially emerging ones) and complex mixtures found in air, soil and water, developing innovative methods to evaluate their toxicity and biomonitor our environment. The ToxLearn4EU project comprises a consortium of seven Higher Education Institutes (HEIs), three Research Institutes and several laboratories from all over Europe. It intends to modernise education in Toxicology and other related areas by developing innovative educational content, such as interactive courses and case studies, which will be available online and with free access. The main goal is to produce quality digital education on current topics in this field. The project meets the needs of: • Students: by stimulating their interest and limiting school dropout through active pedagogies, also adapted to digital practice. • Teachers: by proposing stimulating online courses that can be integrated into their courses or by motivating them to design and create new innovative courses. • Partners: by modernising part of their curricula. • Stakeholders of toxicology and ecotoxicology: by developing student and lifelong learner competencies adapted to the job market and up-to-date to recent changes at the European Green Deal. • European Commission: by modifying curricula and developing courses adapted to the Green Deal. This information is presented on behalf of the ToxLearn4EU consortium. Project founded by the Erasmus+ program (KA220-HED - Cooperation partnerships in higher education) with reference 2021-1-FR01-KA220-HED-000030081. [ABSTRACT FROM AUTHOR]

The choice of agronomic practices for phytomanagement of metal-contaminated soils is of crucial importance to optimize plant biomass yields and to mitigate both environmental and health risks due to metal exposure. The present study aimed to assess the effects of agronomic practices on shoot yield and on metal (Cd, Pb, and Zn) accumulation in the organs of the energy crop miscanthus ( Miscanthus × giganteus ) during the first three years since the plantation on metal-contaminated agricultural soils. Three miscanthus cultivars, hereafter named Mis-A, Mis-B and Mis-I, were planted at low and high density. An inoculum of arbuscular mycorrhizal fungi-AMF ( Glomus LPA Val 1) was added during plantation, and nitrogen fertilization was applied during the third growing season. Metal accumulation in miscanthus organs was determined during the second growing season, whereas shoot yields and their metal concentrations were determined during both the second and the third growing seasons. Based on metal concentrations and bioconcentration factors, the three cultivars mainly accumulated metals in their roots. The shoot yields increased from 3.7 to 10.3 t DW ha −1 in the second growing season to 15.8–23.3 t DW ha −1 in third growing season. There were no or very few significant differences in metal concentrations and shoot yields within treatments comprising the same cultivar. The addition of AMF inoculum increased metal (mainly Cd and Zn) accumulation in miscanthus organs and in the shoot yields and this was more observed in both Mis-B and Mis-I which presented a higher root mycorrhization level than in Mis-A. Shoot yields in treatments comprising different cultivars depended not on fertilization but on the interactions between cultivar and planting density, and between cultivar, planting density and AMF inoculum. Whatever the treatment and the sampling period, Pb concentrations did not significantly differ in shoot yields. The interaction between cultivar and planting density resulted in higher Cd concentrations in the yields of Mis-B planted at low density during the third growing season. Zn concentrations increased with fertilization in all treatments, and with the addition of the AMF inoculum in Mis-B and in Mis-I. Overall, the results demonstrated that the three cultivars could be potential candidates for coupling phytostabilization and biomass production on metal-contaminated soils. [ABSTRACT FROM AUTHOR]

European countries have been particularly affected by the Covid 19 pandemic. Most universities had to close their doors and face many challenges. With the closure of universities and the confinement of the population, universities had to change their teaching by using digital technology tools massively. Despite the lack of preparation, the absence of adapted tools and experience in digital pedagogy, they have adapted remarkably well. Regarding students, several studies have shown a negative impact of confinement and distance learning on mood, as well as the occurrence of health disorders. Moreover, although students highlight the responsiveness of their teachers and the quality of the work done, most of them consider that the overall quality of teaching has deteriorated. The RALDE consortium involves several European higher education institutions (UNED, University of Navarra, University of Maastricht and INP Toulouse) and small specialised companies (Adict and Tom's Content Shop) and focuses on Biology and Environmental Sciences. Its main objective is to develop and use innovative and free educational resources to provide high-quality, inclusive digital education, focusing on implementing hybrid, cutting-edge, distance and online learning to address the problems of Higher Education Institutions (HEIs) revealed during the pandemic crisis. Another essential objective is the development of digital skills in teachers and educators. Direct and tangible expected results are: • European study on the pedagogical practices of HEIs during the pandemic lockdown. • Development of guidelines for online evaluation. • Creation of innovative online pedagogical content: virtual • A series of tutorials for teachers to develop their own innovative online pedagogical content. • Training on digitisation and online presentation skills. This information is presented on behalf of the RALDE consortium. Project founded by the Erasmus+ program (KA226 - Partnerships for Digital Education Readiness) with reference KA2020-1-FR01-KA226-HE-095581. [ABSTRACT FROM AUTHOR]

