Toxicology in the 21st Century Program (Tox21) Genomic Toxicology

Tox21项目是由来自美国国立卫生研究院的研究人员，包括NCATS和国家环境健康科学研究所（NIEHS）的国家毒理学计划（NTP），环境保护局（EPA）和食品和药物管理局（FDA）的研究人员之间的联邦合作。这些机构共同努力推进体外毒理学试验。Tox21项目由以下专业组成：基因组毒理学、系统毒理学和计算毒理学。基因组毒理学团队开发了高通量基因表达技术，目标是每年从数十万个样本中生成大量人类基因的数据。RNA-seq技术在独特的固态384孔板格式下进行了优化和强大的自动化。实验成功地对384个样本中的每一个样本中代表347个细胞应激基因的1,041个分析进行了多路处理，这些分析依次汇集/多路处理，用于单个NextGen测序反应，包括大约2亿个序列。对NCATS RASL-Seq平台的全面评估显示，该平台具有出色的重复性（平均CV值为10-11%）、高通量（样品：一周内6 x 384孔板）、与qPCR的一致性为90%，且成本中等（< 12美元/个样品）。RASL-seq平台分析了61种线粒体膜调节剂、19种烟草成分和19种抗病毒核苷类似物药物的剂量效应。数据分析被编写成一系列自动化步骤，或建立RASL-seq数据分析和RNA-seq全基因组基因表达数据分析的流水线。

The Tox21 program is a federal collaboration among researchers from NIH, including NCATS and the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS), the Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA). These agencies work together to advance in vitro toxicological testing. The Tox21 program is comprised of the following specialties: Genomic Toxicology, Systems Toxicology, and Computational Toxicology. The Genomic Toxicology team has developed high-throughput gene expression technologies with the goal of annually generating data from hundreds of thousands of samples across a vast number of human genes. RNA-seq technology was optimized and robustly automated in the unique solid-state 384-well plate format. Experiments successfully multiplexed 1,041 assays representing 347 cellular stress genes in each of 384 samples, which were in turn pooled/multiplexed for a single NextGen sequencing reaction comprising approximately 200 million sequences. A thorough evaluation of the NCATS RASL-Seq platform showed excellent reproducibility (average 10-11% CV), high-throughput (samples: 6 x 384-well plates in one week), 90% concordance with qPCR, and moderate cost (<$12/sample). The RASL-seq platform was used to analyze dose-responses for 61 mitochondrial membrane modulators, 19 tobacco components and 19 antiviral nucleoside analog drugs. Data analyses were scripted into a series of automated steps, or pipeline for both RASL-seq data analyses and RNA-seq whole-genome gene expression data analyses established. The Tox21 Genomic Toxicology team also has improved cellular models for hepatocytes, neurons and vascular endothelial cells. Tox21 Phase II includes detailed analysis of compounds that are found to be active in primary quantitative high-throughput screening. This secondary screening will require improved cellular models, typically immortalized cells, or stem cells, which are differentiated in vitro into functional hepatocytes, neurons and cardiomyocytes. For neuronal toxicity, the team compared SH-SY5Y neuroblastoma cells, LUHMES conditionally immortalized dopaminergic human neurons and neuronal stem cells as models of neuronal toxicity. They found that LUHMES cells are most sensitive to most of these compounds, and most surprising, that neurons differentiated from LUHMES cells were more sensitive to 11 toxicants that the parent undifferentiated cell line. Lastly, the Tox21 Genomic Toxicology team has used induced pluripotent stem cell (iPSC) technology to generate disease-in-a-dish models. For example, in the tobacco project, both RASL-seq and RNA-seq were used to help the team understand the effects of tobacco components on vascular endothelial cells. By producing these cells using iPSC from smokers with and without vascular disease, the team was able to determine whether some patients are genetically susceptible or resistant to the disease. The Tox21 Genomic Toxicology team discovered that iPSC-derived endothelial cells (iECs) in fact responded to smoke very similarly to primary endothelial cells from three vascular beds, validating the iEC model. They repeated an RNA-seq experiment that establishes that genetic/epigenetic differences between four individuals govern distinct responses to tobacco smoke. This experiment utilized 12 iPSC-derived endothelial cell lines from the four individuals and found that the three cell lines from each individual were very reproducible. They also assessed the dose-dependent cytotoxicity and gene expression responses to 19 selected tobacco components on human umbilical vein endothelial cells as well as normal iECs.