Citation:

Lee, F., I. Shah, Y. Soong, J. Xing, I. Ng, F. Tasnim, AND H. Yu. Reproducibility and Robustness of High-Throughput S1500+ Transcriptomics on Primary Rat Hepatocytes for Chemical-Induced Hepatotoxicity Assessment. Current Research in Toxicology. Elsevier B.V., Amsterdam, Netherlands, 2:282-295, (2021). https://doi.org/10.1016/j.crtox.2021.07.003

Impact/Purpose:

Here we present a new high-throughput transcriptomics (HTTr) assay for hepatotoxicity prediction that is based on using rat primary hepatocytes and the TempO-Seq platform. Our results demonstrate this platform to be highly reproducible across batches and between technology platforms (specifically, Affymetrix RAE 2302). A key finding is that using the S1500+ assay ("surrogate transcriptome") delivers almost equal value as the "whole transcriptome" assay. The HTTr platform presented in this work offers a new approach methodology (NAM) for cost-effectively testing environmental chemicals for hepatic liabilities, which are one of the most frequent outcomes in guideline testing studies.

Description:

Cell-based in vitro models coupled with high-throughput transcriptomics (HTTr) are increasingly utilized as alternative methods to animal-based toxicity testing. Here, using a panel of 14 chemicals with different risks of human drug-induced liver injury (DILI) and two dosing concentrations, we evaluated an HTTr platform comprised of collagen sandwich primary rat hepatocyte culture and the TempO-Seq surrogate S1500+ (ST) assay. First, the HTTr platform was found to exhibit high reproducibility between technical and biological replicates (r greater than 0.85). Connectivity mapping analysis further demonstrated a high level of inter-platform reproducibility between TempO-Seq data and Affymetrix GeneChip data from the Open TG-GATES project. Second, the TempO-Seq ST assay was shown to be a robust surrogate to the whole transcriptome (WT) assay in capturing chemical-induced changes in gene expression, as evident from correlation analysis, PCA and unsupervised hierarchical clustering. Gene set enrichment analysis (GSEA) using the Hallmark gene set collection also demonstrated consistency in enrichment scores between ST and WT assays. Lastly, unsupervised hierarchical clustering of hallmark enrichment scores broadly divided the samples into hepatotoxic, intermediate, and non-hepatotoxic groups. Xenobiotic metabolism, bile acid metabolism, apoptosis, p53 pathway, and coagulation were found to be the key hallmarks driving the clustering. Taken together, our results established the reproducibility and performance of collagen sandwich culture in combination with TempO-Seq S1500+ assay, and demonstrated the utility of GSEA using the hallmark gene set collection to identify potential hepatotoxicants for further validation.

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