Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme.

BACKGROUND: Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). METHODS: RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. RESULTS: Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. CONCLUSION: Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.

Parental diuron-exposure alters offspring transcriptome and fitness in Pacific oyster Crassostrea gigas.

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.

A mesocosm study of fate and effects of CuO nanoparticles on endobenthic species (Scrobicularia plana, Hediste diversicolor).

The fate and effects of CuO nanoparticles (CuO NPs) were examined in endobenthic species (Scrobicularia plana , Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms (exposure to Cu at 10 µg L(-1) in particulate (CuO NPs) or soluble salt (CuNO(3)) forms) for 21 days. Labile Cu was determined in water and sediment by using diffusive gradient in thin films. No labile Cu being detected from CuO NPs; the observed effects in invertebrates exposed to CuO NPs were mainly attributed to the toxicity of nanoparticulate rather than dissolved Cu toxicity. Bioaccumulation of CuO NPs was observed in both species. Biomarkers were examined at different levels of biological organization: biochemical markers of defense and damage, biomarkers of genotoxicity (comet assay), and behavioral biomarkers (feeding and burrowing). Behavioral biomarkers, antioxidant defenses (catalase, glutathion S-transferase, metallothionein), and genotoxicity are the most sensitive tools to highlight the effect of soluble or nanoparticulate metal forms. Concerning other biomarkers of defense (superoxide dismutase, lactate dehydrogenase, laccase) and damage (thiobarbituric acid reactive substances, acetylcholinesterase, acid phosphatase), no significant effects were detected. This experiment shows the suitability of mesocosms for studying the environmental effects of nanoparticles.

Molecular and phenotypic effects of early exposure to an environmentally relevant pesticide mixture in the Pacific oyster, Crassostrea gigas.

Early life stages are crucial for organism development, especially for those displaying external fertilization, whose gametes and early stages face environmental stressors such as xenobiotics. The pacific oyster, Crassostrea gigas, is considered a model species in ecotoxicology because of its ecological characteristics (benthic, sessile, filter feeding). So far studies have investigated the impact of xenobiotics at embryotoxic, genotoxic and physiological endpoints, sometimes at the multigenerational scale, highlighting the role of epigenetic mechanisms in transmitting alterations induced by exposure to single xenobiotics. However, to date, little is known about the impact of environmentally-mimicking contaminants cocktails. Thus, we examined the impact of an early exposure to environmentally relevant mixture on the Pacific oyster life history. We studied transcriptomic, epigenetic and physiological alterations induced in oysters exposed to 18 pesticides and metals at environmental concentration (nominal sum concentration: 2.85 µg.L-1, measured sum concentration: 3.74 ± 0.013 µg.L-1) during embryo-larval stage (0-48 h post fertilization, hpf). No significant differences in embryo-larval abnormalities at 24 hpf were observed during larval and spat rearing; the swimming behaviour of exposed individuals was disturbed, while they were longer and heavier at specific time points, and exhibited a lower epinephrine-induced metamorphosis rate as well as a higher survival rate in the field. In addition, RNA-seq analyses of gastrula embryos revealed the differential expression of development-related genes (e.g. Hox orthologues and cell cycle regulators) between control and exposed oysters. Whole-genome DNA methylation analyses demonstrated a significant modification of DNA methylation in exposed larvae marked by a demethylation trend. Those findings suggest that early exposure to an environmentally relevant pesticide mixture induces multi-scale latent effects possibly affecting life history traits in the Pacific oyster.

Differences in chemical contaminants bioaccumulation and ecotoxicology biomarkers in Mytilus edulis and Mytilus galloprovincialis and their hybrids.

The Mytilus mussels are spread all over the world and many related species coexist in several areas and can produce hybrid offspring. Mussels have been used for decades in national and international programs to monitor chemical contamination in the environment. Differences in bioaccumulation and biotransformation abilities between species and their hybrids should be evaluated to assess the comparability of the results obtained within the international biomonitoring programs. The objective of this study was to characterize bioaccumulation abilities and biomarker responses in Mytilus edulis, Mytilus galloprovincialis and their hybrids via an in situ transplantation experimentation on their progenies. Four mussel groups (M. edulis, M. galloprovincialis and two hybrids batches) issued from genetically characterized parents were transplanted for one year in Charente Maritime (France) to ensure their exposure to identical sources of contamination. The bioaccumulation of several families of contaminants (trace metals, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers, polychlorinated biphenyls), the response of several biomarkers (DNA strand breaks level, lysosomal membrane stability, metallothionein content, acetylcholine esterase activity) and some physiological parameters (growth, mortality, gonadal development), were analyzed. Differences were observed between species, however they were contaminant-specific. Variations in contaminants levels were observed between progenies, with higher levels of Cu, PBDE, PCB in M. edulis, and higher levels of Cd, Hg, Zn in M galloprovincialis. This study demonstrated that variations in contaminant bioaccumulation and different biomarker responses exist between Mytilus species in the field. Data on species or the presence of hybrid individuals (or introgression) is an important additional parameter to add to biomonitoring programs databases.

