Dataset Information

Comprehensive Analyses and Prioritization of Tox21 10K Chemicals Affecting Mitochondrial Function by in-Depth Mechanistic Studies.

ABSTRACT:

Background

A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput in vitro methods using target/pathway-specific, mechanism-driven assays.

Objectives

Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial function.

Methods

We used a tiered testing approach to prioritize for more extensive testing 622 compounds identified from a primary, quantitative high-throughput screen of 8,300 unique small molecules, including drugs and industrial chemicals, as potential mitochondrial toxicants by their ability to significantly decrease the mitochondrial membrane potential (MMP). Based on results from secondary MMP assays in HepG2 cells and rat hepatocytes, 34 compounds were selected for testing in tertiary assays that included formation of reactive oxygen species (ROS), upregulation of p53 and nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE), mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode Caenorhabditis elegans.

Results

A group of known mitochondrial complex inhibitors (e.g., rotenone) and uncouplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and uncouplers, were detected. From this study, we identified four not well-characterized potential mitochondrial toxicants (lasalocid, picoxystrobin, pinacyanol, and triclocarban) that merit additional in vivo characterization.

Conclusions

The tier-based approach for identifying and mechanistically characterizing mitochondrial toxicants can potentially reduce animal use in toxicological testing. https://doi.org/10.1289/EHP2589.

SUBMITTER: Xia M

PROVIDER: S-EPMC6112376 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Publications

Comprehensive Analyses and Prioritization of Tox21 10K Chemicals Affecting Mitochondrial Function by in-Depth Mechanistic Studies.

Xia Menghang M Huang Ruili R Shi Qiang Q Boyd Windy A WA Zhao Jinghua J Sun Nuo N Rice Julie R JR Dunlap Paul E PE Hackstadt Amber J AJ Bridge Matt F MF Smith Marjolein V MV Dai Sheng S Zheng Wei W Chu Pei-Hsuan PH Gerhold David D Witt Kristine L KL DeVito Michael M Freedman Jonathan H JH Austin Christopher P CP Houck Keith A KA Thomas Russell S RS Paules Richard S RS Tice Raymond R RR Simeonov Anton A

Environmental health perspectives 20180726 7

<h4>Background</h4>A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput <i>in vitro</i> methods using target/pathway-specific, mechanism-driven assays.<h4>Objectives</h4>Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial functi ...[more]

PMID: 30059008

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Project description:Since 2009, the Tox21 project has screened ∼8500 chemicals in more than 70 high-throughput assays, generating upward of 100 million data points, with all data publicly available through partner websites at the United States Environmental Protection Agency (EPA), National Center for Advancing Translational Sciences (NCATS), and National Toxicology Program (NTP). Underpinning this public effort is the largest compound library ever constructed specifically for improving understanding of the chemical basis of toxicity across research and regulatory domains. Each Tox21 federal partner brought specialized resources and capabilities to the partnership, including three approximately equal-sized compound libraries. All Tox21 data generated to date have resulted from a confluence of ideas, technologies, and expertise used to design, screen, and analyze the Tox21 10K library. The different programmatic objectives of the partners led to three distinct, overlapping compound libraries that, when combined, not only covered a diversity of chemical structures, use-categories, and properties but also incorporated many types of compound replicates. The history of development of the Tox21 "10K" chemical library and data workflows implemented to ensure quality chemical annotations and allow for various reproducibility assessments are described. Cheminformatics profiling demonstrates how the three partner libraries complement one another to expand the reach of each individual library, as reflected in coverage of regulatory lists, predicted toxicity end points, and physicochemical properties. ToxPrint chemotypes (CTs) and enrichment approaches further demonstrate how the combined partner libraries amplify structure-activity patterns that would otherwise not be detected. Finally, CT enrichments are used to probe global patterns of activity in combined ToxCast and Tox21 activity data sets relative to test-set size and chemical versus biological end point diversity, illustrating the power of CT approaches to discern patterns in chemical-activity data sets. These results support a central premise of the Tox21 program: A collaborative merging of programmatically distinct compound libraries would yield greater rewards than could be achieved separately.

Project description:Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is possible to formulate mechanism-based hypotheses on the cytotoxic properties of the tested chemicals.

Dataset Information

Comprehensive Analyses and Prioritization of Tox21 10K Chemicals Affecting Mitochondrial Function by in-Depth Mechanistic Studies.

Background

Objectives

Methods

Results

Conclusions

Publications

Comprehensive Analyses and Prioritization of Tox21 10K Chemicals Affecting Mitochondrial Function by in-Depth Mechanistic Studies.

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