Exposure to different copper forms – nanoparticles, nanowires, salt and field aged: gene expression profiling in Enchytraeus crypticus
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ABSTRACT: The testing of NMs under the currently available standard toxicity tests does not cover many of the NMs specificities. One of the current recommended approaches forward lays on understanding the mechanisms of action as these can help predicting long term effects and safe-by-design production. Copper nanomaterials (Cu-NMs) usage has been highly increasing with the concern in terms of exposure, effect and associated risks. In the present study we used the high-throughput gene expression tool developed for Enchytraeus crypticus (44Kx4 Agilent microarray) to study to the effect of exposure to several copper forms. The copper treatments include two NMs (spherical and wires) and two copper-salt treatments (CuNO3 spiked and Cu field historical contamination). Testing was done based on reproduction effect concentrations (EC20, EC50) using 3 and 7 days exposure periods.
Project description:The effect of nanomaterials (NMs) is less understood in light of the implemented and existing methodologies for regular chemicals. To understand the mode of action of NMs is one of the alternatives to improve predictions and environmental risk assessment (ERA). In the present work the high-throughput gene expression tool (4x44K microarray for Enchytraeus crypticus) was used to investigate the mechanisms activated by Ni exposure. Ni nanoparticles (Ni-NPs) were investigated together with Ni-salt (NiNO3). Testing was done based on reproduction effect concentrations (EC20, EC50) using 3 and 7 days exposure periods.
Project description:The transcriptome of the ecotoxicological model Enchytraeus crypticus is well studied but the downstream changes at the protein level remained a gap. Changes in the protein regulation following exposure to CuO nanomaterial (NM) and Cu salt (CuCl2) were investigated. High performance liquid chromatography with tandem mass spectrometry using tandem mass tags was used. CuO NM elicited higher number of differentially expressed proteins compared to CuCl2 with little to no overlap of proteins. CuO NM caused more stress response mechanisms, with good agreement between differentially expressed proteins, genes and metabolites. CuCl2 caused higher impact in shorter time periods, but organisms have conserved mechanisms (constitutive genes) that allow Cu handling and detoxification. CuO NM caused higher impact after a longer exposure period, inducing regulation of facultative genes with a whole differentiated paradigm and cascade. This could be due to different issues: 1) the cell uptake route is different for Cu NM and Cu ions 2) internalized Cu NM can result in a “Trojan- horse” effect 3) the cascade of events occurs in a different time order 4) the organism uptake is different between life stages, i.e., cocoons thickened surface protects the entry of NM and juveniles have facilitated entry via tegument. Protein responses are of key importance when trying to understand the link between exposure and the related adverse biological effects. We here used advanced proteomic profiling techniques to describe how the protein responses differ when an organism is exposed to Copper (Cu) nanomaterial compared to when it is exposure to Cu ions. We observed that when organisms are exposed to the nano-form they have longer-term changes in protein patterns, patterns that are different from the responses following exposure to the ionic form. On the level of individual proteins we identified nano-specific mechanisms.
Project description:Silver nanomaterials (AgNMs) are broadly used in many products and also rate among the most studied nanoscaled materials. Their ecotoxicological impact in soil invertebrates has been covered, mostly using standard testing, where endpoints like survival and reproduction are assessed. The underlying molecular mechanisms have been assessed to a much less extent. Hence, we here assessed differentially expressed proteins (DEPs) and metabolites (DEMs) by high-throughput (HTP) techniques (HPLC-MS/MS with tandem mass tags for proteome analysis, as well as reversed-phase (RP)- or hydrophilic interaction liquid chromatography (HILIC) with mass spectrometric detection for metabolome analysis. The standard soil model Enchytraeus crypticus was exposed to Ag NM300K and soluble AgNO3, at the reproduction EC20 and EC50, in a time series of 0, 7, and 14 days. The impact was clearly larger after 14 days. Ag NM300K caused more upregulated DEPs/DEMs, and more so at the EC20, compared to the EC50, whereas AgNO3 caused a dose response increase of DEPs/DEMs. Similar pathways were activated, although often via opposite regulation (up vs down) of DEPs hence dissimilar mechanisms underlie the apical impact. Affected pathways include e.g. energy metabolism transport proteins, detoxifying enzymes, histidine (e.g. neurotransmission by gamma-aminobutyric acid (GABA)) and lipid metabolism. Uniquely affected by AgNO3 were catalase, malate dehydrogenase and ATP-citrate synthase, and by Ag NM300K were heat shock proteins (HSP70) and ferritin. The gene expression-based data in Adverse Outcome Pathway (AOP) was confirmed and additional key events were added. Evidences support that toxicity of Ag NM increases in longer-term exposure.