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.

Metal stable isotopes in transplanted oysters as a new tool for monitoring anthropogenic metal bioaccumulation in marine environments: The case for copper.

Metal release into the environment from anthropogenic activities may endanger ecosystems and human health. However, identifying and quantifying anthropogenic metal bioaccumulation in organisms remain a challenging task. In this work, we assess Cu isotopes in Pacific oysters (C. gigas) as a new tool for monitoring anthropogenic Cu bioaccumulation into marine environments. Arcachon Bay was taken as a natural laboratory due to its increasing contamination by Cu, and its relevance as a prominent shellfish production area. Here, we transplanted 18-month old oysters reared in an oceanic neighbor area into two Arcachon Bay mariculture sites under different exposure levels to continental Cu inputs. At the end of their 12-month long transplantation period, the oysters' Cu body burdens had increased, and was shifted toward more positive δ65Cu values. The gradient of Cu isotope compositions observed for oysters sampling stations was consistent with relative geographic distance and exposure intensities to unknown continental Cu sources. A binary isotope mixing model based on experimental data allowed to estimate the Cu continental fraction bioaccumulated in the transplanted oysters. The positive δ65Cu values and high bioaccumulated levels of Cu in transplanted oysters support that continental emissions are dominantly anthropogenic. However, identifying specific pollutant coastal source remained unelucidated mostly due to their broader and overlapping isotope signatures and potential post-depositional Cu isotope fractionation processes. Further investigations on isotope fractionation of Cu-based compounds in an aqueous medium may improve Cu source discrimination. Thus, using Cu as an example, this work combines for the first time a well-known caged bivalve approach with metal stable isotope techniques for monitoring and quantifying the bioaccumulation of anthropogenic metal into marine environments. Also, it states the main challenges to pinpoint specific coastal anthropogenic sources utilizing this approach and provides the perspectives for further studies to overcome them.

Realistic environmental exposure to microplastics does not induce biological effects in the Pacific oyster Crassostrea gigas.

The aim of the present study was to evaluate the presence and potential toxic effects of plastic fragments (<400 µm) of polyethylene and polypropylene on the Pacific oyster Crassostrea gigas. Oysters were exposed to environmentally relevant concentrations (0, 0.008, 10, 100 µg of particles/L) during 10 days, followed by a depuration period of 10 days in clean seawater. Effects of microplastics were evaluated on the clearance rate of organisms, tissue alteration, antioxidant defense, immune alteration and DNA damage. Detection and quantification of microplastics in oyster's tissues (digestive gland, gills and other tissues) and biodeposits using infrared microscopy were also conducted. Microplastics were detected in oyster's biodeposits following exposure to all tested concentrations: 0.003, 0.006 and 0.05 particles/mg of biodeposits in oysters exposed to 0.008, 10 and 100 µg of particles/L, respectively. No significant modulation of biological markers was measured in organisms exposed to microplastics in environmentally relevant conditions.

Toxicity of binary mixtures of pesticides to the marine microalgae Tisochrysis lutea and Skeletonema marinoi: Substance interactions and physiological impacts.

This study screened binary mixtures of pesticides for potential synergistic interaction effects on growth of the marine microalgae Tisochrysis lutea and Skeletonema marinoi. It also examined the single and combined effects of three of the most toxic substances on microalgal physiology. Single substances were first tested on each microalgal species to determine their respective EC50 and concentration-response relationships. The toxicity of six and seven binary mixtures was then evaluated in microplate experiments on the growth of T. lutea and S. marinoi, respectively, using two mixture modelling approaches: isobolograms and the MIXTOX tool, based on Concentration Addition (CA) or Independent Action (IA) models. Significant cases of antagonism (for both species) and synergism (for S. marinoi) were observed for the mixtures of isoproturon and spiroxamine, and isoproturon and metazachlor, respectively. These two mixtures, together with that of isoproturon and diuron, for which additivity was observed, were further studied for their impacts on the physiology of each species. Exposures were thus made in culture flasks at three concentrations, or concentration combinations for mixtures, selected to cause 25%, 50% and 75% growth rate inhibition. The effects of the selected pesticides singly and in combination were evaluated at three perceived effect concentrations on esterase metabolic activity, relative lipid content, cytoplasmic membrane potential and reactive oxygen species (ROS) content by flow cytometry, and on photosynthetic quantum yield (ϕ'M) by PAM-fluorescence. Isoproturon and diuron singly and in mixtures induced 20-40% decreases in ϕ'M which was in turn responsible for a significant decrease in relative lipid content for both species. Spiroxamine and metazachlor were individually responsible for an increase in relative lipid content (up to nearly 300% for metazachlor on S. marinoi), as well as cell depolarization and increased ROS content. The mixture of isoproturon and metazachlor tested on S. marinoi caused a 28-34% decrease in ϕ'M that was significantly higher than levels induced by each of substances when tested alone. This strong decrease in ϕ'M could be due to a combined effect of these substances on the photosynthetic apparatus, which is likely the cause of the synergy found for this mixture.

