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Development and validation of molecular toxicological test methods for chemical safety: design and establishment of new in vitro and non-vertebrate testassays
Project
Project code: BfR-TOX-08-9-003
Contract period: 01.01.2015
- 31.12.2019
Purpose of research: Experimental development
Increasing concerns regarding potential adverse health effects caused by hormonally active chemicals, biocides or plant protection products (endocrine disruptors, ED) has led to particularly strict regulations for ED in Europe. For this reason, there is currently a great need for predictive test methods for ED identification and assessment. One focus of the experimental work in the Unit 94 is therefore the development of animal-free test methods according to the 3R principle with a special focus on ED. Our experimental work is based on two main pillars: high-resolution fluorescence microscopy (BioImaging Unit) and robotics-assisted high-content / high-throughput (HC / HT) screening (Screening Unit). With the help of the BioImaging Unit, we first seek to identify potential molecular modes-of-action of ED on a cellular level and define cell biologically relevant endpoints for the development of animal-free test methods. With the help of the Screening Unit, we then optimize the developed test methods with regard to their capability for high throughput screening and automation, and finally validate their reliability and relevance in relation to existing animal-based test methods. Currently, two test methods, the MCF-7 Assay and the C. elegans Assay, are in transition from development to automation phase. The MCF-7 Assay is based on quantitative changes in the cell morphology of a human breast cancer cell line, which are associated with the activity of the estrogen receptor signaling pathway. Our current findings indicate that this correlation can also be observed in tissue sections of patients with diagnosed metastatic breast cancer. For this reason, we use the estrogen-dependent modulation of cell morphology in MCF-7 cell culture as a cell biologically relevant endpoint for the identification and characterization of ED that influence the estrogen receptor signaling pathway and thus possibly influence breast cancer progression. The C. elegans Assay is based on the interference of ED with growth and reproduction of the C. elegans invertebrate model organism. Our data on a group of pesticides, the azole fungicides, indicate a good comparability of the assay with experiments performed in male rats as well as in cell-based test systems. The C. elegans Assay combines the benefits of current in vivo and in vitro test systems and further enables the efficient identification and characterization of combination effects of ED using cell biologically relevant endpoints. In the fiscal period 2018, we aim to further optimize both assays and to automate standardized procedures with the help of the Screening Unit to facilitate robotics-assisted HC / HT screening. The automation phase includes the establishment of staining protocols as well as the generation of efficient image analysis pipelines using integrated software, and automated data processing by open source analysis platforms such as KNIME. At the end of the 2018 fiscal period, we will probably also be able to validate the reliability and relevance of these assays using a chemical library of currently 444 potentially endocrine-disrupting environmental chemicals. The findings on the effects of these substances on cellular model systems up to an entire organism, such as C.elegans, will then in turn inform our analyses of underlying molecular modes-of-action of ED. A key aspect of the fiscal period 2018 and beyond will be the further development of the required IT infrastructure as well as the identification and establishment of appropriate means for the efficient and traceable organization of the data generated by the BioImaging and Screening Units. In addition, the anticipated completion of the equipment room and the installation of the microscopes in the previous fiscal period has not yet been fully achieved, so that familiarization with the functionality of the new equipment and software and integration of current research projects is also a focus of this fiscal period. Our primary focus on imaging technologies to develop non-animal test methods is further complemented by a series of molecular biology methods at the genome, transcriptome, and proteome levels to identify relevant molecular modes-of-action.
Section overview
Subjects
- Biotechnology
- Toxicology
Framework programme
Funding programme
Excutive institution
BfR - Department 9: Experimental Toxicology and ZEBET (BfR - TOX)
