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

21 世纪毒理学计划 (Tox21) - 系统毒理学

• 批准号: 10469238
• 负责人: Menghang Xia
• 金额: $ 29.79万
• 依托单位: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10469238
• 关键词: 3-Dimensional; Acetylcholine; Ache; Advocate; Agonist; Anabolism; Animal Testing; Biochemical Pathway; Bioenergetics; Biological; Biological Assay; Biomedical Engineering; Biotechnology; CYP2B6 gene; CYP3A4 gene; Caenorhabditis elegans; Caffeine; Cardiac Myocytes; Cell Line; Cell model; Cell physiology; Cells; Cellular Assay; Chemical Warfare Agents; Chemicals; Chlorides; Chlorpyrifos; Cholinergic Receptors; Cholinesterases; Cilostazol; Colchicine; Collaborations; Collection; Consumption; Cosmetics; Cytidine Diphosphate Diglycerides; Data; Development; Docking; Dopamine; Drug toxicity; Elements; End Point Assay; Engineered skin; Epithelial; Ethers; European Union; Evaluation; Exposure to; Genomics; Gluconeogenesis; Glutamates; Goals; Gonadotropin-Releasing Hormone Receptor; Green Fluorescent Proteins; Guidelines; Health; Hour; Human; Immune response; In Vitro; Knock-out; Label; Laws; Lead; Leadership; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Membrane Potentials; Mercury; Metabolic Activation; Metabolism; Methods; Mission; Mitochondria; Modeling; Molecular; Motor Neurons; Muscarinic M1 Receptor; Muscarinics; National Institute of Environmental Health Sciences; National Toxicology Program; Nematoda; Nervous system structure; Neurites; Neurons; Neurotoxins; Neurotransmitters; Nuclear Receptors; Organelles; Oxygen Consumption; Parathion; Pathway interactions; Pattern; Pesticides; Pharmaceutical Preparations; Pharmacology Study; Phase; Phosphatidylglycerols; Phytochemical; Pilot Projects; Proteins; Proteomics; Publishing; Research; Research Personnel; Rotenone; Safety; Services; Signal Transduction; Skin; Specificity; Spinal; Structure; System; TP53 gene; Tacrine; Technology; Testing; Thick; Tight Junctions; Time; Tissues; Topical application; Toxic effect; Toxicology; Translational Research; Triglycerides; United States Environmental Protection Agency; United States Food and Drug Administration; United States National Institutes of Health; Validation; Work; base; bioinformatics tool; biological systems; bioprinting; computational toxicology; consumer product; cytokine; design; developmental neurotoxicity; donepezil; drug metabolism; environmental chemical; environmental toxicology; follow-up; frontier; hazard; high throughput screening; in silico; in vivo; induced pluripotent stem cell; inhibitor/antagonist; irritation; lipid biosynthesis; lipid metabolism; mitochondrial dysfunction; mitochondrial membrane; neurotoxicity; parkin gene/protein; predictive modeling; programs; protein expression; receptor; response; robotic system; screening; three dimensional cell culture; tool; toxicant; two-dimensional

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?

