Jui-Hua Hsieh

The concordance between RNA-seq and microarray data depends on chemical treatment and transcript abundance

The concordance of RNA-sequencing (RNA-seq) with microarrays for genome-wide analysis of differential gene expression has not been rigorously assessed using a range of chemical treatment conditions. Here we use a comprehensive study design to generate Illumina RNA-seq and Affymetrix microarray data from the same liver samples of rats exposed in triplicate to varying degrees of perturbation by 27 chemicals representing multiple modes of action (MOAs). The cross-platform concordance in terms of differentially expressed genes (DEGs) or enriched pathways is linearly correlated with treatment effect size (R20.8). Furthermore, the concordance is also affected by transcript abundance and biological complexity of the MOA. RNA-seq outperforms microarray (93% versus 75%) in DEG verification as assessed by quantitative PCR, with the gain mainly due to its improved accuracy for low-abundance transcripts. Nonetheless, classifiers to predict MOAs perform similarly when developed using data from either platform. Therefore, the endpoint studied and its biological complexity, transcript abundance and the genomic application are important factors in transcriptomic research and for clinical and regulatory decision making.

Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway

The U.S. Tox21 program has screened a library of approximately 10,000 (10K) environmental chemicals and drugs in three independent runs for estrogen receptor alpha (ERα) agonist and antagonist activity using two types of ER reporter gene cell lines, one with an endogenous full length ERα (ER-luc; BG1 cell line) and the other with a transfected partial receptor consisting of the ligand binding domain (ER-bla; ERα β-lactamase cell line), in a quantitative high-throughput screening (qHTS) format. The ability of the two assays to correctly identify ERα agonists and antagonists was evaluated using a set of 39 reference compounds with known ERα activity. Although both assays demonstrated adequate (i.e. >80%) predictivity, the ER-luc assay was more sensitive and the ER-bla assay more specific. The qHTS assay results were compared with results from previously published ERα binding assay data and showed >80% consistency. Actives identified from both the ER-bla and ER-luc assays were analyzed for structure-activity relationships (SARs) revealing known and potentially novel ERα active structure classes. The results demonstrate the feasibility of qHTS to identify environmental chemicals with the potential to interact with the ERα signaling pathway and the two different assay formats improve the confidence in correctly identifying these chemicals.

Neurite outgrowth in human induced pluripotent stem cell-derived neurons as a high-throughput screen for developmental neurotoxicity or neurotoxicity

Due to the increasing prevalence of neurological disorders and the large number of untested compounds in the environment, there is a need to develop reliable and efficient screening tools to identify environmental chemicals that could potentially affect neurological development. Herein, we report on a library of 80 compounds screened for their ability to inhibit neurite outgrowth, a process by which compounds may elicit developmental neurotoxicity, in a high-throughput, high-content assay using human neurons derived from induced pluripotent stem cells (iPSC). The library contains a diverse set of compounds including those that have been known to be associated with developmental neurotoxicity (DNT) and/or neurotoxicity (NT), environmental compounds with unknown neurotoxic potential (e.g., polycyclic aromatic hydrocarbons (PAHs) and flame retardants (FRs)), as well as compounds with no documented neurotoxic potential. Neurons were treated for 72h across a 6-point concentration range (∼0.3-100μM) in 384-well plates. Effects on neurite outgrowth were assessed by quantifying total outgrowth, branches, and processes. We also assessed the number ofviable cells per well. Concentration-response profiles were evaluated using a Hill model to derive benchmark concentration (BMC) values. Assay performance was evaluated using positive and negative controls and test replicates. Compounds were ranked by activity and selectivity (i.e., specific effects on neurite outgrowth in the absence of concomitant cytotoxicity) and repeat studies were conducted to confirm selectivity. Among the 80 compounds tested, 38 compounds were active, of which 16 selectively inhibited neurite outgrowth. Of these 16 compounds, 12 were known to cause DNT/NT and the remaining 4 compounds included 3 PAHs and 1 FR. In independent repeat studies, 14/16 selective compounds were reproducibly active in the assay, of which only 6 were selective for inhibition of neurite outgrowth. These 6 compounds were previously shown in the literature to be neurotoxic. These studies shed light on the current status of human iPSCs in DNT/NT screening and their utility in identifying, ranking, and prioritizing compounds with DNT/NT potential for further in vivo testing.

Quantitative High-Throughput Profiling of Environmental Chemicals and Drugs that Modulate Farnesoid X Receptor

The farnesoid X receptor (FXR) regulates the homeostasis of bile acids, lipids, and glucose. Because endogenous chemicals bind and activate FXR, it is important to examine which xenobiotic compounds would disrupt normal receptor function. We used a cell-based human FXR β-lactamase (Bla) reporter gene assay to profile the Tox21 10K compound collection of environmental chemicals and drugs. Structure-activity relationships of FXR-active compounds revealed by this screening were then compared against the androgen receptor, estrogen receptor α, peroxisome proliferator-activated receptors δ and γ, and the vitamin D receptor. We identified several FXR-active structural classes including anthracyclines, benzimidazoles, dihydropyridines, pyrethroids, retinoic acids, and vinca alkaloids. Microtubule inhibitors potently decreased FXR reporter gene activity. Pyrethroids specifically antagonized FXR transactivation. Anthracyclines affected reporter activity in all tested assays, suggesting non-specific activity. These results provide important information to prioritize chemicals for further investigation, and suggest possible modes of action of compounds in FXR signaling.

Development of Novel Cell Lines for High-Throughput Screening to Detect Estrogen-Related Receptor Alpha Modulators.

