High-Throughput Screening to Advance In Vitro Toxicology: Accomplishments, Challenges, and Future Directions.

Traditionally, chemical toxicity is determined by in vivo animal studies, which are low throughput, expensive, and sometimes fail to predict compound toxicity in humans. Due to the increasing number of chemicals in use and the high rate of drug candidate failure due to toxicity, it is imperative to develop in vitro, high-throughput screening methods to determine toxicity. The Tox21 program, a unique research consortium of federal public health agencies, was established to address and identify toxicity concerns in a high-throughput, concentration-responsive manner using a battery of in vitro assays. In this article, we review the advancements in high-throughput robotic screening methodology and informatics processes to enable the generation of toxicological data, and their impact on the field; further, we discuss the future of assessing environmental toxicity utilizing efficient and scalable methods that better represent the corresponding biological and toxicodynamic processes in humans.

Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders.

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Predicting binding between 55 cannabinoids and 4,799 biological targets by in silico methods.

Recently, there has been an increase in cannabis-derived products being marketed as foods, dietary supplements, and other consumer products. Cannabis contains over a hundred cannabinoids, many of which have unknown physiological effects. Since there are large numbers of cannabinoids, and many are not commercially available for in vitro testing, an in silico tool (Chemotargets Clarity software) was used to predict binding between 55 cannabinoids and 4,799 biological targets (enzymes, ion channels, receptors, and transporters). This tool relied on quantitative structure activity relationships (QSAR), structural similarity, and other approaches to predict binding. From this screening, 827 cannabinoid-target binding pairs were predicted, which included 143 unique targets. Many cannabinoids sharing core structures (cannabinoid "types") had similar binding profiles, whereas most cannabinoids containing carboxylic acid groups were similar without regards to their core structure. For some of the binding predictions (43), in vitro binding data were available, and they agreed well with in silico binding data (median fourfold difference in binding concentrations). Finally, clinical adverse effects associated with 22 predicted targets were identified from an online database (Clarivate Off-X), providing important insights on potential human health hazards. Overall, in silico biological target predictions are a rapid means to identify potential hazards due to cannabinoid-target interactions, and the data can be used to prioritize subsequent in vitro and in vivo testing.

Trends using biological target-based assays for drug detection in complex sample matrices.

In vivo, drug molecules interact with their biological targets (e.g., enzymes, receptors, ion channels, transporters), thereby eliciting therapeutic effects. Assays that measure the interaction between drugs and bio-targets may be used as drug biosensors, which are capable of broadly detecting entire drug classes without prior knowledge of their chemical structure. This Trends article covers recent developments in bio-target-based screening assays for detecting drugs associated with the following areas: illicit products marketed as dietary supplements, food-producing animals, and bodily fluids. General challenges and considerations associated with using bio-target assays are also presented. Finally, future applications of these assays for drug detection are suggested based upon current needs.

Picamilon, a γ-aminobutyric acid (GABA) analogue and marketed nootropic, is inactive against 50 biological targets.

Picamilon is an analogue of the neurotransmitter γ-aminobutyric acid (GABA), which is marketed as a nootropic claiming to enhance cognition. There is a lack of in silico, in vitro and in vivo data on the safety of picamilon. Therefore, to ascertain potential physiological effects of picamilon, it was screened against 50 safety-related biological targets (receptors, ion channels, enzymes and transporters) by in silico and in vitro methods. Using two in silico tools, picamilon was not predicted to bind to the targets. Similarly, picamilon exhibited weak or no binding to the targets when measured in vitro at 10 µM. Overall, this data shows that picamilon, although structurally similar to other GABA analogues, has a different biological target binding profile. Picamilon's lack of binding to the 50 targets fills important data gaps among GABA analogues, a group of structurally related substances found in drugs and other consumer products.

Recent applications of phosphodiesterase (PDE5) inhibition assays for detecting adulterated sexual enhancement products.

Consumer products marketed for sexual enhancement are frequently adulterated with erectile dysfunction (ED) drugs and analogs; consuming these undisclosed adulterants can pose significant health hazards. Although ED drugs/analogs have unpredictable and diverse structures that pose challenges for detecting them, they all share the ability to inhibit phosphodiesterase-5 (PDE5) activity, a pharmacological mechanism responsible for their effects. Consequently, several PDE5 inhibition assays have been recently applied as screening methods to detect ED drug/analogs in products. Here, the successes and challenges are highlighted for screening sexual enhancement products by PDE5 inhibition assays.

