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 1,751 unique chemicals from the EPA's ToxCast collection in MCF7 cells using 8 concentrations and an exposure duration of 6 h. We hypothesized that concentration-response modeling of signature scores could be used to identify putative molecular targets and cluster chemicals with similar bioactivity.

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.

An automated computational data pipeline to rapidly acquire, score, and rank toxicological data for ecological hazard assessment.

Biological Evaluations support Endangered Species Act (ESA) consultation with the US Fish and Wildlife Service and National Marine Fisheries Service by federal action agencies, such as the USEPA, regarding impacts of federal activities on threatened or endangered species. However, they are often time-consuming and challenging to conduct. The identification of pollutant benchmarks or guidance to protect taxa for states and tribes when USEPA has not yet developed criteria recommendations is also of importance to ensure a streamlined approach to Clean Water Act program implementation.

Exploring the effects of experimental parameters and data modeling approaches on in vitro transcriptomic point-of-departure estimates.

Multiple new approach methods (NAMs) are being developed to rapidly screen large numbers of chemicals to aid in hazard evaluation and risk assessments. High-throughput transcriptomics (HTTr) in human cell lines has been proposed as a first-tier screening approach for determining the types of bioactivity a chemical can cause (activation of specific targets vs. generalized cell stress) and for calculating transcriptional points of departure (tPODs) based on changes in gene expression.

Navigating Transcriptomic Connectivity Mapping Workflows to Link Chemicals with Bioactivities.

Screening new compounds for potential bioactivities against cellular targets is vital for drug discovery and chemical safety. Transcriptomics offers an efficient approach for assessing global gene expression changes, but interpreting chemical mechanisms from these data is often challenging. Connectivity mapping is a potential data-driven avenue for linking chemicals to mechanisms based on the observation that many biological processes are associated with unique gene expression signatures (gene signatures).

FutureTox IV Workshop Summary: Predictive Toxicology for Healthy Children.

FutureTox IV, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in November 2018. Building upon FutureTox I, II, and III, this conference focused on the latest science and technology for in vitro profiling and in silico modeling as it relates to predictive developmental and reproductive toxicity (DART). Publicly available high-throughput screening data sets are now available for broad in vitro profiling of bioactivities across large inventories of chemicals.

High-Throughput Transcriptomics Platform for Screening Environmental Chemicals.

New approach methodologies (NAMs) that efficiently provide information about chemical hazard without using whole animals are needed to accelerate the pace of chemical risk assessments. Technological advancements in gene expression assays have made in vitro high-throughput transcriptomics (HTTr) a feasible option for NAMs-based hazard characterization of environmental chemicals. In this study, we evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for HTTr concentration-response screening of a small set of chemicals in the human-derived MCF7 cell model.

Comparison of Approaches for Determining Bioactivity Hits from High-Dimensional Profiling Data.

Phenotypic profiling assays are untargeted screening assays that measure a large number (hundreds to thousands) of cellular features in response to a stimulus and often yield diverse and unanticipated profiles of phenotypic effects, leading to challenges in distinguishing active from inactive treatments. Here, we compare a variety of different strategies for hit identification in imaging-based phenotypic profiling assays using a previously published Cell Painting data set. Hit identification strategies based on multiconcentration analysis involve curve fitting at several levels of data aggregation (e.

Development of a prioritization method for chemical-mediated effects on steroidogenesis using an integrated statistical analysis of high-throughput H295R data.

Synthesis of 11 steroid hormones in human adrenocortical carcinoma cells (H295R) was measured in a high-throughput steroidogenesis assay (HT-H295R) for 656 chemicals in concentration-response as part of the US Environmental Protection Agency's ToxCast program. This work extends previous analysis of the HT-H295R dataset and model by examining the utility of a novel prioritization metric based on the Mahalanobis distance that reduced these 11-dimensional data to 1-dimension via calculation of a mean Mahalanobis distance (mMd) at each chemical concentration screened for all hormone measures available. Herein, we evaluated the robustness of mMd values, and demonstrate that covariance and variance of the hormones measured appear independent of the chemicals screened and are inherent to the assay; the Type I error rate of the mMd method is less than 1%; and, absolute fold changes (up or down) of 1.

