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The transcriptomic effects of Ibuprofen (IBU) in the digestive gland tissue of Mytilus galloprovincialis Lam. specimens exposed at low environmental concentrations (250 ng L −1 ) are presented. Using a 1.7 K feature cDNA microarray along with linear models and empirical Bayes statistical methods 225 differentially expressed genes were identified in mussels treated with IBU across a 15-day period. Transcriptional dynamics were typical of an adaptive response with a peak of gene expression change at day-7 (177 features, representing about 11% of sequences available for analysis) and an almost full recovery at the end of the exposure period. Functional genomics by means of Gene Ontology term analysis unraveled typical mussel stress responses i.e. aminoglycan (chitin) metabolic processes but also more specific effects such as the regulation of NF-κB transcription factor activity. [ABSTRACT FROM AUTHOR]

Gene expression studies provided a major step forward in ecotoxicology and ecological functional genomics, among other areas. How to classify changes in gene expression to be significant compared to its normal operating range (NOR) requires a number of biological and statistical criteria. Doubts have been casted about when a change in gene expression has ecologically relevant meaning and what is the normal biological variation or NOR. Because genes are highly sensitive and self-regulating pieces, how can we design our experiments to control normal variation? The main aim of the present paper is to discuss such questions, while providing suggestions for improvement and interpretation. We have analysed several gene control data sets of Enchytraeus albidus comparing standard control test conditions, i.e. (1) from experiments performed at different occasions in time, (2) to culture media, (3) to acetone control, (4) from exposure time series (2, 4 and 8 days) and (5) between replicates. We discriminated between some normal variation sources. For instance, the transference of organisms from cultures to standard testing condition suggests a lowered general metabolism. Chemical carriers like acetone seem to cause changes related with soil structure and hypoxia conditions. Organisms have a “time-like” transcription signature when sampled along a time series. The coefficient of variance (CV) between biological replicates was 0.1 < CV < 1.3 for 17% of the genes. Variability will partly be inherent to the species, but most important is to understand the ground for interpretations as discussed. Here, the first attempt for the establishment of a NOR for E. albidus gene expression was made which represents a naturally varying ecological window – this contributes for the discrimination between stress responses and natural ecological variations in soil studies. [ABSTRACT FROM AUTHOR]

Metals are among the most common contaminants in soils in Europe. Although their effects are relatively well known regarding survival and reproduction to soil invertebrates, their mode of action is poorly understood. Enchytraeus albidus is a model organism in ecotoxicology and with the development of a gene library for this species, transcriptomic studies are now possible. The main aim of this study is to understand the Cu and Ni mechanisms of response in E. albidus, in comparison with Cd and Zn (already studied). E. albidus were exposed to Cu and Ni for 4 days to the reproduction effect concentrations EC50 and EC90. Results indicate that Cu and Ni have similar mechanisms of toxicity. When comparing four elements (hierarchical clustering) it was possible to observe a clear separation of Cd from all other metals. This separation correlates with the available information from other species regarding the toxicokinetics of the tested elements. [Copyright &y& Elsevier]

Metal ecotoxicity to soil organisms (for example, in enchytraeids) has been addressed mainly by assessing effects on survival and reproduction, but very little is known about the underlying molecular mechanisms of responses. The main purpose of the present study was to assess and compare the transcriptional responses of Enchytraeus albidus to an essential (Zn) and a nonessential (Cd) metal. Exposure was performed with two concentrations with a known effect on reproduction (effective concentration for 50% [EC50] and 90% [EC90]) at three time points (2, 4, and 8 d). Results showed that transcriptional responses were influenced by exposure duration but, independently of that, the mechanisms of response to Cd and Zn were consistently different. Both metals affected pathways related to the regulation of gene expression, calcium homeostasis, and cellular respiration. Mechanisms of toxicity that were exclusively associated with Cd exposures were the inhibition of DNA repair and the impairment of ubiquitin-mediated proteolysis. The microarray for E. albidus was a useful tool for detecting molecular pathways affected by metal exposures. Transcriptional responses strongly correlated with known mechanisms of Cd and Zn responses in other organisms, suggesting cross-species conserved mechanisms of action. It should be highlighted not only that the authors could retrieve mechanistic information but also that genes responded within 2 to 8 d of exposure. This represents an additional advantage of using such molecular endpoints as a complement to the traditional, more time-consuming endpoints. Environ. Toxicol. Chem. 2012; 31: 2289-2299. © 2012 SETAC [ABSTRACT FROM AUTHOR]

