Subcellular Distribution of Dietary Methyl-Mercury in Gammarus fossarum and Its Impact on the Amphipod Proteome.

The transfer of methyl-Hg (MeHg) from food is central for its effects in aquatic animals, but we still lack knowledge concerning its impact on invertebrate primary consumers. In aquatic environments, cell walls of plants are particularly recalcitrant to degradation and as such remain available as a food source for long periods. Here, the impact at the proteomic level of dietary MeHg in Gammarus fossarum was established and linked to subcellular distribution of Hg. Individuals of G. fossarum were fed with MeHg in cell wall or intracellular compartments of Elodea nuttallii. Hg concentrations in subcellular fractions were 2 to 6 times higher in animals fed with cell wall than intracellular compartments. At the higher concentrations tested, the proportion of Hg in metal-sensitive fraction increased from 30.0 ± 6.1 to 41.0 ± 5.7% for individuals fed with intracellular compartment, while biologically detoxified metal fraction increased from 30.0 ± 6.1 to 50.0 ± 2.8% when fed with cell wall compartment. Data suggested that several thresholds of proteomic response are triggered by increased bioaccumulation in each subcellular fraction in correlation with Hg exclusively bound to the metal-sensitive fraction, while the increase of biologically detoxified metal likely had a cost for fitness. Proteomics analysis supported that the different binding sites and speciation in shoots subsequently resulted in different fate and cellular toxicity pathways to consumers. Our data confirmed that Hg bound in cell walls of plants can be assimilated by G. fossarum, which is consistent with its feeding strategy, hence pointing cell walls as a significant source for Hg transfers and toxicity in primary consumers. The high accumulation of Hg in macrophytes makes them a risk for food web transfer in shallow ecosystems. The present results allowed gaining new insights into the effects and uptake mechanisms of MeHg in aquatic primary consumers.

Biotic formation of methylmercury: A bio-physico-chemical conundrum.

Mercury (Hg) is a natural and widespread trace metal, but is considered a priority pollutant, particularly its organic form methylmercury (MMHg), because of human's exposure to MMHg through fish consumption. Pioneering studies showed the methylation of divalent Hg (HgII) to MMHg to occur under oxygen-limited conditions and to depend on the activity of anaerobic microorganisms. Recent studies identified the hgcAB gene cluster in microorganisms with the capacity to methylate HgII and unveiled a much wider range of species and environmental conditions producing MMHg than previously expected. Here, we review the recent knowledge and approaches used to understand HgII-methylation, microbial biodiversity and activity involved in these processes, and we highlight the current limits for predicting MMHg concentrations in the environment. The available data unveil the fact that HgII methylation is a bio-physico-chemical conundrum in which the efficiency of biological HgII methylation appears to depend chiefly on HgII and nutrients availability, the abundance of electron acceptors such as sulfate or iron, the abundance and composition of organic matter as well as the activity and structure of the microbial community. An increased knowledge of the relationship between microbial community composition, physico-chemical conditions, MMHg production, and demethylation is necessary to predict variability in MMHg concentrations across environments.

Molecular Effects, Speciation, and Competition of Inorganic and Methyl Mercury in the Aquatic Plant Elodea nuttallii.

Mercury (Hg) remains hazardous in aquatic environments because of its biomagnification in food webs. Nonetheless, Hg uptake and impact in primary producers is still poorly understood. Here, we compared the cellular toxicity of inorganic and methyl Hg (IHg and MeHg, respectively) in the aquatic plant Elodea nuttallii. IHg and MeHg regulated contigs involved in similar categories (e.g., energy metabolism, development, transport, secondary metabolism), but MeHg regulated more contigs, supporting a higher molecular impact than IHg. At the organism level, MeHg induced antioxidants, while IHg decreased chlorophyll content. The uptake of Hg and expression of a subset of contigs was subsequently studied in complex media. Measured uptake pointed to a contrasted impact of cell walls and copper (Cu) on IHg and MeHg. Using a speciation modeling, differences in uptake were attributed to the differences in affinities of IHg and MeHg to organic matter in relation to Cu speciation. We also identified a distinct gene expression signature for IHg, MeHg, and Cu, further supporting different molecular toxicity of these trace elements. Our data provided fundamental knowledge on IHg and MeHg uptake in a key aquatic primary producer and confirmed the potential of transcriptomics to assess Hg exposure in environmentally realistic systems.

