Gene Expression Profile Assessment in Zebrafish (Danio rerio)

The Zebrafish Danio rerio, in the last decade, has been recognized as the best model among the vertebrate model organisms. The exquisite features and the high homology with the mammalian system have facilitated its application in many field e.g. eco-toxicology, biomedicine, genetics and physiology. The complete sequencing of the genome provided enormous value giving the possibility to develop a DNA microarray for gene expression analysis. Gene expression represents a unique way of characterizing how cells and organisms adapt to changes in the external environment (Lettieri, EHP, 2006). The measurements of gene expression levels, upon exposure to a chemical, can be used both to provide information about the mechanism of action of the toxicant, and also to form a sort of ¿genetic signature¿ for the identification of toxic products. Benzo[a]pyrene (BaP) is a well-known chemical pollutant, which belongs to the family of polycyclic aromatic hydrocarbons (PAHs) which are formed during combustion processes. Extensive studies have been published on the metabolism and the toxicity of PAHs, as well as epidemiologic studies which suggest a role of the PAHs in carcinogenetic and mutagenic effects in many species. BaP as well as other PAHs is included in the Priority Hazardous Substances adopted in November 2001 by the new Water Framework Directive (2000). Our studies focused on the analysis of gene expression changes in a liver cell line of Zebrafish upon exposure to BaP concentrations close to environmental level using DNA Microarrays. In our gene expression analysis, we identified nine genes, among them the cyctochrome P450 CYP1A involved in the xenobiotic response and the cyb5 whose role in the activation of human CYP1A2 was recently reported in the literature.JRC.H.5-Rural, water and ecosystem resource

Proteomic analysis of the marine diatom Thalassiosira pseudonana upon exposure to benzo(a)pyrene

Abstract Background The polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants ubiquitously distributed. They are generated by incomplete combustion of organic materials such as wood or fossil fuels. Due to their carcinogenic, mutagenic effects and to their wide distribution in the environment, these pollutants pose many concerns to researchers and regulators. In our laboratories we investigated the effect of Benzo(a)pyrene (BaP) exposure in the marine diatom Thalassiosira pseudonana, which has become an important model organism in aquatic toxicology studies. Results In order to investigate the mechanism of action of PAHs, we exposed the diatoms for 24 h to 36.45 µg/L of B(a)P which inhibits the growth by about 30 % and analysed the relative protein expression profile by a quantitative proteomics approach based on iTRAQ labels. The proteomics profile analysis showed that around 10 % of the identified proteins were regulated and one fourth of them confirmed the gene expression changes seen by DNA microarray. Particularly interesting was the down regulation of the Silicon transporter 1 (SIT1), an enzyme that is responsible for the uptake of silica from the media into the cell of diatoms. Regulation of SIT1 upon BaP treatment was also confirmed at the gene expression level. Conclusions The potential use of the regulated proteins found in this study as early indicators of environmental exposure to PAHs is discussed. In particular, SIT1 is considered a promising biomarker and SIT1 expression changes were confirmed also when the diatoms were exposed to field samples, e.g. marine surface sediments contaminated by PAHs.JRC.DDG.H.5-Rural, water and ecosystem resource

Microbial Biodiversity and Molecular Approach

Biodiversity is given by the variety of species on Earth resulting from billions of years of evolution. Molecular-phylogenetic studies have revealed that the main diversity of life is microbial and it is distributed among three domains: Achaea, Bacteria, and Eukarya. The functioning of whole biosphere depends absolutely on the activities of the microbial world. Due to their versatility microbes are the major natural providers of ecological services as well play major role in semi-artificial systems such as sewage treatment plants, landfills, and in toxic waste bioremediation. As for other organisms many pressures and drivers are causing decrease of microbial biodiversity. Several publications document the effect of chemical pollutants e.g. Polycyclic Aromatic Hydrocarbons (PAHs), of atmospheric pollution, of temperature change and of fertilization on microbial community structure. These studies are now possible because sequencing technologies are in ongoing revolution allowing massive de novo sequencing producing millions of bases in a single day. Metagenomics, metatranscriptomics, metaproteomics and single-cell sequencing are approaches providing a view not only of the community structure (species phylogeny, richness, and distribution) but also of the functional (metabolic) potential of a community because virtually about all genes are captured and sequenced. Unfortunately, although microrganisms are very important for the functioning of whole biosphere public knowledge, awareness and political actions did not yet deal with microbes when biodiversity and its decrease are highlighted.JRC.DDG.H.5-Rural, water and ecosystem resource

Transcriptomics Responses in Marine Diatom Thalassiosira pseudonana Exposed to the Polycyclic Aromatic Hydrocarbon Benzo[a]pyrene

Diatoms are unicellular, photosynthetic, eukaryotic algae with a ubiquitous distribution in water environments and they play an important role in the carbon cycle. Molecular or morphological changes in these species under ecological stress conditions are expected to serve as early indicators of toxicity and can point to a global impact on the entire ecosystem. Thalassiosira pseudonana, a marine diatom and the first with a fully sequenced genome has been selected as an aquatic model organism for ecotoxicological studies using molecular tools. A customized DNA microarray containing probes for the available gene sequences has been developed and tested to analyze the effects of a common pollutant, benzo(a)pyrene (BaP), at a sub-lethal concentration. This approach in diatoms has helped to elucidate pathway/metabolic processes involved in the mode of action of this pollutant, including lipid metabolism, silicon metabolism and stress response. A doseresponse of BaP on diatoms has been made and the effect of this compound on the expression of selected genes was assessed by quantitative real time-PCR. Up-regulation of the long-chain acyl-CoA synthetase and the anti-apoptotic transmembrane Bax inhibitor, as well as down-regulation of silicon transporter 1 and a heat shock factor was confirmed at lower concentrations of BaP, but not the heat-shock protein 20. The study has allowed the identification of molecular biomarkers to BaP to be later on integrated into environmental monitoring for water quality assessment.JRC.H.1-Water Resource

