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Mechanical analysis of liver-toxic substances in a hepatic co-culture model

Project

Food and consumer protection

This project contributes to the research aim 'Food and consumer protection'. Which funding institutions are active for this aim? What are the sub-aims? Take a look:
Food and consumer protection


Project code: BfR-CPS-08-1329-529
Contract period: 01.10.2014 - 31.03.2015
Purpose of research: Experimental development
Keywords: toxicology, chemical risk assessment, co-culture test

General major scientific and political goals are to improve the understanding of the mode of action and to judge the potential risk of hepatotoxic chemicals on a molecular level. In particular substances being identified to migrate from consumer-related products and thus could come in contact with consumers are of main interest. o-anisidine and ketoconazole were selected and their effects were intensively studied using proteomics, transcriptomics and additional biochemical assays. In this study we established and utilized a co-culture model composed of hepatocytes (HepG2) and monocyte-like cells (THP-1) in order to minimize animal experiments. The model system allows to simultaneously investigate the reaction of hepatocytes being stimulated by immune cells as well as to study the effects of degradation products being formed by hepatocytes on monocyte-like cells. In this project we quantified more than 1,000 Proteins in THP-1 as well as in HepG2 cells after treatment with 3 different concentrations of ketoconazole (typical hepatotoxic substance) and o-anisidine (example of migrating substance) in single as well as in the co-culture model. Additionally, ketoconazole and ketoconazole metabolites were quantified in HepG2 und THP-1 cells as well as the supernatants to draw conclusions on the exposition scenarios. Based on these data cellular response mechanism towards the chemicals as well as differences between single and co-culture were determined and validated using targeted biochemical assays.

Liver injury as a result of a sterile inflammation is closely linked to the activation of immune cells, including macrophages, by damaged hepatocytes. This interaction between immune cells and hepatocytes is as yet not considered in any of the in vitro test systems applied during the generation of new drugs. Here, we established and characterized a novel in vitro co-culture model with two human cell lines, HepG2 and differentiated THP-1. Ketoconazole, an antifungal drug known for its hepatotoxicity, was used as a model compound in the testing of the co-culture. Single cultures of HepG2 and THP-1 cells were studied as controls. Different metabolism patterns of ketoconazole were observed for the single and co-culture incubations as well as for the different cell types. The main metabolite N-deacetyl ketoconazole was found in cell pellets, but not in supernatants of cell cultures. Global proteome analysis showed that the NRF2-mediated stress response and the CXCL8 (IL-8) pathway were induced by ketoconazole treatment under co-culture conditions. The upregulation and ketoconazole-induced secretion of several pro-inflammatory cytokines, including CXCL8, TNF-α and CCL3, was observed in the co-culture system only, but not in single cell cultures. Taking together, we provide evidence that the co-culture model applied might be suitable to serve as tool for the prediction of chemical-induced sterile inflammation in liver tissue in vivo.

Arch Toxicol. 2017 Feb;91(2):799-810. doi: 10.1007/s00204-016-1686-y. Epub 2016 Mar 10.

 

 

 

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BMEL Frameworkprogramme 2008

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