Miscanthus × giganteus demonstrated good phytostabilization potentials by decreasing the trace elements (T.E.s) mobility and enhancing the degraded soil quality. Nevertheless, most of the published work was performed under controlled conditions in ex situ pot experiments and/or with soils being spiked. Hence, data about the plant's tolerance to increased T.E. concentrations in real conditions is still scarce and requires further investigation. For this sake, a field experiment was established by cultivating miscanthus plants in three different agricultural plots representing gradient trace element (Cd, Pb and Zn) concentrations. Another uncontaminated plot was also introduced. Results showed that T.E. concentrations in the leaves were tolerable to the plant. In addition, no variations were detected between the miscanthus cultivated in the contaminated and uncontaminated soils at the level of antioxidant enzymatic activities (ascorbate peroxidase and superoxide dismutase), photosynthetic pigments (chlorophyll a and b and carotenoids), and secondary metabolites (phenolic compounds, flavonoids, anthocyanins, and tannins). These outcomes validate the high capacity of miscanthus to resist and tolerate contaminated conditions. Such results may contribute to further understanding of the miscanthus tolerance mechanisms.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility., (© 2023. Springer Nature Limited.)

Bosnia and Herzegovina (B&H) is among the European countries with the highest rate of air pollution-related death cases and the poorest air quality. The main causes are solid fuel consumption, traffic, and the poorly developed or implemented air pollution reduction policies. In addition, the city of Sarajevo, the capital of B&H, suffers temperature inversion episodes in autumn/winter months, which sustain air pollution. Human biomonitoring studies may be confounded by the lifestyle of subjects or possible metabolic alterations. Therefore, this study aimed to evaluate Ligustrum vulgare L. as a model for air pollution monitoring by measuring DNA damage at one rural and two urban sites. DNA damage was measured as tail intensity (TI) in L. vulgare leaves, considering seasonal, sampling period, leaf position and staging, and spatial (urban versus rural) variation. Effects of COVID-19 lockdown on TI were assessed by periodical monitoring at one of the selected sites, while in-house grown L. vulgare plants were used to test differences between outdoor and indoor air pollution effects for the same sampling period. Significantly higher TI was generally observed in leaves collected in Campus in December 2020 and 2021 compared with March (P < 0.0001). Outer and adult leaves showed higher TI values, except for the rural site where no differences for these categories were found. Leaves collected in the proximity of the intensive traffic showed significantly higher TI values (P < 0.001), regardless of the sampling period and the stage of growth. In regards to the COVID-19 lockdown, higher TI (P < 0.001) was registered in December 2020, after the lockdown period, than in periods before COVID-19 outbreak or immediately after the lockdown in 2020. This also reflects mild air pollution conditions in summer. TI values for the in-house grown leaves were significantly lower compared to those in situ. Results showed that L. vulgare may present a consistent model for the air pollution biomonitoring but further studies are needed to establish the best association between L. vulgare physiology, air quality data, and air pollution effects., (© The Author(s) 2022. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The positive impact on restoring soil functionality, decreasing toxic elements (TE) bioaccessibility, and enhancing soil physicochemical and biological parameters established a consensus on considering a Miscanthus × giganteus convenient species for phytomanaging wide TE contaminated areas. Nevertheless, information about the plant's mode of reaction to elevated soil multi-TE concentrations is still scarce. For the sake of investigating the miscanthus response to stressful TE concentrations, an ex-situ pot experiment was initiated for 18 months, with three miscanthus cultivars referred to as B, U, and A planted in soils with gradient Cd, Pb, and Zn concentrations. A non-contaminated control soil was introduced as well, and plants were cultivated within. Results revealed that the long exposure to increasing soil TE concentrations caused the number of tillers per plant to decline and the TE concentrations in the leaves to boost progressively with the soil contamination. The photosynthetic pigments (chlorophyll a, b, and carotenoids) were negatively affected as well. However, the phenolic compounds, flavonoids, tannins, and anthocyanins, along with the antioxidant enzymatic activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase elevated progressively with the TE concentration and exposure duration. Conclusively, miscanthus plants demonstrated an intensified and synchronized antioxidative activity against the TE concentration.

