Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2... Show moreAdaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI. Show less
Hiemstra, S.; Ramaiahgari, S.C.; Wink, S.; Callegaro, G.; Coonen, M.; Meerman, J.; ... ; Water, B. van de 2019
Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2... Show moreAdaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI. Show less
We have studied shape with a particular focus on the zebrafish model system. The shape is an essential appearance of the phenotype of a biological specimen and it can be used to read out a... Show moreWe have studied shape with a particular focus on the zebrafish model system. The shape is an essential appearance of the phenotype of a biological specimen and it can be used to read out a current state or response or to study gene expression. So accurate shape analysis requires a precise shape description. Moreover, a sufficiently large sampling size of the specimens is necessary to ensure a justified and unbiased shape analysis. The latter is very important for high-throughput in compound screening. Therefore, top performance in zebrafish analysis requires high-throughput imaging (HTI). To deal with HTI, we aim to design an elaborate and well-performing HTI architecture. For the essential operations we need computational approaches to obtain the 2D/3D shape representations that are precise and yet can be acquired fast. The quality of the obtained shape descriptions will be validated in a straightforward manner with scalar primitives, i.e., the volume and surface area of a 3D shape. These primitives serve as 3D measurements for a robust primary shape assessment in the phenotype characterisation. Using only shape description is not sufficient, e.g., for high-resolution imaging on tissue and cellular level, so texture should be considered to complement and enhance the shape analysis. Show less