Silver (Ag) is amongst the most well studied nanomaterials (NMs), although most studies have only dealt with a single AgNM at a time and one biological endpoint. We here integrate the results of various testing-tools (endpoints) using a terrestrial worm, the standard ecotoxicological model organism Enchytraeus crypticus. Exposure spanned both water and soil exposure, it covered all life stages (cocoons, juveniles and adults), varying exposure durations (1-2-3-4-5-21 days), and covered 5 biological endpoints: hatching success, survival, reproduction, avoidance and gene expression (qPCR target genes GABA and Acetyl cholinesterase). We tested 4 Ag materials: PVP coated (PVP-AgNM), non-coated (NC-AgNM), the JRC reference Ag NM300K and AgNO 3. Results showed that short-term exposure via water to assess impact on cocoons' hatching predicted longer term effects such as survival and reproduction. Moreover, if we extended the exposure from 11 to 17 day this allowed discrimination between hatch delay and impairment. Exposure of juveniles and adults via water showed that juveniles were most sensitive with survival affected. Across materials the following toxic ranking was observed: AgNO 3 ≥ Ag NM300K ≫ NC-AgNM ≥ PVP-AgNM. E. crypticus avoided AgNO 3 in a dose-response manner, avoiding most during the first 24 h. Avoidance of Ag NM300K and NC-AgNM only occurred during the first 24 h and the PVP coated AgNM were not avoided at all. The up-regulation of the GABA triggering anesthetic effects, indicated the high ecological impact of Ag materials in soil: Ag affects the GABAergic system hence organisms were not able to efficiently avoid and became intoxicated, this caused impacts in terms of survival and reproduction. Image 1 • Novel multi-endpoint approach for mechanistic based hazard assessment in E. crypticus. • Ag exposed organisms cannot avoid Ag after 24 h. • Ag affects the GABBAergic correlating to avoidance. • Ag affects primarily embryo development and juvenile stages. • High ecological impact of Ag long term exposure expected. Multi-endpoint approach advances the understanding of effects of (nano)materials: towards a mechanistic based risk assessment strategy. [ABSTRACT FROM AUTHOR]

Injuries can impact the overall well-being and function of the animals. Nevertheless, the healing capacity after mechanical trauma (e.g. perforation) is sparsely studied in soil ecotoxicology models. In this study we investigated the recovery capacity from inflicted wounding in the soil ecotoxicology model Enchytraeus crypticus (Oligochaeta). The process involved perforation of E. crypticus, using a microneedle, using three treatments (corresponding to different wounding locations): After (A), Before (B), and After and Before (A + B) the clitellum, in addition to controls. After wounding, the animals were kept in LUFA 2.2 soil, up to 42 (21 + 21) days, to evaluate survival and reproduction. Further, the expression of three target genes (mitochondrial calcium uniporter regulator 1 (Mcu-1), superoxide dismutase (Sod), and Rho family GTPase 1 (Rho-1)) was analysed by qPCR to understand if the healing mechanisms in E. crypticus are similar to those reported for the nematode Caenorhabditis elegans. Results showed that A + B wounding affected animals' survival (day 21) but a recovery/healing mechanism seems to occur, as observed with time in terms of reproduction (day 42) performance. Rho-1 gene was down-regulated, in line with what is described necessary for wound closure. The optimized test procedures can be further used as described to assess the impacts of chemical exposure on injured animals. [Display omitted] • Virtually all animals undergo physical injuries during their life. • Wound healing mechanisms were investigated in E. crypticus (A: After; B: Before clitellum). • Survival, reproduction and gene expression were analysed after perforating wounding. • Animals' performance was affected by wounding, although recovered. • Wounding closure probably requires Rho-1 inhibition (qPCR confirmed). [ABSTRACT FROM AUTHOR]

Abstract Gene expression can vary with the organisms' life stage. It is known that embryos can be more sensitive to toxicant exposure, as previously demonstrated for Enchytraeus crypticus (Oligochaeta) exposed to cadmium (Cd), known to cause embryotoxicity and hatching delay. It was shown that Ca enters embryos via the L-type Ca channels in the cocoon membrane, this being affected in Cd exposed embryos (Cd-Ca competition is well-known). In the present study, the embryotoxic mechanisms of Cd were studied via high-throughput gene expression for E. crypticus. Cocoons (1–2 days old), instead of the adult organism, were exposed in Cd spiked LUFA 2.2 soil during 1 day. Results showed that Cd affected Ca homeostasis which is implicated in several other molecular processes. Several of the major modulators of Cd toxicity (e.g., impaired gene expression, cell cycle arrest, DNA and mitochondrial damage) were identified in the embryos showing its relevancy as a model in ecotoxicogenomics. The draft Adverse Outcome Pathway was improved. Previously was hypothesized that gene regulation mechanisms were activated to synthesize more Ca channel proteins – this was confirmed here. Further, novel evidences were that, besides the extracellular competition, Cd competes intracellularly which causes a reduction in Ca efflux, and potentiates Cd embryotoxicity. Graphical abstract Image 1 Highlights • E. crypticus embryos are more sensitive to Cd than adults. • Embryos' mechanisms were studied using high-throughput gene expression. • Major modulators of Cd toxicity were identified in the embryos. • Cd competes with Ca inside the cells (novel), besides extracellular (known). • Adverse Outcome Pathway (AOP) was improved. [ABSTRACT FROM AUTHOR]