Miscanthus × giganteus demonstrated good phytostabilization potentials in toxic element (TE) contaminated soils. However, information about its tolerance to elevated concentrations is still scarce. Therefore, an ex-situ pot experiment was launched using three cultivars (termed B, U, and A) grown in soils with a gradient Cd, Pb and Zn concentrations. Control plants were also cultivated in non-contaminated soil. Results show that the number of tillers per plant, stem diameter as well as leaf photosynthetic pigments (chlorophyll a , b and carotenoids) were negatively impacted by soil contamination. On the other hand, phenolic compounds, flavonoids, tannins, and anthocyanins levels along with the antioxidant enzymatic activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase increased in the plants grown on contaminated soils. Altogether, these data demonstrate that miscanthus is impacted by concentrations of toxic elements yet is able to tolerate high levels of soil contamination. These results may contribute to clarifying the miscanthus tolerance strategy against high contamination levels and its efficiency in phytoremediation.

Phytomanagement is proposed as a cost-effective and environmentally-friendly suggestion for sustainable use of large metal-contaminated areas. In the current work, the energy crop miscanthus (Miscanthus × giganteus) was grown in ex situ conditions on agricultural soils presenting a Cd, Pb and Zn contamination gradient. After 93 days of culture, shoot and root growth parameters were measured. Soils and plants were sampled as well to study the TE accumulation in miscanthus and the effects of this plant on TE mobility in soils. Results demonstrated that miscanthus growth depended more on the soils silt content rather than TE-contamination level. Moreover, soil organic carbon at T93 increased in the soils after miscanthus cultivation by 25.5-45.3%, whereas CaCl 2 -extractible TEs decreased due to complex rhizosphere processes driving plant mineral uptake, and organic carbon inputs into the rhizosphere. In the contaminated soils, miscanthus accumulated Cd, Pb and Zn mainly in roots (BCF in roots: Cd " Zn > Pb), while strongly reducing the transfer of these elements from soil to all organs and from roots to rhizomes, stems and leaves (average TFs: 0.01-0.06, 0.11-1.15 and 0.09-0.79 corresponding to Cd, Pb and Zn respectively). Therefore, miscanthus could be considered a TE-excluder, hence a potential candidate crop for coupling phytostabilization and biomass production on the studied Metaleurop TE-contaminated soils., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Miscanthus x giganteus is suggested as a good candidate for phytostabilization of metal-polluted soils. Its late harvest in winter generates large amounts of leaf litter on the soil surface. However, little is known about the mobility and the bioavailability of metals following leaf decomposition and the consequences on the succeeding culture. Ex situ artificial aging for 1, 3, and 6 months was conducted with miscanthus leaf fragments incorporated into three agricultural soils displaying a gradient concentration in Cd (0.6, 3.1 and 7.9 mg kg -1 ), Pb (32.0, 194.6 and 468.6 mg kg -1 ), and Zn (48.4, 276.3 and 490.2 mg kg -1 ) to simulate the leaf litter input over 20 years of miscanthus culture. We investigated the impacts on physicochemical and biological soil parameters, CaCl 2 -extractable metal, and their subsequent ryegrass shoot concentrations, and hence on ryegrass health. The results showed that the amended soils possessed higher pH along with greater available phosphorous and soil organic carbon values. The respiratory activity and microbial biomass carbon in the amended soils increased mainly after 1 month of aging, and decreased afterwards. Despite the higher Pb- and Zn-CaCl 2 extractability in the amended soils, the phytoavailability slightly increased only in the most contaminated soils. Moreover, leaf incorporation did not affect the ryegrass biomass, photosynthetic pigment contents, nor the antioxidative enzyme activities. Conclusively, leaf incorporation induced slight variations in soil physicochemical and biological parameters, as well as metal extractability, but not to an extent that might cause a considerable threat to the subsequent culture. Nevertheless, these results are preliminary data that require confirmation by long-term in-situ experimentations as they reflect the modelization of long-term impact of leaf decomposition on soil-plant system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

