Project description:There is a need to move from binary hazard assessment to more quantitative assessment of genotoxicity to better inform human health risk assessment and understand the relevance of positive in vitro genotoxicity findings. New approach methodologies (NAMs), including transcriptomic biomarkers combined with high-throughput technologies, enable the testing of a broad concentration range, which allows quantitative assessment of in vitro results. Initial work with the transcriptomic GENOMARK and TGx-DDI biomarkers demonstrate their potential use for hazard identification and chemical prioritization; however, no study has evaluated the concordance and complementarity of GENOMARK and TGx-DDI. The overall aim of this study is to examine if a combined approach of integrating both transcriptomic biomarkers for genotoxicity in human relevant HepaRGTM cells increases the certainty in hazard calls and potency rankings of chemicals. A sub-aim is to investigate whether GENOMARK is applicable to the TempO-Seq® high-throughput sequencing technology, to collect concentration-response data to rapidly perform hazard classification and potency ranking. Therefore, HepaRGTM cells were exposed to 10 chemicals (i.e. eight known in vivo genotoxicants and two in vivo non-genotoxicants) in increasing concentrations over 72h. TempO-Seq® was used to obtain concentration-response data for both biomarkers. Benchmark concentration (BMC) modelling of chemicals that were classified positive was conducted to obtain BMCs and transcriptomic points of departure (tPODs) for potency ranking. The results confirm that GENOMARK is applicable to TempO-Seq® since it achieved 100% predictive accuracy. In addition, a high concordance was observed in the hazard classifications and potency rankings between both biomarkers. Overall, our findings show that in vitro transcriptomic data can be used to rapidly and effectively identify genotoxic hazards while simultaneously providing additional insights on potency that is more informative in a modern hazard assessment paradigm. The results of this case study support the high value of integrating these NAMs in a weight of evidence evaluation of genotoxicity using the important human-liver cell line.

Project description:The United States Environmental Protection Agency has proposed a tiered testing strategy for chemical hazard evaluation based on new approach methods (NAMs). The first tier includes in vitro profiling assays applicable to many (human) cell types, such as high-throughput transcriptomics (HTTr) and high-throughput phenotypic profiling (HTPP). The goals of this study were to: (1) harmonize the seeding density of U-2 OS human osteosarcoma cells for use in both assays; (2) compare HTTr- versus HTPP-derived potency estimates for 11 mechanistically diverse chemicals; (3) identify candidate reference chemicals for monitoring assay performance in future screens; and (4) characterize the transcriptional and phenotypic changes in detail for all-trans retinoic acid (ATRA) as a model compound known for its adverse effects on osteoblast differentiation. The results of this evaluation showed that (1) HTPP conducted at low (400 cells/well) and high (3000 cells/well) seeding densities yielded comparable potency estimates and similar phenotypic profiles for the tested chemicals; (2) HTPP and HTTr resulted in comparable potency estimates for changes in cellular morphology and gene expression, respectively; (3) three test chemicals (etoposide, ATRA, dexamethasone) produced concentration-dependent effects on cellular morphology and gene expression that were consistent with known modes-of-action, demonstrating their suitability for use as reference chemicals for monitoring assay performance; and (4) ATRA produced phenotypic changes that were highly similar to other retinoic acid receptor activators (AM580, arotinoid acid) and some retinoid X receptor activators (bexarotene, methoprene acid). This phenotype was observed concurrently with autoregulation of the RARB gene. Both effects were prevented by pre-treating U-2 OS cells with pharmacological antagonists of their respective receptors. Thus, the observed phenotype could be considered characteristic of retinoic acid pathway activation in U-2 OS cells. These findings lay the groundwork for combinatorial screening of chemicals using HTTr and HTPP to generate complementary information for the first tier of a NAM-based chemical hazard evaluation strategy.

Project description:High-throughput transcriptomics (HTTr) uses gene expression profiling to characterize the biological activity of chemicals in in vitro cell-based test systems. As an extension of a previous study testing 44 chemicals, HTTr was used to screen an additional 1751 unique chemicals from the EPA’s ToxCast collection in MCF7 cells using eight concentrations and an exposure duration of 6 hours. We hypothesized that concentration-response modeling of signature scores could be used to identify putative molecular targets and cluster chemicals with similar bioactivity. Clustering and enrichment analyses were conducted based on signature catalog annotations and ToxPrint chemotypes to facilitate molecular target prediction and grouping of chemicals with similar bioactivity profiles. Enrichment analysis based on signature catalog annotation identified known mechanisms-of-action (MeOAs) associated with well-studied chemicals and generated putative MeOAs for other active chemicals. Chemicals with predicted MeOAs included those targeting estrogen receptor (ER), glucocorticoid receptor (GR), retinoic acid receptor (RAR), the NRF2/KEAP/ARE pathway, AP-1 activation and others. Using reference chemicals for ER modulation, the study demonstrated that HTTr in MCF7 cells was able to stratify chemicals in terms of agonist potency, distinguish ER agonists from antagonists, and cluster chemicals with similar activities as predicted by the ToxCast ER Pathway model. Uniform manifold approximation and projection (UMAP) embedding of signature-level results identified novel ER modulators with no ToxCast ER Pathway model predictions. Finally, UMAP combined with ToxPrint chemotype enrichment was used to explore the biological activity of structurally-related chemicals. The study demonstrates that HTTr can be used to inform chemical risk assessment by determining in vitro points-of-departure, predicting chemicals’ molecular mechanism(s)-of-action (MeOA) and grouping chemicals with similar bioactivity profiles.

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High-Throughput Transcriptomics Platform for Screening Environmental Chemicals

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