Cyclosporine A treated in vitro models induce cholestasis response through comparison of phenotype-directed gene expression analysis of in vivo Cyclosporine A-induced cholestasis

• Cholestasis is modeled in mice using cyclosporin A.
• Progression of serum phenotype is correlated to liver gene expression changes.
• Phenotype-directed analysis retrieves in vivo cholestasis-related gene sets.
• In vitro model evaluation for cholestasis can be performed at the gene set level.
• Cholestasis-related genes should be expected to differ between in vivo and in vitro.

In vitro models for hepatotoxicity testing are a necessity for advancement of toxicological research. Assessing the in vitro response requires in vivo validated gene sets reflective of the hepatotoxic phenotype. Cholestasis, the impairment of bile flow, is induced in C57BL/6J mice treated with cyclosporine A (CsA) to identify phenotype reflective gene sets. CsA treatment through oral gavage for 25 days induced cholestasis, as confirmed by histopathology and serum chemistry. Over 1, 4, and 11 days of CsA exposure gradual increases in serum markers were correlated to gene expression. This phenotype-directed analysis identified gene sets specific to the onset and progression of cholestasis, such as PPAR related processes and drug metabolism, by circumventing other effects of CsA, such as immunosuppression, found in dose*time group analysis. In vivo gene sets are enriched in publicly available data sets of CsA-treated HepaRG and primary mouse hepatocytes. However, genes identified within these gene sets did not overlap between in vivo and in vitro. In vitro regulated genes represent the initial response to cholestasis, whereas in vivo genes represent the later adaptive response. We conclude that the applicability of in vitro models for hepatotoxicity testing fully depends on a solid in vivo phenotype anchored analysis.