Characterisation and validation of an in vitro transactivation assay based on the 22Rv1/MMTV_GR-KO cell line to detect human androgen receptor agonists and antagonists.

We describe the characterisation and validation of an androgen receptor (AR) transactivation assay for detection of AR agonists and antagonists using a stably transfected human prostate cancer cell line. This 22Rv1/mouse mammary tumour virus glucocorticoid knock-out cell line based AR transactivation assay was validated by criteria in Organisation for Economic Cooperation and Development Guidance Document 34 to determine if the assay performed equally well to the AR EcoScreen Assay included in Test Guideline for AR Transactivation (OECD TG 458). There was no Glucocorticoid Receptor (GR) crosstalk, and no changes in the AR DNA sequence in cells after the successful knock out of GR. Subsequently, the concordance of classifications of the 22 test chemicals was 100% in all laboratories. The AR agonistic and antagonistic inter-laboratory coefficients of variation based on log[10% effect for 10 nM DHT, PC10] and log[inhibitory response of 800 pM DHT by at 30%, IC30] from comprehensive tests were 2.75% and 2.44%, respectively. The AR agonist/antagonist test chemical classifications were consistent across AR EcoScreen ARTA assay data for 82/89%, and the balanced accuracy, sensitivity, and specificity were 83/90%, 88/100% and 78/80%, respectively. This assay was successfully validated and was approved for inclusion in TG 458 in 2020.

Assessing bioactivity-exposure profiles of fruit and vegetable extracts in the BioMAP profiling system.

The ToxCast program has generated in vitro screening data on over a thousand chemicals to assess potential disruption of important biological processes and assist in hazard identification and chemical testing prioritization. Few results have been reported for complex mixtures. To extend these ToxCast efforts to mixtures, we tested extracts from 30 organically grown fruits and vegetables in concentration-response in the BioMAP® assays. BioMAP systems use human primary cells primed with endogenous pathway activators to identify phenotypic perturbations related to proliferation, inflammation, immunomodulation, and tissue remodeling. Clustering of bioactivity profiles revealed separation of these produce extracts and ToxCast chemicals. Produce extracts elicited 87 assay endpoint responses per item compared to 20 per item for ToxCast chemicals. On a molar basis, the produce extracts were 10 to 50-fold less potent and when constrained to the maximum testing concentration of the ToxCast chemicals, the produce extracts did not show activity in as many assay endpoints. Using intake adjusted measures of dose, the bioactivity potential was higher for produce extracts than for agrichemicals, as expected based on the comparatively small amounts of agrichemical residues present on conventionally grown produce. The evaluation of BioMAP readouts and the dose responses for produce extracts showed qualitative and quantitative differences from results with single chemicals, highlighting challenges in the interpretation of bioactivity data and dose-response from complex mixtures.

Confirmation of high-throughput screening data and novel mechanistic insights into VDR-xenobiotic interactions by orthogonal assays.

High throughput screening (HTS) programs have demonstrated that the Vitamin D receptor (VDR) is activated and/or antagonized by a wide range of structurally diverse chemicals. In this study, we examined the Tox21 qHTS data set generated against VDR for reproducibility and concordance and elucidated functional insights into VDR-xenobiotic interactions. Twenty-one potential VDR agonists and 19 VDR antagonists were identified from a subset of >400 compounds with putative VDR activity and examined for VDR functionality utilizing select orthogonal assays. Transient transactivation assay (TT) using a human VDR plasmid and Cyp24 luciferase reporter construct revealed 20/21 active VDR agonists and 18/19 active VDR antagonists. Mammalian-2-hybrid assay (M2H) was then used to evaluate VDR interactions with co-activators and co-regulators. With the exception of a select few compounds, VDR agonists exhibited significant recruitment of co-regulators and co-activators whereas antagonists exhibited considerable attenuation of recruitment by VDR. A unique set of compounds exhibiting synergistic activity in antagonist mode and no activity in agonist mode was identified. Cheminformatics modeling of VDR-ligand interactions were conducted and revealed selective ligand VDR interaction. Overall, data emphasizes the molecular complexity of ligand-mediated interactions with VDR and suggest that VDR transactivation may be a target site of action for diverse xenobiotics.

Screening the ToxCast phase II libraries for alterations in network function using cortical neurons grown on multi-well microelectrode array (mwMEA) plates.

Methods are needed for rapid screening of environmental compounds for neurotoxicity, particularly ones that assess function. To demonstrate the utility of microelectrode array (MEA)-based approaches as a rapid neurotoxicity screening tool, 1055 chemicals from EPA's phase II ToxCast library were evaluated for effects on neural function and cell health. Primary cortical networks were grown on multi-well microelectrode array (mwMEA) plates. On day in vitro 13, baseline activity (40 min) was recorded prior to exposure to each compound (40 µM). Changes in spontaneous network activity [mean firing rate (MFR)] and cell viability (lactate dehydrogenase and CellTiter Blue) were assessed within the same well following compound exposure. Following exposure, 326 compounds altered (increased or decreased) normalized MFR beyond hit thresholds based on 2× the median absolute deviation of DMSO-treated wells. Pharmaceuticals, pesticides, fungicides, chemical intermediates, and herbicides accounted for 86% of the hits. Further, changes in MFR occurred in the absence of cytotoxicity, as only eight compounds decreased cell viability. ToxPrint chemotype analysis identified several structural domains (e.g., biphenyls and alkyl phenols) significantly enriched with MEA actives relative to the total test set. The top 5 enriched ToxPrint chemotypes were represented in 26% of the MEA hits, whereas the top 11 ToxPrints were represented in 34% of MEA hits. These results demonstrate that large-scale functional screening using neural networks on MEAs can fill a critical gap in assessment of neurotoxicity potential in ToxCast assay results. Further, a data-mining approach identified ToxPrint chemotypes enriched in the MEA-hit subset, which define initial structure-activity relationship inferences, establish potential mechanistic associations to other ToxCast assay endpoints, and provide working hypotheses for future studies.