The enzyme-modified comet assay is a commonly used method to detect specific DNA lesions. However, still a lot of errors are made by many users, leading to dubious results and even misinterpretations. This technical note describes some critical points in the use of the enzyme-modified comet assay, such as the enzyme concentration, the time of incubation, the format used and the equipment. To illustrate the importance of these conditions/parameters, titration experiments of formamidopyrimidine DNA glycosylase (Fpg) were performed using the 2 gels/slide and the 12 minigels/slide formats (plus the 12-Gel Comet Assay Unit™). Incubation times of 15 and 30 min, and 1 h were used. Results showed that the 12 minigels/slide system requires a lower volume and concentration of Fpg. A longer time of incubation has a bigger impact when using such format. Moreover, the paper describes how to perform and interpret a titration experiment when using the enzyme-modified comet assay., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The comet assay has become one of the methods of choice for the evaluation and measurement of DNA damage. It is sensitive, quick to perform and relatively affordable for the evaluation of DNA damage and repair at the level of individual cells. The comet assay can be applied to virtually any cell type derived from different organs and tissues. Even though the comet assay is predominantly used on human cells, the application of the assay for the evaluation of DNA damage in yeast, plant and animal cells is also quite high, especially in terms of biomonitoring. The present extensive overview on the usage of the comet assay in animal models will cover both terrestrial and water environments. The first part of the review was focused on studies describing the comet assay applied in invertebrates. The second part of the review, (Part 2) will discuss the application of the comet assay in vertebrates covering cyclostomata, fishes, amphibians, reptiles, birds and mammals, in addition to chordates that are regarded as a transitional form towards vertebrates. Besides numerous vertebrate species, the assay is also performed on a range of cells, which includes blood, liver, kidney, brain, gill, bone marrow and sperm cells. These cells are readily used for the evaluation of a wide spectrum of genotoxic agents both in vitro and in vivo. Moreover, the use of vertebrate models and their role in environmental biomonitoring will also be discussed as well as the comparison of the use of the comet assay in vertebrate and human models in line with ethical principles. Although the comet assay in vertebrates is most commonly used in laboratory animals such as mice, rats and lately zebrafish, this paper will only briefly review its use regarding laboratory animal models and rather give special emphasis to the increasing usage of the assay in domestic and wildlife animals as well as in various ecotoxicological studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The comet assay, also called single cell gel electrophoresis, is a sensitive, rapid and low-cost technique for quantifying and analysing DNA damage and repair at the level of individual cells. The assay itself can be applied on virtually any cell type derived from different organs and tissues of eukaryotic organisms. Although it is mainly used on human cells, the assay has applications also in the evaluation of DNA damage in yeast, plant and animal cells. Therefore, the purpose of this review is to give an extensive overview on the usage of the comet assay in animal models from invertebrates to vertebrates, covering both terrestrial and water biota. The comet assay is used in a variety of invertebrate species since they are regarded as interesting subjects in ecotoxicological research due to their significance in ecosystems. Hence, the first part of the review (Part 1) will discuss the application of the comet assay in invertebrates covering protozoans, platyhelminthes, planarians, cnidarians, molluscs, annelids, arthropods and echinoderms. Besides a large number of animal species, the assay is also performed on a variety of cells, which includes haemolymph, gills, digestive gland, sperm and embryo cells. The mentioned cells have been used for the evaluation of a broad spectrum of genotoxic agents both in vitro and in vivo. Moreover, the use of invertebrate models and their role from an ecotoxicological point of view will also be discussed as well as the comparison of the use of the comet assay in invertebrate and human models. Since the comet assay is still developing, its increasing potential in assessing DNA damage in animal models is crucial especially in the field of ecotoxicology and biomonitoring at the level of different species, not only humans., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human activities generate environmental stresses that can lead plant populations to become extinct. Population survival would require the evolution of adaptive responses that increase tolerance to these stresses. Thus, in pseudometallophyte species that have colonized anthropogenic metalliferous habitats, the evolution of increased metal tolerance is expected in metallicolous populations. However, the mechanisms by which metal tolerance evolves remain unclear. In this study, parent populations were created from non-metallicolous families of Noccaea caerulescens. They were cultivated for one generation in mesocosms and under various levels of zinc (Zn) contamination to assess whether Zn in soil represents a selective pressure. Individual plant fitness estimates were used to create descendant populations, which were cultivated in controlled conditions with moderate Zn contamination to test for adaptive evolution in functional traits. The number of families showing high fitness estimates in mesocosms was progressively reduced with increasing Zn levels in soil, suggesting increasing selection for metal tolerance. In the next generation, adaptive evolution was suggested for some physiological and ecological traits in descendants of the most exposed populations, together with a significant decrease of Zn hyperaccumulation. Our results confirm experimentally that Zn alone can be a significant evolutionary pressure promoting adaptive divergence among populations.

