Lehre

Background
Chemical pollution in aquatic ecosystems is recognized as a major environmental threat, resulting in significant alterations to aquatic biodiversity. However, organisms are equipped with different mechanisms of defense that allow species to cope with chemical exposure. Among these mechanisms, biotransformation processes are of particular importance due to their role in detoxification and in reducing the bioaccumulation potential of chemicals. Nonetheless, previous studies in fish suggest that biotransformation ability differs significantly among taxonomic groups, leading to differential sensitivity to pollution across species.
Recently, the moderlieschen (Leucaspius delineatus) has been proposed as a potential model species in ecotoxicology, as it corresponds to an endemic fish species inhabiting temperate ecosystems in Europe with significant ecological value. However, almost no information exists regarding the performance and sensitivity of this fish species when challenged with chemical exposure. Therefore, this project aims to gain insight into the biotransformation ability of the moderlieschen and advance its applicability in assessing responses to chemical exposure in fish.
Aim
Determine the biotransformation ability of the moderlieschen (Leucaspius delineatus) towards micropollutants (e.g. pharmaceuticals and pesticides) using well-established in vitro methodologies.
Methods
The proposed project is established at the interface of toxicology, chemistry, cell biology, and ecology, and aims to implement well-established in vitro systems for the evaluation of biotransformation ability.
The activity of different biotransformation pathways will be evaluated by determining chemical clearance rates of select micropollutants, including pharmaceuticals and pesticides. Chemical clearance will be estimated via substrate depletion experiments using S9 sub-cellular (enzymatic) fractions isolated from fresh tissue as well as from cell lines established from the moderlieschen. Chemical analyses will be then conducted using state-of-the-art instrumentation, including high performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry (HRMS).
The methodologies in the proposed work will involve (i) isolation of enzymatic fractions, (ii) routine cell culture techniques, (iii) protein quantification, (iv) spectrophotometry, and (v) instrumental analysis (e.g. HPLC-HRMS).
Suitable candidates for this project are expected to hold a bachelor (BSc) degree in chemistry, biology, biochemistry, environmental science, or a related discipline. Moreover, candidates should have a strong interest for advancing animal alternatives in (eco)toxicology.
If you wish to apply, please send your most recent CV to Dr. Marco E. Franco (marco.franco@cluttereawag.ch). This work will be performed at the Department of Environmental Toxicology at Eawag in Dübendorf, Switzerland.

Background
There are numerous chemicals in circulation that can contribute to variable adverse outcomes in different species, and assessing all these possible combinations using traditional toxicity testing methods is an impossible task. Therefore, computational methods are needed to determine the potential for chemicals to lead to a variety of adverse effects in different species. Adverse outcome pathways (AOPs) describe a sequence of events from the molecular to cellular, to organism response level resulting from exposure to a stressor. AOPs provide a framework for knowledge organization of toxicity mechanisms and adverse outcomes across organisms, but their specificity can limit the number of different stressors and species that can be mapped to existing AOPs. By building information from external databases into the AOP framework, new stressors and species can be mapped onto existing AOPs. This will help contribute to high throughput assessment of potential stressor – toxicity mechanism – adverse outcome relationships across species.

Aim
We would like to expand the application of AOPs by building external data sources into existing AOPs to enable prediction of stressor mechanisms contributing to adverse outcomes in different species.

Methods
Data will be curated from a variety of public databases (e.g., the AOP Database) and sources will be integrated together using common ontologies. As a preliminary case study, pesticides will be the chemical class under study, neurotoxicity will be the adverse outcome, and zebrafish will be the test species. From the set of developed AOPs with expanded data points, stressor predictions will be made following a weight of evidence approach.

The techniques applied include data curation, harmonization, and integration, and subsequent data analysis and visualization.

Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline and should have experience coding (e.g., in R or Python).

For further information please contact Marissa Kosnik.
This work will be performed at Eawag at the Department of Environmental Toxicology in Dübendorf.

Background

The aquatic environment is significantly impacted by a constant introduction of chemical contaminants. Upon exposure, aquatic animals, like fish, display different mechanisms of defense, collectively known as the chemical defensome. Among these mechanisms, biotransformation pathways are key components of an organism’s ability to cope with pollution. For example, biotransformation directly influences the bioaccumulation of chemicals. To date, the evaluation of biotransformation pathways and the assessment of bioaccumulation relies extensively on the use of live animals (e.g. OECD TG305 and TG319), despite the availability of novel animal alternatives, specifically fish cell lines. It is important, therefore, to explore the biotransformation ability of established fish cell lines to advance their application in the assessment of bioaccumulation and overall responses to chemical exposure, while continuing to support the reduction, refinement, and potential replacement of whole-animal models.

Aim

Characterize the kinetics and magnitude of activity of different phase I and II biotransformation enzymes in established fish cell lines, and increase their applicability in the evaluation of bioaccumulation potential.