High-throughput screening for identifying acetylcholinesterase inhibitors: Insights on novel inhibitors and the use of liver microsomes.

Rapid, higher throughput, and predictive toxicological methods are needed to assess vast numbers of chemicals with unknown safety profiles. A current effort towards this goal is Toxicology in the 21st Century (Tox21), a United States government consortium using a battery of in vitro assays to screen a library of 10,000 compounds relevant to food, drug, and environmental safety. Recently, we implemented in vitro assays for measuring acetylcholinesterase (AChE) inhibition, a mechanism of toxicity, into Tox21's high-throughput screening campaign (Li S., et al. Environ Health Persp 2021;129:047008, doi:10.1289/EHP6993). In this Commentary, we provide detailed insights on two topics related to our article: (1) prioritizing recently discovered AChE inhibitors from our screening based upon physiological relevance and (2) incorporating human liver microsomes into the AChE inhibition assay to identify metabolically active AChE inhibitors.

Acetylcholinesterase Inhibition Assays for High-Throughput Screening.

Acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh), a vital neurotransmitter that regulates muscle movement and brain function, including memory, attention, and learning. Inhibition of AChE activity can cause a variety of adverse health effects and toxicity. Identifying AChE inhibitors quickly and efficiently warrants developing AChE inhibition assays in a quantitative, high-throughput screening (qHTS) platform. In this chapter, protocols for multiple homogenous AChE inhibition assays used in a qHTS system are provided. These AChE inhibition assays include a (1) human neuroblastoma (SH-SY5Y) cell-based assay with fluorescence or colorimetric detection; (2) human recombinant AChE with fluorescence or colorimetric detection; and (3) combination of human recombinant AChE and liver microsomes with colorimetric detection, which enables detection of test compounds requiring metabolic activation to become AChE inhibitors. Together, these AChE assays can help identify, prioritize, and predict chemical hazards in large compound libraries using qHTS systems.

Temporal resolution in electrochemical imaging on single PC12 cells using amperometry and voltammetry at microelectrode arrays.

Carbon-fiber-microelectrode arrays (MEAs) have been utilized to electrochemically image neurochemical secretion from individual pheochromocytoma (PC12) cells. Dopamine release events were electrochemically monitored from seven different locations on single PC12 cells using alternately constant-potential amperometry and fast-scan cyclic voltammetry (FSCV). Cyclic voltammetry, when compared to amperometry, can provide excellent chemical resolution; however, spatial and temporal resolution are both compromised. The spatial and temporal resolution of these two methods have been quantitatively compared and the differences explained using models of molecular diffusion at the nanogap between the electrode and the cell. A numerical simulation of the molecular flux reveals that the diffusion of dopamine molecules and electrochemical reactions both play important roles in the temporal resolution of electrochemical imaging. The simulation also reveals that the diffusion and electrode potential cause the differences in signal crosstalk between electrodes when comparing amperometry and FSCV.

Trends in computational simulations of electrochemical processes under hydrodynamic flow in microchannels.

Computational modeling and theoretical simulations have recently become important tools for the development, characterization, and validation of microfluidic devices. The recent proliferation of commercial user-friendly software has allowed researchers in the microfluidics community, who might not be familiar with computer programming or fluid mechanics, to acquire important information on microsystems used for sensors, velocimetry, detection for microchannel separations, and microfluidic fuel cells. We discuss the most popular computational technique for modeling these systems--the finite element method--and how it can be applied to model electrochemical processes coupled with hydrodynamic flow in microchannels. Furthermore, some of the limitations and challenges of these computational models are also discussed.

Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition.