Highlights: [•] Toxicity of Ag-NPs and AgNO3 was studied in the terrestrial worm (E. albidus). [•] Effects were assessed for gene expression, survival and reproduction. [•] AgNO3 was more toxic than Ag-NPs. [•] Toxicity could not be fully explained by NPs ionization. [•] Gene profile suggests similar mechanisms of toxicity involved for AgNPs and AgNO3. [ABSTRACT FROM AUTHOR]

Abstract: Despite increased utilization of copper (Cu) nanoparticles, their behaviour and effect in the environment is largely unknown. Enchytraeids are extensively used in studies of soil ecotoxicology. Ecotoxicogenomic tools have shown to be valuable in nanotoxicity interpretation. A cDNA microarray for Enchytraeus albidus has recently been developed, which was used in this study. We compared the gene expression profiles of E. albidus when exposed to Cu-salt (CuCl2) and Cu-nanoparticles (Cu-NP) spiked soil. Exposure time was 48h with a concentration range of 400 to 1000mg Cu/kg. There were more down-regulated than up-regulated genes. The number of differently expressed genes (DEG) decreased with increasing concentration for CuCl2 exposure, whereas for Cu-NP, the number did not change. The number of common DEG decreased with increasing concentration. Differences were mainly related to transcripts involved in energy metabolism (e.g. monosaccharide transporting ATPase, NADH dehydrogenase subunit 1, cytochrome c). Overall, our results indicated that Cu-salt and Cu-NP exposure induced different gene responses. Indirect estimates of Cu-NP related ion-release indicated little or no free Cu2+ activity in soil solutions. Hence, it was concluded that the Cu-NP effects were probably caused by the nanoparticles themselves and not by released ions. [Copyright &y& Elsevier]

Abstract: Changes in soil properties (e.g. pH, organic matter content, granulometry) can influence chemical toxicity to organisms and act alone as stressors. Previous studies on Enchytraeus albidus showed that changes in soil properties caused effects on reproduction and avoidance behavior and also oxidative stress. In addition, results at the transcritptomic level indicated changes in gene expression profile due to soil properties changes. In this study, E. albidus was exposed to modified versions of the artificial standard OECD soil (higher clay and lower sand proportion, lower pH, and lower organic matter content) in different exposure times (2, 4 and 8 days). The gene expression profile was characterized using a class comparison statistical analysis. Results indicated that the transcriptional response was time dependent, with different genes being affected at different time points. Results also showed some genes (and biological functions) being affected in a soil specific way. The unknown gene function in the data-set makes further discrimination difficult. [Copyright &y& Elsevier]

• First study on nanomaterials epigenetic effects in environmental species. • Multigenerational exposure to nanomaterials induced epigenetic changes. • Expression of epigenetic related genes was significantly regulated. • The study links epigenetic and highly ecological relevant endpoints. • Global DNA methylation showed a similar trend with reproduction. Chemical stressors can induce epigenomic changes, i.e., changes that are transferred to the next generation, even when the stressor is removed. Literature on chemical induced epigenetic effects in environmental species is scarce. We here provide the first results on epigenetic effects caused by nanomaterials with an environmental OECD standard soil model species Enchytraeus crypticus species. We assessed the epigenetic potential in terms of global DNA methylation, gene-specific methylation via bisulfite sequencing and MS-HRM (Methylation Sensitive - High Resolution Melting), and gene expression qPCR for genes involved in DNA methylation, histone modifications, non-coding RNA and stress response mechanisms). We have exposed E. crypticus in a multigenerational (MG) test design to Cu (copper oxide nanomaterials (CuO NMs) and copper salt (CuCl 2)). To link possible epigenetic effects to population changes, we used exposure concentrations (ECx) that caused a 10% and 50% reduction in the reproductive output (10% and 50% are the standards for regulatory Risk Assessment), the organisms were exposed for five consecutive generations (F1–F5) plus two generations after transferring to clean media (F5–F7), 7 generations in a total of 224 days. Results showed that MG exposure to Cu increased global DNA methylation and corresponded with phenotypic effects (reproduction). Gene expression analyses showed changes in the epigenetic, stress and detoxification gene targets, depending on the generation and Cu form, also occurring in post-exposure generations, hence indicative of transgenerational effects. There were in general clear differences between organisms exposed to different Cu-forms, hence indicate nanoparticulate-specific effects. [ABSTRACT FROM AUTHOR]