Signature Analysis of High-Throughput Transcriptomics Screening Data for Mechanistic Inference and Chemical Grouping.

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.

High-Throughput Transcriptomics Screen of ToxCast Chemicals in U-2 OS Cells.

New approach methodologies (NAMs) aim to accelerate the pace of chemical risk assessment while simultaneously reducing cost and dependency on animal studies. High Throughput Transcriptomics (HTTr) is an emerging NAM in the field of chemical hazard evaluation for establishing in vitro points-of-departure and providing mechanistic insight. In the current study, 1201 test chemicals were screened for bioactivity at eight concentrations using a 24-h exposure duration in the human- derived U-2 OS osteosarcoma cell line with HTTr. Assay reproducibility was assessed using three reference chemicals that were screened on every assay plate. The resulting transcriptomics data were analyzed by aggregating signal from genes into signature scores using gene set enrichment analysis, followed by concentration-response modeling of signatures scores. Signature scores were used to predict putative mechanisms of action, and to identify biological pathway altering concentrations (BPACs). BPACs were consistent across replicates for each reference chemical, with replicate BPAC standard deviations as low as 5.6 × 10-3 µM, demonstrating the internal reproducibility of HTTr-derived potency estimates. BPACs of test chemicals showed modest agreement (R2 = 0.55) with existing phenotype altering concentrations from high throughput phenotypic profiling using Cell Painting of the same chemicals in the same cell line. Altogether, this HTTr based chemical screen contributes to an accumulating pool of publicly available transcriptomic data relevant for chemical hazard evaluation and reinforces the utility of cell based molecular profiling methods in estimating chemical potency and predicting mechanism of action across a diverse set of chemicals.

Transcriptomics-Based Points of Departure for Daphnia magna Exposed to 18 Per- and Polyfluoroalkyl Substances.

Per- and polyfluoroalkyl substances (PFAS) represent a large group of contaminants of concern based on their widespread use, environmental persistence, and potential toxicity. Many traditional models for estimating toxicity, bioaccumulation, and other toxicological properties are not well suited for PFAS. Consequently, there is a need to generate hazard information for PFAS in an efficient and cost-effective manner. In the present study, Daphnia magna were exposed to multiple concentrations of 22 different PFAS for 24 h in a 96-well plate format. Following exposure, whole-body RNA was extracted and extracts, each representing five exposed individuals, were subjected to RNA sequencing. Following analytical measurements to verify PFAS exposure concentrations and quality control on processed cDNA libraries for sequencing, concentration-response modeling was applied to the data sets for 18 of the tested compounds, and the concentration at which a concerted molecular response occurred (transcriptomic point of departure; tPOD) was calculated. The tPODs, based on measured concentrations of PFAS, generally ranged from 0.03 to 0.58 µM (9.9-350 µg/L; interquartile range). In most cases, these concentrations were two orders of magnitude lower than similarly calculated tPODs for human cell lines exposed to PFAS. They were also lower than apical effect concentrations reported for seven PFAS for which some crustacean or invertebrate toxicity data were available, although there were a few exceptions. Despite being lower than most other available hazard benchmarks, D. magna tPODs were, on average, four orders of magnitude greater than the maximum aqueous concentrations of PFAS measured in Great Lakes tributaries. Overall, this high-throughput transcriptomics assay with D. magna holds promise as a component of a tiered hazard evaluation strategy employing new approach methodologies. Environ Toxicol Chem 2024;00:1-16. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Application of Cell Painting for chemical hazard evaluation in support of screening-level chemical assessments.

