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

Tox 21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。Tox 21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。 系统毒理学团队已确定、开发、优化和/或筛选了10多种试验。亮点包括进行5次在线筛选，包括多巴胺受体D2、针对Tox 21 10 K化合物库的激动剂和拮抗剂模式下的β-2肾上腺素能受体，以及针对Tox 21机器人系统上的LOPAC库的激动剂和拮抗剂模式下的β-1肾上腺素能受体测定。 美国Tox 2计划利用定量高通量筛选（qHTS）方法，使用一系列基于细胞的体外测定来分析数千种环境化学品。这些测定的局限性，特别是那些测量与DNA损伤和修复相关的事件（即，遗传毒性）是缺乏异生物质代谢能力。为了克服这一限制，我们研究了将代谢组分（例如，肝微粒体）引入现有的Tox 21测定中。在以前的研究中，我们已经成功地将肝微粒体纳入p53 β-内酰胺酶报告基因和AChE检测。目前，我们正在优化雄激素受体测定与肝微粒体添加。在优化过程开始时使用人肝微粒体;然而，包括氟替卡松在内的阳性对照化合物均未显示任何活性。当Aroclor 1254诱导的大鼠肝微粒体（RLM）与氟替卡松共同处理时，与无微粒体共同处理相比，发生了明显的左移。然而，Aroclor 1254诱导的RLM由于其毒性已被禁止而不可用。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。这项工作正在进行中。 评估刺激和致敏潜力是局部药物和其他消费品（如化妆品）安全性评价的关键要素。为了评估化合物的刺激和致敏潜力，我们使用单层皮肤细胞和三维培养模型（包括重建的人上皮和全层皮肤模型）测试了约500种局部应用的化合物，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作用。这项研究代表了倡导用生物工程皮肤模型取代当前动物试验的第一步。为了开发用于评估化合物致敏潜力的直接肽反应性测定（DPRA）的HTS可比方法，我们将测量游离半胱氨酸或赖氨酸肽的量的DPRA测定从传统的使用高效液相色谱平台修改为高通量串联质谱系统，这大大提高了筛选通量。最近，我们已经针对Tox 21 10 K化合物库验证和筛选了KeratinoSens Nrf 2-ARE-Luc测定，以鉴定具有致敏潜力的化合物。初步筛选后，我们确定了一组Nrf 2/ARE激活剂，并在体外试验，包括DPRA，IL-8，和人类细胞系激活试验（hCLAT）电池进一步评估他们。该手稿目前处于修订阶段。 我们已经通过使用在HEK 293-GnRHR、HEK 293-KISS 1 R和HEK 293-WT细胞上筛选的对Tox 21 10 K化合物文库进行了在线筛选。初步筛选后，我们确定并挑选了一组GnRHR和KISS 1 R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KISS 1 R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KISS 1 R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，我们已经确定了一组新的GnRHR和KISS 1 R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一旦进入人体就可能像激素一样起作用，它们可能会以与雌二醇相同的方式诱导DNA损伤，这对BRCA 1突变的女性来说非常重要。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，用于测量DNA损伤的生物标志物H2 AX。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，可能是环境中的化学物质在人体内起激素作用，特别是那些可以模拟雌二醇的化学物质，也可能损伤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以前的Tox 21筛选中鉴定出的生物活性。我们确定了四种诱导DNA损伤的化合物，这些化合物可以被ER抑制剂抑制。采用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，来鲁替尼结合ER配体结合位点，但不结合其他三种化合物。最后，我们发现，来鲁替尼通过ER激活诱导癌基因c-MYC的表达。这项研究确定了来替尼作为一种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《当前毒理学研究》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.