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

21 世纪毒理学计划 (Tox21) - 系统毒理学

基本信息

批准号：
10901692
负责人：
Menghang Xia
金额：
$ 76.51万
依托单位：
NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
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/10901692
关键词：
3-Dimensional Ache Advocate Agonist Androgen Receptor Animal Testing Aroclor 1254 BRCA1 Mutation Binding Binding Sites Biochemical Pathway Biological Biological Assay Biological Markers Biomedical Engineering Cell Survival Cells Cellular Assay Chemical Actions Chemicals Collaborations Consumption Cosmetics Cysteine DNA Damage DNA Repair Docking Dopamine D2 Receptor Elements Epithelium Estradiol Estrogen Receptors Evaluation Event Female Flutamide Genomics Goals Health High Pressure Liquid Chromatography Hormones Human Human Cell Line Human body IL8 gene Image In Vitro Journals Lactamase Leadership Left Libraries Ligand Binding Liver Microsomes Lysine Manuscripts Mass Spectrum Analysis Measures Metabolic Methods Microsomes Mission Modeling Molecular National Center for Advancing Translational Sciences National Institute of Environmental Health Sciences National Toxicology Program Oncogenes Pathway interactions Peer Review Peptides Pharmaceutical Preparations Phenobarbital Preparation Publishing Rattus Receptor Activation Receptors, Adrenergic, beta-1 Reporter Genes Research Research Personnel Safety Skin Specificity System TP53 gene Technology Testing Thick Tight Junctions Time Topical application Toxic effect Toxicology United States Environmental Protection Agency United States Food and Drug Administration United States National Institutes of Health Work Xenobiotic Metabolism antagonist beta-2 Adrenergic Receptors beta-Lactamase biological systems c-myc Genes cell dimension computational toxicology consumer product cytokine design environmental chemical environmental toxicology genotoxicity high throughput screening in vitro Assay in vivo inhibitor irritation monolayer novel predictive modeling process optimization programs receptor binding response robotic system screening screening program

项目摘要

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 5 online screenings, including dopamine receptor D2, beta-2 adrenergic receptor in both agonist and antagonist modes against the Tox21 10K compound library and beta-1 adrenergic receptor assay in both agonist and antagonist modes against the LOPAC library on the Tox21 robotic system. The US Tox2 Program has utilized a quantitative high throughput screening (qHTS) approach to profile thousands of environmental chemicals using a battery of in vitro cell-based assays. The limitation of these assays, particularly those that measure events associated with DNA damage and repair (i.e., genotoxicity), is the absence of a xenobiotic metabolism capability. To overcome this limitation, we investigated methods to incorporate a metabolic component (e.g., liver microsomes) into existing Tox21 assays. In the previous studies, we have successfully incorporated liver microsomes into p53 beta-lactamase reporter gene and AChE assays. Currently, we are optimizing androgen receptor assay with liver microsomes addition. Human liver microsomes were used in the beginning of the optimization process; however, none of the positive control compounds including flutamide showed any activity. When Aroclor 1254-induced rat liver microsomes (RLM) were co-treated with flutamide, a clear left shift occurred when compared to no microsome co-treatment. However, Aroclor 1254-induced RLM was not available due to its toxicity that has been banned. Therefore, we are currently trying other compound (e.g., phenobarbital) induced RLM to test our assay system. This effort is ongoing. Assessing irritation and sensitization potential is a key element in the safety evaluation of topical drugs and other consumer products such as cosmetics. To evaluate the compounds for their irritation and sensitization potential, we tested about 500 topically applied compounds by using monolayer skin cells and three-dimensional culture models including reconstructed human epithelial and full-thickness skin models by measuring tight junctions, cell viability, and cytokine secretions for assessing chemical irritation and sensitization. This study represents the first step in advocating for replacement of current animal tests with bio-engineered skin models. To develop an HTS comparable method of direct peptide reactivity assay (DPRA) that has been used for assessing compound sensitization potential, we modified DPRA assay measuring the amount of free cysteine or lysine peptide from traditionally using a high-performance liquid chromatography platform to a high throughput tandem mass spectrum system, which greatly increases the screening throughput. Recently, we have validated and screened KeratinoSens Nrf2-ARE-Luc assay against the Tox21 10K compound library to identify the compounds with sensitization potential. After primary screening, we identified a group of Nrf2/ARE activators and further evaluated them in a battery of in vitro assays including DPRA, IL-8, and human cell line activation test (hCLAT). The manuscript is currently in revision stage. We have performed online screening against the Tox21 10K compound library by using screened on HEK293-GnRHR, HEK293-KISS1R, and HEK293-WT cells. After primary screening, we identified, and cherry picked a group of GnRHR and KISS1R agonists that were further tested in the secondary confirmation study. Among potential GnRHR and KISS1R agonists, 78% of compounds were confirmed as GnRHR agonists and 81% compounds were confirmed as KISS1R agonists. To further validate the activity of these agonists, we performed a p-ERK assay using HTRF technology. From this study we have identified a group of novel GnRHR and KISS1R agonists. The manuscript is under preparation. Recent studies indicate estradiol induces DNA damage through the activation of the estrogen receptor (ER). Given that many environmental chemical compounds may act like hormones once they enter the human body, it is possible that they induce DNA damage in the same way as estradiol, which is of great concern to females with the BRCA1 mutation. In this study, we developed and optimized an imaging-based high-throughput assay measuring H2AX, a biomarker for DNA damage. As recent research indicates that estradiol induces DNA damage by activating estrogen receptors (ER), it is possible that environmental chemicals that act as hormones in the human body, particularly those that can mimic estradiol, may also damage DNA. Using the newly developed assay, we screened a subset of 907 chemical compounds that showed bioactivity identified from previous Tox21 screenings. We identified four compounds that induced DNA damage that can be suppressed by an ER inhibitor. The ER binding activity of these compounds was investigated using ER lactamase reporter gene assay and molecular docking analysis. These analyses indicated that lestaurtinib bind to ER ligand binding site but not the other three compound. Finally, we found that lestaurtinib induced the expression of an oncogene, c-MYC, through ER activation. This study identified lestaurtinib as a DNA damage inducer that acts through ER activation. The manuscript has been published in a peer-reviewed journal, Current Research in Toxicology.

