Citation:

Villeneuve, D., M. Le, M. Hazemi, A. Biales, D. Bencic, K. Bush, R. Flick, J. Martinson, M. Morshead, K. Santana Rodriguez, K. Vitense, AND K. Flynn. Pilot testing and optimization of a larval fathead minnow high throughput transcriptomics assay. Current Research in Toxicology. Elsevier B.V., Amsterdam, Netherlands, 4:100099, (2022). https://doi.org/10.1016/j.crtox.2022.100099

Impact/Purpose:

The next generation blue-print of computational toxicology at the US EPA proposes to use high throughput assays incorporating measurements of global gene expression changes in cells or small organisms as a first tier assay for hazard screening. To date, these high throughput gene expression assays have been developed with human cell lines. However, the screening battery currently lacks representation of biological pathways that are unique to other taxonomic groups of organisms including lower vertebrates such as fish, invertebrates, plants, etc. The present research reports initial efforts to develop and evaluate high throughput, gene expression-based, tier 1 screening assays for ecological hazard. Pilot results indicate that the effect concentrations estimates are generally protective relative to concentrations that produce adverse outcomes, making the approach favorable for screening. Additionally, the collected data were used to evaluate various assay design and acceptance criteria to further refine and improve the assays. This research represents a significant step toward ensuring the ecological hazards are not overlooked in EPA’s overall strategy. Additionally, evaluation of the strengths and limitations of these novel assays will help to define their appropriate application to support EPA decision-making regarding data poor chemicals.

Description:

Concentrations at which global gene expression profiles in cells or animals exposed to a test substance start to differ significantly from those of controls have been proposed as an alternative point of departure for use in screening level hazard assessment. The present study describes pilot testing of a high throughput compatible transcriptomics assay with larval fathead minnows. One day post hatch fathead minnows were exposed to eleven different concentrations of three metals, three selective serotonin reuptake inhibitors, and four neonicotinoid-like compounds for 24 h and concentration response modeling was applied to whole body gene expression data. Transcriptomics-based points of departure (tPODs) were consistently lower than effect concentrations reported in apical endpoint studies in fish. However, larval fathead minnow-based tPODs were not always lower than concentrations reported to elicit apical toxicity in other aquatic organisms like crustaceans or insects. Random in silico subsampling of data from the pilot assays was used to evaluate various assay design and acceptance considerations such as transcriptome coverage, number of replicate individuals to sequence per treatment, and minimum number of differentially expressed genes to produce a reliable tPOD estimate. Results showed a strong association between the total number of genes for which a concentration response relationship could be derived and the overall variability in the resulting tPOD estimates. We conclude that, for our current assay design and analysis pipeline, tPODs based on fewer than 15 differentially expressed genes are likely to be unreliable for screening and that interindividual variability in gene expression profiles appears to be a more significant driver of tPOD variability than sample size alone. Results represent initial steps toward developing high throughput transcriptomics assays for use in ecological hazard screening.

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