Cardiotoxicity is a serious complication of anticancer therapy by anthracycline antibiotics. Except for intercalation into DNA/RNA structure, inhibition of DNA-topoisomerase and histone eviction from chromatin, the main mechanism of their action is iron-mediated formation of various forms of free radicals, which leads to irreversible damage to cancer cells. The most serious adverse effect of anthracyclines is, thus, cardiomyopathy leading to congestive heart failure, which is caused by the same mechanisms. Here, we briefly summarize the basic types of free radicals formed by anthracyclines and the main processes how to scavenge them. From these, the main attention is paid to metallothioneins. These low-molecular cysteine-rich proteins are introduced and their functions and properties are reviewed. Further, their role in detoxification of metals and drugs is discussed. Based on these beneficial roles, their use as a new therapeutic agent against oxidative stress and for cardioprotection is critically evaluated with respect to their ability to increase chemoresistance against some types of commonly used cytostatics.

Arbuscular mycorrhizal fungus (AMF)-assisted phytoremediation could constitute an ecological and economic method in polluted soil rehabilitation programs. The aim of this work was to characterize the trace element (TE) phytoremediation potential of mycorrhizal Miscanthus × giganteus. To understand the mechanisms involved in arbuscular mycorrhizal symbiosis tolerance to TE toxicity, the fatty acid compositions and several stress oxidative biomarkers were compared in the roots and leaves of Miscanthus × giganteus cultivated under field conditions in either TE-contaminated or control soils. TEs were accumulated in greater amounts in roots, but the leaves were the organ most affected by TE contamination and were characterized by a strong decrease in fatty acid contents. TE-induced oxidative stress in leaves was confirmed by an increase in the lipid peroxidation biomarker malondialdehyde (MDA). TE contamination decreased the GSSG/GSH ratio in the leaves of exposed plants, while peroxidase (PO) and superoxide dismutase (SOD) activities were increased in leaves and in whole plants, respectively. AMF inoculation also increased root colonization in the presence of TE contamination. The mycorrhizal colonization determined a decrease in SOD activity in the whole plant and PO activities in leaves and induced a significant increase in the fatty acid content in leaves and a decrease in MDA formation in whole plants. These results suggested that mycorrhization is able to confer protection against oxidative stress induced by soil pollution. Our findings suggest that mycorrhizal inoculation could be used as a bioaugmentation technique, facilitating Miscanthus cultivation on highly TE-contaminated soil., (Copyright © 2015. Published by Elsevier B.V.)

The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.

The in situ stabilization of metals in soils using plants with great biomass value is a promising, cost-effective, and ecologically friendly alternative to manage metal-polluted sites. The goal of phytostabilization is to reduce the bioavailable concentrations of metals in polluted soil and thus reduce the risk to the environment and human health. In this context, this study aimed at evaluating Miscanthus × giganteus efficiency in phytostabilizing metals on three contaminated agricultural sites after short-term exposure under greenhouse conditions and after long-term exposure under field conditions. Particular attention was paid to the influence of Miscanthus cultivation on (i) Cd, Pb, and Zn fractionation using sequential extractions and (ii) metal bioaccessibility using an in vitro gastrointestinal digestion test. Data gave evidence of (i) different behaviors between the greenhouse and the field; (ii) metal redistribution in soils induced by Miscanthus culture, more specifically under field conditions; (iii) higher environmental availability for Cd than for Pb and Zn was found in both conditions; and (iv) overall, a higher bioaccessible fraction for Pb (about 80 %) and Cd (65-77 %) than for Zn (36-52 %) was recorded in the gastric phase, with a sharp decrease in the intestinal phase (18-35 % for Cd, 5-30 % for Pb, and 36-52 % for Zn). Compared to soils without culture, the results showed that phytostabilization using Miscanthus culture provided evidence for substantial effects on oral bioaccessibility of Cd, Pb, and Zn.

