Toxicoproteomics applied to in vitro investigation of liver toxicity using HepaRG cells

The NOTOX project 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 glucoronidation 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.