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

National Center For Advancing Translational Sciences

Investigators

Linked publications, trials & patents

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 Division of Translational Toxicology (DTT) 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 5 online screenings and validations, including androgen receptor in the presence of microsomes, peroxisome proliferator-activated receptor alpha in antagonist mode, and Wnt signaling pathway in both agonist and antagonist modes against the Tox21 10K compound library and the LOPAC library on the Tox21 robotic system. In collaboration with scientists from DTT/NIEHS, we have successfully screened the Tox21 10K compound library using calcium flux assays on HEK293-GnRHR, HEK293-KISS1R, and HEK293-WT cells. This effort identified and subsequently validated 78% of potential GnRHR agonists and 81% of KISS1R agonists, with their activity further confirmed by p-ERK assays utilizing HTRF technology to narrow down compounds activating downstream gene expression. Our research led to the pivotal discovery of musk ambrette as a novel KISS1R agonist, demonstrating concentration-dependent Gnrh1 expression stimulation in murine/human hypothalamus cells and zebrafish brains, alongside new GnRHR agonists including acetylcholine, methacholine, carbachol, bethanechol, and 2-(dimethylamino)ethyl acrylate. The effects of these agonists on GnRH pathway downstream genes (Egr1, c-Fos, c-Jun) were investigated via qRT-PCR, and molecular docking was performed to predict their binding positions. The significant findings from this agonist study were published in Endocrinology, garnered extensive media coverage from prominent outlets like Science Daily1, The Hill2, Endocrine Society3, Neuroscience News4, Newsweek5, NIH Research Matters6, and NBC News7, and were recognized as one of the top publications of 2024 by the journal's editor-in-chief, additionally earning the NIEHS Intramural Paper of the Month for December 2024, while current efforts are focused on advancing KISS1R antagonist studies. 1Science Daily: https://www.sciencedaily.com/releases/2024/09/240910120957.htm 2The Hill: https://thehill.com/policy/equilibrium-sustainability/4871684-chemicals-hormone-disruptors-puberty/ 3Endocrine Society: https://www.endocrine.org/news-and-advocacy/news-room/2024/girls-may-start-puberty-early-due-to-chemical-exposure 4Neuroscience News: https://neurosciencenews.com/endocrinology-puberty-chemicals-27643/ 5Newsweek: https://www.newsweek.com/girls-may-start-puberty-early-toxic-chemicals-1952718 6NIH Research Matters: https://www.nih.gov/news-events/nih-research-matters/certain-chemicals-may-trigger-early-puberty-girls 7NBC News: https://www.nbcnews.com/health/womens-health/early-puberty-may-linked-common-chemical-used-personal-care-products-rcna169967 Sites contaminated with hazardous materials often have polluted soil, air, and water, posing significant health risks to those living and working nearby. Individuals in these environments are frequently exposed to multiple metals and other hazardous substances simultaneously. However, the combined effects of these exposures are not well understood. In this study, we examined the toxicity of metal mixtures using five categories of in vitro assays: cytotoxicity, oxidative stress, genotoxicity, cytokine release, and angiogenesis. In the cytotoxicity assay with primary human cells, we observed that mixtures containing nickel/arsenic/cadmium and beryllium/arsenic/cadmium demonstrated greater cytotoxicity than the single components, indicating that the mixtures amplify toxic effects. To further explore the mechanisms involved, we assessed metal-induced oxidative stress, which is a common pathway for metal toxicity. We found that oxidative stress caused by cadmium was further increased when combined with other metal compounds. The enhanced oxidative stress further elevated DNA damage, inhibited DNA synthesis, and activated the p53 pathway. Additionally, they significantly increased the secretion of interleukin-8 and promoted angiogenesis more than the component compounds. These findings, published in Environmental Science & Technology, provide new insights into the risks associated with metal contamination in environmental settings. The Cytochrome P450 (CYP) family of enzymes are membrane-bound