Expression profiling by high throughput sequencing
Summary
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.
Overall design
Male HSD rats were housed five animals per cage and randomly assigned to treatment and control groups. Animals were divided into five testing blocks each consisting of four test articles administered at eight or nine doses plus a control group. Four animals per dose group receive the test article via gavage for five consecutive days and euthanized 24 hours following the last dose. Dosing for a four-chemical block was conducted simultaneously in the same room. Kidney and liver were removed from each animal and up to three samples (~250 mg total) from the left liver lobe and right kidney were taken. Each sample was cut into smaller pieces (approximately 5 mm3) and aliquoted into screw-cap tubes. Two of the samples from each animal were preserved in RNA later, one of which was used for RNA extraction at Battelle and submission to BioSpyder for transcriptomic screening by TempO-Seq analysis as previously described (Yeakley, et al., 2017). The remaining samples from each animal were snap frozen in liquid nitrogen and stored at -80 degrees C.
Less...
More...