Methods

Fish cell lines represent a robust in vitro model for the evaluation of chemical exposure. Therefore, well-established cell lines such as the rainbow trout (Oncorhynchus mykiss) RTL-W1 (liver), RTgill-W1 (gill), RTgutGC (gut) and RTbrain (brain) will be assessed for their ability to display different phase I and II biotransformation enzymes. Additional cell lines from other organs and fish species will be considered.

The characterization of the kinetics and overall activity of biotransformation enzymes will follow standardized colorimetric and fluorescence-based bioassays to explore real-time biotransformation activity in a short time.

The methodologies in the proposed work will involve (i) routine cell culture techniques, (i) protein quantification, (iii) spectrophotometry, and (iv) instrumental analysis (e.g. HPLC-FLD, LC-MS).

Suitable candidates for this project are expected to hold a BSc degree in biology, biochemistry, environmental science, or a related discipline. Moreover, candidates should have a strong interest for advancing animal alternatives in toxicology.

For further information, please contact Dr. Marco E. Franco
This work will be performed at the Department of Environmental Toxicology at Eawag in Dübendorf, Switzerland.

Background
In agricultural areas, insecticides comprise a considerable part of the chemical mixture present in the aquatic environment: they reach water bodies via run-off or leaching after spraying events. While designed to be neurotoxic to insects, insecticides can have adverse effects on a multitude of organisms due to the high conservation of the nervous system among phyla. To assess the risk of insecticides for the environment and estimate their long-term ecological effects, it is important to investigate their impact on non-target organisms such as fish. Toxic effects elicited by insecticides can occur at different organizational levels and may range from easily observable lethal to very subtle behavioral effects. The molecular pathways behind these effects, however, are still largely unknown, but are important to understand in order to develop predictive models, biomarkers and in vitro tests.

Aim
We would like to gain a better understanding of neurotoxic effects in fish by linking observed effects on organism-level to underlying molecular events.

Methods
Due to its exceptional suitability for molecular and developmental studies, the zebrafish, Danio rerio, will be used as a test organism. Based on previous experiments, in which the effects of insecticides on the behavior and neuromuscular structure of zebrafish larvae were investigated, gene expression analysis will be conducted focusing on genes involved in development and functioning of the nervous system and muscles.

The techniques applied include (i) breeding and handling of zebrafish, (ii) design of primers, (iii) RNA isolation, (iv) qPCR, and eventually (v) CRISPR-Cas9 gene editing (if time allows).

Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline and should have a genuine interest in neuroscience and molecular biology.

For further information please contact Colette vom Berg and Sarah Könemann.
This work will be performed at Eawag at the Department of Environmental Toxicology in Dübendorf.

Background:

Tire and Road Wear Particles (TRWP), which are generated by the erosion of tires while driving, have recently been estimated as representing up to 28.3% of all primary microplastics released in the aquatic environment, for an estimated mass of 424 Ktons/year. This finding raises questions regarding the toxicological impact of these TRWP and associated chemicals on aquatic organisms.

Aim:

This study aims at determining the toxicity mechanisms induced by TRWP and associated chemicals to fish using different rainbow trout cell lines (Oncorhynchus mykiss).

Methods:

We will use several rainbow trout cells lines (e.g., from gill, gut and brain), which will be subject to different exposure scenarios.

You will gain experience with routine cell culture techniques (how to work in sterile conditions and culture cells). You will moreover learn how to set up and perform different assays: cell viability assays to determine the cytotoxicity of chemicals (seeding cells using different substrates, exposure to chemicals and multi-endpoint viability assay), bioavailability assays to quantify how much of a chemical has entered the cells (mass spectrometry) and gene expression analysis (RT-PCR) to identify molecular mechanisms induced. Finally, you will learn how to produce, interpret and present scientific data.

If you are interested in this particular research topic and are excited to know more about environmental toxicology research in general, please contact Anna Toso or Kristin Schirmer.

This research will be performed at the department of Environmental Toxicology, Eawag, in Dübendorf.

Background

In mammals, mechanistic target of rapamycin (mTOR) pathway is known to be a central cellular hub that mediates regulation of cell growth and proliferation in response to a host of endogenous and exogenous stimuli. The mTOR is a serine-threonine kinase that, upon activation, initiates a cascade of phosphorylation events for its downstream targets, which are further involved in regulating transcription, translation, autophagy, metabolism, and cytoskeletal organization, among many other essential cellular processes. Interestingly, exposure to chemicals can influence many of the upstream signals sensed by the mTOR, such as energy levels, oxidative stress, proteotoxic stress, genotoxicity, or inflammation. The role of the mTOR in regulating cellular responses to stress has indeed been recognized, but so far rarely considered with regard to chemical toxicity, in fish even less so than in mammals. Therefore, the research in my group seeks to understand the role of mTOR in regulating fish growth in response to chemical exposures and other environmental factors such as temperature. The project described here will characterize the baseline relationships between the mTOR activity levels and growth responses in a fish cell line.

