The thyroid hormone (TH) system is involved in several important physiological processes, including regulation of energy metabolism, growth and differentiation, development and maintenance of brain function, thermo-regulation, osmo-regulation, and axis of regulation of other endocrine systems, sexual behaviour and fertility and cardiovascular function. Therefore, concern about TH disruption (THD) has resulted in strategies being developed to identify THD chemicals (THDCs). Information on potential of chemicals causing THD is typically derived from animal studies. For the majority of chemicals, however, this information is either limited or unavailable. It is also unlikely that animal experiments will be performed for all THD relevant chemicals in the near future for ethical, financial and practical reasons. In addition, typical animal experiments often do not provide information on the mechanism of action of THDC, making it harder to extrapolate results across species. Relevant effects may not be identified in animal studies when the effects are delayed, life stage specific, not assessed by the experimental paradigm (e.g., behaviour) or only occur when an organism has to adapt to environmental factors by modulating TH levels. Therefore, in vitro and in silico alternatives to identify THDC and quantify their potency are needed. THDC have many potential mechanisms of action, including altered hormone production, transport, metabolism, receptor activation and disruption of several feed-back mechanisms. In vitro assays are available for many of these endpoints, and the application of modern '-omics' technologies, applicable for in vivo studies can help to reveal relevant and possibly new endpoints for inclusion in a targeted THDC in vitro test battery. Within the framework of the ASAT initiative (Assuring Safety without Animal Testing), an international group consisting of experts in the areas of thyroid endocrinology, toxicology of endocrine disruption, neurotoxicology, high-throughput screening, computational biology, and regulatory affairs has reviewed the state of science for (1) known mechanisms for THD plus examples of THDC; (2) in vitro THD tests currently available or under development related to these mechanisms; and (3) in silico methods for estimating the blood levels of THDC. Based on this scientific review, the panel has recommended a battery of test methods to be able to classify chemicals as of less or high concern for further hazard and risk assessment for THD. In addition, research gaps and needs are identified to be able to optimize and validate the targeted THD in vitro test battery for a mechanism-based strategy for a decision to opt out or to proceed with further testing for THD.

In vitro high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. Here we discuss streamlining the validation process, specifically for prioritization applications. By prioritization, we mean a process in which less complex, less expensive, and faster assays are used to prioritize which chemicals are subjected first to more complex, expensive, and slower guideline assays. Data from the HTS prioritization assays is intended to provide a priori evidence that certain chemicals have the potential to lead to the types of adverse effects that the guideline tests are assessing. The need for such prioritization approaches is driven by the fact that there are tens of thousands of chemicals to which people are exposed, but the yearly throughput of most guideline assays is small in comparison. The streamlined validation process would continue to ensure the reliability and relevance of assays for this application. We discuss the following practical guidelines: (1) follow current validation practice to the extent possible and practical; (2) make increased use of reference compounds to better demonstrate assay reliability and relevance; (3) de-emphasize the need for cross-laboratory testing; and (4) implement a web-based, transparent, and expedited peer review process.

Tetra-n-octylammonium bromide and benzethonium chloride are synthetic quaternary ammonium salts that are widely used in hospitals and industries for the disinfection and surface treatment and as the preservative agent. Recently, the activities of HERG channel inhibition by these compounds have been found to have potential risks to induce the long QT syndrome and cardiac arrhythmia, although the mechanism of action is still elusive. This study was conducted to investigate the mechanism of HERG channel inhibition by these compounds by using whole-cell patch clamp experiments in a CHO cell line stably expressing HERG channels. Tetra-n-octylammonium bromide and benzethonium chloride exhibited concentration-dependent inhibitions of HERG channel currents with IC(50) values of 4nM and 17nM, respectively, which were also voltage-dependent and use-dependent. Both compounds shifted the channel activation I-V curves in a hyperpolarized direction for 10-15mV and accelerated channel activation and inactivation processes by 2-fold. In addition, tetra-n-octylammonium bromide shifted the inactivation I-V curve in a hyperpolarized direction for 24.4mV and slowed the rate of channel deactivation by 2-fold, whereas benzethonium chloride did not. The results indicate that tetra-n-octylammonium bromide and benzethonium chloride are open-channel blockers that inhibit HERG channels in the voltage-dependent, use-dependent and state-dependent manners.

