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

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

Expert survey on identification of gaps in available test methods for evaluation of endocrine disruptors

According to the 2012 WHO/UNEP publication 'State of the Science of Endocrine Disrupting Chemicals' research into endocrine disrupting chemicals over the last decade has indicated that, despite the progress achieved in development and validation of test methods for evaluation of endocrine disruptors, there are still several gaps that need to be addressed. Considering the expected significant amount of work needed to fill the gaps and the limited resources available, it will be important to set priorities for the upcoming period (next 5-10 years) for the development and validation of test methods. Thus there is a need to focus the European input to the OECD test guideline programme to effectively enhance the identification of chemical substances with endocrine disrupting properties whilst making best use of existing resources. With this objective in mind, DG Environment, supported by JRC, is organising a European expert workshop on setting priorities for further development and validation of test methods for evaluating endocrine disruption. The workshop will take place on 30 May - 01 June 2017 in Brussels. The deliberations will focus on what is necessary and achievable in the context of resources, timescales and animal welfare considerations. In preparation for the workshop, JRC has drawn up a questionnaire to gather input from experts in the field on key issues to be used as a basis for the further discussions at the workshop. An online survey with the title "Identifying gaps in available test methods for evaluation of endocrine disruptors" was performed on the EU Survey platform and open for commenting from 19/05/2015 until 15/06/2015. A selected group of experts (EFSA Scientific Committee and WG on EDs, ECHA ED WG and RAC, WNT (European members from OECD webpage), Experts identified in Annex 3 of the "Roadmap for setting priorities for further development and validation of test methods and testing approaches for evaluating endocrine disruptors") was invited to participate in the survey. Experts were asked to rank endocrine related diseases/disorders regarding the possibility to predict them with existing test methods (TMs). They were further asked to rank diseases/disorders regarding the need to develop new test methods to better cover those. Experts were then requested to provide their views on including further tests based on those discussed in the OECD (2012) "Detailed Review Paper on the state of the science on novel in vitro and in vivo screening and testing methods and endpoints for evaluating endocrine disruptors" and their views on the current OECD Conceptual Framework and proposals for improvements. Forty experts representing 15 countries and different stakeholder groups (authorities; academia; civil society organisation; industry) replied. The purpose of this report is to present the detailed survey results. Multiple choice questions were evaluated and where possible quantitative rankings were performed. In addition, the survey respondents provided a lot of valuable information in numerous free text comments. Those are included in the report in tables as they were received, without editing them, unless personal information had to be removed. Brief summaries of the main points raised are added after each section.JRC.F.3-Chemicals Safety and Alternative Method

Strategic aims for improving the regulatory assessment of Developmental Neurotoxicity (DNT) using non-animal methods

Currently, the identification of chemicals that have the potential to induce developmental neurotoxicity (DNT) is based on animal testing, since there are no regulatory accepted alternative methods for this purpose. Since at the regulatory level, systematic testing of DNT is not a standard requirement within the EU legislation of chemical safety assessment, DNT testing is only performed in higher tiered tests triggered based on structure activity relationships or evidence of neurotoxicity in systemic adult studies. However, these triggers are rarely used and in addition do not always serve as reliable indicators of DNT as they are observed in an adult rodent animal. Consequently, to date only a limited amount of chemicals (Grandjean and Landrigan, 2006; Smirnova et al., 2014), mainly pesticides (Bjørling-Poulsen et al., 2008) have been tested under US EPA (OPPTS 870.630) or OECD DNT TG 426. Therefore, there is the pressing need for developing alternative methodologies that can more rapidly and cost-effectively screen large numbers of chemicals for their potential to cause DNT. In this report we propose that in vitro studies could contribute to the identification of potential triggers for DNT evaluation since existing cellular models permit the evaluation of a chemical impact on key neurodevelopmental processes, mimicking different windows of human brain development, especially if human models derived from induced pluripotent stem cells are applied. Furthermore, the battery of currently available DNT alternative test methods anchored to critical neurodevelopmental processes and key events identified in DNT Adverse Outcome Pathways (AOPs) could be applied to generate in vitro data useful for various regulatory purposes. Incorporation of in vitro mechanistic information would increase scientific confidence in decision making, by decreasing uncertainty and leading to refinement of chemical grouping according to biological activity. In this report development of IATA (Integrated Approaches to Testing and Assessment) based on key neurodevelopmental processes and AOP-informed is proposed as a tool for not only speeding up chemical screening, but also providing mechanistic data in support of hazard assessment and in the evaluation of chemical mixtures. Such mechanistically informed IATA for DNT evaluation could be developed integrating various sources of information (e.g., non-testing methods, in vitro approaches, as well as in vivo animal and human data), contributing to screening for prioritization, hazard identification and characterization, and possibly safety assessment of chemicals, speeding up the evaluation of thousands of compounds present in industrial, agricultural and consumer products that lack safety data on DNT potential. It is planned that the data and knowledge generated from such testing will be fed into the development of an OECD guidance document on alternative approaches to DNT testing.JRC.F.3-Chemicals Safety and Alternative Method