BACKGROUND: Inhibition of acetylcholinesterase (AChE), a biomarker of organophosphorous and carbamate exposure in environmental and occupational human health, has been commonly used to identify potential safety liabilities. So far, many environmental chemicals, including drug candidates, food additives, and industrial chemicals, have not been thoroughly evaluated for their inhibitory effects on AChE activity. AChE inhibitors can have therapeutic applications (e.g., tacrine and donepezil) or neurotoxic consequences (e.g., insecticides and nerve agents). OBJECTIVES: The objective of the current study was to identify environmental chemicals that inhibit AChE activity using in vitro and in silico models. METHODS: To identify AChE inhibitors rapidly and efficiently, we have screened the Toxicology in the 21st Century (Tox21) 10K compound library in a quantitative high-throughput screening (qHTS) platform by using the homogenous cell-based AChE inhibition assay and enzyme-based AChE inhibition assays (with or without microsomes). AChE inhibitors identified from the primary screening were further tested in monolayer or spheroid formed by SH-SY5Y and neural stem cell models. The inhibition and binding modes of these identified compounds were studied with time-dependent enzyme-based AChE inhibition assay and molecular docking, respectively. RESULTS: A group of known AChE inhibitors, such as donepezil, ambenonium dichloride, and tacrine hydrochloride, as well as many previously unreported AChE inhibitors, such as chelerythrine chloride and cilostazol, were identified in this study. Many of these compounds, such as pyrazophos, phosalone, and triazophos, needed metabolic activation. This study identified both reversible (e.g., donepezil and tacrine) and irreversible inhibitors (e.g., chlorpyrifos and bromophos-ethyl). Molecular docking analyses were performed to explain the relative inhibitory potency of selected compounds. CONCLUSIONS: Our tiered qHTS approach allowed us to generate a robust and reliable data set to evaluate large sets of environmental compounds for their AChE inhibitory activity. https://doi.org/10.1289/EHP6993.

Temporal analysis of protozoan lysis in a microfluidic device.

A microfluidic device was fabricated and characterized for studying cell lysis of Arcella vulgaris, a nonpathogenic amoeba, over time. The device contains a series of chambers which capture cells allowing them to be subsequently exposed to a constant flow of biocidal agent. With this microfluidic system, individual cells are observed as they undergo lysis. This allows high-throughput measurements of individual lysis events, which are not possible with conventional techniques. Differences in lysis and decay times for Arcella were seen at different flow rates and concentrations of benzalkonium chloride, a biocidal detergent. The efficacy of benzalkonium chloride, chlorhexidine digluconate, phenol, sodium dodecyl sulfate, and Triton X-100 were compared, revealing information on their mechanisms of action. The presented device allows cell capture, controlled exposure to chemical biocides, and observation of lysis with single-cell resolution. Observations at the single cell level give insight into the mechanistic details of the lysis of individual Arcella cells vs. the population; decay times for individual Arcella cells were much shorter when compared to a population of 15 cells.

Hybrid capillary-microfluidic device for the separation, lysis, and electrochemical detection of vesicles.

The primary method for neuronal communication involves the extracellular release of small molecules that are packaged in secretory vesicles. We have developed a platform to separate, lyse, and electrochemically measure the contents of single vesicles using a hybrid capillary-microfluidic device. This device incorporates a sheath-flow design at the outlet of the capillary for chemical lysis of vesicles and subsequent electrochemical detection. The effect of sheath-flow on analyte dispersion was characterized using confocal fluorescence microscopy and electrochemical detection. At increased flow rates, dispersion was minimized, leading to higher separation efficiencies but lower detected amounts. Large unilamellar vesicles (diameter approximately 200 nm), a model for secretory vesicles, were prepared by extrusion and loaded with an electroactive molecule. They were then separated and detected using the hybrid capillary-microfluidic device. Determination of size from internalized analyte concentration provides a method to characterize the liposomal suspension. These results were compared to an orthogonal size measurement using dynamic light scattering to validate the detection platform.

Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides.

The inhibition of acetylcholinesterase (AChE) has pharmaceutical applications as well as potential neurotoxic effects. The in vivo metabolites of some chemicals including organophosphorus pesticides can become more potent AChE inhibitors compared to their parental compounds. To account for the effects of biotransformation, we have developed and characterized a high-throughput screening method for identifying AChE inhibitors that become active or more potent following xenobiotic metabolism. In this study, an enzyme-based assay was developed in 1536-well plates using recombinant human AChE combined with human or rat liver microsomes. The AChE activity was measured by two methods with different readouts: colorimetric and fluorescent. The assay exhibited exceptional performance characteristics including large assay signal window, low well-to-well variability and high reproducibility. The performance of the assays with microsomes was characterized by testing a group of known AChE inhibitors including parent compounds and their metabolites. Large potency differences between the parent compounds and the metabolites were observed in the assay with microsome addition. Both assay readouts were required for maximal sensitivity. These results demonstrate that this platform is a promising method to profile large numbers of chemicals that require metabolic activation for inhibiting AChE activity.

