High throughput microscopy of mechanism-based reporters in druginduced liver injury

  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.  Toxicolog

Dynamic imaging of adaptive stress response pathway activation for prediction of drug induced liver injury

Drug-induced liver injury remains a concern during drug treatment and development. There is an urgent need for improved mechanistic understanding and prediction of DILI liabilities using in vitro approaches. We have established and characterized a panel of liver cell models containing mechanism-based fluorescent protein toxicity pathway reporters to quantitatively assess the dynamics of cellular stress response pathway activation at the single cell level using automated live cell imaging. We have systematically evaluated the application of four key adaptive stress pathway reporters for the prediction of DILI liability: SRXN1-GFP (oxidative stress), CHOP-GFP (ER stress/UPR response), p21 (p53-mediated DNA damage-related response) and ICAM1 (NF-κB-mediated inflammatory signaling). 118 FDA-labeled drugs in five human exposure relevant concentrations were evaluated for reporter activation using live cell confocal imaging. Quantitative data analysis revealed activation of single or multiple reporters by most drugs in a concentration and time dependent manner. Hierarchical clustering of time course dynamics and refined single cell analysis allowed the allusion of key events in DILI liability. Concentration response modeling was performed to calculate benchmark concentrations (BMCs). Extracted temporal dynamic parameters and BMCs were used to assess the predictive power of sub-lethal adaptive stress pathway activation. Although cellular adaptive responses were activated by non-DILI and severe-DILI compounds alike, dynamic behavior and lower BMCs of pathway activation were sufficiently distinct between these compound classes. The high-level detailed temporal- and concentration-dependent evaluation of the dynamics of adaptive stress pathway activation adds to the overall understanding and prediction of drug-induced liver liabilities.Toxicolog

Dynamic modeling of Nrf2 pathway activation in liver cells after toxicant exposure

Cells are exposed to oxidative stress and reactive metabolites every day. The Nrf2 signaling pathway responds to oxidative stress by upregulation of antioxidants like glutathione (GSH) to compensate the stress insult and re-establish homeostasis. Although mechanisms describing the interaction between the key pathway constituents Nrf2, Keap1 and p62 are widely reviewed and discussed in literature, quantitative dynamic models bringing together these mechanisms with time-resolved data are limited. Here, we present an ordinary differential equation (ODE) based dynamic model to describe the dynamic response of Nrf2, Keap1, Srxn1 and GSH to oxidative stress caused by the soft-electrophile diethyl maleate (DEM). The time-resolved data obtained by single-cell confocal microscopy of green fluorescent protein (GFP) reporters and qPCR of the Nrf2 pathway components complemented with siRNA knock down experiments, is accurately described by the calibrated mathematical model. We show that the quantitative model can describe the activation of the Nrf2 pathway by compounds with a different mechanism of activation, including drugs which are known for their ability to cause drug induced liver-injury (DILI) i.e., diclofenac (DCF) and omeprazole (OMZ). Finally, we show that our model can reveal differences in the processes leading to altered activation dynamics amongst DILI inducing drugs.Toxicolog

A Dutch MYH7 founder mutation, p.(Asn1918Lys), is associated with early onset cardiomyopathy and congenital heart defects

Background Mutations in the myosin heavy chain 7 (MYH7) gene commonly cause cardiomyopathy but are less frequently associated with congenital heart defects. Methods In th

High throughput microscopy of mechanism-based reporters in druginduced liver injury

  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.  </div

Mapping the dynamics of Nrf2 antioxidant and NFκB inflammatory responses by soft electrophilic chemicals in human liver cells defines the transition from adaptive to adverse responses

A comprehensive understanding of the dynamic activation and crosstalk between different cellular stress response pathways that drive cell adversity is crucial in chemical safety assessment. Various chemicals have electrophilic properties that drive cell injury responses in particular oxidative stress signaling and inflammatory signaling. Here we used bacterial artificial chromosome-based GFP cellular stress reporters with live cell confocal imaging, to systematically monitor the differential modulation of the dynamics of stress pathway activation by six different soft electrophiles: sulforaphane, andrographolide, diethyl maleate, CDDO-Me, ethacrynic acid and tert-butyl hydroquinone. The various soft electrophiles showed differential potency and dynamics of Nrf2 activation and nuclear translocation. These differences in Nrf2 dynamics correlated with distinct activation pattern of Nrf2 downstream targets SRNX1 and HMOX1. All soft electrophiles caused a strong dose dependent suppression of a cytokine-induced NFĸB response represented by suppression of NFĸB nuclear oscillation and inhibition of the downstream target gene activation A20 and ICAM1, which followed the potency of Nrf2 modulation but occurred at higher concentration close to saturation of Nrf2 activation. RNAi-based depletion of RelA resulted in a prolonged presence of Nrf2 in the nucleus after soft electrophile treatment; depletion of Nrf2 caused the induction of NFĸB signaling and activation of its downstream targets A20 and ICAM1. A systematic transcriptome analysis confirmed these effects by soft electrophiles on Nrf2 and NFκB signaling crosstalk in human induced-pluripotent stem cell-derived hepatocyte-like cells. Altogether our data indicate that modulation of Nrf2 by soft electrophiles may have consequences for efficient inflammatory signaling.</p