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Toxicoproteomics applied to in vitro investigation of liver toxicity using HepaRG cells
S30
Abstracts / Toxicology Letters 238S (2015) S30–S31
Sponsored Symposium SpS: Predicting Long Term Toxic Effects Using Computer Models Based on Systems Characterization of Organotypic Cultures – The NOTOX Project SpS-1 Improved in vitro systems for prediction of hepatotoxicity
SpS-2 Toxicoproteomics applied to in vitro investigation of liver toxicity using HepaRG cells F. Bertile 1,∗ , G. Tascher 1 , D. Müller 2 , S. Klein 2 , L. Fredricksson 3 , I. Johansson 3 , V. Shevchenko 4 , C. Chesne 4 , M. Ingelmann-Sundberg 3 , E. Heinzle 2 , F. Noor 2 , A. Van Dorsselaer 1 1
CNRS-IPHC, Université de Strasbourg, Strasbourg, France Saarland University, Biochemical Engineering, Saarbruecken, Germany 3 Karolinska Institutet, Physiology & Pharmacology, Stockholm, Sweden 4 Biopredic International, St Gregoire, France 2
M. Ingelman-Sundberg 1,∗ , L. Fredriksson Puigvert 1 , S. Klein 2 , P. Peters 3 , S. Moro 1 , C. Bell 1 , D. Hendriks 1 , D. Müller 2 , V. Schweitzer 2 , F. Noor 2 , E. Heinzle 2 1
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden 2 Saarland University, Biochemical Engineering, Saarbruecken, Germany 3 Institute of Nanoscopy, University of Maastricht, Maastricht, Netherlands Liver toxicity is a major societal burden and hepatotoxicity is difficult to foresee due to species differences and the difficulties to keep hepatocyte cultures functionally appropriate because of rapid de-differentiation of the cells. In order to mimic true in vivo human hepatotoxicity it is also evident that diseased models would be of importance for the in vitro models to better comply with different situations in vivo in man. Within the NOTOX project we have further developed and applied 3D models based on HepaRG and human primary hepatocytes to improve predictability of primary human hepatocyte (PHH) based in vitro systems for measuring long-term toxicity. Within the project we monitor the changes induced by different compounds at the transcriptomic, epigenomic, proteomic and metabonomic levels. We have characterized the spheroids morphologically and functionally and initially found that 3D HepaRG spheroids can be kept active for at least 30 days even without addition of serum. In many cases they can better predict hepatotoxicity than the corresponding 2D models. We have also found that the PHH spheroids are of good functionality with respect to urea and albumin production and expression of CYP enzymes for at least 35 days and are suitable for determining chronic drug toxicity during at least 28 days. In addition we are developing modified diseased spheroid models mimicking inflammation, viral infection, cholestasis and steatosis. In particular the HepaRG and PHH derived cholestatic models have been found to be effective and to be able to discriminate between cholestatic and non-cholestatic toxicity of drugs in the presence of bile acids. In the current presentation recent data from the hepatocyte models with respect to effects of drugs on cytotoxicity, transcriptional and metabonomic pathways under acute and chronic conditions will be presented. http://dx.doi.org/10.1016/j.toxlet.2015.08.081
The NOTOX project (www.notox-sb.eu/) develops systems biology approaches for in vitro toxicity studies using the human hepatic cell line HepaRG. Because the toxicoproteomics field has been boosted by recent technological advances, we notably took advantage of the wide diversity of proteomic platforms (GeLC–MS/MS, 2DE-MS) to show that HepaRG cells exhibit a phenotype very similar to that of functional liver cells (approx. 3500 proteins identified). We also showed good batch to batch reproducibility of the production/differentiation process of the cell line, by comparing different HepaRG-batches using 2D-DIGE and label-free quantitative proteomics. Because secreted proteins are a valuable source for biomarkers, we also examined the secretome of HepaRG cells using shotgun proteomics (approx. 200 proteins identified). We found that acute acetaminophen (APAP) exposure alters cell’s viability and proteome. A targeted proteomics strategy (LC-SRM) revealed that APAP appears predominately detoxified by glucouronidation rather than sulfation. Using a spectral-count-based proteomics strategy, abundance of 189 proteins was altered either due to culture conditions or APAP treatment, with energy metabolism being especially affected. We also evaluated the impact of chronic valproic acid (VPA) exposure both on monolayer and 3D-spheroid cultures of HepaRG cells. Using a label-free proteomics approach (XICs), 250 were found differentially expressed as a function of time and/or VPA dose. Pathway analysis indicated alteration, among others, of the lipid metabolic process. The effects on the proteome were visible already when commonly applied endpoint assays like e.g. cell viability or ATP content did not yet indicate toxicity. In a systems biology perspective, proteomics results were integrated with the transcriptomics and metabolomics data obtained by the NOTOX consortium. Simultaneous visualization of different omics data using home-developed bioinformatics tools was helpful to better understand these data. In summary, HepaRG cells represent a good model system for in vitro toxicity studies, provided that culture conditions are sufficiently optimized. Applied to HepaRG cells, toxicoproteomics is powerful enough to produce new data of importance for gaining insights into adverse outcome pathways. http://dx.doi.org/10.1016/j.toxlet.2015.08.082
