Investigation of Troglitazone Toxicity using Transcriptomic Analysis in In Vivo Mouse and In Vitro Stem Cell Based Models

Troglitazone (TRO), an anti-diabetic drug, was withdrawn from the market due to incidences of idiosyncratic hepatotoxicity and to a lesser extent cardiotoxicity in a small sub-set of the patient population. Post-marketing drug withdrawal emphasises the requirement for better predictive toxicity assays for use during drug development. Stem cells can potentially be differentiated into any cell type and thus could be utilised in novel in vitro assays to predict toxicity during preclinical drug development. The hypothesis for this thesis was that transcriptomic changes in mouse embryonic stem (mES) cell derived cardiomyocytes and hepatocytes are signatures for cardiotoxic and hepatotoxic effects of troglitazone in the mouse. Standardised and reproducible protocols were developed for chemical induced differentiation of mES cells into cardiomyocytes and hepatocytes. The differentiated cells expressed key cardiac and hepatic marker genes, however, cell cultures were heterogeneous and contained a mixture of immature and terminally differentiated cells. Transcriptomic signatures of TRO toxicity in vivo were obtained using normal and high fat diet induced diabetes mouse models. These signatures were compared with those obtained from TRO treatment of mES cell derived cardiomyocytes and hepatocytes. In vitro transcriptomic signatures implicated an endoplasmic reticulum (ER) stress based mechanism for TRO induced toxicity in both hepatocytes and cardiomyocytes. In vivo transcriptomic data also indicated that TRO induced hepatic ER stress. In particular, the up-regulation of Trib3 was observed in vivo, in the livers of normal and diabetic mice and in vitro, in mES cell derived hepatocytes and cardiomyocytes. Increased expression of genes involved in cardiac remodelling due to TRO induced plasma volume increase were observed in vivo, however, in vitro, these genes were down-regulated. These data indicate that there is some correlation between in vitro and in vivo hepatic transcriptomic signatures of TRO toxicity but no correlation between the cardiac signatures.