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

Abstract

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 ‘TPD-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). We applied the TPD-seq workflow 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 (∼9 k for fish cells; ∼6 k 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 31–155 mM across the cell lines. OECD test guideline 249 (RTgill-W1 cells) recommends the use of DCA and here we calculated a tPOD of ∼5 to 76 μM. Finally, exposure of the two human cell lines to H₂O₂ resulted in tPOD values that ranged from 0.7 to 1.1 mM in Caco-2 cells and 5–30 μM 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 TPD-seq workflow can be determined with additional case studies.