Abstracts / Toxicology Letters 238S (2015) S30–S31
Sponsored Symposium SpS: Predicting Long Term Toxic Effects Using Computer Models Based on Systems Characterization of Organotypic Cultures – The NOTOX Project SpS-1 Improved in vitro systems for prediction of hepatotoxicity
SpS-2 Toxicoproteomics applied to in vitro investigation of liver toxicity using HepaRG cells F. Bertile 1,∗ , G. Tascher 1 , D. Müller 2 , S. Klein 2 , L. Fredricksson 3 , I. Johansson 3 , V. Shevchenko 4 , C. Chesne 4 , M. Ingelmann-Sundberg 3 , E. Heinzle 2 , F. Noor 2 , A. Van Dorsselaer 1 1
CNRS-IPHC, Université de Strasbourg, Strasbourg, France Saarland University, Biochemical Engineering, Saarbruecken, Germany 3 Karolinska Institutet, Physiology & Pharmacology, Stockholm, Sweden 4 Biopredic International, St Gregoire, France 2
M. Ingelman-Sundberg 1,∗ , L. Fredriksson Puigvert 1 , S. Klein 2 , P. Peters 3 , S. Moro 1 , C. Bell 1 , D. Hendriks 1 , D. Müller 2 , V. Schweitzer 2 , F. Noor 2 , E. Heinzle 2 1
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden 2 Saarland University, Biochemical Engineering, Saarbruecken, Germany 3 Institute of Nanoscopy, University of Maastricht, Maastricht, Netherlands Liver toxicity is a major societal burden and hepatotoxicity is difficult to foresee due to species differences and the difficulties to keep hepatocyte cultures functionally appropriate because of rapid de-differentiation of the cells. In order to mimic true in vivo human hepatotoxicity it is also evident that diseased models would be of importance for the in vitro models to better comply with different situations in vivo in man. Within the NOTOX project we have further developed and applied 3D models based on HepaRG and human primary hepatocytes to improve predictability of primary human hepatocyte (PHH) based in vitro systems for measuring long-term toxicity. Within the project we monitor the changes induced by different compounds at the transcriptomic, epigenomic, proteomic and metabonomic levels. We have characterized the spheroids morphologically and functionally and initially found that 3D HepaRG spheroids can be kept active for at least 30 days even without addition of serum. In many cases they can better predict hepatotoxicity than the corresponding 2D models. We have also found that the PHH spheroids are of good functionality with respect to urea and albumin production and expression of CYP enzymes for at least 35 days and are suitable for determining chronic drug toxicity during at least 28 days. In addition we are developing modified diseased spheroid models mimicking inflammation, viral infection, cholestasis and steatosis. In particular the HepaRG and PHH derived cholestatic models have been found to be effective and to be able to discriminate between cholestatic and non-cholestatic toxicity of drugs in the presence of bile acids. In the current presentation recent data from the hepatocyte models with respect to effects of drugs on cytotoxicity, transcriptional and metabonomic pathways under acute and chronic conditions will be presented. http://dx.doi.org/10.1016/j.toxlet.2015.08.081
The NOTOX project (www.notox-sb.eu/) develops systems biology approaches for in vitro toxicity studies using the human hepatic cell line HepaRG. Because the toxicoproteomics field has been boosted by recent technological advances, we notably took advantage of the wide diversity of proteomic platforms (GeLC–MS/MS, 2DE-MS) to show that HepaRG cells exhibit a phenotype very similar to that of functional liver cells (approx. 3500 proteins identified). We also showed good batch to batch reproducibility of the production/differentiation process of the cell line, by comparing different HepaRG-batches using 2D-DIGE and label-free quantitative proteomics. Because secreted proteins are a valuable source for biomarkers, we also examined the secretome of HepaRG cells using shotgun proteomics (approx. 200 proteins identified). We found that acute acetaminophen (APAP) exposure alters cell’s viability and proteome. A targeted proteomics strategy (LC-SRM) revealed that APAP appears predominately detoxified by glucouronidation rather than sulfation. Using a spectral-count-based proteomics strategy, abundance of 189 proteins was altered either due to culture conditions or APAP treatment, with energy metabolism being especially affected. We also evaluated the impact of chronic valproic acid (VPA) exposure both on monolayer and 3D-spheroid cultures of HepaRG cells. Using a label-free proteomics approach (XICs), 250 were found differentially expressed as a function of time and/or VPA dose. Pathway analysis indicated alteration, among others, of the lipid metabolic process. The effects on the proteome were visible already when commonly applied endpoint assays like e.g. cell viability or ATP content did not yet indicate toxicity. In a systems biology perspective, proteomics results were integrated with the transcriptomics and metabolomics data obtained by the NOTOX consortium. Simultaneous visualization of different omics data using home-developed bioinformatics tools was helpful to better understand these data. In summary, HepaRG cells represent a good model system for in vitro toxicity studies, provided that culture conditions are sufficiently optimized. Applied to HepaRG cells, toxicoproteomics is powerful enough to produce new data of importance for gaining insights into adverse outcome pathways. http://dx.doi.org/10.1016/j.toxlet.2015.08.082