Phosphodiesterase (PDE5) inhibition assay for rapid detection of erectile dysfunction drugs and analogs in sexual enhancement products.

Products marketed as dietary supplements for sexual enhancement are frequently adulterated with phosphodiesterase-5 (PDE5) inhibitors, which are erectile dysfunction drugs or their analogs that can cause adverse health effects. Due to widespread adulteration, a rapid screening assay was developed to detect PDE5 inhibitors in adulterated products. The assay employs fluorescence detection and is based on measuring inhibition of PDE5 activity, the pharmacological mechanism shared among the adulterants. Initially, the assay reaction scheme was established and characterized, followed by analysis of 9 representative PDE5 inhibitors (IC50 , 0.4-4.0 ng mL-1 ), demonstrating sensitive detection in matrix-free solutions. Next, dietary supplements serving as matrix blanks (n = 25) were analyzed to determine matrix interference and establish a threshold value; there were no false positives. Finally, matrix blanks were spiked with 9 individual PDE5 inhibitors, along with several mixtures. All 9 adulterants were successfully detected (≤ 5 % false negative rate; n = 20) at a concentration of 1.00 mg g-1 , which is over 5 times lower than concentrations commonly encountered in adulterated products. A major distinction of the PDE5 inhibition assay is the ability to detect adulterants without prior knowledge of their chemical structures, demonstrating a broad-based detection capability that can address a continuously evolving threat of new adulterants. The PDE5 inhibition assay can analyze over 40 samples simultaneously within 15 minutes and involves a single incubation step and simple data analysis, all of which are advantageous for combating the widespread adulteration of sex-enhancement products.

Flow characterization of a microfluidic device to selectively and reliably apply reagents to a cellular network.

A three-dimensional microfluidic device has been successfully fabricated and the flow streams characterized for eventual use in studying communication in an in vitro network of nerve cells. The microfluidic system is composed of two layers of channels: a lower layer for the delivery of pharmacological solutions and an upper layer of channels used to direct the flow of the pharmacological solution streams and perfuse the cells with media and nutrients. Flow profiles have been characterized with computational fluid dynamics simulations, confocal fluorescence microscopy, and carbon-fiber amperometry, which have been used to map changes in flow profiles at different bulk flow rates. Ultimately, the microfluidic system and incorporated cell network will show how networked neurons adapt, compensate, and recover after being exposed to different chemical compounds.

Identification of acetylcholinesterase inhibitors using homogenous cell-based assays in quantitative high-throughput screening platforms.

Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of acetylcholine, a neurotransmitter associated with muscle movement, cognition, and other neurobiological processes. Inhibition of AChE activity can serve as a therapeutic mechanism, but also cause adverse health effects and neurotoxicity. In order to efficiently identify AChE inhibitors from large compound libraries, homogenous cell-based assays in high-throughput screening platforms are needed. In this study, a fluorescent method using Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) and the Ellman absorbance method were both developed in a homogenous format using a human neuroblastoma cell line (SH-SY5Y). An enzyme-based assay using Amplex Red was also optimized and used to confirm the potential inhibitors. These three assays were used to screen 1368 compounds, which included a library of pharmacologically active compounds (LOPAC) and 88 additional compounds from the Tox21 program, at multiple concentrations in a quantitative high-throughput screening (qHTS) format. All three assays exhibited exceptional performance characteristics including assay signal quality, precision, and reproducibility. A group of inhibitors were identified from this study, including known (e.g. physostigmine and neostigmine bromide) and potential novel AChE inhibitors (e.g. chelerythrine chloride and cilostazol). These results demonstrate that this platform is a promising means to profile large numbers of chemicals that inhibit AChE activity.