The class III peroxidase PRX17 is a direct target of the MADS-box transcription factor AGAMOUS-LIKE15 (AGL15) and participates in lignified tissue formation.

Several physiological functions have been attributed to class III peroxidases (PRXs) in plants, but the in planta role of most members of this family still remains undetermined. Here, we report the first functional characterization of PRX17 (At2g22420), one of the 73 members of this family in Arabidopsis thaliana. Localization of PRX17 was examined by transient expression in Nicotiana benthamiana. Loss- and gain-of-function mutants in A. thaliana were studied. Regulation at the gene and protein levels was analyzed using ß-glucuronidase (GUS) activity, quantitative reverse transcriptase (qRT)-PCR, zymography, and chromatin immunoprecipitation. Phenotypes were characterized including lignin and xylan contents. PRX17 was expressed in various tissues, including vascular tissues, and PRX17 was localized to the cell wall. In prx17, the lignin content was reduced in the stem and siliques and bolting was delayed, while the opposite phenotype was observed in 35S:PRX17 plants, together with a significant increase of lignin and xylan immunofluorescence signal. Finally, we demonstrated that the transcription factor AGAMOUS-LIKE15 (AGL15) binds to the PRX17 promoter and regulates PRX17 expression level. This converging set of structural, transcriptomic and physiological data suggests that PRX17, under the control of AGL15, contributes to developmental programs by playing an essential role in regulating age-dependent lignified tissue formation, including changes in cell wall properties.

Transcriptomic and Physiological Responses of the Green Microalga Chlamydomonas reinhardtii during Short-Term Exposure to Subnanomolar Methylmercury Concentrations.

The effects of short-term exposure to subnanomolar methyl-mercury (MeHg) concentrations, representative of contaminated environments, on the microalga Chlamydomonas reinhardtii were assessed using both physiological end points and gene expression analysis. MeHg bioaccumulated and induced significant increase of the photosynthesis efficiency, while the algal growth, oxidative stress, and chlorophyll fluorescence were unaffected. At the molecular level, MeHg significantly dysregulated the expression of genes involved in motility, energy metabolism, lipid metabolism, metal transport, and antioxidant enzymes. Data suggest that the cells were able to cope with subnanomolar MeHg exposure, but this tolerance resulted in a significant cost to the cell energy and reserve metabolism as well as ample changes in the nutrition and motility of C. reinhardtii. The present results allowed gaining new insights on the effects and uptake mechanisms of MeHg at subnanomolar concentrations in aquatic primary producers.

Role of Settling Particles on Mercury Methylation in the Oxic Water Column of Freshwater Systems.

As the methylation of inorganic mercury to neurotoxic methylmercury has been attributed to the activity of anaerobic bacteria, the formation of methylmercury in the oxic water column of marine ecosystems has puzzled scientists over the past years. Here we show for the first time that methylmercury can be produced in particles sinking through oxygenated water column of lakes. Total mercury and methylmercury concentrations were measured in the settling particles and in surface sediments of the largest freshwater lake in Western Europe (Lake Geneva). While total mercury concentration differences between sediments and settling particles were not significant, methylmercury concentrations were about ten-fold greater in settling particles. Methylmercury demethylation rate constants (kd) were of similar magnitude in both compartments. In contrast, mercury methylation rate constants (km) were one order of magnitude greater in settling particles. The net potential for methylmercury formation, assessed by the ratio between the two rate constants (km kd-1), was therefore up to ten fold greater in settling particles, denoting that in situ transformations likely contributed to the high methylmercury concentration found in settling particles. Mercury methylation was inhibited (∼80%) in settling particles amended with molybdate, demonstrating the prominent role of biological sulfate-reduction in the process.

Dendrochemical assessment of mercury releases from a pond and dredged-sediment landfill impacted by a chlor-alkali plant.

Although current Hg emissions from industrial activities may be accurately monitored, evidence of past releases to the atmosphere must rely on one or more environmental proxies. We used Hg concentrations in tree cores collected from poplars and willows to investigate the historical changes of Hg emissions from a dredged sediment landfill and compared them to a nearby control location. Our results demonstrated the potential value of using dendrochemistry to record historical Hg emissions from past industrial activities.

