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Identifying suitable experimental designs to determining differentially expressed genes and benchmark doses from transcriptomic dose-response studies in zebrafish embryos

ABSTRACT: Identifying suitable experimental designs to determining differentially expressed genes and benchmark doses from transcriptomic dose-response studies in zebrafish embryos

PROVIDER: PRJNA957853 | ENA |

REPOSITORIES: ENA

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Identifying suitable experimental designs to determining differentially expressed genes and benchmark doses from transcriptomic dose-response studies in zebrafish embryos

Project description:To identify suitable experimental designsfor toxicology experiments (i.e., sample size, pool size, number of biological replicates, and number of doses), we exposed zebrafish embryos to 0.1% dimethyl sulfoxide for 4-120 hours post fertilization. We then created 96 samples to examine the impact of pool size comprised of 1, 5, 10, and 20 zebrafish embryos each (n = 24). From these, total RNA extraction was extracted and transcriptomic analyses were done using the TempO-Seq S1500+ Surrogate Assay platform. Samples were sequencing on an Illumina NextSeq 2000. The data were simulated to conduct power analyses exploring suitable study designs for identification of differentially expressed genes and for benchmark dose analysis. The aim was to identify a feasible pool size and sample size that would yield an acceptable false discovery rate for these analyses.

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Toxic Responses Induced at High Doses May Affect Benchmark Doses.

Project description:To derive reference points (RPs) for health-based guidance values, the benchmark dose (BMD) approach increasingly replaces the no-observed-adverse-effect level approach. In the BMD approach, the RP corresponds to the benchmark dose lower confidence bounds (BMDLs) of a mathematical dose-response model derived from responses of animals over the entire dose range applied. The use of the entire dose range is seen as an important advantage of the BMD approach. This assumes that responses over the entire dose range are relevant for modeling low-dose responses, the basis for the RP. However, if part of the high-dose response was unnoticed triggered by a mechanism of action (MOA) that does not work at low doses, the high-dose response distorts the modeling of low-dose responses. Hence, we investigated the effect of high-dose specific responses on BMDLs by assuming a low- and a high-dose MOA. The BMDLs resulting from modeling fictitious quantal data were scattered over a broad dose range overlapping with the toxic range. Hence, BMDLs are sensitive to high-dose responses even though they might be irrelevant to low-dose response modeling. When applying the BMD approach, care should be taken that high-dose specific responses do not unduly affect the BMDL that derives from low doses.

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Evaluation of 5-Day In Vivo Rat Liver and Kidney with High-Throughput Transcriptomics for Estimating Benchmark Doses of Apical Outcomes

Project description:The Division of the National Toxicology Program (DNTP) is currently evaluating high-throughput transcriptomics (HTT) as an approach to provide estimates of chemical exposure that may pose minimal risk. HTT was evaluated in a 5-day in vivo rat model (with repeated dosing) with the objective to determine if benchmark doses (BMD) values for transcriptional pathway changes in the liver and kidney could estimate BMD values for traditional toxicological (apical) endpoints. Eighteen chemicals, most having been tested by the NTP in 2-year bioassays, were chosen for this study. Some of these chemicals are known to be potent hepatotoxicants (e.g. DE71, PFOA, furan, and methyl eugenol) in rodents, some exhibit toxicity but have minimal effects on the liver (e.g. acrylamide and α,β-thujone), and some exhibit little overt toxicity (e.g. ginseng and milk thistle extract) based on traditional toxicological evaluations. Male Sprague Dawley rats were exposed daily for 5 consecutive days by oral gavage to 8 to 10 dose levels of each chemical. Liver and kidney were collected 24 hours after the final exposure and assayed using HTT with the rat S1500+ platform. HTT dose-response data were analyzed using BMD Express 2.2 to determine transcriptional BMD values for liver and kidney. BMDS Wizard was used to determine apical BMD values for histopathological effects from historical chronic or sub-chronic toxicity studies. For many of the chemicals, the lowest transcriptional BMDs from the 5-day assays were within a factor of 5 of the lowest histopathological BMDs estimated from the traditional toxicity studies. These data suggest that using HTT in a 5-day in vivo model provides reasonable estimates of BMD values for traditional apical endpoints. This approach may be useful to prioritize chemicals for further testing while providing actionable data in a timely and cost-effective manner.

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