Tox21 计划的联邦合作伙伴包括环境保护局 (EPA)、食品和药物管理局 (FDA) 和 NIH，并由 NCATS 和国家环境健康科学研究所 (NIEHS) 的国家毒理学计划 (NTP) 领导。这些机构共同努力推进体外毒理学测试。 Tox21 计划由三个 NCATS 团队组成：系统毒理学、基因组毒理学和计算毒理学。 系统毒理学团队已鉴定、开发、优化和/或筛选了 10 多种检测方法。亮点包括针对 Tox21 机器人系统上的 LOPAC 化合物库以激动剂和拮抗剂模式进行 6 项在线验证，包括促性腺激素释放激素受体、多巴胺 D2、毒蕈碱 Ach 受体 M1 和 5 羟色胺受体 2A 测定。 线粒体是重要的细胞器，参与重要的细胞过程，包括生物能、代谢和信号传导。作为美国 Tox21 计划第二阶段 Tox21 工作的一部分，系统毒理学团队使用一系列测定（包括线粒体膜电位 (MMP)、ROS 形成），从之前针对 Tox21 10K 化合物集合的筛选中鉴定出了一组线粒体毒物、p53、Nrf2/ARE、线粒体耗氧量、细胞 Parkin 易位以及线虫线虫中的幼虫发育和 ATP 状态。为了进一步研究复合作用机制，我们对人类 AC16 人心肌细胞中从我们之前的研究 (Xia et al., 2018) 中鉴定出的几种鲜为人知的线粒体毒物进行了全局蛋白质组学分析。暴露于这些线粒体毒物后，使用基于质谱（MS）的组学技术和生物信息学工具，一些脂质代谢相关途径中的一组蛋白质的表达水平发生了显着变化，包括CDP-二酰基甘油、三酰基甘油和磷脂酰甘油生物合成。这些参与新陈代谢和能量使用重定向的蛋白质表达变化也与线粒体功能障碍有关。这项试点研究将有助于推进转化科学中的药物/毒性目标验证，例如总结表明线粒体功能障碍的蛋白质变化的一般模式。 评估刺激和致敏潜力是外用药物和化妆品等其他消费品安全性评估的关键要素。欧盟 (EU) 法律已强制要求使用先进的细胞模型来替代动物测试的产品和消费品成分。为了评估这些化合物的刺激性和致敏潜力，我们使用皮肤细胞的二维 (2D) 和三维 (3D) 培养作为替代方法，测试了约 500 种局部应用的化合物。重建人上皮 (RhE) 和全层皮肤 (FTS) 的检测终点包括活力； TEER，皮肤紧密连接的衡量标准；和细胞因子分泌物以评估局部化合物的刺激和致敏作用。这项研究代表了倡导生物工程皮肤模型取代当前动物测试的第一步。这项研究的结果发表在《生物工程和生物技术前沿》上。为了分析化合物的致敏潜力，我们使用 OECD 测试指南建议的 KeratinoSens 测定法在 qHTS 平台中筛选 Tox21 10K 化合物库。初步筛选后，我们确定了一组活性物质，并将使用一系列后续测定（包括使用 3D 生物打印组织）进一步测试它们的致敏潜力。 AChE 是体内主要的胆碱酯酶，负责代谢关键的神经递质乙酰胆碱。抑制 AChE 活性可导致神经毒性，已知的抑制剂包括有机磷农药、化学战剂、药物和各种植物化学物质。为了使用体外和计算机模型来识别抑制 AChE 活性的环境化学物质，我们识别了一组已知的 AChE 抑制剂，以及许多以前未报道的 AChE 抑制剂，例如氯化白屈菜红碱和西洛他唑。其中许多化合物，例如吡唑磷、伏杀磷和三唑磷，需要代谢活化。这项研究确定了可逆抑制剂（例如多奈哌齐和他克林）和不可逆抑制剂（例如毒死蜱和乙基溴磷）。进行分子对接分析以解释所选化合物的相对抑制效力。 随着越来越多的环境化合物被引入商业用途，需要开发可靠且有效的筛选方法来识别可能对神经系统产生不利影响的化合物。神经突生长可以作为评估化合物对神经发育的毒性的终点之一。在这项研究中，我们使用诱导多能干细胞（iPSC）衍生的人类脊髓运动神经元和皮质谷氨酸能神经元，以高含量高通量形式开发了绿色荧光蛋白（GFP）标记的神经突生长测定。我们通过筛选一组 84 种独特的化合物来验证该测定，这些化合物之前已在其他神经突生长测定中筛选过。该库由已知的发育神经毒物、未知毒性的环境化合物和阴性对照组成。神经元培养 40 小时，然后用 1.56 nM 至 92 M 不同浓度的化合物处理 24 和 48 小时。在 84 种测试化合物中，皮质神经元和运动神经元的神经突生长分别被 36 种和 31 种化合物选择性抑制。秋水仙碱、鱼藤酮和氯化甲基汞 (II) 抑制皮质和运动神经元的神经突生长。有趣的是，一些化合物，如对硫磷和双酚 AF，对皮层神经元中的神经突生长有抑制作用，而其他化合物，如 2,2',4,4'-四溴二苯醚和咖啡因，则抑制皮层神经元中的神经突生长。运动神经元。这些研究收集的数据表明，GFP 标记的 iPSC 衍生的人类神经元是一种很有前途的工具，可用于识别和优先考虑具有发育神经毒性潜力的化合物，以进一步进行危险表征。 PXR是调节药物代谢的重要核受体；最近还被证明对糖异生、癌症、脂肪生成和免疫反应有影响。为了分析激活 PXR 的化合物，我们筛选了 hPXR-luc 细胞系。确定了 4 个结构簇可高度激活 PXR，同时选择 21 种化合物根据效力、功效、结构簇和新颖性进行进一步评估。然后将这些选定的化合物在 HepaRG 细胞中进行处理，以分析 CYP3A4 和 CYP2B6 的诱导作用。十一种化合物显着诱导 CYP3A4，然后进一步分析其 PXR 活性。 HepaRG-PXR-KO 细胞也用于鉴定这 11 种化合物是否为真正的 PXR 激活剂，因为当 PXR 从细胞系中敲除时，所有 11 种化合物的活性均受到显着抑制。还进行了一项药理学研究，使用已知的选择性 PXR 抑制剂 SPA70 与所有 11 种化合物共同治疗。当用 0.5 M SPA70 处理时，每种化合物的活性向右移动，然后当与 0.75 M SPA 70 共同处理时再次向右移动。通过鉴定 23 中显示的活性，还对 11 种化合物的选择性进行了分析。其他 Tox21 检测。 该项目的研究是否产生了任何商业化或正在开发的产品或服务？