Integrative cross-omics analysis in primary mouse hepatocytes unravels mechanisms of cyclosporin A-induced hepatotoxicity.

The liver is responsible for drug metabolism and drug-induced hepatotoxicity is the most frequent reason for drug withdrawal, indicating that better pre-clinical toxicity tests are needed. In order to bypass animal models for toxicity screening, we exposed primary mouse hepatocytes for exploring the prototypical hepatotoxicant cyclosporin A. To elucidate the mechanisms underlying cyclosporin A-induced hepatotoxicity, we analyzed expression levels of proteins, mRNAs, microRNAs and metabolites. Integrative analysis of transcriptomics and proteomics showed that protein disulfide isomerase family A, member 4 was up-regulated on both the protein level and mRNA level. This protein is involved in protein folding and secretion in the endoplasmic reticulum. Furthermore, the microRNA mmu-miR-182-5p which is predicted to interact with the mRNA of this protein, was also differentially expressed, further emphasizing endoplasmic reticulum stress as important event in drug-induced toxicity. To further investigate the interaction between the significantly expressed proteins, a network was created including genes and microRNAs known to interact with these proteins and this network was used to visualize the experimental data. In total 6 clusters could be distinguished which appeared to be involved in several toxicity related processes, including alteration of protein folding and secretion in the endoplasmic reticulum. Metabonomic analyses resulted in 5 differentially expressed metabolites, indicative of an altered glucose, lipid and cholesterol homeostasis which can be related to cholestasis. Single and integrative analyses of transcriptomics, proteomics and metabonomics reveal mechanisms underlying cyclosporin A-induced cholestasis demonstrating that endoplasmic reticulum stress and the unfolded protein response are important processes in drug-induced liver toxicity.