Development of the First Watch List under the Environmental Quality Standards Directive

According to Directive 2008/105/EC (the Environmental Quality Standards Directive, EQSD), a new mechanism is needed to provide high-quality monitoring information on the concentrations of polluting substances in the aquatic environment across the EU. The aim of this mechanism is to support the identification of priority substances for regulation under the Water Framework Directive. A restricted number of substances (up to 10) are to be included in a dynamic Watch List, remaining there for limited time. Three compounds, i.e. diclofenac, 17-beta-estradiol (E2), and 17-alpha-ethinylestradiol (EE2) have already been identified for inclusion in the first Watch List, for the specific purpose of better informing the determination of suitable risk reduction measures. Therefore, up to seven additional substances should be identified for inclusion. This report describes the procedure to identify a short-list of substances, based on the suspected risk to or via the aquatic environment, as well as on the unavailability of sufficient monitoring data or data of sufficient quality to identify the risk posed by those substances, and to prioritise them at EU level. From the short-list, seven additional substances are proposed for inclusion in the first Watch List.JRC.H.1-Water Resource

Cyanotoxins: methods and approaches for their analysis and detection

Cyanotoxins are secondary metabolites produced by cyanobacteria, a group of photosynthetic prokaryota especially found in freshwater. In favourable conditions (i.e. high nutrient levels, light intensity, water temperature), cyanobacteria can form blooms, a natural phenomenon characterised by an algal biomass accumulation and the possible release of cyanotoxins in water ecosystems. Toxins represent an emerging threats for the aquatic organisms which can bioaccumulate these compounds and transfer them throughout the food chain to wildlife and humans. Other ways of exposure for humans include the oral, dermal and inhalation route. The consumption of contaminated drinking water, skin contact and swallowing water during recreational activities are among the most frequently reasons for human poisonings caused by cyanotoxins. The associated symptoms usually range from severe headache to fever, respiratory paralysis and in rare case, death. The World Health Organization (WHO) has issued a provisional guideline value of 1 µg/L in drinking water for Microcystin-LR (MC-LR), the most toxic, widespread and common toxin in water supplies. Due to the lack of complete toxicological data for a range of cyanotoxins, their concentration in drinking water is not yet well regulated even in countries belonging to the European Union (EU). In this report, attention is focused on the methodologies commonly used to detect cyanotoxins in water environments. These applications can be grouped in: I) microscopy analysis II) physicochemical methods III) molecular-based methods IV) biochemical-based methods V) chemical methods. Each technique shows specific limitations in terms of sensitivity, reliability and limit of detection. The choice of the best one to use is determined in accordance with the information they provide, the availability of facilities and the technical expertise of the operators. Most of the research about cyanotoxins has been mainly focused on microcystins (MCs). The other cyanotoxins have been much less investigated and more tools need to be developed to overcome this problem. Notwithstanding there is no a single analytical application able to detect all cyanotoxin variants in an environmental sample. Some current methods described in this report show great promise in terms of being simple, cost-effective, specific and sensitive for the analysis of a defined toxin.JRC.D.2-Water and Marine Resource

Phylogenetic and Functional Metagenomic Profiling for Assessing Microbial Biodiversity in Environmental Monitoring

Environmental management decisions have to base on holistic understanding of biodiversity and functional capability in ecosystems. Environmental metagenomics is an emerging and powerful approach allowing rapidly and reliably determine and compare microbial biodiversity and functional profiles. Advances in next generation sequencing technologies and bioinformatic tools allow set up analysis pipelines which are useful for well designed and targeted monitoring exercises already today. In the paper we demonstrate how direct sequencing of the total community DNA and analysis of the data are applicable to assess anthropogenic impact on the coastal marine ecosystems.JRC.H.1-Water Resource

Review of the 1st Watch List under the Water Framework Directive and recommendations for the 2nd Watch List

The surface water Watch List (WL) under the Water Framework Directive (WFD) is a mechanism for obtaining high-quality Union-wide monitoring data on potential water pollutants for the purpose of determining the risk they pose and thus whether Environmental Quality Standards (EQS) should be set for them at EU level. According to the EQS Directive (article 8b) , this list should be updated every 2 years. The main objectives of this report are: • To present an overview of the data gathered during the 1st year of monitoring of the 1st WL (also called WL dataset in this report), • To assess whether this WL dataset is sufficient to determine the risk posed by the WL substances, and consequently to determine whether any of these substances can be taken out of the WL, • To propose new substance(s) to be included in the second WL, using the information and results from the latest review of the list of priority substances, as well as any other relevant information available at the time of this report. The executive summary first explains the context for the assessment. Then, mirroring the report itself, it presents an overview of the WL dataset for the different WL substances, it specifies the criteria for taking substances out of the WL and the substances proposed on the basis of these criteria, and finally it presents the criteria for including new substances in the WL and the new proposed WL candidates.JRC.D.2-Water and Marine Resource