Something from nothing? Ensuring the safety of chemical mixtures

Headlines - Humans and the environment are exposed to a cocktail of chemicals from different sources. - Combined exposure to multiple chemicals can lead to health/ environmental effects even if single substances in the mixture do not exceed safe levels. - The assessment and management of mixtures is only partly covered by current legislation, which focuses on single substances in isolated sectors. - Methodology to address mixture risks is available, yet many knowledge gaps need to be filled. In particular, real co-exposure patterns are mostly unknown. - JRC is performing research on new strategies to assess the combination effects of chemicals.JRC.F.3-Chemicals Safety and Alternative Method

Scientific methodologies for the assessment of combined effects of chemicals - a survey and literature review

Exposure of humans and wildlife to chemicals via food, consumer products, the environment etc. can imply exposure to an infinite number of different combinations of chemicals in mixtures. It is practically impossible to test all these possible mixtures experimentally and it is therefore needed to find smart strategies to assess the potential hazards using new tools that rely less on in vivo testing and incorporate instead alternative experimental and computational tools. In this report the current state of the art for the application of these alternative tools for assessing the hazard of chemical mixtures is briefly reviewed. The focus is hereby on the adverse outcome pathway (AOP) concept, in vitro methods, omics techniques, in silico approaches such as quantitative structure activity relationships (QSARs) and read-across, toxicokinetic and dynamic energy budget (DEB) modelling, and on integrated approaches to testing and assessment (IATA). Furthermore, an expert survey was performed to collect up to date information and experience on the current use of different approaches for assessing human and environmental health risks from exposure to chemical mixtures, with a view to informing the development of a consistent assessment approach. An online survey was performed among experts in the field of combined exposure assessment in the period of January to March 2014, addressing both, human health and environmental risk assessment. Fifty-eight experts from 21 countries, different stakeholder groups and sectors of legislation participated in the survey. The main sectors where most experience is already gained in assessing mixtures are in the area of plant protection products and chemicals. These were also rated highest regarding the priority for performing mixture assessments. Experts have experience with the whole mixture as well as the component-based approaches applying them to both, intentional and unintentional mixtures. Mostly concentration addition (CA) based methods are used for predicting mixture effects. Regarding the use of novel and alternative tools in the risk assessment of mixtures, expert opinions are split between those applying them (often more in a research context) and those that generally think these tools are valuable but their use is currently limited because of lack of guidance, lack of data, or lack of expertise. A general need for clear guidance for combined exposure assessments was identified. Overall, a high potential in applying novel tools and scientific methodologies for the assessment of chemical mixtures can be identified. They allow deriving meaningful information on individual mixture components or whole mixtures, enabling a better understanding of the underlying mechanisms of mixture effects. Their main strengths lie in their integrated use and smart combination to put different aspects regarding the hazard from combined exposure to multiple chemicals into context. In order to benefit from these tools in the hazard assessment of mixtures, more guidance on their use is needed to facilitate a more widespread application.JRC.I.5-Systems Toxicolog