In plants, an increasing interest for the comet assay was shown in the last decade. This versatile technique appears to be promising to detect the genotoxic effect of pollutants and to monitor the environment. However, the lack of a standardised protocol and the low throughput of the assay limit its use in plants. The aims of this paper are to identify key factors affecting comet assay performance and to improve its reliability and reproducibility. We examined the effect of varying several parameters on four different plant species: broad bean (Vicia faba), white clover (Trifolium repens), English ryegrass (Lolium perenne) and miscanthus (Miscanthus x giganteus). The influence of both internal (different nucleus isolation methods, presence or absence of filtration and lysis steps) and external (room temperature, light intensity) parameters were evaluated. Results clearly indicate that short chopping is more efficient to isolate nuclei than the standard slicing method. Filtration and lysis steps were shown to be unnecessary and thus should be skipped. Data also demonstrate that high room temperatures and light could induce DNA damage in isolated nuclei. Calibration tests with H2O2 or ethyl methanesulfonate revealed that a special attention should be paid to plant growing stage, leaf position and exposure duration., (© The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The mitigation of potential health hazards and land scarcity due to land use change can be addressed by restoring functional and ecosystem services of contaminated land. Physico-chemical remediation options are criticized as being costly and not providing environment-friendly solutions. The use of plants and associated microorganisms could be a sustainable, cost-effective option to reduce pollutant exposure. Phytomanagement aims at using valuable non-food crops to alleviate environmental and health risks induced by pollutants, and at restoring ecosystem services. Suitable plant species must be tolerant to contaminants, reduce their transfer into the food chain, and efficiently produce marketable biomass. Based on Miscanthus' capacity to sequestrate inorganic contaminants into the root system and to induce dissipation of persistent organic contaminants in soil, these plant species are favorable for phytostabilization and phytodegradation. Among Miscanthus species, the noninvasive hybrid Miscanthus × giganteus, with a high lignocellulosic content, is a promising biomass crop for the bio-economy, notably the biorefinery and bioenergy industries. Planting this species on contaminated and marginal land is a promising option to avoid changes in arable land use to mitigate the food vs. biofuel controversy. Key issues in promoting sustainable management of Miscanthus sp. on contaminated land are: (a) crop suitability, integration, and sustainability in a region with a potential local market; (b) site suitability in relation to the species' requirements and potential, (c) biotic interactions in the landscape diversity; and (d) increase in shoot yields in line with various stressors (e.g., pollutants, drought, cold temperatures), and with minimal inputs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

As a result of the industrial revolution, anthropogenic activities have enhanced there distribution of many toxic heavy metals from the earth's crust to different environmental compartments. Environmental pollution by toxic heavy metals is increasing worldwide, and poses a rising threat to both the environment and to human health.Plants are exposed to heavy metals from various sources: mining and refining of ores, fertilizer and pesticide applications, battery chemicals, disposal of solid wastes(including sewage sludge), irrigation with wastewater, vehicular exhaust emissions and adjacent industrial activity.Heavy metals induce various morphological, physiological, and biochemical dysfunctions in plants, either directly or indirectly, and cause various damaging effects. The most frequently documented and earliest consequence of heavy metal toxicity in plants cells is the overproduction of ROS. Unlike redox-active metals such as iron and copper, heavy metals (e.g, Pb, Cd, Ni, AI, Mn and Zn) cannot generate ROS directly by participating in biological redox reactions such as Haber Weiss/Fenton reactions. However, these metals induce ROS generation via different indirect mechanisms, such as stimulating the activity of NADPH oxidases, displacing essential cations from specific binding sites of enzymes and inhibiting enzymatic activities from their affinity for -SH groups on the enzyme.Under normal conditions, ROS play several essential roles in regulating the expression of different genes. Reactive oxygen species control numerous processes like the cell cycle, plant growth, abiotic stress responses, systemic signalling, programmed cell death, pathogen defence and development. Enhanced generation of these species from heavy metal toxicity deteriorates the intrinsic antioxidant defense system of cells, and causes oxidative stress. Cells with oxidative stress display various chemical,biological and physiological toxic symptoms as a result of the interaction between ROS and biomolecules. Heavy-metal-induced ROS cause lipid peroxidation, membrane dismantling and damage to DNA, protein and carbohydrates. Plants have very well-organized defense systems, consisting of enzymatic and non-enzymatic antioxidation processes. The primary defense mechanism for heavy metal detoxification is the reduced absorption of these metals into plants or their sequestration in root cells.Secondary heavy metal tolerance mechanisms include activation of antioxidant enzymes and the binding of heavy metals by phytochelatins, glutathione and amino acids. These defense systems work in combination to manage the cascades of oxidative stress and to defend plant cells from the toxic effects of ROS.In this review, we summarized the biochemiCal processes involved in the over production of ROS as an aftermath to heavy metal exposure. We also described the ROS scavenging process that is associated with the antioxidant defense machinery.Despite considerable progress in understanding the biochemistry of ROS overproduction and scavenging, we still lack in-depth studies on the parameters associated with heavy metal exclusion and tolerance capacity of plants. For example, data about the role of glutathione-glutaredoxin-thioredoxin system in ROS detoxification in plant cells are scarce. Moreover, how ROS mediate glutathionylation (redox signalling)is still not completely understood. Similarly, induction of glutathione and phytochelatins under oxidative stress is very well reported, but it is still unexplained that some studied compounds are not involved in the detoxification mechanisms. Moreover,although the role of metal transporters and gene expression is well established for a few metals and plants, much more research is needed. Eventually, when results for more metals and plants are available, the mechanism of the biochemical and genetic basis of heavy metal detoxification in plants will be better understood. Moreover, by using recently developed genetic and biotechnological tools it may be possible to produce plants that have traits desirable for imparting heavy metal tolerance.