'Cell Painting' is an imaging-based high-throughput phenotypic profiling (HTPP) method in which cultured cells are fluorescently labeled to visualize subcellular structures (i.e., nucleus, nucleoli, endoplasmic reticulum, cytoskeleton, Golgi apparatus / plasma membrane and mitochondria) and to quantify morphological changes in response to chemicals or other perturbagens. HTPP is a high-throughput and cost-effective bioactivity screening method that detects effects associated with many different molecular mechanisms in an untargeted manner, enabling rapid in vitro hazard assessment for thousands of chemicals. Here, 1201 chemicals from the ToxCast library were screened in concentration-response up to ∼100 µM in human U-2 OS cells using HTPP. A phenotype altering concentration (PAC) was estimated for chemicals active in the tested range. PACs tended to be higher than lower bound potency values estimated from a broad collection of targeted high-throughput assays, but lower than the threshold for cytotoxicity. In vitro to in vivo extrapolation (IVIVE) was used to estimate administered equivalent doses (AEDs) based on PACs for comparison to human exposure predictions. AEDs for 18/412 chemicals overlapped with predicted human exposures. Phenotypic profile information was also leveraged to identify putative mechanisms of action and group chemicals. Of 58 known nuclear receptor modulators, only glucocorticoids and retinoids produced characteristic profiles; and both receptor types are expressed in U-2 OS cells. Thirteen chemicals with profile similarity to glucocorticoids were tested in a secondary screen and one chemical, pyrene, was confirmed by an orthogonal gene expression assay as a novel putative GR modulating chemical. Most active chemicals demonstrated profiles not associated with a known mechanism-of-action. However, many structurally related chemicals produced similar profiles, with exceptions such as diniconazole, whose profile differed from other active conazoles. Overall, the present study demonstrates how HTPP can be applied in screening-level chemical assessments through a series of examples and brief case studies.

Hands-on immunology: Engaging learners of all ages through tactile teaching tools.

Ensuring the public has a fundamental understanding of human-microbe interactions, immune responses, and vaccines is a critical challenge in the midst of a pandemic. These topics are commonly taught in undergraduate- and graduate-level microbiology and immunology courses; however, creating engaging methods of teaching these complex concepts to students of all ages is necessary to keep younger students interested when science seems hard. Building on the Tactile Teaching Tools with Guided Inquiry Learning (TTT-GIL) method we used to create an interactive lac operon molecular puzzle, we report here two TTT-GIL activities designed to engage diverse learners from middle schoolers to masters students in exploring molecular interactions within the immune system. By pairing physical models with structured activities built on the constructivist framework of Process-Oriented Guided Inquiry Learning (POGIL), TTT-GIL activities guide learners through their interaction with the model, using the Learning Cycle to facilitate construction of new concepts. Moreover, TTT-GIL activities are designed utilizing Universal Design for Learning (UDL) principles to include all learners through multiple means of engagement, representation, and action. The TTT-GIL activities reported here include a web-enhanced activity designed to teach concepts related to antibody-epitope binding and specificity to deaf and hard-of-hearing middle and high school students in a remote setting and a team-based activity that simulates the evolution of the Major Histocompatibility Complex (MHC) haplotype of a population exposed to pathogens. These activities incorporate TTT-GIL to engage learners in the exploration of fundamental immunology concepts and can be adapted for use with learners of different levels and educational backgrounds.

Combining phenotypic profiling and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: Retinoic acid as an example.

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.

Building the lac Operon: A Guided-Inquiry Activity Using 3D-Printed Models.

Undergraduate biology courses rely heavily on visual representation of information. Students view images of plants, animals, and microbes, interpret data presented in graphs, and use drawings to understand how cells and molecules interact in three dimensions. Traditional teaching approaches exclude students with visual impairments and disadvantage students with disabilities that affect their interpretation and processing of visual and spatial information, and also students who simply do not identify as "visual learners." By using new technologies to develop tactile teaching tools (TTTs) that can be employed in classrooms, we aim to create inclusive learning environments and more effectively instruct diverse learners. The advent of affordable and accessible 3D printing technology makes it possible to create tactile models that represent molecules, cells, and entire organisms more accurately than traditional visual representations. We describe the assessment of a 3D gene expression puzzle as a guided inquiry learning activity in which students must correctly assemble a series of components in order to achieve an output. Upon completion of the puzzle, the TTT provides tactile feedback through vibration to signal transcriptional activation. Analysis of pre- and postassessment performance demonstrated statistically significant increases in individual students' paired assessment scores in two different classroom implementations, with a greater effect size at a rural minority-serving institution than an urban R1 university. These encouraging preliminary data suggest that TTTs with guided-inquiry learning disproportionately benefit disadvantaged student populations and could serve as a tool in leveling the playing field when teaching abstract biological concepts in diverse educational settings.