Primary Cell Phenotypic Screening Illuminates ADRs and AOPs.

Preclinical, in vitro screening for adverse drug reactions continues to present challenges in the field of drug development. In this issue of Cell Chemical Biology, Shah et al. (2017) employ a phenotypic screening strategy using a panel of human primary cells to define a signature response and an adverse outcome pathway for delayed type IV skin hypersensitivity.

Incorporating High-Throughput Exposure Predictions With Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing.

We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compared against regulatory exposure estimates, providing an activity-to-exposure ratio (AER) useful for a risk-based ranking strategy. As ToxCast efforts expand (ie, Phase II) beyond food-use pesticides toward a wider chemical domain that lacks exposure and toxicity information, prediction tools become increasingly important. In this study, in vitro hepatic clearance and plasma protein binding were measured to estimate OEDs for a subset of Phase II chemicals. OEDs were compared against high-throughput (HT) exposure predictions generated using probabilistic modeling and Bayesian approaches generated by the U.S. Environmental Protection Agency (EPA) ExpoCast program. This approach incorporated chemical-specific use and national production volume data with biomonitoring data to inform the exposure predictions. This HT exposure modeling approach provided predictions for all Phase II chemicals assessed in this study whereas estimates from regulatory sources were available for only 7% of chemicals. Of the 163 chemicals assessed in this study, 3 or 13 chemicals possessed AERs < 1 or < 100, respectively. Diverse bioactivities across a range of assays and concentrations were also noted across the wider chemical space surveyed. The availability of HT exposure estimation and bioactivity screening tools provides an opportunity to incorporate a risk-based strategy for use in testing prioritization.

Understanding mechanisms of toxicity: insights from drug discovery research.

Toxicology continues to rely heavily on use of animal testing for prediction of potential for toxicity in humans. Where mechanisms of toxicity have been elucidated, for example endocrine disruption by xenoestrogens binding to the estrogen receptor, in vitro assays have been developed as surrogate assays for toxicity prediction. This mechanistic information can be combined with other data such as exposure levels to inform a risk assessment for the chemical. However, there remains a paucity of such mechanistic assays due at least in part to lack of methods to determine specific mechanisms of toxicity for many toxicants. A means to address this deficiency lies in utilization of a vast repertoire of tools developed by the drug discovery industry for interrogating the bioactivity of chemicals. This review describes the application of high-throughput screening assays as experimental tools for profiling chemicals for potential for toxicity and understanding underlying mechanisms. The accessibility of broad panels of assays covering an array of protein families permits evaluation of chemicals for their ability to directly modulate many potential targets of toxicity. In addition, advances in cell-based screening have yielded tools capable of reporting the effects of chemicals on numerous critical cell signaling pathways and cell health parameters. Novel, more complex cellular systems are being used to model mammalian tissues and the consequences of compound treatment. Finally, high-throughput technology is being applied to model organism screens to understand mechanisms of toxicity. However, a number of formidable challenges to these methods remain to be overcome before they are widely applicable. Integration of successful approaches will contribute towards building a systems approach to toxicology that will provide mechanistic understanding of the effects of chemicals on biological systems and aid in rationale risk assessments.

The hypolipidemic natural product guggulsterone is a promiscuous steroid receptor ligand.

Guggulsterone (GS) is the active substance in guggulipid, an extract of the guggul tree, Commiphora mukul, used to treat a variety of disorders in humans, including dyslipidemia, obesity, and inflammation. The activity of GS has been suggested to be mediated by antagonism of the receptor for bile acids, the farnesoid X receptor (FXR). Here, we demonstrate that both stereoisomers of the plant sterol, (E)- and (Z)-GS, bind to the steroid receptors at a much higher affinity than to FXR. Both stereoisomers bind to the mineralocorticoid receptor (MR) with a Ki value of approximately 35 nM, which is greater than 100 times more potent than their affinity for FXR. Both (E)- and (Z)-GS also displayed high affinity for other steroid receptors, including the androgen (AR), glucocorticoid (GR), and progesterone receptors (PR) with Ki values ranging from 224 to 315 nM. In cell-based functional cotransfection assays, GSs behaved as antagonists of AR, GR, and MR, but as agonists of PR. Agonist activity was also demonstrated with estrogen receptor (ER) alpha; however, the potency was very low (EC50 > 5000 nM). In addition, GS displayed activity in functional assays in cell lines expressing endogenous AR, GR, ER, and PR. These data suggest that the variety of pharmacological effects exhibited by GS may be mediated by targeting several steroid receptors.