We introduce a new high-throughput transcriptomics (HTTr) platform comprised of a collagen sandwich primary rat hepatocyte culture and the TempO-Seq assay for screening and prioritizing potential hepatotoxicants. We selected 14 chemicals based on their risk of drug-induced liver injury (DILI) and tested them in hepatocytes at two treatment concentrations. HTTr data was generated using the TempO-Seq whole transcriptome and S1500+ assays. The HTTr platform exhibited high reproducibility between technical replicates (r>0.9) but biological replication was greater for TempO-Seq S1500+ (r>0.85) than for the whole transcriptome (r>0.7). Reproducibility between biological replicates was dependent on the strength of transcriptional effects induced by a chemical treatment. Despite targeting a smaller number of genes, the S1500+ assay clustered chemical treatments and produced gene set enrichment analysis (GSEA) scores comparable to those of the whole transcriptome. Connectivity mapping showed a high-level of reproducibility between TempO-Seq data and Affymetrix GeneChip data from the Open TG-GATES project with high concordance between the S1500+ gene set and whole transcriptome. Taken together, our results provide guidance on selecting the number of technical and biological replicates and support the use of TempO-Seq S1500+ assay for a high-throughput platform for screening hepatotoxicants. FASTQ files and read counts data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) (GSE152128). This dataset is associated with the following publication: Lee, F., I. Shah, Y.T. Soong, J. Xing, I.C. 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).

Gene expression data from the Fluidigm qRT-PCR arrays was analyzed in R (v3.6.1; R Foundation for Statistical Computing, 2019). Prior to processing through the tcpl package, each qRT-PCR primer set was annotated as an individual assay endpoint (aeid) for analyses. For each plate, well types were designated for test compound wells (t), positive controls (c), (that is phenobarbital) and neutral controls (n, DMSO). Fold-change in the number of amplification cycles needed to pass the background threshold (Ct) for 96 transcripts to (ftp://newftp.epa.gov/COMPTOX/CCTE_Publication_Data/CCED_Publication_Data/Wambaugh/ToxCast_LTEA, file LTEA_Level2_20191119.zip) were normalized to the geometric mean of three housekeeping genes (ACTB, GAPDH, POLR2A) to generate ΔCt values (cval). Prior to calculating the response values (rval), or ΔΔCt, for each transcript (n = 96) per well, the baseline value (bval), the plate-wise median of the neutral control wells, was generated for each plate (the normalization process is described in detail in supplemental file SupFile4-DeltaCTCalculation.docx). The bval was subtracted from the cval to yield the rval or log2 Fold Change per transcript. Gene expression data from the Fluidigm qRT-PCR arrays was analyzed in R (v3.6.1; R Foundation for Statistical Computing, 2019). Prior to processing through the tcpl package, each qRT-PCR primer set was annotated as an individual assay endpoint (aeid) for analyses. For each plate, well types were designated for test compound wells (t), positive controls (c), (that is phenobarbital) and neutral controls (n, DMSO). Fold-change in the number of amplification cycles needed to pass the background threshold (Ct) for 96 transcripts to (ftp://newftp.epa.gov/COMPTOX/CCTE_Publication_Data/CCED_Publication_Data/Wambaugh/ToxCast_LTEA, file LTEA_Level5_20191119.zip) were normalized to the geometric mean of three housekeeping genes (ACTB, GAPDH, POLR2A) to generate ΔCt values (cval). Prior to calculating the response values (rval), or ΔΔCt, for each transcript (n = 96) per well, the baseline value (bval), the plate-wise median of the neutral control wells, was generated for each plate (the normalization process is described in detail in supplemental file SupFile4-DeltaCTCalculation.docx). The bval was subtracted from the cval to yield the rval or log2 Fold Change per transcript. Supplemental File LTEA_Inucyte_Images.zip is comprised of 20,493 images totaling more than 15 gigabytes. Cell morphology images were acquired for each well/plate with an Essen IncuCyte™ FLR automated phase-contrast microscope located inside a tissue culture incubator. Six 96-well culture plates were loaded into the instrument and imaged for an elapsed time (~24 minutes). The IncuCyte™ software was used for image capturing and export of images in JPEG format. This dataset is associated with the following publication: Franzosa, J., J. Bonzo, J. Jack, N.C. Baker, P. Kothiya, R. Witek, P. Hurban, S. Siferd, S. Hester, I. Shah, S. Ferguson, K. Houck, and J. Wambaugh. High-throughput toxicogenomic screening of chemicals in the environment using metabolically competent hepatic cell cultures. npj Systems Biology and Applications. Springer Nature Group, New York, NY, 7: Article 7, (2021).

Datasets used in RD-023418: Adverse Outcome Pathway-Driven Identification of Rat Hepatocarcinogens in Short-Term Assays. This dataset is associated with the following publication: Rooney, J., T. Hill, C. Qin, F. Sistare, and C. Corton. Adverse outcome pathway-driven identification of rat liver tumorigens in short-term assays. TOXICOLOGY AND APPLIED PHARMACOLOGY. Academic Press Incorporated, Orlando, FL, USA, 356: 99-113, (2018).

