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

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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.