In previous studies at our laboratory it was demonstrated that drug exposure of HepG2 cells can lead to an altered TNFα-induced NF-κB oscillatory phenotype, concurrent with a synergistically... Show moreIn previous studies at our laboratory it was demonstrated that drug exposure of HepG2 cells can lead to an altered TNFα-induced NF-κB oscillatory phenotype, concurrent with a synergistically increased sensitivity for TNFα-induced apoptosis. We have also shown that synergistic drug/ TNFα-induced cell death is dependent on concurrent cellular stress responses. To monitor these stress responses, we have developed a fluorescent protein stress response reporter platform, feasible for high throughput approaches. In this thesis, we focus on an in vitro HepG2 cell model, in which the addition of TNFα reflects inflammatory stress. We studied inflammatory signaling, both for use in improved in vitro testing approaches and to gain mechanistic understanding of TNFα-induced signaling in hepatocytes. Show less
Drug Induced liver injury (DILI) is a major problem in the clinic and a big economic treat to the pharmaceutical industry. DILI is often missed in preclinical stages of drug development as... Show more Drug Induced liver injury (DILI) is a major problem in the clinic and a big economic treat to the pharmaceutical industry. DILI is often missed in preclinical stages of drug development as it occurs only in rare cases. Adaptive cellular stress responses are paramount in the healthy control of cell and tissue homeostasis and generally activated during toxicity in a chemical-specific manner. Only in susceptible individuals drug exposure can not only activate stress responses, but also switch on cell death signaling leading to liver injury. Therefore, monitoring adaptive stress response activation is key in prediction of DILI. In this thesis we established a platform containing a panel of distinct adaptive stress response reporter cell lines based on BAC-transgenomics GFP tagging in HepG2 cells. We validate and test these reporters in chapters 2 and 3. In chapter 4 we validate the system in a 3D environment where HepG2 cells show an enhanced liver like phenotype. In chapter 5 and 6 we show this reporter system can also be used for mechanistic research by unraveling crosstalk between DNA damage and Nrf2 signaling and by identifying novel regulators of Nrf2 signaling. Together, this thesis contributes to a more elaborate understanding of DILI. Show less
Bringing safe medicines to the market has remained a major challenge to the pharmaceutical industry. Recent years have seen increased drug attrition rates due to toxicity - even after rigorous... Show moreBringing safe medicines to the market has remained a major challenge to the pharmaceutical industry. Recent years have seen increased drug attrition rates due to toxicity - even after rigorous testing in both in vitro and in vivo test models. This is partly due to poor prediction of human-specific responses in these models. This thesis aims to address the issue by developing advanced in vitro models and methods that can complement and improve the predictive power of in vitro assays at preclinical level. Liver and kidneys are often susceptible to drug insult due to their respective roles in drug metabolism and reabsorption. We have developed a robust 3D in vitro model for liver toxicity studies, this model shows many hallmarks of in vivo hepatocytes, is applied in a 384-micro-well format and is compatible with standard medium- and high-throughput lab infrastructure for routine drug screening. This thesis also discusses the role of immune mediators in aggravating kidney toxicity and use of sophisticated high-content screening approach to measure apoptosis and necrosis in real time. These models are promising new tools for preclinical drug safety testing Show less
