An integrated approach to determine interactive genotoxic and global gene expression effects of multiwalled carbon nanotubes (MWCNTs) and benzo[a]pyrene (BaP) on marine mussels: evidence of reverse ‘Trojan Horse’ effects

International audienc

Antagonistic Interactions between Benzo[a]pyrene and Fullerene (C60) in Toxicological Response of Marine Mussels

This study aimed to assess the ecotoxicological effects of the interaction of fullerene (C60) and benzo[a]pyrene (B[a]P) on the marine mussel, Mytilus galloprovincialis. The uptake of nC60, B[a]P and mixtures of nC60 and B[a]P into tissues was confirmed by Gas Chromatography–Mass Spectrometry (GC–MS), Liquid Chromatography–High Resolution Mass Spectrometry (LC–HRMS) and Inductively Coupled Plasma Mass Spectrometer (ICP–MS). Biomarkers of DNA damage as well as proteomics analysis were applied to unravel the interactive effect of B[a]P and C60. Antagonistic responses were observed at the genotoxic and proteomic level. Differentially expressed proteins (DEPs) were only identified in the B[a]P single exposure and the B[a]P mixture exposure groups containing 1 mg/L of C60, the majority of which were downregulated (~52%). No DEPs were identified at any of the concentrations of nC60 (p < 0.05, 1% FDR). Using DEPs identified at a threshold of (p < 0.05; B[a]P and B[a]P mixture with nC60), gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that these proteins were enriched with a broad spectrum of biological processes and pathways, including those broadly associated with protein processing, cellular processes and environmental information processing. Among those significantly enriched pathways, the ribosome was consistently the top enriched term irrespective of treatment or concentration and plays an important role as the site of biological protein synthesis and translation. Our results demonstrate the complex multi-modal response to environmental stressors in M. galloprovincialis

Étude chez l'huître creuse, Crassostrea gigas, des anomalies génomiques provoquées par l'exposition à des concentrations environnementales de diuron : caractérisation des atteintes, étude de leur héritabilité et conséquences pour la survie et la croissance des naissains

The aims of this PhD were to determine (1) the effects of the herbicide diuron on the genome of the oyster, Crassostrea gigas, (2) the possible transmission to the offspring of damaged DNA and (3) its consequences on oyster physiology. The genotoxicity of diuron and the vertical transmission of DNA damage were highlighted after parental exposure to environmental concentrations of diuron during the gametogenesis. Primary DNA lesions were detected in both somatic cells and germ cells of exposed genitors. Detection of the oxidized base 8-oxodGuo in gonads shows that exposure to diuron led to an oxidative stress which likely explains the observed DNA strand breaks in spermatozoa. The transmission of these DNA damage was confirmed by fluorescent in situ hybridization: aneuploid nuclei were detected in embryos from diuron-exposed genitors. This hyper- and hypodiploidy particularly concerns 5S and 18-5.8-28S ribosomal genes localized on chromosomes 4, 5 and 10. Flow cytometry analysis showed survival of hypodiploid individuals at spat stage. This vertical transmission of damaged genetic material is associated with deleterious effects on the physiology of the offspring (developmental defects, growth retardation). Data acquired during this PhD contribute to a better understanding of the long-term effects of exposure to genotoxic pollutants. In the context of massive mortality outbreaks, the presence of chemical pollutants may contribute to a weakening of oysters in case of the presence of pathogens or adverse physicochemical environmental conditions.les effets d’un herbicide, le diuron, sur le génome de l'huître creuse, (2) la possible transmission des dommages à l'ADN à la descendance ainsi que (3) les conséquences physiologiques associées. La génotoxicité du diuron et la transmission verticale des dommages à l'ADN ont été démontrées après une exposition parentale à des concentrations environnementales de diuron pendant la période de gamétogenèse. Des lésions primaires à l’ADN ont été détectées aussi bien dans les cellules somatiques que dans les cellules germinales des géniteurs exposés. La détection de la base oxydée 8-oxodGuo dans les gonades montre que l'exposition au diuron a conduit à un stress oxydant qui vraisemblablement explique les cassures à l’ADN observées dans les spermatozoïdes. La transmission de ces dommages à l’ADN a été confirmée par la technique d’hybridation in situ : des noyaux aneuploïdes ont été détectés chez les embryons issus des géniteurs exposés au diuron. Cette hyper- et hypodiploïdie concerne notamment les gènes ribosomaux 5S et 18-5.8-28S présents sur les chromosomes 4, 5 et 10. Les analyses de cytométrie en flux montrent la survie d’individus hypodiploïdes au stade naissain. Cette transmission verticale de matériel génétique endommagé s'accompagne d'effets délétères au niveau de la physiologie des descendants (anomalies de développement, retard de croissance). Les données acquises au cours de cette thèse contribuent à une meilleure connaissance des effets à long terme de l'exposition à des polluants génotoxiques. Dans le contexte des mortalités massives, la présence de polluants chimiques pourrait contribuer à un affaiblissement des huîtres face à la présence de pathogènes ou à des conditions physicochimiques du milieu défavorable

