J. Sidaway

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.

The metabonomic effects of hepatotoxic doses of pravastatin on the urinary metabolic profiles of female rats have been investigated using ultra performance liquid chromatography (UPLC)-oa-TOF-MS and, independently, by (1)H NMR spectroscopy. UPLC was performed using a 1 mm microbore column packed with 1.7 microm particles. Examination of the data obtained from the individual animals, aided by statistical interpretation of the data, made it possible to identify potential markers for toxicological effects, with both NMR and UPLC-MS analysis highlighting distinct changes in the urinary metabolite profiles. These markers, which included elevated taurine and creatine, as well as bile acids, were consistent with hepatotoxicity in some animals, and this hypothesis was supported by histopathological and clinical chemistry findings. The analytical data from both techniques could be used to define a metabolic "trajectory" as toxicity developed and to provide an explanation for the lack of hepatotoxicity for one of the animals. The two analytical approaches (UPLC-MS and NMR) were found to be complementary whilst the use of a 1mm i.d. x 100 mm column reduced the amount of sample required for analysis to 2 microL, compared with 10 microL for a 2.1mm i.d. x 100 mm column. The 1mm i.d. column also provided increased signal-to-noise without loss of chromatographic efficiency.