Estrogen-related receptor alpha (ERRα), the first orphan nuclear receptor discovered, is crucial for the control of cellular energy metabolism. ERRα and its coactivator, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), are required for rapid energy production in response to environmental challenges. They have been implicated in the etiology of metabolic disorders such as type 2 diabetes and metabolic syndrome. ERRα also plays a role in the pathogenesis of breast cancer. Identification of compounds that modulate ERRα signaling may elucidate environmental factors associated with these diseases. Therefore, we developed stable cell lines containing an intact ERRα signaling pathway, with and without the coactivator PGC-1α, to use as high-throughput screening tools to detect ERRα modulators. The lentiviral PGC-1α expression constructs and ERRα multiple hormone response element (MHRE) reporters were introduced into HEK293T cells that express endogenous ERRα. A cell line expressing the reporter alone was designated “ERR.” A second cell line expressing both reporter and PGC-1α was named “PGC/ERR.” Initial screenings of the Library of Pharmacologically Active Compounds (LOPAC) identified 33 ERR and 22 PGC/ERR agonists, and 54 ERR and 15 PGC/ERR antagonists. Several potent ERRα agonists were dietary plant compounds (e.g., genistein). In conclusion, these cell lines are suitable for high-throughput screens to identify environmental chemicals affecting metabolic pathways and breast cancer progression.

Assessment of the DNA damaging potential of environmental chemicals using a quantitative high-throughput screening approach to measure p53 activation: Screening for p53 Activation to Identify Genotoxicants

Genotoxicity potential is a critical component of any comprehensive toxicological profile. Compounds that induce DNA or chromosomal damage often activate p53, a transcription factor essential to cell cycle regulation. Thus, within the US Tox21 Program, we screened a library of ∼10,000 (∼8,300 unique) environmental compounds and drugs for activation of the p53‐signaling pathway using a quantitative high‐throughput screening assay employing HCT‐116 cells (p53+/+) containing a stably integrated β‐lactamase reporter gene under control of the p53 response element (p53RE). Cells were exposed (‐S9) for 16 hr at 15 concentrations (generally 1.2 nM to 92 μM) three times, independently. Excluding compounds that failed analytical chemistry analysis or were suspected of inducing assay interference, 365 (4.7%) of 7,849 unique compounds were concluded to activate p53. As part of an in‐depth characterization of our results, we first compared them with results from traditional in vitro genotoxicity assays (bacterial mutation, chromosomal aberration); ∼15% of known, direct‐acting genotoxicants in our library activated the p53RE. Mining the Comparative Toxicogenomics Database revealed that these p53 actives were significantly associated with increased expression of p53 downstream genes involved in DNA damage responses. Furthermore, 53 chemical substructures associated with genotoxicity were enriched in certain classes of p53 actives, for example, anthracyclines (antineoplastics) and vinca alkaloids (tubulin disruptors). Interestingly, the tubulin disruptors manifested unusual nonmonotonic concentration response curves suggesting activity through a unique p53 regulatory mechanism. Through the analysis of our results, we aim to define a role for this assay as one component of a comprehensive toxicological characterization of large compound libraries. Environ. Mol. Mutagen. 58:494–507, 2017.

Gender differences in cancer susceptibility: role of oxidative stress

Cancer is a leading cause of death worldwide and environmental factors, including chemicals, have been suggested as major etiological incitements. Cancer statistics indicates that men get more cancer than women. However, differences in the known risk factors including life style or occupational exposure only offer partial explanation. Using a text mining tool, we have investigated the scientific literature concerning male- and female-specific rat carcinogens that induced tumors only in one gender in NTP 2-year cancer bioassay. Our evaluation shows that oxidative stress, although frequently reported for both male- and female-specific rat carcinogens, was mentioned significantly more in literature concerning male-specific rat carcinogens. Literature analysis of testosterone and estradiol showed the same pattern. Tox21 high-throughput assay results, although showing only weak association of oxidative stress-related processes for male- and female-specific rat carcinogens, provide additional support. We also analyzed the literature concerning 26 established human carcinogens (IARC group 1). Oxidative stress was more frequently reported for the majority of these carcinogens, and the Tox21 data resembled that of male-specific rat carcinogens. Thus, our data, based on about 600000 scientific abstracts and Tox21 screening assays, suggest a link between male-specific carcinogens, testosterone and oxidative stress. This implies that a different cellular response to oxidative stress in men and women may be a critical factor in explaining the greater cancer susceptibility observed in men. Although the IARC carcinogens are classified as human carcinogens, their classification largely based on epidemiological evidence from male cohorts, which raises the question whether carcinogen classifications should be gender specific.

Correction: Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage

[This corrects the article DOI: 10.1371/journal.pone.0177902.].

Tox21 Enricher: Web-based Chemical/Biological Functional Annotation Analysis Tool Based on Tox21 Toxicity Screening Platform

The US Toxicology Testing in the 21st Century (Tox21) program was established to develop more efficient and human‐relevant toxicity assessment methods. The Tox21 program screens >10,000 chemicals using quantitative high‐throughput screening (qHTS) of assays that measure effects on toxicity pathways. To date, more than 70 assays have yielded >12 million concentration‐response curves. The patterns of activity across assays can be used to define similarity between chemicals. Assuming chemicals with similar activity profiles have similar toxicological properties, we may infer toxicological properties based on its neighbourhood. One approach to inference is chemical/biological annotation enrichment analysis. Here, we present Tox21 Enricher, a web‐based chemical annotation enrichment tool for the Tox21 toxicity screening platform. Tox21 Enricher identifies over‐represented chemical/biological annotations among lists of chemicals (neighbourhoods), facilitating the identification of the toxicological properties and mechanisms in the chemical set.