Cytochrome P450 2D6 and 3A4 enzyme inhibition by amine stimulants in dietary supplements.

A number of dietary supplements used for weight loss and athletic performance enhancement have been recently shown to contain a variety of stimulants, for which there is a lack of pharmacological and toxicological information. One concern for these emerging compounds is their potential to inhibit metabolic enzymes in the liver such as cytochromes P450 (CYP), which can lead to unexpected interactions among dietary supplements, drugs, and other xenobiotics. In this study, inhibition of human recombinant CYP2D6 and CYP3A4 by 27 amine stimulants associated with dietary supplements and their analogs was evaluated by luminescence assays. The strongest CYP2D6 inhibitors were coclaurine (IC50 = 0.14 ± 0.01 µM) and N-benzylphenethylamine (IC50 = 0.7 ± 0.2 µM), followed by several other relatively strong inhibitors (IC50 , 2-12 µM) including ß-methylphenethylamine, N,ß-dimethylphenethylamine (phenpromethamine), 1,3-dimethylamylamine (DMAA), N,α-diethylphenethylamine, higenamine (norcoclaurine) and N,N-diethylphenethylamine. Only nine compounds inhibited CYP3A4 by 20-55% at 100 µM. Results of this study illustrate that several amine stimulants associated with dietary supplements inhibit CYP2D6 and CYP3A4 in vitro, and these compounds may participate in adverse drug-dietary supplement interactions in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

A fluorescence assay for measuring acetylcholinesterase activity in rat blood and a human neuroblastoma cell line (SH-SY5Y).

Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of the neurotransmitter acetylcholine, and inhibition of AChE can have therapeutic applications (e.g., drugs for Alzheimer's disease) or neurotoxic consequences (e.g., pesticides). A common absorbance-based AChE activity assay that uses 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) can have limited sensitivity and be prone to interference. Therefore, an alternative assay was developed, in which AChE activity was determined by measuring fluorescence of resorufin produced from coupled enzyme reactions involving acetylcholine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). The Amplex Red assay was used for two separate applications. First, AChE activity was measured in rat whole blood, which is a biomarker for exposure to AChE inhibitor pesticides. Activity was quantified from a 10(5)-fold dilution of whole blood, and there was a linear correlation between Amplex Red and DTNB assays. For the second application, Amplex Red assay was used to measure AChE inhibition potency in a human neuroblastoma cell line (SH-SY5Y), which is important for assessing pharmacological and toxicological potential of AChE inhibitors including drugs, phytochemicals, and pesticides. Five known reversible inhibitors were evaluated (IC50, 7-225 nM), along with irreversible inhibitors chlorpyrifos-oxon (ki=1.01 nM(-1)h(-1)) and paraoxon (ki=0.16 nM(-1)h(-1)). Lastly, in addition to inhibition, AChE reactivation was measured in SH-SY5Y cells incubated with pralidoxime chloride (2-PAM). The Amplex Red assay is a sensitive, specific, and reliable fluorescence method for measuring AChE activity in both rat whole blood and cultured SH-SY5Y cells.

Sex hormone modulation of both induction and inhibition of CYP1A by genistein in HepG2/C3A cells.

Genistein is a widely consumed phytoestrogen in dietary supplements and has been reported to play roles in both cancer prevention and promotion. These conflicting effects may be complicated by sex differences. Cytochrome P450 1A (CYP1A) participates in carcinogen activation and detoxification, and the enzyme may interact with genistein. Therefore, modulation of CYP1A by a combination of genistein and sex hormones could be responsible for sex differences related to cancer prevention and promotion. In the current study, a human liver cell line, HepG2/C3A, cultured in sex hormone-supplemented media was used to investigate the modulatory effect of genistein on CYP1A gene expression and activity. Genistein exerted both long-term (72 h) induction and short-term (immediate) inhibition of CYP1A activity in HepG2/C3A cells. In the long-term study, CYP1A gene expression and enzyme activity were induced to a greater extent in male hormone-supplemented cells than female ones. In the short-term study, CYP1A activity was inhibited more strongly by genistein in the male hormone-supplemented cells than in the female hormone-supplemented cells. These significant differences suggest that male hormones can modulate the effects of genistein on CYP1A gene expression and activity.