Towards Mechanistic Understanding of Mercury Availability and Toxicity to Aquatic Primary Producers.

The present article reviews current knowledge and recent progress on the bioavailability and toxicity of mercury to aquatic primary producers. Mercury is a ubiquitous toxic trace element of global concern. At the base of the food web, primary producers are central for mercury incorporation into the food web. Here, the emphasis is on key, but still poorly understood, processes governing the interactions between mercury species and phytoplankton, and macrophytes, two representatives of primary producers. Mass transfer to biota surface, adsorption to cell wall, internalization and release from cells, as well as underlying toxicity mechanisms of both inorganic mercury and methylmercury are discussed critically. In addition, the intracellular distribution and transformation processes, their importance for mercury toxicity, species-sensitivity differences and trophic transfer are presented. The mini-review is illustrated with examples of our own research.

Editorial trend: adverse outcome pathway (AOP) and computational strategy - towards new perspectives in ecotoxicology.

The adverse outcome pathway (AOP) has been conceptualized in 2010 as an analytical construct to describe a sequential chain of causal links between key events, from a molecular initiating event leading to an adverse outcome (AO), considering several levels of biological organization. An AOP aims to identify and organize available knowledge about toxic effects of chemicals and drugs, either in ecotoxicology or toxicology, and it can be helpful in both basic and applied research and serve as a decision-making tool in support of regulatory risk assessment. The AOP concept has evolved since its introduction, and recent research in toxicology, based on integrative systems biology and artificial intelligence, gave it a new dimension. This innovative in silico strategy can help to decipher mechanisms of action and AOP and offers new perspectives in AOP development. However, to date, this strategy has not yet been applied to ecotoxicology. In this context, the main objective of this short article is to discuss the relevance and feasibility of transferring this strategy to ecotoxicology. One of the challenges to be discussed is the level of organisation that is relevant to address for the AO (population/community). This strategy also offers many advantages that could be fruitful in ecotoxicology and overcome the lack of time, such as the rapid identification of data available at a time t, or the identification of "data gaps". Finally, this article proposes a step forward with suggested priority topics in ecotoxicology that could benefit from this strategy.

Analysis of the Elodea nuttallii transcriptome in response to mercury and cadmium pollution: development of sensitive tools for rapid ecotoxicological testing.

Toxic metals polluting aquatic ecosystems are taken up by inhabitants and accumulate in the food web, affecting species at all trophic levels. It is therefore important to have good tools to assess the level of risk represented by toxic metals in the environment. Macrophytes are potential organisms for the identification of metal-responsive biomarkers but are still underrepresented in ecotoxicology. In the present study, we used next-generation sequencing to investigate the transcriptomic response of Elodea nuttallii exposed to enhanced concentrations of Hg and Cd. We de novo assembled more than 60 000 contigs, of which we found 170 to be regulated dose-dependently by Hg and 212 by Cd. Functional analysis showed that these genes were notably related to energy and metal homeostasis. Expression analysis using nCounter of a subset of genes showed that the gene expression pattern was able to assess toxic metal exposure in complex environmental samples and was more sensitive than other end points (e.g., bioaccumulation, photosynthesis, etc.). In conclusion, we demonstrate the feasibility of using gene expression signatures for the assessment of environmental contamination, using an organism without previous genetic information. This is of interest to ecotoxicology in a wider sense given the possibility to develop specific and sensitive bioassays.

Anaerobic mercury methylators inhabit sinking particles of oxic water columns.

Increased concentration of mercury, particularly methylmercury, in the environment is a worldwide concern because of its toxicity in severely exposed humans. Although the formation of methylmercury in oxic water columns has been previously suggested, there is no evidence of the presence of microorganisms able to perform this process, using the hgcAB gene pair (hgc+ microorganisms), in such environments. Here we show the prevalence of hgc+ microorganisms in sinking particles of the oxic water column of Lake Geneva (Switzerland and France) and its anoxic bottom sediments. Compared to anoxic sediments, sinking particles found in oxic waters exhibited relatively high proportion of hgc+genes taxonomically assigned to Firmicutes. In contrast hgc+members from Nitrospirae, Chloroflexota and PVC superphylum were prevalent in anoxic sediment while hgc+ Desulfobacterota were found in both environments. Altogether, the description of the diversity of putative mercury methylators in the oxic water column expand our understanding on MeHg formation in aquatic environments and at a global scale.

