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.
Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were ...recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new 'STressREsponse Microarray' (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzoapyrene (BaP) at 5, 50, and 100 μg/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of BaP was also determined demonstrating exposure in both tissues. In addition to the well-known effects of BaP on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers (Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose.DNA adducts measurements using 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-BPDE). Lipofiscin content increased significantly in BaP exposed mussels. Immunohistochemical analysis of tubulin and actin showed changes in cytoskeleton organisation. Our results adopting an integrated approach confirmed that the combination of newly developed transcriptomic approcah, classical biomarkers along with chemical analysis of water and tissue samples should be considered for environmental bioimonitoring and ecotoxicological studies to obtain holistic information to assess the impact of contaminants on the biota.
The EU directive 2001/18/EC requires any genetically modified (GM) event to be stable. In the present work, a targeted Next-Generation Sequencing (NGS) approach using barcodes to specifically tag ...each individual DNA molecules during library preparation was implemented to detect mutations taking into account the background noise due to amplification and sequencing errors. The method was first showed to be efficient in detecting the mutations in synthetic samples prepared with custom-synthesized mutated or non-mutated P35S sequences mixed in different proportions. The genetic stability of a portion of the P35S promoter targeted for GM detection was then analyzed in GM flour samples. Several low frequency mutations were detected in the P35S sequences. Some mutated nucleotides were located within the primers and probes used in the P35S diagnostic test. If present not as somatic mutations but as the consensus sequence of some individuals, these mutations could influence the efficiency of the P35S real time PCR diagnostic test. This methodology could be implemented in genetic stability studies of GM inserts but also to detect single nucleotide mutant GM plants produced using "new breeding techniques".
This study aimed to assess the ecotoxicological effects of the interaction of fullerene (C
) and benzoapyrene (BaP) on the marine mussel,
. The uptake of
C
, BaP and mixtures of
C
and BaP 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 BaP and C
. Antagonistic responses were observed at the genotoxic and proteomic level. Differentially expressed proteins (DEPs) were only identified in the BaP single exposure and the BaP mixture exposure groups containing 1 mg/L of C
, the majority of which were downregulated (~52%). No DEPs were identified at any of the concentrations of
C
(
< 0.05, 1% FDR). Using DEPs identified at a threshold of (
< 0.05; BaP and BaP mixture with
C
), 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
.
Toxicology 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, benzoapyrene, cyclophosphamide, 7,12-dimethylbenzaanthracene, 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.
Nanomaterials (NMs) are defined as materials with at least one external dimension below 100 nm. Their small size confers them interesting unique physico-chemical properties, hence NMs are ...increasingly used in a diversity of applications. However, the specific properties of NMs could also make them more harmful than their bulk counterparts. Therefore, there is a crucial need to deliver efficient NM hazard assessment in order to sustain the responsible development of nanotechnology. This study analysed the genotoxic potential of several NMs: one titanium dioxide (TiO2) and two zinc oxide NMs (ZnO) that were tested up to 100 μg/mL on 2D and 3D hepatic HepaRG models. Genotoxicity analysis was performed comparing the alkaline comet assay in classical and high throughput formats. Moreover, oxidative DNA lesions were investigated with the Fpg-modified comet assay. Results showed that TiO2 NMs were not cytotoxic and not genotoxic in either cell model, although a small increase in the % tail DNA was observed in 3D HepaRG cells at 100 μg/mL in the classical format. The two ZnO NMs (ZnO S. NMs a commercial suspension and NM110 provided by the European Union Joint Research Centre) induced a concentration-dependent increase in cytotoxicity that was more pronounced in the 2D (>20% cytotoxicity was observed for ZnO S. at concentrations greater than 25 μg/mL, and for NM 110 at 50 μg/mL) than in the 3D model (more than 20% cytotoxicity for ZnO S. NMs at 50 μg/mL). While ZnO S. NMs induced DNA damage associated with cytotoxicity (at 25 and 50 μg/mL in 2D and 50 μg/mL in 3D), NM110 showed a clear genotoxic effect at non-cytotoxic concentrations (25 μg/mL in 2D and at 25 and 50 μg/mL in 3D). No major differences could be observed in the comet assay in the presence or absence of the Fpg enzyme. High throughput analysis using CometChip® mostly confirmed the results obtained with the classical format, and even enhanced the detection of genotoxicity in the 3D model. In conclusion, this study demonstrated that new approach methodologies (NAMs), 3D models and the high throughput format for the comet assay, were more efficient in the detection of genotoxic effects, and are therefore promising approaches to improve hazard assessment of NMs.
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•Three nanomaterials were tested for genotoxicity in the liver HepaRG cell line.•Both 2D and 3D HepaRG models were used.•Traditional and high throughput versions of the comet assay were compared.•Both high throughput format and 3D model enhanced the detection of genotoxicity.
Diffuse pollution of the environment by pesticides has become a major soil threat to non-target organisms, such as earthworms for which declines have been reported. However some endogeic species are ...still abundant and persist in intensively cultivated fields, suggesting they become tolerant to long-term anthropogenic pressure. We thus considered the working hypothesis that populations of Aporrectodea caliginosa earthworms from conventionally managed fields developed a tolerance to pesticides compared with those from organically managed fields. To investigate this hypothesis, we studied earthworm populations of the same genetic lineage from soils that were either lowly or highly contaminated by pesticides to detect any constitutive expression of differentially expressed molecular pathways between these populations. Earthworm populations were then experimentally exposed to a fungicide—epoxiconazole—in the laboratory to identify different molecular responses when newly exposed to a pesticide. State-of-the-art omics technology (RNA sequencing) and bioinformatics were used to characterize molecular mechanisms of tolerance in a non-targeted way. Additional physiological traits (respirometry, growth, bioaccumulation) were monitored to assess tolerance at higher levels of biological organization. In the present study, we generated the de novo assembly transcriptome of A. caliginosa consisting of 64,556 contigs with N50 = 2862 pb. In total, 43,569 Gene Ontology terms were identified for 21,593 annotated sequences under the three main ontologies (biological processes, cellular components and molecular functions). Overall, we revealed that two same lineage populations of A. caliginosa earthworms, inhabiting similar pedo-climatic environment, have distinct gene expression pathways after they long-lived in differently managed agricultural soils with a contrasted pesticide exposure history for more than 22 years. The main difference was observed regarding metabolism, with upregulated pathways linked to proteolytic activities and the mitochondrial respiratory chain in the highly exposed population. This study improves our understanding of the long-term impact of chronic exposure of soil engineers to pesticide residues.
