Oxidative stress is the consequence of an abnormal increase of reactive oxygen species (ROS). ROS are generated mainly during the metabolism in both normal and pathological conditions as well as... Show moreOxidative stress is the consequence of an abnormal increase of reactive oxygen species (ROS). ROS are generated mainly during the metabolism in both normal and pathological conditions as well as from exposure to xenobiotics. Xenobiotics can, on the one hand, disrupt molecular machinery involved in redox processes and, on the other hand, reduce the effectiveness of the antioxidant activity. Such dysregulation may lead to oxidative damage when combined with oxidative stress overpassing the cell capacity to detoxify ROS. In this work, a green fluorescent protein (GFP)-tagged nuclear factor erythroid 2-related factor 2 (NRF2)-regulated sulfiredoxin reporter (Srxn1-GFP) was used to measure the antioxidant response of HepG2 cells to a large series of drug and drug-like compounds (2230 compounds). These compounds were then classified as positive or negative depending on cellular response and distributed among different modeling groups to establish structure-activity relationship (SAR) models. A selection of models was used to prospectively predict oxidative stress induced by a new set of compounds subsequently experimentally tested to validate the model predictions. Altogether, this exercise exemplifies the different challenges of developing SAR models of a phenotypic cellular readout, model combination, chemical space selection, and results interpretation. Show less
Braak, S.J. ter; Klip, J.E.; Wink, S.; Hiemstra, S.W.; Cooper, S.L.; Middleton, A.; ... ; Water, B. van de 2022
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... Show moreA 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. Show less
Hiemstra, S.W.; Fehling-Kaschek, M.; Kuijper, I.A.; Bischoff, L.J.M.; Wijaya, L.S.; Rosenblatt, M.; ... ; Kaschek, D. 2022
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... Show moreCells 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. Show less
Formation of the reactive amodiaquine quinoneimine (AQ-QI) and N-desethylamodiaquine quinoneimine (DEAQ-QI) plays an important role in the toxicity of the anti-malaria drug amodiaquine (AQ).... Show more Formation of the reactive amodiaquine quinoneimine (AQ-QI) and N-desethylamodiaquine quinoneimine (DEAQ-QI) plays an important role in the toxicity of the anti-malaria drug amodiaquine (AQ). Glutathione conjugation protects against AQ-induced toxicity and GSTP1 is able to conjugate its quinoneimine metabolites AQ-QI and DEA-QI with glutathione. In this study, HepG2 cells transiently transfected with the human GSTP1 construct were utilized to investigate the protective effect of GSTP1 in a cellular context. HepG2 cells were exposed to synthesized QIs, which bypasses the need for intracellular bioactivation of AQ or DEAQ. Exposure was accompanied by decreased cell viability, increased caspase 3 activity, and decreased intracellular GSH levels. Using high-content imaging-based BAC-GFP reporters, it was shown that AQ-QI and DEAQ-QI specifically activated the endoplasmic reticulum (ER) stress response. In contrast, oxidative stress, DNA damage, or inflammatory stress responses were not activated. Overexpression of GSTP1 resulted in a two-fold increase in GSH-conjugation of the QIs, attenuated QI-induced cytotoxicity especially under GSH-depletion condition, abolished QIs-induced apoptosis but did not significantly inhibit the activation of the ER stress response. In conclusion, these results indicate a protective role of GSTP1 by increasing enzymatic detoxification of AQ-QI and DEAQ-QI and suggest a second protective mechanism by interfering with ER stress induced apoptosis. Show less
Wink, S.; Hiemstra, S.W.; Huppelschoten, S.; Klip, J.E.; Water, B. van de 2018
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... Show moreDrug-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. 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