Project description:Exposure to different copper forms – nanoparticles, nanowires, salt and field aged: gene expression profiling in Enchytraeus crypticus
Project description:Understanding the mode of action of nanomaterials (NMs) aids in improving predictions and environmental risk assessment. In the present study, a high-throughput (HTP) microarray was used to study Enchytraeus crypticus gene expression. Four Ag materials (Ag NM300K, PVP-coated AgNPs, AgNPs, and AgNO3) were tested at reproduction effect concentrations, EC20 and EC50, to anchor gene expression responses to higher effect level. The results showed that while PVP-AgNPs and AgNPs had similar responses, Ag NM300K caused effects via a differentiated transcriptomic profile, with uniquely affected processes (e.g. transcytosis). For the AgNPs, the EC50 negatively affected apoptosis, which can lead to accumulation of abnormal cells and cause apical damage (reproduction). Mechanisms which are known to be related to Ag toxicity and which were observed here for the various Ag forms included apoptosis regulation, cell redox homeostasis, impairment of energy production and response to DNA damage. This HTP genomic tool enabled discrimination between Ag materials, which is not possible via standard tests (i.e. survival and reproduction endpoints). Moreover, gene expression analysis provided information regarding the mechanisms of toxicity of NMs and the pathways uniquely affected by NMs. An adverse outcome pathway (AOP) was drafted for the first time for Ag NMs; this AOP can and should be used as a basis for further research.
Project description:Titanium dioxide (TiO2) based nanomaterials (NMs) are among the most produced NMs worldwide. When irradiated with light, particularly UV, TiO2 is photoactive, a property that is explored for several purposes. There is an increasingly number of reports on the negative effects of photoactivated TiO2 to non-target organisms. We have here studied the effect of a suite of reference type TiO2 NMs i.e. NM103, NM104, NM105 and compared these to the Bulk) with and without UV radiation to the oligochaete Enchytraeus crypticus. High-throughput gene expression was used to assess the molecular mechanisms, while also anchoring it to known effects at organism level (i.e., reproduction). Results showed that the photoactivity of TiO2 (UV exposed) played a major role in enhancing TiO2 toxicity, activating transcription of oxidative stress, lysosome damage and apoptosis mechanisms. For non-UV activated TiO2, where toxicity at organism level (reproduction) was lower, results showed the potential for long-term effects (i.e., mutagenic and epigenetic). NM specific mechanisms were identified: NM103 affected transcription and translation, NM104_UV negatively affected reproductive system/organs; and NM105_UV activated superoxide anion response. Results provided mechanistic information for UV-related phototoxicity of TiO2 materials and evidences of the potential long-term effects.
Project description:Despite the increased utilization of nanoparticles, the behavior and effect in the environment is largely unknown and few resources are available for health and environmental effect studies. Enchytraeids are extensively used in studies of soil ecotoxicology and recently, a cDNA microarray for Enchytraeus albidus was developed, allowing also toxicogenomic studies in this species. These organisms are ecologically relevant small worms that indirectly contribute to the regulation and degradation of organic matter. In this study we compared the gene expression profiles of E. albidus when exposed to copper-salt (CuCl2) and copper nanoparticles (Cu-NP) spiked soil. The worms were exposed for 48 hours in soil to a range of concentrations. Microarray hybridizations revealed different response patterns between copper-salt and copper nanoparticles exposed organisms, these differences are mainly related with transcripts involved in the energy metabolism of the organisms. Despite unknown gene function in the data-set there are indications that Cu-salt and Cu-NP exposure induced specific gene level responses. Fluorescently labelled cDNA, from enchytraeids exposed during 2 days to control soil (from Hygum site in Denmark) and to the different exposure conditions (Cy5), was synthesized for microarray analysis and hybridizations were performed. After scanning, spots were identified and ratios quantified using the QuantArray (Packard Biochip Technologies). Statistical analysis of the microarrays was performed using limmaGUI package (1.18.0) (Smyth, 2005) in the R (2.8.0) software environment (http://www.R-project.org/). After being submitted to local background subtraction, microarrays were normalized using global loess method. To statistically evaluate the differential gene expression between the different conditions, a gene-per-gene linear model (limma – linear model for microarray analysis) and empirical Bayes methods were applied. The results were then corrected for multiple testing using the Benjamini-Hochberg’s method (adjusted p<0.05 was considered significant) (Benjamini and Hochberg, 1995).