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

Tox21项目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，NCATS和国家环境健康科学研究所(NIEHS)的国家毒理学计划(NTP)领导下的合作伙伴。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。 系统毒理学团队已经确定、开发、优化和/或筛选了10多种分析方法。亮点包括在Tox21机器人系统上进行6次在线筛选，包括MSTI/电泳法，使用或不使用肝微粒体的P53分析，针对Tox21 10K化合物文库的CREB，CYP2C9和CYP2D6。 线粒体是参与重要细胞过程的重要细胞器，包括生物能量学、新陈代谢和信号传递。作为美国Tox21计划第二阶段Tox21工作的一部分，系统毒理学团队已经针对Tox21 10K化合物集合使用了一系列分析方法，包括线粒体膜电位(MMP)、ROS形成、P53、Nrf2/ARE、线粒体耗氧量、细胞Parkin易位以及线虫幼虫发育和ATP状态。为了进一步研究复合作用的机制，我们在人AC16人心肌细胞中对我们之前的研究(夏等人，2018年)中发现的几种鲜为人知的线粒体毒物进行了全球蛋白质组学研究。利用基于MS的组学技术和生物信息学工具，在线粒体毒物作用下，CDP-二酰甘油、三酰甘油和磷脂酰甘油生物合成途径中一组蛋白质的表达水平发生了显著变化。这些参与代谢和能量使用重新定向的蛋白质表达变化也与线粒体功能障碍有关。这项先导性研究将有助于推进翻译科学中的药物/毒性靶点验证，例如总结表明线粒体功能障碍的蛋白质变化的一般模式。描述这项研究的手稿已被接受发表在《遗传学前沿》上。 评估刺激性和致敏潜力是局部药物和其他消费品(如化妆品)安全性评估中的一个关键因素。欧洲联盟(EU)的法律已经规定，在消费品的产品和成分中使用先进的细胞模型作为动物试验的替代品。为了评估这些化合物的刺激性和致敏潜力，我们使用二维(2D)和三维(3D)皮肤细胞培养作为替代方法，测试了大约500种局部应用的化合物。重建的人体上皮(RHE)和全层皮肤(FTS)中的检测终点包括活性；TEER，皮肤中发现的紧密连接的衡量标准；以及细胞因子分泌，以评估局部化合物的刺激性和致敏性。这项研究是倡导生物工程皮肤模型取代当前动物试验的第一步。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，我们使用了OECD测试指南建议的角蛋白Sens试验在qHTS平台上筛选Tox21 10K化合物文库。在初步筛选后，我们确定了一组活性物质，并将使用包括使用3D生物打印组织在内的一系列后续测试来进一步测试它们的致敏潜力。 AChE是体内主要的胆碱酯酶，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。在CFSAN/FDA、NTP和EPA的合作下，系统毒理学团队从初步筛选中确定了一组AchE抑制剂，并进行了一组后续试验，以进一步研究化合物的作用机制。这份手稿总结了实验结果，已提交出版。 PXR是一种重要的核受体，调节药物代谢；最近也被证明对糖异生、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个高活性PXR的结构簇，选择了20个化合物进行进一步的效力、有效性、结构簇和新颖性评估。这些选定的化合物然后在HepaRG细胞中处理，以分析CYP3A4和CYP2B6的诱导。11个化合物对细胞色素P3A4有明显的诱导作用，并对其PXR活性进行了分析。这些化合物进一步在HepaRG-PXR-KO细胞系中进行测试，以确认PXR的参与。手稿目前正在准备中。

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 screenings, including MSTI/electrophile assay, p53 assay with or without liver microsomes, CREB, CYP2C9 and CYP2D6 against the Tox21 10K 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. The manuscript describing this study has been accepted for publication in Frontiers in Genetics. 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. In collaboration with the CFSAN/FDA, NTP and EPA, the Systems Toxicology team has identified a group of AChE inhibitors from the primary screening and performed a group of follow-up assays to further study the mechanism of compound action. The manuscript summarized the experimental findings has been submitted for the publication. 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 20 compounds were selected 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 analyzed for their PXR activity. These compounds were further tested in a HepaRG-PXR-KO cell line to confirm the involvement of PXR. The manuscript is currently under preparation.