Sara C. Novais

Effect of Cu-nanoparticles versus one Cu-salt: Analysis of stress biomarkers response in Enchytraeus albidus (Oligochaeta)

Abstract In the present study, the main goal was to compare the effects of ionic copper versus copper nanoparticles in Enchytraeus albidus assessing the effect at the biomarker level, testing different concentrations and exposure times. Measured parameters were lipid peroxidation (LPO), total, reduced and oxidized glutathione content (TG, GSH and GSSG), the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferases (GSTs) and cholinesterases (ChEs). Results showed that both salt- and nano-copper caused oxidative stress and damage to E. albidus, as confirmed by LPO levels, and effects could be discriminated between the copper forms. Nevertheless and despite the visible discrimination between nano and the salt form (time and exposure dependent), there was no single or a set of biomarkers that provided the best discrimination.

Effect of Cu-nanoparticles versus Cu-salt in Enchytraeus albidus (Oligochaeta): differential gene expression through microarray analysis.

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 (CuCl(2)) and Cu-nanoparticles (Cu-NP) spiked soil. Exposure time was 48 h with a concentration range of 400 to 1000 mg Cu/kg. There were more down-regulated than up-regulated genes. The number of differently expressed genes (DEG) decreased with increasing concentration for CuCl(2) 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 Cu(2+) 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.

Biochemical characterization of cholinesterases in Enchytraeus albidus and assessment of in vivo and in vitro effects of different soil properties, copper and phenmedipham

Enchytraeus albidus are important organisms of the soil biocenosis, used as standard test species in environmental risk assessment. The inhibition of cholinesterases (ChE) activity of several species has been widely used to assess the exposure and effects of anti-cholinesterase environmental contaminants. Several studies have shown the association between ChE activity inhibition and adverse effects on behaviour and survival. Extensive studies addressing survival and behavioural endpoints, as well as other biomarkers, have been done in E. albidus with different types of soil contaminants. The main objectives of this study were: (1) to characterize biochemically the ChE present in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, using different substrates and selective inhibitors; (2) to assess the in vivo effects of copper, phenmedipham and different soil properties (pH, organic matter, clay) on the ChE activity; (3) to assess the in vitro effects of copper and phenmedipham on the ChE activity. The results suggest the presence of one ChE in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, which displays properties of both acetylcholinesterase and pseudocholinesterase considering the typical mammalian enzymes. It is also shown that ChE activity is not inhibited by exposure to different soil properties and that copper and phenmedipham inhibited ChE activity both in in vivo and in in vitro conditions and therefore ChE inhibition seems to be a robust biomarker for this herbicide and this heavy metal. This study showed that ChE activity in E. albidus might be correlated to previously determined higher level effects like survival and reproduction, as well as avoidance behaviour.

Gene Expression Responses Linked to Reproduction Effect Concentrations (EC10,20,50,90) of Dimethoate, Atrazine and Carbendazim, in Enchytraeus albidus

Background Molecular mechanisms of response to pesticides are scarce and information on such responses from soil invertebrates is almost inexistent. Enchytraeus albidus (Oligochaeta) is a standard soil ecotoxicology model species for which effects of many pesticides are known on survival, reproduction and avoidance behaviour. With the recent microarray development additional information can be retrieved on the molecular effects. Methodology/Principal Findings Experiments were performed to investigate the transcription responses of E. albidus when exposed to three pesticides – dimethoate (insecticide), atrazine (herbicide) and carbendazim (fungicide) – in a range of concentrations that inhibited reproduction by 10%, 20%, 50% and 90% (EC10, EC20, EC50 and EC90, respectively). The goal of this study was to further identify key biological processes affected by each compound and if dose-related. All three pesticides significantly affected biological processes like translation, regulation of the cell cycle or general response to stress. Intracellular signalling and microtubule-based movement were affected by dimethoate and carbendazim whereas atrazine affected lipid and steroid metabolism (also by dimethoate) or carbohydrate metabolism (also by carbendazim). Response to DNA damage/DNA repair was exclusively affected by carbendazim. Conclusions Changes in gene expression were significantly altered after 2 days of exposure in a dose-related manner. The mechanisms of response were comparable with the ones for mammals, suggesting across species conserved modes of action. The present results indicate the potential of using gene expression in risk assessment and the advantage as early markers.

Exposure of **Enchytraeus albidus** to Cd and Zn : changes in cellular energy allocation (CEA) and linkage to transcriptional, enzymatic and reproductive effects

Cellular energy allocation (CEA) is a measure of the energy status of an organism. The effects of Cd and Zn (reproduction EC(50)s and EC(90)s) on the total energy budget of Enchytraeus albidus (Oligochaeta) were assessed through CEA determination, over periods of time from 0 to 8 d. Results showed reduction on the energy reserves for both metals after 2 d exposure. Lipids were the first reserves to be used and carbohydrates were reduced exclusively after Cd exposure. Electron transport system (ETS) activities were enhanced, suggesting increased metabolism and higher energy requirements for metal detoxification. This was supported by previous results at transcription level, where an up-regulation of genes involved in the mitochondrial oxidative phosphorylation was verified. Additionally, the reduction of CEA may be related with the decrease on the reproductive output. These results showed the relevance of integrating various endpoints, which enabled an overview of various processes and to unravel mechanisms of action of chemicals.