Demonstrating the need for chemical exposure characterisation in a microplate test system: toxicity screening of sixteen pesticides on two marine microalgae.

Pesticides used in viticulture create a potential risk for the aquatic environment due to drift during application, runoff and soil leaching. The toxicity of sixteen pesticides and one metabolite were evaluated on the growth of two marine microalgae, Tisochrysis lutea and Skeletonema marinoi, in 96-h exposure assays conducted in microplates. For each substance, concentrations of stock solutions were analytically measured and abiotic assays were performed to evaluate the chemical stability of pesticides in microplates. For two chemicals, microalgae exposures were run simultaneously in microplates and culture flasks to compare EC50 calculated from the two exposure systems. Results from chemical analyses demonstrated the low stability of hydrophobic pesticides (log KOW > 3). For such chemicals, EC50 values calculated using measured pesticide concentrations were two-fold lower than those first estimated using nominal concentrations. Photosystem II inhibitors were the most toxic herbicides, with EC50 values below 10 µg L-1 for diuron and around double this for isoproturon. Chlorpyrifos-methyl was the only insecticide to significantly affect the growth of T. lutea, with an EC50 around 400 µg L-1. All fungicides tested were significantly toxic to both species: strobilurins showed low overall toxicity, with EC50 values around 400 µg L-1, whereas quinoxyfen, and spiroxamine, showed high toxicity to both species, especially to T. lutea, with an EC50 below 1 µg L-1 measured for spiroxamine in culture flasks. This study highlights the need to perform chemical analyses for reliable toxicity assessment and discusses the advantages and disadvantages of using microplates as a toxicity screening tool.

The interplay at the replisome mitigates the impact of oxidative damage on the genetic integrity of hyperthermophilic Archaea.

8-oxodeoxyguanosine (8-oxodG), a major oxidised base modification, has been investigated to study its impact on DNA replication in hyperthermophilic Archaea. Here we show that 8-oxodG is formed in the genome of growing cells, with elevated levels following exposure to oxidative stress. Functional characterisation of cell-free extracts and the DNA polymerisation enzymes, PolB, PolD, and the p41/p46 complex, alone or in the presence of accessory factors (PCNA and RPA) indicates that translesion synthesis occurs under replicative conditions. One of the major polymerisation effects was stalling, but each of the individual proteins could insert and extend past 8-oxodG with differing efficiencies. The introduction of RPA and PCNA influenced PolB and PolD in similar ways, yet provided a cumulative enhancement to the polymerisation performance of p41/p46. Overall, 8-oxodG translesion synthesis was seen to be potentially mutagenic leading to errors that are reminiscent of dA:8-oxodG base pairing.

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.

Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos.

