New Presentation: Optimising the algal toxicity test towards generation of multi-omics data and adverse outcome pathway discovery

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The adverse outcome pathway (AOP) concept represents a framework to organize mechanistic understanding of toxicological interactions by causally linking critical molecular key events (KE) to apical endpoints relevant for chemical risk assessment. Currently, only few methodologies can be considered for an accurate and reliable discovery and quantification of KEs in an exhaustive approach, commonly requiring sustained research effort. In this context, the objective of our presented proof-of-concept study was to showcase the identification and characterisation of molecular KEs from the molecular stress response of Chlamydomonas reinhardtii to toxic insult, applying a novel setup of high-throughput genome-wide scaling multi-omics technologies. The approach towards achieving this end was a suite of untargeted (direct-infusion mass-spec, DIMS; RNA sequencing) and targeted (LC-MS/MS, -UV, qRT-PCR) metabolomics, lipidomics and transcriptomics technologies. This methodology enabled us to profile the concentration- and time-response profiles of molecular signatures from algae exposed to non-specific mechanism (baseline toxicity) and target-specific mechanism (carotenoid biosynthesis inhibition) toxicants. To enable this work, a rigorously controlled algal culturing and testing system was optimised regarding growth rate, final cell density, pH stability, cell cycle synchronisation, reproducible exposure to volatile chemicals, and rapid quenching and harvesting of biomass for omics data collection. Furthermore, a unique multi-phase experimental design was developed for rapid identification (untargeted), characterisation and verification (targeted) of putative KEs over a time-course design. Multi-omics data from toxicant-exposed C. reinhardtii were collected and initial progress made towards computational analysis, putative KE designation, and targeted verification of identified biomarkers. With this study, a powerful experimental approach for hypothesis-free KE discovery and AOP hypothesis is being developed, employing omics-driven algal phenotyping to advance the integration of omics data into AOP development and ultimately, to provide mechanism-based support for regulatory decision-making in environmental risk assessment.

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