Development of a microarray for Enchytraeus Albidus (oligochaeta): preliminary tool with diverse applications

Standard bioassays allow hazard assessment at the population level, but much remains to be learned about the molecular level response of organisms to stressors. The main aim of this study was the development of a DNA microarray for Enchytraeus albidus, a common soil worm species. Further, this microarray was tested using worms exposed to Cu, phenmedipham, and different soil types. Hybridization onto the developed microarray revealed several genes with homology to known sequences. Genes of interest were confirmed through real-time polymerase chain reaction. It was possible to discriminate between natural and chemical stressors and chemical concentrations. Gene responses were detected under conditions known to have effects in the reproduction of individuals. It was confirmed that the integration of different endpoints improves the assessment process and enhances the understanding of the modes of action of stressors. The chemical stress-induced genes were related to factors such as immune response, stress response, metabolic processes, and/or signal transduction. The present study represents the first step of a gene-level study in the ecologically relevant and standard test species E. albidus. It demonstrates the usefulness of cDNA normalization in the production of cDNA libraries of ecotoxicological standard organisms that are not genome models like E. albidus.

Changes in cellular energy allocation in Enchytraeus albidus when exposed to dimethoate, atrazine, and carbendazim

Cellular energy allocation (CEA) is a methodology developed to evaluate the effects of toxic stress on the metabolic balance of organisms. It consists of the integration of the energy reserves available (Ea; total carbohydrate, protein and lipid content) and energy consumption (Ec) estimated by measuring electron transport system (ETS) activity. The main goal of the present study was to evaluate the effects on the energy budget of the soil invertebrate Enchytraeus albidus (Oligochaeta) after exposure to dimethoate, atrazine, and carbendazim (by testing the reproduction 10% effective concentration, 20% effective concentration, 50% effective concentration and 90% effective concentration) over periods of time from 0 d to 2 d, 4 d, and 8 d. Significant changes in energy reserves were observed with all pesticides, together with effects on energy consumption. Carbohydrates were the first energy source to be used, and clear depletions occurred with all pesticides. Energy consumption increased generally over longer exposures and with higher concentrations of the pesticides. Although clear changes were seen in the individual energy reserve budgets and on Ec, CEA was only significantly reduced with atrazine exposures longer than 4 d. The nearly absent effects on CEA at concentrations known to affect reproduction indicate that the reduction in reproduction is not likely to be caused by a reduction in the total energy budget during the first 8 d of exposure. The present study showed the importance of complementing CEA interpretation with the individual Ea and Ec parameters, in particular if these show opposite balances. The Ea and Ec results were in good agreement with gene transcription results from a parallel study, hence suggesting translation and showing the advantage of combining various effect levels to advance the understanding of mechanisms.

Transcriptional responses in Enchytraeus albidus (Oligochaeta): Comparison between cadmium and zinc exposure and linkage to reproduction effects

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.

Nickel response in function of temperature differences: effects at different levels of biological organization in Daphnia magna.

In this study, gene transcription profiling in combination with the assessment of systemic parameters at individual and population levels were applied to study the (toxic) effects induced through temperature stress in the presence or the absence of an additional chemical stressor (nickel) in Daphnia magna. It was illustrated that lower temperatures were mainly characterized by a reduction of growth and lipid content, while higher temperatures caused an increase of both endpoints. Many of the differentially regulated transcripts could be correlated with processes affected at higher hierarchical levels of biological organization. Gene clusters with probable roles in producing offspring (peak expression at 22°C), enhancing the metabolic rate (temperature related expression) and translational processes (increased expression at 14°C) were identified. However, it was not possible to pinpoint a specific subset of genes, exclusively responding to temperature or nickel and allowing a retrospective identification of the particular stressor. Overall, extreme temperatures caused a higher level of stress in the organisms in comparison to nickel exposure. Moreover, organisms subjected to the natural stressor appeared to be less capable of dealing with the additional chemical stressor and as a result activate or repress more gene pathways.

Sensitivity of the sea snail Gibbula umbilicalis to mercury exposure--linking endpoints from different biological organization levels.

Mercury contamination is a common phenomenon in the marine environment and for this reason it is important to develop cost-effective and relevant tools to assess its toxic effects on a number of different species. To evaluate the possible effects of Hg in the sea snail Gibbula umbilicalis, animals were exposed to increasing concentrations of the contaminant in the ionic form for 96 h. After this exposure period, mortality, feeding and flipping behavior, the activity of the biomarkers glutathione S-transferase, superoxide dismutase, catalase, lactate dehydrogenase and cholinesterase, the levels of lipid peroxidation and cellular energy allocation were measured. After 96 h of exposure to the highest Hg concentration (≈LC20), there was a significant inhibition of the cholinesterase activity as well as impairment in the flipping behavior and post-exposure feeding of the snails. Cholinesterase inhibition was correlated with the impairment of behavioral responses also caused by exposure to Hg. These endpoints, including the novel flipping test, revealed sensitivity to Hg and might be used as relevant early warning indicators of prospective effects at higher biological organization levels, making these parameters potential tools for environmental risk assessment. The proposed test species showed sensitivity to Hg and proved to be a suitable and resourceful species to be used in ecotoxicological testing to assess effects of other contaminants in marine ecosystems.