Poster: The identification of toxicological markers in Adverse Outcome Pathway discovery in chlamydomonas reinhardtii

Current regulatory toxicity testing methods have become unsuitable for the thorough assessment of chemicals for commercial use, as lack of insight into toxicological mechanism prevents accurate predictive risk assessment. Adverse outcome pathways (AOPs) offer a framework for collating mechanistic data from a diverse range of methodologies, including in silico and in vitro approaches, for use in regulatory decision making. The aim of this work was to develop and test a more comprehensive experimental design, for the targeted characterisation of key events in the toxicological response of Chlamydomonas reinhardtii upon herbicide exposure, thereby contributing to the development of a quantitative AOP. Here we present how an initial hypothesis for an AOP was created from available literary evidence, with focus on `omics and multiple-endpoint assay data, for the selected herbicide, norflurazon. This hypothesised AOP allowed development of targeted assays for investigation of predicted key events in a time- and concentration-response methodology. LC-UV was used to monitor suspected toxicological markers of the carotenoid biosynthesis pathway (phytoene, phytofluene, ¿-carotene). qPCR was used to identify differential mRNA expression of chloroplast-specific thioredoxin PRX1, and a lipid peroxidation assay was applied for determining downstream effects of non-specific oxidative stress. A concentration- and time-dependent response in phytoene accumulation was observed, whilst concentration dependent ¿-carotene depletion was shown at later time-points (24 hours post exposure). Increased PRX1 expression was identified within an hour of exposure, whilst lipid peroxidation occurred between 4 and 24 hours post-exposure. Significant (p = < 0.01) effects on cell number, an adverse outcome, were observed at 2000µg/L after 24 hours. This study highlighted the necessity for use of synchronous algal cultures for accurately understanding mechanism, as this would enable more accurate determination of time- and concentration- responses due to diurnal algal life cycles. Ultimately, this work has shown proof-of-concept and laid the foundation for development of a quantitative AOP for norflurazon.

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