WP8 Functional analyses
Description
Changes in mitochondrial ROS formation as a consequence of mitochondrial dysfunction represents a relevant AOP in liver and heart toxicity. Increased cellular ROS formation can induce cellular signalling pathways or, as a more detrimental effect, at severely increased levels result in oxidative damage to cellular constituents like proteins and DNA. As continuously released by the Electron TransportChain, mitochondria are the main ROS-producing cellular organelles. To analyse this, Electron Spin Resonance spectroscopy, the only technique available that can directly identify and quantify radical formation, and the most effective and direct method fordetecting free radicals, will be used by partner UM.
Due to the ability of many in vitro systems to circumvent mitochondrial impartment, oxygen consumption is the key early indicator of ETC dysfunction for standard glucosegrown cells. To facilitate such oxygen measurements on standard microtitre plates, partner LuxCel has developed a series of oxygen-sensitive fluorescent probes for the assessment of both isolated mitochondria and whole cells. Using this platform, mitochondrial liabilities have been identified for a wide variety of drug classes, including thiazolidonediones, statins, antidepressants and biguanid. This has recently been advanced through the development of an extracellular acidification assay compatible with parallel oxygen measurements and both probes have been combined to facilitate parallel oxygen consumption and extracellular acidification screening. LuxCel has also developed a series of probes for assessing intracellular oxygen which can be a keymetabolic indicator particularly when analysing transient metabolic events. As mentioned before, next to mitochondria, inflammation is a key aspect in the course of drug-induced liver and heart toxicity. The nuclear factor-kappaB (NF-kappaB) transcriptional regulatory system is essential to the control of inflammation and cell survival, and is a target for modulation by drugs. Recent work indicates that modification of crucial proteinthiols on inhibitor of NF-kappaB (IkappaB) kinase-beta by electrophiles might control the responsiveness of this system in inflammation and oxidative stress. A series of drugtoxicities are mediated through stimuli exerted by tissue-specific cells such as macrophages, stellate or endothelial cells which induce inflammatory cascades upon treatment with a toxicant. For example, the prototypical hepatotoxicant acetaminophen has been shown to induce processes involving resident macrophages (Kupffer cells) which aggravate liver damage. Further, asymptomatic inflammation is suggested to be a key factor contributing to idiosyncratic liver injury of otherwise ‘clean’ drugs. Thus, assessment of inflammation as a contributing factor in drug-induced toxicity is key for the work proposed here. Unlike conventional cell models including only a single celltype, our multi cell-type 3d spheroids encompass inflammatory cells as well, and thus offer the possibility to capture drug-induced effects leading to activation thereof. A potential immune cell activation in vitro can thus be measured by quantification of cytokines in the cell culture supernatant over time and will be applied to both liver and heart tissue cultures, while inflammatory markers can be analysed in organ biopsies.
