Project description:Per- and polyfluoroalkyl substances (PFAS) are a very large (thousands of chemicals) category; these substances have important industrial and consumer product applications. PFAS are highly persistent in the environment, and some are known to pose human health hazard. Regulatory agencies worldwide are considering restrictions and outright bans of PFAS; however, little data exists to make informed decisions. Therefore, a prioritization strategy is urgently needed for evaluation of potential hazards of PFAS. Structure-based grouping could expedite selection of PFAS for testing; still, the hypothesis that structure-effect relationships exist requires confirmation. We tested 26 structurally diverse PFAS from 8 groups in two human cell types from organs that are thought to be targets for PFAS. We used human induced pluripotent stem cell-derived hepatocytes and cardiomyocytes and tested concentration-response effects on both cell function and gene expression. Few phenotypic effects were observed in hepatocytes, but negative chronotropy was observed for 8 of 26 PFAS. Substance- and cell-dependent transcriptomic changes were more pronounced; however, little evidence of group-specific effects was observed. In hepatocytes, we found up-regulation of stress-related and extracellular matrix organization pathways, and down-regulation of fat metabolism. In cardiomyocytes, contractility-related pathways were most affected. Using these data, we derived phenotypic and transcriptomic point of departure estimates and compared them to predicted PFAS exposures. The conservative estimates for bioactivity and exposure were used to derive margin-of-exposure (MOE) for each PFAS. We found that most (23 of 26) PFAS had MOE>1. Overall, our data suggests that chemical structure-based grouping of PFAS may not be an appropriate strategy to predict their biological effects. This means that testing of the individual PFAS would be needed for confident decision-making. Our proposed strategy of using two human cell types and considering both phenotypic and transcriptomic effects, combined with dose-response analysis, may be used for prioritization of PFAS.

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.

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Informing Hazard Identification and Risk Characterization of Environmental Chemicals by Combining Transcriptomic and Functional Data from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

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