Genotoxic effects of lead (0-20μM) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10μM. In addition, at this concentration, DNA damage time-dependently increased until 12h. Then, a decrease in DNA damages was recorded. The significant induction of micronucleus formation also reinforced the genotoxic character of this metal. Direct interaction of lead with DNA was also evaluated with the a-cellular comet assay. The data showed that DNA breakages were not associated with a direct effect of lead on DNA. In order to investigate the relationship between lead genotoxicity and oxidative stress, V. faba were exposed to lead in the presence or absence of the antioxidant Vitamin E, or the NADPH-oxidase inhibitor dephenylene iodonium (DPI). The total inhibition of the genotoxic effects of lead (DNA breakage and micronucleus formation) by these compounds reveals the major role of reactive oxygen species (ROS) in the genotoxicity of lead. These results highlight, for the first time in vivo and in whole-plant roots, the relationship between ROS, DNA strand-breaks and chromosome aberrations induced by lead., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aided phytostabilisation is a cost-efficient technique to manage metal-contaminated areas, particularly in the presence of extensive pollution. Plant establishment and survival in highly metal-contaminated soils are crucial for phytostabilisation success, as metal toxicity for plants is widely reported. A relevant phytostabilisation solution must limit metal transfer through the food chain. Therefore, this study aimed at evaluating the long-term efficiency of aided phytostabilisation on former agricultural soils highly contaminated by cadmium, lead, and zinc. The influence of afforestation and fly ash amendments on reducing metal phytoavailability was investigated as were their effects on plant development. Before being planted with a tree mix, the site was divided into three plots: a reference plot with no amendment, a plot amended with silico-aluminous fly ash and one with sulfo-calcic fly ash. Unlike Salix alba and Quercus robur, Alnus glutinosa, Acer pseudoplatanus and Robinia pseudoacacia grew well on the site and accumulated, overall, quite low concentrations of metals in their leaves and young twigs. This suggests that these three species have an excluder phenotype for Cd, Zn and Pb. After 8 years, metal availability to A. glutinosa, A. pseudoplatanus and R. pseudoacacia, and translocation to their above-ground parts, strongly decreased in fly ash-amended soils. Such decreases fit well together with the depletion of CaCl(2)-extractable metals in amended soils. Although both fly ashes were effective to decrease Cd, Pb and Zn concentrations in above-ground parts of trees, the sulfo-calcic ash was more efficient., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Due to anthropogenic activities, large extends of soils are highly contaminated by Metal Trace Element (MTE). Aided phytostabilisation aims to establish a vegetation cover in order to promote in situ immobilisation of trace elements by combining the use of metal-tolerant plants and inexpensive mineral or organic soil amendments. Eight years after Coal Fly Ash (CFA) soil amendment, MTE bioavailability and uptake by two plants, Lolium perenne and Trifolium repens, were evaluated, as some biological markers reflecting physiological stress. Results showed that the two plant species under study were suitable to reduce the mobility and the availability of these elements. Moreover, the plant growth was better on CFA amended MTE-contaminated soils, and the plant sensitivity to MTE-induced physiological stress, as studied through photosynthetic pigment contents and oxidative damage was lower or similar. In conclusion, these results supported the usefulness of aided phytostabilisation of MTE-highly contaminated soils., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Sustainable management of large surface areas contaminated with trace elements is a real challenge, since currently applied remediation techniques are too expensive for these areas. Aided phytostabilisation appears to be a cost efficient technique to reduce metal mobility in contaminated soils and contaminated particle spread. In this context, this study aimed at evaluating the long-term efficiency of aided phytostabilisation on former agricultural soils highly contaminated with trace elements. The influence of afforestation and fly ash amendments to reduce metal mobility was investigated. Before being planted with a tree mix, the study site was divided into three plots: a reference plot with no amendment, the second amended with silico-aluminous fly ash and the third with sulfo-calcic fly ash. After eight years, some soil physico-chemical parameters, including cadmium (Cd), lead (Pb) and zinc (Zn) extractability were modified. In particular, pH decreased on the whole site while organic carbon content increased. The alteration of these parameters influencing trace element