Copper contamination is widespread along coastal areas and exerts adverse effects on marine organisms such as mollusks. In the Pacific oyster, copper induces severe developmental abnormalities during early life stages; however, the underlying molecular mechanisms are largely unknown. This study aims to better understand whether the embryotoxic effects of copper in Crassostrea gigas could be mediated by alterations in gene expression, and the putative role of DNA methylation, which is known to contribute to gene regulation in early embryo development. For that purpose, oyster embryos were exposed to 4 nominal copper concentrations (0.1, 1, 10 and 20 µg L-1 Cu2+) during early development assays. Embryotoxicity was monitored through the oyster embryo-larval bioassay at the D-larva stage 24 h post fertilization (hpf) and genotoxicity at gastrulation 7 hpf. In parallel, the relative expression of 15 genes encoding putative homeotic, biomineralization and DNA methylation proteins was measured at three developmental stages (3 hpf morula stage, 7 hpf gastrula stage, 24 hpf D-larvae stage) using RT-qPCR. Global DNA content in methylcytosine and hydroxymethylcytosine were measured by HPLC and gene-specific DNA methylation levels were monitored using MeDIP-qPCR. A significant increase in larval abnormalities was observed from copper concentrations of 10 µg L-1, while significant genotoxic effects were detected at 1 µg L-1 and above. All the selected genes presented a stage-dependent expression pattern, which was impaired for some homeobox and DNA methylation genes (Notochord, HOXA1, HOX2, Lox5, DNMT3b and CXXC-1) after copper exposure. While global DNA methylation (5-methylcytosine) at gastrula stage didn't show significant changes between experimental conditions, 5-hydroxymethylcytosine, its degradation product, decreased upon copper treatment. The DNA methylation of exons and the transcript levels were correlated in control samples for HOXA1 but such a correlation was diminished following copper exposure. The methylation level of some specific gene regions (HoxA1, Hox2, Engrailed2 and Notochord) displayed changes upon copper exposure. Such changes were gene and exon-specific and no obvious global trends could be identified. Our study suggests that the embryotoxic effects of copper in oysters could involve homeotic gene expression impairment possibly by changing DNA methylation levels.

Combined effects of antifouling biocides on the growth of three marine microalgal species.

The toxicity of the antifouling compounds diuron, irgarol, zinc pyrithione (ZnPT), copper pyrithione (CuPT) and copper was tested on the three marine microalgae Tisochrysis lutea, Skeletonema marinoi and Tetraselmis suecica. Toxicity tests based on the inhibition of growth rate after 96-h exposure were run using microplates. Chemical analyses were performed to validate the exposure concentrations and the stability of the compounds under test conditions. Single chemicals exhibited varying toxicity depending on the species, irgarol being the most toxic chemical and Cu the least toxic. Selected binary mixtures were tested and the resulting interactions were analyzed using two distinct concentration-response surface models: one using the concentration addition (CA) model as reference and two deviating isobole models implemented in R software; the other implementing concentration-response surface models in Excel®, using both CA and independent action (IA) models as reference and three deviating models. Most mixtures of chemicals sharing the same mode of action (MoA) were correctly predicted by the CA model. For mixtures of dissimilarly acting chemicals, neither of the reference models provided better predictions than the other. Mixture of ZnPT together with Cu induced a strong synergistic effect on T. suecica while strong antagonism was observed on the two other species. The synergy was due to the transchelation of ZnPT into CuPT in the presence of Cu, CuPT being 14-fold more toxic than ZnPT for this species. The two modelling approaches are compared and the differences observed among the interaction patterns resulting from the mixtures are discussed.

Effects of a parental exposure to diuron on Pacific oyster spat methylome.

Environmental epigenetic is an emerging field that studies the cause-effect relationship between environmental factors and heritable trait via an alteration in epigenetic marks. This field has received much attentions since the impact of environmental factors on different epigenetic marks have been shown to be associated with a broad range of phenotypic disorders in natural ecosystems. Chemical pollutants have been shown to affect immediate epigenetic information carriers of several aquatic species but the heritability of the chromatin marks and the consequences for long term adaptation remain open questions. In this work, we investigated the impact of the diuron herbicide on the DNA methylation pattern of spat from exposed Crassotrea gigas genitors. This oyster is one of the most important mollusk species produced worldwide and a key coastal economic resource in France. The whole genome bisulfite sequencing (WGBS, BS-Seq) was applied to obtain a methylome at single nucleotide resolution on DNA extracted from spat issued from diuron exposed genitors comparatively to control spat. We showed that the parental diuron exposure has an impact on the DNA methylation pattern of its progeny. Most of the differentially methylated regions occurred within coding sequences and we showed that this change in methylation level correlates with RNA level only in a very small group of genes. Although the DNA methylation profile is variable between individuals, we showed conserved DNA methylation patterns in response to parental diuron exposure. This relevant result opens perspectives for the setting of new markers based on epimutations as early indicators of marine pollutions.

Integrated monitoring of chemicals and their effects on four sentinel species, Limanda limanda, Platichthys flesus, Nucella lapillus and Mytilus sp., in Seine Bay: A key step towards applying biological effects to monitoring.