PURPOSE: The constitutive androstane receptor (CAR, NR1I3) is a xenobiotic sensor governing the transcription of numerous hepatic genes associated with drug metabolism and clearance. Recent evidence suggests that CAR also modulates energy homeostasis and cancer development. Thus, identification of novel human (h) CAR activators is of both clinical importance and scientific interest. METHODS: Docking and ligand-based structure-activity models were used for virtual screening of a database containing over 2000 FDA-approved drugs. Identified lead compounds were evaluated in cell-based reporter assays to determine hCAR activation. Potential activators were further tested in human primary hepatocytes (HPHs) for the expression of the prototypical hCAR target gene CYP2B6. RESULTS: Nineteen lead compounds with optimal modeling parameters were selected for biological evaluation. Seven of the 19 leads exhibited moderate to potent activation of hCAR. Five out of the seven compounds translocated hCAR from the cytoplasm to the nucleus of HPHs in a concentration-dependent manner. These compounds also induce the expression of CYP2B6 in HPHs with rank-order of efficacies closely resembling that of hCAR activation. CONCLUSION: These results indicate that our strategically integrated approaches are effective in the identification of novel hCAR modulators, which may function as valuable research tools or potential therapeutic molecules.

BACKGROUND: Over the past 20 years, an increased focus on detecting environmental chemicals that pose a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. Environmental Protection Agency (EPA) Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP; thus, processing these chemicals using current test batteries could require millions of dollars and decades. A need for increased throughput and efficiency motivated the development of methods using in vitro high throughput screening (HTS) assays to prioritize chemicals for EDSP Tier 1 screening (T1S). OBJECTIVE: In this study we used U.S. EPA ToxCast HTS assays for estrogen, androgen, steroidogenic, and thyroid-disrupting mechanisms to classify compounds and compare ToxCast results to in vitro and in vivo data from EDSP T1S assays. METHOD: We implemented an iterative model that optimized the ability of endocrine-related HTS assays to predict components of EDSP T1S and related results. Balanced accuracy was used as a measure of model performance. RESULTS: ToxCast estrogen receptor and androgen receptor assays predicted the results of relevant EDSP T1S assays with balanced accuracies of 0.91 (p < 0.001) and 0.92 (p < 0.001), respectively. Uterotrophic and Hershberger assay results were predicted with balanced accuracies of 0.89 (p < 0.001) and 1 (p < 0.001), respectively. Models for steroidogenic and thyroid-related effects could not be developed with the currently published ToxCast data. CONCLUSIONS: Overall, results suggest that current ToxCast assays can accurately identify chemicals with potential to interact with the estrogenic and androgenic pathways, and could help prioritize chemicals for EDSP T1S assays.

The 5-methylcytosine (5-mC) derivative 5-hydroxymethylcytosine (5-hmC) is abundant in the brain for unknown reasons. Here we characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC by using glucosylation coupled with restriction-enzyme digestion and microarray analysis. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary in human and mouse. This boundary change was mainly due to 5-hmC in the brain but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent data sets and with single-molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC relative to alternatively spliced exons. Our study suggests a new role for 5-hmC in RNA splicing and synaptic function in the brain.

Drug-induced phospholipidosis (PLD), characterized by an intracellular accumulation of phospholipids and formation of concentric lamellar bodies, has raised concerns in the drug discovery community, due to its potential adverse effects. To evaluate the PLD induction potential, 4,161 nonredundant drug-like molecules from the National Institutes of Health Chemical Genomics Center (NCGC) Pharmaceutical Collection (NPC), the Library of Pharmacologically Active Compounds (LOPAC), and the Tocris Biosciences collection were screened in a quantitative high-throughput screening (qHTS) format. The potential of drug-lipid complex formation can be linked directly to the structures of drug molecules, and many PLD inducing drugs were found to share common structural features. Support vector machine (SVM) models were constructed by using customized atom types or Molecular Operating Environment (MOE) 2D descriptors as structural descriptors. Either the compounds from LOPAC or randomly selected from the entire data set were used as the training set. The impact of training data with biased structural features and the impact of molecule descriptors emphasizing whole-molecule properties or detailed functional groups at the atom level on model performance were analyzed and discussed. Rebalancing strategies were applied to improve the predictive power of the SVM models. Using the undersampling method, the consensus model using one-third of the compounds randomly selected from the data set as the training set achieved high accuracy of 0.90 in predicting the remaining two-thirds of the compounds constituting the test set, as measured by the area under the receiver operator characteristic curve (AUC-ROC).

