Screening for drug-induced hepatotoxicity in primary mouse hepatocytes using acetaminophen, amiodarone, and cyclosporin a as model compounds: an omics-guided approach

Abstract

Drug-induced hepatotoxicity is a leading cause of attrition for candidate pharmaceuticals in development. New preclinical screening methods are crucial to predict drug toxicity prior to human studies. Of all in vitro hepatotoxicity models, primary human hepatocytes are considered as 'the gold standard.' However, their use is hindered by limited availability and inter-individual variation. These barriers may be overcome by using primary mouse hepatocytes. We used differential in gel electrophoresis (DIGE) to study large-scale protein expression of primary mouse hepatocytes. These hepatocytes were exposed to three well-defined hepatotoxicants: acetaminophen, amiodarone, and cyclosporin A. Each hepatotoxicant induces a different hepatotoxic phenotype. Based on the DIGE results, the mRNA expression levels of deregulated proteins from cyclosporin A-treated cells were also analyzed. We were able to distinguish cyclosporin A from controls, as well as acetaminophen and amiodarone-treated samples. Cyclosporin A induced endoplasmic reticulum (ER) stress and altered the ER-Golgi transport. Moreover, liver carboxylesterase and bile salt sulfotransferase were differentially expressed. These proteins were associated with a protective adaptive response against cyclosporin A-induced cholestasis. The results of this study are comparable with effects in HepG2 cells. Therefore, we suggest both models can be used to analyze the cholestatic properties of cyclosporin A. Furthermore, this study showed a conserved response between primary mouse hepatocytes and HepG2 cells. These findings collectively lend support for use of omics strategies in preclinical toxicology, and might inform future efforts to better link preclinical and clinical research in rational drug development.

FIG. 1.

Hierarchical cluster analysis of the experimental groups (control, acetaminophen, amiodarone, and cyclosporin A). The clustering is based on the log standard abundance of the significant differential spots (p≤0.05) with a hierarchical clustering algorithm in the EDA module of the Decyder software.

FIG. 2.

PCA analysis of the experimental groups (control, acetaminophen, amiodarone, and cyclosporin A), based on the significant differential spots (One-Way Anova p≤0.05).

FIG. 3.

Proteome map of the differentially expressed proteins (One-Way Anova p≤0.05). All the identified spots are indicated with a number which corresponds to the numbers used in Table 1.

FIG. 4.

Classification of the differential expressed proteins in HepG2 and primary mouse hepatocytes after exposure to cyclosporin A with the Panther classification system (http://www.pantherdb.org).

FIG. 5.

Expression differences by western blotting of (A) perilipin2/adipophilin and (B) bile salt sulfotransferase from primary mouse hepatocytes treated with acetaminophen, amiodarone, or cyclosporin A.

FIG. 6.

Venn diagram of the significant differentially expressed proteins in HepG2 and primary mouse hepatocytes. Overlaps contain the number of significant differentially expressed proteins in both systems induced by the used hepatotoxicants.