Abstract
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 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.
Highlights
Drug-induced liver injury (DILI) is a major problem in the clinic, as 50% of all liver failures are caused by drugs (Ostapowicz et al 2002)
Delta-like 1 homolog (DLK1) was > 100-fold higher in 3D HepG2 spheroids compared to Primary human hepatocytes (PHH) and HepaRG
As a likely consequence of this enhanced differentiation, the terminal hepatocyte differentiation marker Glucose-6-phosphatase (G6PC) gene was 4- to 11-fold higher in 3D HepG2 spheroids compared to PHH and HepaRG
Summary
Drug-induced liver injury (DILI) is a major problem in the clinic, as 50% of all liver failures are caused by drugs (Ostapowicz et al 2002). Idiosyncratic DILI occurs only in rare cases, in approximately 0.1–0.01% of patients administrating the drug, and has a variable latency time, ranging from a few days up to a few years after first administration of the drug. These properties make it complicated to predict which drug will eventually cause idiosyncratic DILI in the clinic. In vitro tools have been used during preclinical toxicity to screen for cell viability in simple end-point measurements These assays do not reflect the early cell state changes occurring when cells adapt to the insult induced by the candidate chemical entity. Using high-content live cell confocal microscopy, we can quantitatively evaluate the dynamics of the activation of individual stress response pathway components at the individual cell level (Wink et al 2016, 2018)
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