Towards better prediction of xenobiotic genotoxicity: CometChip technology coupled with a 3D model of HepaRG human liver cells

International audienceToxicology is facing a major change in the way toxicity testing is conducted by moving away from animal experimentation towards animal-free methods. To improve the in vitro genotoxicity assessment of chemical and physical compounds, there is an urgent need to accelerate the development of 3D cell models in high-throughput DNA damage detection platforms. Among the alternative methods, hepatic cell lines are a relevant in vitro model for studying the functions of the liver. 3D HepaRG spheroids show improved hepatocyte differentiation, longevity, and functionality compared with 2D HepaRG cultures and are therefore a relevant model for predicting in vivo responses. Recently, the comet assay was developed on 3D HepaRG cells. However, this approach is still low throughput and does not meet the challenge of evaluating the toxicity and risk to humans of tens of thousands of compounds. In this study, we evaluated the performance of the high-throughput in vitro CometChip assay on 2D and 3D HepaRG cells. HepaRG cells were exposed for 48 h to several compounds (methyl methanesulfonate, etoposide, benzo[a]pyrene, cyclophosphamide, 7,12-dimethylbenz[a]anthracene, 2-acetylaminofluorene, and acrylamide) known to have different genotoxic modes of action. The resulting dose responses were quantified using benchmark dose modelling. DNA damage was observed for all compounds except 2-AAF in 2D HepaRG cells and etoposide in 3D HepaRG cells. Results indicate that the platform is capable of reliably identifying genotoxicants in 3D HepaRG cells, and provide further insights regarding specific responses of 2D and 3D models

Genotoxic and epigenetic effects of diuron in the Pacific oyster: in vitro evidence of interaction between DNA damage and DNA methylation

Recently, research has contributed to better knowledge on the occurrence of pesticides in coastal water by identifying frequently detected substances, their concentration range and their acute and chronic toxicity for organisms. Pesticide pollution is of particular concern in France due to important agricultural activities and presence of several exoreic catchment areas that vehicle pesticides up to coastal waters, impacting non-target marine species. Several ecotoxicology questions remain to be addressed concerning the long-term effects of chronic pesticide exposure and the mechanisms involved in adaptation to chemical stress. In the present study, we brought new insights on the genetic and epigenetic effects of the herbicide diuron in oyster genitors. During gametogenesis, we exposed Crassostrea gigas to environmentally realistic herbicide concentrations (0.2–0.3 μg L−1 during two 7-day periods at half-course and end of gametogenesis). Diuron exposure was shown to decrease global DNA methylation and total methyltransferase activity in whole oyster tissue; this is consistent with the previous observation of a significant decrease in DNMT1 gene expression. Diuron effect seemed to be tissue-specific; hypermethylation was detected in the digestive gland, whereas diuron exposure had no effect on gill and gonad tissue. The genotoxicity of diuron was confirmed by the detection of one adduct in gonad DNA. By using in vitro approaches and human DNMT1 (DNMT1 has not been purified yet in bivalves), the presence of DNA lesions (adduct, 8-oxodGuo) was shown to interfere with DNMT1 activity, indicating a complex interaction between DNA damage and DNA methylation. Based on our results, we propose mechanisms to explain the effect of diuron exposure on DNA methylation, a widespread epigenetic mark

Tolerance of soil engineer to pesticide residual contamination in agrosystems: an omic strategy for studying the molecular mechanisms at play.

International audienc

Tolerance of soil engineer to pesticide residual contamination in agrosystems: an omic strategy for studying the molecular mechanisms at play.

International audienc

Tolérance de la faune ingénieure du sol à la contamination résiduelle par les produits phytosanitaires dans les agroécosystèmes : approches omiques des mécanismes moléculaires en jeu

National audienc

Parental exposure to environmental concentrations of diuron leads to aneuploidy in embryos of the Pacific oyster, as evidenced by fluorescent in situ hybridization

Changes in normal chromosome numbers (i.e. aneuploidy) due to abnormal chromosome segregation may arise either spontaneously or as a result of chemical/radiation exposure, particularly during cell division. Coastal ecosystems are continuously subjected to various contaminants originating from urban, industrial and agricultural activities. Genotoxicity is common to several families of major environmental pollutants, including pesticides, which therefore represent a potential important environmental hazard for marine organisms. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to short-term exposure to the herbicide diuron at environmental concentrations during gametogenesis. In this paper, Fluorescent in situ hybridization (FISH) was used to further characterize diuron-induced DNA damage at the chromosomal level. rDNA genes (5S and 18-5.8-28S), previously mapped onto C. gigas chromosomes 4, 5 and 10, were used as probes on the interphase nuclei of embryo preparations. Our results conclusively show higher aneuploidy (hypo- or hyperdiploidy) level in embryos from diuron-exposed genitors, with damage to the three studied chromosomal regions. This study suggests that sexually-developing oysters are vulnerable to diuron exposure, incurring a negative impact on reproductive success and oyster recruitment