The International workshop on Integrated Assessment of CONtaminants impacts on the North sea (ICON) provided a framework to validate the application of chemical and biological assessment thresholds (BACs and EACs) in the Seine Bay in France. Bioassays (oyster larval anomalies, Corophium arenarium toxicity assay and DR Calux) for sediment and biomarkers: ethoxyresorufin-O-deethylase (EROD) activity, acetylcholinesterase (AChE) activity, lysosomal membrane stability (LMS), DNA strand breaks using the Comet assay, DNA adducts, micronucleus (MN), PAH metabolites, imposex, intersex and fish external pathologies were analysed in four marine sentinel species (Platichthys flesus, Limanda limanda, Mytilus sp. and Nucella lapilus). Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metals were analysed in biota and sediment. Results for sediment and four species in 2008-2009 made it possible to quantify the impact of contaminants using thresholds (Environmental Assessment Criteria/EAC2008: 70% and EAC2009: 60%) and effects (EAC2008: 50% and EAC2009: 40%) in the Seine estuary. The Seine estuary is ranked among Europe's most highly polluted sites.

Comparative embryotoxicity and genotoxicity of the herbicide diuron and its metabolites in early life stages of Crassostrea gigas: Implication of reactive oxygen species production.

Herbicides are one of the major classes of pollutants contaminating coastal waters over the world. Among them, diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a phenylurea herbicide frequently detected in oyster-producing area, known to be toxic for this important exploited non-target species. With the aim to investigate the mechanisms by which diuron displays its toxicity in oyster, the implication of both biotransformation and oxygen reactive species (ROS) production was studied considering embryotoxicity and genotoxicity as endpoints. Comparative embryotoxicity and genotoxicity of diuron and its main metabolites (DCPMU, DCPU and 3,4-DCA) were thus studied on oyster larvae by the embryo-larval bioassay on D larvae and the comet assay on trochophore larvae, respectively. Exposures were also performed in presence and absence of known ROS scavenger compounds - ascorbic acid and N-acetylcysteine, to evaluate the involvement of oxyradicals in the toxic responses. In the case of diuron, the production of ROS on exposed oyster larvae was also measured using 2',7'-dichlorodihydrofluorescein diacetate as a probe for flow cytometric analysis. The results we obtained showed the embryotoxicity and genotoxicity of diuron and its metabolites in early life stages of the Pacific oyster. For concentrations ranging from 0.05 to 0.5µgL(-1), diuron appeared significantly more embryotoxic than DCPMU and DCPU (p<0.001). Embryotoxicity decreased with diuron metabolism as follows: diuron≥DCPMU=DCPU, highlighting that biotransformation can constitute a true detoxication pathways in oyster larvae by decreasing the toxicity of the parent compound. In the opposite, no difference was observed between diuron and its metabolites concerning larval development when considering a lower and more environmentally realistic range of concentrations (0.002-0.050µgL(-1)). 3,4-DCA was the only compound that did not show any sign of embryotoxicity, even at concentrations up to 5µgL(-1). Concerning genotoxicity, no significant difference was observed between diuron and all of its metabolites including 3, 4 DCA with damages detected from the concentration of 0.05µgL(-1). As for diuron, the toxicity of the metabolites seems to be mediated in some part by ROS production as clearly demonstrated by the decrease in genotoxicity and developmental abnormalities in the presence of the oxidant scavenger, ascorbic acid.

Toxicity assessment of water-accommodated fractions from two different oils using a zebrafish (Danio rerio) embryo-larval bioassay with a multilevel approach.

Petroleum compounds from chronic discharges and oil spills represent an important source of environmental pollution. To better understand the deleterious effects of these compounds, the toxicity of water-accommodated fractions (WAF) from two different oils (brut Arabian Light and Erika heavy fuel oils) were used in this study. Zebrafish embryos (Danio rerio) were exposed during 96h at three WAF concentrations (1, 10 and 100% for Arabian Light and 10, 50 and 100% for Erika) in order to cover a wide range of polycyclic aromatic hydrocarbon (PAH) concentrations, representative of the levels found after environmental oil spills. Several endpoints were recorded at different levels of biological organization, including lethal endpoints, morphological abnormalities, photomotor behavioral responses, cardiac activity, DNA damage and exposure level measurements (EROD activity, cyp1a and PAH metabolites). Neither morphological nor behavioral or physiological alterations were observed after exposure to Arabian Light fractions. In contrast, the Erika fractions led a high degree of toxicity in early life stages of zebrafish. Despite of defense mechanisms induced by oil, acute toxic effects have been recorded including mortality, delayed hatching, high rates of developmental abnormalities, disrupted locomotor activity and cardiac failures at the highest PAH concentrations (∑TPAHs=257,029±47,231ng·L(-1)). Such differences in toxicity are likely related to the oil composition. The use of developing zebrafish is a good tool to identify wide range of detrimental effects and elucidate their underlying foundations. Our work highlights once more, the cardiotoxic action (and potentially neurotoxic) of petroleum-related PAHs.

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas.

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.

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 Crassostrea 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.