Chemical genomics profiling of environmental chemical modulation of human nuclear receptors

Abstract

Background: The large and increasing number of chemicals released into the environment demands more efficient and cost-effective approaches for assessing environmental chemical toxicity. The U.S. Tox21 program has responded to this challenge by proposing alternative strategies for toxicity testing, among which the quantitative high-throughput screening (qHTS) paradigm has been adopted as the primary tool for generating data from screening large chemical libraries using a wide spectrum of assays.

Objectives: The goal of this study was to develop methods to evaluate the data generated from these assays to guide future assay selection and prioritization for the Tox21 program.

Methods: We examined the data from the Tox21 pilot-phase collection of approximately 3,000 environmental chemicals profiled in qHTS format against a panel of 10 human nuclear receptors (AR, ERα, FXR, GR, LXRβ, PPARγ, PPARδ, RXRα, TRβ, and VDR) for reproducibility, concordance of biological activity profiles with sequence homology of the receptor ligand binding domains, and structure-activity relationships.

Results: We determined the assays to be appropriate in terms of biological relevance. We found better concordance for replicate compounds for the agonist-mode than for the antagonist-mode assays, likely due to interference of cytotoxicity in the latter assays. This exercise also enabled us to formulate data-driven strategies for discriminating true signals from artifacts, and to prioritize assays based on data quality.

Conclusions: The results demonstrate the feasibility of qHTS to identify the potential for environmentally relevant chemicals to interact with key toxicity pathways related to human disease induction.

Figure 1

Distributions of compound activity outcomes in the agonist-mode (A) and antagonist-mode (B) NR assays. (C) Distribution of active agonists and antagonists.

Figure 2

Intralibrary (A) and interlibrary (B) compound reproducibility across different NR assays. Intralibrary reproducibility is calculated by comparing the activity of copies of each compound replicated within the U.S. EPA or NTP compound library. Interlibrary reproducibility is calculated by comparing the activity of the NTP copy and U.S. EPA copy of each compound presented in both the U.S. EPA and NTP libraries.

Figure 3

Comparison of the human NR LBD similarity and compound activity-pattern similarity. (A) and (B) Hierarchical clustering (Spotfire DecisionSite, version 8.2; Spotfire Inc., Cambridge, MA) of the agonist-mode (A) and antagonist-mode (B) NR assays using the correlation of the compound curve ranks (from the ratio readout) as the similarity metric, where each row represents a compound and each column represents an NR assay. The heat maps are colored based on compound activity: compounds that showed apparent activation (red) and inhibition (blue); less conclusive activators or inhibitors are colored a lighter shade of red or blue; inactive compounds are shown in white. (C) Phylogram of the LBDs of the nine human NRs tested. Amino acid sequences of the LBDs were downloaded from the PubMed protein database (PubMed 2010) and aligned using ClustalW2 (European Molecular Biology Laboratory–European Bioinformatics Institute 2009).

Figure 4

Example structure classes with consistent NR activity patterns or signatures. Compounds were clustered by structure similarity using the SOM algorithm. Compounds in the same cluster belong to the same structure class. The structure classes shown contain compounds with similar activity patterns as well. DDT, dichlorodiphenyltrichloroethane.