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The Tox21 robotic platform for the assessment of environmental chemicals – from vision to reality

https://doi.org/10.1016/j.drudis.2013.05.015Get rights and content

Highlights

  • The Tox21 effort is to advance in vitro toxicological testing in the 21st century.

  • Tox21 chemical library contains approximately 10,000 environmental chemicals.

  • A battery of in vitro assays will be validated and screened in a qHTS platform.

  • Tox21 robotic system is capable of screening the Tox21 chemical library in triplicates in a week.

Since its establishment in 2008, the US Tox21 inter-agency collaboration has made great progress in developing and evaluating cellular models for the evaluation of environmental chemicals as a proof of principle. Currently, the program has entered its production phase (Tox21 Phase II) focusing initially on the areas of modulation of nuclear receptors and stress response pathways. During Tox21 Phase II, the set of chemicals to be tested has been expanded to nearly 10,000 (10K) compounds and a fully automated screening platform has been implemented. The Tox21 robotic system combined with informatics efforts is capable of screening and profiling the collection of 10K environmental chemicals in triplicate in a week. In this article, we describe the Tox21 screening process, compound library preparation, data processing, and robotic system validation.

Introduction

Toxicity assessment of environmental chemicals has traditionally relied on animal-based toxicological methods, but such studies are low-throughput, costly, and inconsistently predictive of human toxicity. To potentially overcome these limitations and to rapidly and efficiently evaluate the thousands of environmental chemicals with little or no prior toxicological information, the National Toxicology Program (NTP) [1] proposed a roadmap for toxicology testing in the 21st century promoting the advancement of toxicology from a mainly observational science at the level of disease-specific and animal-based models to a predictive science focused upon a broad inclusion of target-specific mechanistic approaches [1]. In 2005, the National Research Council (NRC) [2] was commissioned by the U.S. Environmental Protection Agency [EPA, http://www.epa.gov/ncct (accessed 02.04.13)] and the National Institute of Environmental Health Sciences to develop a long-range vision and strategic plan for toxicity testing [3]. This plan emphasized the use of new tools in molecular toxicology, computational sciences, and information technology to address the challenge of evaluating the thousands of untested chemicals present in the environment [2], stating that traditional toxicity testing methods are inadequate due to the high cost and the vast number of animals that would be needed to characterize these chemicals. Both the NTP and NRC visions rely on the use of chemical profiling strategies to study crucial cellular responses to xenobiotics using primarily high-throughput screening (HTS)-based in vitro human cell models. The use of cellular targets or ‘toxicity’ pathway perturbations as new, discrete toxicological endpoints constitutes the first step to identify mechanisms of toxicity for each xenobiotic and to provide predictive potential. Mechanistic findings would be used to prioritize chemicals for more in-depth, targeted in vitro or in vivo testing leading to the development and refinement of predictive toxicological models [4].

To accomplish this vision, the Tox21 collaboration was formed between the NTP, the EPA National Center for Computational Toxicology (NCCT), and the NIH Chemical Genomics Center (NCGC) in 2008 [3] with the addition of the Food and Drug Administration (FDA) in 2010. Each organization brings a different set of complementary skills including expertise in experimental toxicology, computational toxicology, and HTS technologies. During Tox21 Phase I (proof of principle), over 75 biochemical- and cell-based assays were successfully used to screen the initial Tox21 collection of approximately 2800 compounds in a quantitative high-throughput screening (qHTS) platform using 1536-well plate format. A comprehensive list and thorough description of the Phase I assays can be found elsewhere 5, 6, 7. Briefly, these assays interrogated different aspects of cellular physiology including overall cellular health (cytotoxicity and apoptosis induction, DNA damage), perturbation of cell signaling pathways [e.g. ARE/Nrf2, antioxidant response element/NF-E2 related factor 2; CREB, cAMP response element binding; hypoxia-inducible factor 1 alpha (HIF-1α)], inflammatory response induction [e.g. nuclear factor kappa B (NFκB); tumor necrosis factor alpha (TNFα); interleukin-8 (IL-8)], nuclear receptor modulation [e.g. androgen receptor (AR); estrogen receptor (ER)] as well as specific isolated cellular targets including enzyme inhibition, membrane transport inhibition, hERG (human ether-a-go-go) channel inhibition, receptor binding and specific protein–protein interaction disruption 5, 6, 7.

