Toxicology in the 21st Century Program (Tox21) - Systems Toxicology

National Center For Advancing Translational Sciences

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Abstract

The Tox21 programs federal partners include the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) and NIH, with leadership from NCATS and the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS). These agencies work together to advance in vitro toxicological testing. The Tox21 Program is comprised of three NCATS teams: Systems Toxicology, Genomic Toxicology, and Computational Toxicology. The Systems Toxicology team has identified, developed, optimized, and/or screened more than 10 assays. Highlights range from performing 4 online screenings, including gonadotropin-releasing hormone receptor, Muscarinic Ach receptor M1, Kisspeptin receptor, and 5Hydroxytryptamine receptor 2A assays in both agonist and antagonist modes against the Tox21 10K compound library on the Tox21 robotic system. The US Tox2Program has utilized a quantitative high throughput screening (qHTS) approach to profile thousands of environmental chemicals using a battery of in vitro cell-based assays. The limitation of these assays, particularly those that measure events associated with DNA damage and repair (i.e., genotoxicity), is the absence of a xenobiotic metabolism capability. To overcome this limitation, we investigated methods to incorporate a metabolic component (e.g., liver microsomes) into existing Tox21 assays. We used a p53 beta-lactamase reporter gene assay (p53-bla) as a model system to incorporate metabolic capability into qHTS assays. We screened the Tox21 10K compound library using a p53-bla assay alone or with rat liver microsomes (RLM) or human liver microsomes (HLM) supplemented with NADPH, to identify compounds that induce p53 signaling after biotransformation. Two hundred seventy-eight compounds were identified as active under any of these three conditions. Of these 278 compounds, 73 gave more potent responses in the p53-bla plus RLM assay, and 2 were more potent in the p53-bla plus HLM assay compared with the responses they generated in the p53-bla assay without microsomes. To confirm the role of metabolism in the differential responses, we retested these 75 compounds in the absence of NADPH or with heat-attenuated microsomes. Forty-four compounds treated with RLM, but none with HLM, became less potent under these conditions, confirming the role of RLM in metabolic activation. Further evidence of biotransformation was obtained by measuring the half-life of the parent compounds in the presence of microsomes. Taken together, our study indicates that this approach can identify compounds that are not be detected by standard screening methods that lack metabolic capability. Assessing irritation and sensitization potential is a key element in the safety evaluation of topical drugs and other consumer products such as cosmetics. To evaluate the compounds for their irritation and sensitization potential, we tested about 500 topically applied compounds by using monolayer skin cells and three-dimensional culture models including reconstructed human epithelial and full-thickness skin models by measuring tight junctions, cell viability, and cytokine secretions for assessing chemical irritation and sensitization. This study represents the first step in advocating for replacement of current animal tests with bio-engineered skin models. To develop an HTS comparable method of direct peptide reactivity assay (DPRA) that has been used for assessing compound sensitization potential, we modified DPRA assay measuring the amount of free cysteine or lysine peptide from traditionally using a high-performance liquid chromatography platform to a high throughput tandem mass spectrum system, which greatly increases the screening throughput. Recently, we have validated and screened KeratinoSens Nrf2-ARE-Luc assay against the Tox21 10K compound library to identify the compounds with sensitization potential. After primary screening, we identified a group of Nrf2/ARE activators and further evaluated them in a battery of in vitro assays including DPRA, IL-8, and human cell line activation test (hCLAT). AChE is the primary cholinesterase in the body that metabolizes a key neurotransmitter, acetylcholine. Inhibition of AChE activity can lead to neurotoxicity and known inhibitors include organophosphorus pesticides, chemical warfare agents, drugs, and various phytochemicals. To identify environmental chemicals that inhibit AChE activity using in vitro models, we have used enzyme- and cell-based AChE inhibition assay to screen the Tox21 10K compound library. From the primary screening, over 100 AChE inhibitors have been identified. These compounds were further studied their AChE inhibition in SH-SY5Y and human neuron stem cells (hNSC) in monolayer culture vs spheroid culture. Some AChE inhibitors showed more potency in hNSC spheroid culture compared to monolayer culture, while higher expression levels of CYP3A4 and CYP2D6 were found in spheroids than in monolayer culture, suggesting CYP enzymes were involved in bioactivation. To explore the interactions between AChE and their inhibitors, we used a molecular docking analysis to study the binding mode of these AChE inhibitors and found that all the AChE inhibitors were predicted to bind the active sites of the AChE. Using this approach, we identified a group of known AChE inhibitors and many previously not reported AChE inhibitors including some AChE inhibitors that need metabolic activation. Many of these AChE inhibitors were marketed drugs, withdrawn drugs, or unapproved drug candidates. They compared IC50s of these AChE inhibitors with human plasma concentrations (Cmax) from the literature and calculated the ratios of IC50 over Cmax. A common benchmark indicating physiological relevance is IC50/Cmax < 10. Using these ratio values can prioritize AChE inhibitors for further in-depth study. Since the outbreak of a global pandemic coronavirus disease-19 (COVID-19), various potential therapeutic agents for COVID-19 are being investigated worldwide mainly through the drug repurposing approach. Many drugs employed as anti-viral may exert unwanted side effects (i.e., toxicity) via unknown mechanisms. To quickly assess these drugs for their potential toxicological effects and mechanisms, we used the Tox21 in vitro assay datasets generated from screening 10,000 compounds consisting of approved drugs and environmental chemicals against multiple cellular targets and pathways. Many these drugs were shown to be active modulators in our nuclear receptor signaling pathway assays. Several signaling pathways including AR, Nrf2/ARE, ER, FXR, ER stress, and targets like HDAC were reported for their role in regulating ACE2 and TMPRSS2 expression, which are the main host cell factors that aid in SARS-CoV-2 pathogenicity. Some of the drugs in our study were shown to be cytotoxic against a wide range of cells and they include chlorpromazine, curcumin, emetine, lopinavir, ritonavir, niclosamide, and nitazoxanide. The cytotoxicity of these compounds might be due to their target promiscuity as shown by their activities in multiple Tox21 in vitro assays. Although in vitro cytotoxicity assays were used to screen chemicals/drugs for their relative toxicities at micro-molar concentrations, these assays can identify a potential hazard due to the multiple uses or high doses of the drugs when administered in humans. These qHTS data are publicly available and can provide valuable information on drug activities and their off-target effects, which can be further investigated for their potential uses in treating COVID-19 infection.