Immunotoxicity of relevant mixtures of pesticides and metabolites on THP-1 cells.

Pesticides are used to combat agricultural pests but also trigger side effects on non-target organisms. Particularly, immune system dysregulation is a major concern due to the organism's increased vulnerability to diseases, including cancer development. Macrophages play essential roles in innate and adaptive immunity and can undergo classical (M1) or alternative (M2) activation. The M1 pro-inflammatory phenotype has an antitumor role, while M2 favors tumor promotion. Although previous studies have linked pesticide exposure to immune compromise, macrophage polarization is still poorly studied. Here, we investigated the effects of 72 h-long exposure to the mixture of four pesticides widely used in Brazil (glyphosate, 2,4-D, mancozeb, and atrazine), and their main metabolites (aminomethylphosphonic acid, 2,4-diclorophenol, ethylenethiourea, and desethylatrazine) on human leukemia monocytic THP-1 cell line at concentrations based on the Acceptable Daily Intake (ADI) value established in the country. The data revealed immunotoxicity related to impaired cell metabolism in all exposed groups, decreased cell attachment (Pes: 10-1; Met: 10-1; Mix: all concentrations), and disturbance in nitric oxide (NO) levels (Met: 10-1, 101; Mix: all concentrations). The polarization of macrophages towards a more pro-tumor M2-like phenotype was also supported by decreased secretion of the pro-inflammatory cytokine TNF-α (Pes 100, 101) and increased IL-8 (Pes 101). These outcomes alert about the risk of pesticide exposure in the Brazilian population.

Bioaccumulation and molecular effects of carbamazepine and methylmercury co-exposure in males of Dreissena polymorpha.

Dreissena polymorpha is a bivalve promising for biomonitoring in freshwater ecosystems thanks to its abundance and high filtration activity allowing rapid uptake of toxicants and identification of their negative effects. Nonetheless, we still lack knowledge on its molecular responses to stress under realistic scenario, e.g. multi-contamination. Carbamazepine (CBZ) and Hg are ubiquitous pollutants sharing molecular toxicity pathways, e.g. oxidative stress. A previous study in zebra mussels showed their co-exposure to cause more alterations than single exposures, but molecular toxicity pathways remained unidentified. D. polymorpha was exposed 24 h (T24) and 72 h (T72) to CBZ (6.1 ± 0.1 µg L-1), MeHg (430 ± 10 ng L-1) and the co-exposure (6.1 ± 0.1 µg L-1CBZ and 500 ± 10 ng L-1 MeHg) at concentrations representative of polluted areas (~10× EQS). RedOx system at the gene and enzyme level, the proteome and the metabolome were compared. The co-exposure resulted in 108 differential abundant proteins (DAPs), as well as 9 and 10 modulated metabolites at T24 and T72, respectively. The co-exposure specifically modulated DAPs and metabolites involved in neurotransmission, e.g. dopaminergic synapse and GABA. CBZ specifically modulated 46 DAPs involved in calcium signaling pathways and 7 amino acids at T24. MeHg specifically modulated 55 DAPs involved in the cytoskeleton remodeling and hypoxia-induced factor 1 pathway, without altering the metabolome. Single and co-exposures commonly modulated proteins and metabolites involved in energy and amino acid metabolisms, response to stress and development. Concomitantly, lipid peroxidation and antioxidant activities were unchanged, supporting that D. polymorpha tolerated experimental conditions. The co-exposure was confirmed to cause more alterations than single exposures. This was attributed to the combined toxicity of CBZ and MeHg. Altogether, this study underlined the necessity to better characterize molecular toxicity pathways of multi-contamination that are not predictable on responses to single exposures, to better anticipate adverse effects in biota and improve risk assessment.

A consensus protocol for the recovery of mercury methylation genes from metagenomes.