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。亮点包括进行 5 项在线筛查，包括针对 Tox21 10K 化合物库的激动剂和拮抗剂模式下的多巴胺受体 D2、β-2 肾上腺素受体测定，以及针对 Tox21 上的 LOPAC 库的激动剂和拮抗剂模式下的 β-1 肾上腺素受体测定机器人系统。美国 Tox2 计划采用定量高通量筛选 (qHTS) 方法，通过一系列体外细胞分析来分析数千种环境化学物质。这些检测的局限性，特别是那些测量与 DNA 损伤和修复（即基因毒性）相关的事件的检测，是缺乏外源代谢能力。为了克服这一限制，我们研究了将代谢成分（例如肝微粒体）纳入现有 Tox21 检测的方法。在之前的研究中，我们已成功将肝微粒体纳入p53 β-内酰胺酶报告基因和AChE检测中。目前，我们正在通过添加肝微粒体来优化雄激素受体测定。在优化过程的开始阶段使用了人肝微粒体；然而，包括氟他胺在内的阳性对照化合物均未显示出任何活性。当 Aroclor 1254 诱导的大鼠肝微粒体 (RLM) 与氟他胺联合治疗时，与没有微粒体联合治疗相比，出现了明显的左移。然而，Aroclor 1254 诱导的 RLM 由于其毒性已被禁止而无法使用。因此，我们目前正在尝试其他化合物（例如苯巴比妥）诱导 RLM 来测试我们的测定系统。这项努力正在进行中。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。为了评估这些化合物的刺激性和致敏潜力，我们通过测量紧密连接、细胞活力和细胞因子分泌，使用单层皮肤细胞和三维培养模型（包括重建的人类上皮和全层皮肤模型）测试了约 500 种局部应用的化合物用于评估化学刺激性和致敏性。这项研究代表了倡导用生物工程皮肤模型取代当前动物测试的第一步。为了开发一种可用于评估化合物致敏潜力的直接肽反应性测定 (DPRA) 的 HTS 类似方法，我们将测量游离半胱氨酸或赖氨酸肽量的 DPRA 测定法从传统使用高效液相色谱平台修改为高效液相色谱平台。通量串联质谱系统，大大提高了筛选通量。最近，我们针对 Tox21 10K 化合物库验证和筛选了 KeratinoSens Nrf2-ARE-Luc 检测，以识别具有致敏潜力的化合物。经过初步筛选后，我们鉴定了一组 Nrf2/ARE 激活剂，并通过一系列体外测定（包括 DPRA、IL-8 和人类细胞系激活测试 (hCLAT)）进一步评估它们。目前手稿正处于修改阶段。我们通过对 HEK293-GnRHR、HEK293-KISS1R 和 HEK293-WT 细胞进行筛选，对 Tox21 10K 化合物库进行了在线筛选。经过初步筛选后，我们确定并挑选了一组 GnRHR 和 KISS1R 激动剂，并在二次确认研究中进行了进一步测试。在潜在的GnRHR和KISS1R激动剂中，78%的化合物被确认为GnRHR激动剂，81%的化合物被确认为KISS1R激动剂。为了进一步验证这些激动剂的活性，我们使用 HTRF 技术进行了 p-ERK 测定。从这项研究中，我们鉴定了一组新型 GnRHR 和 KISS1R 激动剂。手稿正在准备中。最近的研究表明雌二醇通过激活雌激素受体 (ER) 诱导 DNA 损伤。鉴于许多环境化学化合物进入人体后可能会像激素一样发挥作用，它们可能会像雌二醇一样引起DNA损伤，这是BRCA1突变女性非常关心的问题。在这项研究中，我们开发并优化了一种基于成像的高通量测定法，用于测量 H2AX（DNA 损伤的生物标志物）。最近的研究表明，雌二醇通过激活雌激素受体 (ER) 来诱导 DNA 损伤，因此，在人体内充当激素的环境化学物质，特别是那些可以模仿雌二醇的化学物质，也可能会损伤 DNA。使用新开发的检测方法，我们筛选了 907 种化合物的子集，这些化合物显示出从之前的 Tox21 筛选中鉴定出的生物活性。我们鉴定了四种可诱导 DNA 损伤的化合物，而这些损伤可以被 ER 抑制剂抑制。使用 ER 内酰胺酶报告基因测定和分子对接分析研究了这些化合物的 ER 结合活性。这些分析表明来他替尼与 ER 配体结合位点结合，但不与其他三种化合物结合。最后，我们发现来他替尼通过 ER 激活诱导癌基因 c-MYC 的表达。这项研究确定 Lestaurtinib 是一种 DNA 损伤诱导剂，通过 ER 激活发挥作用。该手稿已发表在同行评审期刊《毒理学当前研究》上。

