Comparison of in vitro transcriptomic points of departure with fish acute and chronic toxicity values: A case study of rainbow trout cells exposed to pesticides. Comparison of in vitro transcriptomic points of departure with fish acute and chronic toxicity values: A case study of rainbow trout cells exposed to pesticides

There is growing scientific and regulatory interest in transcriptomic points of departure (tPOD) values from in vitro experiments as an alternative to animal test method. The objective of this study was to calculate tPOD values in rainbow trout gill cells (RTgill-W1 following OECD 249) exposed to 20 pesticides, and to evaluate how these values compare to fish acute and chronic toxicity data from the ECOTOX database. Cells were exposed to one fungicide (chlorothalonil), ten herbicides (atrazine, glyphosate, imazethapyr, metolachlor, diquat, s-metolachlor, AMPA, dicamba, dimethenamid-P, metribuzin), eight insecticides (chlorpyrifos, diazinon, permethrin, carbaryl, clothianidin, imidacloprid, thiamethoxam, chlorantraniliprole), and OECD 249 positive control 3,4-dichloroaniline. Sequencing libraries were prepared with UPXome, and tPODs calculated with ExpressAnalyst. The method identified 44,262 unique genes, with 1,115 genes having >5 counts in the 576 samples sequenced. For all chemicals, tPODs were derived and tPOD mode values ranged from 0.003 to 141µM with an average of 37µM. There were significant correlations between these tPOD mode values (x-value) and in vitro cytotoxicity EC50s from RTgill-W1 cells (y=1.1x+0.89, R2=0.85, p<0.001; n=11), rainbow trout acute toxicity LC50s (y=0.9x+0.59, R2=0.56, p<0.001; n=20), fish chronic sub-lethal effect concentrations (y=0.62x+0.38, R2=0.36, p=0.01; n=16) and fish chronic lethal effect concentrations (y=0.77x-0.28, R2=0.57, p=0.002; n=14). Bland–Altman plot statistical analyses of these comparisons also showed good agreement. Overall, these data demonstrate that tPOD values can be derived from an experimental platform that links together elements of OECD 249 with a transcriptomic method with high throughput potential. The findings support the notion that tPODs from short-term in vitro studies may be comparable to effect concentration data from in vivo studies of fish exposed for chronic durations. Overall design: To dervie transcriptomic points of departure (tPODs) from the in vitro Rainbow trout gill (RTgill-W1) cell line assay following OECD 249 guidelines. RTgill-W1 cells were exposed to 20 pesticides for 24 hours in 96-well plates over 8 concentrations (3-fold serial dilutions). Pesticide concentrations were prepared in DMSO (solvent control) or water. Exposures were repeated 3 times for each chemical over 3 different passages (n=3 biological replicates) for a total of six 96-well plates, and 576 samples. Pesticides were split into two groups (Batch 1; n=10 pesticides, and Batch 2; n=10 pesticides). 3 wells were also designated as unexposed control RTgillW1 cells, referred to as ExpressRef. Following the 24 hour exposure, cells exposed to pesticides and the solvent control DMSO were lysed from the wells and then were prepared for RNA-sequencing using the QIAGEN UPXome protocol. The UPXome protocol individually tags each well in a 96-well plate with a unique code that allows for 24 samples to be pooled, and then tagged with a unique library code. Samples in a library can then be run on a single column for RNA-sequencing. Afterward analysis, R1 and R2 files are paired and demultiplexed according to their unique codes to separate individual samples and allow for subsequent analysis using ExpressAnalyst to derive tPODs.