Otoferlin Depletion Results in Abnormal Synaptic Ribbons and Altered Intracellular Calcium Levels in Zebrafish.

The protein otoferlin plays an essential role at the sensory hair cell synapse. Mutations in otoferlin result in deafness and depending on the species, mild to strong vestibular deficits. While studies in mouse models suggest a role for otoferlin in synaptic vesicle exocytosis and endocytosis, it is unclear whether these functions are conserved across species.

Considerations for Strategic Use of High-Throughput Transcriptomics Chemical Screening Data in Regulatory Decisions.

Recently, numerous organizations, including governmental regulatory agencies in the U.S. and abroad, have proposed using data from New Approach Methodologies (NAMs) for augmenting and increasing the pace of chemical assessments.

Transcriptomic and phenotypic profiling in developing zebrafish exposed to thyroid hormone receptor agonists.

There continues to be a need to develop in vivo high-throughput screening (HTS) and computational methods to screen chemicals for interaction with the estrogen, androgen, and thyroid pathways and as complements to in vitro HTS assays. This study explored the utility of an embryonic zebrafish HTS approach to identify and classify endocrine bioactivity using phenotypically-anchored transcriptome profiling. Transcriptome analysis was conducted on zebrafish embryos exposed to 25 estrogen-, androgen-, or thyroid-active chemicals at concentrations that elicited adverse malformations or mortality at 120 h post-fertilization in 80% of animals exposed.

High-Throughput H295R Steroidogenesis Assay: Utility as an Alternative and a Statistical Approach to Characterize Effects on Steroidogenesis.

The U.S. Environmental Protection Agency Endocrine Disruptor Screening Program and the Organization for Economic Co-operation and Development (OECD) have used the human adrenocarcinoma (H295R) cell-based assay to predict chemical perturbation of androgen and estrogen production.

Comparative Toxicogenomic Responses to the Flame Retardant mITP in Developing Zebrafish.

Monosubstituted isopropylated triaryl phosphate (mITP) is a major component of Firemaster 550, an additive flame retardant mixture commonly used in polyurethane foams. Developmental toxicity studies in zebrafish established mITP as the most toxic component of FM 550, which causes pericardial edema and heart looping failure. Mechanistic studies showed that mITP is an aryl hydrocarbon receptor (AhR) ligand; however, the cardiotoxic effects of mITP were independent of the AhR.

Phenotypically anchored transcriptome profiling of developmental exposure to the antimicrobial agent, triclosan, reveals hepatotoxicity in embryonic zebrafish.

Triclosan (TCS) is an antimicrobial agent commonly found in a variety of personal care products and cosmetics. TCS readily enters the environment through wastewater and is detected in human plasma, urine, and breast milk due to its widespread use. Studies have implicated TCS as a disruptor of thyroid and estrogen signaling; therefore, research examining the developmental effects of TCS is warranted.

TBBPA exposure during a sensitive developmental window produces neurobehavioral changes in larval zebrafish.

Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame retardants (BFRs), is a ubiquitous contaminant in the environment and in the human body. This study demonstrated that zebrafish embryos exposed to TBBPA during a sensitive window of 8-48 h post-fertilization (hpf) displayed morphological malformations and mortality. Zebrafish exposed exclusively between 48 and 96 hpf were phenotypically normal.

Advanced morphological - behavioral test platform reveals neurodevelopmental defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants.

The increased use of flammable plastics and electronic devices along with stricter fire safety standards has led to the heavy use of flame retardant chemicals in many consumer, commercial, and industrial products. Although flame retardant use has increased, a great deal of uncertainty surrounds their safety with some evidence showing toxicity and risk to human and environmental health. Recent efforts have focused on designing high-throughput biological platforms with nonmammalian models to evaluate and prioritize chemicals with limited hazard information.