Mercury (Hg) methylation genes (hgcAB) mediate the formation of the toxic methylmercury and have been identified from diverse environments, including freshwater and marine ecosystems, Arctic permafrost, forest and paddy soils, coal-ash amended sediments, chlor-alkali plants discharges and geothermal springs. Here we present the first attempt at a standardized protocol for the detection, identification and quantification of hgc genes from metagenomes. Our Hg-cycling microorganisms in aquatic and terrestrial ecosystems (Hg-MATE) database, a catalogue of hgc genes, provides the most accurate information to date on the taxonomic identity and functional/metabolic attributes of microorganisms responsible for Hg methylation in the environment. Furthermore, we introduce "marky-coco", a ready-to-use bioinformatic pipeline based on de novo single-metagenome assembly, for easy and accurate characterization of hgc genes from environmental samples. We compared the recovery of hgc genes from environmental metagenomes using the marky-coco pipeline with an approach based on coassembly of multiple metagenomes. Our data show similar efficiency in both approaches for most environments except those with high diversity (i.e., paddy soils) for which a coassembly approach was preferred. Finally, we discuss the definition of true hgc genes and methods to normalize hgc gene counts from metagenomes.

Effects of carbamazepine in aquatic biota.

Carbamazepine (CBZ) is one of the most common pharmaceuticals found in the aquatic environment. Here, we reviewed studies in aquatic animals highlighting that CBZ affected ROS homeostasis but also the neuroendocrine system, cell viability, immunity, reproduction, feeding behavior and growth. Notably, the acetylcholinesterase activity was modified by concentrations of the order of ng L-1 CBZ. At ≥10 µg L-1, data pointed that CBZ triggered the production of ROS, modifying the activity of antioxidant enzymes and produced a significant cellular stress at concentrations ≥100 µg L-1. However, the response appeared species-, organ- and time-dependent, and was impacted by different experimental conditions and the origin of animals. In this context, this review discusses the available data and proposes future research priorities.

Metabolic, cellular and defense responses to single and co-exposure to carbamazepine and methylmercury in Dreissena polymorpha.

Carbamazepine (CBZ) and Hg are widespread and persistent micropollutants in aquatic environments. Both pollutants are known to trigger similar toxicity mechanisms, e.g. reactive oxygen species (ROS) production. Here, their effects were assessed in the zebra mussel Dreissena polymorpha, frequently used as a freshwater model in ecotoxicology and biomonitoring. Single and co-exposures to CBZ (3.9 µg L-1) and MeHg (280 ng L-1) were performed for 1 and 7 days. Metabolomics analyses evidenced that the co-exposure was the most disturbing after 7 days, reducing the amount of 25 metabolites involved in protein synthesis, energy metabolism, antioxidant response and osmoregulation, and significantly altering cells and organelles' structure supporting a reduction of functions of gills and digestive glands. CBZ alone after 7 days decreased the amount of α-aminobutyric acid and had a moderate effect on the structure of mitochondria in digestive glands. MeHg alone had no effect on mussels' metabolome, but caused a significant alteration of cells and organelles' structure in gills and digestive glands. Single exposures and the co-exposure increased antioxidant responses vs control in gills and digestive glands, without resulting in lipid peroxidation, suggesting an increased ROS production caused by both pollutants. Data globally supported that a higher number of hyperactive cells compensated cellular alterations in the digestive gland of mussels exposed to CBZ or MeHg alone, while CBZ + MeHg co-exposure overwhelmed this compensation after 7 days. Those effects were unpredictable based on cellular responses to CBZ and MeHg alone, highlighting the need to consider molecular toxicity pathways for a better anticipation of effects of pollutants in biota in complex environmental conditions.

Transcriptome analysis of various flower and silique development stages indicates a set of class III peroxidase genes potentially involved in pod shattering in Arabidopsis thaliana.