Aims

This master thesis will investigate the correlation between the mTOR pathway activity and growth in cultured fish cells. For this, fish cells will be exposed to specific pharmacological inhibitors or activators of the mTOR pathway and its activity will be monitored by measuring changes in phosphorylation status of mTOR substrate proteins. In parallel, growth and several other endpoints will be assessed in the exposed cells to understand their correlation with the observed phosphorylation-based signaling dynamics.

Methods

Experimental work on the proposed master thesis will provide training in basic cell culture techniques, chemical exposure, fish cell growth assay, and microscopy/imaging. The central analytical task will include performing targeted phosphoproteomic analysis of selected proteins using mass spectrometry. Our phosphoproteomic assays constitute a novel tool to study phosphorylation-based signaling in fish, where this mechanistic aspect has been so far largely neglected due to challenges with obtaining species-specific antibodies for (de)phosphorylated proteins of interest. The workflow consists of protein extraction from cultured cells, protein digestion, phosphopeptide enrichment, and measurement on our state of the art LC-MS/MS instrumentation.

If you are interested in this project please contact: Ksenia Groh.

PROJECT SUMMARY

Permanent fish cell lines of rainbow trout (Oncorhynchus mykiss) have great potential as alternatives to conventional fish tests in chemical safety testing. While several strategies and assay procedures are being developed that use fully defined media for chemical exposure, the routine culture of these cells, and certain chemical exposure assays, still require fetal calf serum (FBS) for cell maintenance and proliferation.

AIMS

On this background, we aim to develop a fully transparent serum-free cell culture medium for cell lines from rainbow trout. Cultures of a rainbow trout gill cell line (RTgill-W1) are known to be anchorage dependent cells, meaning attachment to solid surface is prerequisite for successful cell proliferation. Optimal cell attachment of RTgill-W1 is dominantly achieved by culturing cells in FBS. Whereas omission of serum leads to poor cell attachment and stopped cell proliferation. While desirable combination of externally supplemented growth factors may contribute to overcoming cell proliferation limitation under serum-free conditions, appropriate attachment of cells on substrate still needs to happen prior to cell grow. Thus, identification of supplements or coating materials that promote appropriate cell attachment are still major events that needs to be unravelled on the road to chemically defined serum-free media formulation.

APPROACH/EXPERIMENTAL OUTLINE

For this Master Project, we propose to identify cell attachment factors and develop a cell attachment assay for the RTgill-W1 cell line using the 96-well plate format. A high-throughput screening approach that quantifies impact of selected attachment supplements, alone and in combination, on fish cell attachment over seven days will be used to identify the optimal composition of the serum-free media. Big emphases will be put on time-dependent studying of cell attachment and cell lose during 7 days period of cell cultivation of either serum-free or FBS-containing media.

Throughout this master thesis, the candidate will first learn how to routinely culture RTgill-W1 cell lines, gain experience in cell viability assay and/or cell-growth assay. Subsequently the candidate will have the opportunity to apply various protocols for cell staining and have some training in basic techniques, such as fluorescent microscopy and Cell Imagining Multi-Mode Reader imaging and high-content/through-put screening. Experimental design, data presentation and analyses are an integral part of this project as well.

Interested? If you are excited about this line of research please contact Kristin Schirmer (Kristin.Schirmer@eawag.ch) or Barbara Jozef (Barbara.Jozef@eawag.ch). The work will be performed at Eawag in the department of Environmental Toxicology in Dübendorf.

Kurstitel	Kursnummer	Personen
Ecotoxicology (ETHZ)	701-1312-00L	Kristin Schirmer, Elisabeth Janssen
Ecotoxicology Laboratory (ETHZ)	701-1330-00P	Kristin Schirmer, Colette vom Berg, Ksenia Groh
Introduction to Toxicology (ETHZ)	752-1300-00L	Kristin Schirmer, Colette vom Berg (zusammen mit Shana Sturla)
Sustainability and Water Resources (ETHZ)	118-0111-00L	Darcy Molnar, Paolo Burlando, Kristin Schirmer,

Abteilung Umwelttoxikologie

Aktuelle Master Projekte

Micropollutant biotransformation in the moderlieschen (Leucaspius delineatus), an endemic species of temperate ecosystems in Europe

Expansion of AOP linkages to predict diverse stressor effects

Characterization of phase I and II biotransformation enzymes in fish cell lines

Identification of molecular pathways mediating the toxicity of insecticides in fish

Mechanistic insights into the toxicity of tire particles applying fish cell lines

Influence of mTOR pathway modulation on growth in fish cells

Identification of attachment factors for the development of serum-free medium of RTgill-W1 cell line

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