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。亮点包括执行5个在线筛选，包括针对Tox21 10K化合物文库的激动剂和拮抗剂模式的多巴胺受体D2、β-2肾上腺素能受体，以及针对Tox21机器人系统上的Lopac文库的激动剂和拮抗剂模式的β-1肾上腺素能受体分析。 美国Tox2计划利用一种定量高通量筛选(QHTS)方法，使用一组基于体外细胞的分析方法来描述数千种环境化学物质。这些检测方法的局限性，特别是那些测量与DNA损伤和修复(即遗传毒性)相关的事件的检测方法，是缺乏异种生物代谢能力。为了克服这一限制，我们研究了在现有的Tox21分析中加入代谢成分(例如，肝脏微粒体)的方法。在以前的研究中，我们已经成功地将肝微粒体整合到P53β-内酰胺酶报告基因中，并进行了AChE检测。目前，我们正在优化雄激素受体的测定，加入肝脏微粒体。在优化过程的开始使用了人肝微粒体；然而，包括氟他胺在内的阳性对照化合物都没有表现出任何活性。当Aroclor 1254诱导的大鼠肝微粒体(RLM)与氟他胺联合处理时，与不联合处理相比，发生了明显的左移。然而，由于Aroclor 1254的毒性已被禁用，因此无法获得Aroclor 1254诱导的RLM。因此，我们目前正在尝试其他化合物(如苯巴比妥)诱导的RLM来测试我们的检测系统。这一努力正在进行中。 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。为了评估这些化合物的刺激性和致敏性，我们使用单层皮肤细胞和三维培养模型测试了大约500种局部应用的化合物，包括重建的人类上皮和全层皮肤模型，通过测量紧密连接、细胞活力和细胞因子分泌来评估化学刺激和致敏性。这项研究是倡导用生物工程皮肤模型取代目前的动物试验的第一步。为了建立一种HTS可比的直接肽反应性分析(DPRA)方法，用于评估化合物的敏化潜力，我们对DPRA方法进行了改进，将传统的使用高效液相色谱平台测量游离半胱氨酸或赖氨酸肽的方法改进为高通量串联质谱仪，大大提高了筛选的吞吐量。最近，我们针对Tox21 10K化合物文库验证和筛选了角蛋白感受器Nrf2-Are-Luc实验，以鉴定具有致敏潜力的化合物。经过初步筛选，我们确定了一组Nrf2/ARE激活剂，并在包括DPRA、IL-8和人类细胞系激活试验(HCLAT)在内的一系列体外试验中对它们进行了进一步评估。这份手稿目前正在修改阶段。 我们在HEK293-GnRHR、HEK293-KISS1R和HEK293-WT细胞上进行了Tox21 10K化合物文库的在线筛选。经过初步筛选，我们鉴定并挑选了一组GnRHR和KISS1R激动剂，这些激动剂在二次确证研究中进行了进一步的测试。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检测。从这项研究中，我们发现了一组新的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)来诱导DNA损伤。鉴于许多环境化合物一旦进入人体，可能会起到荷尔蒙的作用，因此它们可能会像雌二醇一样导致DNA损伤，雌二醇是BRCA1突变女性非常担心的问题。在这项研究中，我们开发并优化了一种基于成像的高通量检测方法，用于检测DNA损伤的生物标志物--H_2AX。由于最近的研究表明，雌激素通过激活雌激素受体(ER)而导致DNA损伤，因此在人体内充当激素的环境化学物质，特别是那些能够模拟雌激素的环境化学物质，可能也会损伤DNA。使用新开发的分析方法，我们筛选了907种化合物的子集，这些化合物显示了从之前的Tox21筛选中鉴定出的生物活性。我们鉴定了四种可以诱导DNA损伤的化合物，这些损伤可以被ER抑制剂抑制。用ER内酰胺酶报告基因分析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，来妥替尼与ER配体结合部位，而不是其他三个化合物。最后，我们发现来妥替尼通过ER激活诱导了癌基因c-myc的表达。本研究确定来妥替尼是一种DNA损伤诱导剂，通过内质网激活发挥作用。这篇手稿已经发表在同行评议的期刊《当代毒理学研究》上。

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.