tPOD-seq: a high throughput RNA-seq method to derive transcriptomic points of departure from cell lines

There is growing scientific and regulatory interest in transcriptomic points of departure (tPOD) values from high-throughput in vitro experiments. To further help democratize tPOD research, here we outline ‘tPOD-seq’ which links microplate-based exposure methods involving cell lines for human (Caco-2, Hep G2) and environmental (rainbow trout RTgill-W1) health, with a commercially available RNA-seq kit, with a cloud-based bioinformatics tool (ExpressAnalyst.ca). To demonstrate tPOD-seq’s utility, we applied it to derive tPODs for solvents (dimethyl sulfoxide, DMSO; methanol) and positive controls (3,4-dichloroaniline, DCA; hydrogen peroxide, H2O2) commonly used in toxicity testing. The majority of reads mapped to protein coding genes (~10.5k for fish cells; ~7k for human cells), and about 50% of differentially expressed genes were curve-fitted from which 90% yielded gene benchmark doses. The most robust transcriptomic responses were caused by DMSO exposure, and tPOD values were 31-136.6 mM across the cell lines. OECD test guideline 249 (RTgill-W1 cells) recommends the use of DCA and here we calculated a tPOD of ~0.005 to ~0.076 mMmg/L. Finally, exposure of the two human cell lines to H2O2 resulted in similar tPOD values that ranged from 0.68-1.1 in Caco-2 cells and 0.0048-0.03 in Hep G2 cells. The methods outlined here are designed to be performed in laboratories with basic molecular and cell culture facilities, and the performance and scalability of the tPOD-seq approach can be determined with additional case studies. Caco-2, Hep G2 and RTgill-W1 cell lines were exposed to solvents and positive controls commonly used for in vitro assays at various concentrations. Endpoints examined were cytotoxicity and transcriptomics