Quantitative phosphoproteomics reveal cellular responses from caffeine, coumarin and quercetin in treated HepG2 cells.

Protein phosphorylation is the most common type of post-translational modification where serine, threonine or tyrosine are reversibly bound to the phosphate group of ATP in a reaction catalyzed by protein kinases. Phosphorylation plays an important role in regulation of cell homeostasis, including but not limited to signal perception and transduction, gene expression and function of proteins. Protein phosphorylation happens on a fast time scale and represents an energy-efficient way for the cell to adapt to exposure to chemical stressors. To understand the cascade of cellular signaling induced by exposure to chemicals, we have exposed HepG2 cells to three chemicals with different modes of action, namely, caffeine, coumarin, and quercetin in a concentration and time response manner. Significantly upregulated and downregulated phosphosites were screened to analyze the activation/deactivation of signaling pathways by protein kinases. In total, 69, 44 and 12 signaling pathways were found enriched in caffeine, coumarin and quercetin treated cells, respectively, of which 9 pathways were co-enriched with 11 jointly responded kinases. Among identified co-responded kinases, CDK1, MAPK1 and MAPK3 play important roles in cell cycle and insulin signaling pathways. Quantitative phosphoproteomics can sensitively distinguish the effects of different chemicals on cells, allowing the assessment of chemical safety through changes in substrates and metabolic pathways at the cellular level, which is important for the development of non-animal approaches for chemical safety assessment.

Using transcriptomics, proteomics and phosphoproteomics as new approach methodology (NAM) to define biological responses for chemical safety assessment.

Omic-based technologies are of particular interest and importance for hazard identification and health risk characterization of chemicals. Their application in the new approach methodologies (NAMs) anchored on cellular toxicity pathways is based on the premise that any apical health endpoint change must be underpinned by some alterations at the omic levels. In the present study we examined the cellular responses to two chemicals, caffeine and coumarin, by generating and integrating multi-omic data from multi-dose and multi-time point transcriptomic, proteomic and phosphoproteomic experiments. We showed that the methodology presented here was able to capture the complete chain of events from the first chemical-induced changes at the phosphoproteome level, to changes in gene expression, and lastly to changes in protein abundance, each with vastly different points of departure (PODs). In HepG2 cells we found that the metabolism of lipids and general cellular stress response to be the dominant biological processes in response to caffeine and coumarin exposure, respectively. The phosphoproteomic changes were detected early in time, at very low doses and provided a fast, adaptive cellular response to chemical exposure with 7-37-fold lower points of departure comparing to the transcriptomics. Changes in protein abundance were found much less frequently than transcriptomic changes. While challenges remain, our study provides strong and novel evidence supporting the notion that these three omic technologies can be used in an integrated manner to facilitate a more complete understanding of pathway perturbations and POD determinations for risk assessment of chemical exposures.