Lead-induced DNA damage in Vicia faba root cells: Potential involvement of oxidative stress

Genotoxic effects of lead (0–20 µM) 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 10 µM. In addition, at this concentration, DNA damage time-dependently increased until 12 h. 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

Lead Uptake, Toxicity, and Detoxification in Plants

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 phytoavailable. 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, Lead Uptake, Toxicity, and Detoxification in Plants 131 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

Lead-induced genotoxicity to Vicia faba L.roots in relation with metal cell uptake and initial speciation

Formation of organometallic complexes in soil solution strongly influence metals phytoavailability. However, only few studies deal with the influence of metalspeciation both on plant uptake and genotoxicity. In the present study, Viciafaba seedlings were exposed for 6 h in controlled hydroponic conditions to 5 μM of lead nitrate alone and chelated to varying degrees by different organic ligands. Ethylenediaminetetraacetic acid and citric acid were, respectively, chosen as models of humic substances and low weight organic acids present in natural soil solutions. Visual Minteq software was used to estimate free lead cations concentration and ultimately to design the experimental layout. For all experimental conditions, both micronucleus test and measure of leaduptake by plants were finally performed. Chelation of Pb by EDTA, a strong chelator, dose-dependently increased the uptake in V. faba roots while its genotoxicity was significantly reduced, suggesting a protective role of EDTA. A weak correlation was observed between total lead concentration absorbed by roots and genotoxicity (r2=0.65). In contrast, a strong relationship (r2=0.93) exists between Pb2+ concentration in exposure media and genotoxicity in the experiment performed with EDTA. Citric acid induced labile organometallic complexes did not demonstrate any significant changes in leadgenotoxicity or uptake. These results demonstrate that metalspeciation knowledge could improve the interpretation of V. fabagenotoxicity test performed to test soil quality

Implication du stress oxydatif dans la toxicité du plomb sur une plante modèle, Vicia faba

Le plomb est un des métaux les plus utilisés par l'Homme mais c'est aussi l'un des plus toxiques. Dans les écosystèmes, la teneur en plomb d'origine anthropique n'a cessé d'augmenter depuis plusieurs siècles. Dans plusieurs régions en France, le plomb pose actuellement des problèmes importants en matière de contamination de l'environnement. Les plantes, du fait de leur immobilité, sont particulièrement exposées. De nombreuses études ont déjà évalué la toxicité de ce métal sur les végétaux, mais la très grande majorité a utilisé des concentrations en plomb très largement supérieures à celles pouvant se retrouver dans des sites pollués. Le but de ces travaux a été d'évaluer sur une espèce sentinelle, Vicia faba, la toxicité de doses de plomb réalistes. Nous avons montré que le plomb pénétrait rapidement dans le système racinaire et générait, après seulement quelques heures, un important stress oxydatif à l'origine d'une peroxydation lipidique et de l'induction d'effets génotoxiques dans les cellules racinaires : cassures au niveau de l'ADN et induction d'aberrations chromosomiques. Nous avons pu mettre en évidence que la source de ce stress oxydatif était directement liée à l'activation de NADPH oxydases racinaires via une calmoduline kinase. L'utilisation d'inhibiteurs de cette enzyme et d'antioxydants a permis de montrer que la production d'espèces réactives de l'oxygène, initiée par la NADPH oxydase, était impliquée dans la toxicité du plomb et plus particulièrement dans sa génotoxicité. ABSTRACT : Lead contamination of ecosystems from human sources has increased for centuries. Owing to their immobility, plants are specially exposed. The goal of this study was to assess the toxicity of realistic lead doses on Vicia faba. We demonstrated that lead uptake in roots was quick. After only few hours, lead uptake induced an important oxidative stress in Vicia faba roots. This oxidative stress caused lipid peroxidation and genotoxic effects on roots cells: DNA strand breaks and chromosomal aberrations. We also demonstrated that NADPH oxidase activation via a calmodulin kinase was implicated in oxidative stress generation. The use of antioxidant and NADPH oxidase inhibitors highlighted that NADPH oxidase-mediated ROS production was implicated in lead toxicity and genotoxicit

Assessment of the genotoxicity of Cu and Zn in raw and anaerobically digested slurry with the Vicia faba micronucleus test

Genotoxicity of Cu and Zn was assessed by use of the micronucleus (MN) test on Vicia faba roots. Plants were exposed to various leachates of rawand anaerobically digested pig slurry, with maximum total concentrations of 200MCu and 600MZn. The results indicated stabilisation of the organic matter during anaerobic digestion of the slurry and bioconversion of some phytotoxic organic compounds (e.g. phenols or p-cresol), but did not showa relationship between Cu and Zn concentrations and MN frequency. Exposure of Vicia plants to binary inorganic solutions of Cu and Zn (CuSO4/ZnSO4, 1:3) showed a significant micronucleus induction at concentrations of 40M Cu and 120M Zn and higher. When MN frequency was plotted against dissolved Cu (<0.45m), applied as slurry or as CuSO4, a single curve was obtained. At concentrations lower than 10M, modulation of the genotoxic effect of Cu was found. At concentrations up to 150M,MNinduction increased significantly, while phytotoxic symptoms appeared at higher concentrations

Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots

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 leadinduced 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 extracellular medium, our data highlighted the implication of Ca 2+ channel in leadinduced oxidative burst

Author Correction: DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death (Scientific Reports, (2021), 11, 1, (16793), 10.1038/s41598-021-95976-7)

Link to the corrected article: [https://farfar.pharmacy.bg.ac.rs/handle/123456789/3944

DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death

The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan–Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06–1.90) for overall mortality, and 1.94 (1.04–3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.This article has been corrected. Link to the correction: [https://farfar.pharmacy.bg.ac.rs/handle/123456789/3975

Implication du stress oxydatif dans la toxicité du plomb sur une plante modèle, Vicia faba

Lead contamination of ecosystems from human sources has increased for centuries. Owing to their immobility, plants are specially exposed. The goal of this study was to assess the toxicity of realistic lead doses on Vicia faba. We demonstrated that lead uptake in roots was quick. After only few hours, lead uptake induced an important oxidative stress in Vicia faba roots. This oxidative stress caused lipid peroxidation and genotoxic effects on roots cells: DNA strand breaks and chromosomal aberrations. We also demonstrated that NADPH oxidase activation via a calmodulin kinase was implicated in oxidative stress generation. The use of antioxidant and NADPH oxidase inhibitors highlighted that NADPH oxidase-mediated ROS production was implicated in lead toxicity and genotoxicityLe plomb est un des métaux les plus utilisés par l'Homme mais c'est aussi l'un des plus toxiques. Dans les écosystèmes, la teneur en plomb d'origine anthropique n'a cessé d'augmenter depuis plusieurs siècles. Dans plusieurs régions en France, le plomb pose actuellement des problèmes importants en matière de contamination de l'environnement. Les plantes, du fait de leur immobilité, sont particulièrement exposées. De nombreuses études ont déjà évalué la toxicité de ce métal sur les végétaux, mais la très grande majorité a utilisé des concentrations en plomb très largement supérieures à celles pouvant se retrouver dans des sites pollués. Le but de ces travaux a été d'évaluer sur une espèce sentinelle, Vicia faba, la toxicité de doses de plomb réalistes. Nous avons montré que le plomb pénétrait rapidement dans le système racinaire et générait, après seulement quelques heures, un important stress oxydatif à l'origine d'une peroxydation lipidique et de l'induction d'effets génotoxiques dans les cellules racinaires : cassures au niveau de l'ADN et induction d'aberrations chromosomiques. Nous avons pu mettre en évidence que la source de ce stress oxydatif était directement liée à l'activation de NADPH oxydases racinaires via une calmoduline kinase. L'utilisation d'inhibiteurs de cette enzyme et d'antioxydants a permis de montrer que la production d'espèces réactives de l'oxygène, initiée par la NADPH oxydase, était impliquée dans la toxicité du plomb et plus particulièrement dans sa génotoxicitéTOULOUSE-ENSAT-Documentation (315552324) / SudocSudocFranceF

Development of an automated scoring system for plant comet assay

In plants, an increasing interest for the Comet Assay was shown in the last decade and this versatile technique appears to be promising to detect the genotoxic effect of pollutants and to monitor the environment. However, its use in plant studies was rather limited compared to animal studies because of (i) the difficulty to isolate intact nuclei compared to animal systems and the lack of a standardized protocol, (ii) the low throughput of current nucleus extraction, and (iii) the lack of a high throughput comet assay scoring method. In order to deal with these issues, we recently identified the key steps of the comet assay on plant models and proposed an optimized protocol to increase its reliability (Pourrut et al.; 2015). At the same time, in the frame of the French-Norwegian project ComPack (2014-2017), we have worked on: - the development a new nucleus extraction technique compatible with the high-throughput comet assay scoring methods; - the automation of the scoring method based on the automated scoring system Pathfinder™, developed by IMSTAR. Details and results from the preliminary experiments will be presented and discussed. Major issues have been: - scoring system: specific adaptation of the automated scoring system Pathfinder™ is crucial as it was initially set up for human/animal cells; - background: optimization of the protocol to reduce the presence of debris and increase background quality; - nucleus density: increase the density of nuclei is of importance to increase scoring reliability (Sharma et al., 2012). In conclusion, increasing plant nucleus extraction yield and automated scoring of nuclei do represent big challenges. However, our promising preliminary results open up the perspective of an automated high-throughput scoring of plant nuclei