hemeproteins that play a crucial role in the metabolism of drugs and xenobiotics. Individual CYPs are classified into families and subfamilies based on their amino acid sequence similarities. Over 50 CYPs have been identified in humans, with the CYP1, 2, and 3 families primarily responsible for metabolizing approximately 80% of clinical drugs. To assess the impact of environmental chemicals on the activities of these key CYP enzyme families, we screened the Tox21 10K compound library to identify inhibitors of CYP1A2, 2C9, 2C19, 2D6, and 3A4 enzymes. The data obtained from these screenings were analyzed to determine the structural classes responsible for inhibiting multiple and/or selective CYPs. Known structural classes exhibiting pan-CYP inhibition, such as azole fungicides, were confirmed, along with established clinical inhibitors of CYPs, including erythromycin and verapamil for CYP3A4, and paroxetine and terbinafine for CYP2D6. Additionally, previously unknown but potent selective CYP inhibitors (IC50 values < 1 µM) were identified, such as yohimbine, an indole alkaloid, inhibiting CYP2D6, and loteprednol, a corticosteroid, inhibiting CYP3A4. These findings suggest that evaluating a candidate compound's impact on CYP function can help preemptively mitigate potential adverse reactions and toxicity during drug development or the toxicological characterization of environmental chemicals. ICRF193 is a catalytic inhibitor of Topoisomerase 2 (TOP2), one of the major targets in cancer therapy. Although ICRF193 has not been approved for clinical use, it has potential implications in chemotherapy. In this study, we explored the potential of ICRF193 in combination with other drugs to enhance therapeutic outcomes. We optimized a high throughput cytotoxicity assay using a 1536-well plate format and screened 2,678 compounds, including both approved and investigational drugs, for their cytotoxic effects in the presence and absence of ICRF193. We found that etoposide, a known TOP2-targeting drug, has a synergistic effect with ICRF193 across several cancer cell lines, including HCT116, MCF7, and T47D. Interestingly, at higher concentrations (>10 µM), ICRF193 reduced the cytotoxicity of etoposide. Follow-up studies showed that the combination of ICRF193 and etoposide synergistically induced DNA double-strand breaks and led to G2 phase cell cycle accumulation. Notably, this synergistic effect was unique to etoposide and was not observed with other TOP2 inhibitors in our compound library. Overall, our results suggest that ICRF193 interacts specifically with etoposide to enhance its genotoxic effects. The pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, plays a critical role in regulating drug metabolism and clearance. Recent studies have also implicated PXR in the modulation of cell proliferation, apoptosis, immune response, and energy homeostasis. Identifying compounds that modulate PXR activity is crucial for preventing drug-drug interactions, identifying chemicals that could potentially cause toxicity, and developing therapeutic agents. In this study, we screened the National Center for Advancing Translational Sciences (NCATS) Pharmacologically Active Chemical Toolbox library, consisting of 5,099 unique synthetic and naturally derived small molecules, using a HepG2-CYP3A4-hPXR cell line to identify PXR antagonists. From the primary screening, 94 compounds were identified as potential PXR antagonists, and 66 were confirmed in subsequent validation experiments. Out of these, 20 potential PXR antagonists were selected for further investigation, including pharmacological shift assays, mRNA gene expression analysis, and molecular docking studies. Four compounds, GSM2, JNJ-31020028, fusidic acid, and PU-H71, were found to inhibit CYP3A4 mRNA expression in HepaRG cells, a physiologically relevant cell line. Among these, GSM2 and fusidic acid demonstrated the necessary pharmacological shift results in both HepG2 and HepaRG cells, confirming their PXR antagonistic activity. Additionally, these two compounds successfully docked into the PXR ligand binding domain, indicating their binding capability. GSM2 and fusidic acid have been identified as novel PXR antagonists, providing valuable insights for further investigation into drug-drug interactions and the potential therapeutic or toxic effects of these compounds