Toxicity assessment of degradation products using zebrafish and advanced LC-MS techniques

To investigate the toxicity of the degradation products formed during different eAOP treatments, zebrafish larvae with an age of 3 days postfertilization will be exposed to samples collected before, during and after eAOP treatment via immersion. Zebrafish is a novel and promising small-animal model for toxicity studies. The strength of this in vivo model lies in its high genetic, physiologic and pharmacologic homology to humans. Their high fecundity, optical clarity and small size moreover allow performing tests in a medium to high-throughput fashion using microgram-scale quantities of compound. In this way, a unique system is obtained that combines the throughput of in vitro techniques (required to screen the large number of samples that will be generated in this project) with the bio-relevance of the whole animal. Zebrafish will first be exposed to the entire sample obtained before, during or after eAOP treatment. When toxicity is observed, the compounds responsible for the toxic effects will be separated and identified using advanced LC-MS techniques. Due to the expected complexity of the samples, the potential of multi-dimensional LC in combination with quadrupole time-of-flight MS will be evaluated for this purpose. Once potentially toxic compounds are identified, these compounds will be purified using (preparative) chromatography and zebrafish will be exposed to these compounds separately to confirm their toxicity. 10 larvae per sample will be live-screened at 24 and 48h post-treatment for lethality or symptoms of toxicity, including, lethality (as determined by cardiac arrest), edema, impaired circulation, change in heart rate, hemorrhaging, tissue necrosis, loss of posture, impaired motility (diminished or absent touch response), body curvature, swim bladder and jaw defects. This assessment will be carried out through visual observation of treated larvae using a standard light stereomicroscope. When toxicity is observed, a more specific, organ-related assessment of the toxicity will be made using different transgenic zebrafish lines. In this way, compounds will be screened for hepato-, cardio-, and neurotoxicity via high-throughput behavioral and seizure assays, cell imaging and 2D image based morphometric analysis.