项目成果

期刊论文数量（73）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Identification of Angiogenesis Inhibitors Using a Co-culture Cell Model in a High-Content and High-Throughput Screening Platform.

在高内涵和高通量筛选平台中使用共培养细胞模型鉴定血管生成抑制剂。

DOI：
发表时间：
2018-06
期刊：
影响因子：
2.7
作者：
Li, Shuaizhang;Hsu, Chia;Sakamuru, Srilatha;Zou, Chaozhong;Huang, Ruili;Xia, Menghang
通讯作者：
Xia, Menghang

High-Throughput Screening to Predict Chemical-Assay Interference.

高通量筛选以预测化学测定干扰。

DOI：
发表时间：
2020-03-04
期刊：
影响因子：
4.6
作者：
Borrel, Alexandre;Huang, Ruili;Sakamuru, Srilatha;Xia, Menghang;Simeonov, Anton;Mansouri, Kamel;Houck, Keith A;Judson, Richard S;Kleinstreuer, Nicole C
通讯作者：
Kleinstreuer, Nicole C

Two-Dimensional Cellular and Three-Dimensional Bio-Printed Skin Models to Screen Topical-Use Compounds for Irritation Potential.

二维细胞和三维生物打印皮肤模型，用于筛选局部使用化合物的潜在刺激性。

DOI：
发表时间：
2020
期刊：
影响因子：
0
作者：
Wei, Zhengxi;Liu, Xue;Ooka, Masato;Zhang, Li;Song, Min Jae;Huang, Ruili;Kleinstreuer, Nicole C;Simeonov, Anton;Xia, Menghang;Ferrer, Marc
通讯作者：
Ferrer, Marc

Resources for Developing Reliable and Reproducible In Vitro Toxicological Test Methods.

用于开发可靠且可重复的体外毒理学测试方法的资源。

DOI：
发表时间：
2021-06-21
期刊：
影响因子：
4.1
作者：
Petersen, Elijah J;Reipa, Vytautas;Xia, Menghang;Sharma, Monita
通讯作者：
Sharma, Monita

Predictive Models to Identify Small Molecule Activators and Inhibitors of Opioid Receptors.

识别阿片受体小分子激活剂和抑制剂的预测模型。

DOI：
发表时间：
2021
期刊：
影响因子：
0
作者：
Sakamuru, Srilatha;Zhao, Jinghua;Xia, Menghang;Hong, Huixiao;Simeonov, Anton;Vaisman, Iosif;Huang, Ruili
通讯作者：
Huang, Ruili