Dataset for Robarts et al., 'Hepatic Transcriptome Comparative In Silico Analysis Reveals Similar Pathways and Targets Altered by Legacy and Alternative Per- and Polyfluoroalkyl Substances in Mice' published in Toxics, DOI https://doi.org/10.3390/toxics11120963, PMCID 10748317. This dataset is associated with the following publication: Robarts, D., J. Dai, C. Lau, U. Apte, and J. Corton. Hepatic Transcriptome Comparative In Silico Analysis Reveals Similar Pathways and Targets Altered by Legacy and Alternative Per- and Polyfluoroalkyl Substances in Mice. Toxics. MDPI, Basel, SWITZERLAND, 11(12): 963, (2023).

This repository contains the necessary data, python source code and jupyter notebooks to reproduce the results from our manuscript, "Estimating Hepatotoxic Doses Using High-content Imaging in Primary Hepatocytes." Using in vitro data to estimate point of departure (POD) values is an important component of new approach method (NAM)-based chemical risk assessments. In this case study we evaluated a NAM for hepatotoxicity based on rat primary hepatocytes, high-content imaging (HCI) and in vitro to in vivo extrapolation (IVIVE). This dataset is associated with the following publication: Shah, I., T. Antonijevic, B. Chambers, J. Harrill, and R. Thomas. Estimating Hepatotoxic Doses Using High-content Imaging in Primary Hepatocytes. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 183(2): 285-301, (2021).

This dataset provides measured in vitro intrinsic clearance rates for 54 chemicals tested using isolated hepatocytes from humans, rats, and rainbow trout. The test chemicals were selected to provide broad coverage across the industrial and pesticidal chemical space while also prioritizing chemicals of interest to EPA’s Endocrine Disruptor Screening Program (EDSP). A data evaluation framework was developed to identify results suitable for rate reporting. Acceptable results were then used to evaluate the chemical domain of applicability of the applied methods, the influence of starting substrate concentration on measured rates of intrinsic clearance, and differences in metabolic activity among species. These findings provide data for chemicals of specific interest to the EDSP. More importantly, the results provide critical guidance on future use of in vitro biotransformation assays to support high-throughput chemical risks assessments. This dataset is associated with the following publication: Black, S., J. Nichols, K. Fay, S. Matten, and S. Lynn. Evaluation and comparison of in vitro intrinsic hepatic clearance rates measured using cryopreserved hepatocytes from humans, rats, and rainbow trout. TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 457: 152819, (2021).

This dataset was prepared from the US Environmental Protection Agency's (EPA) Toxicity Reference Database (ToxRefDB) that contains information for 1,142 chemicals and 5,960 studies. Curations include information regarding the study design, chemical identity, dosing, treatment group parameters, treatment-related (significantly different from control) and critical (adverse) effects for all dose treatment groups, as well as endpoint testing status according to guideline specifications. ToxRefDB data was examined for all subchronic (SUB) studies with complete curations, which included registrant-submitted toxicity studies from the US EPA’s Office of Pesticide Programs (OPP) and guideline studies sourced from the National Toxicology Program (NTP). Statistically significant differences between treatment and control group data at p<0.05 within the source documents was extracted and denoted with a “treatment-related” Boolean indicator “true”. Across the studies with absolute liver weights and relative-to-body (RLW) liver weights, the treatment-related mean effect values at the lowest effect (LE) dose levels as well as mean control liver weights were determined for all chemical-study-sex-species-exposure route groupings. The LE-ALW and LE-RLW changes were quantified as effect size differences from control using the following equation: Effect_size = 100 x (LE Effect_value – Control Effect_Value) / Control Effect_Value Any microscopic liver pathology effects occurring at the corresponding LE dose level of weight change were also identified and listed in the dataset. Histopathology terms were presented as they appeared in ToxRefDB without harmonizing different hierarchical levels and aggregating multiple terms used to depict the same lesions. The final dataset that includes chemical stressor information, study source identifiers, study type, sex, species, strain, administration route, administration method, dose level, mg/kg/day value, qualitative and quantitative effect information, effect size from control, and pathology effects if present. The dataset includes data from 389 subchronic studies on 273 chemicals. This dataset is associated with the following publication: Mezencev, R., M. Feshuk, L. Kolaczkowski, G. Peterson, Q. Zhao, S. Watford, and J. Weaver. The association between histopathologic effects and liver weight changes induced in mice and rats by chemical exposures: an analysis of the data from Toxicity Reference Database (ToxRefDB). TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 200(2): 404-413, (2024).

RNA-seq data from HepaRG cells exposed for 2 hours to microcystin-LR and -RR at multiple concentrations each. This dataset is associated with the following publication: Biales, A., D. Bencic, R. Flick, A. Delacruz, D. Gordon, and W. Huang. Global transcriptomic profiling of microcystin-LR or -RR treated hepatocytes (HepaRG).. Toxicon: X. Elsevier B.V., Amsterdam, NETHERLANDS, 8: 100060, (2020).