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Data Protection Declaration
Imprint
Development of methods for detection of obesogenic substances
Project
Project code: BfR-ZEBET-08-1322-707
Contract period: 01.12.2017
- 31.12.2018
Purpose of research: Experimental development
Endocrine disruptors (ED) may alter hormonally regulated processes and consequently cause adverse health effects in humans (WHO/IPCS 2002). Substances with ED properties are therefore strictly regulated considering precautionary health based consumer protection. However, to adequatly identfy EDs it is necessary to develop new in vitro test systems that are able to analyse the endocrine mechanism (e.g. activation of hormone receptors). In this context several methods for detection of substances with estrogenic or androgenic properties or acting on biosynthesis of steroid hormones or influencing the thyroid have been developed in the past decade. However, a lack of methods for detection of ED influencing other parts of the endocrine system (non-EATS) has recently been recognized (OECD DRP; EC Meeting Report 2017). This is especially problematic because among the most frequent endocrine mediated disease are obesity, metabolic syndrome and diabetes mellitus the most prominent. These conditions are certainly caused by multiple factors (lack of excersise, high caloric intake). However an influence of specific EDC, so called obesogens, has recently been discussed and substantiated (Grün and Bloomberg 2006). A challenge in the context of developing methods for detection of obesogenic compounds is the complexity of the hormonal regulation of energy and lipid homeostasis. There are not only multiple organs (stomach, gut, pancreas, liver, fatty tissue, hypothalamus etc.) and signalling molecules (ghrelin, leptin, insulin, glucagon, adiponektin, IL6 etc) involved but also several receptors and signalling pathways(LXR, FXR, PXR, PPARs, FOXO1 u.a.). For a recent overview see Lempradl et al 2015. Consequently it is not possible to establish a simple mechanistic assay to detect obesogenes but to develop a battery of methods covering multiple mechanisms and endpoints. The central organ in energy and fatty acid metabolism is the liver. It is well known, that obesity and metabolic syndrome are associated with hepatic steatosis. Previous work has shown, that some substances causing steatosis in vivo by interference with certain receptors (PXR) and related gene expression changes (FAT/CD36, FASN) (Heise et al 2015 and 2017) do also cause a related effect (formation of lipid vacuoles, gene expression changes) in apropriate liver cell models (Zahn, Knebel et al., in preparation). Additionaly several molecular initiating events on the level of receptor activation and key events on the gene expression level of the adverse outcome pathway (AOP) of steatosis could be observed in vivo and in vitro (Heise et al 2015). Within this project, work on HepaRG and HepG2 cells by use of High-Content-Screening- (HCS) and High-Throughput-Screening- (HTS) methods should be conducted. Additionally extra-hepatic cell-lines from other tissues involved in regulation of fatty acid metabolism and energy homeostasis (like e.g. 3T3-L1 Zellen, differentiating to adipocytes) should be used and tested whether they are responsive to known and potential obesogenes. These systems could be an important part of a testbattery based on an AOP for steatosis and obesity.
The murine cell line 3T3 L1 has been established that allows a direkt analysis of adipocyte differentiation. A protocoll to differentiate these cells in adipocytes has been optimized as well as a protocoll to detect fatty acid accumulation in the cells based on the AdipoRed Assay. This method was further develop to allow the quantification of fatty acid accumulation with fluorecence microscopie. Thus, the established methology allows screening for substances that affect adipocyte differentiation and / or fatty acid synthesis.
Section overview
Subjects
- Biotechnology
- Toxicology
Framework programme
Funding programme
Excutive institution
BfR - Centre for Documentation and Evaluation of Alternatives to Animal Experiments (BfR - ZEBET)
