In-plate toxicometabolomics of single zebrafish embryos

Toxicometabolomic studies involving zebrafish embryos have become
increasingly popular for linking apical endpoints to biochemical
perturbations as part of adverse outcome pathway determination. These
experiments involve pooling embryos to generate sufficient biomass for
metabolomic measurement, which adds both time and cost. To address this
limitation, we developed a high-throughput toxicometabolomic assay
involving single zebrafish embryos. Incubation, microscopy, embryo
extraction, and instrumental metabolomic analysis were all performed in
the same 96-well plate, following acquisition of conventional
toxicological endpoints. The total time for the assay (including testing
of 6 doses/n = 12 embryos per dose plus positive and negative
controls, assessing conventional endpoints, instrumental analysis, data
processing and multivariate statistics) is <14 days. Metabolomic
perturbations at low dose were linked statistically to those observed at
high dose and in the presence of an adverse effect, thereby
contextualizing omic data amongst apical endpoints. Overall, this assay
enables collection of high resolution metabolomic data in a high
throughput manner, suitable for mode of action hypothesis generation in
the context of pharmaceutical or toxicological screening.