Pilot testing and optimization of a larval fathead minnow high throughput transcriptomics assay

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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 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.

Dan Villeneuve Aug 03, 2022 16:57:17
Table 3 File Key • Files of the form “---.filtered.+1.12-16.normFinal.txt” are the original data matrix files submitted to BMDExpress2 for analysis. Three letter abbreviation before the first period indicates the test chemical. • All_Chemicals_Filtered_Median_BMD_and_tPOD_calculations.csv contains a summary of the BMD and tPOD calculations for each chemical. • Files of the form “---filtered.+1.12-16.normFinal_sampleRep8_williams…..” are an export of the Williams trend test results for each chemical. The three letter abbreviation before the first period indicates the test chemical. • All .bm2 files are BMDExpress2 files containing the full BMDExpress2 analysis, including the dose-response modeling results. Files can be opened with BMDExpress2 software, but not with other word processing or spreadsheet programs. Chemical names are spelled out. Other names, are the name of the analyst that completed the analysis.


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