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 6 online validations, including gonadotropin-releasing hormone receptor, Dopamine D2,, Muscarinic Ach receptor M1, and 5Hydroxytryptamine receptor 2A assays in both agonist and antagonist modes against the LOPAC compound library on the Tox21 robotic system. Mitochondria are essential cellular organelles that participate in important cellular processes, including bioenergetics, metabolism, and signaling. As part of the Tox21 effort in the phase II of U.S. Tox21 program, the Systems Toxicology team has identified a group of mitochondria toxicants from previous screen against the Tox21 10K compound collection using a panel of assays including mitochondrial membrane potential (MMP), ROS formation, p53, Nrf2/ARE, mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode C. elegans. To further study the mechanism of compound action, we have performed a global proteomic profiling of several lesser-known mitochondria toxicants identified from our previous study (Xia et al., 2018) in human AC16 human cardiomyocytes. After expose to these mitochondria toxicants, the expression level of a group of proteins has been significantly changed in several lipid metabolism related pathways including CDP-diacylglycerol, triacylglycerol and phosphatidylglycerol biosynthesis using Mass spectrometry (MS)-based omics technology and bioinformatics tools. These protein expression changes involved in metabolism and redirection of energy usage were also related to mitochondria dysfunction. This pilot study will help to advance drug/toxicity target validation in translational sciences such as summarized a general pattern of protein changes indicating mitochondrial dysfunction. Assessing irritation and sensitization potential is a key element in the safety evaluation of topical drugs and other consumer products such as cosmetics. The use of advanced cellular models as alternatives to animal testing for both products and ingredients in consumer products is already mandated by law in the European Union (EU). To evaluate the compounds for their irritation and sensitization potential, we tested about 500 topically applied compounds by using two-dimensional (2D) and three-dimensional (3D) culture of skin cells as an alternative method. The assay endpoints in reconstructed human epithelial (RhE) and full-thickness skin (FTS) include viability; TEER, a measure of the tight junctions found in skin; and cytokine secretions to assess irritation and sensitization of topical compounds. This study represents the first steps in advocating bio-engineered skin models to replace current animal tests. The findings from this study have been published in Frontiers of Bioengineering and Biotechnology. To profile compounds for their sensitization potential, we have used KeratinoSens assay suggested by the OECD test guideline to screen the Tox21 10K compound library in a qHTS platform. After the primary screening, we identified a group of actives and will further test them for their sensitization potential using a panel of the follow-up assays including use of the 3D-bioprinted tissues. 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 and in silico models, we have identified a group of known AChE inhibitors, as well as many previously not reported AChE inhibitors, such as chelerythrine chloride and cilostazol. 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. With the increasing number of environmental compounds introduced into commercial use, there is a need to develop reliable and efficient screening methods to identify compounds that may adversely impact the nervous system. Neurite outgrowth can serve one of the end points to assess compound toxicity on neuro development. In this study we developed a green fluorescent protein (GFP) labeled neurite outgrowth assay in a high-content high-throughput format using induced pluripotent stem cell (iPSC) derived human spinal motor neurons and cortical glutamatergic neurons. We validated this assay by screening a set of 84 unique compounds that have previously been screened in other neurite outgrowth assays. This library consists of known developmental neurotoxicants, environmental compounds with unknown toxicity, and negative controls. Neurons were cultured for 40 hours and then treated with compounds at various concentrations ranging from 1.56 nM to 92 M for 24 and 48 hours. Among the 84 tested compounds, neurite outgrowth in cortical neurons and motor neurons were selectively inhibited by 36 and 31 compounds, respectively. Colchicine, rotenone, and methyl mercuric (II) chloride inhibited neurite outgrowth in both cortical and motor neurons. It is interesting to note that some compounds like parathion and bisphenol AF had inhibitory effects on neurite outgrowth specifically in the cortical neurons, while other compounds, such as 2,2',4,4'-tetrabromodiphenyl ether and caffeine, inhibited neurite outgrowth in motor neurons. The data gathered from these studies show that GFP-labeled iPSC-derived human neurons are a promising tool for identifying and prioritizing compounds with developmental neurotoxicity potential for further hazard characterization. PXR is an important nuclear receptor that regulates drug metabolism; it has also recently been shown to have an impact on gluconeogenesis, cancer, lipogenesis, and the immune response. To profile the compounds that activate PXR, we have screened the hPXR-luc cell line. Four structural clusters were identified to highly activate PXR, while 21 compounds were chosen for further evaluation based on potency, efficacy, structural clustering, and novelty. These chosen compounds were then treated in HepaRG cells to analyze the induction of CYP3A4 and CYP2B6. Eleven compounds significantly induced CYP3A4 and were then further analyzed for their PXR activity. HepaRG-PXR-KO cells were also used to identify these 11 compounds as true PXR activators, as the activity of all 11 were significantly inhibited when PXR was knocked out of the cell line. A pharmacological study was also performed, using the known selective PXR inhibitor SPA70, to co-treat with all 11 compounds. Each of the compound activity shifted to the right when treated with 0.5 M of SPA70, and then shifted again to the right when co-treated with 0.75 M of SPA 70. The 11 compounds were also profiled for selectivity by identifying the activity displayed in 23 other Tox21 assays. Are any products or services commercially available or being developed that have arisen from the research in this project?