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•Earthworms chronically exposed to high pesticide concentrations in agriculture soils.•Tolerance to pesticide investigated in the endogeic earthworm A.caliginosa.•de novo assembly transcriptome of A. caliginosa was generated.•Specific gene expression revealed in populations differently exposed in natura.•Proteolytic activities and the respiratory chain pathways differently expressed.
One of the primary challenges in ecotoxicology is to contribute to the assessment of the ecological status of ecosystems. In this study, we used Pacific oyster Crassostrea gigas to explore the ...effects of a parental exposure to diuron, a herbicide frequently detected in marine coastal environments. The present toxicogenomic study provides evidence that exposure of oyster genitors to diuron during gametogenesis results in changes in offspring, namely, transcriptomic profile alterations, increased global DNA methylation levels and reduced growth and survival within the first year of life. Importantly, we highlighted the limitations to identify particular genes or gene expression signatures that could serve as biomarkers for parental herbicide-exposure and further for multigenerational and transgenerational effects of specific chemical stressors. By analyzing samples from two independent experiments, we demonstrated that, due to complex confounding effects with both tested solvent vehicles, diuron non-specifically affected the offspring transcriptome. These original results question the potential development of predictive genomic tools for detecting specific indirect impacts of contaminants in environmental risk assessments. However, our results indicate that chronic environmental exposure to diuron over several generations may have significant long term impacts on oyster populations with adverse health outcomes.
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•Effects of parental diuron exposure were evidenced in an exploited marine bivalve.•Growth and survival rates of offspring were transiently affected.•Global DNA methylation levels were increased in genitor sperm and in offspring.•Great alterations occurred in transcriptomic profiles.•Diuron impact was not gene-specific due to confounding effects with solvents used.
The new challenges in toxicology demand novel and innovative in vitro approaches for deriving points of departure (PODs) and determining the mode of action (MOA) of chemicals. Therefore, the aim of ...this original study was to couple in vitro studies with untargeted metabolomics to model the concentration-response of extra- and intracellular metabolome data on human HepaRG cells treated for 48 h with three pyrrolizidine alkaloids (PAs): heliotrine, retrorsine and lasiocarpine. Modeling revealed that the three PAs induced various monotonic and, importantly, biphasic curves of metabolite content. Based on unannotated metabolites, the endometabolome was more sensitive than the exometabolome in terms of metabolomic effects, and benchmark concentrations (BMCs) confirmed that lasiocarpine was the most hepatotoxic PA. Regarding its MOA, impairment of lipid metabolism was highlighted at a very low BMC (first quartile, 0.003 µM). Moreover, results confirmed that lasiocarpine targets bile acids, as well as amino acid and steroid metabolisms. Analysis of the endometabolome, based on coupling concentration-response and PODs, gave encouraging results for ranking toxins according to their hepatotoxic effects. Therefore, this novel approach is a promising tool for next-generation risk assessment, readily applicable to a broad range of compounds and toxic endpoints.
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•PA effects on HepaRG cells were studied by concentration-response modelling and BMC.•The endometabolome was found to be more sensitive than the exometabolome.•LAS had the highest hepatotoxic potency, followed by HEL and RET.•The bell shape is the most frequent trend in response to LAS.•Lipids, bile acids and amino acids were especially affected by LAS.
The effects of C
on mTOR (mechanistic Target of Rapamycin) activity in mussel digestive gland were investigated. mTOR is a kinase that senses physiological and environmental signals to control ...eukaryotic cell growth. mTOR is present in two complexes: the phosphorylated mTORC1 regulates cell growth by activating anabolic processes, and by inhibiting catabolic processes (i.e. autophagy); mTORC2 also modulates actin cytoskeleton organization. Mussels were exposed to C
(0.01, 0.1 and 1 mg/L) for 72 h. Immunocytochemical analysis using a specific antibody revealed the cellular distribution of C
in mussel digestive gland, already at the lowest concentration. In exposed mussels, the dephosphorylation of mTORC1 and mTORC2 may explain the C
effects, i.e. the reduction of lysosomal membrane stability, the enhancement of LC3B protein, and the increase of lysosomal/cytoplasmic volume ratio; as well the cytoskeletal alterations. No oxidative stress was observed. Multivariate analysis was used to facilitate the interpretation of the biomarker data. Finally, a low density oligo-microarray was used to understand the cellular responses to fullerene. Transcriptomics identified a number of differentially expressed genes (DEGs) showing a maximum in animals exposed to 0.1 mg/L C
. The most affected processes are associated with energy metabolism, lysosomal activity and cytoskeleton organization. In this study, we report the first data on the subcellular distribution of C
in mussel's cells; and on the involvement of mTOR inhibition in the alterations due to nanoparticle accumulation. Overall, mTOR deregulation, by affecting protein synthesis, energy metabolism and autophagy, may reduce the capacity of the organisms to effectively grow and reproduce.
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