Regulatory assessment of chemical mixtures: Requirements, current approaches and future perspectives

n/

Thresholds of Contaminants: A Synthesis

A fundamental problem in ecotoxicology is the prediction of long term population and ecosystem-level effects of contaminant exposure based on dose response data of few individuals obtained over a short time period. In addition, environmental fluctuations will always affect significantly the population/ecosystem resilience. However, these fluctuations are not taken into account under dose-response experiments on individuals. In the Thresholds project we have analyzed some of these questions by using experiments, data analysis tools and modelling approaches. Several important finding may be summarized as follows: 1 Molecular level effects are detected even at concentrations that did not affect the macroscopic end point studied, i.e. growth rate. 2 Natural populations are more sensitive that populations in cultures. 3 There are differences for the same species at different environments, e.g. Mediterranean, Black Seas and Atlantic Ocean. 4 The environmental conditions and the time of release of the contaminant cause a variability of the response at ecosystem level that can reach 50%. 5 At the actual level of knowledge it is difficult to assess if the legal approach, based on the precautionary principle, is over or under conservative, when considering molecular and its long term effects, the combined effects of mixtures and the environmental fluctuations that affect all ecosystems. 6 A similar colour code to the one adopted for biological quality elements should be adopted for the definition of EQS, with values higher than the EQS as orange (poor). This will allow assessing contamination trends and an early detection of a chemical contamination problems. 7 In aquatic environments ecosystem experience the combined effects of mixtures. Ecotoxicological risk assessment should be performed taking this aspect into account. However, with the amount of new chemicals being produced and the detection limits required it is clear that new integrated indicators are necessary. Limiting the levels of certain chemicals in the environment is one step to improve ecosystem health but alone it will not prevent further deterioration. 8 Due to practical limitations, knowledge on ecotoxicology is only available for a small fraction of the anthropogenic chemical pressure. The importance of this simplification has not been comprehensibly assessed and introduce uncertainty in the appropriate outcome of current legislation and managing practices.JRC.H.5-Rural, water and ecosystem resource

Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line

Background: A broad spectrum of cytotoxicity assays is currently used in the fields of (eco)toxicology and pharmacology. To choose an appropriate assay, different parameters like test compounds, detection mechanism, specificity, and sensitivity have to be considered. Furthermore, tissue or cell line can influence test performance. For zebrafish (Danio rerio), as emerging model organism, cell lines are now increasingly used, but few studies examined cytotoxicity in these cell systems. Therefore, we compared four cytotoxicity assays in the zebrafish liver cell line, ZFL, to test four differently acting model compounds. The tests comprised two colorimetric assays (MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity) and two fluorometric assays (alamarBlue® using resazurin, and CFDA-AM based on 5-carboxyfluorescein diacetate acetoxymethyl ester). Model compounds were the pharmaceutical Tamoxifen, its metabolite 4-Hydroxy-Tamoxifen, the fungicide Flusilazole and the polycyclic aromatic hydrocarbon Benzo[a]pyrene. Results: All four assays performed well in the ZFL cells and led to reproducible dose-response curves for all test compounds. Effective concentrations causing 10% or 50% loss of cell viability (EC10 and EC50 values) varied by a maximum factor of 7.0 for the EC10 values and a maximum factor of 1.8 for the EC50 values. The EC values were not statistically different between the four assays, which is due to the assessed unspecific effects of the compounds. However, most often, the MTT assay and LDH assay showed the highest and lowest EC values, respectively. Nevertheless, the LDH assay showed the highest intra- and inter-assay variabilities and the lowest signal-to-noise ratios. In contrast to MTT, the other three assays have the advantage of being non-destructive, easy to handle, and less time consuming. Furthermore, AB and CFDA-AM can be combined on the same set of cells without damaging the cells, allowing later on their use for the investigation of other endpoints. Conclusions: We recommend the alamarBlue and CFDA-AM assays for cytotoxicity assessment in ZFL cells, which can be applied either singly or combined.JRC.H.5-Rural, water and ecosystem resource