The use of qHTS to produce high quality and reliable data greatly reduces the frequency of false positives and false negatives, which is crucial in computational modeling, low dose extrapolation, and risk assessment. In 2010, Phase II (production phase) of Tox21 was started, with the initial focus on nuclear receptor and stress response pathway assays. Most assays are multiplexed with cell viability measurements to distinguish between true positive responses and those due to increased cytotoxicity (e.g. nuclear receptor antagonism). The Tox21 Phase II library contains approximately 10,000 (10K) compounds. In 2011, a dedicated Tox21 robotic system was installed and made fully operational, which is capable of screening the Tox21 ‘10K’ compound library in 15-point concentration-responses in triplicate each week.

Section snippets

Screening process within the Tox21

To ensure that the Tox21 Phase II is successful, a thorough screening process has been implemented (Fig. 1). The assays nominated by the researchers from government, private, academic, and non-governmental organizations are reviewed by the Tox21 Pathways/Assays working group, one of four working groups (Chemical Selection, Pathways/Assays, Informatics, and Targeted Testing) in Tox21 collaboration, and approved by the Tox21 leadership 5, 8. The assays are selected based on their biological and

Preparation of the Tox21 10K compound library

The Tox21 chemical library represents a wide range of compounds with structural diversity in relation to toxicology and commonly used in the environment (pesticides, industrial, food-use, drugs, among others). Three Tox21 partners – EPA [http://www.epa.gov/ncct/dsstox/sdf_tox21s.html (accessed 02.04.13)], NTP [http://www.epa.gov/ncct/dsstox (accessed 02.04.13)], and NCGC [12] – each contributed over 3000 physical compounds primarily procured from commercial sources. The library totals over

The Tox21 robot

The Tox21 robotic system consists of a high-precision robotic arm (Stäubli, Duncan, SC, USA) surrounded by workstations that include compound plate storage units, assay plate incubators, liquid handlers, and readers (Fig. 3). The six-axis robotic arm with a specially designed gripper and barcode reader [14] is used to transport plates to different stations with a high degree of accuracy and precision. Assay plates are stored in two climate-controlled rotating incubators that can hold 1080

Data processing and analyses

Gross assay performance is assessed initially using quality metrics from each plate and also visually. For each plate, CV, S/B, and Z′-factor are calculated and examined during the execution of the primary screen. ‘Failed plates’ identified by abnormally poor values are inspected visually and, if necessary, excluded from further data analysis. Analysis of compound concentration–response data was performed as previously described [15]. Briefly, raw plate reads for each titration point were first

Validation of Tox21 robotic system

To validate the Tox21 robotic system, a well characterized cell viability assay (CellTiter-Glo® luminescence cell viability assay, Promega, Madison, WI, USA) was run in parallel using either the robotic system (termed online run) or similar standalone equipment (offline run). This assay determines the viability based on intracellular adenosine triphosphate (ATP) content, which is directly linked to metabolically active cells. The assay is conducted by a single addition of the CellTiter-Glo

Concluding remarks

The Tox21 collaboration is a multiagency effort among the EPA, FDA, NTP and NCGC to advance in vitro toxicological testing in the 21st century. After completion of proof of principle in Tox21 Phase I, the focus of Tox21 Phase II is on developing and evaluating a battery of in vitro assays with target-specific and mechanism-based readouts adoptable to a high throughput and high-content screening platform. All of the cell-based pathway/target assays can be run multiplexed together with a cell

Conflicts of interest

None.

Acknowledgments

We gratefully acknowledge Danielle VanLeer and Tongan Zhao for developing the compound plate tracking system. This work was supported by the Intramural Research Programs of the National Toxicology Program (Interagency agreement #Y2-ES-7020-01), the National Institute of Environmental Health Sciences, and the U.S. Environmental Protection Agency.

The views expressed in this paper are those of the authors and do not necessarily reflect the statements, opinions, views, conclusions, or policies of

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