Xenopus oocytes in environmental toxicology : a promising tool ?

Les amphibiens constituent le groupe le plus menacé d’extinction parmi les vertébrés. Néanmoins, peu de travaux en toxicologie des amphibiens tiennent compte des stades précoces de leur cycle de vie. Pourtant, un individu est exposé directement aux substances présentes dans le milieu aquatique dès l’émission des gamètes. Cette thèse de doctorat a pour objectifs d’apporter de nouvelles données sur les effets d’expositions à des xénobiotiques d’ovocytes de Xenopus laevis, de rechercher des cibles au sein de ce gamète et de participer au développement d’un nouveau modèle en écotoxicologie pour évaluer la qualité de milieux aquatiques.Les avantages, que présentent ces ovocytes, nous ont permis de développer des protocoles efficaces pour appréhender la toxicité de substances. Des endpoints ont pu être définis autour de la maturation ovocytaire, de la fécondation, du développement embryonnaire et de la formation de jeunes têtards. Les effets d’expositions au cadmium, au plomb, au cuivre, à la bouillie bordelaise, au glyphosate, au RoundUp® GT Max et à la deltaméthrine ont été déterminés. Des essais ont été également conduits pour des échantillons de milieux soumis à différentes pressions anthropiques.Il est apparu que l’ovocyte de xénope est sensible aux expositions, notamment au cadmium et au glyphosate et différentes signatures d’expositions sont apparues, comme la formation de doubles structures cytologiques induites par le glyphosate.Les réponses mises en évidence prouvent que l’ovocyte de X. laevis est un modèle pertinent et permettent de recommander l’étude des premières étapes du cycle de vie de l’amphibien en toxicologie aquatique.Amphibians are one of the most imperiled group of extinction. Nevertheless, few toxicological studies are interested in the earliest steps of their life cycle, even if gamete emission, fertilization and embryogenesis are directly exposed to water pollution. In this context, this PhD thesis aims to bring new data about xenobiotic exposure effects on Xenopus laevis oocytes, to highlight targets inside this germ cells and to contribute to the elaboration of a new model in ecotoxicology to assess aquatic environment quality.As a well-known gamete, the xenopus oocyte makes possible to establish suitable experimental designs to assess toxicity. Many endpoints were defined regarding the oocyte maturation, the fertilization and also the development. The experiments were conducted in metal (cadmium, lead, copper) and in phytopharmaceutical (Bordeaux mixture, glyphosate, RoundUp® GT Max, deltamethrin) contaminated conditions, but also in environmental samples from various aquatic habitats.The xenopus oocyte appeared to be sensitive to contaminant exposures and specially to cadmium and both formulations of glyphosate. Never observed effects were reported. Pollutant signatures were also pointed up, like the double cytological structures induced by glyphosate exposures.The observed responses and results from environmental water experimentations show that X. laevis oocyte is a pertinent model in ecotoxicology and allow to recommend the first steps of the amphibian life cycle in aquatic toxicology

Adverse effects of fly ashes used as immobilizing agents for highly metal-contaminated soils on Xenopus laevis oocytes survival and maturation—a study performed in the north of France with field soil extracts

International audienceAmphibians are now recognized as the most endangered group. One of this decline causes is the degradation of their habitat through direct contamination of water, soil leaching, or runoff from surrounding contaminated soils and environments. In the North of France, the extensive industrial activities resulted in massive soil contamination by metal compounds. Mineral amendments were added to soils to decrease trace metal mobility. Because of the large areas to be treated, the use of inexpensive industrial by-products was favored. Two types of fly ashes were both tested in an experimental site with the plantation of trees in 2000. Aim of the present work was to investigate the effects of extracts from metal-contaminated soils treated or not for 10 years with fly ashes on Xenopus laevis oocyte using cell biology approaches. Indeed, our previous studies have shown that the Xenopus oocyte is a relevant model to study the metal ion toxicity. Survival and maturation of oocyte exposed to the soil extracts were evaluated by phenotypic approaches and electrophysiological recordings. An extract derived from a metal-contaminated soil treated for 10 years with sulfo-calcic ashes induced the largest effects. Membrane integrity appeared affected and ion fluxes in exposed oocytes were changed. Thus, it appeared that extracted elements from certain mineral amendments used to prevent the mobility of metals in the case of highly metal-contaminated soils could have a negative impact on X. laevis oocytes

Effects of Aluminium Contamination on the Nervous System of Freshwater Aquatic Vertebrates: A Review

International audienceAluminium (Al) is the most common natural metallic element in the Earth’s crust. It is released into the environment through natural processes and human activities and accumulates in aquatic environments. This review compiles scientific data on the neurotoxicity of aluminium contamination on the nervous system of aquatic organisms. More precisely, it helps identify biomarkers of aluminium exposure for aquatic environment biomonitoring in freshwater aquatic vertebrates. Al is neurotoxic and accumulates in the nervous system of aquatic vertebrates, which is why it could be responsible for oxidative stress. In addition, it activates and inhibits antioxidant enzymes and leads to changes in acetylcholinesterase activity, neurotransmitter levels, and in the expression of several neural genes and nerve cell components. It also causes histological changes in nerve tissue, modifications of organism behaviour, and cognitive deficit. However, impacts of aluminium exposure on the early stages of aquatic vertebrate development are poorly described. Lastly, this review also poses the question of how accurate aquatic vertebrates (fishes and amphibians) could be used as model organisms to complement biological data relating to the developmental aspect. This “challenge” is very relevant since freshwater pollution with heavy metals has increased in the last few decades

Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage

International audienceAmong the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration

Barrage fishponds, a funnel effect for metal contaminants on headwater streams

International audienceFishponds are man-made shallow water bodies that are still little studied because of their small size. They represent high value ecosystems, both environmentally (biodiversity hotspot) and economically (fish production). They can have a high place on the hydrographic network, so their influence on water quality is of first importance for rivers and water bodies located downstream and monitored under the Water Framework Directive. These small water bodies can be a source of contaminants during draining period or an efficient buffer for pesticides. We wanted to evaluate whether these ponds could also be a remediation tool against metals by following the annual evolution of upstream/downstream flows. Cadmium, copper, lead and zinc concentrations were quantified in the dissolved phase upstream and downstream of three ponds, each one having a specific agricultural environment (traditional or organic). Metal concentration was quantified in sediments and water. For the dissolved phase, the predictive non-effect concentration was often exceeded, suggesting an environmental risk. Results highlighted also greater quantity of metals at the downstream of the pond compared to the upstream, suggesting remobilization into the ponds or direct cross-sectional contributions from the watershed (e.g. runoff from crops) or even remobilization. Regarding sediments, minimal contamination was shown but a high mineralogical variability. No buffer effect of ponds, which could reduce the risk of acute or chronic toxicity, was detected

Effects of glyphosate and a commercial formulation Roundup® exposures on maturation of Xenopus laevis oocytes

International audiencePesticides are often found at high concentrations in small ponds near agricultural field where amphibians are used to live and reproduce. Even if there are many studies on the impacts of phytopharmaceutical active ingredients in amphibian toxicology, only a few are interested in the earlier steps of their life cycle. While their populations are highly threatened with extinction. The aim of this work is to characterize the effects of glyphosate and its commercial formulation Roundup® GT Max on the Xenopus laevis oocyte maturation which is an essential preparation for the laying and the fertilization. Glyphosate is an extensively used herbicide, not only known for its effectiveness but also for its indirect impacts on non-target organisms. Our results showed that exposures to both forms of glyphosate delayed this hormone-dependent process and were responsible for spontaneous maturation. Severe and particular morphogenesis abnormalities of the meiotic spindle were also observed. The MAPK pathway and the MPF did not seem to be affected by exposures. The xenopus oocyte is particularly affected by the exposures and appears as a relevant model for assessing the effects of environmental contamination

Development of a simple multiresidue extraction method for the quantification of a wide polarity range list of pesticides and transformation products in eggs by liquid chromatography and tandem mass spectrometry

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Miniaturization of an extraction protocol for the monitoring of pesticides and polar transformation products in biotic matrices

The authors sincerely thank the fish farmers and owners for granting access to their ponds. They are also grateful to Alain Iurétig (sampling and pre-treatment, Univ. of Lorraine), Pamela Hartmeyer (pre-treatment, Univ. of Lorraine), Aisha Nunoo (extractions, Univ. of Lorraine/ISA), and Maud Dessein-Lepasteur (extractions and analysis, Univ. of Lorraine/ISA) for their work, as well as to Arnaud Chaumot and Laura Garnero from the Ecotoxicology Team of the UR RIVERLY (INRAE, Centre de Lyon-Villeurbanne) for providing chironomid larvae. They would also like to thank ABC Translation for proofreading the manuscript.International audienceMonitoring pesticides in the environment requires the use of sensitive analytical methods. However, existing methods are generally not suitable for analyzing small organisms, as they require large matrix masses. This study explores the development of a miniaturized extraction protocol for the monitoring of small organisms, based on only 30 mg of matrix. The miniaturized sample preparation was developed using fish and macroinvertebrate matrices. It allowed the characterization of 41 pesticides and transformation products (log P from −1.9 to 4.8) in small samples with LC-MS/MS, based on European guidelines (European Commission DG-SANTE, 2019). Quantification limits ranged from 3 to 460 ng g−1 dry weight (dw) for fish and from 0.1 to 356 ng g−1 dw for invertebrates, with most below 60 ng g−1 dw. Extraction rates ranged from 70% to 120% for 35 molecules in fish. Recoveries ranged from 70% to 120% for 37 molecules in macroinvertebrates. Inter-day precision was below 30% for 32 molecules at quantification limits. The method was successfully applied to 17 fish and 19 macroinvertebrates collected from two ponds of the French region of Dombes in November and May 2018, respectively. Both sample matrices were nearly always contaminated with benzamide, imidacloprid-desnitro, and prosulfocarb at respective concentrations of 42–237, 3, and 30–165 ng g−1 dw in fish, and 62–438, 2–6, and 15–29 ng g−1 dw in macroinvertebrates. Results show that this method is an effective tool for characterizing polar pesticides in small biotic samples

Distribution of pesticides and some of their transformation products in a small lentic waterbody: Fish, water, and sediment contamination in an agricultural watershed

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