mobility is explained by afforestation. Over time, concentrations of CaCl(2)-extractable metals increased and were correlated with the soil pH decrease. In the amended soils, extractable Cd, Pb and Zn concentrations were lower than in the reference soil. The results indicated that the two fly ashes buffered natural soil acidification due to vegetation development and limited trace element mobility and thus could limit their bioavailability. For long-term phytostabilisation, special attention should be focused on the soil pH, metal mobility and phytoavailability analysis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Lead has gained considerable attention as a persistent toxic pollutant of concern,partly because it has been prominent in the debate concerning the growing anthropogenic pressure on the environment. The purpose of this review is to describe how plants take lead up and to link such uptake to the ecotoxicity of lead in plants.Moreover, we address the mechanisms by which plants or plant systems detoxify lead.Lead has many interesting physico-chemical properties that make it a very useful heavy metal. Indeed, lead has been used by people since the dawn of civilization.Industrialization, urbanization, mining, and many other anthropogenic activities have resulted in the redistribution of lead from the earth's crust to the soil and to the environment.Lead forms various complexes with soil components, and only a small fraction of the lead present as these complexes in the soil solution are phyto available. Despite its lack of essential function in plants, lead is absorbed by them mainly through the roots from soil solution and thereby may enter the food chain. The absorption of lead by roots occurs via the apoplastic pathway or via Ca2+-permeable channels.The behavior of lead in soil, and uptake by plants, is controlled by its speciation and by the soil pH, soil particle size, cation-exchange capacity, root surface area,root exudation, and degree of mycorrhizal transpiration. After uptake, lead primarily accumulates in root cells, because of the blockage by Casparian strips within the endodermis. Lead is also trapped by the negative charges that exist on roots' cell walls.Excessive lead accumulation in plant tissue impairs various morphological, physiological, and biochemical functions in plants, either directly or indirectly, and induces a range of deleterious effects. It causes phytotoxicity by changing cell membrane permeability, by reacting with active groups of different enzymes involved in plant metabolism and by reacting with the phosphate groups of ADP or ATP,and by replacing essential ions. Lead toxicity causes inhibition of ATP production, lipid peroxidation, and DNA damage by over production of ROS. In addition, lead strongly inhibits seed germination, root elongation, seedling development, plant growth, transpiration, chlorophyll production, and water and protein content. The negative effects that lead has on plant vegetative growth mainly result from the following factors: distortion of chloroplast ultrastructure, obstructed electron transport,inhibition of Calvin cycle enzymes, impaired uptake of essential elements, such as Mg and Fe, and induced deficiency of CO2 resulting from stomatal closure.Under lead stress, plants possess several defense strategies to cope with lead toxicity. Such strategies include reduced uptake into the cell; sequestration of lead into vacuoles by the formation of complexes; binding of lead by phytochelatins,glutathione, and amino acids; and synthesis of osmolytes. In addition, activation of various antioxidants to combat increased production of lead-induced ROS constitutes a secondary defense system.

The mechanism of oxidative burst induced by lead in Vicia faba excised roots was investigated by luminol-dependent chemiluminescence. Results showed that lead triggered a rapid and dose-dependent increase in chemiluminescence production. In this study, specific inhibitors of putative reactive oxygen species (ROS) sources were used to determine the mechanism of lead-induced ROS generation. This generation was sensitive to dephenylene iodonium (DPI), quinacrine and imidazole, some inhibitors of the NADPH-oxidase and not inhibited by other putative ROS sources inhibitors. Data reported in this work clearly demonstrated the pivotal role of NADPH-oxidase-like enzyme in early steps of lead-induced oxidative burst. To investigate the respective implication of calmodulin and protein kinase (PK) in lead-induced NADPH-oxidase activation, excised roots were treated with the calmodulin inhibitor W7 or with the PK inhibitor staurosporine. The chemiluminescence generation inhibition by these inhibitors illustrated the role of PK in lead-induced NADPH-oxidase activation and revealed a calmodulin-dependent step. Using the calcium entry blocker La(3+) or different concentrations of calcium in the extra-cellular medium, our data highlighted the implication of Ca(2+) channel in lead-induced oxidative burst.