BACKGROUND: Oxidative stress has been implicated in the pathogenesis of a variety of diseases ranging from cancer to neurodegeneration, highlighting the need to identify chemicals that can induce this effect. The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of oxidative stress. Thus, assays that detect the up-regulation of this pathway could be useful for identifying chemicals that induce oxidative stress. OBJECTIVES: We used cell-based reporter methods and informatics tools to efficiently screen a large collection of environmental chemicals and identify compounds that induce oxidative stress. METHODS: We utilized two cell-based ARE assay reporters, β-lactamase and luciferase, to screen a U.S. National Toxicology Program 1,408-compound library (NTP 1408, which contains 1,340 unique compounds) for their ability to induce oxidative stress in HepG2 cells using quantitative high throughput screening (qHTS). RESULTS: Roughly 3% (34 of 1,340) of the unique compounds demonstrated activity across both cell-based assays. Based on biological activity and structure-activity relationship profiles, we selected 50 compounds for retesting in the two ARE assays and in an additional follow-up assay that employed a mutated ARE linked to β-lactamase. Using this strategy, we identified 30 compounds that demonstrated activity in the ARE-bla and ARE-luc assays and were able to determine structural features conferring compound activity across assays. CONCLUSIONS: Our results support the robustness of using two different cell-based approaches for identifying compounds that induce ARE signaling. Together, these methods are useful for prioritizing chemicals for further in-depth mechanism-based toxicity testing.

The limitations of traditional toxicity testing characterized by high-cost animal models with low-throughput readouts, inconsistent responses, ethical issues, and extrapolability to humans call for alternative strategies for chemical risk assessment. A new strategy using in vitro human cell-based assays has been designed to identify key toxicity pathways and molecular mechanisms leading to the prediction of an in vivo response. The emergence of quantitative high-throughput screening (qHTS) technology has proved to be an efficient way to decompose complex toxicological end points to specific pathways of targeted organs. In addition, qHTS has made a significant impact on computational toxicology in two aspects. First, the ease of mechanism of action identification brought about by in vitro assays has enhanced the simplicity and effectiveness of machine learning, and second, the high-throughput nature and high reproducibility of qHTS have greatly improved the data quality and increased the quantity of training datasets available for predictive model construction. In this review, the benefits of qHTS routinely used in the US Tox21 program will be highlighted. Quantitative structure-activity relationships models built on traditional in vivo data and new qHTS data will be compared and analyzed. In conjunction with the transition from the pilot phase to the production phase of the Tox21 program, more qHTS data will be made available that will enrich the data pool for predictive toxicology. It is perceivable that new in silico toxicity models based on high-quality qHTS data will achieve unprecedented reliability and robustness, thus becoming a valuable tool for risk assessment and drug discovery.

Decreases in mitochondrial membrane potential (MMP) have been associated with mitochondrial dysfunction that could lead to cell death. The MMP is generated by an electrochemical gradient via the mitochondrial electron transport chain coupled to a series of redox reactions. Measuring the MMP in living cells is commonly used to assess the effect of chemicals on mitochondrial function; decreases in MMP can be detected using lipophilic cationic fluorescent dyes. To identify an optimal dye for use in a high-throughput screening (HTS) format, we compared the ability of mitochondrial membrane potential sensor (Mito-MPS), 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide, rhodamine 123, and tetramethylrhodamine to quantify a decrease in MMP in chemically exposed HepG2 cells cultured in 1,536-well plates. Under the conditions used, the optimal dye for this purpose is Mito-MPS. Next, we developed and optimized a homogenous cell-based Mito-MPS assay for use in 1,536-well plate format and demonstrated the utility of this assay by screening 1,280 compounds in the library of pharmacologically active compounds in HepG2 cells using a quantitative high-throughput screening platform. From the screening, we identified 14 compounds that disrupted the MMP, with half-maximal potencies ranging from 0.15 to 18 μM; among these, compound clusters that contained tyrphostin and 3'-substituted indolone analogs exhibited a structure-activity relationship. Our results demonstrate that this homogenous cell-based Mito-MPS assay can be used to evaluate the ability of large numbers of chemicals to decrease mitochondrial function.