BACKGROUND: Plant class III peroxidases exist as a large multigenic family involved in numerous functions suggesting a functional specialization of each gene. However, few genes have been linked with a specific function. Consequently total peroxidase activity is still used in numerous studies although its relevance is questionable. Transcriptome analysis seems to be a promising tool to overcome the difficulties associated with the study of this family. Nevertheless available microarrays are not completely reliable for this purpose. We therefore used a macroarray dedicated to the 73 class III peroxidase genes of A. thaliana to identify genes potentially involved in flower and fruit development. RESULTS: The observed increase of total peroxidase activity during development was actually correlated with the induction of only a few class III peroxidase genes which supports the existence of a functional specialization of these proteins. We identified peroxidase genes that are predominantly expressed in one development stage and are probable components of the complex gene networks involved in the reproductive phase. An attempt has been made to gain insight into plausible functions of these genes by collecting and analyzing the expression data of different studies in plants. Peroxidase activity was additionally observed in situ in the silique dehiscence zone known to be involved in pod shattering. Because treatment with a peroxidase inhibitor delayed pod shattering, we subsequently studied mutants of transcription factors (TF) controlling this mechanism. Three peroxidases genes -AtPrx13, AtPrx30 and AtPrx55- were altered by the TFs involved in pod shatter. CONCLUSIONS: Our data illustrated the problems caused by linking only an increase in total peroxidase activity to any specific development stage or function. The activity or involvement of specific class III peroxidase genes needs to be assessed. Several genes identified in our study had not been linked to any particular development stage or function until now. Notably AtPrx13, which is one of the peroxidase genes not present on commercially available microarrays. A systematic survey of class III peroxidase genes expression is necessary to reveal specific class III peroxidase gene functions and the regulation and evolution of this key multifunctional enzyme family. The approach used in this study highlights key individual genes that merit further investigation.

Effects of cadmium, inorganic mercury and methyl-mercury on the physiology and metabolomic profiles of shoots of the macrophyte Elodea nuttallii.

Macrophytes are known to bioaccumulate metals, but a thorough understanding of tolerance strategies and molecular impact of metals in aquatic plants is still lacking. The present study aimed to compare Hg and Cd effects in a representative macrophyte, Elodea nuttallii using physiological endpoints and metabolite profiles in shoots and cytosol. Exposure 24 h to methyl-Hg (30 ng L-1), inorganic Hg (70 ng L-1) and Cd (280 µg L-1) did not affect photosynthesis, or antioxidant enzymes despite the significant accumulation of metals, confirming a sublethal stress level. In shoots, Cd resulted in a higher level of regulation of metabolites than MeHg, while MeHg resulted in the largest number of regulated metabolites and IHg treatment regulated no metabolites significantly. In cytosol, Cd regulated more metabolites than IHg and only arginine, histidine and mannose were reduced by MeHg exposure. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of data suggested that exposure to MeHg resulted in biochemical changes including aminoacyl-tRNA biosynthesis, glycine, serine and threonine metabolism, nitrogen metabolism, arginine and proline metabolism, cyanoamino acid metabolism, while the treatment of Cd stress caused significant variations in aminoacyl-tRNA biosynthesis and branched-chain amino acids pathways. Data supports an impact of MeHg on N homeostasis, while Cd resulted in an osmotic stress-like pattern and IHg had a low impact. Marked differences in the responses to MeHg and IHg exposure were evidenced, supporting different molecular toxicity pathways and main impact of MeHg on non-soluble compartment, while main impact of IHg was on soluble compartment. Metabolomics was used for the first time in this species and proved to be very useful to confirm and complement recent knowledge gained by transcriptomics and proteomics, highlighting the high interest of multi-omics approaches to identify early impact of environmental pollution.

Mercury cycling in freshwater systems - An updated conceptual model.

The widely accepted conceptual model of mercury (Hg) cycling in freshwater lakes (atmospheric deposition and runoff of inorganic Hg, methylation in bottom sediments and subsequent bioaccumulation and biomagnification in biota) is practically accepted as common knowledge. There is mounting evidence that the dominant processes that regulate inputs, transformations, and bioavailability of Hg in many lakes may be missing from this picture, and the fixation on the temperate stratified lake archetype is impeding our exploration of understudied, but potentially important sources of methylmercury to freshwater lakes. In this review, the importance of understudied biogeochemical processes and sites of methylmercury production are highlighted, including the complexity of redox transformations of Hg within the lake system itself, the complex assemblage of microbes found in biofilms and periphyton (two vastly understudied important sources of methylmercury in many freshwater ecosystems), and the critical role of autochthonous and allochthonous dissolved organic matter which mediates the net supply of methylmercury from the cellular to catchment scale. A conceptual model of lake Hg in contrasting lakes and catchments is presented, highlighting the importance of the autochthonous and allochthonous supply of dissolved organic matter, bioavailable inorganic mercury and methylmercury and providing a framework for future convergent research at the lab and field scales to establish more mechanistic process-based relationships within and among critical compartments that regulate methylmercury concentrations in freshwater ecosystems.